ABSTRACT
Exposure to high altitude results in hypobaric hypoxia which is considered as an acute physiological stress and often leads to high altitude maladies such as high altitude pulmonary edema (HAPE) and high altitude cerebral edema (HACE). The best way to prevent high altitude injuries is hypoxic preconditioning which has potential clinical usefulness and can be mimicked by cobalt chloride. Preconditioning with cobalt has been reported to provide protection in various tissues against ischemic injury. However, the effect of preconditioning with cobalt against high altitude induced pulmonary edema has not been investigated in vivo. Therefore, in the present study, rats pretreated with saline or cobalt (12.5mg/kg body weight) for 7days were exposed to hypobaric hypoxia of 9142m for 5h at 24°C. Formation of pulmonary edema was assessed by measuring transvascular leakage of sodium fluorescein dye and lung water content. Total protein content, albumin content, vascular endothelial growth factor (VEGF) and cytokine levels were measured in bronchoalveolar lavage fluid. Expression of HO-1, MT, NF-κB DNA binding activity and lung tissue pathology were evaluated to determine the effect of preconditioning on HAPE. Hypobaric hypoxia induced increase in transvascular leakage of sodium fluorescein dye, lung water content, lavage total protein, albumin, VEGF levels, pro-inflammatory cytokine levels, tissue expression of cell adhesion molecules and NF-κB DNA binding activity were reduced significantly after hypoxic preconditioning with cobalt. Expression of anti-inflammatory protein HO-1, MT, TGF-ß and IL-6 were increased after hypoxic preconditioning. These data suggest that hypoxic preconditioning with cobalt has protective effect against HAPE.
Subject(s)
Altitude , Cobalt/pharmacology , Hypoxia/complications , Pulmonary Edema/etiology , Pulmonary Edema/prevention & control , Albumins/metabolism , Animals , Bronchoalveolar Lavage Fluid , Capillary Permeability/drug effects , Cell Adhesion Molecules/metabolism , Cytokines/metabolism , DNA/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Heme Oxygenase-1/metabolism , Hypoxia/metabolism , Hypoxia/pathology , Hypoxia/physiopathology , Lung/blood supply , Lung/drug effects , Male , Metallothionein/metabolism , Rats , Rats, Sprague-Dawley , Transcription Factor RelA/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The present study was carried out to evaluate the immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae, by expressing full length GroEL in heterologous host Escherichia coli BL21(DE3). PCR-amplified groEL was ligated in pQE 30 expression vector and subsequently transformed in E. coli DH5alpha strains. Cloning of groEL was confirmed by double digestion, followed by DNA sequencing. The His-tag containing recombinant GroEL was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of GroEL, the mice were immunized by injecting 40 microg GroEL protein per mouse intraperitoneally. The results showed a significant increase in antibody titre and lymphocyte proliferation in animals immunized with GroEL as compared with control. Further, there was an appreciable increase in interleukin-2 (IL-2) and IL-4 production in lymphocytes isolated from immunized mice as compared with control. To determine the efficacy of GroEL in eliciting protection, the mice were challenged with the lethal dose of S. pneumoniae A66 type 3 capsular strain intranasally after the seventh day of the last immunization. In the GroEL-immunized mice the onset of death was insignificantly delayed and all the mice died by the seventh day postinfection.
Subject(s)
Chaperonin 60/immunology , Pneumococcal Infections/immunology , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/therapeutic use , Streptococcus pneumoniae/immunology , Animals , Antibodies, Bacterial/blood , Chaperonin 60/biosynthesis , Chaperonin 60/genetics , Cloning, Molecular , Female , Immunoglobulin G/blood , Interleukin-2/biosynthesis , Interleukin-4/biosynthesis , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Pneumococcal Infections/metabolism , Pneumococcal Vaccines/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Streptococcus pneumoniae/genetics , Vaccines, Subunit/immunology , Vaccines, Subunit/therapeutic useABSTRACT
Heat shock proteins (Hsps) represent dominant antigens in numerous microbial infections, suggesting a potential use of pathogen-derived Hsps for vaccination. The present study evaluates the immunogenicity and protective efficacy of groEL (Hsp60) of Salmonella enterica serovar Typhi against lethal challenge by S. Typhi Ty2 and Salmonella enterica serovar Typhimurium in mice. The groEL gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with groEL resulted in a significant increase in antibody titers. Antibody isotyping revealed that groEL immunization induces both IgG1 and IgG2a antibodies. There was a significant increase in lymphocyte proliferation, interleukin-4 and interferon-gamma levels in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant groEL protein with or without adjuvant conferred 70-90% protection against lethal infections either by S. Typhi Ty2 or S. Typhimurium. Passive immunization with anti-groEL sera also protected 50% mice against lethal infection.
