RESUMO
We adapted the RNA FISH Stellaris method to specifically detect the expression of Plasmodium genes by flow cytometry and ImageStream (Flow-FISH). This new method accurately quantified the erythrocytic forms of (1) Plasmodium falciparum and Plasmodium vivax and (2) the sexual stages of P vivax from patient isolates. ImageStream analysis of liver stage sporozoites using a combination of surface circumsporozoite protein (CSP), deoxyribonucleic acid, and 18S RNA labeling proved that the new Flow-FISH is suitable for gene expression studies of transmission stages. This powerful multiparametric single-cell method offers a platform of choice for both applied and fundamental research on the biology of malaria parasites.
Assuntos
Malária , Esporozoítos , Animais , Expressão Gênica , Humanos , Malária/parasitologia , Plasmodium falciparum/genética , Plasmodium vivax/genética , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , RNARESUMO
We generated a new humanized mouse model to study HLA-restricted immune responses. For this purpose, we created unique murine hosts by enforcing the expression of human SIRPα by murine phagocytes in murine MHC-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of nonpermissive mice following injection of human hematopoietic stem cells. We showed that these mouse/human chimeras were able to generate HLA-restricted responses to immunization. These new humanized mice may offer attractive models to study immune responses to human diseases, such as HIV and EBV infections, as well as to assay new vaccine strategies.
Assuntos
Antígenos HLA/administração & dosagem , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Quimera por Radiação/imunologia , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/administração & dosagem , Antígenos de Diferenciação/sangue , Antígenos de Diferenciação/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Modelos Animais de Doenças , Feminino , Antígenos HLA/genética , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Quimera por Radiação/genética , Receptores Imunológicos/administração & dosagem , Receptores Imunológicos/sangue , Receptores Imunológicos/genéticaRESUMO
Plasmodium vivax is the most widespread human malaria parasite. Due to the presence of extravascular reservoirs and relapsing infections from dormant liver stages, P. vivax is particularly difficult to control and eliminate. Experimental research is hampered by the inability to maintain P. vivax cultures in vitro, due to its tropism for immature red blood cells (RBCs). Here, we describe a new humanized mice model that can support efficient human erythropoiesis and maintain long-lasting multiplication of inoculated cryopreserved P. vivax parasites and their sexual differentiation, including in bone marrow. Mature gametocytes were transmitted to Anopheles mosquitoes, which led to the formation of salivary gland sporozoites. Importantly, blood-stage P. vivax parasites were maintained after the secondary transfer of fresh or frozen infected bone marrow cells to naïve chimeras. This model provides a unique tool for investigating, in vivo, the biology of intraerythrocytic P. vivax.