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Tumor immunosuppression is a limiting factor for successful cancer therapy. The lipid sphingosine-1-phosphate (S1P), which signals through 5 distinct G protein-coupled receptors (S1PR1-5), has emerged as an important regulator of carcinogenesis. However, the utility of targeting S1P in tumors is hindered by S1P's impact on immune cell trafficking. Here, we report that ablation of the immune cell-specific receptor S1PR4, which plays a minor role in immune cell trafficking, delayed tumor development and improved therapy success in murine models of mammary and colitis-associated colorectal cancer through increased [CD8.sup.+] T cell abundance. Transcriptome analysis revealed that S1PR4 affected proliferation and survival of [CD8.sup.+] T cells in a cell-intrinsic manner via the expression of Pik3ap1 and Lta4h. Accordingly, PIK3AP1 expression was connected to increased [CD8.sup.+] T cell proliferation and clinical parameters in human breast and colon cancer. Our data indicate a so-far-unappreciated tumor-promoting role of S1P by restricting [CD8.sup.+] T cell expansion via S1PR4., Introduction The impact of the immune system on tumor progression is multifaceted. Although inflammation may promote cancer progression, data obtained from animal models and clinical studies demonstrate that the immune [...]

Bruton tyrosine kinase (BTK) is present in a wide variety of cells and may thus have important non-B cell functions. Here, we explored the function of this kinase in macrophages with studies of its regulation of the NLR family, pyrin domaincontaining 3 (NLRP3) inflammasome. We found that bone marrow-derived macrophages (BMDMs) from BTK-deficient mice or monocytes from patients with X-linked agammaglobulinemia (XLA) exhibited increased NLRP3 inflammasome activity; this was also the case for BMDMs exposed to low doses of BTK inhibitors such as ibrutinib and for monocytes from patients with chronic lymphocytic leukemia being treated with ibrutinib. In mechanistic studies, we found that BTK bound to NLRP3 during the priming phase of inflammasome activation and, in doing so, inhibited LPS- and nigericin-induced assembly of the NLRP3 inflammasome during the activation phase of inflammasome activation. This inhibitory effect was caused by BTK inhibition of protein phosphatase 2A-mediated (PP2A-mediated) dephosphorylation of Ser5 in the pyrin domain of NLRP3. Finally, we show that BTK-deficient mice were subject to severe experimental colitis and that such colitis was normalized by administration of anti-IL-[beta] or anakinra, an inhibitor of IL-1[beta] signaling. Together, these studies strongly suggest that BTK functions as a physiologic inhibitor of NLRP3 inflammasome activation and explain why patients with XLA are prone to develop Crohn's disease., Introduction The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, when appropriately activated and enabled to release mature IL-1[beta] and IL-18, serves as a powerful mechanism for the induction of the [...]

Immunotherapy has transformed the treatment landscape for a wide range of human cancers. Immune checkpoint inhibitors (ICIs), monoclonal antibodies that block the immune-regulatory 'checkpoint' receptors CTLA-4, PD-1, or its ligand PD- L1, can produce durable responses in some patients. However, coupled with their success, these treatments commonly evoke a wide range of immune-related adverse events (irAEs) that can affect any organ system and can be treatment-limiting and life-threatening, such as diabetic ketoacidosis, which appears to be more frequent than initially described. The majority of irAEs from checkpoint blockade involve either barrier tissues (e.g., gastrointestinal mucosa or skin) or endocrine organs, although any organ system can be affected. Often, irAEs resemble spontaneous autoimmune diseases, such as inflammatory bowel disease, autoimmune thyroid disease, type 1 diabetes mellitus (T1D), and autoimmune pancreatitis. Yet whether similar molecular or pathologic mechanisms underlie these apparent autoimmune adverse events and classical autoimmune diseases is presently unknown. Interestingly, evidence links HLA alleles associated with high risk for autoimmune disease with ICI-induced T1D and colitis. Understanding the genetic risks and immunologic mechanisms driving ICI-mediated inflammatory toxicities may not only identify therapeutic targets useful for managing irAEs, but may also provide new insights into the pathoetiology and treatment of autoimmune diseases., Introduction The 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison and Tasuku Honjo for their work on the role of inhibitory immune 'checkpoint' receptors in [...]

