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Background: Metabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function., Methods: Intestinal organoids derived from mouse colon were grown in normal glucose media (5 mM) or high glucose media (25 mM) to study the impact of hyperglycemic stress on the intestinal barrier. Additionally, organoids were pretreated with 35 kDa hyaluronan (HA35) to investigate the effect of hyaluronan on epithelial barrier under high glucose stress. Immunoblotting as well as confocal imaging was used to understand changes in barrier proteins, quantitative as well as spatial distribution, respectively. Alterations in barrier function were measured using trans-epithelial electrical resistance and fluorescein isothiocyanate flux assays. Untargeted proteomics analysis was performed to elucidate mechanisms by which HA35 exerts a protective effect on the barrier. Intestinal organoids derived from receptor knockout mice specific to various HA receptors were utilized to understand the role of HA receptors in barrier protection under high glucose conditions., Results: We found that high glucose stress decreased the protein expression as well as spatial distribution of two key barrier proteins, zona occludens-1 (ZO-1) and occludin. HA35 prevented the degradation or loss of ZO-1 and maintained the spatial distribution of both ZO-1 and occludin under hyperglycemic stress. Functionally, we also observed a protective effect of HA35 on the epithelial barrier under high glucose conditions. We found that HA receptor, layilin, was involved in preventing barrier protein loss (ZO-1) as well as maintaining spatial distribution of ZO-1 and occludin. Additionally, proteomics analysis showed that cell death and survival was the primary pathway upregulated in organoids treated with HA35 under high glucose stress. We found that XIAP associated factor 1 (Xaf1) was modulated by HA35 thereby regulating apoptotic cell death in the intestinal organoid system. Finally, we observed that spatial organization of both focal adhesion kinase (FAK) as well as F-actin was mediated by HA35 via layilin., Conclusion: Our results highlight the impact of hyperglycemic stress on the intestinal barrier function. This is of clinical relevance, as impaired barrier function has been observed in individuals with metabolic syndrome. Additionally, we demonstrate barrier protective effects of HA35 through its receptor layilin and modulation of cellular apoptosis under high glucose stress., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The integrity and permeability of epithelial and endothelial barriers depend on the formation of tight junctions, adherens junctions, and a junction-associated cytoskeleton. The establishment of this junction-cytoskeletal module relies on the correct folding and oligomerization of its protein components. Molecular chaperones are known regulators of protein folding and complex formation in different cellular compartments. Mammalian cells possess an elaborate chaperone network consisting of several hundred chaperones and co-chaperones. Only a small part of this network has been linked, however, to the regulation of intercellular adhesions, and the systematic analysis of chaperone functions at epithelial and endothelial barriers is lacking. This review describes the functions and mechanisms of the chaperone-assisted regulation of intercellular junctions. The major focus of this review is on heat shock protein chaperones, their co-chaperones, and chaperonins since these molecules are the focus of the majority of the articles published on the chaperone-mediated control of tissue barriers. This review discusses the roles of chaperones in the regulation of the steady-state integrity of epithelial and vascular barriers as well as the disruption of these barriers by pathogenic factors and extracellular stressors. Since cytoskeletal coupling is essential for junctional integrity and remodeling, chaperone-assisted assembly of the actomyosin cytoskeleton is also discussed.

Introduction: The aim of this case-control study was to determine if exposure to tumor necrosis factor alpha inhibitors (TNFIs) or immunomodulators (thiopurines or methotrexate) was associated with development of primary gastrointestinal lymphoma (PGIL) in patients with chronic inflammatory conditions., Methods: Patients with PGIL and controls evaluated at a tertiary care center over 20 years were matched 1:3 using a medical record informatics search engine based on their chronic inflammatory condition (Crohn's disease [CD], ulcerative colitis [UC], rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis) and duration of follow-up. Patients who started on TNFI within 3 months of PGIL diagnosis were excluded. We extracted demographics, medical history, and medications used. Univariate models using conditional logistic regression were used due to the small number of matched pairs., Results: Twenty PGIL cases matched with 60 controls were followed for a mean 9.9 ± 6.9 and 9.7 ± 8.6 years, respectively. Mean age at time of PGIL diagnosis was 47.5 ± 22.0 (standard deviation) years and the majority (75%) were males. The most common inflammatory diagnosis was inflammatory bowel disease (80% of cases; 45% with UC and 35% with CD). Development of PGIL was not associated with TNFI (odds ratio [OR] = 2.6; 95% confidence interval [CI] 0.69-11.01; P  = .18), but with use of TNFI in combination with thiopurines (OR = 8.93; 95% CI 1.43-80.25; P  = .014). Risk of PGIL increased with every additional TNFI (2.277 (1.002-5.713); P  = .0494). All cases exposed to multiple TNFI were also exposed to thiopurines. Use of thiopurines (alone or in combination) was the greatest risk factor (OR = 6.32; 95% CI 1.55-37.05; P  = 0.006) to develop PGIL., Conclusions: TNFI therapy was not associated with increased risk for PGIL unless used in combination with thiopurines and with every switch to a different TNFI., Competing Interests: The authors have no relevant conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Crohn's & Colitis Foundation.)

