CpG ODN在OVA诱导的哮喘小鼠模型中的治疗作用
摘要
支气管哮喘(简称哮喘)是一种常见的气道慢性炎症性疾病。T淋巴细胞及其分泌的多种细胞因子在哮喘的发病过程中起重要作用,表现为Th1/Th2细胞功能失衡且Th2型免疫反应占优势。大量哮喘动物实验研究表明,CpG ODN能够增强Th1型免疫反应进而抑制哮喘的特征性表现,包括减少气道嗜酸性粒细胞的炎性浸润、降低气道高反应性、下调Th2类细胞因子的表达以及降低血清中变应原特异性的IgE的水平。
CpG ODN是一种含未甲基化CpG基序的寡脱氧核苷酸,通过模拟细菌的DNA片段经人工合成,具有广泛免疫调节作用。根据不同的结构特点和体外刺激作用,CpG ODN可分为A、B和C三种类型。Dynavax公司研制的ISS 1018是一种B型CpG ODN,临床前和临床试验已证实了ISS 1018对过敏性疾病有治疗作用。
在本课题中,我们应用卵清白蛋白(OVA)诱导的急性哮喘小鼠模型,探讨了本科室自行设计的三种新型ODN(含CpG基序的B型CpG ODN和C型CpG ODN,不含CpG基序的抑制性ODN)对哮喘的治疗作用,并以ISS 1018为阳性对照。结果表明,不论是腹腔注射还是鼻腔滴入,C型CpG ODN均能显著减轻气道炎症反应、减少嗜酸性粒细胞的浸润和粘液分泌。在变应原致敏的同时给予C型CpG ODN,能够增强Th1型细胞因子的表达并促进IgG2a抗体的产生,还可能激活调节性T细胞发挥免疫抑制作用,进而下调了哮喘的Th2型优势免疫反应。经比较得出,C型CpG ODN对哮喘的治疗效果优于ISS 1018,提示本室拥有自主知识产权的C型CpG ODN可能成为一种治疗哮喘的新型有效的免疫调节剂。
Background
Bronchial asthma is a kind of chronic inflammatory airway disease which involves multiple cells and cellular components. The inflammatory cells include mast cells, eosinophils and T lymphocytes, and the cellular components consist of cytokines, chemokines, inflammatory mediators and so on. T lymphocytes and a variety of cytokines play an important role in the process of asthma. The immune changes of asthma is considered to be Th1/Th2 imbalance and Th2 immune response.dominated
CpG ODN, mimicing bacterial DNA fragments and containing unmethylated CpG motif, is a kind of synthetic oligodeoxynucleotide with immune stimulating effects. CpG ODN can be divided into three types including A, B and C. They are recognized by TLR9 expressed in endosomes of human B cells and plasmacytoid dendritic cells (pDC), and capable of stimulating NK cells, macrophages and cytotoxic T lymphocytes (CTL) indirectly. Thus CpG ODN could induce and enhance the innate immune response and acquired immune response, including Th1 biased immune response and T regulatory immune response. Animal experiments of asthma show that CpG ODN, either used alone or combined with allergen systemicly or locally, can prevent the occurrence of asthma by inhibiting the development of airway eosinophilic inflammatory infiltration, airway hyper-responsiveness (AHR) and the secretion of allergen-specific IgE and Th2-type cytokines. Therefore, CpG ODN displays promising prospect in treating asthma.
Purpose
In this study, novel B- type CpG ODN, C-type CpG ODN and suppressive ODN designed by our lab were used in OVA-induced mouse model of acute asthma. By intraperitoneal injection, select a candidate CpG ODN with best therapeutic effect on asthma and preliminary explore the possible mechanism. In the following study, through nasal instillation, evaluate the effect of the candidate CpG ODN on treating asthma.
Methods
1 Establishment of asthma model and intervention by ODN.
In the first part, thirty-six BALB/c mice were randomly divided into six groups, including OVA group, C-CpGgroup, B-CpG group, S-ODN group, ISS 1018 group and NS group,. On day 0, 7 and 14, by intraperitoneal injection, the mice were sensitized with OVA/Alum (10μg/1 mg, OVA group) or OVA/Alum/ODNs (10μg/1 mg/5μg, ODN groups), or NS/Alum (NS group) in 200μl of saline, respectively. From day 21, mice were challenged with 1%OVA solution in a self-made glass container for 20min each time during five consecutive days. Then mice were killed on day 26. In the second part, eighteen BALB/c mice were randomly divided into three groups, including OVA group, C-CpG group and NS group. On day 0, 7 and 14, mice in OVA group and C-CpG group were sensitized with OVA/Alum (10μg/1 mg) in 200μl of saline, and NS group with NS/Alum instead. On day 21, 22 and 23, mice in C-CpG group were treated by nasal instillation of C-CpG (15μg), NS and OVA group treated by NS instead. From day 25～28, mice were challenged using the previous method. Then mice were killed on day 29.
