Implantação da rede laboratorial para realização do ensaio de liberação de interferon-gama (IGRA) para detecção de tuberculose latente no Estado de São Paulo: primeiros passos e desafios / Implementation of the laboratory network to carry out the interferon-gamma release assay (IGRA) to detect latent tuberculosis in the State of Sao Paulo: first steps and challenges

A tuberculose (TB) continua sendo um grande desafio para a saúde pública mundial e, para um controle eficiente, também é essencial identificar pessoas com tuberculose latente (ILTB). O ensaio de liberação de interferon-gama (IGRA), incorporado pelo SUS em 2021, permitirá ampliar o diagnóstico de ILTB, em complemento à prova tuberculínica. Para essa implantação, as coordenações do Programa Estadual e da Rede de Laboratórios de TB/SP iniciaram a identificação de executores do IGRA a partir da rede de laboratórios de TB e/ou CD4, para verificar possíveis barreiras para implantação do teste. Foram avaliados os insumos e os profissionais para execução do ensaio, a infraestrutura laboratorial e a disponibilidade de equipamentos. Dez laboratórios avaliaram amostras de sangue total com o kit QuantiFERON®-TB Gold Plus e relataram sua experiência quanto à logística de amostras, execução do ensaio e liberação de laudos. Para otimizar o exame, a coleta ocorreu em tubos heparinizados (sódio ou lítio). Foi sugerida a logística da rede de laboratórios de CD4, que foi utilizada por 20% dos laboratórios participantes, enquanto 50% optaram pelo agendamento. Não foram reportadas dificuldades na liberação de laudo. Dois laboratórios avaliaram o número de células T CD4+ prévio e no momento do IGRA, observando diferença em 10% dos pacientes, fator que pode ser relevante na análise do resultado. Ao todo, foram analisadas 383 amostras, 81 (21,1%) reagentes, 297 (77,5%) não reagentes e cinco (1,3%) indeterminados. Foi observada grande variação de positividade (3,6-50,0%) entre os laboratórios, provavelmente devido à população atendida. Apesar dos desafios encontrados, consideramos que a taxa média de positividade (~20%) sugere que a oferta do IGRA na rede pública possibilitará o aumento do diagnóstico de ILTB e melhor controle da TB.

Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review

The liver is primarilyresponsible for energy homeostasis and the regulation of lipid, carbohydrate and protein metabolism. Lipid metabolism consists of distributing lipids to peripheral tissues or ensuring their return to the liver to be reprocessed. Additionally, cellular metabolism isregulated by several molecules in different signaling pathways. Lipid homeostasis in the liver is mainly regulated by AKT, AMPK, SREBP, PPAR, and JNK. The PI3K/AKT/mTOR signaling pathway results in the biosynthesis of macromolecules and regulates lipogenesis and the expression of lipogenic genes. AMPK is an energy sensor that regulates metabolism and is activated when stored ATP is depleted, and it is responsible for the suppression of several key lipogenic factors in the liver related to cholesterol and fatty acid synthesis. SREBPs control lipogenic geneexpressionandcholesterol metabolism and actin the nutritional regulation of fatty acids and triglycerides. The continued activation of SREBPs is associated with cellular stress, inflammation and ultimately steatosis. PPARs are intrinsically important regulators of lipid metabolism. These genes are essential tovarious metabolic processes, especially lipid and glucose homeostasis, and can play a role in cell differentiation. JNK signaling is related to insulin resistance and its activation results in decreased mitochondrial activity and fat accumulation. Therefore, the study of cell signaling pathways related to lipid metabolism and liver function may help to identify abnormalities and develop strategies to manage and regulate metabolic disorders and resulting complications.

Potential application of rLc36 protein for diagnosis of canine visceral leishmaniasis.

Visceral leishmaniasis (VL) is fatal if left untreated. Infected dogs are important reservoirs of the disease, and thus specific identification of infected animals is very important. Several diagnostic tests have been developed for canine VL (CVL); however, these tests show varied specificity and sensitivity. The present study describes the recombinant protein rLc36, expressed by Leishmania infantum, as potential antigen for more sensitive and specific diagnosis of CVL based on an immunoenzymatic assay. The concentration of 1.0 µg/mL of rLc36 enabled differentiation of positive and negative sera and showed a sensitivity of 85% and specificity of 71% (with 95% confidence), with an accuracy of 76%.

