Events associated with susceptibility to invasive Salmonella enterica serovar Typhi in BALB/c mice previously infected with Plasmodium berghei ANKA.

Numerous mechanisms have been proposed to explain why patients with malaria are more susceptible to bloodstream invasions by Salmonella spp., however there are still several unknown critical factors regarding the pathogenesis of coinfection. From a coinfection model, in which an S. enterica serovar Typhi (S_Typhi) was chosen to challenge mice that had been infected 24 h earlier with Plasmodium berghei ANKA (P.b_ANKA), we evaluated the influence of malaria on cytokine levels, the functional activity of femoral bone marrow-derived macrophages and neutrophils, and intestinal permeability. The cytokine profile over eight days of coinfection showed exacerbation in the cytokines MCP-1, IFNγ and TNFα in relation to the increase seen in animals with malaria. The cytokine profile was associated with a considerably reduced neutrophil and macrophage count and a prominent dysfunction, especially in ex vivo neutrophils in coinfected mice, though without bacterial modulation that could influence the invasion capacity of ex vivo S_Typhi obtained from liver macerate in non-phagocyte cells. Finally, irregularities in the integrity of intestinal tissue evidenced ruptures in the enterocyte layer, a presence of mononuclear leukocytes in the enterocyte layer, an increase of goblet cells in the enterocyte layer and a high volume of leukocyte infiltrate in the sub-mucosa were greatly increased in coinfected animals. Increases of mononuclear leukocytes in the enterocyte layer and volume of leukocyte infiltrate in the sub-mucosa were also seen in monoinfected animals with P. berghei ANKA. Our findings suggest malaria causes a disarrangement of intestinal homeostasis, exacerbation of proinflammatory cytokines and dysfunction in neutrophils that render the host susceptible to bacteremia by Salmonella spp.

A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry.

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.

A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.