Expression of recombinant heat-shock protein 70 of MCAN/IR/96/LON-49, a tool for diagnosis and future vaccine research

Heat shock protein 70 [HSP70] is present in all organisms studied so far, and is a major immunogen in infections caused by pathogens including Leishmania spp. The aim of this study was to clone and express HSP70 from L. infantum strain MCAN/IR/96/LON-49 and evaluate antibody response against HSP70 in visceral leishmaniasis [VL]. The L. infantum HSP70 gene segment was amplified by specific primers. It was cloned into pTZ57R vector and subcloned into pET32a [+] expression vector. The new construct was transformed in the E.coli Rosetta strain, and HSP70 protein was expressed in the presence of 1 mM IPTG and purified using a Hi- Trap chelating column. Antibody responses against HSP70 were determined by ELISA in 37 patients with visceral leishmaniasis and 63 healthy controls. Expression of HSP70 protein was confirmed using SDS-PAGE electrophoresis and dot blot with an anti-His tag antibody. There was no difference between the sequence of nucleotides of the HSP70 gene in the present study and other reported sequences. The ELISA results indicated that the sera of 81.1% [30/37] of the patients and 6.3% [5/63] of controls reacted to L. infantum HSP70. The conservative nature of the HSP70 molecule is an advantage in vaccine studies, because of minor differences [6%] between the nucleotide sequences and consequently the similarity in amino acid sequences in various strains of L. infantum. It could therefore be used in vaccine research against leishmaniasis and also as a tool for serodiagnosis

Interleukin-10 gene polymorphisms and susceptibility to brucellosis in Iranian patients

Interleukin-10 [IL-10] is a Th2-type cytokine that inhibits macrophage activation. It is known that production of IL-10 is affected by its gene promoter poiymorphisms. To investigate the relationship between IL- 10 gene promoter polymorphisms and susceptibility to brucellosis. One hundred and ninety patients with brucellosis and 81 healthy animal husbandmen who owned infected animals and consumed their contaminated dairy products were included in this study. All individuals were genotyped for three bi-allelic IL-b gene promoter polymorphisms at positions -1082[G/A], -819[T/C], and -592[A/C] using polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP]. The distribution of C alleles at positions -592 and -819 of IL-10 were significantly higher in patients than in the healthy animal husbandmen [p = 0.034 and p = 0.0086, respectively]. IL-10 ATA single and double haplotypes were significantly higher in controls, compared to the patients [p = 0.0278 and p = 0.013, respectively]. According to the results higher frequency of C alleles at positions -592 and -819 of IL- 10 in patients may be considered as genetic factors for susceptibility to brucellosis

Polymorphism in the first intron of interferon gamma gene [+874T-A] in Iranian patients with brucellosis

Brucella is a gram-negative bacterium, causing acute and chronic infection in humans and animals. Cell-mediated immunity is the main protective immune response against Brucella spp. Activation of macrophages by IFN-gamma and generation of reactive oxygen intermediates and nitric oxide are the main immunologic mechanisms responsible for control of Brucella infection. To investigate the correlation between IFN-gamma gene polymorphism and brucellosis. 195 patients with brucellosis, 186 healthy patients' family members and 82 healthy farmers who kept infected animals and consumed their contaminated dairy products were selected to take part in the study. IFN-gamma genotyping at position +874 [T to A] was carried out by allele specific polymerase chain reaction [AS-PCR] method. The frequency of AT and TT genotypes significantly increased in farmers compared to patients with rucellosis [P=0.03] while there was no significant difference in genotype distribution between patients and their healthy family members. IFN-gamma [+874] AA genotype is probably a genetic factor that contributes to the susceptibility of the individuals to brucellosis