Tumour necrosis factor-alpha polymorphism as one of the complex inherited factors in pemphigus.

The aim of our study was to analyse a significance of tumour necrosis factor (TNF)-alpha promoter gene polymorphisms in relation to the HLA-DR locus in genetic predisposition to pemphigus. TNF-alpha gene polymorphisms in position -238 and -308 were identified using a modified polymerase chain reaction-restriction fragment length polymorphism method in 53 patients with pemphigus (38 with pemphigus vulgaris, 15 with pemphigus foliaceus) and 87 healthy controls. The HLA-DRB1 locus was typed using the polymerase chain reaction SSO method in all the patients and 152 population controls. Carriers of the TNF-alpha polymorphic -308 A allele were found to be more frequent in the pemphigus foliaceus group in comparison with the control group (odds ratio (OR) = 8.12; p = 0.0005). A significant association between HLA-DRB1*04 (OR = 3.86; pcor = 0.0001) and DRB1*14 (OR = 8.4; pcor = 0.0001) and pemphigus vulgaris was found. In this group of patients a decreased frequency of HLA-DRB1*07 (OR = 0.08; pcor = 0.006) was also identified. We have shown for the first time a positive association of TNF-alpha polymorphism in position -308 with pemphigus foliaceus

Towards a 21st-century roadmap for biomedical research and drug discovery:consensus report and recommendations

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and nongovernmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathway-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this

Concentration-Dependent Noncysteinyl Leukotriene Type 1 Receptor-Mediated Inhibitory Activity of Leukotriene Receptor Antagonists

BACKGROUND: The use of leukotriene antagonists (LTRAs) for asthma therapy has been associated with a significant degree of inter-patient variability in response to treatment. Some of that variability may be attributable to non-cysteinyl leukotriene type 1 receptor (CysLT(1)) mediated inhibitory mechanisms that have been demonstrated for this group of drugs. OBJECTIVE: We have used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and CysLT(1)-independent anti-inflammatory activity of two chemically different, clinically relevant, LTRAs (montelukast and zafirlukast). RESULTS: Using receptor desensitization experiments in monocytes and CysLT(1) transfected HEK293 cells, and IL-10 and CysLT(1) siRNA induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists, LTD(4) and uridine diphosphate (UDP), signal through calcium mobilization, acting on separate receptors and that both pathways were inhibited by montelukast and zafirlukast. However, 3 logs higher concentrations of LTRAs were required for inhibition of UDP induced signaling. In monocytes, UDP, but not LTD(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. Both LTRAs, at low micromolar concentrations, also inhibited calcium ionophore induced leukotriene (LTB(4) and LTC(4)) production, indicating 5-lipoxygenase inhibitory activities. CONCLUSION: We report here that montelukast and zafirlukast, acting in a concentration dependent manner, can inhibit non-CysLT(1) mediated, proinflammatory reactions, suggesting activities potentially relevant for inter-patient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma

Characterisation of P2Y12 Receptor Responsiveness to Cysteinyl Leukotrienes

Leukotriene E4 (LTE4), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y12 is required for LTE4 mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y12 to cysteinyl leukotrienes. Models of human CysLT1, CysLT2 and P2Y12 overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and β-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT1 and CysLT2 but failed to induce any calcium response in cells expressing P2Y12 or P2Y12+ Gα16. In contrast, selective P2Y12 agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y12+ Gα16. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y12 when intracellular cAMP and β-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y12 to analyse potential signalling and cell activation through P2Y12 receptor or receptor heterodimers but no specific LTE4 responses were observed. These results show that LTE4 as well as other cysLTs do not activate intracellular signalling acting through P2Y12 and suggest that another LTE4 specific receptor has yet to be identified

Cytosolic phospholipase A2α activation induced by S1P is mediated by the S1P3 receptor in lung epithelial cells

Cytosolic phospholipase A2α (cPLA2α) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA2α in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA2α inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA2α, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA2α in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA2α activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA2α and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA2α activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung

Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells

Background : Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. Objective : To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. Methods : ASM cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and western blotting. Receptor signalling and function was determined by mRNA knockdown and intracellular calcium mobilisation experiments. Results : S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P[subscript 2] and S1P[subscript 3] receptors activated intracellular calcium mobilisation and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. Conclusion : S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma