Genetic polymorphisms of NAT2, CYP2E1 and GST enzymes and the occurrence of antituberculosis drug-induced hepatitis in Brazilian TB patients

Isoniazid (INH), one of the most important drugs used in antituberculosis (anti-TB) treatment, is also the major drug involved in hepatotoxicity. Differences in INH-induced toxicity have been attributed to genetic variability at several loci, such as NAT2, CYP2E1, GSTM1 and GSTT1, that code for drug-metabolising enzymes. Our goal was to examine the polymorphisms in these enzymes as susceptibility factors to anti-TB drug-induced hepatitis in Brazilian individuals. In a case-control design, 167 unrelated active tuberculosis patients from the University Hospital of the Federal University of Rio de Janeiro, Brazil, were enrolled in this study. Patients with a history of anti-TB drug-induced acute hepatitis (cases with an increase to 3 times the upper limit of normal serum transaminases and symptoms of hepatitis) and patients with no evidence of anti-TB hepatic side effects (controls) were genotyped for NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms. Slow acetylators had a higher incidence of hepatitis than intermediate/rapid acetylators [22% (18/82) vs. 9.8% (6/61), odds ratio (OR), 2.86, 95% confidence interval (CI), 1.06-7.68, p = 0.04). Logistic regression showed that slow acetylation status was the only independent risk factor (OR 3.59, 95% CI, 2.53-4.64, p = 0.02) for the occurrence of anti-TB drug-induced hepatitis during anti-TB treatment with INH-containing schemes in Brazilian individuals.

Validação de métodos de estimativa da gordura corporal em portadores do HIV/Aids

OBJETIVO: Validar métodos de estimativas da gordura corporal (somatória de espessura de dobras cutâneas, circunferência da cintura (CC) e razão cintura-quadril (RCQ)) em portadores do HIV/Aids, tendo como padrão ouro a absortometria por dupla emissão de raios-X (Dexa) e a tomografia computadorizada de abdômen (TCA). MÉTODOS: Foram estudados 15 portadores do HIV/Aids tratados em uma unidade de saúde coligada a um hospital público universitário, São Paulo. Foram medidas a gordura subcutânea total (GST) mediante a somatória da espessura de sete dobras (bíceps, tríceps, subescapular, axilar média, supra-ilíaca, abdominal e panturrilha medial), a gordura subcutânea central (GSC) (somatória da espessura de quatro dobras) e a gordura subcutânea de membros (GSM) (somatória da espessura de três dobras). A GST, GSC e GSM foram comparadas com as medidas de gordura obtidas pela Dexa. A CC, a RCQ e a GSC foram comparadas com as medidas de gordura obtidas pela TCA. Na análise estatística, utilizou-se o coeficiente de correlação de Pearson (r) e foi utilizado o teste de Mann-Whitney. RESULTADOS: A gordura medida pela Dexa foi correlacionada com GST, a GSC e GSM, mesmo após o ajuste pela idade (r>0,80 para todos). A gordura total de abdômen medida pela TCA foi correlacionada com a CC, RCQ e a GSC após o ajuste pela idade (r>0,80 para todos). CONCLUSÕES: Os métodos de estimativa da gordura corporal devem ser escolhidos de acordo com o tipo de gordura a ser avaliada e podem ser utilizados em pesquisas e nos serviços de saúde como alternativa à Dexa e TCA para portadores do HIV/Aids.

ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.

Human IgG responses against the N-terminal region of Merozoite Surface Protein 1 of Plasmodium vivax

The complete primary structure of the gene encoding the Merozoite Surface Protein 1 of Plasmodium vivax (PvMSP-1) revealed the existence of interspecies conserved regions among the analogous proteins of other Plasmodia species. Here, three DNA recombinant clones expressing 50, 200 and 500 amino acids from the N-terminal region of the PvMSP-1 protein were used on ELISA and protein immunoblotting assays to look at the IgG antibody responses of malaria patients from the Brasilian amazon region of Rondônia. The results showed the existance of P. vivax and P. falciparum IgG antibodies directed against PvMSP-1 antigenic determinants expressed in the clones containing the first 200 and the following 500 amino acids of the molecule, but not within the one expressing the most N-terminal 50 amino acids. Interestingly, there was no correlation between the levels of these IgG antibodies and the previous number of malaria infections.

Vaccine strategies against schistosomiasis

In this review the authors analyze the effector and regulatory mechanisms in the immune response to schistosomiasis. To study these mechanisms two animal models were used, mouse and rat. The mouse totaly permissive host like human, show prominent-T cell control in the acquisition of resistance. But other mechanisms like antibody mediated cytotoxity (ADCC) involving eosinophils and IgG antibodies described in humans, are observed in rats. Also in this animal, it is observed specific IgE antibody high production and blood and tisssue eosinophilia. Using the rat model and schistosomula as target, some ADCC features have emerged: the cellular population involved are bone marrow derived inflammatory cell (mononuclear phagocytes, eosinophils and platelets), interacting with IgE through IgE Fc receptors. Immunization has been attempted using the recombinant protein Sm28/GST. Protection has been observed in rodents with significant decrease of parasite fecundity and egg viability affecting the number, size and volume of liver egg granulomas. The association of praziquantel and immunization with with Sm28/GST increases the resistance to infection and decreases egg viability. The authors suggest the possibility of the stablishment of a future vaccine against Schistosoma mansoni.

Vaccine strategies against schistosomiasis

Schistosomiasis, the second major parasitic disease in the world after malaria affects at least 200 million people, 500 million being exposed to the risk of infection. It is widely agreed that a vaccine strategy wich could lead to the induction of effector mechanisms reducing the level of reinfection and ideally parasite fecundity would deeply affect the incidence of pathological manifestations as well as the parasite transmission potentialities. Extensive studies performed in the rat model have allowed the identification of novel effector mechanisms involving IgE antibodies and various inflammatory cell populations (eosinophils, macrophages and platelets) whereas regulation of immune response by blocking antibodies has been evidencial. Recent epidemiological studies have now entirely confirmed in human populations the the role of IgE antibodies in the acquisition of resistance and the association of IgG4 blocking antibodies with increased susceptibility. On the basis of these concepts, several schistosome glutathion S-transferase (Sm 28 GST) appears as a pronising vaccine candidate. Immunization experiments have shown that two complementary goals can be achieved: (a) a partial but significant reduction of the worm population (up to 60//in rats); (b) a significant reduction of parasite fecundity (up in the mice and 85//in cattle) and egg viability (up to 80//). At least two distinct immunological mechanisms account for these two effects. IgE antibodies appear as a major humoral component of acquired resistance whereas IgA antibodies appear as a major humoral factor affecting parasite fecundity. These studies seem to represent a parasite diseases through the identification of potentially protective antigens and of the components of the immune response which vaccination should aim at inducing.

Development of a vaccine strategy against human and bovine schistosomiasis: background and update

Schistosomiasis is a chronic and debilitating parasitic disease that affects over 200 million people throughout the world and causes about 500,000 deaths annually. Two specific characteristics of schistosome infection are of primordial importance to the development of a vaccine: schistosomes do not multiply within the tissues of their definitive hosts (unlike protozoan parasites) and a partial non-sterilizing immunity can have a marked effect on the incidence of pathology and on disease transmission. Since viable eggs are the cause of disease pathology, a reduction in worm fecundity whether or not accompanied by a reduction in parasite burden is a sufficient goal for vaccine induced immunity. We originally showed that IgE antibodies played in experimental models a pivotal role for the development of protective immunity. These laboratory findings have been now confirmed in human populations. Following the molecular cloning and expression of a protein 28 kDa protein of Schistosoma mansoni and its identification as a glutathion S-transferase, immunization experiments have been undertaken in several animal species (rats, mice, baboons). Together with a significant reduction in parasite burden, vaccination with Sm28 GST was recently shown to reduce significantly parasite fecundity and egg viability leading to a decrease in liver pathology. Whereas IgE antibodies were shown to be correlated with protection against infection, IgA antibodies have been identified as one of the factors affecting egg laying and viability. In human populations, a close association was found between IgA antibody production to Sm28 GST and the decrease of egg output. The use of appropriate monoclonal antibody probes has allowed the demonstration that the inhibition of parasite fecundity following immunization was related to the inhibition of enzymatic activity of the molecule. Epitope mapping of Sm28 GST has indicated the prominent role of the N and C terminal domains. Immunization with the corresponding synthetic peptides was followed by a decrease of 70% of parasite fecundity and egg viability. As a preliminary step towards phase I human trials, vaccination experiments have been performed in cattle, a natural model for Schistosoma bovis. Vaccination of calves with the S. bovis GST has led to a reduction of ever 80% of egg output and tissue egg count. Significant levels of protection were also observed in goats after immunization with the recombinant S. bovis GST. Increasing evidence of the participation of IgA antibodies in protective immunity has prompted us toward the development of mucosal immunization. Preliminary results indicate that significant levels of protection can be achieved following oral immunization with live attenuated vectors or liposomes. These studies seem to represent a promising approach towards the future development of a vaccine strategy against one of major human parasitic diseases.

