| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 98 === House dust mites and storage mites are present in residence throughout the world, and the importance and clinical relevance have grown rapidly. Tyrophagus putrescentiae (Tp) is one of the most dominant species of storage mites. The first part of this study was the survey of Tp infestation from the storage stuff in Taiwan, the results showed there are approximately 15% samples contaminated with Tp. Among them the pet food, mushroom and farina appeared to be most easily contaminated. The beans and rice are rarely contaminated. The factors involved in the contamination include package breakage, types of storage stuff, long storage time, and storage environment (temperature, humidity and tightness of closeness).
The second part was to investigate the prevalence of Tp-sensitization, identify its major allergens and clinical relevance in elderly subjects with age over 70 years. There was a significantly higher prevalence of Tp-hypersensitivity in elderly subjects in comparison with the young adult population. In the age-association study of Tp and Dermatophagoides pteronyssinus(Dp) sensitization, the elderly subjects were more sensitized to Tp than to Dp (p=0.02). The major allergens, Tyr p 2 and Tyr p 3, were identified with molecular weights of 16 kDa (53%) and 26 kDa (50%) may play important roles in the Tp hypersensitivity. The elderly subjects with chronic obstructive pulmonary disease (COPD) were higher percentage of Tp-sensitive and the non-COPD subjects were less sensitive rate to mites. The third part was to investigate the sensitization to Tp or Dp and to identify the cross-reactivity in patients with allergic rhinitis. The data showed there were higher subject skin test positive reactions to Dp (69.2%) than to Tp (29.1%). Among the Tp sensitive subjects, most of them were derived from the cross-reactivity of Dp. The immunoblot inhibition demonstrated that the cross-reactivity was coming from the Der p 2. Among these subjects, the higher frequency of IgE responses were group 2 allergens (rDer f 2、rDer p 2、rTyr p 2)about 73~82%. The high level of cross-reactivity between Der p 2 and Tyr p 2, it may be due to IgE- binding epitopes were similar. And the titers of IgE antibodies to rTyr p 2 and rDer p 2 were well correlated (R2=0.89) but not rTyr p 3 and rDer p 3 (R2=0.35). It indicated that the rTyr p 3 can be used as a marker for diagnosis of Tp hypersensitivity. The fourth part was to clone and purify the group 3 allergen in Tp. The rTyr p 3 is a 26-kDa protein and homologous to trypsin-like serine protease. It demonstrates Tyr p 3 is one of the major allergen (58%) in Tp-sensitized patients. A limited level (10~20%) of cross-reactivity has been found between rTyr p 3 and Der p 3. The nTyr p 3 can activate the protease activated receptor-2 of human lung epithelial cells-A549. After the treatment with nTyr p 3, the expression of IL-6 and IL-8 were significantly increased.
The fifth part was to use Balb/c mice to study the mechanism of pathogenesis of allergic asthma caused by Tp in the murine model. After the sensitization with crude extract of Tp / Al(OH)3 , the titer of the IgE and IgG1 were significantly increased. After the challenge with Tp via intratrachea, the neutrophils, eosinophils and cytokines (IL-4, IL-5 and IL-13) were significantly increased in the BALF(bronchoalveolar lavage fluid). The Penh value of AHR was enhanced, so it showed the blocked level in the trachea was serious. A great amount of mucosa appeared near to the nose and mouth in the sensitized mice. After the sensitization and challenge with Tp, the percentage of Th1 cells was decreased but the Th2 cells increased. It indicated that the immune response was shifted to the Th2-type reaction. The bronchial columnar epithelium of the sensitized mice was thick and destroyed, and infiltration of inflammatory cells in lung increased. This model will provide the application for the immunotherapy or vaccination in the future.
The sixth part was to evaluate the effects of intranasal (IN) immunotherapy with Tyr p 3 + CpG on Balb/c mice as an animal model to study the allergic asthma caused by Tp. After sensitization of the crude extract of Tp, we found Tp-specific antibodies of IgE and IgG1 increased in sensitized mice. But after the treatment with Tyr p 3 + CpG, the Tp-specific IgE or IgG1 synthesis might be inhibited but IgG2a production raised efficiently by the IN immunotherapy. The leukocyte subpopulation in the BALF was detected and the results showed the infiltrations of lymphocytes, neutrophils and eosinophils were significantly increased in BALF after the Tp sensitization. The number of lymphocytes, neutrophils and eosinophils was significantly decreased after immunotherapy with rTyr p 3 mixed with CpG-ODNs compared with PBS control. These results showed that rTyr p 3 mixed with CpG-ODNs had the best anti-inflammatory effect on the airway. After the immunotherapy, the Th2 derived cytokines- IL-5 and IL-13 were significantly decreased but increased of IL-12, IgA, IL-10, Foxp 3 and TGF-β secretion in groups of rTyr p 3 mixed with CpG-ODNs in comparison with PBS control. These results support that this kind of immunotherapy is an effective strategy for the treatment of allergic asthma.
The seventh part was to evaluate the effects of IN immunotherapy with rTyr p 2+ CpG or rDer p 2 + CpG on Balb/c mice model to study the allergic asthma caused by group2 allergens. After sensitization of rTyr p 2 or rDer p 2, antibodies of IgE and IgG1 increased in sensitized mice. But after the treatment with rTyr p 2 + CpG or rDer p 2 + CpG, the group2-IgE or IgG1 synthesis might be inhibited but IgG2a production raised efficiently by the IN immunotherapy. After the therapy the immune response was shifted to the Th1-type reaction. It could provide an effective improvement in the airway and the effects was similar to the treatment with DEX. The neutrophils and eosinophils were significantly decreased in BALF after the therapy. The bronchia columnar epithelium and infiltration of inflammatory cells in lung were significantly improved.
In conclusion, the mites of Tp usually present in the contaminated storage stuff. Different age population has different prevalence of mite hypersensitivity. The prevalence of sensitization to Tp was significantly higher in the elderly subjects. Most of the subjects with allergic rhinitis sensitive to Tp were originated from the cross-reactivity of Der p 2. The major allergens in Tp we identified were Tyr p 2 and Tyr p 3. The treatment with rTyr p 3 + CpG provides an effective strategy for the immunotherapy of airway inflammation triggered by Tp. In the group 2-sensitized mice, the treatment with rTyr p 2 + CpG or rDer p 2 + CpG could supply immunomodulatory effects caused by any type of group-2 allergens. This concept of the immunotherapy can provide an effective improvement in the hypersensitivity via the cross-reactivity of pan-allergen.
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