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Infecção por Cryptosporidium parvum: resposta imunológica e tecnologia de anticorpos monoclonais para o diagnóstico
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Cryptosporidium parvum é um protozoário intracelular, de distribuição ubiquitária, que causa enterite em humanos e animais. A diarreia é autolimitada em indivíduos imunocompetentes, podendo ser fatal em imunocomprometidos.
Actualmente não existem terapêuticas específicas ou preventivas disponíveis, e o elevado custo dos actuais métodos de diagnóstico, sustentam a importância do
desenvolvimento de novas abordagens.
Vários estudos salientam a importância das células T CD4+ na resposta imunitária à infecção por C. parvum; outros direccionam a sua atenção para as células reguladoras (Treg); e outros tentam esclarecer qual o papel das citocinas específicas produzidas pelas células Th1 e Th2, na regulação da resposta imune, acompanhada pela
produção de imunoglobulinas particulares. Na área do diagnóstico, vários e diferentes métodos têm sido utilizados, variando entre a rapidez de execução, especificidade e
custo. Várias questões colocam-se, relacionadas com as características das células envolvidas na resposta à infecção e com a especificidade/sensibilidade de cada técnica.
Com o objectivo de estudar a resposta imunitária de longo-termo à infecção por C. parvum, no modelo animal imunocompetente, murganhos Balb/c foram inoculados
por via oral com oocistos de C. parvum, e efectivadas colheitas e análise de amostras fecais, intestino, sangue e baço, segundo um protocolo previamente definido. A
caracterização das populações celulares do sangue e baço foi feita por citometria de fluxo e a identificação e a quantificação de imunoglobulinas e citocinas no soro, por
tecnologia xMAP® Luminex. A análise por citometria de fluxo não revelou diferenças estatisticamente significativas entre os murganhos infectados e os controlos, tendo sido
apenas observado um aumento do número de neutrófilos e eosinófilos circulantes, e a sua posterior diminuição, no primeiro grupo de murganhos. Associada à elevada
variabilidade observada após a reinfecção, tais variações são sugeridas como sendo o perfil exibido por estas populações de células no contexto de infecção por C. parvum no organismo imunocompetente, particularmente os eosinófilos, que apresentam um comportamento idêntico em infecção por outros parasitas.
O aumento da secreção de TNF-α e IFN-γ (citocinas Th1) nos murganhos infectados, comparativamente com os grupos controlo, para além da secreção de citocinas Th2, como IL-4, IL-5 e IL-10, após a reinfecção, sugere um balanço entre as respostas Th1, para controlar o crescimento do parasita, e Th2, para limitar a patologia.
A IgG1 foi o isotipo predominante ao longo de toda a infecção e reinfecção, tendo-se observado um pico de IgG2a após a reinfecção, seguido de diminuição. Esta variação poderá estar relacionada com a função da IgG1 e da IgG2a, ao nível da opsonização e neutralização dos agentes patogénicos, respectivamente.
A obtenção de hibridomas secretores de anticorpos específicos para antigénios de C. parvum, por fusão celular, permitiu obter anticorpos e testar a sua aplicação à
detecção de oocistos de C. parvum, em amostras fecais humanas e de animais (bovinos).
Em resumo, os resultados obtidos permitem sugerir o perfil de imunoglobulinas e citocinas envolvido na resposta à infecção por C. parvum no modelo roedor imunocompetente, assim como desenvolver um futuro “kit” para detecção de C. parvum em amostras biológicas, por técnicas de imunofluorescência.
