Fluctuations in butyrate-producing bacteria in ulcerative colitis patients of North India

AIM: To study the interplay between butyrate concentration and butyrate-producing bacteria in fecal samples of ulcerative colitis (UC) patients vs control individuals. METHODS: Fecal samples were collected from 14 control individuals (hemorrhoid patients only) and 26 UC patients (severe: n = 12, moderate: n = 6, remission: n = 8), recruited by the gastroenterologist at the Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India. Disease activity in UC patients was determined by clinical colitis activity index. We employed fluorescent in situ hybridization in combination with flow cytometry to enumerate the clostridium cluster population targeted by 16S rRNA gene probe. Major butyrate-producing species within this cluster were quantified to see if any change existed in control vs UC patients with different disease activity. This observed change was further validated by quantitative polymerase chain reaction. In addition to this,we carried out gas chromatography to evaluate the changes in concentration of major short chain fatty acids (SCFAs), namely acetate, n -butyrate, iso -butyrate, in the above samples. Student t test and Graph pad prism-6 were used to compare the data statistically. RESULTS: There was a significant decrease of Clostridium coccoides (control, 25.69% ± 1.62% vs severe, 9.8% ± 2.4%, P = 0.0001) and Clostridium leptum clusters (control, 13.74% ± 1.05% vs severe, 6.2% ± 1.8%, P = 0.0001) in fecal samples of UC patients. Furthermore, we demonstrated that some butyrateproducing members of the clostridial cluster, like Fecalibacterium prausnitzii (control, 11.66% ± 1.55% vs severe, 6.01% ± 1.6%, P = 0.0001) and Roseburia intestinalis (control, 14.48% ± 1.52% vs severe, 9% ± 1.83%, P = 0.02) were differentially present in patients with different disease activity. In addition, we also demonstrated decreased concentrations of fecal SCFAs, especially of n -butyrate (control, 24.32 ± 1.86 mmol/μL vs severe, 12.74 ± 2.75 mmol/μL, P = 0.003), iso -butyrate (control, 1.70 ± 0.41 mmol/μL vs severe, 0.68 ± 0.24 mmol/μL, P = 0.0441) and acetate (control, 39.51 ± 1.76 mmol/μL vs severe, 32.12 ± 2.95 mmol/ μL,P = 0.047), in the fecal samples of UC patients. The observed decrease of predominant butyrate producers of clostridial clusters correlated with the reduced SCFA levels in active UC patients. This was further confirmed by the restoration in the population of some butyrate producers with simultaneous increase in the level of SCFA in remission samples. CONCLUSION: Our observations indicate that decreases in members of the clostridial cluster resulting in reduced butyrate levels contribute to the etiology of UC.

Differentiating Crohn's disease from intestinal tuberculosis

Differentiating Crohn's disease(CD) and intestinal tuberculosis(ITB) has remained a dilemma for most of the clinicians in the developing world, which are endemic for ITB, and where the disease burden of inflammatory bowel disease is on the rise. Although, there are certain clinical(diarrhea/hematochezia/perianal disease common in CD; fever/night sweats common in ITB), endoscopic(longitudinal/aphthous ulcers common in CD; transverse ulcers/patulous ileocaecal valve common in ITB), histologic(caseating/confluent/large granuloma common in ITB; microgranuloma common in CD), microbiologic(positive stain/culture for acid fast-bacillus in ITB), radiologic(long segment involvement/comb sign/skip lesions common in CD; necrotic lymph node/contiguous ileocaecal involvement common in ITB), and serologic differences between CD and ITB, the only exclusive features are caseation necrosis on biopsy, positive smear for acid-fast bacillus(AFB) and/or AFB culture, and necrotic lymph node on cross-sectional imaging in ITB. However,these exclusive features are limited by poor sensitivity, and this has led to the development of multiple multi-parametric predictive models. These models are also limited by complex formulae, small sample size and lack of validation across other populations. Several new parameters have come up including the latest Bayesian meta-analysis, enumeration of peripheral blood T-regulatory cells, and updated computed tomography based predictive score. However, therapeutic anti-tubercular therapy(ATT) trial, and subsequent clinical and endoscopic response to ATT is still required in a significant proportion of patients to establish the diagnosis. Therapeutic ATT trial is associated with a delay in the diagnosis of CD, and there is a need for better modalities for improved differentiation and reduction in the need for ATT trial.

