Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment / Acumulación celular y efecto anti- Leishmania de la protoporfirina IX exógena y endógena después del tratamiento fotodinámico

Introduction: Photodynamic therapy (PDT) using 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) constitutes an interesting alternative for cutaneous leishmaniasis treatment. Objective: To evaluate the production of PpIXbased on the administration of ALA and MAL and the effect of ALA-PDTat cellular level on non-infected and infected THP-1 cells using Leishmania ( Viannia ) panamensis or Leishmania ( Leishmania ) infantum (syn Leishmania chagasi ) parasites. Materials and methods: Protoporphyrin IX (PpIX) production and mitochondrial colocalization were evaluated by confocal microscopy. Cell toxicities were evaluated after treatment with the compounds, followed by light irradiation (597-752 nm) at 2.5 J/cm 2 fluency using a colorimetric MTT assay for THP-1 cells and a standard microscopic analysis of parasites. Results were expressed as compound concentration activity against 50% of cells or parasites (CC 50 or IC 50 ). Results: ALA or MAL induced an endogenous PpIX with a red fluorescence localized mainly in the mitochondria inside human cells. ALA and MAL-PDT induced a similar range of toxicities on THP-1 cells (CC 50 0.16±0.01mM and 0.33±0.019 mM, respectively) without any apparent inhibition of intracellular parasites in the infected cells as compared to untreated controls. Exogenous PpIX-PDT was toxic to THP-1 cells (CC 50 0.00032±0.00002 mM), L. (L.) infantum (IC 50 0.003±0.0001 mM) and L. (V.) panamensis (IC 50 0.024±0.0001 mM) promastigotes. Conclusions: Despite the effectiveness of exogenous PpIX on promastigotes and the production of PpIX by human infected cells, treatment with ALA or MAL before irradiation was unable to completely destroy L. (L.) infantum or L. (V.) panamensis intracellular amastigotes.

Introducción. El tratamiento fotodinámico con ácido 5-aminolevulínico como inductor de la protoporfirina IX (ALA-PpIX) constituye una alternativa interesante en el tratamiento de la leishmaniasis cutánea. Objetivo. Evaluar la producción de protoporfirina IX (PpIX) a partir de la administración de ALA o MAL y el efecto de la PDT con ALA a nivel celular en células THP-1 no infectadas e infectadas con Leishmania ( Viannia ) panamensis o Leishmania ( Leishmania ) infantum (syn. Leishmania chagasi ). Materiales y métodos. La producción de protoporfirina IX y su ‘colocalización´ mitocondrial se evaluaron mediante microscopía ‘confocal´. Se evaluó la toxicidad celular después del tratamiento con los compuestos y la aplicación de irradiación de luz (597-752 nm) en una fluencia de 2,5 J/cm 2 mediante el empleo de la prueba colorimétrica con metil-tiazol-tetrazolio (MTT) en las células, y de métodos microscópicos estándar en los parásitos. Los resultados se expresaron como la concentración del compuesto activo en el 50 % de las células o parásitos (CC 50 o CI 50 ). Resultados. El ácido aminolevulínico o el metil-5-aminolevulinato indujeron la protoporfirina IX endógena en células humanas, y se observó fluorescencia de color rojo en las mitocondrias. La actividad del ácido aminolevulínico y del metil-5-aminolevulinato utilizados con terapia fotodinámica fue similar en las células THP-1 (CC 50 0,16±0,01 mM y 0,33±0,019 mM, respectivamente) y, aparentemente, no inhibió los parásitos en las células infectadas, en comparación con los controles. El tratamiento exógeno con protoporfirina IX y terapia fotodinámica fue tóxico para las células THP-1 (CC 50 0,00032 ±0,00002 mM) y para los promastigotes de L. (L .) infantum (IC 50 0,003±0,0001 mM) y L. ( V .) panamensis (CI 50 0,024±0,0001 mM). Conclusiones. A pesar de la ‘fotoactividad´ del tratamiento con protoporfirina IX en promastigotes y de su producción después del tratamiento con ácido aminolevulínico y metil-5-aminolevulinato en las células infectadas con Leishmania , no se observó daño en los amastigotes presentes en las células de L. ( L .) infantum o L . ( V .) panamensis .

In vitro efficacy of Coriandrum sativum, Lippia sidoides and Copaifera reticulata against Leishmania chagasi / Eficácia in vitro de Coriandrum sativum, Lippia sidoides e Copaifera reticulata sobre Leishmania chagasi

The increased incidence of visceral leishmaniasis (VL) in Brazil is due to a lack of effective disease control measures. In addition to that, no effective treatment exists for canine VL in response to synthetic drugs. Thus, the objective of this study was to evaluate the effect of the essential oils of Coriandrum sativum and Lippia sidoides, and oleoresin from Copaifera reticulata, on Leishmania chagasi promastigotes and amastigotes. We also examined the toxicity of these treatments on the murine monocyte cell line RAW 264.7. To determine the IC50 a MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on promastigotes, and an in situ ELISA assay was conducted on amastigotes. Here, we demonstrate that oleoresin from C. reticulata was effective against both promastigotes (IC50 of 7.88 µg.mL-1) and amastigotes (IC50 of 0.52 µg.mL-1), and neither of the two treatments differed significantly (p > 0.05) from pentamidine (IC50 of 2.149 µg.mL-1) and amphotericin B (IC50 of 9.754 µg.mL-1). Of the three plant oils tested, only oleoresin showed no toxicity toward monocyte, with 78.45% viability after treatment. Inhibition of promastigote and amastigote growth and the lack of cytotoxicity by C. reticulata demonstrate that oleoresin may be a viable option for analyzing the in vivo therapeutic effects of leishmanicidal plants.

