Allergen exposure chambers: harmonizing current concepts and projecting the needs for the future - an EAACI Position Paper.

BACKGROUND: Allergen exposure chambers (AECs) are clinical facilities allowing for controlled exposure of subjects to allergens in an enclosed environment. AECs have contributed towards characterizing the pathophysiology of respiratory allergic diseases and the pharmacological properties of new therapies. In addition, they are complementary to and offer some advantages over traditional multicentre field trials for evaluation of novel therapeutics. To date, AEC studies conducted have been monocentric and have followed protocols unique to each centre. Because there are technical differences among AECs, it may be necessary to define parameters to standardize the AECs so that studies may be extrapolated for driving basic immunological research and for marketing authorization purposes by regulatory authorities. METHODS: For this task force initiative of the European Academy of Allergy and Clinical Immunology (EAACI), experts from academia and regulatory agencies met with chamber operators to list technical, clinical and regulatory unmet needs as well as the prerequisites for clinical validation. RESULTS: The latter covered the validation process, standardization of challenges and outcomes, intra- and interchamber variability and reproducibility, in addition to comparability with field trials and specifics of paediatric trials and regulatory issues. CONCLUSION: This EAACI Position Paper aims to harmonize current concepts in AECs and to project unmet needs with the intent to enhance progress towards use of these facilities in determining safety and efficacy of new therapeutics in the future.

Double-blind, placebo-controlled, dose-ranging study of new recombinant hypoallergenic Bet v 1 in an environmental exposure chamber.

BACKGROUND: Recombinant allergens offer a tool for improving specific immunotherapy (SIT). OBJECTIVE: To find the optimal dose of a new hypoallergenic folding variant of recombinant Bet v 1 (rBet v 1-FV) as SIT for patients with birch pollen allergy. METHODS: Before SIT, thirty-seven adult patients were exposed for eight hours in an environmental exposure chamber (EEC) to birch pollen at an average concentration of 3500 ± 500 grains/m(3) , then randomized to four maintenance dose groups of rBet v 1-FV and one placebo group: 20 µg (n = 7), 80 µg (n = 8), 160 µg (n = 7), 320 µg (n = 8), and placebo (n = 7). Patients were treated for 10 weeks with weekly injections and then re-exposed in the EEC. The optimal dose for SIT was assessed using efficacy results from the EEC, IgG responses, and tolerability. RESULTS: Thirty-six patients were evaluable for efficacy assessment. The total symptom score significantly decreased in all active groups compared with placebo (-18.8% for placebo patients; -71.9%, P = 0.0022 for 20 µg; -75.6%, P = 0.0007 for 80 µg; -81.8%, P = 0.0009 for 160 µg; -78.3%, P = 0.0003 for 320 µg). IgG1 increased significantly in all active groups compared to placebo. All four active doses were well tolerated, no serious adverse event occurred; two Grade II reactions, according to EAACI classification, were observed, one in each of the 160- and 320-µg groups. CONCLUSIONS: Considering efficacy, immunological response, and tolerability, a maintenance dose of 80 µg of rBet v 1-FV appears to be the ideal dose for allergen immunotherapy in birch pollen allergic patients.

Supramaximal cholecystokinin displaces Munc18c from the pancreatic acinar basal surface, redirecting apical exocytosis to the basal membrane.

Exocytosis at the apical surface of pancreatic acinar cells occurs in the presence of physiological concentrations of cholecystokinin (CCK) but is inhibited at high concentrations. Here we show that Munc18c is localized predominantly to the basal membranes of acinar cells. Supramaximal but not submaximal CCK stimulation caused Munc18c to dissociate from the plasma membrane, and this displacement was blocked by protein kinase C (PKC) inhibitors. Conversely, whereas the CCK analog CCK-OPE alone failed to displace Munc18c from the membrane, this agent caused Munc18c displacement following minimal PKC activation. To determine the physiological significance of this displacement, we used the fluorescent dye FM1-43 to visualize individual exocytosis events in real-time from rat acinar cells in culture. We showed that supramaximal CCK inhibition of secretion resulted from impaired apical secretion and a redirection of exocytic events to restricted basal membrane sites. In contrast, CCK-OPE evoked apical exocytosis and could only induce basolateral exocytosis following activation of PKC. Infusion of supraphysiological concentrations of CCK in rats, a treatment that induced tissue changes reminiscent of mild acute pancreatitis, likewise resulted in rapid displacement of Munc18c from the basal membrane in vivo.

Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action.

In pancreatic beta-cells, glucose metabolism signals insulin secretion by altering the cellular array of messenger molecules. ATP is particularly important, given its role in regulating cation channel activity, exocytosis, and events dependent upon its hydrolysis. Uncoupling protein (UCP)-2 is proposed to catalyze a mitochondrial inner-membrane H(+) leak that bypasses ATP synthase, thereby reducing cellular ATP content. Previously, we showed that overexpression of UCP-2 suppressed glucose-stimulated insulin secretion (GSIS) in isolated islets (1). The aim of this study was to identify downstream consequences of UCP-2 overexpression and to determine whether insufficient insulin secretion in a diabetic model was correlated with increased endogenous UCP-2 expression. In isolated islets from normal rats, the degree to which GSIS was suppressed was inversely correlated with the amount of UCP-2 expression induced. Depolarizing the islets with KCl or inhibiting ATP-dependent K(+) (K(ATP)) channels with glybenclamide elicited similar insulin secretion in control and UCP-2-overexpressing islets. The glucose-stimulated mitochondrial membrane ((m)) hyperpolarization was reduced in beta-cells overexpressing UCP-2. ATP content of UCP-2-induced islets was reduced by 50%, and there was no change in the efflux of Rb(+) at high versus low glucose concentrations, suggesting that low ATP led to reduced glucose-induced depolarization, thereby causing reduced insulin secretion. Sprague-Dawley rats fed a diet with 40% fat for 3 weeks were glucose intolerant, and in vitro insulin secretion at high glucose was only increased 8.5-fold over basal, compared with 28-fold in control rats. Islet UCP-2 mRNA expression was increased twofold. These studies provide further strong evidence that UCP-2 is an important negative regulator of beta-cell insulin secretion and demonstrate that reduced (m) and increased activity of K(ATP) channels are mechanisms by which UCP-2-mediated effects are mediated. These studies also raise the possibility that a pathological upregulation of UCP-2 expression in the prediabetic state could contribute to the loss of glucose responsiveness observed in obesity-related type 2 diabetes in humans.

Mutations to the third cytoplasmic domain of the glucagon-like peptide 1 (GLP-1) receptor can functionally uncouple GLP-1-stimulated insulin secretion in HIT-T15 cells.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone with powerful antidiabetogenic effects that are thought to be mediated by adenylyl cyclase (AC). Recently, we generated two GLP-1 receptor mutant isoforms (IC3-1 and DM-1) that displayed efficient ligand binding and the ability to promote Ca2+ mobilization from intracellular stores but lacked the ability to couple to AC. In the present study, the wild-type rat GLP-1 receptor (WT-GLP-1 R) or the IC3-1 and DM-1 mutant forms were expressed for the first time in the insulin-producing HIT-T15 cells. Only cells expressing WT-GLP-1 R displayed dramatically elevated GLP-1-induced cAMP responses and elevated insulin secretion. The increase in GLP-1-stimulated secretion in cells expressing WT-GLP-1 R, however, was not accompanied by differences in glucose-stimulated insulin release. Prolonged exposure to GLP-1 (10 nM, 17 h), not only led to an increase in insulin secretion but also increased insulin mRNA levels, but only in cells expressing the WT-GLP-1 R and not the mutant isoforms. Electrophysiological analyses revealed that GLP-1 application enhanced L-type voltage-dependent Ca2+ channel (VDCC) currents > 2-fold and caused a positive shift in VDCC voltage-dependent inactivation in WT-GLP-1R cells only, not control or mutant (DM-1) cells. This action on the Ca2+ current was further enhanced by the VDCC agonist, BAYK8644, suggesting GLP-1 acts via a distinct mechanism dependent on cAMP. These studies demonstrate that the GLP-1 receptor efficiently couples to AC to stimulate insulin secretion and that receptors lacking critical residues in the proximal region of the third intracellular loop can effectively uncouple the receptor from cAMP production, VDCC activity, insulin secretion, and insulin biosynthesis.

Members of the Kv1 and Kv2 voltage-dependent K(+) channel families regulate insulin secretion.

In pancreatic beta-cells, voltage-dependent K(+) (Kv) channels are potential mediators of repolarization, closure of Ca(2+) channels, and limitation of insulin secretion. The specific Kv channels expressed in beta-cells and their contribution to the delayed rectifier current and regulation of insulin secretion in these cells are unclear. High-level protein expression and mRNA transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and insulinoma cells. Inhibition of these channels with tetraethylammonium decreased I(DR) by approximately 85% and enhanced glucose-stimulated insulin secretion by 2- to 4-fold. Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit, specifically eliminating Kv2 family currents, reduced delayed rectifier currents in these cells by 60-70% and enhanced glucose-stimulated insulin secretion from rat islets by 60%. Expression of a C-terminal truncated Kv1.4 subunit, abolishing Kv1 channel family currents, reduced delayed rectifier currents by approximately 25% and enhanced glucose-stimulated insulin secretion from rat islets by 40%. This study establishes that Kv2 and 1 channel homologs mediate the majority of repolarizing delayed rectifier current in rat beta-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.

Myogenic nitric oxide synthase activity in canine lower oesophageal sphincter: morphological and functional evidence.

1. Studies on canine lower oesophageal sphincter (LOS) evaluated the existence and function of a myogenic, nitric oxide synthase (NOS) by use of immunocytochemistry for NOS isozymes, NADPH-d histochemistry, [3H]-L-arginine to [3H]-L-citrulline transformation. In addition, functional studies in the muscle bath were performed. 2. Smooth muscle bundles or freshly isolated smooth muscle cells of LOS were NADPH-d reactive but did not recognize some antibodies against neural, endothelial or inducible NOS. NADPH-d reactivity and immunoreactivity to a neural NOS antibody were colocalized in LOS enteric nerves. Muscle plasma membrane-enriched fractions from fresh and cultured LOS cells converted [3H]-L-arginine to [3H]-L-citrulline; activity was mostly Ca2+/calmodulin-dependent. 3. N-Nitro-L-arginine (L-NOARG) persistently increased tone (blocked by L-arginine) in muscle strips despite blockade of nerve function. Nifedipine prevented or abolished L-NOARG-induced, but not carbachol-induced, contraction showing that tone increase by L-NOARG required functional L-Ca channels. 4. Membrane-bound, myogenic NOS in canine LOS may release NO continuously when Ca2+ entry through L-Ca channels occurs under physiological conditions and thereby modulate tone in LOS.

Arginosuccinate synthetase, arginosuccinate lyase and NOS in canine gastrointestinal tract: immunocytochemical studies.

Nitric oxide synthase (NOS) requires the substrate L-arginine for NO production to support multiple gastrointestinal functions. We asked, 'Where do enzymes to regenerate L-arginine from L-citrulline exist?'. We examined loci of immunoreactivities in the canine gastrointestinal tract for arginosuccinate synthetase and arginosuccinate lyase, enzymes that resynthesize L-arginine from L-citrulline, in relation to the distribution of nNOS immunoreactivity or NADPH-diaphorase histochemistry. Arginosuccinate synthetase and lyase were present in many neurones and nerve fibres in the myenteric plexus of the lower oesophageal sphincter (LOS), antrum, pylorus, ileum and colon; in the submucosal plexus of ileum and colon; in longitudinal muscle of ileum and colon; and in nerve bundles in circular muscle everywhere. LOS muscle was also immunoreactive for both enzymes. Circular and longitudinal muscle cells of the ileum and colon and cells resembling interstitial cells of Cajal in the deep muscular plexus of the ileum and the submuscular plexus of the colon also appeared immunoreactive. In neurones, arginosuccinate synthetase and nNOS were usually co-localized. NADPH diaphorase activity was present in LOS and likely in pylorus, but not in muscularis externa of ileum or colon. We conclude that resynthesis of L-arginine probably occurs in enteric nerves, interstitial cells of Cajal (ICC) and LOS muscle; also apparently in some cells without NOS to utilize it.

