Identification of hepatitis B virus A1762T/G1764A double mutant strain in patients in Southern Brazil

Abstract Infection by hepatitis B virus (HBV) is a worldwide public health problem. Chronic HBV infection with high viral replication may lead to cirrhosis and/or hepatocellular carcinoma. Mutant HBV strains, such as the HBV A1762T/G1764A double mutant, have been associated with poor prognosis and higher risk of the patient for developing cirrhosis and/or hepatocellular carcinoma. This study analyzed the presence of the HBV A1762T/G1764A double mutant in patients with chronic HBV and its association with clinical parameters such as viral load, aminotransferases, and HBV antigens. A total of 49 patients with chronic hepatitis B were included in the study, and the HBV A1762T/G1764A double mutant strain was detected in four samples (8.16%) by polymerase chain reaction followed by restriction fragment length analysis (PCR-RFLP). The viral load was not significantly different between patients with or without the double mutant strain (p = 0.43). On the other hand, carriers of the HBV A1762T/G1764A double mutant had higher levels of ALT (p = 0.0028), while AST levels did not differ between groups (p = 0.051). In this study, 75% of the samples with the HBV A1762T/G1764A double mutation were HBeAg negative and anti-HBe positive, reflecting seroconversion even though they still displayed high viral loads. Our study has shown that the HBV A1762T/G1764A double mutant strain circulates in Brazilian patients, and is associated with elevated levels of ALT and HBeAg seroconversion.

Identification of hepatitis B virus A1762T/G1764A double mutant strain in patients in Southern Brazil.

Infection by hepatitis B virus (HBV) is a worldwide public health problem. Chronic HBV infection with high viral replication may lead to cirrhosis and/or hepatocellular carcinoma. Mutant HBV strains, such as the HBV A1762T/G1764A double mutant, have been associated with poor prognosis and higher risk of the patient for developing cirrhosis and/or hepatocellular carcinoma. This study analyzed the presence of the HBV A1762T/G1764A double mutant in patients with chronic HBV and its association with clinical parameters such as viral load, aminotransferases, and HBV antigens. A total of 49 patients with chronic hepatitis B were included in the study, and the HBV A1762T/G1764A double mutant strain was detected in four samples (8.16%) by polymerase chain reaction followed by restriction fragment length analysis (PCR-RFLP). The viral load was not significantly different between patients with or without the double mutant strain (p=0.43). On the other hand, carriers of the HBV A1762T/G1764A double mutant had higher levels of ALT (p=0.0028), while AST levels did not differ between groups (p=0.051). In this study, 75% of the samples with the HBV A1762T/G1764A double mutation were HBeAg negative and anti-HBe positive, reflecting seroconversion even though they still displayed high viral loads. Our study has shown that the HBV A1762T/G1764A double mutant strain circulates in Brazilian patients, and is associated with elevated levels of ALT and HBeAg seroconversion.

Frequency of the mdr1 gene polymorphism rs1045642 (c3435t) in hcv-hiv co-infected patients / Frequência do polimorfismo rs1045642 (C3435T) do gene MDR1 em pacientes coinfectados HCV-HIV

ABSTRACT Background Due to the high prevalence of co-infection by hepatitis C virus (HCV) and human immunodeficiency virus (HIV) and the severity of these infections, the understanding of the biological mechanisms involved in these processes, including viral behavior and host genetic profile, is of great importance for patient treatment and for public health policies.Some single nucleotide polymorphisms (SNPs) in the human genome, such as the SNP rs1045642 (C3435T) in the MDR1 gene, have been reported to be associated to the sustained virological response (SVR) to HCV treatment in HCV-HIV co-infected patients. Objective The present study analyzes the MDR1 gene C3435T polymorphism in HCV-HIV co-infected patients. Methods A total of 99 HCV-HIV patients were included in the study. The DNA was extracted from blood samples, and the SNP rs1045642 was assessed by Real Time PCR (qPCR). Risk factors for acquiring the virus and the SVR after HCV treatment with pegylated interferon-alpha and ribavirin were also analyzed. Results Among the patients, 54 (54.5%) were male and 45 (45.5%) were female. The average age was 46.1±9.8 years. The SVR after HCV treatment was 40%. The frequencies of MDR1 genotypes CC, CT and TT were 28.3%, 47.5% and 24.2%, respectively. Allele frequencies were 52% for the C allele and 48% for the T allele. No association was found for SNP rs1045642 (C3435T) regarding response to treatment (P=0.308). Conclusion - In this study, the C3435T polymorphism in the MDR1 gene appears not to be associated with SVR in HCV-HIV co-infected individuals.

RESUMO Contexto Em virtude da elevada prevalência da coinfecção pelos vírus da hepatite C (HCV) e da imunodeficiência humana (HIV) e às inúmeras complicações que esses vírus acarretam, é fundamental o maior entendimento do comportamento biológico dos mesmos. O polimorfismo de nucleotídeo único rs1045642 C3435T do gene de resistência a múltiplas drogas MDR1, no qual ocorre modificação do códon ATC para ATT, parece estar relacionado à resposta virológica sustentada ao tratamento do HCV em coinfectados HCV-HIV. Objetivo Mapear o polimorfismo C3435T do gene MDR1 em pacientes coinfectados HCV-HIV e correlacionar com dados clínicos e laboratoriais. Métodos Foram analisados 99 pacientes coinfectados HCV-HIV. A identificação molecular do polimorfismo de nucleotídeo único rs1045642 do gene MDR1 foi realizada pela técnica de PCR em tempo real (qPCR) alelo-específico com primers e sondas específicos para a identificação desse polimorfismo. Fatores de risco para a aquisição do HCV e a resposta virológica sustentada ao tratamento do HCV com interferon-alfa peguilado e ribavirina foram analisados. Resultados Dentre os pacientes avaliados, 54 (54,5%) eram do gênero masculino e 45 (45,5%) do gênero feminino. A média de idade foi de 46,1 anos (±9,8). As frequências dos genótipos CC, CT e TT foram 28,3%, 47,5% e 24,2% respectivamente, e as frequências alélicas foram 52% para alelo C e 48% para alelo T. Não houve associação entre o gene MDR1 e a resposta virológica sustentada (P=0,308). Conclusão Neste estudo, o polimorfismo C3435T no gene MDR1 não apresentou associação com a resposta virológica sustentada ao tratamento em indivíduos coinfectados HCV-HIV.

Cationic Nanoemulsions as a Gene Delivery System: Proof of Concept in the Mucopolysaccharidosis I Murine Model.

Mucopolysaccharidosis I (MPS I) is an autosomal recessive lysosomal storage disease due to deficient a-L-iduronidase (IDUA) activity. It results in the accumulation of the glycosaminoglycans (GAGs) heparan and dermatan sulfate and leads to several clinical manifestations. This study describes the use of cationic nanoemulsions as a non-viral carrier for the plasmid named pIDUA, which has the gene that encodes for the IDUA enzyme. Cationic nanoemulsions, composed by a medium chain triglycerides oil core stabilized by DOTAP, DOPE and DSPE-PEG, were prepared by high pressure homogeneization. pIDUA was complexed with nanoemulsions in the end of manufacturing process. Physicochemical properties of complexes were influenced by the charge ratio used. From a charge ratio of +2/-, it was observed a total complexation of pIDUA with formulation as well as a protection of plasmid against DNAse I digestion. In vitro assay in fibroblasts of one MPS I patient presented greater and significant trasfection efficiency for pIDUA complexed to formulation in the +4/- charge ratio. This formulation was administered via the tail vein and the portal vein. Animals were compared to untreated MPS I mice. Transfection efficiency was measured as IDUA enzyme activity. After intravenous administration, IDUA activity was significantly higher in lungs and liver. The set of results shows the formulation obtained at the +4/- charge ratio as a promising non-viral gene delivery system, once showed increased enzyme activity both in vitro and in vivo.

Evaluation of the C3435T polymorphism in the MDR1 gene in patients with hepatocellular carcinoma.

