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.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

Anti-Helicobacter pylori activity and oxidative burst inhibition by the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin from Paepalanthus latipes

Helicobacter pylori is a bacterium recognized as the major cause of chronic gastritis and peptic ulcers. Infection by H. pylori induces inflammatory responses and pathological changes in the gastric microenvironment. The host Keywords: immune cells (especially neutrophils) release inflammatory mediators and large 5-methoxy-3,4-dehydroxanthomegnin amounts of reactive oxygen species (ROS), which are associated with an increased Helicobacter pyloririsk of developing gastric cancer. In this study, we evaluated the anti-H. pylori and oxidative burst antioxidantactivitiesofa1,4-naphthoquinone-5-methoxy-3,4-dehydroxanthomegnin. Paepalanthus latipes The antimicrobial activity was assessed using a spectrophotometric microdilution technique, and antioxidant activity was assessed by noting the effect of 5-methoxy3,4-dehydroxanthomegnin on the neutrophil oxidative burst using luminol-and lucigenin-amplified chemiluminescence. The results showed that 5-methoxy-3,4dehydroxanthomegnin is a potent anti-H. pylori compound (MIC 64 µg/mL and MBC 128 µg/mL) and a strong antioxidant. 5-Methoxy-3,4-dehydroxanthomegnin decreased luminol- and lucigenin-amplified chemiluminescence, with ED50 values of 1.58±0.09 µg/mL and 5.4±0.15 µg/mL, respectively, reflecting an inhibitory effect on the oxidative burst. These results indicate that 5-methoxy-3,4-dehydroxanthomegnin is a promising compound for the prevention and treatment of diseases caused by H. pylori infection, such as gastritis, peptic ulceration, and gastric cancer, because reactive oxygen intermediates are involved in the pathogenesis of gastric mucosal injury induced by H. pylori infections.

The triad Indole-3-Acetic Acid Ethyl Ester/Esterase/Horseradish Peroxidase as a new cytotoxic Prodrug/Enzyme combination

The antibody-directed enzyme prodrug therapy (ADEPT) is a means of restricting the action of toxic drugs to the tumor site. The enzyme/prodrug pair horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) has been studied as a combination with potential application in ADEPT strategies. In this combination, the non-toxic plant hormone IAA is activated to cytotoxic species by the catalytic action of HRP. Objective: We studied the use of the ethyl ester of IAA as a new prodrug that could be activated by two enzymes, HRP and esterase. Methods: The oxidation of IAA and its ethyl ester, catalyzed by HRP, was monitored by the consumption of dioxygen and liquid chromatography. The cytotoxicity of IAA and its ethyl ester in combination with HRP and esterase was assessed using the lineage McCoy cells through the trypan blue and neutral red assays. Results: We found that HRP was not able to catalyze the oxidation of IAA-ethyl ester in the absence of an additional esterase. Hence, the potential cytotoxicity of the IAA-ethyl ester could be controlled by sequential treatment with esterase, to liberate the carboxyl group, and HRP, for oxidation and generation of cytotoxic species. We present evidence for the potential application of the combination IAA-ethyl ester/esterase/horseradish peroxidase as a new ADEPT, GDEPT or related strategy. Conclusions: We suggest that this technique could provide more selectivity in the generation of cytotoxic drugs at tumor sites.

A complexa fisiopatologia dos episódios vaso-oclusivos na anemia falciforme / The complex pathophysiology of te vasoocclusive episodes in the sickle cell anemia

Anemia hemolítica e vaso-oclusão são achados clínicos característicos da anemia falciforme. A vaso-oclusão é um processo complexo que envolve não apenas a polimerização dos tetrâmeros de hemoglobina S desoxigenada, mas também interações entre os eritrócitos falciformes, o endotélio vascular, plaquetas, leucócitos e proteínas plasmáticas. A aderência aumentada dos eritrócitos falciformes ao endotélio tem sido implicada como o passo inicial da vaso-oclusão. Outros pesquisadores têm destacado a interferência dos leucócitos e das plaquetas no fluxo sangüíneo. A oclusão microvascular resulta em crises dolorosas agudas, enquanto a oclusão macrovascular parece ser a causa da falência de órgãos. A anemia resulta da diminuição acentuada da sobrevida dos eritrócitos falciformes, associada a uma resposta eritropoética limitada. A eritropoese aumenta intensamente, mas não é suficiente para compensar a destruição eritrocitária e manter concentrações normais de eritrócitos e hemoglobina, devido, principalmente, à baixa afinidade da hemoglobina S pelo oxigênio e ao aumento do 2,3-Difosfoglicerato. É muito difícil separar os processos que conduzem a anemia daqueles que conduzem a vaso-oclusão. A compreensão do envolvimento de múltiplos componentes do sangue na vaso-oclusão pode elucidar as manifestações clínicas e complicações da anemia falciforme e fornecer novas perspectivas para o tratamento preventivo e curativo.

