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Background and aims: Persistent intestinal damage is associated with higher complication rates in celiac disease. We aimed to assess the potential modifiers of mucosal recovery. Materials and methods: We screened databases (PubMed, Embase, Cochrane Trials, and Web of Science) for papers on celiac disease. Papers discussing (1) celiac patients (2) follow-up biopsy and (3) mucosal recovery after commencement of a gluten-free diet were included. The primary outcome was to produce a comprehensive analysis of complete mucosal recovery (i.e., Marsh 0 on follow-up). We compared children’s recovery ratios to those of adults. Patients following a strict gluten-free dietary regimen were included in a subgroup. Summary point estimates, 95% confidence intervals (CIs), and 95% predictive intervals (PIs) were calculated. Heterogeneity was tested with I2-statistic. The PROSPERO registration number is CRD42016053482. Results: The overall complete mucosal recovery ratio, calculated from 37 observational studies, was 0.36 (CI: 0.28–0.44, PI: -0.12–0.84; I2: 98.4%, p<0.01). Children showed higher complete mucosal recovery ratio than adults (p<0.01): 0.65 (CI: 0.44–0.85, PI: -0.10–1.39; I2: 96.5%, p<0.01) as opposed to 0.24 (CI: 0.15–0.33, PI: -0.19–1.08; I2: 96.3%, p<0.01). In the strict dietary adherence subgroup, complete mucosal recovery ratio was 0.47 (CI: 0.24–0.70, PI: -0.47–1.41; I2: 98.8%, p<0.001). On meta-regression, diagnostic villous atrophy (Marsh 3) ratio (-8.97, p<0.01) and male ratio (+6.04, p<0.01) proved to be a significant determinant of complete mucosal recovery, unlike duration of gluten-free diet (+0.01, p = 0.62). The correlation between complete mucosal recovery ratio and age on diagnosis is of borderline significance (-0.03, p = 0.05). Conclusions: There is considerable heterogeneity across studies concerning complete mucosal recovery ratios achieved by a gluten-free diet in celiac disease. Several celiac patients fail to achieve complete mucosal recovery even if a strict dietary regimen is followed. Younger age on diagnosis, less severe initial histologic damage and male gender predispose for achieving mucosal recovery. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer survival rates in Africa are low as many cases are diagnosed late. Currently, there are no inexpensive, non-invasive and simple techniques that can be employed in poor resource settings for early case detection. In this study, we explored the possibility using blood in gastric juice as a screening tool to identify patients requiring referral for endoscopy. Methods: The study was conducted at the University Teaching Hospital endoscopy unit in Lusaka, Zambia. During esophagogastroduodenoscopy, gastric juice was aspirated and the pH determined using pH paper test strips. The presence of blood was tested using urinalysis reagent strips. Results: We enrolled 276 patients; 147(53%) were female and median age was 49 years (IQR 40–64 years). The presence of blood was associated with mucosal lesions, [OR 2.1; 95% CI 1.2–3.7, P = 0.004]. It was also associated with gastric cancer, [OR 6.7; 95% CI 2–35, P = 0.0005], even at 1:10 and 1:100 dilutions, [OR 5.4; 95% CI 2.3–13.8, P<0.0001] and [OR 9.1; 95% CI 3.5–23, P<0.0001] respectively. The sensitivity for gastric cancer detection using blood in gastric juice was 91% and the specificity was 41%. Analysis using the intensity of blood in gastric juice yielded an area under the receiver operating characteristic curve of 0.78; 95% CI 0.71–0.86 with a sensitivity of 79% and a specificity of 77%. Conclusions: The presence of blood in gastric juice is associated with gastric mucosal lesions. It has a high sensitivity but low specificity for gastric cancer detection. [ABSTRACT FROM AUTHOR]

Progression of systemic scleroderma (SSc), a chronic connective tissue disease that causes a fibrotic phenotype, is highly heterogeneous amongst patients and difficult to accurately diagnose. To meet this clinical need, we developed a novel three-layer classification model, which analyses gene expression profiles from SSc skin biopsies to diagnose SSc severity. Two SSc skin biopsy microarray datasets were obtained from Gene Expression Omnibus. The skin scores obtained from the original papers were used to further categorize the data into subgroups of low (<18) and high (≥18) severity. Data was pre-processed for normalization, background correction, centering and scaling. A two-layered cross-validation scheme was employed to objectively evaluate the performance of classification models of unobserved data. Three classification models were used: support vector machine, random forest, and naive Bayes in combination with feature selection methods to improve performance accuracy. For both input datasets, random forest classifier combined with correlation-based feature selection (CFS) method and naive Bayes combined with CFS or support vector machine based recursive feature elimination method yielded the best results. Additionally, we performed a principal component analysis to show that low and high severity groups are readily separable by gene expression signatures. Ultimately, we found that our novel classification prediction model produced global gene signatures that significantly correlated with skin scores. This study represents the first report comparing the performance of various classification prediction models for gene signatures from SSc patients, using current clinical diagnostic factors. In summary, our three-classification model system is a powerful tool for elucidating gene signatures from SSc skin biopsies and can also be used to develop a prognostic gene signature for SSc and other fibrotic disorders. [ABSTRACT FROM AUTHOR]

This paper addresses the problem of quantifying biomarkers in multi-stained tissues based on the color and spatial information of microscopy images of the tissue. A deep learning-based method that can automatically localize and quantify the regions expressing biomarker(s) in any selected area on a whole slide image is proposed. The deep learning network, which we refer to as Whole Image (WI)-Net, is a fully convolutional network whose input is the true RGB color image of a tissue and output is a map showing the locations of each biomarker. The WI-Net relies on a different network, Nuclei (N)-Net, which is a convolutional neural network that classifies each nucleus separately according to the biomarker(s) it expresses. In this study, images of immunohistochemistry (IHC)-stained slides were collected and used. Images of nuclei (4679 RGB images) were manually labeled based on the expressing biomarkers in each nucleus (as p16 positive, Ki-67 positive, p16 and Ki-67 positive, p16 and Ki-67 negative). The labeled nuclei images were used to train the N-Net (obtaining an accuracy of 92% in a test set). The trained N-Net was then extended to WI-Net that generated a map of all biomarkers in any selected sub-image of the whole slide image acquired by the scanner (instead of classifying every nucleus image). The results of our method compare well with the manual labeling by humans (average F-score of 0.96). In addition, we carried a layer-based immunohistochemical analysis of cervical epithelium, and showed that our method can be used by pathologists to differentiate between different grades of cervical intraepithelial neoplasia by quantitatively assessing the percentage of proliferating cells in the different layers of HPV positive lesions. [ABSTRACT FROM AUTHOR]

Introduction: There is no consensus on how to investigate men with negative transrectal ultrasound guided prostate biopsy (TRUS-B) but ongoing suspicion of cancer. Three strategies used are transperineal (TP-B), transrectal saturation (TS-B) and MRI-guided biopsy (MRI-B). We compared cancer yields of these strategies. Methods: Papers were identified by search of Pubmed, Embase and Ovid Medline. Included studies investigated biopsy diagnostic yield in men with at least one negative TRUS-B and ongoing suspicion of prostate cancer. Data including age, PSA, number of previous biopsy episodes, number of cores at re-biopsy, cancer yield, and Gleason score of detected cancers were extracted. Meta-regression analyses were used to analyse the data. Results: Forty-six studies were included; 12 of TS-B, 14 of TP-B, and 20 of MRI-B, representing 4,657 patients. Mean patient age, PSA and number of previous biopsy episodes were similar between the strategies. The mean number of biopsy cores obtained by TP-B and TS-B were greater than MRI-B. Cancer detection rates were 30·0%, 36·8%, and 37·6% for TS-B, TP-B, and MRI-B respectively. Meta-regression analysis showed that MRI-B had significantly higher cancer detection than TS-B. There were no significant differences however between MRI-B and TP-B, or TP-B and TS-B. In a sensitivity analysis incorporating number of previous biopsy episodes (36 studies) the difference between MRI-B and TP-B was not maintained resulting in no significant difference in cancer detection between the groups. There were no significant differences in median Gleason scores detected comparing the three strategies. Conclusions: In the re-biopsy setting, it is unclear which strategy offers the highest cancer detection rate. MRI-B may potentially detect more prostate cancers than other modalities and can achieve this with fewer biopsy cores. However, well–designed prospective studies with standardised outcome measures are needed to accurately compare modalities and define an optimum re-biopsy approach. [ABSTRACT FROM AUTHOR]

Introduction: The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature. Methods: Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed. Results: A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal’s temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model. Conclusion: We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials. [ABSTRACT FROM AUTHOR]

Crohn’s Disease (CD) results from inappropriate response toward commensal flora. Earlier studies described CD as a Th1 mediated disease. Current models view both phenotypes as a continuum of various permutations between Th1, Th2 and Th17 pathways compounded by a range of Treg disfunctions. In the present paper, we develop a mathematical model, by a system of differential equations, which describe the dynamic relations among these T cells and their cytokines. The model identities four groups of CD patients according to up/down regulation of Th1 and Th2. The model simulations show that immunosuppression by TNF-α blockage benefits the group with Th1High/Th2Low while, by contrast, the group with Th1Low/Th2High will benefit from immune activation. [ABSTRACT FROM AUTHOR]

Diagnosis of liver cirrhosis is essential in the management of chronic hepatitis C virus (HCV) infection. Liver biopsy is invasive and thus entails a risk of complications as well as a potential risk of sampling error. Therefore, non-invasive diagnostic tools are preferential. The aim of the present study was to create a model for accurate prediction of liver cirrhosis based on patient characteristics and biomarkers of liver fibrosis, including a panel of non-cholesterol sterols reflecting cholesterol synthesis and absorption and secretion. We evaluated variables with potential predictive significance for liver fibrosis in 278 patients originally included in a multicenter phase III treatment trial for chronic HCV infection. A stepwise multivariate logistic model selection was performed with liver cirrhosis, defined as Ishak fibrosis stage 5–6, as the outcome variable. A new index, referred to as Nordic Liver Index (NoLI) in the paper, was based on the model: Log-odds (predicting cirrhosis) = −12.17+ (age×0.11) + (BMI (kg/m2)×0.23) + (D7-lathosterol (μg/100 mg cholesterol)×(−0.013)) + (Platelet count (x109/L)×(−0.018)) + (Prothrombin-INR×3.69). The area under the ROC curve (AUROC) for prediction of cirrhosis was 0.91 (95% CI 0.86–0.96). The index was validated in a separate cohort of 83 patients and the AUROC for this cohort was similar (0.90; 95% CI: 0.82–0.98). In conclusion, the new index may complement other methods in diagnosing cirrhosis in patients with chronic HCV infection. [ABSTRACT FROM AUTHOR]

Background: The confirmatory diagnosis of Osteogenesis Imperfecta (OI) requires invasive, commonly bone biopsy, time consuming and destructive methods. This paper proposes an alternative method using a combination of two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) microscopies from easily obtained human skin biopsies. We show that this method can distinguish subtypes of human OI. Methodology/Principal Findings: Different aspects of collagen microstructure of skin fresh biopsies and standard H&E-stained sections of normal and OI patients (mild and severe forms) were distinguished by TPEF and SHG images. Moreover, important differences between subtypes of OI were identified using different methods of quantification such as collagen density, ratio between collagen and elastic tissue, and gray-level co-occurrence matrix (GLCM) image-pattern analysis. Collagen density was lower in OI dermis, while the SHG/autofluorescence index of the dermis was significantly higher in OI as compared to that of the normal skin. We also showed that the energy value of GLCM texture analysis is useful to discriminate mild from severe OI and from normal skin. Conclusions/Significance: This work demonstrated that nonlinear microscopy techniques in combination with image-analysis approaches represent a powerful tool to investigate the collagen organization in skin dermis in patients with OI and has the potential to distinguish the different types of OI. The procedure outlined in this paper requires a skin biopsy, which is almost painless as compared to the bone biopsy commonly used in conventional methods. The data presented here complement existing clinical diagnostic techniques and can be used as a diagnostic procedure to confirm the disease, evaluate its severity and treatment efficacy. [ABSTRACT FROM AUTHOR]

In this study we use artificial intelligence (AI) to categorise endometrial biopsy whole slide images (WSI) from digital pathology as either "malignant", "other or benign" or "insufficient". An endometrial biopsy is a key step in diagnosis of endometrial cancer, biopsies are viewed and diagnosed by pathologists. Pathology is increasingly digitised, with slides viewed as images on screens rather than through the lens of a microscope. The availability of these images is driving automation via the application of AI. A model that classifies slides in the manner proposed would allow prioritisation of these slides for pathologist review and hence reduce time to diagnosis for patients with cancer. Previous studies using AI on endometrial biopsies have examined slightly different tasks, for example using images alongside genomic data to differentiate between cancer subtypes. We took 2909 slides with "malignant" and "other or benign" areas annotated by pathologists. A fully supervised convolutional neural network (CNN) model was trained to calculate the probability of a patch from the slide being "malignant" or "other or benign". Heatmaps of all the patches on each slide were then produced to show malignant areas. These heatmaps were used to train a slide classification model to give the final slide categorisation as either "malignant", "other or benign" or "insufficient". The final model was able to accurately classify 90% of all slides correctly and 97% of slides in the malignant class; this accuracy is good enough to allow prioritisation of pathologists' workload. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the impact of glycemic control of diabetes mellitus (DM) on prostate cancer detection in a biopsy population. Patients and Methods: We retrospectively reviewed the records of 1,368 men who underwent prostate biopsy at our institution. We divided our biopsy population into three groups according to their history of DM, and their Hemoglobin A1c (HbA1c) level: a no-DM (DM−) group; a good glycemic control (DM+GC) group (HbA1c <6.5%); and a poor glycemic control (DM+PC) group (HbA1c ≥6.5%). For sub-analyses, the DM+PC group was divided into a moderately poor glycemic control (DM+mPC) group (6.5≤ HbA1c <7.5%) and a severely poor glycemic control (DM+sPC) group (HbA1c ≥7.5%). Results: Among 1,368 men, 338 (24.7%) had a history of DM, and 393 (28.7%) had a positive biopsy. There was a significant difference in prostatic specific antigen density (PSAD) (P = 0.037) and the frequency of abnormal DRE findings (P = 0.031) among three groups. The occurrence rate of overall prostate cancer (P<0.001) and high-grade prostate cancer (P = 0.016) also presented with a significantly difference. In the multivariate analysis, the DM+PC group was significantly associated with a higher rate of overall prostate cancer detection in biopsy subjects compared to the DM− group (OR = 2.313, P = 0.001) but the DM+PC group was not associated with a higher rate of high-grade (Gleason score ≥7) diseases detected during the biopsy (OR = 1.297, P = 0.376). However, in subgroup analysis, DM+sPC group was significantly related to a higher risk of high-grade diseases compared to the DM− group (OR = 2.446, P = 0.048). Conclusions: Poor glycemic control of DM was associated with a higher risk of prostate cancer detection, including high-grade disease, in the biopsy population. [ABSTRACT FROM AUTHOR]

We tested the underlying assumption that citation counts are reliable predictors of future success, analyzing complete citation data on the careers of ~ 150,000 scientists. Our results show that i) among all citation indicators, the annual citations at the time of prediction is the best predictor of future citations, ii) future citations of a scientist's published papers can be predicted accurately (r²=0:80 for a 1-year prediction, Pv0:001) but iii) future citations of future work are hardly predictable. [ABSTRACT FROM AUTHOR]

Background and aim: Trials of treatments for non-alcoholic steatohepatitis require endpoint assessment with liver biopsies. Previous large-scale trials have calculated their sample size expecting high retention but on average did not achieve this. We aimed to quantify the proportion of participants with a valid follow-up biopsy. Methods: We conducted a systematic review of MEDLINE and Embase until May 2020 and included randomized clinical trials of any intervention in non-alcoholic steatohepatitis with at least 1-year follow-up. We were guided by Cochrane methods to run a meta-analysis with generalized linear mixed models with random effects. Results: Forty-one trials (n = 6,695) were included. The proportion of participants with a valid follow-up biopsy was 82% (95%CI: 78%-86%, I2 = 92%). There was no evidence of a difference by location, trial length, or by allocated treatment group. Reasons for missing follow-up biopsies were, in ranked order, related to participants (95 per 1,000 participants (95%CI: 69–129, I2 = 92%), medical factors, protocol, trial conduct, and other/unclear. Biopsy-related serious adverse events occurred in 16 per 1,000 participants (95% CI: 8–33, I2 = 54%). No biopsy-related deaths were reported. Conclusions: The proportion of participants with a valid follow-up biopsy in therapeutic trials in non-alcoholic steatohepatitis is on average 82%, with around 1 in 10 participants declining a follow-up biopsy. These findings can inform adequately-powered trials. [ABSTRACT FROM AUTHOR]

Chronic rhinosinusitis (CRS) is characterized according to the presence or absence of nasal polyps (NPs) and displays nasal microbiota dysbiosis. However, optimal sampling methods of the nasal microbiome in CRS have not been identified. We aimed to assess the microbial composition in patients with CRS, comparing different sampling methods (swab and tissue biopsy), tissue types (uncinate tissue and NP), and disease subtypes. Samples were obtained by swabbing the middle meatus and taking a biopsy of uncinate tissue (UT) in patients with CRS with (CRSwNP, N = 8) or without NP (CRSsNP, N = 6) and controls (N = 8). NPs were also harvested in CRSwNP. DNAs were extracted from fifty-two samples and analyzed by 16S rRNA gene amplicon sequencing. As a result, a great interpersonal variance was observed in nasal swabs, while UT samples presented distinct microbiome with low inter-personal differences. Moreover, the UT microbiomes were further differentiated into three clusters which are associated with disease status (control, CRSsNP, and CRSwNP). Compared to UT, NP revealed a unique microbiome profile with significantly less bacterial diversity. Prevotella was the genus whose abundance was negatively correlated with disease severity in NP. In conclusion, tissue samples are better specimens than nasal swabs for assessing the microbiomes of CRS patients. Several bacteria in UT and NP tissues revealed an association with clinical severity of CRSwNP. [ABSTRACT FROM AUTHOR]

Background: Personalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection. Methods: The Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days. Results: A total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence. Conclusions: Highly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management. [ABSTRACT FROM AUTHOR]

Objective: To identify risk factors associated with unfavorable outcomes in children with IgA vasculitis with nephritis (Henoch-Schőnlein purpura nephritis)(IgA-VN) Methods: PubMed, Embase, and Web of Science databases were searched for studies, published in English through February 2019. The data were extracted to perform pooled analysis, heterogeneity testing, subgroup analysis, sensitivity analysis, and publication bias analysis. Results: This meta-analysis showed that, older age at onset (WMD 1.77, 95% CI 0.35–3.18, p = 0.014), lower glomerular filtration rate (GFR; WMD -23.93, 95% CI -33.78- -14.09, p<0.0001), initial renal manifestations with nephrotic syndrome (OR 1.74, 95% CI 1.12–2.70, p = 0.013), with nephritic-nephrotic syndrome (OR 4.55, 95% CI 2.89–7.15, p<0.0001) and renal biopsy with crescentic nephritis (International Study of Kidney Disease in Children [ISKDC] grades III-V) (OR 3.85, 95% CI 2.37–6.28, p<0.0001) were significant risk factors associated with poor outcomes in IgA-VN, whereas initial clinical features with hematuria (OR 0.33, 95% CI 0.16–0.69, p = 0.003) and mild proteinuria±hematuria (OR 0.46, 95% CI 0.28–0.75, p<0.0001) were associated with progression to good outcomes. By contrast, gender, hypertension and initial renal manifestations of acute nephritic syndrome were not significantly associated with poor outcomes in IgA-VN. Conclusion: This meta-analysis showed that older age at onset, lower GFR, initial renal features of nephrotic syndrome and nephritic-nephrotic syndrome and renal biopsy with crescentic nephritis (ISKDC grades III-V) were predictive of poor prognosis in children with IgA-VN. [ABSTRACT FROM AUTHOR]

The bovine dry period is a non-lactating period between consecutive lactations characterized by mammary gland involution and redevelopment phases to replace senescent mammary epithelial cells with active cells primed for the next lactation. Dairy cows exposed to heat stress during the dry period experience milk yield reductions between 3–7.5 kg/d in the next lactation, partially attributed to processes associated with mammary cell growth and turnover during the dry period. However, the carry-over impact of dry period heat stress on mammary morphology during lactation has yet to be determined. In the current study, we hypothesized that exposure to heat stress during the dry period would alter alveolar microstructure and cellular turnover (i.e. proliferation and apoptosis) during lactation. Cows were either subjected to heat stress (HT, access to shade; n = 12) or cooling (CL, access to shade, fans, and soakers; n = 12) for a 46 d dry period. Upon calving, all cows were treated similarly with access to cooling for their entire lactation. Six cows per treatment were randomly selected for mammary gland biopsies at 14, 42, and 84 days in milk. Tissues were sectioned and stained for histological analysis. During lactation, HT cows produced 4 kg less colostrum and 3.7 kg less milk compared with CL cows. Lactating mammary gland microstructure was impacted after exposure to dry period heat stress; HT cows had fewer alveoli and a higher proportion of connective tissue in the mammary gland relative to CL cows, however alveolar area was similar between treatments. Rates of mammary epithelial cell proliferation and apoptosis were similar between treatment groups. This suggests that heat stress exposure during the dry period leads to reductions in milk yield that could be caused, in part, by a reduction in alveoli number in the lactating mammary gland but not to dynamic alterations in cellular turnover once lactation is established. [ABSTRACT FROM AUTHOR]

Globally, HIV/AIDS is a leading cause of morbidity worldwide among reproductive-aged cisgender women, highlighting the importance of understanding effects of contraceptives on HIV-1 risk. Some observational studies suggest there may be an increased risk of HIV-1 acquisition among women using the long-acting injectable progestin contraceptive, depo-medroxyprogesterone acetate. The potential mechanism of this susceptibility is unclear. There are few data on the role of the upper female reproductive tract in HIV-1 transmission, and the mechanisms of HIV-1 infection are likely to differ in the upper compared to the lower reproductive tract due to differences in tissue composition and variable effects of sex steroids on mucosal immune cell distribution and activity. In this study, we measured the susceptibility of mucosal immune cells from the upper female reproductive tract to HIV-1 entry using the virion-based HIV-1 fusion assay in samples from healthy female volunteers. We studied 37 infectious molecular clones for their ability to fuse to cells from endometrial biopsies in three participants and found that subtype (B or C) and origin of the virus (transmitted founder or chronic control) had little influence on HIV-1 fusion susceptibility. We studied the effect of contraceptives on HIV-1 susceptibility of immune cells from the cervix, endometrium and peripheral blood by comparing fusion susceptibility in four groups: users of the copper intrauterine device (IUD), levonorgestrel-containing oral contraceptive, levonorgestrel-containing IUD and unexposed controls (n = 58 participants). None of the contraceptives was associated with higher rates of HIV-1 entry into female reproductive tract cells compared to control samples from the mid-luteal phase. [ABSTRACT FROM AUTHOR]

Background: Whether differences in outcome between primary (pIgAN) and secondary IgA nephropathy (sIgAN) exist is uncertain. Methods: We conducted a retrospective, observational study that included all histologically diagnosed IgAN patients between 2010–2017 (N = 306), 248 with pIgAN and 58 with sIgAN. To obtain samples with similar risk of progression, sIgAN patients were grouped as liver disease and autoimmune/viral disease and propensity score matched to corresponding pIgAN samples. Univariate (Kaplan Meier) and multivariate time-dependent (Cox modelling) analyses were performed to identify predictors of the composite end-point (doubling of serum creatinine, end-stage kidney disease or death). Results: Of the whole cohort, 20% had sIgAN (6% alcoholic cirrhosis, 6% autoimmune disease and 8% viral infections). sIgAN patients were older, had more comorbidities, lower proteinuria and higher haematuria, but similar distribution in MESTC lesions and eGFR as those with pIgAN. They reached the end-point in similar proportions with those with pIgAN (43 vs. 30%; p = 0.09) but their mortality was higher (19 vs. 3%; p<0.0001). Both in unmatched (HR 0.80, 95%CI 0.42–1.52; p = 0.5) and matched samples (log-rank test: liver disease-IgAN vs. pIgAN, p = 0.1; autoimmune/viral-IgAN vs. pIgAN, p = 0.3), sIgAN was not predictive for end-point. In analyses restricted only to sIgAN, those with viral infections (HR, 10.98; 95% CI, 1.12–107.41; p = 0.03) and lower eGFR (HR, 0.94; 95%CI, 0.89–0.98; p = 0.007) had a worse prognosis. Immunosuppression did not influence outcome. Conclusions: The differences in MESTC score and outcome between pIgAN and sIgAN seems to be minimal, suggesting that “associated” describes better than “secondary” the relationship among the two. Immunosuppression did not to influence outcome of sIgAN. [ABSTRACT FROM AUTHOR]