The tight junction protein claudin-2 is upregulated in inflammatory bowel disease, and yet its deficit worsens infectious and chemical colitis. In this issue of the JCI, Raju and Shashikanth et al. examined the contribution of claudin-2 to immune-mediated colitis. The authors used transgenic mouse models to show that claudin-2 deficiency attenuated colitis progression as well as a leak barrier defect, albeit at the risk of intestinal obstruction. Further, inhibition of claudin-2 by targeting casein kinase 2 (CK2) also ameliorated colitis. The findings reveal unsuspected links between the pore and leak pathways of intestinal permeability and immune responses leading to colitis. They additionally suggest potential targets for therapeutic intervention in inflammatory bowel disease., Intestinal barrier The intestinal epithelium maintains a semipermeable barrier between the gut lumen and the body as a physiological imperative. This barrier allows the uptake of needed nutrients and reclamation [...]

Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N-ethyl-N-nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLR[right arrow]MyD88[right arrow]AREG/EREG[right arrow]EGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis. epidermal growth factor receptor signaling | inflammatory bowel disease | Toll-like receptor signaling | ENU mutagenesis | intestinal homeostasis doi/ 10.1073/pnas.1014669107

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [[sup.3]H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [[sup.3]H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo. colitis; growth inhibition; intestine nitric oxide; neuronal nitric oxide synthase; cGMP; cAMP doi: 10.1152/ajpgi.00259.2009.

Mizushima T, Sasaki M, Ando T, Wada T, Tanaka M, Okamoto Y, Ebi M, Hirata Y, Murakami K, Mizoshita T, Shimura T, Kubota E, Ogasawara N, Tanida S, Kataoka H, Kamiya T, Alexander JS, Joh T. Blockage of angiotensin II type 1 receptor regulates TNF-[alpha]-induced MAdCAM-1 expression via inhibition of NF-[kappa]B translocation to the nucleus and ameliorates colitis. Am J Physiol Gastrointest Liver Physiol 298: G255-G266, 2010. First published November 25, 2009; doi: 10.1152/ajpgi.00264.2009.-- Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is an important target in the treatment of inflammatory bowel disease (1BD). Recently, treatment of IBD with an antibody to [alpha]4137-integrin, a ligand for MAdCAM-1, has been an intense focus of research. Our aim was to clarify the mechanism by which MAdCAM-1 is regulated via angiotensin II type 1 receptor (AT1R), and to verify if AT1R might be a novel target for IBD treatment. The role of AT1R in the expression of MAdCAM-1 in SVEC (a murine high endothelial venule cell) and MJC-1 (a mouse colonic endothelial cell) was examined following cytokine stimulation. We further evaluated the effect of AT1R on the pathogenesis of immunemediated colitis using AT1R-deficient (AT1R-/-) mice and a selective AT1R blocker. AT1R blocker significantly suppressed MAdCAM-1 expression induced by TNF-[alpha], but did not inhibit phosphorylation of p38 MAPK or of I[kappa]B that modulate MAdCAM-1 expression. However, NF-[kappa]B translocation into the nucleus was inhibited by these treatments. In a murine colitis model induced by dextran sulfate sodium, the degree of colitis, judged by body weight loss, histological damage, and the disease activity index, was much milder in AT1R-/- than in wild-type mice. The expression of MAdCAM-I was also significantly lower in AT1R-/- than in wild-type mice. These results suggest that AT1R regulates the expression of MAdCAM-1 under colonic inflammatory conditions through regulation of the translocation of NF-[kappa]B into the nucleus. Furthermore, inhibition of ATIR ameliorates colitis in a mouse colitis model. Therefore, AT1R might be one of new therapeutic target of IBD via regulation of MAdCAM-1. mucosal addressin cell adhesion molecule-l; inflammatory bowel disease; nuclear factor-[kappa]B doi: 10.1152/ajpgi.00264.2009

