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Biomedical subjects

G J Gleich

Publications and source records attributed to G J Gleich.

At least 253 records · Page 14Linked to original sources
PubMed1989

Conjunctival deposition of eosinophil granule major basic protein in vernal keratoconjunctivitis and contact lens-associated giant papillary conjunctivitis.

To investigate the role of the eosinophil in vernal keratoconjunctivitis and contact lens-associated giant papillary conjunctivitis, we assessed the presence of eosinophil granule major basic protein in conjunctival tissues by immunofluorescence. Biopsy specimens of conjunctiva were taken from nine patients with vernal keratoconjunctivitis, seven patients with giant papillary conjunctivitis, and five control subjects. We performed a masked semiquantitative assessment of immunofluorescence on sections from each specimen. The vernal keratoconjunctivitis and giant papillary conjunctivitis groups had significantly (P less than .05) more major basic protein deposition than controls. No significant correlation between severity of disease and degree of major basic protein deposition was found. We found extracellular eosinophil granules in one of three vernal keratoconjunctivitis specimens examined by transmission electron microscopy. Thus, eosinophil degranulation commonly occurs in vernal keratoconjunctivitis and giant papillary conjunctivitis with release of eosinophil granule major basic protein and presumably other toxic granule proteins onto affected tissues. These cationic proteins are potent cytotoxins and are able to stimulate mast cell degranulation.

Blood Proteins↗
PubMed1989

Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein.

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.

Blood Bactericidal Activity↗
PubMed1989

Molecular cloning of the human eosinophil peroxidase. Evidence for the existence of a peroxidase multigene family.

Human eosinophil peroxidase (EPO) was purified from eosinophil granules derived from the peripheral blood of patients with eosinophilia. The molecular mass of the H and L subunits was determined by gel filtration to be 57,000 and 11,000 daltons, respectively. The partial amino acid sequences of both subunits were used to construct oligonucleotides for the screening of several cDNA libraries, including one derived from human-induced umbilical cord mononuclear cells. A cDNA clone was isolated corresponding to EPO. The nucleotide sequence revealed an open reading frame of 2,106 bp, corresponding to a prosequence, L chain, and H chain, in this order. Comparison of the EPO nucleotide sequence with other peroxidases, such as myeloperoxidase, suggests the existence of a multigene family.

Amino Acid Sequence↗
PubMed1989

IgA-induced eosinophil degranulation.

Eosinophils play an important role as effector cells in allergic, parasitic, and other conditions. The mechanism(s) by which eosinophils mediate their effector functions was studied by incubation of human normodense eosinophils with Sepharose beads coupled to various Ig isotypes as targets. Controls included eosinophils incubated alone or incubated with uncoated beads, human serum albumin-, or OVA-coated beads. An eosinophil granule protein, the eosinophil-derived neurotoxin (EDN), was measured as an indicator of eosinophil degranulation. Eosinophils released eosinophil-derived neurotoxin when incubated with Sepharose beads coupled to Ig of the IgG or IgA isotypes, as well as IgA-Fc fragments. Mixtures of IgG and IgA on beads did not act synergistically. Secretory IgA (sIgA) provided the most potent signal for eosinophil degranulation and was two to three times more potent than IgG. Furthermore, 2 to 17% of the normodense eosinophils bound to IgG- or IgA-coated beads, whereas 24 to 27% of the eosinophils bound to sIgA-coated beads. Thus, sIgA may be the principal Ig mediating eosinophil effector function at mucosal surfaces in helminth infections and hypersensitivity diseases, especially bronchial asthma.

Cell-Free System↗
PubMed1989

Comparison of antifading agents used in immunofluorescence.

