Sangeeta Khare - Academia.edu (original) (raw)

Papers by Sangeeta Khare

ALTEX, 2015

Models of the outer epithelia of the human body - namely the skin, the intestine and the lung - h... more Models of the outer epithelia of the human body - namely the skin, the intestine and the lung - have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance ...

International Journal of Leprosy and Other Mycobacterial Diseases

The serum concentrations of the phagocytosis stimulating the tetrapeptide, tuftsin, were determin... more The serum concentrations of the phagocytosis stimulating the tetrapeptide, tuftsin, were determined by competitive enzyme immunoassay in borderline tuberculoid/tuberculoid (BT/TT, 16 cases), borderline lepromatous/lepromatous (BL/LL, 16 cases), and in healthy controls (20 cases). Using checkerboard titration, 10 ng/well of diphtheria toxoid-p-aminophenylacetyl tuftsin (DTPT) conjugate when incubated with tuftsin antisera at 1:15,000 dilution with a preincubation time of 60 min with the competitor (tuftsin) followed by a further 60-min incubation onto the DTPT-coated wells gave consistent results with a sensitivity of 5 ng/well tuftsin. The mean serum tuftsin concentration was significantly lower in BL/LL patients (134.42 +/- 48.7 ng/ml, p less than 0.01) than in healthy controls (262.86 +/- 59.8 ng/ml), while BT/TT sera (210.94 +/- 75.5 ng/ml) showed slightly decreased levels than did normals, which was not statistically significant. The mean serum IgG levels in BL/LL and BT/TT patients (37.26 +/- 10.99 mg/ml; 28.08 +/- 6.57 mg/ml, respectively) showed significantly (p less than 0.001) higher concentrations than did healthy controls (12.3 +/- 3.6 mg/ml). These observations on the serum concentrations of tuftsin and IgG in leprosy individuals suggest that there is splenic dysfunction in BL/LL patients in terms of the processing of leukokinin to release the free, active molecule tuftsin.

Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and p... more Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrheaassociated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a DsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-b, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-b signaling pathways and the regulation of actin cytoskeleton pathway. and modeling that helped to provide a more system level perspective of host immune response to the Texas A&M University researchers. Data were processed by KD, utilizing Seralogix's proprietary computational pipeline for biological systems analysis. The relation between Seralogix and Texas A&M University, College of Veterinary Medicine is strictly on a collaborative (mutually beneficial) research basis with no financial arrangements, commitments or interests. KD's motivation is to see his computational tools produce results that contribute to the improved understanding of host response to pathogen invasions (an objective of his National Health Institute research grants). KD contributed to the interpretation of the analysis results provided to the Texas A&M University researchers. Seralogix has no ownership of the data, nor results produced by their tools. RE is currently employed by Sequenom Inc. SK is currently employed by the United States Food and Drug Administration. This does not alter the authors9 adherence to all the PLoS ONE policies on sharing data and materials. *

Medical Research Archives, 2015

Molecular and cellular biochemistry, 1997

Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) is known to potentiate the immunogenic activity of anti... more Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) is known to potentiate the immunogenic activity of antigen-fed macrophages. The present study describes the mechanism of action of tuftsin in leprosy patients throughout the spectrum of the disease in vitro as a function of culture age in terms of (A) involvement of second messengers cAMP, cGMP and [Ca2+]i and (B) number of tuftsin binding sites/and their relative affinities on the monocytes/macrophages. There is apparently no direct involvement of either cAMP or cGMP while comparing the stimulated and unstimulated cultures during in vitro differentiation of monocytes (days 1, 3 and 7) or with the spectrum of the disease. Inhibition of superoxide anion release either by verapamil or with Quin 2 clearly demonstrated the involvement of [Ca2+]i as a second messenger during activation of monocytes/macrophages with tuftsin. Scatchard analysis of radiolabelled tuftsin binding data showed only one type of tuftsin receptor (low affinity) on BL/ LL m...

