Ricardo Alberto Moliterno

Association of human leukocyte antigen DQ1 and dengue fever in a white Southern Brazilian population

Dengue is an infectious disease of viral etiology transmitted by the mosquitoes Aedes aegypti, A. albopictus, and A. scutellaris. It can develop either as a benign form or as a severe hemorrhagic form. Previous work showed an association of the hemorrhagic form with human leukocyte antigens (HLA), suggesting a role of genetic factors in disease susceptibility. Nevertheless, data on HLA association with the classical form of the disease is scarce in literature. Sixty-four patients and 667 normal individuals, living in the state of Parana, Southern Brazil, were used as test and control group, respectively. The patients developed the disease during a virus 1 dengue outbreak either in Maringa city in 1995 (47) or in Paranavai city in 1999 (17). The diagnostic was confirmed through serology and/or viral culture. HLA class I and II typing was performed by the classical microlynfocitotoxicity test using monoclonal antisera and fluorobeads. Qui-square statistical analysis confirmed a positive association with HLA-DQ1 (76.6% vs 57.7%; p = 0.005243; pc = 0.026215). HLA-DR1 also presented an increased frequency in the test group, not statistically significant after p correction though (32.8% vs 15.9%; p = 0.005729; pc = 0.080206). In conclusion, genetic factors may play a role on the susceptibility to the classical dengue, virus 1, in the Brazilian population. Further independent studies should be performed in the Brazilian population to confirm these preliminary data.

Association of cytokine genetic polymorphism with hepatites B infection evolution in adult patients

The infection by the hepatitis B virus (HBV) has different forms of evolution, ranging from self-limited infection to chronic hepatic disease. The objective of this study was to evaluate the influence of cytokine genetic polymorphisms in the disease evolution. The patients were divided into two groups, one with chronic HBV (n = 30), and the other with self-limited infection (n = 41). The genotyping for TNF (-308), TGFB1 (+869, +915), IL-10 (1082, -819, and -592), IL-6 (-174), and IFNG (+874) was accomplished by the PCR-SSP (polymerase chain reaction with sequence specific primers technique using the One Lambda kit. Although no statistically significant differences were found between the groups, the combination of TNF -308GG and IFNG +874TA was found in a lower frequency in chronic patients than in individuals with self-limited infection (26.7 versus 46.3%; P = 0.079; OR = 0.40; IC95% = 0.14-1.11). In chronic patients with histological alterations it was not observed the genotype TGFB1+869 C/C, against 24.4% in the self limited infection group (100 versus 75.6%; P = 0.096; OR = 7.67; IC95% = 0.42-141.63). Further studies in other populations, and evaluation of a greater number of individuals could contribute for a better understanding of the cytokine genetic polymorphism influence in HBV infection evolution.

KIR genes and their human leukocyte antigen ligands in the progression to cirrhosis in patients with chronic hepatitis C

Natural killer (NK) cells play pivotal roles in immune responses against infection with viruses, such as hepatitis C virus (HCV), and killer cell immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells. The aim of this study was to investigate the possibility that KIR genes and their human leukocyte antigen (HLA) ligands influence progression to cirrhosis in patients infected with genotype 1 of HCV. A total of 145 Brazilian patients with confirmed chronic hepatitis C grouped from F0 to F4 according to fibrosis progression to cirrhosis were evaluated. Genotyping of KIR and HLA genes was performed by polymerase chain reaction with sequence-specific oligonucleotide probes. The HLA-C2 KIR ligand was more frequent in patients than in healthy controls (74.5% vs 64.3%, p = 0.04, odds ratio (OR) = 1.6, 95% confidence interval (CI) = 1.03-2.52). Moreover, the HLA-C1C2 genotype was more frequent in patients with advanced fibrosis or cirrhosis (F3-F4 group) than in patients in the F0-F2 group (61.6% vs 44.7%, p = 0.06) and in the F4 group compared with the F0-F3 group (65.7% vs 46.7%, p = 0.05, OR = 2.19, 95% CI = 1.01-4.73). NK and KIR ligands may contribute to the development of liver damage in patients chronically infected by HCV.

Influence of KIR genes and their HLA ligands in susceptibility to dengue in a population from southern Brazil.

