Epithelial TLR4 Signaling Activates DUOX2 to Induce Microbiota-Driven Tumorigenesis.

BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.

Low-Fat, High-Fiber Diet Reduces Markers of Inflammation and Dysbiosis and Improves Quality of Life in Patients With Ulcerative Colitis.

BACKGROUND & AIMS: A high-fat diet has been associated with an increased risk of ulcerative colitis (UC). We studied the effects of a low-fat, high-fiber diet (LFD) vs an improved standard American diet (iSAD, included higher quantities of fruits, vegetables, and fiber than a typical SAD). We collected data on quality of life, markers of inflammation, and fecal markers of intestinal dysbiosis in patients with UC. METHODS: We analyzed data from a parallel-group, cross-over study of 17 patients with UC in remission or with mild disease (with a flare within the past 18 mo), from February 25, 2015, through September 11, 2018. Participants were assigned randomly to 2 groups and received a LFD (10% of calories from fat) or an iSAD (35%-40% of calories from fat) for the first 4-week period, followed by a 2-week washout period, and then switched to the other diet for 4 weeks. All diets were catered and delivered to patients' homes, and each participant served as her or his own control. Serum and stool samples were collected at baseline and week 4 of each diet and analyzed for markers of inflammation. We performed 16s ribosomal RNA sequencing and untargeted and targeted metabolomic analyses on stool samples. The primary outcome was quality of life, which was measured by the short inflammatory bowel disease (IBD) questionnaire at baseline and week 4 of the diets. Secondary outcomes included changes in the Short-Form 36 health survey, partial Mayo score, markers of inflammation, microbiome and metabolome analysis, and adherence to the diet. RESULTS: Participants' baseline diets were unhealthier than either study diet. All patients remained in remission throughout the study period. Compared with baseline, the iSAD and LFD each increased quality of life, based on the short IBD questionnaire and Short-Form 36 health survey scores (baseline short IBD questionnaire score, 4.98; iSAD, 5.55; LFD, 5.77; baseline vs iSAD, P = .02; baseline vs LFD, P = .001). Serum amyloid A decreased significantly from 7.99 mg/L at baseline to 4.50 mg/L after LFD (P = .02), but did not decrease significantly compared with iSAD (7.20 mg/L; iSAD vs LFD, P = .07). The serum level of C-reactive protein decreased numerically from 3.23 mg/L at baseline to 2.51 mg/L after LFD (P = .07). The relative abundance of Actinobacteria in fecal samples decreased from 13.69% at baseline to 7.82% after LFD (P = .017), whereas the relative abundance of Bacteroidetes increased from 14.6% at baseline to 24.02% on LFD (P = .015). The relative abundance of Faecalibacterium prausnitzii was higher after 4 weeks on the LFD (7.20%) compared with iSAD (5.37%; P = .04). Fecal levels of acetate (an anti-inflammatory metabolite) increased from a relative abundance of 40.37 at baseline to 42.52 on the iSAD and 53.98 on the LFD (baseline vs LFD, P = .05; iSAD vs LFD, P = .09). The fecal level of tryptophan decreased from a relative abundance of 1.33 at baseline to 1.08 on the iSAD (P = .43), but increased to a relative abundance of 2.27 on the LFD (baseline vs LFD, P = .04; iSAD vs LFD, P = .08); fecal levels of lauric acid decreased after LFD (baseline, 203.4; iSAD, 381.4; LFD, 29.91; baseline vs LFD, P = .04; iSAD vs LFD, P = .02). CONCLUSIONS: In a cross-over study of patients with UC in remission, we found that a catered LFD or iSAD were each well tolerated and increased quality of life. However, the LFD decreased markers of inflammation and reduced intestinal dysbiosis in fecal samples. Dietary interventions therefore might benefit patients with UC in remission. ClinicalTrials.gov no: NCT04147598.

Intestinal exposure to PCB 153 induces inflammation via the ATM/NEMO pathway.