INTRODUCTION: Considering the high prevalence of liver tumors and the impact on patient survival, a greater understanding of the biological behavior of those tumors if of great importance. The multidrug resistance gene (MDR1) may present as single nucleotide polymorphism (SNP) which can affect the expression and activity of P-glycoprotein (Pgp), and high expression of Pgp has been associated with a worse prognosis in affected patients. OBJECTIVE: To correlate the C3435T polymorphism in the MDR1 gene with the immunohistochemical expression of Pgp. MATERIAL AND METHODS: A total of 67 samples from patients with diagnosis of hepatocellular carcinoma (HCC), collected in the period from 2000 to 2009, were analyzed. The polymorphism in the MDR1 gene was determined by the technique of allele-specific real time PCR using TaqMan assay, and the expression of protein Pgp was evaluated by immunohistochemistry. RESULTS: Among the samples evaluated, 56 (83.6%) were from male patients and 11 (16.4%) from females. Mean age was 60.6 years (± 8.8), ranging from 37 to 85 years. The etiology of the HCC was related to hepatitis C virus infection (HCV) in 31 (46.3%) of cases, followed by hepatitis C virus infection + alcohol in 24 cases (35.8%), alcohol in 4 cases (6)%, hepatitis B virus (HBV) in 4 cases (6%) and other factors in 4 cases (6%). Liver transplantation was performed in 48 cases (71.6%) and hepatectomia in 19 cases (28.4%). The genotypes CC, CT and TT showed frequencies of 25.4%, 41.8% and 32.8%, respectively, and the allele frequencies were 46.3% for allele C and 53.7% for allele T. The expression of Pgp in over 75% of the cells was significantly more frequent in tumor tissue. On the other hand, a low expression of Pgp, in less than 25% of the cells, was significantly more frequent in non-tumor tissue. The Pgp expression in more than 50% of tumor cells of individuals with genotypes CC, CT and TT was 15.7%, 51.0% and 33.3%, respectively, and was significantly higher when in the presence of allele T (p = 0.002). CONCLUSION: The presence of the polymorphic allele T is related to increased expression of Pgp protein in patients with HCC.

Expression of cell growth negative regulators MEG3 and GADD45γ is lost in most sporadic human pituitary adenomas.

We aimed at the evaluation of MEG3 and GADD45γ expression in sporadic functioning and clinically non-functioning human pituitary adenomas, morphologically characterized by immunohistochemistry analysis and their association with clinical features. Thirty eight patients who had undergone hypophysectomy at São José Hospital of Irmandade Santa Casa de Misericórdia in Porto Alegre, Brazil, were included in this study. We evaluated tumor-type specific MEG3 and GADD45γ expression by qRT-PCR in the pituitary adenomas, and its association with clinical features, as age, gender and tumor size, obtained from medical records. The patients consisted of 21 males and 17 females and the mean age was 47 ± 14 (mean ± SD), ranging from 18 to 73 years-old. Of these 14 were clinically non-functioning, 10 GH-secreting, 9 PRL-secreting, and 5 ACTH-secreting pituitary adenomas. All samples were macroadenomas, except four ACTH-secreting tumors, which were microadenomas. In summary, MEG3 and GADD45γ expression was significantly lost in most clinically non-functioning adenomas (78 and 92%, respectively). Other assessed pituitary tumor phenotypes expressed both genes at significantly different levels, and, in some cases, with overexpression. There was no significant association between gene expression and the analyzed clinical features. Our results confirm the previous report, which indicated that MEG3 and GADD45γ expression is lost in the majority of human pituitary tumors, mainly in clinically-nonfunctioning adenomas. Functioning tumors had differences of relative expression levels. The two groups of tumors are probably genetically different and may have a different natural history.

Anal pressure in experimental diabetes.

PURPOSE: Diabetes mellitus is a metabolic endocrine disorder that affects many systems, the gastrointestinal system often being among the affected systems. This experimental study work was designed to demonstrate altered anal sphincter pressures in an experimental model of diabetes mellitus (DM). MATERIALS AND METHODS: Male Wistar rats (mean weight = 250 g) were used and randomized in two groups (n = 10): CO = control and DM. DM was induced by administering a single dose of streptozotocin. Glycemic levels were measured at the start (time = 0) and end (time = 60) of the experiment and anorectal manometry at the end. RESULTS: DM rats presented a significant increase in glycemia at day 60 (DM = 407.14 +/- 73.76) as compared to the control group (time 0 = 175.7 +/- 18.62 and time 60 = 198.04 +/- 28.66). Anorectal manometry showed a significant decrease in anal pressure in the DM group at day 60 (DM = 34.2 +/- 4.97) as compared to the CO group at the same time (CO = 67.4 +/- 2.06), with P < 0.01 and P < 0.001 (Student's t test). CONCLUSIONS: The results suggest that DM, due to the high glycemic levels, lead to alterations such as anal sphincter hypotony, which may cause complications such as fecal incontinence.

O papel do óxido nítrico na pressão anal esfincteriana de ratos submetidos à colite experimental / The role of nitric oxide in sphincteric anal pressure of rats with experimental colitis

O óxido nítrico (NO) é um radical livre sintetizado endogenamente por várias células do nosso organismo. Apresenta um amplo espectro de ações fisiológicas, sendo as mais importantes o seu mecanismo de ação parácrino no relaxamento da musculatura lisa, sua atividade neurotransmissora em vários sistemas e seu envolvimento no processo inflamatório. O NO é sintetizado em diferentes tecidos através da conversão da L-arginina em L-citrulina pela ação da enzima óxido nítrico sintase (NOS). OBJETIVOS: Este estudo tem por objetivo demonstrar o envolvimento do óxido nítrico no processo intestinal inflamatório de ratos Wistar submetidos à colite experimental com ácido acético. MATERIAL E MÉTODOS: Foram utilizados 20 ratos machos Wistar, com peso entre 250 e 350 gramas divididos em dois grupos de 10 animais. Os animais do grupo em estudo foram submetidos à administração intracolônica, por enema, de uma solução de ácido acético diluído a 7 por cento e com volume de 3 ml. O grupo controle recebeu apenas enema de solução salina. Foram avaliados os índices histológicos, a expressão da enzima óxido nítrico sintase (iNOS) e a pressão anal esfincteriana. RESULTADOS: Os índices histológicos apresentaram uma significativa elevação no grupo colite quando comparados ao grupo controle, tanto na avaliação macroscópica quanto na microscópica. A expressão da enzima iNOS também foi significativamente maior no grupo colite quando comparada ao grupo controle. A pressão anal esfincteriana foi significativamente mais baixa no grupo colite na comparação ao grupo controle. CONCLUSÃO: Os animais submetidos à colite experimental apresentam um aumento da expressão da enzima óxido nítrico sintase induzível (i-NOS). Este aumento, associado ao conseqüente aumento do nível de óxido nítrico, ocasiona uma diminuição dos níveis de pressão anal esfincteriana.

The nitric oxide (NO) is a free radical synthesized from some cells of our organism. It presents with an ample specter of physiological actions being the most important its mechanism of action in the relaxation of the smooth musculature, its neurotransmissor activity in some systems and its involvement in the inflammatory process. The NO is synthesized in different tissues by the conversion of the L-arginine in L-citruline with the action of the enzyme nitric oxide sintase(NOS). OBJECTIVES: the aim of this study is to demonstrate the involvement of nitric oxide in the inflammatory intestinal process of Wistar rats submitted to experimental colitis with ascetic acid. MATERIAL AND METHODS: 20 male Wistar rats had been used with weight between 250 and 350 g divided in two groups of 10 animals. The animals of the group in study had been submitted to intracolonic administration, by enema, of a solution with acid ascetic diluted to 7 percent - 3 ml. The control group received only enema with saline solution. The histological scores, the expression of the enzyme nitric oxide sintase (iNOS) and the sphincteric anal pressure had been evaluated. RESULTS: The histological scores had presented a significant rise in the group colitis when compared with the control group in the macroscopic as well as in the microscopical evaluation. The expression of the enzyme iNOS was also significantly higher in the colitis group when compared to the control group. The sphincteric anal pressure was significantly lower in the group colitis when compared to control group. CONCLUSION: The animals submitted to the experimental colitis presented an increase of the iNOS expression. This increase, associated with the consequent increase in nitric oxide level, causes a reduction of the sphincteric anal pressure levels.