Combination of flow cytometry and qPCR to study the immune response of american cutaneous leishmaniasis patients.

American Cutaneous leishmaniasis (ACL) is a public health problem. The immunological response is mainly dependent on T cell cytokine responses and might influence disease presentation, susceptibility and development. The understanding of the host immune response role in the control and in the pathology of leishmaniasis is relevant and has implications on diagnosis, follow-up and vaccine development. In this study, the differences in the immune response and T cell profile of patients before treatment was investigated through flow cytometry and real time PCR in peripheral blood mononuclear cells after different antigenic stimulations. Among the main findings are the significant presence of TNF and IFN-γ gene expression after 24 h of in vitro stimulation, and 48 h later the presence of CD4+ T and CD8+ T cells producing IL-10 and IL-4. This may be due to the differences in cytokine release over time and the presence of cells other than lymphocytes influencing the mRNA transcript detection. Evaluation of the immune response of individuals with leishmaniasis or other diseases should associate different technologies and times points for a clear and more reliable assessment of the immune response. This would help in the design of vaccine strategies/immunotherapies.

Cytokines and NO in American tegumentary leishmaniasis patients: profiles in active disease, after therapy and in self-healed individuals.

Studies suggest the influence of immune response on the successful treatment of American tegumentary leishmaniasis (ATL), and indicate the existence of protective immunity in self-healed patients. Thus, the aim of this work was to quantify interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL-) 10, IL-17, IL-22 and nitric oxide (NO) in culture supernatants of PBMC from patients with active disease (AD), after treatment (AT), and from self-healed (SH) and healthy subjects (CT), in response to Leishmania (Viannia) braziliensis insoluble antigen (AgIns). All groups of patients produced IFN-γ, indicating a predominant proinflammatory profile. AD and AT patients presented TNF-α levels, with a slight increase after therapy, whereas it was weakly quantified in SH. Interestingly, NO secretion was significant in these individuals, whereas IL-17 appeared in low levels and seems to be regulated by NO. Although IL-22 was detected in AD, its role is still questionable. The presence of IL-10 in all groups of patients suggests that the cytokine plays distinct roles in the disease. These results indicate that specific cellular immunity takes part against Leishmania, but with some similarities between the different clinical states herein described; these mediators seem to be necessary for the cure to occur.

Cellular immune response evaluation of cutaneous leishmaniasis patients cells stimulated with Leishmania (Viannia) braziliensis antigenic fractions before and after clinical cure.

American cutaneous leishmaniasis (ACL) is a disease where susceptibility or resistance is dependent on T cell response. This is characterized by an increased in CD4⁺ T cells, capable of inducing opposite disease profiles, and CD8⁺ T cells, that are related to immuno protection. We characterized T lymphocytes from patients before and after treatment, patients that spontaneously healed and controls, also evaluating their production of IL-10, IL-4, TNF-α and IFN-γ, after stimulation with soluble/insoluble antigenic fractions of Leishmania (Viannia) braziliensis. We observed the production of suppressive cytokines in the early phase of leishmaniasis with significant presence CD4⁺ T cells, suggesting their connection with disease progression. After healing, the immune pattern observed was a type 1 response, what seems to be associated with cure and/or protection in the ACL. The results also showed that both fractions induced a specific immune response, contributing to the search for relevant antigens in this disease.

Comparison of flow cytometry and indirect immunofluorescence assay in the diagnosis and cure criterion after therapy of American tegumentary leishmaniasis by anti-live Leishmania (Viannia) braziliensis immunoglobulin G.

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve)=0.8 (95% CI=0.64-0.89) and AUC=0.90 (95% CI=0.75-0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL.