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The zebrafish model system is helping researchers improve the health and welfare of people and animals and has become indispensable for advancing biomedical research. As genetic engineering is both resource intensive and time-consuming, sharing successfully developed genetically modified zebrafish lines throughout the international community is critical to research efficiency and to maximizing the millions of dollars in research funding. New restrictions on importation of zebrafish into Canada based on putative susceptibility to infection by the spring viremia of carp virus (SVCV) have been imposed on the scientific community. In this commentary, we review the disease profile of SVCV in fish, discuss the findings of the Canadian government's scientific assessment, how the interpretations of their assessment differ from that of the Canadian research community, and describe the negative impact of these regulations on the Canadian research community and public as it pertains to protecting the health of Canadians.

Background The current model of rheumatoid arthritis management is treat to target, which involves intensive treatment that is titrated until remission from disease activity is attained. Another treatment target that might be more important to patients is normalising health-related quality of life (HRQoL). HRQoL is a multidimensional concept; whether remission and intensive treatment improve all dimensions equally is uncertain. We evaluated the impact of intensive treatment and remission on HRQoL in rheumatoid arthritis. Methods Patients enrolled to the CARDERA and TACIT trials were evaluated. CARDERA randomised 467 patients to 6 months of either active or placebo high-dose corticosteroids; TACIT randomised 205 patients to 1 year of combination disease-modifying antirheumatic drugs (DMARDs) or tumour necrosis factor α inhibitors (TNFi). We measured HRQoL with the Short Form 36 (SF-36), capturing information on physical and mental health across eight domains. The effect of treatment and remission on SF-36 scores was evaluated by linear regression and spydergrams, respectively. Findings Corticosteroids resulted in significant improvements in physical health, relative to placebo (physical component summary [PCS] score β=3·78, p=0·0001); no effects were seen on mental health (as judged by mental component summary [MCS] score). TNFi therapy provided a greater improvement in physical health than did combination DMARDs (PCS β=3·04, p=0·034); no difference in mental health improvement was observed. In both trials, patients in remission at the final timepoint had physical health scores substantially below those seen in the normal UK population. Normalised PCS and MCS scores (which have a mean score of 50 in the general population) for patients in remission were, respectively, 46 (SD 9·3) and 52 (11·5) in CARDERA and 40 (10·5) and 53 (9·8) in TACIT. Interpretation We found that intensive rheumatoid arthritis treatment does not improve all aspects of HRQoL: corticosteroids and TNFi provided improvements in physical but not mental health, relative to placebo and combination DMARDs, respectively. Although remission improved HRQoL, it did not normalise physical health. Therefore, measures specifically targeting HRQoL are needed. The study's strength is its replication of key findings across two trials; that it did not test a prespecified hypothesis is a key limitation. Funding National Institute for Health Research.

Molecular signals are not the only forces that pattern and shape the developing embryo. Mechanical stresses sensed by cells also seem to be involved in creating the body plan.

Delivering treat-to-target strategies in rheumatoid arthritis (RA) involves commitments from both providers and payers for healthcare. We have summarised the perspectives of payers from England, where the National Health Service (NHS) provides universal care that is without cost for patients.We reviewed the literature - including that from the NHS and National Institute for Health and Clinical Excellence (NICE) - concerning payers views on the clinical effectiveness and cost-effectiveness of treat-to-target strategies for RA.Commissioners pay for government-funded English healthcare and providers (divided between General Practitioners (GPs) and Hospital Consultants) and deliver it according to NICE guidance. Treat-to-target using intensive disease-modifying drug (DMARD) combinations with glucocorticoids are recommended for early active RA. Treatment tapering is recommended when disease control is achieved. Some aspects of treat-to-target are recommended in established RA, including the early management of flares and the use of biologics in persistently active RA that is non-responsive to DMARDs. However, treat-to-target is not widely recommended in established RA, mainly because the evidence base is incomplete. English healthcare is moving towards quality care becoming the main driver and is adopting 'integrated care' involving both GPs and consultants for most long-term disorders; RA is likely to be included within these approaches, which are unlikely to focus specifically on treat-to-target.Payers strongly support treat-to-target in early RA. In established disease there is limited enthusiasm; without stronger evidence for efficacy and cost-effectiveness this is unlikely to change.

Background During aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis. Methods Satellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56+ cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised. Results Large-scale isolation of CD56+ cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers. Conclusions We have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function. Electronic supplementary material The online version of this article (doi:10.1007/s13539-012-0097-z) contains supplementary material, which is available to authorized users.

Development of the heart, like that of other organs, requires the specification of progenitor cell populations that will ultimately form the differentiated cell types of the functional organ. A relatively recent and exciting advance in cardiac research has been the identification of cardiovascular progenitor cells (CPCs), which have the potential to form the major cell types of the heart (cardiomyocytes, smooth muscle, and endothelium/endocardium). This suggests that a common progenitor is responsible for much of heart development and has spurred great interest in use of CPC-like cells for cardiac repair. In this review, CPC development is discussed, with a focus on early events prior to the initiation of cardiac gene expression. In particular, I discuss evidence that CPC fate is established during gastrulation, well before a time when heart development has typically been studied. Pathways regulating CPC specification are examined. The relationship between CPC specification and migration is further discussed. Finally, how CPCs may be related to efforts to promote cardiac development by approaches including reprogramming is discussed.

