Usefulness of Dried Blood Spots (DBS) to perform hepatitis C virus genotyping in drug users in Senegal.

The aim of this pilot study was to analyze the Hepatitis C Virus (HCV) genotypes circulating in Senegal among Drug User (DUs), using Dried Blood Spots (DBS) as RNA source for molecular assays. Heroin and/or cocaine users (n = 506) were recruited in Dakar from April to July 2011, using a Respondent Driven Sampling (RDS) method. DBS preparation consisted of five drops of whole blood from finger applied to a Whatman paper card. HCV infection was screened by the detection of anti-HCV antibodies, using a rapid immune-chromatographic test. HCV RNA was quantified on anti-HCV positive DBS, using the Abbott RealTime HCV® Genotyping was performed on DBS with detectable viral load with Versant® HCV Genotype 2.0 Assay (LiPA) and Abbott RealTime HCV Genotype II assay®. Among the 506 participants, 120 were tested as positive for anti-HCV antibodies and their samples were analyzed for HCV RNA viral load and genotype. Out of the 120 DBS tested, HCV RNA was detected on 25 (20.8%). The median viral load was 15,058 IU/ml (ranging from 710 to 766,740 IU/ml). All positive DBS were suitable for the genotyping assay, that showed a predominance of genotype 1 (21/25) including 16 genotypes 1a and 5 genotypes 1b. HCV genotype 1 prevails in a DU population in Dakar. DBS could be useful for HCV RNA genotyping, but optimal storage conditions should required avoiding RNA impairment. Acknowledging this limitation, DBS could be a great interest for detecting and genotyping HCV viremic patients. J. Med. Virol. 89:484-488, 2017. © 2015 Wiley Periodicals, Inc.

[Does exercise therapy for chronic lower-back pain require daily isokinetic reinforcement of the trunk muscles?]. / Le réentraînement à l'effort de la lombalgie chronique nécessite-t-il un renforcement musculaire isocinétique quotidien du tronc?

OBJECTIVE: The goal of this study was to determine the benefits of a functional retraining programme (with or without daily isokinetic reinforcement of the trunk muscles) in patients with lower-back pain. METHOD: Two groups of 30 patients took part in the study. The control group (CG) underwent a four-week reconditioning program in a day hospital, whereas a second interventional group (IG) additionally performed daily isokinetic training of the trunk muscles. Three evaluations were carried out: before hospitalization (T1), immediately after hospitalization (T2) and three months postrehabilitation (T3). RESULTS: We observed an improvement in each parameter after rehabilitation, regardless of the group. A decrease in the DALLAS scores revealed a reduced impact of lower-back pain on the patients' lives. Pain experienced fell by 24%, analgesic treatment was significantly decreased (CG: -53%; IG: -56%), muscle endurance was improved (quadriceps: +30%, abdominal muscles: +20%, paraspinal muscles: +23%, quadratus lumborum: +33%) and the patients were more supple, as revealed by a decrease in the finger-to-ground distance (at T1, CG: 12.9+/-6.1cm; IG: 13.6+/-5.5 cm at T1; CG: 2.2+/-5.4 cm; IG: 2.8+/-5.1cm at T2). The sole difference for CG and IG at T2 resulted from an improvement in the performance of the trunk extensor muscles, which was significantly greater in the IG (CG: +14%; IG: +20%). Three months after rehabilitation, the benefits were still present for the two groups and, indeed, were even greater for certain parameters. CONCLUSION: Regardless of the protocol, the patients improved in both physical and psychological terms and these improvements were maintained over a short period, at least. Our results confirmed that one functional recovery programme is not superior to another for patients with lower-back pain.

Cd/Zn exposure interactions on metallothionein response in Eisenia fetida (Annelida, Oligochaeta).

