The role of bile acid retention and a common polymorphism in the ABCB11 gene as host factors affecting antiviral treatment response in chronic hepatitis C.

Summary.  The outcome of hepatitis C virus (HCV) infection and the likelihood of a sustained virological response (SVR) to antiviral therapy depends on both viral and host characteristics. In vitro studies demonstrated that bile acids (BA) interfere with antiviral interferon effects. We investigate the influence of plasma BA concentrations and an ABCB11 polymorphism associated with lower transporter expression on viral load and SVR. Four hundred and fifty-one Caucasian HCV-patients treated with PEG-interferon and ribavirin were included in the study. ABCB11 1331T>C was genotyped, and plasma BA levels were determined. The 1331C allele was slightly overrepresented in HCV-patients compared to controls. In HCV-patients, a significant difference between patients achieving SVR vs non-SVR was observed for HCV-2/3 (5 vs 9 μm; P = 0.0001), while median BA levels in HCV-1 were marginally elevated. Normal BA levels <8 μm were significantly associated with SVR (58.3%vs 36.3%; OR 2.48; P = 0.0001). This difference was significant for HCV-2/3 (90.7%vs 67.6%; P = 0.002) but marginal in HCV-1 (38.7%vs 27.8%; P = 0.058). SVR rates were equivalent between ABCB11 genotypes for HCV-1, but increased for HCV-2/3 (TT 100%vs CC 78%; OR 2.01; P = 0.043). IL28B genotype had no influence on these associations. No correlation between BA levels and HCV RNA was detected for any HCV genotype. The higher allelic frequency of ABCB11 1331C in HCV-patients compared to controls may indirectly link increased BA to HCV chronicity. Our data support a role for BA as host factor affecting therapy response in HCV-2/3 patients, whereas a weaker association was found for HCV-1.

Virologic and immunologic responses in treatment-naive patients to ritonavir-boosted atazanavir or efavirenz with a common backbone.

Background: Atazanavir boosted with ritonavir (ATV/r) and efavirenz (EFV) are both recommended as first-line therapies for HIV-infected patients. We compared the 2 therapies for virologic efficacy and immune recovery. Methods: We included all treatment-naïve patients in the Swiss HIV Cohort Study starting therapy after May 2003 with either ATV/r or EFV and a backbone of tenofovir and either emtricitabine or lamivudine. We used Cox models to assess time to virologic failure and repeated measures models to assess the change in CD4 cell counts over time. All models were fit as marginal structural models using both point of treatment and censoring weights. Intent-to-treat and various as-treated analyses were carried out: In the latter, patients were censored at their last recorded measurement if they changed therapy or if they were no longer adherent to therapy. Results: Patients starting EFV (n = 1,097) and ATV/r (n = 384) were followed for a median of 35 and 37 months, respectively. During follow-up, 51% patients on EFV and 33% patients on ATV/r remained adherent and made no change to their first-line therapy. Although intent-to-treat analyses suggest virologic failure was more likely with ATV/r, there was no evidence for this disadvantage in patients who adhered to first-line therapy. Patients starting ATV/r had a greater increase in CD4 cell count during the first year of therapy, but this advantage disappeared after one year. Conclusions: In this observational study, there was no good evidence of any intrinsic advantage for one therapy over the other, consistent with earlier clinical trials. Differences between therapies may arise in a clinical setting because of differences in adherence to therapy.

Regulation of gammadelta versus alphabeta T lymphocyte differentiation by the transcription factor SOX13.

alphabeta and gammadelta T cells originate from a common, multipotential precursor population in the thymus, but the molecular mechanisms regulating this lineage-fate decision are unknown. We have identified Sox13 as a gammadelta-specific gene in the immune system. Using Sox13 transgenic mice, we showed that this transcription factor promotes gammadelta T cell development while opposing alphabeta T cell differentiation. Conversely, mice deficient in Sox13 expression exhibited impaired development of gammadelta T cells but not alphabeta T cells. One mechanism of SOX13 function is the inhibition of signaling by the developmentally important Wnt/T cell factor (TCF) pathway. Our data thus reveal a dominant pathway regulating the developmental fate of these two lineages of T lymphocytes.

