Kyoung Yong Jeong

Allergenicity of recombinant Bla g 7, German cockroach tropomyosin

Background: Cockroach infestation may sensitize and elicit allergic responses to genetically predisposed individuals. Invertebrate tropomyosins are a frequent cause of allergy and highly cross‐reactive in nature. In this study, we aimed to produce recombinant German cockroach tropomyosin and investigate its allergenicity.

Allergenic Tropomyosins and Their Cross-Reactivities

The ingestion or inhalation of some proteins may lead to adverse immune reactions. Allergens may trigger allergic reactions in genetically predisposed individuals when they are absorbed through the skin or make contact with mucous membranes. An allergic disease often deteriorates the quality of life and may sometimes be life-threatening due to anaphylactic shock. A number of allergens have been characterized from various multicellular organisms to date. It is thought to be reasonable to pay a special attention to the substance which is highly cross-reactive and which causes adverse responses in the molecules that are not sensitized but similar to the sensitized allergen. Tropomyosin has been described as an important food allergen in shrimp, lobster, crab, oysters, squid, and other invertebrates. Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins among invertebrates, but vertebrate tropomyosins are not known to be allergenic. Several tropomyosins from domestic arthropods have been reported to be allergenic. Recently, it was suggested that an infection of helminthic parasites might lead to sensitization to tropomyosin and elicit allergic reactions to other invertebrates. Much effort has been made to characterize these allergenic tropomyosins from various sources. We will discuss the physicochemical characteristics and the potential application of tropomyosin for the diagnosis and therapeutics of allergic disorders.

Regulation of German cockroach extract‐induced IL‐8 expression in human airway epithelial cells

Background Cockroaches have been known as a cause of respiratory allergies such as asthma. IL‐8 plays an integral role in the coordination and persistence of the inflammatory process in the chronic inflammation of the airways in asthma.

Optimization of Allergen Standardization

Preparation of high quality allergen extracts is essential for the diagnosis and immunotherapy of allergic disorders. Standardization of allergen extracts concerns determination of the allergen unit, development of reference material and measurement of the overall IgE binding capacity of an allergen extract. Recently, quantification of individual allergens has been the main focus of allergen standardization because the allergenicity of most allergen extracts is known to be mainly dependent on the content of a small number of allergen molecules. Therefore, characterization of major allergens will facilitate the standardization of allergens. In this article, we review the current state of allergen standardization. In addition, we briefly summarize the components of allergen extracts that should be under control for the optimization of allergen standardization, since its adjuvant-like activities could play an important role in allergic reactions even though the molecule itself does not bind to the IgE antibodies from subjects.

Molecular Cloning and Characterization of Tropomyosin, a Major Allergen of Chironomus kiiensis, a Dominant Species of Nonbiting Midges in Korea

ABSTRACT Chironomids are widely and abundantly distributed in the vicinity of standing waters. Larvae of Chironomus and some other genera are known to contain hemoglobins, which have been described as a major allergen, and the adults that have no hemoglobins also have been reported to contain allergens. In this study, we tried to establish the role of chironomid allergy and characterize the allergen of Chironomus kiiensis adults. Skin tests using C. kiiensis adult extracts were performed on patients with allergic symptoms. A cDNA library of C. kiiensis adults was screened with C. kiiensis immune mouse sera to identify allergens, and results were confirmed using skin test-positive human sera. Recombinant allergen was expressed in Escherichia coli and purified by affinity chromatography using nickel-nitrilotriacetic acid agarose to investigate its allergenic properties. Out of 275 allergic patients 14.2% showed a positive reaction to C. kiiensis adult crude extracts in the skin test. The tropomyosin was cloned by immunoscreening and expressed in Escherichia coli. C. kiiensis tropomyosin has a high homology at the amino acid level with tropomyosins which were previously known to be allergens in various arthropods (Periplaneta americana, 86.3%; Panulirus stimpson, 78.9%; Dermatophagoides pteronyssinus, 76.5%). Specific immunoglobulin E antibodies reacting to recombinant tropomyosin were detected in 17 (81%) of 21 patients whose skin test results were positive. Cross-reactivity against house dust mites and other insects was noticed with mouse anti-recombinant tropomyosin immune serum. C. kiiensis adults were shown to be an important source of inhalant allergens in Korea. Molecular cloning of C. kiiensis tropomyosin was performed and IgE reactivity was demonstrated using skin test-positive human sera. Recombinant tropomyosin will be useful for further studies or clinical applications.

