Use of the indirect hemagglutination inhibition reaction for evaluating the specific activity of allergens

To evaluate the specific activity of house-dust allergen, the passive hemagglutination inhibition test was used. Nine commercial batches of house-dust allergen were studied by means of this test. The results of the determination of the activity of house-dust allergen in the passive hemagglutination inhibition test, the skin tests and the indirect mast-cell degranulation test were in good correlation.     

Inhibition of NO-synthase and degranulation of rat omental mast cells in vitro

Mast cell amines, platelet-activating factor (PAF), thromboxanes and leukotrienes have been shown to be released during nitric oxide-synthase inhibition in the rat intestine. Mast cells in rat isolated omentum (OMCs) or isolated from the rat peritoneal cavity (PMCs) have been used here to investigate the relationship(s) between these agents. N-nitro-L-arginine methyl ester (L-NAME, 100 muM) caused some degranulation of OMCs, but no enhancement of histamine release from PMCs. PAF (5 muM) and U46619 (1 muM) degranulated OMCs and enhanced histamine release from PMCs. Pre-treatment of the omentum with BN52021 (10 muM) inhibited degranulation of OMCs in response to L-NAME, PAF or U46619. Pretreatment with 1-benzylimidazole (5 or 50 muM) inhibited the effect of L-NAME but not that of PAF. Indomethacin (1 muM) or sodium nitroprusside (10 muM) also inhibited the effects of L-NAME, but nordihydroguaiaretic acid (30 muM) did not. In PMCs BN52021 inhibited PAF-induced, but not U46619-induced, release of histamine. These results suggest that inhibition of nitric oxidesynthase in the omentum by L-NAME allows thromboxanes to release PAF, which in turn degranulates and releases histamine from OMCs.     

Endothelin-1 induces mucosal mast cell degranulation in the rat small intestine

The enhanced production of endothelial cell-derived vasoactive mediators and the activation of mast cells (MCs) have been implicated in the pathogenesis of mucosal damage during ischemia and reperfusion injuries. The first objective of our study was to define the in vivo relation between endothelin-1 (ET-1) and the MC system. Secondly, we determined whether pretreatment with ET receptor antagonists would attenuate MC responses to exogenous ET-1. In the first series of experiments, increasing doses of ET-1 (0. 1, 1 and 3 nmol/kg i.v.) were administered to anesthetized rats. In the second series, the animals were pretreated with equimolar doses of the ET-A receptor antagonist BQ-610 or ETR-P1/fl peptide, and the ET-B receptor antagonist IRL-1038. Intestinal perfusion changes and macrohemodynamics were recorded, and the proportion of degranulated MCs was determined in ileal biopsies. The average mucosal thickness was recorded with an image analysis system. ET-1 induced dose-dependent alterations in the hemodynamic and morphological parameters and caused pronounced mucosal injury, with a significant reduction in villus height. The ratio of degranulated MCs was similar in all ET-treated groups (77%, 82% and 86%) to that observed in animals subjected to 15-min ischemia and 60-min reperfusion (85% degranulation). Pretreatment with BQ-610 and ETR-P1/fl peptide attenuated the ET-1 induced alterations in the hemodynamic parameters and decreased structural injury to the mucosa. ET-induced MC degranulation was significantly inhibited by the ET-A receptor antagonists, but not by IRL-1038. These results indicate that elevated levels of circulating ET-1 might induce intestinal mucosal tissue injury and MC degranulation via activation of ET-A receptors, and raise the possibility that ET-A receptor antagonist administration could exert a potentially beneficial effect through a mechanism other than the blockade of vasoconstriction in pathologies associated with an increased ET-1 release.     

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.     

Role of reactive oxygen species in mast cell degranulation

Mast cells are a heterogeneous multifunctional cellular population that promotes connective tissue homeostasis by slow release of biologically active substances, affecting primarily the permeability of vessels and vascular tone, maintenance of electrolyte and water balance, and composition of the extracellular matrix. Along with this, they can rapidly release inflammatory mediators and chemotactic factors that ensure the mobilization of effector innate immune cells to fight against a variety of pathogens. Furthermore, they play a key role in initiation of allergic reactions. Aggregation of high affinity receptors to IgE (FcεRI) results in rapid degranulation and release of inflammatory mediators. It is known that reactive oxygen species (ROS) participate in intracellular signaling and, in particular, stimulate production of several proinflammatory cytokines that regulate the innate immune response. In this review, we focus on known molecular mechanisms of FcεRI-dependent activation of mast cells and discuss the role of ROS in the regulation of this pathway.     

Disodium cromoglycate stabilizes mast cell degranulation while reducing the number of hemoglobin-induced microvascular leaks in rat mesentery

Blood substitutes, such as diaspirin cross-linked Hb (DBBF-Hb), have been considered for use during blood transfusions. Unfortunately, bolus injection of modified Hb has been shown to rapidly increase the leakage of microvessels to plasma albumin. This effect may result from production of excess reactive oxygen species (ROS) and could be linked to the observed increase in degranulated mast cells (DMC). Disodium cromoglycate (cromolyn) stabilizes mast cells and therefore might minimize the venular permeability in the rat mesentery. In 10 anesthetized Sprague-Dawley rats, the mesenteric preparation was continuously suffused with cromolyn while the microvasculature was filled with DBBF-Hb solution (10 mg/ml) for 10 min. Six animals received cromolyn pretreatment [two intravascular injections over 30 min (experiment A)] and four animals received pretreatment with 2% HEPES-buffered saline (HBS)-BSA (experiment B). Two more animals were pretreated with HBS-BSA without DBBF-Hb infusion but with cromolyn suffusion (experiment C). Another set of experiments was performed on five animals without cromolyn suffusion or any pretreatment but with DBBF-Hb infusion (experiment D). All groups then received a 1-min perfusion of FITC-albumin, fixation for 60 min, and microscopic examination. Experiments A and B demonstrated a significant reduction in the number of venular leaks and DMC compared with experiment D, but not in the area of venular leaks. These results suggest mast cell degranulation is not a major contributor to microvascular leakage induced by DBBF-Hb.     

Mycophenolic acid inhibits the degranulation of rat peritoneal mast cells.

The effect of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase, on the degranulation of rat peritoneal mast cells (RMC) was studied. RMC were pretreated for 48 hr with 0.1-10 microM MPA before the cells were sensitized with IgE and triggered with specific Ag. The net amount of [3H]5-HT released from granules was decreased by 44 and 32% with 1 and 10 microM MPA treatment, respectively. MPA inhibition of degranulation was completely reversed by the addition of 30 microM guanosine to the incubation medium. There was no difference in the apparent number or affinity of IgE binding sites between control and MPA-treated RMC. MPA pretreatment also had no effect on the IgE receptor-mediated production of PGD2 in RMC. These results suggest that depletion of intracellular GTP pools by MPA can disrupt the signaling between the IgE receptor and the secretory granules and that, under these same conditions, the release and metabolism of arachidonic acid are unaffected.     

Use of the passive hemagglutination inhibition reaction for the purpose of determining the antigenic activity of anatoxins in vitro]

The work deals with the study of the possibility of using the passive hemagglutination inhibition test (antibody neutralization) for the determination of the antigenic activity of botulinum toxoids, types A, B and E. Erythrocytic diagnostic preparations were shown to allow the determination of up to 0.1 g of antigen. At the same time in vitro determinations were found to satisfactorily correlate with the results of in vivo determinations of the antigenic activity of these toxoids in the antitoxin-fixation test.     

Cytochemical localization of glycoconjugates in rat peritoneal mast cells during degranulation

Summary Cytochemical methods for the localization of glycoconjugates including concanavalin A-horseradish peroxidase (ConA-HRP) and dialysed iron were used to study the distribution of glycoconjugates in mast cell granules during degranulation. The ConA-HRP method revealed intense staining of discharged mast cell granules. Dialysed iron staining was seen at the granule periphery, with extruded granules exhibiting more intense staining than undischarged granules.Some of the work reported herein was performed in partial fulfillment of the requirements for a Ph.D. degree.     

Alterations in mast cell degranulation and ovarian histamine in the proestrous hamster

Mast cells in the ovary of cyclic hamsters were observed exclusively in the hilum and in the vicinity of blood vessels that enter and exit the ovary. Ovaries were collected on proestrus from hamsters at 0900 h preluteinizing hormone (LH) surge, 1500 h (peak LH surge), and 2100 h (post-LH surge) and processed for routine histologic staining with toluidine blue. A significant increase in the percentage of extensively degranulating mast cells was observed coincident with the gonadotropin surge (0900 h: 5.39 +/- 0.97%; 1500 h: 20.39 +/- 2.76%). At the peak of the LH surge the ovarian histamine concentration was also significantly higher than those before and after the surge (1500 h: 5.13 +/- 0.94 ng/mg ovary; 0900 h and 2100 h: 2.84 +/- 0.35 and 3.02 +/- 0.48 ng/mg, respectively). The results indicate that a major source of ovarian histamine may be mast cells residing in the ovarian hilum and surrounding the ovarian blood vessels that enter and exit the ovary. In addition, the gonadotropin surge on the day of proestrus may be a trigger for release of mast cell histamine.     

Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation

Molecular and Cellular Biochemistry - Calcium ion (Ca2+) uptake into the mitochondrial matrix influences ATP production, Ca2+ homeostasis, and apoptosis regulation. Ca2+ uptake across the...     

The initiation of mast cell degranulation: activation at the cell membrane.

The low molecular weight mast cell activator, polymyxin B, has been covalently bound to an insoluble matrix of Sepharose 4B. It has been demonstrated that mast cells in preparations of rat peritoneal cells bind to Sepharose 4B-polymyxin B beads but not to control beads. The bound cells are stimulated to degranulate by this interaction at the cell membrane with the resultant release of biogenic amines.     

Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

Response of bronchial smooth muscle to mast cell degranulation in situ

We studied the response of bronchial smooth muscle to mast cell degranulation with Ascaris suum antigen (AA) and compound 48/80 (48/80) in 26 mongrel dogs in situ. Bronchial smooth muscle response was measured isometrically in situ from a segment of the right middle lobe bronchus; tracheal response was monitored isometrically as a control. After intra-arterial (ia) injection of AA into the bronchial circulation, bronchial contraction preceded tracheal contraction by 19.2 +/- 4.6 s (P less than 0.002). Bronchial contraction to AA (21.7 +/- 3.4 g) was substantially greater than to 48/80 (10.5 +/- 1.8 g, P less than 0.05) corresponding to differences in maximal systemic histamine concentrations (146 +/- 24.1 vs. 1000 +/- 236 ng/ml, P less than 0.01). In 5 dogs, the effect of leukotriene D4 (LTD4) and FPL 55712 was studied. Injection of 10(-8) mol ia LTD4 caused no bronchial contraction. In four dogs, 10(-7) mol FPL 55712 caused no bronchial relaxation after initial precontraction during immune degranulation with AA; intravenous chlorpheniramine (5 mg/kg) caused 69.7 +/- 9.4% relaxation. We demonstrate a model that permits selective immune degranulation of a single major resistance bronchus in situ. We conclude that AA-induced degranulation in dogs caused bronchial contraction predominantly by secretion of preformed mediator.     

Inhibitory effect of polyphenol-enriched apple extracts on mast cell degranulation in vitro targeting the binding between IgE and FcepsilonRI

Extracts from immature fruit of the apple (Rosaceae, Malus sp.), which contain procyanidins (polymers of catechins) as the major ingredients, are known to inhibit histamine release from mast cells. We analyzed in this study the mechanism for the anti-allergic activity of two polyphenol-enriched apple extracts. These extracts, termed "crude apple polyphenol (CAP)" and "apple condensed tannin (ACT)", reduced the degranulation of mast cells caused by cross-linking of the high-affinity receptor for IgE (FcepsilonRI) with IgE and the antigen in a dose-dependent manner. Furthermore, western blotting revealed that phosphorylation of the intracellular signal-transduction molecules caused by cross-linking of FcepsilonRI was markedly decreased by the addition of CAP or ACT. We then analyzed the effects of CAP and ACT on the binding of the IgE antibody to FcepsilonRI on mast cells, which is the first key step in the allergic reaction mediated by mast cells, and found that this binding was markedly inhibited by both CAP and ACT. These results indicate that the inhibition of binding between FcepsilonRI and IgE by either CAP or ACT was the probable cause of the suppression of mast cell activation. This is the first report demonstrating the molecular mechanism for the anti-allergic effect of procyanidin-enriched extracts from apples.     

Characterization of an antiserum specific for cell surface antigens of rat mast cells.

Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.