We studied metallothionein (MT) response in the manure worm Eisenia fetida after exposures to cadmium (Cd), zinc (Zn) or cadmium and zinc spiked media. MT was studied both at the protein level by Dot Immunobinding Assay, (DIA) and at the expression level by Northern blotting. Cd was highly accumulated by worms whereas Zn body concentration was regulated. In addition, Zn would limit Cd accumulation in worms exposed to low Cd concentrations (1 and 8 mg Cd kg(-1) of dry soil). Exposure to a mixture of Cd and Zn at high concentrations increased cytosolic MT levels. This increase would allow worms to regulate body Zn concentrations and also to limit Cd toxicity. Cd exposures increased gene expression of Cd-binding MT isoform (MT 2A) whereas Zn did not. However, when both metals were at high concentrations in the exposure medium, this expression was further increased. Several hypotheses are proposed to explain the results and the best approach to estimate metal exposure of this earthworm species is given. Further experiments have now to be performed to evaluate the usefulness of these MT responses for field contaminated soils toxicity assessment using this earthworm species.

The strong induction of metallothionein gene following cadmium exposure transiently affects the expression of many genes in Eisenia fetida: a trade-off mechanism?

Metal pollution causes disturbances at various levels of biological organization in most species. Important physiological functions could be affected in the exposed individuals and among the main physiological functions, immunity may provide one (or more) effector(s) whose expression can be directly affected by a metal exposure in various macroinvertebrates. Protein expressions were studied in order to test them as molecular biomarkers of metal exposure in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, transcriptionally controlled tumor protein, protein kinase C, ubiquitin and cyclophilin-A. The level of expression of each gene was analysed in whole organism following exposures to cadmium in soil using real-time PCR. Metallothionein, transcriptionally controlled tumor protein and cyclophilin-A expression were also measured following copper exposures in soil because these genes seemed to be sensitive to copper. This work enabled to distinguish metallothionein and cyclophilin-A among the 15 selected effectors. A strong decrease of the number of transcripts was also detected for most effectors soon after the exposure to cadmium suggesting that a trade-off mechanism occurs.

Cloning and real-time PCR testing of 14 potential biomarkers in Eisenia fetida following cadmium exposure.

Important biological activities could be affected in metal exposed species, and amongthe main physiological functions, immunity may provide one (or more) effector(s) which expression can be directly affected by a metal exposure in various macroinvertebrates. As many proteinic effectors showed a high degree of homology between species, we have developed a PCR approach to characterize partial mRNA sequences of selected effectors in the laboratory model, Eisenia fetida. After cloning, levels of expression of each gene were analyzed following exposures (80 and 800 mg/kg) to cadmium spiked soils using real-time PCR. An implemented approach was allowed to test quickly potential biomarkers in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, trancriptionally controlled tumor protein, protein kinase C, and ubiquitin. Most of the selected effectors did not show variations of expression level after exposure. Others expressed weak changes of expression as heat shock proteins. At lastfor catalase and metallothionein, early suitable variations of expression were observed.

Fluorescence fingerprints of Eisenia fetida and Eisenia andrei.

We describe a fluorescent method that allows to differentiate the worms Eisenia fetida and Eisenia andrei. In fact, the coelomic fluid of E. andrei displays specific fluorescence absent in that of E. fetida. The two species do not metabolize the same types of molecules and thus can be differentiated at the molecular level. Each species has specific fluorescence fingerprints.

Quantification of proviral load of human immunodeficiency virus type 2 subtypes A and B using real-time PCR.

