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

Experimental model of pulmonary allergic inflammation induced by latex in Balb/c

The prevalence of allergic airway diseases has dramatically increased in recent years all over the world. Murine models of allergic airway inflammation have provided helpful information about treatment and cellular and molecular mechanisms of the disease. Previous published works using murine models to investigate latex allergy did not introduce a complete characteristic eosinophilic allergic airway inflammation. Latex allergy is important due to serious health impacts and widespread use of its products. Thus, the aim of this study was to establish a new mouse model of latex allergic airway inflammation using aerosol inhalation. Initially, four groups of mice were injected intraperitoneally [IP] with 0, 10, 50, or 200 mg of latex extract and their serum anti-latex IgE titers were compared using ELISA to find out the optimum dose for IP injection. In the second stage, a standard protocol of inhalation was designed and three doses of latex extract solutions including 1%, 0.1%, and 0.01% were used to induce allergic airway inflammation. Characteristics of this model were shown by studying different parameters including bronchoalveolar lavage [BAL], cytokines [Interleukin-5 [IL-5] and interleukin-13 [IL-13]] and serum anti-latex IgE and IgG1 titers by ELISA, specific histologic changes in the lung and eosinophilia of the bone marrow, peripheral blood, BAL, and lung inflammatory foci. The aerosol inhalation of 1% latex allergens solution and pre-sensitization with 50 mg of latex in this study resulted in the development of characteristic allergic airway inflammation in BALB/c mice. These features included elevated allergen specific IgE and IgG1, peripheral blood, BAL and bone marrow eosinophilia and characteristic inflammatory response in lung with eosinophil infiltration. Elevated levels of IL-5 and IL-13 can be a sign of this type of inflammation. This paper describes a latex aerosol inhalational challenge model of eosinophilic airway inflammation in latex pre-sensitized BALB/c mice