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Article in Chinese | WPRIM | ID: wpr-942823


Objective: To evaluate the immunostimulatory potential of cross-reactive molecule heat shock protein 60 (HSP60) of filarial parasite Brugia malayi and Leishmania donovani. Methods: HSP60 of Brugia malayi (BmHSP60) was amplified using gene-specific primer, cloned in pTriEx4 vector, expressed in BL21-DE3 cells, and recombinant HSP60 (rHSP60) of 65 kDa was purified by affinity chromatography using Ni-NTA column. The recombinant protein was desalted by the dialysis membrane, and the presence of endotoxin level was determined by Limulus amebocyte lysate assay. The recombinant protein was tested for cell proliferation, nitric oxide release, expression of Th1 and Th2 cytokines, and transcription factors (STATs) in vitro using murine macrophage cell line (J774A.1). Results: Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential. rBmHSP60 exposure upregulated the expression of iNOS, STAT1, STAT4, Th1 cytokines (IFN-γ, TNF-α, IL-12), and nitric oxide release. In addition, no remarkable change was observed in the expression of IL-6, IL-10, and STAT3 in macrophage cell line J774A.1. The ELISA analysis showed the levels of IFN-γ, TNF-α, and IL-12 were upregulated while IL-10 level was downregulated, revealing that BmHSP60 triggered a Th1 immune response. Conclusions: Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses, and can be used as an immunoprophylactic agent against leishmaniasis. Furthermore, in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.

Article in English | WPRIM | ID: wpr-819553


OBJECTIVE@#To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.@*METHODS@#The human macrophage/monocyte cell line THP-1, the mouse macrophage cell line RAW 264.7 and naive peritoneal macrophages (PM) from the rodent host Mastomys coucha (M. coucha) were incubated at 37 °C in 5% CO(2) atmosphere with extracts of microfilariae (Mf), third stage infective larvae (L(3)) and adult worms (Ad) of Brugia malayi. After 48 hr post exposure, IL-1β, IL-6, TNF-α, IL-10 and nitric oxide (NO) in cell-free supernatants were estimated.@*RESULTS@#Extracts of all the life stages of the parasite were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines in both the cell lines and peritoneal macrophages of M. coucha. Mf was the strongest stimulator of pro-inflammatory cytokines followed by L(3) and Ad; however, Ad was a strong stimulator of IL-10 release. Mf was found to have potential to modulate LPS-induced NO release in RAW cells. Ad-induced NO release was concentration dependent with maximum at 20 μg/mL in both RAW and PMs.@*CONCLUSIONS@#The results show that parasites at all life stages were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines and NO release from macrophages of susceptible host M. coucha, human and mouse macrophage cell lines. Mf can suppress the LPS-induced NO release in RAW cells. The findings also show that the two cell lines may provide a convenient in vitro system for assaying parasite-induced inflammatory mediator release.

Animals , Brugia malayi , Allergy and Immunology , Cells, Cultured , Culture Media , Chemistry , Female , Humans , Inflammation Mediators , Metabolism , Interleukin-10 , Metabolism , Interleukin-1beta , Metabolism , Interleukin-6 , Metabolism , Macrophages, Peritoneal , Allergy and Immunology , Parasitology , Male , Mice , Murinae , Allergy and Immunology , Parasitology , Tumor Necrosis Factor-alpha , Metabolism