Addition of metronidazole to azathioprine for the prevention of postoperative recurrence of Crohn's disease: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Endoscopic recurrence occurs in up to 80% of patients with Crohn's disease 1 year after intestinal resection. Imidazole antibiotics, thiopurines, and particularly their combination have proven efficacy in preventing endoscopic recurrence. The aim of the study was to compare the efficacy of the addition of metronidazole (for 3 months after the surgical treatment) to azathioprine for the prevention of postsurgical endoscopic recurrence. METHODS: A pilot study was made of 50 patients with Crohn's disease undergoing intestinal resection with ileocolic anastomosis and treated with 2 to 2.5 mg/kg of azathioprine per day for 1 year. The patients were randomized to receive additional 15 to 20 mg/kg of metronidazole per day or placebo for the first 3 months (n = 25 per arm). Endoscopic assessment was performed 6 and 12 months after the surgical resection. The primary end point was the prevention of endoscopic recurrence as defined by a Rutgeerts score of <2 at 6 months. The initial sample size had an 80% statistical power in detecting an absolute risk reduction of ≥30%. RESULTS: Endoscopic recurrence occurred in 28% and 44% of the patients at 6 months (P = 0.19) and in 36% and 56% (P = 0.15) at 12 months in the metronidazole and placebo groups, respectively. No statistically significant differences were found between the treatment groups regarding severe endoscopic recurrence (Rutgeerts score ≥ 3) at 6 and 12 months. Likewise, there were no differences in the rate of adverse events between the treatment groups. CONCLUSIONS: The addition of metronidazole to azathioprine did not significantly reduce the risk of endoscopic recurrence beyond azathioprine alone in this study but does not worsen its safety profile.

Omega-3 fatty acids and inflammatory bowel diseases - a systematic review.

BACKGROUND & AIM: Despite their well known anti-inflammatory actions, the clinical usefulness of omega-3 PUFA in inflammatory bowel disease is controversial. We aimed to systematically review the available data on the performance of omega-3 PUFA as therapeutic agents in these patients. METHODS: Electronic databases were systematically searched for RCT of fish oil or omega-3 PUFA therapy in both active and inactive ulcerative colitis or Crohn's disease, without limitation on either the length of therapy or the form it was given, including nutritional supplements and enteral formula diets. Eligible articles were assessed for methodological quality on the basis of the adequacy of the randomisation process, concealment of allocation, blinding of intervention and outcome, possible biases, and completeness of follow-up. The five-point Oxford quality score was calculated. RESULTS: A total of 19 RCT were finally selected for this review. Overall, available data do not allow to support the use of omega-3 PUFA supplementation for the treatment of both active and inactive inflammatory bowel disease. Negative results are quite consistent in trials assessing the use of omega-3 PUFA to maintain disease remission, particularly ulcerative colitis, and to a lesser extent Crohn's disease. Trials on their use in active disease do not allow to draw firm conclusions mainly because the heterogeneity of design (ulcerative colitis) or their short number (Crohn's disease). In most trials, the appropriateness of the selected placebo is questionable. CONCLUSION: The present systematic review does not allow to make firm recommendations about the usefulness of omega-3 PUFA in inflammatory bowel disease.

Bacteria and spontaneous experimental colitis: immunological changes.

BACKGROUND: Intestinal commensal flora seems to be a requisite for both human and experimental intestinal inflammation. Our aim was to assess the immunological changes in the colon of IL-10(-/-) mice depending on the environmental conditions. MATERIALS AND METHODS: Twelve wild-type (WT) and 24 IL-10(-/-) 4-week-old mice were kept under specific pathogen-free (SPF) conditions for 4 weeks. Half of them were transferred to a conventional environment. Mice were sacrificed at 12 weeks of age, and the incidence and severity of colitis was assessed. Intraepithelial (IEL) and lamina propria (LPL) lymphocytes were assessed for phenotype and apoptosis by flow cytometry. Toll-like receptors 2 (TLR2) and TLR9 expression was assessed by real-time PCR. Immunohistochemical analyses for cell apoptosis, TLR2 and MyD88 were also performed. RESULTS: IL-10(-/-) mice shifted to conventional conditions showed a greater incidence (66% vs. 50%) and severity of colitis than animals kept under SPF conditions (P = 0·009). The number of CD3+ IEL was higher and their apoptosis rate lower in IL-10(-/-) than in their WT counterparts, regardless of the environment. In LPL, however, these differences were only observed in mice shifted to conventional conditions. TLR2 expression was significantly increased in SPF-housed IL-10(-/-) mice when compared to WT controls. Immunohistochemistry demonstrated the loss of TLR2 and MyD88 in damaged areas. CONCLUSIONS: In SPF conditions, IL-10 deficiency appears to be compensated by an increased epithelial TLR2 expression, thus resulting in a milder colonic damage. However, in conventional conditions, this compensatory mechanism would be exceeded inducing a more severe colonic damage with activation of LPL immune cells.

