Adenosine induces histamine release from human bronchoalveolar lavage mast cells

Previous studies have shown that in vitro adenosine enhances histamine release from activated human lung mast cells obtained by enzymic dispersion of lung parenchyma. However, adenosine alone has no effect on histamine release from these cells. Given the evidence for direct activation of mast cells after endobronchial challenge with adenosine and previous studies indicating that mast cells obtained at bronchoalveolar lavage are a better model for asthma studies than those obtained by enzymic dispersion of lung tissue, the histamine-releasing effect of adenosine was examined on lavage mast cells. Bronchoalveolar lavage fluid was obtained from patients attending hospital for routine bronchoscopy (n = 54). Lavage cells were challenged with adenosine or adenosine receptor agonists (20 min, 37 degrees C) and histamine release determined using an automated fluorometric assay. Endogenous adenosine levels were also measured in lavage fluid (n = 9) via an HPLC method. Adenosine alone caused histamine release from ravage mast cells in 37 of 54 patients with a maximal histamine release of 20.56 +/- 2.52% (range 5.2-61 %). The adenosine receptor agonists (R)-N-6-(2-phenylisopropyl)adenosine, 5'-N-ethylcarboxamido-adenosine and CGS21680 also induced histamine release from lavage mast cells. Preincubation of lavage mast cells with the adenosine receptor antagonist xanthine amine congener caused significant inhibition of the response to adenosine (P = 0.007). There was an inverse correlation between endogenous adenosine levels in the lavage fluid and the maximal response to in vitro adenosine challenge of the lavage cells. The findings of the present study indicate a means by which adenosine challenge of the airways can induce bronchoconstriction and support a role for adenosine in the pathophysiology of asthma. The results also suggest that cells obtained from bronchoalveolar ravage fluid may provide the ideal model for the testing of novel, adenosine receptor, targeted therapies for asthma.

Histamine release from bronchoalveolar lavage cells from asthmatic subjects after allergen challenge and relationship to the late asthmatic response

Background


Metachromatic cells obtained from asthmatic subjects demonstrate increased spontaneous and stimulated histamine release in vitro. Their ability to synthesize and store proinflammatory cytokines has focused renewed interest on their role in asthma.


Objective: The late asthmatic response provides a useful model of clinical asthma. The aim of the study was to examine metachromatic cell derived mediators and histamine releasability in vitro after in vivo allergen exposure in atopic subjects with and without asthma and relate them to the type of physiological response observed.

Methods: Bronchoalveolar lavage (BAL) cells were obtained 4 h after challenge from asthmatics exhibiting a single early response (EAR, n = 5), a dual response (LAR, n = 7), unchallenged (basal, n = 5), atopic non-asthmatic (ANA, n = 6) and non-atopic non-asthmatics (normal, n = 5). BAL histamine and tryptase concentrations and in vitro histamine release (HR) after stimulation with anti-IgE, allergen, A23187, conconavalin A and substance P were compared.

Results:Metachromatic cell numbers were lower in normal controls compared with all asthmatic groups and in LAR compared with EAR. Metachromatic cell derived mediators were higher in asthmatic compared with normal subjects. Spontaneous HR in LAR (20.5 ± 5.0%) was lower than EAR (29.5 ± 3.9%) and ANA (30.2 ± 1.4%) (P < 0.05). No differences were seen in stimulated HR between EAR and LAR. HR in ANA stimulated with anti-IgE was greater than LAR (P < 0.05). HR in ANA stimulated with anti-IgE was greater than LAR (P < 0.05). After stimulation with ionophore A23187 (1 μM), release was greater in LAR compared with basal (P < 0.05) and no different at 5 μM. All subject groups responded to substance P (SP) but was significantly more in the asthmatic subjects compared to normal controls (P < 0.05). Allergen challenge did not modify the response of asthmatic subjects to SP.

Conclusion: Functional differences in metachromatic cell reactivity are present in atopic subjects 4 h after in vivo allergen exposure which relate to the physiological response observed after this time and suggest that there is ongoing metachromatic cell degranulation subjects who subsequently develop LAR.

Substance P induces histamine release from human pulmonary mast cells.

Substance P elicits histamine release from human skin and rodent mast cells. Since neuropeptide-mediated reflexes may be important in asthma, we examined the ability of substance P to stimulate human mast cells obtained at bronchoalveolar lavage (BAL). BAL samples were obtained at routine bronchoscopy from 35 non-preselected patients. Histamine release experiments were performed in a standard manner using substance P and the calcium ionophore A23187. Both substance P (50 μM) and A23187 caused histamine release (median 26.7%, range 6.2–62.8% and 32.1%, 7.7–56.8% respectively) which was significantly greater (P < 0.0001) than the spontaneous release (median 15.6%, range 4.1–33.4%), i.e. that in the absence of any stimulus. Substance P induced histamine release was via an energy dependent process and was blocked by preincubation with antimycin A. A significant correlation was observed between substance P induced release and spontaneous release but was not observed with A23187 induced release. Mast cell counts correlated significantly with substance P induced release but not with spontaneous or A23187 induced release. The substance P induced histamine secretion was elicited at similar concentrations to those used with rodent and human skin mast cells. Asthma is associated with increased numbers of mast cells which have both increased spontaneous and stimulated secretory responses. Thus, in vivo, the bronchoconstrictor action of substance P may in part result from activation of mast cells in the bronchial lumen.

Post-release GPS tracking of hand-reared Irish hare Lepus timidus hibernicus leverets, Slemish, Co. Antrim, Northern Ireland.

Animal rescue centres release large numbers of captive-bred, rehabilitated or translocated animals into the wild annually but little is known about their post-release survival and behaviour. We developed a novel and innovative coupling of traditional radio-tags with new GPS loggers to track hand-reared Irish hare Lepus timidus hibernicus leverets after release into the wild. Cyanoacrylate SuperGlue® proved a poor fixative with two out of three leverets managing to detach their tags within 24 hours. Nevertheless, a total of 2,505 GPS locations were recorded every 60 seconds for one leveret over three nights (approx. 835 per night). The leveret dispersed

Modulation of gel formation and drug-release characteristics of lidocaine-loaded poly(vinyl alcohol)-tetraborate hydrogel systems using scavenger polyol sugars

Polyol sugars, displaying a plurality Of hydroxyl groups, were shown to modulate tetra hydroxyborate (borate) cross-linking in lidocaine hydrochloride containing poly(vinyl alcohol) scini-solid hydrogels. Without polyol, demixing of borate cross-linked PVA hydrogels into two distinct phases was noticeable upon lidocaine hydrochloride addition, preventing further use as a topical System. D-Mannitol incorporation was found to be particularly suitable in cicumventing network constriction induced by ionic and pH effects upon adding the hydrochloride salt of lidocaine. A test formulation (4% w/v lidocaine HCl, 2% W/V D-mannitol, 10% w/v PVA and 2.5%, w/v THB) was shown to constitute an effective delivery system, which was characterised by an initial burst release and a drug release mechanism dependent on temperature, changing from a diffusion-controlled system to one with the properties of a reservoir system. The novel flow properties and innocuous adhesion of PVA-tetrahydroxyborate hydrogels Support their application for drug delivery to exposed epithelial surfaces, Such as lacerated wounds. Furthermore, addition of a polyol, such as mannitol, allows incorporation of soluble salt forms of active therapeutic agents by modulation of cross-linking density. (C) 2008 Elsevier B.V. All rights reserved.

Endothelin 1 Stimulates Ca2+-Sparks and Oscillations in Retinal Arteriolar Myocytes via IP3R and RyR-Dependent Ca2+ Release

PURPOSE:
To investigate endothelin 1 (Et1)-dependent Ca(2+)-signaling at the cellular and subcellular levels in retinal arteriolar myocytes.
METHODS:
Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using confocal laser microscopy.
RESULTS:
Basal [Ca(2+)](i), subcellular Ca(2+)-sparks, and cellular Ca(2+)-oscillations were all increased during exposure to Et1 (10 nM). Ca(2+)-spark frequency was also increased by 90% by 10 nM Et1. The increase in oscillation frequency was concentration dependent and was inhibited by the EtA receptor (Et(A)R) blocker BQ123 but not by the EtB receptor antagonist BQ788. Stimulation of Ca(2+)-oscillations by Et1 was inhibited by a phospholipase C blocker (U73122; 10 µM), two inhibitors of inositol 1,4,5-trisphosphate receptors (IP(3)Rs), xestospongin C (10 µM), 2-aminoethoxydiphenyl borate (100 µM), and tetracaine (100 µM), a blocker of ryanodine receptors (RyRs).
CONCLUSIONS:
Et1 stimulates Ca(2+)-sparks and oscillations through Et(A)Rs. The underlying mechanism involves the activation of phospholipase C and both IP(3)Rs and RyRs, suggesting crosstalk between these Ca(2+)-release channels. These findings suggest that phasic Ca(2+)-oscillations play an important role in the smooth muscle response to Et1 within the retinal microvasculature and support an excitatory, proconstrictor role for Ca(2+)-sparks in these vessels.

Sustained release of proteins from a modified vaginal ring device

A new vaginal ring technology, the insert vaginal ring (InVR), is presented. The InVR overcomes the current shortfall of conventional vaginal rings (VRs) that are generally ineffectual for the delivery of hydrophilic and/or macromolecular actives, including peptides, proteins and antibodies, due to their poor permeation characteristics in the hydrophobic polymeric elastomers from which VRs are usually fabricated. Release of the model protein BSA from a variety of insert matrices for the InVR is demonstrated, including modified silicone rods, directly compressed tablets and lyophilised gels, which collectively provided controlled release profiles from several hours to beyond 4 weeks. Furthermore, the InVR was shown to deliver over 1 mg of the monoclonal antibody 2F5 from a single device, offering a potential means of protecting women against the transmission of HIV.