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Biomedical subjects

R M Helm

Publications and source records attributed to R M Helm.

At least 37 records · Page 2Linked to original sources
PubMed1995

Comparison of commercial peanut skin test extracts.

BACKGROUND: Skin prick testing is a major tool for diagnosing food allergy. Food allergen extracts have not been standardized; this may lead to great variability in the predictive accuracy of skin prick tests. METHODS: Six commercial peanut skin test extracts were compared in vitro with RAST inhibition assays, ELISA, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with sera from peanut-allergic adults and in vivo by skin prick testing. RESULTS: ELISA showed that the content of peanut allergens Ara h I and Ara h II in the extracts ranged from 0.0015 to 0.0236 and 0.0001 to 0.0164 mg Eq/ml, respectively. RAST inhibition studies showed that the extracts produced curves of similar slope, suggesting conservation of allergenic epitopes. SDS-PAGE revealed differences in protein profiles because roasted extracts generally possessed the same number and proportion of major protein bands but raw extracts varied more in both respects. SDS-PAGE and immunoblotting showed that two of the extracts contained major IgE-binding protein bands that did not appear in the others. One roasted extract gave little protein banding and consequently little IgE binding. CONCLUSIONS: Skin testing results showed no differences in the ability of the extracts to provoke a positive skin test response in peanut-sensitive subjects.

Arachis↗
PubMed1995

Epitope specificity of the major peanut allergen, Ara h II.

The antigenic and allergenic structure of Ara h II, a major allergen of peanuts, was investigated with the use of four monoclonal antibodies obtained from BALB/c mice immunized with purified Ara h II. Our previous studies with monoclonal antibodies generated to peanut allergens showed this method to be useful for epitope mapping. When used as a solid phase in an ELISA, these monoclonal antibodies captured peanut antigen, which bound human IgE from patients with positive peanut challenge responses. The Ara h II monoclonal antibodies were found to be specific for peanut antigens when binding for other legumes was examined. In ELISA inhibition studies with the monoclonal antibodies, we identified two different antigenic sites on Ara h II. In similar studies with pooled human IgE serum from patients with positive challenge responses to peanuts, we identified two closely related IgE-binding epitopes. These characterized monoclonal antibodies to Ara h II will be useful for future studies to immunoaffinity purify the Ara h II allergen and to use in conjunction with recombinant technology for determining structure-function relationships.

2S Albumins, Plant↗
PubMed1995

Frequency of food allergy in a pediatric population from Spain.

We evaluated the prevalence and characteristics of the principal foods implicated in 355 children diagnosed with IgE-mediated food allergy. Diagnosis was established on the basis of positive clinical history for the offending food, positive specific IgE by skin prick test and RAST, and open food challenge. Our results showed the principal foods involved in allergic reactions are: eggs, fish, and cow's milk. These are followed in frequency by fruits (peaches, hazelnuts and walnuts), legumes (lentils, peanuts and chick peas) and other vegetables (mainly sunflower seeds). The legumes demonstrated the highest degree of clinical cross-reactivity. Most patients with food allergy reacted to one or two foods (86.7%). Only 13.3% of patients reacted to 3 or more foods, mostly to legumes and fruits. We found that food allergy begins most frequently in the first (48.8%) and second (20.4%) years of life. Allergy to proteins of cow's milk, egg, and fish begins predominantly before the second year, demonstrating a clear relationship with the introduction of these foods into the child's diet. Allergy to foods of vegetable origin (fruits, legumes and other vegetables) begins predominantly after the second year.

Age Factors↗
PubMed1995

Recombinant peanut allergen Ara h I expression and IgE binding in patients with 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 E. 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

Adult↗
PubMed1994

The production of interferon-gamma in response to a major peanut allergy, Ara h II correlates with serum levels of IgE anti-Ara h II.

