59 research outputs found
Intracellular sphingosine kinase activity as a regulator of endothelial cell inflammatory and angiogenic potential.
The normal endothelium is in a non-activated state evidenced by its inability to support leukocyte adhesion and its lack of angiogenesis. I present evidence in this thesis that the intracellular levels and activity of Sphingosine Kinase (SK) regulate the inflammatory potential of the endothelium and are also an important determinant of the ability of the endothelium to undergo angiogenesis. Moderate (three to fivefold) elevations in SK activity in human umbilical vein endothelial cells resulted in endothelial activation, as indicated by the heightened expression of adhesion molecules, and further sensitized the cells to subliminal doses of inflammatory cytokines. This corresponded with enhanced neutrophil binding in the un-stimulated and stimulated states. Over-expression of a dominant-negative SK (G82D, containing the substitution glycine to aspartate and which blocks agonist-induced activation of SK) inhibited the adhesion molecule response to inflammatory cytokine and inhibited leukocyte adhesion. Over-expression of SK increased cell survival under the stressful conditions of serum deprivation and loss of attachment with extracellular matrix, which was associated with suppression of apoptotic mechanisms. Raised SK activity also stimulated cell migration and cellular remodeling, additional measures of angiogenesis. Over-expression of SK enabled activation of the phosphatidyl inositol-3kinase (PI3K1Akt) pathway in response to serum deprivation, and this pathway was obligatory in mediating SK-induced cell survival. Activation of the PI-3K1Akt pathway in cells with raised SK activity was mediated by the cell junctional molecule platelet endothelial cell adhesion molecule-l (PECAM-l), which was upregulated and dephosphorylated and critical in SK-induced cell survival. Thus raised intracellular SK activity enables a PECAM-l-dependent activation of the PI-3K1Akt pathway to augment cell survival, thus exposing a hitherto unexplored pathway of endothelial cell survival which may be manipulated therapeutically. The findings suggest a possible role for SK levels in the regulation of angiogenic phenomena as well as in the capacity of endothelial cells to survive in suspension (circulating endothelial cells). Thus SK could be considered as a novel target for therapeutic manipulation in diseases of aberrant inflammation and angiogenesis.Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 200
Meloxicam-Induced Rhabdomyolysis in the Context of an Acute Ross River Viral Infection
Acute rhabdomyolysis is a clinical and laboratory syndrome resulting from the breakdown of skeletal muscle, with the release of intracellular contents into the circulatory system, which can cause potentially lethal complications. Here, we present the case of a patient who developed acute rhabdomyolysis after consumption of meloxicam for jaw pain and experienced generalized myalgias in the context of an acute febrile illness with generalized urticaria. Further investigation indicated elevated muscle enzymes and acute renal failure. Serological analysis revealed that the patient was positive for Ross River virus (RRV) IgM. Genetic studies to detect CYP2C9 polymorphisms were negative. Meloxicam was discontinued. He responded to conservative measures within 2 weeks. Oral aspirin challenge was negative, suggesting a drug-specific effect of meloxicam rather than a class effect. Our case indicates a causative role for meloxicam and/or acute RRV in rhabdomyolysis
Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus
<p>Abstract</p> <p>Background</p> <p>Chandipura virus (CHPV), a member of family <it>Rhabdoviridae </it>was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.</p> <p>Methods</p> <p>Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [<it>in vivo </it>(mice) <it>in ovo </it>(eggs), <it>in vitro </it>(Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.</p> <p>Results</p> <p>Real-time one step RT-PCR was optimized using <it>in vitro </it>transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 10<sup>2</sup>-10<sup>10 </sup>(r<sup>2 </sup>= 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 10<sup>0 </sup>PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 10<sup>2 </sup>PFU/ml). Vero and PS cell-lines (1.2 × 10<sup>3 </sup>PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.</p> <p>Conclusion</p> <p>On account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.</p
Genome-wide imputation identifies novel associations and localises signals in idiopathic inflammatory myopathies.
OBJECTIVES
The idiopathic inflammatory myopathies (IIM) are heterogeneous diseases, thought to be initiated by immune activation in genetically predisposed individuals. In this study we imputed variants from the Immunochip array using a large reference panel to fine-map associations and identify novel associations in IIM.
METHODS
We analysed 2,565 Caucasian IIM samples collected through the Myositis Genetics Consortium (MYOGEN) and 10,260 ethnically-matched controls. We imputed 1,648,116 variants from the Immunochip array using the Haplotype Reference Consortium panel and conducted association analysis on IIM, and clinical and serological subgroups.
RESULTS
The human leukocyte antigen (HLA) locus was consistently the most significantly associated region. Four non-HLA regions reached genome-wide significance, three in the whole IIM cohort (SDK2 and LINC00924 - both novel, and STAT4), with evidence of independent variants in STAT4, and NAB1 in the polymyositis (PM) subgroup. We also found suggestive evidence of association with loci previously associated with other autoimmune rheumatic diseases (TEC and LTBR). We identified more significant associations than those previously reported in IIM, for STAT4 and DGKQ in the total cohort, for NAB1 and FAM167A-BLK loci in PM, and CCR5 in inclusion body myositis. We found enrichment of variants among DNase I hypersensitivity sites and histone marks associated with active transcription within blood cells.
CONCLUSIONS
We report novel and strong associations in IIM and PM, and localise signals to single genes and immune cell types. This article is protected by copyright. All rights reserved
Identification of Novel Associations and Localization of Signals in Idiopathic Inflammatory Myopathies Using Genome-Wide Imputation
OBJECTIVES: The idiopathic inflammatory myopathies (IIM) are heterogeneous diseases, thought to be initiated by immune activation in genetically predisposed individuals. In this study we imputed variants from the Immunochip array using a large reference panel to fine-map associations and identify novel associations in IIM. METHODS: We analysed 2,565 Caucasian IIM samples collected through the Myositis Genetics Consortium (MYOGEN) and 10,260 ethnically-matched controls. We imputed 1,648,116 variants from the Immunochip array using the Haplotype Reference Consortium panel and conducted association analysis on IIM, and clinical and serological subgroups. RESULTS: The human leukocyte antigen (HLA) locus was consistently the most significantly associated region. Four non-HLA regions reached genome-wide significance, three in the whole IIM cohort (SDK2 and LINC00924 - both novel, and STAT4), with evidence of independent variants in STAT4, and NAB1 in the polymyositis (PM) subgroup. We also found suggestive evidence of association with loci previously associated with other autoimmune rheumatic diseases (TEC and LTBR). We identified more significant associations than those previously reported in IIM, for STAT4 and DGKQ in the total cohort, for NAB1 and FAM167A-BLK loci in PM, and CCR5 in inclusion body myositis. We found enrichment of variants among DNase I hypersensitivity sites and histone marks associated with active transcription within blood cells. CONCLUSIONS: We report novel and strong associations in IIM and PM, and localise signals to single genes and immune cell types
Focused HLA analysis in Caucasians with myositis identifies significant associations with autoantibody subgroups
Objectives: Idiopathic inflammatory myopathies (IIM) are a spectrum of rare autoimmune diseases characterised clinically by muscle weakness and heterogeneous systemic organ involvement. The strongest genetic risk is within the major histocompatibility complex (MHC). Since autoantibody presence defines specific clinical subgroups of IIM, we aimed to correlate serotype and genotype, to identify novel risk variants in the MHC region that co-occur with IIM autoantibodies.
Methods: We collected available autoantibody data in our cohort of 2582 Caucasian patients with IIM. High resolution human leucocyte antigen (HLA) alleles and corresponding amino acid sequences were imputed using SNP2HLA from existing genotyping data and tested for association with 12 autoantibody subgroups.
Results: We report associations with eight autoantibodies reaching our study-wide significance level of p<2.9x10(-5). Associations with the 8.1 ancestral haplotype were found with anti-Jo-1 (HLA-B*08:01, p=2.28x10(-53) and HLA-DRB1*03:01, p=3.25x10(-9)), anti-PM/Scl (HLA-DQB1*02:01, p=1.47x10(-26)) and anti-cN1A autoantibodies (HLA-DRB1*03:01, p=1.40x10(-11)). Associations independent of this haplotype were found with anti-Mi-2 (HLA-DRB1*07:01, p=4.92x10(-13)) and anti-HMGCR autoantibodies (HLA-DRB1*11, p=5.09x10(-6)). Amino acid positions may be more strongly associated than classical HLA associations; for example with anti-Jo-1 autoantibodies and position 74 of HLA-DRB1 (p=3.47x10(-64)) and position 9 of HLA-B (p=7.03x10(-11)). We report novel genetic associations with HLA-DQB1 anti-TIF1 autoantibodies and identify haplotypes that may differ between adult-onset and juvenile-onset patients with these autoantibodies.
Conclusions: These findings provide new insights regarding the functional consequences of genetic polymorphisms within the MHC. As autoantibodies in IIM correlate with specific clinical features of disease, understanding genetic risk underlying development of autoantibody profiles has implications for future research
The effects of markedly raised intracellular sphingosine kinase-1 activity in endothelial cells
The enzyme sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which is an important survival factor for endothelial cells (EC). Modest increases in intracellular SK1 activity in the EC are known to confer a survival advantage upon the cells. Here, we investigated the effects of more dramatic increases in intracellular SK1 in the EC. We found that these cells show reduced cell survival under conditions of stress, enhanced caspase-3 activity, cell cycle inhibition, and cell-cell junction disruption. We propose that alterations in the phosphorylation state of the enzyme may explain the differential effects on the phenotype with modest versus high levels of enforced expression of SK1. Our results suggest that SK1 activity is subject to control in the EC, and that this control may be lost in conditions involving vascular regression.Vidya Limaye, Mathew A. Vadas, Stuart M. Pitson and Jennifer R. Gambl
Leopard detection matrix and covariates
This file contains the detection matrix and detection covariates used for leopard in our logistic regression models
all species occupancy covariate file
This contains the data on occupancy covariates used for all three species in our logistic regression models
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