臺灣博碩士論文加值系統

漂浮在空氣的過敏原中，蟑螂是重要過敏原之一，會引起許多IgE媒介之過敏疾病而且已知和氣喘疾病有密切的關係。傳統的臨床檢驗和治療蟑螂過敏均是使用未經標準化的蟑螂粗萃物，由於其中含有許多非過敏原之雜蛋白，導致臨床診斷上的敏感度和特異性不足，因此，還有改善的空間。在本研究的第一部份，首先製備美國蟑螂主要過敏原 Per a 1.0104、Per a 3.0203和Per a 7.0101之重組蛋白質。收集118個蟑螂過敏的病人血清並且分析血清中 specific IgE 對於 Per a 1.0104、3.0203和 7.0101 之盛行率。由 IgE ELISA 的統計結果顯示，Per a 1.0104、3.0203 和 7.0101在118個蟑螂過敏的病人血清之盛行率分別為 66.1、67.8 和 57.6%，表示 Per a 1.0104、Per a 3.0203和Per a 7.0101是美國蟑螂的主要過敏原。希望將來這些重組過敏原可以做為台灣地區美國蟑螂過敏減敏治療疫苗之來源。期待能利用混合多種重組過敏原以取代傳統之天然萃取物，應用於臨床檢驗，建立病患之IgE反應圖譜 (profile)，稱為 component-resolved diagnosis。如此一來，比起傳統的過敏原檢測更能精確診斷出病患之致敏來源以及臨床嚴重度，利於將來可以針對個體之過敏原圖譜進行專一性免疫治療。另一方面，呼吸道上皮細胞是如何受到蟑螂過敏原 (Cra A) 所活化，當中的過敏機轉還不清楚。目前，許多文獻報導指出 interleukin-8 (IL-8) 在過敏疾病中扮演一個重要角色，可以引起呼吸道上皮細胞持續性之發炎反應。因此，第二部份的研究目的是探討 Cra A 引起呼吸道上皮細胞釋放 IL-8之分子機轉。首先，利用人類肺癌上皮細胞株 A549作為分析平台，在Cra A刺激之下，A549會釋放IL-8，而且IL-8的mRNA和蛋白質的表現皆和處理濃度和時間成正相關。其次，Cra A引起 A549釋放 IL-8的作用，會受到絲胺酸蛋白酶抑制劑 (serine protease inhibitor)-PMSF以及 aprotinin 所阻斷但是不會受到半胱氨酸蛋白質酶抑制劑 (cysteine protease inhibitor) 以及天門冬胺酸蛋白質酶抑制劑 (aspartic protease inhibitor) 所阻斷，這個結果證實 Cra A 具有絲胺酸蛋白質酶之酵素活性而且和細胞激素IL-8之生成有關。接著，我們利用real-time PCR觀察接受器Protease-Activated Receptors (PARs) mRNA 之變化情形，發現 A549在 Cra A 處理兩小時之後，PAR-2 和 PAR-3 的 mRNA 相較於未處理組會達到1.73 和1.94倍的變化。最後，我們探討 PAR-2 和 PAR-3被活化之後，下游是否藉由活化 MAPK 途徑造成細胞激素 IL-8生成。由 Western blotting 的結果顯示，在 Cra A 短時間處理之後，會增加 ERK1/2 and JNK 磷酸化的情形。我們也利用抑制劑U0126 (ERK 1/2 kinase inhibitor) 或是 SP600125 (JNK kinase inhibitor) 前處理A549兩小時，分別100% 和45% 抑制 IL-8 釋放。綜合上述的結果，我們提出可能的致敏模式為:當人類呼吸道上皮細胞經美國蟑螂粗萃物刺激後，將活化細胞膜的受器 PAR-2和 PAR-3，經由 ERK1/2 和 JNK MAPK 訊息傳遞途徑，誘發 IL-8的生成。

Cockroaches are known to produce potent aeroallergens which elicit IgE-mediated allergy and strongly associated with asthma. Traditional methods of cockroach allergy diagnosis and treatment are based on the use of crude extracts containing wide variety of undesirable protein, which leaves rooms for improvability regarding the test sensitivity and specificity. In the Part I of this study, we would like to utilize recombinant proteins (rPer a 1.0104, 3.0203 and 7.0101) to replace cockroach crude extracts in clinical diagnosis and immunotherapy. A total of 118 sera from cockroach-allergic patients were collected and analyzed for their specific IgE against rPer a 1.0104, 3.0203 and 7..0101 IgE ELISA results showed that the prevalence of IgE antibodies to rPer a 1.0104, 3.0203 and 7.0101 were 66.1%, 67.8% and 57.6%, respectively. Current data demonstrated that Per a 1.0104, 3.0203 and 7.0101are important P. americana major allergens that should be considered for clinical use. The use of component-resolved diagnostics may be useful to evaluate the allergen cocktails for immunotherapy by monitoring patient’s IgE directed to relevant allergens. Beside, the mechanisms underlying epithelial cell activation