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Biomedical subjects

S P Nair

Publications and source records attributed to S P Nair.

At least 19 recordsLinked to original sources
PubMed2005

Protective immune response against foot-and-mouth disease virus challenge in guinea pigs vaccinated with recombinant P1 polyprotein expressed in Pichia pastoris.

Vaccination of the susceptible livestock with potent, safe and cost effective vaccine is the primary requirement to control foot-and-mouth disease (FMD) in an endemic country. In this study, an alternative approach was used in which structural protein genes of all the four serotypes of FMDV (O, Asia 1, A22 and C) were expressed separately in methylotrophic yeast Pichia pastoris. The recombinant polyproteins (P1) were characterized by SDS-PAGE and in Western Blot analysis. Partially purified protein was used for immunization in guinea pigs with different adjuvant formulations and immune response studied. Ninety micrograms of the recombinant protein per monovalent dose was used for immunization. A single injection of a monovalent or polyvalent vaccine was given to guinea pigs with various adjuvant combinations viz., Monovalent recombinant protein either adjuvanted with Montanide-ISA50V or Indigenous oil, Monovalent recombinant protein mixed with 1/10th dose of inactivated oil-adjuvanted virus vaccine and Polyvalent recombinant protein with Montanide ISA50V. FMDV specific humoral immune response was observed at about 28th day post vaccination. The immune response as assessed by indirect ELISA and Serum neutralization test titres was found to be 320-640 and 16-32, respectively. When challenged with virulent homologous type 'O' virus, the guinea pigs showed protective C index of 2.01,1.81, 2.56 and 2.48, respectively, with above said adjuvant combinations. The study has shown that yeast-expressed FMDV P1 polyprotein in a single dose could elicit a protective immune response in guinea pigs, and this could be a possible future vaccine candidate in homologous host.

Animals↗
PubMed2005

The interaction between staphylococcal superantigen-like proteins and human dendritic cells.

Staphylococcus aureus produce a family of exotoxins (staphylococcal superantigen like proteins, SSLs) with structural, but not functional, homology to superantigens. These proteins have previously been shown to interact selectively with antigen presenting cells, including dendritic cells. The functional consequences of this interaction are now explored. SSL7 and 9 had no effect on viability or morphology of dendritic cells. The proteins did not induce dendritic cell maturation, as measured by cell surface phenotype. Exposure to SSL did not alter the ability of dendritic cells to take up FITC-dextran. Finally, exposure to SSLs did not impair the ability of the dendritic cells to stimulate allogeneic or antigen specific T cell responses. However, dendritic cells loaded with SSL7 or 9 were able to stimulate a T cell proliferative response in 3/8 healthy individuals tested. Sera from nine out of 10 individuals tested contained antibodies against both SSL7 and SSL9, and the response to each SSL was specific and not cross-reactive. The results demonstrate that SSLs are immunogenic in humans at both the B and T cell level, but it remains unclear whether this response is to the benefit of the bacterium or the host.

Antibody Formation↗
PubMed2002

Early antibody responses of cattle for foot-and-mouth disease quadrivalent double oil emulsion vaccine.

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals. The multiplicity of FMDV serotypes in animals poses a central problem in the policy of vaccination and is of much concern to health authorities. Hence it is the practice of vaccination with polyvalent vaccine for prophylactic measure. In the present report, we analysed the early antibody responses elicited by FMDV quadrivalent (FMDV O, A, C and Asia 1 serotypes) double emulsion (Montanide ISA 206) vaccines in cattle. We observed variations between various viral serotypes in eliciting early antibody response although neutralizing antibody response against all the four serotypes were detected as early as fourth day following vaccination. The duration of immunity also appeared to maintain for long period. The neutralizing antibody titres were maintained well above 2log(10) even after 6 months of vaccination irrespective of serotypes. Thus, allows the possibilities of two vaccinations per year for the maintenance of herd immunity.

Adjuvants, Immunologic↗
PubMed2002

Staphylococcus aureus fibronectin binding proteins A and B possess a second fibronectin binding region that may have biological relevance to bone tissues.