2 Record of asthmatic symptoms in the process of OVA challenge.
In the process of OVA atomization challenge, a variety of clinical symptoms of mice including irritability, shortness of breath, head and face itching, arched back, and nodding motion were observed during five consecutive days. And times of touching noses, times of spasm, latency of spasm and mean duration of each spasm were recorded under the blind principle.
3 Detection of mRNA expression of cytokines by RT-PCR.
Right lungs of mice were isolated, shredded and stored in Trizol reagent. Then transcription factors GATA-3 and T-bet, cytokines including IL-4、IFN-γ、IL-10 and TGF-βmRNA expression were detected by RT-PCR.
4 Related detection of lung histopathology.
The rest of lungs were fixed in 10% Formalin and prepared for pathlogical sections by HE staining and PAS staining. Pathological scores and eosinophils counts were analyzed in HE staining sections through the light microscope.
5 Detection of OVA specific antibodies by indirect ELISA.
Blood were collected by tail vein the day before each OVA sensitization and initial challenge, and by removing eyes on the final day. After sera were isolated, OVA specific IgG, IgG1 and IgG2a antibodies were detected by indirect ELISA.
Results
The results of symptoms during the process of OVA challenge indicated that times of touching noses in model group had no significance compared with control group in two models. In the first model, times of spasm in model group become more, mean duration of each spasm become longer, and latency of spasm become shorter. While in the second model, times of spasm and latency of spasm in model group had no significance compared with control group. Using the quantitative indicators of symptoms to evaluate the success of asthma model should be questioned.
Through histopathology of HE staining, the model group showed obvious bronchial epithelial damage, mucosal edema, bronchial mucus secretion and inflammatory cell infiltration peri-bronchial and peri-blood vessels. By PAS staining, the model group is strongly positive, with marked hyperplasia of goblet cells, accompanied by mucus hyper-secretion and mucus plug formation. In contrast, the control group showed no epithelial thickening, no airway inflammatory cell infiltration but structural integrity of the alveolar wall. And PAS staining is negative with no mucus secretion. Pathological scores and EOS counts in the model group were obviously higher than the control group. It is indicated that the established asthma model was a success and could be used to study the effect of CpG ODN on asthma.
Compared with the model group, C-CpG and B-CpG group both significantly reduced airway EOS infiltrated inflammation and mucus secretion. The effects were better than ISS 1018 and S-ODN. C-CpG alone by nasal instillation also significantly reduced allergic airway inflammation in asthma.
C-CpG and B-CpG induced lower levels of IL-4 and higher levels of T-bet、IFN-γ、IL-10 and TGF-β. C-CpG upregulated the ratio of T-bet/GATA-3 and IFN-γ/IL-4.
At the time of OVA sensitization, intraperitoneal injection of all ODNs significantly increased the levels of OVA specific IgG. B-CpG、ISS 1018 and S-ODN increased IgG1. C-CpG and B-CpG increased IgG2a. C-CpG upregulated the ratio of IgG2a/IgG1. However, before OVA challenge, intranasal instillation of C-CpG had little effect on levels of IgG, IgG1 and IgG2a.
Conclusion
At the time of OVA sensitization, intraperitoneal injection of C type CpG ODN, significantly reduces airway eosinophils infiltrated inflammation of asthma, and inhibit Th2-type cytokines. It increases Th1-type transcription factors and cytokines and induce high levels of OVA specific IgG2a. It could also stimulate T regulatory inhibited cytokines to regulate Th1/Th2 imbalanced immune response. C type CpG ODN show better effect than B type CpG ODN. Before OVA challenge, intranasal instillation of C type CpG ODN alone also reduces airway eosinophils infiltrated inflammation of asthma. Therefore, C type CpG ODN might be a novel immunomodulator to treat asthma.
CpG ODN是一种含未甲基化CpG基序的寡脱氧核苷酸,通过模拟细菌的DNA片段经人工合成,具有广泛免疫调节作用。根据不同的结构特点和体外刺激作用,CpG ODN可分为A、B和C三种类型。Dynavax公司研制的ISS 1018是一种B型CpG ODN,临床前和临床试验已证实了ISS 1018对过敏性疾病有治疗作用。
在本课题中,我们应用卵清白蛋白(OVA)诱导的急性哮喘小鼠模型,探讨了本科室自行设计的三种新型ODN(含CpG基序的B型CpG ODN和C型CpG ODN,不含CpG基序的抑制性ODN)对哮喘的治疗作用,并以ISS 1018为阳性对照。结果表明,不论是腹腔注射还是鼻腔滴入,C型CpG ODN均能显著减轻气道炎症反应、减少嗜酸性粒细胞的浸润和粘液分泌。在变应原致敏的同时给予C型CpG ODN,能够增强Th1型细胞因子的表达并促进IgG2a抗体的产生,还可能激活调节性T细胞发挥免疫抑制作用,进而下调了哮喘的Th2型优势免疫反应。经比较得出,C型CpG ODN对哮喘的治疗效果优于ISS 1018,提示本室拥有自主知识产权的C型CpG ODN可能成为一种治疗哮喘的新型有效的免疫调节剂。
Background
Bronchial asthma is a kind of chronic inflammatory airway disease which involves multiple cells and cellular components. The inflammatory cells include mast cells, eosinophils and T lymphocytes, and the cellular components consist of cytokines, chemokines, inflammatory mediators and so on. T lymphocytes and a variety of cytokines play an important role in the process of asthma. The immune changes of asthma is considered to be Th1/Th2 imbalance and Th2 immune response.dominated
CpG ODN, mimicing bacterial DNA fragments and containing unmethylated CpG motif, is a kind of synthetic oligodeoxynucleotide with immune stimulating effects. CpG ODN can be divided into three types including A, B and C. They are recognized by TLR9 expressed in endosomes of human B cells and plasmacytoid dendritic cells (pDC), and capable of stimulating NK cells, macrophages and cytotoxic T lymphocytes (CTL) indirectly. Thus CpG ODN could induce and enhance the innate immune response and acquired immune response, including Th1 biased immune response and T regulatory immune response. Animal experiments of asthma show that CpG ODN, either used alone or combined with allergen systemicly or locally, can prevent the occurrence of asthma by inhibiting the development of airway eosinophilic inflammatory infiltration, airway hyper-responsiveness (AHR) and the secretion of allergen-specific IgE and Th2-type cytokines. Therefore, CpG ODN displays promising prospect in treating asthma.
Purpose
In this study, novel B- type CpG ODN, C-type CpG ODN and suppressive ODN designed by our lab were used in OVA-induced mouse model of acute asthma. By intraperitoneal injection, select a candidate CpG ODN with best therapeutic effect on asthma and preliminary explore the possible mechanism. In the following study, through nasal instillation, evaluate the effect of the candidate CpG ODN on treating asthma.
Methods
1 Establishment of asthma model and intervention by ODN.
In the first part, thirty-six BALB/c mice were randomly divided into six groups, including OVA group, C-CpGgroup, B-CpG group, S-ODN group, ISS 1018 group and NS group,. On day 0, 7 and 14, by intraperitoneal injection, the mice were sensitized with OVA/Alum (10μg/1 mg, OVA group) or OVA/Alum/ODNs (10μg/1 mg/5μg, ODN groups), or NS/Alum (NS group) in 200μl of saline, respectively. From day 21, mice were challenged with 1%OVA solution in a self-made glass container for 20min each time during five consecutive days. Then mice were killed on day 26. In the second part, eighteen BALB/c mice were randomly divided into three groups, including OVA group, C-CpG group and NS group. On day 0, 7 and 14, mice in OVA group and C-CpG group were sensitized with OVA/Alum (10μg/1 mg) in 200μl of saline, and NS group with NS/Alum instead. On day 21, 22 and 23, mice in C-CpG group were treated by nasal instillation of C-CpG (15μg), NS and OVA group treated by NS instead. From day 25～28, mice were challenged using the previous method. Then mice were killed on day 29.
2 Record of asthmatic symptoms in the process of OVA challenge.
In the process of OVA atomization challenge, a variety of clinical symptoms of mice including irritability, shortness of breath, head and face itching, arched back, and nodding motion were observed during five consecutive days. And times of touching noses, times of spasm, latency of spasm and mean duration of each spasm were recorded under the blind principle.
3 Detection of mRNA expression of cytokines by RT-PCR.
Right lungs of mice were isolated, shredded and stored in Trizol reagent. Then transcription factors GATA-3 and T-bet, cytokines including IL-4、IFN-γ、IL-10 and TGF-βmRNA expression were detected by RT-PCR.
4 Related detection of lung histopathology.
The rest of lungs were fixed in 10% Formalin and prepared for pathlogical sections by HE staining and PAS staining. Pathological scores and eosinophils counts were analyzed in HE staining sections through the light microscope.
5 Detection of OVA specific antibodies by indirect ELISA.
Blood were collected by tail vein the day before each OVA sensitization and initial challenge, and by removing eyes on the final day. After sera were isolated, OVA specific IgG, IgG1 and IgG2a antibodies were detected by indirect ELISA.
Results
The results of symptoms during the process of OVA challenge indicated that times of touching noses in model group had no significance compared with control group in two models. In the first model, times of spasm in model group become more, mean duration of each spasm become longer, and latency of spasm become shorter. While in the second model, times of spasm and latency of spasm in model group had no significance compared with control group. Using the quantitative indicators of symptoms to evaluate the success of asthma model should be questioned.
Through histopathology of HE staining, the model group showed obvious bronchial epithelial damage, mucosal edema, bronchial mucus secretion and inflammatory cell infiltration peri-bronchial and peri-blood vessels. By PAS staining, the model group is strongly positive, with marked hyperplasia of goblet cells, accompanied by mucus hyper-secretion and mucus plug formation. In contrast, the control group showed no epithelial thickening, no airway inflammatory cell infiltration but structural integrity of the alveolar wall. And PAS staining is negative with no mucus secretion. Pathological scores and EOS counts in the model group were obviously higher than the control group. It is indicated that the established asthma model was a success and could be used to study the effect of CpG ODN on asthma.
