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1.  Evidence questioning cromolyn’s effectiveness and selectivity as a “mast cell stabilizer” in mice 
Cromolyn, widely characterized as a “mast cell stabilizer”, has been used in mice to investigate the biological roles of mast cells in vivo. However, it is not clear to what extent cromolyn can either limit the function of mouse mast cells or influence biological processes in mice independently of effects on mast cells. We confirmed that cromolyn (at 10 mg/kg in vivo or 10 – 100 μM in vitro) can inhibit IgE-dependent mast cell activation in rats in vivo (measuring Evans blue extravasation in passive cutaneous anaphylaxis and increases in plasma histamine in passive systemic anaphylaxis) and in vitro (measuring peritoneal mast cell β-hexosaminidase release and prostaglandin D2 synthesis). However, under the conditions tested, cromolyn did not inhibit those mast cell-dependent responses in mice. In mice, cromolyn also failed to inhibit the ear swelling or leukocyte infiltration at sites of passive cutaneous anaphylaxis. Nor did cromolyn inhibit IgE-independent degranulation of mouse peritoneal mast cells induced by various stimulators in vitro. At 100 mg/kg, a concentration ten times higher than that which inhibited passive systemic anaphylaxis in rats, cromolyn significantly inhibited the increases in plasma concentrations of mouse mast cell protease-1 (but not of histamine) during passive systemic anaphylaxis, but had no effect on the reduction in body temperature in this setting. Moreover, this concentration of cromolyn (100 mg/kg) also inhibited LPS-induced TNF production in genetically mast cell-deficient C57BL/6-KitW-sh/W-sh mice in vivo. These results question cromolyn’s effectiveness and selectivity as an inhibitor of mast cell activation and mediator release in the mouse.
doi:10.1038/labinvest.2012.116
PMCID: PMC3580174  PMID: 22906983
Anaphylaxis; cromolyn; disodium cromoglycate; DSCG; mast cell; mouse; rat
2.  Effects of Sodium Cromoglycate on Iranian Asthmatic Subjects Without Exposure to any Bronchoconstrictor agent 
Cromolyn sodium, a mast cell stabilizing agent, provides an immediate protective effect against the exercise-induced bronchoconstriction while being used before the exercise. However, cromolyn is ineffective in reversing asthmatic bronchospasm; it is used as a maintenance therapy and has a prophylactic role in chronic asthma.
The purpose of this study was to determine the extent of change in baseline lung function tests following a single dose of cromolyn sodium in adult asthmatics.
Forty volunteers (33 women and 7 men) with moderate to severe persistent asthma were randomly assigned to receive 20 mg cromolyn, 40 mg cromolyn or cromolyn-placebo. The percent of improvement in lung function parameters was compared among the groups, during 1 h of inhalation.
Low dose of cromolyn induced more improvement in most lung function parameters such as forced expiratory flow volume in one second, forced vital capacity and peak expiratory flow compared with other groups. After 15 min, the improvement percentage of baseline forced expiratory flow volume in one second was 3.35 ± 1.5, for sodium cromoglycate-20 mg group compared with 0.98 ± 1.43 and - 0.68 ± 1.2 for sodium cromoglycate-placebo and sodium cromoglycate-40 mg, groups respectively. However, the differences between means were not significant. Furthermore, based on the definition of American Thoracic Society (ATS) for a ”significant post-bronchodilator response” developed in a few patients 15 min after the inhalation of 20 mg cromolyn sodium.
It is suggested that probably the inhalation of 20 mg of cromolyn sodium could immediately improve the lung function in few adults with asthma.
PMCID: PMC3832161  PMID: 24250478
Cromolyn sodium; Lung function tests; Forced expiratory flow volume in one second; Bronchial asthma; Adults
3.  Benzydamine Oral Spray Inhibiting Parasympathetic Function of Tracheal Smooth Muscle 
Objectives
Benzydamine is a nonsteroidal anti-inflammatory agents agent with anti-inflammatory and local anesthesia properties that is available in the entire world as an oral spray for oral mucositis patients who are suffering from radiation effects. The effect of benzydamine on oral mucositis in vivo is well known; however, the effect of the drug on tracheal smooth muscle has rarely been explored. During administration of the benzydamine for oral symptoms, it might affect the trachea via oral intake or inhalation.
Methods
We examined the effectiveness of benzydamine on isolated rat tracheal smooth muscle. The following assessments of benzydamine were performed: effect on tracheal smooth muscle resting tension; effect on contraction caused by 10-6M methacholine as a parasympathetic mimetic; and effect of the drug on electrically induced tracheal smooth muscle contractions.
Results
Addition of methacholine to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of benzydamine at doses of 10-5M or above elicited a significant relaxation response to 10-6M methacholine-induced contraction. Benzydamine could inhibit electrical field stimulation-induced spike contraction. It alone had a minimal effect on the basal tension of trachea as the concentration increased.
Conclusion
This study indicated that high concentrations of benzydamine might actually inhibit parasympathetic function of the trachea. Benzydamine might reduce asthma attacks in oral mucositis patients because it could inhibit parasympathetic function and reduce methacholine-induced contraction of tracheal smooth muscle.
doi:10.3342/ceo.2015.8.1.65
PMCID: PMC4338094  PMID: 25729498
Benzydamine; Anti-Inflammatory Agents, Non-Steroidal; Trachea; Smooth Muscle; In Vitro Study
4.  Double-masked, randomized, parallel-group study comparing olopatadine 0.1% ophthalmic solution with cromolyn sodium 2% and levocabastine 0.05% ophthalmic preparations in children with seasonal allergic conjunctivitis 
Background: It is estimated that >50% of medications have not been tested in children. Physicians need pediatric data to guide them in treating children. Olopatadine hydrochloride ophthalmic solution 0.1% is a topical antiallergic agent that is both an antihistamine with high affinity and selectivity for the histamine H1 receptor and a mast cell stabilizer that inhibits the release of histamine and other proinflammatory mediators from human conjunctival mast cells. The efficacy and tolerability of olopatadine has been demonstrated by comparative studies in adults and children with seasonal allergic conjunctivitis (SAC).
Objective: Pediatric patient data were extracted from 2 clinical trials to assess the efficacy and tolerability of olopatadine hydrochloride ophthalmic solution 0.1% compared with those of cromolyn sodium ophthalmic solution 2% and levocabastine ophthalmic solution 0.05% as treatment for SAC in children.
Methods: In study 1, conducted at 15 centers in 7 countries (Europe and Australia) from October 1995 to December 1997, olopatadine was instilled BID and placebo (vehicle) BID for 6 weeks and compared with cromolyn instilled QID. Study 2, conducted at 17 centers in 8 countries (Europe and Australia) from November 1998 to June 2000, compared olopatadine BID with levocabastine BID. In both studies, children of either sex and any race, aged 4 to 11 years, and having proven grass pollen allergies were assigned to treatment in a double-masked, randomized fashion. Slit-lamp examination, the physician's impression scale, and self-ratings were used to obtain efficacy data. Data analyses were based on pollen concentrations. The tolerability assessments were based on visual acuity, pupil diameter, intraocular pressure, and a dilated fundus examination.
Results: Study 1 comprised 30 children (olopatadine [n = 13] and cromolyn sodium [n = 17]; 18 boys, 12 girls; mean age, 7.9 years [range, 4–11 years]). Study 2 comprised 22 children (olopatadine [n = 10] and levocabastine [n = 12]; 12 boys, 10 girls; mean age, 8.6 years [range, 5–11 years]). In study 1, ocular itching (P = 0.010), redness seen on slit-lamp examination (P = 0.003), and eyelid swelling (P = 0.034) were significantly less intense with olopatadine than with cromolyn sodium during the peak pollen period. In study 2, redness seen on slit-lamp examination (P = 0.040) and self-rated ocular redness (P = 0.024) were significantly less intense with olopatadine than levocabastine during the peak pollen period. Olopatadine was well tolerated.
Conclusion: Olopatadine hydrochloride ophthalmic solution 0.1% was more effective than both cromolyn sodium 2% and levocabastine 0.05% ophthalmic preparations in controlling ocular signs and symptoms of SAC in children and was well tolerated when administered twice daily for 6 weeks.
doi:10.1016/S0011-393X(04)90032-X
PMCID: PMC3997093  PMID: 24764588
seasonal allergic rhinoconjunctivitis; eye drops; olopatadine; placebo; levocabastine; cromolyn sodium
5.  Mast Cell Inhibition Attenuates Myocardial Damage, Adverse Remodeling and Dysfunction during Fulminant Myocarditis in Rat 
Background
Myocarditis is a life-threatening heart disease characterized by myocardial inflammation, necrosis and chronic fibrosis. While mast cell inhibition has been suggested to prevents fibrosis in rat myocarditis, little is known about its effectiveness in attenuating cardiac remodeling and dysfunction in myocarditis. Thus, we sought to test the hypothesis that mast cell inhibition will attenuate the inflammatory reaction and associated left ventricular (LV) remodeling and dysfunction after fulminant autoimmune myocarditis.
Methods and Results
To induce experimental autoimmune myocarditis, we immunized 30 rats with porcine cardiac myosin twice at a 7-day interval. On day 8 animals were randomized into treatment either with an intraperitoneal (IP) injection of 25mg/kg of cromolyn sodium (n=13), or an equivalent volume (~0.5ml IP) of normal saline (n=11). All animals were scanned by serial echocardiography studies before treatment (baseline echocardiogram) and after 20 days of cromolyn sodium (28 days after immunization). Furthermore, serial cardiac magnetic resonance was performed in a subgroup of 12 animals. After 20 days of treatment (28 days from first immunization), hearts were harvested for histopathological analysis. By echocardiography, cromolyn sodium prevented LV dilatation and attenuated LV dysfunction, compared with controls. Postmortem analysis of hearts showed that cromolyn sodium reduced myocardial fibrosis, as well as the number and size of cardiac mast cells in the inflamed myocardium, compared with controls.
Conclusions
Our study suggests that mast cell inhibition with cromolyn sodium attenuates adverse LV remodeling and dysfunction in myocarditis. This mechanism-based therapy is clinically relevant and could improve the outcome of patients at risk for inflammatory cardiomyopathy and heart failure.
doi:10.1177/1074248412458975
PMCID: PMC3968541  PMID: 23172937
myocarditis; cardiac remodeling; mast cells; fibrosis
6.  Anti-inflammatory effects of theophylline, cromolyn and salbutamol in a murine model of pleurisy. 
British Journal of Pharmacology  1996;118(3):811-819.
1. The aim of this study was to examine the effect of theophylline, cromolyn and salbutamol, three well-known anti-asthmatic drugs, on the early (4 h) and late (48 h) phases of cell migration and fluid leakage induced by carrageenin in the pleural cavity of mice. 2. In the first set of experiments, animals were pretreated (30 min) with different doses of theophylline (0.5-50 mg kg-1, i.p.), cromolyn (0.02-0.2 mg per pleural cavity) or salbutamol (0.05-50 mg kg-1, i.p.); the total and differential cell content, and also the exudate were analysed 4 h after carrageenin (1%) administration. Afterwards, in order to evaluate the time course effects of these drugs on both phases of the inflammatory reaction, one dose employed in the above protocol was chosen, to pretreat (0.5-24 h) different groups of animals. The studied parameters were evaluated 4 and 48 h after pleurisy induction. 3. Acute administration of theophylline (1-50 mg kg-1, i.p.) cromolyn (0.02-0.2 mg per pleural cavity) and salbutamol (0.5-50 mg kg-1, i.p.), 30 min prior to carrageenin, caused significant inhibition of total cell and fluid leakage in the pleural cavity at 4 h (P < 0.01). All drugs exerted a long-lasting inhibitory effect on both exudation and cell migration (P < 0.01) when administered 0.5-8 h before pleurisy induction. However, the temporal profile of the inhibitory effect induced by these drugs on the first phase of the inflammatory reaction was clearly different. Thus, the inhibitory effect induced by theophylline and cromolyn on exudation was significantly longer (up to 24 h) in comparison to their effects on cell migration (only up to 8 h). In contrast, although salbutamol when administered 30 min before pleurisy induction abolished fluid leakage (P < 0.01), this effect was not sustained in the groups pretreated for 4-8 h. In these latter groups, a significant but much smaller reduction of exudation was observed (P < 0.01), whereas the magnitude of cell migration inhibition did not vary. 4. The second phase (48 h) of the inflammatory reaction induced by carrageenin (1%) was significantly inhibited by cromolyn (0.02 mg per pleural cavity) when this drug was administered 0.5-24 h before pleurisy induction (P < 0.01). Similar results were observed when theophylline (50 mg kg-1, i.p.) was administered 0.5-4 h before the injection of the phlogistic agent (P < 0.01). Treatment of the animals with salbutamol (5 mg kg-1, i.p.), 0.5-24 h before pleurisy induction, did not inhibit either cell migration or fluid leakage. In this condition, a significant increase of these parameters was observed in the group pretreated with salbutamol 8-24 h before pleurisy induction (P < 0.01). 5. These results indicate that theophylline and cromolyn were able to inhibit the early (4 h) and late (48 h) phases of the inflammatory reaction induced by carrageenin in a murine model of pleurisy. Salbutamol was effective only against the early phase. The inhibitory effects of theophylline, cromolyn and salbutamol on the early phase of this inflammatory reaction were long-lasting, although a distinct profile of inhibition was observed among them. These findings confirm and extend previous results described in other models of asthma and support both clinical and experimental evidence suggesting that these anti-asthmatic agents exhibit marked anti-inflammatory properties.
PMCID: PMC1909745  PMID: 8762112
7.  Evaluation of Thioperamide Effects Using Rat's Trachea Model 
Objectives
Thioperamide is used as an antagonist to the histamine H3 receptor. During administration of the drug, the trachea may be affected via nasal or oral inhalation. This study was to determine the effects of thioperamide on the trachea of rats in vitro.
Methods
We tested the effectiveness of thioperamide on isolated rat trachea submersed in Kreb's solution in a muscle bath. Changes in tracheal contractility in response to the application of parasympathetic mimetic agents were measured. The following assessments of thioperamide were performed: 1) effect on tracheal smooth muscle resting tension; 2) effect on contraction caused by 10-6 M methacholine as a parasympathetic mimetic; 3) effect of the drug on electrically-induced tracheal smooth muscle contractions.
Results
Thioperamide induced a significant relaxation response at a preparation concentration up to 10-4 M. The drug also inhibited the electrical field stimulation induced spike contraction. However, thioperamide alone had a minimal effect on the basal tension of the trachea at increasing concentrations.
Conclusion
The study indicated that high concentrations of thioperamide might actually antagonize cholinergic receptors and block parasympathetic function of the trachea.
doi:10.3342/ceo.2013.6.1.12
PMCID: PMC3604264  PMID: 23526076
Trachea; Asthma; In vitro; Thioperamide
8.  Effect of Cromolyn on S100P Interactions With RAGE and Pancreatic Cancer Growth and Invasion in Mouse Models 
Background
We previously found that S100P, a member of the S100 protein family, is expressed in more than 90% of pancreatic tumors and is associated with tumor growth and invasion. In the current study, we investigated the ability of the antiallergy drug, cromolyn, to block S100P function.
Methods
Interactions between cromolyn and S100P were investigated using a drug affinity column and by examining cromolyn’s effects on coimmunoprecipitation of S100P and receptor for advanced glycation end-products (RAGE). The effects of cromolyn on cell growth, invasion, and nuclear factor- κ B (NFκB) activity of pancreatic cancer cells with (BxPC-3 and MPanc-96) and without (Panc-1) endogenous S100P were investigated by cell proliferation assay, by cell invasion assay, and by luciferase reporter gene assay, respectively. The effects of cromolyn on tumor growth in vivo were investigated in three orthotopic models (n = 20 mice per model) by administration of cromolyn (5 mg/kg body weight, daily) with and without gemcitabine (125 mg/kg body weight, biweekly), the drug currently used to treat pancreatic cancer. Tumor growth was assayed by reporter gene expression. All statistical tests were two-sided.
