RESUMO
Chagasic cardiomyopathy (CC) is the main manifestation of Chagas Disease (CD). CC is a progressive dysfunctional illness, in which transforming growth factor beta (TGF-ß) plays a central role in fibrogenesis and hypertrophy. In the present study, we tested in a three-dimensional (3D) model of cardiac cells culture (named cardiac spheroids), capable of mimicking the aspects of fibrosis and hypertrophy observed in CC, the role of TGF-ß pathway inhibition in restoring extracellular matrix (ECM) balance disrupted by T. cruzi infection. Treatment of T. cruzi-infected cardiac spheroids with SB 431542, a selective inhibitor of TGF-ß type I receptor, resulted in a reduction in the size of spheroids, which was accompanied by a decrease in parasite load and in fibronectin expression. The inhibition of TGF-ß pathway also promoted an increase in the activity of matrix metalloproteinase (MMP)-2 and a decrease in tissue inhibitor of matrix metalloproteinase (TIMP)-1 expression, which may be one of the mechanisms regulating extracellular matrix remodeling. Therefore, our study provides new insights into the molecular mechanisms by which inhibition of TGF-ß signaling reverts fibrosis and hypertrophy generated by T. cruzi during CC and also highlights the use of cardiac spheroids as a valuable tool for the study of fibrogenesis and anti-fibrotic compounds.
Assuntos
Cardiomiopatias/tratamento farmacológico , Doença de Chagas/tratamento farmacológico , Coração/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Benzamidas/farmacologia , Cardiomiopatias/genética , Cardiomiopatias/parasitologia , Cardiomiopatias/fisiopatologia , Doença de Chagas/genética , Doença de Chagas/parasitologia , Doença de Chagas/fisiopatologia , Dioxóis/farmacologia , Matriz Extracelular/genética , Fibronectinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/parasitologia , Humanos , Metaloproteinase 2 da Matriz/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta/genética , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/patogenicidadeRESUMO
We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.
Assuntos
Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Parasitemia/diagnóstico , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , DNA de Protozoário/sangue , DNA de Protozoário/química , Humanos , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reação em Cadeia da Polimerase/normas , RNA Ribossômico 18S/genética , Recidiva , Sensibilidade e EspecificidadeRESUMO
Mycobacterium leprae, an obligate intracellular pathogen, shows a unique tropism for Schwann cells (SC). This leads to the peripheral neuropathy disorder observed in leprosy. In this study, we investigated signal transduction events and the intracellular fate of M. leprae during the interaction of the microorganism with SC. First, we demonstrated that the human schwannoma cell line ST88-14 readily phagocytized the bacteria as observed by time-lapse microscopy, actin staining and electron microscopy. The effect of specific kinase inhibitors on M. leprae internalization was then investigated showing that functional protein tyrosine kinase, calcium-dependent protein kinase and phosphatidylinositol 3-kinase, but not cAMP-dependent protein kinase are essential for phagocytosis of the bacteria. Similar results were obtained when irradiated and live bacteria were compared and when M. leprae was pre-coated with recombinant histone-like-protein/laminin binding protein, a bacterial adhesin. In addition, experiments were performed to analyze the bacterial trafficking within the endosomal network by labeling the acidified intracellular compartments of M. leprae-infected SC with the Lysotracker acidotrophic probe. Acidification of vesicles containing live M. leprae was minimal in both RAW murine macrophages and SC, although phagosomes containing heat-killed bacteria seem to follow normal endocytic maturation. These data indicate that the invading bacteria interfere with normal endocytic pathway maturation of bacteria-containing phagosomes within SC.