Compared with the model group, C-CpG and B-CpG group both significantly reduced airway EOS infiltrated inflammation and mucus secretion. The effects were better than ISS 1018 and S-ODN. C-CpG alone by nasal instillation also significantly reduced allergic airway inflammation in asthma.
C-CpG and B-CpG induced lower levels of IL-4 and higher levels of T-bet、IFN-γ、IL-10 and TGF-β. C-CpG upregulated the ratio of T-bet/GATA-3 and IFN-γ/IL-4.
At the time of OVA sensitization, intraperitoneal injection of all ODNs significantly increased the levels of OVA specific IgG. B-CpG、ISS 1018 and S-ODN increased IgG1. C-CpG and B-CpG increased IgG2a. C-CpG upregulated the ratio of IgG2a/IgG1. However, before OVA challenge, intranasal instillation of C-CpG had little effect on levels of IgG, IgG1 and IgG2a.
Conclusion
At the time of OVA sensitization, intraperitoneal injection of C type CpG ODN, significantly reduces airway eosinophils infiltrated inflammation of asthma, and inhibit Th2-type cytokines. It increases Th1-type transcription factors and cytokines and induce high levels of OVA specific IgG2a. It could also stimulate T regulatory inhibited cytokines to regulate Th1/Th2 imbalanced immune response. C type CpG ODN show better effect than B type CpG ODN. Before OVA challenge, intranasal instillation of C type CpG ODN alone also reduces airway eosinophils infiltrated inflammation of asthma. Therefore, C type CpG ODN might be a novel immunomodulator to treat asthma.
引文
[1]. Alexey Fedulov, Eric Silverman, Yuhong Xiang, et al. Immunostimulatory CpG Oligonucleotides Abrogate Allergic Susceptibility in a Murine Model of Maternal Asthma Transmission[J]. The Journal of Immunology, 2005, 175: 4292–4300.
[2]. Beatrice Jahn-Schmid, Ursula Wiedermann, Barbara Bohle, et al. Oligodeoxynucleotides containing CpG motifs modulate the allergic Th2 response of BALB/c mice to Bet v 1, the major birch pollen allergen[J]. Allergy Clin Immunol, 1999, 1015–23.
[3]. Bhattacharjee RN, Akira S, et al. Modifying toll- like receptor 9 signaling for therapeutic use[J]. Mini Rev Med Chem, 2006, 6(3):287–291.
[4]. Brewer JP, Kisselgof AB, Martin TR.Genetic variability in pulmonary physiologic, cellular, and antibody responses to antigen in mice[J]. Am J Respir Crit Care Med, 1999;160:1150–1156.
[5]. Buhl R, et al. Omalizumab (Xolairs) improves quality of life in adult patients with allergic asthma: a review[J]. Respir Med, 2003, 97:123–129.
[6]. David Broide, Jurgan Schwarze, et al. Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice[J]. The Journal of Immunology 1998, 161: 7054–7062.
[7]. David E. Fonseca, Joel N. Kline. Use of CpG oligonucleotides in treatment of asthma and allergic disease[J]. Advanced Drug Delivery Reviews 2009, 61: 256–262.
[8]. DAVID H. BROIDE, GREG STACHNICK, DIEGO CASTANEDA, et al. Systemic administration of immunostimulatory DNA sequences mediates reversible inhibition of Th2 responses in a mouse model of asthma[J]. Journal of Clinical Immunology, 2001, 21(3):175–182.
[9]. Denise Serebrisky, Ariel A. Teper, Chih-Kang Huang, et al. CpG Oligodeoxynucleotides can reverse Th2-associated allergic airway responses and alter the B7.1/B7.2 expression in a murine model of asthma[J]. The Journal of Immunology, 2000, 165: 5906–5912.
[10]. Dian-Jung Chiang, Yi-Ling Ye, Wei-Li Chen, et al. Ribavirin or CpG DNASequence–Modulated Dendritic Cells Decrease the IgE Level and Airway Inflammation[J]. Am J Respir Crit Care Med, 2003, 168: 575–580.
[11]. Diego Alignani, Belkys Maletto, Miriam Liscovsky, et al. Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice[J]. Leukoc. Biol, 2005, 77: 898–905.
[12]. DONG Liang, CHEN Ming, ZHANG Qi, et al. T-bet/GATA-3 ratio is a surrogate measure of Th1/Th2 cytokine profiles and may be novel targets for CpG ODN treatment in asthma patients[J]. Chin Med J, 2006, 119(16):1396–1399.
[13]. F. Estelle R. Simons, Yasufumi Shikishima, Gary Van Nest, et al. Selective immune redirection in humans with ragweed allergy by injecting Amb a 1 linked to immunostimulatory DNA[J]. Allergy Clin Immunol, 2004, 113: 1144–51.
