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During the past three decades, the life expectancy for individuals with hemophilia a (a hereditary deficiency of coagulation factor viii) and hemophilia b (a hereditary deficiency of coagulation factor ix) has markedly increased, primarily due to advances in medical care, as well as the introduction and availability of clotting factor replacement products and effective treatments for infectious diseases [110]. However, with this increased longevity and improved quality of life, comes a generation of middle - aged and elderly hemophiliacs that are experiencing age - related health conditions that have subsequently not been seen in this population [1, 6, 10]. Prior to the availability of factor replacement therapy, the majority of patients with hemophilia died at an early age from bleeding problems [2, 4, 11]. Throughout the 1980s and 1990s, high death rates were observed, due to blood - borne viral infections, specifically human immunodeficiency virus (hiv) and the hepatitis c virus (hcv), and replaced bleeding complications as the primary cause of death among hemophiliacs [5, 12]. Nevertheless, barring the increased mortality rates discerned during this period, the life expectancy for patients with hemophilia, particularly those with mild or moderate hemophilia, in high and middle income nations, is currently drawing near to that of the general male population [13, 14]. While a variety of age - related co - morbidities have been reported among males in the general population, only a modest amount of information exists regarding these new causes of age - related morbidity and mortality and more specifically how to deal with these conditions among individuals with hemophilia [1, 5, 6, 11]. The primary focus of this study was to identify and estimate the prevalence of co - morbid conditions that were associated with middle - aged and elderly hemophilia patients attending at texas treatment center and evaluate the implications these conditions may have on future care among individuals in this population . We conducted a retrospective study among adult patients, 40 years of age or greater, who had a clinical diagnosis of hemophilia a or hemophilia b, and attended the gulf states hemophilia and thrombophilia center (gshtc) at least once between january 2007 and august 2010 . The gshtc is the largest treatment center in the southwest and one of the largest in the country . Data were obtained from the patient's medical charts using a standardized data abstraction form, and information regarding the patient's demographic characteristics, as well as clinical and co - morbid conditions, was examined . Laboratory findings, in conjunction with physician notes as well as medication and problem lists from the patient's medical charts, were used to determine whether or not they had a specific comorbidity . Furthermore, the diagnostic criteria used to define individual co - morbid conditions are described in table 1 . Patients who met the inclusion criteria but died during the specified observation range were also included in the study . In addition, we defined comorbidity as the presence of one or more diseases / conditions, other than the patient's hemophilia disease . Differences between patients with hemophilia a and hemophilia b were assessed using fischer's exact test . In addition, the prevalence of co - morbid conditions was examined among this patient cohort and compared to that of general us male population when data were available . Characteristics of the study sample according to their disease status are summarized in table 2 . Between january 2007 and august 2010, the gshtc saw a total of 404 hemophilia patients, roughly 16% were 40 years of age or greater . Of the 63 patients who met the inclusion criteria, the majority had hemophilia a, 71%, respectively . The mean age of the cohort was approximately 53 years for patients with hemophilia a and 54 years among patients with hemophilia b. in addition, the majority of the sample visited the treatment center within one year of the observation period was treated on demand and had mild hemophilia . Patients with hemophilia a and hemophilia b did not differ significantly on any of the variables examined with the exception of race / ethnicity and employment status . Table 3 presents the prevalence of specific co - morbidities among the study sample, including comparisons to the age - matched, general us male population when data were available . Confidence limits were also estimated in order to draw comparisons to that of the general us population . However, due to the small sample size of the study cohort, it is difficult to draw significant comparisons of the representativeness of the prevalence estimates reported between our patient population and that of the general us male population . All patients in the study had at least one co - morbid condition other than their hemophilia disease, with the majority having between 3 and 6 conditions (mean number of conditions in the sample was 4). As expected, more than half of the sample suffered from hemophilic arthropathy, 55% of which had severe hemophilia . Only 21% of the cohort (13 patients) did not have any blood - borne viral infections: 6% had hepatitis b virus (hbv) infection, 25% had hiv, 78% had hcv, 25% were coinfected with hiv and hcv, and 5% had both hbv and hcv . Additionally, differences in co - morbidities between hemophilia a and hemophilia b patients were also examined . While the results did show differences between the groups with respect to their hiv, hcv, cardiovascular disease, renal disease, and arthropathy status, it is difficult to provide any meaningful statistically significant comparisons due to the small sample size of the study cohort . Causes of death included hepatocellular carcinoma (hcc), acute myeloid leukemia, and lung cancer, and 2 patients died from accidental, nonmedical factors . Additionally, 5 patients in the sample, all of whom had hemophilia a (1 patient had mild hemophilia a, and 4 patients had severe hemophilia a), received a liver transplant during the observation period . Among patients receiving a liver transplant, 80% were diagnosed with end - stage liver disease due to hcv . Over three - fourths (78%) of the study population had at least one cardiovascular risk factor including hypertension (46%), a body mass index (bmi) of 25.0 or higher (65% of the sample was overweight or obese), high cholesterol (16%), and diabetes mellitus (14%). In addition, the prevalence of other co - morbidities that were examined among patients in this population are reported in table 2 and include renal disease (8%), thyroid disease (6%), depression (8%), neurological disease (5%) including parkinson's disease, epilepsy, and dementia, respiratory disease (11%) including asthma, sleep apnea, sarcoidosis, and chronic obstructive pulmonary disease, and gastrointestinal disease (5%) including peptic ulcer disease and acid reflux disease . This study revealed a variety of notable differences with regard to comparing the prevalence of multiple co - morbid conditions among middle - aged and elderly hemophilia a and hemophilia b patients to that of the general us male population . Previous research has suggested that as much as 62% of the us population over the age of 65 have more than one co - morbid condition . Results from this study indicated that all of the patients in this sample had more than one co - morbid condition other than their underlying hemophilia disease, and roughly 19% of the cohort over the age of 65 had at minimum 3 or more co - morbid conditions . As noted in previous studies, the prevalence of hiv and hcv infections are considerably higher among hemophiliacs than in the general male population [4, 5] and are a leading cause of morbidity and mortality in this population [2, 22, 23]. It is estimated that greater than 90% of hemophiliacs who were treated with plasma - derived factor concentrates before 1985 became infected with hcv, and greater than 55% of these patients were also coinfected with hiv . Among patients in this cohort who were between 40 and 49 years of age (n = 29), the prevalence of hiv infection was approximately 38% compared to 0.74% among age - matched males in the us . In addition, the prevalence of hcv infection in our patient sample was 12 to 90 times higher than in the age - matched male population (4049 years: 72% versus 6%; 5059 years: 83% versus 1.6%; 60 years: 81% versus 0.9%). Furthermore, chronic hcv infection is a major risk factor in the development of liver cirrhosis and hcc [11, 25], and, subsequently, hcc has emerged as a significant cause of mortality among hcv - infected hemophiliacs . Roughly 11% of patients in this sample who were diagnosed with hcv progressed to hcc; one patient died, and more than half (57%) received a liver transplant . Since the risk of hcc among hcv - infected hemophilia patients increases with older age and the presence of hiv infection [11, 22, 23], it is expected that many of the hcv - infected patients in this sample will progress to end - stage liver disease or hcc . While cardiovascular disease (cvd) has been the primary cause of death among individuals in the us for the past eight decades, several studies have reported that mortality from cardiac events among individuals with hemophilia is lower than in the general population [7, 8, 13, 27, 28] and hemophilia may actually have a protective effect against cvd [4, 11, 28]. The prevalence of cvd among patients between 40 and 59 years of age in this study was similar compared to the age - matched us male population (40% versus 39%), but slightly lower among those patients of 60 years of age or greater (69% versus 71%). However, it should be noted that in order to provide comparisons between this cohort to that of age - matched males in the us, patients with cvd were defined as having any of the following conditions (based on the national center for health statistics definition): congestive heart disease, heart failure, stroke, and hypertension . If we excluded hypertension from the analysis and defined cvd as having only one of the following conditions: congestive heart disease, heart failure and/or stroke, the prevalence of cvd among all patients in the study was approximately 11% . Despite conflicting reports regarding a possible protective effect against cvd among individuals with hemophilia [4, 11, 28], common risk factors for cvd including hypertension, high cholesterol, diabetes mellitus (dm), and overweight / obesity the prevalence of hypertension was lower compared to the us population among all age groups except among patients between 55 and 64 years of age . No differences were observed between our patient population and the age - matched general population with respect to high cholesterol . While over 65% of this cohort was overweight or obese, these rates were lower compared to the age - matched population . In addition, overweight and obesity were more prevalent among patients with mild hemophilia (72%), which is similar to a finding reported in a previous study . An increased bodyweight is an important risk factor associated not only with cvd, but also in the development of dm and chronic arthropathy . Only a limited amount of information exists regarding the prevalence of dm among hemophiliacs . The study conducted by walsh et al . Estimated that the prevalence of dm among mild hemophiliacs was 24% compared to 6% in control males (mean age of subjects in both groups was 46 years of age). The prevalence of dm among all patients in this sample was 14%, and roughly 89% of those patients who had dm also had a bmi greater than 25 . The majority of hemophiliacs born prior to the availability of prophylactic therapy suffer from hemophilic arthropathy, which continues to be the primary cause of morbidity among individuals in this population . As expected, half of the patients in this cohort suffer from chronic arthropathy, of which 56% had severe hemophilia and 53% had a bmi of 25 or higher . In addition, overweight and obesity may have greater implications on persons with hemophilia due to the fact that an increased bodyweight may cause additional damage to already deteriorated joints . Furthermore, since the dosage of factor replacement treatment is based on bodyweight, the costs of care are much higher among overweight and obese patients compared with patients with a normal bodyweight . Renal disease is another age - related medical condition that affects hemophiliacs, who are reported to have up to a 50-fold increase in mortality due to renal failure compared to the general population . Previous research has indicated that risk factors for renal failure among individuals with hemophilia include increased age, hypertension, and hiv co - infection [1, 32]. Approximately 8% of patients in this sample had chronic renal disease, 20% of which also had hiv, and 40% of which had hypertension . With the mean age of all patients in this cohort being 53 years of age, it is likely that more cases of renal disease will be observed as the population continues to age . While hemophilia care has undergone substantial advancements during the past three decades, a variety of healthcare needs arising from many of the age - related co - morbidities mentioned in this paper pose significant challenges for the treatment and management of these conditions among aging hemophiliacs . Currently, there are few evidence - based guidelines that direct medical professionals on how to most effectively manage the comprehensive care needs of hemophilia patients with these age - related co - morbidities [1, 11]. As a result, optimal treatment and care for hemophiliacs with multiple co - morbid conditions has been challenging for both healthcare providers and the patient . Further studies are needed to document the safety and efficacy of certain drug therapies, procedures, and lifestyle changes among aging hemophiliacs with multiple co - morbid conditions . For example, no explicit guidelines are available for addressing cvd risk in persons with hemophilia . While there are modifiable lifestyle factors that can be addressed including diet, exercise, and smoking cessation programs, safety of frequent aspirin use needs to be evaluated as this could cause an increase in the bleeding frequency among hemophiliacs . In addition, with regard to lifestyle factors such as exercise and activity programs to help reduce the risk of cvd as well as other conditions such as overweight / obesity and diabetes, these may not be practical and/or may be difficult particularly for patients with chronic arthropathy . Support from physical therapists at treatment centers may play a critical role in promoting an active lifestyle within the boundaries of a patient's abilities . Given the complexities associated with how to best care for and treat hemophiliacs with multiple co - morbid conditions, successful management must entail a multifaceted approach, which encompasses drawing from expertise not only from hematology, but that of cardiology, oncology, urology, infectious disease, orthopedics, hepatology, nephrology, and internal medicine . A lack of coordination between these various departments may affect the delivery of appropriate and timely healthcare services . Hemophilia treatment centers should play an essential role in coordinating the care for these patients to ensure they are knowledgeable of the services they need as well as understand the implications associated with seeking timely care from these services . However, management and treatment requirements for specific co - morbidities need to be an interdisciplinary effort, and hemophilia treatment centers cannot be the sole caregivers for hemophiliacs with multiple co - morbidities . As such, lifestyle issues as well as general screening programs should be incorporated in the management and treatment plans, including assistance with timely referrals and followup with appropriate specialty services for aging hemophilia patients attending treatment centers . Individuals with multiple co - morbid conditions tend to receive suboptimal care, which can lead to poorer health outcomes and increased treatment costs . In addition, the challenge of providing adequate care increases in complexity as the number of chronic conditions increases [21, 33], resulting in disproportionately high health care costs . It is estimated that over 75% of health care expenditures in the us are spent on care for persons with multiple co - morbidities and the average spending per person with co - morbidities is roughly five times greater compared with individuals with no chronic conditions . As the world population of persons with hemophilia ages, an increase in age - related health conditions not previously seen in this population are likely to become more prevalent . Hemophilia is an already costly disease, and understanding both treatment care needs and its related costs among aging hemophiliacs with multiple co - morbidities is critical in order to provide optimal care and manage their comprehensive health needs effectively . While this study identified a variety of co - morbidities associated with middle - aged and elderly hemophiliacs, only 63 patients were included in the cohort, thus making it difficult to provide any statistically significant comparisons . Moreover, due to the small sample size of the cohort, our estimates of co - morbidities make it difficult to draw significant comparisons between our hemophilia patient population and the general us male population . In addition, since patients were classified as having a specific condition based on information available in their medical charts, the possibility of underestimating certain co - morbidities may be of concern . Further research, particularly larger, multicenter, prospective studies are needed to identify how to best care for and treat aging hemophilia patients with multiple co - morbidities.
Patient 1 was a 52-year - old woman with a history of hypertension who became ill on may 1, 2014, and was admitted to hospital a on may 11 with high fever (temperature> 38c), cough, dyspnea, diarrhea, and anorexia . Her condition deteriorated, and she was transferred to an intensive care unit (icu). Her condition remained poor, and on may 29, 18 days after her symptoms began, she died of progressive respiratory failure . Patient 1 had not traveled to saudi arabia, but she had had close contact with a woman who had influenza - like illness and who had traveled to saudi arabia 2 weeks before her symptoms began . This contact of patient 1 is suspected of being the index case - patient, but when throat swab and sputum samples were collected from her, she had no symptoms, and pcr results were negative . A serum sample was not tested because serologic testing for mers - cov was not available . Patient 2 was the 50-year - old sister of patient 1 and also had a history of hypertension . She became ill on may 11, 2014, with fever (temperature> 38c), cough, hemoptysis, nausea, vomiting, and anorexia . She was admitted to hospital a on may 17; her condition improved, and she was discharged on may 30, 19 days after onset of symptoms . Patient 3 was a 35-year - old female nurse assistant at hospital a who had no underlying medical conditions . Her symptoms of sore throat and productive cough were detected on may 26 as part of the investigation of the first 2 cases; co - infection with influenza a(h1n1)pdm09 was detected . Patient 3 was advised to stay home and follow infection control precautions until respiratory samples tested negative . Patient 4 was a 44-year - old male physician at hospital a with a history of chronic heart disease who had contact with patient 1 during her hospitalization in icu . Mild respiratory symptoms developed in patient 4 on june 6; his condition deteriorated, and he was admitted to a hospital in tehran, iran, on june 17 with fever (temperature> 38c), sore throat, cough, dyspnea, chills, anorexia, and myalgia . Patient 4 s symptoms were initially severe, but his condition improved, and he was discharged on june 21 . Patient 5 was a 67-year - old woman who was admitted to hospital a on june 6 because of exacerbation of chronic obstructive pulmonary disease . She was discharged from the hospital on june 14 and was in stable condition until severe acute respiratory infection (sari) developed . She was readmitted to hospital a with fever (temperature> 38c), cough, and dyspnea on june 25 . During her first hospitalization, the patient had close contact with another patient who had sari but had tested negative for mers - cov . A respiratory sample from patient 5 was obtained on june 30, and she died on july 5 . All 5 patients were residents of kerman province and had no history of travel or contact with animals in the 14 days before becoming ill . Throat swab specimens and sputum samples were collected and analyzed by using real - time reverse transcription pcr (rt - pcr) performed on the basis of a previously reported method by targeting the upstream e region and open reading frame 1b of the virus (5). The samples from patients 1, 2, and 4 yielded n gene sequences positive for mers - cov . Phylogenetic analysis showed differences between these sequences and a consensus sequence retrieved from genbank (accession no . All 3 sequences from these cases had polymorphisms at positions 28880 (tc), 28941 (gc), and 29097 (tg). For the isolate from patient 4, another nonsynonymous mutation was observed at position 29329 (c to t), which resulted a change of tyrosine to isoleucine . In all 3 sequences, nucleotide c was detected at position 29147, as was the case with the first identified isolate of mers - cov . For some sequences in genbank, this position contains t. phylogenic sequence analysis of 3 middle east respiratory syndrome coronavirus (mers - cov) isolates from patients in kerman province, iran (boldface), 2014, compared with sequences from genbank (accession numbers shown). Mega 5.2 (http://www.megasoftware.net) was used for construction of neighbor - joining tree by using the kimura 2-parameter model with uniform rates and 1,000 bootstrap replicates . We identified a cluster of mers - cov infections in iran (table), showing apparent person - to - person transmission but with unclear transmission routes for some patients . In this cluster, patient 1 was in close contact with a person suspected of being the index case - patient, but we were unable to verify the infection status of this patient . The source of infection for patients 3 and 4 was patient 1 or 2, but the source for patient 5 s infection remains unknown . However, subclinical cases of mers - cov infection have been reported to the world health organization (7); exposure to a person with subclinical infection could explain an active infection that has an unknown route of transmission . Throat swab specimens and sputum samples were collected from all close contacts of the 5 patients in this cluster, including family members, other patients in the hospital, and health care workers . Patient 1 had a pregnant daughter who was a frequent visitor during her hospitalization but who tested negative for mers - cov by real - time rt - pcr . Before patient 1 was hospitalized, none of her contacts showed signs of mers - cov infection, but after her hospitalization (during her second week of her illness), her sister became ill and subsequently tested positive for the virus . This finding suggests that, as with severe acute respiratory syndrome, mers - cov is not readily transmitted during the early phases of the disease (3), in contrast to the other human coronaviruses, which are transmitted early in the infection (2). Early recognition of confirmed mers - cov infections and investigation of the contacts of these patients are critical for effective epidemic control . Because saudi arabia has reported the highest number of mers - cov infections, one approach for limiting the transmission of this virus may be to screen travelers from iran who report sari to detect mers - cov . However, screening of pilgrims from iran who traveled to mecca during the 2013 hajj did not detect mers - cov infections (national influenza center iran, unpub . First, some persons who may have had mers - cov infection were not tested, such as the probable index case - patient with whom patient 1 had contact, the patient with sari with whom patient 5 had contact, and the contacts of these persons . Second, we performed n gene pcrs on samples from all 5 case - patients, but results were negative for patients 3 and 5, which suggests that these samples should be tested with more specific primers . In summary, we identified 5 cases of mers - cov in the same province in iran; for several of these cases, virus transmission routes were not clearly defined . Future research should focus on clarifying routes of transmission for this virus, including the possibility of transmission from persons with subclinical infection.
A central aim of untargeted metabolomics and metabonomics studies is the identification of marker metabolites which play a crucial role in the experimental context [1, 2]. Mass spectrometry combined with either gas chromatography (gc / ms) or liquid chromatography (lc / ms) has become a key technology for metabolome analysis under different experimental conditions [3, 4]. A typical data set after peak detection and sample alignment [57] consists of several thousand marker candidates which are characterized by a retention time (rt), a mass - to - charge value (m / z), and a multivariate intensity profile of abundance levels per condition, respectively . The experimental conditions are represented by replicate samples and may correspond to environmental disease or genetic perturbations [911]. In order to obtain a high - quality data set of experiment - related marker candidates, the raw data set is usually ranked and filtered using supervised machine learning techniques such as random forest classification [12, 13] or statistical analysis based on anova or kruskal - wallis tests [1416]. The filtered marker candidates are then annotated according to known metabolites from public biological and biomedical compound databases [1721]. A central task of annotation is the calculation of actual molecular masses corresponding to each marker candidate by correcting the m / z ratios according to the ionization mode, potential adduct formation, and included natural isotopes . This problem can be addressed by applying the ionization rules [xm+y], where x denotes the number of combined target molecules, y the mass of attached molecules (adduct formation), and z the degree of ionization (e.g., single or double). Additionally, the number of included isotopes has to be estimated in order to query databases which contain monoisotopic compound masses . Based on a potential ionization rule with parameters x, y, z and the number of included isotopes, the corresponding compound mass can be calculated . For the corrected masses which cannot be assigned to particular compounds, the number of considered formulas can be significantly reduced by incorporating information from preprocessing as well as rules for heuristic filtering of molecular formulas, respectively . A major step in this process is the estimation of the number of included carbon atoms based on the intensity profiles of previously detected isotopologues . There are a great number of software packages available, which provide tools for statistical analysis of multivariate experimental data [25, 26]. A number of tools for peak detection and sample alignment of mass spectrometry data, such as metalign or openms, also support the deconvolution of isotopologues and statistical analysis [27, 28]. For the xcms platform, a package for the annotation of lc / esi - ms mass signals based on adduct rules has been implemented . The calculation of possible ionization products and the rule - based heuristic filtering of molecular formulas is provided by several software packages [22, 24]. However, to the best of our knowledge, there is no software available which incorporates all of these methods in a single user - friendly tool as offered by marvis - filter . In the following sections, the algorithm for adduct / isotope correction and the implementation of marvis - filter are described in detail . The algorithm is based on the input of the retention times, m / z ratios, and raw intensity profiles of all marker candidates in a data set and calculates as output the potential monoisotopic mass, ionization rule, and number of included c - isotopes for every candidate . The approach is based on a greedy strategy which minimizes the number of potential molecular masses and simultaneously maximizes the similarity of intensity profiles between candidates with a similar retention time and actual mass . This concept follows the paradigm that in mass spectrometry analysis a metabolite is usually represented by several marker candidates with a similar retention time and intensity profile, but different m / z ratios according to the various possibilities of ionization and number of included isotopes . As parameters, the algorithm expects a list of ionization / adduct rules sorted according to their relevance, the assumed maximal number of c - isotopes per marker candidate, a mass tolerance, an rt tolerance, and a minimal cosine similarity of intensity profiles . The isotopologues correction is restricted to the detection of c. for storage of pairwise cosine similarities between candidate profiles, the algorithm utilizes a five - dimensional matrix m. each entry m(m, a1,i1,a2,i2) corresponds to the maximal cosine similarity between the intensity profile of candidate m, assuming ionization rule a1 and i1c - isotopes, and another candidate, which has a similar retention time (within tolerance) and corrected mass (within tolerance) assuming ionization rule a2 and i2c - isotopes . For each candidate m, the algorithm then chooses the ionization rule and number of c - isotopes which is supported by the highest sum of cosine similarities . In the following calculate all possible masses by applying all ionization rules and number of c - isotopes to all candidate m / z ratios . Consider all pairs of potential masses under the following constraints and fill m with pairwise cosine similarities of corresponding candidate profiles . Consider only pairs of different marker candidates.consider only pairs within the mass and rt tolerance.consider only pairs with at least the requested cosine similarity . For each entry in m hold only the maximum cosine similarity . Consider only pairs of different marker candidates . Calculate the reduced three - dimensional matrix m with summed entries: (1)m(m, a1,i1)red=a2,i2m(m, a1,i1,a2,i2). Choose for each candidate m: the adduct rule and isotope number with the maximal sum of similarities cmax = maxa1,i1(m(m, a1,i1)). If cmax = 0, use the first ionization rule and zero c - isotopes as default . Calculate the masses according to chosen rules and isotope numbers . In order to avoid apparently false associations between marker candidates, negative cosine similarities are disregarded . If for a given candidate different selections of the ionization rule and the number of isotopes maximize the sum of cosine similarities, the ionization rules with the highest relevance and the minimal number of c - isotopes are selected . Following the annotation of the ionization rules and c - isotopes, the number of carbon atoms per candidate is estimated by comparing the raw intensities of marker candidates with zero predicted c - isotopes (i m) and the respective marker candidates including one c - isotope (im+1) according to the following formula: (2)nc=98.9 im+11.1 i m, corresponding to the natural abundances of carbon isotopes . Given a pair of candidates, annotated as isotopologues (m and m + 1) and with the same ionization rule, a robust estimation of the number of carbon atoms is obtained by calculating the median nc over all samples included in both intensity profiles . Marvis - filter is implemented in the matlab and c programming language and has been compiled together with the marvis - cluster tool for microsoft windows xp / vista/7 . Execution of the software requires installation of the matlab compiler runtime, which is provided with the software . The installation packages, the documentation, and example data sets can be downloaded from the project home page http://marvis.gobics.de/. For data import and export marvis - filter uses the csv (comma separated values) file format, which can easily be processed by statistical analysis software and spreadsheet applications . Marvis - filter also supports the direct import of aligned mass spectrometry samples from markerlynx application manager of masslynx (waters corporation, milford). For interactive analysis, ranking and filtering of multivariate intensity profiles marvis - filter provides the well - known one - way anova and kruskal - wallis tests combined with methods for p value adjustment for multiple - hypothesis testing [30, 31]. Based on customizable lists of ionization rules, the adduct / isotope correction can be performed on raw or filtered data sets . The ionization rules are imported as text files and can easily be adapted or extended . (1) displays the adjusted p values (y - axis) of all candidate intensity profiles in the current data set sorted in ascending order . The data set can interactively be filtered according to a user - defined significance level by selecting a marker, sliding the red separator line or jumping to a predefined level . The profile plot (2) shows the raw intensity profile of the currently selected marker candidate . Intensity values of replicated samples belonging to the same experimental conditions are marked in the same color . (3) displays information about all marker candidates of the data set arranged according to the p values and characterized by the m / z ratio, rt and additional user - defined scores, which can be imported along with the data set . After adduct and isotope correction, the additional annotations are displayed in this listbox as well . Data set clipboard listbox (4) shows data sets which are currently held in the marvis clipboard . The current (filtered or unfiltered) data set can simply be added or removed to / from this list . The data set clipboard supports an adduct and isotope correction of selected data sets in a batch mode . Data sets which were corrected based on different sets of ionization rules (e.g., positive and negative ionization) may be combined into one single data set . For selected candidate profiles, bar plots, standard error plots, and boxplots can easily be inspected and exported in various image formats . For detailed analysis, the user can zoom into all plots . Additionally, marvis - filter provides a convenient interface for quick candidate search based on the i d, rt, m / z, or mass value . Marvis - filter also provides a molecular formula calculator, which is based on the seven golden rules and utilizes the estimated number of carbon atoms per marker candidate obtained after adduct and isotope correction . Marvis - filter and marvis - cluster are combined in the marvis - suite which features the direct data exchange between preprocessing in marvis - filter and convenient visualization of multivariate intensity profiles and high - level cluster analysis in marvis - cluster . The functionality of marvis - filter is demonstrated using two data sets of a metabolomic case study for plant wounding experiments . The data sets are available on the project homepage http://marvis.gobics.de/ together with a detailed description of the extraction and uplc - tof method . Additionally, the data sets are available for import in marvis - filter after installation of the marvis - suite (wound_neg_raw.csv and wound_pos_raw.csv in the examples directory). The case study reflects a wounding time course of arabidopsis thaliana wild - type (wt) plants as well as of mutant plants (dde 2 - 2), which are deficient in the biosynthesis of the plant wound hormone jasmonic acid and its derivatives . The first four conditions reflect the metabolic situation within a wounding time course of wild - type (wt) plants, starting with the unwounded control plants (abbreviation wt_0) followed by the plants harvested 0.5 (wt_30), 2 (wt_2), and 5 hours past wounding (wt_5). The conditions 5 to 8 represent the analogous time course for the jasmonate deficient mutant plant dde 2 - 2 (aos_0, aos_30, aos_2, aos_5). The two data sets are imported sequentially in marvis - filter using the import raw csv data entry in the file menu with the following options: delimiter:,; start row: 5, start column: 3; i d label: i d; generate ids: activated; x column: 2; x label: rt; y column: 3; y label: m / z; condition identifiers: wt_0, wt_30, wt_2, wt_5, aos_0, aos_30, aos_2, aos_5 . After data import, the marker candidates are sorted and ranked according to the p values of a kruskal - wallis test and the bonferroni - holm adjustment for multiple hypothesis testing by selecting the corresponding checkboxes in the filter dialog and the adjustment for multiple testing dialog . Adduct and isotope correction are performed on the full data sets separately using predefined sets of adduct rules for the negative (table 2) and positive ionization mode (table 3), an rt tolerance of 0.04 minutes, a mass tolerance of 0.005 da, a minimal cosine similarity of 0.75, and a maximum number of two c - isotopes per candidate . The adduct rules had been determined in previous targeted uplc - tof - ms experiments . After correction, the data sets are filtered according to a significance level for adjusted p values of 0.01 (goto level entry in selection table 1 shows the initial number of imported marker candidates and the number of high - quality marker candidates after filtering . Finally, the two data sets in the marvis clipboard are concatenated using the combine button . The combined data set can be sorted according to a user - defined method once again and is then presented in a new marvis - filter window . After selecting the whole data set, the combined subset of 3504 high - quality marker candidates can be exported as a csv file, and clustered as well as visualized using marvis - cluster (goto marvis - cluster entry in the marvis - suite menu). Figure 2 shows the results from clustering of the filtered and combined data in marvis - cluster . The corrected, filtered, and combined data sets were used to identify metabolites which show a significant change of abundance in the wound time course in wt and/or jasmonate deficient mutant plants . First, the corrected masses of marker candidates were matched to molecular masses of all compounds recorded in the kegg and aracyc database or literature based on a tolerance of 0.005 da . The identity of marker candidates was confirmed based on the isotopic pattern and coelution with identical standards or ms / ms fragmentation . Thus, a number of oxylipins could be identified as wound - induced metabolite markers (see table 4). Oxylipins are metabolites deriving from lipid peroxidation and are involved in regulating developmental processes as well as environmental responses, like the inflammatory or wound response, in nearly every organism . Among these bioactive lipids, mammals use predominantly c20 fatty acids (eicosanoids), while in plants c18 fatty acids are most abundantly used for the biosynthesis of oxylipins or so - called octadecanoids . The identified oxylipins (see table 4) are part of the -linolenic acid metabolism or members of the compound class of mono- and digalactosyldiacylglycerols . They are described in the context of plant wounding [33, 34, 36]. Thirteen of the fifteen identified oxylipins could only be detected in either the negative or the positive ionization mode . The findings are supported by very low adjusted p values from the kruskal - wallis test of the intensity profiles (see previous section and table 4). Marvis - filter combines essential preprocessing tools for mass spectrometry data analysis within a single user - friendly tool . Large data sets from the negative and positive ionization mode can easily be imported, corrected, filtered, and combined . Lists of ionization rules for adduct correction can be customized, extended, and commented in a convenient way using a standard text editor . Within the marvis - suite filtered and combined data sets can directly be clustered, visualized, and analyzed in detail using the marvis - cluster tool . In a case study 75 high - quality marker candidates could be clearly assigned to fifteen compounds of the oxylipin class based on the adduct and isotope correction in marvis - filter . The combination of data sets deriving from the negative and positive ionization mode is an important step for further data analysis . In the case study, most of the identified metabolites the significance of the selected wound markers is supported by a high number of annotated and assigned ions / marker candidates and by very low adjusted p values from the kruskal - wallis test . The statistical filtering of marker candidates reduced the complexity of the data sets from about 48000 to 3500 significant candidates (about 7 percent).
Women differ from men in coronary artery disease epidemiology, symptoms, pathophysiology, and clinical outcome [13]. Several studies have reported that women are associated with an increased mortality rate following coronary interventions compared with men . The worse outcomes in women might be partly explained by some clinical factors such as delayed onset of disease, older age, smaller body surface area, smaller vessel, and comorbidities . However, whether women have smaller vessel and different plaque characteristics on atherosclerotic segment have not been clearly documented . And it remains uncertain whether plaque components of coronary stenotic lesion differ between men and women . Recently, virtual histology - intravascular ultrasound (vh - ivus) using spectral analysis of the radiofrequency ultrasound backscatter signals was introduced to clinical practice to characterize the plaque composition . The purpose of this study was to examine the gender differences of the plaque characteristics in culprit lesions of stable angina patients as assessed by vh - ivus . Between january 2006 and december 2006, preinterventional vh - ivus was performed in 240 patients with stable angina undergoing elective coronary intervention . Stable angina was defined as no change in the frequency and duration of cardiac ischemic symptoms within 4 weeks before the intervention . In the present study, inclusion criteria were defined as follows: (1) a de novo lesion with> 75% angiographic stenosis and (2) over 60-year old patients for matching age difference between gender . We excluded patients with bypass graft lesions, hemodialysis, acute coronary syndrome, or old myocardial infarction . Finally, 148 patients (88 men and 60 women) with stable angina were eligible and enrolled in this study . A 20 mhz, 3.2 f, vh - ivus catheter (eagle eye gold, volcano therapeutics, rancho cordova, ca, usa) was placed distal to the target lesion and was pulled back with a motorized transducer pullback system at a rate of 0.5 mm / s . A single observer who was blinded to the clinical / angiographic findings analyzed ivus images of culprit segment with> 50% diameter stenosis . Manual contour detection of both the lumen and the media - adventitia interface was performed . Volumetric data were automatically determined by the software, a summation of measured cross - sectional areas in all frames of the pullback region based on simpson's rule . The raw radiofrequency data was captured at the top of the r wave during auto - pullback and reconstructed the color - coded map that classified coronary plaque into 4 different components automatically by the software (ivus lab software, volcano therapeutics). And the volume and percentage of each plaque component were also automatically calculated by the software . Fibrous tissue was shown in green, fibrofatty tissue in greenish - yellow, dense calcium in white, and necrotic core in red based on mathematical autoregressive spectral analysis of ivus backscatter . The accuracy for each plaque component between in vivo vh - ivus and in vitro histopathology plasma high - sensitive c - reactive protein (hs - crp) was measured by elisa using an immunonephelometric assay kit (dade behring, illinois). Glomerular filtration rate (gfr) was calculated by modification of diet in renal disease study equation . Statistical analysis was performed with statview (sas institute, cary, north carolina). Continuous variables were compared by unpaired student's t - test or mann - whitney u statistic test . Volumetric ivus data was presented as total volume per lesion length (mm / mm) for correcting the differences of lesion length among the subjects . For all analyses, a p value <.05 was defined as statistically significant . Women had less diabetes and had higher level of high - density lipoprotein (table 1). No women received hormone replacement therapy in the present study . Women had significantly smaller vessel volume compared with men (women versus men; 12.7 3.9 versus 14.5 4.2 mm / mm, p = .01) and smaller plaque volume (women versus men; 8.4 3.5 versus 9.7 3.5 mm / mm, p = .04). However, these differences were no longer significant when corrected for body surface area (bsa). In vh - ivus analysis after correcting for bsa (table 3), the volume and percentage of dense calcium were significantly higher in women compared with men (women versus men; 0.44 0.40 versus 0.32 0.26 mm / mm / bsa p = .03, 11.2 7.6 versus 8.2 6.1%, p = .009). Though there was no significant difference in the volume of fibrofatty tissue between women and men (women versus men; 0.50 0.39 versus 0.62 0.50 mm / mm / bsa, p = .11), the percentage of fibrofatty tissue was significantly less in women (women versus men; 12.1 5.4 versus 15.1 8.7%, p = .03). In multivariate analysis (table 4), gender was the only independent factor for the percentage of dense calcium (odds ratio = 2.5, p = .05). In the present study, using preinterventional vh - ivus images, women had a higher amount of calcium in the culprit lesion plaque compared with men, though there were no quantitative differences of plaque burden when adjusted for body surface area . Several studies suggest that women are associated with an increased mortality rate following coronary interventions compared with men, although not consistently observed [710]. The worse outcomes in women might be partly explained by some clinical factors such as older age, smaller body surface area, and comorbidities at the time of presentation . However, whether women have different plaque characteristics on atherosclerotic segment has not been clearly documented . An angiographic study in patients without visual evidence of coronary atherosclerosis has reported that lumen size in women was smaller than in men, and an autopsy study has reported that this difference in coronary size is due to the mass of the heart . A previous study using grey - scale ivus has reported that women and men also had similar reference and plaque burden and eccentricity . Similarly, in our study, there were not any gender - specific differences of quantitative variables such as vessel volume and plaque volume when adjusted for body surface area . Taken together with our results, the gender differences of vessel size might be dependent on body size . Coronary calcified plaques presumably reflect the pathological process of plaque formation and progression from simple fatty streaks to complex plaques [1517] and are associated with the long - term mortality and ischemic event [18, 19]. The use of computed tomography for detection of coronary artery calcium has been studied previously, and this method could evaluate the whole coronary arteries and nonculprit segments . Whereas a study using multidetector - row computed tomography has reported that calcium concentration of individual calcified plaque is independent of age and sex, our data using vh - ivus showed that women have more calcified plaque components in the culprit lesion compared with men . It is difficult to distinguish between intimal atherosclerotic calcification and medial calcification by the present imaging modality like ct, grey - scale ivus, and vh - ivus . Further studies will be needed to clarify the impact of gender differences on atherosclerotic calcification . Estrogen has multiple biologic effects that might vary according to the underlying state of the vasculature and other tissue [21, 22]. A recent randomized clinical study has reported that estrogen could reduce calcified plaque burden of coronary arteries in young menopausal women . Though there was no women who received hormone replacement therapy in the present study, these results might have suggested that estrogen inhibits the progression of atherosclerosis . The biological explanations for gender differences in cardiovascular diseases are more complex, so we think that the synthesis of the underlying mechanisms that explain these differences has been impossible . This is a study with a limited number of patients with stable angina undergoing coronary intervention, possibly posing a risk of patient selection bias . Our analysis included only the culprit lesions; therefore, the results may not be applicable to other parts of coronary arteries . The vh - ivus technology is unable to differentiate thrombus from other plaque are components . Although we have investigated the patients with stable angina, there is a possibility that small thrombi in the plaque classified into the incorrect tissue . The differences in comorbid conditions and lipid profiles may have influenced the results . In previous studies using vh - ivus analysis, the presence of diabetes and lower hdl level contributes to the increment of dense calcium and necrotic core [24, 25]. Although diabetes patients were less and high - density lipoprotein levels were higher in women, absolute and percentage of dense calcium volume were higher in women compared with men . And gender was an independent factor for the percentage of dense calcium in multivariate analysis . Thus, we think that gender difference might contribute to the calcification of coronary arteries independently . As we have also excluded the patients younger than 60 years old, these results cannot be generalized to all age groups of women due to the different risk profile in pre- versus postmenopausal women . In conclusion, the present study using preinterventional vh - ivus images has suggested that women had more calcium in the composition of the culprit lesion plaque compared with men in elderly patients with stable angina . While it is yet to be proven if the management of coronary calcification has a significant clinical relevance, further studies with a gender - specific approach are necessary for the better understanding of coronary heart disease.
Following approval by our institutional review board, the medical charts of patients who received stsg for treatment of foot and leg ulcers between 2002 and 2010 were identified and retrospectively reviewed . Inclusion criteria included those patients who had a documented history of diabetes mellitus and an ulceration of the foot or leg distal to the tibial tuberosity . Patients were excluded from the study if they did not have a history of diabetes mellitus and/or less than 6 months follow up from the time of the application of the stsg . A total of 183 patients received stsg treatment by the primary author during the selected time frame . One hundred seven (n=107) met the inclusion criteria and were included in the study . Information regarding comorbidities and potential risk factors for healing was also collected from each patient's medical record including age, history of smoking, history of alcohol use, history of intravenous (iv) drug use, wound size, rheumatoid arthritis, end - stage renal disease, cardiac disease (coronary artery disease or congestive heart failure), peripheral vascular disease (pvd), history of fracture and history of charcot neuroarthropathy . Prior to application of the stsg, all patients underwent conservative local wound care with or without negative - pressure wound therapy in an attempt to promote a uniform granular wound bed with minimal wound exudate, fibrin, or slough . Stsg application was delayed if there were any local signs of infection, malodor, purulent drainage, or edema . Any patient with questionable peripheral vascular status was referred to vascular surgery for workup and cleared prior to surgery . All stsg were performed in an operating room setting under either general or local regional anesthesia . Surgical preparation of the wound was achieved by sharp or mechanical debridement of all non - viable tissue from the wound bed and wound edges in a sterile environment . The wound was copiously irrigated with at least 1000 ml of normal saline and local hemostasis was achieved by a combination of direct pressure and/or topical thrombin . All donor sites for patients in this study were from the anterior aspect of the ipsilateral thigh (fig . Marcaine 0.5% plain was infiltrated in the subcutaneous tissue surrounding the donor site and the appropriately sized area was prepped with sterile mineral oil to enhance gliding of the dermatome . The donor stsg site was then harvested utilizing a power dermatome set to 0.018 inch thickness and a width of two to four inches . For larger recipient areas, additional passes of the dermatome the donor site was dressed with povidone - iodine soaked non - adherent gauze and sterile dry dressing . The stsg was then meshed in a 1:1.5 ratio and applied directly to the wound bed taking care to smooth any wrinkles and allow maximum apposition of the graft with the wound surface (fig . The stsg was secured with staples around the edges under minimal tension and a non - adherent dressing was placed directly over the graft surface . In order to minimize graft migration and limit shear forces, a bolster dressing consisting of the foam portion of a surgical scrub brush and multiple layers of cast padding were secured firmly over the stsg (fig . All patients were placed in a bulky splint until after the first post - operative visit . The patients were followed clinically at two weeks post - operatively and then on a weekly basis for dressing changes and to assess healing progress . One week follow - up intervals were chosen in order to allow a quantifiable degree of healing to occur between clinic visits . Dressing changes consisted of re - application of the bolster dressing and roll gauze with continuation of the short leg cast or boot . Once healed, patients were seen in the outpatient clinic every four weeks until a minimum of 6 months follow up from the time of stsg application (fig . Graft site with stsg meshed, smoothed, and secured stsg covered with non - adherent dressing in place with staples . Fifty - eight year old male with type 2 diabetes mellitus treated for ulceration on the lateral aspect of his ankle . His ulceration showed no improvement following conservative treatment with compressive dressings and local wound care . Multiple applications of human fibroblast - derived dermal substitute were also performed with no improvement . After 6 months of treatment, he was taken to the operating room for an autogenous stsg . (a) pre - debridement, (b) post - debridement, (c) postoperative day 0, (d) postoperative day .10, (e) 4 weeks postoperative, (f) 8 weeks postoperative, (g) 6 months postoperative the frequencies and mean time to healing were calculated for all variables of interest . Each variable was analyzed for healing time using a two - sample independent t - test . A total of 107 consecutive diabetic patients met the inclusion criteria for this study ranging in age from 28 to 88 years (average age, 59.1). One - hundred - and - seven stsg were applied in total, with 29/107 (27.1%) to the right foot, 28/107 (26.2%) to the left foot, 25/107 (23.4%) to the right leg and 25/107 (23.4%) to the left leg . Six patients (5.6%) were current smokers, two (1.9%) admitted to current chewing tobacco use, and three patients (2.8%) admitted to iv drug abuse . Nine patients (8.4%) had end - stage renal disease, defined as a glomerular filtration rate <15 ml / min, with all nine patients receiving ongoing dialysis treatment . Three patients (2.8%) had a significant history of cardiac disease which included a diagnosis of coronary artery disease and/or congestive heart failure . Seven patients (6.5%) had a documented history of peripheral vascular disease which included at least one abnormal non - invasive vascular test (ankle - brachial index, transcutaneous oximetry, doppler wave - forms, segmental pressures, and pulse volume recordings) and/or non - palpable pedal pulses on examination . All patients with pvd received a workup by vascular surgery prior to application of stsg and were deemed to have sufficient blood supply for healing . In addition, all patients included in the study were deemed to have adequate blood flow to heal a stsg before undergoing the procedure . One patient (0.9%) had a history of charcot neuroarthropathy and received a stsg . Sixty patients (56.1%) had no other comorbidities besides diabetes mellitus, 29 patients (27.1%) had one comorbidity, and 18 patients (16.8%) had two or more comorbidities in addition to diabetes . Among all patients, approximately half the patients (53/107 patients, or 49.5%) had a starting wound area of less than 50 cm, 37/107 (34.6%) had a wound area of 50 to 100 cm, and 17/107 (15.9%) had a wound area greater than 100 cm . The number of weeks to complete wound healing ranged from 3 to 16 weeks with an average healing time of 5.1 weeks . The vast majority of patients (97/107, or 90.1%) were completely healed by 6 weeks post - operatively while only four patients (3.7%) required 10 weeks or more to completely heal (graph 1). Our analysis demonstrated that none of the comorbidities or risk factor variables had an independent effect on time to complete wound healing (table 1). Similarly, age, wound location and wound size each appeared to have had no effect on time to heal (table 2) the patients who had 100% graft take had a shorter healing time than those with graft take of less than 95% (p<0.001) (table 3). Those patients with 100% stsg take had a mean healing time of 4.8 weeks compared to 7.9 weeks for those patients with less than 95% stsg take (graph 2). The range of stsg take percentage among all groups was between 40 and 100% with a mean take, overall, of 97% . Time to complete wound healing (n=107) sample size too small for calculation of p value . Time to complete wound healing for patient age, wound size, and wound location percent of stsg take and time to complete wound healing represents statistical significance from other values . Percent graft take and mean healing time only three of the patients (2.8%) had a complication with the original stsg . In two patients (1.9%), the stsg dressing was removed too early and both patients required re - grafting and eventually healed . The other patient had underlying osteomyelitis and required revisional resection of the bone and re - grafting which also eventually healed completely . Patients with complications had a mean time to complete wound healing of 12.0 weeks compared to 4.9 weeks for patients without complications (table 4). Despite success with stsg in surgery and wound care, there remain relatively few studies addressing its use in diabetic lower extremity wounds . In our study of diabetic patients, the mean time to complete wound healing was 5.1 weeks with a range of 316 weeks . This average is comparable to that of other studies of wound healing in diabetic populations . Recently, ramanujam et al . Retrospectively reviewed 83 diabetic patients treated with stsgs for diabetic foot and ankle wounds and reported a median time to healing of 6.9 weeks among those patients without complications (10). Mahmoud et al . Prospectively studied patients with stsg versus conservative wound care for diabetic foot wounds and found a statistically significant reduction in mean hospital stay and healing time for those patients treated with stsg (11). Compared the healing rates of meshed vs non - meshed stsg in 42 patients and found no significant difference (12). The mean healing time for the meshed group was 19.84 and 20.36 days for the non - meshed group . Most stsg studies in non - diabetic studies report healing times between 2 and 4 weeks . Impaired healing in diabetic patients is well - studied and can be attributed to multiple factors including impaired macro and microcirculation, peripheral neuropathy, endothelial dysfunction, and poor glycemic control (1416). One shortcoming of our study is that we did not quantitatively analyze preoperative glycemic control . However, ramanujam et al . Did not find a statistically significant difference in preoperative hemoglobin a1c levels and healing time, despite high average preoperative hemoglobin a1c values in their patients (10). Conversely, a study by marston found a direct correlation between hyperglycemia and wound healing . In this study, we found that the specific preoperative risk factors showed no effect on healing time (13). Likewise, age, wound size, and wound location did not seem to have a significant effect on healing time . Surprisingly, there was no significant effect of wound size on difference in healing time . The mean time to healing for those with wound size> 100 cm was 5.7 weeks compared to 4.7 weeks for wounds 50 to 100 cm and 5.2 weeks for wounds <50 cm . Thus, those patients with a wound size between 50 and 100 cm actually healed faster, on average, than those with a wound size of <50 cm . Patients in our study with complications took longer to heal by more than 7 weeks on average than those without complications . Of the three patients who had complications, two had healing times of 10 weeks and the other had a healing time of 16 weeks . The mean healing time for those patients with complications was 12.0 weeks versus 4.9 weeks for those without complications . Only one patient among those without complications had a healing time longer than 7 weeks . Two patients in our study had the stsg pulled off at 2 weeks by the nursing facility . Both patients required re - grafting and healed without further complications by week 10 . The other patient had osteomyelitis of the underlying bone which required resection . This patient eventually went on to heal the wound with aggressive local wound care and re - grafting by week 16 . It stands to reason that post - operative complications such as infection, noncompliance, seroma, swelling and stsg pressure delay healing time by disruption of the graft and interfering with the healing process . Likewise, patients who must undergo revisional surgery would also be expected to have a delay in healing time . This is in contrast to several other studies that have reported higher rates of complications . Ramanujam et al . Reported a post - graft complication rate of 35% with 16 patients experiencing an infection (10). They similarly noted a significant increase in time to complete wound healing in those patients who experienced complications . Similarly, mahmoud et al . Reported that 38% of their diabetic patients who received a stsg failed to heal by post - operative week eight (11). The patients in our study who had less than 95% graft take had an average healing time of more than 3 weeks slower than those with a graft take of 100% (7.9 weeks vs 4.8 weeks), and almost 3 weeks slower than those with a graft take between 95 and 99% (7.9 weeks vs 5 weeks). Skin graft healing and incorporation is a complex biological process involving various stages of adherence, nourishment, revascularization, and final incorporation (6). Once harvested, the skin graft is deprived of its native nutrients and blood supply and can only survive by adherence to the wound bed and diffusion of nutrients from the underlying vascular supply until revascularization occurs . It stands to reason that any mechanical or biological disruption of this process puts the graft at risk for failure or prolonged healing . In our study, we took measures to prevent disruption to the graft by securing it place with staples and applying a bolster dressing to minimize shearing or compressive forces . Care was also taken to prepare the wound bed prior to graft placement in a manner that wound maximize the probability of incorporation . The vast majority of our patients (90.7%) had greater than 95% graft take and, of those, 73.8% had 100% graft take . Only 10 patients (9.3%) had less than 95% graft take, but this group took longer to heal . Clearly, the amount of graft take is an indication of the underlying healing process . Stsg with poor graft take can naturally be expected to take significantly longer to heal . Our study is only descriptive in nature, therefore other statistical methods such as tests for association and regression analysis would likely provide better information regarding the effects of each variable of interest as well as their additive effects on stsg healing times . On the same note, this study does not take into account possible interactions among the variables themselves which can influence the results . A major limitation of our study was the small sample size . Only having one patient with charcot neuroarthropathy, for example, was inadequate for analysis with respect to healing time . In addition, our study was retrospective in design which prevented inclusion of some data (e.g. Type of diabetes and preoperative hemoglobin a1c levels) which might have been useful . A prospective, multicenter study is needed to more accurately investigate and determine the effects of certain risk factors and comorbidities on stsg wound healing time in the diabetic population . Our study demonstrated a very low complication rate of 2.8% and an average wound healing time of 5.1 weeks for all patients who received a stsg for treatment of a diabetic foot or leg ulcer . None of the patient characteristics or comorbidities in this study appeared to affect stsg healing times, but we did find an average increase in healing time among patients with complications (12.0 weeks) versus those without complications (4.9 weeks). Finally, in our group of subjects, those patients with decreased graft take had prolonged healing times . This underscores the importance of optimizing the stsg for incorporation by minimizing any mechanical or biological barriers to healing . We conclude that autologous stsg are a safe and reliable alternative for the treatment of non - healing diabetic foot and leg wounds . The authors have received no funding or benefits from industry to conduct this literature review.
Phospholipids containing polyunsaturated fatty acids are highly prone to modification by reactive oxygen species, thereby generating a plethora of biologically active oxidized phospholipids (oxpls). A variety of (patho-) physiological effects ascribed to oxpls underline their relevance, e.g., in inflammation, atherosclerosis,(6) or immune response. (7) the modes of action are diverse . First, different families of receptors were described to become activated due to oxpl binding. (1) second, oxpls were shown to interact with drugs, thereby influencing their pharmacokinetics. (8) finally, one may expect the exceptional structure of an oxpl molecule to affect its biophysical properties in the lipid membrane . In this report bioactive oxpls contain extensively modified or truncated acyl chains in the sn-2 position, typically terminated by polar carboxylic or aldehydic groups . As was shown by nuclear magnetic resonance, langmuir balance, and molecular dynamics (md) simulations, the truncated polar moiety can protrude into the aqueous phase, despite the energy penalty associated with the exposition of the nonpolar chain regions . As a consequence, bilayer properties such as permeability, mobility, and headgroup hydration we have recently studied the diffusional properties of a fluorescent oxpl analogue, 1-palmitoyl-2-glutaroyl - sn - glycero-3-phospho - n - alexa647-ethanolamine (pgpe - alexa647), in the live cell plasma membrane, and found exceptionally high mobility of 2 m / s,(15) in agreement with the expected lysolipid - like behavior . Moreover, we observed transient immobilization at endocytic sites, which we attributed to a preferential partitioning within highly curved membrane regions due to the inverted cone - like shape of the pgpe - alexa647. (15) to better understand the behavior of oxpl - molecules in membranes we decided to further address its properties in well - defined model systems . We used pgpe - alexa647 as a representative carboxylated oxpl; we have previously shown that this fluorescent analogue mimics closely the behavior of the nonlabeled molecule in living cells. (16) all data were referenced against a conventional headgroup labeled fluorescent phospholipid, dihexadecanoylphosphoethanolamine (dhpe)-bodipy . First, we were interested whether the probe displays any preference with respect to the phase state of the membrane . The cellular plasma membrane is believed to be segregated into domains,(17) where a more ordered (raft-) phase shall coexist with a disordered phase. (18) generation of phase - separated model membranes has become standard in many laboratories (for reviews see, e.g., refs (1921)) and provided a wealth of insights into the thermodynamics of lipid bilayers . Indeed, a few reports confirmed the presence of ordered environments also in the cellular plasma membrane . In this study, we found a moderate preference of the pgpe - alexa647 for the liquid - disordered phase but also significant partitioning into the liquid ordered phase, indicating a rather promiscuous localization . Second, in order to explain the high mobility of pgpe - alexa647 observed in the live cell plasma membrane, we further characterized the diffusion constant in various model membranes . Experiments were performed on supported lipid bilayers (slbs) using line - scan fluorescence correlation spectroscopy (fcs)(25) and single molecule tracking. (26) substantially higher oxpl mobility was observed consistently; it could be diminished by increasing cholesterol content and intensified by introducing artificial obstacles . 1,2-dioleoyl - sn - glycero-3-phosphocholine (dioleoylphosphatidylcholine; dopc), sphingomyelin from porcine brain (brsm), n - octadecanoyl - d - erythro - sphingosine (c18 ceramide; cer), and cholesterol were purchased from avanti polar lipids (alabaster, al, u.s.a .) And used without further purification . Two different fluorescent lipids were used as probes: n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a - diaza - s - indacene-3-propionyl)-1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine and triethylammonium salt (bodipy fl dhpe, invitrogen, carlsbad, ca, u.s.a . ); the oxidized phopholipid 1-palmitoyl-2-glutaroyl - sn - glycero-3-phospho - n - alexa647-ethanolamine (pgpe - alexa647) was synthesized as described previously. (16) optical adhesive 88, used to glue the mica on coverslips, was purchased from norland products inc . Two different buffers were used for sample preparation: buffer a (150 mm nacl, 10 mm hepes, and 3 mm nan3, ph 7.4) and buffer b (pbs - buffer; paa pasching, austria). Buffer a was filtered through a 0.2 m filter (nalgene, rochester, ny, u.s.a .) Prior to use . 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(cap biotinyl) (sodium salt) (18:1 biotinyl cap pe; dope - biotin) was purchased from avanti polar lipids (alabaster, al, u.s.a . ). Glass slides (menzel, #1, braunschweig, germany) were incubated in a 3:1 piranha solution of sulfuric acid (j.t . Baker, 9597%, new jersey, u.s.a .) And hydrogen peroxide (merck, 30%, new jersey, usa) for 20 min, rinsed with deionized water and ethanol, and dried by nitrogen . Glass slides were then glued on a measurement chamber (lab - tek, nunc, thermo fisher scientific, rochester) from which the glass support has been removed . A total of 10 mg of dopc was dissolved in a mixture of methanol and chloroform (1:3). Then, 10 l of a 10 mg / ml dopc solution was evaporated under nitrogen stream and diluted with 100 l buffer b. vesicle solutions were prepared by ultrasonicating for 20 min . The accrued vesicle solution was put on a glass slide or avidin - coated surface . After 20 min the bilayer had been formed and was washed with buffer b. fluorescently labeled lipids were incorporated after bilayer formation by incubation from the aqueous subphase: for this, bilayers were incubated with a 2,5 nm solution of dhpe - bodipy and with a 5 nm solution of pgpe - alexa647 for another 20 min, followed by thorough washing with buffer b. avidin - coated glass surfaces were prepared by incubating a piranha - cleaned glass - coverslip with a 10 mg / ml avidin solution for 20 min, and subsequent washing with buffer b. dopc, bsm, and cholesterol were mixed in organic solution (1:3/methanol: chloroform) in a molar ratio of 2:2:1 . After solvent evaporation, the lipid film thus obtained was slowly rehydrated using buffer b at 10 mg / ml lipid concentration and resuspended through vigorous vortexing . After sonicating the suspension at 60 c, a small aliquot was diluted in buffer a and deposited in the presence of 2 mm cacl2 on a 10 m thick, freshly cleaved mica glued onto a glass coverslip . The coverslip was sealed with a home - built polypropylene chamber and incubated at 55 c for 5 min . After that, the sample was rinsed at the same temperature at least 10 times with buffer a and allowed to cool down to 25 c . At this stage, fluorescently labeled lipids could be incorporated into the bilayer by addition from the aqueous subphase . The concentration of dhpe - bodipy was varied between 0.1 to 0.01 mol% and for pgpe - alexa647 from 0.5 to 0.01 mol% . After 20 min, the sample was rinsed again to remove the fluorescent lipids from the buffer solution . Single molecule experiments were performed as described previously. (27) briefly, a zeiss axiovert 200 microscope was equipped with a 100 na=1.46 plan - apochromat objective (zeiss, oberkochen germany). Samples were illuminated in objective - type total internal reflection (tir) configuration via the epiport using 488 nm light from an ar laser (model 2017 - 05ar, spectra physics, mountain view, ca, u.s.a .) With an intensity of typically 911 kw / cm and 647 nm light from a kr laser (stabilite 2017-kr, spectra physics) with intensities of typically 510 kw / cm . A slit aperture (zeiss) with a width of 7 m in the object plane was used as field stop to confine the illumination area . After appropriate filtering (z488 647mv2, z488 647rpc, chroma, vt, u.s.a . ), emitted signals were split into two color channels using a custom - made dichroic wedge (chroma) and imaged on the same back - illuminated, liquid - nitrogen - cooled ccd camera (micro max 1300-pb, roper scientific, trenton, nj, u.s.a . ). For the precise control of all laser pulse trains, an acoustic - optical modulator (1205c, isomet, springfield, va, u.s.a .) Was used . Timing protocols were generated and controlled by an in - house program package implemented in labview (national instruments, austin, tx, u.s.a . ). Experiments were performed in a home - built incubator box equipped with a heating unit, a temperature - adjustable stage insert and an objective heater (pecon, erbach, germany). Signals were analyzed by fitting a two - dimensional gaussian profile, yielding the position with an accuracy of 50 nm . The sample was mounted on a high - precision xy - stage (scan i m 120 100, mrzhuser, germany). For single molecule analysis, images were analyzed using in - house algorithms implemented in matlab (mathworks, natick, ma, u.s.a.). (27) individual diffraction limited signals were selected and fitted with a gaussian profile, yielding the single molecule position, the brightness b and the full width at half - maximum (fwhm) of the gaussian function . Single molecule mobility was analyzed as described previously. (27) in brief, trajectories are specified by a sequence of positions x(t), with i ranging from 1 to the number of observations of this trajectory . The mean square displacements r were calculated as a function of the time - lag tlag = n(till + tdelay) according to r = x(t + tlag) x(t)), with n denoting the difference in frame index . Estimated data were analyzed by fitting with the functionyielding the lateral diffusion constant d and the single molecule localization precision . Only the first two data - points were used for the linear fit. (27) line - scan fluorescence correlation spectroscopy (lsfcs) was performed at room temperature (25 c) on a lsm 510 meta (zeiss, jena, germany) as described in ref (25). The fiber output was coupled to a home - built fcs detection unit, consisting of an emission filter and an achromatic doublet (linos photonics, goettingen, germany), to image the internal pinhole onto the optical fiber connected to an avalanche photodiode (apd) (perkin - elmer, boston, ma, u.s.a . ). Correlation curves were obtained with a hardware correlator (correlator.com, bridgewater, nj, u.s.a . ). For confocal fluorescence microscopy, the excitation light of an argon laser at 488 nm (or a hene laser at 543 nm) was reflected by a dichroic mirror (hft 488/543/633) and focused onto the sample by a zeiss c - apochromat 40, na) 1.2, uvvisir water immersion objective . The fluorescence signal was then recollected by the same objective and, after passing through a 525/50 bandpass filter (or a 580/60 bandpass filter), measured by a photomultiplier (pmt). The confocal geometry was ensured by a 70 m (80 m) pinhole in front of the pmt . Fcs measurements were performed using the same optical path described for the fluorescence imaging, the signal from the sample being collected in this case by the avalanche photodiodes in the fcs unit . The membranes simulated in this work consisted of 2500 lipids per leaflet and contained 50 water molecules per lipid (corresponding to 12.5 water beads in the coarse grained representation). The content of cholesterol was increased from 0% to 50% by substituting dopc with cholesterol . Additionally, 1% of the lipid molecules was the oxidized lipid 1-palmitoyl-2-glutaroyl - sn - glycero-3-phosphocholine (pgpc) introduced by substituting dopc with pgpc (figure 4a). The lipids were randomly placed in the plain of the bilayer and rotated using an axis normal to the membrane . Atom overlaps were resolved by first expanding the bilayer in the membrane plane, followed by a size reduction in small steps to the typical area per lipid of a dopc - cholesterol mixture, involving energy minimization and geometry optimization for each deflation step. (28) the systems was further equilibrated by first carrying out 10 and 50 ns equilibration runs with first restraining in z the phosphate of the phospholipids and the polar oxygen of the cholesterol and then only restraining the phospholipids . After 100 ns of unconstrained equilibration we carried out a production run of 200 ns . The parameters of the oxidized tail of the pgpc lipid were extracted from all atom simulations of pgpc in 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine (popc) using berger lipids,(31) missing parameters were taken from the force field and from wong - ekkabut et al. (14) and using the spc water model. (32) the truncated oxidized glycero - lipid tail was coarse grained into two beads, one containing the charged carboxy terminus, one consisting of the aliphatic carbons of the chain . Bead distances and bond and dihedral angles were measured and added to the coarse grained description of pgpc . A constant temperature of 310 k was maintained using the velocity rescale (v - rescale) algorithm(35) with a coupling time of 0.3 ps, coupling independently the water and the lipids plus ions to an external bath . A pressure of 1 bar was maintained in both the membrane plane and the membrane normal, using semi - isotropic berendsen pressure coupling scheme(36) with a time constant of 3 ps . A shift function was applied to the van der waals interactions between 0.9 and 1.2 nm, and the electrostatic interactions were shifted over the entire range from 0 to 1.2 nm . Diffusion constants were calculated by fitting the linear part of the mean square displacment curves with r = 4dtlag, after removing overall translation by fitting of the dopc molecules . It is a known limitation of the coarse grained martini force field that on an absolute scale the velocity of molecules are overestimated and scaling up to an order of magnitude has been suggested. (29) we applied a scaling factor of 15 to obtain accordance on the absolute scale of the experimentally observed diffusion rates . The density profiles were calculated using standard procedures after removing translational shifts of the membranes, which were determined from the mean position of all dopc headgroups . The positions of the pgpc and dopc phosphate groups along the membrane normal were extracted from the density plots by fitting the phosphate group density peak with a gaussian function (figure 5). Distances between each phospholipid molecule (represented by its phosphate group) and the nearest cholesterol molecule (represented by its hydroxyl group) were calculated for each time point within a trajectory (figure 6). The resulting histograms (bin - width 0.02 nm) were averaged for all phospholipids in the simulation . To address the phase preference of pgpe - alexa647, we studied its partitioning in supported lipid bilayers containing bsm: dopc: chol in a molar ratio of 2:2:1 (figure 1), which shows separation in liquid ordered (l.o .) And disordered (l.d .) Phase. (39) pgpe - alexa647 and dhpe - bodipy were added from the aqueous phase to ensure that the probes inserted only in the upper leaflet; flip - flop is too slow to yield probe redistribution between leaflets during the experimental time frame of less than 1 h.(40) while the dhpe - bodipy was largely excluded from the ordered phase (green color channel), we observed only weak contrast for pgpe - alexa647 . We performed line - scan fcs to determine accurate values of the surface densities in the ordered (lo) versus the disordered phase (ld), yielding the partition coefficients k = lo/ld for pgpe - alexa647 (k = 0.75) and for dhpe - bodipy (k = 0.32) (see table 1). We also calculated the diffusion constants of the probe molecules, yielding higher mobility of the oxidized phospholipid analogue versus dhpe - bodipy in both phases (table 1). Partitioning and mobility of pgpe - alexa647 and dhpe - bodipy in phase - separated supported lipid bilayers . Panels ac show confocal fluorescence images of a dopc / bsm / chol 2:2:1 supported bilayer doped with trace amounts dhpe - bodipy and pgpe - alexa647: an overlay, the bodipy - channel and the alexa647-channel are shown in panels ac, respectively . We performed line - scan fcs to quantify partition coefficients and mobilities (sketch in the inset to a: the blue cone shows the detection volume, the blue and green encircled areas correspond to the l.o . And (d) representative averaged autocorrelation curves measured in different lipid phases of the bilayer at room temperature . Shown phase (upper figures) and the l.o . Phase (bottom figures) for the two different probes dhpe - bodipy (left) and pgpe - alexa647 (right). Data were fitted for two - dimensional diffusion, yielding the diffusion constants d and the surface densities . The obtained fit values are listed in table1 . The rather high l.o .- phase partitioning of pgpe - alexa647 lead us to the hypothesis that ordered phases in general may be well accessible by the oxpl . We thus investigated a bilayer composed of bsm: dopc: chol: cer in a molar ratio of 0.64:1:1:0.36, which yields a two phase equilibrium between a ceramide - enriched gel - like phase and a liquid disordered phase. (41) previous data revealed efficient exclusion of all investigated fluorescent analogues. (42) we indeed confirmed reduced partitioning into the ceramide - enriched phase for both probes . Due the low partitioning, we estimated the surface densities directly from the confocal images: a partition coefficient of cer/ld = 0.004 was calculated for dhpe - bodipy; yet, also in this case ceramide - phase partitioning of pgpe - alexa647 was significantly higher (cer/ld = 0.01). Phase was reduced compared to the ternary system for both probes, and the mobility difference basically vanished (table 1). Probe mobility in the ceramide - enriched gel - like phase was too low to be measurable . In summary, the oxidized lipid analogue shows rather weak phase preference; in general, its mobility is higher than the mobility of a conventional phospholipid . We continued by putting our focus on the mobility difference between pgpe - alexa647 and dhpe - bodipy . Glass - supported lipid bilayers of dopc were prepared and studied at 25 or 37 c . After bilayer formation, pgpe - alexa647 and dhpe - bodipy were added from the aqueous phase . We used single molecule tracking to determine the diffusion constant of both lipid analogues in the same bilayer regions . To eliminate potential influences due to the immobilization of molecules at surface defects we started each recording sequence by a photobleaching pulse, which irreversibly destroyed all fluorophores in the field of view (figure 2a). After a recovery time of 0.52 s, unbleached fluorescent lipid molecules have entered the field of view and could be tracked . By this measurement mode, we could restrict the analysis to a clean fraction of freely diffusing probe molecules . For our experiments, we used stroboscopic illumination with a time delay of tdel = 2 ms between two consecutive illumination pulses and an illumination time till = 1 or 3 ms . From the single molecule trajectories, the distribution of step sizes was determined and further analyzed to assess potential heterogeneity in the sample mobility, which would yield deviations from a monoexponential behavior . However, we found here for all bilayers and probe molecules purely monoexponential functions (exemplified in figure 2b). We also tested for anomalous subdiffusion behavior, being indicative for confinements of the tracer or nonequilibrated systems:(27) when plotting the mean square displacement (r) as function of the time - lag (tlag), anomalous subdiffusion would yield a sublinear increase . However, for all data sets we found perfectly linear relationships (exemplified in figure 2c). We found substantially higher mobility for pgpe - alexa647 compared to dhpe - bodipy (figure 3): at 25 c the oxpl showed a 1.3-fold higher diffusion constant than the conventional phospholipid, at 37 c we found even a ratio of 2.2 . Interestingly, this difference was reduced and finally disappeared for dopc bilayers containing increasing amounts of cholesterol (figure 3). (a) the upper row sketches the applied laser timing protocol, the lower row an example of an image sequence . After recording a prebleach image the according area was totally photobleached by applying a laser pulse for tbleach250 ms . The photobleaching efficiency was controlled by recording an image immediately after the bleach pulse . After a recovery time of trec2000 ms, single fluorescently labeled lipids entering the field of view can be resolved as diffraction limited signals . By recording an image sequence with high time resolution (till = 3 ms, tdelay = 2 ms) panel b shows the cumulative density function cdf of square displacements between consecutive images at tlag = 10 ms for pgpe - alexa647 . (c) exact values for diffusion constants d were calculated by plotting r versus tlag, and fitting with eq 1 . A clear difference for d is observed by comparing pgpe - alexa647 (dark gray line, d = 7.2 0.22 m / s) and dhpe - bodipy (gray line, d = 3.1 0.09 m / s). Diffusion constant of dhpe and pgpe in a dopc / cholesterol bilayer as a function of cholesterol content, determined by single molecule tracking . Dopc bilayers containing up to 50% cholesterol were labeled with pgpe - alexa647 (full circles) and dhpe - bodipy (open circles). Experiments were performed at 37 c (a) or 25 c (b). To understand this behavior, we performed coarse - grained md simulations of a dopc matrix containing low concentrations of pgpc and varying concentrations of cholesterol . Figure 4a shows a snapshot of the membrane: consistent with previous all - atom simulations on similar compounds, we observed the reversal of the truncated acyl - chain in the sn-2 position of pgpc, which frequently protruded out of the membrane into the aqueous subphase . No aggregation or alignments of the analyzed lipids was observable on length scales> 5 nm, indicating that influences of potential membrane undulations can be neglected. (46) we determined the diffusion coefficient for both dopc and pgpc by calculating r and fitting to the linear region according to r = 4dtlag . At zero cholesterol we observed a 1.4-fold higher mobility of the oxpl pgpc compared to the matrix lipid dopc, similar to our experimental data recorded at 25 c . Since pgpc carries essentially only one chain that is inserted into the hydrophobic core of the membrane, the consequentially small area of the lipid appears to be responsible for its high mobility . Dopc is show in gray, pgpc is show in blue (phosphate group in light blue), and cholesterol is show in orange . (b) effects of increasing cholesterol on the lateral diffusion of pgpc (full circles) and dopc (open circles). Strikingly, the simulations also revealed a convergence of the mobility ratio to 1 with increasing cholesterol concentrations (figure 4b), in perfect agreement to our experimental data . Electron density profiles gave a first hint on the origin of the effect (figure 5). At zero cholesterol content, the pgpc headgroup region (indicated by the phosphate group) is shifted away from the bilayer center by 1.5 compared to the dopc headgroup region . This effect diminished with increasing cholesterol concentration down to a shift of 0.8 at 40% cholesterol . The mean interaction energy between pgpc and the host matrix increased linearly with increasing cholesterol concentration (figure 5c), indicating an improved accommodation of the oxpl in the membrane . Taken together, the simulations indicate that increasing cholesterol content leads to a shift of pgpc toward the bilayer center, which is accompanied by facilitated contacts and stronger interactions with the host lipids and consequentially an adaption of the mobility values between probe and matrix molecules . (a) total electron density profiles of dopc and cholesterol are shown along with the electron density of the phosphate groups of pgpc and dopc, which are scaled for better visualization . (b) we calculated the peak - shift in the maxima of the phosphate groups for dopc versus pgpc, which is shown as function of cholesterol concentration . Error bars were determined by comparing peak position of the first half of the trajectory with the second half and by differences in peak to peak distances between upper and lower leaflets . (c) the cumulative potential energy of interaction with all membrane components per pgpc molecule is shown relative to the value obtained at 0% cholesterol content . Addition of cholesterol to the membrane increases the interaction strength while no changes where observed for interaction with the aqueous phase (not shown). We also analyzed the average distance of dopc or pgpc to their nearest cholesterol molecule (figure 6). Two peaks are clearly visible: the first one representing probe molecules with directly bound cholesterol and the second one representing probe molecules that are shielded by dopc from the nearest cholesterol . For dopc, two effects can be observed when increasing the cholesterol concentration: the first peak gets more pronounced, as the likelihood for direct contact with cholesterol increases, and the distance to the second peaks gets smaller, reflecting the condensing effect of cholesterol . First, in contrast to dopc, the first peak is substantially lower than the second peak at low cholesterol concentrations; the ratios approach the dopc - case at high cholesterol content . This means that pgpc has a smaller preference for being associated with cholesterol than with dopc, most visible at low cholesterol content . Second, the location of the second peak does not shift as strongly as for dopc, indicating that the condensing effect in the immediate proximity of pgpc is less pronounced . Distribution of distances r between each phospholipid molecule (represented by its phosphate group) and the nearest cholesterol molecule (represented by its hydroxyl group). Plots represent averages over the entire trajectory and over all phospholipids of (a) pgpc and (b) dopc . High cholesterol concentrations level out the differences in the mobility of oxpl and conventional lipids . It is thus difficult to reconcile the exceptionally high mobility of pgpe - alexa647 in the plasma membrane (d = 1.32.4 m / s(15)) with the high native cholesterol concentrations of 3040% . In comparison, standard lipids or lipid - anchored proteins show a mobility which is 5- to 10-fold lower . We suspected that the diffusion matrix in the plasma membrane may be highly structured, providing essentially a percolation matrix . To enforce this effect in our model system, we artificially introduced obstacles in the proximal leaflet by immobilizing a substantial fraction of lipids . Interleaflet coupling should transmit the effect to the distal leaflet. (53) for this, a dopc bilayer containing 4.2 mol% biotin - dope was prepared on a glass surface coated with avidin . While pgpe - alexa647 mobility was reduced only approximately 2-fold, dhpe - bodipy became basically immobile (figure 7a, c). Consistently, addition of 40 mol% cholesterol substantially decreased the mobility of pgpe - alexa647 (figure 7d). The presence of dope - biotin did not influence the mobility of pgpe - alexa647 or dhpe - bodipy in a dopc bilayer on pure glass (figure 7b). Bilayers were prepared on glass supports (a and b) or glass supports coated with avidin (c and d). Diffusion constants were determined for pgpe - alexa647 (light gray bars) and dhpe - bodipy (dark gray bars). Experiments were performed on bilayers of dopc (a), dopc containing 4.2 mol% biotin - dope (b and c), or bilayers containing 4.2% biotin - dope and 40% cholesterol (d). We studied partitioning and mobility of the oxidized phospholipid pgpe - alexa647 with reference to the conventional phospholipid dhpe - bodipy in different lipid membranes . In summary, we made the following observations: pgpe - alexa647 shows only marginal preference for fluid versus ordered phases . In general, conventional phospholipids show altered phase partitioning compared to one chain lipids (compare refs (54) and (55)). It is tempting to follow an argument of the vaz group, who ascribed the rate - limiting step for association of a lyso - lipid with a bilayer to the formation of free area with appropriate size in the membrane surface. (55) for the oxpl, single chain insertion is sufficient for thermodynamic equilibrium,(11) therefore the formation of a marginal free area will be sufficient for insertion of pgpe - alexa647 . Phase show similar susceptibility for the formation of free area on the length scale of a single acyl chain cross sectional area . Md simulations revealed that pgpc slightly sticks out of the headgroup region, thereby essentially reducing its effective area, which most likely causes the enhanced mobility. (56) the experimental data show that the effect was more pronounced at elevated temperature, indicating that the shift of pgpc out of the bilayer plane is entropically favored . Increased cholesterol concentration md simulations of a dopc matrix containing varying cholesterol content and low amounts of pgpc allowed us to further elucidate the effect of cholesterol on pgpc mobility . At low cholesterol concentrations, pgpc is preferentially surrounded by dopc, and its headgroup protrudes out of the headgroup region of the dopc matrix . With increasing cholesterol content, a higher rate of association of pgpc with cholesterol could be observed . Thereby the pgpc headgroup is pulled into the bilayer, with the effect of increasing its interactions with the host lipids and decreasing its mobility . Moreover, in a four component mixture of bsm: dopc: chol: cer (0.64:1:1:0.36), we found hardly any difference in the l.d . We attribute the disappearance of mobility differences to a higher cholesterol concentration in the l.d . Phase; indeed, ceramide is known to displace cholesterol from the l.gel phase,(57) thereby increasing the cholesterol content in the fluid phase . The mobility of pgpe - alexa647 is substantially higher than the mobility of dhpe - bodipy in ordered or obstructed matrices . In the liquid ordered phase of a ternary system, a mobility ratio of r = 2.3 was obtained . Moreover, when we artificially increased the obstacle density in a fluid supported lipid bilayer by immobilizing biotin - dope in the proximal bilayer leaflet on avidin, the ratio dramatically increased to r = 51.1 . Deverall et al . Described the effect of random obstacles on lipid mobility by extending the free area theory(56) and reported nonlinear dependence on the tracer size. (58) essentially, large tracers are more easily stuck between obstacles than small tracers . This effect appears directly relatable to the plasma membrane, where immobilized lipids or acylated proteins on the cytosolic leaflet provide a similar obstructed matrix for tracers located at the exoplasmic leaflet . In summary, we have found that the oxidized phospholipid pgpc and its fluorescent analogue pgpe - alexa647 move with increased mobility compared to conventional phospholipids . The lysolipid - like character enables the probe to enter ordered environments, thereby providing a means for spreading rapidly and homogeneously over complex matrices like the cellular plasma membrane . Mobility is not only determined by the fluidity of the matrix, but also by immobile obstacles or by the localization of the probe along the bilayer normal, which can be influenced by the cholesterol concentration.
Ilio - iliac arteriovenous fistula (avf) is a rare disease that occurs in less than 1% of all common iliac artery aneurysms (ciaas) and in 27% of ruptured aneurysms . Surgical repair of an avf resulting from a ruptured abdominal aortic aneurysm (aaa) was first described by cooley in 1955, and several similar cases have since been reported . Nevertheless, perioperative mortality in patients with aaas with avfs is still as high as 67% in some series . Preoperative diagnosis of avf is difficult, and control of venous bleeding during open surgery can affect surgical outcome . Here we report a case of a contained rupture of a left ciaa with an ilio - iliac avf that was diagnosed preoperatively and successfully treated via surgery . An 86-year - old man was admitted to a local hospital because of a temporary loss of consciousness and left leg edema . His medical history was unremarkable . Plain computed tomography (ct) showed enlargement of the abdominal aorta and left common iliac artery (cia), and he was transferred to our institution for further evaluation and treatment . Upon admission, the patient s blood pressure was 130/76 mmhg, and his heart rate was 88 beats / min . A pulsatile abdominal mass with vascular murmur and thrill was noted . The left lower leg was considerably swollen compared with the lower right leg (left: 37.0 cm, right: 34.4 cm). No clinical signs of high - output heart failure, including hepatomegaly, jugular vein dilatation, and pleural effusion, were observed . Blood test results (hemoglobin, 10.4 g / dl; hematocrit, 30.9%) indicated anemia . Arterial phase ct (ge optima ct 660, ge healthcare, tokyo, japan) of the abdominal and pelvic vessels using a contrast agent (omnipaque, 300 mg / ml; daiichi sankyo pharma, tokyo, japan) showed an infrarenal aaa (maximum diameter, 60 mm) and a left ciaa (70 mm) complicated by a pressed left common iliac vein (civ). Early appearance of contrast medium in the dilated left civ was notable and indicated the presence of an ilio - iliac avf (fig . 1). An abdominal echo revealed a mosaic flow, suggesting a shunt between the left cia and civ (fig . The clinical presentation and imaging findings led to the diagnosis of a contained rupture of the left ciaa exploding into the left civ . After proximal and distal control of the aneurysm was obtained, the aneurysm was opened longitudinally, and intramural thrombi were removed . Opening venous bleeding was controlled via balloon occlusion (tmp balloon catheter 9 fr, tokai medical products inc ., the avf was approximately 10 mm in diameter and was closed directly via continuous suture using 40 monofilament, non - absorbable polypropylene thread (ethicon inc ., somerville, nj, usa). A dacron bifurcated graft [intergard 16 8 mm, intervascular s.a . (maquetcardiovascular, la ciotat, france)] was anastomosed proximally to the infrarenal aorta and distally to the right cia and the left external iliac artery . The left internal iliac artery was closed directly, and the inferior mesenteric artery was reconstructed by anastomosing the left leg of the graft . Postoperative contrast - enhanced ct revealed a patent graft and absence of the avf (fig . 3a); however, magnetic resonance venography showed left iliac vein occlusion and significant collateral circulation (fig . Abdominal avfs are anomalous passageways between the abdominal aorta, iliac artery, or renal artery and the inferior vena cava, iliac vein, or renal vein . They result not only from aneurysmal diseases (primary cause), but also from iatrogenesis, malignancy, and trauma such as gunshot wounds (secondary causes). Although rare, abdominal avfs can be life - threatening, and intensive care management and urgent treatment consisting of open surgery or endovascular repair are occasionally required . In their review of case reports, mcauley et al . Identified three symptoms consistently associated with the presence of an avf: (1) high - output cardiac failure with a precipitous onset, (2) a pulsatile abdominal mass accompanied by a thrill and a bruit, and (3) unilateral lower - extremity ischemia or venous engorgement . However a variety of symptoms appear depending on the size and location of the avf, time after the rupture, cardiopulmonary function, and bleeding into the retroperitoneum . Several case reports recommend the use of ct or duplex ultrasound as a means of diagnosing avfs . Any combination of imaging modalities, such as contrast - enhanced ct and abdominal ultrasonography, as used in this case, are useful, especially for regional diagnosis of avf . Unresolved issues in open repair, which was performed in our study, include the control of venous bleeding and the prevention of pulmonary embolisms caused by debris or air . In the present case, a balloon occlusion catheter was used to minimize intraoperative bleeding from the fistula, as previously reported . However, we suggest that the balloon technique be considered as a stand - by procedure in open repair . It may be particularly useful when the avf is large or the vessel wall is fragile . Treatment of aneurysms with avfs via endovascular repair has become increasingly more common in recent years . Compared with open repair, it is less invasive, and the risk of major blood loss during treatment is low . However, its long - term performance is still unknown . In a systematic review, the longest follow - up time after endovascular repair was 24 months, and the mean follow - up time was only 9 months . Endovascular repair also carries the risk of type 2 endoleak due to venous bleeding through the avf and consequent expansion of the aneurysm . Conclusions regarding this procedure require long - term follow - ups and randomized controlled trials with large case numbers . In the current case, the patient s anatomy was unsuitable for endovascular treatment, and technical difficulties due to avf coil embolization were envisioned . If his anatomy had been suitable, endovascular repair would have been considered as an initial therapy in view of his age . Notably, in the present case, there was no sign of high - output heart failure throughout the treatment course . Postoperative magnetic resonance venography indicated blood flow failure of the left iliac vein due to compression of the aneurysm . We conclude that the main cause of the swelling in the left leg was not due to left iliac vein occlusion but to an avf, because the swelling was immediately reduced after surgery . We report the successful surgical treatment of an ilio - iliac avf associated with a ruptured ciaa . Using the balloon occlusion technique definitive diagnosis of avf is sometimes difficult, because subjective symptoms and objective findings can be caused by various conditions . Use of multiple imaging modalities facilitates correct preoperative diagnosis in early disease stages and consequently improves surgical outcome . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Makoto iijima: study design, data collection, data analysis, and manuscript preparation.
Percutaneous coronary intervention (pci) has become the first - line treatment for patients suffering from obstructive coronary artery disease (cad). Drug - eluting stents (des) have significantly reduced the risk of restenosis and the need for repeat revascularization when compared to bare metal stent (bms).1 in the ravel and sirius trials,2,3 when compared to bms, des improved restenosis rates and late lumen loss, and decreased target lesion revascularization (tlr) from 16.6% to 4.1% (p<0.01). Although first - generation des (1st gen des) were introduced to disrupt neointimal growth by the use of antiproliferative drugs, this benefit was acquired at the expense of a substantial delay in vascular healing and the clinical consequences of late and very late stent thrombosis (lst / vlst).4 second - generation des (2nd gen des) were developed with newer alloys, biocompatible polymers, thinner struts, and different drugs kinetics, resulting in a reduction of lst / vlst . Small vessel cad accounts for up to 30% of all pci5 and remains an independent predictor of angiographic restenosis and tlr, even after the introduction of des.6 the cobalt chromium everolimus - eluting stent (cocr - ees) (xience v; abbott vascular, santa clara, california, usa) has been reported as one of the most frequently used 2nd gen des with better event - free survival rates in small vessels.7 this review article discusses the preclinical, clinical, and pathological performance of cocr - ees in small vessel cad . The antiproliferative drug used is everolimus, a hydroxyethyl derivative of sirolimus which acts as an immunosuppressant . It induces cell cycle arrest in the g1 phase by inhibiting the mammalian target of rapamycin, a serine / threonine protein kinase that regulates cell growth, proliferation, motility, protein synthesis, and transcription, among others.8,9 the polymer is a thin (7.8 m) bio - inert, non - erodible, and ultra - pure fluorinated copolymer (poly - n - butyl methacrylate [pbma] and poly - vinylidene fluoride and hexafluoropropylene [pvdf - hfp]) that provides both elasticity and stability . Pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp, which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus, respectively, and no top - coat layer is applied . The polymer composition provides mechanical integrity after stent deployment, followed by the controlled release of everolimus at a total dose of 100 g / cm, delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability, and at the same time, increasing its radiopacity, radial strength, and fracture resistance.12 the xience v and xience nano share the same platform design, delivery system, drug, and coating materials . The differentiating features of the xience nano pertain to a balloon diameter of 2.25 mm with a nominal inner stent diameter of 2.25 mm, as compared to xience v, which is available at diameters of 2.5 mm, 2.75 mm, 3.0 mm, 3.5 mm, and 4.0 mm . There are several stent - related factors that have been associated with lst / vlst, such as strut thickness, polymer characteristics, coating integrity, and drug dose, among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard, des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model . Two main factors were reported to determine acute thrombogenicity: 1) strut thickness; and 2) polymer coating . Thrombogenicity within the various bms designs correlated with strut thickness; stents with thicker struts were 49% more thrombogenic than stents with thinner struts (0.880.38 for struts <100 m versus 1.440.65 for struts> 100 m; p=0.036). After 3 days of implantation in porcine coronary arteries, stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts (0.210.041 mm versus 0.130.019 mm; p=0.004); also, neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts (1.560.40 versus 0.830.41; p=0.016). The authors also evaluated overlapping stents, which were more thrombogenic than single length - matched controls, more so for thicker than thinner struts stents (2.320.96 and 3.250.11 versus 1.000.17; p<0.001). Moreover, overlapping thinner strut des (0.510.019) were less thrombogenic than overlapping bms (p<0.001) and even the single bms controls (p<0.001).17 in this landmark study, kolandaivelu et al17 also found that coated stents were less thrombogenic than corresponding bms (0.760.02 versus 1.000.15; p<0.002), and clot mass was also significantly lower for des when compared with bms (0.670.35 versus 1.030.54; p=0.011). Hypersensitivity reactions induced by the durable polymers used in 1st gen des were suggested to contribute to the occurrence of lst.1820 recent advances in stent technology, with the introduction of more biocompatible polymers, have reduced the risk of this complication.21,22 chin - quee et al23 evaluated two different polymers currently available for des in rabbit iliac arteries where cocr stents were coated with pvdf - hfp or phosphorylcholine polymer (without drug) and assessed for endothelialization at 14 days by confocal and scanning electron microscopy (sem). Endothelialization was equivalent and near complete for pvdf - hfp versus phosphorylcholine polymer - coated stents (> 80% by sem). Also, acute thrombogenicity was assessed in a chandler loop model using porcine blood; thrombus adherence was similar for both polymers (0.940.23 versus 0.990.20). These results suggest that the polymers examined here did not impede endothelization.23 polymer coating defects can potentially change the drug elution kinetics of des, or they can lead to chronic inflammatory reactions.17 furthermore, the nonuniform coating of stent struts may impact platelet adhesion and endothelialization . Finally, coating fragments may embolize downstream, resulting in myocardial ischemia . Yazdani et al24 evaluated 48 des for coating integrity in cocr - ees, zotarolimus - eluting stent (zes), paclitaxel - eluting stent (pes), and biolimus a-9-eluting stent (bes) in a rabbit iliofemoral stent model for durations of 7 days, 28 days, 90 days, and 180 days . The cocr - ees and zes had the least amount of coating defects as compared to the pes and bes . However, coating defects were shown to increase over time within the zes, whereas in the cocr - ees, the amount of irregularity remained constant over time . Newer generation des were designed to outperform first - generation devices in this regard . In our laboratory,25 we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees (64.0%27.5%), followed by zes (30.2%14.2%), pes (26.8%15.8%), and sirolimus - eluting stent (ses) (6.4%4.2%), with a statistically significant difference versus cocr - ees (p<0.003) and bms (p<0.0001) as a control stent (figure 1). At 28 days, all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees, but without statistically significant differences among groups . Furthermore, cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry, the greatest frequency of uncovered struts was observed in the middle stented segment, while proximal and distal segments showed overall greater coverage . We also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule, pecam-1, as a surrogate, and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts, which demonstrates a functionally and biologically active endothelium.25 the antiproliferative drug used is everolimus, a hydroxyethyl derivative of sirolimus which acts as an immunosuppressant . It induces cell cycle arrest in the g1 phase by inhibiting the mammalian target of rapamycin, a serine / threonine protein kinase that regulates cell growth, proliferation, motility, protein synthesis, and transcription, among others.8,9 the polymer is a thin (7.8 m) bio - inert, non - erodible, and ultra - pure fluorinated copolymer (poly - n - butyl methacrylate [pbma] and poly - vinylidene fluoride and hexafluoropropylene [pvdf - hfp]) that provides both elasticity and stability . Pbma serves as a base coat for the stent and facilitates anchorage of pvdf - hfp, which serves as a matrix layer containing the drug at a ratio of 83%/17% for polymer / everolimus, respectively, and no top - coat layer is applied . The polymer composition provides mechanical integrity after stent deployment, followed by the controlled release of everolimus at a total dose of 100 g / cm, delivering up to 80% of the drug after 4 weeks.10 the platform is the multilink vision l-605 cobalt chromium alloy with a strut thickness of 81 m mounted on a compliant tapered vision balloon,11 structurally designed to improve deliverability and conformability, and at the same time, increasing its radiopacity, radial strength, and fracture resistance.12 the xience v and xience nano share the same platform design, delivery system, drug, and coating materials . The differentiating features of the xience nano pertain to a balloon diameter of 2.25 mm with a nominal inner stent diameter of 2.25 mm, as compared to xience v, which is available at diameters of 2.5 mm, 2.75 mm, 3.0 mm, 3.5 mm, and 4.0 mm . There are several stent - related factors that have been associated with lst / vlst, such as strut thickness, polymer characteristics, coating integrity, and drug dose, among others.13,14 it is known that strut thickness affects the angiographic and clinical outcome after pci.15 in this regard, des with thinner struts have been reported to provide improvement in outcomes with respect to target vessel revascularization when compared to thicker strut des in calcified lesions.16 kolandaivelu et al17 evaluated the impact of strut thickness on thrombogenicity in a chandler loop model . Two main factors were reported to determine acute thrombogenicity: 1) strut thickness; and 2) polymer coating . Thrombogenicity within the various bms designs correlated with strut thickness; stents with thicker struts were 49% more thrombogenic than stents with thinner struts (0.880.38 for struts <100 m versus 1.440.65 for struts> 100 m; p=0.036). After 3 days of implantation in porcine coronary arteries, stents with thicker struts demonstrated significantly more thrombus and 62% more clots compared to their thinner strut counterparts (0.210.041 mm versus 0.130.019 mm; p=0.004); also, neointimal fibrin accumulated to a greater extent around the thicker struts compared to the thinner struts (1.560.40 versus 0.830.41; p=0.016). The authors also evaluated overlapping stents, which were more thrombogenic than single length - matched controls, more so for thicker than thinner struts stents (2.320.96 and 3.250.11 versus 1.000.17; p<0.001). Moreover, overlapping thinner strut des (0.510.019) were less thrombogenic than overlapping bms (p<0.001) and even the single bms controls (p<0.001).17 in this landmark study, kolandaivelu et al17 also found that coated stents were less thrombogenic than corresponding bms (0.760.02 versus 1.000.15; p<0.002), and clot mass was also significantly lower for des when compared with bms (0.670.35 versus 1.030.54; p=0.011). Hypersensitivity reactions induced by the durable polymers used in 1st gen des were suggested to contribute to the occurrence of lst.1820 recent advances in stent technology, with the introduction of more biocompatible polymers, have reduced the risk of this complication.21,22 chin - quee et al23 evaluated two different polymers currently available for des in rabbit iliac arteries where cocr stents were coated with pvdf - hfp or phosphorylcholine polymer (without drug) and assessed for endothelialization at 14 days by confocal and scanning electron microscopy (sem). Endothelialization was equivalent and near complete for pvdf - hfp versus phosphorylcholine polymer - coated stents (> 80% by sem). Also, acute thrombogenicity was assessed in a chandler loop model using porcine blood; thrombus adherence was similar for both polymers (0.940.23 versus 0.990.20). These results suggest that the polymers examined here did not impede endothelization.23 polymer coating defects can potentially change the drug elution kinetics of des, or they can lead to chronic inflammatory reactions.17 furthermore, the nonuniform coating of stent struts may impact platelet adhesion and endothelialization . Finally, coating fragments may embolize downstream, resulting in myocardial ischemia . Yazdani et al24 evaluated 48 des for coating integrity in cocr - ees, zotarolimus - eluting stent (zes), paclitaxel - eluting stent (pes), and biolimus a-9-eluting stent (bes) in a rabbit iliofemoral stent model for durations of 7 days, 28 days, 90 days, and 180 days . The cocr - ees and zes had the least amount of coating defects as compared to the pes and bes . However, coating defects were shown to increase over time within the zes, whereas in the cocr - ees, the amount of irregularity remained constant over time . Newer generation des were designed to outperform first - generation devices in this regard . In our laboratory,25 we compared 1st gen des and 2nd gen des in new zealand white rabbits and reported at 14 days that re - endothelialization above struts was variable among stents with significantly greater coverage in cocr - ees (64.0%27.5%), followed by zes (30.2%14.2%), pes (26.8%15.8%), and sirolimus - eluting stent (ses) (6.4%4.2%), with a statistically significant difference versus cocr - ees (p<0.003) and bms (p<0.0001) as a control stent (figure 1). At 28 days, all evaluated stents had more than 60% endothelial cell coverage above struts in favor of cocr - ees, but without statistically significant differences among groups . Furthermore, cocr - ees had the least percentage of struts lacking endothelial coverage compared to other des.25 based on morphometry, the greatest frequency of uncovered struts was observed in the middle stented segment, while proximal and distal segments showed overall greater coverage . We also evaluated endothelium integrity using the platelet endothelial cell adhesion molecule, pecam-1, as a surrogate, and found that cocr - ees had significantly greater cell - to - cell contact sites above the struts, which demonstrates a functionally and biologically active endothelium.25 randomized controlled clinical trials have established differential outcomes in the safety and efficacy of des used in distinct clinical settings, and stent - related factors may play an important role in the scenery of small vessel pci.13,14,26 cannon et al27 evaluated the safety of xience nano in vessels> 2.25 mm but <2.5 mm at 1-year follow - up . The authors established a performance goal (pg) at 20.4% for target lesion failure (tlf) based on clinical trials and registries, which evaluated 2.25 mm diameter des.14,28,29 the 1-year tlf rate was 8.1%, with an upper one - sided limit (95% confidence interval) of 13.0%, meeting the pg of 20.4% (p<0.0001). The 1-year tlf rate was mainly driven by low cardiac death and myocardial infarction (mi) rates . The most important difference noted in this study was a higher tlf rate for the reference vessel diameter (rvd) 2.12 mm (number [n] = 72), which reached 13.89%, compared to 1.56% for a rvd> 2.12 mm (n=64). In a post clinically, no statistically significant differences were found for tlf (5.7% versus 4.9%; p=1.0) and major adverse cardiovascular event rates (mace)/tlf rates (7.5% versus 8.5%; p=1.0) in diabetics versus nondiabetics, respectively . Also, angiographically, in - stent and in - segment late loss showed no difference between diabetics and nondiabetics (0.220.47 mm versus 0.190.36 mm, p=0.83; and 0.140.48 mm versus 0.170.38 mm, respectively, p=0.76).27 small vessel cad represents a challenge for interventional cardiologists, with higher restenosis and stent thrombosis (st) rates.30,31 hermiller et al32 evaluated the safety of cocr - ees in small and nonsmall vessels in a real - world scenario, applying a 2.5 mm diameter cut - off . The mean rvd for small vessels was 2.550.36 mm and 3.250.46 mm for the nonsmall vessel group (p<0.001). Definite or probable st rates were low and not significantly different between the groups at 0.37% versus 0.40% (p=0.88) for the small and nonsmall vessel groups, respectively . The composite rate of cardiac death or mi was comparable for the small and nonsmall vessel group (4.5% versus 5.1%, respectively; p=0.57). The 1-year tlr rate was also comparable in the small vessel group (small group 3.8% versus nonsmall group 3.0%; p=0.35). This study demonstrated the safety of cocr - ees in small vessels despite the fact that this group consisted of more females, those with a higher rate of diabetes, and those with more complex lesion characteristics.32 in a separate study, ito et al33 compared cocr - ees and pes for small vessel revascularization by pooling the data from the spirit iii and iv trials.34,35 from 4,689 patients, two groups were analyzed: the small vessel group (rvd: 2.250.19 mm; n=1,019) and the large vessel group (rvd: 2.990.35 mm; n=2,586). After 1-year follow - up, in patients with small vessels disease, the tlf (cocr - ees 4.4% versus pes 7.9%; p=0.03) and mace (cocr - ees 4.5% versus pes 7.9%; the clinical endpoint, tlf, was composed of cardiac death, target vessel mi, and ischemia driven - tlr (id - tlr). Amid the others, only id - tlr showed a significant reduction at 1 year (everolimus - eluting stents [ees] 2.4% versus pes 5.5%; p=0.02). Although, st showed higher rates in small vessel revascularization, the authors found that st was significantly lower in patients with small vessels treated with cocr - ees than in those treated with pes (0.2% versus 1.2%, respectively; p=0.04).33 currently, the factors predictive of in - stent restenosis can be divided into patient - related, procedure - related, and lesion - related factors . Patient - related factors such as diabetes, a history of restenosis, and genetic factors have been reported as risk factors of in - stent restenosis.33 procedure - related factors include the number of stents implanted, the total stent length, and stent overlap . Lesion - related characteristics, which impact the rate of restenosis, include small vessel size, long lesion length, and the severity of pretreatment as well as posttreatment lesion stenosis, among others.36 claessen et al37 collected data from spirit ii, iii, and iv,34,38,39 and combined three groups: short lesions in large vessels (group a); long lesions in large vessels or short lesions in small vessels (group b); and long lesions in small vessels (group c) to evaluate the safety and efficacy of cocr - ees versus pes . The mace rate after 2 years of follow - up was lower in group a, intermediate in group b, and highest in group c (5.6% versus 8.2% versus 10.4%, respectively; p<0.0001). Also, a similar trend was observed for mi (3.3% versus 3.0% versus 4.5%, respectively; p=0.02) and id - tlr (2.9% versus 5.0% versus 6.3%, respectively; p=0.0002). The authors also evaluated mace rates by stent type (pes and cocr - ees), and found that the higher the lesion complexity, the greater the mace incidence (7.0%, 11.2%, and 12.8% for pes, respectively, p=0.007; versus 4.8% versus 6.6% versus 9.1% for cocr - ees, respectively, p=0.001). On the other hand, the 2-year rate of definite or probable st (academic research consortium, arc definition) also increased with greater lesion complexity after pes implantation (group a 0.7% versus group b 1.9% versus group c 2.8%; p=0.03), but that relationship was not present after cocr - ees implantation (0.9% versus 0.6% versus 0.6%; p=0.65). Cocr - ees were associated with significantly lower rates of mace, mi, id - tlr, and st in groups b and c, but no statistical significance was found in the less complex group (group a). Multivariate analysis found that the use of cocr - ees rather than pes was an independent predictor of freedom from mace in group b (p<0.0001) and group c (p=0.004), but not in group a (p=0.19).37 recently, we reported40 the pathologic findings of 2nd gen des and compared these to 1st gen des . A total of 204 lesions (ses = 73; pes = 85; cocr - ees = 46) from 149 autopsy cases with implant duration> 30 days and 3 years were pathologically analyzed to determine differences . The observed frequency of lst and vlst was less for cocr - ees (4%) compared with ses (21%; p=0.029) and pes (26%; p=0.008). The prevalence of restenosis for cocr - ees (17%) did not differ significantly from that observed in ses (14%) and pes (12%). The frequency of uncovered struts was markedly lower for cocr - ees (2.6%) as compared to ses (18.0%; p<0.0005) and pes (18.7%; p<0.0005). The prevalence of des with> 30% uncovered struts was also significantly lower in cocr - ees (20%) than in ses (60%; p<0.0005) and pes (67%; p<0.0005). In terms of inflammation, the overall prevalence of neoatherosclerosis after cocr - ees implantation in native coronary arteries was 29%, which did not differ significantly from ses (35%; p=0.62) and pes (19%; p=0.47).40 complex lesion characteristics and unstable plaques are associated with a greater delay in arterial healing when compared to pci of a simple and stable plaque by pathology.41 therefore, we evaluated the prevalence of> 30% uncovered struts in the setting of off - label versus on - label clinical indications . Cocr - ees compared with ses and pes showed greater strut coverage for both on - label (14% versus 50% versus 57%, respectively) and off - label (25% versus 68% versus 75%, respectively) indications . When analyzing the cvpath stent database composed of 865 cases, 68 had cocr - ees implanted and 12 cases were found with a stent diameter of 2.5 mm or less (table 1). From those 12 cases, the mean stent length was 35.624.9 mm and the mean stent diameter was 2.30.27 mm . Case 1: a 45-year - old woman with obesity, hypertension, and diabetes who presented with mi secondary to involvement of the left circumflex artery, which was revascularized; 5 days after the procedure, she died suddenly with st . Case 2: a 58-year - old male with a history of obesity and cad presented with non - st segment elevation mi, and left anterior descending artery occlusion in the region of the left diagonal branch; bifurcation stenting was performed . The patient died suddenly 7 days after the procedure; at autopsy, there was a large infarction and mild to moderate thrombus in the stented region . The other three patients had stable cad and only mild inflammation was observed with moderate peristrut fibrin and platelet deposition (figure 2). One of the three cases had thrombosis and stent fracture (20%); the other two were nonstent - related deaths . The remaining seven cases (table 1) had a duration> 30 days (242.9222.6 days). Acute coronary syndrome was the indication in one case (14.3%), and the cause of death was stent - related a 72-year - old woman with obesity, hypertension, diabetes, atrial fibrillation, and a history of multiple revascularizations . She died from st 210 days later with underlying restenosis . For the rest of the cases (n=6) with duration> 30 days, there were no differences in the principal histopathological findings among those presenting with acute coronary syndrome versus stable cad . Overall, the histopathological analysis showed mild to moderate chronic peristrut inflammation consisting of monocytes, t - lymphocytes, and macrophages (figure 3) without any significant eosinophils . Restenosis was observed in one of the six cases; however, the xience case was sandwiched between two vision stents, both of which had total occlusion . Nevertheless, we must recognize the limited number of cases analyzed at autopsy . A greater number of cases and matched control groups will be required to understand the full scope of histopathological findings of cocr - ees in small vessel disease . Des have progressively improved clinical outcomes, but the potential risk of st is still a concern and limits the use of des, especially in small vessel cad . Overall, lst and vlst have been reported with an incidence of 0.2% and 0.4% per year, respectively.42 however, considering the large amount of stents implanted worldwide, those numbers are still high . Several reports of st have been associated with 1st gen des, especially after dual anti - platelet therapy termination.43,44 over time, great effort has been made to improve the technology, thus reducing strut thickness from 140 m to approximately 7080 m, resulting in a dramatic reduction of thrombogenicity in bench studies.17,45 advances in polymer technology have been enormous; new biocompatible polymers (pbma or pvdf - hfp) result in less inflammation after stent implantation, with the consequence of more complete and functional endothelization.46,47 also, cocr - ees show fewer coating defects after implantation when compared to different des, and this result was maintained over time and may improve vascular biocompatibility.25 clinical data confirmed the outstanding performance of cocr - ees, with lower rates of definitive / probable st, tlr, and mace . The pg for tlf was overperformed with cocr - ees when compared with a competitor des in small vessel disease . Mace and tlf rates were similar among diabetics and nondiabetics.27 at pathology, cocr - ees revealed less inflammation and greater strut coverage when compared to 1st gen des, while maintaining similar efficacy in reducing neointimal growth . Specifically, in small vessel disease, cocr - ees have been shown to be less thrombogenic compared to 1st gen des; however, inflammation and restenosis remain a problem in this setting, and further technological and procedural progress is needed to improve patient outcomes . In the des era, small vessel cad remains a great challenge for interventional cardiologists . Stent design and the material combination may provide better outcomes, especially in small vessel disease . Cocr - ees with thinner struts, biocompatible polymers, reduced drug load, and better radiopacity and trackability have shown excellent results from preclinical, clinical, and pathological studies in small vessel cad.
The reconstruction of multiple missing teeth with dental implants is a predictable and proven treatment technique for edentulous patients in both anterior and posterior regions.12 however, the molar teeth are often shown to be a troublesome area for implant restorations from mechanical and biological aspects,3 which is due to complicated and complex factors of implant prosthetic components and the load - related bone contact area.4 the strong occlusal forces exert harmful effects on an implant prosthesis and alveolar bone in the posterior area,56 which results in marginal bone loss and decreased implant stability, and can lead to complications in an implant fixture and its suprastructure.78 natural teeth are traditionally splinted in order to decrease the stress and increase the stability of prosthesis . This can also result in a smaller horizontal load being transferred to the supporting teeth, and can compensate for the crown - root ratio increasing in various alveolar bone - loss regions and periodontally compromised patients.39 several studies have recommended that adjacent implants should be splinted with the fixed retained prosthesis.3 when off - axis forces are applied to an implant, they induce an adverse loading that can cause mechanical failure of a restored implant and biological failure of the surrounding bone that could lead to implant failure.101112 the aim of a splinted implant restoration is to favorably distribute the stress between the implants in order to minimize the transmission of horizontal forces to the bone - implant contact area.31314 in particular, two - implant splinting (2-is) in the posterior region can promote the stability in the mesiodistal direction and relieve the stress in the buccolingual direction.9 2-is can also be considered as an important treatment option in patients without anterior guidance or with parafunctional oral habits.15 nevertheless, several procedures of multiple - implant restoration splinting are highly technique - sensitive, and the accuracy of the final prosthesis is mainly limited.15 alveolar bone loss is common in patients with periodontitis, which will lead to an unfavorable crown - implant ratio (c / i ratio). An off - axis force acting on an implant restoration with an increasing c / i ratio and crown height space (chs) of the implant - defined as the distance from the alveolar bone crest to the occlusion plane - can induce a detrimental load at the implant restoration neck area, and result in surrounding bone loss and eventual prosthetic failure.1617 according to grossmann et al.,3 the splinting technique can be an appropriate treatment option for periodontitis patients who have an impaired occlusal relationship due to the loss of alveolar bone and multiple teeth . In spite of splinted implant restoration being beneficial for periodontitis patients with severe alveolar bone loss and excessive occlusal forces in the posterior region, most of the studies have been theoretical, with insufficient clinical analyses and negative long - term results.3 several studies of splinted prostheses have involved short - term investigations and shown limited efficacies and controversial results, and so further investigations and long - term studies are required.151819 the aim of this retrospective study was to determine the efficacies of 2-is, and compare them with those of single implant restoration (1-ir) in the first and second molar regions, which has been demonstrated to produce good results in previous studies . This study has also identified the appropriate clinical considerations for splinted implant restoration of the molar region . This study was approved by the institutional review board of the ilsan hospital, national health insurance service (nhis) (approval no . All surgical treatment procedures were performed by periodontists at the department of periodontology, ilsan hospital, nhis . The study was limited to the posterior region, including patients with missing first and second molars only, in order to minimize the effects of position and occlusal force . The internal connection implant fixtures comprising a sand - blasted, large - grit, acid - etched surface (implantium, dentium, seoul, korea; straumann, institut straumann, basel, switzerland) were placed using a one- or two - stage surgical procedure as the manufacturer's protocol . The prosthesis type [i.e., occlusal screw (os), lateral screw (ls), cementation (cm), and screw - cement - retained prosthesis (scrp)] was selected depending on the condition of the patient and the preferences of the prosthodontist, and occlusal adjustment was carried out to obtain the optimal centric and eccentric contact forces . Maintenance care that emphasized scaling and oral hygiene instruction was provided every 3 - 6 months, and intraoral periapical or panorama radiographs were obtained every 12 months . Patients who had undergone implant surgery at the department of periodontology, ilsan hospital, nhis during 2005 - 2014 were reviewed over a mean functional loading period (flp) of 40 months . The following inclusion criteria were applied: sex ratio of 1: 1, aged 20 - 80 years (mean age 58.5 years), and good systemic health condition (including well - controlled systemic diseases). The implant prosthesis had been functioning for 1.1 - 102.8 months, with a mean loading period of 41.4 months . Single - implant restorations in the first or second molar region were classified into the 1-ir group, while restorations involving two splinted implants in the first and second molar regions were classified into the 2-is group . Patients with severe systemic disease, advanced or aggressive periodontitis, or parafunctional oral habits (e.g., excessive occlusal force, heavy clenching, or bruxism) were excluded from this research . In total, 408 implants in 234 patients who conformed to the inclusion and exclusion criteria were investigated . All data related to these patients with implant treatments were based on the clinical treatment records, clinical photographs, and radiographs of the patients . The following clinical factors of the patients were considered: sex, mean age, implant location, flp, bone grafting, clinical c / i ratio, chs, horizontal distance (hd) between the two implants (hdi, the first and second molar positions), and hd between the natural tooth in the mesial position and the implant in the distal position (hdni, the first or second molar position).2021 based on previous studies, the clinical c / i ratio was defined as the distance ratio measured from the clinical crown to the implant fixture (standard fulcrum located at the marginal bone), and chs was measured as the distance from the alveolar bone crest to the occlusion plane.22 hdi and hdni were measured as the distance at the marginal bone level on the day of implant placement . A pacs workstation (centricity ge healthcare, waukesha, wi, usa) was used to calculate the clinical c / i ratio, chs, hdi, and hdni on the radiographs, and distortion caused by magnification was corrected using a calibration based on the known interthread pitch of the implant (implantium, dentium: 0.6 mm; straumann, institut straumann: 1.25 mm) as a reference . Mechanical complications [i.e., sl, screw fracture (sf), cf, and repeated sl] and biological complications [i.e., peri - implant mucositis (pm) and periimplantitis (pi)] were evaluated for each patient . The biological complications were examined based on mobility, suppuration, probing depth, bleeding on probing, and alveolar bone loss . A reversible inflammation of peri - implant mucosa was diagnosed as pm, and loss of alveolar bone was diagnosed as pi.23 all measurements were performed using a unc periodontal probe (hu - friedy, chicago, il, usa). In comparisons and analyses of two groups, the chi - square test and student's t - test (two - tailed with independent samples) were used to identify the relationships between the clinical factors (i.e., sex, mean age, implant location, flp, clinical c / i ratio, chs, hdi, and hdni) and the complication rates (i.e., mechanical and biological complications). The chi - square test was applied to noncontinuous variables and student's t - test (two - tailed with independent samples) was applied to continuous variables . The optimal cutoff value for flp related to the complications was evaluated using receiver operating characteristics (roc) analysis . The results obtained in all of the investigations were analyzed using spss software (version 19.0, spss, chicago, il, usa). The cutoff for statistical significance was set at p the subjects of this study comprised the 1-ir group, which contained 124 patients (69 males, 55 females) with a mean age of 56.26 years (range, 23 - 77 years), and the 2-is group, which contained 110 patients (53 males, 57 females) with a mean age of 59.01 years (range, 34 - 91 years). The implants in the 1-ir group were distributed in the posterior region as follows: maxillary first molar, n = 22 (15.9%); maxillary second molar, n = 10 (7.3%); mandibular first molar, n = 43 (31.2%); and mandibular second molar, n = 63 (45.6%). Totals of 32 (23.2%) and 106 (76.8%) implants were positioned in the maxilla and mandible, respectively . In the 2-is group, 134 (67 pairs, 49.6%) and 136 (68 pairs, 50.4%) implants were positioned in the maxilla and mandible, respectively . The mean flp was 42.87 months (range, 1.84 - 101.25 months) for the 1-ir group and 39.86 months (range, 1.08 - 102.75 months) for the 2-is group . In addition, bone grafting was performed for 32 (23.3%) implants in the 1-ir group and for 108 (54 pairs, 40%) implants in the 2-is group, showing an intergroup difference of about threefold . For the 1-ir group, the clinical c / i ratio was 1.11 0.47, chs was 9.65 1.98 mm, and hdni was 2.80 1.17 mm; the corresponding values for the 2-is group were 1.07 0.21, 9.62 1.75 mm, and 3.26 1.30 mm, respectively . These results are consistent with a previous study finding that in order to minimize bone loss, hdi (i.e., interimplant distance) should be longer than hdni (i.e., distance from the adjacent natural tooth to the implant).24 most of the data represents a mean value of normal distribution curve; the deviation is observed in individual clinical situation of a patient . Since periodontist performed the treatment in the controlled clinical setting, it can be said that the position of implant is appropriate in this study (table 1). Mechanical complications were found in 31 (22.6%) of the 138 implants in the 1-ir group, with sl (n = 23, 16.7%) being the most common complication . This was followed by sf and cf (n = 3, 2.2%), and then repeated sl (n = 2, 1.5%). Mechanical complications were found in 30 (11.1%) of the 270 implants in the 2-is group, corresponding to approximately half the rate in the 1-ir group . Cf (n = 14, 5.2%) was the most common complication, followed by sl (n = 10, 3.7%) and then sf (n = 6, 2.2%), while repeated sl did not occur in any of the implants . The rate of mechanical complications differed significantly between the two groups (p = .020), with only sl showing a clearly significant increase in the 1-ir group (p <.001). The rate of biological complications was markedly higher in the 2-is group than in the 1-ir group . Only pi (n = 5, 3.6%) was found in the 1-ir group . In contrast, out of the 44 (16.3%) implants with biological complications in the 2-is group, pi was found in 26 (9.6%) implants, followed by pm, which was found in 18 (6.7%) implants . The rate of biological complication differed significantly between the two groups (p <.001), with significantly elevated incidence rates of pm (p = .002) and pi (p = .046) in the 2-is group . In 1-ir group, sl, sf, and cf occurred simultaneously on one implant . Also, sl and cf occurred simultaneously on another implant, and sl and pi occurred simultaneously on the other implant . In 2-is group, sl and sf occurred simultaneously on one implant, and sl, cf, and pi occurred simultaneously on another implant (table 2). Patients in the 2-is group who did or did not experience complications at least once were classified into the complication and success groups, respectively . Mechanical and biological complications showed no statistically significant associations with sex, age, implant location in the jaw, and bone grafting . The flp was the only clinical factor to show a statistically significant difference with complications and success in the 2-is group (p = .049). 2-is remained relatively successful up to a mean of 38 months, while biological and mechanical complications arose after mean time periods of approximately 49 and 56 months, respectively . Implant - supported fixed dental prostheses (isfdps) located in the posterior region can induce stress in the implant and marginal bone when there is an unfavorable c / i ratio (anatomical and/or clinical c / i ratio of 2).25 in the present study, the overall clinical c / i ratio in the 2-is group was 1.07 0.21 (first molar region = 1.06 0.18, second molar region = 1.09 0.23), and the maximum value was 1.84 (table 1), confirming that a favorable c / i ratio had been achieved . Moreover, the clinical c / i ratio was close to 1: 1 in both the mechanical complication and success groups (1.05 0.14 and 1.08 0.21) and the biological complication and success groups (1.05 0.23 and 1.08 0.20), demonstrating favorable clinical c / i ratios.25 correspondingly, there were no statistically significant differences in the clinical c / i ratios . Recent studies262728 have found that chs values exceeding 15 mm indicate an increased risk of implant prosthesis failure due to a vertical cantilever effect . In this study, the overall chs in the 2-is group was 9.62 1.75 mm (first molar region = 9.82 1.65 mm, second molar region = 9.43 1.84 mm), with a maximum value of 14.28 mm (table 1). Given that this is within the acceptable chs range (8 - 12 mm) and below 15 mm, chs is not expected to have a negative effect on the prognosis of the implant prosthesis . Chs did not differ significantly among the mechanical complication and success groups (9.67 1.31 and 9.62 1.80 mm, respectively) and the biological complication and success groups (10.04 1.94 and 9.53 1.70 mm, respectively). Hdi also did not differ significantly among the mechanical and biological complication groups (3.36 1.27 and 3.51 1.51 mm, respectively) and the mechanical and biological success groups (3.25 1.30 and 3.21 1.25 mm, respectively) (table 3). Ls was the most commonly used type of prosthesis in the 1-ir group (n = 84, 60.9%), and was associated with the following rates of mechanical complications: sl, n = 14 (16.7%); repeated sl, n = 1 (1.2%); sf, n = 2 (2.4%); and cf, n = 2 (2.4%). The next most common type of prosthesis was cm (n = 23, 16.7%; sl, n = 5, 21.7%; repeated sl, n = 0, 0%; sf, n = 1, 4.4%; cf, n = 1, 4.4%), followed by scrp (n = 19, 13.8%; sl, n = 2, 10.5%; repeated sl, n = 0, 0%; sf, n = 0, 0%; cf, n = 0, 0%) and os (n = 12, 8.7%; sl, n = 2, 16.7%; repeated sl, n = 1, 8.3%; sf, n = 0, 0%; cf, n = 0, 0%). Sl was the most frequent mechanical complication, and was associated with the prosthesis types as follows: os, n = 2 (16.7%); ls, n = 14 (16.7%); cm, n = 5 (21.7%); and scrp, n = 2 (10.5%). Although sl occurred proportionally the most often in cm, in terms of absolute numbers it occurred the most often in ls . However, there were no statistically significant associations between prosthesis types and mechanical complication rates (p = .304). Ls was the most commonly used type of prosthesis in the 2-is group (n = 150, 55.6%), and was associated with the following rates of mechanical complications: sl, n = 8 (5.3%); repeated sl, n = 0 (0%); sf, n = 4 (2.7%); and cf, n = 6 (4.0%). The next most common type of prosthesis was cm (n = 35, 25.9%; sl, n = 2, 2.9%; repeated sl, n = 0, 0%; sf, n = 2, 2.9%; cf, n = 4, 11.4%), followed by scrp (n = 17, 12.6%; no complications) and os (n = 8, 5.9%; no complications). The overall mechanical complication rates were lower for all prosthesis types in the 2-is group than in the 1-ir group, with sl being particularly rare . Os and scrp, which were used proportionally less, showed no complications at all . As with the 1-ir group, there were no statistically significant differences (p = .425) (table 4). There was no case of pm for any of the prosthesis types in the 1-ir group . Pi occurred in os (n = 3, 25.0%), ls (n = 1, 1.2%), and cm (n = 1, 4.4%), but was not observed in scrp . Conversely, biological complications occurred for all prosthesis types in the 2-is group, with the following rates: os (pm, n = 0, 0%; pi, n = 4, 25.0%), ls (pm, n = 4, 2.7%; pi, n = 18, 12.0%), cm (pm, n = 12, 17.1%; pi, n = 4, 5.7%), and scrp (pm, n = 2, 5.9%; pi, n = 0, 0%). Pm was most common in cm, while pi showed the highest rate in os but the highest absolute frequency in ls . In both groups, there were no statistically significant associations between prosthesis types and biological complications (p = .385 and 0.385, respectively) (table 4). The results listed in table 3 indicate that flp was the only variable that had an important impact on mechanical and biological success . The roc curve for flp of mechanical and biological complications is shown in fig . The area under the roc curve (auc) for flp of mechanical complications is 0.725, which indicates a reliable result since the value exceeds 0.5 . The optimal cutoff value was 46.57 months (95% confidence interval, 0.61 - 0.84), which gave a sensitivity of 69.2% and a specificity of 69.7% . In addition, the auc for flp of biological complications was 0.615, which is also a reliable result (i.e.,> 0.5). The optimal cutoff value was 39.80 months (95% confidence interval, 0.49 - 0.74), which gave a sensitivity of 54.5% and a specificity of 54.9% . While 1-ir has been predictable treatment modality in the posterior edentulous region, mechanical and biological complications occur frequently.2930 these complications include sl, cf, implant fixture fracture, de - cementation, pm, and pi, and also, they often occur in multiple - implant restoration splinting.31 in addition, the implant success rate of 1-ir (94.3%) was not significantly lower than that of multiple - implant restoration splinting (97.1%).32 therefore, these two types of the implant restoration have been reported to have similar success rates . However, there were some differences between the 1-ir and 2-is groups in the present study in the characteristics of mechanical and biological complication rates related to clinical factors . There was no significant association between the implant position (first or second molar region) in the 1-ir group and the occurrence of mechanical and biological complications (p = .243 and p = .746, respectively). These results were identical to those of a previous study.33 also, prosthesis types were not associated with complication rates in the 1-ir and 2-is groups, and did not affect the comparison of the two groups (p = .276). Compared to 2-is sl was reported as the most common mechanical complication of implant - supported single crowns (isscs),2934 and also in the present study this was the only major complication associated with a statistically significant increase in occurrence (p <.001). Sex was the only clinical factor exerting an important influence on the occurrence of mechanical complications in 1-ir (p = .040). Finally, 91.3% (21 implants) of all sl cases occurred in male patients, which is possibly due to the biting force and occlusal contact area both being greater than in female patients.3536 conversely, in 2-is, there was a significant increase in the rates of biological complications (p <.001). One possible explanation is that although regular dental hospital visits following the completion of the final implant prosthesis enabled examinations during the early stages, the intervals between the dental hospital visits became longer over time, resulting in a reduced awareness about oral hygiene management . Similarly, the significant increases in pm (p = .002) and pi (p = .046) are due to the difficulty of performing adequate oral hygiene management in splinted implant restorations . Several studies have found that nonsplinted implant restoration was advantageous for oral hygiene management.3 these results emphasize the need to provide patients with specific instructions about the use of dental floss and interdental brushes, especially for 2-is . Previous studies2737 found that sl induced changes in centric and eccentric contact forces and nonideal occlusion, and that this was a major cause of sf and cf . Moreover, food impaction on the inferior aspect of the implant prosthesis was caused by sl, and the resulting gingival redness and swelling not only increased the occurrence rates of pm and pi but also decreased the survival and success rates of the implants.38 in the present study we also observed the simultaneous occurrence of sf, cf, and pi with sl in three implants from the 1-ir group and in two implants from the 2-is group . The superiority of 1-ir in the first and second molar regions has been reported previously.3940 2-is was advantageous over 1-ir in terms of mechanical complications but disadvantageous in terms of biological complications . The decreased incidence of mechanical complications is probably attributable to the improved stress distribution resulting from the splinting technique reducing the transfer of excessive forces to the implant fixture and surrounding bone31314 . In addition, the splinting technique is able to promote the retention and resistance of the prosthesis, and successful outcomes can also be expected under certain limiting conditions such as insufficient abutment length, abnormal loading, or long treatment period.3 the increased incidence of biological complications is probably due to structural aspects of the splinting technique . It is often difficult to ensure the formation of appropriate paths and distances when placing fixtures in patients with periodontal disease . This makes it difficult to form an appropriate contour and embrasure between the inferior aspect of the splinted implant restoration and the interproximal region, which is a limitation in oral hygiene management . Given that biological complications cause inflammation and the loss of tissue and bone in the surrounding implant, and increase the risk of implant failure, these complications need to be managed carefully . Conversely, the disconnection and reconnection are more convenient for 1-ir, and examining and ensuring the hygiene of the prosthesis are easier than for the splinted implant restoration.4142 the only clinical factor that strongly affected the mechanical and biological complications associated with 2-is was the flp . In 2-is, relatively successful outcomes were maintained for up to 38 months on mean after the placement of the implant prosthesis . However, biological and mechanical complications occurred after mean flp of approximately 49 and 56 months, respectively . These findings contrast with a previous study finding that mechanical complications usually occurred sooner than biological complications during follow - up periods of 1 - 2 years.43 we attribute this difference to our clinical protocol of implant treatment for preventing mechanical complications, since patients were encouraged to visit the dental hospital continuously at short intervals after completing the treatment so that their implant prostheses could be examined regularly . Pm and pi were previously reported to occur in 50% and 10 - 43% of all implants, respectively.4445 however, the incidence of biological complications was considerably lower in the present study, with pm and pi occurring in 0% and 3.6% of 1-ir, respectively, and in 6.7% and 9.6% of 2-is . The dental hospital visits occurred regularly at intervals of 3 - 6 months during the 3 years after implant placement in the present study . However, after 3 years the interval between the dental hospital visits increased to 1 year, and some cases were lost to follow - up due to cancelled appointments . This led to neglect of oral hygiene management, which resulted in increased rates of biological complications due to the deposition of plaque and calculus and of mechanical complications due to the lack of regular examinations approximately 1 - 2 years later . Therefore, within the limitations of this study, the optimal cutoff values for flp with significant effects on the mechanical and biological success of 2-is were calculated using roc analysis . This information can be used to determine the optimal period for follow - up by predicting the time at which complications might occur . Such a strategy will help prevent complications, while making it possible to explain the importance of regular dental hospital visits to patients and also providing them with motivation . In the previous study, an excessively long hdni (> 3.7 mm) was a significant factor affecting the prognosis of isscs located in the first and second molar regions.33 however, in the present study, hdi was not an important cause of the complications in 2-is . This is probably due to hdi has to be longer than hdni in order to reduce the alveolar bone loss,24 and the reduction in the cantilever effect due to dispersal of the occlusal forces when the implant prosthesis is splinted . Moreover, careful consideration should be given to a certain degree of hdi needing to be established for an interproximal design in order to simplify the use of oral hygiene products such as dental floss and interdental brushes . The results of this study need to be analyzed carefully because they were limited by the lack of investigations of various clinical factors that could affect implant restoration, such as the individual physiological characteristics, occlusal relationships, and parafunctional oral habits of each patient.464748 therefore, more reliable results can be expected from future investigations of restorations involving two or more splinted implants to analyze the effect of the above factors on mechanical and biological complications and from long - term evaluations . In addition, a careful prospective study of flp should be performed, since the present study found flp to be an important factor affecting mechanical and biological complications in 2-is . Flp are the most significant clinical factor for the mechanical and biological complication rates of 2-is . Biological complications come about in flp of 39.80 months and mechanical complications in flp of 46.57 months . Therefore, clinical consideration of flp will help to prevent the mechanical and biological complications of 2-is.
Worldwide, approximately 1.38 million women are diagnosed with breast cancer each year . In developed countries, 80% of women with breast cancer will survive at least 60 months due to early detection techniques and effective anti - cancer treatments . In the uk, there are currently around 550,000 breast cancer survivors . Breast cancer treatments can cause chronic side effects such as oestrogen deprivation symptoms, athralgias, fatigue, lymphoedema, peripheral neuropathy, reduced bone health, upper extremity functional impairments and overall functional decline . A considerable number of breast cancer survivors experience some of these side effects although there is currently no accurate quantitative data on the incidence of these symptoms . The evidence that exercise is effective in treating many of these chronic or late appearing side effects is compelling: a recent systematic review and meta - analysis supported the use of exercise to prevent or treat fatigue and lymphoedema and to improve functional status and upper body range of movement . In addition, prospective observational studies suggest that around 3 h of aerobic activity per week can significantly reduce the risk of cancer recurrence and breast cancer mortality . There is now a need for randomised controlled trials (rcts) to examine the long - term effects of exercise interventions for improving outcomes such as quality of life, symptom management and ultimately cancer recurrence and mortality . To date, the longest follow - up with cancer survivors after an exercise intervention is 2 years, with most rcts only following participants up to 6 months post intervention . The aim of this study was to follow - up participants from a rct during adjuvant treatment 18 and 60 months after the exercise intervention . The original study s aim was to determine if there were functional and psychological benefits of a 12-week supervised group exercise programme during treatment for early stage breast cancer, including a 6-month follow - up . The study was designed as a pragmatic randomised controlled prospective open trial and was set in three oncology clinics in scotland for recruitment and in community facilities for the exercise intervention . The participants were 203 women with breast cancer with 177 completing the 6-month follow - up . The intervention incorporated a variety of safe cardiovascular, muscular strength and flexibility exercises and group discussion of exercise behaviour change techniques, in addition to usual care . The control group received usual care until the 6-month follow - up when they had a one to one discussion about how to incorporate physical activity into their lifestyle . The main outcome measures were: quality of life (fact) questionnaire, beck depression inventory (bdi), positive and negative affect scale (panas), body mass index (bmi), 7-day recall of physical activity from the scottish physical activity questionnaire-2 (spaq), 12-min walk test and assessment of shoulder mobility . The results showed significant intervention effects at 12 weeks and 6 months follow - up for metres walked in 12 min, minutes of moderate intensity activity reported in a week, shoulder mobility, breast cancer specific subscale of quality of life and for positive mood . It was concluded that a supervised group exercise programme provided functional and psychological benefit after a 12-week intervention and six months later . To determine if intervention effects continued after the 6-month follow - upto determine if women who had higher levels of activity after diagnosis and treatment had a different functional or psychological profile than women who had lower levels of activity and to elicit views from the women concerning their experience of physical activity post intervention to determine if intervention effects continued after the 6-month follow - up to determine if women who had higher levels of activity after diagnosis and treatment had a different functional or psychological profile than women who had lower levels of activity and to elicit views from the women concerning their experience of physical activity post intervention however, in this paper, we will not report on the qualitative analysis of the women s views . All women who had participated in the original study and who had agreed to being contacted again (n = 148) were contacted at 18 months after the intervention and invited to participate in the follow - up study . Sixty months after the intervention, all women (regardless of 18-month participation) were contacted again and invited to participate in a further follow - up . Each woman s general practitioner (gp) was contacted to ensure it was appropriate to write to the participant . Women who agreed to take part were then contacted by telephone to arrange for reassessment at the local sports facility where the original assessments had been carried out . All procedures and outcome measures were identical to the original study . Each appointment lasted approximately 2 h. all procedures were approved by the local nhs research ethics committee and informed consent obtained . In the original study, the participants were seen at the beginning and end of the exercise intervention period (i.e. Baseline and 3 months) and at the 6-month follow - up (i.e. 6 months from the end of the intervention period). In this study, we add 18 and 60-month follow - up time points . At each time point, the participants self - reported the total number of minutes of leisure time activity undertaken in the previous week, using a validated questionnaire (spaq). At each time point, they were classified as more or less active if they were above or below, respectively, the sample median leisure time activity for their age group . Baseline demographics were summarised and compared for those who had and had not dropped out of the study at each follow - up time point . The effect of the intervention on change from baseline in each outcome was modelled over time using a linear mixed effects model with a random intercept for subject, adjusting for study site, therapy received at baseline and age . The mean difference in change from baseline between the intervention groups at each time point was estimated with a 95% confidence interval (ci) and p value . The model implicitly accounts for missing data by considering the individual trends over time as well as the observed group means at each time point to give a more accurate estimate (than the observed group mean alone) of the population group means over time . For example, if women with lower scores at earlier time points tend to be more likely to drop out later, then the estimated mean at later time points will be adjusted downwards slightly to account for this . The differences in the outcomes at each time point between the more and less active group were estimated using similar models, additionally adjusting for intervention group . Note that it was not possible to consider change from baseline for this comparison because women were not necessarily in the same activity groups at different time points and so the outcomes were the actual scores rather than change from baseline . All women who had participated in the original study and who had agreed to being contacted again (n = 148) were contacted at 18 months after the intervention and invited to participate in the follow - up study . Sixty months after the intervention, all women (regardless of 18-month participation) were contacted again and invited to participate in a further follow - up . Each woman s general practitioner (gp) was contacted to ensure it was appropriate to write to the participant . Women who agreed to take part were then contacted by telephone to arrange for reassessment at the local sports facility where the original assessments had been carried out . All procedures and outcome measures were identical to the original study . Each appointment lasted approximately 2 h. all procedures were approved by the local nhs research ethics committee and informed consent obtained . In the original study, the participants were seen at the beginning and end of the exercise intervention period (i.e. Baseline and 3 months) and at the 6-month follow - up (i.e. 6 months from the end of the intervention period). In this study, we add 18 and 60-month follow - up time points . At each time point, the participants self - reported the total number of minutes of leisure time activity undertaken in the previous week, using a validated questionnaire (spaq). At each time point, they were classified as more or less active if they were above or below, respectively, the sample median leisure time activity for their age group . Baseline demographics were summarised and compared for those who had and had not dropped out of the study at each follow - up time point . The effect of the intervention on change from baseline in each outcome was modelled over time using a linear mixed effects model with a random intercept for subject, adjusting for study site, therapy received at baseline and age . The mean difference in change from baseline between the intervention groups at each time point was estimated with a 95% confidence interval (ci) and p value . The model implicitly accounts for missing data by considering the individual trends over time as well as the observed group means at each time point to give a more accurate estimate (than the observed group mean alone) of the population group means over time . For example, if women with lower scores at earlier time points tend to be more likely to drop out later, then the estimated mean at later time points will be adjusted downwards slightly to account for this . The differences in the outcomes at each time point between the more and less active group were estimated using similar models, additionally adjusting for intervention group . Note that it was not possible to consider change from baseline for this comparison because women were not necessarily in the same activity groups at different time points and so the outcomes were the actual scores rather than change from baseline . One hundred and fourteen women attended follow - up at 18 months and 87 women attended at 60 months . The flow of participants through this follow - up study is shown in fig . 1 (see reference 8 for the flow diagram for the original study). Baseline demographic characteristics of those that took part in the study at the 18 and 60-month follow - up versus those that did not are shown in table 1 . Those who participated in the follow - up at 60 months were, at baseline, 3 years older and 5 kg lighter on average and were faster walkers (i.e. Probably fitter); and may have been slightly less depressed and with less negative mood than those who did not participate at 60 months . Women in work prior to diagnosis and those that were less deprived were more likely to participate than those who were housewives or more deprived . There were no differences in the proportions of control and exercise group women that responded at either 18 or 60 months.fig . 1participant flow through follow - up studytable 1demographics at baseline for women that took part in the follow - up study (responders) and women that did not take part in the follow - up study (non - responders) at each subsequent time point: summary statistics and p values for differences between responders and non - responders (wilcoxon / fisher s test)18 months5 yearsresponderpresponderpnoyesnoyesage (years)n87114<0.01114870.03mean (sd)49.0 (9.2)53.5 (9.3)50.3 (9.5)53.2 (9.3)baseline weight (kg)n85114<0.0111287<0.01mean (sd)74.2 (15.5)68.3 (13.3)73.2 (15.2)67.8 (13.2)height (cm)n871120.20114850.14mean (sd)161.0 (6.3)159.9 (6.0)160.9 (6.3)159.7 (5.9)bdi scoren851120.06112850.06mean (sd)13.3 (7.2)11.4 (7.1)13.1 (7.5)11.1 (6.6)panas positiven871120.10113860.13mean (sd)26.5 (8.4)28.9 (9.0)26.8 (8.6)29.2 (8.9)panas negativen871120.05113860.09mean (sd)19.5 (7.9)17.2 (6.8)19.1 (7.8)17.0 (6.5)12-min walk (m)n851140.18112870.03mean (sd)973.4 (220.8)996.0 (224.8)958.1 (242.4)1,022.7 (190.0)spaq leisure time activity (min)n851100.32110850.71mean (sd)367.0 (330.3)365.1 (267.9)375.1 (323.2)354.1 (257.7)srm total scoren871140.80114870.98mean (sd)30.7 (5.8)30.8 (5.3)30.6 (5.7)30.9 (5.4)bmi (kg / m)n851120.01112850.02mean (sd)28.6 (6.0)26.8 (5.3)28.3 (5.9)26.6 (5.3)exercise groupcontrol46/102 (45.1)56/102 (54.9)0.6759/102 (57.8)43/102 (42.2)0.78exercise41/99 (41.4)58/99 (58.6)55/99 (55.6)44/99 (44.4)study centregri16/33 (48.5)17/33 (51.5)0.5521/33 (63.6)12/33 (36.4)0.57boc62/151 (41.1)89/151 (58.9)85/151 (56.3)66/151 (43.7)other9/17 (52.9)8/17 (47.1)8/17 (47.1)9/17 (52.9)therapychemotherapy7/15 (46.7)8/15 (53.3)0.527/15 (46.7)8/15 (53.3)0.67radiotherapy21/57 (36.8)36/57 (63.2)32/57 (56.1)25/57 (43.9)combination59/129 (45.7)70/129 (54.3)75/129 (58.1)54/129 (41.9)surgery typemast only31/57 (54.4)26/57 (45.6)0.1838/57 (66.7)19/57 (33.3)0.20lump only48/116 (41.4)68/116 (58.6)65/116 (56.0)51/116 (44.0)lump and mast1/2 (50.0)1/2 (50.0)1/2 (50.0)1/2 (50.0)lump and recon0/1 (0.0)1/1 (100.0)0/1 (0.0)1/1 (100.0)mast and recon6/22 (27.3)16/22 (72.7)9/22 (40.9)13/22 (59.1)other1/2 (50.0)1/2 (50.0)1/2 (50.0)1/2 (50.0)tamoxifen usedno57/117 (48.7)60/117 (51.3)0.0872/117 (61.5)45/117 (38.5)0.15yes30/83 (36.1)53/83 (63.9)42/83 (50.6)41/83 (49.4)highest education levelschool40/92 (43.5)52/92 (56.5)1.0054/92 (58.7)38/92 (41.3)0.77other43/99 (43.4)56/99 (56.6)55/99 (55.6)44/99 (44.4)employment status (prior to diagnosis)ft / pt10/29 (34.5)19/29 (65.5)0.0111/29 (37.9)18/29 (62.1)0.02sick52/111 (46.8)59/111 (53.2)66/111 (59.5)45/111 (40.5)housewife17/26 (65.4)9/26 (34.6)20/26 (76.9)6/26 (23.1)retired8/35 (22.9)27/35 (77.1)17/35 (48.6)18/35 (51.4)occupation (prior to diagnosis)professional17/48 (35.4)31/48 (64.6)0.7221/48 (43.8)27/48 (56.2)0.39managerial14/35 (40.0)21/35 (60.0)18/35 (51.4)17/35 (48.6)clerical25/55 (45.5)30/55 (54.5)32/55 (58.2)23/55 (41.8)manual15/33 (45.5)18/33 (54.5)20/33 (60.6)13/33 (39.4)carstairs deprivation1217/58 (29.3)41/58 (70.7)0.0423/58 (39.7)35/58 (60.3)0.013542/86 (48.8)44/86 (51.2)53/86 (61.6)33/86 (38.4)6726/53 (49.1)27/53 (50.9)35/53 (66.0)18/53 (34.0)periodsno70/169 (41.4)99/169 (58.6)0.3695/169 (56.2)74/169 (43.8)0.74irregular8/17 (47.1)9/17 (52.9)9/17 (52.9)8/17 (47.1)regular9/15 (60.0)6/15 (40.0)10/15 (66.7)5/15 (33.3)hysterectomyno80/179 (44.7)99/179 (55.3)0.36101/179 (56.4)78/179 (43.6)1.00yes7/22 (31.8)15/22 (68.2)13/22 (59.1)9/22 (40.9)hrtnever58/124 (46.8)66/124 (53.2)0.3173/124 (58.9)51/124 (41.1)0.69former24/67 (35.8)43/67 (64.2)35/67 (52.2)32/67 (47.8)current5/10 (50.0)5/10 (50.0)6/10 (60.0)4/10 (40.0) participant flow through follow - up study demographics at baseline for women that took part in the follow - up study (responders) and women that did not take part in the follow - up study (non - responders) at each subsequent time point: summary statistics and p values for differences between responders and non - responders (wilcoxon / fisher s test) to determine if there were any lasting effects of the intervention, comparisons were made between the original treatment and control groups . Table 2 shows descriptive statistics of the outcome data at 18 and 60 months with corresponding treatment effect estimates . There were significant differences between the intervention and control groups at 60 months for spaq leisure time activity over the previous week and panas positive mood score with the intervention group reporting higher activity and more positive mood . Even for outcomes for which there was no significant difference at 60 months, the intervention group was consistently observed to do better than the control group throughout the entire 5-year follow - up period . The treatment effect estimates at 18 and 60 months are also displayed in units of 1 standard deviation in fig . 2 for all outcomes measured and are of similar magnitude at both time points . At 5 years, the intervention group achieved on average around 200 min of activity each week more than the control group (see table 2 for related data). In general, our analyses suggested that 5 years subsequent to taking part in such an exercise intervention similar patients would be likely to achieve on average 50 to 350 min of extra physical activity per week than patients treated as usual . This is a substantial difference which could lead to considerable health benefit.table 2main outcomes: summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baselinesummaries at each time pointeffect estimate (exercise control)baseline18 months5 years18 m baseline5y baselinefact - galln20111487mean74.480.787.1(sd)(14.3)(14.6)(11.1)controln10256432.20.9mean72.779.685.7(1.8, 6.2)(3.4, 5.2)(sd)(15.6)(14.7)(11.4)0.2860.683exercisen995844mean76.181.788.5(sd)(12.6)(14.6)(10.7)bdi scorealln19711487mean12.29.37.00.80.2(sd)(7.2)(7.7)(6.7)(3.1, 1.5)(2.8, 2.4)controln9856430.4950.857mean12.99.77.5(sd)(7.5)(7.7)(6.7)exercisen995844mean11.58.96.6(sd)(6.9)(7.8)(6.7)panas positivealln19911487mean27.831.034.2(sd)(8.8)(9.8)(8.3)controln10056431.53.4mean28.030.633.1(1.4, 4.3)(0.2, 6.7)(sd)(9.2)(10.1)(8.7)0.3120.040exercisen995844mean27.731.335.2(sd)(8.4)(9.6)(7.8)panas negativealln199114870.80.5mean18.216.715.4(2.9, 1.4)(2.9, 1.8)(sd)(7.4)(7.5)(5.3)0.4870.655controln1005643mean19.117.416.3(sd)(7.7)(8.1)(5.6)exercisen995844mean17.316.014.5(sd)(6.9)(6.9)(5.0)12-min walkalln1999583mean9861,0851,065(sd)(223)(192)(158)controln10047402040mean9751,0661,031(33, 74)(16, 97)(sd)(235)(169)(163)0.4630.164exercisen994843mean9971,1041,096(sd)(211)(213)(147)spaq leisure time activity (min)alln1951118479204mean366533557(48, 206)(54, 354)(sd)(296)(355)(321)0.2220.008controln995541mean365500462(sd)(288)(334)(263)exercisen965643mean367565648(sd)(306)(373)(347)srm total scorealln20111086mean30.732.432.8(sd)(5.5)(5.3)(5.1)controln10253430.31.2mean30.331.731.8(1.1, 1.7)(0.3, 2.7)(sd)(5.7)(5.6)(5.9)0.6520.109exercisen995743mean31.233.133.8(sd)(5.4)(5.0)(3.9)bmialln197111850.30.6mean27.627.527.3(1.2, 0.7)(1.6, 0.4)(sd)(5.7)(5.1)(5.4)0.5460.222controln1005643mean27.728.028.0(sd)(6.1)(6.4)(6.7)exercisen975542mean27.426.926.7(sd)(5.3)(3.3)(3.7)18 m 18 months follow - up, 5y 60 months follow - upeffects estimates are displayed as the mean estimate, 95% confidence interval and p value . 2exercise treatment effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side main outcomes: summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baseline 18 m 18 months follow - up, 5y 60 months follow - up effects estimates are displayed as the mean estimate, 95% confidence interval and p value . Model adjusted for study site, baseline therapy and age exercise treatment effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side adjusting for other baseline variables (such as deprivation category, occupation prior to diagnosis, hysterectomy status, work status) had negligible effect on the group differences for any of the outcomes, despite some of these baseline variables showing strong relationships with the outcomes and/or differing between women that were followed up at 18 and 60 months and those that were not . To determine if there were differences between those women who self - reported themselves as being less active at each follow - up point, comparisons were made between these two categories of women, adjusting for original treatment group (as well as baseline study site, therapy and age). The model - estimated trends for the main outcomes over all time points, with confidence intervals, are given in figs . Figure 3 illustrates that the more active group was observed to walk a slightly longer distance in 12 min at every follow - up time point, though the differences were not significant.fig . 3model - estimated mean 12-min walk distance, beck depression inventory score, bmi and shoulder range of motion score over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time pointfig . 4model - estimated mean fact - g, fact - b subscale, panas positive and panas negative scores over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time point model - estimated mean 12-min walk distance, beck depression inventory score, bmi and shoulder range of motion score over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time point model - estimated mean fact - g, fact - b subscale, panas positive and panas negative scores over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time point the bdi score was marginally significantly different between the groups at baseline and decreased for both groups over time . A larger decrease in depression levels for the group identifying as active was associated with significant differences at all follow - up points . Bmi scores were on average slightly lower for the active group throughout the study, though the difference was statistically significant only at baseline and 12 weeks . There were statistically significant differences between the activity groups for total shoulder range of motion at baseline and 6 months follow - up only, and the observed difference was not consistent over time . Figure 4 shows similar increases in fact - g average scores (and therefore quality of life improvements) for both activity groups over time . In general, by 60 months follow - up there were no statistically significant differences in any of the quality of life scales, despite the consistency of the observed difference over time . Panas negative was significantly lower in the more active group at the end of the original study period and this persisted out to 6 months follow - up, despite there being no difference at baseline . This difference was not, however, statistically significant at 18 or 60 months, though the observed difference remained similar over time . Similarly, the more active group had significantly higher panas positive at baseline and at the end of the original study period and though the magnitude of this difference was similar at 60 months, it was marginally non - significant (0.078). The physical activity effects estimates, in units of 1 standard deviation, are displayed in fig . 5physical activity effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side physical activity effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side one hundred and fourteen women attended follow - up at 18 months and 87 women attended at 60 months . The flow of participants through this follow - up study is shown in fig . 1 (see reference 8 for the flow diagram for the original study). Baseline demographic characteristics of those that took part in the study at the 18 and 60-month follow - up versus those that did not are shown in table 1 . Those who participated in the follow - up at 60 months were, at baseline, 3 years older and 5 kg lighter on average and were faster walkers (i.e. Probably fitter); and may have been slightly less depressed and with less negative mood than those who did not participate at 60 months . Women in work prior to diagnosis and those that were less deprived were more likely to participate than those who were housewives or more deprived . There were no differences in the proportions of control and exercise group women that responded at either 18 or 60 months.fig . 1participant flow through follow - up studytable 1demographics at baseline for women that took part in the follow - up study (responders) and women that did not take part in the follow - up study (non - responders) at each subsequent time point: summary statistics and p values for differences between responders and non - responders (wilcoxon / fisher s test)18 months5 yearsresponderpresponderpnoyesnoyesage (years)n87114<0.01114870.03mean (sd)49.0 (9.2)53.5 (9.3)50.3 (9.5)53.2 (9.3)baseline weight (kg)n85114<0.0111287<0.01mean (sd)74.2 (15.5)68.3 (13.3)73.2 (15.2)67.8 (13.2)height (cm)n871120.20114850.14mean (sd)161.0 (6.3)159.9 (6.0)160.9 (6.3)159.7 (5.9)bdi scoren851120.06112850.06mean (sd)13.3 (7.2)11.4 (7.1)13.1 (7.5)11.1 (6.6)panas positiven871120.10113860.13mean (sd)26.5 (8.4)28.9 (9.0)26.8 (8.6)29.2 (8.9)panas negativen871120.05113860.09mean (sd)19.5 (7.9)17.2 (6.8)19.1 (7.8)17.0 (6.5)12-min walk (m)n851140.18112870.03mean (sd)973.4 (220.8)996.0 (224.8)958.1 (242.4)1,022.7 (190.0)spaq leisure time activity (min)n851100.32110850.71mean (sd)367.0 (330.3)365.1 (267.9)375.1 (323.2)354.1 (257.7)srm total scoren871140.80114870.98mean (sd)30.7 (5.8)30.8 (5.3)30.6 (5.7)30.9 (5.4)bmi (kg / m)n851120.01112850.02mean (sd)28.6 (6.0)26.8 (5.3)28.3 (5.9)26.6 (5.3)exercise groupcontrol46/102 (45.1)56/102 (54.9)0.6759/102 (57.8)43/102 (42.2)0.78exercise41/99 (41.4)58/99 (58.6)55/99 (55.6)44/99 (44.4)study centregri16/33 (48.5)17/33 (51.5)0.5521/33 (63.6)12/33 (36.4)0.57boc62/151 (41.1)89/151 (58.9)85/151 (56.3)66/151 (43.7)other9/17 (52.9)8/17 (47.1)8/17 (47.1)9/17 (52.9)therapychemotherapy7/15 (46.7)8/15 (53.3)0.527/15 (46.7)8/15 (53.3)0.67radiotherapy21/57 (36.8)36/57 (63.2)32/57 (56.1)25/57 (43.9)combination59/129 (45.7)70/129 (54.3)75/129 (58.1)54/129 (41.9)surgery typemast only31/57 (54.4)26/57 (45.6)0.1838/57 (66.7)19/57 (33.3)0.20lump only48/116 (41.4)68/116 (58.6)65/116 (56.0)51/116 (44.0)lump and mast1/2 (50.0)1/2 (50.0)1/2 (50.0)1/2 (50.0)lump and recon0/1 (0.0)1/1 (100.0)0/1 (0.0)1/1 (100.0)mast and recon6/22 (27.3)16/22 (72.7)9/22 (40.9)13/22 (59.1)other1/2 (50.0)1/2 (50.0)1/2 (50.0)1/2 (50.0)tamoxifen usedno57/117 (48.7)60/117 (51.3)0.0872/117 (61.5)45/117 (38.5)0.15yes30/83 (36.1)53/83 (63.9)42/83 (50.6)41/83 (49.4)highest education levelschool40/92 (43.5)52/92 (56.5)1.0054/92 (58.7)38/92 (41.3)0.77other43/99 (43.4)56/99 (56.6)55/99 (55.6)44/99 (44.4)employment status (prior to diagnosis)ft / pt10/29 (34.5)19/29 (65.5)0.0111/29 (37.9)18/29 (62.1)0.02sick52/111 (46.8)59/111 (53.2)66/111 (59.5)45/111 (40.5)housewife17/26 (65.4)9/26 (34.6)20/26 (76.9)6/26 (23.1)retired8/35 (22.9)27/35 (77.1)17/35 (48.6)18/35 (51.4)occupation (prior to diagnosis)professional17/48 (35.4)31/48 (64.6)0.7221/48 (43.8)27/48 (56.2)0.39managerial14/35 (40.0)21/35 (60.0)18/35 (51.4)17/35 (48.6)clerical25/55 (45.5)30/55 (54.5)32/55 (58.2)23/55 (41.8)manual15/33 (45.5)18/33 (54.5)20/33 (60.6)13/33 (39.4)carstairs deprivation1217/58 (29.3)41/58 (70.7)0.0423/58 (39.7)35/58 (60.3)0.013542/86 (48.8)44/86 (51.2)53/86 (61.6)33/86 (38.4)6726/53 (49.1)27/53 (50.9)35/53 (66.0)18/53 (34.0)periodsno70/169 (41.4)99/169 (58.6)0.3695/169 (56.2)74/169 (43.8)0.74irregular8/17 (47.1)9/17 (52.9)9/17 (52.9)8/17 (47.1)regular9/15 (60.0)6/15 (40.0)10/15 (66.7)5/15 (33.3)hysterectomyno80/179 (44.7)99/179 (55.3)0.36101/179 (56.4)78/179 (43.6)1.00yes7/22 (31.8)15/22 (68.2)13/22 (59.1)9/22 (40.9)hrtnever58/124 (46.8)66/124 (53.2)0.3173/124 (58.9)51/124 (41.1)0.69former24/67 (35.8)43/67 (64.2)35/67 (52.2)32/67 (47.8)current5/10 (50.0)5/10 (50.0)6/10 (60.0)4/10 (40.0) participant flow through follow - up study demographics at baseline for women that took part in the follow - up study (responders) and women that did not take part in the follow - up study (non - responders) at each subsequent time point: summary statistics and p values for differences between responders and non - responders (wilcoxon / fisher s test) to determine if there were any lasting effects of the intervention, comparisons were made between the original treatment and control groups . Table 2 shows descriptive statistics of the outcome data at 18 and 60 months with corresponding treatment effect estimates . There were significant differences between the intervention and control groups at 60 months for spaq leisure time activity over the previous week and panas positive mood score with the intervention group reporting higher activity and more positive mood . Even for outcomes for which there was no significant difference at 60 months, the intervention group was consistently observed to do better than the control group throughout the entire 5-year follow - up period . The treatment effect estimates at 18 and 60 months are also displayed in units of 1 standard deviation in fig . 2 for all outcomes measured and are of similar magnitude at both time points . At 5 years, the intervention group achieved on average around 200 min of activity each week more than the control group (see table 2 for related data). In general, our analyses suggested that 5 years subsequent to taking part in such an exercise intervention similar patients would be likely to achieve on average 50 to 350 min of extra physical activity per week than patients treated as usual . This is a substantial difference which could lead to considerable health benefit.table 2main outcomes: summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baselinesummaries at each time pointeffect estimate (exercise control)baseline18 months5 years18 m baseline5y baselinefact - galln20111487mean74.480.787.1(sd)(14.3)(14.6)(11.1)controln10256432.20.9mean72.779.685.7(1.8, 6.2)(3.4, 5.2)(sd)(15.6)(14.7)(11.4)0.2860.683exercisen995844mean76.181.788.5(sd)(12.6)(14.6)(10.7)bdi scorealln19711487mean12.29.37.00.80.2(sd)(7.2)(7.7)(6.7)(3.1, 1.5)(2.8, 2.4)controln9856430.4950.857mean12.99.77.5(sd)(7.5)(7.7)(6.7)exercisen995844mean11.58.96.6(sd)(6.9)(7.8)(6.7)panas positivealln19911487mean27.831.034.2(sd)(8.8)(9.8)(8.3)controln10056431.53.4mean28.030.633.1(1.4, 4.3)(0.2, 6.7)(sd)(9.2)(10.1)(8.7)0.3120.040exercisen995844mean27.731.335.2(sd)(8.4)(9.6)(7.8)panas negativealln199114870.80.5mean18.216.715.4(2.9, 1.4)(2.9, 1.8)(sd)(7.4)(7.5)(5.3)0.4870.655controln1005643mean19.117.416.3(sd)(7.7)(8.1)(5.6)exercisen995844mean17.316.014.5(sd)(6.9)(6.9)(5.0)12-min walkalln1999583mean9861,0851,065(sd)(223)(192)(158)controln10047402040mean9751,0661,031(33, 74)(16, 97)(sd)(235)(169)(163)0.4630.164exercisen994843mean9971,1041,096(sd)(211)(213)(147)spaq leisure time activity (min)alln1951118479204mean366533557(48, 206)(54, 354)(sd)(296)(355)(321)0.2220.008controln995541mean365500462(sd)(288)(334)(263)exercisen965643mean367565648(sd)(306)(373)(347)srm total scorealln20111086mean30.732.432.8(sd)(5.5)(5.3)(5.1)controln10253430.31.2mean30.331.731.8(1.1, 1.7)(0.3, 2.7)(sd)(5.7)(5.6)(5.9)0.6520.109exercisen995743mean31.233.133.8(sd)(5.4)(5.0)(3.9)bmialln197111850.30.6mean27.627.527.3(1.2, 0.7)(1.6, 0.4)(sd)(5.7)(5.1)(5.4)0.5460.222controln1005643mean27.728.028.0(sd)(6.1)(6.4)(6.7)exercisen975542mean27.426.926.7(sd)(5.3)(3.3)(3.7)18 m 18 months follow - up, 5y 60 months follow - upeffects estimates are displayed as the mean estimate, 95% confidence interval and p value . 2exercise treatment effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side main outcomes: summary statistics at 18 and 60 months for the control and exercise intervention groups and model effect estimates of treatment effect differences for change from baseline 18 m 18 months follow - up, 5y 60 months follow - up effects estimates are displayed as the mean estimate, 95% confidence interval and p value . Model adjusted for study site, baseline therapy and age exercise treatment effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side adjusting for other baseline variables (such as deprivation category, occupation prior to diagnosis, hysterectomy status, work status) had negligible effect on the group differences for any of the outcomes, despite some of these baseline variables showing strong relationships with the outcomes and/or differing between women that were followed up at 18 and 60 months and those that were not . To determine if there were differences between those women who self - reported themselves as being less active at each follow - up point, comparisons were made between these two categories of women, adjusting for original treatment group (as well as baseline study site, therapy and age). The model - estimated trends for the main outcomes over all time points, with confidence intervals, are given in figs . Figure 3 illustrates that the more active group was observed to walk a slightly longer distance in 12 min at every follow - up time point, though the differences were not significant.fig . 3model - estimated mean 12-min walk distance, beck depression inventory score, bmi and shoulder range of motion score over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time pointfig . 4model - estimated mean fact - g, fact - b subscale, panas positive and panas negative scores over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time point model - estimated mean 12-min walk distance, beck depression inventory score, bmi and shoulder range of motion score over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time point model - estimated mean fact - g, fact - b subscale, panas positive and panas negative scores over time for the more and less active groups, adjusted for original study site, therapy received at baseline and baseline age, with p values for tests of differences between the groups at each time point the bdi score was marginally significantly different between the groups at baseline and decreased for both groups over time . A larger decrease in depression levels for the group identifying as active was associated with significant differences at all follow - up points . Bmi scores were on average slightly lower for the active group throughout the study, though the difference was statistically significant only at baseline and 12 weeks . There were statistically significant differences between the activity groups for total shoulder range of motion at baseline and 6 months follow - up only, and the observed difference was not consistent over time . Figure 4 shows similar increases in fact - g average scores (and therefore quality of life improvements) for both activity groups over time . In general, by 60 months follow - up there were no statistically significant differences in any of the quality of life scales, despite the consistency of the observed difference over time . Panas negative was significantly lower in the more active group at the end of the original study period and this persisted out to 6 months follow - up, despite there being no difference at baseline . This difference was not, however, statistically significant at 18 or 60 months, though the observed difference remained similar over time . Similarly, the more active group had significantly higher panas positive at baseline and at the end of the original study period and though the magnitude of this difference was similar at 60 months, it was marginally non - significant (0.078). The physical activity effects estimates, in units of 1 standard deviation, are displayed in fig . 5 for all of the outcomes at 18 and 60 months.fig . 5physical activity effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side physical activity effect estimates for all outcomes at 18 and 60 months, adjusted for original study site, therapy received at baseline and baseline age, with 95% confidence intervals (cis) and corresponding p values at the right hand side this is the first study to examine the long - term effects of an exercise intervention in a rct with cancer survivors . The number of women lost to follow - up at 18 (44%) and 60 months (58%) is higher than that observed in a 2-year follow - up of a 6-month rehabilitation programme to reduce lymphoedema after breast cancer surgery (27%) but similar to that found in a longitudinal study of older breast cancer survivors in a 6-year follow - up (50%). Five years after taking part in the study, women who were assigned to the original intervention group, who had received the opportunity to attend a 12-week programme of supervised group exercise and group discussion of behaviour change issues, self - reported more leisure time activity and more positive mood than those women originally assigned to the control group condition . This is very encouraging as our trial was designed to promote long - term exercise behaviour change . Both the intervention and control groups self - reported high levels of physical activity at 5 years exceeding current public health recommendations of achieving 150 min of moderate activity in a week . Thus even the control group has benefitted from being involved in this project 5 years after diagnosis . However, as the data from table 2 shows, the intervention group reported around 200 min of activity more than the control group at 5 years, and although there is also high variability in these data, this is a statistically significant difference . The possibility of over reporting physical activity because of the self - report nature of the data must be acknowledged but the difference between the two groups in terms of physical activity is substantial . This suggests that the experience of attending the group exercise sessions had influenced the ability to sustain physical activity at a high level for the intervention group . This increase in physical activity could have important additional physical and mental well - being effects such as improved mood which we have observed, and reduced risk of recurrence of breast cancer, improved bone health and biomarker levels (e.g. Insulin pathway and inflammation) which were not measured in the original study . An important element of the exercise programme was the group discussions that happened at the end of each class . Each week, for 6 weeks, a specific theme was covered in group discussion after the exercise (for example, the health benefits of exercise, enhancing self - efficacy and setting goals) and supported with specifically constructed materials . These themes were guided by a model of behaviour change and were designed to promote independent exercise after the intervention . The six week block was repeated on a rolling basis, allowing all participants to hear the same themes . At the end of the 12-week intervention the control group received a personal consultation after the 6-month follow - up about how to increase physical activity levels . After the final data collection, women from both groups who expressed an interest in a local exercise referral scheme were given information on how to attend . The results show that the original intervention had a long lasting effect on helping the intervention group maintain a more physically active life . The difference in physical activity level that we see at 60 months between intervention and control group can be attributed to the experience of the class and group discussion of behaviour change challenges and solutions . In a study of cancer survivors diagnosed more than 5 years ago, over 53% reported difficulties in crouching, standing for 2 h, carrying 10 pounds and walking quarter of a mile compared to 21% of a matched sample with no cancer history . This demonstrates the importance of helping cancer survivors maintain basic levels of physical performance for simple activities of daily living . Positive mood is an indication of psychological well - being and may also be linked to increased activity levels . Williams et al . Found that an acute positive affective response to a single bout of moderate intensity exercise predicted physical activity participation levels 6 and 12 months later . This is consistent with other follow - up studies and recent meta - analysis which suggest positive effects of exercise on psychosocial parameters . Overall the pattern of results suggests a range of benefits of participating in the supervised exercise programme providing that the programme includes discussion of behaviour change challenges and solutions . The results therefore support the implementation of exercise opportunities into cancer rehabilitation in the same way that exercise is now a mainstream component of cardiac rehabilitation . Irrespective of original group allocation, those who self - reported as engaging in higher levels physical activity recorded benefits on many of the quality of life and mood variables in comparison to those who self - reported that they were less active . This suggests that being active, regardless of original group allocation to intervention or control conditions, was associated with quality of life and mood benefits . A 13-year follow - up of 374 women diagnosed with breast cancer at a young age (<40) showed that the women whose exercise activity increased following diagnosis scored significantly higher (p = 0.005) on the sf-36 physical health quality of life scale . Likewise a prospective study investigating physical activity and quality of life in 545 breast cancer survivors showed that greater physical activity levels 3 years post diagnosis were related to less fatigue and better physical functioning . In general, statistically significant differences are more apparent at 18-month follow - up than at 60 months, though it is important to note that the number of women responding at 60 months was lower and the magnitude of the effect for several outcomes is similar to the corresponding 18-month effect . This is first study to follow an intervention group for 60 months after an exercise intervention for women with early stage breast cancer and our response rate is similar to other studies of this length . A limitation is that there were some differences in baseline demographics and outcome scores between those that did and did not return for follow - up and a reasonably high rate of dropout at 60 months . However, we used statistical modelling methods that appropriately accounted for such missing data to give reliable estimates of the population group means and corresponding differences over time, and we adjusted the models for baseline demographics . Physical activity measures in this study were self - reported and future studies should attempt objective monitoring of physical activity patterns including sedentary time . This is first study to follow an intervention group for 60 months after an exercise intervention for women with early stage breast cancer and our response rate is similar to other studies of this length . A limitation is that there were some differences in baseline demographics and outcome scores between those that did and did not return for follow - up and a reasonably high rate of dropout at 60 months . However, we used statistical modelling methods that appropriately accounted for such missing data to give reliable estimates of the population group means and corresponding differences over time, and we adjusted the models for baseline demographics . Physical activity measures in this study were self - reported and future studies should attempt objective monitoring of physical activity patterns including sedentary time . Some of the benefits of a supervised exercise programme that incorporated discussion of behaviour change techniques, which were reported 6 months following the original intervention, have remained 60 months after the original study ended . These include higher levels of self - reported leisure time activity and more positive mood for the intervention group in comparison to the original control group . Categorising the women by self - reported activity status, rather than by original allocation to intervention and control conditions, also shows benefits over time in terms of lower levels of depression and higher levels of mood and quality of life for those who report being more active . Cancer survivors should be encouraged to engage in regular physical activity and to work towards achieving the public health recommendations for sufficient physical activity during and after treatment for early stage breast cancer [15, 16]. Services to support regular physical activity might include supervised exercise sessions in early stages, similar to that provided for cardiac rehabilitation, and encouragement to make use of local physical activity opportunities . This article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
Toll - like receptors (tlrs) are a family of transmembrane receptors which play an important role in the host defense against microorganisms . Tlrs are mainly expressed in human immune - related cells such as monocytes, neutrophils, macrophages, dendritic cells, t cells, b cells and nk cells . They initiate inflammatory response including the production of cytokines, chemokines and some adhesion molecules . About eleven human tlrs have been identified up to now and each of them participates in a specific intracellular signaling pathway . Tlrs 1, 2, 4, 5 and 6 are typical for bacterial products, tlrs 3, 7 and 8 are characteristic for viral infection and tlr-9 is associated with bacterial and viral inflammatory response . Sepsis is a complex clinical syndrome which is connected with activation and dysfunction of the immune system . Many features of the immunopathology of sepsis are still unclear . To date, the outcome of sepsis and sepsis shock is poor despite the development of antibiotics and other supportive care therapies . Toll - like receptors play a key role in the mediation of systemic responses to pathogens during sepsis . . Showed that the expression of tlr-2 and tlr-4 on monocytes in septic patients is higher than in healthy individuals . Sepsis remains a common cause of mortality in patients with acute myeloid leukemia treated with intensive induction chemotherapy . The expression of tlrs and their association with the development of sepsis in patients with acute myeloid leukemia remains unspecified . The aim of our study was to investigate the possible associations between expression of tlr2, tlr4 and tlr9 and occurrence of sepsis in patients treated with intensive induction chemotherapy for aml . A total of 103 patients with newly diagnosed acute myeloid leukemia (aml) were examined (47 females and 56 males). There were 57 patients (55%) with acute myeloid leukemias (minimally differentiated, without maturation and with maturation) and 46 patients (45%) with acute myelomonocytic and monoblastic leukemia . A total of 17 patients (17%) had favorable cytogenetic / molecular risk, 52 (50%) patients had intermediate cytogenetic / molecular risk and 34 patients (33%) had poor cytogenetic / molecular risk . The healthy control group included 20 age - matched individuals (9 females and 11 males). Samples were collected after informed consent from patients and control group . Using quantitative reverse transcriptase pcr, the mrna expression of genes tlr2, tlr4 and tlr9 was measured . To establish the expression of tlrs transcripts in cd34 + cells, mrnas were isolated with trizol, and cdnas were prepared with moloney murine leukemia virus reverse transcriptase . The pcr was performed using ampli - taq dna polymerase with denaturation, annealing and elongation . The ct value of the target genes was normalized (ct) to the ct value of the gus gene of the samples . Statistica 8.0. statistical analysis was performed by means of mann whitney u test, and p <0.05 indicated a significant difference . To determine the independent factors of sepsis occurrence, the analysis involved expression of tlr2, tlr4 and tlr9, cytogenetic risk, age, gender of patients and type of leukemia . Clinical characteristics of patients are summarized in table 1.table 1clinical data of patients with aml103 patientsgender47 f/56 mmedian age51 (range 1885)diagnosisaml with minimally differentiated6aml without maturation22aml with maturation29aml myelomonocytic34aml monoblastic12cytogenetic / molecular riskfavorable risk17intermediate risk52poor risk34neutropenic fever98 patientssepsis20 patientsinfectious agent62 patientsbacterial45 patientsfungal15 patientsviral2 patientssources of infectionrespiratory system30 patientsabdomen20 patientsurinary tract10 patientsskin2 patients clinical data of patients with aml neutropenic fever occurred in 98 patients (95%). In 62 patients, (60%) infectious agent was found . In 36 patients, we identified 20 episodes of severe sepsis (20%). A total of 10 patients with symptoms of severe sepsis died from infection . All patients with symptoms of sepsis after induction chemotherapy had poor cytogenetic / molecular risk . Gram - negative bacteria were more commonly found in patients with sepsis and included: escherichia coli in 4 patients, klebsiella pneumoniae in 6 patients and pseudomonas aeruginosa in 5 patients . Tlr2 and tlr4 mrna expression was higher in patients with neutropenic fever than in the group with asymptomatic neutropenia after chemotherapy, although the difference was not statistically significant . Among the patients with neutropenic fever, the mrna expression of tlr2 and tlr4 was significantly higher in septic patients than in patients without sepsis symptoms . Moreover, we observed that expression of tlr2 and tlr4 was significantly higher in patients with aml and bacterial infection in comparison with the group with isolated fungal infection . In comparison with control group, tlr2 and tlr4 mrna expression was higher in aml patients than in healthy individuals although there was no statistically significant difference (ct tlr2 0.9 0.85 vs 0.82 0.87 and ct tlr4 0.33 0.23 vs 0.29 0.32). The results are shown in table 2.table 2correlation between mrna expression of tlrs and sepsis in aml patientssepsisn = 20no sepsisn = 83 p ct tlr20.93 0.820.78 0.85<0.01ct tlr40.38 0.290.34 0.25<0.01ct tlr90.002 0.0010.004 0.003nsbacterial infectionn = 42fungal infectionn = 15 p ct tlr21.15 1.060.66 0.51<0.01ct tlr40.45 0.380.21 0.19<0.01ct tlr90.002 0.0010.003 0.002ns n number of patients, ns not significant correlation between mrna expression of tlrs and sepsis in aml patients n number of patients, ns not significant multivariate logistics regression model revealed that type of leukemia was an independent factor for sepsis occurrence . Patients with myelomonocytic and monoblastic leukemia had higher risk of manifestation of sepsis than other patients . Patients with response after induction chemotherapy (complete remission and partial remission) had significantly lower risk of sepsis occurrence than patients with no response after treatment . The results are shown in table 3.table 3multivariate logistics regression analysis results for sepsis occurrence in aml patientsn = 103const botlr2tlr4tlr9agesexriskresponsetype of leukemia0.1630.2490.6250.4690.0000.0290.5461.4291.415standard deviation0.9090.4001.091165.1430.0160.5010.5550.5100.542t(94)0.1790.6230.5730.0030.0040.0580.9842.8032.609 p 0.8580.5350.5680.9980.9970.9540.3280.0060.01195% cl1.9681.0432.791328.3650.0310.9651.6482.4410.338 + 95% cl1.6420.5451.540327.4260.0311.0230.5560.4172.492chi - square0.0320.3880.3290.0000.0000.0030.9687.8596.804 p 0.8580.5330.5660.9980.9970.9540.3250.0050.009or0.8500.7790.5350.6251.0001.0290.5790.2404.11695% cl0.1400.3520.0610.0000.9690.3810.1920.0871.402 + 95% cl5.1661.7254.6651.0322.7811.7440.65912.081or0.3240.3070.9921.0041.0290.5790.2404.11695% cl0.0090.0050.0050.1250.3810.1920.0871.402 + 95% cl11.76018.387206.6108.0562.7811.7440.65912.081 cl confidence limit, or odds ratio multivariate logistics regression analysis results for sepsis occurrence in aml patients cl confidence limit, or odds ratio toll - like receptors play an important role in host defenses and participate in some of pathological processes including sepsis . Tlr2 and tlr4 are expressed on the cell surface, and tlr9 works in intracellular space [6, 7]. Tlr2 recognizes some of ligands located on a variety of microorganisms such as bacteria, fungi, viruses and parasites . Tlr4, in association with co - receptor cd14, identifies lipopolysaccharide (lps). Recognition of pathogens ligands by tlrs leads to the activation of some signaling pathways and the secretion of proinflammatory cytokines, such as tumor necrosis factor alpha (tnf-), interleukin-1 (il-1) and interleukin-8 (il-8). Sepsis is a systemic inflammatory response to a microbial infection and remains one of the leading causes of death . The outcome of sepsis and sepsis shock is poor despite the development of new antibiotics and supportive care therapies . Many molecular mechanisms of sepsis are unclear, and it is essential to understand some immunological processes associated with the development of sepsis [4, 9]. In septic patients, the tlrs recognize some specific ligands of pathogens and initiate immune response due to tlr - dependent and tlr - independent pathways . Over the recent years, many studies analyzed the role tlrs in sepsis and sepsis shock [8, 11]. Showed that expression of tlrs on monocytes from septic patients was significantly up - regulated in comparison with healthy individuals . Patients with aml who are newly diagnosed or relapsed and who are receiving cytotoxic chemotherapy are predisposed to sepsis due to bacterial or fungal infections . The explanation of some mechanisms of sepsis could be useful in risk stratification of development of sepsis or sepsis shock in patients with aml during intensive chemotherapy . The exact role of tlrs in the development of sepsis in aml patients is unknown . The aim of our study was to analyze the expression of tlr2, tlr4 and tlr9 in patients with symptoms of sepsis after intensive induction chemotherapy for aml . We detected mrna expression of tlr2, tlr4 and tlr9 in 103 patients with aml before the beginning of treatment . We demonstrate that the mrna expression of tlr2 and tlr4 before induction chemotherapy was significantly higher in septic patients than in patients without sepsis symptoms . From among 20 patients with sepsis, it is interesting that in multivariate analysis type of leukemia was an independent factor for sepsis occurrence, and patients with myelomonocytic and monoblastic leukemia had higher risk of the manifestation of sepsis than other patients . It was impossible to compare our observations with other results because there is no data on the role of tlrs in the development of sepsis in aml patients treated with intensive chemotherapy . Perhaps the high expression of tlrs may initiate the systemic response to pathogens during sepsis and promote proliferation and expansion of immune system cells . The high expression of tlrs in patients with symptoms of sepsis could be connected with an increase in the production of inflammatory cytokines and chemokines . Some studies suggest that the outcome of sepsis depends on genetic influence [14, 15]. Septic patients with tlr-4 polymorphism had increased risk of bacterial infection . A better understanding of tlrs biology may disclose new therapeutic approaches for sepsis . In conclusion, our results suggest that tlrs could become potential biological markers for the development of sepsis in patients with acute myeloid leukemias after intensive induction chemotherapy
Rhegmatogenous retinal detachment (rd) is caused by liquefied vitreous passing through a retinal break into the subretinal space, separating the neurosensory retina from the retinal pigment epithelium . Low myopes [0.75 to 2.75 diopters (dpt)] show an odds ratio of 3.14 for rd, and the odds ratio was shown to rise steeply with increasing myopic refractive errors in the population . Refractive surgeries, like laser in situ keratomileusis (lasik) and laser - assisted subepithelial keratomileusis, have been popularized for correction of low to moderate myopia . Vision - threatening posterior segment complications may occur after refractive surgeries, including macular hemorrhages, macular holes and rhegmatogenous rd . The reported incidence of rhegmatogenous rd in lasik patients is not high, ranging from 0.033 to 0.25% [9, 131415]. However, many have regarded a suction ring application during lasik to be a potential risk factor for rhegmatogenous rd, because this procedure may induce vitreous traction and detachment resulting from sudden decompression of the eye [16, 17]. It has been reported that retinal breaks were more commonly located in the inferotemporal quadrant in rhegmatogenous rd after lasik . Here, we report on a case with bilateral simultaneous rhegmatogenous rd following lasik surgery . She was wearing a correction for myopia with a prescription of 6.5 dpt in the right eye and 7.00 dpt in the left eye . Her past medical history was unremarkable . On initial examination, visual acuity was counting fingers at 1 m on the right and 10/10 on the left . Dilated fundus examination revealed a total rd secondary to a retinal tear at approximately 11 o'clock in the right eye . There was a localized rd secondary to retinal tear at approximately 13 o'clock in the left eye, and the posterior pole was attached (fig . The patient underwent pars plana vitrectomy surgery combined with endolaser photocoagulation and silicone oil tamponade in the right eye . A week later, pneumatic retinopexy was done in the left eye . As the retinal tear did not seal, a 360 scleral buckling surgery was performed and the retina was attached . On the last visit, risk of rd is 10-fold higher in eyes over 3.0 dpt and is 0.075% in eyes exceeding 10 dpt . There are several reports on post - lasik rd and most of these cases have high myopia (> 10 dpt) [21, 22]. Increased vitreous liquefaction, earlier posterior vitreous detachment and higher incidence of vitreoretinal degeneration, such as lattice degeneration, are thought to be attributable to the higher prevalence in rhegmatogenous rd in myopes . In our case, there was bilateral rhegmatogenous rd in both eyes at the same time . As far as we know, no such case has been reported previously . Ozdamar et al . Reported a bilateral rd with giant retinal tear following lasik surgery . Reviglio et al . Presented a case with high myopia (13.00 + 3.00 15 dpt in the right eye and 13.00 + 3.00 170 dpt in the left eye) who developed rd 14 h after lasik surgery . Arevalo et al . Evaluated 1,1594 lasik patients with myopia in a 10-year follow - up . They found rd in 22 eyes of 19 patients during the course (ranging between 1 month and 10 years). Therefore, lasik surgery could not be the only causative factor for rd in these eyes . There is a positive correlation between rd and the amount of myopia . In a study by ogawa and tanaka, patients with> 15 qin et al . Reported 6 rd cases in 9,598 lasik patients (0.033%). As known, myopia increases the risk of rd (from 0.70 to 6%). The difference may be due to detailed fundus examination and preventive interventions prior to lasik surgery . It was in accordance with two previous large series which reported 16.3 and 27.3 months, respectively [22, 27]. All the previously reported cases in the literature were either unilateral rd or bilateral rd presenting at separate times following lasik . Simultaneous rd patients, as a subgroup of bilateral rd, may have more severe retinal weakness . Younger patients with myopia and coexisting retinal degenerations may be predisposed to simultaneous rd as in this case . To conclude, although lasik surgery is still an effective and safe method used to correct myopia, bilateral simultaneous rd should be included in the postoperative lasik complications . A thorough and careful retinal examination should be done before surgery, particularly in young myopic patients.
We report an unusual case of bilateral vertical lacquer crack with no history of ocular trauma and with progressive marked enlargement and consequent visual loss . Three - year follow - up was completed using best - corrected visual acuity, serial fundus photographs, intravenous fluorescein angiography, and optical coherence tomography . We report the occurrence of lacquer crack in a 43-year - old woman with no history of trauma except for laser in situ keratomileusis surgery for mild myopia (as reported by the patient) in the past 5 years and habitual ocular rubbing . Lacquer crack started in the right eye and became evident 1 year later in the left eye . Serial photography after repeated intravitreal injections of ranibizumab for subfoveal choroidal new vessel showed the lacquer crack widened gradually in both eyes . We hypothesize that a thin bruch s membrane in high myopia is prone for small rupture initially either spontaneously or following laser in situ keratomileusis and subsequent widening of the rupture by oculopression and intravitreal injections from rise in intraocular pressure . Three diseases that involve bruch s membrane give somewhat similar fundus appearance in different clinical settings: choroidal rupture (trauma),13 angioid streaks (pseudoxanthoma elasticum),4,5 and lacquer cracks (high myopia). Choroidal rupture is a break in the retinal pigment epithelium, bruch s membrane, and throughout the choroid following blunt trauma . Clinically, it appears as single (or multiple) white or yellowish crescent - shaped subretinal streak concentric to the optic disc.1,2 during a closed globe injury, the eyeball is initially compressed followed by rapid rebound expansion . The sclera s tensile strength resists this compression . The retina is elastic and stretches during such an injury . However, bruch s membrane lacks elasticity and breaks, taking with it the overlying retinal pigment epithelium and underlying choroid . On the other hand, angioid streaks usually emanate from the disc, tend to be straighter, and are reddish in color . Angioid streaks are associated with several systemic diseases, the most common being pseudoxanthoma elasticum.46 bruch s membrane in pseudoxanthoma elasticum is markedly thickened and calcified losing its elasticity and, hence, becomes brittle with dehiscence after minor trauma.5 the third disease, lacquer crack, is typically found in high myopia (with a prevalence ranging from 4.3% to 9.2%).710 experimentally and histologically, lacquer cracks represent healed and mechanical breaks of the retinal pigment epithelium, bruch s membrane, and choriocapillaris complex.11 we present a case of progressive sequential bilateral very wide single vertical macular angioid streak - like lacquer crack with no history of blunt trauma and with gradual visual loss after intravitreal injections for choroidal new vessel (cnv) formation . A 43-year - old married syrian woman presented at the age of 40 with distortion of central vision in the right eye . Choroidal dystrophy was suspected in damascus (syria) (figures 1 and 2). Rheumatology and infectious workup was negative (syphilis, tuberculosis, anti - double - stranded dna, anti - single - stranded dna, rheumatoid factor). Cnv was also detected in the macular area in dubai (united arab emirates) and intravitreal injections were advised . She subsequently received three monthly intravitreal ranibizumab injections in barcelona (spain) (figure 3a and b). She had undergone bilateral refractive surgery (excimer laser 5 years ago followed by laser in situ keratomileusis [lasik] 3 years ago). Fifteen months after initial right eye involvement, the patient expressed the same symptoms in the left eye, such as seeing broken lines and asymmetric faces . She received one injection in beirut (lebanon), and subsequently, three monthly injections in dubai to the left eye . Consultation in london (uk) revealed a best - corrected visual acuity of 20/80 in the right eye and 20/30 in the left eye . Dilated fundoscopy revealed bilateral areas of vertically linear atrophy, more extensive in the right eye, without subretinal fluid or hemorrhage by optical coherence tomography . The reported clinical impression then was myopic chorioretinal degeneration versus lacquer crack - associated myopic degeneration . She was advised to have regular checkup without intravitreal injections as long as the macula stays dry . The patient was frustrated because of both inability to pinpoint a definite diagnosis and gradual decline in vision bilaterally . Fundoscopy revealed definite widening of the vertical macular choroidal rupture bilaterally (figure 4a and b). Axial length measurement with the iol master (carl zeiss meditec ag, jena, germany) was 27.35 mm in the right eye and 27.31 mm in the left eye . Keratometry readings were 38.6 d in the right eye and 38.2 d in the left eye . The skin of the neck had tiny pinkish papules suspicious for early goose flesh (figure 5). Two separate dermatology consultations and skin biopsies did not favor the diagnosis of pseudoxanthoma elasticum . Our working diagnosis was lacquer crack from high myopia (without pseudoxanthoma elasticum) (figure 6) possibly initiated by lasik and exacerbated by ocular rubbing and intravitreal injections (figure 7). The patient was instructed to avoid ocular rubbing, head - down position, and intravitreal injection (unless an exudative component was present) and to have prior paracentesis before any future intravitreal injections . We propose the following pathophysiology to explain several findings in the present case (figure 6). Macular lacquer cracks can occur spontaneously7,8 or just after lasik procedures.9 in these high myopes that undergo lasik, mechanical stress caused by intraocular pressure (iop) elevation from the pneumatic suction ring may induce tangential stress at the posterior segment (sudden increase in iop to 6070 mmhg).7 another potential cause of stress might be the impact of excimer laser on the cornea producing stress waves along the axis of the eye . Stress wave amplitudes during photoablation reach a maximal pressure focus of up to 100 atmospheres located in the posterior lens and anterior vitreous and then decrease to below 10 atmospheres at the retina.12 in addition, iop increases by an average of 30 mmhg in eyes without reflux, 1 minute after intravitreal injections.13 this acute iop elevation puts stress on a weak, thin, and brittle bruch s membrane leading to cracks . Via a similar mechanism, oculopression (ocular massage) can cause dehiscence of bruch s membrane as reported by ruderman et al,14 or as in our case, widening of the bruch s membrane choroid dehiscence . Rishi et al15 described a 12-year - old girl with osteogenesis imperfecta, with sudden visual loss in the left eye from choroidal neovascular membrane . Follow - up at 1 month revealed the development of lacquer crack running through the macula, underlying the fovea . The patient received two re - treatments at 1-month intervals with further progression of lacquer cracks, while cnv had regressed and vision stabilized at the 20/200 level . It is also conjectured that the further widening of the lacquer crack occurred as a result of transient increase in iop following the intravitreal injection . There has been a similar event of enlargement of angioid streaks following intravitreal bevacizumab in an eye with cnv.6 also, choroidal ruptures were observed after intravitreal injection of bevacizumab for aggressive retinopathy of prematurity.3 we presented a case of extremely wide lacquer crack that resembled an angioid streak and led to some difficulty in diagnosis and management, especially with the absence of a history of high myopia . Many high myopes have progressive enlargement of the posterior sclera causing passive stretch on a brittle, thinned out bruch s membrane . Hence, these eyes are at moderate risk for bruch s membrane rupture and these breaks can enlarge with time . Care needs to be exerted in such eyes in order to avoid iop rise from blunt ocular trauma, ocular rubbing, and ocular surgery such as lasik, intravitreal injections, and phakic lens implant.
To estimate the effect of the dioxin crisis on the number of campylobacter infections, a model was designed by which the number of expected cases for 1999 could be calculated . This model was based on the data collected by the sentinel laboratory surveillance network from 1994 to 1998 . The cumulative numbers per week were used to drive a monthly chronologic series for 1994 to 1998 and modeled according to the fourier transformation (18,19). This procedure explains the cyclic patterns of data by spectral analysis; a complex time series with cyclic components is decomposed into sine and cosine terms describing the seasonal changes and a linear term to identify the trend (figure 1). The final model has three cyclical terms (52, 26, and 13 weeks), i.e., a strong yearly variation and two harmonics at the half and quarter year . The linear trend shows a yearly increase from 1994 to 1998, with a slope of 15% . When the cyclical contributions are included, r=0,86, that is, 86% of the variation in the number of the campylobacter infections can be explained by the model . The epidemic threshold is set at a distance of 1.96 standard deviations (sd)(95% confidence interval [ci]), which has shown to be useful for distinguishing epidemic increases from random variation (18), or (as for the dioxin crisis) exceptional, epidemic decrease . Figure 2 shows the model, including the 95% ci and the actual numbers by which it was calculated . Poultry production per workday for 1998 and 1999 is also shown, representing the production per workday in indices with 1995 as reference year (1995 = 100) (20). Campylobacteriosis in belgium at period of dioxin crisis, model 1994 - 1998 and poultry production index . Age and gender distribution during the crisis was compared with the monthly distribution of age and gender in previous years . The model was made with ms excel 2000; spss version 9.0 was used to compare distributions . Figure 2 shows the model, including the 95% ci as calculated from the numbers in the previous years during a period of 20 weeks (middle of april to the end of august, weeks 15 to 35) as well as the actual count for this period in 1999 . The number of campylobacter infections analyzed by the sentinel laboratory surveillance network fit the 95% ci except during the dioxin crisis (from week 21), when all poultry and eggs were taken off the supermarket shelves . The number of campylobacter infections from week 23 to week 26 in 1999 is on average 94 cases per week or almost 40% lower than the expected average of 153 cases per week (sdmodel=15 cases / week; sd = standard deviation). Overall, for the month of june (week 23 to 26), the expected number of infections was 643 (stmodel=61 cases / month) while the actual number of cases in 1999 was 375 . The monthly calculated numbers follow the same trend as campylobacter infections in 1999 and a similar decrease in numbers during the dioxin crisis . After 4 weeks, the ban was lifted, and the number of campylobacter infections returned to the interval calculated by the model . Poultry production, even though over a longer period, also returned to levels comparable with the month of the previous year (1998). There was no difference in age or gender distribution of campylobacter infections during the crisis compared with the rest of the year . The economic impact is probably the easiest to determine, as this aspect is data driven . However, the dioxin crisis had a tremendous impact on health and health - related matters, including food consumption . The sudden change in food consumption, related to the withdrawal of poultry and eggs, had an immediate effect on the number of foodborne diseases, e.g., campylobacter . The fitted model shows an unexpected decline in june 1999 . Even though the decline in numbers is not exceptional, looking at other declines in the number of campylobacter infections, as in february 1996 or february 1997, these happen during a downward trend in numbers, while the drop in june 1999 occurs when numbers would normally be increasing . The decline in campylobacteriosis and the lack of poultry in shops lasted 4 weeks, exactly the period from seizing all belgian chicken and egg products from the supermarket shelves until the return of these items . This supports a direct link and contradicts a possible ecological fallacy as the time frame, which has an abrupt beginning and end, is similar for both the dioxin crisis and the decline in number of campylobacter infections (21). During this period a major concern with ecologic studies is often the flue line (generally, the geographic boundaries of the occurrence of the risk factor and the occurrence of the illness), as was the case in the european study of the association between olive oil and cancer (22). The dioxin crisis differs from the commonly analyzed ecologic studies in this geographic aspect, since the borders of the impact of the dioxin crisis are the same as the borders of a well - established surveillance system . Campylobacter is associated with several risk factors and risk behaviors, such as contact with farm and domesticated animals (mainly cats and kittens) (1012) or recent history of antibiotic use (14). However, the dioxin crisis would not have an immediate effect on these factors, as it was primarily a food scare . Drinking raw milk, occasionally found to be a risk factor (12,16), is also unlikely to have a contributed to the decline in numbers since milk products were not taken off the shelves . Meat, in particular pork prepared on the barbecue, is generally accepted to be an important risk factor (10,16). Pork meat is associated with c. coli, which accounted for 12% (in 1998) and 21% (in 1999) of all campylobacter infections (4). Meat remained available during the crisis, which might explain the small increase in c. coli infections, as it is imaginable that chicken, which is the main source of c. jejuni infections, was replaced by pork during the crisis . Eggs were not on sale during the crisis, but they are generally not associated with campylobacter contamination . The withdrawal of chicken and all related products from the supermarket during the dioxin crisis is the most likely reason for the sudden decline in campylobacter infections . Chicken is found to be the principal source of infection in most case - control studies (11,13,14,16). In seattle, rare, raw, and cooked chicken were all significantly associated with campylobacter infection (8). Undercooked chicken was found to be a risk factor in a colorado study (12). Handling raw and even frozen chicken, possibly because of cross - contamination in the kitchen, has also been significantly associated with campylobacteriosis (10). As all belgian poultry was withdrawn, our data allow an estimation of the number of campylobacter infections directly related to poultry . The decline in the number of campylobacter infections in belgium by 40% was due to the withdrawal of belgian poultry from the market . In 1999, 199,251 tons of poultry meat was available for human consumption; 81,261 tons (41%) was imported (23). Foreign poultry remained available on the market . According to a marketing bureau that investigates trends in shopping behavior of 3,000 families in belgium, eating habits have changed little because of the dioxin crisis (24); moreover, the overall purchase of poultry in 1999 increased by almost 9% . However, a shift was seen in the quality of the poultry sold; after the crisis, consumers preferred chicken with some sort of quality label, even though the current labels do not specifically address contamination issues . Besides the 40% decrease in campylobacter infections during the dioxin crisis, this experiment also highlights the remaining baseline of 60%, averaging 75 infections a month . According to an analysis of foodborne disease information in the united states (1999), only 80% of the campylobacter spp . Furthermore, as only belgian chicken was banned and non - belgian poultry was still on sale, a number of poultry - related infections are still present in the reported numbers . With at least 40% of the campylobacter infections in belgium explained by poultry and 20% by non - foodborne causes, the source of the remaining infections should be further explored and investigated . The use of a disaster as an epidemiologic tool offers a unique opportunity to observe exceptional changes in the occurrence of infections or other diseases . The causes or consequences of the crisis can serve as treatment in an uncontrolled natural experiment . The dioxin crisis as experiment showed that> 40% of the campylobacter infections can be attributed to poultry.
Pain in the region of the pes anserinus (pa) insertion is a common rheumatologic condition diagnosed as pes anserinus tendinitis or bursitis (patb) syndrome . Its diagnosis is entirely based on clinical manifestations, which is marked by spontaneous medial knee pain on climbing or descending stairs, tenderness at the pa insertion, and occasionally local swelling (1). The anserine bursa lies underneath the pa, the name given to the conjoined tendon of the sartorius, gracilis, and semitendinosus muscle at its insertion into the upper medial aspect of tibia, about 5 centimeters distal to the medial aspect of the knee joint (2). Patb syndrome is frequently associated with osteoarthritis (oa) and vice versa . In a previous report, anserine bursitis was clinically diagnosed in 29 (46.8%) among 62 korean patients with knee oa (3). In contrast, 20 (83.3%) among 24 patb patients were reported to have radiographic evidence of knee oa (4). As a result, it has been taken for granted that " pain of oa could have a cause due to periarticular problems, such as anserine bursitis " (5). In addition, patb syndrome is also associated with obesity or type ii diabetes mellitus (dm) (2, 6). Ultrasonographic (us) evaluation is useful for diagnosing a variety of regional pain syndrome and soft tissue rheumatism and has been increasingly employed in the rheumatologic practice . The objectives of this study were to assess the us findings in patients with knee oa with patb syndrome and to determine the correlation between the us findings and the response to local corticosteroid injection . We prospectively studied 26 patients with knee oa diagnosed at a university affiliated rheumatology clinic (all women, mean age 63.4 yr, range 53 - 77). Sex, age, height, weight, body mass index, and radiographic knee osteoarthritis graded i - iv according to kellgren and lawrence (7) were recorded . Patb syndrome was clinically diagnosed, as described previously with minor modification (6). Briefly, each patient completed a questionnaire regarding knee pain as follows: 1) have you had knee pain in the last 2 weeks? All of the patients underwent a complete history and examination, including deep palpation of the anserine bursa at the same location over the medial aspect of the tibia below the knee joint . Patb syndrome was diagnosed if the patient responded positively to the questionnaire 1) and one of 2), 3), or 4) and tenderness was elicited on examination of anserine area . Patients with the clinical diagnosis of patb syndrome were enrolled into the us study . For the purpose of comparison between symptomatic and asymptomatic side, patients who complained of tenderness in only one anserine area were included . A linear array 7 mhz transducer (hdi 5000, atl ultrasound, bothell, wa, u.s.a .) Was used . Us examination of the knee and pa was performed as previously reported with minor modification by a radiologist trained in musculoskeletal us (8). The pa was located 2.5 - 3 cm distal to the medial joint line with the transducer placed longitudinally . The following us examinations were performed: measurement of the thickness of the pa (mean of 3 measures); morphologic intratendinous pa tissue characteristics; presence of anserine tendinitis (both thickening and loss of normal fibrillar echotexture); presence of bursitis (circumscribed anechoic fluid collection of 2 mm or greater). A mixture of 40 mg triamcinolone acetonide and 1 ml of 2% lidocaine was infiltrated at the point of maximal tenderness in the anserine bursa area . Response to local corticosteroid injection was evaluated by pain visual analog scale (vas) related to the knee and global patient / physician assessment using likert scale 2 weeks after corticosteroid injection (0=best response, complete relief of pain, 1=good response, pain decreased markedly, but pain remains, 2=fair, pain decreased slightly, 3=the same, pain was the same as that before the treatment, 4=worse, pain was worse than that before the treatment). Culturally adapted and validated korean version of western ontario and macmaster (womac) osteoarthritis index was also used (9). Data are presented as means.d . For continuous variables and as frequency (%) for categorical variables . A p - value was calculated by student's t - test for continuous variables or by chi - square test for categorical variables . A p value of p<0.05 was considered statistically significant and all the statistical analyses were performed using spss for windows (version 10.0, chicago, il, u.s.a . ). Examination, only 2 patients (7.7%) showed us evidence of patb . In one patient, marked thickening and loss of normal fibrillar echotexture of symptomatic side compared to asymptomatic side was observed (fig . 1). In the other patient, anechoic fluid collection in the symptomatic anserine bursa area 2). Positive us finding of patb did not correlate with age, duration of osteoarthritis, body mass index or radiographic grading of oa . The thickness of pa tendon was significantly greater in symptomatic side compared to asymptomatic side, but when the 2 patients with us evidence of patb were excluded, the difference was not statistically significant (table 2). Other us findings in these patients included the following: suprapatellar pouch effusion 22 (84.6%), degenerative spur change 16 (61.5%), popliteal cyst 4 (15.4%), infrapatellar bursitis 1 (3.8%). The demographic and clinical characteristics of these 17 patients were not different from those of 9 patients who were not injected with corticosteroids except that those who were injected were older than those who were not injected (table 1). Pain vas significantly decreased from 73.1 mm before corticosteroid injection to 50.7 mm after injection (p=0.0004, table 3). Womac pain index and womac physical function index also improved significantly after corticosteroid injection (10.6 and 33.8 before corticosteroid injection and 7.5 and 27.4 after injection, p=0.007 and 0.006, respectively). Global patient assessment using likert scale revealed that 2 patients showed best response, 6 good, 1 fair, 8 the same, and none worse (table 4). Global physician assessment revealed that 2 patients showed best response, 4 good, 5 fair, 1 the same, and 5 worse . It is of note that the 2 patients who showed the best response were those who showed us evidence of patb . It is also of note that after the corticosteroid injection, tenderness on deep anserine palpation disappeared in only 5 patients (29%) including the 2 patients that showed us evidence of patb . Currently, it is a common practice to diagnose soft tissue rheumatic problem clinically and to treat it empirically, sometimes using local corticosteroid injections . Many of these soft tissue rheumatic problems, however, are elusive to diagnose by imaging modalities or pathological examination . Previous reports on radiographic finding of patb are rare . In a report by hall and joffe (10), computed tomography (ct) imaging revealed a sharply defined low - attenuation cyst lying immediately beneath the pes anserinus tendon in a patient with non - tender mass on the anteromedial aspect of the leg below knee . Notably, the authors pointed out that distension of the anserine bursa is not synonymous with the clinical syndrome of anserine bursitis, because the case patient did not have any symptoms pertaining to the knee, and that the clinical syndrome of anserine bursitis actually may be a tendinitis or a fascitis involving the insertions of the pes anserinus tendon . In another report, anserine bursa was visualized both by ultrasonography and ct (11). In accordance with our result, ultrasonography demonstrated a cystic mass located on the anteromedial surface of the proximal tibia . Ct guided aspiration of bursa was perfomed along with intrabursal injection of methylprednisolone, which led to resolution of symptoms . Few studies systematically elucidate the morphological characteristics of the pa insertion and bursa in clinically diagnosed patb syndrome by imaging modalities . Because us examination is an excellent and a widely used technique for imaging superficial soft tissues, it can be routinely used as a diagnostic tool to improve the assessment of joints and soft tissues . One study examined us features of the pa and subcutaneous medial knee fat in patients with clinically diagnosed patb syndrome (8). Of 37 patients, anserine bursitis was diagnosed in only 3 knees and pa tendinitis in 1 . Another study which systemically assessed the musculoskeletal us findings in patients with type 2 diabetes mellitus (dm) showed that 28.6% of clinical patb syndrome had pa tendinitis findings (12). In line with these reports, our study showed that only a minority of clinically diagnosed patb syndrome patients had us evidence of patb . First, us examination may not delineate the pa tendon abnormalities involved in the patb syndrome . In this regard, however, because tissues with few mobile protons emit little or no signal and therefore the internal architecture of the tendon may not be well demonstrated by mri, this limitation in visualization of tendon abnormality may also happen with mri . Second, the tenderness elicited by deep palpation of anserine area may not stem from pa tendon or anserine bursa, but from other structures not adequately visualized by us . In our patients, the most common us finding was suprapatellar effusion followed by degenerative spur change, all of which are primary findings of underlying knee oa . Small numbers of patients had popliteal cyst, and infrapatellar bursitis, but these lesions are not likely to be responsible for the tenderness around the anserine area in the majority of our patients . Third, the point of tenderness by deep palpation may be another tender point with unusually low threshold for perception of tenderness . For instance, about 30% of normal controls elicited tenderness on deep palpation of anserine area (unpublished observation). There are a paucity of data regarding the effectiveness of local corticosteroid injection in patients with patb syndrome . In a report by larsson and baum (13), there was a significantly better result when steroids were used compared to when lidocaine alone was used 1 month after the injections . Seventy - one percent of patients injected had improved significantly after a follow up of 2 - 61 months . However, another report revealed that only 24% of patients showed good response and among these only a third experienced total relief of pain (14). This discrepancy probably results from the differences in methodology, such as retrospective or prospective design, or the definition of response . Although pain vas and womac index improved significantly in our patients after injection, less than half of patients responded that they had more than good response and only 11.8% reported complete relief of their discomfort . Because all of our patients had knee oa, relief of symptoms of patb alone may not be sufficient for alleviation of knee discomfort . In addition, womac scale has not been validated for the assessment of symptom change in patb syndrome previously . Despite this limitation, it is of note that only those patients who had us evidence of patb showed complete relief of discomfort after corticosteroid injection . In conclusion, among knee oa patients with clinically diagnosed patb syndrome, only a minority had us findings indicative of patb . Local corticosteroid injection resulted in complete relief of pain only in those patients with us findings of patb . Recently, evidence that us exam can serve as a useful tool to determine the usage of non - steroidal anti - inflammatory drug versus acetaminophen in knee oa has been suggested (15). Also for optimal management of soft tissue problems in knee oa, assessment by imaging modalities such as us examination is useful.
The expression of the disease results from the interaction of host defense mechanisms, microbial agents, environmental, and genetic factors . Various compounds, such as cytokines, have been detected in gingival crevicular fluid (gcf) and may be especially beneficial for diagnosing current periodontal status and addressing the effects of periodontal treatment . Il-8 belongs to the interleukin-8 supergene family that includes small peptides with chemotactic activity for specific types of leukocyte populations . This cytokine is induced and secreted by many cells, such as monocytes, lymphocytes, fibroblasts, epithelial, and endothelial cells [7, 8] as well as by synovial cells . Il-8 attracts and activates polymorphonuclear leukocytes (pmn) in inflammatory regions [9, 10]. It induces the adhesion of pmn to endothelial cells and their transendothelial migration as well as the release of granule enzymes from these cells . In periodontal patients, mcgee et al . Found that il-8 concentrations were significantly higher in gingiva adjacent to probing pocket depth 3 mm and lowest adjacent to> 6 mm sulci . In gcf, chung et al . Suggested that the absence of a direct relationship between il-8 and pmn recruitment may characterize individuals at risk for progression of periodontitis, while in another study, no significant difference in gcf il-8 levels between localized juvenile periodontitis and healthy subjects was shown . According to mathur et al ., the total amount of il-8 was significantly higher in diseased compared to healthy sites . Interleukin-6 (il-6) is an important cytokine involved in the regulation of host response to tissue injury and infection . It is produced by a variety of cells, such as monocytes, fibroblasts, osteoblasts, and vascular endothelial cells in response to inflammatory challenges . It plays an important role in b - cell differentiation and in t - cell proliferation, while il-6, synergistic with interleukin-1 (il-1), induces bone resorption . However, it has also been reported that it can increase the production of tissue inhibitors of matrix metalloproteinases (timp) [24, 25], suppresses il-1 expression, while it can induce the synthesis of il-1 receptor antagonist (il-1ra) and the release of soluble tnf receptors suggesting its anti - inflammatory properties . In patients with refractory periodontitis, active sites those displaying loss of attachment> 2.1 mm in 3 months revealed significantly higher gcf il-6 levels than inactive ones . In hiv-1-infected patients, gcf il-6 levels were increased compared to uninfected periodontal patients . According to guillot et al ., in periodontal sites requiring surgery (unresolved sites), gcf il-6 levels were significantly lower compared to those at resolved sites (not requiring surgery). In gingival tissues, prabhu et al . Reported that expression of il-6 m rna was significantly higher in diseased tissues compared to healthy ones in periodontitis patients . The purpose of this study was to examine the gcf levels of il-6 and il-8 in periodontal sites with varying degrees of destruction and inflammation of periodontal patients prior to and following surgical and/or nonsurgical periodontal therapy . Patients with chronic periodontitis were recruited into this randomised, longitudinal, split - mouth, interventional study, from patients referred to the department of periodontology, aristotle university of thessaloniki . The selection criteria were (1) patients aged 3565 years for males and 3545 years for females; (2) good general health with no history of systemic disease; (3) no medication was taken; (4) no periodontal therapy received in the preceding 1 year; (5) more than 20 remaining teeth; (6) moderate to advanced periodontal disease as evidenced by multiple sites with a probing depth of 5 mm or more, extensive radiographic bone loss and bleeding on gentle probing; (7) pregnant or lactating females were excluded . Informed consent was obtained from each patient prior to enrolment in this study, and ethical approval was obtained from the aristotle university of thessaloniki ethics committee . In each patient, two quadrants of either the mandible or maxilla were randomly assigned as experimental . In each experimental quadrant, 4 periodontal interproximal sites in single - rooted teeth were selected . Three sites displaying probing pocket depths (pd) 5 mm and a gingival index (gi) of 2 or 3 were defined as diseased sites and 1 site with pd 3 mm and gi = 0 or 1 was defined as a nondiseased control site . A total of 96 test sites were included in the study, 72 of them as diseased and 24 as nondiseased sites . Sites in one experimental quadrant received nonsurgical periodontal treatment consisting of oral hygiene instructions, scaling and root surface debridement, while the contralateral sites received nonsurgical followed by surgical periodontal treatment, using a modified widman flap . At 6, 16, and 32 weeks following treatment, the dentition received supragingival polishing with a rubber cup and pumice . Prior to as well as 6, 16, and 32 weeks following periodontal therapy, a gcf sample was taken from each test site, and il-6 and il-8 were quantified . The following clinical measurements were also evaluated: (1) plaque index (pli), according to silness and le, (2) gingival index (gi), according to le, (3) probing pocket depth (pd), and (4) clinical attachment loss (cal), to the nearest millimeter with a williams probe . The pd score in each site individual acrylic stents were fabricated with reference grooves as reference points for the above clinical measurements and for gcf sampling . Patients were all asked about their smoking habits and were classified as smokers or never - smokers . Former smokers, that is, patients who had stopped the habit, were not included in this study . Both experimental quadrants were isolated with cotton rolls, and clinically detectable supragingival plaque was removed using a curette without touching the marginal gingiva . Sites were gently dried with an air syringe, and a single sterile paper strip (periopaper, oraflow, plain view, ny, 11803, usa) for each examined site was inserted into the gingival crevice, until mild resistance was felt and was kept there for 30 s. strips contaminated by bleeding were discarded and gcf sampling was repeated the following day . The amount of gcf collected was quantitated using periotron 6000 (siemens medical systems, inc ., iselin, nj, usa), which had been calibrated with 1: 5 diluted serum . Each paper strip was placed into a coded sealed plastic tube containing 250 l phosphate buffered saline (pbs). The samples were left at 4c for 2 h and, then, they were frozen at 70c and stored until cytokine analysis . Gcf was eluted from each filter paper strip into pbs as follows: before the il-6 and il-8 assays were performed, samples were left at 4c for 2 h. then, each strip was lifted to the surface of the eluent, and another 350 l of pbs was added to the strip (600 l final volume). Samples were, then, refrigerated at 4c for another 20 min and centrifuged at 10,000 rpm for 10 min . Finally, the strips were discarded . Commercial elisa kits (r and d systems, abingdon, oxon, uk) were used to analyse il-6 and il-8 levels . A murine antihuman monoclonal antibody specific for il-6 and il-8 was precoated onto a 96-well microplate . After washing of unbound proteins, an enzyme - linked (horseradish peroxidase) polyclonal antibody (200 l) specific for il-6 or il-8 (goat antihuman) was added to each well . Then, 200 l of a substrate solution was added and any colour developed was proportional to the amount of il-6 or il-8, respectively, bound in the initial step . The intensity of the colour (optical density) was measured using a microplate reader at 450 nm (wavelength correction set to 540 nm) within 30 min . A standard curve was prepared by plotting the concentration of the il-6 (standards 300, 100, 50, 25, 12.5, 6.25, 3.12, and 0 pg / ml) or the concentration of the il-8 standards (2000, 1000, 500, 250, 125, 62.5 31.2, and 0 pg / ml) against their optical density and the concentration of il-6 or il-8, respectively, was determined . Then, the pg of il-6 or il-8 in each sample (total amount) was calculated and the il-6 or il-8 concentration (pg / ml) was determined by dividing the amount of il-6 or il-8 by the gcf volume (l). The elisa assays were run in duplicate; mean values were used to calculate total amounts and concentrations of each cytokine . The minimum detectable level of il-6 (sensitivity of elisa) was typically less than 0.70 pg / ml, while the minimum detectable level of il-8 was less than 10 pg / ml ., chicago illsion, usa). For all time intervals, the mean cytokine and clinical values from the 2 healthy and the 6 diseased sites in each patient were used for the purposes of analysis . Differences in cytokine levels between diseased and nondiseased sites as well as between smokers and nonsmokers were evaluated by the mann - whitney test . In each case, the level of significance was set at p <0.05 . Comparison of the clinical measurements prior to and following therapy was performed by wilcoxon signed ranks test . The same test was used to investigate the differences in gcf volume and in gcf il-6 or il-8 levels before as well as 6, 16, and 32 weeks following periodontal therapy . Finally, the kendall's correlation coefficient was used to study the correlation between il-6, il-8 levels, and clinical parameters . A bonferroni correction for all multiple comparisons was applied . To assess the distribution of the data, the kolmogorov - smirnov test was used . Twelve volunteers (7 females and 5 males, mean age 45.4 years) took part in this study . Interleukin-6 was detected in gcf samples from 63/72 diseased sites (87.5%) and from 23/24 (95.83%) nondiseased sites, at baseline . The respective values at the 6th week following therapy were 60/72 (83.33%) and 22/24 (91.66%), at the 16th week 57/72 (79.17%) and 23/24 (95.83%), while at the 32nd week were 64/72 (88.89%) and 23/24 (95.83%). At baseline, interleukin-8 was detected in gcf samples from 71/72 diseased sites (98.61%) and from all the nondiseased sites (100%). The respective values following therapy were: 71/72 (98.61%) and 23/24 (95.83%) at the 6th week, 70/72 (97.22%) and 23/24 (95.83%) at the 16th week, and, finally, 71/72 (98.61%) and 23/24 (95.83%) at the 32nd week . The gcf volume, expressed in l, of 72 diseased and 24 nondiseased sites both prior to and following therapy are presented in table 1 . Mean gcf values were significantly higher in diseased compared to nondiseased sites (p <0.01 at baseline, p <0.05 following therapy). Periodontal therapy resulted in a significant decrease in gcf volume in both diseased and nondiseased sites (p <0.01 and p <0.05, resp . ). The concentration (il-6, il-8) and the total amount (tail-6, tail-8) of interleukin-6 and -8 in gcf expressed in pg/l and pg/30 s, respectively, are presented in table 2 . Mean il-6 concentration values were significantly higher in nondiseased compared to diseased sites both prior to and following periodontal treatment (at baseline p <0.01, post - treatment p <0.05). Periodontal treatment resulted in significant increase of il-6 concentration in diseased sites (p <0.01 at 6 weeks), while in nondiseased sites il-6 levels remained almost unchanged . Following therapy, tail-6 levels were slightly reduced at 6 weeks, but at 32 weeks they were increased in almost pretreatment levels . Mean il-8 concentration was significantly higher in nondiseased compared to diseased sites (p <0.01 at baseline and at 6 weeks). Following therapy, il-8 concentration increased significantly (p <0.05 at 6 and 16 weeks) in diseased sites, while in nondiseased it displayed only a slight increase . Both treatment modalities, surgical and nonsurgical periodontal treatment, resulted in similar il-6 as well as il-8 total amounts and concentrations at each interval following treatment . A strong correlation (p <0.05) was also found between il-6 and il-8 concentrations (table 3). In diseased sites, periodontal treatment led to improvements in all clinical parameters (table 2). At 6 weeks, mean pd, cal, pli, and gi scores the pli and gi scores were also decreased (p <0.05) at 16 weeks . However, by 32 weeks after - therapy an increase in pd, cal, gi, and pli scores was noted (p <0.05 for gi and pli). In nondiseased sites, periodontal therapy resulted in a significant decrease only in pli and gi scores at 6 weeks (p <0.01) and at 16 weeks (p <0.05). At 32 weeks, an increase in pli score was noted (p <0.05). The comparison between il-6, il-8 levels, and clinical parameters revealed strong negative correlations between il-6 concentration and gi, pd and cal (table 4). The concentration and the total amount of il-6 and il-8 as well as the mean values of pd and cal in seven smokers and five nonsmokers are presented in table 5 . In all sites, tail-6 levels were higher in nonsmokers than in smokers both prior to and following treatment (significantly in diseased sites at 6 weeks, p <0.05). Similarly, il-6 concentration was higher in nonsmokers compared to smokers without reaching statistical significance . At baseline, in all sites tail-8 levels were higher in nonsmokers (significantly p <0.01 in nondiseased sites). Periodontal treatment led to reduced tail-8 levels in nonsmokers, while it did not seem to influence them in smokers . Following therapy, tail-8 levels were higher in smokers compared to nonsmokers (significantly at 32 weeks in diseased and at 6 weeks in nondiseased sites). In diseased sites, il-8 concentration in smokers increased following therapy, while it remained almost unchanged in nonsmokers . Thus, il-8 was significantly higher in smokers post - treatment (p <0.01). In nondiseased sites, il-8 at baseline was higher in nonsmokers (p <0.01), while following therapy il-8 was higher in smokers (p <0.01). The major pathophysiological role of interleukin-8 lies in affecting neutrophils [15, 37]. Il-8 levels in gcf, therefore, from patients during periodontal therapy could be helpful in monitoring the progression of periodontal disease . In our study, no clear differences in the total amount of il-8 were observed, when diseased sites were compared with nondiseased ones implying either the inflammatory status of healthy sites or the role of il-8 to the steady state of the gingival . Additionally, the findings of previous studies [14, 39] suggested an inverse relationship between pmn recruitment responsible for the periodontal status and il-8 levels in gcf ., on the contrary, found that the total amount of il-8 was higher in diseased compared to healthy sites . Periodontal treatment resulted in a significant decrease of mean tail-8 in diseased and nondiseased sites at 6 weeks . However, at that time, in 23 of the 72 diseased sites an increase of tail-8 was noted . . Found that in some patients scaling and root planing led to decreased and in some others to increased levels of il-8 and of -glucuronidase, a pmn indicator, and tried to correlate them with individuals at risk for progression of periodontitis . In our study, the sites with increased il-8 levels following therapy were not characterized by significant loss of attachment or inflammation . Weak correlations between tail-8 and clinical parameters were observed, positive with gi and negative with pd . This could be explained by the fact that clinical parameters, such as probing depth, clinical attachment loss, and bleeding on probing do not necessarily reflect current disease activity as well as by the small number of patients and their heterogeneity . Both treatment modalities, surgical and nonsurgical, improved the clinical indices and resulted in lower tail-6 and tail-8 levels 6 weeks following therapy . At 32 weeks, these levels increased . At that time these data suggest that in this split - mouth design research, where factors influencing the one half of the mouth might also influence the other half, the treatment modality did not influence the amounts of il-6 and il-8 in gcf 32 weeks following therapy . The concentrations of both il-6 and il-8 were significantly higher in nondiseased compared to diseased sites, while following periodontal treatment they increased significantly . It has been suggested that in gcf the total cytokine amount might be more representative of the disease status as compared to the concentration . According to chapple et al . Thus, it was proposed that the total marker activity per 30 s gcf sample rather than the concentration of the marker might provide a better correlation with health or disease status . Il-6 has direct stimulatory effects on bone resorption, although this is controversial . On the other hand, il-6 was suggested to have anti - inflammatory properties . In our study, neither disease severity nor inflammatory status seemed to influence significantly tail-6 levels . There was a weak negative correlation between tail-6 and pd or gi, while in nondiseased sites, mean tail-6 was numerically higher compared to diseased sites . Following treatment, tail-6 levels were slightly reduced at 6 and at 16 weeks . According to our findings, the total amounts of gcf il-6 could hardly be correlated with either periodontal destruction or inflammation . In agreement with our results, bozkurt et al . Suggested that in patients with adult periodontitis no correlation between gcf il-6 levels and clinical parameters was found . . Found significant positive correlations between gingival bleeding as well as pd and il-6 . Atilla and ktkler detected higher gcf il-6 levels in sites with gingivitis than in healthy ones, while lee et al . The diversity of the results in different studies support the idea that the production of inflammatory mediators differs from site to site and from subject to subject and their levels may be influenced by several factors, such as genetic factors and bacterial composition . Smoking is an important environmental risk factor for the initiation and progression of periodontitis [5153]. In agreement with previous findings, our results showed that smoking did not influence significantly il-6 levels in gcf, although there was a trend for higher il-6 levels in nonsmokers compared to smokers, significantly at 6 weeks following therapy . Before treatment, when the antigenic stimuli from the bacterial plaque in untreated periodontal pockets were high, the total amounts of il-8 were higher in nonsmokers than in smokers . Following therapy, il-8 levels decreased significantly in nonsmokers; on the contrary, they did not seem to be influenced in smokers . As a consequence, tail-8 levels were higher in smokers following treatment compared to nonsmokers, as it was suggested from a previous study on experimental gingivitis . The concentration of il-8 in nonsmokers remained almost unchanged following therapy, while in smokers it increased steadily and became significantly higher than in nonsmokers . Although one could consider the small number of patients studied, when interpreting the results, our observations suggest that smoking may keep the gcf content of il-8 in high levels influencing the response to periodontal therapy . Additionally, in diseased sites a better clinical result was achieved in nonsmokers following treatment, as evident from the pd and cal values . This is in agreement with previous observations that the outcome of periodontal therapy was significantly compromised in smokers [51, 52, 55]. Both treatment modalities improved significantly the clinical indicies; this improvement was accompanied by a down regulation of the mean total amount of il-8 in gcf . The total amounts of il-6, however, were not significantly influenced during the 32 weeks following therapy . A strong relationship between il-6 or il-8 levels in gcf and periodontal destruction or inflammation was not found . Moreover, the smoking status seemed to influence both the total amount and the concentration of il-8 in diseased and nondiseased sites as well as il-6 levels to a lesser extend . The value of monitoring gcf il-6 and il-8 was not evident in our study; further studies are required to clarify the exact role of these cytokines in periodontal disease and to evaluate other factors that in conjunction with local ones may influence their levels in gcf.
Biomethylation of metals and metalloids by microorganisms is a widespread phenomenon in anaerobic habitats including waste deposits, sewage sludge, and alluvial soils [13]. The stepwise methylation results in both partly methylated nonvolatile species as well as fully methylated volatile metal(loid) compounds . Considering the direct exposure to humans, the formation of volatile metal(loid) compounds by the intestinal biocenosis has attained considerable attention in the last years . In vivo studies showed that after ingestion of bismuth subcitrate, the metal will be methylated by microbes in the gut and volatile trimethylbismuth (me3bi) can be detected in blood and breath . Furthermore, arsenic, selenium, tellurium, and antimony were volatilized by the microbiocenosis of an in vitro model of the human intestinal microbiota . Have shown that colloidal bismuth subcitrate (cbs) as well as bismuth cysteine is methylated by human liver cells in vitro . Whereas for the toxicity of nonvolatile methylated metal(loid) species, research has successively intensified in particular for arsenic [812] and mercury [1315], little conclusive data are available in case of volatile species . Dimethylarsine (me2ash) induced dna damage in human embryonic cells by formation of a peroxyl radical (ch3)2asoo . Furthermore, kato et al . Showed that trimethylarsine (me3as) induced micronuclei in the bone marrow of mice after intraperitoneal injections of 8.5 and 14.7 mg / kg . These findings were confirmed by andrewes et al . Who investigated the dna - damaging potential of me2ash and me3as using supercoiled dna . They concluded that the latter two arsines are about 100 times more potent than the most genotoxic nonvolatile arsenical, dimethylarsinous acid (me2asoh). In comparison to nonvolatile species, moreover, most studies focus on the toxicity of one compound or several compounds from one element, which makes a comparison between volatile organometal(loid) species difficult due to the different experimental systems used . In this study, we aimed to comparatively investigate the cytotoxic and genotoxic effects of the volatile metal(loid) compounds trimethylbismuth (me3bi), dimethylarsenic iodide (me2asi), trimethylarsine (me3as), tetramethyltin (me4sn), and dimethylmercury (me2hg). For our studies, we developed an exposure system dedicated for the exposure to volatile organometal(loid) species . Three different cell types were chosen for toxicity testing: cho-9 cells an established cell system for toxicity testing, caco cells same cell types were used in previous studies investigating cellular uptake and toxicity of nonvolatile organic and inorganic metal(loid) compounds [1923]. To the best of our knowledge, this is the first study testing the toxicity of these volatile metal(loid) species in vitro . (hepg2) (atcc, hb 8065) were cultured in minimal essential medium (mem) with earle's bss and sodium bicarbonate (cc, pro, germany) supplemented with 10% heat - inactivated fcs (gibco), nonessential amino acids (0.1 mm), sodium pyruvate (1 mm), and 100 iu / ml penicillin / streptomycin (cc, pro). (caco-2) (atcc 169) were cultured in 75% mem with 20% fcs, 5% nonessential amino acids (0.1 mm), 1% l - glutamine, and 0.5% gentamycin . 85050302) and grown in ham's f12 medium (cc, pro) supplemented with 10% fcs, and 100 iu / ml penicillin / streptomycin (cc, pro). All the adherent growing cell lines were kept at 37c in a 5% co2 atmosphere . Prior to exposure approximately 2 10 cells were placed on the membrane of cell culture inserts (thincerts, 0.4 m membrane, transparent; greiner bio - one, germany) with 3 ml of their respective medium for 24 h. all volatile organic metal(loid) compounds were of analytical grade unless stated otherwise and were either synthesized in the institute of environmental analytical chemistry or purchased from the following suppliers: trimethylbismuth (me3bi) from vezerf (idar - oberstein, germany), trimethylarsine (me3as) from sigma - aldrich (taufkirchen, germany), tetramethyltin (me4sn) from strem chemicals (kehl, germany), and dimethylmercury (me2hg) from acros organics (geel, belgium). Briefly, to 30 ml of an aqueous solution of dimethylarsenic acid ((ch3)2aso(oh)) and potassium iodide (ki) concentrated sulphuric acid was added . For the reduction step, so2 was bubbled through the mixture and a yellow oil ((ch3)2asi) was separated after distillation . Identification was performed by h - nmr and gc - ms analysis (data not shown). Boiling points of all used metal(loid) for exposure of cells to the volatile organometal(loid) species, the thincert cell culture inserts were placed in 1000 ml glass flasks equipped with a teflon screw cap and two plug valves in order to allow purging of the gas phase . Additionally, a septum screw cap for injection of the volatile test substances was fitted at the lower end of the glass flasks . To fix a thincert cell culture insert into the headspace of the exposure glass flask the culture medium was buffered with hepes (25 mm) (ccpro gmbh, oberdorla, germany). Before exposure, the glass flask was closed and purged with argon for at least 3 minutes to purge oxygen out of the bottle because especially trimethylbismuth is extremely oxygen sensitive . Afterwards different amounts of one metal(loid) were injected through the septa screw cap and cells were exposed for 1 h. this time point was chosen because of results from previous studies which showed that longer exposure times than 1 h caused a high degree of cytotoxicity (data not shown). The concentration range was evaluated in pre - experiments (data not shown). After exposure, treated cells were harvested with trypsin (0.05%) (sigma) for the trypan blue test and the comet assay . Control experiments with me3as verified that the cells are exposed through the membrane and not through the culture medium, as no cytotoxic effect was observable when a nonpermeable cover was placed below the membrane (data not shown). To detect cytotoxicity in exposed cell cultures, the cell suspension was mixed with an equivalent volume of 0.4% trypan blue solution (sigma) and subsequently evaluated under the light microscope . The membrane of dead cells is permeable to trypan blue (blue stained cells), whereas living cells remain unstained . Cell viability is expressed as percentage of surviving cells compared to the total number of cells: (1)% viable cells = unstained cellsunstained+stained cells100 . All experiments were repeated at least twice and significance was calculated by the student's t - test . To compare the toxicity of the different metal(loid) compounds, lc50 values (lethal concentration to 50% of the cells) were calculated . Dna damage was tested using the alkaline comet assay, first described by ostling and johanson . The comet assay is a sensitive microgel electrophoresis technique to detect dna damage in single cells . The assay was performed as described by singh et al . With minor modifications . In short, microgels were prepared by sticking a chamber slide (chamber slides lab - tek ii, nalgene nunc international, rochester, usa) with eight chambers to a gelbond film (lonza gmbh, cologne, germany). Each chamber was sealed by adding 50 l of 0.75% low melting point (lmp) agarose (invitrogen gmbh, invitrogen gmbh, germany). 45 l of lmp agarose were mixed with 20 l cell suspension containing 8,000 cells . After solidification, cells were lysed overnight at 4c in freshly prepared lysis solution . Prior to electrophoresis then the slides were kept in neutralisation solution for 30 min and further transferred to absolute ethanol for 2 h before the gels were left to dry overnight . The dna was stained for 15 min using sybr green and the extent of dna damage was analysed at a 40x magnification using the comet assay iv software (perceptive instruments, uk) and a ccd camera attached to a leica microscope . The data of three individual experiments have been summarized and are plotted using their mean value and the standard error of mean . In comparison to the tested metal(oid) compounds, me2hg was the most cytotoxic and induced 50% cell death (lc50) in cho-9 cells already at the lowest concentration tested (10.8 mol / lgv) (figure 2). The comet assay was not applicable in cho-9 cells because the lowest tested concentration of me2hg was already cytotoxic to the cells . Due to the technical limitation of the minimal applicable droplet size because of its extraordinary toxicity, not all cell lines were exposed to dimethyl mercury . Then, we abstained from exposure of the other cell lines to dimethyl mercury . The lc50 value for me2hg in caco cells was higher than in cho-9 cells (40 mol / lgv), indicating a higher resistance of colon cells to the toxic compound than fibroblasts (table 2). Me2asi was highly cytotoxic in hepg2 cells (lc50: 10.8 mol / lgv) and cho-9 cells (lc50: 11 mol / lgv), whereas cytotoxicity in caco cells was considerably lower (lc50: 335 mol / lgv) (figure 3, table 2). Similar to me2hg, testing of genotoxicity was not possible because of technical limitations in application of lower concentrations . Hepg2 cells were most sensitive (lc50: 86 mol / lgv) followed by caco cells (lc50: 129 mol / lgv) and cho-9 cells (lc50: 450 mol / lgv) (figure 4, table 2). There were no significant genotoxic effects in cho-9 cells detectable up to a concentration of 334 mol / lgv (figure 5). The highest tested concentration of 557 mol / lgv induced significantly elevated tail moments in the comet assay, however, the cytotoxicity was reduced below 50% in these experiments . The volatile me3bi was cytotoxic in all three tested cell lines (figure 6). Caco cells were the most sensitive cell line (lc50: 110 mol / lgv), followed by cho-9 cells (lc50: 128 mol / lgv) and hepg2 cells (lc50: 194 mol / lgv) (table 2). Results of the comet - assay revealed that me3bi was genotoxic at concentrations> 108 mol / lgv (figure 7). However, at higher concentrations (162 and 216 mol / lgv) me3bi was cytotoxic and thus genotoxic results were not evaluable anymore . Me4sn did not show a high level of cytotoxicity and induced 50% cell death (lc50) just in caco cells at a concentration of 170.7 mol / lgv . In cho-9 and hepg2 cells, the cell viability was not reduced below 50% up to a tested concentration of 429.4 mol / lgv and 161.7 mol / lgv, respectively (figure 8, table 2). Genotoxic effects in cho-9 cells measured by comet - assay were not significantly elevated after me4sn exposure compared to the untreated control (figure 9). From the metal(loid)s tested in this study, mercury is undoubtedly the most intensively investigated species, but this applies only to elemental and monomethyl mercury but not to the dimethylated species . In our study, me2hg was highly cytotoxic in cho-9 and caco cells . The extraordinary toxicity of dimethylmercury is at least known since the death of karen wetterhahn in 1997, months after spilling no more than a few drops of this compound on her latex - gloved hand . The reason for its extraordinary toxicity is the ability of this lipophilic compound to penetrate the cell membrane . Numerous studies have implicated a molecular mimicry in the uptake of thiol conjugates in selective target cells . Reported a negligible mercury concentration of mercury inside cho cells after treatment with dimethyl - mercury . The authors suggest from their study that the volatile mercury species escapes from the treatment solution before it can pass the cell membrane . In our experimental setup, the toxicity of the volatile arsenic compounds me2asi and me3as were studied in the present experiments . In both cell lines (cho-9 and caco-2 cells), me2asi exhibited a very high cytotoxicity similar to me2hg . In comparison to nonvolatile me2asoh, which is among the most toxic arsenic species reported, similar levels of toxicity were found when comparing the lc50-concentrations of gas (gv) and liquid (lv) volumes, respectively (table 3). Furthermore, me3as showed a significant cytotoxicity and genotoxic effects in contrast to the nonvolatile pentavalent form, me3aso, which was not cytotoxic at the concentrations tested (table 3). Unexpectedly, we found significant differences between the different cell lines used . In particular, the cytotoxicity of caco cells towards me2asi (lc50: 335 mol / lgv) was a factor of 30 lower than that found in cho-9 and hepg2 cells . Contrary to me2asi, cho-9 cells were a factor of 4 to 5 less susceptible to me3as than caco and hepg2 cells, respectively . The low susceptibility of caco towards me2asi could be attributed to the ability of caco cell to express mrp2, a multidrug resistance protein capable of catalysing as efflux . The different behaviours of me2asi and me3as indicate different mechanisms of their toxicological action . Methylated arsenic (iii) species have been shown to be genotoxic in several test systems [18, 3133] and are potent clastogens . In the present experiments, we could not evaluate the genotoxicity of me2asi and me3as because of its cytotoxicity at minimal applicable concentrations . Me3as showed significantly elevated tail moments only at cytotoxic concentrations, thus a genotoxicity testing was also not possible . The nonvolatile bismuth species monomethylbismuth was already tested for cyto- and genotoxicity in human cells in an earlier study . The results showed that the trivalent monomethylbismuth (mebi(iii)) exerted cytotoxicity even in micromolar concentrations in human hepatocytes (lc50: 350 m) after 1 h exposure . In the present study, the cytotoxic effect of the volatile me3bi in caco, cho, and hepg2 cells confirmed the observation that methylated bismuth compounds are more toxic than inorganic bismuth compounds . The lc50 value in hepg2 cells was 194 mol / lgv for me3bi compared to 350 mol / llv for mebi(iii). There seems to be a trend to an increased toxicity of methylated bi compounds with augmented methyl groups in hepg2 cells . Cytotoxicity of a trialkylated bismuth compound has been detected until now only with triphenyl - bismuth in human embryonic lung fibroblasts . In the experiments of von recklinghausen et al . With mebi(iii), the authors demonstrated that the compound is able to induce genomic damage in human lymphocytes by induction of a significant number of chromosomal aberrations and sister chromatid exchanges after 24 h exposure time . In the present experiments, we also detected dna damage after an exposure of cho-9 cells to me3bi for 1 hour only . Also here recent studies with methylated tin compounds in vitro revealed a considerable toxicological potential of some organotin species but demonstrated clearly that the toxicity is modulated by the cellular uptake capability . The highly hydrophobic and volatile compound me4sn induced neither cytotoxicity detected by using the trypan blue test nor genotoxicity evaluated with the comet assay in cho-9 cells up to a tested concentration of 429 mol / lgv . In summary, the present study indicates that some volatile organometal(loid) compounds are able to exhibit a significant toxicity to mammalian cells . While exposure to volatile organometal(loid)s in the environment is relatively rare, the formation of these compounds in the intestine may contribute to the toxicity of ingested metal(loid)s . In accordance to methylated volatile arsenic species, recent studies of our group indicated that the induction of cyto- and genotoxic effects caused by the nonvolatile trivalent methylated arsenic species is primarily dependent upon their ability to penetrate the cell membrane . Likewise, we assume that the high cyto- and genotoxicity for volatile organometal(loid) compounds found in this study can be attributed to their ability to pass cell membranes . The observation that the toxicity highly depends both upon the metal(loid) species and the exposed cell type indicates different mechanisms of their toxicological action, which need to be subject of further studies.
A 56-year - old hispanic male presented to the emergency department for gradually progressive shortness of breath and chest tightness of 4-hour duration . He was recently diagnosed with stage iv mantle cell lymphoma and had received his first cycle of r - chop (rituximab, cyclophosphamide, doxorubicin, hydrochloride (hydroxydaunomycin), vincristine sulfate (oncovin), and prednisone) chemotherapy 2 days prior to presentation . Rituximab infusion was postponed by 1 day due to inclement weather, and he received this 1 day prior to presentation . He also received one dose of intravenous (iv) rasburicase 2 days prior to presentation . He had been using emla (eutectic mixture of topical 2.5% lidocaine and 2.5% prilocaine) cream, 30 gm topically, around his port to minimize pain during infusions, the last application being 1 day prior to the presentation . He denied any cough, fever, chills, sputum production, headache, or dizziness . Significant past medical history included corneal dystrophy, hyperlipidemia, hypertension, and gastro - esophageal reflux disease . Blood pressure was 146/81 mmhg, pulse rate was 92/min, respiratory rate was 24/min, and temperature was 98.4f . He was alert and oriented to time, place, and person, and not in respiratory distress . He had a port - a - cath in the right anterior chest with no erythema or discharge around the catheter entry site . Laboratory investigation showed a white cell count of 77,900/mm (with differential count of neutrophils 85.9%, lymphocytes 0.8%, 1.8%), platelet count of 394,000/mm, and hb of 9.3 g / dl . Leukocytosis was thought to be secondary to the dexamethasone and pegfilgrastim received 2 days ago . Serum uric acid was 0.6 mg / dl (3.47.8 mg / dl), creatinine 0.77 mg / dl (0.51.5 mg / dl), and potassium 3.9 meq / l . The patient was placed on non - invasive, positive pressure ventilation because of persistent hypoxia via facemask, but his pulse oximetry remained at 86% with 100% fractional inspiration of oxygen (fio2). Arterial blood gas (abg) on 100% fio2 revealed pao2 215.6 (normal 7590 mmhg), ph 7.45 (normal 7.367.46), pco2 42.6 (normal 3446 mmhg), bicarbonate 29.5 (normal 2227 meq / l), base excess 5 (normal 2 to 2 t changes and cardiac enzymes were within normal range . Computed tomography scan of the chest was negative for pulmonary embolism and other abnormalities . Saturation gap between the oxygen saturation in pulse oximetry and arterial blood gas analysis led to a high suspicion of acquired methemoglobinemia . Methemoglobin level (methb) was 8.4% (normal 0.41.5%) and carboxyhemoglobin level was 3% (normal 12%). Although his symptoms were improved with oxygen therapy, he was given one dose of 100 mg (1 mg / kg) of intravenous methylene blue in the emergency department due to persistent hypoxia, and transferred to intensive care unit . Repeat methb level was still significantly elevated at 7.3% 2 hours later; hence, a repeat dose of methylene blue was given . Although a third dose was planned, his hemoglobin (hb) level dropped to 6.6 g% from 9.3 g% . Iu / l), haptoglobin <1.0 (normal 36195 mg / dl), total bilirubin 2.4 mg / dl (normal 0.21.1 mg / dl), and direct bilirubin 0.5 mg / dl (normal 00.4 mg / dl). The third dose of methylene blue was held and he was transfused with four units of packed red blood cells . Given the clinical situation of methemoglobinemia, unresponsive to iv methylene blue, and hemolysis after iv methylene blue in the background of recent use of rasburicase, occult g6pd deficiency was suspected . G6pd level was low (1.5 /g hb) (normal 8.813.4 /g hb), which is diagnostic for g6pd deficiency . Methb level normalized the next day and he was transferred out of the intensive care unit and then discharged from the hospital . Methemoglobin (methb) is constantly produced in the red blood cells by autoxidation of hemoglobin . Its level is maintained in a steady state of less than 2% of total hemoglobin (3). The major pathway is cytochrome b5 reductase pathway (also called nicotinamide adenine dinucleotide - dependent methb reductase). The second one is nicotinamide adenine dinucleotide phosphate (nadph)dependent methb reductase, which requires a cofactor such as methylene blue or riboflavin for activation . The latter is clinically important for the treatment of methemoglobinemia by intravenous methylene blue (4). Exposure to oxidizing agents can result in excessive formation of methb, which can lead to clinically significant methemoglobinemia, resulting in tissue hypoxia and death (4, 5). Patients usually develop cyanosis without any symptoms in levels of 1020%, with confusion starting above these levels and progressively worsening symptoms as the level increases resulting in coma, cardiovascular collapse, and death in levels above 70% (1, 6). The most common oxidizing agents are dapsone, topical anesthetic agents (benzocaine, lidocaine, and prilocaine), nitrites, and aniline dyes (7). Rasburicase is a recombinant uric acid oxidase and converts uric acid to water - soluble allantoin, and hydrogen peroxide, which is responsible for methemoglobinemia and hemolysis, especially with concurrent g6pd deficiency (8). Onset can be as early as 90 min from administration of rasburicase to beyond 6 hours (9). As our patient did not have any evidence of tumor lysis syndrome, and as rituximab is not listed as an oxidizing agent, a combination of application of emla and recent use of rasburicase might have precipitated methb . The lack of improvement in oxygen saturation with high - flow oxygen without any apparent causes and the gap in oxygen saturation> 5% between abg and pulse oximetry (saturation gap) are considered to be the diagnostic clues . The diagnosis is confirmed by the level of methemoglobin in blood (1, 6). The treatment with specific antidote is usually recommended in patients with blood methemoglobin level of> 20% in symptomatic patients and> 30% in asymptomatic patients . Patients with significant other comorbid conditions, including severe anemia, heart disease, lung disease, or carbon monoxide poisoning, should be treated even if the blood methemoglobin level is as low as 10% . However, symptoms may not always correlate with the level and clinical judgment is important for decision making (1, 2). Our case had occult g6pd deficiency and developed hemolysis after a second dose of iv methylene blue . Although the patient had received rasburicase almost 60 hours prior to the hemolytic episode, the long half - life of rasburicase might have played a role in the setting of occult g6pd deficiency . Screening for g6pd deficiency before rasburicase therapy would have cautioned against the use of methylene blue and subsequently prevented hemolysis . It could have encouraged physicians to use an alternative therapy such as high - dose vitamin c, especially in the setting of methemoglobinemia . In fact, it is the drug of choice in g6pd deficiency, acting independent of the nadph pathway (9). The diagnosis of g6pd deficiency in the suspected patient would require repeat testing in 23 months after the acute hemolysis episode resolves, if initial test is normal or borderline (10). The food and drug administration has a black box warning for both hemolysis and methemoglobinemia as contraindications to the use of rasburicase . It is suggested that high - risk ethnicity for g6pd deficiency, including people of african american, african, mediterranean, and south asian descent, should be screened for g6pd deficiency before the administration of rasburicase (10). However, it may not be practical in an emergency situation, such as severe tumor lysis syndrome and severe methemoglobinemia, because it takes 2448 hours for test results to be available (8). As our patient's symptoms were improved on oxygen even before the first dose of iv methylene blue, he could have been simply observed with oxygen . The decision was based on the basis of pulse oximetry, even though he did not have any tissue hypoxia and did not have any symptoms . This would have avoided the treatment - related complications, including hemolysis and blood transfusion, which can be devastating . Non - improvement in oxygenation with high - flow oxygen without apparent causes and the saturation gap of> 5% between abg and pulse oximetry are considered to be the diagnostic clues . The diagnosis is made by blood methemoglobin level . In patients with mild asymptomatic methemoglobinemia without any comorbid conditions, it would be reasonable to simply observe and treat symptomatically to avoid severe treatment - related complications, especially in patients with suspected g6pd deficiency . If we opt to treat these patients, treating with vitamin c would be a better choice . The authors have not received any funding or benefits from industry or elsewhere to conduct this study.
The global incidence of malignant diseases has significantly increased during the last decades in parallel with the rapid industrialization of most countries . In iran, as the third cause of death and the second prevalent chronic diseases, cancers pose a significant health burden . Malignancies are considered primarily as environmental diseases with 90 - 95% of cases attributed to environmental factors and 5 - 10% to genetic factors . Common environmental factors that are attributable to cancer death include tobacco use (25 - 30%), dietary habits and obesity (30 - 35%), infections (15 - 20%), radiation (ionizing and non - ionizing, up to 10%), tensions, and environmental pollutants . Environmental pollution is considered as one of the main causes for some types of cancers . For instance, each year nearly one million ton of lead (pb) is added to the soil at global level . This contains large quantities of atmospheric dust, scattering ash, chemical fertilizers used in agriculture, and industrial and urban wastes . Although inorganic contaminants and extensive distribution of metals are considered as one of the most likely potential exposure ways for populations, still enough attention is not paid to the environmental factors affecting health . Isfahan, a province with an area of about 107,045 square kilometers, equivalent to 6.3% of the total area of iran, is located between 30 degrees 43 minutes and 34 degrees 27 minutes north latitude and 49 degrees 38 minutes and 55 degrees 32 minutes east of the greenwich meridian . This province is also considered as one of the major agricultural poles of the country and the usage of chemical fertilizers in agriculture has been added plenty of pb into the province's soils . Pb is up taken by the plants from contaminated land, thus agricultural products are the most important way to enter this element in the food chain . Pb is not biodegradable, does not undergo dissociation, and cannot be metabolized in human body . Therefore, after entering the body, pb will be deposited and accumulated in blood, skin, organs, and tissues such as fat, muscles, bones, and joints . This study aimed to map pb distribution and the spatial distribution of the ten most common types of cancers in the province . The ten most common cancers in isfahan were determined by using data recorded from 2007 to 2009 by the isfahan cancer registry program of the isfahan provincial health center . We obtained the distribution map of pb in the province of isfahan during 2007 to 2009 from the mineral exploration organization, and the information on soil pb concentration from the isfahan agricultural jihad organization . The pb concentration was documented in three fields of agricultural, non - agricultural, and urban and industrial land . The geographic mapping of cancers and soil pb was then incorporated using geographic information system (gis) software . After producing the spatial distribution model, overall 10,142 medical records of patients with documented pathology report of cancer in 2007 to 2009 were recruited from the cancer registry data . Most prevalent cancers in the province of isfahan during 2007 - 2009 spatial distribution of pb in 2007 - 2009 is depicted in figure 1 . It shows that pb was more abundant in south, west, center, north, and east of the province . Skin cancer was more prevalent in cities of isfahan, najaf abad, kashan, naeen, ardestan, and natanz (figure 2). The rate of skin cancer was correlated with pb distribution in the province, i.e. It was lower in areas away from pb - contaminated regions . Soil lead distribution map in the province of isfahan, iran (values are presented in km .) Spatial distribution of skin cancer in the province of isfahan, iran (values are expressed as number of persons .) Spatial distribution of breast cancer in the province of isfahan, iran (values are expressed as number of persons .) Spatial distribution of hematological malignancies in the province of isfahan, iran (values are expressed as number of persons .) Spatial distribution of bladder cancer in the province of isfahan, iran (values are expressed as number of persons .) Spatial distribution of prostate cancer in the province of isfahan, iran (values are expressed as number of persons .) As shown in figure3, the prevalence of breast cancer was also directly related with the soil pb concentration . The cities of isfahan, najaf abad, kashan, borkhar, meymeh, naeen, ardestan, and natanz had the highest prevalence of breast cancer (figure3). The spatial distribution of hematological malignancies was consistent with the distribution of soil pb throughout the province (figure4). Mapping of bladder and prostate cancers were not associated with soil pb distribution (figures 5 and 6). Plotting the spatial distribution of the most common types of malignant diseases and soil pb concentration showed significant associations between mapping for skin, breast, and hematological malignancies and pb distribution in the province of isfahan . This finding may suggest that soil contamination by pb, and possibly other industrial metals, may contribute in the incidence of cancers . We should acknowledge that in addition to the possible association of soil pb with malignancies, the higher prevalence of diagnosed cancers might have been a result of better medical facilities and health centers in larger cities of the province . However, our data comprised the records from the whole province including smaller cities for three years, and earlier diagnosis of malignancies in larger cities would not affect our findings . Although pb naturally exists in the environment, most pb concentrations found in the environment are accumulated from human activities . The industrial pollutants, fertilizers, and other agricultural items are the main sources of pb added to the environment by humans . Pb distribution in the areas where the population and human activities are higher is more prominent . The main reason can be contamination by industrial pollution, pb - acid batteries, electronic components, cable sheathing, ammunition, glass, ceramics, pb pipes, paints, alloys, connections, and joints in roofs used for protection against rain . The larger pb particles drop to the ground straightaway and will pollute soils or surface waters, whereas the smaller particles will be transported long distances through air and may remain in the atmosphere . This pb - cycle caused by human production is more extended than the natural pb - cycle . Although western lifestyle, obesity, smoking, and epidemiologic transition have crucial roles in the development of non - communicable diseases, including cancers, they cannot solely explain the worldwide rapid increase in the incidence of cancers . Environmental pollution, including soil contamination by toxic metals during industrialization may be an important contributing factor. [810] in 1980, barrett depicted the spatial distribution as the possible etiology of disease, and proposed the importance of medical geography on human health . He suggested for the first time that a geographic area represents the complex physical, biological, and cultural processes . By analyzing the elements and patterns, it is possible to determine and track the spatial distribution of various diseases . A large body of evidence supports the health hazards of environmental pb exposure. [1217] it is estimated that reducing the concentrations of environmental pb can significantly decrease the standardized death rate . A study in taiwan revealed that living in high pb - polluted areas can result in an increased incidence rate of brain cancer . An ecologic study conducted in 2000 - 2007 among chinese population living in nine agricultural villages with high environmental pollution showed significant associations between exposure to multiple heavy metals, including pb, and cancer mortality . Our findings on the probable association of pb exposure with breast cancer are consistent with some previous studies. [2123] in addition to ecological studies, such association has also been documented in animal models . It is suggested that pb may accelerate tumor growth rates, and may interact with some protective factors against breast cancer development . A growing body of evidence proposed that environmental metal exposure may be associated with changes in epigenetic factors . This association may lead to a possible link between heritable changes in gene expression and disease susceptibility and development. [2527] to solve the global problem, soil resources should be tested and evaluated periodically . Preventive measures, such as using filters, lime, and other things to prevent excessive entry of sewage into rivers and freshwater resources and soil, should be considered . Cultivation of plants like borage or oxtongue plant that absorb toxic elements may be beneficial for reducing pb levels . However, products of such plants should not be consumed in areas with pb - contaminated soil . An intersectoral collaboration is necessary for reducing environmental pb production . On the other hand, increasing public knowledge on how to prevent exposure is another important strategy in this regard . Health professionals have a distinctive capacity to increase the knowledge of the whole population through educating their patients . According to the results of this study, not all diseases are caused by inheritance or genetic factors . In fact, environmental factors could also be responsible for some diseases such as malignant . Control pb producing industries to improve work - related environmental health and increasing the knowledge of health professionals and the general population in this regard are also of high importance . Programs aiming at lowering the cancer risk will thus have to consider effective measures to reduce the production of and exposures to pb and other industrial metals that are currently contaminating the environment.
The dr - tb stat task force comprises stakeholders working to introduce new medications for treating mdr tb (table 3). Dr - tb stat officially became a task force of the gdi in july 2015 . Although task force status was pending, dr - tb stat held its initial meeting in april 2015, and since then has met monthly by conference call to discuss progress and challenges in new drug introduction generally and in regard to specific countries and programs . Information was collected from multiple sources: national tb programs, country reports, who reports, information from janssen therapeutics and otsuka pharmaceuticals, a variety of donors (us agency for international development [usaid], unitaid, and the global fund), order data from the global drug facility, and reports from nongovernment organizations (e.g., partners in health, mdecins sans frontires, treatment action group, and kncv tuberculosis foundation). Data were collected and updated monthly by using a standard collection form that included the following variables: 1) the number of patients treated with each drug as part of compassionate use / expanded access programs, 2) the number of patients treated with each drug under programmatic conditions, 3) the number of countries using each drug under programmatic conditions, 4) the number of orders placed for each drug, 5) the number of countries in which each drug has been registered or in which registration is pending, and 6) the projected number of patients and countries that will be receiving each drug in 2016 . Summary data were tabulated to describe the overall assessment of global progress that is described here . For this report, compassionate use was defined as use of the drug accessed from the company for a specific patient; expanded access was defined as use of the drug accessed from the company for a group of patients who meet certain criteria (18). In addition to focusing on global progress, dr - tb stat also helps identify and address barriers to new drug introduction both generally and in program - specific settings . Detailed field notes documented the challenges reported by participants in the dr - tb stat discussions . These notes were then reviewed to identify barriers to introduction of bdq and dlm under programmatic conditions by using a thematic analysis (19). For the thematic analysis, notes were reviewed by a trained qualitative scientist (j.f .) To identify patterns and recurring content until saturation was reached . The dr - tb stat task force comprises stakeholders working to introduce new medications for treating mdr tb (table 3). Dr - tb stat officially became a task force of the gdi in july 2015 . Although task force status was pending, dr - tb stat held its initial meeting in april 2015, and since then has met monthly by conference call to discuss progress and challenges in new drug introduction generally and in regard to specific countries and programs . Information was collected from multiple sources: national tb programs, country reports, who reports, information from janssen therapeutics and otsuka pharmaceuticals, a variety of donors (us agency for international development [usaid], unitaid, and the global fund), order data from the global drug facility, and reports from nongovernment organizations (e.g., partners in health, mdecins sans frontires, treatment action group, and kncv tuberculosis foundation). Data were collected and updated monthly by using a standard collection form that included the following variables: 1) the number of patients treated with each drug as part of compassionate use / expanded access programs, 2) the number of patients treated with each drug under programmatic conditions, 3) the number of countries using each drug under programmatic conditions, 4) the number of orders placed for each drug, 5) the number of countries in which each drug has been registered or in which registration is pending, and 6) the projected number of patients and countries that will be receiving each drug in 2016 . Summary data were tabulated to describe the overall assessment of global progress that is described here . For this report, compassionate use was defined as use of the drug accessed from the company for a specific patient; expanded access was defined as use of the drug accessed from the company for a group of patients who meet certain criteria (18). In addition to focusing on global progress, dr - tb stat also helps identify and address barriers to new drug introduction both generally and in program - specific settings . Detailed field notes documented the challenges reported by participants in the dr - tb stat discussions . These notes were then reviewed to identify barriers to introduction of bdq and dlm under programmatic conditions by using a thematic analysis (19). For the thematic analysis, notes were reviewed by a trained qualitative scientist (j.f .) To identify patterns and recurring content until saturation was reached . As of october 1, 2015,> 700 persons had received bdq through compassionate use and expanded access . In addition, 1,258 persons had received bdq under programmatic conditions in 9 countries plus the european union (eu). Orders for bdq had been placed through the gdf for 1,680 persons in 24 additional countries, and these programmatic treatments are expected to begin in the next 6 months (table 4; figure 1). Global progress on programmatic use of bedaquiline (bdq) to treat multidrug - resistant tuberculosis . Gray indicates countries that have not reported using bdq under program conditions . In terms of registration, bdq has been registered in the eu (28 countries) and 13 additional countries . Based on countries reported plans and the plans of other implementing groups, bdq possibly could be given to a cumulative total of> 7,000 persons under programmatic conditions by the end of 2016 . Turnaround time from placement of order to drug delivery was 36 months despite repeated requests, otsuka pharmaceuticals did not provide specific information about dlm access, citing this information as proprietary . Therefore, data on the use of dlm were compiled from other sources . According to the company, as of october 1, 2015,> 100 patients had received dlm through compassionate use . Dlm orders cannot be placed through the gdf, and otsuka pharmaceuticals did not provide information about pending drug orders (figure 2). Global progress on the programmatic use of delamanid (dlm) to treat multidrug - resistant tuberculosis . Reported plans and the plans of other implementing groups, dlm could potentially be given to> 550 persons under program conditions by the end of 2016 . As of october 1, 2015,> 700 persons had received bdq through compassionate use and expanded access . In addition, 1,258 persons had received bdq under programmatic conditions in 9 countries plus the european union (eu). Orders for bdq had been placed through the gdf for 1,680 persons in 24 additional countries, and these programmatic treatments are expected to begin in the next 6 months (table 4; figure 1). Global progress on programmatic use of bedaquiline (bdq) to treat multidrug - resistant tuberculosis . Gray indicates countries that have not reported using bdq under program conditions . In terms of registration, bdq has been registered in the eu (28 countries) and 13 additional countries . Based on countries reported plans and the plans of other implementing groups, bdq possibly could be given to a cumulative total of> 7,000 persons under programmatic conditions by the end of 2016 . Despite repeated requests, otsuka pharmaceuticals did not provide specific information about dlm access, citing this information as proprietary . Therefore, data on the use of dlm were compiled from other sources . According to the company, as of october 1, 2015,> 100 patients had received dlm through compassionate use . Dlm orders cannot be placed through the gdf, and otsuka pharmaceuticals did not provide information about pending drug orders (figure 2). Global progress on the programmatic use of delamanid (dlm) to treat multidrug - resistant tuberculosis . No information was found about pending registration in any additional countries . According to countries reported plans and the plans of other implementing groups, dlm could potentially be given to> 550 persons under program conditions by the end of 2016 . Data collected on barriers to use of new drugs that were discussed during dr - tb stat meetings revealed a variety of reported challenges to using new drugs under programmatic conditions . In general, the problems reported fell into 10 areas: 1) lack of awareness of drug availability and procurement process; 2) limited availability of adequate technical expertise; 3) confusion around who requirements, most notably pharmacovigilance; 4) limited availability of quality clinical trials data supporting the use of new drugs under programmatic conditions; 5) challenges in sharing rapidly changing information about new drugs with key stakeholders and incorporating such information into national guidelines; 6) concerns that the process of new drug introduction is too complicated under programmatic conditions; 7) prolonged turnaround time for drug procurement; 8) difficulties in import and customs clearance; 9) limited access to companion mdr tb medications, especially linezolid and clofazimine; and 10) lack of high - level national government support . Many of the goals toward achieving the targets in the global call to action have been met for bdq . However, the goals set for dlm are unlikely to be met (table 5). * bdq, bedaquiline; dlm, delamanid . Although mdr tb is treatable and curable, managing it under program conditions has long been characterized by a host of problems . These include challenges in the detection of resistance, especially to second - line drugs; delays in prompt initiation of appropriate therapy; and insufficient mechanisms for ongoing monitoring for toxicity and continued engagement of patients throughout the 1824-month course of treatment . Furthermore, challenges exist to staff training and retention, information management, and infection control . Access to new drugs will not erase these barriers, but access does offer hope for improving individual patient outcomes and decreasing the transmission of mdr tb . Uptake of bdq and dlm under programmatic conditions, however, has not kept pace with need . According to who recommendations for the use of these medications, 25%50% of mdr tb patients meet criteria to receive these drugs because of their high - level resistance, intolerance to drugs currently used to treat mdr tb, or risks for poor outcomes (20). The occurrence of 500,000 new cases of mdr tb each year means that 125,000250,000 new patients would benefit from the use of either of these drugs each year, a number that does not include the large number of prevalent mdr tb patients who might benefit from new treatment options . More conservative estimates of the need for new drugs using the actual numbers of mdr tb patients placed on treatment each year show that 24,25048,500 of the 96,000 persons started on treatment (21) would benefit from access . Even using these less ambitious numbers, it is clear that more work needs to be done to improve access to bdq and dlm . The findings of dr - tb stat show that more persons with mdr tb who have an indication for new drugs have access to bdq than to dlm . Wider access to bdq is occurring even though the who recommendations for dlm use are broader and despite the association of bdq with a higher death rate in its phase iib trial . For several reasons bdq was conditionally approved by a stringent regulatory authority and recommended by who> 1 year before dlm was approved (22); over time, access to both drugs might become more comparable . First, the compassionate use / expanded access program for bdq started nearly 3 years earlier and was more extensive and systematic than that for dlm . As a result, multiple countries and clinical providers have had experience with bdq and thus might be more comfortable using it on a broader scale (23). Second, bdq is registered in more countries than dlm; thus programmatic use might be more straightforward . Third, bdq also is available through the gdf, whereas dlm can be obtained directly only from the company, although the company s website contains no information about how the drug can be purchased (24). Finally, in march 2015, usaid began implementing a bdq donation program in which 30,000 treatment courses are provided free of charge for 4 years, along with the technical assistance that countries might need to implement the drug (25). Although otsuka pharmaceuticals announced a 20 by 2020 access initiative meaning 20% of mdr tb patients worldwide would receive dlm by 2020 (26)the company has not provided information about how this program will work . Stakeholders participating in the dr - tb stat meetings discussed multiple barriers and challenges to new drug implementation that also appear to be slowing new drug introduction . Several of these seem to be misconceptions about how to access or use the drugs, including confusion about processes for procurement and import, the specific who recommendations especially around pharmacovigilance for the use of new drugs, and beliefs that the drugs cannot be used under programmatic conditions . Indeed, all stakeholders participating in dr - tb stat listed lack of access to technical assistance from experienced providers, including provision of rapidly changing information about new drug introduction, as a major challenge . These challenges could be addressed by providing global training for programs and their implementing partners . One approach to filling this gap would be using innovative training tools, including web - based training . Plans for offering such training globally are now being coordinated by the dr - tb stat participants with the hope that much of the confusion about how to optimally use bdq and dlm under programmatic conditions can be eased . Other challenges in new drug implementation stem from lack of access to bdq and dlm and the other drugs needed to form complete treatment regimens . Increased registration and additional experience importing the medications also should help the drugs clear customs more rapidly . The inclusion of both drugs, along with linezolid, on the who model essential drugs list in april 2015 might help boost country confidence and registration for their use in additional countries . Finally, improved access to companion drugs used with bdq and dlm, especially linezolid and clofazimine, is urgently needed . Until sufficient evidence enables bdq and dlm to be combined, linezolid and clofazimine (and other group 5 drugs) are used to ensure that the new drugs are not added singly to a failing regimen . These companion drugs are expensive in the case of linezolid, more so than bdq and are not registered with an mdr tb indication in most of the countries using bdq and dlm . Similar attention needs to be given to these drugs, and countries need to be supported in accessing them . Dr - tb stat is likely to begin formally monitoring and assisting countries with access to these medications in 2016 . Access to new drugs alone will not solve the ongoing crisis of mdr tb, and much more comprehensive action is needed to improve care for persons with this disease on a global level (27). These medications, however, are one of the few current hopes for more effective and less toxic therapy for mdr tb affected persons worldwide (28). Coordinated efforts can help ensure timely and equitable access to bdq and dlm for persons who need them most.
A couple s intentions to have a number of descendants can be thwarted in a number of ways . The most difficult of factors are those that are restrictive and outside of a couple s sphere of influence or resources . The need to have a child can be reflected in motives such as happiness, well - being (family relationships), identity, parenthood (life - fulfilment), social control and continuity (dyer et al ., 2008). Parenthood - motives may vary according to gender, societies and cultures, but neither the level of education nor wealth can substitute the inherent need for a biological child . In developing countries particularly in the sub - saharan african region, human immunodeficiency virus (hiv) infection together with limited resources adds to the barriers in becoming a parent . Although the south african s bill of rights (constitution of the republic of south africa act no 108 of 1996) decree that south africans can make decisions concerning reproduction; access to and the use of assisted reproduction technology (art) are viewed in general as prohibitively expensive and only accessible to the privileged few . Resources available in the public / tertiary art units and to private practices and its clients, affect reproductive healthcare screening and concurrent diagnostic and therapeutic decisions which include; scheduling for intra - uterine inseminations (iui), in vitro fertilization (ivf) and intra - cytoplasmic sperm injection (icsi), combined with semen decontamination for hiv - seropositive males . Several factors can impact on the financial health of an art programme . Cost - drivers with emphasis on art laboratory set - up and procedures in south africa will be discussed in this review, structured on a previous examination of cost - drivers in south africa (huyser and boyd, 2012), i.e. (1) art procedures, (2) detection and prevention of infections, (3) sperm preparations, and (4) laboratory facilities, including supplies needed to perform procedures (fig . The first two tertiary art institutions in south africa were established in pretoria and cape town in 1982 (fourie et al ., 1988; kruger et al ., 1985), with the first test tube different forms of art services are provided in the country, i.e. Public service academic - centred art units or private ventures with independent specialists utilizing office - based or corporate pathology laboratories, and larger established art associates consisting of clinical and laboratory art specialists . It is questionable if the current (approximately) 28 national art service providers (cross - referenced with known providers in provinces and www.ivf-worldwide.com) are providing an adequate reproductive health service within a nation of 52 million people with a variety of cultures and languages (http://www.southafrica.info/about/people/population). The speciality, repertoire and type of art structure, i.e. Private vs. public / tertiary, will impact on capacity, services offered, revenue generated and patient population of the unit . South africa has four national tertiary art units, situated in cape town (groote schuur and tygerberg / vincent palotti, www.aevitas.co.za), bloemfontein (femspes group - www.femspes.co.za) and in pretoria (steve biko academic hospital formerly known as pretoria academic hospital, and previously as the hf verwoerd hospital) (http://www.pah.org.za/departments/endocrine.html). Two out of the four national tertiary art units, situated in cape town and pretoria are entirely dependent on public funding . The reproductive and endocrine unit, as part of the department of obstetrics and gynaecology at the university of pretoria provides both diagnostic and therapeutic art procedures, including semen decontamination for hiv+ patients, and general cryopreservation (huyser and fourie, 2010), and is an accredited training unit for clinical technologists and medical biological scientists in reproductive biology . The majority of patients presently attending the art program at the unit for diagnostic purposes are from the lower to middle income groups with an average gross household income of 1,405 967 (1) per month . Patients that participate in an art attempt have a slightly higher average gross household income of 2,260 1,730 per month (random sampling of 100 non - overlapping patients from 2012 in the diagnostic and therapeutic groups, respectively). Two different art cost groupings, i.e. State subsidized and private patient structured categories are available at our unit, with a third low cost option that can be accessed by both categories: [a] a subsidized category through state funding, for couples without medical aid and an annual income of less than 4,205 per household; with patients contributing towards registration fees (4.00 per consultation / visit), medications with partial media costs . The actual procedures will cost 97.00 for an iui and 1,269.00 for ivf / icsi in this category . [b] a private category (including medication, clinical-, pathology- and laboratory fees) for couples with a medical aid and/or a household income above 8,410 p.a . A fee of up to 435 (case - dependent) is payable for an iui attempt and a maximum of 1,830 for ivf / icsi procedures . [c] an affordable low cost option for ivf / icsi is also available to patient categories [a] & [b], and is based on initiatives for accessible ivf (ombelet and campo, 2007; ombelet et al ., 2008; ombelet, 2009); this includes basic medication, minimal clinical-, pathology- and laboratory fees . Approximate costs range from 401 to 962 (category [a] & [b] dependent) for an ivf or icsi cycle . Patients can select the low cost option, but need to comply with criteria based on aetiology, age and case history . Access to the subsidized category is subjected to budget allocation, and only a small number of patients qualify for a subsidy in this category per year . The patient ratio of 1:6:1 for the cost categories a to b to c, respectively in 2011 - 2012 (huyser and boyd, 2012), vs. a patient ratio of 1:4:2 for 2012- early 2013 could indicate a personal budget decline in the present time . A retrospective cost analysis in 1986 for the set - up of our art unit indicated that the cost for an ivf cycle amounted to 135.50 (fourie et al ., 1988). The average cost for procedures from 20 private south african art units (mostly from gauteng), were obtained telephonically (in april 12 with a follow up in april 13) (fig . Total cost estimations including medications, ultrasound scans and laboratory fees were obtained for a standard iui, ivf and icsi procedure . In the private sector, ivf procedures increased in 2012 - 13 on average from 2,930 to 3,255 with a similar trend found for the icsi procedures . The average costs (standard deviation) per procedure in the private sector are: (i) iui: 542 159, (ii) ivf: 3,255 576 and (iii) icsi: 3,302 625 . The cost for an iui procedure can vary depending on the number of inseminations . Dyer and kruger (2012) referred to general out - of - pocket costs for a standard ivf cycle of 841 (subsidized in the public sector) and 2,944 (within the private sector) in south africa, which reflects the previous mentioned fee structure in 2012 . The average percentage of the major cost - drivers of an ivf cycle at an active private practice in south africa in 2012 were the following: 8% of costs are allocated for clinic fees, 28% to medication, 29% to clinicians fees & consultations, and 35% for laboratory fees (for use of equipment and the laboratory, disposables, culture media and staff expenditures) (huyser and boyd, 2012). Interestingly, laboratory expenses accounted for 39.2% of the total ivf cycle cost at our unit in 1986 (fourie et al ., 1988). Converting from an ivf procedure to an icsi procedure items used in the laboratory can amount to nearly 48% of all costs per icsi cycle . Approximately 70% (14 out of 20 practices) of the south african art units that were contacted during the present study (fig . Iui on the other hand can be viewed as the most cost - effective art procedure (garceau et al ., 2002), and is revealed in the low comparative costs for medications, 8% of the total expenses, vs. 23 - 28% for an ivf or icsi cycle (proportional data communicated by one of the largest art units in south africa) (huyser and boyd, 2012). Chambers and co - workers (2009) indicated that the cost of (art) treatment reflects the costliness of the underlying healthcare system rather than the regulatory or funding environment . Within south africa, access to art is restrictive due to limited health insurance coverage of art procedures, restrained access to a few art units within the public sector (dyer and kruger, 2012), and limited funding to public sector art providers . Funding of art centers and treatment of infertility competes with a range of health priorities . Similar to the debate on prevention vs. treatment of infertility (dyer and pennings, 2010), the detection and thus prevention of pathogen transmission will be less expensive and more beneficial to a large number of people than treatment alone . Screening of the couple should however, be directed by the incidence of disease(s) in the specific patient population, medical history and physical examination of the couple (elder et al ., 2005). With the prevalence of hiv in sub - saharan africa, the question arises should all art participants be screened / re - screened for blood borne viruses (bbv)? Correspondingly, since a variety of bacteria species are present in approximately 50% of all semen samples obtained for art procedures, with gram - negative species present in only a fraction of samples (fourie et al ., 2012), should all semen samples be submitted for bacteriological culture and sensitivity? An answer could be prophylactic or empiric anti - microbial treatment options, with concurrent costs (huyser and boyd, 2012) especially in a rural setting in the absence of pathology services or due to logistical reasons . Without a south african technical directive regarding the screening and treatment of art patients for bbv / pathogens it is doubtful if requirements of the european union tissue and cells directives (www.eur-lex.europa.eu) for art units in the eu, whereby biological screening must be carried out at the time of donation i.e. Of sperm or oocytes, can be used as a blue print for south african art units . The repeated screening (for hiv, hbv, hcv) of patients was probed by wingfield and cotell (2010), who suggested an initial baseline screening with appropriate risk reduction measures to prevent cross - contaminations during an art procedure . With rapid screening technology available at affordable costs (approximately 1.30 per hiv rapid test) in south africa, all art patients currently the cost for a single hiv rapid test (2) is approximately 1.5% of a rt - pcr quantitative (hiv-1 rna) and 8% of an elisa hiv-1 test . Rapid tests are inexpensive, simple to perform, individually packaged with a shelf - life of approximately 12 months (world health organization, 2004) and are well suited for resource constrained settings . Preliminary experiences in our laboratory on using rapid tests (hiv, hcv, hbv) as a first - line screening indicates that the tests are easy to execute, and takes approximately 20 minutes per test to perform . All hiv positive results were confirmed with a secondary more extensive rapid test and patients are counselled to undergo a confirmatory viral validation (preferably with cd4 and viral load analysis). No false positive or false negative results were encountered for the rapid tests up to date . Eight percent of patients that have never undergone an hiv - test previously, tested positive with the rapid tests (n = 100 individuals), with a 3:1 ratio for females to males (stander, 2013, personnel communication). A layered risk - reduction approach is practised at our unit when dealing with contaminants in semen prior to an art cycle, i.e. Provide guidelines on sample collection in native languages to male patients to reduce skin contaminants; prescribe suitable treatment based on susceptible testing of semen prior to an assisted reproduction procedure (opposed to prophylactic antibiotic treatment); use semen washing / decontamination procedures combined with a physical device (e.g. The proinsert, [nidacon, sweden]) together with discontinuous density gradients to diminish microbe re - contamination (huyser and fourie, 2010; fourie et al ., 2012). Semen quality is taken as a surrogate measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments (cooper et al ., 2010). The appropriateness of sperm preparation techniques and costs to obtain purified sperm should be considered, since the post - processed sperm sample s quality greatly governs the choice of art procedure to follow . Also, if further washing steps after density gradient centrifugation (dgc) will be sufficient to wash sperm free of hiv . A cochrane based review by boomsma et al . (2011) showed that no specific semen preparation technique (i.e. Dgc; swim - up; as well as wash and centrifugation techniques) improved clinical outcome with reference to iui procedures . The reason being that a minimum threshold of> 1 million motile spermatozoa is needed for successful conception through iui, irrespective of the type of sperm preparation method used (ombelet et al ., 2003). One such option is an office - based semen preparation device called sep - d (surelife media technologies (cat no: sl 001)) with a current price tag of 23.00/device, consisting of a semen preparation kit containing a syringe pre - filled with a buffered culture media . Motile sperm is separated from seminal plasma through a direct swim - up technique, where after the motile sperm fraction is retained in approximately 300 medium within the device, which is then connected to an iui catheter (7.00/catheter when purchased separately, cat no: sl 002 - 12), and used for insemination (www.surelifeivf.com). Within the south african market a couple who qualifies for an iui procedure and resides in a rural town, could travel to a local general practitioner or clinic for repeated inseminations . Gentis and co - workers (2012) reported a good clinical outcome for iui patients in a randomized controlled study at a south african art unit while using the sep - d semen processing device . The risks associated with sperm preparation techniques should be discussed with patients (who, 2010; eke and oragwu, 2011). Data on sperm washing for hiv - seropositive patients are merely observational in nature according to a cochrane review by eke and oragwu (2011). Sperm washing refers to a sequential three phase procedure, i.e. Dgc, washing of the sperm pellet and swim - up step; as was initiated by semprini and co - workers (1992) to prepare semen samples for art from hiv - positive males . The bold undertaking was prior to the initiation of highly active antiretroviral treatment or validation of viral particles in the washed sperm sample (semprini et al . This safety record is backed by published data from centers worldwide using iui, ivf and icsi procedures for hiv - positive males . Various factors may contribute to the lack of randomized trials in this area of art, including hiv - regulations, inequalities in art treatment modalities, as well as costs of sperm washing and art procedures (with particular reference to resource - poor countries in africa) (eke and oragwu, 2011). The choice of a sperm preparation technique is however vital when processing sub - optimal semen samples for ivf or icsi, with discontinuous dgc being the preferred method to optimize samples . Quotes for seven known brands of gradients and wash solutions were received from south african agencies in april 2013 . Five of the seven brands are included in the listed culture media products (see section: laboratory facilities, supplies and environmental aspects), together with purespermmedia (nidacon, sweden; www.nidacon.com) and sil - select (fertipro n.v ., belgium; www.fertipro.com). A single processing (2 ml semen sample) will cost on average 16.82 3.34 (ranging from 10.11 to 19.33). The term sperm decontamination was coined at our laboratory to distinguish sperm washing from the decontamination procedure used for samples possibly containing various infectious microbes e.g. Bacteria, hiv, hcv and cmv (huyser and fourie, 2010). This involves the layering of density gradients and the semen using a proinsert kit (www.tekevent.com/nidacon/proinsert), at a cost of 10.10 for the device (cat no: ni - p115 - 5, nidacon). The kit consists of two conical tubes with two elongated pipettes and a proinsert device . The purified motile sperm pellet (after centrifugation) is retrieved using the elongated pipette without re - contaminating the pellet with infectious micro - organisms . A final washing step follows to get rid of density particles and a portion of the purified sperm sample can be submitted for testing (hiv-1 proviral dna and rna using a sensitive molecular based technique such as reverse transcription polymerase chain reaction (rt - pcr)). More than a decade of research in the treatment of hiv+ semen samples culminated in the clinical application of procedures during art treatment of patients at our unit . A 100% and 98.1% success rate in the removal of hiv-1 rna and dna respectively, is maintained for the decontamination procedure at our laboratory (n = 100 semen samples, post - processing pcr validations, 2011 - 2012). Since the costs of rt - pcr viral validations on purified sperm samples are expensive especially in a developing country, (86.20 per test for dna or rna at a national pathology laboratory) the question arises if all purified samples should be tested? Due to restricted access to pathology laboratories and cost implications, only qualitative viral validations are available in most developing countries . Within south africa, molecular viral testing is more accessible, and in our experiences up to 32% of patients with an undetectable hiv blood load can have a positive hiv-1 rna seminal viral load (n = 100 patients). Patients are informed of the procedure failure rate, hiv - related health screening (cd4 + cell levels) and additional infectious disease tests, general risk - reduction methods, extra costs for viral validations and cryopreservation of semen samples prior to the initiation of an art attempt (huyser and boyd, 2012). Financial pressures can result in procedural shortcuts and the demand to maximize patient throughput in some private practices and laboratories offering sperm processing procedures . In the absence of national directives on the art treatment of hiv+ patients within a developing country, best practice frameworks and directives from developed countries could be adapted and used as guidelines for assisted reproduction laboratories in the developing world . All art related laboratory items except for general pharmacy articles are imported from various parts of the world to south africa . Equipment, sperm processing solutions, embryo culture media and disposables have to be couriered with concomitant imported taxes . Setting - up an art laboratory in any part of the world depends on economics, availability and optimal maintenance of items . Procedures should be best - practice - based with reliable equipment, disposables, and techniques within a risk - reduction environment . This section will discuss the costs applicable to selected art equipment and mainstream embryo culture media that is commercially available and currently in use within south africa . The price tag for six different benchtop incubators and five media brands in south africa is compared . The costs to purchase six different benchtop incubators (listed alphabetically): i.e. Bt37(origio / planer scanlab equipment a / s, lynge, denmark; www.origio.com); g85 and g185 standard (k - systems kivex biotec ltd, birkerd, denmark; www.k-systems.dk); k - minc-1000 (cookmedical, brisbane, australia; www.cookmedical.com), labo c - top (labor - technik - gttingen, germany; www.labotect.com); miri multi - room incubator for ivf (esco medical, singapore; www.medical.escoglobal . Table i demonstrates the south african price range from 3,769.80 to 22,763.57 for benchtop incubators which can accommodate 8 to 48 petri dishes (35 mm), respectively . The costs are vat inclusive and may consist of installation, a start - up kit or humidification container where applicable . For more details see the individual websites . When the same incubators are purchased in belgium, three out of the six incubators are currently between 6.59 - 28.69% less expensive, and three incubators are 1.44 - 25.98% more expensive compared to purchase prices in south africa . . A similar cost extrapolation should be applicable to all art equipment and disposables imported into south africa . A cost analysis for the set - up of the art programme at our unit in the eighties, indicated that the total cost for laboratory equipment amounted to 11,606 . A single inverted microscope, two water jacketed upright incubators, a stereo as well as a light microscope, a single laminar flow and biological safety cabinet, hygrometer, dry - oven, centrifuge, refrigerator, osmometer, ph - meter and electronic balance constituted the laboratory to initiate the art programme (fourie et al ., 1988). More than 90% of this equipment was still fully functional twenty years on (2005/6), when the laboratory moved to new purpose built laboratories within steve biko academic hospital (pretoria, south africa). Similar equipment to date will cost between 67,283 and 92,515, depending on the model size and/or brand type (huyser and boyd, 2012). The previously mentioned historical cost analysis article by fourie and co - workers (1988) did not refer to a micro - manipulator (for icsi procedures), or any cryopreservation equipment during the initiation phase of the laboratory . Five different brands of culture media are currently used in south africa: i.e. Globalmedia (lifeglobal one - step protocol; ivfonline, usa, www.lifeglobal.com), medicult media (embryoassist & blastassist two - step protocol; origio, denmark, www.origio.com), quinns advantage media (sage media products two - step protocol; coopersurgical, (usa, www.coopersurgical.com); sydneyivf (k - sicm & k - sibm two - step protocol; cookmedical ireland, www.cookmedical.com); and vitrolife - g - series media (g-1 & g-2, two - step protocol; scandinavia ivf science, gteborg, sweden; www.vitrolife.com). Quotes for the media brands were obtained in april / may in 2013 from south african distributers . Total costs per media brand for a full art cycle including dgc, ivf, and icsi are indicated in fig . Costs were calculated per ml of medium used per patient, per cycle attempt according to laboratory protocol, with embryo culture in micro - drops under oil . A similar cost analyses were performed in 2012 (huyser and boyd, 2012). Culture media for an ivf procedure for six oocytes amounts to 40.26 8.76 (ranging from 27.25 to 51.79) and costs approximately15% less than media used in an icsi procedure (mean cost of 47.14 11.04 (ranging from 31.22 to 60.32)). Cryopreservation solutions for non - vitrification procedures cost on average 19.96 9.20 (ranging from 10.18 to 28.74), with a 37% difference in cost for a vitrification attempt (mean cost of 31.85 10.37 (ranging from 14.55 to 41.38)). An average annual increase of 7% for vitrification cryopreservation, whereas an annual average decrease of 3% for non - vitrification solutions was noted . These costs exclude the cryopreservation carriers / devices available as open or closed single - straw systems, cryo - storage tanks, or liquid nitrogen . Courier services and/or company representatives, which manage the transport and delivery of art culture media timeously, play a vital role in failures or successes in the art laboratory . Durable packaging and protection of temperature sensitive culture media while in transit through developing countries are also extremely important during (occasional) protracted customs clearance, especially in summertime . The first two tertiary art institutions in south africa were established in pretoria and cape town in 1982 (fourie et al ., 1988; kruger et al ., 1985), with the first test tube different forms of art services are provided in the country, i.e. Public service academic - centred art units or private ventures with independent specialists utilizing office - based or corporate pathology laboratories, and larger established art associates consisting of clinical and laboratory art specialists . It is questionable if the current (approximately) 28 national art service providers (cross - referenced with known providers in provinces and www.ivf-worldwide.com) are providing an adequate reproductive health service within a nation of 52 million people with a variety of cultures and languages (http://www.southafrica.info/about/people/population). The speciality, repertoire and type of art structure, i.e. Private vs. public / tertiary, will impact on capacity, services offered, revenue generated and patient population of the unit . South africa has four national tertiary art units, situated in cape town (groote schuur and tygerberg / vincent palotti, www.aevitas.co.za), bloemfontein (femspes group - www.femspes.co.za) and in pretoria (steve biko academic hospital formerly known as pretoria academic hospital, and previously as the hf verwoerd hospital) (http://www.pah.org.za/departments/endocrine.html). Two out of the four national tertiary art units, situated in cape town and pretoria are entirely dependent on public funding . The reproductive and endocrine unit, as part of the department of obstetrics and gynaecology at the university of pretoria provides both diagnostic and therapeutic art procedures, including semen decontamination for hiv+ patients, and general cryopreservation (huyser and fourie, 2010), and is an accredited training unit for clinical technologists and medical biological scientists in reproductive biology . The majority of patients presently attending the art program at the unit for diagnostic purposes are from the lower to middle income groups with an average gross household income of 1,405 967 (1) per month . Patients that participate in an art attempt have a slightly higher average gross household income of 2,260 1,730 per month (random sampling of 100 non - overlapping patients from 2012 in the diagnostic and therapeutic groups, respectively). Two different art cost groupings, i.e. State subsidized and private patient structured categories are available at our unit, with a third low cost option that can be accessed by both categories: [a] a subsidized category through state funding, for couples without medical aid and an annual income of less than 4,205 per household; with patients contributing towards registration fees (4.00 per consultation / visit), medications with partial media costs . The actual procedures will cost 97.00 for an iui and 1,269.00 for ivf / icsi in this category . [b] a private category (including medication, clinical-, pathology- and laboratory fees) for couples with a medical aid and/or a household income above 8,410 p.a . . A fee of up to 435 (case - dependent) is payable for an iui attempt and a maximum of 1,830 for ivf / icsi procedures . [c] an affordable low cost option for ivf / icsi is also available to patient categories [a] & [b], and is based on initiatives for accessible ivf (ombelet and campo, 2007; ombelet et al ., 2008; ombelet, 2009); this includes basic medication, minimal clinical-, pathology- and laboratory fees . Approximate costs range from 401 to 962 (category [a] & [b] dependent) for an ivf or icsi cycle . Patients can select the low cost option, but need to comply with criteria based on aetiology, age and case history . Access to the subsidized category is subjected to budget allocation, and only a small number of patients qualify for a subsidy in this category per year . The patient ratio of 1:6:1 for the cost categories a to b to c, respectively in 2011 - 2012 (huyser and boyd, 2012), vs. a patient ratio of 1:4:2 for 2012- early 2013 could indicate a personal budget decline in the present time . A retrospective cost analysis in 1986 for the set - up of our art unit indicated that the cost for an ivf cycle amounted to 135.50 (fourie et al . The average cost for procedures from 20 private south african art units (mostly from gauteng), were obtained telephonically (in april 12 with a follow up in april 13) (fig . Total cost estimations including medications, ultrasound scans and laboratory fees were obtained for a standard iui, ivf and icsi procedure . In the private sector, ivf procedures increased in 2012 - 13 on average from 2,930 to 3,255 with a similar trend found for the icsi procedures . The average costs (standard deviation) per procedure in the private sector are: (i) iui: 542 159, (ii) ivf: 3,255 576 and (iii) dyer and kruger (2012) referred to general out - of - pocket costs for a standard ivf cycle of 841 (subsidized in the public sector) and 2,944 (within the private sector) in south africa, which reflects the previous mentioned fee structure in 2012 . The average percentage of the major cost - drivers of an ivf cycle at an active private practice in south africa in 2012 were the following: 8% of costs are allocated for clinic fees, 28% to medication, 29% to clinicians fees & consultations, and 35% for laboratory fees (for use of equipment and the laboratory, disposables, culture media and staff expenditures) (huyser and boyd, 2012). Interestingly, laboratory expenses accounted for 39.2% of the total ivf cycle cost at our unit in 1986 (fourie et al ., 1988). Converting from an ivf procedure to an icsi procedure items used in the laboratory can amount to nearly 48% of all costs per icsi cycle . Approximately 70% (14 out of 20 practices) of the south african art units that were contacted during the present study (fig . Iui on the other hand can be viewed as the most cost - effective art procedure (garceau et al ., 2002), and is revealed in the low comparative costs for medications, 8% of the total expenses, vs. 23 - 28% for an ivf or icsi cycle (proportional data communicated by one of the largest art units in south africa) (huyser and boyd, 2012). Chambers and co - workers (2009) indicated that the cost of (art) treatment reflects the costliness of the underlying healthcare system rather than the regulatory or funding environment . Within south africa, access to art is restrictive due to limited health insurance coverage of art procedures, restrained access to a few art units within the public sector (dyer and kruger, 2012), and limited funding to public sector art providers . Funding of art centers and treatment of infertility competes with a range of health priorities . Similar to the debate on prevention vs. treatment of infertility (dyer and pennings, 2010), the detection and thus prevention of pathogen transmission will be less expensive and more beneficial to a large number of people than treatment alone . Screening of the couple should however, be directed by the incidence of disease(s) in the specific patient population, medical history and physical examination of the couple (elder et al ., 2005). With the prevalence of hiv in sub - saharan africa, the question arises should all art participants be screened / re - screened for blood borne viruses (bbv)? Correspondingly, since a variety of bacteria species are present in approximately 50% of all semen samples obtained for art procedures, with gram - negative species present in only a fraction of samples (fourie et al ., 2012), should all semen samples be submitted for bacteriological culture and sensitivity? An answer could be prophylactic or empiric anti - microbial treatment options, with concurrent costs (huyser and boyd, 2012) especially in a rural setting in the absence of pathology services or due to logistical reasons . Without a south african technical directive regarding the screening and treatment of art patients for bbv / pathogens it is doubtful if requirements of the european union tissue and cells directives (www.eur-lex.europa.eu) for art units in the eu, whereby biological screening must be carried out at the time of donation i.e. Of sperm or oocytes, can be used as a blue print for south african art units . The repeated screening (for hiv, hbv, hcv) of patients was probed by wingfield and cotell (2010), who suggested an initial baseline screening with appropriate risk reduction measures to prevent cross - contaminations during an art procedure . With rapid screening technology available at affordable costs (approximately 1.30 per hiv rapid test) in south africa, all art patients currently the cost for a single hiv rapid test (2) is approximately 1.5% of a rt - pcr quantitative (hiv-1 rna) and 8% of an elisa hiv-1 test . Rapid tests are inexpensive, simple to perform, individually packaged with a shelf - life of approximately 12 months (world health organization, 2004) and are well suited for resource constrained settings . Preliminary experiences in our laboratory on using rapid tests (hiv, hcv, hbv) as a first - line screening indicates that the tests are easy to execute, and takes approximately 20 minutes per test to perform . All hiv positive results were confirmed with a secondary more extensive rapid test and patients are counselled to undergo a confirmatory viral validation (preferably with cd4 and viral load analysis). No false positive or false negative results were encountered for the rapid tests up to date . Eight percent of patients that have never undergone an hiv - test previously, tested positive with the rapid tests (n = 100 individuals), with a 3:1 ratio for females to males (stander, 2013, personnel communication). A layered risk - reduction approach is practised at our unit when dealing with contaminants in semen prior to an art cycle, i.e. Provide guidelines on sample collection in native languages to male patients to reduce skin contaminants; prescribe suitable treatment based on susceptible testing of semen prior to an assisted reproduction procedure (opposed to prophylactic antibiotic treatment); use semen washing / decontamination procedures combined with a physical device (e.g. The proinsert, [nidacon, sweden]) together with discontinuous density gradients to diminish microbe re - contamination (huyser and fourie, 2010; fourie et al ., 2012). Semen quality is taken as a surrogate measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments (cooper et al ., 2010). The appropriateness of sperm preparation techniques and costs to obtain purified sperm should be considered, since the post - processed sperm sample s quality greatly governs the choice of art procedure to follow . Also, if further washing steps after density gradient centrifugation (dgc) will be sufficient to wash sperm free of hiv . A cochrane based review by boomsma et al . (2011) showed that no specific semen preparation technique (i.e. Dgc; swim - up; as well as wash and centrifugation techniques) improved clinical outcome with reference to iui procedures . The reason being that a minimum threshold of> 1 million motile spermatozoa is needed for successful conception through iui, irrespective of the type of sperm preparation method used (ombelet et al ., 2003). One such option is an office - based semen preparation device called sep - d (surelife media technologies (cat no: sl 001)) with a current price tag of 23.00/device, consisting of a semen preparation kit containing a syringe pre - filled with a buffered culture media . Motile sperm is separated from seminal plasma through a direct swim - up technique, where after the motile sperm fraction is retained in approximately 300 medium within the device, which is then connected to an iui catheter (7.00/catheter when purchased separately, cat no: sl 002 - 12), and used for insemination (www.surelifeivf.com). Within the south african market a couple who qualifies for an iui procedure and resides in a rural town, could travel to a local general practitioner or clinic for repeated inseminations . Gentis and co - workers (2012) reported a good clinical outcome for iui patients in a randomized controlled study at a south african art unit while using the sep - d semen processing device . The risks associated with sperm preparation techniques should be discussed with patients (who, 2010; eke and oragwu, 2011). Data on sperm washing for hiv - seropositive patients are merely observational in nature according to a cochrane review by eke and oragwu (2011). Sperm washing refers to a sequential three phase procedure, i.e. Dgc, washing of the sperm pellet and swim - up step; as was initiated by semprini and co - workers (1992) to prepare semen samples for art from hiv - positive males . The bold undertaking was prior to the initiation of highly active antiretroviral treatment or validation of viral particles in the washed sperm sample (semprini et al . This safety record is backed by published data from centers worldwide using iui, ivf and icsi procedures for hiv - positive males . Various factors may contribute to the lack of randomized trials in this area of art, including hiv - regulations, inequalities in art treatment modalities, as well as costs of sperm washing and art procedures (with particular reference to resource - poor countries in africa) (eke and oragwu, 2011). The choice of a sperm preparation technique is however vital when processing sub - optimal semen samples for ivf or icsi, with discontinuous dgc being the preferred method to optimize samples . Quotes for seven known brands of gradients and wash solutions were received from south african agencies in april 2013 . Five of the seven brands are included in the listed culture media products (see section: laboratory facilities, supplies and environmental aspects), together with purespermmedia (nidacon, sweden; www.nidacon.com) and sil - select (fertipro n.v . A single processing (2 ml semen sample) will cost on average 16.82 3.34 (ranging from 10.11 to 19.33). The term sperm decontamination was coined at our laboratory to distinguish sperm washing from the decontamination procedure used for samples possibly containing various infectious microbes e.g. Bacteria, hiv, hcv and cmv (huyser and fourie, 2010). This involves the layering of density gradients and the semen using a proinsert kit (www.tekevent.com/nidacon/proinsert), at a cost of 10.10 for the device (cat no: ni - p115 - 5, nidacon). The kit consists of two conical tubes with two elongated pipettes and a proinsert device . The purified motile sperm pellet (after centrifugation) is retrieved using the elongated pipette without re - contaminating the pellet with infectious micro - organisms . A final washing step follows to get rid of density particles and a portion of the purified sperm sample can be submitted for testing (hiv-1 proviral dna and rna using a sensitive molecular based technique such as reverse transcription polymerase chain reaction (rt - pcr)). More than a decade of research in the treatment of hiv+ semen samples culminated in the clinical application of procedures during art treatment of patients at our unit . A 100% and 98.1% success rate in the removal of hiv-1 rna and dna respectively, is maintained for the decontamination procedure at our laboratory (n = 100 semen samples, post - processing pcr validations, 2011 - 2012). Since the costs of rt - pcr viral validations on purified sperm samples are expensive especially in a developing country, (86.20 per test for dna or rna at a national pathology laboratory) the question arises if all purified samples should be tested? Due to restricted access to pathology laboratories and cost implications, only qualitative viral validations are available in most developing countries . Within south africa, molecular viral testing is more accessible, and in our experiences up to 32% of patients with an undetectable hiv blood load can have a positive hiv-1 rna seminal viral load (n = 100 patients). Patients are informed of the procedure failure rate, hiv - related health screening (cd4 + cell levels) and additional infectious disease tests, general risk - reduction methods, extra costs for viral validations and cryopreservation of semen samples prior to the initiation of an art attempt (huyser and boyd, 2012). Financial pressures can result in procedural shortcuts and the demand to maximize patient throughput in some private practices and laboratories offering sperm processing procedures . In the absence of national directives on the art treatment of hiv+ patients within a developing country, best practice frameworks and directives from developed countries could be adapted and used as guidelines for assisted reproduction laboratories in the developing world . All art related laboratory items except for general pharmacy articles are imported from various parts of the world to south africa . Equipment, sperm processing solutions, embryo culture media and disposables have to be couriered with concomitant imported taxes . Setting - up an art laboratory in any part of the world depends on economics, availability and optimal maintenance of items . Procedures should be best - practice - based with reliable equipment, disposables, and techniques within a risk - reduction environment . This section will discuss the costs applicable to selected art equipment and mainstream embryo culture media that is commercially available and currently in use within south africa . The price tag for six different benchtop incubators and five media brands in south africa is compared . The costs to purchase six different benchtop incubators (listed alphabetically): i.e. Bt37(origio / planer scanlab equipment a / s, lynge, denmark; www.origio.com); g85 and g185 standard (k - systems kivex biotec ltd, birkerd, denmark; www.k-systems.dk); k - minc-1000 (cookmedical, brisbane, australia; www.cookmedical.com), labo c - top (labor - technik - gttingen, germany; www.labotect.com); miri multi - room incubator for ivf (esco medical, singapore; www.medical.escoglobal . Table i demonstrates the south african price range from 3,769.80 to 22,763.57 for benchtop incubators which can accommodate 8 to 48 petri dishes (35 mm), respectively . The costs are vat inclusive and may consist of installation, a start - up kit or humidification container where applicable . For more details see the individual websites . When the same incubators are purchased in belgium, three out of the six incubators are currently between 6.59 - 28.69% less expensive, and three incubators are 1.44 - 25.98% more expensive compared to purchase prices in south africa . . A similar cost extrapolation should be applicable to all art equipment and disposables imported into south africa . A cost analysis for the set - up of the art programme at our unit in the eighties, indicated that the total cost for laboratory equipment amounted to 11,606 . A single inverted microscope, two water jacketed upright incubators, a stereo as well as a light microscope, a single laminar flow and biological safety cabinet, hygrometer, dry - oven, centrifuge, refrigerator, osmometer, ph - meter and electronic balance constituted the laboratory to initiate the art programme (fourie et al ., 1988). More than 90% of this equipment was still fully functional twenty years on (2005/6), when the laboratory moved to new purpose built laboratories within steve biko academic hospital (pretoria, south africa). Similar equipment to date will cost between 67,283 and 92,515, depending on the model size and/or brand type (huyser and boyd, 2012). The previously mentioned historical cost analysis article by fourie and co - workers (1988) did not refer to a micro - manipulator (for icsi procedures), or any cryopreservation equipment during the initiation phase of the laboratory . Five different brands of culture media are currently used in south africa: i.e. Globalmedia (lifeglobal one - step protocol; ivfonline, usa, www.lifeglobal.com), medicult media (embryoassist & blastassist two - step protocol; origio, denmark, www.origio.com), quinns advantage media (sage media products two - step protocol; coopersurgical, (usa, www.coopersurgical.com); sydneyivf (k - sicm & k - sibm two - step protocol; cookmedical ireland, www.cookmedical.com); and vitrolife - g - series media (g-1 & g-2, two - step protocol; scandinavia ivf science, gteborg, sweden; www.vitrolife.com). Quotes for the media brands were obtained in april / may in 2013 from south african distributers . Total costs per media brand for a full art cycle including dgc, ivf, and icsi are indicated in fig . Costs were calculated per ml of medium used per patient, per cycle attempt according to laboratory protocol, with embryo culture in micro - drops under oil . A similar cost analyses were performed in 2012 (huyser and boyd, 2012). Culture media for an ivf procedure for six oocytes amounts to 40.26 8.76 (ranging from 27.25 to 51.79) and costs approximately15% less than media used in an icsi procedure (mean cost of 47.14 11.04 (ranging from 31.22 to 60.32)). Cryopreservation solutions for non - vitrification procedures cost on average 19.96 9.20 (ranging from 10.18 to 28.74), with a 37% difference in cost for a vitrification attempt (mean cost of 31.85 10.37 (ranging from 14.55 to 41.38)). An average annual increase of 7% for vitrification cryopreservation, whereas an annual average decrease of 3% for non - vitrification solutions was noted . These costs exclude the cryopreservation carriers / devices available as open or closed single - straw systems, cryo - storage tanks, or liquid nitrogen . Courier services and/or company representatives, which manage the transport and delivery of art culture media timeously, play a vital role in failures or successes in the art laboratory . Durable packaging and protection of temperature sensitive culture media while in transit through developing countries are also extremely important during (occasional) protracted customs clearance, especially in summertime . The dual demand for art in south africa is mirrored in the provision of low cost accessible art services to lower - income nationals including middle - income private patients in the public service . The country s foreign - exchange rate can be two sides of the same coin; i.e. Equipment is more expensive to import than the purchase price within a confederacy, however comparative cut - rate art costs can lure non - nationals to south africa as a choice destination for reproductive tourism . The downturn in the global economy on the other hand has a sobering effect transnationally and should influence manufacturers of art products to develop products that can stand the test of time . Art procedures need not be propelled towards the must - have and cannot - do - without approach, but providers should also reflect on the validity of the techniques and equipment, without compromising treatment virtue . Art treatments should be globally within the reach of a much larger part of the population.
Acute heart failure (ahf) is associated with an adverse prognosis with an all cause mortality (acm) of 1020% at 23 months and 1737% at 1 year.1, 2, 3, 4, 5 single biomarkers on top of clinical assessment are useful for risk stratification in patients with chronic heart failure (hf).6, 7 however in ahf, the incremental predictive value of a single biomarker is small when added to a clinical score . Results from the multinational observational cohort on acute heart failure (moca) study suggest improved risk prediction when miscellaneous biomarkers are assessed simultaneously.5 this concept accounts for the absence of one single relevant pathomechanism in ahf while accepting that interpretation of test results is more difficult . Red cell distribution width (rdw) is a readily available biomarker increasing with cytokine activation, impaired iron mobilization, and decreasing haemoglobin level.8, 9, 10 rdw, thus, integrates various pathophysiological mechanisms associated with hf progression, which can explain why elevated rdw is a robust marker of risk for mortality in acute and chronic hf.11, 12, 13 this prospective registry of consecutive patients hospitalized for ahf investigates whether elevated rdw remains predictive in ahf patients when patients are grouped by lvef 50% or lvef <50% . This is a prospective registry of consecutive patients (n = 408) hospitalized for ahf at a tertiary centre . Patients presenting with ahf at the emergency wards were included when hospitalized for heart failure at our institution and after signing the consentment . Patients with acute coronary syndrome, or cardiogenic shock in need of intensive care were not included . Patients without outcome (n = 6) were excluded from final analysis . Echocardiographic data derive from standard transthoracic studies signed by a boardcertified cardiologist at our institution; all exams were performed during index hospitalization . Physicians' diagnosis of comorbidity followed the respective guidelines.14, 15, 16 rdw was measured using the advia 120 hematology analyzer (siemens healthcare diagnostics). Rdw was reported as coefficient of variation (in percent) of erythrocyte cell volume . Continuous variables are presented as mean (sd) or median (interquartile range; iqr). Rdw quartiles were defined in accordance with the literature13, 17, 18 with the lowest quartile serving as the reference group . The distribution of rdw was skewed; therefore, logarithmically transformed values of rdw (ln rdw) were used as dependent variable for multiple linear regressions as reported elsewhere.13 age, gender, and rdw quartiles were forced into the model predicting acm; independent explanatory variables were identified by backward elimination of variables associated with rdw at a probability (p) of <0.1 in univariate analysis . Stepwise cox proportional hazards models calculated the hazard ratio (hr) selecting covariables on the basis of backward stepwise analysis with removal set at p <0.1 . Survival curves were calculated using the kaplan meier method; comparison of survival curves used the logrank test . All tests were twosided and used a significance level of p <0.05 . Analyses were performed using the r statistical software (version r 3.1.0) (r development core team). Continuous variables are presented as mean (sd) or median (interquartile range; iqr). Rdw quartiles were defined in accordance with the literature13, 17, 18 with the lowest quartile serving as the reference group . The distribution of rdw was skewed; therefore, logarithmically transformed values of rdw (ln rdw) were used as dependent variable for multiple linear regressions as reported elsewhere.13 age, gender, and rdw quartiles were forced into the model predicting acm; independent explanatory variables were identified by backward elimination of variables associated with rdw at a probability (p) of <0.1 in univariate analysis . Stepwise cox proportional hazards models calculated the hazard ratio (hr) selecting covariables on the basis of backward stepwise analysis with removal set at p <0.1 . Survival curves were calculated using the kaplan meier method; comparison of survival curves used the logrank test . All tests were twosided and used a significance level of p <0.05 . Analyses were performed using the r statistical software (version r 3.1.0) (r development core team). Patients with high rdw were older, had a higher incidence of previous hfrelated hospitalizations, and chronic hf was more prevalent by trend (p = 0.0612). Patients in the high rdw quartile had lower lvef, lower systolic and diastolic blood pressure, and lower haemoglobin; creatinine, creactive protein, and ntprobnp levels were higher . Patients with high rdw received more often loop diuretics and oral anticoagulation (table 1). Hhf, previous hospitalization for heart failure; in hosp mortality, in hospital mortality; los, length of stay; cs, cardiogenic shock; pe, pulmonary edema; rhf, right heart failure; htn, hypertension; decomp hf: decompensated heart failure; lvef, left ventricular ejection fraction; hr, heart rate; sr, sinus rhythm; sbp, systolic blood pressure; dbp, diastolic blood pressure; bmi, body mass index; copd, chronic obstructive pulmonary disease . Correlation was poor between rdw and lvef (p = 0.21), egfr (p = 0.12), or length of stay (p = 0.10). Rdw levels and haemoglobin levels correlated with strength of 0.0021 0.0002 (p = 0.021). Strength of the correlation between rdw (ln rdw) and different variables table 3 shows the results of a multivariable cox model (maximal logistical likelihood 614.37; lr test statistic: 79.74; p = 1.2 10). The following variables were associated with increased hr (listed in increasing order): platelets, age, diastolic blood pressure, valvular pathology, chronic hf, elevated rdw, male sex, and cardiogenic shock . Variables with a decreased hr are listed in decreasing order: lower rdw, weight, and systolic blood pressure . Adjusted proportional hazards model for all cause 1year mortality female gender and lowest rdw group were used as reference; valvular hd, valvular heart disease . During followup 114 patients (28.4%) died . The kaplan meier analysis including all patients showed a graded increased probability of mortality with increasing quartile of rdw (figure 1 (a); 18; p = 0.0004). In patients with reduced lvef, mortality was not significantly different between rdw quartiles (figure 1 (b); 6.6; p = 0.084). Study patients with preserved lvef showed a graded increased probability of mortality with rising rdw quartile (figure 1 (c); 9.9; p = 0.0195). (b) rdw quartiles and 1year survival in ahf patients with lvef <50% . (c) rdw quartiles and 1year survival in ahf patients with lvef 50% . Ahf patients with preserved lvef were older, the number of previous hospitalizations for hf was lower, and the prevalence of female gender and diagnosis of de novo hf was higher . Ahf patients with preserved lvef more often presented with hypertensive crisis whereas prevalence of ischemic aetiology and diabetes was lower . The prevalence of valvular pathology was not significantly different between hf patients with preserved and reduced systolic ejection fraction . In ahf patients with preserved lvef systolic blood pressure was higher while heart rate and ntprobnp levels were lower . Demographics and characteristics of ahf patients with reduced and preserved ejection fraction see legend table 1, nipv, noninvasive pulmonary ventilation, for others see table 1 . Cad, coronary artery disease; mr, mitral regurgitation; ar, aortic regurgitation . Overall 19.9% of all patients presented with high rdw, 65% of these patients had a transthoracic echocardiography during hospitalization; 63.4% of these presented a lvef <50% . Patients with lvef 50% and high rdw were older (80 vs. 75 y; p = 0.013), heart rate was lower (80 vs. 90 bpm p = 0.04), and history of hf was less frequent (47 vs. 79%, p = 0.04). The burden of comorbidities (i.e. Diabetes, arterial hypertension, chronic obstructive bronchopulmonary disease, bmi, renal dysfunction by egfr) was not significantly different between patients with high rdw and lvef of <50% or 50% . The results of the present study add to the available data regarding the predictive relevance of elevated rdw in acute hf . First, the results confirm the relevance of high rdw for prediction of mortality in ahf.11, 12, 13 second, the present study population shows that high rdw is associated with increased mortality in ahf patients with lvef 50% while there is no interaction between high rdw and mortality in ahf patients with lvef <50% . The small size of the present study population raises the concern whether the sample is representative . However, demographic, clinical, and biological characteristics of the present study collectively correspond with respective characteristics reported from larger studies in ahf.19, 20 in addition, the present study replicates associations between rdw and outcome as described elsewhere, in particular, the negative correlation of rdw with haemoglobin levels as reported in the candesartan in heart failure: assessment of the reduction in mortality and morbidity (charm),8 and in the staminahfp (study of anemia in hf population) or the unitehf (united investigators to evaluate heart failure) registry.9 in addition, this study replicates the relation of high rdw with increased mortality already described in other ahf study populations.11, 12, 13 finally, high rdw was retained in the model for prediction of acm similar to other studies in acute or chronic hf.8, 13, 17 in the general population, rdw increases with conditions of ineffective red cell production (such as iron deficiency, folate or b12 deficiency, and hemglobinopathy), increased red cell destruction, or after blood transfusion . Anaemia is a strong predictor of mortality in heart failure suggesting that rdw is a strong predictor for mortality and morbidity8, 10, 12 because of the high prevalence of anaemia.8 in fact, some heart failure studies link elevated rdw with inflammation, malnutrition, and renal dysfunction that may all impact on erythropoiesis.18, 21 however, results from the charm north america study cohort (n = 2697) indicate a prognostic role of rdw that is independent from haemoglobin level based on the observation that both parameters were retained in the final model for prediction of acm but without reciprocal interaction.8 this observation was replicated in the duke databank cohort providing supportive evidence.8 additional evidence for a distinct role of rdw derives from the european prospective investigation into cancer and nutrition (epic) norfolk study and the malm cancer and diet study which show an association of rdw with incident heart failure while rdw was in this middleaged healthy population without relation to iron metabolism or inflammation.22, 23 high rdw is associated with increased interleukin6 levels (il6) as shown in the staminahfp and the unitehf registries.9 in patients with mild or moderate hf with reduced lvef, increased il6 levels are associated with increased risk of disease progression.24 furthermore, il6 are likely to be relevant in hf with preserved hf as suggested in a study by collier et al . Which showed an increase of il6 levels in patients with lvef 50% and heart failure symptoms while il6 was low in patients without hf symptoms.25, 26 complementary evidence of a pathophysiological role of il6 in hf derives from the health, aging, and body composition (abc) study that showed a correlation between increased il6 levels and incident hf with preserved lvef.27 because blood samples were not retained from the present study population, we cannot correlate rdw with il6 levels; however, increased crp levels in patients with high rdw suggest increased il6 levels because il6 levels correlate with crp levels patients with ahf and chronic hf.24, 28 in the present study, high rdw was associated with increased mortality in patients with lvef 50% while mortality was not different between rdw groups of patients with reduced lvef . Several characteristics of the two subgroups may explain this finding: first, evidencebased pharmacological treatment6 was increased in the majority of patients with reduced lvef <50% during hospitalization (inhibitors of the renin angiotensin system: 70% to 92%; blocker treatment: 35 to 58%; antagonists of the mineralocorticoid receptor: 21 to 38%). It is well known that increase of pharmacological hf treatment improves 1year survival;6, 13 therefore, mortality difference between the rdw quartiles in the present study may have decreased because of improved drug therapy; second, more patients with preserved lvef and low rdw had developed ahf because of hypertensive crisis . Hypertensive crisis is associated with a low mortality29 suggesting accentuation of the mortality difference between low and high rdw quartiles; third, recent results from the swedish heart failure registry suggest that blocker treatment reduces acm in hf patients with preserved lvef.30 only 42% of the study patients with preserved lvef 50% were on blocker treatment suggesting an increased hazard for mortality in patients without blocker in accordance with the proportional hazards model of this study . In fact, blocker treatment decreased from 52% to 41% in patients with preserved lvef and high rdw . The singlecentre study design, the sample size, and the outcome parameter of 1year allcause mortality without consideration of cardiovascular mortality or rehospitalization rate represent limitations of the study . Furthermore, echocardiograms were not obtained in all patients, which might have introduced a selection bias . The singlecentre study design, the sample size, and the outcome parameter of 1year allcause mortality without consideration of cardiovascular mortality or rehospitalization rate represent limitations of the study . Furthermore, echocardiograms were not obtained in all patients, which might have introduced a selection bias . We have shown for the first time the prognostic relevance of rdw in ahf with preserved ejection suggesting that rdw may help to stratify risk already at admission . In a next step, this observation should be confirmed in a larger ahf population with echocardiographic exams obtained during index hospitalization . In addition, the results of this study merit further workup of the interaction of rdw and il6 in ahf with preserved ejection fraction, and thus, increase our understanding of clinically relevant pathomechanisms in these patients . Cardiomet, an initiative of the chuv to improve quality of care in cardiovascular and metabolic disease; swiss national science foundation (320030_147121/1), swissheart foundation, to r.h.
Native hawaiians are descendents of the aboriginal peoples inhabiting the hawaiian archipelago prior to western contact in 1778 and exercising sovereign governance prior to the 1892 overthrown of the hawaiian kingdom by the united states (usa) [1, 2]. The 2000 us census enumerated 401,000 americans (0.1% of the total population) of full or part - hawaiian ethnicity, about 60% of whom reside in the state of hawaii . Native hawaiians comprise about 24.3% of the state's current population . As in other states, the population of hawaii is aging, with an increasing number of residents living into old age . Although life expectancy in hawaii exceeds that of other us states, studies conducted within the state reveal continuing ethnic differences in life expectancy . As depicted in figure 1, over six decades (19502000) native hawaiians have consistently had the lowest life expectancy when compared to the state's three other largest groups, namely, caucasians, filipinos (americans), and japanese (americans). Notably, the magnitude of this disparity about 10 years lower than the longest lived group has not changed over time . About 16% of deaths among native hawaiians in 2005 occurred before 45 years, which is at least two times higher than for any other ethnic group living in the state . Mortality disparities are particularly significant when comparing native hawaiians with japanese; in 2005, 60% of deaths among japanese occurred at age 80 + years, compared to only 25% of native hawaiians . As a result, native hawaiians are underrepresented in the older age groups . In 2008, 21.4% of the state's overall population was 60 + years of age but only 11.1% of native hawaiians are in this age group . To look at it another way, native hawaiians comprised 24.3% of the total state population in 2008, but only 12.6% of residents age 60 + are native hawaiians . Table 1 displays population totals and age distributions of the state's four largest ethnic groups . Other investigators have examined data for the native hawaiian population in general and have found that native hawaiians have a higher prevalence of obesity, numerous chronic conditions, and greater impoverishment than other ethnic groups living in the state [57]. The relatively poor health status of the native hawaiian population overall has been of significant interest since the publication of the seminal e ola mau: the native hawaiian health needs study report . Historic difficulty in native hawaiians' use of western mainstream healthcare services due to socioeconomic disadvantage, discrimination, and cultural misunderstanding are highlighted . Subsequently, social welfare researchers in gerontology and native hawaiian health have documented a number of socioeconomic disparities (e.g., higher rates of poverty, lower educational levels, homelessness, and incarceration) as well as health disparities (e.g., higher rates of diabetes and certain types of cancer, lower utilization of health services). To improve native hawaiian health and well - being, these researchers have underscored the need to consider more distal factors such as historical trauma or the cumulative effect of negative physical, sociocultural, political, and economic changes on current health disparities [712]. Concomitantly, they articulate the strengths of traditional native hawaiian culture, including those cultural values and practices on health, care giving, and social support that might be integrated into interventions which offer the prospect of increased longevity and enhanced quality of life [8, 10, 11]. Importance of this research notwithstanding, no recent studies have compared health indicators by ethnicity for older adults in hawaii with a specific focus on native hawaiian elders . Research described here attempts to address this gap in the current knowledge and was conducted by researchers associated with h kpuna: national resource center for native hawaiian elders . Funded by the us administration on aging (aoa), h kpuna is one of three resource centers for native elders in the usa . The name derives from the native hawaiian cultural belief that one's life essence (spiritual energy, ancestral knowledge) is transmitted to others through sharing of the h (breath of life). It is believed that such sharing allows kpuna (elders) to pass on vital knowledge and wisdom to subsequent generations, thus perpetuating valued cultural traditions and a positive sense of identity . Grounded in these and other traditional native hawaiian cultural values, h kpuna seeks to assure the transmission of h from kpuna (elders) to younger generations by achieving parity in life expectancy and good health among native hawaiian older adults comparable to that of other older americans . As a center dedicated to the health of native hawaiian elders, h kpuna develops and disseminates knowledge to inform policy and service innovations . In this paper, we examine proximal influences to the health and longevity of native hawaiian elders, specifically describe the causes of premature mortality among native hawaiians, and identify the ways in which sociodemographic and behavioral factors of hawaii's elders vary by ethnicity . Our research was guided by these questions: (1) what are causes of premature mortality? (2) how does this vary by ethnicity? And (3) how do sociodemographic and health behavioral indicators vary by ethnicity? To address these questions we reviewed relevant statistical data collected by two hawaii department of health surveillance programs . Written requests for data were submitted to two surveillance programs of the hawaii department of health (doh): vital records and the hawaii behavioral risk factor surveillance system (hbrfss). Data sources are briefly described, as well as methods used to arrive at population - based statistics relevant for examining the underrepresentation of native hawaiian elders in the state's older (60 years) adult population . We requested data on the state's largest ethnic groups from the vital records program, which routinely gathers information on births, deaths, and marriages that take place in the state . Based on death record data, we calculated the years of productive life lost (ypll) for the state's largest ethnic groups . Ypll is an index that measures the extent of premature mortality by giving a weight to each premature death from the predetermined cutoff age, with proportionally higher weights for younger deaths . Because ypll is especially sensitive to the age distribution of the population, it cannot be used in cross - ethnic - group comparisons . However, ypll can be converted to a rate that is independent of sample size and population distribution following a method proposed by lee . These rates, herein called total population life lost per person in a lifetime (tpll), can be compared across ethnic groups . To construct tpll, we obtained resident death data from hawaii for the 3-year period, 19992001, for each age group by ethnicity, sex, and underlying cause of death of the deceased . Averaging 3 years of death data was done to smooth annual fluctuations in death . Cause of death is coded following the international classification of disease 10th revision (icd-10). We analyzed six causes of death common among older adults cancer, heart disease, cerebrovascular disease, unintentional injuries, suicide, and diabetes in addition to total death . Both population and death data were classified into age groups, <1, 14 years, and then in 5-year intervals . All deaths in a given age group are assumed to have occurred at the midpoint age of the interval . We set age 70 as the cutoff for premature mortality in conformance with recommendations of the national center for health statistics . With the upper limit for the premature death at 70, the ypll owing to premature mortality is given by (1)ypll=(70ci)di, where, ci is the midpoint of the ith age interval and di is the number of deaths in the ith age interval . Thus (70-ci) is the weight given to the deaths in the ith age group . The summation runs from age 0 to 70 . For the cause - specific ypll, di is the number of deaths from that particular cause of death . Ypll, as a function of di, is to a large extent determined by the size and age distribution of the population as well as premature mortality . To convert ypll to a rate, we applied the index proposed by lee, which is independent of population size and age distribution . In lee's method, the age - specific weight of ypll is multiplied by the age - specific death rate of the population, that is, (2)(70cj)djpj, where pj is the population of age j. the resultant is the annual number of ypll expected to occur per person in age j. originally, lee named this measure as the cumulative rate of potential life lost (crpll). But here we call it the total potential life lost per person in lifetime (tpll), since it also presents the number of premature deaths . When the age is grouped, tpll is expressed by (3)tpll=ni(70ci)dipi, where ni is the length of the interval of the age group . This is the total number of ypll expected per person before age 70 in the study population . To establish 95% confidence intervals (cis), we estimated the standard deviation of tpll in each ethnic group (4)[ni(70ci)]2pidipipidipi . Further, as the 3-year average of death was used for di, the variance of our tpll is the above quantity divided by 3 . We requested special data runs from the hawaii behavioral risk factor surveillance system (hbrfss) to examine ethnic variation in sociodemographic, clinical, and behavioral factors in the state's elders . Part of the behavioral risk factor surveillance system (brfss) of the centers for disease control and prevention (cdc), the hbrfss gathers data by telephone from about 6,000 randomly selected adults (18 years of age). Respondents are queried about behaviors that directly or indirectly affect health and health - related topics . For example, brfss solicits information on height and weight to calculate body mass index (bmi), health behaviors (e.g., smoking), health screening (e.g., use of breast, cervical and colorectal cancer early detection screening), and chronic disease (e.g., diabetes, hypertension). Sample data are adjusted and weighted based on ethnicity distributions, and estimates are produced for native hawaiians, caucasians, filipinos, and japanese . We requested a special tabulation that averaged responses from three years of data (2005, 2006, and 2007) relevant to older adults (60 years of age) residing in hawaii . After years of data were combined, hbrfss provided data tables for older adults as a whole, and for the four largest ethnic groups . The sample size of older adults over three years included 658 native hawaiians, 2,652 caucasians, 561 filipinos, and 1,840 japanese, for a total of 6,346 elders . For hawaii, the overall tpll before age 70 per person from premature mortality was estimated at 3.3 years in 2000, but tpll varied by ethnic group (table 2). Among the groups we analyzed, the smallest number of years lost was observed for japanese at 2.6 years and, as expected, the largest was for native hawaiians, at 5.3 years . The difference in tpll between japanese and native hawaiians is twofold, meaning that, on average, native hawaiians are losing twice as many years of potential life as japanese . Among the six causes of death, cancer caused the largest number of potential years lost before age 70, with 0.74 years per person, accounting for more than 22% of overall tpll . Heart disease, ranking second (0.59 years), accounted for 18% of overall tpll . Unintentional injuries ranked third (0.40 years), accounting for 12% of overall tpll . Death from suicide (0.21 years), stroke (0.13 years), and diabetes (0.06 years) ranked fourth, fifth, and sixth, respectively . As illustrated in figure 2, the importance of cause of death varied considerably by ethnic group . Cancer was the most important cause of tpll in all populations, except native hawaiians, while heart disease was top - ranked for native hawaiians . While cancer and heart disease were the top - leading causes of tpll in most ethnic groups, accidents ranked second for caucasians, while accidents ranked third for other groups . Suicide, cerebrovascular disease, and diabetes ranked fourth, fifth, and sixth in each ethnic group . Native hawaiians had the highest tpll for each of the six causes of death, almost twofold higher for cancer than the other groups, two - to - four times higher for heart disease, and two - to - three times higher for diabetes . Sociodemographic data from hbrfss indicate that native hawaiian older adults have the largest proportion of females to males; women account for 61.5% of hawaiian elders compared to caucasian females who account for 50.9% of caucasian elders . This suggests that a disproportionate amount of native hawaiian men are dying before the age of 60 years . About 38.2% of native hawaiian elders attended at least one year of college . This percent is similar to that of filipino elders (39.7%), but much lower than for japanese (56.1%) and caucasians (72.7%). Further, about 24% of native hawaiian elders are employed (perhaps indicating that they have had to delay retirement due to higher impoverishment rates), compared to only 18% of japanese elders . According to hbrfss, the percentage of elders with health insurance was relatively high overall (97%) although the percentage of native hawaiian elders reporting access to health insurance was 95% . Approximately 7.2% of native hawaiian elders reported not being able to see a physician in the past year due to cost, compared to less than 1% of japanese elders . Native hawaiian elders reported the highest prevalence of asthma (20% versus 11% overall) and diabetes (25% versus 17% overall). However, native hawaiians reported prevalence of heart attached, angina / chd, and stroke similar to caucasians . Hbrfss data indicate a relatively high prevalence of behaviors associated with increased disease risk among native hawaiian elders . For example, native hawaiian elders were most likely of the four ethnic groups to smoke every day or occasionally, about 14.6% compared to only 6.9% of japanese elders (although native hawaiian elders with a history of smoking were most likely to state that they were trying to quit or have stopped smoking at least for one day in the past year). Native hawaiian and caucasian elders were most likely to report drinking more than four or five alcoholic beverages on one occasion (about 9% in each group). About one - third of all ethnic groups reported that they were overweight, however 36% of native hawaiian elders were obese compared to only 8% of japanese elders . Data from hbrfss indicate that 28% of filipinos, 26% of native hawaiians, 23% of japanese, and 19% of caucasian elders rated their personal health as fair or poor . About 18% of hawaiian elders responded that they were most likely to need special equipment, in comparison to 13% of all elders . About 80% of female elders reported having a mammogram within the last two years, including 78% of native hawaiian female elders . Among male elders, native hawaiians and filipinos were the least likely to have had a prostate - specific antigen (psa) test for prostate cancer in the past 12 months (all male elders = 54%, native hawaiians = 37.7%, filipinos = 33.7%). About 47% of filipino elders and about 53% of hawaiian elders have ever had a sigmoidoscopy or colonscopy to check for colorectal cancer, compared to 71% of caucasians and 73% of japanese . Data from two state surveillance programs highlight ethnic differences in cause of death, health, and behavioral indicators that may help to explain some of the ethnic differences in life expectancy . For example, native hawaiian elders are least likely to have attended college and most likely to have incomes below 200% of poverty . They are least likely to have access to a primary care physician, have routine checkups, or to participate in early detection cancer screening . Further, they are most likely to smoke and be obese . In comparison to other major ethnic groups, native hawaiian elders have the highest or second highest prevalence of a number of chronic diseases, including asthma and diabetes . Heart disease is the leading cause of premature death for native hawaiians, and native hawaiians lose significantly more years of productive life to heart disease, cancer, injuries, suicide, stroke, and diabetes than other ethnic groups . These findings for older adults are consistent with earlier research on the general hawaiian population that documents serious health and social disparities [36, 9]. There were four major limitations to our research; these limitations point to a number of remaining and critical gaps in knowledge about native hawaiian elders . First, the study describes, but does not explain, the relationship of ethnicity and health indicators . Thus, caution must be exercised in using findings to directly inform policy or practice innovations . Continued research is needed to explain the relationship of native hawaiian ethnicity and proximal health indicators . Second, the study captured ethnic variation in health outcomes; the role of gender as it interacts with ethnicity was not explored . Research across the life course consistently exposes disparities by gender, and this is especially so in later life [19, 20]. For example, poverty rates for older women are nearly twice as high as for men . Our understanding of the lives of native hawaiian elders will be strengthened by the inclusion of gender in analyses of social and health disparities . Third, this study used surveillance data collected from hawaii residents only; thus, findings cannot be generalized to populations outside the state, including native hawaiians living on the north american continent . At present, our knowledge of hawaiian elders residing in the continental usa is very limited . This is an important omission since nearly 40% of native hawaiians live in the contiguous states, primarily california, washington, and oregon . Extending life expectancy and improving quality of life requires that we understand life expectancies, health status, health care needs, preferences for care, and utilization patterns of all native hawaiian elders . Future research is needed to determine if native hawaiians on the continent experience similar disparities, such as shorter life expectancies than other ethnic groups in their new communities . Fourth and finally, the current study relied on surveillance data that did not address distal factors, such as the influence of historical trauma and systemic discrimination on current health disparities . Native hawaiian and other indigenous health researchers consistently emphasize the influence of historical trauma and intergenerational marginalization on current health disparities [1, 5, 812, 2226]. Sotero's conceptual model of historical trauma posits that subjugation of a population by a dominant group has a cumulative effect on the physical, sociocultural, political, and economic well - being of the oppressed group . The trauma is felt by first - generation survivors (i.e., those who directly experienced the traumatic events), as well as by successive generations of their descendents . Across succeeding generations, the traumatic impact may be mitigated to some degree by cultural resiliency and other group protective factors . However, the result is an excess of social and physical ills that ultimately lead to population - specific health disparities . In the case of native hawaiians, historical records document the precipitous decline in population numbers and health status of native hawaiians following contact with the west . At the earliest known point of contact in 1778, members of the cooke exploratory expedition describe natives of the islands as hardy, robust, and capable of great physical activity . However, during the first 150 years of western contact, native hawaiians suffered disability and premature death from foreign diseases such as influenza, measles, small pox, syphilis, and mumps [1, 23]. Depopulation was severe; in the first century of contact the native population declined from an estimated 800,000 to 50,000 . Depopulation was accompanied by cultural degradation with the native language and many traditional practices outlawed and/or subordinated as inferior to the english language and western practices, respectively [23, 24]. Among the most devastating changes were those related to shifts in the land tenure system from one of collective stewardship to one of private ownership and monopoly capitalism . This shift assured the rise of westerner - owned plantations and eventually, led to the overthrow of the hawaiian kingdom in 1892 and us annexation in 1898 . The exponential growth of the plantation economy and subsequent loss of native hawaiian sovereignty coupled with severe depopulation caused a collective grief among native hawaiian survivors [9, 23, 24]. Na makaainana (those who tend the land, common people) were alienated from their aina (land), the source of their spiritual, social, and economic well - being . As natives lost access to their land, there was a mass exodus to port cities where they became wage laborers and in some cases, debtors, paupers, and na paahao (prisoners, convicts) [12, 24]. In line with sotero, native hawaiian health researchers are linking the poor health of native hawaiians in contemporary times to this cascade of adverse historic events and intergenerational social marginalization [9, 10, 12]. Despite the relative success of native hawaiian organizations and groups to build cultural pride, positive identity, and holistic health in communities, the social marginalization of native hawaiians for example, native hawaiian children are overrepresented in the state's child welfare population, native hawaiian youth are disproportionately represented in the state's juvenile justice system, and native hawaiian adult men and women are overrepresented at every stage of hawaii's criminal justice system [2, 27]. The overall picture remains one of a population with more social and health disparities in comparison to the other large ethnic groups in the state . In the last few decades some social policies have been enacted to redress past wrongs and support programs aimed at reducing the disparate health outcomes affecting native hawaiians (e.g., public law 100579, the native hawaiian health care act of 1988). However, it is clear that more must be done to increase the longevity of native hawaiians and enhance the quality of life among native hawaiian elders . Our findings underscore the ongoing need to target behavioral risks affecting native hawaiian longevity across the life span . Health promotions grounded in evidence - based strategies and tailored on native hawaiian cultural preferences, as well as socio - economic circumstances have been recommended to reduce smoking and sedentary lifestyle, improve dietary practices, and increase participation in early detection screening [913]. In the past, health promotions that disregard relevant cultural, socio - economic factors, and systemic barriers have been received with distrust and even resentment by native hawaiian consumers who have experienced such efforts as cultural impositions [1, 12]. Thus, in targeting behavioral risks affecting longevity, it is crucial to develop interventions that adhere to principles of community - based participatory research and meaningfully involve native hawaiian communities in identification of barriers and assets salient to intervention development and delivery . Based on results from the current study, we conclude with three service and policy implications for native hawaiian elders . First, all hawaii residents need primary health care that includes health education to promote optimal health practices, as well as chronic disease prevention and control . The inclusion of health promotion and disease prevention and control in primary care is especially important for native hawaiians who as a group have shorter life expectancies than other ethnic groups and who enter their senior years with more chronic conditions and poorer health habits than most other ethnic groups . Health promotion and disease management programs are needed to both inform native hawaiians of their risk for heart disease, and other chronic conditions, as well as to reduce the adverse impact of these conditions on their overall well - being . Contributors to heart disease, such as hypertension and high cholesterol, can be controlled by diet, exercise, and medications . Although many cancers cannot be prevented, they can be cured and/or controlled if diagnosed and treated early . Thus, enrollment in evidence - based health promotion and disease management programs should be encouraged financially and programmatically . Second, all hawaii residents need health care to be affordable and this seems especially important for native hawaiian elders . Unfortunately, the high costs of deductibles, copayments, and noninsured treatments lead to delays in seeking necessary health care and may discourage elders from completing treatment or taking medications as recommended . Compounding the problem is the fragmented system of long - term care, both in the provision and funding of services . This issue has critical implications for native hawaiians, who may require long - term care services early in life due to earlier onset of disability . Nationally, long - term care services and financing are undergoing major programmatic changes because of the demand for cost - effective and efficient practices for improving quality of life of individuals in need of long - term care . Improvements to the long - term care system include aging and disability resource centers (adrcs), which offer one - stop shopping for individuals in need of long - term care services, cash, and counseling programs (through which elders and caregivers are provided vouchers to pay for long - term care services and providers of their choice), expansion of community residential care models (such as assisted living, small group homes, and geriatric foster care), and the culture change movement (i.e., to make nursing homes more homelike and less institutional). The adaptation of these or other new models that aim to streamline and humanize long - term care, while reducing costs, should be balanced and sensitive to the health profile and needs of each kpuna or native hawaiian elder . Third, services need to reflect older adult preferences . For native hawaiian elders this may include preferences for culturally based programs and services . As all professionals will find themselves working with increasing numbers and proportions of diverse older adults with chronic disease, this becomes an increasingly important care component . Similar to all older adults, quality care for native hawaiian elders acknowledges their desire to remain in their own homes with an array of assistance from families, friends, and home and community - based services that honor and reflect their culture . Not surprisingly, quality of life is influenced by the education and training of professionals and other service workers who provide the care . Successful interventions for native hawaiian elders are predicated on practitioners having an understanding of cultural values and traditions that influence elder's health practices, with cultural preferences that may include involvement of the elder's extended family and use of health promotion approaches reflecting holistic wellness [911, 13]. Continued effort is needed to develop an affordable and culturally responsive health care system that supports acute care, as well as health education and promotion, disease prevention, early disease management and treatment, and community - based long - term care . Further, services must be offered to people across the life span, thereby offering the opportunity for parity in life expectancy and the hopeful prospect of good health to native hawaiian elders comparable to that of other older adults . The information in this paper does not reflect the option of the us administration on aging.
