Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection.

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.

Effect of adjuvants on immune response and protective immunity elicited by recombinant Hsp60 (GroEL) of Salmonella typhi against S. typhi infection.

Heat shock proteins (Hsps) have been reported to be dominant antigens for the host immune response to various pathogens and thus, have great potential for use in vaccination. In the present study, we evaluated the immunogenicity and protective efficacy of GroEL of Salmonella enterica serovar Typhi against lethal infection by S. typhi Ty2 in mice with or without adjuvants. Anti GroEL-IgG titers were significantly higher in mice immunized with either GroEL-alone or in combination with alum/Complete Freund's adjuvant (CFA) as compared to the control. Analysis of antibody isotypes suggested predominance of Th2 type immune response in GroEL + alum immunized animals as revealed by higher IgG1/IgG2a ratio. Whereas, immunization of animals with GroEL + CFA or GroEL-alone shifted the immune response toward Th1 phenotype. Mice immunized with GroEL with or without adjuvants, showed a significant increase in lymphocyte proliferation and cytokine levels. The animals immunized with GroEL + CFA or GroEL-alone showed higher IFN-gamma and IL-2 levels than alum group, indicating Th1 response whereas IL-4 levels (Th2 response) were found to be highest in alum group as compared to other two immunized groups. Immunization of mice with GroEL-alone, GroEL + alum, and GroEL + CFA provided 70, 50 and 80% protection, respectively, against lethal challenge by S. typhi in mice. The differences in the percentage protection among various groups were attributed to the differences in the immune responses generated by respective immunizations. The present study shows that GroEL forms an ideal candidate molecule to develop a recombinant protein based vaccine against human typhoid.

Sub-chronic oral toxicity study in Sprague-Dawley rats with hypoxia mimetic cobalt chloride towards the development of promising neutraceutical for oxygen deprivation.

The present study evaluates the toxicity from sub-chronic administration of CoCl(2) (12.5mg cobalt kg(-1) BW for 7 days) to male Sprague-Dawley rats in view of the beneficial effects of CoCl(2) in animals and for developing efficacious therapeutic regimen in humans. 32 rats weighing 200+/-25 g were used for all experiments. Blood was collected for hematological and biochemical analysis and various organs were dissected after perfusion of animals under anesthesia for other analyses. Mean feed consumption and feed conversion efficiency values were comparable across all study groups; however, hematological analysis depicted a significant increase in hemoglobin, hematocrit and RBC in the entire cobalt-supplemented groups, which are a component of its beneficial effect. There was a significant increase in monocytes, granulocytes and WBC after 1 and 24h, which were comparable with control after 7 days. Other biochemical analyses also showed no change with respect to control. Though the metal content increased significantly in liver initially (1 and 24h) after treatment, it was equivalent to control after 7 days. Moreover, histopathological analysis revealed no evidence of changes that could be attributed to cobalt pretreatment. It is therefore reasonable to conclude that the present study supports further use of the present dose of CoCl(2), which was found to be nontoxic.

Hypoxic preconditioning with cobalt attenuates hypobaric hypoxia-induced oxidative damage in rat lungs.

Shukla, Dhananjay, Saurabh Saxena, Purushotman Jayamurthy, Mustoori Sairam, Mrinalini, Singh, Swatantra Kumar Jain, Anju Bansal, and Govindaswamy Ilavazaghan. High Alt. Med. Biol. 10:57-69, 2009.-Hypoxic preco759nditioning (HPC) provides robust protection against injury from subsequent prolonged hypobaric hypoxia, which is a characteristic of high altitude and is known to induce oxidative injury in lung by increasing the generation of reactive oxygen species (ROS) and decreasing the effectiveness of the antioxidant defense system. We hypothesize that HPC with cobalt might protect the lung from subsequent hypobaric hypoxia-induced lung injury. HPC with cobalt can be achieved by oral feeding of CoCl(2) (12.5 mg kg(-1)) in rats for 7 days. Nonpreconditioned rats responded to hypobaric hypoxia (7619 m) by increased reactive oxygen species (ROS) generation and a decreased GSH/GSSG ratio. They also showed a marked increase in lipid peroxidation, heat-shock proteins (HSP32, HSP70), metallothionins (MT), levels of inflammatory cytokines (TNF-alpha, IFN-gamma, MCP-1), and SOD, GPx, and GST enzyme activity. In contrast, rats preconditioned with cobalt were far less impaired by severe hypobaric hypoxia, as observed by decreased ROS generation, lipid peroxidation, and inflammatory cytokine release and an inceased GSH/GSSG ratio. Increased expression of antioxidative proeins Nrf-1, HSP-32, and MT was also observed in cobalt- preconditioned animals. A marked increase in the protein expression and DNA binding activity of hypoxia-inducible transcriptional factor (HIF-1alpha) and its regulated genes, such as erythropoietin (EPO) and glucose transporter-1 (glut-1), was observed after HPC with cobalt. We conclude that HPC with cobalt enhances antioxidant status in the lung and protects from subsequent hypobaric hypoxia-induced oxidative stress.

Neuroprotective effect of cobalt chloride on hypobaric hypoxia-induced oxidative stress.

Hypobaric hypoxia, characteristic of high altitude is known to increase the formation of reactive oxygen and nitrogen species (RONS), and decrease effectiveness of antioxidant enzymes. RONS are involved and may even play a causative role in high altitude related ailments. Brain is highly susceptible to hypoxic stress and is involved in physiological responses that follow. Exposure of rats to hypobaric hypoxia (7619 m) resulted in increased oxidation of lipids and proteins due to increased RONS and decreased reduced to oxidized glutathione (GSH/GSSG) ratio. Further, there was a significant increase in superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) levels. Increase in heme oxygenase 1 (HO-1) and heat shock protein 70 (HSP70) was also noticed along with metallothionein (MT) II and III. Administration of cobalt appreciably attenuated the RONS generation, oxidation of lipids and proteins and maintained GSH/GSSH ratio similar to that of control cells via induction of HO-1 and MT offering efficient neuroprotection. It can be concluded that cobalt reduces hypoxia oxidative stress by maintaining higher cellular HO-1 and MT levels via hypoxia inducible factor 1alpha (HIF-1alpha) signaling mechanisms. These findings provide a basis for possible use of cobalt for prevention of hypoxia-induced oxidative stress.

Immunomodulatory effects of Premna tomentosa (L. Verbenaceae) extract in J 779 macrophage cell cultures under chromate (VI)-induced immunosuppression.

OBJECTIVE: In the present study, the immunomodulatory effects of Premna tomentosa extract against chromate (VI)-induced toxicity was assessed in J 779 macrophage cell line. DESIGN: The cells were analyzed for cytotoxicity, phagocytosis, oxidant burst, antioxidant status, and cell proliferation. RESULT: Chromate treatment resulted in a significant increase in cytotoxicity and free radical production. Furthermore, there is a significant decrease in reduced glutathione (GSH) levels and glutathione peroxidase activity (GPx). There was an appreciable decrease in cell proliferation and phagocytosis by macrophages in the presence of chromate. However, pretreatment of the cells with P. tomentosa extract (500 microg concentration), 30 minutes prior to chromate (VI) treatment resulted in a significant inhibition of chromate-induced cytotoxicity and reactive oxygen species production. The extract also restored the antioxidant status, cell proliferation, and phagocytosis similar to that of control cells. CONCLUSION: The results confirm the cytoprotective and immunomodulatory effects of the leaves of P. tomentosa and its possible usage in immunosuppressed conditions.