Killer cell immunoglobulin-like receptors (KIR) form a group of regulatory molecules that specifically recognise human leukocyte antigen (HLA) class I molecules, modulating the cytolytic activity of natural killer cells. The purpose of this study was to investigate the influence of KIR genes and their class I HLA ligands in susceptibility to dengue fever in a population from southern Brazil through a case-control study. One hundred four subjects with confirmed diagnoses of dengue participated in this study, along with a control group of 172 individuals from the same geographic area. HLA and KIR genotyping was performed by polymerase chain reaction with sequence-specific oligonucleotide probes (PCR-SSOP) and with sequence-specific primer (PCR-SSP) techniques, respectively. Data analysis showed significant differences for the KIR2DS1 (54.8% vs 40.7%, P = 0.03), KIR2DS5 (50.0% vs 36.0%, P = 0.03) and KIR2DL5 (76.0% vs 56.4%, P = 0.001) genes. With regard to KIR-ligand pairs, positive associations with dengue were observed in KIR3DS1-Bw4 (45.2% vs 29.7%, P = 0.01), KIR3DL1-Bw4 (80.7% vs 65.1%, P < 0.001), KIR2DL1-C2 (75.0% vs 62.2%, P = 0.03) and KIR2DS1-C2 (40.4% vs 25.6%, P = 0.01) interactions, and a negative association in KIR2DL3-C1/C1 (18.2% vs 33.1%, P = 0.01). Furthermore, the analysis of KIR haplogroups showed a possible protective factor against dengue fever in individuals with the AA genotype. Taken together, these results suggest the existence of genetic predisposition to dengue fever in the population from southern Brazil.

Association of cytokine genetic polymorphisms with the humoral immune response to recombinant vaccine against HBV in infants

The prevention of hepatitis B by vaccination is one the most efficient tools to avoid the transmission of the virus, although a considerable variability to the anti‐HBsAg antibody response has been described. Recently, polymorphisms of cytokine regulating genes have been described which seem to influence the immune response to various antigens. This articles objective was to evaluate the influence of cytokine genetic polymorphisms onto the humoral immune response to hepatitis B vaccine in infants. Vaccinated children were classified according to the level of anti‐HBsAg antibody titles. The genotyping for TNF (−308), TGFB1 (+869, +915), IL‐10 (−1082, −819, −592), IL‐6 (−174), and IFNG (+874) was accomplished by the PCR‐SSP technique. The TNF (−308) allele A presented a lower but not statistically significant frequency at 5% level in high responder patients (3.7% vs. 12.3%, P = 0.0919). The same was seen for the TNF (−308) genotype GA (7.4% vs. 24.5%, P = 0.0757). Further studies in other populations and evaluation of a greater number of individuals may contribute for a better understanding of the cytokine gene polymorphism influence in general and TNF polymorphism more specifically in the humoral immune response to the HBsAg vaccination in newborn children. J. Med. Virol. 82:929–933, 2010.

DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors

The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience column kit (One Lambda Inc., San Diego, CA, USA) and a modified salting-out method. Only the salting-out method was able to extract high concentrations of DNA (mean, 180 ng/(1/4)microl), which were measured using the Qubit fluorescence detector (Invitrogen, USA). This method enabled amplification of HLA (human leukocyte antigen) genes using the Luminex PCR-SSO (polymerase chain reaction - sequence-specific oligonucleotide) technology, which demands good quality DNA, and amplification of KIR (killer-cell immunoglobulin-like receptor) genes using an in-house PCR-SSP (polymerase chain reaction - sequence-specific primer) technique, which demands a specific concentration of DNA (10 ng/(1/4)microl). We concluded that the modified salting-out technique was very efficient, simple and fast for DNA extraction from human coagulated blood samples, with the aim of genotyping the HLA and KIR genes.

Protection against hepatitis B by the Butang® recombinant vaccine in newborn children in South Brazil

The prevention of hepatitis B by vaccination is one of the most efficient tools to avoid the transmission of the virus. This study evaluated the immunogenicity of the national vaccine Butang in children born in Campo Mourão City, state of Paraná, Brazil, aged 7 to 12 months, by determining the anti-HBsAg antibodies levels after completion of the National Immunization Program Protocol for hepatitis B. All 70 children evaluated by the MEIA method (immune-enzymatic micro particles) showed seroconversion to the Butang vaccine. Nine children (12.9%) presented a low response, with anti-HBs titers between 11 and 100 mUI/ml; 39 children (55.7%) showed a good response to the vaccine, with titers between 101 and 1000 mUI/ml; and 22 children (31.4%) showed antibodies titers higher than 1000 mUI/ml. The mean titer of the anti-HBs antibody titers was 1408.1 +/- 2870.26 mUI/ml (15.7 to 19560.0 m UI/ml). The levels of antibodies produced by the prematurely-born children were not statistically different from those found in the newborns. Fifty-five children were also evaluated through the ELFA method (ELISA with a final detection in fluorescence), which presented similar results. The results obtained in our study corroborated the effectiveness of the national vaccine Butang in newborn children of Campo Mourão City, Paraná, even if they were premature.

