Screening Genes for Association with Loci for Nitrogen-Use Efficiency in Perennial Ryegrass by Pyrosequencing\u3csup\u3eTM\u3c/sup\u3e

The application of marker-assisted selection to improve quantitative traits in perennial ryegrass (Lolium perenne) is cumbersome. It requires a priori knowledge on the association of markers and genes. The knowledge on the chromosomal location of major genes for quantitative traits as well as on gene sequences is rapidly growing. However, determination of the genetic constitution of parents prior to their use in breeding still is impractical. More realistic is to collect association data along with the testing activities needed for breeding new varieties. This study uses changes in allele frequency due to selection as a criterion for gene-trait association. Selection-dependent changes are detected with single nucleotide polymorphisms (SNPs) of candidate genes using DNA-pools of F2 plants differing in nitrogen-use efficiency (NUE). The procedure and its feasibility are outlined for one locus

Selection for Nitrogen Use Efficiency in Perennial Ryegrass using Hydroponics

A selection method was tested for improving nitrogen use efficiency of perennial ryegrass (Lolium perenne L.) using a hydroponics system that controls the plant nitrogen concentration. Divergent selection for plant production, leaf area increase and dry matter distribution in six segregating populations under limiting nitrogen supply resulted in differences between offspring of upward and downward selections larger than 20 % in most characteristics. Moderately high realized heritabilities were found for some characteristics (up to 0.88). It is concluded that early selection for nitrogen use efficiency is feasible using this hydroponics system

Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis

Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)–matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4+ T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19L-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19L-specific CD4+ T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8+ mHag-specific T cells. They also lysed CD19L-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19L-derived HLA class II–restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings

A Uniform Genomic Minor Histocompatibility Antigen Typing Methodology and Database Designed to Facilitate Clinical Applications

BACKGROUND: Minor Histocompatibility (H) antigen mismatches significantly influence the outcome of HLA-matched allogeneic stem cell transplantation. The molecular identification of human H antigens is increasing rapidly. In parallel, clinical application of minor H antigen typing has gained interest. So far, relevant and simple tools to analyze the minor H antigens in a quick and reliable way are lacking. METHODOLOGY AND FINDINGS: We developed a uniform PCR with sequence-specific primers (PCR-SSP) for 10 different autosomal minor H antigens and H-Y. This genomic minor H antigen typing methodology allows easy incorporation in the routine HLA typing procedures. DNA from previously typed EBV-LCL was used to validate the methodology. To facilitate easy interpretation for clinical purposes, a minor H database named dbMinor (http://www.lumc.nl/dbminor) was developed. Input of the minor H antigen typing results subsequently provides all relevant information for a given patient/donor pair and additional information on the putative graft-versus-host, graft-versus-tumor and host-versus-graft reactivities. SIGNIFICANCE: A simple, uniform and rapid methodology was developed enabling determination of minor H antigen genotypes of all currently identified minor H antigens. A dbMinor database was developed to interpret the genomic typing for its potential clinical relevance. The combination of the minor H antigen genomic typing methodology with the online dbMinor database and applications facilitates the clinical application of minor H antigens anti-tumor targets after stem cell transplantation

The HERMIT in the machine: a plugin for the interactive transformation of GHC core language programs

The importance of reasoning about and refactoring programs is a central tenet of functional programming. Yet our compilers and development toolchains only provide rudimentary support for these tasks. This paper introduces a programmatic and compiler-centric interface that facilitates refactoring and equational reasoning. To develop our ideas, we have implemented HERMIT, a toolkit enabling informal but systematic transformation of Haskell programs from inside the Glasgow Haskell Compiler’s optimization pipeline. With HERMIT, users can experiment with optimizations and equational reasoning, while the tedious heavy lifting of performing the actual transformations is done for them. HERMIT provides a transformation API that can be used to build higher-level rewrite tools. One use-case is prototyping new optimizations as clients of this API before being committed to the GHC toolchain. We describe a HERMIT application - a read-eval-print shell for performing transformations using HERMIT. We also demonstrate using this shell to prototype an optimization on a specific example, and report our initial experiences and remaining challenges

A Human Minor Histocompatibility Antigen Specific for B Cell Acute Lymphoblastic Leukemia

Human minor histocompatibility antigens (mHags) play an important role in the induction of cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene encoding the antigenic peptide recognized by HB-1–specific CTLs. Interestingly, expression of the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus–transformed B cells. The HB-1 gene–encoded peptide EEKRGSLHVW is recognized by the CTL in association with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid substitution is critical for recognition by HB-1–specific CTLs. The restricted expression of the polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1–specific CTLs in vitro using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD

Pathogenesis of bovine spongiform encephalopathy in sheep

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrPSc) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrPSc was detected after 6 months in the tonsil and the ileal Peyer’s patches. At 9 months postinfection, PrPSc accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrPSc accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrPSc was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7–L1. At subsequent time points, PrPSc was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrPSc involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy

Spring emergence and canopy development strategies in miscanthus hybrids in Mediterranean, continental and maritime European climates

Due to its versatility and storability, biomass is an important resource for renewable materials and energy. Miscanthus hybrids combine high yield potential, low input demand, tolerance of certain marginal land types and several ecosystem benefits. To date, miscanthus breeding has focussed on increasing yield potential by maximising radiation interception through: 1) selection for early emergence, 2) increasing the growth rate to reach canopy closure as fast as possible, and 3) delayed flowering and senescence. The objective of this study is to compare early season re-growth in miscanthus hybrids cultivated across Europe. Determination of differences in early canopy development on end-of-year yield traits is required to provide information for breeding decisions to improve future crop performance. For this purpose, a trial was planted with four miscanthus hybrids (two novel seed-based hybrids M. sinensis×sinensis (M sin×sin) and M. sacchariflorus×sinensis (M sac×sin), a novel rhizome-based M sac×sin and a standard Miscanthus×giganteus (M×g) clone) in the UK, Germany, Croatia and Italy, and was monitored in the third and fourth growing season. We determined differences between the hybrids in base temperature, frost sensitivity and emergence strategy. M×g and M sac×sin mainly emerged from belowground plant organs, producing fewer but thicker shoots at the beginning of the growing season, but these shoots were susceptible to air frosts (determined by recording 0°C 2 m above ground surface). By contrast, M sin×sin emerged 10 days earlier, avoiding damage by late spring frosts and producing a high number of thinner shoots from aboveground shoots. Therefore, we recommend cultivating M sac×sin at locations with low risk and M sin×sin at locations with higher risk of late spring frosts. Selecting miscanthus hybrids that produce shoots throughout the vegetation period is an effective strategy to limit the risk of late frost damage and avoid reduction in yield from a shortened growing season

Phenotype Frequencies of Autosomal Minor Histocompatibility Antigens Display Significant Differences among Populations

Minor histocompatibility (H) antigens are allogeneic target molecules having significant roles in alloimmune responses after human leukocyte antigen–matched solid organ and stem cell transplantation (SCT). Minor H antigens are instrumental in the processes of transplant rejection, graft-versus-host disease, and in the curative graft-versus-tumor effect of SCT. The latter characteristic enabled the current application of selected minor H antigens in clinical immunotherapeutic SCT protocols. No information exists on the global phenotypic distribution of the currently identified minor H antigens. Therefore, an estimation of their overall impact in human leukocyte antigen–matched solid organ and SCT in the major ethnic populations is still lacking. For the first time, a worldwide phenotype frequency analysis of ten autosomal minor H antigens was executed by 31 laboratories and comprised 2,685 randomly selected individuals from six major ethnic populations. Significant differences in minor H antigen frequencies were observed between the ethnic populations, some of which appeared to be geographically correlated