BACKGROUND: Patients who have completed initial cancer treatment (cancer survivors) have been relatively neglected. We need data to help us better understand the needs of this group and to underpin evidence-based service development.METHODS: Scoping reviews of research published in the last two decades focussing on the problems faced by cancer survivors, and the effectiveness of interventions for these problems were undertaken. The aim was to identify what we know, what we do not know and opportunities where research could provide new information. We searched for, retrieved and rapidly appraised systematic reviews sourced from the most common electronic databases supplemented by more recently published individual studies.RESULTS: The research evidence is surprisingly limited. We have some knowledge of the prevalence and nature of depression, pain and fatigue in cancer survivors. We know much less about cognitive and physical impairment, employment, financial well-being and relationships. Even where we have evidence, it is mostly of only moderate quality, is most often only for breast cancer and focuses almost exclusively on the early phase of survivorship. We have good evidence for the effectiveness of drug treatments for pain and moderate evidence for fatigue and depression, but not for other symptoms. Interventions based on rehabilitative and self-management approaches remain in the early stages of evaluation.INTERPRETATION: There has been a substantial amount of research describing many of the problems experienced by the cancer survivors. This is strongest in the area of symptoms in the period soon after treatment. However, the quality of the evidence is often poor, and some topics have been little examined. We urgently need data on the natural evolution and scale of the problems of cancer survivors obtained from well-designed, large-scale cohort studies and the robust testing of interventions in clinical trials. Given the current financially constrained research funding environment, we suggest areas in which strategic investment might give findings that have the potential to make a major impact on patient well-being in a 5-year time scale. British Journal of Cancer (2011) 105, S82 - S94; doi: 10.1038/bjc.2011.425 www.bjcancer.com (C) 2011 Cancer Research UK

Rheumatoid arthritis (RA) is considered to occur when genetic and environmental factors interact to trigger immunopathological changes and consequently an inflammatory arthritis. Over the last few decades, epidemiological and genetic studies have identified a large number of risk factors for RA development, the most prominent of which comprise cigarette smoking and the shared epitope alleles. These risks appear to differ substantially between anti-cyclic citrullinated peptide (ACPA)-positive and ACPA-negative disease. In this article, we will summarise the risk factors for RA development that have currently been identified, outlining the specific gene-environment and gene-gene interactions that may occur to precipitate and perpetuate autoimmunity and RA. We will also focus on how this knowledge of risk factors for RA may be implemented in the future to identify individuals at a high risk of disease development in whom preventative strategies may be undertaken.

Rheumatoid arthritis (RA) is associated with excess cardiovascular (CV) disease. Many studies have shown subclinical atherosclerosis in RA is associated with CV risk factors and inflammation. Their relationship with CV events has however received less attention. Furthermore, except for hypertension CV risk factor management has not been examined in a UK RA population. We therefore evaluated the contribution of RA specific and CV risk factors to CV events alongside the management of CV risk factors in RA patients.We assessed the prevalence, screening and treatment of CV risk factors in a cross-sectional survey of RA patients consecutively attending specialist clinics. We used binary logistic regression to examine relationships between CV events and RA and CV risk factors.We enrolled 309 patients (81% female; median age 60 years; median disease duration 8 years). 27 (9%) had previous CV events. 56% had hypertension, 42% hyperlipidaemia, 11% diabetes, 52% were ex/current smokers and 26% obese. Lipid status was unknown in one third. 47% of patients on anti-hypertensive agents were undertreated. CV events were associated with hyperlipidaemia (OR 13.5; 95% CI 3.9, 45.9), hypertension (OR 6.4; 95% CI 1.9, 21.9), having ever smoked (OR 2.7; 95% CI 1.1, 6.5), RA duration (OR 1.09; 95% CI 1.06, 1.13) and erosions (OR 2.9; 95% CI 1.1, 8.2).CV events are prevalent in RA. They are associated with CV risks and RA factors. Despite this burden we found CV risk factors were inadequately managed. A robust system to identify and treat CV risks in RA is required.

Cerebral cavernous malformations (CCMs) are vascular anomalies of the central nervous system that arise due to mutations in genes encoding three unrelated proteins: CCM1 (KRIT1); CCM2 (Malcavernin/OSM) and CCM3 (PDCD10). Both biochemical and mutant studies suggest that CCM1 and CCM2 act as part of a physical complex to regulate vascular morphogenesis and integrity. In contrast, mouse Ccm3 mutant and in vitro cell culture data suggests an independent role for Ccm3. In this study, we sought to use the zebrafish model system to examine for the first time the role of ccm3 in cranial vessel development. We report that inhibition of zebrafish ccm3a/b causes heart and circulation defects distinct from those seen in ccm1 (santa) and ccm2 (valentine) mutants, and leads to a striking dilation and mispatterning of cranial vessels reminiscent of the human disease pathology. ccm3, but not ccm2, defects can be rescued upon overexpression of stk25b, a GCKIII kinase previously shown to interact with CCM3. Morpholino knockdown of the GCKIII gene stk25b results in heart and vasculature defects similar to those seen in ccm3 morphants. Finally, additional loss of ccm3 in ccm2 mutants leads to a synergistic increase in cranial vessel dilation. These results support a model in which CCM3 plays a role distinct from CCM1/2 in CCM pathogenesis, and acts via GCKIII activity to regulate cranial vasculature integrity and development. CCM3/GCKIII activity provides a novel therapeutic target for CCMs, as well as for the modulation of vascular permeability.

Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2–5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs., Inherited congenital heart defects are prevalent in the human population, but the molecular mechanisms are poorly understood. In this article, deficiency in the chromatin remodelling factor, Brg1, is shown to alter cardiac development in both mouse and zebrafish laboratory models.

