Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting- induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms.

BACKGROUND: Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. OBJECTIVE: To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. METHODS: Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. RESULTS: sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. CONCLUSION: We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

Haptoglobin interacts with the human mast cell line HMC-1 and inhibits its spontaneous proliferation.

Haptoglobin (Hp) is an acute phase reactant produced by hepatocytes. There is evidence for an immunomodulatory potential of Hp, though there is no clear evidence yet about the mechanisms of this action. We have previously shown that Hp interacts with the beta2-integrin CD11b/CD18. In addition, other investigators reported the binding of Hp to B lymphocytes through the CD22 receptor, and to neutrophils through two different receptors. In the present study, we investigated the interaction of haptoglobin with the human mast cell line HMC-1. We report that fluorescein isothiocyanate (FITC)-labelled haptoglobin binds to this cell line and that binding is increased by calcium in a dose- and time-dependent manner. Hp binding sites on HMC-1 were upregulated upon stimulation with phorbol myristate acetate (PMA)/A23187 and after treatment with anti-CD43 and anti-CD44 monoclonal antibodies (MoAbs). HMC-1 cells do not express either CD11b/CD18 or CD22 receptors, indicating that the haptoglobin-binding receptor on this cell line is different from the known receptors. Assessment of cell function showed that Hp inhibits the spontaneous growth of HMC-1 up till 40% at higher Hp concentrations, but it did not exhibit any effect on the expression of CD54 on the release of either tryptase or IL-1ra. In conclusion, haptoglobin binds specifically to human mast cells via a receptor different from CD11b/CD18 and CD22, and may play a role in the modulation of mast cell functions. Exploration of Hp effects in mast cell-dependent diseases such as allergic rhinitis and urticaria seems warranted.

Composition and stability of allergenic extracts made from gamma-irradiated rye grass (Lolium perenne) pollen.

BACKGROUND: Phenol is commonly added to allergenic extracts as a bacteriostatic agent, but it is poisonous and also detrimental to proteins, which accelerates extract degradation. Sterilization by gamma-irradiation of the source material could be an alternative to the use of phenol. OBJECTIVE: To analyse the potential effects of gamma-irradiation of pollen on the composition, potency, and stability of the resulting extract, and compare them with those of phenol. METHODS: Ryegrass (Lolium perenne) pollen was sterilized by gamma-irradiation at a dose of 25 kGy. Extracts prepared from the irradiated pollen were then compared by electrophoresis techniques and RAST inhibition to extracts, without or with 0.5% phenol, from nonirradiated pollen. In addition, proteolytic activity was compared in extracts from irradiated and nonirradiated pollen. To evaluate the stability of extracts on storage, they were analysed after forced degradation for up to 7 days at 37 degrees C. RESULTS: When fresh extracts were analysed, there were no noticeable differences between the three types, as judged by immunoblotting and RAST inhibition experiments. However, on storage, extracts from irradiated pollen appeared to be superior to extracts from nonirradiated pollen, as some proteins were more stable in the former. This could be related to the lower proteolytic activity we have also observed in extracts from irradiated pollen. In contrast, extracts containing phenol degraded much faster, as proven by all our methods of investigation. CONCLUSION: Gamma-irradiation of pollen did not influence the IgE-binding capacity of the resulting extracts, but did yield extracts with somewhat improved stability, probably by reducing the proteolytic activity. It may be concluded that gamma-irradiation of the source material represents a good alternative to the use of phenol for the preparation of allergenic extracts.

Allergenic and antigenic activity of peptide fragments in a whey hydrolysate formula.

BACKGROUND: Milk hydrolysates, although frequently used as substitutes in cases of cow's milk allergy, show a reduced but never a complete abolishment of antigenicity and allergenicity. OBJECTIVE: Our purpose was to determine the lower molecular weight limit of peptides to elicit skin reactions and to bind IgE antibodies in vitro. METHODS: Using FPLC, an ultrafiltrated whey hydrolysate, was fractionated in different molecular weight fractions. Skin-prick tests were performed with the hydrolysate and its fractions in five cow's milk allergic children, and RAST inhibition tests were done using the serum of these children. RESULTS: On the basis of the lowest extinction values between two peaks of the chromatogram, seven fractions with molecular weights between 15000 and 125 Da were obtained. Peptides of > 2600 Da elicited a clearly positive skin reaction and inhibited IgE-binding, while peptides of < 1400 Da did not give any positive skin reaction but were still able to inhibit to a small extent IgE-binding to the hydrolysate. CONCLUSION: Our findings suggest that for skin reactivity peptides of > 1400 Da are needed. The minimal molecular mass for IgE binding in vitro appears to be situated between 1400 and 970 Da. Such peptides might be used to develop a safe formula for patients reacting to milk hydrolysates or even for tolerance induction.

Allergenic proteins in different brands of latex and synthetic medical examination gloves.

The steady increase in anaphylactic reactions to latex medical gloves has raised increasing awareness in the medical community. Even gloves claimed to be hypo-allergenic still may contain substantial amounts of IgE-binding proteins. We have studied non-powdered latex and synthetic examination glove extracts for their allergenicity using the immunoblot technique. Protein levels varied considerably among glove extracts and the amount did not always correlate with the presence of allergenic proteins. IgE binding proteins were found in 2 of the 7 powder-free latex glove brands. Synthetic glove extracts did not contain allergens. The study demonstrates that immunoblot analysis is a useful technique in order to select gloves with a minimal risk to raise IgE antibodies.

Determination of independent risk factors and comparative analysis of diagnostic methods for immediate type latex allergy in spina bifida patients.

