Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

Metabolismo de microemulsoes semelhantes a fase lipidica da LDL em pacientes portadores de leucemias agudas / Metabolism of microemulsions with similar composition to that of the lipid phase of LDL in patients acute leukemia

No presente trabalho, 15 pacientes portadores de LMA e 8 de LLA receberam por via endovenosa uma emulsao cuja a fase lipidica e similar a LDL e destituida de apoproteinas, com o objetivo de se verificar se esta emulsao apresentava comportamento similar a LDL natural nestes pacientes. Pacientes com LMA revelaram uma taxa de remocao da emulsao 3 vezes mais rapida que os pacientes com LLA, observou-se tambem uma correlacao negativa entre a taxa de remocao e os niveis de LDL-colesterol nos pacientes com LMA (P>0.003), indicando que tanto a lipoproteina natural como a emulsao sao removidas pela mesma via metabolica. Esta correlacao nao foi vista nos pacientes com LLA. Quando o experimento foi repetido em 10 pacientes com LMA apos remissao da doenca, observou-se uma diminuicao significativa na taxa de remocao da emulsao comparado aos pacientes pre-tratamento. Esta diferenca nao foi observada quando o mesmo experimento foi feito em 5 pacientes com LLA

Metabolismo de emulsoes semelhantes a fase lipidica da LDL em individuos normais e hiperlipidemicos / Metabolism of emulsion similar to lipidic phase in normals and hyperlipidemics individuals

Emulsoes de composicao semelhante a das lipoproteinas plasmaticas de baixa densidade (LDL), sem apolipoproteinas, preparadas por sonicacao dos constituintes lipidicos, com adicao de (1-14C)-oleato de colesterol e purificadas por ultracentrifugacao, foram injetados em dez pacientes com hipercolesterolemia, quatro com hipertrigliceridemia e dez individuos normolipidemicos