Therapeutic effect of antisecretory factor-rich egg yolk on the late phases of 2,4,6-trinitrobenzenesulphonic acid colitis in mice.

Antisecretory factor (AF) is expressed in all tissues of mammals, inhibits intestinal hypersecretion and has anti-inflammatory properties as well. Endogenous AF synthesis may be stimulated by feeding hydrothermally processed cereals. Alternatively, freeze-dried egg yolk can be used as a source of exogenous AF. Several reports have suggested that AF from freeze-dried egg yolk may be useful in inflammatory bowel disease. We assessed the effect of freeze-dried, AF-rich egg yolk intake on 2,4,6-trinitrobenzenesulphonic acid (TNBS) colitis. Balb/c mice were randomised to receive (1) AF in sterile drinking-water (4 g/l, n 38) and (2) sterile drinking-water alone (vehicle, n 38) from TNBS or saline administration onwards. Different subsets of mice were killed at weeks 1-3 after TNBS or saline administration. Macroscopic and microscopic damage was assessed in colonic specimens. Eicosanoid and cytokine production was evaluated in supernatants of 24 h-incubated colonic explants. Myeloperoxidase activity was measured in frozen colonic samples, while apoptosis was assessed in paraffined samples by the in situ oligoligation method. AF-treated mice showed a milder colonic damage compared with the vehicle group, which became statistically significant at week 3. This was accompanied by decreased IL-2, IL-1 and leukotriene B4 production at weeks 2 and 3, as well as increased interferon-γ at week 1, in AF-treated mice compared with vehicle-treated mice. AF-treated mice had significantly increased counts of apoptotic cells in the lamina propria at weeks 1 and 2 post-TNBS. In conclusion, the administration of AF-rich egg yolk has a therapeutic effect in the late phases of TNBS colitis in Balb/c mice.

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

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

Resequencing of positional candidates identifies low frequency IL23R coding variants protecting against inflammatory bowel disease.

Genome-wide association studies (GWAS) have identified dozens of risk loci for many complex disorders, including Crohn's disease. However, common disease-associated SNPs explain at most ∼20% of the genetic variance for Crohn's disease. Several factors may account for this unexplained heritability, including rare risk variants not adequately tagged thus far in GWAS. That rare susceptibility variants indeed contribute to variation in multifactorial phenotypes has been demonstrated for colorectal cancer, plasma high-density lipoprotein cholesterol levels, blood pressure, type 1 diabetes, hypertriglyceridemia and, in the case of Crohn's disease, for NOD2 (refs. 14,15). Here we describe the use of high-throughput resequencing of DNA pools to search for rare coding variants influencing susceptibility to Crohn's disease in 63 GWAS-identified positional candidate genes. We identify low frequency coding variants conferring protection against inflammatory bowel disease in IL23R, but we conclude that rare coding variants in positional candidates do not make a large contribution to inherited predisposition to Crohn's disease.

Regulation of colorectal cancer cell apoptosis by the n-3 polyunsaturated fatty acids Docosahexaenoic and Eicosapentaenoic.

Several studies have suggested that the n-3 fatty acids Docosahexaenoic (DHA) and Eicosapentaenoic (EPA) have an important protective effect on colorectal cancer, and this could be at least partly due to their proapoptotic activity. It is unclear, however, how this phenomenon is triggered and what mechanisms are implicated. Here, we show that both DHA and EPA have an important proapoptotic effect on colorectal cancer cells with different molecular phenotypes but not in noncancerous cells. Apoptosis is caspase dependent, and both intrinsic and extrinsic pathways are implicated. The dimerization of Bax and Bak, the depolarization of the mitochondrial membrane, and the subsequent release of cytochrome c and Smac/Diablo to the cytosol evidence the activation of the intrinsic pathway. The implication of the extrinsic pathway is shown by the activation of caspase-8, along with the down-regulation of FLIP. The timing of caspase-8 activation, and the oligomerization of Bid with Bax, suggest a cross-talk with the intrinsic pathway. None of the death receptors that commonly initiate the extrinsic pathway: FAS, TNF-R1, and TRAIL-R2 are found to be responsible for triggering the apoptosis cascade induced by DHA and EPA. Neither PPARgamma nor cyclooxygenase-2, two likely candidates to regulate this process, play a significant role. Our findings suggest that the down-regulation of two key regulatory elements of the extrinsic and intrinsic pathways, FLIP and XIAP, respectively, is determinant in the induction of apoptosis by DHA and EPA. These fatty acids could potentially be useful adjuvant anticancer agents in combination with other chemotherapeutic elements.

Prospective, observational, cross-sectional study of intestinal infections among acutely active inflammatory bowel disease patients.

BACKGROUND: Intestinal infections have been claimed to precipitate or aggravate flares of inflammatory bowel disease (IBD). The reported incidence of such infections among IBD patients varies between 9 and 13%, but only a few prospective studies have been conducted. AIMS: To evaluate the incidence of intestinal infections by enteropathogens in patients with active IBD, their impact on clinical outcome, and to identify associated risk factors. PATIENTS AND METHODS: Consecutive patients admitted because of a relapse or suspected onset of IBD were prospectively included. At admittance, stool samples for culture, examination for intestinal parasites, and cytotoxin assay for Clostridium difficile were collected. Baseline clinical characteristics, potential risk factors for gastrointestinal infections, and clinical outcome were recorded. RESULTS: Ninety-nine episodes were included. Six intestinal infections were diagnosed in 6 patients (5 ulcerative colitis, 1 ileocolonic Crohn's disease), Campylobacter jejuni being the most frequent isolated microbe (n = 5). None of the patients with intestinal infection needed surgery, but two of them required second-line therapies. CONCLUSIONS: Gastrointestinal infections among IBD patients do not exceed 10% and occur mostly in patients with extensive involvement of the colon. Infection by enteropathogenic bacteria does not appear to be associated with a poorer clinical outcome of the IBD flare.

Lactobacillus fermentum CECT 5716 prevents and reverts intestinal damage on TNBS-induced colitis in mice.

BACKGROUND: Probiotics attenuate gut inflammation when administered before experimental colitis, but data on their effect after colitis induction are scarce. We aimed to evaluate the effects of Lactobacillus fermentum CECT 5716 on gut injury when administered either before or after trinitrobencene sulfonic acid (TNBS) colitis in Balb/c mice. METHODS: In a preventive study, probiotic or vehicle was administered for 2 weeks before colitis. Then mice were allocated to: probiotic + TNBS, probiotic + sham, vehicle + TNBS, or vehicle + sham, and sacrificed 72 hours later. In a therapeutic study, mice were allocated into the same groups as before. Probiotic or vehicle were administered for 3 weeks. Mice were sacrificed at weeks 1, 2, and 3 after TNBS. Histological score, myeloperoxidase activity, and eicosanoid and cytokine production in colonic explant cultures were measured. Immunohistochemistry for nitrotyrosine and MyD88 was also performed. RESULTS: In the preventive study, colitis was milder with probiotic than with vehicle (P = 0.041). This was associated with increased PGE(2), IL-2, and IL-4 production, as well as attenuated nitrotyrosine staining in the former. In the therapeutic study, histological score at week 1 post-TNBS was higher in probiotic than in vehicle fed mice (P = 0.018). However, at weeks 2 and 3 the histological score was significantly lower-with decreased IL-6 production and increased MyD88 staining-in mice receiving the probiotic. CONCLUSIONS: Pretreatment with L. fermentum CECT 5716 attenuates TNBS colitis, an effect that seems to be due to its antioxidant abilities. When administered after TNBS, this probiotic is also effective in accelerating colitis recovery, and this is associated with an enhanced Toll-like receptor function.