The current study was undertaken to examine the potential role of T cells in the pathogenesis of peanut allergy. Peripheral blood mononuclear cells (PBMCs) from patients with peanut allergy, patients with asthma, and nonatopic normal control subjects were assessed for proliferation after stimulation with a 17 kd major peanut allergen (Ara h II), ovalbumin, casein, soy, and Candida albicans. We found that Ara h II and C. albicans induced significantly higher levels of proliferation than ovalbumin, casein, and soy. Because interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) play critical roles in IgE regulation, we assessed the production of these cytokines after stimulation with C. albicans and Ara h II. C. albicans stimulated similar levels of IFN-gamma in all three study groups. In contrast, after stimulation with Ara h II, culture supernatants from PBMCs of subjects with peanut allergy contained significantly lower levels of IFN-gamma than did the PBMCs of the two control groups (p = 0.02). More important, there was a significant (p = 0.05) inverse correlation between the serum IgE anti-Ara h II levels and IFN-gamma production by PBMCs from the respective peanut-allergic patients. IL-4 protein was not detected in culture supernatants of PBMCs stimulated with Ara h II. However, amplification of cytokine gene transcripts by polymerase chain reaction did demonstrate IL-4 expression in Ara h II-stimulated PBMCs from both patients with peanut allergy and control subjects. These data suggest that the level of IFN-gamma production in response to Ara h II may be an important factor in determining the development of peanut-specific IgE responses.

2S Albumins, Plant↗
PubMed1994

Epitope specificity and immunoaffinity purification of the major peanut allergen, Ara h I.

The antigenic and allergenic structure of Ara h I, a major allergen of peanuts, was investigated with the use of seven monoclonal antibodies obtained from BALB/c mice immunized with purified Ara h I. Previous work with monoclonal antibodies produced to allergens has primarily been done with inhalant allergens. Only recently have the major allergens of various foods been determined so that investigations with monoclonal antibodies into the allergenic epitopes could begin. When used as a solid phase in an ELISA, these monoclonal antibodies captured peanut antigen, which bound human IgE from patients with positive results to challenges to peanuts. The Ara h I monoclonal antibodies were found to be specific for peanut antigens when binding for other legumes was examined. In ELISA inhibition studies with the monoclonal antibodies, we identified four different antigenic sites on Ara h I. In related studies with pooled human IgE serum from patients with positive results to challenges to peanuts, we identified three similar IgE-binding epitopes. As a means of purifying the Ara h I allergen, we prepared an immunoaffinity column with monoclonal antibody 8D9. We eluted from this column the allergen Ara h I, which had a mean molecular weight of 63.5 kd and which bound human IgE from individual and pooled serum of patients with peanut sensitivity.

Allergens↗
PubMed1994

Isolation of the 36-kD German (Blattella germanica) cockroach allergen using fast protein liquid chromatography.

Cockroach allergens have been established as important sensitizing agents in the induction of asthma. In the present investigation, we identified a 36-kD allergen from German cockroach (Blattella germanica) using fast protein liquid chromatography that reacted in the Bla g II monoclonal assay developed by Pollart et al. Defatted whole body German cockroaches were extracted in phosphate-buffered saline and fractionated using gel filtration (Superdex 75) and anion exchange (MonoQ) chromatography. The 36-kD allergen was isolated from this enriched allergen preparation by excision from modified preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, electroelution, dialysis and lyophilization. SDS-PAGE/Western immunoblot analysis using a serum pool of cockroach-sensitive individuals revealed a single band. The lyophilized 36-kD allergen was then analyzed for amino acid composition and digested for peptide amino acid sequencing.

Allergens↗
PubMed1994

Identification of peanut agglutinin and soybean trypsin inhibitor as minor legume allergens.