[14]. Finotto S, Sanctis GT, Lehr HA, et al. Treatment of allergic airway inflammation and hyper-responsiveness by anti-sense induced local blockade of GATA-3 expression[J]. Exp Med, 2001, 193(10): 1247–1260.
[15]. Finotto S, Neurath MF, Glickman JN, et al. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet[J]. Science, 2002, 295(11): 336–338.
[16]. Gail M. Gauvreau, Edith M. Hessel, et al. Immunostimulatory Sequences Regulate Interferon-Inducible Genes but not Allergic Airway Responses[J]. Am J Respir Crit Care Med, 2006, 174(1):15–20.
[17]. G. Senti, P. Johansen, S. Haug, et al. Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trial[J]. Clinical & Experimental, Allergy, 2009, 39: 562–570.
[18]. Gail M. Gauvreau , Edith M. Hessel, et al. Immunostimulatory Sequences Regulate Interferon-Inducible Genes but not Allergic Airway Responses[J]. American Journal of Respiratory and Critical Care Medicine, 2006, 174: 15–20.
[19]. Herz U, Braun A, Ruckert R, Renz H. Various immunological phenotypes are associated with increased airway responsiveness[J]. Clin Exp Allergy, 1998, 28: 625–634.
[20]. Hessel EM, Van Oosterhout AJ, Hofstra CL, et al. Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice[J]. Eur J Pharmacol,1995, 293: 401–412.
[21]. Jae Youn Cho, Marina Miller, Kwang Je Baek, et al. Immunostimulatory DNA Inhibits transforming growth factor-βexpression and airway remodeling [J]. Am. J. Respir. Cell Mol. Biol, 2004, 30: 651–661.
[22]. Jae Youn Cho, Marina Miller, Kwang Je Baek, et al. Immunostimulatory DNA sequences inhibit respiratory syncytial viral load, airway inflammation, and mucus secretion [J]. Allergy Clin Immunol, 2001, 108: 697–702.
[23]. J-C Renauld, et al. New insights into the role of cytokines in asthma[J]. Clin Pathol, 2001, 54: 577–589.
[24]. J.G. McHutchison, B.R. Bacon, S.C. Gordon, E. Lawitz, M. Shiffman, et al. Phase 1B, randomized, double-blind, dose-escalation trial of CpG 10101 in patients with chronic hepatitis C virus[J]. Hepatology, 2007, 46: 1341–1349.
[25]. Joanna V. Santeliz, Gary Van Nest, Paula Traquina, et al. Amb a 1–linked CpG oligodeoxynucleotides reverse established airway hyperresponsiveness in a murine model of asthma[J]. Allergy Clin Immunol, 2002, 109: 455–62.
[26]. JOEL N. KLINE, KUNIHIKO KITAGAKI, THOMAS R. BUSINGA, et al. Treatment of established asthma in a murine model using CpG oligodeoxynucleotides[J]. Am J Physiol Lung Cell Mol Physiol, 2002, 283: 170–179.
[27]. Joel N. Kline, Thomas J. Waldschmidt, et al. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma[J]. The Journal of Immunology, 1998, 160: 2555–2559.
[28]. Hadama K, Suzaki Y, Goldman A, et al. Allergy independent maternal transmission of asthma susceptibility[J]. Immunol, 2003, 170: 1683–1689.
[29]. Hall G, Houghton CG, Rahbek JU, et al. Suppression of allergen reactive Th2-mediated responses and pulmonary eosinophilia by intranasal administration of an immunodominant peptide is linked to IL-10 production[J]. Vaccine, 2003, 21(526): 549–561.
[30]. Hayashit, Gong X, Rossetto C, et al. Induction and inhibition of the Th2 phenotype spread: implications for childhood asthma[J]. Immunology, 2005, 174(9): 5864–5873.
[31]. Helen Tighe, Kenji Takabayashi, David Schwartz, et al. Conjugation of immunostimulatoryDNA to the short ragweed allergen Amb a 1 enhances its immunogenicity and reduces its allergenicity[J]. Allergy Clin Immunol, 2000, 106: 124–34.
[32]. Hidekazu Shirota, Kunio Sano, Tadashi Kikuchi, et al. Regulation of murine airway eosinophilia and Th2 cells by antigen-conjugated CpG Oligodeoxynucleotides as a novel antigen-specific immunomodulator[J]. The Journal of Immunology, 2000, 164: 5575–5582.
[33]. Hidekazu Shirota, Kunio Sano, Tadashi Kikuchi, et al. Regulation of T-helper Type 2 cell and airway eosinophilia by transmucosal coadministration of antigen and Oligodeoxynucleotides containing CpG motifs[J]. Am. J. Respir. Cell Mol. Biol, 2000, 22: 176–182.