Protective effect of HLA-DRB1 11 and predisposition of HLA-C 04 in the development of severe liver damage in Brazilian patients with chronic hepatitis C virus infection.

The objective of this study was to investigate human leucocyte antigen (HLA) genes in patients chronically infected with hepatitis C virus (HCV) and to analyse the possible role of these genes in the progression of chronic hepatitis C. One hundred and forty‐five (145) Brazilian patients infected only with HCV genotype 1 were evaluated. HLA class I (A*, B*, C*) and class II (DRB1*, DQA1*, DQB1*) typing were carried out by PCR‐SSO, through Luminex technology. Associations were found with protection against development of liver damage by both DRB1*11 (5.0% versus 18.2%, P = 0.0016, OR = 0.23, CI 95% = 0.09–0.58; Pc=0.0208) and DRB1*11‐DQA1*05‐DQB1*03 haplotype (4.2% versus 15.3%, P = 0.0032; OR = 0.24, CI 95% = 0.08‐0.64). Liver damage was associated with HLA‐C*04 in patients with <20 years of infection (38.4% versus 9.1%, P = 0.002, OR = 6.25, CI 95% = 1.97–19.7; Pc=0.0238). It is concluded that HLA alleles can influence the development of liver damage in HCV type‐1 chronically infected Brazilian patients.

Influence of HLA alleles in response to treatment with pegylated interferon-alpha and ribavirin in patients with chronic hepatitis C

The objective of this study was to analyse the possible role of HLA polymorphism of chronically infected hepatitis C virus patients in the response outcome to treatment with pegylated interferon‐alpha plus ribavirin. To that end, 144 Brazilian patients infected only with genotype 1 of the virus were treated with pegylated interferon‐alpha at 1.5 μg kg−1 in conjunction with ribavirin (1000 mg if patient weight was <75 kg and 1250 mg if >75 kg) for 48 weeks. The patients did not have concomitant HBV or HIV infections or liver disease, did not undergo previous antiviral treatment, and were followed up for 24 weeks after the end of treatment to assure they presented a sustained virological response. Patients were classified according to response to treatment in responsive (SVR), nonresponsive (NRS) and relapsers (REL). HLA class I and class II typing were carried out through PCR‐SSO using Luminex technology. A statistically higher frequency of DRB1*11 patients was observed in the SVR group (39.6% vs. 14.3%P = 0.0012; Pc = 0.0156; OR = 3.94; 95% CI = 1.8–8.8). HLA‐DQB1*03 patients were also more frequent in the SVR group, but the P value lost significance after Bonferroni correction (62.3% vs. 41.7%P = 0.024; Pc = 0.14, OR = 2.3; 95% CI = 1.14–4.60). HLA class II antigens can positively influence the response to treatment with pegylated interferon‐alpha and ribavirin.

Influence of cytokine and cytokine receptor gene polymorphisms on the degree of liver damage in patients with chronic hepatitis C

Hepatic fibrosis may be the result of repetitive injury to hepatocytes caused by HCV infection and the immune response to it. Cytokines regulate the inflammatory response to injury and modulate hepatic fibrogenesis. Single nucleotide polymorphisms (SNPs) located in cytokine genes may influence the cytokine expression and secretion that may contribute to hepatic fibrogenesis in HCV infection. The aim of this study was to determine the genotype of 22 SNPs found in the genes of 13 cytokines/cytokine receptors to assess the influence of polymorphic variants on the stage of liver damage in Brazilian patients chronically infected with HCV genotype 1 only. 141 unrelated patients were grouped according to their stage of fibrosis: absence of fibrosis or patients in the initial stages of fibrosis (F0-F2, n = 84), patients with advanced stages of fibrosis or cirrhosis (F3-F4, n = 57), without cirrhosis (F0-F3, n = 103), and with cirrhosis (F4, n = 38). The comparison of frequencies in each sub-sample was performed by 2 × 2 contingency tables using the chi-square or Fishers exact test. Stepwise logistic regression was also used to assess independent associations between cirrhosis or fibrosis with polymorphic variants. The TNFA-308G:A genotype conferred increased risk of fibrosis and cirrhosis. The TNFA-238G:G genotype was associated with protection from cirrhosis. The IL10-819C:T genotype conferred protection from fibrosis and the IL1B-511C:T genotype conferred increased risk of cirrhosis. Some of these genotypes showed results on the borderline of statistical significance in the bivariate analysis. We conclude that gene variants of cytokines/receptors may influence liver damage in patients chronically infected by HCV genotype 1.