Background: The development of Crohn's disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated., Materials and Methods: Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells., Results: CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites., Conclusions: The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Upadacitinib is a selective Janus kinase inhibitor approved for the management of ulcerative colitis and is under evaluation for the management of Crohn's disease [CD] in Phase 3 clinical trials., Aims: Our goal was to describe our real-world experience with upadacitinib in CD., Methods: This is a two-centre retrospective cohort study of adult patients with moderate to severe CD on upadacitinib. The primary outcome was clinical response and remission as determined by stool frequency and abdominal pain scores. Secondary endpoints included endoscopic response and remission as determined by change in Simple Endoscopic Score for CD. Outcomes were assessed at 3 months after starting upadacitinib and at the patient's most recent follow-up. We further evaluated adverse events and dose-related response., Results: A total of 45 CD patients received upadacitinib and were included in the safety analysis. Thirty-six patients received upadacitinib for CD, whereas nine received it for inflammatory arthritis [n = 8] or pyoderma [n = 1]. Thirty-three patients received upadacitinib for 3 months or longer and were included in the efficacy analysis. At the 3-month follow-up, 21 patients achieved clinical response [63.6%] and nine achieved clinical remission [27.2%]. At time of last follow-up, 23 patients had clinical response [69.7%], ten achieved clinical remission [30.3%] and four [28.6%] achieved endoscopic remission. Adverse events occurred in 12 patients [26.7%]. Two patients had a serious adverse event [4.5%] without associated mortality., Conclusion: In this real-world cohort of highly refractory CD patients, upadacitinib was effective in inducing remission and had an acceptable safety profile., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Disruption of the intestinal epithelial barrier is a hallmark of mucosal inflammation. It increases exposure of the immune system to luminal microbes, triggering a perpetuating inflammatory response. For several decades, the inflammatory stimuli-induced breakdown of the human gut barrier was studied in vitro by using colon cancer derived epithelial cell lines. While providing a wealth of important data, these cell lines do not completely mimic the morphology and function of normal human intestinal epithelial cells (IEC) due to cancer-related chromosomal abnormalities and oncogenic mutations. The development of human intestinal organoids provided a physiologically-relevant experimental platform to study homeostatic regulation and disease-dependent dysfunctions of the intestinal epithelial barrier. There is need to align and integrate the emerging data obtained with intestinal organoids and classical studies that utilized colon cancer cell lines. This review discusses the utilization of human intestinal organoids to dissect the roles and mechanisms of gut barrier disruption during mucosal inflammation. We summarize available data generated with two major types of organoids derived from either intestinal crypts or induced pluripotent stem cells and compare them to the results of earlier studies with conventional cell lines. We identify research areas where the complementary use of colon cancer-derived cell lines and organoids advance our understanding of epithelial barrier dysfunctions in the inflamed gut and identify unique questions that could be addressed only by using the intestinal organoid platforms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lechuga, Braga-Neto, Naydenov, Rieder and Ivanov.)

Background & Aims: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored., Methods: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models., Results: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3 + T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis., Conclusion: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The implementation of biologic therapy has improved the treatment and clinical course of patients with inflammatory bowel disease since the initial approval of infliximab for Crohn's disease in 1998. However, the efficacy and safety profiles of currently available therapies are still less than optimal in several ways, highlighting the need for novel therapeutic targets. Several new drug classes (Janus kinase inhibitors, anti-integrins, sphingosine-1-phosphate receptor modulators, anti-interleukin-23 antibodies, and stem cell therapies) are currently being studied in Crohn's disease and ulcerative colitis with promising results. This article reviews the current literature and provides an updated overview of the emerging therapies., (Copyright © 2022, Gastro-Hep Communications, Inc.)

Background: Microscopic colitis (MC) is suspected to result from increased immune activity in gut mucosa. Immune checkpoint inhibitors (ICIs) treat cancer by activating the immune system, and further investigation is needed regarding their role in the development of MC., Methods: A retrospective case series investigated cases of endoscopically and histologically confirmed MC developing after administration of ICIs. Clinical notes and medication administration records were reviewed for demographics, symptom duration, and treatment response., Results: Nineteen cases of de novo MC were identified, with 95% of cases requiring steroid treatment, 53% presenting with hospitalization, and colitis-related mortality in 1 individual. Symptom onset occurred a median of 160 days after initiation of ICI therapy and 53 days after their most recent dose of therapy. Patients had a median of 125 days of symptoms, and ICI therapy was held in 70% of individuals due for treatment., Conclusions: MC can develop after ICI administration, and presents with severe symptoms, often requiring hospitalization and steroid treatment. In certain individuals this can require a prolonged treatment course of steroid therapy or immunomodulators. Individuals developing diarrhea after ICI therapy warrant thorough workup including endoscopy and rapid treatment initiation given the disease severity observed in this series., (© The Author(s) 2022. Published by Oxford University Press on behalf of Crohn's & Colitis Foundation.)

Background and Aims: First-degree relatives of gastric cancer patients are at increased risk of developing gastric cancer. Increased oxidative stress, including lipid peroxidation, has been associated with gastric carcinogenesis. Whether first-degree relatives of gastric cancer patients have increased oxidative stress remains unknown. We aimed to compare oxidative stress in patients with gastric cancer, their first-degree relatives, and dyspeptic controls., Methods: A total of 155 patients undergoing upper endoscopy were prospectively enrolled, including 50 with gastric cancer, 49 first-degree relatives of gastric cancer patients, and 56 controls. Serum concentrations of malondialdehyde (MDA) and glutathione) and activities of superoxide dismutase (SOD) and catalase were measured. Multivariate analysis adjusting for sex, age, smoking status, and alcohol consumption was performed., Results: Lipid peroxidation, as measured by concentration of MDA (nmol/mL), was higher ( p = 0.04), and glutathione levels were lower ( p < 0.001) in the gastric cancer group compared to controls. There was no difference in the catalase activity among the groups. There was no difference in glutathione and MDA concentration or catalase activity between the different stages of gastric cancer based on the TNM classification. Relatives of gastric cancer patients had higher glutathione concentration ( μ mol/mL) compared to gastric cancer patients (262.5 vs. 144.6; p = 0.018), while there was no difference in MDA concentration. Catalase and superoxide dismutase activity were lower in the gastric cancer group (3.82 vs. 0.91; p < 0.001 and 1.04 vs. 0.6; p < 0.001) compared to their first-degree relatives. Interestingly, MDA concentration in the first-degree relative group was higher than in the control group (7.9 vs. 5.1; p = 0.03)., Conclusions: In this study, similarly to gastric cancer patients, their first-degree relatives were found to have increased oxidative stress compared to controls. Further studies are warranted to validate this observation and to better understand the role of oxidative stress as a possible biomarker in this population., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Manuel B. Braga-Neto et al.)

