Toll-like receptor 3 gene polymorphisms are not associated with the risk of hepatitis B and hepatitis C virus infection

INTRODUCTION: The present study investigated the prevalence of two single-nucleotide polymorphisms (SNPs) in the Toll-like receptor 3 (TLR3) gene in patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). METHODS: Samples collected from HCV (n = 74) and HBV (n = 35) carriers were subjected to quantitative real-time PCR (qPCR) to detect the presence of the SNPs rs5743305 and rs3775291 in TLR3 and to measure the following biomarkers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and prothrombin time (PT). A healthy control group was investigated and consisted of 299 HCV- and HBV-seronegative individuals. RESULTS: No significant differences in allele, genotype and haplotype frequencies were observed between the investigated groups, and no association was observed between the polymorphisms and histopathological results. Nevertheless, genotypes TA/AA (rs5743305) and GG (rs3775291) appear to be associated with higher levels of ALT (p<0.01), AST (p<0.05) and PT (p<0.05). In addition, genotypes TT (rs5743305; p<0.05) and GG (rs3775291; p<0.05) were associated with higher GGT levels. CONCLUSIONS: This genetic analysis revealed the absence of an association between the polymorphisms investigated and susceptibility to HBV and HCV infection; however, these polymorphisms might be associated with a greater degree of biliary damage during the course of HCV infection.

Hepatitis B: epidemiological, immunological, and serological considerations emphasizing mutation

The global prevalence of hepatitis B virus is estimated to be 350 million chronic carriers, varying widely from low (<2%, as in Western Europe, North America, New Zealand, Australia, and Japan) to high (>8% as in Africa, Southeast Asia, and China). The overall prevalence in Brazil is about 8%. There are currently 7 genotypic variations, from A to G, and also 4 main surface antigen subtypes: adw, ayw, adr, and ayr. There has been great interest in identifying the geographic distribution and prognosis associated with the various genotypes and subtypes. Although the serologic test is highly sensitive and specific, it does not detect cases of mutant hepatitis B, which is increasingly common worldwide due to resistance and vaccine escape, antiviral therapy, and immunosuppression, among other causes. Alterations in surface, polymerase, X region, core, and precore genes have been described. The main mutations occur in surface and in core/precore genes, also known as occult hepatitis, since its serologic markers of active infection (HBsAg) and viral replication (HBeAg) can be negative. Thus, mutation should be suspected when serologic tests to hepatitis B show control of immunity or replication coincident with worsened clinical status and exclusion of other causes of hepatitis.

Simultaneous LC-MS/MS quantification of P-glycoprotein and cytochrome P450 probe substrates and their metabolites in DBS and plasma.

BACKGROUND: An LC-MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. RESULTS: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. CONCLUSION: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

CYP3A activity measured by the midazolam test is not related to 3435 C >T polymorphism in the multiple drug resistance transporter gene.

A recent study with 69 Japanese liver transplants treated with tacrolimus found that the MDR13435 C >T polymorphism, but not the MDR12677 G >T polymorphism, was associated with differences in the intestinal expression level of CYP3A4 mRNA. In the present study, over 6 h, we measured the kinetics of a 75 microg oral dose of midazolam, a CYP3A substrate, in 21 healthy subjects genotyped for the MDR13435 C >T and 2677 G >T polymorphism. No statistically significant differences were found in the calculated pharmacokinetic parameters between the three 3435 C >T genotypes (TT, CT and CC group, respectively: Cmax (mean +/- SD: 0.30 +/- 0.08 ng/ml, 0.31 +/- 0.09 ng/ml and 0.31 +/- 0.11 ng/ml; Apparent clearance: 122 +/- 29 l/h, 156 +/- 92 l/h and 111 +/- 35 l/h; t1/2: 1.9 +/- 1.1 h, 1.6 +/- 0.90 h and 1.7 +/- 0.7 h). In addition, the 30-min 1'OH midazolam to midazolam ratio, a marker of CYP3A activity, determined in 74 HIV-positive patients before the introduction of antiretroviral treatment, was not significantly different between the three 3435 C >T genotypes (mean ratio +/- SD: 3.65 +/- 2.24, 4.22 +/- 3.49 and 4.24 +/- 2.03, in the TT, CT and CC groups, respectively). Similarly, no association was found between the MDR12677 G >T polymorphism and CYP3A activity in the healthy subjects or in the HIV-positive patients. The existence of a strong association between the activity of CYP3A and MDR13435 C >T and 2677 G >T polymorphisms appears unlikely, at least in Caucasian populations and/or in the absence of specific environmental factors.

Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study.

BACKGROUND:HIV-1-infected patients vary considerably by their response to antiretroviral treatment, drug concentrations in plasma, toxic events, and rate of immune recovery. This variability could have a genetic basis. We did a pharmacogenetics study to analyse the association between response to antiretroviral treatment and allelic variants of several genes. METHODS:In 123 patients, we did PCR analyses of the gene for the multidrug-resistance transporter (MDR1), which codes for P-glycoprotein, of genes coding for isoenzymes of cytochrome P450, CYP3A4, CYP3A5, CYP2D6, and CYP2C19, and of the gene for the chemokine receptor CCR5. We measured concentrations in plasma of the antiretroviral agents efavirenz and nelfinavir by high-performance liquid-chromatography, and measured levels of P-glycoprotein expression, CD4-cell count, and HIV-1 viraemia. FINDINGS: Median drug concentrations in patients with the MDR1 3435 TT, CT, and CC genotypes were at the 30th, 50th, and 75th percentiles, respectively (p=0.0001). In patients with CYP2D6 extensive-metaboliser or poor-metaboliser alleles, median drug concentrations were at percentiles 45 and 62.5, respectively (p=0.04). Patients with the MDR1 TT genotype 6 months after starting treatment had a greater rise in CD4-cell count (257 cells/microL) than patients with the CT (165 cells/microL) and CC (121 cells/microL) genotype (p=0.0048), and the best recovery of naïve CD4-cells. INTERPRETATION:The polymorphism MDR1 3435 C/T predicts immune recovery after initiation of antiretroviral treatment. This finding suggests that P-glycoprotein has an important role in admittance of antiretroviral drugs to restricted compartments in vivo.

Inhibition of glial cell proinflammatory activities by peroxisome proliferator-activated receptor gamma agonist confers partial protection during antimyelin oligodendrocyte glycoprotein demyelination in vitro.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.

A Study of Cryptosporidium parvum Genotypes and Population Structure

Genetic evidence for the occurrence of two Cryptosporidium parvum subgroups is presented. This evidence is based on restriction fragment length polymorphism analysis of several independent loci. Sequence analysis of the b -tubulin intron revealed additional polymorphism. The stability of the genetic profiles following passage of C. parvum isolates between different hosts was investigated.

Association of CYP3A5 genotypes with blood pressure and renal function in African families.

OBJECTIVE: Renal cytochrome P450 3A5 (CYP3A5) activity has been associated with blood pressure and salt sensitivity in humans. We determined whether CYP3A5 polymorphisms are associated with ambulatory blood pressure (ABP) and with glomerular filtration rate (GFR) in African families. METHODS: Using a cross-sectional design, 375 individuals from 72 families, each with at least two hypertensive siblings, were recruited through a hypertension register in the Seychelles (Indian Ocean). We analyzed the association between the CYP3A5 alleles (*1, *3, *6 and *7) and ABP, GFR and renal sodium handling (fractional excretion of lithium), from pedigree data, allowing for other covariates and familial correlations. RESULTS: CYP3A5*1 carriers increased their daytime systolic and diastolic ABP with age (0.55 and 0.23 mmHg/year) more than non-carriers (0.21 and 0.04 mmHg/year). CYP3A5*1 had a significant main effect on daytime systolic/diastolic ABP [regression coefficient (SE): -29.6 (10.0)/-8.2 (4.1) mmHg, P = 0.003/0.045, respectively] and this effect was modified by age (CYP3A5*1 x age interactions, P = 0.017/0.018). For night-time ABP, the effect of CYP3A5*1 was modified by urinary sodium excretion, not by age. For renal function, CYP3A5*1 carriers had a 7.6(3.8) ml/min lower GFR (P = 0.045) than non-carriers. Proximal sodium reabsorption decreased with age in non-carriers, but not in CYP3A5*1 carriers (P for interaction = 0.02). CONCLUSIONS: These data demonstrate that CYP3A5 polymorphisms are associated with ambulatory BP, CYP3A5*1 carriers showing a higher age- and sodium- related increase in ABP than non-carriers. The age effect may be due, in part, to the action of CYP3A5 on renal sodium handling.

