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Kabeerdoss J.,Wellcome Trust Research Laboratory | Sankaran V.,Wellcome Trust Research Laboratory | Pugazhendhi S.,Wellcome Trust Research Laboratory | Ramakrishna B.S.,Wellcome Trust Research Laboratory
BMC Gastroenterology | Year: 2013

Background: Alterations in the fecal bacterial flora occur in inflammatory bowel disease (IBD). We examined the abundance and diversity of Clostridium leptum group, an important group of carbohydrate-fermenting bacteria, in the feces of patients with IBD and compared them with healthy controls.Methods: Seventeen healthy controls (HC), 20 patients with Crohn's disease (CD) and 22 patients with ulcerative colitis (UC) participated in the study. DNA extracted from fecal samples was amplified by PCR targeting 16S rRNA gene sequences specific to C. leptum group. The PCR product was subjected to temporal temperature gradient electrophoresis (TTGE) and the number and position of individual bands were noted and diversity was estimated. The identity of bands at different positions was confirmed by cloning and sequencing. Real time quantitative PCR with Mesa Green, targeted at specific 16S rRNA gene sequences, was used to quantitate C. leptum group and its most prominent constituent, Faecalibacterium prausnitzii.Results: Twenty five different operational taxonomic units (OTUs, equivalent to species) were identified constituting the C. leptum group in these participants. Their sequences were deposited in GenBank [accession numbers GQ465348 to GQ465370]. OTU number was significantly reduced in CD (7.7±3.7, mean±SD) and UC (9.0±3.0) compared to HC (11.9±2.2) (P=0.0005). The Simpson D index of alpha diversity was not significantly different between the three groups. Total numbers of C. leptum group bacteria and F. prausnitzii were reduced in both CD and UC compared to HC (P=0.0036 and P<0.0001 respectively). Disease activity did not influence numbers of C. leptum or F. prausnitzii in patients with CD or UC.Conclusion: C. leptum numbers and diversity were significantly reduced in both CD and UC suggesting that alterations noted were not specific to one disease. This could contribute to reduced short chain fatty acid production in IBD. © 2013 Kabeerdoss et al.; licensee BioMed Central Ltd.

Gladstone B.P.,Wellcome Trust Research Laboratory | Gladstone B.P.,Albert Ludwigs University of Freiburg | Ramani S.,Wellcome Trust Research Laboratory | Mukhopadhya I.,Wellcome Trust Research Laboratory | And 16 more authors.
New England Journal of Medicine | Year: 2011

BACKGROUND: More than 500,000 deaths are attributed to rotavirus gastroenteritis annually worldwide, with the highest mortality in India. Two successive, naturally occurring rotavirus infections have been shown to confer complete protection against moderate or severe gastroenteritis during subsequent infections in a birth cohort in Mexico. We studied the protective effect of rotavirus infection on subsequent infection and disease in a birth cohort in India (where the efficacy of oral vaccines in general has been lower than expected). METHODS: We recruited children at birth in urban slums in Vellore; they were followed for 3 years after birth, with home visits twice weekly. Stool samples were collected every 2 weeks, as well as on alternate days during diarrheal episodes, and were tested by means of enzyme-linked immunosorbent assay and polymerase-chain-reaction assay. Serum samples were obtained every 6 months and evaluated for seroconversion, defined as an increase in the IgG antibody level by a factor of 4 or in the IgA antibody level by a factor of 3. RESULTS: Of 452 recruited children, 373 completed 3 years of follow-up. Rotavirus infection generally occurred early in life, with 56% of children infected by 6 months of age. Levels of reinfection were high, with only approximately 30% of all infections identified being primary. Protection against moderate or severe disease increased with the order of infection but was only 79% after three infections. With G1P[8], the most common viral strain, there was no evidence of homotypic protection. CONCLUSIONS: Early infection and frequent reinfection in a locale with high viral diversity resulted in lower protection than has been reported elsewhere, providing a possible explanation why rotavirus vaccines have had lower-than-expected efficacy in Asia and Africa. (Funded by the Wellcome Trust.) Copyright © 2011 Massachusetts Medical Society. All rights reserved.

Pugazhendhi S.,Wellcome Trust Research Laboratory | Santhanam S.,Wellcome Trust Research Laboratory | Venkataraman J.,Government Stanley Medical College | Creveaux I.,University of Clermont Ferrand | Ramakrishna B.S.,Wellcome Trust Research Laboratory
Gene | Year: 2013

Background: Three mutations (two missense and one frameshift) in the NOD2 gene are associated with Crohn's disease (CD) in a proportion of patients with Crohn's disease in North America, Europe and Australia. These three mutations are not found in Indian patients with CD. We undertook new studies to identify polymorphisms in the NOD2 gene in the Indian population and to detect whether any of these were associated with inflammatory bowel disease (IBD) in this population. Methods: Individual exons of the NOD2 gene were amplified by PCR and subjected to denaturing high performance liquid chromatography (DHPLC) to detect heteroduplex formation. All 12 exons of the NOD2 gene were amplified and Sanger-sequenced to detect polymorphisms in the NOD2 gene. 310 patients with CD, 318 patients with ulcerative colitis (UC) and 442 healthy controls (HC) were recruited for association studies. DNA from these participants was evaluated for the identified eight polymorphisms by Sequenom analysis. Results: Heteroduplex formation was noted by DHPLC in exons 2 and 4 of the NOD2 gene. Sequencing of the entire NOD2 gene data revealed eight polymorphisms - rs2067085, rs2066842, rs2066843, rs1861759, rs2111235, rs5743266, rs2076753, and rs5743291 - of which the latter four were described for the first time in Indians. None of these polymorphisms was associated with CD. The SNPs rs2066842 and rs2066843 were in significant linkage disequilibrium. Both SNPs showed a significant association with UC (P = 0.03 and 0.04 respectively; odds ratio 1.44 and 1.41 respectively). Conclusion: Four NOD2 polymorphisms were identified for the first time in the Indian population. Of 8 NOD2 polymorphisms, none were associated with CD but two were weakly associated with UC. NOD2 polymorphisms do not play a major role in CD genesis in India. © 2012 Elsevier B.V.

Rajendran P.,Wellcome Trust Research Laboratory | Kang G.,Wellcome Trust Research Laboratory
Vaccine | Year: 2014

Rotaviruses are enteric pathogens causing acute, watery, dehydrating diarrhea in various host species, including birds and mammals. This study collected data on the disease burden and strain prevalence of Group A rotavirus in animals and humans in Vellore and investigated interspecies transmission by comparison of circulating genotypes. Stool samples from children aged less than 5 years, admitted to the hospital between January 2003 and May 2006 for diarrhea and diarrheal samples from animals that were collected from a veterinary clinic and several dairy farms near Vellore between February 2007 and May 2008 were processed and subjected to RNA extraction and reverse-transcription PCR for genotyping of VP7 and VP4. Of 394 children with diarrhea, 158 (40%) were positive for rotavirus and the common G types identified were G1 (47, 29.7%), G2 (43, 27.2%), G9 (22, 13.9%), G10 (2, 1.2%), G12 (1, 0.6%) and mixed infections (27, 17.8%). The common P types were P[4] accounting for 57 (36%) samples, P[8] 57 (36%), P[11] 3 (1.8%) and P[6] 2 (1.2%). Of 627 animals, 35 (1 bullock, 2 goats, 32 cows) were found to be infected with rotavirus (5.5%). The common G types identified in order of frequency were G6 (17, 48.5%), G2 (10, 28%), G10 (4, 11%), G8 (2, 5.7%) and mixed infections (2, 5.7%). The common P types were P[6] accounting for 16 (46%) samples, P[4] 7 (20%), P[1] 3 (8.5%) and P[8] 3 (8.5%). An unusual P type P[15] was seen in one sample in combination with G10. The finding of G2 infections which are rarely identified in animals implies anthroponotic transmission since this genotype is predominantly associated with infection in humans. © 2014.

