Hyperglycemic environments directly compromise intestinal epithelial barrier function in an organoid model and hyaluronan (∼35 kDa) protects via a layilin dependent mechanism.

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.

Risk of Primary Gastrointestinal Lymphoma in Patients With Inflammatory Conditions Exposed to Tumor Necrosis Factor Alpha Inhibitors and Immunomodulators: A Case-Control Study.

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 ±â€…6.9 and 9.7 ±â€…8.6 years, respectively. Mean age at time of PGIL diagnosis was 47.5 ±â€…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.

Hypomethylation and Overexpression of Th17-Associated Genes is a Hallmark of Intestinal CD4+ Lymphocytes in Crohn's Disease.

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.

Understanding disruption of the gut barrier during inflammation: Should we abandon traditional epithelial cell lines and switch to intestinal organoids?

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.

Multicentre Real-world Experience of Upadacitinib in the Treatment of Crohn's Disease.

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.

Clinical Course and Impact of Immune Checkpoint Inhibitor Colitis Resembling Microscopic Colitis.

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.

Increased Oxidative Stress in Gastric Cancer Patients and Their First-Degree Relatives: A Prospective Study from Northeastern Brazil.

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 (µmol/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.

BMI1 maintains the Treg epigenomic landscape to prevent inflammatory bowel disease.

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.

Use of Immune Checkpoint Inhibitors in Patients With Pre-established Inflammatory Bowel Diseases: Retrospective Case Series.

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.

Impact of obesity on disease activity and disease outcome in inflammatory bowel disease: Results from the Swiss inflammatory bowel disease cohort.

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/m2) 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.

Impact of Bariatric Surgery on the Long-term Disease Course of Inflammatory Bowel Disease.

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.

Deregulation of Long Intergenic Non-coding RNAs in CD4+ T Cells of Lamina Propria in Crohn's Disease Through Transcriptome Profiling.

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_000261 and XLOC_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_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_000261 appears to negatively regulate RORγt protein expression in Th17 cells.

Disruption of FOXP3-EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation.

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.

Clinical characteristics of distal gastric cancer in young adults from Northeastern Brazil.

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.

De-novo Inflammatory Bowel Disease After Bariatric Surgery: A Large Case Series.

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.

Glutamine and alanyl-glutamine increase RhoA expression and reduce Clostridium difficile toxin-a-induced intestinal epithelial cell damage.

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.

Helicobacter pylori virulence genes detected by string PCR in children from an urban community in northeastern Brazil.

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.

Protective effects of alanyl-glutamine supplementation against nelfinavir-induced epithelial impairment in IEC-6 cells and in mouse intestinal mucosa.

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.

Higher frequency of cagA EPIYA-C phosphorylation sites in H. pylori strains from first-degree relatives of gastric cancer patients.

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.