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D A Loegering

Publications and source records attributed to D A Loegering.

At least 55 records · Page 3Linked to original sources
PubMed1981

Purification of human eosinophil-derived neurotoxin.

Human and animal eosinophils contain a powerful neurotoxin that causes selective neuronal and axonal damage to white matter of cerebellum and spinal cord of experimental animals when injected intrathecally. This reaction is termed the "Gordon phenomenon." We purified the eosinophil-derived neurotoxin from eosinophil-rich leukocyte suspensions or eosinophil granules from four patients with various hypereosinophilic syndromes. A single protein with an average molecular weight of 18,400 was isolated by sequential chromatography on Sephadex G-50 columns and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of column fractions. The purified eosinophil-derived neurotoxin from the cells of these patients retained the potent neurotoxic activity of the crude eosinophil or eosinophil granule extracts in experimental animals. These animals developed the syndrome of stiffness and ataxia progressing to severe paralysis characteristic of the Gordon phenomenon. Histologic examination of the brains of animals injected with purified eosinophil-derived neurotoxin confirmed the characteristic widespread loss of Purkinje cells and severe spongiform vacuolation in the white matter of cerebellum, brain stem, and spinal cord. We have established the location of eosinophil-derived neurotoxin in the eosinophil granule and have shown that it is distinct from several other eosinophil proteins, the granule major basic protein, and the Charcot-Leyden crystal protein (lysophospholipase).

Animals↗
PubMed1981

Elevated serum levels of the eosinophil granule major basic protein in patients with eosinophilia.

A radioimmunoassay was established for the human eosinophil granule major basic protein (MBP). The mean level of MBP in sera from 105 normal control patients was 454 ng/ml, whereas in a sample of 188 patients with various forms of diseases, including the hypereosinophilic syndrome, levels as high as 14,000 ng/ml were measured. Serum levels of MBP did not correlate with eosinophil counts in normal subjects, but a positive correlation was seen in patients with eosinophilia; the patients with eosinophil counts greater than 350/mm3 generally showed increased levels of MBP. Many patients with skin disease and normal eosinophil counts had elevated levels of serum MBP. Monomer MBP has a molecular weight of 9,300, but in sera of patients with eosinophilia, the MBP activity was of high molecular weight, greater than 50,000. Analyses of serum by Sephadex G-200 and by electrofocusing suggest that MBP is not simply polymerized, but rather is bound to a larger carrier molecule. Monomeric MBP can be isolated from serum by reduction of serum with dithiothreitol, alkylation with iodoacetamide, and acidification to pH 2 followed by fractionation on Sephadex G-50 at pH 2. Under these conditions, up to 80% of the MBP emerges in monomeric form. The results indicate that eosinophil granule proteins circulate in blood covalently bound to serum proteins, and that elevated concentrations of serum MBP are present in some diseases associated with eosinophilia.

Alkylation↗
PubMed1981

Elevated levels of the eosinophil granule major basic protein in the sputum of patients with bronchial asthma.

The eosinophil granule major basic protein (MBP) is toxic to parasites and mammalian cells. Because eosinophilia is characteristic of asthma, we tested the effect of MBP on bronchi and assayed sputa for this protein. We found that MBP damaged bronchial epithelium in vitro and produced changes that mimicked those in asthma. Radioimmunoassay of sputa from 100 consecutive patients with respiratory diseases revealed MBP levels above 0.1 mug/ml in 13 patients, and 11 of these had asthma. In 15 patient hospitalized for asthma, MBP levels of sputum were markedly elevated. Treatment with bronchodilators and glucocorticoids caused an increase peak expiratory flow rate, a reduction in blood eosinophils, and a decrease in the serum and sputum levels of MBP. The results indicate that eosinophil granule constituents are released into the bronchi in asthma and that measurement of sputum MBP may be useful in identifying asthma. The possibility that the eosinophil damages bronchial epithelium in asthma is discussed.

Asthma↗
PubMed1979

The effect of ablation of eosinophils on immediate-type hypersensitivity reactions.

The effect of ablation of eosinophils on hypersensitivity reactions in guinea-pigs was tested by administration of rabbit antiserum to the eosinophil (AES) and by administration of glucocorticoids. Both AES and methylprednisolone ablated eosinophils from the blood and peritoneal cavity of test animals. Neither administration of AES nor methylprednisolone, however, altered passive cutaneous or systemic anaphylactic reactions when compared to reactions occurring in control animals treated with normal rabbit serum (NRS). Also there was no consistent effect of AES on the intensity of the Arthus reaction. The effect of ablation of eosinophils on histamine release in the passively sensitized peritoneal cavity of the guinea-pig was also tested. In five experiments a significant reduction in histamine release was seen in AES-treated animals. Ablation of eosinophils by cortisone acetate also resulted in a marked reduction in the quantity of histamine released into the peritoneal cavity following passive sensitization and antigen challenge. Histamine release following intraperitoneal injection of compound 48/80 was not affected by either the prior administration of AES or cortisone acetate, suggesting that the stores of histamine were not depleted by these agents. Overall these results suggest that eosinophils do not play a prominent role in initial expression of immediate-type hypersensitivity reactions where the density of these cells in tissues is low. When present in larger numbers, however, eosinophils may contribute to histamine release in immediate-type reactions.

Anaphylaxis↗
PubMed1978

Localization of the guinea pig eosinophil major basic protein to the core of the granule.

