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Pore D.,Indian National Institute of Cholera and Enteric Diseases | Pore D.,Cleveland Clinic | Chakrabarti M.K.,Indian National Institute of Cholera and Enteric Diseases
Vaccine | Year: 2013

Shigellosis is the leading cause of childhood mortality and morbidity. Despite many years of extensive research a practical vaccine is not yet available against the disease. Recent studies illustrate that bacterial outer membrane proteins are budding target as vaccine antigen. Outer membrane proteins A (OmpA) are among the most immunodominant antigens in the outer membrane of gram negative bacteria and possess many characteristics desired of a vaccine candidate. We observe that OmpA of Shigella flexneri 2a is crossreactive and common antigen among Shigella spp. and the epitope is widely exposed on the cell surface as well as capable of evoking protective immunity in mice. The protective immunity involves participation of both the humoral and cellular immune responses, since OmpA boosts rapid induction of IgG and IgA in both the systemic and mucosal compartments and also activates Th1 cells. The immunopotentiating activity of OmpA is mediated by its ability to bind and stimulate macrophages and up-regulate the surface expression of MHCII, CD80 and CD40, leading to activation of CD4+ T cells to secrete cytokines and express chemokine receptor and IL-12Rβ2, thereby orchestrating the bridge between innate and adaptive immune responses. This ability is dependent on Toll-like receptor 2 (TLR2), as demonstrated by lack of response by TLR2 knockdown macrophages to OmpA. Hence this property of OmpA to link innate and adaptive immunity via TLR2 offers a novel vista to develop vaccine against shigellosis. © 2013 Elsevier Ltd.

Dutta P.,Indian National Institute of Cholera and Enteric Diseases | Das S.,Indian National Institute of Cholera and Enteric Diseases
Current Topics in Medicinal Chemistry | Year: 2016

Antimicrobial peptides (AMPs) are integral components of the host innate immune system and functional throughout the plant and animal kingdoms. AMPs are short cationic molecules and lethal against a wide range of bacteria, viruses, fungi, yeast and protozoa due to their membranolytic effects on the negatively-charged microbial membranes. In addition, they exert multiple immunomodulatory roles like chemotaxis, modulation of cytokine and chemokine expression, leukocyte activation etc. Since AMPs suffer loss of microbicidal properties under serum and tissue environments, their capacity to modulate the immune system may predominates under the physiological conditions. Discovery of new antibiotics is lagging far behind the rapidly spreading drug resistance among the microorganisms. Both natural and synthetic AMPs have shown promise as ‘next generation antibiotics’ due to their unique mode of action, which minimises the chance of development of microbial resistance. In addition, they have therapeutic potential against non-infectious diseases like chronic inflammation and cancer. Many of the synthetic AMPs are currently undergoing clinical trials for the treatment of debilitating diseases, such as catheter-related infections, diabetic foot ulcers, chemotherapy-associated infections etc. Some of them have already entered the market as topical preparations. In this review, we synopsise the current literature of natural and synthetic AMPs in different infectious and inflammatory diseases of human microfloral habitats, especially the gastrointestinal, respiratory and genitourinary tracts and the skin. We also discuss the classification of AMPs, their mode action and antimicrobial spectrum, including the pathogen evasion mechanisms. In short, we tried to present the locus standi of AMPs in relation to human diseases and highlight the most promising synthetic peptides emerging from the clinical trials. Finally, we focused on the limitations and hurdles in terms of cost of production, bioavailability, pharmacokinetic stability and toxicity faced by commercial development and clinical use of the AMPs and strategies to overcome these hurdles. © 2016 Bentham Science Publishers.

Ghosh A.,Indian National Institute of Cholera and Enteric Diseases | Ramamurthy T.,Indian National Institute of Cholera and Enteric Diseases
Indian Journal of Medical Research | Year: 2011

Antimicrobial resistance poses a major threat in the treatment of infectious diseases. Though significant progress in the management of diarrhoeal diseases has been achieved by improved hygiene, development of new antimicrobials and vaccines, the burden remains the same, especially in children below 5 yr of age. In the case of cholera, though oral rehydration treatment is the mainstay, antimicrobial therapy is mandatory at times to reduce the volume of stool and shorten the duration of the disease. Though for many pathogens, antimicrobial resistance emerged soon after the introduction of antibiotics, Vibrio cholerae remained sensitive to most of the antibiotics for quite a long period. However, the scenario changed over the years and today, V. Cholerae strains isolated world over are resistant to multiple antibiotics. A myriad number of mechanisms underlie this phenomenon. These include production of extended-spectrum beta-lactamases, enhanced multi-drug efflux pump activity, plasmid-mediated quinolone and fluoroquinolone resistance, and chromosomal mutations. Horizontal transfer of resistance determinants with mobile genetic elements like integrons and the integrating conjugative elements (ICEs), SXTs help in the dissemination of drug resistance. Though all strains isolated are not resistant to all antibiotics and we are not as yet ''stranded'', expanding spectrum of drug resistance is a definite cause for concern. Pipelines of discovery of new antibiotics are drying up as major pharmaceutical companies are losing interest in investing money in this endeavour, mainly due to the short shelf-life of the antibiotics and also due to the fast emergence of drug resistance. To address this issue, attempts are now being made to discover drugs which are pathogen specific and target their ''virulence mechanisms''. It is expected that development of resistance against such antibiotics would take much longer. This review briefly focuses on all these issues.

Ghosh A.,Cleveland Clinic | Chawla-Sarkar M.,Indian National Institute of Cholera and Enteric Diseases | Stuehr D.J.,Cleveland Clinic
FASEB Journal | Year: 2011

Maturation of NOS enzymes requires that they incorporate heme to become active, but how this cellular process occurs is unclear. We investigated a role for chaperone heat shock protein 90 (hsp90) in enabling heme insertion into the cytokine-inducible mouse NOS. We used macrophage cell line RAW 264.7 and human embryonic kidney HEK293T cells and studied insertion of native heme during iNOS expression and insertion of exogenous heme into preformed apo-iNOS. Pulldown experiments showed that the hsp90-iNOS complex was present in cells, but the extent of their association was inversely related to iNOS heme content. Hsp90 was primarily associated with apo-iNOS monomer and was associated 11-fold less with heme-containing iNOS monomer or dimer in cells. Kinetic studies showed that hsp90 dissociation occurred coincident with cellular heme insertion into apo-iNOS (0.8 h-1). The hsp90 inhibitor radicicol or coexpression of an ATPase-defective hsp90 blocked heme insertion into apo-iNOS by 90 and 75%, respectively. The ATPase activity of hsp90 was not required for complex formation with iNOS but was essential for heme insertion to occur. We conclude that hsp90 plays a primary role in maturation of iNOS protein by interacting with the apoenzyme in cells and then driving heme insertion in an ATP-dependent manner. © FASEB.

Safa A.,City University of Hong Kong | Nair G.B.,Indian National Institute of Cholera and Enteric Diseases | Kong R.Y.C.,City University of Hong Kong
Trends in Microbiology | Year: 2010

Vibrio cholerae typically contains a prophage that carries the genes encoding the cholera toxin, which is responsible for the major clinical symptoms of the disease. In recent years, new pathogenic variants of V. cholerae have emerged and spread throughout many Asian and African countries. These variants display a mixture of phenotypic and genotypic traits from the two main biotypes (known as 'classical' and 'El Tor'), suggesting that they are genetic hybrids. Classical and El Tor biotypes have been the most epidemiologically successful cholera strains during the past century, and it is believed that the new variants (which we call here 'atypical El Tor') are likely to develop successfully in a manner similar to these biotypes. Here, we describe recent advances in our understanding of the epidemiology and evolution of the atypical El Tor strains. © 2009 Elsevier Ltd. All rights reserved.

Mukherjee S.,Indian National Institute of Cholera and Enteric Diseases | Biswas T.,Indian National Institute of Cholera and Enteric Diseases
Cellular Signalling | Year: 2014

Interferon (IFN)-γ and tumor necrosis factor (TNF)-α cause chronic inflammation of the intestine leading to progression of inflammatory bowel disease (IBD), which is manifested through rapid apoptosis of the intestinal epithelial cells (iECs). Here, we show inhibition of IFN-γ and TNF-α-induced apoptosis of INT-407 cells by porin, a microbe-associated molecular pattern (MAMP) with affinity for toll-like receptor (TLR)2 and commonly present in Gram-negative bacteria. Proinflammatory cytokines induce apoptosis by activation of caspase 8 that triggers caspase 9 through Bax finally leading to activation of caspase 3, the executioner caspase. Interestingly, while IFN-γ and TNF-α promotes Bax expression, in contrast porin up-regulates anti-apoptotic Bcl-xL resulting in iEC survivability. We show elevated expression of TLR2 is a key requisite for IFN-γ and TNF-α mediated caspase 8 up-regulation that contributes to apoptosis of iECs. Down-regulation of TLR2 expression is central for checking apoptosis which is achieved by elevated level of toll-interacting protein (TOLLIP) in presence of porin. Attempts to limit IBD is in progress with anti-IFN-γ and anti-TNF-α Abs or use of IL-10. Although probiotic bacterial proteins have shown to successfully reduce IFN-γ and TNF-α mediated apoptosis, the exact mechanism of their action has remained elusive. This study identifies the underlying sequential events of transient TLR2 stimulation followed by its blocking in response to the bacterial outer membrane protein, which advocates intervention at TLR-juncture is crucial for controlling IBD. © 2014 Elsevier Inc.