The relationship between a predisposition to obesity and the development of colitis is not well understood. Our aim was to characterize the adipokine response and the extent of colitis in diet-induced obese (DIO) rats. DIO and control, diet-resistant (DR) animals were administered either saline or trinitrobenzene sulfonic acid (TNBS) to induce colitis. Macroscopic damage scores and myeloperoxidase (MPO) activity were measured to determine the extent of inflammation. Trunk blood was collected for the analysis of plasminogen activator inhibitor-1 (PAI-1) as well as leptin, ghrelin, and adiponectin. Colonic epithelial physiology was assessed using Ussing chambers. DIO rats had a modestly increased circulating PAI-1 before TNBS treatment; however, during colitis, DR animals had more than a fourfold increase in circulating PAI-1 compared with DIO rats. Circulating leptin was higher in DIO rats compared with DR animals, in the inflamed and noninflamed states. These changes in TNBS-induced adipokine profile were accompanied by decreased macroscopic tissue damage score in DIO animals compared with DR tissues. Furthermore, TNBS-treated DR animals lost significantly more weight than DIO rats during active inflammation. Colonic epithelial physiology was comparable between groups, as was MPO activity. The factors contributing to the decreased colonic damage are almost certainly multifold, driven by both genetic and environmental factors, of which adipokines are likely to play a part given the increasing body of evidence for their role in modulating intestinal inflammation. diet-induced obese; body weight; leptin; plasminogen activator inhibitor-1; adiponectin; ghrelin doi: 10.1152/ajpgi.00164.2009.

Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production. colitis | endoplasmic reticulum | mucin | goblet cell | protein processing

Treatment with anti-TNF-[alpha] MAb has been accepted as a successful maintenance therapy for patients with inflammatory bowel diseases (IBD). Moreover, it has been recently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoietic cells may prevent the development of colitis-associated cancer (CAC). However, it remains unclear whether the TNF-[alpha] signaling in epithelial cells is involved in the development of CAC. To investigate this, we studied the effects of anti-TNF-[alpha] MAb in an animal model of CAC by administration of azoxymethane (AOM) followed by sequential dextran sodium sulfate (DSS) ingestion. We observed that the NF-[kappa]B pathway is activated in colonic epithelia from DSS-administered mice in association with upregulation of TNFR2 rather than TNFR1. Immunoblot analysis also revealed that the TNFR2 upregulation accompanied by the NF-[kappa]B activation is further complicated in CAC tissues induced in AOM/DSS-administered mice compared with the nontumor area. Such NF-[kappa]B activity in the epithelial cells is significantly suppressed by the treatment of MP6-XT22, an anti-TNF-[alpha] MAb. Despite inability to reduce the severity of colitis, sequential administration of MP6-XT22 reduced the numbers and size of tumors in association with the NF-[kappa]B inactivation. Taken together, present studies suggest that the TNFR2 signaling in intestinal epithelial cells may be directly involved in the development of CAC with persistent colitis and imply that the maintenance therapy with anti-TNF-[alpha] MAb may prevent the development of CAC in patients with long-standing IBD. tumor necrosis factor-[alpha]; colitis-associated cancer; intestinal epithelial cells; carcinogenesis; TNFR2

Phosphate homeostasis is critical for many physiological functions. Up to 85% of phosphate is stored in bone and teeth. The remaining 15% is distributed in cells. Phosphate absorption across the brush-border membrane (BBM) of enterocytes occurs mainly via a sodium-dependent pathway, which is mediated by type IIb sodium-phosphate cotransporters (NaPi-IIb). Patients of inflammatory bowel diseases (IBDs) suffer not only from diarrhea and nutrient malabsorption but also from bone loss. About 31-59% of patients with IBD develop bone disorders. Since the intestine is a primary location for dietary phosphate absorption, it is logical to postulate that there is an inverse relationship between gastrointestinal disorders and phosphate transport, which, in turn, contributes to bone disorders observed in patients with IBD. Phosphate absorption and NaPi-IIb expression was studied with BBM vesicles isolated from trinitrobenzene sulphonic acid (TNBS) animals as well as in Caco-2 cells. The mechanism of TNF-[alpha] downregulation of NaPi-IIb expression was investigated by luciferase assay, gel mobility shift assay (GMSA), and coimmunoprecipitation. Intestinal phosphate absorption mediated by NaPi-IIb was reduced both in TNBS colitis and in TNF-[alpha]-treated cells. Transient transfection indicated that TNF-[alpha] inhibits NaPi-IIb expression by reducing NaPi-IIb basal promoter activity. GMSAs identified NF1 protein as an important factor in TNF-[alpha]-mediated NaPi-IIb downregulation. Signaling transduction study and coimmunoprecipitation suggested that TNF-[alpha] interacts with EGF receptor to activate ERK1/2 pathway. Intestinal phosphate absorption mediated by NaPi-IIb protein is reduced in colitis. This inhibition is mediated by the proinflammatory cytokine TNF-[alpha] through a novel molecular mechanism involving TNF-[alpha]/EGF receptor interaction. trinitrobenzene sulphonic acid colitis; Caco-2 cells; type IIb sodium-phosphate cotransporters; EGF receptor