FOXP3+ Tregs are expanded within the inflamed intestine of human Crohn's disease, yet FOXP3-mediated gene repression within these cells is lost. The polycomb repressive complexes play a role in FOXP3 target gene regulation, but deeper mechanistic insight is incomplete. We have now specifically identified the polycomb-repressive complex 1 (PRC1) family member, BMI1 in the regulation of a proinflammatory enhancer network in both human and murine Tregs. Using human Tregs and lamina propria T cells, we inferred PRC1 to regulate Crohn's associated gene networks through assays of chromatin accessibility. Conditional deletion of BMI1 in murine FOXP3+ cells led to systemic inflammation. BMI1-deficient Tregs beared a TH1/TH17-like phenotype as assessed by assays of genome wide transcription, chromatin accessibility and proteomic techniques. Finally, BMI1 mutant FOXP3+ cells did not suppress colitis in the adoptive transfer model of human inflammatory bowel disease. We propose that BMI1 plays an important role in enforcing Treg identity in vitro and in vivo. Loss of Treg identity via genetic or transient BMI1 depletion perturbs the epigenome and converts Tregs into Th1/Th17-like proinflammatory cells, a transition relevant to human Crohn's disease associated CD4+ T cells.

The etiology of inflammatory bowel disease (IBD) has yet to be fully understood; however, it is thought to be a result of genetic, immunologic, and environmental factors, including changes in the gut microbiome. 1 , 2 Immune checkpoint inhibitors (ICI) have revolutionized treatment of advanced cancer. They activate the immune system by promoting cytotoxic T-cell survival and antitumor effects. A total of 7 ICIs currently are approved by the United States Food and Drug Administration, and target cytotoxic T lymphocyte-associated protein 4 (ipilimumab); anti-programmed cell death 1 (PD-1) (nivolumab, pembrolizumab, and cemiplimab), or anti-PD-ligand 1 (atezolizumab, durvalumab, and avelumab). However, by activating the immune system, these medications also can lead to off-target inflammation and autoimmunity, including ICI-induced colitis, which has been reported in up to 13.6% of patients. 3 ., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Certolizumab pegol (CZP) has been successfully used for the treatment of Crohn disease (CD); however, real-world data regarding the utility of CZP trough levels (CTLs) are lacking. We aimed to correlate CTL with CD outcomes and to determine frequency of CZP antibodies., Methods: Retrospective evaluation of all CD patients on maintenance CZP with CTL obtained between 2016 and 2019. Outcomes included: median CTL, presence of anti-CZP antibodies, biochemical response (BR), clinical response (CR), radiologic response (RR), radiologic healing (RH), and mucosal healing (MH)., Results: Seventy-seven CD patients were included. Median CTL was 18.9 µg/mL (interquartile range, 7.6-35.4). Twenty-three patients (27.3%) had positive antibody levels, with lower median CTL compared to patients with no antibodies (0.0 vs 29.8; P < 0.0001). Median CTL levels were higher in patients with vs without CR (30.4 vs 10.3 µg/mL; P = 0.0015) and RR (29.6 vs 5.8 µg/mL; P = 0.006). CZP dosing at least every 2 weeks was associated with higher odds of achieving MH (odds ratio, 3.2; 95% confidence interval, 1.03-9.97). CTL resulted in change in clinical management in 62.7% of cases and presence of CMZ antibodies was associated with an odds ratio of 5.83 (95% confidence interval, 1.57-21.73) of change in management. Receiver operating characteristic curve and quartile analysis suggested that CTL >19 µg/mL is associated with increased rates of CR and RR., Conclusions: Higher CTL was significantly associated with CR and RR. The rate of CZP antibodies was 27.3%. Our data suggest maintenance CTL of ≥19 µg/mL should be achieved in order to optimize outcomes in clinical practice., (© The Author(s) 2021. Published by Oxford University Press on behalf of Crohn's & Colitis Foundation.)

Objective: The purpose of this study was to investigate the impact of obesity on disease activity and disease outcome in patients with inflammatory bowel disease., Patients and Methods: The impact of obesity on inflammatory bowel disease disease activity and outcome was retrospectively assessed in 3075 patients enrolled in the prospective nation-wide Swiss inflammatory bowel disease cohort between July 2006 and September 2018. Baseline characteristics, disease activity and disease course in 325 obese inflammatory bowel disease patients (body mass index ≥30 kg/m 2 ) were compared to 1725 normal weight inflammatory bowel disease individuals (body mass index 18.5-24.9)., Results: Among 3075 patients in the prospective Swiss inflammatory bowel disease cohort, 325 patients (10.6%) were obese, namely, 194 Crohn's disease patients, 131 ulcerative colitis, and inflammatory bowel disease-unclassified patients. Disease activity scores were elevated in obese Crohn's disease (Crohn's Disease Activity Index 33 vs 20, p  = 0.001), but not ulcerative colitis patients. Obese Crohn's disease, but not ulcerative colitis patients were less likely to be in remission based on a Crohn's Disease Activity Index less than 100 and a calprotectin less than 100 ug/g. In a multivariate regression model, obesity was negatively associated with disease remission in Crohn's disease (odds ratio 0.610, 95% confidence interval 0.402-0.926, p  = 0.020), but not ulcerative colitis. Increased soft stool frequency was observed in both obese Crohn's disease and ulcerative colitis patients. Adjusted Cox regression models revealed increased risk of complicated disease course in obese Crohn's disease patients (hazard ratio 1.197, 95% confidence interval 1.046-1.370, p  = 0.009). No association between obesity and disease progression, index treatment failure was seen neither in Crohn's disease nor ulcerative colitis., Conclusion: Obesity is associated with decreased rates of disease remission and increased risk of complicated disease course in Crohn's disease over a six-year follow-up period. No effects were seen on disease progression and index treatment failure neither in Crohn's disease nor ulcerative colitis.