Immunoglobulin E Reactivity of Recombinant Allergen Tyr p 13 from Tyrophagus putrescentiae Homologous to Fatty Acid Binding Protein

ABSTRACT The storage mite, Tyrophagus putrescentiae, is one of the important causes of allergic disorders. Fifteen allergenic components were demonstrated in storage mite by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, but only the group 2 allergen Tyr p 2 has been cloned and characterized. In this study, we attempted to identify and characterize new allergens from T. putrescentiae, which is a dominant species of storage mite in Korea. Expressed sequence tags were analyzed to identify possible storage mite allergens, and the cDNA sequence encoding a protein homologous to fatty acid binding protein, a mite group 13 allergen, was identified and named Tyr p 13. Its deduced amino acid sequence showed 61.1 to 85.3% identity with other mite group 13 allergens. The recombinant protein was expressed in Escherichia coli using a pET 28b vector system, and its allergenicity was investigated by enzyme-linked immunosorbent assay (ELISA). The recombinant allergen was detected in 5 of 78 (6.4%) T. putrescentiae-positive sera tested, and it inhibited 61.9% of immunoglobulin E binding to crude extract at an inhibitor concentration of 10 μg/ml by inhibition ELISA using serum from the patient who showed the strongest reaction by ELISA. In this study, a novel allergen was identified in T. putrescentiae. This allergen could be helpful for more-detailed characterizations of storage mite allergy.

Identification of Novel Allergenic Components from German Cockroach Fecal Extract by a Proteomic Approach

Background: Cockroaches produce potent allergens, and cockroach feces are known to be especially rich in allergens. In this study, we analyze the allergenic components from cockroach feces and evaluate allergenicity of recombinant α-amylase identified from fecal extract. Methods: IgE-reactive proteins from German cockroach fecal extract were analyzed by proteomic analysis and immunoblotting. Recombinant α-amylase was produced and its allergenicity was evaluated by ELISA. Results: Analysis of German cockroach fecal extracts identified 12 IgE-reactive components. Most of these allergens were found to be digestive enzymes such as α-amylase, trypsin, chymotrypsin, metalloprotease, and midgut carboxypeptidase A, but the identity of 3 IgE-reactive proteins is still unknown. Glycinin-like proteins, which were likely derived from the cockroach diet, were also identified. German cockroach α-amylase shares the highest identity with pig α-amylase (55.8%), followed by mite group 4 allergens (Blo t 4, 50.4%; Der p 4, 49.8%; Eur m 4, 47.4%). In this study, recombinant α-amylase from German cockroach was expressed, and its allergenicity was examined by ELISA. Specific IgE against recombinant amylase was detected in 41.4% (12/29) of serum samples from German cockroach-sensitized subjects. Recombinant α-amylase was able to inhibit 55% of specific IgE to German cockroach whole-body extract. Conclusions: Amylase was found to be an important novel allergen in cockroach feces. It is hoped that recombinant α-amylase will be useful for further studies and clinical applications.

Enzymatic activities of allergen extracts from three species of dust mites and cockroaches commonly found in Korean home.

Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.

Characterization of the major allergens of Pachycondyla chinensis in ant sting anaphylaxis patients

Background The ant species Pachycondyla chinensis, which has spread from Far Eastern Asia to New Zealand and North America, induces anaphylactic reactions in human with its sting. However, the major allergens of P. chinensis have not yet been characterized.

Molecular Cloning and the Allergenic Characterization of Tropomyosin from Tyrophagus putrescentiae

Storage mites have been recognized as a cause of asthma and rhinitis. Studies from several countries have shown that the IgE-mediated allergy to storage mites is of considerable importance, especially in rural populations. This study aimed to identify and characterize new allergens from Tyrophagus putrescentiae. A partial cDNA sequence encoding tropomyosin was isolated from the cDNA library by immunoscreening using anti-mouse IgG1 sera raised against T. putrescentiae whole body extract. The deduced amino acid sequence shares 64-94% identity with previously known allergenic tropomyosins. Its recombinant protein was produced by using a pET 28b expression system and purified by affinity chromatography using Ni-NTA agarose. The IgE reactivities of tropomyosins from T. putrescentiae and Dermatophagoides farinae were compared by enzyme linked immunosorbent assay (ELISA). Recombinant Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity against the same sera from storage mite-sensitized and house dust mite-sensitized subjects. Both recombinant Tyr p 10 and Der f 10 showed little inhibition of IgE binding to T. putrescentiae crude extract by ELISA. Tropomyosin seems to contribute only a small portion of the cross-reactivity with house dust mites.