[34]. Jason D. Marshall, Simin Abtahi, Joseph J. Eiden, et al. Immunostimulatory sequence DNA linked to the Amb a 1 allergen promotes TH1 cytokine expression while downregulating TH2 cytokine expression in PBMCs from human patients with ragweed allergy[J]. Allergy Clin Immunol, 2001, 108: 191–7.
[35]. Kazuhisa Asai, Susan C Foley, et al. Amb a 1-immunostimulatory oligodeoxynucleotide conjugate immunotherapy increases CD4+CD25+ T cells in the nasal mucosa of subjects with allergic rhinitis[J]. Allergol Int, 2008, 57(4): 377–81
[36]. Kenji Takabayashi, Lev Libet, Dugald Chisholm, et al. Intranasal immunotherapy is more effective than intradermal immunotherapy for the induction of airway allergen tolerance in Th2-sensitized mice[J]. The Journal of Immunology, 2003, 170: 3898–3905.
[37]. K. Kitagaki, T. R. Businga and J. N. Kline. Oral administration of CpG-ODNs suppresses antigen-induced asthma in mice[J]. Clinical and Experimental Immunology, 2005;143: 249–259.
[38]. Kline JN, Krieg AM, Waldschmidt TJ, et al. CpG oligodeoxynucleotides do not require Th1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma[J]. Allergy Clin Immunol, 1999, 104(6): 1258–64.
[39]. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM. CpG motifs in bacterial DNA trigger direct B-cell activation[J]. Nature, 1995, 374: 546–549.
[40]. Kumar RK, Foster PS: Modeling allergic asthma in mice-pitfalls and opportunities[J]. Am JRespir Cell Mol Biol, 2002, 27: 267–272.
[41]. Kunihiko Kitagaki, Vipul V. Jain, Thomas R. Businga, et al. Immunomodulatory effects of CpG Oligodeoxynucleotides on established Th2 responses[J]. Clinical and Diagnostic Laboratory Immunology, 2002, p. 1260–1269.
[42]. Michelle V. Fanucchi, Edward S. Schelegle, Gregory L. Baker, et al. Immunostimulatory oligonucleotides attenuate airways remodeling in allergic monkeys[J]. Am J Respir Crit Care Med, 2004, 170: 1153–1157.
[43]. Peter S. Creticos, John T. Schroeder, et al. Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis[J]. N Engl J Med, 2006, 355(14): 1445–55.
[44]. Reid K. Ikeda, Jyothi Nayar, Jae Youn Cho, et al. Resolution of airway inflammation following ovalbumin inhalation comparison of ISS DNA and Corticosteroids[J]. Am. J. Respir. Cell Mol. Biol, 2003, 28: 655–663.
[45]. Repa, A. et al. In?uence of the route of sensitization on local and systemic immune responses in a murine model of type I allergy[J]. Clin. Exp.Immunol, 2004, 137: 12–18.
[46]. Sanjiv Sur, James S. Wild, Barun K. Choudhury, et al. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG Oligodeoxynucleotides[J]. The Journal of Immunology, 1999;162: 6284–6293.
[47]. Schelegle ES, Gershwin LJ, Miller LA, Fanucchi MV, et al. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae) [J]. Am J Pathol, 2001, 158: 333–341.
[48]. Sel S, Wegmann M, Dicke T, et al. Effective prevention and therapy of experimental allergic asthma using a GATA-3 specific DNAzyme[J]. Allergy Clin Immunol, 2008, 121(4): 910–916.
[49]. Shigeru Ashino1, Daiko Wakita1, Yue Zhang, et al. CpG-ODN inhibits airway inflammation at effector phase through down-regulation of antigen-specific Th2-cell migration into lung[J]. International Immunology, 2007, 20(2): 259–266.
[50]. Sook Young Lee, Jae Youn Cho, Marina Miller, et al. Immunostimulatory DNA inhibits allergen induced peribronchial angiogenesis in mice [J]. Allergy Clin Immunol, 2006, 117: 597–603.
[51]. S. Underwood, M. Foster, D. Raeburn, et al. Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness[J]. Eur Respir J, 1995, 8: 2104–2113.
[52]. Takeahita F, Gursel I , Ishii KJ, et al. Signal transduction pathways mediated by the interaction of CpG DNA with Toll - like receptor 9 [J]. Sem in Immnol, 2004, 16(1): 17–21.
[53]. Takeda K, Hamelmann E, Joetham A, Shultz LD, Larsen GL, Irvin CG, Gelfand EW. Development of eosinophilic airway infammation and airway hyperresponsiveness in mast cell-defcient mice[J]. Exp Med, 1997, 186: 449–54.
[54]. Tahereh Mousavi, Nader Tajik, Maziar Moradi, et al. CpG Immunotherapy in Chenopodium album sensitized mice: The comparison of IFN-gamma, IL-10 and IgE responses in intranasal and subcutaneous administrations[J]. Clinical and Molecular Allergy, 2008, 6: 10–15.