Insomnia is the most common sleep disorder, characterized by difficulty in falling asleep and/or maintaining sleep . It can also lead to emotional consequences, which further lead to affective disorders, such as, depression and anxiety . The prevalence of insomnia in the general population, by strictly applying the diagnostic and statistical manual of mental disorders - fourth edition (dsm - iv) criteria, is six percent, but the symptoms of insomnia are much more common, which trouble nearly one - third of the global adult population . The pharmacological treatments used for treating insomnia are mainly benzodiazepines and non - benzodiazepine drugs, such as, zolpidem and zaleplon, both acting on benzodiazepine receptors . Antihistamines, antidepressants, antipsychotics, anticonvulsants, and melatonin receptor agonists have also been used to treat insomniac patients with different comorbidities . Newer targets for treating insomnia have been persistently explored because none of the currently used drugs can be called ideal in their therapeutic role of promoting sleep throughout the night, maintaining a normal sleep architecture, and being free of any residual morning adverse after effects . One such target of interest, for developing a potential insomnia therapy has been the orexin receptor . Suvorexant, the first in a new class of drugs to treat insomnia by targeting the orexin receptors, has been approved recently, on 13 august, 2014, by the us food and drug administration (fda). It has been developed by the pharmaceutical company merck, under the brand name belsomra . Orexins, also referred to as hypocretins, are neuropeptides secreted from the lateral hypothalamus neurons . Two orexin neuropeptides, orexin - a (oxa) and orexin - b (oxb), have been identified, which act with different affinities through two g - protein coupled receptors, ox1r and ox2r . The orexin secreting neurons in the lateral hypothalamus are less than 100,000 in number, but their receptors are expressed in many areas of the brain with a suggested role in arousal, appetite, metabolism, reward, stress, and autonomic function . The projections of the orexin system are particularly extensive in the regions of the brain, which regulate various aspects of arousal and motivation such as the noradrenergic neurons of the locus coeruleus, the histaminergic neurons of the tuberomammillary nucleus, the serotonergic neurons of the raphe nuclei, and the dopaminergic neurons of the ventral tegmental area . During the initial studies on the orexin system, it was seen that loss of orexinergic neurons resulted in severe sleepiness in animals, with an inability to maintain wakefulness . Orexinergic neurons were found to be reduced significantly in the brain in humans suffering from narcolepsy, a disorder with excessive sleepiness . Once the role of orexins in maintaining normal wakefulness was established, it was followed with an exciting quest for an antagonist, which would be a novel manner in which to promote sleep and treat insomnia . Suvorexant was the first selective dual orexin receptor antagonist (dora) to be approved in recent times . It would bind reversibly with both the orexin receptors, ox1r and ox2r, and inhibit the activation of the arousal system, thus, facilitating sleep induction and maintenance . Suvorexant is described chemically as a [(7r) - 4 - (5 - chloro - 1,3 - benzoxazol - 2 - yl) - 7- methyl - 1,4- diazepan - 1 - yl] [5 - methyl- 2- (2h - 1,2,3- triazol- 2- yl) phenyl] methanone . The initial compound had a fluoroquinazoline ring, but it produced reactive metabolites in the microsomal incubations, so it was modified by replacing fluoroquinazoline with a chlorobenzoxazole moiety, and the modified compound, which was named mk- 4305, underwent the subsequent trials . It is well - absorbed orally, producing peak plasma concentrations in about two hours . It has a high plasma protein binding capacity and is mainly metabolized by the cyp3a4 system . The recommended dose is 10 mg once at night, which can be increased to a maximum of 20 mg . Suvorexant was evaluated for safety and efficacy in a randomized, double - blind, placebo - controlled polysomnography study . In one period comprising of total 243 patients, four different groups received suvorexant 10, 20, 40, and 80 mg . Polysomnography was done on the first night and at the end of the period, that is, after four weeks . Suvorexant showed significant dose - related sleep efficiency on the first night and at the end of four weeks, as compared to the placebo . In another randomized, double - blind, placebo - controlled, crossover polysomnography study, which was conducted on healthy adults, the overnight sleep parameters were recorded and the morning after the residual effects were assessed for 10, 50 or 100 mg doses of suvorexant, as compared to the placebo . Suvorexant has been found to be generally safe and well - tolerated in patients . At the recommended therapeutic dose of less than 20 mg study on healthy volunteers has shown that there is no residual effect with suvorexant 10 mg on the following day, but the higher doses significantly reduce subjective alertness and prolong the reaction time . In a phase 2, a randomized, double - blind trial on 229 patients of insomnia, change in the patient's neurophysiology on account of drugs or placebo, was assessed by electroencephalographic power spectral density . Suvorexant, at 10 mg, produced minimal neurophysiological disturbance as compared to placebo in healthy subjects and other treatments such as zolpidem . In a phase 3, randomized, double - blind, placebo - controlled trial of suvorexant for a one - year treatment of insomnia, 362 (69%) of a total of 521 patients experienced some adverse effects . Somnolence was the most common adverse event, reported for 69 (13%) patients . However, all the patients in this study had received a dose of 30 to 40 mg suvorexant, which was higher than the current approved dose of 10 mg . Doses higher than 20 mg have also been reported to be associated with rapid onset of daytime somnolence, motor impairment, driving impairment, and unconscious night time activity, such as, sleep walking, suicidal ideation, hypnagogic hallucinations, abnormal dream pattern, and effects resembling mild cataplexy . It may have a potential additive effect when used with antidepressants and other drugs with a sedative action . On account of low - abuse liability, similar to zolpidem, it has been placed in schedule iv of the controlled substances act . Did not appear to produce physical dependence and withdrawal syndrome on discontinuation after chronic therapy of one year . This may be a point in favor of suvorexant over the other traditional drugs for insomnia, which act through benzodiazepine receptors and carry the risk of physical dependence on chronic use . Suvorexant has not yet been compared to other drugs approved for insomnia, so its relative advantages in terms of efficacy or adverse effect profile, will emerge more clearly in future, after head to head comparative trials with the already available drugs . This is not the first attempt at developing and marketing an orexin receptor antagonist for insomnia in recent years . Almorexant, a dual orexin receptor antagonist was being tried as a potential blockbuster drug by the pharmaceutical companies glaxosmithkline (gsk) and actelion, but after the phase iii clinical trial in november 2009, the project was aborted citing tolerability issues, which were not disclosed . Suvorexant has been well - tolerated, without major safety issues, in all the studies so far, at the 10 mg dose . Suicidal ideation seen at higher doses in the clinical trials could be an adverse consequence of antagonism at the orexin receptors that are present in mesolimbic pathways regulating affect, reward, and motivation . Some of the other adverse effects at higher than 20 mg doses, such as, sleep walking, hypnagogic hallucinations, abnormal dreams, and cataplexy seem related to the rapid eye movement (rem) sleep interference caused by suvorexant . These adverse effects may not appear with a 10 mg dose; but larger studies are needed to confirm this . It has been suggested that there is differential contribution of the two orexin receptors in regulating sleep / wakefulness, with a more important role of ox2r antagonism in inducing sleep as compared to ox1r . Pharmacology of a more selective antagonism of either ox1r or ox2r alone has not been properly elucidated in the clinical studies because of the lack of suitable subtype - selective, orally bioavailable ligands . In preclinical studies, comparing the effects of single orexin receptor antagonist (sora) for the subtype ox2r, with the dual antagonist suvorexant, both decreased the wakefulness time with a similar efficacy in a dose - dependent manner . However, there was a slight difference in the type of effect on the sleep architecture, as the ox2r antagonist primarily increased the total non - rapid eye movement (nrem) sleep time with minimal effects on rem sleep, whereas, suvorexant increased both the total nrem and rem sleep time, with predominant effects on rem sleep . It is to be seen in further trials whether ox2r subtype - selective antagonists are better in maintaining the normal sleep architecture . Suvorexant is described chemically as a [(7r) - 4 - (5 - chloro - 1,3 - benzoxazol - 2 - yl) - 7- methyl - 1,4- diazepan - 1 - yl] [5 - methyl- 2- (2h - 1,2,3- triazol- 2- yl) phenyl] methanone . The initial compound had a fluoroquinazoline ring, but it produced reactive metabolites in the microsomal incubations, so it was modified by replacing fluoroquinazoline with a chlorobenzoxazole moiety, and the modified compound, which was named mk- 4305, underwent the subsequent trials . It is well - absorbed orally, producing peak plasma concentrations in about two hours . It has a high plasma protein binding capacity and is mainly metabolized by the cyp3a4 system . The recommended dose is 10 mg once at night, which can be increased to a maximum of 20 mg . Suvorexant was evaluated for safety and efficacy in a randomized, double - blind, placebo - controlled polysomnography study . It was a two - period (four weeks per period) crossover study . In one period comprising of total 243 patients, four different groups received suvorexant 10, 20, 40, and 80 mg . Polysomnography was done on the first night and at the end of the period, that is, after four weeks . Suvorexant showed significant dose - related sleep efficiency on the first night and at the end of four weeks, as compared to the placebo . In another randomized, double - blind, placebo - controlled, crossover polysomnography study, which was conducted on healthy adults, the overnight sleep parameters were recorded and the morning after the residual effects were assessed for 10, 50 or 100 mg doses of suvorexant, as compared to the placebo . Suvorexant has been found to be generally safe and well - tolerated in patients . At the recommended therapeutic dose of less than 20 mg study on healthy volunteers has shown that there is no residual effect with suvorexant 10 mg on the following day, but the higher doses significantly reduce subjective alertness and prolong the reaction time . In a phase 2, a randomized, double - blind trial on 229 patients of insomnia, change in the patient's neurophysiology on account of drugs or placebo, suvorexant, at 10 mg, produced minimal neurophysiological disturbance as compared to placebo in healthy subjects and other treatments such as zolpidem . In a phase 3, randomized, double - blind, placebo - controlled trial of suvorexant for a one - year treatment of insomnia, 362 (69%) of a total of 521 patients experienced some adverse effects . Somnolence was the most common adverse event, reported for 69 (13%) patients . However, all the patients in this study had received a dose of 30 to 40 mg suvorexant, which was higher than the current approved dose of 10 mg . Doses higher than 20 mg have also been reported to be associated with rapid onset of daytime somnolence, motor impairment, driving impairment, and unconscious night time activity, such as, sleep walking, suicidal ideation, hypnagogic hallucinations, abnormal dream pattern, and effects resembling mild cataplexy . It may have a potential additive effect when used with antidepressants and other drugs with a sedative action . On account of low - abuse liability, similar to zolpidem, it has been placed in schedule iv of the controlled substances act . Did not appear to produce physical dependence and withdrawal syndrome on discontinuation after chronic therapy of one year . This may be a point in favor of suvorexant over the other traditional drugs for insomnia, which act through benzodiazepine receptors and carry the risk of physical dependence on chronic use . Suvorexant has not yet been compared to other drugs approved for insomnia, so its relative advantages in terms of efficacy or adverse effect profile, will emerge more clearly in future, after head to head comparative trials with the already available drugs . This is not the first attempt at developing and marketing an orexin receptor antagonist for insomnia in recent years . Almorexant, a dual orexin receptor antagonist was being tried as a potential blockbuster drug by the pharmaceutical companies glaxosmithkline (gsk) and actelion, but after the phase iii clinical trial in november 2009, the project was aborted citing tolerability issues, which were not disclosed . Suvorexant has been well - tolerated, without major safety issues, in all the studies so far, at the 10 mg dose . Suicidal ideation seen at higher doses in the clinical trials could be an adverse consequence of antagonism at the orexin receptors that are present in mesolimbic pathways regulating affect, reward, and motivation . Some of the other adverse effects at higher than 20 mg doses, such as, sleep walking, hypnagogic hallucinations, abnormal dreams, and cataplexy seem related to the rapid eye movement (rem) sleep interference caused by suvorexant . These adverse effects may not appear with a 10 mg dose; but larger studies are needed to confirm this . It has been suggested that there is differential contribution of the two orexin receptors in regulating sleep / wakefulness, with a more important role of ox2r antagonism in inducing sleep as compared to ox1r . Pharmacology of a more selective antagonism of either ox1r or ox2r alone has not been properly elucidated in the clinical studies because of the lack of suitable subtype - selective, orally bioavailable ligands . In preclinical studies, comparing the effects of single orexin receptor antagonist (sora) for the subtype ox2r, with the dual antagonist suvorexant, both decreased the wakefulness time with a similar efficacy in a dose - dependent manner . However, there was a slight difference in the type of effect on the sleep architecture, as the ox2r antagonist primarily increased the total non - rapid eye movement (nrem) sleep time with minimal effects on rem sleep, whereas, suvorexant increased both the total nrem and rem sleep time, with predominant effects on rem sleep . It is to be seen in further trials whether ox2r subtype - selective antagonists are better in maintaining the normal sleep architecture . With the approval of suvorexant as a treatment for insomnia, a novel method to regulate the arousal pathway in the brain by orexin antagonism has been introduced . The traditional use of drugs acting on benzodiazepine receptors for treating insomnia has its own limitations, especially in the elderly, because of the associated adverse effects, such as, amnesia, confusion, and gait disturbance . It also appears to be suitable for the chronic therapy of insomnia because of minimal physical dependence and can be a good alternative in patients who have a history of substance abuse . The availability of this first drug in the class of orexin receptor antagonists, with a potential to provide more peaceful sleep and less troublesome mornings, is an important and welcome development in insomnia management.
The etiology was presumed to be tubercular if there was (a) corroborative evidence (such as a positive tuberculin skin test or quantiferon - tb gold test), (b) exclusion of all other known causes of infectious uveitis except tuberculosis and noninfectious uveitic syndromes, and (c) a favorable therapeutic response to antitubercular therapy . All patients underwent chest radiography that was normal . Besides a complete clinical evaluation that included best - corrected visual acuity (bcva), intraocular pressure (iop), slit lamp biomicroscopic examination, and conventional imaging methods (digital photography and fa, when required, on visupac 450 plus, carl zeiss, jena, germany), these patients additionally underwent spectralis hra+oct (heidelberg engineering, heidelberg, germany) imaging with simultaneously obtained faf and oct images at all follow - ups . The spectralis system uses heidelberg eye explorer software version 1.5.0 with image capture module version 1.1.0 . An infrared fundus image was acquired parallel to oct scan to ensure correct placement of image before acquiring faf images simultaneous with oct scans . The spectralis scans along with the faf images were obtained before doing fa, using the 30 field - of - view mode . The image acquisition was done by selecting the dense volume scan type over a scan angle of 20 or 30 and by adjusting the width / height of the oct scan (depending upon the extent of the lesions in the fundus). We use infrared mode to focus the fundus image, and once focused, we switch to combined faf and sd - oct mode and capture the images simultaneously . An integrated eye tracking allowed for live averaging of faf images and the sd - oct scans . The baseline faf - oct images were defined as the reference images to enable acquisition of images at the same site during follow - up visits . In addition to oral corticosteroids, all patients received four - drug antitubercular therapy including isoniazid (5 mg / kg / day), rifampicin (450 mg / day if body weight was 50 kg and 600 mg / day if body weight was> 50 kg), ethambutol (15 mg / kg / day), and pyrazinamide (25 to 30 mg / kg / day) initially for 3 to 4 months . All patients were receiving antitubercular therapy until their last visit and did not show any recurrence of inflammation . The oct scans were analyzed and correlated with faf / fa images in acute as well as healing stages . During the course of the disease in patients with slc, we observed a progressively changing pattern on sd - oct scans that was consistent with the abnormal faf signals detected simultaneously.in an acute lesion of slc, there was an ill - defined area of increased autofluorescence around the lesion . The sd - oct passing through the area showed a localized, fuzzy area of hyperreflectivity in the outer retinal layers involving the rpe, photoreceptor outer segment tips (post), photoreceptor inner segment outer segment (is / os) junction, external limiting membrane (elm), and the outer nuclear layer (onl). The lesion was localized external to the outer plexiform layer with a mild distortion of the inner retinal layers . There was no increased backscattering from the inner choroid.as the lesions started to heal, they became well defined and acquired a thin border of hypoautofluorescence while remaining predominant hyperautofluorescent centrally . The sd - oct scan through the hyperautofluorescent area showed disappearance of the hyperreflective fuzzy areas that were replaced by irregular, hyperreflective knobbly elevations of the outer retinal layers . The rpe, the post, is / os junction, and the elm could not be distinguished . Photoreceptor complex.as the lesions healed further over the next 36 months, they appeared stippled with predominantly hypoautofluorescence . The sd - oct scan showed loss of rpe, post, is / os junction, and elm . The sd - oct passing through the area showed a localized, fuzzy area of hyperreflectivity in the outer retinal layers involving the rpe, photoreceptor outer segment tips (post), photoreceptor inner segment outer segment (is / os) junction, external limiting membrane (elm), and the outer nuclear layer (onl). The lesion was localized external to the outer plexiform layer with a mild distortion of the inner retinal layers . There was no increased backscattering from the inner choroid . As the lesions started to heal, they became well defined and acquired a thin border of hypoautofluorescence while remaining predominant hyperautofluorescent centrally . The sd - oct scan through the hyperautofluorescent area showed disappearance of the hyperreflective fuzzy areas that were replaced by irregular, hyperreflective knobbly elevations of the outer retinal layers . The rpe, the post, is / os junction, and the elm could not be distinguished ., there was an increased reflectance from the choroidal layers due to attenuating rpe photoreceptor complex . As the lesions healed further over the next 36 months, they appeared stippled with predominantly hypoautofluorescence . The sd - oct scan showed loss of rpe, post, is / os junction, and elm . The clinical details and the findings on faf and sd - oct during the healing of lesions are listed in table 1 . These changes are illustrated in figs . 1 and 2.table 1clinical details of patients with active tubercular serpiginouslike choroiditis along with findings on combined fundus autofluorescence and spectralis domain optical coherence tomography imaging as the lesions evolved from an acute stage up to healed stagepatientsexageeyeinitial visual acuityas inflammationvitreous cellstype of slc lesionststquantiferon - tb gold testfaf of acute lesionsd - oct of acute lesionfaf of healing lesionsd - oct of healing lesionfaf of healed lesionsd - oct of healed lesionfollow - up (months)final visual acuity1m20rightcf 1 ftnil++placoidpositivenddiffuse, feeble hyperautofluorescentfuzzy, hyperreflective areas involving rpe, post, photoreceptor is os junction, elm and onlcentral hyperautofluorescent with hypoautofluorescent borderirregular, knobbly elevations of outer retinal layers that are indistinct . The onl appears normal.predominantly hypoautofluorescentloss of rpe, post, is os junction, and elm5cf 1 m2m19right6/9nil+multifocalnegativepositive36/9left6/6nil+multifocal6/63m35left6/9nilnilmultifocalpositivend66/6as anterior segment, slc serpiginouslike choroiditis, tst tuberculin skin test, m male, cf counting fingers, nd not done, af autofluorescence, sd - oct spectral domain optical coherence tomography, rpe retinal pigment epithelium, post photoreceptor outer segment tips, is os junction inner segment outer segment junction, elm external limiting membrane, onl outer nuclear layerfig . 1a f right eye fundus picture of patient #2 with inactive and active (arrows) lesions of serpiginouslike choroiditis (a) that appeared hypofluorescent in early (b) and hyperfluorescent in late - phase fluorescein angiogram (c). 2combined fundus autofluorescence (faf) and spectral domain optical coherence tomography (sd - oct) images through the active lesion(s). The green frame (left panel) indicates the borders of the scanned area . The position marker corresponds to the retinal location through which the displayed oct scan is obtained . In the right eye (a c), in acute stage (a), there is an ill - defined area of increased autofluorescence (left panel) with fuzzy area of hyperreflectivity in outer retinal layers (white arrows) involving the retinal pigment epithelium (rpe), photoreceptor outer segment tips (post), photoreceptor inner segment outer segment (is os) junction, external limiting membrane (elm), and outer nuclear layer (onl) (right panel). About 2 weeks later (b), as the lesions started healing, they became well defined with a thin hypoautofluorescent border and predominantly hyperautofluorescence centrally in the right eye (left panel). The sd - oct showed irregular knobby elevations (white arrows) of the outer retinal layers (right panel). The rpe, post, is os junction, and elm could not be distinguished . There was an increased reflectance from the choroidal layers (red arrows) due to disappearing rpe three months later (c), as the lesions healed further, they appeared stippled with predominantly hypoautofluorescence (left panel). The sd - oct scan showed loss of rpe, post, is os junction, and elm (white arrows) (right panel). D f similar changes were seen in the left eye in acute (d), healing (e), and healed (f) stages of active lesions of slc clinical details of patients with active tubercular serpiginouslike choroiditis along with findings on combined fundus autofluorescence and spectralis domain optical coherence tomography imaging as the lesions evolved from an acute stage up to healed stage as anterior segment, slc serpiginouslike choroiditis, tst tuberculin skin test, m male, cf counting fingers, nd not done, af autofluorescence, sd - oct spectral domain optical coherence tomography, rpe retinal pigment epithelium, post photoreceptor outer segment tips, is os junction inner segment outer segment junction, elm external limiting membrane, onl outer nuclear layer a f right eye fundus picture of patient #2 with inactive and active (arrows) lesions of serpiginouslike choroiditis (a) that appeared hypofluorescent in early (b) and hyperfluorescent in late - phase fluorescein angiogram (c). The left eye showed similar lesions (d f) combined fundus autofluorescence (faf) and spectral domain optical coherence tomography (sd - oct) images through the active lesion(s). The position marker corresponds to the retinal location through which the displayed oct scan is obtained . In the right eye (a c), in acute stage (a), there is an ill - defined area of increased autofluorescence (left panel) with fuzzy area of hyperreflectivity in outer retinal layers (white arrows) involving the retinal pigment epithelium (rpe), photoreceptor outer segment tips (post), photoreceptor inner segment outer segment (is os) junction, external limiting membrane (elm), and outer nuclear layer (onl) (right panel). About 2 weeks later (b), as the lesions started healing, they became well defined with a thin hypoautofluorescent border and predominantly hyperautofluorescence centrally in the right eye (left panel). The sd - oct showed irregular knobby elevations (white arrows) of the outer retinal layers (right panel). The rpe, post, is os junction, and elm could not be distinguished . There was an increased reflectance from the choroidal layers (red arrows) due to disappearing rpe three months later (c), as the lesions healed further, they appeared stippled with predominantly hypoautofluorescence (left panel). The sd - oct scan showed loss of rpe, post, is os junction, and elm (white arrows) (right panel). D f similar changes were seen in the left eye in acute (d), healing (e), and healed (f) stages of active lesions of slc patient 2: a 19-year - old male presented with decreased vision in both eyes since 3 months . On examination, the bcva was 6/9 and 6/6 in the right and left eyes, respectively . The iop were 14 and 12 mmhg in the right and left eyes, respectively . Both eyes showed unremarkable anterior segment and multifocal lesions of active as well as inactive choroiditis in the posterior pole (fig . Simultaneous faf and sd - oct imaging of the right eye revealed findings as explained in the results section (1 .) About 2 weeks later, the lesions started to heal and appeared as described in three months later, the lesions healed further and appeared as explained in results section (3 .) Likewise, left eye imaging also showed a similar pattern of faf and sd - oct changes during acute, healing, and healed stages of the lesions (fig . Patient 2: a 19-year - old male presented with decreased vision in both eyes since 3 months . On examination, the iop were 14 and 12 mmhg in the right and left eyes, respectively . Both eyes showed unremarkable anterior segment and multifocal lesions of active as well as inactive choroiditis in the posterior pole (fig . Simultaneous faf and sd - oct imaging of the right eye revealed findings as explained in the results section (1 .) About 2 weeks later, the lesions started to heal and appeared as described in results section (2 .) (fig . Three months later, the lesions healed further and appeared as explained in results section (3 .) Likewise, left eye imaging also showed a similar pattern of faf and sd - oct changes during acute, healing, and healed stages of the lesions (fig . Tubercular slc can be clinically differentiated from sc by the frequent presence of vitritis and multifocal choroiditis lesions in posterior pole and periphery, often sparing the juxtapapillary region . The lesions in sc are, however, usually around the optic disk and spread contiguously to the macula . People with slc are from areas endemic for tuberculosis, have positive uveitis work - up for tuberculosis, and respond favorably to antitubercular therapy with oral corticosteroids . The main histological findings described in sc are atrophy of the choriocapillaries, the rpe, and the photoreceptors [4, 7]. While the choriocapillaries was reported to be the most affected layer that appeared acellular, the large choroidal vessels were unremarkable . Moderate, diffuse lymphocytic infiltration of the choroid has been reported, with predominant rpe atrophy . Occasionally, rpe hypertrophy has been seen correlating with areas of pigment clumping clinically [4, 7]. On the other hand, however, isolation of mycobacterium tuberculosis from the rpe in an eye with tubercular panuveitis has strongly suggested preferential localization of the mycobacteria in the rpe, even in eyes with panuveitis or related intraocular inflammation, including multifocal choroiditis or serpiginouslike choroiditis . High - speed, high - resolution oct, by providing unprecedented details, has enhanced our understanding of the ultrastructure of the retina . Distinct scattering bands correspond to photoreceptor is / os junction, photoreceptor outer segment tips, and the rpe and represent the thick scattering bands of outer retina . The sd - oct changes in healed scars of slc have shown disruption of the outer retina at the site of scars with loss of junction between the inner and outer segments of photoreceptors and thinning of rpe / bruch membrane complex and a correspondent increase in light reflectivity from the choroid . Areas of thickening of rpe / bruch membrane complex have also been shown in the regions of scars . However, there is no report of sd - oct changes in any active and healing stages of slc lesions . All eyes with active lesions of slc in our patients illustrate the progressive changes in the outer retinal layers on oct scans that correlated with the faf changes . The faf images obtained simultaneously demonstrated the transition from initial hyperautofluorescent of acute lesions to predominant hypoautofluorescent in the healed stage . Absence of any demonstrable changes in the inner choroid during the active stage of the lesion on oct scans may suggest a primary involvement of the rpe and not the choroid in tubercular slc lesions . The faf signals provide a strong clue to the status of rpe cells in various degenerative, inflammatory, and neoplastic disease processes . The faf is increased (hyperautofluorescence) in the presence of increased metabolic activity of the rpe and decreased (hypoautofluorescence) when there is loss of the rpe . We observed that the structural changes on oct scans occurring during the course of slc followed a stepwise orderly sequence, similar to those as seen on the faf images . Increased autofluorescence in the acute lesions seen as diffuse, subtle, feeble hyperautofluorescent probably reflects retinal edema which was structurally evident as hyperreflectivity spreading into the outer retinal layers in the oct scans of our patients . This possibly suggests cellular infiltration or extracellular fluid accumulation in these layers due to inflammation . As the lesions started healing, hyperautofluorescence decreased and hypoautofluorescence increased due to loss of rpe . The outer retinal layers on oct scans of our patients showed attenuation and progressive loss in the affected areas as the lesions healed . This was seen as an irreversible, collective loss of the outer retinal layers involving the rpe, photoreceptor outer and inner segments, and the elm in oct scans . Acute inflammatory lesions involving the rpe often cause a thickening at the level of rpe . It is believed that the lesions in sc arise deep in the retina, and the overlying retina appears edematous . The edema subsides as the lesions heal, and the rpe choriocapillaries undergo atrophy . Yeh et al . Have hypothesized that rpe may be the site of primary insult and hence, more severely damaged in presumed tuberculosis - associated serpiginouslike choroidopathy . The size, number, or content of the fluorophores in the rpe cells may be altered by inflammation which increases the fluorophores content by inducing certain prooxidative pathways . Increased autofluorescence in other inflammatory conditions also such as white dot syndromes has been correlated with areas of rpe elevation on oct image during active disease . The faf abnormalities (hyperautofluorescent in active stage progressively becoming hypoautofluorescent in healed stage) have been well recognized (unpublished data). We observed that in the very initial stages of disease occurrence, when there was feeble hyperautofluorescence in the areas of new lesions, the oct showed hyperreflectivity in the outer retinal layers that was fuzzy and ill defined . This is an important oct finding during the acute stage of slc that may reflect the site of primary insult in slc . However, sd - oct technology is not yet able to image the choroid similarly to the retina, and hence, the absence of choroid changes on sd - oct is not enough to definitely exclude its primary involvement in slc . Hyperreflectivity in the outer retinal layers in an active sc lesion is believed to be suggestive of acute inflammation involving deeper retinal and choroidal structures . From the oct findings of our patients, we speculate that in an acute lesion of slc, there is an increased metabolic activity caused by primary inflammation of the rpe cells . Release of inflammatory mediators into the retinal layers adjacent to the rpe causes a fuzzy, hyperreflective appearance on sd - oct . Following an acute inflammatory episode, the rpe cells undergo hyperplasia and hypertrophy which is evident as hyperautofluorescence on faf due to increased collection of lipofuscin . This corresponds to the localized, knobbly elevations of the outer retinal layers which represents clumping of the inflamed rpe cells . Once these damaged rpe cells undergo atrophy, there is an irreversible loss of photoreceptors giving rise to the loss of the outer retinal layers on oct . The late pigmentation of the retinal scar associated with rpe hypertrophy or hyperplasia also leads to a decreased faf signal (especially when photoreceptors are disrupted), as can also be seen in figs . 1 and 2 . The baring of choroidal vessels in healed lesions of sc in contrast to their masking by hyperpigmentary changes of the rpe in slc may also be due to entirely different entities affecting the inner choroid and the rpe cells, respectively . The limitations of our study include a small number of cases and lack of clinicopathologic correlation . The outer retinal bands on sd - oct, particularly is / os junction and post, have not been so far correlated to the histological structures, and such correlation is still mostly presumed . However, the sequential ultrastructural changes in the outer retinal morphology on sd - oct scans as seen in our patients provide important information that may add a new dimension in understanding the primary site of pathology in inflammatory conditions affecting the choroid and the rpe photoreceptor complex.
Lipopolysaccharide (lps) is a major component of the cell wall of gram - negative bacteria and is a well - known potent inducer of inflammation and inflammatory bone loss [15]. Lps is known to induce the production of many local factors, including proinflammatory cytokines, such as tnf- and il-1, from macrophages or other cells involved in mediating the inflammatory response in tissues . There is reason to suggest that osteoclast recruitment could be central to diseases involving bone erosion, such as rheumatoid arthritis, periprosthetic bone loss, postmenopausal osteoporosis, and periodontal disease . Such osteoclast formation and activation require the expression of two factors: receptor activator of nf-b ligand (rankl) and macrophage colony stimulating factor (m - csf). Furthermore, tumor necrosis factor- (tnf-) has also been reported to induce osteoclast formation in vitro [1214] and in vivo [15, 16]. These inflammatory cytokines have been linked with lps - induced osteoclast formation and bone destruction in vivo and in vitro [2, 1720]. Muramyl dipeptide (mdp), the minimal essential structural unit responsible for the immunological activity of pgns, is distributed ubiquitously in the cell walls of both gram - negative and gram - positive bacteria . It has been reported that mdp can enhance the production of tnf- when injected into mice and can cause lethal shock in mice challenged with lps . In addition, mdp has been shown to synergistically enhance lps - induced proinflammatory cytokine production in human monocyte cells . Mdp alone cannot induce osteoclast formation in mouse cocultures of primary osteoblasts and hematopoietic cells; however, it can enhance osteoclast formation induced by lps, il-1, and tnf- but not by 1,25-dihydroxy - vitamin - d3 (1,25(oh)2d3) or prostaglandin - e2 (pge2). Indeed, it has been shown that mdp can upregulate rankl expression in osteoblasts treated with lps or tnf- but not those treated with 1,25(oh)2d3 . In this study, we show that mdp enhances lps - induced osteoclast formation in vivo and increases the expression of rankl in vivo and in stromal cell cultures in vitro . Mdp also enhances the lps - induced expression of tlr4a signal transducing receptor for lps both in vivo and in stromal cells in vitro . Two- to 10-week - old male c57bl6/j mice were purchased from clea japan (tokyo, japan) for use in this study . Escherichia coli lps was purchased from sigma - aldrich (st . Louis, mo). (beverly, ma): polyclonal anti - phospho - p44/42erk, anti - phospho - jnk, anti - phospho - p38, anti--actin, and anti - rabbit igg horseradish peroxidase- (hrp-) linked antibodies . Mice calvariae were injected daily for 5 days with pbs, lps alone (10 g / day or 100 g / day, referred to as low or high, resp . ), mdp (100 g / day) alone, or lps (10 g / day) and mdp (100 g / day) (lps + mdp). The mice were then sacrificed, and the calvariae were immediately harvested and fixed overnight in 4% paraformaldehyde at 4c . Samples were then demineralized in 14% ethylene - diaminetetraacetic acid for 3 days at 4c . The sections were stained for trap activity and counterstained with hematoxylin for analysis of osteoclast formation . In addition, the percentage of interface of bone marrow space covered by osteoclasts was histomorphometrically determined in specimens derived from each sample . Serum was obtained from mice after 5 days of daily lps administration with or without mdp . The levels of tracp 5b were determined using a mouse trap assay kit (ids, tyne and wear, uk). Tracp 5b levels were measured at 405 nm using an absorption microplate reader (model 550; bio - rad, richmond, ca). The levels of c - terminal telopeptide fragments of type i collagen were determined using a mouse ctx assay kit (ids, tyne and wear, uk). C - terminal telopeptide fragments of type i collagen levels were measured at 450 nm using an absorption microplate reader (model 550; bio - rad, richmond, ca). For in vitro experiments, bone marrow cells from the femora and tibiae of mice were flushed with culture medium . The harvested cells were incubated in dulbecco's modified eagle's medium (dmem; sigma - aldrich) containing 10% fetal bovine serum, 100 iu / ml penicillin g (life technologies, carlsbad, ca), and 100 g / ml streptomycin (life technologies). After 2 weeks of culture, cells were washed with pbs to remove floating cells . Adherent cells from these cultures were used as bone marrow stromal cells in this study . Adherent bone marrow stromal cells were incubated in culture medium supplemented with high or low lps alone, lps + mdp, or mdp alone . After 3 days of culture, total rna was isolated from adherent cells using an rneasy mini kit (qiagen, valencia, ca). For in vivo experiments, harvested calvariae were frozen in liquid nitrogen, ground using a micro smash ms-100r (tomy seiko, tokyo, japan), and then centrifuged in 800 l of trizol reagent (invitrogen, carlsbad, ca). All cdna was synthesized from 2 g of total rna using reverse transcriptase and oligo - dt primers (invitrogen) in a reaction volume of 20 l . The mrna expression levels of trap, cathepsin k, rankl, and tlr4 were quantified by real - time rt - pcr using a thermal cycler dice real time system (takara, shiga, japan). Reactions were performed in a 25 l volume containing 2 l of cdna, 12.5 l of sybr premix ex taq (takara), and 25 pmol/l primers . The cycling conditions were as follows: 95c for 10 s for initial denaturation followed by 45 cycles of amplification, with each cycle consisting of a denaturation step at 95c for 5 s and an annealing step at 60c for 30 s. gene expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (gapdh) mrna . The following primers were used: for gapdh, 5-ggtggagccaaaagggtca-3 and 5-gggggctaagcagttg - gt-3; cathepsin k, 5-gcagaggttgtactatga-3 and 5-gcaggcgttgttcttatt-3; trap, 5-aacttgcgaccattgtta-3 and 5-ggggacctttcgttgatgt-3; rankl, 5-cctgaggccagccattt-3 and 5-cttggcccagcct-3; and tlr4, 5-cactgttcttctcctgcctgac-3 and 5-tggttgaagaaggaatgtcatc-3. Calvariae were harvested and the soft tissues were carefully removed . Calvariae were then fixed in pbs - buffered formaldehyde (4%) for 3 days at 4c and then washed with pbs for radiological analysis . Microfocus computed tomography (scanxmate - e090; comscan, kanagawa, japan) was used to assay the bone resorption pits in the calvariae, and tri/3d - bon64 software (ratoc system engineering, tokyo, japan) was used to build three - dimensional reconstruction images of the calvariae . The ratio of bone destruction to total area was calculated using imagej (nih, bethesda, md). Stromal cells were cultured in serum - free dmem for 3 h before treatment with lps and/or mdp for the various durations, as indicated . Treated cells were washed twice with ice - cold pbs and then lysed in lysis buffer (cell signaling technology) containing a protease inhibitor mixture . Cell lysates (30 g) were boiled in the presence of lithium dodecyl sulfate sample buffer (life technologies) for 5 min and subjected to sds polyacrylamide gel electrophoresis using 415% mini - protean tgx gels (bio - rad, hercules, ca). Proteins were transferred to nitrocellulose membranes using trans - blot turbo (bio - rad) and incubated in blocking solution (5% bovine serum albumin in tris - buffered saline containing 0.05% tween-20) for 1 h to reduce nonspecific binding . Membranes were then exposed to primary antibodies for 1 h at 4c, washed four times, and then incubated with anti - rabbit igg hrp - conjugated secondary antibody for 30 min . Membranes were again washed extensively and then incubated with enhanced chemiluminescence detection using supersignal west femto maximum sensitivity substrate (thermo fisher scientific, wilmington, de). Lps was administered with or without mdp into the supracalvariae of mice to analyze the effect of mdp on lps - induced osteoclastogenesis in vivo . In the high lps (100 g / day) group and the lps + mdp group, numerous osteoclasts were observed . In comparison, significantly fewer osteoclasts were observed in the low lps (10 g / day), mdp alone, or pbs groups (figures 1(a), 1(b), 1(c), and 1(d)). Real - time rt - pcr was undertaken to analyze cathepsin k and trap mrna levels two markers of osteoclasts . We found that both cathepsin k and trap mrna were significantly higher in the lps + mdp group and the high lps group as compared with the low lps group (figure 1(e)). To further analyze the effect of mdp on lps - induced osteoclast formation in vivo, lps (10 g / day) was injected into mouse calvariae with increasing concentrations of mdp (0, 1, 10, and 100 g). We found that higher mdp concentrations led to an increase in osteoclast number in a dose - dependent manner (figure 2). We next used microfocus computed tomography to assess the degree of bone destruction observed in the calvariae of mice administered with lps (figure 3(a)). We found significantly more bone destruction in the high lps group as compared with the pbs group . In addition, bone destruction in the lps + mdp group was higher than that in the low lps group (figure 3(b)). This increased bone destruction was corroborated by the tracp 5b serum analysis, where we found that tracp 5b was increased in the high lps group as compared with that in the pbs, low lps, and mdp only groups . Moreover, tracp 5b serum levels were higher in the lps + mdp group than in the pbs, low lps, and mdp only groups (figure 3(c)). C - terminal telopeptide fragments of type i collagen serum levels were also higher in the lps + mdp group than in the pbs, low lps, and mdp only groups (figure 3(d)). We found that rankl mrna was elevated in the high lps and lps + mdp groups as compared with pbs, low lps, and mdp alone groups . Mdp was thus able to enhance lps - induced rankl expression in vivo (figure 4). Bone marrow stromal cells were cultured for 3 days in the presence of lps with or without mdp to ascertain the effect of these two additives on rankl expression in stromal cell cultures in vitro . We found elevated rankl mrna expression in the high lps group as compared with the pbs, low lps, and mdp alone groups . Similarly, rankl mrna was significantly higher in the lps + mdp group as compared with the pbs and low lps groups (figure 5). Pth (100 g / day) was administered with or without mdp into mouse supracalvaria to analyze the effect of mdp on pth - induced osteoclastogenesis in vivo . We observed numerous osteoclasts with the higher concentration of pth (10 g / day), which was significantly diminished in mice treated with low - dose pth (1 g / day), pth (1 g / day) + mdp, mdp alone, or pbs (figures 6(a), 6(b), 6(c), and 6(d)). Cathepsin k and trap mrna levels were significantly increased in the high pth group as compared with the pth + mdp, pbs, low pth, and mdp alone groups (figure 6(e)). Mice calvariae were injected daily for 5 days with pth (1 g) + mdp (100 g) in a 100 l volume of pbs or separately with high pth (10 g), low pth (1 g), mdp (100 g), or pbs alone to ascertain the effect of these compounds on rankl . We found that rankl mrna was higher in the high pth group than in the pth + mdp, pbs, low pth, or mdp alone groups (figure 6(f)). We next determined the effect of mdp on lps- and pth - induced tlr4 expression, a receptor for lps . We found that tlr4 mrna expression levels were higher in the high lps and lps + mdp groups than in the pbs, low lps, and mdp alone groups . On the other hand, pth did not induce tlr4 mrna and mdp did not enhance tlr4 mrna in the presence of pth (figure 7). Bone marrow stromal cells were cultured for 3 days in lps or pth with or without mdp . In these cultures, we show that tlr4 mrna with high lps (100 ng / ml) was higher than that in the pbs, low lps (10 ng / ml), or mdp alone groups . In addition, tlr4 mrna expression in the lps (10 ng / ml) + mdp group was significantly higher than that in the pbs and low lps (10 ng / ml) groups . As seen in the in vivo analysis, pth was also unable to induce tlr4 mrna in stromal cells and this could not be recovered with the coadministration of mdp (figure 8). Finally, we sought to explore the molecular mechanisms through which mdp enhances lps - activated signaling . We showed that lps activated erk, p38, and jnk in mouse bone marrow stromal cells after 15 min incubation . Mdp alone was unable to induce phosphorylation of any of the kinases; however, mdp enhanced lps - induced phosphorylation of all three kinases after just 15 min of incubation (figure 9). In this study, we evaluated the effect of mdp in lps - induced osteoclast formation and bone resorption in vivo . To our knowledge, this is the first time that this analysis has been reported . We found that mdp enhances lps - induced osteoclast formation and bone resorption and also enhances lps - induced rankl and tlr4 expression in vivo and in stromal cell in vitro . Furthermore, mdp enhanced lps - induced phosphorylation of erk, p38, and jnk kinases in stromal cells, although mdp alone could not induce their activity . It has been reported that lps can induce osteoclast formation and bone resorption in certain clinical conditions, such as periodontal diseases [2, 25]. We have previously shown that osteoclasts can be induced in calvariae and in periodontal membrane tissues in the presence of lps . Yang et al . Showed that mdp enhances lps - induced osteoclast formation when cocultured with osteoblasts in vitro . In the present study, we evaluated whether mdp could enhance lps - induced osteoclast formation and bone resorption in vivo . We found that a daily injection of 100 g / day for 5 days was sufficient to induce osteoclasts in vivo, but not with injections of 10 g / day for 5 days . Next, to analyze the effect of mdp on lps - induced osteoclastogenesis in vivo, the lower concentration of lps was administered with or without mdp into mouse supracalvaria . We found increased numbers of osteoclasts and an elevated expression of osteoclast markers (cathepsin k and trap) with high lps (100 g / day) and with low lps (10 g / day) plus mdp but not with low lps (10 g / day) or mdp alone or with the vehicle, pbs . Serum tracp 5b levels with lps (10 g) plus mdp were higher than that in the lps only group . Investigated the effect of pgn on lps - induced osteoclast formation and bone resorption and found that pgn significantly induced osteoclast formation and bone resorption in mice coinjected with lps . Thus, it is likely that mdp might be the key component in lps - induced osteoclast formation and bone resorption as mediated by pgn . Lps has also been reported to stimulate osteoblast production / secretion of rankl . In the present study, we, too, found elevated rankl mrna levels in the high - dose lps group as compared with the control groups both in vivo and in vitro, indicating that lps induced rankl expression in stromal cells . Yang et al . Also examined osteoblasts cultured in the presence of lps with or without mdp . However, we showed that mdp alone could not induce rankl expression either in vitro or in vivo, suggesting that mdp enhances the effect of lps . We also evaluated whether mdp could enhance pth - induced osteoclast formation and bone resorption . In the present study, the results suggested that although mdp affects lps - induced signaling it cannot affect pth - induced signaling . Tlr4 induces the natural host defense system by rapidly triggering proinflammatory processes [3638]. Lps is recognized by tlr4 on the cell surface [39, 40]. In this study, we found that lps enhances tlr4 expression in mouse calvariae and in stromal cell culture . Furthermore, we found that mdp could enhance lps - induced tlr4 expression in vivo and in stromal cells . These results provide further support for the premise that mdp enhances lps signaling, and its signaling through tlr4 may be how mdp enhances the effects of lps . Cyclooxygenase- (cox-) 2 and pge2 are reportedly increased in dental pulp fibroblasts by costimulation with nod1 or nod2 ligands and tlr2 or tlr4 ligands . Furthermore, the production of il-1, il-6, and il-8 in these fibroblasts is accelerated by costimulation with these ligand combinations through the increased expression of traf6 . It has been reported that mdp synergistically enhances osteoclast induction by lps, il-1, and tnf- through increased rankl expression in osteoblasts . Have also shown that lps activates the phosphorylation of erk, p38, and jnk in osteoblasts . We corroborated these results, showing that lps activates all three kinases in mouse bone marrow stromal cells . . Also showed that lps stimulated erk1/2 phosphorylation in osteoblasts and that this could be enhanced by mdp . However, they did not check the effect of lps on other mapks, such as p38 and jnk . We found that mdp enhanced the phosphorylation of erk, p38, and jnk that was induced by lps in stromal cells . Although these results provide some insight into the signaling pathways activated by lps, the exact mechanism by which mdp enhances lps signaling is unclear, and further studies are needed to clarify this point . We found that mdp enhances lps - induced osteoclast formation, as measured by increased rankl and tlr4 expression in vivo and in vitro . Our findings suggest that mdp might play an important role in pathological bone resorption in diseases with associated bacterial infections, such as periodontitis.
Ambulatory peritoneal dialysis is the preferred form of dialysis in countries where the facility of maintenance hemodialysis is limited . Infections in the form of peritonitis, exit site infections and tunnel infections are the most common problems with peritoneal dialysis . Although gram - positive bacilli are the most common cause of peritonitis, they can also be caused by gram - negative bacilli, while fungi accounts for about 5 - 10% cases of peritonitis . Peritonitis secondary to zygomycosis has been reported only as case reports and till date about 16 cases have been reported in the world literature . We report a case of peritonitis secondary to zygomycetes infection, which was managed successfully with the help of surgery and amphotericin b. earlier, two cases of zygomycetes associated peritoneal dialysis infection have been reported from india; however, both of them succumbed, making our case probably the first from india to have survived this infection . A 46-year - old male with end stage renal disease secondary to iga nephropathy had been started on continuous ambulatory peritoneal dialysis (capd) 6 years back . He was using the twin bag system (baxter healthcare, mcgaw park, illinois, usa), and was doing 4 exchanges / day . 3 years after starting on capd, he developed an episode of bacterial peritonitis, which was managed successfully with antibiotics . Currently, he presented with 3 days history of severe abdominal pain with high grade fever and turbid effluent . Meropenem and vancomycin which he received for 48 h before presenting to us . At presentation his capd fluid analysis showed 1050 cells with 75% polymorphs while the fluid culture was sterile . Antibiotics with no response . Repeat capd effluent bacterial and fungal cultures, acid fast staining and mycobacterial culture (collected later) were sterile . After 72 h, due to non - response, his capd catheter was removed and he was continued on i.v . Antibiotics . Despite these measures, he continued to have high grade fever . At the end of 1 week, a usg guided ascitic tap drained thick pus, following which a pigtail catheter was inserted in the largest of loculi, which failed to drain the collection . Due to persistence of fever and loculated pus collections, he underwent open laparotomy . Intra - operatively, multiple loculated fluid collections with dense adhesions between bowel loops and peritoneum were noted [figure 1a]. The peritoneal and omental biopsy taken intra - operatively revealed large areas of necrosis along with broad aseptate fungal hyphae consistent with zygomycosis [figure 1b]. He received a total of 3 g amphotericin b and was doing fine at 6 months follow - up . (a) intra - operative photograph showing dense adhesions between bowel loops and peritoneum . Fungal peritonitis, accounts for about 5 - 10% of capd peritonitis across different series . About 80 - 90% of the episodes are caused by candida species, while among the filamentous fungi, aspergillus is the most common . Fungal peritonitis poses a real problem to the nephrologists and mycologists with its relatively high mortality rate (5 - 15%) and significantly high permanent capd discontinuation rate (about 40% patients shifted to hemodialysis). Infection by zygomycetes group is extremely rare and till date about 16 cases have been described [table 1]. Risk factors include previous antibiotic use, uncontrolled diabetes mellitus and desferrioxamine therapy . Since our patient was on i.v . Antibiotics for about 1 week only, we do nt think that this may be a predisposing factor for development of fungal peritionitis in our case . Due to the same reason, our patient did not receive any antifungal prophylaxis . Clinical profile and outcome of published cases of zygomycetes peritonitis the clinical presentation of these patients is not different from an episode of usual bacterial peritonitis with pain abdomen, fever and cloudy effluent, however, these patients fails to improve after adequate empirical antibiotic therapy for suspected bacterial peritonitis . The diagnosis is most cases was made by culturing the organism from the capd effluent, however, only in few cases a tissue invasion was documented . In our case, direct tissue invasion was documented and the large amount of tissue necrosis noted was consistent with the capacity for vascular invasion of this fungus . In all the other previous cases, amphotericin b, either conventional or liposomal was used as an initial treatment with duration of therapy of 12 weeks [table 1]. There is now some evidence that in amphotericin resistant cases, posaconazole is an effective drug and at least 60% of the amphotericin resistant cases can be salvaged by posaconazole therapy . Whatever be the antifungal therapy, the role of source removal in the successful management of these patients cannot be overemphasized . In our case also, quick removal of the capd catheter as well as laparotomy and drainage of the intra - abdominal abscesses were helpful in the successful outcome . The outcome of zygomycetes peritonitis is very dismal, with 11 of the 16 cases succumbing to the infection, while none of the surviving patient was able to go back on peritoneal dialysis . Our case is probably first from india documenting a successful outcome with the help of surgery and conventional amphotericin b. thus, a high index of suspicion in non - resolving peritonitis is required for diagnosing zygomyceteal peritonitis . If culture negative peritonitis gets complicated, we may need to suspect other unusual causes of peritonitis like an invasive fungal infection or mycobacterial infection . Early documentation of fungi with the help of peritoneal dialysis effluent culture or peritoneal biopsy, rapid institution of antifungal therapy and a low threshold for surgery are key parameters for a successful outcome of these patients.
Hepatitis b virus (hbv) and hepatitis c virus (hcv) infections are still among the important health issues, in turkey . However, human immunodeficiency virus (hiv) infections are less frequently observed, in the country . Although the modes of transmission are through parenteral, horizontal and vertical routes, while its incidence may vary between different countries and regions . The individuals who had blood transfusions, patients undergoing hemodialysis, intravenous drug addicted individuals, people who had tattoos / piercings, communal living environments, contamination of a family member and prisoners are the main risk groups (1) it is known that 400 million people worldwide are infected with hbv and approximately 175 million people are infected with hcv . These infections become chronic and cause hepatic failure, cirrhosis and hepatocellular cancer (1, 2). According to a 2014 report of the world health organization (who), 35 million people in the world are infected with hiv and approximately 39 million people have died because of this disease since it was defined in 1981 . According to the data stated in june 2014 by turkish ministry of health, it is thought that this number does not reflect the reality since there is insufficient admissions to health institutions of sexually transmitted infections and inaccurate recording (3). Determination of hepatitis infections and implementing preventive measures in communal living environments, such as prisons, is of high importance . Furthermore, the high rates of drug addiction among prison inmates make this issue more challenging . Although there are several worldwide studies dealing with this risky group, few studies are conducted in turkey . The current study aimed to discuss the prevalence and genotypes of hepatitis, and prevalence of hiv among the prison inmates regarding the literature . The study was approved in the ethics committee of kahramanmaras sutcu imam university medical faculty, kahramanmaras, turkey (date: 02.06.2014, number: 07) and conducted with the permission of the public prosecutor of kahramanmaras (date: 29.08.2014, no . Two - hundred and sixty - six cases sentenced in kahramanmaras prison due to certain crimes such as robbery, sexual assault, assault, substance abuse or selling were recruited for the study . These cases were evaluated in polyclinics of necip fazil city hospital from may 2014 to may 2015 . The age, gender and risk factors were considered, and hbsag, anti - hbs, hbeag, anti - hbe, anti - hbcigg, anti - hcv and anti - hiv levels were examined . Medical record files issued by infectious diseases department of necip fazil hospital, a clinic providing healthcare service for prisoners, for the individuals were investigated . The relationship between diagnosis and individuals characteristics was evaluated using pearson s chi - square test with yates continuity correction and fisher s exact test . Data were expressed as the mean sd and the distribution of frequencies as percentage . Out of 266 subjects participated in the study, 89.5% (n = 238) were male and 10.5% (n = 28) were female with a mean age of 31.21 8.99 years (min = 18, max = 63). Regarding the examined risk factors, 72.6% (n = 193) of the subjects were free from risk factors; while, 27.4% (n = 73) showed risk factors . Out of 73 subjects with the risk factors, 20.3% had intravenous substance use, 3.8% had a history of operation / transfusion and 1.9% had a history of indentation; while, 1.5% of subjects had previous unprotected sexual contact . When the subjects were examined according to the presence of hepatitis b, hbsag positivity was detected in 2.6% (n = 7) and hbv dna level in only two subjects was above 2,000 iu / ml . No subject had received chronic hepatitis b treatment . The ratio of anti - hbs positive subjects was 35.0% (n = 93) and 43% (n = 40) of them had been immunized by vaccination (57% were anti - hbs and anti - hbc igg positive), isolated anti - hbc igg positivity was detected in only one subject . Anti - hcv was positive in 17.7% (n = 47) of the kahramanmaras prison inmates and genotypes 3 and 1 were 68.1% (n = 32) and 2.1% (n = 1), respectively . The ratio of anti - hcv positive and hcv rna negative individuals who had not received treatment was 29.8% (n = 14). It is not clear whether these 14 subjects demonstrated the presence of infection or it was false anti - hcv positivity . While there was a history of intravenous substance use in eight subjects and a history of dental treatment in one, however, no risks were found in five subjects . The ratio of anti - hcv positive and hcv rna negative individuals who had received treatment constituted 3.8% (n = 10) of the study population . When the prisons were examined in general; the prison inmates had risky behavioral patterns, violent tendencies, deviant sexual behavior and substance abuse; and it was found that the cleaning and hygiene procedures were not properly followed in the environments where such crowded groups live together with continued risky behaviors in prisons (4). Continued substance abuse in prisons by most of the drug addicts, common use of injection materials, tattoos and other circumstances that result in blood contact increase the risk of infections (2). It is reported that 60% - 80% of the individuals in prisons previously had an hbv infection and only 5% - 10% of them carry the disease; however, the relation of hbv infections to substance use could not be fully demonstrated (5 - 7). It is stated that hcv transmission is observed among intravenous drug addicts, especially in industrially developed countries (8 - 11). Hcv seroprevalence is 30% among the prison inmates and in a meta - analysis including 30 studies, a relation was found between hcv infection and intravenous substance use (8, 12, 13). The subjects included in the current study were primarily young (mean age 31 years) and male . The higher ratio of males when compared to females was due to the fact that the number of female prisoners was less than the male ones . When the risk factors were examined, there was a history of intravenous substance use in 20.3% of the prisoners included in the study . In the previously conducted studies, it was reported that hcv transmission was a result of intravenous substance use (8, 12, 13). In the studies on prisoners with a history of intravenous substance use, the ratio of hbsag positivity in the current study was 2.6%, which was lower compared to the related literature . A study from iran reported that 12.37% of the individuals in the specific groups were immunized (isolated hbsab positive) by vaccination (15). Therefore, it is demonstrated that the rate of immunization among the individuals who demonstrate risky behaviors is low and vaccination is necessary to protect public health . According to a report of who in 2013 the reported stated that this ratio was 3.6% (3.2% - 4.1%) in north africa / middle east region, in which turkey is located (18). In a study, dealing with general european population, by esteban et al . The prevalence of hcv was reported 0.5% in western europe, 2.5% in southern europe and 6% in eastern europe (19). In a meta - analysis conducted by vescio the prevalence of hcv was 30% - 40% (2% - 58%) among the prisoners (8). According to the literature, the rate of hcv infection among prisoners was higher compared to the general population . In turkey, the rate of anti - hcv positivity among the general population varies from 0.1% to 0.9% (20, 21). It is thought that the data do not reflect the worldwide populations as the studies are conducted in relatively limited regions . Thus, authors believe that the current study could represent the statistics of turkey . Worldwide examination of the patients with hcv based on genotype revealed that the most common subtypes were genotypes 1 and 3; however, it was found that genotype 3 was particularly higher among prisoners (22 - 27). Anti - hcv was positive in 17.7% of kahramanmaras prison inmates and positivity rates of genotypes 3 and 1 were 68.1% and 2.1%, respectively . The ratio of anti - hcv positive, hcv rna negative subjects, who had never received treatment, was 29.8% . The study by roman et al . Which included patients and prisoners who had been hospitalized for treatment (28.4%), found that 53.4% of hcv positive cases among the general population were genotype 1 and in prisoners, 46.5% of the hcv positive cases were genotype 3 (28). When the studies on prisoners were examined, an hcv genotype 3 coupling was 13.4% in germany (29); 22% - 63% in france (30, 31) and 29.3% in the netherlands (32). Many studies have revealed that the most important risk factor among the individuals with hcv genotype 3 was intravenous substance use (33 - 35). In the studies previously carried out in various states of usa, hiv positivity was 0.7% - 7.0% among the prisoners and this ratio was 0.4% in australia (2, 36). In the current study, hiv positivity was not detected . In general, the disease has an asymptomatic duration of approximately 8 - 10 years if the virus is transmitted through unprotected sexual contact or substance use . A failed registry system that does not work properly and the patients do not apply to a health center because of fear of being stigmatized make the diagnosis, treatment and follow up of the disease more difficult . In conclusion, the current study provides an approximate hcv prevalence and genotypic distribution among the prisoners in turkey . Similar to european countries, intravenous drug use is a significant risk factor in hcv transmission . It is important to prevent intravenous drug user (ivdu) or develop preventive measures for hcv transmission among the individuals in prisons . It is essential to consider this situation both to develop preventive public health policies for human health and effective use of economic resources and also plan the diagnosis and early treatment . It is necessary to conduct large scaled studies to determine hcv prevalence and substance abuse in turkey . In conclusion, the current study provides an approximate hcv prevalence and genotypic distribution among the prisoners in turkey . Similar to european countries, it is important to prevent intravenous drug user (ivdu) or develop preventive measures for hcv transmission among the individuals in prisons . It is essential to consider this situation both to develop preventive public health policies for human health and effective use of economic resources and also plan the diagnosis and early treatment . It is necessary to conduct large scaled studies to determine hcv prevalence and substance abuse in turkey.
Traditional culture techniques and the use of agar plates remain important tools in the identification of bacterial infections and are still in common use within the field of diagnostic microbiology . In other areas of the clinical laboratory, a combination of diagnostically effective screening tests and automation has led to improvements in throughput, precision, data handling, and patient safety (1, 2). These technologies are now assisting laboratories in meeting the many challenges being faced by diagnostic facilities around the world . While automated specimen, plate handling, and plate imaging devices are now being introduced into the microbiology laboratory (3, 4), the reporting of cultures has remained a predominantly manual process . This contrasts with technologies now routinely applied within diagnostic devices in the fields of cytopathology and hematology (5, 6) where some prescreening using image analysis technologies has been introduced to streamline workflow and produce efficiencies . South australia) is an image analysis device dedicated to screening agar plates for growth . It is able to detect colonies, enumerate the various colony types present, and, with use of an interpretive algorithm, apply standard rules to assign each plate and case into convenient categories for further processing . As infections of the urinary tract are common (7) and contribute to a significant proportion of the effort and resource use within clinical microbiology laboratories (8, 9, 10, 11, 12), the performance of apas was assessed against the traditional manual reading method for culture plates inoculated with urine . Clean catch and catheter urine samples submitted for routine cultures to a clinical laboratory (healthscope pathology, wayville, south australia) over a 4-week period were included in the study . Referrals were sourced from community clinics and hospitals with a variety of age groups and clinical presentations represented . Specimens were collected in compliance with the laboratory's protocols, stored under laboratory controlled conditions, and tested on the day of receipt . The apas system used in this study consisted of a plate - handling mechanism, a lighting module, a digital camera, and analytical software . A high - quality monitor linked to the system the device estimated the total colony numbers and converted these to a cfu per milliliter value appropriate to the 1-l inoculum used in this study . All colonies detected by the device were differentiated into one of the morphology groups listed in table 1 . Differentiation was based on the physical characteristics and color reactions associated with the agar formula, with the groupings designed as a guide for interpretation and not as a replacement for formal identification procedures . Colony identification performance by apas compared with that of a reference panel additional information such as operator alerts for critical specimens and positive complementary tests can be used by the device for consideration within the decision algorithm . In this study, urine leukocyte counts performed by the laboratory were uploaded to apas from the laboratory's information system and used to screen for cases of sterile pyuria . The image analysis and leukocyte counts were collated by the device, and an interpretive algorithm was applied using rules based on published guidelines (13, 14). Cases were then segmented by the device into one of three groups: positive for plates requiring further work, negative for plates with a low probability of requiring further work, and review where an on - screen assessment by a microbiologist was required before a workflow or reporting decision was made . All results were presented for on - screen review and electronically transferred to a database . Each report included the enumeration of growth and a summary of the colony morphologies present . Urine cultures were prepared by inoculating 1 l of well - mixed urine onto standard 90-mm plates of trypticase soy agar with sheep blood and macconkey agar with crystal violet (remel, lenexa, ks, usa). A reference panel of three experienced microbiologists independently and outside the routine workflow recorded the amount of growth and the colony morphologies found on the plates . The growth enumeration was estimated for the 1-l inoculum and recorded in cfu per milliliter, while the colony morphologies present on each plate were reported as described in table 1 . The urine leukocyte counts obtained by the laboratory's standard method were electronically transferred to the device for use within the decision algorithm . A pyuria flag, defined as 50 10 leukocytes / ml, was used by the device to indicate possible cases of sterile pyuria . These cases were then reported by apas as requiring further review by a microbiologist if growth was not detected . Results generated by the laboratory from the routine workflow for the urine samples enrolled in the study were filed for comparison with the apas results ., brisbane, australia) analyzed the results by comparing the apas findings with the consensus results from the reference panel . Where differences in individual panel member reports were present, a majority finding was used . Performance values for growth detection, enumeration, and the differentiation of colony morphologies using sensitivity and specificity calculations were obtained . Sensitivity was defined as the number of true positives divided by the number of true positives plus false negatives expressed as a percentage, and specificity was the number of true negatives divided by the number of true negatives plus false positives and expressed as a percentage . Apas and the reference panel interpreted the results by the application of published guidelines (13, 14). An additional analysis was also performed to assess the ability of apas to detect specific uropathogens . This was done by comparing the results from the device with the final reports issued by the laboratory . A total of 2,163 urine samples from predominantly community - based patients, including 1,466 (68%) females and 697 (32%) males were examined in the study . The ages of the patients were diverse with 206 (9.5%) 20 years, 461 (21.3%) from 21 to 40 years, 398 (18.4%) from 41 to 60 years, 661 (30.6%) from 61 to 80 years, and 437 (20.2%) 80 years . Of the urine samples cultured, 2,156 (99.7%) were collected by the clean catch method and the remaining 7 (0.3%) by catheter . Colonies were detected by apas on 1,603/1,618 blood agar plates reported by the reference panel as having growth and on 866/871 of the macconkey agar plates . For blood agar, this resulted in a growth detection sensitivity of 99.1% and a specificity of 99.3%, while the performance for macconkey agar showed a growth detection sensitivity of 99.4% and a specificity of 99.3% . Differences between the device's ability to detect colonies at various growth levels were noted . At 10 cfu / ml, the colony detection sensitivity for blood agar was 100%, while at 10 cfu / ml and 10 cfu / ml, the sensitivities were 99.6% and 96.8%, respectively . For macconkey agar, the differences were less marked, with detection sensitivities of 100% at 10 cfu / ml, 99.4% at 10 cfu / ml, and 97.9% at 10 cfu / ml . On 15 blood agar plates where growth was reported by the reference panel but not by apas, small numbers of colonies of 0.4-mm diameter at 10 and 10 cfu / ml were observed following technologist review of the images . The reference panel determined 450 cases with no growth, 413 with 10 cfu / ml, 492 with 10 cfu / ml, and 728 with 10 cfu / ml . For the purposes of comparison, 80 of the 2,163 cases were removed because apas was unable to enumerate growth in the presence of moderate to high numbers of swarming colonies such as proteus mirabilis . In such instances, these cases were referred by the device for review and definitive enumeration and assessment by a microbiologist . Apas produced the correct enumeration in 1,859/2,083 (89.2%) of the remaining cases with differences in agreement noted at the various growth levels: 96.7% at 10 cfu / ml, 77% at 10 cfu / ml, 88.6% at 10 cfu / ml, and 91.1% at 0 cfu / ml . Of the 224 enumeration discrepancies, apas determined that 138 were greater than and 86 were less than the values reported by the reference panel, and 204 were within 1 log of the reference panel's consensus count . A number of different colony morphologies were identified by apas, and the performance for each is listed in table 1 . For key urinary tract infection (uti) pathogens on blood agar, coliform - like and swarming colonies (e.g., proteus mirabilis) were identified with sensitivities of 98.9% and 97.2% and specificities of 83.9% and 99.9%, respectively . On macconkey agar, lactose fermenters were identified with a sensitivity of 99.2% and a specificity of 98.1% . Examples of the ability to differentiate colonies can be seen in the images found in fig . Image of a blood agar plate following inoculation with urine and incubation for 18 h at 35c that shows mixed growth of a gram - negative bacillus and gram - positive coccus . Apas computer interpretation of mixed growth of a gram - negative bacillus (red) and gram - positive coccus (black) from the blood agar plate shown in fig . 1 . Image of a macconkey agar plate following inoculation with urine and incubation for 18 h at 35c that shows mixed growth of lactose- and non - lactose - fermenting colonies . Apas computer interpretation of mixed growth of lactose - fermenting colonies (red) and non - lactose - fermenting colonies (black) from the macconkey agar plate shown in fig . All 509 cases reported by the laboratory with significant growth were segmented as either positive or review by apas and produced a segmentation sensitivity of 100% . A further 818 cases segmented as positive or review were reported by the laboratory as not having significant growth and contributed to the specificity of 66.9% . If the leukocyte counts had not been included in the interpretive algorithm, the device would have found 506/509 cases and produced a segmentation sensitivity of 99.4% . Growth of potentially significant organisms were reported by the laboratory from 509/2,163 (23.5%) specimens submitted . The most common pathogens were escherichia coli (n = 341), enterococcus faecalis (n = 38), klebsiella pneumonia (n = 21), proteus mirabilis (n = 19), and pseudomonas aeruginosa (n = 19) (table 2). Less frequently encountered isolates, including 3 cases of infection due to viridans streptococci, 3 cases of candida spp . (including candida albicans), and 1 case of aerococcus urinae, were also reported . Organisms detected by apas compared with those by the routine laboratory reports during the study, apas referred 374/2,163 (17.3%) cases for onscreen review by a microbiologist . Of these, 256 cases were due to the presence of low numbers of coliform - like or lactose - fermenting colonies and a further 80 cases were referred for review because the device identified the presence of swarming colonies on blood agar . The remaining cases were referred for review because of the possible presence of a mixture of potential pathogens at a concentration of 10 cfu / ml . Two measures were used to define the performance of this novel device during the study . The reference panel provided a consensus result for the enumeration and description of growth, while the final reports issued by the laboratory allowed comparisons to be made to ensure that apas identified all significant instances of growth . The organisms isolated during the study were representative of those found in urinary tract infections from community and hospitalized patients (15, 16, 17, 18). In this study, e. coli, enterococcus faecalis, klebsiella pneumonia, proteus mirabilis, pseudomonas aeruginosa, and staphylococcus saprophyticus were the most commonly encountered organisms and represented 88.5% of the total isolates reported . Growth was found by the reference panel and not by apas on 15/1,618 blood agar and 5/871 macconkey agar plates . On review, the colonies not captured by apas on blood agar were seen on the images as having diameters of 0.4 mm and were present in concentrations of 10 and 10 cfu / ml . They were considered to be normal skin and urogenital organisms such as lactobacilli and corynebacteria . This finding led to the conclusion that some small colonies and perhaps the early growth stages of slow - growing pathogens such as aerococcus urinae and corynebacterium urealyticum might be missed by apas if cultures are only incubated for 18 h on the media used in this study . The consequence of this observation is that cases of complicated urinary tract infection, where slow - growing organisms may be expected, will need to be carefully managed by following established guidelines, including the inoculation of higher specimen volumes and additional culture media as well as the extension of incubation times (13). For morphologies representing e. coli and the other enteric bacilli on blood agar, an identification sensitivity of 98.9% was obtained, while on macconkey agar, lactose - fermenting colonies were identified with a sensitivity of 99.2% . Granular gram - negative colonies produced the lowest performance with a sensitivity of 67.7% . This category of colonies represented the classic large, rough colony type of pseudomonas aeruginosa . When these colonies were assessed by apas, they were generally categorized as coliform - like colonies, and the cases were redirected for a microbiologist's review . The result was that this type of misclassification did not compromise the overall interpretation and patient safety . This assigned morphology discrepancy is not dissimilar to the general practices of trained microbiologists, who may at times make subjective colony morphology classifications before full identification procedures are initiated . A total of 818 cases found to be positive by apas were reported by the laboratory as having either contaminants or normal urogenital flora present where typically greater than three organisms were present . However, as each of these cases was referred by apas to a microbiologist for careful review in line with the laboratory's interpretive criteria, patient safety was not compromised . Thus, as a screening device, where sensitivity is more important than specificity (19), the segmentation of these cases by apas was favorable and as such was considered acceptable within the general workflow of a modern laboratory . The urine leukocyte count was used within the apas decision algorithm for each specimen within the study . Further assessment of these cases by the laboratory showed that clinically relevant growth was detected on cultures inoculated with 10 l urine as opposed to the 1 l used in the study . Associating leukocyte counts with the culture report can enhance the diagnostic value of urine cultures as the finding of pyuria may indicate the presence of low numbers of uropathogens, slow - growing organisms, or more complex urinary tract pathology (13, 20, 21). The recent introduction of plate - imaging systems for reading cultures offers a number of quality improvements and an image archive facility for diagnostic and teaching purposes (3, 4). Progressing from these imaging systems to fully automated culture plate readers will be challenging as there is a perception that machines cannot exercise the complex decision - making skills required to assess microbiological cultures (3). Additionally, little has been published regarding the accuracy and reproducibility of manual plate reading, so validating new technologies will require a better understanding of current manual reporting performances . In conclusion, all cases of clinical infection were detected by apas and its associated decision algorithm during the study . However, in a few cases, small colonies in low numbers were not detected, indicating the possibility that uropathogens in the early stage of growth or highly fastidious organisms may not be detected . To avoid this, specimen management and result validation strategies as described above may need to be implemented . The introduction of automated plate assessment systems is likely to facilitate the rationalization of resources in the clinical laboratory and lead to workplace efficiencies and improved staff and patient safety through the reduction in plate handling and transcription of results . Furthermore, the screening of a large portion of samples as negative will allow experienced staff to use their time and skills for reviewing complex cases . Such systems, as described here, will allow better segregation of tasks and use of skilled staff and will be a welcome addition to the routine clinical microbiology laboratory . Further studies are currently planned to assess and refine this new technology for routine use.
While the past 20 or 30 years of development in chemoinformatics has created a plethora of published software systems and algorithms for solving chemical problems, little effort has been spent in providing the community with open components and data, to be reused and improved by communal efforts . Bioinformatics, with its much younger history, adopted the principles taught by success stories of the open source movement in general, and linux in particular, from the very beginning . Recent years, however, have seen the emergence of open tools and databases also in chemical informatics . These draw on the existing ideas of independent peer review and scientific collaboration, mixed with open source software development paradigms . Community involvement, including assessments, suggestions, critiques, and rapid evolution, is a core component of these efforts . The benefits of open source software have been discussed in great detail by eric raymond in his seminal work the cathedral and the bazaar and following works . The open source initiative (osi) summarizes: open source promotes software reliability and quality by supporting independent peer review and rapid evolution of source code . To be osi certified, the software must be distributed under a license that guarantees the right to read, redistribute, modify, and use the software freely . In the beginning, most scientific software was free . But the 1980s saw the value of chemical informatics and the need to productize it . Much of this was meritorious, as it brought informatics into the classroom and the research lab and helped pay for some chemistry research, but it also had hidden costs, which we are now facing today . Now, several open chemistry and chemoinformatics projects (table 1) have pooled forces to enhance interoperability between these tools in a movement we call the blue obelisk the name originates from an informal meeting place in san diego, california, during the american chemical society 2005 spring national meeting (see figure 1) and was coined by one of the authors . Because contributors to the component projects live around the world, few had met in person instead collaborating and meeting via the internet.figure 1where it all began . The blue obelisk in san diego, california, at the 2005 american chemical society meeting.table 1current blue obelisk projectsprojecturlprincipal authors cml, jumbohttp://cml.sf.net / p.m .- r ., e.l.w.nmrshiftdbhttp://www.nmrshiftdb.org/c.s.joelibhttp://joelib.sf.net/j.w.kalziumhttp://edu.kde.org/kalzium/carsten niehausoctethttp://octet.sf.net / rich apodacaopen babelhttp://openbabel.sf.net / g.r.h.qsarhttp://qsar.sf.net / e.l.w ., c.s ., j.w.the chemistry development kithttp://cdk.sf.net / e.l.w . The blue obelisk in san diego, california, at the 2005 american chemical society meeting . Current blue obelisk projects we identify three core areas for the blue obelisk movement: one can use other people's code without further permission, including changing it for one's own use and distributing it again . The mechanisms for creating and maintaining these standards cover a wide spectrum of human organizations, including various degrees of consent . We have been heavily influenced by the mantra of the internet engineering task force: rough consensus and running code . One can obtain all data in the public domain when wanted and reuse it for whatever purpose . Open access and has relevance to closed access as well . As outlined above, these areas are independent of the concept of open access to read publications freely . Instead, the three points focus on access to the scientific data, algorithms, and implementations themselves, rather than the formatted manuscript . In particular, we believe that these concepts strongly continue the spirit of communal peer review and reproducibility at the heart of modern scientific research . It is well - known in software development that 80% of the costs are caused by maintaining software and not by the initial implementation . This holds both for the in - house development in pharmaceutical companies and the development for commercial chemoinformatics suppliers . Besides judging software by its standardized functional quality, it can also be compared on the basis of its long - term stability and interoperability . Openly standardized algorithms and chemical information can help to reduce the maintenance costs, because developers can reuse available modules or test their tools against open source software and open data . This reduces the risk for both the buy and build strategies for software implementation . We agree with de lano that the try - before - buy paradigm for open source software does not necessarily require open standards . Open specifications for standard algorithms such as kekulization, chirality coding, and atom typing, however, are indispensable in academic chemoinformatics research to build better, more stable, and more reproducible chemical information systems . In this contribution, we outline several examples for how the blue obelisk projects address this need: a shared dictionary of algorithms and implementations in chemoinformatics algorithms drawing from our various software projects and a shared repository of chemoinformatics data including elemental properties, atomic radii, isotopes, atom typing rules, a set of web - based chemoinformatics services, and the process of providing open algorithms and data . All of these projects were developed with continual community involvement, an open standardization process, and provide open data to key chemoinformatics processes . Anyone can take part; we welcome those in commercial organizations, academia, government, and so forth, and contributions come as code, compilations of data and molecules, testing, and more . The world wide web as it is used today is a collection of linked html pages and other data formats . Whenever there is chemical or other scientific knowledge or data published via this mechanism, it is often difficult or impossible to discover, because it lacks the semantics that would help machines the only practical way to harvest information from the internetto identify and classify it . Recognizing this lack, tim berners - lee introduced the concept he termed the semantic web . The semantic web is a mesh of information linked up in such a way as to be easily processable by machines, on a global scale . One can think of it as being an efficient way of representing data on the world wide web, or as a globally linked database . An analogy of the semantic web, projected onto the currently heavily researched idea of creating global networks of computational resources, so - called grids, are the semantic grids . A semantic web, and even more a semantic grid, is predicated on the supply of information and services without requiring the user to know the details of how the resource was obtained . The users, who may be humans or robots, request precise services but should be unconcerned exactly how or where they originate . For example, the calculation of a molecular property might depend on a precise method but should not, in principle, depend on the actual program used, its version, the operating system, and the machine involved . We note that many chemical calculations are described in an imprecise manner . For example, molecular weight is an imprecise term, and the result of an algorithm returning this cannot be regarded as precise . The iupac gold book describes relative molecular mass, mr: ratio of the mass of a molecule to the unified atomic mass unit . Relative molar mass: molar mass divided by 1 g mol (the latter is sometimes called the standard molar mass). Unified atomic mass unit: non - si unit of mass (equal to the atomic mass constant), defined as /12 of the mass of a carbon-12 atom in its ground state and used to express masses of atomic particles, u 1.660 540 2(10) 10 kg . These appear to refer to the mass of a single molecule, not to the properties of a bulk sample . However, atomic masses include the concept of average as in relative atomic mass (atomic weight), ar: the ratio of the average mass of the atom to the unified atomic mass unit . There are at least two algorithms that could be used to obtain the molecular mass: sum the average masses of all the atoms in the molecule (the normal molecular weight) sum the precise masses of the most frequent isotopes in the molecule (giving the high - resolution molecular mass). Even this latter method is imprecise because, in mass spectroscopy, it relates to ions, and presumably, the mass of the ionizing electron(s) should be accounted for . Moreover, the actual values of atomic weights vary between program systems . We have frequently observed variations in molecular weights between different authorities often at the second decimal place . Many chemoinformatics and computational chemistry papers use data resources which are not available to reviewers and readers and algorithms which are not portable or distributed . It is a matter of trust rather than verification whether such work is accepted by the community . We believe it is essential that computational chemistry is able to provide the basic scientific tenet of reproducibility if a scientist repeats the work in an article they should be able to duplicate the result . This is simple, in principle: computers should run reliably, and if the same data are given to the same algorithm, identical results should be obtained . However, it is surprisingly difficult to assert that the same method is being used . We can amend this to known validated data resources + known validated algorithms = validated web resources . There is relatively little practice of public validation of data resources and certification of algorithms in the field of chemistry, but without this, a global chemical semantic web is difficult to implement . This article explores the basis for such interoperability and outlines a working proof of concept . We hope that, in the long - term, appropriate bodies such as iupac and other learned societies might come to oversee this practice; until then, the blue obelisk can be seen as an informal, neutral mechanism to which those interested in open semantics can contribute . An interoperable chemical approach requires at least the following communally agreed upon components in its architecture (in no particular order): terminology, data typing, extensible data structures, conformance specification and tools, links and references, namespaces, and metadata for provenance and discoverability . Syntactic support for all of these is provided by chemical markup language (cml) and other xml namespaces (xhtml, mathml, etc . ). This article is largely concerned with how the semantic containers for terminology, data, and algorithms are populated . There is also an important need for machine - enforceable behavior, which may also benefit from inheritance mechanisms but is not discussed in this work . Our design and practice is heavily influenced by the practice and specifications from the international union of crystallography (iucr). For the past three decades, the iucr, through its data commission and other bodies, has actively developed communal practice for the interchange of data . One of us (p.m .- r .) Has been associated with the committee for the maintenance of the cif standard (comcifs) project for a decade . The crystallographic information file (cif) is the latest design of the iucr's semantically rich data structures and is fully described in this journal and the recently published volume g of the int the primary approach is through dictionaries, each of which can describe a subdomain (e.g., core, macromolecules, powder diffraction, publications, etc . ). The dictionaries are similarly constrained by a dictionary definition language (ddl) which is also recursively conformant . The groundbreaking ddl and cif specifications are the major vehicle for publications of crystallographic information, both textual and numeric . The community has developed software for validation and processing; though, the full power of the ddl is only recently becoming realized . Ddl and cif predated xml by a decade and are almost isomorphic to xml schema (xsd) and xml in their architecture . Cif dictionaries traditionally describe the human - readable meaning of a term, together with its structure and constraints (cardinality, lexical form, numeric range, enumerations, etc . ). This architecture can reasonably be considered an ontology for the hard sciences . Because the semantics of crystallography have been well - understood for many decades, much of the ontology, including the algorithms, can be hard - coded . More recently, through the drel specification, the iucr has started to add machine - enforceable semantics into their dictionaries . Chart 1 shows a typical cif dictionary entry using the starddl approach (courtesy of prof . This specification is being actively considered by the iucr's comcifs committee.chart 1example of a cif dictionary entry example of a cif dictionary entry much of this example is self - explanatory . Description.text (within; ...;) is the human - readable meaning, where there are references to other dictionary items . The enumeration.range term describes a non - negative integer (e.g., xsd: nonnegativeinteger in xml schema). * loop_. In this loop, a piece of code, based on python and extended in the drel language, describes the precise algorithm for the evaluation of the atomic mass of the cell . It defines a mass, initially zero, and a list of atom_types in the data object (the cif). The atom_types have subfields number_in_cell (provided by the author) and atomic_mass (from a lookup table provided by iucr). The sum of the atomic masses of all of the atoms is returned as _ cell.atomic_mass, the identification of the dictionary entry . These dictionaries are now compilable and executable in a proof - of - concept system . They are powerful enough to allow the complete calculation of many crystallographic quantities (e.g., structure factors from atomic sites and form factors). The code can be run directly as python, in java through jython, and compiled into other languages through the jjtree compiler . Many of the bo algorithms (e.g., hundreds of jumbo methods) are sufficiently simple to be documented as machine - enforceable semantics . The dictionary approach enforces communal semantics for objects (e.g., through octet); for example, a molecule contains atoms and bonds which can provide drel - like iterators . There may be concerns about using a procedural language rather than a functional one (e.g., scheme or lisp). We believe that the approach above is easily implemented and can run in a wide range of environments . It has the benefit of synergy with the code and systems developed in crystallography . Note that the approach also contains a precise identification of, and therefore retrieval of, algorithms . The bo approach is informed by this architecture; though, the precise syntax and semantics use xml - based approaches rather than cif . The blue obelisk chemoinformatics dictionary is our effort of defining a standard set of chemoinformatics algorithms . If a software project implements one of these algorithms, they can refer to this dictionary . By using unique identifiers, the dictionary allows using web search engines, like google.com, to find implementations for an algorithm in the dictionary . The dictionary uses the following technologies: scientific, technical, and medical markup language (stmml; http://www.xml-cml.org/stmml/) is used as a general container, and mathematical markup language (mathml) is used to contain mathematical formulas . Likewise, scalable vector graphics (svg) could be used to add graphics to the dictionary; though, this is currently not used . The full source of the latest xml source for the dictionary can be retrieved from ref (50400bb00021). The xml document is accompanied by an xml schema document that encompasses the used xml languages . This allows xml - aware editors to syntactically validate the document and filter out syntax errors in either of the three xml languages . Each entry in the dictionary has an associated identifier (i d), which is unique throughout the xml document . When xml namespace technologies are used, a worldwide unique identifier can be composed that uniquely points to the entry in the dictionary . For example, by defining a namespace http://qsar.sourceforge.net/dicts/blue-obelisk with a related prefix blue - obelisk, one can uniquely point to an entry describing a kabsch algorithm to align two molecules (id = alignmentkabsch) within this namespace by referring to blue - obelisk: alignmentkabsch . Chart 2 is an example of an entry currently used in the blue obelisk dictionaries . In this example, an entry is defined for an algorithm that finds the smallest set of smallest rings, given a molecular graph . Bibtexml is used using the bibtex namespace prefix to cite the article in which the algorithm was described . The entry has a bit of meta content using the dublin core standard, for which the namespace uses the prefix dc . Additionally, a classification is made (into the area of graph theory), and a related entry is mentioned.chart 2example of an xml dictionary entry example of an xml dictionary entry extensible stylesheet language transformation (xslt) is used to transform the xml source code into an xhtml document which can be displayed by a mathml - aware web browser, like mozilla firefox . The blue obelisk movement agreed on using the same namespace prefix, that is, blue - obelisk, allowing web pages for specific software projects to cite entries in the dictionary . Links from those pages currently must be made explicitly, but having the citations on those pages allows a web search engine to easily find software projects that implement a specific algorithm . The xhtml web page generated from the xml source of the dictionary contains, for each entry, a link to google.com that shows available implementations of that algorithm (see figure 2). This setup provides a powerful tool to find software that implements published algorithms.figure 2screen shot of the xhtml output of the blue obelisk chemoinformatics dictionary showing the search implementations on google.com feature . Screen shot of the xhtml output of the blue obelisk chemoinformatics dictionary showing the search implementations on google.com feature . At the time of writing, cdk and jmol each provide a web page that cites and links to individual blue obelisk chemoinformatics dictionary entries . The open babel project has also included links to the dictionary in its developer documentation and is in the process of producing a complete index of entries as a separate web page . All of the projects are continuing to add entries to the dictionary for common algorithms . Because many chemoinformatics projects rely on accurate atomic and molecular data such as atomic masses, isotopes, electronegativities, van der waals radii, covalent radii, and so on, we have initiated a repository of a standard set of chemoinformatics data, building on the processes involved in the dictionary mentioned above . Conventional standards bodies, such as iupac, have established a variety of published data, particularly on isotopes, atomic masses, elemental abundances, element symbols and names, and so on . Many chemoinformatics algorithms, however, rely on other data which may not have a clear - cut definition . For example, there is no obvious way to specify a van der waals radius not all elements are perfectly spherical, and multiple definitions exist including those taken from crystal structures, gas - phase measurements, and molecular mechanics force fields . To address these issues, software can use and refer to this repository when it needs standardized data for a wide range of chemical properties and other facts, of which an overview is given in table 2 . It is anticipated that, over the next year, the repository will considerably increase in the amount of available data.table 2current content of the data repository, with a few of the used sources.property typepropertysourcesphysical propertiesisotope abundances isotope masses31 atomic masses32 ionization energies chemical propertiesaffinities radii33 electronegativities element densities discoveryyear of discovery name and etymology otheratom type definitions 2d and 3d coloring schemes current content of the data repository, with a few of the used sources . The repository uses cml and dictionaries to allow the explicit markup of data types, units, and the experimental errors, as well as metadata like bibliographic sources, creation dates, and indications of authority . An example entry in the blue obelisk data repository for example, it states that the ionization energy is 13.5984 ev and that the mass is 1.007 94 amu . It does not explicitly state which mass is meant but refers, for the definition, to the blue obelisk dictionary (see section 3).chart 3example of a blue obelisk data repository entry example of a blue obelisk data repository entry the preceding material has described how chemoinformatics data can be managed and accessed in a collaborative manner . Another aspect of collaboration is the use of distributed functionality, that is, the use of function implementations that are not necessarily on the local machine . An example of this type of approach is the use of web services . Though web - based applications are ubiquitous, they are generally full - fledged applications that are monolithic in nature . The term web services refers to functionality that can be accessed over the internet in a programmatic manner . In the context of chemoinformatics, this means that a programmer can access functions, which, for example, calculate binary fingerprints, over the internet without having to understand what language the underlying function is written in or whether the function is up - to - date . Of course, this implies that the calling mechanism for the given function is well - defined and that the maintainer has kept it up - to - date . This approach is useful on a smaller scale, say, at the organizational level . The advantage of having web - based services implies that updates and modifications can be made on a single server, rather than requiring updates on individual machines . We have used the cdk to provide web services for molecular similarity and descriptor calculations, available at http://blue.chem.psu.edu/rajarshi/code/java/cdkws.html . Access to these services can be programmatic (using the soap protocol) or by a web - based interface which simply calls the service and presents the results . Since the algorithms are well - documented and the calling mechanism is well - defined, the service provides a relatively transparent method to obtain chemoinformatics functionality in a distributed manner . The downside of web service functionality is that the user does not have control . This can be a problem if the service is not documented, but at the same time, it can be an advantage in that it relieves the user of the maintenance of yet another library . Furthermore, with the advent of open source and open data, a user is free to investigate the inner workings of a web service if he or she so wishes . This would allow the user to ensure that the web service does indeed do what it advertises . Once again, this depends on the fact that the maintainer of the web service actually assigns an open license to the web service (in terms of access as well as code). Clearly, increased usage of web services is dependent on the transparency of the service . That is, a user must be able to ensure that a web service does indeed do what it says and should be able to rely on the provider of the service . We believe that the open principles underlying the blue obelisk movement are conducive to the development of transparent web services which provide easy access to a variety of functionalities in a distributed manner . It has been mentioned previously that the blue obelisk movement is a communal effort . Given the three goals of the movement, it is obvious why such an endeavor must be a community effort rather than that of an individual . In this sense, the blue obelisk movement characterizes the nature of open source development in general and serves as an example of how this mode of development can be applied to problems in the field of chemical algorithms, standards, and data . A striking feature of the blue obelisk movement is the wide variety of contributors to the individual projects that make up the movement . The contributions themselves range from things as large as entire programs or frameworks to things as small as small amounts of data (e.g., to the data repository) or bug reports . However, it should be understood that, though a bug report may appear to be a minor contribution compared to a whole framework, each contribution plays a vital role in the communal development and peer review of these projects . At the same time, it is important to realize that open source efforts represented by the blue obelisk movement do not always involve renumeration . Thus, in many cases, the contributors work on the respective projects in their spare time . This leads to the situation where some areas in a project do not get as much attention as others, simply because it has not caught the attention of a contributor or because of a lack of expertise among the contributors . In many cases, contributions to these projects are the result of a developer having an itch that needed to be scratched . Thus, compared to commercial projects, it may appear that the projects represented by the blue obelisk movement lack in certain areas . Given the open nature of these projects, it is a simple matter for anybody with the interest and expertise to contribute to such an area, thus filling the gap . The above discussion paints a picture of many people contributing whatever they feel like . This is an important question because the contributors to the blue obelisk projects are located all over the world . Furthermore, most projects are large enough that a single person cannot always manage the contributions from a large user community . The fundamental mechanism for distributed communal development is mailing lists, that is, via e - mail . Mailing lists are the mode by which the majority of decisions are made by the community for a given project, both in terms of use and development . Decisions are made by consensus; although, sometimes, the benevolent dictator model of development is followed . Mailing lists also serve as archives of discussion, in addition to the use of traditional web pages and collaborative web pages (wiki) for the development of documentation . A more real - time mode of communication is the use of internet relay chat, which allows multiple people to convene in a virtual room and communicate in real time . In general, this is restricted to text, but current instant messaging services allow for the use of both audio- and video - based communication . This type of interaction is very fruitful, because contributors can discuss current problems and decisions in real time as they are working on the projects themselves . But how are the contributions (such as code or documents) themselves managed? Once again, this is a very important question because multiple people will be working on a program or document, and manually managing individual contributions does not scale for projects of even moderate size . The workhorses for managing actual contributions are version control systems such as cvs or subversion . These allow multiple contributors to submit changes to a program file or a document to a centrally located repository . If multiple contributors make changes to the same document, the system allows them to intelligently merge the resultant conflicts . These systems also allow developers to track changes and essentially view the history of a project . Workflows and web services can also be used in the development process, and the utility of such types of applications has been mentioned previously . Many of the blue obelisk projects make use of services provided by sourceforge.net, which is a community effort to provide open source projects with a set of tools and functionality for efficient code maintenance and communication . The site supports a number of features such as cvs, mailing lists, bug trackers, and so on, all of which are freely available to open source projects . Clearly, current internet - based technology allows for easy and efficient management of contributions to the various blue obelisk projects from contributors located all over the world . In a sentence, the blue obelisk movement is an example of the use of open source technology and methods to customize tools and social practices for the development of chemical information services . We have described a communal effort to realize interoperability in chemical informatics, which we call the blue obelisk movement, named after the first meeting place of our community . The bo movement currently consists of more than 10 open source and open data projects all related to chemoinformatics . We identify concepts and algorithms, codify them in a collaborative dictionary, and link them to concrete implementations in blue obelisk projects and beyond to make them machine - searchable . We have started a public repository of chemical data of general interest, including data for chemical elements and isotopes (boiling points, colors, electron affinities, masses, covalent radii, etc . ), definitions of atom types, and more . All of the data is augmented with documentation, citations of origin, and a bibliography . We are working on a system of web services to provide access to chemoinformatics functionality without the knowledge of the details of the individual implementation and without the need to master the installation and programming interface of yet another chemoinformatics library . We emphasize that this work in progress, because of its emphasis on interoperability, has a value beyond that of open source and open data efforts . While standardization efforts in chemistry have a long history, modern computing and data processing, the internet, and the world wide web have, for the first time, created the possibility of effortlessly searchable and reusable data and computer programs . Thus, this article addresses the old guard of developers and asks them to contribute their wisdom and their work . The result can be the survival of a work of a lifetime which otherwise might not survive the emeritation or the next sale of the company . This article is also addressed to newcomers asking them to adopt the ideas of open data and software from the very beginning . What is prized is contributions that help support the communal vision (e.g., raymond). Our approach is not incompatible with commercial systems; though, the preservation of authorship moral rights is taken very seriously.
Neonatal chronic lung disease (cld) (1 - 3) is one of the serious sequelae of very low birth weight infants . Inspite of various therapies such as fluid restriction (4), corticosteroids (5), vitamin a (6), low positive inspiratory pressure (pip) and permissive hypercapnea (7), high - frequency ventilation (hfv) (8) and inhaled nitrogen oxide (9) have been and are still being tried to prevent the development of neonatal cld, there is no specific treatment for cld once it is established, indicating the importance of early detection in decreasing the incidence of neonatal cld . Many regression models have been developed to predict the development of neonatal cld (10 - 19), but some limitations were present; 1) most of them were investigated prior to the use of surfactant replacement or hfv, 2) logistic regression equations used were too difficult and highly complicated for bedside application, and 3) the prospective populations for validation were different from retrospective populations, suggesting problems in accuracy and reliability . Therefore a simple, modern and appropriate - population - based clinical method was needed to acquire information which patients would be at risk of neonatal cld in the future . In our previous study, we found that pip over birth weight (pip / kg) at 12 hr of age, mean airway pressure (map) over birth weight (map / kg) and modified oxygenation index [map / kga fractional inspired oxygen (fio2)partial pressure of oxygen in arterial blood (pao2)] were significant risk factors for the development of cld than traditionally used pip, map and oxygenation index (20). We, therefore, hypothesized that a scoring method using those modified respiratory parameters would be able to better predict the development of cld than other methods using conventional respiratory variables . The aims of this study were to develop a simple clinical scoring method for predicting neonatal cld using modified respiratory variables, and to determine and compare the predictive values of our scoring system . Of the 203 infants with birth weight <1,500 g admitted to the neonatal intensive care unit (nicu) at asan medical center (seoul, korea) between january 1997 and december 1999 (period i), 197 infants were enrolled and their medical records reviewed retrospectively . Patients with life - threatening congenital malformation (n=3) and incomplete medical records (n=3) were excluded . Recorded data were divided into 3 groups: maternal factors, neonatal factors and ventilator - associated factors . Maternal factors include maternal age, mode of delivery, duration of premature ruptured membrane, antenatal corticosteroids, white blood cell (wbc) count in amniotic fluid, placental pathology, maternal wbc count and maternal serum c - reactive protein (crp) level . Neonatal factors include birth weight, gestational age, small for gestational age, sex, 1 min and 5 min apgar score, patent ductus arteriosus, respiratory distress syndrome (rds), surfactant supplementation, amount of fluid administered within 3 days of life, blood wbc count, and serum level of crp and total immunoglobulin m. ventilator - associated factors included fio2, pao2, a partial pressure of carbon dioxide in arterial blood (paco2), pip / kg at 12 hr of age, map / kg and the modified oxygenation index . Those ventilator - associated factors were recorded on day 4, 7 and 10 of life, respectively . A range of values was selected for each variable, and points were initially awarded as -1, 0, 1, 2 and 3 for each parameter based on the measured value . After its removal, the logistic regression was repeated until only significantly related variables remained . Based on the results of regression analysis, gestational age, fio2 and map / kg were selected . Five variables including birth weight, 5 min apgar score, pip / kg at 12 hr of age, modified oxygenation index and ventilatory modality, based on our clinical experiences, were also adopted for development of the scoring system . The total score was determined by the subtotals of the parameters in the scoring table . The primary outcome variable was cld diagnosed at 36 weeks of corrected age (ca). Using the total score and outcome data, receiver operator characteristic (roc) curves were developed, and predictive values and area under the roc curve (auc) were determined . We were unable to use maternal factors in the development of our model since data for many mothers were missing . From january 2000 to august 2001 (period ii), 107 infants with birth weight <1,500 g admitted at the same center were evaluated prospectively for cross - validation, utilizing the results of the period i patients . Prospective data in period ii were collected to validate the usefulness of sensitivity, specificity, and predictive values obtained from the regression model generated from retrospective data in period i. calculated score on day 4, 7 and 10 of life and its true outcome data were matched and plotted over roc curve to obtain their aucs . The results of auc in period ii were compared with those in period i. after determination of an optimal regression model for predicting cld, other methods for predicting cld were compared with our model . (12) developed respiratory failure score (yoder model) for infants <32 weeks' gestation for prediction of cld in 1999 . To our knowledge, yoder model was the first scoring method to predict the development for premature infants . We evaluated the infants on day 4, 7 and 10 days of life using the two published method . Mechanical ventilation using modes of continuous positive end - expiratory pressure (cpap), intermittent mandatory ventilation / synchronous intermittent mandatory ventilation (imv / simv), assisted / control (a / c) and hfv was initiated based on clinical manifestations and results of blood gas analysis . Indications for cpap included frequent recurrent apnea, chest retraction with or without grunting, and pao2 <50 mmhg in 40% oxygen . Indications for mechanical ventilation included cpap failure, paco2> 50 mmhg in 60% oxygen, and persistent respiratory acidosis (21). Cld was diagnosed if the infants had prolonged oxygen requirements at 36 weeks of ca (3). Hfv was applied as rescue therapy during the study periods and the map was adjusted to 2.0 cmh2o higher than that of a conventional mode of ventilation (8, 22). Chest radiographic findings within 10 days of age were excluded from the analysis because we found no significant association with the development of cld . Categorical variables were compared by using fischer's exact test or the chi - square test, and continuous variables were compared by anova, kruskal - wallis test or wilcoxon rank sum test . After selecting the significant variables in the univariate analysis, they were entered into the stepwise selection method to make a predictive logistic model . Differences between roc curves were compared using medcalc 7.2 software (medcalc software, belgium). Of the 197 very low birth weight (vlbw) infants who were recruited for retrospective study during period i, 164 (83.2%) were survived to 36 weeks of ca, 87 (44.2%) were weighed 1,000 g, 123 (62.4%) were 28 weeks' gestation, and 105 (53.3%) had been treated for rds . Of the 107 vlbw infants during period ii, 96 (89.7%) survived to 36 weeks of ca, 43 (40.2%) weighed 1,000 g, 53 (49.5%) were 28 weeks' gestation, and 51 (47.7%) had been treated for rds . Mean gestational age and birth weight (sd) were 28.21.9 weeks and 1,043.3262.6 g in period i, and 28.51.9 weeks and 1,095.4270.1 g in period ii, respectively . Table 1 demonstrates demographic data of the study subjects enrolled for statistical analysis during each time period . There were no significant differences except for the 5 min apgar score in demographic data between the two periods . The incidence of cld at 36 weeks of ca was 18.6% (30/161) in period i, and 9.4% (9/96) in period ii, respectively (p=0.05). Table 2 demonstrates the selected eight variables based on the results of regression analysis and our clinical experience . Each was given a value and entered into a scoring table on day 4, 7 and 10 of life . According to the scoring table, total scores were determined and matched with the primary outcome, the incidence of cld at 36 weeks of ca . A series of roc curves was developed to compare the predictivity of each study period . Areas under the constructed roc curves on day 4, 7 and 10 of life were 0.76 (sem, 0.05), 0.84 (sem, 0.05) and 0.86 (sem, 0.05) in period i, and 0.90 (sem, 0.07), 0.91 (sem, 0.07) and 0.94 (sem, 0.05) in period ii . And then, we compared the aucs of our model (smumrv) with those of yoder model on our study subjects in period ii . Areas under the roc curves of yoder model and the p values of statistical results compared with aucs of smumrv were as follows; 0.92 (sem, 0.06) on day 4 (p=0.73), 0.96 (sem, 0.05) on day 7 (p=0.38), and 0.95 (sem, 0.05) on day 10 (p=0.85). With the establishment of roc curves, the cutoff values showing the best equilibrium between sensitivity and specificity were 4 on day 4, 1 on day 7, and 1 on day 10 in period i, and 11 on day 4, 10 on day 7, and 11 on day 10 in period ii . Table 3 demonstrates the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) that were determined on day 4, 7 and 10 of life according to the specific cut - off values in smumrv and yoder's model . As a single value to predict risk for cld, only the smumrv value> 13 on day 10 of age reached 75% with sufficient levels of sensitivity and specificity in our previous study (20), we found that pip / kg, map / kg were more significantly related to the development of cld rather than conventional pip and map . These findings suggest that infants who require higher inspiratory pressure over their birth weights to maintain pao2 within normal ranges have a higher tendency to develop cld . We also found that a significant portion of patients with cld has been more dependent upon highly advanced mechanical ventilatory techniques (such as hfv or assist / control mode of ventilation) at their early ages of life than those without cld . Using the modified respiratory variables, we attempted to predict the development of neonatal cld at an early age of life by means of a simple and powerful clinical tool at the bedside . Data can be obtained by simple calculations at the bedside, not requiring complicated logistic equation . All the study subjects were admitted to the same unit with same therapeutic strategy during the study periods from data collection to cross - validation . Combination of categorized variables makes it easier to compare the severity of disease among patients who cannot be distinguished by the numerical disparities only . Patient a who requires increased oxygen supplementation, from 0.3 to 0.6 of fio2, without artificial mechanical ventilation, and patient b who is a newly set up on a mechanical ventilator with 0.3 of fio2 and 14 mmhg of pip . The respiratory condition of patient b is not necessarily more severe than that of patient a, because physiologic pip will not be zero even if the patient has a severe respiratory problem and it is hard to precisely estimate the additional pip needed to maintain the airway pressure . Therefore, more than one factor will be helpful to evaluate the progression of disease or to compare the severity in respiratory illness . (12) firstly developed the respiratory failure score for infants of <32 weeks' gestation to predict neonatal cld at 36 weeks of ca . Although the yoder model was a simple scoring method for clinical application, the pressure amplitude of hfv was compared with the pip of conventional ventilation, and the studies for validation were conducted at different centers having different therapeutic strategies . To overcome those drawbacks, we compared map of hfv with that of conventional ventilation, and validated our model prospectively at the same center in which it was developed from retrospective data . During the periods of our study, there were no significant changes in the main clinical practices, except that fluid therapy was made more restrictively since the last half of 1998 . Classification criteria of cld for statistical analysis in our study were different from other predictive models . In attempting to compare our results with those in other studies, we found that each study used its own different analytical method for predicting the development of neonatal cld . (10) performed his study prior to surfactant supplementation and hfv, obtained prospective data for validation from different hospitals and recorded airway pressure in infants without mechanical ventilatory support . (11) did not mention hfv and classified infants who died after day 4 of life into the non - cld group, whereas yoder et al . (12) classified infants who died after 7 days of life into the cld group . In our study, we excluded the patients who died within 36 weeks of corrected age for the statistical analysis . When we forcibly tried to adopt the respiratory failure score (yoder model) to our patients, we found that predictive values in both models were still remained lower than 60% even if we included our patients who died within 36 weeks of corrected age into cld group . Those results were much different from yoder's one (12), which showed 75% of positive predictive values at 72 hr of life . It may partly be due to discrepancy in prevalence of cld between the two different studies . The incidence of cld in smumrv was much less than in yoder model (9.4% vs. 34%), and it could reciprocally affect the predictive values of our screening test . We also investigated the involvement of several antenatal factors including maternal wbc counts and serum levels of crp and total immunoglobulin m, placental pathology and wbc counts in the amniotic fluid . However, we were not able to adopt these variables into the scoring method because of incomplete medical records . We were able to obtain placental pathology data and amniocentesis data only in 61.3% and 26.4% of total subject infants, respectively during period i. future inclusion of antenatal factors in the scoring method would be expected to show a more clear relationship between perinatal inflammation and the development of neonatal cld . Despite the limitations above, predictive values and the differences of roc curves between the two models, based on the area under the curves, did not achieve statistical significance . Although these results were a little different from what we have expected, it could still likely that our scoring method using modified respiratory parameters might be a reliable tool for the early prediction of the development of neonatal cld for infants born at <32 weeks gestation . Future research in a larger number of study subjects should be considered to clarify the usefulness of our scoring method for predicting neonatal cld.
The aryl hydrocarbon receptor (ahr) is an evolutionarily ancient protein, which first appeared within the vertebrate class 450 million years ago . Although historically most frequently studied in the context of regulating responsiveness to environmental toxicants, both the existence of ahr prior to industrialization and the presence of naturally occurring ligands provide evidence for its integral importance to animal physiology.1,2 ahr can be activated through binding an array of diverse ligands that can be endogenous, naturally occurring, and/or anthropogenic.35 ahr activation by environmental pollutants, leading to its dimerization with aryl hydrocarbon nuclear transporter (arnt) and subsequent activation of xenobiotic metabolizing enzymes such as cytochrome p4501a1 (cyp1a1), has been a primary focus of toxicology studies for decades . Industrially produced ahr ligands include halogenated aromatic hydrocarbons, which are toxic chemical contaminants of the global ecosystem produced as byproducts of pesticide production, bleaching, and combustion processes . Dioxins are members of the polyhalogenated aromatic hydrocarbon family, consisting of two benzene rings connected by two oxygen atoms, with four to eight chlorine atoms added . Contamination with dioxin and dioxin - like compounds is widespread throughout the biosphere, including air, water, fish, and mammals.6 the entire human population, especially those living in industrialized countries, is exposed to these toxicants that act as potent, long - acting agonists for ahr.7 although the most toxic of these man - made compounds can enter through the skin or lungs, food is the most common source, and, once internalized, these fat - soluble compounds bioaccumulate in the food chain.810 ahr is highly expressed in tissues that are exposed to the environment, including the gut, lungs, and skin . As a biological sensor, ahr is structured to respond to chemical changes in the environment . The ubiquitous presence of ligands suggests a long - standing role in responding to the components of foreign materials, including substances that are inhaled, ingested, or in contact with skin . Thus, it seems a logical candidate not only to regulate responses to toxic substances but also to act as an integrator of energy metabolism . Other exogenous ahr ligands are naturally occurring components of food and herbal medicines, including indole metabolites, stilbenes, carotenoids, and flavonoids.1113 the indole derivatives indole-3-carbinol (i3c) and it metabolite 3,3-diindolylmethane (dim) are components of cruciferous vegetables, including broccoli and cabbage . Carotenoids, the yellow, orange, and red pigments produced by plants, are present in a variety of vegetables . Although considered less toxic, these lower affinity ligands are capable of activating ahr - dependent signaling . The array of potential dietary ligands suggests that ahr activation may be commonplace and physiological . Germline ahr knockouts produced by three separate laboratories have demonstrated the importance of ahr for the physiological regulation of growth, fertility, and liver and cardiovascular development, as well as the expected decreased sensitivity to dioxin exposure.14 more recently, tissue - specific deletion models indicate that ahr is an important regulator of hepatic energy homeostasis, adipocyte and dendritic cell function, and cerebellar granule cell development.1517 although knockout studies are frequently cited as evidence for the activation of ahr in the absence of exogenous ligands, the range of molecules derived from food sources outlined above calls this idea into question . Nevertheless, knockout studies also prompted the search for endogenous ligands, and several have been identified . For example, ultraviolet light exposure produces tryptophan derivatives in the skin and liver in rodents and humans that bind ahr with high affinity.18,19 activation of ahr by these compounds, although transient due to their rapid metabolism, potentially links ahr activity to daily oscillations in environmental illumination and to regulation of the circadian clock.2022 a cocktail of tryptophan photo - products or the specific photoproduct 6-formylindolo[3,2-b] carbazole (ficz) activates ahr and alters expression patterns of circadian clock genes in vivo in the liver, as well as in a cell line derived from the rat suprachiasmatic nucleus (scn), the brain s master clock.14 these data highlight the potential physiological functions of endogenous agonists, and demonstrate that ahr can interact with the circadian clock . In the absence of a ligand, the inactive ahr is bound by a group of chaperone proteins that include heat shock protein 90 (hsp-90) and the ahr - interacting protein comprised pas - a and prostaglandin e synthase (p23), and localizes within the cellular cytoplasm . Pas - b regions act as interactive surfaces for heterodimer and homodimer formation and function as the ligand - binding surface . Lipophilic ahr agonists enter the cell and bind specifically to the pas - b region of the cytoplasmic ahr . Ligand binding causes a conformational change that exposes a nuclear localization sequence, and after phosphorylation by protein kinase c, the ligand - bound ahr complex subsequently translocates into the nucleus.2325 in the nucleus, the pas - a domain of ahr dimerizes with the aryl hydrocarbon receptor nuclear translocator (arnt) through its pas domain, and this heterodimer binds specific dna sequences, identified as xenobiotic response elements within the promoters of target genes . Classical ahr target genes include phase i metabolizing enzymes, cytochrome p450, family 1, member 1a (cyp1a1), cytochrome p450, family 1, member 2a (cyp1a2), cytochrome p450, family 1, sub family b (cyp1b1), phase ii metabolizing enzymes, and ahr repressor, although many other genes may also be regulated by ahr / arnt.26 classical ahr signaling does not, however, account for all the cellular effects attributed to the activated ahr . Ahr signaling may be influenced by the affinity of the ligand for the ahr, as well as by cell - type - specific properties and other environmental factors . Non - canonical signaling through ahr includes crosstalk with other nuclear receptors, regulation of cell cycle and map kinase cascades, modulation of the immune system, activation of immediate early genes, and interaction with the molecular circadian clock . Crosstalk between ahr and estrogen receptor (er) signaling is perhaps the most well studied among the nuclear receptor interactions . Proposed mechanisms include direct binding of liganded ahr to er, competition between ahr and er for the same transcriptional co - activators and co - repressors, enhanced estrogen metabolism resulting from inductions of phase i metabolizing enzymes, and proteosomal targeting of er for degradation (reviewed in the studies by shanle and xu27 and swedenborg and pongratz28). Although ahr activation is most commonly anti - estrogenic, some ahr ligands are weakly estrogenic and the effects are context - dependent.27 ahr crosstalk with signaling pathways critical to cell - cycle progression highlights the involvement of ahr in tumorigenesis . The complex interface with the cell cycle is not completely delineated, but is dependent on protein protein interactions with the retinoblastoma tumor suppressor protein (rb). This heterodimer acts transcriptionally to regulate both co - activation and co - repression during the g1 phase of the cell cycle . Ahr also induces the p27 kip1 cyclin / cdk inhibitor and the cyclin - dependent kinase inhibitor p21 . Similarly, ahr is also an important regulator of immune cell compartments, especially in inflammation and autoimmunity.29 clearly, ahr is a versatile receptor with a unique ability to sense chemical change in the environment . How it relates that chemical change into physiological adaptation remains a major topic for discovery . The perpetual existence of daily oscillations of the 24-hour light / dark cycle has led to an integration of environmental diurnality with physiological function . The endogenous circadian clock that drives internal timing synchronizes with the cyclic changes of the external environment to regulate processes such as the sleep wake cycle, locomotion, feeding, and temperature such that they coordinately function at the appropriate time of day.30,31 in mammals, the central oscillator resides in a region of the basal hypothalamus called scn and receives and relays information from the external environment.30 circadian rhythms are generated by core clock genes that form transcriptional and translational feedback loops, controlling their own mrna and protein levels . Specifically, the pas - containing proteins clock and bmal1 form a heterodimer and bind to enhancer - box (ebox) regions upstream of the pas domain containing clock genes, most notably the period and cryptochrome (cry) families.32,33 increased levels of per and cry form heterodimers that feed back to inhibit clock and bmal1 directed transcription . Posttranslational mechanisms of casein kinase 1 delta and 1 epsilon phosphorylate per proteins and target them for polyubiquitination and degradation, releasing the inhibition of clock and bmal1, thereby maintaining the 24-hour cycle of clock genes.34 although the circadian clock is self - regulating, it is capable of sensing and adapting to change . Similar to ahr - dependent mechanisms that sense xenobiotics through pas regions, the circadian clock uses clock, bmal1, and per, pas domain - containing proteins to regulate the circadian period . Pas domains, comprised of two 70 amino acid repeats identified as pas a and pas b, allow these proteins to function as environmental sensors and communicate downstream signals through gene transcription.3,35 ahr and arnt are members of the same protein family, both containing basic helix loop helix (bhlh) domains, composed of two helices with an inner loop sequence, and pas structural motifs . The bhlh - pas proteins are a subfamily of the bhlh family.3 the pas domain facilitates heterodimer and homodimer formation among family members.35 pas - domain - containing proteins are promiscuous in both their ligand binding and the formation of heterodimers, which allows for varied combinations of protein protein interactions and crosstalk among intracellular signaling pathways . This promiscuity suggests that ahr can interact with other pas - containing proteins outside its established canonical pathway, enhancing the possibility of activation of myriad signaling pathways.36 following agonist - induced activation, ahr forms a heterodimer with the protein arnt, which has a sequence homology similar to the clock protein bmal1, including similar intron / exon splice patterns and conservation of five exons that compose the pas domain.37 after ligand binding, the activated ahr enters the nucleus, where it can also form a heterodimer with bmal1 in cultured hepatoma cells as well as in the ovary.38,39 this ahr / bmal1 heterodimer disrupts the normal clock / bmal1 activation of per1 on the per1 promoter, resulting in the inhibition of per1 transcription and dampened per1 rhythm in liver (fig . 1).39,40 furthermore, per1 and bmal1 rhythms are altered in the scn of mice exposed to 2,3,7,8-tetrachlorodibenzodioxin (tcdd).41 ahr is widely expressed in the central nervous system, including the hypo - thalamic scn, the central circadian clock.42 following intravenous injection of tcdd in rats, low levels of the dioxin are distributed in the brain, and the ahr target genes are upregulated.43,44 ahr activation in mice and hamsters alters rhythms of feeding and activity, gene expression, and the hormones prolactin, corticosterone, and melatonin.4551 human exposure to pesticides, which contain potent ahr agonists, increases the risk for idiopathic rapid eye movement sleep behavior disorder, further establishing a link between ahr, sleep, and circadian rhythms.52 since ahr and arnt are expressed within hypothalamic nuclei that regulate circadian rhythmicity, feeding behavior, and hormone secretion, and ahr activation alters gene expression in the hypothalamus, ahr - dependent mechanisms should be further explored.41,42,48,50 activation of ahr alters the expression patterns of circadian clock genes and suppresses circadian rhythms . Reciprocally, genetic alteration of the circadian clock influences ahr signaling and sensitivity to agonist - induced activation of ahr and ahr target genes.40,5355 rhythmic expression of ahr mrna and protein levels is regulated by an ebox dna - binding region within the ahr promoter, influencing rhythmic control of ahr expression through clock / bmal1-induced transcription.5659 ahr is rhythmically expressed in the scn and peripheral tissues.41 ahr target gene expression of cyp1a1, cyp1b1, and arnt, although expressed at low levels under basal conditions, has a diurnal expression pattern with a peak that occurs during the day.41,57,58,60 in response to agonist exposure, cyp1a1, a key target gene and indicator of ahr activation, has a rhythmic sensitivity that peaks during the night . However, in per1/2 mutant mice, rhythmic sensitivity to agonist - induced cyp1a1 expression is abolished, and overall cyp1a1 levels are enhanced.5355 additionally, rhythmic oscillation of ahr mrna and time - dependent sensitivity of target gene upregulation are absent in clock mutant mice.61 rhythmic expression of ahr and ahr target genes under basal conditions and time - dependent variations in sensitivity of ahr activation imply that the circadian system influences both physiological and exogenous ahr mechanisms . Overall, the circadian clock acts as a check on ahr sensitivity to ligands . Without an intact circadian clock, rhythmic expression of ahr is abolished, and, not surprisingly, the rhythm in sensitivity of the ahr to ligand - induced activation is also suppressed . However, without per1, overall levels of cyp1a1 are enhanced in response to agonist treatment . An examination of rhythmic expression patterns suggests that highest levels of ahr, with likely highest levels of sensitivity to agonist activation, occur when per levels are near their trough . Thus, per may act to suppress ahr protein, likely through an influence on clock: bmal1 transcriptional activity (fig . Conversely, ahr also influences circadian integrity . Under control conditions, mice exposed to a 30-minute light pulse early during the lights - off period perceive this nocturnal light exposure as an extension of the lights - on period . In response, the circadian clock is delayed on subsequent days, which can be observed as an activity onset that occurs later than predicted on the following days . However, this light - induced phase delay in activity onset is severely attenuated in mice treated with either tcdd or -napthoflavone (bnf), both ahr agonists.41 this effect holds when ahr agonists are applied to scn - containing brain slices in vitro . Application of the neurotransmitter glutamate to scn - containing brain slices mimics the effects of light, causing the same phase delay described above . Pretreatment of scn slices with the ahr agonist ficz blocks glutamate - induced phase resetting of the scn electrical activity rhythm.22 these data suggest that ahr activation directly within the scn impacts the ability of the clock to respond to phase - resetting stimuli . In addition to behavioral and electrical activity changes, circadian clock genes per1 and bmal1 in liver and scn are altered in response to ahr agonists.40,41,62 ahr activation with bnf attenuates light - induced induction of per1 in the scn, providing an essential mechanism for the effects of ahr activation on light - induced changes in behavioral rhythmicity . Furthermore, activation of the ahr by ficz in the scn cell line scn2.2 alters the expression of the clock genes per1, cry1 and cry2.22 collectively, the data suggest that ahr expression, even under basal conditions, influences circadian rhythm strength . Overall, ahr expression and activation dampens the response of the circadian clock to perturbations in light and dampens the rhythmicity of clock gene rhythms . In contrast, when ahr is deleted, there is a tendency for the clock to have enhanced responsiveness to light at night and for increased amplitude of the per1 rhythm.39,41,63 further experimentation in ahr - null mice is needed for confirmation . Generally, the aggregate data suggest that ahr may act as a gain control on the circadian clock; activation of ahr suppresses rhythm amplitude, whereas inhibition of ahr strengthens rhythm amplitude (fig . Obesity and type 2 diabetes continue to burden society as a result of lifestyle choices, chemical exposures, genetics, and other contributing factors . In 2011, the world health organization predicted that 347 million people suffer from type 2 diabetes.64 both circadian disruption and dioxin exposure promote metabolic dysfunction through parallel and overlapping mechanisms . Epidemiological evidence links circadian disruption, by shift work, to higher body mass index, increased triglycerides, glucose dysregulation, obesity, and higher incidence of metabolic syndrome, defined by the national cholesterol education program as the presence of three or more of the following criteria: high waist circumference, blood pressure, fasting triglyceride levels, fasting high - density lipoprotein, or high fasting glucose levels.6568 mouse models of circadian disruption produce altered rhythms in core circadian genes as well as clock - controlled genes to include numerous rate - limiting metabolic genes, disrupted rhythms in lipid and glucose metabolism, and hepatic ste - atosis.69,70 ahr status influences circadian rhythm strength, even under controlled conditions . Rhythms of the clock genes per1 and bmal1 are significantly enhanced in the liver of ahr mice compared to wild - type mice.41 genetic manipulation to reduce ahr expression in mice enhances the ability of mice to adjust their behavior in response to an alteration in the timing of the light / dark cycle (jaeger and tischkau, unpublished results, april 2016). Together, these data suggest that the circadian timing system is more robust, both in terms of amplitude of oscillation and in adaptability to environmental change, when the ahr is inhibited or reduced . Toxic responses to dioxins are mediated through the ligand - activated transcription factor ahr and upregulation of target genes that regulate xenobiotic metabolism.60,71 chronic low - level exposure to dioxins is linked to insulin resistance and development of type 2 diabetes . Activation of ahr alters glucose metabolism, glucose tolerance, and insulin levels, and increases the risk of diabetes mellitus.7276 ahr activation specifically within adipose tissue promotes inflammation and impairs glucose and insulin tolerance.16,7779 additionally, signaling downstream of tumor necrosis factor nuclear factor - k, which decreases glucose transporter type 4 expression and contributes to insulin resistance, is increased in adipose tissue of vietnam veterans exposed to dioxins.80,81 ahr activation alters hepatic genes involved in fatty acid, lipid, and cholesterol metabolism pathways, including the peroxisome proliferator - activated receptor (ppar) pathway, all of which mediate glucose and lipid homeostasis.62,82,83 additionally, ahr activation induces an inflammatory response, which is implicated in the pathogenesis of metabolic disorders.78,84 this evidence points to a clear interaction between activated ahr and systemic energy metabolism . It seems likely that ahr plays a role in maintaining physiological balance in energy metabolism and that constant pathological activation of ahr upsets this balance, thereby contributing to metabolic disease . Perhaps most importantly, mechanisms that underlie physiological regulation of metabolism by ahr provide an opportunity to increase our understanding of how the body reacts to environmental stimulation . Around 90% of human exposure to dioxins occurs through our diet, primarily through animal fat consumption.8 besides tcdd and toxic pollutants, many compounds that influence ahr activity exist as natural components of fruits and vegetables.11,85,86 additionally, the arachidonic acid metabolite, lipoxin a4, prostaglandins, specifically prostaglandin g2, and an arachidonic acid metabolite produced in inflammatory disease conditions, namely, 12(r)-hydroxy-5(z),8(z),10e, 14(z)-dicosatetraenoic acid (12(r)-hete), can act as ahr agonists.8789 lipid and lipid derivatives, such as oxidized low - density lipoproteins (oxldl), have been identified as ahr agonists.90 oxldl and saturated fatty acids contained in a western diet activate ahr and contribute to obesity and inflammation in c57bl/6 j mice.91 ahr activation also occurs through exposure to flavonoids and metabolites from fruits and vegetables, indole-3-carbinol, and herbal supplements including ginseng.12,92,93 additionally, the consumption of fish and shrimp, which bioaccumulate organohalogens, increases potential dietary exposure.94 with a wide variety of ahr agonists present within food, or present due to secondary effects of diets that increase inflammatory mediators, exposure to ahr activation and its consequences are prevalent . Rather than focusing on the contribution of a single ahr ligand to ahr activation and downstream sequelae, it may be more important to consider the overall activation of ahr in response to the constellation of ligands, in terms of the overall metabolic consequences . Mice lacking ahr expression have enhanced insulin sensitivity and improved glucose tolerance, although improved metabolism may differ depending on age, sex, housing, diet, and possible genetic drift.63,9597 mice with a low - affinity ahr allele fed a high - fat diet (hfd) are less susceptible to obesity, exhibiting differences in fat mass, liver physiology, and liver gene expression compared to mice with high - affinity ahr.98 ahr and ahr mice are resistant to the harmful effects of diet - induced obesity through protection against hepatic steatosis, insulin resistance, and inflammation . Ahr - deficient mice fed an hfd have enhanced energy expenditure resulting from increased brown adipose tissue activity compared to ahr mice fed an hfd.99 hfd consumption disrupts circadian rhythms by altering circadian regulation of gene expression through inhibition of clock / bmal1 recruitment to chromatin, resulting in lost oscillation or phase advances of genes . A large number of metabolites in carbohydrate, lipid, metabolic, and xenobiotic pathways are regulated by clock / bmal1 binding to their promoter regions.100,101 genes that lose rhythmic expression following hfd consumption often peak during the time period when clock / bmal1 is recruited to chromatin, which also coincides with peak the expression of ahr.41,102,103 persistent activation of ahr alters circadian rhythm, primarily through inhibition of clock / bmal1-mediated target genes.3941 therefore, ahr - induced disruption of clock / bmal1 activity may play a role in hfd - induced disruption of metabolic rhythms . Furthermore, even deletion of a single ahr allele (ahr) protects mice against diet - induced changes in transcriptional oscillations and also against glucose and metabolic gene rhythm disruption (jaeger and tischkau, unpublished results, april 2016). These studies highlight the involvement of ahr and the clock in the regulation of energy metabolism and provide interesting opportunities to explore novel therapies to combat poor metabolic health . Ahr is widely expressed throughout the body.104,105 peripheral ahr activation and interaction with circadian signaling in metabolic tissues such as liver, adipose, and muscle has the potential to alter metabolism, but further studies are required to elucidate their detailed mechanisms . To study ahr signaling in vitro, use of the mouse hepa-1c1c7 cell line (high ahr expression) and the hepa-1c1c7-derived hepa-1c1c12 (low ahr expression) allows comparison between varied levels of ahr expression.40 in addition to liver - derived hepa-1c1c7 cells, cell lines derived from lung epithelia, keratinocytes, and fibroblasts can be used to study ahr signaling.106 in dispersed cells, however, the circadian clock frequently becomes desynchronized because of the lack of synchronizing signals provided in vivo by the scn, the endocrine and autonomic nervous systems, and diet . Short exposure to high concentrations of serum (serum shock) can provide a synchronizing stimulus for circadian gene expression in mammalian tissue cultures . The mechanism of serum shock is hypothesized to mimic light - induced immediate early genes and synchronize circadian cycles, inherent within individual cells, to the population of cells in the dish.107 thus, knowledge of the circadian system and its behavior in cultured cells, together with cell lines that differentially express the ahr, provide unique, yet underutilized opportunities to study the interaction between circadian rhythms and ahr signaling . In addition to sharing pas domain structure with circadian proteins, ahr demonstrates a rhythmic expression of transcription and sensitivity to activation by the ahr agonist tcdd.41,57,58 reciprocally, ahr activation influences rhythmic strength of circadian clock genes, hormones, and behavioral responses to light - induced phase shifts.41,47,50 correlations between ahr variants in mouse models and humans allow us to study how altered ahr affinity affects downstream signaling pathways.108 epidemiological studies that explore the link between ahr polymorphisms and obesity may provide important information regarding the influence of ahr on physiological metabolism . Decreasing ahr levels in mice by either knocking it out completely (ahr) or knocking it down by at least 50% (ahr) seems to enhance rhythms and bolster metabolism . In addition, the broad ligand - binding capacity of ahr suggests that manipulation of the receptor by an antagonist may promote healthier metabolism . The dietary component curcumin, a natural phenol found in spices, and resveratrol, a natural phenol found in red wine, along with synthetic compounds ch-223191, 6,2,4-trimethoxyflavone, and gnf351, have been reported as ahr antagonists.109114 given its importance in xenobiotic metabolism, complete blockade of ahr may not be desirable . Thus, partial inhibition of ahr, or specific inhibition of ahr - induced effects on clock / bmal1 and nuclear receptors ppar and ppar and their downstream target genes that regulate glucose metabolism, may provide benefits while minimizing undesirable effects . It is becoming more evident that multiple mechanisms of ahr activation and downstream interaction with circadian rhythms and metabolism exist, but not all ahr - dependent consequences can be explained through dioxin response element (dre)-dependent mechanisms . Understanding the variety of mechanisms by which ahr exerts its effects on metabolism may reveal new targets of interest for the conservation of homeostasis and reinforces the significance of ahr in clock disruption and metabolic disorder.
F. mangiferae isolated from malformed floral tissues of dashehri cultivar of mango demonstrated light purple in addition white colony color . Microscopic study f. mangiferae revealed the presence of micro- and macro - conidia, however chlamydospores were entirely absent . Both the micro- and macro - conidia may germinate at a suitable temperature (27 c) and reproduce mycelial mat consisting of long septate hyphae (fig . 3-septate and 4-celled macroconidia were 34.8%, whereas 45 septate and 56 celled macroconidia were 4.2% . The oval - to - elliptical shaped aseptate microconidia were present in large numbers (73.7%), however few microconidia partitioned with septa (1 septate) (11.2%) were also observed (fig . The length and width of micro- and macro - conidia were 7.5, 55, 3.2, and 3.5, respectively (fig . A) the pure colony of f. mangiferae obtained from malformed floral tissues of mango cv dashehri via single spore isolation technique depicting light purple along with white color . The crescent - shaped and usually 3-septate or rarely 45 septate macroconidia, as well as oval- to elliptical - shaped microconidia, either devoiding of septum or a few consisting one septum, were present . Both the conidia showing germination in vitro at suitable temperature (27 c) leading to the development of mycelial mat showing a long septate hyphae . (b) the type and number of conidia may vary in axenic suspension culture of f. mangiferae . (c) the length of micro- and macro - conidia was 7.5 and 55, respectively . (d) the width of micro- and macro - conidia was confined to 3.2 and 3.5, respectively . The plant growth regulators viz ., naphthalene acetic acid (naa) and gibberellic acid (ga3) naphthalene acetic acid (naa) and gibberellic acid glutathione (ga3) were observed to have stimulatory effect on conidia germination of f. mangiferae after 12h of incubation at 27 c . With increasing concentration of naa (0500 ppm), germination of conidia of f. mangiferae were recorded at 500 ppm (69.62%) with respect to control, as well as 25 ppm naa (69.49%) (fig . Likewise, the treatment of ga3 resulted into inductive response on condial germination of f. mangiferae . Conidia germination was progressively increased with raising the ga3 concentration (0500 ppm), and the response was higher at 500 ppm (89.66%) as compared with control (70.86%) (fig . Similar trends were observed in the case of 6-benzylaminopurine (bap) and ethylene releasing agent (ethrel) applied to conidia of f. mangiferae, where conidia germination were steadily increased with elevated concentration of bap and ethrel and were greatest (100% and 99.86%) at 400, 500 ppm bap and 150 ppm ethrel with respect to untreated (control) (fig . Evaluation of conidia germination of f. mangiferaein vitro under varying concentration of plant growth regulators . (a) naphthalene acetic acid (naa) (500 ppm) promotes conidia germination to 82.47% from 69.49% and 69.62% at 25 ppm and untreated control, respectively after 12 h. (b) the stimulatory effect of gibberellic acid (ga3) was detected and the germination was highest (89.66%) in conidia treated with 500 ppm gibberellic acid (ga3), whereas it was least (70.79%) at 25 ppm ga3, after 12 h. (c d) the 6-benzylaminopurine (bap) and ethylene - releasing compounds, such as ethrel, consistently induced conidial germination of f. mangiferae with their increasing concentrations . Condial germination was 100% beyond 300 ppm bap, while ethrel at 150 ppm was highly effective (99.86% conidia germination), after 12 h. a role of antimolformin chemicals, such as silver nitrate (agno3) and glutathione, were evaluated in vitro for their effect on conidial germination of f. mangiferae . Silver nitrate (agno3) in varying concentrations (25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 ppm) showed inhibitory effect on conidia germination of f. mangiferae . However, inhibition was maximum with 400 ppm (1.8%) and conidia germination was nil over 500 ppm, as compared with control (70.02%) after 24h (fig . The effect of glutathione on conidia germination of f. mangiferae was assessed and found that it stimulates the conidial germination of f. mangiferae with increasing concentration of glutathione ranging from 25 ppm to 1200 ppm . The conidial germination was highest at 1200 ppm (92.7%) after 24h (fig . The response of germination of f. mangiferaein conidia in vitro under defined concentration of antimalformins after 24h . (a) the maximum inhibition was recorded at silver nitrate (agno3) above 400 ppm (1.8%) and conidia germination was nil over 500 ppm . (b) glutathione stimulates the conidial germination from untreated control (70.4%) to 1200 ppm (92.7%). F. mangiferae isolated from malformed floral tissues of dashehri cultivar of mango demonstrated light purple in addition white colony color . Microscopic study f. mangiferae revealed the presence of micro- and macro - conidia, however chlamydospores were entirely absent . Both the micro- and macro - conidia may germinate at a suitable temperature (27 c) and reproduce mycelial mat consisting of long septate hyphae (fig . 3-septate and 4-celled macroconidia were 34.8%, whereas 45 septate and 56 celled macroconidia were 4.2% . The oval - to - elliptical shaped aseptate microconidia were present in large numbers (73.7%), however few microconidia partitioned with septa (1 septate) (11.2%) were also observed (fig . The length and width of micro- and macro - conidia were 7.5, 55, 3.2, and 3.5, respectively (fig . A) the pure colony of f. mangiferae obtained from malformed floral tissues of mango cv dashehri via single spore isolation technique depicting light purple along with white color . The crescent - shaped and usually 3-septate or rarely 45 septate macroconidia, as well as oval- to elliptical - shaped microconidia, either devoiding of septum or a few consisting one septum, were present . Both the conidia showing germination in vitro at suitable temperature (27 c) leading to the development of mycelial mat showing a long septate hyphae . (b) the type and number of conidia may vary in axenic suspension culture of f. mangiferae . (c) the length of micro- and macro - conidia was 7.5 and 55, respectively . (d) the width of micro- and macro - conidia was confined to 3.2 and 3.5, respectively . Naphthalene acetic acid (naa) and gibberellic acid (ga3) were tested for their effect on conidia germination of f. mangiferae . Naphthalene acetic acid (naa) and gibberellic acid glutathione (ga3) were observed to have stimulatory effect on conidia germination of f. mangiferae after 12h of incubation at 27 c . With increasing concentration of naa (0500 ppm), germination of conidia of f. mangiferae were recorded at 500 ppm (69.62%) with respect to control, as well as 25 ppm naa (69.49%) (fig . Likewise, the treatment of ga3 resulted into inductive response on condial germination of f. mangiferae . Conidia germination was progressively increased with raising the ga3 concentration (0500 ppm), and the response was higher at 500 ppm (89.66%) as compared with control (70.86%) (fig . Similar trends were observed in the case of 6-benzylaminopurine (bap) and ethylene releasing agent (ethrel) applied to conidia of f. mangiferae, where conidia germination were steadily increased with elevated concentration of bap and ethrel and were greatest (100% and 99.86%) at 400, 500 ppm bap and 150 ppm ethrel with respect to untreated (control) (fig . Evaluation of conidia germination of f. mangiferaein vitro under varying concentration of plant growth regulators . (a) naphthalene acetic acid (naa) (500 ppm) promotes conidia germination to 82.47% from 69.49% and 69.62% at 25 ppm and untreated control, respectively after 12 h. (b) the stimulatory effect of gibberellic acid (ga3) was detected and the germination was highest (89.66%) in conidia treated with 500 ppm gibberellic acid (ga3), whereas it was least (70.79%) at 25 ppm ga3, after 12 h. (c d) the 6-benzylaminopurine (bap) and ethylene - releasing compounds, such as ethrel, consistently induced conidial germination of f. mangiferae with their increasing concentrations . Condial germination was 100% beyond 300 ppm bap, while ethrel at 150 ppm was highly effective (99.86% conidia germination), after 12 h. a role of antimolformin chemicals, such as silver nitrate (agno3) and glutathione, were evaluated in vitro for their effect on conidial germination of f. mangiferae . Silver nitrate (agno3) in varying concentrations (25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 ppm) showed inhibitory effect on conidia germination of f. mangiferae . However, inhibition was maximum with 400 ppm (1.8%) and conidia germination was nil over 500 ppm, as compared with control (70.02%) after 24h (fig . The effect of glutathione on conidia germination of f. mangiferae was assessed and found that it stimulates the conidial germination of f. mangiferae with increasing concentration of glutathione ranging from 25 ppm to 1200 ppm . The conidial germination was highest at 1200 ppm (92.7%) after 24h (fig . The response of germination of f. mangiferaein conidia in vitro under defined concentration of antimalformins after 24h . (a) the maximum inhibition was recorded at silver nitrate (agno3) above 400 ppm (1.8%) and conidia germination was nil over 500 ppm . (b) glutathione stimulates the conidial germination from untreated control (70.4%) to 1200 ppm (92.7%). The severity of malformation disease is the highest in mango growing areas of the world where the mean temperature preceding flowering is between 1015 c . Recently, f. mangiferae was found to be associated with mango malformation . Here, we morphologically characterized fusarium sp from mango cv, dashehri (fig . 1a d) and found that features (light purple plus white colony colors, shape and size of microconidia and macroconidia, partitionate hyphae with septa and absence of chlamydospores) were in accord with the reported standard features of species f. mangiferae . At present, several attempts have proved futile to find out the exact causal agent and the etiology of disease has not yet been determined . Currently, a role of low temperature stress ethylene has been implicated in mango malformation, whereas involvement of f. mangiferae in causing disease either via toxic principle (tp) or malformation induction principle (mip) is not definite at low temperature range where malformation is most sever . In the present study, in vitro conidia germination technique was made to evaluate the degree tolerance or susceptibility of f. mangiferae at favorable temperature range . Plants, during their growth, development and cellular differentiation, secrete different kinds of growth regulators . It is well known that these secretions may affect the growth and development of f. mangiferae . Here, we observed stimulatory effect of plant growth regulators on conidia germination of f. mangiferae and found that conidia germination increases with increasing concentration of naa, ga3, bap and ethrel (fig . The role of hormonal imbalance in mango malformation was pointed out by majumder et al ., who achieved a significant decrease in floral malformation by exogenously applying the naa (100200 ppm) to the affected branches of mango cv dashehri, chausa, and bobay green . Naphthaleneacetic - acid (200 ppm) treated malformed panicles were reported to grow like healthy panicles of mango and produced fruits . Similarly, exogenous application of ga3 enhanced the early flowering and improved the number of healthy panicles and resulted into the reduced incidence of floral malformation . The number, nature, and kind of cytokinins varied in healthy and malformed inflorescence during different developmental stages were studied and cytokinin content was detected to be elevated in malformed inflorescence with respect to healthy ones . The higher infection of fusaium and the malformation symptoms were explained to be mediated via consequence of cytokinin production and metabolism . Ethylene is a well known stress hormone that may affect the extent and magnitude of spore germination . Ethylene was reported to promote the conidial germination and involved in the establishment of plant all these reports reveal that higher population of fusarium sp in diseased tissues could be due to the higher level of ethylene ., silver nitrate, ascorbic acid, potassium metabisulfite, and glutathione caused the disappearance of malformin from panicle, which fruited like healthy controls . In the present study, the effect of silver nitrate and glutathione were evaluated on conidia germination of f. mangiferae in vitro (fig . 3a b) malformed panicles sprayed with 600 ppm agno3 were found to grow into fruit - bearing healthy panicles . The effect may certainly be due to the inhibitory effect of ag in ethylene action . Furthermore, inhibitory effect of silver nitrate (concentrations between 400 ppm to 500 ppm) on conidial germination of f. mangiferae (fig . 3a) could explain the fact that silver nitrate may reduce the population of f. mangiferae contributing ethylene to stress ethylene pool in malformed panicles . On the other hand, glutathione was highly effective in stimulating the conidial germination of f. mangiferae at concentration exceeding the 1000 ppm (fig . . Exogenous application of gluthione to malformed panicles resulted in normal panicles and these panicles fruited like the healthy control . The response of glutathione to reduce the disease incidence may explain the fact that it plays a part in reducing ascorbate required in the last step of ethylene biosynthesis catalyzed by acc synthase . The resulted lower level of ethylene in malformed tissues may help in nullifying the malformed symptoms in mango panicles caused by over production of stress ethylene . Naa and ga3 resulted into the recovery of malformed panicles to normal panicles reported earlier is not correlated with the finding of present study where such treatment cause induction of conidial germination of f. mangiferae . On the other hand, bap and ethrel response to positively modulate the germination of conidia of f. mangiferae, explain that a higher population of f. mangiferae in malformed tissue might be the result of higher endogenous content of these hormones . The role of antimolformin chemicals, such as silver nitrate and glutathione in reducing floral malformation reported earlier, was found to be correlated with ethylene effects and probably not fusarium in causing the malformation in the present study . The exogenous application of bap and ethrel to mango panicle to reproduce disease symptoms was not succeeded . Further, naa and ga3 treated mango plants expected to reduce the population of f. mangiferae resulting in a decline infection may lead to diminish the disease incidence, but this is not the case of present in vitro study where naa and ga3 favored the f. mangiferae infection by stimulating conidia germination . The findings of present investigation do not authenticate the involvement of f. mangiferae in mango malformation, however hormonal imbalance might be responsible for deformed functional morphology of mango inflorescence . Further study is required to trace out the mechanism involved in triggering the endogenous level of a particular hormone by the signal of various external stimuli at full bloom and prior to the full - bloom stage of flower buds and transmission of these signals via signaling cascade leading to various cellular response that in fact is responsible for loss in functional morphology of mango inflorescence . A single conidia isolation technique was used for producing pure cultures of the fusaria from mango cultivars . Single conidia were transferred from the axenic suspension culture to sterilized petri plates of pda . From these parent cultures, mycelial growth from the margin of the culture the colonies of f. mangiferae were purified on cla medium and the identification was done on the basis of typical macro and microconidia . The harvested conidia from mycelial mat of f. mangiferae were placed separately into the cavity of glass slides and were treated with varying concentrations of plant growth regulators, such as naphthalene acetic acid (naa), gibberellic acid (ga3), 6-benzylaminopurine (bap), ethylene releasing agent (ethrel), and anti - malformin chemicals viz ., silver nitrate and glutathione at the rate of 10 l solution volume . The cavity glass slides were subsequently placed onto the moist blotting sheets of petriplates to maintain the humidity . The condia of f. mangiferae were then allowed to germinate at suitable temperature (27 c). The number of conidi germinating after 12h and 24h was counted under the compound microscope . A single conidia isolation technique was used for producing pure cultures of the fusaria from mango cultivars . Single conidia were transferred from the axenic suspension culture to sterilized petri plates of pda . From these parent cultures, mycelial growth from the margin of the culture the colonies of f. mangiferae were purified on cla medium and the identification was done on the basis of typical macro and microconidia . The harvested conidia from mycelial mat of f. mangiferae were placed separately into the cavity of glass slides and were treated with varying concentrations of plant growth regulators, such as naphthalene acetic acid (naa), gibberellic acid (ga3), 6-benzylaminopurine (bap), ethylene releasing agent (ethrel), and anti - malformin chemicals viz ., silver nitrate and glutathione at the rate of 10 l solution volume . The cavity glass slides were subsequently placed onto the moist blotting sheets of petriplates to maintain the humidity . The condia of f. mangiferae were then allowed to germinate at suitable temperature (27 c). The number of conidi germinating after 12h and 24h was counted under the compound microscope.
The online version of this article (doi:10.1007/s13555 - 016 - 0093-x) contains supplementary material, which is available to authorized users . Onychomycosis is a common infectious disease, which if inappropriately treated can lead to spread of the infection, complications, diminished patient quality of life and may result in stigmatization . A fungal nail psychosocial perception study carried out in hong kong revealed wide - ranging misconceptions about onychomycosis treatments since 26% of respondents thought disinfectant solutions, such as vinegar or alcohol, were effective treatments, 42% incorrectly believed that antibiotics were an effective treatment, and 14% thought the only cure was complete removal of the nail . Another survey indicated that many people with mild, uncomplicated onychomycosis may never consult a physician, and hence are likely to rely on topical self - medication . To be effective, a topical treatment for onychomycosis must have potent fungicidal activity and be able to penetrate the nail plate, consisting of dense cross - linked keratin fibers held together by cysteine - rich proteins and disulphide bonds . Amorolfine 5% nail lacquer (loceryl, galderma sa, lausanne, switzerland) is a topical antifungal with proven clinical efficacy in the treatment of onychomycosis caused by dermatophytes, yeasts and moulds [57]. The active substance, amorolfine hydrochloride (5.574 g in 100 ml ethanol - based nail lacquer), has a fungicidal effect and broad antimycotic spectrum . A wide range of acid - based medical devices are commercialized to treat onychomycosis and common ingredients include acids to inhibit fungal growth, glycerin to hydrate and moisturize the nail, and urea to hydrate and gently dissolve the intercellular matrix of the nail plate, in addition to penetration enhancers and film - forming agents . However, there is a notable lack of published reports on their exact composition, mechanism of action and whether they can penetrate through the nail down to the nail bed . The objective of this study was to compare the antifungal activity of amorolfine 5% nail lacquer with three different commercially available acid - based medical devices using an in vitro nail penetration assay . Four products were tested: (a) amorolfine 5% nail lacquer; (b) ethyl lactate and acetic acid (excilor, vemedia, diemen, the netherlands); (c) citric acid and urea (scholl fungal nail treatment, bayer healthcare ag, leverkusen, germany); and (d) ethyl lactate, glycerin, lactic acid, and citric acid (nailner, youmedical, amsterdam, the netherlands). A total of 6 non - diseased big toe nails were taken from six fresh human cadavers and washed before use . For each test product, 3 pieces of nail plate from different donors were treated with 25 l test product / cm . The compound was applied to the center of uncut nails and allowed to spread evenly . After air - drying for 24 h, the nail was inverted and two disks of 4 mm diameter (biopsy punch) were cut from the center of each piece of nail (a total of 6 assays per test product). Each disk was placed, with the treated side facing upwards, at the center of a seeded agar plate of trichophyton rubrum (atcc mya-4438, manassas, va, usa) (25 10 conidia / ml). Disks from untreated nails were used as controls . Following 4-day incubation at 30 c, the mean zone of inhibition the standard error of the mean (sem) was calculated and compared between groups . A mean zone of inhibition> no formal statistical testing was considered necessary to compare the amorolfine 5% nail lacquer group with the other treated groups (which gave values of zero without any variability). This article does not contain any new studies with human or animal subjects performed by any of the authors . The mean nail thickness ranging from 0.86 to 1.09 mm was similar for assays for all test products (table 1). Nail disks treated with amorolfine 5% nail lacquer showed a mean zone of inhibition sem of 59.2 3.4 mm in diameter (table 1 and fig . 1e) all showed no zones of inhibition (mean effective zones of 0 0 mm) (table 1). Amorolfine 5% nail lacquer demonstrated potent antifungal activity when compared to the three medical devices tested (fig . 2).table 1measurement of zones of inhibitiontest compounddonornail disknail thickness (mm)inhibition zone diameter (mm)(a) amorolfine 5% nail lacquerdonor 110.8370donor 121.0951donor 231.0853donor 241.0968donor 351.2552donor 361.2561 mean (sem)1.0959.2 3.4(b) ethyl lactate and acetic aciddonor 410.50donor 420.540donor 231.020donor 240.980donor 550.980donor 561.110 mean (sem)0.860 0(c) citric acid and ureadonor 110.690donor 120.830donor 631.430donor 641.460donor 351.160donor 360.960 mean (sem)1.090 0(d) ethyl lactate, glycerin, lactic acid, and citric aciddonor 410.50donor 420.50donor 631.010donor 641.010donor 551.150donor 561.090 mean (sem)0.880 0(e) untreated controldonor 111.260donor 121.090donor 231.330donor 240.870donor 350.750donor 361.020 mean (sem)1.050 0fig . 1representative plates showing zones of inhibition of trichophyton rubrum growth in the nail penetration assay with a amorolfine 5% nail lacquer; b ethyl lactate and acetic acid; c citric acid and urea; d ethyl lactate, glycerin, lactic acid, and citric acid; e untreated controlfig . 2effect of amorolfine 5% nail lacquer and three medical devices against trichophyton rubrum in a nail penetration assay measurement of zones of inhibition representative plates showing zones of inhibition of trichophyton rubrum growth in the nail penetration assay with a amorolfine 5% nail lacquer; b ethyl lactate and acetic acid; c citric acid and urea; d ethyl lactate, glycerin, lactic acid, and citric acid; e untreated control effect of amorolfine 5% nail lacquer and three medical devices against trichophyton rubrum in a nail penetration assay the disk - diffusion method has been widely used to demonstrate antifungal activity of various drugs [8, 9]. In this study, amorolfine 5% nail lacquer demonstrated effective antifungal activity (inhibition zone 59.2 mm in diameter), corroborating previous findings showing that amorolfine 5% nail lacquer penetrates the nail to the site of infection in the subungual compartment [5, 10, 11]. Due to its high potency, even small amounts of amorolfine reaching the nail bed inhibit or kill the pathogens and amorolfine concentrations deep in the nail bed still exceeded the minimum inhibitory concentrations of t. rubrum (<0.0010.13 g / ml). Conversely, the three acid - based medical products tested did not show any antifungal activity in the human nail penetration model used . This easy - to - use in vitro nail penetration model was developed taking care to ensure that the antifungal effects witnessed were from the test compound penetrating the nail plate and the test substance could not spread over the edge of the nail biopsy to influence the growing fungi beneath the nail plate . The compound was applied to the center of uncut nails, allowed to spread evenly and dry before biopsy punches were taken from the center of the nail specimen to ensure there was no spillage of the test compound . By inverting the nail before taking the biopsy, the disks were cut in the direction of unexposed nail surface to exposed nail surface to ensure that the test compound was not artificially dragged from the biopsy punch . It is noteworthy that it would also be possible to increase the biopsy size or seal the margins of the biopsied nail plate . However, presumably there was no spreading over the edges since no zones of inhibition were observed with the acid - based medical devices under identical assay conditions in this head - on comparative study . The role of acidity (low ph) in the pathogenesis of dermatophytes is complex . Transmission electron microscopy has shown that many fungal cells were necrotic when t. rubrum or candida albicans was treated for 60 min in direct contact with 50% k101 nail solution (moberg pharma, bromma, sweden), a topical formulation of ph 4 containing 50 g propylene glycol, 15 g lactic acid and 10 g urea . The most prominent changes were observed with t. rubrum; the cell wall was clearly damaged, the membrane was disrupted and the content in the cytoplasm was degraded . The authors suggested that the presence of a diol (propylene glycol) disturbed cell wall integrity by an unspecific mode of action and the low ph may have contributed to the efficacy . Although ph 4 is not low enough to inhibit growth, the production of t. rubrum arthroconidia has been shown to be dependent on ph with 85% less arthroconidia produced at ph 4.5 compared to ph 7.5 . A ph of 3 was found to be fungicidal when sabouraud dextrose broths of different ph values were inoculated with t. rubrum and incubated for 14 days at 25 c . However, a penetration test in an in vitro porcine nail model showed that even after 120 applications of acetic acid (excilor), the ph measured in the nails was only 3.37, i.e., not low enough to be fungicidal . Furthermore, the lowest ph of 3.37 was only measured in the superficial parts of the nail (0.5 mm depth). Although acid - based solutions may penetrate and acidify the nails, as stated by the manufacturers, the difficulty in obtaining a low enough ph throughout the nail and in the nail bed may explain why the three acid - based medical devices demonstrated no fungicidal activity in the nail penetration model . Possible limitations of this in vitro study are that nails only received a single application of test product, and extrapolating from in vitro to in vivo may not be entirely accurate as in the nail environment in vivo, natural compounds may influence fungal growth . However, this in vitro nail penetration model has been shown to closely simulate in vivo testing and visually demonstrates both nail penetration and antifungal activity after a single application of amorolfine 5% nail lacquer . Clinical evidence of efficacy of acid - based solutions against onychomycosis remains scarce [17, 18]. The k101 nail solution (a mixture of propylene glycol, lactic acid and urea, ph 4) has been clinically demonstrated to be effective in the treatment of distal subungual onychomycosis [1820]. In a randomized placebo - controlled study (n = 346 k101, n = 147 placebo), more patients with 50% nail involvement achieved mycological cure after 26 weeks in the k101 group (27.2%) than in the placebo group (10.4%; p = 0.0012). This study confirms penetration through the nail of amorolfine 5% nail lacquer with potent antifungal activity in an in vitro human nail penetration model following a single application, whereas the three acid - based products tested had no antifungal activity under identical assay conditions . Below is the link to the electronic supplementary material . Supplementary material 1 (pdf 239 kb) supplementary material 1 (pdf 239 kb) this article does not contain any new studies with human or animal subjects performed by any of the authors . 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After the introduction of the laminoplasty by hirabayashi in 19779), the expansive open door laminoplasty has become a widely adopted surgical procedure for treating multilevel cervical spondylotic myelopathy (csm), ossification of posterior longitudinal ligament (opll), spinal cord injury without fracture and dislocation with cervical spinal stenosis . Thereafter, various techniques for cervical laminoplasty were reported by several authors . Based on the results in these reports, laminoplasty is usually classified as open door (single door) or double door (french door). Since then, various modifications and supplementary procedures have been devised for further improvement of safety and efficacy of the decompression and for stability of the spine . Traditional laminoplasty has proven effective in resolving neurological symptoms . However, because of the absence of rigid fixation, secondary narrowing of the spinal canal and neurological deterioration over the long - term follow - up2,14), several types of rigid fixation have been introduced in an attempt to improve the surgery . Plate fixation combined with bone struts and ceramic spacers, or spacers alone, or plate fixation alone has also been used during laminoplasty to construct a complete laminar arch5). However, the absence of bone healing on the open side, cannot achieve the goal of recreating a stable laminar arch without solid bony union on the hinge side28). Bone struts and ceramic spacers have a benefit that they can recreate a lamina arch, as bony healing can occur on both the grafted side . However, it has a risk associated with graft kickout, which causes reclosure of the lamina and root or cord compression by spacer dislodgement into the spinal canal12,23). We have used a modified unilateral open - door laminoplasty using hydroxyapatite (ha: hoya corporation pentax, tokyo, japan) spacers and malleable titanium miniplates (fig . We retrospectively reviewed the outcome of consecutive 38 patients who underwent modified unilateral open - door laminoplasty using ha spacers and malleable titanium miniplates between june 2008 and may 2012 . There were 29 men and 9 women who ranged in age from 38 to 77 years (mean 55.2 years). Of these, 17 patients had a history of trauma and 21 patients showed symptoms of cord compression without a history of trauma . For patients with a history of trauma, those who do not have a fracture or dislocation of the cervical spine were selected for the study . Patient's neurological status was evaluated using the frankel scale3) just before surgery and at the final visit . In this scale, the maximum score is' e' (normal motor, sensory function) and the minimum score is' a' (absent motor, sensory function). We calculated a scale of 1 to 5 (1=a, 2=b, 3=c, 4=d and 5=e). The presence of myelopathy was assessed using japanese orthopaedic association (joa) scale just before surgery and postoperative final visit . In this scale, recovery from myelopathy at postoperative final visit was calculated using the formula: (postoperative final visit joa scale - just before surgery joa scale)/(17-just before surgery joa scale)8). Radiologic evaluations of the cervical spine included plain radiography at preoperative, 1, 2, 4, 6, 12 months after surgery to assess for curvature of the cervical spine (fig . 2), and 3-dimensional computed tomography (3-d ct) scans at 12 months after surgery to assess for dimension of the cervical spinal canal and ha position, implant - related complications (fig . Evaluation of the cervical spine magnetic resonance imaging was performed at 12 months after surgery to assess for expansion of dural sac and decompression of spinal cord . The curvature of the cervical spine was measured by the angle formed by two lines extending from the posterior borders of the c2 and c7 vertebral bodies in the neutral position13), and the range of motion (rom) was measured by the summation of the cervical angles in flexion and extension (fig . The axial dimension of the cervical spinal canal was measured at the same lamina level on the axial ct image at preoperative and 12 months after surgery, and the number of pixels of spinal canal was calculated by image j program version 1.47 (nih, usa) (fig . All surgical procedures were performed with patients in prone position in slightly flexion of neck in a mayfield head fixator . A standard posterior midline approach allowed exposure of the cervical laminae at the targeted operative levels and laterally to the facet joints . Trough preparation in the hinged and opening sides of the laminofacet junction was performed with high - speed drill and kerrison punch . From rostral to caudal, each lamina was gently lifted using raney appliers . And after selecting proper size of ha laminar spacer (10 mm or 12 mm), the selected ha spacer and a malleable titanium miniplate was assembled using a miniscrew(6 mm) (fig . Each lamina was secured with the assembled ha spacer and miniplate complex using another miniscrews (6 mm) (fig . Analyses were performed using the ibm statistical package for the social sciences version 18.0 (spss inc ., chicago, il). All surgical procedures were performed with patients in prone position in slightly flexion of neck in a mayfield head fixator . A standard posterior midline approach allowed exposure of the cervical laminae at the targeted operative levels and laterally to the facet joints . Trough preparation in the hinged and opening sides of the laminofacet junction was performed with high - speed drill and kerrison punch . From rostral to caudal, each lamina was gently lifted using raney appliers . And after selecting proper size of ha laminar spacer (10 mm or 12 mm), the selected ha spacer and a malleable titanium miniplate was assembled using a miniscrew(6 mm) (fig . Each lamina was secured with the assembled ha spacer and miniplate complex using another miniscrews (6 mm) (fig . Analyses were performed using the ibm statistical package for the social sciences version 18.0 (spss inc ., chicago, il). The 38 patients who underwent' modified unilateral opendoor laminoplasty using ha spacers and malleable titanium miniplates' are summarized in table 1 . In total, 125 cervical laminae were operated in 38 patients . And the number of laminoplasty levels ranged from 3 to 4 (mean 3.29). Of these, 9 patients underwent additional anterior cervical discectomy and fusion (acdf) due to marked herniated nucleus pulposus . The mean operation time was 226.1min (110 - 465min), and the mean volume of intraoperative blood loss was 715.5 cc (350 - 1,500 cc). Postoperatively, the mean frankel scale was improved from 3.97 to 4.55 (p=0.002) (fig . 5). And mean joa scale was also increased from 12.76 to 14.63 with mean calculated recovery rates of 62.8240.57% . There was a statistically significant increase of postoperative final visit joa scale (p<0.001) (fig . Neuroimaging data were obtained in the 38 patients who were followed up more than 6 months postoperatively . Postoperative mr imaging revealed good expansion of dural sac and decompression of spinal cord in minimum follow up period of 1 year in all patients . There were no implant - related complications such as breakdown or dislocation of ha implants, delayed dural laceration in any case . And stability / fusion of the reconstructed laminae was found at minimum follow up period of 1 year in all patients . Postoperatively, the overall cervical rom was changed from 43.939.20 to 31.6010.24 (p<0.001). The lordotic curvature of the cervical spine was decreased from 19.09 to 15.60 (p=0.025). The difference of pre- and postoperative curvature of the cervical spine was ranged -3.508.64 (fig . The axial dimension of the cervical spinal canal was significantly improved from 1.750.48 cm to 2.700.58 cm (p<0.001) (fig . Postoperatively, the mean frankel scale was improved from 3.97 to 4.55 (p=0.002) (fig . 5). And mean joa scale was also increased from 12.76 to 14.63 with mean calculated recovery rates of 62.8240.57% . There was a statistically significant increase of postoperative final visit joa scale (p<0.001) (fig . Neuroimaging data were obtained in the 38 patients who were followed up more than 6 months postoperatively . Postoperative mr imaging revealed good expansion of dural sac and decompression of spinal cord in minimum follow up period of 1 year in all patients . There were no implant - related complications such as breakdown or dislocation of ha implants, delayed dural laceration in any case . And stability / fusion of the reconstructed laminae was found at minimum follow up period of 1 year in all patients . Postoperatively, the overall cervical rom was changed from 43.939.20 to 31.6010.24 (p<0.001). The lordotic curvature of the cervical spine was decreased from 19.09 to 15.60 (p=0.025). The difference of pre- and postoperative curvature of the cervical spine was ranged -3.508.64 (fig . The axial dimension of the cervical spinal canal was significantly improved from 1.750.48 cm to 2.700.58 cm (p<0.001) (fig . Laminoplasty is usually classified as open door (single door) or double door (french door)25). The single open door laminoplasty can be divided into classical suture fixation and titanium miniplate fixation25). There have been various modifications and supplementary instruments for laminoplasty to maintain hinge patency and provide secure fixation . For example, there are ha spacer, ceramic lamina, miniplate osteosynthesis, and allogenic or autogenous bone grafting33). The classical suture fixation is not only technically difficult but also do not provide rigid fixation . Furthermore, the neurological deterioration due to re - closure of opened lamina which is associated with cutout, breakage or stretching over time has been reported8,25,28). In one series using only suture fixation, up to 34% of patients demonstrated some degree of re - closure at one or more levels19). Also, the titanium miniplate fixation has a risk of dislodgement19). Allogenic or autogenous bone graft and a ceramic block have been used for the maintenance of opened lamina . These spacers have advantages such as a bony healing of the grafted side and maintenance of the laminar arch . But, they have a disadvantage such as root or cord compression due to graft kickout12,23,30). We found that our surgical technique was relatively easy compared with other techniques such as classical open - door laminoplasty and double - door laminoplasty5,8,9,24). Because making " small " holes and sutures on the lateral masses in the deep operative field and bone grafting was not necessary, laminoplasty - associated morbidities like graft kickout and spacer dislodgement were not observed in our series . While laminoplasty can provide sufficient decompression of the cervical spinal cord, preserving the motion of cervical spine, consistent reconstruction of the expanded laminae of the vertebral arches is needed for clinical recovery from the symptoms and to prevent postoperative kyphosis of the cervical spine and adhesion of scar tissue . A sinking or nonunion of the expanded laminae may lead to neurologic regression, segmental motor paralysis . A laminoplasty method should be technically simple, safe and provide immediate strong and rigid fixation . Though, laminoplasty relatively preserves the posterior elements compared with laminectomy, postoperative decrease of rom was frequently described27). Seichi et al . Reported that only 22% of preoperative rom was maintained after laminoplasty . These authors also mentioned that the loss of rom after laminoplasty was caused by unexpected fusion of the facet joints29). The high fusion rate in their series may have occurred because they used iliac crest as struts for the laminoplasty . Reported that 88% of preoperative rom was maintained after a laminoplasty preserving the c2 muscle and active rehabilitation18). Fujimori et al . Reported that rom preservation rate was 75% in the csm group, and 61% in the opll group . In their study, the loss of rom was caused mainly by a decrease in the extension angle in both groups . They assumed that this restriction of extension may have occurred partly as a result of impingement of the opened lamina because the spinous processes were preserved in all their cases . Meyer et al . Noted that plated laminoplasty led to a loss of rom in extension20). In our study, the percentage of rom preservation was 73.3222.39% . We assume that this decrease of rom may have occurred partly as a result of impingement of the opened lamina and leading to mild restriction of extension . Recently, several authors have presented new technique of cervical laminoplasty using hydroxyapatite laminar spacers and titanium miniplates . Tanaka et al . Reported that hardware failure or screw loosening did not occur during 36.3 months mean follow - up period after a single open door laminoplasty using hydroxyapatite laminar spacers and titanium miniplates in 22 patients31). Goto et al . Reported that hardware failure or screw loosening did not occur during 24.3 months mean follow - up period after a single open door laminoplasty using hydroxyapatite laminar spacers and titanium miniplates in 25 patients5). We first fixated the lifted lamina with spacer at the laminofacet junction before screw fixation . Due to stable lamina manipulation during the operation, it has less possibility of cord injury and prevents over - lifting of laminae . As it is once more fixed with miniplate, it helps to maintain continuous expansion of lamina . We suggest that it is not only technically safe, but also provides stable reconstruction, which can be an alternative method to avoid re - closure and dislodgement . The main component of ha spacer is hydroxyl compound of calcium phosphate that produces natural bone matrix . In the last 20 years, it has been used efficiently as oral, plastic, otological and orthopedic surgery instead of bone . Some experimental studies reported successful bone healing around ha by bone ingrowth into the pores and formation of bridging bone on the surface of the implant, proving osteoconduction1,32). In comparison with autografts, ha space has many advantages such as good biomechanical stability, reductions in operation time, blood loss, and donor site morbidity24). The laminae could be solidly fixed without using the soft malleable miniplates, because there existed no space between bone edge of lamina and ha spacer . Since there is no gap between bone edge of lamina and ha spacer, the laminar space is the main structure that takes the mechanical force compressing laminae vertically or laterally . Goto et al . Described that the screw loosening was more frequently occurred in cases in which only hard miniplates were used for laminar fixation without spacers because the miniplate and screws directly bear the stability of the laminae5). In our study, we found no implant - related complications in any case documented during the follow - up period . Our technique has drawbacks, such as additional cost of spacer, supplement time for spacer and plate assembly . Another shortcomings of this study include the small patient population and the brief follow - up duration . Nevertheless, it has advantage compared with' plate alone technique' and' spacer alone technique' . Ha spacer generates bone healing at the open side of lamina and it can reduce complication due to' graft kickout' as ha spacer fixed to plate . Future studies, enrolling more patients with longer periods of follow - up, will be necessary to evaluate postoperative axial pain and the longterm stability . Unilateral open - door laminoplasty using ha spacer and miniplates appears to be not only a safe, rapid, and easy procedure but also an efficient method by which to obtain an immediate, rigid stabilization of the posterior elements of the cervical spine after laminoplasty.
Hydrogen peroxide is a chemical compound that has a single peroxide chain which is unstable and splits into reactive radicals . In the past it was used for therapeutic enemas to relieve meconium ileus in infants and fecal impaction in adults for nearly 100 years, but this practice has largely been abandoned after it was proven that it can cause colonic damage similar to that of ulcerative colitis or pseudomembranous colitis . There are some case reports of chemical colitis resulting from self - administered hydrogen peroxide enema in naturopathic therapy in adults, but no case has been reported of caustic injury after ingesting hydrogen peroxide in children . We report a rare case of severe, acute colitis after administration of a hydrogen peroxide enema in a child . A 2-year old girl was referred to our emergency department complaining of bloody stools and cramping abdominal pain for a 12-hour period . The hydrogen peroxide solution was stored in a home refrigerator for cleansing properties, but it was mistaken for a glycerin suppository . A small amount of hematochezia was noted . On arrival at the emergency department, she was hemodynamically stable and in no respiratory distress . The abdominal physical examination revealed a soft abdomen with mild, diffuse tenderness and hyperactive bowel sounds . All results of blood tests were normal except for mild leukocytosis; white blood cell count was 10 900/l and the hemoglobin (hb) level was 11.4 g / dl . Her growth and development were normal and there was no evidence of physical or sexual child abuse . A flexible sigmoidoscopy was performed to assess the severity of the mucosal injury; it showed diffuse mucosal hemorrhage and marked edema with friability in the rectal mucosa (figure 2). Pathologic findings revealed erosion of the surface and sloughing with lymphocyte aggregation (figure 3). On subsequently obtaining a history, the caregivers indicated that the 35% hydrogen peroxide was used for natural health purposes . It was kept in a home refrigerator for cleansing properties, but it was mistaken for a glycerin suppository by them . The patient was managed conservatively with nothing per mouth and antibiotics (metronidazole, 30 mg / kg / d). Hematochezia resolved within six hospital days and abdominal pain was improved on the third hospital day . It has concentrations ranging from 3% to 90%, and is widely available in a variety of medical and household products . It is most often used as an irrigation and disinfecting solution, and 3% solutions are used as common household disinfectants . Therefore, it can be a common source of accidental poisonings, especially in children . Because of its instability and decomposability into water and oxygen in the presence of alkali or enzyme catalase, it can affect mucous membranes such as those in the liver, kidney, red blood cells and bone marrow . So, the quantity of oxygen produced exceeds maximum blood solubility; venous gas formation within the mesenteric and portal venous system can occur, resulting in systemic embolization and lipid peroxidation . Chemical colitis is a type of colitis, an inflammation of the large intestine, caused by the introduction of chemicals to the colon by an enema or other procedures . Like other injury, the pathogenesis of hydrogen peroxide colitis is thought to be secondary to the chemical reaction resulting in penetration of highly reactive oxygen species, resulting in damage to the colonic mucosa . The volume of oxygen liberated from the decomposition of hydrogen peroxide can be considerable, with 30 ml of 35% hydrogen peroxide yielding 3.5 l of oxygen, and it is thought to be secondary to absorption of hydrogen peroxide into the epithelial interstices and capillaries . Hydrogen peroxide enteritis with 3% solution can cause instant bubbling on the mucosal surface followed by a whitening of the mucosa termed the snow white sign . After exposure, the colon becomes distended within moments . Despite the potential for severe injury from hydrogen peroxide, many cases have occurred after accidental contamination of endoscopes with hydrogen peroxide in adults . Clinical sequelae range from mild, self - limited colitis to strictures, perforations requiring surgery or even fatality . For example, there is a report about a patient who developed chemical colitis with rectal bleeding after self - administration of a hydrogen peroxide enema in korea, and the clinical course was good with conservative management . But in another case report, the clinical presentation was consistent with acute ulcerative colitis symptoms sudden onset of abdominal pain with bloody diarrhea and the patient developed shock and died on the fourth hospital day . Endoscopy should be performed in all hemodynamically stable patients to assess for injury because signs and symptoms do not consistently correlate with the extent of injury . The recommended treatments include bowel rest, broad spectrum antibiotics and fluid resuscitation . In this case, the patient presented with symptoms that included abdominal pain and hematochezia . Clinical manifestations were similar to other cases of hydrogen peroxide enema in adults, and the radiologic and endoscopic findings were similar to other chemical colitis including mucosal friability and hemorrhage, but the snow white sign was not seen . Pathologic findings were also similar to other cases of chemical colitis by hydrogen peroxide enema in adults . Because this is the first report of chemical colitis caused by 35% hydrogen peroxide in children, we chose the recommended treatments for adults, and the clinical course was good in this child . However, she should be monitored for late - onset complications because there is no exact data about long - term complications of chemical colitis in children . Hydrogen peroxide is readily available from pharmacies in korea, and several websites are available to the public that describe its use as an enema . Therefore, we believe clinicians and caregivers should be aware of this potentially lethal chemical culprit and pay attention to handling hydrogen peroxide because the storage and use of 35% hydrogen peroxide for natural health benefits can result in an emerging source for more a serious accident in children.
Intraductal papillary neoplasms of the breast form a wide spectrum of pathological changes, with benign intraductal papilloma on one end of the spectrum and papillary carcinoma at the other end . Intracystic papillary carcinoma (ipc) is a variant of papillary carcinoma and accounts for 0.5 - 1% of breast cancers [2 - 4]. Physical examination and imaging findings are not usually sufficient to distinguish between benign tumors and this malignant intracystic lesion . The imaging technique which provides the greatest information about these tumors is ultrasonography [1, 2, 5]. Fine needle aspiration biopsy (fnab) can show a malignant lesion, but it is often inaccurate and excisional biopsy is usually necessary for definitive diagnosis [2, 4, 6]. Ipc was originally reported as a localized non - invasive carcinoma, but is occasionally associated with ductal carcinoma in situ (dcis) or invasive carcinoma around the main tumor [2, 3, 7, 8]. Complete excision of the cyst which should include the intracystic tumor is the treatment of choice [1, 2]. Here, a case of ipc in a 48-year - old woman a 50-year - old woman presented to the breast diagnostic clinic with 10 months history of an enlarging right breast mass . The mass was painless, when the patient was presented for physical examination . On clinical examination, she had a firm, well - circumscribed mass in the central part of the right breast at 2 o'clock, 2.03.0 cm . The mass was mobile and there was no evidence of nipple retraction, abnormal nipple discharge or skin thickening . Bilateral mammography using a dedicated free - standing unit showed an oval - shaped mass with smooth circumscribed margins in the central part of the right breast, measuring 2.0 3.0 cm, without calcification . There were no associated findings of skin thickening, microcalcification or parenchymal distortion (figure 1). Ultrasonography using a dedicated breast ultrasound unit showed a well - circumscribed regular- shaped cystic mass with an internal hypoechoic solid component projecting into the cyst from its wall measuring 22 18 28 mm (figure 2). Fine needle aspiration biopsy cytology of the cyst revealed a bloodstained fluid that was suspicious, papanicolaou grade iii cytology . Macroscopic examination of the specimen showed a cyst 23 cm with an intracystic papillary projection (figure 3). Histopathological examination revealed a cyst with a fibrotic wall and a papillary frond with a fibrovascular core and pleomorphic epithelial cells (figure 4). As regards the histologic findings, the breast cystic lesion corresponded to an intracystic papillary carcinoma . The breast tissue around the margins of the cystic lesion showed no evidence of malignancy . P53, cyclin p1, and c- erb- br expression was negative in our patient . A partial mastectomy was performed for the patient and there was no evidence of dcis or invasive carcinoma on histological examination at the surgical margin . Cystic carcinoma of the breast includes a heterogeneous spectrum of tumors . These include ipc with or without invasion, ductal carcinoma with cystic degeneration, and cystic hypersecretory ductal adenocarcinoma [3, 9]. Ipc is a rare neoplasm of the breast, so far classified as a histologic variant of dcis . However, this is no debated since the overall clinical and radiological presentation of ipc is different from dcis and metastatic cases have been reported . According to haris, nearly 4% to 70% of patients with ipc show dcis or invasive carcinoma around the main tumor . Ipc can be present as a pure form or associated with ductal carcinoma in situ or ductal carcinoma invasive around the tumor . The frequency of lymph node involvement, local recurrence and distant recurrence is 0% to 11%, 3% to 70% and 0% to 4%, respectively [6, 7, 11]. Ipcs are reported in patients from 25 to 80 years of age, with a peak incidence in patients aged 40 to 75 years old [2, 4]. These tumors may have a wide spectrum of presentations varying from a focally invasive lesion to a large mass located within a cystically dilated duct . The tumor may be small and detected only on mammography or may be large and palpable in clinical examination [2, 6, 12]. It may be seen anywhere in the breast, and not only in the retroareolar region . Recently, imaging diagnostic methods and less invasive histopathological examinations have made it possible to diagnose ipc before excisional biopsy and radical surgery . The mammographic finding of ipc is usually a well - circumscribed, high - density mass . Ipc usually presents as a smooth sharply circumscribed mass without an irregular or nodular contour, exceptwhen the tumor breaks through the wall of the cyst to invade the adjacent parenchyma [2, 12, 13]. Ultrasonography is the preferred imaging method to distinguish cystic from solid masses of the breast and to differentiate simple cyst from cysts with intracystic tumoral lesions [12 - 15]. Ultrasonography usually shows complex cystic and solid masses that have mild to moderate posterior acoustic shadowing or posterior acoustic enhancement [5, 13, 16]. In rare cases, color doppler ultrasound is a useful diagnostic method to distinguish between the solid portion of the cystic lesion and echogenic internal debri . Magnetic resonance (mr) imaging of the ipc shows mural nodules and internal septa . Mr imaging of the large ipc might show a multicystic appearance [13, 14, 18, 19]. It is well documented that mr imaging is more sensitive in detecting dcis around the ipc than other imaging methods . In younger patients, however, the presence of high background signals sometimes make it difficult to demonstrate the dcis with mr because of the high contrast of mr [19, 20]. It is feasible to perform a preoperative ultrasound - guided fine needle aspiration biopsy cytology (fnabc) or core biopsy of the cystic and solid component for cytological and histological studies . The aspirated fluid is often bloody, although the bloody aspirate is not pathoganomonic for ipc . Although ultrasound and aspiration biopsy are usually the first steps in the diagnosis of ipc, cytological examination also has high false - negative results due to necrotic materials, degenerative changes in the diagnostic cells and abundant obscuring blood in the cystic lesion [14 - 16].core needle biopsy is a useful tool for diagnosis of ipc, although it is important to keep in mind that core needle biopsy of the central solid portion of the mass cannot distinguish between in - situ and invasive lesions at the periphery of ipc . On pathologic examination, most tumors have a soft or friable consistency, and have a spherical circumscribed contour . Ipc usually appears as a mass with cystic component, which might have a papillary, nodular or shaggy internal surface . There is usually fibrotic tissue in the wall of the cyst, which limits invasion into the surrounding parenchyma . Most ipcs are characterized by well- circumscribed nodules surrounded by a fibrous capsule and large vessels might be seen within the papillary nodules or the internal septa of intracystic lesion . Hemorrhagic areas within the solid components of the tumor and blood within the cystic spaces are often recognized . Pathologically, intracystic papillary carcinoma can show four cellular patterns: cribriform, compact columnar epithelial, stratified spindle cell, or a transitional cell form resembling urothelium, or a combination of two or more of these patterns may be seen . Ipc may be associated with foci of dcis or invasive cancer, and necrosis is often a prominent feature when an associated invasive component is present [2, 7, 8]. The treatment of ipc is similar to other forms of breast cancer in which lumpectomy, segmentectomy and mastectomy are reliable actions . Sentinel node biopsies or axillary dissections are often performed for evaluation of axillary lymph nodes . It is important for the surgeon to know that approximately half of all patients with ipc are associated with dcis or invasive carcinoma to design an appropriate surgical treatment [2, 7]. Partial mastectomy without axillary lymph node dissection is the standard treatment for patients with non - invasive ipc, while patients with invasive ipc usually undergo mastectomy with lymph node dissection . The prognosis for ipc is usually very good with disease - specific survival rates approximately 100% . In cases with a large amount of nuclear atypia
The recent development of a radioreceptor assay for thyrotrophin has made it possible to detect immunoglobulins that inhibit the binding of thyrotropin to its receptor in some patients with autoimmune thyroid diseases . Although these immunoglobulins have been detected primarily in patients with graves disease, in whom their relation with thyroid stimulating antibodies has been extensively studied, they have also been found in a small portion of hypothyroid patients with hashimoto s thyroiditis . These immunoglobulins, originally called thyroid - stimulating immunoglobulins by smith and hall, are more appropriately termed thyrotrophin - binding inhibitor immunoglobulins, and they are now considered to be autoantibodies to portions of the thyroid plasma membrane, including the thyrotrophin receptor . In the present study, we investigated the activity of thyrotrophin binding inhibitor immunoglobulins in graves disease and various types of thyroiditis, and analyzed the clinical and laboratory features of patients who have these inhibitors . Thirty patients with graves disease, 13 patients with hashimoto s thyroiditis, 20 patients with lt - srh, 5 patients with postpartum thyroiditis, and 7 patients with subacute thyroiditis (sat) diagnosed inclusively between november, 1985 and october, 1986 have been studied (table 6). The diagnosis of graves disease was based on the following criteria: (1) nervousness, profuse sweating, palpitation, fatigue and weakness, weight loss, increased appetite, thyroid enlargement and exopthalmos, (2) elevation of serum thyroxine (t4), and (3) increased radioactive iodine uptake . The diagnosis of hashimoto s thyroiditis was based on the follwoing criteria: (1) hypothyroidism, enlarged, firm or hard thyroid gland, (2) decreased serum t4 and t3, (3) diffuse lymphocytic infiltration, often with a considerable admixture of plasma cells by the examination of fine needle aspiration cytology or biopsy, (4) decreased raiu . The clinical diagnosis of lt - srh was based on the following criteria: (1) painless, non - tender goiter, (2) elevated serum t4, t3, and (3) decreased raiu . The diagnosis of sat was based on the following criteria: (1) painful, tender thyroid gland, (2) fever, (3) elevation of the erythrocyte sedimentation rate (esr), (4) normal or elevcated serum t4, t3, and (5) decreased raiu . The clinical diagnosis of post - partum thyroiditis was based on (1) a non - tender diffuse enlarged thyroid gland, puffy face, (2) normal or decreased serum t4, (3) history of recent delivery, and (4) decreased raiu . Thyroid hormone concentrations were measured by radioimmunoassay (ria) with commercially available kits and t4 by tetrabead- from abbott . The serum thyroid stimulating hormone (tsh) was measured by immunoradiometric assay with the tsh riabead kit . Thyrotrophin binding inhibitor immunoglobulins (tbii) was measured utilizing the radioreceptor assay method of shewring and smith . Radioidine uptake was measured at 2 and 24 hours after oral administration of 50 cii . Laboratory findings in 10 normal controls, 1 male and 9 females, show serum t4 10.11.6 g / dl, serum tsh 1.870.94 iu / ml, tbii 3.03.0%, and i uptake at 2 hours 8.03.0% and at 24 hours 23.08.0% respectively (table 6). In 30 patients with graves disease, 8 males and 22 females, serum t4, tsh, and tbii were 19.21.8 g / dl, 0.760.06 iu / ml, and 44.98.7% respecitively . I uptake at 2 hours was 44.77.36% and at 24 hours 57.56.95% (table 1, 6). In 13 patients with hashimoto s thyroiditis, 2 males and 11 females, serum t4, tsh, and tbii were 3.564.37 g / dl, 24.0514.20 iu / ml, 8.698.06% respectively . I uptake shows 2 hour levels of 9.9 7.2% and 24 hour levels of 20.9 15.1% (table 2, 6) (fig . 1, 2). Laboratory data in 20 patients with lt - srh, 1 male and 19 females, shows serum t4 10.573.74 g / dl, tsh 1.730.95 iu / ml, tbii 7.632.32% respectively, and i uptake shows 2 hours levels of 4.72.9%, and 24 hours levels of 12.89.6% (table 3, 6). In postpartum thyroiditis, serum t4, tsh, and tbii were 7.183.53 g / dl, 1.741.14 iu / ml, 3.331.16% respecitively, while i uptake at 2 hours was 3.661.15% and at 24 hours was 10.337.64% (table 4, 6). In subacute thyroiditis, 7 patients all female, serum t4, tsh, and tbii were 10.472.56 g / dl, 1.460.65 iu / ml, 2.672.33% respectively . I uptake levels at 2 hours were 6.863.13% and at 24 hours 16.012.0% respectively (table 5, 6). There is almost universal agreement that thyroid autoantibodies exist and belong to the igg class of immunoglobins . These are antibodies against components of the thyroid plasma membrane, possibly including the tsh receptor . These immunoglobulins are thought to bind to their complementary antigenic regions on the plasma membrane and activate adenylate cyclase, thereby initiating a chain of reactions that leads to thyroid growth, increased vascularity, and hypersecretion of hormone . The recent development of a radioreceptor assay for thyrotrophin has made it possible to detect immunoglobulins that inhibit the binding of thyrotropin to its receptor in patients with autoimmune thyroid disease . Although these immunoglobulins have been principally detected in patients with graves disease, they have also been found in a small proportion of hypothyroid patients with hashimoto s thyroiditis . These immunoglobulins, originally called thyroid - stimulating immunoglobulins by smith and hall, are more appropriately termed thyrotrophin - binding inhibitor immunoglobulins and are now considered to be antibodies to portions of the thyroid plasma membranes, including the thyrotrophin receptor . Radioreceptor techniques are employed to demonstrate that igg is capable of inhibiting the binding of i - labeled bovine tsh to specific binding sites on the humane or porcine thyroid membrane . The present study shows that thyrotrophin inhibitor immunoglobulin levels are significantly elevated in all 30 patients with untreated graves disease (table 1, 6). Tbii activity was detected in 110 of 132 patients (83.3%) with untreated graves disease by cho et al .. in 9 cases of untreated graves disease, tbii activities ranged from 1476%, and were abnormally high in all cases when compared with 0.82% tbii activity in the normal controls by kim et al .. in the present study, the mean serum tbii activities were 8.698.06% in hashimoto s thyroiditis and 7.632.32% in lymphocytic thyroiditis with spontaneously resolving hyperthyroidism (lt - srh) (fig . 1, 2). Lt - srh is characterized by a painless, non - tender goiter, transient hyperthyroidism, decreased thyroid radioactive iodine uptake and focal or diffuse lymphocytic infiltration on biopsy (table 3, 6). Lt - srh has heen classified as a variant of subacute thyroiditis (sat), because the clinical course of each is so similar . However, dorfman et al . And nikolai et al . Have reported that lt - srh was a similar form of the chronic lymphocitic thyroiditis (clt), on the basis of the findings of positive thyroid auto - antibodies and lymphocytic infiltration on biopsy specimen . The tbii activity findings of our studies on hashimoto s thyroiditis and lt - srh, suggest that those diseases were a similar form of the disease from an immunological basis . Nikolai et al . Observed 54 patients with a history of lt - srh over a 1 to 15 year period follow - up . 23 patients (42.6%) were found to have a goiter after returning to normal thyroid hormone levels and persistent lymphocytic infiltration was noted on biopsy specimen . 5.6% of the patients showed permanent hypothyroidism while 32% of them had persistent antithyroid antibodies and 11% of the patients experienced recurrence . These finding suggest that lt - srh may be as a persistent and progressive disease as clt with recurrent episodes . Gorman et al . Pointed out the lack of oxyphilic change, as a suggestive histologic difference from clt, but nikolai et al . Were against this finding by the observation of focal oxyphilic changes in the biopsy specimens of lt - srh . One cannot conclusively confirm a diagnosis of lt - srh by only fine needle aspiration cytology of the thyroid gland but we noted numerous lymphocytes in 20 patients of the lt - srh group . The tbii acitivities in subacute thyroiditis (sat) were 2.672.33%, and were very low when compared with 3.03.0% in the normal control values in this study (table 5, 6). These findings suggested that the etiology of sat is rather than a classical autoimmune thyroiditis but of viral origin . Have described four functional stages of sat . In the first stage, acute inflammation causes the release of preformed stores of thyroid hormones to the inflamed thyroid gland . The patients show signs of clinical hyperthyroidism, and thyroid raiu is absent or quite low . In the second stage, over a period of a few weeks, serum levels of thyroid hormones decline to normal and clinical evidence of hyperthyroidism ceases . In the third stage, serum thyroid hormone values decline to the hypothyroid level, serum tsh rises above normal levels, raiu rises to normal or increased levels, and the patients may be clinically hypothyroid . The tbii acitivities in postpartum thyroiditis of this study were 3.331.16%, and were similar values compared with 3.03.0% or normal control (table 4, 6). Even though the tbii activities were almost equal between postpartum and subacute thyroiditis, but disease entity of these amino and co - workers reported cases of transient postpartum hypothyroidism with positive antithyroid antibodies . Nikolai et al ., as well as hamburger, report an unusually high frequency of postpartum silent thyroiditis patiensts in the population . About half of the amino group women with silent thyroiditis had episodes within 6 months of pregnancy . Fine needle biopsy in the thyrotoxic phase showed lymphocytic thyroiditis and needle biopsy showed focal involution of epithelium without pseudogranulomas . Moreover, antithyroid antibody titers fall during pregnancy and rebound at postpartum . A reduction of goitrous hypothyroidism during pregnancy has been reported . Patients with autoimmune thyroiditis, k - cells, and antithyroid antibodies increased after delivery . These findings indirectly support a possible autoimmune etiology for silent postpartum thyroiditis, involving on antibody - dependent cell mediated cytotoxic process.
Ms incidence has increased, particularly among females [14], indicating the influence of environmental factors . The integration of magnetic resonance imaging (mri) in diagnostic criteria since the 1990s and progress in immunomodulatory drug treatments have contributed to these increasing rates [5, 6]. Studies performed since 1964 confirm this observation in finland [810], which is located in northern europe between the latitudes 60 and 70n . High - risk areas in the western districts, seinjoki and vaasa, are characterized by an irregular incidence pattern, and an increased male risk was observed in seinjoki in 19791993 . We aimed to analyze the gender - specific incidence in high- and medium - risk areas in 19812010 to make inferences on the etiological factors correlated with high - risk groups . The incidence in the former medium - risk area pirkanmaa is studied for the first time . Incidence is regarded as the most important indicator of disease frequency, and changes in incidence reflect environmental factors in genetically stable populations . The total population in 2010 was 850630 . The pirkanmaa central hospital (population 485911) the seinjoki and vaasa central hospitals serve populations of 198469 and 166250, respectively . Both seinjoki and vaasa are mainly rural districts, while pirkanmaa is more urbanized, which is reflected in the age - structure and distribution of its populations . From 1981 to 2010, the population increased by 19% in pirkanmaa and 9% in vaasa, while a 2% decrease was reported in seinjoki . A 21% decrease in the age group 039 years was observed in seinjoki and vaasa . Mri scans have been used in diagnostics from 1990 in pirkanmaa and 1993 in seinjoki and vaasa . The national institute for health and welfare and the local ethical standards committee approved the retrospective examination of identified patient records in the hospitals in this study . Patients with multiple sclerosis in the health care districts of pirkanmaa, seinjoki and vaasa, including jacobstad, were recruited from hospital registries from january 1, 1981 to december 31, 2010 according to multiple sclerosis or morbus demyelinans diagnoses and optic- or retrobulbar neuritis (340, 341, and 377 in the international classification of diseases, icd versions 8 and 9, g35, g37, and h46 in icd-10). The patient records were then examined by the authors (markus holmberg, annukka murtonen, and marja - liisa sumelahti). Patient information was collected on the date, and the quality of the first symptoms and clinical findings were recorded . Confirmatory positive results at the time of diagnosis were obtained from patient records for mri, cerebrospinal fluid (csf), and evoked potentials (ep). Patient cases were included in the analyses when fulfilling the criteria of clinically (cd) or laboratory supported definite (lsd) ms as previously described by poser during january 1, 1981 to december 31, 2010, and when the patients resided in the study districts at the year of diagnosis . The patient's residence was updated using a personalized identification number and the year of diagnosis at statistics finland . The age - adjusted and gender - specific incidence per 10 person - years was calculated from january 1, 1981 to december 31, 2010 for three 10-year - periods (19811990, 19912000, and 20012010) with a 95% confidence interval (ci) by district . The incidence of the 5-year - periods did not change our overall conclusions (data not shown). Furthermore, to avoid chance variation, we used 10-year - periods in our calculations . Due to the small number of cases in the age - specific strata, indirect standardization was used to study regional risk during 19812010 . In this study, the incidence rates in pirkanmaa from 1981 to 2010 in each 10-year - age group were used to calculate the expected numbers of cases for both the seinjoki and vaasa populations . The resulting expected number of cases was then compared to the actual observed numbers of cases in seinjoki and vaasa, resulting in the standardized incidence rate (sir), which was the ratio of observed and expected cases . The onset symptoms were evenly distributed (the district - specific figures are not shown), as were the total f / m ratio (2.2), median age at onset (32.0), and age at diagnosis (37.0 years). The median diagnostic delay decreased from 4.0 years to 2.0 years (chi - square test p <0.001) during the study periods . The csf (including either the igg index, immunoelectrophoresis, or both) was performed in 8992% of the cases . The decreasing use of evoked potentials (27% to 19%) and the increasing use of mri (36% to 98%) was observed during the follow - up evaluation . The second scan, which was used to study dissemination in time and space in 65% of the cases, was performed during the next 18 months after the first scan in 58% of the cases, 94% of which were positive for ms . The delay to the first mri from the date of onset of symptoms was significantly decreased during the follow - up evaluation (figure 2). From january 1, 1981 to december 31, 2010, the age - adjusted incidence was 6.7 10 (95% ci: 6.27.2) in pirkanmaa, 12.5 10 (95% ci: 11.513.5) in seinjoki, and 8.3 10 (95% ci: 7.49.2) in vaasa . The sir was 1.9 (95% ci: 1.72.0) in seinjoki and 1.2 (95% ci: 1.11.4) in vaasa, compared to pirkanmaa (sir 1.0). The incidence for 10-year - periods (19811990, 19912000, and 20012010) is shown in table 3 . A steady increase in pirkanmaa (from 5.1 to 8.2) and a two - fold increase in both seinjoki (from 7.1 to 14.7) and vaasa (from 5.7 to 11.7) were observed . The f / m ratios remained stable in pirkanmaa (2.5, 2.1, 2.2) and increased 1.3-fold (1.8, 1.9, and 2.3) in seinjoki and 1.6-fold (1.8, 2.1, and 2.8) in vaasa . The f / m ratios in the 10-year - age groups according to district are shown in figure 3 . The highest f / m ratio of 3.5 was observed in the 1019-year - age group . In pirkanmaa, the ratio subsequently remained stable, whereas peaks were observed in seinjoki (age group: 3049-years) and vaasa (age group: 3039-years). Female risk values in seinjoki (sir 1.8, 95% ci: 1.72.0) and vaasa (1.5, 95% ci: 1.31.7) were similar and the age - specific incidence peaked at 3039 years (figure 4). Male risk in seinjoki was 2-fold compared to pirkanmaa (sir 2.00, 95% ci: 1.72.3) and the incidence was high in the large age group (2059 year olds). In vaasa, male risk (1.4, 95% ci: 1.21.7) peaked in the older age groups . Regional differences in ms epidemiology were among the earliest observations made in finland [810]. Earlier observations on high - risk ms in seinjoki have also been confirmed here, because the 30-year - incidence rate of 12.5/10 person - years in 2010 is the highest reported [8, 10]. However, the regional ms risk in seinjoki remains nearly two - fold (sir 1.9) compared to vaasa (1.2) and pirkanmaa (1.0) in finland . The southern and urbanized uusimaa district, which locates the capital city helsinki, and pirkanmaa (formerly hme) are regarded as regions with similar and standard ms risk in finland [8, 10]. This perception holds true, because an incidence of 5.1 in uusimaa was reported in 1993, which was similar to the rate of 5.1 in pirkanmaa in 19811990 . Consistent with these findings, contemporary studies performed in ireland and wales [2, 14] revealed an approximately 1.5-fold increase in the f / m ratio, observed also in this study in districts of seinjoki and vaasa from 19812010 . Ratios of 2.3 in seinjoki and 2.8 in vaasa in 2010 exceeded the corresponding ratios of 1.6 and 2.2 in an earlier study from 19791993 . However, the ratio in pirkanmaa remained stable, which was consistent with other previous studies [15, 16]. Ms affects women during their most active years of life . In this study, we showed the highest f / m ratio of 3.5 in the 1019-year - age group, where the total incidence remains low . Thus, the high f / m ratio in the youngest age group may indicate the presence of risk factors affecting this female subpopulation, independent of a geographically associated risk . The local risk of multiple sclerosis in seinjoki may be explained by genetic factors because hla characterization has demonstrated increased frequencies for b7, b12, and dr2 among both patients and their healthy relatives . The patients' families were subsequently examined for the myelin basic protein (mbp) gene on chromosome 18, which is a candidate gene involved in multiple sclerosis . Genetic linkage and association analyses suggested that a genetic predisposition to multiple sclerosis was closely linked to the mbp gene in this population . Despite this finding, the genetic background was similar in rural areas of partially swedish - speaking vaasa, finnish - speaking seinjoki, and the more urbanized pirkanmaa [1921]. Risk among males was increased in seinjoki despite the presence of a declining male incidence . The sir for males remained 2-fold (2.0, 95% ci: 1.72.3), with an sir of 2.8 (95% ci: 2.33.5) reported in an earlier study in 1979 - 93 . Although the sirs for males in both seinjoki and vaasa (1.4) were higher compared to pirkanmaa, they did not differ from the female ratios . Given that the populations examined were genetically homogeneous and stable and that the genetic changes in these populations were slow, the gender - specific incidence trends observed in this study indicated that environmental factors affecting the increased male ms risk in high - risk districts were less powerful than factors affecting the increasing female preponderance in the 1990s . According to this observation, the factors affecting males and females may be different in general, and some of these factors may act temporally and locally . The common awareness of ms and the availability of mri from 1993 in all districts, together with revised criteria and justified early dmt start in rrms have also precipitated ms diagnosis in finland [22, 23]. The diagnostic evaluation performed in this study was on the basis of the international and national current care guidelines [2426]. Due to a highly centralized health care system, we were able to obtain full coverage of the ms patients in this population - based study . The diagnosis - based incidence was used to avoid proportional incidence and underestimated risk, in addition to problems with recognition and the definition of the historical and often arbitrary onset symptoms in long - term follow - up evaluations . Active csf sampling in finland promoted the application of poser criteria, in which a positive finding is essential . Csf s considered fundamental in differential diagnostics for ms . Because purely clinical diagnoses were nearly nonexistent in 20012010, diagnostic specificity may be considered high in this cohort . However, the number of cases was fairly low, and the population in central parts of the country was younger compared to seinjoki and vaasa, which was among the factors that may confound a regional and temporal comparison of incidence . Thus, because small numbers were generally present in the age- and gender - specific strata, we decided to use indirect standardization in the risk calculations performed in this study . We observed a significant decrease in the diagnostic delay, which was consistent with rapid mr imaging after the first symptoms in 20012010 in each district . Despite this finding, the incidence during the last ten - year period in both vaasa and seinjoki showed a stabilization after a two - fold increase in 19812000 . In 19812010, the population at risk in the 1039-year - age groups decreased to 11% and 26% in vaasa and seinjoki, respectively . The number of ms cases in 20012010 decreased by 21% in both seinjoki (a decrease from 122 to 96 patients) and vaasa (from 80 to 63 patients), which was observed in males and caused the recent stabilization and observed incidence decline . In pirkanmaa, the population only decreased by 3% and the number of new ms cases increased by 23% (from 130 to 168 patients), which was accompanied by a steadily increasing trend . Despite these recent differences in population structure, the current sources of livelihood were similar in the districts, including those of the swedish - speaking population (22%) in the vaasa district, which may indicate that the environmental risk factors may be connected to changes and differences in lifestyle . In conclusion, seinjoki and vaasa represented high ms risk areas, where risk was observed to be high among females, peaking in the youngest age group, and among males in a large age group . However, high risk in general reflects both genetic and environmental effects . These effects may also be shared among other autoimmune diseases, such as type 1 diabetes mellitus, which predominantly affects males . The incidence of type 1 diabetes mellitus closely follows ms, both geographically and temporally, in finland . Thus, population - based case control studies are required to further characterize these factor effects, which may include a role for vitamin d, several lifestyle factors (including smoking, obesity, and hormone replacement therapy), and later childbirth among females.
Distal median nerve masses may be developed post - traumatic or non - traumatic . In this paper, we aim to present a 52 year old female case with a postraumatic neuroma of the median nerve in the left wrist . A 52-year - old female patient had accidental incised wound over her left wrist which was primarily sutured . Intraoperatively the mass was seen to arise from medial nerve and careful excision was done protecting the nerve . At one year follow up the patient is relived of her symptoms with no sensorimotor deficit . Post traumatic neuroma present as unrelieved pain and progressive swelling . A high index of suspicion should be kept in cases of wound that are primarily sutured over an area with superficial nerves . Soft tissue, skin or bone tumors should be considered in the differential diagnosis of the hand tumors . Neuromas are the most common solid peripheral nerve tumor frequently seen in 30 - 60 ages . Neuromas can originate after trauma to the nerves and such cases are rarely reported in literature [4 - 6]. In this paper, we aim to present a case with post - traumatic neuroma of the median nerve on the left wrist diagnosed by usg - clinical findings and confirmed with pathological reports . A 52-year - old female had accidental incised wound over her left wrist which was primarily sutured elsewhere . She was reffered to our centre 6 months after injury with unrelieved wrist pain and progressively growing mass on the wrist . Usg showed a solid tumor of size 2 x 3 cms and a decision for excision of the mass was taken . Local anaesthesia was given over the area and skin and subcutaneous tissues were dissected to isolate the tumor . The tumor was carefully excised from the surrounding tissue trying to keep the nerve intact . Intraoperative examination was possible as patient was under local anesthesia and minimal motor disability and hypoesthesia were noted (fig . Histopathological examinations revealed wallerian degeneration in evolutionary phase as revealed by disorganized growth in axonal and perineural structures (fig 4). The patient had a normal outcome with no pathological findings after one year follow - up median nerve neurinomma pathological findings hand injuries must be carefully examined because of anatomic and neurovascular complexity of hand and wrist . Sharp devices, gunshot injuries, occupational injuries and traffic accidents are the most common causes of hand injuries[7 - 9] in a study, seddon divided nerve fascicle injuries into three types; noropraxia, aksonothemesis and norothemises . It is stated in this study that noropraxia, does not need any surgical treatment, wallerian degeneration does not occur and the injury can recover completely . In axonothemesis, axonal integrity can be broken but endoneural tubes remain contact . Motor, sensorial and otonomic disfunctions can be present with complete wallerian degeneration . In norothemesis, when the nerve is completely disfonctioned, internal anatomy of the nerve can also be damaged and motor, sensorial and otonomic disfunctions at the distal region of the injury become significantly present . Clinical and electroneuromyographical (enmg) study results can be the same with axonothemesis . When the nerves are partially or completely damaged after injury, axonal regeneration initiated by proximal part of the nerve is mostly resulted as neuroma . In adults, mean regeneration rate is 1 - 2 mm / day . If fibrous barrier has been developed in injury site, than the axons cannot be able to find enough room to grow up and form a mass with fibrous tissue which is called neuroma . There are some articles about post - taumatic or non - traumatic median nerve masses in the literature . In 1969, a two cm sized neuroma occured after distal radius fracture and trapped in fracture line was disgussed . After having median nerve excision for neuroma, nerve ends were sutured and the patients showed remarkable improvement in the four - mounth follow - up . In the recent studies; chen et al . Reported a 42-year - old female cut the wrist in a suicide attempt and had subsequent tendon and median nerve repairment followed by rehabilitation . After neuroma on the median nerve region was confirmed by the excision of the lesion in the surgery, nerve repairment with a nerve graft was performed and the patient's symptoms improved significantly . Reported that distal traumatic median nerve neuromas are mostly painful and related with recurrent carpal tunnel syndrome which is generally managed with hypothenar fat pad flap . Hubert et al . Reported a schwannoma sized 4.0x0.5x1.2 cm and located in carpal tunnel . Bagatur and yalinkaya reported two giant lipomas in carpal tunnel . In a study of zdemir et al ., 10 of 14 patients had tumours located in the median nerve distribution area, whereas 4 were found on the ulnar nerve distribution . Four tumours were at the wrist level, 3 at the palm level, and 7 at the digital level . Our case was a post - traumatic neuroma of the median nerve on the left wrist and the diagnose was confirmed by clinical, ultrasonographical and electrodiagnostic examinations . After one - year follow - up, the patient had normal clinical findings without any pain or neuromotor loss . In addition, opposition loss is frequently present in distal level median nerve injuries . However, in our case, the patient had complete nerve injury with no opposition loss or any motor deficits . In conclusion, the masses on the wrists and hands are not rare and can be seen as posttraumatic or non - traumatic . However, trauma related soft - tissue masses on the wrist have usually been reported as case reports in the literature . Therefore, hand injuries, must be carefully inspected by experienced orthopedic surgeons because of anatomic and neurovascular complexity of hand and all possible outcomes should be considered in the management of these cases . Incised wounds over nerves should be carefully inspected for nerve injuries and primary suturing of such wounds can lead to post traumatic neuromas . These neuromas are cause of pain and discomfort, however they can be easily treated by excision of the lesion
While pancreatic resection is the only effective treatment with prolonged survival in operable pancreatic cancer and peri - ampullary cancer, it is also a procedure of significant morbidity and complications . The most commonly reported postoperative complications after pancreatic resection are pancreatic fistula, delayed gastric emptying, intra - abdominal sepsis and postoperative hemorrhage . Many vessels are encountered during pancreatic resection in particular pancreaticoduodenectomy (pd), especially superior mesenteric and portal veins, superior mesenteric and hepatic arteries, and celiac trunk . Hepatic artery is usually the only arterial supply of the liver, any injury of common hepatic artery (cha) carries the hazard of deprivation of the liver of arterial blood which may lead to acute liver failure, sepsis or liver abscess, and biliary complications in form of stricture or leak at site of anastomosis or bile duct vanishing syndrome in the most devastating scenario, . We describe a case of 70-year - old male, with eight weeks history of jaundice and repeated vomiting . No other relevant history was available . The significantly altered lab parameters showed a total serum bilirubin 5.7 mg / dl, direct of 4.7 mg / dl, inr 1.2, with normal alt, ast, serum electrolyte and serum creatinine . An ultrasound of abdomen was done which showed mild hepatomegaly with dilated intra and extra hepatic biliary channels, no features of cirrhosis of liver or portal hypertension and identified a hypo - echoic lesion in the head of pancreas . Based on these data we ordered contrast enhanced computed tomography (cect) scan for the abdomen, which revealed soft tissue mass arising from head of pancreas with 3 5 cm size with slight haziness of fat planes between it and stomach, superior mesenteric artery, portal and superior mesenteric vein are free from any invasion, with mildly dilated stomach with enlarged supra - pancreatic and peri - portal lymph nodes 2 3 cm with no distant metastasis . Patient was diagnosed as cancer head of pancreas with possibility of gastric wall invasion and decision was taken for surgical intervention and preoperative plan was for pancreaticodudenctomy . He was evaluated by cardiac, pulmonary and anesthesia physician and was found fit for surgery . During dissection of supra - pancreatic lymph nodes, common hepatic artery was accidentally injured and repair with proline 8/0 using surgical loups 3.5 was done . Intraoperative doppler on hepatic artery shows normal flow with ri 0.6 and psv 100 cm / s pre anastomotic and 125 cm / s post anastomotic and sat 71 ms . Postoperative day (pod) one, patient was stable, but doppler on hepatic artery revealed no detectable flow . (normal range; alt <41 u / l, ast <38 u / l) cect was done and revealed completely thrombosed common hepatic artery with no detected contrast beyond site of repair . Interestingly intrahepatic small accessory left hepatic artery with hardly detectable middle hepatic artery was found which followed to be arised from left gastric artery (fig . 1). Decision was taken to follow the patient for few days to detect the trend of laboratory findings . Surprisingly alt, ast, and bilirubin start to decrease significantly from pod2 with gradual improvement of all liver function parameters . Patient was shifted from icu to word on pod3; another doppler on hepatic artery did not detect any flow in the hepatic artery after the site of repair . On pod 10, u / l; ast 34 u / l and total bilirubin was 1.3 mg / dl (fig . Follow up two weeks later in out patient clinic revealed normalization of liver function tests with non - detectable hepatic artery flow by liver doppler and no relevant significant complaint . Follow up six months later revealed normal lab parameters, cect scan was done again and revealed completely thrombosed hepatic artery with small accessory left hepatic artery with hardly detectable middle hepatic artery (fig . Hepatic artery thrombosis following pd in not a commonly encountered complication . Although thrombosis of common hepatic artery may pass without significant morbidity due to compensatory flow of arterial blood to the hepatic artery proper through gastrodudenal artery (gda), but this is not the situation in pd as gda is ligated as a step of the procedure, keeping the hazards any thrombosis of cha will lead to complete deprivation of the liver from its arterial blood supply . The typical course of such a situation is hepatic infarction followed by acute liver failure and septicemia . Impairment of hepatic blood supply was termed as ischemic hepatitis (ih) which indicates inadequate hepatic perfusion, one of the possible causes of ischemic hepatitis is common hepatic artery thrombosis, it is characterized by significant rise in liver enzymes and bilirubin with undetectable doppler signals on common hepatic artery, cect is a confirmatory imaging modality is such a situation showing hypo perfusion of the liver, abrupt of the contrast at site of obstruction and hypo dense filling defect in the arterial lumen . Clinical presentations of ih ranges from devastating course which is fulminant acute liver failure due to ischemic hepatic necrosis and bile duct vanishing syndrome to less catastrophic courses as biliary stricture or liver abscess . All these undesirable sequel encourage early intervention and trial to revascularize the obstructed cha to avoid these morbidities,, . Here in our case we noticed small intra hepatic arterial vessels in spite of absence of any contrast in hepatic artery in cect scan with apparent thrombus at site of repair . A hardly detectable small accessory left hepatic artery with middle hepatic artery mostly arised from left gastric artery are noticed . Also faint hepatic contrast was noticed in the arterial phase in cect, this encourage our team to take the decision of not to operate the patient again to re - vascularize the artery as we believed, these small vessels were sufficient to give enhancement to the liver so they may be sufficient to supply the liver itself . Surprisingly liver enzymes and liver functions improve significantly starting from pod 2 which supported our believe regarding sufficiency of these small vessels to give arterial blood supply to the liver, six months later follow up cect scan showed a completely thrombosed cha with no detected liver infarction, abscess or biliary complications . Although hepatic artery is the only arterial supply of the liver, occasionally small accessory arteries may give significant arterial blood supply, which may compensate any blockage or thrombosis of hepatic artery, especially after ligation of gda as in pd . In such a situation liver enzymes and liver enhancement in cect scan act as surrogate markers to assess the sufficiency of this flow to the liver . Ramy hassan and ahmed zidan were involved in writing manuscript . Ahmed taha and bashir fadel were involved in patient management, and tameem ibraheem revise and approve manuscript for submission.
The natural history of idiopathic pulmonary fibrosis (ipf) is characterized by decreasing pulmonary function over time . Some patients may experience acute exacerbations (ae)sudden worsening of pulmonary function, increased oxygen requirements, new opacities on chest imaging, and a diffuse alveolar damage (dad) pattern of lung injury without an identifiable etiology . Estimates vary widely, but each year, a significant minority of ipf patients experience an ae (ae - ipf); about 50% of these patients require icu admission, and 80% die within 30 days [24]. A circulating biomarker is needed to allow clinicians to more precisely evaluate disease activity, in particular, to identify patients at risk for developing ae - ipf and to predict outcomes in patients who suffer ae . Serum carbohydrate antigen-6 (krebs von den lungen-6 or kl-6), lactate hydrogenase (ldh), and surfactant proteins a and d (sp - a and sp - d) levels have been evaluated as biomarkers of ipf; each possesses some predictive ability, but none is perfect [2, 58]. It is a versatile 16 kda peptide hormone product of the obese (ob) gene, and its main function is to regulate energy balance [9, 10]. Recently, studies have demonstrated that multiple tissues and cells can produce and secrete leptin, such as liver stellate cells, placenta, gastric fundic mucosa, pancreas, and lung tissue [1013]. The leptin receptor (lr) exists on hemopoietic precursors, immune cells, vascular endothelium, liver, adipose, and lung tissues [1014]. Beyond its metabolic function in energy regulation, leptin is implicated in various other physiological processes, including the immune response, inflammatory reactions, and the development of carcinomas, cardiovascular, nervous system, chronic liver, and several respiratory diseases [1215], including obstructive sleep apnoea (osa), obesity hypoventilation syndrome (ohs), chronic obstructive pulmonary disease (copd), and acute lung injury (ali) [13, 1618]. To our knowledge, in addition to leptin's potential role in ali, evidence suggests it has a critical role in fibrogenesis programs in the liver, kidney, and lung [14, 19, 20]. For example, leptin - resistant mice are protected from bleomycin - induced pulmonary fibrosis . In normal human lung fibroblasts, leptin augments the transcription of profibrotic genes induced by transforming growth factor - beta 1 (tgf-1). Among patients with ali or acute respiratory distress syndrome (ards), levels of leptin in bronchoalveolar lavage (bal) fluid, we speculate that leptin maybe an important factor in the development of aes and a biomarker for ae - ipf . The purpose of this study was to measure the expression of leptin in peripheral blood of subjects with ae - ipf and s - ipf . We aimed to begin to build the case for leptin as a biomarker of ae - ipf occurrence and severity by comparing plasma leptin levels in patients with ae - ipf versus those with stable ipf (s - ipf) and by examining correlations between plasma leptin and clinical variables among ipf patients . The study sample consisted of 62 patients with ipf (30 with ae - ipf and 32 with s - ipf) and 12 healthy controls evaluated at nanjing drum tower hospital, nanjing, university medical school from october 2009 to september 2014 . The diagnoses of ipf and ae - ipf were made in accordance with published criteria [1, 2]. Stable ipf (s - ipf) means the clinical symptoms, pulmonary function tests, and chest imaging are stable at least one month before collecting peripheral blood . The study was approved by the ethics committee at nanjing drum tower hospital and conducted in accordance with the principles set forth under the declaration of helsinki (1989). Peripheral blood samples were collected in evacuated tubes containing ethylene diamine tetraacetic acid (edta) from ae - ipf, s - ipf patients and healthy controls in the early morning on empty stomachs . The date of blood collection for patients was the day following hospital admission (including stable ipf patients who were admitted for the diagnoses or following up). The plasma concentrations of leptin (millipore corporation, usa) were measured by enzyme linked immunosorbent assay (elisa) according to the manufacturer's protocols . Kl-6 plasma levels (fujirebio inc, japan) were assayed by chemiluminescent enzyme immunoassay according to the manufacturer's instructions . Vital status was ascertained from medical records or follow - up telephone calls on november 24, 2014 . Survival time was calculated from the date of blood collection to the date of death or vital status ascertainment . Data are presented as mean standard deviation (sd) or as counts as appropriate . Differences in values for continuous variables between ae - ipf, s - ipf, and healthy controls were compared by using the kruskal - wallis test . The correlations between plasma levels of leptin and clinical variables were analyzed by spearman correlations . Survival curves were generated for each group using kaplan - meier estimates and compared by using the log - rank test . A multivariate cox regression model was built to examine leptin as a predictor of time - to - death while controlling for age and oxygenation . With only 30 deaths, including more than three predictors would lead to model over fit; thus, we selected clinically relevant variables to control while examining the effects of leptin on the outcome . Roc curves were used to assess the performance of leptin as a marker of ae - ipf (versus s - ipf) or death ., chicago il, usa) and prism version 5 (graphpad, san diego, ca, usa). The mean age of the 12 healthy controls (males / females: 10/2) was 59.50 4.89 years . In subjects with ae - ipf, leptin concentration was significantly greater than in subjects with s - ipf or healthy controls (figure 1(a)). Leptin concentration correlated with several clinical variables (table 2). Of the 62 subjects with ipf, 33 died, 23 survived, and in 6, vital status was unable to be ascertained . There was no difference (p = 0.71) in plasma leptin between decedents (n = 26, 22.41 12.29 ng / ml) and survivors (n = 3, 20.08 13.51 ng / ml) in the subgroup of subjects with ae - ipf, nor did plasma leptin discriminate between decedents (n = 7) and survivors (n = 21) in the s - ipf subgroup (13.57 7.16 versus 10.86 8.43 ng / ml, p = 0.203). Survival of subjects with ae - ipf was significantly worse than subjects with s - ipf (log - rank, p <0.001) (figure 2(b)). According to the cut - off value for leptin from roc curve predicting mortality was 13.79 ng / ml . Compared to subjects with leptin levels 13.79 ng / ml, those with levels> 13.79 ng / ml had shorter survival (log - rank, p = 0.003) (figure 2(c)). For survival among subjects whose kl-6 level was greater than the standard, accepted cut - off value (500 u / ml) was no different from subjects whose kl-6 level was 500 u / ml (p = 0.286) (figure 2(d)). In a multivariable cox model that included leptin and controlled for two other clinically important predictors, leptin was the independent predictor of time - to - death (table 3). The cut - off values of plasma leptin predicting ae and death for ipf patients were 15.52 ng / ml (sensitivity 67.86%, specificity 75%) and 13.79 ng / ml (sensitivity 68.75%, specificity 75%), respectively . The area under the roc curve for leptin in distinguishing ae - ipf from s - ipf was 0.761 (95% ci, 0.6440.879; p <0.001) (figure 3(a)) and for distinguishing decedents from survivors was 0.729 (95% ci, 0.5960.862; p = 0.004) (figure 3(b)). Crp is also significantly elevated in patients with ae - ipf than s - ipf (shown in table 1). By comparing the areas under roc curves, we found plasma leptin (0.761, p = 0.000; 0.729, p = 0.003) was a better biomarker of ipf acute exacerbation and survival than crp (0.734, p = 0.002; 0.690, p = 0.010) (supplementary figures 1(a) and 1(b) in supplementary material available online at http://dx.doi.org/10.1155/2016/6940480). In this study, we assessed plasma leptin concentrations in patients with ipf and in healthy controls and found the following: leptin concentration was higher in subjects with ae - ipf than those with s - ipf and higher in subjects who died than in those who survived to vital status ascertainment . Survival was significantly shorter for patients with leptin levels above, compared with those below, 13.79 ng / ml, and leptin was an independent predictor of survival when controlling for age and oxygenation . The causes and mechanism of acute exacerbation in patients with ipf remain unclear, although it is well known that patients with ae - ipf have significant morbidity and high mortality [1, 4]. Identifying a biomarker to predict ipf disease activity (particularly the occurrence of ae) and outcome of the disease is needed . Elevated serum levels of sp - a, sp - d, kl-6, and ccl18 have been found to be associated with acute exacerbation of ipf [8, 22]. However, these tests are not readily available, and their performance characteristics, while decent, are not perfect . Leptin is a proinflammatory cytokine and plays an important role in the pathogenesis of ards, liver, and lung fibrosis [14, 18, 23]. Peripheral blood leptin is a poor predictor of hepatitis c virus - related fibrosis and represents a negative prognostic factor for response to lamivudine monotherapy in these patients [24, 25]. Furthermore, in patients with ards, increased levels of bal fluid leptin are associated with adverse outcomes . Studies have identified the lung as a leptin - responsive and leptin - producing organ . Suggestions that leptin could play a key role in lung fibrosis include that it augments tgf-1 signaling in lung fibroblasts; it does so by inhibiting ppar. In addition, leptin signaling is required for bleomycin - induced lung fibrosis . Leptin resistance can protect ali - susceptible animals from a leptin - mediated inflammatory response to hyperoxia . Additional compelling data have emerged from the copd literature: during acute exacerbation of copd, circulating leptin levels increased inappropriately . This is hypothesized as being related to temporary disturbances in the energy balance and the systemic inflammatory response [26, 27]. We suspect leptin may be involved in the development of acute exacerbations of ipf, perhaps by mediating the inflammatory response to injury . The mortality of ae - ipf subjects was significantly higher than s - ipf subjects, consistent with the published reports [24]. In addition, subjects with plasma leptin levels above the cut - off value had significantly shorter survival than those under cut - off value . In contrast, kl-6 was unable to discriminate survivors from decedents in our study, although results from other studies suggest it may be useful for evaluating disease activity and predicting the clinical outcomes . The multivariate analysis showed that even while controlling for other predictors known to possess prognostic value, leptin remained an independent predictor of time - to - death . Roc curves also demonstrated that leptin can differentiate ae - ipf patients from s - ipf patients and predict the survival of ipf patients . First, the number of patients and normal controls enrolled was small, and the sample was entirely asian (chinese). These may substantially limit the ability to generalize results to the ipf universe, including the 13.79 ng / ml cut - off value . We have no data on the within - subject variability of leptin; levels from the same patient in different clinical circumstances (e.g., before, during, and after ae and when entirely stable) would be informative and provide data to further support the utility of leptin as a biomarker of disease . A prospective, multicenter, and multinational study of a larger patient cohort would be useful to help provide additional data on leptin . In summary, we found that leptin was elevated in ae - ipf and that high plasma leptin concentrations are associated with poor survival . Additional research is needed to confirm and extend these results, to determine whether and how leptin plays a role in the pathogenesis of ae, and to delineate the utility of plasma leptin as a biomarker of ae - ipf occurrence and predictor of survival in ipf patients.
Pharmacies assume an imperative part in the provision of health care and well - being at the community level . Generally, pharmacies have extended working hours, are commonly visited, and conveniently placed within the heart of community dwellings . The pharmacist's contribution is significant in health promotion, health maintenance, and health improvement of the communities in which they serve . Additionally, pharmacists are in a unique position to understand the needs of community members through daily interaction with patients and customers . In a few areas, pharmacists are often the patient's first point of contact, and for some their only contact with the health care professional . Pharmacists are capable of providing oral health information based on request by the patient, and a few did it proactively . Most of the pharmacists believed that providing oral health advice is within the realm of their profession . Additionally, individuals with lack of access to dental services and those from lower socioeconomic conditions are more likely to seek oral health - related advice from pharmacists . At present, a range of products effective in curing several of the oral health ailments and meeting the patient's expectations are available in the market, especially in pharmacies . There are products for managing dental decay, tartar buildup, gingivitis, dental hypersensitivity, teeth staining, and dental erosion, etc . Self - medication with over - the - counter drugs has for quite some time been a typical practice in communities . Presently, people have easy access to certain dental treatments because of the availability of numerous over - the - counter dental remedies, which were available only through the dental professionals in the past . Due to changes in conveyance of primary health care the role played by pharmacists in the provision of overall and oral health cares has been acknowledged as an important and relevant issue by the government of the united kingdom . Recently, the pharmacist's function evolved from dispenser of drugs to the approved member of the health care team . A study of pharmacists found that the pharmacist has to face at least one inquiry each week on some mouth - related issue; almost 50% of these inquiries were identified with oral sores or ulcers . Pharmacists provided some oral health advice, and in the coming years the demand for such advice is going to increase for which pharmacists have expressed their readiness to expand their knowledge related to oral health . Data from saudi arabia suggested that almost 34% of the pharmacists reported 10 daily requests for oral health advice mainly related to toothache, mouth ulcers, and mouth malodor . These complaints were mainly managed by medications and only very small percentages of the dental patients were referred to the dentist . Most of the recommendations of oral health products made by pharmacists in riyadh, riyadh province, saudi arabia were mainly based on personal experience and patient inputs rather than scientific information highlighting deficiencies in oral health knowledge . There are many ways in which the pharmacist can come forward to provide services such as encouraging the use of fluoridated tooth pastes and soft - bristle toothbrushes, advising on a healthy diet and eating habits, encouraging the use of preventive and therapeutic oral care services, and providing the needed information, skills, and motivation to individuals and caregivers about the prevention of oral diseases . By doing all these, pharmacists can actively participate in oral disease identification, assessment, management, referral, and prevention . Hence, there is a need for the pharmacist to be included as a member of a multidisciplinary oral health squad . Identifying and addressing gaps in oral health knowledge, attitudes, and practices of pharmacists is of utmost importance before they can be considered as a member of oral health promotion team . Hence, the aim of the study was to determine the prevailing oral health knowledge, attitude, and self - care practices among a sample of pharmacists from riyadh, riyadh province, saudi arabia . A cross - sectional study was conducted among a sample of pharmacists working in community- and hospital - based pharmacies in riyadh, riyadh province, saudi arabia . A list of pharmacies and hospitals in riyadh was obtained using online business and health insurance directories . From a total of 379 pharmacies, 100 pharmacies and 200 pharmacists were selected in the study by employing a convenience sampling approach based on the ease of accessibility . A sample size of 200 was determined by considering a value of 0.05, required power of 0.82, and effect size of 0.18 for one - tailed test for pearson product coefficient, which is computationally similar to spearman's rank correlation coefficient . (franz faul, universitat kiel, germany) a self - administered questionnaire was distributed to the study participants by trained dental interns and the data were collected . In addition, dental interns provided the needed explanation requested by the respondents . The study questionnaire was designed by a team of dental professionals after a thorough literature review . After an initial draft of the questionnaire was prepared in english, it was validated in two steps . First, the questionnaire was sent to dental public health professionals to obtain their expert opinion with regard to its simplicity, relativity, and importance . Second, a pilot study was conducted by selecting a small sample (n = 20) of pharmacists, and content authenticity was pretested to determine practicability, cogency, and rendition of responses . Moreover, all the amendments were incorporated into the questionnaire while ensuring its consistency with the published literature . After an in - depth discussion, questionnaire was finalized and subsequently distributed to the pharmacists for their response . The first part consisted of demographic information (gender, nationality, qualification, experience, and type of pharmacy) of the respondents . The second part elicited the basic knowledge of oral health, by asking 15 questions about dental plaque, causes, symptoms, consequences, prevention, and treatment of common oral diseases . The third part determined the attitude of the pharmacists toward oral health by asking five questions, in which their responses were assessed through a 5-point likert scale (strongly agree, agree, indifferent, disagree, and strongly disagree) of agreement . The fourth part assessed the oral self - care behavior by considering three questions on toothbrushing frequency, timing, and material / method . Every correct response in the knowledge section was scored 1, creating a scale in range of 015 . The overall knowledge scores were categorized into poor (05), average (5.110), and good (10.115). Similarly, for the attitude section strongly agree and agree responses were scored 1 while indifferent, disagree, and strongly disagree responses were scored 0 . A mean score of less than 4 was considered as negative attitude while a score of 45 was taken as positive attitude . In the oral self - care practices section, more appropriate responses of twice daily brushing, the morning and night after food and toothbrushing with paste were scored 1 with all other responses being scored 0 . A mean practice score of less than 2.4 was considered as inadequate and a score of 2.4 and above was deemed to be adequate for oral self - care practice . The higher the score, the better the respondent's oral health knowledge, attitude, and self - care practices . The study was approved by the research center of riyadh colleges of dentistry and pharmacy . Additionally, written informed consent was obtained from the respondents prior to participation in the study . Data collected through the questionnaire were entered and statistical analysis was performed by using ibm spss statistics for windows, version 21.0 . The mean and standard deviations for knowledge, attitude, and practice scores were calculated . Normality distribution of the data was assessed by applying kolmogorov smirnov and shapiro wilk's tests, which showed significant p value (p <0.05) indicating nonnormal distribution of the data . Whitney u and kruskal wallis tests were applied as a part of inferential statistics . Additionally, spearman's rank correlation coefficient was used to assess the association between knowledge attitude, knowledge practice, and attitude practice . For all statistical purposes, p <a cross - sectional study was conducted among a sample of pharmacists working in community- and hospital - based pharmacies in riyadh, riyadh province, saudi arabia . A list of pharmacies and hospitals in riyadh was obtained using online business and health insurance directories . From a total of 379 pharmacies, 100 pharmacies and 200 pharmacists were selected in the study by employing a convenience sampling approach based on the ease of accessibility . A sample size of 200 was determined by considering a value of 0.05, required power of 0.82, and effect size of 0.18 for one - tailed test for pearson product coefficient, which is computationally similar to spearman's rank correlation coefficient . A self - administered questionnaire was distributed to the study participants by trained dental interns and the data were collected . The study questionnaire was designed by a team of dental professionals after a thorough literature review . After an initial draft of the questionnaire was prepared in english, it was validated in two steps . First, the questionnaire was sent to dental public health professionals to obtain their expert opinion with regard to its simplicity, relativity, and importance . Second, a pilot study was conducted by selecting a small sample (n = 20) of pharmacists, and content authenticity was pretested to determine practicability, cogency, and rendition of responses . Moreover, the pharmacists' opinions were sought to make the questionnaire simpler and shorter . All the amendments were incorporated into the questionnaire while ensuring its consistency with the published literature . Data from the pilot study were not utilized in the final analysis . After an in - depth discussion, questionnaire was finalized and subsequently distributed to the pharmacists for their response . The first part consisted of demographic information (gender, nationality, qualification, experience, and type of pharmacy) of the respondents . The second part elicited the basic knowledge of oral health, by asking 15 questions about dental plaque, causes, symptoms, consequences, prevention, and treatment of common oral diseases . The third part determined the attitude of the pharmacists toward oral health by asking five questions, in which their responses were assessed through a 5-point likert scale (strongly agree, agree, indifferent, disagree, and strongly disagree) of agreement . The fourth part assessed the oral self - care behavior by considering three questions on toothbrushing frequency, timing, and material / method . Every correct response in the knowledge section the overall knowledge scores were categorized into poor (05), average (5.110), and good (10.115). Similarly, for the attitude section strongly agree and agree responses were scored 1 while indifferent, disagree, and strongly disagree responses were scored 0 . A mean score of less than 4 was considered as negative attitude while a score of 45 was taken as positive attitude . In the oral self - care practices section, more appropriate responses of twice daily brushing, the morning and night after food and toothbrushing with paste were scored 1 with all other responses being scored 0 . A mean practice score of less than 2.4 was considered as inadequate and a score of 2.4 and above was deemed to be adequate for oral self - care practice . The higher the score, the better the respondent's oral health knowledge, attitude, and self - care practices . The study was approved by the research center of riyadh colleges of dentistry and pharmacy . Additionally, written informed consent was obtained from the respondents prior to participation in the study . Data collected through the questionnaire were entered and statistical analysis was performed by using ibm spss statistics for windows, version 21.0 . The mean and standard deviations for knowledge, attitude, and practice scores were calculated . Normality distribution of the data was assessed by applying kolmogorov smirnov and shapiro wilk's tests, which showed significant p value (p <0.05) indicating nonnormal distribution of the data . Hence, nonparametric mann whitney u and kruskal wallis tests were applied as a part of inferential statistics . Additionally, spearman's rank correlation coefficient was used to assess the association between knowledge attitude, knowledge practice, and attitude practice . For all statistical purposes, p <a total of 200 pharmacists responded to the questionnaire, making the response rate 100% . A mjority of them were males (67.5%) of non - saudi nationality (65.5%). Most of the respondents had (90.0%) a bachelor of pharmacy qualification with 40% having 610 years of experience . Moreover, a majority of the pharmacists (65%) were working with chains of pharmacies, as mentioned in table 1 . Characteristics of the study participan more than 90% of the study participants had knowledge about the purpose of toothbrushing, prevention of tooth decay and gum disease, that bleeding gum indicated gum disease, that the effect of sweet retention on dentition led to tooth decay, methods of prevention of tooth decay, impact of oral health on general health, and consequences teeth loss interfering with speech . Around 83% of the study participants knew about tobacco chewing or smoking as the cause of oral cancer and 87.5% agreed about the possibility of moving irregularly placed teeth into the correct position . Nearly 77.5% of the participants said that improper toothbrushing was the reason behind gum diseases and 72.5% said regular toothbrushing and flossing could prevent gum diseases . About 72.5% of the participants mentioned 13 months interval as the duration for changing the toothbrush . Less than half of the study participants knew about that tooth decay and gum disease due to dental plaque and 47% pointed out that the bacteria in dental plaque was the reason for tooth decay . Only 12.5% of the study participants could recognize dental plaque as soft deposit on the teeth and 30% reported that gum disease was the common reason for tooth loss in old age as shown table 2 . Correct responses to the knowledge questions by pharmacists (n=200) when enquired about the attitude of study participants toward a regular visit to the dentist and replacing missing teeth by artificial teeth, 62.5% and 42.5% responded positively . A very high percentage (94.5%) of the participants strongly agreed that the smoking was a bad habit and 27.5% of the study participants showed a strong disagreement with the statement that dentists care only about treatment and not prevention . Similarly, 88.5% of the study participants showed a strong agreement with the statement that treatment of toothache was similar to the treatment of any other organ in the body, as mentioned in table 3 . Attitude toward oral health oral self - care practices among the study participants varied with less than half brushing their teeth twice daily and just 23% brushing in the morning and at night . Additionally, 92.5% of them brushed their teeth by using toothpaste and toothbrush, as shown in figure 1 . Oral self - care practices among pharmacists (%) the association of demographic characteristics and mean knowledge, attitude, and practices questions are expressed in table 4 . Male participants showed significantly more knowledge (5.44 vs 4.92, p = 0.001) and practice scores (2.18 vs 1.94, p = 0.008) toward oral health compared to their female counterparts . Similarly, non - saudis and those working in chain pharmacies showed significantly higher knowledge (5.42 vs 4.99, p = 0.003 and 5.42 vs 5, p = 0.005) and practice scores (2.18 vs 1.94, p = 0.007 and 2.19 vs 1.93, p = 0.003) compared to their saudi counterparts and those working in hospital - based pharmacies . Mean and standard deviations of knowledge, attitude, and practice scores pharmacists with master's degree qualification showed significantly higher mean knowledge scores (6.43 vs 5.83 vs 5.16, p = 0.001) compared to those with diploma and bachelor's qualifications . On the contrary, the mean attitude score was significantly higher among (3.99 vs 3 vs 2.93, p = 0.001) pharmacists with bachelor's degree qualification as compared to those with diploma and master's degree qualifications . But the mean practice score was found to be significantly higher among (3.00 vs 2.64 vs 2.03, p = 0.001) pharmacists with diploma compared to those with master's or bachelor's degree qualifications . Similarly, pharmacists with 1115 years of experience showed a significantly higher mean knowledge score as compared to those with 1620 years, 610 years, and 05 years' experience (5.96 vs 5.20 vs 5.19 vs 5.14, p = 0.008). The mean practice score was significantly higher among pharmacists with 05 years of experience (2.27 vs 2.04 vs 2.03 vs 1.92, p = 0.027) as compared to those with 1620 years, 610 years, and 1115 years' experience as shown in table 4 . The overall mean scores of oral health knowledge, attitude, and self - care practices were reported to be 5.27 1.05, 3.89 0.83, and 2.1 0.61, respectively, suggesting average oral health knowledge, negative attitude, and inadequate oral self - care practices as shown in the figure 2 . Mean and standard deviations of knowledge, attitudes, and practice scores among pharmacists the spearman correlation test revealed a significant positive correlation between knowledge and practice (r = 0.262, p <0.01), whereas knowledge and attitude (r = -0.149, p <0.05) as well as attitudes and practices (r = -0.196, p <0.01) were negatively correlated as shown in the table 5 . Spearman's correlation coefficients between knowledge - attitude (k - a), knowledge - practice (k - p), and attitude - practice (a - p) practice scor riyadh is rapidly growing capital city with many extension areas built to accommodate the increasing population . Pharmacies were the first to open and provide 24-h service to cater to the health needs of the population before hospitals or polyclinics were opened in such areas . Many of the residents staying in such localities receive some health- and oral health - related advices from the pharmacists . There are a variety of reasons as to why people seek health and oral health advices from the pharmacist in their vicinity . These include financial limitations, nonavailability of hospitals or polyclinics nearby, busy schedule, and difficulty in making appointment with the dentist or physician . The pharmacist's own knowledge, attitude, and oral self - care practices are major determinants for his / her role as an oral health promoter in a community setting . Hence, this study disclosed the knowledge, attitude, and self - care practices toward oral health among a sample of pharmacists from riyadh, riyadh province, saudi arabia . The resent study revealed average oral health knowledge among a sample of pharmacists from riyadh, riyadh province, saudi arabia . This could be due to several factors such as the lack of the pharmacist's education and training in oral health, lesser degree of motivation by pharmacists to know about oral health, lack of time, limited interaction with dental professionals, and lack of opportunities for professional development . Additionally, it has been reported that 50% of the pharmacists never met the dental professionals practicing close to their pharmacies though 90% of the pharmacies were located near dental clinics . Knowledge of dental plaque was limited and this finding was lower than that reported among other health professionals . A similar finding of poor oral health knowledge has been reported among bachelor of pharmacy students from malaysia . In general, the possible explanation for such a finding could be that most of the male pharmacists work in community - based pharmacies in which many people seek oral health advice and information about oral health care products . On the contrary, female pharmacists predominantly work in hospital - based pharmacies dispensing mainly doctor - prescribed medications to a limited number of patients . This could have resulted in less exposure to oral health information by female pharmacists unlike community - based pharmacists . Moreover, frequent interaction between oral health professionals and male pharmacists might be the reason for increased oral health knowledge among male pharmacists . Another possible factor could be frequent contact between medical representatives and male pharmacists with the former explaining about the oral health care products . The present study result is a contrast to other reported studies in which females demonstrated higher oral health literacy . In saudi arabia, community pharmacies are either owned by a single person or attached to a network of pharmacies scattered across a city, province, or the country . Most of the community pharmacies are managed by non - saudi pharmacists while saudi pharmacists prefer to work in the government sector . In the present study, non - saudi pharmacists showed significantly higher oral health knowledge as compared to saudi pharmacists . The probable reason could have been a higher content of oral health information in pharmacy education in the institutes from which non - saudis graduated . Attitude refers to the inclination to react in a certain way to a certain situation, and to see and interpret events according to certain predispositions . In the present study, it can be speculated that the average oral health knowledge among pharmacists could be the main reason for such a finding . In the present study, a significant positive correlation between the pharmacist's knowledge and practice was observed . On the contrary, a significantly negative correlation between knowledge attitude and attitude practice was observed . From this finding, it can be interpreted that lack of knowledge adversely influences the attitude leading to poor oral self - care practices of the pharmacists . However, higher oral health knowledge has a direct impact on oral self - care practices by improving the individual's self - awareness, self - protection, and personal hygiene performances . The strengths of the present study was a sufficient sample size and the varied oral health - related questions designed to disclose an existing level of oral health knowledge, attitude, and self - care practices . The limitations of the present study include the lack of a standard questionnaire for measuring the oral health knowledge and the nonavailability of any reported comparable study instrument . Therefore, the results of the present study were compared to the oral health knowledge of other health professionals . Moreover, the results of present study rely on self - reported data; the oral health information may have been biased through over- and underreporting due to social desirability . Dental publications offer conflicting results on the impact of the oral health knowledge, attitude, and self - care practices, and oral diseases . However, gathering such data has been useful in planning an oral health education program targeted toward pharmacists . There is scarce data available regarding oral health knowledge of pharmacists toward their own oral health knowledge, attitudes, and practices . To develop a sound strategy for improving the oral health of pharmacists, for this, additional studies are needed to be conducted in a wide geographic area, preferably by considering a nationally representative sample of pharmacists from saudi arabia by using reliable and indigenously developed measures . With enhanced oral health knowledge and practices, pharmacists can serve as oral health promoters in community pharmacy setups by catering to the oral health needs of the society . Pharmacists who were considered in the present study demonstrated an average knowledge, negative attitude, and inadequate self - care practices toward oral health . However, increasing oral health knowledge can have a profound improvement in oral self - care practices . Hence, there is a need to increase the basic oral health knowledge of the pharmacist to improve his / her own oral self - care practices and empower him / her to be utilized as a member of the oral health care team, thereby enabling him / her to provide evidence - based advice and guidance to clients . To achieve this, professional organizations such as the saudi dental society and saudi pharmaceutical society should take proactive steps to provide regular oral health educational programs to pharmacists.
Molar pregnancies represent a significant burden of disease on the spectrum of gestational trophoblastic diseases . The incidence appears to be higher in women from south asia, including a trend towards recurrent molar pregnancies [1, 2]. This higher trend in some populations has been attributed to nutritional and socioeconomic status . The purpose of our study was to review all the molar pregnancies at our institution . Our specific aims were then to determine the incidence, the associated morbidity, presentation, risk factors, and complications noted at our institution . This data would really be helpful in the context of the city which serves as the tertiary referral center for all the cases from the largest province of the country of pakistan . Ethical approval for the study was obtained from the department of obstetrics and gynaecology, bolan medical college, quetta, pakistan, as well as the local ethical committee for research, and the research conducted was performed according to the declaration of helsinki . The study was a prospective study carried out at the largest tertiary care government hospital in the city of quetta in balochistan, the largest province in pakistan . People belonging to different castes live here along with many refugees who were from the adjacent war - torn country of afghanistan and migrated during the early 1980s and 1990s . This represents one of the major teaching / tertiary care centers for the province . Due to lack of computerized medical records, we started our study from 1994 and were able to collect data for all cases that needed eph in gynae units i and ii at sandeman medical college hospital, quetta, pakistan, over period of 2 years from september 25th 1994 to september 1st 1996 . F. mahrukh's fcps degree and now is being sent for publication after completion of her degree . Furthermore, records after transfer of the gynecology department to another institution were not available for review . During this period, there were a total of 16,625 patients admitted to the both units of obstetrics and gynecology at our institution . 85 patients were diagnosed and confirmed with histopathological findings to have a molar pregnancy . Following identification of these patients, data regarding their basic demographics, risk factors, associated complications and followup were then collected and entered into a database developed in microsoft access 2000 . An attempt to follow up all 85 patients this data was then imported into the statistical package for social sciences version 14.0 (spss inc, chicago, ill, usa) for further analysis . As noted above, 85 patients (0.51%) were diagnosed with a molar pregnancy from a total of 16,625 patients admitted to the institution . This translates into an incidence of ~5.1 per 1,000 patients admitted to the institution . In majority of the patients, the classical presentation was that of delayed menstrual periods suggestive of pregnancy and vaginal bleeding . When combined with findings of an out of proportion enlargement of the uterus and absent fetal heart tones, the diagnosis of a molar pregnancy was suspected . This was then confirmed by measuring the serum beta - hcg (-hcg) and sonography . All sonograms were performed by author f. mahrukh herself and were noted to be helpful in aiding the diagnosis in 79.5% of the cases . Whereas, -hcg was noted to be elevated in all patients and was more than 50,000 miu / ml in 85.8% of the cases . Table 1 outlines the incidence of hydatiform mole in relation to parity and gravidity along with other associated factors . Although hydatidiform mole is more common in primigravidas, in our study most patients were multigravida . Among 85 cases of hm, the range of parity was from 0 to 17 . As outlined in table 1, in our study more than 70% of cases had size of uterus 412 weeks greater than gestational age; and more than 17% cases had size of uterus more than 12 weeks greater . Excessive uterine size is usually associated with markedly elevated levels of human chorionic gonadotropin (hcg) from trophoblastic overgrowth . Likewise, excessive uterine size was noted in 21/74 (28%) of patients at the new england trophoblastic center . Similarly, theca lutein cysts develop almost exclusively in patients with very high hcg levels which induce ovarian hyper stimulation and produce bilateral multilocular ovarian cysts . In this study, cysts were found in about 39% of cases out of which 17.64% patients had cysts greater than 6 cms in size . They usually produce symptoms like pelvic pressure and discomfort in many patients . In most of the cases, they regress spontaneously within 8 weeks . Depending on how the diagnosis of molar pregnancy was made (clinical versus sonographic), the incidence of theca lutein has been quoted to be between 20 and 46% of patients with molar pregnancy [57]. Since the advent and frequent use of ultrasound, larger sizes of theca lutein cysts and larger uterine sizes have become less common . Even though in our subset of patients, the cysts were present in about 39% of the cases, none of them needed emergency surgery for torsion . This is similar to another series from turkey, where only 1 patient needed emergency surgery for torsion . Vaginal bleeding was the commonest symptom (94.2%); apart from amenorrhea which was present in all the cases . This was also noted to be the commonest symptom by a large series by goldstein where it was present in 97% of their patients; and also in a series from china were it was present in 83.2% of the patients with hydatidiform mole [10, 11]. Likewise, preeclampsia and hyperemesis were reported in 12 to 27 percent and 20 to 26 percent of patients and occurred almost exclusively in those with markedly elevated human chorionic gonadotropin values and excessive uterine size . These patients were then followed up to see the management they received and their outcomes . Suction, dilatation, and curettage were noted to be the preferred method of management in 62 (72.9%) of the cases . 12 women underwent an elective hysterectomy as primary therapy for intact hydatidiform mole (hm); 5 of whom also underwent with bilateral salpingo - oophorectomy (bso) and 4 with unilateral salpingo - oophorectomy . All these patients were noted to be older than 40 and had complete their family planning . In patients who underwent salpingo - oophrectomies, reason was noted to be the large size of associated ovarian cyst on one and/or both sides . Both suction and sharp curettage specimens were submitted to the department of pathology for histopathological examination and confirmed the diagnosis in all of our cases . Persistence of uterine bleeding as a complication was noted in 73 (85.9%) of the patients; with it persisting in 13 (15.3%) patients for more than 2 weeks . The need for transfusion of packed red blood cells (prbcs) was noted in all patients; with a mean of 2.58 units of prbcs (range 16). Postmolar trophoblastic disease was diagnosed on the basis of a rise of hcg after the initial plateau or with the detection of metastases . 2 patients in our study period developed postmolar trophoblastic disease after complete molar pregnancy . After their initial management, patients were noted to be classified into low risk or high risk group based on goldstein's mole prognosis scoring system and received prophylactic single - agent chemotherapy with methotrexate if they fell into the high risk group (score of more than 4). Several investigators have reported that prophylactic chemotherapy at the time of molar evacuation reduces the frequency of postmolar tumor [12, 13]. Kim and colleagues reported in a prospective randomized trial that prophylactic methotrexate reduced the incidence of postmolar tumor from 47 to 14% in patients with high - risk complete mole . Prophylactic chemotherapy may be particularly beneficial in patients with high - risk complete moles when hormonal followup is either unavailable or unreliable . In patients at our institution, 70 (82.4%) of the patients fell into high risk group; and given the considerable lack of followup of patients presenting at our institution alongside the fact that a bulk of them are afghan refugees, single - agent prophylactic chemotherapy with methotrexate is usually employed at our institution if patients fall in the high risk group . Serial measurement of serum hcg levels is used to monitor the behavior of resident trophoblastic tissue after surgical evacuation of hydatidiform mole . A plateau is a pattern where there is neither decrease nor an increase> 10% or 50% of serum levels over 3 weeks on the basis of three or four consecutive weekly measurement over 3 weeks . Similarly, a rise is defined as increasing levels on the basis of two or more consecutive weekly measurements . Sharp regression is when levels immediately fall after evacuation; while slow regression is when serum levels have regress slowly to normal within 8 to 9 weeks from uterine evacuation . In our study population as noted in table 2, most of the patients in the high risk group who received single - agent chemotherapy after surgical evacuation had a sharp regression curve while most of the patients in group b had a slow fall over 68 weeks . Followup of these patients was attempted for a period of at least 2 years from the time of surgical evacuation . None of these patients developed persistent trophoblastic disease, invasive mole, or choriocarcinoma during the follow - up period . Even though, an attempt was made to follow these patients for 2 years, at government hospitals and due to the fact that a bulk of these patients are refugees, followup is a challenging task . This is also a factor in the government hospitals in the rest of the country as well, and would remain a continuous ongoing challenge . In our study, thus we have identified not only the incidence of molar pregnancies at our institution which serves as the largest tertiary care facility for the entire province of pakistan and neighboring afghanistan, but also highlighted the significant morbidity, management strategies, and associated complications . Limitation of our study includes limited followup for some of these patients, which as noted would remain a continuous ongoing challenge for the tertiary care hospital, since it serves not only the city but essentially the entire province and neighboring afghanistan . Single - agent chemotherapy is well tolerated in patients classified as high risk . As noted earlier by goldstein et al ., prophylactic chemotherapy may be particularly beneficial in patients with high - risk complete moles when hormonal followup is either unavailable or unreliable . Given our institutional setting and the demographics, we would advocate the prophylactic chemotherapy for the high risk group since followup is not entirely reliable.
Pentacene was deposited on a muscovite mica surface by physical vapor deposition from a knudsen cell in an ultrahigh vacuum chamber . The cell temperatures were adjusted between 450 and 490 k, in order to realize deposition rates between 0.01 ml / min and 4.5 ml / min, respectively . The mica samples (10 10 0.01 mm) were attached to a steel plate via tantalum wires . The steel plate was heated resistively, and its temperature was controlled by a ni, the temperature of the mica sample could be varied between 100 and 1000 k. typically, the sample holder was cooled during the experiments for a better residual gas pressure, but the sample was hold at 300 k during deposition by proper sample heating . For a quantitative determination of the deposited material a quartz microbalance was used, which was located next to the mica substrate . The mica substrate was cleaved with adhesive tape prior to installation into the vacuum chamber and subsequently gently sputtered by argon ions . Ten minutes of sputtering with 500 ev ar+ ions at an argon pressure of 5 10 mbar were sufficient to change the 5a film morphology from needle like islands, composed of lying molecules, to compact islands composed of standing molecules . The surface chemical composition was analyzed by auger electron spectroscopy and x - ray photoelectron spectroscopy . Thermal desorption spectroscopy was applied to determine the thermal stability of the pentacene film and the sticking coefficient . Ex - situ atomic force microscopy (nanosurf, easyscan2) was used to analyze the film morphology.
Moyamoya disease (mmd) is characterized by a chronic progressive steno - occlusive change of the distal internal carotid artery and abnormal development of a fine vascular network (moyamoya vessels) at the base of the brain . Takeuchi and shimizu11) had initially reported this disease in 1957, followed by suzuki11) in 1969 who named the disease as moyamoya (the japanese word for puff of smoke). The disease was initially known to be endemic where it was limited to japan but is now found worldwide.4 - 9)12)15) several studies have indicated a high prevalence of mmd in asian countries, particularly in japan, korea, and china.2)8)9) japan has the highest outbreak of illness frequency, followed by korea and china . The national prevalence of mmd in japan is well - documented.7)13) until this investigation, the epidemiology of mmd in korea has not been reported . We reviewed the national health insurance corporation (nhic) data to study the epidemiological features of mmd in korea . The authors requested data from the nhic of mmd patients who were treated from 2004 until 2008 in korea . The data in this study were standardized based on data of the south korean population in 2004 - 2008, which was provided by statistic korea . In the present investigation, these data gathered from nhic may not adhere strictly to the criteria for mmd proposed by yonekawa et . Al.14) and we could not gather detailed data including symptoms, diagnostic and treatment methods of the mmd . The nhic data revealed that in 2004, 2,539 mmd patients were treated in korea, representing a prevalence rate of 5.2 per 100,000 people . The respective numbers of patients and prevalence rate were 2,987 and 6.3 in 2005, 3,429 and 7.0 in 2006, 4,051 and 8.6 in 2007, and 4,517 and 9.1 in 2008, representing an annual increase of 15% . In 2008, 466 people were newly diagnosed with mmd, representing an incidence rate of 1 per 100,000 persons . The prevalence of mmd in korea increased from 5.2 per 100,000 in 2004 to 9.1 per 100,000 in 2008 (fig . 1, 2). The gender balance was 1,547 men (34%) and 2,970 women (66%). 3 . There is a bimodal peak pattern, first on teenagers (10 - 19 years old) and second among those in their forties (40 - 49 years old). The nhic data revealed that in 2004, 2,539 mmd patients were treated in korea, representing a prevalence rate of 5.2 per 100,000 people . The respective numbers of patients and prevalence rate were 2,987 and 6.3 in 2005, 3,429 and 7.0 in 2006, 4,051 and 8.6 in 2007, and 4,517 and 9.1 in 2008, representing an annual increase of 15% . In 2008, 466 people were newly diagnosed with mmd, representing an incidence rate of 1 per 100,000 persons . The prevalence of mmd in korea increased from 5.2 per 100,000 in 2004 to 9.1 per 100,000 in 2008 (fig . 1, 2). The gender balance was 1,547 men (34%) and 2,970 women (66%). 3 . There is a bimodal peak pattern, first on teenagers (10 - 19 years old) and second among those in their forties (40 - 49 years old). Mmd was first described in japan, and originally considered a disease that predominantly affected asian populations . Its prevalence is highest in japan, followed by korea and china.8) in japan, four national surveys were conducted: 1986, 1990, 1995, and 2003.7) in 2003, the total number of patients treated in japan was estimated at 7,700 and the annual rate of newly diagnosed cases in 2003 was 0.54 per 100,000 population . The estimated prevalence of mmd in japan has almost doubled during the most recent decade where data is available (3,900 in 1994 and 7,700 in 2003).7) in korea, mmd was first reported in 1969, based on case reports of hemangiomatous malformation of the brain.2) in korea, two co - operative studies on mmd patients treated at several neurological institutes were reported . However, to date, the national epidemiological features of mmd in korea have not been reported.2)3)5) this is the first study to report the korean national epidemiology of mmd . The authors obtained data from the nhic on mmd patients who were treated from 2004 to 2008 . These data included both symptomatic and asymptomatic cases . In 2008, 466 people were newly diagnosed with mmd, representing an incidence rate of 1 per 100,000 persons . The prevalence of mmd in korea has increased from 5.2 per 100,000 in 2004 to 9.1 per 100,000 in 2008 . The annual increase may reflect both an actual increase in new cases and an increased detection of asymptomatic existing cases due to an improved diagnostic capability and developing brain check - up system . Increasing numbers of mmd cases have been reported worldwide.1)7)15) in japan, the prevalence and annual rate in 1994 were 3.16 and 0.35 per 100,000 people and in 2003, it was reported as 6.03 and 0.54 respectively.7) the prevalence rate almost doubled in 10 years because of the increase in the occurrence rate . The higher detection rate and prevalence of mmd in japan may have contributed to the increase in newly diagnosed cases . After an induction of recently developed noninvasive diagnostic tools, asymptomatic mmd is being diagnosed more frequently.7) in korea, the prevalence rate of the mmd was 6.03 per 100,000 in 2003 . The well - known specific features of mmd are a bimodal pattern of age distribution and female prevalence . This study revealed a bimodal age distribution where the highest was observed among teenagers, followed by those in their forties . National figures for mmd in china have not been reported, but there is data on the epidemiological and clinical features in nanjing, a provincial capital city.9) the annual average detection rate was 0.43 per 100.000 and the prevalence rate was 3.92 per 100,000 . This was lower than the prevalence of 6.3 found in japan and 5.2 found in korea, but similar to taiwan.4) mmd has been observed throughout the world and it affects individuals from different ethnic backgrounds.9) but it is rarely observed among americans and europeans . A recent european study has reported an incidence of approximately 1/10 th of that in japan.15) studies in the united states suggest an incidence of 0.086/100,000 persons.12) compared to whites, ethnicity - specific incidence rate ratios were 4.6 for asian americans, 2.2 for african americans, and 0.5 for hispanics.12) based on the japanese national data, the prevalence rate of mmd between japan and korea are similar . In conclusion, the prevalence rate of mmd was 5.2 per 100,000 in 2004 and it increased at a rate of 15% annually through 2008.
The question which diagnostic test should be used in case of abnormal uterine bleeding is still a matter of debate . Diagnostic algorithms for intrauterine disease are usually based on studies and meta - analyses published in the literature and mostly include one or more of the following diagnostic modalities: office endometrial sampling (dijkhuizen et al ., 2000; hysteroscopy (clark et al ., 2002b), ultrasound (smith - bindman et al ., 1998; gupta et al ., 2002; tabor et al ., 2002), fluid contrast sonohysterography (shg) (de kroon et al ., 2003). Office endometrial sampling is accurate in the diagnosis of endometrial cancer (clark et al ., 2002a), but misses most focal lesions, such as polyps (van den bosch et al ., 1995). Hysteroscopy is considered the gold standard to diagnose focal intracavity lesions, but performs somewhat less in the detection of malignancy (clark et al ., 2002b). Ultrasound is useful in the triage of postmenopausal patients at risk for endometrial disease, by measuring the endometrial thickness (tabor et al ., 2002; smith - bindman et al ., sonohysterography (shg) has been proposed as first step examination in the diagnosis of focal lesions such as endometrial polyps and intracavity fibroids (de kroon et al ., 2003). The choice of a diagnostic examination may also be influenced by the personal skills and preference of the clinician, as well as by the availability of the diagnostic tools . Each patient is described by a set of attributes: variables with numeric (e.g. Endometrium thickness in mm) or symbolic values (e.g. Menopausal or premenopausal). Each non - terminal node of a decision tree contains a test on one or more attributes (e.g. Sis) which result (e.g. Lesion or no lesion) is used to select the branch to follow from that node . The terminal nodes reflect the decision outcomes (e.g. Normal or abnormal) (quinlan, 1993). In this study, we built decision trees to predict intrauterine disease, based on a clinical data set, and using mathematical software . A dataset of 402 consecutive patients evaluated between october 2004 and november 2006 at the one stop bleeding clinic of the university hospital leuven were included . (2008). At the one stop bleeding clinic the patients underwent a grey scale ultrasound (n = 402) followed by colour doppler examination (n = 402), followed by contrast sonohysterography (n = 398), office hysteroscopy (n = 381) and endometrial sampling (n = 243). If indicated the patients underwent operative hysteroscopy (n = 131) or hysterectomy (n = 14). At grey scale ultrasound the total endometrial thickness was measured in the midsagittal plane . The ultrasound examiner also reported the presence or absence of an intracavitary lesion, and if applicable, the type of lesion (e.g. Endometrial polyp, intracavitary myoma). At color doppler examination the total endometrial thickness was measured again and the presence or absence of a pedicle artery (timmerman et al ., 2003) was recorded . The presence or absence of an intracavitary lesion, and the type of lesion the histology results at endometrial sampling were classified as nominal variable: abnormal (including endometrial polyps, intracavitary myoma, endometrial hyperplasia and endometrial malignancy), normal (including endometrial atrophy, proliferative- and secretory changes of the endometrium) or no histology (in the absence of a histology result). (including endometrial atrophy, proliferative- and secretory changes of the endometrium) or abnormal (including endometrial polyps, intracavitary myoma, endometrial hyperplasia and endometrial malignancy). The final diagnosis was based on ultrasound with sis, hysteroscopy, endometrial biopsy, operative hysteroscopy and hysterectomy findings in 8.0%, 16.2%, 39.8%, 32.6% and 3.5%, respectively . Sensitivity is defined as the proportion of patients with an abnormal diagnosis that are correctly identified as such, while specificity refers to normal diagnosis . Two groups of parameters were considered in the prediction of the final diagnosis: pre - test parameters (including patient s age, weight, height, parity, menopausal status; presence or absence of abnormal bleeding symptoms; the result of a cervical cytology smear within the last 6 months) and post - test parameters (including the total endometrial thickness as measured at grey scale ultrasound, the presence or absence of an intracavitary lesion at grey scale ultrasound, the type of intracavitary lesion seen at ultrasound, the presence or absence of a pedicle artery sign, the endometrial thickness measured at color doppler imaging, the presence or absence of an intracavitary lesion at sis, the type of intracavitary lesion seen at sis, the presence or absence of an intracavitary lesion at office hysteroscopy, the type of intracavitary lesion seen at hysteroscopy, the histology of the endometrial sampling). The data set was split into a training set (first 70%: 281 patients) and a test set (last 30%: 121 patients). The reported performance values are presented for the test set on which the decision trees are validated . To make the test results comparable, only 94/121 test patients without missing values for any of the variables included in the studied decision trees the decision trees are built using an iterative process: a decision tree is first built on the complete training set of 281 patients with and without a cross - validation strategy with 10 folds . The important variables singled out in these trees are selected, and in the next round patients with missing values for these selected variables are excluded and a new decision tree is built on the reduced training set . Again resulting decision trees will incorporate some of the variables, while others are not used . In the next round the patients with missing values for the latest selected variables are excluded and another decision tree is built that will select some of the variables . The final decision tree obtained with cross - validation is chosen based on its highest cross - validation training performance with a small difference in full and cross - validation training performance to avoid overfitting . Furthermore, reduced - error pruning is applied to reduce the chance on overfitting the training data, which means that a large tree is grown before replacing some branches by a terminal node . The training set is split into three folds of which one is used for pruning and the rest for growing the tree . After building a decision tree on the training set, patients with missing values for one or more of the included variables are removed from the test set before validating the decision trees . First a decision tree has been built using both pre - test and post - test parameters . Second a decision tree only based on post - test parameters was built . Finally a decision tree was designed without using the hysteroscopy variables . The waikato environment for knowledge analysis (weka) software (version 3.4.8, university of waikato, new zealand) was used for the development of decision trees with the j48 algorithm, a slightly modified version of c4.5 (quinlan, 1993). The average (sd) patient s weight and height was 69.9 kg (14.2) and 163.8 cm (6.1), respectively . Fifty - three percent of women were premenopausal and 12.7% were nulliparous (mean parity 1.9; sd 1.2). The mean endometrial thickness at grey scale ultrasound examination was 9.6 mm (sd 6.8). In 11 patients (2.7%) endometrial cancer was diagnosed, in 24 (6.0%) endometrial hyperplasia, in 111 (27.6) an endometrial polyp and in 48 (11.9%) an intracavitary myoma . The first decision tree using both pre - test and post - test parameters (tree #1) was built on the training set containing 254 patients . The selected variables were: the presence or absence of an intracavitary lesion on hysteroscopy, the parity, the menopausal status, the histology result at office endometrial sampling and the endometrial thickness as measured at grey scale ultrasound examination (fig . 1). The test set contains 121 patients . After removing patients with missing values (for the presence or absence of an intracavitary lesion on hysteroscopy, the histology result at office endometrial sampling and the endometrial thickness at grey scale ultrasound), ninety cases (89.1%) were correctly classified by tree #1, the sensitivity was 95.8% and the specificity 83.0% . ($) pre - test parameters include patient s age, weight, height, parity, menopausal status; bleeding symptoms; cervical cytology . () post - test parameters include ultrasound-, color doppler-, sis-, hysteroscopy- findings as well as the histology results after endometrial sampling . () abnormal was defined as the presence of benign or malignant intracavitary pathology; normal was defined as the absence of any intracavitary lesion, and includes endometrial atrophy as well as proliferative- and secretory endometrial changes . Post - test parameters (tree #2) was built on the training set containing 243 patients . The selected variables were: the presence or absence of an intracavitary lesion on hysteroscopy, the histology results of the office endometrial sampling, the presence or absence of an intracavitary lesion on sis and the presence or absence of a pedicle artery at color doppler imaging (fig . 2). After removing test patients with missing values (for the presence of an intracavitary lesion on hysteroscopy, the histology results of endometrial sampling, the presence of an intracavitary lesion on sis), 98 patients were left in the test set . Eighty - seven cases (88.8%) were correctly classified by tree #2, the sensitivity was 97.9% and the specificity 80.4% . () post - test parameters include ultrasound-, color doppler-, sis-, hysteroscopy- findings as well as the histology results after endometrial sampling . () abnormal was defined as the presence of benign or malignant intracavitary pathology; normal was defined as the absence of any intracavitary lesion, and includes endometrial atrophy as well as proliferative- and secretory endometrial changes . The third decision tree was designed using both pre - test and post - test parameters but without using the hysteroscopy variables (tree #3). The selected variables were: the presence or absence of an intracavitary lesion on sis, the histology results at office endometrial sampling and the patient s parity (fig . Ninety cases (84.1%) were correctly classified by tree #3, the sensitivity was 92.3% and the specificity 76.4% . ($) pre - test parameters include patient s age, weight, height, parity, menopausal status; bleeding symptoms; cervical cytology . () post - test parameters include ultrasound-, color doppler-, sis-, hysteroscopy- findings as well as the histology results after endometrial sampling () sis = saline infusion sonography . () abnormal was defined as the presence of benign or malignant intracavitary pathology; normal was defined as the absence of any intracavitary lesion, and includes endometrial atrophy as well as proliferative- and secretory endometrial changes . To be able to compare the performance of the different decision trees, only those patients without missing values for any of the selected variables the diagnostic accuracy was 88.3%, 88.3% and 84.0% for tree #1, #2 and #3 respectively, the sensitivity and specificity was 95.5% and 82%, 97.7% and 80.0, 93.2 and 76.0%, respectively (table i). (tree #1 using both pre - test$and post - test parameters; tree #2 using only post - test parameters; tree #3 using pre - test and post - test parameters without hysteroscopy data). () intracavitary pathology includes both benign- and malignant disease (endometrial polyps, intracavitary fibroids, endometrial hyperplasia and endometrial cancer). ($) pre - test parameters include patient s age, weight, length, parity, menopausal status; bleeding symptoms; cervical cytology . () post - test parameters include ultrasound-, color doppler-, sis-, hysteroscopy- findings as well as the histology results after endometrial sampling . Unlike the one - to - one comparison between two tests, the method used in this study enables the comparison between different decision trees containing multiple tests . Our study used the data of individual patients to build and to validate the diagnostic algorithms . The presented decision trees were built by a non - biased mathematician and are therefore not influenced by the clinician s preferences . Other studies have built decision trees based on hypothetical likelihood ratios and assumptions extracted from other series (clark et al ., 2006). Basically, all 3 trees start with an imaging technique: hysteroscopy or sis). If no lesion was seen at first evaluation, other diagnostic steps are proposed to lower the false negative rate . Most clinical algorithms also propose an imaging technique as cornerstone examination in the diagnosis of intracavitary lesions (van den bosch, 2007). Imaging, beit hysteroscopy or sis, selects who needs endometrial sampling (e.g. In case of a diffusely thickened endometrium), who should undergo operative hysteroscopy (e.g. In case of an endometrial polyp) and who does not need further testing (i.e. In case of a thin and regular endometrium). Imaging may also act as quality control during subsequent endometrial sampling procedure: e.g. If a thickened endometrium had been seen on ultrasonography or hysteroscopy, and if endometrial sampling hardly yields any tissue, the lesion most probably has been missed during the sampling . The decision trees were built to diagnose intrauterine disease including both benign and malignant lesions . The algorithm is expected to depend on the prevalence of the endpoint (i.e. Benign and malignant intracavitary disease) in the study population . In our series, consisting mostly of perimenopausal women presenting with abnormal bleeding, the prevalence of focal intracavitary lesions, such as endometrial polyps, was relatively high, while the prevalence of cancer was low . In another population the resulting decision tree may be different . The results may also be influenced by the choice of the reference test in the diagnosis of intracavitary lesions . Histology together with diagnostic hysteroscopy is usually considered the gold standard in the diagnosis of intracavitary lesions . However, both histology and hysteroscopy have their limitations too: e.g. A resected endometrial polyp may get lost during the processing of the specimen (duffy et al ., 2003), or a sessile endometrial lesion may remain unseen at hysteroscopy . Because of the lack of any infallible gold standard, any decision tree will be prone to some bias . Tree #2 included the pedicle artery sign as last step in those women with a focal lesion seen at sis, but not on hysteroscopy: a vessel seen at color doppler inside a focal thickening is indicative for intracavitary pathology, whereas in the absence of any color doppler signal an artifact (e.g. A blood clot or some endometrial tissue pushed up while treading the sis - catheter) is more probable . The selection of parity and menopausal status was somewhat unexpected . In tree #1, if no lesion was seen at hysteroscopy, an endometrial biopsy was proposed straight away in the parous women, whereas, in nulliparous women, endometrial sampling was restricted to premenopausal patients: nulliparous, postmenopausal women did not seem to benefit from further testing . In tree #3 the endometrial thickness is used in the last step of tree #1: an endometrial thickness above 7.4 mm is considered abnormal . It must be emphasized that this cut - off value cannot be extrapolated for use as a single test outside the algorithm, but only in the very selected cases of premenopausal nulliparous women in whom hysteroscopy failed to show a lesion and in whom endometrial sampling showed a normal histology . If used as single test the cut - off value for endometrial thickness above which malignancy is to be ruled out lies between 3 and 5 mm (tabor et al ., 2002; smith - bindman et al ., 1998; gupta et al ., 2002; epstein & valentin, 2004; timmermans, 2009). Decision tree #3 is very simple, but still has a reasonable accuracy without third or fourth line tests to lower the false negative rate . Allowing hysteroscopy to be used in second or third line examination we do not pretend that decision trees built by mathematician are superior to algorithms based on good clinical judgement . However, the - sometimes unexpected - results of the mathematical decision trees may lead the clinician to interesting reflection as to the current clinical practice . In practice, the choice of a diagnostic algorithm will also be influenced by other factors, such as the personal preference and skills of the clinician, the availability of the different diagnostic methods, the possibility to use the decision tree in a one stop clinic setting, the patient s preference for one test (van den bosch et al.
This randomized, examinerblind, paralleldesign, controlled, singlecentre study was conducted in the usa between 1 november and 2 december 2011 . The primary objective was to compare the efficacy in reducing gingivitis and plaque of an experimental mouthrinse containing 0.15% lae with that of a mouthrinse containing 5% hydroalcohol (negative control) after 4 weeks of use as adjuncts to tooth brushing . The secondary objective was to compare the efficacy of these mouthrinses in reducing gingivitis and plaque after 2 weeks use and their ability to reduce gingival bleeding after 2 and 4 weeks use . The study was conducted in accordance with the protocol, the abbreviated investigational device exemption regulations (21 cfr part 812), international conference on harmonisation harmonised tripartite guideline for good clinical practice (1996), the declaration of helsinki (2000) and applicable local regulatory requirements and laws ., subjects made three visits to the clinic: on day 1 (screening / baseline visit 1), day 15 1 day (visit 2) and day 29 1 day (visit 3). Subjects were required to refrain from oral hygiene practices for 1218 h and from eating, drinking or smoking for 4 h before study visits . Subjects were to refrain from using unassigned oral care products (including interdental cleaning devices except for the removal of impacted food) or having any dental work done (except for emergency procedures) throughout the study . Subjects received test materials in blinded packaging, although taste differences were perceivable upon product use . The sponsor provided blinded test materials; personnel dispensing the test products or supervising their use did not participate in the examination of subjects . At the screening visit, qualifying subjects provided written informed consent before their participation in the study . Subjects completed a medical / dental history questionnaire and underwent an oral examination and evaluation for plaque, gingivitis and bleeding . Subjects then received dental prophylaxis to remove plaque (confirmed by disclosure), stains and calculus . The subjects were randomly assigned to one of two treatment groups to receive either an experimental 0.15% laecontaining mouthrinse or a 5% hydroalcohol negativecontrol mouthrinse (both manufactured by johnson & johnson healthcare products division of mcneilppc inc ., skillman, nj, usa). Subjects were assigned a unique randomization number, allocated sequentially by site staff, based on a randomization schedule provided by the study sponsor . All subjects received a standard fluoride toothpaste (colgate cavity protection toothpaste; manufactured by colgate palmolive company, new york, ny, usa) and a softbristled toothbrush (reach advanced design toothbrush; distributed by johnson & johnson healthcare products division of mcneilppc, inc . ), instruction on oral hygiene, and diary cards . Subjects were instructed to brush their teeth twice daily in their usual manner and to rinse for 30 s after each brushing with 20 ml of their assigned mouthrinse . Use of the mouthrinse was supervised at visits 1 and 2 . At visits 2 and 3 subjects subjects compliance with studyproduct usage instructions was assessed by review of their completed diary cards and by collecting and weighing mouthrinse bottles at visits 2 and 3 . Three indices were used to assess clinical efficacy: the turesky modification of the quigleyhein plaque index (pi; turesky et al . 1982), the modified gingival index (mgi; lobene et al . 1986) and the bleeding index (bi; ainamo & bay 1975, saxton & van der ouderaa 1989). The coprimary endpoints of the study were wholemouth mean pi score at visit 3 (week 4) and wholemouth mean mgi score at visit 3 . Secondary endpoints included wholemouth mean pi and mgi scores at visit 2 (week 2); wholemouth mean bi scores at visits 2 and 3; and microbiological absolute and log counts for oral microbes derived from plaque samples collected at visit 1 and visit 3 . Men and women aged 18 years and in good general health with signs of adequate oral hygiene (i.e. Daily tooth brushing and no signs of oral neglect) were eligible for inclusion in the study . All subjects had to have: 20 natural teeth with scorable surfaces; a mean mgi score 1.95; a baseline mean pi score 1.95 for overnight plaque accumulation; and an absence of significant oral soft tissue pathology, periodontitis or extensive subgingival calculus . Exclusion criteria included: a history of significant adverse events following use of oral hygiene products; conditions requiring prophylactic use of antibiotics before dental surgery according to united states clinical practice; use of antibiotics, antiinflammatory or anticoagulant therapy or any medication that might interfere with efficacy evaluations in the 4 weeks before the study; regular use of antiplaque/gingivitis dental products within 2 weeks of the study; and any severe acute or chronic medical condition or laboratory abnormality that might pose a risk to the participant or interfere with interpretation of the results of the study . Subjects were assessed for adverse events, gingivitis, gingival bleeding and plaque, in that order . Plaque accumulation was scored at all visits using the pi (turesky et al . 1982) for six surfaces (distobuccal, midbuccal, mesiobuccal, distolingual, midlingual and mesiolingual) of all scorable teeth after disclosing (0: no plaque; 1: separate flecks or discontinuous band of plaque at the gingival margin; 2: thin [up to 1 mm] continuous band of plaque at the gingival margin; 3: band of plaque wider than 1 mm but less than onethird of surface; 4: plaque covering more than onethird but less than twothirds of surface; 5: plaque covering more than twothirds of surface). Gingivitis was assessed at all visits on the buccal and lingual marginal gingivae and interdental papillae of all scorable teeth using the mgi (0: normal; 1: mild inflammation of any point of the gingival unit; 2: mild inflammation of the entire gingival unit; 3: moderate inflammation of the gingival unit; 4: severe inflammation of the gingival unit; lobene et al . Gingival bleeding was assessed at all visits . A periodontal probe (0.5 mm diameter tip) was inserted into the gingival crevice and swept distal to mesial at a 60 angle while maintaining contact with the sulcular epithelium . Four areas around each tooth were assessed (distobuccal, midbuccal, midlingual and mesiolingual). Bleeding was recorded using the gingival bi (0: absence after 30 s; 1: bleeding after 30 s; 2: immediate bleeding; ainamo & bay 1975, saxton & van der ouderaa 1989) 30 s after an entire surface (e.g., buccal) in each quadrant was probed . Plaque samples were collected, using a sterile curette, from the buccal surfaces of teeth 28 in the upper right quadrant by a trained dental professional at visits 1 and 3 following the clinical assessments . Microbiological assessment of the plaque samples using dna: dna hybridization was conducted to assess shifts in the oral microflora . Dna isolated from the bacteria in the plaque samples was fixed in lanes to nylon membranes using a minislot 60 device (immunetics, cambridge, ma, usa) and hybridized by checkerboard hybridization (socransky et al . 2004) in a miniblotter 45 (immunetics) to digoxigeninlabelled whole genomic dna probes for 41 species . The bacterial species recognized by the probes were categorized as shown in table 1 . Dna probes were detected using an antibody to digoxigenin conjugated with alkaline phosphatase, and chemifluorescence detection . Signals were detected using attophos substrate (amersham life sciences, arlington heights, il, usa) and read with a storm fluoroimager (molecular dynamics, sunnyvale, ca, usa). Standards for each species were included at a concentration of 10 and 10 cells on the membrane as controls . The assay sensitivity was adjusted to permit detection of 10 cells of a given species by adjusting the concentration of the dna probe . Signals were converted to absolute counts by comparison with the standards on the same membrane . Bacterial species detected by genomic dna probes in dna: dna hybridization analysis of dental plaque samples oral examinations of the buccal and sublingual mucosa, lips / labial mucosa, mucobuccal fold, gingiva, tongue, hard and soft palate, uvula, oropharynx, teeth and dental restorations were conducted at each visit to monitor oral tolerability . Emergent or worsening adverse events were recorded at each recall visit, after a review of medical history and a thorough examination of the oral cavity . Abnormalities, such as lip bites, food burns and traumatic ulcers, that were not considered clinically significant by the investigator were not recorded as adverse events . The safety analysis was conducted on all randomized subjects who used at least one dose of the study products . The number and proportion of subjects experiencing adverse events throughout the study period were summarized according to the medical dictionary for regulatory activities system organ class and preferred term version 14.1 . The planned sample size of 40 subjects in each treatment group was based on estimates of standard deviations and means from a 4week pilot study, and provides 95% power to detect a difference in means of 0.102 (assuming a standard deviation of 0.125) for wholemouth mean pi and wholemouth mean mgi at the 0.05 level of significance (twosided). Demographics and baseline characteristics were compared betweenv treatment groups using analysis of variance or a chisquared test . Fisher's exact test was used instead of a chisquared test if the number of subjects was sufficiently small . The primary and secondary efficacy analyses were based on the full analysis set (i.e. Randomized subjects who used at least one dose of study product and had at least one postbaseline efficacy assessment) using an analysis of covariance with treatment as a factor and corresponding baseline value as the covariate . Microbiological counts for each category of oral microbe were reported using summary statistics . For this study, data imputations were not performed as the number of missing data was expected to be negligible . This randomized, examinerblind, paralleldesign, controlled, singlecentre study was conducted in the usa between 1 november and 2 december 2011 . The primary objective was to compare the efficacy in reducing gingivitis and plaque of an experimental mouthrinse containing 0.15% lae with that of a mouthrinse containing 5% hydroalcohol (negative control) after 4 weeks of use as adjuncts to tooth brushing . The secondary objective was to compare the efficacy of these mouthrinses in reducing gingivitis and plaque after 2 weeks use and their ability to reduce gingival bleeding after 2 and 4 weeks use . The study was conducted in accordance with the protocol, the abbreviated investigational device exemption regulations (21 cfr part 812), international conference on harmonisation harmonised tripartite guideline for good clinical practice (1996), the declaration of helsinki (2000) and applicable local regulatory requirements and laws ., subjects made three visits to the clinic: on day 1 (screening / baseline visit 1), day 15 1 day (visit 2) and day 29 1 day (visit 3). Subjects were required to refrain from oral hygiene practices for 1218 h and from eating, drinking or smoking for 4 h before study visits . Subjects were to refrain from using unassigned oral care products (including interdental cleaning devices except for the removal of impacted food) or having any dental work done (except for emergency procedures) throughout the study . Subjects received test materials in blinded packaging, although taste differences were perceivable upon product use . The sponsor provided blinded test materials; personnel dispensing the test products or supervising their use did not participate in the examination of subjects . At the screening visit, qualifying subjects provided written informed consent before their participation in the study . Subjects completed a medical / dental history questionnaire and underwent an oral examination and evaluation for plaque, gingivitis and bleeding . Subjects then received dental prophylaxis to remove plaque (confirmed by disclosure), stains and calculus . The subjects were randomly assigned to one of two treatment groups to receive either an experimental 0.15% laecontaining mouthrinse or a 5% hydroalcohol negativecontrol mouthrinse (both manufactured by johnson & johnson healthcare products division of mcneilppc inc ., skillman, nj, usa). Subjects were assigned a unique randomization number, allocated sequentially by site staff, based on a randomization schedule provided by the study sponsor . All subjects received a standard fluoride toothpaste (colgate cavity protection toothpaste; manufactured by colgate palmolive company, new york, ny, usa) and a softbristled toothbrush (reach advanced design toothbrush; distributed by johnson & johnson healthcare products division of mcneilppc, inc . ), instruction on oral hygiene, and diary cards . Subjects were instructed to brush their teeth twice daily in their usual manner and to rinse for 30 s after each brushing with 20 ml of their assigned mouthrinse . Use of the mouthrinse was supervised at visits 1 and 2 . At visits 2 and 3 subjects subjects compliance with studyproduct usage instructions was assessed by review of their completed diary cards and by collecting and weighing mouthrinse bottles at visits 2 and 3 . Three indices were used to assess clinical efficacy: the turesky modification of the quigleyhein plaque index (pi; turesky et al . 1982), the modified gingival index (mgi; lobene et al . 1986) and the bleeding index (bi; ainamo & bay 1975, saxton & van der ouderaa 1989). The coprimary endpoints of the study were wholemouth mean pi score at visit 3 (week 4) and wholemouth mean mgi score at visit 3 . Secondary endpoints included wholemouth mean pi and mgi scores at visit 2 (week 2); wholemouth mean bi scores at visits 2 and 3; and microbiological absolute and log counts for oral microbes derived from plaque samples collected at visit 1 and visit 3 . Men and women aged 18 years and in good general health with signs of adequate oral hygiene (i.e. Daily tooth brushing and no signs of oral neglect) were eligible for inclusion in the study . All subjects had to have: 20 natural teeth with scorable surfaces; a mean mgi score 1.95; a baseline mean pi score 1.95 for overnight plaque accumulation; and an absence of significant oral soft tissue pathology, periodontitis or extensive subgingival calculus . Exclusion criteria included: a history of significant adverse events following use of oral hygiene products; conditions requiring prophylactic use of antibiotics before dental surgery according to united states clinical practice; use of antibiotics, antiinflammatory or anticoagulant therapy or any medication that might interfere with efficacy evaluations in the 4 weeks before the study; regular use of antiplaque/gingivitis dental products within 2 weeks of the study; and any severe acute or chronic medical condition or laboratory abnormality that might pose a risk to the participant or interfere with interpretation of the results of the study . Subjects were assessed for adverse events, gingivitis, gingival bleeding and plaque, in that order . Plaque accumulation was scored at all visits using the pi (turesky et al . 1982) for six surfaces (distobuccal, midbuccal, mesiobuccal, distolingual, midlingual and mesiolingual) of all scorable teeth after disclosing (0: no plaque; 1: separate flecks or discontinuous band of plaque at the gingival margin; 2: thin [up to 1 mm] continuous band of plaque at the gingival margin; 3: band of plaque wider than 1 mm but less than onethird of surface; 4: plaque covering more than onethird but less than twothirds of surface; 5: plaque covering more than twothirds of surface). Gingivitis was assessed at all visits on the buccal and lingual marginal gingivae and interdental papillae of all scorable teeth using the mgi (0: normal; 1: mild inflammation of any point of the gingival unit; 2: mild inflammation of the entire gingival unit; 3: moderate inflammation of the gingival unit; 4: severe inflammation of the gingival unit; lobene et al . Gingival bleeding was assessed at all visits . A periodontal probe (0.5 mm diameter tip) was inserted into the gingival crevice and swept distal to mesial at a 60 angle while maintaining contact with the sulcular epithelium . Four areas around each tooth were assessed (distobuccal, midbuccal, midlingual and mesiolingual). Bleeding was recorded using the gingival bi (0: absence after 30 s; 1: bleeding after 30 s; 2: immediate bleeding; ainamo & bay 1975, saxton & van der ouderaa 1989) 30 s after an entire surface (e.g., buccal) in each quadrant was probed . Plaque samples were collected, using a sterile curette, from the buccal surfaces of teeth 28 in the upper right quadrant by a trained dental professional at visits 1 and 3 following the clinical assessments . Microbiological assessment of the plaque samples using dna: dna hybridization was conducted to assess shifts in the oral microflora . Dna isolated from the bacteria in the plaque samples was fixed in lanes to nylon membranes using a minislot 60 device (immunetics, cambridge, ma, usa) and hybridized by checkerboard hybridization (socransky et al . 2004) in a miniblotter 45 (immunetics) to digoxigeninlabelled whole genomic dna probes for 41 species . The bacterial species recognized by the probes were categorized as shown in table 1 . Dna probes were detected using an antibody to digoxigenin conjugated with alkaline phosphatase, and chemifluorescence detection . Signals were detected using attophos substrate (amersham life sciences, arlington heights, il, usa) and read with a storm fluoroimager (molecular dynamics, sunnyvale, ca, usa). Standards for each species were included at a concentration of 10 and 10 cells on the membrane as controls . The assay sensitivity was adjusted to permit detection of 10 cells of a given species by adjusting the concentration of the dna probe . Signals were converted to absolute counts by comparison with the standards on the same membrane . Bacterial species detected by genomic dna probes in dna: dna hybridization analysis of dental plaque samples oral examinations of the buccal and sublingual mucosa, lips / labial mucosa, mucobuccal fold, gingiva, tongue, hard and soft palate, uvula, oropharynx, teeth and dental restorations were conducted at each visit to monitor oral tolerability . Emergent or worsening adverse events were recorded at each recall visit, after a review of medical history and a thorough examination of the oral cavity . Abnormalities, such as lip bites, food burns and traumatic ulcers, that were not considered clinically significant by the investigator were not recorded as adverse events . The safety analysis was conducted on all randomized subjects who used at least one dose of the study products . The number and proportion of subjects experiencing adverse events throughout the study period were summarized according to the medical dictionary for regulatory activities system organ class and preferred term version 14.1 . The planned sample size of 40 subjects in each treatment group was based on estimates of standard deviations and means from a 4week pilot study, and provides 95% power to detect a difference in means of 0.102 (assuming a standard deviation of 0.125) for wholemouth mean pi and wholemouth mean mgi at the 0.05 level of significance (twosided). Demographics and baseline characteristics were compared betweenv treatment groups using analysis of variance or a chisquared test . Fisher's exact test was used instead of a chisquared test if the number of subjects was sufficiently small . The primary and secondary efficacy analyses were based on the full analysis set (i.e. Randomized subjects who used at least one dose of study product and had at least one postbaseline efficacy assessment) using an analysis of covariance with treatment as a factor and corresponding baseline value as the covariate . Microbiological counts for each category of oral microbe were reported using summary statistics . For this study, data imputations were not performed as the number of missing data was expected to be negligible . Eightyseven subjects were randomized to the two study treatments: 43 to receive the 0.15% lae mouthrinse and 44 to receive the negative control . One subject in the control group experienced a serious adverse event (testicular swelling, unrelated to the mouthrinse) and discontinued study treatment . The demographic and baseline variables of the two study groups are shown in table 2; no statistically significant differences were noted between the groups . Demographics and baseline characteristics (all randomized subjects) bi, bleeding index; lae, ethyl lauroyl arginate; mgi, modified gingival index; pi, plaque index; sd, standard deviation . The 0.15% laecontaining mouthrinse was associated with a statistically significantly greater reduction from baseline in the wholemouth mean pi score compared with the control mouthrinse, with a betweentreatment difference in the least squares means of 1.23 (95% confidence interval [95% ci]: 1.07, 1.39), equating to a 42.6% greater reduction versus control (p <0.001; table 3). The reduction in the wholemouth mean mgi score was also statistically significantly greater with the lae mouthrinse (difference: 0.23 [95% ci: 0.19, 0.28]; 10.7% reduction compared with the control; p <0.001). Statistically significant differences in wholemouth mean pi and mgi scores between the 0.15% lae mouthrinse and control mouthrinse, in favour of the lae mouthrinse, were also evident after 2 weeks of treatment (table 3). Wholemouth mean pi, mgi and bi scores (full analysis set) p <0.001 versus control (based on analysis of covariance model). Bi, bleeding index; ci, confidence interval; lae, ethyl lauroyl arginate; mgi, modified gingival index; pi, plaque index; sd, standard deviation; se, standard error . In the gingival bleeding assessment (table 3), the 0.15% laecontaining mouthrinse was associated with statistically significantly greater reductions from baseline in wholemouth mean bi score (p <0.001) at both weeks 2 and 4 compared with the control . At week 2, a 36.3% reduction in the proportion of bleeding sites was observed compared with the control group (0.077 versus 0.121; difference 0.04 [95% ci: 0.03, 0.06]). At week 4, a 50.9% reduction in the proportion of bleeding sites was observed compared with the control group (0.058 versus 0.119; difference 0.06 [95% ci: 0.04, 0.08]). All bleeding scores were either 0 or 1 in both treatment groups across all study visits . Therefore, in this study, the mean scores were equivalent to proportions of bleeding sites . Microbiological analysis of the plaque samples collected at baseline and week 4 (visit 3) revealed no significant compositional changes in the oral microflora . Total microbiological counts (log10) of complexes at baseline and week 4 were similar for both treatment groups, with most differences within 0.5 log (fig . Proportions of each complex were also similar at baseline and week 4 (fig . 2b), with the exception of the purple complex, which was slightly less common at week 4 than at baseline following treatment with both the laecontaining mouthrinse (4.19% versus 5.43% respectively) and the control mouthrinse (5.12% versus 7.86% respectively); and the orange complex, which was slightly more common at week 4 than at baseline (23.82% versus 16.87% respectively) in the laecontaining mouthrinse group . Microbiological analysis of supragingival plaque collected at baseline and after 4 weeks of treatment with 0.15% laecontaining mouthrinse or 0.5% hydroalcohol control mouthrinse . (a) total microbiological counts (log10) for each complex of bacterial species; (b) summary of percentage of counts represented by each complex . For details of bacterial species detected by each category of probe, see table 1 . No adverse events related to oral soft tissue were recorded during the study in either treatment group . One subject who received the control mouthrinse experienced a serious adverse event of testicular swelling requiring hospitalization and discontinued from the study . This was related to testicular cancer and not to the use of the control mouthrinse . No incidences of tooth staining were recorded as adverse events . In both groups there were minor protocol deviations, but no violations associated with subject compliance . The findings of this 4week randomized controlled study show that twicedaily use of an experimental 0.15% laecontaining mouthrinse as an adjunct to tooth brushing resulted in statistically significant reductions from baseline in plaque accumulation, gingivitis and bleeding after 2 and 4 weeks of use in subjects with mildtomoderate gingivitis . Plaque was reduced by 42.6% (difference in least squares means of scores: 1.23 [95% ci: 1.07, 1.39] relative to the negative control by week 4, and gingivitis reduced by 10.7% (difference 0.23 [95% ci: 0.19, 0.28]). Notably, a reduction in bleeding of 50.9% versus the negative control was evident after 4 weeks of use (difference 0.06 [95% ci: 0.04, 0.08]). The 0.15% laecontaining mouthrinse was well tolerated, with no adverse events affecting the oral soft tissue . Microbiological analysis of plaque samples obtained throughout the study indicated no microbial shift in the oral microflora tested, suggesting that there are no safety concerns with use of the 0.15% laecontaining mouthrinse when used over a 4week period . A longer study (e.g. 6 months) would provide more information on the efficacy and safety of 0.15% laecontaining mouthrinse when used over the long term . In addition, no formal assessment of tooth staining or calculus was included in this study . The effects of the mouthrinse are consistent with its proposed action of reducing the adhesion of dental plaque to the dental pellicle (giertsen et al . 2007). In a previous in situ study, in which subjects wore acrylic appliances containing proteincoated discs on the buccal surface of their teeth, use of a 0.5% laecontaining mouthrinse threetimes daily significantly reduced the total number of bacteria and most of the taxa tested in plaque collected from the discs (giertsen et al . 2007). The control of plaque, and the subsequent reduction in gingivitis, is a key goal in the maintenance of gingival and oral health (american academy of periodontology 2005). Many factors influence oral health; some are controllable, e.g. Toothbrushing time, frequency and duration, while others are difficult to control or change, e.g. Host response, motivation and dexterity . For example, of those individuals who use dental floss, less than half utilize it correctly (lang et al . The efforts of dentists and hygienists to improve their patients oral hygiene habits could be significantly assisted by the use of adjunctive oral care products, which are easy to use and help to mitigate the compliance / technique issues associated with interdental cleaning (van der ouderaa 1991, warren & chater 1996). Ideally, an oral antiplaque agent should prevent biofilm formation yet have no adverse effects on the oral microflora . In this study, the negligible changes in plaque and gingivitis levels in the control group effectively confirmed the lack of change in the subjects mechanical plaquecontrol practices . Any meaningful change in these levels (worsening or improvement) would suggest a change from their prestudy habits; worsening in the control group would suggest that study instructions inhibited subjects usual oral hygiene practices, while improvement in the control group would likely suggest greater compliance with the mechanical regimen the fact that the control group started and completed the study with significant (and nearly identical) levels of plaque and gingivitis indicates that, in this population, brushing twice daily in their usual manner was not sufficient . The results of this study thus demonstrate that mechanical oral hygiene alone does not adequately improve the gingival health of individuals with gingivitis . It is well recognized that mechanical cleaning is the most important component of oral hygiene; however, it only appears to maintain control at a certain level of plaque and gingivitis . Therefore, to improve upon an individual's baseline level, a mouthrinse is a useful adjunct to mechanical home care . However, as with any therapeutic intervention, adequate compliance with recommended product usage is of paramount importance . It is recognized that compliance with rinsing twice a day, to achieve the added benefit, may represent a challenge to some individuals . Therapeutic components of oral care rinses have been shown to be effective in reducing gingivitis in numerous clinical trials and systematic reviews (wu & savitt 2002, zimmer et al . More generalized use of chlorhexidinecontaining mouthrinses for plaque and gingivitis control is limited by the potential for poor compliance due to tooth staining and calculus formation with daily use (charles et al . There is therefore a need for dailyuse products with comparable efficacy but which lack these side effects . The results of this study suggest that the laecontaining mouthrinse may be a suitable alternative adjunctive treatment for the control of mildtomoderate gingivitis in adults . The mechanism of action of lae differs from chlorhexidine in that lae has a physical effect by preventing attachment of bacteria to the pellicle (giertsen et al . 2007) rather than bactericidal or bacteriostatic effects . The surfaceactive compound delmopinol, which is available as an antimicrobial mouthrinse, also binds to the pellicle and prevents adherence of dental plaque (vassilakos et al . This study show that a mouthrinse containing 0.15% lae used as an adjunct to tooth brushing was effective in the reduction of plaque, gingivitis and bleeding at both 2 and 4 weeks of use in subjects with mildtomoderate gingivitis, and was well tolerated.
Human herpes virus 4 (hhv-4), also called epstein - barr virus (ebv), as a member of herpesviridae, is one of the most common viruses in humans . It is commonly associated with non - specific clinical signs and is usually presented by painful sore throat, swollen glands, chills, fever and chronic fatigue syndrome . The primary ebv infection in africa, southeast asia and latin america occurs in early childhood, while in developed countries the first peak of infection seems to be before the first five years of life while the second peak is during adolescence (3). According to epidemiological studies the average age of ebv primary infection is increasing (4), e.g. In us, it ranges from 50% for 5 year olds to 90% for 25 years old (5). In brazil the pattern of positive ebv igg ab showed a higher prevalence with increasing age, reaching a peak in the second decade of life (3). In spain, the distribution of primary infection has shown two peaks, one at the age of two to four years and another at the age of 14 - 18 years (6). A recent study of 94 children in the republic of china demonstrated that 78.6% had ebv - vca igg by the end of the first year of life and 80.7% were seropositive by the age of three (7, 8). A similar study in chile revealed that 50% of children from low and medium socioeconomic level had been primarily infected by the age of two in comparison with 5.9% of high socioeconomic children of the same age (9). In malaysian children the presence of ebv - igm antibody, occurred at four to six months and by eight years many children became infected with ebv (10). Since the clinical picture of ebv primary infection could be challenging, and because this infection usually causes no symptoms and can be indistinguishable from other mild, brief infections of children such as streptococcal throat infection, it is important to use the best clinical means for diagnosis (6, 10). The routine diagnosis of ebv primary infection is based on several serological tests such as indirect fluorescent antibody (ifa), rapid monospot tests (for heterophile antibodies) and enzyme immune assay (eia) for detection of early antigens (ea), the viral capsid antigens (vca) or the ebv nuclear antigen (ebna) (11 - 13). To the best of our knowledge, there is no study on the incidence of ebv primary infection in iran . The aim of this study was to determine the incidence of ebv primary infection, among iranian suspected patients who had referred to namazi hospital of fars province (south of iran) by the elisa method . The studied population consisted of 346 suspected patients who had referred to the professor alborzi clinical microbiology research center, namazi hospital, shiraz, southern iran, during march 2007 to march 2011 . The major clinical manifestations of these patients included painful sore throat, swollen glands, chills, fever and tiredness . There were 211 males (61%) and 135 females (39%) with an age range of 0 to 20 years (6.31 4.66: 10.97 years). The study population was distributed to four age groups, as follow: group i (0 - 5 years, n = 192), group ii (6 - 10 years, n = 96), group iii (11 - 15 years, n = 38), and group iv (16 - 20 years, n = 20). A 5 ml blood sample was collected from each individual; sera was separated and stored at -20c for further examination . The igm antibodies against ebv vca were evaluated by a standard commercially available elisa kit (euroimmun, lubeck, germany). Descriptive analysis and chi - square tests were used to explain the results and compare between the incidence of ebv infection in different age and sex groups . The data were analyzed by the spss software (spss for windows, version 16, spss inc ., chicago, il, usa). The studied population consisted of 346 suspected patients who had referred to the professor alborzi clinical microbiology research center, namazi hospital, shiraz, southern iran, during march 2007 to march 2011 . The major clinical manifestations of these patients included painful sore throat, swollen glands, chills, fever and tiredness . There were 211 males (61%) and 135 females (39%) with an age range of 0 to 20 years (6.31 4.66: 10.97 years). The study population was distributed to four age groups, as follow: group i (0 - 5 years, n = 192), group ii (6 - 10 years, n = 96), group iii (11 - 15 years, n = 38), and group iv (16 - 20 years, n = 20). A 5 ml blood sample was collected from each individual; sera was separated and stored at -20c for further examination . The igm antibodies against ebv vca were evaluated by a standard commercially available elisa kit (euroimmun, lubeck, germany). Descriptive analysis and chi - square tests were used to explain the results and compare between the incidence of ebv infection in different age and sex groups . The data were analyzed by the spss software (spss for windows, version 16, spss inc ., chicago, il, usa). Out of 346 patients, 104 (30.00%) were positive for ebv vca - igm, from whom 62.5% and 37.5% were males and females, respectively . There was no significance difference in the incidence of ebv primary infection between males and females (p> 0.05) (figure 1). Our results showed that 61.5% of ebv vca - igm positive patients belonged to age group i, 30% to age group ii, 5.8% to age group iii and finally 1.9% to age group iv . Statistical analysis revealed that there were no significant differences between age group i (64/192) and ii (32/96), ii (32/96) and iii (6/38), iii (6/38) and iv (2/20) (p> 0.05). However, the incidence of ebv primary infection was significantly different between age groups i and iii (p <0.05) and also between age groups i and iv (p <0.05) (figure 2). Symptomatic acute ebv primary infection is clinically associated with infectious mononucleosis (i m) accompanied by fever, tonsillopharyngitis, hepatosplenomegaly, lymphadenopathy, and the increase of mononuclear blood cell number (1, 14, 15). Fever, pharyngitis and lymphadenopathy are the classic triad of i m (2, 14). In the present study, the most common symptoms of our cases included fever, temperature reaching 40c, sore throat with and without exudates and painful cervical lymphadenopathy . Since the monospot test has an approximate sensitivity and specificity of 85% and 94% for diagnosis of ebv infection, and it may be negative in about 50% of cases under age of 12 (1, 3), definitive diagnosis for ebv infection is based on vca - igm and vca - igg antibodies detection (8, 14, 16, 17). The presence of vca - igm in more than 80% of acute sera is the best indication for confirming ebv primary infection within four to eight weeks since infection, while after this period such indication disappears . Its shortened presence in very young children is related to the lower titer achieved (14, 17 - 19). In an earlier study in the united kingdom, 92% of ebv infected patients were anti vca - igm positive (14, 16). Our study showed that 30% of 346 cases with typical symptomatic ebv infection were vca - igm positive . Different population - based studies have reported various prevalence rates of ebv infection in children, from 20% to 80% at age two to three years in different regions . In industrialized and developed countries with high standards of living, it was shown that in malaysian children, primary infection indicated by emergence of ebv igm antibody, occurred at four to six months of age, while all children were seropositive by age of eight (10). In another study in japan, anti vca - igm positive rate in infants with acute primary infection was 25% yet 80% in those four years of age and over (23). In this study, the highest incidence of ebv primary infection belonged to the age group i (0 - 5 years) with a rate of 61.5% . This percentage decreased with age and reached 1.9% by the age 16 - 20 years . Previous studies declared that there is no difference in ebv seroprevalence between different genders in children (24 - 26). In the present study, there was also no significance difference in ebv primary infection incidence between males and females . To sum up, we can conclude that accurate and on time diagnosis of ebv primary infection in both children and adolescents will help prevent unnecessary hospitalization, medication, and incorrect medical decisions . Moreover, in most cases, the best means to differentiate ebv primary infection from other infections and malignancies is by detection of igm specific antibodies against viral capsid antigen (vca - igm) in the sera . Among different serological assays,
Mongolia has the highest incidence of hepatocellular carcinoma (hcc) and liver cirrhosis (lc) worldwide (fig . Hcc occurs in 61.9 patients per 100,000 population each year, and lc occurs in 350 patients per 100,000, and its high occurrence has been attributed to the high prevalence of chronic viral hepatitis . The hepatitis b (hbv) and c viruses (hcv) are highly prevalent in mongolia . The seroprevalence of hbv is 11.8% in the unvaccinated population (meta - analysis of eight studies), and that of hcv is 15.6% in the apparently healthy population . The prevalence of chronic hcv infection in mongolia is strikingly high considering that the global hcv carrier prevalence is estimated to be about 3%, ranging from 0.1 to 10% or more . Therefore, mongolia has the highest prevalence (> 15%) of hcv and (> 10%) hbv infection, along with egypt and tanzania . This high prevalence is attributed to improper sterilization and disinfection of medical and dental equipment that might contribute to the spread of hepatitis viruses . Interestingly, most hcv infections are caused by genotype 1 (98.8%) in patients with chronic hepatitis, and genotype 2 infection is very rare (1.2%). In this regard, improvements in blood safety as well as the development and execution of strict disinfection and sterilization guidelines for health - related procedures such as phlebotomy, injection, and dental and surgical manipulations are required to control the spread of hepatitis viruses in mongolia . Besides chronic hepatitis c, mongolia is confronted with a high prevalence of chronic hepatitis b (table 1). The hbv surface antigen (hbsag) prevalence is 9 - 10% of healthy individuals in mongolia . With such a high hbv carrier rate in the general population, hbv infection and its associated complications pose serious health problems . To reduce the rate of hbv infection via mother - to - baby transmission during infancy, the mongolian government launched a universal hbv immunization program in 1991 . Genotypes a, b, and c infection are rare at 0.9, 0.9, and 6.0% respectively . One study demonstrated that 10 - 50% of hbv immunization attempts failed and that hbv vaccine failure is not associated with hbv mutants but is often associated with an inability to simultaneously administer the vaccine and immunoglobulin at birth . Most lc is attributable to hcv, hbv, and hepatitis d virus (hdv), and a small portion is due to alcohol (fig . (95% confidence interval [ci], 37 to 43) are hbsag+; 39% (95% ci, 36 to 42) are antihcv+, 20% (95% ci, 18 to 23) are dual+, and 1% are negative (fig ., cancers have been second among the causes of mortality in mongolia . According to the 2010 national statistics, hcc is the most common malignancy, representing 44.2% of all cancers in mongolia (fig . An increase in the incidence and deaths from hcc has been observed in recent years (fig . 5). 6). In europe, the usa, and japan, hcv is the predominant cause of development of hcc, whereas hbv infection is the main cause of hcc in korea . Mortality in males is mainly due to cancers of the liver and stomach and in females it is most commonly due to liver and cervical cancers . Higher incidences of cancer are observed in adults aged 45 to 60 years (table 3). The 1-year hcc survival rate is 21 - 24%, that at 3 years is 10 - 11%, and at 5 years is 6 - 8% according to an unpublished natioanal cancer center report . Liver biopsy is not routine in mongolia, only 40 - 60 are performed each year, mainly for diagnosis of hcc . Noninvasive methods, such as fibroscan, were not introduced until 2011, and> 500 patients have undergone this test to - date . Such methods will decrease the diagnostic errors associated with other nonspecific conventional methods such as ultrasonography (us) and increase the detection of lc, because both patients and doctors prefer noninvasive methods . Early diagnosis of hcc is of extreme importance in mongolia . The number of high - quality us machines is very low, particularly in rural areas, and the quality of the us and examiners is unsatisfactory . Most family doctors do not classify their patients as high-, moderate-, and low - risk, which makes it difficult to refer patients for dynamic computed tomography (ct) due to a fear of frequent radiation exposure . Low - quality us machines operated by poorly trained technicians diagnose hcc lesions that have grown> 3 cm in most cases . Many patients tend to seek medical attention only after the development of jaundice or liver failure . High - resolution imaging diagnostic modalities, including ct and magnetic resonance imaging, are essential for a proper hcc diagnosis . Unfortunately, only two hospitals perform 2 - 3 phase contrast cts at this time . In our recent as - yet unpublished study, the most - used diagnostic imaging modality for the first examination was us in all patients . The second - most common imaging modality was contrast - enhanced ct in 58 (44.9%) patients followed by contrast - enhanced ct and hepatic angiography in 32 (24.8%). The majority of patients had advanced hcc, with 63 (48.8%) in stage iii and 49 (38.0%) in stage iv . It is important to note that no patient had stage i hcc and only 17 (13.2%) patients had stage ii hcc . Surgical resection was performed in 16% of patients, and trans - arterial chemoembolization (tace) was carried out in 27% of patients with multiple or large tumors . Radiofrequency ablation (rfa) is considered curative in conjunction with resection, but only 2% of patients received rfa as a first - line treatment and 55% received palliative care . Liver resection and tace remain the mainstream treatment options in mongolia (fig . 7). Two surgical methods are used for major hepatectomies: the conventional and glissonian pedicle approaches (fig . The glissonian pedicle approach was introduced in the last few years and has already produced positive results . While average blood loss was 358.5 ml in the glissonian pedicle approach group, it was 747.5 ml during conventional liver resection . Patients who underwent liver resection using a glissonian pedicle approach survived longer than those who received the conventional method (fig . Viral hepatitis b, c, and alcohol consumption remain the main causes of chronic liver diseases, such as chronic hepatitis, lc, and hcc, in mongolia . Insufficient data limits our knowledge of the exact burden of hdv as a cause of lc and liver tumors . There has been no study of the genetic traits of mongolians as a causative factor for hcc . Although the need for such research is debatable, genetic factors cannot be disregarded without careful investigation . The high incidence of hcc and end - stage liver disease must be addressed at all levels - etiology, diagnosis, treatment, and prevention . Delayed diagnosis contributes greatly to the high hcc and lc mortality rate in mongolia . The national program, which includes high - risk patients and frequent follow - up examinations, must be implemented by the government as soon as possible . Lc previously raised little attention in mongolia, but it certainly deserves more attention, because lc mortality is higher than that of gastric cancer . There is a need to train more liver pathologists and interventional radiologists, because there is currently no distinction made between early and advanced hcc in mongolia . Liver fibrosis staging is not assessed by pathologists . With the introduction of noninvasive methods such as fibroscan in 2012, lc and fibrosis diagnoses only 40 - 60 liver biopsies are performed annually due to the shortage of interventional radiologists . The number of rfas must be increased or the procedures should be performed in conjunction with other treatment methods such as tace . Technical obstacles must be removed and the infrastructure available for rfa treatment should be improved . Surgical resection must be provided as the first - line treatment option for patients who satisfy the criteria . Sorafenib, though not officially registered in mongolia, has been under clinical trial in five patients with unresectable hcc at ncc, and the results will be disclosed in the near future . Concerns of severe side effects and high cost as well as low survival benefit remain obstacles to hcc chemotherapy . Palliative treatment is the only care for patients not qualified for surgery, rfa, or tace, and 55% of hcc patients receive palliative care due to delayed diagnosis . Liver transplantation is an attractive option for curative treatment but is currently not performed in hcc patients . Only five living donor liver transplantations (ldlt) have been performed, all for lc . The infrastructure, legal, and socioeconomic environment for ldlt must be improved greatly, so that as many as 500 additional procedures can be performed annually . Hcc can be greatly reduced with proper treatment of alcohol addiction and hepatitis viruses b, c, and d. standard treatment for hcv, including peg - interferon plus ribavirin, was registered in mongolia only 2 - 3 years ago . However, the price is exceedingly high for those with middle and lower class incomes . We calculated the total cost of hcv diagnosis and treatment on per patient and per country bases . An estimated 516,994 people are infected with hcv and 150 us$/person is needed for a full set of diagnostic tests . A total of 12,267 us$/person is needed for 48-week peginterferon treatment including 1 month of hospital stay, whereas classic interferon treatment costs 2,953 us$/person . Currently, with no insurance coverage for viral treatment, a total of 6.34 billion us$is required to treat every citizen with pegylated interferon, and 1.52 billion us$is needed to treat using conventional interferon therapy . The financial burden of hcv is so great that 41 life - years are needed for middle - class mongolian patients to save enough money to have the latest hepatitis treatment . Hcv treatment will cost more than the country's gross domestic product, which stands at 4.2 billion us$as of 2009 . As more oral treatment agents become available in the coming years, thus, it is crucial for the world health organization, pharmaceutical companies, and other international organizations to help countries like mongolia fight hcv . Lamivudine was registered 3 years ago, and tenofovir disoproxil fumarate is undergoing registration by the mongolian federal drug administration . Adefovir, telbivudine, and entecavir are not yet registered . Despite the affordable price of lamivudine for most patients, although there is no cure for hdv, upcoming trials show promise for peg - interferon only, peg - interferon in combination with tenofovir, or tenofovir alone treatments . Alcohol consumption remains one of the major causes of hcc in mongolia, and this issue has to be addressed by both doctors and the government . The flourishing alcohol business has to be controlled by the government and anti - alcohol laws must be implemented . With the elimination of causes and improvements in diagnosis and treatment, we believe that a reduction in mortality from hcc and lc by at least by 30% is possible in the near future.