Leprosy review, 1997

The production of reactive nitrogen intermediates (RNI) by macrophages is critical to host defenc... more The production of reactive nitrogen intermediates (RNI) by macrophages is critical to host defence, particularly for exerting the bactericidal and tumoricidal properties. Nitric oxide (NO) were measured in the peripheral blood-derived monocytes/macrophages of normal and leprosy patients (BT/TT and BL/LL) in the presence and absence of 'tuftsin' as a function of in vitro culture age (on 1, 3, 7 days). Macrophages from both groups of leprosy patients were able to produce NO during the unstimulated state but only BL/LL macrophages could be activated by tuftsin to produce significantly high levels of NO. This increase was highest on day 1, then gradually decreased with in vitro culture age. Surprisingly, tuftsin was unable to enhance the NO production in normal macrophages above the basal level. Further, normal and BT/TT macrophages had only Cu-Zn derived superoxide dismutase (SOD) activity whereas BL/LL cultures has Cu-Zn and Mn derived SOD activity. These studies indicate that...

Leprosy review, 1993

Phagocytic cells respond to a variety of membrane stimulants by producing reactive oxygen interme... more Phagocytic cells respond to a variety of membrane stimulants by producing reactive oxygen intermediates (ROI), i.e. O2-, H2O2 and OH.metabolites. Plasma membrane activation is associated with superoxide generating NADPH oxidase, thereby causing the production of these toxic species. Stimulation of phagocytic cells also results in activation of purine catabolism, which directs the metabolic flux through xanthine oxidase to produce the superoxide anion. We previously observed that BL/LL macrophages (M phi) exhibited a premature inability to undergo tuftsin stimulated phagocytosis and microbicidal activity. The present study was undertaken to measure ROI levels in the absence and presence of 'tuftsin' pulsing as a function of in vitro culture age and also correlated these levels with adenosine deaminase (ADA) activity. The latter is known to be a contributor of O2- generation and is also involved in the maturation of the monocyte/macrophage system. The behaviour of normal and t...

Indian Journal of Clinical Biochemistry, 1998

In this study, we have examined the effect of linking of bioactive fragment of human IL-113 (163-... more In this study, we have examined the effect of linking of bioactive fragment of human IL-113 (163-171) or polytuftsin (PT, a synthetic polymer of natural immunomodulator 'luftsin") with synthetic peptides of HIV on the induction of immune response to the synthetic peptides. A panel of synthetic peptides representing defined region of gp41, gp120 and gag were used as antigens. Immunomodulators linked peptides (i.e. peptide-lL-11~ or peptide-PT) or peptide dimers were employed for immunization in Balb/c mice. Mice immunized with the peptideimmunomodulator develop effective T-cell proliferation, in vib'o cytoldne release and higher anUbody production, but not with peptide dimers. We also found that peptide-immunomodulators induced high level of IgG2a antibody production. Furthermore, there was a positive correlation between the levels of cytokine (IL-2 & IFN-3') and IgG isotype production. Thus it would appear that incorporation of IL-1 ~ fragment or PT selectively enhances the Thl type response to these peptides and may therefore be important for virus neutralization and clearance. However, the effect of IL-I~ fragment was found to be more pronounced than polytuftsin. Such an approach may provide effective vaccination against other infectious diseases.

PloS one, 2013

Salmonella enterica serovar Heidelberg (S. Heidelberg) can cause foodborne illness in humans foll... more Salmonella enterica serovar Heidelberg (S. Heidelberg) can cause foodborne illness in humans following the consumption of contaminated meat and poultry products. Recent studies from our laboratory have demonstrated that certain S. Heidelberg isolated from food-animal sources harbor multiple transmissible plasmids with genes that encode antimicrobial resistance, virulence and a VirB4/D4 type-IV secretion system. This study examines the potential role of these transmissible plasmids in bacterial uptake and survival in intestinal epithelial cells and macrophages, and the molecular basis of host immune system modulation that may be associated with disease progression. A series of transconjugant and transformant strains were developed with different combinations of the plasmids to determine the roles of the individual and combinations of plasmids on virulence. Overall the Salmonella strains containing the VirB/D4 T4SS plasmids entered and survived in epithelial cells and macrophages to a...

