Soo-Ray Wang

Identification of allergens and antigens of Bermuda grass (Cynodon dactylon) pollen by immunoblot analysis

Allergens and antigens of Bermuda grass pollen fractionated by SDS‐polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes were identified using twenty‐one sera of Bermuda grass pollen‐allergic patients. The IgE‐ and IgG‐binding pollen components transferred to nitrocellulose were detected by reaction with enzyme‐labelled anti‐human IgE and anti‐human IgG, respectively. There was heterogeneity in both IgE‐ and IgG‐binding patterns of the allergic sera tested. Fourteen pollen components, ranging in molecular weight from 16000 to 88000 daltons, bound to IgE antibodies. Only two of the fourteen allergens identified reacted with IgE antibodies of more than 50% of the twenty‐one allergic sera. The pollen component with a molecular weight of 32000 daltons showed by far the highest frequency of IgE binding, being recognized by sixteen (76%) of the twenty‐one sera examined. Fifteen (71 %) of the twenty‐one sera tested had IgE antibodies that reacted with more than one of the fourteen allergenic components identified. Pollen components recognized by IgE antibodies also reacted with IgG antibodies, and there were components only recognized by IgG antibodies. Results obtained from this study should be useful both clinically and in research.

Alkaline Serine Proteinase: A Major Allergen of Aspergillus oryzae and Its Cross-Reactivity with Penicillium citrinum

Background:Aspergillus species are common indoor airborne fungi and have been considered as causative agents of human allergic disorders. However, allergens of different Aspergillus species have not been effectively characterized. The object of this study was to identify and characterize IgE-binding components of Aspergillus oryzae. Methods. Allergens of A. oryzae were identified by immunoblot analysis using sera from asthmatic patients. The N-terminal amino acid sequences of allergens thus identified were determined by Edman degradation. The antigenic and the allergenic cross-reactivities between allergens of different fungi were analyzed by immunoblotting and immunoblot inhibition analysis, respectively, using a monoclonal antibody (MoAb) 55A against the 33-kD major allergen of Penicillium citrinum and a mixture of IgE-containing asthmatic serum samples. Results: Thirteen components of A. oryzae ranging in apparent molecular weight from 16 to 42 kD react with IgE antibodies. A 34-kD component that showed intense IgE-binding reactivity and was detectable in the highest frequency in our asthmatic serum samples tested was considered a major allergen of A. oryzae. The 34-kD component also reacted with MoAb 55A. Results from immunoblot inhibition studies also demonstrated the IgE cross-reactivity between the 34-kD major allergens of A. oryzae and P. citrinum. In addition, the sequence of the N-terminal 18 amino acid residues of the 34-kD major allergen of A. oryzae was found to be identical to that of the alkaline serine proteinase from the same Aspergillus species. Conclusion: The 34-kD major allergen of A. oryzae is an alkaline serine proteinase. There is IgE cross-reactivity between the major serine proteinase allergens of A. oryzae and P. citrinum.

Sensitization to Blomia tropicalis among Asthmatic Patients in Taiwan

House dust mites have been reported as one of the most important allergens in Taiwan especially in asthmatic patients. This study was conducted to determine the allergenicity of Blomia tropicalis and sensitization of asthmatic patients in Taiwan. Serial dust samples were collected every month between July 1993 and June 1994 from 13 houses of mite-allergic patients. About 1 m2 surface area of a quilt was vacuumed. The floating method was used to collect mites, then identification and counting were performed. Results showed that Dermatophagoides pteronyssinus and B. tropicalis were the two most common species of mites found in allergenic patients’ houses in Taipei. D. pteronyssinus accounted for 52.1% of the total number of mites and was found in every house. B. tropicalis, although not present in every sample, accounted for 44.3% of the total number of mites. The skin test positive reaction to B. tropicalis, D. pteronyssinus and Dermatophagoides farinae were 73.3, 88.3 and 85.0% as determined from 60 allergic patients who attended our allergy clinics. The extract prepared from B. tropicalis was used to determine the allergenicity and contained at least 30 protein components when silver stained. The most frequently detected allergens were proteins with molecular weights of 14.3, 106.5, 94.0, 72.0, 91.9, 63.7, 100.3, 43.6, 27.3, 62.0, 34.7, 18.3, 41.1 and 21.9 kD. The frequency of IgE binding of patient sera to those proteins were 87.0, 65.2, 56.5, 43.4, 39.1, 39.1, 34.8, 30.4, 30.4, 17.4, 17.4, 17.4, 13.0 and 8.7%. The results from immunoblot inhibition showed that there was IgE cross-reactivity among the B. tropicalis and D. pteronyssinus. However, there were two major allergenic components of B. tropicalis not inhibited by D. pteronyssinus with molecular weights of about 14.3 and 27.3 kD. The use of B. tropicalis extract for diagnostic purposes to identify patients with specific sensitivity should be considered in Taiwan.

