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Previous issue date: 2002 / Resumo: A anemia falciforme é uma alteração genética das hemácias, caracterizada por hemólise crônica e episódios de crises de oclusão vascular. O defeito genético consiste em uma mutação na posição 6 da cadeia f3da hemoglobina, que confere às hemácias alterações na sua forma e estrutura, tornando-as mais rígidas, deformadas e distorcidas, com tendência para a formação de polúneros e conseqüente obstrução do fluxo sanguíneo. Um simples ponto de mutação transforma a anemia falciforme em uma entidade clínica heterogênea, sugerindo a idéia de que diferentes mecanismos possam estar envolvidos. O mecanismo exato, pelo qual o fluxo sanguíneo normal é interrompido pelas hemácias, ainda não é totalmente esclarecido. Numa doença como a anemia falciforme, na qual a velocidade da passagem das
hemácias rígidas pelos vasos sanguíneos tem importância relevante, o tônus e a regulação vascular assumem papel primordial, sendo este controle mediado, em parte, pelo óxido nítrico, cuja ação é inibidapelo superóxido. Diversos estudos têm indicado o envolvimento da lesão endotelial e do processo inflamatório subclínico no desencadeamento e progressão dos processos da oclusão vascular. A participação dos leucócitos no dano aos tecidos dos pacientes com anemia falciforme é objeto de investigação, sendo a leucocitose um fator de mau prognóstico na doença. Investigou-se inicialmente a liberação do óxido nítrico pelos leucócitos, analisando sua capacidade de inibir a agregação de plaquetas lavadas induzida pela trombina. Os resultados mostram que os granulócitos de pacientes com anemia falciforme (n=9) liberam óxido nítrico de maneira similar aos granulócitos de controles sadios (n=9), pois foi necessário um número semelhante de células para inibir a agregação plaquetária (mediana de 4,4 x 106e 4,3 x 106,respectivamente). Na ausência de SOD, não foi detectada liberação de óxido nítrico pelos leucócitos mononucleares de pacientes com anemia falciforme (n=9), pois eles não afetaram a agregação de plaquetas nesta situação. Na
presença de SOD, não houve diferença na liberação do óxido nítrico entre leucócitos mononucleares de pacientes com anemia falciforme e controles, pois o número de leucócitos mononucleares necessário para inibir a agregação de plaquetas foi semelhante entre os pacientes (mediana de 3,4 x lO6ml, n=9) e os controles sadios (mediana de 3,3 x lO6ml,n=9). Os fagócitos contêm uma nicotinamida adenina inuc1eotídeofosfato (NADPH) oxidase (forma reduzida) associada à sua membrana citoplasmática, que gera superóxido e outros reativos intermediários tóxicos do oxigênio, os quais apresentam potencial de injúria aos tecidos. O sistema NADPH-oxidase é composto por cinco subunidades: p40-pho / Abstract: Sickle cell anemia is a genetic disorder of red blood cells characterized by chronic hemolysis and episodic vaso-oclusive crisis. The molecular defect is caused by a point mutation at the 6thposition ofthe f3chain ofhemoblobin, which leads erythrocytes to abnormal sickle shape, rigidity, deformability and distortion with consequent blood flow obstruction. A single point mutation leads sickle cell disease to a clínical heterogenous disorder, supporting the idea that a different set of mechanisms might be involved in this disease. It is still not completely understood how sickle erythrocytes interrupt blood flow. Current evidence indicate that endothelium damage, and a subclínical inflammatoryprocess take place in the initiation and progression of vascular occlusion. The role of leukocytes in the tissue injury of sickle cell anemia patients is a current subject of investigation. The NADPH-oxidase system of professional phagocytes catalyzes the production of superoxide by the one-eletron reduction of oxigen (respiratory burst). This is
the starting point for the production of reactive oxidants species, which are potential tissueinjury mediators. The NADPH-oxidase system is composed by tive components: p40-Phox p4TPh°x,p6Tphoxexist in the cytosol as a complexoThe other two components gp91-phoxandp22-PhOX form the cytocrome b558in the cell membrane and function as the terminal electron carrier ofthe oxidase. Afier activation, the cytosolic complex migrate to membrane binding to citocrome b558and assemblythe active oxidase. The involvement of the leukocyte NADPH-oxidase system has not been investigated before in sickle cell anemia. The experimental approach was focused on: a) the release of nitric oxide by neutrophils and mononuclear leukocytes ITom sickle cell anemia patients. It was assayed by the ability of leukocytes to inhibit thrombin-induced washed platelet aggregation. Neutrophils ITom sickle cell anemia patients released nitric oxide in a similar manner to those ITomhealthy controls. The inhibition of platelet aggregation by neutrophils ftom sickle cell anemia or ftom hea1thycontrols was blocked by L-NAME (300JlM), but not by D-NAME (300JlM), and was reversed by L-arginine (lmM). Additionaly, a similar number of neutrophils ftom sickle cell anemia patients and írom healthy controIs was required to inhibit platelet aggregation. Mononuc1ear leukocytes írom sickle cell anemia patients inhibited platelet aggregation only in the presence of superoxide dismutase (60 U rnl-I) b) the release of superoxide by neutrophils and mononuc1earsickle cell anemia leukocytes. lt was assassed by the SOD specific inhibition of cytochrome c reduction assay. PMA (30nM) or ZYM (100 partic1es/cell)-induced release of superoxide by mononuc1ear leukocytes :lTomsickle cell anemia patients was significant1yhigher than that observed in mononuc1ear leukocytes írom
healthy controIs (p<0,001 e p<O,Ol, respectively, Mann-Whitney test). The levels of superoxide released by neutrophils írom sickle cell anemia patients was similar to those írom healthy controIs.
