Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.

Production of nitric oxide by murine macrophages induced by lipophosphoglycan of Leishmania major

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is the surface glycoconjugate lipophosphoglycan (LPG). In addition, LPG is shown to be useful as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we made attempts to characterize functions of the culture supernatant, and membrane LPG isolated from metacyclic promastigotes of Leishmania major. The purification scheme included anion-exchange chromatography, hydrophobic interaction chromatography and cold methanol precipitation. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by SDS-PAGE and thin layer chromatography. The effect of mLPG and sLPG on nitric oxide (NO) production by murine macrophages cell line (J774.1A) was studied. Both sLPG and mLPG induced NO production in a dose dependent manner but sLPG induced significantly higher amount of NO than mLPG. Our results show that sLPG is able to promote NO production by murine macrophages.

Identification of novel Leishmania major antigens that elicit IgG2a response in resistant and susceptible mice

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.

Effect of immunomodulator pyrimethamine and cimetidine on immunosuppression induced by burn blister fluid

Despite recent advances in burn wound management, sepsis remains the main cause of death in patients resuscitated after major thermal injury. Increased susceptibility to infections has been related to severe suppression of the immune system. The aim of this study was to induce immune suppression with blister fluid injection, and to modulate immune response by use of cimetidine and pyrimethamine in animal model. Male Balb/c mice were injected with blister fluid intrapritoneally [ip]. Fluids were collected from parital-thickness burn blisters and then the delayed type hypersensitivity [DTH] to sheep red blood cell [SRBC] and the effects of different doses of immunomodulators [Cimetidine and Pyrimethamine] on this response were quantitated. A marked suppression of DTH was observed in mice injected with blister fluid. Pyrimethamine and Cimetidine at all three doses caused a significant enhancement of DTH response to SRBC compared with blister fluid injected in control group. This finding represents evidence of a host defense defect within the burn wound and also indicates the blister fluid exhibit immunosuppressor factor that can modulate with immunomadulatory drugs like cimetidine and pyrimethamine

Restorative effect of vitamin E supplemintation on hepatic lipid peroxidation and lipid profile changes induced by sublethal Gamma-radiation in BALB/c mice

In order to find a prophylactic supplementation for individuals who are at risk of exposure to ionizing radiation, we attempted to evaluate the effect of Vitamin E [Vit-E], a biological free radical scavenger, on restoration of hepatic lipid peroxidation [LPO] and lipid profile [LP] changes induced by sublethal gamma- radiation in BALB/c mice. The concentrations of cholesterol and phospholipid were determined in control and irradiated mice. Also, changes in lipid peroxidation and lipid profile were assessed by measuring the level of malonyldialdehyde, lipid hydroperoxides, and conjugated dienes. Our results showed that sublethal gamma -radiation caused significant changes in hepatic lipid peroxidation and lipid profile. However, Vit-E supplementation was able to restore the changes of lipid peroxidation and lipid profile in irradiated mice. We have concluded that the mice that received Vit-E supplementation were able to tolerate biomembrane damage provoked by 1.09 Gy for 3 days gamma -radiation. This supports the hypothesis that Vit-E may afford an efficient protection against ionizing radiation. However additional studies using higher doses of gamma -irradiation should be performed