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Notes: Background There is general agreement that proteins eluting from different natural rubber latex products can cause immediate type hypersensitivity reactions in latexallergic patients. However, there is as yet no consensus as to what are the most important allergens in natural rubber latex.Objective We wanted to purify and characterize at the primary structure level three natural latex proteins, suggested to represent significant allergens.Methods Proteins were purified from ultracentrifuged bottom fraction of natural rubber latex using high performance liquid chromatography gel filtration and reversed phase chromatography. Purified proteins were subjected to tryptic cleavage, peptide separation and amino acid sequencing. Immunoblotting was used to demonstrate IgE antibodies to the purified proteins in sera from latex-allergic patients.Results A 20 kDa protein was identified by amino acid sequencing as prohevein, a major protein in the rubber tree Hevea brasiliensis, and a 30 kDa natural rubber latex protein as hevamine. another essential rubber tree protein. A third, previously undescribed natural rubber latex protein, showed high homology to several plant endo-1,3-β-glucosidases. In immunoblolting, the purified prohevein bound IgE antibodies from 24/29 (83%) sera of latex-allergic patients including positive results in 4/6 latex-allergic children with spina bifida or other congenital anomalies. The purified prohevein elicited positive skin-prick test reactions in all six latex-allergic patients showing IgE to prohevein. The purified 36 kDa protein bound IgE from 6/29 (21%) latex-allergic sera, and the purified hevamine from only 1/29 patient sera.Conclusion The observed high frequency of IgE antibodies to prohevein suggests that this protein is a major natural rubber latex allergen

Notes: Background Quantification of natural rubber latex (NRL) allergens of NRL glove extracts has been reported in several studies. Similarly, immunoassay studies reporting the level of NRL aeroallergens in air samples have been published. When studying the NRL allergens of gloves, however, little attention has been focused on identifying the relationship between extractable NRL allergens of medical gloves and NRL aeroallergens in indoor air.Objective In an experimental study we analysed NRL aeroallergens of medical gloves in joint relation to total airborne dust concentration and NRL allergen concentration in gloves.Methods NRL aeroallergen level was measured using a chamber setting with 18 lots of powdered medical gloves. In each setting 10 pairs of powdered NRL gloves were swinging in an unventilated chamber (9 m3). Air samples were collected using airflow through Millipore filters (pore size 0.8 µm). The filters were weighed before and after the experiment, and total airborne dust concentration in the chamber was calculated. The filter samples were then extracted and the NRL allergen level measured by IgE ELISA-inhibition assay. Furthermore, cut NRL gloves were extracted and analysed by the same method. Finally, levels of two major NRL allergens, Hev b1 and Hev b 6.02, were measured in three selected NRL glove brands.Results The NRL aeroallergen level in the chamber air ranged from 〈 0.9 to 2.9 allergen units (AU)/m3. The total airborne dust concentration in the chamber air remained low with all lots of gloves measured (range 〈 20 to 80 µg/m3). The NRL allergen level in cut glove extracts varied over 100-fold (〈 10 to 1050 AU/mL). Statistically significant correlation between aeroallergen concentration and airborne dust (r = 0.8, P = 0.0015) concentration was found. Moreover, significant correlation between aeroallergen levels and allergen content of cut glove pieces was observed (r = 0.59, P 〈 0.05). Hev b 1 levels varied from 9 to 25 ng/mL and the levels of Hev b 6.02 from 1720 to 14460 ng/mL in the glove extracts. In the extracts from airborne dust samples, Hev b 6.02 content varied from 61 to 183 ng/m3, whereas Hev b 1 levels were very low (0.4 to 3 ng/m3).Conclusion An elevated NRL aeroallergen level is rather related to a high level of airborne glove powder than to a high concentration of extractable NRL allergen in medical gloves.

