In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195.

BACKGROUND: Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE: To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS: The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS: Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS: PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Oxygen supplementation increases the total work and muscle damage markers but reduces the inflammatory response in COPD patients.

INTRODUCTION: Oxygen supplementation (O2-Suppl) is recommended for pulmonary rehabilitation with higher exercise intensities. However, high-intensity exercise tends toward muscle damage and a greater inflammatory response. We aimed to investigate the effect of O2-Suppl during exercise test (EET) on CRP level and muscle damage (CPK, LDH, lactate) in non-hypoxemic COPD patients. METHODS: Eleven non-depleted patients with COPD (FEV1 65.5 ± 4.3 %) performed two EET (room-air or O2-Suppl-100 %), through a blind, randomized, and placebo-controlled crossover design. CPK, LDH and CRP were measured before, immediately after and 24 h after EET. RESULTS: Exercise time was higher with O2-Suppl (49.9 ± 37.3 %; p = 0.001) and increases in CPK and LDH were observed compared to basal values in the O2-Suppl (28.4UI/L and 28.3 UI/L). The O2-Suppl protocol resulted in a lower increase in CRP (92.1 ± 112.4 % vs. 400.1 ± 384.9 %; p = 0.003). CONCLUSIONS: O2-Suppl increases exercise-tolerance, resulting in increased muscle injury markers in COPD. However, oxygen supplementation attenuates the inflammatory response, even upon increased physical exercise.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Caracterização fenotípica e funcional das células T CD4+ e CD8+ Vß12 na resposta imune em paciente com Leishmaniose Cutânea localizada.

As leishmanioses constituem um complexo de doenças causada pelo protozoário intracelular, do gênero Leishmania, sendo a resposta imune celular essencial para controle, eliminação e proteção contra a infecção. A teoria clonal da imunidade celular propõe que as respostas imunológicas são estabelecidas através do aumento na frequência de clones específicos ao antígeno. Para avaliar a resposta das células T à infecção por Leishmania, investigamos, por citometria de fluxo, a expressão de cadeias Vß de receptores de células T (TCRs), estado de ativação, capacidade de adesão ao endotélio e potencial funcional de clones específico. Em um grupo de pacientes com Leishmaniose Cutânea Localizada (LCL), avaliamos diferentes subpopulações de células T através da expressão da região Vß, no sangue periférico e na biópsia da lesão. Utilizamos células mononucleares de sangue periférico (CMSPs), de pacientes LCL e controles saudáveis, nas quais avaliamos, ex vivo, a expressão de moléculas de ativação (CD25, CD69 e HLA-DR), adesão (LFA-1, VLA-4 e CD62L), co-estimulatória (CD27 e CD28) e marcadores de memória (CCR7, CD45RA, CD45RO). In vitro, as CMSPs de pacientes com LCL foram estimuladas com SLA para avaliar a expansão do clone Vß12 e a produção de citocinas intracelulares (IL-2, IFN-y e TNF). Nossos resultados mostram: (1) diminuição no sangue das células T CD4+ que expressam Vß2, Vß5, Vß12 e CD8+ Vß5, Vß12, Vß14 nos pacientes com LCL quando comparado com os controles saudáveis; (2) um perfil de ativação prévia de células T CD4+ Vß2, Vß5 e CD8+ Vß5, Vß14, com expressão mais elevada HLADR, enquanto que o clone Vß12 apresentou diferentes níveis de ativação; (3) uma correlação positiva entre as frequências das células T CD4+ e CD8 Vß12+ ativadas e o tamanho das lesões; (4) alta expressão de moléculas de adesão, indicando direcionamento das células T CD4+ e CD8+ expressando Vß12 para o local da lesão, confirmado através da (5) alta freqüência de células Vß12 CD62Llow no sangue dos pacientes quando comparado aos controles; (6) os pacientes apresentaram frequência mais elevada de células CD4+ Vß12 com fenótipo efetor (CD45RA+CCR7-; CCR7-CD45RO+CD28-; CD45RO-CD27-) enquanto as células CD8+ Vß12 apresentaram o fenótipo de memória efetora (CD45RA-CCR7-, CCR7-CD45RO-CD28-, CD45RO+CD27-), esse perfil apresenta correlação positiva entre a frequência de células no sangue, com o tamanho da lesão; (7) CMSPs de pacientes com LCL, estimuladas com SLA, apresentaram aumento na frequência das células T CD4+ e CD8+ expressando Vß12; (8) in vitro, as células T CD4+ Vß12 produziram predominantemente IFN-y, enquanto células CD8+ Vß12 produzem IFN-y e TNF; (9) Ambas as células Vß12 têm uma subpopulação dupla positiva para IFN-y e TNF. O Vß12 contribui para a resposta inflamatória através produção de IFN-γ e TNF, assim como para manutenção da lesão devido a presença das células T CD8+ de memória efetora com capacidade de produção simultânea de IFN-γ e TNF