Physical stimuli can significantly alter cellular behavior, giving rise to biochemical signals involved in molecular response.1 this process is called mechanotransduction, and the responsible structures sensitive to mechanical forces are most probably cytoskeleton elements.2 a number of studies have demonstrated that cells are sensitive to several kinds of physical cues (shear stress, topography, mechanical deformation, etc), influencing cell migration,3 differentiation,4 and proliferation.5 among these stimuli, gravity is required for the correct development of land - based organisms, and in particular for the skeleton and for the muscle and nervous systems.6 an increasing amount of research is focused on the effects of gravity alterations on the physiological processes, but also on the possibility to exploit this stimulus as a potential therapeutic cue.79 as an example, improved regeneration of infarcted myocardium has been achieved after injection of stem cells differentiated following a 2 g hypergravity treatment, which enhanced the activities of cardiac marker mef-2 by promoting the nuclear export of histone deacetylase 5.10 chang et al investigated altered gravity effects on human lung adenocarcinoma, demonstrating the ability of simulated microgravity to decrease the metastatic potential of this tumor cell line.11 other researchers used microgravity stimulation as an approach for the development of a large amount of -cell spheroids, which once transplanted in mice are able to improve the symptoms of diabetes.12 among different tissues, bone is particularly affected by altered gravity conditions: evidence regarding bone regeneration suggests that hypergravity exposure conversely to microgravity, which negatively affects osteogenesis may enhance the osteogenic potential of osteoblast precursors.13 the ability of mesenchymal stem cells (mscs) to differentiate into osteoblasts is well known, but the osteogenic potential of mscs decreases with the prolonged culture duration necessary to obtain an appropriate number of cells for clinical applications.14 some countermeasures to this issue could come from nanotechnology, which proposes many different typologies of nanoparticles (nps) for stem cell labeling, tracking, delivery, and stimulation,15 including several examples of nanomaterials able to foster osteogenesis in mscs.1618 our group, as an example, successfully exploited barium titanate nps (btnps; figure 1a) as a possible agent for the improvement of osteogenic differentiation of mscs.19 btnps belong to a class of ferroelectric materials showing high piezoelectricity,20 and with regard to biomedical applications, they demonstrate high cytocompatibility,21 excellent properties as nonlinear imaging probes,22 and the ability to deliver doxorubicin in cancer cells by improving drug uptake.23 moreover, as previously mentioned, btnps were proven to enhance the osteogenesis of mscs, as demonstrated by an increment of hydroxyapatite deposition . Starting from these findings, we decided to develop a protocol for msc stimulation by combining incubation with btnps with treatment in hypergravity, with the goal of improving the differentiation process toward osteoblasts, and thus to obtain stem cells with enhanced osteogenic differentiation potential . 2013 campaign at the european space agency (esa; noordwijk, the netherlands), taking advantage of the large - diameter centrifuge for the hypergravity treatment . With the proposed experimental protocol, we had the aims of 1) investigating cell morphology and differentiation (at gene, protein, and phenotype level) following the combination of hypergravity treatment and btnp administration, and 2) evaluating np uptake by stem cells in altered gravity conditions, thus investigating the possibility of enhancing cellular internalization by simply exploiting an increased gravitational force . Rat mscs (scr027; millipore) were used at the second passage in all the experiments . For the maintenance of mscs, the medium was composed of dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum, 100 u / ml penicillin, 100 mg / ml streptomycin, and 200 mm glutamine (all these reagents from gibco). Cultures were maintained in an incubator at standard culture conditions (37c, 5% co2, and 100% humidity). For all the experiments, mscs were trypsinized and seeded on glass slides (diameter 13 mm) 48 hours before hypergravity treatment at 10,000/cm for tests in proliferation conditions and at 30,000/cm for tests under osteogenic differentiation . Osteogenesis was induced in the differentiation samples immediately before the hypergravity treatment by supplementing the medium with 100 nm dexamethasone, 200 m ascorbic acid 2-phosphate, and 10 mm glycerol 2-phosphate (all these reagents from sigma - aldrich). Some samples (both proliferating and differentiating) were moreover provided with btnps (20 g / ml), also in this case immediately before the hypergravity treatment . This dose was selected based on our previous results of an analysis of btnp effects on mscs, where 20 g / ml was found to be the optimal concentration at which nps did not negatively affect cellular functions.19 the nps, obtained from nanostructured and amorphous materials (houston, tx, usa), were about 150 nm in radius, and were administered to the cell culture upon stabilization in gum arabic (sigma - aldrich). Images of the final dispersion of btnps were acquired after gold - sputtering by scanning electron microscopy through a dual - beam system (fei helios 600; figure 1a). Further details on np characterization have been previously reported.19 glass slides supporting the cell cultures were transferred in cylindrical vials filled with biocompatible polydimethylsiloxane (10:1 base: cross - linking agent ratio, curing temperature 60c), and fully covered with 800 l of the appropriate cell - culture medium to exclude shear - stress effects (figure 1b). For the positioning of the samples, we used a delrin structure designed to support 30 cylindrical vials (figure 1c). It is composed of four large rotating arms with a swing gondola at each extremity (figure 1d), and can support hypergravity levels between 1 g and 20 g. we performed analyses on proliferating and differentiating rat mscs provided with and without 20 g / ml of btnps immediately before the altered gravity treatment, as previously described . The sample support was put in an incubator inside a gondola and accelerated at 20 g for 3 hours at 32c . At the end of the hypergravity stimulation, the proliferating samples were immediately processed for subsequent analysis, while differentiating samples were incubated for further 48 hours in differentiating conditions (in incubator, 37c, 5% co2) before the assessment of osteogenesis . Analogous experiments, as normal - gravity controls, were performed, maintaining cells at the same conditions of atmosphere and temperature for 3 hours at 1 g. all the procedures were performed in duplicate . In summary, we examined the following experimental classes: 1) proliferating cells at 1 g without btnps, 2) proliferating cells at 1 g with btnps, 3) proliferating cells at 20 g without btnps, 4) proliferating cells at 20 g with btnps, 5) differentiating cells at 1 g without btnps, 6) differentiating cells at 1 g with btnps, 7) differentiating cells at 20 g without btnps, and 8) differentiating cells at 20 g with btnps . Immediately after the treatment, some proliferative samples were processed in order to evaluate the effects of hypergravity and btnps on msc adhesion and shape . To assess changes in morphology, cytoskeleton conformation, and adhesion, immunofluorescence with the cytoskeleton / focal cells were incubated for 45 minutes with a vinculin primary monoclonal antibody (mouse antirat, diluted 1:100 in 10% goat serum in phosphate - buffered saline [pbs]) after fixation with 4% paraformaldehyde in pbs for 20 minutes at 4c, permeabilization for 15 minutes with 0.1% triton x-100 (sigma - aldrich), and treatment with a blocking solution (10% goat serum in pbs) for 1 hour . Thereafter, a staining solution composed of a green fluorescent labeled secondary antibody (goat antimouse, diluted 1:50 in 10% goat serum), 100 m tetramethylrhodamine isothiocyanate phalloidin for f - actin labeling, and 1 m 4,6-diamidino-2-phenylindole (dapi) for nucleus counterstaining were added . Samples were finally washed several times with pbs before observation under a confocal laser - scanning microscope (c2s; nikon). Cell shape was analyzed on proliferative samples stained with coomassie brilliant blue (0.2% for 5 minutes). Cell area and different shape descriptors were calculated with imagej software (http://rsb.info.nih.gov/ij) analyzing at least 50 well - distinct cells, acquired with an inverted optical microscope (nikon eclipse ti). In particular, the solidity (s), circularity (c), and roundness (r) of cells were investigated according to the following equations:24 roundness = r = ab(1)where a and b are the width and length of the minimum bounding (the smallest rectangle enclosing the selection), respectively, circularity = c=4ap2(2)where p is the perimeter and a is the cell area, and solidity = s = aconvexa(3)where convexa is the area enclosed by the smallest shell that borders all the points of the cell . Np internalization has been investigated by a multimodal microscope with an in - plane resolution of approximately 300 nm and a resolution of 1 m along the optical axis . Coherent anti - stokes raman scattering (cars) has been exploited to obtain images of cells, based on a degenerate pump - and - probe beam (papb) created by a ti sa pulsed laser (chameleon vision ii; coherent) and a supercontinuum generator (photonic crystal fiber scg-800; newport) that produces a broadband stokes beam . The beams were chirped through their transmission by two sf6 glass blocks a 10 cm - long one for the papb, and a 15 cm - long one for the stokes radiation in order to optimize the spectral resolution . For the excitation of ch2 bonds at a raman shift of 2,850 cm and thus for the cell imaging, we adjusted the delay between the papb and the stokes beam . For the localization of btnps, an 806 nm papb was combined with stokes photons producing a sum - frequency generation (sfg) signal from btnps at approximately 452 nm . The images were subject to analysis with imagej software, and the amount of btnp internalization was calculated as the ratio of the area occupied by the nps inside the cells and the total cell area, on at least 50 cells for each experimental treatment . Quantitative real - time reverse - transcription polymerase chain reaction (qpcr) was used to investigate the messenger ribonucleic acid (mrna) transcription of osteogenesis - marker genes (runt related transcription factor 2 [runx2], collagen type i alpha-1 [col1a1], and alkaline phosphatase [alpl]) and of rhoa (ras homolog gene family, member a), which regulates osteogenesis - activating runx2 following mechanical stimulation.25 after the experimental procedures, cells were trypsinized and centrifuged, and the total rna was extracted from the samples with the high pure rna isolation kit (roche) according to the manufacturer s instructions . The quantity and purity of rna was verified through spectrophotometric analysis (nanodrop; thermo scientific). Thereafter, complementary deoxyribonucleic acid was obtained from the reverse transcription of 100 ng of rna through iscript reverse transcription supermix (bio - rad). The following protocol was used for the retrotranscription: 25c for 5 minutes, 42c for 45 minutes, 48c for 15 minutes, and finally 85c for 5 minutes . The amplification was carried out on the cfx connect real - time pcr detection system (bio - rad) thermocycler with the ssoadvanced sybr green supermix (bio - rad). The temperature steps for the amplification reaction were: one cycle at 98c for 30 seconds, 40 cycles at 98c for 3 seconds, and 60c for 7 seconds, a temperature ramp from 65c to 95c, with 0.5c / second increments (for melting curve generation). The housekeeping genes adopted were gusb and rpl19; the cycle threshold (ct) value relative to the control sample (cultures performed at 1 g, without np treatment) was considered as the reference for the calculation of ct (difference between ct values deriving from difference between ct of target and housekeeping genes) for the subsequent samples.26 primer sequences (forward and reverse) of the investigated genes are reported in table 1 . The analysis was focused on the main proteins involved in the early stage of osteogenesis (alpl and col1; glyceraldehyde-3-phosphate dehydrogenase [gapdh] was adopted as the reference). For protein extraction, after trypsinization and centrifugation, cell pellets were lysed in 50 mm tris - hcl (ph 7.6), 2 mm ethylenediaminetetraacetic acid, 100 mm nacl, 1% nonidet p40, and antiproteases 1 (all these reagents from sigma). Protein quantification was performed through the pierce bca protein assay kit (thermo scientific) following the manufacturer s protocol . Twenty micrograms of protein for each sample was separated on a 4%15% mini - protean tgx stain - free gel (bio - rad) under reducing conditions, and then transferred to nitrocellulose membrane . The membranes were saturated for 45 minutes with 4% nonfat dry milk in pbs for blocking nonspecific binding sites, and then probed with primary antibodies against alpl (rabbit antirat, diluted 1:8,000, abcam biotechnology), col1 (rabbit antirat, diluted 1:1,000, abcam biotechnology), or gapdh (mouse antirat, diluted 1:2,000, abcam biotechnology) overnight at 4c . Finally, specific secondary horseradish peroxidase - conjugated anti - rabbit or antimouse antibodies (kpl, final concentration 0.2 g / ml) were used, and the immunocomplexes were detected by chemiluminescence (ecl clarity; bio - rad) using the chemi - doc xrs+ system (bio - rad). The intensity of the bands was quantified through the chemi - doc xrs+ software by adopting the 1 g sample values as reference for the relative expression and by normalizing to the gapdh values . Alizarin red solution (millipore) was used to stain in orange red the calcium deposits of the msc cultures induced to differentiate . Following paraformaldehyde fixation, cells were incubated with 500 l of alizarin red solution at room temperature for 30 minutes . After extensive washing steps with deionized water, cells were visualized in bright field under the optical microscope for image acquisition . Imagej was used to detect and automatically quantify the size of alizarin red - positive areas for each experimental group, analyzing at least ten random fields per sample . All the described experiments were carried out at least in triplicate, and all procedures were replicated twice . With regard to image analysis, data were tested with the nonparametric kruskal each box plot is composed of whiskers indicating the minimum and maximum of the data, while the top and the bottom of the box are the first and third quartiles, respectively; the band inside the box is the median . Qpcr data were analyzed with bio - rad cfx manager software . In all cases, rat mscs (scr027; millipore) were used at the second passage in all the experiments . For the maintenance of mscs, the medium was composed of dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum, 100 u / ml penicillin, 100 mg / ml streptomycin, and 200 mm glutamine (all these reagents from gibco). Cultures were maintained in an incubator at standard culture conditions (37c, 5% co2, and 100% humidity). For all the experiments, mscs were trypsinized and seeded on glass slides (diameter 13 mm) 48 hours before hypergravity treatment at 10,000/cm for tests in proliferation conditions and at 30,000/cm for tests under osteogenic differentiation . Osteogenesis was induced in the differentiation samples immediately before the hypergravity treatment by supplementing the medium with 100 nm dexamethasone, 200 m ascorbic acid 2-phosphate, and 10 mm glycerol 2-phosphate (all these reagents from sigma - aldrich). Some samples (both proliferating and differentiating) were moreover provided with btnps (20 g / ml), also in this case immediately before the hypergravity treatment . This dose was selected based on our previous results of an analysis of btnp effects on mscs, where 20 g / ml was found to be the optimal concentration at which nps did not negatively affect cellular functions.19 the nps, obtained from nanostructured and amorphous materials (houston, tx, usa), were about 150 nm in radius, and were administered to the cell culture upon stabilization in gum arabic (sigma - aldrich). Images of the final dispersion of btnps were acquired after gold - sputtering by scanning electron microscopy through a dual - beam system (fei helios 600; figure 1a). Further details on np characterization have been previously reported.19 glass slides supporting the cell cultures were transferred in cylindrical vials filled with biocompatible polydimethylsiloxane (10:1 base: cross - linking agent ratio, curing temperature 60c), and fully covered with 800 l of the appropriate cell - culture medium to exclude shear - stress effects (figure 1b). For the positioning of the samples, we used a delrin structure designed to support 30 cylindrical vials (figure 1c). It is composed of four large rotating arms with a swing gondola at each extremity (figure 1d), and can support hypergravity levels between 1 g and 20 g. we performed analyses on proliferating and differentiating rat mscs provided with and without 20 g / ml of btnps immediately before the altered gravity treatment, as previously described . The sample support was put in an incubator inside a gondola and accelerated at 20 g for 3 hours at 32c . At the end of the hypergravity stimulation, the proliferating samples were immediately processed for subsequent analysis, while differentiating samples were incubated for further 48 hours in differentiating conditions (in incubator, 37c, 5% co2) before the assessment of osteogenesis . Analogous experiments, as normal - gravity controls, were performed, maintaining cells at the same conditions of atmosphere and temperature for 3 hours at 1 g. all the procedures were performed in duplicate . In summary, we examined the following experimental classes: 1) proliferating cells at 1 g without btnps, 2) proliferating cells at 1 g with btnps, 3) proliferating cells at 20 g without btnps, 4) proliferating cells at 20 g with btnps, 5) differentiating cells at 1 g without btnps, 6) differentiating cells at 1 g with btnps, 7) differentiating cells at 20 g without btnps, and 8) differentiating cells at 20 g with btnps . Immediately after the treatment, some proliferative samples were processed in order to evaluate the effects of hypergravity and btnps on msc adhesion and shape . To assess changes in morphology, cytoskeleton conformation, and adhesion, immunofluorescence with the cytoskeleton / focal adhesion staining kit (millipore) was performed . Cells were incubated for 45 minutes with a vinculin primary monoclonal antibody (mouse antirat, diluted 1:100 in 10% goat serum in phosphate - buffered saline [pbs]) after fixation with 4% paraformaldehyde in pbs for 20 minutes at 4c, permeabilization for 15 minutes with 0.1% triton x-100 (sigma - aldrich), and treatment with a blocking solution (10% goat serum in pbs) for 1 hour . Thereafter, a staining solution composed of a green fluorescent labeled secondary antibody (goat antimouse, diluted 1:50 in 10% goat serum), 100 m tetramethylrhodamine isothiocyanate phalloidin for f - actin labeling, and 1 m 4,6-diamidino-2-phenylindole (dapi) for nucleus counterstaining were added . Samples were finally washed several times with pbs before observation under a confocal laser - scanning microscope (c2s; nikon). Cell shape was analyzed on proliferative samples stained with coomassie brilliant blue (0.2% for 5 minutes). Cell area and different shape descriptors were calculated with imagej software (http://rsb.info.nih.gov/ij) analyzing at least 50 well - distinct cells, acquired with an inverted optical microscope (nikon eclipse ti). In particular, the solidity (s), circularity (c), and roundness (r) of cells were investigated according to the following equations:24 roundness = r = ab(1)where a and b are the width and length of the minimum bounding (the smallest rectangle enclosing the selection), respectively, circularity = c=4ap2(2)where p is the perimeter and a is the cell area, and solidity = s = aconvexa(3)where convexa is the area enclosed by the smallest shell that borders all the points of the cell . Np internalization has been investigated by a multimodal microscope with an in - plane resolution of approximately 300 nm and a resolution of 1 m along the optical axis . Coherent anti - stokes raman scattering (cars) has been exploited to obtain images of cells, based on a degenerate pump - and - probe beam (papb) created by a ti sa pulsed laser (chameleon vision ii; coherent) and a supercontinuum generator (photonic crystal fiber scg-800; newport) that produces a broadband stokes beam . The beams were chirped through their transmission by two sf6 glass blocks a 10 cm - long one for the papb, and a 15 cm - long one for the stokes radiation in order to optimize the spectral resolution . For the excitation of ch2 bonds at a raman shift of 2,850 cm and thus for the cell imaging, we adjusted the delay between the papb and the stokes beam . For the localization of btnps, an 806 nm papb was combined with stokes photons producing a sum - frequency generation (sfg) signal from btnps at approximately 452 nm . The images were subject to analysis with imagej software, and the amount of btnp internalization was calculated as the ratio of the area occupied by the nps inside the cells and the total cell area, on at least 50 cells for each experimental treatment . Quantitative real - time reverse - transcription polymerase chain reaction (qpcr) was used to investigate the messenger ribonucleic acid (mrna) transcription of osteogenesis - marker genes (runt related transcription factor 2 [runx2], collagen type i alpha-1 [col1a1], and alkaline phosphatase [alpl]) and of rhoa (ras homolog gene family, member a), which regulates osteogenesis - activating runx2 following mechanical stimulation.25 after the experimental procedures, cells were trypsinized and centrifuged, and the total rna was extracted from the samples with the high pure rna isolation kit (roche) according to the manufacturer s instructions . The quantity and purity of rna was verified through spectrophotometric analysis (nanodrop; thermo scientific). Thereafter, complementary deoxyribonucleic acid was obtained from the reverse transcription of 100 ng of rna through iscript reverse transcription supermix (bio - rad). The following protocol was used for the retrotranscription: 25c for 5 minutes, 42c for 45 minutes, 48c for 15 minutes, and finally 85c for 5 minutes . The amplification was carried out on the cfx connect real - time pcr detection system (bio - rad) thermocycler with the ssoadvanced sybr green supermix (bio - rad). The temperature steps for the amplification reaction were: one cycle at 98c for 30 seconds, 40 cycles at 98c for 3 seconds, and 60c for 7 seconds, a temperature ramp from 65c to 95c, with 0.5c / second increments (for melting curve generation). The housekeeping genes adopted were gusb and rpl19; the cycle threshold (ct) value relative to the control sample (cultures performed at 1 g, without np treatment) was considered as the reference for the calculation of ct (difference between ct values deriving from difference between ct of target and housekeeping genes) for the subsequent samples.26 primer sequences (forward and reverse) of the investigated genes are reported in table 1 . The analysis was focused on the main proteins involved in the early stage of osteogenesis (alpl and col1; glyceraldehyde-3-phosphate dehydrogenase [gapdh] was adopted as the reference). For protein extraction, after trypsinization and centrifugation, cell pellets were lysed in 50 mm tris - hcl (ph 7.6), 2 mm ethylenediaminetetraacetic acid, 100 mm nacl, 1% nonidet p40, and antiproteases 1 (all these reagents from sigma). Protein quantification was performed through the pierce bca protein assay kit (thermo scientific) following the manufacturer s protocol . Twenty micrograms of protein for each sample was separated on a 4%15% mini - protean tgx stain - free gel (bio - rad) under reducing conditions, and then transferred to nitrocellulose membrane . The membranes were saturated for 45 minutes with 4% nonfat dry milk in pbs for blocking nonspecific binding sites, and then probed with primary antibodies against alpl (rabbit antirat, diluted 1:8,000, abcam biotechnology), col1 (rabbit antirat, diluted 1:1,000, abcam biotechnology), or gapdh (mouse antirat, diluted 1:2,000, abcam biotechnology) overnight at 4c . Finally, specific secondary horseradish peroxidase - conjugated anti - rabbit or antimouse antibodies (kpl, final concentration 0.2 g / ml) were used, and the immunocomplexes were detected by chemiluminescence (ecl clarity; bio - rad) using the chemi - doc xrs+ system (bio - rad). The intensity of the bands was quantified through the chemi - doc xrs+ software by adopting the 1 g sample values as reference for the relative expression and by normalizing to the gapdh values . Alizarin red solution (millipore) was used to stain in orange red the calcium deposits of the msc cultures induced to differentiate . Following paraformaldehyde fixation, cells were incubated with 500 l of alizarin red solution at room temperature for 30 minutes . After extensive washing steps with deionized water, cells were visualized in bright field under the optical microscope for image acquisition . Imagej was used to detect and automatically quantify the size of alizarin red - positive areas for each experimental group, analyzing at least ten random fields per sample . All the described experiments were carried out at least in triplicate, and all procedures were replicated twice . With regard to image analysis, data were tested with the nonparametric kruskal wallis analysis followed by the nemenyi damico wolfe each box plot is composed of whiskers indicating the minimum and maximum of the data, while the top and the bottom of the box are the first and third quartiles, respectively; the band inside the box is the median . Qpcr data were analyzed with bio - rad cfx manager software . In all cases, immunofluorescent staining of cytoskeleton (f - actin in red, vinculin in green) of proliferating mscs qualitatively showed evident effects following hypergravity treatment (figure 2). In particular, 20 g - treated cells appeared more stretched and with f - actin organized in parallel fibers with respect to the 1 g control cultures . In order to gain quantitative data about the influence of hypergravity and nps on cell morphology, we calculated cell area and cell - shape descriptors (solidity, circularity, roundness) on coomassie blue - stained cultures in proliferative conditions immediately after the hypergravity treatment (figure 3a). Concerning area evaluation (figure 3b), we observed a significant increase when cells were treated with btnps (8,6482,419 m), hypergravity (9,7352,795 m), or with the combination of the two stimuli (9,7332,739 m), compared to the control at 1 g not treated with the nps (6,1551,602 m) (p<0.05 in all the three treatments with respect to the control). The solidity of the cells (figure 3c) after hypergravity stimulation, independently of the presence of btnps (0.830.05 and 0.810.04 for 20 g and 20 g + btnps, respectively) was significantly lower than that of the cells grown at 1 g (0.930.02 and 0.980.02 for 1 g and 1 g + btnps, respectively; p<0.05 in the 20 g treatments with respect to the 1 g treatments). A similar trend was observed for circularity (figure 3d), the distributions of which denoted a significant decrease of the values of proliferating mscs subjected to 20 g acceleration (0.440.10 and 0.460.06 for 20 g and 20 g + btnps, respectively) with respect to 1 g conditions (0.720.07 and 0.770.08 for 1 g and 1 g + btnps, respectively) (p<0.05). Finally, we found an analogous trend in the reduction of the roundness (figure 3e) for the cells that underwent hypergravity treatment (0.420.13 and 0.540.10 for 20 g and 20 g + btnps, respectively) when compared to the controls at 1 g (0.710.11 and 0.670.08 for 1 g and 1 g + btnps, respectively). Nevertheless, only the roundness reduction at 20 g was statistically significant with respect to 1 g (p<0.05). Results of nonlinear microscopy on proliferating and differentiating cells internalizing btnps at 1 g and 20 g are reported in figure 4 . Figure 4a depicts images representative of the four experimental conditions, highlighting a perinuclear cytoplasmic accumulation of btnps . The cars signal from cells is represented in green, the sfg signal from nps in red: we can appreciate the emission spectrum from a bundle of nps when irradiated with the papb and the stokes beam in figure 4b . Four individual bands are visible, originating from: a second harmonic generation shg signal from the 806 nm pump beam, sfg from the combination of the 806 nm pump beam and the portion of the stokes beam temporarily overlapping to it (ie, corresponding to approximately 1,045 nm), broadband second harmonic generation from the stokes beam, and nonresonant cars background . A quantitative evaluation of np uptake (figure 4c) performed in terms of percentage of the cytoplasmic area occupied by the btnps revealed a strong enhancement of btnp internalization in cells that underwent hypergravity stimulation (p<0.05), both in differentiation (6.80.8% at 20 g, 3.60.3% at 1 g) and in proliferation (2.70.3% at 20 g, 1.40.1% at 1 g) conditions . The generally higher internalization in differentiation conditions was simply due to a longer btnp incubation time (3 + 48 hours for differentiation samples versus 3 hours for proliferation samples). The evaluation of the gene transcription through qpcr of samples under differentiation conditions is shown in the plots of figure 5a . Runx2 was significantly upregulated in 20 g - treated samples (1.5-fold at 20 g, 1.8-fold at 20 g + btnps) with respect to the 1 g and 1 g + btnp cultures . Col1a1 transcription was significantly enhanced (1.5-fold) only in the double - stimulation 20 g + btnps, while down - regulation (0.6-fold) was noticed in cultures performed at 1 g in the presence of the nps . Finally, once again alpl was significantly upregulated (1.6-fold) when cells were synergistically stimulated with hypergravity and nps with respect to all the other treatments . To evaluate how hypergravity affected the osteogenic pathway, we focused also on the transcription of rhoa in proliferating cells (thus without any osteoinductive chemical cue) immediately after the hypergravity treatment, since rhoa codes for a small guanosine triphosphatase protein known to play a key role in osteogenesis following mechanical stimulation.27 figure 5b shows a significant upregulation (p<0.05) of rhoa mrna transcription in all treatments (1 g + btnps 2.0-fold, 20 g 2.5-fold, 20 g + btnps 2.8-fold) with respect to the 1 g control not treated with btnps . Moreover, this is interesting to highlight, as the 20 g + btnp group revealed a significant upregulation of rhoa with respect to the 1 g + btnp group (1.4-fold, p<0.05), thus suggesting a role of both nps and hypergravity in the enhancement of msc differentiation . In order to assess the effects of our stimulation procedures at the protein - expression level also, western blotting of collagen type i and alkaline phosphatase was performed, and the results are reported in figure 6a . Quantitative values obtained evaluating band intensities (figure 6b) proved an enhancement of collagen type i expression in all the experimental groups with respect to the control at 1 g not treated with btnps, but only hypergravity conditions were statistically significant (20 g 1.3-fold, 20 g + btnps 1.5-fold; p<0.05). Moreover, the increasing expression of collagen type i in the synergic stimulation hypergravity + nps is significant (p<0.05) when compared to the 1 g + btnp treatment (1.3-fold, figure 6b). With regard to alkaline phosphatase, no significant expression differences were detected among any of the treatments . The alizarin red assay (figure 7a) revealed an increase of the calcium deposition (in terms of size of calcium nodules) in samples treated with btnps both at 20 g (11,9091,691 m, 45% higher with respect to 20 g without btnps 8,1921,988 m) and at 1 g (8,8132,615 m, 74% higher with respect to 1 g without btnps 5,0712,265 m). Interestingly, there was also around a 35% increment in 20 g + btnp cultures with respect to the 1 g + btnp ones; however, statistically significant differences were only detected in the samples stimulated at 20 g + btnps with respect both to 1 g (134%, p<0.005) and 20 g (45%, p<0.005; figure 7b). Immunofluorescent staining of cytoskeleton (f - actin in red, vinculin in green) of proliferating mscs qualitatively showed evident effects following hypergravity treatment (figure 2). In particular, 20 g - treated cells appeared more stretched and with f - actin organized in parallel fibers with respect to the 1 g control cultures . In order to gain quantitative data about the influence of hypergravity and nps on cell morphology, we calculated cell area and cell - shape descriptors (solidity, circularity, roundness) on coomassie blue - stained cultures in proliferative conditions immediately after the hypergravity treatment (figure 3a). Concerning area evaluation (figure 3b), we observed a significant increase when cells were treated with btnps (8,6482,419 m), hypergravity (9,7352,795 m), or with the combination of the two stimuli (9,7332,739 m), compared to the control at 1 g not treated with the nps (6,1551,602 m) (p<0.05 in all the three treatments with respect to the control). The solidity of the cells (figure 3c) after hypergravity stimulation, independently of the presence of btnps (0.830.05 and 0.810.04 for 20 g and 20 g + btnps, respectively) was significantly lower than that of the cells grown at 1 g (0.930.02 and 0.980.02 for 1 g and 1 g + btnps, respectively; p<0.05 in the 20 g treatments with respect to the 1 g treatments). A similar trend was observed for circularity (figure 3d), the distributions of which denoted a significant decrease of the values of proliferating mscs subjected to 20 g acceleration (0.440.10 and 0.460.06 for 20 g and 20 g + btnps, respectively) with respect to 1 g conditions (0.720.07 and 0.770.08 for 1 g and 1 g + btnps, respectively) (p<0.05). Finally, we found an analogous trend in the reduction of the roundness (figure 3e) for the cells that underwent hypergravity treatment (0.420.13 and 0.540.10 for 20 g and 20 g + btnps, respectively) when compared to the controls at 1 g (0.710.11 and 0.670.08 for 1 g and 1 g + btnps, respectively). Nevertheless, only the roundness reduction at 20 g was statistically significant with respect to 1 g (p<0.05). Results of nonlinear microscopy on proliferating and differentiating cells internalizing btnps at 1 g and 20 g are reported in figure 4 . Figure 4a depicts images representative of the four experimental conditions, highlighting a perinuclear cytoplasmic accumulation of btnps . The cars signal from cells is represented in green, the sfg signal from nps in red: we can appreciate the emission spectrum from a bundle of nps when irradiated with the papb and the stokes beam in figure 4b . Four individual bands are visible, originating from: a second harmonic generation shg signal from the 806 nm pump beam, sfg from the combination of the 806 nm pump beam and the portion of the stokes beam temporarily overlapping to it (ie, corresponding to approximately 1,045 nm), broadband second harmonic generation from the stokes beam, and nonresonant cars background . A quantitative evaluation of np uptake (figure 4c) performed in terms of percentage of the cytoplasmic area occupied by the btnps revealed a strong enhancement of btnp internalization in cells that underwent hypergravity stimulation (p<0.05), both in differentiation (6.80.8% at 20 g, 3.60.3% at 1 g) and in proliferation (2.70.3% at 20 g, 1.40.1% at 1 g) conditions . The generally higher internalization in differentiation conditions was simply due to a longer btnp incubation time (3 + 48 hours for differentiation samples versus 3 hours for proliferation samples). The evaluation of the gene transcription through qpcr of samples under differentiation conditions is shown in the plots of figure 5a . Runx2 was significantly upregulated in 20 g - treated samples (1.5-fold at 20 g, 1.8-fold at 20 g + btnps) with respect to the 1 g and 1 g + btnp cultures . Col1a1 transcription was significantly enhanced (1.5-fold) only in the double - stimulation 20 g + btnps, while down - regulation (0.6-fold) was noticed in cultures performed at 1 g in the presence of the nps . Finally, once again alpl was significantly upregulated (1.6-fold) when cells were synergistically stimulated with hypergravity and nps with respect to all the other treatments . To evaluate how hypergravity affected the osteogenic pathway, we focused also on the transcription of rhoa in proliferating cells (thus without any osteoinductive chemical cue) immediately after the hypergravity treatment, since rhoa codes for a small guanosine triphosphatase protein known to play a key role in osteogenesis following mechanical stimulation.27 figure 5b shows a significant upregulation (p<0.05) of rhoa mrna transcription in all treatments (1 g + btnps 2.0-fold, 20 g 2.5-fold, 20 g + btnps 2.8-fold) with respect to the 1 g control not treated with btnps . Moreover, this is interesting to highlight, as the 20 g + btnp group revealed a significant upregulation of rhoa with respect to the 1 g + btnp group (1.4-fold, p<0.05), thus suggesting a role of both nps and hypergravity in the enhancement of msc differentiation . In order to assess the effects of our stimulation procedures at the protein - expression level also, western blotting of collagen type i and alkaline phosphatase was performed, and the results are reported in figure 6a . Quantitative values obtained evaluating band intensities (figure 6b) proved an enhancement of collagen type i expression in all the experimental groups with respect to the control at 1 g not treated with btnps, but only hypergravity conditions were statistically significant (20 g 1.3-fold, 20 g + btnps 1.5-fold; p<0.05). Moreover, the increasing expression of collagen type i in the synergic stimulation hypergravity + nps is significant (p<0.05) when compared to the 1 g + btnp treatment (1.3-fold, figure 6b). With regard to alkaline phosphatase the alizarin red assay (figure 7a) revealed an increase of the calcium deposition (in terms of size of calcium nodules) in samples treated with btnps both at 20 g (11,9091,691 m, 45% higher with respect to 20 g without btnps 8,1921,988 m) and at 1 g (8,8132,615 m, 74% higher with respect to 1 g without btnps 5,0712,265 m). Interestingly, there was also around a 35% increment in 20 g + btnp cultures with respect to the 1 g + btnp ones; however, statistically significant differences were only detected in the samples stimulated at 20 g + btnps with respect both to 1 g (134%, p<0.005) and 20 g (45%, p<0.005; figure 7b). It is widely recognized that several kinds of cells, including endothelial cells,28 osteoblasts,29 myoblasts,30 and stem cells,31 are sensitive to a change of gravity - force intensity . In particular, it was found that hypergravity treatments could enhance osteogenesis in osteoblast - like cells.32 in this paper, we have reported on the hypothesis that upon hypergravity stimulation and btnp administration, mscs enhance their commitment toward osteogenesis . Mechanical forces are well known to induce cytoskeleton rearrangements, and these conformation changes deeply affect stem cell behavior.33 to analyze these phenomena in our experimental conditions, we performed a quantitative evaluation of several cell - shape descriptors . The morphometric analysis demonstrated that following a hypergravity treatment, cells were less circular, more spread, and cover a larger area when compared to the 1 g control . Overall, the characterization of cell shape and morphology showed that an increment of gravitational force, both in the presence or not of btnps, provided as a consequence a more irregular and spread morphology . This effect, consistent with other results available in the literature,34 could contribute to the maturation toward osteoblasts, given that spread cells are committed to osteogenesis, and conversely, a rounded shape promotes adipogenesis.35 furthermore, the f - actin organization in parallel and well - defined stress fibers qualitatively observed after hypergravity stimulation is a further hint of cytoskeleton - tension enhancement, and consequently of the mechanotransduction leading to osteogenesis (mediated by rhoa and runx2).36 among the impressive variety of inorganic nanomaterials investigated in nanomedicine, btnps are still relatively unexplored yet most promising, owing to their good biocompatibility,21 osteoinductive properties,19 and nonlinear optical properties.22 nonlinear microscopy performed thanks to these particular features allowed the enhancement of np uptake following hypergravity treatment to be assessed . Our results proved that hypergravity enhances np internalization, both in proliferation and differentiation cultures . A number of studies in the literature have aimed to enhance nanocarrier internalization by using physical approaches, such as ultrasounds37 or magnetic fields.38 however, these methods suffer some disadvantages related to cell damage and/or undesired effects on metabolism and functions . Hypergravity could therefore represent a valid alternative for enhanced drug and gene delivery mediated by nps for plenty of in vitro applications . Since osteogenic differentiation was induced for just 2 days (because of technical constrains at the esa facilities), the mrna - expression analysis was focused on genes involved in the early stage of osteogenesis (ie, runx2, col1a1, alpl). The obtained results, which indicated general gene upregulation following hypergravity and btnp treatments, are particularly interesting, since runx2 encodes for the key transcription factor that induces osteogenic differentiation,39 col1a1 product is the major component of the bone organic matrix,40 and alpl is involved in the mineralization process.41 corroborating our findings, similar results were obtained following msc stimulation at 2 g and at 10 g.42,43 our results highlight that the performed stimulations (hypergravity and incubation with btnps) can synergistically act in improving the early stage maturation of mscs toward osteoblasts . Western blotting showed an increment of col1 expression in hypergravity - stimulated cultures, these being data in line with other findings on osteoblast - like cells treated for 24 hours at 13 g, which demonstrated a significant increment in collagen expression.44 in our case, moreover, the combination of hypergravity with btnp incubation further increased collagen production . Therefore, increased col1 mrna expression was also translated to enhanced protein production; instead, concerning alpl, we observed upregulation of the mrna, but not enhancement of the protein expression, most probably because of a too - early stage of differentiation.45 obviously, gene activation does not always result in an increase in protein levels, because of the translation - regulation and protein - degradation mechanisms; however, even though this point is interesting, it is out of the scope of this work to deeply analyze the molecular mechanisms of transduction regulation involved in this process . Finally, we investigated the osteoblast phenotype through the alizarin red assay, which revealed an enhancement of calcium deposits in the presence of btnps and hypergravity treatment with respect to the other experimental groups . Indeed, the size of calcium nodules in samples under hypergravity conditions and btnp treatment was found to be significantly higher when compared to the hypergravity stimulation alone, thus further suggesting a synergic effect of the double stimulation . These data are consistent with the results obtained by prodanov et al who demonstrated an increase of calcium content in mscs cultured on nanotextured substrate and subjected to a 10 g treatment.43 taken together, all the obtained results suggest two hypotheses about the mechanism of msc osteogenesis enhancement following hypergravity treatment and btnp incubation . The first hypothesis is based on the already proven osteoinduction effectiveness of btnps:46 as np uptake is increased following hypergravity treatment, osteogenic maturation is enhanced because of a higher number of internalized btnps . However, we have to consider also improvements of msc maturation when hypergravity is applied without the presence of nps . In order to understand whether hypergravity could itself act as a mechanical cue able to induce osteogenesis, we performed an analysis of the transcription of rhoa, as the activity of this gene is altered following cell - shape changes, and more in general following mechanical stimulation of cells.47 our results provide the evidence that a 3-hour 20 g stimulation makes cells more spread and elongated, reducing their circularity . Several studies have reported that cell shape affects rhoa regulation:4850 spread cells cause rhoa activation that responds to mechanical stimuli enhancing cell tension and stiffness, and upregulating biochemical factors for the induction of the osteogenic pathway, while adipogenesis is inhibited . We therefore analyzed rhoa transcription in the absence of any osteoinductive chemical stimulation and immediately after the treatments . A significant increase of rhoa mrna was observed in both 1 g + btnp and in 20 g treatments, thus demonstrating that both stimuli are involved in the activation of runx2 transcription (triggered by rhoa product), and thus that both stimuli are osteoinductive . In a recent work,19 it was indeed demonstrated that btnps are able to mechanically stimulate mscs by remodeling their cytoskeleton and by increasing their stiffness . Therefore, the rhoa upregulation we observed after btnp treatment at 1 g is consistent with the reported mechanical stimulation induced by the nps . Interestingly, the combination of btnps and hypergravity had an even more pronounced effect on the upregulation of rhoa with respect to the single treatments, thus indicating that their combination intensifies osteogenesis . We can thus deduce that hypergravity acts as an osteoinductuive stimulus per se, and at the same time, by enhancing np internalization, further increases the osteoinductive potential of the btnps, thus achieving a double - level and synergic effect following the applied treatments . The goal of this research was to propose new strategies to overcome difficulties in the osteogenic differentiation of mscs . Collected data demonstrated a short treatment (3 hours) at 20 g combined with incubation with 20 g / ml of btnps synergistically promoted the osteogenesis of mscs, evaluated both at the gene and phenotype levels . Hypergravity, in addition to providing per se osteogenic stimulation, is able to promote np uptake, thus further enhancing np effects at low doses . All the collected results, even if preliminary, are promising for the elaboration of new approaches in several biomedical fields, including drug delivery, tissue engineering, and regenerative medicine.
Subarachnoid hemorrhage (sah) is one of the most common radiological features of traumatic brain injury (tbi) occurring in 30%40% of moderate severe head injuries . Vasospasm and resulting cerebral ischemia as well as cerebral hyperemia can cause delayed neurological recovery and poor outcome . A 56-year - old male suffered a moderate head injury following an alleged road traffic accident and presented to the neuro intensive care unit with a glasgow coma scale (gcs) score of 11/15 . The computerized tomography (ct) scan of brain showed bilateral frontal and right temporal contusion with left sylvian and temporal sah . He was treated with antiedema measures, namely, mannitol 1 g / kg and hypertonic saline (3%) at 30 ml / h infusion . Ct scan was repeated when gcs did not improve after 48 h. it showed a relative decrease in cerebral edema compared to the earlier scan . In view of this, antiedema measures were continued for further 2 days . However, there was no improvement in gcs . A transcranial doppler (tcd) done at this time was suggestive of cerebral hyperemia [figure 1] with a lindegaard ratio (lr) of 4 . The possibility of vasospasm in view of traumatic sah was ruled out with a normal digital subtraction angiography (dsa) [figure 2] done on the 5 posttrauma day . Conservative management was continued and the patient started improving clinically to gcs score of 13/15 on day 7 . Transcranial doppler flow with hyperemia of brain angiographic image of bilateral internal carotid arteries ruling out vasospasm transcranial doppler flow when hyperemia reduced tcd can be an important bedside tool in the management of head injury . In patients with unanticipated delay in improvement of gcs, tcd can play a vital role . The lr calculated as the ratio of mean flow of middle cerebral artery (mca) and mean flow of extracranial internal carotid artery helps diagnose or rule out vasospasm as a cause . The mean flow velocities of mca coupled with lr differentiates vasospasm from hyperemia [table 1]. Transcranial doppler grading criteria for middle cerebral artery vasospasm as in our case with lr of 4, a definitive dsa was needed to rule out vasospasm . The significance of hyperemia on the management and clinical outcome of tbi is still unknown . The effect of hyperemia on intracranial pressure (icp) is a key to predicting clinical outcome . Found hyperemia associated with raised icp to be a predictor of poor outcome and needing aggressive treatment while mild hyperemia without raised icp was linked to favorable outcome . In our case, also, mild moderate hyperemia suggested by lr of 4 and pulsatility index of 0.8 relating to low icp had a good outcome . The doppler waveform suggestive of hyperemia was described by chan et al . As an absent diastolic notch similar to our recording . The clinical implications of the above - mentioned findings are of uncertain significance . However, as observed in our patient, the resolution of hyperemia coincided with clinical improvement and may just be the temporal profile of the injury . The popularity of tcd as a bedside tool is hampered by the limitation that it is extremely operator dependent . The angle of insonation and detailed knowledge of cerebral vascular anatomy and its variant limits the interpretation . This case report highlights the importance of evaluating with tcd for an unexplained persistent low gcs . Cerebral hyperemia may be one of the causes of nonimproving gcs in the early phases of tbi . Once diagnosed, this entity may not need any further treatment other than close neurological observation.
Angiolipoma (al) is a relatively rare tumor of the head and neck region, although it occurs more commonly in the forearm and the trunk regions . This tumor has been rarely reported in the oral cavity, and to best of our knowledge, it has only been reported twice in the lip . The histological characteristics of angiolipoma are a mixture of mature adipocytes and interspersed connective tissues with vascular vessels containing fibrin thrombi and mast cell infiltration, which are distinct from usual lipomas . We present, a rare case of non - infiltrating angiolipoma occurring on the upper lip in a 9-year - old female . A 9-year - old female presented with an asymptomatic swelling of upper lip since birth . She complained of a slight increase in size of swelling for the past 1 month . Clinical examination revealed a swelling of 2 2 cm in diameter at the midline of the upper lip [figure 1]. Swelling was well circumscribed, soft in consistency, movable, non - tender, non - fluctuant, and no bruit was present . The mass was dissected free of the adjacent tissue and the lesion was surgically removed under local anesthesia . On gross examination, the mass was multilobulated, grayish white in color, soft in consistency and measuring about 2 2 cm in diameter [figure 2]. Clinical photograph of patient gross specimen showing multilobulated mass histopathologically, h and e stained section under low magnification revealed an encapsulated tumor mass consisting of adipose tissue separated by branched vascular network [figure 3]. Thin walled small capillary like vessels were present at the periphery of the tumor mass [figure 4]. Under higher magnification numerous mature adipocytes with variable sized endothelial lined blood vessels were seen [figure 5]. Toluidine blue staining showed an increased mast cells density in and around blood vessels [figure 7] with degranulation [figure 8]. . Encapsulated tumor mass consisting of adipose tissue separated by branched vascular network (h and e stain, original magnification, 10) numerous mature adipocytes with variable sized endothelial lined blood vessels (h and e stain, original magnification, 10) thin walled capillary like vessels present at the periphery of the tumor mass (h and e stain, original magnification, 40) presence of fibrinous microthrombi (h and e stain, original magnification, 40) tumor tissue showing increased mast cells density in and around blood vessels (toluidine blue stain, original magnification 40) tumor tissue showing degranulating mast cell in and around blood vessels (toluidine blue stain, original magnification 40) it is widely assumed that benign lipomatous tumors represent a common group of neoplasm's that cause few complaints or complications and present little diagnostic difficulty . . Thirteen percent of all lipomas occur in head and neck, including cheek, tongue, palate, parotid gland, neck and larynx . They are usually asymptomatic, slow - growing, soft and well - circumscribed submucosal or superficial lesion, mainly located on the buccal mucosa . The bulk of lipomatous tumors may be grouped into four categories: superficial lipoma, a tumor composed of mature fat and arising in the superficial (subcutaneous) soft tissues, represents by far the most common mesenchymal neoplasm . Deep lipomas arise from or are intimately associated with tissues deep to the subcutis or with specific anatomic sites . The main subdivisions of this group are angiomyolipoma, intramuscular and intermuscular lipoma, lipoma of the tendon sheath, neural fibrolipoma with or without macrodactyly and lumbosacral lipoma . Infiltrating or diffuse neoplastic or non - neoplastic proliferations of mature fat may cause compression of vital structures or may be confused with atypical lipomatous neoplasm / well - differentiated liposarcoma . This group is composed of six entities: diffuse lipomatosis, pelvic lipomatosis, symmetric lipomatosis, adiposis dolorosa, steroid lipomatosis, and nevus lipomatosus . Variants of lipoma are much less common and differ from ordinary lipoma by characteristic microscopic picture and specific clinical setting . These include angiolipoma (al), myolipoma, angiomyolipoma, myelolipoma, chondroid lipoma, spindle cell / pleomorphic lipoma, hibernoma and lipoblastoma / lipoblastomatosis . Al is a benign mesenchymal tumor made up of mature lipocytes and proliferating blood vessels . They are benign subcutaneous lesions most common in young male patients in their second or third decades of life . They can be multiple in nature, and are most commonly seen on forearm (two - third of cases) followed by the trunk and upper arm . Only 38 cases of head and neck als have been reported . To the best of our knowledge there are only two cases of al of the lip reported in the english literature . When seen in the oral cavity, it has been noted to be on the lip, tongue, palatal tissue, cheek being the most common site . In a review of al of head and neck, alvi et al . Mentioned certain distinct characteristics which are present between head and neck al and non - head and neck al [table 1]. Characteristics of hand neck versus non - head and neck al differential diagnosis of a soft mobile mass in the head and neck, and especially in the upper lip, should alert the clinician to consider other entities such as canalicular adenoma, basal cell adenoma, pleomorphic adenoma, angioleiomyoma, schwannoma, neurofibroma, other benign mesenchymal tumors and xanthogranuloma . Different theories suggest that a lipoma differentiates because of some unknown stimulus; the tumor is of neurogenic origin; and it is congenital . Possible causes include fatty metamorphosis of a central hemangioma, hyperplasia of fat with an associated increase in vascular channels, or a true neoplasm . There is support for the theory that an al originates as a congenital lipoma, which later undergoes vascular proliferation . Howard and helwig think that embryonic sequestration of multipotential cells become activated at puberty by hormones and differentiate into a simple lipoma . Further stimuli such as trauma can cause vascular infiltration of the lesion, but trauma is not present in many cases of al . It has been speculated that mast cells might play a role in their increased vascularity . Mast cells around blood vessels strongly express vascular endothelial growth factor (vegf) in angiolipoma, which is known to be an essential growth factor for endothelial cells in angiogenesis . In the present case, toluidine blue staining showed an increased mast cells density around blood vessels, with degranulation, speculating its role in vasculogenesis . However, there has been no direct demonstration of molecular mechanisms for angiogenesis participation by mast cells in angiolipoma . Based on studies by gonzales - crussi et al . It presents as painless or tender subcutaneous nodules, generally in pubescent patients and is rare before puberty . Histologically it is encapsulated, and is a mixture of mature adipocytes and a proliferation of thin - walled vascular channels . They extend into the surrounding tissue and are characterized by a non - encapsulated tumor mass . The present case was that of non - infiltrating al of 2 2 cm in diameter located in the upper lip of young female . Following are the current histologic guidelines for diagnosis of al: well encapsulated (non - infiltrating als) or poorly encapsulated (infiltrating als).evidence of 50% mature adipocytes in the tumor.interspersed angiomatous proliferation in the tumor.fibrinous microthrombi.absence of other mesenchymal elements (smooth muscle) or pleomorphism . Well encapsulated (non - infiltrating als) or poorly encapsulated (infiltrating als). Evidence of 50% mature adipocytes in the tumor . Interspersed angiomatous proliferation in the tumor . Histopathological differential diagnosis includes hemangioma, lipoma, angiomyolipoma, infiltrating lipoma, angiofibrolipoma, angiomyxolipoma, and liposarcoma . Angiomyolipoma is composed of varying amounts of blood vessels, smooth muscles and fat cells . Infiltrating lipoma consists of lesional fat tissue infiltrating in the deeper tissue, in the form of long thin streaks radiating from the intratumoral mass . Angiofibrolipoma is composed of varying amounts of blood vessels, fibrous tissue and fat cells . It is the most common benign tumor of the kidney and is strongly associated with tuberous sclerosis . The hypovascular lesions may be difficult to distinguish from ordinary lipomas, although the identification of microthrombi allows this distinction . Liposarcoma can be confused with al but can usually be differentiated by the presence of embryonal adipose tissue, pleomorphism, increased number of mitosis, and metastasis . The al has not been shown to spontaneously regress and has been shown to continue to enlarge as opposed to other entities such as a hemangioma . The treatment of choice for a non - infiltrating al is surgical excision, whereas a wide local excision with free margins has been advocated for the infiltrating type . In the case of inadequate resection the recurrence of al has been reported to be dependent on whether the mass is infiltrating . Infiltrating als have been reported to be more common in an older age group . Their recurrence has been reported to be as high as 62.5% in areas outside the head and neck . Noninfiltrating types are seen more commonly in a younger age group and have no tendency to recur . A successful treatment of a giant infiltrating angiolipoma with interferon alfa also has been reported . In conclusion, lipomas represent about 1%4% of all neoplasm's of the oral cavity . They are usually asymptomatic, slow - growing, soft and well - circumscribed submucosal or superficial lesion . It consists of mature adipocytes and interspersed connective tissues with vascular vessels containing fibrin thrombi and mast cell infiltration . Wide surgical excision with free margins is the treatment of choice, offering a good prognosis and an almost negligible relapse rate.
Superior semicircular canal dehiscence syndrome (scds) is a recently recognized clinical condition, which was initially described by minor, et al.1) the syndrome usually encompasses a constellation of vestibular and audiological symptoms, such as sound and/or pressure induced vertigo and oscillopsia, along with conductive hearing loss and autophony, and typically manifests as sound and/or pressure induced nystagmus at the plane of the superior semicircular canal (ssc).2 - 4) the proposed underlying mechanism involves the existence of a dehiscence at the apex of the ssc (third mobile window), in addition to the round and oval windows of the osseous cochlea, which in effect potentiates the transmission of sudden changes in the middle and/or intracranial pressure, thus altering the related neural firing rates of the vestibular system, and may also alter inner ear fluid dynamics, causing dissemination of the acoustic energy.5,6) while the classic presentation can be suspected on clinical and audiometric data, imaging plays an important role in the evaluation of these patients . Advances in computed tomography (ct) now allow high resolution images to demonstrate the bony defect, while multiplanar reformations can also aid in the radiologic diagnosis.7 - 9) despite the several investigations on this condition, there are no clear causes of scds . Certainly, a congenital / developmental basis for the condition has been described.9 - 13) there was a report describing the development of scds from an intact ear, confirmed by serial follow - up ct scans.14) in that study, osteomyelitis and increased bony absorption associated with chronic brain pulsation might be one of the possible causes of scds . Because inflammatory process is related to the decreased mastoid air - cell volume,15,16) we wanted to compare the mastoid air - cell volume of the patients with scds and that of the age- and sex - matched patients with otosclerosis and temporal bone (tb) fracture, who are supposed to represent normal mastoid pneumatization . 10 patients with scds confirmed by symptoms, signs, vestibular evoked myogenic potential and tb ct imaging were enrolled . To compare the mastoid air - cell volume, the otosclerosis and tb fracture patients identified in the tb ct were selected as control groups . The otosclerosis was identified in both sides, and with no other pathology, such as chronic otitis media (com) and middle ear effusion . This study was approved by the institutional review board of our institute and followed the recommended guidelines . High - resolution tb ct scans were performed using a 64 row detector ct scanner (siemens, medical system, erlanger, germany). The axial plane images were acquired using a 0.6 mm section thickness at 200 ma and 120.0 kv . The coronal and oblique sagittal reconstruction images, which were parallel to the superior semicircular canal, were acquired using 0.4 mm interval reconstructions . All of the images were evaluated on a diagnostic petavision pacs station (emsoma, seoul, korea). To measure the mastoid air - cell volume, we used 3d reconstruction software . The imaging data were stored in a digital imaging and communication in medicine file and then imported to a personal computer running vworks 4.0 software (cybermed, seoul, korea). When performing reconstruction using a surface rendering algorithm, the selection of the window thresholds were -1024 to -318 hounsfield units . The volume of mastoid air - cell was automatically calculated in the 3d reconstruction (fig . Statistical analysis was conducted using wilcoxon signed rank test to compare volume between the scds side and the normal side in scds patients, and left and right side of the otosclerosis patients . The differences of mastoid air - cell volume in the scds, otosclerosis and tb fracture were determined using the kruskal - wallis test and mann - whitney u test . All statistical analyses were performed using spss 20.0 software (spss, ibm corp ., armonk, ny, usa), and the p values <0.05 were considered significant . Mean mastoid air - cell volume in scds side was 3319.9 mm, whereas 4177.2 mm in the normal side (table 1). There was a significant difference of the mastoid air - cell volume between the lesion side and the normal side of scds patients (p=0.022, wilcoxon signed rank test). Mean mastoid air - cell volume of otosclerosis patients was 6594.3 mm in the right side and 6380.5 mm in the left side (table 1). And there was no significant difference of mastoid air - cell volume between the right and left sides (p=0.445, wilcoxon signed rank test). Mean mastoid air - cell volume in normal side of tb fracture was 6477.2 mm (table 1). There is no difference between the mastoid air - cell volume of the otosclerosis and that of tb fracture patients (p=0.684). The mastoid air - cell volume of the scds (both sides) showed significant difference compared to that in any side of the otosclerosis patients (p=0.013, kruskal - wallis test). In the post - hoc analysis, the mastoid air - cell volume in scds side was significantly smaller than those of otosclerosis patients (p=0.009, mann - whitney u test)(fig . 2). However, the difference between the mastoid air - cell volume in the normal side of scds patients and that of otosclerosis patients was not significant (p=0.063, mann - whitney u test). Similarly, the mastoid air - cell volume of the scds (both sides) showed significant difference compared to that in tb fracture patients (p=0.006, kruskal - wallis test). In the post - hoc analysis, the mastoid air - cell volume in scds side and normal side of scds patients were significantly smaller than that of tb fracture patients (p=0.002, p=0.019, respectively, mann - whitney u test)(fig . The decrease of mastoid air - cell volume could be seen several conditions, such as otitis media with effusion, com, and cholesteatoma.15,16) many studies reported that the inflammatory process is related to the decreased mastoid air - cell volume . Our results demonstrated significantly smaller mastoid air - cell volume in the scds side than in that of the otosclerosis patients and tb fracture patients (p=0.009, p=0.002, respectively). The mastoid air - cell volume (4177.2 mm) in the normal side of scds patients was smaller than that (6594.3 mm in the right ear and 6380.5 mm in the left ear) of the otosclerosis patients, although it was not significant (p=0.063). But, the mastoid air - cell volume in the normal side of scds patients significantly smaller than tb fracture patients (4177.2 mm vs. 6477.2 mm, p=0.019). Because no patients with scds in this study had soft tissue density in the mastoid in tb ct scans, suggesting there is no active inflammation in the mastoid, inflammatory process (acute otitis media or middle ear effusion) in the early childhood could have had a role in the decreased mastoid air - cell volume in the scds ear . It remains unclear whether scds is a congenital / developmental disorder or whether it is acquired . It has been reported that tb specimens from infants show uniformly thin bone over the superior canal in the middle fossa at birth, with gradual thickening until 3 years of age, and that scds may arise from failure of postnatal bone development.9) tsunoda and terasaki17) has described the embryological basis for dehiscence . If the otocyst is situated close to the developing brain during this stage, there is inadequate space for the growth of the superior surface of the superior semicircular canal as the otocyst may lie against the dura . Hindering gradual thickening of the superior canal roof by otitis media before the age of 3, associated with the decreased mastoid pneumatization, additionally, scds may be generated by chronic brain pulsation and the pressure exerted by the temporal lobe on the middle cranial fossa.9) however, another study demonstrated that the radiologic prevalence of scds among older age groups increases, suggesting that scds is more commonly an acquired rather than developmental condition.18) chronic bone inflammation by previous otitis media in childhood, which can be identified as having decreased mastoid volume, may render the bone overlying the superior canal susceptible to brain pulsation or the pressure from the temporal lobe, resulting in the generation of scds . Our findings revealed that the mastoid air - cell volume in the scds side was significantly smaller than that of otosclerosis and tb fracture patients, furthermore the volume in the normal side of scds patients was significantly smaller than that of tb fracture patients, which suggest that the decreased mastoid pneumatization is closely related to the generation of scds, whether scds is a congenital / developmental disorder or whether it is acquired.
The malaria parasite, plasmodium, is a major public health burden in the developing world, and despite the existence of antimalarial treatment active in blood stages of infection, there is a continual need for novel drug design as the parasite develops resistance to current treatments . Additionally, treatment for the hypnozoite - causing species, plasmodium vivax, requires primaquine, which has severe side effects and causes hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency . Discovery of novel compounds in antimalarial drug development is essential for future intervention strategies . Atg8 is the ubiquitin - like (ubl) protein necessary for formation and maturation of autophagosomes in autophagy in eukarya . In yeast and mammals, atg8 is lipidated to the autophagosome membrane, but in plasmodium, atg8 is partially conjugated to the membrane of the apicoplast under nonstarvation conditions . The apicoplast is a nonphotosynthetic chloroplast - like organelle present in apicomplexans and is essential for isoprenoid synthesis . Under starvation conditions, atg8 relocates to acidic vesicles with rab7 in and near the food vacuole . Atg8 is essential to the plasmodium parasite and has been proposed as a target for antimalarial drug design . In most eukaryotes, lipidation of atg8 to phosphatidylethanolamine (pe) in membranes normally requires proteolytic processing of the c - terminus of atg8 by atg4 and activation via adenosine 5-triphosphate (atp) followed by intermediate thioester bond formation with the e1-activating enzyme atg7 . Atg8 is then transferred to its e2-like conjugating enzyme atg3, forming a second thioester intermediate before being conjugated to the nitrogen of pe (figure 1). This process also requires noncovalent interaction between atg8 and atg3 through a well - characterized atg8-interacting motif (aim) in atg3 and two hydrophobic pockets, termed the w and l - site, in atg8 . Notably, in plasmodium, atg8 is synthesized with a c - terminal glycine and therefore does not require activation by atg4 . Recently published studies showed a drastic growth defect in plasmodium falciparum when levels of pfatg7 were reduced . This along with the essentiality of plasmodium atg8 suggests that targeting pfatg8 lipidation is a good strategy for drug intervention . We previously elucidated the protein crystal structure of p. falciparum atg8 bound to a peptide corresponding to pfatg3 s aim (pdb code 4eoy). Regions of diversity exist between the human and plasmodium system that may be exploitable through small molecule inhibition . Our mutational and interaction studies suggest that the plasmodium atg8-atg3 interaction requires atg8 s w / l site as well as the apicomplexan loop on atg8 (residues 6776), termed the a - loop . Here, we report the identification of a class of compounds that inhibit the plasmodium atg8-atg3 interaction and that inhibit in vitro growth of p. falciparum in blood- and liver - stage assays, presumably through prevention of pfatg8 lipidation . Previously, we developed a surface plasmon resonance (spr)-based competition assay to identify compounds that disrupt the pfatg8-pfatg3 noncovalent interaction.pfatg3 is immobilized onto an spr chip, and pfatg8 is injected in the presence of dimethyl sulfoxide (dmso, control) or a compound (dissolved in dmso), and binding is measured by the spr response . The medicines for malaria venture (mmv) malaria box of 200 druglike and 200 probelike molecules was screened at 5 m in a primary spr screen (figure 2a). Six compounds met the cutoff for at least 25% inhibition of the pfatg8-pfatg3 interaction: (n-(4-methylphenyl)-4-pyridin-2-yl-1,3-thiazol-2-amine) (1), (2-methylsulfanyl - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide) (2), (2-bromo - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide) (3), n-[2-chloro-5-(trifluoromethyl)phenyl]-2-[2-(4-methylphenyl)pyrazolo[1,5-a]pyrazin-4-yl]sulfanylacetamide (4), 1-[4-(dimethylamino)phenyl]-6,6-dimethyl-1,3,5-triazine-2,4-diamine (5), and 2-n,3-n - bis(4-bromophenyl)quinoxaline-2,3-diamine (6) (figure 2a,2b, table 1). In subsequent dose - dependent studies, 46 demonstrated a constant level of inhibition independent of concentration of the small molecule and were not further investigated . Compounds 13 led to dose - dependent inhibition with an spr inhibitory concentration (ic50 spr) ranging from 6 to 18 m (figure 2c). Interestingly, these compounds shared a common scaffold: 4-pyridin-2-yl-1,3-thiazol-2-amine (pta) incorporated as the n - substituent in various anilines or benzamides . 13 were tested for their effect on the stability of pfatg8 using fluorescence - based thermal shift assays (tsas). None of the compounds significantly affected the melting temperature (tm) indicating that they most likely did not disturb the tertiary structure of pfatg8 (figure 2d). Pfatg8 was immobilized onto an spr ni - nitrilotriacetic acid (nta) chip via a 12 histidine n - terminal tag . 1, chosen for its better solubility, was injected over the chip, and binding was measured . Compound 1 led to a dose - dependent increase in spr response, indicating binding to pfatg8 (figure 3a). We next sought to determine the binding site for the pta compounds with in silico docking . The openeye software package (www.eyesopen.com) was used to dock conformers of the compounds against the x - ray structure of pfatg8 (pdb code 4eoy). All three compounds docked to the w - site of pfatg8 (figure 3b). 2 and 3 bound with the pyridine ring in the w - site, whereas 1 was predicted to bind with the pyridine ring in the l - site, the thiazole ring positioned between the pockets, and the methylbenzene group in the w - site . An alternate pose was enriched within the top 10 poses output by the docking study in which 1 binds solely within the w - site in a more compact conformation . 1 is missing a donor oxygen compared to 2 and 3 and, therefore, may adopt a different mode of binding to the w- and l - sites of pfatg8 . 2 docked with the pyridine ring in the w - site, and the methylsulfanylbenzene group bound to the l - site of pfatg8 . Docking was repeated using a version of the receptor for which the carbonyl of lys47 was input as a hydrogen bond acceptor constraint (figure 3b inset). 1 had a similar binding pose to the highest ranked pose from the unconstrained docking, while 2 was slightly different with the pyridine ring rotated 90 in the w - site and the benzene ring positioned just below and to the left of the l - site pocket with the methylsulfanyl group reaching into the mostly hydrophobic l - site . The half maximal inhibitory concentrations (ic50) for these compounds in p. falciparum 3d7 blood stages were previously reported and are located on the ncbi pubchem database (http://pubchem.ncbi.nlm.nih.gov). 1 has a reported ic50 of 350400 nm (pubchem bioassay i d (aid): 660866 and 449703). The reported ic50 for 2 ranged from 0.20 to 6.8 m, while 3 ranged from 1.36 to 4.52 m (pubchem aid: 660866 and 449707). We focused on compound 1 for further studies because the reported cytoxicity in human cell lines is much lower than that of compounds 2 or 3 (pubchem aid: 660872, 685525, and 449705). Pfatg8 is expressed and lipidated during the liver stage where it partially localizes to the apicoplast . Treatment of early liver stage parasites with the autophagy inhibitor 3-methyladenine is reported to delay conversion of the parasite into its trophozoite form . 1 was previously tested in plasmodium yoelii liver stage cultures and did not display> 50% inhibition at the screening concentration of 10 m; an ic50 was not reported (pubchem aid: 602118 and 602156).p . Yoelii and plasmodium berghei are often used to test drugs for liver stage inhibition as they are easier to culture . However, these are rodent malaria models and may not be indicative of activity in p. falciparum . Analysis of the w / l - site in these three species revealed differences in the amino acid composition that could affect drugs predicted to bind in that region (figure 4). We utilized a recently established p. falciparum liver stage in vitro model in which sporozoites isolated from infected mosquitos salivary glands invade hc-04 hepatocytes . Hc-04 is a unique immortalized cell line that exhibits the expression of biochemical markers characteristic for normal hepatocytes and allows for the full development of the human malaria parasite, p. Falciparum. (2022) using this system, we assessed the effect of 1 on the development of p. falciparum 3d7-green fluorescent protein (gfp) parasites in human hepatocytes in vitro . No change in the viability of hc-04 cells was detected in response to treatment with 3 m or 30 m of 1 for 96 h (figure 5a). Using flow cytometry, we observed about a 50% decrease in the proportion of hepatocytes infected with p. falciparum 3d7-gfp sporozoites (gfp+/propidium iodide (pi)- cells) in response to treatment with 30 m, but not with 3 m of 1 (figure 5b, c). Additionally, there was a dose - dependent reduction in the intensity of gfp fluorescence at both concentrations of 1, indicating inhibition of parasite development within hepatocytes, at least in vitro (figure 5d). Because 1 did not affect cell survival or cell growth of hc-04 cells (figure 5a), the compound s effect on the parasite is unlikely to result from host cell cytotoxicity . We next sought to determine whether 1 had an effect on pfatg8 in p. falciparum blood stage cultures . In immunoblot assays, very low levels of endogenous pfatg8 were detected in dmso - treated control cells . Incubation of cells in minimal media lacking human serum for 5 h led to a very slight increase in pfatg8 . In contrast, treatment with 50 m cytocidal levels of compound 1 led to a drastic increase in pfatg8 protein levels as well as to a shift in mobility, likely corresponding to the unlipidated form of pfatg8 . Overall protein levels were unchanged, indicating an up - regulation or accumulation of pfatg8 in the presence of 1 under conditions that are likely leading to cell death (figure s1 of the supporting information). After treating the parasites for 6 hours, a dose - dependent increase in delipidation of pfatg8 is already observed at 12.5 m of 1, and at 25 m, unlipidated pfatg8 is the predominant species (figure 6a, figures s2s4 of the supporting information). When investigating the soluble versus insoluble membrane fraction, pfatg8 could only be detected in the soluble fraction with the number of parasites used per lane . We attribute this to the low amount of pfatg8 present in the untreated control and to reaching the detection limit of our assay (data not shown). At the treatment concentration and duration used in the study, our studies indicated that the pta scaffold is a good platform for hit - optimization . We synthesized a pta - benzaldehyde derivative, 4-formyl - n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide (7), with a functional handle extending off the common hydrophobic ring system (table 1, scheme 1). 7 was prepared from commercially available pta and 4-formyl benzoic acid through a dicyclohexylcarbodiimide (dcc)-promoted amide coupling . Compound 7 can be tethered through a dialkoxyamine linker to a library of aldehydes and can be screened using our primary spr competition assay against the pfatg8-pfatg3 interaction . In docking studies, 7 bound the w- and l - site of pfatg8 in a fashion similar to compound 2 with the functional handle positioned toward the a - loop pocket (figure 7a). A gain in parasite selectivity for such bifunctional analogues is expected as the a - loop is missing in the human atg8 homologues . To confirm binding to pfatg8, we tethered 7 to (+) -biotinamidohexanoic acid hydrazide (bach) through its reactive aldehyde group and tested binding with spr . His12-pfatg8 injected at various concentrations led to a dose - dependent increase in spr response indicating pfatg8 directly binds 8 with a kd of 540 nm . In contrast, the human atg8 homologue, microtubule - associated protein light chain 3 (hlc3) showed much lower affinity for 8 with a kd of 18 m, indicating specificity of the pta scaffold for p. falciparum (figure 7b). Additionally, recombinant pfatg3 did not bind immobilized 8 (data not shown) in agreement with our docking studies suggesting binding to the w - site of pfatg8 . Compound 7 was converted to the hydrochloride salt and was subjected to acid - catalyzed acetal formation with methanol and trimethyl orthoformate under microwave irradiation to provide the dimethyl acetal compound 9, an unreactive derivative, to confirm the inhibitory activity of the starting platform (table 1, scheme 1). 4-(dimethoxymethyl)-n-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide 9 has a similar shape and distribution of the acceptor and donor pairs as the original pta compounds (figure 7c) and docked onto pfatg8 in a fashion similar to 7 (figure 7a). Spr studies confirmed that 9 inhibited the pfatg8-pfatg3 interaction with an ic50 of 2.86 m in spr (figure 8a). We next measured growth inhibition of p. falciparum 3d7 by 1 using the sybr green i assay . This assay exploits the absence of nuclei in erythrocytes with a fluorescent dye that is unquenched upon binding to nucleic acids, preferentially double - stranded dna . In two of three independent experiments, the ic50 of 1 was 768 nm, similar to previously published results, while in a third experiment, the ic50 was 3.3 m, resulting in an average ic50 of 1.61 1.47 m . Using this assay, 9 had a potency similar to that of compound 1 against the blood stage of p. falciparum with an average ic50 of 1.48 0.6 m (figure 8b). We identified compound 1 through a screen for inhibitors against the plasmodium atg8-atg3 protein protein interaction (ppi). Targeting protein protein interactions has long been overlooked in the drug development field . It was thought to be difficult because of the shallower nature of many pockets and the more extensive network of residues involved in the interaction compared to an enzymatic site . However, ppis also offer the opportunity for more selectivity, an important concept when targeting a eukaryotic parasitic protein within a eukaryotic host . Great methodological and technological advances have been made recently using this approach with promising leads in cancer drug development . In the case of our inhibitor, in addition to its potential as a therapeutic drug, any 1-derived inhibitor could serve as a tool to elucidate the function of pfatg8 during different stages of the malaria life cycle as its function is currently unclear . Treatment of p. falciparum with high levels of 1 led to a drastic increase in pfatg8 protein levels, presumably the unlipidated form as judged by its migration in sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page). This increase could be due to an up - regulation of pfatg8 synthesis to compensate for inhibition or due to a buildup of existing protein levels because of a blockade in autophagic degradation . In yeast, nitrogen starvation leads to induction of atg8 expression while inhibition of later stages of autophagy leads to accumulation and even greater protein levels of atg8 . Further studies are necessary to determine if pfatg8 is up - regulated at the transcriptional, translational, or degradation level in response to treatment with 1 . This could be due to an accumulation of the drug inside the parasite or could be because even slight inhibition of pfatg8 lipidation has drastic effects on parasite growth, similar to reaching the tipping point on a balance . An alternative explanation is the result of off - target effects; however, the observed delipidation of pfatg8 could not be attributed to an off - target effect . 1 was previously reported to have low cytotoxicity with an ld50 (lethal dose) of 18.2 m in hepg2 cells and half maximal cytotoxicity concentration (cc50) and ic50 of 32 m in huh7 cells (pubchem aid: 685525, 660872, 449705). In our study together, this indicates 1 may be a good starting scaffold for antimalarial drug design . Compound 1 had lower activity in the liver stage than in the blood stage, which could either indicate that pfatg8 is less important in the liver stage or that less compound is delivered to the parasite in liver cells . There is precedence for this variability in the efficacy of antimalarials between the liver and blood stages . Compound 1 showed 50% inhibition of liver stage parasites at 30 m, which is within the range for cytotoxicity in human cells . We suggest the use of 1 and the pta scaffold for further probe development to increase specificity and potency in both the liver and blood stages . All pta - containing mmv compounds had a ligand efficiency by atom number (lean) score greater than 0.3 in the blood stage assay, indicative of good ligand efficiency and good potential for future drug optimization . This combined with the higher affinity of 8 for pfatg8 compared to human lc3 makes the pta scaffold a promising starting point for expansion . We are currently pursuing optimization through a combinatorial oxime library approach using 7 with the goal to extend the inhibitor into the a - loop pocket and to gain selectivity toward pfatg8 . Our spr data confirm that 1 directly binds to pfatg8, while our docking studies suggest that the pta scaffolds of compounds 13, 7, and 9 bind the w - site . The w - sites of atg8 homologues in mammals and yeast participate in numerous protein protein interactions through binding to the aromatic residue of an aim, including nonautophagic proteins . Therefore, our inhibitor and its derivatives could be used to identify novel plasmodium atg8 interactions as well as to confirm paralogous interactions known to occur in yeast and mammalian cells . Taken together, our bioinformatics analysis of the pta - binding site and the spr binding studies strongly suggest that the amino acid differences near the l- and w - site may have contributed to the failure of 1 in the p. yoelii liver stage drug - screening assay . Two residue differences (i8v, p9s) adjacent to the w - site may lead to a widening of the w - site in p. yoelli because of their shorter side chains, and one residue (v62i) participating directly in the l - site results in an extended side chain and therefore may decrease the volume of the l - site pocket . Additionally, l115 m just below the l - site binding pocket may also contribute to a decreased volume as a secondary shell residue . Care and emphasis on choice of model system should be taken into account when studying protein ligand interactions, as single amino acid substitutions may have drastic effects on binding . His12-pfatg8 variants were expressed and purified with cobalt - nta affinity columns similar to his6-pfatg8, as previously published with the exception that proteins were eluted from cobalt - charged talon resin (clonetech) in buffer containing 50 mm ethylenediaminetetraacetic acid (edta) rather than imidazole . Spr runs were conducted on a biacore 3000 instrument (ge healthcare) at 25 c with a flow rate of 50 l / min, unless otherwise specified . Running buffer (rb) consisted of 1 phosphate - buffered saline (pbs) (1 mm kh2po4, 5.6 mm na2hpo4, 154.5 mm nacl, ph 7.4), 0.01% v / v p20, and varying amounts of dmso (quality biologicals). A double referencing method was applied to correct for nonspecific binding to the chip with interspersed blank injections correcting for baseline drifts . Changes in refractive index because of dmso were accounted for with a dmso calibration curve . Mbp - pfatg3 was immobilized onto a cm5 chip (biacore) as described previously with mbp immobilized on a reference flowcell . Compounds were added to 300 nm his6-pfatg8 in rb at a final concentration of 5 m, and 40 l was injected, followed by a 12.5 l injection of 2 m mgcl2 for dissociation and regeneration of the spr chip surface . Thirty microliters of pfatg8 at 200 nm was injected in the presence of a 2-fold dilution series of compound (highest concentration of 50 m) or equivalent volume of dmso (final dmso concentration was 1%). His12-pfatg8 was injected over an nta - chip (ge healthcare) preconditioned with nickel, leading to capture of 3000 response units (rus). Running a 2-fold - dilution series of compounds, highest concentration of 75 m, was injected over pfatg8 variants at 40 l / min . 8 was injected over a neutravidin - coated spr chip (ge healthcare) on one flowcell, with 130 rus immobilized, while bach was injected over a reference flowcell (150 rus immobilized). His6-pfatg8, human lc3, or his6-pfatg3 was injected in duplicate in running buffer containing 10 mm hepes ph 7.5, 150 mm nacl, 0.05% p20 . Protein was dissociated from the chip after each cycle with 10 l injection of 20 mm hepes ph 7.4, 1% w / v sds regeneration solution . All reagents were obtained from commercial suppliers and were used without further purification . Acetonitrile was distilled after drying on cah2 and then was stored over 3 molecular sieves . Dynamic adsorbents 3263 m silica gel was used for flash column chromatography, and 250 m f254 plates were used for thin layer chromatography (tlc). Microwave - assisted reactions were carried out using a biotage initiator microwave synthesizer (300 w). H and c nmr spectra were acquired on a bruker avance iii 500 spectrometer operating at 500 mhz for h and 125 mhz for c. chemical shift values are reported as (ppm) relative to chcl3 at 7.27 ppm and dmso at 2.50 ppm for h nmr and chcl3 at 77.0 ppm and dmso at 39.51 ppm for c nmr . Mass spectrometry analysis was carried out at university of illinois at urbana - champagne, school of chemical sciences, mass spectrometry laboratory . The purity of synthesized compounds was 95% as analyzed by high - performance liquid chromatography (hplc, beckman gold nouveau system gold) on a c18 column (grace alltima 3 m c18 analytical rocket column, 53 mm 7 mm) using triethylammonium acetate buffer (50 mm, ph 7) and acetonitrile (acn) as eluent, flow rate 3 ml / min, and detection at 300 nm . To a solution of 4-(pyridin-2-yl)-1,3-thiazol-2-amine (0.059 g, 0.33 mmol) in acetonitrile (2.0 ml) was added sequentially dicyclohexylcarbodiimide (0.076 g, 0.37 mmol), 4-formylbenzoic acid (0.050 g, 0.33 mmol), and n, n - dimethylamino pyridine (0.012 g, 0.10 mmol). The mixture was heated at 50 c for 17 h and then was allowed to cool to ambient temperature . Solids were removed by vacuum filtration, and the resulting filtrate was condensed under reduced pressure . The resulting yellow solid was redissolved in chcl3 (5 ml), and 1 m hcl (5 ml) was added to give a yellow - tan emulsion at the liquid this solid was collected by centrifugation at 4000 rpm for 5 min followed by manual collection of the resulting cake (this acid precipitation was necessary to remove closely eluting impurities). The solid was then purified by silica flash column chromatography (dichloromethane(dcm):meoh: triethylamine 94:5:1) rf = 0.32 . The product was obtained as a yellow powder (23 mg, 22% yield). H nmr (500 mhz, dmso - d6) (ppm) = 12.95 (br . S., 1h), 10.13 (s, 1h), 8.63 (d, j = 3.93 hz, 1h), 8.31 (d, j = 8.17 hz, 2h), 8.07 (d, j = 8.33 hz, 2h), 8.03 (d, j = 7.86 hz, 1h), 7.92 (s, 1h) 7.91 (td, j = 2.00 hz, 8.75 hz, 1h), 7.35 (ddd, j = 1.10, 4.79, 7.47 hz, 1h) c nmr (500 mhz, dmso - d6) (ppm) = 192.90, 164.74, 158.91, 152.03, 149.54, 149.38, 138.50, 137.30, 137.15, 129.41, 128.94, 122.88, 120.06, 112.30 . High - resolution mass spectrometry (hrms, electrospray ionization, esi) m / z: calcd 310.0650 (m h); found 310.0651 (m h). Twenty - five microliters 50 mm 7 dissolved in dmso was incubated with 20 l 50 mm bach (5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a - hexahydrothieno[3,4-d]imidazol-4-yl]-n-(6-hydrazinyl-6-oxohexyl)pentanamide) (sigma - aldrich) dissolved in dmso and 10 l sodium acetate (ph 4.5) with 0.02% sodium azide at 37 c overnight . 7 dissolved in chcl3 was treated with 1 m hcl as described above to form the hydrochloride salt . 7 hcl (0.040 g, 0.12 mmol) was suspended in meoh (0.5 ml), and trimethylorthoformate (0.010 ml, 0.91 mmol) was added followed by p - toulene sulfonic acid monohydrate (0.003 g, 0.012 mmol). This solution was heated by microwave irradiation at 130 c in a sealed vial for 5 min and then was stirred at ambient temperature for 36 h at which time a precipitate formed . The solvent was removed under reduced pressure, and the residue was dissolved in dcm (10 ml) and was washed with saturated nahco3 (10 ml) and brine (10 ml) and was dried with na2so4 . Condensation under reduced pressure yielded the product as a yellow powder (26 mg, 61% yield). H nmr (500 mhz, cdcl3) (ppm) = 9.81 (br s, 1h), 8.64 (d, j = 4.24 hz, 1h), 7.96 (d, j = 8.17 hz, 2h), 7.91 (d, j = 7.86 hz, 1h), 7.74 (s, 1h), 7.74 (td, j = 1.73, 7.70 hz, 1h), 7.62 (d, j = 8.17 hz, 2h), 7.22 (dd, j = 4.95, 6.84 hz, 1h), 5.47 (s, 1h), 3.35 (s, 6h) c nmr (500 mhz, cdcl3) (ppm) = 164.24, 158.12, 152.24, 149.85, 149.62, 143.32, 136.84, 131.80, 127.51, 127.29, 122.69, 120.50, 112.21, 102.08, 52.71 . Hrms (esi) m / z: calcd 356.1069 (m h); found 356.1070 (m h). Assays were conducted in 1 pbs with 1:1800 final dilution of sypro orange dye (invitrogen). Fluorescence was measured from 20 to 80 c in a biorad c1000 thermal cycler . One hundred microliters of pta compounds or equivalent volume of dmso was added to his6-pfatg8 . The hc-04 cell line (atcc, manassas, va, u.s .) Was maintained in complete medium (imdm containing 2.5% fcs, 100 units / ml penicillin, 100 g / ml streptomycin, and 2 mm l - glutamine, all from gibco, life technologies, grand island, ny). P. falciparum 3d7-gfp parasite strain was propagated in the parasitology core facility, the johns hopkins malaria research institute . In vitro infection of human hepatocytes briefly, salivary glands were sequestered from infected anopheles gambiae mosquitoes at day 17 after exposure to infective blood meal, and homogenates were separated on an optiprep density gradient (sigma - aldrich, st . Sporozoites were collected from the gradient interface, were washed in complete medium, were counted using a hemocytometer, and were incubated with hc-04 cells at 3:1 sporozoite to hepatocyte ratio for 2 h at 37 c . Infected cultures were further propagated in complete medium alone or in medium supplemented with 3 m or 30 m of 1 . Flow cytometry based detection of infected cells was done 72 h post infection using facscalibur flow cytometer (bd biosciences) and was analyzed using flowjo software (tree star, inc ., effect of 1 on the viability of in vitro propagated hc-04 cells was monitored as follows: 0.3 10 cells per well were seeded into the 24-well plate and were treated with 3 m or 30 m of 1 in complete medium for 96 h, and a relevant amount of dmso was used as a vehicle control . Detection of annexin - v positive and pi - positive cells in hepatocyte cultures was done by flow cytometry according to the manufacturer s instruction (invitrogen, life technologies, grand island, ny, u.s . ). P. falciparum 3d7 and fcr3 cultures were maintained using modified, previously published methods at 37 c, 2% hematocrit of human red blood cells . Complete culture media consisted of sterile rpmi 1640 media (life technologies) supplemented with 10% human serum and 0.005% hypoxanthine and buffered with final concentrations of 0.6% hepes and 0.26% nahco3 . The fcr3 strain was maintained at 3% co2 and 5% o2, 92% n2 atmosphere, while the 3d7 strain was maintained at 5% co2, 5% o2, and 90% n2 atmosphere . J. smith, seattle biomed) asynchronous culture, 25% parasitemia, was washed in starvation media lacking human serum and was resuspended in complete media with 50 m compound 1 or equivalent dmso or in starvation media with equivalent dmso for 5 h. rbcs were harvested with centrifugation and were lysed with 0.2% saponin, and rbc lysate was removed through three 1 pbs washes . Parasites were harvested by centrifugation and were washed in 1 pbs with complete edta - free protease inhibitors (roche) and were lysed by repeated vortexing and boiling in sds reducing sample buffer . Lysates were separated with sds - page on a 420% polyacrylamide gel and were subjected to western blotting with 1:400 -tgatg8, demonstrated to be cross - reactive with pfatg8 (generously provided by dr . Hrp - conjugated secondary antibodies (southern biotech) were detected by supersignal west femto (thermo scientific) or amersham ecl prime (ge healthcare) chemiluminescent substrate . Ap - conjugated secondary antibodies (emd millipore) were detected using nbt / bcip (promega) colorimetric stain . Total protein levels were visualized with proact membrane stain (amresco) and were quantified with imagej . Parasite morphology at time of harvesting was visualized with light microscopy at 100 magnification on olympus bx53 system microscope (olympus america, inc . ). Ten microliters of 10 compound diluted in rpmi 1640 media (gibco) with a constant concentration of 1% dmso was added to a 96 well plate (costar), 90 l of 1.5% ring stage, synchronized with 5% w / v sorbitol p. falciparum 3d7 parasites, 1% hematocrit, in culture media with 10% v / v human serum with 10 g / ml gentamycin . Each compound concentration and 1% v / v dmso controls were run in triplicate . Plates were incubated at 37 c in 5% o2, 5% co2, and 90% n2 for 72 h. plates were frozen, thawed, and incubated with 100 l 2 sybr green in lysis buffer (20 mm tris ph 7.5, 5 mm edta, 0.008% saponin, 0.08% tritonx-100) in the dark for at least 1 h. fluorescence was measured with a plate reader (hts 7000, perkinelmer) at excitation / emission wavelengths of 485/535 nm . Docking was conducted using the openeye software package using standard parameters if not specified otherwise (www.eyesopen.com). A receptor for pfatg8 was made with make_receptor from oedocking toolkit without constraints covering the whole molecule to detect potential binding pockets on the surface . Three main pockets (w - site, l - site, a - site) were detected with 377, 271, and 513 volumes, used in first docking studies . A second receptor was generated with specific constraints to the carbonyl of lys47 as hydrogen bond donor . A maximum of 2000 conformers for each compound from the mmv malaria box was prepared with omega2 . Fred was used to dock these conformers onto both the constrained and unconstrained receptor . Docking results were visualized using the openeye visualization software, vida . Using itasser, models of p. yoelii and p. berghei atg8 were generated with pdb code 4eoy as the parent molecule . Default values as suggested by the web server were used to generate these models . Sequences for p. yoelii (pyym_0504500) and p. berghei (pbanka_050410) were obtained from plasmodb . Atg8, represented by the rectangle, is essential to the elongation of the autophagosomal membrane . (b) generic conjugation pathway shown for yeast system . In plasmodium, atg8 is synthesized with a c - terminal glycine that does not require proteolytic processing for activation . Identification of a common scaffold that inhibits atg8-atg3 from the mmv malaria box screen . (b) bar graph showing inhibition of hits in primary screen, denoted by compound number . Inhibition was measured with increasing amount of compound in spr competition screen . Mean and standard deviation (sd) of three injections are shown . Error bars show sd of three measurements . Identification of 1 binding site on pfatg8 . 1 was injected over immobilized pfatg8-variants in two separate runs at four concentrations . (b) in silico docking of pta compounds to pfatg8 (pdb code 4eoy). Overall structure of pfatg8 with w- and l - site and a - loop demarcated . Docking was also performed with a hydrogen bond constraint to the carbonyl of lys47, located between the w- and l - site . Predicted pose for constrained docking is shown in green . For 1, an alternate pose, it is highly ranked in the unconstrained docking and is enriched in the top 10 poses as shown in magenta . Amino acid changes between the species are shown in red with p. falciparum letter and numbering followed by p. yoelii . W - site, l - site, and a - loop pockets are shown in mesh in cyan, purple, and green, respectively . Falciparum atg8 pocket sizes were calculated with openeye vida visualization software (www.eyesopen.com). Effect of 1 treatment on the development of p. falciparum 3d7 gfp parasite in hc-04 cells in vitro . (a) flow cytometry based detection of annexin - v positive and pi - positive cells in hepatocyte cultures treated with 1 (as described in experimental section). Dot plot graphs demonstrate representative pattern of staining, and bar graphs show summary (mean sd) of viable cell detection obtained in three independent hepatocyte cultures . (b) viable infected hepatocytes (gfp+/pi) were detected by flow cytometry in hc-04 cultures 72 h post infection with p. falciparum . Dot plot graphs demonstrate representative pattern of staining, and numbers reflect percentages of gfp positive cells in total pi negative cell populations . (c) summary (mean sd) of viable infected cell detection obtained in three independent hepatocyte cultures . (d) gfp - specific fluorescence was assessed in infected cultures exposed to 1 or dmso for 72 h. histograms demonstrate one representative staining pattern, and numbers reflect mean fluorescence intensity (mfi) in gfp - positive populations . Bar graphs reflect gfp - specific mfi (mean sd) in viable cell population detected in three independent hepatocyte cultures . (a) dose - dependent immunoblot analysis of p. falciparum treated with dmso or 3.375, 6.75, 12.5, or 25 m 1 for 6 h. chloroquine (cq) at 50 nm was used as a positive control of autophagy inhibition . The blot was probed with antibody against tgatg8, demonstrated to be cross reactive against pfatg8 . (b) blood smears of p. falciparum after treatment with dmso or 50 m 1 for 5 h, observed at 100 magnification . Representative images for different stages are shown, progressing from ring stage on the left to late schizont on the right . 7 (yellow) and 9 (green) were docked onto the constrained receptor of pfatg8 (pdb code 4eoy) with openeye docking suite . Pfatg8 and hlc3 were injected over immobilized 8, and binding was measured with spr . (c) superposition of small molecule hits derived from mmv malaria box with 9 . The individual molecular surfaces with their corresponding electrostatic potential are depicted in side and top view . All four molecules share the pta moiety and were superimposed using rocs via shape complementarity and tanimoto color scoring function . The figure was prepared with vida and was rendered in povray (www.povray.org). (a) inhibition of pfatg8-pfatg3 interaction by 9 . Spr response of pfatg8 injected over immobilized pfatg3 was measured in the presence of increasing concentration of 9 . Sybr green i assays were used to measure inhibition by chloroquine (cq), 1, and 9 . Growth inhibition curves are shown for one experiment with ic50 values from two to three experiments in table inset . Pta - containing compounds used in studies listed with pubchem compound identification (cid), mmv i d, chemical structure, molecular weight (g / mol), ic50 in spr and blood stage assays, and lean score, calculated as log (ic50)/number of heavy atoms . The organisms are maintained in licensed bsl2 facilities, and approvals are obtained annually for all consortia laboratories . Human erythrocytes are obtained either commercially or from healthy volunteers under johns hopkins irb - approved protocols . Because these cells are provided to the lab without identifiers
This retrospective study included patients who had undergone ct between january 1, 2010, and december 31, 2010, and met the following criteria: they were over the age of 20 years, they gave us their informed consent and they underwent ct examinations to assess abdominal or urological lesions for reasons unrelated to lbp . The ct scans ordered by the departments of general surgery and urology were included . To prevent a result bias, we excluded patients in whom a chief complaint of lbp was the primary indication for the ct examination ordered by the departments of orthopaedic surgery, neurologic surgery, rehabilitation and pain clinic . A total of 472 participants who were aged from 20 to 84 years were consecutively enrolled . All the participants who had undergone multidetector ct scanning were asked to complete two questionnaires, which were administrated by senior trained nurses who were not involved in this study . The question on lbp that was translated into korean in the questionnaire was " have you had lbp that needed medication almost every day for at least 1 month in the last 12 months? " This was modified from nordic low back pain questionnaire.17) the individual's answers of " yes " or " no " to the above question was used in the present study as the lbp outcome . The question has been widely used by several authors18,19) for work - related compensation . Ct was performed on one of three 16-multidetector computed tomography (mdct) machines or a dual source 64-mdct system (lightspeed ultra, ge healthcare, milwaukee, wi, usa). The axial slice thickness varied from 0.75 to 2.5 mm because the images were obtained from patients with different indications and by different protocols . All the ct scans were analyzed in a blinded fashion by two orthopedic surgeons independently . The images were reviewed on a secure - access picture - archiving communication system (philips sectra, linkping, sweden). All the ct images that were initially reviewed were the axial images at the intervertebral disc level . Lumbar facet joints were graded on both sides at levels of l1 - 2, l2 - 3, l3 - 4, l4 - 5, and l5-s1 . Four grades of facet joint osteoarthritis were defined using criteria similar to those suggested by pathria et al.20) and weishaupt et al.21) (table 1). Lsfjoa was defined as at least one joint affected by facet joint disease between the spinal levels l1 and s1 (grade 2). Before the analysis, the study population was dichotomized on the basis of the presence of lsfjoa (grade we analyzed lsfjoa in 5 different age groups (<40, 40 - 49, 50 - 59, 60 - 69, and 70 years) and according to gender . The prevalence of lsfjoa in males and females each was compared according to the age group and according to the involved spinal level using chi - square () test for trend or multiple binary logistic regression analysis . Multiple binary logistic regression analysis was used to show the association between lbp and lsfjoa after adjusting for age, gender and spinal level . All the statistical analyses were performed with ibm spss ver . 19.0 (ibm co., armonk, ny, usa). This retrospective study included patients who had undergone ct between january 1, 2010, and december 31, 2010, and met the following criteria: they were over the age of 20 years, they gave us their informed consent and they underwent ct examinations to assess abdominal or urological lesions for reasons unrelated to lbp . The ct scans ordered by the departments of general surgery and urology were included . To prevent a result bias, we excluded patients in whom a chief complaint of lbp was the primary indication for the ct examination ordered by the departments of orthopaedic surgery, neurologic surgery, rehabilitation and pain clinic . A total of 472 participants who were aged from 20 to 84 years were consecutively enrolled . All the participants who had undergone multidetector ct scanning were asked to complete two questionnaires, which were administrated by senior trained nurses who were not involved in this study . The question on lbp that was translated into korean in the questionnaire was " have you had lbp that needed medication almost every day for at least 1 month in the last 12 months? " This was modified from nordic low back pain questionnaire.17) the individual's answers of " yes " or " no " to the above question was used in the present study as the lbp outcome . The question has been widely used by several authors18,19) for work - related compensation . Ct was performed on one of three 16-multidetector computed tomography (mdct) machines or a dual source 64-mdct system (lightspeed ultra, ge healthcare, milwaukee, wi, usa). The axial slice thickness varied from 0.75 to 2.5 mm because the images were obtained from patients with different indications and by different protocols . All the ct scans were analyzed in a blinded fashion by two orthopedic surgeons independently . The images were reviewed on a secure - access picture - archiving communication system (philips sectra, linkping, sweden). All the ct images that were initially reviewed were the axial images at the intervertebral disc level . Lumbar facet joints were graded on both sides at levels of l1 - 2, l2 - 3, l3 - 4, l4 - 5, and l5-s1 . Four grades of facet joint osteoarthritis were defined using criteria similar to those suggested by pathria et al.20) and weishaupt et al.21) (table 1). Lsfjoa was defined as at least one joint affected by facet joint disease between the spinal levels l1 and s1 (grade 2). Before the analysis, the study population was dichotomized on the basis of the presence of lsfjoa (grade 2) on any side at any level . We analyzed lsfjoa in 5 different age groups (<40, 40 - 49, 50 - 59, 60 - 69, and 70 years) and according to gender . The prevalence of lsfjoa in males and females each was compared according to the age group and according to the involved spinal level using chi - square () test for trend or multiple binary logistic regression analysis . Multiple binary logistic regression analysis was used to show the association between lbp and lsfjoa after adjusting for age, gender and spinal level . All the statistical analyses were performed with ibm spss ver . 19.0 (ibm co., armonk, ny, usa). Fifty - three have lsfjoa but 209 have no lsfjoa in men (20.23%) and thirty have lsfjoa but 180 have no lsfjoa in women (14.29%). There is no statistically significant difference between men and women on the prevalence of lsfjoa (p = 0.092). The increasing age demonstrated a higher prevalence of facet joint osteoarthritis with statistical significance (p = 0.015). In men, the difference in the prevalence of lsfjoa according to the spinal level was statistically significant (p = 0.001) and the highest prevalence of lsfjoa was found at l4 - 5 . In women, the different in the prevalence of lsfjoa across spinal levels was statistically significant (p = 0.003) and the highest prevalence of lsfjoa was found at l5-s1 . In all population, the difference in the prevalence of lsfjoa according to the spinal level was statistically significant (p = 0.000) and the l5-s1 level exhibited highest prevalence . The gender difference at each level was not statistically significant (l1 - 2, p = 0.377; l2 - 3, p = 0.741; l3 - 4, p = 0.567; l5-s1, p = 0.893) except at l4 - 5 (p = 0.002). Men demonstrated a higher prevalence of lsfjoa compared to women at l4 - 5 level (table 4). The prevalence of lsfjoa according to spinal level in individuals with or without lbp is listed in table 5 . The prevalence of lsfjoa was not associated with lbp in men (p = 0.093), whereas the prevalence of lsfjoa was associated with lbp in women (p = 0.003). The prevalence of lsfjoa according to age in individuals with or without lbp is shown in table 6 . The prevalence of lsfjoa was not statistically significantly associated with lbp (all, p> 0.05). Lsfjoa at l3 - 4 and l5-s1 was related to lbp in women at a statistically significant level (p = 0.018 and p = 0.026, respectively). No significant difference in the prevalence of lsfjoa was identified between individuals with and without lbp in the study population as a whole or in the subgroup analysis based on age and gender except for the above - mentioned cases . Regarding the multiple logistic regression analysis, lbp was a dependent variable and lsfjoa at each spinal level and gender were included as independent variables . Lbp was not associated with spinal level and age group but was more common in women than men (p = 0.002) (table 7). Fifty - three have lsfjoa but 209 have no lsfjoa in men (20.23%) and thirty have lsfjoa but 180 have no lsfjoa in women (14.29%). There is no statistically significant difference between men and women on the prevalence of lsfjoa (p = 0.092). The increasing age demonstrated a higher prevalence of facet joint osteoarthritis with statistical significance (p = 0.015). In men, the difference in the prevalence of lsfjoa according to the spinal level was statistically significant (p = 0.001) and the highest prevalence of lsfjoa was found at l4 - 5 . In women, the different in the prevalence of lsfjoa across spinal levels was statistically significant (p = 0.003) and the highest prevalence of lsfjoa was found at l5-s1 . In all population, the difference in the prevalence of lsfjoa according to the spinal level was statistically significant (p = 0.000) and the l5-s1 level exhibited highest prevalence . The gender difference at each level was not statistically significant (l1 - 2, p = 0.377; l2 - 3, p = 0.741; l3 - 4, p = 0.567; l5-s1, p = 0.893) except at l4 - 5 (p = 0.002). Men demonstrated a higher prevalence of lsfjoa compared to women at l4 - 5 level (table 4). The prevalence of lsfjoa according to spinal level in individuals with or without lbp is listed in table 5 . The prevalence of lsfjoa was not associated with lbp in men (p = 0.093), whereas the prevalence of lsfjoa was associated with lbp in women (p = 0.003). The prevalence of lsfjoa according to age in individuals with or without lbp the prevalence of lsfjoa was not statistically significantly associated with lbp (all, p> 0.05). Lsfjoa at l3 - 4 and l5-s1 was related to lbp in women at a statistically significant level (p = 0.018 and p = 0.026, respectively). No significant difference in the prevalence of lsfjoa was identified between individuals with and without lbp in the study population as a whole or in the subgroup analysis based on age and gender except for the above - mentioned cases . Regarding the multiple logistic regression analysis, lbp was a dependent variable and lsfjoa at each spinal level and gender were included as independent variables . Lbp was not associated with spinal level and age group but was more common in women than men (p = 0.002) (table 7). This is the first study to describe the prevalence of lsfjoa identified by mdct in an adult community - based korean population . The prevalence of lsfjoa in this study was lower than that in the study by kalichman et al.11) this study also evaluated the association between lsfjoa and lbp in the adult community - based korean population . Several authors have reported that lsfjoa is more common in the most caudal motion segments.22,23,24) fujiwara et al.25) found that the median grade of lsfjoa at l4 - 5 was significantly higher than that at l3 - 4, while no significant differences were found between l3 - 4, l5-s1, and between l4 - 5 and l5-s1 . Kalichman and hunter23) stated that the possible reason for the high prevalence and severity of lsfjoa at the l4 - 5 spinal level may be its position as a transition between the more mobile lumbar segments and the relatively stiff l5-s1 segment . Unlike previous studies, the highest prevalence of lsfjoa was found at the l4 - 5 spinal level in men and at l5-s1 in women . Approximately 15%-40% of chronic lbp is attributed to lsfjoa.10) the histologic basis for facet joint pain has been scientifically established, but the precise clinical etiology remains undetermined.10) hyperextension increases the load on the lumbar facet joint and stretches the capsule . This mechanical deformation may stimulate nociceptors in the joint capsule causing pain.26,27) the observation that the age is associated with the incidence of lsfjoa is not surprising.11) lewin22) stated that facet joints showed only minor cartilage changes before the age of 45 years and that the osteoarthritis advanced with age . The prevalence of lsfjoa in our study was more common in subjects less than 40 years of age than 40 - 49 years . Gender has not been associated with the prevalence of lsfjoa in other studies.23,24) fujiwara et al.25) found that motion segments in women showed significantly greater motion in lateral bending, flexion, and extension, but not in axial rotation, than in men in a cadaveric study . In our study, the prevalence of lsfjoa was not different between men and women . In terms of specific spinal level, the present study did reveal statistically significant differences in the prevalence of lsfjoa between men and women at l4 - 5 spinal level where men demonstrated a significantly higher prevalence of lsfjoa than women . The result is same as the study by eubanks et al.16) but is in contrast to the study of kalichman et al.11) the relationship of radiographic lsfjoa to the clinical syndrome of lbp is inconsistent.3) the cardinal role of facet joint abnormalities in patients with lbp is still debated.5,6,7) schwarzer et al.13) even questioned the clinical importance of facet joint osteoarthritis . They were not able to demonstrate a significant correlation between the degree of osteoarthritis observed on ct and the pain score during the facet block . In our study, the prevalence of lsfjoa was not associated with lbp . One of the limitations of this study is that it was a cross - sectional investigation without any longitudinal follow - up . In addition, we did not adjust for the bias such as occupation, which should be addressed in further analysis . The prevalence of lsfjoa based on ct imaging was 17.58% (20.23% in men and 14.29% in women), was not associated with gender, increased with age, and was the highest at the l5-s1 spinal level in an adult community - based korean population . At the l4 - 5 spinal level, lsfjoa was more common in men than in women . No significant association was observed between lsfjoa and lbp at any spinal level and age except at l3 - 4 and l5-s1 levels where lsfjoa was related to lbp in women.
Kawasaki disease (kd) is an acute multisystem vasculitis that afflicts mostly young children . Since the first report of kd in 1967 (1) however, the clinical and epidemiologic studies have proposed that kd is closely related to an infectious disease (2, 3). The acute onset of a self - limited course, the prevalent population (rare in <6 months of age and> 5 yr of age), the existence of clusters or epidemics with a wave - like spread, all suggest that kd is related to infectious agents, particularly of viral origin . On the other hand, the elevated levels of the inflammatory indices including the white blood cell (wbc) and neutrophil count and the c - reactive protein (crp) suggest that bacterial agents including superantigens are involved in kd (4). We previously found that the igm and iga levels increased at 1 week and 2 weeks after intravenous immunoglobulin (ivig) treatment with a statistical significance (5). Many therapeutic modalities have been attempted in order to prevent the coronary artery lesions (cal) as the major complication of kd . Ivig therapy is known to decrease the numbers of cal, and is now accepted as standard treatment for kd (6, 7). The total duration of fever in kd in the era before ivig therapy was reported to be approximately 1 - 2 weeks (mean 10 days) regardless of treatments with aspirin or steroids (1, 8). We evaluated the inflammatory indices including the wbc count and crp according to the onset of fever in children with kd, and postulated that inflammatory processes in kd reach a peak at the sixth day of fever . The subjects of this study were 152 children who had been diagnosed with kd (82 boys and 70 girls) between july 1999 and december 2002 at the catholic university of korea, daejeon st . The diagnostic criteria were based on the diagnostic guidelines of kawasaki disease presented by the japan kawasaki disease research committee (9). One hundred nineteen children met the criteria for kd and 33 cases of an incomplete kd were included . Incomplete kd patient was defined as those who do not fulfil the recommended criteria at presentation regardless of echocardiographic findings . All patients with incomplete presentation who did not show elevated crp (<2.0 mg / dl), and wbc and neutrophil (<10,000/l and <5,000/l, respectively) levels with repeated examinations in the earlier days of illness or those who did not show an increased platelet count 7 days after ivig treatment were excluded (4 cases). Children were treated with ivig (i.v .- globulin s, green cross, korea: 5% liquid preparation containing only maltose, igg: maltose=1:2) at a dose of 2 g / kg over 12 hr and a dose of aspirin (30 - 40 mg / kg) during the febrile period . After obtaining parental consent, the serial examinations were performed three times during admission: before ivig administration, and 24 hr and 7days after ivig administration . Cal were defined and classified as follows: ectasia was defined when coronary arterial dilatation with the diameter 4 mm was seen or when the diameter was less than 1.5 times than that of adjacent artery diameter; aneurysm, when dilatation> 4 mm ectasia with or without, multiple, pyramidal / fusiform aneurysm was present . Twenty - six of the 152 children had cal (21 in ectasias and 5 in aneurysms). Thirteen children showed a resistance to ivig therapy (a fever for more than 48 hr after initiating the ivig infusion). The ethics committee on clinical research, the catholic university of korea, approved this study . The means of all continuous variables were compared using one way anova, fisher's extact test and chi - square test . There were no significant differences in terms of age (mean 28.416.2 months) and sex distribution (male to female ratio, 82:70) among the groups . The mean incidence of cal evaluated within 2 - 3 weeks of the onset of fever was 17.1% . There was a trend for the incidence of cal to be higher in the sixth day (40%) and the ninth day groups (42.9%) than the other groups . Twenty - two percent of cases had fever with less than 4 of the diagnostic criteria for kd at presentation (incomplete kd). There was a trend for the incidence of incomplete kd to be higher in the third day (35%), the eighth day (33%) and the ninth day groups (43%). Wbc and neutrophil counts, crp and creatine phosphokinase (cpk) values were the highest in the sixth day group, and the last two indices showed a bell - shaped distribution pattern based on the peak sixth day values . In contrast, albumin, and hdl - cholesterol values were the lowest in the sixth day group, and they showed a pattern of u - shaped distribution . The platelet count and total cholesterol value showed a trend for increase with subsequent days of fever . The hemoglobin, esr and ldh values did not change significantly . The ast and alt values were the highest in the third day group, but were not statistically significant . The inflammatory processes of an infection progress to a peak stage, then regress to a convalescence by host immune response . The total duration of fever in most uncomplicated viral infections is approximately 1 week, including measles (5 days) and epstein - barr virus infection (6 days). On the other hand, the duration of fever in an untreated bacterial infection differs according to the causative agent . However causative disease caused by intracellular organisms such as typhoid fever lasts much longer (> 2 weeks). Early studies revealed that the total duration of kd without ivig therapy was 1 - 2 weeks (mean 10 days) (1, 8). Therefore, it is postulated that the peak inflammatory process in kd is at the fifth to sixth day after the onset of fever . The results in this study shows that the levels of inflammatory indices reach a peak or nadir on the sixth day of the fever, which agrees with the above postulation . Wbc and neutrophil counts, esr and crp levels are commonly used as indices of severity of inflammation . Crp increases rapidly within 24 hr in various conditions, like bacterial infections, trauma, tissue necrosis, and malignant neoplasm . The decreased albumin or hdl - cholesterol values have been reported in inflammatory diseases including kd (11, 12). Although, this study could not show difference of statistical significance in any of the indices between groups, the distribution patterns of these indices strongly support our postulation . Ast and alt values appeared to be higher in the early days of the natural course in kd . The cases with elevated ast and alt above two folds the normal values were also higher in the early days of the fever (4 day, 20 of 50 cases) than in the later days (7 days, 5 of 38 cases). A decreased total cholesterol level in the earlier days tends to increase with days of the fever . The platelet counts also tended to increase by days of fever in the acute stage . Recently, two studies reported the relationship between the initial day of the ivig treatment and the clinical or laboratory outcomes in kd (13, 14). Although the two studies are similar in study design, the results of cal frequency and laboratory findings were not identical . (13) reported that the group receiving ivig treatment prior to the fifth day of illness showed a higher cal at 1 month, and no differences in the laboratory findings except for alt and ast when compared to those given ivig after the fifth day . In contrast, tse et al . (14) reported a case - control study showing that early ivig treatment at day 5 or earlier showed a lower incidence of cal at 1 yr, and some differences in the laboratory findings including higher hemoglobin, alt, and albumin values and lower platelet count . If our postulation is correct, different results of laboratory study between the two groups might result from differences in the number of patients in each different fever day . Earlier studies have reported that the risk of cal in kd is associated with some demographic or laboratory factors such as the prolonged fever duration, or an increased crp (15 - 17). Ivig is quite effective for improving the clinical symptoms as well as the laboratory findings including crp, and for preventing coronary complications (6, 7). We previously repored that a high dose ivig treatment (2 g / kg) lowered the values of various proteins including albumin and lipoproteins, and also values of the inflammatory indices within 24 hr except esr (5, 18). In addition, we also found that ivig - resistant patients showed a sustained high values of crp and wbc on 24 hr after ivig with a higher risk of cal (19, 20). If the risk of cal is associated with the intensity of the inflammation reflected by laboratory indices, early treatment prior to the peak stage of inflammation can help prevent cal in kd . There are some limitations in interpreting our results . Because the data had a large number of variables (seven variables) and an uneven distribution of the number of variables, a diagnosis of kd in patients before 5 days illness could not fulfil the diagnostic criteria and those with incomplete kd were included in this study . All patients with incomplete kd were selected on the basis of the laboratory findings as described earlier as well as the clinical criteria . However, there was no significant difference in laboratory values between the incomplete kd group and typical kd group (data not shown). In conclusion, it is very important to determine when the inflammatory processes of kd reach a peak as a self - limiting disease . As for the coronary complications in kd, it is believed that the more severe inflammatory processes, which are reflected by prolonged fever duration or higher values of the inflammatory indices, have a higher risk . Therefore a higher single - dose ivig treatment before the peak stage of kd may help reduce the intensity of inflammation and the frequency of cal . Repeated examination of the inflammatory indices can help decide the timing for appropriate ivig treatment for children with an incomplete presentation in the earlier days, and for evaluating the effects of ivig treatment.
Advances in adjuvant and neoadjuvant therapies and accurate preoperative imaging techniques have improved the prognosis of patients with sarcoma . Tumors in the diaphysis are relatively uncommon, and in some patients it may be possible to achieve adequate margins without sacrificing the adjacent articular surface . Available methods include the use of autogeneous extracorporeally irradiated bone [13], massive allograft, distraction osteogenesis and intercalary custom - made endoprostheses [6, 7, 8]. Previously, long - term results of intercalary endoprosthetic reconstruction are not clear, and we have used extracorporeal radiation and re - implantation in patients with primary or metastatic diaphyseal bone tumors . Custom - made diaphyseal implants allow immediate weight bearing, but the complications include loosening, wear and breakage of the implants [6, 7]. However, intraoperative extracorporeal autogeneous irradiated bone grafting (iorbg) after femoral tumor resection also sometimes fails to achieve long - term survival due to non - union or fracture of bones and breakage of implant [1, 2]. Most especially, a pathologic fracture in a femoral lesion has a marked effect on the patient's quality of life . Here, we present a useful salvage surgery to reconstruct femoral diaphyseal defects due to iorbg fracture with custom - made intercalary endoprostheses in two elderly patients . In 2002 and 2005, two patients with femoral intercalary resection of malignant tumor underwent iorbg reconstruction with intramedullary nail or plate . At 32 and 96 months after iorbg reconstruction, both patients had iorbg fractures . We used custom - made endoprostheses in these two patients to reconstruct femoral diaphyseal bone defect after excision of failed iorbg . The intercalary endoprosthetic system (k - max, kyocera medical corporation, kyoto, japan) for femoral and tibial diaphyseal bone tumors is custom made with titanium alloy . The prosthesis took 3 weeks to manufacture, and the cemented stems were proximally and distally linked by a taper locking system with a bolt passing through parts of the prosthesis . Resection of the graft bone at the appropriate level and removal of the implant were carried out . Active physiotherapy was started on the second postoperative day, wherein, the patient was allowed to partially weight bear with gradual progression to full weight bearing by the time of discharge . Functional outcome was assessed using the musculoskeletal tumor society (msts) functional evaluation system for reconstructive procedures after skeletal resection . The msts score is composed of pain, function, emotional acceptance, walking ability, gait and use of walking aids, with a higher score indicating better functional outcome . Case 1: a 61-year - old male was treated at the age of 54 for primary soft - tissue sarcoma of the thigh which invaded the femur (fig . Wide en bloc resection of the tumor with involved femoral diaphysis, isolation of the tumor with involved bone, extracorporeal irradiation with 50 gy as a single bolus dose to the isolated bone and reimplantation of the irradiated bone into the host with intramedullary nail were performed (fig . 2a). At 74 months after primary surgery, graft bone fracture and intramedullary nail breakage occurred at the proximal diaphysis (fig . Segmental intercalary resection, implant removal and reconstruction with custom - made intercalary prosthesis were performed (fig . Plain radiograph (a) and ct (b) showing lytic destructive lesion in left femoral diaphyseal bone . T1- and t2-weighted magnetic resonance imaging (c) showing a soft tissue mass (20 20 12 cm) around femoral bone . Figure 2:radiographs showing extracorporeal irradiation after wide resection and stabilization with intramedullary nail (a). At 74 months after operation, radiograph showing fracture of graft bone with broken implant (b and c) and revision with intercalary endoprosthesis (d). A 54-year - old male with primary soft tissue sarcoma . Plain radiograph (a) and ct (b) showing lytic destructive lesion in left femoral diaphyseal bone . T1- and t2-weighted magnetic resonance imaging (c) showing a soft tissue mass (20 20 12 cm) around femoral bone . Radiographs showing extracorporeal irradiation after wide resection and stabilization with intramedullary nail (a). At 74 months after operation, radiograph showing fracture of graft bone with broken implant (b and c) and revision with intercalary endoprosthesis (d). Case 2: a 71-year - old female was treated at the age of 68 for metastatic synovial sarcoma of the femur (fig wide en bloc resection of the tumor and reconstruction with the use of iorbg were performed (fig . Distal thigh pain due to fracture of the graft and breakage of plate occurred (fig . . Failed graft bone resection, implant removal and reconstruction with custom - made intercalary endoprosthesis were performed (fig . Aseptic loosening occurred at 29 months after the first endoprosthetic replacement which required a revision procedure (fig . Figure 3:a 68-year - old female with metastatic synovial sarcoma in right femoral diaphyseal bone . Radiograph (b) showing wide resection included femoral diaphyseal bone and reconstruction with iorbg and plate . At 32 months after surgery, radiograph (c) showing graft bone fracture (arrow) and reconstruction with intercalary endoprosthesis (d). Aseptic loosening occurred 29 months after surgery (e), and revision surgery was performed (f). Radiograph (b) showing wide resection included femoral diaphyseal bone and reconstruction with iorbg and plate . At 32 months after surgery, radiograph (c) showing graft bone fracture (arrow) and reconstruction with intercalary endoprosthesis (d). Aseptic loosening occurred 29 months after surgery (e), and revision surgery was performed (f). There are several options for reconstruction of defects in the femoral diaphysis after resection of the tumor . However, the optional methods for reconstruction of defects are unclear . Hanna et al . Reviewed 23 patients who underwent limb salvage by endoprosthetic replacement of the femoral diaphysis for primary bone tumor with a reconstruction survival of 85% at 5 years and 68% at 10 years . Biological reconstruction is believed to be more time consuming than endoprosthetic replacement with a prolonged period of immobilization after surgery and a significant risk of non - union and/or fracture . Generally, the patients with implant failure and non - union and fracture of graft bone require open reduction and internal fixation with bone grafting . For our two elderly patients with fractures of the graft bones, the latter were salvaged using custom - made intercalary prostheses . When the patients were subjected to reconstruction with repeat bone graft for implant and iorbg failure, it is difficult for elderly patients to require a lengthy period of non - weight bearing for union . Prolonged immobilization is required for graft union, and there are high rates of failure after biological reconstruction, which makes its use less favorable for these patients, especially those receiving palliative treatment . Endoprosthetic reconstruction of the femoral diaphysis allows patients to recover early weight bearing and function without a lengthy period of no weight bearing . Although both patients resumed daily life early, one patient underwent revision to an intercalary replacement at 29 months for aseptic loosening of the distal stem . As a risk of later failure and subsequent need for surgery, the indication on young patients should be considered . Custom - made intercalary endoprosthesis is a useful surgical treatment for fracture of an iorbg reconstruction in limb salvage surgery especially in elderly patients.
Diabetes mellitus is a chronic metabolic disorder that can result in multiple long - term micro- and macrovascular complications . Diabetes is well known as the leading cause of blindness, end - stage renal disease (esrd) and limb amputation . In korea, approximately 18.6% of diabetic patients have retinopathy, 27.3% albuminuria, and 33.5% diabetic neuropathy . In a nationwide survey in 2012, microvascular complications significantly affect the quality of life and impose a major burden on the healthcare system and economy . The development of microvascular complications is related to several environmental risk factors, including duration of diabetes, degree of hyperglycemia, blood pressure, and dyslipidemia . In the landmark u.k . Prospective diabetes study, which enrolled newly diagnosed type 2 diabetes mellitus (t2 dm) patients, participants randomized to intensive glucose control (median hemoglobin a1c [hba1c] 7.0%) had a 25% reduction in microvascular complications including vitreous hemorrhage, retinal photocoagulation and esrd compared with those in the conventional treatment group (median hba1c 7.9%) after 10 years of follow - up . In the recent action to control cardiovascular risk in diabetes (accord) trial, intensive glycemic control targeting hba1c <6.0% resulted in 23% reduction of retinopathy progression and delayed onset of albuminuria and peripheral neuropathy . Treatment with fenofibrate also decreased the risk of retinopathy in the accord eye study . Despite various interventions to control these environmental factors, large individual variations in the outcome of diabetic microvascular complications exist . Some patients with a short duration of diabetes develop microvascular complications although they had relatively good glycemic control . In contrast, some people do not develop microvascular complications even with a prolonged disease duration and with poor glycemic control . These clinical findings suggest that genetic factors play a role in the pathogenesis of microvascular complications . For example, the heritability for diabetic retinopathy was estimated at 18% and for proliferative diabetic retinopathy (pdr) at 52% . The development of diabetic nephropathy also differs based on ethnicity and african americans and asians have 1.9- and 1.8-fold increased risks of esrd, respectively, compared with european diabetic patients . Consequently, efforts have been made to identify genetic risk factors for diabetic microvascular complications using candidate gene approach, linkage analysis and the recent genome - wide association studies (gwass). Based on biological or positional plausibility, numerous candidate genes were selected for genetic association studies . The most thoroughly investigated genes include vascular endothelial growth factor a (vegfa), aldo - keto reductase family 1, member b1 (akr1b1), and erythropoietin (epo) for diabetic retinopathy and angiotensin 1 converting enzyme (ace), protein kinase c (prkcb), and erythropoietin (epo) for diabetic nephropathy . Although significant associations were initially reported, subsequent studies frequently showed inconsistent results . Most of the candidate gene association studies were conducted using a small sample size and had less stringent statistical thresholds . In addition, a meta - analysis showed that these candidate variants were not significantly associated on a genome - wide basis . The advances in genotyping technology and publicly available databases of reference genomes and human genetic variations, including the international hapmap project, have contributed to understanding the genetic risk factors of common metabolic disorders using gwass . In gwass, hundreds of thousands or more of single - nucleotide variants are genotyped and tested for association with a disease or a continuous trait in several hundred or more subjects . Recently, gwass have increased the number of genetic markers to more than one million by imputation methods and the sample size has increased to more than one hundred thousand by using meta - gwass . The first successful gwas on t2 dm was published in 2007 . Since then, at least 77 confirmed genetic loci for t2 dm have been identified, providing a better understanding of diabetes pathophysiology and there are ongoing efforts to use this genetic information in risk prediction and tailoring of individualized therapy . In parallel, attempts have been made to unravel the genetic risk factors for diabetic microvascular complications using gwass . In this article, we reviewed the recent gwass on diabetic retinopathy, nephropathy and neuropathy and discussed their limitations and future directives . Diabetic retinopathy is clinically defined by the retinal microvascular lesions in diabetic patients and broadly classified into nonproliferative diabetic retinopathy (npdr) and pdr . Diabetic macular edema can result in moderate visual loss and can be present at any stage of diabetic retinopathy, although more common in advanced retinopathy . The gold standard for classification of diabetic retinopathy severity diabetic retinopathy increases as the duration of diabetes increases . According to the wisconsin epidemiologic study of diabetic retinopathy in t2 dm patients, diabetic retinopathy is present in 20% of patients at the time of diagnosis, which increases to 60% to 85% after 15 years . Whether the pathophysiology of retinopathy differs between type 1 diabetes mellitus (t1 dm) and t2 dm remains unknown . All heterogeneity factors - including the severity, duration, and type of diabetes - should be considered to understand the genetic risk factors for diabetic retinopathy . Currently, five gwass on diabetic retinopathy have been published . The first gwas performed included 283 mexican - american t2 dm retinopathy patients and controls (table 1). . Found two potential loci in the introns of calcium / calmodulin - dependent protein kinase iv (camk4) and formin 1 (fmn1) genes . Huang et al . Reported a gwas on t2 dm diabetic retinopathy including 749 taiwanese patients and found seven independent loci with potential significance (p<1.010). However, they reported the lowest p value among the six genetic models (genotype, allele, trend, additive, dominant, and recessive) and did not adjust for multiple comparisons . The third study is the largest gwas conducted to date and is a meta - analysis of two gwass, genetics of kidneys in diabetes (gokind) and epidemiology of diabetes interventions and complications (edic) studies . The most significant variant was rs476141 located in a long non - coding rna (loc339529) in chromosome 1 with p values of 1.2010 . The study by sheu et al . Used a two - stage gwas with follow - up genotyping in an independent population . Although they found three potential genetic variants in stage 1 gwas in taiwanese subjects, these findings were not replicated in the hispanic population . The latest gwas on diabetic retinopathy was a three - stage design performed by awata et al . In japanese subjects . Among the eight variants that were followed - up to the third stage, none reached a genome - wide significance threshold . The most significant variant was located in rp1 - 90l14.1, a long non - coding rna gene, with a p value of 1.7010 in the meta - analysis of the three - stage results . Overall, three studies were performed with asian subjects, one with mexican - american and one with european subjects . One study included t1 dm patients and the remaining four studies included t2 dm patients . The earliest two studies performed by fu et al . And huang et al . Were single - stage gwass with a small sample of fewer than 1,000 subjects . The majority of the genetic variants reported from the five studies did not pass the conventional significance threshold of p<5.010, except for several variants in the study by huang et al . . However, the latter study reported the best p value among various genetic models and did not correct for multiple comparisons . None of the genetic variants reported overlapped among the five studies; however, cases and controls were defined differently, which could be a crucial point when performing a genetic study . Regarding case groups, several studies included subjects with either npdr or pdr, whereas others included only subjects with pdr . Regarding the control group, only in the study by sheu et al . The subjects were limited to those with a diabetes duration of more than 8 years without any diabetic retinopathy . The heterogeneity in study design and relatively small sample sizes could explain the inconsistencies in the genetic variants identified in the five gwass on diabetic microvascular complications . A clear and rational definition of cases and controls is necessary to enhance genetic contrast as well as a large sample size to ensure sufficient statistical power . Diabetic nephropathy is clinically defined as an increase in urinary albumin excretion and a decrease in kidney function . Classification of diabetic nephropathy using the kidney disease: improving global outcomes group criteria is based on estimated glomerular filtration rate (egfr) and the degree of proteinuria . The egfr is generally calculated using the modification of diet in renal disease formula and is divided into five stages . The egfr reflects the current kidney function and proteinuria reflects the extent of pathological kidney damage . Proteinuria is a hallmark of diabetic nephropathy and precedes the decline in kidney function, but is not a prerequisite in some cases . A large body of evidence indicates that treatments to prevent or delay its progression should include intensive glycemic and blood pressure control . In the action in diabetes and vascular disease (advance) trial, intensive glycemic control resulted in the risk reduction for microalbuminuria (30 to 300 mg / g), macroalbuminuria (> 300 mg / g) and esrd, by 9%, 30%, and 65%, respectively . In the pivotal study of diabetes control and complications trial (dcct), intensive glucose control in t1 dm patients resulted in 39% reduced occurrence of microalbuminuria . However, 25% of participants in the intensive treatment group eventually developed microalbuminuria during the 6.5-year follow - up period . Therefore, individual variations in the risk of diabetic nephropathy exist and genetic factors likely play an important role . Consequently, efforts have been made to understand the genetic risk factors for diabetic nephropathy . The first large - scale genotyping of more than 80,000 gene - based single nucleotide polymorphisms was performed in 2005 by shimazaki et al . In 920 japanese t2 dm patients . They identified an intronic variant, rs741301, of the engulfment and cell motility 1 (elmo1) gene to be significantly associated with diabetic nephropathy . In vitro experiments suggested its role in the overaccumulation of extracellular matrix proteins and progression of glomerulosclerosis . Subsequently, a gwas by hanson et al . Used pooled dna and validated the top signals using individual genotyping in 207 pima indian t2 dm esrd cases and controls . A variant in plasmacytoma variant translocation (pvt1) gene was suggestively associated with esrd . They used two - stage gwas with follow - up genotyping in 1,705 t1 dm cases and controls . The four loci having a potential association signal of p<1.010 included ferm domain containing 3 (frmd3), cysteinyl - trna synthetase (cars), chimerin 2 (chn2), and carboxypeptidase (cpvl). The rs1888747 variant in frmd3 and rs451041 variant in cars showed associations with time to onset of diabetic nephropathy in an independent cohort of dcct / edic study with p<0.05 . They reported suggestive loci for esrd on zinc finger, miz - type containing 1 (zmiz1) and musculin (msc) genes as well as the association signals in six previously reported genetic loci of diabetic nephropathy (p0.0006). A relatively large - scale gwas involving 3,393 african - american t2 dm subjects was performed by mcdonough et al . In 2011 . However, it was uncommon for african - americans to have normal albuminuria after a diabetes duration of 10 years and the authors used nondiabetic controls for the control group . Therefore, analysis was performed to discriminate association signals between t2dm - associated esrd, t2 dm and all - cause esrd using additional genotyping in african - americans . Although none of the genetic variants reached genome - wide significance, several genes - including sam and sh3 domain containing 1 (sash1), ribosomal protein s12 gene (rps12), and lim kinase 2 (limk2)-were suggested as strong candidates for diabetic nephropathy in t2 dm patients . As previous studies were not fully powered for gwas and the phenotype definition was different, a collaborative effort was made to conduct the genetics of nephropathy: an international effort (genie) study on t1 dm diabetic nephropathy . In the first stage, a gwas was meta - analyzed in three cohorts with a sample size of 5,783 subjects . In the second stage, de novo genotyping the study was adequately powered and two variants were found associated with esrd in af4/fmr2 family, member 3 (aff3) and in the intergenic region between repulsive guidance molecule family member a (rgma) and multiple c2 domains, transmembrane 2 (mctp2) that reached a genome wide significance threshold of p<5.010 . In a gwas on finnish diabetic nephropathy study and genie consortium, sandholm et al . Identified a gender - specific variant, rs4972593, which was associated with the risk of t1 dm esrd only in females . A gwas was also conducted on urinary albumin excretion rate in 5,675 t1 dm patients . This study identified rs2410601 in the intergenic region between pleckstrin and sec7 domain containing 3 (psd3) and sh2 domain containing 4a (sh2d4a) as the most significantly associated with albumin excretion rate (p=3.8510). Among the nine gwass, six included participants of european origin and one study each of japanese subjects, pima indians and african - americans . Only three studies were performed on t2 dm, and the remaining six on t1 dm subjects . Only a few studies, including those from the genie consortium, had sufficient statistical power for gwas and most of the earlier studies were limited in terms of sample size . Genetic variants in earlier reports, such as in elmo1, were analyzed in subsequent studies and an association with diabetic nephropathy was confirmed in several, but not all, studies . Whether the pathophysiology of diabetic nephropathy differs between t1 dm and t2 dm patients remains unknown . Nonglycemic factors, such as insulin resistance and dyslipidemia, in t2 dm may modulate the development of diabetic nephropathy and certain genetic risk factors could be involved in this process . Most of the well - powered gwass were performed on t1 dm patients and the genetic variants of diabetic nephropathy in t2 dm patients should be elucidated . Diabetic sensorimotor polyneuropathy is one of the most common complications in diabetic patients with an estimated lifetime prevalence of up to 50% . In 2009, the toronto consensus panel on diabetic neuropathies updated its definition and diagnostic criteria for diabetic polyneuropathy . Diagnosis of distal symmetric polyneuropathy is categorized into possible, probable, confirmed, and subclinical, according to the certainty based on symptoms and signs . The confirmation of neuropathy requires typical symptoms, signs, and positive nerve conduction studies . Currently, only one gwas on neuropathic pain in diabetic patients has been published (table 3). Using the genetics of diabetes audit and research tayside (godarts) study, meng et al . The case control status was defined based on the prescription of medications frequently used for diabetic sensorimotor polyneuropathy, including duloxetine, gabapentin, pregabalin, capsaicin, and lidocaine patch . In a single - stage gwas without follow - up genotyping, rs17428041 located in the intergenic region between the gdnf family receptor alpha 2 (gfra2) and docking protein 2 (dok2) in addition, the narrow sense heritability of diabetic neuropathic pain was 11%, excluding the effect of gene - gene and gene - environment interactions . Further studies are required to replicate this finding and to identify additional genetic variants of diabetic neuropathy . During the past several years, the identification of genetic risk factors for diabetic microvascular complications has improved . However, most of the studies were not fully powered for gwass, with the exception of the genie study . Therefore, most of the results associated with the genetic risk factors were below the genome - wide significance threshold and inconsistent among studies . In addition, the definition of cases and controls differed, thereby introducing significant heterogeneity . Based on the findings reported, these genetic association results should be validated in other populations . In addition, a collaborative effort to harmonize phenotype definitions and to increase sample size is necessary . Whether certain microvascular complications are caused by specific genetic risk factors, or common genetic risk factors are shared by different microvascular complications should be clarified . Additionally, a possible difference in genetic risk factors for microvascular complications between t1 dm and t2 dm patients should be explored . Whether confirmed genetic variants for t1 dm or t2 dm per se have significant effects on the development of microvascular complications remains unclear . Finally, a metabolic memory or legacy effect, as shown by the dcct / edic trial, should be considered; this might be mediated by epigenetic change . Compared to t2 dm, genetic studies on diabetic microvascular complications are still in the early stages and have further challenges to overcome . Further genetic studies of microvascular complications will enhance understanding of their pathogenesis and facilitate the development of effective preventive and therapeutic measures.
Retinitis pigmentosa (rp) is the term used for a group of retinal diseases that are characterized by inherited, progressive degeneration of retinal tissue, mainly rod and secondarily cone photoreceptors . The clinical features are night blindness and visual field (vf) loss due to loss of rod photo - receptors . Many patients fall into a classical pattern of difficulties with dark adaptation and night blindness and loss of midperipheral vision field . As the disease advances, they lose peripheral vision, eventually developing tunnel vision with the remaining cone photoreceptors, and finally lose central vision and visual acuities (vas) as these cones secondarily degenerate in the macular region.1,2 typically, it takes several years until the patients lose their central vision; therefore, it is important to protect the cone photoreceptors in the macular area for rp patients . Because rp is caused by various mutations in any of> 45 responsible genes, the processes of degeneration are considered to be not uniform, and no effective treatment other than nutritional supplementation of vitamin a currently exists . Recently, noorwez et al3 reported that valproic acid (vpa) can increase the yield of properly folded rp mutant rhodopsins; by using their high - throughput screening method involving in silico, cell - based, and in vitro assays, the authors were able to identify pharmacological chaperones of misfolded rhodopsin . Based on the data, clemson et al4 reported in their retrospective study that treatment with vpa in patients with rp improved vas and vfs . However, there were controversies over that study,57 and another group, bhalla et al,8 not only claimed no improvement in va and vf in their study but also stated that vpa may facilitate some adverse side effects . To date, various clinical studies have been performed,9,10 but no conclusion has been reached regarding the efficacy of using vpa in patients with rp . The pharmacological basis of the antiepileptic action of vpa has been related to the regulation of the glutamate excitatory neurotransmission and/or gamma - aminobutyric acid (gaba) inhibitory neurotransmission.11 recent studies showed that vpa is an effective inhibitor of histone deacetylases, the key enzymes for the control of histone acetylation state and hence for the epigenetic regulation of gene expression . Mainly through inhibition of histone deacetylases, vpa induces apoptosis of microglia cells12 and activates bdnf promoter.13 moreover, vpa induces neuronal differentiation but suppresses astrocytic and oligodendrocytic differentiation of neural stem cells14 and promotes neurite outgrowth.15 in this prospective study, we examined the efficacy and safety of vpa use in japanese patients with rp . This study is registered with the clinical trials registry of the japan medical association center for clinical trials, number jma - iia00053 . In this prospective, interventional, noncomparative case study ethics committee approval was granted by the ethics committee at the institute of biomedical research and innovation . All the patients were seen at the institute of biomedical research and innovation hospital (kobe, japan) from december 2010 to january 2013 . The diagnosis of rp was based on the patients symptoms of night blindness, ring scotoma or concentric constriction of vfs, markedly reduced or nonrecordable a- and b - wave amplitudes on electroretinogram (erg) testing, and ophthalmoscopic findings (ie, characteristic fundus changes in attenuated retinal vessels and bone - spicule - like pigment clumping). The inclusion criteria were the following: 20 years old or older, best - corrected visual acuities (bcvas) of> 20/200 and <20/30, and vfs of 10 measured using goldmann perimeter with isopter i4 . The exclusion criteria were the following: patients with retinal diseases other than rp, including retinal degeneration secondary to inflammation or infection and retinal vascular or macular diseases; cataractous lens gradings of more than n1, c2, or p1 according to lens opacities classification system iii grading; previous intraocular surgery except for uncomplicated cataract extraction; women of childbearing potential who were pregnant, nursing, or planning a pregnancy; presentation of liver disease or a urea cycle disorder; patients who had drug hypersensitivity; patients who had attempted suicide or had suicidal thoughts with manic depressive illness; and patients who were using contraindication medicine . Prior to treatment at the initial study visit, each patient underwent ophthalmic examinations, including bcva measured using a landolt chart, intraocular pressure measurements, vf measured with the humphrey field analyzer (hfa; zeiss - humphrey systems, dublin, ca, usa) 10 - 2 program, slit - lamp biomicroscopy, and dilated indirect fundus ophthalmoscopy . After baseline measurements were obtained, all the patients were instructed to receive oral supplementation of 400 mg (the lowest dosage used for anticonvulsant therapy) of vpa (depakene - r; kyowa hakko kirin, tokyo, japan) daily for 6 months . The patients returned to our clinic for follow - up visits and were asked to report on the development of any subjective visual changes as well as any systemic adverse events . Bcvas, vfs, and ophthalmic findings were collected throughout the entire study at months 1, 3, 6 (end of supplementation), 9, and 12 . Blood samples were collected to check blood counts, clinical biochemistry, and the blood concentration of vpa at months 1, 2, 3, 4, and 6 (end of supplementation). Bcva was converted to the value of logarithm of the minimum angle of resolution (logmar) for all analyses . Vf test results were summarized using the mean deviation (md) value calculated by the hfa provided software . The calculation of the md value involved averaging the differences between the measured sensitivities and the age - adjusted normal sensitivities (total deviations) at each test point . Although not included as routine tests in our study design, microperimetry-1 (mp-1; nidek, gamagori, japan) or multifocal erg (veris; electro - diagnostic imaging, inc ., redwood, ca, usa and le-4000; tomey, nagoya, japan) were also recorded in some patients at pretreatment and at 6 months or at 6 months and 12 months . The primary end point of this study was improvement in bcva after the 6 months of treatment with vpa, and the secondary end points were vf and the occurrence of adverse events . The variance approximation for the estimation of sample size was obtained from previous studies that have tested a similar hypothesis.4 a sample size calculation was performed before the study, assuming a maximum dropout rate of 30% . Accordingly, we assumed that at least 30 patients were going to fail power of 80% (-1) to detect a logmar 0.2 difference in va between patients before and after receiving vpa . All parameters obtained prior to vpa treatment and at 1 month, 3 months, 6 months, 9 months, and 12 months after treatment initiation were compared using the wilcoxon signed - rank test with bonferroni correction . We utilized a bonferroni adjustment for multiple comparisons, after which p - values 0.01 were considered as statistically significant . All statistical analyses were performed using the statistical analysis software (spss inc ., chicago, il, usa). This study is registered with the clinical trials registry of the japan medical association center for clinical trials, number jma - iia00053 . In this prospective, interventional, noncomparative case study, the study protocols adhered to the tenets of the declaration of helsinki . Ethics committee approval was granted by the ethics committee at the institute of biomedical research and innovation . All the patients were seen at the institute of biomedical research and innovation hospital (kobe, japan) from december 2010 to january 2013 . The diagnosis of rp was based on the patients symptoms of night blindness, ring scotoma or concentric constriction of vfs, markedly reduced or nonrecordable a- and b - wave amplitudes on electroretinogram (erg) testing, and ophthalmoscopic findings (ie, characteristic fundus changes in attenuated retinal vessels and bone - spicule - like pigment clumping). The inclusion criteria were the following: 20 years old or older, best - corrected visual acuities (bcvas) of> 20/200 and <20/30, and vfs of 10 measured using goldmann perimeter with isopter i4 . The exclusion criteria were the following: patients with retinal diseases other than rp, including retinal degeneration secondary to inflammation or infection and retinal vascular or macular diseases; cataractous lens gradings of more than n1, c2, or p1 according to lens opacities classification system iii grading; previous intraocular surgery except for uncomplicated cataract extraction; women of childbearing potential who were pregnant, nursing, or planning a pregnancy; presentation of liver disease or a urea cycle disorder; patients who had drug hypersensitivity; patients who had attempted suicide or had suicidal thoughts with manic depressive illness; and patients who were using contraindication medicine . Prior to treatment at the initial study visit, each patient underwent ophthalmic examinations, including bcva measured using a landolt chart, intraocular pressure measurements, vf measured with the humphrey field analyzer (hfa; zeiss - humphrey systems, dublin, ca, usa) 10 - 2 program, slit - lamp biomicroscopy, and dilated indirect fundus ophthalmoscopy . After baseline measurements were obtained, all the patients were instructed to receive oral supplementation of 400 mg (the lowest dosage used for anticonvulsant therapy) of vpa (depakene - r; kyowa hakko kirin, tokyo, japan) daily for 6 months . The patients returned to our clinic for follow - up visits and were asked to report on the development of any subjective visual changes as well as any systemic adverse events . Bcvas, vfs, and ophthalmic findings were collected throughout the entire study at months 1, 3, 6 (end of supplementation), 9, and 12 . Blood samples were collected to check blood counts, clinical biochemistry, and the blood concentration of vpa at months 1, 2, 3, 4, and 6 (end of supplementation). Bcva was converted to the value of logarithm of the minimum angle of resolution (logmar) for all analyses . Vf test results were summarized using the mean deviation (md) value calculated by the hfa provided software . The calculation of the md value involved averaging the differences between the measured sensitivities and the age - adjusted normal sensitivities (total deviations) at each test point . Although not included as routine tests in our study design, microperimetry-1 (mp-1; nidek, gamagori, japan) or multifocal erg (veris; electro - diagnostic imaging, inc ., redwood, ca, usa and le-4000; tomey, nagoya, japan) were also recorded in some patients at pretreatment and at 6 months or at 6 months and 12 months . The primary end point of this study was improvement in bcva after the 6 months of treatment with vpa, and the secondary end points were vf and the occurrence of adverse events . The variance approximation for the estimation of sample size was obtained from previous studies that have tested a similar hypothesis.4 a sample size calculation was performed before the study, assuming a maximum dropout rate of 30% . Accordingly, we assumed that at least 30 patients were going to fail power of 80% (-1) to detect a logmar 0.2 difference in va between patients before and after receiving vpa . All parameters obtained prior to vpa treatment and at 1 month, 3 months, 6 months, 9 months, and 12 months after treatment initiation were compared using the wilcoxon signed - rank test with bonferroni correction . We utilized a bonferroni adjustment for multiple comparisons, after which p - values 0.01 were considered as statistically significant . All statistical analyses were performed using the statistical analysis software (spss inc ., chicago, il, usa). Two patients were lost to follow - up after the 6 months visit; the data for these patients were not included in the analysis . Overall, 29 patients (12 males and 17 females) with rp completed the 12 months study period . The patients ages ranged from 30 years to 72 years (mean sd: 52.511.5 years). Mendelian inheritance studies disclosed 13 sporadic, eleven autosomal recessive, and five autosomal dominant patterns . The age, sex, mendelian inheritance, bcva, md value of the vf, and mean blood vpa concentration are shown in table 1 . First, we evaluated the changes in the visual function during the vpa administration period (from baseline to 6 months) and the cessation period (from 6 months to 12 months). The median changes in the value of log - mar bcva per month were 0.00 (interquartile range [iqr], 0.180.00) during administration and 0.00 (iqr, 0.000.20) during cessation, which showed statistically significant difference (p=0.001; figure 1a). The median changes in the md value of vf per month were 0.11 db (iqr, 0.180.03) during administration and 0.48 db (iqr, 0.170.28) during cessation, which also showed statistically significant difference (p=0.001; figure 1b). Next, we evaluated the shift in va (logmar) and vf (db) over time (figure 2). The median logmar bcva values were 0.39 (iqr, 0.300.69) at baseline, 0.39 (iqr, 0.300.52; p=0.08) at 1 month, 0.39 (iqr, 0.300.61; p=0.02) at 3 months, 0.39 (iqr, 0.300.52; p=0.006) at 6 months, 0.39 (iqr, 0.300.69; p=0.14) at 9 months, and 0.39 (iqr, 0.300.76; p=0.62) at 12 months . Compared with baseline, the logmar bcva value was significantly improved at 6 months, during the period of vpa treatment (figure 2a). The median md values of vf were 28.89 db (iqr, 20.2633.18) at baseline, 27.54 db (iqr, 18.9232.52; p=0.001) at 1 month, 28.14 db (iqr, 18.2932.60; p=0.004) at 3 months, 28.16 db (iqr,18.5931.96; p=0.004) at 6 months, 27.97 db (iqr, 19.5133.11; p=0.88) at 9 months, and 29.15 db (iqr, 18.6833.06; p=0.97) at 12 months . Compared with baseline, the median md values of vf were significantly improved at 1 month, 3 months, and 6 months, corresponding to the period of vpa treatment (figure 2b). The mean blood concentration of vpa increased to 43.0416.96 g / ml at 1 month after intake . During the internal use period, the blood concentration of vpa was stable in each patient and the total mean blood concentration value of vpa within 6 months was 39.0012.47 g / ml . As shown in figure 3, there were no significant relations between the mean blood vpa concentration values of each patient and the changes in bcva (r=0.06, p=0.73) and vf (r=0.18, p=0.33) at 6 months . The patients subjective visual symptoms reported during and after vpa treatment are summarized in table 2 . During intake of vpa, eleven of 29 (38%) patients felt no change in their vision, eight (28%) patients felt clearer color vision, four (14%) patients felt legibleness, and four (14%) patients felt brightness . After the cessation of vpa intake, eight (28%) patients reported having blurred vision, four (14%) patients reported seeing dimness, three (10%) patients reported having photophobia, two (7%) patients reported difficulty seeing colors, and one (3%) patient experienced fatigue . Although not included in the study design and statistical analysis, we performed mp-1 and multifocal erg tests on some patients, and the preliminary results are shown in the supplementary materials section . Some patients gained sensitivity by mp-1 at 6 months after vpa treatment (figure s1). With multifocal erg, the amplitudes of (p1-n1) in rings 3 and 4 (perifoveal area)16 were relatively unchanged between the two periods, whereas (p1-n1) amplitudes of ring 1 (center of the fovea) seem to increase in some patients after 6 months of treatment, whereas the changes after vpa cessation seem to be insignificant (figure s2). Throughout the study period, no systemic drug - related serious adverse events were observed in the study participants; checked blood counts from collected blood samples and clinical biochemistry were within normal lesion . During the period of vpa intake, dizziness stomatitis, alopecia, and diarrhea were each reported in one (3%) patient . While several reports showed that oral vpa treatment improved vas and vfs of rp patients, the opposite results were shown in other reports . The results of existing reports in vpa treatment are summarized in table 3 . In the present study of rp patients, the bcva and md values in the hfa 10 - 2 program significantly improved on average after 6 months of vpa intake and returned to the baseline levels after the cessation of the vpa treatment (figure 2). Based on a large cohort study of natural courses of rp, the mean annual rates of decline of remaining bcva and vf were estimated to be 1.0%8.6% and 2.6%13.5%, respectively.17 compared to these data, our results showed some beneficial effect of vpa on rp patients at the 12-month time point even with its confined periodic effect . We cannot deny the possibility of placebo effect, but two previous studies using docosahexaenoic acid or 9-cis -carotene had a randomized placebo treatment group without provision of multivitamin, and the placebo group in both of these studies worsened in vf by 1.41.32 db with hfa between years 0 and 4 or by 0.54.5 cm with goldmann perimetry within 90 days.18,19 indeed, we cannot make a direct comparison between our results and these past studies, but we think that the vpa effect observed here is worthy of further investigation with a controlled study design in the future . Nevertheless, the effect of vpa on bcva observed in our study may not seem to be remarkable as one might expect (0.2 log units). A large cohort study showing the significant effect to slow the progression of rp by nutritional supplements such as vitamin and docosahexaenoic acid presented its efficacy by vf and erg amplitudes but not by bcva.20,21 altogether, these results indicate that the change in bcva may not be sufficiently sensitive to evaluate the efficacy of a treatment for rp patients . We also performed mp-1 and, more objectively, multifocal ergs on a limited number of patients . Although the data collection was performed in the limited number of patients, we observed the increased p1-n1 amplitudes specifically in ring 1 central fovea in some patients after 6 months of vpa treatment . This may imply the effect of vpa on the area of remaining photo - receptors, but the overall amplitudes were very small . We need more data to objectively evaluate the effect of vpa on foveal function . In this study, there were no significant relationship between the mean blood vpa concentrations of each patient and the changes in bcva and vf . First of all, vpa dosage used in this study was relatively low in order to minimize adverse effect of vpa, and the plasma vpa levels in our patients were below the therapeutic range (45100 mg / l). With this low concentration, albumin binding sites on vpa were unsaturated, and therefore, vpa binds to albumin at variable degrees, making its pharmacological behavior difficult to estimate.2224 if we use the therapeutic range of vpa, it might be able to show clearer effect of vpa on bcva or vf . We also observed a significant improvement in md values of hfa 10 - 2 programs with vpa treatment . In the original report by clemson et al, their studies were based on an in vitro experiment that vpa acts as a molecular chaperone of rhodopsin proteins that increases the yield of properly folded mutant rhodopsins . Therefore, these authors suggested a potential effect of vpa on autosomal dominant rp, targeting improperly folded mutant rhodopsins, in rod photoreceptors . Our current study included rp patients with seemingly various causal genes with 13 sporadic, eleven autosomal recessive, and five autosomal dominant hereditary patterns . Our hfa examination with central 10 - 2 program may also represent cone photoreceptor functions . Additionally, close hearing of patient s subjective symptoms also suggested some improvement in cone photoreceptor function: 16 of 29 (55%) patients felt it was easier to see during the period of vpa intake, whereas after cessation, eye discomfort was registered in 18 of 29 (62%) patients and half of the patients made some description related to color vision . Stasheff et al25,26 found that after degeneration started, ganglion cells exhibited hyperactivity, firing spontaneously at rates many times greater than normal in rd1 and rd10 mice, strains with closely related rp . Because the pharmacological basis of the antiepileptic action of vpa has been related to reduction in neuronal excitability by the increase in gabaergic activity, it is possible that vpa reduced hyperactivity of ganglion cells . Kimura et al27 also reported that vpa reduced retinal ganglion cell death in a mouse model of normal tension glaucoma . In this report, they indicated that vpa exerts neuroprotective effects through suppression of oxidative stress and stimulation of cell survival signaling . Therefore, patients may have felt that it was easier to see with reduced visual noise from spontaneous firing of ganglion cells or with neuroprotective effects . This phenomenon may also explain why bcva and vf were improved in our study with various types of genetic patterns . However, this hypothesis was based on subjective symptoms of vpa - treated rp patients, and we need further objective evaluation of cone - related functions, including color vision or contrast sensitivity, in vpa - treated patients . Vpa has been widely used as an antiepileptic drug for several decades, and the use of vpa monotherapy in the treatment of epilepsy was not associated with vf defects.28,29 however, abnormal color visions in epileptic adolescents treated with vpa were reported.30,31 sisk7 reported three cases with complications of vpa treatment, two of which had severe decrease in bcva; the two patients were 8 years and 15 years of age and received 10 mg / kg / d of vpa for 45 months . Bhalla et al8 reported that 12 (39%) of 31 vpa - treated patients reported systemic side effects, of whom nine (29%) discontinued vpa intake due to side effects . In this study, we did not observe a severe decline in visual functions or systemic adverse events to discontinue vpa treatment . One possible explanation is that our patients were all at the age of 30 years or older and the dosage of vpa was relatively low (400 mg / d). It is still necessary to carefully observe the patients systemic conditions during vpa treatment and to exercise caution when using vpa for young rp patients . In this prospective study, we found the following: 1) while in use, oral intake of vpa was suggestive of a short - term benefit to patients with rp and 2) regardless of the genotype, there were no systemic drug - related adverse events . It is necessary to examine the effect of a longer vpa supplementation in a controlled study design . The changes in the retinal sensitivity during the vpa administration period and the cessation period . Notes: scatter plots show changes in retinal sensitivity during the vpa administration period (from baseline to 6 months; n=24) (a) and the cessation period (from 6 months to 12 months; n=24) (b). Retinal sensitivities of the central 2 the changes in the (p1-n1) amplitude during the vpa administration period and the cessation period . Notes: scatter plots showing changes in retinal sensitivities during the vpa administration period (from baseline to 6 months; n=21) (a) and the cessation period (from 6 months to 12 months; n=9) (b). Multifocal ergs of the central 20 were measured with veris (electro - diagnostics, inc .,) and le-4000 (tomey). Abbreviations: p1-n1, first positive wave minus first negative wave; vpa, valproic acid; erg, electroretinogram; deg, degree.
Alzheimer's disease (ad) is the most common form of dementia in the elderly . It is characterized by neuronal cell loss and progressive accumulation of neurofibrillary tangles (nft) in neurons, and amyloid fibers in neuritic (senile) plaques and in the walls of blood vessels . As the disease progresses, ad patients experience changes in personality, behavior, and social interactions . Amyloid beta peptide (a - beta) is the major component of amyloid plaques in the brain of individuals affected by ad . The formation of the plaques is due to an overproduction of a - beta through enzymatic cleavage of the larger amyloid protein precursor (app). While the monomeric a - beta is not neurotoxic, under specific conditions it is able to misfold from its soluble form into small oligomers and highly ordered fibrillar aggregates . This phenomenon is known as the aggregation of proteins and it is a characteristic feature of several neurodegenerative diseases . Although neuronal degeneration occurs near the amyloid plaques, some studies have suggested that intermediates such as protofibrils or simple oligomers are also involved in ad pathogenesis and even appear to be the more dangerous species in the onset of the pathology . Genetic researches have demonstrated that only a small fraction (about 5%) of all ad cases is caused by inherited alterations with precocious symptom appearance (before age 65). The early ad onset is, in most cases, originated by mutations in three genes: app, psen1, and psen2 . As a result, a large amount of a - beta toxic fragments is produced and deposited as plaques . The great majority of all ad cases is sporadic in origin, with old age (> 65) as the main risk factor . The amyloid cascade hypothesis has tried to explain the origin of the disease . This theory postulates that the deposition of a - beta is the origin of the pathology and that cell loss, dementia, and vascular damage are strictly linked with this deposition . This hypothesis can give an explanation for the ad early onset but it is not viable for the ad sporadic form . Indeed, patients affected by ad late - onset very rarely present app, psen1, and psen2 gene mutations . Further, the presence of the plaques can also be observed in the elderly without ad development . Type 2 diabetes is associated to a reduced ability of insulin to stimulate glucose utilization (insulin resistance). It has been recently recognized that ad is closely linked to the diabetes mellitus in a way that it is still unclear and a deficiency in the glucose metabolism can be considered a risk factor in the sporadic ad onset [68]. Further some diabetes drugs appear to slow the cognitive decline associated with ad . It has been also demonstrated that extracellular injection of insulin is able to protect neurons against a - beta induced cell death . It has been reported that insulin can protect cultured rat neurons against a - beta induced toxicity . Experimental data have demonstrated that a - beta competes for binding of insulin to its receptor . However, it is not understood if this effect was originated through the binding of a - beta to insulin or directly to the insulin receptor . By using mature cultures of hippocampal neurons it was found that a - beta soluble oligomers (also known as addls) caused loss of the activation of insulin receptors (ir) on the neuronal surface, and this event was linked to disruption of insulin signaling . Other studies indicate that insulin, interacting with a - beta, inhibits its fibrillar growth as shown in a cell - free assay and in the cell surface of human brain pericytes reducing the a - beta toxic effect . Recently, in two different model systems, sea urchin embryo and neuroblastoma cell line, it has been demonstrated that a - beta oligomers are more toxic than larger and highly structured fibrils [14, 15]. In particular, oligomers and fibrils, involved in the neurodegenerative process the authors postulate that the large fibrils, remaining on the extracellular space and obstructing the membrane functional channels, can induce an extrinsic programmed cell death pathways through the activation of caspase 8 only . On the contrary, the small oligomers are able to penetrate into the cells . They, damaging the mitochondria, cause a release of the cytochrome c that, in turn, induces activation of the intrinsic apoptotic pathway by the intervention of the caspase 9 . The present study is addressed to investigate whether insulin is able to protect cells by a - beta oligomers toxicity, through inhibition of specific apoptotic pathways activation . Lan5 human neuroblastoma cell lines were plated onto 96-well plates at a density of 6 10 per well and cultured with rpmi 1640 medium (celbio) supplemented with 10% fetal bovine serum (fbs) (gibco) and 1% antibiotics (50 /ml penicillin and 50 g / ml streptomycin) and antimycotics (sigma). Cells were maintained in humidified 5% co2 atmosphere at 37c . In order to obtain the oligomers, recombinant a42 (ra42) was dissolved in 0.01 m trishcl buffer at ph 7.2 and insulin (sigma) in hepes buffer at ph 8.2 . Cells were treated with two different concentrations 25 and 40 m of oligomeric ra42 for 1 h. after this treatment ra42 was removed and the cell were incubated without or with insulin at different concentrations (50, 100, 200 m) in medium serum free at 37c for 20 h. for successive experiments we selected the a - beta oligomer and insulin concentrations at 40 and 100 m, respectively . In caspase assays, the incubation time with insulin was 4 h. the treated cultured cells and the controls were morphologically analysed by microscopy inspection using an axioscop 2 microscope (zeiss, usa) or used for specific assays . Depending on the experiments we also utilized, as controls, both the untreated cells and the cultured cells with buffers at the concentration utilized to dissolve ra42 or insulin . After treatments of the cells, 20 l of the mts solution were added to each well, and the incubation was prolonged for 4 h at 37c, 5% co2 . The absorbance was read at 490 nm on the microplate reader victor 1420 multilabel counter (perkin elmer). Briefly, cells untreated or after treatment with ra42 alone or with insulin were fixed with 4% paraformaldehyde in pbs for 30 min . Triton x-100 in pbs for 5 min, rinsed with pbs and incubated with tunel reaction mixture (enzyme, nucleotides) in an humidified atmosphere at 37c for 1 hour . Staining was obtained by using a peroxidase substrate, hydrogen peroxide and the stable chromogen, diaminobenzidine (dab). After this procedure, samples were rinsed three times with pbs and analysed under zeiss axioscop microscope . One vial of mito red was dissolved in dmso according to the manufacturer instructions (sigma). Living cells were incubated with 20 nm mito red for 5 min and red staining indicates that the mitocondrial is active . For nuclear staining, cells were incubated in the hoechst 33258 (5 g / ml) for 30 min and intense blue staining indicates nuclear fragmentation . Nuclear morphology and mitochondrial activity were analyzed by microscopic inspection using a leica dhl fluorescent microscope at excitation / emission wavelengths of 350/450 nm, respectively . Caspase-8, -9, and -3 activities in cells were measured using commercially available luminescent assays (caspase - glo 8, caspase - glo 9 and caspase - glo 3/7 assay systems, promega). Caspase reagent specific for each kit was added directly to the cells in white 96-well plates and after mixing, they were incubated for 1530 min before recording luminescence with victor 1420 multilabel counter (perkin elmer) apparatus . Total proteins were prepared by dissolving in solubilizing buffer (50 mm tris - hcl ph 8.0, 150 mm nacl, 0.5% triton x-100, 2 mm pmsf, 10 g / ml protease inhibitor, 1 mm na3vo4, and 1 mm naf) lan5 cells untreated (control) or treated with oligomers, alone or with insulin . Protein samples (20 g) were electrophoretically separated using 10% sds - page gel and transferred onto nitrocellulose filters for immunoblotting . After blocking in 3% bsa in tbst, the western blot was incubated with antiphosphorylated hsp70 (hsp70) (1: 1000; cell signalling) or anticonstitutive hsp70 (hsc70) (1: 500; cell signalling) or anti -actin (1: 1000). Primary antibodies were detected using the ecl chemiluminescence kit (amersham) according to the manufacturer's instructions and using secondary antibodies conjugated to horseradish peroxidase (1: 1500; amersham). Statistical evaluation was conducted by anova, followed by student's t - test for analysis of significance . As a first step to evaluate the amount of insulin necessary to interfere with cell damage induced by a - beta oligomers, a dose - response study was performed . Lan5 neuroblastoma cells were treated before with different amounts of ra42 oligomers (25, 40 m) and then with different insulin concentrations (50, 100, 200 m). As shown in figure 1(a), cells treated with ra42, 25 m or 40 m showed a mortality of about 60% and 85%, respectively, if compared to the control . When insulin was added, in the sample previously treated with oligomers, a recovery of cell viability of about 20% for the minor concentration and 100% for the higher concentration was observed, indicating that insulin plays a protective effect against a - beta toxicity (figure 1(a)). Moreover, to visualize the results obtained by the viability assay, the morphological effect compared to the corresponding controls was examined by microscopic inspection . Neurons treated with ra42 oligomers at different degeneration steps were observed and some representative images are shown in figure 1(b). Morphological changes resulted in a reduction of the cellular body, neuritis, and cell numbers . Cells treated with ra42 oligomers and insulin appeared to recover the regular morphology of neurons . In apoptosis a biochemical cascade activates proteases that destroy biomolecules required for cell survival . During this process the cytoplasm condenses, organelles aggregate, chromatin condenses, and nucleus fragments . After cell treatment with ra42 oligomers, we detected in the survived cells morphological modifications, typical hallmarks of the apoptosis process . In particular granules, resembling apoptotic bodies, were observable (data not shown). To investigate if insulin can revert this effect, lan5 cells were put through to the tunel assay after treatment with oligomers alone or with oligomers and insulin . An intense brown nuclear staining is visible in the cells treated with ra42 (figure 2(b)), indicating that the apoptotic process has been triggered, whereas no staining is detectable in the cells treated with oligomers and insulin (figure 2(c)), as seen for the control cells (figures 2(a) and 2(d)). The major executioners in the apoptotic program are proteases known as caspases . In some forms of apoptosis, the extrinsic apoptotic pathway is initiated by activation of caspase 8 after death receptor binding; in other forms, activation of the intrinsic apoptotic pathway is initiated by signaling molecules, recruited by mitochondria . They lead a release of cytochrome c from mitochondrial matrix to cytoplasm where it binds to apaf-1 protein to form the apoptosome that activates caspase 9 . Both these pathways are able to activate the executrix caspase-3 involved in the final part of death process . To identify in which ra42-oligomer induced apoptotic pathway insulin interferes, we performed caspase 8 and caspase 9 luminometric assays . No activation of capsase 8 by oligomers stimulus occurred and the same result for insulin stimulus was obtained (figure 3(a)). Instead, as can be seen in figure 3(b), a - beta oligomers activate caspase 9 and this activation is noticeably reduced by the presence of insulin, indicating, once again, that insulin is able to produce a positive protective effect . In order to confirm this result, lan5 cells treated as described above were put through to caspase 3 assay, the typical executrix caspase . As expected, in the lan5 cells treated with oligomers and insulin a reduction in the caspase 3 activation with respect to the a - beta oligomer treated cells these results confirm that a - beta oligomers induce intrinsic apoptotic pathway and the insulin is able to down - regulate caspases 9 and 3 activation . Caspase 9 activation is considered an event subsequent to mitochondrial damage . In order to confirm that a dysfunction of this organelle occurred after a - beta stimulus and this dysfunction is replaced or avoided by insulin treatment, we tested the metabolic activity of the mitochondria by using mito red, a specific dye that permits to measure the respiratory activity . Once the dye is introduced into the cells, it freely diffuses through the outer mitochondrial membrane and enters exclusively into the mitochondrial matrix of the metabolically active mitochondrials . Moreover, the nucleus integrity was visualized by staining the samples with the specific hoechst dye . In figure 4 an intense red staining, comparable to the control cells, was observed in cell treated with a - beta oligomers and insulin and no intense blue staining, indicating nuclear fragmentation, was observed . In contrast, absence of red staining is visible in the cells treated with a - beta oligomers alone, while an intensive blue staining is evident, stating the presence of dna nicks, a typical apoptosis hallmark . This analysis confirms that the ra42 induced degeneration, via mitochondrial damage, has been counteracted by insulin addition . Hsp70 has a negative regulatory role in apoptosis and is activated, by phosphorylation process, in response to a variety of stress stimuli . To test whether the insulin can play its protective role by activating or improving activation of hsp70, same amounts of proteins extracted from cells, (i) untreated, (ii) a - beta treated, (iii) treated with insulin alone, and (iv) previously treated with a - beta, were loaded on a sds - page . The immunoblot was incubated with antibodies against both the constitutive and inducible hsp70 stress proteins and antiactin for normalizing gel loading . Levels of the constitutively expressed hsp70 form (hsc70) do not change after any stimulus . In agreement with other studies, a - beta presence increased the hsp70 stress - inducible form (hsp70) as compared with the control (figure 5). Moreover, insulin enhanced a - beta mediated activation of hsp70 suggesting that this major induction could be necessary to complete a cell survival program . An increasing number of reports suggest that a - beta accumulates inside neurons with aging and that a - beta aggregation can be an initial event for the characteristic cell degeneration present in the alzheimer's pathology . A - beta aggregated molecules alter normal cell homeostasis including impaired glucose / energy metabolism, mitochondrial dysfunction and oxidative stress . Recently, several studies suggest that disturbance in insulin metabolism, especially insulin resistance, plays a role in the onset and development of ad . Thus, growing evidence begins to find links between ad and type 2 diabetes and, for this reason, ad has been considered the brain - type diabetes [5, 19]. Here we provide evidence that insulin can protect cells against damage induced by a - beta oligomers avoiding the apoptosis program . We found that insulin inhibits a - beta cell death in lan5 neuroblastoma cells in a dose - dependent manner . According to several results different are the hypothesis reported to explain a mechanism underlying the protective role played by insulin to contrast the impairment produced by a - beta oligomers . Among the first studies, it has been demonstrated that specifically dibutyryl camp or insulin inhibit toxic effect of a - beta2235 in cultured rat hippocampal neurons and a recover of the intracellular signal transduction disorder was suggested . More recently, on the basis that a - beta and insulin are both amyloidogenic peptides sharing a common sequence recognition motif, it has been reported that a - beta hinders the insulin binding to its receptor (ir), leading to an ir reduced autophosphorylation . The authors suggest that the link between a - beta and insulin is associated to the impairment of glucose utilization, and in agreement to this hypothesis, a recovery of cell viability, through insulin administration could be due to the retrieval of normal glucose metabolism . Other authors suggest that insulin inhibits a - beta fibril network formation, an essential step in exerting its toxicity, at the cell surface of human brain pericytes (hbp). In fact, the hbp line does not express ir, therefore in this case the protective effect of insulin cannot be due to its reduced binding to ir . All the results reported above underline that insulin is able to recover an intracellular damage induced by a - beta, whose effect is to produce cellular stress and degeneration . Apoptosis has been often associated to neurodegeneration and to find a mechanism that control caspase activation could be a promising approach to hinder the cell death process . By specific assays, we demonstrate that insulin opposes to apoptotis and particularly caspase 9 and 3 activation after a - beta induced toxicity . According to previous results caspase 8 was not activated by a - beta oligomers, because the extrinsic pathway is a preferential process activated by larger a - beta fibrils . Caspase 9 activation is a consequence of mitochondrial dysfunction often provoked by oxidative stress, and increased oxidative stress has been implicated in the etiology of several pathology including diabetes and ad [22, 23]. Mitochondria are both the major generators and direct targets of reactive oxygen species (ros) and this had led to the idea that oxidative stress and mitochondrial damage are contributory factors to several disorders . Moreover, it has been demonstrated that, in the presence of a - beta, insulin prevents the decline in mitochondrial oxidative phosphorylation efficiency and avoids an increase in oxidative stress . These data are in agreement with the result here shown that the mitochondrial activity is impaired when the neuroblastoma cells are treated with a - beta oligomers and that this effect is recovered when insulin is added . Moreover, insulin prevents apoptotic pathway activation as observable by the absence of nuclear fragmentation, that instead is well evident in the oligomers treated cells . Thus, we suggest that insulin prevents mitochondrial dysfunction probably by inhibition of ros formation and activating specific cell signaling (work in progress). Further, it is known that insulin activates the serine - threonine kinase akt, a protein downstream of pi3k, involved in survival pathway [2527]. Akt has been well demonstrated to phosphorylating and increasing the expression of a number of proteins involved in apoptotic signaling cascade such as the bcl-2 family [28, 29]. Presence of a - beta produces a toxic effect, responsible for the observed neuron death . Some evidences point out the role of molecular chaperones in neurodegenerative processes [30, 31]. We show that a stress response is activated by the increased expression of inducible hsp70 . Interestingly, a - beta is able to activate hsp70 but, in the present experimental conditions, the endogenous stress response is insufficient to revert the apoptotic pathway . Upregulation of hsp70 has been found in neurons treated with a - beta and a modulator role in a - beta toxicity has been proposed . The authors hypothesize that an imbalance between the neuronal hsp protective capacity and the toxic accumulation of a - beta could be the cause of the neuronal death . The presence of insulin enhances the activation of the stress proteins, suggesting that an increased survival program has been activated and the neurons recover their viability . Moreover, it has been demonstrated that hsp70 is induced by pi3/akt activation that in turn is activated by insulin . Heat shock proteins can neutralize the neurotoxicity in animal models suggesting potential therapeutic approaches in neurodegeneration associated with abnormal folding and toxicity [33, 34]. Hsp70 plays a role in preventing protein aggregation and degradation [36, 37]. Helped by insulin, that increases its expression and activation, we can assume that hsp70 could prevent a - beta oligomers by successive aggregation steps or promote oligomers degradation . The present data point out the protective action of insulin in inhibiting a - beta cell toxicity and they could be an initial step to understand as insulin resistance or disturbance in insulin metabolism can contribute to neurodegeneration . Moreover, insulin may constitute a therapeutic agent against ad, activating cell survival signaling pathways.
Hsp can affect multiple organs presenting with a characteristic rash in most of the patients . Familial mediterranean fever (fmf) is an inherited inflammatory disease common in mediterranean populations . A 16 year old boy was referred with history of abdominal pain lasting for 20 days . He was hospitalized and had appendectomy . Due to the persistence of his abdominal pain after surgery, the patient was diagnosed as hsp with renal, gastrointestinal tract and skin involvement . We performed dna analysis in our patient because he had diagnosis of vasculitis with severe symptoms and found that he was carrying heterozygote p369s mutation . Our case is noteworthy as it indicates that it may be important not to overlook presence of fmf mutations in patients with a diagnosis of severe vasculitis . The annual incidence of hsp is 22.1/100000 children and 75% of cases are seen between 3 and 10 years of age . The diagnostic criteria include palpable purpura with at least one of the following manifestations: abdominal pain, iga deposition, arthritis or arthralgia, or renal involvement . The disease is characterized by deposition of immunglobulin a (iga) containing immune complexes and complements within small vessel walls, and often within renal mesangium . Renal involvement occurs in 20 - 60% of patients with hsp, usually manifesting as hematuria, and often associated with proteinuria . Most of the patients with renal involvement have a good prognosis . However, some patients progress to end - stage renal disease, and renal involvement in hsp is one of the major causes of chronic renal failure in childhood . Also some patients may develop serious complications, such as intestinal intussusception, perforation or obstruction . Familial mediterranean fever (fmf) is an inherited inflammatory disease common in mediterranean populations . It is characterized by recurrent episodes of fever, peritonitis, pleurisy, rashes and arthritis and may be complicated by renal amyloidosis . Fmf is caused by mutations in the gene mefv, which encodes pyrin / marenostrin, a protein implicated in the regulation of neutrophil activity . Some types of vasculitis are more frequent in fmf, including hsp, polyarteritis nodosa (pan) and behet's disease . We present a 16-year old boy with henoch - schnlein vasculitis who had severe renal and gastrointestinal involvement and rarely seen heterozygote p369s mutation in mefv gene a 16-year old boy was referred to hospital with a history of abdominal pain for 20 days . He was hospitalized and had appendectomy . Due to the persistence of abdominal pain after surgery he had also sudden onset of palpable purpuric rashes, first on periumblical and gluteal zone, then on the extensor surface of his lower extremities . He also reported coffee ground vomiting and dark color urine on the day of admission . There was no history of recent drug exposure, immunization, or upper respiratory tract infection . In physical examination blood pressure was 140/90 mmhg (9599 percentile), other vital signs were normal . Laboratory tests showed an erythrocyte sedimentation rate (esr) of 55mm / h (normal: <20mm / h), c - reactive protein: 1,7 mg / dl (normal: <1 mg / dl), fibrinogen 501 mg / dl (normal: 200400 mg / dl), hemoglobin 12.1g / dl, hematocrit 35.3%, white blood cell count (wbc) 23.500/mm, platelet count 944.000/mm, serum urea 23 mg / dl, creatinine 0.6 mg / dl, albumin 2.8 g / dl, alanine aminotransferase 47 u / l, aspartate aminotransferase 91 u / l, amylase 70 mg / dl, lipase 29 mg / dl . The anti - streptolysin - o titer was 100 iu / ml (normal: 0150 iu / ml). Antinuclear antibody (ana), anti dsdna, antineutrophil cytoplasmic antibody (anca), and anticardiolipine antibody were negative . Urinalysis revealed macroscopic hematuria and proteinuria with 24-h urinary protein excrection of 104.7 mg / m / h . Abdominal ultrasound, renal doppler ultrasound and renal magnetic resonance angiography (mra) were normal . On histopathological examination, 25 glomeruli were seen in routine stains including hematoxylen and eosine, periodic acid schiff and masson - trichrome stains . In two of the glomeruli, slightly increased mesangial matrix was noted, and other glomeruli were normal in appearance . Coarsely granular iga deposits were seen diffusely, i.e. In all glomeruli in mesangial area, while as far as igm is concerned, mesangial deposits were detected in two glomeruli . Based on these clinic findings, the patient was diagnosed as having hsp with renal, gastrointestinal tract and skin involvement . Since there are reports of increasing frequency of accompanying mefv mutations in patients with hsp and alterations in the mefv gene is an important susceptibility factor for the development of vasculitis and, also since it affects clinical presentation and is associated with a more severe course, we performed dna analysis in our patient who had severe vasculitic involvement and found that he was carrying heterozygote p369s mutation . Alternate day pulse steroid treatment (30mg / kg) was administered to the patient for 3 times and followed by oral maintenance steroid treatment (2mg / kg / day). Colchicine treatment was also initiated after detection of the fmf mutation . Because no amelioration in proteinuria was achieved with this therapy, cyclophosphamide (2mg / kg / day) hsp is an immunologically mediated systemic vasculitis of small blood vessels . Despite being one of the most common vasculitides of childhood, definitive data on the etiology remains unknown, although many antigens, such as infective agents, vaccinations, drugs, and insect bites have been found to trigger hsp . Hsp is mediated by immune deposits (typically with iga), resulting in necrosis of the wall of small and medium - sized arteries with extravasation of erythrocytes, infiltration of tissue with neutrophils, and deposition of nuclear fragments from degenerating neutrophils, a picture called leukocytoclastic vasculitis (lcv). Renal involvement determines the long - term prognosis, and the prevalence ranges from 20% and 60% according to the different reports . The most common clinical sign of henoch - schnlein nephropathy is isolated microscopic hematuria, often associated to proteinuria . The presence of renal failure, arterial hypertension, nephrotic proteinuria, and histological findings at renal biopsy (proportion of glomeruli with crescents) have traditionally represented a poor prognostic factor . There have been some reports in which the renal involvement in hsp has been associated with several factors, such as the age at onset, abdominal symptoms, the recurrence of purpura, and treatment with corticosteroids and plasma coagulation factor xiii concentrate . The risk of chronic renal failure is related to the initial clinical presentation, being less than 2% in those with hematuria and/or minimal proteinuria to 19% when both nephritic and nephrotic syndromes are found . Gastrointestinal disease occurs in up to 85% of patients with varying syptoms like abdominal pain, sometimes associated with nausea, vomiting, or bleeding . Mucosal lesions can develop anywhere in the gi tract, but the duodenum and small bowel are the most commonly involved sites . Complications of involvement of gastrointestinal tract in hsp include intramural hematomas, intussusceptions, bowel infarction, bowel perforation, pancreatitis, appendicitis, and cholecystitis . Intussusception is the most common surgical complication of hsp in childhood, occuring in 0.7 - 13.6% of patients . In our patient, due to the renal biopsy findings consistent with hsp, negative anca and normal renal doppler ultrasound and renal mra, diagnosis of pan was ruled out . The presence of typical rash, severe abdominal pain, nephrotic proteinuria and iga deposition on kidney biopsy confirmed the diagnosis of hsp . Fmf is an autosomal recessive disease affecting people of mediterranean ancestry with recurrent self - limited attacks of fever and inflammatory serositis . Fmf is caused by mutations in mefv gene, which encodes pyrin / marenostrin, a protein implicated in the regulation of neutrophil activity . The incidence of fmf is as high as 1/1000 - 2000, and the estimated carrier rate is 1/5 . The four most commonly reported mutations in mefv gene are m694v, m680i, v726a and e148q . Several types of vasculitis are associated with fmf, pan, hsp, behet's disease and protracted febrile myalgia . The overall incidence of fmf vasculitis in patients with pan is 1%, and in 5% hsp patients fmf vasculitis is detected, and it is significantly higher in fmf than in the general population . The occurrence of circulating immune complexes in 50% of patient with fmf, complement consumption, defective inhibition of complement activation and uncontrolled release of tnf during the attacks have been described . Therefore an immune - related mechanism has been suggested to be involved in the pathogenesis of fmf and fmf - associated vasculitis . The diagnosis of fmf is based on the clinical criteria, family history, exclusion of other hereditary periodic fever syndromes and the patient's response to colchicine treatment . The demonstration of mefv gene mutations is necessary in only suspected patients to establish a definitive diagnosis . There are subjects who are homozygotes, or compound heterozygotes, but not having any symptom of fmf . On the other hand, there are fmf patients who respond to colchicine treatment, in whom no mutation in the mefv gene has been demonstrated . Fmf and vasculitis have remarkable similarity: fever, abdominal pain, arthritis, skin lesions and blood in stool and urine . It has been reported that in some rheumatic diseases mefv mutations (in a single allele) were increased suggesting that the mutated mefv allele was acting as a susceptibility factor, also mefv mutations are known to promote inflammation and especially to favor the development of severe vasculitis . Our patient did not demonstrate characteristic features of fmf such as abdominal pain, fever and family history of fmf . Recently, increased number of studies reporting mefv mutations in patients with hsp suggests association of this gene with clinical presentation, especially with a severe course . Depending on these results we performed dna analysis in our patient and detected heterozygote p369s mutation . P369s mutation has been reported in turks, syrians, armenians, lebanese, palestinians, and japanese [15,2428]. In a study performed in turkey, frequency of rarely seen p369s mutation was reported as 2.55% . Association of behet's disease, which is one of the fmf associated vasculitis, with p369s mutation was reported in the literature but there are no reports regarding association of this mutation with hsp . Our patient who was diagnosed to have hsp with severe renal and gastrointestinal involvement was found to have the rarely encountered p369s mutation in mefv gene upon screening . This case which demonstrates the association of p369s mutation in fmf patients with hsp, is remarkable as it indicates the importance of this minor mutation with development of severe vasculitis . In conclusion our case is noteworthy as it shows that it may be important not to overlook presence of mefv mutations in patients with a diagnosis of severe vasculitis.
