Beatrice Flogerzi

Effects of inhibition of prostaglandin endoperoxide synthase-2 in chronic gastro-intestinal ulcer models in rats

1 In the stomach, prostaglandins protect the gastric mucosa against injuries. One rate‐limiting step in prostaglandin synthesis is mediated by prostaglandin endoperoxide synthase (PGHS), the target enzyme of non‐steroidal anti‐inflammatory drugs (NSAIDs). Two isoforms of PGHS exist: a constitutive (PGHS‐1) and an inducible (PGHS‐2) enzyme. PGHS‐1 is the major source of gastric prostaglandins under physiological conditions. Inhibition of prostaglandin synthesis by traditional NSAIDs such as indomethacin and diclofenac which non‐selectively inhibit both PGHS‐1 and PGHS‐2, causes gastric and intestinal ulceration and delays gastric ulcer healing in chronic models. It has been shown that selective PGHS‐2 inhibitors such as L‐745,337 (5‐methanesulphonamide‐6‐(2,4‐difluorothio‐phenyl)‐1‐indanone) are not ulcerogenic and do not inhibit gastro‐intestinal prostaglandin synthesis. However, minimal information is available on the long‐term effects of PGHS‐2 inhibitors on the healing of previously established gastric injuries. We assessed the cellular localization and expression of PGHS‐1 and PGHS‐2 during gastric ulcer healing and assessed the effects of L‐745,337 on previously established cryoulcers in the rat gastric stomach. 2 PGHS‐1 and PGHS‐2 were located and quantified by immunohistochemistry during experimental gastric ulcer healing. PGHS‐2 immunoreactivity was only negligible in the normal gastric wall, but after gastric ulcerations, it was strongly detected in monocytes, macrophages, fibroblasts and endothelial cells below and between the regenerative glands. PGHS‐1 immunoreactivity detected in normal gastric mucosa, disappeared after gastric ulceration in the mucosa adjacent to the ulcer crater. However, it reappeared in the regenerative glands from day 5 onwards. Thus, PGHS‐1 and PGHS‐2 were located at different sites and their maximal expression followed a different time‐sequence. 3 We assessed the effects of L‐745,337, indomethacin and diclofenac on gastric ulcer healing and histological healing parameters in rats. L‐745,337, indomethacin and diclofenac dose‐dependently decreased the healing of gastric ulcers. L‐745,337, indomethacin and diclofenac decreased epithelial cell proliferation in the ulcer margin and microvessel density in the ulcer bed on day 8 and increased the thickness of the granulation tissue below the ulcer crater and the gap between both edges of the muscularis mucosae on day 15. Indomethacin and diclofenac, but not L‐745,337, decreased synthesis of 6‐keto‐PGF1α and PGE2 in tissue fragments from the stomach and terminal ileum and decreased platelet thromboxane B2 synthesis in clotting whole blood. 4 Dose‐response curves for the inhibition of chronic gastric ulcer healing by L‐745,337 (administered twice daily intragastrically) showed an ID50 value of 1.7 mg (4.3 μmol) kg−1. Dose‐response curves for the inhibition of PGE2 synthesis in inflammatory exudates in the acute carrageenin sponge rat model, showed ID50 values of 1.1  mg (3.1  μmol) kg−1 and 1.3 (3.3  μmol) mg kg−1 for indomethacin and L‐745,337, respectively. Thus, inhibition of chronic gastric ulcer healing by L‐745,337 occurs within a potentially therapeutic dose‐range. 5 In summary, PGHS‐2 is markedly accumulated after gastric ulceration in monocytes, macrophages, fibroblasts and endothelial cells in regions of maximal repair activity. Selective inhibition of PGHS‐2 by L‐745,337 delayed gastric ulcer healing though interference with epithelial cell proliferation, angiogenesis and maturation of granulation tissue in a potentially therapeutic dose range. PGHS‐2‐derived prostaglandins seem to have an important role in gastric ulcer healing.