Notes: Background Wheat-dependent, exercise-induced anaphylaxis (WDEIA) is a severe allergy where wheat ingestion together with physical exercise induces anaphylaxis. We have previously shown that patients with WDEIA have IgE antibodies against gliadin proteins and identified ω-5 gliadin (Tri a 19) as a major allergen.Objective The aim of this study was to examine gliadin-specific IgG subclass, IgA and IgE antibodies, basophil histamine release and cell-mediated responses in WDEIA.Methods Sera and peripheral blood mononuclear cells (PBMC) were obtained from patients with WDEIA and from controls without wheat allergy. Serum antibodies to crude gliadin extract (CGE) and purified ω-5 gliadin were measured by ELISA and basophil reactivity by histamine-release test. Gliadin-induced cell-mediated responses were assessed by lymphocyte proliferation assay, and cytokine mRNA expression with real-time quantitative PCR.Results All patients with WDEIA, but none of the controls, had IgE antibodies to CGE and ω-5 gliadin. Both allergens released high levels of histamine from the basophils of patients with WDEIA. Levels of IgA antibodies to CGE and ω-5 gliadin were significantly elevated in the patients, but the distribution of IgG subclass antibodies showed no statistically significant differences between the two groups. Proliferative responses of PBMC to CGE were increased in patients with WDEIA, and stimulation of PBMC with CGE caused, both in patients and in controls, a clear induction of IL-10 mRNA. Compared with the controls, induction of IL-10 mRNA expression in patients with WDEIA was significantly (P 〈 0.01) suppressed.Conclusion These results suggest that, in addition to IgE antibodies against ω-5 gliadin, specific IgA antibodies may be involved in the pathogenesis of WDEIA. Decreased expression of IL-10 mRNA in PBMC during gliadin stimulation may facilitate the development of gliadin-specific T cell responses.

Notes: Background An association between allergic reactions to natural rubber latex and to banana has been reported but the immunochemical properties of the putative crossreacting allergens remain unknown.Obfective To study extracts of banana and natural rubber latex and sera from latex-allergic patients for possible crossreacting allergens and IgE antibodies.Methods Sera from 22 latex-allergic patients and 22 control subjects with no evidence of allergy to latex or to banana were studied. All patients had positive and controls negative reactions in skin-prick testing using an eluate of latex gloves. IgE antibodies to natural rubber latex and to banana were evaluated by immunoblotting and by radioailergosorbent test (RAST) and crossreactivity between allergens in banana and natural rubber latex by immunoblot inhibition. Skin-prick testing was used to examine in vivo reactivity to banana.Results Ten of the 22 (45%) latex-allergic patients sera recognized altogether 14 allergens in banana by immunoblotting. The most frequently identified banana allergens were 23, 32, 36, 39 and 47kDa proteins. The banana skin-prick test was positive in 14 of 18 (78%) latex-allergic patients studied and banana RAST in 12 of 14 patient sera tested. Fourteen of 21 interviewed patients reported symptoms from eating or handling bananas. In immunoblot inhibition studies a dose-dependent inhibition of IgE binding to banana extract with natural rubber latex proteins was observed in all five patient sera tested and, likewise, the binding of IgE to natural rubber latex extract was inhibited with banana proteins in four of the five patient sera.Conclusions The present results confirm the existence of crossreacting allergens in natural rubber latex and banana and provide new information on the immunochemical nature and heterogeneity of these allergens.

Notes: Background Stachybotrys chartarum is a damp building mould and a potent toxin producer that has been related to serious cases of respiratory health problems. However, the direct link between exposure and health symptoms has not been established.Objective To examine the mechanism by which exposure to spores of satratoxin producing and non-producing S. chartarum strains induce inflammatory responses in murine lungs.Methods BALB/c mice were intranasally exposed for 3 weeks to spores of a satratoxin-producing and a non-producing S. chartarum strain. Inflammatory cell infiltration was characterized from bronchoalveolar lavage (BAL) fluid. Cytokine and chemokine mRNA expression in lung tissue was measured with real-time PCR. Bronchial responsiveness to methacholine (MCh) was determined by whole-body plethysmography and serum antibody levels by ELISA.Results A dose-dependent increase in monocytes, neutrophils and lymphocytes was observed in BAL fluid after intranasal (i.n.) instillation of S. chartarum spores. There was no difference in the BAL between exposure to the satratoxin-producing and the non-producing strains. Infiltration of inflammatory cells was associated with an induction of pro-inflammatory cytokine (IL-1β, IL-6 and TNF-α) and chemokine (CCL3/MIP-1α, CCL4/MIP-1β and CCL2/MCP-1) mRNA levels in the lungs. Interestingly, CXCL5/LIX was the only chemokine that showed significantly higher mRNA levels after exposure to the satratoxin-producing strain compared with the non-producing strain. MCh-induced bronchial responsiveness was not altered significantly after mould instillation. Moreover, no significant increase in total or specific IgE, IgG2a and IgG1 antibody levels were found after S. chartarum exposure.Conclusion These results indicate that lung inflammation induced by i.n. instillations of S. chartarum spores is regulated by the induction of pro-inflammatory cytokines and leucocyte-attracting chemokines. The data also imply that S. chartarum-derived components, other than satratoxins, are mediating the development of this inflammatory response.