by American cockroach allergens (Cra A) are unclear. Interleukin (IL) -8 plays a critical role in the persistence of the inflammatory process in allergy. In the Part II, we aimed to investigate the mechanism by which Cra A triggers IL-8 released from human airway epithelial cells. Firstly, we determined the effects of Cra A on IL-8 expression and secretion in A549 cells. Cra A stimulated the production of IL-8 at protein and mRNA levels with dose- and time-dependent manners. Secondly, the effect of Cra A on IL-8 release was blocked by serine-protease inhibitors, PMSF and aprotinin; while it could not be suppressed by cysteine- or aspartic protease inhibitors. It demonstrated that serine protease activities were found in the extract of American cockroach. Thirdly, A549 cells expressed all four protease-activated receptors (PARs) at mRNA levels as assessed by real-time PCR. When epithelial cells were stimulated with Cra A, PAR-2 and PAR-3 mRNAs reached up to 1.73 and 1.94-folds increase than nonstimulated cells. Finally, we investigated the role of mitogen-activated protein kinases (MAPK) from PAR-2 or PAR-3 activation in IL-8 synthesis. Western blotting showed that an increase in ERK1/2 and JNK phosphorylation after Cra A stimulation. Furthermore, when A549 cells were preincubated with U0126 (ERK 1/2 kinase inhibitor) or SP600125 (JNK kinase inhibitor), it resulted in a reduction 100% and 44% of IL-8 production, respectively. We conclude that Cra A induced IL-8 release in airway epithelial cells and this is dependent on activation of PAR-2 and-3, and coordinating with the ERK1/2 and JNK signaling pathways.

中文摘要 I
英文摘要 (abstract) III
目錄 V
圖目錄 X
表目錄 XII
縮寫對照 (abbreviations) XIII
第一章 前言 1
第一節 過敏疾病的簡史 1
第二節 過敏原的定義 2
第三節 過敏反應的致病機轉 2
第四節 過敏疾病的診斷 3
1.皮膚穿刺試驗 (Skin Prick Test) 4
2.血清測試 4
第五節 過敏疾病的預防及治療 5
第六節 蟑螂過敏症 5
第七節 美國蟑螂過敏原的特性介紹 6
1.Per a 1 6
2.Per a 3 7
3.Per a 7 7
4.美國蟑螂粗萃物具備絲胺酸蛋白酶酵素活性 7
第八節 Protease-Activated Receptors (PARs) 之介紹 8
第九節 Mitogen-activated protein kinase (MAPK) 之介紹8
第十節 Interleukin 8 (IL-8) 之介紹 9
第十一節 研究目的 9
第二章 實驗材料與方法 11
第一節 實驗材料 11
1.美國蟑螂粗萃物 11
2.蟑螂過敏病人篩選方法及血清來源 11
3.A549細胞株 11
第二節 化學藥品與器材 11
1.一般化學試劑 11
2.限制酶及核酸修飾酵素 11
3.DNA電泳相關藥品 12
4.蛋白質電泳相關藥品 12
5.引子 12
第三節 重要器材及儀器 12
1.桌上型離心機 12
2.電泳設備 12
3.其他器材 12
第四節 實驗方法 13
1.微量質體DNA的萃取 13
2.表現重組蛋白質及純化 14
3.蛋白質濃度定量 17
4.蛋白質電泳分析 17
5.西方墨漬法 (Western blotting) 19
6.IgE ELISA 20
7.以Azocoll方法測定美國蟑螂粗萃物之蛋白酶活性 21
8.細胞培養 21
9.A549細胞之RNA 萃取 (RNA extraction) 22
10.即時定量聚合酶鏈鎖反應 (real-time PCR) 23
11.以 Sandwich ELISA 測定IL-8濃度 24
12.蛋白質酶抑制 (protease inhibition) 實驗 25
13.A549細胞之蛋白質萃取以及西方墨點法 25
14.MAPK pathway 抑制實驗 26
15.統計分析 27
第三章 結果 28
1.Per a 1.0104、Per a 3.0203與 Per a 7.0101質體之確認 28
2.蛋白質電泳與免疫轉漬分析 28
3.IgE ELISA 測試 29
4.IgE ELISA 之 OD 值和臨床上 i6 ImmunoCAP值兩者相關性比較 29