Comparison of interferon-gamma release assay versus tuberculin skin test for tuberculosis screening in inflammatory bowel disease

OBJECTIVES: Reactivation of latent tuberculosis (TB) in inflammatory bowel disease (IBD) patients treated with antitumor necrosis factor-alpha medication is a serious problem. Currently, TB screening includes chest x-rays and a tuberculin skin test (TST). The interferon-gamma release assay (IGRA) QuantiFERON-TB Gold In-Tube (QFT-G-IT) shows better specificity for diagnosing TB than the skin test. This study evaluates the two test methods among IBD patients.METHODS: Both TST and IGRA were performed on 212 subjects (114 Crohns disease, 44 ulcerative colitis, 10 indeterminate colitis, 44 controls).RESULTS: Eighty-one percent of IBD patients were under immunosuppressive therapy; 71% of all subjects were vaccinated with Bacille Calmette Guérin; 18% of IBD patients and 43% of controls tested positive with the skin test (P < 0.0001). Vaccinated controls tested positive more often with the skin test (52%) than did vaccinated IBD patients (23%) (P= 0.011). Significantly fewer immunosuppressed patients tested positive with the skin test than did patients not receiving therapy (P= 0.007); 8% of patients tested positive with the QFT-G-IT test (14/168) compared to 9% (4/44) of controls. Test agreement was significantly higher in the controls (P= 0.044) compared to the IBD group.CONCLUSIONS: Agreement between the two test methods is poor in IBD patients. In contrast to the QFT-G-IT test, the TST is negatively influenced by immunosuppressive medication and vaccination status, and should thus be replaced by the IGRA for TB screening in immunosuppressed patients having IBD.

Roles of hepatocyte growth factor and its receptor Met during gastric ulcer healing in rats

BACKGROUND & AIMS It is unclear which growth factors are primarily responsible for stimulating gastric ulcer healing. The roles of hepatocyte growth factor (HGF) and Met/HGF receptor during gastric ulcer healing were studied in rats. METHODS HGF and Met/HGF receptor were located and quantified by in situ hybridization and immunohistochemistry during experimental gastric ulcer healing. The in vivo effects of exogenous recombinant human HGF on cell proliferation and ulcer healing were assessed and compared with those of placebo and omeprazole treatment. RESULTS Compared with intact oxyntic mucosa, messenger RNA (mRNA) of HGF and met was substantially greater in the ulcerated region on days 3 and 15. HGF mRNA was located in stromal cells between the regenerative glands and in the arterial vessels of submucosal tissue, whereas met mRNA was located in the epithelial cells of the regenerative glands. After cryoinjury, immunoreactivity for the Met/HGF receptor was absent on day 3, reappeared on day 8, and was overexpressed on day 15. Exogenous recombinant human HGF had no effect on the ulcer healing parameters over days 3-8, but it did increase epithelial cell proliferation in the ulcer margin over days 8-15. CONCLUSIONS These data suggest that HGF mediates specific tissue interactions between mesenchyme and epithelia during gastric ulcer healing.

Immune sensitization to yeast antigens in ASCA-positive patients with Crohn's disease.

BackgroundAlimentary antigens may play a role in the perpetuation of inflammation in Crohn’s disease (CD). Yeast antigens are widespread components of food. A proportion of CD patients develop antibodies against the yeast Saccharomyces cerevisiae (ASCA), but little is known about the cellular immune reactivity against food antigens in antibody-positive and -negative patients. MethodsLymphocytes from patients with CD, ulcerative colitis, and healthy controls were tested for their proliferative response after stimulation with the yeast antigen mannan and ovalbumin. The cellular phenotypes and activation markers were analyzed via FACS. Cytokine concentrations and antibody titers were determined by ELISA. ResultsOnly lymphocytes of ASCA-positive patients with CD proliferated after stimulation with mannan. These lymphocytes expressed increased activation markers (CD25, CD69). Activation of T cells was mediated by antigen-presenting cells and was associated with increased tumor necrosis factor-&agr; (TNF-&agr;) levels. The immune reactivity to ovalbumin was predominantly found in CD patients. It was weaker compared with mannan, independent of ASCA status, and also present in healthy controls. ConclusionsA disturbed humoral and cellular response to the yeast antigen mannan is specifically seen in a subgroup of CD patients. This phenomenon may be due to a loss of tolerance toward yeast and is possibly genetically determined.