Results
S100P was retained on a cromolyn affinity column. Cromolyn blocked the coimmunoprecipitation of S100P and RAGE. In vitro, cromolyn (100 µ M) inhibited S100P-stimulated Panc-1 cell proliferation (S100P, mean = 0.93 U, versus S100P + cromolyn, mean = 0.56 U, difference = 0.37 U; 95% confidence interval [CI] = 0.24 to 0.49 U; P = .001, n = 3), invasion (S100P, mean = 58.0%, versus S100P + cromolyn, mean = 9.4%, difference = 48.6%; 95% CI = 38.8% to 58.8%; P <.001, n = 3), and NFκB activity (S100P, mean = 14 460, versus S100P + cromolyn, mean = 7360 photons/s, difference = 7100 photons/s; 95% CI = 3689 to 10 510 photons/s; P = .005, n = 3). In vivo, cromolyn inhibited tumor growth in mice bearing tumor with endogenous S100P (BxPC-3: control, mean = 1.6 × 109 photons/s, versus cromolyn, mean = 4.4 × 108 photons/s, difference = 1.2 × 109 photons/s; 95% CI = 6.2 × 108 to 1.6 × 109 photons/s; P <.001, n = 5; MPanc-96: control, mean = 1.1 × 1010 photons/s, versus cromolyn, mean = 4.8 × 109 photons/s, difference = 6.2 × 109 photons/s; 95% CI = 1.9 × 109 to 1.0 × 1010 photons/s; P = .009, n = 5) and increased the effectiveness of gemcitabine (BxPC-3: gemcitabine, mean = 9.2 × 108 photons/s, versus combination, mean = 1.8 × 108 photons/s, difference = 7.4 × 108 photons/s; 95% CI = 4.5 × 108 to 1.0 × 109 photons/s; P <.001; MPanc-96: gemcitabine, mean = 4.1 × 109 photons/s, versus combination, mean = 2.0 × 109 photons/s, difference = 2.1 × 109 photons/s; 95% CI = 4.4 × 108 to 3.8 × 109 photons/s; P <.001). However, cromolyn had no effect on growth of tumors lacking S100P (Panc-1).
Conclusion
Cromolyn binds S100P, prevents activation of RAGE, inhibits tumor growth, and increases the effectiveness of gemcitabine in experimental models.
doi:10.1093/jnci/djj498
PMCID: PMC4461034  PMID: 17179482
9.  Chondroitin sulphate inhibits connective tissue mast cells 
British Journal of Pharmacology  2000;131(6):1039-1049.
Mast cells derive from the bone marrow and are responsible for the development of allergic and possibly inflammatory reactions. Mast cells are stimulated by immunoglobulin E (IgE) and specific antigen, but also by a number of neuropeptides such as neurotensin (NT), somatostatin or substance P (SP), to secrete numerous pro-inflammatory molecules that include histamine, cytokines and proteolytic enzymes.Chondroitin sulphate, a major constituent of connective tissues and of mast cell secretory granules, had a dose-dependent inhibitory effect on rat peritoneal mast cell release of histamine induced by the mast cell secretagogue compound 48/80 (48/80). This inhibition was stronger than that of the clinically available mast cell ‘stabilizer' disodium cromoglycate (cromolyn). Inhibition by chondroitin sulphate increased with the length of preincubation and persisted after the drug was washed off, while the effect of cromolyn was limited by rapid tachyphylaxis.Immunologic stimulation of histamine secretion from rat connective tissue mast cells (CTMC) was also inhibited, but this effect was weaker in umbilical cord-derived human mast cells and was absent in rat basophilic leukemia (RBL) cells which are considered homologous to mucosal mast cells (MMC). Oligo- and monosaccharides were not as effective as the polysaccharides.Inhibition, documented by light and electron microscopy, involved a decrease of intracellular calcium ion levels shown by confocal microscopy and image analysis. Autoradiography at the ultrastructural level showed that chondroitin sulphate was mostly associated with plasma and perigranular membranes.Chondroitin sulphate appears to be a potent mast cell inhibitor of allergic and nonimmune stimulation with potential clinical implications.
doi:10.1038/sj.bjp.0703672
PMCID: PMC1572430  PMID: 11082109
Chondroitin sulphate; disodium cromoglycate; heparin; histamine; mast cells; proteoglycans; secretion
10.  Mast Cells Mediate Hyperoxia-Induced Airway Hyper-reactivity in Newborn Rats 
Pediatric research  2010;68(1):70-74.
Premature infants are at increased risk of developing airway hyper-reactivity following oxidative stress and inflammation. Mast cells contribute to airway hyper-reactivity partly by mediator release, so we sought to determine if blocking mast cell degranulation or recruitment prevents hyperoxia-induced airway hyper-reactivity, mast cell accumulation, and airway smooth muscle changes. Rats were exposed at birth to air or 60% O2 for 14 days, inducing significantly increased airway hyper-reactivity (AHR) in the latter group, induced by nebulized methacholine challenge, measured by forced oscillometry. Daily treatment (postnatal days 1-14) with intraperitoneal cromolyn prevented hyperoxia-induced AHR, as did treatment with imatinib on postnatal days 5-14, compared with vehicle treated controls. Cromolyn prevented mast cell degranulation in the trachea but not hilar airways, and blocked mast cell accumulation in the hilar airways. Imatinib treatment completely blocked mast cell accumulation in tracheal/hilar airway tissues. Hyperoxia-induced AHR in neonatal rats is mediated, at least in part, via the mast cell.
doi:10.1203/PDR.0b013e3181e0cd97
PMCID: PMC3061400  PMID: 20386143
11.  Mast cell involvement in the adenosine mediated airway hyper-reactivity in a murine model of ovalbumin-induced lung inflammation 
British Journal of Pharmacology  2005;145(7):845-852.