Partial replacement of dietary (n-6) fatty acids with medium-chain triglycerides decreases the incidence of spontaneous colitis in interleukin-10-deficient mice.

Enteral nutrition has a primary therapeutic effect in active Crohn's disease. It is unknown which nutrient(s) account for this action, but a role for both the amount and type of dietary fat has been postulated. Some clinical and experimental data suggest that medium-chain triglycerides (MCT) may reduce intestinal inflammation. We aimed to assess the effect of replacing part of the dietary fat with MCT on the incidence and severity of colitis in interleukin (IL)-10(-/-) mice under specific pathogen-free conditions. Twenty-four IL-10(-/-) 4-wk-old mice were randomized to receive a control diet based on sunflower oil [(n-6) fatty acids (FA)] and an experimental isocaloric, isonitrogenous diet with 50% sunflower and 50% coconut oil (MCT diet). When the mice were 12 wk old, they were killed and the colon was examined for the presence of colitis, lymphocyte subpopulations and apoptosis, ex vivo cytokine production in supernatant of colon explants, toll-like receptor (TLR)-2 and TLR-9 mRNA, and FA profile in colonic tissue homogenates. Colitis incidence was lower in the IL-10(-/-) mice fed the MCT diet (1/12) than in the mice fed the control diet (8/12; P = 0.03). The histological damage score was also lower in the former (P < 0.0005). Feeding the MCT diet resulted in fewer total and apoptotic intraepithelial CD3+ and lamina propria CD3+CD4+ lymphocytes, as well as downregulated production of IL-6 and interferon-gamma, and reduced TLR-9 mRNA. We conclude that partial replacement of dietary (n-6) FA with MCT decreases the incidence of colitis in a model of spontaneous intestinal inflammation and provide experimental arguments for a possible primary therapeutic effect of MCT in human Crohn's disease.

Cytomegalovirus infection in ulcerative colitis: a prospective, comparative study on prevalence and diagnostic strategy.

BACKGROUND: Cytomegalovirus (CMV) infection has been reported in ulcerative colitis (UC), especially in severe, steroid-refractory disease. However, its role in steroid-refractoriness remains unknown. Our goals were to evaluate the prevalence of CMV disease in UC, the best diagnostic strategy, and the influence of disease activity and/or treatment in its development. METHODS: Prospective, observational study including 114 subjects with active UC requiring intravenous steroids, steroid-refractory UC, inactive UC on mesalamine, inactive UC on azathioprine, and healthy controls. CMV antibodies, pp65-antigenemia, and rectal biopsies for hematoxylin and eosin staining, immunohistochemistry, and CMV-pp67 mRNA were performed. These procedures were repeated after medical treatment only in patients with active UC. CMV disease was defined by the presence of inclusion bodies and/or positive immunohistochemistry in colonic biopsies. RESULTS: CMV disease was found in 6 steroid-refractory, CMV-IgG-positive UC patients but not among controls, inactive UC, or steroid-responding UC patients. In 5 out of the 6 patients, CMV disease was diagnosed after 7-10 days on cyclosporine. CONCLUSIONS: CMV disease in UC only affects seropositive, steroid-refractory UC patients. Steroid/cyclosporine treatment together with disease activity may predispose to latent colonic CMV reactivation. The impact of antiviral therapy on the clinical outcome of these patients remains to be elucidated.

Tuberculous chemoprophylaxis requirements and safety in inflammatory bowel disease patients prior to anti-TNF therapy.

BACKGROUND: Preventive actions are advised since the use of anti-tumor necrosis factor (TNF) agents is known to increase the risk of tuberculosis (TB). No data related to the effectiveness and safety of the preventive chemoprophylaxis (ChP) for TB in inflammatory bowel disease (IBD) patients are available. The goal was to evaluate the requirements, effectiveness, and safety profile of ChP in IBD patient candidates for anti-TNF therapy. METHODS: All IBD patients diagnosed with latent TB while evaluated for anti-TNF therapy from the IBD database of 9 Spanish centers were included. Epidemiological and clinical data, risk factors for hepatotoxicity, ChP regimens, and side effects were registered. RESULTS: Sixty-three out of 497 IBD evaluated patients (12.5%) had latent TB. Sixty-eight percent were on immunomodulators and 42% on systemic corticosteroids when a TB skin test (TST) was performed. The detection of a positive TST was done in 86% after a single exposure, but 14% needed a booster. All but 1 were treated with isoniazid alone for 6 or 9 months, and only 1 case required ChP discontinuation because of hepatotoxicity. No risk factors for hepatotoxicity were found. No cases of active TB were noticed in the 67 patients further treated with anti-TNF therapy. CONCLUSIONS: More than 10% of Spanish IBD patients who are candidates for anti-TNF therapy have latent TB. TST retest is required to identify at least 14% of such patients; therefore, it should be considered if the initial TST is negative. ChP is safe in IBD patients even in those taking concomitant, potentially hepatotoxic drugs.

Long-term methotrexate for Crohn's disease: safety and efficacy in clinical practice.

GOALS: To assess the efficacy and safety profile of methotrexate (MTX) for the treatment of Crohn's disease (CD) in clinical practice. BACKGROUND: MTX is widely used for some chronic immunologic diseases. Although some randomized controlled trials suggest its efficacy in CD, this drug remains a second-line, underused, immunomodulator. STUDY: Medical records of all patients treated with MTX for CD in our center (n=44) were reviewed. Clinical and epidemiologic parameters, including risk factors for hepatotoxicity, were registered. RESULTS: MTX was prescribed mainly for steroid-dependency (n=22) and as concomitant treatment to infliximab (n=18). Mean duration of treatment was 22.9+/-19 months, with a mean cumulative dose of MTX of 1169+/-784 mg. Thirty-nine percent of patients developed drug-related side effects, hepatotoxicity being the most frequent [13 patients (30%)]. However, only 5 patients (11%) had to discontinue MTX. In steroid-dependent CD patients, disease remission and complete steroid withdrawal was achieved in 77% of cases. Seven patients lost their initial response to MTX during follow-up, leading to a cumulative probability of remission of 39% after 3 years of treatment. CONCLUSIONS: MTX is well tolerated in most CD patients. Although a great proportion of steroid-dependent CD patients achieve disease remission and steroid withdrawal, there is a trend to a loss of efficacy with time. Larger, long-term studies are necessary to establish the role of MTX in the management of CD.

Impact of azathioprine on the prevention of postoperative Crohn's disease recurrence: results of a prospective, observational, long-term follow-up study.

BACKGROUND: Postoperative recurrence (PR) occurs early after intestinal resection in >75% of Crohn's disease (CD) patients. No well-established strategy for long-term PR prevention is available. The aim was to prospectively evaluate the long-term endoscopic and clinical outcomes of postoperative CD on maintenance treatment with azathioprine (AZA), especially in patients who developed endoscopic lesions confined to the ileocolic anastomosis. METHODS: Long-term AZA therapy (2-2.5 mg/kg/day) was initiated immediately after surgery in 56 consecutive patients who underwent a curative intestinal resection. Clinical and biological assessments every 3 months, as well as yearly endoscopic evaluation, were performed until the end of the study or clinical PR (CPR). RESULTS: Thirty-seven patients (70%) showed mucosal lesions at endoscopy after a median of 12 months (range 12-60); however, in 15 of these patients lesions were confined to the anastomosis and only 6 showed endoscopic progression, but none of them developed CPR. Among the remaining 22 patients with endoscopic PR (EPR), 23% suffered a CPR during follow-up. Thirty percent of patients remained free of EPR after a median follow-up of 33 months (range 12-84). The cumulative probability of EPR was 44%, 53%, 69%, and 82%, at 1, 2, 3, and 5 years, respectively. No predictive factors of EPR were found. CONCLUSIONS: Early postoperative use of AZA seems to delay EPR development in comparison to historical series or placebo groups in randomized controlled trials. Although usually considered as endoscopic recurrence, those lesions confined to the ileocolonic anastomosis are not likely to progress or to become symptomatic in the short term.