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1.  Autotaxin Production of Lysophosphatidic Acid Mediates Allergic Asthmatic Inflammation 
Rationale: Bioactive lipid mediators, derived from membrane lipid precursors, are released into the airway and airspace where they bind high-affinity cognate receptors and may mediate asthma pathogenesis. Lysophosphatidic acid (LPA), a bioactive lipid mediator generated by the enzymatic activity of extracellular autotaxin (ATX), binds LPA receptors, resulting in an array of biological actions on cell proliferation, migration, survival, differentiation, and motility, and therefore could mediate asthma pathogenesis.
Objectives: To define a role for the ATX-LPA pathway in human asthma pathogenesis and a murine model of allergic lung inflammation.
Methods: We investigated the profiles of LPA molecular species and the level of ATX exoenzyme in bronchoalveolar lavage fluids of human patients with asthma subjected to subsegmental bronchoprovocation with allergen. We interrogated the role of the ATX-LPA pathway in allergic lung inflammation using a murine allergic asthma model in ATX-LPA pathway–specific genetically modified mice.
Measurements and Main Results: Subsegmental bronchoprovocation with allergen in patients with mild asthma resulted in a remarkable increase in bronchoalveolar lavage fluid levels of LPA enriched in polyunsaturated 22:5 and 22:6 fatty acids in association with increased concentrations of ATX protein. Using a triple-allergen mouse asthma model, we showed that ATX-overexpressing transgenic mice had a more severe asthmatic phenotype, whereas blocking ATX activity and knockdown of the LPA2 receptor in mice produced a marked attenuation of Th2 cytokines and allergic lung inflammation.
Conclusions: The ATX-LPA pathway plays a critical role in the pathogenesis of asthma. These preclinical data indicate that targeting the ATX-LPA pathway could be an effective antiasthma treatment strategy.
doi:10.1164/rccm.201306-1014OC
PMCID: PMC3826286  PMID: 24050723
asthma; lysophosphatidic acid; autotaxin; allergic airway inflammation
2.  Induced expression and functional effects of aquaporin-1 in human leukocytes in sepsis 
Critical Care  2013;17(5):R199.
Introduction
Gene expression profiling was performed via DNA microarrays in leukocytes from critically ill trauma patients nonseptic upon admission to the ICU, who subsequently developed either sepsis (n = 2) or severe sepsis and acute respiratory distress syndrome (n = 3). By comparing our results with published expression profiling studies in animal models of sepsis and lung injury, we found aquaporin-1 to be differentially expressed across all studies. Our aim was to determine how the water channel aquaporin-1 is involved in regulating the immune response in critically ill patients during infection acquired in the ICU.
Methods
Following the results of the initial genetic screening study, we prospectively followed aquaporin-1 leukocyte expression patterns in patients with ICU-acquired sepsis who subsequently developed septic shock (n = 16) versus critically ill patients who were discharged without developing sepsis (n = 13). We additionally determined aquaporin-1 expression upon lipopolysaccharide (LPS) exposure and explored functional effects of aquaporin-1 induction in polymorphonuclear granulocytes (PMNs).
Results
Leukocyte aquaporin-1 expression was induced at the onset of sepsis (median 1.71-fold increase; interquartile range: 0.99 to 2.42, P = 0.012 from baseline) and was further increased upon septic shock (median 3.00-fold increase; interquartile range: 1.20 to 5.40, P = 0.023 from sepsis, Wilcoxon signed-rank test); no difference was observed between baseline and discharge in patients who did not develop sepsis. Stimulation of PMNs by LPS led to increased expression of aquaporin-1 in vitro, which could be abrogated by the NF-κB inhibitor EF-24. PMN hypotonic challenge resulted in a transient increase of the relative cell volume, which returned to baseline after 600 seconds, while incubation in the presence of LPS resulted in persistently increased cell volume. The latter could be abolished by blocking aquaporin-1 with mercury and restored by incubation in β-mercaptoethanol, which abrogated the action of mercury inhibition.
Conclusions
Aquaporin-1 is induced in leukocytes of patients with ICU-acquired sepsis and exhibits higher expression in septic shock. This phenomenon may be due to LPS-triggered NF-κB activation that can also lead to alterations in plasma membrane permeability.
doi:10.1186/cc12893
PMCID: PMC4056620  PMID: 24028651
3.  A Metabolically-Stabilized Phosphonate Analog of Lysophosphatidic Acid Attenuates Collagen-Induced Arthritis 
PLoS ONE  2013;8(7):e70941.
Rheumatoid arthritis (RA) is a destructive arthropathy with systemic manifestations, characterized by chronic synovial inflammation. Under the influence of the pro-inflammatory milieu synovial fibroblasts (SFs), the main effector cells in disease pathogenesis become activated and hyperplastic while releasing a number of signals that include pro-inflammatory factors and tissue remodeling enzymes. Activated RA SFs in mouse or human arthritic joints express significant quantities of autotaxin (ATX), a lysophospholipase D responsible for the majority of lysophosphatidic acid (LPA) production in the serum and inflamed sites. Conditional genetic ablation of ATX from SFs resulted in attenuation of disease symptoms in animal models, an effect attributed to diminished LPA signaling in the synovium, shown to activate SF effector functions. Here we show that administration of 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl-phosphonate (BrP-LPA), a metabolically stabilized analog of LPA and a dual function inhibitor of ATX and pan-antagonist of LPA receptors, attenuates collagen induced arthritis (CIA) development, thus validating the ATX/LPA axis as a novel therapeutic target in RA.
doi:10.1371/journal.pone.0070941
PMCID: PMC3726599  PMID: 23923032
4.  Autotaxin expression from synovial fibroblasts is essential for the pathogenesis of modeled arthritis 
Synovial fibroblasts from patients and mice with arthritis express autotaxin, and ablation of autotaxin in fibroblasts ameliorates disease.
Rheumatoid arthritis is a destructive arthropathy characterized by chronic synovial inflammation that imposes a substantial socioeconomic burden. Under the influence of the proinflammatory milieu, synovial fibroblasts (SFs), the main effector cells in disease pathogenesis, become activated and hyperplastic, releasing proinflammatory factors and tissue-remodeling enzymes. This study shows that activated arthritic SFs from human patients and animal models express significant quantities of autotaxin (ATX; ENPP2), a lysophospholipase D that catalyzes the conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA). ATX expression from SFs was induced by TNF, and LPA induced SF activation and effector functions in synergy with TNF. Conditional genetic ablation of ATX in mesenchymal cells, including SFs, resulted in disease attenuation in animal models of arthritis, establishing the ATX/LPA axis as a novel player in chronic inflammation and the pathogenesis of arthritis and a promising therapeutic target.
doi:10.1084/jem.20112012
PMCID: PMC3348105  PMID: 22493518
5.  CreZOO—the European virtual repository of Cre and other targeted conditional driver strains 
The CreZOO (http://www.crezoo.org/) is the European virtual repository of Cre and other targeted conditional driver strains. These mice serve as tools for researchers to selectively ‘switch off’ gene expression in mouse models to examine gene function and disease pathology. CreZOO aims to capture and disseminate extant and new information on these Cre driver strains, such as genetic background and availability information, and details pertaining promoter, allele, inducibility and expression patterns, which are also presented. All transgenic strains carry detailed information according to MGI's official nomenclature, whereas their availability [e.g. live mice, cryopreserved embryos, sperm and embryonic stem (ES) cells] is clearly indicated with links to European and International databases and repositories (EMMA, MGI/IMSR, MMRRC, etc) and laboratories where the particular mouse strain is available together with the respective IDs. Each promoter/gene includes IDs and direct links to MGI, Entrez Gene, Ensembl, OMIM and RGD databases depending on their species origin, whereas allele information is presented with MGI IDs and active hyperlinks to redirect the user to the respective page in a new tab. The tissue/cell (special) and developmental (temporal) specificity expression patterns are clearly presented, whereas handling and genotyping details (in the form of documents or hyperlinks) together with all relevant publications are clearly presented with PMID(s) and direct PubMed links. CreZOO's design offers a user-friendly query interface and provides instant access to the list of conditional driver strains, promoters and inducibility details. Database access is free of charge and there are no registration requirements for data querying. CreZOO is being developed in the context of the CREATE consortium (http://www.creline.org/), a core of major European and international mouse database holders and research groups involved in conditional mutagenesis.
Database URL: http://www.crezoo.org/; alternative URL: http://www.e-mouse.org/
doi:10.1093/database/bas029
PMCID: PMC3381224  PMID: 22730454
6.  Finding and sharing: new approaches to registries of databases and services for the biomedical sciences 
The recent explosion of biological data and the concomitant proliferation of distributed databases make it challenging for biologists and bioinformaticians to discover the best data resources for their needs, and the most efficient way to access and use them. Despite a rapid acceleration in uptake of syntactic and semantic standards for interoperability, it is still difficult for users to find which databases support the standards and interfaces that they need. To solve these problems, several groups are developing registries of databases that capture key metadata describing the biological scope, utility, accessibility, ease-of-use and existence of web services allowing interoperability between resources. Here, we describe some of these initiatives including a novel formalism, the Database Description Framework, for describing database operations and functionality and encouraging good database practise. We expect such approaches will result in improved discovery, uptake and utilization of data resources.