Publisher Summary This chapter reviews the utility of zebrafish for the study of heart development. It summarizes the experimental approaches that have been particularly useful for analysis of heart formation and outlines the major phases of heart development in zebrafish, emphasizing current understanding of the molecular and cellular regulation of each step. It also reflects on the application of the zebrafish model to the study of human heart disease and highlights the important impact of the combinatorial use of imaging, embryology, and genetics in zebrafish on the understanding of how genes regulate cardiac development. By combining multiple genetic and embryological experimental approaches, investigators have taken considerable strides toward elucidating the mechanisms that drive heart patterning and morphogenesis, including and in addition to the work encapsulated here. Notably, the ability to study development at the cellular level in the zebrafish embryo has been especially useful for linking essential genes to the specific cell behaviors that they regulate. As the number of investigators using zebrafish increases and the sophistication of zebrafish technology advances, the pace of discovery will undoubtedly continue to accelerate. In particular, emerging methods for targeted gene inactivation, enhanced strategies for spatial and temporal manipulation of gene function, and new techniques for automated image analysis are likely to facilitate high-throughput analysis of phenotypes generated by forward, reverse and chemical genetics.

A 33-year-old man with recessive dystrophic epidermolysis bullosa presented with a 3-month history of an enlarging mass within scarring on the posterior aspect of the right shoulder. The clinical appearance of the mass with an almost cobbled, verrucous surface, and its rapid evolution suggested the development of a squamous cell carcinoma (SCC) in a chronically scarred site. Histopathological examination of a biopsy taken from the lesion subsequently revealed it to be a verruciform xanthoma. This case shows that benign phenomena can mimic SCC and underlines the need for a biopsy to be taken promptly.

Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)s878, that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblins634 revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)s878 line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS., Author Summary Aberrant electrical activity of the heart, otherwise known as cardiac arrhythmia, may disrupt heart contractions, leading to loss of consciousness and sudden death. Every year, approximately 450,000 individuals in the United States die suddenly from this event. Currently, the only proven preventive therapy for sudden cardiac death is the automatic implantable cardioverter defibrillator, which carries a significant burden and cost to the patient. Greater understanding of the cardiac conduction system, which coordinates rhythmic beating of the heart, may lead to novel and safer therapeutic options for these patients. Working with zebrafish, a productive model system for understanding human disease, we have developed a cardiac-specific fluorescent calcium indicator zebrafish transgenic line to analyze the formation of the cardiac conduction system. Using this fluorescent transgenic line, we have observed four distinct physiologic cardiac conduction stages that correspond to cellular and anatomic changes of the developing heart. Furthermore, we have designed and performed a new, physiology-based, forward genetic screen to identify cardiac conduction mutants that would have escaped discovery in previous screens. Overall, these studies may prove rewarding toward developing therapeutic options aimed at maintaining and/or improving overall cardiac health., By characterizing the genetic underpinnings of the vertebrate cardiac conduction system, a new study sheds light on the mechanisms underlying cardiac arrhythmias, which cause the sudden death of hundreds of thousands of people each year.

Serum response factor (SRF) is a single copy, highly conserved transcription factor that governs the expression of hundreds of genes involved with actin cytoskeletal organization, cellular growth and signaling, neuronal circuitry and muscle differentiation. Zebrafish have emerged as a facile and inexpensive vertebrate model to delineate gene expression, regulation, and function, and yet the study of SRF in this animal has been virtually unexplored. Here, we report the existence of two srf genes in zebrafish, with partially overlapping patterns of expression in 3 and 7 day old developing animals. The mammalian ortholog (srf1) encodes for a 520 amino acid protein expressed in adult vascular and visceral smooth muscle cells, cardiac and skeletal muscle, as well as neuronal cells. The second zebrafish srf gene (srf2), encoding for a presumptive protein of only 314 amino acids, is transcribed at lower levels and appears to be less widely expressed across adult tissues. Both srf genes are induced by the SRF coactivator myocardin and attenuated with a short hairpin RNA to mammalian SRF. Promoter studies with srf1 reveal conserved CArG boxes that are the targets of SRF-myocardin in embryonic zebrafish cells. These results reveal that SRF was duplicated in the zebrafish genome and that its protein expression in all three muscle cell types is highly conserved across vertebrate animals suggesting an ancient code for transcriptional regulation of genes unique to muscle cell lineages.

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)s843 transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos.We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.

Low- and middle-income countries suffer disproportionately from reduced life expectancy and quality of life. Injuries are overlooked as contributors to global inequities in health, yet the long-term disabilities they frequently produce represent a significant burden. The Fogarty International Center of the National Institutes of Health convened a panel of experts in trauma and injury from the United States and low- and middle-income nations to identify research gaps in this area and opportunities to create new knowledge. Panel members identified sustainable programs of research established through stable linkages between institutions in high-income nations and those in low- and middle-income nations as a priority. The resulting benefits of addressing the growing burden of trauma and injury to communities in resource-constrained settings around the world would be substantial.

