Biomedical subjects

Gerald J Gleich

Publications and source records attributed to Gerald J Gleich.

31 records · Page 2Linked to original sources

PubMed2003

Regulation of eosinophil membrane depolarization during NADPH oxidase activation.

Protein kinase C (PKC) activation in human eosinophils increases NADPH oxidase activity, which is associated with plasma membrane depolarization. In this study, membrane potential measurements of eosinophils stimulated with phorbol ester (phorbol 12-myristate 13-acetate; PMA) were made using a cell-permeable oxonol membrane potential indicator, diBAC4(3). Within 10 minutes after PMA stimulation, eosinophils depolarized from -32.9+/-5.7 mV to +17.3+/-1.8 mV. The time courses of depolarization and proton channel activation were virtually identical. Blocking the proton conductance with 250 microM ZnCl2 (+43.0+/-4.2 mV) or increasing the proton channel activation threshold by reducing the extracellular pH to 6.5 (+44.4+/-1.4 mV) increased depolarization compared with PMA alone. Additionally, the protein kinase C (PKC) delta-selective blocker, rottlerin, inhibited PMA-stimulated depolarization, indicating that PKCdelta was involved in regulating depolarization associated with eosinophil NADPH oxidase activity. Thus, the membrane depolarization that is associated with NADPH oxidase activation in eosinophils is sufficient to produce marked proton channel activation under physiological conditions.

Cell Membrane↗

PubMed2003

Effect of eosinophil adhesion on intracellular signaling in cholinergic nerve cells.

Eosinophil localization to cholinergic nerves occurs in a variety of inflammatory conditions, including asthma. This localization is mediated by interactions between eosinophil integrins and neuronal vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Eosinophil-nerve cell interactions lead to generation of neuronal reactive oxygen species and release of eosinophil proteins. The effects of eosinophil adhesion on neuronal intracellular signaling pathways were investigated. Eosinophil adhesion to IMR32 cholinergic nerves led to a rapid and sustained activation of the nuclear transcription factors nuclear factor (NF)-kappaB and activator protein (AP)-1 in the nerve cells. Eosinophil binding to neuronal ICAM-1 led to a rapid activation of ERK1/2 in nerve cells. Inhibition of ERK1/2 prevented NF-kappaB activation. Eosinophil adhesion to VCAM-1 resulted in AP-1 activation, mediated partially by rapid activation of the p38 mitogen-activated protein kinase. These data show that adhesion of eosinophils induces mitogen-activated protein kinase-dependent activation of the transcription factors NF-kappaB and AP-1 in nerve cells, indicating that eosinophil adhesion may control nerve growth and phenotype.

Acetylcholine↗

PubMed2003

Effects of eosinophils on nerve cell morphology and development: the role of reactive oxygen species and p38 MAP kinase.

The adhesion of eosinophils to nerve cells and the subsequent release of eosinophil products may contribute to the pathogenesis of conditions such as asthma and inflammatory bowel disease. In this study we have separately examined the consequences of eosinophil adhesion and degranulation for nerve cell morphology and development. Eosinophils induced neurite retraction of cultured guinea pig parasympathetic nerves and differentiated IMR32 cholinergic neuroblastoma cells. Inhibition of eosinophil adhesion to IMR32 cells attenuated this retraction. Eosinophil adhesion to IMR32 cells led to tyrosine phosphorylation of a number of nerve cell proteins, activation of p38 MAP kinase, and generation of neuronal reactive oxygen species (ROS). Inhibition of tyrosine kinases with genistein prevented both the generation of ROS in the nerve cells and neurite retraction. The p38 MAP kinase inhibitor SB-239063 prevented neurite retraction but had no effect on the induction of ROS. Thus eosinophils induced neurite retraction via two distinct pathways: by generation of tyrosine kinase-dependent ROS and by p38 MAP kinase. Eosinophils also prevented neurite outgrowth during differentiation of IMR32 cells. In contrast to their effect on neurite retraction, this effect was mimicked by medium containing products released from eosinophils and by eosinophil major basic protein. These results indicate that eosinophils modify the morphology of nerve cells by distinct mechanisms that involve adhesion and released proteins.

Animals↗

PubMed2003

Interactions of eosinophil granule proteins with skin: limits of detection, persistence, and vasopermeabilization.

BACKGROUND: Eosinophil granule proteins, including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO), and major basic protein (MBP), are prominently deposited in skin in several cutaneous disorders and likely contribute to disease pathology. OBJECTIVE: We sought to determine the limit of detection, persistence, and vasopermeabilization activity of the eosinophil granule proteins in skin. METHODS: The eosinophil granule proteins were injected intradermally. Their minimum detectable concentrations in human surgical waste skin and their persistence in guinea pig skin were determined by indirect immunofluorescence. Vasopermeabilization activity in the guinea pig without and with H1 antihistamine (pyrilamine maleate) pretreatment was assessed by extrusion of Evans blue dye-treated plasma. RESULTS: The lowest detectable cutaneous concentrations were 0.05 micromol/L EPO, 0.1 micromol/L MBP, 0.25 micromol/L ECP, and 1 micromol/L EDN. Granule proteins persisted in guinea pig skin in vivo for 1 week (EPO), 2 weeks (ECP), 2.5 weeks (EDN), and 6 weeks (MBP). Each of the eosinophil granule proteins increased cutaneous vasopermeability in a concentration-dependent manner. The potency of vasopermeabilization induced by each granule protein was comparable with that of histamine. Pyrilamine maleate pretreatment of guinea pigs did not alter increased vasopermeability induced by ECP and EDN but significantly inhibited that induced by EPO and MBP. CONCLUSIONS: Micromolar concentrations of eosinophil granule proteins are often deposited in skin in eosinophil-associated cutaneous disorders such as atopic dermatitis. These pathophysiologically relevant concentrations of eosinophil granule proteins cause increased cutaneous vasopermeability (both by means of histamine-independent and histamine-dependent mechanisms) and might alter cutaneous function for days to weeks.

Animals↗

PubMed2003

Localization and cellular distribution of pregnancy-associated plasma protein-a and major basic protein in human ovary and corpora lutea throughout the menstrual cycle.

OBJECTIVE: To assess the expression and cellular distribution of pregnancy-associated plasma protein-A (PAPP-A) and major basic protein (MBP) in human ovarian tissue during the menstrual cycle. DESIGN: Ovarian tissues (n = 50) and corpora lutea (n = 18) were obtained from patients undergoing hysterectomy/oophorectomy for benign conditions and tissue sections were immunostained for MBP and PAPP-A. SETTING: University medical center. INTERVENTION(S): Immunostaining of tissue sections using antibodies to PAPP-A and MBP. MAIN OUTCOME MEASURE(S): Microscopic evaluation to assess the presence, distribution, and cellular co-localization of MBP and PAPP-A and to describe any variations in their expression during the menstrual cycle. RESULT(S): Major basic protein (MBP) is found in several ovarian cell types throughout the menstrual cycle. The MBP immunostaining of ovarian follicles varied depending on the size, with primordial follicles staining in the ooplasm with a lack of staining in the granulosa and theca cells. In the intermediate/mature follicles, MBP was immunolocalized in theca, but not in granulosa cells except in the mature follicles. Pregnancy-associated plasma protein-A (PAPP-A) was immunolocalized in primordial follicle ooplasm, theca externa of intermediate/mature follicles, and in granulosa cells with increased intensity as luteinization progressed. The luteal tissue is the major site of MBP and PAPP-A with highest intensity found during the midluteal phase associated with both small and large luteal cells. CONCLUSION(S): The expression and distinct pattern of MBP and PAPP-A cellular localization in human ovarian tissue during folliculogenesis and in luteal tissue suggest that their individual and combined actions in a cell specific fashion may play a role in growth and differentiation of theca, granulosa, and luteal cells.

Blood Proteins↗

PubMed2003

Structural characterization of a case-implicated contaminant, "Peak X," in commercial preparations of 5-hydroxytryptophan.

OBJECTIVE: To determine the chemical structure of a contaminant, X1, previously found in eosinophilia myalgia syndrome case-implicated 5-hydroxytryptophan (5-OHTrp), and also present in over-the-counter (OTC) commercially available 5-OHTrp. METHODS: Case-implicated 5-OHTrp as well as 6 OTC samples were subjected to accurate mass HPLC-mass spectrometry and HPLC-electrochemical detection, and reacted with reduced glutathione. Peak X1 was subsequently subjected to HPLC-tandem mass spectrometry (MS/MS), as well as the resulting nucleophilic glutathione product. All these data were compared with analysis carried out under identical conditions on authentic 4,5-tryptophan-dione (Trp-4,5D). RESULTS: Based on accurate mass, tandem mass spectrometric analysis, and comparision with authentic standard compound analysis, X1 was determined to be 4,5-tryptophan-dione, a putative neurotoxin. The presence of X1 in OTC samples varied from 0.5 to 10.3% of the amount of Trp-4,5D present in case-implicated 5-OHTrp. CONCLUSION: Peak X1 was identified as the putative neurotoxin Trp-4,5D. It was found in case-implicated 5-OHTrp as well as 6 OTC samples. This gives some cause for concern in terms of the safety of such commercial preparations of 5-OHTrp.

5-Hydroxytryptophan↗

PubMed2002

Non-IgE-dependent activation of human lung- and cord blood-derived mast cells is induced by eosinophil major basic protein and modulated by the membrane form of stem cell factor.

The allergic reaction begins with the antigen-induced aggregation of occupied high-affinity IgE receptors expressed on mast cell surface, their activation, and the release of proinflammatory mediators that cause the "early phase" of this process. In addition, mast cell activation induces the onset of a "late phase" reaction characterized by the tissue infiltration of inflammatory cells, mainly eosinophils. We have hypothesized that during the late phase mast cells interact with and are activated by eosinophils. Here we report that highly purified human lung mast cells became responsive to eosinophil major basic protein (MBP) when in coculture with human lung fibroblasts. In addition, cord blood-derived mast cells maintained in coculture with 3T3 fibroblasts released more histamine and prostaglandin D(2) (PGD(2)) compared with cells maintained in suspension. The fibroblast-derived membrane form of stem cell factor (SCF) was found to be involved in the mast cell increased responsiveness to MBP. In fact, cord blood-derived mast cells cocultured with 3T3 in the presence of antisense for SCF or cocultured with fibroblasts that do not express the membrane form of SCF were inhibited in their histamine-releasing activity toward MBP. In addition, this form of SCF induced the expression of a pertussis toxin-sensitive G(i) protein, G(i3) that interacts with MBP to trigger mast cell non-IgE-dependent activation in a manner similar to other cationic compounds such as compound 48/80. Mast cell responsiveness to eosinophil mediators is a potentially novel evidence for an alternative pathway of allergen-independent activation able to contribute to the perpetuation of allergy.

3T3 Cells↗

PubMed2002

Treatment of hypereosinophilic syndrome with imatinib mesilate.

Patients with hypereosinophilic syndrome show persistent eosinophilia without recognised cause. We treated five such patients with 100 mg imatinib mesilate (formerly STI-571) daily; four male patients with normal serum interleukin 5 showed complete haematological responses; a female patient who did not respond had raised serum interleukin-5 concentrations. One patient developed leucopenia after 4 days of treatment; counts returned to normal when treatment was stopped. After 1 month, eosinophilia recurred; with further treatment for 2 days, eosinophil counts again became normal. All patients who responded stopped other treatments and reduced imatinib mesilate to 200 mg per week. This drug effectively controls eosinophilia in patients with hypereosinophilic syndrome and normal interleukin-5 concentrations.

Administration, Oral↗

PubMed2002

Human eosinophils produce neurotrophins and secrete nerve growth factor on immunologic stimuli.

Neurotrophins, such as nerve growth factor (NGF) and neurotrophin-3 (NT-3), are essential for development, function, and survival of peripheral sympathetic and sensory neurons. Most eosinophilic leukocytes in the human body are localized in mucosal tissues; however, the roles of eosinophils in human diseases are not fully understood. We found that human eosinophils constitutively express messenger RNA for NGF and NT-3, synthesize and store these proteins intracellularly, and continuously replenish them. Incubation of eosinophils with a transcription inhibitor, actinomycin D, for 8 hours completely depletes intracellular NGF and NT-3. New synthesis of NGF is enhanced by Fc-receptor-mediated stimuli, such as immunoglobulin (Ig)A and IgG immune complexes; in contrast, production of NT-3 is not affected by these stimuli. Notably, supernatants of eosinophils stimulated with IgA immune complex and interleukin 5 promote neurite extension of the PC-12 pheochromocytoma cell line; this effect is abolished by pretreatment of the supernatants with anti-NGF-neutralizing antibody. By enzyme-linked immunosorbent assay, substantial amounts of NGF protein are also detected in the supernatants of stimulated eosinophils. Furthermore, in patients with seasonal allergic rhinitis, the concentrations of NGF in nasal secretions correlate with the magnitudes of eosinophilic inflammation in the airway, suggesting a potential clinical implication of eosinophil NGF. Our observations propose a new pathologic mechanism by which eosinophils may contribute to enhanced neurologic responses in patients with allergic diseases and other eosinophilic disorders. Alternatively, eosinophils may play important roles in maintenance and restoration of homeostatic functions of mucosal tissues through the pleitropic activities of NGF.

Adolescent↗

PubMed2002

Biological activities of ecalectin: a novel eosinophil-activating factor.

Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for beta-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the beta-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation.

Antibodies↗

PubMed2002

Effects of eosinophils on mast cells: a new pathway for the perpetuation of allergic inflammation.

Mast cells have a clear-cut pathologic role in allergy, participating in a number of chronic inflammatory conditions, in helmintic parasitosis, and in some solid tumor reactions, but also in physiological situations, such as wound healing and innate immunity. Mast cells release a large number of proinflammatory, immunoregulatory, and tissue regulatory mediators after activation induced by either IgE-dependent or IgE-independent mechanisms. While much information has been gathered on the immunological mast cell activation both in rodent and human systems, only minimal knowledge exists on the non-immunological activation especially in human mast cells. Mast cell IgE-independent activation occurs through G(i3alpha) which has been identified as the pertussis toxin (Ptx)-sensitive heterotrimeric G protein that interacts with cationic secretagogues inducing PLC-independent mast cell exocytosis. Mast cell IgE-independent activation in allergy probably occurs when mast cells encounter eosinophils, the main inflammatory cells of the allergic reactions that persist throughout the late phase and when the inflammatory condition becomes chronic. This review summarizes regarding the influence of eosinophils on mast cell activation, thus demonstrating that IgE-independent activation has a relevant role in pathophysiological processes as well as in mast cell IgE-dependent activation.

Blood Proteins↗

PubMed2002

Evidence for pathogenic involvementof eosinophils and neutrophilsin Churg-Strauss syndrome.

BACKGROUND: Churg-Strauss syndrome (CSS) is a multi-organ disease with tissue and blood eosinophilia. OBJECTIVE: Our aim was to study eosinophil and neutrophil involvement in CSS. METHODS: Eight lesional skin biopsy specimens from 6 patients with CSS and serum and blister fluid from one patient were tested for eosinophil and neutrophil activity. Indirect immunofluorescence on skin specimens used antibodies to eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and neutrophil elastase (NE). Serum and blister fluid specimens were analyzed for granule protein levels and for eosinophil-activating cytokines. RESULTS: Indirect immunofluorescence showed prominent cellular and extracellular staining for EDN in skin biopsy specimens; MBP staining was less extensive. Five biopsy specimens showed marked cellular NE staining; 4 showed prominent extracellular NE. Serum and blister fluid specimens contained elevated MBP, EDN, and interleukin 5 levels and enhanced eosinophil survival in culture. Granulocyte-macrophage colony-stimulating factor and interleukin 5 were detected in blister fluid. Blister fluid contained more NE than normal serum. CONCLUSIONS: Both eosinophils and neutrophils likely participate in skin lesion development in CSS.

Adult↗

PubMedDate not supplied

A modular on-line three-dimensional liquid chromatography-tandem mass spectrometry approach to characterization of organelle proteomes.

In this work we describe the use of a modular multidimensional chromatography-tandem mass spectrometry approach for rapid identification of proteins. In particular we highlight the use of a strong cation exchange cartridge in conjunction with a membrane postconcentration cartridge and nano-HPLC on-line with tandem mass spectrometry to characterize the eosinophil granule organelle proteome. Details are provided of the analytical approach we have developed and we discuss some of the advantages compared with previously reported analyses, as well as providing some specific examples of novel proteins identified.

Amino Acid Sequence↗