Database URL: http://www.casimir.org.uk/casimir_ddf
doi:10.1093/database/baq014
PMCID: PMC2911849  PMID: 20627863
7.  Mouse Resource Browser—a database of mouse databases 
The laboratory mouse has become the organism of choice for discovering gene function and unravelling pathogenetic mechanisms of human diseases through the application of various functional genomic approaches. The resulting deluge of data has led to the deployment of numerous online resources and the concomitant need for formalized experimental descriptions, data standardization, database interoperability and integration, a need that has yet to be met. We present here the Mouse Resource Browser (MRB), a database of mouse databases that indexes 217 publicly available mouse resources under 22 categories and uses a standardised database description framework (the CASIMIR DDF) to provide information on their controlled vocabularies (ontologies and minimum information standards), and technical information on programmatic access and data availability. Focusing on interoperability and integration, MRB offers automatic generation of downloadable and re-distributable SOAP application-programming interfaces for resources that provide direct database access. MRB aims to provide useful information to both bench scientists, who can easily navigate and find all mouse related resources in one place, and bioinformaticians, who will be provided with interoperable resources containing data which can be mined and integrated.
Database URL: http://bioit.fleming.gr/mrb
doi:10.1093/database/baq010
PMCID: PMC2911845  PMID: 20627861
8.  The RNA-Binding Protein Elavl1/HuR Is Essential for Placental Branching Morphogenesis and Embryonic Development▿ †  
Molecular and Cellular Biology  2009;29(10):2762-2776.
HuR is an RNA-binding protein implicated in a diverse array of pathophysiological processes due to its effects on the posttranscriptional regulation of AU- and U-rich mRNAs. Here we reveal HuR's requirement in embryonic development through its genetic ablation. Obligatory HuR-null embryos exhibited a stage retardation phenotype and failed to survive beyond midgestation. By means of conditional transgenesis, we restricted HuR's mutation in either embryonic or endothelial compartments to demonstrate that embryonic lethality is consequent to defects in extraembryonic placenta. HuR's absence impaired the invagination of allantoic capillaries into the chorionic trophoblast layer and the differentiation of syncytiotrophoblast cells that control the morphogenesis and vascularization of the placental labyrinth and fetal support. HuR-null embryos rescued from these placental defects proceeded to subsequent developmental stages but displayed defects in skeletal ossification, fusions in limb elements, and asplenia. By coupling gene expression measurements, data meta-analysis, and HuR-RNA association assays, we identified transcription and growth factor mRNAs controlled by HuR, primarily at the posttranscriptional level, to guide morphogenesis, specification, and patterning. Collectively, our data demonstrate the dominant role of HuR in organizing gene expression programs guiding placental labyrinth morphogenesis, skeletal specification patterns, and splenic ontogeny.
doi:10.1128/MCB.01393-08
PMCID: PMC2682039  PMID: 19307312
9.  Models for financial sustainability of biological databases and resources 
Following the technological advances that have enabled genome-wide analysis in most model organisms over the last decade, there has been unprecedented growth in genomic and post-genomic science with concomitant generation of an exponentially increasing volume of data and material resources. As a result, numerous repositories have been created to store and archive data, organisms and material, which are of substantial value to the whole community. Sustained access, facilitating re-use of these resources, is essential, not only for validation, but for re-analysis, testing of new hypotheses and developing new technologies/platforms. A common challenge for most data resources and biological repositories today is finding financial support for maintenance and development to best serve the scientific community. In this study we examine the problems that currently confront the data and resource infrastructure underlying the biomedical sciences. We discuss the financial sustainability issues and potential business models that could be adopted by biological resources and consider long term preservation issues within the context of mouse functional genomics efforts in Europe.
doi:10.1093/database/bap017
PMCID: PMC2790311  PMID: 20157490
10.  Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis 
Respiratory Research  2009;10(1):14.
Background
Recent evidence has underscored the role of hypoxia and angiogenesis in the pathogenesis of idiopathic fibrotic lung disease. Inhibitor of growth family member 4 (ING4) has recently attracted much attention as a tumor suppressor gene, due to its ability to inhibit cancer cell proliferation, migration and angiogenesis. The aim of our study was to investigate the role of ING4 in the pathogenesis of pulmonary fibrosis both in the bleomycin (BLM)-model and in two different types of human pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP).
Methods
Experimental model of pulmonary fibrosis was induced by a single tail vein injection of bleomycin in 6- to 8-wk-old C57BL/6mice. Tissue microarrays coupled with qRT-PCR and immunohistochemistry were applied in whole lung samples and paraffin-embedded tissue sections of 30 patients with IPF, 20 with COP and 20 control subjects.
Results
A gradual decline of ING4 expression in both mRNA and protein levels was reported in the BLM-model. ING4 was also found down-regulated in IPF patients compared to COP and control subjects. Immunolocalization analyses revealed increased expression in areas of normal epithelium and in alveolar epithelium surrounding Masson bodies, in COP lung, whereas showed no expression within areas of active fibrosis within IPF and COP lung. In addition, ING4 expression levels were negatively correlated with pulmonary function parameters in IPF patients.
Conclusion
Our data suggest a potential role for ING4 in lung fibrogenesis. ING4 down-regulation may facilitate aberrant vascular remodelling and fibroblast proliferation and migration leading to progressive disease.
doi:10.1186/1465-9921-10-14
PMCID: PMC2662808  PMID: 19250543
11.  Soluble TNF Mediates the Transition from Pulmonary Inflammation to Fibrosis 
PLoS ONE  2006;1(1):e108.
Background
Fibrosis, the replacement of functional tissue with excessive fibrous tissue, can occur in all the main tissues and organ systems, resulting in various pathological disorders. Idiopathic Pulmonary Fibrosis is a prototype fibrotic disease involving abnormal wound healing in response to multiple sites of ongoing alveolar epithelial injury.
Methodology/Principal Findings
To decipher the role of TNF and TNF-mediated inflammation in the development of fibrosis, we have utilized the bleomycin-induced animal model of Pulmonary Fibrosis and a series of genetically modified mice lacking components of TNF signaling. Transmembrane TNF expression is shown to be sufficient to elicit an inflammatory response, but inadequate for the transition to the fibrotic phase of the disease. Soluble TNF expression is shown to be crucial for lymphocyte recruitment, a prerequisite for TGF-b1 expression and the development of fibrotic lesions. Moreover, through a series of bone marrow transfers, the necessary TNF expression is shown to originate from the non-hematopoietic compartment further localized in apoptosing epithelial cells.
Conclusions
These results suggest a primary detrimental role of soluble TNF in the pathologic cascade, separating it from the beneficial role of transmembrane TNF, and indicate the importance of assessing the efficacy of soluble TNF antagonists in the treatment of Idiopathic Pulmonary Fibrosis.
doi:10.1371/journal.pone.0000108
PMCID: PMC1762410  PMID: 17205112
13.  Cytoskeletal Rearrangements in Synovial Fibroblasts as a Novel Pathophysiological Determinant of Modeled Rheumatoid Arthritis 
PLoS Genetics  2005;1(4):e48.
Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology–assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease.
Synopsis
Rheumatoid arthritis (RA) is a chronic destructive disease that affects 1–3% of the general population, exacting substantial personal, social, and economic costs. Current treatments alleviate the symptoms and offer immediate relief for many patients but do not cure the disease. While the cause of the disease remains poorly understood, the completion of the Human Genome Project and the emergence of functional genomics and high-throughput technologies offer intriguing new possibilities. For example, expression profiling creates a molecular fingerprint of the disease status by quantifying the expression levels of thousand of genes simultaneously. Similarly, reverse genetics (the genetic modification of a particular gene in search of its function) allow for the creation of animal models of disease. To discover novel genes and/or cellular pathways involved in the development of the disease, the authors used two methods in an animal model of RA for large-scale expression profiling. They identified a large number of genes and molecular processes that are deregulated in the disease. Using this information, the authors described pathophysiologic determinants of RA and created a unique list of disease modifiers of potential therapeutic value.
doi:10.1371/journal.pgen.0010048
PMCID: PMC1270006  PMID: 16254600
14.  Effect of phospholipase A2 inhibitory peptide on inflammatory arthritis in a TNF transgenic mouse model: a time-course ultrastructural study 
Arthritis Research & Therapy  2004;6(3):R282-R294.
We evaluated the therapeutic effect of secretory phospholipase A2 (sPLA2)-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA2 inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA2 detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide- and TNF-induced release of prostaglandin E2 from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA2 inhibition.
doi:10.1186/ar1179
PMCID: PMC416452  PMID: 15142275
peptide; secretory phospholipase A2 inhibition; rheumatoid arthritis; TNF transgenic mouse model; ultrastructural alterations
15.  Functional analysis of an arthritogenic synovial fibroblast 
Arthritis Research & Therapy  2003;5(3):R140-R157.
Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellullar matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.
doi:10.1186/ar749
PMCID: PMC165045  PMID: 12723986
fibroblast; gene expression; migration; rheumatoid arthritis; tumor necrosis factor
16.  The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2 
Molecular and Cellular Biology  1999;19(10):6532-6542.
V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.
PMCID: PMC84623  PMID: 10490593

Results 1-16 (16)