We determined mouse skin FA composition after 2, 4 and 8 weeks of consuming a control diet or a diet supplemented with fish oil. Fish oil markedly enhanced EPA and DHA in mouse skin within 2 weeks, and this increase plateaued after 4 weeks. The FA composition in mouse skin was different from that of serum, indicating that skin has homeostatic control of FA metabolism. Mice fed the control diet designed to simulate Western human diet displayed similar skin FA composition as that of humans. The present study presents a validated method for FA quantification that is needed to investigate the mechanisms of actions of dietary treatments in both mouse and human skin.Altering the fatty acid (FA) composition in the skin by dietary fish oil could provide therapeutic benefits. Although it has been shown that fish oil supplementation enhances EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) abundance in the skin, comprehensive skin FA profiling is needed. We established a gas chromatography-mass spectrometry method, which allows precise quantification of FA profile using small (<24 mm The other limitation is that only a small size of skin specimen can be obtained from live human and experimental animals. These obstacles demand an effective extraction method and a sensitive quantification method to determine FA profiles in the skin.FA quantification procedures include lipid extraction followed by transesterification of FAs into corresponding fatty acid methyl ester (FAME) via derivatization1022Herein, we established a method of FA extraction coupled with gas chromatography-mass spectrometry (GC-MS) for quantifying skin FA profiles, which comprised eleven FAs. These eleven FAs encompassed three saturated FAs, including lauric acid LAA, 12:0), myristic acid  and palmitic acid ; and one monounsaturated FA, oleic acid , which are abundant in human and rodent skin. In addition, we examined four n-3 FAs and three n-6 FAs, which are directly relevant to the actions of fish oil supplementation:0, myrisStearic acid (18:0) is one of the most abundant FAs in biological samples. In our preliminary studies, we quantified stearic acid in the skin together with the eleven FAs shown in the present study. Surprisingly, we found that the abundance of stearic acid was as low as lauric acid (12:0), which was the least abundant FA examined. We also found that abundance of stearic acid was not affected by fish oil supplementation. Since the aim of our study was to investigate the influence of fish oil on the composition of major FAs, we did not include the stearic acid in the present study.We determined time course of changes of FA compositions in the skin of mice fed a diet supplemented with or without fish oil. The diet without supplementation was designed to simulate the Western human fat intake, which contains high fat and is low in n-3 FAs. We also quantified FA composition in human skin specimens.The FA standards were analyzed as FAMEs by GC-MS. All FA standards were well resolved. A representative chromatograph is shown in Eleven fatty acids in experimental diets were quantified using GC-MS. The FA composition (expressed as mole%) is shown in We next established a method for extracting FAs in skin specimens. Skin specimens were lysed by pulverization and a subsequent 10-minute proteinase K digestion as described in the Material and Methods section. Proteinase K digestion significantly increased FA extractability but did not affect FA composition (data not shown).Skin FA composition was quantified at 2, 4 and 8 weeks after treatment of experimental diets . In compSerum was collected from mice fed the experimental diets for 8 weeks. The results  indicateThe effects of dietary FAs on skin FA composition is secondary to its effects on FA composition in circulating blood. In order to determine the correlations in FA changes between the skin and blood, fold-changes (fish oil versus control) of FAs in the skin and serum are presented in the When comparing control diet fed mice per se, there were also important differences in the FA composition between the skin and serum. The pronounced differences included: PA and OA were major FAs in the serum, compromising 29% and 28%, respectively, of total measured FAs, whereas OA was the predominant FA in the skin, and accounted for 53% of total measured FAs. In addition, AA comprised 18% of FAs in the serum, whereas it comprised 3% of FAs in skin.In order to compare skin FA composition between mice and humans, we quantified FA composition in skin specimens obtained from individuals who did not consume fish oil Supplements . We founSkin FAs play an important role in maintaining skin homeostasis15Fish oil supplementation markedly and rapidly enhanced EPA and DHA in skin at 2 weeks. Significant reduction (−60%) of AA occurred at 4 weeks. Increasing EPA and a reduction in AA presumably will alter production of bioactive eicosanoids and thus affect skin biology3Our results have shown that fish oil supplementation reduces abundance of AA in both the skin and serum in comparison to the control diet. Reduced AA in the skin can be attributed to the AA reduction in the serum. AA levels in the serum are determined by diet and AA synthesis in the liver2424In addition to extensively studied EFAs, such as EPA, DHA and AA, we have quantified DPA and DGLA. DPA, an intermediate product between EPA and DHA, has been shown to possess functions different from EPA and DHA2728We compared serum and skin FA composition in mice fed with or without fish oil for 8 weeks. We found that abundance of many FAs between skin and serum is significantly different, indicating skin controls FA accretion, which is particularly true for AA. AA comprised approximately 3% of FAs in mouse skin but 18% of FAs in mouse serum. Similarly in humans, we found that AA comprises 2.4% of FAs in human skin  in the pThe functional impacts of dietary EPA and DHA on the skin have often been investigated in mice fed diets supplemented with fish oil versus corn oil or soybean oil34The aim of the present study was to establish an accurate and reliable method that can quantify changes of FA profiles brought by fish oil treatment using small skin specimens. Such a method is a prerequisite for future investigations into the potential beneficial effects of fish oil supplementation on prevention and treatment of certain skin disorders. These investigations would be performed on patients and/or animal models with defective skin. Healthy animals were utilized in the present study. Fish oil did not cause histological alteration in healthy mouse skin, indicating that fish oil does not possess unwanted side effects on healthy skin. The method established using healthy skin provides a reference for pertinent studies of dermatological research.To our knowledge, comprehensive FA profiles in human skin have not been reported previously, although FA compositions in the sebum have been studied36The processes of collection and extraction of FAs from the sebum and skin cells are completely different. Sebum on the skin’s surface can be readily collected using non-invasive methods, such as tape stripingThe differences between FAs in the sebum and skin cells described above dictate the research focuses on sebum and skin FAs are different. Our main focus was to determine the changes of AA, EPA, DPA, DHA brought by fish oil treatment. In addition, we examined how fish oil affects major FAs in skin cells. We focused on eleven major FA species because FAs extracted from a small skin specimen are not sufficient for detecting FAs with low abundance. In contrast, sebum which is much more accessible than skin biopsies can be collected in a sufficient amount to allow determination of FAs of trace amountIt has been shown that dietary fish oil can reduce triacylglycerol (DAG) in circulating blood in part by reducing the production of very low density lipoprotein (VLDL) in the liverFAs in cells predominantly exist as acyl chains in phospholipids. Thus, fish oil treatment-induced changes of FAs shown in this study reflect the alterations of esterified FAs bound with phospholipids. Since esterified AA, EPA and DHA in membrane phospholipids can be hydrolyzed to free FAs, which can be further converted to various lipid signaling moleculesFemale C57BL/6 mice were purchased from Charles River Laboratory . Mice were housed five per cage under a 12 hour light–dark cycle and maintained at 22 ± 1 °C. Mice were randomized to receive a control or fish oil diet, which was custom compounded by Dyets Inc. . The compositions of diets were adapted from our previous studies34Mice were fed one of these two diets ad libitum for various times. Diet was stored at −80 °C. Approximately five grams of the respective diet were dispensed daily per animal. At the end of the study, animals were sacrificed by isoflurane inhalation and decapitation. No significant difference in body weights has been observed in animals receiving either of the diet. All animal experiments were conducted in accordance with the guidelines of the National Institutes of Health. All animal protocols were approved by the University Committee on Use and Care of Animals at the University of Michigan.All procedures involving human subjects were approved by the University of Michigan Institutional Review Board and were carried out according to the principles of the Declaration of Helsinki. Written informed consent was obtained from each subject. Volunteers who did not consume fish oil supplements were recruited. The age and sex of five volunteers were: 54, female; 30, female; 44, male; 59, male; and 33, male; respectively. Skin samples (4 mm in diameter) were obtained from buttock/hip by punch biopsies and stored in −80 °C until processing as described previously40Fatty acid standards were purchased from NuChek Prep . These included: dodecanoic , tetradecanoic  hexadecanoic , octadeca-9-enoic , octadeca-9,12-dienoic acid , octadeca-9,12,15-trienoic , eicosa-8,11,14-trienoic , eicosa-5,8,11,14-tetraenoic , eicosa-5,8,11,14,17-pentaenoic , docosa-7,10,13,16,19-pentaenoic , docosa-4,7,10,13,16,19-hexaenoic . The margaric acid (17:0) was used as an internal standard.MethPrep II (methanolic m-trifluoromethylphenyltrimethylammonium hydroxide) derivatization reagent was obtained from Alltech Inc. . All other reagents were purchased from Sigma-Aldrich .Each FA standard was dissolved in hexane:dichloromethane (1:1) at concentrations ranging from 0.5–2 mg/ml to make stock solutions. Stock solutions then were diluted to make six mixtures of standards with concentrations ranging from 1.25–50 μg/ml. The concentration of the internal standard (17:0) was kept constant at 10 μg/ml in all samples. Standard mixtures were derivatized and analyzed by GC-MS as described below. The calibration curves were calculated by linear regression. The intra-day and inter-day relative standard deviations (RSD) were quantified using standards with concentration of 1.25 μg/ml, which was the lowest concentration used for making calibration curves. Injection volume was 1 μl.At various times after feeding the experimental diets, mice were anesthetized and hair was removed using an electric clipper. Skin specimens (4 mm in diameter) were obtained using a biopsy punch . Two frozen skin punch specimens were homogenized using a metal pulverizer . Pulverized samples were placed in 400 μl PBS and sonicated by Ultrasonic Cell Disruptor  followed by proteinase K (200 ng/ml) incubation at 56 °C for 10 minutes to lyse skin tissue and cells. The resultant lysate was centrifuged at 12,000 rpm to remove residue hair and tissue debris that were resistant to protease K digestion. To extract fatty acids, supernatant (200 μl) were mixed with the solvent composed of dichloromethane (800 μl), methanol (400 μl) and water (200 μl)The same method was used for FA extraction from human skin specimens. FAs extracted from one human skin biopsy (4 mm in diameter) were sufficient for quantification by GC-MS. Mouse blood samples were obtained using cardiac puncture. Fatty acids were extracted from serum (50 μl) using the method described above.Dried fatty acid extracts were derivatized by adding 70 μl of hexane:dichloromethane (1:1) and 30 μl Meth-Prep II. Samples were vortexed briefly and incubated at room temperature for 30 minutes to allow formation of FAMEs. FAs exist as esterified and free FAs in a cell. The transesterification procedure used by our study converted both esterified and free FAs into FAMEs. Thus, the method utilized in our study examined total FAs.GC-MS method was adapted from our previous publicationThe significances of differences between groups of samples were determined by ANOVA followed by post hoc analysis using Tukey’s test. Differences were considered significant when the p-value was less than 0.05. Data are expressed as mean ± standard error of mean (SEM).How to cite this article: Wang, P. et al. Gas chromatography-mass spectrometry analysis of effects of dietary fish oil on total fatty acid composition in mouse skin. Sci. Rep.7, 42641; doi: 10.1038/srep42641 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The complete coding sequences of three melon necrotic spot viruses (MNSVs) were obtained from viral metagenomics of stool samples from patients with acute gastroenteritis. These genomes were most similar to Spanish strains sequenced in 2003 and a novel MNSV watermelon strain in 2014. The human gastrointestinal (GI) tract is inhabited by a wide variety of viruses, including enteric viruses, bacteriophages, and plant and insect viruses acquired through diet. Metagenomic analyses of human enteric RNA viruses have shown that plant viruses are highly abundant in gut viromes and remain infectious to plants even after transit through the gut . While tGammacarmovirus (family Tombusviridae) that infects melons and cucumbers. It is endemic in Japan (4Next-generation sequencing (NGS) was utilized to investigate clinical stool specimens from three separate norovirus outbreaks in 2012 , 2014 , and 2016 . Because a metagenomic sequencing approach was employed , which an Japan 46, the Amn Japan 4, and Eurn Japan 4, 9 and hn Japan 4 and Asian Japan 4. The genn Japan 4.The three sequenced MNSV genomes range from 4,204 to 4,240 nucleotides (nt) and include all coding sequences, with short regions missing at the 5′ and 3′ ends. Genomes assembled from the 2012 and 2014 specimens, MNSV/USA/MD/2012 and MNSV/USA/OR/2014, respectively, are 100% identical to one another despite the stool samples being collected roughly 1.5 years apart and in different states. BLASTn comparison of the MNSV genomes to sequences available in the NCBI database showed that genomes MNSV/USA/MD/2012 and MNSV/USA/OR/2014 shared ≤69.3% nucleotide identity with other MNSV genomes, with the greatest nucleotide identity to watermelon sequence MNSV-Kochi (AB232926). Interestingly, these genomes shared a high degree of identity (95.7%) to a partial genome (2211 nt) of a novel watermelon MNSV strain detected during a 2014 outbreak in a commercial greenhouse in Spain , the 3′ KY124135 to KY124137.Genomic sequences for MNSV/USA/MD/2012, MNSV/USA/OR/2014, and MNSV/USA/2016 have been deposited in GenBank under the accession numbers


ST-segment elevation in lead aVR (STEaVR) anticipates left main and/or three-vessel disease (LM/3VD) in patients with acute coronary syndromes. STEaVR is generally reciprocal to and accompanied by ST-segment depression (STD) in the precordial leads. SYNTAX score (SS) is an angiographic scoring system and is widely used to evaluate the severity and complexity of coronary artery disease. The purpose of our study was to assess the relationship between STEaVR and SS.We performed a retrospective analysis of 117 patients with non-ST-segment elevation acute coronary syndrome (NSTEACS). Electrocardiograms at presentation were reviewed, especially for ST-segment elevation of ≥ 0.05 mV in lead aVR and STD of ≥ 0.05 mV in more than two contiguous leads. All lesions causing ≥ 50% stenosis in a coronary artery with a diameter of ≥ 1.5 mm were included in the SS calculation. SS was divided into two groups: ≥ 23: high, < 23: low.1–V4  were independent predictors of a high SS.Among the 117 patients, 80 (68.4%) had STEaVR and 37 (31.6%) did not. Patients with STEaVR had a higher SS and higher rate of LM/3VD  than those without STEaVR. On multivariate analysis, STEaVR  and STD in leads VThis study demonstrated that STEaVR was an independent predictor of a high SS. Previous studies have shown the independent predictive value of ST-segment elevation in lead aVR (STEaVR) for left main and/or three-vessel disease LM/3VD) in non-ST-segment elevation acute coronary syndrome (NSTEACS).VD in non2SYNTAX score (SS) is a recently developed angiographic grading tool to evaluate the complexity of coronary artery disease. It is widely used for determining the optimal revascularisation strategy. It is also a powerful stratification mechanism, allowing uniform, standardised assessment of the extent and severity of coronary artery disease.6 The purpose of this study was to assess the relationship between STEaVR and SS in patients with NSTEACS.A retrospective analysis was performed on all patients who had undergone coronary angiography and coronary artery bypass grafting (CABG) between January 2013 and January 2016 at the Near East University Hospital. Myocardial infarction (MI) was diagnosed according to the criteria of the European Society of Cardiology and American College of Cardiology.7Inclusion criteria for the study were troponin level greater than the 99th percentile reference value before cardiac catheterisation, chest pain or ischaemic changes on the electrocardiogram (ECG), including horizontal or down-sloping STD (≥ 0.05 mV), and absence of ST-segment elevation on the ECG. Exclusion criteria were previous CABG, bundle branch block or ventricular pace rhythm, severe aortic stenosis, hypertrophic cardiomyopathy, cardiac arrest on presentation, ventricular tachycardia, supraventricular tachycardia with heart rate greater than 160 beats per min, implantable cardioverter defibrillator shock, subsequent documented diagnosis of Takotsubo cardiomyopathy, myocarditis or pulmonary embolism.The study was approved by the local ethics committee. Patients’ demographic data and risk factors, including current smoking, diabetes mellitus (DM), hypertension (HT), hyperlipidaemia, previous MI, and previous percutaneous coronary intervention were obtained from medical records.Cardiac troponin T (cTnT) levels were measured using the electrochemiluminescence immunoassay method (Roche Cobas E601). The upper limit of normal for cTnT was 0.014 ng/ml, which represented the 99th percentile reference value. cTnT was measured serially at intervals of approximately four hours, both before and after catheterisation as clinically indicated, with the highest level noted as the peak cTnT.Two independent, blinded physicians reviewed ECGs obtained at presentation. In the event of an interpretative discrepancy, a consensus between reviewers was reached through discussion.1–V4), lateral  and inferior  region. STEaVR of ≥ 0.05 mV was recorded. Transthoracic echocardiography was performed in a standard manner during hospitalisation, and left ventricular ejection fraction (LVEF) was calculated using the biplane Simpson’s method.ST-segment shifts were measured at the J point for ST-segment elevation and depression. STD of ≥ 0.05 mV in more than two contiguous leads was recorded. A cut-off value of ≥ 0.05 mV for STD was chosen, in line with the current universal definition of MI.8 The location of STD was recorded as the anterior  was used.Coronary angiography was performed by the Judkins technique. All lesions causing ≥ 50% stenosis in a coronary artery with a diameter of ≥ 1.5 mm were included in the SS calculation. For calculation, the website software  software package. Continuous variables are expressed as mean ± standard deviation (mean ± SD) and categorical variables are expressed as percentage (%). The Kolmogorov–Smirnov test was used to evaluate the distribution of variables. Student’s t-test was used to evaluate continuous variables showing a normal distribution, and the Mann–Whitney U-test was used to evaluate variables that did not show a normal distribution. A p-value < 0.05 was considered statistically significant.To identify predictors of increased SS, the following variables were initially assessed in a univariate model: age, hypertension, diabetes, STD in anterior, lateral and inferior leads, and STEaVR. Significant variables in univariate analysis were then entered into a multivariate logistic regression analysis using backward stepwise selection.A total of 117 patients who underwent coronary angiography within five days and CABG within two weeks of presentation with the diagnosis of NSTEACS were included in the analysis. Among the 117 patients, 80 (68.4%) had a STEaVR of ≥ 0.05 mV.The patients’ characteristics are summarised and presented in Patients with STaVR had a significantly higher rate of LM/3VD and higher SS than those without STEaVR  . The resOur study showed that STEaVR and STD in the anterior leads were independently associated with a high SS and higher rates of LM/3VD in patients with NSTEACS. To our knowledge, this is the first study to evaluate STEaVR in patients with NSTEACS who underwent coronary angiography followed by CABG surgery.Previous studies have reported the independent predictive value of STEaVR for LM/3VD in NSTEACS. Barbares et al.3, V4 and V5. In addition to the predictive value for LM/3VD, STD carries a significant prognostic value in patients with NSTEMI.4–V6 has been reported to be an independent predictor for short-term mortality in patients with inferior ST-elevation myocardial infarction (STEMI).Atie et al.Janata et al.Although major STEaVR > 0.1 mV) remained an independent predictor of LM/3VD, minor (0.05–0.1 mV) and major STEaVR were not independent predictors of in-hospital or six-month death, after adjusting for other validated prognosticators in the GRACE risk model. mV remaiSS, which is used in the evaluation of angiographic severity and extent of coronary lesions, has been shown to predict mortality in addition to its role in the decision-making process of interventional procedure.Nabati et al.Several studies have shown a different ratio of STEaVR in patients with NSTEACS; Barrabés et al.In our study, patients with STEaVR had a significantly higher rate of LM/3VD than those without STEaVR . High rates of STEaVR and LM/3VD in our study are thought to have resulted from the inclusion of CABG patients into the study.1–V4, which is equivalent of STEMI. Therefore, our study group may have included patients with posterior STEMI. Fourth, we could not access death records in our country, therefore we could not evaluate mortality rates in this study.There are several limitations in this study. First, our study was a retrospective, observational study. Second, the sample size was small. Third, we did not exclude patients with posterior infarction presenting with STD in VThis study demonstrates that STEaVR and STD in the anterior leads were independent predictors of a higher SS and higher rate of LM/3VD in patients with NSTEACS.


To investigate the relationship between two distinct measures of disability: self-reported functional limitations and objectively-screened clinical impairments.We undertook an all age population-based survey of disability in two areas: North-West Cameroon (August/October 2013) and Telangana State, India (Feb/April 2014). Participants were selected for inclusion via two-stage cluster randomised sampling (probability proportionate to size cluster selection and compact segment sampling within clusters). Disability was defined as the presence of self-reported functional limitations across eight domains, or presence of moderate or greater clinical impairments. Clinical impairment screening comprised of visual acuity testing for vision impairment, pure tone audiometry for hearing impairment, musculoskeletal functioning assessment for musculoskeletal impairment, reported seizure history for epilepsy and reported symptoms of clinical depression (depression adults only). Information was collected using structured questionnaires, observations and examinations.Self-reported disability prevalence was 5.9% (95% CI 4.7–7.4) and 7.5% (5.9–9.4) in Cameroon and India respectively. The prevalence of moderate or greater clinical impairments in the same populations were 8.4% (7.5–9.4) in Cameroon and 10.5% (9.4–11.7) in India. Overall disability prevalence  was 10.5% in Cameroon and 12.2% in India, with limited overlap between the sub-populations identified using the two types of tools. 33% of participants in Cameroon identified to have a disability, and 45% in India, both reported functional limitations and screened positive to objectively-screened impairments, whilst the remainder were identified via one or other tool only. A large proportion of people with moderate or severe clinical impairments did not self-report functional difficulties despite reporting participation restrictions.Tools to assess reported functional limitation alone are insufficient to identify all persons with participation restrictions and moderate or severe clinical impairments. A self-reported functional limitation tool followed by clinical screening of all those who report any level of difficulty would identify 94% of people with disabilities in Cameroon and 95% in India, meeting the study criteria. Initially, disability was viewed as a purely medical phenomenon determined by an individual having an impairment in body functioning or structure  . Later, For example, the disease poliomyelitis  may affect leg muscle weakness (body function and structure) limiting the individual’s ability to walk (activities) and thus attend school (participation restrictions). This “dysfunctioning” can be mediated by environmental factors (e.g. assistive devices) and personal factors (e.g. family support).Disability data based on the ICF are crucial for appropriate service-planning and evidence-based advocacy. Despite this, few robust population-level disability surveys exist globally. Amongst those that do, non-comparable and non-comprehensive methodologies are used .There are three broad measurement approaches in disability. The most rapid is direct questioning, e.g. “Do you consider yourself to have a disability?” , 10. ThiA second approach is to ask people to report whether they experience activity limitations in core domains of function e.g. whether they have difficulties in seeing or hearing , 13. ThiEfforts to assess disability status in this way includes work by several international organisations. The Washington Group on Disability Statistics was established as a United Nations Statistical Commission City Group in 2001, and has developed a short and extended set of questions on functioning for adults, and a functioning module for children aged 2–17 , 16. TheA third methodology is to objectively measure clinical impairments or the presence of specific, potentially disabling health conditions, e.g. visual acuity or seizure history. This approach focuses on objectively determining whether the individual has impairments or health conditions that affect the “body function and structure” component of the ICF. Objective screening criteria on cause and severity can aid service planning and produces reliable and comparable data . HoweverComprehensive surveys of disability that assess both reported activity/functional limitations and clinical impairments or health conditions are absent from the literature. The aim of this study was to explore the interaction between measures of clinical impairment and reported functional limitation in estimating disability prevalence within the ICF, through studies in India and Cameroon.We undertook a population-based disability survey in one district each of Cameroon  and India . We screened for disability using i) self-reported functional limitation and ii) clinical assessment of impairment  and potentially disabling health conditions (epilepsy and depression).We conservatively estimated the all-age prevalence of disability  to be 4% in both India and Cameroon , 19. ThiUsing probability proportionate to size sampling, 51 clusters of 80 people were selected using the most recent census data  in each country –22.HouseEnumerated participants were invited to attend the survey screening at a central community location over two consecutive days. Those who were not able to attend the central location (e.g. due to mobility restriction) were examined in their homes at the end of the second day.All participants were screened for both i) self-reported functional limitations and ii) clinical impairments/disabling health conditions.Self-reported functional limitations were assessed using the Washington Group Extended Set on Functioning (ten core/ four non-core domains) and the draft UNICEF/Washington Group Extended Set on Child Functioning and Disability (eight core/ four non-core domains) with response options as described in Clinical impairments and two potentially disabling health conditions (epilepsy and depression) were assessed using pre-existing tools. The tool, methodology and severity threshold for each are described in Participants identified as having a vision, hearing or musculoskeletal impairment (henceforth MSI) were examined by the relevant clinician in the team to determine cause and refer to services as appropriate. In Cameroon, each team consisted of one Ear Nose and Throat nurse, one physiotherapist or orthopaedic clinical officer, one ophthalmic nurse and seven non-clinical fieldworkers. In India, each team comprised one audiologist, one physiotherapist, one vision technician or ophthalmic assistant and seven non-clinical fieldworkers.Mild MSI and mild hearing impairment were recorded in both settings. In India, mild visual impairment was also recorded.A participant was classified as having a disability in this study if they:Screened positive to any moderate/severe clinical impairment  or severe potentially disabling health condition (epilepsy or depression)–“clinical” cases.Reported significant functional limitations (“a lot of difficulty” or “cannot do”) in any core functional domains. Children aged 2–17 years: seeing, hearing, walking, self-care, understanding, being understood, learning, remembering; Adults 18+ years: seeing, hearing, walking or climbing steps, understanding, being understood, remembering, concentrating, self-care, upper body strength, fine motor dexterity.)–“self-reported” cases.All participants who were classified as having a disability (“cases”) were further interviewed about socio-demographics, poverty, livelihoods, education, health, water and sanitation, activities and participation.Participation scores were generated using a question set developed by SINTEF which assesses ability to perform a range of activities in the respondents’ current environment. DomainsIn India, all tools were translated into Telegu and back-translated by an independent translator. Any differences between the translated and original version were discussed and the translations were modified accordingly. The primary language in the Cameroon site was English, however the population in the study area also spoke both Pidgin English and Nkom. Interviewers were recruited who spoke all three languages, and the quality of their verbal translation into these languages was assessed: the interviewers asked the question in the local language and an independent person translated this back into English. Differences were noted and discussed, and a phonetic phrase-sheet of standard translations of terms  was developed to ensure consistency. Cognitive testing of the questionnaires was then carried out in each site to assess feasibility and understanding.Three teams per country were trained for ten days on disability sensitivity and project protocols. This included formal inter-observer variation testing of all clinical screens, and an observed practice of the full protocol on thirty volunteers.Data were analysed using STATA 12.0. The ‘svy’ command was used to derive prevalence estimates accounting for cluster sampling. Predictors of the agreement between the different disability measures were analysed using logistic regression. Specifically, we assessed demographic  and impairment-related  predictors of people with clinical impairments also reporting functional limitations. Mean participation scores among participants screening positive for the different disability measures were compared using the student t-test. Cross-tabulations were conducted