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Background and Aims To inform their future use in regulated clinical trials to evaluate treatment efficacy hypotheses, the measurement properties of three histologic indices, Geboes Score (GS), Robarts Histopathology Index (RHI), and Nancy Index (NI), were evaluated among patients with ulcerative colitis. Methods Analyses were conducted on data from a Phase 3 clinical trial of adalimumab (M14-033, n=491) and focused on evaluating the measurement properties of the GS, RHI, and NI. Specifically, internal consistency and inter-rater reliability, convergent, discriminant, and known-groups validity, and sensitivity to change were assessed at Baseline, and Weeks 8 and 52. Results Internal consistency for the RHI showed lower alpha (α) values at Baseline (α=0.62) relative to Weeks 8 (α=0.82) and 52 (α=0.81). The inter-rater reliability values of RHI (0.91), NI (0.64), and GS (0.53) were excellent, good, and fair, respectively. Regarding validity, Week 52 correlations were moderate to strong between full and partial Mayo scores and Mayo subscale scores and the RHI and GS, and were weak to moderate for the NI. Significant differences between mean scores of all three histologic indices were observed across known-groups based on Mayo endoscopy subscores and full Mayo scores at Weeks 8 and 52 (p Conclusions The GS, RHI, and NI are each capable of producing reliable and valid scores that are sensitive to changes in disease activity over time, in patients with moderately to severely active ulcerative colitis. While all three indices demonstrated relatively acceptable measurement properties, the GS and RHI performed better than the NI.

Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts—namely high background—IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.

Ly108, a glycoprotein of the signaling lymphocytic activation molecule family of cell surface receptors expressed by T, B, NK, and APCs has been shown to have a role in NK cell cytotoxicity and T cell cytokine responses. In this study, we describe that CD4+ T cells from mice with a targeted disruption of exons 2 and 3 of Ly108 (Ly108ΔE2+3) produce significantly less IL-4 than wild-type CD4+ cells, as judged by in vitro assays and by in vivo responses to cutaneous infection with Leishmania mexicana. Surprisingly, neutrophil functions are controlled by Ly108. Ly108ΔE2+3 mice are highly susceptible to infection with Salmonella typhimurium, bactericidal activity of Ly108ΔE2+3 neutrophils is defective, and their production of IL-6, IL-12, and TNF-α is increased. The aberrant bactericidal activity by Ly108ΔE2+3 neutrophils is a consequence of severely reduced production of reactive oxygen species following phagocytosis of bacteria. Thus, Ly108 serves as a regulator of both innate and adaptive immune responses.

The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate-buffered saline (PBS) developed clinical evidence of colitis at 6-8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Th1 and macrophage-derived cytokines including interferon gamma, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-12, and IL-18 lymphotoxin-beta. In addition, message levels and vascular surface expression of ICAM-1, VCAM-1, and MAdCAM-1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.

The b2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) is important for lymphocyte trafficking and activation as well as recruitment to sites of tissue inflammation. The objective of this study was to assess the role of ‘T-cell-associated’ LFA-1 in the pathogenesis of chronic colitis in vivo. Transfer of CD4 1 CD25 � T cells isolated from wild-type (wt) mice into immunodeficient recipients [recombinase-activating gene-1-deficient (RAG-1 � /� )] produced moderate to severe colitis, whereas RAG-1 � /� mice injected with CD11a-deficient (CD11a � /� ; LFA-1 � /� ) donor T cells displayed minimal macroscopic and histological evidence of colitis. Surface expression of L-selectin, a4, a4b7 and chemokine receptor-7 were similar for wt and CD11a � /� donor T cells. Attenuated disease in the CD11a � /� ! RAG-1 � /� animals was associated with decreased numbers of CD4 1 T cells in the mesenteric lymph nodes (MLNs), spleen and intestinal lamina propria (LP). In addition, significant reductions in Th1 cytokines were observed following ex vivo stimulation of mononuclear cells obtained from the MLNs and colonic LP. Interestingly, mononuclear cells obtained from the spleens of CD11a � /� ! RAG-1 � /� exhibited enhanced pro-inflammatory cytokine production compared with splenocytes obtained from wt ! RAG-1 � /� colitic mice. Taken together, our data suggest that T-cell-associated CD11a (LFA-1) expression plays a dual role in the initiation of chronic gut inflammation by facilitating naive T-cell priming/activation and expansion within MLNs and by augmenting pro-inflammatory cytokine production following secondary stimulation by antigenpresenting cells in the colonic interstitium.

