ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.

IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production

Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMC in vitro. Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcɛRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-α, IL-5, IL-13, macrophage inflammatory protein 1α, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcɛRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcɛRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.

Calcium regulation of a slow post-spike hyperpolarization in vagal afferent neurons

Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca2+-dependent K+ current. This current produces a post-spike hyperpolarization (AHPslow) that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHPslow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from <0.1 to >10 Hz. After a single action potential, the AHPslow in nodose neurons displays a slow rise time to peak (0.3–0.5 s) and a long duration (3–15 s). The slow kinetics of the AHPslow are due, in part, to Ca2+ discharge from an intracellular Ca2+-induced Ca2+ release (CICR) pool. Action potential-evoked Ca2+ influx via either L or N type Ca2+ channels triggers CICR. Surprisingly, although L type channels generate 60% of action potential-induced CICR, only Ca2+ influx through N type Ca2+ channels can trigger the CICR-dependent AHPslow. These observations suggest that a close physical proximity exists between endoplasmic reticulum ryanodine receptors and plasma membrane N type Ca2+ channels and AHPslow potassium channels. Such an anatomical relation might be particularly beneficial for modulation of spike-frequency adaptation in vagal afferent neurons.

Neurotrophins and hyperalgesia

Nerve growth factor (NGF), a member of the neurotrophin family, is crucial for survival of nociceptive neurons during development. Recently, it has been shown to play an important role in nociceptive function in adults. NGF is up-regulated after inflammatory injury of the skin. Administration of exogenous NGF either systemically or in the skin causes thermal hyperalgesia within minutes. Mast cells are considered important components in the action of NGF, because prior degranulation abolishes the early NGF-induced component of hyperalgesia. Substances degranulated by mast cells include serotonin, histamine, and NGF. Blockade of histamine receptors does not prevent NGF-induced hyperalgesia. The effects of blocking serotonin receptors are complex and cannot be interpretable uniquely as NGF losing its ability to induce hyperalgesia. To determine whether NGF has a direct effect on dorsal root ganglion neurons, we have begun to investigate the acute effects of NGF on capsaicin responses of small-diameter dorsal root ganglion cells in culture. NGF acutely conditions the response to capsaicin, suggesting that NGF may be important in sensitizing the response of sensory neurons to heat (a process that is thought to operate via the capsaicin receptor VR1). We also have found that ligands for the trkB receptor (brain-derived neurotrophic factor and neurotrophin-4/5) acutely sensitize nociceptive afferents and elicit hyperalgesia. Because brain-derived neurotrophic factor is up-regulated in trkA positive cells after inflammatory injury and is transported anterogradely, we consider it to be a potentially important peripheral component involved in neurotrophin-induced hyperalgesia.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

Amyloid beta peptide potentiates cytokine secretion by interleukin-1 beta-activated human astrocytoma cells.

Neurodegenerative processes in Alzheimer disease (AD) are thought to be driven in part by the deposition of amyloid beta (A beta), a 39- to 43-amino acid peptide product resulting from an alternative cleavage of amyloid precursor protein. Recent descriptions of in vitro neurotoxic effects of A beta support this hypothesis and suggest toxicity might be mediated by A beta-induced neuronal calcium disregulation. In addition, it has been reported that "aging" A beta results in increased toxic potency due to peptide aggregation and formation of a beta-sheet secondary structure. In addition, A beta might also promote neuropathology indirectly by activating immune/inflammatory pathways in affected areas of the brain (e.g., cortex and hippocampus). Here we report that A beta can modulate cytokine secretion [interleukins 6 and 8 (IL-6 and IL-8)] from human astrocytoma cells (U-373 MG). Freshly prepared and aged A beta modestly stimulated IL-6 and IL-8 secretion from U-373 MG cells. However, in the presence of interleukin-1 beta (IL-1 beta), aged, but not fresh, A beta markedly potentiated (3- to 8-fold) cytokine release. In contrast, aged A beta did not potentiate substance P (NK-1)- or histamine (H1)-stimulated cytokine production. Further studies showed that IL-1 beta-induced cytokine release was potentiated by A beta-(25-35), while A beta-(1-16) was inactive. Calcium disregulation may be responsible for the effects of A beta on cytokine production, since the calcium ionophore A23187 similarly potentiated IL-1 beta-induced cytokine secretion and EGTA treatment blocked either A beta or A23187 activity. Thus, chronic neurodegeneration in AD-affected brain regions may be mediated in part by the ability of A beta to exacerbate inflammatory pathways in a conformation-dependent manner.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.