Peanuts and soybeans are frequent causes of food hypersensitivity reactions in children. Sera from 12 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to peanut and sera from 5 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to soybean were used to identify and characterize specific legume allergens. Identification of a minor allergen from peanut and a minor allergen from soybean was accomplished using various physicochemical techniques. The peanut fraction, peanut agglutinin, isolated by anion-exchange chromatography and electrolution and confirmed by amino acid sequencing, bound IgE in only 50% of the peanut challenge positive patients. The soybean fraction, soybean trypsin inhibitor, identified by gel filtration and electroelution and confirmed by amino acid sequencing, bound IgE in only 20% of the soy challenge positive patients. The identification of these two known legume proteins as minor allergens should allow further immunologic and structural investigations to compare the major and minor legume allergens.

Allergens↗
PubMed1993

Identification of cockroach aeroallergens from living cultures of German or American cockroaches.

The Air Sentinel and polytetrafluoroethylene (PTFE) membranes were used to capture airborne particles over living colonies of German or American cockroaches. Silver-stained SDS-PAGE gels revealed protein bands at 80, 55, 36, and several bands below the 33-kD marker. SDS-PAGE/immunoblots of PTFE eluates from German cockroach colonies incubated with serum from cockroach-sensitive individuals revealed IgE-binding bands with apparent molecular weights of 36 and 80 kD. Only the 36-kD allergen and allergens below the 33-kD marker were evident in the American PTFE eluate. ELISA analysis with a monoclonal antibody assay identified the presence of both Bla g I and Bla g II in the German PTFE eluate. No Bla g I or Bla g II could be identified in the American PTFE eluate. These studies demonstrate that in addition to Bla g I and Bla g II, several other aerosolized allergens become airborne over cockroach colonies and may be important in the environment where cockroaches are abundant.

Air Pollution↗
PubMed1992

Allergenicity of peanut and soybean extracts altered by chemical or thermal denaturation in patients with atopic dermatitis and positive food challenges.

Peanuts and soybeans are two of the six most common foods to cause food hypersensitivity reactions in children. We used the serum of 10 patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to peanut and two patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to soybean to investigate the change in IgE-specific and IgG-specific binding to these proteins altered by either chemical or thermal denaturation. We used IgE- and IgG-specific ELISA-inhibition analyses to compare these effects on the crude peanut and crude soy extracts, as well as on the major allergenic fractions of both proteins. Heating the soy proteins at various temperatures and time intervals did not significantly change the IgE- or IgG-specific binding of the soy positive pooled serum. When the peanut proteins were subjected to similar heating experiments, the IgE- and IgG-specific binding did not change. When these same proteins were treated with enzymes in the immobilized digestive enzyme assay system used to mimic human digestion, the binding of IgE to the crude peanut and crude soy extracts was reduced; 100-fold for peanut and 10-fold for soybean. Therefore it appears that thermal denaturation of peanut and soybean protein extracts does not enhance or reduce IgE- and IgG-specific binding activity. Chemical denaturation appears to minimally reduce the binding of these proteins.

Adolescent↗
PubMed1992

Identification and characterization of a second major peanut allergen, Ara h II, with use of the sera of patients with atopic dermatitis and positive peanut challenge.

Peanuts are frequently a cause of food hypersensitivity reactions in children. Serum from nine patients with atopic dermatitis and a positive double-blind, placebo-controlled, food challenge to peanut were used in the process of identification and purification of the peanut allergens. Identification of a second major peanut allergen was accomplished with use of various biochemical and molecular techniques. Anion exchange chromatography of the crude peanut extract produced several fractions that bound IgE from the serum of the patient pool with positive challenges. By measuring antipeanut specific IgE and by IgE-specific immunoblotting we have identified an allergic component that has two closely migrating bands with a mean molecular weight of 17 kd. Two-dimensional gel electrophoresis of this fraction revealed it to have a mean isoelectric point of 5.2. According to allergen nomenclature of the IUIS Subcommittee for Allergen Nomenclature this allergen is designated, Ara h II (Arachis hypogaea).

2S Albumins, Plant↗
PubMed1991

Identification of a major peanut allergen, Ara h I, in patients with atopic dermatitis and positive peanut challenges.

Peanuts are among the most common causes of immediate hypersensitivity reactions to foods. Serum from nine patients with atopic dermatitis and a positive double-blind, placebo-controlled, food challenge to peanut were used to begin the process of identification and purification of the major peanut allergens. Identification of a major peanut allergen was accomplished by use of anion-exchange column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ELISA, thin-layer isoelectric focusing, and IgE-specific immunoblotting. Anion-exchange chromatography revealed several fractions that bound IgE from the serum of the challenge-positive patient pool. By measuring antipeanut-specific IgE in the ELISA and in IgE-specific immunoblotting, we identified an allergenic component with two Coomassie brilliant blue staining bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a mean molecular weight of 63.5 kd. Examination of this fraction by the IgE antipeanut ELISA with individual serum and by the ELISA-inhibition assay with pooled serum, we identified this fraction as a major allergen. Thin-layer isoelectric focusing and immunoblotting of this 63.5 kd fraction revealed it to have an isoelectric point of 4.55. Based on allergen nomenclature of the IUIS Subcommittee for Allergen Nomenclature, this allergen is designated, Ara h I (Arachis hypogaea).

Allergens↗
PubMed1990

Identification and partial characterization of the soybean-dust allergens involved in the Barcelona asthma epidemic.

Asthma epidemics in Barcelona, Spain, have been attributed to dust generated by the unloading of soybeans in the harbor. Sera of four different groups of 10 subjects in each group were studied: (1) patients attending an emergency room in Barcelona for an asthma attack on epidemic days, group A, (2) patients attending an emergency room for an attack on nonepidemic days, group B, (3) patients with asthma from other cities, group C, and (4) patients without asthma from Barcelona matched by age and sex with group A, group D. All subjects in group A had IgE to allergens in extracts of various soybean samples. In contrast, only one of the 10 subjects in each of groups B and C and none of those subjects in group D had IgE to uncleaned bean and hull extracts. Radioimmunoassay demonstrated that in sera from patients with asthma during an asthma outbreak reacted primarily to soybean hull and dust extracts. Sodium dodecyl sulfate-polyacrylamide and gel electrophoresis thin-layer isoelectrofocusing demonstrated protein bands of 97.4 to less than 14.4 kd and isoelectric point between 6 and 3.5. By Western blot and thin layer isoelectrofocusing/blotted radioimmunoisoelectrofocusing, IgE of patients with asthma during an asthma outbreak reacted weakly to two protein bands of molecular weight ranging from 42 to 21 kd, strongly to glycoprotein bands with molecular weight less than 14.4 kd, and isoelectric point less than 6, which appeared to be the major allergens.

Adolescent↗
PubMed1990

Biochemical and immunochemical comparison of Africanized and European honeybee venoms.

Africanized honeybees (HBs) pose a hazard to both normal and sting-sensitive subjects in certain areas of Central and South America, and it is predicted that they will soon be present in the southern United States as well. Using an electrical stimulation device, we collected Africanized HB venom (AHV) in Venezuela and European HB venom (EHV) in Louisiana. These venoms, along with commercial European HB venom (CHV), were compared by thin-layer isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Coomassie brilliant blue and silver-stained banding patterns of AHV and EHV were essentially identical to CHV. Western blots were prepared from SDS-PAGE gels and tested with pooled sera from EHV-sensitive subjects and then radiolabeled antihuman IgE. The resulting autoradiographs revealed similar banding patterns among EHV, AHV, and CHV. RAST-inhibition studies were performed with solid-phase CHV and pooled sera from EHV-sensitive subjects. The specific allergenic activities of the three HB venoms (allergy units per milligram of protein) were comparable. By RAST-inhibition assay with solid-phase, highly purified individual venom components, AHV and EHV both contained phospholipase A2, hyaluronidase, and high-molecular-weight allergens. The increased morbidity after Africanized HB stings is likely related to their more defensive behavior during which many bees react by stinging rather than to biochemical or allergenic differences between AHV and EHV.