Background: An association between inflammatory bowel disease (IBD) and obesity has been observed. Little is known about the effect of weight loss on IBD course. Our aim was to determine the impact of bariatric surgery on long-term clinical course of obese patients with IBD, either Crohn's disease (CD) or ulcerative colitis (UC)., Methods: Patients with IBD who underwent bariatric surgery subsequent to IBD diagnosis were identified from 2 tertiary IBD centers. Complications after bariatric surgery were recorded. Patients were matched 1:1 for age, sex, IBD subtype, phenotype, and location to patients with IBD who did not undergo bariatric surgery. Controls started follow-up at a time point in their disease similar to the disease duration in the matched case at the time of bariatric surgery. Inflammatory bowel disease medication usage and disease-related complications (need for corticosteroids, hospitalizations, and surgeries) among cases and controls were compared., Results: Forty-seven patients met inclusion criteria. Appropriate matches were found for 25 cases. Median follow-up among cases (after bariatric surgery) and controls was 7.69 and 7.89 years, respectively. Median decrease in body mass index after bariatric surgery was 12.2. Rescue corticosteroid usage and IBD-related surgeries were numerically less common in cases than controls (24% vs 52%; odds ratio [OR], 0.36; 95% confidence interval [CI], 0.08-1.23; 12% vs 28%; OR, 0.2; 95% CI, 0.004-1.79). Two cases and 1 control were able to discontinue biologics during follow-up., Conclusions: Inflammatory bowel disease patients with weight loss after bariatric surgery had fewer IBD-related complications compared with matched controls. This observation requires validation in a prospective study design., (© 2019 Crohn's & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The aetiology of Crohn's disease [CD] involves immune dysregulation in a genetically susceptible individual. Genome-wide association studies [GWAS] have identified 200 loci associated with CD, ulcerative colitis, or both, most of which fall within non-coding DNA regions. Long non-coding RNAs [lncRNAs] regulate gene expression by diverse mechanisms and have been associated with disease activity in inflammatory bowel disease. However, disease-associated lncRNAs have not been characterised in pathogenic immune cell populations., Methods: Terminal ileal samples were obtained from 22 CD patients and 13 controls. RNA from lamina propria CD4+ T cells was sequenced and long intergenic non-coding RNAs [lincRNAs] were detected. Overall expression patterns, differential expression [DE], and pathway and gene enrichment analyses were performed. Knockdown of novel lincRNAs XLOC&#95;000261 and XLOC&#95;000014 was performed. Expression of Th1 or Th17-associated transcription factors, T-bet and RORγt, respectively, was assessed by flow cytometry., Results: A total of 6402 lincRNAs were expressed, 960 of which were novel. Unsupervised clustering and principal component analysis showed that the lincRNA expression discriminated patients from controls. A total of 1792 lincRNAs were DE, and 295 [79 novel; 216 known] mapped to 267 of 5727 DE protein-coding genes. The novel lincRNAs were enriched in inflammatory and Notch signalling pathways [p <0.05]. Furthermore, DE lincRNAs in CD patients were more frequently found in DNA regions with known inflammatory bowel disease [IBD]-associated loci. The novel lincRNA XLOC&#95;000261 negatively regulated RORγt expression in Th17 cells., Conclusions: We describe a novel set of DE lincRNAs in CD-associated CD4+ cells and demonstrate that novel lincRNA XLOC&#95;000261 appears to negatively regulate RORγt protein expression in Th17 cells., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Clostridium difficile causes intestinal inflammation, which increases adenosine. We compared the expression of adenosine receptors (AR) subtypes A1, A2A, A2B, and A3 in HCT-8, IEC-6 cells, and isolated intestinal epithelial cells, challenged or not with Clostridium difficile toxin A and B (TcdA and TcdB) or infection (CDI). In HCT-8, TcdB induced an early A2BR expression at 6 h and a late A2AR expression at 6 and 24 h. In addition, both TcdA and TcdB increased IL-6 expression at all time-points (peak at 6 h) and PSB603, an A2BR antagonist, decreased IL-6 expression and production. In isolated cecum epithelial cells, TcdA induced an early expression of A2BR at 2s and 6 h, followed by a late expression of A2AR at 6 and 24 h and of A1R at 24 h. In CDI, A2AR and A2BR expressions were increased at day 3, but not at day 7. ARs play a role in regulating inflammation during CDI by inducing an early pro-inflammatory and a late anti-inflammatory response. The timing of interventions with AR antagonist or agonists may be of relevance in treatment of CDI.

Background: Gastric cancer (GC) is an important cause of morbidity and mortality worldwide. However, population-based data on GC mortality dynamics in low and middle income countries are scarce., Methods: We analyzed GC mortality in Brazil based on all GC-related deaths registered 2000-2015., Results: A total of 17,374,134 deaths were recorded, with GC identified in 214,808 (1.24%) cases-203,941 (94.9%) as underlying cause, and 10,867 (5.1%) as associated cause of death. Adjusted rates for age and sex was 6.85 deaths/100,000 inhabitants [95% confidence interval (CI) 6.73-6.97]. The highest mortality rates were found in males [10.00; rate ratio (RR) 1.85; 95% CI 1.78-1.91; p < 0.0001] and patients ≥ 45 years of age (24.98; RR 3.79; 95% CI 3.55-4.05; p < 0.0001). The South (7.56; RR 1.62; 95% CI 1.50-1.76; p < 0.0001) and Southeast (7.36; RR 1.59; 95% CI 1.48-1.71; p < 0.0001) regions had the highest regional rates. Spatial and spatiotemporal high-risk mortality areas in 2004-2007 were located mainly in the South, Southeast, and Central-West regions. After 2008, the Northeast region became a high-risk area, especially Ceará State., Conclusion: GC remains a significant public health problem with high mortality burden and unequal distribution in Brazilian states. The new patterns in poorer regions and the high risk in some specific populations show a clear process of epidemiological transition over time. There is a need to strengthen nationwide epidemiological monitoring, surveillance, prevention, and control for GC in the country.

T cell lineage decisions are critical for the development of proper immune responses to pathogens as well as important for the resolution of inflammatory responses. This differentiation process relies on a combination of intrinsic and extrinsic factors converging upon epigenetic regulation of transcriptional networks relevant to specific T cell lineages. As these biochemical modifications represent therapeutic opportunities in cancer biology and autoimmunity, implications of writers and readers of epigenetic marks to immune cell differentiation and function are highly relevant. Given the ready adoption of histone methyltransferase inhibitors in the clinic, we focus this review on the role of three histone modifying complexes: PRC-1, PRC-2, and G9A in modulating T cell fate decisions. Furthermore, we explore the role of long non-coding RNAs in regulating these processes, and discuss recent advances and challenges of implementing epigenetic therapies into clinical practice.

