Mei-Whey Hung

Isolation and characterization of a novel 98-kd Dermatophagoides farinae mite allergen

BACKGROUNDnExposure to allergens from house dust mites is a significant cause of immediate hypersensitivity. Thus far, the active mite allergens defined are low molecular weight (MW) proteins or glycoproteins. However, other important mite allergens remain to be investigated. In this study a high MW mite antigen with a high IgE-binding activity was characterized.nnnMETHODSnAn anti-Dermatophagoides farinae (Df) monoclonal antibody, mAb642, which recognized a 98-kd allergenic mite protein, was used for affinity chromatography. The purified Df642 was characterized biochemically and immunologically.nnnRESULTSnCompetitive ELISA demonstrated that mAb642 was inhibited by the interaction between serum IgE from allergic patients and Df642 antigen in a dose-dependent fashion. The IgE reactivity to both 98-kd and 92-kd components was removed or diminished by preincubation of asthmatic sera with Df642-coated CNBr-activated cellulose-4B gel. Two-dimensional immunoblot analysis revealed that there are at least 4 isoforms of Df642 that represent a minor component in the crude mite extract. The allergenicity of Df642 was assayed by IgE immunoassay with a large panel of 67 sera from asthmatic patients with positive skin reactions, and Df 642 showed positive IgE reactivity with more than 80% of the sera tested. Thus it should be classified as an important allergen. In addition, amino acid sequence analysis revealed that Df642 shares more than 50% homology with paramyosin from invertebrates.nnnCONCLUSIONnWe have identified and characterized a 98-kd house dust mite allergen that showed greater than 80% IgE reactivity with sera from patients allergic to mites. This is the first high MW allergen characterized to date, and it shares high sequence homology with paramyosins in invertebrates.

Protein sequence analysis and mapping of IgE and IgG epitopes of an allergenic 98-kDa Dermatophagoides farinae paramyosin, Der f 11

Background: A 98‐kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11.

Molecular cloning and characterization of full‐length cDNAs encoding a novel high‐molecular‐weight Dermatophagoides pteronyssinus mite allergen, Der p 11

Background:u2002 Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high‐MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application.

EXPRESSION OF SERUM IL-2, IL-2R, AND CD8 LEVELS DURING HYPOSENSITIZATION IN HOUSE-DUST-SENSITIVE ASTHMATICS

In this study, we used the enzyme-linked immunosorbent assay (ELISA) to evaluate the changes of serum interleukin-2 (IL-2), interleukin-2 receptor (IL-2R), and suppressor/cytotoxicity factor (CD8) in house dust-sensitive asthmatic children during hyposensitization. Patients before immunotherapy presented significantly higher serum levels of IL-2 and IL-2R than normal subjects (p less than 0.001), but these levels became normal after three years of hyposensitization. No significant difference of serum CD8 level was noted between pretreated patients and normal controls. Although the serum CD8 level in treated patients also decreased after three years of immunotherapy, this decrease was not significant compared with the pretreated patients (p greater than 0.05). This study suggests that serum IL-2 and IL-2R markers might be helpful in analyzing allergic states associated with immune activation and in evaluating the therapeutic effects of hyposensitization.

Detection of House Dust Mite Allergens and Immunoblot Analysis in Asthmatic Children

The mite allergen extracts (Dermatophagoides pteronyssinus and D. farinae), were fractionated by polyacrylamide gel electrophoresis and autoradiography. The molecular weight of the most apparent proteins of D. pteronyssinus was between 27,000 and 35,000, and the protein components of D. farinae were in the 9-100-kilodalton range. Specific IgE antibodies directed against mite allergens were utilized as probes to detect the allergens. Using immunoblot analysis and radiostained technique, D. pteronyssinus-sensitive patient sera displayed the major protein of allergens with molecular weights of 15,800 and 27,000-31,000, while D. farinae-sensitive patients sera displayed the major protein of allergens with molecular weights of 12,000, 15,800 and 26,000-29,000. The electroblotting technique is fast, convenient, and highly suitable for both allergen composition studies and screening of antibody specificities.

Correlation between serum IgE and specific IgE antibody titer to house dust mite in children with asthma.

Using the enzyme-linked immunosorbent assay (ELISA) for IgE specific to mites, we evaluated the relationship between total serum IgE levels and IgE antibodies specific to mite in 58 asthmatic children. Our results showed that there was a positive correlation between total serum IgE and IgE antibody specific to Dermatophagoides pteronyssinus (r = 0.57; p less than 0.001) and Dermatophagoides farinae (r = 0.59; p less than 0.001). There was also a significant correlation between D. pteronyssinus-specific IgE and D. farinae-specific IgE (r = 0.68; p less than 0.001). This suggests that there are common allergens between the two species. The close correlation between the ELISA assay and skin test suggests that the former will be useful for the diagnosis of mite allergy in asthma.

Changes of serum-specific IgE antibody titer during hyposensitization in mite-sensitive asthmatic children

In this study, we used the enzyme-linked immunosorbent assay (ELISA) to evaluate the changes of IgE antibody titer against Dermatophagoides pteronyssinus (D. pteronyssinus) or Dermatophagoides farinae (D. farinae) in asthmatic children after immunotherapy. According to ELISA analysis, a significantly higher mean level of IgE antibody titer to D. pteronyssinus (or D. farinae) was found in nonhyposensitized asthmatic children than in the pediatric control group (p less than 0.001), but there was no significant difference between the group receiving short course (one year or less) immunotherapy and the group without immunotherapy (p greater than 0.05). We also noted the significant reduction of specific IgE antibody to D. farinae and D. pteronyssinus in the hyposensitized group after long-term immunotherapy; namely, 1.5 and 3 years, respectively, compared with the nontreated group (p less than 0.01). Although D. pteronyssinus-specific IgE antibody decreased less rapidly than D. farinae-specific IgE antibody, both kept decreasing throughout the period of immunotherapy. This study also indicates that the ELISA test may be helpful in screening specific IgE antibodies, diagnosing allergic disease, and evaluating therapeutic effects of hyposensitization.