5.美國蟑螂粗萃物 (Cra A) 具備蛋白質酶之活性 30
6.美國蟑螂萃取物 (Cra A) 對 A549 細胞之細胞型態影響 30
7.美國蟑螂萃取物 (Cra A ) 會刺激 A549 細胞釋放interleukin 8 (IL-8) 30
8.美國蟑螂粗萃物 (Cra A) 刺激 A549 細胞釋放 IL-8，不論是在蛋白質或是基因層次都和時間成正相關 31
9.A549細胞受到Cra A處理而分泌interleukin 8 (IL-8) 會受到 serine protease inhibitor (絲胺酸蛋白質酶抑制劑) 所抑制 32
10.人類肺癌呼吸道上皮細胞株 (A549) 在Cra A處理之下，會活化PAR-2以及PAR-3 32
11.利用Western blotting 偵測 MAP kinase (ERK1/2, p38, and JNK)在 A549細胞內之活化情形 33
12.人類肺癌呼吸道上皮細胞株 (A549) 受到 Cra A刺激而分泌IL-8確實經由 MAPK 之 ERK1/2或是 JNK 訊號傳遞路徑之活化 34
第四章 討論 35
第一節 重組蛋白質之表現與純化 35
第二節 重組過敏原之應用 36
第三節 美國蟑螂致敏機轉之探討 37
第四節 未來展望 39
第五章 參考文獻 41
圖 46
Fig. 1: Identifications of Per a 1.0104、Per a 3.0203 and Per a 7.0101 recombination plasmid 46
Fig. 2: SDS-PAGE (A) and immunoblotting (B) of purified recombinant Per a 1.0104, 3.0203 and 7.0101 47
Fig. 3: Using IgE ELISA assay, 66.1, 67.8 and 57.6% of 118 sera were found to contain the specific IgE to recombinant Per a 1.0104, 3.0203 or 7.0101, respectively 48
Fig. 4: Correlation between the value of i6 CAP and IgE ELISA 49
Fig. 5: A549 cells’ morphology changed upon Cra A stimulation 50
Fig. 6: Cra A stimulation of IL-8 secretion in A549 cells 51
Fig. 7: Time course of Cra A on IL-8 production 53
Fig. 8: Effects of protease inhibitors on Cra A-induced IL-8 release by A549 cells 55
Fig. 9: Proteinase-activated receptors (PARs) expression levels were determined in human A549 cells by real-time PCR 56
Fig. 10: Effect of Cra A on PAR-2 protein total expression assesses by Western blotting 57
Fig. 11: The effect of Cra A and PAR-2 AP on MAPK activation 58
Fig. 12: Requirement of MAP kinases for IL-8 production in response to Cra A 59
Fig. 13: Proposed model for Cra A-induced IL-8 expression via PAR-2 and PAR-3 activation in A549 cell 60

表 61
Table 1: Primers for PCR amplication of IL-8 and beta-actin cDNA 61
Table 2: Clinical and IgE ELISA data of cockroach allergic patient 62

附錄 66
Fig. 1: Identifications of Per a 1.0104、Per a 3.0203 and Per a 7.0101 recombination plasmid 46
Fig. 2: SDS-PAGE (A) and immunoblotting (B) of purified recombinant Per a 1.0104, 3.0203 and 7.0101 47
Fig. 3: Using IgE ELISA assay, 66.1, 67.8 and 57.6% of 118 sera were found to contain the specific IgE to recombinant Per a 1.0104, 3.0203 or 7.0101, respectively 48
Fig. 4: Correlation between the value of i6 CAP and IgE ELISA 49
Fig. 5: A549 cells’ morphology changed upon Cra A stimulation 50
Fig. 6: Cra A stimulation of IL-8 secretion in A549 cells 51
Fig. 7: Time course of Cra A on IL-8 production 53
Fig. 8: Effects of protease inhibitors on Cra A-induced IL-8 release by A549 cells 55
Fig. 9: Proteinase-activated receptors (PARs) expression levels were determined in human A549 cells by real-time PCR 56
Fig. 10: Effect of Cra A on PAR-2 protein total expression assesses by Western blotting 57
