In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated. Copyright (C) 2003 John Wiley Sons, Ltd.

Amino acids 1-20 of the hepatitis C virus (HCV) core protein specifically inhibit HCV IRES- dependent translation in Hep G2 cells, and inhibit both HCV IRES- and cap-dependent translation in HuH7 and CV-1 cells.

A self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES- and cap-dependent translation in an in vitro system. This effect was duplicated in a dose-dependent manner with a synthetic peptide representing amino acids 1-20 of the HCV core protein. This peptide was able to bind to the HCV IRES as shown by a mobility shift assay. In contrast, a peptide derived from the hepatitis B virus (HBV) core protein that contained a similar proportion of basic residues was unable to inhibit translation or bind the HCV IRES. A recombinant vaccinia-HCV core virus was used to examine the effect of the HCV core protein on HCV IRES-dependent translation in cells and this was compared with the effects of an HBV core-recombinant vaccinia virus. In CV-1 and HuH7 cells, the HCV core protein inhibited translation directed by the IRES elements of HCV, encephalomyocarditis virus and classical swine fever virus as well as cap-dependent translation, whereas in HepG2 cells, only HCV IRES-dependent translation was affected. Thus, the ability of the HCV core protein to selectively inhibit HCV IRES-dependent translation is cell-specific. N-terminal truncated (aa 1-20) HCV core protein that was expressed from a novel recombinant vaccinia virus in cells abrogated the inhibitory phenotype of the core protein in vivo, consistent with the above in vitro data.

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor ( GlyR) alpha(1) subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha(1) subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His(107) from one subunit and His(109) from an adjacent subunit. This was tested by co-expressing alpha(1) subunits containing the H107A mutation with alpha(1) subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha(1) homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha(1) subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha(1)beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha(1) homomers. The binding of zinc at the interface between adjacent alpha(1) subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

Effect of a sardine supplement on C-reactive protein in patients receiving hemodialysis

Objective - The study evaluated the effect of a canned sardine supplement in C-reactive protein (CRP) in patients on hemodialysis (HD) and the compliance and adherence to this supplement. Design - This was a quasi-experimental study: Participants with a serum CRP of 5 mg/dL or less volunteered to consume a sardine supplement or were maintained on the usual cheese/ham sandwich supplement. Setting - The study took place in two outpatient dialysis units in Lisbon, Portugal. Patients - The study comprised 63 patients receiving maintenance HD three times per week for at least 6 months and an initial CRP concentration of 5 mg/dL or less. Exclusion criteria included the presence of graft vascular access or history of cancer. Intervention - After a 4-week washout period, the nutritional intervention included a canned sardine sandwich for the case group (n = 31) and a cheese or ham sandwich for the control group (n = 32), to be ingested during each routine HD session, 3 times per week, for 8 weeks. Main outcome measure - Serum levels of high-sensitivity CRP were the outcome measure. Results - Only 65 patients from the invited 186 patients met the inclusion criteria and agreed to eat the sardine sandwich supplement three times per week and were involved in the study. A significant proportion of 48% (n = 31, case group) consumed the sardine sandwich supplement three times per week for 8 weeks, fulfilling the requirements and completing the study. The present investigation showed that a sardine sandwich supplement had no effect on CRP levels among patients on HD. However, when participants were stratified according to tertiles of CRP distribution values at baseline, a reduction in CRP levels was found for those in the higher tertile, being higher for the case group (P = .047). Although diabetic patients were excluded from the analysis (eight in the sardine supplementation group and seven in the control group) a significant CRP reduction was found (P = .034). Conclusion - Although a supplement of low-dose n-3 long-chain polyunsaturated fatty acids had no effect on the plasma high-sensitivity CRP of the supplemented group, a reduction in CRP levels was found when patients were stratified for tertiles of CRP (for the upper tertile) and diabetic status (for nondiabetic patients). These findings need to be further confirmed. This canned sardine supplement was accepted by an important proportion of patients, enhancing diet variety and contributing for a greater n-3 long-chain polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid intake.