The localization of the guinea pig eosinophil major basic protein (MBP) within the cell was investigated by the use of immunoelectron microscopy and by isolation of the granule crystalloids. First, by immunoperoxidase electron microscopy, we found that the MBP of eosinophil granules is contained within the crystalloid core of the granule. Specific staining of cores was present when rabbit antiserum to MBP was used as the first stage antibody in a double antibody staining procedure, whereas staining was not seen when normal rabbit serum was used as the first stage antibody. Second, crystalloids were isolated from eosinophil granules by disruption in 0.1% Triton X-100 and centrifugation through a cushion of 50% sucrose. Highly purified core preparations yielded essentially a single band when analyzed by electrophoresis on polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The E1%1cm of the core protein was 26.8 +/- 1.0 (X +/- SEM); the E1%1cm for the MBP was 26.3. The core protein could not be distinguished from the MBP by radioimmunoassay (RIA) and essentially all of the protein in the core preparations could be accounted for as MBP. The results indicate that the MBP is contained in the core of the guinea pig eosinophil granule and that it is probably the only protein present in the core.

Animals↗
PubMed1976

Comparative properties of the Charcot-Leyden crystal protein and the major basic protein from human eosinophils.

Guinea pig eosinophil granules contain a protein, the major basic protein (MBP), which accounts for more than half of the total granule protein, has a high content of arginine, and displays a remarkable tendency to form disulfide-linked aggregates. In this study we have purified a similar protein from human eosinophil granules and have compared the human MBP to the protein comprising the Charcot-Leyden crystal (CLC). Eosinophils from patients with various diseases were purified and disrupted, and the granule fraction was obtained. Examination of the granule fraction by transmission electron microscopy showed numerous typical eosinophil granules. Analyses of granule lysates by gel filtration and by polyacrylamide gel electrophoresis revealed the presence of peroxidase and MBP with properties similar to that previously found in guinea pig eosinophil granules. The human MBP had a molecular weight of 9,200, contained less than 1% carbohydrate, was rich in arginine, and readily formed disulfide-bonded aggregates. CLC were prepared from eosinophil-rich cell suspensions by homogenization in hypotonic saline. The supernates following centrifugation of cell debris spontaneously formed CLC. Analysis of CLC revealed the presence of a protein with a molecular weight of 13,000 containing 1.2% carbohydrate. The protein displayed a remarkable tendency to aggregate even in the presence of 0.2 M acetic acid. Human MBP and CLC protein differed in their molecular weights, carbohydrate compositions, and amino acid analyses. Mixtures of the MBP and the CLC protein yielded two bands in polyacrylamide gel electrophoresis. Neither eosinophil protein increased vascular permeability in the guinea pig skin or contracted the guinea pig ileum. The results indicate that the human MBP and the CLC are distinct substances with properties such that one cannot be derived from the other.

Animals↗
PubMed1976

Isolation and partial characterization of a major basic protein from rat eosinophil granules.

The rat eosinophil granule possesses a major basic protein (MBP) similar to that previously isolated from human and guinea pig eosinophils. This conclusion is based on demonstration that the major protein of the rat eosinophil granule is a low molecular weight, highly basic material with an amino acid composition similar to that of the human and guinea pig MBP.

Amino Acids↗
PubMed1975

Reactivity of rabbit antiserum to guinea pig eosinophils.

Although the eosinophil has been recognized as a distinctive cell type for almost 100 years, the major functions of these cells remain unknown. As an approach to defining these functions we have treated guinea pigs with rabbit antiserum to eosinophils (AES) in an attempt to ablate these cells from tissues. Rabbits were immunized thrice with purified eosinophils and the antisera were absorbed with peripheral blood cells from guinea pigs made eosinopenic with methyprednisolone to remove antibodies reactive with serum proteins and erythrocytes. The resulting sera reacted strongly with eosinophils in cytotoxicity tests and had weak or no reactivity with neutrophils. However, absorption of AES with purified neutrophils removed antieosinophil activity. Intraperitoneal injection of potent AES into guinea pigs resulted in complete absence of eosinophils from the peripheral blood and from the peritoneal cavity with only transient or no reduction in circulating neutrophils. Eosinophils were also reduced in bone marrow, spleen, and intestine. The ability of neutrophils to absorb AES activity in spite of weak reactivity in cytotoxicity tests may reflect a quantitative difference in antigenic determinants between eosinophils and neutrophils.

Animals↗
PubMed1974

Physiochemical and biological properties of the major basic protein from guinea pig eosinophil granules.

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

Amino Acids↗
PubMed1973

Identification of a major basic protein in guinea pig eosinophil granules.

Elucidation of the functions of the eosinophil might be accomplished by analysis of the granule constituents. We have purified eosinophils (93% or greater) from the peritoneal cavity of the guinea pig and have investigated a variety of methods to disrupt cells and liberate intact granules. Lysis in 0.34 M sucrose gave the best yield of granules and these had the characteristic morphology of eosinophil granules when examined by electron microscopy. Granules were solubilized by a variety of treatments and the solutions analyzed by polyacrylamide electrophoresis at pH 3 in 6 M urea. Comparison of the electrophoretic patterns of solubilized eosinophil and neutrophil granules revealed a difference: a major portion (53+/-3%; x +/-1 SE) of the protein from the eosinophil granule migrated as a single component. This major band protein has a molecular weight between 6,000 and 12,000 daltons and a pI of 10 or greater. Analysis of eosinophil granule constituents on Sephadex G-50 revealed two main peaks; peak 1 possessed peroxidase activity and peak 2 contained the major band protein. These studies indicate that eosinophil granules contain a cationic protein of low molecular weight which lacks peroxidase activity and which accounts for greater than 50% of granule protein.

Animals↗