MicroRNA exhibit altered expression in the inflamed colonic mucosa of ulcerative colitis patients

AIM To investigate the miRNA expression in colonic mucosal biopsies from endoscopically inflamed and non inflamed regions of ulcerative colitis(UC) patients. METHODS Colonic mucosal pinch biopsies were analyzed from the inflamed and non inflamed regions of same UC patient. Total RNA was isolated and differential miRNA profiling was done using microarray platform. Quantitative Real Time PCR was performed in colonic biopsies from inflamed(n = 8) and non-inflamed(n = 8) regions of UC and controls(n = 8) to validate the differential expression of miRNA. Potential targets of dysregulated miRNA were identified by using in silico prediction tools and probable role of these miRNA in inflammatory pathways were predicted.RESULTS The miRNA profile of inflamed colonic mucosa differs significantly from the non-inflamed. Real time PCR analysis showed that some of the miRNA were differentially expressed in the inflamed mucosa as compared to non inflamed mucosa and controls(mi R-125 b, mi R-223, miR-138, and mi R-155), while(miR-200a) did not show any significant changes. In contrast to microarray, where mi R-378 d showed downregulation in the inflamed mucosa, q RT-PCR showed a significant upregulation in the inflamed mucosa as compared to the non inflamed. The in silico prediction analysis revealed that the genes targeted by these mi RNAs play role in the major signaling pathways like MAPK pathway, NF-κB signaling pathway, cell adhesion molecules which are all assciated with UC.CONCLUSION The present study reports disease specific alteration in the expression of mi R-125 b, mi R-155, mi R-223 and mi R-138 in UC patients and also predict their biological significance.

Intestinal permeability and its association with the patient and disease characteristics in Crohn's disease

AIM:To assess the intestinal permeability (IP) in patients with Crohn's disease (CD) and study the association of IP with the patient and disease characteristics. METHODS: One hundred and twenty five consecutive patients of CD (Males: 66) were diagnosed on the basis of a combination of standard clinical, endoscopic, imaging and histological features. CD activity index (CDAI) was used to calculate the activity of the disease while the behavior of the disease was assessed by the modified Montreal classification. IP was measured by the ratio of the percentage excretion of ingested doses of lactulose and mannitol in urine (LMR). The upper limit of normality of LMR (0.037) was derived from 22 healthy controls. RESULTS: Thirty six percent of patients with CD had increased IP. There was no significant difference in mannitol excretion (patients vs controls = 12.5% vs 14.2%, P = 0.4652), but lactulose excretion was significantly higher in patients compared to healthy controls (patients vs controls = 0.326% vs 0.293%, P = 0.0391). The mean LMR was also significantly higher in the patients as compared to healthy controls [0.027 (0.0029-0.278) vs 0.0164 (0.0018-0.0548), P = 0.0044]. Male patients had a higher LMR compared to females [0.036 (95% CI 0.029, 0.046) vs 0.022 (95% CI 0.0178, 0.028) (P = 0.0024), though there was no difference in the number of patients with abnormal IP in boththe sexes. Patients with an ileo-colonic disease had a higher LMR than those with only colonic disease [0.045 (95% CI 0.033, 0.06) vs 0.021 (95% CI 0.017, 0.025) (P < 0.001)]. Of patients with ileo-colonic disease, 57.8% had an abnormal IP, compared to 26.7% with colonic and 15.6% with small intestinal disease. Patients with a stricturing disease had significantly higher LMR compared to non-fistulising non-stricturing disease [0.043 (95% CI 0.032, 0.058) vs 0.024 (95% CI 0.019, 0.029) (P = 0.0062)]. There was no correlation of IP with age, disease activity, duration of illness, D-xylose absorption, upper GI involvement, perianal disease, and extra- intestinal manifestations. On multiple regression analysis, male gender and ileo-colonic disease were independent factors associated with increased IP. Gender, location, behavior of the disease and upper GI involvement could explain up to 23% of variability in IP (R2 = 0.23). CONCLUSION: IP was increased in 36% of patients with CD. Male gender and an ileo-colonic disease were the independent factors associated with increased IP.

Low dose oral curcumin is not effective in induction of remission in mild to moderate ulcerative colitis: Results from a randomized double blind placebo controlled trial

AIM To evaluate the role of oral curcumin in inducing clinical remission in patients with mild to moderate ulcerative colitis(UC).METHODS A prospective randomized double-blind placebo-controlled trial comparing the remission inducing effect of oral curcumin and mesalamine 2.4 g with placebo and mesalamine 2.4 g in patients of ulcerative colitis with mild to moderate severity was conducted from January 2003 to March 2005. The included patients received 1 capsule thrice a day of placebo or curcumin(150 mg) for 8 wk. Patients were evaluated clinically and endoscopically at 0,4 and 8 wk. The primary outcome was clinical remission at 8 wk and secondary outcomes were clinical response, mucosal healing and treatment failure at 8 wk. The primary analysis was intention to treat worst case scenario(ITT-WCS).RESULTS Of 300 patients with UC, 62 patients(curcumin: 29, placebo: 33) fulfilled the inclusion criteria and were randomized at baseline. Of these, 21 patients did not complete the trial, 41 patients(curcumin: 16, placebo: 25) finally completed 8 wk. There was no significant difference in rates of clinical remission(31.3% vs 27.3%, P = 0.75), clinical response(20.7% vs 36.4%, P = 0.18), mucosal healing(34.5% vs 30.3%, P = 0.72), and treatment failure(25% vs 18.5%, P = 0.59) between curcumin and placebo at 8 wk.CONCLUSION Low dose oral curcumin at a dose of 450 mg/d was ineffective in inducing remission in mild to moderate cases of UC.

