Lai-Chen Tsai

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

Dexamethasone suppresses apoptosis in a human gastric cancer cell line through modulation of bcl‐x gene expression

Treatment of human gastric cancer TMK‐1 cells with transcription and translation inhibitors rapidly triggered cell apoptosis. Along with cell apoptosis, the Bcl‐xS level was markedly upregulated suggesting a crucial role of this protein in promoting the apoptotic process. In the presence of dexamethasone, however, cell apoptosis was greatly attenuated as demonstrated by DNA histogram shift and DNA fragmentation. Studies using the glucocorticoid receptor antagonist RU486 indicated that attenuation of apoptosis was mediated through glucocorticoid receptors. Dexamethasone not only suppressed the apoptosis‐associated upregulation of Bcl‐xS but also enhanced the basal level of Bcl‐xL in the cells. In addition, bcl‐x mRNA stability was significantly extended in the presence of dexamethasone. These results indicate that dexamethasone exerted a protective effect and delayed apoptosis of TMK‐1 cells by modulating bcl‐x gene expression.

Induction of specific Th1 responses and suppression of IgE antibody formation by vaccination with plasmid DNA encoding Der f 11

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-gamma. 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.

Effects of transcription and translation inhibitors on a human gastric carcinoma cell line: Potential role of Bcl-Xs in apoptosis triggered by these inhibitors☆

The effects of the macromolecular synthesis inhibitors 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), actinomycin D, and cycloheximide on the human gastric cancer TMK-1 cell line were studied. These agents inhibited DNA, RNA, or protein synthesis efficiently and induced cell death rapidly in a wide range of concentrations. After 8 hr of exposure to these agents, the cells exhibited morphological features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptotic bodies. Western blot analysis revealed that these inhibitors altered the protein levels of apoptosis-related gene products such as c-Myc, Bcl-X(S), and the mutant p53 (mp53) in TMK-1 cells markedly. The c-myc mRNA and protein levels were decreased initially and were then induced markedly to a new level after 4 hr of exposure to DRB, a RNA polymerase II inhibitor. The Bcl-X(S) levels were increased rapidly after treatment with all of these agents, whereas the levels of Bcl-X(L) and Bax remained largely unchanged. Northern blot analysis indicated that the c-myc overexpression is concomitant to DRB-induced DNA fragmentation and that the increased mp53 protein level was mainly a posttranscriptional event. Our observations suggest that the up-regulation of Bcl-X(S) may serve as an important mechanism for the apoptosis triggered by these inhibitors. This study also provides evidence for the notion that interference with the cellular survival pathway may lead to apoptosis.

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.

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.

Suppression of the growth of human colorectal carcinoma cells (LS174T) by radiolabeled monoclonal antibody (131I-MAbC27) in tissue culture and nude mice

A monoclonal antibody against carcinoembryonic antigen (CEA), MAbC27, and its F(ab′)2 fragments were prepared and labeled with131I. They effectively suppressed the growth of a human colorectal carcinoma cell line, LS174T, both in culture medium and in inoculated nude mice, whereas131I-labeled normal mouse immunoglobulin or131I itself did not have similar effects. Intravenous injection of131I-MAbC27 or131I-MAbF(ab′)2 following inoculation of carcinoma cells suppressed their growthin vivo. The suppression effect was even more effective when intact antibody rather than its F(ab′)2 fragments was used, especially when the treatment was repeated. This study indicates that radiolabeled MAbC27 may be used as a therapeutic agent in CEA-secreting human colorectal carcinomas.

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.

Denaturation of Ovalbumin Abrogates Oral Induction of Airway Hyperreactivity and IgG1, IgG2 Antibody Responses in Guinea Pigs

Background: The effects of denaturation of ovalbumin (OVA) on the induction of oral sensitization in guinea pigs were examined. Methods: Guinea pig antibody and airway responses were assessed after 10 feedings of chemically or heat–denatured OVA or egg white (EW). Results: Their specific IgG, IgG1 and IgG2 antibody responses were orally sensitized by OVA or EW, but not by chemically or heat–denatured OVA or EW. When further exposed to 0.1% OVA or conalbumin aerosol, those fed OVA or EW, but not denatured OVA or EW, had increased pulmonary resistance and decreased tidal volume. On the other hand, in those fed denatured OVA, boiled EW or saline only, a second sensitization with 1% OVA aerosol generated antibody responses and airway hyperreactivity. Using a sandwich ELISA, guinea pig serum OVA was detected after feeding EW, but not chemically denatured or boiled EW. Conclusions: It is likely that guinea pig gut absorption of OVA may result in oral sensitization. Chemical or heat denaturation of proteins may minimize their intestinal uptake and thus abrogates the induction of oral sensitization in guinea pigs.