In vitro binding and survival assays of Leishmania parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

<p>Abstract</p> <p>Background</p> <p>There are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with <it>Leishmania</it>. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with <it>L. chagasi</it>.</p> <p>Results</p> <p>In the presence of exogenous serum, opsonized <it>Leishmania </it>promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18). In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the <it>L. chagasi </it>experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized <it>L. chagasi </it>were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages.</p> <p>Conclusion</p> <p>These studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after <it>L. chagasi </it>infection using flow cytometry.</p

Avaliação de IFN-γ e IL-10 em cães naturalmente infectados com Leishmania (Leishmania) chagasi com e sem manifestações clínicas

RESUMO: As leishmanioses têm como agentes etiológicos parasitas intracelulares obrigatórios pertencentes ao gênero Leishmania capazes de infectar diferentes espécies de mamíferos e nestes se reproduzirem dentro do sistema fagocítico mononuclear. Os cães domésticos são os principais responsáveis pela manutenção da cadeia epidemiológica da doença, podendo apresentar uma grande variedade de perfis clínicos, desde aparentemente sadios a severamente acometidos. Avaliou-se a expressão das citocinas de cães naturalmente infectados com Leishmania (Leishmania) chagasi. Foram coletadas 50 amostras, sendo 20 de animais positivos e sintomáticos para Leishmaniose Visceral Canina (LVC), 20 de animais positivos e assintomáticos e 10 de animais sabidamente negativos para a LVC. As amostras foram analisadas pelo teste imunocromatográfico rápido Dual Path Platform (DPP/Biomanguinhos®) e pelo ELISA (EIE/Biomanguinhos®) indireto para detecção de anticorpos anti-Leishmania. Após as confirmações dos testes, foi realizado o ELISA de captura (R & D Systems) para quantificação das citocinas IL-10 e IFN-γ. Houve diferença estatística entre os grupos observando um aumento nos níveis de IFN-γ nos animais assintomáticos e um aumento de IL-10 nos sintomáticos

Quantificação da IL-10 e do INF-γ em cães com ou sem sinais clínicos de infecção com Leishmania (Leishmania) chagasi

RESUMO: As leishmanioses compreendem um complexo de doenças causadas por parasitos intracelulares obrigatórios pertencentes ao gênero Leishmania. Consideradas como importante problema de saúde pública, sendo os cães domésticos os principais responsáveis pela manutenção da cadeia epidemiológica da doença, estima-se que mais da metade dos cães infectados não manifestam sinais clínicos da enfermidade. Avaliou-se o perfil de IL-10 e INF- γ de cães naturalmente infectados com Leishmania (Leishmania) chagasi no município de São Luís-MA. Foram coletadas 50 amostras, sendo 20 de animais positivos e sintomáticos para Leishmaniose Visceral Canina (LVC), 20 de animais positivos e assintomáticos e 10 de animais sabidamente negativos para a LVC. As amostras foram analisadas pelo teste imunocromatográfico rápido Dual Path Platform (DPP/Biomanguinhos®) e pelo ELISA (EIE/Biomanguinhos®) indireto para detecção de anticorpos anti-Leishmania. Após as confirmações dos testes, foi realizado o ELISA de captura para quantificação das citocinas IL-10 e INF-γ através do kit Milliplex MAP. Houve diferença estatística entre os grupos, observando um aumento de IL-10 em soros de cães sintomáticos para LVC, comparado com o grupo de animais assintomáticos, sugerindo que animais com essa expressão de IL-10 podem estar associados à susceptibilidade a doença. Assim como o aumento dos níveis de INF-γ observados em cães assintomáticos, comparado com o grupo de cães sintomáticos, poderiam estar relacionados à cronicidade da doença

In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with -1

<p><b>Copyright information:</b></p><p>Taken from "In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with "</p><p>http://www.biomedcentral.com/1746-6148/3/11</p><p>BMC Veterinary Research 2007;3():11-11.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1894629.</p><p></p>e of promastigotes bound to monocytes of animals experimentally infected (AEI) with in the presence or absence of C5D serum (2,5 × 10parasites/well) (*p < 0.01)

In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with -5

<p><b>Copyright information:</b></p><p>Taken from "In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with "</p><p>http://www.biomedcentral.com/1746-6148/3/11</p><p>BMC Veterinary Research 2007;3():11-11.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1894629.</p><p></p>e of C5D serum. (C) Promastigotas adhesion with the absence of serum. (D) Note some amastigotas could be seen 48 hours after interaction with the absence of C5D serum Giemsa. 1000×

In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with -6

<p><b>Copyright information:</b></p><p>Taken from "In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with "</p><p>http://www.biomedcentral.com/1746-6148/3/11</p><p>BMC Veterinary Research 2007;3():11-11.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1894629.</p><p></p>Average of peritoneal macrophages binding to from experimental infected animals with the presence or absence of C5D serum, after 50 min incubation (2,5 × 10parasites/well). (C) Average of infected peritoneal macrophages binding to from naturally infected animals with the presence or absence of C5D serum after, fourth eight hour incubation (5 × 10parasites/well) (*) p < 0.01

In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with -7

<p><b>Copyright information:</b></p><p>Taken from "In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with "</p><p>http://www.biomedcentral.com/1746-6148/3/11</p><p>BMC Veterinary Research 2007;3():11-11.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1894629.</p><p></p>(B) Average of monocyte-derived macrophages binding to from experimental infected animals with the presence or absence of C5D serum, after 50 min incubation (5 × 10parasites/well). (C) Average of infected peritoneal macrophages binding to from experimental infected animals with the presence or absence of C5D serum after, fourth eight hour incubation (5 × 10parasites/well) (*) p < 0.01

In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with -0

<p><b>Copyright information:</b></p><p>Taken from "In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with "</p><p>http://www.biomedcentral.com/1746-6148/3/11</p><p>BMC Veterinary Research 2007;3():11-11.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1894629.</p><p></p>overslips (assay with absence of serum C5D). Giemsa. 1000×

In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with -3

<p><b>Copyright information:</b></p><p>Taken from "In vitro binding and survival assays of parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with "</p><p>http://www.biomedcentral.com/1746-6148/3/11</p><p>BMC Veterinary Research 2007;3():11-11.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1894629.</p><p></p>C) CD11b positive cells; (D) CD11b expression during the binding to mononuclear cells with the presence of C5D serum. () CD11b expression during the binding to mononuclear cells with the absence of C5D serum