to describe the relationship between i) any level reported functional limitation in seeing and any level visual impairment ii) any level reported limitation in hearing and any level hearing impairment iii) any level reported limitation in walking, climbing, upper body strength and fine motor skills and any level muscular-skeletal impairment, iv) any level of reported limitation and any level clinical impairment aggregated across the domains above.Ethical Approval was granted by:The London School of Hygiene & Tropical Medicine (UK)National Ethics Committee for Research in Human Health Cameroon Baptist Convention Health Board Institutional Review Board (Cameroon)Indian Institute of Public Health Hyderabad Institutional Ethics Committee (India)Government of India Health Ministry Screening Committee (India)Medicines for minor health ailments were distributed by clinical team members where appropriate and participants with unmet health needs were referred to relevant services. Written (signature or thumb print) informed consent was obtained from all participants. Caregivers provided consent for participants <18 years in India and <21 years in Cameroon in accordance with country ethics.The survey response rate was 87% in Cameroon (n = 3567) and 88% in India (n = 3574) . There wThe overall population prevalence of disability  was 10.5% (95% CI 9.0–12.2) in Cameroon and 12.2% (10.6–14.1) in India . In bothSignificant functional limitation was reported by 5.9% of participants in Cameroon and 7.5% in India (“self-reported” cases) . In bothOverall, 8.4% (95% CI 7.5–9.4) in Cameroon and 10.5% (9.4–11.7) in India screened positive for one or more impairment/health condition  . PrevaleOne third (33%) of participants in Cameroon who were identified as having a disability, and 45% in India, were both self-reported cases and clinical cases (Category B).A smaller proportion  were self-reported cases, but not clinical cases. This category included people who :Screened positive for mild clinical impairments below the severity threshold defined as “clinical cases” .Reported significant functional limitations in domains not directly screened clinically  Reported functional limitations in domains which when evaluated clinically were found not to be impaired  Almost half of participants who were identified as having a disability in each setting , were clinical cases but not self-reported cases (Category C). The vast majority of people in this group reported at least “some” functional limitations in at least one domain, but no core domains in which they had “a lot of difficulty” or were “unable” to complete the activity (case definition for “self-reported case”). Amongst those in Category C this included: 93% of adults in both countries, 69% of children in Cameroon and 53% of children in India.Expanding the case-definition for reported functional limitations to include either i)“some difficulty” in any basic domain, or ii)“some difficulty in any two domains” substantially increases the all-age prevalence of disability via self-report in Cameroon to 58.3% (95% CI 55.2–61.3) and 35.3% (32.7–38.1) respectively. In India, the prevalence of disability increased to 47.0% (44.0–50.1) and 27.6% (24.9–30.5) respectively. Expanding the case-definition to “some difficulty” in any basic domain would redefine 77.2% of Category C  in Cameroon and 79.3% in India as Category B  but would diminish Category B as a proportion to 12.7% (11.1–14.6) in Cameroon and and 20.4% (18.3–22.7) in India due to the large increase in Category A (self-report only).In India, a single question “Do you consider yourself [your child] to have a disability” was included. Only 3.8% (95% CI 2.9–4.9) of the overall sample answered affirmatively. This included 25.5% of clinical cases, and 42.3% of self-reported cases (data not shown). Amongst those who answered “yes” to the single question, 14.1% were neither a self-reported nor clinical case.In India amongst clinical cases, adults aged ≥ 66years were more likely than adults aged 34–49 years to also be a self-reported case . There was no association with age in Cameroon or sex in either setting. Clinical cases were more likely to also be self-reported cases if their impairments were severe  or profound  compared to moderate. Having multiple  or physical  impairments compared to vision impairment was also significantly associated with also being a self-reported case in both settings.The overall disability prevalence estimated in the study was 10.5% (95% CI 9.0–12.2) in Cameroon and 12.2% (10.6–14.1) in India, reflecting all participants who either self-reported significant functional limitation (5.9% [4.7–7.4] in Cameroon and 7.5% [5.9–9.4] in India) or screened positive clinically to a moderate or severe impairment or disabling health condition (8.4% [7.5–9.4] in Cameroon and 10.5% [9.4–11.7] in India). As expected, disability prevalence in both countries increased exponentially with age, irrespective of how disability was measured. Use of a single question on disability in India identified less than one third (26.5%) of those otherwise classed as having a disability.These figures are higher than other country prevalence estimates of disability. In Cameroon, the 2011 Demographic and Health Survey estimated an all-age prevalence of 5.4%, whilst a 2010 study by Cockburn et al. in North-West Cameroon estimated a regional prevalence of 6.2%, 36. HowVery few previous studies have considered the agreement between measures of self-reported functional limitations and clinical screening. One study by Kempen et al. (1996) found discrepancies between self-reported and performance-based motor and sensory limitations amongst the elderly, related to socio-demographic factors and personality traits . Our stuIn adults, participation restrictions were highest amongst those who both screened positive for clinical impairments and reported significant functional limitations (Category B), although there was no relationship by case category in children.The disparity between the sub-populations identified using a reported functional limitation tool and a battery of impairment screens has several major implications.Firstly, this result provides evidence that clinical impairment tools in isolation do not adequately capture all significant functional limitations, with 21% of those with disability in Cameroon and 14% of those in India not identified via clinical tools.Secondly, that 46% of those considered to have a disability in Cameroon and 41% in India, were identified via objective clinical measures but did not report a significant functional limitation and would therefore be missed by surveys using reported functioning tools only. Specifically, participants with moderate impairments or impairments in vision or hearing, were less likely to self-report functional limitations. From the perspective of universal health coverage and delivery of health and rehabilitative interventions, this may be inadequate. Several studies have highlighted the “hierarchy” of disability , 39. NamThirdly, our study adds to the considerable ongoing debate related to appropriate measures for population-based disability measurement within the ICF, and the theoretical basis for determining the population of interest , 41. It One solution, where resources allow, might therefore be to screen populations first with the Washington Group Questions to measure the magnitude of significant functional limitations (“a lot of difficulty” or “cannot do”) and provide a comparable estimate of disability between countries and over time. Secondly, a simple clinical screen could be administered to all participants who respond to having at least “some” difficulty in a specific domain so that all moderate/severe impairments are identified and the appropriate referrals to maximise functioning offered. This approach would identify 94% of people with disabilities in Cameroon and 95% in India, based on the present study criteria, although further work is needed to address screening for mental health disorders and cognitive impairments. The use of mid-level clinicians as opposed to specialists in this study increases the feasibility of this approach. In addition, recent innovations in mobile tools for impairment screening would decrease the burden on clinical team members, who would only need to visit participants failing the screen criteria to provide any diagnosis or referral as appropriate , 44. FinWe acknowledge that the lack of clinical screens for common mental disorders (other than depression), and for cognitive impairment, makes a comprehensive exploration of tools to measure disability difficult to achieve. This is a critical limitation in the field of disability measurement, particularly in a survey setting, and is considered a priority by leading scholars in global mental health , 45–47.It is also important to acknowledge that different methodologies impact on comparability of disability prevalence estimates and available information, even within the broader classification of self-reported functioning tools. Multiple international agencies, including several United Nations agencies, have agreed to endorse the “short-set” Washington Group Questions for upcoming data collection. However, work is ongoing to further develop and finalise other tools, including the Washington Group Extended Set used in this study and the Model Disability Survey (developed after our study was conducted) . Future The study used a robust sampling methodology to provide estimates of disability compatible with the ICF. The study measured and compared the relationship between different components of disability, adding to the evidence base in this important measurement area.However, tools and diagnostic tests for assessing mental health and cognitive impairments in this study were limited. The findings of the present study will be used to inform the Washington Group Working Group on Mental Health and further this goal.Finally, PTA and OAE testing was affected particularly in Cameroon by environmental conditions and consistently high background noise, increasing the risk of false positives.Tools to assess reported functional limitation alone are insufficient to identify all persons with moderate or severe clinical impairments that impact on participation. A self- reported tool followed by clinical screening of all those who report “some difficulty” in functioning would identify 94% of people with disabilities in Cameroon and 95% in India, based on the study criteria. This would allow data to be collected using the internationally agreed and comparable standard (self-report) whilst also ensuring adequate information on impairments and participation restrictions for service provision. However, further work is needed on field tools for assessment of common mental disorders and cognitive impairment to comprehensively assess disability within the ICF.