The metalloproteinase ADAMTS-2 has procollagen I N-proteinase activity capable of cleaving procollagens I and II N-propeptides in vitro, whereas mutations in the ADAMTS-2 gene in dermatosparaxis and Ehlers-Danlos syndrome VIIC show this enzyme to be responsible in vivo for most biosynthetic processing of procollagen I N-propeptides in skin. Yet despite its important role in the regulation of collagen deposition, information regarding regulation and substrate specificity of ADAMTS-2 has remained sparse. Here we demonstrate that ADAMTS-2 can, like the procollagen C-proteinases, be regulated by transforming growth factor-beta 1 (TGF-beta 1), with implications for mechanisms whereby this growth factor effects net increases in formation of extracellular matrix. TGF-beta 1 induced ADAMTS-2 mRNA approximately 8-fold in MG-63 osteosarcoma cells in a dose- and time-dependent, cycloheximide-inhibitable manner, which appeared to operate at the transcriptional level. Secreted ADAMTS-2 protein induced by TGF-beta 1 was 132 kDa and was identical in size to the fully processed, active form of the protease. Biosynthetic processing of ADAMTS-2 to yield the 132-kDa form is shown to be a two-step process involving sequential cleavage by furin-like convertases at two sites. Surprisingly, purified recombinant ADAMTS-2 is shown to cleave procollagen III N-propeptides as effectively as those of procollagens I and II, whereas processing of procollagen III is shown to be decreased in Ehlers-Danlos VIIC. Thus, the dogma that procollagen I and procollagen III N-proteinase activities are provided by separate enzymes appears to be false, whereas the phenotypes of dermatosparaxis and Ehlers-Danlos VIIC may arise from defects in both type I and type III collagen biosynthesis.

Uterine leiomyomata (UL) are benign tumors affecting many women of reproductive age. Cytogenetic studies have indicated that a significant percentage of leiomyomata have chromosomal rearrangements, including those involving the long arm of chromosome 7. Several candidate genes that map to chromosome 7 have been studied for possible roles in the pathogenesis of these tumors. PCOLCE, a gene whose product is involved in the cleavage of type I procollagen C-propeptide, has been mapped to the critical interval on chromosome 7, band q22. Here we evaluate by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization the expression and deletion status of PCOLCE in a series of karyotyped uterine leiomyomata.

The placenta has been the subject of extensive basic research efforts in two distinct fields. The developmental biology of placenta has been studied because it is the first organ to develop during embryogenesis and because a number of different gene mutations in mice result in embryonic lethality due to placental defects. The trophoblast cell lineage is relatively simple such that only two major, terminally differentiated cell types appear: an "invasive trophoblast" cell subtype such as extravillous cytotrophoblast cells in humans and trophoblast giant cells in mice, and a "transport trophoblast" cell subtype that is a syncytium (syncytiotrophoblast) in humans and mice. These two cell types also have been the focus of endocrinologists because they are the source of major placental hormones. Understanding the transcriptional regulation of placental hormone genes has given insights into the control of specificity of gene expression. Because most placental hormones are produced by very specific trophoblast cell subtypes, the transcriptional details promise to give insights into cell-subtype specification. The fields of developmental biology and molecular endocrinology appear to be meeting on this common ground with the recent discovery of key transcription factors. Specifically, the basic helix-loop-helix (bHLH) transcription factor Hand1 is essential for differentiation of trophoblast giant cells in mice and also regulates the promoter for the giant cell-specific hormone, placental lactogen I gene (Pl1). In contrast, formation of syncytiotrophoblast cells in mice is controlled by a distinct genetic pathway that is governed by the Gcm1 transcription factor, a homologue of the Drosophila glial cells missing gene. Human GCM I has been shown to regulate the activity of the placental-specific enhancer of the aromatase gene (CYP19), which is specifically expressed in syncytiotrophoblast. Together, these findings imply that some key transcription factors have the dual functions of controlling both critical cell fate decisions in the trophoblast cell lineage and later the transcription of cell subtype-specific genes unrelated to development.

Background Advances in imputation methods have enabled association studies of HLA protein amino acid polymorphisms and rheumatoid arthritis (RA) susceptibility. A recent meta-analysis of this region has attributed most of its association with seropositive RA to 5 amino acid positions [1]. Radiological progression in RA has a significant genetic component. Previous studies have linked shared-epitope alleles to radiological damage in RA; the association between HLA amino acid polymorphisms and radiological progression is unexplored. Objectives To evaluate the relationship between HLA amino acid polymorphisms and radiological progression (defined as the development of new erosions over 2 years) in early, active RA patients enrolled to a clinical trial. Methods The Combination Anti-Rheumatic Drugs in Early RA (CARDERA) trial randomised 467 patients to receive methotrexate with or without ciclosporin, corticosteroids or both treatments in a factorial-design. We evaluated 421 European ancestry patients with archived DNA (passing QC procedures) and radiological data available. Radiographs were evaluated 6-monthly (for 2 years) for the development of new erosions. Genotyping was performed on the ImmunoChip; HLA alleles and amino acid polymorphisms were imputed using SNP2HLA [2]. We evaluated 237 amino acid positions in 3 MHC class I and 5 MHC class II proteins and 255 HLA alleles for their association with new erosions. Association testing was performed using UNPHASED version 3.0.7, which utilises a standard retrospective likelihood model for a binary trait and allows the use of polymorphic markers [3]. A P -value threshold of 0.0002 was applied based on permutation testing. Ethical approval was obtained; all patients provided consent. Results One amino acid position, which was position 10 in HLA-DRβ1 ( P =0.0001) passed our pre-defined significance threshold for association with new erosions. Using glutamine as the reference amino acid, the OR for new erosions with tyrosine was 0.443 (95% CI 0.278-0.706). A similar trend was observed for HLA alleles; the strongest association was seen with HLA-DRB1 *04 ( P -value=0.0002), which encodes glutamine at position 10. The absence of *04 alleles provided an OR for new erosions of 0.513 (95% CI 0.360-0.730). Conclusions We demonstrate an association between amino acid polymorphisms at position 10 in the HLA-DRβ1 protein and the development of radiological erosions in early active RA. Unlike amino acid polymorphisms associating with RA susceptibility (located in binding grooves) this polymorphism influences β- and α-chain binding; it could therefore influence radiological damage through attenuating antigen presentation within the joint. Replication of our finding in other cohorts is required to confirm its validity. References Raychaudhuri S et al. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis. Nat Genet 2012 29; 44: 291-6. Jia X et al. Imputing amino acid polymorphisms in human leukocyte antigens. PLoS One 2013; 8: e64683. Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data. Hum Hered 2008; 66: 87-98. Acknowledgements Arthritis Research UK (Grant Reference Number 19739 to ICS); National Institute for Health Research (NIHR) Biomedical Research Centre at Guy9s and St Thomas9 NHS Foundation Trust in partnership with King9s College London. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4069

