The impact of HLA class I and EBV latency-II antigen-specific CD8(+) T cells on the pathogenesis of EBV(+) Hodgkin lymphoma.

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.

Investigating preference-performance relationships in aboveground-belowground life cycles: a laboratory and field study with the vine weevil (Otiorhynchus sulcatus).

The preference-performance hypothesis has principally considered insect herbivores with aboveground lifecycles, although the hypothesis could be equally relevant to insects with life stages occurring both aboveground and belowground. Moreover, most studies have focussed on either laboratory or field experiments, with little attempt to relate the two. In this study, the preference-performance hypothesis was examined in an aboveground-belowground context in the laboratory using the vine weevil (Otiorhynchus sulcatus (F.)) (Coleoptera: Curculionidae) and two cultivars of red raspberry (Rubus idaeus), Glen Rosa and Glen Ample. A two-year field study (2008-2009) was also undertaken to characterise the population dynamics of adult weevils on the two raspberry cultivars. Larval performance (abundance and mass) differed significantly between Glen Rosa and Glen Ample, with Glen Rosa resulting in 26% larger but 56% fewer larvae compared to Glen Ample. Larval abundances were significantly and positively correlated with root nitrogen and magnesium concentrations, but negatively correlated with root iron. However, concentrations of these minerals were not significantly different in the two cultivars. Adult weevils did not preferentially select either of the two cultivars for egg laying (laying 3.08 and 2.80 eggs per day on Glen Ample and Glen Rosa, respectively), suggesting that there was no strong preference-performance relationship between adult vine weevils and their belowground offspring. Field populations of adult vine weevils were significantly higher on Glen Ample than Glen Rosa, which may reflect the higher larval survival on Glen Ample observed in laboratory experiments.

Multi-environment QTL mapping in blackcurrant (Ribes nigrum L.) using mixed models.

The first genetic linkage map of blackcurrant, published by Brennan et al. (Euphytica 161:19-34, 2008), identified regions where quantitative trait loci (QTLs) for some important traits were located. The analysis was complicated by the fact that the mapping population was found to contain two subgroups, with segregation ratios consistent with these being crossed and selfed offspring. The QTL analysis was based on the trait mean over 3 years and focused on the crossed offspring. Here we proposed a mixed model multi-environment approach for this population. The 3 years are considered as three separate environments, the data from both the selfed and crossed offspring are combined and different residual terms are explored to model the correlation between the years. This permits tests for interactions between the QTLs, the year and the type of offspring (selfed or crossed). This is applied to re-analyse two important traits, anthocyanin concentration and budbreak. Several additional QTLs were identified, some affecting the trait in both the selfed and crossed offspring, others in just one.

Strains of Epstein-Barr virus infecting multiple sclerosis patients.

Both epidemiological and experimental studies have indicated that the ubiquitous herpesvirus Epstein-Barr virus (EBV) plays a role in the pathogenesis of multiple sclerosis (MS). Some features of MS epidemiology, such as the decline in risk among migrants from high to low MS prevalence areas, suggest the presence of variant EBV strains that increase MS risk. The objective of this study was to investigate whether genetic variability in EBV is associated with MS. Genes encoding for two EBV antigens (EBNA1 and BRRF2) were sequenced in EBV isolates from 40 MS patients and a similar number of control subjects. These viral antigens were chosen for analysis because they are known to stimulate atypical immune responses in MS. Extensive sequence polymorphism was observed within the EBNA1 and BRRF2 genes in isolates from both MS patients and controls. Interestingly, several single nucleotide polymorphisms within the EBNA1 gene, and one within the BRRF2 gene, were found to occur at marginally different frequencies in EBV strains infecting MS patients versus controls. Although this study does not find a simple causal relationship between EBV strains and the occurrence of MS, the existence of haplotypes that occur at different frequencies in MS patients versus controls may provide an area for future study of the role of EBV strain variation in multiple sclerosis.

The isolation of genomic DNA from blackcurrant (Ribes nigrum L.).

A method is described for isolating DNA of high molecular mass (M(r)) from blackcurrant and other softfruit species. Following a hexacethylytimethyl ammonium bromide (CTAB)-based extraction procedure, samples are treated with a glycosidic hydrolase mixture and RNase, and then purified. The suitability of this DNA for Southern analysis and genomic-library construction is demonstrated.

Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit.

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.

Non-destructive visualisation in three dimensions of freezing events in flowers of blackcurrant (Ribes nigrum L.) using spin-echo NMR microscopy.

Flowers of blackcurrant (Ribes nigrum L.) were subjected to freezing stress in vivo, and the resulting damage examined in three dimensions using a spin-echo nuclear magnetic resonance imaging sequence. Increased signal intensity was detected in the damaged flowers, particularly at the base of the style, in T2-weighted images. This is thought to be the result of intracellular freezing, which causes membrane damage and leakage of cellular contents. It is proposed that this represents the main site of damage within the flowers. The imaging of flowers of differing developmental ages showed larger increases in signal from fully open flowers after freezing damage compared with those in the initial stages of bud, suggesting that the enclosed nature of the flower buds may have a protective effect on the sensitive stylar base. The use of three-dimensional nuclear magnetic resonance imaging provides a rapid and effective means for the visualisation of freezing events within floral tissues; the effective resolution of the images enables greater accuracy and clarity in interpretation than hitherto possible in two dimensions.

An approach to the determination of winter chill requirements for different Ribes cultivars.

Winter chilling is critical for flowering and fruiting of many temperate fruits, with evidence that blackcurrant (Ribes nigrum L.) cropping has been adversely affected by warm winters. Accurate models of chill accumulation in blackcurrant are required so that breeding strategies can be formulated for the generation of new cultivars with resilience to future climates. Existing models for chill accumulation have largely been derived from statistical correlation; here we report the derivation of improved models for blackcurrant using controlled environment treatments. Hardwood cuttings from a diverse set of cultivars were exposed to constant or varying chilling temperatures and the effects on bud break after transfer to a warm, permissive environment evaluated. The impact of different combinations of temperature and chilling periods were described in terms of their overall 'Effectiveness' (E). Clear genotypic differences were found, with excessive chilling often inhibiting bud break. There was a significant interaction between observed chilling response and the period of low temperature exposure. A number of chilling models to explain observed interactions between chilling temperature and time of exposure on bud break were compared; the most effective involved an optimal response to increasing chill accumulation. The effects of varying temperatures during chilling on bud break were complex, with warm temperature breaks substantially inhibiting bud development and cooler temperature breaks tending to enhance bud burst. The relevance of these models to generic studies of endodormancy is discussed, together with their potential application to the development of phenotyping screens for future breeding using diverse blackcurrant germplasm.

Gall mite molecular phylogeny and its relationship to the evolution of plant host specificity.

The phylogenetic relationships of all seven known species of Cecidophyopsis mites (Acari: Eriophyidae) with Ribes hosts have been inferred from ribosomal DNA sequences. This analysis found groups of closely related mites. The five gall-forming species, four of which are monophagous and one which has two hosts, were found in two groups. Another group consisted of the two non gall-forming species, one of which has two hosts, while the other is monophagous. The molecular phylogeny of their known Ribes host plants was calculated using the equivalent ribosomal regions as the mites. The structure of the two trees (mites vs hosts) was clearly different, implying that mite speciation did not closely follow speciation events in the plant hosts. Instead, the three groups of Ribes-infesting Cecidophyopsis mites have derived from a common galling ancestor millions of years ago. Each mite group has recently diversified onto different primary hosts. One group of mites has also lost the galling habit. The results have implications for host range changes and the durability of mite-resistance genes in cultivated Ribes.

RAPD fingerprinting of blackcurrant (Ribes nigrum L.) cultivars.

Ribes nigrum germplasm was screened for random amplified polymorphic DNA (RAPD) markers. Fiftyfour markers were identified which generated individual fingerprints for each of 21 cultivars. Genetic variation within R. nigrum germplasm, as detected by RAPDs, demonstrated that the genetic basis for improvement of blackcurrant is narrower than would be expected by the analysis of parentage.