c) the activity of SOD or cellular glutathione peroxidase in neutrophils and mononuc1ear leukocytes írom sickle cell anemia patients was assayed by the xantine-oxidase induced cytochrome c reduction and the cellular glutathione peroxidase activity was asseyed by the NADPH oxidation induces by tbuthyl hydroperoxide. The activites of SOD and GHS-Px in neutrophils and mononuc1ear sickle cell anemia leukocytes were similar to those ITom healthy controIs d) the gene expression of gp91-phox, p22-phoxand p6Tphoxin mononuc1ear sickle cell anemia leukocytes. Monocytes' RNA :lTom steady state sickle cell
disease patients and healthy control subjects were used in a quantitativerelative reverse transcriptase-polymerase chain reaction (RT-PCR). The results revealed that gp91-phoxrelative gene expression in mononucIear leukocytes írom sickle cell anemia patients (n=21) was significant1yhigher compared to healthy controIs (n=23, p=0,03, Mann-Whitney test) and that p2Tphox(n=24 patients, 24 controls) and p6Tphox(n=22 patients, 22 controls) relative gene expression in mononuc1ear sickle cell anemia leukocytes was similar to healthy controIs.e) the phosphorylation of a group of 47kDa cell proteins. It was assayed by the incorporation of 32P-Labeledby sickle cell (n=4, 4 x1O7cells/rnl) or healthy controls (n=6, 4 xl07 cells/rnl) mononuclear leukocytes, in basal state or after stimulation with PMA (30nM). We found a higher phosphorylation in 47kDa protein sickle cell mononuclear leukocytes than in healthy controls. Conclusions The results have shown that leukocytes :&om sickle cell anemia patients released nitric oxide in a similar manner to those :&omhealthy controls; sickle cell anemia mononuclear leukocytes release more superoxide, when stimulated with PMA or ZYM, than mononuclear leukocytes ITom healthy controls; gp91-PhoXgene expression is upregulated in monocytes :&omsickle cell patients compared to healthy controls, and the phosphorylation of 47kDa proteins in sickle cell anemia mononuclear leukocytes is more
intense than healthy controls. The NADPH-system is activated in sickle cell anemia mononuclear leukocytes and may be responsible, at least in part, for the inactivation of nitric oxide. This may contribute to inflammationand vascular dysregulation in sickle cell disease / Doutorado / Pediatria / Doutor em Saude da Criança e do Adolescente
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unicamp.br:REPOSIP/308288 |
| Date | 13 June 2002 |
| Creators | Motta, Pericles Mendonça Dias da, 1964- |
| Contributors | UNIVERSIDADE ESTADUAL DE CAMPINAS, Condino Neto, Antonio, 1961-, Vasconcelos, Dewton de Moraes, Sannomiya, Paulina, Saad, Sara Terezinha Ollala |
| Publisher | [s.n.], Universidade Estadual de Campinas. Faculdade de Ciências Médicas, Programa de Pós-Graduação em Ciências Médicas |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | 177p. : il., application/pdf |
| Source | reponame:Repositório Institucional da Unicamp, instname:Universidade Estadual de Campinas, instacron:UNICAMP |
| Rights | info:eu-repo/semantics/openAccess |
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