Biomedical subjects

Horng-Der Shen

Publications and source records attributed to Horng-Der Shen.

14 recordsLinked to original sources

PubMed2006

Keloid-derived fibroblasts have a diminished capacity to produce prostaglandin E2.

Keloids result from pathological wound healing responses. However, the pathogenesis of keloids is still poorly understood. PGE2 was shown to decrease fibroblast proliferation, inhibit collagen synthesis and enhance the expression of matrix-metalloproteinases (MMPs). This study sought to delineate the production of PGE2 by normal and keloid-derived dermal fibroblasts. Human normal and keloid dermal fibroblasts were cultured in vitro. Cell proliferation and viability were determined based on WST-1 assay. IL-1beta-induced PGE2 production and effects of PGE2 on the synthesis of procollagen by culture-derived fibroblasts were determined by using enzyme-linked immunosorbant assay (ELISA) kits. IL-1beta-induced MMP-1 production by culture-derived fibroblasts was determined with an MMP-1 immunoassay kit. Our results showed that normal and keloid-derived fibroblasts exhibited a statistically significant increase (p<0.05) in cell proliferation when the cells were cultured in media with an increase in the concentrations (0%, 2% and 10%) of fetal bovine serum (FBS). In culture medium without FBS, an increase in cell proliferation of keloid-derived fibroblasts was detectable when compared with those of control fibroblasts. IL-1beta (1 ng/ml and 10 ng/ml) stimulated statistically significant production (p<0.01) of PGE2 by both normal and keloid-derived fibroblasts. However, lower levels of PGE2 produced by keloid-derived fibroblasts were detectable compared with those produced by normal-derived fibroblasts (p<0.05). In this study, although not statistically significant, inhibition of procollagen production by PGE2 in a dose-dependent manner was found. In addition, decreased production of MMP-1 by keloid-derived fibroblasts compared with those of control fibroblasts was also observed. In conclusion, keloid-derived fibroblasts produced less PGE2 than those produced by control fibroblasts. The role of diminished capacity of PGE2 production in keloid formation is presently unknown and needs further study.

Adult↗

PubMed2005

A vacuolar serine protease (Rho m 2) is a major allergen of Rhodotorula mucilaginosa and belongs to a class of highly conserved pan-fungal allergens.

BACKGROUND: Rhodotorula mucilaginosa is one of the most frequently encountered species of yeasts in our environment. We reported here a major allergen of R. mucilaginosa. METHODS: A major R. mucilaginosa allergen (Rho m 2) was characterized by two-dimensional (2D) immunoblotting, protein sequencing, cDNA cloning and IgE cross-reactivity with fungal serine proteases. RESULTS: Fourty-four sera (28%) from 157 bronchial asthmatic patients showed IgE-immunoblot reactivity against R. mucilaginosa extract. Among these 44 sera, 25 (57%) demonstrated IgE binding against a 31-kDa protein of R. mucilaginosa. Protein sequencing results suggest that it is a vacuolar serine protease. The corresponding cDNA clone encoding a mature protein of 312 residues was isolated. It shares 67-68% sequence identity with vacuolar serine protease allergens from three different Penicillium species (Pen ch 18, Pen o 18 and Pen c 18) and designated as Rho m 2 by the Allergen Nomenclature Committee. The native and recombinant Rho m 2 react with IgE antibodies and monoclonal antibody (MoAb) FUM20 against fungal serine proteases. IgE cross-reactivity between nRho m 2 and nPen ch 18 was observed. It was also detectable between rRho m 2 and rPen o 18. CONCLUSION: Our results suggest that R. mucilaginosa may also be a significant causative agent of human respiratory allergic disorders. We identified a vacuolar serine protease as a major allergen of R. mucilaginosa (Rho m 2) and a pan allergen of prevalent airborne fungal species. We detected IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens.

Allergens↗

PubMed2004

Comparison of different adjuvants of protein and DNA vaccination for the prophylaxis of IgE antibody formation.