Mannan-binding lectin deficiency results in unusual antibody production and excessive experimental colitis in response to mannose-expressing mild gut pathogens

Background In Crohns disease (CD) the deficiency of mannan-binding lectin (MBL) is associated with an increased prevalence of anti-Saccharomyces cerevisiae antibodies (ASCA) and with complicated phenotypes of the disease. However, the role of MBL in intestinal inflammation is currently unclear. A study was undertaken to analyse local MBL expression in human intestine and the consequences of MBL deficiency in experimental colitis and yeast infection. Methods ASCA were measured by ELISA. MBL was assessed by ELISA and quantitative PCR. Wild type and MBL-deficient mice were administered dextran sulfate sodium (DSS) in the presence or absence of viable Candida albicans or adhesive invasive Escherichia coli (AIEC). Mice were infected with C albicans to assess generation of anti-yeast mannan antibodies. Results MBL expression was virtually undetectable in the intestinal mucosa of both healthy controls and patients with CD, irrespective of macroscopic inflammation, indicating that systemic MBL must be responsible for the reduced risk of complicated disease in MBL-competent patients with CD. MBL-deficient mice showed enhanced DSS colitis upon oral challenge with C albicans or AIEC. C albicans could be recovered from the kidneys of colitic/C albicans-fed MBL-deficient, but not wild type mice. Infection with C albicans induced high titres of anti-C albicans mannan IgM and IgG in MBL-deficient mice but only a modest and transient IgM response with no class switch to IgG in wild type mice. Cross-reactive ASCA IgM continuously increased in MBL-deficient mice but rapidly declined after transient induction in wild type mice. In MBL-deficient mice, increased C albicans dissemination correlated with reduced early retention in the circulation. Conclusions These results suggest that systemic MBL helps to prevent excessive inflammation upon access of normally mild pathogens across the damaged intestinal epithelium. Lack of this innate defence promotes antibody responses with cross-reactive potential against common mannan epitopes. These interpretations are compatible with the increased prevalence of ASCA and complicated disease phenotypes in MBL-deficient patients with CD.

Galectin-3 modulates T cell activity and is reduced in the inflamed intestinal epithelium in IBD

Background: Galectins are involved at different stages in inflammation. Galectin‐3, although mostly described as proinflammatory, can also act as an immunomodulator by inducing apoptosis in T cells. The present study aims to determine galectin‐3 expression in the normal and inflamed intestinal mucosa and to define its role in T cell activity. Materials and Methods: Galectin‐3 was detected by quantitative polymerase chain reaction with total RNA from endoscopic biopsies and by immunohistochemistry. Biopsies and peripheral blood mononuclear cells (PBMC) were stimulated in vitro and were used to assess the functional consequences of inhibition or exogenous addition of galectin‐3. Results: Galectin‐3 is expressed at comparable levels in controls and inflammatory bowel disease (IBD) patients in remission. In the normal mucosa, galectin‐3 protein was mainly observed in differentiated enterocytes, preferentially at the basolateral side. However, galectin‐3 was significantly downregulated in inflamed biopsies from IBD patients. Ex vivo stimulation of uninflamed biopsies with tumor necrosis factor led to similar galectin‐3 messenger RNA downregulation as in vivo. When peripheral blood mononuclear cells (PBMC) were analyzed, galectin‐3 was mainly produced by monocytes. Upon mitogen stimulation, we observed increased proliferation and decreased activation‐induced cell death of peripheral blood T cells in the presence of galectin‐3‐specific small interfering RNA. In contrast, exogenous addition of recombinant galectin‐3 led to reduced proliferation of mitogen‐stimulated peripheral blood T cells. Conclusions: Our results suggest that downregulation of epithelial galectin‐3 in the inflamed mucosa reflects a normal immunological consequence, whereas under noninflammatory conditions, its constitutive expression may help to prevent inappropriate immune responses against commensal bacteria or food compounds. Therefore, galectin‐3 may prove valuable for manipulating disease activity.

Phenotypic associations of Crohn's disease with antibodies to flagellins A4-Fla2 and Fla-X, ASCA, p-ANCA, PAB, and NOD2 mutations in a swiss cohort†

Background: Distinct Crohns disease (CD) phenotypes correlate with antibody reactivity to microbial antigens. We examined the association between antibody response to 2 new flagellins called A4‐Fla2 and Fla‐X, anti‐Saccharomyces cerevisiae antibodies (ASCA), anti‐neutrophil cytoplasmic antibodies (p‐ANCA), anti‐pancreas antibodies (PAB), NOD2 mutations (R702W, G908R, and L1007fsinsC), and clinical CD phenotypes (according to Vienna criteria). Methods: All the above‐mentioned antibodies as well as NOD2 mutations were determined in 252 CD patients, 53 with ulcerative colitis (UC), and 43 healthy controls (HC) and correlated with clinical data. Results: A seroreactivity for A4‐Fla2/Fla‐X/ASCA/p‐ANCA/PAB (in percent) was found in 59/57/62/12/22 of CD patients, 6/6/4/51/0 of UC patients, and 0/2/5/0/0 of healthy controls. CD behavior: 37% B1, 36% B2, and 27% B3. In multivariate logistic regression, antibodies to A4‐Fla2, Fla‐X, and ASCA were significantly associated with stricturing phenotype (P = 0.027, P = 0.041, P < 0.001), negative associations were found with inflammatory phenotype (P = 0.001, P = 0.005, P < 0.001). Antibodies to A4‐Fla2, Fla‐X, ASCA, and NOD2 mutations were significantly associated with small bowel disease (P = 0.013, P = 0.01, P < 0.001, P = 0.04), whereas ASCA was correlated with fistulizing disease (P = 0.007), and small bowel surgery (P = 0.009). Multiple antibody responses against microbial antigens were associated with stricturing (P < 0.001), fistulizing disease (P = 0.002), and small bowel surgery (P = 0.002). Conclusions: Anti‐flagellin antibodies and ASCA are strongly associated with complicated CD phenotypes. CD patients with serum reactivity against multiple microbes have the greatest frequency of strictures, perforations, and small bowel surgery. Further prospective longitudinal studies are needed to show that antibody‐based risk stratification improves the clinical outcome of CD patients. (Inflamm Bowel Dis 2009)

Amelioration of murine colitis by feeding a solution of lysed Escherichia coli.

Background: Immune reactivity towards the bacterial intestinal flora plays an important part in the pathogenesis of inflammatory bowel disease. Disease activity can be positively influenced by the administration of living probiotic bacteria. We investigated the effect of soluble bacterial antigens extracted from Escherichia coli (strain Laves) on the disease activity of murine colitis. Methods: C3H.IL-10-/- and BALB/c mice with dextran sulphate sodium-induced colitis were treated with either a bacterial lysate from E. coli or with a placebo. Mice were monitored and inflammation was assessed by histological scoring, analysis of fecal IL-1β and measurement of cytokine production by ELISA. T cell proliferation was quantified by 3 H-thymidine incorporation. Results: Clinically and histologically, bacterial-lysate-treated mice revealed significantly (P < 0.05) fewer signs of colitis than placebo-treated mice. Fecal IL-1β and mucosal TNF-α and IFN-γ concentrations were significantly lower (P < 0.05) in verum-treated mice than in the placebo group. Furthermore, lymphocyte proliferation after stimulation with lipopolysaccharide or caecal bacterial antigen was significantly (P < 0.05) reduced in verum-treated mice. Conclusion: The use of E. coli lysate is effective in the amelioration of murine colitis. This effect may be due to a decreased Th1 reaction and to an induction of tolerance against bacterial antigens.