Staphylococcus aureus is a major human pathogen that has a propensity for targeting to bone tissues and thereby causing bone disease. A plausible hypothesis is that S. aureus targets to bone using the MSCRAMM family of surface proteins possessed by this organism. Two such proteins that have recently been shown to be important in bone infections are the S. aureus fibronectin binding proteins (FnBP) A and B. To identify fibronectin-binding domains from S. aureus that have biological relevance to bone, a phage display library of S. aureus genomic DNA was constructed and panned sequentially against immobilized fibronectin and cultured osteoblasts. Using this system, phage displaying a second fibronectin-binding region within the N-terminal part of FnBPA and FnBPB, which is distinct from the primary fibronectin-binding domain located within the D repeat region of these proteins, was isolated. Phage displaying this second region bound to both immobilized fibronectin and to osteoblasts and/or the extracellular matrix synthesized by these cells, thereby suggesting a biological relevance for these regions in S. aureus binding to bone tissues. Analysis of these binding regions for their ability to bind to other extracellular matrix proteins revealed a preference for fibronectin, with slight binding to fibrinogen and no binding to collagen or laminin.

Adhesins, Bacterial↗
PubMed2002

Expression of the S. aureus hysA gene in S. carnosus from a modified E. coli-staphylococcal shuttle vector.

We have modified an E. coli-staphylococcal shuttle vector for use in the general cloning and expression of genes from pathogenic staphylococci in Staphylococcus carnosus. As S. carnosus is non-pathogenic, this expression system will facilitate the study of the roles of individual gene products in the disease process. To evaluate the use of this expression system, a DNA fragment containing the Staphylococcus aureus hyaluronate lyase (hysA) gene was cloned into the modified vector, pNW21, and introduced into S. carnosus. Hyaluronate lyase was both produced and secreted by S. carnosus. In addition, the secreted HysA protein was enzymatically active, as determined using a zymographic assay.

Base Sequence↗
PubMed2001

Analysis of the effect of changing environmental conditions on the expression patterns of exported surface-associated proteins of the oral pathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans has been specifically implicated in the aetiology of one or more of the periodontal diseases, conditions in which inflammation of the gums is associated with destruction of the alveolar bone supporting the teeth. In these diseases there is loss of attachment of the gums (gingivae) to the teeth forming a periodontal pocket. The microenvironment of this pocket is extremely complex and it is likely that there will be substantial variation in the environmental conditions operating in this habitat. The aim of the current investigation was to study the effect of disease-relevant environmental factors on the production and release of secreted surface- associated proteins of A. actinomycetemcomitans. These secreted proteins contain many of the virulence determinants of this organism. A range of environmental conditions were investigated: growth in a CO(2)-enriched aerobic atmosphere vs anaerobic growth, presence of serum or blood, biofilm vs planktonic mode of growth and iron depletion. Differential expression of a number of the secreted surface-associated proteins was observed under different growth conditions and these included the glycolytic enzyme triose phosphate isomerase. An ability to adapt to prevailing environmental conditions may facilitate the survival of the organism in the changing microIenvironment of the periodontal pocket.

Adaptation, Biological↗
PubMed2001

Identification of photolabile outer membrane proteins of Porphyromonas gingivalis.

As the prevalence of antibiotic-resistant strains of bacteria increases, novel ways of treating infections need to be developed. This is particularly pertinent with respect to the periodontal diseases--the most common chronic bacterial infections of man. The use of a photosensitizer in combination with red light has been demonstrated to be effective in killing several human pathogens, including the oral bacterium, Porphyromonas gingivalis, a major pathogen in periodontitis. Killing was associated with alterations in the molecular masses of several outer membrane and plasma membrane proteins and these may be therapeutic targets for photodynamic therapy and other antimicrobial approaches. To identify these photolabile proteins, we have used a panel of monoclonal antibodies raised to whole P. gingivalis. A number of the antibodies recognized various photolabile proteins. Using a combination of Western blotting and protein sequencing the predominant photolabile proteins in P. gingivalis have been identified as the major secreted/cell surface proteases--Lys and Arg gingipain.

Adhesins, Bacterial↗
PubMed2001

Staphylococcus aureus accessory regulators: expression within biofilms and effect on adhesion.

Many of the genes encoding the virulence factors for Staphylococcus aureus are controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). This regulation may be affected by the environment in which the organisms are grown. In the majority of ecosystems, bacteria grow attached to surfaces and form biofilms. We used S. aureus strains containing mutations inactivating agr and sar to determine whether the presence of these genes influences the attachment of the bacterium to a surface. We also used strains harbouring reporter constructs of the agr and sar operons to determine their expression in biofilms. The attachment study results showed that the sarA mutant strain adhered better to glass than did the agrA mutant or the wild type. There was an increased adherence to fibronectin-coated glass for all three strains compared to glass. Thus, these adhesion studies demonstrate that agr and sar have pleiotrophic effects on the surface expression of molecules responsible for binding to different substrata. In the biofilms higher numbers of bacteria and the greatest expression were observed at the base, but there were no observable differences between the reporter constructs. Expression of the agr and sar reporter fusions was significantly higher in the deepest layers of the biofilms where the greatest numbers of bacteria were also observed, perhaps as one might expect for genes that are regulated in a cell density dependent fashion.