Little information is available about molecular markers for sarcopenia and osteoporosis in Asian populations. ACTN3 polymorphism with sarcopenia and osteoporotic status in older Korean adults. This study investigated the association of the ACTN3 R/X genotyping was determined using TaqMan probes. Older Korean 62 men and 270 women (mean age 73.7 ± 6.6 years) participated in this study. Body mass index, percent body fatness, appendicular skeletal muscle mass, and bone mineral density of the lumbar spine, femur, and total body were analyzed with dual-energy X-ray absorptiometry.p = 0.043) and osteoporosis  than RR homozygotes . The OR of XX homozygotes for having sarcopenia remained significant  after adjustments for age, gender, body fatness, and serum vitamin D. The OR of XX homozygotes for having osteoporosis was no longer significant  after adjustments for the covariates. Determination of odds ratios (ORs) and 95% confidence intervals (CIs) using binary logistic regression analyses showed that XX homozygotes were at a significantly higher risk of sarcopenia (OR = 2.056, 95%  CI = 1.024–4.127,  ACTN3 R577X genotype may influence decline in muscle and bone health phenotypes in older Korean adults. Our findings suggest that the Sedentary aging involves structural and functional changes that are conducive to mobility limitations, falls, fractures, and disability . Many ofThe global population aged 60 years or older has increased in nearly all countries in conjunction with decreased mortality and declining fertility. Due to the same reasons in conjunction with increasing average life expectancy at birth, Korea is moving toward an aged society at the fastest pace in the world. In 2010, Koreans aged 65 years and older reached 11% in the population, and the number is projected to increase to 24.3% in 2030 and reach 37.4% in 2050 [Individual differences in LBM and BMD are primarily determined by differences in genetic makeup . Heritab (ACTN3), myostatin, ciliary neurotrophic factor, and vitamin D receptor. Among the well-known candidate genes for physical fitness and/or performance is the ACTN3 gene. The human ACTN3 gene encodes α-actinin-3 protein, which is specifically expressed in fast glycolytic Type IIx fibers and 50% of fast oxidative Type IIa fibers [The molecular bases of sarcopenia and osteoporotic status have been actively explored to identify candidate genes; however, these identifications have been at best tentative and occasionally inconsistent. A few genes have been established as contributing to variation in LBM , 14 and a fibers , and is a fibers . A commoa fibers . Actn3 knockout mouse, MacArthur et al. [In previous animal studies involvingr et al. , 18 and r et al. , 20 showr et al.  in a recr et al.  and animr et al. . ACTN3 genotypes and LBM and BMD phenotypes in older adults are controversial and are mainly from Caucasian populations [ ACTN3 genotypes with sarcopenia and osteoporotic status in Asian populations. This study investigated the association between ACTN3 genotype and susceptibility to sarcopenia and osteoporotic status in older Korean adults.Unlike for physical fitness and performance, however, published data onulations –26. Few n = 62) or women (n = 270) aged 65 years or older; no self-reported difficulty walking; no difficulty performing basic activities of daily living; no reported cane use, walker, crutches, or other special equipment to get around; no active treatment for cancer in the prior three years; and no enrollment in a lifestyle intervention trial. All participants had a homogeneous geographical ancestry wherein both parents were from the Korean Peninsula. The Sungkyunkwan University Institutional Review Board, in accordance with the World Medical Association Declaration of Helsinki, reviewed and approved the study protocol. Informed consent was obtained from all participants prior to the study.A total of 332 older adults were recruited via advertisements  in local community centers in the city of Suwon located in the Northwestern Gyeonggi Province, Republic of Korea, between December 2014 and August 2016. Eligibility criteria included men (2). Fasting serum and whole-blood samples were collected for assessment of vitamin D and ACTN3 genotyping, respectively. Bone mineral density and LBM were measured as described below.Data were collected during two separate visits. During the first visit, demographic characteristics and general health status were assessed with a standardized self-administered questionnaire modified from the ACSM Guidelines for Exercise Testing and Prescription . InterviDEXA measurements were made using the Lunar DPX Pro total body pencil beam densitometer , while only exposing subjects to one scan. Before participation, the subjects were asked to disclose information about weight and pregnancy status, as both could exclude them from having the scan performed due to safety and health reasons. Subjects were also instructed to disclose any information about implanted metal that could affect the analysis of the scan. The subjects were contained within the imaging field of the DEXA scanner. Additionally, the subjects were generally healthy, and weight was stable at the time of measurement. All the subjects met the inclusion criteria. The exclusion criteria included current patient's report of any disease that affects bone metabolism, including hyperthyroidism, hyperparathyroidism, chronic renal failure, bronchial asthma, rheumatoid arthritis, and cancer, and objects which are being taken, such as glucocorticoids or thyroid hormone.One day before the DEXA scan, the subjects were advised not to participate in any strenuous physical activity, to avoid any caffeine-containing beverages, and to drink water as usual. On the morning of the exam, the subjects arrived at the laboratory in 8–10-hr overnight fasting. The subjects were asked to remove their shoes, all objects from their pockets, and any jewelry or metal before being scanned. Then the subjects lied supine with the face and torso facing up in the center of the DEXA platform with their entire body within the field of the scan as outline on the DEXA platform. The arms rested by the subject's side with palms of the hands resting on the table top and fingers slightly separated, but the thumbs were not placed under the buttocks. X rays were then delivered at two energies from a low current X-ray located beneath the DEXA machine. The two X-ray energies were then passed through the subject, posterior to anterior, and collected in the detector arm above. The duration of the scan ranged from 10 to 15 minutes depending on the thickness of the subject. The effective dosage of the DEXA scan is 0.001 mSv , which is comparable to natural background radiation for 3 hours. DEXA measurement methods and validation have been reported elsewhere .2. We used the Asian Working Group for Sarcopenia (AWGS) to determine the prevalence of sarcopenia in this study sample [2 for men and women, respectively.Appendicular skeletal muscle mass (ASM) was obtained by adding the LBM of the four limbs. The skeletal mass index (SMI) was defined as ASM/heighty sample . The AWG2 and T-score, as calculated by the device. Osteopenia (low BMD) was defined as a T-score at the femoral neck of between −1.0 standard deviation (SD) and −2.5 SD below the young adult mean (T-score < −1 and >−2.5). Osteoporosis was defined as a T-score at the femoral neck of 2.5 SD or more below the young adult mean (T-score ≤ −2.5) according to World Health Organization criteria [T-score reflects a value compared with that of persons matched for age and sex in Korean general population aged 20–40 years old .BMDs at the lumbar spine (L1–L4), femur , and total body were measured by the DEXA scan. Results of the bone densitometry were expressed in g/cmcriteria . T-scoreSerum vitamin D levels were determined using LIAISON 25(OH) Vitamin D TOTAL Assays, which are direct competitive chemiluminescence immunoassays for human serum that use the DiaSorin LIAISON automated analyzer . Intra-assay coefficients of variation for serum vitamin D were 3 to 6% and interassay coefficients were 7 to 11%. ACTN3 R577X (rs1815739) polymorphism was genotyped using TaqMan real-time PCR , according to the protocol provided by the manufacturer. PCR reactions contained 10 ng genomic DNA, 12.5 μl TaqMan Genotyping Master-mix (2x), 0.625 μl Custom TaqMan genotyping assay mix (20x), and various amount of DNase-free, RNasefree water so that the final PCR volume was brought up to 25 μl per reaction. The amplification protocol (95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min) was performed using Applied Biosystems 7500 instruments (Applied Biosystems).Blood samples for genotyping were collected in tubes containing disodium-EDTA as an anticoagulant and stored at −70°C until extraction. Total DNA was extracted from whole-blood samples using QIAmp DNA Mini Kits , following the kit instructions. Thehttps://www.broadinstitute.org/haploview/haploview) was used to check Hardy-Weinberg equilibrium (HWE) on the genotype distribution of the ACTN3 gene and establish whether the sample was from a normally distributed population. One-way analyses of variance followed by Tukey post hoc tests, if necessary, were used to test significant differences in dependent variables across the ACTN3 genotypes. With respect to BMDs of lumbar spine (L1–L4), femur , and total body, the analyses were performed for each variable in duplicate in the units of g/cm2 and T-score. The overall significance level was set to alpha 0.05. To correct for multiple testing, therefore, we used the Bonferroni method to obtain the significance level (p = 0.025) used for every test.Haploview 4.1  and recessive (XX versus any R) effects. Associations with osteoporotic status and sarcopenia were examined by binary logistic regressions, with correction for measured covariates. Results were expressed as odds ratio (OR) and 95% confidence interval (95% CI) with reference to normal group with respect to sarcopenia and osteoporotic status (osteopenia plus osteoporosis), respectively. Statistical significance was set at  ACTN3 gene (XX) was found in 20.8% of 332 study participants. The ancestral genotypes were 26.5% for homozygous (RR) and 52.7% for heterozygous (RX). The genotype frequencies for the ACTN3 gene were in HWE (p = 0.293)  in XX  or RR  or RX  genotype. No significant differences were observed in mean age (p = 0.716), height (p = 0.150), weight (p = 0.067), BMI (p = 0.456), or body fat percentage (p = 0.831) across the genotypes (The mutant genotype of the= 0.293) . There wenotypes .T-scores are calculated using different normal reference data. Our study used a manufacturer-provided reference population based on a healthy Korean-adult matched for age and