Cryptosporidium parvum is an intracellular protozoan, with a ubiquitous distribution, causing enteritis in humans and animals. Diarrhea is self-limited in immunocompetent individuals, becoming life-threatening in immunocompromised hosts. Currently, specific therapeutic or preventive interventions are not yet available, and the high cost of existing diagnosis methods, support the importance of developing new approaches. Several studies highlight the importance of CD4+ T cells in the immune response to infection by C. parvum; others address their attention to the immunoregulatory cells (Treg); and even others attempt to clarify the role of specific cytokines produced by Th1 and Th2 cells to regulate the immune response, together with the production of specific immunoglobulins. In the diagnostic field, several and different methods have been used, varying between speed of execution, specificity and price. Several questions arise related to the type of cells involved in the response to infection and the specificity/sensitivity of each technique. With the aim of studying the long-term immune response to infection by C. parvum, in immunocompetent animal, Balb/c mice were orally inoculated with C. parvum oocysts, and faeces, small intestines, blood and spleens were collected for analysis, according to a previously defined protocol. Peripheral blood and spleen cell populations were characterized by flow cytometry, and Luminex xMAP® technology assay was used to quantify immunoglobulins and cytokines in serum. The flow cytometry analysis did not show statistically significant differences between the control and infected mice, having only been observed an increase in the number of circulating neutrophils and eosinophils, and their subsequent decrease, in the latter group. Associated with the high variability observed after reinfection, such variations are suggested as the profile exhibited by these cell populations in the context of an infection by C. parvum in immunocompetent organism, especially eosinophils, which have a similar behavior upon infection by other parasites. The increased secretion of TNF-α and IFN-γ (Th1 cytokines) in infected mice, when compared with the control group, beyond the secretion of Th2 cytokines (IL-4, IL- 5 and IL-10), after reinfection, suggests a balance between Th1 cells to control parasite growth, and Th2 to limit the pathology. IgG1 was the predominant isotype along infection and reinfection, with a peak of IgG2a after reinfection, followed by its decrease. This variation may be related to the function of IgG1 and IgG2a, namely in pathogens opsonization and their neutralization, respectively. The obtaining of hybridomas secreting antibodies specific for C. parvum antigens by cell fusion, allowed us to obtain antibodies and test them in the detection of oocysts of C. parvum in faecal samples from human and cattle. In brief, the obtained results suggest the profile of immunoglobulins and cytokines involved in the response to infection by C. parvum in immunocompetent mice, as well as developing a future "kit" for detecting C. parvum in biological samples by immunofluorescence.
Cryptosporidium parvum is an intracellular protozoan, with a ubiquitous distribution, causing enteritis in humans and animals. Diarrhea is self-limited in immunocompetent individuals, becoming life-threatening in immunocompromised hosts. Currently, specific therapeutic or preventive interventions are not yet available, and the high cost of existing diagnosis methods, support the importance of developing new approaches. Several studies highlight the importance of CD4+ T cells in the immune response to infection by C. parvum; others address their attention to the immunoregulatory cells (Treg); and even others attempt to clarify the role of specific cytokines produced by Th1 and Th2 cells to regulate the immune response, together with the production of specific immunoglobulins. In the diagnostic field, several and different methods have been used, varying between speed of execution, specificity and price. Several questions arise related to the type of cells involved in the response to infection and the specificity/sensitivity of each technique. With the aim of studying the long-term immune response to infection by C. parvum, in immunocompetent animal, Balb/c mice were orally inoculated with C. parvum oocysts, and faeces, small intestines, blood and spleens were collected for analysis, according to a previously defined protocol. Peripheral blood and spleen cell populations were characterized by flow cytometry, and Luminex xMAP® technology assay was used to quantify immunoglobulins and cytokines in serum. The flow cytometry analysis did not show statistically significant differences between the control and infected mice, having only been observed an increase in the number of circulating neutrophils and eosinophils, and their subsequent decrease, in the latter group. Associated with the high variability observed after reinfection, such variations are suggested as the profile exhibited by these cell populations in the context of an infection by C. parvum in immunocompetent organism, especially eosinophils, which have a similar behavior upon infection by other parasites. The increased secretion of TNF-α and IFN-γ (Th1 cytokines) in infected mice, when compared with the control group, beyond the secretion of Th2 cytokines (IL-4, IL- 5 and IL-10), after reinfection, suggests a balance between Th1 cells to control parasite growth, and Th2 to limit the pathology. IgG1 was the predominant isotype along infection and reinfection, with a peak of IgG2a after reinfection, followed by its decrease. This variation may be related to the function of IgG1 and IgG2a, namely in pathogens opsonization and their neutralization, respectively. The obtaining of hybridomas secreting antibodies specific for C. parvum antigens by cell fusion, allowed us to obtain antibodies and test them in the detection of oocysts of C. parvum in faecal samples from human and cattle. In brief, the obtained results suggest the profile of immunoglobulins and cytokines involved in the response to infection by C. parvum in immunocompetent mice, as well as developing a future "kit" for detecting C. parvum in biological samples by immunofluorescence.
Descrição
Palavras-chave
Parasitologia médica Protozoários Células Imunoglobinas Hidromas Imunofluorescência
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical