Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 April 2011-31 May 2011

This article documents the addition of 92 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anopheles minimus, An. sinensis, An. dirus, Calephelis mutica, Lutjanus kasmira, Murella muralis and Orchestia montagui. These loci were cross-tested on the following species: Calephelis arizonensi, Calephelis borealis, Calephelis nemesis, Calephelis virginiensis and Lutjanus bengalensis.     

Water deficits affect caffeate O-methyltransferase, lignification, and related enzymes in maize leaves. A proteomic investigation

Drought is a major abiotic stress affecting all levels of plant organization and, in particular, leaf elongation. Several experiments were designed to study the effect of water deficits on maize (Zea mays) leaves at the protein level by taking into account the reduction of leaf elongation. Proteomic analyses of growing maize leaves allowed us to show that two isoforms of caffeic acid/5-hydroxyferulic 3-O-methyltransferase (COMT) accumulated mostly at 10 to 20 cm from the leaf point of insertion and that drought resulted in a shift of this region of maximal accumulation toward basal regions. We showed that this shift was due to the combined effect of reductions in growth and in total amounts of COMT. Several other enzymes involved in lignin and/or flavonoid synthesis (caffeoyl-CoA 3-O-methyltransferase, phenylalanine ammonia lyase, methylenetetrahydrofolate reductase, and several isoforms of S-adenosyl-l-methionine synthase and methionine synthase) were highly correlated with COMT, reinforcing the hypothesis that the zone of maximal accumulation corresponds to a zone of lignification. According to the accumulation profiles of the enzymes, lignification increases in leaves of control plants when their growth decreases before reaching their final size. Lignin levels analyzed by thioacidolysis confirmed that lignin is synthesized in the region where we observed the maximal accumulation of these enzymes. Consistent with the levels of these enzymes, we found that the lignin level was lower in leaves of plants subjected to water deficit than in those of well-watered plants.     

Mapping of a spontaneous mutation for early flowering time in maize highlights contrasting allelic series at two-linked QTL on chromosome 8

Only a few mutations affecting flowering time have been detected in maize. We analyzed a spontaneous early mutation, vgt-f7p, which appeared during production of the inbred line F7. This mutation shortens the time from planting to flowering by about 100 growing degree days (GDD), and reduces the number of nodes. It therefore seems to affect the timing of meristem differentiation from a vegetative to a reproductive state. It was mapped to a 6 cM confidence interval on chromosome 8, using a QTL mapping approach. QTL analysis of a mapping population generated by crossing the mutant F7 line (F7p) and the Gaspé flint population showed that vgt-f7p is probably allelic to vgt1, a QTL described in previous studies, and affects earliness more strongly than the Gaspé allele at vgt1. Global analysis of the QTL in the region suggested that there may be two consensus QTL, vgt1 and vgt2. These two QTL have contrasting allelic effects: rare alleles conferring extremely early flowering at vgt1 vs. greater diversity and milder effects at locus vgt2. Finally, detailed syntenic analysis showed that the vgt1 region displays a highly conserved duplicated region on chromosome 6, which also plays an important role in maize flowering time variation. The cloning of vgt1 should, therefore, also facilitate the analysis of the molecular basis of variation due to this second region.     

Surfactant protein D decreases pollen-induced IgE-dependent mast cell degranulation

Mast cells play a key role in allergy and asthma. They reside at the host-environment interface and are among the first cells to make contact with inhaled microorganisms and particulate antigens. Pulmonary surfactant proteins A and D (SP-A and SP-D) function in lung host defense by enhancing microbe phagocytosis and mediating other immune cell functions, but little is known about their effects on mast cells. We hypothesized that SP-A and/or SP-D modulate IgE-dependent mast cell functions. Pollen starch granules (PSG) extracted from Dactylis glomerata and coated with trinitrophenol (TNP) were used as a model of an inhaled organic particulate allergen. Our data revealed that SP-D inhibited by 50% the release of beta-hexosaminidase by peritoneal mast cells sensitized with IgE anti-TNP and stimulated with TNP-PSG. In contrast, SP-A had no effect. Furthermore, SP-D aggregated PSG in a dose-dependent manner, and this aggregation was mediated by SP-D's carbohydrate recognition domain. A single arm SP-D mutant (RrSP-Dser15,20) neither aggregated PSG nor inhibited degranulation, suggesting that multimerization of SP-D is required for maximal PSG aggregation and inhibition of PSG-induced mast cell degranulation. This study is the first to demonstrate that SP-D modulates IgE-mediated mast cell functions, which are important in asthma and allergic inflammation.     