Allergens↗
PubMed1990

Shared allergenic activity in Asian (Blattella asahinai), German (Blattella germanica), American (Periplaneta americana), and Oriental (Blatta orientalis) cockroach species.

Whole-body extracts of the feral and peridomestic Asian cockroach (Blattella asahinai) and the three domestic cockroach species, German (Blattella germanica), American (Periplaneta americana), and Oriental (Blatta orientalis), were compared allergenically using an IgE serum pool from 4 German cockroach sensitive individuals. In crossover radioallergosorbent inhibition analysis, the Asian cockroach shared allergenic activity primarily with the German cockroach polymer and to a lesser extent with either the American or Oriental cockroach polymers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thin-layer isoelectric focusing analysis of the extracts showed similar although varying intensities of Coomassie blue stained banding patterns among five extracts analyzed. Electroblotting analysis with 12.5% SDS-PAGE of the whole-body German cockroach extract and IgE serum from individuals sensitive to German cockroach revealed eight allergenic components with apparent molecular weights of 92, 80, 67, 48, 36, 27, 25 and 18 kD. Five components could be identified in the whole-body extract of the Asian cockroach corresponding to apparent molecular weights of 92, 67, 48, 40, and 32 kD. Analysis of individual serum by immunoblot analysis with each of the cockroach extracts showed considerable heterogenicity in the IgE-binding pattern. Although the Asian cockroach demonstrated considerable cross-reacting allergenic components to German and relatively fewer cross-reacting allergenic components to either the Oriental or American, it is too early to establish genus- or species-specific cockroach allergens. It is important to point out that German cockroach sensitive individuals should be made aware of the potential exposure of Asian cockroach aeroallergens both indoors and outdoors in areas with high infestations of Asian cockroaches.

Allergens↗
PubMed1988

Production of a proposed international reference standard Alternaria extract. II. Results of a collaborative trial.

A lyophilized Alternaria extract prepared from a defined eight-strain source material was compared with three other Alternaria extracts to assess its potential suitability as an international standard (IS). Twenty-two laboratories in 12 different countries collaborated. Assay methods included RAST inhibition, quantitative immunoelectrophoresis, thin-layer isoelectric focusing, skin testing, leukocyte histamine release, and various other methods for thorough characterization of the extracts. In addition, three laboratories quantitated specific allergen in each extract. The proposed IS extract could be used to assign a relative potency to other test extracts. In separate studies, the proposed IS extract was demonstrated to be stable for at least 21 months when it is stored at -70 degrees C, -20 degrees C, and 4 degrees C.

Alternaria↗
PubMed1988

Identification of a German cockroach-specific allergen by human IgE and rabbit IgG.

To further study the immunology of insect hypersensitivity, we identified and partially characterized the principal allergens in whole body German cockroach (WBGCR) (Blattella germanica) and compared this extract to whole body antigens prepared from other insects. WBGCR extract was fractionated over a calibrated Sephadex G-200 column; peak allergenic activity was contained in fraction 3 (GCR3), containing components with apparent molecular weights ranging from 12,500 to 75,000 daltons. The antigenicity, allergenicity, and specificity of GCR3 components were tested by using rabbit antisera raised to GCR3 or true armyworm (Pseudaletia unipuncta). Radioimmunoassay, cross-inhibition and immunoblot studies revealed, particularly in the IgE system, that GCR3 contained an allergen with a pI of 6.7 and MW of 36,000 daltons that was unique to WBGCR extracts and not present in other insect species, including true armyworm, caddis fly, lakefly, yellow jacket, or honeybee. This GCR3 component may represent a specific marker for the diagnosis of cockroach hypersensitivity in an insect-sensitive population of individuals.

Allergens↗