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent organic pollutants that adversely affect human health. PCBs bio-accumulate in organisms important for human consumption. PCBs accumulation in the body leads to activation of the transcription factor NF-κB, a major driver of inflammation. Despite dietary exposure being one of the main routes of exposure to PCBs, the gut has been widely ignored when studying the effects of PCBs. OBJECTIVES: We investigated the effects of PCB 153 on the intestine and addressed whether PCB 153 affected intestinal permeability or inflammation and the mechanism by which this occurred. METHODS: Mice were orally exposed to PCB 153 and gut permeability was assessed. Intestinal epithelial cells (IECs) were collected and evaluated for evidence of genotoxicity and inflammation. A human IEC line (SW480) was used to examine the direct effects of PCB 153 on epithelial function. NF-кB activation was measured using a reporter assay, DNA damage was assessed, and cytokine expression was ascertained with real-time PCR. RESULTS: Mice orally exposed to PCB 153 had an increase in intestinal permeability and inflammatory cytokine expression in their IECs; inhibition of NF-кB ameliorated both these effects. This inflammation was associated with genotoxic damage and NF-кB activation. Exposure of SW480 cells to PCB 153 led to similar effects as seen in vivo. We found that activation of the ATM/NEMO pathway by genotoxic stress was upstream of NF-kB activation. CONCLUSIONS: These results demonstrate that oral exposure to PCB 153 is genotoxic to IECs and induces downstream inflammation and barrier dysfunction in the intestinal epithelium.

Intestinal Epithelial Toll-Like Receptor 4 Signaling Affects Epithelial Function and Colonic Microbiota and Promotes a Risk for Transmissible Colitis.

Evidence obtained from gene knockout studies supports the role of Toll-like receptor 4 (TLR4) in intestinal inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in patients with inflammatory bowel disease. However, little is known of the effect of increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that overexpress TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are characterized by increases in the density of mucosa-associated bacteria and bacterial translocation. Furthermore, increased epithelial TLR4 signaling was associated with an impaired epithelial barrier, altered expression of antimicrobial peptide genes, and altered epithelial cell differentiation. The composition of the colonic luminal and mucosa-associated microbiota differed between villin-TLR4 and wild-type (WT) littermates. Interestingly, WT mice cohoused with villin-TLR4 mice displayed greater susceptibility to acute colitis than singly housed WT mice did. The results of this study suggest that epithelial TLR4 expression shapes the microbiota and affects the functional properties of the epithelium. The changes in the microbiota induced by increased epithelial TLR4 signaling are transmissible and exacerbate dextran sodium sulfate-induced colitis. Together, our findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately the host's susceptibility to colitis.

Innate immunity in the small intestine.

PURPOSE OF REVIEW: This article reviews the most recent publications on innate immunity in the small intestine. We will go over the innate immune receptors that act as sensors of microbial presence or cell injury, Paneth cells as the main epithelial cell type that secrete antimicrobial peptides, and mucosal production of immunoglobulin A (IgA). In addition, we will give an update on examples of imbalance of the innate immune response resulting in clinical disease with the most relevant example being Crohn's disease. RECENT FINDINGS: Toll-like receptors (TLRs) are involved in B-cell homing to the intestine, rejection of small intestinal allografts, and recruitment of mast cells. The TLR adaptor Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-ß is necessary to activate innate immunity after Yersinia enterocolitica infection. Moreover, MyD88 is required to keep the intestinal microbiota under control and physically separated from the epithelium, and RegIIIγ is responsible for the bacterial segregation from the lining epithelial cells. In Crohn's disease, ATG16L1 T300A variant promotes a proinflammatory response; and miR-196 downregulates a protective immunity-related GTPase family M protein (IRGM) polymorphism leading to impaired clearance of adherent Escherichia coli in the intestine. SUMMARY: The intestine is continuously exposed to dietary and microbial antigens. The host has to maintain intestinal homeostasis to keep the commensal and pathogenic bacteria under control. Some of the mechanisms to do so are by expression of innate immune receptors, production of antimicrobial peptides, secretion of IgA, or autophagy of intracellular bacteria. Unfortunately, in some cases the innate immune response fails to protect the host and chronic inflammation, transplant rejection, or other disorders may occur.

Cancer in inflammatory bowel disease: lessons from animal models.

PURPOSE OF REVIEW: Human colitis-associated cancers (CAC) represent a heterogeneous group of conditions in which multiple oncogenic pathways are involved. In this article, we review the latest studies using genetic, chemical, bacterial and innate immune-mediated experimental models of CAC. RECENT FINDINGS: Using the azoxymethane-dextran sodium sulfate model, wound healing pathways seem to be required in the development of CAC. There is also an emerging understanding that commensal and/or pathogenic bacteria can promote tumorigenesis, through T cell and TLR-mediated inflammation. Using specific transgenic mice (villin-CD98, T cell SMAD7, villin-TLR4) or specific knockout mice, investigators have determined that derangements in epithelial or innate and adaptive immune pathways can result in CAC. Subtle perturbations in epithelial repair - both too little or too exuberant - can render mice susceptible to tumorigenesis. SUMMARY: With the aid of animal models, we have witnessed a rapid expansion of our knowledge of the molecular and immunologic mechanisms underlying inflammatory cancers. Though animal models have contributed a discrete amount of information to our understanding of tumorigenesis in the setting of intestinal inflammation, it is clear that no single animal model will be able to adequately recapitulate the pathogenesis of complex colorectal cancers, but each model gets us one step closer to comprehending the nature of CAC.

Innate immunity in the small intestine.

PURPOSE OF REVIEW: In this article, we provide an update of the latest findings related to the innate immunity in the small intestine. In particular, we will focus on innate immune receptors and antimicrobial strategies that keep luminal bacteria and viral pathogens under control to avoid mucosal damage. These strategies include IgA secretion and antimicrobial peptides produced by Paneth cells, and downregulation or anergy of the innate immune receptors themselves. RECENT FINDINGS: Pattern-recognition receptors are the main target in the study of innate immunity in the intestinal mucosa due to their involvement in the regulation of host-commensal interactions. It has been shown that TLR5-deficient mice develop metabolic syndrome and have altered intestinal microbiota. On the contrary, NOD2 has been associated with the activation of autophagy and the inhibition of TLR4. Moreover, NOD2 has been described to be essential to keep a feedback loop in the host-commensal homeostasis, through the kinase Rip-2. SUMMARY: Innate immunity in the small intestine is mainly characterized by IgA secretion and Paneth cell antimicrobial function. In both cases pattern-recognition receptors, Toll-like receptors and nucleotide-binding and oligomerization domain-like receptors, are involved. A better understanding of the innate immunity in the small intestine would provide valuable information to develop vaccines against pathogens.

Paucicellular lymphocytic colitis: is it a minor form of lymphocytic colitis? A clinical pathological and immunological study.

OBJECTIVES: It has been suggested that paucicellular lymphocytic colitis (PLC) should be considered to be part of the morphological spectrum of microscopic colitis. The aim of the study was to evaluate whether PLC may be considered to be a true microscopic colitis, and in this case, whether it is a minor form of lymphocytic colitis (LC) or a different entity. METHODS: All incident cases of PLC, LC, and collagenous colitis (CC) during the period 2004-2006 were included. The incidence rate and the clinical, histopathological, and immunological features of PLC were assessed and compared with those of both LC and CC. Immunoreactivities to CD25, c-Kit, and FOXP3 in lamina propria were assessed. RESULTS: In all, 19 patients with CC, 19 with LC, and 26 with PLC were identified. CD25+FOXP3+ expression was seen only in classical forms of microscopic colitis: 12 of 19 LC, 14 of 20 CC, and none of 20 PLC cases (P < 0.0001). Diarrhea ceased in 21 of the 26 patients, with a decrease in the daily stool number from 5.08 +/- 0.44 to 1.7 +/- 0.2 (P < 0.005). The five patients with no response to therapy fulfilled the Rome II criteria of irritable bowel syndrome (IBS). CONCLUSIONS: The incidence rate of PLC, identified using objective histological criteria, was higher than those of CC and LC. The lack of expression of CD25+FOXP3+ cells in PLC, in contrast to those seen in both LC and CC, would suggest the existence of different pathophysiological mechanisms and does not support that PLC is a minor form of LC.

