Search

Abstract Anastomotic leakage (AL) is a potentially life-threatening complication following colorectal cancer (CRC) resection. In this study, we aimed to unravel longitudinal changes in microbial structure before, during, and after surgery and to determine if microbial alterations may be predictive for risk assessment between sufficient anastomotic healing (AS) and AL prior surgery. We analysed the microbiota of 134 colon mucosal biopsies with 16S rRNA V1-V2 gene sequencing. Samples were collected from three location sites before, during, and after surgery, and patients received antibiotics after the initial collection and during surgery. The microbial structure showed dynamic surgery-related changes at different time points. Overall bacterial diversity and the abundance of some genera such as Faecalibacterium or Alistipes decreased over time, while the genera Enterococcus and Escherichia_Shigella increased. The distribution of taxa between AS and AL revealed significant differences in the abundance of genera such as Prevotella, Faecalibacterium and Phocaeicola. In addition to Phocaeicola, Ruminococcus2 and Blautia showed significant differences in abundance between preoperative sample types. ROC analysis of the predictive value of these genera for AL revealed an AUC of 0.802 (p = 0.0013). In summary, microbial composition was associated with postoperative outcomes, and the abundance of certain genera may be predictive of postoperative complications.

Abstract Background Despite extensive research on microbiome alterations in ulcerative colitis (UC), the role of the constituent stable microbiota remains unclear. Results This study, employing 16S rRNA-gene sequencing, uncovers a persistent microbial imbalance in both active and quiescent UC patients compared to healthy controls. Using co-occurrence and differential abundance analysis, the study highlights microbial constituents, featuring Phocaeicola, Collinsella, Roseburia, Holdemanella, and Bacteroides, that are not affected during the course of UC. Co-cultivation experiments, utilizing commensal Escherichia coli and Phocaeicola vulgatus, were conducted with intestinal epithelial organoids derived from active UC patients and controls. These experiments reveal a tendency for a differential response in tight junction formation and maintenance in colonic epithelial cells, without inducing pathogen recognition and stress responses, offering further insights into the roles of these microorganisms in UC pathogenesis. These experiments also uncover high variation in patients’ response to the same bacteria, which indicate the need for more comprehensive, stratified analyses with an expanded sample size. Conclusion This study reveals that a substantial part of the gut microbiota remains stable throughout progression of UC. Functional experiments suggest that members of core microbiota – Escherichia coli and Phocaeicola vulgatus – potentially differentially regulate the expression of tight junction gene in the colonic epithelium of UC patients and healthy individuals.

The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air–liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1β, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.

Multiple myeloma (MM) progression is closely dependent on cells in the bone marrow (BM) microenvironment, including fibroblasts (FBs) and immune cells. In their BM niche, MM cells adhere to FBs sustaining immune evasion, drug resistance and the undetectable endurance of tumor cells known as minimal residual disease (MRD). Here, we describe the novel bi-specific designed ankyrin repeat protein (DARPin) α-FAPx4–1BB (MP0310) with FAP-dependent 4–1BB agonistic activity. The α-FAPx4–1BB DARPin simultaneously binds to FAP and 4–1BB overexpressed by activated FBs and immune cells, respectively. Although flow cytometry analysis showed that T and NK cells from MM patients were not activated and did not express 4–1BB, stimulation with daratumumab or elotuzumab, monoclonal antibodies (mAbs) currently used for the treatment of MM, significantly upregulated 4–1BB both in vitro and in MM patients following mAb-based therapy. The mAb-induced 4–1BB overexpression allowed the engagement of α-FAPx4–1BB that acted as a bridge between FAP+FBs and 4–1BB+NK cells. Therefore, α-FAPx4–1BB enhanced both the adhesion of daratumumab-treated NK cells on FBs as well as their activation by improving release of CD107a and perforin, hence MM cell killing via antibody-mediated cell cytotoxicity (ADCC). Interestingly, α-FAPx4–1BB significantly potentiated daratumumab-mediated ADCC in the presence of FBs, suggesting that it may overcome the BM FBs’ immunosuppressive effect. Overall, we speculate that treatment with α-FAPx4–1BB may represent a valuable strategy to improve mAb-induced NK cell activity fostering MRD negativity in MM patients through the eradication of latent MRD cells.

Abstract Introduction During the SARS-CoV-2 pandemic, the training of medical students was severely disrupted. Government authorities imposed a second national lockdown, which lasted from December 2020 to May 2021, forcing medical schools to adapt their teaching strategies. Several surveys have assessed students’ perspectives, but there is limited data available to objectively assess and compare different strategies between medical faculties. The aim of this study was to evaluate the lecturer-based perspective to assess the impact on the theoretical and practical education of medical students before and during the second lockdown. Methods We developed a questionnaire focusing on theoretical and practical training during pandemic before and during the second lockdown. The questionnaire was sent by personal invitation in January 2021 to all responsible lecturers of gastroenterology and hepatology departments at university hospitals in Germany. Results The complete response rate was obtained from 52.5% (21/40) teaching centres representative of all regions in Germany. While the majority of respondents (62%) reported a limited impact of the lockdown on theoretical training, 90.5% reported a significant impact on practical training during the SARS-CoV-2 pandemic response. Several centres adapted virtual or hybrid education early during the pandemic, bedside teaching was down to 38.1% (8/21), particularly for students in the early phase of education, suggesting a substantial long-term impact on students' skills. Conclusion Our questionnaire-based data provide one of the first systematic evaluation of faculty-based assessments of the potential impact of the pandemic during lockdown on medical education. We observed considerable heterogeneity in strategies for maintaining teaching activities during the SARS-CoV-2 pandemic in Germany.

Abstract The aerobic granular biomass technology was optimized for treating nitrate-polluted groundwater based on the biological denitrification processes in order to provide drinking water. Reactors inoculated with granular biomass were operated at progressively lower C/N rate using acetate and methanol to encourage heterotrophic denitrification, in order to meet the recommended requirements described by European Drinking Water Framework Directive. The granulation and long-term stability of granular biomass under low C/N were successful for all stages, demonstrated compactness of granules and absence of filamentous microorganisms. The nitrate removal was similar in methanol- and acetate-fed reactors, occurring in both cases nitrate removal ratios > 80%, and fact allows the selection of one of both depending groundwater polluted case. Also, feeding reactors with 2 C/N ratio showed nitrate removal values of ≥ 95%, treating highly polluted groundwater (100 mg·L−1). The microbial diversity was higher in the methanol-fed reactor with representative phylotypes as Flavobacterium, Cytophagaceae, NS9 marine group, while species richness was higher in the acetate-fed reactor, which was mainly represented by Flavobacterium genus. Statistical analyses revealed the higher resilience of bacterial population on granules fed with acetate, showing more resistance under drop C/N ratio. Oscillating pollution in groundwater during seasonal periods should be treated using acetate as carbon source for denitrification carried out by granular biomass, while stable pollution concentrations over time allow the use of methanol as a carbon source since the greater microbial diversity allows the elimination of other contaminants present in groundwater.

Abstract Background and aims We aimed to identify mucin-microbiome signatures shaping the tumor microenvironment in gastric adenocarcinomas and clinical outcomes. Methods We performed high-throughput profiling of the mucin phenotypes present in 108 gastric adenocarcinomas and 20 functional dyspepsia cases using validated mucin-based RT-qPCRs with subsequent immunohistochemistry validation and correlated the data with clinical outcome parameters. The gastric microbiota was assessed by 16S rRNA gene sequencing, taxonomy, and community composition determined, microbial networks analyzed, and the metagenome inferred in association with mucin phenotypes and expression. Results Gastric adenocarcinomas with an intestinal mucin environment or high-level MUC13 expression are associated with poor survival. On the contrary, gastric MUC5AC or MUC6 abundance was associated with a more favorable outcome. The oral taxa Neisseria, Prevotella, and Veillonella had centralities in tumors with intestinal and mixed phenotypes and were associated with MUC13 overexpression, highlighting their role as potential drivers in MUC13 signaling in GC. Furthermore, dense bacterial networks were observed in intestinal and mixed mucin phenotype tumors whereas the lowest community complexity was shown in null mucin phenotype tumors due to higher Helicobacter abundance resulting in a more decreased diversity. Enrichment of oral or intestinal microbes was mucin phenotype dependent. More specifically, intestinal mucin phenotype tumors favored the establishment of pro-inflammatory oral taxa forming strong co-occurrence networks. Conclusions Our results emphasize key roles for mucins in gastric cancer prognosis and shaping microbial networks in the tumor microenvironment. Specifically, the enriched oral taxa associated with aberrant MUC13 expression can be potential biomarkers in predicting disease outcomes. Video Abstract

Abstract Helicobacter pylori (H. pylori) infection has been considered as the main causal factor in gastric carcinogenesis, but other bacterial species may also play an important role in pathophysiology of gastric cancer. The aim of the study was to explore the link between gastric cancer prognosis and the mucosal microbial community in tumorous and adjacent gastric tissue. The bacterial profile was analysed using 16S sequencing (V1–V2 region). Microbial differences were mostly characterized by lower relative abundances of H. pylori in tumorous gastric tissues. Bacterial community and outcome data analysis revealed the genus Fusobacterium and Prevotella significantly associated with worse overall survival in gastric cancer patients. In particular, Fusobacterium was associated with significant increase in hazard ratio in both univariable and multivariable analysis and independently validated using TCMA data. Phylogenetic biodiversity of Fusobacterium species in the stomach revealed F. periodonticum as the most prevalent in healthy subjects, while F. nucleatum was most abundant in patients with gastric cancer. Bacterial community network analysis in gastric cancer suggests substantial complexity and a strong interplay between F. nucleatum and Prevotella. In summary, mucosal microbial community in the stomach was associated with worse overall survival in gastric cancer patients. Strongest negative impact on prognosis was linked to the abundance of F. nucleatum in tumorous specimens, suggesting its translational relevance in management of gastric cancer patients.

The host genetic background for hepatocellular carcinoma (HCC) is incompletely understood. We aimed to determine if four germline genetic polymorphisms, rs429358 in apolipoprotein E (APOE), rs2642438 in mitochondrial amidoxime reducing component 1 (MARC1), rs2792751 in glycerol‐3‐phosphate acyltransferase (GPAM), and rs187429064 in transmembrane 6 superfamily member 2 (TM6SF2), previously associated with progressive alcohol‐related and nonalcoholic fatty liver disease, are also associated with HCC. Four HCC case‐control data sets were constructed, including two mixed etiology data sets (UK Biobank and FinnGen); one hepatitis C virus (HCV) cohort (STOP‐HCV), and one alcohol‐related HCC cohort (Dresden HCC). The frequency of each variant was compared between HCC cases and cirrhosis controls (i.e., patients with cirrhosis without HCC). Population controls were also considered. Odds ratios (ORs) associations were calculated using logistic regression, adjusting for age, sex, and principal components of genetic ancestry. Fixed‐effect meta‐analysis was used to determine the pooled effect size across all data sets. Across four case‐control data sets, 2,070 HCC cases, 4,121 cirrhosis controls, and 525,779 population controls were included. The rs429358:C allele (APOE) was significantly less frequent in HCC cases versus cirrhosis controls (OR, 0.71; 95% confidence interval [CI], 0.61‐0.84; P = 2.9 × 10−5). Rs187429064:G (TM6SF2) was significantly more common in HCC cases versus cirrhosis controls and exhibited the strongest effect size (OR, 2.03; 95% CI, 1.45‐2.86; P = 3.1 × 10−6). In contrast, rs2792751:T (GPAM) was not associated with HCC (OR, 1.01; 95% CI, 0.90‐1.13; P = 0.89), whereas rs2642438:A (MARC1) narrowly missed statistical significance (OR, 0.91; 95% CI, 0.84‐1.00; P = 0.043). Conclusion: This study associates carriage of rs429358:C (APOE) with a reduced risk of HCC in patients with cirrhosis. Conversely, carriage of rs187429064:G in TM6SF2 is associated with an increased risk of HCC in patients with cirrhosis.

The gastrointestinal (GI) tract is home to a complex and dynamic community of microorganisms, comprising bacteria, archaea, viruses, yeast, and fungi. It is widely accepted that human health is shaped by these microbes and their collective microbial genome. This so-called second genome plays an important role in normal functioning of the host, contributing to processes involved in metabolism and immune modulation. Furthermore, the gut microbiota also is capable of generating energy and nutrients (eg, short-chain fatty acids and vitamins) that are otherwise inaccessible to the host and are essential for mucosal barrier homeostasis. In recent years, numerous studies have pointed toward microbial dysbiosis as a key driver in many GI conditions, including cancers. However, comprehensive mechanistic insights on how collectively gut microbes influence carcinogenesis remain limited. In addition to their role in carcinogenesis, the gut microbiota now has been shown to play a key role in influencing clinical outcomes to cancer immunotherapy, making them valuable targets in the treatment of cancer. It also is becoming apparent that, besides the gut microbiota’s impact on therapeutic outcomes, cancer treatment may in turn influence GI microbiota composition. This review provides a comprehensive overview of microbial dysbiosis in GI cancers, specifically esophageal, gastric, and colorectal cancers, potential mechanisms of microbiota in carcinogenesis, and their implications in diagnostics and cancer treatment.

Microbiota research has received an increasing attention for its role in disease development and fungi are considered as one of the key players in the microbial niche. Various sequencing approaches have been applied to uncover the role of fungal community in health and disease; however, little is known on the performance of various primers and comparability between the studies. Motivated by the recent publications, we performed a systematic comparison of the 18S and ITS regions to identify the impact of various primers on the sequencing results. Using four pairs of primers extensively used in literature, fungal community was retrieve from 25 fecal samples, and applying high throughput sequencing; and the results were compared in order to select the most suitable primers for fungal detection in human fecal samples. Considering the high variability between samples, primers described in the Earth microbiome project detected the broadest fungal spectrum suggesting its superior performance in mycobiome research.

BackgroundAntibiotic susceptibility of Helicobacter pylori to antibiotics may vary among different niches of the stomach. The progression of chronic H. pylori gastritis to atrophy changes intragastric physiology that may influence selection of resistant strains.AimTo study the antibiotic resistance of H. pylori taking the severity of atrophic gastritis in antrum and corpus into account.MethodsHelicobacter pylori-positive patients (n = 110, m = 32, mean age 52.6 ± 13.9 years) without prior H. pylori eradication undergoing upper gastrointestinal (GI) endoscopy for dyspeptic symptoms were included in a prospective study. Patients were stratified into three groups depending on the grade of atrophy: no atrophy (OLGA Stage 0), mild atrophy (OLGA Stage I–II) and moderate/severe atrophy (OLGA Stage III–IV). Two biopsies each from the antrum and the corpus and one from the angulus were taken and assessed according to the updated Sydney system. H. pylori strains were isolated from antrum and corpus biopsies and tested for antibiotic susceptibility (AST) for amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline, and rifampicin by the agar dilution methods. A Chi-square test of independence with a 95% confidence interval was used to detect differences in the proportion of patients with susceptible and resistant H. pylori strains.ResultsAmong 110 patients, primary clarithromycin resistance (R) was 30.0%, both in the antrum and corpus; metronidazole resistance accounted for 36.4 and 34.5% in the antrum and corpus; and levofloxacin was 19.1 and 22.7% in the antrum and corpus, respectively. Resistance rates to amoxicillin, tetracycline, and rifampicin were below 5%. Dual antibiotic resistance rate was 21.8%, and triple resistance rate was 9.1%. There was a significant difference in the resistance rate distribution in antrum (p