Genotypic features of lentivirus transgenic mice.

Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

New insights into dendritic cell biology and histiocytosis through a transgenic tumor model

SUMMARY The effective development of an immune response depends on the careful interplay and the regulation between innate and adaptive immunity. As the dendritic cells (DCs) are equipped with many receptors, such as Toll-like receptors, which can detect the presence of infection by recognizing different component of bacteria, fungi and even viruses, they are the among the first cells to respond to the infection. Upon pathogen challenge, the DCs interpret the innate system activation as a maturation signal, resulting in the migration of the DCS to a draining lymph node site. There, activated DCs present efficiently antigens to naïve T cells, which are in turn activated and initiate adaptive immunity. Therefore, DCs are the main connectors between innate and adaptive immune systems. In addition to be the most efficient antigen- presenting cells, DCs play a central role in the regulation of immune responses and immune tolerance. Despite extensive research, many aspects related to DC biology are still unsolved and/or controversial. The low frequency of DCs in vivo often hamper study of DC biology and in vitro-derived DCs are not suited to address certain questions, such as the development of DC. We sought of transforming in vivo the DCs through the specific expression of an oncogene, in order to obtain unlimited numbers of these cells. To achieve this goal, transgenic mouse lines expressing the SV40 Large T oncogene under the control of the CD1 1 c promoter were generated. These transgenic mice are healthy until the age of three to four months without alterations in the DC biology. Thereafter transgenic mice develop a fatal disease that shows features of a human pathology, named histiocytosis, involving DCs. We demonstrate that the disease development in the transgenic mice correlates with a massive accumulation of transformed DCs in the affected organs. Importantly, transformed DCs are immature and fully conserve their capacity to mature in antigen presenting cells. We observe hyperproliferation of transformed DCs only in the sick transgenic mice. Surprisingly, transformed DCs do not proliferate in vitro, but transfer of the transformed DCs into immunodeficient or tolerant host leads to tumor formation. Altoghether, the transgenic mouse lines we have generated represent a valuable tumor model for human histiocytosis, and provide excellent tools to study DC biology. RESUME Le développement d'une réponse immunitaire efficace dépend d'une minutieuse interaction et régulation entre l'immunité innée et adaptative. Comme les cellules dendritiques (DCs) sont équipées de nombreux récepteurs, tels que les récepteurs Toll-like, qui peuvent détecter la présence d'une infection en reconnaissant différents composants bactériens, issus de champignons ou même viraux, elles sont parmi les premières cellules à répondre à l'infection. Suite à la stimulation induite par le pathogène, les DCs interprètent l'activation du système immunitaire inné comme un signal de maturation, résultant dans la migration des DCs vers le ganglion drainant le site d'infection. Là, les DCs actives présentent efficacement des antigènes aux cellules T, qui sont à leur tour activées et initient les systèmes d'immunité adaptative. Ainsi, les DCs forment le lien principal entre les réponses immunitaires innées et adaptatives. En plus d'être les cellules présentatrices d'antigènes les plus efficaces, les DCs jouent un rôle central dans la régulation du système immunitaire et dans le phénomène de tolérance. Malgré des recherches intensives, de nombreux aspects liés à la biologie des DCs sont encore irrésolus et/ou controversés. La faible fréquence des DCs in vivo gêne souvent l'étude de la biologie de ces cellules et les DCs dérivées in vitro ne sont pas adéquates pour adresser certaines questions, telles que le développement des DCs. Afin d'obtenir des quantités illimitées de DCs, nous avons songé à transformer in vivo les DC grâce à l'expression spécifique d'un oncogène. Afin d'atteindre ce but, nous avons généré des lignées de souris transgéniques qui expriment l'oncogène SV40 Large T sous le contrôle du promoter CD1 le. Ces souris transgéniques sont saines jusqu'à l'âge de trois à quatre mois et ne présentent pas d'altération dans la biologie des DCs. Ensuite, les souris transgéniques développent une maladie présentant les traits caractéristiques d'une pathologie humaine nommée histiocytose, qui implique les DCs. Nous montrons que le développement de cette maladie corrèle avec une accumulation massive des DCs transformées dans les organes touchés. De plus, les DCs transformées sont immatures et conservent leur capacité à différencier en cellules présentatrices d'antigène. Nous observons une hyper-prolifération des DCs transformées seulement dans les souris transgéniques malades. Etonnament, les DC transformées ne prolifèrent pas in vitro, par contre, le transfert des DCs transformées dans des hôtes immuno-déficients ou tolérant conduit à la formation de tumeurs. Globalement, les lignées de souris transgéniques que nous avons générées représentent un modèle valide pour l'histiocytose humaine, et de plus, offrent d'excellents outils pour étudier la biologie des DCs.

