Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.<br>A diferenciação de formas epimastigotas (proliferativas) do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas), pode ser reproduzida em laboratório incubando-se as células em um meio quimicamente definido que imita a urina do inseto vetor deste parasita. Os epimastigotas têm um núcleo esférico, o flagelo se projeta da metade do corpo do protozoário e o cinetoplasto (organela que possui o DNA mitocondrial) possui formato de disco. Os tripomastigotas metacíclicos têm um núcleo alongado com o flagelo emergindo da extremidade posterior da célula associado ao cinetoplasto esférico. Neste trabalho descrevemos as mudanças morfológicas que ocorrem durante essa transformação e caracterizamos uma nova forma intermediária do parasita usando reconstrução tridimensional de cortes seriados, visualizados por microscopia eletrônica de transmissão. Essa nova forma intermediária é caracterizada pela compressão do cinetoplasto contra o núcleo alongado, indicando que a metaciclogênese envolve movimentos ativos do cinetoplasto associado à estrutura flagelar em relação ao corpo celular. Como tripomastigotas metacíclicos transcrevem menos que as formas epimastigotas proliferativas, verificamos a presença da RNA polimerase II e medimos a atividade transcricional durante o processo de diferenciação. A presença da enzima e a atividade transcricional permanecem inalteradas durante todas as etapas da metaciclogênese, desaparecendo apenas quando as formas metacíclicas são formadas. Sugerimos que a diferenciação requer uma atividade transcricional, necessária para uma intensa remodelação da célula, que acontece até o cinetoplasto e o flagelo atingirem uma posição posterior do corpo do tripomastigota metacíclico

Management of HIV-associated tuberculosis in resource-limited settings: a state-of-the-art review.

The HIV-associated tuberculosis (TB) epidemic remains a huge challenge to public health in resource-limited settings. Reducing the nearly 0.5 million deaths that result each year has been identified as a key priority. Major progress has been made over the past 10 years in defining appropriate strategies and policy guidelines for early diagnosis and effective case management. Ascertainment of cases has been improved through a twofold strategy of provider-initiated HIV testing and counseling in TB patients and intensified TB case finding among those living with HIV. Outcomes of rifampicin-based TB treatment are greatly enhanced by concurrent co-trimoxazole prophylaxis and antiretroviral therapy (ART). ART reduces mortality across a spectrum of CD4 counts and randomized controlled trials have defined the optimum time to start ART. Good outcomes can be achieved when combining TB treatment with first-line ART, but use with second-line ART remains challenging due to pharmacokinetic drug interactions and cotoxicity. We review the frequency and spectrum of adverse drug reactions and immune reconstitution inflammatory syndrome (IRIS) resulting from combined treatment, and highlight the challenges of managing HIV-associated drug-resistant TB