Introduction: Diet is one of the most important factors contributing to the multistep process of carcinogenesis. The clinical relevance of exogenous food-derived xeno-microRNAs (miRNAs) in human diseases is poorly understood. In this study, we aimed to evaluate the potential clinical relevance of the xeno-miRNA miR-168 in the gastric mucosa along the preneoplastic conditions and gastric carcinogenesis. Methods: For a systematic analysis, we included stomach tissues from patients with different pathologies, including normal mucosa (N), chronic non-atrophic (CNAG) and atrophic gastritis (CAG) and intestinal metaplasia (IM) (n = 72), matched non-tumorous (NT) and tumorous (T) gastric cancer (GC) tissues (n = 81), matched colorectal cancer (CRC) tissues (n = 40), and colon mucosa and faeces from controls and IBD patients. Results: miR-168 was reproducibly detectable in all samples studied, with the highest levels in the proximal upper GI and in non-tumorous compared to tumorous tissues in both GC and CRC. There was no difference related to H. pylori positivity or inflammation grade, while higher miR-168 levels were observed in patients with moderate or severe AG/IM or OLGIM3/4. Survival analysis showed only a small, non-significant trend towards worse overall survival for patients with the highest to lowest miR-168 levels, while no differences were related to Lauren‘s classification. Conclusions: Food-derived xeno miRNAs are reproducibly detectable in the gastric and colonic mucosa. Although the clinically relevant function remains to be elucidated, higher levels of miR-168 in patients with moderate and severe IM merit further investigation.

Summary: Background: Human gut microbiome composition is influenced by genetics, diet and environmental factors. We investigated the microbial composition in several gastrointestinal (GI) compartments to evaluate the impact of genetics, delivery mode, diet, household sharing and aging on microbial similarity in monozygotic and dizygotic twins. Methods: Fecal, biopsy and saliva samples were obtained from total 108 twins. DNA and/or RNA was extracted and the region V1-V2 of the 16S rRNA gene was amplified and sequenced. Bray-Curtis similarity was used for further microbiome comparisons, Mann-Whitney test was applied to evaluate the significant differences between groups and Spearman test was applied to reveal potential correlations between data. Findings: The global bacterial profiles were grouped into two clusters separating the upper and lower GI. The upper GI microbiome composition was strictly dependent on the Helicobacter pylori status. With a positivity rate of 55%, H. pylori completely colonized the stomach and separated infected twins from non-infected twins irrespective of zygosity status. Lower GI microbiome similarity between the twins was defined mainly by household-sharing and aging; whereas delivery mode and host genetics had no influence. There was a progredient decrease in the bacterial similarity with aging. Shared vs. non-shared phylotypes analysis showed that in both siblings the shared phylotypes progressively diminished with aging, while the non-shared phylotypes increased. Interpretation: Our findings strongly highlight the aging and shared household as they key determinants in gut microbial similarity and drift in twins irrespective of their zygotic state. Funding: This work was supported by the grant of the Research Council of Lithuania (Project no. APP-2/2016) and also partially supported by the funds of European Commission through the “European funds for regional development” (EFRE) as well as by the regional Ministry of Economy, Science and Digitalization as part of the “LiLife” Project as part of the “Autonomy in old Age” research group (Project ID: ZS/2018/11/95324).

Inflammatory bowel disease (IBD) is a chronic recurrent inflammatory disease with unknown etiology. Dextran sulfate sodium (DSS) induced colitis is a widely used mouse model in IBD research. DSS colitis involves activation of the submucosal immune system and can be used to study IBD-like disease characteristics in acute, chronic, remission and transition phases. Insight into colon inflammatory parameters is needed to understand potentially irreversible adaptations to the chronification of colitis, determining the baseline and impact of further inflammatory episodes. We performed analyses of non-invasive and invasive colitis parameters in acute, chronic and remission phases of the DSS colitis in C57BL/6 mice. Non-invasive colitis parameters poorly reflected inflammatory aspects of colitis in chronic remission phase. We found invasive inflammatory parameters, positively linked to repeated DSS-episodes, such as specific colon weight, inflamed colon area, spleen weight, absolute cell numbers of CD4+ and CD8+ T cells as well as B cells, blood IFN-γ level, colonic chemokines BLC and MDC as well as the prevalence of Turicibacter species in feces. Moreover, microbial Lactobacillus species decreased with chronification of disease. Our data point out indicative parameters of recurrent gut inflammation in context of DSS colitis.

Gastric carcinogenesis is associated with alterations of microRNAs (miRNAs) and reversal of these alterations may be a crucial element in cancer prevention. Here we evaluate the influence of H. pylori eradication, low-dose aspirin (LDA), non-steroidal anti-inflammatory drugs (NSAIDs) and proton-pump inhibitors (PPI) on modification of inflammatory mucosal miRNAs miR-155 and miR-223 in Helicobacter pylori-infected and non-infected subjects. The study was performed in two parts: 1) interventional study in 20 healthy subjects with and without H. pylori infection or following eradication (each n = 10) where LDA (100 mg) was given daily for 7 days; 2) prospective case-control observational study (n = 188). MiR-155 and miR-223 expression was strongly linked to H. pylori-infection and in short-term view showed a trend for reversal after eradication. Daily LDA as well as regular NSAIDs showed no influence on miRNAs expression both in healthy subjects and patients, while regular PPI intake was associated with lower miR-155 expression in antrum of patients with chronic gastritis independent of density of neutrophils and mononuclear infiltrate. In summary, PPI but not LDA or NSAIDs were associated with modification of inflammatory miRNAs miR-155 and miR-223 in an H. pylori dependent manner. The functional role of inflammatory miR-155 and miR-223 in understanding of H. pylori-related diseases needs further evaluation.

The adaption of gut microbiota (GM) throughout human life is a key factor in maintaining health. Interventions to restore a healthy GM composition may have the potential to improve health and disease outcomes in the elderly. We performed a comprehensive characterization of changes in the luminal and mucosa-associated microbiota composition in elderly compared with younger healthy individuals. Samples from saliva and feces, and biopsies from the upper and lower gastrointestinal tract (UGIT, LGIT), were collected from 59 asymptomatic individuals grouped by age: 40–55, 56–70, and 71–85 years). All underwent anthropometric, geriatric, and nutritional assessment. RNA was extracted and reverse-transcribed into complementary DNA; the V1–V2 regions of 16S ribosomal RNA genes were amplified and sequenced. Abundances of the taxa in all taxonomic ranks in each sample type were used to construct sample-similarity matrices by the Bray–Curtis algorithm. Significant differences between defined groups were assessed by analysis of similarity. The bacterial community showed strong interindividual variations and a clear distinction between samples from UGIT, LGIT, and feces. While in saliva some taxa were affected by aging, this number was considerably greater in UGIT and was subsequently higher in LGIT. Unexpectedly, aging scarcely influenced the bacterial community of feces over the age range of 40–85 years. The development of interventions to preserve and restore human health with increased age by establishing a healthy gut microbiome should not rely solely on data from fecal analysis, as the intestinal mucosa is affected by more significant changes, which differ from those observed in fecal analyses.

Celiac disease (CeD) is a chronic autoimmune disorder characterized by an intolerance to storage proteins of many grains. CeD is frequently associated with liver damage and steatosis. Bile acid (BA) signaling has been identified as an important mediator in gut–liver interaction and the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Here, we aimed to analyze BA signaling and liver injury in CeD patients. Therefore, we analyzed data of 20 CeD patients on a gluten-free diet compared to 20 healthy controls (HC). We furthermore analyzed transaminase levels, markers of cell death, BA, and fatty acid metabolism. Hepatic steatosis was determined via transient elastography, by MRI and non-invasive scores. In CeD, we observed an increase of the apoptosis marker M30 and more hepatic steatosis as compared to HC. Fibroblast growth factor 19 (FGF19) was repressed in CeD, while low levels were associated with steatosis, especially in patients with high levels of anti-tissue transglutaminase antibodies (anti-tTG). When comparing anti-tTG-positive CeD patients to individuals without detectable anti-tTG levels, hepatic steatosis was accentuated. CeD patients with significant sonographic steatosis (defined by CAP ≥ 283 db/m) were exclusively anti-tTG-positive. In summary, our results suggest that even in CeD patients in clinical remission under gluten-free diet, alterations in gut–liver axis, especially BA signaling, might contribute to steatotic liver injury and should be further addressed in future studies and clinical practice.

Background/Aims: L-arginine is an important mediator of cell division, wound healing, and immune function. It can be transformed by the nitric oxide synthase (NOS) to nitric oxide (NO), an important cell signaling molecule. Recent studies from our laboratory demonstrate specific effects of L-arginine (10mM) exposure on gastric acid secretion in rat parietal cells. Methods: Studies were performed with isolated gastric glands and the pH sensitive dye BCECF-AM +/- L-arginine to examine its effects on acid secretion. The direct NO-donor diethylamine NONOate sodium salt hydrate, was also used while monitoring intracellular pH. The specific inhibitor of the intracellular NO signal cascade ODQ was also used. Results: We found that gastric proton extrusion was activated with application of L-arginine (10mM), in a separate series when L-arginine (10mM) + L-NAME (30µM) were added there was no acid secretion. Addition of the NO-donor diethylamine NONOate sodium salt hydrate (10µM) also induced acid secretion. When the selective sGC-inhibitor ODQ was added with NONOate we did not observe acid secretion. Conclusion: We conclude that L-arginine is a novel secretagogue, which can mediate gastric acid secretion. Furthermore, the intake of L-arginine causes direct activation of the H+, K+ ATPase and increased proton extrusion from parietal cells resulting in the increased risk for acid-related diseases. The NO/sGC/cGMP pathway has never been described as a possible intracellular mechanism for H+, K+ ATPase activation before and presents a completely new scientific finding. Moreover, our studies demonstrate a novel role for L-NAME to effectively eliminate NOS induced acid secretion and thereby reducing the risk for L-arginine inducible ulcer disease.

A transcriptome-wide analysis of human liver for demonstrating differences between young and old humans has not yet been performed. However, identifying major age-related alterations in hepatic gene expression may pinpoint ontogenetic shifts with important hepatic and systemic consequences, provide novel pharmacogenetic information, offer clues to efficiently counteract symptoms of old age, and improve the overarching understanding of individual decline. Next-generation sequencing (NGS) data analyzed by the Mann–Whitney nonparametric test and Ensemble Feature Selection (EFS) bioinformatics identified 44 transcripts among 60,617 total and 19,986 protein-encoding transcripts that significantly (p = 0.0003 to 0.0464) and strikingly (EFS score > 0.3:16 transcripts; EFS score > 0.2:28 transcripts) differ between young and old livers. Most of these age-related transcripts were assigned to the categories ‘regulome’, ‘inflammaging’, ‘regeneration’, and ‘pharmacogenes’. NGS results were confirmed by quantitative real-time polymerase chain reaction. Our results have important implications for the areas of ontogeny/aging and the age-dependent increase in major liver diseases. Finally, we present a broadly substantiated and testable hypothesis on a genetically governed ‘aging cascade’, wherein PPP1R10 acts as a putative ontogenetic master regulator, prominently flanked by IGFALS and DUSP1. This transcriptome-wide analysis of human liver offers potential clues towards developing safer and improved therapeutic interventions against major liver diseases and increased insights into key mechanisms underlying aging.

T and B cell compartmentalization is a hallmark of secondary lymphoid organs and is maintained by chemokine-expressing stromal cells. How this stromal cell network initially develops and differentiates into two distinct subsets is poorly known, especially for the splenic white pulp (WP). Here, we show that perivascular fibroblast precursors are triggered by LTα1β2 signals to expand, express CCL19/21, and then differentiate into two functionally distinct fibroblast subsets responsible for B and T cell clustering and WP compartmentalization. Failure to express or sense CCL19 leads to impaired T zone development, while lack of B cells or LTα1β2 leads to an earlier and stronger impairment in WP development. We therefore propose that WP development proceeds in multiple steps, with LTα1β2+ B cells acting as major inducer cells driving the expansion and gradual differentiation of perivascular fibroblasts into T and B zone organizer cells.

Abstract LINE-1 hypomethylation is widely accepted as marker for global genomic DNA hypomethylation, which is a frequent event in cancer. The aim of the study was to evaluate LINE-1 methylation status at different stages of gastric carcinogenesis and evaluate its prognostic potential in clinical settings. LINE-1 methylation was analyzed in 267 tissue samples by bisulfite pyrosequencing including primary colorectal cancer tissues (T-CRC) with corresponding adjacent colon mucosa (N-CRC), gastric cancer tissues (T-GC) with corresponding gastric mucosa (N-GC), normal gastric tissues (N), chronic non-atrophic and atrophic gastritis (CG). LINE-1 methylation level was lower in both T-GC and T-CRC when compared to paired adjacent tissues. No difference was observed for LINE-1 methylation status between patients with normal gastric mucosa, CG and N-GC. LINE-1 methylation in T-GC but not N-GC tended to correlate with age. Subgroup stratification analysis did not reveal significant differences in LINE-1 methylation status according to tumor stage, anatomical location, histological subtype, differentiation grade. We observed similar overall survival data between patients with high or low LINE-1 levels. In summary, LINE-1 hypomethylation is a characteristic feature in GC but not very common in early preneoplastic stages of gastric carcinogenesis. Prognostic role of LINE-1 hypomethylation in GC patients could not be confirmed in this cohort.

Aspirin has been widely recommended for acute and chronic conditions for over 2,000 years. Either single or repetitive doses are commonly used for analgesic and antipyretic reasons and to prevent heart attacks, stroke, and blood clot formation. Recent studies show that it can also be used chronically to dramatically reduce the risk of a variety of cancers. However, prolonged usage of aspirin can cause severe damage to the mucosal barrier, increasing the risk of ulcer formation and GI-bleeding events. In the present study, we show the effects of acute low-dose aspirin exposure as an active secretagogue-inducing gastric acid secretion. Studies were carried out with isolated gastric glands using the pH-sensitive dye BCECF-AM to assess acid secretion. The non-selective NOS inhibitor L-NAME (30 μM), or the specific inhibitor ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) was applied while monitoring intracellular pH. The effects of basolateral exposure to aspirin (acetylsalicylic acid, ASA) caused activation of gastric acid secretion via the H+, K+-ATPase. Our data suggest that aspirin increases nitric oxide (NO) production, which in turn activates acid secretion. Exposure of gastric glands to either the non-selective NOS inhibitor L-NAME, and the highly selective, soluble guanylyl cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) effectively inhibited aspirin-dependent gastric acid secretion. Aspirin can be considered as a novel secretagogue, in the way that it activates the H+, K+-ATPase. With increased daily aspirin consumption, our findings have important implications for all individuals consuming aspirin even in low doses and the potential risks for increased acid secretion.

Two aerobic granular sludge (AGS) sequential batch reactors were operated at a mild (15 °C) temperature for 180 days. One of those bioreactors was exposed to a mixture of diclofenac, naproxen, trimethoprim, and carbamazepine. The AGS system, operating under pressure from emerging contaminants, showed a decrease in COD, BOD5, and TN removal capacity, mainly observed during the first 100 days, in comparison with the removal ratios detected in the control bioreactor. After an acclimatisation period, the removal reached high-quality effluent for COD and TN, close to 95% and 90%, respectively. In the steady-state period, trimethoprim and diclofenac were successfully removed with values around 50%, while carbamazepine and naproxen were more recalcitrant. The dominant bacterial OTUs were affected by the presence of a mixture of pharmaceutical compounds, under which the dominant phylotypes changed to OTUs classified among the Pseudomonas, Gemmobacter, and Comamonadaceae. The RT-qPCR and qPCR results showed the deep effects of pharmaceutical compounds on the number of copies of target genes. Statistical analyses allowed for linking the total and active microbial communities with the physico-chemical performance, describing the effects of pharmaceutical compounds in pollution degradation, as well as the successful adaptation of the system to treat wastewater in the presence of toxic compounds.

Long intergenic non-coding RNAs (lincRNAs) are >200 nucleotides long non-coding RNAs, which have been shown to be implicated in carcinogenic processes by interacting with cancer associated genes or other non-coding RNAs. However, their role in development of rare gastrointestinal stromal tumors (GISTs) is barely investigated. Therefore, the aim of this study was to define lincRNAs deregulated in GIST and find new GIST-lincRNA associations. Next-generation sequencing data of paired GIST and adjacent tissue samples from 15 patients were subjected to a web-based lincRNA analysis. Three deregulated lincRNAs (MALAT1, H19 and FENDRR; adjusted p-value < 0.05) were selected for expression validation in a larger group of patients (n = 22) by RT-qPCR method. However, only H19 and FENDRR showed significant upregulation in the validation cohort (adjusted p < 0.05). Further, we performed correlation analyses between expression levels of deregulated lincRNAs and GIST-associated oncogenes or GIST deregulated microRNAs. We found high positive correlations between expression of H19 and known GIST related oncogene ETV1, and between H19 and miR-455-3p. These findings expand the knowledge on lincRNAs deregulated in GIST and may be an important resource for the future studies investigating lincRNAs functionally relevant to GIST carcinogenesis.

Perioperative chemotherapy improves overall survival in patients with oesophagogastric adenocarcinoma (OAC) and locoregional disease. The mainstay of perioperative chemotherapy in these patients is a platinum/fluoropyrimidine combination. The phase III FLOT4 trial has shown that the FLOT triplet regimen (oxaliplatin, infusional 5-FU, and docetaxel) improves the outcome of patients with OAC and locoregional disease as compared to the ECF triplet (epirubicin, cisplatin, and infusional 5-FU). Targeted therapies have currently no role in the perioperative setting for the treatment of patients with OAC. For patients with oligometastatic disease, upfront gastrectomy followed by chemotherapy did not show any survival benefit compared with chemotherapy alone and thus should be discouraged. Whether surgery should be offered to patients with metastatic OAC achieving a systemic control after upfront chemotherapy is under scrutiny in the phase III FLOT5/Renaissance trial. After neoadjuvant treatment, lymph node status but not pathologic tumor response is an independent factor in the prediction of overall survival. Growing evidence suggests that perioperative chemotherapy may be associated with an increased mortality risk in patients with microsatellite instable (MSI)/mismatch repair-deficient (MMRD) adenocarcinoma, thus validating poor responsiveness to chemotherapy in MSI patients with locoregional disease.

BackgroundMicroRNAs (miRNAs) are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC) and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues.MethodsThe study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA). In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs.ResultsProfiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients' plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression.ConclusionsOur study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers. Target gene analysis demonstrated that BCL2 and DNMT3B expression in GC tissue correlated with their targeting miRNA expression.

BACKGROUND AND AIMS: MicroRNAs (miRNAs) are known for their function as translational regulators of tumor suppressor or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNAs related genes have been shown to affect the regulatory capacity of miRNAs and were linked with gastric cancer (GC) and premalignant gastric conditions. The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of GC or high risk atrophic gastritis (HRAG) in European population. METHODS: Gene polymorphisms were analyzed in 995 subjects (controls: n = 351; GC: n = 363; HRAG: n = 281) of European descent. MiR-27a T>C (rs895819), miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913), miR-492 G>C (rs2289030) and miR-608 C>G (rs4919510) SNPs were genotyped by RT-PCR. RESULTS: Overall, SNPs of miRNAs were not associated with the presence of GC or HRAG. We observed a tendency for miR-196a-2 CT genotype to be associated with higher risk of GC when compared to CC genotype, however, the difference did not reach the adjusted P-value (odds ratio (OR) - 1.46, 95% confidence interval (CI) 1.03-2.07, P = 0.032). MiR-608 GG genotype was more frequent in GC when compared to controls (OR -2.34, 95% CI 1.08-5.04), but significance remained marginal (P = 0.029). A similar tendency was observed in a recessive model for miR-608, where CC + CG vs GG genotype comparison showed a tendency for increased risk of GC with OR of 2.44 (95% CI 1.14-5.22, P = 0.021). The genotypes and alleles of miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 SNPs had similar distribution between histological subtypes of GC and were not linked with the presence of diffuse or intestinal-type GC. CONCLUSIONS: Gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of HRAG, GC or different histological subtypes of GC in European subjects.

