Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR). RESULTS: The range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R(2 )= 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards. CONCLUSION: Both the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement

The Impairment of CD8 Responses Limits the Selection of Escape Mutations in Acute Hepatitis C Virus Infection

Abstract Evasion from protective CD8 responses by mutations within immunodominant epitopes represents a potential strategy of HCV persistence. To investigate the pathogenetic relevance of this mechanism, a careful search for immunodominant CD8 epitopes was conducted in six patients with chronic evolution of HCV infection by analyzing their global CD8 response with a panel of overlapping synthetic peptides covering the overall HCV sequence and by studying the CD8 frequency by tetramer staining. Immunodominant responses were followed longitudinally from the time of acute onset in relation to the evolution of the epitopic sequences. Although intensity of CD8 responses and frequency of HCV-specific CD8 cells declined over time in all patients, mutations emerged in only three of the six acute patients studied. Variant sequences were less efficiently recognized by CD8 cells than parental epitopes and were poorly efficient in inducing a CD8 response in vitro. CD8 epitopes undergoing mutations were targeted by high avidity CD8 cells more efficient in effector function. Our data support the view that immunodominant CD8 responses are affected by inhibitory mechanisms operating early after infection and that the emergence of escape mutations represents an additional mechanism of virus evasion from those CD8 responses that are functionally preserved

A multidisciplinary approach to describe population structure of Solea solea in the Mediterranean Sea

Investigating marine species population structure in a multidisciplinary framework can reveal signatures of potential local adaptation and the consequences for management and conservation. In this study we delineate the population structure of common sole (Solea solea) in the Mediterranean Sea using genomic and otolith data, based on single nucleotide polymorphism (SNPs) markers, otolith shape and otolith trace element composition data. We correlated SNPs with environmental and spatial variables to evaluate the impact of the selected features on the actual population structure. Specifically, we used a seascape genetics approach with redundancy (RDA) and genetic-environmental association (GEA) analysis to identify loci potentially involved in local adaptation. Finally, putative functional annotation was investigated to detect genes associated with the detected patterns of neutral and adaptive genetic variation. Results from both genetic and otolith data suggested significant divergence among putative populations of common sole, confirming a clear separation between the Western and Eastern Mediterranean Sea, as well as a distinct genetic cluster corresponding to the Adriatic Sea. Evidence of fine-scale population structure in the Western Mediterranean Sea was observed at outlier loci level and further differentiation in the Adriatic. Longitude and salinity variation accounted for most of the wide and fine spatial structure. The GEA detected significant associated outlier loci potentially involved in local adaptation processes under highly structured differentiation. In the RDA both spatial distribution and environmental features could partially explain the genetic structure. Our study not only indicates that separation among Mediterranean sole population is led primarily by neutral processes because of low connectivity due to spatial segregation and limited dispersal, but it also suggests the presence of local adaptation. These results should be taken into account to support and optimize the assessment of stock units, including a review and possible redefinition of fishery management units

Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the management of patients.</p> <p>Methods</p> <p>475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay.</p> <p>Results </p> <p>136 out of 475 consecutive samples derived from 48 patients showed evidence of HCMV infection. HCMV DNA was detected in 106 samples, pp65 antigen in 3, and both markers in 27. pp65 antigen detection was associated with higher HCMV DNA levels. The cut-off HCMV DNA level that best predicted pp65 antigenemia in this series of samples was 11,500 copies/ml, but different threshold levels could be observed for specific groups of patients. HCMV disease was observed in 5 out of 48 patients with active HCMV infection. The presence of clinical symptoms was associated with positive pp65 and with higher antigenemia levels. Higher HCMV DNA load at the onset of viral replication was correlated to the development of clinical symptoms.</p> <p>Conclusion</p> <p>Both pp65 antigenemia and HCMV DNA load can be useful for the prospective monitoring of immunocompromised subjects. Specific cut-off levels capable of triggering preemptive antiviral treatment should be determined in accordance to the type of test used and the characteristics of patients and prospectively validated.</p

Immunological and Molecular Correlates of Disease Recurrence after Liver Resection for Hepatocellular Carcinoma

The definition of the risk of hepatocellular carcinoma (HCC) recurrence after resection represents a central issue to improve the clinical management of patients. In this study we examined the prognostic relevance of infiltrating immune cell subsets in the tumor (TIL) and in nontumorous (NT) liver (LIL), and the expression of immune-related and lineage-specific mRNAs in HCC and NT liver derived from 42 patients. The phenotype of infiltrating cells was analyzed by flow cytometry, and mRNA expression in liver tissue was examined by real-time reverse transcription (RT)-PCR. The tumor immune microenvironment was enriched in inhibitory and dysfunctional cell subsets. Enrichment in CD4+ T-cells and in particular CD4 and CD8+ memory subsets within TIL was predictive of better overall survival (OS) and time to recurrence (TTR). Increased programmed death ligand 1 (PDL1) mRNA content and higher prevalence of invariant NKT (iNKT) cells were associated with shorter OS and TTR, respectively. By combined evaluation of infiltrating cell subsets along with mRNA profiling of immune and tumor related genes, we identified the intratumoral frequency of memory T-cells and iNKT-cells as well as PDL1 expression as the best predictors of clinical outcome. HCC infiltrate is characterized by the expression of molecules with negative regulatory function that may favor tumor recurrence and poor survival