Background & Aims: Forkhead box protein 3 (FOXP3) + regulatory T cell (Treg) dysfunction is associated with autoimmune diseases; however, the mechanisms responsible for inflammatory bowel disease pathophysiology are poorly understood. Here, we tested the hypothesis that a physical interaction between transcription factor FOXP3 and the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is essential for gene co-repressive function., Methods: Human FOXP3 mutations clinically relevant to intestinal inflammation were generated by site-directed mutagenesis. T lymphocytes were isolated from mice, human blood, and lamina propria of Crohn's disease (CD) patients and non-CD controls. We performed proximity ligation or a co-immunoprecipitation assay in FOXP3-mutant + , interleukin 6 (IL6)-treated or CD-CD4 + T cells to assess FOXP3-EZH2 protein interaction. We studied IL2 promoter activity and chromatin state of the interferon γ locus via luciferase reporter and chromatin-immunoprecipitation assays, respectively, in cells expressing FOXP3 mutants., Results: EZH2 binding was abrogated by inflammatory bowel disease-associated FOXP3 cysteine 232 (C232) mutation. The C232 mutant showed impaired repression of IL2 and diminished EZH2-mediated trimethylation of histone 3 at lysine 27 on interferon γ, indicative of compromised Treg physiologic function. Generalizing this mechanism, IL6 impaired FOXP3-EZH2 interaction. IL6-induced effects were reversed by Janus kinase 1/2 inhibition. In lamina propria-derived CD4 + T cells from CD patients, we observed decreased FOXP3-EZH2 interaction., Conclusions: FOXP3-C232 mutation disrupts EZH2 recruitment and gene co-repressive function. The proinflammatory cytokine IL6 abrogates FOXP3-EZH2 interaction. Studies in lesion-derived CD4 + T cells have shown that reduced FOXP3-EZH2 interaction is a molecular feature of CD patients. Destabilized FOXP3-EZH2 protein interaction via diverse mechanisms and consequent Treg abnormality may drive gastrointestinal inflammation.

Background and Aims: Gastrointestinal bleeding (GIB) in the setting of thrombocytopenia raises concerns about endoscopic procedure risk. We aimed to assess the safety and outcomes of endoscopy for overt GIB in the setting of severe thrombocytopenia in liver cirrhosis (LC) and non-liver cirrhosis (NLC)., Methods: This is a retrospective study on inpatients who underwent endoscopy within 24 hours of presentation for overt GIB with a platelet count (PC) of 20 to <50 × 10 3 /mL. Outcomes included diagnostic and therapeutic yields, procedural adverse events, packed red blood cell (pRBC) and platelet transfusions, recurrent bleeding rate, and all-cause and GIB-related mortality., Results: One hundred forty-four patients were identified. The median PC was 41 × 10 3 /mL and 61% had LC. The diagnostic yield was 68% (LC = 61%, NLC = 79%, P = .04). Therapeutic yield was 60% (59% vs 60%, P = 1.00). The initial hemostasis rate was 94% with one adverse event. The median number of pRBC and platelet transfusions decreased after intervention in the entire cohort. Recurrent bleeding rates were 22% at 1 month and 30% at 1 year, with no differences between groups. An increased international normalized ratio (INR) >2 was a predictor of recurrent bleeding. All-cause mortality was 19% at 1 month and 37% at 1 year, whereas GIB-associated mortality in our cohort was only 3% at 1 month and 4% at 1 year, with no significant difference between LC and NLC. Predictors of mortality were INR >2, activated partial thromboplastin time >38 seconds, hypotension, intensive care unit admission, and pulmonary comorbidities., Conclusion: In this study cohort, we observed that endoscopy for overt GIB in the setting of severe thrombocytopenia in patients with LC and NLC appears safe, has moderate diagnostic and therapeutic yields with high initial hemostasis rate, and is associated with a significant decrease in pRBC and platelet transfusions. Recurrent bleeding and all-cause mortality rates remain high., (Copyright © 2018 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Aim: To characterize isolated non-obstructive sinusoidal dilatation (SD) by identifying associated conditions, laboratory findings, and histological patterns., Methods: Retrospectively reviewed 491 patients with SD between 1995 and 2015. Patients with obstruction at the level of the small/large hepatic veins, portal veins, or right-sided heart failure were excluded along with history of cirrhosis, hepatic malignancy, liver transplant, or absence of electrocardiogram/cardiac echocardiogram. Liver histology was reviewed for extent of SD, fibrosis, red blood cell extravasation, nodular regenerative hyperplasia, hepatic peliosis, and hepatocellular plate atrophy (HPA)., Results: We identified 88 patients with non-obstructive SD. Inflammatory conditions (32%) were the most common cause. The most common pattern of liver abnormalities was cholestatic (76%). Majority (78%) had localized SD to Zone III. Medication-related SD had higher proportion of portal hypertension (53%), ascites (58%), and median AST (113 U/L) and ALT (90 U/L) levels. Nineteen patients in our study died within one-year after diagnosis of SD, majority from complications related to underlying diseases., Conclusion: Significant proportion of SD and HPA exist without impaired hepatic venous outflow. Isolated SD on liver biopsy, in the absence of congestive hepatopathy, requires further evaluation and portal hypertension should be rule out., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest relevant to this article were reported.

Background: Case reports of inflammatory bowel diseases [IBD] have been reported in patients with a history of bariatric surgery. Our aim was to characterize patients who were diagnosed with IBD after having undergone bariatric surgery., Methods: Electronic medical records were reviewed at two institutions to identify patients who developed de-novo Crohn's disease or ulcerative colitis [UC] after bariatric surgery. Data on demographics, type of bariatric surgical procedure, IBD subtype, phenotype and medication usage were obtained. The incidence rate of de-novo IBD after bariatric surgery [per 100000 person-years] and standardized incidence ratio [SIR] were estimated from a prospective bariatric surgery database., Results: A total of 44 patients with de-novo IBD after bariatric surgery were identified [31 Crohn's disease, 12 UC, one IBD unclassified]. Most patients were female [88.6%], with median age at IBD onset of 44 years [IQR, 37-52] and median time to IBD diagnosis after bariatric surgery of 7 years [IQR, 3-10]. Sixty-eight per cent underwent Roux-en-Y gastric bypass. In the prospective database, the incidence of IBD in patients who underwent bariatric surgery was 26.7 per 100000 person-years [4.5 for UC and 22.3 for Crohn's disease]. The age-adjusted SIR ranged from 3.56 in the 40-49 year age group to 4.73 in the 30-39 year age group., Conclusion: We described a case series of patients developing de-novo IBD after bariatric surgery. There appears to be a numerically higher incidence of Crohn's disease in this population. Confirmation of causality is required in larger patient cohorts.