Assessment of neural inhibition of the lower esophageal sphincter in cats with esophagitis.

BACKGROUND: The aim of this study was to investigate the inhibitory innervation of the lower esophageal sphincter in the presence of esophagitis. METHODS: Esophagitis was produced in five anesthetized cats with intraesophageal perfusion of HCl. Sphincter pressure responses were assessed with a sleeve catheter after administration of bethanechol, cholecystokinin octapeptide, and McNeil-A343 and with intraesophageal balloon distension. RESULTS: In the presence of esophagitis (1) resting lower esophageal sphincter pressure decreased; (2) the excitatory response to bethanechol was maintained; (3) there was a reduction in the excitatory response to McNeil-A343 and cholecystokinin at the highest dosages; (4) there was an increase in the potency of cholecystokinin and McNeil-A343 to produce an inhibitory response; (5) the inhibitory response to intraesophageal balloon distension was maintained; and (6) increased inhibitory responses took longer to normalize than the reduced excitatory responses. CONCLUSIONS: Esophagitis decreases cholinergic excitation, but neural inhibition to the LES remains intact. These findings suggest that blocking intact inhibition may be a new therapeutic approach for esophagitis caused by gastroesophageal reflux.

Inwardly rectifying K(+) channels in esophageal smooth muscle.

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.

Mechanism of action of cholecystokinin octapeptide on cat lower esophageal sphincter.

In five cats we examined 1) the role of a cholinergic mechanism in cholecystokinin octapeptide (CCK-OP) excitation of the lower esophageal sphincter (LES) and 2) the interaction between CCK-OP-induced excitation and inhibition of the LES. Under ketamine anesthesia, LES pressure was monitored with a sleeve catheter. With CCK-OP, LES excitation was seen in four of five cats, and inhibition was seen in three of five cats. (4-Hydroxy-2-butynyl)trimethylammonium chloride (McNeil-A343) produced similar responses in the same cats but was less potent than CCK-OP. Atropine and/or hexamethonium reduced the response to CCK-OP. Pirenzepine had no effect on any CCK-OP response but reduced relaxation and enhanced excitation produced by McNeil-A343. Phentolamine increased CCK-OP-induced relaxation. In conclusion, CCK-OP can produce LES contraction through a preganglionic cholinergic mechanism involving a nicotinic synapse; however, induction of relaxation occurs predominantly at a postganglionic site involving adrenergic modulation. There is animal-to-animal variability in the balance of excitatory and inhibitory mechanisms to the LES, which determines the effect of a drug capable of activating both mechanisms.

Calcium source diversity in canine lower esophageal sphincter muscle.

Tonic contraction of the lower esophageal sphincter (LES) prevents gastroesophageal reflux. LES tone is produced both by cholinergic nerve and myogenic activities. The Ca++ sources for LES tone and carbachol-induced contraction in canine LES strips were determined from the effect on contractile activity of extracellular Ca++ level modulation, Ca++ entrance blockade or enhancement with nifedipine or BayK8644 respectively, and/or inhibition of Ca++ store refilling using the sarcoplasmic reticulum Ca++ pump inhibitor, cyclopiazonic acid. LES tone disappeared when a Ca++-free physiological saline solution or nifedipine was applied. Sustained Ca++ free contractions to carbachol were prevented/abolished by nifedipine or increased Ca++ chelation and enhanced by BayK8644. Inhibition of sarcoplasmic reticulum Ca++ pumps by cyclopiazonic acid reduced Ca++ free contractions to carbachol; BayK8644 restored cyclopiazonic acid-reduced Ca++ free contractions to carbachol. Therefore, some Ca++ stores can be refilled by mechanisms not requiring activity of the sarcoplasmic reticulum Ca++ pump. A preferred pathway may exist whereby Ca++ enters stores directly through L-Ca++ channels. The proposed Ca++ store refilling mechanism involves continuous Ca++ entry through L-Ca++ channels from sites not equilibrated with external Ca++. Therefore, diverse Ca++ stores exist in canine LES which are dependent on Ca++ influx through L-Ca++ channels.

Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions.

Depolarization elicited outward K+ currents from canine lower esophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitive Ca(2+)-dependent K+ channels. Current magnitudes varied with pipette Ca2+ concentration (EC50 = 108.5 nM). NG-nitro-L-arginine (L-NNA, 10(-4)M), IbTX (10(-8)M), or buffering intracellular Ca2+ to 8 nM decreased outward currents > 80%. Sodium nitroprusside (NaNP, 10(-4)M) restored L-NNA-inhibited or low intracellular Ca2+ concentration (not IbTX)-inhibited currents. L-NNA or IbTX application depolarized LES cells from -43 to -35 mV. NaNP restored the membrane potential to -46 mV after L-NNA but not after IbTX application. Nifedipine (30 microM) reduced outward currents and abolished or reduced L-NNA or NaNP effects, respectively. Immunocytochemistry revealed the presence of both argininosuccinate synthetase and argininosuccinate lyase in LES muscle cells. L-Citrulline, like L-arginine, reversed L-NNA inhibition of outward currents; only L-arginine reversed inhibition of outward currents by an antibody to argininosuccinate synthetase. Therefore, endogenous nitric oxide production, activated by Ca2+ entrance involving L-type Ca2+ channels, may continuously enhance outward currents to modulate LES muscle cell membrane potential and excitability.

Nitric oxide from enteric nerves acts by a different mechanism from myogenic nitric oxide in canine lower esophageal sphincter.

In canine lower esophageal sphincter, myogenic constitutive nitric-oxide (NO) synthase (NOS) in plasma membrane limits tone by opening large conductance Ca(2+)-dependent K(+) channels (BK(Ca) channels) and hyperpolarizing the membrane. We examined whether K(V) channels were involved and whether NO from enteric nerves and from NO donors used the same mechanisms. With nerves inactive, 100 nM iberiotoxin, like N-nitro-L-arginine (L-NOARG), increased tone but less. 4-Aminopyridine (4-AP) at 5 mM behaved similarly. Tetraethyl ammonium (TEA) at 20 mM equaled the effect of L-NOARG and occluded any tone increase from any combination of these agents. More than iberiotoxin or 4-AP, TEA decreased relaxations in response to sodium nitroprusside (SNP) or 3-morpholino-sydnonimine (Sin-1) by approximately 50%. In whole-cell patch-clamp recordings, TEA and 4-AP reduced outward K(+) currents additively by >90% at depolarization of +90 mV. Thus, K(+) channels in addition to BK(Ca) channels are opened by myogenic NO, and exogenous NO had relaxing effects both related and unrelated to K(+) channel openings. TEA (20 mM) increased tone but did not inhibit relaxations to electrical field stimulation (EFS) of enteric nerves. 4-AP relaxed tone, an effect that was abolished and reversed by L-NOARG. 4-AP apparently released NO and acetylcholine from nerves. The putative Cl(-) channel blocker niflumic acid (NFA; 30-100 microM) dose dependently reduced tone, but tone, restored by 10(-6) M carbachol or 20 mM TEA, was still relaxed by EFS and by SNP. 4,4'-Diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) at 500 to 1000 microM did not inhibit relaxation to EFS or SNP. The addition of TEA (20 mM) to DIDS (1000 microM) induced tonic and phasic activity and markedly inhibited relaxations to EFS. DIDS plus TEA reduced the relaxations to SNP like TEA alone. Reduction in extracellular ¿Cl(-) by isethionate substitution reduced tone but did not reduce relaxations when tone was restored. The combination of reduced extracellular ¿Cl(-) and TEA did not abolish relaxation to EFS until DIDS was added. Thus, multiple K(+) channels are opened by myogenic NO, and openings of these channels, as well as DIDS-sensitive, undefined mechanisms, are induced when NO is released from nerves or SNP.