Inflammatory bowel diseases (IBD) can involve widespread gastrointestinal dysfunction, even in cases in which inflammation is localized to a single site. The underlying pathophysiology of dysfunction in noninflamed regions is unclear. We examined whether colitis is associated with altered electrogenic ion transport in the ileal mucosa and/or changes in the properties of ileal submucosal neurons. Colitis was induced by administration of trinitrobenzene sulfonic acid (TNBS), and the uninflamed ileum from animals was examined 3, 7, and 28 days later. Electrogenic ion transport was assessed in Ussing chambers. Intracellular microelectrode recordings were used to examine the neurophysiology of the submucosal plexus of the ileum in animals with colitis. Noncholinergic secretion was reduced by 33% in the ileum from animals 7 days after the induction of colitis. The epithelial response to vasoactive intestinal peptide (VIP) was unaltered in animals with colitis, but the response to carbachol was enhanced. Slow excitatory synaptic transmission was dramatically reduced in VIP-expressing, noncholinergic secretomotor neurons. This change was detected as early as 3 days following TNBS treatment. No changes to fast synaptic transmission or the number of VIP neurons were observed. In addition, cholinergic secretomotor neurons fired more action potentials during a given stimulus, and intrinsic primary afferent neurons had broader action potentials in animals with colitis. These findings implicate changes to enteric neural circuits as contributing factors in inflammation-induced secretory dysfunction at sites proximal to a localized inflammatory insult. submucosal plexus; electrogenic secretion; ion transport; synaptic transmission; vasoactive intestinal peptide

NHE8 transporter is a member of the sodium/hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of epithelial cells of kidney and intestine and contributes to vectorial [Na.sup.+] transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of [Na.sup.+]/[H.sup.+] exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from trinitrobenzene sulfonic acid (TNBS) colitis rats or lipopolysaccharide (LPS) rats for RNA extraction and brush-border membrane protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF-[alpha]-mediated NHE8 expression down-regulation in Caco-2 cells. Results from Western blot and real-time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF-[alpha] produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The downregulation of NHE8 expression mediated by TNF-[alpha] could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF-[alpha] inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF-[alpha] decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation. trinitrobenzene sulfonic acid colitis; sodium/hydrogen exchanger 8; intestine

The goal of this study was to investigate the role of the adipokine adiponectin (APN) in development of spontaneous colitis in IL-10 knockout (KO) mice. To this aim, we generated double IL-10 APN KO mice and compared their disease development to that of single IL-10 KO mice. Both IL-10 KO and double IL-10 APN KO mice spontaneously developed colitis of comparable severity. No significant differences in inflammatory infiltrate or crypt elongation were observed in colonic tissue obtained from IL-10 KO and double IL-10 APN KO mice at either 12 or 20 wk of age. A comparable increase in circulating levels of serum amyloid A and IFN-[gamma] was observed in IL-10 KO and double IL-10 APN KO mice as disease progressed. In vitro stimulation of lymphocytes from mesenteric lymph nodes with anti-CD3 and anti-CD28 induced a significantly higher production of IL-17 and TNF-[alpha] in IL-10 KO and double IL-10 APN KO mice compared with their healthy litterrnates. No significant differences in cytokine production from lymphocytes or colonic mRNA expression of cytokines were observed between IL-10 KO and double IL-10 APN KO mice. Both IL-10 KO and double IL-10 APN KO mice had a similar decrease in body weight and bone mass compared with their respective healthy littermates. Finally, APN deficiency did not lead to development of insulin resistance, either in APN KO or double IL-10 APN K0 mice. In conclusion, lack of APN does not play a significant role in the pathogenesis of spontaneous colonic inflammation in the IL-10 KO model. adipocytes; colon; cytokines; inflammation

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 [micro]M) but did constrict in the presence of its nonhydrolyzable analog [alpha],[beta]-methylene ATP (1 p[micro]M). [alpha],[beta]-Methylene ADP (100 [micro]M), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis. purinergic neurotransmission; ectonucleotidase; inflammation; sympathetic; vasoconstriction