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that can lead to cirrhosis, liver transplant, and even hepatocellular carcinoma. While liver biopsy remains the reference standard for disease diagnosis, analytical and clinical development of non-invasive soluble biomarkers of NASH are of great importance to advance the field. To this end, we performed analytical and clinical validation on a series of pro-inflammatory cytokines and chemokines implicated hepatic inflammation; IL-6, CRP, TNFα, MCP-1, MIP-1β, eotaxin, VCAM-1. Biomarker assays were validated for accuracy and precision. Clinical performance was evaluated in a random sample of 52 patients with biopsy-proven NAFLD/NASH. Patients were categorized into three groups according to their fibrosis stage; advanced (F3-F4), mild (F1-2) and no (F0) fibrosis. Serum IL-6 was increased in patients with advanced fibrosis (2.71 pg/mL; 1.26 pg/mL; 1.39 pg/mL p<0.01) compared to patients with mild or no fibrosis respectively. While, there was no significant difference noted in CRP, TNFα, MCP-1, MIP-1β, eotaxin among the three groups, VCAM-1 levels were increased by 55% (p<0.01) and 40% (p<0.05) in the advanced cohort compared to the mild and no fibrosis groups respectively. VCAM-1 also displayed good clinical performance as a biomarker of advanced fibrosis with an area under the receiver operating curve of 0.87. The VCAM-1 assay demonstrated robust accuracy and precision, and VCAM-1 outperformed IL-6, CRP, TNFα, and the chemokines MCP-1, MIP-1β, and eotaxin as a biomarker of advanced fibrosis in NASH. Addition of biomarkers such as IL-6 and VCAM-1 to panels may yield increased sensitivity and specificity for staging of NASH. [ABSTRACT FROM AUTHOR]

In this non-randomized extension study of a randomized controlled trial we converted 87 stable long-term kidney transplant recipients (KTR) from either ciclosporin (CSA, n = 28) or tacrolimus (TAC, n = 59) to TAC modified release (TAC MR4) to study the characteristics of TAC trough levels after conversion with the primary endpoint graft function after 12 months. TAC MR4 consumption was calculated by level-to-dose ([ng/mL]/[mg/d]) and concentration-to-dose ([mg/kg])/d) ratios. Influences of ABCB1 single nucleotide polymorphisms (2677G>T/A, 1236C>T, 3435C>T) on TAC metabolism were studied. Graft function of KTR converted from CSA to TAC MR4 significantly declined over 12 months, and remained unchanged after conversion from TAC to TAC MR4. Conversion from CSA to TAC MR4 resulted in supra therapeutic- and conversion from TAC to TAC MR4 in low trough levels. We could not find associations of ABCB1 genotypes and TAC MR4 trough levels. Adverse events and errors with TAC/TAC MR4 intake were common. In stable long-term KTR conversion from TAC to TAC MR4 is feasible. For conversion from CSA we suggest a rate of 1:40 for a rough estimation of TAC MR4 target doses. Trial registration: PEP Study: Ethics committee N° 393/2004, EudraCT 2004-004209-98. PEP-X Study: Ethics committee amendment application N° 154/01/2008. ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

Background: In recent months, multiple publications have demonstrated the use of convolutional neural networks (CNN) to classify images of skin cancer as precisely as dermatologists. However, these CNNs failed to outperform the International Symposium on Biomedical Imaging (ISBI) 2016 challenge which ranked the average precision for classification of dermoscopic melanoma images. Accordingly, the technical progress represented by these studies is limited. In addition, the available reports are impossible to reproduce, due to incomplete descriptions of training procedures and the use of proprietary image databases or non-disclosure of used images. These factors prevent the comparison of various CNN classifiers in equal terms. Objective: To demonstrate the training of an image-classifier CNN that outperforms the winner of the ISBI 2016 CNNs challenge by using open source images exclusively. Methods: A detailed description of the training procedure is reported while the used images and test sets are disclosed fully, to insure the reproducibility of our work. Results: Our CNN classifier outperforms all recent attempts to classify the original ISBI 2016 challenge test data (full set of 379 test images), with an average precision of 0.709 (vs. 0.637 of the ISBI winner) and with an area under the receiver operating curve of 0.85. Conclusion: This work illustrates the potential for improving skin cancer classification with enhanced training procedures for CNNs, while avoiding the use of costly equipment or proprietary image data. [ABSTRACT FROM AUTHOR]

Background: Knowledge about the relationship between PSA, age and ISUP grade group (ISUP) 1 prostate cancer can improve clinical and biological understanding of prostate cancer. We aimed to investigate the associations between PSA and age and the risk of ISUP 1 and ISUP ≥ 2 prostate cancer, respectively. Methods: We included 6 083 men aged 50–69 biopsied with a total of 72 996 individual biopsy cores from the prospective and population based Stockholm3 diagnostic study. We computed the risk of ISUP 1 and ISUP ≥ 2 prostate cancer and their respective associations with PSA and age. Since lower Gleason grades often are masked by higher grades in the overall Gleason score, we compared associations both for overall Gleason score and for Gleason on individual biopsy cores. Results: ISUP 1 prostate cancer was not significantly associated with PSA at diagnosis: odds ratios ranged from 0.82 (95%CI: 0.68–1.00) for PSA 3–4 ng/mL, 0.96 (95%CI: 0.79–1.16) for PSA 4–6 ng/mL, 0.95 (95%CI: 0.75–1.21) for PSA 6–10 ng/mL, and 0.92 (95%CI: 0.58–1.45) for PSA 10–15 ng/mL compared with PSA 2–3 ng/mL. Age was not significantly associated with risk of ISUP 1 cancer. This contrasts to the strong relationship between ISUP ≥ 2 prostate cancer and its respective associations with PSA and age. Conclusions: We find no significant association between the risk of ISUP 1 prostate cancer and PSA and age at diagnosis indicating that PSA contribution from ISUP 1 prostate cancer is closer to that of benign prostate tissue than to that of ISUP ≥ 2 prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Recurrent Respiratory Papillomatosis (RRP) is a rare disease characterized by the growth of papillomas in the airway and especially the larynx. The clinical course is highly variable among individuals and there is poor understanding of the factors that drive an aggressive vs an indolent course. Methods: A convenience cohort of 339 affected subjects with papillomas positive for only HPV6 or HPV11 and clinical course data available for 1 year or more, from a large multicenter international study were included. Exploratory data analysis was conducted followed by inferential analyses with frequentist and Bayesian statistics. Results: We examined 339 subjects: 82% were diagnosed prior to the age of 18 years, 65% were infected with HPV6, and 69% had an aggressive clinical course. When comparing age at diagnosis with clinical course, the probability of aggressiveness is high for children under five years of age then drops rapidly. For patients diagnosed after the age of 10 years, an indolent course is more common. After accounting for confounding between HPV11 and young age, HPV type was minimally associated with aggressiveness. Fast and Frugal Trees (FFTs) were utilized to determine which algorithms yield the highest accuracy to classify patients as having an indolent or aggressive clinical course and consistently created a branch for diagnostic age at ~5 years old. There was no reliable strong association between clinical course and socioeconomic or parental factors. Conclusion: In the largest cohort of its type, we have identified a critical age at diagnosis which demarcates a more aggressive from less aggressive clinical course. [ABSTRACT FROM AUTHOR]

Introduction: Diffuse parenchymal lung diseases (DPLD) constitute a heterogeneous group of disorders, sometimes requiring surgical lung biopsies (SLB) to obtain a definite diagnosis. Transbronchial cryobiopsy (TBCB) is a new promising interventional bronchoscopic method of obtaining lung tissue that is gaining ground against SLB. Methods: Fifty consecutive patients with indeterminate DPLD (definite/possible UIP excluded), after expert panel review referral, were retrospectively analyzed from January 2016 to August 2018. Patients underwent TBCB under deep sedation with endotracheal intubation and spontaneous breathing at a single, tertiary-care, reference hospital. Results: A total of 110 TBCBs (2.7 per patient, range 1 to 4) were performed. Frequent complications included mild pneumothorax in 5 patients (10%), requiring only oxygen supplementation, and bleeding in 31 patients (62%) that was mild in 19 patients and moderate in 12 patients. No serious bleeding was observed. There was zero mortality and no serious adverse events. Adequate samples for diagnostic purposes were obtained in 46 patients (92%) and pathologic histologic diagnosis was reached in 40 patients (80%). The most frequent histopathological patterns were organizing pneumonia (OP) (25%) and non-specific interstitial pneumonia (NSIP) (15%). After an expert panel review of all cases a final diagnosis was achieved in 38 patients, corresponding to a diagnostic yield of 76% for TBCB. Conclusion: Our single center cohort demonstrates that establishing TBCBs as a new technique is safe and feasible after proper training in specialized centers, resulting in low complication rates and adequate diagnostic yields. [ABSTRACT FROM AUTHOR]