A high-molecular-weight mite allergen Der f11 that was hardly purified for immunotherapy was used to develop the DNA vaccine pDf11. We have shown that vaccination of mice with pDf11 induces Th1 responses characterized by suppression of IgE responses. In the present study, effects of different adjuvants on pDf11 were first studied. Mice receiving pDf11 +/- CpG, bestatin, and bupivacaine had better suppression of IgE responses than those receiving pDf11 +/- lipofectin or alum. Bestatin could greatly boost IgG2a responses. Immunomodulating effects of different adjuvants between protein and DNA vaccines were further elucidated. CpG was the best for both protein and DNA vaccines to profoundly suppress IgE responses, but alum, bestatin and lipofectin were useless for rDf11 to induce IgE inhibition. Neither did the combination of rDf11 and pDf11 have further IgE suppression. In conclusion, CpG is the unique adjuvant for the protein vaccine rDf11 to inhibit IgE responses. In contrast, the DNA vaccine pDf11 +/- CpG, bestatin, or bupivacaine induces profound suppression of IgE responses.

Adjuvants, Immunologic↗

PubMed2004

Elevated levels of soluble adhesion molecules in sera of patients with acute bronchiolitis.

The mechanisms of migration of neutrophils into the airway lumen are crucial in the development of airway injury of acute bronchiolitis and are mediated by adhesion molecules. In this study, we have attempted to evaluate the role of serum concentrations of the soluble form of intercellular adhesion molecule-1 (sICAM-1) in the disease activity in acute bronchiolitis and in respiratory syncytial virus (RSV) infection. Circulating levels of sICAM-1 in sera from 10 normal control subjects, and from 47 hospitalized acute bronchiolitis patients at admission, and from 25 patients on the day of discharge were determined by use of commercially available enzyme-linked immunosorbent assay kits. The mean serum level of sICAM-1 in bronchiolitis patients was significantly higher than in the 10 healthy control infants (345.8 +/- 99.7 microg/mL vs 237.1 +/- 81.7 microg/mL; p<0.05). However, the mean sICAM-1 concentration was similar between RSV-positive and RSV-negative patients (337.5 +/- 99.6 microg/mL vs 350.9 +/- 101.1 microg/mL; p=0.65). Although the mean clinical severity score of RSV-positive patients was significantly higher than that of RSV-negative patients (5.94 +/- 1.83 vs 3.48 +/- 1.70; p<0.05). The improvement of clinical severity score was not well correlated with the change of sICAM-1 level (r=0.22). This study provides evidence that serum levels of sICAM-1 are increased in acute bronchiolitis and further confirms the role of adhesion molecules involved in the pathogenesis of the disease. However, the serum concentrations of the soluble adhesion molecules could not reliably reflect the clinical severity of the disease.

Acute Disease↗

PubMed2003

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.

Adolescent↗

PubMed2003

Detection of IgE reactivity to fungus antigens by immunoblotting in allergic diseases in children.

BACKGROUND: Fungi are well-known as sources for allergens that cause allergic rhinitis and allergic asthma. The aim of this study was to evaluate the prevalence of sensitization to airborne fungi in allergic diseases in children. METHODS: The presence of IgE antibodies to common allergens in serum samples from 77 atopic and 5 non-atopic children was determined by multiple allergosorbent (MAST) chemiluminescent assay. The allergic extracts, Penicillium notatum, Penicillium oxalicum, and Aspergillus fumigatus, were prepared. IgE-binding fungal components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-immunoblotting, using sera from the above-mentioned patients. RESULTS: The MAST data demonstrated that 62 (81%) of the 77 sera tested showed IgE antibodies to both Dermatophagoides pteronyssinus and Dermatophagoides farinae. Only 2 and 1 of the 77 atopic sera were positive to Penicillium and Aspergillus, respectively. However, 13 (17%) sera showed positive IgE-immunoblotting. Eight and 6 of them demonstrated IgE antibodies to more than 10 different components of P. notatum and P. oxalicum respectively. Ten of the 13 sera showed IgE-immunoblot reactivity to A. fumigatus, and only 1 of the 10 positive samples showed a positive MAST of class I to Aspergillus. All the other sera showed negative results in the MAST test to Aspergillus. The discrepancy observed between the results of IgE-immunoblotting using self-prepared extracts and the commercial MAST test of the serum samples tested may be due to the variations in the fungal strains and the allergenic extracts used, and the different assay systems employed. CONCLUSIONS: The results confirmed the importance of airborne fungi in association with allergic disorders in children.

Adolescent↗

PubMed2002

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Allergens↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

cDNA cloning and immunological characterization of a newly identified enolase allergen from Penicillium citrinum and Aspergillus fumigatus.