cDNA Cloning, Biological and Immunological Characterization of the Alkaline Serine Protease Major Allergen from Penicillium chrysogenum

Background:Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. Methods: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species – P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). Results: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116–125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12–73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. Conclusions: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

Identification of vacuolar serine proteinase as a major allergen of Aspergillus fumigatus by immunoblotting and N-terminal amino acid sequence analysis.

Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning.

Alkaline Serine Proteinase Is a Major Allergen of Aspergillus flavus, a Prevalent Airborne Aspergillus Species in the Taipei Area

Background: Aspergillus species are prevalent indoor airborne fungi and have been identified to be a causative agent of human allergic disorders. In the present study, we identified, purified and characterized the allergen(s) from Aspergillus flavus, a predominant airborne Aspergillus species in the Taipei area. Methods: The IgE–binding components of A. flavus were identified by SDS–PAGE immunoblotting with sera from asthmatic patients. The N–terminal amino acid sequences of the major allergens were determined by Edman degradation. The allergenic cross–reactivity among allergens from different fungi was analyzed by immunoblot inhibition using sera from asthmatic patients. The detected major allergen was purified from the culture medium. It was further characterized in terms of its N–terminal amino acid sequence, its IgE–binding activity and its enzymatic activity. Results: The results of the immunoblot analysis indicate that a 34–kD component that has high IgE–binding (63%) frequency is a major allergen of A. flavus. The N–terminus of this 34–kD major allergen (GLTTQKSAP) has high sequence identity with that of the 34–kD alkaline serine proteinase major allergen of A. oryzae. Results from immunoblot inhibition studies indicate that IgE cross–reactivity occurs among the 34–kD major allergens of A. flavus, A. fumigatus and Penicillium citrinum. The 34–kD major allergen of A. flavus was purified from the culture medium by ammonium sulfate precipitation and DEAE ion exchange chromatography. The N–terminal amino acid sequence of the purified allergen (Asp fl 13) is identical to that determined previously for the 34–kD major allergen in the crude extract of A. flavus. The IgE immunoblot reactivity to the 34–kD major allergen in the crude extract can be dose–dependently inhibited by the purified Asp fl 13. The degree of IgE binding to the 34–kD major allergen in the crude extract correlates with that of the purified Asp fl 13 in sera of 8 asthmatic patients. The purified Asp fl 13 has proteolytic activity with casein as substrate at pH 8.0. This enzymatic activity can be inhibited by either phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Conclusion: Our results suggest that the 34–kD alkaline serine proteinase is a major allergen of A. flavus. There was IgE cross–reactivity among allergens of A. flavus, A. fumigatus and P. citrinum.

The prevalence of IgE antibody reactivity against the alkaline serine protease major allergen of Penicillium chrysogenum increases with the age of asthmatic patients

BACKGROUND Penicillium species are prevalent airborne fungi. However, the prevalence of allergic sensitization to Penicillium antigens and the true impact of these ubiquitous fungi on atopic respiratory disorders remain to be determined. OBJECTIVE The purpose of this study was to analyze the prevalence of immunoglobulin (Ig)E and IgG antibodies against Penicillium chrysogenum (Pen ch 13), the alkaline serine protease major allergen of P. chrysogenum, in asthmatic patients of different age groups. METHODS Pen ch 13 was purified from a culture medium of P. chrysogenum. The reactivity of IgE and IgG antibodies to Pen ch 13 in the serum samples of 212 asthmatic patients was analyzed by immunoblotting methods. RESULTS Sixty-nine (33%) of the 212 sera analyzed showed IgE and/or IgG immunoblot reactivity to Pen ch 13. Significant differences in the prevalence of IgE and/or IgG antibody reactivity to Pen ch 13 were found among eight different age groups of 212 asthmatic patients. The frequency of IgE-binding reactivity to Pen ch 13 increased significantly with the age of the patients. It was 7% for the group less than 10 years old and 42% for the group older than 70 years old. In addition, a significant difference between the prevalence of IgE (7%) and IgG (33%) antibodies against Pen ch 13 in the group aged 10 or less was also found. CONCLUSIONS Our study demonstrates that IgE and IgG antibodies specific for Pen ch 13 were detected in approximately one-third of the 212 asthmatic patients analyzed. Our results suggest that allergic sensitization to Pen ch 13, and possibly to other airborne Penicillium species, is more common in older asthmatic patients.

Relationship between Nasal Resistance and Airway Hyperreactivity following Nasal Provocation with Dermatophagoides pteronyssinus in Allergic Rhinitis

To determine whether nasal allergic symptoms can cause bronchial hyperresponsiveness to methacholine, 30 subjects with allergic rhinitis (22 with allergic rhinitis and 8 with allergic asthmatic rhinitis) were studied. All subjects were skin test positive to Dermatophagoides pteronyssinus (DP) and underwent nasal allergic provocation with DP. After provocation, there was a severe nasal allergic reaction in the challenged nostril with a significant increase in nasal resistance both immediately and long (7 h) after DP exposure. There were no significant changes in the forced expiratory volume in 1 s and the forced expiratory flow rate between 25 and 75% of the forced vital capacity and in both allergic rhinitis and allergic asthmatic rhinitis patients. There was also no change in bronchial hyperresponsiveness to methacholine. The eosinophil counts, leukotriene B4, leukotriene C4 and platelet-activating factor levels in nasal discharges also showed no differences in both groups of patients. Our studies suggest that nasal provocation with limited allergen is a safe diagnostic technique. However, the relationship between nasal resistance and airway hyperreactivity is not obvious in this study. The similar concentrations of nasal inflammatory mediators in both allergic rhinitis and allergic asthmatic rhinitis indicate that bronchial hyperreactivity is not solely due to nasal drainage of inflammatory mediators.

Methotrexate Pneumonitis in Bronchial Asthma

Methotrexate pneumonitis is one of the most unpredictable and potentially serious adverse effects associated with the use of low-dose pulse methotrexate in treating rheumatoid arthritis. However, its occurrence in treating bronchial asthma has never been reported. A patient with steroid-dependent bronchial asthma developed interstitial pneumonitis during methotrexate therapy. Dyspnea, fever and oral ulcer occurred successively during the initial 4 months of intermittent low-dose methotrexate pulse therapy. Despite severe hypoxemia and interstitial infiltration in both lung fields, the lung lesions disappeared after treatment with corticosteroid and discontinuation of methotrexate therapy. In conclusion, methotrexate pneumonitis might develop after treatment. Once pneumonitis is suspected, methotrexate should be withdrawn.

Plasma arginase concentration measured by an enzyme-linked immunosorbent assay (ELISA) in normal adult population.

Human liver arginase has many biological effects on lymphocytes, macrophages, liver cells, and tumor cells, in addition to its major role in the liver urea cycle. We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) method to quantitate arginase concentrations in plasma that can be applied to various body fluids. The sensitivity was 2.5 ng/mL. The coefficients of variation were good both in intra- and inter-assay. Using this method to study the stability of an arginase preparation, we found that plasma arginase was stable for only 1 or 2 days even at temperatures as low as 4 degrees C. The mean plasma level was 41.0 +/- 3.3 ng/mL (mean +/- SE) in 143 normal subjects. There was no age difference in the general population and in the male group. However, in the female group, the plasma arginase level increased with age (p = 0.05). Its biological significance was unclear. As a whole, the ELISA method for the measurement of arginase concentration in the body fluid is convenient and reliable.