The use of 2D fingerprint methods to support the assessment of structural similarity in orphan drug legislation.

In the European Union, medicines are authorised for some rare disease only if they are judged to be dissimilar to authorised orphan drugs for that disease. This paper describes the use of 2D fingerprints to show the extent of the relationship between computed levels of structural similarity for pairs of molecules and expert judgments of the similarities of those pairs. The resulting relationship can be used to provide input to the assessment of new active compounds for which orphan drug authorisation is being sought

Systems Biology Approaches Reveal a Specific Interferon-Inducible Signature in HTLV-1 Associated Myelopathy

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that persists lifelong in the host. In ∼4% of infected people, HTLV-1 causes a chronic disabling neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is unknown and treatment remains ineffective. We used gene expression microarrays followed by flow cytometric and functional assays to investigate global changes in blood transcriptional profiles of HTLV-1-infected and seronegative individuals. We found that perturbations of the p53 signaling pathway were a hallmark of HTLV-1 infection. In contrast, a subset of interferon (IFN)-stimulated genes was over-expressed in patients with HAM/TSP but not in asymptomatic HTLV-1 carriers or patients with the clinically similar disease multiple sclerosis. The IFN-inducible signature was present in all circulating leukocytes and its intensity correlated with the clinical severity of HAM/TSP. Leukocytes from patients with HAM/TSP were primed to respond strongly to stimulation with exogenous IFN. However, while type I IFN suppressed expression of the HTLV-1 structural protein Gag it failed to suppress the highly immunogenic viral transcriptional transactivator Tax. We conclude that over-expression of a subset of IFN-stimulated genes in chronic HTLV-1 infection does not constitute an efficient host response but instead contributes to the development of HAM/TSP

Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome

HTLV-1 Tax Mediated Downregulation of miRNAs Associated with Chromatin Remodeling Factors in T Cells with Stably Integrated Viral Promoter

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type

Oral abstracts 3: RA Treatment and outcomesO13. Validation of jadas in all subtypes of juvenile idiopathic arthritis in a clinical setting

Background: Juvenile Arthritis Disease Activity Score (JADAS) is a 4 variable composite disease activity (DA) score for JIA (including active 10, 27 or 71 joint count (AJC), physician global (PGA), parent/child global (PGE) and ESR). The validity of JADAS for all ILAR subtypes in the routine clinical setting is unknown. We investigated the construct validity of JADAS in the clinical setting in all subtypes of JIA through application to a prospective inception cohort of UK children presenting with new onset inflammatory arthritis. Methods: JADAS 10, 27 and 71 were determined for all children in the Childhood Arthritis Prospective Study (CAPS) with complete data available at baseline. Correlation of JADAS 10, 27 and 71 with single DA markers was determined for all subtypes. All correlations were calculated using Spearman's rank statistic. Results: 262/1238 visits had sufficient data for calculation of JADAS (1028 (83%) AJC, 744 (60%) PGA, 843 (68%) PGE and 459 (37%) ESR). Median age at disease onset was 6.0 years (IQR 2.6-10.4) and 64% were female. Correlation between JADAS 10, 27 and 71 approached 1 for all subtypes. Median JADAS 71 was 5.3 (IQR 2.2-10.1) with a significant difference between median JADAS scores between subtypes (p < 0.01). Correlation of JADAS 71 with each single marker of DA was moderate to high in the total cohort (see Table 1). Overall, correlation with AJC, PGA and PGE was moderate to high and correlation with ESR, limited JC, parental pain and CHAQ was low to moderate in the individual subtypes. Correlation coefficients in the extended oligoarticular, rheumatoid factor negative and enthesitis related subtypes were interpreted with caution in view of low numbers. Conclusions: This study adds to the body of evidence supporting the construct validity of JADAS. JADAS correlates with other measures of DA in all ILAR subtypes in the routine clinical setting. Given the high frequency of missing ESR data, it would be useful to assess the validity of JADAS without inclusion of the ESR. Disclosure statement: All authors have declared no conflicts of interest. Table 1Spearman's correlation between JADAS 71 and single markers DA by ILAR subtype ILAR Subtype Systemic onset JIA Persistent oligo JIA Extended oligo JIA Rheumatoid factor neg JIA Rheumatoid factor pos JIA Enthesitis related JIA Psoriatic JIA Undifferentiated JIA Unknown subtype Total cohort Number of children 23 111 12 57 7 9 19 7 17 262 AJC 0.54 0.67 0.53 0.75 0.53 0.34 0.59 0.81 0.37 0.59 PGA 0.63 0.69 0.25 0.73 0.14 0.05 0.50 0.83 0.56 0.64 PGE 0.51 0.68 0.83 0.61 0.41 0.69 0.71 0.9 0.48 0.61 ESR 0.28 0.31 0.35 0.4 0.6 0.85 0.43 0.7 0.5 0.53 Limited 71 JC 0.29 0.51 0.23 0.37 0.14 -0.12 0.4 0.81 0.45 0.41 Parental pain 0.23 0.62 0.03 0.57 0.41 0.69 0.7 0.79 0.42 0.53 Childhood health assessment questionnaire 0.25 0.57 -0.07 0.36 -0.47 0.84 0.37 0.8 0.66 0.4