Efficacy of a Nasal Spray from Citrus limon and Cydonia oblonga for the Treatment of Hay Fever Symptoms - A Randomized, Placebo Controlled Cross-Over Study

Nasal spray from lemon and quince (LQNS) is used to treat hay fever symptoms and has been shown to inhibit histamine release from mast cells in vitro. Forty-three patients with grass pollen allergy (GPA) were randomized to be treated either with placebo or LQNS for one week, respectively, in a cross-over study. At baseline and after the respective treatments patients were provoked with grass pollen allergen. Outcome parameters were nasal flow measured with rhinomanometry (primary), a nasal symptom score, histamine in the nasal mucus and tolerability. In the per protocol population absolute inspiratory nasal flow 10 and 20 min after provocation was higher with LQNS compared to placebo (-37 ± 87 mL/s; p = 0.027 and -44 ± 85 mL/s; p = 0.022). The nasal symptom score showed a trend (3.3 ± 1.8 in the placebo and 2.8 ± 1.5 in the LQNS group; p = 0.070) in favor of LQNS; the histamine concentration was not significantly different between the groups. Tolerability of both, LQNS and placebo, was rated as very good. LQNS seems to have an anti-allergic effect in patients with GPA. Copyright © 2016 John Wiley & Sons, Ltd.

Biogenic amines in Australian animal by-product meals

Biogenic amines were determined in Australian by-product meals including fish meals, poultry meals, meat meals, feather meals and blood meals. They were extracted with 0.1 mol/L HCl, derivatised with dansyl chloride and analysed using HPLC with UV detection at 254 nm. Median levels of putrescine, cadaverine and histamine were determined in the by-product meals, with the ranges being 13-102 mg/kg, 7-220 mg/kg and 4-570 mg/kg, respectively. High levels of biogenic amines were found particularly in fish meals. The results indicate that further studies are required to determine the maximum limits for biogenic amines in by-product meals that will not impair production when fed to poultry.

Challenge-induced plasma exudation and mucinous secretion in human airways

Secretion of mucins and exudation of plasma are distinct processes of importance to innate immunity and inflammatory disease. Yet, little is known about their relation in human airways. The objective of the present study was to use the human nasal airway to determine mucinous secretion and plasma exudation in response to common challenge agents and mediators. Ten healthy volunteers were subjected to nasal challenge-lavage procedures. Thus, the nasal mucosa was exposed to increasing doses of histamine (40 and 400 mu g ml(-1)), methacholine (12.5 and 25 mg) and capsaicin (30 and 300 ng ml(-1)). Fucose was selected as a global marker of mucinous secretion and alpha(2)-macroglobulin as an index of exudation of bulk plasma. All challenge agents increased the mucosal output of fucose to about the same level (P < 0.01-0.05). Once significant secretion had been induced the subsequently increased dose of the challenge agent, in the case of histamine and methacholine, failed to further increase the response. Only histamine increased the mucosal output of alpha(2)-macroglobulin (P < 0.01). We conclude that prompt but potentially rapidly depleted mucinous secretion is common to different kinds of airway challenges, whereas inflammatory histamine-type mediators are required to produce plasma exudation. Along with the acknowledged secretion of mucins, a practically non-depletable, pluripotent mucosal output of plasma emerges as an important component of the innate immunity of human airways.

