Abstract-Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (nϭ980), with no association in the low-risk cohort (nϭ2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age-and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. [1][2][3][4] and the development of cardiovascular disease in predisposed prostacyclin receptor knockout mice, 5,6 underscores the necessity to better understand the effects of COX-2-derived metabolites on cardiovascular health. Endothelial prostacyclin synthesis requires the COX-2 enzyme 7 and may serve a role in protection from atherothrombosis. 8,9 This cardioprotective role has been supported by recent prostacyclin receptor knockout mice studies showing that the absence of the prostacyclin receptor (IP) (International Union of Pharmacology Receptor classification) leads to intimal hyperplasia, atherosclerosis, and hypercoagulability, 5,6 as well as reperfusion injury, 10 and premenopausal atherogenesis. 11 Despite such accumulating information, controversy remains as to whether prostacyclin deficiency is the etiology of the cardiovascular events observed with COX-2 inhibition, 12 particularly as no human studies have directly implicated defective prostacyclin signaling in the development of cardiovascular disease.The human prostacyclin receptor (hIP) gene (PTGIR) spans approximately 7000 bases along chromosome 19 (locus Original received October 11, 2007; revision received February 12, 2008; accepted February 21, 2008 19q13.3) and is comprised of 3 exons separated by 2 introns, 1 intron...

Background: Chondroitin sulfate (CS) and heparan sulfate (HS) are important for cartilage formation. Results: Analyses of zebrafish mutants demonstrate interplay between HS and CS biosynthesis in vivo.Conclusion: HS biosynthesis is prioritized over CS biosynthesis. The balance between extl3 and csgalnact1/csgalnact2 function determines the HS/CS ratio. Significance: Disease progression caused by HS deficiency may be affected by altered CS biosynthesis.

Objective Rituximab-mediated late-onset neutropenia (LON) has been described in various diseases. We investigated its occurrence, consequences and contributing factors in patients with systemic lupus erythematosus (SLE). Methods Rituximab-treated patients from the Karolinska University Hospital ( n = 107) were surveyed. LON was defined as an absolute neutrophil count <1500 cells/μl, occurring four weeks to two years following rituximab treatment, or later during sustained B-cell depletion. Serum levels of B-cell-related cytokines and growth factors of the myeloid lineage were determined using enzyme-linked immunosorbent assay. Results Thirty-two patients (29.9%) developed LON after a median time of 201.5 days. Thirteen patients were admitted to the hospital; 10 due to fever. Three patients developed critical conditions. BAFF levels increased from baseline (median: 0.62 ng/ml) to the post-treatment evaluation (median: 1.16 ng/ml; p < 0.001); post-treatment levels were higher in the LON group ( p = 0.021). APRIL levels were higher in the LON group both at baseline (median: 1.54 versus 1.15 ng/ml; p = 0.027) and post-treatment (median: 2.39 versus 1.11 ng/ml; p = 0.011). IL-6 and GM-CSF levels decreased in the non-LON group ( p < 0.001), but not in LON patients. High baseline disease activity predicted LON development (OR: 4.1; 95% CI: 1.1-15.2 for SLEDAI-2K > 8). No association with neutropenia prior to rituximab treatment was documented. Conclusion Post-rituximab LON was a common complication. Although the phenomenon was predominantly self-limiting, several patients developed severe conditions. Distinct roles of BAFF and APRIL are implicated: BAFF may contribute to LON development, whereas high APRIL levels may be predictive. Rituximab-treated SLE patients should be monitored for neutrophil counts, fever and infections.

Objective Heparan sulfate proteoglycans (HSPGs) regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between HSPGs and chondroitin sulfate proteoglycans (CSPGs) during sprouting angiogenesis. Methods and Results Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of HS biosynthesis. Cells unable to produce HS instead increase their production of CS that binds key angiogenic growth factors such as VEGFA, TGFβ and PDGFB. Lack of HSPG production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of TGFβ and PDGFB signal transduction. Conclusions The present study provides direct evidence for a previously undefined functional overlap between CSPGs and HSPGs during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of PGs in the vasculature.

Neutrophils are the most abundant immune cells found in actively inflamed joints of patients with rheumatoid arthritis (RA), and most animal models for RA depend on neutrophils for the induction of joint inflammation. Exogenous IL-4 and IL-13 protect mice from antibody-mediated joint inflammation, although the mechanism is not understood. Neutrophils display a very strong basal expression of STAT6, which is responsible for signaling following exposure to IL-4 and IL-13. Still, the role of IL-4 and IL-13 in neutrophil biology has not been well studied. This can be explained by the low neutrophil surface expression of the IL-4 receptor α-chain (IL-4Rα), essential for IL-4– and IL-13–induced STAT6 signaling. Here we identify that colony stimulating factor 3 (CSF3), released during acute inflammation, mediates potent STAT3-dependent neutrophil IL-4Rα up-regulation during sterile inflammatory conditions. We further demonstrate that IL-4 limits neutrophil migration to inflamed joints, and that CSF3 combined with IL-4 or IL-13 results in a prominent neutrophil up-regulation of the inhibitory Fcγ receptor (FcγR2b). Taking these data together, we demonstrate that the IL-4 and CSF3 pathways are linked and play important roles in regulating proinflammatory neutrophil behavior.

Twenty-five years have passed since Vane and colleagues proposed a prostacyclin and thromboxane balance as critical to cardiovascular homeostasis. Prostacyclin prevents platelet aggregation and promotes vasodilatation, opposing the effects of thromboxane. Possible compensation by redundant functions, such as nitric oxide, long prevented appreciation of this balance. Effective use of low-dose aspirin in the secondary prevention of atherothrombosis suggested a clinical importance for the balance. However, it was not until the cyclooxygenase-2 inhibitor rofecoxib was withdrawn because of increased cardiovascular events that this critical balance was confirmed in humans. Moreover, clinical observations are supported by elegant animal receptor knockout experiments and subsequent human genetic variant studies. Combined, these findings provide valuable insight into the roles of these prostanoids in the development of atherothrombosis, emphasizing the need to reevaluate the use of selective prostacyclin- and thromboxane-based therapies in cardiovascular disease.

Prostacyclin (PGI(2)) is a major product of COX-2 catalyzed metabolism of arachidonic acid in the endothelium. Recent studies have demonstrated that PGI(2) protects against atherothrombosis. The prostacyclin receptor knockout mice exhibit increased atherosclerosis, enhanced thrombosis, and enhanced proliferative response to carotid vascular injury with increased intima to media ratios [1-3]. Moreover, the recent withdrawal of rofecoxib (Vioxx) due to increased cardiovascular events further supports the critical role of prostacyclin in inhibiting atherothrombosis in humans. Such studies have paralleled intense chemical biology studies to develop more stable prostacyclin analogues. Indeed a number of these analogues are currently being successfully used for the treatment of pulmonary hypertension. In this review we will summarize the current literature on some principles of prostacyclin analogue development, our current understanding of the receptor, and recent developments which implicate prostacyclin in atherothrombotic protection. More than 68 million Americans suffer from cardiovascular disease, which causes more deaths, disability and economic loss than any other group of diseases. Further clinical investigations of orally stable prostacyclin analogues for treatment of cardiovascular diseases other than pulmonary hypertension may now be warranted.

Recent advances in single-cell sequencing technologies enable the generation of large-scale data sets of paired TCR sequences from patients with autoimmune disease. Methods to validate and characterize patient-derived TCR data are needed, as well as relevant model systems that can support the development of antigen-specific tolerance inducing drugs. We have generated a pipeline to allow streamlined generation of ‘artificial’ T cells in a robust and reasonably high throughput manner for in vitro and in vivo studies of antigen-specific and patient-derived immune responses. Hereby chimeric (mouse-human) TCR alpha and beta constructs are re-expressed in three different formats for further studies: ( i ) transiently in HEK cells for peptide-HLA tetramer validation experiments, ( ii ) stably in the TCR-negative 58 ​T cell line for functional readouts such as IL-2 production and NFAT-signaling, and lastly ( iii ) in human HLA-transgenic mice for studies of autoimmune disease and therapeutic interventions. As a proof of concept, we have used human HLA-DRB1∗04:01 restricted TCR sequences specific for a type I diabetes-associated GAD peptide, and an influenza-derived HA peptide. We show that the same chimeric TCR constructs can be used in each of the described assays facilitating sequential validation and prioritization steps leading to humanized animal models.