Background: 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.Methods: The full‐length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5′–3′ rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR‐derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE‐binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite‐allergic children.Results: Der p 11 cDNA consists of a 2625‐bp open reading frame encoding a 103‐kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp‐sensitive asthmatic children, rDer p 11 showed positive IgE‐binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE‐binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG‐binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross‐inhibition between rDer p 11 and rDer f 11 was up to 73–80% at concentrations of 100 μg/ml.Conclusions: This study confirms that the newly identified recombinant Der p 11 is a novel and important high‐MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE‐binding major epitopes are scattered over the entire molecule of Der p 11.

BACKGROUND:The overexpression of Ck19 antigen occurs frequently in human carcinomas. The strategy and mechanism of radioimmunotherapy using Re-188-mAbCx-99 to Ck19 on human cervical carcinoma cells was investigated in this study.MATERIALS AND METHODS:Using mAbCx-99, the overexpression of Ck19 protein in lysates of cell lines and tissues from various patients' cervixes were verified by immunobinding and immunoblot analysis. The therapeutic effect of Re-188-mAbCx-99 on ME180 cells was examined in vitro by cell proliferation, apoptosis, DNA fragmentation and intemucleosomal levels.RESULTS:A relatively high expression of Ck19 was found in all human cervical carcinoma cell lines (4- to 44-fold) and in tissue lysates (26.8- to 79-fold) from patients (31 out of 34) with cervical, endometrial or ovarian carcinomas compared with that of benign or normal control samples. The growth inhibition of ME180, CC7T and Hela cells were significantly higher (p < 0.001) in the Re-188-mAbCx-99-treated (60-80%) than in the Re-188-MOPCIgG1-treated lines (8-18%) after 72-h treatment. After 48 h of treatment with Re-188-mAbCx-99, ME180 cells significantly exhibited DNA fragmentation and morphological features of apoptosis. This effect markedly elevated the expression of p21, p53 and Bcl-xS protein, while the Mcl-1 and Caspase-8 proteins were down-regulated.CONCLUSION:We suggest that an elevated Ck19 level is associated with disease stage in most patients with cervical cancer. The therapeutic effect of Re-188-mAbCx-99 was verified through apoptosis on targeting the enriched Ck19 protein of carcinoma cells.

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.

Background House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11.Objective The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients.Methods The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema.Results Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)(-) allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative.Conclusion The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.

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.

DNA vaccines encoding low-molecular-weight allergens have been used to prevent IgE responses. A high-molecular-weight mite allergen Der f 11 that was hardly to be purified for immunotherapy was used to develop a DNA vaccine here. Vaccination of mice with plasmid DNA encoding Df11 (pDf11) induced Th1 responses characterized by IgG2a responses and spleen cell secretion of IFN-γ. In contrast, sensitization with recombinant Der f 11 (rDf11) and alum induced Th2 responses characterized by IgE responses and spleen cell secretion of IL-4 and IL-5. Vaccination with pDf11 prevented the induction of IgE responses. Moreover, it could inhibit on-going IgE responses. The debate whether CD4+ or CD8+ T cells were the regulatory cells to inhibit IgE responses by DNA vaccination was also examined. First, sensitization of pDf11-vaccinated mice after depletion of CD8+ T cells still showed suppression of IgE responses. Secondly, adoptive transfer of either CD4- or CD8-depleted spleen cells from pDf11-vaccinated mice suppressed IgE responses. In conclusion, this is the first report to confirm the therapeutic effect of a DNA vaccine encoding a strong allergen on specific IgE responses. Both CD4+ and CD8+ T cells are crucial for the immunomodulation of IgE responses by pDf11.

This study was designed to examine the prevalence of positive serum IgE reactivity to the recombinant group 11 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.

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.METHODS:The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays.RESULTS:Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642.CONCLUSIONS:Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.

BACKGROUND:Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df), the major components of house dust, are considered to be etiologic factors of extrinsic asthma. The relationships between immunoglobulin (Ig) G subclass antibodies specific for Dp (or Df) were compared in specific IgE-positive and -negative asthmatic children.METHODS:Serum levels of IgG and IgE subclass antibodies specific for Dp and Df were studied in 52 children (age, 3-13 years; mean age, 8.4 years) with asthma using enzyme-linked immunosorbent assays. The skin prick test was also used in diagnosis of the reactivity of allergic disease.RESULTS:The levels of serum-specific IgG1 and IgG2 to Dp and Df in mite-specific IgE-(or skin-test) positive asthmatic children were significantly higher than those in mite-specific IgE- (or skin test) negative children (p < 0.01). Significant correlations between the level of the specific IgE and IgG1 (r = 0.6067; p = 0.0001) or IgG2 (r = 0.5851; p = 0.0002) to Dp, and IgG1 (r = 0.3823; p = 0.0214) or IgG2 (r = 0.5057; p = 0.0017) to Df were found. The specific IgE level and skin test reactivity showed a high correlation of greater than 96% (50/52).CONCLUSIONS:The levels of mite-specific IgG subclasses were partially compatible to specific IgE levels and skin test reactivity. We conclude that house dust mite allergy is significantly associated with specific IgG1, IgG2 and IgE responses.

Background The important dust mite allergens identified to date are of molecular weights ranging from 14 to 60 kDa. Our previous protein study indicated that the 98-kDa native paramyosin in Dermatophagoides farinae mite showed IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesting that it is a novel major mite allergen. This study described the isolation and characterization of the cDNA clone encoding the 98-kDa mite allergen.Methods A Dermatophagoides farinae cDNA library was constructed in lambda ZAPII vector and the library was immunoscreened with a monoclonal antibody 642. The cDNA insert was sub-cloned into M13 sequencing vector for single-stranded sequencing. The whole cDNA insert was expressed in pGEX-2T Escherichia coli expression system as a fusion protein with GST. The allergenicity of the recombinant peptides was tested by skin tests and IgE immunoassay. The IgE and IgG immunoassays were performed with sera from 20 mite-allergic patients.Results The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slightly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmatic patients reacted positively with the recombinant Dermatophagoides farinae paramyosin, respectively.Conclusion The study indicated that 98-kDa mite paramyosin is an important allergen.

Background: Exposure 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. Methods: An 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. Results: Competitive 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. Conclusion: We 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. (J Allergy Clin Immunol 1998;102:295-303.)

The effects of estradiol, tamoxifen, and retinoic acid on the proliferation of breast and cervical cancer cells were investigated. Estrogen stimulated only MCF-7 cell growth, whereas tamoxifen and retinoic acid inhibited the proliferation of all cells studied. Northern blot analysis indicated that estradiol up-regulates c-myc mRNA level in all cell lines studied regardless of the estrogen receptor status in the cells. On the contrary, tamoxifen inhibits c-myc gene expression in all cell lines studied except in MCF-7 cells where the c-myc transcript was not affected. The inhibitory effect of tamoxifen on c-myc gene expression and cell proliferation in estrogen receptor-negative cells suggest an estrogen receptor-independent mechanism. The results also suggest that different mechanisms are involved in the regulation of cell growth and c-myc gene expression in different cancer cells by estrogen and tamoxifen.