[55]. Tulic MK, Fiset PO, Christodoulopoulos P, et al. Amb a 1-immunostimulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal in?ammatory response[J]. Allergy Clin Immunol, 2004, 113: 235–41.
[56]. Victor Matheu, Alexandra Treschow, Ingrid Teige, et al. Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-βknock-out mice[J]. Respiratory Research, 2005, 6: 25–37.
[57]. Vipul V. Jain, Kunihiko Kitagaki, Thomas Businga, et al. CpG-oligodeoxy nucleotides inhibit airway remodeling in a murine model of chronic asthma[J]. Allergy Clin Immunol, 2002, 110:867–72.
[58]. Vipul V. Jain, Thomas R. Businga, Kunihiko Kitagaki, et al. Mucosal immunotherapy with CpG oligodeoxynucleotides reverses a murine model of chronic asthma induced by repeated antigen exposure[J]. Am J Physiol Lung Cell Mol Physiol, 2003, 285: 1137–1146.
[59]. Vollmer J, Weeratna R, Payette P, Jurk M, Schetter C, Laucht M, Wader T, Tluk S, Liu M, Davis HL, Krieg AM. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities[J]. Eur. J. Immunol, 2004, 34: 251–262
[60]. Wen Xu, Toshiki Tamura, Kiyoshi Takatsu, et al. CpG ODN mediated prevention from ovalbumin-induced anaphylaxis in mouse through B cell pathway[J]. InternationalImmunopharmacology, 2008, 8: 351–361.
[61]. Wilson DR, Nouri Aria KT, Walker SM, et al. Grass pollen immunotherapy: symptomatic improvement correlates with reductions in eosinophils and IL -5 mRNA expression in the nasal mucosa during the pollen season[J]. Allergy Clin Immunol, 2001, 107(6): 971–976.
[62]. Zosky GR, Sly PD. Animal models of asthma[J]. Clin Exp Allergy, 2007, 37: 973–988.
[63]. Zu-qun WU, Yi-ping XU, Hua XIANG, Hua-hao SHEN. Effects of CpG oligodeoxynucleotide on transcription factors GATA-3 and T-bet mRNA expression in asthmatic mice[J]. Acta Pharmacologica Sinica, 2005, 26(9): 1117–1122.
[2]. Beatrice Jahn-Schmid, Ursula Wiedermann, Barbara Bohle, et al. Oligodeoxynucleotides containing CpG motifs modulate the allergic Th2 response of BALB/c mice to Bet v 1, the major birch pollen allergen[J]. Allergy Clin Immunol, 1999, 1015–23.
[3]. Bhattacharjee RN, Akira S, et al. Modifying toll- like receptor 9 signaling for therapeutic use[J]. Mini Rev Med Chem, 2006, 6(3):287–291.
[4]. Brewer JP, Kisselgof AB, Martin TR.Genetic variability in pulmonary physiologic, cellular, and antibody responses to antigen in mice[J]. Am J Respir Crit Care Med, 1999;160:1150–1156.
[5]. Buhl R, et al. Omalizumab (Xolairs) improves quality of life in adult patients with allergic asthma: a review[J]. Respir Med, 2003, 97:123–129.
[6]. David Broide, Jurgan Schwarze, et al. Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice[J]. The Journal of Immunology 1998, 161: 7054–7062.
[7]. David E. Fonseca, Joel N. Kline. Use of CpG oligonucleotides in treatment of asthma and allergic disease[J]. Advanced Drug Delivery Reviews 2009, 61: 256–262.
[8]. DAVID H. BROIDE, GREG STACHNICK, DIEGO CASTANEDA, et al. Systemic administration of immunostimulatory DNA sequences mediates reversible inhibition of Th2 responses in a mouse model of asthma[J]. Journal of Clinical Immunology, 2001, 21(3):175–182.
[9]. Denise Serebrisky, Ariel A. Teper, Chih-Kang Huang, et al. CpG Oligodeoxynucleotides can reverse Th2-associated allergic airway responses and alter the B7.1/B7.2 expression in a murine model of asthma[J]. The Journal of Immunology, 2000, 165: 5906–5912.
[10]. Dian-Jung Chiang, Yi-Ling Ye, Wei-Li Chen, et al. Ribavirin or CpG DNASequence–Modulated Dendritic Cells Decrease the IgE Level and Airway Inflammation[J]. Am J Respir Crit Care Med, 2003, 168: 575–580.
[11]. Diego Alignani, Belkys Maletto, Miriam Liscovsky, et al. Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice[J]. Leukoc. Biol, 2005, 77: 898–905.
[12]. DONG Liang, CHEN Ming, ZHANG Qi, et al. T-bet/GATA-3 ratio is a surrogate measure of Th1/Th2 cytokine profiles and may be novel targets for CpG ODN treatment in asthma patients[J]. Chin Med J, 2006, 119(16):1396–1399.
[13]. F. Estelle R. Simons, Yasufumi Shikishima, Gary Van Nest, et al. Selective immune redirection in humans with ragweed allergy by injecting Amb a 1 linked to immunostimulatory DNA[J]. Allergy Clin Immunol, 2004, 113: 1144–51.
