Quantitation of human herpesvirus-6A, -6B and -7 DNAs in whole blood, mononuclear and polymorphonuclear cell fractions from healthy blood donors

Background

Diagnosis of human herpesvirus-6A (HHV-6A), -6B (HHV-6B) or -7 (HHV-7) infections is often based on the measure of viral load in blood.

Objectives

The aim of this study was to define usual values of HHV-6A, HHV-6B and HHV-7 loads in blood fractions (whole blood [WB], mononuclear cells [PBMCs], polymorphonuclear leukocytes [PMNLs]) of blood donors.

Study design

HHV-6A, HHV-6B and -7 DNAs were quantitated using real-time PCR assays in WB, PBMCs and PMNLs separated on Ficoll or dextran gradients, respectively, for 200 blood donors. Viral loads were expressed as the number of viral genomic copies per million cells (Cop/M) for all fractions, and also per milliliter for WB.

Results

HHV-6B DNA was rarely detected in WB (8%), PBMCs (16.5%), and PMNLs (10.5%), HHV-6A was never detected, whereas HHV-7 DNA was often present in WB (51.5%), PBMCs (62%) and PMNLs (51.5%). Median loads were low with 81 Cop/M in WB, 62 Cop/M in PBMCs and 34.5 Cop/M in PMNLs for HHV-6B, and 129 Cop/M in WB, 225 Cop/M in PBMCs and 62 Cop/M in PMNLs for HHV-7. Viral load expression per million cells and per mL were equivalent. One subject had chromosomally integrated HHV-6 with high viral loads ranging from 2.23 × 10⁶ to 3.21 × 10⁶ Cop/M in all compartments and plasma.

Conclusions

These results allow to propose viral load in WB as a sensitive and suitable marker, with values for healthy subjects at approximately 100 Cop/M for both viruses. The prevalence of chromosomally integrated HHV-6 was 0.5%.     

Ischemia is the prime but not the only cause of human multipotent stromal cell death in tissue-engineered constructs in vivo

Local tissue ischemia is a prime cause responsible for the massive cell death in tissue-engineered (TE) constructs observed postimplantation. To assess the impact of ischemia on the death of implanted human multipotent stromal cells (hMSCs), which have great potential for repairing damaged tissues, we hereby investigated the in vivo temporal and spatial fate of human Luc-GFP-labeled MSCs within fibrin gel/coral scaffolds subcutaneously implanted in nude mice. In vivo bioluminescence imaging monitoring and histological analyses of the constructs tested confirmed the irremediable death of hMSCs over 30 days postimplantation. The kinetics of expression of three hypoxic/ischemic markers (HIF-1α, LDH-A, and BNIP3) was also monitored. Our results provided evidence that hMSCs located within the core of implanted constructs died faster and predominantly and strongly expressed the aforementioned ischemic markers. In contrast, cells located in the outer regions of TE constructs were reperfused by neovascularization and were still viable (as evidenced by their ex-vivo proliferative potential) at day 15 postimplantation. These results support the explanation that in the central part of the constructs tested, death of hMSCs was due to ischemia, whereas in the periphery of these constructs, cell death was due to another mechanism that needs to be elucidated.     

A potent and selective metabotropic glutamate receptor 4 positive allosteric modulator improves movement in rodent models of Parkinson's disease

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGluR4) have been proposed as a novel therapeutic approach for the treatment of Parkinson's disease. However, evaluation of this proposal has been limited by the availability of appropriate pharmacological tools to interrogate the target. In this study, we describe the properties of a novel mGluR4 PAM. 5-Methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine (ADX88178) enhances glutamate-mediated activation of human and rat mGluR4 with EC(50) values of 4 and 9 nM, respectively. The compound is highly selective for mGluR4 with minimal activities at other mGluRs. Oral administration of ADX88178 in rats is associated with high bioavailability and results in cerebrospinal fluid exposure of >50-fold the in vitro EC(50) value. ADX88178 reverses haloperidol-induced catalepsy in rats at 3 and 10 mg/kg. It is noteworthy that this compound alone has no impact on forelimb akinesia resulting from a bilateral 6-hydroxydopamine lesion in rats. However, coadministration of a low dose of l-DOPA (6 mg/kg) enabled a robust, dose-dependent reversal of the forelimb akinesia deficit. ADX88178 also increased the effects of quinpirole in lesioned rats and enhanced the effects of l-DOPA in MitoPark mice. It is noteworthy that the enhancement of the actions of l-DOPA was not associated with an exacerbation of l-DOPA-induced dyskinesias in rats. ADX88178 is a novel, potent, and selective mGluR4 PAM that is a valuable tool for exploring the therapeutic potential of mGluR4 modulation. The use of this novel tool molecule supports the proposal that activation of mGluR4 may be therapeutically useful in Parkinson's disease.     

Competitive allele specific TaqMan PCR for KRAS, BRAF and EGFR mutation detection in clinical formalin fixed paraffin embedded samples

Background

The development of targeted therapies has created a need for robust molecular characterization of cancer and it has become a challenge to validate methods to ensure accuracy in tumor mutation testing.

Methods

The current study was designed to evaluate KRAS, BRAF and EGFR genotyping by Competitive Allele Specific hydrolysis probes (TaqMan) PCR technology (CAST), on suboptimal formalin fixed paraffin embedded (FFPE) tumor samples. Assays were calibrated on FFPE samples and a minimal quantification cycle (Cq) cut-off was determined to standardize analyses and avoid over-interpretation of degraded material. Sensibility, specificity and blinded clinical sample screenings (n = 63) were evaluated.

Results

CAST PCR allowed efficient amplification of FFPE samples, probes were highly specific and all assays had a sensibility inferior to 1% except for the EGFR p.T790M assay. 60/63 samples were correctly typed. The three missed mutations were EGFR exon 19 deletions that were not recognized by the DEL19 assays that were used.

Conclusions

This technology is less laborious and prevent crossover of PCR products as compared to multistep methods. TaqMan® Mutation Detection assay is an important technology to consider in the field of mutation detection for KRAS, BRAF and EGFR point mutation screening. Assay calibration on FFPE samples may prevent erroneous interpretations that will ultimately harm clinical oncology practice.     

Analytical quality of assays and comparison of procedures for the sweat test

Sweat test measuring the chloride ion (Cl(-)) concentration in sweat is a tool for the cystic fibrosis (CF) diagnosis. We evaluated analytical criteria of different available methods and compared them into five hospitals and throught a national quality control program. Sweat tests were performed by stimulation using pilocarpine iontophoresis, sweat collection and measurement of sweat Cl(-) (mmol/L) by titration (colorimetric or coulometric end-point) or by in situ direct potentiometry using a chloride-selective electrode. Indirect determination by sweat conductivity measurement was expressed in mmol/L sodium chloride (NaCl) equivalents (Eq). Linearity range was demonstrated for all measurement procedures in the range 10 to 120 mmol/L. Intra-laboratory coefficients of variation (CVs) were <5% for values between 10 and 100 mmol/L. Inter-laboratory CVs were <3% only for conductivity measurement whatever the range. The comparison of results obtained for a same sweat sample, simultaneously by coulometric and conductivity measurements, demonstrated a first degree linear distribution between 30 to 60 mmol/L Cl(-) allowing us to establish an analytical correspondence table for this range. Thus, calculated values for 30, 40 and 60 mmol/L Cl(-) were respectively 57, 66 and 84 mmol/L NaCl Eq. In conclusion, comparison of methods highlighted that the less the sweat test is automatically controlled, the more the operator influence on results quality is important. Our study supports that sweat test result <50 mmol/L NaCl Eq is unlikely with CF diagnosis in absence of clinical arguments.     

Evaluation of the antiproliferative activity of diterpene isonitriles from the sponge Pseudoaxinella flava in apoptosis-sensitive and apoptosis-resistant cancer cell lines

One new (1) and three known (2-4) isonitrile diterpenes, isolated from the Caribbean sponge Pseudoaxinella flava, were assayed in human cancer cell lines in vitro using an MTT colorimetric assay and quantitative videomicroscopy. Compounds 1-4 displayed activity for human PC3 prostate apoptosis-sensitive cancer cell lines. Compounds 3 and 4 demonstrated similar growth inhibitory effects for three apoptosis-sensitive and three apoptosis-resistant cancer cell lines. Quantitative videomicroscopy analysis revealed that compounds 1 and 2 exerted their activity through cytotoxic effects, and compounds 3 and 4 through cytostatic effects. These results identify marine diterpene isonitriles as potential lead compounds for anticancer drug discovery.     

Long-term quality of life after breast cancer: a French registry-based controlled study

Population-based studies on quality of life (QOL) of long-term breast cancer survivors are quite recent and insufficient attention has been paid to the effect of time since diagnosis. We compared long-term QOL of population-based breast cancer survivors 5, 10, and 15 years after diagnosis with that of healthy controls. Breast cancer survivors were randomly selected from three population-based cancer registries (Bas-Rhin, Calvados and Doubs, France) along with healthy controls, stratified for age and place of residence, randomly selected from electoral rolls. Participants completed five self-administered questionnaires: the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30), Short Form-36 (SF-36), Spielberger State-Trait Anxiety Inventory (STAI), Multidimensional Fatigue Inventory (MFI) and a life conditions questionnaire. An analysis of variance was used to compare QOL scores of breast cancer survivors by period (5, 10, or 15 years) of diagnosis with those of controls, adjusted for sociodemographic data and comorbidities. Six hundred and fifty-two cases and 1,188 controls participated in the study. For many QOL scales, scores were significantly different between cancer survivors and controls. A clinically significant difference was evidenced for the fatigue scales, the SF36 physical functioning, role-physical, and role-emotional scales, with more favorable results for controls. Differences decreased with time and 15-year cancer survivors were generally not different from controls. Scores were particularly influenced by age and mean household income. More efforts should be made, specifically during the first 5 to 10 years after diagnosis, to help women with breast cancer to overcome their impairment in QOL.