Molecular bording of recogniton the antigen for T lymphocites

O estudo dos mecanismos de reconhecimento de epitopos por linfocitos T e fundamental para a manipulacao de processos imunologicos, tais como imunizacao protetora contra patogenos e a inducao de tolerancia em doencas auto-imunes. Neste trabalho almejamos identificar epitopos imunodominantes e caracterizar a interface MHC-peptideo-TCR em antigenos de duas doencas endemicas: a Paracoccidioidomicose e doenca de Chagas. Vacinas eficazes sao capazes de induzir uma resposta imune protetora em uma populacao geneticamente distinta. 0 primeiro modelo abordado neste trabalho foi o estudo de epitopos da glicoproteina gp43 de Paracoccidioides brasiliensis, agente causador de uma micose sistemica que atinge toda a America Latina: a Paracoccidioidomicose (PCM). Analisamos a resposta a gp43, capaz de conferir protecao contra o desafio letal em camundongos, em busca de epitopos imunodominantes em humanos. Para tal, utilizamos o algoritmo TEPITOPE para selecionar sequencias que poderiam se ligar a multiplas moleculas HLA-DR e testamos o reconhecimento por celulas T de pacientes tratados e curados de PCM. A real promiscuidade dos cinco peptideos previstos como os mais promiscuos selecionados da sequencia da gp43 foi avaliada por ensaios de ligacao a 9 diferentes moleculas HLA-DR prevalentes na populacao. Os peptideos tambem foram testados em ensaio de proliferacao primaria com PBMC de 29 pacientes PCM e 13 individuos normais. PBMC de 100 por cento dos pacientes reconheceram a gp43 e 72 por cento (21/29) reconheceram pelo menos um peptideo. 0 peptideo gp43(181-195) se ligou a 100 por cento das moleculas de HLA-DR em ensaio de ligacao e foi reconhecido por 48 por cento (14/29) dos pacientes testados, enquanto que os demais peptideos selecionados se ligaram a 22-78 por cento das moleculas HLA-DR testadas foram reconhecidos por 28-41 por cento dos pacientes. A combinacao desses peptideos em um pool aumentou a frequencia de reconhecimento para 75 por cento. Observamos correlacao positiva entre a promiscuidade na previsao pelo TEPITOPE, frequencia de ligacao e o reconhecimento. 0 mimetismo molecular entre o T. cruzi e antigenos do coracao e um dos mecanismos patogenicos hipotetizados para a cardiopatia chagasica cronica, que ocorre em 30 por cento dos cerca de 10 milhoes de individuos infectados pelo parasita em toda a America Latinaa(au)BV UNIFESP: Teses e dissertaçõe

Proteomic Study of Response to Copper, Cadmium, and Chrome Ion Stress in <i>Yarrowia lipolytica</i> Strains Isolated from Andean Mine Tailings in Peru

Mine tailings are produced by mining activities and contain diverse heavy metal ions, which cause environmental problems and have negative impacts on ecosystems. Different microorganisms, including yeasts, play important roles in the absorption and/or adsorption of these heavy metal ions. This work aimed to analyze proteins synthesized by the yeast Yarrowia lipolytica AMJ6 (Yl-AMJ6), isolated from Andean mine tailings in Peru and subjected to stress conditions with common heavy metal ions. Yeast strains were isolated from high Andean water samples impacted by mine tailings from Yanamate (Pasco, Peru). Among all the isolated yeasts, the Yl-AMJ6 strain presented LC50 values of 1.06 mM, 1.42 mM, and 0.49 mM for the Cr+6, Cu+2, and Cd+2 ions, respectively. Proteomic analysis of theYl-AMJ6 strain under heavy metal stress showed that several proteins were up- or downregulated. Biological and functional analysis of these proteins showed that they were involved in the metabolism of proteins, nucleic acids, and carbohydrates; response to oxidative stress and protein folding; ATP synthesis and ion transport; membrane and cell wall; and cell division. The most prominent proteins that presented the greatest changes were related to the oxidative stress response and carbohydrate metabolism, suggesting the existence of a defense mechanism in these yeasts to resist the impact of environmental contamination by heavy metal ions

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension

Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells

Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans-sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host–parasite interaction