CYP3A5 and ABCB1 genes influence blood pressure and response to treatment, and their effect is modified by salt.

The permeability-glycoprotein efflux-transporter encoded by the multidrug resistance 1 (ABCB1) gene and the cytochromes P450 3A4/5 encoded by the CYP3A4/5 genes are known to interact in the transport and metabolism of many drugs. Recent data have shown that the CYP3A5 genotypes influence blood pressure and that permeability-glycoprotein activity might influence the activity of the renin-angiotensin system. Hence, these 2 genes may contribute to blood pressure regulation in humans. We analyzed the association of variants of the ABCB1 and CYP3A5 genes with ambulatory blood pressure, plasma renin activity, plasma aldosterone, endogenous lithium clearance, and blood pressure response to treatment in 72 families (373 individuals; 55% women; mean age: 46 years) of East African descent. The ABCB1 and CYP3A5 genes interact with urinary sodium excretion in their effect on ambulatory blood pressure (daytime systolic: P=0.05; nighttime systolic and diastolic: P<0.01), suggesting a gene-gene-environment interaction. The combined action of these genes is also associated with postproximal tubular sodium reabsorption, plasma renin activity, plasma aldosterone, and with an altered blood pressure response to the angiotensin-converting enzyme inhibitor lisinopril (P<0.05). This is the first reported association of the ABCB1 gene with blood pressure in humans and demonstration that genes encoding for proteins metabolizing and transporting drugs and endogenous substrates contribute to blood pressure regulation.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Hepatitis B and C in the hemodialysis unit of Tocantins, Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of time on hemodialysis were associated with HBV and HCV positivity, respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14 HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes 1a and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.

Sequence-Based Hepatitis B Virus Antiviral Resistance Testing in Switzerland

BACKGROUND: A growing number of patients with chronic hepatitis B is being treated for extended periods with nucleoside and/or nucleotide analogs. In this context, antiviral resistance represents an increasingly common and complex issue. METHODS: Mutations in the hepatitis B virus (HBV) reverse transcriptase (rt) gene and viral genotypes were determined by direct sequencing of PCR products and alignment with reference sequences deposited in GenBank. RESULTS: Plasma samples from 60 patients with chronic hepatitis B were analyzed since March 2009. The predominant mutation pattern identified in patients with virological breakthrough was rtM204V/I ± different compensatory mutations, conferring resistance to L-nucleosides (lamivudine, telbivudine, emtricitabine) and predisposing to entecavir resistance (n = 18). Complex mutation patterns with a potential for multidrug resistance were identified in 2 patients. Selection of a fully entecavir resistant strain was observed in a patient exposed to lamivudine alone. Novel mutations were identified in 1 patient. Wild-type HBV was identified in 9 patients with suspected virological breakthrough, raising concerns about treatment adherence. No preexisting resistance mutations were identified in treatment-naïve patients (n = 13). Viral genome amplification and sequencing failed in 16 patients, of which only 2 had a documented HBV DNA > 1000 IU/ml. HBV genotypes were D in 28, A in 6, B in 4, C in 3 and E in 3 patients. Results will be updated in August 2010 and therapeutic implications discussed. CONCLUSIONS: With expanding treatment options and a growing number of patients exposed to nucleoside and/or nucleotide analogs, sequence-based HBV antiviral resistance testing is expected to become a cornerstone in the management of chronic hepatitis B.

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.