ABSTRACT. Primary cultures of heart muscle cells provide powerful tools for cardiac cell biological research that permits both physiological and biochemical approaches. In the present study we analyzed the endocytosis of cardiac cells and presented morphological characterization of the endocytic machinery using markers, which enabled us to follow the fluid-phase, receptor-mediated endocytosis and the internalization of large particles. Our results demonstrated the route of the internalized cargo to early endosomes followed or not by its discharge in the late compartments. We also confirmed the ability of cardiac muscle cells to ingest large particles such as the mannosylated ligand zymosan A, and even internalize whole eukaryotic cells such as the protozoan parasite Trypanosoma cruzi. Since endocytosis is involved in many important cellular functions, the present work contributes to the knowledge of possible additional roles played by cardiac muscle cells besides their well known ability to act as physically energetic cells in the body, constantly contracting without tiring.
Chagas disease is an incurable illness caused by the protozoan Trypanosoma cruzi. Cardiomyocytes represent important targets for the parasite infection and alterations in their physiology were reported. Because endocytosis is involved in different cellular events and guanosine triphosphatase (GTPase) Rab proteins play important roles in various aspects of the membrane traffic, our aim was to characterize the expression of Rab proteins in T. cruzi-infected cardiomyocytes, which displayed a downregulation of Rab7 and Rab11, whereas the expression of Rab5a was maintained in the infected cultures even after longer periods of parasite internalization, but early endosome antigen 1 was partially downregulated. The parasite infection also decreased the uptake of fluid phase ligands by the cardiac cultures. The regulation of GTPase proteins and effector molecules can contribute to the altered physiology of the host cells by modifying the normal incoming of nutrients as well as interfering with other important events related to the endocytic pathway.
Objective: We have previously reported that mannose receptors participate and are regulated during Trypanosoma cruzi cardiomyocyte (CM) infection. Our present aim is to characterize the endocytosis of mannosylated ligands like zymosan A (Zy) in uninfected and T. cruzi-infected CM. Methods: CM infected or not by T. cruzi wereincubated with Zy for different periods of time and their internalization was analyzed at light microscopy level. Fluorescent approaches were performed by treating Zy with concanavalin-A-TRITC and washing it exhaustively prior to incubation with CM. The cultures were further stained with phalloidin-FITC and DAPI for actin and DNA visualization, respectively. Results: CM internalized Zy particles in a time-dependent fashion. The ligand specificity was confirmed by the addition of mannan, which efficiently blocked the Zy endocytosis. Designed fluorescent approaches extended and confirmed the Zy internalization by striated cells. Infected cultures displayed impairment in Zy endocytosis, which seems to be directly related to host infection rates. Conclusions: Altogether, our results show the ability of CM to ingest large particles such as the mannosylated ligand Zy. During their infection with T. cruzi, there is a loss in Zy internalization possibly due to the negative modulation of mannose receptors.
Introdução:A Leishmaniose Visceral Canina (LVC) continua sendo um problema de saúde pública. Esta doença é causada pelo protozoário Leishmania, sendo a Leishmania chagasi responsável por esta infecção no Brasil. Cães domésticos são um dos reservatórios do parasito e quando são picados pelo flebótomo, vetor desta doença, podem infectar os seres humanos. Apesar das medidas de controle, as notificações por Leishmaniose têm aumentado constantemente. O diagnóstico precoce e correto é muito importante, pois ajuda na prevenção e controle da doença. Bio-Manguinhos/Fiocruz-RJ, desde 2004, vem produzindo o conjunto diagnóstico para atender à demanda do Ministério da Saúde. Com a alteração do algoritmo de testagem da Leishmaniose Canina, houve a necessidade da manutenção do fornecimento do teste Elisa que passou a ser usado como confirmatório, sendo imprescindível a otimização nos processos de estocagem e transporte, facilitando a sua utilização pelo usuário final e possibilitando a redução de custos.Objetivo: Neste estudo testamos reagentes comerciais que possibilitem a armazenagem e o transporte de todo o kit entre 2 e 80 C. Atualmente, parte dos reagentes é mantida a -200C e outra sob refrigeração, o que implica em maior complexidade logística tanto de transporte como de estocagem e, consequentemente, maior custo.Metodologia: Resultados obtidos em ensaios com placas de Elisa sensibilizadas com antígeno de Leishmaniose (L.Major) e mantidas a -200C foram comparados aos obtidos com placas sensibilizadas com o mesmo antígeno e mantidas entre 2 e 80C. Nestas últimas, adicionouse um estabilizante que previne degradação e desnaturação, além de bloquear qualquer sítio livre na superfície da placa, minimizando possíveis reações cruzadas. Para mantermos o conjugado entre 2 e 80C, o mesmo foi diluído 1:10 em estabilizante e testado em ensaio imunoenzimático, comparando-se os resultados obtidos com os resultados do conjugado diluído em PBS-glicerol 80% e mantido a -200C. Para a conservação dos controles positivo e negativo, adicionouse 0,2% de azida sódica a fim de evitar contaminações no kit mantido entre 2 e 80C. Estes controles foram testados para avaliarmos possíveis interferências da azida sódica e a performance em relação aos mantidos à -200C.
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