Induction of Glutathione S-transferase Activity in Triatoma infestans

Several synthetic pesticides and allelochemicals used to treat Triatoma infestans adults by topic application showed some degree of cytosolic glutathione S-transferase (GST) induction. General inducers of detoxication systems such as phenobarbital and 3-methylcholantrene topically applied on T. infestans resulted in no GST induction. Meanwhile, general insecticide synergist such as piperonyl butoxide (160 mg/insect) increased the GST-activity in the range of 120-140%. Insects injected with reduced glutathione (300 mg/insect) presented at the forth day elevated GST activity

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Variabilidade genética em populações naturais de Ziziphus joazeiro Mart., por meio de marcadores moleculares RAPD

Os estudos de diversidade genética em populações naturais são imprescindíveis para a elaboração de estratégias de conservação. Assim, este trabalho foi realizado com o objetivo de caracterizar geneticamente, por meio de marcadores Random Amplified Polymorphic DNA (RAPD), populações naturais de Ziziphus joazeiro Mart., localizadas na região do Baixo São Francisco sergipano. Foram empregados 20 oligonucleotídeos e, a partir do polimorfismo observado, foram estimadas a porcentagem de polimorfismo, a variabilidade genética e a similaridade genética (Sgij), por meio do coeficiente de Jaccard. O teste de Mantel foi realizado para avaliar a correlação entre a similaridade genética e a distância geográfica; sendo o fluxo gênico também estimado. O polimorfismo observado nas populações de Z. joazeiro variou de 58,1 a 66,5% e a similaridade genética, de 44 a 54%. A similaridade genética não está correlacionada com a distância geográfica, e os valores observados para o índice de diversidade genética de Nei, para o índice de Shannon e para os parâmetros HS, HT e GST foram considerados altos e semelhantes aos encontrados em outras espécies arbóreas. A porcentagem de locos polimórficos foi considerada baixa. Maior identidade genética foi encontrada entre as populações de Canindé do São Francisco e Santana do São Francisco; e a maior distância genética entre as populações de Canhoba e Canindé do São Francisco. O fluxo gênico foi maior que 1. Com base nos resultados, pode-se afirmar que há alta variabilidade genética entre as populações e que estas podem estar geneticamente estruturadas.

Genetic structure and diversity of Copaifera langsdorffii Desf. in Cerrado fragments of the São Paulo State, Brazil

The loss of large areas of Cerrado (Brazilian savanna) in Brazil can lead to reduced biodiversity and to the extinction of species. Therefore, the present study aimed to investigate the genetic fragility of populations of Copaifera langsdorffii Desf exposed to different anthropic conditions in fragments of Cerrado in the state of São Paulo. The study was carried out in two Experimental Stations operated by the Forest Institute (Assis and Itirapina), in one fully protected conservation unit (Pedregulho) and in one private property (Brotas). Analyses were conducted using leaf samples from 353 adult specimens and eight pairs of microsatellite loci. The number of alleles per locus ranged from 13 to 15 in all populations, but the mean number of effective alleles was approximately half this value (7.2 to 9-1). Observed heterozygosity was significant and lower than the expected in all populations. Consequently, all populations deviated from Hardy-Weinberg expected frequencies. Fixation indexes were significant for all populations, with the Pedregulho population having the lowest value (0.189) and Itirapina having the highest (0.283). The analysis of spatial genetic structure detected family structures at distance classes of 20 to 65 m in the populations studied. No clones were detected in the populations. Estimates of effective population size were low, but the area occupied by each population studied was large enough for conservation, medium and long term. Recent reductions or bottlenecks were detected in all four populations. Mean Gst’ (genetic divergence) indicated that most of the variation was within populations. Cluster structure analysis based on the genotypes detected K= 4 clusters with distinct allele frequencies patterns. The genetic differentiation observed among populations is consistent with the hypothesis of genetic and geographic isolation. Therefore, it is essential to adopt conservation strategies that raise the gene flow between fragments.

Atividade de glutationa S-transferase na degradação do herbicida glyphosate em plantas de milho (Zea mays)

A glutationa S-transferase (GST, EC 2.5.1.18) desempenha um papel importante na resposta do estresse causado por herbicidas nas plantas; é considerada uma enzima de desintoxicação, por metabolizar grande variedade de compostos xenobióticos, por meio da conjugação destes com glutationa reduzida, formando substâncias de baixa toxicidade. O milho (Zea mays) foi escolhido neste trabalho por apresentar problemas de injúrias quando submetido ao controle químico de plantas daninhas, por meio do uso de herbicidas. Esta pesquisa teve como objetivo determinar as alterações na atividade desta enzima em plantas de milho submetidas ao tratamento pelo herbicida glyphosate. O delineamento experimental utilizado foi o inteiramente casualizado, em esquema fatorial 4x4, com quatro tratamentos herbicidas (glyphosate nas concentrações de 1.000, 2.500 e 5.000 ppm e as plantas-controle tratadas com água) e quatro estádios de desenvolvimento (9, 16, 23 e 30 dias após a emergência), com cinco repetições. O herbicida foi aplicado na parte aérea das plântulas de milho. A parte aérea foi coletada às 24, 48 e 72 horas após a aplicação do herbicida e utilizada para a determinação da atividade da GST e do teor de lipoperóxidos. Foi verificado que os teores de lipoperóxidos não foram alterados pelo tratamento com o glyphosate, porém a atividade de GST aumentou na maioria dos tratamentos utilizados, indicando ter ação na degradação do herbicida glyphosate em plantas de milho.

Avaliações fisiológicas e bioquímicas de plantas de aguapé (Eichhornia crassipes) cultivadas com níveis excessivos de nutrientes

Plantas de aguapé foram cultivadas em solução nutritiva de Hoagland & Arnon n.2, cujo aumento dos níveis de N, P e Cu estabeleceu as diferenças entre os tratamentos. Utilizou-se o delineamento experimental inteiramente casualizado, com quatro repetições. As variáveis fisiológicas avaliadas foram área foliar, peso de matéria seca e taxa de crescimento absoluto, taxa de crescimento relativo, taxa assimilatória líquida, razão de área foliar, peso específico de folha, área foliar específica. Foram determinados também os teores de açúcares totais e redutores e de aminoácidos totais e a atividade das enzimas glutationa S-transferase e superóxido dismutase. Os extratos enzimáticos foram obtidos da matéria fresca da parte aérea das plantas. Após a coleta, foram determinados os pesos de material seco de raízes, pecíolos e folhas, que foram utilizados para a determinação de açúcares solúveis totais e redutores e de aminoácidos. O excesso de nitrogênio causou aumento de açúcares nas folhas e de aminoácidos nas raízes. Já o tratamento com excesso de fósforo levou ao aumento de açúcares nas raízes. Os resultados apresentados sugerem que, entre os nutrientes em excesso avaliados, o cobre (0,12 mg L-1) foi o maior indutor da atividade da GST e SOD, sugerindo que este elemento induziu estresse nas plantas de aguapé.

Mefenpyr-diethyl action on fenoxaprop-p-ethyl detoxification in wheat varieties

Safeners protect crops against herbicide injury. The aim of this study was to examine the differential susceptibility of five wheat (Triticum aestivum) varieties to the herbicide fenoxaprop-p-ethyl, as well as the performance of mefenpyr-diethyl on minimizing herbicide injury and on lipid contents. Varieties BRS 49, CD 104, CEP 24, IAPAR 78 and Rubi were sprayed with fenoxaprop-p-ethyl (69 g ha¹), fenoxaprop-p-ethyl + mefenpyr-diethyl (69 g + 18.75 g ha-1), or mefenpyr-diethyl (18.75 g ha-1). Plants were evaluated visually for injury at 7 and 14 days after treatment (DAT). Glutathione S-transferase (GST) activity was assayed in aerial parts at 7 DAT, and lipid content was measured at 14 DAT. Varieties CEP 24, IAPAR 78 and Rubi were more tolerant to fenoxaprop-p-ethyl than BRS 49, and CD 104 rapidly recovering from the slight phytotoxicity symptoms produced by the herbicide. Mefenpyr-diethyl prevented crop injury associated with the herbicide. GST activity did not correlate directly with fenoxaprop-p-ethyl detoxification. However, lipid content was related to the susceptibility of wheat to fenoxaprop-p-ethyl treatment.