Use of Cancer Stem Cells to Investigate the Pathogenesis of Colitis-associated Cancer.

Colitis-associated cancer (CAC) can develop in patients with inflammatory bowel disease with long-term uncontrolled inflammation. The mutational history and tumor microenvironment observed in CAC patients is distinct from that observed in sporadic colon cancer and suggests a different etiology. Recently, much attention has been focused on understanding the cellular origin of cancer and the cancer stem cells, which is key to growth and progression. Cancer stem cells are often chemo-resistant making them attractive targets for improving patient outcomes. New techniques have rapidly been evolving allowing for a better understanding of the normal intestinal stem cell function and behavior in the niche. Use of these new technologies will be crucial to understanding cancer stem cells in both sporadic and CAC. In this review, we will explore emerging methods related to the study of normal and cancer stem cells in the intestine, and examine potential avenues of investigation and application to understanding the pathogenesis of CAC.

Diet and Inflammatory Bowel Disease.

Patients with inflammatory bowel disease (IBD) are increasingly becoming interested in nonpharmacologic approaches to managing their disease. One of the most frequently asked questions of IBD patients is what they should eat. The role of diet has become very important in the prevention and treatment of IBD. Although there is a general lack of rigorous scientific evidence that demonstrates which diet is best for certain patients, several diets-such as the low-fermentable oligosaccharide, disaccharide, monosaccharide, and polyol diet; the specific carbohydrate diet; the anti-inflammatory diet; and the Paleolithic diet-have become popular. This article discusses the diets commonly recommended to IBD patients and reviews the supporting data.

The Viral Mimetic Polyinosinic:Polycytidylic Acid Alters the Growth Characteristics of Small Intestinal and Colonic Crypt Cultures.

BACKGROUND & AIMS: The intestinal epithelium is the first line of defense against enteric pathogens. We investigated the response of small intestinal and colonic crypt cultures to a panel of toll-like receptor ligands to assess the impact of microbial pattern recognition on epithelial growth. METHODS: Primary murine jejunal enteroids and colonoids were cultured with lipopeptide Pam3CSK4, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) for 4 to 6 days. Surface area, budding and survival were assessed. Proliferation and numbers of lysozyme positive cells were quantified by flow cytometry. Gene expression was assessed by Nanostring and qRT-PCR. RESULTS: Exposure to Pam3CSK4 and LPS had minimal impact on either enteroids or colonoids. In contrast, Poly I:C increased the surface area of enteroids, while colonoids demonstrated decreased budding. Survival was decreased by Poly I:C in enteroids but not in colonoids. Both enteroids and colonoids exhibited upregulated gene expression of chemokines, but these were increased in magnitude in enteroids. Decreases in gene expression associated with epithelial differentiation and lysozyme positive cells were more apparent in enteroids than in colonoids. Baseline gene expression between enteroids and colonoids differed markedly in levels of stem cell and inflammatory markers. The changes in morphology induced by Poly I:C were mediated by the toll-like receptor adaptor molecule 1 (Ticam1) in enteroids but not in colonoids. CONCLUSIONS: Poly I:C alters the molecular program of epithelial cells and shifts from absorption and digestion towards defense and inflammation. Diversity of responses to microbial patterns in enteroids and colonoids may underlie differences in susceptibility to infection along the intestinal tract.

In silico and Ex vivo approaches identify a role for toll-like receptor 4 in colorectal cancer.

BACKGROUND: Inflammation increases the risk of colorectal cancer (CRC). We and others have described a role for TLR4, the receptor for LPS, in colon cancer. To explore the relationships between TLR4 expression and CRC, we combined the strength of transcriptome array data and immunohistochemical (IHC) staining. METHODS: TLR4 signal intensity was scored in the stromal and epithelial compartments. Detection of differential expression between conditions of interest was performed using linear models, Cox proportional hazards models, and empirical Bayes methods. RESULTS: A strong association between TLR4 expression and survival was noted, though a dichotomous relationship between survival and specific TLR4 transcripts was observed. Increasing TLR4 expression was seen with advancing tumor stage and was also over-expressed in some adenomas. IHC staining confirmed the positive relationship between TLR4 staining score in the CRC tumor stroma and epithelium with tumor stage, with up to 47% of colon cancer stroma positive for TLR4 staining. Increased TLR4 expression by IHC was also marginally associated with decreased survival. We now also describe that pericryptal myofibroblasts are responsible for a portion of the TLR4 stromal staining. CONCLUSIONS: Increased TLR4 expression occurs early in colonic neoplasia. TLR4 is associated with the important cancer-related outcomes of survival and stage.

Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa.

An ideal method of immune cell isolation should provide maximum cell yield without disturbing functional properties. Intestinal endoscopic biopsies, in contrast to surgical samples, allow the study of all disease stages but have the drawback of a minimum amount of tissue available, making protocol optimization mandatory. We compared for the first time two methods of separation of colonic epithelium and five methods of lamina propria cell isolation for colonic biopsy specimens (mechanical, enzymatic and organ culture protocols). Lymphocyte number, viability and phenotype (CD45+, CD103+, CD3+, CD4+, CD8+, CD19+, CD16-56+) were analyzed by flow cytometry. Neither of the two epithelial detachment protocols achieved proper epithelial separation, though the high intensity ion chelation method was more accurate. Maximum cell yield of lamina propria lymphocytes without phenotypic modification was obtained with overnight smooth enzymatic digestion. High dose collagenase incubation caused a marked decrease in CD4+ lymphocytes of the lamina propria as compared to low enzymatic method (p=0.004). Mechanical and biopsy culture are not advisable methods because of the low cell yield, and phenotypic alterations and high contamination rate, respectively.

TLR4 activates the ß-catenin pathway to cause intestinal neoplasia.

Colonic bacteria have been implicated in the development of colon cancer. We have previously demonstrated that toll-like receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS), is over-expressed in humans with colitis-associated cancer. Genetic epidemiologic data support a role for TLR4 in sporadic colorectal cancer (CRC) as well, with over-expression favoring more aggressive disease. The goal of our study was to determine whether TLR4 played a role as a tumor promoter in sporadic colon cancer. Using immunofluorescence directed to TLR4, we found that a third of sporadic human colorectal cancers over-express this marker. To mechanistically investigate this observation, we used a mouse model that over-expresses TLR4 in the intestinal epithelium (villin-TLR4 mice). We found that these transgenic mice had increased epithelial proliferation as measured by BrdU labeling, longer colonic crypts and an expansion of Lgr5+ crypt cells at baseline. In addition, villin-TLR4 mice developed spontaneous duodenal dysplasia with age, a feature that is not seen in any wild-type (WT) mice. To model human sporadic CRC, we administered the genotoxic agent azoxymethane (AOM) to villin-TLR4 and WT mice. We found that villin-TLR4 mice showed an increased number of colonic tumors compared to WT mice as well as increased ß-catenin activation in non-dysplastic areas. Biochemical studies in colonic epithelial cell lines revealed that TLR4 activates ß-catenin in a PI3K-dependent manner, increasing phosphorylation of ß-catenin(Ser552), a phenomenon associated with activation of the canonical Wnt pathway. Our results suggest that TLR4 can trigger a neoplastic program through activation of the Wnt/ß-catenin pathway. Our studies highlight a previously unexplored link between innate immune signaling and activation of oncogenic pathways, which may be targeted to prevent or treat CRC.

Host innate recognition of an intestinal bacterial pathogen induces TRIF-dependent protective immunity.

Toll-like receptor 4 (TLR4), which signals through the adapter molecules myeloid differentiation factor 88 (MyD88) and toll/interleukin 1 receptor domain-containing adapter inducing IFN-ß (TRIF), is required for protection against Gram-negative bacteria. TRIF is known to be important in TLR3-mediated antiviral signaling, but the role of TRIF signaling against Gram-negative enteropathogens is currently unknown. We show that TRIF signaling is indispensable for establishing innate protective immunity against Gram-negative Yersinia enterocolitica. Infection of wild-type mice rapidly induced both IFN-ß and IFN-γ in the mesenteric lymph nodes. In contrast, TRIF-deficient mice were defective in these IFN responses and showed impaired phagocytosis in regional macrophages, resulting in greater bacterial dissemination and mortality. TRIF signaling may be universally important for protection against Gram-negative pathogens, as TRIF-deficient macrophages were also impaired in killing both Salmonella and Escherichia coli in vitro. The mechanism of TRIF-mediated protective immunity appears to be orchestrated by macrophage-induced IFN-ß and NK cell production of IFN-γ. Sequential induction of IFN-ß and IFN-γ leads to amplification of macrophage bactericidal activity sufficient to eliminate the invading pathogens at the intestinal interface. Our results demonstrate a previously unknown role of TRIF in host resistance to Gram-negative enteropathogens, which may lead to effective strategies for combating enteric infections.

Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis.

BACKGROUND: Chronic intestinal inflammation culminates in cancer and a link to Toll-like receptor-4 (TLR4) has been suggested by our observation that TLR4 deficiency prevents colitis-associated neoplasia. In the current study we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis-associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. METHODS: Mice transgenic for a constitutively active TLR4 under the intestine-specific villin promoter (villin-TLR4 mice) were treated with dextran sodium sulfate (DSS) for acute colitis and azoxymethane (AOM)-DSS TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis (UC) and UC-associated cancer. The effect of an antagonist TLR4 antibody (Ab) was tested in prevention of colitis-associated neoplasia in the AOM-DSS model. RESULTS: Villin-TLR4 mice were highly susceptible to both acute colitis and colitis-associated neoplasia. Villin-TLR4 mice had increased epithelial expression of COX-2 and mucosal PGE2 production at baseline. Increased severity of colitis in villin-TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all colitis-associated cancer and progressively increased with grade of dysplasia. As a proof of principle, a TLR4/MD-2 antagonist antibody inhibited colitis-associated neoplasia in the mouse model. CONCLUSIONS: Our results show that regulation of TLRs can affect the outcome of both acute colitis and its consequences, cancer. Targeting TLR4 and other TLRs may ultimately play a role in prevention or treatment of colitis-associated cancer.

Apoptosis resistance of mucosal lymphocytes and IL-10 deficiency in patients with steroid-refractory Crohn's disease.

BACKGROUND: Apoptosis resistance of T-cells is considered an abnormality of immune pathways in Crohn's disease (CD). It has been previously shown that corticosteroids induce apoptosis of cells involved in inflammation. Thus, our aim was to assess the apoptosis of mononuclear cells and pro/antiinflammatory cytokines in the intestinal mucosa of patients with active CD, related to steroid response, and identify cellular and molecular factors that may predict this response to therapy. METHODS: Patients with CD (n = 26), ulcerative colitis (UC) (n = 32), and controls (n = 10) were prospectively studied with mucosal biopsies before and 7-10 days after corticosteroid treatment. Immunophenotype and apoptosis of T and B lymphocytes, plasma cells, and macrophages were assessed by flow cytometry, immunohistochemistry, and immunofluorescence. The cytokine expression pattern was evaluated by quantitative polymerase chain reaction (PCR). RESULTS: Apoptosis resistance of T and B lymphocytes was observed only in steroid-refractory and -dependent CD patients as compared to responsive patients (P = 0.032; P = 0.004, respectively), being evident after steroid treatment. Interleukin (IL)-10 was markedly increased at baseline in steroid-responsive patients compared to the nonresponders (P = 0.006; sensitivity: 88.8%; specificity: 66.6% to predict steroid response). CONCLUSIONS: Apoptosis resistance of mucosal T and B cells in steroid-refractory and -dependent CD patients appears during the evolution of the acute phase, limiting its clinical application as a predictor marker. In contrast, increased expression of IL-10 at an early stage of active steroid-sensitive CD patients supports its usefulness at predicting a good steroid response. Steroid-dependent and -refractory CD patients share similar molecular and cellular pathophysiological mechanisms.

Impact of mass screening for gluten-sensitive enteropathy in working population.

AIM: To assess: (1) frequency and clinical relevance of gluten sensitive enteropathy (GSE) detected by serology in a mass screening program; (2) sensitivity of antitransglutaminase (tTGA) and antiendomysium antibodies (EmA); and (3) adherence to gluten-free diet (GFD) and follow-up. METHODS: One thousand, eight hundred and sixty-eight subjects recruited from an occupational health department underwent analysis for tTGA and EmA and, if positive, duodenal biopsy, DQ2/DQ8 genotyping, clinical feature recording, blood tests, and densitometry were performed. Since > 98% of individuals had tTGA < 2 U/mL, this value was established as the cut-off limit of normality and was considered positive when confirmed twice in the same sample. Adherence to a GFD and follow up were registered. RESULTS: Twenty-six (1.39%) subjects had positive tTGA and/or EmA, and 21 underwent biopsy: six Marsh III (one IIIa, four IIIb, one IIIc), nine Marsh I and six Marsh 0 (frequency of GSE 1:125). The sensitivity of EmA for GSE was 46.6% (11.1% for Marsh I, 100% for Marsh III), while for tTGA, it was 93.3% (88.8% for Marsh I, 100% for Marsh III). All 15 patients with abnormal histology had clinical features related to GSE. Marsh I and III subjects had more abdominal pain than Marsh 0 (P = 0.029), and a similar trend was observed for distension and diarrhea. No differences in the percentage of osteopenia were found between Marsh I and III (P = 0.608). Adherence to follow-up was 69.2%. Of 15 GSE patients, 66.7% followed a GFD with 80% responding to it. CONCLUSION: GSE in the general population is frequent and clinically relevant, irrespective of histological severity. tTGA is the marker of choice. Mass screening programs are useful in identifying patients who can benefit from GFD and follow-up.

Intestinal spirochetosis and chronic watery diarrhea: clinical and histological response to treatment and long-term follow up.

BACKGROUND: The clinical significance of intestinal spirochetosis is uncertain, therefore the aim of the present paper was to assess the prevalence of histological intestinal spirochetosis in patients with and without chronic watery diarrhea and to evaluate its clinical relevance. METHODS: A prospective diagnostic work-up of intestinal spirochetosis was made on biopsy samples taken from patients with chronic watery diarrhea submitted between 1994 and 2004 (1174 colonoscopies with multiple biopsies). Three other positive cases identified from routine endoscopic biopsies also were reviewed. In addition, samples from 100 asymptomatic control patients and a random sample of another 104 colonic specimens were reviewed for intestinal spirochetosis. The diagnosis was established by light and electron microscopy. Polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA and reduced nicotinamide adenine dinucleotide (NADH) oxidase genes of the intestinal spirochetes Brachyspira aalborgi and Brachyspira pilosicoli was performed on tissue biopsies of the 11 positive patients. After diagnosis, treatment with penicillin benzatine (PB) or metronidazole was offered to all symptomatic patients and they were followed for a mean of 45.4 months (range: 37-113 months). RESULTS: Eight patients with chronic watery diarrhea were positive for intestinal spirochetosis. Intestinal spirochetosis was not diagnosed in the controls. Histological resolution of the infection paralleled clinical recovery in six patients (following metronidazole treatment in three). Most patients showed mild, non-specific colonic inflammation. Invasion by the spirochetes was not demonstrated by electron microscopy. Brachyspira aalborgi and B. pilosicoli each were identified by PCR in two cases. CONCLUSIONS: Histological intestinal spirochetosis appears to be relatively uncommon in Catalonia (Spain) compared to previous reports from other countries, but was identified in patients (0.7%) with chronic watery diarrhea. Sustained clinical recovery after spontaneous or drug-induced spirochetal disappearance in these individuals suggests that intestinal spirochetosis may play a pathogenic role in chronic watery diarrhea. Treatment with metronidazole is advisable in patients with persistent symptoms.