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.

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.

Evaluation of the lipid profile between individuals with hepatitis C

Metabolic profiles correlate with hepatitis C virus (HCV) infection and are prognostic for the viral response. However, little is known about the association between lipid profiles and viral load in chronic patients carrying HCV genotypes 1, 2 and 3. The aim of this study was to investigate the influence of the viremia and viral genotype on lipid metabolism by observing the variations in serum lipoprotein and apolipoprotein B, to assess whether HCV predisposes individuals to lipid imbalance and favors the appearance of vascular complications. A sample group of 150 chronic HCV patients with viral genotypes 1, 2 or 3 and a control group of 20 healthy adults (10 men and 10 women), all aged from 20 to 50 years were studied. The serum lipid profile of the chronic patients was analyzed and compared to that of the control group. The high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) and triglyceride levels of the sample group were lower than those of the control group, while the low-density lipoprotein (LDL) and apolipoprotein B levels of the patients were higher. These differences were more significant in patients carrying genotype 3a. There was a positive correlation between the viremia and the changes in apolipoprotein B levels in patients carrying genotype 1b. It was inferred that the risk of developing vascular complications raised in HCV patients. As 90% of LDL protein is composed of apolipoprotein B, the plasmatic concentration of the latter indicates the number of potentially atherogenic particles. Therefore, the lipid profile monitoring may aid in the diagnosis of hepatic infection severity and equally act as a good prognostic marker.

Perfis metabólicos correlacionam-se com infecção pelo vírus da hepatite C (VHC) e são prognósticos da resposta viral em pacientes crônicos. Porém, pouco se sabe a respeito da associação entre perfis lipídicos e a carga viral entre infecções dos genótipos 1, 2 e 3. O objetivo foi estudar a influência da viremia e dos genótipos virais sobre o metabolismo lipídico através das variações de lipoproteínas séricas e apolipoproteína B em hepatopatas crônicos, avaliando se o vírus predispõe os indivíduos a complicações vasculares. O grupo amostral constituiu-se de 150 pacientes crônicos e grupo controle de 20 indivíduos saudáveis. Níveis séricos de HDL, VLDL e triglicérides mostraram-se diminuídos em relação ao grupo controle, enquanto os níveis de LDL e apolipoproteína B mostraram-se elevados. Observou-se correlação positiva entre a viremia e alterações de LDL e apolipoproteína B nos portadores do genótipo 1b. Assim, foi pressuposto que o risco de pacientes portadores do VHC desenvolverem complicações vasculares é elevado, uma vez que cerca de 90% da proteína na LDL constitui-se de apolipoproteína B, sua concentração plasmática indica o número de partículas aterogênicas. Portanto, o monitoramento do perfil lipídico pode auxiliar no diagnóstico da severidade da infecção hepática causada pelo VHC e atuar como bom sinal prognóstico.

[Seropositivity for Chagas disease among blood donors in Araraquara, State of São Paulo, between 2004 and 2008]. / Soropositividade para doença de Chagas entre doadores de sangue em Araraquara, Estado de São Paulo, no período de 2004 a 2008.

INTRODUCTION: Blood transfusion is an important potential source of Chagas disease transmission. METHODS: Files from the Araraquara Regional Blood Center were checked regarding the tests results for Chagas' disease between January 2004 and December 2008. RESULTS: Positive serology was diagnosed in 0.04% of 49,541 blood donations that were performed. Seropositive individuals were aged between 51 and 60 years-old. CONCLUSIONS: The low rate of seropositive donors may reduce the risk of transfusion transmission of Chagas disease. The high occurrence of inconclusive results indicates that the diagnostic methods must be improved.

Soropositividade para doença de Chagas entre doadores de sangue em Araraquara, Estado de São Paulo, no período de 2004 a 2008 / Seropositivity for Chagas disease among blood donors in Araraquara, State of São Paulo, between 2004 and 2008

INTRODUÇÃO: A transfusão sanguínea é uma fonte potencial importante para transmissão da doença de Chagas. MÉTODOS: Foi verificado, nos arquivos do Hemonúcleo Regional de Araraquara, o resultado dos exames para doença de Chagas entre janeiro de 2004 e dezembro de 2008. RESULTADOS: Foram diagnosticadas com sorologia positiva 0,04 por cento das 49541 doações de sangue realizadas. A idade dos soropositivos situou-se entre 51 e 60 anos. CONCLUSÕES: O baixo índice de doadores soropositivos pode reduzir o risco de transmissão via transfusional da doença de Chagas. A alta ocorrência de resultados inconclusivos indicam que os métodos diagnósticos devem ser melhorados.