Caracterização fenotípica e funcional das células T CD4+ e CD8+ VB12 na resposta imune em paciente com Leishmaniose Cutânea Localizada / Phenotypic and functional characterization of CD4 + and CD8 + VB12 T cells in the immune response in a patient with Localized Cutaneous Leishmaniasis

As leishmanioses constituem um complexo de doenças causada pelo protozoário intracelular, do gênero Leishmania, sendo a resposta imune celular essencial para controle, eliminação e proteção contra a infecção. A teoria clonal da imunidade celular propõe que as respostas imunológicas são estabelecidas através do aumento na frequência de clones específicos ao antígeno. Para avaliar a resposta das células T à infecção por Leishmania, investigamos, por citometria de fluxo, a expressão de cadeias Vβ de receptores de células T (TCRs), estado de ativação, capacidade de adesão ao endotélio e potencial funcional de clones específico. Em um grupo de pacientes com Leishmaniose Cutânea Localizada (LCL), avaliamos diferentes subpopulações de células T através da expressão da região Vβ, no sangue periférico e na biópsia da lesão. Utilizamos células mononucleares de sangue periférico (CMSPs), de pacientes LCL e controles saudáveis, nas quais avaliamos, ex vivo, a expressão de moléculas de ativação (CD25, CD69 e HLA-DR), adesão (LFA-1, VLA-4 e CD62L), co-estimulatória (CD27 e CD28)...

Leishmaniasis is a desease caused by infection with the Leishmania protozoan parasite. The cellular immune response is essential for controlling, eliminating and protection of the Leishmania infection. The clonal theory of cellular immunity proposes that immunological responses are established by increasing the frequency of antigen-specific clones. In order to measure the host T cell response to Leishmania infection, we have investigated by flow cytometry, the expression of Vβ chains of T-cell receptors (TCRs), activation state, adhesion to endothelium of capacity and functional potential of specific T. In a group of localized cutaneous leishmaniasis (LCL) patients, we evaluated different T cell subpopulations as identified by their Vβ region expression, in peripheral blood and biopsy. We used peripheral blood mononuclear cells (PBMCs), from CL patients and healthy volunteers, in which we evaluate, ex vivo, the expression of activation molecules (CD25, CD69 and HLA-DR), adhesion (LFA-1, VLA-4 and CD62L), co-stimulatory (CD27 and CD28)...

Avaliação de células T de memória imunológica na Leishmaniose Recidiva Cútis

As leishmanioses são doenças parasitárias causadas pelo protozoário do gênero Leishmania transmitida ao hospedeiro vertebrado pelo inseto vetor, do gênero Phlebotomus (Velho Mundo) ouLutzomiya (Novo Mundo). Apresentam amplo espectro de manifestações clínicas comprometendo pele, mucosa (Leishmaniose Cutânea- LC) e vísceras (Leishmaniose Visceral- LV).