Matrix metalloproteinases (MMP) play an important role in pathogenesis of inflammatory bowel disease (IBD). Two known gelatinases, MMP-2 and MMP-9, are upregulated during IBD. Epithelial-derived MMP-9 is an important mediator of tissue injury in colitis, whereas MMP-2 protects against tissue damage and maintains gut barrier function. It has been suggested that developing strategies to block MMP-9 activity in the gut might be of benefit to IBD. However, given that MMP-2 and MMP-9 are structurally similar, such approaches would also likely inhibit MMP-2. Thus, to gain insight into outcome of inhibiting both MMP-2 and MMP-9, [MMP-2.sup.-/-]/[MMP-9.sup.-/-] double knockout mice (dKO) lacking both MMP-2 and MMP-9 were used in this study. Three models of murine colitis were used: dextran sodium sulfate (DSS), Salmonella typhimurium (S.T.), and trinitrobenzene sulfonic acid (TNBS). Our data demonstrate that MMP-2 and MMP-9 activities were highly upregulated in wild-type (WT) mice treated with DSS, S.T., or TNBS whereas dKO mice were resistant to the development of colitis. WT mice had extensive inflammation and tissue damage compared with dKO mice as suggested by histological assessment and myeloperoxidase activity. In conclusion, these results suggest an overriding role of MMP-9 in mediating tissue injury compared with the protective role of MMP-2 in development of colitis. Thus inhibition of MMP-9 may be beneficial in treatment of colitis even if resulting in inhibition of MMP-2. gelatinase; inflammation; myeloperoxidase activity; cytokines; chemokines

The inflammatory bowel diseases (Crohn' s disease; ulcerative colitis) are idiopathic chronic inflammatory disorders of the intestine and/or colon. A major advancement in our understanding of the pathogenesis of these diseases has been the development of mouse models of chronic gut inflammation. One model that has been instrumental in delineating the immunological mechanisms responsible for the induction as well as regulation of intestinal inflammation is the T cell transfer model of chronic colitis. This paper presents a detailed protocol describing the methods used to induce chronic colitis in mice. Special attention is given to the immunological concepts that explain disease pathogenesis in this model, considerations and potential pitfalls in using this model, and finally different 'tricks' that we have learned over the past 12 years that have allowed us to develop a more simplified version of this model of experimental IBD. adaptive immune system; T cell trafficking; inflammation; inflammatory bowel disease; cytokines; IL-10-deficient mice

Recent evidence implicating tissue factor and the protein C pathway in the hypercoagulable state associated with intestinal inflammation suggests that thrombin is likely to contribute to this response. The objective of this study was to assess the role of thrombin in the extraintestinal thrombosis associated with experimental colitis. Thrombus formation was quantified in microvessels of the cremaster muscle in mice with dextran sodium sulfate (DSS)-induced colonic inflammation. The light/dye endothelial injury model was used to elicit thrombus formation in DSS colitic mice treated with either hirudin, heparin, or antithrombin III. The initiation and propagation/stabilization phases of thrombus formation were quantified using the time of onset of the thrombus and time to blood flow cessation, respectively. Thrombus formation was accelerated in arterioles of DSS colitic mice, as exhibited by significant reductions in the time of thrombus initiation and propagation/stabilization. Colitic mice treated with hirudin, heparin, or antithrombin III did not exhibit a significant change in the time of onset of the thrombus compared with untreated colitic mice. However, all three antithrombin agents largely prevented the DSS-induced reduction in the time to flow cessation following light/dye injury, with hirudin offering complete protection. These findings indicate that thrombin plays a major role in the extraintestinal thrombus formation associated with experimental colitis. Thrombin appears to contribute to the propagation/stabilization, rather than initiation, phase of the colitis-associated thrombogenesis at the distant vascular site. The results support the therapeutic use of antithrombin agents for reducing the risk of thromboembolism in patients with inflammatory bowel disease. coagulation; heparin; hirudin; antithrombin III