MyD88 is an adaptor protein for the TLR family of proteins that has been implicated as a critical mediator of innate immune responses to pathogen detection. In this study, we report that MyD88 plays a crucial role in killing Gram-negative bacteria by primary macrophages via influencing NADPH oxidase function. Peritoneal macrophages from MyD88−/− mice exhibited a marked inability to kill Escherichia coli (F18) or an attenuated strain of Salmonella typhimurium (sseB) in vitro. This defect in killing was due to diminished NADPH oxidase-mediated production of superoxide anion in response to bacteria by MyD88−/− phagocytes as a consequence of defective NADPH oxidase assembly. Defective oxidase assembly in MyD88-deficient macrophages resulted from impaired p38 MAPK activation and subsequent phosphorylation of p47phox. Together these data demonstrate a pivotal role for MyD88 in killing Gram-negative bacteria via modulation of NADPH oxidase activity in phagocytic cells.

CD4+25+ regulatory T (Treg) cells maintain immunological self-tolerance through mechanisms that are only in part understood. Previous studies suggest that the glucocorticoid-induced TNFR-related protein (GITR), which is preferentially expressed on the surface of Treg cells, potentially provides a signal that abrogates Treg suppression. In this study, we show that a soluble form of mouse GITR ligand (sGITR-L) induces GITR-dependent NF-κB activation and blocks in vitro suppression mediated by both resting and preactivated polyclonal and Ag-specific Treg cells. Since sGITR-L along with rIL-2 induces proliferation of CD4+25+ cells, it appears that sGITR-L can break the anergic state of Treg cells. Because sGITR-L also up-regulates IL-2 secretion by activated CD4+25 −T cells, these two sGITR-L induced signals synergize to interfere with suppressor activity by CD4+25+ Treg cells.

The inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.

There is growing clinical evidence suggesting that certain secondary lymphoid tissues (e.g., appendix and spleen) contribute to the initiation and/or perpetuation of ulcerative colitis. In this study, the importance of secondary lymphoid tissues in inducing colitis was assessed experimentally by removing the spleen and/or appendix (or sham operation) prior to inducing colitis in mice. Feeding 2.5% dextran sulphate sodium (DSS) in drinking water over 7 days induced colitis. Clinical disease activity was assessed based on weight loss, stool consistency, and presence of blood in stools. Additional measurements included white blood cell count and hematocrit, and myeloperoxidase activity (MPO) in colon samples. Colonic injury was assessed by histology and computerized image analysis. DSS treatment in sham-operated mice produced colitis associated with weight loss, bloody diarrhea, and mucosal ulceration. Clinical assessment of DSS-treated mice subjected to appendectomy or combined appendectomy/splenectomy exhibited a delayed onset and course of disease activity. Histomorphologic examination revealed significantly lower damage scores and a reduction in ulcerated mucosal surface area. Colonic MPO activity, which correlated with tissue injury and disease activity, was lowest in appendectomized mice. No beneficial effects of splenectomy were observed after 7 days of colitis. These findings support the hypothesis that appendicular lymphoid tissue, but not the spleen, contributes to the development of colitis.

The mucosal interstitia of the intestine and colon are continuously exposed to large amounts of dietary and microbial antigens. Fortunately, the mucosal immune system has evolved efficient mechanisms to distinguish potentially pathogenic from nonpathological antigens. There are, however, situations in which this immune regulation fails, resulting in chronic gut inflammation.

Several reports have implicated reactive oxygen and nitrogen metabolites (RONS) in the initiation and/or progression of inflammatory bowel diseases (IBDs). We have investigated the role of three key RONS-metabolizing enzymes (inducible nitric oxide synthase [iNOS], superoxide dismutase [SOD], nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in a murine model of IBD. Mice genetically deficient (−/−) in either iNOS or the p47phox subunit of NADPH oxidase, transgenic (Tg) mice that overexpress SOD, and their respective wild-type (WT) littermates were fed dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. In addition, the specific iNOS inhibitor 1400W was used in DSS-treated WT and p47phox−/− mice. WT mice responded to DSS feeding with progressive weight loss, bloody stools, elevated serum NOX and colonic mucosal injury with neutrophil infiltration. Both the onset and severity of colitis were significantly attenuated in iNOS−/− and 1400W-treated WT mice. While the responses to DSS did not differ between WT and p47phox−/− mice, enhanced protection was noted in 1400W-treated p47phox−/− mice. Interestingly, SODTg mice exhibited more severe colitis than their WT littermates. These findings reveal divergent roles for superoxide and iNOS-derived NO in intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the intestine and/or colon of unknown etiology in which patients suffer from severe diarrhea, rectal bleeding, abdominal pain, fever, and weight loss. Active episodes of IBD are characterized by vasodilation, venocongestion, edema, infiltration of large numbers of inflammatory cells, and erosions and ulcerations of the bowel. It is becoming increasingly apparent that chronic gut inflammation may result from a dysregulated immune response toward components of the normal intestinal flora, resulting in a sustained overproduction of proinflammatory cytokines and mediators. Many of these Th1 and macrophage-derived cytokines and lipid metabolites are known to activate microvascular endothelial cells, thereby promoting leukocyte recruitment into the intestinal interstitium. This review discusses the basic immune mechanisms involved in the regulation of inflammatory responses in the gut and describes how a breakdown in this protective response initiates chronic gut inflammation.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that regulates lymphocyte trafficking to Peyer's patches and lymph nodes. Although it is widely agreed that MAdCAM-1 induction is involved in chronic gut inflammation, few studies have investigated regulation of MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)-alpha using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-alpha induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-kappa B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1 is regulated via MAPKs, we examined mitogen/extracellular signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that MEK-1/2 is activated by TNF-alpha within minutes and is dependent on TK and p42/44 MAPKs. Similarly, TNF-alpha activated NF-kappa B through TK, p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also shown to be frequently distributed to endothelial junctions both in vitro and in vivo. Cytokines like TNF-alpha stimulate MAdCAM-1 in high endothelium via TK, p38, p42/22 MAPKs, and NF-kappa B/PARP. MAdCAM-1 expression requires NF-kappa B translocation through both direct p42/44 and indirect p38 MAPK pathways in high endothelial cells.

