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Biomedical subjects

D A Loegering

Publications and source records attributed to D A Loegering.

At least 37 records · Page 2Linked to original sources
PubMed1989

Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases.

Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.

Amino Acid Sequence↗
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

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

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↗
PubMed1988

Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein.

Eosinophil granule major basic protein (MBP) is a relatively low molecular weight cationic (pI greater than 10) protein present in the crystalloid core of the eosinophil granule. Amino acid sequence analysis of this protein was undertaken as part of an analysis of the structural basis of the potent cytotoxic activities of MBP on parasites and mammalian cells. Many conventional sequencing strategies were unworkable because of the unusual amino acid composition of MBP and its insolubility in solutions buffered at neutral pH. Less conventional chemical reactions, including cyanogen bromide-induced cleavage at tryptophan and acid-induced cleavage at aspartic acid, were used successfully to obtain peptides which allowed definition of the amino acid sequence of MBP. Characterization of MBP by reverse-phase high pressure liquid chromatography and two-dimensional gel analysis showed no microheterogeneity that might be attributed to post-translational modifications. Comparison of the MBP sequence with a protein sequence data base showed that MBP has no significant sequence homology with other characterized proteins. The basicity (pI 10.9) and hydrophobicity predicted from the MBP sequence are likely responsible for the observed affinity of this cytotoxic molecule for cell surfaces and some serum proteins.

Amino Acid Sequence↗
PubMed1988

Effect of human eosinophil major basic protein on ion transport in dog tracheal epithelium.

Eosinophil major basic protein (MBP) is a granule-associated cytotoxic protein found in sputum and deposited on airway tissues of patients with acute asthma. We therefore studied the effect of human MBP on ion transport in dog tracheal epithelium. We mounted the posterior tracheal membranes of mongrel dogs in Ussing chambers and measured potential differences across the membranes and short-circuit current. Using 22Na+ and 36Cl- as tracers, we determined net ion movements. The addition of MBP (5 X 10(-6) M) to the mucosal, but not to the serosal, side of the membranes produced an increase in short-circuit current from 2.25 +/- 0.19 (mean +/- SE) to 2.78 +/- 0.23 muEq.cm-2h-1 (p less than 0.0001) and in net chloride secretion from 1.57 +/- 0.22 to 2.31 +/- 0.24 muEq.cm-2h-1 (p less than 0.01). There was no change in net sodium movement. Pretreatment with indomethacin (10(-5) M) attenuated, but did not abolish, the increase in short-circuit current. After exposure to MBP, prostaglandin E2 release into the serosal bathing solution increased from 10.0 +/- 4.2 to 17.0 +/- 6.9 ng.cm-2h-1 (p less than 0.05). The results of this study indicate that MBP simulates prostaglandin E2 production and chloride secretion by dog tracheal epithelium. Thus, eosinophils in the airways, through release of MBP, may affect mucociliary clearance by changing the volume and composition of respiratory tract fluid.

Animals↗
PubMed1987

The injurious effect of eosinophil peroxidase, hydrogen peroxide, and halides on pneumocytes in vitro.

Recent data suggest that eosinophils may cause lung injury. To determine if the eosinophil peroxidase (EPO)-hydrogen peroxide (H2O2)-halide system could mediate this injury, we added human EPO, H2O2 (or glucose and glucose oxidase as a continuous source of H2O2), and various halides to monolayers of 51Cr-labeled human A549 and rat type II pneumocytes. Cell lysis was measured as soluble 51Cr release. In initial experiments, EPO in solution did not induce lysis under these conditions. Therefore, in subsequent experiments, pneumocytes were preincubated with EPO for 15 minutes, washed to remove unbound enzyme, and then glucose, glucose oxidase, and the halides were added. EPO alone was not injurious, nor was the addition of glucose and glucose oxidase in the absence of EPO. In contrast, the combined addition of EPO, glucose, glucose oxidase, and chloride produced marked target-cell lysis. This effect was time and EPO dose dependent and was enhanced by the addition of iodide. Catalase and azide substantially inhibited the lysis produced by the EPO-H2O2-halide system, suggesting that EPO-catalyzed products of halide oxidation mediated this form of injury. Finally, the addition of eosinophil major basic protein at 10(-5) mol/L to EPO-coated pneumocytes incubated with glucose, glucose oxidase, and halides failed to enhance or inhibit lysis. We hypothesize that the EPO-H2O2-halide system may injure the lung in asthma and eosinophilic pulmonary syndromes.

Animals↗
PubMed1987

The effect of purified human eosinophil major basic protein on mammalian ciliary activity.