Bacterial Adhesion↗
PubMed2001

Cloning and expression of the Actinobacillus actinomycetemcomitans thioredoxin (trx) gene and assessment of cytokine inhibitory activity.

Thioredoxin is a ubiquitous redox control and cell stress protein. Unexpectedly, in recent years, thioredoxins have been found to exhibit both cytokine and chemokine activities, and there is increasing evidence that this class of protein plays a role in the pathogenesis of inflammatory diseases. In spite of this evidence, it has been reported that the oral bacterium and periodontopathogen Actinobacillus actinomycetemcomitans secretes an immunosuppressive factor (termed suppressive factor 1 [SF1] [T. Kurita-Ochiai and K. Ochiai, Infect. Immun. 64:50-54, 1996]) whose N-terminal sequence, we have determined, identifies it as thioredoxin. We have cloned and expressed the gene encoding the thioredoxin of A. actinomycetemcomitans and have purified the protein to homogeneity. The A. actinomycetemcomitans trx gene has 52 and 76% identities, respectively, to the trx genes of Escherichia coli and Haemophilus influenzae. Enzymatic analysis revealed that the recombinant protein had the expected redox activity. When the recombinant thioredoxin was tested for its capacity to inhibit the production of cytokines by human peripheral blood mononuclear cells, it showed no significant inhibitory capacity. We therefore conclude that the thioredoxin of A. actinomycetemcomitans does not act as an immunosuppressive factor, at least with human leukocytes in cultures, and that the identity of SF1 remains to be elucidated.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Identification of the exported proteins of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans by using alkaline phosphatase fusions.

A phoA fusion library of Actinobacillus actinomycetemcomitans genomic DNA has been screened to identify genes encoding exported and secreted proteins. A total of 8,000 colonies were screened, and 80 positive colonies were detected. From these, 48 genes were identified with (i) more than half having homology to known or hypothetical Haemophilus influenzae genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. The proteins encoded by these genes may, by virtue of their presence on the cell surface, be novel virulence determinants.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria.

Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

Adhesins, Bacterial↗
PubMed2001

Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis.

It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.

Actinobacillus Infections↗
PubMed2000

Prevalence of obesity and diabetes in patients with cryptogenic cirrhosis: a case-control study.

It has recently been suggested that nonalcoholic steatohepatitis (NASH) is an under-recognized cause of cryptogenic cirrhosis (CC) on the basis of higher prevalence of obesity and type II diabetes among these patients. To test this hypothesis, we studied 65 consecutive patients with advanced cirrhosis (Child-Pugh Score >/= 7) of undetermined etiology (CC) from our active waiting list for liver transplantation in January 1993, 1996, and 1999. For each patient, we selected 2 age- and sex-matched controls from the corresponding lists. The prevalence of obesity (defined as body mass index [BMI] >/= 30) and diabetes were compared between the groups. Sixteen patients (and their 32 controls) with CC were excluded as further review of records suggested other possible etiologies. Thus, the final analysis included 49 patients and 98 controls. The etiology of cirrhosis in the control group was alcohol in 16.3%, chronic viral hepatitis in 30.6%, autoimmune hepatitis in 8.2%, and primary biliary cirrhosis (PBC) or primary sclerosing cholangitis in 35.7%. The prevalence of obesity (55% vs. 24%) and type II diabetes (47% vs. 22%) was significantly higher in patients with CC compared with controls. Twenty-three percent of patients with CC had both obesity and diabetes compared with 5% among controls (P =.002). There was no difference in the prevalence of hypercholesterolemia (serum cholesterol > 200 mg/dL) between the groups. In conclusion, patients with advanced CC are more likely to be obese and diabetic compared with age- and sex-matched patients with advanced cirrhosis. This supports the hypothesis that NASH may be an etiological factor in some of the patients with CC.

Adult↗
PubMed2000

Identification of the secreted macromolecular immunogens of Staphylococcus aureus by analysis of serum.