Padronizaçäo da determinaçäo dos parâmetros plaquetários em amostras sangüíneas coletadas com EDTA / Standardization of platelet parameters determination in blood samples collected with EDTA

Novos parâmetros plaquetários estäo disponíveis, rotineiramente, com a introduçäo dos contadores de células automatizados e podem ser muito importantes para a avaliaçäo da funçäo plaquetária. O contador de células Cell-Dyn 3000 (Abbott) prepara um histograma do volume plaquetário que é determinado pelo método de condutividade elétrica e através deste histograma estabelece a contagem de plaquetas (PLT), o volume plaquetário médio (VPM) e o coeficiente de variaçäo do volume plaquetário médio (PDW). Calcula também o plaquetócrito (PCT), que é o produto da contagem plaquetária e do VPM. As plaquetas dilatam em contato com sais do ácido etilenodiaminotetra-acético (EDTA), com o tempo de armazenamento das amostras sagüíneas e geram resultados de VPM elevados. Para avaliar o efeito do tempo de armazenamento no VPM, PDW, PCT e PLT foram coletadas amostras de sangue de 23 pacientes com anemia falciforme durante a fase estável (Grupo I) e 50 controles normais do mesmo sexo, idade e raca (Grupo II) em Vacutainersâ com EDTA K2 e determinados repetidamente durante 24 horas, nos seguintes intervalos de tempo: imediatamente após a punçäo venosa (tempo 0), 15, 30, 60, 120, 240, 360, 480 e 1440 minutos. Os valores médio de VPM e PCT aumentaram (p<0,0001) ao longo do tempo de armazenamento das amostras em ambos os Grupos. Os valores médio de PLT e PDW foram praticamente estáveis ao longo do tempo de armazenamento nos dois Grupos

Quimiluminescencia em celulas blasticas leucemicas e sua aplicaçao no diagnostico diferencial de leucemias agudas / Chemiluminescence in leukemia blasts cells and it application in the differential diagnostic of acute leukemias

E descrito um metodo quimiluminescente simples e sensivel para a caracterizaçao da mieloperoxidase intracelular, utilizavel para a diferenciaçao entre blastos comprometidos com a linhagem mieloide e linfoide de paciente com leucemia aguda. Estabelecendo-se o ponto de "cut-off" em 13 mV de quimiluminescencia, todos os casos de leucemia mieloide aguda puderam ser diferenciados dos casos de leucemia linfoide aguda. A tecnica proposta demontrou a atividade peroxidasica inclusive em blastos do tipo MO e M7 (classificaçao FAB) os quais usualmente nao se coram nas preparaçoes citoquimicas classicas e requerem estudos com microscopia eletronica para a detecçao da mieloperoxidade

Interferência do chumbo na atividade enzimática da anidrase carbônica / Interference of lead in the enzymatic activity of carbon anhydrase

Foram estudadas as variaçöes ocorridas com as atividades enzimáticas do ácido delta-aminolevulínico desidratase, e da anidrase carbônica eritrocitária, em grupos de operários. Determinou-se que operários expostos, ocupacionalmente, ao chumbo, apresentam, além da diminuiçäo da atividade da ALA-D, também a diminuiçäo da atividade da anidrase carbônica

Influência do hábito de fumar na atividade da anidrase carbônica eritrocitária / Influence of smoking habit in erythrocyte carbon anhydrase activity

A atividade da anidrase carbônica eritrocitária foi medida em três grupos de indivíduos: um näo fumante, outro fumante moderado e outro fumante imoderado. Os valores obtidos encontram-se significativamente aumentados no grupo fumante em comparaçäo aos abstêmios mesmo no subgrupo de fumantes moderados. A intensidade do hábito de fumar parece näo influir nos valores da anidrase carbônica entre os tabagistas. Valores do hematócrito e da hemoglobina encontram-se significativamente aumentados no grupo fumante