Jaeschke H.,University of Kansas Medical Center | Williams C.D.,University of Kansas Medical Center | McGill M.R.,University of Kansas Medical Center | Xie Y.,University of Kansas Medical Center | And 2 more authors.
Food and Chemical Toxicology | Year: 2013

Extracts from medicinal plants, many of which have been used for centuries, are increasingly tested in models of hepatotoxicity. One of the most popular models to evaluate the hepatoprotective potential of natural products is acetaminophen (APAP)-induced liver injury, although other hepatotoxicity models such as carbon tetrachloride, thioacetamide, ethanol and endotoxin are occasionally used. APAP overdose is a clinically relevant model of drug-induced liver injury. Critical mechanisms and signaling pathways, which trigger necrotic cell death and sterile inflammation, are discussed. Although there is increasing understanding of the pathophysiology of APAP-induced liver injury, the mechanism is complex and prone to misinterpretation, especially when unknown chemicals such as plant extracts are tested. This review discusses the fundamental aspects that need to be considered when using this model, such as selection of the animal species or in vitro system, timing and dose-responses of signaling events, metabolic activation and protein adduct formation, the role of lipid peroxidation and apoptotic versus necrotic cell death, and the impact of the ensuing sterile inflammatory response. The goal is to enable researchers to select the appropriate model and experimental conditions for testing of natural products that will yield clinically relevant results and allow valid interpretations of the pharmacological mechanisms. © 2013 Elsevier Ltd.

Vivek R.,Wellcome Trust Research Laboratory | Kang G.,Wellcome Trust Research Laboratory
American Journal of Tropical Medicine and Hygiene | Year: 2011

Hepatitis E virus (HEV) in industrialized countries is zoonotically transmitted, and swine act as a major reservoir of HEV. Serum samples from 102 swine and plasma from 34 swine handlers in Vellore, India were tested by using a reverse transcription-polymerase chain reaction to detect and genotype HEV. We measured levels of IgG against HEV in swine handlers and in age and geographically matched controls from rural and urban populations in Vellore. HEV was amplified from two pigs and both viruses belonged to genotype 4. No HEV RNA was amplified from any swine handler, but 94.1% of swine handlers were positive for antibodies against HEV, a seroprevalence rate significantly higher than in rural and urban controls. The HEV genotype circulating in swine in India is different from that of humans, but the higher antibody levels in swine handlers support the possibility that zoonotic infections may occur. Copyright © 2011 by The American Society of Tropical Medicine and Hygiene.

Santhanam S.,Wellcome Trust Research Laboratory | Rajamanickam S.,Wellcome Trust Research Laboratory | Motamarry A.,Wellcome Trust Research Laboratory | Ramakrishna B.S.,Wellcome Trust Research Laboratory | And 4 more authors.
Inflammatory Bowel Diseases | Year: 2012

Background: Ulcerative colitis (UC) is characterized by an energy deficiency state of the colonic epithelium. This study evaluated mitochondrial electron transport chain (ETC) complex activity in normal and disease mucosa in patients with UC. Alterations in ETC complexes were also investigated in experimental colitis in mice. Methods: Biopsies were obtained from macroscopically normal and diseased colonic mucosa of 43 patients with UC and 35 controls undergoing screening colonoscopy and ETC complex activity was assayed biochemically. ETC complex activities were also assayed in colonic epithelial cells isolated from Swiss albino mice with dextran sodium sulfate (DSS)-induced colitis at various stages of induction of colitis. Mucosal nitrite levels and protein carbonyl content were determined. Results: The activity of Complex II was significantly decreased in colonic biopsies from UC patients compared with controls, while activities of other mitochondrial complex were normal. Complex II activity was equally decreased in diseased and normal mucosa in UC; the degree of reduction did not correlate with clinical, endoscopic, or histological grading of disease activity. In DSS-fed mice, a reduction in activity of Complex IV and Complex II was observed. Activity of other complex was not affected. Administration of aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, attenuated all parameters of colitis as well as the reductions in Complex IV and Complex II activity. Conclusions: Reduction in Complex II activity appears to be a specific change in UC, present in quiescent and active disease. Mitochondrial complex dysfunction occurs in DSS colitis in mice and appears to be mediated by nitric oxide. (Inflamm Bowel Dis 2012;) Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

Xie Y.,University of Kansas Medical Center | Ramachandran A.,University of Kansas Medical Center | Ramachandran A.,Wellcome Trust Research Laboratory | Breckenridge D.G.,Gilead Sciences Inc. | And 4 more authors.
Toxicology and Applied Pharmacology | Year: 2015

Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun- N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300. mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24. h observation period. Pretreatment with 30. mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5. h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. © 2015 Elsevier Inc.

