Interaction between peripheral blood mononuclear cells and Trypanosoma cruzi-infected adipocytes: implications for treatment failure and induction of immunomodulatory mechanisms in adipose tissue.

Background/Introduction: Adipose tissue (AT) has been highlighted as a promising reservoir of infection for viruses, bacteria and parasites. Among them is Trypanosoma cruzi, which causes Chagas disease. The recommended treatment for the disease in Brazil is Benznidazole (BZ). However, its efficacy may vary according to the stage of the disease, geographical origin, age, immune background of the host and sensitivity of the strains to the drug. In this context, AT may act as an ally for the parasite survival and persistence in the host and a barrier for BZ action. Therefore, we investigated the immunomodulation of T. cruzi-infected human AT in the presence of peripheral blood mononuclear cells (PBMC) where BZ treatment was added. Methods: We performed indirect cultivation between T. cruzi-infected adipocytes, PBMC and the addition of BZ. After 72h of treatment, the supernatant was collected for cytokine, chemokine and adipokine assay. Infected adipocytes were removed to quantify T. cruzi DNA, and PBMC were removed for immunophenotyping. Results: Our findings showed elevated secretion of interleukin (IL)-6, IL-2 and monocyte chemoattractant protein-1 (MCP-1/CCL2) in the AT+PBMC condition compared to the other controls. In contrast, there was a decrease in tumor necrosis factor (TNF) and IL-8/CXCL-8 in the groups with AT. We also found high adipsin secretion in PBMC+AT+T compared to the treated condition (PBMC+AT+T+BZ). Likewise, the expression of CD80+ and HLA-DR+ in CD14+ cells decreased in the presence of T. cruzi. Discussion: Thus, our findings indicate that AT promotes up-regulation of inflammatory products such as IL-6, IL-2, and MCP-1/CCL2. However, adipogenic inducers may have triggered the downregulation of TNF and IL-8/CXCL8 through the peroxisome proliferator agonist gamma (PPAR-g) or receptor expression. On the other hand, the administration of BZ only managed to reduce inflammation in the microenvironment by decreasing adipsin in the infected culture conditions. Therefore, given the findings, we can see that AT is an ally of the parasite in evading the host's immune response and the pharmacological action of BZ.

Study of association of the rs2275913 IL-17A single nucleotide polymorphism and susceptibility to cutaneous leishmaniasis caused by Leishmania braziliensis.

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is the most spread clinical form of leishmaniasis in Brazil. However, only a few part of the people infected develop clinically perceptive disease, suggesting the influence of human genetic components in the CL pathogeny. The rs2275913 SNP is the nucleotide variant of the IL17A gene. The A allele is associated with a vast number of infectious and non-infectious diseases. Here, we investigated the association of the rs2275913 SNP (G/A) from IL-17A and two forms of susceptibility to CL in Brazil by case-control study. Furthermore, we evaluated the functional relevance of this SNP during the immune response of the host and analyzed its impact in the parasite elimination. Weak associations of A allele with susceptibility to L. braziliensis infection or to symptomatic CL were observed, and a tendency of A allele carriers to be more susceptible to infection and cutaneous disease. Functional analysis of the Th17 cell phenotypes revealed lower frequencies of CD4+ IL-17+ cells in samples of infected people with AA/AG genotypes. Furthermore, people carrying the A allele maintain higher parasite loads, reinforcing the genetic susceptibility findings. This study adds knowledge about the influence of a significant genetic variation on IL-17 promoter on CL pathogenesis, and may contribute to enhance the knowledge about the role of IL-17 in the L. braziliensis infections.

Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

BACKGROUND: Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. METHODS: qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. RESULTS: Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing and qPCR (p = 0.2566). In contrast, a highly significant difference was observed for qPCR and RFLP (p < 0.001). CONCLUSIONS: In this study, we demonstrated the potential use of qPCR as a tool for Leishmania species identification using two Tm ranges.