Nanotoxicology, 2014

Silver nanoparticles (AgNP) are widely used for their antibacterial properties. Incorporation of ... more Silver nanoparticles (AgNP) are widely used for their antibacterial properties. Incorporation of AgNP into food-related products and health supplements represents a potential route for oral exposure to AgNP; however, the effects of such exposure on the gastrointestinal system are mostly unknown. This study evaluated changes in the populations of intestinal-microbiota and intestinal-mucosal gene expression in Sprague-Dawley rats (both male and female) that were gavaged orally with discrete sizes of AgNP (10, 75 and 110 nm) and silver acetate. Doses of AgNP (9, 18 and 36 mg/kg body weight/day) and silver acetate (100, 200 and 400 mg/kg body weight/day) were divided and administered to rats twice daily (10 h apart) for 13 weeks. The results indicate that AgNP prompted size-and dose-dependent changes to ilealmucosal microbial populations, as well as, intestinal gene expression and induced an apparent shift in the gut microbiota toward greater proportions of Gram-negative bacteria. DNA-based analyses revealed that exposure to 10 nm AgNP and low-dose silver acetate caused a decrease in populations of Firmicutes phyla, along with a decrease in the Lactobacillus genus. Analysis of host gene expression demonstrated that smaller sizes and lower doses of AgNP exposure prompted the decreased expression of important immunomodulatory genes, including MUC3, TLR2, TLR4, GPR43 and FOXP3. Gender-specific effects to AgNP exposure were more prominent for the gut-associated immune responses. These results indicate that the oral exposure to AgNP alter mucosa-associated microbiota and modulate the gut-associated immune response and the overall homeostasis of the intestinal tract.

DNA and cell biology, 2015

Cytolethal distending toxin B (cdtB) is a conserved virulence factor in Salmonella enterica serov... more Cytolethal distending toxin B (cdtB) is a conserved virulence factor in Salmonella enterica serovar Typhi. Here we report the presence and functionality of cdtB in some nontyphoidal Salmonella (NTS) serovars, including Salmonella Javiana (cdtB+wt S. Javiana), isolated from imported food. To understand the role of cdtB in NTS serovars, a deletion mutant (cdtB(-)ΔS. Javiana) was constructed. Macrophages were infected with cdtB+wt S. Javiana (wild type), cdtB(-)Δ S. Javiana (mutant), and cdtB-negative NTS serovar (S. Typhimurium). Cytotoxic activity and transcription level of genes involved in cell death (apoptosis, autophagy, and necrosis) were assessed in infected macrophages. The cdtB+wt S. Javiana caused cellular distension as well as high degree of vacuolization and presence of the autophagosome marker LC3 in infected macrophages as compared with cdtB(-)ΔS. Javiana. The mRNA expression of genes involved in the induction of autophagy in response to toxin (Esr1 and Pik3C3) and coreg...

BMC Proc, 2011

Multi-comparative systems biology analysis reveals time-course biosignatures of in vivo bovine pa... more Multi-comparative systems biology analysis reveals time-course biosignatures of in vivo bovine pathway responses to B.melitensis, S.enterica Typhimurium and M.avium paratuberculosis Abstract Background: To decipher the complexity and improve the understanding of host-pathogen interactions, biologists must adopt new system level approaches in which the hierarchy of biological interactions and dynamics can be studied. This paper presents the application of systems biology for the cross-comparative analysis and interactome modeling of three different infectious agents, leading to the identification of novel, unique and common molecular host responses (biosignatures).

Preharvest and Postharvest Food Safety, 2004

... 1999, Naser et al. 1999, Prantera and Scribano 1999, Suenaga et al. 1999, Collins et al. 2000... more ... 1999, Naser et al. 1999, Prantera and Scribano 1999, Suenaga et al. 1999, Collins et al. 2000). ... Deutsche Zeitschrift fuer Thiermedizin und vergleichende Pathologie 21:438454. Kanazawa, K., Haga, Y., Funakoshi, O., Nakajima, H., Munakata, A. and Yoshida, Y. 1999. ...

Veterinary Pathology, 2010

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium-induced ruminant a... more The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium-induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18-), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18- calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18- calves--including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration--were similar to the ones reported in the literature for CD18- calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18- calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene alpha in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha were poorly correlated with the presence or absence of tissue neutrophils.