BACKGROUND: Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders. It is important to understand the allergenic profile of these fungal species. The purpose of the present study is to characterize a newly identified enolase allergen from P. citrinum and A. fumigatus. METHODS: Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation. The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5'- and 3'-rapid amplification cDNA end reactions. RESULTS: Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P. citrinum and A. fumigatus. The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases. The corresponding enolase cDNA from P. citrinum contains 1,552 bp and encodes a protein of 438 residues. In A. fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame. The deduced amino acid sequences of these two enolases have 94% identity. These enolases from P. citrinum and A. fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively. Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P. citrinum component (Pen c 22) and rPen c 22. In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A. fumigatus component (Asp f 22) and rAsp f 22. A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22. In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A. fumigatus and Alternaria alternata was also detected by immunoblot inhibition. CONCLUSIONS: These results demonstrated that a novel enolase allergen from P. citrinum (Pen c 22) and A. fumigatus (Asp f 22) was identified. In addition, IgE cross-reactivity between enolase allergens from A. fumigatus and P. citrinum and between enolases from A. fumigatus and A. alternata was also detected. Results obtained provide more information on fungal enolase allergens.

Allergens↗

PubMed2002

Alkaline serine proteinase from Aspergillus fumigatus has synergistic effects on Asp-f-2-induced immune response in mice.

BACKGROUND: Exposure to Aspergillus fumigatus allergens results in the sensitization and the development of allergic bronchopulmonary aspergillosis in susceptible individuals. Aspergillus antigen consists of a number of chemically diverse components and their cumulative or synergistic effect may result in disease. When mice were challenged with individual recombinant allergens, there was only reduced inflammation and immunological responses compared to the whole antigen. Various enzymes identified from A. fumigatus have been thought to cause airway damage. In the present study, we evaluated the effect of exposure to Asp f 13, an alkaline serine proteinase, and Asp f 2 in mice. METHODS: BALB/c mice were challenged intranasally with Asp f 2 and Asp f 13 alone and in combination. The antibody response, pulmonary inflammation, and airway hyperreactivity were studied. RESULTS: Results demonstrated no major difference in antibody response and airway responses among the different groups. The inflammatory responses in the lungs, however, showed marked differences in the various groups. CONCLUSION: In spite of the similar immunological responses in the different groups of mice studied, the results demonstrate enhanced inflammation in the lungs of mice exposed to a combination of both allergens. Allergens with proteinase activity have been found to be involved in airway inflammation and remodeling, which may also apply for Aspergillus-induced allergy.

Animals↗

PubMed2002

Immunobiology of fungal allergens.

In recent years, considerable attention has been paid in obtaining purified relevant allergens from fungi associated with allergy. Using molecular biology techniques, a number of mold allergens have been obtained by cloning the genes encoding the allergens. Currently, about 70 fungal allergens have been approved by the International Allergen Nomenclature Committee. In this review, we have presented major allergens from Aspergillus, Penicillium, Cladosporium, and Alternaria and discussed their immunochemical characteristics and their role in the diagnosis of allergy and possible usefulness in understanding the pathogenesis of the disease. The structure-function properties and the potential role of these recombinant allergens in the immunomodulatory therapy also are presented.

Air Pollutants↗

PubMed2002

Therapeutic effect of Bacillus Calmette-Guerin with allergen on human allergic asthmatic patients.

Epidemiological studies have suggested that either environmental exposure or immunization with Mycobacterium bovis-Bacillus Calmette-Guerin provides protection against atopy and asthma in Japanese school children. Tuberculin skin tests and tests of airway hyperresponsiveness were performed in asthmatic patients in the course of investigating the effect of Bacillus Calmette-Guerin and Dermatophagoides pteronyssinus immunotherapy. A total of 16 asthmatics and 20 normal individuals were recruited in this study. The results show that 8 (57.1%) of 14 asthmatic patients and 11 (55%) of 20 normal individuals had a positive tuberculin response. Sixteen asthmatic patients received immunotherapy (6 with D. pteronyssinus, 6 with D. pteronyssinus in conjunction with Bacillus Calmette-Guerin, 4 with buffered saline). Results show that there were no statistically significant changes in airway hyperreactivity to methacholine, the ratio of forced expiratory volume (in 1 sec) to forced vital capacity, and D. pteronyssinus-specific immunoglobulin E before and 1 month after immunotherapy in the response to therapy in all 3 groups. The prevalence of positive tuberculin responses in both normal individuals and asthmatic patients was high in Taiwan. Immunotherapy with D. pteronyssinus in conjunction with Bacillus Calmette-Guerin did not alter the non-specific airway hyperreactivity or D. pteronyssinus-specific immunoglobulin E in D. pteronyssinus-sensitive allergic asthmatic patients.