Notes: Patients with wheat-dependent, exercise-induced anaphylaxis experience severe allergic reactions when exercising after ingestion of wheat. The major wheat allergen associated with these reactions is a ω-5 gliadin, and patients following a gluten-free diet have remained free of symptoms.The aim of this study was to examine whether allergens cross-reacting with wheat ω-5 gliadin are present in rye, barley and oats.Sera from 23 adult patients with wheat-dependent, exercise-induced anaphylaxis were examined. Cereal allergens cross-reacting with wheat ω-5 gliadin were identified by immunoblot inhibition. The cross-reactive allergens were purified by gel filtration and reversed-phase chromatography and submitted to amino acid sequencing. Cross-reactivity was further studied by IgE ELISA and ELISA inhibition, and in vivo reactivity by skin prick testing.In immunoblotting rabbit anti-ω-5 gliadin antibodies bound to 70 kDa and 32 kDa proteins in rye and a 34-kDa protein in barley, but not to proteins in oats. N-terminal sequencing identified these proteins as rye γ-70 secalin, rye γ- 35 secalin and barley γ-3 hordein, correspondingly. In ELISA 21/23 (91%) patients with wheat-dependent, exercise-induced anaphylaxis showed IgE antibodies to purified γ-70 secalin, 19/23 (83%) to γ-35 secalin and 21/23 (91%) to γ-3 hordein. In ELISA inhibition ω-5 gliadin inhibited over 90% of the IgE binding of pooled patient sera to solid-phase γ-secalins and γ-3 hordein. Skin prick testing gave positive reactions to γ-70 secalin in 10/15 (67%) patients, to γ-35 secalin in 3/15 (20%) patients and to γ-3 hordein in 7/15 (47%) patients.The results of this study show that γ-70 and γ-35 secalins in rye and γ-3 hordein in barley cross-react with ω-5 gliadin, a major allergen in wheat-dependent, exercise-induced anaphylaxis. These findings suggest that also rye and barley may elicit symptoms in patients with wheat-dependent, exercise-induced anaphylaxis.

Notes: Natural rubber latex (NRL) allergy occurs frequently in children with spina bifida and other children with disorders requiring multiple operations. Also atopic children who have not undergone surgery can be sensitized to NRL, but the outcome of these children has not been studied.〈section xml:id="abs1-2"〉〈title type="main"〉ObjectiveTo study how NRL-allergic children manage at home and whether their skin prick test (SPT) reactivity, latex RAST or IgE antibody levels to NRL allergens change during the follow-up.〈section xml:id="abs1-3"〉〈title type="main"〉MethodsTwenty-four NRL-allergic children who had not undergone surgery and eight children with histories of multiple operations were followed up for a mean of 2.8 years. Clinical symptoms were recorded and all children were re-examined with SPT, latex RAST and ELISA for IgE antibodies to prohevein (Hev b 6.01), hevein (Hev b 6.02) and rubber elongation factor (REF, Hev b 1).〈section xml:id="abs1-4"〉〈title type="main"〉ResultsNineteen of the 24 NRL-allergic children (79%) who had not undergone surgery had occasionally contacts to balloons and other NRL products at home, and 10 of them experienced symptoms ranging from contact urticaria to systemic reactions. Three of the eight NRL-allergic children with a history of multiple operations had contacts to rubber balloons without any symptoms, and five children underwent 1–8 uneventful operations in a latex-free environment. SPT reactivity to NRL allergens, latex-RAST or IgE antibody levels to prohevein or hevein did not change in either group of NRL-allergic children during the follow-up.〈section xml:id="abs1-5"〉〈title type="main"〉ConclusionsOccurrence of clinical symptoms and no decrease in SPT reactivity or IgE levels to NRL allergens in the course of the present follow-up study imply that more attention should be paid to the protection of NRL-allergic children from rubber contacts in the home environment.

Notes: Background:  The characteristic feature of chronic rhinosinusitis with nasal polyposis (CRSwNP) is eosinophilic inflammation of the sinus mucosa; a type of inflammation also seen in asthmatic airways. Similar histopathologic findings of airway remodelling are present in both diseases. Remodelling is tightly controlled by matrix metalloproteinases (MMP). Increase of collagenase-2 (MMP-8) expression in the bronchial epithelial cells has been described in asthmatic patients, but it has not been studied in CRSwNP.Methods:  The concentrations and degree of activation of MMP-8 were analysed by immunofluorometric assay and Western blotting, respectively, in sinus mucus samples from CRSwNP patients and in nasal lavages from healthy controls in relation to inductive cytokines interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α).Results:  Significantly elevated levels of MMP-8 and IL-8 but not TNF-α were found in CRSwNP patients relative to controls. In particular, the activation of mesenchymal-type MMP-8 but not polymorphonuclear-type MMP-8 was associated with elevated IL-8 levels.Conclusions:  The IL-8 and MMP-8 seemingly form an inductive cytokine-proteinase cascade in CRSwNP pathogenesis. Together they provide a target for novel therapies and a diagnostic tool for monitoring CRSwNP treatment.

Notes: Exposure to natural rubber latex (NRL) medical gloves poses risks to patients sensitized to NRL and to users of protective gloves. Previous studies have shown that extractable allergen levels of the gloves vary widely. Since most of the available laboratory methods of NRL allergen measurement lack adequate validation, we wanted to evaluate the performance of a recently developed competitive IgE-ELISA-inhibition method in relation to the skin prick test (SPT) and RAST inhibition, as well as to extractable protein quantification and an immunochemical latex antigen assay (LEAP). Twenty samples of surgical (n = l4) and examination gloves (n=6), covering 〉90% of medical gloves marketed in Finland in 1994–5. were collected by the Finnish National Research and Development Centre for Welfare and Health, coded, extracted, and analyzed by the five methods. The IgE-ELISA inhibition correlated highly significantly with SPT (r=0.94) and RAST inhibition (r=0.96). Likewise, ELISA inhibition and RAST inhibition showed highly significant correlation (P=0.96, P〈0.0001 in all three instances). Protein quantification by a modified Lowry method also correlated highly significantly with SPT (r=0.80). RAST inhibition (r=0.82), and ELISA inhibition (r=0.81. P〈O.OOOI in all three instances). Clearly weaker correlation, though statistically significant (r=0.48. P=0.03), was found between SPT and the LEAP assay. An NRL standard preparation was assigned an arbitrary content of 100OOO allergen units (AU) per ml. In relation to this standard, the NRL allergen level was considered low (〈10 AU/ml) in 11. moderate (10–100 AU/ml) in two, and high (〉100 AU/ml) in seven of the 20 glove brands analyzed. In conclusion, the results of a novel IgE-ELISA-inhibition method of measuring NRL allergen levels in medical gloves correlated highly significantly with those of SPT TTie ELISA method was found to be sensitive, reproducible, technically easy, inexpensive, and suitable for the analysis of large numbers of NRL products. The results of extensive market surveys in 1994 and 1995, communicated to the medical community in Finland, appear to have had a clear effect in moving glove purchasing policies toward the use of low-allergen gloves.

Notes: For the diagnosis of IgE-mediated (immediate) hypersensitivity to natural rubber latex (NRL), skin prick testing with extracts of latex gloves has been widely used, but such extracts are difficult to standardize. The present study aimed to produce on an industrial scale an NRL extract from freshly collected NRL and to evaluate, calibrate, and standardize the extract by both in vivo and in vitro testing. The source material, latex of the rubber tree, Hevea brasiliensis (clone RRIM 600), was frozen immediately after collection in Malaysia and shipped in dry ice to Stallergènes SA, France. Protein and allergen profiles were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, isoelectric focusing (IEF), crossed immunoelectrophoresis (CIE), and crossed radioimmunoelectrophoresis (CRIE). Allergen quantification was effected by RAST inhibition. The capacity of the preparation to elicit immediate hypersensitivity reactions in vivo was measured by skin prick testing in 46 latex-allergic patients and 76 nonallergic control subjects. SDS-PAGE and immunoblot profiles of the extract and an NRL standard (E8) provided by the US Food and Drug Administration were almost identical, disclosing several distinct IgE-binding proteins with apparent molecular weights of 14, 20, 27, 30, and 45 kDa, conforming to reported molecular weights of several significant NRL allergens. An arbitrary index of reactivity (IR) of 100 was assigned to the extract at 1:200 dilution (w/v), having a protein content of 22 μg/ml. Skin prick testing of latex-allergic patients and controls using the extract at 100 IR revealed 93% sensitivity, 100% specificity, 100% negative predictive value, and 96% positive predictive value. In conclusion, a skin prick test reagent for diagnosis of type I NRL allergy was successfully standardized. The reagent was demonstrated to contain most, if not all, of the currently known clinically significant NRL allergens, and it showed high sensitivity and specificity.