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R M Helm

Publications and source records attributed to R M Helm.

53 records · Page 3Linked to original sources
PubMed1987

Production of an international reference standard alternaria extract. I. Testing of candidate extracts.

As part of a program to establish international standards of selected allergens, 6 coded extracts of Alternaria were assessed in 6 laboratories by immunochemical, biochemical and physicochemical procedures. Direct RAST, RAST inhibition, quantitative skin tests and leukocyte histamine release were used to assign relative orders of potency to the 6 extracts. The composition and major allergen content was tested by thin-layer isoelectric focusing and quantitative immunoelectrophoresis (crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis). Three laboratories determined the quantity of purified allergens in each of the preparations. In addition, source materials were sent to an expert Alternaria taxonomist for independent identification. The results showed considerable variation with respect to total allergenic potency and content of individual allergens. Source materials could not be confirmed as Alternaria in some instances. Based on fulfillment of written specifications and assay results, extract No. 6 was recommended by the Alternaria Working Group as the candidate international standard to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies.

Allergens↗
PubMed1986

The preparation and testing of the proposed International Reference (IRP) Bermuda grass (Cynodon dactylon)-pollen extract.

A lyophilized candidate extract of Bermuda-grass (Cynodon dactylon) pollen, intended for use as an International Reference Standard, was prepared by pooling four individual candidate extracts. In preliminary investigations, the four candidate extracts encompassed a variety of extraction methods. The collaborative testing program simultaneously analyzed the proposed reference and the individual extracts and included 11 laboratories performing RAST inhibition, histamine release, crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, isoelectric focusing, sodium dodecyl sulfate gel electrophoresis, and protein determinations with a variety of reagents and methods. The four candidate extracts and the pooled reference were found to be equivalent. The stability of this extract has also been studied. This International Reference Preparation of Bermuda grass-pollen extract should be useful for research and industry.

Allergens↗
PubMed1986

Variability of IgE protein measurement in cell-culture supernatants: results from a multicenter collaborative study.

The sensitivity, specificity, and precision of immunoassays for quantitation of IgE in cell-culture supernatants were tested in a multicenter trial involving 22 laboratories. Fourteen coded test samples included cell-culture medium alone, culture medium with varying concentrations of polyclonal or myeloma IgE, and medium from unstimulated or pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cell (PBMC)-culture supernatants. Two laboratories reported measurable IgE in non-IgE-containing control samples. Although the IgE content of a single 0.50 ng/ml polyclonal IgE sample should have been measured easily by the claims of all laboratories, only 13 laboratories measured IgE in this sample in 22 of the 48 assays. Most laboratories could measure both polyclonal and myeloma IgE at 5.0 ng/ml; however, the IgE determinations for the myeloma proteins were nearer the predicted value. Although 15 laboratories found measurable IgE in the PWM-stimulated PBMC-culture supernatants from a single nonatopic donor, the levels did not differ significantly from that measured in unstimulated PBMC-culture supernatants. Three laboratories reported considerably higher IgE levels in the PWM-stimulated PBMC-culture supernatants than in other PBMC-culture supernatants. Only 13 laboratories could quantitate IgE in each of coded duplicate samples containing 0.50 ng/ml polyclonal IgE. These findings indicate a wide variability in the sensitivity, specificity, and precision of assays used to quantitate low levels of IgE protein. Investigators should be encouraged to make greater use of existing national and international reference materials in the standardization and performance of their IgE immunoassays.

Double-Blind Method↗
PubMed1984

Production and testing of an international reference standard of short ragweed pollen extract.

A lyophilized candidate International Reference Standard of short ragweed pollen extract was prepared by use of defined source material. In preliminary experiments, this extract was demonstrated by RAST inhibition and crossed radioimmunoelectrophoresis assays to contain several well-characterized ragweed allergens and to contain multiple antigenic bands by crossed immunoelectrophoresis analysis. In a subsequent multinational collaborative study involving 12 laboratories in five countries, the candidate extract was compared with existing national reference or commercial ragweed extracts by a variety of immunochemical, biochemical, and physicochemical procedures. The candidate extract could be used to assign relative orders of potency to the comparison-test extracts. In separate studies, the candidate extract was demonstrated to be stable when it was stored at either -20 degrees C or +5 degrees C for at least 2 yr. The candidate extract has been accepted as an International Reference Standard with an assigned arbitrary potency of 100,000 units per ampule .

Humans↗
PubMed1984

Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells.

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.

Actins↗
PubMed1982

Comparison of the receptors for IgE of various rat basophilic leukaemia cell lines. I. Receptors isolated by IgE-sepharose and IgE and anti-IgE.

REceptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of IgE-Sepharose or IgE and anti-IgE, and characterized by SDS-polyacrylamide gel electrophoresis. All cell lines were found to be associated with a receptor molecule (R) which could be isolated either with IgE-Sepharose or IgE and anti-IgE and a second receptor (H) which could only be isolated with the aid of IgE-Sepharose. The relative amounts of these two molecules, as isolated from surface iodinated cells, varied from the RBL cell line to the other and their apparent molecular weights were not identical on all cell lines. Since comparisons were made on the same gel using receptors isolated from cells labelled with different isotopes of iodine, differences in molecular weight must be considered as being intrinsic and not due to methodological variations. These results provide an explanation why differences were observed among receptors for IgE as characterized in various laboratories. In spite of the fact that the various RBL cell lines originated from the same chemically-induced tumour they have, over the years, undergone changes which are reflected in the receptors for IgE.