Background Several studies have tested the hypothesis that RA susceptibility variants associate with radiological progression. Their findings are often not replicated; additionally they have examined only a proportion of risk loci and evaluated each locus separately, despite limited power. Two recent meta-analyses of genome-wide association studies have expanded the number of genetic loci and HLA amino acid polymorphisms associated with RA susceptibility [1, 2]. Our aim was to establish if these variants correlated with radiological progression in early active RA patients enrolled to a clinical trial. We evaluated their association both individually and cumulatively, using a weighted genetic risk score (wGRS) combining risk loci. Objectives To establish if genetic predictors of RA susceptibility also associate with radiological progression in early active RA. Methods We evaluated 421 RA patients previously enrolled to the Combination Anti-Rheumatic Drugs in Early RA (CARDERA) trial. Hand/feet radiographs were read for modified Larsen scores every 6 months for 2 years. We evaluated the association of validated genetic risk variants (69 SNPs, 17 four-digit HLA-DRB1 alleles and polymorphisms in 6 HLA amino acid positions) with log-transformed Larsen scores over time using a linear mixed-effects model. Log e (Larsen score + 1) was the response variable. Time, treatment, age, disease duration, ACPA status, genotype and a genotype*time interaction, were the fixed-effects predictor variables. Genotype was coded additively. All β-estimates were back-transformed to the original Larsen scale. The genotype*time β provided information on the annual increase in Larsen score per risk allele copy relative to the reference genotype; P -values for this term provided information on the variant9s role in radiological progression. We assessed the association between a wGRS incorporating susceptibility SNPs and radiological progression by replacing the linear model9s genotype term with a wGRS. Ethical approval was obtained; all patients provided consent. Results Two SNPs, one HLA-DRB1 allele and two HLA-DRβ1 protein amino acid polymorphisms had P -values P =0.0680). Conclusions RA susceptibility loci did not have a clinically relevant association with radiological progression in European ancestry, early, active RA patients recruited to this trial. This suggests the genetic architectures of RA susceptibility and severity differ. References Okada Y et al. Genetics of rheumatoid arthritis contributes to biology and drug discovery. Nature 2013; [Epub ahead of print]. Raychaudhuri S et al. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis. Nat Genet 2012;44:291-6. Acknowledgements Arthritis Research UK (Grant Reference Number 19739 to ICS); National Institute for Health Research (NIHR) Biomedical Research Centre at Guy9s and St Thomas9 NHS Foundation Trust in partnership with King9s College London. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5018

Background Prescribing patterns in Rheumatoid Arthritis have evolved over recent decades. There is now clear evidence that combination DMARDs and biologics can acheive better disease control. This is guided by the treat to target initiative aiming for remission in all patients. Objectives We have used observational data to determine changes in prescribing patterns since 1996. We set out to describe the % of patients with RA being treated with individual DMARDs, combination therapy, systemic steroids and biologics. Methods Our study included 1492 RA patients attending two rheumatology centres. They comprised four groups of clinic attenders attending between 1996 and 2013. We collected data on the use of DMARDs, systemic steroids and biologics. Data were also collected on the proportion of patients not receiving any immunosuppressive therapy. Results More patients received immunosuppressive treatment with 14% of all RA patients and 16% of intermediate disease activity group not on any treatment in 2012-13 compared with 32% and 23% in the 1996-97 cohort. Methotrexate remains the most frequently prescribed DMARD across all cohorts, however the more contemporaneous cohorts have higher exposure to hydroxychloroquine, sulfasalazine and leflunomide. Conversely, few patients ( The % of patients treated with combination therapy has increased, however almost half of patients remain on a single DMARD (46%). The proportion of patients treated with biologics has steadily increased from 0 to 1996-97, 4% in 2001-03 to 17% and 21% in the two later cohorts. Systemic steroid use has fluctuated over time, prescribed in 14% in 2012-13. Conclusions Increasing numbers of patients are being treated with immunosuppression. The choice of DMARD has changed from fewer patients being prescribed gold, penicillamine and azathioprine, to more patients being treated with methotrexate, hydroxychloroquine, sulfasalazine and leflunomide. Whilst the uptake of combination DMARD therapy and biologics has increased, approximately half of patients remain on a single DMARD only. This may be due to a number of reasons including physician choice, patient choice and drug intolerance. Further research is needed to explore this issue. There are still patients not on immunosuppression, suggesting that we are not treating to target, and not achieving tighter targeted control in all patients. Further research into why patients who have not achieved remission are not being treated with combination therapy is needed. The role of biologics in moderate disease activity patients to further try and achieve remission needs to be explored. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5399

Ian C. Scott, Jemma Walker, Jelmar Quist, Sarah Spain, Rachael Tan, Yuki Okada, Soumya Raychaudhuri, Sophia Steer, Andrew P. Cope and Cathryn M. Lewis Department of Rheumatology, King’s College London, London, Department of Medical and Molecular Genetics, King’s College London, London, UK, Division of Genetics, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA, Department of Rheumatology, King’s College Hospital, London, UK

Background UK guidelines recommend that all patients with early active rheumatoid arthritis are offered combination disease-modifying anti-rheumatic drugs (DMARDs) and short-term corticosteroids. This heterogeneous disease has several subsets; one crucial subdivision is defined by the presence or absence of anti-citrullinated protein antibodies (ACPA). These subsets may differ in their treatment responses. We used data from a published randomised controlled trial to assess whether responses to intensive combination treatments in early rheumatoid arthritis differ by ACPA status. Methods The Combination Anti-Rheumatic Drugs in Early Rheumatoid Arthritis trial randomised 467 patients with early active disease to receive: methotrexate, methotrexate plus ciclosporin, methotrexate plus prednisolone, or methotrexate plus ciclosporin plus prednisolone in a factorial-design. Patients were assessed 6-monthly for 2 years. In this secondary analysis we assessed 431 patients with available ACPA status. To minimise multiple testing we used a mixed-effects repeated measures ANOVA model to test for an interaction between ACPA and treatment on mean changes from baseline to 2 years for each outcome (Larsen score, disease activity score in 28 joints [DAS28], health assessment questionnaire, EuroQol, and physical component and mental component summary scores). When a significant interaction was present, mean changes in outcomes were compared between treatment groups at each time-point using t tests stratified by ACPA status. Odds ratios (ORs) for the onset of new erosions with treatment were calculated stratified by ACPA. Findings Of the 431 rheumatoid arthritis cases, 310 (72%) were ACPA-positive and 121 (28%) were ACPA-negative. Effect of treatment on radiological progression differed according to ACPA status, with a significant interactive effect between ACPA and treatment on Larsen score changes observed (p vs 9·58 [6·76—12·39]; OR for new erosions 0·32 [0·14–0·72], p=0·003). ACPA-negative patients had negligible radiological progression irrespective of treatment (2-year mean Larsen score increase 1·70 [0·29–3·10] with triple therapy vs 2·72 [1·15–4·29] with monotherapy; 7% developed new erosions). The beneficial effects of high-dose corticosteroids on reducing disease activity and improving physical health were confined to ACPA-positive disease. Interpretation In our study, combination DMARDs and corticosteroids provided radiological, disease activity, and physical health benefits only to patients with ACPA-positive early rheumatoid arthritis. These findings suggest that ACPA is an important biomarker for guiding treatment decisions in rheumatoid arthritis. They indicate that current guidelines advocating combination DMARDs and corticosteroids in all patients with early active disease might require reconsideration. Funding Arthritis Research UK, National Institute for Health Research, NIHR Biomedical Research Centre.

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.

Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH(2)-terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH(2) terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycan-cleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes.

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (alpha3beta3gamma2) within the alpha3 and gamma2 chains (). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first alpha3 chain cleavage (200-l65 kDa alpha3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa alpha3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The gamma chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the gamma2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (). Recombinant BMP-1 cleaves gamma2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant gamma2 short arm. alpha3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin alpha3 and gamma2 chains to be substrates for BMP-1 in vitro. We speculate that gamma2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c.embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1−/− embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.

The procollagen C-proteinase enhancer (PCPE) is a glycoprotein that potentiates enzymatic cleavage of the type I procollagen C-propeptide by bone morphogenetic protein-1 (BMP-1). The human PCPE gene (PCOLCE) was previously mapped to 7q22, an area frequently disrupted in uterine leiomyomata, while disruption of the rat PCPE gene leads to anchorage-independent growth and loss of contact inhibition in rat fibroblasts. Here we describe the entire intron/exon organizations of PCOLCE and the mouse PCPE gene (Pcolce) and analyze expression of PCOLCE RNA in various human adult and fetal tissues and of Pcolce RNA at various stages of mouse development. PCOLCE and Pcolce are shown to be small genes 6.0 and 6.5 kb, respectively, with a conserved intron/exon structure comprising 9 exons. A notable difference between the two genes derives from insertion of multiple Alu sequences immediately upstream and downstream and within PCOLCE. Temporal expression of PCPE mRNA is shown to differ from that of BMP-1 and type I procollagen during mouse development, consistent with possible additional functions for PCPE beyond enhancement of C-proteinase activity. Consistent with a possible role in leiomyomata, PCOLCE is shown to be expressed at relatively high levels in uterus.

The trophoblast cell lineage is the first to be specified during mammalian embryogenesis. Although trophoblast derivatives do not contribute to the embryo proper, they are essential from a very early stage for several complex processes. They mediate the attachment to and implantation into the uterus of the conceptus, secrete factors that regulate the fetal and maternal environments, modulate the maternal immune response to “foreign” embryonic tissue, invade the decidual circulation, and provide an interface where the fetal/ maternal exchange of nutrients and waste products can occur (1, 2). Proper development of this cell type is therefore crucial for embryonic survival and growth, and defects in these early events are likely causes of a significant proportion of failures in the successful maintenance of pregnancy. For this reason, there has been considerable interest in determining the factors that regulate normal trophoblast differentiation. Development of cell lineages ultimately involves changes in programs of gene expression, which are in turn regulated by transcription factors. A large number of transcription factors that are expressed in trophoblast have now been identified (Table 15.1). This catalogue has increased recently because of the sequencing of expressed sequence tags (EST) found in placental and purified trophoblast cDNA libraries.

KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3H275R). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3H275R mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3H275R mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans., Author Summary Cardiac defects are among the most common malformations in humans. Most causative genetic mutations remain unknown. To discover new causative genes important in cardiovascular development and function, we examined 1770 mice with randomly mutated genes and found a mutant with aortic valvular stenosis, and increased risk of fetal and neonatal death. Using linkage analysis and sequencing, we identified a protein-altering point mutation in the gene regulatory protein KLF3. Mice that survived into adulthood with one mutant copy of the Klf3 gene had low arterial blood pressure, enlarged hearts, and increased mortality due to heart failure. When both copies of the Klf3 gene was mutant, then embryos had heart defects, and all died before birth. KLF3 had no previously known role in heart development so to confirm these findings, we (1) knocked down klf3 expression in zebrafish embryos and (2) examined mice with a mutation that effectively eliminated the KLF3 protein. In both cases, cardiovascular dysfunction was observed. In conclusion, we have discovered that KLF3 plays diverse and important roles in cardiovascular development and function in mice. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.

Background Rheumatoid arthritis is the most common chronic inflammatory disease in the UK. Serological status such as rheumatoid factor (RF) and anti-citrullinated peptide antibody (ACPA) positivity predict poor outcomes. Early intensive treatment regimens targeting remission reduce disease activity, structural damage, and long-term disability. However, we do not know whether all patients with active disease should have such intensive treatment regimens. Can serological status be used to predict the need for intensive therapy? Methods We analysed samples from a published randomised controlled trial which compared four treatment regimens in patients with early active rheumatoid arthritis (disease duration Findings 402 (86%) patients were positive for IgM RF, 346 (74%) for IgA RF, and 346 (74%) for ACPA. 98 (21%) patients achieved remission at 24 months. In RF IgM negative cases (n=65) the proportion of patients achieving remission at 24 months was similar in all treatment groups (25%, 22%, and 30% for monotherapy, double therapy, and triple therapy, respectively). In RF IgM positive cases, significantly fewer patients achieved remission with monotherapy (13/65, 17%) and double therapy (24/157, 15%) than with triple therapy (27/80, 34%) (p=0·001). There were similar, consistent findings with IgA RF and ACPA, with significantly more seropositive patients achieving remission with triple therapy than with monotherapy. Interpretation Contemporary treatment of rheumatoid arthritis emphasises the use of intensive therapy to achieve remission. However, we have shown that not all patients require such an aggressive approach to therapy. Given the heterogeneity of the diease, treatment should be personalised to the individual, which would minimise costs of treatment as well as potentially toxic side-effects. Our study shows that only seropositive patients with rheumatoid arthritis should be given more intensive therapies. Funding National Institute for Health Research.

Background Improved characterisation of the risk factors for rheumatoid arthritis raises the possibility that they could be combined to identify individuals at high risk of disease in whom preventive strategies can be evaluated. Our aim was to develop a prediction model capable of identifying such individuals with sufficient accuracy to enable the assessment of preventive treatments. Methods Our prediction model combines odds ratios for 15 HLA-DRB1 alleles, 31 non-HLA single nucleotide polymorphisms (SNPs), and smoking status to classify an individual's risk of seropositive rheumatoid arthritis. Our novel modelling technique employs confidence intervals to classify disease risks using a computer-simulated population. We developed several models (HLA, HLA-10/20/31 SNP, HLA-smoking models) to evaluate the impact of different factors on prediction. The ability of each model to discriminate between rheumatoid arthritis and controls was evaluated in two European cohorts: the Wellcome Trust Case Control Consortium (WTCCC: 1542 cases, 1226 controls) and the UK Rheumatoid Arthritis Genetics Consortium (UKRAG: 2623 cases, 1503 controls). Findings HLA-DRB1 alleles conferred most prediction: the WTCCC HLA-only model classified 50% antibodies to citrullinated protein antigens (ACPA)-positive rheumatoid arthritis versus 17% controls as high risk and 60% controls versus 25% ACPA-positive rheumatoid arthritis as reduced risk. Adding smoking information improved prediction (p=0·00033); SNPs provided no significant benefits. The highest area under the curve was 0·81 (95% CI 0·78–0·85). Only a minority had substantially elevated risks of rheumatoid arthritis: 6·9% ACPA-positive cases and 0·31% controls in WTCCC had an odds ratio for rheumatoid arthritis of more than 20 when evaluated with the HLA-31 SNP model. Interpretation Combined information on HLA-DRB1 alleles and smoking provides informative risk prediction for rheumatoid arthritis. Since only a minority of individuals are at substantially elevated risks, modelling may be best focused in a-priori high-risk groups such as those with family histories of rheumatoid arthritis. Further work is needed to define risk factors with large effect sizes for incorporation within our modelling framework. Funding Arthritis Research UK.

A series of homologated 1 alpha-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 molecules with one to three extra carbons in the side chain were used to examine the substrate preferences and hydroxylation site selection mechanisms of the liver vitamin D3-25-hydroxylase (CYP27) and the target cell 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Cultured and transfected cell models, used as sources of these hydroxylases, gave 23-, 24-, 25-, and 27-hydroxylated metabolites which were identified by their high performance liquid chromatography and GC-MS characteristics. Lengthening the side chain is tolerated by each cytochrome P450 isoform such that 25-hydroxylation or 24-hydroxylation continues to occur at the same rate as in the native side chain, while the site of hydroxylation remains the same for the liver enzyme in that CYP27 continues to hydroxylate at C-25 and C-27 (minor) despite the two-carbon-atom extension. Somewhat surprising is the finding that C-24 and C-23 (minor) hydroxylations also do not change as the side chain is extended by as much as three carbons. We conclude that CYP24 must be directed to its hydroxylation site(s) by the distance of carbon 24 from the vitamin D ring structure and not as in CYP27 by the distance of the hydroxylation site from the end of the side chain.

