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Background and purpose: P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). Experimental approach: Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. Key results: RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 mM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by abmethylene ATP (10 mM, 94% cells, n = 31) and was antagonized in LAD2 cells by NF 449 (1 mM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 mM). A P2X7-like nondesensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n = 24; 54% HLMCs, n = 24) which was antagonized by AZ11645373 (1 mM). P2X7-like responses were also evoked in LAD2 cells by 2′(3′)-0-(4-benzoylbenzoyl)ATP (300 mM). A P2X4-like current was evoked by 100 mM ATP (80% LAD2, n = 10; 21% HLMCs, n = 29), the amplitude and duration of which was potentiated by ivermectin (3 mM).

Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-016-9497-4) contains supplementary material, which is available to authorized users.

Targeting the complement component 3a receptor (C3aR) with selective agonists or antagonists is believed to be a viable therapeutic option for several diseases such as stroke, heart attack, reperfusion injuries, and rheumatoid arthritis. We designed a number of agonists, partial agonists, and antagonists of C3aR using our two-stage de novo protein design framework. Of the peptides tested using a degranulation assay in C3aR-transfected rat basophilic leukemia cells, two were prominent agonists (EC50 values of 25.3 and 66.2 nM) and two others were partial agonists (IC50 values of 15.4 and 26.1 nM). Further testing of these lead compounds in a calcium flux assay in U937 cells yielded similar results although with reduced potencies compared to transfected cells. The partial agonists also displayed full antagonist activity when tested in a C3aR inhibition assay. In addition, the electrostatic potential profile was shown to potentially discriminate between full agonists and partial agonists.

BackgroundRandomised controlled trials (RCTs) are considered the gold standard form of evidence for assessing treatment efficacy, but many factors can influence their reliability including methodological quality, reporting quality and funding source.The aim of this study was to examine the relationship between funding source and positive outcome reporting in veterinary RCTs published in 2011 and to assess the risk of bias in the RCTs identified.MethodsA structured search of PubMed was used to identify feline, canine, equine, bovine and ovine clinical trials examining the efficacy of pharmaceutical interventions published in 2011. Funding source and outcomes were extracted from each RCT and an assessment of risk of bias made using the Cochrane risk of bias tool.ResultsLiterature searches returned 972 papers, with 86 papers (comprising 126 individual RCTs) included in the analysis. There was found to be a significantly higher proportion of positive outcomes reported in the pharmaceutical funding group (P) compared to the non-pharmaceutical (NP) and ‘no funding source stated’ (NF) groups (P = 56.9%, NP = 34.9%, NF = 29.1%, p < 0.05). A high proportion of trials had an unclear risk of bias across the five criteria examined.ConclusionsWe found evidence that veterinary RCTs were more likely to report positive outcomes if they have pharmaceutical industry funding or involvement. Consistently poor reporting of trials, including non-identification of funding source, was found which hinders the use of the available evidence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-017-1146-9) contains supplementary material, which is available to authorized users.