The Liver Plays a Major Role in Clearance and Destruction of Blood Trypomastigotes in Trypanosoma cruzi Chronically Infected Mice

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5×106 trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1+, CD8+ and CD4+ cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4+ and CD8+ cells were activated, increased frequencies of CD69+CD8+, CD69+CD4+ and CD25+CD122+CD4+ cells were observed at 24 and 48 h after challenge, and of CD25−CD122+CD4+ cells at 48 h. The major role of CD4+ cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-γ-producing CD4+ cells 24 h after challenge. In contrast, liver CD8+ cells produced little IFN-γ, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge

Genes from Chagas Susceptibility Loci That Are Differentially Expressed in T. cruzi-Resistant Mice Are Candidates Accounting for Impaired Immunity

Variation between inbred mice of susceptibility to experimental Trypanosoma cruzi infection has frequently been described, but the immunogenetic background is poorly understood. The outcross of the susceptible parental mouse strains C57BL/6 (B6) and DBA/2 (D2), B6D2F1 (F1) mice, is highly resistant to this parasite. In the present study we show by quantitative PCR that the increase of tissue parasitism during the early phase of infection is comparable up to day 11 between susceptible B6 and resistant F1 mice. A reduction of splenic parasite burdens occurs thereafter in both strains but is comparatively retarded in susceptible mice. Splenic microarchitecture is progressively disrupted with loss of follicles and B lymphocytes in B6 mice, but not in F1 mice. By genotyping of additional backcross offspring we corroborate our earlier findings that susceptibility maps to three loci on Chromosomes 5, 13 and 17. Analysis of gene expression of spleen cells from infected B6 and F1 mice with microarrays identifies about 0.3% of transcripts that are differentially expressed. Assuming that differential susceptibility is mediated by altered gene expression, we propose that the following differentially expressed transcripts from these loci are strong candidates for the observed phenotypic variation: H2-Eα, H2-D1, Ng23, Msh5 and Tubb5 from Chromosome 17; and Cxcl11, Bmp2k and Spp1 from Chromosome 5. Our results indicate that innate mechanisms are not of primary relevance to resistance of F1 mice to T. cruzi infection, and that differential susceptibility to experimental infection with this protozoan pathogen is not paralleled by extensive variation of the transcriptome

The Trypanosoma cruzi vitamin C dependent peroxidase confers protection against oxidative stress but is not a determinant of virulence.

BACKGROUND: The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS: To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo. CONCLUSIONS/SIGNIFICANCE: TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design

BF Integrase Genes of HIV-1 Circulating in São Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naïve patients from the São Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naïve and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population