Background: The recently established association between higher levels of DNA-incorporated thioguanine nucleotides and lower relapse risk in childhood acute lymphoblastic leukaemia (ALL) calls for reassessment of prolonged 6-thioguanine (6TG) treatment, while avoiding the risk of hepatotoxicity. Objectives: To assess the incidence of hepatotoxicity in patients treated with 6TG, and to explore if a safe dose of continuous 6TG can be established. Data sources: Databases, conference proceedings, and reference lists of included studies were systematically searched for 6TG and synonyms from 1998–2018. Methods: We included studies of patients with ALL or inflammatory bowel disorder (IBD) treated with 6TG, excluding studies with 6TG as part of an intensive chemotherapy regimen. We uploaded a protocol to PROSPERO (registration number CRD42018089424). Database and manual searches yielded 1823 unique records. Of these, 395 full-texts were screened for eligibility. Finally, 134 reports representing 42 studies were included. Results and conclusions: We included data from 42 studies of ALL and IBD patients; four randomised controlled trials (RCTs) including 3,993 patients, 20 observational studies including 796 patients, and 18 case reports including 60 patients. Hepatotoxicity in the form of sinusoidal obstruction syndrome (SOS) occurred in 9–25% of the ALL patients in two of the four included RCTs using 6TG doses of 40–60 mg/m2/day, and long-term hepatotoxicity in the form of nodular regenerative hyperplasia (NRH) was reported in 2.5%. In IBD patients treated with 6TG doses of approximately 23 mg/m2/day, NRH occurred in 14% of patients. At a 6TG dose of approximately 12 mg/m2/day, NRH was reported in 6% of IBD patients, which is similar to the background incidence. According to this review, doses at or below 12 mg/m2/day are rarely associated with notable hepatotoxicity and can probably be considered safe. [ABSTRACT FROM AUTHOR]

Focal segmental glomerulosclerosis (FSGS) is frequently found in biopsies of patients with steroid resistant nephrotic syndrome (SRNS). The pathogenesis of SRNS/FSGS is often unknown and the disease will recur in up to 50% of patients post-transplant, indicating the presence of circulating podocyte-toxic factor(s). Several studies have reported clinical improvement after anti-TNFα therapy. However, prediction of the clinical outcome in SRNS/FSGS is difficult, and novel predictive biomarkers are needed. An image-based assay, which measures disassembly of focal adhesion complexes in cultured podocytes, was used to ascertain the presence of podocyte toxic activity in SRNS/FSGS sera. Expression of TNFα pathway genes was analysed in the Nephroseq FSGS cohort and in cultured podocytes treated with SRNS/FSGS sera. Podocyte toxic activity was detected in 48/96 SRNS/FSGS patients. It did not correlate with serum TNFα levels, age, sex, ethnicity or glomerular filtration rate. In ~25% of the toxic samples, the toxicity was strongly inhibited by blockade of TNFα signaling. Transcriptional profiling of human FSGS biopsies and podocytes treated with FSGS sera revealed significant increases in expression of TNFα pathway genes. We identified patients with serum podocyte toxic activity who may be at risk for FSGS recurrence, and those patients in whom serum podocyte toxicity may be reversed by TNFα blockade. Activation of TNFα pathway genes occurs in podocytes of FSGS patients suggesting a causative effect of this pathway in response to circulating factor(s). In vitro analyses of patient sera may stratify patients according to prognostic outcomes and potential responses to specific clinical interventions. [ABSTRACT FROM AUTHOR]

Poor solute transport through the cartilage endplate (CEP) impairs disc nutrition and could be a key factor that limits the success of intradiscal biologic therapies. Here we demonstrate that treating the CEP with matrix metalloproteinase-8 (MMP-8) reduces the matrix constituents that impede solute uptake and thereby improves nutrient diffusion. Human CEP tissues harvested from four fresh cadaveric lumbar spines (age range: 38–66 years old) were treated with MMP-8. Treatment caused a dose-dependent reduction in sGAG, localized reductions to the amount of collagen, and alterations to collagen structure. These matrix modifications corresponded with 16–24% increases in the uptake of a small solute (376 Da). Interestingly, the effects of MMP-8 treatment depended on the extent of non-enzymatic glycation: treated CEPs with high concentrations of advanced glycation end products (AGEs) exhibited the lowest uptake compared to treated CEPs with low concentrations of AGEs. Moreover, AGE concentrations were donor-specific, and the donor tissues with the highest AGE concentrations appeared to have lower uptake than would be expected based on the initial amounts of collagen and sGAG. Finally, increasing solute uptake in the CEP improved cell viability inside diffusion chambers, which supports the nutritional relevance of enhancing the transport properties of the CEP. Taken together, our results provide new insights and in vitro proof-of-concept for a treatment approach that could improve disc nutrition for biologic therapy: specifically, matrix reduction by MMP-8 can enhance solute uptake and nutrient diffusion through the CEP, and AGE concentration appears to be an important, patient-specific factor that influences the efficacy of this approach. [ABSTRACT FROM AUTHOR]

This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue. [ABSTRACT FROM AUTHOR]

Olfactory ensheathing cells are thought to support regeneration and remyelination of damaged axons when transplanted into spinal cord injuries. Following transplantation, improved locomotion has been detected in many laboratory models and in dogs with naturally-occurring spinal cord injury; safety trials in humans have also been completed. For widespread clinical implementation, it will be necessary to derive large numbers of these cells from an accessible and, preferably, autologous, source making olfactory mucosa a good candidate. Here, we compared the yield of olfactory ensheathing cells from the olfactory mucosa using 3 different techniques: rhinotomy, frontal sinus keyhole approach and rhinoscopy. From canine clinical cases with spinal cord injury, 27 biopsies were obtained by rhinotomy, 7 by a keyhole approach and 1 with rhinoscopy. Biopsy via rhinoscopy was also tested in 13 cadavers and 7 living normal dogs. After 21 days of cell culture, the proportions and populations of p75-positive (presumed to be olfactory ensheathing) cells obtained by the keyhole approach and rhinoscopy were similar (~4.5 x 106 p75-positive cells; ~70% of the total cell population), but fewer were obtained by frontal sinus rhinotomy. Cerebrospinal fluid rhinorrhea was observed in one dog and emphysema in 3 dogs following rhinotomy. Blepharitis occurred in one dog after the keyhole approach. All three biopsy methods appear to be safe for harvesting a suitable number of olfactory ensheathing cells from the olfactory mucosa for transplantation within the spinal cord but each technique has specific advantages and drawbacks. [ABSTRACT FROM AUTHOR]

Many aspects of the laboratory environment are not tailored to the needs of rodents, which may cause stress. Unpredictable stressors can cause ulcers, prolonged pituitary-adrenal activation, and anhedonia. Similarly, pain has been demonstrated to slow wound healing, and mice experiencing pain exhibit altered behavior. However it is unknown how husbandry, which occurs when the mice are inactive, and lack of analgesia, specifically in a punch biopsy procedure, effects animal physiology, behavior, and welfare, particularly as it relates to sleep fragmentation. We hypothesized that sleep fragmentation, induced by unpredictable husbandry and lack of pain management will slow wound healing. Two main treatments were tested in a factorial design in C57BL/6 mice of both sexes (64 mice total); 1) analgesia (carprofen and saline) and 2) sleep disruptions (random and predictable). Mice were singly housed in a non-invasive sleep monitoring apparatus on arrival (Day -4). Disruption treatments were applied from Day -3 to 2. All mice received a punch biopsy surgery (Day 0) with topical lidocaine gel and their analgesic treatment prior to recovery, and on Days 1 and 2. Nesting behavior was assessed daily and a sugar cereal consumption test, as a measure of anhedonia, was conducted on Days -1 to 2. On Day 3, mice were euthanized and wound tissue and adrenal glands were collected. We found that the disruption predictability had no effect on mouse sleep, wound healing, or adrenal cortex:medulla ratio. It’s possible that the disruption period was not long enough to induce chronic stress. However, male mice who received analgesia slept more than their female counterparts; this may be related to sex differences in pain perception. Overall, it does not appear that the predictability of disturbance effects sleep fragmentation or stress responses, indicating that husbandry activities do not need to occur at set predictable times to improve welfare. [ABSTRACT FROM AUTHOR]

Liver sinusoidal endothelial cells (LSECs) play a pivotal role in hepatic function and homeostasis. LSEC dysfunction has been recognized to be closely involved in various liver diseases, including non-alcoholic steatohepatitis (NASH), but not much is known about the fate of the scavenger receptors in LSECs during NASH. Fc gamma receptor IIb (FcγRIIb), known as a scavenger receptor, contributes to receptor-mediated endocytosis and immune complexes clearance. In this study, to elucidate the fate of FcγRIIb in the progression of non-alcoholic fatty liver disease (NAFLD), we examined FcγRIIb levels in NAFLD biopsy specimens by immunohistochemistry, and investigated their correlation with the exacerbation of biological indexes and clinicopathological scores of NASH. The FcγRIIb expression levels indicated significant negative correlations with serum levels of blood lipids (triglyceride, total cholesterol, high-density lipoprotein-cholesterol), type 4 collagen and hyaluronic acid, which are involved in hepatic lipid metabolism disorder, fibrosis, and inflammation, respectively. However, there was no significant difference of FcγRIIb expression levels among the pathological grades of NAFLD. During NAFLD progression, inflammation and fibrosis may influence the expression of FcγRIIb and their scavenger functions to maintain hepatic homeostasis. [ABSTRACT FROM AUTHOR]