We have developed and evaluated a new method to quantify human immunodeficiency virus type 2 (HIV-2) proviral DNA based on LightCycler real-time PCR. The assay has a detection limit of 5 copies/10(5) peripheral blood mononuclear cells (PBMC) and is insensitive to HIV-2 strain variability: HIV-2 subtypes A and B are both recognized and quantified. The intra- and interassay coefficients of variation range from 16 to 40% for high provirus concentrations (5 x 10(5) copies) and from 41 to 39% for low concentrations (5 copies). We used this method to compare the proviral DNA load and viral RNA load in plasma with clinical and immunological status for 29 patients infected by HIV-2 (subtype A in 17 and subtype B in 12). The proviral load (median, 201 copies/10(5) PBMC) was similar to that reported for HIV-1 infection. The median proviral loads did not correlate with the CD4(+) cell count categories and were as follows for CD4(+) cell counts of >400, 200 to 400, and <200 cells/mm(3), respectively: 121 copies/10(5) PBMC (n = 8; range, <5 to 712 copies/10(5) PBMC); 114 copies/10(5) PBMC (n = 9; range, <5 to 1,907 copies/10(5) PBMC); and 285 copies/10(5) PBMC (n = 12; range, 53 to 2,524 copies/10(5) PBMC). Proviral load did not correlate with plasma HIV-2 RNA positivity. As HIV-2 is considered to replicate less efficiently than HIV-1, these high proviral loads might be explained by the proliferation of infected cells.

Variability of human immunodeficiency virus type 2 (hiv-2) infecting patients living in france.

To determine the prevalence of human immunodeficiency virus type 2 (HIV-2) subtypes circulating in France and to identify possible relationships between these subtypes and pathogenesis, we studied 33 HIV-2-infected patients living in France. HIV-2 DNA was directly amplified from peripheral blood mononuclear cells by nested PCR with specific HIV-2 env primers, and the env gene was sequenced. The serological consequences of antigenic variability were studied by using a panel of peptides and by Western blotting. Phylogenetic analysis classified the 33 HIV-2 strains as subtype A (n = 23) or B (n = 10). There were no significant clinical or epidemiological differences between patients infected with either of these two subtypes. There was some evidence for geographical clustering. Subtype A strains from patients originating from the Cape Verde Islands and Guinea Bissau clustered together. The majority of patients infected with subtype B strains originated from the Ivory Coast or Mali. Strains from patients originating in Mali also clustered in subtype A but distinctly from the Cape Verde or Guinea Bissau strains. The subtype B strains showed greater diversity and included some highly divergent strains relative to those previously characterized. The V3 loop of HIV-2 subtypes A and B was found to be quite conserved in comparison with HIV-1. A strong HIV-2 subtype B serological cross-reactivity was found on HIV-1 env antigen by Western blot mostly in the gp41 transmembrane glycoprotein. This could partly explain the double HIV-1 and HIV-2 reactive profiles found in countries where HIV-2 subtype B is prevalent.

New polyfunctional magnesium reagents for organic synthesis

The iodine-magnesium exchange reaction allows the preparation of polyfunctional aryl, heteroaryl, or alkenyl magnesium reagents at low temperature. These reagents display the typical reactivity of Grignard compounds and undergo various copper-catalyzed reactions such as allylation or 1,4-addition. Using this halogen-metal exchange reaction, it was possible to generate polyfunctional magnesium reagents on the solid phase.

H19 overexpression in breast adenocarcinoma stromal cells is associated with tumor values and steroid receptor status but independent of p53 and Ki-67 expression.

In a previous study we described the expression of the H19 gene by in situ hybridization (ISH) in normal breast and in benign or malignant breast tumors (Dugimont T, Curgy JJ, Wernert N, Delobelle A, Raes MB, Joubel A, Stehelin D, Coll J: Biol Cell 1995, 85:117-124). In the present work, 1) we extend the previous one to a statistically useful number of adenocarcinomas, including 10 subclasses, 2) we provide information on the precise ISH localization of the H19 RNA by using, on serial tissue sections, antibodies delineating specifically the stromal or the epithelial component of the breast, and 3) we consider relationships between the H19 gene expression and various clinicopathological information as tumor values (T0 to T4), grades, steroid receptors, lymph node status, and molecular features as the p53 gene product and the Ki-67/MIB1 protein, which is specific to proliferating cells. Data indicate that 1) in 72.5% of studied breast adenocarcinomas an overall H19 gene expression is increased when compared with healthy tissues, 2) the H19 gene is generally overexpressed in stromal cells (92.2%) and rarely in epithelial cells (2.9% only), 3) an up-regulation of the H19 gene is significantly correlated with the tumor values and the presence of both estrogen and progesterone receptors, and 4) at the cellular level, the H19 gene demonstrates an independent expression versus accumulation of both the p53 protein and the Ki-67/MIB-1 cell-cycle marker.

Highly sensitive method for amplification of human immunodeficiency virus type 2 DNA.

We evaluated a new human immunodeficiency virus type 2 (HIV-2) DNA amplification strategy based on peripheral blood mononuclear cell long PCR (XL PCR) followed by nested PCR amplification. The primers used were located in the highly conserved long terminal repeat and in the pol regions of the genome. Five primer pairs corresponding to different regions of the HIV-2 env gene were used in the nested step. Samples from 42 patients were tested, which yielded positive amplification with at least two primer pairs in 40 (95%) samples. A primer pair (EB2/EB5) located on the V3 region succeeded in amplifying proviral DNA in 40 samples.

Comparison of the IgE titers to bovine colostral G immunoglobulins and their F(ab')2 fragments in sera of patients allergic to milk.

Colostral G immunoglobulins (IgGs) are described in many recent studies as having a beneficial effect for the treatment of viral, bacterial and parasitic diarrhea in animals and humans. The specific IgE titers to bovine colostral IgG, to bovine serum IgG, and to F(ab')2 fragments of IgG were immunoenzymatically quantified in sera of patients allergic to milk, to statistically evaluate and compare their relative immunoreactivity towards these purified antigens. The results clearly indicated that 36% of the population tested was potentially allergic to colostral IgG, and serum IgG globally elicited significantly lower IgE titers. The F(ab')2 fragments lead to a significantly decreased immunoreactivity as compared to colostral IgG. This study shows the interesting use of peptic hydrolysis of IgG in producing fragments with preserved therapeutic immunoactivity and reduced potential allergenicity.

[Orosomucoid prealbumin ratio in bronchopulmonary cancers]. / Rapport orosomucoïde sur préalbumine dans les cancers broncho-pulmonaires.

Lung cancer is a common pathology with high mortality due to late diagnosis. The 1987 TNM classification clearly defines the different steps and their prognosis. Although the prognostic value of some biological parameters (mainly serum LDH, sodium and/or albumin) has been established, these are not much used. We have prospectively studied the serum levels of seven proteins (RBP, prealbumin, albumin, transferrin, haptoglobin, orosomucoid, CRP) and we demonstrate the predominant value of prealbumin for the establishment of the prognosis of lung cancer; determination of orosomucoid increases the prognostic value of prealbumin. We confirm, for lung cancer, the prognostic value of the orosomucoid-prealbumin ratio, already known for other cancers.

Effects of corticotropin-releasing factor, arginine vasopressin and oxytocin on the polymorphism of plasma adrenocorticotropic hormone in the rat fetus in late pregnancy.

The effects of rat corticotropin-releasing factor (rCRF, 1.25 pmol/50 microliters/fetus), arginine vasopressin (AVP, 5 pmol/50 microliters/fetus) and oxytocin (OT, 12.5 pmol/50 microliters/fetus) alone or in association were investigated in 21-day-old rat fetuses injected intravenously through the umbilical vein. Blood samples were collected 15 and 30 min after injection for the determination of corticosterone concentration and the different plasma molecular adrenocorticotropic hormone (ACTH) forms isolated by chromatography on Sephadex G50 fine. All the plasma samples chromatographed 15 and 30 min after injection of the different peptides showed 3 different molecular ACTH forms: big ACTH (> 20,000 molecular weight), intermediate ACTH (= 13,000) and little ACTH (= 4,500). The injection of rCRF or AVP alone and rCRF in association with AVP or OT increased the concentrations of big ACTH 15 min and little ACTH 30 min after injection. The injection of OT alone or in association with AVP did not change the concentration of the 3 molecular ACTH forms 15 and 30 min after injection. The rise of big ACTH 15 min after injection was not associated with a significant increase in plasma corticosterone concentration, whereas the increase in little ACTH 30 min later enhanced plasma corticosterone concentration. Our results suggest that rCRF or AVP alone and rCRF in association with AVP or OT injected intravenously in the fetal rat produced a selective release of the molecular ACTH forms and the increase in the plasma corticosterone concentration occurred when the proportion of little ACTH which is the predominant ACTH form in the fetal rat was enhanced.

[Visceral leishmaniasis in HIV infection. A totally opportunistic infection]. / Leishmaniose viscérale au cours de l'infection par le VIH. Une infection opportuniste à part entière.

Visceral leishmaniasis occurring in immunocompromised patients, and in particular during HIV infection, has been described in recent years and differs from the usual Mediterranean kala-azar as encountered in France. In order to define the clinical, diagnostic and therapeutic features of the HIV-Leishmania spp. co-infection, we report 8 new cases and compare them with data from the literature. The co-infection occurs at any stage of HIV infection, usually in drug addicts using intravenous injections. Clinical manifestations, such as fever, weight loss, liver and spleen enlargement and polyadenopathy, and laboratory findings (cytoponia, inflammatory syndrome) are generally present but not specific during the HIV infection course. Moreover, some gastrointestinal and pleuropulmonary forms of the co-infection are misleading. Leishmaniasis serology is negative in 50 percent of the patients. In most cases the diagnosis is provided by detection of the parasite in bone marrow samples. Culture must be systematic, and samplings must be repeated if they are negative. The first-line treatment consists of pentavalent antimony. Almost 80 percent of the patients respond to this treatment, but relapses occur in 50 percent of the cases. This high risk of relapse and the opportunistic behaviour of leishmaniasis justify a prophylaxis of relapses.

Effect of rat atrial natriuretic factor on in vivo and in vitro aldosterone and corticosterone secretions in the rat during the perinatal period.

The effect of rat atrial natriuretic factor (rANF) on aldosterone and corticosterone secretion was investigated in vivo in 21-day-old rat fetuses injected intravenously through the umbilical vein and in vitro on isolated adrenal cells from 17-, 19- and 21-day-old fetuses and 1-, 2-, 3- and 4-week-old rats. In vivo, rANF (50 pmol/50 microliters/fetus) inhibited both basal levels and secretion of aldosterone stimulated by adrenocorticotropin hormone (ACTH(1-24), 0.25 pmol/50 microliters/fetus), but not corticosterone secretion. In vitro, the addition of graded concentrations of rANF (0.001, 0.01 and 10 nmol/l) to the incubation medium did not affect the basal aldosterone and corticosterone secretions of fetal and neonatal adrenal cells. ACTH(1-24) (0.1 nmol/l) stimulated productions of both corticosterone and aldosterone by the adrenal cells at all stages studied. The addition of graded concentrations of rANF to the incubation medium containing ACTH(1-24) (0.1 nmol/l) induced a dose-dependent inhibition of aldosterone secretion by the adrenal cells from 21-day-old fetuses and newborn rats. In contrast, no effect was observed on cells from 17- and 19-day-old fetuses. At all stages investigated, the three doses of rANF were unable to affect ACTH-induced corticosterone secretion in vitro. In isolated adrenal cells from 2-week-old rats, rANF (10 nmol/l) inhibited the secretion of aldosterone induced by ACTH(1-24) (0.1 nmol/l), and by different steroids of the aldosterone synthetic pathway (progesterone, 11-deoxycorticosterone, corticosterone, 1 mumol/l for each steroid). These results suggest that rANF is a specific inhibitor of aldosterone synthesis in the perinatal period of the rat and that the inhibitory effect of rANF occurs both during the early and late pathways of aldosteroidogenesis.