Biomedical subjects

R Nayak

Publications and source records attributed to R Nayak.

At least 19 recordsLinked to original sources

PubMed2007

Maintenance of antigen-specific immunological memory through variable regions of heavy and light chains of anti-idiotypic antibody.

Immunological memory is characterized by a quick and enhanced immune response after re-exposure to the same antigen. To explain the mechanism involved in generation and maintenance of immunological memory, we had earlier proposed a hypothesis involving the relay of memory by idiotypic and anti-idiotypic B cells. The peptidomimic present in the hypervariable region of anti-idiotypic antibody was hypothesized to carry forward immunological memory. In the present work, we provide evidence supporting a role for the anti-idiotypic antibody in eliciting antigen-specific B-cell and T-cell responses. Employing the idiotypic monoclonal antibody (Ab(1)) specific for haemagglutinin (H) protein of rinderpest virus, Ab(2beta) was generated, which possesses an internal image of the H protein in the region between amino acids 527 and 556. We demonstrate that antigen-specific memory is perpetuated by immunization with Ab(2), as shown by maintenance of antigen-specific T-cell responses upon restimulation in vitro of Ab(2) immune splenocytes by antigen-presenting cells expressing H protein or pulsed with H-protein-derived peptides. We have also shown that boosting with antigen-specific anti-idiotypic B cells generates a memory response in antigen-primed mice. Evidence has been provided for the existence of an antigen-specific B-cell idiotypic network in the body that supports the perpetuation of immunological memory as proposed in the relay hypothesis.

Amino Acid Sequence↗

PubMed2006

A combined immuno-informatics and structure-based modeling approach for prediction of T cell epitopes of secretory proteins of Mycobacterium tuberculosis.

The role of secretory proteins of Mycobacterium tuberculosis in pathogenesis and stimulation of specific host responses is well documented. They are also shown to activate different cell types, which subsequently present mycobacterial antigens to T cells. Therefore identification of T cell epitopes from this set of proteins may serve to define candidate antigens with vaccine potential. Fifty-two secretory proteins of M. tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nonameric peptides. All possible overlapping nonameric peptide sequences from 52 secretory proteins were generated in silico and analyzed for their ability to bind to 33 alleles belonging to A, B and C loci of HLA class I. Fifteen percent of generated peptides are predicted to bind to HLA with halftime of dissociation T(1/2) >or=100 min and 73% of the peptides predicted to bind are mono-allelic in their binding. The structural basis for recognition of no-namers by different HLA molecules was studied employing structural modeling of HLA class I-peptide complexes and there exists a good correlation between structural analysis and binding prediction. Pathogen peptides that could behave as self- or partially self-peptides in the host were eliminated using a comparative study with the human proteome, thus reducing the number of peptides for analysis. The implications of the finding for vaccine development are discussed vis-à-vis the limitations of the use of subunit vaccine and DNA vaccine.

Amino Acid Sequence↗

PubMed2006

Evaluation of the buffering capacity of powdered cow's, goat's and soy milk and non-prescription antacids in the treatment of non-ulcer dyspepsia.

BACKGROUND: Non-ulcer dyspepsia (NUD) is the term most commonly used to describe a heterogeneous and often ill-defined group of dyspepsia patients whose symptoms of upper abdominal pain, discomfort or nausea persist in the absence of identifiable cause. Treatment choice commonly includes over the counter medicines and home remedies, e.g. milk. OBJECTIVE: To determine the relative buffering capacity of goat's, cow's and soy milk, non-prescription antacid drugs and combinations thereof. METHODS: The buffering capacities of 25 ml aliquots of each of the powdered milk products, the antacids alone and the combination of antacid and milk were determined. Statistical analysis was used to determine any significant differences in buffering capacity. RESULTS: When the antacids were examined alone, significant differences in buffering capacity were observed. When powdered milk products were examined alone, cow's milk had a significantly higher buffering capacity than either goat's or soy milk. There was no significant difference between goat's and soy milk. In the combination of cow's milk with each of the antacids, brand A and B had a similar buffering capacity, significantly higher than that observed with brand C. CONCLUSIONS: The combination with best observed buffering capacity was brand A with cow's milk, and the weakest buffering capacity was observed with brand C with soy milk. The results obtained can be attributed to the chemical constituents of the antacids and the milk products.

Acid-Base Equilibrium↗

PubMed2005

Defining putative T cell epitopes from PE and PPE families of proteins of Mycobacterium tuberculosis with vaccine potential.