INTRODUCTION:JC virus (JCV), a human polyomavirus that causes progressive multifocal leukoencephalopathy (PML), has been linked to colorectal cancer (CRC). However, determination of JCV infection and its role in carcinogenesis has been challenging, highlighting the need for better diagnostic strategies for this virus. JCV-specific microRNAs (miRNAs) were identified and shown to negatively regulate oncogenic JCV T-Ag. Herein, we determined the pattern of JCV miRNA expression in clinical specimens from healthy subjects and CRC patients. MATERIAL AND METHODS:JCV miRNA expression was validated in CRC cell lines transfected with the JCV T-Ag. Results were confirmed using CRC tissues that were expressed T-Ag. Expression of JCV-specific miR-J1-5p was measured in fresh stool samples from healthy volunteers, and samples from fecal occult blood test kits from healthy subject, and patients with colorectal neoplasms. RESULTS:JCV miR-J1-5p was detected in JCV-transfected, but not vector-transfected, CRC cells, and was stable between cell passages. MiR-J1-5p was present in all six JCV T-Ag+ CRC samples. Surprisingly, JCV miRNA was detectable in all normal tissues, but the expression was much lower in CRC tissues. Similarly, miR-J1-5p expression was present in all fecal samples, but expression was lower in CRCs compared to controls or adenoma patients. CONCLUSION:JC virus-specific miR-J1-5p miRNA is a potential biomarker for viral infection, and the lower expression in patients with colonic neoplasia highlights its biological role regulating oncogenic T-Ag expression in CRC. IMPACT:JCV-specific miRNA is a candidate for the development of a non-invasive screening test, as well as therapeutic intervention for JCV-associated diseases.

Recently, there has been a growing interest in an expanding group of cytokines known as "IL-12 family". The so far gained knowledge about these cytokines, as crucial playmakers in mucosal immunity, has not yet been sufficiently investigated in the context of Helicobacter pylori infection. All genes encoding the monomeric components of these cytokines and their corresponding receptors were examined in gastric epithelial cell lines (AGS and MKN-28) after being infected with 4 H. pylori strains: BCM-300, P1 wild-type, and P1-derived isogenic mutants lacking cytotoxin-associated gene A (cagA) or virulence gene virB7 (multiplicity of infection=50). Both infected and uninfected samples were analyzed after 24h and 48h using real-time quantitative polymerase chain reaction (RT-qPCR). Gene expression analysis demonstrated a strong upregulation of IL23A (encodes p19) by infection, whereas IL23R, Epstein-Barr virus-induced gene 3 (EBI3), IL6ST, IL12A, and IL27RA were found to be expressed, but not regulated, or to a lesser extent. Transcripts of IL12RB2, IL12B, IL12RB1, and IL27A were not detected. Interestingly, P1 resulted in stronger alterations of expression than CagA mutant and BCM-300, particularly for IL23A (59.7-fold versus 32.4- and 6.7-fold, respectively in AGS after 48h, P

Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.

IntroductionPancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer.Materials and methodsTotal RNA was extracted from feces using Qiagen's miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays.ResultsAll selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (pConclusionOur data provide novel evidence for the differential expression of miRNAs in feces of patients with PCA. If successfully validated in large-scale prospective studies, the fecal miRNA biomarkers may offer novel tools for PCA screening research.

INTRODUCTION:JC Virus (JCV), a human polyomavirus, is frequently present in colorectal cancers (CRCs). JCV large T-Ag (T-Ag) expressed in approximately half of all CRC's, however, its functional role in CRC is poorly understood. We hypothesized that JCV T-Ag may mediate metastasis in CRC cells through increased migration and invasion. MATERIAL AND METHODS:CRC cell lines (HCT116 and SW837) were stably transfected with JCV early transcript sequences cloned into pCR3 or empty vectors. Migration and invasion assays were performed using Boyden chambers. Global gene expression analysis was performed to identify genetic targets and pathways altered by T-Ag expression. Microarray results were validated by qRT-PCR, protein expression analyses and immunohistochemistry. Matching primary CRCs and liver metastases from 33 patients were analyzed for T-Ag expression by immunohistochemistry. RESULTS:T-Ag expressing cell lines showed 2 to 3-fold increase in migration and invasion compared to controls. JCV T-Ag expression resulted in differential expression of several genetic targets, including genes that mediate cell migration and invasion. Pathway analysis suggested a significant involvement of these genes with AKT and MAPK signaling. Treatment with selective PI3K/AKT and MAPK pathway inhibitors resulted in reduced migration and invasion. In support of our in-vitro results, immunohistochemical staining of the advanced stage tumors revealed frequent JCV T-Ag expression in metastatic primary tumors (92%) as well as in their matching liver metastasis (73%). CONCLUSION:These data suggest that JCV T-Ag expression in CRC associates with a metastatic phenotype, which may partly be mediated through the AKT/MAPK signaling pathway. Frequent expression of JCV T-Ag in CRC liver metastasis provides further clues supporting a mechanistic role for JCV as a possible mediator of cellular motility and invasion in CRC.