肺臟是我們體內氣體交換的器官，所以肺臟經常要接觸空氣中的病原菌、過敏原和污染物質，肺臟內的防禦系統必須要有效且快速的清除這些病原菌。Der p 1塵蟎過敏原是來自Dermatophagoid pteronyssinus，屬於木瓜蛋白酶半胱氨酸蛋白酶家族的一員其主要是引起過敏性鼻炎和哮喘。然而，如何引起的作用機制尚不清楚。肺泡表面蛋白D (pulmonary surfactant protein D, SP-D)，是由第二型肺泡細胞所分泌，在肺臟中先天免疫防禦系統機制扮演重要角色。研究氣管肺泡的灌洗液 (BAL) 和血液中SP-D的基因多型性及蛋白質分析，已經發現SP-D的多型性和許多肺發炎反應疾病有關聯。目前已知在人類SP-D的編碼序列上有三個單核甘酸變異點 (SNPs)，分別是位在N端的編碼序列第11個的胺基酸，編碼序列第160個的胺基酸，及位於C端的編碼序列第270個的胺基酸。然而SP-D的基因變異體和氣喘的臨床症狀表現及家塵蟎Dermatophagoides ptyronysinnus (Der p)的過敏原感受性的關聯性仍然是未知。本論文的研究主題主要是利用蛋白質體學,免疫學及基因學分析塵蟎過敏原和SP-D引發的過敏氣喘機轉之研究，並分為以下兩大研究主軸來進行。首先，我們先分離及純化anti-Der p 1 的單株抗體， mAb W108，再利用蛋白質體學及免疫學的分析來評估mAb W108在因Der p過敏原所引起的過敏性氣喘中的治療潛能。第二，由我們研究族群中SP-D基因多型性及兒童氣喘的臨床相關研究，我們從哺乳動物的細胞中表現出四個不同基因型的重組SP-D蛋白，進而比較這四個不同基因型的重組SP-D蛋白和家塵蟎的結合親和力來探究SP-D蛋白和因Der p所引起的兒童氣喘的相關性及SP-D蛋白在其中的功能。
在我們研究的第一個部分，我們使用 predictive algorithms 和 site-directed mapping來研究藉由老鼠的單株抗體所找出的Der p 1中和IgE結合的epitopes位置及過敏原多肽的排列。而mAb W108的治療評估則是使用塵蟎刺激所引起氣喘的老鼠來做動物實驗模式。同時，mAb W108是由可強烈抑制 human anti-Der p-specific IgE antibodies 和 the cysteine protease activity of the Der p 1結合的 Der p-specific producing hybridoma clones中所挑選及分離出來。對未處理的Der p蛋白萃取物使用2 D電泳 (2-DE) 及蛋白質體學分析可得到275個明顯的蛋白點，其中四個 (36 kDa, pI values from 4 to 6) 蛋白點屬於 Der p 1 precursor isotypes且可和Der p 1 敏感的病人血清及mAb W108產生反應。在Der p 1 的connected loops (胺基酸殘基151-197和286-320)中有兩個不連續的epitopes 可被辨識出和mAb W108的結合親和力及3D結構模型刺激有相關性。在過敏性氣喘的老鼠模式中，給予 mAb W108可減輕Der p過敏原所引起的呼吸道發炎反應的症狀及反轉Th2的過敏性免疫反應。
在第二部份的研究，我們將參與研究的460名學齡期兒童依據他們的臨床症狀及家塵蟎感受性分為四組(過敏性氣喘[AA]、非過敏性氣喘[NA]、過敏性但不具有氣喘[AN]和不過敏也沒有氣喘[NN])。 SP-D SNPs是用 sequence specific primer-PCR的方法來定出，並利用酵素免疫分析法(ELISA)來分析血清中SP-D蛋白的濃度，而相關性分析是用SPSS來表現。我們的結果顯示 SP-D aa 160的變異體和Der p 感受性有關，但是和氣喘的表型無關。我們同時利用 Flp-In system中的哺乳類細胞來表現四種具有不同基因型的全長度的SP-D重組蛋白，分別為 TG、CG、TA、CA。由 solid-phase結合分析，我們發現SP-D CA的基因變異型和塵蟎過敏原有最高程度的結合親合力，這個結果和我們利用病人的基因研究所發現SP-D CA的基因變異型在兒童的家塵璊所引起的過敏性氣喘中具有保護性是有其相關性的。
總結本篇論文的結論，我們證明在過敏性氣喘的老鼠模式中，給予 mAb W108可減輕Der p過敏原所引起的呼吸道發炎反應的症狀及Th2 cytokines 的免疫反應，可顯示出mAb W108在臨床使用上的治療潛力。而從不同基因變異型的SP-D和塵蟎過敏原的結合親和力試驗中，我們發現SP-D有作用的胺基酸變異型可能在有基因傾向會有過敏原敏感性的人及兒童氣喘患者中扮演著重要的角色。這些發現可提供新的觀點來了解 IgE-mediated疾病和為未來過敏原專一性的免疫治療提供修改過的過敏原疫苗及為過敏性氣喘提供新的治療形式。

As a gas-exchange organ, lung is inevitably exposed to air that is contaminated with pathogens, allergens and pollutants. Hence, the host-defense mechanism within the lung is vital to facilitate clearance of inhaled pathogens as well as to prevent persistent inflammation. The group 1 allergen from Dermatophagoid pteronyssinus (Der p 1), belongs to the papain-like cysteine protease family and is a major allergen of Der p that causes rhinitis and asthma. However, details of pathophysiological mechanism of Der p 1 in allergic diseases are unclear. Pulmonary surfactant protein D (SP-D), as an endogenous secreted C-type lectin from alveolar type II cells, plays important role in the innate immunity of lung. Studies on SP-D polymorphisms and protein levels in bronchoalveolar lavage (BAL) and blood have been indicated associated with a multitude of pulmonary inflammatory diseases. There are three single nucleotide polymorphisms (SNPs) in the coding sequence of human SP-D at SP-D aa11 in the N-terminal region, SP-D aa 160 in the collagen-like domain, and SP-D aa 270 in the carbohydrate recognition domain. Whether the genetic variants of SP-D are in association with clinical phenotypes of asthma and house dust mite, Dermatophagoides ptyronysinnus (Der p) allergen sensitizatioln is still also unknown. Here, in this study, we focused our studies in proteomic, immunologic, and genetic studies in the protective mechanisms of surfactant protein (SP)-D in house dust mite induced-allergic asthma. In this research, we have used two separate approaches to explore this study question. Firstly, we isolated and purified anti-Der p 1 monoclonal antibody, mAb W108, and using proteomic and immunologic analysis to evaluate the therapeutic potential of this mAb W108 in Der p allergen-induced allergic asthma. Secondly, from the clinical association study of SP-D gene polymorphisms and childhood asthma in our population, we have expressed four genetic variant of recombinant SP-D protein from mammalian cells, and compared the mite allergen binding affinity among these four recombinant of SP-D, to explore the association and function of genetic polymorphism of the surfactant protein (SP)-D in mite-sensitization childhood asthma.
In the first part of our studies, we used predictive algorithms and site-directed mapping to investigate the IgE binding epitopes of the Der p 1 using a mouse monoclonal antibody (mAb) and allergen peptides array. The therapeutic use of blocking anti-Der p 1 mAb, W108 was evaluated in a mite-sensitized murine model of asthma. mAb W108 was selected and isolated from Der p-specific producing hybridoma clones, which strongly inhibited the binding of human anti-Der p-specific IgE antibodies and the cysteine protease activity of the Der p 1. Using two-dimensional electrophoresis (2-DE) and proteomic approach on the crude extracts of the Der p revealed about 275 distinct protein spots, four of which (36 kDa, pI values from 4 to 6) belonged to the Der p 1 precursor isotypes that interacted with sera from Der p-sensitive patients and mAb W108. Two discontinuous epitopes on the connected loops (residues 151-197 and 286-320) of the Der p1 were identified according to the relative binding affinity of the peptides to mAb W108 and in a 3D structural model simulation. In the mouse model of allergic asthma, administration of mAb W108 could alleviate the symptoms of Der p allergen-induced airway inflammation and reversed Th2 allergic immune response.
In the second part of study, we have enrolled 460 school-age children in Taiwan, and divided into 4 groups (allergic asthma (AA), non-allergic asthma (NA), allergic non-asthma (AN), and controls (NN)) according to their clinical symptoms and host dust mite sensitization. SP-D SNPs was genotyped by a sequence specific primer-PCR methodology and their serum SP-D levels were analyzed by ELISA. Association analyses were performed by SPSS. Our results showed that SP-D aa 160 variant was associated with Der p sensitization, but not with asthma phenotype. We had also expressed the recombinant protein with TG, CG, TA and CA of full length SP-D from mammalian cells in Flp-In system. From the solid-phase binding assay, we found genetic variants of CA type of SP-D has the highest binding affinity to mite allergen, which was correlated with the genetic finding of our study patients that SP-D CA genetic type had the protective effect in mite-sensitive allergic asthma in children.
In conclusion, we have showed that the administration of mAb W108 in the Der p-sensitized murine model of asthma alleviated allergen-induced airway inflammation and the Th2 cytokines immune response, suggesting its therapeutic potential. From the study of the mite allergen binding affinity among different genetic variants of SP-D, we found the functional amino acid variants in SP-D may play a major role in the genetic pre-disposition to allergen sensitization and childhood asthma. These findings can provide new insights into understand IgE-mediated disease and the design of modified allergen-vaccines for future allergen-specific immunotherapy as well as new therapeutic modality for allergic asthma.

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