Estrogen receptor dependent genetic and epigenetic factors of tamoxifen resistance

Resumo: A decisão da terapêutica hormonal no tratamento do cancro da mama baseiase na determinação do receptor de estrogénio alfa por imunohistoquímica (IHC). Contudo, a presença deste receptor não prediz a resposta em todas as situações, em parte devido a limitações do método IHC. Investigámos se a expressão dos genes ESR1 e ESR2, bem como a metilação dos respectivos promotores, pode estar relacionada com a evolução desfavorável de uma proporção de doentes tratados com tamoxifeno assim como com a perda dos receptores de estrogénio alfa (ERα) e beta (ERß). Amostras de 211 doentes com cancro da mama diagnosticado entre 1988 e 2004, fixadas em formalina e preservadas em parafina, foram utilizadas para a determinação por IHC da presença dos receptores ERα e ERß. O mRNA total do gene ESR1 e os níveis específicos do transcrito derivado do promotor C (ESR1_C), bem como dos transcritos ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 foram avaliados por Real-time PCR. Os promotores A e C do gene ESR1 e os promotores 0K e 0N do gene ESR2 foram investigados por análise de metilação dos dinucleotidos CpG usando bisulfite-PCR para análise com enzimas de restrição, ou para methylation specific PCR. Atendendo aos resultados promissores relacionados com a metilação do promotor do gene ESR1, complementamos o estudo com um método quantitativo por matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) suportado pelo software Epityper para a medição da metilação nos promotores A e C. Fez-se a avaliação da estabilidade do mRNA nas linhas celulares de cancro da mama MCF-7 e MDA-MB-231 tratadas com actinomicina D. Baixos níveis do transcrito ESR1_C associaram-se a uma melhor sobrevivência global (p = 0.017). Níveis elevados do transcrito ESR1_C associaram-se a uma resposta inferior ao tamoxifeno (HR = 2.48; CI 95% 1.24-4.99), um efeito mais pronunciado em doentes com tumores de fenótipo ERα/PgR duplamente positivo (HR = 3.41; CI 95% 1.45-8.04). A isoforma ESR1_C mostrou ter uma semi-vida prolongada, bem como uma estrutura secundária da região 5’UTR muito mais relaxada em comparação com a isoforma ESR1_A. A análise por Western-blot mostrou que ao nível da 21 proteína, a selectividade de promotores é indistinguivel. Não se detectou qualquer correlação entre os níveis das isoformas do gene ESR2 ou entre a metilação dos promotores do gene ESR2, e a detecção da proteína ERß. A metilação do promotor C do gene ESR1, e não do promotor A, foi responsável pela perda do receptor ERα. Estes resultados sugerem que os níveis do transcrito ESR1_C sejam usados como um novo potencial marcador para o prognóstico e predição de resposta ao tratamento com tamoxifeno em doentes com cancro da mama. Abstract: The decision of endocrine breast cancer treatment relies on ERα IHC-based assessment. However, ER positivity does not predict response in all cases in part due to IHC methodological limitations. We investigated whether ESR1 and ESR2 gene expression and respective promoter methylation may be related to non-favorable outcome of a proportion of tamoxifen treated patients as well as to ERα and ERß loss. Formalin-fixed paraffin-embedded breast cancer samples from 211 patients diagnosed between 1988 and 2004 were submitted to IHC-based ERα and ERß protein determination. ESR1 whole mRNA and promoter C specific transcript levels, as well as ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 transcripts were assessed by real-time PCR. ESR1 promoters A and C, and ESR2 promoters 0N and 0K were investigated by CpG methylation analysis using bisulfite-PCR for restriction analysis, or methylation specific PCR. Due to the promising results related to ESR1 promoter methylation, we have used a quantification method by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) together with Epityper software to measure methylation at promoters A and C. mRNA stability was assessed in actinomycin D treated MCF-7 and MDA-MB-231 cells. ERα protein was quantified using transiently transfected breast cancer cells. Low ESR1_C transcript levels were associated with better overall survival (p = 0.017). High levels of ESR1_C transcript were associated with non-favorable response in tamoxifen treated patients (HR = 2.48; CI 95% 1.24-4.99), an effect that was more pronounced in patients with ERα/PgR double-positive tumors (HR = 3.41; CI 95% 1.45-8.04). The ESR1_C isoform had a prolonged mRNA half-life and a more relaxed 5’UTR structure compared to ESR1_A isoform. Western-blot analysis showed that at protein level, the promoter selectivity is undistinguishable. There was no correlation between levels of ESR2 isoforms or ESR2 promoter methylation and ERß protein staining. ESR1 promoter C CpG methylation and not promoter A was responsible for ERα loss. We propose ESR1_C levels as a putative novel marker for breast cancer prognosis and prediction of tamoxifen response.

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

C-reactive protein at 24 hours after hospital admission may have relevant prognostic accuracy in acute pancreatitis: a retrospective cohort study

Introduction: C-reactive protein (CRP) and Bedside Index for Severity in Acute Pancreatitis (BISAP) have been used in early risk assessment of patients with AP. Objectives: We evaluated prognostic accuracy of CRP at 24 hours after hospital admission (CRP24) for in-hospital mortality (IM) in AP individually and with BISAP. Materials and Methods: This retrospective cohort study included 134 patients with AP from a Portuguese hospital in 2009---2010. Prognostic accuracy assessment used area under receiver---operating characteristic curve (AUC), continuous net reclassification improvement (NRI), and integrated discrimination improvement (IDI). Results: Thirteen percent of patients had severe AP, 26% developed pancreatic necrosis, and 7% died during index hospital stay. AUCs for CRP24 and BISAP individually were 0.80 (95% confidence interval (CI) 0.65---0.95) and 0.77 (95% CI 0.59---0.95), respectively. No patients with CRP24 <60 mg/l died (P = 0.027; negative predictive value 100% (95% CI 92.3---100%)). AUC for BISAP plus CRP24 was 0.81 (95% CI 0.65---0.97). Change in NRI nonevents (42.4%; 95% CI, 24.9---59.9%) resulted in positive overall NRI (31.3%; 95% CI, − 36.4% to 98.9%), but IDI nonevents was negligible (0.004; 95% CI, − 0.007 to 0.014). Conclusions: CRP24 revealed good prognostic accuracy for IM in AP; its main role may be the selection of lowest risk patients.

ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Biochemistry, 2004, 43 (46), pp 14566–14576 DOI: 10.1021/bi0485833

C-reactive protein in early risk assessment in acute pancreatitis

ABSTRACT:C-reactive protein (CRP) has been widely used in the early risk assessment of patients with acute pancreatitis (AP), but unclear aspects about its prognostic accuracy in this setting persist. This project evaluated first CRP prognostic accuracy for severity, pancreatic necrosis (PNec), and in-hospital mortality (IM) in AP in terms of the best timing for CRP measurement and the optimal CRP cutoff points. Secondly it was evaluated the CRP measured at approximately 24 hours after hospital admission (CRP24) prognostic accuracy for IM in AP individually and in a combined model with a recent developed tool for the early risk assessment of patients with AP, the Bedside Index for Severity in AP (BISAP). Two single-centre retrospective cohort studies were held. The first study included 379 patients and the second study included 134 patients. Statistical methods such as the Hosmer-Lemeshow goodness-of-fit test, the area under the receiver-operating characteristic curve, the net reclassification improvement, and the integrated discrimination improvement were used. It was found that CRP measured at approximately 48 hours after hospital admission (CRP48) had a prognostic accuracy for severity, PNec, and IM in AP better than CRP measured at any other timing. It was observed that the optimal CRP48 cutoff points for severity, PNec, and IM in AP varied from 170mg/l to 190mg/l, values greater than the one most often recommended in the literature – 150mg/l. It was found that CRP24 had a good prognostic accuracy for IM in AP and that the cutoff point of 60mg/l had a negative predictive value of 100%. Finally it was observed that the prognostic accuracy of a combined model including BISAP and CRP24 for IM in AP could perform better than the BISAP alone model. These results might have a direct impact on the early risk assessment of patients with AP in the daily clinical practice.--------- RESUMO: A proteina c-reactiva (CRP) tem sido largamente usada na avaliação precoce do risco em doentes com pancreatite aguda (AP), mas aspectos duvidosos acerca do seu valor prognóstico neste contexto persistem. Este projecto avaliou primeiro o valor prognóstico da CRP para a gravidade, a necrose pancreática (PNec) e a mortalidade intra-hospitalar (IM) na AP em termos do melhor momento para efectuar a sua medição e dos seus pontos-de-corte óptimos. Em segundo lugar foi avaliado o valor prognóstico da proteína c-reactiva medida aproximadamente às 24 horas após a admissão hospitalar (CRP24) para a IM na AP isoladamente e num modelo combinado, que incluiu uma ferramenta de avaliação precoce do risco em doentes com AP recentemente desenvolvida, o Bedside Index for Severity in Acute Pancreatitis (BISAP). Dois estudos unicêntricos de coorte retrospectivo foram realizados. O primeiro estudo incluiu 379 doentes e o segundo estudo incluiu 134 doentes. Metodologias estatísticas como o teste de Hosmer-Lemeshow goodness-of-fit, a area under the receiver-operating characteristic curve, o net reclassification improvement e o integrated discrimination improvement foram usadas. Verificou-se que a CRP medida às 48 horas após a admissão hospitalar (CRP48) teve um valor prognóstico para a gravidade, a PNec e a IM na AP melhor do que a CRP medida em qualquer outro momento. Observou-se que os pontos de corte óptimos da CRP48 para a gravidade, a PNec e a IM na AP variaram entre 170mg/l e 190mg/l, valores acima do valor mais frequentemente recomendado na literatura – 150mg/l. Verificou-se que a CRP medida aproximadamente às 24 horas após a admissão hospitalar (CRP24) teve um bom valor prognóstico para a IM na AP e que o ponto de corte 60mg/l teve um valor preditivo negativo de 100%. Finalmente observou-se que o valor prognóstico de um modelo combinado incluindo o BISAP e a CRP24 para a IM na AP pode ter um desempenho melhor do que o do BISAP isoladamente. Estes resultados podem ter um impacto directo na avaliação precoce do risco em doentes com AP na prática clínica diária.

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Novel affinity pairs "tag-receptor" for the purification of fusion proteins

Fundação para a Ciência e Tecnologia - SFRH/BD/48804/2008 and the project PTDC/BI/65383/2006 assigned to Prof. Cecíla Roque and also to Associate Laboratory REQUIMTE (Pest-C/EQB/LA0006/2011)

In vitro detection of hepatitis C virus in platelets from uninfected individuals exposed to the virus

IntroductionDespite hepatocytes being the target cells of hepatitis C virus (HCV), viral ribonucleic acid RNA has been detected in other cells, including platelets, which have been described as carriers of the virus in the circulation of infected patients. Platelets do not express cluster differentiation 81 CD81, the main receptor for the virus in hepatocytes, although this receptor protein has been found in megakaryocytes. Still, it is not clear if HCV interacts with platelets directly or if this interaction is a consequence of its association with megakaryocytes. The aim of this study was to evaluate the interaction of HCV with platelets from non-infected individuals, after in vitro exposure to the virus.MethodsPlatelets obtained from 50 blood donors not infected by HCV were incubated in vitro at 37°C for 48h with serum containing 100,000IU∕mL of genotype 1 HCV. After incubation, RNA extracted from the platelets was assayed for the presence of HCV by reverse transcription – polymerase chain reaction RT-PCR.ResultsAfter incubation in the presence of virus, all samples of platelets showed HCV RNA.ConclusionsThe results demonstrate that, in vitro, the virus interacts with platelets despite the absence of the receptor CD81, suggesting that other molecules could be involved in this association.

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.