The identification of T cell epitopes from immune relevant antigens of Mycobacterium tuberculosis is a critical step in the development of a vaccine covering diverse populations. Two multigene families, PE-PGRS and PPE make up about 10% of the M. tuberculosis genome. However, the functions of the proteins coded by these large numbers of genes are unknown. All possible nonameric peptide sequences from PE and PPE proteins were analysed in silico for their ability to bind to 33 alleles of class I HLA. These results reveal that of all PE and PPE proteins, a significant number of these peptides are predicted to be high-affinity HLA binders, irrespective of the length of the protein. The pathogen peptides that could behave as self or partially self-peptides in the host were eliminated using a comparative study with human proteome, thus reducing the number of peptides for analysis. The structural basis for recognition of the nonamers by the respective HLA molecules thus predicted was analyzed by molecular modeling. The structural analysis showed good correlation with the binding prediction. The analysis also led to an understanding of the binding profile of the peptides with respect to different alleles of class I HLA. The predicted epitopes can be tested experimentally for their inclusion in a potential vaccine against tuberculosis that is HLA haplotype-specific.

Alleles↗

PubMed2004

Genetic diversity and virulence gene determinants of antibiotic-resistant Salmonella isolated from preharvest turkey production sources.

This study evaluated the molecular diversity of 29 Salmonella serotypes isolated from turkey ceca and the production environment. Isolates were resistant to bacitracin (100%), erythromycin (100%), novobiocin (100%), rifampin (100%), streptomycin (62%), gentamicin (52%), spectinomycin (48%), tetracycline (31%), sulfamethoxazole/trimethoprim (SXT) (3%) and tobramycin (3%). The minimum inhibitory concentration (MIC) values ranged from 32 to >/=1024 microg/ml. The pulsed-field gel electrophoresis (PFGE) and ribotyping patterns were identical within each of the serotypes Heidelberg, Worthington and Muenster. The plasmid profiles were identical within each of the Salmonella serotypes. Two different clones of Salmonella anatum were differentiated by PFGE typing but not by ribotyping. Heidelberg isolates from nine turkey ceca and three drinker samples had identical antibiotic resistance, PFGE, ribotype and plasmid patterns, suggesting that transmission of this particular clone may have occurred between the birds and the drinkers. Identical PFGE, ribotype and plasmid patterns were observed in one Salmonella worthington isolate from turkey ceca in one flock and two S. worthington isolates from feeder contents and drinkers from a subsequent flock, suggesting transmission of this pathogen between flocks. Individual and multiple polymerase chain reaction (PCR) analyses revealed the presence of the virulence genes invA, aceK and sopB and the absence of the h-1i gene in all isolates. A combination of genotypic and phenotypic markers can be useful in studying genetic variation among natural salmonellae populations in turkey production and delineating possible transmission pathways.

Animal Husbandry↗

PubMed2004

Cytotoxic T cell epitope in cattle from the attachment glycoproteins of rinderpest and peste des petits ruminants viruses.

The surface glycoproteins of rinderpest virus (RPV) confer protective immunity in cattle. We demonstrated that cattle immunized with a recombinant extracellular baculovirus expressing the hemagglutinin (H) protein of RPV (rECV-H) generate virus neutralizing antibody responses, bovine leukocyte antigen (BoLA) class II restricted helper T cell responses and BoLA class I restricted cytotoxic T cell (CTL) responses against RPV-H and hemagglutinin-neuraminidase (HN) glycoprotein of closely related Peste des petits ruminants virus (PPRV). In this study, employing autologous skin fibroblasts transiently expressing truncations of H and HN in a BoLA class I restricted lymphoproliferation assay, we have mapped a highly homologous domain (amino acids 400-423) on these proteins harboring a CTL epitope. Subsequently, based on sequence comparison with available BoLA class I binding motifs, we have identified a BoLA-A11 binding motif (amino acids 408-416) in the stimulatory domain. Autologous cells pulsed with a synthetic peptide corresponding to this sequence stimulated CTLs from rECV-H immunized as well as tissue culture attenuated RPV vaccinated cattle of different breeds and parentage. This is the first epitope identified in cattle on the attachment glycoproteins of RPV and PPRV.

Animals↗

PubMed2003

Isolation and characterisation of Salmonella in a turkey production facility.

1. A comprehensive ecological survey was conducted from April 1997 to June 1999 on 4 turkey flocks (F1 to F4) to identify key pre-harvest sources/vectors of Salmonella colonisation. 2. Turkey caecal and crop content, litter, drinker, air, feed, feeder and environmental swab samples were collected. Conventional microbiological and serological procedures were used to isolate, identify, and confirm the presence or absence of Salmonella. 3. Salmonella was isolated from 13% of litter, 11% of turkey caeca, 10% of drinker, 5% of environmental swab, 3% of feed and 1% of feeder samples. Salmonella heidelberg (65%), S. senftenberg (19%), S. muenster (10%), S. anatum (3%), and S. worthington (3%) were identified. 4. Identifying environmental sources associated with Salmonella colonisation and characterising serotypes would assist in designing pre-harvest controls for this poultry-borne pathogen. Integrators and poultry producers may be able to design hazard analysis and critical control point (HACCP) protocols to reduce the incidence of Salmonella arriving at the processing plant.

Animal Husbandry↗

PubMed2003

Molecular characterization of fluoroquinolone-resistant Campylobacter spp. isolated from poultry.

Campylobacteriosis, an infectious disease caused by Campylobacter jejuni and Campylobacter coli, is treated by fluoroquinolone antibiotics in clinical practices. However, use of these drugs in animal husbandry may select for fluoroquinolone-resistant campylobacters and, thereby, compromise the clinical treatment of infection. In this study, 21 fluoroquinolone-resistant campylobacters were isolated from poultry samples. Morphological and biochemical characteristics indicated that 19 isolates were C. jejuni and two were C. coli. All isolates were resistant to multiple antibiotics but sensitive to chloramphenicol and gentamicin. These isolates were characterized at the molecular level by amplifying the flagellin gene (flaA) by PCR. The PCR protocol amplified a 1.7-kb flaA gene from all isolates. RFLP analysis of the 1.7-kb amplicons after digestion with DdeI yielded four distinct patterns. The 21 fluoroquinolone-resistant campylobacter isolates were further characterized by pulsed-field gel electrophoresis (PFGE) and compared with the PFGE patterns of nine fluoroquinolone-sensitive campylobacter strains. Four of the 21 fluoroquinolone-resistant isolates were untypable by the PFGE protocol. The PFGE analysis with SalI or SmaI indicated that seven or five, respectively, of the 17 resistant isolates had identical macrorestriction profiles (mrps). However, PFGE analysis with a combination of SalI and SmaI indicated that four of the 17 isolates had similar macrorestriction profiles. The PFGE patterns of the 17 fluoroquinolone-resistant isolates were different from the nine sensitive campylobacter strains.

Animals↗

PubMed2002

Tubedown-1 in remodeling of the developing vitreal vasculature in vivo and regulation of capillary outgrowth in vitro.

Tubedown-1 (tbdn-1) is a mammalian homologue of the N-terminal acetyltransferase subunit NAT1 of Saccharomyces cerevisiae and copurifies with an acetyltransferase activity. Tbdn-1 expression in endothelial cells becomes downregulated during the formation of capillary-like structures in vitro and is regulated in vivo in a manner which suggests a functional role in dampening blood vessel development. Here we show that tbdn-1 is expressed highly in the vitreal vascular network (tunica vasculosa lentis and vasa hyaloidea propria) during the pruning and remodeling phases of this transient structure. The vitreal blood vessels of mice harboring a targeted inactivation of TGF-beta2 fail to remodel and abnormally accumulate, a phenomenon reminiscent of the ocular pathology resembling persistent fetal vasculature (PFV) in humans. Since suppression of normal tbdn-1 expression has been previously observed in retinal vessel proliferation, we analyzed vitreal vascular changes and tbdn-1 expression in TGF-beta2(-/-) eyes. The nuclei of vitreal vessel endothelial cells in TGF-beta2(-/-) eyes express proliferating cell nuclear antigen (PCNA) and exhibit increased levels of active (P42/44)mitogen-activated protein kinase (phospho-(P42/44)MAPK), characteristics consistent with proliferative endothelial cells. In contrast to normal vitreal vessels, collagen IV expression exhibited a disorganized pattern in the TGF-beta2(-/-) vitreal vessels, suggesting vessel disorganization and possibly a breakdown of vessel basal laminae. Moreover, vitreal vessels of TGF-beta2(-/-) mice lack expression of pericyte markers (CD13, alpha smooth muscle actin) and show ultrastructural changes consistent with pericyte degeneration. The accumulating vitreal blood vessels of TGF-beta2(-/-) mice, while maintaining expression of the endothelial marker von Willebrand Factor, show a significant decrease in the expression of tbdn-1. We addressed the functional role of tbdn-1 in the regulation of vitreal blood vessels using an in vitro model of choroid-retina capillary outgrowth. Clones of the RF/6A fetal choroid-retina endothelial cell line showing suppression of tbdn-1 levels after overexpression of an antisense TBDN-1 cDNA display a significant increase in the formation of capillary-like structures in vitro compared with controls. These findings suggest that tbdn-1 inhibits capillary-like formation in vitro and may serve to dampen vitreal blood vessel formation preceding the regression of the vitreal vasculature during development. Our results also suggest that tbdn-1 may participate with TGF-beta2 in regulating normal development of the vitreal vasculature.

