Resposta de Anticorpos à Aplicação Intra-articular de Células Tronco Mesenquimais Alogênicas em Equinos
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Data
2019-05-21
Autores
Krieck, André Massahiro Teramoto
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Universidade Estadual Paulista (Unesp)
Resumo
O transplante de células tronco mesenquimais (CTMs) em doenças articulares vem se demonstrando eficaz. O objetivo desse estudo foi avaliar a resposta imune de repetidas aplicações intra-articulares de CTMs alogênicas e a imunogenicidade destas, utilizando ensaios de microcitotoxicidade para avaliar as respostas de anticorpos citotoxicos dependentes de complemento. Quinze equinos adultos foram distribuídos aleatoriamente entre três grupos. Seis animais no grupo ALO onde articulações hígidas receberam CTMs alogênicas, seis no grupo ALO LPS em que as articulações foram previamente inflamadas, pela indução experimental da sinovite com 0,5ng de LPS e três animais no grupo AUTO que foi mantido como controle negativo e receberam CTMs autólogas. Foram realizadas 2 aplicações na mesma articulação de células tronco mesenquimais derivadas de membrana sinovial com intervalo de 4 semanas. Foram colhidas amostras seriadas nos 3 dias após cada aplicação e a cada 7 dias por 8 semanas do liquido sinovial para análise e soro sanguíneo para os ensaios de microcitotoxicidade e exame ultrassonográfico para avaliar efusão articular através da distancia entre a superfície do talus e a capsula articular. Para análise estatística foi utilizado os testes Two Way Repeated Measures ANOVA e teste Turkey's. Foi evidenciada uma reação inflamatória moderada pelo aumento da celularidade do liquido sinovial após as aplicações. Também, foi observado que após a segunda aplicação alogênica, as articulações apresentaram efusão até 28 dias após aplicação quando comparada às aplicações autólogas e em relação à primeira aplicação alogênica. Foi observada a produção de anticorpos citotóxicos a partir de 7 dias em 2 animais do grupo ALO atingindo pelo menos 80% de morte celular e no grupo ALO LPS 3 animais. No grupo AUTO não foi observada a produção de anticorpos citotóxicos. Portanto os resultados demostraram que as CTMs alogênicas foram capazes de desencadear respostas de anticorpos citotóxicos especificos contra as CTMs doadoras, além de elucidar o aumento do infiltrado celular nas articulações sinoviais, assim, as respostas imunes poderiam impactar negativamente na longevidade, na viabilidade e na eficácia celular sendo estes resultados relevantes para utilização das CTMs injetadas no ambiente articular. Desta forma, a terapia de CTMs alogênicas deve ser empregada com cautela, principalmente em repetidas aplicações na mesma articulação, para assim se obter melhor potencial biológico desta terapia.
Transplantation of mesenchymal stem cells (MSCs) in joint diseases has been shown to be effective. The objective of this study was to evaluate the immune response of repeated intra-articular applications of allogeneic MSCs and their immunogenicity using microcytotoxicity assays to evaluate complement dependent cytotoxic antibody responses. Fifteen adult horses were randomly assigned to three groups. Six animals in the ALO group where healthy joints received allogeneic MSCs, six in the ALO LPS group in which the joints were previously inflamed, by the experimental induction of synovitis with 0.5ng of LPS and three animals in the AUTO group that was maintained as negative control and received Autologous MSCs. Two applications were performed in the same articulation of mesenchymal stem cells derived from synovial membrane with interval of 4 weeks. Serial samples were taken 3 days after each application and every 7 days for 8 weeks of synovial fluid for analysis and blood serum for the microcytotoxicity and ultrasound examination to evaluate joint effusion through the distance between the surface of the talus and the joint capsule. Two-way ANOVA and Turkey's test were used for statistical analysis. A moderate inflammatory reaction was evidenced by increased synovial fluid cellularity after the applications. Also, it was observed that after the second allogeneic application, the joints presented effusion until 28 days after application when compared to the autologous applications and in relation to the first allogeneic application. The production of cytotoxic antibodies was observed from 7 days in 2 animals of the ALO group reaching at least 80% of cell death and in the ALO LPS 3 animal group. In the AUTO group, the production of cytotoxic antibodies was not observed. Therefore the results demonstrated that the allogeneic MSCs were capable of triggering specific cytotoxic antibody responses against the donor MSCs, in addition to elucidating the increase of the cellular infiltrate in the synovial joints, the immune responses could negatively impact longevity, viability and efficacy. These results are relevant for the use of MSCs injected in joint environment. Therefore allogeneic MSC therapy should be used with caution, especially in repeated applications in the same joint, in order to obtain a better biological potential of this therapy.
Transplantation of mesenchymal stem cells (MSCs) in joint diseases has been shown to be effective. The objective of this study was to evaluate the immune response of repeated intra-articular applications of allogeneic MSCs and their immunogenicity using microcytotoxicity assays to evaluate complement dependent cytotoxic antibody responses. Fifteen adult horses were randomly assigned to three groups. Six animals in the ALO group where healthy joints received allogeneic MSCs, six in the ALO LPS group in which the joints were previously inflamed, by the experimental induction of synovitis with 0.5ng of LPS and three animals in the AUTO group that was maintained as negative control and received Autologous MSCs. Two applications were performed in the same articulation of mesenchymal stem cells derived from synovial membrane with interval of 4 weeks. Serial samples were taken 3 days after each application and every 7 days for 8 weeks of synovial fluid for analysis and blood serum for the microcytotoxicity and ultrasound examination to evaluate joint effusion through the distance between the surface of the talus and the joint capsule. Two-way ANOVA and Turkey's test were used for statistical analysis. A moderate inflammatory reaction was evidenced by increased synovial fluid cellularity after the applications. Also, it was observed that after the second allogeneic application, the joints presented effusion until 28 days after application when compared to the autologous applications and in relation to the first allogeneic application. The production of cytotoxic antibodies was observed from 7 days in 2 animals of the ALO group reaching at least 80% of cell death and in the ALO LPS 3 animal group. In the AUTO group, the production of cytotoxic antibodies was not observed. Therefore the results demonstrated that the allogeneic MSCs were capable of triggering specific cytotoxic antibody responses against the donor MSCs, in addition to elucidating the increase of the cellular infiltrate in the synovial joints, the immune responses could negatively impact longevity, viability and efficacy. These results are relevant for the use of MSCs injected in joint environment. Therefore allogeneic MSC therapy should be used with caution, especially in repeated applications in the same joint, in order to obtain a better biological potential of this therapy.
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Palavras-chave
Células Precursoras, MHC, Microcitotoxicidade, Resposta Imune Humoral, Humoral Immune Response, Mesenchymal Stromal Cells, Microcytotoxicity