sex in Korean general population aged 20–40 years old .In this study population, we found that prevalence of sarcopenia was 46.8% in men (mean aged 74.4 ± 4.6 years) and 23.3% in women (mean age of 74.4 ± 6.6 years) (data not provided). Prevalence of sarcopenia for men was similar to that of a previous study done by Kim et al. , whereasp = 0.007) and femur total (p = 0.022) and in T-scores for femur neck (p = 0.007) and total body (p = 0.017), with similar trends in femur total (p = 0.022) and total body (p = 0.030) across the genotypes  than XX homozygotes, with no such difference between RX and XX genotypes. RR homozygotes had a significantly higher BMD for femur total (p = 0.018) than XX homozygotes, with no such difference between RR and RX genotypes or between RX and XX genotypes. Similarly, post hoc tests showed that RR homozygotes and RX heterozygotes had significantly higher T-scores for femur neck  than XX homozygotes, with no such difference between RR and RX genotypes. In addition, RR homozygotes and RX heterozygotes had significantly higher T-scores for total body  than XX homozygotes, with no such difference between XX and RX genotypes. With respect to sarcopenia, there was a significant difference (p = 0.016) in ASM index across ACTN3 genotypes. Tukey post hoc test showed that RR homozygotes had a significantly higher ASM than XX homozygotes (p = 0.012), with no such difference between RR and RX genotypes or between RX and XX genotypes. No significant differences were found in serum vitamin D levels across ACTN3 genotypes.With respect to bone health, significant differences in ACTN3 genotypes were seen in relative BMDs for femur neck (enotypes . Tukey pp = 0.037) or two copies  of 577X, independent of the covariates (p = 0.043) only when unadjusted for the covariates. Under a recessive model, however, the risk of 577X to sarcopenia remained significant  even when adjusted for the covariates. Osteoporotic status was significantly associated with the presence of one  and two copies  of 577X. The ORs were not significant  when adjusted for the covariates (p = 0.019), which was no longer significant (p = 0.319) when adjusted for the covariates.Binary logistic regression analyses showed that sarcopenia was significantly associated with the presence of one , reducing a chance of detecting a true effect. In addition, they did not include sarcopenia as an outcome variable. However, the current study extends the previous finding by reporting that the ACTN3 genotype is significantly associated with variability of osteoporotic status as well as sarcopenia in a relatively large sample size of Korean older adults. In addition, the current findings show that the susceptibility of the X allele to sarcopenia and bone health may be somewhat modulated by modifiable risk factors such as body fatness and serum vitamin D levels, implying the significance of a healthy lifestyle consisting of a good nutrition and physical activity in minimizing the susceptibility of the ACTN3 genotype to the detrimental health consequences with aging.With respect to elderly Koreans, there is one previous study reporting an association between then et al.  reportedα-actinin-3 protein [ Actn3 KO mice have altered expression of several genes regulating bone mass and osteoblast/osteoclast activity, contributing to the regulation of bone mass through alterations in bone turnover. In addition, growing evidence shows that the ACTN3 gene X allele is associated with sensitivity to reduced anabolic hormone secretion [ ACTN3 gene X allele may share common pathways mediating susceptibility to sarcopenia and osteoporosis associated with aging. Yet, the biological mechanism(s) by which the ACTN3 R557X SNP contributes to loss of BMD with aging remains to be further investigated.Several explanations are possible for the protective role of the R allele against loss of LBM and BMD. The R allele may function against muscle atrophy with sedentary aging, since it is associated with higher testosterone levels  in Russi protein , which m protein  and bene protein . Using m protein  showed tecretion , anaboliecretion , 23, redecretion , and altecretion . Collect ACTN3 genotype and sarcopenia and osteoporosis could not be determined due to the cross-sectional nature of the study. Second, the ACTN3 R577X polymorphism and its association with sarcopenia may be gender-dependent. Our data were not analyzed in women and men separately because we did not have sufficient power to test the hypothesis. Therefore, whether gender modulates the association between the ACTN3 genotype and sarcopenia and osteoporosis remains to be determined. Third, lifestyle risk factors such as physical inactivity, poor fitness, and malnutrition may be significant modulators in determining the susceptibility of people with the ACTN3 genotype to sarcopenia and osteoporosis. Lastly, ACTN3 gene is one of the candidate genes contributing to variations in muscle and bone health phenotypes. The current findings need to be confirmed in a future study using several variants and a larger sample size.This study has some limitations. First, a cause-effect relationship of the ACTN3 R577X polymorphism and sarcopenia and osteoporotic status in older Korean adults and found increased risk for sarcopenia and osteoporosis with the X allele. Future studies should investigate the extent to which the ACTN3 R577X polymorphism affects alterations in LBM and BMD with aging and the mechanisms by which ACTN3 deficiency interacts with modifiable risk factors such as malnutrition and physical inactivity to influence loss of LBM and BMD in older adults.In summary, we examined the association between the


We present a case of metastatic recurrence of carcinoid tumor accompanied by carcinoid syndrome in a 68-year-old Japanese man, 12 years after resection of typical pulmonary carcinoid. Histopathologic examination from percutaneous liver biopsy revealed metastatic typical carcinoid. Clinical symptoms gradually improved after administration of octreotide LAR. Two years after starting treatment, the disease remains well controlled. This case report illustrates the possibility of antiproliferative effects of octreotide LAR on typical pulmonary carcinoid. Typical pulmonary carcinoid is known as a low-grade subtype of lung neuroendocrine tumor (NET) and is characterized by an indolent nature. Recurrence of typical pulmonary carcinoid after complete resection is very rare 3–5%) . The fre–5% . The2 and absence of necrosis were consistent with typical carcinoid. The hilar and mediastinal lymph nodes were negative for any tumor, and the surgical margins as well were free of any tumor.A 68-year-old Japanese man was referred to our hospital because of multiple hyperechoic lesions in the liver without any symptoms. The patient was a former smoker with a 45-pack-year history. Twelve years prior to this presentation, he had undergone thoracoscopic left upper lobectomy for typical pulmonary carcinoid . Histopathologic examination of the surgical specimen revealed finely granular nuclear chromatin, eosinophilic cytoplasm, and rosette formation . The imm2 and no necrosis were also demonstrated, which was compatible for metastatic typical carcinoid. During 6 months of close observation, the patient developed chronic diarrhea resulting in 12 kg of weight loss. His general condition worsened, and he was hospitalized.The patient had a history of atrial fibrillation. Physical examination revealed no abnormalities. Computed tomography (CT) with intravenous contrast of the abdomen revealed multiple low-density areas. Percutaneous liver biopsy of one of the lesions demonstrated well-differentiated tumor with positive expressions of chromogranin A, synaptophysin, and CD56 . The preα-fetoprotein were both elevated, at 24.7 ng/ml  < 16.3 ng/ml) and 56 ng/ml (NR < 10 ng/ml), respectively. Blood 5-hydroxyindole acetate (5-HIAA), urine 5-HIAA, and serotonin degradation metabolite levels were elevated at 1121 ng/ml (NR 57–230 ng/ml), 472 ng/ml (NR 1.8–6.1 ng/ml), and 86.9 mg/13 h (NR 1–6 mg/24 h), respectively, confirming the diagnosis of carcinoid syndrome. Chest CT showed no abnormalities. Abdominal contrast-enhanced CT revealed multiple tumors in the liver and spleen, as well as hepatomegaly , subcutaneous octreotide therapy was started at a dose of 100 μg/day . Blood s2 and/or the presence of necrosis [2 (10 high-power field (HPF)), nuclear pleomorphism, and irregularity with hyperchromatism and prominent nucleoli, areas of increased cellularity with disorganization, and necrosis. Travis et al. reported increased mitotic activity as the only independent predictor of prognosis in the criteria described by Arrigoni [2 (10 HPF) in viable tumor or the presence of necrosis should be the histologic criterion for atypical carcinoid, because atypical carcinoid would be underestimated if the mitotic range is set between 5 and 10 mitoses (10 high-powered fields), and the presence of necrosis is indicative of poor prognosis.Pulmonary carcinoid tumor is a rare NET that accounts for less than 2% of all lung tumors. Good prognosis can be expected following complete surgical resection of pulmonary carcinoid tumor. The recurrence rate for typical carcinoid is lower 3.6%) than that of atypical carcinoid (33.3%) . Histolo% than thnecrosis . As for necrosis  first deOnly a small number of patients with typical carcinoid experience recurrences, with a median time to recurrence of 4 years , longer than that for atypical carcinoid . As a reThe primary lesion was diagnosed as typical carcinoid. However, part of the primary lesion showed conspicuous nucleoli and high cellularity, consistent with atypical carcinoid according to the former criteria . These fWhether liver metastases originated from the lung in our case represents an important issue, because there is a possibility of the presence of another primary site. When liver metastases are demonstrated, CT of the chest and abdomen, abdominal ultrasonography, upper gastrointestinal endoscopy, and colonoscopy are performed to exclude primary tumors other than those of the lung, but no other gastroenteropancreatic neuroendocrine tumors were found in our case. Brain metastasis without pulmonary lesions is not common for primary sites other than the lung.Patients with carcinoid syndrome display a shorter overall survival  than patients without carcinoid syndrome (5.6 years) . PatientSomatostatin receptor scintigraphy (SRS) is useful for the diagnosis of neuroendocrine tumors . AccumulThe LAR formulation of octreotide inhibits tumor growth in patients with metastatic midgut neuroendocrine tumors . OctreotIn summary, this case report illustrates the possibility of antiproliferative effects of octreotide LAR on typical pulmonary carcinoid.


The spleen is a highly perfused organ involved in the immunological control and elimination of vector-borne pathogens (VBP), which could have a fundamental role in the pathogenesis of splenic disease. This study aimed to evaluate certain VBP in samples from dogs with splenic lesions.Babesia spp., Bartonella spp., Ehrlichia/Anaplasma spp., Hepatozoon canis, Leishmania infantum, hemotropic Mycoplasma spp. and Rickettsia spp. were targeted using PCR assays. Sixty EDTA-blood samples from dogs without evidence of splenic lesions were included as a control group.Seventy-seven EDTA-blood and 64 splenic tissue samples were collected from 78 dogs with splenic disease in a Mediterranean area. Babesia canis (n = 3), Babesia gibsoni (n = 2), hemotropic Mycoplasma spp. (n = 2), Rickettsia massiliae (n = 1) and “Babesia vulpes” (n = 1), in blood; and for B. canis, B. gibsoni, Ehrlichia canis and L. infantum (n = 1 each), in spleen. Two control dogs (3.3%) were positive for B. gibsoni and H. canis (n = 1 each). Benign lesions were detected in the 61.54% of infected dogs (8/13); the remaining 38.46% were diagnosed with malignancies (5/13). Infection was significantly associated to the presence of splenic disease (P = 0.013). There was no difference in the prevalence of infection between dogs with benign and malignant splenic lesions (P = 0.69); however B. canis was more prevalent in dogs with hemangiosarcoma (P = 0.006).More than half (51.56%) of the biopsies (33/64) were consistent with benign lesions and 48.43% (31/64) with malignancy, mostly hemangiosarcoma (25/31). PCR yielded positive results in 13 dogs with spleen alterations (16.67%), for B. canis infection were diagnosed with hemangiosarcoma in the present survey. As previously reported, results support that VBP diagnosis could be improved by analysis of samples from different tissues. The sample size included here warrants further investigation.VBP infection could be involved in the pathogenesis of splenic disease. The immunological role of the spleen could predispose to alterations of this organ in infected dogs. Interestingly, all dogs with The online version of this article (doi:10.1186/s13071-017-2074-z) contains supplementary material, which is available to authorized users. Vector-borne pathogens (VBP) are arthropod-transmitted agents, mainly parasites and bacteria, which cause a significant impact on the health of dogs. In addition to their veterinary importance, dogs can act as reservoirs for VBP of major zoonotic concern . The risBabesia bovis was remarkably similar to that reported in a mouse model of malarial infection indicating that dynamic changes in the distribution of splenic cells is common in the acute response to the hemoparasitic infection  and Th2 anti-inflammatory/regulatory cytokines , throughout the course of natural and experimental visceral leishmaniosis , 12. Thezed dogs . Theilerthe host .Bartonella spp. infection has been associated to the presence of granulomatous splenic lesions, abscess, and vasoproliferative tumors, such as hemangiopericytoma and hemangioendothelioma in dog and human patients, respectively [Bartonella spp. compared to Babesia spp. and hemotropic Mycoplasma spp. in dogs with fibrohistiocytic nodules (FHN) and hemangiosarcoma (HSA) in the spleen [Bartonella henselae [Ehrlichia spp. and Anaplasma spp. described infiltration of perivascular mononuclear cells into the spleen, more severe for ehrlichial infections [Leishmania infantum and Hepatozoon canis were identified in the spleen of dogs with splenitis [On the other hand, VBP infection can induce changes in the architecture of the spleen. Conditions such as babesiosis that increase the size of this organ elevate the risk of parenchymal disruption . Bartoneectively –19. Molee spleen . Mechanie spleen . Additiohenselae . Moreovefections . Leishmaplenitis ; and proplenitis .Babesia spp., Bartonella spp., Ehrlichia/Anaplasma spp., H. canis, L. infantum, hemotropic Mycoplasma spp. and Rickettsia spp., in dogs with splenic disease and to investigate potential associations of these pathogens with the development of splenic lesions.The present study aimed to evaluate the prevalence of certain VBP commonly found in the Mediterranean area, such as Fresh splenic tissue and/or ethylene diamine tetraacetic acid (EDTA)-blood samples from dogs undergoing splenectomy in two Referral Veterinary Hospitals located in a Mediterranean area, were collected and stored immediately after surgery (-20 °C), between January 2010 and June 2016. Diagnosis of splenic disease was based on a combination of clinical signs, laboratory testing, diagnostic imaging assessment, findings on surgical exploration of the abdominal cavity, and cytological and/or histopathological evaluation of the spleen. For 17 cases in which fresh splenic tissue samples were not collected, available paraffin-embedded surgical biopsy samples were retrieved from pathology archive storage facilities, using the Pathology Data Base. Although not at the same time, splenic tissue samples were processed and paraffin-embedded with the same equipment and techniques. Board-certified veterinary pathologists performed the histopathological evaluation of the splenic tissue samples.Control EDTA-blood samples were prospectively collected and stored (-20 °C) from dogs with or without clinical signs attending the hospitals for unrelated reasons during the same period, but whose spleens were deemed within normal limits on imaging assessment (ultrasound and/or computed tomography). All splenic tissue and EDTA-blood samples were identified and processed for polymerase chain reaction (PCR).Signalment was recorded and medical information was reviewed for every dog. Verbal owner consent was received for all dogs prior to their enrolment in the study. This study was carried out in accordance with the International Guiding Principles for Biomedical Research Involving Animals, issued by the Council for the International Organizations of Medical Sciences.Babesia spp., Bartonella spp., Ehrlichia/Anaplasma spp., H. canis, hemotropic Mycoplasma spp. and Rickettsia spp. were targeted using specific PCR assays (Table https://blast.ncbi.nlm.nih.gov/Blast.cgi). The eukaryotic 18S ribosomal ribonucleic acid (rRNA) (Thermo Fisher Scientific) was used as an endogenous control to ensure proper DNA extraction. Quantitative L. infantum PCR was performed as described by Francino and collaborators [DNA from splenic tissue was extracted using the Purelink® Genomic DNA Kit following manufacturer instructions . DNA was extracted from 400 μl of whole blood as previously described . Collectys Table . Real tiborators .Table 1PBabesia spp., Bartonella spp., Ehrlichia/Anaplasma spp., H. canis, L. infantum, hemotropic Mycoplasma spp. and Rickettsia spp. were established. Chi-square and Fisher’s exact test were used to compare proportions of positivity. The level of statistical significance considered was P < 0.05.Statistical analysis was performed using SPSS® 20.0 software. Values for the prevalence of A total of 138 dogs were enrolled in this study. Additional file n = 15), German Shepherd (n = 13), Boxer (n = 6), English Cocker Spaniel (n = 5), Golden Retriever (n = 4), Beagle (n = 3), French Bulldog (n = 3), Great Dane (n = 3), Labrador Retriever (n = 3), and others (n = 22). Age of dogs in this group ranged from 3 to 16 years, with a mean age of 9 years. Signalment was partially unknown for four dogs. The control group included 60 dogs, 35 female (58.3%) and 25 male (41.7%). Twenty-four different breeds were represented including: crossbreed dogs (n = 14), Yorkshire Terrier (n = 9), Golden Retriever (n = 5), Boxer (n = 3), German Shepherd (n = 3), Miniature Schnauzer (n = 3), and others (n = 23). Age of dogs in this group ranged from 1 to 16 years, with a mean age of 8.3 years. Histopathology of the spleen was performed in 64 out of 78 dogs with spleen alterations. Benign lesions were observed in the 51.56% (33/64). The most common were lymphoid nodular hyperplasia (LNH) (n = 10), congestion (n = 9), necrosis (n = 7), hematoma (n = 5), extramedullary hematopoiesis (n = 4), and hemosiderosis (n = 3). Some dogs were histopathologically diagnosed with more than one benign splenic