Balamurugan R.,Wellcome Trust Research Laboratory | Mary R.R.,Wellcome Trust Research Laboratory | Chittaranjan S.,Wellcome Trust Research Laboratory | Jancy H.,Wellcome Trust Research Laboratory | And 2 more authors.
British Journal of Nutrition | Year: 2010

Fe deficiency in women contributes significantly to maternal and child morbidity in India. The intestinal bacterial flora may facilitate absorption of Fe from the caecum and proximal colon. The present study investigated the possibility that intestinal microbiota of anaemic young women may differ from that of women with normal Hb levels. The microbiota was quantified by real-time PCR in faeces of eight anaemic (Hb≤100g/l) and twenty-six normohaemic (Hb≥120g/l) women aged 18-25 years. Sequences of 16S ribosomal DNA (rDNA) specific to Bifidobacterium genus, Lactobacillus acidophilus group, Bacteroides-Prevotella-Porphyromonas group, Clostridium leptum group and Eubacterium rectale were amplified and expressed (as relative difference) relative to the universally conserved bacterial 16S rDNA sequences. Dietary intakes of energy, carbohydrate, fibre and Fe were ascertained by maintenance of a diet diary for a week. Faecal lactobacilli were significantly lower in anaemic women (median 6610-8, relative difference compared with total bacteria) than in the reference group (2910-6; P=0001, unpaired t test with logarithmic transformation). There was no difference between the two groups with respect to any of the other bacteria that were examined. Intakes of energy, carbohydrate, fibre, Fe and milk were similar in both the groups. Fe deficiency in young women in south India was associated with low levels of lactobacilli in the faeces. The relationship between lactobacilli and Fe deficiency needs to be explored further. © The Authors 2010.

Giri S.,Wellcome Trust Research Laboratory | Kindo A.J.,Sri Ramachandra Medical College and Research Institute | Kalyani J.,Sri Muthukumaran Medical College
Journal of Postgraduate Medicine | Year: 2013

Background: The occurrence of candidemia is on a rise worldwide. Non-albicans Candida species have emerged as major causes of candidemia in many countries. Added to it is the problem of antifungal resistance in Candida isolates. Objectives: To find out the prevalence of candidemia in our intensive care unit (ICU) setup along with the antifungal susceptibility pattern of Candida isolates and various risk factors associated with candidemia. Materials and Methods: All Candida isolates from blood stream infections of ICU patients were included in the 1 year study period (November 2008-October 2009). The isolates were speciated using various phenotypic tests. Antifungal susceptibility testing was done by disk diffusion methods according to Clinical and Laboratory Standards Institute guidelines and also using CANDIFAST. Various risk factors associated with the development of candidemia were looked into. Results: A total of 39 Candida isolates were isolated during the study period of 1 year (prevalence of 0.65%). Candida tropicalis (74.35%) was the most common isolate followed by Candida albicans, Candida parapsilosis, Candida krusei and Candida glabrata. All the 39 Candida isolates (100%) were sensitive to amphotericin B while 12 isolates (30.8%) were resistant to fluconazole. The risk factors commonly associated with candidemia patients were long term antibiotic therapy (64.1%), use of central venous catheters (56.4%), urinary catheters (53.9%), steroid therapy (35.9%) and diabetes mellitus (33.3%). Conclusion: Candidemia is emerging as a significant problem in hospitalized patients, especially in ICU setups. Non-albicans Candida species are the major cause of candidemia as found in our study and few other studies in India. Multicentric studies involving many hospitals are required to know the true prevalence of candidemia and the status of antifungal drug resistance among Candida isolates in our country.

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