Peripheral blood mononuclear cell supernatants from asymptomatic dogs immunized and experimentally challenged with Leishmania chagasi can stimulate canine macrophages to reduce infection in vitro.

Leishmania chagasi is the causative agent of visceral leishmaniasis in both humans and dogs in the New World. The dog is the main domestic reservoir and its infection displays different clinical presentations, from asymptomatic to severe disease. Macrophages play an important role in the control of Leishmania infection. Although it is not an area of intense study, some data suggest a role for canine macrophages in parasite killing by a NO-dependent mechanism. It has been proposed that control of human disease could be possible with the development of an effective vaccine against canine visceral leishmaniasis. Development of a rapid in vitro test to predict animal responses to Leishmania infection or vaccination should be helpful. In this study, an in vitro model was established to test whether peripheral blood mononuclear cell (PBMC) supernatants from dogs immunized with promastigote lysates and infected with L. chagasi promastigotes could stimulate macrophages from healthy dogs in order to control parasite infection. PBMC from a majority of the immunized and experimentally infected dogs expressed IFN-gamma mRNA and secreted IFN-gamma when stimulated with soluble L. chagasi antigen (SLA) in vitro. Additionally, the supernatants from stimulated PBMC were able to reduce the percentage of infected donor macrophages. The results also indicate that parasite killing in this system is dependent on NO, since aminoguanidine (AMG) reversed this effect. This in vitro test appears to be useful for screening animal responses to parasite inoculation as well as studying the lymphocyte effector mechanisms involved in pathogen killing by canine macrophages.

Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells.

BACKGROUND: Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs. RESULTS: Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells. CONCLUSIONS: The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.

Ativação de células mononucleares caninas por interleucina-2 e interleucina-12 recombinantes homólogas / Canine mononuclear cells activation by homologous recombinant interleukin-2 and interleukin-12

Visando avaliar futuramente o potencial de citocinas na indução de resposta imune celular específica do tipo Th1 quando associadas a antígeno(s) recombinante(s) de Leishmania chagasi/infantum no cão, a combinação de IL-2 e IL-12 caninas recombinantes é analisada no presente trabalho. Aqui, descrevemos a clonagem do DNA complementar (cDNA) e pela primeira vez, a expressão de IL-2 canina recombinante biologicamente ativa em Escherichia coli e por células de mamífero. Para expressão em E. coli, utilizou-se a construção pRSET-caIL-2, anteriormente gerada por nosso grupo. Para expressão em células de mamíferos, foi realizada a clonagem do cDNA de IL-2, sintetizado por reação de transcrição reversa seguida de reação da polimerase em cadeia (RT-PCR) a partir o RNA total de células monucleares do sangue periférico (CMSP) de cão estimuladas com concanavalina A (Com-A), no vetor pcDNA3.1, gerando a construção pcDNA3.1-caIL-2. (...) Diante dos resultados obtidos, a construção pcDNA3.1-caIL-2 e ambas as formas de IL-2 canina recombinante obtidas, assim como as condições experimentais aqui descritas, poderão ser utilizadas no futuro para o estudo do potencial de IL-2, associada ou não a IL-12, como modulador da resposta imune in vitro e in vivo de cães, durante o desenvolvimento de uma vacina ou imunoterapia para leishmaniose visceral canina.