Adolescent↗

PubMed2002

Prevention of Der p2-induced allergic airway inflammation by Mycobacterium-bacillus Calmette Guerin.

Epidemiologic studies suggest an inverse correlation between infection and development of allergy. The purpose of this study was to test the hypothesis whether a preexisting T helper 1 (Th1)-type immune response elicited by Mycobacterium bovis-bacillus Calmette-Guerin (BCG) immunization could suppress allergic airway inflammation induced by the mite allergen Dermatophagoides pteronyssinus group 2 (Der p2) in an animal model. C57BL/6 mice were immunized with subcutaneous injection of BCG, then intraperitoneal Der p2 emulsified in alum. Der p2-specific immunoglobulin G1 and cytokine production from splenocytes were measured after Der p2 sensitization, and pulmonary function and airway inflammation were determined after inhalation challenge with Der p2. The intraperitoneal Der p2 with alum injection was able to induce Der p2-specific immunoglobulin G1 production, which could be downregulated by the pretreatment with BCG + Der p2. The inoculation of BCG + Der p2 caused splenocytes to produce more interferon-gamma, and this level was higher than that elicited by Der p2 or buffer alone. The positive interferon-gamma-staining CD4 cells were also increased after activation by phorbol myristate acetate and ionomycin. Lung pathology examination found decreased airway inflammation (associated with the best pulmonary function and least airway desquamation) in the mice inoculated with BCG + Der p2. In this Der p2-induced allergy model, BCG inoculation with Der p2 can cause a Th1-type immune response that hinders Der p2-induced allergic sensitization and the development of airway inflammation.

Animals↗

PubMedDate not supplied

Characterization of enolase allergen from Rhodotorula mucilaginosa.

Rhodotorula mucilaginosa (also known as R. rubra) is among the most commonly found yeast strains in our environment. However, allergens from R. mucilaginosa have not yet been characterized at the molecular level. The purpose of this study was to characterize the enolase allergen from R. mucilaginosa and examine the allergenic/antigenic cross-reactivity among fungal enolases. The full-length cDNA encoding the R. mucilaginosa enolase was isolated through the reverse transcriptase-polymerase chain reaction in conjunction with the 5'-end and 3'-end rapid amplification cDNA end reactions. The corresponding natural enolase from R. mucilaginosa was identified using two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis. The results showed that the enolase from R. mucilaginosa is a protein of 439 residues and is encoded by a cDNA of 1497 bp. It shares high sequence identity with enolase allergens from Candida albicans (85%), Saccharomyces cerevisiae (76%), Penicillium citrinum (76%), Aspergillus fumigatus (76%), Cladosporium herbarum (76.5%), and Alternaria alternata (74%). A 47-kD component in the R. mucilaginosa extracts was found to react with IgE or rabbit anti-enolase antiserum and has an N-terminal amino acid sequence identical to that deduced from the isolated enolase cDNA. Sera from three (21%) of 14 allergic patients sensitized to R. mucilaginosa showed IgE binding to this 47-kD R. mucilaginosa component and the His-tagged recombinant enolase. A rabbit antiserum against the P. citrinum enolase and a monoclonal antibody (MoAb; Afueno 8) against the A. fumigatus enolase reacted with all 5 fungal enolases tested. However, an MoAb (E2a) generated by using the Saccharomyces enolase as antigen could only recognize the immunizing enolase. In addition, heterogeneity in immunoblot profiles of IgE antibodies in serum samples from 9 allergic patients against 5 different fungal enolases tested was also observed. The presence of IgE cross-reactivity among enolase allergens from R. mucilaginosa, C. albicans and P. citrinum was detected by immunoblot inhibition. In conclusion, a new and cross-reactive enolase allergen from R. mucilaginosa (Rho m 1) was identified. Although enolases are highly conserved allergens among different fungal species, most of the allergic patients examined in this study differed in their IgE reactivity to the 5 different fungal enolases tested. The results obtained will be of value in understanding the role of enolase allergen in clinical mould allergy.

Allergens↗