Animals↗
PubMed1982

Comparison of the receptors for IgE of various rat basophilic leukaemia cell lines. II. Studies with different anti-receptor antisera.

Receptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of various anti-receptor antisera, and characterized by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Most antisera were shown to react with a receptor designated H and which, as shown previously, could be isolated by IgE-Sepharose but not by a combination of IgE and anti-IgE. Only two highly purified anti-receptor antibody preparations, which had been purified by adsorption to and elution from rat basophilic leukaemia cells, reacted primarily with a second kind of receptor molecule which had previously been designated R. Some indirect evidence was obtained which suggests that H is a highly immunogenic molecule. It was confirmed that the apparent molecular weight of H-like molecules varied on different RBL cell lines.

Animals↗
PubMed1981

Binding of the receptors for IgE by various lectins.

Two receptors for IgE, having apparent molecular weights of 55,000 and 45,000 daltons were isolated from rat basophilic leukaemia cells by means of IgE-Sepharose. Both molecules were bound by concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin (castor bean lectin). The lectins of pea and gorse origin only bound the 45,000 dalton receptor.

Animals↗
PubMed1981

The incorporation of tritiated precursors into receptors for IgE of rat basophilic leukemia cells.

It has previously been shown that two receptors, H and R, having apparent mol. wt of 55,000 and 45,000 daltons, can be isolated from surface iodinated RBL cells. In addition, small amounts of a third molecule of 71,000 daltons (71 K) can now be detected in receptor preparations isolated by means of IgE-Sepharose. The SDS-PAGE patterns show that the three surface molecules were not significantly altered when cell solubilization was carried out in the presence of protease inhibitors. The same three molecules were also observed when cells were biosynthetically labelled with [3H]-fucose of [3H]-galactose. When cells were labelled with [3H]-leucine or [3H]-tyrosine, several additional molecules were observed in IgE-Sepharose purified preparations. When cells were exposed to free IgE- before solubilization, significant inhibition of binding to IgE-Sepharose could only be seen among molecules corresponding to H, R and 71 K. When receptors were isolated by means of DNP-IgE and anti-DNP-Sepharose, fewer molecules were found. One, which was frequently observed, had a mol. wt of 26,000 daltons. This molecule may be non-covalently associated with receptors for IgE.

Amino Acids↗
PubMed1979

Lentil-lectin affinity chromatography of surface glycoproteins and the receptor for IgE from rat basophilic leukemia cells.

Surface proteins and glycoproteins of RBL cells were labelled enzymatically with 125I and then solubilized with Nonidet P-40. Analysis by polyacrylamide-gel electrophoresis in SDS on 10% gel revealed 10 distinctive peaks ranging in molecular weights from 17,000 to 200,000 daltons. Mainly components of higher molecular weights were bound by lentil-lectin Sepharose and could be eluted with alpha-methyl mannoside. The receptor for IgE was clearly shown to bind to the lentil-lectin. A second cell surface component which previously had been shown to bind to IgE-Sepharose as well, was found to bind only slightly to lentil-lectin. Thus, it can be concluded that the receptor for IgE is a glycoprotein with mannose and/or N-acetylglucosamine in the carbohydrate moiety(s).

Animals↗
PubMedDate not supplied

Flow cytometric analysis of lymphocytes from rats following chronic ethanol treatment.

The current investigation focused on lymphoid cell populations of adult male Sprague-Dawley rats fed a total enteral nutrition diet in which ethanol provided 38% of the total calories. Rats received the National Research Council (NRC) recommended daily intake of nutrients 35 days. An evaluation of lymphocyte populations from peripheral blood demonstrated a decrease in the absolute number of B cells (p < or = 0.007) and absolute numbers of CD4 T cells (p < or = 0.06) in the ethanol-treated animals. Spleen and thymus weights were significantly reduced (p = 0.0001) in the ethanol-treated rats and the CD4/CD8 ratio of splenic lymphocytes decreased in the ethanol group (p < or = 0.03). Thymus T-cell recovery from the ethanol-treated group was significantly reduced with no apparent redistribution in subset numbers with the exception of a minor, yet significant, decrease (p < or = 0.05) in the CD4/CD8 ratio. These data are the first to demonstrate that chronic alcohol intake alters lymphoid cell populations in the peripheral blood and primary organs of the immune systems in the presence of adequate nutrition.

Animals↗
PubMedDate not supplied

Isolation, identification, and characterization of clones encoding antigens responsible for peanut hypersensitivity.

Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens. One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I-specific oligonucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly A+RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in Escherichia coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity. With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.

Allergens↗
PubMedDate not supplied

Fatal anaphylactic reactions induced by peanuts.

Peanuts are a common cause of food allergy, but they have infrequently been documented as causing fatal anaphylactic reactions. We review five previously reported fatalities caused by peanut allergy, along with data on IgE binding proteins in extracts of a commercial product containing peanuts that have been deflavored and reflavored and colored to resemble walnuts, pecans, or almonds. Ingestion of this product may pose hazards to peanut-sensitive persons. Finally, we identify several factors that may contribute to the severity and possible lethality of food-induced anaphylaxis.

Adolescent↗