Eosinophil granulocyte infiltration in a variety of lung disorders may directly damage local tissue by release of granule contents. A principal constituent of eosinophil granules is the major basic protein (MBP). Previous light and electron microscopic observations have indicated that guinea pig and human MBP produce detachment of tracheal epithelial cells and cessation of ciliary activity. To quantitate the damage, selected regions of the epithelial surface of rabbit tracheal explants were videotaped before and after treatment with human MBP. Tapes were analyzed for ciliary beat frequency and the extent of zones along the epithelial surface displaying ciliary activity. The MBP at 0.1 mg/ml reduced beat frequency and significantly reduced the measured zones of ciliary activity. Also, MBP at 0.7 mg/ml significantly reduced beat frequency and the measured zone of ciliary activity on the epithelial surface. Beat frequency was lowered by 27% within 10 min, with only 1% further decrease by 60 min. The zones of ciliary activity on the epithelium were continuously decreased throughout the 60 min to 29% of the initially active zone. To examine whether MBP was capable of direct inhibition of ciliary activity, isolated porcine tracheal ciliary axonemes, the structural organelles of individual cilia, were treated with human MBP. Concentrations above 67 micrograms/ml of MBP were completely inhibitory to reactivated isolated axonemes, 67 micrograms/ml stopped activity within 10 min, and 27 micrograms/ml stopped activity within 15 min. Pretreatment of isolated axonemes with increasing concentrations of MBP resulted in decreasing ATPase activity. These effects were not attributable to pH alteration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1987

Comparative toxicity of purified human eosinophil granule proteins for newborn larvae of Trichinella spiralis.

Eosinophils have been implicated in both in vivo and in vitro destruction of helminths. One approach toward elucidating the role of the eosinophil in parasite killing has been to test the toxicity of purified eosinophil granule proteins for parasites in vitro. Previously, major basic protein (MBP) and eosinophil cationic protein (ECP) were shown to be toxic for schistosomules of Schistosoma mansoni, while eosinophil-derived neurotoxin (EDN) was only marginally so. We tested the toxicity of MBP, ECP, and EDN over a range of concentrations (0.006-5 X 10(-4) M) for newborn larvae of Trichinella spiralis. Our observations confirm previous reports of toxicity of mildly reduced and alkylated (R & A) MBP. At concentrations of 5 X 10(-5) M and above, R & A MBP killed 75% or more of the larvae within the first hour of culture. ECP was an effective toxin for these larvae after 3 hr of culture, and by 12 hr, dose-related toxicity was evident. After 24 hr, 100% of the larvae were killed at 5 X 10(-5) M ECP. EDN was much less toxic; after 12 hr, 90% of the larvae survived at concentrations of 1 X 10(4) M, while 5 X 10(-4) M EDN killed all the larvae. At the optimal toxic concentrations of 5 X 10(-5) M ECP and 5 X 10(-4) M EDN, kinetics of killing by these 2 proteins were essentially the same. Thus, on a molecular basis, both MBP and ECP appear to be potent helminthotoxins whereas EDN is much less so.

Animals↗
PubMed1986

Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein.

The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.

Blood Proteins↗
PubMed1986

Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease.

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) were isolated from lysates of human eosinophil granules by gel filtration and ion exchange chromatography on heparin-Sepharose. Radioimmunoassay, using monoclonal antibodies, of fractions from the heparin-Sepharose chromatography showed one peak of EDN activity and two peaks of ECP activity (termed ECP-1 and ECP-2). EDN, ECP-1, and ECP-2 each exhibited heterogeneity in charge and molecular weight when analyzed by two-dimensional nonequilibrium pH gradient electrophoresis and NaDodSO4/PAGE. Digestion of EDN with endoglycosidase F (endo F) decreased its molecular weight and charge heterogeneity. Thus, END likely contains a single complex oligosaccharide. Endo F digestion of ECP-1 and ECP-2 decreased the molecular weight of both polypeptides, indicating that both likely contain at least one complex oligosaccharide. Amino acid sequence analyses showed that ECP-1 and ECP-2 are identical from residue 1 through residue 59 and that the sequences of EDN and ECP are highly homologous (37 of 55 residues identical). Both EDN and ECP NH2-terminal sequences showed significant homology to RNase, especially in regions of the RNase molecule involved in ligand binding. EDN, ECP-1, and ECP-2 had neurotoxic activity, causing the Gordon phenomenon at doses down to 0.15 micrograms when injected into the cisterna magna; the proteins were comparable in their activities. These results indicate that EDN and ECP are related proteins and suggest that they derived from genes associated with the RNase family.

Amino Acid Sequence↗
PubMed1985

Eosinophil- and eosinophil granule-mediated pneumocyte injury.

The function of the eosinophil in eosinophilic pulmonary syndromes and asthma is uncertain. To determine if eosinophils might play a harmful role in these conditions, we cocultured purified human eosinophils, eosinophil major basic protein (MBP), and chromatographically eluted eosinophil granule fractions with human A549 and rat type II pneumocytes. Damage to these target cells was measured as cell lysis and nonlethal cell detachment. We found that unstimulated intact eosinophils affected minimal lysis or detachment of either pneumocyte target, but eosinophils stimulated with phorbol myristate acetate and other activators produced time- and dose-dependent nonlytic detachment of both targets. In contrast, supernatants from activated eosinophils did not produce significant injury, suggesting that close apposition of the effector and target cells was required. Catalase and superoxide dismutase did not inhibit the detaching activity of eosinophils, suggesting that hydrogen peroxide and superoxide anion were not activity of eosinophils, suggesting that hydrogen peroxide and superoxide anion were not responsible for mediating this form of injury. In contrast to our findings with intact eosinophils, we observed that the addition of purified eosinophil MBP to pneumocytes caused marked cytolysis with little detachment. When sequential fractions of eosinophil granules separated by Sephadex G-50 chromatography were added to A549 and rat type II pneumocyte targets, it was found that different fractions produced distinct forms of injury. Higher molecular weight fractions containing lysosomal enzymes and eosinophil peroxidase produced predominantly detachment, whereas fractions enriched in MBP produced lysis. These results indicate that intact eosinophils can produce nonlytic detachment of alveolar pneumocytes that is probably not dependent on the generation of toxic oxygen radicals but rather appears to be mediated by granule-associated products, possibly lysosomal enzymes. Furthermore, although intact eosinophils are not capable of lysing alveolar epithelial cells under the conditions of our assay, MBP has the potential to do so when the protein is released in high enough concentrations. The in vivo relevance of these findings in eosinophilic lung diseases may be that eosinophils, by producing both desquamation and death of alveolar epithelium cells, may increase the permeability of the alveolus to fluid and cells. Moreover, these forms of damage might also enhance the ingress of inhaled antigens across the pulmonary epithelial barrier, thus increasing immunologic sensitization.

Animals↗
PubMed1985

Comparative toxicity of purified human eosinophil granule cationic proteins for schistosomula of Schistosoma mansoni.

The human eosinophil granule contains several distinctive cationic proteins that have been purified to homogeneity, including major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). Two earlier studies have shown that MBP and ECP both damage schistosomula of Schistosoma mansoni in vitro in a dose-dependent fashion. The present study expands upon these observations by comparing the toxicity of MBP, ECP, as well as EDN when tested at equimolar concentrations (0.03-2 X 10(-5) M). On a molar basis, ECP was 8 to 10 times more potent than MBP, and the ECP-mediated killing of schistosomula was qualitatively different than that of MBP. Purified ECP produced complete fragmentation and disruption of schistosomula, whereas MBP produced a distinctive ballooning and detachment of the tegumental membrane. In contrast, EDN was only marginally toxic at high concentrations and caused crinkling of the tegumental membrane. Heating MBP and ECP for four hr at 56 degrees C caused precipitation and loss of toxicity for MBP, but not for ECP. Native MBP (with reactive sulfhydryl groups intact) and stabilized, reduced and alkylated MBP had comparable toxicity. To determine the relative contribution of MBP, ECP and other potentially helminthotoxic eosinophil granule constituents to schistosomulum damage, fractions of acid soluble granule extracts prepared by chromatography on Sephadex G-50 columns were analyzed for toxicity to schistosomula and for MBP and ECP levels by radioimmunoassay. Schistosomula were killed by fractions containing MBP, and to a much lesser and more variable extent by fractions containing EDN and a 21,000 dalton protein, but not by fractions coincident with the elution of ECP, which contained concentrations of ECP below that required to produce significant killing of schistosomula by the purified protein. Therefore, although ECP is a more potent helminthotoxin for schistosomula than MBP on a molar basis, MBP, by virtue of its abundance in the granule, accounts for the bulk of the toxicity in fractions of acid solubilized granules obtained from eosinophils of patients with marked eosinophilia.

Animals↗
PubMed1983

Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin.

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.

Binding, Competitive↗
PubMed1981

Recovery of eosinophils from the peritoneal cavity of the guinea pig.

We studied the effects of various conditions on the recovery of eosinophils from the peritoneal cavity of guinea pigs repeatedly lavaged with saline. We compared the effects of ether and halothane on eosinophil production in guinea pigs either lavaged with saline alone or receiving an injection of polymyxin B before saline lavage. With both anesthetics polymyxin B caused a rapid and consistent increase in eosinophil production, although neutrophils were present. With halothane anesthesia, saline lavage alone yielded mean eosinophil values near those found in the polymyxin group. In contrast, saline lavage alone with ether anesthesia yielded a significantly lower mean eosinophil value than in the polymyxin groups with either anesthetic and the saline lavage alone with halothane anesthesia (P less than 0.05). Additional studies showed that females guinea pigs produced greater numbers of peritoneal eosinophils than male guinea pigs and that peritoneal eosinophilia was maintained for up to 20 weeks by weekly peritoneal lavage with saline alone. Castration of male guinea pigs did not result in eosinophil production comparable to female guinea pigs. Infection with Trichinella spiralis did not enhance peritoneal eosinophilia commensurate with that seen in the peripheral blood. These results indicate that saline lavage alone is a sufficient stimulus for eosinophil production in guinea pigs anesthetized with halothane, that greater numbers of eosinophils are produced in females than males and, finally, that eosinophil production continues at high levels for more than 20 weeks.

Animals↗