Backgroundand objectives The female preponderance of rheumatoid arthritis (RA) implies that reproductive factors may play an important role in its pathogenesis. Although several studies have shown an inverse association between previous pregnancy and RA development this finding has not been consistently reproduced and the precise relationship remains uncertain. The authors therefore undertook a systematic review and meta-analysis of observational studies examining pregnancy as a risk factor for RA. Our primary aim was to establish if pregnancy is associated with the risk of RA. Our secondary aim was to evaluate if this risk varies by rheumatoid factor (RF)/anticyclic citrullinated peptide antibody (ACPA) status. Materials and methods Search strategy: the authors searched Medline/EMBASE (1947 November 2011) using the search terms ‘rheumatoid arthritis.mp’ or ‘arthritis, rheumatoid/’ and ‘pregnancy.mp’ or ‘pregnancy/’ (limited to ‘humans’). The Scottish Intercollegiate Guidelines Network observational study filter was applied. Included manuscript bibliographies were reviewed. Inclusion Criteria: 1) case-control/cohort studies; 2) examining relationships between pregnancy and RA risk; 3) reporting effect size data as OR/RR with 95% CIs in parous versus nulliparous females (or providing crude data allowing their calculation). Statistics: Due to the low prevalence of RA, ORs/RRs were used interchangeably. Due to heterogeneity a random effects model was used to estimate summary ORs/RRs. Crude and adjusted ORs/RRs were evaluated separately. Results 248 citations were identified. 14 studies were included – 10 case-control (evaluating 1967 cases and 6113 controls) and four cohort studies (evaluating 8118 incident RA cases from 2 308 533 individuals). Crude and adjusted ORs/RRs were available for eight studies; three cohort studies reported adjusted and three case-control reported crude ORs/RRs only. Previous pregnancy was inversely associated with RA. The unadjusted summary OR for RA in parous versus nulliparous women was 0.76 (0.63–0.91). The adjusted summary OR for RA was also significant at 0.81 (0.68–0.96). Only a single study stratified risk by RF status- no significant relationship was identified with either subset. No studies evaluated risk by ACPA status. Conclusions Our systematic review shows an inverse relationship between previous pregnancy and RA. Although this indicates a likely protective effect of parity on the risk of RA development caution is required because the largest risks were observed in case-control studies, which are open to recall bias and cannot prove causality. The authors consider that further prospective cohort studies are required, evaluating cases by ACPA status, to better discern what risk factors are important in RA subset pathogenesis.

Approaches to repairing damaged heart tissue have focused on the promise of multipotential stem cells. However, repairing damaged tissue through regeneration--a strategy adopted by animals such as the newt--may also prove beneficial. In their Perspective, Scott and Stainier discuss new work (Poss et al .) that reveals the regenerative capacity of the injured zebrafish heart.

The trophoblast cell lineage is an interesting model system because it is composed of a limited number of cell types that are spatially patterned. Trophoblast stem (TS) cells reside within a layer called the chorion and either remain as stem cells or differentiate into spongiotrophoblast (SpT), trophoblast giant (TG) cells or syncytiotrophoblast cells (SynT) of the labyrinth. Maintenance of the TS phenotype is dependent on stimulation by FGF4, whereas differentiation and/or maintenance of the differentiated derivatives are dependent on key transcription factors: Mash2 for SpT, Hand1 for TG cells and Gcm1 for SynT cells. TS cells proliferate and retain their stem cell phenotype in culture in response to FGF4 and an additional factor(s) that can be provided by conditioned medium from embryonic fibroblast feeder cells (CM). To understand the functions of Hand1, Mash2 and Gcm1 at a cellular level, we tested the effects of their ectopic and over-expression on the ability of TS cells to either continue to proliferate or differentiate into their alternative fates. Expression of Mash2 alone had no effects on TS cell differentiation. However, Mash2-transfected cells continued to divide longer after withdrawal of FGF/CM. Hand1 promoted TGC differentiation, even in the continued presence of FGF4/CM. Stra13, another bHLH factor gene that is expressed in TG cells, also induced TG differentiation. Gcm1 induced a rapid arrest of TS proliferation but, in contrast to Hand1 and Stra13, blocked TG cell differentiation. Although Gcm1 was not sufficient to promote SynT formation, expression of an antisense Gcm1 transcript blocked SynT differentiation. These data suggest that Mash2 functions to promote transient FGF4-independent amplification of trophoblast cells that are progressing towards the SpT and TG cell phenotype. By contrast, Hand1 and Stra13 promote cell cycle exit and restrict cells towards the TG fate, whereas Gcm1 promotes cell cycle exit and restriction towards the SynT fate.

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal–ventral patterning by releasing TGFβ-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis.

International audience; While many factors that modulate the morphogenesis and patterning of the embryonic heart have been identified, relatively little is known about the molecular events that regulate the differentiation of progenitor cells fated to form the myocardium. Here, we show that zebrafish grinch (grn) mutants form a reduced number of myocardial progenitor cells, which results in a profound deficit in cardiomyocyte numbers in the most severe cases. We show that grn encodes the G protein-coupled receptor (GPCR) Agtrl1b, a known regulator of adult cardiovascular physiology. Ectopic expression of Apelin, an Agtrl1b ligand, results in the complete absence of cardiomyocytes. Data from transplantation and transgenic approaches indicate that Agtrl1 signaling plays a cell-autonomous role in myocardial specification, with activity being required coincident with the onset of gastrulation movements. These results support a model in which agtrl1b regulates the migration of cells fated to form myocardial progenitors.