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.

The objectives of this study were to quantify cytokine mRNA levels and endothelial cell adhesion molecule message and protein expression in healthy wild-type and interleukin-10-deficient (IL-10−/−) mice that develop spontaneous and chronic colitis. We found that colonic message levels of IL-1, IL-6, tumor necrosis factor-α, interferon-γ, lymphotoxin-β, and transforming growth factor-β were elevated in colitic mice 10- to 35-fold compared with their healthy wild-type controls. In addition, colonic message levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) were found to be increased 10-, 5-, and 23-fold, respectively, in colitic IL-10−/−mice compared with their wild-type controls. Immunoradiolabeling as well as immunohistochemistry revealed large and significant increases in vascular surface expression of colonic ICAM-1, VCAM-1, and MAdCAM-1 in the mucosa as well as the submucosa of the colons of colitic mice. These data are consistent with the hypothesis that deletion of IL-10 results in the sustained production of proinflammatory cytokines, leading to the upregulation of adhesion molecules and infiltration of mononuclear and polymorphonuclear leukocytes into the cecal and colonic interstitium.

The objective of this study was to quantify E-selectin surface expression in the colon as well as other tissues in a CD4(+) T-cell model of chronic colitis in mice using the newly developed dual radiolabel monoclonal antibody technique. Male SCID mice were reconstituted with either 5 x 10(5) CD4(+) CD45RB(low) or CD45RB(high) T-cells isolated from normal CB-17 donor mouse spleens and subsequently monitored for clinical signs of colitis. We found that animals injected with CD45RB(high) but not CD45RB(low) T-cells nor PBS developed colitis at 6-8 weeks following reconstitution as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of E-selectin compared to SCID mice injected with PBS or reconstituted with CD45RB(low) T-cells, both of which did not develop colitis. We also observed significant increases in E-selectin expression in cecum, small intestine, mesentery, and liver of colitic mice. Our data confirm that reconstitution of SCID mice with CD45RB(high) but not CD45RB(low) T-cells induces chronic colitis and demonstrate that this chronic colitis is associated with enhanced expression of an endothelial cell-specific adhesion molecule. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RB(high) T-cells enhances E-selectin expression in a variety of tissues distant from the site of active inflammation.

The objectives of this study were to assess the role of the inducible isoform of nitric oxide synthase (iNOS) on vascular cell adhesion molecule 1 (VCAM-1) expression in vivo in an acute model of inflammation induced in iNOS-deficient (iNOS−/−) mice and compare these data to those obtained by pharmacological inhibition of iNOS in a CD4+ T lymphocyte-dependent model of chronic colitis. VCAM-1 expression was quantified in vivo using the dual radiolabel monoclonal antibody technique. We found that intraperitoneal injection of 10 μg/kg tumor necrosis factor-α (TNF-α) enhanced VCAM-1 expression by approximately twofold in the colon, cecum, and stomach but not small intestine in iNOS−/−mice compared with TNF-α-injected wild-type mice. Injection of wild-type mice with 25 μg/kg TNF-α further enhanced VCAM-1 expression by approximately twofold compared with wild-type mice injected with 10 μg/kg TNF-α; however, VCAM-1 expression was not further enhanced in any gastrointestinal organ system in iNOS−/− mice. In a second series of experiments, we found that continuous inhibition of iNOS using oral administration of N G-iminoethyl-l-lysine did not alter the enhanced levels of VCAM-1 expression in the colon nor did it alter the severity of colonic inflammation in SCID mice reconstituted with CD4+, CD45RBhigh T cells. We conclude that iNOS may regulate VCAM-1 expression in acute inflammation; however, this effect is modest and tissue specific and occurs only when VCAM-1 expression is submaximal. iNOS does not appear to modulate VCAM-1 expression in an immune model of chronic colitis.