Airway hyper-reactivity to inhaled adenosine, mediated via mast cell activation, is a cardinal feature of asthma. Animal models have been developed in several species to mimic this phenomenon, but only in the rat has a mast cell involvement been clearly defined. In this study, a model of ovalbumin-induced adenosine hyper-reactivity was developed in BALB/c mice to determine whether mast cells are involved in this phenomenon.Sensitised mice were challenged one, two or three times, on a daily basis, and airway responses to the stable adenosine analogue NECA (5′-N-ethylcarboxamido adenosine) determined 4 and 24 h after each challenge. Airway hyper-reactivity was observed in ovalbumin-challenged mice 4 h after a single challenge and to a minor extent 24 h after a single challenge and 4 h after two challenges.Cromolyn (20 mg ml−1), given by aerosol an hour before the NECA provocation, fully inhibited the airway hyper-reactivity observed 4 h after a single allergen challenge, suggesting a role for mast cells in this response. The airway space cellular inflammation was not affected by cromolyn.As observed in human asthma, an acute treatment with steroid (budesonide 3 mg kg−1, given an hour before the allergen challenge) inhibited the NECA airway hyper-reactivity and significantly inhibited the airway space cellular inflammation.These data suggest that the ovalbumin-challenged BALB/c mice can be considered as a suitable model to study the adenosine-induced airway hyper-reactivity phenomenon observed in human asthma.
doi:10.1038/sj.bjp.0706271
PMCID: PMC1576219  PMID: 15912130
Asthma; adenosine airway hyper-reactivity; mice; mast cell
12.  Contributions of Histamine, Prostanoids, and Neurokinins to Edema Elicited by Edema Toxin from Bacillus anthracis▿  
Infection and Immunity  2007;75(4):1895-1903.
Bacillus anthracis edema toxin (ET), composed of protective antigen and an adenylate cyclase edema factor (EF), elicits edema in host tissues, but the target cells and events leading from EF-mediated cyclic-AMP production to edema are unknown. We evaluated the direct effect of ET on several cell types in vitro and tested the possibility that mediators of vascular leakage, such as histamine, contribute to edema in rabbits given intradermal ET. ET increased the transendothelial electrical resistance of endothelial monolayers, a response that is mechanistically inconsistent with the in vivo vascular leakage induced by ET. Screening of several drugs by intradermal treatment prior to toxin injection demonstrated reduced ET-induced vascular leakage with a cyclo-oxygenase inhibitor (indomethacin), agents that interfere with histamine (pyrilamine or cromolyn), or a neurokinin antagonist (spantide). Systemic administration of indomethacin or celecoxib (cyclo-oxygenase inhibitors), pyrilamine, aprepitant (a neurokinin 1 receptor antagonist), or indomethacin with pyrilamine significantly reduced vascular leakage associated with ET. Although the effects of pyrilamine, cromolyn, or aprepitant on ET-induced vascular leakage suggest a possible role for mast cells (MC) and sensory neurons in ET-induced edema, ET did not elicit degranulation of human skin MC or substance P release from NT2N cells in vitro. Our results indicate that ET, acting indirectly or directly on a target yet to be identified, stimulates the production/release of multiple inflammatory mediators, specifically neurokinins, prostanoids, and histamine. These mediators, individually and through complex interactions, increase vascular permeability, and interventions directed at these mediators may benefit hosts infected with B. anthracis.
doi:10.1128/IAI.01632-06
PMCID: PMC1865696  PMID: 17261611
13.  Quercetin Is More Effective than Cromolyn in Blocking Human Mast Cell Cytokine Release and Inhibits Contact Dermatitis and Photosensitivity in Humans 
PLoS ONE  2012;7(3):e33805.
Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell “stabilizer”, is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 µM) can effectively inhibit secretion of histamine and PGD2. Que and cromolyn also inhibit histamine, leukotrienes and PGD2 from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption.
doi:10.1371/journal.pone.0033805
PMCID: PMC3314669  PMID: 22470478
14.  ROLE OF MENINGEAL MAST CELLS IN INTRATHECAL MORPHINE EVOKED GRANULOMA FORMATION 
Anesthesiology  2013;118(3):664-678.
Background
Intrathecal morphine forms granulomas that arise from the adjacent arachnoid membrane. We propose that these inflammatory cells exit the meningeal vasculature secondary to meningeal mast cell degranulation.
Methods
Three sets of experiments were accomplished in dogs. 1) Ex vivo Meningeal mast cell degranulation. Histamine release was measured ex vivo from canine dura incubated with opiates. 2) In vivo cutaneous mast cell degranulation. Flare areas on the dog abdomen were measured after subcutaneous opiates. 3) In vivo granuloma pharmacology. Dogs with lumbar intrathecal catheters received infusion of intrathecal saline or intrathecal morphine. Intrathecal morphine dogs received: i) No other treatment (Control); ii) Twice daily subcutaneous naltrexone; iii) Intrathecal co-infusion of cromolyn; or, iv) Twice daily subcutaneous cromolyn for the 24–28 day study course.
Results
1) Morphine but not fentanyl evoked dural histamine release, which was blocked by cromolyn but not naloxone. 2) Wheal/flare was produced by subcutaneous morphine, methadone, hydromorphone, but not fentanyl, and was unaffected by naltrexone but prevented by cromolyn. 3) Granulomas occurred in all dogs receiving intrathecal morphine (15/15); subcutaneous naltrexone had no effect on granulomas (6/6), but was reduced by concurrent intrathecal cromolyn (0/5) or twice daily subcutaneous cromolyn (1 of 5).
Conclusions
The pharmacology of cutaneous/dural MC degranulation and intrathecal granulomas are comparable, not mediated by opioid receptors, and reduced by agents preventing MC degranulation. If an agent produces cutaneous MC degranulation at concentrations produced by intrathecal delivery, the agent may initiate granulomas.
doi:10.1097/ALN.0b013e31828351aa
PMCID: PMC3788115  PMID: 23426209
15.  Cromolyn ameliorates acute and chronic injury in a rat lung transplant model 
BACKGROUND
Mast cells have been associated with obliterative bronchiolitis (OB) in human pulmonary allografts, although their role in the development of OB remains unknown.
METHODS
In this study, we evaluated the role of mast cells in pulmonary allograft rejection using an orthotopic rat pulmonary allograft model that utilizes chronic aspiration of gastric fluid to reliably obtain OB. Pulmonary allograft recipients (n = 35) received chronic aspiration of gastric fluid with (n = 10) and without (n = 16) treatment with a mast cell membrane stabilizer, cromolyn sodium, or chronic aspiration with normal saline (n = 9) as a control.
RESULTS
The acute graft injury associated with long ischemic time in the model (6 hours total ischemic time; typical acute graft injury rate ~30%) was apparently blocked by cromolyn, because peri-operative mortality associated with the acute graft injury was not observed in any of the animals receiving cromolyn (p = 0.045). Further, the rats receiving cromolyn developed significantly fewer OB lesions than those treated with gastric fluid alone (p < 0.001), with a mean reduction of 46% of the airways affected.
CONCLUSIONS
These findings provide impetus for further studies aimed at elucidating the effects of cromolyn and the role of mast cells in pulmonary allotransplantation.
doi:10.1016/j.healun.2014.03.004
PMCID: PMC4336160  PMID: 24768366
mast cells; aspiration; pulmonary allograft; gastric fluid; obliterative bronchiolitis
16.  Pancreatic and pulmonary mast cells activation during experimental acute pancreatitis 
AIM: To study the activation of pancreatic and pulmonary mast cells and the effect of mast cell inhibition on the activation of peritoneal and alveolar macrophages during acute pancreatitis.
METHODS: Pancreatitis was induced by intraductal infusion of 5% sodium taurodeoxycholate in rats. The mast cell inhibitor cromolyn was administered intraperitoneally (i.p.) 30 min before pancreatitis induction. The pancreatic and pulmonary tissue damage was evaluated histologically and mast cells and their state of activation were evaluated. Peritoneal and alveolar macrophages were obtained and the expression of tumor necrosis factor α was determined. Myeloperoxidase activity was measured to evaluate the effect of mast cell inhibition on the progression of the inflammatory process. Finally, the effect of plasma on cultured mast cells or macrophages was evaluated in vitro.
RESULTS: The mast cell stabilizer significantly reduced inflammation in the pancreas and lung and the activation of alveolar macrophages but had no effect on peritoneal macrophages. Mast cell degranulation was observed in the pancreas during pancreatitis but no changes were observed in the lung. Plasma from rats with pancreatitis could activate alveolar macrophages but did not induce degranulation of mast cells in vitro.
CONCLUSION: Pancreatic mast cells play an important role in triggering the local and systemic inflammatory response in the early stages of acute pancreatitis. In contrast, lung mast cells are not directly involved in the inflammatory response related to pancreatic damage.
doi:10.3748/wjg.v16.i27.3411
PMCID: PMC2904888  PMID: 20632444
Cytokines; Inflammation; Macrophages; Mast cells; Pancreatitis
17.  Pretreatment of cromolyn sodium prior to reperfusion attenuates early reperfusion injury after the small intestine ischemia in rats 
AIM: To investigate the effects of Cromolyn Sodium (CS) pretreated prior to reperfusion on the activity of intestinal mucosal mast cells (IMMC) and mucous membrane of the small intestine in ischemia-reperfusion (IR) injury of rats.
METHODS: Thirty-two Sprague-Dawley (SD) rats were randomly divided into four groups: sham group (group S), model group (group M), high and low dosage of CS groups, (treated with CS 50 mg/kg or 25 mg/kg, group C1 and C2). Intestinal IR damage was induced by clamping the superior mesenteric artery for 45 min followed by reperfusion for 60 min. CS was intravenouly administrated 15 min before reperfusion. Ultrastructure and counts of IMMC, intestinal structure, the expression of tryptase, levels of malondisldehyde (MDA), TNF-α, histamine and superoxide dismutase (SOD) activity of the small intestine were detected at the end of experiment.
RESULTS: The degranulation of IMMC was seen in group M and was attenuated by CS treatment. Chiu’s score of group M was higher than the other groups. CS could attenuate the up-regulation of the Chiu’s score, the levels of MDA, TNF-α, and expression of tryptase and the down-regulation of SOD activity and histamine concentration. The Chiu’s score and MDA content were negatively correlated, while SOD activity was positively correlated to the histamine concentration respectively in the IR groups.
CONCLUSION: Pretreated of CS prior to reperfusion protects the small intestine mucous from ischemia-reperfusion damage, the mechanism is inhibited IMMC from degranulation.
doi:10.3748/wjg.v13.i38.5139
PMCID: PMC4434646  PMID: 17876882
Ischemia; Reperfusion injury; Intestinal mucosal mast cells; Histamine; Tumor necrosis factor-α
18.  Management of Allergic Rhinitis 
Allergic rhinitis is the most common chronic childhood disease. Reduced quality of life is frequently caused by this IgE-mediated disease, including sleep disturbance with subsequent decreased school performance. Asthma and exercise-induced bronchospasm are commonly seen concurrently with allergic rhinitis, and poorly controlled allergic rhinitis negatively affects asthma outcomes. Nonsedating antihistamines or intranasal azelastine are effective agents to manage allergic rhinitis, often in combination with oral decongestants. For moderate to severe persistent disease, intranasal corticosteroids are the most effiective agents. Some patients require concomitant intranasal corticosteroids and nonsedating antihistamines for optimal management. Other available agents include leukotriene receptor antagonists, intranasal cromolyn, intranasal ipratropium, specific immunotherapy, and anti-IgE therapy.
doi:10.5863/1551-6776-10.3.159
PMCID: PMC3468067  PMID: 23118635
allergic rhinitis; drug therapy; management; pediatrics
19.  CRF Induces Intestinal Epithelial Barrier Injury via the Release of Mast Cell Proteases and TNF-α 
PLoS ONE  2012;7(6):e39935.
Background and Aims
Psychological stress is a predisposing factor in the onset and exacerbation of important gastrointestinal diseases including irritable bowel syndrome (IBS) and the inflammatory bowel diseases (IBD). The pathophysiology of stress-induced intestinal disturbances is known to be mediated by corticotropin releasing factor (CRF) but the precise signaling pathways remain poorly understood. Utilizing a porcine ex vivo intestinal model, the aim of this study was to investigate the mechanisms by which CRF mediates intestinal epithelial barrier disturbances.
Methodology
Ileum was harvested from 6–8 week-old pigs, mounted on Ussing Chambers, and exposed to CRF in the presence or absence of various pharmacologic inhibitors of CRF-mediated signaling pathways. Mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) and transepithelial electrical resistance (TER) were recorded as indices of intestinal epithelial barrier function.
Results
Exposure of porcine ileum to 0.05–0.5 µM CRF increased (p<0.05) paracellular flux compared with vehicle controls. CRF treatment had no deleterious effects on ileal TER. The effects of CRF on FD4 flux were inhibited with pre-treatment of tissue with the non-selective CRF1/2 receptor antagonist Astressin B and the mast cell stabilizer sodium cromolyn (10−4 M). Furthermore, anti-TNF-α neutralizing antibody (p<0.01), protease inhibitors (p<0.01) and the neural blocker tetrodotoxin (TTX) inhibited CRF-mediated intestinal barrier dysfunction.
Conclusion
These data demonstrate that CRF triggers increases in intestinal paracellular permeability via mast cell dependent release of TNF-α and proteases. Furthermore, CRF-mast cell signaling pathways and increases in intestinal permeability require critical input from the enteric nervous system. Therefore, blocking the deleterious effects of CRF may address the enteric signaling of mast cell degranulation, TNFα release, and protease secretion, hallmarks of IBS and IBD.
doi:10.1371/journal.pone.0039935
PMCID: PMC3386952  PMID: 22768175
20.  The Effect of Cromolyn Sodium and Nedocromil Sodium Administered by A pressurized Aerosol with A spacer Device on Exercise-Induced Asthma in Children 
Mediators of Inflammation  1994;3(7):S35-S37.
To compare the effectiveness of cromolyn sodium (CS) (10 mg) and nedocromil sodium (NS) (4 mg) administered by a metered dose inhaler (MDI) with a spacer device in preventing exercise-induced asthma (EIA), eight asthmatic children with EIA were studied in a randomized double-blind, cross-over, placebo-controlled study, CS and NS provided significant, comparable protection from EIA and both were better than placebo. We conclude that CS and NS administered by a pressurized aerosol with a spacer device provide equal protection against EIA in children.
doi:10.1155/S0962935194000736
PMCID: PMC2365597  PMID: 18475602
21.  A nebulized complex traditional Chinese medicine inhibits Histamine and IL-4 production by ovalbumin in guinea pigs and can stabilize mast cells in vitro 
Background
Traditional Chinese medicines have been used for anti-asthma treatment for several centuries in many Asian countries, and have been shown to effectively relieve symptoms. Our previous study demonstrated that a complex traditional Chinese medicine (CTCM) administered in nebulized form through the intratracheal route is effective against early-phase air-flow obstruction and can inhibit IL-5 production in ovalbumin (OVA)-sensitized guinea pigs. However, the antiasthmatic mechanisms of CTCMs are still unclear.
Methods
In this study, we examined the underlying mechanism of a CTCM that we used in our previous study in order to ascertain its function in the early-phase response to OVA challenge.
In each group, 10–12 unsensitized or OVA-sensitized guinea pigs were treated with nebulized CTCM before OVA challenge, and the airway responses of the animals to OVA were recorded. Bronchoalveolar lavage fluid (BALF) samples were collected 5 min after OVA challenge, and the histamine and IL-4 contents in the BALF were measured. P815 cells (a mouse mast cell line) were untreated or pretreated with CTCM or cromolyn sodium (a mast cell stabilizer), and incubated with Compound 48/80 (mast cell activator) for 9 hr. The levels of histamine and IL-4 released from the cells were quantified.
Results
We found that the inhibition of bronchoconstriction by the CTCM was attenuated by pretreatment with propranolol, suggesting that the CTCM has a bronchodilator effect that is associated with beta-adrenergic receptor. Our results also showed that the CTCM inhibited histamine and IL-4 secretion in the OVA-induced airway hypersensitivity in guinea pigs at 5 min post-OVA challenge, and in vitro study revealed that the CTCM is able to stabilize mast cells.
Conclusion
In conclusion, our results suggested that the CTCM is a kind of bronchodilator and also a mast cell stabilizer. Our findings provide useful information regarding the possible mechanism of the CTCM, and show its potential for application in the treatment of allergenic airway disease.
doi:10.1186/1472-6882-13-174
PMCID: PMC3716888  PMID: 23849630
Asthma; Nebulization; Drug delivery; Chinese medicine; Ovalbumin
22.  Effects of (R,R)- and (R,R/S,S)-Formoterol on Airway Relaxation and Contraction in an Experimental Rat Model 
Background: Racemic (R,R/S,S)-formoterol is a long-acting β-agonist composed of a 50:50 mixture of (R,R)- and (S,S)-enantiomers.
Objective: The aim of this study was to determine whether (R,R)-formoterol and (R,R/S,S)-formoterol have differing effects on airway contraction and relaxation in vitro.