Variance components analyses of multiple asthma traits in a large sample of Australian families ascertained through a twin proband

Background: Intermediate phenotypes are often measured as a proxy for asthma. It is largely unclear to what extent the same set of environmental or genetic factors regulate these traits. Objective: Estimate the environmental and genetic correlations between self-reported and clinical asthma traits. Methods: A total of 3073 subjects from 802 families were ascertained through a twin proband. Traits measured included self-reported asthma, airway histamine responsiveness (AHR), skin prick response to common allergens including house dust mite (Dermatophagoides pteronyssinus [D. pter]), baseline lung function, total serum immunoglobulin E (IgE) and eosinophilia. Bivariate and multivariate analyses of eight traits were performed with adjustment for ascertainment and significant covariates. Results: Overall 2716 participants completed an asthma questionnaire and 2087 were clinically tested, including 1289 self-reported asthmatics (92% previously diagnosed by a doctor). Asthma, AHR, markers of allergic sensitization and eosinophilia had significant environmental correlations with each other (range: 0.23-0.89). Baseline forced expiratory volume in 1 s (FEV1) showed low environmental correlations with most traits. Fewer genetic correlations were significantly different from zero. Phenotypes with greatest genetic similarity were asthma and atopy (0.46), IgE and eosinophilia (0.44), AHR and D. pter (0.43) and AHR and airway obstruction (-0.43). Traits with greatest genetic dissimilarity were FEV1 and atopy (0.05), airway obstruction and IgE (0.07) and FEV1 and D. pter (0.11). Conclusion: These results suggest that the same set of environmental factors regulates the variation of many asthma traits. In addition, although most traits are regulated to great extent by specific genetic factors, there is still some degree of genetic overlap that could be exploited by multivariate linkage approaches.

Aspects of signal transduction in the gastrointestinal tract

This study was undertaken to increase knowledge of the mechanisms of inter- and intracellular signalling in the gastrointestinal tract. Specific aims were: to use cell lines to elucidate factors affecting growth of gastric cells, to investigate the distribution and aspects of function of isoforms of protein kinase C in a gastric cell line and in the rat gastrointestinal tract and to determine the presence and regulation of nitric oxide synthase in gastrointestinal tissues from the rat and in cell lines. The gastric cancer cell line HGT-1 was used to investigate control of growth. Increases in cell number were found to be dependent on the seeding density of the cells. In cells plated at low density insulin, epidermal growth factor and gastrin all increased cell number. Gastrin produced a bell-shaped dose response curve with a maximum activity at 5nM. No effect of gastrin was apparent in cells plated at high density. α and β isoforms of protein kinase C were found, by immunoblotting procedures, to be widespread in the gastrointestinal tract of the rat, but protein kinase Cε was confined to the gastric mucosa and gastrointestinal smooth muscle. HGT-1 cells contained protein kinase C α and ε but β or γ were not detected. Preincubation of HGT-1 cells for 24h with 1μM phorbol-12,13-dibutyrate down-regulated protein kinase C α but not ε. The inhibition by the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) of the histamine-stimulated increase in cAMP in HGT-1 cells was down regulated by phorbol-12,13-dibutyrate. Inhibition of histamine-stimulation of adenylate cyclase by TPA was Ca2+-dependent and inhibited by the addition of an antibody to protein kinase C α. A role for protein kinase C α in modulating the effect of histamine on adenylate cyclase in HGT-1 cells is suggested. No nitric oxide synthase activity was detected in the gastrointestinal cell lines HGT-l, MKN-45 or CaCo-2. Ca2+-dependent nitric oxide synthase activity was observed in the gastric mucosa and the gastrointestinal smooth muscle from stomach to colon. The gastric: mucosal enzyme was soluble and showed half-maximal activity at 400nM Ca2+. Pretreatment of rats with endotoxin (3mg/kg body weight) induced nitric oxide synthase activity in both jejunal, ileal and colonic mucosa and muscle. A major portion of the induced activity in ileal and colonic mucosa was Ca2+-independent. Nitric oxide synthase activity in a high-density fraction of gastric mucosal cells was inhibited in a dose-dependent fashion by L-nitroarginine, NG-monomethyl-L-arginine, trifluoperazine and L-canavanine (in descending order of potency). Preincubation with okadaic acid and addition of ATPlMg2+ to the homogenisation buffer inhibited enzyme activity, which implies that phosphorylation inhibits gastric mucosal nitric oxide synthase.