Management of acute severe ulcerative colitis

The management strategy of acute severe ulcerative colitis has evolved over the past decade from being entirely restricted to twin choices of intravenous steroids or colectomy to include colon rescue therapies like cyclosporin as well as infliximab. However it still remains a medical emergency requiring hospitalization and requires care from a multidisciplinary team comprising of a gastroenterologist and a colorectal surgeon. The frame shift in management has been the emphasis on time bound decision making with an attempt to curtail the mortality rate to below 1%. Intravenous corticosteroids are the mainstay of therapy. Response to steroids should be assessed at day 3 of admission and partial/non-responders should be considered for alternative medical therapy/surgery. Medical rescue therapies include intravenous cyclosporin and infliximab. Cyclosporin is administered in a dose of 2 mg/kg per day and infliximab is administered as a single dose intravenous infusion of 5 mg/kg. Approximately 75% patients have short term and 50% patients have long term response to cyclosporin. Long term response to cyclosporin is improved in patients who are thiopurine na?ve and are started on thiopurines on day 7. Infliximab also has a response rate of approximately 70% in short term and 50% in long term. Both cyclosporin and infliximab are equally efficacious medical rescue therapies as demonstrated in a recent randomized control trial. Patientsnot responding to infliximab or cyclosporin should be considered for colectomy.

Who to screen and how to screen for celiac disease

Celiac disease(CeD)is a chronic gluten-induced enteropathy with plethoric manifestations.The typical manifestations of CeD such as chronic diarrhea and malabsorption are widely recognized,however,many patients have atypical manifestations like iron deficiency anemia,idiopathic short stature,hypertransaminesemia or infertility,etc.These patients often present to the primary care physicians and/or non-gastrointestinal specialties.However,due to a lack of awareness among the healthcare professionals about the various atypical manifestations,many patients are not screened for CeD.In this review,we have summarized the available literature about the prevalence of CeD in various gastrointestinal(chronic diarrhea)and non-gastrointestinal conditions(iron deficiency anemia,short stature,cryptogenic hypertransaminesemia,cryptogenic cirrhosis or idiopathic ataxia etc.)where the diagnosis of CeD should be considered.In addition,we also discuss special scenarios where screening for CeD should be considered even in absence of symptoms such as patients with type 1 diabetes,Down’s syndrome,and first-degree relatives of patients with CeD.Further,we discuss the diagnostic performance and limitations of various screening tests for CeD such as IgA anti-tissue transglutaminase antibodies,antiendomysial antibodies and anti-deamidated gliadin antibodies.Based on the current recommendations,we propose a diagnostic algorithm for patients with suspected CeD.

肠结核与克罗恩病差异表达基因的全基因分析及FOXP3 mRNA表达的鉴别价值

背景:克罗恩病(CD)和肠结核(ITB)是有着相似表型的慢性肉芽肿性疾病,因此,找到一种生物标志物来鉴别这两种疾病是必要的。本研究旨在对ITB和CD的结肠标本进行全基因表达分析。同时,为了评价调节性T细胞的作用,我们对血清标本及结肠标本进行FOXP3 mRNA定量检测。方法:收集33名研究对象(CD患者、ITB患者及正常对照)血清和结肠组织配对样本,提取标本中的总RNA。采用Illumina HumanWG-6 BeadChip试剂盒对3组研究对象中的6种总RNA样本进行人类全基因组表达芯片分析。采用real-time PCR对具有合格RNA样本的病例(CD 4例,ITB 5例,正常对照4例)血清和组织标本进行FOXP3 mRNA表达水平检测。结果:在CD和ITB患者中,分别有92和382个基因表达上调1.5倍,91和256个基因表达下调1.5倍。过氧化物酶体增殖物介导的PPARγ信号通路在CD中下调最为显著(P<0.005);此外,IL4/5/6信号通路和Toll样受体信号通路也存在显著差异表达,上调2倍以上。而在ITB患者上调最为显著是补体活化通路尤其是经典通路。ITB患者结肠组织标本中FOXP3 mRNA表达水平明显高于CD患者(4.7062.21 vs 1.4860.31,P=0.016)。结论:结肠黏膜FOXP3 mRNA表达可作为鉴别ITB与CD的一项标志物。ITB患者补体激活通路的上调提示ITB的发病机制与肺结核相似。CD患者中PPARγ通路的下调提示PPARγ依赖的微生物屏障功能可能成为新的治疗靶点。