Methods: Cylindrical airway segments 3-mm long were isolated from the mid-trachea of healthy Sprague-Dawley rats and placed in a modified Krebs-Henseleit solution. Dose-response curves of bethanechol-induced contraction (measured as milligrams of tension) and the concentration of bethanechol that elicited 50% to 75% of maximal contraction (EC50–75) were determined. The air-way cylinders were then precontracted with bethanechol at the EC50–75 and exposed to different concentrations of (R,R)-formoterol (0.0001–1.0 μM) or (R,R/S,S)-formoterol (0.0002–2.0 μM). Each concentration of the 2 formoterol formulations contained the same amount of (R,R)-enantiomer (eg, [R,R]-formoterol 0.0001 μM and [R,R/S,S]-formoterol 0.0002 1JM contained the same amount of [R,R]-enantiomer). The relaxation percentage in response to formoterol was calculated as a reduction in tension (in milligrams) in relation to baseline tension in the precontracted state, with each tracheal cylinder serving as its own control. To determine the effect of (R,R)-formoterol on airway contraction, tracheal cylinders were incubated with (R,R)- or (R,R/S,S)-formoterol before electrical field stimulation (EFS).
Results: Tracheae from 56 three-week-old Sprague-Dawley rats were used in the study. The relaxation percentage of precontracted trachea was significantly greater after exposure to (R,R)-formoterol than to (R,R/S,S)-formoterol at a 2-fold higher concentration (P = 0.03; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in a post hoc analysis, the mean (SE) relaxation percentage of precontracted trachea was significantly greater only after exposure to (R,R)-formoterol 0.01 μM than to (R,R/S,S)-formoterol 0.02 μM (15.6% [5.8%] vs 39.0% [5.6%]; P < 0.05, unpaired t test). EFS-induced airway contraction was significantly less in tracheal cylinders incubated in (R,R)-formoterol compared with those incubated in (R,R/S,S)-formoterol at a 2-fold higher concentration (P = 0.05; general linear model with repeated measures analysis comparing the 2 groups of animals). However, in the post hoc analysis, mean (SE) EFS-induced tracheal contraction was significantly less only in (R,R)-formoterol 0.01 μM compared with (R,R/S,S)-formoterol 0.02 μM at 10 V (1070 [55] mgvs 1225 [28] mg; P < 0.05, unpaired t test).
Conclusion: We found that (R,R)-formoterol may induce greater relaxation of precontracted airway smooth muscle cells than (R,R/S,S)-formoterol and that (R,R)-formoterol may have a greater inhibitory effect on the endogenous cholinergic and excitatory nonadrenergic, noncholinergic contractile airway responses than (R,R/S,S)-formoterol. We speculate that the presence of the (S,S)-enantiomer in (R,R/S,S)-formoterol may impair airway relaxation of pre-contracted trachea in rats.
doi:10.1016/j.curtheres.2007.08.005
PMCID: PMC3967278  PMID: 24683215
(R,R)-formoterol; (R,R/S,S)-formoterol; electrical field stimulation; airway contraction; airway relaxation
23.  The Mast Cell Degranulator Compound 48/80 Directly Activates Neurons 
PLoS ONE  2012;7(12):e52104.
Background
Compound 48/80 is widely used in animal and tissue models as a “selective” mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents.
Methodology/Principal Findings
We used in vivo recordings from extrinsic intestinal afferents together with Ca++ imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca++ and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca++ transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H1 and H2 antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca++ transients in mast cell-free enteric neuron cultures.
Conclusions/Significance
The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn.
doi:10.1371/journal.pone.0052104
PMCID: PMC3525567  PMID: 23272218
24.  Involvement of mast cells in monocrotaline-induced pulmonary hypertension in rats 
Respiratory Research  2011;12(1):60.
Background
Mast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored.
Methods
Pulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies.
Results
There was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling.
Conclusions
The accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT- rats.
doi:10.1186/1465-9921-12-60
PMCID: PMC3104382  PMID: 21535881
25.  Cromoglycate drugs suppress eicosanoid generation in U937 cells by promoting the release of Anx-A1 
Biochemical Pharmacology  2009;77(12):1814-1826.
Graphical abstract
Using biochemical, epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the anti-inflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment. GCs alone produced a rapid (within 5 min) concentration-dependent activation of PKCα/β (Protein Kinase C; EC 2.7.11.13) and phosphorylation of Anx-A1 on Ser27. Both phosphoproteins accumulated at the plasma membrane and Anx-A1 was subsequently externalised thereby inhibiting thromboxane (Tx) B2 generation. When administered alone, cromoglycate or nedocromil had little effect on this pathway however, in the presence of a fixed sub-maximal concentration of GCs, increasing amounts of the cromoglycate-like drugs caused a striking concentration-dependent enhancement of Anx-A1 and PKCα/β phosphorylation, membrane recruitment and Anx-A1 release from cells resulting in greatly enhanced inhibition of TxB2 generation. GCs also stimulated phosphatase accumulation at the plasma membrane of U937 cells. Both cromoglycate and nedocromil inhibited this enzymatic activity as well as that of a highly purified PP2A phosphatase preparation. We conclude that stimulation by the cromoglycate-like drugs of intracellular Anx-A1 trafficking and release (hence inhibition of eicosanoid release) is secondary to inhibition of a phosphatase PP2A (phosphoprotein phosphatase; EC 3.1.3.16), which probably forms part of a control loop to limit Anx-A1 release. These experiments provide a basis for a novel mechanism of action for the cromolyns, a group of drugs that have long puzzled investigators.
doi:10.1016/j.bcp.2009.03.010
PMCID: PMC2888050  PMID: 19428336
Sodium nedocromil; Glucocorticoids; Okadaic acid; PKC; PP2A phosphatase

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