lesion. Malignant lesions were observed in the remaining 48.43% (31/64). The most common was HSA (n = 25), followed by carcinoma (n = 2), lymphoma (n = 2), FHN (n = 1), and undifferentiated sarcoma (n = 1). Nine out of the 78 dogs with spleen alterations were diagnosed with splenic torsion, two with splenic rupture due to a blunt abdominal trauma, and three were euthanized. The owners refused further investigation in these 14 cases, so histopathological assessment of the spleen could not be performed.Seventy-eight dogs were included in the group with spleen alterations, 40 female (51.94%) and 37 male (48.05%). Twenty-seven different breeds were represented including crossbreed dogs (n = 4) or blood (n = 9) was tested. On the other hand, only 2 out of 60 control dogs (3.3%) were found PCR-positive for VBP. In the 13 positive dogs, agreement was not found for PCR outcome between splenic tissue and EDTA-blood samples obtained from the same dog. PCR results, type of biological samples analyzed in positive cases, and histopathological diagnosis of the spleen when it was available, are shown in Table χ2 = 6.223, df = 1, P < 0.05). Relationship between VBP infection and the character of the splenic lesions was clinically presumed . Indeed, all dogs infected with B. canis were diagnosed with HSA .PCR was positive for 15 out of 138 dogs included (10.9%). For dogs with spleen alterations, 13 out of 78 (16.67%) were PCR-positive for at least one VBP when either splenic tissue  as compared with dogs without evidence of splenic changes (3.3%), which supports a potential relationship between the pathogenesis of CVBD and splenic diseases.Bartonella spp. DNA was more frequently detected in FHN (29.7%), HSA (26%) and LNH (10%) compared to Babesia spp.  and hemotropic Mycoplasma spp. , while all of the paraffin-embedded splenic tissues from dogs with histologically unremarkable spleens were PCR negative [Bartonella spp., Babesia spp. and Mycoplasma spp. disagree greatly. Differences in epidemiological conditions and populations tested could explain such discrepancy. However, the absence of Bartonella spp. DNA in dogs with splenic disease, mainly vasoproliferative tumor-like lesions, was unexpected in our investigation. Molecular evidence supports that four species of Bartonella, alone or in co-infection, could potentially induce vasoproliferative lesions [Bartonella infection induces overlapping mechanisms in the development of vasoproliferative tumors, such as direct mitogenic effect on endothelial cells, inhibition of endothelial cell apoptosis and activation of hypoxia inducible factor-Iα, resulting in increased expression of the vascular endothelial growth factor [Bartonella spp. exposure and molecular detection of the genera have been reported in dogs [Bartonella spp. between the study by Varanat et al. [Bartonella spp. in Bartonella alpha proteobacteria growth medium (BAPGM) and PCR may enhance the diagnostic confirmation of bartonellosis in blood or tissue samples of naturally infected dogs [To the best of our knowledge, the literature contains only one report by Varanat and collaborators on molecular prevalence of certain VBP in dogs with specific splenic conditions . In the negative . Differenegative  are in a lesions . Bartoneh factor –28. In Sd in FHN .7%, HSA  in dogs . However in dogs  and coult et al.  and the ted dogs , 31, 32.Babesia spp. showed the highest prevalence in the present survey (6.52%) with nine out of 138 dogs infected. Similar rate of molecular detection for Babesia spp. in EDTA-blood samples (5%) was reported for dogs from the same area presenting for compatible clinical signs and/or criteria with ehrlichiosis during 2011 [Babesia spp. by ingestion and clearance of infected red blood cells [B. canis and, interestingly, all of them were diagnosed with HSA. On the other hand, a potential causative role in the development of splenitis has been attributed to the presence of mesenchymal neoplastic diseases [In contrast, ing 2011 . The splod cells . Infecteod cells . Damagedod cells . Moreoveod cells –39. It iod cells . In the diseases . As prevdiseases .Mycoplasma ovis was PCR-detected in one out of two dogs diagnosed with splenic carcinoma. Many studies have indicated that persistent exposure to Mycoplasma spp. is closely associated with oncogenic transformation in human cancers and have found that the Mycoplasma p37 protein alone is sufficient to increase the invasiveness and metastases of cancer cells [Mycoplasma spp. DNA was successfully extracted and amplified from multiple forms of human carcinoma [Mycoplasma spp. cause chromosomal changes and cell transformations in vitro through progressive chromosomal loss and translocations. However, the relationship between Mycoplasma spp. infection and cancer progression is yet to be disclosed [Hemotropic er cells –44. Mycoarcinoma . Experimisclosed .Evidence supports that infiltration and activation of inflammatory cells confer favorable conditions for the progression of cancer, inducing angiogenesis, tumor growth and invasion . To elucB. gibsoni. In contrast to reported data, breed predisposition was not observed for this infection [B. gibsoni [B. gibsoni-infected dog with suspected splenic torsion. In such case, histopathological evaluation of the spleen revealed white and red pulp hyperplasia, associated with a diffuse granulocytic infiltrate, hemorrhages and massive necrosis with vascular thrombosis, suggestive of splenic infarct [L. infantum was also reported [Remaining VBP infected dogs showed benign splenic diseases or were control dogs. Four of these were infected with nfection . Histopa gibsoni . Trotta  gibsoni  describe infarct . In addireported . In thisreported . Based oAnaplasma spp. was not detected in any dog. Furthermore, in the present study, prevalence of Ehrlichia canis, L. infantum, H. canis and Rickettsia massilliae was lower than expected given previous reports conducted in the same area [ame area , 30. Theame area , 31, 51.L. infantum caused by lymphoid organs makes them important for detecting this protozoan [Serological and PCR assays used in parallel could have maximized CVBD diagnosis . Howeverrotozoan . It has rotozoan . Moreoverotozoan . Severalrotozoan .None of the PCR-positive dogs in the present study showed VBP infection on histopathology of the spleen. As previously described, it is possible that some splenic macrophages had phagocytosed DNA fragments, but intact microorganisms were not detectable, so the amplification methods used with PCR allowed positive results with very small amounts of DNA, while this was not achievable by means of histopathology .There were some limitations in the present study, which have still to be mentioned. Due to the partially retrospective nature of this research and the fact that dogs attended at different institutions, information gathered from available medical records was sometimes incomplete. Furthermore, obtaining optimal age and sex-matched groups was not entirely possible because splenectomy is performed only when there is overt evidence of splenic alterations and splenic biopsies are infrequently obtained. In addition, it is not possible to rule out differences in the processing of the paraffin-embedded tissue samples, retrieved from the pathology archive storage facilities, which were obtained before the frozen-samples collection period. Moreover, control dogs without evidence of splenic alterations were selected based on diagnostic imaging assessment; although ultrasonography and/or radiological splenic changes have shown a good correlation with other laboratory examinations in CVBD, histopathology could have been useful to further confirm the absence of splenic disease in such cases , 61. In B. canis infection in dogs with splenic HSA suggests a potential role for this pathogen in the development of vasoproliferative tumors. Furthermore, based on the results obtained here, the presence of splenic disease should prompt further investigation for detection of VBP infection, mainly in endemic areas. Detection of VBP infection is improved by sampling of different tissues.This study provides additional evidence that CVBD could be involved in the pathogenesis of splenic alterations. The high prevalence of