The ability of sera to recognise secreted macromolecules of Staphylococcus aureus was examined by ELISA and Western immunoblotting. Individual secreted proteins were also studied using both human sera and sera from rabbits immunised with secreted macromolecules. Patients sera showed a wide range of IgG antibody titres to secreted macromolecules and whole bacteria. Controls showed a significantly lower IgG response. Western immunoblotting revealed that a significant number of secreted proteins were recognised by circulating IgG antibodies. Surprisingly, both the sera from controls and from patients recognised similar macromolecules including a number of potential virulence factors. The major difference was in the IgG binding to a 16-kDa component, which was recognised by the majority of the sera from infected individuals, but only by a small number of sera from healthy controls. The higher incidence of antibodies recognising the 16 kDa component may be related to our earlier finding that the major bone resorbing component of S. aureus is a heterodimeric protein containing a 16-kDa subunit, the activity of which could be blocked by sera.

Adult↗
PubMed2000

Identification of a novel gene cluster encoding staphylococcal exotoxin-like proteins: characterization of the prototypic gene and its protein product, SET1.

We report the discovery of a novel genetic locus within Staphylococcus aureus that encodes a cluster of at least five exotoxin-like proteins. Designated the staphylococcal exotoxin-like genes 1 to 5 (set1 to set5), these open reading frames have between 38 and 53% homology to each other. All five proteins contain consensus sequences that are found in staphylococcal and streptococcal exotoxins and toxic shock syndrome toxin 1 (TSST-1). However, the SETs have only limited overall sequence homology to the enterotoxins and TSST-1 and thus represent a novel family of exotoxin-like proteins. The prototypic gene in this cluster, set1, has been cloned and expressed. Recombinant SET1 stimulated the production of interleukin-1beta, interleukin-6, and tumor necrosis factor alpha by human peripheral blood mononuclear cells. PCR analysis revealed that set1 was distributed among other strains of S. aureus but not in the other staphylococcal species examined. Sequence analysis of the set1 genes from different strains revealed at least three allelic variants. The protein products of these allelic variants displayed a 100-fold difference in their cytokine-inducing potency. The distribution of allelic variants of the set genes among strains of S. aureus may contribute to differences in the pathogenic potential of this bacterium.

Amino Acid Sequence↗
PubMed2000

Rapid screening for putative exported proteins from Staphylococcus aureus using alkaline phosphatase as a reporter molecule.

Staphylococcus aureus causes a wide range of infections in humans, ranging from superficial skin infections to the more serious toxin-mediated diseases such as toxic shock syndrome. Owing to the increasing resistance of this bacterium to a wide range of antibiotics, the need to determine the virulence factors involved in infection is becoming more important as these molecules are potential therapeutic targets. In this study, we have screened for putative exported proteins from S. aureus on the basis that these proteins are likely to be the first point of contact between the bacterium and host during infection. We have constructed gene fusions between S. aureus DNA and a truncated version of the Escherichia coli phoA gene, and we report on the characterization of the recombinants exhibiting alkaline phosphatase activity. As well as known S. aureus proteins, we have identified a number of putative open reading frames that encode proteins similar to those from nonstaphylococcal species and also unique proteins that do not have any homologues on the current databases.

Alkaline Phosphatase↗
PubMed1999

Molecular chaperones stimulate bone resorption.

Molecular chaperones, also known as heat shock proteins (hsp), are intracellular proteins found in all cells that catalyze protein folding. We have discovered that one class of bacterial molecular chaperone, the chaperonins, are potent inducers of bone resorption. To address the question of whether the osteolytic activity of the chaperonins was unique to this protein class, or was a common attribute of molecular chaperones generally, we have examined a number of bacterial and mammalian molecular chaperones for activity in the murine calvarial bone resorption assay. All the Escherichia coli molecular chaperones (groEL, groES, and dnaK) were active. The osteolytic activity of groEL was inhibited by indomethacin and the natural antagonist of interleukin-1 receptor antagonist (IL-1ra) but was unaffected by neutralization of tumor necrosis factor (TNF) or inhibition of 5-lipoxygenase. Mammalian molecular chaperones of molecular mass 27, 47, 70, and 90 kDa were also tested and, with the exception of the 47 kDa protein, all showed activity in the murine calvarial assay. Molecular chaperones appear, therefore, to have the capacity to modulate the cellular processes in bone explant cultures, resulting in resorption of the calcified matrix. The possibility that these proteins could play a role in the normal or pathological remodeling of bone is discussed.

Animals↗