Studies aimed at elucidating the function of the protein synthesis factor eukaryotic initiation factor 4E (elF-4E) have demonstrated that overexpression of this protein results in marked cell phenotypic and proliferative changes, including neoplastic transformation of cells. These data suggest that elF-4E may somehow participate in the development and progression of tumors in vivo. In order to determine how elF-4E exerts its transforming effects, we examined vascular permeability factor (VPF) levels in cells transfected with an elF-4E vector. Cells overexpressing elF-4E showed an increase in intracellular, and an average 130-fold increase in secreted VPF protein levels (CHO 0.13 ± 0.12 ng/ml; CHO-4E 20.5 ± 12.5 ng/ml) over control cells. HUVEC growth induction revealed these VPF levels to be biologically active. Northern analysis revealed no difference in VPF transcript between the 2 cell lines. Polysome analysis showed that the VPF message in elF-4E-transfected cells was associated with the heavy polysomal regions, whereas the VPF message was associated with light polysomes in control cells. These data strongly suggest that enhanced VPF expression is achieved through translational regulation rather than transcriptional regulation in cells overexpressing elF-4E. This indicates that elF-4E-induced VPF expression may be an important factor in some forms of tumor angiogenesis and development. © 1996 Wiley-Liss, Inc.

Several recent studies have suggested that nitric oxide (NO) derived from the inducible isoform of NO synthase (NOS) may act as an endogenous modulator of the inflammatory response by inhibiting adhesion of leukocytes to endothelial cellsin vitro.Few studies have addressed specifically the role of iNOS in regulating leukocyte recruitmentin vivoin a model of acute inflammation. Thus, the objective of this study was to assess the role of iNOS in modulating neutrophil (PMN) extravasation in an oyster glycogen-induced model of acute peritonitis in rats. Data obtained in the present study demonstrates that injection (IP) of oyster glycogen induces massive and selective PMN recruitment into the peritoneal cavity of rats at 6 hrs following OG administration. These extravasated cells were found to contain significant amounts of iNOS protein as assessed by Western blot analysis. Treatment of rats with the selective iNOS inhibitor L-iminoethyl-lysine (L-NIL) dramatically reduced NO levels in lavage fluid as measured by decreases in nitrate and nitrite concentrations without significantly affecting iNOS protein levels. Although L-NIL inhibited NO production by >70%, it did not alter oyster glycogen-induced PMN recruitment when compared to vehicle-treated rats. We conclude that PMN-associated, iNOS-derived NO does not play an important role in modulating extravasation of these leukocytes in this model of acute inflammation.

Originally described by Uhlig et. al., the anti-CD40 agonist acute model of gut inflammation is a useful tool for interrogating myeloid-driven gut inflammation. Previous studies have shown that this model is sensitive to intervention with anti-TNFα, anti-IFNγ and anti-IL12/ 23p40. Here we present data that extend previous findings in this model and demonstrate an anti-CD40 dose dependent severity of disease pathogenesis as well as dose response curves for treatment with anti-TNFα and anti-IL12/23p40. In addition, we have identified both circulating and fecal biomarkers of disease. Methods: RAG2 deficient mice on the C57BL/6 background were injected i.p. with a single dose of anti-CD40 agonist antibody (FGK45) on day 0 and monitored daily for body weight loss until day 7. Prophylactic treatment with either anti-TNFα or anti-IL12/23p40 was performed using i.p. injections on day -1 and day 2 prior to sacrifice at day 7. Spleens were collected and weights recorded, plasma and feces were also collected for drug exposure levels and biomarker analysis. The colon was removed, flushed with PBS and buffered formalin prior to histological sectioning and scoring. Results: Mice treated with anti-CD40 (single dose 1.25-20mpk) exhibited a dose-dependent increase in splenomegaly, colonic inflammation and liver pathology with an induction ED50 of 2.5mpk. In addition, plasma levels of CXCL-10, MCP-1, MIG and other inflammatory mediators were also increased dose-dependently with CXCL-10 levels highly correlating with histology scores (r2 = 0.85). Fecal calprotectin (S100A8-S100A9 dimer), a biomarker routinely used in the clinic, also increased dose-dependently and correlated well with histologic scores (r2 = 0.72 and 0.68 respectively). As significant liver necrosis was observed at doses of anti-CD40 above 5mpk, this dose was selected for intervention studies. Prophylactic treatment with either anti-IL12/23p40 or anti-TNFα attenuated colonic pathology in a dose-dependent manner at day 7 of disease with an EC50 of 100 μg/mL for anti-p40. Therapeutic intervention (i.e. a single injection given at day 3 of disease) with anti-TNFα also significantly reduced colon histopathology scores while antiIL-12/23p40 only showed therapeutic significance at the highest dose (EC50 of 7 and 1.3 μg/mL respectively). Reduced colonic inflammation in all cases was accompanied by a reduction in circulating CXCL-10 levels. In summary, we have shown that the anti-CD40 acute model of gut inflammation is a useful drug discovery model for interrogating myeloid mechanisms involved in colitis and have identified circulating CXCL-10 and fecal calprotectin as biomarkers related to both pathogenesis and intervention in this model. We have also generated dose-efficacy relationships for clinically validated therapies such as anti-TNF and anti-IL12/23p40.

