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Inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production was demonstrated in J774-G8 macrophages infected with Leishmania (L.) amazonensis promastigotes. The downmodulation of NO production observed in infected and LPS-stimulated J774-G8 cells correlated with a reduction in inducible nitric oxide synthase (iNOS) activity. Reduction in iNOS activity was not paralleled by decreased iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis. Supplementation with L-arginine or tetrahydrobiopterin did not increase NO production, suggesting that inhibition is not due to an insufficiency of substrate or co-factor. Treatment with anti-IL-10, anti-IL-4 or anti-TGF-beta neutralizing antibodies also failed to increase NO production, indicating that these cytokines are not involved in the observed parasite-induced inhibition of NO synthesis. However, treatment of the cultures with IFN-gamma resulted in a marked increase in NO production by infected LPS-stimulated cells. These results show that although L.(L.) amazonensis infection inhibits iNOS activity and NO production by J774-G8 cells, activation by IFN-gamma is capable of overriding the suppression of NO synthesis.

SUMMARYIsolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNg KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNg KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNg KO mice to improve the possibility to isolate New World Leishmania species.

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