Background: It has been suggested that distal gastric carcinoma (GC) in younger patients has a more aggressive outcome than in older patients, however this is a controversial issue. The aim of this study was to compare clinicopathological features between younger and older patients with GC in Northeastern Brazil., Methods: A total of 207 patients with distal GC (41 patients ≤45 years, considered younger group, and 166 > 45 years, considered older group) were evaluated prospectively during a 6 year period., Results: The mean patient age in the young group was 37.41 years old and 64.43 years in the older group. No significant difference was found regarding gender, area of residence, history of alcohol consumption, chronic tobacco smoking. Prevalence of first-degree GC history was 12.5% (7.3% in younger group vs. 13.9% in older; p <  0.46). The most frequent symptom was gastric pain and weight loss. Diffuse infiltrative cancer was more frequently seen in younger patients (70.70% vs. 33.70%, respectively; p <  0.01), as was histologically less differentiated tumors (63.40% vs. 33.10%; p <  0.01) and stage IV of GC (48.80% vs. 30.70%; p <  0.015). Five-year survival, evaluated in 82 patients, was lower in younger patients (p = 0.045); however, after adjusting for stage of GC in the multivariate analysis, this association did not remain significant. Family history of GC and gender had no impact on survival., Conclusions: Younger patients showed higher prevalence of diffuse type of Lauren and lower survival that was attributed to higher rate of advanced stage of GC. Gastric cancer screening strategies should also be considered in younger individuals, especially in areas of high prevalence. Further studies are warranted to determine risk factors associated with gastric cancer in young adults.

Background: Clostridium difficile is an anaerobic bacterium that causes antibiotic-associated diarrhea. It produces toxin A and toxin B (TcdB), which cause injury to the gut epithelium. Glutamine is a fundamental fuel for enterocytes, maintaining intestinal mucosal health. Alanyl-glutamine (AQ) is a highly soluble dipeptide derivative of glutamine. We studied whether administration of AQ ameliorates the effects of TcdB in the intestinal cells and improves the outcome of C. difficile infection in mice., Methods: WST-1 proliferation and cell-wounding-migration assays were assessed in IEC-6 cells exposed to TcdB, with or without AQ. Apoptosis and necrosis were assessed using Annexin V and flow cytometry. C57BL/6 mice were infected with VPI 10463 and treated with either vancomycin, AQ, or vancomycin with AQ. Intestinal tissues were collected for histopathologic analysis, apoptosis staining, and determination of myeloperoxidase activity., Results: AQ increased proliferation in intestinal cells exposed to TcdB, improved migration at 24 and 48 hours, and reduced apoptosis in intestinal cells challenged with TcdB. Infected mice treated with vancomycin and AQ had better survival and histopathologic findings than mice treated with vancomycin alone., Conclusions: AQ may reduce intestinal mucosal injury in C. difficile-infected mice by partially reversing the effects of TcdB on enterocyte proliferation, migration, and apoptosis, thereby improving survival from C. difficile infection.

The accuracy of a nested PCR in gastric DNA obtained by a string test for the diagnosis of Helicobacter pylori infection in asymptomatic children was 94.0%. The cagA-positive toxigenic vacAs1m1 strains were the most prevalent strains, indicating that this population is colonized early by the strains associated with gastric cancer.

Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression.

Purpose: Human Immunodeficiency Virus (HIV) protease inhibitors (PI) remain a crucial component of highly active therapy (HAART) and recently have been demonstrated to have potent antitumor effect on a wide variety of tumor cell lines. However, discontinuation of therapy is an important issue, which may be related to various side-effects, especially diarrhea. The aim of this study was to evaluate the effects of nelfinavir (NFV), an HIV PI, and of alanyl-glutamine (AQ) supplementation, on intestinal cell migration, proliferation, apoptosis and necrosis, using IEC-6 cells and on intestinal crypt depth, villus length, villus area, mitotic index and apoptosis in Swiss mice., Methods: Migration was evaluated at 12 and 24 h after injury using a wound healing assay. Cellular proliferation was measured indirectly at 24 and 48 h using tetrazolium salt WST-1. Apoptosis and necrosis were measured by flow cytometry using the Annexin V assay. Intestinal morphometry and mitotic index in vivo were assessed following a seven-day treatment with 100 mg/kg of NFV, given orally. In vivo proliferation and apoptosis were evaluated by intestinal crypt mitotic index and immunohistochemistry, respectively., Results: In vitro, AQ supplementation enhanced IEC-6 cell migration and proliferation, following challenge with NFV. In vivo, AQ increased intestinal villus length, villus area, crypt depth and cell proliferation and cell migration, following treatment with NFV. AQ did not decrease cell death induced by NFV both in vivo and in vitro., Conclusions: AQ supplementation is potentially beneficial in preventing the effects of PIs, such as NFV, in the intestinal tract.

Background: To evaluate the prevalence of more virulent H. pylori genotypes in relatives of gastric cancer patients and in patients without family histories of gastric cancer., Methods: We evaluated prospectively the prevalence of the infection by more virulent H. pylori strains in 60 relatives of gastric cancer patients comparing the results with those obtained from 49 patients without family histories of gastric cancer. H. pylori status was determined by the urease test, histology and presence of H. pylori ureA. The cytotoxin associated gene (cagA), the cagA-EPIYA and vacuolating cytotoxin gene (vacA) were typed by PCR and the cagA EPIYA typing was confirmed by sequencing., Results: The gastric cancer relatives were significant and independently more frequently colonized by H. pylori strains with higher numbers of CagA-EPIYA-C segments (OR = 4.23, 95%CI = 1.53-11.69) and with the most virulent s1m1 vacA genotype (OR = 2.80, 95%CI = 1.04-7.51). Higher numbers of EPIYA-C segments were associated with increased gastric corpus inflammation, foveolar hyperplasia and atrophy. Infection by s1m1 vacA genotype was associated with increased antral and corpus gastritis., Conclusions: We demonstrated that relatives of gastric cancer patients are more frequently colonized by the most virulent H. pylori cagA and vacA genotypes, which may contribute to increase the risk of gastric cancer.