Fig. 11: The effect of Cra A and PAR-2 AP on MAPK activation 58
Fig. 12: Requirement of MAP kinases for IL-8 production in response to Cra A 59
Fig. 13: Proposed model for Cra A-induced IL-8 expression via PAR-2 and PAR-3 activation in A549 cell 60
Table 1: Primers for PCR amplication of IL-8 and beta-actin cDNA 61
Table 2: Clinical and IgE ELISA data of cockroach allergic patient 62

1. von Pirquet, C. Allergie. Med Wehnschr 53(1906).
2. Kay, A.B. Overview of 'allergy and allergic diseases: with a view to the future'. Br Med Bull 56, 843-864 (2000).
3. Jackson. Allergy and history. Stud Hist Phil Biol & Biomed Sci 34, 383-398 (2003).
4. Valenta, R. Recombinant allergen-based concepts for diagnosis and therapy of type I allergy. Allergy 57 Suppl 71, 66-67 (2002).
5. Deinhofer, K., et al. Microarrayed allergens for IgE profiling. Methods 32, 249-254 (2004).
6. Spahn, J., Covar, R. & Stempel, D.A. Asthma: addressing consistency in results from basic science, clinical trials, and observational experience. J Allergy Clin Immunol 109, S490-502 (2002).
7. 江伯倫，臺灣大學醫學院臨床醫學研究所. 科學發展 (2002).
8. 上野川修一. 圖解過敏與免疫機制（施聖茹譯）. (2002).
9. Matsuda, T., Matsubara, T. & Hino, S. Immunogenic and allergenic potentials of natural and recombinant innocuous proteins. J Biosci Bioeng 101, 203-211 (2006).
10. Becker, W.M. & Reese, G. Immunological identification and characterization of individual food allergens. J Chromatogr B Biomed Sci Appl 756, 131-140 (2001).
11. Ticha, M., Pacakova, V. & Stulik, K. Proteomics of allergens. J Chromatogr B Analyt Technol Biomed Life Sci 771, 343-353 (2002).
12. Allergen nomenclature. WHO/IUS Allergen Nomenclature Subcommittee World Health Organization, Geneva, Switzerland. Clin Exp Allergy 25, 27-37 (1995).
13. Descotes, J. & Choquet-Kastylevsky, G. Gell and Coombs's classification: is it still valid? Toxicology 158, 43-49 (2001).
14. Wynn, T.A. IL-13 effector functions. Annu Rev Immunol 21, 425-456 (2003).
15. Thomas, W.R., Hales, B.J. & Smith, W.A. Recombinant allergens for analysing T-cell responses. Methods 32, 255-264 (2004).
16. Janeway, C.P.T., Mark Walport, and Mark Shlomchik Immunology, Fifth Edition. (2001).
17. http://people.rit.edu/japfaa/physiology.html.
18. Scolozzi, R., Boccafogli, A., Vicentini, L., Baraldi, A. & Bagni, B. Correlation of MAST chemiluminescent assay (CLA) with RAST and skin prick tests for diagnosis of inhalant allergic disease. Ann Allergy 62, 193a-193b (1989).
19. Dreborg, S. The skin prick test in the diagnosis of atopic allergy. J Am Acad Dermatol 21, 820-821 (1989).
20. Holgate. Allergy,2nd Edition, 9-11. 41
21. Costongs, G.M. & Bas, B.M. The first fully automated allergy analyser UniCAP: comparison with IMMULITE for allergy panel testing. Eur J Clin Chem Clin Biochem 35, 885-888 (1997).
