Glycotope structures and intramolecular affinity factors of plant lectins for Tn/T antigens

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Ganglioside GM3 Has an Essential Role in the Pathogenesis and Progression of Rheumatoid Arthritis

Rheumatoid arthritis (RA), a chronic systemic inflammatory disorder that principally attacks synovial joints, afflicts over 2 million people in the United States. Interleukin (IL)-17 is considered to be a master cytokine in chronic, destructive arthritis. Levels of the ganglioside GM3, one of the most primitive glycosphingolipids containing a sialic acid in the structure, are remarkably decreased in the synovium of patients with RA. Based on the increased cytokine secretions observed in in vitro experiments, GM3 might have an immunologic role. Here, to clarify the association between RA and GM3, we established a collagen-induced arthritis mouse model using the null mutation of the ganglioside GM3 synthase gene. GM3 deficiency exacerbated inflammatory arthritis in the mouse model of RA. In addition, disrupting GM3 induced T cell activation in vivo and promoted overproduction of the cytokines involved in RA. In contrast, the amount of the GM3 synthase gene transcript in the synovium was higher in patients with RA than in those with osteoarthritis. These findings indicate a crucial role for GM3 in the pathogenesis and progression of RA. Control of glycosphingolipids such as GM3 might therefore provide a novel therapeutic strategy for RA

An Introduction to Sphingolipid Metabolism and Analysis by New Technologies

Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or “sphingolipidomic” analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine

Proliferation and survival of human amniotic epithelial cells during their hepatic differentiation

Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.Fil: Maymo, Julieta Lorena. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Riedel, Rodrigo Nicolas. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Pérez Alcázar, Germán Antonio. Hospital Universitario Virgen Macarena;Fil: Magatti, Marta. Istituto Ospedaliero;Fil: Maskin, Bernardo. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Dueñas, José Luis. Hospital Universitario Virgen Macarena;Fil: Parolini, Ornella. Istituto Ospedaliero;Fil: Sánchez-Margalet, Víctor. Hospital Universitario Virgen Macarena;Fil: Varone, Cecilia Laura. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Linear and Branched Glyco-Lipopeptide Vaccines Follow Distinct Cross-Presentation Pathways and Generate Different Magnitudes of Antitumor Immunity

Glyco-lipopeptides, a form of lipid-tailed glyco-peptide, are currently under intense investigation as B- and T-cell based vaccine immunotherapy for many cancers. However, the cellular and molecular mechanisms of glyco-lipopeptides (GLPs) immunogenicity and the position of the lipid moiety on immunogenicity and protective efficacy of GLPs remain to be determined.We have constructed two structural analogues of HER-2 glyco-lipopeptide (HER-GLP) by synthesizing a chimeric peptide made of one universal CD4(+) epitope (PADRE) and one HER-2 CD8(+) T-cell epitope (HER(420-429)). The C-terminal end of the resulting CD4-CD8 chimeric peptide was coupled to a tumor carbohydrate B-cell epitope, based on a regioselectively addressable functionalized templates (RAFT), made of four alpha-GalNAc molecules. The resulting HER glyco-peptide (HER-GP) was then linked to a palmitic acid moiety, attached either at the N-terminal end (linear HER-GLP-1) or in the middle between the CD4+ and CD8+ T cell epitopes (branched HER-GLP-2). We have investigated the uptake, processing and cross-presentation pathways of the two HER-GLP vaccine constructs, and assessed whether the position of linkage of the lipid moiety would affect the B- and T-cell immunogenicity and protective efficacy. Immunization of mice revealed that the linear HER-GLP-1 induced a stronger and longer lasting HER(420-429)-specific IFN-gamma producing CD8(+) T cell response, while the branched HER-GLP-2 induced a stronger tumor-specific IgG response. The linear HER-GLP-1 was taken up easily by dendritic cells (DCs), induced stronger DCs maturation and produced a potent TLR- 2-dependent T-cell activation. The linear and branched HER-GLP molecules appeared to follow two different cross-presentation pathways. While regression of established tumors was induced by both linear HER-GLP-1 and branched HER-GLP-2, the inhibition of tumor growth was significantly higher in HER-GLP-1 immunized mice (p<0.005).These findings have important implications for the development of effective GLP based immunotherapeutic strategies against cancers