Background: Non-alcoholic fatty liver disease (NAFLD) may be associated with changes in bile acid (BA) metabolism. Hepatic BA production, measured by serum levels of the precursor 7α-hydroxy-4-cholesten-3-one (C4), is regulated by the farnesoid-X-receptor (FXR)-dependent ileal hormone fibroblast growth factor 19 (FGF19). Low FGF19 and high C4 are features of chronic BA diarrhea. Obeticholic acid, an FXR agonist, stimulates FGF19 and has shown therapeutic potential in both BA diarrhea and in NAFLD. We hypothesized there are associations of FGF19, C4 and BA diarrhea with NAFLD. Methods and findings: 127 patients with known NAFLD were recruited prospectively. Clinical features, including metformin use, markers of NAFLD severity and BA synthesis were analyzed. The overall incidence of chronic diarrhea was 25%, with features of BA diarrhea in 12%. FGF19 negatively correlated with C4 (rs = -0.43, p = 0.001) and with alanine aminotransferase (rs = -0.22, p = 0.03), but not with either NAFLD fibrosis or Fibroscan scores. High C4 was associated with a higher NAFLD fibrosis score (p < 0.05), and with diarrhea (p = 0.001). The median NAFLD fibrosis score was higher in those with diarrhea (p = 0.002). Metformin use, in 44% overall, was particularly associated with diarrhea (in 36% vs 17%, p = 0.02), and a lower median FGF19 (74 vs 105 pg/mL, p < 0.05). Conclusions: Increased hepatic BA production and diarrhea, but not low FGF19, were associated with increased NAFLD fibrosis score, indicating dysregulation of the FXR-FGF19 axis and suggesting hepatic FGF19 resistance. Metformin use was an important factor in a subgroup, lowering FGF19, and resulting in bile acid diarrhea. [ABSTRACT FROM AUTHOR]

Objective: A standardization of minor salivary gland (MSG) histopathology in primary Sjögren’s syndrome (pSS) has been recently proposed. Although there is strong agreement that germinal center (GC)-like structures should be routinely identified, due to their prognostic value, a consensus regarding the best protocol is still lacking. Aim of this study was to compare the performance of different histological techniques and operators to identify GC-like structures in pSS MSGs. MSG biopsies from 50 pSS patients were studied. Methods: Three blinded operators (one pathologist and two rheumatologists with different years of experience in pSS MSG assessment) assessed 50 MSGs of which one slide was stained with haematoxylin and eosin (H&E) and consecutive slides were processed to investigate CD3/CD20, CD21 and Bcl-6 expression. Results: By assessing 225 foci, the best agreement was between H&E-stained sections evaluated by the rheumatologist with more years of experience in pSS MSG assessment and CD3/CD20 segregation. In the foci with CD21 positivity, the agreement further increased. Bcl-6- foci could display a GC, detected with other staining, but not vice versa. Conclusion: GC assessment on H&E-stained sections should be performed with caution, being operator-dependent. The combination of H&E with CD3/CD20 and CD21 staining should be recommended as it is reliable, feasible, able to overcome the bias of operator experience and easily transferrable into routine practice. [ABSTRACT FROM AUTHOR]

The burden of liver disease continues to increase in the UK, with liver cirrhosis reported to be the third most common cause of premature death. Iron overload, a condition that impacts liver health, was traditionally associated with genetic disorders such as hereditary haemochromatosis, however, it is now increasingly associated with obesity, type-2 diabetes and non-alcoholic fatty liver disease. The aim of this study was to assess the prevalence of elevated levels of liver iron within the UK Biobank imaging study in a cohort of 9108 individuals. Magnetic resonance imaging (MRI) was undertaken at the UK Biobank imaging centre, acquiring a multi-echo spoiled gradient-echo single-breath-hold MRI sequence from the liver. All images were analysed for liver iron and fat (expressed as proton density fat fraction or PDFF) content using LiverMultiScan. Liver iron was measured in 97.3% of the cohort. The mean liver iron content was 1.32 ± 0.32 mg/g while the median was 1.25 mg/g (min: 0.85 max: 6.44 mg/g). Overall 4.82% of the population were defined as having elevated liver iron, above commonly accepted 1.8 mg/g threshold based on biochemical iron measurements in liver specimens obtained by biopsy. Further analysis using univariate models showed elevated liver iron to be related to male sex (p<10−16, r2 = 0.008), increasing age (p<10−16, r2 = 0.013), and red meat intake (p<10−16, r2 = 0.008). Elevated liver fat (>5.6% PDFF) was associated with a slight increase in prevalence of elevated liver iron (4.4% vs 6.3%, p = 0.0007). This study shows that population studies including measurement of liver iron concentration are feasible, which may in future be used to better inform patient stratification and treatment. [ABSTRACT FROM AUTHOR]

Cutaneous mast cell tumours are one of the most common canine cancers. Approximately 25% of the tumours metastasise. Activating c-kit mutations are present in about 20% of tumours, but metastases occur in the absence of mutations. Tumour metastasis is associated with significantly diminished survival in spite of adjuvant chemotherapy. Available prognostic tests do not reliably predict whether a tumour will metastasise. In this study we compared the global expression profiles of 20 primary cutaneous mast cell tumours that metastasised with those of 20 primary tumours that did not metastasise. The objective was to identify genes associated with mast cell tumour metastatic progression that may represent targets for therapeutic intervention and biomarkers for prediction of tumour metastasis. Canine Gene 1.1 ST Arrays were employed for genome-wide expression analysis of formalin-fixed, paraffin-embedded biopsies of mast cell tumours borne by dogs that either died due to confirmed mast cell tumour metastasis, or were still alive more than 1000 days post-surgery. Decreased gene expression in the metastasising tumours appears to be associated with a loss of cell polarity, reduced cell-cell and cell-ECM adhesion, and increased cell deformability and motility. Dysregulated gene expression may also promote extracellular matrix and base membrane degradation, suppression of cell cycle arrest and apoptosis, and angiogenesis. Down-regulation of gene expression in the metastasising tumours may be achieved at least in part by small nucleolar RNA-derived RNA and microRNA-effected gene silencing. Employing cross-validation, a linear discriminant analysis-based classifier featuring 19 genes that displayed two-fold differences in expression between metastasising and non-metastasising tumours was estimated to classify metastasising and non-metastasising tumours with accuracies of 90–100% and 70–100%, respectively. The differential expression of 9 of the discriminator genes was confirmed by quantitative reverse transcription-PCR. [ABSTRACT FROM AUTHOR]

To clarify the roles of vitamin D and calcium as potential chemopreventive agents against colorectal cancer in humans, and to develop “treatable”, pre-neoplastic, phenotypic biomarkers of risk for colorectal neoplasms, we estimated the effects of supplemental vitamin D3 (1,000 IU/day [25 μg/day]) and calcium (1,200 mg/day), alone and in combination, on biomarkers of proliferation (mib-1), differentiation (p21), and apoptosis (bax [apoptosis-promoting] and bcl-2 [apoptosis-inhibiting]), in the normal-appearing rectal mucosa in a subsample of participants (n = 104) in a larger randomized, double-blind, placebo-controlled clinical trial among colorectal adenoma patients. The biomarkers were measured in rectal biopsies at baseline and after one year of follow up, using automated immunohistochemistry and quantitative image analysis. In the vitamin D plus calcium group relative to control, in the crypt differentiation zone (upper 40% of crypts), mib-1 expression decreased 24% (P = 0.28); p21 expression alone and relative to mib-1 expression increased 29% (P = 0.06) and 73% (P = 0.06), respectively; and bax expression relative to mib-1 expression increased 58% (P = 0.21). The estimated vitamin D alone treatment effects were similar but of lesser magnitudes, and those for calcium alone were mixed. All estimated treatment effects on bcl-2 expression were close to the null. These pilot study results support further investigation of whether 1) vitamin D and calcium promote colorectal epithelial cell differentiation, reduce proliferation, and promote apoptosis in the normal-appearing human colorectal mucosa, 2) vitamin D and calcium act as chemopreventive agents against colorectal neoplasms, and 3) mib-1, p21, and bax are potential “treatable”, pre-neoplastic, biomarkers of risk for colorectal neoplasms. [ABSTRACT FROM AUTHOR]

The incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in the UK. Patients with HPV-positive OPSCC generally show superior clinical responses relative to HPV-negative patients. We hypothesised that these superior responses could be associated with defective repair of DNA double strand breaks (DSB). The study aimed to determine whether defective DNA repair could be associated with sensitivity to inhibition of DNA repair using the PARP inhibitor Olaparib. Sensitivity to Olaparib, and induction and repair of DNA damage, were assessed in a panel of 8 OPSCC cell-lines, including 2 novel HPV-positive lines. Effects on cell cycle distribution and levels of PARP1 and p53 were quantified. RNA-sequencing was used to assess differences in activity of DNA repair pathways. Two HPV-positive OPSCC lines were sensitive to Olaparib at potentially therapeutic doses (0.1–0.5 μM). Two HPV-negative lines were sensitive at an intermediate dose. Four other lines, derived from HPV-positive and HPV-negative tumours, were resistant to PARP inhibition. Only one cell-line, UPCISCC90, showed results consistent with the original hypothesis i.e. that in HPV-positive cells, treatment with Olaparib would cause accumulation of DSB, resulting in cell cycle arrest. There was no evidence that HPV-positive tumours exhibit defective repair of DSB. However, the data suggest that a subset of OPSCC may be susceptible to PARP-inhibitor based therapy. [ABSTRACT FROM AUTHOR]

Wooden Breast Disease (WBD), a myopathy in commercial broiler chickens characterized by abnormally firm consistency of the pectoral muscle, impacts the poultry industry negatively due to severe reduction in meat quality traits. To unravel the molecular profile associated with the onset and early development of WBD in broiler chickens, we compared time-series gene expression profiles of Pectoralis (P.) major muscles between unaffected and affected birds from a high-breast-muscle-yield, purebred broiler line. P. major biopsy samples were collected from the cranial and caudal aspects of the muscle belly in birds that were raised up to 7 weeks of age (i.e. market age). Three subsets of biopsy samples comprising 6 unaffected (U) and 10 affected (A) from week 2 (cranial) and 4 (caudal), and 4U and 11A from week 3 (cranial) were processed for RNA-sequencing analysis. Sequence reads generated were processed using a suite of bioinformatics programs producing differentially expressed (DE) genes for each dataset at fold-change (A/U or U/A) >1.3 and False Discovery Ratio (FDR) <0.05 (week 2: 41 genes; week 3: 618 genes and week 4: 39 genes). Functional analysis of DE genes using literature mining, BioDBnet and IPA revealed several biological processes and pathways associated with onset and progress of WBD. Top among them were dysregulation of energy metabolism, response to inflammation, vascular disease and remodeling of extracellular matrix. This study reveals that presence of molecular perturbations involving the vasculature, extracellular matrix and metabolism are pertinent to the onset and early pathogenesis of WBD in commercial meat-type chickens. [ABSTRACT FROM AUTHOR]

After sequential treatment with first- and third-generation EGFR tyrosine kinase inhibitors (TKIs), EGFR-mutant non-small cell lung cancers frequently harbor multiple resistance mutations in exon 20 of EGFR including T790M, mediating resistance to first-generation TKIs, and at codons 792, 796, or 797 mediating resistance to third-generation TKIs. However, whether these resistance mutations are in cis or trans has therapeutic implications for patients. We analyzed a cohort of 29 patients with NSCLC harboring EGFR mutations at codons 792, 796, or 797 to establish the configuration of these mutations. We performed hybrid capture-based, next-generation sequencing on formalin-fixed paraffin-embedded biopsy tissue or liquid biopsy. 27 samples had both a T790M mutation and a mutation at codons 792, 796, or 797. In all of these cases, the mutations were found in the cis configuration; the trans configuration was not observed. Two patients’ samples harbored a mutation at codon 797 but no T790M mutation. In these two cases, longitudinal analysis showed earlier biopsies harbored EGFR T790M, which was undetectable following osimertinib treatment. Treatment of one these patients with both first- and third-generation EGFR TKIs resulted in a mixed response. Here we describe multiple configurations of EGFR T790M and third-generation TKI resistance mutations at codons 792, 796, and 797. These mutations are most commonly found in cis, which confers resistance to all current EGFR TKIs. We also describe two patients that exhibited T790M loss with acquisition of a mutation at codon 797. In addition, one of these patients, with an EGFR C797S in a lung biopsy was subsequently found to have EGFR C797N in a later biopsy of pleural fluid, highlighting the dynamic multiclonal nature of advanced NSCLC. [ABSTRACT FROM AUTHOR]

Pathology images are color in nature due to the use of chemical staining in biopsy examination. Aware of the high color diagnosticity in pathology images, this work introduces a compact rotation-invariant texture descriptor, named quantized diagnostic counter-color pattern (QDCP), for digital pathology image understanding. On the basis of color similarity quantified by the inner product of unit-length color vectors, local counter-color textons are indexed first. Then the underlined distribution of QDCP indexes is estimated by an image-wise histogram. Since QDCP is computed based on color difference directly, it is robust to small color variation usually observed in pathology images. This study also discusses QDCP’s extraction, parameter settings, and feature fusion techniques in a generic pathology image analysis pipeline, and introduces two more descriptors QDCP-LBP and QDCP/LBP. Experimentation on public pathology image sets suggests that the introduced color texture descriptors, especially QDCP-LBP, outperform prior color texture features in terms of strong descriptive power, low computational complexity, and high adaptability to different image sets. [ABSTRACT FROM AUTHOR]

Objectives: Idiopathic interstitial pneumonia (IIP) and connective tissue disease -associated interstitial pneumonia (CTD-IP) are the two most common types of interstitial pneumonia. IIP and CTD-IP share common histological features, yet their clinical management is different. Separation of the two conditions based solely on histology can be challenging, and there are no established criteria. Materials and methods: We selected 105 consecutive cases of IIP (79 usual interstitial pneumonia and 26 non-specific interstitial pneumonia) and 49 cases of CTD-IP for derivation and 32 cases of IIP and 10 cases of CTD-IP for validation. Fourteen histological parameters were evaluated independently by two pathologists for derivation group and graded into 0 to 3. The association between the score for each marker and a diagnosis of CTD was investigated using Fisher’s exact test and stepwise logistic regression analysis. A formula for calculating the probability of IIP and CTD-IP was constructed by the markers identified in the regression test with coefficients for each finding. The formula was confirmed using validation case group. Results: Stepwise logistic regression analysis showed that plasmacytosis, lymphoid follicle with germinal center, and airspace fibrin were suggestive of CTD-IP and that fibroblastic foci, smooth muscle hyperplasia, cellular IP, dense perivascular collagen, and fat metaplasia were suggestive of IIP. The formula used to calculate the probabilities based on estimated values for each finding was created, and user-friendly web based app was composed at . On the validation study, 30 out of 32 IIP and eight out of 10 CTD-IPs were distinguished correctly by the app (Specificity: 93%, Sensitivity: 80%). Conclusions: We identified histological markers and derived a practical formula and user-friendly app to distinguish CTD-IPs from IIP. [ABSTRACT FROM AUTHOR]

Our aim is to describe variations in the incidence rates of glomerular disease diagnosed by renal biopsies performed in Uruguay over the last 25 years in relation to sex, age, clinical presentation and histological diagnosis. We analyzed all renal biopsies performed in Uruguay during the 25 years period and estimated incidence rates per million people per year (pmp/yr) for the population older than 14 years. Mann Kendall's trend analysis was used to assess incidence trends. In order to identify changes in trends, we compared annual incidence rates with the Joinpoint method. From 1990 to 2014, 3390 biopsies of native kidneys corresponding to glomerular disease were performed in patients older than 14 years. The average biopsy rate was 58 per pmp/yr. The glomerular disease incidence rate increased progressively over the period (p<0.05). Trends analysis over five-year periods demonstrated a progressive increase of IgA nephropathy (3.08 pmp/yr 1990–1994 to 12.53 pmp/yr 2010–2014 p<0.05), membranous nephropathy (2.38 pmp/yr 1990–1994 to 8.04 pmp/yr 2010–2014 p< 0.05) and lupus nephritis (4,23 pmp/yr 1990–1994 to 7,81 pmp/yr 2010–2014 p<0.05). There was a change in the trend of focal segmental glomerular sclerosis (FSGS) which increased until 1996 and decreased afterwards. The incidence rates of glomerular disease have doubled globally in the last quarter of a century in Uruguay, mainly related to the increase of IgA nephropathy, membranous nephropathy and lupus nephritis. There was a change in the slope of the incidence rate of FSGS. [ABSTRACT FROM AUTHOR]

Aim: To develop a patient derived xenograft (PDX) model of cervical cancer and cervical dysplasia using the subrenal capsule. Methods: Cervical cancer (12 Squamous Cell Carcinoma, 1 Adenocarcinoma, 1 Adenosquamous Carcinoma), 7 cervical dysplasia biopsy and normal cervical tissues were transplanted beneath the renal capsule of immunocompromised NOD/SCID/gamma mice. Resulting tumours were harvested and portions serially transplanted into new recipient mice for up to three in vivo passages. Parent and xenograft tumours were examined by immunohistochemistry for p16INK41, HPV, and CD-45. Single cell suspensions of mixed mouse and human, or human only cell populations were also transplanted. Results: The overall engraftment rate for the primary cervical cancer PDX model was 71.4 ±12.5% (n = 14). Tumours maintained morphological, histoarchitecture and immunohistochemical features of the parent tumour, and demonstrated invasiveness into local tissues. Single cell suspensions did not produce tumour growth in this model. Mean length of time (32.4 +/- 3.5 weeks) for the transplanted tissue to generate a tumour in the animal was similar between successive transplantations. Three of four xenografted cervical dysplasia tissues generated microscopic cystic structures resembling dysplastic cervical tissue. Normal cervical tissue (4 of 5 xenografted) also developed microscopic cervical tissue grafts. Conclusion: The subrenal capsule can be used for a PDX model of human cervical cancer with a good engraftment rate and the ability to model in vivo characteristics of cervical cancer. For the first time we have demonstrated that cervical dysplasia and normal cervical tissue generated microscopic tissues in a PDX model. [ABSTRACT FROM AUTHOR]

Telomere length has been associated with risk of several cancers. However, studies of the relationship between telomere length and colorectal cancer risk have been inconsistent. This study examined the relationship between telomere length in normal colon tissue and the prevalence of colorectal adenoma, a precursor to colorectal cancer. This nested case-control study consisted of 85 patients aged 40 to 65 undergoing a screening colonoscopy: 40 cases with adenoma(s) detected at colonoscopy and 45 controls with normal colonoscopy. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. Relative telomere length (rTL) was quantified in DNA extracted from colon mucosa using quantitative real-time PCR. Logistic regression was used to assess the relationship between telomere length and adenoma prevalence and estimate odds ratios and 95% confidence intervals. rTL was significantly longer in colon tissue of individuals with adenomas compared to healthy individuals (p = 0.008). When rTL was categorized into quartiles according to the distribution of rTL among controls, individuals with the longest telomeres had increased odds of adenoma when compared to individuals with shortest telomeres (OR = 4.58, 95% CI: 1.19, 17.7). This study suggests that long telomeres in normal colon tissue are associated with increased colorectal cancer risk. [ABSTRACT FROM AUTHOR]

Shear wave elastography is emerging as a clinically valuable diagnostic tool to differentiate between benign and malignant breast masses. Elastography techniques assume that soft tissue can be modelled as a purely elastic medium. However, this assumption is often violated as soft tissue exhibits viscoelastic properties. In order to explore the role of viscoelastic parameters in suspicious breast masses, a study was conducted on a group of patients using shear wave dispersion ultrasound vibrometry in the frequency range of 50–400 Hz. A total of 43 female patients with suspicious breast masses were recruited before their scheduled biopsy. Of those, 15 patients did not meet the data selection criteria. Voigt model based shear elasticity showed a significantly (p = 7.88x10-6) higher median value for the 13 malignant masses (16.76±13.10 kPa) compared to 15 benign masses (1.40±1.12 kPa). Voigt model based shear viscosity was significantly different (p = 4.13x10-5) between malignant (8.22±3.36 Pa-s) and benign masses (2.83±1.47 Pa-s). Moreover, the estimated time constant from the Voigt model, which is dependent on both shear elasticity and viscosity, differed significantly (p = 6.13x10-5) between malignant (0.68±0.33 ms) and benign masses (3.05±1.95 ms). Results suggest that besides elasticity, viscosity based parameters like shear viscosity and time constant can also be used to differentiate between malignant and benign breast masses. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer incidence is significant in countries, such as South Africa, with high burdens of both HIV and human papillomavirus (HPV). Cervical cancer is largely preventable if dysplasia is diagnosed and treated early, but there is debate regarding the best approaches for screening and treatment, especially for low-resource settings. Currently South Africa provides Pap smears followed by colposcopic biopsy and LEEP if needed in its public health facilities. We estimated the costs and cost-effectiveness of two approaches for treating cervical intraepithelial neoplasia grade 2 or higher (CIN2+) among HIV-infected women, most of whom were taking antiretroviral treatment, at a public HIV treatment facility in Johannesburg, South Africa. Methods: Method effectiveness was derived from an intention-to-treat analysis of data gathered in a clinical trial completed previously at the study facility. In the trial, women who were diagnosed with CIN2+ and eligible for cryotherapy were randomized to cryotherapy or LEEP. If women were CIN2+ at six months as determined via Pap smear and colposcopic biopsy, all women—regardless of their original treatment assignment—received LEEP. “Cure” was then defined as the absence of disease at 12 months based on Pap smear and colposcopic biopsy. Health service costs were estimated using micro-costing between June 2013 and April 2014. Capital costs were annualized using a discount rate of 3%. Two different service volume scenarios were considered, and results from an as-treated analysis were considered in sensitivity analysis. Results: In total, 166 women with CIN2+ were enrolled (86 had LEEP; 80 had cryotherapy). At 12 months, cumulative loss to follow-up was 12.8% (11/86) for the LEEP group and 13.8% (11/80) for cryotherapy. Based on the unadjusted intention-to-treat analysis conducted for this economic evaluation, there was no significant difference in efficacy. At 12 months, 83.8% (95% CI 73.8–91.1) of women with CIN2+ at baseline and randomized to cryotherapy were free of CIN2+ disease. In contrast, 76.7% (95% CI 66.4–85.2) of women assigned to LEEP were free from disease. On average, women initially treated with cryotherapy were less costly per patient randomized at US$ 118.00 (113.91–122.10), and per case “cured” at US$ 140.90 (136.01–145.79). Women in the LEEP group cost US$ 162.56 (157.90–167.22) per patient randomized and US$ 205.59 (199.70–211.49) per case cured. In the as-treated analysis, which was based on trial data, LEEP was more efficacious than cryotherapy; however, the difference was not significant. Cryotherapy remained more cost-effective than LEEP in all sensitivity and scenario analyses. Conclusions: For this cost-effectiveness analysis, using an intention-to-treat approach and taking into consideration uncertainty in the clinical and cost outcomes, a strategy involving cryotherapy plus LEEP if needed at six months was dominant to LEEP plus LEEP again at six months if needed for retreatment. However, compared to other studies comparing LEEP and cryotherapy, the efficacy results were low in both treatment groups–possibly due to the HIV-positivity of the participants. Further research is needed, but at present choosing the “right” treatment option may be less important than ensuring access to treatment and providing careful monitoring of treatment outcomes. [ABSTRACT FROM AUTHOR]

A challenge in the clinical adoption of cell-free DNA (cfDNA) liquid biopsies for cancer care is their high cost compared to potential reimbursement. The most common approach used in liquid biopsies to achieve high specificity detection of circulating tumor DNA (ctDNA) among a large background of normal cfDNA is to attach molecular barcodes to each DNA template, amplify it, and then sequence it many times to reach a low-error consensus. In applications where the highest possible specificity is required, error rate can be lowered further by independently detecting the sequences of both strands of the starting cfDNA. While effective in error reduction, the additional sequencing redundancy required by such barcoding methods can increase the cost of sequencing up to 100-fold over standard next-generation sequencing (NGS) of equivalent depth. We present a novel library construction and analysis method for NGS that achieves comparable performance to the best barcoding methods, but without the increase in sequencing and subsequent sequencing cost. Named Proximity-Sequencing (Pro-Seq), the method merges multiple copies of each template into a single sequencing read by physically linking the molecular copies so they seed a single sequencing cluster. Since multiple DNA copies of the same template are compared for consensus within the same cluster, sequencing accuracy is improved without the use of redundant reads. Additionally, it is possible to represent both senses of the starting duplex in a single cluster. The resulting workflow is simple, and can be completed by a single technician in a work day with minimal hands on time. Using both cfDNA and cell line DNA, we report the average per-mutation detection threshold and per-base analytical specificity to be 0.003% and >99.9997% respectively, demonstrating that Pro-Seq is among the highest performing liquid biopsy technologies in terms of both sensitivity and specificity, but with greatly reduced sequencing costs compared to existing methods of comparable accuracy. [ABSTRACT FROM AUTHOR]