Title:	Anticorpos IgG murinos de diferentes isotipos e região variável idêntica se ligam de forma diferente à cápsula de Cryptococcus neoformans e a receptores Fc-gama
Authors:	Oliveira, Diane Sthefany Lima de
Orientador(es)::	Felipe, Maria Sueli Soares
Assunto::	Meningite criptococócica AIDS (Doença) - pacientes
Issue Date:	15-May-2016
Data de defesa::	22-Feb-2016
Citation:	OLIVEIRA, Diane Sthefany Lima de. Anticorpos IgG murinos de diferentes isotipos e região variável idêntica se ligam de forma diferente à cápsula de Cryptococcus neoformans e a receptores Fc-gama. 2016. 65 f., il. Dissertação (Mestrado em Biologia Molecular)—Universidade de Brasília, Brasília, 2016.
Abstract:	It is estimated there are 625,000 deaths worldwide per year caused by cryptococcalmeningitis, the most affects people infected by HIV. Cryptococcusneoformans is responsible for the majority of these deaths. Since treatment of cryptococcos is using conventional antifungal has been less effective than ideal, monoclonal antibodies against fungus capsule were developed in the last three decades in order to create new therapeutic tools. However, treating mice with different isotypes of antibody were observed different protective responses. For example, treatment of C. neoformans infected mice with monoclonal antibody 3E5 IgG1 resulted in increased survival, where as the disease was aggravated in animal streated with IgG3 antibody of identical variable region. Immunofluorescence as say showed that the binding patterns of these two 3E5 isotypes are diferentes tothe capsule, which became questionable the classical concept of the antigen binding that defines it is exclusively determined by antibody variable region. In addition to these antigen binding differences, many studies published in 1980s suggest that the IgG3 isotype interacts with an Fc receptor on the surface of macro phages differently from other isotypes, which could also explain differences in protection provided by the 3E5 antibodies of different IgG isotypes. Considering the therapeutic importance of properly knowing the mechanisms by which the same IgG antibody may be protective or not, depending on the irisotype, this study aimed to produce new recombinant monoclonal antibodies, assessing its binding standard antigen and its ability to mediate phagocytosis by Fcreceptors. Two pairs of IgG1and IgG3 antibodies were produced by heterologous expression in mammalian cells. The first pair was derived from monoclonal antibody 2H1, the antibody against C. neoformans capsule who secrystal structure has been resolved. The second pair was derived from the 4-4-20 antibody that recognizes the hapten FITC and was generated to evaluate whether receptor-mediate deffector response isunique to antibodies againstC. neoformans. The heavy and light chains variable domains of the antibodies were generated by chemical synthesis and cloned into expression vectors for IgG1and IgG3 murine, then they were transfected into CHO and NSO cells. The transfection and purification conditions were optimized and led to the batch production of antibodies until it was sufficient for functional testing. The binding of the antibodiestoC. Neoformans cells was tested by indirect immunofluorescence and its ability to mediate phagocytosisby J774 macrophagesand CHO-K1 cells. The 2H1 antibody showed binding pattern similar tothat for 3E5, showing annular and punctuate pattern for IgG1and IgG3 respectively. Both 2H1 and 4-4-20 antibodies were also functional on phagocytes is assay by macrophages. The IgG1 isotype showed exclusive dependence on Fcγ receptor, whereas 2H1 IgG3 was able to mediate phagocytes is even when this receptor was blocked. Therefore, the produced recombinant antibodies were functional and allowed th eattainmen to fimportant experiments which showed that heavy chain canal terantibody binding to the antigen and that IgG3isotype can mediate phagocytosis through an unknown receptor.
metadata.dc.description.unidade:	Instituto de Ciências Biológicas (IB) Departamento de Biologia Celular (IB CEL)
Description:	Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.
metadata.dc.description.ppg:	Programa de Pós-Graduação em Biologia Molecular
Licença::	A concessão da licença deste item refere-se ao termo de autorização impresso assinado pelo autor com as seguintes condições: Na qualidade de titular dos direitos de autor da publicação, autorizo a Universidade de Brasília e o IBICT a disponibilizar por meio dos sites www.bce.unb.br, www.ibict.br, http://hercules.vtls.com/cgi-bin/ndltd/chameleon?lng=pt&skin=ndltd sem ressarcimento dos direitos autorais, de acordo com a Lei nº 9610/98, o texto integral da obra disponibilizada, conforme permissões assinaladas, para fins de leitura, impressão e/ou download, a título de divulgação da produção científica brasileira, a partir desta data.
DOI:	http://dx.doi.org/10.26512/2016.02.D.20236
Appears in Collections:	Teses, dissertações e produtos pós-doutorado

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