Periodontal plastic surgery procedures address these esthetic and functional demands and have become an integral part of the periodontal treatment . Several therapeutic modalities such as free gingival autografts, pedicle grafts, connective tissue graft, grafts combining the two modalities, and guided tissue regeneration have been used for covering the denuded roots and to augment the width and thickness of the keratinized gingival . These procedures have resulted in reduction or elimination of root hypersensitivity, improved esthetics, and facilitation of plaque control . Among various surgical techniques, subepithelial connective tissue (sect) grafts remain the most commonly used and most successful root coverage procedures . The esthetic and functional success of sect graft techniques is highly predictable and reliable which has been documented in several longitudinal studies . Thickness and volume of the tissue to be harvested from the donor site are among the important factors in determining the appropriate treatment method . Variations of size and shape of the palatal vault may also affect the dimensions of the donor tissue harvested . Although an ample number of studies have evaluated the results of utilizing sect graft at the recipient site, a very few studies are focused on evaluating the wound healing and assessing patient - centered outcomes at the palatal donor area . Furthermore, the oral cavity provides a unique environmental challenge for the healing wounds produced during various periodontal surgical procedures . The ideal technique for procuring a connective tissue graft should harvest an adequate graft, be user - friendly, produce minimum palatal discomfort, have minimal operative complications, and create a wound in the donor area that heals quickly with minimal postoperative complications . Various techniques have been developed for harvesting soft tissue grafts from the palate such as trap door technique, parallel incision technique, and single incision technique . In the present study, a new instrument unigraft knife (also called the free gingival graft knife from ace surgical supplies) was used which when activated elevates a partial thickness flap beneath which the connective tissue graft is procured . The healing of the palatal wound created with this knife was compared with the wound created by langer and langer trap door technique . The purpose of the present study was to evaluate and compare the healing of the wound at the palatal donor site and root coverage results of the two different techniques . Sixteen systemically healthy patients with 30 sites (gingival recession 2 mm) were recruited from the outpatient department of periodontology and implantology who presented with miller class i and ii recession (2 mm). Noncomplaint patients, patients with root surface restoration, current smokers, or tobacco users were excluded from the study . Thirty recession sites were divided into two equal groups with 15 recession sites each, that is, group i, which received the graft procured with the unigraft knife (ace surgical supply co., ma, figure 1c) and group ii, which received the graft harvested by the langer and langer technique . The randomization was done by a coin flip technique as the study involved only two groups . (k) postoperative view of recession coverage at recipient site after 6 months all the enrolled patients underwent phase 1 periodontal therapy and were given oral hygiene instructions to ensure that they would adopt the correct brushing technique . Full mouth plaque scores (fmps) and full mouth bleeding scores (fmbs) were recorded initially and after scaling and root planing . Surgery was not carried out till the patients reached fmps <20% and fmbs <20% . Parameters implied for evaluation of healing pattern at the donor site included the measurement of wound size (ws), immediate bleeding (ib) and delayed bleeding (db), complete wound epithelialization (ce), sensibility disorders (sd), and postoperative pain (pp) at baseline, 1, 4, and 12 week postoperatively . Ws measurements were made by measuring the surface area of the palate from where the graft was procured that appeared to be granulating in or clinically did not appear to be covered by epithelium . Measurements were made with a periodontal probe (unc-15) to the nearest measurement of 0.5 mm . Ib and db were assessed and ib was recorded as positive if the donor area presented with bleeding after 2 min application of external pressure with a sterile gauze . Db was measured as positive if the patient presented with prolonged hemorrhage from the palate during the postsurgical period . Ce wound was assessed clinically by means of colored photographs taken at each postsurgical visit . The scores were assigned as follows: 0 = no color match with adjacent tissues, 1 = partial color match with the adjacent tissues, and 2 = complete color match with the adjacent tissues . Sd was assessed by means of a periodontal probe (unc-15, hu - friedy) using a 4-point discrimination scale (coronal, apical, mesial, and distal) around the donor area before and after the surgical procedure and the follow - up visits . Identical assessment was made at the same time in the corresponding contralateral area to collect the most reliable data possible . Objective sensory loss was recorded using a rubbing movement and a pin - pressure nociception . Patients were asked to give a rating of their loss of sensibility based on a 3-point verbal descriptor scale (none, mild or moderate, severe). Pp: at the subsequent postoperative appointments, the patients were asked to rate their discomfort level in the palate for the previous week . The numbers of pills taken for pain were also noted down for each patient . Visual analog scale (vas) was used, and pain was assessed by asking the patients to rate the intensity of the pain perceived in the palate donor area at 1 and 4 week using 100 mm horizontal scale with the left endpoint marked worst pain imaginable as the primary efficacy parameter . The patient was asked to move a finger on the vas tip which coincided with the level of pain experienced . Verbal rating scale (vrs) (no pain, mild pain, moderate pain, severe pain, and very severe pain) was used to rate the discomfort level in the palate donor area at the postoperative appointments . All the clinical measurements were made by the same examiner only, to avoid any inter examiner bias . Except for the evaluation of colored photographs for ce where three examiners scored the photographs separately, on evaluation of healing pattern at the recipient site, the clinical parameters were assessed at the baseline, 3, and 6 month . These included the clinical attachment level (measured as the distance from cementoenamel junction to the base of the pocket), vertical recession (vr measured as the distance from cementoenamel junction to the free gingival margin at the mid - buccal level), and width of keratinized gingiva (kt measured as the distance from most apical position of gingival margin to the mucogingival border at the buccal tooth surface). Initially, in both groups at the recession site, a full mucoperiosteal flap using horizontal and vertical incisions was raised according to the langer and langer technique, sparing the proximal papillae . In the apical areas, all the recipient areas were treated in a similar fashion so that the only difference in the therapy each group received was the method used to obtain the graft . Further, for the donor site, one of the two techniques for harvesting the graft (unigraft knife method or langer and langer technique) was used to harvest the required amount of sect graft for the recession sites as per the randomization table . A palatal region from the first molar to canine was utilized for procuring the required amount of sect graft . The donor area was sounded with a periodontal probe to ensure that there was a minimum of 3 mm soft tissue thickness . Unigraft knife was assembled in a conventional manner to permit cutting in a pulling motion . The knife was assembled with a cutting shoe reversed so that the cutting shoe would cut in a pushing direction . It was then used to elevate a partial thickness trap door flap by pushing the knife, under control, distally across the palate . This trap door flap was retracted mesially to permit access to the connective tissue beneath it . The knife was then assembled in a conventional manner to permit cutting in the pull motion . Now, starting at the distal edge of the trap door flap, the knife was then used to elevate a connective tissue flap [figure 1a - k]. This secondary flap, made up of connective tissue, was incised at the mesial edge . Palatal donor area was sounded with a periodontal probe to ensure 3 mm soft tissue thickness . A pair of parallel incisions (1.5 mm apart) were made into the palate in the area of the first molar to canine . The incisions were made with a single 1012 mm deep pass of no 15 blade mounted on bp handle . The parallel incision was made at least 23 mm from the gingival margin in the palate . Vertical incisions were placed at the mesial and distal end of the most external incision . A 4 - 0 silk suture was placed through the palatal tissue to retract the palatal tissue and to provide access to the tissue between the initial incisions . The tissue was then removed by incising the mesial, distal, and medial edges between the parallel incisions . Pressure was applied with wet gauze to the donor area [figure 2a - i]. The palatal wound was closed with sutures in the vertical incision and also using the suture that had been used to retract the palatal tissue for access . (i) postoperative view of recession coverage at recipient site - after 6 months in both the groups, the procured graft from the palate was secured over the recipient site using vicryl 4 - 0 sutures [figures 1k and 2i]. The overlying flap was sutured as coronally as possible to cover the connective tissue graft . After closure of the vertical incisions, a mild compression with gauge soaked with sterile saline solution was done for 5 min to reduce the size of the clot . A periodontal dressing was placed over the recipient site and on donor site to protect the underlying tissue for 10 days postoperatively . Postoperative instructions included the use of 0.2% chlorhexidine gluconate rinse twice daily and avoidance of trauma to the surgical areas . Initially, in both groups at the recession site, a full mucoperiosteal flap using horizontal and vertical incisions was raised according to the langer and langer technique, sparing the proximal papillae . In the apical areas, all the recipient areas were treated in a similar fashion so that the only difference in the therapy each group received was the method used to obtain the graft . Further, for the donor site, one of the two techniques for harvesting the graft (unigraft knife method or langer and langer technique) was used to harvest the required amount of sect graft for the recession sites as per the randomization table . A palatal region from the first molar to canine was utilized for procuring the required amount of sect graft . The donor area was sounded with a periodontal probe to ensure that there was a minimum of 3 mm soft tissue thickness . Unigraft knife was assembled in a conventional manner to permit cutting in a pulling motion . The knife was assembled with a cutting shoe reversed so that the cutting shoe would cut in a pushing direction . It was then used to elevate a partial thickness trap door flap by pushing the knife, under control, distally across the palate . This trap door flap was retracted mesially to permit access to the connective tissue beneath it . The knife was then assembled in a conventional manner to permit cutting in the pull motion . Now, starting at the distal edge of the trap door flap, the knife was then used to elevate a connective tissue flap [figure 1a - k]. This secondary flap, made up of connective tissue, was incised at the mesial edge . Palatal donor area was sounded with a periodontal probe to ensure 3 mm soft tissue thickness . A pair of parallel incisions (1.5 mm apart) were made into the palate in the area of the first molar to canine . The incisions were made with a single 1012 mm deep pass of no 15 blade mounted on bp handle . The parallel incision was made at least 23 mm from the gingival margin in the palate . Vertical incisions were placed at the mesial and distal end of the most external incision . A 4 - 0 silk suture was placed through the palatal tissue to retract the palatal tissue and to provide access to the tissue between the initial incisions . The tissue was then removed by incising the mesial, distal, and medial edges between the parallel incisions . Pressure was applied with wet gauze to the donor area [figure 2a - i]. The palatal wound was closed with sutures in the vertical incision and also using the suture that had been used to retract the palatal tissue for access . (i) postoperative view of recession coverage at recipient site - after 6 months in both the groups, the procured graft from the palate was secured over the recipient site using vicryl 4 - 0 sutures [figures 1k and 2i]. The overlying flap was sutured as coronally as possible to cover the connective tissue graft . After closure of the vertical incisions, a mild compression with gauge soaked with sterile saline solution was done for 5 min to reduce the size of the clot . A periodontal dressing was placed over the recipient site and on donor site to protect the underlying tissue for 10 days postoperatively . Postoperative instructions included the use of 0.2% chlorhexidine gluconate rinse twice daily and avoidance of trauma to the surgical areas . The statistical analysis was done using spss version 15.0 statistical analysis software (statistical package for the social sciences (spss), version 15.0, ibm, chicago, il). The values were represented in number (%) and mean standard deviation and to test the significance between two means (group i and ii) the student's t - test was used . For comparison of change in parameters in two groups at different time intervals, paired t - test was used . Since this study was aimed to assess the early healing of the wound at the palatal donor site by comparing langer and langer trap door technique and a unigraft knife method for obtaining the connective tissue graft for the treatment of buccal gingival recession, the recipient site was treated in a similar fashion in both the groups . All the patients completed the study period, and there was no drop - out . No complications were observed in any patient, and all patients responded well to the treatment and follow - up visits . Patients in both the groups exhibited pain at 1-week follow - up which was higher in group i than group ii, but it was statistically not significant . As per vas measurements, the patients reported mild to moderate pain at the donor site [table 1]. Comparison of complete wound epithelisation, delayed bleeding, sensibility disorders, at different time intervals on evaluation of scores from vrs scale, the pain reported by group i patients was of moderate intensity whereas for group ii, dull pain was reported by the patients at 1-week interval . However, at subsequent follow - up visits, none of the patients in either of the groups reported pain, till the conclusion of the study . Pp was also assessed as per the number of nsaid pills taken by the patients in both the groups . It was found to be similar at 1 week postoperative interval, and no analgesic pill intake was reported by any patient at 4 and 12 weeks follow - up intervals . When all the parameters of the pp, i.e. Vas, vrs, and nsaids pills taken were analyzed collectively, the amount of pain reported at 1 week in both the groups was significantly higher than at subsequent time intervals [table 2]. Comparing the vas, vrs, pills in both the groups at different time intervals reduction in ws from baseline to all follow - up time intervals was significantly faster in both the groups, but the rate of ws reduction was much faster in group ii compared to group i [table 3]. Comparison of wound size (ws) at different time intervals complete wound epithelization was also achieved at a faster rate in group ii as compared to group i. the difference in the rate of ce was significantly higher (p <0.001) from baseline to 1 week in both the groups [table 4]. Db was seen at 1-week follow - up in both the cases which was higher in group i, but was not statistically significant (p = 0.825). No postoperative bleeding was observed on further follow - up visits in both the groups [table 1]. Comparison of mean change in wound size and complete wound epithelisation at different time intervals in group i and group ii sd was noticed in group ii at 1 week, but was not seen in group i and also no sd was observed at any of the further follow - up visits in either of the groups [table 1]. On evaluation of root coverage parameters at the recipient site, results showed difference in the mean vr, mean root coverage in both the groups to be nonsignificant (p> 0.05; table 5). Intragroup comparison of mean width of keratinized gingiva (kt) showed no statistically significant difference (p = 0.419; table 5) at all - time intervals . Intragroup comparison of mean kt showed that both the groups gained statistically significant amount of tissue at 3 and 6 month postsurgically as compared to baseline (p <0.001) [table 6]. Comparison of vertical recession, probing depth, clinical attachment level and width of keratinized gingiva in two groups at different time intervals . (group i - unigraftknife method and group ii - langer and langer method) comparison of mean change in width of keratinized gingiva (kt) in two groups at different time intervals there are several techniques available to obtain suitable sect graft . Among the techniques used for sect graft harvestation, langer and langer trapdoor method langer and langer method along with most of the techniques relies on free hand dissection of the palate and, therefore, is a highly technique sensitive procedure . The aim of the present study was to compare healing at the palatal donor area using two different surgical techniques to harvest a sect graft for a root coverage procedure . The recipient sites were treated in a similar manner so that the only difference in the therapy each group received was the method used to obtain the graft . Thirty sites in 16 patients were selected (gingival recession 2 mm) for root coverage procedures . This comparative, clinical, randomized study was designed to assess the differences in healing pattern and patient discomfort between the two groups . The connective tissue graft from the palatal site was harvested 23 mm away from the gingival margin for both the techniques . Harris in 1997 also advocated that at least 3 mm of palatal mucosa (thickness) was needed for harvesting a connective tissue graft; therefore in our study, sounding with a periodontal probe was done in both the groups . A uniform connective tissue graft with a thickness of 1.5 mm was obtained using unigraft knife . For langer and langer trap door technique, freehand incisions were made 1.5 mm apart, and an effort was made to keep the thickness of the graft uniform . Two releasing vertical incisions were also given to facilitate the removal of connective tissue graft and to aid in wound closure . At recipient site, in both the groups, attempt was made to completely cover the connective tissue graft by overlying flap . Studies have shown that total vascularization in the part of the connective tissue graft occurred when the flap at the recipient site completely covered it . As per the best of our knowledge, no data have been reported in the literature regarding the assessment of depth of wound at the palatal donor area . Visual cues, such as wound bed color and wound measurements, cannot be relied upon . Therefore, apart from measuring the wound using unc-15, clinical photographs were taken at each postoperative visit to access complete wound epithelization . The ws was measured with unc-15 (to the nearest of 0.5 mm) at 1 week postoperative visit was significantly larger in group i as compared to group ii (p <0.001; table 3). This could have been the result of the high rate of sloughing of the flap seen with the technique using unigraft knife . If the knife was engaged superficially, a thin trap door flap was obtained which might have resulted in high rate of sloughing . Making the incisions freehand could have permitted a deeper trap door incision or a wider base on the trap door flap which may have helped to reduce the sloughing seen . At 4 weeks too, the mean ws was larger in group i as compared to that in group ii (p <0.001; table 4). A deeper incision or a wider base in the unigraft knife group might have helped to reduce the sloughing . This was not possible as there are limited no of sizes of cutting shoe available for this knife . At 4 weeks follow - up visit, although the wound area was epithelized, there was a depression seen at all the palatal sites in the group i, whereas in the group ii, wound area was fully diminished at all palatal sites at 4 weeks follow - up except for 1 patient . By 12 weeks, in both the groups, complete epithelization of the palatal wound area had occurred and clinically wound area was fully diminished . The percentage of complete palatal wound epithelization using langer and langer trap door technique cited in the relevant literature is quite similar to that seen in our study . The rate of sloughing of the primary flap using the langer and langer trap door technique was similar to those seen in previous reports . Edel who performed this technique also reported that degeneration of the primary flap in most patients takes about 1 week . Harris, 1997, in a comparative study of the clinical healing in the donor area demonstrated that a high rate of sloughing occurs in the superficial flap when free gingival graft knife was employed . Unigraft knife method resulted in a larger wound area at 1-week postoperative visit than the langer and langer trap door method . This could have resulted due to a variation in the ws created by the unigraft knife where the flap design was such that the distal border instead of the medial one served as the base of the reflected primary flap . The base of the primary reflected flap in the langer and langer trap door was toward the mid - palate in the region of first molar and canine and was broader than the base of the primary reflected flap in the unigraft knife method . Intragroup comparison of ce showed that in group i, it was completed at 12 weeks postsurgery, whereas in group ii, it was completed at 4 weeks except for one patient where it was observed at 12 weeks period . The results could be correlated to a study by del pizzo et al . Who also found complete palatal wound epithelization by 4 weeks after surgery . Kahnberg and thilander in a study on palatal healing in rats, noted that epithelization progressed from the wound borders, and reduction of the wound surface preceded by contraction of the wound margins and by epithelial cell migration . At none of the time db at 1-week postoperative visit was higher in group i as compared to group ii, but the difference was not statistically significant (p = 0.825; table 4). According to griffin et al ., 2006, proper care was taken in both the groups to ensure that postoperative instructions are abided by . No statistically significant differences were observed regarding the return of sensibility in the palatal donor site in both the groups . Literature supports that transient - postoperative sensory dysfunction is a possible complication after harvesting the graft from the palatal region . Halata et al . Were able to show different nerve endings in the hard palate . In addition to free nerve endings within the epithelium and lamina propria, they also found merkel nerve endings, as well as meissner and ruffini corpuscles, inside the basal lamina and the adjacent connective tissue, all of which are sensitive to touch and pressure . Although sd is not an objective measurement, in our study, both 2-point discrimination and soft - touch discrimination were used which have been reported to be reliable methods to detect the function of these mechanoreceptors . In both groups, mean vas and number of analgesic pills intake in the group at 1 week were higher as compared to that in group ii, yet the difference was not statistically significant . No pain or number of analgesic pills intake was reported by any patient in both the groups after 4 and 12 weeks follow - up intervals . Lengthy surgical procedures may create extensive tissue injury, prolong vasodilation that permits more fluid to accumulate in the interstitial spaces, and results in higher level of biologic mediators released by inflammatory and resident cells . However, differences in patient perception can also influence the levels of reported pp . Both the unigraft knife method and the langer and langer trap door method simplify the technique for obtaining the sect graft from the palate . Undoubtedly, in certain clinician's hands, with certain skill levels and in certain situations, one technique with or without on assessing the root coverage parameters at the recipient site, it was found that the difference in the mean vr in both the groups at baseline, 3, and 6 months was not statistically significant . Mean root coverage achieved for group i was 54%, whereas for group ii, it was 68.13% at final (6 months) postoperative visit . This could be explained by the fact that recipient area in both the groups was treated in a similar fashion . Harris, 1997, also observed similar trends on comparison of root coverage at the recipient site using the unigraft knife method and langer and langer trap door technique . Intergroup comparison of mean width of keratinized gingiva (kt) showed no statistically significant (p = 0.419) difference at all time intervals . Intragroup comparison of mean kt showed that both the groups gained a statistically significant amount of tissue at 3 months and 6 months postsurgically as compared to baseline . Transplanted sect of subsequent size has shown the availability to result in predictable root coverage and increased width of attached gingival . Wennstrm and zucchelli reported that transplanted connective tissue from palate has an ability to alter the differentiation of epithelial cells of the thin covering coronally advanced flap to become keratinized cells . Granulation tissue formation derived from the periodontal ligament also contributes to the increased width of keratinized gingiva . Mucogingival junction regains its genetically defined position following its coronal dislocation with the coronally repositioned flap resulting in the increased gingival dimension . The increase in width of keratinized gingiva observed between 3 and 6 months has been attributed to creeping attachment . Therefore, this study suggests that in terms of operator factors, both the techniques yielded sufficient sect graft suitable for periodontal plastic procedure with minimal operative complication . However, unigraft knife technique created a slightly larger wound area at the palate as compared to the free - hand incisions made in langer and langer technique . The wound in the langer and langer trap door technique method healed slightly more quickly as compared to the unigraft knife method, thus might have accounted for less patient discomfort . During the study, it was observed that the use of unigraft knife was technique - sensitive and it was difficult to adapt the instrument in situations where the palatal vault was high and narrow . Because of only a few sizes of cutting shoes available for unigraft knife, its adaptation to a variety of anatomic forms of palate is limited . The langer and langer trapdoor technique was more user - friendly as the angles of the free - hand incisions could be easily adjusted if some anatomic variations were encountered.
Myasthenia gravis (mg), an autoimmune disease, impedes the postsynaptic acetylcholine receptors at the neuromuscular junction.1 ocular mg is a representative manifestation of this entity in which blepharoptosis and/or eye movement disturbances are often encountered.2 in general, up to 85% of all myasthenic patients show the anticholinesterase receptor antibody in serum, but only about 50% of patients demonstrate ocular muscle weakness.3 although cholinesterase inhibitors with or without steroids are commonly used as a treatment modality, they are, occasionally, less effective for ocular symptoms.4 in such a case, especially for ptosis without any eye movement disorders, ptosis surgery is often performed . This paper presents an ocular mg case with eyelid dysfunction, in which cholinesterase inhibitors and steroids did not work sufficiently, but surgical treatment successfully improved the symptom . A 39-year - old woman was diagnosed with mg when she was 8 years old . With a positive edrophonium test and detectable serum acetylcholine receptor antibody, the diagnosis of ocular mg was confirmed in addition to the unresponsiveness to treatment with pyridostigmine and steroids at 31 years of age . Despite long - term treatment with steroids, she had difficulty opening both eyes, which caused stiff shoulders and headaches . As she showed bilateral 3 mm levator function (figure 1a) without any eye movement disturbances, bilateral frontalis sling procedures were performed with an autologous fascia lata . The skin incision was made 6 mm from the eyelid margin, and the central area of the upper brow margin was also incised (figure 2a). A tunnel was made from the brow incision through the suborbicularis oculi layer, and reached the pretarsal area . The branched fascia lata was sutured with 60 nylon (sigma, tokyo, japan) on the tarsal plate and then the upper eyelid curvature was confirmed by pulling the fascia through the suborbicularis tunnel (figure 2b). After the upper eyelid height was adjusted appropriately with a trial suture at the brow incision, the fascia was fixed at the subcutaneous tissue of the brow . One year after the operation, the upper eyelids showed symmetrically appropriate heights (figure 1b). The patient did not demonstrate exposure keratitis, wound infection, lagophthalmos, or ptosis in the 6 months following the operation . The frontalis sling surgery for ptosis by ocular mg accomplished functionally and cosmetically good outcomes . As the levator function of the patient was bilaterally 3 mm, in general, ptosis with less than 4 mm levator function needs a sling procedure,5 but with more levator function, levator advancement surgery is applied.6 as an excessive advancement of levator often leads to an eyelid - eyeball dissociation, causing dry eyes, a sling procedure should be used in such a case . In the past, numerous materials have been used for slings like silicone rods, gore - tex, and autologous tissue graft.5,79 the autologous graft from fascia lata was chosen rather than the artificial material in terms of its histocompatibility . Although the patient did not show any eye movement disturbances, mg patients often show eye movement disturbances simultaneously with ptosis.10 bilateral ptosis surgery should be avoided in such a case to prevent postoperative diplopia . In this situation, many treatment modalities had been given to the patient, but her condition did not show definite improvement . Generally in mg, anticholinesterases (cholinesterase inhibitors) although steroids are of great short - term benefit in most patients with ocular mg, the side effects associated with steroids may prevent long - term use.10 therapeutic effect of thymectomy is controversial for ocular mg . Therefore, a patient refractory to any medical treatments is a good candidate for ptosis surgery . In conclusion, this paper reports an ocular mg case with eyelid dysfunction, in which cholinesterase inhibitors and steroids did not work sufficiently, but surgical treatment successfully improved the symptom . A sling procedure with an autologous fascia was suitable for correcting poor levator function of an ocular mg case.
Polycystic ovary syndrome (pcos) is a common endocrine problem, which is now recognized as not only a reproductive but also a metabolic disorder with long - term effects on women's health . Pcos is associated with hyperinsulinemia which is associated with infertility and metabolic problems of diabetes and dyslipidemia . However, few studies have looked at carotid intimo - medial wall thickness (cimt) as a reflection of vascular health . Epidemiological studies have demonstrated an association between an increase in the carotid intima - medial wall thickness (cimt) and cardiovascular dysfunction (cvd). Hence, carotid arterial ultrasound is an important tool that could be used to measure the thickness of the intima - media of the common carotid artery which would help us to further characterize the cardiovascular risk in the pcos population . There is not much evidence of study in this part of the state / india to demonstrate such a correlation . Careful carotid intimo - medial wall thickness measurement can help in early detection and management of cardiovascular disorders in patients with pcos . Therefore, the objective of this study is to find an association between cimt and patients with pcos, in an indian setting . The cross - sectional case - control study was conducted in a tertiary care hospital in south india . Fifty - four consecutive women with pcos presenting to the endocrinology outpatient department who signed an informed consent were enrolled into the study and 54 healthy women were also enrolled into the study . The normal healthy group of women consisted of attendants accompanying the patients and volunteers (most of them nursing staff). The inclusion criteria consisted of all subjects in the range of 1635 years and with or without pcos diagnosis as per the rotterdam diagnostic criteria, 2003 . Similarly, all women with nonclassical adrenal 21-hydroxylase deficiency, cushing's syndrome, thyroid dysfunction, hyperprolactinemia, androgen - secreting tumors, diabetes mellitus, patients on medication like a steroid, anti - convulsant and anti - psychotic and history of smoking were excluded from the study . All the information relating the cases and control were collected in predesigned and pretested pro forma . Carotid arterial doppler was performed which evaluates the blood flow through the carotid arteries using the principle of doppler effect to produce pictures of carotid arteries following which the intima and medial thickness of the same were measured in the test group . In the case and control group, intimo - medial thickness (imt) of the carotid artery were measured by a radiologist using a linear 810 mhz ultrasound probe (voluson, ge) in carotid setting, who was blinded about the study groups . In longitudinal view, the distance between the two - echogenic lines parallel to the vessel wall was measured (between the two echogenic lines, a hypoechoic area could be seen, the first echogenic line was the intima and the next one was the contact level of media and adventitia). The measurement was done at two points of common carotid artery (cca) on both the sides [figure 1]. In addition to above, information of age in completed years and height in centimeters up to 0.5 cm and weight in kg up to 0.250 kg . Carotid arterial doppler of the polycystic ovary syndrome subject showing the longitudinal section of the right common carotid artery . Cca: right common carotid artery statistical analysis was performed using spss software 18.0 version . Student's t - test and mann whitney u - test were used to compare the differences in the mean values for various parameters . The mann whitney u - test was employed in case if the data did not follow the normal distribution . . Data collected was stratified into the different groups based on mean cimt, mean age, and mean body mass index (bmi) levels as observed for the control / cases group . Univariate odds ratios along with 95% confidence interval (ci) were computed between the pcos and control group of subjects after segregating the data into two groups based on the above mean values . Further multivariate forward logistic regression was employed to control the effect of bmi and age for finding the odds ratios with regard to cimt in pcos and control group . Student's t - test and mann whitney u - test were used to compare the differences in the mean values for various parameters . The mann whitney u - test was employed in case if the data did not follow the normal distribution . Data collected was stratified into the different groups based on mean cimt, mean age, and mean body mass index (bmi) levels as observed for the control / cases group . Univariate odds ratios along with 95% confidence interval (ci) were computed between the pcos and control group of subjects after segregating the data into two groups based on the above mean values . Further multivariate forward logistic regression was employed to control the effect of bmi and age for finding the odds ratios with regard to cimt in pcos and control group . A total of 54 women with pcos and 54 control subjects underwent carotid ultrasonographic scanning . Mean baseline characteristics of subjects with pcos such as bmi and waist - hip ratio, were obtained [table 1]. Demographic characteristics of polycystic ovary syndrome group and control group the mean age of women with pcos and controls was 24.4 5.3 and 27.7 6.0, respectively, which was found to be statistically significant (p = 0.003). However, bmi was significantly higher (p = 0.001) in pcos compared to controls group (26.9 5.1 vs. 23.3 3.2). Mean carotid imt was significantly higher in pcos subjects than control subjects (0.51 0.07 vs. 0.44 0.06, p <0.001) [table 2]. The cimt range from 0.38 to 0.70 mm in women with pcos and from 0.30 to 0.60 mm in controls group . Carotid intimo - medial wall thickness in polycystic ovary syndrome group and control group on the basis of mean cimt value, data were stratified in two groups, i.e., 0.44 mm in one group and> 0.44 mm in another group . It was noted that 75.9% (41) of pcos cases had cimt> 0.44 mm as compared to only 44.4% (24) in controls group . The differences in proportion were found to be statistically significant (p = 0.001). The corresponding odds ratio was 3.94 (95% ci: 1.738.98) [table 3]. Distribution of cases and controls based on mean carotid intimo - medial wall thickness values since the age and bmi groups of pcos and controls were found to be statistically significant, mean cimt values were estimated for each of the sub - groups [table 4]. The findings indicated statistically significant cimt values for the categories of age 24 and bmi> 24 and age 25 and bmi> 24 . Meanstandard deviation of carotid intimo - medial wall thickness in cases and controls stratified according to age and body mass index categories further odds ratio along with 95% ci were also computed for stratified data [table 5]. Significant odds ratios were found for the categories of age 24 and bmi> 24 and age 25 and bmi> 24 . Odds ratio with 95% confidence interval for polycystic ovary syndrome and controls stratified according to age, body mass index and carotid intimo - medial wall thickness values through the multivariate forward logistic regression analysis attempt was made to control for the confounding effect of bmi and age with regards to cimt values . After controlling for the effect of age and bmi, odds ratio was found to be 3.98 (95% ci: 1.6129.811), which indicates the increased risk for cardiovascular disorders in patients with pcos even after controlling for the effect of confounding factors age and bmi . A woman with pcos encompasses a collective outcome of androgen excess, insulin resistance, and dyslipidemia, which are the strong indicators of cvd . This metabolic syndrome presents early in adolescence, which leads to the development of cvd . The assessment of preclinical vascular disease by noninvasive tests in middle - aged pcos patients with greater imt demonstrated that tendency to atherosclerosis increased in these patients compared with healthy controls . Previous studies suggested that women with pcos face double risk of the metabolic syndrome comparing the nonaffected population of the women in the society . In this study, we investigated cimt thickness in pcos women and control group . Studies of cvd events in women with pcos are limited, but a recent meta - analysis showed that women with pcos had twice the relative risk of coronary heart disease or stroke than controls (de groot et al ., 2011). Some studies discussed the association of pcos with the risk of cvd and its risk factors (legro, 2003; loverro, 2004; cussons et al ., 2006; dokras, 2008; mak and dokras, 2009; wild et al ., 2010). The sustained exposure of women with pcos to higher androgens was not connected with an overload of cvd or mortality even with the rise in cardiovascular risk factors . Another study carried out in taiwan did not show an association between cimt and pcos and the author surmised that might be it was too early to find the difference . Studies have also evaluated the fasting blood glucose quartiles with cimt and found that the correlation was good . In a retrospective study conducted in 2012 by meyer et al ., evaluation of the sum of 36 articles including 1,123 cases of pcos women and 923 healthy women showed that the imt artery in women with pcos was significantly higher than healthy women which was similar to our results that showed imt artery in pcos were significantly higher compared with controls . The study parameters in the two groups - age, height and bmi were significantly equivalent . Reports show that the bmi was higher in the test group than the control group . Fg score was applied in this study to evaluate and quantify hirsutism in women . The mean fg score in the test group was reported implicating that the women with pcos have hirsutism . Increase in total testosterone in longer period impacts in the body fat distribution with an accumulation of fat . The bmi, waist - hip ratio, fg score, and carotid intima - media thickness (cimt) in the test group and control group showed a significant variation akin to the previous studies . This explains the link between the study parameters, which act as potential risk factors for developing the pcos . Higher cimt values were observed in the test group compared to control group (p <0.001). Thus, it is important to measure cimt in women with pcos to predict the risk of cardiovascular disease (cvd). This simple noninvasive test can be done to manage these women with pcos to prevent endothelial function and preserve cardiovascular function . Some limitations of the present study that should be addressed and noted in future studies are included unmeasured variables such as physical activity, diet, socioeconomic backgrounds, lifestyle habits, and association with lipid status . Therefore, we are not able to differentiate the effect of diet, increased physical exercise and weight loss on the features of pcos and metabolic syndrome and different socioeconomic backgrounds or lifestyle habits in our study . Hence, a further study in larger populations with other associated parameters is necessary for better assessment of the results . The limitation of this study was a selection of a small sample size . Some limitations of the present study that should be addressed and noted in future studies are included unmeasured variables such as physical activity, diet, socioeconomic backgrounds, lifestyle habits, and association with lipid status . Therefore, we are not able to differentiate the effect of diet, increased physical exercise and weight loss on the features of pcos and metabolic syndrome and different socioeconomic backgrounds or lifestyle habits in our study . Hence, a further study in larger populations with other associated parameters is necessary for better assessment of the results.
Chronic low back pain is well known as a public health epidemic . In highly developed countries, it is one of the top three causes of degradation in quality - adjusted life - years, along with ischemic heart disease and chronic obstructive pulmonary disease.1 while lumbar spine pathology is an important cause of chronic low back pain, substantial evidence suggests that not all lower - back pain is in fact generated by lumbar spinal structures . The sacroiliac (si) joint has been found to be a pain generator in up to 30% of patients diagnosed with lower - back pain.25 disorders of the si joint may be the result of trauma, pregnancy, inflammatory arthritis, osteoarthritis, or degeneration of the joint either de novo or after lumbar spinal fusion.6,7 diagnosing the si joint as the primary pain generator can be complex, as patients often present with a combination of lower - back, groin, gluteal, and/or leg pain.2,8 furthermore, imaging studies are typically not sensitive to abnormalities in the absence of trauma, ankylosing spondylitis, tumors, or infection.7 si joint pain can be debilitating and treatment with conservative care is often unsuccessful . The economic burden of conservative care in this population is significant for medicare, as well as commercial payer entities, at an estimated 3-year cost of us$1.6 billion per 100,000 commercial covered lives, and 5 year estimated cost of $270 million for medicare beneficiaries.9,10 furthermore, the impact of pain on persons living with the disease is similar to that associated with other prominent orthopedic conditions routinely treated surgically.11 open arthrodesis of the si joint was commonly performed throughout the 1900s.12,13 however, this technique is less common now, as it requires a relatively large incision, significant bone harvesting, and lengthy hospital stay; moreover, patients must avoid weight - bearing for a prolonged period (up to several months) postoperatively.14 a recent study comparing open and minimally invasive surgical (mis) techniques for si joint fusion demonstrated more favorable outcomes in the mis cohort with respect to patient - reported outcomes, operative time, hospital stay, and rate of reoperation.15 herein, we report a patient - level meta - analysis of safety and effectiveness outcomes from a multicenter retrospective study of patients treated with mis si joint fusion using a series of triangular titanium, porous titanium plasma spray - coated implants (ifuse implant system; si - bone, inc ., san jose, ca, usa). Consecutive patients who underwent mis si joint - fusion surgery at six sites were identified . Data extracted from medical charts included demographic information, medical history (including history of prior lumbar spinal fusion), length of hospital stay, surgical operating time, estimated blood loss, complications of surgery, si joint pain measured on a 010 visual analog scale (vas), and satisfaction with surgery . Pain experienced in the lumbar region can arise from various anatomical structures and pathophysiological functions.16 portions of the sacral plexus from s1 and s2 innervate the si joint on the dorsal side, and segments from l3 and s2 innervate the ventral side, resulting in possible dermatomal pain patterns anywhere from l2 to s4.17 therefore, differential diagnosis in this complex population is essential . A detailed clinical history coupled with a positive result on three or more physical provocation maneuvers, such as gaenslen s, flexion abduction external rotation, compression, distraction, and thigh thrust, were used as criteria for further testing of the si joint.18 diagnostic imaging studies, such as x - ray, computed tomography (ct) and magnetic resonance imaging (mri) were performed to assess pathology in the lumbopelvic hip complex . When clinical, physical, and imaging findings were concordant, image - guided diagnostic injections of the si joint were performed as a final step in diagnosing the si joint as the primary pain generator.18,19 a positive result was defined as a 75% reduction in pain immediately following injection of local anesthetic . All patients in this study failed a 6-month course of nonsurgical treatment consisting of a combination of medication optimization, activity modification, physical therapy, and si joint injections . Minimally invasive si joint surgery was performed on all patients using a series of triangular, titanium implants (ifuse implant system) (figure 1). The implants are coated with a porous titanium plasma spray, an osteoconductive substrate that has been routinely used in total joint prostheses for decades to accommodate biologic fixation.20 a radiolucent table was used to facilitate the use of intraoperative fluoroscopy . After general endotracheal anesthesia was administered, the patient was turned prone and prepped in the normal sterile fashion . A lateral incision (3 cm) was made into the gluteal region, positioned over the first sacral body as viewed on a lateral fluoroscopic image . A steinmann pin was passed through the ilium across the si joint to the center of the sacrum (lateral to the neural foramen). After a soft - tissue protector was passed over the pin, a hand drill was used to create a pathway through the ilium, across the si joint, and into the sacrum . Finally, a triangular broach was used to further decorticate the bone and prepare a triangular channel to receive the first implant . Using a pin - guidance system, the most cephalad implant was seated within the sacral ala above the first neural foramen . The second implant was located above or adjacent to the s1 foramen, and the third between the s1 and s2 foramen (figures 2 and 3). Postoperatively, patients were instructed to ambulate with partial weight - bearing using the assistance of a walker . A variable program of gradual return to full weight - bearing was employed based on local practices and patient needs . Baseline demographic variables were summarized, where appropriate, with means, standard deviation, confidence intervals (cis), and frequency tables . Changes in vas pain scores were evaluated across sites using mixed models that accounted for each subject s age, sex, history of prior lumbar fusion, and baseline pain score, and included study site as a random effect . Random - effects models assume that even after controlling for known covariates, outcomes are clustered within sites; models assume that the underlying effect at each site is a random variable rather than a fixed value . Similarly, random - effects logistic regression was used to summarize proportions across sites, controlling for age, sex, and history of prior lumbar fusion . Subgroup analysis was performed similarly, with predefined subgroups: prior lumbar fusion (yes versus no), age greater than or less than 65 years, and sex . When there was significant variation in a baseline characteristic by site, univariate random - effects models taking into account site only were used to report mean values (or proportions) and confidence limits . All analyses were performed using r software.21 clinical improvement was defined using well - accepted values for minimum clinically important difference (mcid) and substantial clinical benefit (scb) available in the literature . Mcid is defined as a change of> 2.0 points, and scb is defined as a 2.5-point decrease or raw score of <3.5.22,23 pain experienced in the lumbar region can arise from various anatomical structures and pathophysiological functions.16 portions of the sacral plexus from s1 and s2 innervate the si joint on the dorsal side, and segments from l3 and s2 innervate the ventral side, resulting in possible dermatomal pain patterns anywhere from l2 to s4.17 therefore, differential diagnosis in this complex population is essential . A detailed clinical history coupled with a positive result on three or more physical provocation maneuvers, such as gaenslen s, flexion abduction external rotation, compression, distraction, and thigh thrust, were used as criteria for further testing of the si joint.18 diagnostic imaging studies, such as x - ray, computed tomography (ct) and magnetic resonance imaging (mri) were performed to assess pathology in the lumbopelvic hip complex . When clinical, physical, and imaging findings were concordant, image - guided diagnostic injections of the si joint were performed as a final step in diagnosing the si joint as the primary pain generator.18,19 a positive result was defined as a 75% reduction in pain immediately following injection of local anesthetic . All patients in this study failed a 6-month course of nonsurgical treatment consisting of a combination of medication optimization, activity modification, physical therapy, and si joint injections . Minimally invasive si joint surgery was performed on all patients using a series of triangular, titanium implants (ifuse implant system) (figure 1). The implants are coated with a porous titanium plasma spray, an osteoconductive substrate that has been routinely used in total joint prostheses for decades to accommodate biologic fixation.20 a radiolucent table was used to facilitate the use of intraoperative fluoroscopy . After general endotracheal anesthesia was administered, the patient was turned prone and prepped in the normal sterile fashion . A lateral incision (3 cm) was made into the gluteal region, positioned over the first sacral body as viewed on a lateral fluoroscopic image . A steinmann pin was passed through the ilium across the si joint to the center of the sacrum (lateral to the neural foramen). After a soft - tissue protector was passed over the pin, a hand drill was used to create a pathway through the ilium, across the si joint, and into the sacrum . Finally, a triangular broach was used to further decorticate the bone and prepare a triangular channel to receive the first implant . Using a pin - guidance system, a total of three implants were placed in the majority of patients . The most cephalad implant was seated within the sacral ala above the first neural foramen . The second implant was located above or adjacent to the s1 foramen, and the third between the s1 and s2 foramen (figures 2 and 3). Postoperatively, patients were instructed to ambulate with partial weight - bearing using the assistance of a walker . A variable program of gradual return to full weight - bearing was employed based on local practices and patient needs . Baseline demographic variables were summarized, where appropriate, with means, standard deviation, confidence intervals (cis), and frequency tables . Changes in vas pain scores were evaluated across sites using mixed models that accounted for each subject s age, sex, history of prior lumbar fusion, and baseline pain score, and included study site as a random effect . Random - effects models assume that even after controlling for known covariates, outcomes are clustered within sites; models assume that the underlying effect at each site is a random variable rather than a fixed value . Similarly, random - effects logistic regression was used to summarize proportions across sites, controlling for age, sex, and history of prior lumbar fusion . Subgroup analysis was performed similarly, with predefined subgroups: prior lumbar fusion (yes versus no), age greater than or less than 65 years, and sex . When there was significant variation in a baseline characteristic by site, univariate random - effects models taking into account site only were used to report mean values (or proportions) and confidence limits . All analyses were performed using r software.21 clinical improvement was defined using well - accepted values for minimum clinically important difference (mcid) and substantial clinical benefit (scb) available in the literature . Mcid is defined as a change of> 2.0 points, and scb is defined as a 2.5-point decrease or raw score of <3.5.22,23 the mean follow - up time was 16 months (range 1226) (table 1). The majority (71%) of patients at all sites were female, and 62% of patients had a history of prior lumbar spinal fusion . Mean patient age varied across sites; the random - effects estimate was 57.7 years (95% ci 53.062.4, range 3089), and 35% were over the age of 65 years . The mean baseline pain score was 8.6 (95% ci 8.19.1) (table 2). At 1 year the mean vas (pain) score dropped to 2.7 (95% ci 1.83.5), representing an improvement of 6.1 points (95% ci 5.76.6). Substantial clinical benefit was achieved in 92% of patients (95% ci 84%96%) and mcid was achieved in 90% (95% ci 82%95%) of patients . When controlling for age, sex, prior lumbar fusion, and study site, the proportion of patients who reported being satisfied or somewhat satisfied at 12 months was 95.7% (95% ci 86.3%98.8%). Regression - coefficient modeling performed to assess the effect of age (> /<65 years), sex, and history of lumbar spinal fusion, was statistically significant (t>2) for sex only . Mean (95% ci) reduction in pain was 5.9 (6.6 to 5.2) for men and 6.2 (6.8 to 5.6) for women; there were no significant differences for age or history of prior lumbar fusion . While the majority of patients experienced significant improvement, a small percentage (15 patients, 10%), reported a change of 1 point or less on vas; nine improved by 1 point, and six had no change . Two of these patients were revisions; prior si joint fusion using percutaneous screws had failed . Both showed improvement on vas scores from 5 to 4, were satisfied with surgery, and indicated they would have the same surgery again for the same result . Operating time, available for 42 patients at three sites, averaged 73 minutes (95% ci 25.4118) (table 3). Estimated blood loss, available for all patients, was minimal: a mean of 31 cc (95% ci 2537) the mean length of hospital stay, available for 109 patients, was 0.8 days (95% ci 0.11.5). A total of 28 postoperative sequelae were reported, the most common were falls (3.5%), trochanteric bursitis (2.8%), facet pain (2.1%), and piriformis syndrome (2.1%) (table 4). Both patients reported complete relief of symptoms after undergoing subsequent mis si joint fusion on the contralateral side . One patient presented with symptoms of nerve - root impingement, confirmed on ct scan . The patient was returned to the operating room, the original implant was removed and replaced with a shorter implant, and the patient recovered without issue . A total of 28 postoperative sequelae were reported, the most common were falls (3.5%), trochanteric bursitis (2.8%), facet pain (2.1%), and piriformis syndrome (2.1%) (table 4). Both patients reported complete relief of symptoms after undergoing subsequent mis si joint fusion on the contralateral side . One patient presented with symptoms of nerve - root impingement, confirmed on ct scan . The patient was returned to the operating room, the original implant was removed and replaced with a shorter implant, and the patient recovered without issue . Intermediate - term (> 1 year) follow - up of a large number of patients who underwent mis si joint fusion using a series of triangular titanium implants showed high rates of pain relief and satisfaction and low rates of perioperative or intermediate - term complications . These findings are consistent with prior reports15,24,25 and a recently published prospective study.26 as with any surgical procedure, an accurate diagnosis is imperative if one is to achieve positive clinical outcomes . Initial clinical presentation in the si joint patient population can be misleading as several pathophysiologic conditions can present similarly . Structures in the lumbopelvic hip complex are interdependent, and kinematic changes in one area can affect surrounding structures.17 a history of sleep disturbance, pain on prolonged sitting, leg instability, and pain in the lower back, buttock, hip, and groin, as well as the si joint, are common . Furthermore, pain and degeneration of the si joint after lumbar spinal fusion is common, with up to 43% of these patients experiencing si joint pain and 75% showing radiographic changes.27,28 an accurate diagnosis requires a combination of history, physical examination maneuvers that stress the si joint, and image - guided intra - articular diagnostic injections . Multiple nonsurgical and surgical treatments for si joint disorders are available . When nonsurgical management fails to provide adequate relief of symptoms, surgical stabilization is an option . A publication summarizing various arthrodesis techniques (both open and mis) reports variable improvements in pain and function, with more invasive approaches reporting moderately high complications and nonunions.15 overall, mis techniques have a record of significant improvements in pain and function, but results vary with implant and patient selection . Similar to other reports, the majority of patients in the present cohort had a history of previous lumbar spinal fusion . It is unclear whether the degradation of the si joint in these patients was a result of adjacent segment disease or de novo degeneration . However, in contrast to other technique reports,29 clinical outcomes using the triangular implants used herein were not diminished in this patient population . Favorable outcomes in patients with prior lumbar spinal fusion underscore the necessity to suspect the si joint as a pain generator in patients with lower - back pain . The low success rate of spinal fusion combined with the high incidence of si joint disorders discovered in patients presenting with lower - back pain leads one to suspect whether the si joint is being overlooked as a pain generator in these patients.3 lower - back pain can obscure si joint disorders, and current imaging technology may not be sensitive in detecting inciting pathology . The type and number of postsurgical adverse events in our study was commensurate with other published studies using this device system.15,2426 the most commonly reported complications in the cohort reported herein were trochanteric bursitis and piriformis syndrome . These events are neither uncommon nor unexpected, and can be a result of altered gait pattern due to lower - back or hip pain, postoperative hip - abductor weakness, increased activity levels, and other trauma in the region . Miller et al conducted an analysis of complaints (adverse events) reported to the device manufacturer (si - bone, inc .) As part of an ongoing postmarket surveillance program.30 they reported a complication rate of 3.8% in 5,319 patients, and events included pain due to nerve impingement, hematoma at the operative site, iliac fracture, wound infection, device migration, and implant malposition . This retrospective chart review lacked patient - reported outcomes, such as the oswestry disability index and short form (sf-36) health survey, available in controlled trials . Radiological outcomes were not assessed; bony bridging cannot be reliably assessed on plain - film radiographs.6 furthermore, in the absence of symptoms requiring further imaging, the cost and radiation exposure of ct scanning precludes such imaging studies from being performed routinely . It was a large multicenter study with intermediate - term (greater than 1 year) outcomes . The patient - level meta - analysis provided a method of examining outcomes that accounted for differences in patient characteristics across sites, subsequently providing more accurate results . For patients with si joint pain recalcitrant to conservative treatment, minimally invasive surgical fusion of the si joint using a series of triangular porous titanium plasma spray - coated implants is a safe surgical option that provides significant symptom relief with a high degree of patient satisfaction.
The diet and lifestyle questionnaire of vip, including a semi - quantitative food frequency questionnaire (ffq), has been completed by almost all participants, with an average recruitment rate for period included in this study (19902008) of 59% within the target age - groups . In support of the population - based nature of the vip cohort are the nearly identical cancer incidence rates in the vip cohort and the population of vsterbotten (39), and the minimal socio - economic selection bias (40). In previous studies less than 1% (n=595) of the vip participants have been defined as sami, and of these the majority are non - reindeer - herding sami (12). From a total of 113,205 health examinations within the vip cohort (19922008), of which 26,491 were repeated measures 10 years apart, a final study population of 77,319 participants was defined, based on the following exclusion criteria: subjects with missing data for more than 10% of the items in the ffq and/or portion size (n=6,715); subjects lacking data for body mass index (bmi) or with bmi<10 kg / m (n=60); subjects with unrealistic food intake level (fil) values, defined as a ratio of total energy intake to estimated basal metabolic rate (41) in the lowest 5th percentile or the highest 2.5th percentile, determined separately for sex and ffq version (n=7,977); and subjects with repeated health surveys (n=21,134), in which the most recent sampling occasion was excluded from all analyses . In the present study, 3 versions of the vip ffq were used: the original, 84-item ffq (n=25,886), an older, nearly identical, 84-item ffq (n=4,083), and the most recent 65-item version, in which most foods are unchanged from the 84-item versions, some food groups have been deleted, and some similar food groups have been merged (n=47,350). All but the oldest version of the ffq have been validated against 24-hour recalls and/or biomarkers for b vitamins fatty acids and beta - carotene (4244). In all ffq versions, meal - time portion sizes were estimated with the aid of 4 colour photographs of a plate containing proportionally increasing amounts of food stuffs representing vegetables and main sources of carbohydrate (for example, rice or pasta) and protein (for example, meat or fish). Calculation of nutrient intakes from the ffq items and portion size estimations is described elsewhere (45). Food items were recalculated into grams / day by multiplying frequency with portion size and adjusting for validated sex- and age - specific intake levels, defined by repeated 24 hour dietary recalls (43). For boiled coffee, the original scale, occasions / day was used, to facilitate comparison with previous studies . Intakes were energy adjusted by the residual method (46). To define low - energy reporters within the data - set, a direct comparison of fil and physical activity level (pal) was used, which is a method appropriate for large sample sizes (47). Pal, available for 94.5% of the subjects, was estimated from 2 questions on general, work - related activity and leisure - time physical activity in exercise clothes (48). Cut - offs for definition of low - energy reporters (61.5% of subjects with pal - data) were calculated by the goldberg method, modified by black (47), and applied to the fil / pal ratio separately for ffq version and sex . A traditional sami diet score was constructed in a similar manner to the mediterranean diet score (1), by adding 1 point for each intake above or below the median intake of several dietary items characteristic or uncharacteristic of the traditional sami diet, respectively . The selection of these dietary items was based on historical references (79), interviews in which elderly sami were asked how they thought that their parents would have filled out the vip ffq in the 1930s1950s (10), and present - time descriptions, including comparisons of diet and lifestyle in present - day reindeer - herding sami, non - herding sami and non - sami in the vip cohort (1013). The score included 1 point for each intake above the median for red meat, fatty fish, total fat, berries and boiled coffee and 1 point for each intake below the median for vegetables, bread and fibre, all calculated separately for sex and ffq version, thus creating a range from 0 to 8 points for each subject . However, the sami participants with the 84-item ffq should overlap almost completely with the dataset from our previous study containing ethnicity data at an individual level (10). In order to provide some indication of the validity of the score, we therefore used that dataset to calculate median traditional sami diet scores for reindeer - herding sami, non - reindeer - herding sami and geographically matched non - sami . A sensitivity analysis was performed by testing models in which the selected traditional sami diet score items were excluded from the score one by one, yielding 8 models of 07 points . Heterogeneity between these reduced models and the original traditional sami diet score were tested by chi - square tests . Furthermore, an extended, 10-point traditional sami diet score was modelled, including intakes of blood dishes and liver / kidney, in the sub - sample of subjects with 84-item ffqs . Mortality end - points up to and including 31 december 2007, were identified by linking the vip database with the swedish national cause - of - death registry . Cancer mortality was defined as underlying cause of death, icd-9 codes 140208, or icd-10 codes c00c97 . Cvd mortality was defined as the main cause of death and/or underlying cause of death, icd-9 codes 390438, or icd-10 likely predictors of mortality, including age, body mass index (bmi, kg / m), current smoking (yes / no), education (lack of post - secondary, yes / no), sedentary lifestyle (no regular physical activity in exercise clothes, yes / no), and intake of alcohol (g / day), fat (g / day), saturated fat (g / day), and total energy (kcal / day), were examined for association with traditional sami diet score categories by kruskal wallis tests (tables i and ii). Baseline characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . Physical activity level was estimated from 2 questions on general, work - related activity and leisure - time physical activity in exercise clothes (48). Low outdoor activity was defined as at most 2 regular leisure time outdoor activities among the following: walking (1 times / day), biking (1 times / day), collecting berries / mushrooms (1 times / week), gardening (1 times / week) or shovelling snow (1 times / week) or fishing / hunting (1 times / month). Sex - specific hazard ratios (hr) for all - cause, cancer (including the most common cancer sites), and cvd mortality were calculated by cox regression . Though all lifestyle variables were significantly associated with traditional sami diet score categories in at least 1 sex, none met our criterion for a confounder: altering the hr for the traditional sami diet score by 10% or more when included in a bivariate model . To facilitate comparison with other studies, common risk factors were kept in the multivariate model 2, which thus included: age, bmi, current smoking, education, sedentary lifestyle, and intake of alcohol and total energy . Proportional hazard assumptions were confirmed by schoenfeld's test . With age categorised into 10-year age groups, all covariates fulfilled the criteria for the proportional hazard assumption in men, but in women the exposure variable, traditional sami diet score, did not, p shoenfeld's test=0.032 . This minor deviation from proportionality indicates an increased risk of unstable results in women, but probably does not otherwise affect our results materially . We have chosen to present results for both men and women, in order to provide as complete a description of the cohort as possible, but our interpretation of the results in women is conservative . The subgroup with low metabolic risk profile included subjects free from hypertension, diabetes, and obesity, whereas a high metabolic risk profile included hypertension and/or diabetes and/or obesity . Hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure 90 mm hg and/or use of medication to lower blood pressure . For classification as non - hypertensive, mmol / l and/or post - load glucose 12.2 mmol / l (measured in capillary plasma). For classification as non - diabetic, measurement of fasting or post - load glucose was required . The cohort was stratified according to pal above or below the median . In order to ensure that dietary changes due to disease did not affect the results, we did analysis with excluding all subjects with follow - up times shorter than 2 years . In addition we replaced the sedentary lifestyle variable in the multivariate model with pal or with a variable representing low physical activity outdoors, the latter encompassing questions on walking, biking, picking berries or mushrooms, gardening, shovelling snow, and hunting and fishing . All tests were 2-sided, and p - values <0.05 were considered statistically significant . The study protocol and data handling procedures were approved by the regional ethical review board of northern sweden (dnr 07 - 165 m). Evaluation by a specific ethical review board representing the sami society would also have been desirable, but such a board does not exist in sweden at present . All study subjects provided written informed consent, and the study was conducted in accordance with the declaration of helsinki . Our study cohort consists of participants from the vsterbotten intervention program (vip), in which health risk measures and anthropometric data are collected from residents of the county of vsterbotten turning 30 (years 19851996), 40, 50 and 60 years of age . The diet and lifestyle questionnaire of vip, including a semi - quantitative food frequency questionnaire (ffq), has been completed by almost all participants, with an average recruitment rate for period included in this study (19902008) of 59% within the target age - groups . In support of the population - based nature of the vip cohort are the nearly identical cancer incidence rates in the vip cohort and the population of vsterbotten (39), and the minimal socio - economic selection bias (40). In previous studies less than 1% (n=595) of the vip participants have been defined as sami, and of these the majority are non - reindeer - herding sami (12). From a total of 113,205 health examinations within the vip cohort (19922008), of which 26,491 were repeated measures 10 years apart, a final study population of 77,319 participants was defined, based on the following exclusion criteria: subjects with missing data for more than 10% of the items in the ffq and/or portion size (n=6,715); subjects lacking data for body mass index (bmi) or with bmi<10 kg / m (n=60); subjects with unrealistic food intake level (fil) values, defined as a ratio of total energy intake to estimated basal metabolic rate (41) in the lowest 5th percentile or the highest 2.5th percentile, determined separately for sex and ffq version (n=7,977); and subjects with repeated health surveys (n=21,134), in which the most recent sampling occasion was excluded from all analyses . In the present study, 3 versions of the vip ffq were used: the original, 84-item ffq (n=25,886), an older, nearly identical, 84-item ffq (n=4,083), and the most recent 65-item version, in which most foods are unchanged from the 84-item versions, some food groups have been deleted, and some similar food groups have been merged (n=47,350). All but the oldest version of the ffq have been validated against 24-hour recalls and/or biomarkers for b vitamins fatty acids and beta - carotene (4244). In all ffq versions, meal - time portion sizes were estimated with the aid of 4 colour photographs of a plate containing proportionally increasing amounts of food stuffs representing vegetables and main sources of carbohydrate (for example, rice or pasta) and protein (for example, meat or fish). Calculation of nutrient intakes from the ffq items and portion size estimations is described elsewhere (45). Food items were recalculated into grams / day by multiplying frequency with portion size and adjusting for validated sex- and age - specific intake levels, defined by repeated 24 hour dietary recalls (43). For boiled coffee, the original scale, occasions / day was used, to facilitate comparison with previous studies . To define low - energy reporters within the data - set, a direct comparison of fil and physical activity level (pal) was used, which is a method appropriate for large sample sizes (47). Pal, available for 94.5% of the subjects, was estimated from 2 questions on general, work - related activity and leisure - time physical activity in exercise clothes (48). Cut - offs for definition of low - energy reporters (61.5% of subjects with pal - data) were calculated by the goldberg method, modified by black (47), and applied to the fil / pal ratio separately for ffq version and sex . A traditional sami diet score was constructed in a similar manner to the mediterranean diet score (1), by adding 1 point for each intake above or below the median intake of several dietary items characteristic or uncharacteristic of the traditional sami diet, respectively . The selection of these dietary items was based on historical references (79), interviews in which elderly sami were asked how they thought that their parents would have filled out the vip ffq in the 1930s1950s (10), and present - time descriptions, including comparisons of diet and lifestyle in present - day reindeer - herding sami, non - herding sami and non - sami in the vip cohort (1013). The score included 1 point for each intake above the median for red meat, fatty fish, total fat, berries and boiled coffee and 1 point for each intake below the median for vegetables, bread and fibre, all calculated separately for sex and ffq version, thus creating a range from 0 to 8 points for each subject . However, the sami participants with the 84-item ffq should overlap almost completely with the dataset from our previous study containing ethnicity data at an individual level (10). In order to provide some indication of the validity of the score, we therefore used that dataset to calculate median traditional sami diet scores for reindeer - herding sami, non - reindeer - herding sami and geographically matched non - sami . A sensitivity analysis was performed by testing models in which the selected traditional sami diet score items were excluded from the score one by one, yielding 8 models of 07 points . Heterogeneity between these reduced models and the original traditional sami diet score were tested by chi - square tests . Furthermore, an extended, 10-point traditional sami diet score was modelled, including intakes of blood dishes and liver / kidney, in the sub - sample of subjects with 84-item ffqs . Mortality end - points up to and including 31 december 2007, were identified by linking the vip database with the swedish national cause - of - death registry . Cancer mortality was defined as underlying cause of death, icd-9 codes 140208, or icd-10 codes c00c97 . Cvd mortality was defined as the main cause of death and/or underlying cause of death, icd-9 codes 390438, or icd-10 codes i00i69 . Likely predictors of mortality, including age, body mass index (bmi, kg / m), current smoking (yes / no), education (lack of post - secondary, yes / no), sedentary lifestyle (no regular physical activity in exercise clothes, yes / no), and intake of alcohol (g / day), fat (g / day), saturated fat (g / day), and total energy (kcal / day), were examined for association with traditional sami diet score categories by kruskal wallis tests (tables i and ii). Baseline characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . Physical activity level was estimated from 2 questions on general, work - related activity and leisure - time physical activity in exercise clothes (48). Low outdoor activity was defined as at most 2 regular leisure time outdoor activities among the following: walking (1 times / day), biking (1 times / day), collecting berries / mushrooms (1 times / week), gardening (1 times / week) or shovelling snow (1 times / week) or fishing / hunting (1 times / month). Sex - specific hazard ratios (hr) for all - cause, cancer (including the most common cancer sites), and cvd mortality were calculated by cox regression . Though all lifestyle variables were significantly associated with traditional sami diet score categories in at least 1 sex, none met our criterion for a confounder: altering the hr for the traditional sami diet score by 10% or more when included in a bivariate model . To facilitate comparison with other studies, common risk factors were kept in the multivariate model 2, which thus included: age, bmi, current smoking, education, sedentary lifestyle, and intake of alcohol and total energy . Categorised into 10-year age groups, all covariates fulfilled the criteria for the proportional hazard assumption in men, but in women the exposure variable, traditional sami diet score, did not, p shoenfeld's test=0.032 . This minor deviation from proportionality indicates an increased risk of unstable results in women, but probably does not otherwise affect our results materially . We have chosen to present results for both men and women, in order to provide as complete a description of the cohort as possible, but our interpretation of the results in women is conservative . The subgroup with low metabolic risk profile included subjects free from hypertension, diabetes, and obesity, whereas a high metabolic risk profile included hypertension and/or diabetes and/or obesity . Hypertension was defined as systolic blood pressure 140 mm hg and/or diastolic blood pressure 90 mm hg and/or use of medication to lower blood pressure . For classification as non - hypertensive, mmol / l and/or post - load glucose 12.2 mmol / l (measured in capillary plasma). For classification as non - diabetic, measurement of fasting or post - load glucose was required . The cohort was stratified according to pal above or below the median . In order to ensure that dietary changes due to disease did not affect the results, we did analysis with excluding all subjects with follow - up times shorter than 2 years . In addition we replaced the sedentary lifestyle variable in the multivariate model with pal or with a variable representing low physical activity outdoors, the latter encompassing questions on walking, biking, picking berries or mushrooms, gardening, shovelling snow, and hunting and fishing . All tests were 2-sided, and p - values <0.05 were considered statistically significant . The study protocol and data handling procedures were approved by the regional ethical review board of northern sweden (dnr 07 - 165 m). Evaluation by a specific ethical review board representing the sami society would also have been desirable, but such a board does not exist in sweden at present . All study subjects provided written informed consent, and the study was conducted in accordance with the declaration of helsinki . Among the 77,319 subjects included in the study, a total of 2,383 deaths occurred, including 975 cancer related and 681 cvd related . Follow - up times ranged from 1 day to 19 years, with a median of 10 years . In the study population, 20.0% of men and 19.7% of women had high traditional sami diet scores (68 points). Table i shows baseline characteristics of the vsterbotten intervention program participants according to traditional sami diet score . A high traditional sami diet score was associated with lower age, education level and pal, and higher prevalence of smoking, sedentary lifestyle and low outdoor activity level . In men, table ii shows the intake levels of various food items, including those making up the traditional sami diet score, as well as total energy and ethanol intakes . The greatest variability was found for vegetables and bread, of which subjects with high traditional sami diet scores had reported intakes of at most half those of subjects with low traditional sami diet scores . Intakes of red meat and fatty fish also varied considerably, with higher intakes associated with higher traditional sami diet scores . Further, a high traditional sami diet score was associated with a lower total energy intake and a higher ethanol intake, especially in men . Baseline dietary characteristics of vsterbotten intervention program participants according to traditional sami diet score calculated separately for ffq version and sex . In table iii, hrs for all - cause, cancer and cvd mortality per 1-point increase in traditional sami diet score are shown . In the crude model 1, adjusted only for age, increasing traditional sami diet score was associated with an elevated all - cause mortality in both men and women [crude hr for men 1.07 (95% ci 1.041.10) p 0.001; crude hr for women 1.06 (95% ci 1.021.11) p 0.001]. In the multivariate model 2, adjusted for age, bmi, current smoking, education, sedentary lifestyle, and intake of alcohol and total energy, risk associations were attenuated and only statistically significant in men [multivariate hr for men 1.04 (95% ci 1.011.07), p=0.018; multivariate hr for women 1.03 (95% ci 0.991.07), p=0.130]. In the subgroup analyses, a statistically significantly elevated all - cause mortality risk was found only in men with a low metabolic risk profile, that is, free from hypertension, diabetes and obesity [multivariate hr 1.06 (ci=1.021.11) p=0.008], and in men with a low pal [multivariate hr 1.05 (ci=1.011.09) p=0.019]. Hazard ratios for all - cause, cancer and cardiovascular disease mortality by traditional sami diet score in participants of the vsterbotten intervention program cohort 19902008 based on intake of red meat, fatty fish, fat, berries, boiled coffee, vegetables, bread and fibre, which are intake variables characteristic of traditional sami culture (1013). Further adjusted for bmi, sedentary lifestyle, education, current smoking, intake of alcohol and total energy . Diabetes and/or hypertension and/or body mass index 30.00 . Physical activity level 1.6 (median). Estimated from 2 questionnaire items on work - related and leisure - time physical activity (48). Estimated from 2 questionnaire items on work - related and leisure - time physical activity (48). For cancer and cvd mortality, hrs were above 1 and statistically significant in both men and women in model 1 . However, associations were not statistically significant in the multivariate model 2, except for cvd mortality in men with a low metabolic risk profile [multivariate hr 1.10 (ci=1.011.20) p=0.023]. Risk associations were above or very close to 1, but not statistically significant, for the most common cancer sites: colorectum (127 deaths), pancreas (93 deaths), breast (80 deaths), prostate (60 deaths), and stomach (52 deaths) (data not shown), with the possible exception of respiratory tract cancer (122 deaths), in which an increased risk with increasing traditional sami diet score of borderline statistical significance was observed [multivariate hr 1.11 (ci = 1.001.24) p = 0.053]. In the subsample of 29,969 subjects with the 84-item ffq (992 male deaths, 673 female deaths), for whom liver / kidney and blood dishes were added to the traditional sami diet score (010 points), results were weaker (data not shown). Results were stronger in subjects who reported a more adequate total energy intake [multivariate hr for all - cause mortality in men 1.07 (ci=1.021.13) p=0.009; and women1.08 (ci=1.001.16) p=0.047, for all - cause mortality]. Excluding subjects with <2 years of follow - up, or replacing the sedentary lifestyle variable with pal or low outdoor physical activity in the multivariate model, had no material effect on the results for all - cause, cancer or cvd mortality, except a reduction in power due to more missing values (data not shown). In table iv, hrs for all - cause mortality for the individual dietary items included in the traditional sami diet score items are presented . For most of the positively defined traditional sami diet score items (1 point for intake exceptions were higher intake of red meat in women [multivariate hr 1.17 (ci=1.021.34) p=0.023] and higher intake of fat in men [multivariate hr 1.12 (ci=1.011.25) p=0.037]. In negatively defined traditional sami diet score items (1 point for intake <median), statistically significant risk associations were observed for low intake of vegetables [multivariate hr 1.14 (ci=1.021.27) p=0.016] and bread [multivariate hr 1.14 (ci=1.031.27) p=0.014] in men . Hazard ratios for all cause mortality according to the traditional sami dietary elements included in the traditional sami diet score hazard ratios were determined by cox regression analyses . Further adjusted for bmi, sedentary lifestyle, education, current smoking, intake of alcohol and total energy . Adjusted as in footnote 3, as well as for the remaining items in the traditional sami diet score . Inclusion in the traditional sami diet score tested in subjects with data on intake of blood dishes and liver / kidney (subjects with a more extensive version of the food frequency questionnaire). The results of the sensitivity analysis are shown in table v. the risk association for all - cause mortality was not materially affected by the removal of any 1 dietary item included in the score . These analyses were performed in men only because of the instability of the traditional sami diet score cox regression model in women, as noted in the materials and methods section . Sensitivity analysis for the traditional sami diet score in men, a comparison of all - cause mortality using 8 reduced models, in which the traditional sami diet score items were excluded from the score one by one hazard ratios were determined by cox regression analyses . Adjusted for age, bmi, sedentary lifestyle, education, current smoking, intake of alcohol and total energy . Tests for heterogeneity between the result presented and the result for the complete traditional sami diet score were performed by a chi - square test . Applying the traditional sami diet score on the data - set used in our previous study (10), yielded a median (1st3rd quartile) score of 6.0 (4.07.0) points in reindeer - herding sami, 4.5 (3.06.0) points in non - reindeer - herding sami, and 4.0 (3.05.0) points in geographically matched non - sami (p 0.001). In this largely non - sami, population - based cohort study of 77,319 men and women in northern sweden, with up to 19 years of follow up, higher traditional sami diet scores were associated with a weak increase in all - cause mortality in men . This increased risk may be equally attributed to cvd and cancer, since hrs above 1 were the general pattern for both endpoints, but it appeared to be particularly pronounced for cvd mortality in men with a low metabolic risk profile (free from diabetes, hypertension and obesity). In women, we found no stable risk associations for traditional sami diet score, though all hrs were 1 . Our findings, in a largely non - sami population, are generally in line with the evidence to date for the individual components of the traditional sami diet score . Although the score is high in some foods widely considered to be healthy, such as fatty fish (25) and berries (26), it is also rich in foods associated with negative health outcomes such as red meat for colorectal and other cancers (19), and lacking in foods associated with positive health outcomes such as vegetables for reduced cvd risk (20). Null associations were also found for fatty fish and berries . In a previous report from the same northern swedish population, no associations between reported fish consumption (lean and fatty fish combined) and risk of acute myocardial infarction were observed (49). In our previous interviews with elderly sami 5070 years ago, we found that intakes of both fatty fish and berries were likely much higher than the intakes of either sami or non - sami in vsterbotten today (10). Thus, the present - day diet in the population of vsterbotten may deviate from the traditional sami diet to such a degree that potential health effects cannot be revealed . Former studies have suggested that the slightly reduced cancer risk in sami populations might be due to traditional sami diets (30). However, not only does the present - day diet in vsterbotten deviate considerably from our definition of traditional sami diet, but it is also possible that our traditional sami diet score does not closely enough reflect the traditional sami diet, which is, in itself difficult to define . Knowledge of the diet prior to the 1700s is limited (7,9), and the centuries since then have been marked by considerable change for the sami population (10). Traditional sami diet, for example by distinguishing between different kinds of red meat, such as wild game and reindeer meat and commercial beef, or fish bought and fish caught in the wild, or wild berries and greens and cultivated ones . Such foods may differ not only in biochemical composition (26,50), but also in related social or behavioural elements not otherwise accounted for in the statistical analyses . Thus, the vip ffq is inevitably an imprecise tool for the construction of a score representing a traditional sami dietary pattern . The traditional sami diet score was not intended to reflect the diet of the sami today . However, we have previously observed differences in the diet of present - day reindeer - herding sami, non - reindeer - herding sami and non - sami groups using the vip ffq (10,12). Furthermore, traditional sami diet scores were high in reindeer - herding sami compared to the other groups in that dataset (10), suggesting some degree of validity for the score, even in the present day . In diet score methodology, overall effects of adherence to a certain dietary pattern are studied, allowing for a single measure encompassing all potential interactions among the dietary components included, but also preventing the detection of effects related to the specific components or interactions . Dietary patterns are thus considered more relevant for public health recommendations, but less relevant for understanding underlying mechanisms (51). Diet scores inevitably provide a rough estimate of diet . To preserve power and limit complexity, they are often restricted to relative, dichotomous, rather than absolute, multiple, cut - offs, and equal weighting of a limited number of dietary components comprising the score . However, as the success of the mediterranean diet illustrates, diet scores can be an important tool in the field of nutritional epidemiology . The intervention built into the vsterbotten intervention program, in which diabetes and hypertension diagnosed through the health survey are treated, may have diluted our results . The weaker risk associations in men with metabolic risk factors at baseline may reflect such an effect . Chance findings may have occurred due to multiple testing, and p - values should therefore be interpreted conservatively . There is also a substantial risk of residual confounding due to factors not, or not adequately, assessed by the vip questionnaire and health survey, such as smoking history, defined only as current smoker, yes / no, and socioeconomic status, represented by education level, in the present study . Physical activity, in particular physical activity patterns of the traditional sami lifestyle, is another important example of residual confounding . Much of the extensive physical activity, while not directly related to occupation, was conducted essentially by need and not as leisure - time or recreational activity (10). Furthermore, recreational physical activity in exercise clothes is a rough measure of physical activity . This question, and the work - related physical activity question, in the vip ffq are therefore probably inadequate for the assessment of physical activity as a confounder or effect modifier of the relationship between traditional sami diet score and mortality . However, replacing the sedentary lifestyle variable with a low outdoor physical activity variable did not materially affect the main findings . The main strengths of this study were the population - based cohort design, the long follow up (up to 19 years), and the large sample size, as well as the rather unique aspect of examining the general population from a minority perspective . Given the inherent limitations of the questionnaire used, and the difficulty defining a score reflecting traditional sami diet, this study should be considered exploratory, a first attempt to address this topic . Further study of cohorts with more detailed information on dietary and lifestyle items relevant for traditional sami culture is warranted . The authors have not received any funding or benefits from industry or elsewhere to conduct this study.
Metachromatic leukodystrophy (mld) (scholz's disease) is an autosomal recessive lysosomal storage disease caused by the lack of arylsulfatase a (asa). This enzyme is essential for the normal metabolism of sulfatides which are vital elements of the myelin sheath . In mld, sulfatides accrue in many organs comprising brain, peripheral nerves, kidneys, liver, and gallbladder . Sulfatide collects in white matter of the central nervous system and peripheral nerves and causes progressive demyelination and lethal neurological symptoms . Mld is diagnosed biochemically by finding low levels of arylsulfatase in peripheral white blood cells and urine . Mld is classified into three main clinical forms on the basis of the age of onset . The most common and lethal form is the late - infantile form, which begins before 4 years of age typically presenting between 12 and 18 months of age, and patients die by the end of the first decade . The juvenile form of mld comprises age onset between 4 and 16 years, whereas symptoms of adult mld begin after puberty . The patients usually present with signs and symptoms of peripheral neuropathy and alterations in intelligence, speech, and coordination . The disorder is progressive with gait disturbance, quadriplegia, decerebration, and mortality by the age of 6 months to 4 years . Magnetic resonance (mr) imaging findings of the brain, especially t2-weighted imaging findings in the form of symmetric t2 hyperintense signal in the periventricular white matter, have been commonly reported in previous reports . However, diffusion - weighted imaging (dwi) findings have been sparsely reported and we present mr imaging, especially dwi findings in a 12-month - old patient of mld . A 12-month - old male presented with regression of milestones and progressive spasticity . Mr imaging examination was performed and t2-weighted images revealed symmetrical hyperintensities, predominantly involving the deep white matter, corpus callosum with sparing of subcortical u - fibers [figure 1]. The tigroid and leopard skin patterns of demyelination, which imply sparing of the perivascular white matter, were evident in the periventricular white matter and centrum semiovale [figure 2]. The corpus callosum (genu and splenium) was also involved . Using the transverse single - shot echo planar diffusion - weighted mr imaging, diffusion mr images were acquired . On b = 1000 mm / s images (heavily dwis), hyperintensities were apparent in the deep white matter and corpus callosum with a signal intensity pattern the same as that of cytotoxic edema . On apparent diffusion coefficient (adc) maps, low levels of arylsulfatase in peripheral white blood cells and urine confirmed the diagnosis of mld . (a and b) bilateral symmetrical butterfly - shaped t2 hyperintensities of the periventricular and deep white matter with relative sparing of the subcortical u - fibers sagittal t2-weighted image showing tigroid pattern of due to hypointense linear bands against the background of hyperintense white matter the corresponding lesions showing restricted diffusion with high diffusion - weighted (a and b) and low apparent diffusion coefficient signal (c and d) symmetric confluent areas of t2 hyperintense signal in the periventricular white matter with sparing of the subcortical u - fibers and with no enhancement in postcontrast images are the most frequent reported mr imaging findings of mld . The tigroid and leopard skin patterns of demyelination are basically due to sparing of perivascular white matter and are visualized as dark spots or dark linear areas against a background of hyperintense white matter, giving the appearance of the skin of a leopard . The disease process also commonly affects the corpus callosum, internal capsule, and corticospinal tracts . The cerebellar white matter may also be affected and appears bright on t2-weighted images . In the advanced stage of mld, it has been reported that the diminished activity of asa leads to dysmyelination (failure of myelin breakdown and reutilization). The corresponding distribution of the lesions on t2-weighted image and dwi in the present case strongly implied that the lesions on dwi were directly linked to the disease process . Restricted diffusion (cytotoxic edema) was seen on the echo planar sequence as hyperintensities on heavily dwis (b = 1000 s / mm) and low adc values on adc maps [figure 3]. Sener reported a similar diffusion mr imaging pattern on echo planar diffusion images of a patient with mld with unchanged pattern on 6-month follow - up . Phillips et al . Also reported a similar pattern of restricted diffusion in patients of phenylketonuria which was hypothesized to be due to impaired myelination leading to reduced mobility of protons . Hence, in the present case of mld, the restricted diffusion pattern perhaps was consistent with some breakdown of the white matter secondary to impaired myelination, which results in restricted mobility of protons leading to a diffusion mr imaging pattern similar to that of cytotoxic edema . To conclude, we report diffusion mr imaging of mld done using the echo planar sequence which has been sparsely reported . More diffusion mr imaging studies of dysmyelinating disorders might improve our interpretation of the imaging features of these diseases.
Patients' active participation in their own care is known to increase motivation and adherence to prescriptions, give better treatment results, create greater satisfaction with received care, and reduce stress and anxiety . Patient participation is an important basis for nursing care and medical treatment and it is also a legal right in many western countries . Studies have established that patients consider participation to be both obvious and important [3, 4], but there are also findings showing the opposite and patients may prefer a passive recipient role [6, 7]. Knowledge of what may influence patients' participation is thus of great importance when it comes to meeting their expectations and demands . Previous research focusing on patient participation from a patient perspective has been performed primarily in medicine and is carried out by physicians [8, 9]. Research on patient participation in nursing care has defined participation in performing clinical or daily living skills . Patient participation has been explored in different situations, for example, discharge planning [1114] and bedside reporting in emergency care and has primarily focused on decision - making in treatment / care (e.g., [1720]). Although nursing theories emphasise participation (e.g.,) and studies have explored patient participation in different contexts and situations, there have not been congruence regarding definition, elements, and processes [8, 22, 23]. The lack of clarity is amplified by the use of several terms: patient / client / consumer / user involvement, collaboration, partnership, and influence [8, 17]. However, when the focus is on the patient perspective, the concept of patient participation is commonly used . . Found that the patient needs to have the intellectual ability to understand and choose between alternatives and make decisions about their own nursing care and the nurse must provide adequate and correct information . Tutton emphasized the significance of developing a relationship between nurse and patient and the importance of understanding the patient as well as gaining and retaining an emotional connection . According to sahlsten et al ., a nurse needs to use strategies including building close co - operation with the patient, getting to know the person, and reinforcing self - care capacity . Factors restricting participation were identified by wellard et al . : limited communication between nurses and patients, task - oriented nursing labour, and environmental constraints limiting patients' privacy . . Found nonparticipation; when patients lack an equal relationship, respect, and information . According to efraimsson et al ., nonparticipation, occurs when professionals are not attuned to the concerns of the patient and individual needs and when they literally silence or disregard the patient's wishes . . Found that a nurse can lack theoretical or practical knowledge required as well as an insight that patient participation requires deliberate and planned interaction between nurse and patient together with adjusted actions within every encounter . Larsson et al . Recently presented barriers for participation from a patient perspective: facing own inability, meeting lack of empathy, meeting a paternalistic attitude, and sensing structural barriers . While several studies have addressed patient participation, few accounts exist based on patients' descriptions of decisive incidents that influenced their participation in nursing care . Accordingly, there is a need to explore situations related to critical incidents that influence patient participation . The aim of this study was to identify incidents and nurses' behaviours that influence patients' participation in nursing care based on patients' experiences from inpatient somatic care . This study is part of a larger project regarding patient participation in nursing care from the perspective of both patient and nurse . A qualitative approach, using the critical incident technique (cit), was employed . The cit is a systematic, inductive, and flexible method where specific descriptions of human behaviour in defined situations are collected . The central concept in cit is a critical incident which is a maior event of great importance to the person involved . The incidents are mostly collected in semistructured face - to - face interviews, the most satisfactory data collection method in cit for insuring that all the necessary details are supplied . The informants are asked to provide descriptions of specific incidents, positive and/or negative, which they perceive as significant . Here, these descriptions were collected within the framework of the interview method in order to generate an adequate depth of response . It is usually sufficient to collect a total of 100 incidents for a qualitative analysis . The participants (n = 17) in this study were recruited from somatic inpatient care . The intention was to have a range of informants able to contribute their experience as patients . The informants were ambulatory patients from three internal medical wards with neither an explicit care philosophy emphasising patient participation, nor a focus on nurse - patient continuity . The wards were focused on (i) stroke, (ii) disorder of kidney and heart, and (iii) lung . All informants were able to communicate in swedish and had no physical or cognitive deficits hampering the ability to describe their experiences as patients . The interviewer assisted the patients to describe the specific incidents that have influenced their participation in nursing care . The interview guide consisted of the following questions: describe a positive significant incident which was successful for your participation in your own nursing care, and describe a negative significant incident where you felt nonparticipation . After the patient had identified an event, the following questions, earlier used by kemppainen, were asked: what were the circumstances leading to that event? The same wording in the questions was kept throughout all interviews, as recommended by flanagan . The informants were recruited from an internal medical clinic in a central hospital in west sweden . All the nurses on the selected wards were sent written information regarding aim and procedure . The nurses were asked to approach patients the day before an interview was scheduled and ask whether they were interested in participating in the study or not . The interviews were held in the patient's own room or adjacent to the wards in a place where there would be no interruption in order to provide a relaxed environment . Each interview was conducted in an open, friendly atmosphere by the main nurse researcher and lasted between 30 and 60 minutes . Each interview was audio - taped and transcribed verbatim by the main researcher (inga e. larsson). The data material was read repeatedly to obtain a sense of the whole . In the data reduction process, an incident, either negative or positive, was identified as critical if it was related to the aim of the study and based on a detailed and discernible narrative of a course of events with a distinct start and end . In the data material, in line with the cit tradition, the classification started with identification and extraction of the incidents . These were analysed, without consideration whether positive or negative, to find similarities . In the second step of analysis, different kinds of nurse behaviours were next identified and classified, followed by patients' responses of these behaviours . Early in the analysis, the number of nurse behaviours increased rapidly and the last three interviews resulted in no new behaviours . To increase credibility, the classifications were discussed by the researchers (inga e. larsson, monika j. m. sahlsten) as no coassessor was involved in the coding . In addition, two researchers (kerstin segesten, kaety a. e. plos) not earlier involved in the study examined the classifications including direct quotations . This final classification system consisted of two main areas and 16 nurse behaviours allocated to six patient responses . The incidents arise in everyday situations and illuminate both positive and negative turning points (table 1). The most frequently described incidents concern situations during medical ward round where the nurse provides no support for patient input, and examples are also given of no preparation ahead of the round . Other incidents concern situations during nursing ward round describing genuine interest and search for patients' experience and views but examples are also given of distance with limited support for patient input . Incidents also describe situations during information session where the nurse provides meaningful and sufficient information but there are also descriptions of missing, insufficient, or inadequate information . The incidents concerning nursing documentation include descriptions of no invitation to participate and examples are also given of no recording of the patients' views . Other incidents concern situations during drug administration where the nurse leaves it to the patient to decide about tablet dosage for pain treatment but there is also examples of when the nurse provides no tablet for sleeping problems as well as routinely interrupts pain treatment infusion with little or none consideration to the individual . The least described type of incidents is meal which include examples of opportunity to choose where and when as well as what to eat and how much . In the next step of analysis, positive incidents were identified as stimulating patient participation and the negative incidents as inhibiting . In table 2, an overview of these two main areas along with patients' responses to nurses' behaviours are provided . The nurses' behaviours are illuminated using direct quotations that illustrate the connection with the narratives . When nurses care about patients and show a genuine interest, they feel treated and accepted as a unique person . The informants emphasised the importance of not being seen solely as an illness or a bed number . Nurses showed that they were accessible: the nurse was there when i needed it . The nurse confirmed the patient by showing that she cared and wanted to get to know me . The fact that the nurse listened and asked questions was considered crucial: she really listened to me and understood my situation . She asked questions to get an overall picture of my condition and find out what i like and want . It helps me to understand my thoughts and how i can process different things . When nurses provide information adapted to the patient's needs, he / she is motivated to actively participate in own care . The nurse gave the necessary explanations: she made sure i got the information i wanted and needed . She explained what the illness meant and how it was all connected, for example, why i took this pill and was given that injection . I was given time to think and ask questions, so i know what it is all about . I was given brochures and books to read, which enabled me to form my own opinion and understand better how it is all connected . Then it was easier for us to talk about my illness and what was going to happen next . It was considered important that the nurse acts as a mediator of contacts: she helped me so that i got to talk with other patients about their experiences and the treatment i was going to begin on . The nurse also took me on a guided tour to say hello on the ward where i was going to be treated and see how it all works . The informants also emphasised the importance of the nurse giving tips about self - care: i was given tips about what to do to make it easier, how to take care of the bandage, give the injections at home, and take care of myself when it comes to food and exercise . When the nurse starts with and utilizes patients' own knowledge, they feel as an asset in their cooperation . The nurse discussed and made agreements: she always included me in discussions because she needs my knowledge, said i was an expert . The nurse also handed over responsibility: i have been allowed to decide on my pain treatment and i take the pills when i need them . That means i do not have to press the call button as soon as it hurts and then i can wait longer so that i do not get so drugged and constipated . When a nurse, who is expected to provide support, seems to view patients in an unreflected way, they feel alone, ignored, and let down . She must have thought that i could do that myself and was just trying to get out of it . You have to dare meet person to person . A nurse was non - supportive during the medical ward round: i tried to give my views during the round and didn't get any help from the nurse . She was silent and didn't dare back me up in front of the physician . They talked about me, but i wasn't asked a single question; i felt ignored and upset . It would have been better to have been backed up directly instead of her coming back afterwards and trying to put everything right . The way a nurse communicates can make patients feel depreciated . A nurse disparaged a patient with baby talk: the nurse talked to me like i was a child; that belittles me as a person and gives an impression of insincerity . A nurse made ironic remarks about an experience: i was told to point at a ruler and got the answer: my dear, you can't be in that much pain . If you were, you'd be both in a cold sweat and more affected . Now, you just think about it one more time . When a nurse seems to want to exercise control and does not attach any importance to patients' views, they feel ignored and unable to participate and exert an influence . When i said we should do like this instead, the nurse said: you don't understand this, what are you making a fuss for . A nurse answered curtly: i am inquisitive and the nurse only answered very briefly . I was constantly being told: we'll have to wait and see what the physician has to say . Surely, it is possible to answer one of my questions reasonably . Maybe she's not allowed to tell me, but she could at least tell me that . A nurse neglected making notes in records: she didn't write what i has asked to be written in my record . When so many people are involved in my care, what is written down is important and it is often wrong; that scares me . When i read the epicrisis of the nursing care plan, i saw that they had copied the old one . It would have been good if i had been allowed to take part in the planning and evaluated my care . This study, based on patients' experiences from inpatient somatic care, provided a picture of incidents, nurses' behaviours that stimulate or inhibit patients' participation and patient reactions on nurses' behaviours . A purposeful sample was used in order to obtain a varied picture of critical incidents of significance for patient participation . In this study, 17 inpatients provided a total of 105 critical incidents which, according to flanagan, may be sufficient for a meaningful analysis . The findings are based on these informants and their ability to describe experiences of patient participation in nursing care . Although a majority of these informants were able to name some of the registered nurses on his / her ward, it is not certain that they in fact were able to distinguish nursing from experiences with other care providers . The sample included informants with different experiences, which increases the possibility of shedding light on the researched question from a variety of perspectives . Various ages, diagnoses, wards, and cultural backgrounds contributed to a rich variation which, taken as a whole, can be regarded as a strength . Actions were taken to enhance credibility in data collection . At the end of each interview, the main conclusions were verbally summarised by the interviewer and the informant supplemented, verified, and further developed the content . When 14 interviews had been conducted, earlier data were replicated and nothing new was added . The interviews were conducted and transcribed verbatim in their entirety by the same person, which enhanced the trustworthiness of the data material collected . Credibility in the analysis was enhanced by continuously switching between the whole and the parts, and comparing and revising until a final classification emerged from the data material . Rigor was ensured by systematically handling the data, repeatedly reading, identifying, and reflecting on the critical incidents . To increase credibility, two of the authors (inga e. larsson and monika j. m. sahlsten) discussed the classifications including direct quotations in order to reduce bias which is recommended by flanagan . Finally, two researchers (kerstin segesten and kaety a. e. plos) not previously involved in the study reviewed and commented on the classifications, which included citations . This study is based exclusively on the patients' experiences . To provide a more complete picture, a future study may include observations of interactions between nurses, physicians, and patients but also interviews afterward to get their perspectives on why they behaved the way they did . Many factors influence each interaction, and asking why could provide more insight and knowledge . Only inpatient somatic care has been highlighted and, obviously, other patients and settings need to be explored . Medical ward rounds still seem to be an incident not conducted in a democratic fashion . States that the rounds serves as a central marketplace for information where the main topic for physicians and nurses is medical information . The patients are only asked in order to reach agreement on decision - making or checking outcomes of treatment . Nursing documentation seems also to be an incident where patients have limited opportunities to exercise influence . The hierarchical nursing classification system carried out in detail may mainly serve organisational and administrative purposes and therefore disregard the patients . The goal has been to record work done by nurses and to provide evidence for performed interventions . Accordingly, nursing documentation is regarded as a matter for nurses and the fact that patients also have views on its content seems to have been noticed earlier in only two studies of patient participation [4, 28]. Drug administration appears to be an incident where ward policies and protocols seem to be emphasised rather than an individual's comfort needs . Pain is an individual experience where patient participation is of uttermost importance for the recovering . The most basic nursing care situations such as participation in daily living skills are not described with the exception of meals, indicating that it may be obvious and/or of minor importance . The findings reveal that stimulating patients' participation occurred when nurses treated the patient as a valuable coworker . This emphasises the importance of a person - centred care and of achieving a genuine connection and trusting companionship, in line with tutton and sahlsten et al . . Each patient's own capacity needs to be reinforced in order to optimise participation where patient and nurse share control and responsibility . To achieve this balance, a nurse ought to develop a personal, ordinary, and spontaneous approach in nursing practice . Our findings highlight that if patients are to feel regarded as unique persons, it is crucial to break free from preconceptions and assumptions of what their needs are and enter into each patient's world . Patients need to feel that the nurse understands their situation and unique prerequisites, which is a starting point for being actively involved in one's own nursing care . According to the informants, it is important to become motivated and engaged through information . It might be helpful to think of the patient as using and trying to implement evidence - based practice, as pointed to by edwards . Patients need to find acceptable interpretations of what is happening to them, which is essential for participation . Patients collect information and take action according to their own assessment of credibility and trustworthiness of information given . If different nurses appear to provide contradictory information or opinions, the patient could be confused as it means that the starting point for coping and action strategies keeps changing . Consequently, information needs to be adequate, individually adjusted, coordinated, and univocal . To meet the patients' needs, nurses have to use pedagogical strategies that promote learning such as focusing on the patient's process of reflection . This implies in - depth questions to induce patients to be self - reflective in order to utilise their own full potential in line with sahlsten et al . . The findings suggest that a patient, who is acknowledged as competent, presupposes stimulation and encouragement as a successful doer and owner of knowledge, in line with hughes and tutton . Patients' desire to do as much as they can by themselves may be seen as a basic human characteristic . Consequently, it is only the patient who can decide what is in his / her own best interest and nurses are then engaged in supporting . If possibilities to choose and make decisions are maximised, this may result in increased motivation to take responsibility, and exert influence and control [36, 39]. According to the informants, this leads to a sense of independence, which increases well - being, but a nurse then needs to relinquish some control, rather than exerting it . The findings reveal that inhibiting patients' participation occurred when nurses treated patients so they felt neglected and as a helpless object of a nurse's actions . This seems to indicate that a person - centred approach is devalued in favour of a task - centred one . A nurse might have a limited understanding of professional nursing care and focus on tasks, which could result in the patient easily becoming a passive object . When patients perceive themselves as being abandoned without backup, this indicates that nurses may use a protective mechanism to screen off emotional or advocacy aspects of their work . This may be due to working under time pressure or an idea that connecting with the patient is risky in a professional relationship . Nurses may need both practical and personal support to reduce a use of blocking behaviours to be able to work in a more responsive and effective way . In order to continuously develop self - awareness and critical monitoring skills, this may increase nurses' ability to reflect and develop their behaviour in patient encounters . To provide sufficient support during medical ward rounds was surprisingly an expectation on nurses by all informants . In order to optimise patient participation, nurses need courage to back up patients to reach self - advocacy and also to be sufficiently confident to question procedures, which are to the patient's disadvantage . However, nurses can see themselves as, and acts as, an intermediary with the physician . Patients rarely get explanations or are encouraged to ask questions, an outdated routine that does not satisfy the demands of patients today . If medical ward rounds should continue in its present shape, patients need information regarding its actual aim, which seems to be to, as physician, get a face on the patient for whom the care planning is done . When patients feel belittled verbally, a nurse may exercise the power of language or behave as a parent figure, also pointed to by hewison . The nurse disparages the patient in order to be in charge and sets the parameters for what is acceptable . Mccabe claims that professional nurses need to be aware of the impact the way they choose to communicate has on their patients . Communication is a powerful tool that mediates ideas, attitudes, and information, but it can also reinforce nurses' authority and hinder or exclude patients so they become increasingly dependent according to kettunen et al . Or result in reluctance . Being ignored without influence mirrors nonrespect and no recognition of patients' requests and their right to participate . By recording the patients' views, things they regard as important will be revealed and made visible, also pointed to by krkkinen and eriksson . This presupposes that the recorder knows the patient, which perhaps was not the case here . When a nurse neglects the importance of written documentation, the informants here felt that they were exposed to risks . Records can be used as working documents for both parties which may improve the content . We recommend that a nurse provide a notebook and encourage patients to keep their own notes . This could support them to remember, prepare for meetings for example, rounds, and ask questions . It can also help patients to participate and take a higher degree of control in their own care . When nurses have a bossy or patronising attitude, this reflects a belief that it is the nurse who knows best what is in the patient's interest . The level of control that nurses themselves have over their practice has been shown to affect the level of active patient participation . If nurses perceive themselves as diminished and not seen, they may repress patients . Empowering the patient this study, based on patients' experiences from inpatient somatic care provided a picture of incidents, nurses' behaviours that stimulate or inhibit patients' participation and patient reactions on nurses' behaviours . In order to promote patient participation, nurses need to be aware of the situations where they could overstep the mark and which of their own behaviours lead to promotion or hindrance . Our findings suggest that there is scope for developing nurses' behaviours in order to activate patients in their own nursing care . The findings may increase understanding of patient participation in nursing practise, education, policymaking, and evaluation . Further verification of the findings is recommended, either by means of replication or other studies in different settings.
Triamcinolone acetonide (ta) injections are often used to treat tendinopathy, but it has substantial adverse effects, such as impaired function of supraspinatus tendon cells and high rate of re - rupture . Previous studies also reported the deleterious effects of ta on histological and biomechanical properties of the injured tendons . Local anesthetics are used for adjunctive pain control in combination with corticosteroids injection in tendinopathies . However, recent studies suggest that local anesthetics of amide derivatives, such as bupivacaine and lidocaine, have deleterious effects on various cell types [611]. It was shown that the cytotoxicity of bupivacaine was dose - dependent in chondrocytes in vitro . A more recent study showed that lidocaine could significantly decrease positive effects of platelet - rich plasma on human tendon cells . While local anesthetics are commonly given with corticosteroids injection in treating tendinopathies, the combined effect of steroids and local anesthetics on tenocytes is largely unknown . Based on these studies, we hypothesized that local anesthetics might potentiate the deleterious effects of ta on tenocytes . The aim of this study was to determine the effects of lidocaine and ta on cultured rat tenocytes and whether there is a synergistic effect . Animal experiments were carried out under the rules and regulations of the animal care and use committee at harbin medical university . Sprague - dawley male rats (weighing 200250 mg) were used for isolation of tenocytes . Each patellar tendon was cut into small sections in aseptic conditions, and then placed and cultured in 6-well culture plates . After 5 min of air - drying for better adherence, dulbecco s modified eagle medium (dmem), 10% fetal bovine serum (fbs), and 1% penicillin and streptomycin were supplemented to each well . The explants were incubated at 37c in a humidified culture chamber supplemented with 5% co2 . Cells began to emerge from the tissue pieces after 34 days in culture and began to attach to the surface of the culture dishes . After the cells reached 80% confluence, they were passaged by washing with phosphate - buffered saline (pbs) and detached with trypsin / ethylene deaminate triacetic acid (edta). After 24 h of culture, attached tenocytes were treated with ta in culture media (regular medium with 0.1 mg / ml of ta). Samples were then washed with pbs solution, and returned to regular culture media with or without ta at 37c in a humidified 5% co2 incubator . There were 4 groups, including the control group, ta group, lidocaine group, and ta + lidocaine group . Each group was cultured in triplicate, and the culture medium was changed every 3 days . Cell viability, cell morphology, and expression of tenocyte - related genes were assayed at day 7 . Cell viability was determined using the cell - counting kit-8 (cck-8) (dojindo, kumamoto, japan) according to the manufacturer s instructions . Tenocytes were seeded in 96-well plates at a density of 510 cells per well for 24 h. cells were exposed to 1% lidocaine for 15 min, followed by removal of each medium and returning to regular culture media with or without ta . Then, tenocytes were allowed to recover in regular medium for 24 h prior to assessing their viability . For the cell viability assay, each well was supplemented with 10 l cck-8 and incubated at 37c for 2 h. the optical density was spectrophotometrically measured at 450 nm . Scleraxis (scx) and tenomodulin (tnmd) are closely related to tendon regeneration . It positively regulates tendon - associated genes such as collagen i and tnmd expression in tendon fibroblasts . As a later differentiation marker of tendon cells, therefore, collagen i, tnmd, and scx were evaluated using rt - pcr in this study . Total rna was extracted using the rneasy mini kit (qiagen, hilden, germany) according to manufacturer s instructions . Cdna was synthesized using the first - strand kit (invitrogen, carlsbad, california). The qrt - pcr was carried out with quantitect sybr green rt - pcr kit (qiagen, hilden, germany). We amplified 2 l total cdna of each sample in a final volume of 50 l reaction mixture . The cycling conditions were: held at 65c for 5 min, snap cooling at 4c for 1 min, 42c for 50 min, and at 72c for 15 min . Rat - specific primers were used for collagen i, tnmd, scx, and gapdh as follows: 5-ccggactgtgaggttaggat-3 (forward) and 5-aacccaaaggacccaaatac-3 (reverse) for collagen i; 5-ccatgctggatgagagaggttac-3 (forward) and 5cacagaccctgcggcagta-3 (reverse) for tnmd; 5-aacacggccttcactgcgctg-3 (forward) and 5-cagtagcacgttgcccaggtg-3 (reverse) for scx; 5-tgactctacccacggcaagttcaa-3 (forward) and 5-acgacatactcagcaccagcatca-3 (reverse) for gapdh . One - way anova with the student - newman - kuels test was used for multiple comparisons . Animal experiments were carried out under the rules and regulations of the animal care and use committee at harbin medical university . Sprague - dawley male rats (weighing 200250 mg) were used for isolation of tenocytes . Each patellar tendon was cut into small sections in aseptic conditions, and then placed and cultured in 6-well culture plates . After 5 min of air - drying for better adherence, dulbecco s modified eagle medium (dmem), 10% fetal bovine serum (fbs), and 1% penicillin and streptomycin were supplemented to each well . The explants were incubated at 37c in a humidified culture chamber supplemented with 5% co2 . Cells began to emerge from the tissue pieces after 34 days in culture and began to attach to the surface of the culture dishes . After the cells reached 80% confluence, they were passaged by washing with phosphate - buffered saline (pbs) and detached with trypsin / ethylene deaminate triacetic acid (edta). Rat patellar tendon - derived tenocytes were plated in culture plates . After 24 h of culture, attached tenocytes were treated with ta in culture media (regular medium with 0.1 mg / ml of ta). Samples were then washed with pbs solution, and returned to regular culture media with or without ta at 37c in a humidified 5% co2 incubator . There were 4 groups, including the control group, ta group, lidocaine group, and ta + lidocaine group . Each group was cultured in triplicate, and the culture medium was changed every 3 days . Cell viability, cell morphology, and expression of tenocyte - related genes were assayed at day 7 . Cell viability was determined using the cell - counting kit-8 (cck-8) (dojindo, kumamoto, japan) according to the manufacturer s instructions . Tenocytes were seeded in 96-well plates at a density of 510 cells per well for 24 h. cells were exposed to 1% lidocaine for 15 min, followed by removal of each medium and returning to regular culture media with or without ta . Then, tenocytes were allowed to recover in regular medium for 24 h prior to assessing their viability . For the cell viability assay, each well was supplemented with 10 l cck-8 and incubated at 37c for 2 h. the optical density was spectrophotometrically measured at 450 nm . Scleraxis (scx) and tenomodulin (tnmd) are closely related to tendon regeneration . It positively regulates tendon - associated genes such as collagen i and tnmd expression in tendon fibroblasts . As a later differentiation marker of tendon cells, therefore, collagen i, tnmd, and scx were evaluated using rt - pcr in this study . Total rna was extracted using the rneasy mini kit (qiagen, hilden, germany) according to manufacturer s instructions . Cdna was synthesized using the first - strand kit (invitrogen, carlsbad, california). The qrt - pcr was carried out with quantitect sybr green rt - pcr kit (qiagen, hilden, germany). We amplified 2 l total cdna of each sample in a final volume of 50 l reaction mixture . The cycling conditions were: held at 65c for 5 min, snap cooling at 4c for 1 min, 42c for 50 min, and at 72c for 15 min . Rat - specific primers were used for collagen i, tnmd, scx, and gapdh as follows: 5-ccggactgtgaggttaggat-3 (forward) and 5-aacccaaaggacccaaatac-3 (reverse) for collagen i; 5-ccatgctggatgagagaggttac-3 (forward) and 5cacagaccctgcggcagta-3 (reverse) for tnmd; 5-aacacggccttcactgcgctg-3 (forward) and 5-cagtagcacgttgcccaggtg-3 (reverse) for scx; 5-tgactctacccacggcaagttcaa-3 (forward) and 5-acgacatactcagcaccagcatca-3 (reverse) for gapdh . One - way anova with the student - newman - kuels test was used for multiple comparisons . The cell density was lower for cells cultured with ta or lidocaine compared to control . The combination of ta and 1% lidocaine induce more obvious morphologic change (figure 1). When the tenocytes were exposed to ta or lidocaine, the viability of the tenocytes was lower than that of the control group . Moreover, we found that viability of the tenocytes cultured with ta and 1% lidocaine was significantly lower than with ta or lidocaine alone (figure 2). Rt - pcr analysis on the cultures exposed to ta and lidocaine was performed as above . We found that the expressions of tenocyte - related genes, collagen i and scx, were decreased by the treatment of ta or 1% lidocaine (figure 3), whereas no significant difference has been detected for the expression of tnmd . When the tenocytes were incubated with the combination of ta and lidocaine, the expressions of tenocyte - related genes were significantly decreased when compared with ta or 1% lidocaine administration alone . The cell density was lower for cells cultured with ta or lidocaine compared to control . The combination of ta and 1% lidocaine induce more obvious morphologic change (figure 1). When the tenocytes were exposed to ta or lidocaine, the viability of the tenocytes was lower than that of the control group . Moreover, we found that viability of the tenocytes cultured with ta and 1% lidocaine was significantly lower than with ta or lidocaine alone (figure 2). Rt - pcr analysis on the cultures exposed to ta and lidocaine was performed as above . We found that the expressions of tenocyte - related genes, collagen i and scx, were decreased by the treatment of ta or 1% lidocaine (figure 3), whereas no significant difference has been detected for the expression of tnmd . When the tenocytes were incubated with the combination of ta and lidocaine, the expressions of tenocyte - related genes were significantly decreased when compared with ta or 1% lidocaine administration alone . Current literature reports indicate that either ta or local anesthetics can have significant detrimental effects on tendon cells . This study shows that ta has cytotoxic effect on tenocytes in vitro, which is synergistically increased when used in conjunction with 1% lidocaine . Many studies have been performed to investigate the effects of glucocorticoids on cultured tendon cells . They showed that ta could decrease cell viability, induce apoptosis, block cell migration, and reduce collagen secretion of tendon cells . Tenocytes showed significantly lower mrna levels of collagen i and scx in the ta - treated group than in the control group . . These results might account for the poor healing in tendons after injection of ta . Ta is commonly used to treat a wide range of tendon tissue disorders; therefore, we selected it for use in our study . Several other investigators have studied the effects of local anesthetics on tendon cells in vitro and in vivo . Lehner et al . Discovered a severe, reactive oxygen species - mediated effect of bupivacaine on tendon cells . Moreover, functional damage of tendon tissue was also elicited by bupivacaine . Because local anesthetics are commonly used with corticosteroids injection, our study attempted to show the combined effect of lidocaine and ta on tenocytes . This study showed that lidocaine affects not only morphology, but also functional activities of tenocytes . We showed that the deleterious effects of ta were enhanced by the administration of lidocaine . Therefore, it we suggest that the combination of ta and lidocaine has greater adverse effects on tendon tissues than ta alone . These results are consistent with previous reports describing the toxicity of lidocaine in other cell types . Future in vivo studies are necessary to determine whether the combination of ta and lidocaine could induce the increased risk for tendon pathology . Ta injection in combination with lidocaine should be used with caution due to the deleterious effects . One limitation of our study is that the underlying molecular mechanisms that are responsible for the deleterious effects of ta and lidocaine in this study are yet to be determined and need to be elucidated in further investigations . Our findings indicate that ta affects the properties of tenocyte by blocking the migration, decreasing the cell viability, and inhibiting the functional activities of the tenocytes . This in vitro study provides information on the detrimental effects of these drugs on tendon tissues . The use of ta with lidocaine in humans should be done with caution given the outcomes of this study.
The development of cancer is a complex multistep process that requires the accumulation of mutations resulting in a cell acquiring the essential hallmarks of cancer: evasion of apoptosis, self - sufficiency in growth signals, insensitivity to antigrowth signals, invasive and metastatic abilities, limitless replicative potential, and sustained angiogenesis . Given that normal adult stem cells already exhibit limitless replicative potential, it is hypothesized that transformed stems cells may be the cells of origin for many cancers [2, 3]. In addition to replicative potential, long - lived stem cells have the opportunity to accumulate oncogenic mutations over years or decades from common mutagenic sources like inflammation, radiation, chemicals, or infection, unlike shorter - lived transit amplifying (ta) cells that rapidly proliferate and differentiate [4, 5]. Like healthy adult stem cells, transformed stem cells these ta cells would be capable of driving tumor formation and generating the heterogeneous combination of populations commonly seen in cancer [6, 7]. Transformed stem cells have been termed cancer stem cells (cscs), also known as cancer initiating cells, and are defined as the fraction of cells within a tumor that are long lived, possess the potential to proliferate indefinitely, and can generate all heterogeneous lineages of the original tumor in xenograft models [6, 8]. Cscs are expected to utilize characteristics commonly found in stem cell populations such as differential metabolic activity, specific signaling pathway activity, and regulation of cell cycling characteristics, albeit with aberrant regulation [7, 9] (table 1). Importantly, cscs that survive treatment could account for tumor recurrence as a result of reactivation of proliferation in surviving cscs . Traditional chemotherapy regimens target proliferating cells, potentially missing slower dividing cscs that must be eradicated to provide long - term disease - free survival . A better understanding of cscs is essential in understanding the biological and clinical consequences of existing regimens and designing new therapies to improve patient outcome . Current methods for isolation and study of cscs rely on cell surface markers found to be enriched in populations with stem cell - like properties . This technique was first used by bonnet and dick in 1997 when they demonstrated that only the cd34cd38 subset of cells were capable of initiating human acute myeloid leukemia (aml) in immune - compromised mouse models . Since the work of these two pioneers, csc populations have been identified in multiple epithelial cancers including the breast, prostate, pancreas, colon [1517], ovaries, and brain . One issue centers on uncertainty of the functional implications of csc markers and is best exemplified by the use of cd133 in the identification of colon cscs . Shortly after ricci - vitiani et al . (2007) demonstrated the use of the cd133 to identify cscs in colon tumor, shmelkov et al . Demonstrated that cd133 expression was not restricted to colon cscs, but that cd133 is expressed on differentiated colonic epithelium in both mice and humans [16, 20]. Reasons behind this contradiction of data still remain unclear, but kemper et al . Methodically evaluated the cd133 antibodies used by both groups and reached the conclusion that the ac133 epitope used by ricci - vitiani et al . Recognized a differentially expressed form of cd133 that is not recognized by the antibody used by shmelkov et al . . It appears that cd133 is expressed in all colon epithelium, while the ac133 epitope is specific for the csc phenotype . Furthermore, kemper et al . Were unable to determine the functional significance of the differentially expressed isoforms of cd133, highlighting another drawback to the use of markers to identify cscs . Very little is known about the function of many of the proposed csc markers, and even less is known about the functional implications they may have for the csc phenotype . At best, markers without functional implications must be viewed as only tools for stem cell enrichment, suggesting the need for a more functionally significant means of csc identification . Given the similarities between normal adult stem cells and cscs, aberrant regulation of self - renewal and quiescence is likely central to csc pathology [9, 10]. Targeting pathways that mediate stem cell quiescence is therefore an intriguing alternate method for csc identification and use in future therapy . The primary objectives of this paper are to place quiescent label - retaining studies in the context of what is currently known about adult stem cells and then review the existing evidence for quiescence in cancer stem cells . We will examine current evidence for the role of quiescence in csc resistance to conventional cancer therapy and recurrence . Finally, we will explore current knowledge of quiescence regulation and how these studies might be considered when developing csc future experiments to develop targeted therapies against cscs . Adult stem cells are critical for continued normal tissue homeostasis and response to wounding for many of the epithelial tissues of the body . Adult stem cells are characterized by their ability to self - renew indefinitely and produce progeny capable of differentiating and repopulating tissue specific lineages . Populations of adult stem cells have been identified in tissues throughout the body, including the skin [2325], mammary glands [26, 27], intestine [28, 29], prostate, brain, and the hematopoietic system [32, 33]. In tissues where cells are frequently lost to the environment, like those of the intestine and skin, new cells are continuously required to replenish those that are lost . To facilitate this constant need for new cells, some epithelial tissues are arranged hierarchically with slowly proliferating stem cells that asymmetrically divide to give rise to a new stem cell and a rapidly dividing transit amplifying (ta) cell . Transit amplifying cells proliferate quickly for a limited number of divisions, allowing for the high degree of cell turnover necessary to sustain adult tissues . Infrequent division or a quiescent nature is not definitive for adult stem cell but is suggested to be important for maintenance of many adult stem cell pools . Evidence suggests that quiescence may play an important role in protecting stem cells from exhausting their proliferative capacity, inhibiting differentiation, and limiting accumulation of mutations during frequent rounds of dna synthesis [3537]. Initial efforts to identify and study adult stem cells took advantage of the slow - cycling nature of stem cell populations in studies employing pulse / chase methodology [23, 28]. In these studies, tritiated thymidine (h - tdr) or 5-bromo-2-deoxy - uridine (brdu) was repeatedly administered to mice or cultured cells that were then followed by an extended period of chase time . During this chase period, rapidly proliferating ta cells divide the label between daughter cells, consequently diluting the label (figure 1). In contrast, slow - cycling stem cells undergo few divisions and retain detectable quantities of label for much longer periods of time . Demonstrated that label retaining cells (lrcs) were exclusively present in the bulge area of the mouse hair follicle . These cells were found to be relatively stem - like: primitive in cytoplasmic contents, structurally similar to other putative stem cell populations, and could be stimulated to proliferate . We utilized human skin xenografted onto immunodeficient mice to show that lrcs were present in an analogous bulge region of human skin delineated by keratin 15 (k15) expression . Cells present in the bulge region have been experimentally shown to be quiescent for up to 1 year, and based on the hair growth cycle of scalp skin can likely remain quiescent for up to 5 years . Using the k15 promoter to drive expression of egfp or lacz, k15 positive cells were found to differentiate into all major epithelial lineages of the mouse skin . We demonstrated that k15 + bulge cells from human skin can differentiate into epidermal, sebaceous, and hair follicle lineages in vitro . Array analysis of the lrc bulge population showed increased activation of smad and inhibitors of the wnt pathway, suggesting the ability for lrcs to organize their niche and communicate with neighboring mesenchymal and epithelial cells, an important characteristic for stem cell function . The work in our lab and others supports a model in which the bulge region of hair follicles represents the stem cell niche in skin . At the onset of the growth phase (anagen) hair follicle stem cells are activated and produce matrix ta cells that proliferate and differentiate into the seven different lineages found within the hair follicle . As matrix ta cells exhaust their proliferative potential they enter a state of destruction (catagen) leading to the loss of the majority of the hair follicle excluding the bulge . Catagen is followed by a period of rest (telogen) in which the bulge stem cells remain quiescent until activation into a new anagen stage . Although likely important for the maintenance of the stem cell pool, quiescence may not be a requirement for adult stem cells . Using a lacz construct under a conditional promoter for the stem cell - associated protein leucine - rich g protein - coupled receptor 5 (lgr-5), jaks et al . Demonstrated a distinct nonlabel retaining subpopulation of bulge cells that overlap with the cd34/k15 at telogen but not anagen . Lineage tracing techniques confirmed that lgr-5 cells actively cycled during normal homeostasis and had a multipotent phenotype . The authors of this paper suggest that the lgr-5 population of cells represents a cycling population of stem cells under normal conditions, whereas the label retaining cd34/k15 stem cells may represent a reserve population that is activated after tissue damage . As yet, a conclusive relationship between these two populations cannot be firmly established . Similar label - retaining methods have been used to study slow - cycling cells in other tissues, such as the small intestine and colon . Work conducted by potten and colleagues identified slow - cycling lrcs at the + 4 position at the base of the colon crypt . These crypt base cells were found to be maintained in a steady state of between four and six cells that go through division approximately once a week . Upon irradiation, these cells demonstrated increased antiapoptotic bcl-2 expression, decreased p53 expression and were highly activated and involved in clonogenic regeneration of the crypt . Detailed biochemical analysis of this population has been limited by the absence of reliable markers and methods capable of sufficiently isolating these cells . Two studies involving the putative stem cell - associated rna binding protein musashi-1 (msi-1) have both demonstrated colocalization of this protein with colon lrcs, but fell short of testing for clonogenicity of this population [44, 45]. Similar to stem cell populations in the skin, 1-integrin was found to be highly expressed in the lower half of the colonic crypt . When sorted via flow cytometry, 1-integrin showed enrichment for clonogenic cells; however, an exact colocalization pattern with lrcs was not evaluated, and therefore, the connection remains only speculative . From the evidence collected in these studies and others, a model has been suggested in which slow - cycling stem cells, found at the base of the crypt, undergo periodic division to give rise to ta cells . Transit amplifying cells low in the crypt undergo rapid division and slowly progress up the crypt, losing replicitative potential and differentiating as they increase in crypt height . These cells are ultimately lost to the environment [6, 43]. As within the hair follicle, there is convincing evidence for an lgr-5 nonlabel retaining population of colon stem cells additionally found at the base of the crypt . While the lrcs reside at the + 4 population, lgr-5 cells are observed as slender wedge - shaped cells at the + 2 position . Again, the exact relationship between the lrcs and the lgr-5 cells is yet to be fully explored, and more data into the lineage potential of both of these cell populations is needed to form a cohesive model . Since the early identification of colon and hair follicle slow - cycling stem cell populations, label - retaining techniques have been used to identify and validate putative stem cell populations in multiple epithelial tissues . In the mammary gland, three separate label - retaining populations have been identified and proposed as possible stem cells . In a study conducted by welm et al ., lrcs were found to comprise a subpopulation of stem cell antigen-1 positive (sca-1) cells . These sca-1 cells were found to be enriched for the ability to form outgrowths, leading the authors to speculate that the lrcs might represent the stem cell population contained within the sca-1 cells . In contrast to this study, shackleton et al . Identified a long - term label - retaining population enriched by the marker combination lincd29cd24 that was able to reconstitute a functional mammary gland from a single cell . The lincd29cd24 did not enrich for the sca-1 population, prompting other groups to suggest a stem cell hierarchy in which multiple layers of stem cells exist within the mammary gland . Using a slightly different methodology, pece used the lipophilic fluorescent dye pkh26 to identify a population of mammary label retaining cells . The use of the pkh26 allows for live sorting of lrcs, which is not possible using the nucleotide analogue brdu and ht - tdr that both require permeabilization of the cell membrane for antibody labeling . Live sorting of pkh26 lrcs demonstrated increased in vitro sphere formation efficiency and regeneration of cleared fat pads over non - lrcs . Pece was also able to conduct transcriptional analysis of the lrc population, from which he created a human normal mammary gland stem cell signature (hnmsc) consisting of the markers cd49f / dner / dll1 . Unfortunately, the exact relationship between the different populations identified by these three groups is not yet clear . In the brain, high doses of h - tdr kill all but one percent of proliferating subependymal . High dose therapeutics did not affect the capacity of quiescent cells to generate spheres in vitro or repopulate the proliferating population in vivo . The ability to survive and re - enter the cell cycle suggests a stem cell phenotype for these quiescent cells . Prostate slow - cycling lrcs located in the proximal ducts demonstrated high proliferative potential and the ability to reconstitute the prostate glandular structure in vitro . This ability singles them out as stem cells over more rapidly cycling ta cells located at the distal region of the ducts . Finally in the pancreas, characterization of lrcs around the acini and ducts suggested a stem cell population by demonstrating increased expression of the putative stem cell marker c - met and activation in response to damage to form duct - like structures . Combined, these data indicate an important role for quiescent lrcs in maintenance and longevity of multiple adult epithelial tissues . If cscs do originate from normal adult stem cells, then it is foreseeable that key stem cell regulatory traits are retained through the oncogenic transition; quiescence is potentially one of these traits . Little research has been done to address how quiescence might play a role in csc biology, but there are some indications that quiescent stem - like populations might contribute to at least some tumors . We previously identified a subpopulation of cells in human sebaceous tumors that expressed the skin stem cell marker keratin 15 (figure 2). These cells appeared to have variable expression of the proliferation marker ki-67, suggesting a low but higher proliferative rate than normal stem cells . In primary ovarian tumors, gao et al . Demonstrated that cd24 cells expressing stem cell - associated genes like nestin, oct4, and both notch1 and notch4 were more slowly proliferating than the bulk tumor cells suggesting a quiescent phenotype . Low numbers of slowly proliferating cd24 cells were shown to produce tumors in a xenograft model where bulk cells were found to be nontumorigenic . Pece also observed a link between cscs and quiescence in breast tumors . Using the hnmsc signature generated with normal mammary lrcs, pece turned his attention to the analysis of primary breast tumors, finding that the hnmsc signature was more commonly found in grade 3 tumors over that of grade 1 . When grade 1 and grade 3 mammospheres were analyzed for pkh label retaining cells, both populations were found to retain label, with grade 3 tumors demonstrating a higher percentage . When evaluated for tumor genicity, breast tumor cells positive for the hnmsc signature were more efficient at forming in vitro spheres and in vivo xenograft tumors that those cells lacking the hnmsc signature . Cultured cancer cell lines are often used to study signaling pathways, invasion, migration, and apoptosis, but are rarely thought of as candidates for csc studies . Many of the most widely used cell lines have been in passage for years, are perceived homogeneous, lack interactions with the appropriate stromal microenvironment, and change characteristics based on alterations in culture conditions . Therefore, cultured cell line studies assessing csc characteristics must be evaluated critically, with data interpreted within the context of the experimental parameters, and results confirmed under biologically relevant conditions . Mcf7, sum149, sum159, sum1315, and mda.mb.231 suggests that these lines may not be as homogenous and void of stem like cells as once thought . Cd44/cd24/esa cells within these lines were found to contain the ability to self - renew, reconstitute the parental line, and to be up to 90% label retaining . If lrcs are found to retain the csc phenotype in cultured cell lines, these cell lines may provide an important resource for future delineation of quiescent pathway regulators . Additional transitive evidence linking quiescence to cscs can be found in the work conducted by roesch et al . In melanoma . This group found that primary melanoma cell lines contained a pkh26 label retaining population that was almost specifically identified by the h3k4 demethylase jarid1b . This population of cells was found to incorporate brdu more slowly but retain it for a longer period of time, lack ki67 staining, and have a doubling time of up to 4 weeks in vitro . When egfp was placed under the control of the jarid1b promoter, gfp cells demonstrated increased sphere forming ability in vitro . Interestingly, gfp cells were able to retain brdu in vivo, but did not show increased tumor initiating ability over gfp cell during the time period analyzed . Small hairpin rna (shrna) knockdown of jarid1b resulted in the in vitro exhaustion of proliferating cells, demonstrating the need for jarid1b cells in maintenance of proliferative capacity but not initiation of tumors . When assessed more fully, both in vitro and in vivo gfp cells gave rise to heterogeneous progeny, including jarid1b gfp+ cells . The most direct evidence to date for quiescence playing a role in cscs comes from a study conducted by dembinski and krauss . In this study vybrant dii cell - labeling solution was used to label pancreatic adenocarcinoma cells and conduct cancer stem studies on flow cytometry sorted label retaining cells . Dii label retaining slow - cycling cells (dii / sccs) comprised ~3% of total cell number . Interestingly, label retaining cells also exhibited an elongated fibroblast shape and an increase in the epithelial - mesenchymal transition markers vimentin, snail, and twist . A fibroblast - like csc is consistent with evidence demonstrating an increase in stem - like properties in cells that have undergone an epithelial - mesenchymal transition . Furthermore, sorted dii / sccs demonstrated a 2.510-fold increase in soft agar colony forming ability, twofold increase in invasive potential, and more than a tenfold increase in xenograft formation over nonlabel retaining cells . Combined, these data suggest that dii / sccs cells represent an enriched csc population . When assessed for common csc marker status, dii / sccs were enriched but only partially overlapped with cd24/cd44 and cd133 populations . It is curious to consider how these commonly used csc markers relate to the lrc populations and what role, if any, these markers play in the slow - cycling phenotype? Like the melanoma study by roesch et al ., dembinski and krauss's study also indicated the ability for lrcs to produce non - lrcs and surprisingly also for non - lrcs to produce lrcs . Such a dynamic suggests two possibilities (1) that the true unknown csc population is favored in the lrcs, but also found in the non - lrcs and can therefore give rise to both populations, or (2) that there exists a dynamic relationship in lrc - csc populations that is context dependent and allows for interconversion between the two states . The dembinski and krauss study argues a dynamic population of cscs that might coincide with an epithelial - mesenchymal transition (emt). Emt plays a central role in embryogenesis and mesoderm differentiation into multiple tissue types during development . The emergence of embryonic stem cell - associated genes like nanog, oct4, sox2, and c - myc in high grade undifferentiated cancers is suggestive that aberrant regulations of emt and other early development pathways might be playing a role in csc characteristics . This data is a further evidence to support a dynamic quiescent slow - cycling model for many types of cancer . Future studies will be important for further development and integration of these observations into the csc model for tumor initiation and propagation . At the present time, we have no clear understanding of why some patients recur and which cancers will have resistance to conventional types of therapy . Tumors from different patients in the same organ are likely to have undergone different oncogenic transitions, leading to a diversity of possible regulatory mechanism and pathway activities that might be contributing to the survival of a specific cancer . While broad patterns like the dysregulation of the wnt pathway in colon carcinomas are commonly observed, the secondary mutations that may accompany these cancers could be vastly different and contribute to survival in different ways . Even within the same tumor, different cscs have the possibility to accumulate unique mutations that may provide added resistance and be passed on to daughter cells . In context with the vast differences in tumorigenesis and heterogeneity with a tumor, it is not surprising that the exact contributors to chemotherapy resistance and consequently which patients will respond optimally to chemotherapy are not well understood . It has been proposed that variations in cell cycle control, antiapoptotic proteins, increased dna damage repair proteins, upregulation of cellular pumps, and increased metabolic activity may all play important roles in chemotherapy resistance [6, 5962]. Conventional chemotherapies and radiotherapies the quiescent nature of many adult stem cell pools is therefore an inherent mechanism for resistance and cell survival to conventional therapies . In the hematopoietic system, normal hematopoietic stem cells (hscs) when treated with the commonly used chemotherapy agent 5-fluorouracil (5-fu), mice that were p21 deficient had a significant decrease in cobblestone area - forming stem cells (10.8%) than normal p21 expressing wild - type mice (60.5%). In the brain, morshead et al . Demonstrated that high doses of tritiated thymidine (h - tdr) killed the constitutively proliferating cells in the adult mouse forebrain, but had no effect on quiescent stem cell ability to generate spheres . This data supports a model in which quiescent mouse forebrain stem cells are able to survive and re - enter the cell cycle to allow for regeneration of the damaged tissue . A similar pattern of stem cell survival and regeneration was observed 72 hours following doxorubicin treatment in mouse intestine . In this experiment, mice intestine demonstrated increased amounts of cell death via apoptosis in the + 36 positions and a parallel disappearance of mitotic activity . This period of relatively nonexistent mitotic activity was followed by stem cell re - entry into the cell cycle and tissue regeneration in the + 4 position stem cell compartment . Furthermore, colon stem cell survival during chemotherapy is aided by increased expression of bh3-only bcl-2 members that inhibit apoptosis, working in parallel with quiescence to increase the likelihood of stem cell survival . In chemotherapy - induced alopecia, the rapidly dividing ta cells in the hair matrix undergo apoptosis, while the stem cells in the bulge region survive to regenerate the follicle after chemotherapy is withdrawn . Potential factors involved in regulating hair follicle stem cell survival such as caveolin-1 are emerging . Similar mechanisms for survival and self - renewal for cscs are plausible in instances of tumor recurrence in human patients where cytotoxic agents kill proliferative cancer cells, leaving quiescent slow - cycling . Cancer stem cells that survive chemotherapy would have the ability to re - enter the cell cycle and produce highly proliferative - rapidly dividing progenitor cells that can re - establish the tumor . It is even probable that successive cycles of chemotherapy would intensify a tumor by weakening the normal stem cell pool and creating therapy resistant cscs that give rise to resistant off - spring . Slow cycling csc populations in the colon, breast, ovaries, and pancreas have been shown to demonstrate both in vivo abilities to survive therapies that kill bulk tumor cells as well as a requirement for doses of up to twice that which are required to kill rapidly proliferating cells in vitro [18, 55, 62, 67]. These data demonstrate how ineffective conventional therapies can be on quiescent cell populations and help to explain why tumors that seem to fully regress during treatment can recur . While large tumor populations may appear to have totally regressed after treatment, single surviving cscs would not be detectable with current diagnostic technology . Populations of cscs that are resistant to chemotherapy or radiation are able to re - enter the cell cycle or never fully undergo cell cycle arrest and are primed to re - establish tumors [53, 68]. Even more devastating to the survival of patients mouse ovarian tumors have been demonstrated to undergo accelerated clonogenic production during radiotherapy regimens, expanding the csc pool and driving development of a more aggressive secondary tumor . Furthermore, these cells would be more likely to produce chemotherapy resistant offspring, rendering the tumor unaffected by later rounds of treatment . While quiescence is likely to contribute to the survival of cscs in response to chemotherapy and radiation, slow cycling is not the sole mechanism and in all likelihood works in parallel with other systems to increase survival . Msi-1 colon cancer cells have been demonstrated to be less sensitive to cytotoxic drugs due to increased il-4 expression and orchestration of antiapoptotic mechanisms . The expression of other antiapoptotic proteins like c - flip and bcl-2 bh-3 only family members is frequently seen in stem cell and csc populations and has been demonstrated to contribute to cell survival during radiation and chemotherapy [59, 60]. Reduced cycling may help to limit cell damage in these cases, decreasing prodeath signals and increasing the potential for csc survival . Additional mechanisms for csc survival include increased dna damage repair, upregulation of cell pumps like the multidrug resistance transporter (mdr1) and the adenosine triphosphate - binding cassette (abcb1), and increased metabolic activity through aldh [61, 62]. Although the quiescence contribution to these mechanisms of resistance is unclear, it is likely that reduced proliferative rate only adds to their effectiveness . Additional time in s or g2 phase of the cell cycle coupled with increased dna repair protein activity may afford a survival advantage over bulk cells that continuously accrue dna damage and ultimately are forced to undergo apoptosis . Reduced cycling speed together with increased pumps would facilitate more drug being removed from cscs, limiting overall cytotoxic effects during the period of treatment . Additionally, quiescence would allow for increased metabolic activity of aldh and other metabolites over that of bulk cells with a shorter cell cycle period . Importantly, there is no reason why combinations or all of these resistance mechanisms could not be playing a role in csc survival . Future therapies may need to address all these issues to be successful in complete tumor eradication . Given the importance of quiescence in the csc contribution to tumor progression and survival, understanding the mechanisms that govern quiescence will prove important in the development of future strategies to better target these cells . Much of our current understanding of the mechanisms controlling quiescence come from studies using conditional induction of quiescence in normal adult fibroblasts . The induction of quiescence in fibroblasts is generally accomplished in one of three ways: mitogen deprivation, contact inhibition, or loss of adhesion . Each method of inducing quiescence in fibroblast appears to yield a different quiescent transcriptional program . The three transcription programs overlap in differential expression of 131 genes that coller et al . This signature is comprised of genes that regulated cell growth and division, suppress apoptosis and differentiation, and govern intercellular communication . Downregulated elements in the quiescence signature consist of genes associated with cell cycle progression including cyclin b1, cdc20, cul-1, and myc . Up regulated genes included important cell cycle regulators like tp53 (p53), cyclin d2, and mxi1 . Also up regulated in this signature are regulators of key stem cell - associated pathways including the wnt pathway (fzd2 and tcf7l2), the bmp pathway (smad1), and the notch pathway (hes1). Notch activation of hes1 is of particular interest as it has been shown to control reversibility of fibroblast quiescence by blocking differentiation and entry in irreversible cell cycle arrest . Notch pathway activity is important in mammary gland development as well as the mammary csc response immediately following irradiation, suggesting that the notch pathway may be a potential target in cscs [5, 71]. Interestingly, there exists a fourth transcriptional program in fibroblasts induced by overexpression of cyclin - dependent kinase inhibitors (cki) like p21 and p16 . The cki p21 has been found to control entry into quiescence and maintenance of the quiescent state, allowing cells to activate a dna damage - like response . Additionally, maintenance of fibroblast quiescence has also been shown to be highly regulated by the retinoblastoma family members rb and p107 . Loss of rb and p107 did not affect the ability of fibroblasts to enter g0, but these cells were unable to maintain the quiescent state . While rb loss is generally associated with the progression of cancer, retention of rb in cscs or contribution of other rb family members like p107 may be important in csc maintenance of quiescence . Developing and studying a quiescence signature in fibroblasts may be important in understanding regulation of the cell cycle, but the exact relevance to quiescent stem cell populations is not very clear . Primarily, quiescence fibroblast studies are conducted on large populations of fibroblasts under biologically stressful conditions like contact inhibition or serum starvation . In contrast, individual stem cells and cscs maintain quiescence while in contact with daughter cells and stromal layers and in the presence of normal mitogenic signals . Additionally, sphere forming assays commonly used for the identification of stem cells and cscs rely specifically on proliferation under nonadherent conditions . If mitogen deprivation, loss of adhesion, and contact inhibition truly activate three different transcriptional programs in quiescent fibroblast populations, it is possible that the transcriptional program facilitated by quiescent stem cells and cscs may be very different . Quiescence regulation of a stem cell population is most comprehensively understood in the hematopoietic system . When compared to differentiated or cycling hscs, quiescent hscs were found to have up - regulated genes associated with cell cycle regulation, translation and rna processing, and metabolic process . Down - regulated genes were generally associated with transcription factors, signaling proteins, cell cycle proteins, and inhibitors of cell cycle progression . In line with these findings, the cki p21 was found to be necessary for quiescence and maintenance of the hsc pool . Mice that are p21 null demonstrate an increase in the number of stem cells present and lose the ability to repopulate the bone marrow in serial transplant experiments, suggesting uncontrolled expansion and eventual exhaustion of the stem cell pool . This deregulation of the stem cell pool is likely due to p21 downstream effects on rb family members: rb, p107, and p130 . Triple knockout of these three family members resulted in hematopoietic progenitor g1/s transition and proliferation, leading to exhaustion of the proliferative potential, similar to that seen in p21 loss . While p21 also appears to play a role in adult neural stem cell regulation and maintenance, other factors occasional exit of neural stem cells from the quiescent state is important for proper tissue maintenance and may be controlled though notch signaling via hes1 oscillations . Down - regulation of hes1 in neural progenitor cells during g1 phase reduced repression of cyclind, ngn2, and dll1, activating notch signaling and driving cell cycle progression and generation of neural progenitors . Neural progenitors and neurons continue to retain low levels of hes1 as they proliferate and differentiate . In neural stem cells, hes1 expression and control of cyclind and notch signaling increase until subsequent g1 entry . Interestingly, p21 loss does not appear to play a significant role during differentiation in the brain, suggesting the need for additional means of cell cycle regulation in differentiated senescent cells . Signaling pathways with interactions to other ckis also play important roles in quiescent adult stem cell regulation . In mammary glands, the hedgehog pathways components gli2 and bmi-1 have been demonstrated to regulate stem cell self - renewal . When injected into cleared mammary fat pads, gli2 or bmi-1 over expressing mammospheres were able to produce substantially more outgrowths than control mammospheres . Bmi-1 has been demonstrated to transcriptionally repress the p16 and p19, suggesting a role for bmi-1 in mammary stem cell cycle control . Additional signaling pathways have been demonstrated to play important roles in stem cell quiescence, specifically the bmp pathway in skin . Bmp and calcineurin signaling up - regulate the transcription factor nfat1c that has been found to highly colocalize with cd34 cells in the hair follicle . Nfat1c represses transcription of cdk4, stalling cells in g1/s phase and maintaining quiescence . Loss of nfat1c permits entry into the cell cycle, shortening telogen and prompting aberrant entry into anagen . While significant advances are being made in understanding quiescence control in normal adult stem cell populations, much less very few studies have been conducted specifically addressing control of quiescent cscs, most likely due to the difficulty of isolating and analyzing pure csc populations . If cscs are truly derived from adult stem cells, then it is possible that hes1, p21, p16, rb family members, bmi-1, and nfat1c play significant roles in csc regulation . Although rare, there are clues that at least some of these regulators are important in cscs . In the colon cancer cell line hct116, p21 null cells were found to produce tenfold smaller tumors in growth assays when compared to normal cells expressing p21 . Under sphere forming conditions, p21 null cells were unable to form spheres, ceased proliferation, and eventually died . This p21 dependence was found to be associated with lack of e - cadherin expression and suppression of apoptosis signals, suggesting a more complex role for p21 in tumor cells than simply regulating cell cycle . Small molecule targeting of p21 or downstream p21 targets may therefore prove to be an effective means of forcing quiescent cscs to cycle or undergo apoptosis . Cancers frequently have aberrant signaling in the wnt, hedgehog (hh), and notch self - renewal pathways that likely contribute to cell cycle control and differentiation . Increased expression of hes1 has been observed in ovarian, breast, and nonsmall cell lung carcinomas, suggesting active regulation of notch signaling . In melanoma, the slow cycling cells identified by rosech et al ., repress notch signaling directly though jarid1b interaction with the notch ligand jagged 1 promoter, consequently reducing intracellular notch and controlling proliferation . Hes1 and jagged1 may therefore be potential targets in future cancer treatments designed to target cscs . Targeted reduction of hes1 would increase notch signaling, driving cscs to proliferate and exhaust their proliferative potential, and making them more susceptible to conventional therapy . In colon cancers, mutations in apc or -catenin in the presence of wnt signal, -catenin is no longer taken up by an apc - dependent degradation complex and translocates to the nucleus where it binds tcf / lef transcriptions factors to control expression of cell cycle target genes . Loss of apc in crypt lrg5 cells has been demonstrated to be an important step towards initiation of intestinal adenomas . Interestingly, cells expressing high wnt downstream transcription factors tcf / lef in primary sphere cultures demonstrated increased clonogenicity and the generation of both cycling and noncycling cells . In tumors, these high wnt expressing cells were located near stromal fibroblasts that provided signals to activate -catenin - dependent transcription . This data suggests that csc cell cycle control may not be entirely cell autonomous and partially regulated by microenvironmental signals . Targeting wnt pathway regulators or the ability for cscs to communicate with their stromal environment may represent potential mechanism for limiting csc expansion and contribution to recurrence . There is also mounting evidence for the requirement of hedgehog signaling in proliferation and survival of both colon and breast tumors . Active hh - gli signaling was found to contribute to the subpopulation of human colon cd133 cells that were able to survive and self - renew in xenograft studies . In breast tumor cd44/cd24 cells, the hh pathway proteins patch (ptch1), gli1, gli2, and bmi-1 all demonstrated like their adult mammary stem cell counterparts, overexpression of bmi-1 in mammary cscs suggests a potential role for p16 and p19 in cell cycle regulation and suggests a potential drug target for improved csc eradication . While p21, p16, notch, wnt, and hedgehog signaling may provide tempting targets for the removal of cscs, targeting of these pathways would require meticulous targeting of csc or titration of inhibitors to act on cscs but not normal stem cell populations additionally, improper application of cell cycle inhibitors like p21 may fuel tumor growth and aggressiveness . The cdk inhibitor p21 acts as a tumor suppressor in dividing cells by protecting against genome instability and working with other tumor suppressors to subdue oncogenes [82, 83]. Loss of p21 combined with chemical induction of carcinogenesis has demonstrated increased induction of tumors and increased aggressiveness in resulting tumors [84, 85]. These data highlight the necessity to be able to selectively target cscs when using cdk inhibitors and add to the challenges ahead in developing treatments to better eradiate cscs . The limited data available on the regulation of quiescence equates to a poor understanding for the role of quiescence in tumor progression and recurrence . Exactly how and to what extent quiescence plays a role in tumor recurrence is at present unclear . What little evidence there is suggests that quiescence might be an important factor in tumor cell survival after conventional therapy . Mechanistically, csc quiescence suggests an inherent means of resistance that when coupled with increased dna repair or metabolic activity could explain the patterns of recurrence and acquired resistance currently observed in posttherapy cancer patients . The functionally relevant identification of quiescent cscs though label - retaining assays may prove to be an important tool in ongoing csc research . Future research must focus on better understanding and targeting of quiescent csc populations, specifically identifying regulators and factors that separate cscs from normal stem cells . General targeting of p21, bmi-1, hes1, and other commonly shared cell cycle regulators might prove disastrous for patients if these treatments eradicate normal stem cell populations as well as cscs . Aberrant regulation of normal stem cell characteristics presents a difficult paradox in fighting cscs: how to target the cancer without harming normal stem cells . Hope exists that careful study of cscs will identify new or differentially expressed targets that will specifically affect tumors, minimizing toxic side effects and leaving patients cancer free.
Are currently classified as overweight or obese (bmi 25 kg / m), and one - third of them are frankly obese (bmi 30 kg / m) (1). Is 26.1 million in 20052010 or 12% of the population (2). Among u.s . Diabetic patients, the prevalence of overweight or obesity has increased to 80% (3). Obesity is associated with increased risks of several cardiometabolic diseases, including hypertension (4), diabetes (5), coronary heart disease (chd) (6,7), heart failure (8), and stroke (9). Cardiovascular diseases, especially chd, are the leading causes of death worldwide . In recent years, several prospective studies have assessed the association between obesity and the risks of total and cvd mortality among diabetic patients, and the results are inconsistent . To date, many studies have reported positive associations (10,11), inverse associations (1214), u - shaped associations (1517), or no associations (18) between bmi and mortality among patients with diabetes . All of these studies were focused on the association between bmi and cvd mortality; however, no studies assessed the association between bmi and the risk of incident chd among diabetic patients . In this study, we examined the association between bmi and the risk of chd among patients with type 2 diabetes in the louisiana state university hospital - based longitudinal study (lsuhls). Between 1997 and 2012, the lsu health care services division (lsuhcsd) operated seven public hospitals and affiliated clinics in louisiana providing quality medical care to the residents of louisiana regardless of their income or insurance coverage (1926). Overall, lsuhcsd facilities have served 1.6 million patients (35% of the louisiana population) since 1997 . Administrative, anthropometric, laboratory, clinical diagnosis, and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . Using these data, a cohort of diabetic patients was established by using the icd-9 (code 250) between 1 january 1999 and 31 december 2009 . Both inpatients and outpatients confirmation of diabetes diagnoses was made by applying the american diabetes association criteria: a fasting plasma glucose level 126 mg / dl; 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test; and one or more classic symptoms plus a random plasma glucose level 200 mg / dl (27,28). The first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . Before diagnosis with diabetes the agreement of diabetes diagnosis was 97%: 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes by using the american diabetes associates diabetes diagnosis criteria (27). The current study included 30,434 newly diagnosed patients (10,955 men and 19,479 women) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables . We only included african americans and whites because the patient numbers of hispanics, asians, and native americans are very small in the lsuhcsd hospitals . Patients were excluded if they were underweight (bmi <18.5 kg / m) because of limited statistical power for this group . Compared with diabetic patients excluded from the present analyses due to missing data, those included in the analyses were younger (51.0 vs. 57.6 years old), had a higher frequency of african americans (58.6 vs. 45.4%), and less males (37.2 vs. 47.1%). The study and analysis plan was approved by the pennington biomedical research center and lsu health sciences center institutional review boards, lsu system . We did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records . The patient s characteristics, including age at diabetes diagnosis, sex, race / ethnicity, family income, smoking status, types of health insurance, bmi, blood pressure, total cholesterol, hdl cholesterol, ldl cholesterol, triglycerides, glycosylated hemoglobin (hba1c), estimated glomerular filtration rate (egfr), and medication (antihypertensive drug, cholesterol - lowering drug, and antidiabetes drug) within a half year before the diabetes diagnosis (baseline), during follow - up after the diabetes diagnosis (follow - up), and the last visit were extracted from the computerized hospitalization records . Height and weight bmi was calculated by dividing weight in kilograms by the square of height in meters . Values of bmi, blood pressure, hba1c, ldl cholesterol, and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements, calculated for each participant from baseline to each year of follow - up . For example, at 1 year, the updated mean is the average of the baseline and 1-year values, and at 3 years, it is the average of baseline, 1-year, 2-year, and 3-year values . In the case of an event during follow - up, the period for estimating updated mean values was from baseline to the year before this event occurred (29). Follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals . The diagnosis of chd was the primary end point of interest of the study and defined according to the following icd-9: chd (icd - codes 410414). Follow - up of each cohort member continued until the date of the diagnosis of chd, the date of the last visit if the subject stopped use of lsuhcsd hospitals, or the date of death, determined from linking to the louisiana office of public health vital records registry, or 31 may 2012 (23,26). The association between bmi and the risk of chd was analyzed by using cox proportional hazards models . Bmi was evaluated in the following two ways: 1) as five weight categories (18.524.9 [reference group], 2529.9, 3034.9, 3539.9, and 40 kg / m) and 2) as a continuous variable . The trend over different categories of bmi was tested in models with the median of each category as a continuous variable . All analyses were adjusted for age (continuous variable) and race (african american and white) (model 1) and further for smoking (never, past, and current), income (continuous variable), and types of insurance (free, self - pay, medicaid, medicare, and commercial) (model 2), and additionally for systolic blood pressure (continuous variable), hba1c (continuous variable), ldl cholesterol (continuous variable), hdl cholesterol (continuous variable), triglycerides (continuous variable), egfr (90, 6089, 3059, 1529, and <15 ml / min/1.73 m), use of antihypertensive drugs (no use, ace inhibitor, angiotensin ii receptor blockers, -blockers, calcium channel blocker, diuretics, and other antihypertensive drugs), use of diabetes medications (no use, oral hypoglycemic agents, and insulin), and use of cholesterol - lowering agents (no use, statins, and other cholesterol - lowering agents) (model 3). We stratified the samples by sex because there was a significant interaction between sex and bmi on the risk of chd . Since the interactions between race and bmi on the risk of chd were not statistically significant, data for white and african americans were combined in some analyses . All statistical analyses were performed with pasw for windows, version 20.0 (ibm spss inc ., between 1997 and 2012, the lsu health care services division (lsuhcsd) operated seven public hospitals and affiliated clinics in louisiana providing quality medical care to the residents of louisiana regardless of their income or insurance coverage (1926). Overall, lsuhcsd facilities have served 1.6 million patients (35% of the louisiana population) since 1997 . Administrative, anthropometric, laboratory, clinical diagnosis, and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . Using these data, a cohort of diabetic patients was established by using the icd-9 (code 250) between 1 january 1999 and 31 december 2009 . Both inpatients and outpatients confirmation of diabetes diagnoses was made by applying the american diabetes association criteria: a fasting plasma glucose level 126 mg / dl; 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test; and one or more classic symptoms plus a random plasma glucose level 200 mg / dl (27,28). The first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . Before diagnosis with diabetes the agreement of diabetes diagnosis was 97%: 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes by using the american diabetes associates diabetes diagnosis criteria (27). The current study included 30,434 newly diagnosed patients (10,955 men and 19,479 women) with type 2 diabetes who were 3095 years of age without a history of chd and stroke at the time of diabetes diagnosis and with complete repeated data on all risk factor variables . We only included african americans and whites because the patient numbers of hispanics, asians, and native americans are very small in the lsuhcsd hospitals . Patients were excluded if they were underweight (bmi <18.5 kg / m) because of limited statistical power for this group . Compared with diabetic patients excluded from the present analyses due to missing data, those included in the analyses were younger (51.0 vs. 57.6 years old), had a higher frequency of african americans (58.6 vs. 45.4%), and less males (37.2 vs. 47.1%). The study and analysis plan was approved by the pennington biomedical research center and lsu health sciences center institutional review boards, lsu system . We did not obtain informed consent from participants involved in our study because we used anonymized data compiled from electronic medical records . The patient s characteristics, including age at diabetes diagnosis, sex, race / ethnicity, family income, smoking status, types of health insurance, bmi, blood pressure, total cholesterol, hdl cholesterol, ldl cholesterol, triglycerides, glycosylated hemoglobin (hba1c), estimated glomerular filtration rate (egfr), and medication (antihypertensive drug, cholesterol - lowering drug, and antidiabetes drug) within a half year before the diabetes diagnosis (baseline), during follow - up after the diabetes diagnosis (follow - up), and the last visit were extracted from the computerized hospitalization records . Height and weight were measured without shoes and with light clothing according to a standardized protocol . Bmi was calculated by dividing weight in kilograms by the square of height in meters . Values of bmi, blood pressure, hba1c, ldl cholesterol, and egfr over time were measured firstly at baseline and secondly as an updated mean of annual measurements, calculated for each participant from baseline to each year of follow - up . For example, at 1 year, the updated mean is the average of the baseline and 1-year values, and at 3 years, it is the average of baseline, 1-year, 2-year, and 3-year values . In the case of an event during follow - up, the period for estimating updated mean values was from baseline to the year before this event occurred (29). Follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals . The diagnosis of chd was the primary end point of interest of the study and defined according to the following icd-9: chd (icd - codes 410414). Follow - up of each cohort member continued until the date of the diagnosis of chd, the date of the last visit if the subject stopped use of lsuhcsd hospitals, or the date of death, determined from linking to the louisiana office of public health vital records registry, or 31 may 2012 (23,26). The association between bmi and the risk of chd was analyzed by using cox proportional hazards models . Bmi was evaluated in the following two ways: 1) as five weight categories (18.524.9 [reference group], 2529.9, 3034.9, 3539.9, and 40 kg / m) and 2) as a continuous variable . The trend over different categories of bmi was tested in models with the median of each category as a continuous variable . All analyses were adjusted for age (continuous variable) and race (african american and white) (model 1) and further for smoking (never, past, and current), income (continuous variable), and types of insurance (free, self - pay, medicaid, medicare, and commercial) (model 2), and additionally for systolic blood pressure (continuous variable), hba1c (continuous variable), ldl cholesterol (continuous variable), hdl cholesterol (continuous variable), triglycerides (continuous variable), egfr (90, 6089, 3059, 1529, and <15 ml / min/1.73 m), use of antihypertensive drugs (no use, ace inhibitor, angiotensin ii receptor blockers, -blockers, calcium channel blocker, diuretics, and other antihypertensive drugs), use of diabetes medications (no use, oral hypoglycemic agents, and insulin), and use of cholesterol - lowering agents (no use, statins, and other cholesterol - lowering agents) (model 3). We stratified the samples by sex because there was a significant interaction between sex and bmi on the risk of chd . Since the interactions between race and bmi on the risk of chd were not statistically significant, data for white and african americans were combined in some analyses . All statistical analyses were performed with pasw for windows, version 20.0 (ibm spss inc ., chicago, il). Baseline characteristics of patients with type 2 diabetes by the outcome during follow - up data represent means or percentages . All data except age are adjusted for age and race . During a mean follow - up period of 7.3 years, 7,414 subjects (2,926 men and 4,488 women) developed chd . Patients who developed chd during follow - up were older and used more glucose - lowering, lipid - lowering, and antihypertensive medication compared with those who did not develop chd . The multivariable - adjusted (age, race, smoking, income, and types of insurance) (model 2) hazard ratios (hrs) for chd at different levels of bmi at baseline (18.524.9 [reference group], 2529.9, 3034.9, 3539.9, and 40 kg / m) were 1.00, 1.14 (95% ci 1.001.29), 1.27 (1.121.45), 1.54 (1.341.78), and 1.42 (1.231.64) (ptrend <0.001) in men and 1.00, 0.95 (0.851.07), 0.95 (0.841.06), 1.06 (0.941.20), and 1.09 (1.001.22) (ptrend <0.001) in women, respectively (table 2). After further adjustment for other confounding factors (systolic blood pressure, hba1c, ldl cholesterol, hdl cholesterol, triglycerides, egfr, use of antihypertensive drugs, use of diabetes medications, and use of cholesterol - lowering agents), this association remained significant among men (ptrend <0.001) and women (ptrend = 0.006). Hrs of chd according to different levels of bmi at baseline, during follow - up, and at last visit among patients with type 2 diabetes adjusted for age and race . Adjusted for age, race, types of insurance, income, and smoking . Adjusted for age, race, types of insurance, income, smoking, systolic blood pressure, ldl cholesterol, hdl cholesterol, triglycerides, hba1c, egfr, use of antihypertensive drugs (none, ace inhibitor, angiotensin ii receptor blockers, -blockers, calcium channel blocker, diuretics, other antihypertensive drugs, and any two or more of above treatments), glucose - lowering agents (none, oral hypoglycemic agents, and insulin), and cholesterol - lowering agents (none, statins, and other cholesterol - lowering agents). When bmi was examined as a continuous variable, the multivariable - adjusted (model 2) hrs of chd for each one - unit increase in bmi at baseline were 1.015 (95% ci 1.0111.020) in men and 1.004 (95% ci 1.0011.008) in women (table 2). There was a significant interaction between sex and bmi on chd risk (= 9.86; 1df, p <0.005), which indicated that this positive association was stronger in men than in women . When we did an additional analysis by using an updated mean value of bmi, we found the same positive association between bmi and chd risk among both men (ptrend <0.001) and women (ptrend <0.001) (table 2). When we did another additional analysis by using the last visit value of bmi, we found a positive association between bmi and chd risk among men (ptrend <0.001) and a u - shaped association between bmi and chd risk among women (ptrend = 0.003) (table 2). Women who were overweight and had class i obesity (bmi 2534.9 kg / m) at last visit had a lower risk of chd compared with normal - weight women (bmi <25 kg / m). After excluding subjects who were diagnosed with chd during the first 2 years of follow - up (n = 3,207), the multivariable - adjusted hrs (model 2) of chd for each one - unit increase in bmi at baseline, during follow - up, and at the last visit were 1.014 (95% ci 1.0101.019), 1.017 (1.0121.022), and 1.015 (1.0091.019) in men and 1.005 (1.0011.009), 1.006 (1.0021.010), and 1.005 (1.0001.008) in women (data not shown), respectively . In stratified analyses, the multivariable - adjusted positive association between bmi and chd risk was present among men with different smoking status (tables 3 and 4). When stratified by age, race, and use of antidiabetic drugs, this positive association of bmi at baseline, during follow - up, and at the last visit with chd risk was still present among men in all subgroups and among women in some of the subgroups (tables 3 and 4). Hrs (95% cis) of chd according to different levels of bmi at baseline among various subpopulations adjusted for age, race, types of insurance, income, and smoking, other than the variable for stratification . Hrs (95% cis) of chd according to different levels of bmi during follow - up and at last visit among various subpopulations adjusted for age, race, types of insurance, income, and smoking, other than the variable for stratification . Our study found a positive association of bmi at baseline and during follow - up with the risk of chd among both men and women with type 2 diabetes, and this association was stronger among men than among women . In addition, we found that a positive association between bmi and the risk of chd was present in both african americans and whites with type 2 diabetes and in nonsmokers and smokers . The positive association did not change among men but changed to a u - shaped association among women with type 2 diabetes when we assessed bmi of the last visit with chd risk . Only a few prospective studies have evaluated the association between obesity and total and cvd mortality among diabetic patients, and the results are controversial including inverse associations (1214), positive associations (10,11), u - shaped associations (1517), or no association (18). The current study was the first, to our knowledge, to assess the association between bmi and the risk of incident chd among diabetic patients . The results of our study indicated a positive association between bmi and the risk of chd among patients with type 2 diabetes . We found this positive association of chd risk by bmi at baseline and during follow - up . In addition, this positive association was present in different race, antidiabetes medication, and smoking groups . It is noteworthy that there was a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes in the current study . Our study found that diabetic women who were overweight and had class i obesity (bmi 2534.9 kg / m) at the last visit had a lower risk of chd compared with normal - weight women (bmi <25 it is well known that women with diabetes have a greater or equal relative risk of chd than men with diabetes (30,31). The current study found a significant positive association of bmi and chd risk among both men and women with type 2 diabetes, and this association is stronger among men than among women . The finding from our study is noteworthy for us to prevent chd among patients with type 2 diabetes . In addition, more studies are needed to confirm the different effect size of bmi with chd risk among men and women with type 2 diabetes . It has been suggested that three potential methodological concerns should be considered when assessing the associations between obesity and health outcomes (32). People with a history of cvd and several other chronic diseases frequently lose weight, and thus, people with a lower weight might increase the estimated risk of death . A recent analysis pooling five longitudinal studies has found that patients who have normal weight at the time of diabetes diagnosis have a higher mortality risk than those who are overweight or obese (12). They suggest that diabetic individuals with metabolically obese normal - weight may reflect underlying illness that predisposes to mortality (33). Despite having a normal bmi, these diabetic individuals have hyperinsulinemia, insulin resistance, and dyslipidemia, and all of these factors predispose individuals to death (33). In the current study, we excluded patients with a history of chd and stroke at time of diabetes diagnosis, which can minimize the influence of reverse causation . Moreover, we performed another sensitivity analysis by excluding the subjects who were diagnosed with chd during the first 2 years of follow - up (n = 3,207), and the positive association of bmi at baseline and during follow - up with chd risk was still present . The second major concern is that confounding factors may distort the association between body weight and chd . Smoking is a particularly important factor because smokers tend to weigh less and have much higher chd risk than nonsmokers . In the current study, smoking status was considered as a confounding factor in the multivariable model, and the positive association between bmi and the risk of chd was found in both never - smokers and smokers . The third methodological concern in some analyses between weight and chd risk is that the physiologic effects of excess fatness, such as hypertension, diabetes, and dyslipidemia, were controlled for statistically, thus artificially removing some of the effects of being overweight . Obesity has been found as a strong risk factor for hypertension (4), high levels of hba1c (5), and high serum cholesterol among diabetic patients (34) and has also been the key or important component of the metabolic syndrome (35). All of these factors are associated with an increased risk of chd (3537) and considered as mediating factors for the physiologic effects of obesity on the chd risk . In the current study, the adjustment for systolic blood pressure, ldl cholesterol, hdl cholesterol, triglycerides, hba1c, egfr, and treatment attenuated the association between bmi and chd risk, but bmi as a continuous variable remained a statistically significant predictor of chd in the multivariable model . There are several strengths in our study, including the large sample size, high proportion of african americans, and the use of administrative databases to avoid differential recall biases . We have used baseline bmi levels, updated mean values of bmi during follow - up, and the last visit value of bmi in the analyses, which can avoid potential bias from a single baseline measurement . In addition, participants in this study used the same public health care system that minimizes the influence of accessibility to health care, particularly in comparing men and women . One limitation of our study is that our analysis was not performed on a representative sample of the population, which limits the generalizability of this study; however, lsuhcsd hospitals are public hospitals and cover> 1.6 million patients, most of whom are low - income persons in louisiana . The results of the current study will have wide applicability for the population with low income and without health insurance in the u.s . Another limitation of our study is that we did not have data on other obesity indicators, such as waist, hip, and thigh circumferences, and did not assess abdominal height, although these adiposity predictors have been shown to be associated with cvd risk (6,38,39). Third, while body weight was measured at each clinic visit, clinically measured bmi might not be as accurate as bmi measured in carefully conducted laboratory studies (40). Fourth, even though our analyses adjusted for an extensive set of confounding factors, residual confounding due to the measurement error in the assessment of confounding factors, unmeasured factors such as heart rates, physical activity, education, and dietary factors, cannot be excluded . In summary, we found a positive association between bmi at baseline and during follow - up with the risk of chd among men and women with type 2 diabetes, and this association was stronger among men than among women . We also found a positive association between bmi at the last visit and the risk of chd among men with type 2 diabetes and a u - shaped association between bmi at the last visit and the risk of chd among women with type 2 diabetes.
Xanthomatous infiltration may be a risk to the rotator cuff muscles and long head of the biceps . We believe it is a rare entity where there is fatty infiltration of the shoulder tendons, predisposing the shoulder to rotator cuff tear . It may be diagnosed on mri, in patients with hyperlipedemia . We describe the mri features of what we suspect is likely the first reported case of rotator cuff tear associated with xanthomatous infiltration, in a patient with hyperlipidemia . A 77 year old with past medical history of mixed (type iia) hyperlipidemia and chronic pain in both shoulders presented with increased left shoulder pain . On physical examination, there was reduced range of motion up to thirty degree s and elicited crepitus with movement . The patient had also recently experienced difficulty sleeping at night due to the intense nature of the pain . The more acute symptoms had started after he had attempted to lift a heavy object . This demonstrated extensive infiltration of the rotator cuff muscles, including the supraspinatus, infraspinatus, and subscapularis . There was also extensive infiltration of the long head of the biceps, as well . (figs . 1 and 2) the long head of the biceps displayed a more stipled appearance, which is characteristic for xanthoma . 3) ultrasound is not routinely performed at our institution, and we do not believe it adds in retrospective diagnosis, once mri has been obtained . Furthermore, there were also complete tears with myotendonous retraction of the supraspinatous and infraspinatous (figs . Conservative management was indicated due to the extent of retracted tendons . Additionally, a full thickness tear of the subscapularis was present, with the long head of the biceps still remaining in the biciptial groove (fig . The patient had initially presented to our institution nearly five years earlier with fasting total cholesterol levels of 270 mg / dl, hdl 40, triglyceride 294, and ldl of 171 . The patient s chart did not document if the patient was on any prior cholesterol medication . The normal cholesterol reference range is <200 for total cholesterol,> 40 for hdl, <150 for triglyceride, and <100 for ldl levels . The patient s other presenting features of hyperlipidemia included pterygium formation over the conjunctiva of his left eye which had extended to the cornea . He also had a history of coronary artery disease and an abdominal aortic aneurysm measuring 5.1 cm, findings which also are commonly seen with hyperlipidemia . Dyslipidemias were traditionally classified by patterns of elevation in lipids and lipoproteins (fredrickson phenotype). A more practical system categorizes dyslipidemias as primary or secondary and characterizes them by increases in cholesterol only (pure or isolated hypercholesterolemia), increases in tgs only (pure or isolated hypertriglyceridemia), or increases in both cholesterol and tgs (mixed or combined hyperlipidemias).1 tendon xanthoma is a nonneoplastic tumor involving tendon and constitutes a significant physical manifestation of hyperlipidemia . These deposits may accompany rapidly progressive atherosclerosis and may signal the presence of of life threatening hyperlipidemia.2 pathogenesis remains unclear, however it is likely that mechanical stress and extensive vascularization are essential factors for xanthoma formation . Moreover, endothelial cells and macrophages cells are principal contributors to the pathogenesis of tendinous xanthomas and to atherogenesis.3 xanthoma can occur in all five types of hypercholesterolemia . In particular, type iia hyperlipidemia and type iii hyperlipidemia have shown a strong association with tendinous xanthomas, which have a predilection to involve the extensors, most notably the achilles and the metacarpal phalangeal extensor tendons of the hands.4,5 the main value of diagnosing tendon xanthomas is establishing the diagnosis of familial hyperlipidemia in patients with hypercholesterolemia . The disease can then be treated appropriately, preventing or retarding atherosclerotic disease.6 once diagnosis is established, lipid level control and excision of lipid deposits shall be a priority . The clinical manifestations of xanthomatosis are broad, and include cataracts, neurological dysfunction, atherosclerosis, as well as tendon xanthoma.7 involved tendons may have a diffuse stippled pattern formed by many low signal round structures of equal size surrounded by high signal material . The uniformly sized, round, low signal structures seen on mr images probably represent collagen fibers, whereas the high signal intensity between the collagen fibers represents infiltrating cholesterol foam cells and an inflammatory response.69 patients with tendinous xanthomas demonstrate focal regions of high signal t1 intensity suggesting high percentage of triglycerides.7,10,11 the diffuse reticulated pattern has been described as a characteristic mr finding of tendinous xanthoma.7,11,12 this reticulated or stippled pattern is clearly depicted within the axial images of the long head of the biceps (fig . 3). A diffusely thickened tendon or one with discreet irregularities is suggestive of a xanthoma.4 this is seen as thickened wavy high t1 and t2 signal within the supraspinatus, infraspinatus, and subscapularis (figs . 1 and 2). Prior to the case presented, tendinous xanthoma has been commonly described in the tendons of the hand, the patellar tendon, the achilles tendon, the plantar aponeurosis, the peroneal tendon, and around the elbow and lower tibia.11,13 the rotator cuff tendons and the long head of the biceps may also now be included on this list and should be reported to aid in the diagnosis of hyperlipidemia . Thus, it is imperative to recognize the imaging features of xanthoma in the shoulder, as well as the more commonly involved tendons . It seems that the principal problem is the conflict of volume of the xanthoma, which in turn may eventually lead to tear . Similarly, cases of tendinous tears have been described with tophaceous gouty infiltration.14 tendon biopsies for demonstration of cholesterol are avoided because it is known that both major and minor direct injuries to any tendon complex can lead to chronic posttraumatic pain syndromes.15 in conclusion, mri may play a more prominent role in diagnosing xanthoma and thus helping to more accurately characterize a patient with familial hyperlipidemia . Perhaps, we may see more shoulder xanthoma or other tendinous xanthoma in lesser known target tendons associated with pathological tears, if patients with high triglyceride levels and family history who have focal pain are more routinely imaged.
Contingent valuation (cv) is a survey - based hypothetical and direct method to estimate monetary valuations of effects of health technologies . The validity of cv to elicit monetary valuations of health care provisions has been adequately established in the literature [2 - 6]. There have been several attempts to use the contingent valuation technique to estimate the willingness to pay for various health services [4,7 - 9]. Many of these studies have tried to estimate the value placed by individuals to protect them from illnesses . Very few studies, however, have looked at the willingness to pay (wtp) for valuing someone else's health state (e.g., parental, household or societal willingness to pay). In nigeria, a study compared the theoretical validity and predictive validity of the binary with follow - up questions technique and the bidding game, using hypothetical and actual wtp for insecticide - treated nets . The study found that consistent slightly higher mean and median wtp amounts were elicited where the bidding game was used . The study suggested that an appropriate wtp elicitation method should be developed to represent the bargaining process in normal market situations in rural nigeria . It recommended that such an indigenous technique would help improve the predictive validity of the contingent valuation method . Agee and crocker calculated parental wtp to minimize the risk of neurological impairments due to lead exposure among children, and used a revealed preference approach based on the parents' decision to obtain a chelation therapy for their child . They, however, failed to estimate wtp to reduce risks of adult neurotoxicity that are smaller than risks among children . Used cv to estimate wtp to avert the risk of injury from using household pesticides . The study indicated that parents place more value on their children's lives compared to their own lives . However, the study failed to recognize the effects of parental altruism and the severity of injuries . A self - administered questionnaire was used, and included questions on demographic information, perceived risk of malignancy, and perceived life expectancy given a diagnosis of malignancy . Finally, liu et al . Used cv to estimate mother's wtp for her own and her child's health in taiwan . The results suggest that mother's wtp for her child's health was higher than that for her own health . The study, however, did not take into account the altruistic maternal nature in the analysis, which could partly justify higher maternal wtp for her child's health . In this paper, we aimed to apply the contingent valuation technique to estimate the parents' willingness to pay for their child's diarrhoeal episode in rural india . The main objective was to explore whether or not parents' systematically valued their children differently on the ground of their child's gender, and if so to what extent does this valuation - based discrepancy exists between genders . It was hypothesized that different parental behaviour towards seeking treatment for their child on the ground of gender would have serious social policy implications . The under - valuation of female children by parents when it comes to treating a disease may elicit the direction and extent of other important parental valuations that may have major impacts on societal development, such as parental valuation about education, human rights, social justice and gender equity . Data were collected during may - june and november - december of 2000 to capture the possible seasonal effects of diarrhoea . The sample cluster was chosen in such a manner that households would be at equidistance from either of the two dispensaries . The sample population was randomly drawn and consisted of 250 households with children from both sexes . Households with two children (one boy and one girl) between five and seven years of age were only included in the sample . It was speculated that there might be differences in valuation if one child was a seven - year old and the other was a three - year old . In total we used a structured questionnaire with open - ended fields to obtain the socioeconomic information including income, employment and household ownership related information, health status including previous history of diarrhoea in the preceding three months, duration and severity, etc . The definition of diarrhoea, its symptoms, duration, and severity were properly explained to each household respondent . Wtp for an episode of diarrhoea was asked to both parents (mother and father), and a mean value was calculated and used as the parental wtp in the analysis . The use of a common initial bid helped prevent any initial biases on the difference between valuations by different households . Three - point bidding was used, which meant that the bidding ended after the third bid with an open - ended question and the amount stated at that bid was the wtp . We estimated the values to parents in rural madras of protecting their children against a minor diarrhoeal episode using the contingent valuation . Parental values were captured through parents' willingness to pay (the mean wtp) to treat a diarrhoeal episode . In our analysis, the willingness to pay was specified as a hedonic function of the duration and severity of a diarrhoeal episode, and of parents' socioeconomic characteristics . Correcting for changes in quality is essential in measuring the value of goods and services . The changes in willingness to pay values with changes in severity and duration of illness were also assessed through sensitivity analysis . In other words, we presented parents with different scenarios by varying the degree of severity and the length of an episode . The gender of child was used as a marker to examine if there was any difference in parental valuation as elicited by contingent valuation between the male and female child . The willingness to pay to avoid an episode of diarrhoea among children was modelled as a linear function of the respondent's characteristics and the severity of illness . We estimated the equation using the maximum likelihood method under the assumption that wtp was distributed log normally . Alternative distributional assumptions, such as logistic, exponential, and weibull were rejected in favor of the lognormal distribution using a likelihood - ratio test . Values for dummy variables are omitted since they can be calculated from (m - m), where m is the fraction in the sample . Additionally, it was expected that education (especially mother's education) and age could have positive effects on wtp for child health . The absence of any chronic disease was a proxy for the overall health status, and was captured by three dummy variables (i.e., f_chronic, m_chronic, c_chronic). It was hypothesized that respondents with chronic disease would exhibit higher wtp to avoid an episode of diarrhoea if the marginal disutility of poor health was increasing . Further, doctor's consultation was included to understand parental responsiveness to treatment of the disease . The duration of illness was measured by the logarithm of the number of days of illness (e.g., log_c_dur). It was assumed that the disutility of illness would increase with the severity and duration . Severity was measured by the number of father's lost working days and the number of child's lost school days (e.g., f_work, c_schl). The regression estimates are provided in table 2 . Estimated parental wtp for treating a diarrhoeal episode in rural india absolute value of asymptotic t - statistics in parentheses . Dependent variable is log (wtp) the results indicated that wtp increased with the household income, and there was gender bias against the male child as income level increased . Education had a positive effect on wtp with increased education level favouring the male child . Wtp also increased with the duration and severity of illness . In order to determine if the effects of illness duration and severity on wtp differed between male and female children, we used a likelihood - ratio test for the equality of illness coefficients between comparable specifications . The chi - square statistic for comparing duration, doctor - visit, father's work loss, and child's school loss coefficients was 23.42 (degrees of freedom = 5). However, the value was much higher than the appropriate critical value for the indicated degrees of freedom . Therefore, the hypothesis of parameter homogeneity between two sexes was rejected . The median wtp for both groups of children were calculated at the corresponding sample means of the independent variables . 54.00 (i.e., us$0.541.15) for male and female child, respectively . Unlike many studies involving contingent valuation technique to estimate wtp for individual health state, we used cv to gauge parental wtp for their child's diarrhoeal episode . We used gender as a marker to distinguish parental wtp between male and female children . The setting of the study was in india, where it was generally perceived that parents favoured male children more than female children . This study shows that in terms of the willingness to pay for child's health care, parents differed on their valuations between sexes and were significantly biased towards male children . This differential valuation on the ground of gender raises some policy questions regarding societal development and broader social equity . In general, we found that educated parents were more willing to pay for their child's health care compared to uneducated parents . In particular, the relationship was found to be stronger in the case of mother being educated . However, the results indicate that gender bias towards male children increased as parental education increased . Although, this result emanated from a small sample of rural households, the result nevertheless insinuates that gender bias does not necessarily diminish with higher educational attainment . If this relationship between parental education and valuation of children (as expressed by wtp) holds true for illness, it may well hold true for education, nutrition, access to information, and other social programmes . In other words, this disturbing trend of gender bias can give rise to an inequitable resource allocation between sexes that may lead to an imbalanced social development . However at the same time, well - off households were willing to pay more to treat the female child than the male child . We did not examine the degree of gender biasness by age of children, i.e., whether the age of the child had any influence on the parental willingness to pay . We did not examine the effect of wtp on family size (i.e., number of children in the household). It may be possible that households with less number of children may be willing to pay more for their children than households with large number of children . Finally, this study should be regarded as an exploratory study to examine parental valuation and gender bias in india . The research was carried out with grant assistance from the commonwealth foundation, united kingdom . The authors would like to thank local interviewers and others who have helped in the realization of the study.
To report a case of radiation - induced macular ischemia where vision and macular perfusion improved after hyperbaric oxygen (hbo) therapy . A 62-year - old male patient developed radiation - induced macular ischemia after he was treated with radiation for brain glioma . The patient presented with best spectacle - corrected visual acuity (bscva) acuity of 20/400 in his right eye . The patient s vision improved from 20/400 to 20/100 after focal laser and intravitreal triamcinolone . His central macular thickness improved from 468 m to 132 m . After receiving hbo therapy,
Diabetes mellitus has long been considered a disease of carbohydrate metabolism, and before the discovery of insulin in 1921, low carbohydrate starvation diets were the default treatment . From the 1930s through to the 1960s, many experts continued to advise strict carbohydrate restriction, with the result that most people with diabetes adopted a high fat, low carbohydrate diet . However, some early work in the 1920s and 1930s had suggested that high carbohydrate diets improved glucose tolerance, and the dramatic increase in deaths from vascular disease in those whose lives were prolonged by insulin treatment led to a volte - face in the 1980s, with authorities now recommending low fat, high carbohydrate diets . The pendulum has again swung the other way, and there is renewed interest in very low carbohydrate diets for the treatment of diabetes, with various physicians extolling the virtues of dietary carbohydrate restriction as the first approach in diabetes management, and some authorities recognizing low carbohydrate diets as a suitable weight - loss strategy for those with type 2 diabetes [5, 6]. Interestingly, the carbohydrate debate seems to be based on strong personal opinion and those working in the area tend to cherry - pick the evidence to support their particular view, whether that of low, moderate, or high carbohydrate . Debates about the issue can become very passionate, and it is worth reminding ourselves that passion in science is an infallible marker of lack of evidence . The evidence available is contradictory at best, and leaves both health professionals and people with diabetes alike wondering if low carbohydrate diets do live up to the hype surrounding them, and whether they should be recommended as a suitable treatment . Recent systematic reviews and meta - analyses including people with type 2 diabetes report that although low carbohydrate diets lead to significantly greater weight loss and improvements in glycated hemoglobin (hba1c) and lipids over the short term [7, 8], there is no greater advantage over the longer term [9, 10]. Despite this evidence, low carbohydrate diets remain an area of controversy and this review aims to provide an overview of the latest evidence, and to explore the role of low carbohydrate diets for people with type 2 diabetes . This article is based on previously conducted studies, and does not involve any new studies of human or animal subjects conducted by the author . One of the issues with the term low carbohydrate is uncertainty about what this means in terms of carbohydrate intake . Ketosis readily occurs at carbohydrate intakes below 50 g / day, and these very low carbohydrate, ketogenic diets (vlckd) appear to have more pronounced effects than other, less restricted carbohydrate diets . The taxonomy for diets containing various amounts of dietary carbohydrate has been suggested in a recent paper, see table 1 . In practice, most atkins - style diets are designed to be very low in carbohydrate (less than 20 g / day initially) and high in protein and fat, and other diets, e.g., the zone and the south beach diet, promote a moderate carbohydrate restriction together with high protein and low fat intakes.table 1taxonomy of diets containing differing amounts of carbohydratedescriptionamount of carbohydrateg / day% total energy intakevery low carbohydrate ketogenic diet205010low carbohydrate<130<26moderate carbohydrate1302302645high carbohydrate>230>45adapted from feinman et al . Treating type 2 diabetes is challenging, encompassing as it does management of glycemia, cardiovascular disease (cvd) risk factors, obesity, and other co - morbidities by a combination of lifestyle strategies (diet and physical activity), behavioral and psychological interventions, pharmaceutical treatment, and bariatric surgery . Medical management of type 2 diabetes has led to cynicism about the efficacy of lifestyle management, particularly dietary strategies, and at present the components of the most effective diet remain unknown . A recent systematic review and meta - analysis suggested that low carbohydrate, low glycemic index (gi), mediterranean, and high protein diets all showed greater improvements in glycemic control than control diets . Despite criticism of the statistical analysis due to heterogeneity of the studies included, this review supports the premise that improvements in glycemic control, cvd risk, and weight loss are achievable with different diets with varying amounts of carbohydrate, and that low carbohydrate diets are not necessarily superior in effect . Treating type 2 diabetes is challenging, encompassing as it does management of glycemia, cardiovascular disease (cvd) risk factors, obesity, and other co - morbidities by a combination of lifestyle strategies (diet and physical activity), behavioral and psychological interventions, pharmaceutical treatment, and bariatric surgery . Medical management of type 2 diabetes has led to cynicism about the efficacy of lifestyle management, particularly dietary strategies, and at present the components of the most effective diet remain unknown . A recent systematic review and meta - analysis suggested that low carbohydrate, low glycemic index (gi), mediterranean, and high protein diets all showed greater improvements in glycemic control than control diets . Despite criticism of the statistical analysis due to heterogeneity of the studies included, this review supports the premise that improvements in glycemic control, cvd risk, and weight loss are achievable with different diets with varying amounts of carbohydrate, and that low carbohydrate diets are not necessarily superior in effect . An electronic search of english language articles was performed using medline (2010may 2015), embase (2010may 2015), and the cochrane central register of controlled trials (2010may 2015) using the search terms low carbohydrate diet and type 2 diabetes . The selection criteria included all randomized controlled trials (rcts) comparing interventions evaluating reduced carbohydrate intake with higher carbohydrate intake in people with diagnosed type 2 diabetes . The title and abstract of each record retrieved from the search were screened by the author (pad), and full articles were retrieved if the information given suggested that the study met the selection criteria . Data were extracted using a specially designed form and included information about authors, country, year of publication, primary and secondary outcomes, intervention, and outcomes . Thirteen of these studies were excluded and eight met the inclusion criteria [1825]. The flow diagram illustrating the search and selection of studies . A descriptive summary of the included trials and main results are shown in tables 2, 3, and 4 . It proved impossible to combine the results of the eight selected studies using statistical methods as the studies were heterogeneous in terms of the dietary intervention (carbohydrate intakes ranged from more than 20 g to 166 g / day), length of follow - up (624 months), data quality, and data reporting . 1study flow diagram showing number of studies screened, assessed for eligibility, and included in the narrative reviewtable 2descriptive summary of recent low carbohydrate trials for people with type 2 diabetesfirst author, yearduration (months)numbersdieticiciqbal, 2010 24707430 g / day carbohydrate30% fat, 500 kcal / day energy deficitelhayany, 2010 128517435% carbohydrate, 45% fat (50% of which was mufa)1 . Mediterranean diet: as ada diet, but mufa fatlarsen, 2011 12534640% carbohydrate, 30% protein, 30% fat55% carbohydrate, 15% protein, 30% fatguldbrand, 2012 24303120% carbohydrate, 30% protein, 50% fat5560% carbohydrate, 1025% protein, 30% fatkrebs, 2013 2420721140% carbohydrate, 30% protein, 30% fat55% carbohydrate, 15% protein, 30% fatmayer, 2014 11.5222420 g / day carbohydrate30% fat, 5001000 kcal / day energy deficit and orlistatyamada, 2014 6121270130 g / day carbohydrate to avoid ketosis5060% carbohydrate, <20% protein, <25% fat, with energy restriction based on japanese recommendationstay, 2015 64647<50 g / day carbohydrate (14%)53% low gi carbohydrate, 17% protein, 30% fat ada american diabetes association, c comparator, gi glycemic index, i intervention, mufa monounsaturated fatty acidstable 3summary of results of recent low carbohydrate trials for people with type 2 diabetes (body weight and glycemic control)first author, yearbody weight loss (kg)changes in hba1c (%) ici c p valueici c p valueiqbal, 2010 1.70.21.20.290.10.20.1nselhayany, 2010 8.97.61.40.5572.01.60.40.88larsen, 2011 2.232.170.070.90.230.280.040.76guldbrand, 2012 2.02.90.90.330.00.20.20.76krebs, 2013 3.96.02.10.730.10.10.00.5mayer, 2014 7.58.10.60.80.70.20.80.045yamada, 2014 2.61.41.20.80.60.20.40.03tay, 2014 12.011.50.50.57nrnrnrnr c comparator, hba1c glycated hemoglobin, i intervention, nr not reported, ns no significant differencetable 4summary of results of recent low carbohydrate trials for people with type 2 diabetes (cardiovascular risk)first author, yeartotal cholesterol (mmol / l)hdl (mmol / l)ldl (mmol / l)triglycerides (mmol / l)systolic bp (mm / hg)diastolic bp (mm / hg)i c p valuei c p valuei c p valuei c p valuei c p valuei c p valueiqbal, 2010 0.03ns0.0ns0.06ns0.14ns6.7ns0.5nselhayany, 2010 0.020.2040.18<0.0010.240.0360.64<0.001nrnrnrnrlarsen, 2011 0.160.320.010.840.010.30.170.344.30.050.40.7guldbrand, 2012 0.20.330.120.150.00.160.10.3520.7430.75krebs, 2013 0.070.030.010.410.030.320.030.0210.870.00.96mayer, 2014 0.230.40.030.50.250.30.280.3110.00660.013yamada, 2014 nrnr0.250.130.080.490.580.081.70.544.60.3tay, 2014 0.00.89nrnr0.00.810.40.0012.30.261.80.1 bp blood pressure, c comparator, hdl high density lipoprotein, i intervention, ldl low density lipoprotein, nr not reported, ns no significant difference study flow diagram showing number of studies screened, assessed for eligibility, and included in the narrative review descriptive summary of recent low carbohydrate trials for people with type 2 diabetes ada american diabetes association, c comparator, gi glycemic index, i intervention, mufa monounsaturated fatty acids summary of results of recent low carbohydrate trials for people with type 2 diabetes (body weight and glycemic control) c comparator, hba1c glycated hemoglobin, i intervention, nr not reported, ns no significant difference summary of results of recent low carbohydrate trials for people with type 2 diabetes (cardiovascular risk) bp blood pressure, c comparator, hdl high density lipoprotein, i intervention, ldl low density lipoprotein, nr not reported, ns no significant difference all eight studies reported weight loss in the group receiving the reduced carbohydrate intervention, with mean weight losses ranging from 1.7 kg to 12.0 kg . The greatest weight loss was reported in the shortest study lasting 6 months . There appeared to be no relationship between degree of carbohydrate restriction and weight loss . However, these studies all included a control group receiving dietary interventions that provided higher carbohydrate intakes but were designed for weight loss, consequently those in the control groups also lost weight during the course of the studies . Mean weight losses in the control group were similar to those in the reduced carbohydrate group and ranged from 0.2 kg to 11.5 kg, with the result that none of the eight studies reported significantly greater weight loss in the group receiving the reduced carbohydrate intervention . Despite no significant differences in weight losses, three of the studies reported significantly greater reductions in hba1c in the reduced carbohydrate intervention group [19, 23, 24]. One of the studies did not report hba1c despite the fact that this measurement was defined as the primary outcome, leading to the speculation that there were no differences in glycemic control between the two groups . This has now been confirmed, with recent publication of a follow - up at 12 months reporting no difference in hba1c reductions between the low and high carbohydrate intakes . Changes in hba1c in the reduced carbohydrate intervention groups were variable between studies, ranging from + 0.1% to 2.0%, with the greatest reduction seen in studies of shorter duration . There appeared to be little correlation between the degree of carbohydrate restriction and changes in glycemic control . Hba1c levels were also reduced in five of the seven control groups, with changes ranging from + 0.1% to 0.3% . In summary, one study failed to report hba1c, three studies showed significant reductions in hba1c in the reduced carbohydrate group [19, 23, 24], and four studies showed no significant differences between the two groups [18, 2022]. All eight studies measured lipid concentrations, and seven studies measured blood pressure [18, 2025]. Most studies reported reductions in lipid concentrations in both the reduced carbohydrate intervention and higher carbohydrate control group, with no significant differences between the two groups . However, significantly greater reductions in the reduced carbohydrate group were reported for total cholesterol concentrations in one study, low density lipoprotein (ldl) and high density lipoprotein (hdl) concentrations in one study, and triglycerides in three studies [19, 22, 25]. Changes in blood pressure were variable and showed no significant differences in six of the seven studies reporting outcomes; four studies reported reductions in systolic blood pressure (sbp) in the reduced carbohydrate group compared to the higher carbohydrate group [18, 20, 23, 25], and three reported increases [21, 22, 24]; for diastolic blood pressure four reported decreases in the reduced carbohydrate intervention group [18, 20, 23, 25] and three reported increases [21, 22, 24]. In summary, although there was no evidence of a deleterious effect of a reduced carbohydrate diet on cvd risk, equally, there was no evidence of superiority over a higher carbohydrate intake . Adherence to the prescribed intervention was assessed by self - reported dietary intake using a variety of methods including 24-hour diet histories and 3-, 4-, and 7-day food diaries . In the majority of the studies, mean intake of carbohydrate in the reduced carbohydrate intervention group was higher than that prescribed; in only two studies did the participants achieve target intakes [24, 25]. Attrition rates were reported for seven studies, and ranged from no dropouts to 60% . There were no differences in attrition rates between the intervention and control groups in any of the studies . In general, lower attrition rates were reported for shorter studies, and for those with fewer participants . All eight studies reported weight loss in the group receiving the reduced carbohydrate intervention, with mean weight losses ranging from 1.7 kg to 12.0 kg . However, these studies all included a control group receiving dietary interventions that provided higher carbohydrate intakes but were designed for weight loss, consequently those in the control groups also lost weight during the course of the studies . Mean weight losses in the control group were similar to those in the reduced carbohydrate group and ranged from 0.2 kg to 11.5 kg, with the result that none of the eight studies reported significantly greater weight loss in the group receiving the reduced carbohydrate intervention . Despite no significant differences in weight losses, three of the studies reported significantly greater reductions in hba1c in the reduced carbohydrate intervention group [19, 23, 24]. One of the studies did not report hba1c despite the fact that this measurement was defined as the primary outcome, leading to the speculation that there were no differences in glycemic control between the two groups . This has now been confirmed, with recent publication of a follow - up at 12 months reporting no difference in hba1c reductions between the low and high carbohydrate intakes . Changes in hba1c in the reduced carbohydrate intervention groups were variable between studies, ranging from + 0.1% to 2.0%, with the greatest reduction seen in studies of shorter duration . There appeared to be little correlation between the degree of carbohydrate restriction and changes in glycemic control . Hba1c levels were also reduced in five of the seven control groups, with changes ranging from + 0.1% to 0.3% . In summary, one study failed to report hba1c, three studies showed significant reductions in hba1c in the reduced carbohydrate group [19, 23, 24], and four studies showed no significant differences between the two groups [18, 2022]. Cardiovascular risk was assessed by changes in lipid concentrations and blood pressure . All eight studies measured lipid concentrations, and seven studies measured blood pressure [18, 2025]. Most studies reported reductions in lipid concentrations in both the reduced carbohydrate intervention and higher carbohydrate control group, with no significant differences between the two groups . However, significantly greater reductions in the reduced carbohydrate group were reported for total cholesterol concentrations in one study, low density lipoprotein (ldl) and high density lipoprotein (hdl) concentrations in one study, and triglycerides in three studies [19, 22, 25]. Changes in blood pressure were variable and showed no significant differences in six of the seven studies reporting outcomes; four studies reported reductions in systolic blood pressure (sbp) in the reduced carbohydrate group compared to the higher carbohydrate group [18, 20, 23, 25], and three reported increases [21, 22, 24]; for diastolic blood pressure four reported decreases in the reduced carbohydrate intervention group [18, 20, 23, 25] and three reported increases [21, 22, 24]. In summary, although there was no evidence of a deleterious effect of a reduced carbohydrate diet on cvd risk, equally, there was no evidence of superiority over a higher carbohydrate intake . Adherence to the prescribed intervention was assessed by self - reported dietary intake using a variety of methods including 24-hour diet histories and 3-, 4-, and 7-day food diaries . In the majority of the studies, mean intake of carbohydrate in the reduced carbohydrate intervention group was higher than that prescribed; in only two studies did the participants achieve target intakes [24, 25]. Attrition rates were reported for seven studies, and ranged from no dropouts to 60% . There were no differences in attrition rates between the intervention and control groups in any of the studies . In general, lower attrition rates were reported for shorter studies, and for those with fewer participants . This review of recent studies evaluating the effects of low carbohydrate diets in people with type 2 diabetes supports previous meta - analyses showing that although there may be greater short - term improvements in glycemic control, weight loss, and cvd risk, this is not sustained over the longer - term . Many studies have attempted to determine the ideal macronutrient (protein, fat, and carbohydrate) intake for people with type 2 diabetes, and evidence to date is inconclusive . One of the best predictors of improved outcomes in people with type 2 diabetes is energy restriction and weight loss, and there are a variety of strategies by which this may be achieved, with no clear indication of the superiority of low carbohydrate diets . This is true for both those with type 2 diabetes and those without [10, 29, 30]. Much of the positive effect of low carbohydrate diets is due to weight loss, and the effect independent of weight change is difficult to assess . In the absence of categorical evidence supporting the use of low carbohydrate diets, one wonders why they have gained such strong support and media attention over the past few years . Many proponents of low carbohydrate diets maintain that recent healthy eating guidelines promoting carbohydrate and restricting fat have been counterproductive and have led to escalating rates of obesity and type 2 diabetes . The cause of obesity is extremely complex and it is unlikely that one factor, that of carbohydrate intake, is the root cause . There is also contrary evidence indicating that diets high in fruit, vegetables, whole grains, and legumes (all of which contain carbohydrate) actually protect against obesity, cvd, and, to a lesser extent, type 2 diabetes . Studies often fail to address the type of carbohydrate included in the diet, and this may affect outcomes . There is now accumulating evidence that unprocessed carbohydrates, including whole grains, fruit, vegetables, and legumes, have health benefits, and those from refined sources, including white bread and white rice and particularly sugar and sugar - sweetened beverages (ssb), are associated with increased risk of obesity, cvd, and type 2 diabetes [3639]. It could be speculated that the benefits of low carbohydrate diets are associated with a reduction in refined carbohydrate and not total carbohydrate per se . For people with type 2 diabetes, there is evidence from a large, long - term rct suggesting that higher carbohydrate diets can improve weight loss, glycemic control, and cvd risk factors (although not cvd mortality). The look ahead trial (clinicaltrials.gov identifier, nct0017953) reported greater weight loss, improvements in glycemia and cvd risk factors, and reduced risk of microvascular complications, depression, sleep apnea, and urinary incontinence at 9.6-year follow - up in those allocated an intensive lifestyle education (ile) program compared to standard diabetes education (des). Those in the ile group were encouraged to increase physical activity and adopt an energy - reduced, low fat, partial meal replacement plan . At 1-year follow - up, they derived a higher proportion of energy from carbohydrate (ile 50.8% vs. des 42.5%) and a lower proportion from fat (ile 34.2% vs. des 39.7%), demonstrating that a higher carbohydrate, lower fat diet was associated with improved outcomes . Concern has been expressed about the long - term health effects of low carbohydrate diets on renal function, calcium metabolism, lack of essential nutrients, and cvd risk, and a systematic review and meta - analysis reported that low carbohydrate diets were associated with a significantly higher risk of all - cause mortality . Reductions in carbohydrate intake may also be associated with an increased risk of hypoglycemia in those treated with insulin or insulin secretagogues, and to reduce this medical supervision, reductions in medication and self - monitoring of blood glucose concentrations are recommended for those adopting a low carbohydrate diet . Low carbohydrate diets tend to be higher in protein, and this may have an adverse effect on renal function . There are very few studies investigating renal function and low carbohydrate diets, although a recent study suggested that improvements in renal function are related to weight loss, and that this occurs to a similar extent with low carbohydrate, mediterranean, and low fat diets . In obese people without diabetes, studies have shown that low carbohydrate diets have no harmful effects on glomerular filtration rate (gfr), albuminuria, fluid or electrolyte balance when compared to a low fat diet [47, 48]. It has been postulated that as very low carbohydrate diets cause ketosis, this induces acidosis, promoting urinary calcium loss and leading to low bone mineral density and increased risk of osteoporosis . There is very little research in this field, and none at all in people with diabetes, making it challenging to draw firm conclusions . One animal study showed that low carbohydrate diets induce low bone mineral density in rats, and two small studies in obese subjects reported deleterious effects on urinary calcium loss and markers for bone formation . Conversely, another study reported no effect of a low carbohydrate diet on bone turnover markers . The long - term effects of low carbohydrate diets on calcium metabolism and bone health are unknown . Other claims about the negative aspects of low carbohydrate diets include that of nutritional deficiencies, namely those commonly found in unprocessed carbohydrate foods including vitamins, minerals, dietary fiber, and phytochemicals with antioxidant properties . There is no evidence to either endorse or refute this suggestion, although a computer - generated analysis showed that low carbohydrate diets are deficient in many micronutrients, and an analysis of four popular diets from the usa (atkins, learn, ornish, and zone) demonstrated that all diets showed a degree of deficiency: specifically thiamine, folic acid, vitamin c, iron, and magnesium in the case of low carbohydrate diets . Low carbohydrate diets may be low in dietary fiber and epidemiological evidence suggests that low intakes of dietary fiber are associated with increased risk of lower gastrointestinal disorders, including colon cancer [34, 55], and this may be further exacerbated by high intakes of red meat and meat products . The most controversial aspect of low carbohydrate diets is that they may increase the risk of cvd as they are associated with higher total and saturated fat intakes . There is little evidence for this in people with type 2 diabetes as there are very few studies; as a result many commentators have extrapolated from studies in the general population . There are some issues with the quality of evidence used to define the relationship between fat intake and cvd risk as most studies are short - term rcts with surrogate end points, or observational and epidemiological studies, where associations do not prove causation . Recent meta - analyses and systematic reviews have reported that there is no association between cvd and type of dietary fat, whether saturated fatty acids (sfa), polyunsaturated fatty acids (pufa), or monounsaturated fatty acids (mufa) [57, 58], leading to headlines stating that scientists have been wrong for decades and have mislead the public with low - fat, healthy eating recommendations . However, both these reviews have been widely criticized for omitting important cohort studies, incorrect extraction of data, incorrect interpretation, and a failure to mention the results of other, superior analyses . Many experts still maintain that there is an association between sfa and cvd, and that the evidence supports substitution of sfa by unsaturated fat . The recently published cochrane review also supports this recommendation, stating that there is a small but potentially important reduction in cvd risk with the reduction of sfa . It is worth remembering that most studies examining the relationship between fat intake and cvd include fat intakes in a fairly narrow range of approximately 3040% of total energy intake, and little is known about the relative effects of intakes above these values . This may be an issue for some individuals adopting a low carbohydrate diet where fat, often sfa, is actively promoted to induce ketosis and increase palatability . As is the case with glycemic control, weight reduction improves cvd risk factors and if weight loss is achieved, there are no significant differences between either low fat, high carbohydrate diets and low carbohydrate diets for primary prevention of cvd . On balance, there is little evidence to support changing current recommendations for fat intake in people with type 2 diabetes . There is a further consideration that is now coming to the fore, and that is the challenge of sustainable nutrition . Sustainable diets, as defined by the food and agriculture organization (fao), are nutritionally adequate, safe, affordable, and culturally acceptable and are sparing of natural and human resources . The carbon footprint of different foodstuffs has been investigated, and the results show that red meat is the most carbon intensive process, followed by dairy, fruit, chicken, and vegetables . Low carbohydrate diets tend to include foods with the biggest carbon footprint and large - scale adoption of these diets will increase greenhouse gas emissions . In terms of cultural acceptance newly industrialized countries such as china and india are experiencing a rapid increase in the prevalence of diabetes [66, 67], and it is estimated that by 2030, 551 billion people (10% of the world s population) will have diabetes . For many of these people, a low carbohydrate diet is either unacceptable for religious or cultural reasons, or simply unaffordable . Concern has been expressed about the long - term health effects of low carbohydrate diets on renal function, calcium metabolism, lack of essential nutrients, and cvd risk, and a systematic review and meta - analysis reported that low carbohydrate diets were associated with a significantly higher risk of all - cause mortality . Reductions in carbohydrate intake may also be associated with an increased risk of hypoglycemia in those treated with insulin or insulin secretagogues, and to reduce this medical supervision, reductions in medication and self - monitoring of blood glucose concentrations are recommended for those adopting a low carbohydrate diet . Low carbohydrate diets tend to be higher in protein, and this may have an adverse effect on renal function . There are very few studies investigating renal function and low carbohydrate diets, although a recent study suggested that improvements in renal function are related to weight loss, and that this occurs to a similar extent with low carbohydrate, mediterranean, and low fat diets . In obese people without diabetes, studies have shown that low carbohydrate diets have no harmful effects on glomerular filtration rate (gfr), albuminuria, fluid or electrolyte balance when compared to a low fat diet [47, 48]. It has been postulated that as very low carbohydrate diets cause ketosis, this induces acidosis, promoting urinary calcium loss and leading to low bone mineral density and increased risk of osteoporosis . There is very little research in this field, and none at all in people with diabetes, making it challenging to draw firm conclusions . One animal study showed that low carbohydrate diets induce low bone mineral density in rats, and two small studies in obese subjects reported deleterious effects on urinary calcium loss and markers for bone formation . Conversely, another study reported no effect of a low carbohydrate diet on bone turnover markers . The long - term effects of low carbohydrate diets on calcium metabolism and bone health are unknown . Other claims about the negative aspects of low carbohydrate diets include that of nutritional deficiencies, namely those commonly found in unprocessed carbohydrate foods including vitamins, minerals, dietary fiber, and phytochemicals with antioxidant properties . There is no evidence to either endorse or refute this suggestion, although a computer - generated analysis showed that low carbohydrate diets are deficient in many micronutrients, and an analysis of four popular diets from the usa (atkins, learn, ornish, and zone) demonstrated that all diets showed a degree of deficiency: specifically thiamine, folic acid, vitamin c, iron, and magnesium in the case of low carbohydrate diets . Low carbohydrate diets may be low in dietary fiber and epidemiological evidence suggests that low intakes of dietary fiber are associated with increased risk of lower gastrointestinal disorders, including colon cancer [34, 55], and this may be further exacerbated by high intakes of red meat and meat products . The most controversial aspect of low carbohydrate diets is that they may increase the risk of cvd as they are associated with higher total and saturated fat intakes . There is little evidence for this in people with type 2 diabetes as there are very few studies; as a result many commentators have extrapolated from studies in the general population . There are some issues with the quality of evidence used to define the relationship between fat intake and cvd risk as most studies are short - term rcts with surrogate end points, or observational and epidemiological studies, where associations do not prove causation . Recent meta - analyses and systematic reviews have reported that there is no association between cvd and type of dietary fat, whether saturated fatty acids (sfa), polyunsaturated fatty acids (pufa), or monounsaturated fatty acids (mufa) [57, 58], leading to headlines stating that scientists have been wrong for decades and have mislead the public with low - fat, healthy eating recommendations . However, both these reviews have been widely criticized for omitting important cohort studies, incorrect extraction of data, incorrect interpretation, and a failure to mention the results of other, superior analyses . Many experts still maintain that there is an association between sfa and cvd, and that the evidence supports substitution of sfa by unsaturated fat . The recently published cochrane review also supports this recommendation, stating that there is a small but potentially important reduction in cvd risk with the reduction of sfa . It is worth remembering that most studies examining the relationship between fat intake and cvd include fat intakes in a fairly narrow range of approximately 3040% of total energy intake, and little is known about the relative effects of intakes above these values . This may be an issue for some individuals adopting a low carbohydrate diet where fat, often sfa, is actively promoted to induce ketosis and increase palatability . As is the case with glycemic control, weight reduction improves cvd risk factors and if weight loss is achieved, there are no significant differences between either low fat, high carbohydrate diets and low carbohydrate diets for primary prevention of cvd . On balance, there is little evidence to support changing current recommendations for fat intake in people with type 2 diabetes . There is a further consideration that is now coming to the fore, and that is the challenge of sustainable nutrition . Sustainable diets, as defined by the food and agriculture organization (fao), are nutritionally adequate, safe, affordable, and culturally acceptable and are sparing of natural and human resources . The carbon footprint of different foodstuffs has been investigated, and the results show that red meat is the most carbon intensive process, followed by dairy, fruit, chicken, and vegetables . Low carbohydrate diets tend to include foods with the biggest carbon footprint and large - scale adoption of these diets will increase greenhouse gas emissions . In terms of cultural acceptance newly industrialized countries such as china and india are experiencing a rapid increase in the prevalence of diabetes [66, 67], and it is estimated that by 2030, 551 billion people (10% of the world s population) will have diabetes . For many of these people, a low carbohydrate diet is either unacceptable for religious or cultural reasons, or simply unaffordable . To date the evidence suggests that low carbohydrate diets are effective for weight loss and improvements in glycemic control and cvd risk, but that they are not superior to other dietary approaches . For this reason, low carbohydrate diets cannot be recommended as the default strategy for people with type 2 diabetes . However, they are another useful tool for those who wish to adopt them, although long - term side effects of these diets remain unknown . The question remains how much carbohydrate should someone with type 2 diabetes eat? Both diabetes uk and the american diabetes association recommend an individualized approach, where health professionals work with the person with diabetes to identify an eating pattern that is based on that individual s lifestyle, culture, and preferences . Both authorities identify carbohydrate management as a key strategy and address both type and amount of carbohydrate, emphasizing unprocessed carbohydrate from whole grains, fruit, and vegetable sources . Perhaps it is time to abandon the macronutrient approach to nutritional advice and begin to talk about specific foods and eating patterns and encourage those associated with health . There is no ideal eating pattern that will benefit all people with diabetes, although total energy intake is an important consideration, especially in those who are overweight or obese . Epidemiological and observational studies show that there are dietary patterns that are associated with better overall health outcomes and which are rich in vegetables, fruit, whole grains, seafood, legumes, and nuts, contain moderate amounts of dairy products, and are lower in red and processed meat, sugar, and refined grains . In summary, although low carbohydrate diets appear to be safe and effective in people with diabetes, there are more sustainable alternatives available and this should be fully explained to all those with type 2 diabetes . This article is based on previously conducted studies, and does not involve any new studies of human or animal subjects conducted by the author . This article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made.
The prevalence of overweight and obesity has increased over the last decade, and current measures have not been able to stem the tide . A wide variety of weight management strategies are presently available, and some involve the use of dietary supplements marketed as slimming aids . Hca is a derivative of citric acid and can be found in plant species native to south asia such as garcinia cambogia, garcinia indica, and garcinia atroviridis . Hca is usually marketed as a weight loss supplement either alone or in combination with other supplements [2, 3]. Some authors have suggested that hca causes weight loss by competitively inhibiting the enzyme adenosine triphosphatase - citrate - lyase [36]. Hca has also been reported to increase the release or availability of serotonin in the brain, thereby leading to appetite suppression . Other postulated weight loss mechanisms include inhibition of pancreatic alpha amylase and intestinal alpha glucosidase, thereby leading to a reduction in carbohydrate metabolism . Animal studies have suggested that hca causes weight loss [3, 9], and human trials involving the use of hca as a weight loss supplement have been carried out . The primary objective of this systematic review was to examine the efficacy of hca in reducing body weight in humans, using data from randomised clinical trials . Electronic searches of the literature were conducted in the following databases: medline, embase, the cochrane library, amed, and cinahl . The search terms used included dietary supplements, antiobesity agents, body weight, hydroxycitrate, garcinia, and derivatives of these . We also searched the internet for relevant conference proceedings and hand searched relevant medical journals, and our own files . The bibliographies of all located articles were also searched . Only randomised, double - blind, placebo - controlled studies were included in this paper . To be considered for inclusion, studies had to test the efficacy of oral hca or any of its salts for weight reduction in obese or overweight humans . Studies which involved the use of hca as part of a combination treatment (dietary interventions containing other supplements in addition to hca), or not involving obese or overweight subjects based on body mass index (bmi) values, were excluded from this paper . Data were extracted systematically by two independent reviewers according to the patient characteristics, interventions, and results . The methodological quality of all included studies was assessed by the use of a quality assessment checklist adapted from the consolidated standard of reporting trials (consort) guidelines [10, 11]. In addition, the jadad score was also used to assess the quality of included studies . Mean changes in body weight were used as common endpoints to assess the differences between hca and placebo groups . Using the standard meta - analysis software, we calculated mean differences (mds) and 95% confidence intervals (cis). Studies included in the meta - analysis were weighted by sd (a proxy for study size). If a trial had 3 arms, only the hca and placebo arms were included in the meta - analysis . Our searches produced 5002 hits of which 23 potentially relevant articles were identified (figure 1). Six trials were excluded because they involved the use of hca in combination with other therapies [7, 1418]. One trial was excluded because it was not blinded, and another because it was single blinded . One of these articles was the same trial published in another journal which had been earlier excluded, while the other article was a report of two individual trials which were included in this systematic review . Thus 12 randomised clinical trials (rcts) including a total of 706 participants met our inclusion criteria, and were included in this systematic review [2, 46, 2431]. None of the included studies reported on how double blinding was carried out, and all studies were also unclear about how the allocation was concealed . The randomization procedure was clear in only a third of included studies [4, 6, 25, 29]. Three rcts [4, 28, 31] did not provide actual values to enable statistical pooling (table 3). One of these rcts reported a nonsignificant difference in bmi or body weight between groups, another reported a significant difference (p <.001) in the hca group compared with placebo . The third rct reported a decrease in body weight and (bmi) from baseline for the hca group, without providing results of intergroup differences . A forest plot (random effect model) for studies with data suitable for statistical pooling is shown in figure 2 . The meta - analysis reveals a statistically significant difference in body weight between the hca and placebo groups . The average effect size was, however, small (md: 0.88 kg; 95% ci: 1.75, 0.00), with a p value of .05 . This translates to about 1% in body weight loss in hca group compared with placebo . The i statistic suggests that there was considerable heterogeneity amongst the trials, the duration of treatment, and the dosages of hca used in the different trials varied widely . A funnel plot of mean difference plotted against trial sample size (figure 3) indicated that most of the studies (which had small sample sizes) were distributed around the mean difference of all the trials . The first included 7 trials [2, 5, 6, 24, 25, 29, 30] with parallel - group design, excluding two studies which were crossover [26, 27]. Meta - analysis of these trials revealed md of 1.22 kg (95% ci: 2.29, 0.14). Heterogeneity was substantial . A second meta - analysis for studies with parallel group designs and dosage ranges of hca between 1 and 1.5 g per day [5, 24, 25, 30] did not reveal a significant difference between hca and placebo; heterogeneity was also substantial in this analysis . A third meta - analysis excluding three studies with outlying data for md [6, 29, 30] did not reveal a significant difference in weight loss between hca and placebo, but heterogeneity was considerable . A further meta - analysis of the two trials with good methodological quality [6, 25] revealed a nonsignificant difference in weight loss (md: 0.88 kg; 95% ci: 0.33, 2.10) between hca and placebo, with i value of 0, suggesting that heterogeneity might not be important . Finally, a meta - analysis of the change in bmi for four studies [6, 24, 29, 31] did not reveal any significant difference between hca and placebo (md: 0.34 kg; 95% ci: 0.88, 0.20), with i value of 0 . One study reported a significant decrease in fat mass in the hca group compared with placebo (p <.05), while two studies [4, 24] reported a significant decrease in visceral, subcutaneous, and total fat areas in the hca group compared with placebo (p <.001). In contrast two other studies [5, 25] found no significant difference in body fat loss between hca and placebo . Adverse events reported in the rcts included headache, skin rash, common cold, and gastrointestinal (gi) symptoms . In most of the studies, there were no major differences in adverse events between the hca and placebo groups . However, in one trial, gi adverse events were twice as frequent in the hca group compared with the placebo group . In total a further 45 participants were reported to have been excluded from the analysis in one trial because they either took a mixture of hca and placebo (28), or were males (17). The objective of this systematic review was to assess the efficacy and effectiveness of hca as a weight reduction agent . The overall meta - analysis revealed a small difference in change in body weight between the hca and placebo groups . The effect is of borderline statistical significance and is no longer significant on the basis of a sensitivity analysis of rigorous rcts . The overall meta - analytic result corroborates the findings from one of the studies without suitable data for statistical pooling, but is at variance with another study . Three studies with small sample sizes [6, 29, 30] seemed to have influenced the overall meta - analytic result in favour of hca over placebo . If these three trials are excluded, the meta - analysis result is no longer significant . The largest and most rigorous rct found no significant difference in weight loss between hca and placebo . The result of our systematic review corroborates the findings from a previous systematic review of weight loss supplements, which reported that the weight reducing effects of most dietary supplements is not convincing . The meta - analysis from this systematic review suggests that hca is not as effective as conventional weight loss pills, for example, orlistat . In a meta - analysis report of 16 studies including over 10 000 participants, overweight and obese patients taking orlistat had a clinically significant reduction in body weight compared to placebo (md: 2.9 kg; 95% ci: 2.5, 3.2). Participants taking orlistat achieved a 5% and 10% weight loss compared to placebo in the results from pooled data . This contrasts quite sharply with the results from the meta - analysis of hca clinical trials . All of the studies included in this review had methodological issues, which are likely to have affected the outcomes in these trials . This is supported by the i values from the overall meta - analysis result which suggested substantial heterogeneity . Larger study sizes with a priori sample size calculation will help eliminate a type ii error (i.e., failure to reject the null hypothesis when it should have been rejected). Only one study performed an intention to treat (itt) analysis, while all the participants in three other studies [24, 26, 27] were reported to have completed the trial . The failure of about 66% of the included studies to report itt analyses casts a doubt as to the validity of their results . In several of the rcts, drop - outs / attrition was unclear . In one study, participants were excluded due to mixed - pill ingestion (an error in coding of pill bottles resulted in some participants receiving a mixture of hca and placebo). Male participants were also excluded from the analysis of this rct because they were too few in number compared with females in the trial . It was also unclear to which intervention group the excluded participants belonged to in this study . The dosage of hca, and the duration of study also varied amongst the rcts . The dosage of hca used ranged from 1 g to 2.8 g daily . Two included studies which differed widely in results [25, 29] also differed widely in dosage of hca . Though one of these studies claimed the bioavailability of the hca used in their trial was high, the dosage of hca used was almost twice that used in the other trial . It is not clear if the higher hca dosage ensures a higher bioavailability of hca . A nonlinear, significant (p <.05) correlation between the dosage of hca and body weight loss seems to exist (figure 4). Garcinia cambogia was the main source of hca in most studies, with garcinia atroviridis being the source of hca in one included study . Furthermore hca is also reported to be found in hibiscus subdariffa, and none of the studies included in this review used hca extracted from this plant species . The duration of the studies included in the review also differed, with a range of 2 to 12 weeks, and mode of 8 weeks . This is probably too short a time to assess the effects of hca on body weight . All of the studies included had parallel - study designs except two which were crossover trials [26, 27]. Four included rcts comprised three intervention groups [6, 26, 27, 29]. None of the included studies indicated whether or not outcome assessors were blinded, and seven studies did not specify the source of funding [2, 4, 6, 24, 28, 29, 31]. The failure of study investigators to adhere strictly to the consort guidelines [10, 11] may have contributed to the variation in methodology (and heterogeneity) of the trials included in the review . Most (7/12) rcts reported adverse events, with headache, nausea, upper respiratory, and gastrointestinal tract symptoms being the most frequent ones . In most of the trials, there were no significant differences in adverse events between hca and placebo . This seems to corroborate the report in another article which suggested that hca is safe for human consumption . A few of the studies reported a positive effect of hca on the blood lipid profile [6, 24, 2931], while one did not find any significant difference between hca and placebo on this blood parameter . However, given the short duration of the studies involving the use of hca, it is unclear how safe this dietary supplement is on the intermediate and long term . In 2009, the food and drug administration (fda) warned consumers about the potential for serious adverse effects associated with the consumption of hydroxycut, a popular hca - containing slimming pill . All of the studies included in this review except two [26, 27] incorporated some form of dietary control into their trials, with participants in one study receiving high fibre diets . The daily caloric intake for participants in the trials included in this review ranged from as low as 1,000 kcal [2, 30], to as high as 3,009 kcal . Half the number of studies in this review did not institute any form of exercise . The extent to which the variation in these lifestyle adjustment factors could have influenced study results is uncertain . Two studies [28, 31] reported a significant reduction in appetite in the hca group (p <.001), but not with placebo . Three other studies did not find any significant difference between hca and placebo groups in terms of satiety effect [5, 26, 27]. All of the studies described their participants as overweight, obese, or both . However, in one rct, the definition of the participants as obese individuals is questionable, because they had a bmi between 2530 kg / m . Based on the world health organisation definition, a bmi between 2529 kg / m is considered overweight, while a bmi 30 kg / m is termed obese . Though our search strategy involved both electronic and non - electronic studies, we may not have identified all the available trials involving the use of hca as a weight loss supplement . Furthermore, the methodological quality of most of the studies identified from our searches is poor, and most studies are of short duration . These factors prevent us from drawing firm conclusions about the effects of hca on body weight . The evidence from rcts suggests that garcinia extracts / hca generate weight loss on the short term . However, the magnitude of this effect is small, is no longer statistically significant when only rigorous rcts are considered, and its clinical relevance seems questionable.
The tumor was named aggressive due to its characteristically slow and insidious growth as well as carrying a moderate - to - high risk of local relapse . Usually, it presents as a vulval polyp clinically and is diagnosed only on histopathology . It is a rare local mesenchymal tumor of unknown etiology usually affecting vulva, perineal region, buttocks, or pelvis of women in reproductive age . Considering its nature of aggression and chance of local relapse, appropriate management and long - term follow - up are necessary to diagnose early recurrence . No single modality of treatment of recurrence has been found to be of proven benefit till now . Radiotherapy and chemotherapy have been used as adjunctive therapies but are unlikely to be useful as it has few mitotic activity . Despite the availability of many options of treatment, recurrence of aa a 40-year - old female para 2 presented with a swelling on the right labia majora with a duration of 3 years growing slowly throughout and increased in size for 6 months [figure 1]. Pedunculated large, partly encapsulated growth from vulva there was no history of any vulval discharge, bleeding, sexual difficulty, or pain, except a sensation of weight hanging while standing . Menstrual cycles were regular with a normal flow . Local examination revealed a well - circumscribed pedunculated fleshy polypoidal mass measuring 18 cm 10 cm . Ultrasonography (usg) of the swelling revealed heterogeneous hyperechoic areas with peripheral vascularity, thick echoes, and nonvascular central areas . Computed tomography (ct) scan of pelvis revealed no pelvic disease inside and had similar findings of the mass like usg . With a clinical diagnosis of a vulvar fibroepithelial polyp or lipofibroma, she underwent local excision of the tumor with ligation of the stalk [figure 2]. The cut surface revealed a glistening, gelatinous, and soft homogeneous appearance [figure 3]. On histopathology, the tumor was composed of spindle- and stellate - shaped cells scattered in a myxoid background [figures 4 and 5]., there would be spindle cells in fatty background with minimum of vessels unlike aa . There were many variable - sized thin - walled and thick - walled vascular channels in the histopathological specimen, which were diagnostic of aa . A 6 monthly follow - up has been done for more than 2 years now without any specific therapy for the prevention of recurrence, yet showing no sign of any relapse so far . Cut open specimen of angiomyxoma showing homogeneous white area high power picture of angiomyxoma, (h and e, 40) immunohistochemistry picture of angiomyxoma, cd34 (low power) postoperative picture of vulva after resection of angiomyxoma the aa tumor commonly presents as an asymptomatic mass in the genital area of women in their reproductive life, but is occasionally reported in men (male - to - female ration being 1:6). The term aggressive denotes its propensity for local aggression and recurrence after excision . Clinically, aa may misdiagnosed as bartholin cyst, lipoma, labial cyst, gartner duct cyst, etc . Superficial angiomyxoma, angiomyofibroblastoma, cellular angiofibroma, and smooth muscle tumors also need to be considered in the differential diagnosis of a polypoidal mass in the perineum . In addition, aa has thick - walled vessels, which are less numerous than thin - walled vessels in angiomyofibroblastoma . On ct scan, these tumors have a well - defined margin with attenuation less than that of muscle . The attenuation on ct and high signal intensity on magnetic resonance imaging (mri) are likely to be due to the presence of loose myxoid matrix and high water content of aa . Usually, this tumor does not metastasize, but there are reports of multiple metastases in women treated initially by excision and ultimately succumbing to it . This hormonally responsive tumor is believed to arise from specialized mesenchymal cells of the pelvic perineal region or from the multipotent perivascular progenitor cells, which often display variable myofibroblastic and fibroblastic features . Immunohistochemically, most aa express different combinations of estrogen and progesterone receptors, vimentin, desmin, smooth muscle actin cd34, and cd44, but all are invariably negative for s-100, carcinoembryonic antigen, and keratin . Recent cytogenetic and molecular studies have revealed a variety of genetic alterations, involving the chromosome 12, in the region 12q13 - 15 . A gene in this region, called high - mobility group protein isoform i - c (hmgi - c), which encodes protens involved in the transcriptional regulation, appears to have a role in the pathogenesis of this tumor . Detection of inappropriate hmgi - c expression using the immunoperoxidase technique with anti - hmgi - c antibody may potentially be a useful marker for microscopic residual disease . Unlike most aas, our case was completely encapsulated without any breach in continuity and without any projections into the neighboring tissues . This may be the reason why there was no recurrence in the past 2 years or so . Considering her very poor economic status and the hpe report, we decided not to start any preventive therapy, i.e., gnrh agonist, etc ., in her case . On h and e staining, the tissue resembled a typical aa, and immunohistochemically, it showed positive for cd34, desmin, and vimentin, hence proving beyond any doubt that it was a case of vulval aa . Our patient required no additional treatment or any investigations postoperatively till date and has been asymptomatic and enjoying good health . Despite this fact, aa is notorious for local recurrence in approximately 70% of the cases after a period of 2 years postoperatively, and it has been reported even 20 years after surgery as well . Proposed the following guideline for treating aa: (1) complete excision of the lesion when possible, avoiding mutilating surgery, (2) adjunct therapy using arterial embolization and/or hormonal treatment needed in case of partial resection of the tumor, and (3) radiotherapy is reserved for cases that are resistant to embolization and/or hormonal therapy and still symptomatic . There are no specific guidelines for postoperative management of vulvar aa; however, due to high recurrence rate and potential morbidity associated with undiagnosed recurrences, several authors recommend a periodic evaluation with physical examination and mri up to 15 years after excision . This case report illustrates the challenges that a physician might face when dealing with a vulvar mass which may be an aa . Though it is a rare entity, it should always be considered, especially when it is an insidious painless lesion, particularly in premenopausal women in their third to fourth decades of life . All relevant hematological and radiological studies including mri or ct scan should help in reducing the number of misdiagnosed cases of aa preoperatively . Once its anatomical location and extension, if any, are defined, any vulvar tumor particularly aacan be optimally treated by surgical excision only, while avoiding any mutilating surgery . If complete resection is possible under the circumstances, one should expect lowest recurrence rate . Aa is rarely life - threatening, and therefore one can afford to have a partial resection when high operative morbidity is anticipated . Irrespective of treatment modalities instituted postsurgery, it is evident that aa requires close and long - term follow - up.
Chronic autoimmune hepatitis (cah) is a serious hepatic disease with an immunological attack against the liver cells . Autoimmune diseases result from immune response toward healthy tissue; hence, they can have protean manifestation with multi - system involvement . Central pontine myelinolysis (cpm) is an uncommon condition with demyelination predominantly in the central portion of the basis pontis . Extrapontine areas such as putamen, caudate, thalamus, cerebellum, splenium of the corpus callosum and sub - cortical white matter are also sometimes involved . It may also be seen in alcoholics, severe liver disease, after liver transplant, malnutrition, hyperemesis gravidarum and autoimmune deficiency syndrome or toxin exposure . In the past however, no case of central and peripheral demyelination in the setting of chronic active hepatitis has been described and it is essential to recognize the symptoms early to prevent the deficits . A 52-year - old male carpenter, vegetarian by diet, normotensive, non - diabetic and euthyroid presented to neurology department with history of progressive quadriparesis since 4 months . He was diagnosed having chronic active hepatitis 15 months back and was initially started on steroids and was tapered to prednisolone 5 mg / day . He attended the neurology department when he developed dysarthria and became drowsy 2 days prior to admission . On examination, patient was drowsy, but arousable, well oriented to time, place and person and would soon drift back to sleep . On general examination, he had no icterus, flaps and sphincter dysfunction or neck stiffness, but had mild pedal edema . Tone was flaccid in the limbs; with power proximally 2 - 3/5 and distally 4/5 . Full blood count, liver enzymes, coagulation screen, electrolytes, serum ammonia and renal function were normal . Ultrasound abdomen showed shrunken liver with irregular margins and diffusely altered echo texture suggestive of chronic liver disease . Immunological tests demonstrated positive smooth muscle antibody and positive anti - nuclear antibody homogenous staining pattern at a titre of 1 in 6 . There was an accompanying hyper - gammaglobulinemia with elevated immunoglobulin g at 2507 (normal range 700 - 1600) and normal immunoglobulin a levels . Double stranded deoxyribonucleic acid antibodies, anti - mitochondrial antibodies and liver / kidney microsomal antibodies were not detected . Thyroid profile, serum b12, ceruloplasmin, alpha fetoprotein levels, protein electrophoresis was normal; lupus anticoagulant, p - anca, c - anca was negative . The positive anti - nuclear and smooth muscle antibody results were strongly suggestive of autoimmune hepatitis and ruled out other autoimmune disorders . Magnetic resonance imaging of brain (t2-weighted) showed hyper intense signals in the basis pontis [figure 1] suggestive of cpm which accounted for his drowsiness . Nerve conduction studies showed prolonged distal latencies in median (5.5 ms), posterior tibial (6.6 ms) and peroneal nerves (6 ms); decreased motor conduction velocity in median (25 m / s), posterior tibial (30 m / s) and peroneal nerves (41 m / s). F waves were prolonged in median (56), post tibial nerves (70) and peroneal nerves (69). Hyperintense signal in t2-weighted images in the central pons suggestive of central pontine myelinolysis with these parameters, patient was diagnosed to have central and peripheral demyelinating disorder . After 10 h of admission, patient sensorium improved and started showing improvement in motor power after 5 days . On discharge, he was able to walk with minimal support on oral corticosteroids . On follow - up after 8 months, he was stable and was maintaining improvement . Our patient had areflexic quadriparesis, eye closure and neck flexor weakness with sphincter sparing, which was suggestive of cidp . The other differential was spinal cord demyelination; however, there was no corticospinal tract involvement clinically as well as on imaging . All the possible secondary causes of cidp were ruled out by appropriate investigations . As patient was drowsy, impending hepatic encephalopathy was thought . The underlying cause is possible molecular mimicry between peripheral nerve glycolipids and myelin proteins with various infectious agent components resulting in an immune response to the peripheral nerves . The cause of cidp is abnormal immune response wherefore the specific triggering mechanism for dysimmune response varies . Cpm is a form of central demyelination with several causes, but the exact pathogenesis is still not known . After, it has been described in patients who underwent too rapid correction of hyponatremia . Demyelination has been postulated due to cellular stress, which is the result of fluctuating osmotic forces and ion shifts that lead to changes in cell volume and cell membrane function . Other clinical studies have shown that myelinolysis may be due to the brain's overshoot of sodium during correction of hyponatremia or compression of myelin by edematous cellular elements . The manifestations of cpm may be mild symptoms like behavioral disturbances, drowsiness, confusion, dysarthria, dysphagia, quadriplegia or seizures . Our patient had mild symptom, there was no pupillary abnormality as tegmentum was spared . As patient was already quadriplegic, the motor weakness due to the cpm could not be ascertained . These syndromes are not exclusive to liver disease and can occur from a variety of causes, most commonly with alcoholic liver disease . Simultaneous central and peripheral demyelination is very rare; though, there have been previous reports in the past . However, in most of the cases reported there is usually a subclinical involvement of one neuroaxis with more overt involvement of the other . We wish to report it as treating physicians should be aware of this potential complication . Our patient highlights the fact that in a susceptible individual, both the central and peripheral myelin can be under the immunological attack . Early diagnosis and prompt treatment can prevent further progression in such cases and preventing morbidity and mortality.
A pregnant 23-year - old female with a six - year history of type 1 diabetes presented with a complaint of increased blur in both eyes for the previous two months . Best corrected visual acuity was 20/40 ou . Slit - lamp examination was entirely unremarkable in both eyes . Dilated funduscopic examination was significant for flame - shaped, dot, and blot hemorrhages in the posterior segment, with associated macular edema and exudates bilaterally (figures 1a and 1b). Optical coherence tomography (oct) revealed a foveal thickness of 578 5 microns and 667 8 microns in the right and left eye, respectively (figures 2a and 2b). All oct scans were performed with the stratus optical coherence tomograph (zeiss - humphrey inc, dublin, ca). Examination findings were consistent with bilateral, nonproliferative diabetic retinopathy and clinically significant macular edema . After careful deliberation with the patient and her obstetrician, a decision was made to treat the bilateral macular edema with intra - vitreal triamcinolone acetonide injection . The patient received 0.05 ml of triamcinolone acetonide 40 mg / ml in the left eye initially and in the right eye one week later . Best - corrected visual acuity was 20/20 and 20/25 in the right and left eye, respectively . Repeat oct revealed a foveal thickness of 159 5 microns and 202 6 microns in the right and left eye, respectively (figures 3a and 3b). Slit - lamp examination and goldman applanation tonometry did not reveal any intraocular hypertension or significant lenticular changes at this visit or at any point after triamcinolone injection . The patient delivered a full - term, healthy baby boy weeks prior to this examination . Progression of diabetic retinopathy during pregnancy has been described previously by several authors.1,2 visual impairment in these cases can result from both proliferative (eg, vitreous hemorrhage, retinal detachment) and nonproliferative etiologies (eg, retinal hemorrhage, papillopathy, macular edema).1 although macular edema may regress in some cases after delivery, in other cases edema can persist and can be associated with severe and persistent visual dysfunction.1 data from large, randomized clinical trials have established the benefits of argon laser photocoagulation for clinically significant macular edema.3 focal laser photocoagulation of actively leaking blood vessels or grid laser for areas of diffuse permeability can decrease clinically significant edema . However, laser photocoagulation in close proximity to the fovea increases the risk of inducing iatrogenic central scotoma as a result of thermal injury to the tissues, or subsequent glial proliferation . There are many reports of off - label use of intravitreal triamcinolone in cases of persistent and refractory diabetic macular edema.4 improved visual acuity and decreased foveal thickness have been documented by serial oct after a single injection . However, these effects do not appear to persist beyond 34 months without repeated administration.5 a medline search using keywords pregnancy revealed no case reports or case series documenting the treatment of clinically significant macular edema with intravitreal corticosteroids in a pregnant patient . Side effects of systemic corticosteroid administration are well known . Major ophthalmic complications of intravitreal corticosteroid injection include, but are not limited to, cataract formation and increased intraocular pressure . Although corticosteroid equivalents can be measured in the aqueous three months after a single intravitreal injection, it is not known how much corticosteroid is released into the systemic circulation after a single intravitreal injection.6 thus, it is hard to estimate the systemic effects of intravitreal corticosteroid therapy . To date, there are no reports of teratogenic outcomes with systemic corticosteroid use in pregnant human females . However, teratogenic effects have been observed in many species receiving equivalent systemic human doses.7 many authors have noted maxillofacial deformity and, in particular, cleft palate in mice which have received corticosteroids early in gestation . The area of greatest thickening was within the fovea of both the right and left eyes, with little edema observed in the extrafoveal area . Laser photocoagulation to this area carried a significant risk of inducing a permanent central scotoma . Given the patient s late stage of pregnancy, we felt that intravitreal steroid posed little risk to either mother or fetus . We were able to achieve resolution of macular edema and improved visual acuity with a single intravitreal dose to each eye . In conclusion, we propose that intravitreal triamcinolone injection may be a viable treatment modality for management of clinically significant macular edema in pregnant patients . The safety profile with administration of this medication is enhanced if the steroid is administered after the first trimester . We suggest a multidisciplinary approach and consultation with an obstetrician whenever using corticosteroid therapy in pregnant patients.
We report choroidal findings by means of enhanced depth imaging spectral - domain optical coherence tomography (edi - oct) in a patient with idiopathic uveal effusion syndrome (iues). A 41-year - old man was referred to us with ciliochoroidal and non - rhegmatogenous retinal detachments . Sclerectomies and sclerostomies were performed at the equator in the lower quadrants, resulting in resolution of the ciliochoroidal and retinal detachments . The subfoveal choroidal thickness measured vertically from the outer border of the rpe to the inner border of the sclera was 787 m which was significantly thicker than the normal value (272 90 m, n = 131) obtained from age - matched normal controls . The findings made by edi - oct have provided additional evidence that choroidal alterations play a role in the pathological process in iues . Idiopathic uveal effusion syndrome (iues) is a rare disease characterized by ciliochoroidal and non - rhegmatogenous retinal detachments associated with an abnormality of trans - scleral diffusion of extravascular proteins in the choroid.1,2 therefore, enhancement of protein diffusion by lamellar sclerectomies and sclerostomies results in resolution of the ciliochoroidal and non - rhegmatogenous retinal detachments.3 enhanced depth imaging spectral - domain optical coherence tomography (edi - oct) has been developed to allow clinicians to evaluate the thickness and structure of the choroid.4 for example, it has been demonstrated in edi - oct images that choroidal thickness is significantly thinner in highly myopic eyes.5 we present our edi - oct findings together with the choroidal angiographic findings in a patient with iues . A 41-year - old man was referred to us in july 2001 complaining of decreased vision in his right eye . He had a history of serous macular detachments in both eyes in 1996, which resolved spontaneously . His best - corrected visual acuity (bcva) was 0.3 od and 1.0 os with mild myopia in both eyes . The axial lengths of the globe were 22.95 mm and 22.65 mm for the right and left eyes, respectively . There were no inflammatory cells in the anterior chamber and vitreous cavity in either eyes . The posterior pole of the ocular fundus showed atrophy of the retinal pigment epithelium (rpe; figure 1a). An annular ciliochoroidal detachment and a shifting bullous retinal detachment were noted in the right eye (figure 1b and c). In the left eye, the vortex veins were only discernible in the temporal superior quadrant in the right eye and in the inferior lower quadrant in the left eye . Sclerectomies and sclerostomies were performed at the equator in the lower quadrants of the right eye in september 2001, resulting in resolution of the ciliochoroidal and retinal detachments . During the surgery the ciliochoroidal and retinal detachments recurred 6 months after the surgery, and the vision decreased to light perception in the right eye because of chorioretinal atrophy . The patient re - visited us complaining of decreased vision in the left eye, in september 2005 . Atrophy of the rpe in the posterior pole and annular ciliochoroidal detachments were noted in the left eye (figure 1d and e). We performed sclerectomies and sclerostomies with application of mitomycin c (mmc) in the inferior quadrants of the left eye in november 2005 . Medical quick absorber (inami co, tokyo, japan) soaked in 0.04% mmc was placed on the sclera after the sclerostomies for 5 minutes to avoid postoperative fibrosis as used for glaucoma filtering surgery . The ciliochoroidal and retinal detachments promptly resolved after surgery with improvement of vision to 1.0 . Oct demonstrated a serous macular detachment and intraretinal fluid . In may 2010, fluorescein angiography (fag) showed marked hyperfluorescence in the macular region of the left eye due to window defect with late subretinal leakage (figure 2a and b). Indocyanine green angiography (icga) revealed diffuse and moderate hyperfluorescence that obscured large choroidal veins in the posterior pole of the ocular fundus in the early phase (figure 2c), which was consistent with previously reported icga findings.6 in the late phase of icga, hypofluorescence was noted throughout the posterior pole (figure 2d). Topical steroid application resulted in a resolution of the serous macular detachment and the intraretinal fluid in the left eye, with improvement in vision . The arrow and line in figure 2c indicates the direction and length of the edi - oct scan . Edi - oct demonstrated low - reflective areas (indicated by asterisks in figure 2e) in the outer choroid (figure 2c and e) in april 2011 . The subfoveal choroidal thickness was measured vertically from the outer border of the rpe to the inner border of the sclera . The choroidal thickness was 787 m (indicated by the arrow in figure 2e) which was significantly thicker than the normal value (272 90 m, n = 131) obtained from age - matched normal controls (indicated by the arrow in figure 2f). Our patient had annular ciliochoroidal detachments with shifting subretinal fluid which was resolved by the sclerectomies and sclerostomies . The patient was a healthy and middle - aged man without nanophthalmos or medical history of any other disease . These characteristics are consistent with those of iues.2 the edi - oct images showed large low - reflective areas in the outer choroid, which could represent dilated choroidal veins or an enlargement of the suprachoroidal space . The icga failed to delineate large choroidal veins in the posterior pole in the early phase because of the diffuse hyperfluorescence throughout the ocular fundus . However, congestion of the choroidal veins could take place because of the abnormality of the vortex veins . Alternatively, the low - reflective areas in the edi - oct images may represent an accumulation of extravascular proteins in the suprachoroidal space . These choroidal findings are comparable to the hypothesized disease mechanism of iues in which extravascular proteins triggered by choroidal congestion accumulate in the suprachoroidal space leading to choroidal and retinal detachments.3 a congenital anomaly of the sclera superimposed on aging and hormonal changes is hypothesized to prevent transport of extravascular proteins across the sclera.3 in fact, the sclera of our patient appeared to be thick and rigid during the surgeries . The findings made by edi - oct have provided additional evidence that choroidal alterations play a role in the pathological process in iues.
Dna topoisomerasesregulate the topological state of dna as required to relieve superhelical density for important biological processes such as replication and transcription [13]. Dna topoisomerase 1 (top1) is expressed at elevated levels during s - phase of the cell cycle and is the topoisomerase primarily responsible for relieving superhelical density generated in front of advancing replication forks in mammalian cells . Top1 preferentially binds superhelical dna and forms a covalent complex as a result of nucleophilic attack by the hydroxyl of tyr 723 on the phosphodiester backbone of the scissile strand of the dna duplex . Dna superhelical density is reduced by controlled rotation of the scissile strand about the nonscissile strand in the cleavage complex [4, 5]. Following release of superhelical tension, the cleavage complex is dissociated by nucleophilic attack of the free 5-oh of the scissile strand to reform the phosphodiester backbone . Dna sequences that have several a - tracts flanking a conserved dna duplex motif are also substrates for dna top1 and serve as a model system for understanding dna recognition and catalysis by top1 . Top1 is the sole target for the camptothecin (cpt) class of anticancer drugs . Cpt forms a stable ternary complex upon binding to the top1:dna covalent cleavage complex . Stabilization of cleavage complexes by cpt converts top1 into a cellular poison since collision of advancing replication forks with trapped top1 cleavage complexes results in dna double - strand breaks . Thus, cpt not only inhibits top1 activity, but also converts the enzymatic activity into dna damage that is potentially lethal to the cell . Over the last decade, it has been shown that a variety of nonnative nucleotide substitutions that may result from oxidative damage to dna (e.g., 8-oxo - dg) or covalent modification of dna nucleobases (e.g., benzpyrene adducts) also cause trapping of top1 cleavage complexes and result in dna dsb formation . Work from our laboratory in collaboration with the pommier lab has shown that misincorporation of deoxyribonucleotide analogs that have anticancer activity, such as fdu and gemcitabine, into top1 cleavage sites also causes trapping of top1 cleavage complexes . Poisoning of top1 by fdu - substituted dna contributes to the cytotoxicity and antitumor activities of fluoropyrimidines . The structural basis for trapping of top1 cleavage complexes by damaged nucleobases or misincorporation of nucleotide analogs into the nonscissile strand of dna remains an area of investigation . Although the dna sequence used in most model studies of top1:dna interactions contains several a - tracts, x - ray crystal structures do not reveal any bending of this dna in either covalent or noncovalent complexes with dna . One question that remains unanswered is how introduction of nonnative nucleotides into the nonscissile strand of dna inhibits the religation reaction . To investigate this issue, we have constructed a model top1 cleavage site consisting of a 39 mer dna hairpin consisting of 13 base pairs with a 10 mer single - stranded overhang (figure 1). We have investigated the thermal stability of this dna hairpin consisting of all native nucleotides and have compared the stability of the native sequence to sequences that contain a single c du, c fdu (5-fluoro-2-deoxyuridine), or c t substitution . These substitutions result in a single mismatched base pair at the site corresponding to the + 1 site relative to the site of top1 cleavage . As expected, introduction of a mismatched base pair decreases the stability of the dna hairpin by approximately 3c . We also investigated the stability of dna duplex formation between these dna hairpins and a dna 10 mer that is complementary to the 10-nucleotide overhang of the hairpin . The interaction of this dna 10 mer with the hairpin provides a model system for the top1 religation reaction . Unexpectedly, we find that the melting temperature for the formation of the 10 mer duplex that would be required for the top1 religation reaction to occur is sensitive to the presence of dna mismatched base pairs in the hairpin even though no mismatched base pairs are present in the 10 mer duplex region . Molecular dynamics simulations of this model system demonstrate that g - du and g - fdu mismatched base pairs increase flexibility and affect coupling with the first 10 base pairs . We conclude that dna mismatched base pairs adjacent to the top1 cleavage site both decrease dna stability and increase flexibility disfavoring formation of the dna conformation required for top1-mediated dna religation . A model top1 cleavage site (figure 1) was designed based upon a dna sequence containing several a - tract motifs that had been previously demonstrated to be a suitable model substrate for the top1 cleavage / religation reaction . The model top1 cleavage site was synthesized in two pieces, a 39 mer dna hairpin containing the gaa sequence that promotes hairpin formation and a 10-nucleotide single - strand dna complementary to the 3-terminus of the hairpin . Upon annealing, the two sequences from a duplex with a single nick in the phosphodiester backbone of one strand corresponding to the scissile strand of the top1 cleavage complex (figure 1). To investigate the effects of deoxynucleotide substitutions on the stability of the model top1 cleavage site, the 39 mer was synthesized four times, once with dc as the putative base pairing partner for the 3-terminal dg and also with t, du, or fdu at this site . In this manner, the base pair at the + 1 site of the model top1 cleavage complex was either the native g - c base pair or was a g - t, g - fdu, or g - du mismatched base pair . These mismatched base pairs occur at the junction corresponding to the site of cleavage for the top1 complex . All dna sequences were synthesized at the university of calgary dna core synthesis facility and purified by gel filtration chromatography . Absorbance versus temperature profiles of each oligonucleotide in buffer were measured at 260 nm using a thermoelectrically controlled aviv model 14ds uv - vis spectrophotometer (lakewood, nj). The temperature was scanned from 20c to 95c for the 39 mer dna hairpins and from 1100c for the model top1 cleavage sites at a heating rate of 0.6c / min . Dna concentrations were 1.52.0 m, and the buffer used was 10 mm sodium phosphate, ph 7.0 with 200 mm nacl added for high - salt conditions . The simulation of the four hairpins is performed using namd with the charmm27 force field [14, 15], with analysis performed using charmm . A normal, matched dna structure was built using predictor to construct 23 base pairs using the same sequence as in the experiments . The resulting overall structure was minimized in charmm with an r - dependent dielectric of 4r, and harmonic restraints to remove bad contacts . The minimization cycle was (1) 100 steps of steepest - descent minimization followed by 100 steps of conjugate gradient minimization both with best - fit harmonic constraints on the hairpin atoms with a mass - weighted force constant of 1 with the remaining bases fixed to relieve any bad contacts with the hairpin; (2) 100 steps of steepest - descent minimization followed by 100 steps of conjugate gradient minimization both with harmonic constraints on all atoms with a mass - weighted force constant of 10; (3) 100 steps of steepest - descent minimization followed by 100 steps of conjugate - gradient minimization both with harmonic constraints on all atoms with a mass - weighted force constant of 1 . The other three hairpins were built by mutating the original matched structure and rebuilding the altered base within charmm followed by the same minimization protocol . Missing parameters for fdu were obtained from our previous quantum mechanical study and the existing fluorine parameters within charmm27 supplemented by two dihedrals created using the parameters from our previous study with force constants from the corresponding unperturbed dihedrals . The resulting structures were fully solvated and charge neutralized with tip3p water in a cubic box using the visual molecular dynamics (vmd) package . The simulation was performed in namd using standard parameters: a 2.0 fs timestep using shake on all bonds to hydrogen atoms, a 12 cutoff, particle mesh ewald with a 1.0 grid determined by namd, langevin constant pressure algorithm with a target pressure of 1.01325 bar, a piston period of 100 fs, a piston decay time of 50/ps, and a piston temperature of 300 k, all as implemented in namd . The simulation protocol consisted of 1000 steps of unconstrained steepest - descent minimization on the fully solvated and ionized system, followed by 250 ps of thermal equilibration to 300 k with temperature reassignment, followed by a 812 ns of production simulation; 10 different simulations with different random initial conditions were performed for each hairpin for a total of 40 simulations . Based on exchange between clusters after 4 ns all - atom clustering and leveling of the all - atom rmsds (data not shown), the first 4 ns of each simulation was discarded as equilibration . The remaining 240 ns in total of simulation data was analyzed with structures saved every 2 ps . Charmm's analysis routines were used to calculate the root - mean - square fluctuations, covariances, and for rmsd - based clustering with a 2.5 cutoff . Matlab was used to perform the clustering analysis of the data with single - linkage euclidean distance measures and an inconsistent value of 0.001 . The effects of mismatched base pairs at the + 1 position on the stability of the model top1 cleavage site were investigated using uv hyperchromicity measurements . Initial studies focused on the stabilities of the four 39 mer dna hairpins (figure 2). The parental hairpin consisting of only watson - crick base pairs in the stem region had a tm of 61.0c . Introduction of a g - t mismatched base pair reduced the stability of the hairpin by 4.4c . The g - du and g - fdu mismatched base pairs also destabilized the hairpin decreasing the tm by 3.0 and 3.5c, respectively . The results demonstrate that introduction of a single mismatched base pair at the + 1 site relative to top1 cleavage destabilizes the duplex by 34.4c . The results are consistent with previous studies indicating similar destabilization effects resulting from introduction of a single mismatched base pair . We next investigated the stability of the model top1 cleavage complex using uv hyperchromicity measurements . The four 39 mer dna hairpins consisting of 13 base pairs with a 10 mer single - stranded overhang were annealed to the 10 mer ssdna complementary to the overhang region (figure 1). The single mismatched base pair corresponding to the + 1 site of a top1 cleavage complex was at the stem terminus, such that annealing of the 10 mer to the overhang extended the stem, albeit with a nicked phosphodiester backbone . The uv hyperchromicity profiles for all four hairpins in the presence of the complementary 10 mer were biphasic, as expected, with release of the 10 mer occurring first followed by melting of the 39 mer hairpins (figure 3). Under low salt conditions (10 mm phosphate buffer, ph 7), the melting temperatures for the two phases of the parental hairpin were 9.7c and 61.1c corresponding to release of the 10 mer and unfolding of the hairpin . The identical melting curve obtained in physiologically relevant salt conditions (10 mm phosphate buffer, ph 7, 200 mm nacl) had a tm of 33.1c for release of the 10 mer and a tm of 77.2c for unfolding of the hairpin . Interestingly, the presence of the mismatched base pairs destabilized both components of the biphasic melting curves . For example, for the physiologically relevant salt conditions with the g - fdu base pair, the tm for release of the 10 mer decreased from 33.1 to 30.1c, and the tm for unfolding of the hairpin decreased from 77.2 to 75.3c . The decrease for the 39 mer hairpin is expected as the mismatched base pair is present in the stem of the hairpin, and the magnitude of destabilization is similar to that observed for the 39 mer hairpin alone . The destabilization of the 10 mer region is somewhat surprising as the sequence does not contain a mismatched base pair . The terminal base pair of the 10 mer, however, stacks upon the site of the mismatched base pair in the 39 mer hairpin, and the observed destabilization likely results from less efficient stacking interactions . The g - t and g - du mismatched base pairs elicited similar degrees of destabilization as the g - fdu mismatch for both release of the 10 mer and melting of the dna hairpin . The results indicate that the presence of a mismatched base pair decreases the stability of the model top1 cleavage complex disfavoring adoption of the geometry required for religation to occur . The results are consistent with decreased stability of the duplex as contributing to the less favorable religation kinetics observed for model top1 cleavage sites containing g - fdu or other mismatched base pairs . Comparing molecular dynamics simulations for the four different 49 mer dna hairpins (39 mer dna hairpin extended without a nick in the phosphodiester backbone) demonstrates that all the mismatches indeed do produce profound effects on the initial 10 base pairs that form the recognition sequence and its complement . Due to the observed effects on the thermal stability and the novel influence of different mismatches occurring outside the recognition sequence, three different atomic measures of structural fluctuations within these first 10 base pairs are used to quantify this influence . First, atomic root - mean - square fluctuations (rmsfs) are calculated for all heavy atoms and averaged on a per - base level . This quantifies the extent to which each atom fluctuates about its equilibrium position and averages these fluctuations at the base - level for comparisons to determine how the different mismatches affect both the overall atomic fluctuations of the first 10 base pairs and the pattern of fluctuations . These determine the extent to which atomic fluctuations, regardless of magnitude, are correlated or anticorrelated and are averaged at the base level for comparison among the four different simulation types . This measure, referred to as the covariance matrix, determines how the different mismatches affect the coupling within the first 10 base pairs . This method has been used by multiple research groups to analyze communication within proteins and the effects of various perturbations on communication . The third and final measure used is cluster analysis, in which the different structural snapshots across all the simulations are clustered together to find the conformations accessed and their populations in the different hairpins . The first noticeable effect of mismatch formation is on the rmsfs of the g - du and g - fdu hairpins; the average rmsf over the first 10 base pairs increases by 13.5% and 12.0% for the g - du and g - fdu hairpins, respectively, relative to the average rmsf of the matched hairpin (table 1). Surprisingly, the g - t mismatch shows almost no overall increase in flexibility (<1%). However, all the hairpins show delocalized changes in flexibility in the first 10 bases relative to the matched hairpin (figure 4). The covariance matrices (figure 5) also exhibit delocalized changes in dynamics, as measured by correlated fluctuations, mostly concentrated within the chain where the mismatches occur (bases 110). The largest such changes occur with the fdu mismatch (figure 5(c)), although more pairs exhibit large perturbations with the du mismatch (figure 5(d)), including bases in the opposite chain near the mismatch . Both the rmsfs and covariances demonstrate that there are delocalized changes in atomic covariances and their correlations in the recognition sequences despite the mismatch occurring outside this sequence; clustering analysis sheds light on the population shifts and conformational changes that may give rise to these variations, and that may perturb the recognition and binding by top1 . Clustering analysis on the first 10 base pairs shows that there are four different conformations that are accessible to each of the four dna sequences . The different mismatches shift population among the different conformations, with the dominate change being increasing population in the rarest conformation found in the normal, matched dna simulation . The population of this conformation increases in the different mismatched from t, to du and to fdu . The actual structural rearrangements that occur during these conformational changes are actually quite modest (figure 6), as is expected in a system as structured as duplex dna . The different mismatches induce subtle shifts in the base backbone and the base interactions these shifts are especially large at the position just downstream from the mismatch (figure 6(b)), and especially so in the conformation that is rarest in the matched dna and dominate in the fdu mismatch (figure 6(c)). One issue that can also be addressed is that of the classification of the different dna sequences, which ones are more similar, and which are more different . With three different measures, the simplest approach to this problem is to cluster the four sequences based on each of these three measures (table 3). By rmsf, two clusters emerge, one consisting of matched and mismatched, and one of fdu and du; by cluster population, three clusters emerge, one consisting of matched and mismatched, one of fdu, and one with du . However, when clustering is based on covariance matrices, although three clusters emerge, the mismatched and fdu sequences are in one cluster with the other two as singleton clusters . These results suggest that the mismatched and matched sequences are most similar, although with the mismatched more similar to fdu in one dynamical measure (covariances), and fdu is most similar to either du or mismatched depending on the measure used . Dna mismatched base pairs occur at high levels in cells both as a consequence of base damage (e.g., cytidine deamination) as well as errors during dna replication . Cancer cells frequently have defects in dna repair processes that may result in greater levels of dna mismatched base pairs being present in the replicated genome . Further, treatment with fluoropyrimidine drugs such as 5fu or fdump results in thymineless conditions and imbalanced deoxynucleotide pools resulting in greater preponderance of mismatched base pairs, including g - fdu mismatched base pairs, introduced during replication and subsequent attempted repair processes . The introduction of such mismatched base pairs has been demonstrated to decrease the stability of duplex dna [23, 24]. The present work demonstrates that mismatched base pairs have a novel effect on the dna component of a top1 cleavage complex by destabilizing the putative religation intermediate that consists of a dna duplex with a nicked phosphodiester backbone . A g - du (or g - fdu) base pair in wobble geometry has two hydrogen bonds as does an a - t watson - crick base pair and thus is a relatively conservative substitution although at elevated ph an ionized g - fdu base pair may form . G - fdu and other mismatched base pairs not repaired prior to initiation of dna replication interfere with the religation step of topoisomerase 1 activity when the mismatched base pair is proximal to the site of top1 cleavage . The structural and thermodynamic basis for the decreased top1-mediated dna religation due to mismatched base pairs remains incompletely understood . While direct interactions between dna and top1 protein may play a major role, the inherent stability of the dna duplex likely the present results demonstrate that g - fdu and other mismatched base pairs destabilize the interaction of the scissile strand with the nonscissile strand of a model top1 cleavage complex . The extent of this destabilization is greater under high salt conditions similar to that which occurs in eukaryotic cells . Molecular dynamics simulations demonstrate that the mismatched base pairs increase the flexibility of the duplex . The extent of this increase in flexibility is dependent upon the type of mismatch with g - du and g - fdu mismatches displaying the greatest increased flexibility . These calculations reveal that the mismatched base pair causes increased atomic fluctuations up to 10 base pairs removed from the site of the mismatch . This increased flexibility makes it less likely that the scissile strand will adopt the correct conformation required for the religation reaction . Thus, the thermodynamic measurements obtained from the uv hyperchromicity data demonstrate that formation of the complex required for religation is disfavored by all dna mismatched base pairs at the + 1 site of the religation complex while the molecular dynamics simulations reveal that the g - du and g - fdu mismatched pairs are especially potent at increasing conformational flexibility and decreasing the likelihood religation will occur . Overall, our results provide new insights into the structural and dynamic process of top1-mediated dna religation and the influence of mismatched base pairs, particularly g - du and g - fdu mismatched base pairs, at disfavoring this process.
Leptospirosis commonly presents as an acute illness occurring in a biphasic pattern of fever followed by an afebrile period and again fever with organ involvement . Neurological involvement (neuroleptospirosis) is rare . Among the several manifestations of neural involvement, myeloradiculopathy, myelopathy, cerebellar dysfunction, transverse myelitis, guillain - barre syndrome, optic neuritis, peripheral neuropathyhave also been described . The hitherto undescribed entity of bilateral abducent nerve palsy as a manifestation of neuroleptospirosis is reported herewith . A 22-year - old male agricultural worker presented with fever, nausea, abdominal pain, yellowish discoloration of the eyes and myalgia . He had noticed the symptoms seven days prior to admission and had been treated symptomatically at a local dispensary . He denied a history of alcohol usage . Since this was during a time when our hospital was getting numerous cases of leptospirosis, and in view of his occupation, a possible clinical diagnosis of leptospirosis injection crystalline penicillin two million units intravenously sixth hourly was commenced while awaiting lab reports for malaria parasite and leptospira, dengue, viral hepatitis serology . Urea 28 mg / dl, serum creatinine 1.4 mg / dl and rbs 112 mg / dl . Chest x - ray was normal . On the second night of hospitalization during routine rounds at 8.30 pm, the patient replied in the affirmative when asked about diplopia, but did not have any pain or headache . The diplopia was worse when he directed his gaze to the left or right (figure 2, diplopia chart). Onset was rapid since evaluation a few hours earlier had not shown any neurological deficit . Limitations of eye movements were confined to abduction in both eyes (figure 3). The size of the convergent squint or esotropia was larger on distant fixation than on near fixation . Cerebrospinal fluid (csf) analysis showed 16 leukocytes / mm - all lymphocytes, glucose of 59 mg / dl and protein level of 39 mg / dl . Diplopia chart depicts that diplopia was worse when gaze was directed to the left or right . Shows extra - ocular movements; it can be seen from this chart that the limitations of eye movements were confined to abduction in both the eyes . Titer was 50 u / ml (<15 u / ml negative, 15 - 20 intermediate,> 20 positive) confirming the clinical diagnosis . He also showed complete recovery from abducent palsy over the next five days and was discharged on the eleventh day after admission . It is an important cause of acute febrile illness in china, the indian subcontinent, southeast asia, africa, south america and central america where malaria, typhoid and dengue are also common . Human infection occurs by direct contact with urine or blood of an infected rodent or animal, or from water or soil contaminated by urine . The organisms can penetrate abraded skin or intact mucous membrane, and enter the circulation and rapidly disseminate to various tissues . The infection is commonly seen in farmers, sewage workers, veterinarians, and animal handlers . After an incubation period of 7 - 14 days the first phase, leptospiremic or septicemic phase, has an abrupt onset, lasts for a week and is characterized by fever, chills, headache, skin rash, myalgia and conjunctival suffusion . The second phase or the immune phase, which can last up to 30 days, is characterized by leptospiruria and the development of anti - leptospira antibodies . . Additional involvement of pulmonary, renal, cardiovascular, and neurological systems can occur . In this phase the rarer clinical presentations described in literature are myeloradiculopathy, myelopathy, cerebellar dysfunction, transverse myelitis, cerebrovascular accident, cerebral venous thrombosis, cerebral arteritis, subarachnoid haemorrhage, optic neuritis, mononeuritis multiplex, peripheral nerve palsy, psychosis, suicidal behaviour, encephalitis, and cerebellitis . Transient paraparesis has been described in a recent report by lih - shinn et al . Although implicated in various neurological manifestations, leptospirosis is often overlooked by clinicians in the evaluation of febrile illnesses with neurological manifestations . In the case reported by kavitha and shastry, their patient had fever, jaundice and lower limb weakness, urinary retention and decreased sensory perceptions below t4 level . As in our case, the confirmation of the diagnosis was by positive anti - leptospira igm serology . In the report by costa et al ., facial palsy was observed following an illness consistent with leptospirosis and in their case, microscopic agglutination test (mat)- the confirmatory test was not done, but seroconversion was observed in the macroagglutination test . In the report by mumford et al ., flaccid paraplegia was observed . In contrast to our patient, this case ended with fatality ascribable to renal and hepatic involvement . The sixth cranial nerve (abducens nerve) innervates the lateral rectus muscle in the ipsilateral eye . Paralyses can be due to direct damage of the sixth cranial nerve, encephalon nuclei or less frequently, diffuse axonal damage . Bilateral sixth nerve palsy is a rare clinical condition . Unlike unilateral palsy, which is often due to local causes such as trauma, increased intracranial pressure, brain stem glioblastoma etc ., the bilateral form is due to systemic causes such as vasculopathy secondary to atherosclerosis and immune mediated vasculitis . Patients with bilateral sixth nerve palsies complain about binocular horizontal diplopia being worse in the gaze direction of the paretic lateral rectus muscle . Currently, there is paucity of knowledge regarding the pathogenetic pathways involved in the above neurological manifestations . The molecular mechanisms by which spirochetes interact with cellular barriers and the chain of events involved in leptospira meningitis and other leptospirosis - related neurological phenomena remain unknown . After invading the bloodstream, leptospira plays a role in the activation of innate immunity cells, such as macrophages, via interaction among the transmembrane toll - like receptor 2 (tlr2), cd14, and leptospiral lipopolysaccharide . The organisms migrate from the bloodstream to the csf by crossing the blood brain barrier . The ensuing neurological disease is dependent on the virulence of strains and on the development of an inflammatory response . Yet, interestingly, most of the current animal models for the spirochetoses do not recreate the manifestations of the neurological spectrum . Experimental approaches to the human neurological manifestations of the spirochetoses have shown inability and difficulty in reproducing aspects of the human disease in animal models . Though reports of other cranial nerve palsies in leptospirosis are made in literature, abducent paralysis has not been mentioned hitherto . In our patient, typical clinical presentation of leptospirosis the other causes of abducent palsy were excluded by medical history and by other investigations . It is also to be noted that the patient developed abducent palsy approximately on the ninth day of symptomatic disease, when the clinical manifestations attributed to leptospirosis had subsided . This is consistent with the concept that cranial palsies, including the present bilateral abducent palsy in patients with leptospirosis is mediated by immunological mechanisms . The novelty of this case is that whereas numerous case reports exist of other cranial nerve palsies occurring in leptospirosis, the present case is probably the first one that exemplifies abducent nerve palsy developing as a result of antecedent leptospira infection . The present case report has highlighted the hitherto unreported manifestation of bilateral abducent nerve palsy . Neuroleptospirosis should be considered in the differential diagnosis of all neuroinfections especially in endemic areas . Practicing physicians in developing countries, where advanced diagnostic facilities are not available, should be aware of the different patterns of clinical presentation and the various rarer manifestations of leptospirosis to be able to make a clinical diagnosis even when confronted with the unusual associations of leptospirosis.
Mood disorders are disturbances in a person s mood categorized into major depressive disorder (mdd) and bipolar disorder (bd). Mdd and bd appear to be the most important risk factors for lifetime suicide in any psychiatric disorder.1 it has been estimated that at the time of suicide, 87.3%98% of psychiatric disorders are from mood disorders and substance abuse.2 among mood disorder patients who attempt suicide, reattempt is more common than the first act.3 the prevalence of reattempted suicide within 1 year after the index suicide attempt is 16%40% nonfatal and 2% fatal.4,5 risk of reattempted suicide following a psychiatric admission is highest during the first months after the attempt and declines over time.6 several risk factors for suicide attempts reported in mdd and bd patients include male sex,7,8 age,5,7 living alone on the day of the index attempt,9 smoking,10 alcohol abuse,9 previous number of suicide attempts,912 method of suicide attempt,9 severity of refractory or recurrent depressions,10,12 somatic illness,9 suicide intent and treatment of mental illness,7,9,11 hospitalization in a psychiatric department, and the use of psychopharmacological drugs during the follow - up period . Other risk factors are impulsivity, stressful life events, hopelessness, being unemployed,13 comorbidity with anxiety and agitation, and reporting suicidal plans or hallucinations at the time of the index episode.14 the current study was undertaken at a setting located in the upper northern part of thailand where the suicide attempt rate of 34.1 per 100,000 population is remarkably high . The suicide completion rate was the highest in the nation at 12.4 per 100,000 population compared with 5.97 per 100,000 population countrywide in 2009 . Mood disorders rank among the top five high - burden diseases in this setting every year . Among those diagnosed with mood disorders, 25% are hospitalized due to suicide attempts.15 suicide reattempts are prevalent in this vulnerable population; however, the incidence and risk factors of suicide reattempts in mood disorder patients have not yet been investigated in this area . Furthermore, consensus on suicide attempt risk factors is still unclear because of their complexity, and the generalizability of previous research may be limited due to discrepancies of cultural context, study populations, design, and data analysis . Thus, there is a need for studies of incidence and risk factors for suicide reattempts in mood disorder patients in thailand . The present study aimed to determine incidence and explored risk factors of suicide reattempts occurring within 1 year after psychiatric hospital discharge in mood disorder patients . Suanprung psychiatric hospital is the largest tertiary psychiatric hospital (700 beds) in the upper northern region of thailand . It is located in chiang mai and has been providing medical care since 1938 . In 2009, mood disorder inpatient and outpatient visits for 2007, 2008, and 2009 were 9069, 10,739, and 11,482, respectively.16 eligible patients were all mood disorder patients diagnosed by psychiatrists and coded by the international statistical classification of diseases and related health problems 10th revision (icd-10). Inclusion criteria were patients diagnosed with icd-10 codes f31.x, f32.x, and f33.x and admitted to suanprung psychiatric hospital owing to suicide attempts between october 2006 and may 2009 . People who reattempted suicide were those mood disorder patients who reattempted nonfatal suicide within 1 year after an admission to suanprung psychiatric hospital owing to attempted suicide . Eligible patients medical charts were examined for the main outcome of the study, which was time to suicide reattempts . Time to suicide reattempts was defined as the number of days within 1 year from the date of hospital discharge to the suicide reattempt date . Patients who did not reattempt suicide during the 1 year of follow - up or with whom contact was lost before the 1-year follow - up were considered a censor group . Further, the national death registration was searched and death date and cause of death were retrieved . A cause of death was further confirmed by telephone calls to the patient s spouse or next of kin . To determine risk factors for suicide reattempts, patients medical files were reviewed from the day of admission and followed for 1 year after hospital discharge . Patient information recorded in hospital files was routinely assessed and documented with a standardized procedure by psychiatrists, trained psychiatric nurses, and pharmacists who interviewed patients and/or their relatives . Five trained nurses working at the study site extracted data and filled in a case record form . To assure validity of the data, two meetings were held by the research team to discuss and clarify each variable in the files before data collection . All factors were selected and divided into categories based on a literature review and the opinion of psychiatrists on the research team . The sociodemographic factors included were sex, age, body mass index, marital status, educational level, occupation, religion, living status (alone or with others), number of children, and summation of number of stressful life events right before or at the index episode . Social support was divided into good or excellent, moderate, and little or very little . Clinical factors included icd-10 diagnoses of bd (coded f31.x) and mdd (coded f32.x and f33.x). Duration of treatment began from the time of the first diagnosis to the index date, and age at onset was the age at the first visit . Previous suicide attempt and previous suicidal ideation were assessed and counted from the first hospital visit to the index date . Psychotic comorbidity was extracted from the icd-10 code and was recorded and grouped into alcohol / substance dependence, which was commonly found, and others . Smoking, alcohol use, and any substance abuse were behaviors at the index date . Medication adherence was assessed by attending pharmacists asking patients or their relatives about consistency in taking medication during the 2 weeks before the index date . It was summarized as highly adhere, meaning the patient always took medication or rarely missed a dose, intermittently adhere, meaning the patient took medication off and on, and poorly adhere, meaning at least seven consecutive days when the patient did not take medication . Other clinical characteristics included family history of mental disorders and family history for attempted or completed suicide . An additional variable collected was the number and type of method(s) used for suicide attempt at the index date . The method was considered a violent method if it was (1) a method other than drug overdose or a single wrist cut or (2) a combination of different methods.17 treatments received during the admission included pharmacotherapy, electroconvulsive therapy, and psychotherapy . Pharmacotherapy was prescribed medication, which we categorized according to the mechanism of action and evidence of relation to suicide . Mood stabilizers were categorized either as lithium or others, such as carbamazepine and valproate . Antidepressants were grouped as norepinephine and/or serotonin reuptake inhibitors, such as amitriptyline and venlafaxine, or as selective serotonin reuptake inhibitors (ssris), such as fluoxitine and fluvoxamine . Antipsychotics were divided into typical, such as zuclopentizol and haloperidol, and atypical, such as clozapine and risperidone . Additionally, data on length of stays and readmission within 28 days were also collected . Because time to suicide reattempts was an outcome of the study, owing to the nature of the fitting exploration model, we determined the best parsimonious model by performing two steps of analysis . First, variables predicting suicide reattempts were explored using univariable cox s proportional - hazards regression . Those with a p - value less than 0.20 were selected for the last step, in which forward elimination multivariable cox s proportional - hazards regression was selected . The global goodness - of - fit test by schoenfeld was used for testing the proportional - hazards assumption.18 in order to assess the model prediction, we used the area under receiver operating characteristic curve method . Two institutional review boards, the faculty of medicine at chiang mai university and suanprung psychiatric hospital, approved the protocol of the study . Suanprung psychiatric hospital is the largest tertiary psychiatric hospital (700 beds) in the upper northern region of thailand . It is located in chiang mai and has been providing medical care since 1938 . In 2009, mood disorder inpatient and outpatient visits for 2007, 2008, and 2009 were 9069, 10,739, and 11,482, respectively.16 eligible patients were all mood disorder patients diagnosed by psychiatrists and coded by the international statistical classification of diseases and related health problems 10th revision (icd-10). Inclusion criteria were patients diagnosed with icd-10 codes f31.x, f32.x, and f33.x and admitted to suanprung psychiatric hospital owing to suicide attempts between october 2006 and may 2009 . People who reattempted suicide were those mood disorder patients who reattempted nonfatal suicide within 1 year after an admission to suanprung psychiatric hospital owing to attempted suicide . Eligible patients medical charts were examined for the main outcome of the study, which was time to suicide reattempts . Time to suicide reattempts was defined as the number of days within 1 year from the date of hospital discharge to the suicide reattempt date . Patients who did not reattempt suicide during the 1 year of follow - up or with whom contact was lost before the 1-year follow - up were considered a censor group . Further, the national death registration was searched and death date and cause of death were retrieved . A cause of death was further confirmed by telephone calls to the patient s spouse or next of kin . To determine risk factors for suicide reattempts, medical files were reviewed from the day of admission and followed for 1 year after hospital discharge . Patient information recorded in hospital files was routinely assessed and documented with a standardized procedure by psychiatrists, trained psychiatric nurses, and pharmacists who interviewed patients and/or their relatives . Five trained nurses working at the study site extracted data and filled in a case record form . To assure validity of the data, two meetings were held by the research team to discuss and clarify each variable in the files before data collection . All factors were selected and divided into categories based on a literature review and the opinion of psychiatrists on the research team . The sociodemographic factors included were sex, age, body mass index, marital status, educational level, occupation, religion, living status (alone or with others), number of children, and summation of number of stressful life events right before or at the index episode . Social support was divided into good or excellent, moderate, and little or very little . Clinical factors included icd-10 diagnoses of bd (coded f31.x) and mdd (coded f32.x and f33.x). Duration of treatment began from the time of the first diagnosis to the index date, and age at onset was the age at the first visit . Previous suicide attempt and previous suicidal ideation were assessed and counted from the first hospital visit to the index date . Psychotic comorbidity was extracted from the icd-10 code and was recorded and grouped into alcohol / substance dependence, which was commonly found, and others . Smoking, alcohol use, and any substance abuse were behaviors at the index date . Medication adherence was assessed by attending pharmacists asking patients or their relatives about consistency in taking medication during the 2 weeks before the index date . It was summarized as highly adhere, meaning the patient always took medication or rarely missed a dose, intermittently adhere, meaning the patient took medication off and on, and poorly adhere, meaning at least seven consecutive days when the patient did not take medication . Other clinical characteristics included family history of mental disorders and family history for attempted or completed suicide . An additional variable collected was the number and type of method(s) used for suicide attempt at the index date . The method was considered a violent method if it was (1) a method other than drug overdose or a single wrist cut or (2) a combination of different methods.17 treatments received during the admission included pharmacotherapy, electroconvulsive therapy, and psychotherapy . Pharmacotherapy was prescribed medication, which we categorized according to the mechanism of action and evidence of relation to suicide . Mood stabilizers were categorized either as lithium or others, such as carbamazepine and valproate . Antidepressants were grouped as norepinephine and/or serotonin reuptake inhibitors, such as amitriptyline and venlafaxine, or as selective serotonin reuptake inhibitors (ssris), such as fluoxitine and fluvoxamine . Antipsychotics were divided into typical, such as zuclopentizol and haloperidol, and atypical, such as clozapine and risperidone . Additionally, data on length of stays and readmission within 28 days were also collected . Because time to suicide reattempts was an outcome of the study, cox s proportional - hazards regression was used for the analysis . Owing to the nature of the fitting exploration model first, variables predicting suicide reattempts were explored using univariable cox s proportional - hazards regression . Those with a p - value less than 0.20 were selected for the last step, in which forward elimination multivariable cox s proportional - hazards regression was selected . The global goodness - of - fit test by schoenfeld was used for testing the proportional - hazards assumption.18 in order to assess the model prediction, we used the area under receiver operating characteristic curve method . Two institutional review boards, the faculty of medicine at chiang mai university and suanprung psychiatric hospital, approved the protocol of the study . A total of 235 patients medical folders were reviewed and included in the analysis (figure 1). Of those, 36 (15.32%) reattempted suicide (median 109.5 days, range 1322), seven (2.98%) completed suicide (median 90 days, range 5185), and 192 (84.21%) neither reattempted nor completed suicide during follow - up . Of all nonfatal suicide reattempts, 14 patients (38.9%) did so within 90 days . Among suicide completers, one (14.3%) did so 5 days after discharge, and four (57.1%) did so within 90 days . Univariable analysis found that stressful life events, psychotic symptoms, and previous suicide attempts were significantly associated with suicide reattempts (tables 2 and 3). Three risk factors that explained 73.3% of the probability of suicide reattempt were attempting suicide more than twice before the index admission (adjusted hazard ratio [hr] 2.48; 95% confidence interval [ci] 1.075.76], concomitantly prescribed typical and atypical antipsychotics (adjusted hr 4.79; 95% ci 1.3916.52), or taking ssri alone (adjusted hr 5.08; 95% ci 1.1422.75) or with norepinephrine and/or serotonin reuptake inhibitors (adjusted hr 6.18; 95% ci 1.1333.65). In addition, readmission within 28 days was significantly higher in the suicide reattempt group (18.8% vs 5.1%, p = 0.008) (data not shown) (table 4). To the best of our knowledge, the present study is the first to determine incidence and explore risk factors of suicide reattempts within 1 year after psychiatric hospital discharge in mood disorder patients . In this study, the incidence of suicide reattempts and suicide completions within 1 year of follow - up after attempted suicide was 15.3% and 3.0%, respectively . This finding is consistent with previous studies that reported suicide reattempts at 13.6%16%6,13 and suicide completions at 1%3%.19 although the median time of suicide reattempts in the present study was longer than in a previous study (109.5 days vs 73.5 days),13 almost 40% of nonfatal suicide reattempts of the patients in this study occurred within 90 days . The reattempt rate is more likely to be clustered in time because the incidence declines throughout the year of follow - up.6 therefore, intensive surveillance, timely assessment, and effective interventions should be emphasized during the first few months and continue for 1 year after hospital discharge . Using the best fit and simple model, we found three risk factors that indicated suicide reattempts: more than two previous suicide attempts before the index admission, concomitantly being prescribed typical and atypical antipsychotics, and taking an ssri alone or concomitantly with norepinephrine and/or serotonin reuptake inhibitors . Previous suicide attempts before the index date is a risk factor associated with suicide reattempts found in several studies.912 in the present study, 74.4% percent of patients had at least one prior suicide attempt before the index date . The hazard ratios increased with an increasing number of previous suicide attempt(s), demonstrating that patients with more prior attempts tended to be more severe, be more impulsive, and take more risks.20 regarding the use of medication, psychopharmacological use prior to and during the follow - up period is a predictor for suicide attempt.6 suicide reattempts increased in mood disorder patients prescribed with antipsychotics or antidepressants . This is in accordance with a meta - analysis study reporting that antidepressants were not beneficial for suicidal patients.21 however, in some previous studies,22 long - term treatment with antidepressants was found to reduce suicides significantly when combined with lithium and antipsychotics . The ability of most antipsychotics to limit the risk of suicidal behavior remains untested in the literature.23 the positive relationship results of antidepressant and antipsychotic use in this study may be due to confounding indications . Patients prescribed with more types of medication were those who were more severe and prone to a higher number of suicide reattempts . Stressful life events and psychotic symptoms were two risk factors associated with suicide reattempts found in the univariable analysis but not in the multivariable analysis . A previous study indicated that stressful life events play an important role in suicide attempts in patients with mood disorders, particularly mdd patients.24 we found that having psychotic symptoms at the time of admission reduced the probability of suicide attempts, which is consistent with a previous study that suggested that psychosis may impair planning for suicide attempts.25 currently, there are a few studies that determine risk factors of suicide reattempts in mood disorders . Most of the research reports risk factors associated with suicide attempts in mdd or bd separately . Therefore, inconsistencies between our findings and previous studies are partially due to the difference in population, methodology, assessment tools, statistical analysis, and small samples of some variables in this study . Other studies have found risk factors for suicide attempts that include male sex;7,8 increasing age (ie, 50 years or older);5,7 living alone on the day of the index attempt;9 the method used in the index attempt;9,26 prior admission for suicide reattempt;8,9,19 family history of mental disorders;27 previous suicidal ideation;25 somatic illness, such as cardiovascular disease and malignancies;7,28 abuse of psychopharmacological agents, alcohol, or substances;28,29 bipolar,30,31 depressed, and mixed episodes among bd patients; recurrent or refractory mdd, including those with psychosis;32 a positive family history of suicidal behaviors;27 not taking lithium; and short - term effects from electroconvulsive therapy.33 there are some limitations of the present study . First, the generalizability of the study results may be limited to mood disorder patients visiting psychiatric hospitals . Those with severe physical injuries resulting from the attempted suicide were likely to be referred to a general hospital . Their characteristics, such as lethality of the method used and severity of psychiatric illness, may be different . Second, data collected from medical folders were subject to incompleteness or missing variables, which caused a lack of power to identify associations . Third, the data relied exclusively on patient interview . Some information, such as drug and alcohol use, can be under - reported . Additionally, the effect of some variables, such as psychotic disease in family members or a family history of suicide, could be underestimated . These variables may be missed because patients were unaware of them, or they may remain unspoken due to stigma . Some factors were not assessed in this study by instruments used in other studies (eg, hopelessness and impulsiveness). Fourth, we used variables at the admission index date as risk factors . However, some factors can change over time and occur during follow - up, such as marital status, employment situation, development of depression, and medication adherence . However, the aim of the study was to examine factors at one specific point in time that predict a high risk of repeated suicide attempts and thus possibly guide treatment . Finally, we found a final model that moderately explained the probability of suicide reattempts (73.3%). A larger sample size is required in future research to increase statistical power and should be prospectively used to confirm the study results . In spite of some limitations, risk factors of suicide reattempts in mood disorder patients were identified . For future research, risk factors for long - term suicide reattempts must be investigated, because they may differ somewhat from the short - term risk factors found in the present study . Furthermore, risk factors categorized by subgroup of mood disorders (ie, mdd and bd) must be separately investigated to enable clinicians to provide intervention specifically directed for each subgroup . Finally, this study lacks statistical power to investigate risk factors for suicide completion and suicidal ideation . Almost 40% of suicide reattempts in mood disorder patients in this study occurred within 90 days after their discharge from a psychiatric hospital . Factors placing patients at high risk for suicide reattempts were over two previous suicide attempts before the index admission, concomitantly being prescribed typical and atypical antipsychotics and antidepressants, and taking an ssri alone or concomitantly with norepinephrine and/or serotonin reuptake inhibitors.
Tears in the retinal pigment epithelium (rpe) occur spontaneously in the course of exudative age - related macular degeneration (amd). Rpe tears also occur following photodynamic therapy (pdt) and laser therapy, and are observed in polypoidal choroidal vasculopathy (pcv) [35]. Eyes with occult exudative amd and retinal pigment epithelial detachment (ped) are especially predisposed to rpe tears . Rpe tears have been reported following intravitreal injections of vascular endothelial growth factor (vegf) inhibitors, such as sodium pegaptanib, bevacizumab, and ranibizumab . The aim of this paper is to report the incidence of rpe tears in patients treated with ranibizumab (lucentis, novartis, basel, switzerland) for fibrovascular retinal pigment epithelial detachment (fvped) due to occult age - related macular degeneration . Functional and morphological results of intravitreal ranibizumab injections and of fvped treatment course during 12-month observation with the analysis a risk factors common to the rpe tears are presented . After approval by the bioethical commission, 30 patients from the retinal clinic at the department of ophthalmology of the military medical institute in warsaw were included in the study . Qualification criteria were as follows: 1/age of over 50 years; 2/ subfoveal fibrovascular retinal pigment epithelial detachment with underlying active occult choroidal neovascularization (cnv) confirmed on fluorescein angiography (fa) (heidelberg engineering hra 2) and optical coherence tomography (slo oct oti), not treated previously; 3/ overall lesion size not exceeding 12 optic disc diameters; 4/ baseline visual acuity (va) of 0.2 logmar-1.0 logmar (3475 letters of early treatment of diabetic retinopathy study etdrs chart). Patients with the following conditions and past treatments were not qualified for ranibizumab treatment: 1/ permanent structural fovea damage (subretinal scar or geographic atrophy); 2/ macular hemorrhage exceeding 50% of lesion size; 3/ retinal detachment; 4/ vitreoretinal or filtration surgery, or transplantation of the cornea; 5/ peripheral retinal photocoagulations within the last month; 6/ macular photocoagulations; 7/ cataract surgery within the last 2 months; 8/ unstabilized glaucoma; 9/ active infection of the eyeball or its protective apparatus; 10/ past or active uveitis; 11/ significant degeneration of peripheral retina . The most important general exclusion criteria were cerebral stroke or myocardial infarction within the last 6 months, severe unstabilized hypertension, and unstable coronary heart disease . Mean age was 74.467.75 years (6186 years); 19 women and 11 men were treated . Treatment was performed according to the following schedule: each patient received 3 intravitreal injections of 0.5 mg ranibizumab in monthly intervals (saturation phase); further treatment was based on activity of the exudative degeneration process . After the end of the saturation phase, each month the patients va was tested with the etdrs chart and oct was performed . The following re - injection criteria were employed: 1/ loss of 5 or more etdrs letters compared to best result during the initial phase of treatment with oct evidence of fluid in the macula; 2/ persistence or appearance of fluid under the retina or intraretinal edema, enlargement of ped on oct; 3/ increase in central retinal point thickness of at least 100 m compared to lowest value during saturation phase; 4/ new macular hemorrhage; 5/ new cnv focus on fa . Each ranibizumab injection was performed in an operation theatre with aseptic rules . Before and after the injection the patients were being monitored for topical and general adverse events and the number of needed ranibizumab injections was counted . Baseline, 1 month after the 1, 2 and 3 ranibizumab injection and at 12 months, va (etdrs letters) and oct parameters (central retinal point thickness crpt-m, fovea volume fv - mm, total macular volume tmv - mm from 3d retinal topography full field 29.2 scans, and ped height pedh-m, ped base pedb greatest linear diameter-m from 6 mm radial scans, manual measurements) were evaluated and compared . 3d retinal topography was performed with option high - resolution frames, high - quality (3s), high - speed (1s) with scan depth 2.3 mm . Data were analyzed (option analyze) and presented as a 3-d topography map of retinal thickness measured between rpe and vitreus surface . In zone analysis by slo oct oti retinal thickness and volume were evaluated: central point retinal thickness, volume of central circle 1 mm diameter (fovea), volume of central circle 6 mm diameter (total macula). Radial scans were performed with option zoom radial, radial increments 15 degrees and scan depth 2.3 mm . For ped base measurement, rpe line was prolonged and section between acute detachment borders was evaluated . For ped height measurement, perpendicular line to the rpe was conducted and section between the highest point of ped and base was evaluated . Lesion (lmps) and leakage (lemps) size in the macular photocoagulation study group disk area were evaluated and compared . Each of the eligible patient s baselines had a ped with subretinal fluid (sf) in oct as a one of the qualification criteria . At one month after the 3 ranibizumab injection and at 12 months, presence of sf was evaluated and compared . After 12 months the results of this therapeutic approach were summed up . To compare mean values of visual acuity, oct parameters at the 5 points included in the study (baseline, after the 1, 2 and 3 injection, and at month 12 of treatment) and fa mean values parameters at the 2 points (baseline and at 12 months), a student s t test was used . For many variables, analysis of variance with anova and least significant differences (lsd) tests were performed or friedman s anova, depending whether the variables had normal distribution . To compare values between subgroups, the student s t test or the mann - whitney u test were used . The associations between the rpe tears and sf were assessed using a chi - square test . Statistical analysis showed that mean values of va (etdrs letters) were significantly different in succeeding intervals and began to improve after the first ranibizumab injection . Mean best corrected visual acuity (bcva) at baseline was 53.2711.52 etdrs letters, after the first injection was 57.3312.35, and after the third injection was 60.6013.10 . Visual improvement of 15 or more letters was observed in 16.7% (5/30) of patients . Visual improvement or stabilization statistical analysis showed that mean values of crpt, fv and tmv significantly differed in succeeding periods and began decreasing after the first ranibizumab injection . Mean crpt at baseline was 354.80159.39 m, after the first injection 289.23113.80 m, after the third injection 205.6765.38 m, and after 12 months 225.6772.77 m . Frequencies of eyes with crpt> 200 m were: baseline 100%, 1 month 80%, 3 months 57%, and 12 months 73% . Mean fv at baseline was 0.260.10 mm, after the first injection 0.210.05 mm, after the third injection 0.180.04 mm, and after 12 months 0.190.067 mm . Mean tmv at baseline was 8.802.27 mm, after the first injection 8.0101.398 mm, after the third injection 7.220.88 mm, and after 12 months 7.351.24 mm . Mean values of pedb were significantly different after the second injection and in succeeding periods compare to baseline . After 12 months there was a significant different in pedb measurements (3503.231143.67 vs. 3089.131347.23 m). Mean values of pedh were significantly different after the second injection and in succeeding periods compared to baseline . Mean pedh at baseline was 460.53159.69 and after 12 months it decreased to 345.60191.61 m . The mean number of injections needed was 6.81.8 (range, 3 to 9). After the third injection, sf was observed in 10 cases (33% of all patients) and after 12 months in 13 cases (43% of all patients). Rpe tears occurred in 8 cases (27% of all patients) and were the main ocular adverse events during the present study, in addition to fovea atrophy in 1 patient with visual loss of 13 letters . In 6 cases, rpe tears were detected after the first ranibizumab injection, and in 2 cases during saturation phase . Two patients with rpe tears reported greater image distortion; the others did not observe this symptom . In all rpe tears cases, one patient after saturation phase refused consecutive ranibizumab injection because of significant visual deterioration, but was observed, and oct and va were monitored . Statistical analysis showed significant differences between the subgroup of 22 patients without rpe tears and the subgroup of 8 patients with rpe tears at the consecutive time intervals in mean values of etdrs, lemps at baseline, etdrs after the 1, 2,3 injection, and after 12 months, lmps, lemps after 12 months, pedh after the 1, 2,3 injection and 12 months (tables 13). There were no significant differences between mean number of ranibizumab injections 7.11.8 vs. 5.91.5, pedb parameters (baseline 3408.141053.37 vs. 3764.751408.30 m p=0.46, after 12 months 3106.181454.46 vs. 3042.251081.52 m p=0.91), and baseline pedh (431 . Analysis of variance revealed significant differences in time course for mean values of etdrs letters (p<0.05) (figure 1) with significant upper parameters for patients without rpe tears, and ped height (p<0.05) with significant upper parameters for rpe tears without baseline (figure 2). There were no significant differences in time course for median values of crpt, fv, tmv, and mean pedb . The mean improvement of + 6.08.1 letters at 12 months was observed in the subgroup without rpe tears, and of + 1.37.5 letters in the rpe tears subgroup without significant difference (p=0.16). Lsd test revealed that in the subgroup without rpe tears, mean values of va significantly differed in succeeding periods compared to baseline (p<0.001). In the subgroup with rpe tears, significant differences in mean values of va were not observed . Visual improvement or stabilization was observed in 90.9% (20/22) of patients without rpe tears (improvement of 15 or more letters in 22.7% 5/22). Visual improvement or stabilization was observed in 87.5% (7/8) of patients with rpe tears (significant improvement was not observed). The chi - square test revealed statistically significant associations between rpe tears and sf in oct at month 12 (p<0.05). In the era of anti - vegf therapy in patients with exudative amd, there has been a resurgence of new publications about rpe tears . Pauleikhoff et al . Reported rpe tears in 12.5% of eyes with vascular ped after 10.7 months . A report by lee et al on rpe tears following ranibizumab therapy is significant in the context of this research . The authors presented the case of a 70-year - old patient with fvped who had been treated with 3 sodium pegaptanib injections . Because of the observed increase in ped and persistent leakage from the cnv, the patient was switched to ranibizumab therapy . There was no tear progression and the ranibizumab therapy was withdrawn . In a 3-month follow - up after the injection complicated by tears, visual acuity was stable at the 20/100 level . In this author s observation, 6 cases of rpe tears were detected after the first ranibizumab injection, and 2 cases during the saturation phase . The timing of the tear onset (within the first month after the injection) suggests a correlation with the effects of the medication . Other reports confirm this correlation, although reports of the onset of tears 4 to 6 weeks following bevacizumab injections predominate in the literature [912]. Described 2 cases of rpe tears after intravitreal injections of ranibizumab in patients with neovascular amd . One patient developed the rpe tears within 2 weeks of the injection, the other within 6 weeks of a second injection . Both patients presented vision loss of 1 line at diagnosis of rpe damage, but during long - term follow - up, visual acuity improved in 1 patient by 1 line and deteriorated in the second patient by 3 lines . The authors suggested that the rapid regression of the fibrovascular membrane promote rpe tears . In the present study, a significant visual deterioration (loss of 12 letters) was observed in 1 patient with rpe tears . In the others (87.5%), stabilization or visual improvement of up to 13 letters in 1 case were noted . The significant differences in mean values of va did not occur during the 12-month observation (lsd test). Smith et al . Reported only 1 incidence (0.61%) of rpe tears in a ranibizumab - treated group of 164 patients with wet amd (occult lesions 60%, subfoveal location 68%, fvped 19 eyes this occurred in a 78-year - old female with fvped (5% of eyes with fvped, 1% of eyes with occult cnv, 1/105 injections in eyes with a fvped 0.95%) with a greatest linear diameter of 3254 m, who returned 4 weeks after ranibizumab injection with visual improvement from 20/200 to 20/100 . She has remained stable over 12 months without further injections . In this interventional case series, the occult lesions containing a fvped tended to be larger (4.5 mps da compared with 3.8), and fvped eyes required 6 injections on average, more than the average for the entire cohort . Additionally, there was an average decrease in vision with a fvped (0.15 logmar) compared with the entire cohort (0.05 logmar) with statistically significant difference . Described in their conclusion that incidence of rpe tears associated with ranibizumab therapy is low and may result from a predisposition (fvped) rather than being an effect of treatment . In the present study, rpe tears occurred in 27% of all fvped patients and this is a high value . There were no significant differences between numbers of ranibizumab injections 6.81.8 for all groups vs. 5.91.5 for the rpe tears subgroup . Visual stabilization or improvement was observed in 90% of patients, with a mean improvement of + 4.78.1 letters at 12 months . The mean improvement of + 6.08.1 letters at 12 months was observed in the subgroup without rpe, and of + 1.37.5 letters in the rpe tears subgroup, but without significant difference (p=0.16). However, in time course mean values of va parameters were significantly lower for rpe tears . Retrospectively analyzed a group of 328 patients with serous ped and documented visual deterioration treated with anti - vegf agents, pdt with steroids . In spite of visual acuity improvement by about 0.066 logmar and retinal thickness reduction in all patients, 41 (12.5%) developed rpe tears . In the authors opinion, tears of rpe or only partial flattening of the ped always indicated a worse prognosis in eyes with exudative amd than in eyes with classic choroidal neovascularization . Rpe tears are an extremely rare complication in patients with classic cnv without rpe detachment . Konstantinidis et al . Observed rpe tears in 4 (5.4%) eyes after a mean of 4 ranibizumab injections . Mean baseline bcva was 0.67 log mar and improved despite the rpe tear to 0.22 logmar . Reported 3 cases of rpe tears in a group of 40 patients with predominantly classic cnv secondary to amd who were treated with 1.25 mg of intravitreal bevacizumab . Arias et al . Found that in cases with rpe tears va is not always affected and is conditioned by localization of rpe damage (in the present study all rpe tears involved fovea). It is most frequently explained by translation of the cnv forces on the rpe or the effect of hydrostatic pressure in the space under the detached rpe . Rapid absorption of the fluid underneath the rpe appears to be a coexisting factor . According to gass, rpe tears are formed as a result of a rapid loss of vascular integrity, which results in an abrupt ped enlargement . Emphasized the role of the traction force associated with the cnv expansion or shrinkage of the cnv / rpe complex as a result of vegf inhibitor activity . Also pointed to the tangents of the forces acting during contraction of the subretinal membrane as the cause of tears . Recurring subretinal fluid due to breakdown of the outer blood - retinal barrier following interruption and folding of the rpe was observed in the patients, despite transient positive visual effects (the study revealed statistically significant associations between rpe tears and sf). Because rpe tears are a significant clinical problem, retina specialists must determine the risk factors . Performed a retrospective evaluation of oct of 393 eyes with cnv (stratus - oct zeiss, jena, germany). The height of the ped that accompanied cnv, thickness of the central retina, and maximal thickness of the retina were measured . In the 15 eyes in which rpe tears occurred following bevacizumab administration, the ped was significantly higher than in the other group . There was no positive correlation between the preprocedural thickness of the central retina, the maximal thickness of the retina, and the accompanying intraretinal swelling . There was a strong correlation between the ped height and the overall size of the lesion, as well as with the size of the cnv focal lesion . The risk for rpe tears increased from 0.5% (100 m) to 14.8% (600 m) with an increase in the rpe detachment height . In the absence of ped prior to the procedure, the present study revealed significant differences in time course for mean values of ped height (p<0.001) with significant upper parameters for rpe tears, but without significant differences in baseline values, fovea and macula volume compared to eyes without rpe tears . Chan et al . Observed rpe tears in 21 fvped eyes among 125 fvped eyes (16.8% of eyes with fvped and 2.1% of all analyzed eyes) after bevacizumab injections . The mean time interval from the bevacizumab injection to the development of an rpe tear was 30.422.7 days . Significantly greater mean fvped height (648.9245 m) with more subretinal fluid was found in the rpe tears subgroup in comparison to the fvped subgroup without rpe damage (338.1201.6 m). Also, in the rpe tears subgroup there was higher mean fvped volume index (9.36.3 vs. 3.54.0 mm) and total mean macular volume (8.01.8 vs. 7.41.2 mm). The present study did not reveal significant differences in fv and tmv between subgroup parameters . Gelisken et al . Reported rpe tears in 3.6% of eyes (15/409) following a single administration of bevacizumab . The exudative lesions classified as overall medium (between 4 and 6 disc diameters, dd) and large (more than 6 dd) had a statistically higher tear incidence . If subretinal hemorrhage was predominant in the entire lesion, the tear incidence was 6.9% . In the present study baseline leakage parameters in fa were significantly different in patients with rpe tears (p=0.03), without significant difference in baseline lesions parameters . The initial condition of the contour of the detached pigment epithelium is important for predicting rpe tears, as pointed out by moroz et al ., who conducted a retrospective evaluation of the course of treatment with bevacizumab in 24 eyes in which serous ped was originally diagnosed . In 6 eyes, rpe tears occurred shortly after the first injection . The second was step - like interruptions or interruptions of the continuity of the rpe line . One of these phenomena was observed in oct prior to bevacizumab administration in every eye in which rpe tears later developed . In the group of 18 eyes in which rpe tears did not develop, corrugation was observed in two eyes and step - like microinterruptions were not observed at all . The differences were statistically significant . Among patients with rpe tears from the present study, 4 had interruptions of the rpe line at baseline oct . Described a case of spontaneous rpe tears . In earlier oct, small interruptions of the rpe line were observed . These interruptions were considered to be microholes in the rpe layer and not full thickness tears . Vegf is currently considered the most important mediator of angiogenesis in physiological and pathological processes, including various eye diseases such as exudative amd . Various preparations, including off - label therapies, such as bevacizumab, are used . Isolated cases of rpe tears following ranibizumab administration are described, and the treatment seems safe . Significant risk factors for rpe tears following ranibizumab and bevacizumab injections, such as occult cnv with ped, have been identified and confirmed . This author s experience demonstrates that in eyes with fvped and rpe tears treated with ranibizumab, stabilization of visual acuity without significant improvement is predictable . One of the risk factors common to rpe tears may be baseline leakage parameter and pretreatment distorted rpe contour in oct . During ranibizumab therapy in eyes with rpe tears, upper parameters of fvped height may occur without significant differences in fovea and macula volume compared to eyes without rpe tears . Nevertheless, tears may cause bothersome image distortions and substantial discomfort, as emphasized by patients . It should be kept in mind that tears may also be generated spontaneously in exudative amd . Prior to initiating anti - vegf therapy, known and confirmed rpe risk factors should be considered and discussed with patients at risk as part of the pre - operative evaluation.
The study site was a deer farm with 900 hinds, including 550 adult hinds and 350 yearling hinds . This farm is located in the los alcornocales natural park in the cdiz province (andaluca, southern spain; 3617n, 547w), an area near the sea that is <500 m above sea level . Abundant wild red deer and moderate densities of roe deer (capreolus capreolus) are present in the area . Blood samples were collected by cervical puncture from 510 living farmed red deer, placed in sterile tubes containing edta, and frozen at 20c . Samples from adult deer hinds (n = 160) were obtained on july 12 and 13, 2007; yearling stags (n = 350) were sampled on august 28, 2007 . We tested 200 serum samples by using a competitive viral protein 7 (vp7) elisa (institute pourquier, montpellier, france). The samples were analyzed in duplicate according to the manufacturer s instructions . After rna extraction from 510 red deer blood samples, pcrs (rt - pcrs): 1) a group - specific rt - pcr detecting a conserved region within the btv nonstructural protein (ns) 1 segment (6); 2) a btv-1 serotype - specific rt - pcr (7); 3) a btv-4 serotype - specific assay (8); and 4) a group - specific rt - pcr that detects epizootic hemorrhagic disease (ehd) (9). Btv-4 pcr was performed as a 1-step real - time rt - pcr, and btv-1, ehd, and the group - specific assays were conducted as gel - based, 1-step rt - pcrs . Prevalence of bt antibodies and btv-1 and btv-4 rna and confidence intervals for prevalence (binomial exact, clopper - pearson) were calculated by using quatitative parasitology 3.0 software (10). Of the analyzed serum samples all yearling deer were elisa negative except for 3 doubtful samples; all of them had negative results in the btv, btv-1 and btv-4 rt - pcr assays (figure 1). Results of elisa to detect bluetongue virus (btv) viral protein 7 in 200 serum samples collected from red deer, spain . Results from yearlings were negative; results from adults showed an age - increasing trend of contact with btv . Bars represent 95% confidence intervals for prevalence (binomial exact, clopper - pearson). Of the adult deer, 25% showed positive results in the btv group - specific pcr . Positive samples were sequenced to confirm the presence of btv nucleic acid and further analyzed for the identification of the serotype . Six rna samples from adult deer were positive for the btv-4specific rt - pcr, and their sequences were confirmed by using blast software (http://blast.ncbi.nlm.nih.gov/blast.cgi). None of the samples from adult deer were positive either for btv-1-specific or ehd - specific rt - pcrs . Yearlings, however, showed a different pattern of results: 16.33% animals showed positive results in the group - specific and the btv-1specific rt - pcrs . This result suggests that, although adult deer maintained circulation of btv-4 rna, this serotype did not infect the yearlings despite the presence of the vector and the optimal conditions for infection in the study area . Surprisingly, several animals were positive to the ehd - specific assay . However, when the pcr products were purified and sequenced, none of the obtained sequences showed homology with published ehd sequences . (11), in which btv-1positive samples cross - reacted with the available ehd primers . The amplified pcr product obtained had approximately the same size as the pcr product expected for ehd, thus giving a false - positive result . Our results agree with what was found in livestock during surveillance programs: adult animals had probably been in contact with btv-4 during the outbreak that started in southern spain in 2004 . In contrast to the vaccinated domestic ruminants, deer were able to maintain btv-4 rna, thus confirming our initial hypothesis . However, detection of btv rna without concurrent virus isolation does not mean that deer are a long term reservoir host of btv (12). Simultaneous evaluation of adjacent cohorts of domestic and wild ruminants by using the same virus detection assays will be required to unambiguously define the precise role of wildlife in the epidemiology of btv infection . Yearling deer were apparently infected with btv-1, which has been present in spain since 2007 . When epidemiologic information about the study area was compared with the information for the deer samples analyzed, evidence was found supporting our results: adult deer were sampled on july 12, 2007, and yearlings were sampled august 20, 2007, i.e., 26 days after btv-1 presence was confirmed at 60 km distance from the deer farm (www.oie.int/wahis/reports/en_imm_0000005799_20070726_123322.pdf) (figure 2). Epidemiologic situation for bluetongue virus (btv) in spain, july august 2007 . The first btv-1 case in spain was reported in tarifa (purple circle), only 60 km west from a deer farm where the samples were collected (blue diamond). Thus, adult deer had been sampled when btv-1 was not present in the country yet . In contrast, yearlings were already positive to btv-1 only 26 days after this serotype was first reported in livestock in the same area . There are 2 explanations for this finding: 1) btv-1 is a highly pathogenic serotype (13), causing high death rates in sheep, that may also cause high death rates in deer; and 2) deer and other wild ruminants may be highly susceptible to btv infection, thus, making them good sentinels for this disease . Regarding ehd, despite the negative results obtained, lack of robust molecular tools for its detection is noteworthy . All available rt - pcrs are based on the sequences of ehd strains that have never been detected in the mediterranean area.
It includes nonoperative management with traction followed by immobilization in halo vest or somi brace or operative management in the form of posterior fixation with c2 pedicle screw only or c2-c3 fusion with c2 pedicle and c3 lateral mass screw and c2-c3 anterior fusion . However, when untreated it poses a complicated problem . The surgical management of untreated hangman fracture in not reported in literature . We report the surgical management of untreated hangman's fracture in a 30-year - old male who reported to us 12 weeks following injury . A 30-year - old man, manual laborer by occupation had fallen from 20 feet height . He had acute pain in the neck and took treatment in the form of over the counter medication . On presentation to the opd, he was having tenderness on the upper cervical spine and severe limitation of cervical spine movements in all directions . He had spasticity and exaggerated deep tendon reflexes in all 4 extremities, however the muscle power and sensation were normal . Radiograph revealed a type iia hangman fracture (levine and edward modification of effendi classification) with localized c2-c3 kyphosis of 33 [figure 1]. Computed tomography (ct) scan revealed fracture of pars interarticularis of c2 with spondylolisthesis of c2 over c3 . Magnetic resonance imaging revealed the compression of epidural space posterior to c3 with intensity changes in the odontoid process [figure 3]. After the application of gardner - wells tongs, gentle reduction maneuver was attempted; however the reduction could not be achieved . . Grade iii spondylolisthesis of c2 over c3 with localized kyphosis of 33 dynamic x rays showing absence of any significant c2-c3 mobility magnetic resonance imaging showing edema of odontoid process with compression of epidural sac posterior to c3 stage 1: with patient in supine position, anterior exposure was done using smith - robinson approach . Wound was closed in layers.stage 2: following anterior release and c2-c3 discectomy, gradual extension resulted in improved c2-c3 alignment . Patient was then carefully turned to prone position and posterior midline exposure was carried out . Further reduction was carried out by using a well - contoured rod pulling the c2 posteriorly . Autologous posterior iliac crest bone grafting was done and wound closed in layers over a drain.stage 3: patient was again placed in supine position and c2 and c3 bodies were exposed via the previous incision . C2-c3 anterior fusion was done using tri - cortical iliac crest bone graft and stabilization was carried out with the anterior cervical plate . Stage 1: with patient in supine position, anterior exposure was done using smith - robinson approach . Stage 2: following anterior release and c2-c3 discectomy, gradual extension resulted in improved c2-c3 alignment . Patient was then carefully turned to prone position and posterior midline exposure was carried out . Further reduction was carried out by using a well - contoured rod pulling the c2 posteriorly . Autologous posterior iliac crest bone grafting was done and wound closed in layers over a drain . Stage 3: patient was again placed in supine position and c2 and c3 bodies were exposed via the previous incision . C2-c3 anterior fusion was done using tri - cortical iliac crest bone graft and stabilization was carried out with the anterior cervical plate . Anterior c2-c3 union was noted at 5 months postoperative and confirmed with x - ray [figure 4] and ct scan [figure 5], following which brace was discontinued . At 18 months postoperative, the patient had normal neurological examination and had resumed his pretrauma occupation . X - ray at 5 months showing acceptable c2-c3 alignment with c2-c3 anterior fusion computed tomography scan at 18 months showing solid c2-c3 interbody fusion neglected spinal injuries, either secondary to overlooked diagnosis or due to the circumstantial and socioeconomic factors, are not uncommon but discussed infrequently in the literature . Management of cervical dislocations is challenging when it is delayed by more than 3 weeks . Formation of fibro cartilaginous tissue around facet joints and contracture of anterior supporting ligament, muscles, and disc make closed reduction extremely difficult and sometimes hazardous . The management of neglected subaxial cervical spine dislocation has been discussed in literature and varying approaches have been suggested . These are posterior release followed by anterior fusion, anterior release followed by posterior fixation and posterior release followed by anterior discectomy, and fusion followed by posterior fixation . The management of neglected type 2a hangman's fracture poses unique problems and guideline for the treatment of neglected subaxial cervical spine dislocation cannot be applied . Preoperative traction was contraindicated in our case as being a type iia hangman fracture which is a flexion distraction injury, damage to annulus and posterior longitudinal ligament is expected and traction can lead to injury to the spinal cord or nerve roots . Moreover, traction on the newly formed fibro cartilaginous tissue within the canal between fractured pars and remaining posterior elements, may lead to kinking and worsening of neurological deficit . Due to these reasons, preoperative traction was avoided . Liu et al ., srivastava et al . And jain et al . Reported satisfactory results by a two - staged procedure consisting of posterior release and partial facetectomy followed by for anterior discectomy and fusion, for neglected subaxial dislocations . In our case, since the c2-c3 facet was already aligned, release of contracted anterior structures and c2-c3 discectomy is to be the logical first step . This ensures feasibility of reduction and neural protection by preventing retropulsion of c2-c3 disc during reduction . In stage ii, posterior fixation is to be done as putting implant on the tensile (posterior) surface provides a biomechanically sound construct . Following reduction and posterior instrumentation, anterior void in c2-c3 disc space the satisfactory outcome in our patient leads us to believe that anterior - posterior - anterior is the appropriate surgical approach for the treatment of such patients.
Idiopathic gingival fibromatosis (igf) is an uncommon, benign, hereditary, slowly progressive, nonhemorrhagic fibrous enlargement of keratinized gingiva . It usually begins at the time of eruption of permanent teeth, but can develop with the eruption of deciduous dentition and rarely present at birth . The hyperplastic gingival tissue is pale - pink, firm; has leathery consistency and presents a characteristic pebbled surface . The enlarged tissues may partially or totally cover the dental crowns, can cause diastemas, delay or impede tooth eruption and periodontitis . In severe cases, it may lead to mastication and speech impediments or lip closure difficulties . An 18-year - old male, accompanied by his father reported to the department with the complaint of enlarged gums in upper and lower arches, which caused difficulties in speech, mastication and complete closure of lips, thereby leading to esthetic impairment . The patient presented with gradual and progressive enlargement of both upper and lower gingival tissues from the age of 6 years . The patient exhibited no signs of hypertrichosis, mental retardation, epilepsy or intake of medication known to cause gingival overgrowth . On examination, patient had bilaterally symmetrical face with incompetent lips with the bimaxillary protrusion . The gingiva was pink in color with melanin pigmentation, and consistency was firm and fibrous . The enlarged gingiva covered the crowns of all the teeth till the incisal or occlusal third region [figure 1]. Dentition revealed all permanent teeth except mandibular premolar (35) and an over retained deciduous molar (75). Preoperative photographs showing generalized gingival enlargement investigations included orthopantomograph, in which no significant alveolar bone loss was observed [figure 2]. Laboratory investigations which included complete hemogram, thyroid tests (t3, t4, and thyroid stimulating hormone), parathyroid test, calcium and alkaline phosphatase levels were made . All the reports except the levels of alkaline phosphatase (which was slightly increased) were within the physiological limits . Panoramic radiograph showing presence of retained deciduous molar in mandibular left region the treatment plan consisted of sextant wise surgical excision of the enlarged gingiva under local anesthesia . The treatment procedure was explained to the patient and parent, and written consent was obtained . Ledge and wedge procedure was done to remove gingival overgrowth over the palatal aspect of the maxillary posterior region and mandibular posterior region [figure 3]. Intrasurgical photographs histopathology showed parakeratinized stratified epithelium, which was acanthotic at places and elongated rete pegs . The bulk of connective tissue was composed of dense fibrous connective tissue and numerous fibroblasts . Histologic section shows hyperparakeratinized hyperplastic squamous epithelium with dense fibrocollagenous tissue the patient was recalled every week for 6 weeks when the periodontal pack was removed, and the next sextant was operated upon . The patient was then placed on a schedule of periodic recall visits for maintenance care . No recurrence of gingival enlargement was observed 6 months after the surgery [figure 5]. Gingival fibromatosis may occur as an inherited condition known as hereditary gingival fibromatosis, or it may be associated with inflammation, leukemic infiltration, and medications . According to gorlin igf is most commonly associated with hypertrichosis, also occasionally associated with mental retardation and epilepsy . It occurs either as an isolated disease or combined with some rare syndromes like zimmerman - laband syndrome (defects of bone, nail, ear, nose and splenomegaly), murray - puretic - drescher syndrome (multiple dental hyaline tumors), rutherford syndrome (corneal dystrophy), cowden syndrome (multiple hamartomas), and cross syndrome (hypopigmentation with athetosis). In the present case, the patient exhibited no signs of hypertrichosis, mental retardation and epilepsy or intake of medication known to cause enlargement . Idiopathic gingival fibromatosis affects the attached gingiva as well as gingival margin and interdental papillae . The cause is unknown, and thus the condition is designated as idiopathic . Some cases have a hereditary basis, but the genetic mechanisms involved are not well understood . A study of several families found the mode of inheritance to be autosomal recessive in some cases and autosomal dominant in others . Histologically, the gingival hyperplasia is mainly due to an increase and thickening of collagen bundles in connective tissue stroma . Igf keratinocytes seem to have an important role in pathogenesis by inducing extracellular matrix accumulation by fibroblasts . Furthermore, it has been reported that increased proliferation and elevated production of extracellular matrix molecules, fibronectin and type i collagen could lead to an increased bulk of gingiva . In the present case, the history revealed that the enlargement started in mixed dentition period . Emerson recommended that the best time for the excision of gingival enlargement is when all the permanent teeth have erupted . Among the suggested treatment protocols, ledge and wedge technique along with internal bevel gingivectomy was selected as it helped in the placement of primary incisions as opposed to the conventional external bevel gingivectomy procedure . Moreover, this procedure does not leave a large external bevel and, therefore, result in less postoperative pain and bleeding . After surgery, recurrence is expected within a few months after surgery and is most commonly seen in children and teenagers rather than adults . Maintenance of good oral hygiene with professional cleaning and home care maintenance is necessary to prevent the recurrence.
Male fvb / n jcl mice (clea japan, tokyo, japan) were studied at 1216 weeks of age . The animals were housed individually in plastic cages at 24 1c with lights on from 0600 to 1800 h, and they were maintained with free access to a laboratory diet (oriental yeast, tokyo, japan) and water . Mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg body mass) and xylazine (10 mg / kg), and a chronic double - walled stainless steel cannula was implanted stereotaxically and unilaterally into the right side of the vmh, arc, dmh, or pvh or into the lateral ventricle according to the atlas of franklin and paxinos (26). The stereotaxic coordinates were ap 1.3 (1.3 mm anterior to the bregma), l 0.3 (0.3 mm lateral to the bregma), and h 5.8 (5.8 mm below the bregma on the surface of the skull) for the vmh; ap 1.6, l 0.2, and h 6.1 for the arc; ap 1.6, l 0.3, and h 5.5 for the dmh; ap 0.75, l 0.2, and h 4.9 for the pvh; and ap 0.3, l 1.0, and h 2.25 for the lateral ventricle . Cannulas were anchored firmly to the skull . In experiments featuring administration of the mcr antagonist shu9119, cannulas were implanted both into vmh and intracerebroventricularly . Three days before determination of tissue glucose uptake, a silicone catheter was implanted into the external jugular vein . Animals were handled repeatedly during the recovery period (2 weeks) after cannula implantation to habituate them to the injection and blood - sampling procedures . Correct placement of the cannula tips was verified microscopically in brain sections in all experiments, with> 90% of animals manifesting correct placement . Tyrosine phosphorylation of signal transducer and activator of transcription 3 (stat3) in each medial hypothalamic nucleus was examined after injection of leptin, as described below . All animal experiments were performed in accordance with institutional guidelines for the care and handling of experimental animals, and they were approved by the ethics committee for animal experiments of the national institute for physiological sciences . Leptin (5 ng) (national hormones and pituitary program, torrance, ca) or mt - ii (10 ng) (phoenix pharmaceuticals, burlingame, ca) dissolved in 0.1 l physiological saline was injected with the use of a hamilton microsyringe into the right side of the vmh, dmh, pvh, or arc of freely moving mice through the unilateral cannula implanted into the corresponding nucleus . The concentrations of leptin (3 mol / l) and mt - ii (100 mol / l) dissolved in 0.1 l physiological saline injected into the medial hypothalamic nuclei are at least 10 times higher than those necessary for the maximum activation of leptin receptor and mcrs (2729). Alternatively, mt - ii (3 g) in 0.5 l saline was injected into the lateral ventricle . Shu9119 (1 g) (phoenix pharmaceuticals) in 0.5 l saline was also injected intracerebroventricularly immediately before leptin injection . The dose of mt - ii and shu9119 injected intracerebroventricularly altered food intake significantly (30,31). Control animals received 0.1 l saline delivered into the various nuclei or 0.5 l saline delivered intracerebroventricularly, respectively . The rate constant of net tissue uptake of 2-[h]deoxy - d - glucose (2[h]dg) in peripheral tissues was determined, as described previously (32,33), by injecting a mixture of 6.25 ci 2[h]dg (10 ci / mmol) and 1.25 ci [c]sucrose (10 ci / mmol) (american radiolabeled chemicals, st . Louis, mo) through the jugular vein catheter 3 or 6 h after the microinjection of leptin or mt - ii . Blood was collected 0, 10, 15, and 20 min after injection of the radioactive tracers . Immediately after collection of the final blood sample (20 min), an overdose of pentobarbital sodium (100 mg / kg) was injected through the jugular vein catheter and the mice were rapidly decapitated . Hypothalamic nuclei were then rapidly dissected as described below and frozen in liquid nitrogen for subsequent immunoblot analysis . Skeletal muscle (soleus, red and white portions of the gastrocnemius, and extensor digitorum longus [edl]) as well as interscapular bat, heart, spleen, and epididymal white adipose tissue (wat) were rapidly dissected, weighed, and assayed for the radioactivity . Plasma samples were also analyzed for glucose (glucose cii test; wako, osaka, japan) and insulin (mouse insulin elisa kit [u - type]; shibayagi, gunma, japan) concentrations . The rate constant of net tissue uptake of 2[h]dg was calculated as described previously (32,33). The radioactivity of [c]sucrose in tissues was used to calculate the 2[h]dg radioactivity remaining in the extracellular space (33). The accuracy of the dissection was assessed by measurement of mrnas for neuropeptides or transcription factors such as corticotropin - releasing factor (crf) mrna for the pvh, pomc and npy mrnas for the arc, sf1 mrna for the vmh, and the absence of these various mrnas for the dmh . The right side of the pvh, arc, vmh, or dmh was dissected from a 1-mm - thick sagittal section prepared from the midline of the fresh brain (online appendix supplemental fig the homogenates were centrifuged, and the resulting supernatants (5 g of protein) were fractionated by sds - page . Immunoblot analysis was then performed with antibodies (1 g / ml), including those to the tyr - phosphorylated or total forms of stat3 (cell signaling technology, danvers, ma), to c - fos (santa cruz biotechnology, santa cruz, ca), or to -actin (cell signaling technology). Immune complexes were visualized with horseradish peroxidase conjugated secondary antibodies (santa cruz biotechnology) and enhanced chemiluminescence reagents (ge healthcare, tokyo, japan). Protein bands were quantified using image j software (national institutes of health, http://rsbweb.nih.gov/ij). Total rna was isolated from hypothalamic and peripheral tissue with the use of isogen (nippon gene, wako, japan), and portions of the rna (300 ng) were subjected to reverse transcription with an oligo(dt) primer and avian myeloblastosis virus reverse transcriptase (takara, shiga, japan). The resulting cdna was subjected to the pcr with la taq (takara) and primers obtained from sigma genosys (ishikari, japan). For quantitative real - time pcr, cdna was amplified using sybr green pcr master mix with an abi 7500 real - time pcr system (applied biosystems, tokyo, japan). Data were normalized by the amount of eukaryotic elongation factor 2 (eef2) mrna . Statistical analysis of stat3 phosphorylation, c - fos expression, and real - time pcr was performed by student t test, and analysis for other experiments was performed by anova followed by dunnett test . A p value of <0.05 was considered statistically significant . Leptin (5 ng) (national hormones and pituitary program, torrance, ca) or mt - ii (10 ng) (phoenix pharmaceuticals, burlingame, ca) dissolved in 0.1 l physiological saline was injected with the use of a hamilton microsyringe into the right side of the vmh, dmh, pvh, or arc of freely moving mice through the unilateral cannula implanted into the corresponding nucleus . The concentrations of leptin (3 mol / l) and mt - ii (100 mol / l) dissolved in 0.1 l physiological saline injected into the medial hypothalamic nuclei are at least 10 times higher than those necessary for the maximum activation of leptin receptor and mcrs (2729). Alternatively, mt - ii (3 g) in 0.5 l saline was injected into the lateral ventricle . Shu9119 (1 g) (phoenix pharmaceuticals) in 0.5 l saline was also injected intracerebroventricularly immediately before leptin injection . The dose of mt - ii and shu9119 injected intracerebroventricularly altered food intake significantly (30,31). Control animals received 0.1 l saline delivered into the various nuclei or 0.5 l saline delivered intracerebroventricularly, respectively . The rate constant of net tissue uptake of 2-[h]deoxy - d - glucose (2[h]dg) in peripheral tissues was determined, as described previously (32,33), by injecting a mixture of 6.25 ci 2[h]dg (10 ci / mmol) and 1.25 ci [c]sucrose (10 ci / mmol) (american radiolabeled chemicals, st . Louis, mo) through the jugular vein catheter 3 or 6 h after the microinjection of leptin or mt - ii . Blood was collected 0, 10, 15, and 20 min after injection of the radioactive tracers . Immediately after collection of the final blood sample (20 min), an overdose of pentobarbital sodium (100 mg / kg) was injected through the jugular vein catheter and the mice were rapidly decapitated . Hypothalamic nuclei were then rapidly dissected as described below and frozen in liquid nitrogen for subsequent immunoblot analysis . Skeletal muscle (soleus, red and white portions of the gastrocnemius, and extensor digitorum longus [edl]) as well as interscapular bat, heart, spleen, and epididymal white adipose tissue (wat) were rapidly dissected, weighed, and assayed for the radioactivity . Plasma samples were also analyzed for glucose (glucose cii test; wako, osaka, japan) and insulin (mouse insulin elisa kit [u - type]; shibayagi, gunma, japan) concentrations . The rate constant of net tissue uptake of 2[h]dg was calculated as described previously (32,33). The radioactivity of [c]sucrose in tissues was used to calculate the 2[h]dg radioactivity remaining in the extracellular space (33). The accuracy of the dissection was assessed by measurement of mrnas for neuropeptides or transcription factors such as corticotropin - releasing factor (crf) mrna for the pvh, pomc and npy mrnas for the arc, sf1 mrna for the vmh, and the absence of these various mrnas for the dmh . The right side of the pvh, arc, vmh, or dmh was dissected from a 1-mm - thick sagittal section prepared from the midline of the fresh brain (online appendix supplemental fig . 1 [available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0638/dc1]). The homogenates were centrifuged, and the resulting supernatants (5 g of protein) were fractionated by sds - page . Immunoblot analysis was then performed with antibodies (1 g / ml), including those to the tyr - phosphorylated or total forms of stat3 (cell signaling technology, danvers, ma), to c - fos (santa cruz biotechnology, santa cruz, ca), or to -actin (cell signaling technology). Immune complexes were visualized with horseradish peroxidase conjugated secondary antibodies (santa cruz biotechnology) and enhanced chemiluminescence reagents (ge healthcare, tokyo, japan). Protein bands were quantified using image j software (national institutes of health, http://rsbweb.nih.gov/ij). Total rna was isolated from hypothalamic and peripheral tissue with the use of isogen (nippon gene, wako, japan), and portions of the rna (300 ng) were subjected to reverse transcription with an oligo(dt) primer and avian myeloblastosis virus reverse transcriptase (takara, shiga, japan). The resulting cdna was subjected to the pcr with la taq (takara) and primers obtained from sigma genosys (ishikari, japan). For quantitative real - time pcr, cdna was amplified using sybr green pcr master mix with an abi 7500 real - time pcr system (applied biosystems, tokyo, japan). Data were normalized by the amount of eukaryotic elongation factor 2 (eef2) mrna . C - fos expression, and real - time pcr was performed by student t test, and analysis for other experiments was performed by anova followed by dunnett test . Microinjection of leptin (5 ng) into the vmh induced a significant increase in the rate constant of 2[h]dg uptake in the red type of skeletal muscle (soleus and red portion of gastrocnemius), mixed type of skeletal muscle (edl), bat, and heart but not in spleen or epididymal wat (fig . 1). Glucose uptake in red or mixed skeletal muscle was significantly increased at 6 h after leptin injection (fig . 1a), whereas that in bat and heart was increased at both 3 and 6 h (fig . Glucose uptake in the white portion of the gastrocnemius showed a tendency to increase in response to leptin, but the change was not statistically significant . Glucose uptake in peripheral tissues at 3 h after saline injection into the vmh did not differ from that apparent at 6 h (data not shown). Injection of leptin into the vmh significantly increased glut4 mrna in bat and heart but not in soleus muscle at 6 h after leptin injection (fig . Hexokinase ii mrna did not change in soleus, bat, or heart (fig . Effects of leptin injection into the vmh on glucose uptake and gene expression in peripheral tissues . The rate constant of 2[h]dg uptake was measured in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) at 3 and 6 h after injection of leptin or saline (control) into the vmh of mice ., saline vmh (6 h);, leptin vmh (3 h);, leptin vmh (6 h). E: the mrna levels of glut4, hexokinase ii (hkii), and ucp1 in soleus, bat, and heart were quantified by real - time pcr . Values are normalized by the level of eef2 mrna . Gastro - r, red portion of gastrocnemius; gastro - w, white portion of gastrocnemius . P <0.05 vs. the corresponding value for saline - injected controls ., saline vmh (6 h);, leptin vmh (6 h). Injection of maximal dose of leptin into the arc induced a small but significant increase in the rate constant of 2[h]dg uptake in bat at 6 h after injection, but it had no effect on glucose uptake in skeletal muscle, heart, spleen, or wat (fig . Injection of leptin into the dmh or pvh had no effect on glucose uptake in peripheral tissues (fig . 2). Glucose uptake in peripheral tissues after saline injection into the dmh or pvh did not differ from that apparent after saline injection into the arc (data not shown). Plasma glucose and insulin concentrations were not affected by leptin injection into the vmh or other hypothalamic nuclei (supplemental table 2), consistent with previous observations (13,15). Effects of leptin injection into the arc, dmh, or pvh on glucose uptake in peripheral tissues . The rate constant of 2[h]dg uptake was measured in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) at 6 h after injection of leptin into the hypothalamic nuclei of mice . Data are means se for six or seven mice . * p <0.05 vs. the corresponding value for control animals injected with saline into arc ., saline arc;, leptin arc; leptin dmh;, leptin pvh . To determine whether leptin activated ob - rb in the hypothalamic nuclei, we examined the tyrosine phosphorylation of stat3 in tissue samples therefrom . Rt - pcr analysis confirmed that the isolated pvh, arc, and vmh specimens were enriched in crf mrna, pomc and npy mrnas, and sf1 mrna, respectively, and that the dmh was largely devoid of these mrnas (fig . Microinjection of leptin into the vmh, dmh, or pvh preferentially increased the tyrosine phosphorylation of stat3 to the similar extent in the corresponding nucleus at 6 h after injection (fig . Injection of leptin into the arc increased the tyrosine phosphorylation of stat3 in the dmh as well as in the arc (fig . 3e), possibly as a result of leakage of leptin into the dmh through the surface of the injection cannula, given that the arc cannula passed through the dmh . Together, these results suggested that the vmh is a key target of leptin in its regulation of glucose uptake in skeletal muscle, heart, and bat, whereas the leptin receptor in the arc mediates stimulation of glucose uptake in bat . Effects of leptin injection into medial hypothalamic nuclei on tyrosine phosphorylation of stat3 and c - fos expression . A: rt - pcr analysis of crf, pomc, npy, sf1, and eef2 (loading control) mrnas in the pvh, arc, vmh, and dmh . B e: immunoblot analysis of the phosphorylation of stat3 on tyr in the medial hypothalamic nuclei at 6 h after leptin injection into the vmh (b), dmh (c), pvh (d), or arc (e). Leptin was injected into the right side of the hypothalamic nuclei, and the same side of the pvh (p), arc (a), vmh (v), and dmh (d) was collected . Representative blots of tyr - phosphorylated stat3 are shown in the upper panels, and quantitative data (means se) from four to six mice are shown in the lower panels . The amount of tyr -phosphorylated stat3 was normalized by that of total stat3, and the normalized values were expressed relative to the corresponding value for the pvh of control mice . F: immunoblot analysis of c - fos expression in the medial hypothalamic nuclei after leptin injection into the vmh . The right side of the pvh, arc, vmh, and dmh was collected at 6 h after injection of leptin or saline into the same side of the vmh . A representative blot is shown in the upper panel, and quantitative data (means se) from four to six mice are shown in the lower panel . The amount of c - fos was normalized by that of -actin, and the normalized values were expressed relative to the corresponding value for the saline - injected control mice . * p <0.05 . We next examined whether leptin injection into the vmh might increase neuronal activity in other hypothalamic nuclei by measuring expression of the transcription factor c - fos . Leptin injection into the vmh significantly increased c - fos expression in the arc as well as in the vmh at 6 h after injection (fig . Leptin injection into the vmh increased the phosphorylation of stat3 in the nucleus preferentially at 1 h after the injection, similar to that at 6 h. however, it did not increase c - fos expression in any nucleus at 1 h (supplemental fig . Leptin injection into the vmh did not increase pomc mrna in the arc at 6 h after injection (supplemental fig . We next examined the role of mcrs in glucose uptake in peripheral tissues induced by injection of leptin into the vmh (fig . Injection of the mcr antagonist shu9119 (1 g) into the lateral ventricle (intracerebroventricularly) abolished the increase in 2[h]dg uptake in peripheral tissues normally apparent at 6 h after the injection of leptin into the vmh . Injection of shu9119 alone did not affect glucose uptake in peripheral tissues . Plasma glucose and insulin levels were also not changed in response to intracerebroventricular injection of shu9119 (supplemental table 2). Effect of intracerebroventricular injection of shu9119 on glucose uptake in peripheral tissues induced by injection of leptin into the vmh . The rate constant of 2[h]dg uptake in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) was measured 6 h after injection of leptin or saline (control) into the vmh . We tested the effects of intracerebroventricular injection of the mcr agonist mt - ii on glucose uptake in peripheral tissues (fig . The intracerebroventricular injection of mt - ii (3 g) increased 2[h]dg uptake in bat, heart, and all types of skeletal muscle, including the white portion of the gastrocnemius, but not in spleen or epididymal wat, at 3 or 6 h after injection . Glucose uptake in peripheral tissues at 3 h after saline injection did not differ from that apparent at 6 h (data not shown). The intracerebroventricular injection of mt - ii increased the plasma glucose level at both 3 and 6 h after injection (supplemental table 2). These results thus suggested that intracerebroventricular injection of mt - ii promotes glucose production as well as glucose uptake in certain peripheral tissues . In contrast, plasma insulin concentration did not change after intracerebroventricular injection of mt - ii, despite the associated hyperglycemia, suggesting that mt - ii inhibits insulin secretion from pancreatic -cells . The rate constant of 2[h]dg uptake in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) was measured at 3 or 6 h after intracerebroventricular injection of mt - ii or saline (control). Finally, we examined the effects of direct injection of mt - ii into the individual medial hypothalamic nuclei on glucose uptake in peripheral tissues (fig . Microinjection of mt - ii (10 ng) into the vmh increased 2[h]dg uptake in bat, heart, and all types of skeletal muscle but not in spleen or epididymal wat . In contrast, injection of mt - ii into the pvh increased glucose uptake only in bat, and that into the dmh or arc did not affect glucose uptake in any of the peripheral tissues examined . Plasma glucose and insulin levels did not change in response to injection of mt - ii into any of the hypothalamic nuclei (supplemental table 2). Effects of mt - ii injection into vmh, pvh, dmh, or arc on glucose uptake in peripheral tissues . The rate constant of 2[h]dg uptake in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) was measured at 6 h after the injection of mt - ii into the individual hypothalamic nuclei ., vmh;, mt - ii vmh;, mt - ii pvh;, mt - ii dmh;, mt - ii arc . Microinjection of leptin (5 ng) into the vmh induced a significant increase in the rate constant of 2[h]dg uptake in the red type of skeletal muscle (soleus and red portion of gastrocnemius), mixed type of skeletal muscle (edl), bat, and heart but not in spleen or epididymal wat (fig . 1). Glucose uptake in red or mixed skeletal muscle was significantly increased at 6 h after leptin injection (fig . 1a), whereas that in bat and heart was increased at both 3 and 6 h (fig . Glucose uptake in the white portion of the gastrocnemius showed a tendency to increase in response to leptin, but the change was not statistically significant . Glucose uptake in peripheral tissues at 3 h after saline injection into the vmh did not differ from that apparent at 6 h (data not shown). Injection of leptin into the vmh significantly increased glut4 mrna in bat and heart but not in soleus muscle at 6 h after leptin injection (fig . Hexokinase ii mrna did not change in soleus, bat, or heart (fig . Effects of leptin injection into the vmh on glucose uptake and gene expression in peripheral tissues . The rate constant of 2[h]dg uptake was measured in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) at 3 and 6 h after injection of leptin or saline (control) into the vmh of mice ., saline vmh (6 h);, leptin vmh (3 h);, leptin vmh (6 h). E: the mrna levels of glut4, hexokinase ii (hkii), and ucp1 in soleus, bat, and heart were quantified by real - time pcr . Values are normalized by the level of eef2 mrna . Gastro - r, red portion of gastrocnemius; gastro - w, white portion of gastrocnemius . P <0.05 vs. the corresponding value for saline - injected controls ., saline vmh (6 h);, leptin vmh (6 h). Injection of maximal dose of leptin into the arc induced a small but significant increase in the rate constant of 2[h]dg uptake in bat at 6 h after injection, but it had no effect on glucose uptake in skeletal muscle, heart, spleen, or wat (fig . Injection of leptin into the dmh or pvh had no effect on glucose uptake in peripheral tissues (fig . 2). Glucose uptake in peripheral tissues after saline injection into the dmh or pvh did not differ from that apparent after saline injection into the arc (data not shown). Plasma glucose and insulin concentrations were not affected by leptin injection into the vmh or other hypothalamic nuclei (supplemental table 2), consistent with previous observations (13,15). Effects of leptin injection into the arc, dmh, or pvh on glucose uptake in peripheral tissues . The rate constant of 2[h]dg uptake was measured in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) at 6 h after injection of leptin into the hypothalamic nuclei of mice . Data are means se for six or seven mice . * p <0.05 vs. the corresponding value for control animals injected with saline into arc ., saline arc;, leptin arc; leptin dmh;, leptin pvh . To determine whether leptin activated ob - rb in the hypothalamic nuclei, we examined the tyrosine phosphorylation of stat3 in tissue samples therefrom . Rt - pcr analysis confirmed that the isolated pvh, arc, and vmh specimens were enriched in crf mrna, pomc and npy mrnas, and sf1 mrna, respectively, and that the dmh was largely devoid of these mrnas (fig . Microinjection of leptin into the vmh, dmh, or pvh preferentially increased the tyrosine phosphorylation of stat3 to the similar extent in the corresponding nucleus at 6 h after injection (fig . Injection of leptin into the arc increased the tyrosine phosphorylation of stat3 in the dmh as well as in the arc (fig . 3e), possibly as a result of leakage of leptin into the dmh through the surface of the injection cannula, given that the arc cannula passed through the dmh . Together, these results suggested that the vmh is a key target of leptin in its regulation of glucose uptake in skeletal muscle, heart, and bat, whereas the leptin receptor in the arc mediates stimulation of glucose uptake in bat . Effects of leptin injection into medial hypothalamic nuclei on tyrosine phosphorylation of stat3 and c - fos expression . A: rt - pcr analysis of crf, pomc, npy, sf1, and eef2 (loading control) mrnas in the pvh, arc, vmh, and dmh . B e: immunoblot analysis of the phosphorylation of stat3 on tyr in the medial hypothalamic nuclei at 6 h after leptin injection into the vmh (b), dmh (c), pvh (d), or arc (e). Leptin was injected into the right side of the hypothalamic nuclei, and the same side of the pvh (p), arc (a), vmh (v), and dmh (d) was collected . Representative blots of tyr - phosphorylated stat3 are shown in the upper panels, and quantitative data (means se) from four to six mice are shown in the lower panels . The amount of tyr -phosphorylated stat3 was normalized by that of total stat3, and the normalized values were expressed relative to the corresponding value for the pvh of control mice . F: immunoblot analysis of c - fos expression in the medial hypothalamic nuclei after leptin injection into the vmh . The right side of the pvh, arc, vmh, and dmh was collected at 6 h after injection of leptin or saline into the same side of the vmh . A representative blot is shown in the upper panel, and quantitative data (means se) from four to six mice are shown in the lower panel . The amount of c - fos was normalized by that of -actin, and the normalized values were expressed relative to the corresponding value for the saline - injected control mice . * p <0.05 . We next examined whether leptin injection into the vmh might increase neuronal activity in other hypothalamic nuclei by measuring expression of the transcription factor c - fos . Leptin injection into the vmh significantly increased c - fos expression in the arc as well as in the vmh at 6 h after injection (fig . Leptin injection into the vmh increased the phosphorylation of stat3 in the nucleus preferentially at 1 h after the injection, similar to that at 6 h. however, it did not increase c - fos expression in any nucleus at 1 h (supplemental fig . Leptin injection into the vmh did not increase pomc mrna in the arc at 6 h after injection (supplemental fig . We next examined the role of mcrs in glucose uptake in peripheral tissues induced by injection of leptin into the vmh (fig . 4). Injection of the mcr antagonist shu9119 (1 g) into the lateral ventricle (intracerebroventricularly) abolished the increase in 2[h]dg uptake in peripheral tissues normally apparent at 6 h after the injection of leptin into the vmh . Injection of shu9119 alone did not affect glucose uptake in peripheral tissues . Plasma glucose and insulin levels were also not changed in response to intracerebroventricular injection of shu9119 (supplemental table 2). Effect of intracerebroventricular injection of shu9119 on glucose uptake in peripheral tissues induced by injection of leptin into the vmh . The rate constant of 2[h]dg uptake in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) was measured 6 h after injection of leptin or saline (control) into the vmh ., saline vmh;, shu9119 i.c.v . ;, leptin vmh;, leptin vmh + shu9119 i.c.v . We tested the effects of intracerebroventricular injection of the mcr agonist mt - ii on glucose uptake in peripheral tissues (fig . The intracerebroventricular injection of mt - ii (3 g) increased 2[h]dg uptake in bat, heart, and all types of skeletal muscle, including the white portion of the gastrocnemius, but not in spleen or epididymal wat, at 3 or 6 h after injection . Glucose uptake in peripheral tissues at 3 h after saline injection did not differ from that apparent at 6 h (data not shown). The intracerebroventricular injection of mt - ii increased the plasma glucose level at both 3 and 6 h after injection (supplemental table 2). These results thus suggested that intracerebroventricular injection of mt - ii promotes glucose production as well as glucose uptake in certain peripheral tissues . In contrast, plasma insulin concentration did not change after intracerebroventricular injection of mt - ii, despite the associated hyperglycemia, suggesting that mt - ii inhibits insulin secretion from pancreatic -cells . The rate constant of 2[h]dg uptake in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) was measured at 3 or 6 h after intracerebroventricular injection of mt - ii or saline (control). Finally, we examined the effects of direct injection of mt - ii into the individual medial hypothalamic nuclei on glucose uptake in peripheral tissues (fig . 6). Microinjection of mt - ii (10 ng) into the vmh increased 2[h]dg uptake in bat, heart, and all types of skeletal muscle but not in spleen or epididymal wat . In contrast, injection of mt - ii into the pvh increased glucose uptake only in bat, and that into the dmh or arc did not affect glucose uptake in any of the peripheral tissues examined . Plasma glucose and insulin levels did not change in response to injection of mt - ii into any of the hypothalamic nuclei (supplemental table 2). Effects of mt - ii injection into vmh, pvh, dmh, or arc on glucose uptake in peripheral tissues . The rate constant of 2[h]dg uptake in skeletal muscle (a), bat and heart (b), spleen (c), and epididymal wat (d) was measured at 6 h after the injection of mt - ii into the individual hypothalamic nuclei . Data are means sem for six or seven mice . * p <0.05 vs. the corresponding value for saline - injected control animals ., vmh;, mt - ii vmh;, mt - ii pvh;, mt - ii dmh;, mt - ii arc . Leptin is a physiologically and clinically important hormone that regulates glucose metabolism in peripheral tissues . We have previously shown that microinjection of leptin into the vmh and nearby medial hypothalamic area preferentially increased glucose uptake in skeletal muscle, heart, and bat (1416). In the present study, we found that activation of the leptin receptor specifically in the vmh, as detected by measurement of the tyrosine phosphorylation of stat3, resulted in a marked increase in glucose uptake in those peripheral tissues, similar to the effects of intracerebroventricular or peripheral administration of leptin (13,36). In contrast, injection of leptin into the arc increased glucose uptake only in bat . Injection of leptin into the dmh or pvh had no effect on glucose uptake in any of the peripheral tissues examined . The present data thus suggest that the leptin receptor in the vmh and arc regulates glucose uptake in different peripheral tissues . Our present results further indicate that mcr activation is necessary for the increase in glucose uptake in peripheral tissues induced by injection of leptin into the vmh . The intracerebroventricular injection of shu9119 thus abolished the effect of leptin injected into the vmh on peripheral glucose uptake, whereas intracerebroventricular injection of mt - ii increased glucose uptake in peripheral tissues . Moreover, injection of mt - ii into vmh, but not that into the dmh or arc, increased glucose uptake in skeletal muscle, heart, and bat, whereas injection of mt - ii into the pvh increased glucose uptake preferentially in bat . Both mcrs in the vmh and pvh may play an important role in the regulation of glucose uptake in peripheral tissues . Pomc neurons in the arc receive strong excitatory input from the dorsomedial region of the vmh (37). The restoration of ob - rb expression in the vmh by adeno - associated virus mediated gene transfer in koletsky rats increased the amount of pomc mrna in the arc (20). We have now shown that injection of leptin into the vmh increased c - fos expression in the arc without an effect on stat3 phosphorylation in the arc . Furthermore, the effect of leptin injected into the vmh on glucose uptake in peripheral tissues was found to be dependent on mcrs in the brain . We therefore propose that stimulation of vmh neurons by leptin results in activation of a set of pomc neurons in the arc and thereby increases glucose uptake in skeletal muscle, heart, and bat . Mcrs in the vmh and pvh contribute to the upregulation of glucose uptake in peripheral tissues induced by injection of leptin into the vmh . Although c - fos expression did not increase in the pvh in response to leptin injection into the vmh, this may have been due to the operation of -aminobutyric acid (gaba)-mediated neurotransmission in the pvh (6). The mechanism by which injection of leptin into the arc increased glucose uptake specifically in bat remains unclear . One possible explanation for this observation is that the injection of leptin into the arc activated a selective set of pomc neurons in the arc that regulate glucose uptake in bat alone, with pomc neurons being abundant in both the anterior and posterior regions of the arc (38). It is also possible that pomc neurons in the arc that regulate glucose uptake in skeletal muscle and heart require excitatory input as well as leptin for their full activation . The intracerebroventricular injection of mt - ii increased glucose uptake in bat to a markedly greater extent than did the maximum dose of injection of mt - ii into the vmh or pvh . Mc4r - expressing neurons in several regions of the brain stem as well as in the hypothalamic nuclei connect polysynaptically with interscapular bat (39). Direct injection of mt - ii into the raphe pallidus induces a thermogenic response in interscapular bat (40). Glucose uptake in bat might thus be regulated by mcrs in multiple brain regions . To date, there is little histological evidence of a connection between vmh neurons and interscapular bat, although injection of leptin into the vmh increases plasma catecholamine levels more effectively than does that into other hypothalamic regions (41). We previously showed that injection of leptin into the medial hypothalamus increased glucose uptake in peripheral tissues through the activation of sympathetic nerves (14,15). Injection of leptin into the medial hypothalamus also increased insulin sensitivity in peripheral tissues by a -adrenergic mechanism (14). Whereas the molecular mechanism remains elusive, a direct effect of -adrenergic receptors expressed in peripheral tissues as well as a -adrenergic receptor dependent increase in blood flow appear to contribute to leptin - induced glucose uptake in these tissues . The dmh is an important hypothalamic nucleus in the regulation of thermogenesis in bat (42). Injection of a gaba typea (gabaa) receptor antagonist into the dmh thus increased thermogenic activity in bat (42). Moreover, injection of a gabaa receptor agonist into the dmh blocked sympathetic, thermogenic, and cardiovascular responses induced either by injection of prostaglandin e2 into the medial preoptic area (43) or by skin cooling (44). Both ob - rb and mc4r are abundant in the dmh (7). Furthermore, dmh neurons, including those expressing mc4r, connect polysynaptically with interscapular bat (39). However, we have now shown that injection of leptin or mt - ii into the dmh did not increase glucose uptake in the peripheral tissues examined . Although we cannot exclude the possibility that injection of leptin or mt - ii activated only a subset of dmh neurons expressing ob - rb or mcr, our results suggest that the leptin receptor and mcr in the dmh have other roles, such as the regulation of food intake or modulation of bat thermogenesis in response to cold stimuli . Our present results indicate that the vmh regulates glucose uptake in skeletal muscle as well as in heart and bat, accompanying increased glut4 mrna in bat and heart . Thus, increased expression of glut4 may involve the acute increase in glucose uptake in bat and heart in response to leptin, while other mechanism might be involved in the leptin - induced glucose uptake in skeletal muscle . Intracerebroventricular injection of mt - ii has been shown to increase glut4 mrna expression in skeletal muscle at 24 h after the injection (22). Gene expression in skeletal muscle in response to leptin and mt - ii may require> 24 h. the present results showed that injection of leptin or mt - ii in some medial hypothalamic nuclei increased glucose uptake in certain peripheral tissues, while it did not alter plasma glucose level . These results suggest that leptin and mt - ii stimulates hepatic glucose production as well as glucose utilization in peripheral tissues . The result was supported by the previous report (13) showing that leptin increased plasma glucose turnover in mice at 6 h after the injection . Moreover, gutierrez - juarez et al . (45) has shown that intracerebroventricular infusion of mcr agonist increased hepatic glucose production in lean rats at 6 h after the start of the infusion, while it inhibited hepatic glucose production at 7 days (46). These results suggest that the effects of leptin and melanocortin receptor agonist on hepatic glucose production alter time dependently . Recent study suggests that mt - ii seems to be a promising agent to bypass leptin resistance and to improve energy homeostasis in diet - induced obese mice (47). We have shown that intracerebroventricular injection of mt - ii but not leptin activates amp - activated protein kinase in skeletal muscle in diet - induced obese mice (48). The pvh and vmh may be target sites of mt - ii to improve glucose metabolism in these mice . Together, our results suggest that the vmh plays a key role in leptin- and mt - ii induced glucose uptake in skeletal muscle, heart, and bat, whereas the leptin receptor in the arc and mcrs in the pvh regulate glucose uptake in bat . The medial hypothalamic nuclei thus appear to play distinct roles in the regulation of glucose uptake in peripheral tissues by leptin and mt - ii.
The street foods play an important socioeconomic role in meeting food and nutritional requirements of city consumers at affordable prices to the lower and middle income groups and are appreciated for their unique flavors and convenience [13]. Street foods also assure food security for low income urban population and livelihood for a significant proportion of the population in many developing countries . Street foods are described as wide range of ready - to - eat foods and beverages or prepared at home and consumed on the streets without further preparation . These food items are usually sold by vendors and hawkers in the streets or other similar public places . While street vended foods are appreciated for their unique flavors as well as their convenience, they are also important in contributing to the nutritional status of the population . In contrast to these potential benefits, it is also recognized that street food vendors are often poor, uneducated, and lack knowledge in safe food handling, environment, sanitation and hygiene, mode of food display, food service and hand washing, sources of raw materials, and use of potable water . Consequently, street foods are perceived to be a major public health risk . Foodborne illnesses of microbial origin are a major health problem associated with street foods [68]. In addition, resistance of foodborne microorganisms in multi - drug made the food safety situation more vulnerable in public health . Diarrheal diseases are the most common food poisoning cases in bangladesh and in some cases, these can cause death . The diseases are caused by either toxin from the microbe or by the human body's reactions to the microbe . The traditional processing methods that are used in the preparation, inappropriate holding temperature, and poor personal hygiene of food handlers are some of the main causes of contamination of street foods [11, 12]. Also the foods are not effectively protected from flies and dust [13, 14]. In bangladesh, street foods are mostly prepared and processed manually and sold to the public at various lorry terminals, by the roadside or by itinerant vendors . A study of the socioeconomic conditions and determination of the hygienic and sanitary practices of street food vendors in dhaka city corporation was carried out by fao 2010 . The study result demonstrated that 25% street food vendors are illiterate and cannot write their names and have no formal education . As street food business requires low investment, most of the vendors (88%) were found to own the business . Most of the vending shops (68%) were located on the footpath irrespective of areas surveyed and 30% vending carts were placed near the municipal drain and 18% near the sewerage . Microbiological study of different foods items, drinking water, and hand swab samples showed the prevalence of overwhelmingly high numbers of aerobic bacteria, coliform bacteria, and pathogens . For the sake of public health, it is important to understand the epidemiology of foodborne illnesses because it will help in prevention and control efforts, appropriately allocating resources to control foodborne illness, monitoring, and evaluation of food safety measures, development of new food safety standards, and assessment of the cost - effectiveness of interventions . The purpose of this study is to see the microbial risk of food poisoning associated with street food in order to determine the magnitude of the problem, risk factors, monitoring and surveillance, and measures of control . The street food vendors of bangladesh are not enumerated in the formal sector of country's economy . They are identified as the informal sector where their businesses are conducted as a form of irregular, unstable, and marginal economic activities . As such there is no systematic documentation of the numbers of street food vendors, their scale of businesses, or the viability of their pursuits . After rickshaw - pulling, street vending is probably the second most important employment opportunity for the urban poor in bangladesh, and particularly important for young and middle - aged men who have migrated to dhaka in the past five to ten years . Dhaka is among the world's cities with the highest number of hawkers: in asia, only mumbai (~250,000), delhi (~200,000), calcutta (~150,000), and bangkok (~100,000) have similarly large numbers of street vendors . However, benjamin conducted a survey on street food vendors in dhaka, over a period of three years (2007 to 2010). This survey and official labour statistics demonstrated that between 90,000 and 100,000 street vendors sell prepared food items, and around 418,000 people or 2.9 percent of dhaka's total population depend on the income generated by street food vendors . This implies that almost eight million people or 55 percent of the population of dhaka take some street food everyday . The significance of street food system of dhaka is beyond doubt and selling street food is not a marginal economic activity, but a normal yet highly visible social practice, that is, economically efficient and deeply embedded in the urban economy and in urban life [2, 4, 20]. A glimpse of the socioeconomic background of the vendors is presented below to help understand who the street food vendors are.both males and females and married and unmarried operate as street food vendors . Their age range is between 25 and 60 years with a majority being in the age group of 3040 years.many street food vendors and their families have their origin in rural backgrounds or have moved to urban centers at a later stage or else live in rural areas and travel daily to the city for their business operations.the level of education achieved by the street food vendors is comparatively low and in the case of a majority, education levels varied between grades 5 and 8.many street food vendors are constrained by the unstable socioeconomic backgrounds in their families.employment history of the street food vendors shows their previous involvement in several urban - based, irregular, and low - paid income generating activities, which required hard manual labor, prior to their involvement in the street food business . Their age range is between 25 and 60 years with a majority being in the age group of 3040 years . Many street food vendors and their families have their origin in rural backgrounds or have moved to urban centers at a later stage or else live in rural areas and travel daily to the city for their business operations . The level of education achieved by the street food vendors is comparatively low and in the case of a majority, education levels varied between grades 5 and 8 . Employment history of the street food vendors shows their previous involvement in several urban - based, irregular, and low - paid income generating activities, which required hard manual labor, prior to their involvement in the street food business . Street food vendors are a self - employed category of small entrepreneurs who are not dependent on any institutional structures to find their livelihoods . Their enterprises evolve exclusively around their own individual strengths and the support extended to them by their immediate social networks such as family members and other close associates . The earnings from their business enterprises are a means of living for the vendors themselves and their dependent family members . As such, these economic activities of the street food vendors have not only provided a source of livelihood to the vendors and their dependent family members but also have reduced the plight of their becoming an economic and social burden on the state . Street food, therefore, not only meets the food requirements particularly of those of the low income categories but also the busy customers who do not have much time either to prepare their own food or to go to other eating houses where probably the food is more expensive and servicing is time consuming . Street vendors face unique kinds of livelihood risks because of the legal, physical, and sociocultural environment in which they work . The most pressing and ongoing risk for many street vendors is the possibility that local government authorities will forcibly remove them from the streets or confiscate their merchandise . This risk of displacement often increases in the context of elections, mega events, or efforts to beautify historic city centers . Just like formal business operators, street vendors are less productive in unstable institutional environments where rules are irregular and unpredictable [5, 19]. Many must lift and haul heavy loads of goods to and from their point of sale each day . The physical environments in which they work typically lack proper infrastructure, such as clean running water, toilets, and solid waste removal (table 1). Street vendors are exposed to physical harm from the improper provision of fire safety equipment and the improper regulation of traffic in commercial areas . They are also exposed to a high concentration of air pollutants and to inclement weather . These physical risks take a particular toll on young children who must accompany their mothers to vend in the streets . Given the growing numbers of the street food vendors and the customers who patronize them, the issues and problems the vendors encounter need special attention of the authorities concerned . Harassment on the part of local authorities including evictions, confiscation of merchandise, and demands for bribes is a common source of income risk for street vendors . Street vendors legal status can act as a bridge between their employment conditions and the range of employment risks they face (table 1 and figure 1). A vendor with a fixed structure in a designated market, for example, may be more likely to hold a license or permit, and in turn would be less exposed to certain kinds of risks . Likewise, a street vendor who works as an employee selling a particular kind of product, such as newspapers, may be better protected by law and therefore less vulnerable . Obtaining legal status of some kind is therefore a key demand of street trading organizations in many cities . The number of people living in the country's capital dhaka almost doubled from 5.3 to 9.3 million . This development has led to an increase in the demand for relatively inexpensive and ready - to - eat foods as many urban residents spend most of the day outside of the house and have little time and money to spend on food . Rapid urbanization also turned street - food vending into an important business; in dhaka alone, around 200,000 people earn their living by selling street foods [4, 23]. The low cost, accessibility, and convenience are the key factors for the growing popularity of street foods . Women play a very vital role in the street food sector through their direct and/or indirect involvement in the business . Additionally a significant number of street vendors are woman - headed households [1, 19]. The diversity that exists among street food vendors is reflected in the type of food they prepare / sell, the scale of their business, the mode in which they are operating, the locations in which they prepare and sell food, the type of clients to whom they sell food, and so forth . There are so many varieties that it is impossible to provide a menu of all the different street foods consumed around the world . In bangladesh, street foods include chola boot (chickpeas), bhelpuri (puffed rice with potatoes), and samucha (deep - fried dough stuffed with vegetables and/or meat) as well as drinks like sugar - cane juice and lassi (yoghurt and water). Other popular snacks are ghugni (boiled and mashed white peas with spices), singara (flour wraps stuffed with vegetables, spices, and occasionally liver), and different types of cakes . The customer surveys undertaken by fao 2006 and other investigators revealed that the main consumers of street foods in most countries were other members of the informal sector, such as fellow hawkers and hustlers and casual wage laborers . Other important categories of customer were children and students, office workers, and housewives . The studies also found that street foods were consumed across all income groups and the proportion of the daily household food budget spent on street foods was high, ranging from 25 percent in bogor to 47 percent in chonburi, thailand . The frequency and regularity of consumption were variable: in some countries, street foods were bought daily and formed an integral part of the diet; in others, notably in bangladesh, they appeared supplementary and few customers bought them daily [6, 26]. Some categories of consumer (students, itinerant unskilled laborers, and the homeless) were found to buy almost all their food from vendors . The cost of street foods is usually competitive compared with that of foods purchased from larger food establishments, such as restaurants and fast food outlets . Also, due to the sometimes high costs of fuel and ingredients in urban contexts, economies of scale can create a street food cheaper than the same food prepared at home . The hygienic aspects of street food vending are a major concern for food control officers . Vending stands are often crude structures, and running water, washing facilities, and toilettes may not be available . Improved safety of street foods can be achieved through awareness raising programmes involving several partners such as local authorities, the food vendors, government departments, consumer organizations, standard setting bodies, and some nongovernmental organizations . In some instances, the vendors are keen to participate in programmes that provide basic facilities that make it possible for them to work in clean environments . For example, in a survey of street food vendors in lusaka and harare, the vendors indicated that they would be willing to pay for basic facilities such as running water and electricity but would want the local authorities to provide the water points, refuse receptacles, and washing facilities . A viable partnership involving local authorities, vendors and policy makers is therefore encouraged as this should lead to the improvement of business conditions and allow for the improvement of the livelihoods of vendors and their families . The battle against foodborne diseases is facing new challenges due to the globalization of the food market, climate change, and changing patterns of human consumption as fresh and convenient foods are currently preferred . As food is biological in nature, it is capable of supporting the growth of microorganisms and foodborne diseases result from the ingestion of contaminated foods and food products . More than 250 different types of viruses, bacteria, parasites, toxins, metals, and prions are associated with foodborne diseases in humans . Although viruses are more responsible for more than 50% of all foodborne illnesses; generally hospitalizations and deaths associated with foodborne infections are due to bacterial agents . The infections range from mild gastroenteritis to life - threatening neurologic, hepatic, and renal syndromes caused by either toxin from the disease - causing microbe or by the human body's reaction to the microbe itself . Of the many thousands different bacterial species, more than 90% of food - poisoning illnesses are caused by species of staphylococcus, salmonella, clostridium, campylobacter, listeria, vibrio, bacillus, and enteropathogenic escherichia coli . For instance, in the us and france, in the last decade of the 20th century, salmonella was the most frequent cause of bacterial foodborne illness (5,70010,200 cases), followed by campylobacter (2,6003,500 cases) and listeria (304 cases). In south africa, species of listeria, enterobacter, and aeromonas were the most prevalent bacteria in ready - to - eat foods . However, no such databases are available for bangladesh as well as other developing countries . Therefore, from october 2012 to the present, our laboratory conducted a series of experiments to assess the microbial quality of street foods of bangladesh . More than 100 street foods samples of 20 kinds including singara, jhal - muri, chatpati, chetoi pitha, chola / bengal gram, jilapi, jar drinking water, pickles, amra, tehari, vegetable rolls, sugarcane juice, raw cucumber slices, milk, other juices, beverages, and bread were analyzed for major foodborne pathogens including, salmonella spp ., escherichia coli o157, o111, o26, and other e. coli, other coliforms, listeria spp ., and staphylococcus spp . The presence of all the above mentioned pathogenic organisms found in the street foods was presented in table 2 . In addition, the isolated pathogenic organisms were found resistant to at least 7 antibiotics . These studies demonstrated that foods sold in the street of dhaka city constitute a potential microbial hazard to human health . In addition, mamun et al . Reported that among different items of street vended foods, 54% of sliced fruits samples, 59% of jhalmuri samples, 29% of chotpotis samples, 53% of vajavuji samples, and all (100%) sharbat samples were found unsatisfactory microbial quality . This finding also reflected poor microbiological quality of the school - based street vended foods indicating a health threat to the school children of dhaka city [1, 8]. Nonfood grade chemical additives, such as colorants and preservatives, and contaminants, such as pesticide residues, have also been found in street foods . A chemical analysis of street foods in bogor found unpermitted coloring agents such as textile dyes and also pesticide residues . Proper use of salt, spices, nitrates, and sugar is an important means of preventing food spoilage, but the drive to keep prices low may lead to the purchase of cheap ingredients containing unpermitted chemical additives from unauthorized suppliers . Chemicals such as colorants may also be added to mask the poor quality of cheap materials . Due to the conditions under which street foods are sold, there is concern that food may be contaminated with heavy metals and pesticide residues . These contaminants may come from the utensils, raw materials, or transport methods used and may also occur due to the lack of appropriate storage facilities . A study carried out in ghana revealed that street food vendors source their pots and other utensils from both formal and informal manufacturers / retailers . Some of the street food samples had higher levels of lead, cadmium, arsenic, mercury, and copper than average food samples, suggesting possible leaching from the utensils . Further tests showed that lead from the pots obtained from informal manufacturers could leach into the food . These pots are manufactured using scrap metal that could come from diverse sources such as derelict cars, car batteries, and industrial machinery, which are obviously not suitable for use with foods . This was attributed to the fact that when police raid these vendors, they usually confiscate their wares, including the pots and utensils . For fear of losing their more expensive pots, the vendors resort to using informally fabricated pots, thereby exposing consumers to the possibility of food contamination by heavy metals . Further work must be done in order to reduce the exposure of consumers to heavy metals and pesticide residues through street - vended food [29, 36]. Purchasing ready - to - eat foods and ingredients from street / market vendors poses a considerable risk to public health, especially due to the poor hygienic practices . In most cases, the vendors do not have adequate washing facilities, and some vendors started their duties without taking a proper bath . Some of the vendors sleep at the vending sites in order to protect their wares . Foods and ingredients are also subjected to repeated contamination from unwashed hands and the materials used for wrapping, such as leaves, old newspapers, and reusable polyethylene bags . However, many vendors are aware of the need to wear clean and appropriate clothes . Some of the female vendors wear headgear and aprons . After a few awareness - raising campaigns for vendors, the absence of water points near their work places and poor drainage facilities make them unable to practice good hygiene . Moreover, some food handlers washed their hands in the same bucket used for cleaning utensils, which may lead to the contamination of food with faecal matter . On the other hand, most food vendors operate their business without health certificates or licenses, which poses additional concern and required extensive training programme on food preparation and handling techniques . Street food vendors use cheap bar soap than liquid soap, which may be more effective, to clean their utensils, but as they use cold water, resulting in inefficient cleaning . Furthermore, washed plates are often stored in an unclean corner, plastic bowl, or cardboard box, leading to recontamination of the plates . This leads to an increased pest population and resulted in an increased risk of food contamination . In many instances, the vending sites are not included within the city or town plans, and therefore amenities such as refuse collection are not available . City authorities are often faced with the dilemma that if they provide services to illegal operations, this will imply recognition of these operations . At the same time, because the vending operations are illegal and vendors do not contribute anything towards the maintenance of infrastructure or provision of public services, therefore, they are not entitled to the service . This contributes to further deterioration of the hygienic condition of the area where the foods are vended . Poor sanitary conditions in the area where foods are vended also contribute to poor food storage and transport conditions . Street food vendors in some cities obtain their vegetables, maize meal, and other condiments from licensed shops, and therefore there is less concern regarding the safety of these raw materials . However, most of the vendors have no fixed stalls where they can store their raw materials on site . They usually store their goods at home overnight and transport them the following day, often improperly covered, to their operating sites . Thus, the food becomes prone to contamination during transportation [18, 19]. A recent review of the situation in asia found great diversity among the legal instruments developed to control the street food trade . Some countries had no specific legislation or control systems at all [19, 22]. In those countries where street food activities were regulated by law, the regulations or by - laws affecting the street food trade were part of a larger body of legislation dealing with food, health, or environmental sanitation . Licensing or registration systems, inspection systems, and codes of practice are other forms of regulation that are in effect in some countries . A number of pieces of legislation relating to the preparation and sale of safe street foods have been established by the bangladesh government . The bangladesh pure food ordinance 1959 (revised 2005) has several sections dealing with the safety of street food: adulteration of food; prohibition of calcium carbide, formalin, and insecticide; selling unwholesome food; uncovered foods; and unhygienic premises and violations of the health code . The other relevant legal measures related to safe street foods are the bangladesh standards and testing institution (bsti) ordinance 37 of 1985; the consumers rights preservation act 2009; and the penal code of 1860, sections 272276 . A mobile court to monitor street food vendors a number of civil society organizations have emerged in recent years in bangladesh to promote safe street foods and overall food safety . For example, the consumers association of bangladesh (cab), vocta (consumer), which has conducted street food surveys and organized awareness, campaigns through rallies, seminars, workshops, and policy advocacy . The electronic and print media are also involved in providing public awareness of safe street foods . In contrast, key constraints to the effective management of street foods are the lack of awareness of personal hygiene and safe food among street food vendors and consumers; insufficient awareness among the consumers about the consumer rights act; lack of clarity in existing legislation and standards on street food; no specific regulatory body for licensing, provision of i d card, medical fitness, or dress codes concerning street food vendors; no demarcation of specific areas by local municipalities for street food vendors; insufficient number of sanitary inspectors; the inability of sanitary inspectors to take penal actions against street food vendors; and absence of appropriate training and supervision of street food vendors . The quality and safety of street foods is determined by numerous factors such as the business organization, regulatory aspects, technical aspects related to the preparation, preservation and display of food sold in the streets, the consumer perspective, and educational programs . In order to improve the conditions of street food vendors and to make sure that the food sold does not jeopardize public health, the first and foremost necessity is to build awareness that food vendor should maintain certain quality standard . In many areas, street foods are sold and food safety issues are not taken into consideration neither on the producer nor on the consumer side . Consumers tend to look mostly at the price and might be already accustomed to the taste of unhealthy meals . Vendors, on the other hand, have a very small margin of profit and are incentivized to keep expenses low by utilizing low quality ingredients and disregarding costly hygienic practices . To break this vicious cycle, governments need to embrace street food vendors as a dynamic economic sector . With their adaptability to the frenetic life in the global cities, street food vendors have a huge potential to quickly fill niches, greatly improving urban access to food . While excessive regulation of the sector carries the risk of suffocating this adaptability and would just shift the problem to a new informal sector consisting of those dodging the regulation, certain minimum standards, especially related to food quality, need to be enforced . Vendors should be given some basic training on how to safely prepare and store food and businesses should be certified accordingly . While some proposed the application of haccp standards, others argued against it stressing the need for much simpler guidelines such as the five keys to safer food . In addition, municipalities should provide vendors with appropriate infrastructure like access to clean water and sewage systems . Street food vendors should be encouraged to partake in awareness raising programmes and given access to microcredit . In order to improve the vendors standing, strengthening their overall position vis - - vis authorities, promoting their organization into cooperatives has been identified as a path to follow . In addition to helping vendors run their business in a more efficient and safe manner, cooperatives would also ease the authorities work in enforcing hygienic and business standards . In general, interventions and programmes can only be successful if they do not focus on one aspect alone . Tackling only food quality, for instance, cannot ensure that street food vendors play the most positive role in realizing food security of the urban population . It is important not to forget that the street foods constitute a very heterogeneous sector and the interventions need to be carefully planned by keeping different aspects such as gender, secondary audience, and local customs into consideration . It is also necessary to differentiate between vendors selling freshly prepared food on the spot or hawking dishes prepared earlier at home, with the second practice being much more risky in terms of foodborne pathogen and spores . Needless to say, general education levels also play an important role in ensuring safe street foods . The more both vendors and patrons will be educated and the more they will know about issues such as nutrition and food safety, the more they will be interested in having the business as clean and the products as healthy as possible.
After institutional review board approval, we did a retrospective chart review of infants 1500 g birth weight and treated for aprop between january 2006 and december 2009 . The neonatal intensive care unit at our institute is a level iii nursery, which also caters infants born at other centers (outborn). Aprop was diagnosed in accordance with the international classification of rop and documented by detailed retinal drawings and retcam (clarity msi, pleasanton, california, usa). We completed a chart review that retrieved various parameters including birth weight, gestational age, setting of birth (inborn / outborn), level of nursery care (level i nursey: basic level nursery, level ii: specialty services like mechanical ventilation, level iii sub - specialty care including sustained life support), neonatal illnesses, and oxygenation . We excluded infants with hydrocephalus, as this condition leads to an exaggerated birth weight . Characteristics of rop including the zone, pattern of neovascularization, and atypical features were noted . All infants underwent confluent laser (less than half burn width apart) application with either a diode laser or 532 nm laser delivered through the indirect ophthalmoscopic system . We noted details of laser treatment including postconceptional age at treatment, number of spots, number of laser sittings, time to regression, and outcome . The criteria for unfavorable outcome at 6 months were: (1) retinal detachment (stage 4a/4b/5), (2) falciform fold involving the macula, and (3) a retro - lental tissue / mass obscuring the view of the posterior pole . Sixteen infants 1500 g birth weight underwent laser photocoagulation for aprop during the study period . Disease characteristics of infants developing aprop the mean birth weight and gestational age were 1791.27 281.86 g (range, 1500 - 2300 g) and 30.7 1.03 weeks (range, 29 - 32 weeks), respectively . All infants were outborn and referred from level i / ii accredited nurseries, which had a limited level of neonatal care with paucity or nonavailability of saturation monitors and oxygen blenders . Duration of oxygen exposure was known for 11 infants and ranged from 7 to 23 days . Systemic risk factors in infants 1500 g developing aprop (n=14) * twenty - nine eyes of 15 infants developed aprop . Of the 29 eyes, 10 (34.5%) had zone 1 and 19 (65.5%) had posterior zone 2 disease . Fundus photographs from a male infant weighing 1500 g at birth and 29 weeks of gestation demonstrate aprop in posterior zone 2 and regression following confluent laser photocoagulation . (a) fundus photograph prior to laser photocoagulation demonstrates plus disease and flat new vessels in posterior zone 2 . (b) fundus photograph at 37 weeks shows complete regression fundus photographs from a male infant weighing 1875 g at birth and 32 weeks of gestation demonstrate aprop in zone 1 with extensive brush like proliferation projecting into the vitreous and covering the optic disc and posterior pole in right eye (a) and left eye (b), respectively . Both eyes had an unfavorable outcome after laser fundus photographs from a male infant weighing 1990 g at birth and 30 weeks of gestation demonstrate aprop in posterior zone 2 and large vascular loops . (a) fundus photograph prior to laser demonstrates plus disease, flat new vessels and hemorrhages in posterior zone 2 . (b) the nasal aspect of same eye demonstrates a large vascular loop (block arrow) enclosing a grayish avascular retina (arrow). (d) the nasal aspect of same eye shows laser scars inside the vascular loop the mean post conceptional age at laser treatment was 35.2 1.54 weeks (range, 33 - 39 weeks). The mean number of spots were 2323.83 1211.20 (range, 840 - 5210). Of the 29 eyes, 22 (75.9%) eyes had complete regression after laser . The mean time to regression was 6.14 2.05 weeks (range, 3 - 10 weeks). Seven (24.1%) eyes had an unfavorable outcome (stage 5: two eyes, stage 4a: two eyes, cataract: two eyes, vitreous hemorrhage: one eye). One eye with stage 5 underwent lensectomy vitrectomy with an unfavorable outcome . The other eye with stage 5 both eyes of an infant had localized (2 clock hours) stage 4a detachments which remained stable until a follow - up of 34 months . Overall, a complete regression was achieved in 25 (86.2%) of the 29 eyes . Various studies from the west and japan describe aprop in infants <30 weeks of gestational age and <1000 g birth weight . However, recent studies from india report aprop in older and heavier infants . In a recent study from north india, 15.91% infants developing aprop had a birth weight above 1500 g. the reported risk factors for zone 1 aprop include extreme prematurity, disruption of vasculogenesis, and a low platelet count . A recent study has observed the use of supplemental unblended oxygen in heavier infants developing aprop . Most of the infants in the present series had multiple co - morbidities and received supplemental oxygen in centers with poor neonatal care . It is plausible that early and excessive exposure to unmonitored oxygen therapy may lead to aprop - like morphology in these infants . However, the present study is too small to prove causal association of any risk factor with aprop in heavier infants . Aprop in heavier infants differs in certain aspects from disease seen in extremely low birth weight premature infants [table 3]. First, there is a preponderance of posterior zone 2 aprop with more mature central vasculature as compared with poorly developed vasculature in zone 1 aprop . Second, vessels extend for a considerable distance into the nasal retina forming large loops and enclosing an underlying avascular retina . Finally, we did not observe relentless fibrovascular proliferation after laser treatment, which might limit outcomes in typical aprop . Clinical features, risk factors and outcomes for various types of rop the present study is retrospective and lacks a control group . A prospective study is difficult, given the fact that infants come from different centers . In conclusion, aprop may be seen in infants 1500 g birth weight . Further studies should evaluate the risk factors, pathogenesis, and genetic mechanisms for aprop in these infants.
Generally speaking, cell transplantation studies anticipate that transplanted cells survive long - term to be integrated into the host spinal cord tissue, serving as a scaffold for the outgrowth of regenerating axons in spinal cord injury . . However, this anticipation is not necessarily applicable to some somatic cells: bone marrow stromal cells (bmscs) did not survive long - term, disappearing from the spinal cord within 23 weeks after transplantation in rats . Nevertheless, the transplantation of bmscs enhanced the outgrowth of regenerating axons and promoted locomotor improvement of rats . This suggests that bmscs release some trophic factors that are effective for tissue repair and functional recovery in spinal cord injury (sci). Transplantation of choroid plexus epithelial cells (cpecs) also enhanced the axonal regeneration and locomotor improvement in rats with sci . However, they disappeared from the spinal cord shortly after transplantation, as in the case of bmscs . Immature cells, such as neural stem / progenitor cells (nspcs), survive long - term, proliferate extensively, and differentiate into glial cells and/or neurons after transplantation . These properties are, although appearing to contribute to tissue repair and the establishment of new neural connections, regarded as deleterious factors from the clinical point of view of safety: no method is available at present to manipulate and control the behaviors of these cells to allow them to appropriately integrate into the host spinal cord . Cell transplantation studies aiming at the regeneration of tissues or organs have significance only when they are clinically applicable under the condition that the transplants are safe and they promote functional improvements of recipients . Bmscs are cells that adhere to the dish on culture of a bone marrow perfusate . In an experiment in which bmscs were transplanted by infusion through the cerebrospinal fluid (csf) via the 4 ventricle, transplanted bmscs attached to the spinal cord surface, and a few of them homed in on the spinal cord lesions . Host spinal cord axons located near the lesions were spared, avoiding secondary degeneration in bmsc - transplanted rats (ohta et al ., 2004). In an experiment in which bmscs were transplanted directly into the spinal cord lesions of rats with sub - acute sci, transplanted bmscs similarly survived short - term (12 weeks after transplantation) as cell assemblies in the lesions, in which no astrocytes were present . Such astrocyte - devoid areas, although appearing empty on immunostaining for glial fibrillary acidic protein (gfap), were filled with extracellular matrices, through which numerous axons extended (figure 1). These axons were myelinated by schwann cells, as in the case of peripheral nerves, indicating that they might not be spared but regenerated axons . Features of axons extending through the astrocyte - devoid areas suggest that they are regrowing axons from axotomized fibers, but not sprouts from spared axons . Cavity formation was reduced, and locomotor functions were improved by cell transplantation (ide et al ., 2010). In another experiment, bmscs were infused through the csf three times in rats with chronic (4 weeks after injury) sci . The findings of axonal outgrowth through the astrocyte - devoid areas and reduced cavity formation were the same as those in the preceding studies of acute and subacute sci . The spinal cord was contusion - injured at t89 in adult rats, and bone marrow stromal cells (bmscs) were transplanted into the spinal cord lesion at 2 weeks post - injury . Rats were fixed at 1 week post - transplantation, and horizontal sections of the spinal cord lesion were observed . The lesion is filled with tissue (s) different from the spinal cord parenchyma (h). (a-3): glial fibrillary acidic protein (gfap) immunohistochemistry (red) in the section adjacent to a-2 . Engrafted bmscs (site indicated by asterisk) are located at the border of the astrocyte - devoid area . Numerous axons (red) extend in a bundle along the total length of the astrocyte - devoid area shown in a-3 . There is no finding suggesting the blocking of extension of growing axons at the transition zones (arrows) on the rostral or caudal side . Bone marrow mononuclear cells (bmncs) were separated by density - gradient centrifugation from the bone marrow perfusate, and used without culture for transplantation to rats with sci (yoshihara et al ., 2007). The short - term survival with no integration into the host spinal cord of bmscs and bmncs suggested that they secrete some trophic factors that promote axonal regeneration and tissue repair, leading to locomotor improvements . The short - term survival of bmscs and bmncs, although appearing to be a disadvantage, guarantees the safety of their transplantation on clinical application . Based on these studies, bmscs and bmncs a total of 5 patients received bmsc transplantation up until 2009, and 10 patients received bmnc transplantation up until 2013 (suzuki et al ., 2014). In both clinical applications, there was no adverse effect, and patients showed varying degrees of improvement in motor, light touch, and pin - prick scores . The choroid plexus (cp) forms the ventricular wall of the brain, and consists of epithelial cells and the underlying pia mater . Choroid plexus epithelial cells (cpecs) are a continuation of ventricular ependymal cells, and the site of csf production . In addition, cpecs have been studied as the primary gate for trafficking of the immune cells from the vascular system to the csf in injuries and diseases of the cns . It has been reported that cpecs secrete many kinds of trophic factors, including insulin - like growth factor (igf), fibroblast growth factor (fgf), and hepatocyte growth factor (hgf), for the maintenance of normal cns functions . The first experiment on cp transplantation was performed using minced cp tissues as a transplant in spinal cord lesions . Numerous regenerating axons extended toward the transplanted cp tissues in the spinal cord, suggesting that the cp might be effective as a transplant for sci . In an in vitro experiment, neurons associated with cpecs extended many long, elaborate neurites on the surface of cpecs . In another in vitro study, the conditioned medium (cm) of cpec culture promoted neuronal survival and neurite extension of hippocampal neurons . On the other hand, matsumoto et al . (2013) studied the effect of the transplantation of cultured cpecs on brain infarction by infusion into the csf via the 4 ventricle, demonstrating that cultured cpecs markedly reduced infarcted lesions caused by carotid artery ligation . This indicated that cpecs did not replace the infarcted tissue, but exerted their effects by releasing diffusible neurotrophic factors that reached the lesion through the csf . . A recent study on the transplantation of cultured cpecs into spinal cord lesions demonstrated that cpecs enhanced the extension of regenerating axons in the lesions, and promoted locomotor improvements (kanekiyo et al ., 2016). Cpecs did not survive long - term, disappearing from the spinal cord 23 weeks after transplantation . There were no findings suggesting the proliferation, differentiation, or migration of transplanted cpecs in the host spinal cord lesions . These characteristics of cpecs as transplants are the same as those of bmscs and bmncs . It has been suggested that cpecs may, as in the case of bmscs and bmncs, secrete some trophic factors effective for the recovery of spinal cord injury by promoting the intrinsic regeneration capacity of the spinal cord . This does not mean that neurotrophic factors work to transform intrinsic cells into new neurons or glial cells, but to enhance tissue repair, including axonal regeneration and cavity suppression . It is hoped that some humoral factors effective for spinal cord injury will be identified in the near future . We conclude that the transplantation of bmscs, bmncs and cpecs arouses and enhances the intrinsic ability of the spinal cord to regenerate . Transplanted cells do not need to survive in the host spinal cord to serve as a scaffold for tissue repair, including axonal regeneration . This thought is not in line with the generally believed concept of cell transplantation studies for the treatment of sci . This proposition is important as a new premise for the study of spinal cord regeneration and the treatment of sci . Since the epoch - making study showing that transplanted peripheral nerve segments provide a favorable environment for the growth of regenerating axons in the cns, schwann cells have been regarded as a key cell for providing an appropriate environment for the growth of regenerating axons in the cns . Schwann cells are obtained from adult peripheral nerves: they are enzymatically dissociated from peripheral nerves, and after treatment with forskolin, cultured for several weeks . Schwann cells serve as a scaffold for regenerating axons . However, after growing through the schwann cell assemblies, regenerating axons do not exit the transplants in the spinal cord lesion (kanno et al ., 2015). It is assumed that axons are blocked from extending through the glial scar formed at the border of the lesion . This disadvantage was overcome by the chondroitinase - digestion of chondroitin sulfate, local injection of growth factors (afgf, gdnf), and/or co - transplantation of schwann cells with other types of cells such as bmscs and olfactory ensheathing cells . Recent studies suggest that the astrocyte scar is necessary for axonal regeneration in the spinal cord lesion (anderson et al ., 2016). It was reported that 6090% of the transplanted schwann cells survived over 3 weeks after transplantation . Schwann cell proliferation peaked at 2 weeks, decreased thereafter, and ceased at 12 weeks post - transplantation . Schwann cells are well - integrated into the host spinal cord tissue (deng et al ., 2015). Similarly, olfactory ensheathing cells are regarded as effective and safe transplants for spinal cord injury . The miami project to cure paralysis at the university of miami received permission from the fda to begin a clinical trial of transplanting human autologous schwann cells to treat patients with sci in 2012 . Schwann cells harvested from the sural nerve of the participant will be autologously transplanted into the epicenter of the participant s spinal cord injury . It was reported that there was a marked infiltration of endogenous schwann cells into the host spinal cord tissue on schwann cell transplantation . This finding is in line with our study showing that axons in the astrocyte - devoid areas were surrounded by schwann cells in bmsc transplantation . In the experiment, in which nspcs were obtained from the fetal cns, and transplanted directly into the spinal cord lesion or indirectly through the csf, nspcs survived, integrated, and migrated extensively . Some of them had differentiated into neurons and astrocytes by 4 weeks post - transplantation . Nspcs infused through the csf via the 4 ventricle attached to the surface of the spinal cord, continued to proliferate, and occupied a large area of the spinal cord 3 weeks after transplantation (bai et al ., 2003). These findings were interesting from the point of view of basic science; however, we wondered at that time whether nspcs with the potent properties of migration and proliferation were safe as transplants from a clinical point of view . The spinal cord was transected at c5, and nspcs in fibrin matrices containing a growth factor cocktail were transplanted 2 weeks after transection . Nspcs filled the lesion cavities, differentiated into neurons, and extended numerous axons rostrally up to the midbrain and even to the cortex . Many axons also extended caudally up to t6, or even to the t12 level . No myelination was noted on regenerated axons extending through the white matter of the host spinal cord . No functional recovery was observed . They suggested that ectopic innervations could potentially be manipulated and shaped with axon - guidance strategies . In addition to ectopic innervations, the proliferation, migration, and differentiation of transplanted cells should be appropriately manipulated in a clinical application . This means that nspcs cannot be used for clinical purposes until methods are developed by which the behaviors of nspcs can be appropriately manipulated and controlled . Regarding functional recovery, nspc transplantation is not necessarily effective for locomotor improvement, which is the essential parameter for the clinical application of cell transplantation (table 1).
Primary adhesive capsulitis is a common shoulder condition characterized by painful loss of both active and passive range - of - motion in all planes of the glenohumeral joint, especially external rotation . Although the pathogenesis progresses through fibrosis and culminates in joint contracture, it is generally recognized as a self - limiting process with an unknown etiology . Despite reports of 96% to 100% of patients returning to normal shoulder function by two - year and four - year follow - up, some authors have described severe limitations in range - of - motion and persistent pain and weakness at similar durations of follow - up. [69] nevertheless, several treatments are recognized and utilized to reduce pain and improve range - of - motion faster than the disease's natural history course . These treatments, in isolation or combined, include intra - articular corticosteroid injection into the glenohumeral joint, subacromial corticosteroid injection, intra - articular saline hydrodilation with distention and eventual rupture of the glenohumeral joint capsule, intra - articular sodium hyaluronate injection into the glenohumeral joint, suprascapular nerve block, shoulder manipulation under anesthesia, physical therapy with modalities, oral corticosteroid tapers, oral nsaids (non - steroidal anti - inflammatory drugs) and analgesics, and open or arthroscopic surgery with synovectomy and glenohumeral capsular releases . Sodium hyaluronate injection into the glenohumeral joint for the treatment of adhesive capsulitis has shown similar clinical improvements as those seen following corticosteroid injection with fewer side effects . The effects seen following hyaluronate injection include, but are not limited to, reduction in pain and improved range - of - motion, anti - inflammation, chondroprotection, and improved synovial fluid characteristics . Primary adhesive capsulitis of the shoulder is usually described as a self - limiting condition that resolves spontaneously by two to four years. [34] however, the natural history is not completely understood and recent studies have shown that longer term disability may occur following resolution of the disease. [69] further, it is not entirely clear how much a specific treatment improves long - term outcomes, if a specific treatment expedites clinical improvement, and if a specific treatment results in faster or better clinical outcomes than other treatments . Sodium hyaluronate is a well - recognized, safe, and minimally invasive treatment that results in improved outcomes in adhesive capsulitis of the shoulder . To the authors knowledge, no study has fully evaluated the literature reporting clinical outcomes following sodium hyaluronate intra - articular glenohumeral joint injection for the treatment of adhesive capsulitis . The purpose of this systematic review was to comprehensively analyze the evidence regarding the effectiveness of intra - articular sodium hyaluronate injections in the treatment of primary adhesive capsulitis . We hypothesized that intra - articular sodium hyaluronate injections would result in significant improvements in passive range - of - motion, shoulder and general clinical outcome measures, and pain scales at short- and mid - term follow - up . To address our hypotheses, we performed a systematic review of the available medical literature using several medical databases, including pubmed, medline, cinahl (cumulative index to nursing and allied health literature), sportdiscus with full text, and cochrane central register of controlled trials / database of systematic reviews / methodology register . The search was independently performed by all three authors (jdh, mjg, glj) on july 18, 2010 . Search terms included frozen shoulder, adhesive capsulitis, stiff shoulder, duplay disease, periarthritis, injection, sodium hyaluronate, hyaluronic acid, synvisc, and hyalgan . Levels i, ii, iii, iv evidence (according to the oxford center for evidence based medicine used by the american version of the journal of bone and joint surgery). Potential inclusive papers were manually reviewed, discussed among authors, and a decision made regarding inclusion or exclusion . In the event of disagreement among authors for study inclusion, the final decision was made by the senior author (glj). The full text article was reviewed and reference list checked for potential studies not identified by our original search . Inclusion criteria were level i, ii, iii, iv evidence studiesenglish language studieshuman subjectsstudy publication date from january 1, 1950 to july 18, 2010studies investigating treatment of primary adhesive capsulitis (including subjects with diabetes mellitus)studies investigating treatment of primary adhesive capsulitis during the freezing or frozen stagestudies investigating intra - articular glenohumeral joint sodium hyaluronate injectionstudies reporting clinical outcomes following intra - articular glenohumeral joint sodium hyaluronate injection level i, ii, iii, iv evidence studies english language studies study publication date from january 1, 1950 to july 18, 2010 studies investigating treatment of primary adhesive capsulitis (including subjects with diabetes mellitus) studies investigating treatment of primary adhesive capsulitis during the freezing or frozen stage studies investigating intra - articular glenohumeral joint sodium hyaluronate injection studies reporting clinical outcomes following intra - articular glenohumeral joint sodium hyaluronate injection exclusion criteria were: level v evidencenon - english language studiesstudies investigating treatment of non - adhesive capsulitis causes of shoulder painstudies investigating treatment of secondary causes of adhesive capsulitisstudies investigating treatment of primary adhesive capsulitis during the thawed stage non - english language studies studies investigating treatment of non - adhesive capsulitis causes of shoulder pain studies investigating treatment of secondary causes of adhesive capsulitis studies investigating treatment of primary adhesive capsulitis during the thawed stage see [table 1] for all database search criteria citation results . Four studies were excluded due to being in a non - english language (korean, japanese). [1417] one study was excluded as it evaluated the use of hyaluronate for supraspinatus tendinosis and not adhesive capsulitis . One study was excluded as it evaluated the use of subacromial hyaluronate injection for rotator cuff tear arthropathy . One study did treat subjects with adhesive capsulitis, however it did not report clinical outcomes for those subjects, therefore was excluded . Seven studies met inclusion criteria and were further analyzed[1102125] the inclusion of diabetic subjects within a study was not specifically excluded from our review . Nevertheless, three studies specifically excluded diabetic subjects,[2325] one study specifically allowed diabetic subjects, and three studies did not report specifically an approval or denial of diabetic subjects. [12122] for the purposes of this review, the control group was defined as no treatment, small volume (<5 ml) intra - articular glenohumeral joint injection of saline or lidocaine . Medical database search strategy citation results subjects were in stage ii adhesive capsulitis, based on classification systems reported by finnoff or not clinically staged. [12125] an attempt was made to identify common outcome measure(s) across all studies analyzed . However, several different clinical outcome measures were used for assessment, including passive range - of - motion (forward elevation, abduction, external rotation, and internal rotation), cho functional score, visual analog scale (vas) for pain intensity, constant score, and japanese orthopaedic association (joa) score . Imaging outcomes (including magnetic resonance imaging and ultrasound) were also recorded when available . Studies, demographic data and treatment data were analyzed with mean, median, mode, and standard deviation calculations, when appropriate . A paired t - test was utilized for outcome data comparison, when possible based on study reporting . The lack of subject - level - specific data and heterogeneity in outcome reporting precluded meta - analysis . Seven studies met criteria for inclusion (four studies were level i evidence; three studies were level iv evidence[212224]) [table 2]. Three hundred and three subjects (308 shoulders) were eligible for enrollment within these seven studies . There were 11 subjects who withdrew for various reasons within two studies, leaving 292 subjects (297 shoulders; 5 bilateral cases) for analysis . Of the four studies that randomized subjects to different treatments, one utilized alternating consecutive selection, one randomized based on date of study entry, and two did not report a randomization method . Six studies[12125] did not report the presence or absence of a financial conflict of interest, while one study denied a financial conflict of interest . Affected shoulder dominance was only reported in one study and this study reported that the affected shoulder was the dominant shoulder in 87 of 90 subjects (97%). Four of seven studies reported the side (right versus left) of the affected shoulder and 104 were right - sided (53%), while 91 were left - sided (47%). Mean pre - treatment duration of symptoms was 7.3 1.8 months (reported in five studies). Mean subject age at the time of treatment initiation was 59.1 4.4 years of age . Study demographic information one - hundred forty subjects underwent either one or multiple isolated sodium hyaluronate injections (120 intra - articular glenohumeral joint and 20 subacromial bursa). Variable sodium hyaluronate preparations were utilized, including both low molecular weight (lmw; <500 kda) and high - molecular weight (hmw;> 500 kda)[121222425] [table 3]. Nine subjects underwent isolated single intra - articular glenohumeral joint lidocaine (1%; 4 ml) injection (control). Forty - three subjects underwent an initial intra - articular glenohumeral joint injection of triamcinolone, followed by five weekly intra - articular glenohumeral joint injections of sodium hyaluronate . Sixteen subjects underwent combined intra - articular glenohumeral joint injections of triamcinolone and sodium hyaluronate . None of the injections were performed with the assistance of fluoroscopy, but 21 were performed with the assistance of ultrasound . Twenty - two subjects underwent isolated physical therapy consisting of range - of - motion, stretching, hot pack, ultrasound, and tens (transcutaneous electrical nerve stimulation) modalities and 20 subjects were instructed on codman exercises and stretches once and asked to perform at home as tolerated as little or as frequently as desired (control). Therefore, two groups (n=29) met the a priori definition of control subjects (small - volume lidocaine intra - articular injection [n=9] and no formal treatment group [n=20]). Sodium hyaluronate preparations within individual studies clinical follow - up after treatment was 9.0 8.3 weeks (range 1 to 26 weeks). All seven studies reported at least one clinical outcome measure, while two utilized post - treatment gadolinium - enhanced shoulder mri[2425] and one utilized post - treatment ultrasound . There were no complications reported after any of the treatments . Following isolated intra - articular glenohumeral joint injection of sodium hyaluronate, constant score significantly improved by 15 days (8 points) and 3 months (20 points) (n=27) (p<0.001); forward elevation significantly improved (22 14 degrees) by 6 weeks (n=91) (p<0.001); abduction significantly improved (21 4.9 degrees) by two to three weeks (n=56) and by two to three months (26 14 degrees) (n=89) (p<0.001, p<0.01); external rotation significantly improved (14 7.1 degrees) by two to three months (n=89) (p<0.001); and internal rotation significantly improved (5 degrees) by two months (n=62). Following isolated intra - articular glenohumeral joint injection of triamcinolone, constant score significantly improved by 15 days (8 points) and 3 months (15 points) (n=26) (p<0.01). The constant score did not improve after triamcinolone injection as much as after sodium hyaluronate injection, however this difference was not statistically significant . Following isolated intra - articular glenohumeral joint injection of triamcinolone, abduction significantly improved by three to six months (25 9.9 degrees) (n=40) (p<0.01); external rotation significantly improved (20 4.5 degrees) (n=40) (p<0.01) by three to six months; and internal rotation improvement (2 degrees) (n=14) (p>0.05) was not significant . At three to six months, the difference in abduction between intra - articular triamcinolone and hyaluronate was not significant (p=0.942), nor was the difference in external rotation (p=0.463) or internal rotation . Following combined intra - articular glenohumeral joint injection of hyaluronate and triamcinolone (either simultaneous or subsequent injections), forward elevation significantly improved 47 degrees by 6 weeks post - injection (p<0.05). The difference in forward elevation was not significantly different between combined triamcinolone and hyaluronate versus isolated hyaluronate (p=0.386). Following combined intra - articular injection of hyaluronate and triamcinolone, abduction significantly improved 34 17 degrees by mean 16 weeks post - injection (p<0.05). The difference in abduction was not significantly different between combined triamcinolone and hyaluronate versus isolated hyaluronate (p=0.660). Following combined intra - articular injection of hyaluronate and triamcinolone by mean 16 weeks post - injection, external rotation significantly improved 28 2.8 degrees (p<0.05) and this was significantly greater improvement than hyaluronate injection alone (p=0.039). Following combined intra - articular injection of hyaluronate and triamcinolone by mean 16 weeks post - injection, internal rotation significantly improved 34 11 degrees (p<0.05) and this was significantly greater improvement than hyaluronate injection alone (p=0.031). Comparison of intra - articular sodium hyaluronate injection and control demonstrated significantly (p<0.05) greater improvement in constant score (20 points versus 10 points) at 3 months post - treatment . The difference in improvement in external rotation by 10 weeks after injection versus control (14 7.1 degrees versus 9 degrees) was not statistically significant (p=0.333), nor was the difference in improvement in abduction by 8 weeks post - injection (25 10 degrees versus 20 degrees) (p=0.346). The purpose of this systematic review was to comprehensively analyze the literature with regard to the efficacy of intra - articular sodium hyaluronate injections in the treatment of primary adhesive capsulitis . We identified seven studies (four of level i evidence) that reported clinical outcomes following treatment of adhesive capsulitis with intra - articular hyaluronate injection . Our hypothesis was confirmed, demonstrating that sodium hyaluronate injection into the glenohumeral joint significantly improves shoulder range - of - motion, constant scores, and pain at short - term follow - up following treatment of adhesive capsulitis . Comparison of isolated intra - articular hyaluronate and triamcinolone injections demonstrated that there were no significant differences in constant scores or range - of - motion . Comparison of isolated intra - articular hyaluronate injection and control demonstrated that hyaluronate injections had significantly greater improvement in constant scores . Improvement in range - of - motion following isolated hyaluronate injection was greater than control; however, the difference was not significant . Following combined intra - articular glenohumeral joint injection of hyaluronate and triamcinolone versus isolated hyaluronate, the improvements in range - of - motion after combined injection were significantly greater than isolated hyaluronate in internal and external rotation, but not in abduction or forward elevation . The intertwined scaffold network between hyaluronate and aggrecan proteoglycans containing keratan sulfate and chondroitin sulfate glycosaminoglycans is a major mechanical structure within joint cartilage . This known biological composition prompted the use of different preparations of viscosupplementation in the treatment of knee osteoarthritis and has demonstrated success . The anti - adhesive properties of hyaluronate have found utility in preventing postoperative adhesions in gynecologic and abdominal surgery. [2932] these lubricating effects of hyaluronate have led to use in orthopedic surgery as well, via prevention of adhesion formation after both wrist and finger flexor tendon repair[3338] and tenolysis . Use of hyaluronate in the treatment of shoulder pain has also yielded success. [11020254041] thus, extrapolation to treatment of stiff shoulder and adhesive capsulitis has demonstrated success and improvements in range - of - motion, pain, and function. [1102125] six different preparations are commonly used in the united states and are both avian- and bacterial - derived products of variable molecular weight and viscosity . The normal hyaluronate molecule (mean 800 kda to 5000 kda molecular weight) has similar molecular weight and viscosity to these commercial products (500 kda to 6000 kda molecular weight). Hyaluronate injection into synovial joints is safe. [204344] our review further supported this, with no complications in any of the seven studies analyzed . Further, intra - articular injection of corticosteroid may elevate blood glucose levels in diabetic patients, thus warranting vigilance and careful monitoring of blood glucose concentrations and likely adjustments of insulin requirements . Hyaluronate may serve as a viable, equally efficacious alternative to corticosteroid to avoid the risk of elevated blood glucose level following injection . Despite incomplete understanding, evidence of mechanism of action in the treatment of osteoarthritis is based on several theories: reduction of friction via increased viscoelasticity with injection of hyaluronate into the joint, coating and protecting damaged cartilage, anti - inflammation and subsequent pain reduction, and improved synovial fluid concentrations and synovium abnormalities. [2425] gadolinium - enhanced mri in the shoulders of adhesive capsulitis patients has shown significant signal enhancement in the synovium and subsequent attenuation of this hyperintensity with treatment. [2425] a calculated value, the coefficient of enhancement (ce), using this latter mri technique, has been shown to negatively correlate with clinical improvements . Glenohumeral intra - articular injection with hyaluronate has shown improved clinical outcome and decreased mri ce values . Since decreased ce is indicative of decreased synovitis, transitivity indicates intra - articular hyaluronate is anti - inflammatory . Despite the level 1 evidence nature of four of the studies within this systematic review, properly conducted randomization attempts to eliminate selection bias and support the internal validity of a study . None of the four studies that randomized patients within this review utilized a valid randomization method . Further, it was not directly stated whether or not the study authors were involved in the randomization of subjects . The control group defined within one study in the analyzed studies in the review and a priori defined for the review yielded a control population of 10% of the overall analyzed subject population within the review . The presence of concurrent intervention (performance bias) confounders within the studies limits conclusions drawn . These include concomitant intra - articular steroid injection preceding five weekly hyaluronate injections, concomitant low - volume intra - articular injections of local anesthetic and saline,[102425] subacromial versus intra - articular glenohumeral joint injection in one study, and different physical therapy regimens following injection among all studies or the presence / absence of physical therapy in one study . The accuracy of blind intra - articular glenohumeral joint injection ranges from 33% to 47%, versus 93% accuracy with assistance of ultrasound . The use of imaging in intra - articular injection may be necessary to optimize true results . Assessment of range - of - motion via goniometer (as was done in only two studies in this review) has high intra - tester reliability (though, is limited by the tester's experience and ability to properly identify bony landmarks) but only fair to moderate inter - tester reliability . Nevertheless, range - of - motion measurement was either not reported or visually assessed in 5 out of the 7 included studies, despite recommendations for calibrated goniometer use in the performance of high - level evidence . Dissimilar doses, molecular weights, and viscosities of the different hyaluronate preparations introduce further bias . Also, incomplete understanding of the true natural history of adhesive capsulitis precludes definitive conclusions on how treatment affects the course of disease . However, independent evaluators of clinical outcome or range - of - motion following treatment were utilized in only three of seven studies . Heterogeneity in outcome measures used in the 7 studies within this review precludes meta - analysis and complete assimilation of data for larger sub - group subject numbers and comparisons . The best assessment of an orthopedic disease process and its response to treatment is via a general health outcome tool and a joint- or body - part - specific outcome tool . The primary outcome measures used in the studies analyzed were shoulder range - of - motion and constant score, cho function score, joa score,[2425] and variable vas pain scores. [1102123] a general health assessment tool was not utilized in any of the studies within this review . A significant limiting factor in this review is the very short - term follow - up duration (minimum one week to maximum 26 week follow - up). Although the natural history of adhesive capsulitis is incompletely understood, it is well - recognized that regardless of treatment most patients achieve their maximal outcome between two to four years after following treatment . Despite this, studies have demonstrated severe loss of motion, inability to perform activities of daily living, and mild residual pain and weakness at longer term follow - up (up to seven years). [69] this systematic review showed that sodium hyaluronate injection into the glenohumeral joint significantly improves shoulder range - of - motion, constant scores, and pain at short - term follow - up following treatment of adhesive capsulitis . Isolated intra - articular hyaluronate injection has equivalent outcomes compared to intra - articular corticosteroid injection . Intra - articular hyaluronate injection was safe, with no reported complications within the studies in this review.
The treatment of chronic inflammatory bowel disease (ibd) is always a challenge for gastroenterologists dealing with this type of condition.1,2 indeed, the variability and complexity of the clinical picture, the possibility that other organs and systems may be involved, and the possible toxicity caused by drugs make both the diagnosis and treatment of this condition particularly complex . In recent years, considerable progress has been made, both in diagnostic techniques and in the range of therapeutic options available . However, with regard to the latter, it has been shown that greater treatment potential is often accompanied by an increased risk of adverse events.3,4 the development of granulocyte - monocyte apheresis (gma)5 appears to be an innovative approach, comprising both treatment safety and therapeutic potential . The adacolumn is the most diffuse device of this type and consists of a column packed with cellulose acetate beads capable of adsorbing granulocytes and monocytes and through which the blood of the patients is run . Extremely positive results have been reported using this method for the treatment of ulcerative colitis in japan,613 but results obtained in europe and the us have been more contradictory.1419 nevertheless, a recent meta - analysis20 pooling data from seven randomized controlled trials 6,9,17,2124 clearly demonstrated the benefits of this method with respect to control treatments for the induction of remission or response at week 12 . On the other hand, the only sham - controlled randomized controlled trial, in a slightly to moderately ill population, did not show a statistically significant difference between sham and active treatment.24 in this report, the current knowledge related to gma in the treatment of ibd has been reviewed by means of an extensive research aimed at retrieving all papers regarding the clinical efficacy and safety of the adacolumn in the treatment of ulcerative colitis . This research was done by means of a medline search using specific key words, including medical subjects headings (mesh) for papers published since 1995 . Additional references were also obtained by hand searches and cross - referencing . In all, only abstracts written in english were retrieved . The most meaningful papers regarding other aspects of the treatment, ie, mechanism(s) of action, identification of ideal patient profile, and pharmacoeconomics were also retrieved and included in the analysis by the authors . Thus, on the basis of each author s personal experience, an expert opinion on this therapeutic approach was drawn up . The underlying mechanism of action in gma comprises the selective removal of cell populations taking part in the induction and perpetuation of intestinal inflammation in ibd . Gma is highly selective for granulocytes, monocytes, and macrophages5 and, in accordance with this finding, it has been shown that the outflow from gma columns has a low (40/60%) monocyte and granulocyte cell count, while the lymphocyte and erythrocyte populations are almost unchanged . Interestingly, a reduction of monocytes and granulocytes can also be seen in the colonic mucosa of patients who respond to treatment.25 in particular, a recent study reported that inflammatory cd14+cd16 + monocytes are considerably reduced after 10 sessions of gma in ulcerative colitis, as well as in crohn s disease.26 a marked reduction in proinflammatory cytokines also accompanies this effect, which is probably triggered by a dual mechanism of action, ie, by their direct adsorption on the column and via reactive immune regulation of nonabsorbed leukocytes.27 in fact, interleukin-6 mrna and interleukin-8 mrna return to normal levels following gma.28 moreover, the clinical efficacy of gma in ibd appears to be associated with an increase in circulating t regulatory lymphocytes, with a higher expression of foxp3 in cd4 + t cells29 and with a reduction of both myeloid and plasmocytoid dendritic populations.30 generally speaking, several elements indicate that, in addition to removal of activated cells, a reactive immunomodulatory effect is one of the mechanisms of action of gma . Many experimental demonstrations of the efficacy of gma in ibd have been derived from uncontrolled studies7,1317,25,3134 performed in patients who do not respond to conventional pharmacological treatment . In this setting, the data are fairly homogeneous, showing favorable responses (remission or partial response) at percentages varying from 60% to 84% of patients treated . Treatment with gma also appears to be more advantageous compared with extension or intensification of conventional pharmacological treatment, which has involved steroids in most studies . In those papers, use of gma has led to a rapid reduction in the steroid dose and/or withdrawal of steroid administration . Furthermore, gma is associated with a good safety profile.20 evaluation of efficacy has been primarily performed using clinical and/or biohumoral parameters, while the healing of the intestinal mucosa has rarely been taken into consideration . According to the very limited amount of data available, mucosal healing would appear to take place in about 25% of cases.35 in ulcerative colitis, the efficacy of gma and leukocyte apheresis, the other apheresis technique currently used to treat ibd, appears to be comparable, with a slight advantage for gma, as shown in a prospective study that compared the two methods.36 while bearing in mind the statistical limits associated with the small number of patients recruited in this study (39 patients), the clinical response for the two procedures was similar (72.2% for gma versus 66.6% for leukocyte apheresis). Due to the obvious difficulty of carrying out double - blind randomized studies with inactive columns, results are available from only two studies of this type (one using leukocyte apheresis and one using gma). In the leukocyte apheresis study, the response to active treatment was significantly higher than in control subjects (80% versus 33%, respectively).37 surprisingly, in the other study, carried out in more than 200 patients treated with gma or an inactive column,24 no significant difference was found (clinical response for 44% versus 39%, respectively). In spite of the theoretically adequate strength of the study, the large number of cases rejected during recruitment or lost to follow - up during the study, together with debatable inclusion criteria, have given rise to some doubts regarding the reliability of these results . Furthermore, a recent meta - analysis20 of the seven randomized, controlled studies performed using the adacolumn, which also included the afore - mentioned shamcontrolled study,24 nevertheless demonstrated the greater efficacy of gma in reducing clinical and endoscopic activity in ulcerative colitis compared with comparator treatment.20 results in over 1000 patients treated in the previously mentioned uncontrolled cohorts would appear to support the efficacy of gma further . The suggestion that the efficacy of gma would appear to continue beyond the actual treatment period, with a carryover effect, is certainly interesting, although controversial . A lower probability of recurrence in the 612 months following effective gma than after pharmacological treatment was documented in other studies.18,20,39 this proved true also in one randomized study showing that gma reduced the probability of relapse of the disease.23 this study, carried out in patients in remission but at a high risk of recurrence (patients with fecal calprotectin> 5 times the upper limit of normal), demonstrated the efficacy of preventive treatment with gma . In fact, 67.7% of patients treated with conventional pharmacological treatment showed a relapse of the disease within 6 months, while a preventive cycle of five sessions of gma reduced the risk by more than half (27.6%). The most commonly used gma treatment schedule consists of one weekly session for 5 weeks . Each apheresis session lasts for 60 minutes and the volume of filtered blood amounts to 1800 ml each session . Alternative schedules have been proposed with the use of intensive gma comprising two sessions per week for a total of 10 sessions, or longer gma, ie, an increase in the duration of each session or in the total number of sessions . It is somewhat difficult to establish whether these latter approaches are more efficacious compared with standard treatment, given that the evidence available so far concerns only open studies, each with a limited number of cases . In a study by sakuraba et al, an intensive gma program was found to be more efficacious than the conventional schedule in achieving remission (71% versus 54%), and was also achieved in a shorter time period (14.9 9.5 days versus 28.1 16.9 days).40 an even more intensive treatment schedule of daily gma (five sessions in five consecutive days) has been reported, with favorable therapeutic results41 and good safety, so that this type of schedule is now used in several japanese institutions . Therefore, a frequency - dependent or dose - dependent response is possible . On the other hand, the safety profile of the procedure remains unchanged when the frequency of the sessions is increased . In another study, increasing the number of sessions was more effective in achieving remission in patients undergoing steroid treatment compared with an increase in the steroid dose.9 however, recent data from a large number of european steroid - dependent or steroid - resistant patients treated openly with either the classic or an extended procedure demonstrate comparable efficacy, as far as both achievement of remission (40% versus 44%) and clinical response (59% versus 56%) are concerned.42 in both cases, the treatment was well tolerated without significant differences in the frequency of adverse events . Use of a combined regimen of intensive extended apheresis (two sessions per week for the first 3 weeks and one session per week for another 8 weeks) in a study by hanai et al in 70 patients, provided responses, as far as percentages are concerned, that were comparable with steroids administered intravenously (83% versus 65%).22 in particular, the gma response appears to be slower but longer - lasting in time (the percentage of response at 2 weeks is 40% for gma and 50% for steroids, respectively, whereas at 6 and 12 weeks, it is 77% and 65%, respectively, and 82% and 61%, respectively). Prolonging the duration of each session to 90 minutes, together with an increase in the number of sessions (one per week for 10 weeks), appears to increase the percentage of remissions (83%) and to reduce the need for steroids.12 in conclusion, the data available on more intensive and/or extended use of gma differ considerably and need to be confirmed in controlled studies . In the meantime, it would seem reasonable to adhere to the recommended treatment schedule of one session per week for 5 weeks . Treatment of ulcerative colitis is characterized by a well defined flow chart with the following sequence: mesalazine (mild and moderate active disease and maintenance treatment); steroids (moderate to severe active disease); and immunosuppressants and biological treatment (steroid - dependent and steroid - resistant disease). Severe forms of the disease are not a major priority indication for gma treatment, because a very rapid response is needed in these cases, as will occur in many cases when using steroids and/or biological drugs . Negative evidence is present in the literature in support of this consideration.24 on the other hand, evaluation of gma in 163 patients with mild to moderate ulcerative colitis, for whom simultaneous treatment with mesalazine and/or azathioprine40 was permitted, showed a high rate of clinical remission (62.4%). A randomized study17 even showed a trend in favour of gma compared with steroids in mild to moderate forms of the disease (90% versus 75% of remission - response, respectively) with a reduction in side effects but with higher costs . In one single report, gma appeared to be more effective in steroid - nave patients and patients treated with low doses and for a short time only, but the data must still be confirmed in larger and more homogeneous studies.43 other possible predictors of response found in these studies are low steroid doses43 and high basal granulocyte levels.35 in this type of patient, gma could be an alternative option to steroid treatment and become a first - line option in conditions for which contraindications to the use of steroids are greater, eg, diabetes, high blood pressure, and glaucoma . However, steroid resistance with mild to moderate activity or steroid dependence appear, at present, to be the main indications for gma . As confirmation of this, in a japanese post - marketing surveillance study of 697 patients treated at 53 centers in the period 19992006, 489 patients were found to be resistant to conventional forms of treatment, especially steroids.34 this report confirmed, in a larger number of cases, the previous retrospective review of the first 100 scandinavian patients (which grouped together ulcerative colitis, crohn s disease, and indeterminate colitis), who were almost all steroid - resistant or steroid - dependent19 and the data contained in the italian registry (92% steroid - resistant/ steroid - dependent patients).44 further, the duration of clinical response to gma treatment, although differing in the various studies, would appear to be long enough to allow the delayed response of azathioprine to be reached, thus suggesting a possible role for gma in bridge therapy as an alternative to infliximab . In addition, because gma has no significant side effects (see safety section), it could be preferable in patients with contraindications to biological treatment (intolerance to infliximab, carriers of specific antibodies, hepatitis b virus carriers, multidrug immunosuppression, or a history of tumors). As far as its possible use in prolonging remission is concerned, the efficacy of gma used monthly has also been reported in a small group of 10 patients.40 the possible role of gma in preventing relapses in high - risk patients, identified by high levels of fecal calprotectin, was reported by maiden et al.23 no comparative studies of treatment with biological drugs and gma or associated treatment using the two methods are available . Gma has been demonstrated to be very safe, with a low percentage of side effects . The first important study assessing the efficacy and safety profile of gma in the treatment of active ulcerative colitis carried out in japan at the end of the 1990s6 reported side effects in only 8% of cases treated with apheresis compared with 43% of adverse events recorded in a group of patients treated with traditional drugs, ie, steroids and/or mesalazine . In fact, the percentage of side effects occurring with gma in the main clinical trials ranged from 5% to 33%, with almost total absence of serious adverse events.712,16,17,24,45 the main adverse events reported were shivering, nausea, headaches, flushing, and fever . Use of painkillers before starting the procedure may prevent onset of headaches, while fever can easily be treated with common antipyretics.46 in extremely rare circumstances, infection of the upper airways16,24 has been reported . Some alterations in hematochemical tests, consisting of an increase in transaminases and leucopenia, have also been very rarely observed.34,47 however, in most cases, side effects have been mild to moderate, almost never requiring discontinuation of treatment . The excellent tolerability of gma reported in clinical studies has also been confirmed in observational studies carried out in large numbers of unselected patients, reflecting the real use of gma in daily clinical practice.19,34 in particular, in the recent post - marketing surveillance study carried out in japan, adverse events, including complications related to the procedure (eg, difficulty in finding venous access, difficulty in preventing coagulation in the apheresis system, difficulty in maintaining a suitable venous return) occurred in 2.3% of treatment sessions and in 8.18% of patients.34 the most frequent events were headache (1.58%) and fever (1.29%), but no serious adverse events were found . No statistically significant differences in the rate of side effects were found between patients undergoing few procedures (fewer than five) and patients undergoing more than six treatments . A relatively higher rate of side effects occurred in women and in hospitalized patients (p <0.05). With regard to the italian experience, the national register of therapeutic apheresis (online at http://www.aferesi.it) shows that almost 95% of gma procedures (94.9%) produced no adverse events, with headaches being reported in 3.9% of cases.44 gma has also been used in the treatment of pediatric forms of ibd, in spite of the lack of data currently available . From the limited evidence so far, which mainly comes from japan, the treatment has proved to be extremely safe also in this context.4851 recently, the results of some european studies have also been published, which confirm the safety of gma in the treatment of pediatric patients with ibd.52,53 in particular, a small spanish study of nine pediatric patients did not report any adverse events (either early or late), despite placement of a central venous catheter,52 while the most frequent side effects found in a larger study in scandinavia (37 patients with chronic ibd), all slight and not long - lasting, were tiredness (reported in most children) and headache (in 30% of subjects undergoing treatment).53 gma has proved to be an effective and safe procedure in the treatment of steroid - dependent and chronically active ulcerative colitis . The main limitations to its use lie in the relatively high cost compared with traditional forms of treatment, and also in the need to carry out this treatment in a hospital environment . A recent spanish pharmacoeconomic study, which evaluated gma in patients suffering from steroid - dependent ulcerative colitis in terms of cost efficacy in comparison with traditional azathioprine treatment showed that the costs involved in the two different approaches were comparable.54 in fact, despite an increase in cost of 5377 per year per patient treated (11,436 versus 6059), the higher response rate obtained using gma (61% versus 38.5%) as well as reduction in side effects and need for surgery indicate that the cost required to obtain remission is not very different between the two types of treatment (18,748 versus 15,738). The results of a recent scandinavian study, published at present only in abstract form, confirm that gma can be considered to be a cost - effective treatment if compared with traditional treatment approaches, given that the increase in cost, calculated by quality - adjusted life - years gained (55,426), is in keeping with that of treatments considered to be cost - effective.55 unfortunately, no pharmacoeconomic study comparing all the treatment approaches used for steroid - dependent/ steroid - resistant forms of ulcerative colitis, including biological drugs, is available as yet . However, if we consider the high cost of this latter type of treatment, gma may in comparison still prove to be economical . However, without the results of appropriate pharmacoeconomic studies, no final conclusions can be drawn on this issue at present . According to published data, gma is a useful technique among the various treatment options available for ulcerative colitis . In this setting, gma has been used primarily in steroid - dependent or steroid - resistant patients . However, promising preliminary results have also been obtained in steroid - nave patients or when gma is used as an alternative treatment to steroids during the acute phases of the disease . Thus, on the basis of the available data, and also due to the fact that the treatment is still rather expensive, we believe that its current use should be restricted mainly to steroid - dependent or steroid - resistant patients . However, gma should also be considered for those patients in whom standard treatment cannot be used, due to lack of efficacy, toxicity, or personal intolerance . These possible indications are also based on the safety profile of gma, which has now been confirmed both in clinical trials and in post - marketing surveillance, and probably also by some pharmacoeconomic data which do not appear to be particularly negative . Although some observations suggest that diversified gma schedules (more intensive and prolonged treatment) may in some instances offer better results, it is advisable at present to adhere to traditional treatment schedules, with one session per week for 5 weeks . We believe that, in the future, as long as research progresses towards an increasing recognition of specific disease patterns, it will become possible to select patient subgroups likely to respond better to specific therapeutic approaches, such as gma.
A recent study demonstrated that a pest - like sequence present at the nh2 terminus of llo and absent in pfo is responsible for the rapid degradation of llo in the host cell cytosol (decatur and portnoy, 2000). Pest - like sequences are thought to target eukaryotic proteins for phosphorylation and degradation, and deletion or specific amino acid substitution of this sequence in llo led to increased cytotoxicity and lower virulence in a mouse model . When the sequence was introduced in pfo and the chimeric toxin expressed in l. monocytogenes, bacteria were less toxic than those expressing wild - type pfo and were able to multiply intracellularly in j774 macrophages . Thus, introduction of a pest motif in llo is a strategy used by l. monocytogenes to restrict the activity of this powerful toxin to the host cell vacuole, thereby preserving the intracellular niche for bacterial multiplication . Intriguingly, another report (lety et al ., 2001) has also shown that l. monocytogenes mutants expressing a pest - deleted hly allele, although fully hemolytic, are strongly impaired in virulence in a mouse model . However, in contrast to the previous report, in which j774 nonbactericidal macrophages were used, the authors of this second report used bone marrow derived macrophages and showed that pest - deleted or -substituted mutants were unable to escape from the phagocytic vacuole raising the interesting possibility that the pest motif may play different roles in different cells . Direct evidence for a link between the optimum activity at low ph and compartment - specific pore - forming activity of llo has been provided by a recent study using a ph - sensitive and membrane - impermeant fluorophore 8-hydroxy - pyrene-1,3,6-trisulfonic acid (hpts). It was shown that l. monocytogenes containing phagosomes (average ph 5.9) rapidly acidify after bacterial uptake, followed by an increase in ph and dye release from the vacuole . Perforation of the vacuole was inhibited by lysosomotropic agents, such as ammonium chloride and bafilomycin a1 (beauregard et al ., 1997). Bafilomycin a1 was also shown to inhibit l. monocytogenes escape from the primary vacuole in epithelial cells (conte et al ., 1996). These experiments indicate that llo activity is maximal in the phagosome lumen, requires this low ph for activity and leads to membrane disruption, increasing ph, and inactivation of llo activity, an auto - switch process . In their recent study, glomski et al . (2002), by swapping dissimilar residues from the ph - insensitive ortholog perfringolysin o into llo, identified leucine 461 of llo as a key residue responsible for the low optimum ph activity of llo . Changing this residue to threonine results in a molecule highly active at ph 7 . If one assumes that llo shares with pfo, a general structure similarity, the l461 t mutation is located in the outer loop of the fourth domain (fig . 1). That a single amino acid residue is sufficient to increase the activity at ph 7.0 is quite astonishing, although it is important to note that activity at ph 5.0 is also increased in the mutant . How this single amino acid change llo l461 t affects ph sensitivity thus awaits further biochemical analysis . Interestingly, the mutation that altered the optimum ph activity of pfo l462f described above was located in the conserved undecapeptide, which is very close to the region of the corresponding l461 t mutation (jones et al ., 1996). It is particularly worth noting that while the llo l461 t mutant can efficiently permeabilize the host cell membrane from the cytosolic compartment, it only promotes efficient escape from the vacuolar compartment if this latter has been acidified, suggesting that additional bacterial or host factors activated by low ph are needed to act in concert with llo to mediate escape from the phagosomal compartment . These findings could also reflect the difference in lipid composition of the internal versus external leaflets of the plasma membrane lipid bilayer, resulting in a difference in the behavior of llo depending on the nature of the first leaflet with which it is in contact . While it is clear that llo is largely responsible for mediating escape from the vacuole in most cell lines, two l. monocytogenes plcs also play a role . Mutants lacking both the broad - range plc (plcb) and a phosphatidylinositol - specific plc (plca) escape from a macrophage vacuole at 50% of the efficiency of the wild - type . Plcb can even replace llo in some cell lines, such as the human epithelial cell lines hela and henle 407 (vazquez - boland et al . Optimal pore formation occurs between ph 5.5 and 6.0, the ph of an early endosome . One consequence of the pore formation is an elevation of the vacuolar ph, which may prevent vacuolar maturation, thus allowing the l. monocytogenes plcs, perhaps in concert with additional host factors, to mediate vacuolar dissolution . Llo activity is then progressively switched off and degraded and bacterial multiplication can take place in the host cytosol . Meanwhile, llo may have induced a series of signaling events since it is now well established that in addition to its role in escape from the phagosomal vacuole, llo is one of the most potent l. monocytogenes signal - inducing molecules (vazquez - boland et al ., 2001).
X - linked severe combined immunodeficiency (x - scid) is a rare, life - threatening disease that is characterized by marked impairment of both cellular and humoral immunity (1). Human x - scid occurs in as many as 50% of patients with primary scid; patients have complete or marked deficiency of t cells but carry a normal or slightly increased number of b cells, despite a deficiency in specific antibody responses (2). Infants with scid have no tonsils, rarely have detectable peripheral lymph nodes, and have a small, poorly differentiated thymus gland . Without bone marrow transplantation (bmt), affected patients suffer severe and persistent infections, often with opportunistic pathogens, and generally die in infancy . The abnormal gene was mapped to the xq13 region, and later identified as the gene encoding the common gamma (c) chain (3, 4) that is common to the cell - surface receptors for six interleukin (il) molecules (il-2, 4, 7, 9, 15, and 21) (2 - 5). Based on the amino acid sequence, the complete cdna clone encoding the 64-kda molecule was isolated and demonstrated to be the cognate il-2r chain (6, 7). The intracellular portion of the c chain is known to interact with janus kinase 3 (jak3), a signaling kinase that cooperates with other jak and stat proteins in a complex signal transduction array (8, 9). Various mutations in the c chain gene have been reported in patients with x - scid (10 - 15). However, little is known about the clinical and immunological characteristics, or the genetic polymorphism in korean patients with x - scid . Here, we report the mutation identified in one korean family with x - scid, along with prenatal diagnosis of the disease by rflp analysis . To our knowledge, this is the first report of the molecular diagnosis of x - scid in a korean patient . A 13-month - old boy was transferred to the department of pediatrics, chonnam national university hospital, because of refractory, progressive pneumonia, and nodular skin lesions . From the age of 7 months the birth history was not contributory, but there was an infant death history of unknown etiology for his uncle on the mother's side ., he was immediately intubated and maintained with an artificial ventilator because of severe, bilateral pneumonia causing respiratory failure . The blood counts on admission were: white blood cell, 45,100/l (neutrophil 75.5%, lymphocyte 15.1%); hemoglobin, 14.4 g / dl; platelets 209,000/l . His serum immunoglobulin profile was: igg, 27.0 mg / dl (normal range, 345 - 1,236 mg / dl); igm, 50.4 mg / dl (41 - 173 mg / dl); iga, 22.9 mg / dl (14 - 159 mg / dl). Flow cytometric analysis of lymphocyte immunophenotype was: cd3 t cells, 2.5% (normal range, 60 - 85%); cd19 b cells, 95.4% (8 - 20%); cd16/cd56 natural killer cells, 0.5% . On physical examination, he had no bcg scar, and biopsy on the nodular skin lesion was later found to be a tuberculous granuloma . He was treated with broad - spectrum antibiotics, anti - tuberculosis agents, prophylactic antifungal agents, and intravenous globulins . The recent blood counts without infection were: 5,300/l (neutrophils, 58.5%; lymphocytes, 27.4%); hemoglobin, 12.5 g / dl; platelets 265,000/l . Blood samples were obtained from the patient and various family members with written informed consent . Heparinized venous blood samples from the patient and his family members were fractionated on a ficoll - hypaque gradient to isolate peripheral blood mononuclear cells (pbmcs). Pbmcs were washed twice in pbs and 0.02% nan3, and analyzed by flow cytometry for c chain expression using the pe - conjugated rat anti - human c chain monoclonal antibody tugh4 (beckton - dickinson, mountain view, ca, u.s.a . ). Incubation with unlabeled tugh4 before the addition of labeled tugh4 provided the background control staining . The method for immunofluorescence staining was the same as that described previously (16). As shown in fig . 1b, pbmcs from the mother showed c chain expression on both cd3- and cd19-positive populations . In contrast, cells from the patient showed a very depressed c chain expression on cd3- and cd19-positive cells, suggesting both t and b cells from the patient did not express c chain on the cell surface (fig . The eight exons of the c chain and surrounding genomic sequences were amplified from genomic dna as described previously (12). As shown in fig . 2a, we found a single base substitution (c690 t) at exon 5, resulting in an amino acid change at codon 226 (r226c) in the patient . His mother was found to have one mutant and one normal allele, indicating that she was a heterozygous carrier . As the c - to - t substitution predicted the creation of a new alui restriction endonuclease site, the sequencing results the patient showed dna cleavage with alui (bands of 179 and 133 bp), while the normal control was resistant to alui . The mother possessed an uncleaved (312 bp) and two smaller bands identical to those in the patient, implying heterozygosity . By rflp analysis, we were able to perform a prenatal diagnosis on a male fetus . At the 24th week of gestation, 3 ml of umbilical cord blood was sampled percutaneously, and analyzed for lymphocyte phenotyping . The fetus had the same t - b+nk- phenotype (cd3 t cells, 1.2%; cd19 b cells, 78.6%; cd16/cd56 natural killer cells, 1.2%). As shown in fig . 2c, the fetal dna showed the same cleaved dna pattern as the patient, while the healthy control dna showed the normal pattern . Rflp analysis demonstrated that the fetus had the same mutation as his brother, and the pregnancy was terminated after lengthy counseling . The patient is now being cared for at our outpatient clinic, awaiting a stem cell transplant from an alternative donor, because he does not have an hla - matched sibling . Here, we report the first mutation analysis of the c chain gene in a presumed x - scid patient from a korean family . The patient had a single point mutation (c690 t) at exon 5, resulting in an amino acid change at codon 226 (r226c). The national institutes of health database of c chain mutations lists 264 mutation entries, comprising 169 unique molecular events (http://www.nhgri.nih.gov/dir/gmbb/scid/il2rgbase.html). Mutations have been identified throughout all eight exons of the c chain, the majority of which are missense mutations, nonsense mutations, or splicing defects that give rise to aberrant transcripts . The missense mutation found in this study is not novel . The available data suggest that the codon 690 may be a hot spot for c chain gene mutation, as reported in several studies (10, 12 - 15). We did not examine the stability of mrna or protein expression of the c chain in this study, but our previous study demonstrated that the c chain containing the point mutation was not expressed on the cell surface because it was trapped in the golgi apparatus and failed to be transported to the cell surface (12). Further identification of the locations and types of mutations in the c chain gene may lead to a better understanding of the functional domains of the protein . In addition, the patient showed a very little expression of c chain on both b and t cells . In a previous study, cell - surface staining of patient - derived b cell lines with an anti-c chain antibody showed no c chain staining in 47% and trace amounts in 32%, but normal intensity of staining in 21% of patients (11). In another study, rapid protein analysis with flow cytometry and immunoblotting were shown to be useful for diagnosis of x - scid and jak3-deficient scid . Although the patients had mutations in the c chain gene, some had trace or normal levels of c chain expression . Thus, these proteinbased assays have led to rapid molecular diagnoses of x - scid that have subsequently been confirmed by genetic analysis . The male x - scid patient in the present study had a classical, severe phenotype, and laboratory data showed low numbers of t cells, relatively well - preserved b cells, and reduced nk cell numbers . X - scid is a disease that is characterized by severe lymphopenia and recurring persistent infections in the first months of life (17). A human jak3-deficiency disease has similar clinical features to x - scid (18), and the c chain and jak3 signaling pathway have been suggested to be crucial for t cell development, and to contribute to the x - scid phenotype (18). In addition, gene disruption of either il-7 (19) or the il-7r subunit (20) has been shown to lead to severe developmental perturbations . Thus, signaling defects in the il-7/il-7r system resulting from changes in the c chain may account for the developmental anomalies that lead to x - scid . We also performed a prenatal diagnosis for this family, based on our genetic findings . Most of the primary immunodeficiency diseases can be diagnosed by means of screening for lymphopenia or for t cell deficiency in cord blood at birth . In addition, fully defining the molecular defects of patients is essential for genetic counseling of family members and prenatal diagnosis . In conclusion, we described here the molecular and cellular identification of an x - scid patient in a korean family . Our data emphasize that flow cytometric analysis is an important and powerful assay that can contribute to diagnosis of x - scid . Further studies of x - scid mutations should be compiled and analyzed to provide data to clarify the correlation between the severity of the disease and the types and locations of the mutations in x - scid patients.
This study was a cross - sectional study of penicillin susceptibility among clinical blood isolates reported at all acute - care hospitals in the delaware valley case control network (dvccn). Dvccn is a population - based network of all adult acute - care hospitals in the five pennsylvania counties around the metropolitan philadelphia region . These counties (bucks, montgomery, chester, delaware, and philadelphia) contain 52 adult acute - care hospitals serving a population of 3.4 million residents . Microbiology laboratory supervisors were contacted by telephone at each area hospital during january 1999 to collect data on all pneumococcal blood isolates during the preceding year . Local laboratory personnel reported cumulative numbers of pneumococcal blood isolates and the proportion of isolates that were penicillin nonsusceptible for the 12-month period . When available, laboratories separately reported susceptibility percentiles as susceptible, intermediate susceptible, and resistant; otherwise, the laboratories reported the combined percentile nonsusceptible (intermediate plus resistant). We collected information on unique isolates but did not confirm that these represented unique episodes of patient infections . However, we think hospitals generally reported only one isolate per patient because guidelines recommend that hospitals report cumulative susceptibility data on the basis of single isolates per patient per reporting period (8). The same hospital laboratories were contacted by mail with follow - up telephone calls requesting the same information on cumulative numbers of pneumococcal blood isolates and the proportion of isolates that were penicillin nonsusceptible for the 12-month period . Annual number of pneumococcal blood isolates and penicillin - susceptibility rates were summarized for each hospital for each year . Standard national committee for clinical laboratory standards (nccls) criteria define three categories of penicillin susceptibility for s. pneumoniae: susceptible (mic <0.1 g / ml), intermediate susceptible (mic 0.11.0 g / ml) and resistant (mic> 2.0 g / ml) (9). Penicillin - nonsusceptible isolates are defined as intermediate susceptible and resistant isolates combined . For all analyses we specifically excluded any hospital site reporting <10 isolates per year as too unreliable to provide point estimates of resistance rates . This level is consistent with nccls guidelines recommending hospital laboratories report cumulative susceptibility data only for organisms for which there is a minimum of 10 isolates per reporting period (8). Similar to prior studies of hospital - level variation (10,11), we report the proportion of hospitals with> 5% deviation from the overall regional mean rate of resistance because lower levels of deviation are unlikely to influence prescribing decisions . To analyze the reliability of individual hospital rates of pneumococcal resistance, we examined the correlation of rates across each of the 3 years of data collection . We restricted these analyses to those hospitals reporting> 10 pneumococcal blood isolates in 1998, 1999, and 2000 . Correlations between any 2 years were calculated with spearman s correlation coefficient, testing the assumption that pneumococcal susceptibility levels at individual hospitals should be correlated year to year, regardless of whether overall susceptibility levels for the region were rising, falling, or remaining constant . In addition, we calculated an intra - class correlation coefficient across all 3 years of data at the hospital level by using a general linear model for analysis of variance (12). Next, we tested the assumption that the proportion of nonsusceptible s. pneumoniae at each hospital should demonstrate an underlying geographic clustering . First, we analyzed whether the location of each hospital at the county level was associated with the proportion of nonsusceptible pneumococci by using the nonparametric kruskal - wallis test . Second, we conducted geographic information system (gis) mapping to visually judge the spatial distribution of hospital susceptibility rates . Hospitals were geographically assigned longitude and latitude coordinates on the basis of the hospital address by using gis mapping software (maptitude, caliper corporation, newton, ma) to display the proportion of nonsusceptible pneumococci at each hospital location on the map . Using data from 1998 only, we graphically represented the proportion of nonsusceptible pneumococci as a proportional symbol map (bubble plot), where each hospital location is represented by a circle whose radius is proportional to the level of penicillin nonsusceptibility among pneumococcal blood isolates . We tested the null hypothesis that no spatial autocorrelation of the nonsusceptibility rates occurred with moran s i statistic, based on a proximity matrix calculated from the city - block distances between the longitude - latitude coordinate points of hospital addresses . Moran s i is a spatial cross - product coefficient that is interpreted similarly to the pearson correlation coefficient (13,14). This study was a cross - sectional study of penicillin susceptibility among clinical blood isolates reported at all acute - care hospitals in the delaware valley case control network (dvccn). Dvccn is a population - based network of all adult acute - care hospitals in the five pennsylvania counties around the metropolitan philadelphia region . These counties (bucks, montgomery, chester, delaware, and philadelphia) contain 52 adult acute - care hospitals serving a population of 3.4 million residents . Microbiology laboratory supervisors were contacted by telephone at each area hospital during january 1999 to collect data on all pneumococcal blood isolates during the preceding year . Local laboratory personnel reported cumulative numbers of pneumococcal blood isolates and the proportion of isolates that were penicillin nonsusceptible for the 12-month period . When available, laboratories separately reported susceptibility percentiles as susceptible, intermediate susceptible, and resistant; otherwise, the laboratories reported the combined percentile nonsusceptible (intermediate plus resistant). We collected information on unique isolates but did not confirm that these represented unique episodes of patient infections . However, we think hospitals generally reported only one isolate per patient because guidelines recommend that hospitals report cumulative susceptibility data on the basis of single isolates per patient per reporting period (8). A second data collection was conducted in early 2001 . The same hospital laboratories were contacted by mail with follow - up telephone calls requesting the same information on cumulative numbers of pneumococcal blood isolates and the proportion of isolates that were penicillin nonsusceptible for the 12-month period . Annual number of pneumococcal blood isolates and penicillin - susceptibility rates were summarized for each hospital for each year . Standard national committee for clinical laboratory standards (nccls) criteria define three categories of penicillin susceptibility for s. pneumoniae: susceptible (mic <0.1 g / ml), intermediate susceptible (mic 0.11.0 g / ml) and resistant (mic> 2.0 g / ml) (9). We specifically excluded any hospital site reporting <10 isolates per year as too unreliable to provide point estimates of resistance rates . This level is consistent with nccls guidelines recommending hospital laboratories report cumulative susceptibility data only for organisms for which there is a minimum of 10 isolates per reporting period (8). Similar to prior studies of hospital - level variation (10,11), we report the proportion of hospitals with> 5% deviation from the overall regional mean rate of resistance because lower levels of deviation are unlikely to influence prescribing decisions . To analyze the reliability of individual hospital rates of pneumococcal resistance, we examined the correlation of rates across each of the 3 years of data collection . We restricted these analyses to those hospitals reporting> 10 pneumococcal blood isolates in 1998, 1999, and 2000 . Correlations between any 2 years were calculated with spearman s correlation coefficient, testing the assumption that pneumococcal susceptibility levels at individual hospitals should be correlated year to year, regardless of whether overall susceptibility levels for the region were rising, falling, or remaining constant . In addition, we calculated an intra - class correlation coefficient across all 3 years of data at the hospital level by using a general linear model for analysis of variance (12). Next, we tested the assumption that the proportion of nonsusceptible s. pneumoniae at each hospital should demonstrate an underlying geographic clustering . First, we analyzed whether the location of each hospital at the county level was associated with the proportion of nonsusceptible pneumococci by using the nonparametric kruskal - wallis test . Second, we conducted geographic information system (gis) mapping to visually judge the spatial distribution of hospital susceptibility rates . Hospitals were geographically assigned longitude and latitude coordinates on the basis of the hospital address by using gis mapping software (maptitude, caliper corporation, newton, ma) to display the proportion of nonsusceptible pneumococci at each hospital location on the map . Using data from 1998 only, we graphically represented the proportion of nonsusceptible pneumococci as a proportional symbol map (bubble plot), where each hospital location is represented by a circle whose radius is proportional to the level of penicillin nonsusceptibility among pneumococcal blood isolates . We tested the null hypothesis that no spatial autocorrelation of the nonsusceptibility rates occurred with moran s i statistic, based on a proximity matrix calculated from the city - block distances between the longitude - latitude coordinate points of hospital addresses . Moran s i is a spatial cross - product coefficient that is interpreted similarly to the pearson correlation coefficient (13,14). For the survey of 1998 susceptibility results, we received responses from 47 of 52 hospitals in the dvccn . However, in two instances, the results from multiple hospitals were combined at a single laboratory and could not be separated for reporting purposes . Of the 45 laboratories reporting results, 33 laboratories reported> 10 pneumococcal blood isolates during 1998 . Among the 33 sites, the median number of pneumococcal blood isolates during 1998 was 19 (range 10 - 92). Figure 1 displays the frequency distribution of the proportion of isolates with nonsusceptibility to penicillin as reported by each hospital in 1998 . The proportion of nonsusceptible isolates ranged from 0% to 67%, with a mean proportion of 21% . If we only included hospitals with> 20 pneumococcal isolates in 1998, a total of 16 hospitals would be included in the analysis; the proportion of nonsusceptible isolates ranged from 0% to 36%, with a mean proportion of 20% . Fifty - six percent of hospitals were> 5% above or below the mean proportion of nonsusceptible isolates . The number of hospitals with each reported level of penicillin nonsusceptibility among all pneumococcal bloodstream isolates at each hospital in 1998 are shown . Penicillin nonsusceptibility was defined as any isolate with a penicillin mic> 0.1 g / ml . Hospitals with <10 isolates in 1998 were excluded . For the 1999 and 2000 survey results of these, 23 hospital laboratories reported> 10 pneumococcal isolates in 1 of the 2 study years . The mean proportion of isolates with reduced susceptibility to penicillin was 19% in 1999 and 24% in 2000 . Fifty - three percent of sites were> 5% above or below the mean in 1999, and 65% of sites were> 5% above or below the mean in 2000 . The table summarizes the relationship between each hospital s annual proportion of nonsusceptible pneumococcal blood isolates during the 3 years of the study . For hospitals reporting> 10 isolates in 1998, 1999, and 2000 (n = 15), the proportion of nonsusceptible isolates at each hospital was poorly correlated between any 2 years . For 1998 to 1999, the spearman correlation coefficient was 0.07 (p = 0.82), for 1999 to 2000, the spearman correlation coefficient was 0.28 (p = 0.32), and for 1998 to 2000, the spearman correlation coefficient was 0.03 (p = 0.91). Location of hospital by county did not correlate with the proportion of pneumococcal blood isolates nonsusceptible to penicillin (p = 0.45, 0.37, 0.08 in 1998, 1999, and 2000, respectively). Figure 2 is a proportional symbol map with each hospital location represented by a circle whose radius corresponds to the proportion of nonsusceptible pneumococcal blood isolates at each hospital in 1998 . Overall, figure 2 shows geographic clustering of the 33 hospitals reporting> 10 pneumococcal blood isolates in 1998, 14 of which are gathered in philadelphia, the most heavily populated county in our five - county study area . No geographic clustering in the proportion of nonsusceptible pneumococci isolated at each hospital was evident from our data . This finding is supported by a moran correlation coefficient of 0.01, demonstrating a lack of statistically significant spatial autocorrelation in the proportion of nonsusceptible hospital pneumococci at hospitals across the five - county region (p = 0.80). Geographic distribution of penicillin nonsusceptibility among pneumococcal isolates at 33 hospitals in the delaware valley in 1998 . Is represented by a circle with an h in the center at the corresponding longitude and latitude of the hospital . The radius of the circle is directly proportional to the proportion of penicillin - nonsusceptible pneumococci at each hospital in 1998 . Most resistant isolates now demonstrate reduced susceptibility to multiple antimicrobial drug classes, including -lactams, macrolides, and tetracyclines . However, substantial geographic variability in the proportion of pneumococci resistant to different antimicrobial drugs at the international and national levels suggests that the impact of resistance on empirical treatment decisions may vary across regions . Indeed, at least one guideline recommends that the selection of empirical therapy for outpatients with community - acquired pneumonia should be influenced by regional antimicrobial drug susceptibility patterns for s. pneumoniae (2). For most physicians, the obvious source of information on community - acquired pneumonia is the microbiologic susceptibility results from their local hospital laboratory . Hospitals frequently provide this information in the form of antibiograms, specifically designed to aid in antimicrobial drug selection . This study demonstrates that substantial variability exists in hospital - reported rates of pneumococcal drug resistance over a small geographic region and that the rates at each site are poorly correlated over time . In addition, using a variety of approaches, we were unable to demonstrate substantial geographic clustering of the data at the individual hospital level, suggesting that the individual hospital rates poorly reflect an underlying rate of resistance in the community . Other studies have demonstrated similar heterogeneity in the proportion of drug - resistant pneumococci reported by hospitals over small geographic areas . The centers for disease control and prevention reported levels of pneumococcal resistance to penicillin among invasive isolates from all hospitals within the seven regions in the united states that comprise their active bacterial core surveillance program . The proportion of penicillin - nonsusceptible s. pneumoniae ranged from 15.3% to 38.3% across the seven regions . The proportion of hospitals within each region that were> 5% below or above the average for the region ranged from 35% to 76% . No hospital characteristics, including proportion of isolates from children or from black patients, predicted deviation from the regional average (11). More recently, 16 hospitals in brooklyn, new york, participated in boroughwide surveillance for s. pneumoniae in 1997 and 1999 . Aggregating the hospitals into the western and eastern ends of the borough demonstrated a significant difference in the proportion of penicillin - susceptible isolates, 57% versus 75% (p = 0.046) (15). Our hypothesis that individual hospital rates of drug - resistant s. pneumoniae should demonstrate geographic clustering is based on the assumption that rates of resistance should reflect the underlying distribution of drug resistance in the source community . Prior research has demonstrated that patients with community - acquired pneumonia are hospitalized on average <5 miles from their place of residence (16). Thus, hospitals within the same geographic region should admit patients with similar underlying rates of drug resistance . Moreover, if the pneumococcal resistance rates reported by individual hospitals should be used to guide physician antibiotic prescribing decisions for patients with community - acquired pneumococcal infections, hospitals serving similar communities should report similar rates of resistance . Beyond reflecting true variability in the underlying community levels of drug resistance, hospital - level variation in the proportion of penicillin - susceptible first, chance error can create significant variability in hospital - level results particularly since many hospitals have only a small number of invasive pneumococcal isolates per year . We excluded hospitals with <10 isolates in each study year from our analyses to reduce the role of chance in our findings, but the relatively small number of isolates at each site undoubtedly contributed to the significant year - to - year variability and lack of geographic clustering . However, a minimum of 10 isolates per year is the nccls - recommended minimum number of isolates for reporting cumulative antimicrobial susceptibility results for any species in any reporting period (8). Second, variation in testing strategies at individual hospitals may create bias in the proportion of isolates identified as nonsusceptible if the thresholds for sending microbiologic tests vary according to characteristics that are likely to influence the probability of a drug - resistant infection . While this may be an important determinant of variability in drug - susceptibility rates among clinical isolates from respiratory and sinus samples, we believe that it is less likely to explain variability in susceptibility rates among blood isolates since thresholds for sending blood cultures are more standardized across clinical practices, particularly in the management of patients hospitalized with community - acquired pneumonia . Third, hospitals may differ in their methodologic approach and quality assurance for pneumococcal susceptibility testing . However, a study based on a nationwide college of american pathologists proficiency survey of hospital laboratories demonstrated that, while the use of specific susceptibility tests varies substantially, few major interpretive errors occurred in assigning the susceptibility to standardized pneumococcal isolates (17). In addition, prior studies have found that the results of susceptibility data generated at local hospital facilities provide a reasonable surrogate for susceptibility data generated at centralized facilities (18). Finally, geographic proximity of hospitals does not ensure that the patient populations served by those hospitals come from the same communities . Indeed, substantial literature has established important variation in hospital referral patterns that creates diversity in the source of patient populations among geographically proximate hospitals . Future research will need to consider the role of referral patterns in explaining some of the hospital - level variability observed in this and other studies . One limitation of this study is our focus on a single geographic region in eastern pennsylvania . Patterns and determinants of hospital - level variation in pneumococcal drug susceptibility may produce different results in other regions . In addition, because we focused on hospitals providing adult care, we cannot generalize our results to pediatric hospitals . Regardless of whether individual hospitals provide valid information on local levels of pneumococcal susceptibility, variability in these levels is only clinically meaningful if the range of susceptibility crosses some threshold for the empirical choice of specific antimicrobial drugs . Currently in the united states, empirical use of penicillins for the treatment of community - acquired pneumonia is uncommon relative to the use of fluoroquinolones and macrolides (19). However, as resistance to these classes of antimicrobial drugs continues to grow, physicians will need to determine appropriate thresholds for switching to yet newer antimicrobial agents . In these settings, valid information on local rates of pneumococcal drug susceptibility ideally, data should be derived from large samples reflective of the region for which the empirical recommendations apply . Continued research is needed to determine whether susceptibility data provided by local hospital microbiology laboratories can ever serve this purpose.
It is essencial to localize the toxic region accurately in hyperthyroid patients before making a decision to remove with operation . Here we report a cese of an intrathoracic toxic thyroid nodule causing hyperthyroidism with a multinodular normal functional cervical thyroid gland . A 62-year - old female presented with a 2 years history of weakness, weight loss, tremulousness palpitations, and heat intolerance . Physical examination revealed a normal weight female in no distress; blood pressure was 120/80 mmhg supine, pulse 96, and regular . Laboratory examination results from complete blood cell count, urinalysis, electrocardiography, and biochemical tests were within the normal limits . Thyroid function tests revealed hyperthyroidism; free t3: 4.5 ng / ml l (normal: 1.85 ng / ml), free t4: 1.83 ng / ml (normal: 0.81.9 ng / ml), and thyroid - stimulating hormone: 0.016 u / ml (normal: 0.44 u / ml . Radionuclide tc-99 m pertechnetate (mtc04) thyroid scan images obtained [figure 1a and c] from neck and mediastinum 20 min after intervenes . Administration of 3 mci tc99 m pertechnetate using a parallel hole low energy all - purpose collimator . The scan showed a normoactive cervical thyroid gland and a hyperactive intrathoracic area on the left side of thyroid . Thyroid radioactive iodine uptake (raiu) measured over the neck and the uptake after oral administration of 7 ci i-131 was 15% at 2 h and 34% at 24 h. a thyroid scan was also obtained with gamma camera at 24 h [figure 1b] using a parallel hole high - energy collimator . Planar images of neck and mediastinum and cervicothoracic single photon emission computed tomography images were taken . Images showed a normal functioning cervical thyroid gland and a hyperfunctioning intrathoracic thyroid tissue on the left side of thyroid . Chest roentgenogram [figure 2] revealed widened upper mediastinum with no tracheal displacement and fibrotic changes in the basal regions that suggested bronchiectasis . Ultrasonographic assessment of thyroid bed showed a normal sized multinodular cervical goiter (right lobe: 43 mm 17 mm 22 mm, left lobe: 46 mm 31 mm 22 mm and isthmus: 5 mm) and did not define a substernal enlargement . Thoracic computed tomographic scans [figure 3] showed a 6 cm 6 cm 4.5 cm diameter, smooth edge mass in the left superior portion of the anterior mediastinum with patchy calcification and heterogeneous contrasting which is related with the left thyroid lobe in a small area . . Tc-99 m pertechnetate images of anterior and posterior thorax and mediastinum (a) showed a normoactive cervical thyroid gland and an activity accumulation in the intrathoracic area on the left side of thyroid (a, arrowheads). Anterior i-131 thyroid scan (b) revealed a normal functioning cervical thyroid gland and a hyperfunctioning intrathoracic thyroid tissue on the left side (b, arrow) and support the accumulation in the upper thoracic area . Anterior tc-99 m pertechnetate images (c, arrow) showed that it was a thyroid tissue, and a marker was located in inferior isthmus region chest x - ray revealed widened upper mediastinum (arrow) with no tracheal displacement and fibrotic changes on the basal regions that suggested bronchiectasis in thoracic computed tomography scans; axial images of lung window showed a smooth edge mass in the left superior anterior mediastinum (upper arrows), and patchy calcification and heterogeneous contrasting were seen in axial images of the bone window (lower arrows). There was no significant tracheal and esophageal compression or displacement due to a hyperfunctioning intrathoracic goiter with a normal multinodular functional cervical thyroid gland, the patient was diagnosed as hyperthyroidism . The patient was treated with graded doses of propylthiouracil, but partial response was provided . Radionuclide scintigraphy is valuable in confirming suspected or clinically evident thyroid tissue that is extending into mediastinum . Although mtc04 has been used for routine thyroid scintigraphy, i-131 is preferred for imaging of substernal goiter because mtc04 activity in goiter remains lower comparing with high level of background activity in the heart and great vessels . Moreover, for imaging structures behind sternum, higher energy photons of i-131 are needed . In this case, our patient has high raiu values indicating clinical hyperthyroidism; however, her cervical thyroid was in normal size without enlargement and abnormal uptake . The possibility of the existence of external thyroidal tissue should be kept in mind in such suspicious cases . The case we present, a hyperthyroid patient with high radioiodine uptake without cervical thyroid enlargement suggest that the radiation is coming from intrathoracicgoiter which was detected recently . Intrathoracic goiter (retrosternal or substernal) is one of the major diagnostic consideration in evaluation of upper anterior mediastinal masses . Because of iodine trapping of thyroid tissue, radioiodine scintigraphy remains an excellent confirmatory test . However, few cases of i-131 accumulations in thoracic region confirmed to be bronchogenic cyst or carcinoma have been reported were confirmed as . Park et al . Analyzed 54 patients and demonstrate that most intrathoracic goiters can be diagnosed by thyroid scintigraphy with a sensitivity of 93% . They conclude that most intrathoracic goiters have thyroid function, and radioiodine scintigraphy is a definitive and cost - effective test for diagnosis . In addition, they recommend that radioiodine scintigraphy should be the first study for further evaluation of upper anterior mediastinal masses . Reported a hyperthyroid patient with intrathoracic goiter that was cured after excision of the mass . They stated that radioactive iodine treatment may be used unless the patient has compression symptoms . Also mentioned the importance of radionuclide scintigraphy with either mtc04 or i-123 in their study including children with thyroid dysgenesis . They use those scintigraphic modalities as a diagnostic tool averting invasive procedure such as biopsy and providing both functional and anatomic information to show where the thyroid tissue is and how effective it works . Aydin et al . Reported a case underlining the value of mtc04 scintigraphy with and without potassium perchlorate administration . Disappearance of tracer accumulation after applying potassium perchlorate suggests that mediastinal mass was an intrathoracic goiter . Pathological improvement supports this technique as a simple, accurate, and cost - effective imaging . In our case, high thyroid hormone levels and isotopic uptake in the intrathoracic goiter with normal uptake of the cervical thyroid were diagnostic for toxic intrathoracic goiter . Higher accumulation in thoracic mass than cervical thyroid in both mtc04 and i-131 scintigraphy reminds us the development of hyperthyroidism in the intrathoracic goiter which had not yet produced feedback suppression of the cervical thyroid . Our patient was cured after excision of mediastinal mass despite the absence of compression symptoms or mass effect . Intrathoracic goiters are generally considered an indication for surgery; radioiodine therapy remains a choice for those who cannot be operable because of advanced systemic disease or other reasons . Our patient did not have significant tracheal deviation or compression symptoms but her hyperthyroidism cannot be controlled by antithyroid drugs and symptomatic improvement was incomplete . This case highlights the importance of mediastinal imaging using i-131 and mtc04 scintigraphy in thyroid detection . Patients should not only be imaged with a pinhole collimator but also their mediastinal region should be viewed with parallel hole collimators.
More than 2 billion people globally are estimated to suffer from micronutrient deficiencies (von grebmer et al . Iron, zinc, vitamin a, iodine, folate and other vitamin b deficiencies are among the most widespread global micronutrient deficiencies (muthayya et al . Factors that contribute to poor micronutrient intake and absorption include poor dietary diversity, poor nutrient density of staplebased complementary foods, antinutritional factors in plantbased foods and environmental enteropathy . Disease conditions resulting from infections can further aggravate micronutrient deficiencies (katona & katonaapte 2008). Strategies such as targeted supplementation with vitamin a, or universal salt iodization, oil fortification with vitamin a, or flour fortification with iron or folic acid, are often part of national strategies to address the problem of micronutrient malnutrition among vulnerable groups throughout the life cycle . Overcoming deficiencies of micronutrients can have a potential benefit in terms of young child survival, growth and development and preventing intrauterine growth restriction and low birth weight (black et al . Reviews such as the copenhagen consensus have consistently ranked micronutrients as the most costeffective development intervention (copenhagen consensus panel 2012). Economic analyses suggest that fortification and/or targeted supplementation with, for instance, iron, zinc, vitamin a, folic acid and iodine can be highly costeffective (horton 2006; horton et al . Fortification will not reach all individuals but can be a costeffective strategy provided that a centrally processed, affordable fortified food vehicle is available and either the deficiency is widespread or only a small group is affected but the adverse effects because of the deficiency are very costly (horton 2006). Targeted micronutrient programs such as home fortification or periodic highdose supplements have the advantage to deliver micronutrients of concern to the vulnerable subpopulation(s) without unnecessarily exposing other groups in the population . In order for supplementation to be costeffective, the defined target group should be readily reached with little compliance issues (horton 2006). When implementing micronutrient programs there is the risk of providing insufficient or excessive amounts, missing those at risk or redundant coverage (coverage of individuals with adequate intake, or coverage by more programs than necessary), potentially resulting in inadequate or excessive intakes and poor costeffectiveness . Designing an effective and safe fortification or supplementation program imposes the dual challenge of reaching the target population groups at most risk of micronutrient deficiency while avoiding overexposure of individuals at the high end of the intake distribution curves (european food safety authority (efsa) 2010). This may pose particular challenges regarding micronutrient interventions that exert benefits in the target population but unintentionally adverse effects in a subgroup of this target population with already high intake or status of the micronutrient (e.g. Targeted vitamin a supplementation) (european food safety authority (efsa) 2010). Increasing micronutrient intakes through universal fortification can deliver potential benefits to deficient population groups, but sometimes also risks in other population groups when consumed in excess (e.g. Folic acid fortification to prevent neural tube defects in the unborn child versus masking of vitamin b12 deficiency in elderly) (european food safety authority (efsa) 2010). Values have been set for the intake of each micronutrient below which the risk for adverse health effects is likely to increase . The risk of an inadequate intake increases as an individual's intake falls further below the recommended intake (food and nutrition board & institute of medicine (iom) 2000). At two standard deviations below the recommended intake, i.e. The estimated average requirement (ear), adverse health consequences because of a micronutrient deficiency condition increase in incidence and/or severity the further the usual intake falls below the ear (renwick et al . 2004). Therefore, reducing the number of individuals with inadequate micronutrient intakes can be expected to have clear benefits on micronutrient deficiencyrelated morbidity especially when the morbidity is severe and/or chronic . However, the adverse health consequences of deficiency are far better understood than the evidence base related to the consequences of excess, if any . The excess level of intake is neither well understood and defined for many micronutrients, nor do biomarkers exist that can detect early adverse effects . Threshold for intake or a biomarker, above which the risk of adverse effects may increase and the dose response relationship (renwick et al . 2004). The tolerable upper intake level (ul) is the highest daily intake still considered to be safe for almost all healthy individuals in a specified group . Depending on the magnitude of the evidence, the ul is obtained by downward adjustment of the noobservedadverseeffect level (if known) or the lowestobservedadverseeffect level by applying an uncertainty factor . This has resulted for a number of micronutrients in an ul that is close to the recommended nutrient intake because of large uncertainty factors used to set this ul (european commission 2006). For some micronutrients, no evidence of risk of adverse effects is established, and hence no safe tolerable upper intake level (ul) is defined (i.e. Biotin, pantothenic acid, chromium, vitamin b1, b2, b12 and k). For other micronutrients, the risk of exceeding the ul is low (i.e. Vitamin b6, c, d and e, niacin, molybdenum, phosphorus and selenium). For some micronutrients, a potential risk of exceeding the maximum safe ul exists (i.e. Vitamin a as preformed retinol, zinc, iron, iodine, copper and calcium). For the latter micronutrients, programs should be cautiously designed and monitored to ensure at risk population groups are reached without any appreciable risk of adverse effects in subgroups that consume high amounts of the micronutrient . Important elements of the program cycle include the gathering of information to assess the nutrition situation, development of a national policy and strategy, program design, implementation and monitoring, review and evaluation, and adjustment if necessary . Before implementing a micronutrient program it is critical to assess the nutrition situation by collecting data among the different population groups that can be referred to as the abcd of nutrition assessment; anthropometric assessments, biomarker or biochemical assessments, clinical assessments (morbidity and mortality) and dietary assessment . These data allow understanding the magnitude of the health problem and the vulnerable population group(s) at risk of micronutrient deficiency . Information on food and micronutrient intake distribution in the different population groups will help predict how micronutrient interventions will work in a variety of contexts . Another critical element in planning (cost) effective and safe programs is the ongoing monitoring and evaluation of the nutrition situation through collection of data . It provides an indication of the effectiveness and safety of the intervention and allows adjusting the type of intervention, amount of provided micronutrients or geographic distribution, if needed . Some countries have implemented one or multiple programs, focusing mostly on vitamin a, iodine and iron . However, availability of data on biomarkers predictive of nutrient intake, status and adverse health effects (combs et al . 2013; raiten & combs 2015) is still sparse, and few countries have conducted detailed nutrition surveys, as their collection can be time and resourceintensive . Often multiple programs with micronutrients are implemented to reach a greater proportion of the atrisk population . In particular, implementation of multiple micronutrient interventions requires coordination and monitoring to ensure that these programs are complementary and not overlapping, which may result in unnecessarily exposure of vulnerable groups . Collecting dietary intake data and biomarkers can be used to correct program designs to ensure that intake ranges in the different lifestage groups are effective and safe . Dietary intake data can be used in software modelling to predict the shift in micronutrient adequacy in different population groups for a selected food vehicle and fortification level . Until recently, dietary intake modelling tools were not able to predict the effect of intake from combined fortification and supplementation programs . As addressed in this symposium report, a new simulation approach allows to predict the effect of implementing fortification combined with supplementation on intakes below the ear and above the ul and related program cost versus the savings (englestone et al . Other software tools have been developed that simulate the effect of changing food or nutrient intakes on prevention of the related burden of disease (i.e. The benefits). In risk benefit approaches, the same principle is used to assess both the preventable burden of disease and the risks related to excessive food or nutrient intake, provided that sufficient evidence is available to simulate the adverse health response . In order to use a risk benefit approach, a better understanding is needed of the relation between micronutrient intake, status and morbidity / mortality outcomes . For some micronutrients the link between micronutrient intake, status and morbidity / mortality is sufficiently established to predict health benefits when overcoming its deficiency (horton 2006). However, with the current dearth of data in many countries on micronutrient intake, status and related disease incidence, program modelling to predict the benefits, and even more so the risks, is still a challenge . It is nevertheless encouraging that in many countries the collection of nutrition data is increasing, which will further help program planners and policymakers to design costeffective and safe micronutrient interventions . Minimum and maximum intake values for micronutrients are relevant for population groups, but do not necessarily reflect adequate or safe intake values for all individuals in that group . The requirement and safety limit for a micronutrient not only varies by age, gender and lifestage, but also by health condition, medication use and genetic profile, making it even more challenging to develop a safe and effective program tailored to all at risk individuals . For instance, chronic disease conditions or genetic polymorphisms may lead to higher micronutrient requirements . Micronutrients do not necessarily provide benefits in all deficiency conditions; complex interactions may exist between the delivered micronutrients, micronutrient status, infections and medication use . Complicating factors include (1) interactions between micronutrients, for instance iron and zinc reducing each other's absorption (lnnerdal 2004), (2) micronutrient drug interactions; micronutrients reducing the absorption or efficacy of a drug or vice versa (scaglione & panzavolta 2014; hathcock 1985), or (3) antagonistic interactions between micronutrients and infections (e.g. Iron and malaria). The benefits and risks of some micronutrients therefore depend on the setting in which they are provided . For instance, the effect of certain micronutrients may not only depend on whether the target population is deficient or not, but also on whether malaria control strategies are implemented in malaria endemic regions . This is exemplified by the pemba study, in which children living on malariaendemic pemba island who received iron and folic acid supplements, were more likely to die or need treatment in hospital for an adverse event (sazawal et al . However, in a substudy of the trial in which children were given malariareduction measures, children who were iron deficient and anaemic at baseline, showed a significant reduction in adverse events, including malaria episodes, suggesting that supplementation with iron and folic acid may especially confer benefits to those who are deficient . These issues, altogether, pose a challenge to program planners in designing micronutrient intervention programs that meet the requirements of the majority of the population at risk while still being safe . There is a need for tools that assist is designing micronutrient intervention programs that are effective in reaching the target groups at risk of developing micronutrient deficiencies, efficient, in terms of reaching the target group and not unnecessarily exposing nontarget groups, and safe, in terms of not overexposing a segment of the population that is already more than adequate, especially vulnerable target groups . There is a need to understand how specific micronutrients, mainly as folic acid and iron, may interact with medication and infection, and how they exert beneficial or antagonistic effects under these conditions . This raises the question of how to achieve the maximum possible public health benefits of a micronutrient intervention strategy with the least risks . Monitoring of data (dietary intake, program coverage, biomarkers and morbidity) among vulnerable population groups is essential to optimize effectiveness, safety, and efficiency of micronutrient programs.modelling tools are needed that predict the effectiveness, safety, and efficiency of micronutrient programs by integrating above data.research is needed to identify biomarkers predictive of micronutrient exposure, status, and potential health consequences of inadequate and especially excessive micronutrient exposure . This may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level, currently often based on scant data, particularly for young children.more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients, drugs, and infections . Monitoring of data (dietary intake, program coverage, biomarkers and morbidity) among vulnerable population groups is essential to optimize effectiveness, safety, and efficiency of micronutrient programs . Modelling tools are needed that predict the effectiveness, safety, and efficiency of micronutrient programs by integrating above data . Research is needed to identify biomarkers predictive of micronutrient exposure, status, and potential health consequences of inadequate and especially excessive micronutrient exposure . This may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level, currently often based on scant data, particularly for young children . More insight is needed into the antagonistic effects resulting from complex interactions between micronutrients, drugs, and infections . Until recently, dietary intake modelling tools were not able to predict the effect of intake from combined fortification and supplementation programs . As addressed in this symposium report, a new simulation approach allows to predict the effect of implementing fortification combined with supplementation on intakes below the ear and above the ul and related program cost versus the savings (englestone et al . Other software tools have been developed that simulate the effect of changing food or nutrient intakes on prevention of the related burden of disease (i.e. The benefits). In risk benefit approaches, the same principle is used to assess both the preventable burden of disease and the risks related to excessive food or nutrient intake, provided that sufficient evidence is available to simulate the adverse health response . In order to use a risk benefit approach, a better understanding is needed of the relation between micronutrient intake, status and morbidity / mortality outcomes . For some micronutrients the link between micronutrient intake, status and morbidity / mortality is sufficiently established to predict health benefits when overcoming its deficiency (horton 2006). However, with the current dearth of data in many countries on micronutrient intake, status and related disease incidence, program modelling to predict the benefits, and even more so the risks, is still a challenge . It is nevertheless encouraging that in many countries the collection of nutrition data is increasing, which will further help program planners and policymakers to design costeffective and safe micronutrient interventions . Minimum and maximum intake values for micronutrients are relevant for population groups, but do not necessarily reflect adequate or safe intake values for all individuals in that group . The requirement and safety limit for a micronutrient not only varies by age, gender and lifestage, but also by health condition, medication use and genetic profile, making it even more challenging to develop a safe and effective program tailored to all at risk individuals . For instance, chronic disease conditions or genetic polymorphisms may lead to higher micronutrient requirements . Micronutrients do not necessarily provide benefits in all deficiency conditions; complex interactions may exist between the delivered micronutrients, micronutrient status, infections and medication use . Complicating factors include (1) interactions between micronutrients, for instance iron and zinc reducing each other's absorption (lnnerdal 2004), (2) micronutrient drug interactions; micronutrients reducing the absorption or efficacy of a drug or vice versa (scaglione & panzavolta 2014; hathcock 1985), or (3) antagonistic interactions between micronutrients and infections (e.g. Iron and malaria). The benefits and risks of some micronutrients therefore depend on the setting in which they are provided . For instance, the effect of certain micronutrients may not only depend on whether the target population is deficient or not, but also on whether malaria control strategies are implemented in malaria endemic regions . This is exemplified by the pemba study, in which children living on malariaendemic pemba island who received iron and folic acid supplements, were more likely to die or need treatment in hospital for an adverse event (sazawal et al . However, in a substudy of the trial in which children were given malariareduction measures, children who were iron deficient and anaemic at baseline, showed a significant reduction in adverse events, including malaria episodes, suggesting that supplementation with iron and folic acid may especially confer benefits to those who are deficient . These issues, altogether, pose a challenge to program planners in designing micronutrient intervention programs that meet the requirements of the majority of the population at risk while still being safe . There is a need for tools that assist is designing micronutrient intervention programs that are effective in reaching the target groups at risk of developing micronutrient deficiencies, efficient, in terms of reaching the target group and not unnecessarily exposing nontarget groups, and safe, in terms of not overexposing a segment of the population that is already more than adequate, especially vulnerable target groups . There is a need to understand how specific micronutrients, mainly as folic acid and iron, may interact with medication and infection, and how they exert beneficial or antagonistic effects under these conditions . This raises the question of how to achieve the maximum possible public health benefits of a micronutrient intervention strategy with the least risks . Monitoring of data (dietary intake, program coverage, biomarkers and morbidity) among vulnerable population groups is essential to optimize effectiveness, safety, and efficiency of micronutrient programs.modelling tools are needed that predict the effectiveness, safety, and efficiency of micronutrient programs by integrating above data.research is needed to identify biomarkers predictive of micronutrient exposure, status, and potential health consequences of inadequate and especially excessive micronutrient exposure . This may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level, currently often based on scant data, particularly for young children.more insight is needed into the antagonistic effects resulting from complex interactions between micronutrients, drugs, and infections . Monitoring of data (dietary intake, program coverage, biomarkers and morbidity) among vulnerable population groups is essential to optimize effectiveness, safety, and efficiency of micronutrient programs . Modelling tools are needed that predict the effectiveness, safety, and efficiency of micronutrient programs by integrating above data . Research is needed to identify biomarkers predictive of micronutrient exposure, status, and potential health consequences of inadequate and especially excessive micronutrient exposure . This may help setting the appropriate noobservedadverseeffect or lowestadverseeffect level, currently often based on scant data, particularly for young children . More insight is needed into the antagonistic effects resulting from complex interactions between micronutrients, drugs, and infections . During the global summit on food fortification on 911 september 2015 in arusha, one of the sessions included a special session coorganized by sight and life and unicef, entitled effective and safe micronutrient interventions: weighing the risks against the benefits. The aim of the session was to identify existing tools and needs to assist policy makers in designing effective micronutrient programs with the highest public health benefits and least risks . The presentations in the session addressed this challenge, based on a number of case studies related to folic acid, iodine and vitamin a. maaike bruins (dsm biotechnology center, delft, the netherlands), with the title assessing the risks and benefits of micronutrients interventions. In order to understand the risks of inadequate and excessive intake of a micronutrient, information is required on the intake distribution of the micronutrient in the population . This enables to assess the proportion of a population lifestage group with intakes below their requirements and above their ul . Software is available, such as pcside and imapp, for the estimation and modelling of food and micronutrient intake distributions, which can assist in selecting optimal food vehicles and fortification levels (iowa state university 2015). Other tools make it possible to estimate the preventable public disease burden because of a micronutrient intervention program, expressed for instance as preventable disabilityadjusted life years (dalys). The daly is a quantitative measure of overall disease burden and can serve as important guide to decisionmaking . The daly is expressed as the number of years lost because of early death or morbidity (e.g. Infections, anaemia, motor and cognitive impairments, or blindness). The daly metric is composed of mortality and incidence, duration and disability of the morbidity (related to micronutrient deficiency). For instance, preventing dalys by overcoming maternal folate deficiency may have a large public health impact; the preventable lifelong and severe disabilities resulting from of neural tube defects are substantial, even if a few neural tube defects in 1000 births could be prevented . The world health organization (who) has developed a source of estimates for costeffectiveness of micronutrient interventions is the choosing interventions that are cost effective (whochoice) database (world health organization (who) 2016). The estimates are based on known intervention costs and effectiveness (in terms of micronutrient status), and links between micronutrient status and morbidity / mortality outcomes . The who has also developed tools that can assist program planners in simulating the public health impact of an increase in micronutrient intake in terms of dalys gained (world health organization (who) 2015). This requires (1) dietary intake data in different population groups and (2) evidence for a dose response relationship between micronutrient intake and morbidity because of deficiency . With the introduction of a micronutrient intervention, for instance, when implementing mandatory fortification, the benefits of preventing frequent, or severe, or lifelong disabilities should be balanced against the possible risks of excess intake in other population groups (hoekstra et al . Benefit assessment software provides the option to quantify the public health risks and benefits, and balance them, using for instance dalys as a common metric (ellis & aspurger 2000). Even though simulating changes in dalys resulting from micronutrient intervention programs can provide useful information to policy makers to set investment priorities, moreover, for many micronutrients the dose response relationship between excessive intake and risks is not well known . Even though simulating changes in dalys resulting from micronutrient intervention programs can provide useful information to policy makers to set investment priorities, next to these sophisticated modelling tools, more userfriendly tools are needed that integrate information on program coverage, dietary intake distribution, biomarkers and morbidity to design effective and safe micronutrient programs in terms of public health impact . Increasing micronutrient intake may confer benefits on those who have inadequate intakes . For instance, in some studies iron supplementation has been associated with an increased risk of malaria and death in children living in malariaendemic regions, but not when regular malaria surveillance and treatment services are provided (world health organization (who) 2015). Another example of a micronutrient that may not necessarily confer benefits under all circumstances is, for instance, folic acid . Folic acid supplementation may be beneficial to the folatedeficient child, but may confer risks when highdose supplements are implemented in malariaendemic settings alongside antimalarial drugs (kupka 2015). The who calls for micronutrient powder (mnp) programs to be implemented alongside malaria control strategies in malaria endemic regions . Current mnp formulations generally provide folic acid and other micronutrients at the recommended nutrient intake level . However, the benefits and risks of providing supplemental folic acid are largely unknown . The limited data available suggest that folate deficiency may not be a major public health problem among children living in subsaharan africa; as a result, supplemental folic acid may not confer health benefits . Furthermore, folic acid provided at supraphysiological, and possibly even physiological, levels may favour the growth of the parasite plasmodium falciparum (responsible for 85% of malaria cases), inhibit clearance of the parasite by sulphadoxinepyrimethamine (sp)treated malaria and increase subsequent recrudescence (nzila et al . 2014). Limiting prophylactic sp use or promoting the use of insecticidetreated bed nets may render the use of folic acid in mnp programs safer, but programmatic barriers to these approaches may remain . The use of 5methyltetrahydrofolic acid concomitantly with sp may be a promising alternative, as it does not affect sp efficacy, although its stability in food and costinuse remain to be confirmed (scaglione & panzavolta 2014). The presentation concluded that more work is needed to characterize the prevalence of folate deficiency among young children worldwide, and to optimize the benefit risk ratio of mnp programs in subsaharan africa . Michael zimmerman from the eth zrich (switzerland) discussed the risks and benefits of salt iodization programs . In general, the relatively small risks of iodine excess are far outweighed by the substantial risks of iodine deficiency (zimmermann 2008). Iodine deficiency has multiple adverse effects on growth and development because of inadequate thyroid hormone production, which are termed the iodine deficiency disorders (idd). Idd remains one of the most common causes of preventable mental impairment worldwide . In nearly all iodinedeficient countries, the best strategy to control idd is salt iodization, one of the most costeffective ways of contributing to economic and social development (zimmermann 2008). In areas of severe iodine deficiency, iodine repletion in pregnant women iodine repletion in newborns reduces infant mortality, and may improve cognitive development and growth . Even in areas of mildtomoderate iodine deficiency, repletion in schoolaged children improves cognition and fine motor skills (zimmermann 2007). On the other hand, the introduction of iodized salt to regions with chronic idd may transiently increase the incidence of thyroid disorders, such as iodineinduced hyperthyroidism and autoimmune hypothyroidism, and programs should therefore include monitoring for both iodine deficiency and excess . More data on the epidemiology of thyroid disorders caused by differences in iodine intake are needed, and should be the focus of future research . The adverse effects of vitamin a deficiency include childhood blindness, and the increased risk of mortality from measles and diarrhoea (stevens et al . Vitamin a supplementation and fortification programs can be a cheap and effective way to reduce morbidity and mortality because of vitamin a deficiency (horton 2006). These programs have significantly improved millions of lives among preschool children in many parts of the world (stevens et al . Longterm excessive vitamin a intakes, on the other hand, can lead to adverse effects, such as hepatotoxicity . Generally, the substantial risks of vitamin a deficiency can be expected to far outweigh the relatively small risks of vitamin a excess (allen & haskell 2002). The lack of coordination of multiple vitamin a interventions and many overlapping schemes in some areas has increased concern about the risks of these programs, as some children could theoretically receive well above their recommended daily dose of the vitamin . The uncertainty around the level of excess for vitamin a has resulted in a small margin of safe intake for young children, between the recommended intake levels and maximum safe ul georg lietz from newcastle university (newcastle upon tyne, uk) discussed how to best assess the window of benefit for vitamin a intake . In his presentation, dr . Lietz highlighted the need for reliable, robust and affordable biomarkers of inadequate and excess status, to enable policy and program makers to adjust the correct level and coverage of their interventions . The appropriate noobservedadverseeffect level or the lowestobservedadverseeffect level for children up to 5 years of age needs to be urgently determined and the uls revised as most uls for children are extrapolated from uls for adults . Dr . Lietz also discussed the benefits and problems associated with the most recent methods to have been developed to enable the monitoring of both fortification and supplementation programs, including the application of stable isotope dilution techniques, and the potential for labonchip technologies for future monitoring . He ended his presentation by highlighting the need to coordinate different methods of risk assessment, to make the monitoring process more efficient, reliable and costeffective . Reina englestone from the university of california (davis, ca, usa) presented a dietary modelling approach to assess the risk of inadequate and excessive intakes under different program scenarios, and their applications to program planning . Her presentation provided an overview of the methodology for the use of dietary data to examine the risk of inadequate and excessive micronutrient intakes using dietary reference values, and given information on current dietary patterns and the reach of program delivery platforms . Examples from cameroon illustrated the use of dietary modelling to set fortification levels, by predicting the effects of each new or modified program on dietary intake (englestone et al . This dietary modelling tool can be used to predict the effect of fortification of different food vehicles and overlapping micronutrient programs on the percentage of the population with intakes below ear or above the ul (brown et al . 2015). Finally, the role of complementary modelling tools was discussed, including the (1) lives saved tool (list), which predicts the mortality reduction from some micronutrient interventions delivered to mothers and children (johns hopkins school of public health 2015), (2) models assessing liver vitamin a concentration, which can assist in the interpretation of risk of dietary intakes above the upper intake level in young children, and (3) program coverage surveys which reveal absent or redundant program coverage and thus help identify subpopulations (e.g. Regions) at risk of inadequate or excessive intake (aaron 2014; spohrer 2015). Population dietary intake data can be used to model the effect of different food vehicles and micronutrient levels to optimize coverage and minimize the risks of inadequate and excessive intakes in relation to dietary cutoff points . Monitoring intake data will enable policy makers to adjust to the correct food vehicle, micronutrient level, and coverage of intervention programs . However, the success of these programs has, inevitably, to be confirmed by applying biomarkers indicative of micronutrient status that are affordable, sensitive and specific, and can be applied in population settings (combs et al . Some methods which are currently applied are inadequate, because of their lack of precision and accuracy, to detect nutrient concentrations in atrisk population groups, or because of insensitivity to changes in status within a certain range (e.g. Serum retinol), or alterations during inflammation (zinc, retinol, ferritin). Thus, improved biomarkers are urgently needed which will enable policy makers to understand the benefits and risks in vulnerable population groups related to increasing micronutrient intakes . Tools are available which assess or predict the effects of micronutrient programs, in terms of dietary intakes against dietary cutoff points . However, there is still need for standardized, userfriendly tools, which predict the public health impact made by micronutrient interventions, by integrating benefits and possible risk . More research is warranted in order to understand the concerns regarding the safety of iron and folic acid supplementation among young children in malaria endemic areas, and their interaction with antimalarials and infections . Research is needed to assess the minimum and maximum intake range for vitamin a outside which intakes might cause concern . In general, the small risks of micronutrient excess are far outweighed by the substantial benefits of overcoming the deficiency . However, there is an urgent need to monitor overlapping micronutrient supplementation and fortification programs, and to evaluate the benefits and risks (if any) of increasing population micronutrient intakes for each setting (population group, region, choice of food vehicle, micronutrient level in food, micronutrient status of the population group, concomitant food and medications, etc . ). Klaus kraemer is the director of sight and life foundation, a nutrition think tank primarily funded by dsm . The authors declare that they have all participated in drafting of the manuscript, and that they have approved the final version.
Cysticercosis, caused by the metacestode stage of taenia solium, is a serious health and veterinary problem in many developing countries [13]. In humans, t. solium cysticerci cause neurocysticercosis, which affects ~50 million people worldwide, and t. solium also infects pigs, its intermediate host, leading to major economic losses [5, 6]. When humans ingest undercooked contaminated pork meat, the adult worm develops in the small intestine . After two months of asymptomatic infection, this tapeworm starts producing thousands of eggs that, once released with the stools, can contaminate the environment, infecting pigs (rapidly differentiating into cysticerci mainly in the muscle) and humans (where most severe symptoms are observed due to the presence of cysticerci in the brain) [1, 7]. Thus, maintenance of the parasite's life cycle depends on the adult tapeworm development . In fact, even in communities which do not rear or consume pigs, human neurocysticercosis can be found, because of the presence of a tapeworm carrier [9, 10]. Furthermore, tapeworm development in turn depends on scolex evagination, the initial step through which a single cysticercus becomes an adult parasite with capability of producing infective eggs . Recent information reveals that sex hormones can affect the course of a parasite infection [1216], as in the case of taeniasis / cysticercosis [1719]. Moreover, frequency of t. solium pig cysticercosis is increased during pregnancy, when there is a significant increase in progesterone levels [19, 20]. It has also been demonstrated that castration in naturally infected male boars, induces an increase in the prevalence of cysticercosis, which highlights the possible role of host androgens to restrict parasite establishment and estrogens to facilitate it . Furthermore, taenia crassiceps (a close relative of t. solium) has shown to be affected by in vivo and in vitro sex steroid treatment . Specifically, 17-estradiol increases the reproduction of t. crassiceps cysticerci in vitro, while testosterone or dihydrotestosterone decreases it . When castrated mice are treated with 17-estradiol, the number of parasites as well as their infective capacity increases up to 200% [22, 23] meanwhile progesterone has the opposite effect in castrated mice of both sexes: a decrease in the parasite loads of almost 100% . Since an mrna sequence similar to that of estrogen receptor has been found in t. crassiceps, it is possible that the direct effect of estradiol on t. crassiceps reproduction could be due to its binding to this receptor . In fact, numerous sex steroid actions in vertebrates are mediated by the binding to their nuclear receptors, which in this form regulate gene expression, as in the case of estrogens, androgens, and progesterone . The latter interacts with two main progesterone receptor (pr) isoforms, whereas other hormones such as androgens only have one specific receptor . Interestingly, a similar mechanism could occur also in parasites [21, 29, 30]. Steroid hormone effects are not restricted only to cestode parasites but also to nematodes such as ancyclostoma dudodenale, whose number of larval and adult stages is increased by sex steroid hormones in several organs of mice . Moerover, adult and muscle larvae of trichinella spiralis are increased in ovarectomized female rats, suggesting that estrogens are restrictive factors for parasite establishment, while androgens should play a permissive role to the infection . For instance, in vitro, testosterone has an antifecundity effect upon male and female schistosoma mansoni adult worms, as well as dehydroepiandrosterone, which in vitro reduces the viability and oviposition of schistosoma mansoni . As it can be seen, direct effects of sex steroids upon helminth parasites (cestodes, nematodes, and trematodes) are not unusual . In fact, previous results suggest that these pathogens are not only directly affected by hormones, but they have also developed several strategies to exploit the host's endocrine microenviroment [33, 34], which include degradation of host proteins as an alternative source of aminoacids, development of parasitic - sex steroid receptors [29, 30], and cross - activation of signal transduction pathways [36, 37]. Taking into consideration this information, the aim of the present study was to explore the role of progesterone on t. solium cysticerci development, evaluating its in vitro effects on scolex evagination and adult worm growth, key processes in the maintenance of the infectious cycle in pigs and humans . The in vitro effect of progesterone on t. solium was studied through pharmacological (the use of ru486, a progesterone antagonist which binds to intracellular pr) and molecular (rt - pcr, western blot, phylogenetic analyses) approaches, in order to figure out the mechanism of progesterone actions in the parasite . T. solium cysticerci were dissected from the muscle of infected pigs, which were euthanized at the veterinary school of the universidad nacional autnoma de mxico . The method was previously evaluated by the university animal care and use committee to ensure compliance with international regulations and guidelines . The fibrous capsule that surrounds each parasite was carefully separated with the use of a dissection microscope . Once dissected, cysticerci were placed in tubes containing sterile pbs (1x) supplemented with 100 u / ml of antibiotics - fungizone (gibco, grand island, ny, usa). Samples were centrifuged for 10 minutes, at 800 g at 4c and the supernatant was discarded . Pellets containing cysticerci were incubated in dulbecco's modified medium (dmem) without fetal serum supplementation (gibco, brl, rockville, md, usa). They were then washed by centrifugation 3 times for 10 minutes at 800 g with dmem . After the final wash, viable parasites (complete and translucent cystic structures) culture grade progesterone and ru486 were obtained from sigma (sigma - aldrich, usa). For in vitro tests, progesterone - water soluble (powder cell culture tested, sigma - aldrich, usa) was dissolved in dmem free - serum culture medium, while ru486 was dissolved in pure ethanol (sigma) to the desired stock concentration, and sterilized by passage through a 0.2 mm millipore filter . For concentration - response curves, the experimental design was as follows, using four wells per treatment: (a) progesterone vehicle (only dmem); (b) ru486 vehicle (ethanol at the final concentration of 0.06% per well); (c) progesterone at 0.06, 0.25, 2.5, 3.175, and 63.5 m; (d) ru486 (at the same concentrations of progesterone); and (e) a combination of progesterone and ru486 in all concentrations described before . For time - response curves, cysticerci from all treatments were cultured during 20 days, with daily inspections of scolex evagination and worm length . Culture wells contained 5ml of dmem - medium and were incubated at 37c and 5% co2 . Progesterone and ru486 were prepared in a final volume of 100 l and added to 5 ml of medium in each well . From concentration - response curves of each steroid, we selected an optimal concentration for progesterone (0.25 m) and ru486 (2.5 m), to be used in the time - response curves . Culture media, as well as hormone treatments, were completely replaced every 24 hours during 20 days of culture . Scolex evagination and worm length were daily determined in all cultured cysticerci using an inverted microscope (olympus, mo21, tokyo) at 10x and 20x magnification . Worm length was considered as the millimetric addition of scolex, neck, and strobila . Total rna was isolated from t. solium cysticerci of each in vitro treatment as well as from uterus of mouse (positive expression control) using trizol reagent (invitrogen, carlsbad, calif, usa). In brief, cysticerci were disrupted in trizol reagent (1ml/0.1 g tissue) and 0.2 ml of chloroform was added per ml of trizol . The aqueous phase was recovered after 10 minute of centrifugation at 14 000 g. rna was precipitated with isopropyl alcohol, washed with 75% ethanol, and redissolved in rnase - free water . Rna concentration was determined by absorbance at 260 nm and its purity was verified after electrophoresis on 1.0% denaturing agarose gel in presence of 2.2 m formaldehyde . Total rna from all treated cysticerci was reverse - transcribed followed by specific pcr amplification of the putative tspr by using one specific pair of primers designed to amplify corresponding fragments of the dna - binding domain (one of the most conserved regions of all pr sequenced genes reported in the nih gene data bank). -actin was used as a control gene of constitutive expression, as we have previously described [21, 39]. Primer sequence of pr was: sense 5-ggaggcagaaattccagacc and antisense 5-gacaacaaccctttggtagc; for -actin, primer sequence was sense 5-gggtcagaaggattcctatg and antisense 5-ggtctcaaacatgatctggg . Pcr products were visualized in 2% agarose gel stained with ethidium bromide . In all cases, a single - band corresponding to the expected base pair size of the amplified gene fragment was detected . Tspr expression is presented as the ratio of the optical density (od) of the studied gene relative to the expression in the same preparation of the -actin gene . Tspr was directly purified from the gel using a commercial kit (dneasy tissue kit, qiagen) and sequenced . Dna sequences were determined by using a thermo sequenase cycle sequencing kit (biorad) and an automatic sequencer (model lic-4200, aloka co.). Sequence data were analyzed by using dnasis software (hitachi software engineering, tokyo, japan). In addition, nucleotide sequences were translated to their corresponding protein sequences by means of expasy molecular server . Untreated cysticerci and those treated with progesterone and/or ru486 were disrupted in tris - hcl (1ml/0.1 g tissue), proteinase k (100 units / ml), and proteases inhibitor cocktail (calbiochem). The supernatant was recovered after 15 minutes of centrifugation at 8000 g. protein concentration was obtained by absorbance at 320 nm using the bradford - lowry method . Total protein of t. solium (50 g per well) was boiled in reducing laemmli sample buffer, separated by sds - page (10% acrylamide) and electro - blotted onto nitrocellulose membranes . Membranes were blocked for 2 h with pbs 1x buffer (137 mm nacl, 2.7 mm kcl, 4.3 mm na2hpo4, 1.47 mm kh2po4) containing 0.25% of bsa . For protein immunodetection, membranes were subjected overnight to immunoblotting with 1 g / ml of anti - pr polyclonal antibody (c-20, santa cruz biotech . ), diluted 1: 1500, followed by hrp - conjugated anti - rat igg (santa cruz biothecnology; diluted 1: 5000) for 1 hour, at room temperature . Next, membranes were washed five times in 1x pbs and bands were visualized using the enhanced chemoluminicensce system, according to manufacturer's instructions (super signal ecl, pierce). Chemiluminiscent signals were captured on kodak bio - max film, and bands were quantitatively analyzed from digitized images captured from the films with the gel - doc system (biorad, richmond, calif, usa), using the bio - rad quantity one software . The content of the protein band corresponding to the tspr is presented as the ratio of the optical density of the studied protein relative to the content of -tubulin in the same preparation, used as a constitutively loading control protein . The tspr protein sequence was aligned to the pr protein sequences of other species (including mammals, birds, fish, one reptilian, and one amphibian) obtained from protein data sets in genbank . The genetic differentiation between taxa was estimated using the mean character difference with the help of paup * 4.0b10 software . It is important to point out that the number of species used for the analysis was selected based on the pr sequence found in the gene data bank (for some species there is only one sequence). The response variable used in statistical analyses was the total number of evaginated scolices that showed worm growth and motility in all wells of each hormone concentration and time of exposure, for every experiment . If anova showed significant differences among treatments, a tukey test was applied for test significance . When t. solium cysticerci were in vitro exposed to progesterone, an increase in the scolex evagination was observed in all treated parasites compared to control groups, where only 40% spontaneously evaginated (figure 1). However, this evagination - promoting effect mediated by progesterone was independent of the tested concentrations (figure 1(a)). Ru486 showed a strong anti - parasite effect, since progressively inhibited scolex evagination, reaching its maximum effect at 2.5 m, even in the presence of progesterone (figure 1). Interestingly, in the case of ru486, a concentration - dependent pattern was evident, and no significant differences were observed between ru486 plus progesterone and ru486 alone - treated groups (figure 1(a)). Concomitantly, the evagination - promoting effect of progesterone (0.25 m) was maintained through all 20 days of in vitro culture, reaching its highest response on day 14 in culture, in relation to untreated parasites (figure 1(b)). Consistently, when cysticerci were exposed to 2.5 m of ru486, scolex evagination was observed neither during the first days of culture nor at the end of the process (figure 1(b)). It is important to mention that viability of evaginated cysticerci was verified daily by means of worm motility in the culture plate, which was constant through all days of in vitro culture . Injured parasites were recognized by a progressive internal disorganization: development of opaque areas in the tegument and loss of translucence of the vesicule (data not shown). Progesterone also affected in vitro worm growth . From the lowest concentration (0.06 m) progesterone duplicated worm length on day 10th (measured as the addition of scolex, neck, and strobila of the developing parasite) with respect to the control group, and reached a plateau (figure 2(a)). The opposed effect was observed with ru486 treatment: when t. solium cysticerci were exposed to 2.5 m and higher concentrations of ru486, a total inhibition of worm development was seen, even in the presence of the highest concentration of progesterone (figure 2(a)). Dissimilar to the results of evagination percentage, the effect of ru486 on worm length was independent of the tested concentration . In addition, the t. solium worm gradually grew up in response to 0.25 m of progesterone (figure 2(b)). Differentiated worms in absence of hormones or anti - hormonal stimulus had a spontaneous development, reaching their maximum length (3.5 mm) at 12 day in culture (figure 2(b)). Once again, in the presence or absence of progesterone, no worm differentiation was observed with 2.5 m of ru486 along all the time of in vitro culture (figure 2(b)). A single band corresponding to the expected molecular weight of the amplified fragment of pr (approximately 206 bp) was detected from tspr and mouse uterus (figure 3(a)). Moreover, progesterone and ru486, separately and/or combined, increased tspr mrna content related to the control group (figure 3(b)). Tspr protein was detected by western blot as a main band of approximately 116 kda (figure 4(a)). This tspr matches to pr - b isoform that has been previously reported for rodents and human cell lines [4346]. Nevertheless, no bands corresponding to pr - a isoform were identified in any of the tested treatments in the parasite (figure 4(a)). On the contrary, by using the same antibody, a couple of bands of 87 and 116 kda were well recognized in the control tissue used (rat uterus), corresponding to pr - a and pr - b, respectively (figure 4(a)). Tspr content was increased in response to ru486 but not when parasites were exposed to both progesterone and ru486 (figure 4(b)). A preliminary sequence of tspr was obtained by pcr product sequencing and then translated to protein sequence (expasy proteomics server). A posterior analysis of this tspr showed homology of around 60% to the protein sequences previously reported for mouse, rat, rabbit, and human pr in the gendatabank . It is important to mention that the analyzed conserved motif was situated in a region of approximately 50 aa, located in the dna - binding domain of the c - terminal motif (from aa position number 110 to 160 of the mammal sequences described earlier) (data not shown). A more precise analysis of the tspr sequence involved a neighbor joint tree (njt) for studying phylogenetic relationships (figure 5). This njt was inferred from the pr dataset, producing a single tree composed by 5 groups . The first contained sequences of 6 species of mammals (including pig and human, both natural hosts of the parasite), the second group consisted of one reptilian and one bird . The fourth group was composed of 3 fish species, and the fifth group was only composed by t. solium . The phylogenetic relationships among the 5 groups received good bootstrap support ranging from 66 to 100% (see figure 5). Additionally, tspr is related to the pr family of vertebrates, more closely associated to reptilian and amphibian (figure 5). This finding suggests that tspr is definitively not a product of host cell contamination, specifically not of pig nor human cells, because of the big distance between t. solium and mammals in the njt . Here we describe the effects of progesterone and its antagonist ru486 upon scolex evagination and adult worm growth . First of all, it was clear that progesterone has a direct stimulatory effect on t. solium in vitro . In fact, progesterone exerts a marked evagination - promoting effect in a concentration - independent pattern, maintained entire time in culture, making that all parasites differentiate at 14 days of in vitro culture . T. solium cysticerci not only showed evagination but they also presented a constant motility in the culture plate, which suggests that progesterone did not affect parasite viability and, therefore, they were alive during the culture process . Moreover, progesterone also induced the growth of the worm in the evaginated parasites by 2 folds, with respect to untreated cysticerci . Interestingly, neither evagination nor worm size depend on progesterone concentrations, which are different from those tipically found in mammals [4750]. This finding highlights that sex steroids could conserve several effects on invertebrate organisms (such as helminth parasites), but their action mechanisms may differ from those reported in rodent models and human cells . We also provide elements for a possible action mechanism through which progesterone exerts its effects upon t. solium differentiation . In fact, this helminth seems to have developed a molecule able to recognize progesterone and mediate its effects, a protein we named tspr (taenia solium progesterone receptor). This putative steroid receptor is expressed in t. solium and was upregulated by progesterone and ru486 . It is important to highlight that tspr has a counterpart sequence, recently identified in the sequencing of the t. solium genome, performed by the consortium of the taenia solium genome project of the universidad nacional autnoma de mxico (data not shown). Interestingly, this tspr matches only to pr - b isoform, suggesting that t. solium presents only one form of pr - like, as it has been also described in other organisms such as rabbits [52, 53]. This result indicates that a single form of tspr is actively expressed and translated to protein, which probably has repercussions on t. solium physiology and its relationship to the host . Furthermore, this tspr showed high degree of relation to their pr counterparts in fish and amphibian, but it is distant to mammalian sequences . This finding has two important connotations: firstly, it suggests that tspr is a close relative of the steroid nuclear receptors that bind to progesterone . Secondly, this pr in t. solium definitively is not a contamination product from pig or human cells because it has a far relation to prs sequenced in these organisms . This remarkable antiparasite effect was maintained even in the presence of the highest concentration of progesterone (63.5 m). Moreover, ru486-treated cysticerci showed internal disorganization; development of opaque areas in the tegument, loss of translucence in the vesicule, and no differentiation along all the in vitro culture time . This result suggests a possible toxic effect of ru486 upon t. solium cysticerci, independent of the tspr that has not yet been reported for this progesterone - antagonist . Ru486 induced tspr expression as well as the concomitant protein production, clearly suggesting that this anti - hormone regulates pr expression in t. solium, as it has been reported in rodent models and human cell lines [54, 55]. Then, although ru486 had upregulatory effects on tspr, the same toxic response in t. solium was observed, indicating that ru486's antiparasite mechanism does not involve tspr nor progesterone - dependent pathways inhibition, but a potent cysticide effect on the parasite, worthy of further investigation . Here, we describe two different effects of progesterone and ru486 that probably have different action mechanisms directly upon t. solium development . Progesterone effects could explain, at least partially, the higher prevalence of cysticercosis in pregnant than in nonpregnant swine . Then, t. solium could respond to progesterone through a tspr with capacity to bind the hormone, and in this way regulate parasite viability and survival inside an immunocompetent host . To the opposite, the ru486 antiparasite effect was lethal for t. solium . In fact, ru486 permanently blocked the evagination of t. solium cysticerci, which has negative consequences to the adult tapeworm development and production of infective eggs . Thus, ru486 could be considered as a potential new agent in the interruption of the t. solium's life cycle, once we already know optimal doses of this antihormone as well as adverse and secondary effects in humans and other animal models . To our knowledge, this is the first report where anticysticercus effects are described for ru486 and open a promissory field in the design of new strategies that include the antihormone therapy in the control of taeniasis / cysticercosis caused by taenia solium.

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