Background: Several transcription factors have been proposed to regulate IBD including the signal transducer and activator of transcription-6 (STAT-6). Methods: The role of STAT-6 was examined in the 5% dextran sulfate sodium (DSS)-induced murine model of colitis using STAT-6−/− and wildtype mice. Results: The disease activity index (DAI) revealed a significant increase in DAI in STAT-6−/− mice over STAT-6+/+ mice given DSS. Both STAT-6−/− and wildtype mice displayed severe inflammation and crypt damage. Additionally, STAT-6−/− mice showed significant injury to the proximal colon compared with their littermate controls. Furthermore, STAT-6−/− mice receiving DSS had dramatically higher levels of serum nitrite/nitrate than all other groups. STAT-6−/− animals also displayed higher levels of inteferon-γ than wildtype mice. Conclusions: Because STAT-6 has been reported to regulate the expression and activity of inducible NO synthase (iNOS), our data suggest that, in DSS colitis, STAT-6 may modulate iNOS, to limit NO formation and control the extent of inflammation in the colon. We conclude that STAT-6 may normally play an important regulatory role in the pathogenesis of inflammatory bowel disease, possibly through modulation of iNOS and interferon-γ.

The objective of this study was to assess the roles of NK cells, B cells and/or intraepithelial lymphocytes (IEL) in suppressing the development of colitis in nude mice reconstituted with CD4(+)CD45RB(high) T cells. BALB/c nude mice were lethally irradiated and reconstituted with bone marrow from different immunodeficient mice to generate athymic chimeras devoid of one or more lymphocyte populations. Transfer of CD4(+)C45RB(high) T cells into chimeric recipients devoid of B cells, T cells and IEL produced severe colitis within 6-8 weeks, whereas transfer of these same T cells into B cell- and T cell-deficient or T cell-deficient chimeras produced little to no gut inflammation. In addition, we found that nude mice depleted of NK cells or RAG-1(-/-) mice reconstituted with IEL failed to develop colitis following transfer of CD45RB(high) T cells. Severe colitis could, however, be induced in nude mice by transfer of activated/T(h)1 CD4(+)CD45RB(low) T cells. Taken together, our data suggest that IEL, but not B cells or NK cells, play an important role in suppressing the development of chronic colitis in this model. In addition, our data demonstrate that suppression of disease may be due to polarization of naive CD4(+) cells toward a non-pathogenic and/or regulatory phenotype.

Reactive oxygen and nitrogen species (RONS), once viewed solely as toxic by-products of aerobic metabolism and components of leukocyte bactericidal defenses, have since gained recognition as important intra- and intercellular signalling molecules in both normal as well as pathological conditions. Reactive oxygen and nitrogen species are formed in cells during oxidant stress, in reperfusion injury, and in response to several growth factors and cytokines (120). Indeed, these species may represent a novel class of second or third messengers. Like other 2nd messenger systems (e.g. Ca2+, cAMP), reactive oxygen and nitrogen species are ideal signalling molecules. They are rapidly formed and highly labile, which may serve to restrict them to specific cellular compartments and improve target specificity while reducing unwanted, non-specific effects. Most importantly, the biochemical modifications induced by modest levels of reactive nitrogen and oxygen species are largely reversible (119).

Central to inflammatory responses are the integrin-mediated adhesive interactions of cells with their ECM-rich environment. We investigated the role of the collagen-binding integrin alpha(1)beta(1) in intestinal inflammation using the mouse model of colitis induced by dextran sodium sulfate (DSS). mAb's directed against murine alpha(1) were found to significantly attenuate inflammation and injury in DSS-treated wild-type mice; similar protection was seen in mice deficient for alpha(1)beta(1) integrin. Blockade or loss of alpha(1)beta(1) was also associated with decreased mucosal inflammatory cell infiltrate and cytokine production. Importantly, we demonstrated that development and alpha(1)-mediated inhibition of DSS-induced colitis occurred independently of lymphocytes (Rag-2(-/-) mice), and identified the monocyte as a key alpha(1)beta(1)-expressing cell type involved in the development of colitis in this model. In response to DSS, both alpha(1) deficiency and anti-alpha(1) mAb treatment significantly reduced monocyte accumulation and activation within the lamina propria. In summary, the data demonstrate that engagement of leukocyte-associated alpha(1)beta(1) receptors with ECM plays a pivotal role in mediating intestinal inflammation via promotion of monocyte movement and/or activation within the inflamed interstitium. Therapeutic strategies designed to disrupt such interactions may prove beneficial in treating intestinal inflammation.

The inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.

One of the most consistent and dramatic findings in both experimental and human inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis) is the enhanced expression of the inducible isoform of nitric oxide synthase (iNOS) and the sustained overproduction of the free radical nitric oxide (NO). The role that iNOS-derived NO plays in the pathophysiology of inflammatory bowel disease remains the subject of intense investigation and active debate. Although several different studies using a variety of animal models of acute and chronic gut inflammation suggest that NO may promote intestinal inflammation, an equally impressive number of investigations suggest that iNOS may play no role or may act to attenuate or to limit the extent of inflammatory tissue injury. This review discusses some of the basic concepts related to the immunoregulation of chronic gut inflammation and summarizes the current state of knowledge of the role that NO may play in modulating inflammatory tissue injury.

Recent studies by a number of different laboratories have implicated nitric oxide (NO) as an important modulator of a variety of acute and chronic inflammatory disorders. A hallmark of inflammation is the adhesion of leukocytes to post-capillary venular endothelium and the infiltration of leukocytes into the tissue interstitium. Leukocyte adhesion and infiltration is known to be dependent on interaction of the leukocytes with the endothelial cell surface via a class of glycoproteins collectively known as endothelial cell adhesion molecules (ECAMs). Several recent studies suggest that NO may modulate cytokine-induced ECAM expression in cultured endothelial cells in vitro by regulating the activation of nuclear transcription factor kappa B (NF-kappaB). This discussion reviews some of the more recent studies that assess the role of the different NOS isoforms on the inflammatory response in vivo.

The inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) are chronic, idiopathic inflammatory disorders of the intestine and/or colon characterized by rectal bleeding, severe diarrhea, abdominal pain, fever and weight loss. Histologic examination of biopsies obtained from patients with active episodes of IBD reveal the infiltration of large numbers of leukocytes such as polymorphonuclear leukocytes (PMNs), monocytes, and lymphocytes into the intestinal interstitium. Extensive mucosal and/or transmural injury and dysfunction including edema, loss of goblet cells, decreased mucous production, crypt cell hyperplasia, erosions and ulcerations, accompany this inflammatory infiltrate. Despite several years of intense investigation, the etiology and specific pathogenetic mechanisms responsible for IBD remain poorly defined. Recent experimental and clinical studies suggest that the initiation and pathogenesis of these diseases are multi-factorial involving interactions among genetic, environmental and immune factors [1]. Regardless of exactly how these interactions ultimately promote chronic gut inflammation, it is becoming increasingly apparent that the immune system plays a crucial role in disease pathogenesis. Because the inflammation is localized primarily to the intestinal tract in IBD, investigators have focused on the intestinal lumen as the site for the antigenic trigger. Indeed, the chronic relapsing nature of IBD coupled to the fact that a large percentage of patients with Crohn’s disease will experience recurrence of the disease following surgical resection of the bowel, suggests that the antigen or antigens that initiate and perpetuate this disease are part of the normal gut flora. Different etio-logic theories have been proposed to account for this apparent mucosal immune system activation, however, data obtained from numerous experimental studies suggest that chronic gut inflammation may result from a dysregulated immune response to components of the normal gut flora [2]. For example, several groups of investigators have shown that targeted deletion of certain genes known to be important in regulating the inflammatory response or immune manipulation in mice induces chronic colitis in these animals [3]. Furthermore, the colonic inflammation observed in virtually all of these animal models is dependent upon the presence of normal gut flora. Animals raised under germ-free conditions either fail to develop disease or develop much milder forms of colitis [3]. These studies have lead investigators to suggest that bacterial/immune interactions in individuals with defects in their immune system may represent an important pathophysiological mechanism for the development of IBD in humans.

Abstract —Previous studies have suggested that oxygen-derived free radicals are involved in the pathophysiology of myocardial ischemia/reperfusion (MI/R) injury. Specifically, neutrophils have been shown to mediate postischemic ventricular arrhythmias and myocardial necrosis. We hypothesized that MI/R injury would be reduced in the absence (−/−) of NADPH oxidase. Heterozygous control mice (n=23) and NADPH oxidase –/– mice (n=24) were subjected to 30 minutes of coronary artery occlusion and 24 hours of reperfusion. Myocardial area at risk per left ventricle was similar in heterozygous control hearts (55±3%) and NADPH oxidase –/– hearts (61±4%). Contrary to our hypothesis, the size of infarct area at risk was similar in the heterozygous control mice (42±4%) and NADPH oxidase –/– mice (34±5%) ( P =not significant). In addition, echocardiographic examination of both groups revealed that left ventricle fractional shortening was similar in NADPH oxidase –/– mice (n=8; 27±2.5%) and heterozygous control mice (n=10; 23.3±3.3%) after MI/R. Superoxide production, as detected by cytochrome c reduction, was significantly impaired ( P –/– mice (n=6) compared with heterozygous mice (n=7) (0.04±0.03 versus 2.2±0.08 nmol O 2 ·min –1 ·10 6 cells –1 ). Intravital microscopy of the inflamed mesenteric microcirculation demonstrated that leukocyte rolling and adhesion were unaffected by the absence of NADPH oxidase. Oyster glycogen-stimulated neutrophil transmigration into the peritoneum was also similar in both the heterozygous control mice and NADPH oxidase –/– mice ( P =not significant). These findings suggest that NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after MI/R.

The primary product of the interaction between nitric oxide (NO) and superoxide ([figure]) is peroxynitrite (ONOO−), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or[figure]can further react with ONOO−to form species which mediate nitrosation. Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and[figure]. Since ONOO−reacts not only with NO and[figure]but also with CO2, the effects of bicarbonate ([figure]) on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3-diaminonaphthalene and reduced glutathione were examined. Nitric oxide and [figure] were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of [figure] did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2does not affect the scavenging of ONOO−by either NO or[figure]. However, an increase in the rate of DHR oxidation by ONOO−in the presence of [figure] suggests that a CO2-ONOO−adduct does play a role in the interaction of NO or[figure]with a product derived from ONOO−. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO−norcis-HOONO. It was concluded that NO reacts with bothtrans-HOONO and a CO2adduct of ONOO−to form nitrosating species which have similar oxidation chemistry and reactivity with[figure]and NO.

The objective of this study was to assess the effects of two structurally distinct yet selective proteasome inhibitors (PS-341 and lactacystin) on leukocyte adhesion, endothelial cell adhesion molecule (ECAM) expression, and nuclear factor-kappaB (NF-kappaB) activation in tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells (HUVEC) and the transformed, HUVEC-derived, ECV cell line. We found that TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclear neutrophil (PMN) adhesion to HUVEC but not to ECV; TNF also significantly enhanced surface expression of vascular cell adhesion molecule 1 and E-selectin (in HUVEC only), as well as intercellular adhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVEC with lactacystin completely blocked TNF-stimulated PMN adhesion, partially blocked U-937 adhesion, and completely blocked TNF-stimulated ECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1 expression in ECV. Pretreatment of HUVEC with PS-341 partially blocked TNF-stimulated leukocyte adhesion and ECAM expression. These effects of lactacystin and PS-341 were associated with inhibitory effects on TNF-stimulated NF-kappaB activation in both HUVEC and ECV. Our results demonstrate the importance of the 26S proteasome in TNF-induced activation of NF-kappaB, ECAM expression, and leukocyte-endothelial adhesive interactions in vitro.

We compared U-937 cell adhesion and adhesion molecule expression in human umbilical venous (HUVECs) and arterial (HUAECs) endothelial cells exposed to tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide (LPS). TNF and LPS stimulated vascular cell adhesion molecule (VCAM)-1 surface expression and adhesion of U-937 monocyte-like cells to HUVECs but not to HUAECs. Antibody studies demonstrated that in HUVECs at least 75% of the adhesion response is VCAM-1 mediated. Interleukin-1 stimulated U-937 cell adhesion to and VCAM-1 surface expression in both HUVECs and HUAECs. Pyrrolidinedithiocarbamate and the proteasome inhibitor MG-132 blocked TNF- and LPS-stimulated U-937 cell adhesion to HUVECs. These agents also significantly decreased TNF- and LPS-stimulated increases in HUVEC surface VCAM-1. TNF increased VCAM-1 protein and mRNA in HUVECs that was blocked by pyrrolidinedithiocarbamate. However, neither TNF or LPS stimulated VCAM-1 expression in HUAECs. TNF stimulated expression of both intercellular adhesion molecule-1 and E-selectin in HUVECs, but in HUAECs, only intercellular adhesion molecule-1 was increased. Electrophoretic mobility shift assays demonstrated no difference in the pattern of TNF-stimulated nuclear factor-κB activation between HUVECs and HUAECs. These studies demonstrate a novel and striking insensitivity of arterial endothelium to the effects of TNF and LPS and indicate a dissociation between the ability of HUAECs to upregulate nuclear factor-κB and VCAM-1.