Characterization of antiproliferative and cytotoxic properties of the HSV-1 immediate-early ICPo protein

BACKGROUND: The identification of novel proteins displaying cytostatic and/or cytotoxic actions that could eventually be used for gene therapy is a major issue in cancer research. Data from the literature suggested that the immediate-early ICP0 protein from herpes simplex virus type 1 (HSV-1) could fulfill these properties as it had been observed that this protein is involved in arrest of cell growth at the G1/S and G2/M phases of the cell cycle and that deletion of ICP0 from HSV-1 or HSV-1-recombinant vectors significantly reduced their cytotoxicity. The molecular basis of its action is likely related to the ability of ICP0, which possesses E3-ubiquitin ligase activity, to target destruction of key cellular proteins, including centromeric proteins, resulting in abnormal chromosome segregation, unusual cytokinesis, and emergence of nuclear morphological aberrations. However, neither the gene therapy potential of ICP0 on its own nor its action on primary quiescent cells has been assessed to date. The aim of this work was therefore to evaluate the antiproliferative and cytotoxic properties of ICP0 on a human glioblastoma cell line and on quiescent primary cells, and to explore whether this protein has a potential for gene therapy of cancer. METHODS: HSV-1-based amplicon particles were generated following a recently described method that produces relatively high titers of vector stocks that are essentially free of helper virus. These vectors express either wild-type ICP0 or FXE, a RING finger mutated inactive form of ICP0, together with reporter green fluorescent protein (GFP). The vectors were used to infect Gli36 cells, which derive from a human glioblastome, and cultured rat primary cardiomyocytes and brain cells, two well-established models of non-dividing cells. Expression and localization of ICP0 and FXE, as well as their action on centromeres and nuclear morphology, were evaluated by Western blotting, indirect immune fluorescence, and confocal microscopy using specific antibodies and DAPI labeling. The impact of ICP0 on cell growth, toxicity, and viability was evaluated in the different cells using a variety of methods, including FACS analysis after propidium iodide and AnnexinV staining, crystal violet staining, clonogenic capability, caspase 3 activation, MTT tests, and release of lactate dehydrogenase, after infection with the different vectors. RESULTS: The three cell types under study showed high levels of transduction by amplicons and strong expression of GFP, ICP0, and FXE transgenic proteins. Wild-type ICP0, but not FXE, induced centromeric disruption, appearance of micronuclei, arrest of proliferation, and significant cell death in glioblastoma Gli36 cells. In contrast, neither micronuclei formation nor any other sign of cell toxicity could be observed in cultured primary cardiomyocytes or brain cells, as evaluated by MTT tests and crystal violet staining. Furthermore, in the case of cardiomyocytes, expression of ICP0 did not interfere with beating as cells continued to beat at the same frequency as non-infected cells for several days post-infection. Neither AnnexinV early staining nor caspase 3 activation was observed in dying infected Gli36 cells, suggesting that these cells were not entering apoptosis. In contrast, release of lactate dehydrogenase by infected Gli36 cells suggested a necrotic way of death. CONCLUSIONS: ICP0 induced a strong cytostatic action followed by significant cell death on the glioblastoma Gli36 cell line. In contrast, neither cell death nor any other evidence of ICP0-induced toxicity affecting major physiological parameters was observed in primary cultured cardiomyoctes and brain cells, two models of primary non-cycling cells. These results suggest that ICP0 has gene therapy potential and could represent the first member of a novel family of directly acting proteins that could be used to treat cancers.