Growth suppression of human colorectal carcinoma in nude mice by monoclonal antibody C27-abrin A chain conjugate

PURPOSE: The aim of this study was to assess an immunotoxin, monoclonal antibody C27-abrin A chain conjugate (MAAC), that might be effective in the treatment of colorectal carcinoma. METHODS: The immunotoxin was prepared by a specific monoclonal antibody against carcinoembryonic antigen (CEA), monoclonal antibody C27, linked toN-succinimidyl-3-(2-pyridyldithio)propionate and then coupled covalently to the toxic abrin-A chain to synthesize MAAC. The therapeutic role of this immunotoxin in suppressing thein vitro andin vivo growth of CEA-secreting human colorectal cancer cells (LS174T) was assayed by methods of protein biosynthesis inhibition, cell colony proliferation, and treatment of tumor cells before and after inoculation in nude mice. RESULTS: We found that MAAC effectively suppressed the growth of LS174T in culture medium and completely eradicated cells in inoculated nude mice. In contrast, irrelevant immunotoxin antiferritin-abrin A chain conjugate and isotype-matched monoclonal immunoglobin (MOPC21IgG1)-abrin A chain conjugate did not cause such effects. Thein vitro toxicity was highly specific because the conjugate (MAAC) inhibitedde novo protein biosynthesis, impeded growth, and caused death of cells possessing surface CEA determinants. The 50 percent inhibition dose values of the conjugate for colonogenic survival and for protein biosynthesis in LS174T cells were 0.09 Μg/ml and 0.06 Μg/ml, respectively. Colony survival was inhibited 96.3 percent after prolonged MAAC treatment. MAAC showed selective cytotoxicity; the inhibitory effect of MAAC to the CEA-secreting LS174T cells over the CEA-nonsecreting human embryonic kidney cells was 16-fold. CONCLUSION: These results indicate that MAAC may be of benefit in therapy during or soon after resection of colorectal carcinoma or in patients who have micrometastasis.

Serum Specific IgE Reactivity to Recombinant Der f 11 in Asthmatic Children

This study was designed to examine the prevalence of positive serum IgE reactivity to the recombinant group11 Dermatophagoides farinae allergen (rDer f 11) in asthmatic children in Taiwan. Using immunoblot analysis in a preliminary study of 18 asthmatic children, 13 (72.2%) reacted positively to rDer f 11 and 16 (88.9%) showed positive reactivity to D. farinae extracts. The allergenicity of rDer f 11 was further evaluated with in vivo skin tests and in vitro IgE immunodot assays in 24 mite skin-test-positive asthmatic children. Whereas 17 (70.8%) had positive skin tests to rDer f 11, 18 (75.0%) had positive serum IgE reactivity to rDer f 11. A good coincidence (87.5%) between the immunodot assay and the skin test was confirmed in these asthmatic children. Moreover, the prevalence of serum IgE reactivity to rDer f 11 was further investigated in a large panel of 49 mite skin-test-positive asthmatic children. Again, 38 (77.6%) had positive serum IgE reactivity to rDer f 11 in immunodot assays. Taken together, the positive IgE reactivity to rDer f 11 in immunodot analysis ranged from 75 to 77.6% in two groups of 73 mite skin-test-positive asthmatic children. High incidence of serum IgE antibodies specific for rDer f 11 in the present study suggests that Der f 11 is a novel major allergen of house dust mites.

Isolation and characterization of a novel major Dermatophagoides pteronyssinus mite allergen, Der p 11

Abstract Rationale Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites have been regarded as a major source of the indoor allergens. To date, two groups of high molecular weight (mw) dust-mite allergens to human have been identified. Isolation of high mw Dp mite allergens is important in characterizing the complete spectrum of mite IgE specificities and providing a suitable substance for clinical application. Methods A full-length Der p 11 gene was isolated by Dp cDNA library immunoscreening, 5-3 rapid amplification of cDNA ends (RACE) and polymerase chain reaction (PCR). Sequence analysis and alignment were scanned with a combination of CGC and BLAST program package. Results The Der p 11 gene is a 2965-bp cDNA gene with 2625-bp open reading frame coding for an 875-amino acid protein. The protein sequence of the newly recognized Dp allergen exhibited significant homology with other invertebrate paramyosins. The whole cDNA insert and PCR-derived fragments were generated and expressed in E. coli. The recombinant proteins including Der p 11 and its derivative peptides (p1-p4) were used for IgE binding immunoassay. An in vitro IgE-binding study showed that 75% (15/20), 65% (13/20), 76% (38/50), 60% (18/30), 73.3% (22/30), 66.7% (20/30), 63.3% (19/30) and 6.7% (2/30) in mite-sensitive asthmatic sera reacted positively with rDer p 2, rDer f 11, rDer p 11, p1, p2, p3, p4 and GST, respectively. IgE reactivity to rDer p11 fragments frequently reacted even in those patients with MAST(Dp)-negative sera. Conclusions Der p 11 is the homology of Der f 11 exhibiting high IgE reactivity to allergic sera from asthmatic children, potentially important house dust mite Dp allergen.