Turnover numbers for 3-O-methylglucose transport by the homologous glucose transporters GLUT1 and GLUT4 were compared to those for truncated and chimeric transporters expressed in Xenopus oocytes to assess potential regulatory properties of the C-terminal domain. The ability of high intracellular sugar concentrations to increase the turnover number for sugar entry ("accelerated exchange") by GLUT1 and not by GLUT4 was maintained in oocytes. Replacing the GLUT1 C terminus with that of GLUT4 stimulated turnover 1.6-fold, but abolished accelerated exchange. Thus, the GLUT1 C terminus permits accelerated exchange by GLUT1, but in doing so must interact with other GLUT1 specific sequences since the GLUT4ctrm1 chimera did not exhibit this kinetic property. Removal of 38 C-terminal amino acids from GLUT4 reduced its turnover number by 40%, whereas removing only 20 residues or replacing its C terminus with that of GLUT1 increased its turnover number 3.5-3.9 fold. Therefore, using mechanisms independent of those which alter transporter targeting to the plasma membrane, C-terminal mutations in either GLUT1 or GLUT4 can activate transport normally restricted by the native C-terminal domain. These results implicate the C termini as targets of physiological factors, which through covalent modification or direct binding might alter C-terminal interactions to regulate intrinsic GLUT1 and GLUT4 transporter activity.

Targeted delivery remains one of the biggest challenges in the development of RNAi-based therapeutics. Cequent has developed a proprietary delivery technique for RNA interference, transkingdom RNAi (tkRNAi), in which non-pathogenic bacteria are engineered to invade specific target cells, produce and release short hairpin RNA (shRNA). We have previously shown tkRNAi to be successful in cell culture assays, in a mouse model for human colon cancer (APCmin), and in non-human primates (cynomolgus monkeys), by suppressing the oncogene, β-catenin. β-catenin is the key oncogene implicated in Colorectal Cancer (CRC) and Familial Adenomatous Polyposis (FAP); the latter being an orphan hereditary disease resulting in the formation of hundreds of polyps in the gastrointestinal tract and ultimately leads to the development of colon cancer without surgical intervention. No pharmaceutical treatment is available for patients with FAP. The non-human primate studies designed to evaluate on-target toxicities were conducted with a precursor (CEQ501) to our clinical candidate, CEQ508. Following optimization, which most importantly included platform engineering to improve hairpin production and processing, we conducted a large GLP mouse bridging toxicity study comparing off-target effects of CEQ508 and CEQ501. No CEQ508 or CEQ501-related adverse responses were identified in the following study parameters analyzed: clinical observations, body weights, serum chemistry, hematology, cytokines, gross or histopathology. As no test article-related changes were identified under the conditions of this study at the highest dose evaluated, the No Observed Effect Level (NOEL) for daily oral administration of either CEQ508 or CEQ501 was 5×109 cfu/day, or 2×1011 cfu/kg/day. (CEQ501 in non-human primates showed a similar safety profile with the No Observed Adverse Effect Level (NOAEL) determined to be 1×1012 cfu/day, or 2×1011 cfu/kg/day). Together with multiple internally conducted pharmacology and pharmacokinetic experiments, Cequent used this data in the nonclinical section of an IND filed in late 2009. In addition to presenting detailed in vivo data from our filing, an update on further development will be presented on CEQ508 for the proposed daily dosing of FAP patients with this oral tkRNAi therapeutic targeting β-catenin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4513.

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NSAID Induce Colitis in ILIO-ISplenocyte-ReconstJtuted, Rag Mice WT SpL Reconst IL104. SpL Reconst, No Ceils Transferred NSAdD 01 3.70.2 0 No NSAiD 0 fl fl inflammatory score, mean SE, 15 mice/groep, from 3 separate expedments

Inflammatory bowel disease (IBD) is associated with Th1/Th2 cytokine dysregulation, leukocyte extravasation, and tissue edema, but the mechanisms for cytokine-mediated vascular dysfunction are not understood. To investigate how cytokines might control edema in IBD, we determined vascular permeability and IFN-gamma expression in two models of murine colitis: SCID mice reconstituted with CD45RB(high T-lymphocytes (CD45RB(high)/SCID mice), and interleukin-10 gene deficient (IL-10(-/-)) mice. We also investigated the in vitro effects of IFN-gamma and IL-10 on human endothelial solute barrier and junction protein expression. Vascular permeability in CD45RB(high)/SCID and IL-10(-/-) mice was quantified using tissue (131)I-IgG accumulation. The IFN-gamma message was quantified using the ribonuclease protection assay. Endothelial barrier integrity in vitro was measured by transmonolayer electrical resistance, and junctional proteins were examined by immunoblotting and fluorescence microscopy. Both CD45RB(high)/SCID and IL-10(-/-) mice exhibit enhanced colonic microvascular leakage and IFN-gamma message levels compared to their respective controls. In vitro, IFN-gamma also reduced endothelial barrier (monolayer electrical resistance, increased albumin permeability) and reduced tight junction (occludin) expression and staining. These effects were reversed by pretreatment of monolayers with IL-10. Therefore, in vivo IFN-gamma and IL-10 may modulate microvascular leakage in IBD partly by controlling the expression of intestinal endothelial tight junctional proteins.