ABSTRACT
The need to evaluate medicines for children is widely acknowledged due to pervasive unlicensed medicine use in the pediatric setting. The EU Paediatric Regulation was developed to address these considerations, which subsequently led to the establishment of the National Institute of Health Research (NIHR) Medicines for Children Research Network (MCRN) in England. MCRN supports public and industry studies, and facilitates feasibility, site setup, recruitment and other services. The MCRN and other networks are members of the European Network of Paediatric Research at the European Medicines Agency (Enpr-EMA). Enpr-EMA was established to foster and coordinate research, and develop collaborations across Europe. MCRN works with Enpr-EMA, industry and others to improve the conduct of research for the benefit of children's health.
Subject(s)
Biomedical Research/organization & administration , Clinical Trials as Topic/methods , Pediatrics/methods , Biomedical Research/legislation & jurisprudence , Biomedical Research/methods , Biomedical Research/trends , Child , Clinical Trials as Topic/legislation & jurisprudence , Clinical Trials as Topic/trends , Government Agencies , Government Regulation , Humans , Pediatrics/legislation & jurisprudence , Pediatrics/organization & administration , Pediatrics/trends , United KingdomABSTRACT
1. Chronic inflammation is a central feature of asthma. The inflammatory cytokine tumour necrosis factor alpha (TNFalpha) has been implicated in this disease, and is known to alter airway smooth muscle functionally. 2. The aim of this study was to investigate the influence of TNFalpha on tachykinin-induced airway relaxation. Mouse tracheae were cultured in the absence and presence of TNFalpha for 1 or 4 days. 3. In the absence of TNFalpha, substance P (SP) and neurokinin A (NKA) induced comparable levels of relaxation in fresh and cultured segments. Functional studies with selective antagonists/inhibitors indicated that the relaxation was mediated by the NK(1) receptor coupled to cyclooxygenase (COX)-2 activation and subsequent release of prostaglandin E(2) (PGE(2)). TNFalpha attenuated SP- and NKA-induced relaxation in a time- and concentration-dependent manner, decreasing the ability of PGE(2) to relax tissues. 4. Further studies indicated that TNFalpha elevated COX-2 activity and that concomitant inhibition of COX-2 reversed TNFalpha-attenuated PGE(2) relaxation. Culture with PGE(2) decreased SP- and PGE(2)-mediated relaxation, further implicating the activity of COX-2 in the attenuation of tachykinin signalling. 5. Gene expression analysis demonstrated that TNFalpha increased the expression of smooth muscle COX-2, PGE(2) synthase and EP(2) receptor mRNA, and decreased the expression of the EP(4) receptor. 6. Overall, these results show that NK(1) receptor-mediated relaxation induced by PGE(2) is attenuated by prolonged TNFalpha stimulation. Increased COX-2 activity induced by TNFalpha appears to be central to this process.
Subject(s)
Dinoprostone/physiology , Muscle Relaxation/physiology , Prostaglandin-Endoperoxide Synthases/metabolism , Tachykinins/physiology , Trachea/enzymology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cyclooxygenase 2 , Dinoprostone/antagonists & inhibitors , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , In Vitro Techniques , Male , Mice , Mice, Inbred BALB C , Muscle Relaxation/drug effects , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/metabolism , Tachykinins/antagonists & inhibitors , Trachea/drug effectsABSTRACT
1. Asthma research is arguably limited by an absence of appropriate animal models to study the pharmacology of inflammatory mediators that affect airway hyperresponsiveness and remodelling. Here we assessed an assay based on mouse tracheal segments cultured for 1-32 days, and investigated contractile responses mediated by muscarinic and 5-hydroxytryptamine (5-HT) receptors following long-term exposure to tumour necrosis factor-alpha (TNFalpha). 2. Following culture, in the absence of TNFalpha, maximum contractile responses to KCl and carbachol were similar, with an increase in response up to day two and a decrease to a stable level after 8 days. Maximal relaxations to isoprenaline were not affected by the culture procedure. The potency of KCl and isoprenaline increased throughout the study. DNA microarray data revealed that global gene expression changes were greater when tissues were introduced to culture than when they were maintained in culture. The morphology of smooth muscle cells was maintained throughout the culture period. 3. 5-HT induced a weak contraction in both fresh and cultured (up to 8 days) segments. Culture with TNFalpha produced a time- and concentration-dependent increase in the maximal contraction to 5-HT, evidently mediated by 5-HT(2A) receptors, whereas, the potency for carbachol was reduced. 4. In conclusion, the phenotype of airway smooth muscle remained largely intact during the culture period, even though minor changes were obtained during the first days of culture. The time-dependent effect of TNFalpha indicates the importance of studying the long-term effect of cytokines on the smooth muscle cells in relation to airway hyperresponsiveness and remodelling.
