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Background To obtain a systematic overview of genes, which may serve therapeutic targets, the CHDI Foundation has recently established the HD Research Crossroads database (http://www.hdresearchcrossroads.org/). With currently over 800 genes, this resource constitutes the most extensive curation of genes relevant to HD, and provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD. Aims Our aims are (1) to obtain a comprehensive overview of therapeutic targets for HD, their functions and associated pathways as well as (2) to identify potential novel disease-relevant mechanisms. Methods We have carried out a variety of bioinformatical and statistical analyses to scrutinise genes curated in the HD Crossroads. For selected processes, we also analysed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Crossroads with previous genome-wide linkage studies. Results Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signalling), but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signalling). For follow-up studies, we provide a compendium of molecular mechanisms that are associated with HD. Additionally, we derived a candidate set of 24 novel genetic modifiers. Details can be obtained at http://hd.sysbiolab.eu. Conclusions Our analyses can be seen as a first step towards a comprehensive list of biological processes, molecular functions, and pathways involved in HD, and may provide a basis for the development of more holistic disease models and new therapeutics.

Abstract 4784 The identification of soluble factors involved in stem cell renewal is a major goal in the assessment of the BM niche. We have previously shown that human endothelial cell (EC) supernatants can induce the proliferation of hematopoietic progenitor cells (HPCs), especially after stimulation with IL-1b. To identify new potential growth factors, we compared the expression profile of IL-1b-stimulated ECs over 4, 8 and 16 h with non-stimulated Ecs using oligonucleotide microarrays covering more than 46 000 transcripts. Most significant changes were detected after 4 h. Utilization of Gene Ontology annotation for the stimulated EC transcriptome indicated multiple upregulated genes encoding extracellular proteins with a cell–cell signaling function. Using flow cytometry, delta, colony and cobblestone assays, we assessed the proliferative capacities of 11 gene products, i.e. IL-8, IL-32, FGF-18, osteoprotegerin, Gro 1–3, ENA78, GCP-2, CCL2 and CCL20, which are not known to induce HPC expansion. Notably, IL-32 and to a lesser degree osteoprotegerin and Gro 3 significantly induced the proliferation of HPCs. Furthermore, IL-32 attenuated chemotherapy-related BM cytotoxicities by increasing the number of colony forming cells in mice (7.4±0.87×103 versus 0.7±0.25×103, p=0.003). Our findings confirm that the combination of microarrays and gene annotation helps to identify new hematopoietic growth factors. Disclosures: No relevant conflicts of interest to declare.

Background The endoplasmic reticulum (ER) is a crucial organelle for correct folding and modification of numerous proteins. Accumulation of unfolded or misfolded protein causes a stress signal in the ER. To ensure fidelity of protein folding, several transcriptional and translational mechanisms are activated during the unfolded protein response (UPR). The main function of the UPR is re-establishing homeostasis by increasing the folding capacity. Recent studies, however, have suggested that ER stress and activated UPR are interconnected with inflammatory processes (K Zhang, RJ Kaufman. Nature 2008). Aims In this study, we explore the intriguing possibility that ER stress is an initiator of neuroinflammation in HD and is thereby crucially contributing to degeneration of neuronal tissue. Methods We have conducted a critical review of the accumulated evidence of inflammation induced by ER stress in neurodegenerative diseases in general and in HD specifically. Additionally, we scrutinised publically accessible data sets for molecular mechanisms linking ER stress, UPR and neuroinflammation. Results Several independent lines of evidence indicate that ER stress can mediate inflammatory responses and can enhance neurodegeneration. Notably, secreted inflammatory cytokines and activated microglia cells could be a possible mechanism leading to axonal damage and extensive neuronal cell death in HD patients. Using a systems biology approach, we developed explicit models for the interconnection between ER stress and neuroinflammation. Based on these models, we propose a defined set of follow-up experiments to further clarify the role of inflammatory signalling in HD. Conclusions The conducted review points to several mechanisms linking ER stress to inflammation and degeneration of neuronal tissue in Huntington9s disease. We hope that the suggested experimental studies can ultimately lead to the development of novel HD drug therapeutics.

Background The use of high throughput technologies has provided us with large amounts of data for Huntington9s disease (HD), especially at the gene and protein levels. As phenotypic changes in HD may not just be due to the activity of individual genes but interrelated alterations of whole molecular networks, it is important to systematically analyse and represent accumulated data in a network based approach. Aim To establish, analyse and visualise protein interaction networks through a dedicated database to HD. This approach will not only help to understand molecular mechanisms better but also help in identifying potential molecular markers involved in HD. Methods A HD specific protein–protein database interaction with additional gene expression information obtained from high throughput gene expression experiments has been constructed. We further identified both pathways as well as gene ontology (GO) annotations that are enriched in HD and constructed networks of the pathways and enriched GO annotations. Results Our integrative network based methodology helped us to identify pathways and networks that are specific to the HD and may lead to the identification of novel molecular targets. Follow-up analyses of molecular changes will distinguish between potential causes and effects in HD. Conclusions Our study demonstrates that network approaches can greatly facilitate the interpretation of large biological data sets and can support the elucidation of the molecular mechanisms underlying HD. We hope that the constructed database will help researchers worldwide to find a cure for HD.

Purpose. The expansion of hematopoietic progenitor cells (HPC) from cord blood is limited so far. For HPC expansion and differentiation we evaluated a coculture model of cord blood-derived HPC and endothelial cells (EC) isolated from the umbilical cord. Methods. CD34(+) HPC were cultured directly in contact with the endothelial monolayer or indirectly using a 0.4μm microporous transmembrane. EC were stimulated by various cytokines including TPO, interleukin (IL) 1β, IL4, and IL6. After 1 and 2 weeks, the HPC were counted and analyzed by flow cytometry. Their plating efficiencies were determined by methylcellulose assays, and their reconstitutive potential was analyzed by cobblestone assays. DNA microarrays on the expanding cells as well as on cytokine-stimulated EC were also performed. Results. EC stimulation with thrombopoietin and IL4 had no significant effect on mononuclear cell expansion. In contrast, IL1β stimulation resulted in a 10 and 100fold increase of the cell number after one and two weeks, respectively. More than 90% of these cells were CD33(+) and had a plating efficiency equivalent to that of HPC post isolation (7.3 ±1.2 versus 8.3±0.68%). The number of 5-wk cobblestone-area forming cells among these expanded cells was diminished, whereas the number of 2-wk cobblestone areas did not change (10.6 versus 8.5). The expanded cells could further mature into functional granulocytes. Time-course DNA microarrays of IL1β-stimulated EC demonstrated an increased expression of genes encoding tumor necrosis factor-induced proteins, interleukins, and endothelial adhesion molecules. When IL1β was replaced by IL6, a 7-fold expansion of morphologically progenitor-like cells took place. 10–13% of these cells were positive for CD133, had a plating efficiency of 10.2±1.7%, and showed on average 14.5 five-week cobblestone areas. Conclusion. Cocultures of cord blood-derived HPC with umbilical cord-derived EC offer alternative methods for the expansion and differentiation of hematopoietic cells.

In this study we present two novel normalization schemes for cDNA microarrays. They are based on iterative local regression and optimization of model parameters by generalized cross-validation. Permutation tests assessing the efficiency of normalization demonstrated that the proposed schemes have an improved ability to remove systematic errors and to reduce variability in microarray data. The analysis also reveals that without parameter optimization local regression is frequently insufficient to remove systematic errors in microarray data.