Patil S.R.,Network for Engineering and Economics Research and Management NEERMAN | Patil S.R.,University of California at Berkeley | Arnold B.F.,University of California at Berkeley | Salvatore A.L.,Stanford University | And 4 more authors.
PLoS Medicine | Year: 2015

Background: Poor sanitation is thought to be a major cause of enteric infections among young children. However, there are no previously published randomized trials to measure the health impacts of large-scale sanitation programs. India’s Total Sanitation Campaign (TSC) is one such program that seeks to end the practice of open defecation by changing social norms and behaviors, and providing technical support and financial subsidies. The objective of this study was to measure the effect of the TSC implemented with capacity building support from the World Bank’s Water and Sanitation Program in Madhya Pradesh on availability of individual household latrines (IHLs), defecation behaviors, and child health (diarrhea, highly credible gastrointestinal illness [HCGI], parasitic infections, anemia, growth). Methods and Findings: We conducted a cluster-randomized, controlled trial in 80 rural villages. Field staff collected baseline measures of sanitation conditions, behaviors, and child health (May–July 2009), and revisited households 21 months later (February–April 2011) after the program was delivered. The study enrolled a random sample of 5,209 children ,5 years old from 3,039 households that had at least one child ,24 months at the beginning of the study. A random subsample of 1,150 children ,24 months at enrollment were tested for soil transmitted helminth and protozoan infections in stool. The randomization successfully balanced intervention and control groups, and we estimated differences between groups in an intention to treat analysis. The intervention increased percentage of households in a village with improved sanitation facilities as defined by the WHO/UNICEF Joint Monitoring Programme by an average of 19% (95% CI for difference: 12%–26%; group means: 22% control versus 41% intervention), decreased open defecation among adults by an average of 10% (95% CI for difference: 4%–15%; group means: 73% intervention versus 84% control). However, the intervention did not improve child health measured in terms of multiple health outcomes (diarrhea, HCGI, helminth infections, anemia, growth). Limitations of the study included a relatively short follow-up period following implementation, evidence for contamination in ten of the 40 control villages, and bias possible in self-reported outcomes for diarrhea, HCGI, and open defecation behaviors. Conclusions: The intervention led to modest increases in availability of IHLs and even more modest reductions in open defecation. These improvements were insufficient to improve child health outcomes (diarrhea, HCGI, parasite infection, anemia, growth). The results underscore the difficulty of achieving adequately large improvements in sanitation levels to deliver expected health benefits within large-scale rural sanitation programs. © 2014 World Bank.

Basu S.,Indian National Institute of Cholera and Enteric Diseases
European Journal of Clinical Microbiology and Infectious Diseases | Year: 2014

Colonization of the neonatal gut takes place immediately after birth. Bacteria that get colonized are considered to be “normal” flora derived principally from the mother and the immediate environment. However, for some neonates, the colonization of the gut, particularly with potential pathogens, may lead to subsequent infections or sepsis. The immune system and the gut barrier in neonates is vulnerable, with decreased acid secretion, low levels of protective mucous, and decreased motility, particularly in those who are premature and of low birth weight. This makes the neonatal gut especially prone to colonization with aerobic Gram-negative bacilli (GNB). And these GNB may later, under circumstances favorable to them, cause disease in the neonates. In developing countries, it is the GNB that cause the majority of the infections. In addition, the use of antibiotics in the neonatal intensive care unit also triggers colonization with antibiotic-resistant bacteria. This review discusses various aspects of neonatal gut colonization, neonatal sepsis, and tries to gather support to understand the connection between the gut and subsequent sepsis in neonates. © 2014, Springer-Verlag Berlin Heidelberg.

Dutta S.,Indian National Institute of Cholera and Enteric Diseases
eLife | Year: 2013

It has long been assumed that resistance to antibiotics reduces the fitness of disease-causing bacteria, but experiments on Salmonella Typhi, the bacteria that causes Typhoid fever, are now challenging this view. © Dellas et al.

Mitra S.,Indian National Institute of Cholera and Enteric Diseases | Chakrabarti M.K.,Indian National Institute of Cholera and Enteric Diseases | Koley H.,Indian National Institute of Cholera and Enteric Diseases
Vaccine | Year: 2013

Recently, we have demonstrated, immunization of adult female mice with outer membrane vesicles (OMVs) of Shigella boydii type 4 protected their offspring passively from shigellosis. In our present study, we have advanced our research by formulating multi-serotype outer membrane vesicles (MOMVs), mixing the OMVs of Shigella dysenteriae 1 δ. stx, Shigella flexneri 2a, 3a and 6, S. boydii type 4 and Shigella sonnei to achieve a broad spectrum protection against shigellosis. Adult mice were immunized orally with 50. μg of MOMVs, four times at weekly intervals. Immunological parameters were observed at various time points, before, during and after immunization, in adult mice. Passive protection was examined in their offspring by measuring protective efficacy and studying intestinal colonization, after challenging with various Shigella strains. Immunized dams exhibited a consistent broad spectrum antibody response. 3-4 day-old offspring of immunized dams showed significant long term passive protection against wild type S. flexneri 2a, 3a, and 6, S. boydii type 2 and S. dysenteriae 1. Their stomach extracts, essentially containing mother's milk, have also exhibited significant levels of anti-MOMVs immunoglobulins. In conclusion, MOMVs formulation represents an easy, safe immunization strategy that was found suitable to provide complete passive protection to the neonatal mice against all four serogroups of Shigellae. It could be exploited for the development of a novel non-living vaccine against human shigellosis in near future. © 2013 Elsevier Ltd.

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