Acetyltransferases↗

PubMed2002

Screening of Salmonella isolates from a turkey production facility for antibiotic resistance.

An ecological survey was conducted from April 1997 to June 1999 on four turkey flocks (F1 to F4). Turkey cecal contents, litter, waterers, feed, feeders, and environmental swabs were analyzed. Presence of Salmonella was determined using conventional microbiological screening techniques and confirmed by serology. Positive isolates were serotyped and screened for antibiotic resistance. From a total of 69 Salmonella isolates 25% were resistant to one or more antibiotics including gentamicin (G), spectinomycin (SP), streptomycin (S), tetracycline (T), tobramycin (TO), and trimethoprim/sulfamethoxazole. Isolates included 45 S. heidelberg, 13 S. senftenberg, 7 S. muenster, 2 S. anatum, and 2 S. worthington. Resistance to antibiotic(s) was highest among waterer isolates (55%) followed by environmental swabs (43%), feeder content samples (33%), turkey cecal contents (26%), and litter samples (5%). Frequencies of antibiotic-resistant Salmonella in F1, F2, and F4 were 27, 13, and 40%, respectively. Salmonella was undetected in F3. In F1, S. heidelberg from cecal content and waterer samples was resistant to G, SP, S, and T, whereas S. anatum from waterer samples was resistant to T and S. In F2, S. worthington from litter and feeder content samples was resistant to T, and in F4, S. muenster from environmental swabs was resistant to TO, S, SP, and G. Identifying preharvest sources and characterizing serotype and antibiotic-resistance profile can assist poultry producers and integrators in tracking movement of Salmonella on turkey farms.

Animal Feed↗

PubMed2001

Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of Peste des petits ruminants virus: identification of a T cell determinant.

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus within the family Paramyxoviridae, causes a fatal disease 'peste des petits ruminants' in goats and sheep. This enveloped virus is antigenically closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants. PPRV harbors two major surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. The surface glycoproteins of morbilliviruses are highly immunogenic and confer protective immunity. In this study, we investigated the immune responses generated in goats immunized with low doses of purified recombinant extracellular baculovirus carrying a membrane bound form of the HN protein of PPRV without any adjuvant. We report that the immunized goats develop both humoral and cell-mediated immune responses. Antibodies generated in the immunized animals could neutralize both PPRV and RPV in vitro. Further, using a combination of Escherichia coli expressed deletion mutants of PPRV-HN and RPV-H proteins, and synthetic peptides corresponding to the highly conserved N-terminal sequences of MV-H protein, we have mapped an N-terminal T cell determinant (amino acids 123-137) and a C-terminal domain (amino acids 242-609) harboring potential T cell determinant(s) in goats.

Amino Acid Sequence↗

PubMed2001

Recombinant hemagglutinin protein of rinderpest virus expressed in insect cells induces humoral and cell mediated immune responses in cattle.

Rinderpest virus causes a highly contagious and often fatal disease in domestic and wild ruminants. The surface glycoproteins, hemagglutinin (H) and fusion (F) proteins of this enveloped virus are known to confer protective immunity in cattle. We have reported the generation of a recombinant baculovirus expressing H protein and studied its protective properties in cattle. In this report, we demonstrate that the recombinant baculovirus encoded H protein expressed in insect cells gets incorporated into extracellular baculovirus. Single administration of low doses of purified recombinant extracellular virus with or without adjuvant induces virus neutralizing antibody responses and bovine leukocyte antigen (BoLA) class II restricted helper T cell responses in cattle.

Animals↗

PubMed2001

Idiotype and antigen-specific T cell responses in mice on immunization with antigen, antibody, and anti-idiotypic antibody.

Idiotypic determinants of immunoglobulin molecules can evoke both CD4(+) and CD8(+) T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell. The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.

Animals↗

PubMed2001

Idiotypic-anti-idiotypic B cell interactions generated against a protective antigen of a morbillivirus in mice.

The idiotypic network theory (N. K. Jerne, Ann. Immunol. 125, 373-389, 1974) predicts that any antibody that can be made by an individual would have its preexisting specific complementary B cells in its germline repertoire. We transplanted syngeneic BALB/c mice with live hybridoma cells and demonstrated the simultaneous presence of interacting idiotypic and anti-idiotypic B cells in an individual animal by immuno-cytoadherence assays. Furthermore, we demonstrate that interacting B cells displaying idiotypic and anti-idiotypic antibodies are subjected to lysis by complement. It is therefore tempting to speculate that this complement-sensitive interaction between idiotypic and complementary anti-idiotypic B cells in vivo may provide a mechanism for the regulation of B cell populations.

Animals↗

PubMed2001

Identification of a cytotoxic T-cell epitope on the recombinant nucleocapsid proteins of Rinderpest and Peste des petits ruminants viruses presented as assembled nucleocapsids.

The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells. We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E. coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells. Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c). A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response. The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted. Based on the prediction for H-2(d)-restricted T-cell motifs we tested the lysis of transfected P815 (H-2(d)) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins. We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins. Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope. This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides.

Amino Acid Motifs↗

PubMed2001

Perpetuation of immunological memory: a relay hypothesis.

A mechanism is proposed which explains the perpetuation of B-cell immunological memory indefinitely without requiring the presence of long-living memory cells or persisting antigen. The salient feature of this model is that immunological memory can be perpetuated indefinitely through the mutual interaction of idiotypic and anti-idiotypic B cells. These cells mutually stimulate and clonally expand with either specific or bystander T-cell help. Because B cells can present antigen, they present 'apparently foreign' idiopeptides to T cells. The idiopeptides of de novo synthesized antibody is presented to CD8+ T cells that recognize the idiopeptide-presenting cell as targets and regulate their population. The recycling of immunoglobulins from surface to endosomal compartment of B cells leads to the presentation of idiopeptides by major histocompatibility complex (MHC) class II to CD4+ T cells. Even if the majority of the clonally expanded cells die because of lack of stimulation, cytotoxic T lymphocyte (CTL) lysis or for other reasons, the surviving cells will be able to carry forward the memory. This mechanism also provides a means for affinity maturation through idiotypic selection of somatically mutated high affinity cells or those from the naïve pool. We have termed these two types of complementary B cells as Burnet B cells: those which recognize the antigen or antigen mimic, and Jerne B cells, which can recognize the idiotypes of antibody and carry antigen mimics. The proposed hypothesis can explain differential duration of memory for different antigens, the shelf space paradox, affinity maturation, repertoire shift, etc.

Animals↗

PubMed2001

Mapping of T-helper epitopes of Rinderpest virus hemagglutinin protein.

Rinderpest virus (RPV) is a highly contagious and often fatal disease of domestic and wild ruminants, caused by rinderpest virus of the genus Morbillivirus under the family Paramyxoviridae. Hemagglutinin (H) and fusion (F) proteins of this enveloped virus confer protective immunity against experimental challenge with virulent rinderpest virus. We have earlier demonstrated that immunization with a single dose of recombinant extracellular baculovirus expressing H protein elicits H-specific humoral and lymphoproliferative responses in cattle. The lymphoproliferative responses are predominantly BoLA class II restricted. In this work, we have analyzed lymphoproliferative responses of peripheral lymphocytes from immunized cattle to truncated H protein fragments expressed in E. coli for locating domains harboring Th epitopes. One region (aa 113-182) recognized by immune T cells is conserved in the H protein of measles virus, which was earlier shown to contain a dominant Th epitope in mouse. Synthetic peptides within this region of measles virus H protein were used to identify a Th epitope conserved in the H protein of RPV virus (aa 123-137) in cattle. A second Th epitope located at the C-terminus of RPV-H was mapped to the region corresponding to aa 512-609 using truncated protein fragments expressed in E. coli. The C-terminal epitope (aa 575-583) was mapped using synthetic peptides corresponding to measles virus H as well as RPV-H protein.

Amino Acid Sequence↗

PubMed2001

Recombinant hemagglutinin protein of rinderpest virus expressed in insect cells induces cytotoxic T-cell responses in cattle.

Rinderpest virus (RPV), a member of the genus Morbillivirus within the Paramyxoviridae family, causes a highly contagious and often fatal disease known as rinderpest in wild and domestic ruminants. The envelope of the virus contains two surface glycoproteins, namely the hemagglutinin (H) and the fusion (F) proteins, both of which have been shown to confer protective immunity in animals. In this paper, we demonstrate that single administration of low doses of recombinant H protein of RPV expressed in insect cells in the form of extracellular virus induces long lasting bovine leukocyte antigen class I restricted cytotoxic T-cell (CTL) responses in cattle in the absence of adjuvant. This is the first report of CTL responses in cattle against one of the protective antigens of RPV.

Animals↗