French consensus: Treatment of newly diagnosed restless legs syndrome.

Treatment of restless legs syndrome (RLS) must only be considered after a definite positive diagnosis. The RLS phenotype must be characterised precisely, iron deficiency always tested for, and aggravating factors eliminated when possible. Medical treatment is considered for severe or very severe forms and based on dopaminergic agonists, α2δ-1 ligands and/or opioids. First line treatment will be a low-dose monotherapy and the choice of treatment depends on the results of the clinical examination and investigations.

The genetic basis for susceptibility to Rift Valley fever disease in MBT/Pas mice.

The large variation in individual response to infection with Rift Valley fever virus (RVFV) suggests that host genetic determinants play a role in determining virus-induced disease outcomes. These genetic factors are still unknown. The systemic inoculation of mice with RVFV reproduces major pathological features of severe human disease, notably the hepatitis and encephalitis. A genome scan performed on 546 (BALB/c × MBT) F2 progeny identified three quantitative trait loci (QTLs), denoted Rvfs-1 to Rvfs-3, that were associated with disease susceptibility in MBT/Pas mice. Non-parametric interval-mapping revealed one significant and two suggestive linkages with survival time on chromosomes 2 (Rvfs-1), 5 (Rvfs-3) and 11 (Rvfs-2) with respective logarithm of odds (LOD) scores of 4.58, 2.95 and 2.99. The two-part model, combining survival time and survival/death, identified one significant linkage to Rvfs-2 and one suggestive linkage to Rvfs-1 with respective LOD scores of 5.12 and 4.55. Under a multiple model, with additive effects and sex as a covariate, the three QTLs explained 8.3% of the phenotypic variance. Sex had the strongest influence on susceptibility. The contribution of Rvfs-1, Rvfs-2 and Rvfs-3 to survival time of RVFV-infected mice was further confirmed in congenic mice.

Efficacy of sustained combination therapy for at least 6 months with thiopurines and infliximab in patients with ulcerative colitis in clinical remission: a retrospective multicenter French experience.

BACKGROUND AND AIMS: Long-term benefits of combination therapy (combotherapy) with infliximab (IFX) and azathioprine (AZA) have been less studied in ulcerative colitis (UC) than in Crohn's disease. The aim of the present study was to determine UC disease activity in patients who received at least 6 months of combotherapy, and whether cotreatment for more than 6 months was useful in these patients. METHODS: A retrospective multicenter study was conducted in seven French academic centers from January 2010 to September 2012, including all UC patients having received at least 6 months of combotherapy in prolonged remission off steroids. During the follow-up period, which was divided into trimesters, scheduled IFX was continued as maintenance and AZA could be withdrawn. Assessment of UC activity by trimester was based on the following events: disease relapse defined by clinical relapse requiring a change of treatment, IFX failure, and colectomy. RESULTS: Eighty-two patients were included (mean age 38 years; male:female ratio 1:1) and followed up for a median of 22.3±14.0 months. Comparing 393 trimesters of combotherapy with 282 trimesters of IFX alone, fewer clinical relapses were observed with combotherapy (p = 0.049). Similar results were observed for IFX failure (p = 0.048). No difference was observed for colectomy. Duration of combotherapy longer than 9 months was inversely associated with clinical relapse (hazard ratio = 0.32 [95% confidence interval 0.15-0.70]). CONCLUSIONS: UC patients treated with combotherapy should maintain IFX and AZA for at least 9 months. Further studies are required to determine the optimal duration of combotherapy before stopping AZA in this situation.

Multicollinearity in spatial genetics: separating the wheat from the chaff using commonality analyses.