Various chemicals including p-phenylenediamine, n-propyl gallate, 1,4-diazobicyclo[2,2,2]-octane and Citifluor were tested to determine their effectiveness in retarding the fading of fluorescein-conjugated antibody bound to eosinophil granule major basic protein in tissues. Immunofluorescence fading and intensity were measured quantitatively using the Zeiss Zonax microphotometer system. All of these agents effectively retarded fluorescence fading; p-phenylenediamine and n-propyl gallate were the most effective. Comparison of glycerol-based and polyvinyl alcohol-based media containing n-propyl gallate or p-phenylenediamine showed that the glycerol-based medium yields higher mean fluorescence intensities than the polyvinyl alcohol-based medium. Slides mounted with p-phenylenediamine-glycerol could be stored for up to 2 weeks without loss of fluorescence intensity. Although p-phenylenediamine has many undesirable chemical properties, we conclude that it is the agent of choice for retarding the fading of tissue preparations stained with fluorescein isothiocyanate conjugates when used in a glycerol-based medium.

Blood Proteins↗
PubMed1989

Blood eosinophils and eosinophil-derived proteins in allergic asthma.

The concentrations of the eosinophil (EOS)-derived proteins, major basic protein (MBP), EOS-derived neurotoxin (EDN), EOS peroxidase (EPO), and EOS cationic protein (ECP), and EOS counts were measured in the peripheral blood of 12 atopic subjects with asthma and 23 normal control subjects. The same measurements were performed in seven subjects with asthma with previously documented dual (early plus late) asthmatic responses after inhalation challenges with methacholine and allergen. EOSs (p less than 0.001), MBP (p less than 0.01), EDN (p less than 0.01), and ECP (p greater than 0.03) were elevated in the subjects with asthma compared with control subjects, whereas EPO (p less than 0.01) concentrations were reduced. There were no significant differences between baseline measurements of FEV1, EOSs, MBP, EDN, EPO, or ECP on the methacholine- and allergen-challenge days. When the changes in these variables after allergen challenge were compared with the corresponding changes after methacholine challenge, there were no significant differences at 0 to 60 minutes or at 3 hours, whereas EDN (p less than 0.025), EPO (p less than 0.05), and ECP (p less than 0.025) were relatively increased at 6 to 12 hours and accompanied the late falls in FEV1 (p less than 0.001). EOSs (p less than 0.025) were elevated at 24 hours when there was a small relative increase in MBP (p less than 0.05). EOSs appear to be "activated" in subjects with allergic asthma, and further activation may occur during late asthmatic responses.

Adult↗
PubMed1989

Major basic protein and eosinophil-derived neurotoxin concentrations in nasal-lavage fluid after antigen challenge: effect of systemic corticosteroids and relationship to eosinophil influx.

The late-phase response to nasal challenge with antigen is associated with a mixed inflammatory cell influx in which the eosinophil demonstrates the earliest and greatest proportionate rise. We investigated the evidence for activation of the eosinophil during the late response by measuring the concentration of the eosinophil-derived mediator major basic protein (MBP) and the eosinophil-derived neurotoxin (EDN) in nasal-lavage fluids before and for 11 hours after antigen challenge in 13 subjects with seasonal allergic rhinitis. The subjects received oral prednisone (20 mg three times daily) or placebo in a double-blind, crossover manner for 2 days before each of two antigen challenges. After placebo pretreatment, significant increases over diluent baseline (4.5 +/- 0.4 ng/ml) occurred in the levels of MBP in nasal-lavage fluid during the early (9.8 +/- 2.9 ng/ml; p less than 0.005) and late (15.3 +/- 4.8 ng/ml; p less than 0.01) responses to antigen challenge. Significant increases (p less than 0.05) in the concentration of EDN also occurred during the late response to antigen that correlated with the levels of MBP (r = 0.48; p less than 0.001). The cumulative late-phase increase in MBP correlated closely (rs = 0.96; p less than 0.005) with the total influx of eosinophils. Oral prednisone pretreatment significantly reduced the mean of each subject's peak late-phase concentration of both MBP (30.7 +/- 5.8 ng/ml versus 13.3 +/- 4.3 ng/ml; p = 0.005) and EDN (885 +/- 659 ng/ml versus 71 +/- 41 ng/ml; p less than 0.05). These data provide evidence for eosinophil degranulation during the late response and inhibition of this response by prednisone, supporting its pathogenetic role.

Blood Proteins↗
PubMed1989

Eosinophil granule major basic protein in acute renal allograft rejection.

Conventional staining techniques to determine the presence of tissue eosinophils underestimate their number and do not usually detect eosinophil degranulation. We have studied the involvement of eosinophils in acute renal allograft rejection by immunofluorescence localization of eosinophil granule major basic protein (MBP) in the kidney and by measurement of MBP in the plasma and urine by radioimmunoassay. Tissue eosinophilia and extracellular deposition of MBP indicative of eosinophil degranulation were observed in 94% and 87%, respectively, of patients with acute rejection as compared with 17% and 17%, respectively, of patients with cyclosporine nephrotoxicity. The urine levels of MBP were significantly elevated in acute rejection but not in cyclosporine nephrotoxicity. Plasma MBP concentrations were within the normal range in both acute rejection and cyclosporine nephrotoxicity. The presence of marked tissue eosinophilia and eosinophil degranulation did not always indicate irreversible rejection. Interleukin-2 and IL-2 receptors were also elevated in the urine during acute rejection. These results support a role for the eosinophil as an effector of tissue damage during rejection and suggest the potential usefulness of urine MBP determinations for the immunologic monitoring of transplanted patients.

Acute Disease↗
PubMed1989

Epithelial augmentation of trachealis contraction caused by major basic protein of eosinophils.

We studied the effect of epithelial removal and intraepithelial administration of human eosinophil granule major basic protein (MBP) on the contraction of underlying canine tracheal smooth muscle in 23 dogs in vivo. A dual in situ tracheal preparation was utilized that allowed sharp excision of epithelium. The response to intra-arterial acetylcholine (ACh) was augmented substantially in five dogs receiving 200 micrograms MBP by intraepithelial instillation. Active tension elicited by 10(-8) mol intra-arterial ACh was 34.0 +/- 2.2 g/cm before and 46.1 +/- 2.6 g/cm 30 min after MBP (P less than 0.002). There was no change in active tension in the control segment in the same dogs after intraepithelial instillation of vehicle only (34.7 +/- 3.2 vs. 34.4 +/- 2.3 g/cm; P = NS). Instillation of MBP directly into the subepithelial tracheal smooth muscle did not alter contraction. To assess whether this augmentation was caused by inhibition of an epithelial-derived relaxant factor, additional studies were performed in nine other dogs in which the epithelium was excised discretely from one of the two tracheal segments. No significant differences in contractile response to ACh or relaxation response to isoproterenol were observed at 2, 15, 30, or 60 min after epithelial excision. We demonstrate that intraepithelial administration of MBP augments the contraction of underlying canine tracheal smooth muscle elicited by ACh. This augmentation is a direct effect of MBP and does not require antagonism of epithelial inhibition.

Animals↗
PubMed1989

Eosinophils and human disease.

The likely roles of the eosinophil leukocyte in human disease are reviewed. The eosinophil is richly endowed with toxic cationic proteins and is able to mount a respiratory burst. Thus, eosinophils have the capability to damage various targets, and evidence exists that they do so during helminth infections and during the course of many hypersensitivity diseases. Here we discuss the role of the eosinophil in human onchocerciasis with particular attention to the Mazzotti reaction. We also discuss other diseases where eosinophil degranulation is seen, especially cutaneous diseases. Finally, the possible role(s) of the granule major basic protein in human pregnancy is noted.

Animals↗
PubMed1989

Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro.