Vaccine, 2007

Bovine tuberculosis is a chronic granulomatous disease caused by Mycobacterium bovis. Lack of def... more Bovine tuberculosis is a chronic granulomatous disease caused by Mycobacterium bovis. Lack of definitive diagnostics and effective vaccines for domestic animals are major obstacles to the control and eradication of bovine tuberculosis. Auxotrophic mutants of Mycobacterium tuberculosis have shown promise as vaccine candidates for preventing human tuberculosis. Similarly, we constructed a leucine auxotroph of M. bovis, by using allelic exchange to delete leuD (encoding isopropyl malate isomerase), creating a strain requiring exogenous leucine for growth in vitro. We vaccinated 10 cattle subcutaneously with 10(9)CFU of M. bovis DeltaleuD and 10 age-matched, gender-matched controls were injected with phosphate-buffered saline. Vaccinated cattle had significantly increased in vitro antigen-specific T-cell-mediated responses. All cattle were challenged intranasally on day 160 post-immunization with 10(6)CFU of virulent M. bovis Ravenel S. On day 160 post-challenge vaccinated cattle had significantly reduced tissue mycobacterial burdens and 6 of 10 had complete clearance of the challenge strain and histopathological lesions were dramatically less severe in the vaccinated group. Thus, a single subcutaneous immunization of the M. bovis DeltaleuD mutant produced highly significantly protective immunity as measured by a reduction in tissue colonization, burden, bacilli dissemination, and histopathology caused by virulent M. bovis Ravenel S challenge.

Vaccine, 2011

The aim of research on infectious diseases is their prevention, and brucellosis and salmonellosis... more The aim of research on infectious diseases is their prevention, and brucellosis and salmonellosis as such are classic examples of worldwide zoonoses for application of a systems biology approach for enhanced rational vaccine development. When used optimally, vaccines prevent disease manifestations, reduce transmission of disease, decrease the need for pharmaceutical intervention, and improve the health and welfare of animals, as well as indirectly protecting against zoonotic diseases of people. Advances in the last decade or so using comprehensive systems biology approaches linking genomics, proteomics, bioinformatics, and biotechnology with immunology, pathogenesis and vaccine formulation and delivery are expected to enable enhanced approaches to vaccine development. The goal of this paper is to evaluate the role of computational systems biology analysis of host:pathogen interactions (the interactome) as a tool for enhanced rational design of vaccines. Systems biology is bringing a new, more robust approach to veterinary vaccine

PLoS ONE, 2013

Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human be... more Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human beings and infects hosts primarily through the oral route. The epithelium covering domed villi of jejunal-ileal Peyer's patches is an important site of entry for several pathogens, including Brucella. Here, we use the calf ligated ileal loop model to study temporal in vivo Brucella-infected host molecular and morphological responses. Our results document Brucella bacteremia occurring within 30 min after intraluminal inoculation of the ileum without histopathologic traces of lesions. Based on a system biology Dynamic Bayesian Network modeling approach (DBN) of microarray data, a very early transient perturbation of the host enteric transcriptome was associated with the initial host response to Brucella contact that is rapidly averted allowing invasion and dissemination. A detailed analysis revealed active expression of Syndecan 2, Integrin alpha L and Integrin beta 2 genes, which may favor initial Brucella adhesion. Also, two intestinal barrier-related pathways (Tight Junction and Trefoil Factors Initiated Mucosal Healing) were significantly repressed in the early stage of infection, suggesting subversion of mucosal epithelial barrier function to facilitate Brucella transepithelial migration. Simultaneously, the strong activation of the innate immune response pathways would suggest that the host mounts an appropriate protective immune response; however, the expression of the two key genes that encode innate immunity anti-Brucella cytokines such as TNF-a and IL12p40 were not significantly changed throughout the study. Furthermore, the defective expression of Toll-Like Receptor Signaling pathways may partially explain the lack of proinflammatory cytokine production and consequently the absence of morphologically detectable inflammation at the site of infection. Cumulatively, our results indicate that the in vivo pathogenesis of the early infectious process of Brucella is primarily accomplished by compromising the mucosal immune barrier and subverting critical immune response mechanisms. Animal and Zoonotic Disease Defense ONR-N00014-04-1-0 grant. CAR was supported by an I.N.T.A.-Fulbright Argentina Fellowship. CLG received support from an NIH cardiology fellowship, Cardiology Department, University of Texas Southwestern Medical Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.