22. Tsai, J.J., Liao, E.C., Tsai, F.H., Hsieh, C.C. & Lee, M.F. The effect of local nasal immunotherapy in allergic rhinitis: using strips of the allergen dermatophagoides
pteronyssinus. J Asthma 46, 165-170 (2009).
23. Baumholtz, M.A., Parish, L.C., Witkowski, J.A. & Nutting, W.B. The medical importance of cockroaches. Int J Dermatol 36, 90-96 (1997).
24. Eggleston, P.A. & Arruda, L.K. Ecology and elimination of cockroaches and allergens in the home. J Allergy Clin Immunol 107, S422-429 (2001).
25. Wu, C.H. & Lee, M.F. Molecular characteristics of cockroach allergens. Cell Mol Immunol 2, 177-180 (2005).
26. Bernton, H.S. & Brown, H. Insect Allergy--Preliminary Studies of the Cockroach. J Allergy Clin Immunol 35, 506-513 (1964).
27. Kang, B., Vellody, D., Homburger, H. & Yunginger, J.W. Cockroach cause of allergic asthma. Its specificity and immunologic profile. J Allergy Clin Immunol 63, 80-86 (1979).
28. Lan, J.L., Lee, D.T., Wu, C.H., Chang, C.P. & Yeh, C.L. Cockroach hypersensitivity: preliminary study of allergic cockroach asthma in Taiwan. J Allergy Clin Immunol 82,
736-740 (1988).
29. Wu, C.H. & Lan, J.L. Cockroach hypersensitivity: isolation and partial characterization of major allergens. J Allergy Clin Immunol 82, 727-735 (1988).
30. Wu, C.H., Lee, M.F., Yang, J.S. & Tseng, C.Y. IgE-binding epitopes of the American cockroach Per a 1 allergen. Mol Immunol 39, 459-464 (2002).
31. Wu, C.H., Lee, M.F., Wang, N.M. & Luo, S.F. Sequencing and immunochemical characterization of the American cockroach per a 3 (Cr-PI) isoallergenic variants. Mol
Immunol 34, 1-8 (1997).
32. Asturias, J.A., et al. Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen. J Immunol 162,
4342-4348 (1999).
33. Pomes, A., Wunschmann, S., Hindley, J., Vailes, L.D. & Chapman, M.D. Cockroach allergens: function, structure and allergenicity. Protein Pept Lett 14, 960-969 (2007).
34. Pomes, A., et al. Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. J Biol Chem 273, 30801-30807 (1998).
35. Wang, N.M., Lee, M.F. & Wu, C.H. Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen. Allergy 54, 119-127 (1999). 42
36. Jomori, T. & Natori, S. Molecular cloning of cDNA for lipopolysaccharide-binding protein from the hemolymph of the American cockroach, Periplaneta americana.
Similarity of the protein with animal lectins and its acute phase expression. J Biol Chem 266, 13318-13323 (1991).
37. Santos, A.B., et al. Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp
allergens. J Allergy Clin Immunol 104, 329-337 (1999).
38. Aki, T., et al. Immunochemical characterization of recombinant and native tropomyosins as a new allergen from the house dust mite, Dermatophagoides farinae. J Allergy Clin Immunol 96, 74-83 (1995).
39. Wu, C.H., Chiang, B.T., Fann, M.C. & Lan, J.L. Production and characterization of monoclonal antibodies against major allergens of American cockroach. Clin Exp
Allergy 20, 675-681 (1990).
40. Sudha, V.T., Arora, N., Gaur, S.N., Pasha, S. & Singh, B.P. Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana. Allergy 63, 768-776 (2008).
41. Adam, E., et al. The house dust mite allergen Der p 1, unlike Der p 3, stimulates the expression of interleukin-8 in human airway epithelial cells via a proteinase-activated
receptor-2-independent mechanism. J Biol Chem 281, 6910-6923 (2006).
42. Lee, K.E., et al. Regulation of German cockroach extract-induced IL-8 expression in human airway epithelial cells. Clin Exp Allergy 37, 1364-1373 (2007).
43. Chiu, L.L., Perng, D.W., Yu, C.H., Su, S.N. & Chow, L.P. Mold allergen, pen C 13, induces IL-8 expression in human airway epithelial cells by activating protease-activated receptor 1 and 2. J Immunol 178, 5237-5244 (2007).
44. Macfarlane, S.R., Seatter, M.J., Kanke, T., Hunter, G.D. & Plevin, R. Proteinase-activated receptors. Pharmacol Rev 53, 245-282 (2001).
45. Steinhoff, M., et al. Proteinase-activated receptors: transducers of proteinase-mediated signaling in inflammation and immune response. Endocr Rev 26, 1-43 (2005).
46. Rallabhandi, P., et al. Analysis of proteinase-ctivated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity. J Biol Chem 283, 24314-24325 (2008).
47. Qi, M. & Elion, E.A. MAP kinase pathways. J Cell Sci 118, 3569-3572 (2005).
48. Pearson, G., et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22, 153-183 (2001).
49. Luscinskas, F.W., et al. C-C and C-X-C chemokines trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Ann N Y Acad Sci 902, 288-293 (2000).
43
50. Hoffmann, E., Dittrich-Breiholz, O., Holtmann, H. & Kracht, M. Multiple control of interleukin-8 gene expression. J Leukoc Biol 72, 847-855 (2002).
51. Mulayim, N., Palter, S.F., Kayisli, U.A., Senturk, L. & Arici, A. Chemokine receptor expression in human endometrium. Biol Reprod 68, 1491-1495 (2003).
52. Wu, C.H., Lee, M.F. & Wang, N.M. Expression of the American cockroach Per a 1 allergen in mammalian cells. Allergy 55, 1179-1183 (2000).
53. Gelber, L.E., et al. Sensitization and exposure to indoor allergens as risk factors for asthma among patients presenting to hospital. Am Rev Respir Dis 147, 573-578
(1993).
54. Chavira, R., Jr., Burnett, T.J. & Hageman, J.H. Assaying proteinases with azocoll. Anal Biochem 136, 446-450 (1984).
55. Bhat, R.K., Page, K., Tan, A. & Hershenson, M.B. German cockroach extract increases bronchial epithelial cell interleukin-8 expression. Clin Exp Allergy 33, 35-42
(2003).
56. Reed, C.E. & Kita, H. The role of protease activation of inflammation in allergic respiratory diseases. J Allergy Clin Immunol 114, 997-1008; quiz 1009 (2004).
57. Middelberg, A.P. Preparative protein refolding. Trends Biotechnol 20, 437-443 (2002).
58. Blight, M.A. & Holland, I.B. Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator. Trends Biotechnol 12, 450-455 (1994).
59. CH, S. Production of soluble recombinant proteins in bacteria. Bio Technology 7, 1141-1149 (1989).
60. Singh, S.M. & Panda, A.K. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 99, 303-310 (2005).
61. Niederberger, V. & Valenta, R. Recombinant allergens for immunotherapy. Where do we stand? Curr Opin Allergy Clin Immunol 4, 549-554 (2004).
62. Mothes, N., Valenta, R. & Spitzauer, S. Allergy testing: the role of recombinant allergens. Clin Chem Lab Med 44, 125-132 (2006).
63. Rosenstreich, D.L., et al. The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma. N Engl J Med 336, 1356-1363 (1997).
64. Valenta, R., et al. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy 29, 896-904 (1999).
65. Scharenberg, A.M. & Kinet, J.P. Initial events in Fc epsilon RI signal transduction. J Allergy Clin Immunol 94, 1142-1146 (1994).
66. Thomas, W.R., Smith, W.A., Hales, B.J., Mills, K.L. & O'Brien, R.M. Characterization and immunobiology of house dust mite allergens. Int Arch Allergy Immunol 129, 1-18 (2002). 44
67. Hashimoto, S., et al. p38 Mitogen-activated protein kinase regulates IL-8 expression in human pulmonary vascular endothelial cells. Eur Respir J 13, 1357-1364 (1999).
68. Ostrowska, E. & Reiser, G. The protease-activated receptor-3 (PAR-3) can signal autonomously to induce interleukin-8 release. Cell Mol Life Sci 65, 970-981 (2008).