Expressão de glicoesfingolipídeos no carcinoma espinocelular do trato aerodigestivo superior Glycosphingolipid expression in squamous cell carcinoma of the upper aerodigestive tract

Os glicoesfingolipídios (GSLs) são importantes componentes da membrana celular, organizados em microdomínios, relacionados a receptores de membrana e comportamento anti-social da célula neoplásica como crescimento descontrolado, invasão e ocorrência de metástases. OBJETIVO: Como a expressão de GSLs no carcinoma espinocelular (CEC) é tema pouquíssimo estudado decidiu-se realizar estudo prospectivo visando avaliar a expressão de GSLs no CEC do trato aerodigestivo superior. MÉTODO: Coletou-se 33 amostras de CEC e mucosa normal e GSLs extraídos e purificados por cromatografia de fase reversa em coluna de C-18 e hidrólise alcalina em metanol. Os GSLs foram quantificados por densitometria das placas de cromatografia de alta resolução em camada delgada coradas com orcinol. RESULTADOS: Observou-se aumento significativo de GSLs no CEC (3,57µg/mg) em comparação à mucosa normal (1,92µg/mg), principalmente do monosialogangliosídeo (GM3), trihexosilceramida (CTH), dihexosilceramida (CDH), globosídeo (Gb4). A expressão de monohexosilceramida (CMH) foi semelhante no CEC e na mucosa normal. O aumento do GM3 no CEC foi demonstrado por métodos imunoquímicos empregando-se MAb DH2 (anti-GM3). Analisando-se os carboidratos do CMH por cromatografia gasosa acoplado a espectrômetro de massa constatou-se que a mucosa normal expressa glucosilceramida e o CEC glucosilceramida e galactosilceramida. CONCLUSÃO: O aumento de GSLs no tecido tumoral pode representar alterações dos microdomínios da membrana celular resultantes do processo de transformação maligna, responsáveis por uma maior interação célula-célula e célula-matriz aumentando seu potencial de infiltração e metástase, possibilitando o emprego dos GSLs e de MAbs no diagnóstico e no tratamento do CEC, a exemplo do que ocorre no melanoma.<br>Glycosphingolipids are integral constituents of cellular membrane, arranged in rafts, and with neoplasic cell anti-social behavior, like uncontrolled cell growth, invasiveness, and metastatic potential. AIM: However, there are few studies about glycosphingolipids (GSL) expression in squamous cell carcinoma (SCC). Since GSL are known to be tumor-associated markers we decided to perform a prospective study on the GSL profiles of SCC. METHOD: Specimens of 33 SCC and normal mucosa were obtained and GSLs were extracted and purified by reverse-phase chromatography on C18 column and alkaline hydrolysis in methanol. GSLs were quantified using densitometry of orcinol-stained HPTLC plates. RESULT: A significant increase of GSLs in SCC (3.57µg/mg) was observed as compared to normal mucosa (1.92µg/mg). In SCC, an increase of 2 to 3 times in the amounts of CDH, CTH, Globoside, and GM3 was observed in comparison to normal mucosa. The identification of GM3 as well as its increased expression in SCC was confirmed unequivocally by HPTLC immunostaining and indirect immunofluorescence using MAb DH2 (anti-GM3). BY analyzing SCC and normal mucosa CMHs by GC/MS, normal mucosa expresses only glucosylceramide whereas SCC cells express both glucosylceramide and galactosylceramide. CONCLUSION: The increase in the amount of GSLs in tumor tissue may represent changes of cell membrane microdomains resulting from the malignant transformation process, which is responsible for greater cell-cell or cell-matrix interaction thereby increasing their potential for infiltration and metastasis