Background and purpose:
The histamine H
4
receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In ...this report, we describe the
in vitro
and
in vivo
anti‐inflammatory properties of a potent histamine H
4
receptor antagonist, A‐940894 (4‐piperazin‐1‐yl‐6,7‐dihydro‐5H‐benzo6,7cyclohepta1,2‐dpyrimidin‐2‐ylamine).
Experimental approach:
We have analysed the pharmacological profile of A‐940894 at mouse native, rat recombinant and human recombinant and native, histamine H
4
receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan‐induced peritonitis.
Key results:
A‐940894 potently binds to both human and rat histamine H
4
receptors and exhibits considerably lower affinity for the human histamine H
1
, H
2
or H
3
receptors. It potently blocked histamine‐evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine‐induced shape change of mouse bone marrow‐derived mast cells and chemotaxis of human eosinophils
in vitro
. In a mouse mast cell‐dependent model of zymosan‐induced peritonitis, A‐940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D
2
levels. Finally, A‐940894 has good pharmacokinetic properties, including half‐life and oral bioavailability in rats and mice.
Conclusions and Implications:
These data suggest that A‐940894 is a potent and selective histamine H
4
receptor antagonist with pharmacokinetic properties suitable for long‐term
in vivo
testing and could serve as a useful tool for the further characterization of histamine H
4
receptor pharmacology.
Background and purpose: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In ...this report, we describe the in vitro and in vivo anti‐inflammatory properties of a potent histamine H4 receptor antagonist, A‐940894 (4‐piperazin‐1‐yl‐6,7‐dihydro‐5H‐benzo6,7cyclohepta1,2‐dpyrimidin‐2‐ylamine).
Experimental approach: We have analysed the pharmacological profile of A‐940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan‐induced peritonitis.
Key results: A‐940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine‐evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine‐induced shape change of mouse bone marrow‐derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell‐dependent model of zymosan‐induced peritonitis, A‐940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A‐940894 has good pharmacokinetic properties, including half‐life and oral bioavailability in rats and mice.
Conclusions and Implications: These data suggest that A‐940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long‐term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.
We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Vα2Cα, Vβ8.2Cβ) expressed in insect cells binds both Vβ8.2-specific bacterial superantigen staphylococcal ...enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak·conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Vα and Vβ domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak·conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mm. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by β-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.
Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be ...stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR beta -chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted alpha beta TCR and its superantigen and MHC/peptide ligands. The extracellular domains of the murine D10 TCR (V alpha 2, V beta 8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble D10 TCR bound to Staphylococcus aureus enterotoxin C2 with an association rate of 1.69 plus or minus 0.12 x 10 super(4) M super(-1) sec super(-1) and dissociation rate of 1.9 plus or minus 0.47 x 10 super(-2) sec super(-1), giving a dissociation constant of 1.1 mu M. Binding of the TCR to S. aureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-A super(k) molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I-A super(k) chimera specifically stimulated proliferation of the D10 T-cell clone, and bound to immobilized soluble D10 TCR with an association rate of 1.07 plus or minus 0.19 x 10 super(4) M super(-1) sec super(-1) and a dissociation rate of 2.2 plus or minus 0.65 x 10 super(-2) sec super(-1), giving a dissociation constant of 2.1 mu M.
GroEL, an Escherichia coli homolog of the heat shock protein 60 family of molecular chaperonins, has been implicated as a target of T cell-mediated immune responses in a broad spectrum of infections. ...In order to produce large quantities of native protein for raising and stimulating GroEL specific T cell lines, we have developed a simple and rapid two-step protocol for purifying native E. coli GroEL heat shock (or stress) protein which takes advantage of the inherent structural and functional properties of the protein. Based on a combination of gel exclusion chromatography, ATPase activity assay, isoelectric focusing, and circular dichroism analyses we conclude that our purification process yields native tetradecameric GroEL.
Summary Background Uncertainties persist about the magnitude of associations of diabetes mellitus and fasting glucose concentration with risk of coronary heart disease and major stroke subtypes. We ...aimed to quantify these associations for a wide range of circumstances. Methods We undertook a meta-analysis of individual records of diabetes, fasting blood glucose concentration, and other risk factors in people without initial vascular disease from studies in the Emerging Risk Factors Collaboration. We combined within-study regressions that were adjusted for age, sex, smoking, systolic blood pressure, and body-mass index to calculate hazard ratios (HRs) for vascular disease. Findings Analyses included data for 698 782 people (52 765 non-fatal or fatal vascular outcomes; 8·49 million person-years at risk) from 102 prospective studies. Adjusted HRs with diabetes were: 2·00 (95% CI 1·83–2·19) for coronary heart disease; 2·27 (1·95–2·65) for ischaemic stroke; 1·56 (1·19–2·05) for haemorrhagic stroke; 1·84 (1·59–2·13) for unclassified stroke; and 1·73 (1·51–1·98) for the aggregate of other vascular deaths. HRs did not change appreciably after further adjustment for lipid, inflammatory, or renal markers. HRs for coronary heart disease were higher in women than in men, at 40–59 years than at 70 years and older, and with fatal than with non-fatal disease. At an adult population-wide prevalence of 10%, diabetes was estimated to account for 11% (10–12%) of vascular deaths. Fasting blood glucose concentration was non-linearly related to vascular risk, with no significant associations between 3·90 mmol/L and 5·59 mmol/L. Compared with fasting blood glucose concentrations of 3·90–5·59 mmol/L, HRs for coronary heart disease were: 1·07 (0·97–1·18) for lower than 3·90 mmol/L; 1·11 (1·04–1·18) for 5·60–6·09 mmol/L; and 1·17 (1·08–1·26) for 6·10–6·99 mmol/L. In people without a history of diabetes, information about fasting blood glucose concentration or impaired fasting glucose status did not significantly improve metrics of vascular disease prediction when added to information about several conventional risk factors. Interpretation Diabetes confers about a two-fold excess risk for a wide range of vascular diseases, independently from other conventional risk factors. In people without diabetes, fasting blood glucose concentration is modestly and non-linearly associated with risk of vascular disease. Funding British Heart Foundation, UK Medical Research Council, and Pfizer.
Summary Background Whether triglyceride-mediated pathways are causally relevant to coronary heart disease is uncertain. We studied a genetic variant that regulates triglyceride concentration to help ...judge likelihood of causality. Methods We assessed the −1131T>C (rs662799) promoter polymorphism of the apolipoprotein A5 ( APOA5 ) gene in relation to triglyceride concentration, several other risk factors, and risk of coronary heart disease. We compared disease risk for genetically-raised triglyceride concentration (20 842 patients with coronary heart disease, 35 206 controls) with that recorded for equivalent differences in circulating triglyceride concentration in prospective studies (302 430 participants with no history of cardiovascular disease; 12 785 incident cases of coronary heart disease during 2·79 million person-years at risk). We analysed −1131T>C in 1795 people without a history of cardiovascular disease who had information about lipoprotein concentration and diameter obtained by nuclear magnetic resonance spectroscopy. Findings The minor allele frequency of −1131T>C was 8% (95% CI 7–9). −1131T>C was not significantly associated with several non-lipid risk factors or LDL cholesterol, and it was modestly associated with lower HDL cholesterol (mean difference per C allele 3·5% 95% CI 2·6–4·6; 0·053 mmol/L 0·039–0·068), lower apolipoprotein AI (1·3% 0·3–2·3; 0·023 g/L 0·005–0·041), and higher apolipoprotein B (3·2% 1·3–5·1; 0·027 g/L 0·011–0·043). By contrast, for every C allele inherited, mean triglyceride concentration was 16·0% (95% CI 12·9–18·7), or 0·25 mmol/L (0·20–0·29), higher (p=4·4×10−24 ). The odds ratio for coronary heart disease was 1·18 (95% CI 1·11–1·26; p=2·6×10−7 ) per C allele, which was concordant with the hazard ratio of 1·10 (95% CI 1·08–1·12) per 16% higher triglyceride concentration recorded in prospective studies. −1131T>C was significantly associated with higher VLDL particle concentration (mean difference per C allele 12·2 nmol/L 95% CI 7·7–16·7; p=9·3×10−8 ) and smaller HDL particle size (0·14 nm 0·08–0·20; p=7·0×10−5 ), factors that could mediate the effects of triglyceride. Interpretation These data are consistent with a causal association between triglyceride-mediated pathways and coronary heart disease. Funding British Heart Foundation, UK Medical Research Council, Novartis.
PDF