Direct gradient analyses in spatial genetics provide unique opportunities to describe the inherent complexity of genetic variation in wildlife species and are the object of many methodological developments. However, multicollinearity among explanatory variables is a systemic issue in multivariate regression analyses and is likely to cause serious difficulties in properly interpreting results of direct gradient analyses, with the risk of erroneous conclusions, misdirected research and inefficient or counterproductive conservation measures. Using simulated data sets along with linear and logistic regressions on distance matrices, we illustrate how commonality analysis (CA), a detailed variance-partitioning procedure that was recently introduced in the field of ecology, can be used to deal with nonindependence among spatial predictors. By decomposing model fit indices into unique and common (or shared) variance components, CA allows identifying the location and magnitude of multicollinearity, revealing spurious correlations and thus thoroughly improving the interpretation of multivariate regressions. Despite a few inherent limitations, especially in the case of resistance model optimization, this review highlights the great potential of CA to account for complex multicollinearity patterns in spatial genetics and identifies future applications and lines of research. We strongly urge spatial geneticists to systematically investigate commonalities when performing direct gradient analyses.

Comparative landscape genetic analyses show a Belgian motorway to be a gene flow barrier for red deer (Cervus elaphus), but not wild boars (Sus scrofa).

While motorways are often assumed to influence the movement behaviour of large mammals, there are surprisingly few studies that show an influence of these linear structures on the genetic make-up of wild ungulate populations. Here, we analyse the spatial genetic structure of red deer (Cervus elaphus) and wild boars (Sus scrofa) along a stretch of motorway in the Walloon part of Belgium. Altogether, 876 red deer were genotyped at 13 microsatellite loci, and 325 wild boars at 14 loci. In the case of the red deer, different genetic clustering tools identified two genetic subpopulations whose borders matched the motorway well. Conversely, no genetic structure was identified in the case of the wild boar. Analysis of isolation-by-distance patterns of pairs of individuals on the same side and on different sides of the motorway also suggested that the road was a barrier to red deer, but not to wild boar movement. While telemetry studies seem to confirm that red deer are more affected by motorways than wild boar, the red deer sample size was also much larger than that of the wild boars. We therefore repeated the analysis of genetic structure in the red deer with randomly sub-sampled data sets of decreasing size. The power to detect the genetic structure using clustering methods decreased with decreasing sample size.

A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml(-1) NS1. Up to 1µgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.

Genetic structure and assignment tests demonstrate illegal translocation of red deer (Cervus elaphus) into a continuous population.

Molecular forensic methods are being increasingly used to help enforce wildlife conservation laws. Using multilocus genotyping, illegal translocation of an animal can be demonstrated by excluding all potential source populations as an individual's population of origin. Here, we illustrate how this approach can be applied to a large continuous population by defining the population genetic structure and excluding suspect animals from each identified cluster. We aimed to test the hypothesis that recreational hunters had illegally introduced a group of red deer into a hunting area in Luxembourg. Reference samples were collected over a large area in order to test the possibility that the suspect individuals might be recent immigrants. Due to isolation-by-distance relationships in the data set, inferring the number of genetic clusters using Bayesian methods was not straightforward. Biologically meaningful clusters were only obtained by simultaneously analysing spatial and genetic information using the program baps 4.1. We inferred the presence of three genetic clusters in the study region. Using partial Mantel tests, we detected barriers to gene flow other than distance, probably created by a combination of urban areas, motorways and a river valley used for viticulture. The four focal animals could be excluded with a high certainty from the three genetic subpopulations and it was therefore likely that they had been released illegally.

Secretion of flaviviral non-structural protein NS1: from diagnosis to pathogenesis.

Flaviviruses are major arthropod-borne human pathogens responsible for life-threatening encephalitis, hepatitis and haemorrhagic fevers. These enveloped, single-stranded, positive-sense RNA viruses encode a polyprotein precursor of about 3400 amino acids, processed into three structural and seven non-structural proteins. The non-structural glycoprotein NS1 is essential for flavivirus viability. During host-cell infection in vitro, NS1 is found associated with intracellular organelles as a requisite for its role in viral replication, or is transported to the cell surface where it may trigger specific signalling pathways. In addition, a secreted form of the protein is released from flavivirus-infected mammalian cells. We have previously shown that the NS1 protein circulates during the acute phase of the disease in the plasma of patients infected with dengue virus type 1 and have extended our retrospective studies to dengue type 2 and type 3 cohorts, confirming the value of the NS1 antigen as an alternative diagnostic marker. Interestingly, detection of the NS1 protein in yellow fever virus and West Nile virus infections suggests that NS1 secretion is a hallmark of human flavivirus infections. The objectives of our current studies are to define the biological properties of the secreted form of the NS1 protein, to evaluate its possible contribution to viral pathogenesis, and to validate this protein as a candidate target for passive immunoprophylaxis against flaviviruses.

Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion.

Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species. This noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kDa and a Stokes radius of 64.4 A. During protein export, one of the two oligosaccharides of NS1 is processed into an endo-beta-N-acetylglucosaminidase F-resistant complex-type sugar while the other remains of the polymannose type, protected in the dimeric subunit from the action of maturation enzymes. Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin. These results, together with observations related to the absence of secretion of NS1 from Den-infected insect cells, suggest that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell.

[Apoptotic cell death in response to dengue virus infection: what are the consequences of viral pathogenesis?]. / La mort cellulaire par apoptose en réponse à l'infection par le virus de la dengue: quelles conséquences dans la pathogénie virale?

Dengue is a human disease of viral etiology which may be fatal in its hemorragic form. It is widely spread in the tropical areas of the different continents and has been dramatically expanding over the past 30 years. Although an immunological disorder is thought to be involved in dengue physiological symptoms, the pathogenesis of dengue hemorragic fever has not yet been elucidated. Whether the immune response is deleterious or beneficial to the host remains a matter of debate. Other factors, related to virus replication in specific host cells, could also contribute to the severity of the disease. Apoptotic cell death is one of the important consequences of dengue virus infection both in vitro and in vivo. Dengue replication triggers apoptotic signals in neurons and hepatocytes although the original effectors and kinetics differ. Implications of the ongoing apoptotic processes in viral pathogenesis will be further discussed.

Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited.

BACKGROUND: Dengue virus infection may be asymptomatic or lead to undifferentiated febrile illness or dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). The major clinical manifestations of DHF/DSS are high fever, haemorrhage, hepatomegaly and circulatory failure. OBJECTIVES: The relatively high level of viraemia only a few days after infection may reflect a large number of replication sites. However, the degree of cell injury in fatal cases of DHF/DSS is not sufficient to explain death and suggests metabolic disturbance rather than tissue destruction. This theory was investigated in this study. RESULTS: We demonstrated that replication of dengue virus in infected cells induces stress leading to apoptotic cell death in vitro and in vivo. CONCLUSIONS: The elimination of apoptotic bodies by phagocytic cells is a previously unsuspected pathway of dengue virus clearance from infected tissues. However, the mechanisms of host defence involving apoptosis and phagocytic cell activation may cause local tissue injury or transient homeostasis imbalance and may trigger further deleterious events.

Effect of dehydroepiandrosterone on vaginal and uterine histomorphology in the rat.

Dehydroepiandrosterone (DHEA) applied on the dorsal skin of ovariectomized animals, at the twice daily dose of 30 mg, resulted in a complete reversal of the vaginal atrophy seen 1, 3 and 6 months after ovariectomy, and induced proliferation and mucification of the vaginal epithelium. A similar mucification of the vaginal epithelium related to androgenic action was observed in intact rats treated with DHEA. While treatment of intact rats with DHEA resulted in a significant increase in uterine weight at 1 month, the value of the same parameter decreased by approximately 30% after 3 and 6 months of DHEA administration. In ovariectomized DHEA-treated animals, uterine weight was increased at all time intervals. At histopathological examination, following DHEA administration to intact animals, stimulation was seen in the myometrial layers of the uterus whereas atrophy, involving especially the endometrium, became apparent after 3 and 6 months of treatment. In ovariectomized animals, the endometrium remained atrophic at all time intervals during DHEA treatment and the uterine epithelium thus remained atrophic under DHEA treatment. Examination of the effect of DHEA on the vaginal epithelium indicates that local application of DHEA on the vaginal mucosa is approximately 10-fold more efficient than application at a distant site on the skin. Reversal of the ovariectomy-induced increase in serum LH levels was also observed after DHEA treatment. The present data suggest that DHEA possesses a tissue-specific action, through its local transformation into active estrogens in the vaginal epithelium while the uterine epithelium remains atrophic. In addition, the site of administration of DHEA appears to be a significant factor, at least for its stimulatory effect on the vaginal mucosa.

Induction of programmed cell death (apoptosis) by dengue virus in vitro and in vivo.

Dengue is a human disease which may be fatal in its hemorrhagic form. How dengue virus- and host-specified factors underlie virulence and pathogenesis is poorly understood. An immunological disorder is thought to be involved in dengue physiological symptoms. Whether the immune response is deleterious or beneficial to the host remains a matter of debate. In this review, we summarized developments in research on viral pathogenesis in the context of apoptosis triggered by dengue virus infection. Apoptosis, an active process of cell destruction, is one of the important consequences of dengue virus infection in vitro and in vivo. Dengue virus replication induces apoptosis in mouse neurons and human hepatocytes. The ability to activate this genetically programmed cell death pathway is dependent on both viral and cellular determinants.

High bioavailability of dehydroepiandrosterone administered percutaneously in the rat.

Dehydroepiandrosterone (DHEA) administered percutaneously by twice daily application for 7 days to the dorsal skin of the rat stimulates an increase in ventral prostate weight with approximately one third the potency of the compound given by subcutaneous injection. The doses required to achieve a 50% reversal of the inhibitory effect of orchiectomy are approximately 3 and 1 mg respectively. By the oral route, on the other hand. DHEA has only 10-15% of the activity of the compound given percutaneously. Taking the bioavailability obtained by the subcutaneous route as 100%, it is estimated that the potencies of DHEA by the percutaneous and oral routes are approximately 33 and 3% respectively. Similar ratios of activity were obtained when dorsal prostate and seminal vesicle weight were used as parameters of androgenic activity. When examined on an estrogen-sensitive parameter, namely uterine weight in ovariectomized rats, the stimulatory effect of DHEA was much less potent than its androgenic activity measured in the male animal, a 50% reversal of the inhibitory effect of ovariectomy on uterine weight being observed at the 3 and 30 mg doses of DHEA administered by the subcutaneous and percutaneous routes respectively. When measured on uterine weight, percutaneous DHEA thus shows a 10% potency compared with the subcutaneous route. The sulfate of DHEA (DHEA-S), on the other hand, was approximately 50% as potent as DHEA at increasing ventral prostate weight after subcutaneous or percutaneous administration. When the effect was measured on dorsal prostate and seminal vesicle weight, percutaneous DHEA-S had 10-25% of the activity of DHEA. DHEA decreased serum LH levels in ovariectomized animals, an effect which was completely reversed by treatment with the antiandrogen flutamide. On the other hand, flutamide had no significant effect on the increase in uterine weight caused by DHEA, thus suggesting a predominant estrogenic effect of DHEA at the level of the uterus and an estrogenic effect on the feedback control of LH secretion. The present data show a relatively high bioavailability of percutaneous DHEA as measured by its androgenic and/or estrogenic biological activity in well-characterized peripheral target intracrime tissues in the rat.

Human isolates of dengue type 1 virus induce apoptosis in mouse neuroblastoma cells.

Human isolates of dengue (DEN) type 1 viruses FGA/89 and BR/90 differ in their membrane fusion properties in mosquito cell lines (P. Desprès et al., Virology 196:209-216, 1993). FGA/89 and BR/90 were assayed for their neurovirulence in newborn mice, and neurons were the major target cells for both DEN-1 virus strains within the central nervous system. To study the susceptibility of neurons to DEN virus infection, DEN virus replication was analyzed in the murine neuroblastoma cell line Neuro 2a. Infection of Neuro 2a cells with FGA/89 or BR/90 induced apoptotic DNA degradation after 25 h of infection. Studies of DEN protein synthesis revealed that accumulation of viral proteins leads to apoptotic cell death. The apoptotic process progressed more rapidly following BR/90 infection than it did after FGA/89 infection. The higher cytotoxicity of BR/90 for Neuro 2a cells was linked to an incomplete maturation of the envelope proteins, resulting in abortive virus assembly. Accumulation of viral proteins in the endoplasmic reticulum may induce stress and thereby activate the apoptotic pathway in mouse neuroblastoma cells.

Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae.

The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectroscopy NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to Z-Dhb-D-Pro-L-Leu-D-Ala-D-Ala-D-Ala-D-Ala-D-Val-Gly-D-Ala-D-Val-D-Ala-D- Val-Z-Dhb-Da-Thr-L-Ala-L-Dab-D-Dab-L-Phe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.

Purification and renaturation of Japanese encephalitis virus nonstructural glycoprotein NS1 overproduced by insect cells.

The nonstructural protein NS1 of Japanese encephalitis virus is a major immunogen produced during flavivirus infection. However, the function of this protein has not been identified. To analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in Spodoptera frugiperda insect cells (Sf9), using a recombinant baculovirus, and purified. As described previously by M. Flamand, V. Deubel, and M. Girard (1992, Virology 191, 826-836), a small fraction of the synthesized recombinant protein could mature into a dimer, whereas the major part was retained in intracellular aggregates. This insolubility was used to recover the protein in a purified form using a two-step procedure. Isolated inclusion bodies, in which NS1 constituted over 60% of the protein, were solubilized in 8 M urea. NS1 was further purified by reverse-phase HPLC and recovered at over 90% purity with an overall yield of over 60%. Conditions promoting reoxidation-renaturation of the purified protein were then investigated at a concentration of 100 micrograms/ml at pH 8. The presence of 8 M urea during reoxidation of NS1 with oxidized glutathione was essential prior to renaturation by dialysis to avoid reaggregation, the main side pathway of refolding in vitro. Three major species, all monomeric, were resolved by nonreducing SDS-PAGE. The form showing the lowest apparent molecular weight comigrated with native unreduced NS1 and was recognized by a monoclonal antibody directed against a conformational epitope strictly dependent on the native structure of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of recombinant baculoviruses expressing the outer capsid protein VP2 of five BTV serotypes and the induction of neutralizing antibodies to homologous and heterologous BTV serotypes.

DNA representing RNA segment L2 of 5 different bluetongue virus (BTV) serotypes (BTV-1, -2, -11, -13 and -17) corresponding to the gene that codes for the BTV neutralization antigen VP2, have been inserted individually into baculovirus transfer vectors in lieu of the 5' coding region of the polyhedrin gene of Autographa california nuclear polyhedrosis virus (AcNPV). After co-transfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived transfer vector DNAs, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with each of these viruses, protein was made similar in size and antigenic properties to the authentic BTV VP2 protein. To evaluate the biological activities of these proteins, antibodies were raised to them in guinea pigs. The neutralization capabilities of these antisera were tested against homologous and, for two of the higher titered sera, heterologous BTV serotypes. Each antiserum neutralized the infectivity of the homologous virus serotype. In heterologous virus challenges, the serum raised to the VP2 protein of BTV-13 also neutralized the infectivity of BTV-1, and to lesser extents BTV-3, -16, -8 and -9 (BTV-2, -10, -11, -15 were not tested). The serum raised to the VP2 of BTV-17 neutralized BTV-20 and -21, and to lesser extents BTV-4 and -8 (again, BTV-2, -10, -11, -15 were not tested).

Variant mitochondrial plasmids of broad bean arose by recombination and are controlled by the nuclear genome.

Various cytoplasms of broad bean contain three mitochondrial plasmids (mtp1, 2 and 3), previously described. In cytoplasm 350 we have observed several additional mitochondrial plasmids, varying in number and in identity according to the nuclear background. Replacement of the nucleus by backcrossing led to the appearance or disappearance of additional plasmids, indicating that the nuclear genome controls either the creation or the copy level of mitochondrial plasmids. Analysis of eight variant additional plasmids (mtp4-11) suggests that they all result from a double recombination event between mtp1 and mtp2. In all cases, one recombination point was located within a 276-bp sequence, identical in both plasmids. For 7 plasmids, the region in which the second recombination event occurred could be narrowed down to a short stretch containing imperfect tandem repeats of a 31-bp motif. The largest sequence shared by the recombination regions was hexanucleotide GCGACG.