INTRODUCTION: Blood transfusion is an important potential source of Chagas disease transmission. METHODS: Files from the Araraquara Regional Blood Center were checked regarding the tests results for Chagas' disease between January 2004 and December 2008. RESULTS: Positive serology was diagnosed in 0.04 percent of 49,541 blood donations that were performed. Seropositive individuals were aged between 51 and 60 years-old. CONCLUSIONS: The low rate of seropositive donors may reduce the risk of transfusion transmission of Chagas disease. The high occurrence of inconclusive results indicates that the diagnostic methods must be improved.

Frequency of class I anti-HLA alloantibodies in patients infected by HIV-1

The aim of this study was to evaluate the presence of class I anti-HLA alloantibodies in patients infected by HIV-1 and relate it with the different clinical courses of the disease. Blood samples were collected in EDTA tubes from 145 individuals. HIV-1 infection was confirmed by ELISA test. The presence of class I anti-HLA alloantibodies and HLA allele's were determined. Clinical evolution was set as fast (<1 year between diagnostic and death), moderate (1-3 years) or slow (>3 years). Class I anti-HLA alloantibodies presence was lower in healthy individuals than in those infected by HIV-1 (4.2 percent against 32.4 percent). However, an equal distribution of these alloantibodies was found among the individuals infected, independent on the clinical evolution. Thus, class I anti-HLA alloantibodies was not a determinant factor for patient worsening.

O objetivo deste estudo foi avaliar a presença de aloanticorpos anti-HLA classe I em pacientes infectados pelo HIV-1 e relacioná-la aos diferentes cursos clínicos da doença. Amostras de sangue de 145 indivíduos HIV positivo foram coletadas em tubos com EDTA. A infecção pelo HIV-1 foi confirmada por teste ELISA e a presença de aloanticorpos anti-HLA classe I determinada em seguida. A evolução clínica foi definida como rápida (<1 ano entre diagnóstico e morte), moderada (1-3 anos) ou lenta (>3 anos). A presença de aloanticorpos anti-HLA classe I foi menor em indivíduos saudáveis em relação aos infectados pelo HIV-1 (4,2 por cento contra 32,4 por cento). Porém, a distribuição destes aloanticorpos entre os indivíduos infectados foi igual, independente da evolução clínica. Deste modo, a presença de aloanticorpos anti-HLA classe I não é um fator determinante na piora clínica do paciente.

'Candidatus Liberibacter americanus', associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil.

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacter asiaticus' and in Africa by 'Candidatus Liberibacter africanus'. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. 'Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the alpha-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the alpha-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus 'Ca. Liberibacter'. However, while the 16S rRNA gene sequences of 'Ca. L. asiaticus' and 'Ca. L. africanus' had 98.4% similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96.0% similarity with the 16S rRNA gene sequences of 'Ca. L. asiaticus' or 'Ca. L. africanus'. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the 'Ca. L asiaticus'/'Ca. L. africanus group', but as a separate branch. Within the genus 'Candidatus Liberibacter' and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of 'Ca. L. asiaticus', from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, 'Ca. L. asiaticus' and 'Ca. L. africanus' had quite different sequences, with similarity between 66.0 and 79.5%. These results confirm that the SPS-HLB liberibacter is a novel species for which the name 'Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the 'American' liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of 'Ca. L. asiaticus', suggesting that this psyllid is also a vector of 'Ca. L. americanus' in SPS. 'Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while 'Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that 'Ca. L. americanus' is the major cause of HLB in SPS.