Anti-Saccharomyces cerevisiae mannan antibodies (ASCA) of Crohn's patients crossreact with mannan from other yeast strains, and murine ASCA IgM can be experimentally induced with Candida albicans.

Background: Anti‐Saccharomyces cerevisiae antibodies (ASCA) present in a subgroup of Crohns disease (CD) patients indicate loss of tolerance against commensal antigens. ASCA can be induced in Candida albicans‐infected rabbits, suggesting their potential crossreactive nature. The present study aimed to determine crossreactivities of ASCA with cell wall mannans from other yeasts, including the opportunistic pathogen C. albicans, and to define the requirements for (crossreactive) ASCA in experimental mice. Methods: ASCA were determined by enzyme‐linked immunosorbent assay (ELISA). ASCA were neutralized by preincubating sera with purified mannans. Binding of ASCA was visualized by Western blot. Mice were immunized with live yeasts and experimental colitis was induced with dextran sodium sulfate (DSS). Results: Seroreactivity of ASCA‐positive CD patients against S. cerevisiae mannan significantly correlates with that against mannans from 5 other yeast species, including C. albicans. This correlation is due to crossreactive IgG, demonstrated by the loss of reactivity after preincubation of sera with mannans from the other yeasts. Immunization of mice with S. cerevisiae or C. albicans fails to induce (crossreactive) ASCA IgM or IgG antibodies. Subsequent chronic experimental colitis concomitant with feeding live yeasts promotes ASCA IgM but not IgG generation, while titers remain modest compared to those in ASCA‐positive CD patients. Conclusions: Correlations of ASCA reactivities against mannans from different yeasts are due to crossreactive IgGs. The inability of mice to readily generate ASCA is in line with the current opinion that genetic predisposition is a prerequisite for the development of this and other unusual immune reactivities in CD.

Low Mannan-Binding Lectin Serum Levels Are Associated With Complicated Crohn's Disease and Reactivity to Oligomannan (ASCA)

OBJECTIVES:Mannan-binding lectin (MBL) acts as a pattern-recognition molecule directed against oligomannan, which is part of the cell wall of yeasts and various bacteria. We have previously shown an association between MBL deficiency and anti-Saccharomyces cerevisiae mannan antibody (ASCA) positivity. This study aims at evaluating whether MBL deficiency is associated with distinct Crohns disease (CD) phenotypes.METHODS:Serum concentrations of MBL and ASCA were measured using ELISA (enzyme-linked immunosorbent assay) in 427 patients with CD, 70 with ulcerative colitis, and 76 healthy controls. CD phenotypes were grouped according to the Montreal Classification as follows: non-stricturing, non-penetrating (B1, n=182), stricturing (B2, n=113), penetrating (B3, n=67), and perianal disease (p, n=65). MBL was classified as deficient (<100 ng/ml), low (100–500 ng/ml), and normal (500 ng/ml).RESULTS:Mean MBL was lower in B2 and B3 CD patients (1,503±1,358 ng/ml) compared with that in B1 phenotypes (1,909±1,392 ng/ml, P=0.013). B2 and B3 patients more frequently had low or deficient MBL and ASCA positivity compared with B1 patients (P=0.004 and P<0.001). Mean MBL was lower in ASCA-positive CD patients (1,562±1,319 ng/ml) compared with that in ASCA-negative CD patients (1,871±1,320 ng/ml, P=0.038). In multivariate logistic regression modeling, low or deficient MBL was associated significantly with B1 (negative association), complicated disease (B2+B3), and ASCA. MBL levels did not correlate with disease duration.CONCLUSIONS:Low or deficient MBL serum levels are significantly associated with complicated (stricturing and penetrating) CD phenotypes but are negatively associated with the non-stricturing, non-penetrating group. Furthermore, CD patients with low or deficient MBL are significantly more often ASCA positive, possibly reflecting delayed clearance of oligomannan-containing microorganisms by the innate immune system in the absence of MBL.