All identified RF1-type strains appear to be introduced from a single source, suggesting that intergenotypic recombination in HCV is sporadic and not associated with cocirculation of different genotypes in a population. The RF1 strain in this study was responsive to interferon in vivo.

SllmmaryThe CD40 surface membrane molecule plays an important role in the activation of mature human B cells, but its role in earlier stages orB lineage development is unknown. Here, we have investigated the effects of triggering the CD40 antigen on B cell precursors (BCP) by crosslinking with anti-CD40 antibody presented by Fc'/-receptor type II-transfected murine Ltk-cells (CD40 system). CD10 + surface immunoglobulin negative (slg-) BCP, freshly isolated from fetal bone marrow or precultured on stromal cells, proliferated in the CD40 system. This effect required the presence of IL-3, which acted as a specific cosignal among a panel of cytokines examined. The association of IL-10 and IL-7 potentiated the observed IL-3 and CD40-dependent BCP proliferation, demonstrating that IL-10 can act on early B lineage cells. CD40-dependent activation of fetal BCP did not favor maturation to slg + B cells, but resulted in the induction of high levels of surface membrane CD23. The emerging CD23 + BCP lacked slg and CD10, and represented an important proportion of the cycling cells in the CD40-dependent cultures. Taken together, our data demonstrate that stimulation of the CD40 antigen induces expression of the CD23 gene, and regulates cell proliferation during normal human B cell ontogeny.T he life history orB lymphocytes can be divided into two major steps. First, an antigen-independent process (B lymphopoiesis) permits constant generation of new immunocompetent surface (s)llgM + slgD + mature B cells in the bone marrow (for reviews see references 1 and 2). Then, mature B cells can be driven by antigens to proliferate and differentiate in secondary lymphoid organs (immunopoiesis) (for a review see reference 3). Both soluble cytokines and cell membrane-associated molecules regulate the various steps of B lymphocyte development. In this context, the CD40 B cell surface membrane molecule has recently been identified as playing a fundamental role in the process of immunopoiesis. Human CD40 is a 50-kD glycosylated phosphoprotein (4), that belongs to a new superfamily of receptors that includes the receptors for nerve growth factor and TNF (5, 6).Anti-CD40 antibody presented by Fc',/receptor type II (Fc'yRII) (CDw32)-transfected murine fibroblastic Ltkcells (CD40 system) induces strong and long-lasting B cell expansion in the presence of IL-4 (7, 8). The proliferation of anti-CD40-activated mature B cells is also enhanced by IL-10 (9). More strikingly, IL-10 allows CD40-activated B cells to produce large quantities of IgM, IgG, and IgA after their differentiation into plasma cells. Culturing CD40-activated naive slgD + slgM + B cells in the presence of IL-4 or IL-10 and TGF-~ induces the production of IgE or IgA, respectively, as a consequence of isotype switching (10-14).Recent studies have indicated that CD40 crosslinking represents the first step of the interaction between T and B cells. A ligand to CD40 has recently been identified and expressed from activated T cells, and CD40-Ig fusion proteins inhibit T cell-dependent B ce...

Hepatitis C virus (HCV) exists as a quasispecies within an infected individual. We have previously reported an in-frame 3 bp insertion event at the N-terminal region of the E2 glycoprotein from a genotype 4a HCV isolate giving rise to an atypical 28 aa hypervariable region (HVR) 1. To further explore quasispecies evolution at the HVR1, serum samples collected over 9.6 years from the same chronically infected, treatment naïve individual were subjected to retrospective clonal analysis. Uniquely, we observed that isolates containing this atypical HVR1 not only persisted for 7.6 years, but dominated the quasispecies swarm. Just as striking was the collapse of this population of variants towards the end of the sampling period in synchrony with variants containing a classical HVR1 from the same lineage. The replication space was subsequently occupied by a second minor lineage, which itself was only intermittently detectable at earlier sampling points. In conjunction with the observed genetic shift, the coexistence of two distinct HVR1 populations facilitated the detection of putative intra-subtype recombinants, which included the identification of the likely ancestral parental donors. Juxtaposed to the considerable plasticity of the HVR1, we also document a degree of mutational inflexibility as each of the HVR1 subpopulations within our dataset exhibited overall genetic conservation and convergence. Finally, we raise the issue of genetic analysis in the context of mixed lineage infections.

Pre-treatment HCV quasispecies complexity and diversity may predict response to interferon based anti-viral therapy. The objective of this study was to retrospectively (1) examine temporal changes in quasispecies prior to the start of therapy and (2) investigate extensively quasispecies evolution in a group of 10 chronically infected patients with genotype 3a, treated with pegylated α2a-Interferon and ribavirin.The degree of sequence heterogeneity within the hypervariable region 1 was assessed by analyzing 20-30 individual clones in serial serum samples. Genetic parameters, including amino acid Shannon entropy, Hamming distance and genetic distance were calculated for each sample. Treatment outcome was divided into (1) sustained virological responders (SVR) and (2) treatment failure (TF).Our results indicate, (1) quasispecies complexity and diversity are lower in the SVR group, (2) quasispecies vary temporally and (3) genetic heterogeneity at baseline can be use to predict treatment outcome.We discuss the results from the perspective of replicative homeostasis.

SummaryWe have recently demonstrated that tumor necrosis factor ol (TNF-ot) potentiates interleukin 3 (IL-3)-and granulocyte/macrophage colony-stimulating factor-induced growth of CD34 + hematopoietic progenitor cells (HPC), and favors the generation of dendritic/Langerhans cells. The stimulatory effect of TNF-ot was detailed in the present study. Thus, CD34 + HPC entering in cycle (S/G2M) after a 48-h pulse with IL-3 expressed the transferrin receptor (TfR), and fluorescence-activated cell sorter-separated TfR + HPC, but not TIP,-HPC, showed a high proliferative response to IL-3. In contrast, TfR-HPC were found to undergo strong proliferation in response to IL-3 + TNF-cz. Limiting dilution experiments indicated that TNF-cz increased both the frequency and the average size of clones generated from TfR-HPC as a result of the development of a higher number of large clones. In contrast, TNF-ot did not enhance the IL-3-dependent proliferation of TfR + HPC. Preculturing CD34 + HPC for 48 h with TNF-ot enhanced the subsequent generation of IL-3-dependent colony-forming units. Precultures with TNF-a or cultures with suboptimal doses of TNF-cz allowed the recruitment of cells with both granulocytic and monocytic differentiation potential. Taken together, our results indicate that TNF-ot recruits a subpopulation of CD34 § HPC hyposensitive to IL-3, with high proliferative capacity and some features of multipotential progenitors, that are likely to be more primitive than those responding to IL-3 alone.

Background: Recombination between hepatitis C single stranded RNA viruses is a rare event. Natural viable intragenotypic and intergenotypic recombinants between 1b-1a, 1a-1c and 2k-1b, 2i-6p, respectively, have been reported. Diagnostically recombinants represent an intriguing challenge. Hepatitis C genotype is defined by interrogation of the sequence composition of the 5' untranslated region [5'UTR]. Occasionally, ambiguous specimens require further investigation of the genome, usually by interrogation of the NS5B region. The original purpose of this study was to confirm the existence of a suspected mixed genotype infection of genotypes 2 and 4 by clonal analysis at the NS5B region of the genome in two specimens from two separate individuals. This initial identification of genotype was based on analysis of the 5'UTR of the genome by reverse line probe hybridisation [RLPH].

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The chromosomal region 13q14.3 is frequently deleted in B cell chronic lymphocytic leukemia (B-CLL) and it is supposed that a tumor suppressor gene, involved in this leukemogenesis, is located in this area. The first exons of two genes, Leu1 and Leu2, mapped in a minimally deleted 13q14.3 region, are systematically lost in B-CLL sharing a 13q14.3 deletion. These two genes have been proposed as strong tumor suppressor gene candidates. However, in a study on 15 13q14.3 deleted B-CLL, we found three patients in which this critical region was not involved. Because of these results and that no mutations were detected on the two genes in a previous study, we think that Leu1 and Leu2 can be excluded as tumor suppressor genes.