The beta-chemokines MIP-1alpha and RANTES and lipoprotein metabolism in HIV-infected brazilian patients

HIV patients are predisposed to the development of hypertriglyceridemia and hypercholesterolemia as a result of both viral infection and HIV infection therapy, especially the protease inhibitors. Chemokines and cytokines are present at sites of inflammation and can influence the nature of the inflammatory response in atherosclerosis. We investigated the correlation between biochemical variables and beta-chemokines (MIP-1alpha and RANTES) and the apolipoprotein E genotype in HIV-infected individuals. The apolipoproteins were measured by nephelometry. Triglycerides and total cholesterol were determined by standard enzymatic procedures. The beta-chemokines were detected by ELISA. The genetic category of CCR5 and apolipoprotein E were determined by PCR amplification and restriction enzymes. Immunological and virological profiles were assessed by TCD4+ and TCD8+ lymphocyte counts and viral load quantification. Positive correlations were found between apo E and CD8+ (p = 0.035), apo E and viral load (p = 0.018), MIP-1alpha and triglycerides (p = 0.039) and MIP-1a and VLDL (p = 0.040). Negative correlations were found between viral load and CD4+ (p = 0.05) and RANTES and CD4+ (p = 0.029). The beta-chemokine levels may influence lipid metabolism in HIV-infected individuals.

Avaliação do polimorfismo no gene da metilenotetrahidrofolato redutase e concentração de folato e vitamina B12 em pacientes portadores do HIV-1 em tratamento com anti-retrovirais / Evaluation of the polymorphisms in methylenetetrahydrofolate reductase gene and the levels of folate and B12 in HIV-infected patients under antiretroviral therapy

Neste trabalho, investigamos concentração da vitamina B12 e folato, considerando-se a influência dos genótipos da metilenotetrahidrofolato redutase, o perfil imunológico e a terapia antiretroviral utilizada na população brasileira portadora do HIV. Um grupo de 86 indivíduos portadores do HIV-1 e 29 doadores de sangue foram recrutados para compor a casuística. Entre os infectados pelo HIV-1, observou-se menor concentração de B12 no grupo com maior número de linfócitos TCD4+. Não encontramos diferença na distribuição genotípica para as mutações MTHFR C677T e A1298C entre infectados e não infectados pelo HIV-1. Indivíduos portadores do HIV, genótipo C677C, apresentaram concentrações menores de B12 em relação ao grupo controle de mesmo genótipo. A terapia antiretroviral não mostrou qualquer influência nos valores de folato e vitamina B12. Estudos adicionais são necessários para reavaliar a prevalência de menores concentrações de B12 e folato e de hiperhomocisteinemia na população portadora do HIV sob a ótica do uso de HAART e da melhoria na sobrevida dos pacientes.

Aspectos imunologicos do sistema enzimatico fenoloxidase de Schistosoma mansoni / Immunological aspects of the phenol oxidase enzymatic system of Schistosoma mansoni

O sistema enzimatico fenoloxidase (EC1.10.3.1, EC1.10.3.2) esta amplamente distribuido entre os seres vivos, tendo sido descrito em diferentes especies do reino animal e vegetal. Apesar de desempenhar um papel fundamental na formacao da capsula ou parede dos ovos de trematodeos, o sistema enzimatico fenoloxidase (PO) tem sido pouco estudado nesses organismos. No presente trabalho sao apresentados os resultados iniciais de imunizacoes de coelhos contra PO de femeas adultas de S. mansoni e tirosinase de cogumelo (Sigma). As analises imunologicas, realizadas atraves de imunodifusao dupla (teste de Ouchterlony) e imunoeletroforese, revelaram identidade imunitaria parcial entre PO de machos e femeas....

South American rattlesnake venom: its hemolytic power

Foi estudado o poder hemolítico do veneno da cascavel (Crotalus durissus terrificus). Encontrou-se grande número de suas fraçöes sem capacidade de hemolisar eritrócitos de carneiro. O veneno "in cura", recentemente extraído, e em estado líquido tem pouca atividade lítica. A cristalizaçäo do veneno aumenta sua concentraçäo e poder lítico. Os resultados de hemólise do sangue de carneiro obtidos em placas e tubos foram comparados evidenciando um grande número de animais com venenos com alto poder heomlítico. Os valores näo foram proporcionais quando os mesmos venenos foram examinados com hemáceas de homem. Neste caso os percentuais de hemólise foram mais baixos. Pode-se verificar que o poder hemolítico do veneno se relaciona com a concentraçäo e pureza de suas fraçöes