Comparative vaccine studies in HLA-A2.1-transgenic mice reveal a clustered organization of epitopes presented in hepatitis C virus natural infection

A polyepitopic CD8(+)-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.     

The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region

Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.     

A simplified method for the detection of Y chromosome microdeletions in infertile men using a multiplex sequence-tagged site-based amplification

Sixteen sequence-tagged sites (STSs) were combined in five amplification reactions, to screen for deletions of DNA fragments located within the AZFa, AZFb, and AZFc regions of the Y chromosome. This multiplex strategy is fast and reliable, and most of the azoospermia-associated deletions reported so far are detected with this simplified method. Internal control STSs are included that allow discrimination between deletion and failure of amplification.     

Differentiation of immune cells challenged by bacteria in the common European starfish,Asterias rubens (Echinodermata)

Amoebocytes are the main effector cells of the echinoderm immune system. In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type. The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria. Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus). Amoebocytes were collected at regular time intervals for 24 h. The cytometric characteristics and the phagocytic activity were studied by flow cytometry. Three amoebocyte groups of different size were identified. The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected. All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed. G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity. Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria. It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.     

Biogenesis and metabolism of Alzheimer's disease Abeta amyloid peptides

Biochemical and genetic evidence indicates the balance of biogenesis/clearance of Abeta amyloid peptides is altered in Alzheimer's disease. Abeta is derived, by two sequential cleavages, from the receptor-like amyloid precursor protein (APP). The proteases involved are beta-secretase, identified as the novel aspartyl protease BACE, and gamma-secretase, a multimeric complex containing the presenilins (PS). Gamma-secretase can release either Abeta40 or the more aggregating and cytotoxic Abeta42. Secreted Abeta peptides become either degraded by the metalloproteases insulin-degrading enzyme (IDE) and neprilysin or metabolized through receptor uptake mediated by apolipoprotein E. Therapeutic approaches based on secretase inhibition or amyloid clearance are currently under development.     

Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3)

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.     

Heat shock proteins and aging in Drosophila melanogaster

Heat shock proteins (Hsps) are conserved molecular chaperones that are upregulated following exposure to environmental stress and during aging. The mechanisms underlying the aging process are only beginning to be understood. The beneficial effects of Hsps on aging revealed in mild stress and overexpression experiments suggest that these proteins are part of an important cell protection system rather than being unspecific molecular chaperones. Among the Hsps families, small Hsps have the greatest influence on aging and the modulation of their expression during aging in Drosophila suggest that they are involved in pathways of longevity determination.     

Biotinylated synthetic chemokines: their use for the development of nonradioactive whole-cell binding assays

A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen.     

Growth and hepatic acetyl coenzyme-A carboxylase activity are affected by dietary protein level in European seabass (Dicentrarchus labrax)

Two trials were undertaken with European seabass (Dicentrarchus labrax) to estimate the protein requirements for maintenance and growth as well as the effect of dietary protein level on the activity of hepatic acetyl coenzyme-A carboxylase (ACoAC). Six diets were formulated to contain graded levels of protein (from 5 to 55% crude protein (CP)) at a constant (12%) lipid level. Three other diets were also formulated to contain 35, 45 and 55% CP, but with a higher lipid level (19%). Groups of 10 individually marked fish (IBW: 100 g) and groups of 8 fish (IBW: 160 g) were used in trial I and II, respectively. Fish were fed to visual satiety and intake was recorded. At the end of both studies, whole body, liver and plasma samples were withdrawn for analyses. Growth rate was improved with increasing dietary CP level. Despite not being the object of a statistical analysis, feed efficiency tended to be enhanced at higher dietary CP level and protein efficiency ratio tended to decrease with increased protein intake. The reduction of the dietary protein/energy ratio, due to the increase of dietary lipids further improved growth and feed utilisation. Data from both experiments indicate 4.5+/-0.5 g kg(-1) d(-1) as the daily protein intake for maximum N gain and 520+/-50 mg kg(-1) d(-1) as the maintenance needs for nitrogen balance. An increase of dietary CP level, up to 25%, increased ACoAC activity. A further increase in dietary CP level (35 to 55%) did not affect liver ACoAC activity. The increase in dietary lipid level depressed significantly liver ACoAC specific activity.