BACKGROUND: Development of allergy to natural rubber latex in spina bifida patients is determined by several risk factors, such as age, number of interventions and atopic disease that are, however, interdependent. Furthermore, several diagnostic procedures have been analysed, but a comprehensive analysis of their diagnostic significance is lacking. OBJECTIVE: To determine the independent major risk factor(s) for development of natural rubber latex allergy and the most valuable diagnostic procedure. METHODS: In aselectively collected spina bifida patients, we correlated existing natural rubber latex allergy with age, sex, atopy and the number of hospitalizations and of surgical interventions in appropriately matched subgroups. Allergy to natural rubber latex was established by application of a latex glove fragment on the skin. Skin-prick tests with glove eluate, a natural latex extract and a commercial latex extract were carried out as were specific IgE measurements by radioimmuno assay (RAST-CAP). The results of the latex application test are compared with the other diagnostic methods. RESULTS: Out of 74 fully evaluated patients, 17 had a positive application test. The number of surgical interventions correlates strongly with the presence of natural rubber latex allergy (P<0.0002), independent of age, sex and presence of atopy. Skin-prick tests with unstandardized allergens made from known high allergenic latex gloves represent the most sensitive diagnostic method, with the highest negative predictive value and a specificity of 0.95. RAST-CAP was the next best method with a specificity of 0.93, a sensitivity of 0.89 and a negative predictive value of 0.97. CONCLUSION: The number of surgical interventions is the major independent determining factor for allergy to natural rubber latex in spina bifida patients. Unstandardized skin-prick tests are the most sensitive and specific diagnostic tool, but RAST-CAP is almost equally performant and therefore a valid alternative.

Identification of haptoglobin as an alternative ligand for CD11b/CD18.

Haptoglobin is an acute phase protein with presumed anti-inflammatory activities. We report that purified fluorescein-labeled haptoglobin 1-1 binds to THP1 and U937 promonocytic cell lines, to monocytes, to granulocytes, and to a subset of CD8+ T cells and to NK cells. Studies with radioiodinated haptoglobin on THP1 cells were consistent with specific binding to one class of receptors with a density of 1.7 x 10(5) binding sites per cell and a low affinity of 6.5 x 10(-6) Kd. Binding was increased by Ca2+ and by Ca2+ and ADP. Binding to THP1 and U937 cells could be inhibited by preincubation with nonfluoresceinated haptoglobin and by fibrinogen, but not by albumin, transferrin, or alpha1-acid glycoprotein. Fibrinogen binds to the CD11b/CD18 integrin. We therefore examined whether haptoglobin has the same receptor. The anti-CD11b mAb44 indeed inhibited the binding of fluoresceinated haptoglobin to THP1 and U937 cell lines, and haptoglobin inhibited the binding of the anti-CD11b mAb anti-Leu15 and mAb44 to both cell lines. An anti-CD18 mAb partly inhibited the binding of fluoresceinated haptoglobin to THP1 and U937, indicating that the beta-chain of MAC-1 is also involved in haptoglobin binding. There was no interference between the binding of anti-CD4, anti-CD11a, or anti-CD11c mAb and haptoglobin binding to THP1 cells. Binding of haptoglobin to purified CD11b/CD18 indicates that it binds directly to the receptor. Haptoglobin is an alternative low affinity ligand for the CD11b/CD18 integrin, suggesting that this acute phase protein might regulate MAC-1-dependent cell function in vivo.

Influence of the pH of the extraction medium on the composition of birch (Betula verrucosa) pollen extracts.

Extracts from birch (Betula verrucosa) pollen were prepared at different pH, with constant pH monitoring and adjustment to preset values in the range 5.5-8.5. The total protein content of these extracts was directly correlated with the pH. Coomassie brilliant blue-stained isoelectric focusing and SDS-PAGE gels and immunoblot analysis demonstrated qualitative differences: some proteins were lost while others appeared when pH was changed. At pH 8.5, formerly unknown birch pollen allergens were detected with pI 9, 9.10, and 9.30 by about 30% of birch pollen-sensitive sera. Birch pollen extracts prepared at a pH close to neutrality, namely, 6.5 and 7.5, showed the greatest protein and different allergen diversity. Thus, extraction pH values are necessary to analyze the whole pattern of allergenic components in an extract.

Analysis of the stabilizing effect of epsilon-aminocaproic acid by electrophoretic techniques and immunoblotting.

The effect of epsilon-aminocaproic acid (EACA) on the degradation of aqueous pollen extracts was studied by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. The extracts were stored at 4 degrees C for 7 days and at 37 degrees C for 1, 4, and 7 days. Addition of 0.1 mol/L of EACA before storage partly protected the extract from degradation. The protective effect of EACA could be demonstrated most clearly by immunoblotting, suggesting that more epitopes were preserved in an antigenic configuration. The stabilizing effect increased with higher EACA concentrations.

Stabilizing effect of epsilon-aminocaproic acid on allergenic extracts.

The effect of epsilon-aminocaproic acid (EACA) on the degradation of an aqueous Lolium perenne extract was studied by intracutaneous tests and by RAST inhibition. Extracts for skin testing stored at 4 degrees C for 12 months and at 37 degrees C for 6 weeks were significantly protected from degradation by addition of 0.1 mol/L of EACA before storage. Extracts were stored for RAST inhibition at 4 degrees C for 6 months and at 37 degrees C for 7 days. Shelf life was twofold to threefold increased when 0.1 mol/L of EACA was added to the dilution medium. EACA also protected the extracts from the effect of freezing and thawing. Comparison with the effect of human serum albumin indicated a rather short activity of human serum albumin, whereas the effect of EACA lasted longer. It is suggested that EACA can be used to increase the stability of aqueous allergen extracts for skin testing and for hyposensitization therapy.