O aumento na incidência da Leishmaníase Visceral (LV) no Brasil deve-se à ineficácia das medidas de controle da doença. Além disso, não há tratamento efetivo para LV canina com drogas sintéticas. Assim, o objetivo deste trabalho foi avaliar o efeito dos óleos essenciais de Coriandrum sativum e de Lippia sidoides e do óleo-resina de Copaiferareticulata sobre promastigotas e amastigotas de Leishmania chagasi e analisar o grau de toxicidade sobre células monocíticas murinas RAW 264.7. Para determinar a CI50 sobre promastigotas foi usado teste MTT (brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio) e sobre amastigotas foi realizado imunoensaio in situ pela técnica de ELISA. Os resultados obtidos comprovaram que o óleo-resina de C. reticulata foi o mais eficaz contra as formas promastigotas (CI50 de 7,88 µg.mL-1) e amastigotas (CI50 de 0,52 µg.mL-1) e em nenhum dos dois testes diferiu do controle pentamidina que obteve CI50 de 2,149 µg.mL-1, no teste sobre promastigotas, e anfotericina B que obteve CI50 de 9,754 µg.mL-1, nos testes com amastigotas (p > 0.05). Quanto à citotoxicidade apenas o óleo-resina não apresentou toxicidade com 78,45% de monócitos viáveis. Os resultados obtidos sobre promastigotas e amastigotas e a ausência de citotoxicidade do óleo-resina de C. reticulata evidenciam que este óleo-resina pode ser viável para a análise de seus efeitos terapêuticos em testes in vivo.

Efeitos da saliva de lutzomyia intermédia sobre monócitos humanos / Effects of the saliva of intermediate lutzomyia on human monocytes

A saliva dos flebotomíneos contém moléculas farmacológicas potentes e complexas que, quando inoculadas na pele do vertebrado, são capazes de modular os sistemas hemostático, inflamatório e imune do hospedeiro. No presente estudo, nós avaliamos os efeitos do conteúdo da glândula salivar de Lutzomyia intermedia, o vetor natural da Leishmania braziliensis, na produção de citocinas e na expressão de moléculas de superfície em monócitos estimulados com LPS, infectados ou não por L. braziliensis. Avaliamos também a carga parasitária em células infectadas e expressão de quimiocinas em monócitos pré-sensibilizados com a saliva de L. intermedia e com a saliva de L. longipalpis. Monócitos selecionados de células mononucleares do sangue periférico (CMSP) de doadores saudáveis foram pré-sensibilizados com SGS de L. intermedia, estimulados com LPS e, em seguida, infectados ou não com L. braziliensis. A produção de citocinas foi avaliada por ELlSA em sobrenadante de cultura; expressão de moléculas de superfície foi determinada por citometria de fluxo e a expressão de quimiocinas foi analisada por PCR em Tempo Real em ensaios de Quantificação Relativa (QR). Monócitos humanos pré-tratados com SGS de L. intermedia mostraram uma redução significante na produção de IL-10 e um aumento significante na expressão de CD86. A infecção com L. braziliensis aumentou significativamente a produção de TNF-a, porém não alterou a expressão de CD86, nem a taxa de infecção em monócitos pré-sensibilizados com SGS. A expressão de IL-8, MIP-1a, MIP-1f3 e Rantes aumentou após estímulo de monócitos humanos com SGS de L. intermedia e L. longipalpis. Nossos dados indicam que a saliva de L. intermedia é capaz de modificar a produção de citocinas, a expressão de moléculas de superfície e a expressão de quimiocinas em monócitos humanos e estas alterações podem afetar a evolução da leishmaniose em infecções naturais.

Phenotypic, functional, and quantitative characterization of canine peripheral blood monocyte-derived macrophages

The yield as well as phenotypic and functional parameters of canine peripheral blood monocyte-derived macrophages were analyzed. The cells that remained adherent to Teflon after 10 days of culture had high phagocytic activity when inoculated with Leishmania chagasi. Flow cytometric analysis demonstrated that more than 80 percent of cultured cells were positive for the monocyte/macrophage marker CD14.

Human immune response to triatomine embryo extract

Dipetalogaster maximus embryo extracts were used to stimulate peripheral blood mononuclear cells (PBMC) and in ELISA with sera either from Trypanosoma cruzi infected or non-infected individuals. The results showed that there was significant proliferative response and high antibody, titers in sera of chagasic patients.

Usefulness of rabbits and mice as experimental laboratory models for Ehrlichia bovis.

Attempts were made to infect mice and immunosuppressed rabbits with Ehrlichia bovis. While evidence of infection could be noticed in rabbits, their identity as E. bovis needs confirmation. Mice appeared to be infected and showed clear inclusions in both blood monocytes and peritoneal macrophages. While symptoms of disease were not observable in rabbits, alopecia, dullness and death were noticed among infected mice. It is concluded that mice are better laboratory models for E. bovis infection; also that infection in mice could be enhanced by immunosuppression.