We tested the effects of four eosinophil granule cationic proteins: major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), on guinea pig tracheal epithelium in vitro. Examination by inverted microscopy revealed that MBP, both the form stabilized by alkylation of sulfhydryl groups as well as the native form of the molecule, ECP, EPO by itself, as well as EPO + H2O2 + halide, but not EDN, cause dose-related damage to the tracheal epithelium. The lowest concentrations of MBP and ECP causing damage were 10 and 100 micrograms/ml, respectively. In contrast, EDN, although biochemically similar to ECP, did not damage the tracheal epithelium in concentrations of up to 200 micrograms/ml. MBP caused exfoliation, as well as bleb formation and ciliostasis. EPO in the presence of the H2O2-producing enzyme glucose oxidase (GO), Cl-, 0.11 M, and iodide caused ciliostasis, bleb formation, and exfoliation of epithelial cells at concentrations as low as 1 U/ml (3.9 micrograms/ml). EPO + GO in the presence of Cl-, 0.11 M, alone or with Cl- and l-, 10(-4) M, or Cl- and Br-, 5 x 10(-5) M, were all toxic to epithelium. Surprisingly, EPO by itself caused partial ciliostasis, bleb formation, and exfoliation of epithelial cells in a dose-related manner at concentrations as low as 10 to 30 U/ml (39 to 121 micrograms/ml). These results confirm prior observations showing the toxicity of MBP to tracheal epithelium and indicate that ECP and EPO alone, as well as EPO + GO + halide, cause damage. Thus, several eosinophil granule proteins are able to damage respiratory epithelium.

Alkylation↗
PubMed1989

Leukocytes and mediators in bronchoalveolar lavage during allergen-induced late-phase asthmatic reactions.

We have measured the total and differential cell counts, histamine, leukotriene (LT) B4 and LTC4, immunoglobulins, complement (C3), eosinophil-derived basic proteins, and monocyte complement rosettes in bronchoalveolar lavage (BAL) 6 h after challenge with either antigen or diluent control in seven patients with antigen-induced single early reactions, and seven with dual (early and late phase) reactions. In both groups, the total cell counts in BAL were similar, irrespective of whether they were challenged with antigen or diluent. However, in the late-phase responders (LPR), there were significant increases in lymphocytes, neutrophils, and eosinophils (p less than 0.05), and significant decreases in the percentage of lung mast cells (p less than 0.05). The eosinophil major basic protein and eosinophil-derived neurotoxin increased in four of five subjects with dual responses and in the majority of single early responders (SER). BAL histamine concentrations increased in five of seven patients with dual responses. There were no consistent changes in LTB4 concentrations in either the LPR or the SER between diluent and antigen days, but a small but significant increase in LTC4 was observed in the LPR. Concentrations of IgG, IgA, IgM, IgE, C3, and albumin did not differ significantly. The percentage of monocyte complement rosettes also increased significantly (p less than 0.05) in LPR, but not in SER. These findings support the hypothesis that eosinophils and their products play a role in tissue injury in LPR and that eosinophil infiltration may be associated with macrophage activation.

Adolescent↗
PubMed1989

Injurious effect of the eosinophil peroxide-hydrogen peroxide-halide system and major basic protein on human nasal epithelium in vitro.

Tissue injury is observed in allergic and nonallergic eosinophilic rhinitis, but the mechanism of this injury is unclear. Because eosinophils are prominent in biopsy specimens in these conditions, we hypothesized that they may participate in the injury process. Initially, we developed techniques to isolate and purify human nasal epithelial cells from turbinate biopsies to use as target cells for eosinophil granule products. Primary cultures from explants were characterized by electron microscopy and indirect immunofluorescence with a panel of primary monoclonal and polyclonal antibodies. These studies revealed the homogeneity of the cells and confirmed their epithelial nature. Cultured nasal epithelial cells were then exposed to either purified human eosinophil peroxidase, bromide, and glucose plus glucose oxidase, as a continuous source of hydrogen peroxide, or eosinophil major basic protein. Neither eosinophil peroxidase alone nor glucose plus glucose oxidase in the absence of eosinophil peroxidase were injurious, but the combined addition of eosinophil peroxidase, glucose/glucose oxidase, and bromide produced marked target cell lysis. This effect was time- and eosinophil peroxidase dose-dependent. Catalase and azide significantly inhibited the lysis of these cells, suggesting the eosinophil peroxidase-catalyzed products of halide oxidation mediated this form of injury. The addition of purified human eosinophil major basic protein also caused dose- and time-dependent lysis of the nasal epithelial cells but required longer incubation periods to effect injury. We hypothesize that the eosinophil peroxidase-hydrogen peroxide-halide system and major basic protein may injure the nasal epithelium in inflammatory conditions such as allergic and nonallergic eosinophilic rhinitis.

Blood Proteins↗
PubMed1989

Immunofluorescent localization of eosinophil granule major basic protein in fatal human cases of Baylisascaris procyonis infection.

We examined eosinophil degranulation in tissues from patients infected with Baylisascaris procyonis as shown by the extracellular deposition of granule major basic protein (MBP). We utilized immunofluorescence to localize MBP in eosinophils and at sites of degranulation to study specimens from 2 fatal cases of B. procyonis infection. Large numbers of intact eosinophils were present in the brain around blood vessels and necrotic migration tracks and in mesenteric granulomata. Extensive extracellular MBP deposition was present in the necrotic migration tracks in the brain and around larvae in the mesenteric granulomata in association with the radiating eosinophilic deposits characteristic of the "Splendore-Hoeppli phenomenon." The Splendore-Hoeppli deposits consist in part of eosinophil granule MBP. Release of the cytotoxic MBP in response to invading larvae may cause tissue damage. Central nervous system tissue damage by cytotoxic eosinophil granule proteins may contribute to the neurologic symptoms of B. procyonis infection.

Animals↗
PubMed1989

Pharmacological control of human basophil histamine release stimulated by eosinophil granule major basic protein.

Eosinophil granule major basic protein (MBP) is a 13,800 MW arginine-rich polypeptide that is unique among basic molecules in its ability to stimulate human basophil histamine release. We examined the Ca2+ requirements and pharmacological regulation of MBP-stimulated histamine release. Minimal MBP-induced histamine release occurred in the absence of extracellular Ca2+, whereas addition of 0.1 mM Ca2+ resulted in 70% of the maximum histamine release response. Maximum histamine release required 0.5 to 1 mM extracellular Ca2+. The MBP-induced histamine release was blocked by a calmodulin antagonist and by theophylline and was partially inhibited by an inhibitor of phospholipase A2. Release was unaffected by inhibition of protein kinase C. Basophil pretreatment with pertussis toxin also resulted in a concentration-dependent inhibition of release, suggesting involvement of a GTP regulatory protein in the activation mechanism. Histamine release stimulated by a 13,900 MW poly-L-arginine exhibited a dissimilar pharmacological profile from that of MBP. These results support the non-cytolytic nature of the MBP activation mechanism and identify pharmacological approaches for control of MBP-induced mediator release.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Acidic precursor revealed in human eosinophil granule major basic protein cDNA.

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and various cell types, is a 13.8-kD single polypeptide rich in arginine with a calculated isoelectric point (pI) of 10.9. A cDNA for human MBP was isolated from a gamma GT10 HL-60 cDNA library. The nucleotide sequence of the MBP cDNA indicates that MBP is translated as a 25.2-kD preproprotein. The 9.9-kD pro-portion of proMBP is rich in glutamic and aspartic acids and has a calculated pI of 3.9, while proMBP itself has a calculated pI of 6.2. We suggest that MBP is translated as a nontoxic precursor that protects the eosinophil from damage while the protein is processed through the endoplasmic reticulum to its sequestered site in the granule core toxic MBP, and we present results from the literature suggesting that other cationic toxins, which damage cell membranes, may also be processed from nontoxic precursors containing distinct anionic and cationic regions.

Amino Acid Sequence↗