Helicobacter pylori causes chronic gastric inflammation and significantly increases the risk of duodenal and gastric ulcer disease and distal gastric carcinoma. In this study, we evaluated the Helicobacter pylori vacA and cagA genotypes in patients from a Brazilian region where there is a high prevalence of gastric cancer. Polymerase chain reaction (PCR) was used to investigate vacA mosaicism and cagA status in the gastric mucosa of 134 H. pylori-positive patients, including 76 with gastritis: 28 with peptic ulcer disease and 30 with gastric cancer. The s1m1 variant was the predominant vacA genotype observed, whereas the s1 allele was more frequently observed in patients with more severe diseases associated with H. pylori infection [p = 0.03, odds ratio (OR) = 5.72, 95% confidence interval (CI) = 1.15-38.60]. Furthermore, all of the s1 alleles were s1b. Mixed vacA m1/m2 strains were found more frequently in patients with gastric cancer and a cagA-positive status was significantly associated with gastric cancer (p = 0.016, OR = 10.36, 95% CI = 1.35-217.31). Patients with gastric cancer (21/21, 100%, p = 0.006) or peptic ulcers (20/21, 95%, p = 0.02) were more frequently colonised by more virulent H. pylori strains compared to gastritis patients (41/61, 67.2%). In conclusion, in the northeastern of Brazil, which is one of the regions with the highest prevalence of gastric cancer in the country, infection with the most virulent H. pylori strains, carrying the cagA gene and s1m1 vacA alleles, predominates and is correlated with more severe H. pylori-associated diseases.

Background: Helicobacter pylori infection is acquired predominantly in childhood. There is also evidence that children loss the infection. Therefore, factors that account for children remain infected need to be investigated because once established the infection persists throughout the life unless treated., Methods: This study aimed to evaluate the H. pylori infection in children of a low-income community at baseline and 8years later to determine the predictor factors linked to the maintenance, acquisition, and loss of the infection using regression models of generalized estimating equations. H. pylori status was determined by (13) C-urea breath test., Results: Data from 37.7% (133/353) of the children were available. No difference between the characteristics of the included and nonincluded children was observed. The prevalence of infection increased from 53.4 to 64.7%. Thirty-nine children (29.3%) remained noninfected, 47.4% remained infected, 17.3% became infected, and 6.0% lost the infection. Factors associated with to remain infected compared with to remain noninfected included the age, increased number of children in the household, and the use of well water instead of municipal water. The acquisition of the infection was associated with the male gender., Conclusion: Factors linked to remain and to gain H. pylori infection in a poor region were increased number of children in the household and the male gender. Also, the acquisition rates were higher than the loss rates, which lead to an increase in the infection prevalence with age., (© 2011 Blackwell Publishing Ltd.)

Background: Methotrexate treatment has been associated to intestinal epithelial damage. Studies have suggested an important role of nitric oxide in such injury. The aim of this study was to investigate the role of nitric oxide (NO), specifically iNOS on the pathogenesis of methotrexate (MTX)-induced intestinal mucositis., Methods: Intestinal mucositis was carried out by three subcutaneous MTX injections (2.5 mg/kg) in Wistar rats and in inducible nitric oxide synthase knock-out (iNOS-/-) and wild-type (iNOS+/+) mice. Rats were treated intraperitoneally with the NOS inhibitors aminoguanidine (AG; 10 mg/Kg) or L-NAME (20 mg/Kg), one hour before MTX injection and daily until sacrifice, on the fifth day. The jejunum was harvested to investigate the expression of Ki67, iNOS and nitrotyrosine by immunohistochemistry and cell death by TUNEL. The neutrophil activity by myeloperoxidase (MPO) assay was performed in the three small intestine segments., Results: AG and L-NAME significantly reduced villus and crypt damages, inflammatory alterations, cell death, MPO activity, and nitrotyrosine immunostaining due to MTX challenge. The treatment with AG, but not L-NAME, prevented the inhibitory effect of MTX on cell proliferation. MTX induced increased expression of iNOS detected by immunohistochemistry. MTX did not cause significant inflammation in the iNOS-/- mice., Conclusion: These results suggest an important role of NO, via activation of iNOS, in the pathogenesis of intestinal mucositis.

Background: This study conducted in Northeastern Brazil, evaluated the prevalence of H. pylori infection and the presence of gastritis in HIV-infected patients., Methods: There were included 113 HIV-positive and 141 age-matched HIV-negative patients, who underwent upper gastrointestinal endoscopy for dyspeptic symptoms. H. pylori status was evaluated by urease test and histology., Results: The prevalence of H. pylori infection was significantly lower (p < 0.001) in HIV-infected (37.2%) than in uninfected (75.2%) patients. There were no significant differences between H. pylori status and gender, age, HIV viral load, antiretroviral therapy and the use of antibiotics. A lower prevalence of H. pylori was observed among patients with T CD4 cell count below 200/mm3; however, it was not significant. Chronic active antral gastritis was observed in 87.6% of the HIV-infected patients and in 780.4% of the control group (p = 0.11). H. pylori infection was significantly associated with chronic active gastritis in the antrum in both groups, but it was not associated with corpus chronic active gastritis in the HIV-infected patients., Conclusion: We demonstrated that the prevalence of H. pylori was significantly lower in HIV-positive patients compared with HIV-negative ones. However, corpus gastritis was frequently observed in the HIV-positive patients, pointing to different mechanisms than H. pylori infection in the genesis of the lesion.

