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Globotriaosylceramide (Gb3) is a resistance factor against human immunodeficiency virus (HIV) infection, but its expression has not been studied on human CD4+ T-cells. It was proposed that CD4+ T-cells may express Gb3 upon in vitro stimulation. To examine this, the optimal method for surface-expressed Gb3 detection was determined by comparing reagents, which showed that natural ligand (VT1B) and rat IgM monoclonal antibody (38-13) were the best methods. Using these, stimulated cells upregulated Gb3 in subsets of CD4+ T-cells, including T regulatory and NKT cell phenotypes, although the expression remained less than 2 percent of total cells. An enrichment method confirmed this. Examination of total Gb3 revealed that stimulated CD4+ T-cells without surface-expressed Gb3 did not express intracellular Gb3. Based on these results, it is concluded that CD4+ T-cells do not express Gb3 at significant levels under the conditions examined and, thus, may not have potential for resistance to HIV infection.

Globotriaosylceramide (Gb3) is a resistance factor against human immunodeficiency virus (HIV) infection, but its expression has not been studied on human CD4+ T-cells. It was proposed that CD4+ T-cells may express Gb3 upon in vitro stimulation. To examine this, the optimal method for surface-expressed Gb3 detection was determined by comparing reagents, which showed that natural ligand (VT1B) and rat IgM monoclonal antibody (38-13) were the best methods. Using these, stimulated cells upregulated Gb3 in subsets of CD4+ T-cells, including T regulatory and NKT cell phenotypes, although the expression remained less than 2 percent of total cells. An enrichment method confirmed this. Examination of total Gb3 revealed that stimulated CD4+ T-cells without surface-expressed Gb3 did not express intracellular Gb3. Based on these results, it is concluded that CD4+ T-cells do not express Gb3 at significant levels under the conditions examined and, thus, may not have potential for resistance to HIV infection.

Fabry disease is an inherited defect of lysosomal α-galactosidase A (α-GALA), causing progressive accumulation of glycosphingolipids with terminal α-galactosyl moieties, especially globotriaosylceramide (Gb3Cer) and in to a small extent also galabiosylceramide (Ga2Cer) and blood group B glycolipids, in most tissues and body fluids. This diploma thesis is an extension of previous laboratory studies and intends to contribute to clarification of some specific features of catabolic pathways of glycolipids substrates in lysosomal storage disorders, especially blood group B glycolipids. Therefore, analysis of human pancreas and lungs tissues was performed using TLC imunodetection and immunohistochemical analysis of these glycolipids. The most striking observation was massive accumulation of B-6-2 glycolipid and of others complex B-glycolipids in the pancreas of the patient with Fabry disease with blood group B. The level of blood group B substrates exceeded significantly storage of Gb3Cer substrate. An important part of this work were metabolic experiments in cell cultures in order to answer the question about participation of related glycosidases - α-galactosidase A and α-N- acetylgalactosaminidase (α -NAGA) in the lysosomal degradation of glycosphingolipids with terminal α-galactose. Loading experiments were...

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Orientador: Tomomasa Yano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T11:52:53Z (GMT). No. of bitstreams: 1 Bernedo-Navarro_RobertAlvin_D.pdf: 2646572 bytes, checksum: 02b50bc28d5be6cddb78abfe621a047c (MD5) Previous issue date: 2013 / Resumo: Escherichia coli produtora de toxina de Shiga (STEC) é um grupo de importantes patógenos para humanos. Essas bactérias são relacionadas a várias doenças, como por exemplo, Síndrome Urêmica Hemolítica e produzem potentes toxinas denominadas toxinas de Shiga. Essas toxinas, tanto Stx1 quanto Stx2, compartilham um receptor celular comum, a globotriaosilceramida (Gb3) e exibem a mesma atividade biológica intracelular. O desenvolvimento de novos agentes neutralizantes dos danos induzidos por Stx pode representar uma estratégia promissora para o tratamento das doenças causadas por STEC em humanos. No presente estudo, nós desenvolvemos peptídeos sintéticos que exibem atividade neutralizante contra a citotoxicidade induzida por Stx tanto in vitro quanto in vivo e, além disso, que se ligam eficientemente ao receptor Gb3. O peptídeo P12-26 compete eficientemente com Stx2 para a ligação ao Gb3 in vitro. Além disso, os peptídeos PC7-12, P12-26 e PC7-30 inibiram a citotoxicidade de Stx1 e Stx2 em células Vero. Nós observamos que o peptídeo PC7-30 em forma de loop e o peptídeo P12-26 que é linear produziram as maiores porcentagens de inibição de Stx1 e Stx2 em células Vero, respectivamente. No entanto, o peptídeo P12-26 não inibiu a letalidade em camundongos, enquanto que o peptídeo PC7-30 inibiu a letalidade causada pela toxina Stx1. Nossos resultados indicam que os peptídeos P12-26 e PC7-30 são candidatos promissores para o desenvolvimento de agentes terapêuticos contra as doenças em seres humanos causadas por STEC / Abstract: Shiga toxin-producing Escherichia coli strains are important pathogens for humans. These bacteria are linked with severe diseases such as hemolytic uremic syndrome and produce potent known as Shiga toxins. These toxins, Stx1 and Stx2, share a common cellular receptor called globotriaosylceramide (Gb3) and exhibit the same intracellular biological activity. The development of new neutralizing agents for Stx-induced damage may represent a promising strategy for the treatment of diseases caused by STEC infections. In this study, we developed synthetic peptides that exhibit neutralizing activity against Stxinduced cytotoxicity both in vitro and in vivo and that bind efficiently to the Gb3 receptor. The peptide P12-26 competed efficiently with Stx2 for binding to Gb3 in vitro. Moreover, the peptides PC7-12, P12-26 and PC7-30 inhibited the cytotoxicity of Stx1 and Stx2 in Vero cells. We observed that the loop-constrained peptide PC7-30 and linear peptide P12-26 produced higher percentages of inhibition of Stx1 and Stx2 in Vero cells, respectively. However, the peptide P12-26 did not inhibit lethality in mice, whereas the loopconstrained peptide PC7-30 inhibited the lethality caused by Stx1. Our results indicate that the peptides P12-26 and PC7-30 are promising candidates for the development of therapeutic agents against diseases caused by STEC in humans / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular