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Biomedical subjects

G J Gleich

Publications and source records attributed to G J Gleich.

At least 91 records · Page 5Linked to original sources
PubMed1998

Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions.

Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied. The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation. To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P. brasiliensis, we used an immunofluorescence technique to localize the P. brasiliensis organisms and eosinophils and major basic protein. Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P. brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed. Nine biopsy tissues from seven patients were analyzed. All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P. brasiliensis. Furthermore, using the polyclonal anti-major basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils). The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P. brasiliensis had extracellular major basic protein deposited on the organisms. These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.

Adult↗
PubMed1997

Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.

In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs.

Acetylcholine↗
PubMed1997

On-line HPLC-tandem mass spectrometry analysis of contaminants of L-tryptophan associated with the onset of the eosinophilia-myalgia syndrome.

The structural characterization of a number of contaminants of L-tryptophan (Trp) associated with eosinophilia myalgia syndrome has been performed for the first time by the powerful structural elucidation technique of tandem mass spectrometry coupled with on-line HPLC (LC-ESI-MS/MS). The identity of the contaminants: peaks UV-5, 3-(phenylamino)alanine, (PAA); E 1,1'-ethylidenebis(tryptophan); 200, 2-(3-indolylmethyl)-L-tryptophan; (all identified as case related) and peaks 1, 3-carboxy-1,2,3,4-tetrahydro-beta-carboline; 2, 3-carboxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline; 100, 2-(2,3 dihydroxy-1-[3-indolyl]propyl)-L-tryptophan; and 300 and 400, diastereomers of 3-carboxy-1-[3-indolyl-methyl]-1,2,3,4-tetrahydro-beta-carboline, have been confirmed by this technique. By comparison of tandem MS (MS/MS) data from these compounds with the MS/MS data of several other impurities, we have structurally characterized peaks CC, KK and OO, as well as two previously unreported components labeled as peak P18 and peak P31. Peak P18 was unresolved from the large Trp peak and has been characterized as indole-3-ethylamine. Peak P31 was previously unresolved from peak 200, a case related compound and therefore its structure is of extreme importance. This compound has been tentatively identified as 2-(3-indolyl)-L-tryptophan.

Chromatography, High Pressure Liquid↗
PubMed1997

Roles of CD9 molecules in survival and activation of human eosinophils.

CD9 is a cell surface glycoprotein belonging to the transmembrane 4 superfamily. In this report, we demonstrate that cross-linking CD9 with different forms of mAb activates distinct functions of human eosinophils. Anti-CD9 mAb (clone ALB6) immobilized onto tissue culture plates induced eosinophil degranulation. This effect of anti-CD9 mAb (clone ALB6) was unique because other immobilized clones of anti-CD9 mAb (clones FMC56 and ML13) or anti-HLA Class I mAb failed to induce degranulation. In addition, neutrophil degranulation was not provoked by the immobilized clone ALB6, consistent with a lack of expression of CD9 on neutrophils. Eosinophil degranulation induced by anti-CD9 mAb (clone ALB6) was abolished by pretreatment of eosinophils with anti-CD18 mAb, suggesting that beta2 integrins are involved in the reaction. In contrast to the results with immobilized clones, all clones of anti-CD9 mAb in soluble form enhanced eosinophil survival. This enhanced survival was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated the enhanced survival. In fact, by ELISA and RT-PCR, GM-CSF protein and mRNA were detected in supernatants and cell lysates of eosinophils stimulated by soluble anti-CD9 mAb, but not by immobilized mAb. These results indicate that CD9 serves as a molecule that delivers stimulation signals on eosinophils. The eosinophil's cellular responses may differ and be dependent on the manner of receptor ligation.

Antigens, CD↗
PubMed1997

Dermal eosinophils in atopic dermatitis undergo cytolytic degeneration.

BACKGROUND: Immunofluorescent staining for eosinophil granule proteins in lesional skin of patients with atopic dermatitis shows extensive extracellular deposition throughout the upper dermis with relatively few intact eosinophils. OBJECTIVE: This study was carried out to determine whether eosinophil granule protein deposition in atopic dermatitis occurs by classical exocytosis, by piecemeal degranulation, or as a result of cytolysis. METHODS: Skin biopsy specimens from 10 patients with atopic dermatitis were examined by electron microscopy. RESULTS: The biopsy specimens showed varying degrees of dermal eosinophil granule major basic protein deposition by indirect immunofluorescence. Specimens from seven patients showed striking alterations of eosinophils by electron microscopy including intact eosinophils with granule alterations (reversal of core staining and/or core lucency) and with uropod processes. Biopsy specimens from six patients showed evidence of eosinophil degeneration with disruption of nuclear and/or plasma membranes. In four patients' specimens, membrane-bound eosinophil granules were present near degenerating eosinophils or were present in the absence of recognizable eosinophils. Evidence of classical exocytotic degranulation was not observed. Two of the specimens were also examined by immunoelectron microscopy for major basic protein localization. In these, major basic protein appeared to be lost from the granule core and distributed in the eosinophil cytoplasm as granules disintegrated and the cell disrupted. CONCLUSION: These findings support the hypothesis that eosinophils undergo cytolysis with release of granule contents and membrane-bound granules; this is likely the usual mechanism of eosinophil granule protein release in atopic dermatitis.

Adult↗
PubMed1997

Increases in cytosolic Ca2+ accompany basophil activation by eosinophil granule major basic protein.

The response of basophil cytosolic free Ca2+ concentrations ([Ca2+]i) to stimulation by eosinophil granule major basic protein (MBP) was assessed using digital video microscopy. MBP stimulated an elevation in basophil [Ca2+]i that, in general, developed slowly and increased monotonically. In some experiments, however, the average rise in [Ca2+]i was biphasic, with an initial transient increase followed by a larger and more sustained elevation. At the single cell level MBP stimulation caused frequent, asynchronous oscillations in [Ca2+]i that ranged up to 300 nM in amplitude. Chelation of extracellular Ca2+ selectively blocked the second, sustained rise in [Ca2+]i as well as MBP-induced histamine release. Within individual experiments, the increase in [Ca2+]i paralleled the level of histamine release. These results demonstrate that, unlike the activation of neutrophils or eosinophils, basophil activation by MBP correlates with an elevation in [Ca2+]i. Further, the MBP induced [Ca2+]i response exhibits several features in common with the IgE-mediated [Ca2+]i response.

Basophils↗
PubMed1997

Localization of eosinophils to airway nerves and effect on neuronal M2 muscarinic receptor function.

Neuronal M2 muscarinic receptors inhibit acetylcholine release from pulmonary parasympathetic nerves but are dysfunctional in antigen-challenged animals and asthmatics. Deletion of pulmonary eosinophils protects M2 receptor function in antigen-challenged guinea pigs. Therefore, the association of eosinophils with airway nerves was investigated. Nerve-associated eosinophils were significantly increased in challenged animals compared with controls (0.75 +/- 0.05 vs. 0.28 +/- 0.05 eosinophils/nerve). In antigen-challenged animals, eosinophil density was greatest around airway nerves, suggesting recruitment to the nerves. M2 receptor function was inversely correlated with the number of eosinophils per nerve, thus eosinophils are associated with airway nerves in antigen-challenged guinea pigs, where they impair M2 receptor function. In airways from three patients with fatal asthma, 196 of 637 eosinophils (30%) were associated with nerves, and release of eosinophil major basic protein was evident; conversely, in three control patients 1 of 11 (9%) eosinophils were in contact with nerves. Thus eosinophils and their granule proteins are also seen in association with airway nerves in patients with asthma.

Acetylcholinesterase↗
PubMed1997

Production of IL-8 and release of eosinophil-derived neurotoxin by normal peripheral blood eosinophils.

To study the regulation of IL-8 production and release of eosinophil-derived neurotoxin (EDN) by normal blood eosinophils, we isolated eosinophils from healthy individuals and stimulated them with immobilized secretory IgA with or without exogenous IL-5 for 3, 12, and 24 h, and with different concentrations of ionomycin for 24 h. Eosinophils cultured with secretory IgA for 3 and 12 h showed strong expression of mRNA for IL-8 by reverse transcription polymerase chain reaction. Exogenous IL-5 enhanced mRNA for IL-8 expression by eosinophils after 24 h of incubation. IL-8 secretion increased in a time-dependent manner throughout the 24 h of observation; in contrast, EDN release reached a plateau value after 12 h. Furthermore, at least 2 microM ionomycin was necessary for induction of IL-8 secretion, whereas 0.5 microM induced detectable EDN release by eosinophils. These results suggest that the mechanism of eosinophil degranulation may be different from those of cytokine secretion.

Dose-Response Relationship, Drug↗
PubMed1997

The immediate and late allergic response to segmental bronchopulmonary provocation in asthma.

The response to antigen is an important factor in the development of airway inflammation. Segmental bronchoprovocation (SBP) with antigen and subsequent bronchoalveolar lavage (BAL) have provided valuable insight into the mechanisms of allergic inflammation. To determine the features of allergic airway response in asthma, 19 subjects with mild asthma underwent antigen SBP in a dose-dependent manner. The amount of antigen used in SBP was 0 (saline), and 1, 5, or 20% of the antigen dose required to drop the FEV1 by 20% (APD20). BAL was done at 5 min and 48 h after SBP. BAL histamine levels increased modestly 5 min after antigen SBP. At 48 h, there was a marked increase in eosinophils and IL-5 concentration even in airway segments where the release of histamine was small. Moreover, eosinophils correlated with IL-5 levels at 48 h (r = 0.63; p < 0.001), but not with BAL histamine concentrations at 5 min. GM-CSF levels did not increase after antigen SBP and did not correlate with eosinophils. These observations indicate that asthmatic subjects can develop a dose-dependent response to antigen SBP that is characterized by a modest increase in histamine immediately after antigen exposure, and marked eosinophilia, which appears proportionately greater than the histamine response and relatively greater than what is seen in allergic nonasthmatic subjects. This feature might be important to the eventual development of airway inflammation in asthma.

Adult↗
PubMed1997

Transmigration of eosinophils through basement membrane components in vitro: synergistic effects of platelet-activating factor and eosinophil-active cytokines.

Migration of eosinophils through the basement membrane barrier is an important step for their infiltration into tissues. To investigate the mechanism of transmigration, we used a chamber fitted with a Matrigel membrane as a model of the basement membrane. In this model, eosinophils treated with cytokines or chemotactic factors alone did not transmigrate from the upper to the lower chamber. However, platelet-activating factor (PAF) strongly induced transmigration of eosinophils stimulated by interleukin (IL)-5, indicating that both a cytokine and a chemotactic factor are required for eosinophil migration through Matrigel. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 also stimulated eosinophil transmigration in the presence of PAF. Of seven eosinophil chemotactic factors tested, leukotriene B4, C5a, RANTES, macrophage inflammatory protein-1alpha, and IL-8 did not induce significant eosinophil transmigration. Only PAF and eotaxin induced transmigration of eosinophils through Matrigel in the presence of IL-5; PAF was more potent than eotaxin at the optimal concentration. In contrast, PAF, eotaxin, and RANTES all potently induced migration of eosinophils through bare membrane in the absence of IL-5. Finally, eosinophil migration through Matrigel was markedly reduced by a combination of anti-CD18 and anti-CD29 monoclonal antibodies, suggesting that it is mediated by beta1- and beta2-integrin adhesion molecules. Our findings demonstrate that eosinophil transmigration through a basement membrane model requires both a specific chemoattractant, such as PAF, and an eosinophil-activating cytokine, such as IL-5. This synergistic effect is likely important in the tissue accumulation of activated eosinophils in allergic and other eosinophil-associated diseases.

Basement Membrane↗
PubMed1997

Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9.

In general, inflammatory cells cross basement membranes by producing proteinases. To investigate the role of proteinases in eosinophil basement membrane migration, we studied peripheral blood eosinophils in Matrigel-coated chemotaxis chambers. Electron microscopy showed degradation of the Matrigel layer when eosinophils, added to the upper chamber, transmigrated the membrane in the presence of both platelet-activating factor (PAF) in the lower chamber and interleukin (IL)-5 in both chambers. In the absence of either or both PAF and IL-5, no changes occurred in the Matrigel layer. Matrigel transmigration of eosinophils induced by PAF and IL-5 was inhibited by 1,10-phenanthroline, batimastat, 3,4-dichloroisocoumarin, chymostatin, and a neutralizing antibody for the matrix metalloproteinase (MMP)-9, indicating that serine proteinase(s) and MMP, specifically MMP-9, were involved in the transmigration of eosinophils through Matrigel. In contrast, eosinophil migration through a bare membrane was not affected by batimastat. Using gelatin zymography and immunoblotting, MMP-9 was detected in the migration upper chamber supernatant of the eosinophil transmigration assay and in the conditioned medium of eosinophils. Release of MMP-9 by eosinophils was increased by IL-5, PAF, or both, but the substrate-degrading activity of MMP-9 was increased only in the presence of both IL-5 and PAF, indicating that the releasing and activating mechanisms of MMP-9 are involved in eosinophil basement membrane migration. This study implicates MMP-9 in basement membrane migration of eosinophils and suggests its involvement in inflammatory diseases where tissue eosinophilia plays a role.

Basement Membrane↗
PubMed1997

Increased eosinophil granule proteins in gut lavage fluid from patients with inflammatory bowel disease.

OBJECTIVE: To explore the potential role of eosinophils in the pathogenesis of inflammatory bowel disease (IBD). DESIGN: We measured the concentrations of eosinophil granule proteins-namely, major basic protein, eosinophil peroxidase, eosinophil cationic protein, and eosinophil-derived neurotoxin-in gut lavage fluid. MATERIAL AND METHODS: Ten healthy persons and 17 patients with IBD (9 with Crohn's disease and 8 with ulcerative colitis) underwent gut lavage. Each study subject submitted an early specimen when lavage effluent became liquid and a late specimen when the output became clear. The concentrations of the granule proteins were measured by immunoassay. RESULTS: The median concentrations of eosinophil-derived neurotoxin and eosinophil cationic protein were significantly higher in patients with IBD than in control subjects for both early and late lavage specimens. Excretion of eosinophil peroxidase was also significantly higher in patients with IBD than in the healthy control subjects, but only in the early specimens. No differences were noted in the concentrations of any of the proteins between patients with ulcerative colitis and those with Crohn's disease. CONCLUSION: Concentrations of eosinophil granule proteins were increased in whole gut lavage fluid from patients with IBD in comparison with healthy control subjects. These results encourage further studies of the role of eosinophils in the pathogenesis of IBD.

Adult↗