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Biomedical subjects

G Marone

Publications and source records attributed to G Marone.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by N-formyl-methionyl-leucyl-phenylalanine.

BACKGROUND: Cyclosporin A (CsA) binds with high affinity to cyclophilin, a critical step in the molecular mechanism of action of cyclosporins, where cyclosporin H (CsH) has extremely low affinity for cyclophilin. CsH differs from CsA by the substitution of the L-methyl valine at position 11 with it D-isomer. METHODS: We compared the effects of CsA and CsH on the release of performed (histamine) and de novo synthesized inflammatory mediators (peptide leukotriene C4) from peripheral blood basophils activated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). RESULTS: CsH (8 to 800 nmol/L) concentration-dependently inhibited histamine and leukotriene C4 release from purified and unpurified basophils activated by FMLP, whereas CsA (8 to 800 nmol/L) had little inhibitory effect on histamine release from basophils challenged with FMLP. Inhibition of histamine release from basophils challenged with FMLP was extremely rapid and was abolished by washing the cells (three times) before challenge. CsH (8 to 800 nmol/L) had no effect on the release of histamine caused by C5a, platelet activating factor, monocyte chemotactic activating factor, RANTES, IL-8, bryostatin 1, and phorbol myristate. Preincubation of basophils with granulocyte-macrophage colony-stimulating factor (30 and 100 pmol/L), but not IL-1 beta (30 and 100 ng/ml), concentration-dependently reversed the inhibitory effect of CsH on FMLP-induced histamine release. CsH competitively inhibited the effect of FMLP on histamine release from basophils. The dissociation constant (Kd) for the CsH-FMLP receptor complex was approximately 9 x 10(-8) mol/L, more than 10-fold lower than that (approximately equal to 1.3 x 10(-6) mol/L) of N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP), a known formyl peptide receptor antagonist. CsH inhibited tritiated FMLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately 5.4 x 10(-7) mol/L, whereas BocMLP was less potent with a concentration required to inhibit binding by 50% of approximately 9.1 x 10(-5) mol/L. Scatchard analysis revealed that the decreased tritiated FMLP binding caused by CsH was due to a decrease in the Bmax (0.22 +/- 0.04 nmol/L/5 x 10(6) cells vs 0.09 +/- 0.01 nmol/L/5 x 10(6) cells; p < 0.05), without a significant difference in the Kd (5.16 +/- 1.22 nmol/L vs 6.32 +/- 2.42 nmol/L; p = NS). CONCLUSION: CsH is a potent and selective inhibitor of mediator release from basophils induced by activation of the formyl peptide receptor; it acts by interfering with agonist binding to FMLP receptors.

Basophils↗
PubMed1996

Human basophil/mast cell releasability. XI. Heterogeneity of the effects of contrast media on mediator release.

BACKGROUND: The activation of basophils and mast cells plays a role in the pathogenesis of anaphylactoid reactions occurring during the administration of iodinated radiocontrast media. METHODS: We compared the effects of three contrast media (CM), Hexabrix (sodium and meglumine salts of ioxaglic acid), Telebrix (sodium and meglumine salts of ioxitalamic acid), and Optiray (ioversol) on the release of preformed (histamine and tryptase) and de novo synthesized (prostaglandin D2 and leukotriene C4) mediators from human basophils and mast cells isolated from lung, skin, and heart tissue. The commercial preparations were evaluated in parallel with the pure substances. Mannitol was used as a positive control inducing histamine release (HR) by hyperosmolar stimulation. RESULTS: Hexabrix (0.1 to 0.3 mol/L), Telebrix (0.1 to 0.5 mol/L), Optiray (0.2 to 0.5 mol/L), and the corresponding pure substances concentration-dependently induced HR from basophils. A positive correlation was found between CM osmolality and HR from basophils. Mast cells isolated from different anatomic sites responded differently to the three CM. Hexabrix and Optiray induced histamine and tryptase release from human lung mast cells, but not from human skin mast cells. No correlation was found between the osmolality of CM and HR from human lung mast cells. There was a significant correlation between the percent of histamine and tryptase release induced by CM from human lung mast cells. Hexabrix, Telebrix, and Optiray also induced histamine and tryptase release from human heart mast cells. None of the CM induced the de novo synthesis of leukotriene C4 or prostaglandin D2 from basophils or mast cells. The kinetics of HR caused by CM differed according to the drug used and the cell (basophils or human lung mast cells) examined. CM-induced HR from basophils and human lung mast cells was temperature-dependent, partially influenced by extracellular Ca2+ concentrations, and not modified by preincubation of basophils with IL-2 or IL-3. CONCLUSIONS: These results provide evidence of the heterogeneity of the effects of CM on mediator release from human basophils and mast cells from different anatomic sites. They also suggest that hyperosmolarity may be an important factor in the activation of basophils by CM, but less relevant for mast cells. CM induce only the release of preformed mediators. The measurement of plasma tryptase might be clinically useful for monitoring adverse reactions caused by CM.

Basophils↗
PubMed1996

Role of mast cells, basophils and their mediators in adverse reactions to general anesthetics and radiocontrast media.

General anesthetics and radiocontrast media (RCM) can cause anaphylactic or anaphylactoid reactions. These are usually underdiagnosed and underreported, but their incidence is apparently rising. Their pathogenesis is complex and not completely understood, but the release of vasoactive mediators from basophils and mast cells plays a central role. The recent development of in vitro techniques to study the release of preformed (histamine and tryptase) and de novo synthesized mediators (PGD2, LTC4, and PAF) from purified basophils and mast cells has made it possible to quantify the mediator-releasing activity of anesthetics such as muscle relaxants, general anesthetics, opioids, and benzodiazepines and RCM on human basophils and mast cells isolated from lung, skin and heart tissues. The majority of general anesthetics and RCM tested induced only the release of preformed mediators (histamine and tryptase), not of the de novo synthesized eicosanoids. There was wide variability in the response of basophils and mast cells from different donors to the same drug or RCM, presumably due to the releasability parameter. Hyperosmolality is probably not the only factor responsible for basophil and mast cell activation by RCM. The in vitro release of histamine induced by anesthetic drugs and RCM was correlated with the release of tryptase. Given the longer half-life of tryptase than histamine in plasma, measurements of plasma tryptase may become a useful diagnostic tool for identifying adverse reactions to anesthetics and RCM.

Anesthetics, General↗
PubMed1996

Oxatomide inhibits the release of proinflammatory mediators from human basophils and mast cells.

Oxatomide (OXA), a histamine H1 receptor antagonist, is effective in the treatment of patients with allergic rhinitis, some allergic skin disorders, and bronchial asthma. We have characterized the effect of OXA on the immunologic release of preformed (histamine and tryptase) and de novo synthesized mediators (leukotriene C4:LTC4 and prostaglandin D2:PGD2) from human basophils and mast cells purified (from 10 to 82%) from human lung parenchyma (HLMC) and skin tissue (HSMC). Preincubation (15 min, 37 degrees C) of basophils with OXA (10(-7)-10(-5) M) before Der p I antigen or anti-IgE challenge concentration-dependently (10-40%) inhibited the immunologic release of histamine and LTC4. OXA (10(-7)-10(-5) M) also inhibited (10-40%) histamine, tryptase and LTC4 release from HLMC activated by anti-IgE. In addition, OXA caused a concentration-dependent inhibition of histamine, tryptase and PGD2 release from HSMC immunologically challenged with a monoclonal antibody against the alpha chain of the high affinity receptor for IgE (anti-Fc epsilon RI) or anti-IgE. These results demonstrate that OXA exerts anti-inflammatory activities by inhibiting the release of preformed and de novo synthesized mediators from human basophils and mast cells.

Adult↗
PubMed1995

Migration of human inflammatory cells into the lung results in the remodeling of arachidonic acid into a triglyceride pool.

Increasing evidence suggests that the metabolism of arachidonic acid (AA) may be different in inflammatory cells isolated from blood or migrating into tissues. To explore the possibility that changes in AA metabolism between blood and tissue inflammatory cells could be due in part to a different content or distribution of AA in glycerolipid classes, we studied these parameters in six human inflammatory cells isolated from blood (eosinophils, monocytes, neutrophils, and platelets) or from the lung tissue (mast cells and macrophages). Lung cells generally had a higher total cellular content of AA than that found in the blood cells. In addition, both mast cells and macrophages had a large endogenous pool of AA associated with triglycerides (TG), containing 45 and 22% of their total cellular AA, respectively. To address the hypothesis that cells migrating into the lung had a higher cellular level of AA and a larger AA pool in TG, we studied neutrophils isolated from the bronchoalveolar lavage (BAL) of patients with adult respiratory distress syndrome. BAL neutrophils had a fourfold increase in cellular AA as compared with blood neutrophils and contained 25% of their AA in TG versus 3% in blood neutrophils. BAL neutrophils also had a higher number of cytoplasmic lipid bodies (8 +/- 3/cell) relative to blood neutrophils (2 +/- 1/cell). High concentrations of free AA were also found in the cell-free BAL fluid of adult respiratory distress syndrome patients. To explore whether changes in BAL neutrophils may be due to the exposure of the cells to high concentrations of exogenous AA found in BAL, we incubated blood neutrophils in culture with AA (10-100 microM) for 24 h. Neutrophils supplemented with AA had a 10-fold increase in the amount of AA associated with TG and a sixfold increase in the number of lipid bodies. In addition, supplementation with AA induced a dose-dependent formation of hypodense cells. Taken together, these data indicate that human inflammatory cells undergo a fundamental and consistent remodeling of AA pools as they mature or enter the lung from the blood. These biochemical and morphological changes can be mimicked in vitro by exposing the cells to high levels of AA. This mechanism may be responsible for the changes in AA mobilization and eicosanoid metabolism observed in tissue inflammatory cells.

Adult↗
PubMed1995

Human skin mast cells express functional beta 1 integrins that mediate adhesion to extracellular matrix proteins.

We have evaluated the adhesion of human cutaneous mast cells to several components of the extracellular matrix (plasma fibronectin, laminin, collagen type I and IV) and studied whether these cells express the beta 1 integrins potentially involved in the adhesion to these proteins. Human skin mast cells (5.1 +/- 1.5% pure) spontaneously adhered to fibronectin and laminin (0.1 to 10 micrograms/ml) immobilized on plastic surfaces (e.g., 14 +/- 7.2% and 14 +/- 4.4% adhesion at 10 micrograms/ml, respectively). Similar results were obtained with a 90% pure mast cell preparation. In contrast, cutaneous mast cells did not adhere to collagen type I (1.6 +/- 0.5% adhesion) or type IV (1.2 +/- 0.8% adhesion). Control adhesion in BSA-coated wells was < 5%. Mast cell adhesion to fibronectin was optimal after an incubation period of 60 to 90 min (t1/2 = 28.2 +/- 6.2 min), whereas adhesion to laminin was faster (t1/2 = 10.1 +/- 1.2 min), being nearly optimal after a 15-min incubation period. Human skin mast cell adhesion to fibronectin and laminin was found to be dependent on the presence of divalent cations in the extracellular medium. Dual-color immunofluorescence and flow cytometry were used to evaluate whether human skin mast cells (51.3 +/- 3.9% pure) express beta 1 integrins that may mediate cell adhesion to extracellular matrix proteins. These mast cells were found to express VLA (very late Ag)-3 (75.3 +/- 35.6 specific fluorescence intensity) and, to lesser degree, VLA-4 and VLA-5 receptors (8.0 +/- 2.5 and 6.9 +/- 3.2 specific fluorescence intensity, respectively). In contrast, VLA-1, VLA-2, and VLA-6 integrins were not expressed significantly. mAb to VLA-3, VLA-4, and VLA-5 each inhibited by 70% skin mast cell adhesion to fibronectin. mAb to VLA-3 nearly abolished mast cells adhesion to laminin, whereas anti-VLA-4 and anti-VLA-5 were ineffective. Finally, immunosuppressant cyclosporin A (100 nM) and FK-506 (10 nM) significantly inhibited mast cell adhesion to both fibronectin and laminin (p < 0.05). Our data demonstrate that human skin mast cells spontaneously adhere to fibronectin and laminin, and that this adhesion is mediated by VLA-3, VLA-4, and/or VLA-5 integrins on these cells. Interactions between these beta 1 integrins and extracellular matrix proteins may be involved in perivascular tissue localization of human mast cells in vivo.

Cations, Divalent↗
PubMed1995

Human heart mast cells. Isolation, purification, ultrastructure, and immunologic characterization.

We have isolated, partially purified, and characterized the mast cells from human heart tissue. The histamine content of left and right ventricles and septum of hearts obtained from 25 patients undergoing heart transplantation was 5.4 +/- 0.6, 5.3 +/- 0.5, and 5.6 +/- 0.5 micrograms/g of wet tissue, respectively. Ultrastructural study of cardiac mast cells revealed scroll, crystal, and mixed granules, homogeneously dense granules, and lipid bodies in the cytoplasm. A mild collagenase digestion was used to disperse the heart mast cells; the average yield was 3.2 +/- 0.6% (range: 0.8 to 13.6%). The average histamine and tryptase content/heart mast cells was 3.3 +/- 0.2 pg (n = 25) and 24.2 +/- 4.3 micrograms/10(6) cells (n = 11), respectively. Survival of cardiac mast cells after overnight culture was 71.9 +/- 5.4% (n = 23). The purification of human heart mast cells can be brought from less than 0.1 to 12% by a combination of low-speed centrifugation over albumin (2%) solution and Percoll gradient. Viability as shown by trypan blue exclusion was greater than 90%. Heart mast cells released histamine in response to immunologic (anti-IgE, anti-Fc epsilon RI, and C5a) and nonimmunologic stimuli (recombinant human stem cell factor, A23187, and compound 48/80) but did not respond to substance P, FMLP, 12-O-tetradecanoylphorbol-13-acetate, or acetylcholine. There was a linear correlation between the percentage of release caused by anti-IgE and anti-Fc epsilon RI, whereas there was no correlation between the release caused by C5a and anti-IgE-mediated stimuli. Cross-linking with anti-IgE of IgE on heart mast cells induced the release of tryptase (10.1 +/- 2.1 micrograms/10(7) cells; n = 10) and the de novo synthesis of PGD2 (17.3 +/- 4.3 ng/10(6) cells; n = 10) and of leukotriene C4 (19.1 +/- 4.5 ng/10(6) cells; n = 10). There was a linear correlation between the percentage of histamine secretion and tryptase release (r = 0.67; p < 0.001) induced by cross-linking of Fc epsilon RI. similarly, there was a significant correlation between percentage of histamine secretion and PGD2 (r = 0.63; p < 0.001) and LTC4 (r = 0.64; p < 0.001) release. Immunoelectron microscopy demonstrated the presence of chymase in cardiac mast cells. Mast cells isolated from human heart can be a useful model with which to study the role of these cells and their mediators in cardiac anaphylaxis and cardiovascular diseases.

Cell Separation↗
PubMed1995

The antineoplastic bryostatins affect human basophils and mast cells differently.

Bryostatins, macrocyclic lactones from the marine bryozoan Bugula neritina, are potent antineoplastic agents and multi-potential stimulators of immune cells. We have examined the effects of bryostatins on mediator release from human basophilic leukocytes and human tissue mast cells. Bryostatins 1, 2, and 5 (10 to 3,000 nmol/L) induced histamine secretion from purified and unpurified peripheral blood basophils, whereas they caused no release of peptide-leukotriene C4 from these cells. The rate of histamine release caused by bryostatin 1 was slower than that caused by anti-IgE (t1/2 +/- SEM = 38.2 +/- 4.7 minutes v 8.9 +/- 0.2 minutes; P < .01), whereas the temperature dependence was similar (optimum release at 37 degrees C, approximately 30% less at 30 degrees C, and no release at 22 degrees C or 4 degrees C). The addition of increasing concentrations of extracellular Ca2+ to the medium caused histamine release in the presence of bryostatins. Subeffective concentrations of bryostatins and anti-IgE produced a synergistic effect on histamine release from basophils. Staurosporine, chelerythrine, and calphostin C (0.1 to 10 nmol/L), which are protein kinase C inhibitors, inhibited the histamine secretion activated by bryostatin 1 and tetradecanoylphorbol-acetate (TPA). Preincubation with granulocyte-monocyte colony-stimulating factor (GM-CSF; 1 and 5 nmol/L) and interleukin-3 (IL-3; 10 ng/mL) potentiated the activation of human basophils induced by bryostatin 1. Neither bryostatin 1 nor bryostatin 2 induced the release of histamine from mast cells isolated from human lung or skin tissues. However, brief (10 minutes) preincubation with bryostatin 1 (3 to 300 nmol/L) potently inhibited the histamine secretion induced by anti-IgE from skin or lung mast cells. Bryostatin 1 was a more potent (by approximately 30 times) inhibitor of IgE-mediated histamine release than was TPA. The heterogeneous effects exerted by bryostatins on human basophils and mast cells can be of interest for those designing therapeutic trials using these agents.

Antibodies, Anti-Idiotypic↗
PubMed1995

Clinical advances in mastocytosis.

Mastocytosis is a disease characterized by an abnormal proliferation of tissue mast cells. The events primarily responsible for mast cell proliferation in mastocytosis are largely unknown, but a derangement of the network involving c-kit receptor and its natural ligand (stem cell factor, which promotes mast cell growth and differentiation in man) is likely to have a primary role in this disease. Mastocytosis comprises a wide spectrum of clinical conditions determined by the degree of mast cell proliferation, the organ systems involved, the age at onset and the association with hematologic diseases. Mastocytosis can occur in a pediatric or an adult form. In both groups of patients, the disease may be limited to the skin (cutaneous mastocytosis) or be systemic, involving predominantly the bone marrow and the gastrointestinal tract. The symptoms in patients with mastocytosis are generally related to the increased release of mast-cell-derived mediators, such as histamine, prostaglandin D2, peptide leukotrienes, platelet-activating factor, heparin and proteolytic enzymes. The measurement of these chemical mediators (histamine, tryptase and prostaglandin D2 and their metabolites) in body fluids is useful for the diagnosis and the laboratory evaluation of patients with systemic mastocytosis. As little is known about the pathogenesis of the different forms of mastocytosis, the treatment of the majority of these patients is largely symptomatic.

Humans↗
PubMed1995

Immunological characterization and functional importance of human heart mast cells.

Mast cells are present in normal and even more abundant in diseased human heart tissue and their localization is of particular relevance to their function. Within heart tissue mast cells lie between myocytes and in close contact with blood vessels. They are also found in the coronary adventitia and in the shoulder regions of a coronary atheroma. The density of cardiac mast cells is markedly higher in some patients with myocarditis and dilated cardiomyopathy than in accident victims without cardiovascular diseases. More importantly, in some of these conditions there is in situ evidence of mast cell activation. We have described an original technique to isolate and purify HHMC for in vitro study. This procedure gives viable cells and after stimulation with immunological or non-immunological stimuli they release performed (histamine and tryptase) and newly generated mediators (PGD2 and LTC4). We have demonstrated that HHMC differ from those in other anatomical districts in that they are activated by specific immunological and non-immunological stimuli, and in their relation to the arachidonic acid metabolism, suggesting that the local microenvironment can influence their phenotypic and biochemical characteristics. Our own and other findings suggest that HHMC have complex and significant roles in different pathophysiological conditions involving the cardiovascular system. Direct activation of HHMC by therapeutic and diagnostic substances injected intravenously explains some of the anaphylactoid reactions caused by these agents. HHMC possess Fc epsilon RI and IgE bound to the surface and C5a receptors, which could explain how cardiac mast cells are involved in systemic and cardiac anaphylaxis. Cardiac mast cells and those in human coronary arteries also play a role in the early and late stages of atherogenesis and during ischemic myocardial injury. In conclusion, although studies of HHMC are in their infancy, their in vitro isolation may be useful in identifying additional mediators synthesized and released, stimuli relevant to human pathophysiology, and pharmacological agents selectively modulating the activation of these cells and their mediators. Drugs specifically acting on HHMC or on their mediators may eventually be useful in treating different cardiovascular diseases.

Anaphylaxis↗
PubMed1995

Bryostatin 1 enhances lymphokine activated killer sensitivity and modulates the beta 1 integrin profile of cultured human tumor cells.

Bryostatin 1 interferes with protein kinase C (PKC) signaling which is involved in the activation of human and murine cytotoxic T lymphocytes, and in the growth and differentiation of tumor cells. Bryostatin 1 has immunomodulating and antitumor properties as demonstrated by preclinical and clinical studies. Here we report that bryostatin 1 increases the susceptibility to lymphokine activated killers and modifies the pattern of beta 1 integrin expression of human tumor cells. On the basis of these results the use of bryostatin 1 in combination with immunostimulating cytokines such as interleukin-2 in the treatment of human cancer is suggested.

Adjuvants, Immunologic↗
PubMed1995

Human heart mast cells: a definitive case of mast cell heterogeneity.

Mast cells and their chemical mediators play a role in cardiac and systemic anaphilaxis. Perivascular and cardiac mast cells have been implicated in the pathogenesis of coronary artery spasm, atherosclerosis, myocardial ischemia, and cardiomyopathy. Despite this, nothing is known about the immunological and biochemical characteristics of the human heart mast cell (HHMC). We have isolated and partially purified HHMC and compared them with mast cells isolated from lung (HLMC) and skin (HSMC) tissues. Cross-linking of the high-affinity receptor for IgE (Fc epsilon RI) by a polyclonal anti-Fc epsilon antibody caused the release of preformed (histamine and tryptase) and de novo synthesized mediators [peptide leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)]. The tryptase content of HHMC (19.4 +/- 1.5 micrograms/10(6) cells) was lower than HSMC (33.4 +/- 2.5 micrograms/10(6) cells) and higher than HLMC (10.6 +/- 1.9 micrograms/10(6) cells). Maximal stimulation of HHMC with anti-IgE led to the release of LTC4 (17.5 +/- 5.1 ng/10(6) mast cells) and PGD2 (17.8 +/- 5.0 ng/10(6) mast cells, whereas HSMC synthesized more PGD2 (65.0 +/- 6.8 ng/10(6) mast cells) and much less LTC4 (< 5 ng/10(6) cells). Recombinant human C5a anaphylatoxin and protamine induced histamine release from HHMC and HSMC, but not from HLMC. Substance P and morphine selectively induced the release of histamine from HSMC, but not from HHMC and HLMC. Compound 48/80 caused histamine release from HSMC and HHMC, but not from HLMC. The pattern of mediators synthesized and the responsiveness of HHMC to different secretagogues appear unique providing strong evidence of human mast cell heterogeneity.

Antibodies, Anti-Idiotypic↗
PubMed1994

Differential roles for triglyceride and phospholipid pools of arachidonic acid in human lung macrophages.

Arachidonic acid (AA) incorporation into and release from cellular glycerolipids are thought to be crucial events for the regulation of eicosanoid biosynthesis in inflammatory cells. The goal of our study was to determine the distribution of AA in the lipid pools of human lung macrophages (HLM) isolated from human lung parenchyma and to define the changes in AA pools occurring during cell activation. Mass spectrometry analysis indicated that the major pools of AA in HLM were located in phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triglycerides (TG), and, to a lesser extent, in phosphatidylinositol/phosphatidylserine (PI/PS) and in a phospholipid similar to bis-(monoacylglyceryl)-phosphate (BMP). Exogenous AA was initially incorporated into TG and subsequently distributed within phospholipids. After 24 h of labeling, the distribution of exogenous AA in glycerolipid classes closely mimicked that of endogenous AA. Under these equilibrium conditions, HLM released 18.7 and 20.2% of cellular AA when stimulated with TPA or A23187, respectively. Free AA was the major product released by TPA- or A23187-stimulated HLM. However, A23187 but not TPA also induced the formation of leukotriene B4 and 5-HETE. AA was released from PC > PI/PS > or = PE > BMP. In contrast to phospholipids, the amount of AA in TG increased 15 to 90 min after cell activation and returned to prestimulation levels after 180 min. These data indicate that AA is stored in several glycerolipid pools of HLM. Among these pools, BMP is unique of macrophages and TG are particularly enriched in AA in HLM. During the early stage of cell activation, phospholipids act as a source of AA, whereas TG function as a reacylation pool for AA released from phospholipids. These data indicate that TG and phospholipid pools have a differential role in the control of free AA levels generated during HLM activation.

Arachidonic Acid↗