Expression du gene codant pour l'insulin-like growth factor 2 (IGF-2) dans le cancer primitif du foie chez l'homme

SIGLEINIST T 70769 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Time for hepatocellular carcinoma immunotherapy: insights for successful clinical applications in this challenging tumor

The multiplicity and phenotype of intratumoral immune infiltrate have been shown to influence the clinical outcome of hepatocellular carcinoma (HCC), thus providing a strong rationale to therapeutic interventions aimed at restoring the dysfunctional immune response against the tumor. Improving the knowledge of the complex interactions between transformed hepatocytes, nonparenchymal resident cells, and infiltrating immune cells (characterizing the HCC microenvironment) will be instrumental to increase the success rate of existing immunotherapeutic strategies and to identify new potential targets for intervention or biomarkers to optimize the selection of candidate patients

A serially coupled stationary phase method for the determination of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine by liquid chromatography ion trap tandem mass spectrometry

Oxidative attack to DNA is of particular interest since DNA modifications can lead to heritable mutations. The most studied product of DNA oxidation is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). While 8-oxodG determination in blood and tissue cells is prone to artifacts, its measurement in urine employing liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained more and more interest for increased reliability. LC-MS/MS can be affected by matrix effects and this is particularly true when ion trap is used as MS analyzer, due to ion accumulation in the trap and related space charge effect. In the present work, we have developed a LC-MS/MS method where the combination of cation exchange and reverse phase solid phases resulted in LC separation optimization. This together with the employment of an isotopically labeled internal standard, allowed the usage of ion trap LC-MS/MS, typically not employed for quantitative measurement in biological samples, for the measurement of 8-oxodG in urine samples from control populations. Four different urine matrices were employed for method validation. Limit of quantitation was set at least at 0.5 ng/ml. While analyzing urine samples from healthy volunteers, 8-oxodG levels reported as ng/ml were statistically different comparing males with females (p<0.05, Mann Whitney test); while comparing results normalized for creatinine no statistical significant difference was found. Mean urinary 8-oxodG level found in healthy volunteers was 1.16±0.46 nmol/mmol creatinine. The present method by enhancing at best the chromatographic performances allows the usage of ion trap LC-MS/MS for the measurement of 8-oxodG in urine samples from control populations

Optimisation of triple therapy for patients with chronic hepatitis C: a systematic review

Background: Triple therapy with Pegylated-Interferon α (PEG-IFNα)/Ribavirin (RBV) and Boceprevir (Boc) or Telaprevir (Tel) significantly improved sustained virological response (SVR) rates for patients with genotype 1 HCV infection compared to PEG-IFNα/RBV alone (dual therapy). However, less is known about factors associated with rates of SVR and of adverse events (AEs). Material and Methods: The aim of this systematic review was to evaluate the evidence regarding the factors affecting response and rate of AEs associated with triple therapy. We performed systematic electronic searches in Medline, Embase, Scopus and Central as well as a list of reference literature. We included randomised controlled trials examining triple therapy compared with dual therapy and reporting data according to patients features and about AEs. Odds ratios (OR) were pooled using either fixed or random effect model, as appropriate. Results: We included data from 14 studies. Treatment with triple therapy increased SVR rate compared to dual therapy especially in patients previously treated with PEG-IFNα/RBV and with increased pretreatment alanine aminotransferase (ALT) levels. Higher rate of serious AEs and treatment discontinuation due to AEs was also observed particularly in treatment-experienced patients. Conclusions: The present study shows how improved results of triple therapy are mainly observed in some patientsâ subsets and are accompanied by increased risk of AEs compared to dual therapy. These results might be useful for optimising treatment of chronic hepatitis C when IFN-free regimens are unavailable

INTRACELLULAR-LOCALIZATION OF FULL-LENGTH AND TRUNCATED HEPATITIS-C VIRUS CORE PROTEIN EXPRESSED IN MAMMALIAN-CELLS

The putative hepatitis C virus core protein has a predicted molecular weight of about 22 kD and contains two carboxy (COOH)-terminal hydrophobic domains. The cleavages generating the hepatitis C virus structural proteins (core, El and E2) are catalyzed by host signal peptidases. In the present study, we investigated the processing and intracellular localization of the hepatitis C virus core protein expressed in mammalian cells. Expression vectors encoding the entire core protein or COOH-terminal deletion mutants under the control of SV40 regulatory sequences were transfected in COS cells. Immunofluorescent staining with either polyclonal immunoglobulin or monoclonal anti-core antibodies showed that fragments containing the COOH-terminal hydrophobic stretch were retained in the cytoplasm of transfected cells, whereas truncated core proteins deleted of 28 or more residues were located in the nucleus. Our results suggest that a putative nuclear targeting sequence is contained in the first 40 residues of the core protein. (C) Journal of Hepatology