Background and Aims: To further evaluate intrafamilial transmission of H. pylori infection during childhood, we investigated the prevalence of H. pylori in family members from a poor H. pylori high-prevalence urban community in the Northeast of Brazil., Methods: H. pylori infection was investigated in 570 members of 128 households, by (13) C-urea breath test in children and by ELISA in mothers and other adult relatives., Results: The overall prevalence of H. pylori infection (376/570) increased with age (p < .001) and ranged from 28.9%, in children aged 6 months to 5 years, to 82% in adults over 40 years. An H. pylori positive mother and the number of infected siblings are independent risk factors for childhood H. pylori infection (OR = 2.2, 95% CI = 1.0-4.6 and OR = 4.3, 95% CI = 2.3-8.1, respectively) The number of siblings, number of younger siblings, and number of infected younger siblings were also associated with the infection in the univariate analysis. The number of infected younger siblings remained independently associated with the infection (p = .000), even after controlling for all the above cited variables, in addition to the H. pylori status of siblings and mothers, age, number of people per room, and number of children in the household., Conclusion: The transmission of H. pylori occurs from infected mothers to their offspring and among siblings, notably from younger siblings to the older ones., (© 2010 Blackwell Publishing Ltd.)

Background: Protease inhibitors (PI's) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable side-effects, such as diarrhea. This study aims to investigate the effects of selected antiretroviral agents on intestinal histopathology and function in vivo and on cell proliferation and death in vitro., Methods: Selected antiretroviral drugs were given orally over 7 days, to Swiss mice, as follows: 100 mg/kg of nelfinavir (NFV), indinavir (IDV), didanosine (DDI) or 50 mg/kg of zidovudine (AZT). Intestinal permeability measured by lactulose and mannitol assays; net water and electrolyte transport, in perfused intestinal segments; and small intestinal morphology and cell apoptosis were assessed in treated and control mice. In vitro cell proliferation was evaluated using the WST-1 reagent and apoptosis and necrosis by flow cytometry analysis., Results: NFV, IDV, AZT and DDI caused significant reductions in duodenal and in jejunal villus length (p < 0.05). IDV and AZT increased crypt depth in the duodenum and AZT increased crypt depth in the jejunum. NFV, AZT and DDI significantly decreased ileal crypt depth. All selected antiretroviral drugs significantly increased net water secretion and electrolyte secretion, except for DDI, which did not alter water or chloride secretion. Additionally, only NFV significantly increased mannitol and lactulose absorption. NFV and IDV caused a significant reduction in cell proliferation in vitro at both 24 h and 48 h. DDI and AZT did not alter cell proliferation. There was a significant increase in apoptosis rates in IEC-6 cells after 24 h with 70 ug/mL of NFV (control: 4.7% vs NFV: 22%) while IDV, AZT and DDI did not show any significant changes in apoptosis compared to the control group. In jejunal sections, IDV and NFV significantly increased the number of TUNEL positive cells., Conclusion: The PI's, NFV and IDV, increased cell apoptosis in vivo, water and electrolyte secretion and intestinal permeability and decreased villus length and cell proliferation. NFV was the only drug tested that increased cell apoptosis in vitro. The nucleoside reverse transcriptase inhibitors, AZT and DDI, did not affect cell apoptosis or proliferation. These findings may partly explain the intestinal side-effects associated with PI's.

In this study, we have examined the role of glutamine derivatives in reducing 5-fluorouracil (5-FU)-induced epithelial damage in an undifferentiated crypt intestinal cell line, IEC-6. In this model, we have investigated proliferation indirectly by detecting the enzyme-derived formazan dye from the tetrazolium salt WST-1 in viable cells at 24 and 48 h after 5-FU treatment. Migration was measured at 12 and 24 h after razor scraping of the cell monolayer. Cell death was measured by quantifying the percentage of apoptotic and necrotic figures by flow cytometry at 12 and 24 h following 5-FU challenge. Neither glutamine nor alanyl-glutamine prevented 5-FU-induced apoptosis and necrosis in IEC-6 cells at 12 and 24 h after 5-FU challenge. However, glutamine and alanyl-glutamine enhanced migration and proliferation when compared with 5-FU-treated controls (P < 0.05). These new findings support our earlier study on the benefit of oral glutamine in enhancing epithelial recovery after 5-FU challenge.

Vitamin A (retinol), a fat-soluble vitamin, is an essential nutrient for the normal functioning of the visual system, epithelial cell integrity and growth, immunity, and reproduction. Our group has investigated the effect of high doses of oral vitamin A on early childhood diarrhea in our prospective community-based studies from Northeast Brazil and found a beneficial role in reducing the mean duration but not incidence of diarrheal episodes. In this study, we explored the role of retinol supplementation in intestinal cell lines following Clostridium difficile toxin A (TxA) challenge. C. difficile is the most common anaerobic pathogen borne with antibiotic-borne diarrhea and pseudomembranous colitis. Since retinol is critical for the integrity of tight junctions and to modulate the cell cycle, we have focused on changes in transepithelial electrical resistance (TEER) in Caco-2, a more differentiated intestinal cell line, and on models of cell proliferation, migration and viability in IEC-6 cells, an undifferentiated crypt cell line, following TxA injury. In this model, retinol therapy reduced apoptosis, improved cell migration and proliferation, and prevented the reduction in TEER, following C. difficile TxA challenge in a glutamine-free medium. These results suggest the role of retinol in protecting intestinal epithelial barrier function from C. difficile TxA enterotoxic damage.

We investigated the prevalence and the risk factors for infection with Helicobacter pylori in a randomly-selected population of adults from a low-income community in Northeastern Brazil. Helicobacter pylori infection was determined by ELISA. Risk factors were assessed using a structured interview. Two hundred and four individuals were included in the study, including 49 males and 155 females, ranging from 18 to 80 years old. Overall, 165 of 204 participants (80%) were H. pylori positive, without significant gender differences (p= 0.49). The infection rate was of 84.7% in subjects 18 to 30 years of age, increasing to 92% in subjects 46-60 years old. Above 60 years old, the prevalence decreased slightly. As a whole, the prevalence of infection did not increase significantly (p=0.147) with age. There were no significant differences in the prevalence of H. pylori infection, when patients were classified by age, smoking habit, educational level, alcohol consumption, the number of persons per room, the number of children per household, the number of adults per household, cup-sharing, household pets, toilet location, number of persons per bed and medical history of antibiotic and raw vegetable ingestion. In conclusion, no risk factors associated with infection was found in these adults, suggesting that the infection, even in a poor population, may be acquired predominantly during childhood; the relatively high prevalence that we observed may be more due to a cohort effect than to acquisition of infection during adulthood.