Deficient immunoregulation by [CD4.sup.+] T cells is an important susceptibility trait for inflammatory bowel disease, but the role of other regulatory cell types is less understood. This study addresses the role and mechanistic interaction of B cells and [CD8.sup.+] T cells in controlling immune-mediated colitis. The genetic requirements for B cells and [CD8.sup.+] T cells to confer protective immunoregulation were assessed by cotransfer with colitogenic [G[alpha]i2.sup.-/-] T cells into immune-deficient mice. Disease activity in [G[alpha]i2.sup.-/-] T cell recipients was evaluated by [CD4.sup.+] T intestinal lymphocyte abundance, cytokine production levels, and large intestine histology. B cells deficient in B7.1/B7.2, CD40, major histocompatibility complex (MHC) II (Abb), or native B cell antigen receptor (MD4) were competent for colitis protection. However, transporter-1-deficient B cells failed to protect, indicating a requirement for peptide MHC I presentation to [CD8.sup.+] T cells. [CD8.sup.+] T cells deficient in native T cell receptor repertoire (OT-1) or cytolysis ([perforin.sup.-/-]) also were nonprotective. These finding reveal an integrated role for antigen-specific perforin-dependent [CD8.sup.+] T cell cytotoxicity in colitis immunoregulatory and its efficient induction by a subset of mesenteric B lymphocytes. CD8-positive T lymphocytes; B lymphocytes; immunoregulation; inflammatory bowel disease; cytolysis

Background and aims: Enteric bacterial and/or food antigens may be crucial in the development of colitis but little is known of the exact mechanism of antigen specific reactions in this [...]

Background Escherichia coli is believed to participate in the etiology of Crohn's disease (CD) and possibly of ulcerative colitis (UC), due at least in part to the observed rise in the number of these bacteria in the gut microbiota of CD and UC patients. Nevertheless, it is not fully understood whether this quantitative variation occurs equally throughout the mucosal and luminal spaces of the gut. To assess this question, stools and mucosa biopsies from distinct intestinal sites were cultured aiming at determining their E. coli concentration. The cultures were additionally screened for the presence of some virulence genes of pathogenic E. coli. Results Analyses of clinical materials from 14 controls (38 biopsies and 14 stools samples), 11 CD (25 biopsies and 11 stools samples) and 7 UC patients (18 biopsies and 7 stools samples) indicated no significant variation in the number of E. coli present in stools, but a rise of at least one log.sub.10 CFU/mg in biopsies from the ileum of CD patients and the sigmoid and rectum of CD and UC patients. The cultures were screened for the presence of E. coli attaching and effacing (eae), invasion plasmid antigen H (ipaH), aggregative adherence transcriptional activator (aggR), Shiga cytotoxins (stx), and heat labile enterotoxin (elt) and the following serine proteases autotransporters of Enterobacteriaceae (SPATE) genes: plasmid encoded toxin (pet), secreted autotransporter toxin (sat), Shigella extracellular protein (sepA), protein involved in intestinal colonization (pic) and Shigella IgA-like protease homolog (sigA). Six of the 10 genes screened were detected in the total of samples investigated: aggR, eae, pet, sat, sepA and sigA. No difference in the prevalence of any of these markers was observed in cultures from different clinical materials or groups of patients. Methods Bacterial quantitation was carried out following cultures of diluted samples suspensions in MacConkey agar, Wilkins Chalgren agar for anaerobes, E. coli/ coliform chromocult agar, and blood agar. Screening for E. coli virulence genes was performed by multiplex PCR of DNA purified from total MacConkey undiluted broth cultures. Conclusion In CD and UC patients only the mucosa associated population of E. coli is augmented and the proliferation is prominent in the ileum of CD and rectum and sigmoid of both UC and CD patients which are sites where the lesions usually are observed. The augmented E. coli population in these sites presented a low number of the virulence markers, possibly meaning that they are not relevant for the disease process. Keywords: Escherichia coli, Bacteria, Virulence, Crohn's disease, Ulcerative colitis, Author(s): Helton Luis de Souza[sup.1] , Vanessa R de Carvalho[sup.1] , Fernando Gomes Romeiro[sup.2] , Ligia Yukie Sassaki[sup.2] , Rogeria Keller[sup.1] and Josias Rodrigues[sup.1,3] Background Intestinal microbiota is one of [...]

Collagenous gastritis, collagenous sprue, and collagenous colitis share striking histologic similarities and occur together in some patients. They also share some drug and disease associations. Pediatric cases of collagenous gastritis, however, lack most of these associations. The etiologies of the collagenous gastroenteritides are not known, so it is not clear whether they are similar because they share pathogeneses, or because they indicate a common histologic response to varying injuries. The features, disease and drug associations, and the inquiries into the pathogenesis of these disorders are reviewed., During the past several decades a set of gastrointestinal inflammations have become recognized that are characterized by subsurface collagen deposition and are so histologically similar that common sense would suggest [...]