Summary
Visceral leishmaniasis (VL) is epidemic in Brazil with an increasing incidence of human cases and canine reservoirs, with host hypergammaglobulinemia. Conventional enzyme‐linked immunosorbent assay (cELISA) based on several parasitic antigens is the main method for diagnosis and indication of treatment. Dissociative ELISA (dELISA) uses acidic treatment to free immunoglobulin G (IgG) from immune complexes, and its use revealed a significant positive fraction of suspected cases with negative serology. Looking for small molecules or haptens that block IgG antibodies, we purified by molecular exclusion chromatography, 1000–3000 MW molecules from promastigote soluble extract, mostly oligosaccharides comprising 6–13 sugar residues using MALDI‐TOF analysis. Glycan‐BSA complex (GBC) was constructed by conjugating promastigote glycans to BSA molecules, allowing their use in the solid support in cELISA or dELISA. Sera from experimentally infected hamsters showed higher levels of blocked monomeric IgG during infection, mostly against GBC, which was also present in lower concentrations in the promastigote soluble extract dELISA. Those data show that most of the specific monomeric IgG in serum are blocked by haptens composed by glycans produced by the parasite, better detected in the high dilution of sera in the dELISA assays. dELISA is a useful technique for detecting blocked monomeric antibodies that could have difficult clearance from blood, which could result in hypergammaglobulinemia.
Ferrão TF. The importance of parasitic peptidases in the supply of amino acids in promastigotes of Leishmania (leishmania) infantum [dissertation]. São Paulo: "Faculdade de Medicina, Universidade de São Paulo"; 2020.Visceral leishmaniasis (VL) is a disease widely distributed in Brazil and worldwide, with high morbidity and mortality. In Brazil, it is caused by the species Leishmania (L.) infantum with vector transmission by females of Lutzomyia longipalpis. The parasite has two forms in its life cycle, the promastigotes present in insect vectors and amastigotes present in mammalian cells. The parasite's nutrition depends on amino acids obtained by interactions with the host. Different peptidases have already been documented in the Leishmania parasites, with four groups according to the active site. In this work, we evaluate the role of parasitic peptidases in nutritional processes, survival and multiplication promastigotes of L. (L.) infantum. Promastigotes were grown in standard medium (MP199). The number of parasites was monitored daily and on the 3rd day of stay in culture medium the parasites were analyzed in relation to its composition. The data obtained were used as a reference to standardize the analytical techniques used later in the parasites, captured by means rich in free amino acids (MRA) and rich in protein (MRP) and their culture supernatants. The comparison between parasites maintained in MRA and MRP demonstrates a higher protein concentration in MRP 26.22 µg / mL compared to MRA 10.71 µg / mL. As the morphological changes show a greater thickening of glycocalyx in promastigotes with greater proteolytic activity applied to MRP, detected with protein substrates marked with fluorochrome FITC. The data suggested here as parasitic peptidases present nutritional activities and methods that cause effects on their composition and cause parasitic changes inducing the actions of peptidases and modifications of the parasite surface to allow greater ease of use of the molecules and samples of amino acids, a different way from The function of these enzymes allows a better understanding of parasitic metabolism and its availability as a target for new drugs. Descriptors: Leishmania; peptidases Peptide Hydrolases; Proteolysis; Metabolism; Amino acids; Glycocalyx.
ISTA DE ABREVIATURAS E SIGLASµg Micrograma µL Microlitro µM Micromolar BSA Albumina de soro bovino (Bovine serum albumin) EDTA Ácido etilenodiamino tetra-acético EPL Extrato de promastigotas de L. (L.) infantum FITC Isotiocianato de fluoresceína (Fluorescein isotiocyanate) HCl Ácido clorídrico IgG Imunoglobulina G KDa Kilodalton LPLC Cromatografia liquida de exclusão molecular (Low Pressure Liquid Chromatography) M Molar mg Miligrama mL Mililitro MP199 Meio padrão 199 suplementado com urina e soro fetal MRA Meio rico em aminoácidos. MRP Meio rico em proteínas NaCl Cloreto de Sódio OVA Ovalbumina PBS Salina tamponada com fosfato (Phosphate buffered saline) pH Potencial hidrogeniônico pM Picomolar SBF Soro Fetal Bovino TLC Cromatografia de camada delgada (...
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