Current cytogenetic map of the common shrew, Sorex araneus L.: localization of 7 genes and 4 microsatellites

Here we present information on the assignment of 7 genes, ACADVL, ADORA3, ATP7A, MTMR4, MYH2, HBB, TSPAN-3, and 4 common shrew microsatellites to chromosomes of the common shrew (Sorex araneus) and on the current status of its cytogenetic map. Comparative mapping data were used for the analysis of evolutionary chromosomal rearrangements in the common shrew genome.

High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in separate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana transformed by the vacuum infiltration method. Co-expression was monitored by the synthesis of three bacterial proteins involved in the production of polyhydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin-resistant transgenic plants analysed, 13 plants were co-transformed with the two distinct T-DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segregation ratio consistent with the presence of a single functional insert. Genetic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-producing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orientation about the left borders. Transformation of A. thaliana by the co-infiltration of two A. tumefaciens containing distinct T-DNAs is, thus, an efficient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of novel metabolic pathways requiring the expression of numerous transgenes.

The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models.

Gene expression data from microarrays are being applied to predict preclinical and clinical endpoints, but the reliability of these predictions has not been established. In the MAQC-II project, 36 independent teams analyzed six microarray data sets to generate predictive models for classifying a sample with respect to one of 13 endpoints indicative of lung or liver toxicity in rodents, or of breast cancer, multiple myeloma or neuroblastoma in humans. In total, >30,000 models were built using many combinations of analytical methods. The teams generated predictive models without knowing the biological meaning of some of the endpoints and, to mimic clinical reality, tested the models on data that had not been used for training. We found that model performance depended largely on the endpoint and team proficiency and that different approaches generated models of similar performance. The conclusions and recommendations from MAQC-II should be useful for regulatory agencies, study committees and independent investigators that evaluate methods for global gene expression analysis.

Expression of mitofusin 2(R94Q) in a transgenic mouse leads to Charcot-Marie-Tooth neuropathy type 2A.

Charcot-Marie-Tooth disease type 2A is an autosomal dominant axonal form of peripheral neuropathy caused by mutations in the mitofusin 2 gene. Mitofusin 2 encodes a mitochondrial outer membrane protein that participates in mitochondrial fusion in mammalian cells. How mutations in this protein lead to Charcot-Marie-Tooth disease type 2A pathophysiology remains unclear. We have generated a transgenic mouse expressing either a mutated (R94Q) or wild-type form of human mitofusin 2 in neurons to evaluate whether the R94Q mutation was sufficient for inducing a Charcot-Marie-Tooth disease type 2A phenotype. Only mice expressing mitofusin 2(R94Q) developed locomotor impairments and gait defects thus mimicking the Charcot-Marie-Tooth disease type 2A neuropathy. In these animals, the number of mitochondria per axon was significantly increased in the distal part of the sciatic nerve axons with a diameter smaller than 3.5 microm. Importantly, the analysis of R94Q transgenic animals also revealed an age-related shift in the size of myelinated axons leading to an over-representation of axons smaller than 3.5 microm. Together these data suggest a link between an increased number of mitochondria in axons and a shift in axonal size distribution in mitofusin 2(R94Q) transgenic animals that may contribute to their neurological phenotype.