[14]. Finotto S, Sanctis GT, Lehr HA, et al. Treatment of allergic airway inflammation and hyper-responsiveness by anti-sense induced local blockade of GATA-3 expression[J]. Exp Med, 2001, 193(10): 1247–1260.
[15]. Finotto S, Neurath MF, Glickman JN, et al. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet[J]. Science, 2002, 295(11): 336–338.
[16]. Gail M. Gauvreau, Edith M. Hessel, et al. Immunostimulatory Sequences Regulate Interferon-Inducible Genes but not Allergic Airway Responses[J]. Am J Respir Crit Care Med, 2006, 174(1):15–20.
[17]. G. Senti, P. Johansen, S. Haug, et al. Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trial[J]. Clinical & Experimental, Allergy, 2009, 39: 562–570.
[18]. Gail M. Gauvreau , Edith M. Hessel, et al. Immunostimulatory Sequences Regulate Interferon-Inducible Genes but not Allergic Airway Responses[J]. American Journal of Respiratory and Critical Care Medicine, 2006, 174: 15–20.
[19]. Herz U, Braun A, Ruckert R, Renz H. Various immunological phenotypes are associated with increased airway responsiveness[J]. Clin Exp Allergy, 1998, 28: 625–634.
[20]. Hessel EM, Van Oosterhout AJ, Hofstra CL, et al. Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice[J]. Eur J Pharmacol,1995, 293: 401–412.
[21]. Jae Youn Cho, Marina Miller, Kwang Je Baek, et al. Immunostimulatory DNA Inhibits transforming growth factor-βexpression and airway remodeling [J]. Am. J. Respir. Cell Mol. Biol, 2004, 30: 651–661.
[22]. Jae Youn Cho, Marina Miller, Kwang Je Baek, et al. Immunostimulatory DNA sequences inhibit respiratory syncytial viral load, airway inflammation, and mucus secretion [J]. Allergy Clin Immunol, 2001, 108: 697–702.
[23]. J-C Renauld, et al. New insights into the role of cytokines in asthma[J]. Clin Pathol, 2001, 54: 577–589.
[24]. J.G. McHutchison, B.R. Bacon, S.C. Gordon, E. Lawitz, M. Shiffman, et al. Phase 1B, randomized, double-blind, dose-escalation trial of CpG 10101 in patients with chronic hepatitis C virus[J]. Hepatology, 2007, 46: 1341–1349.
[25]. Joanna V. Santeliz, Gary Van Nest, Paula Traquina, et al. Amb a 1–linked CpG oligodeoxynucleotides reverse established airway hyperresponsiveness in a murine model of asthma[J]. Allergy Clin Immunol, 2002, 109: 455–62.
[26]. JOEL N. KLINE, KUNIHIKO KITAGAKI, THOMAS R. BUSINGA, et al. Treatment of established asthma in a murine model using CpG oligodeoxynucleotides[J]. Am J Physiol Lung Cell Mol Physiol, 2002, 283: 170–179.
[27]. Joel N. Kline, Thomas J. Waldschmidt, et al. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma[J]. The Journal of Immunology, 1998, 160: 2555–2559.
[28]. Hadama K, Suzaki Y, Goldman A, et al. Allergy independent maternal transmission of asthma susceptibility[J]. Immunol, 2003, 170: 1683–1689.
[29]. Hall G, Houghton CG, Rahbek JU, et al. Suppression of allergen reactive Th2-mediated responses and pulmonary eosinophilia by intranasal administration of an immunodominant peptide is linked to IL-10 production[J]. Vaccine, 2003, 21(526): 549–561.
[30]. Hayashit, Gong X, Rossetto C, et al. Induction and inhibition of the Th2 phenotype spread: implications for childhood asthma[J]. Immunology, 2005, 174(9): 5864–5873.
[31]. Helen Tighe, Kenji Takabayashi, David Schwartz, et al. Conjugation of immunostimulatoryDNA to the short ragweed allergen Amb a 1 enhances its immunogenicity and reduces its allergenicity[J]. Allergy Clin Immunol, 2000, 106: 124–34.
[32]. Hidekazu Shirota, Kunio Sano, Tadashi Kikuchi, et al. Regulation of murine airway eosinophilia and Th2 cells by antigen-conjugated CpG Oligodeoxynucleotides as a novel antigen-specific immunomodulator[J]. The Journal of Immunology, 2000, 164: 5575–5582.
[33]. Hidekazu Shirota, Kunio Sano, Tadashi Kikuchi, et al. Regulation of T-helper Type 2 cell and airway eosinophilia by transmucosal coadministration of antigen and Oligodeoxynucleotides containing CpG motifs[J]. Am. J. Respir. Cell Mol. Biol, 2000, 22: 176–182.
[34]. Jason D. Marshall, Simin Abtahi, Joseph J. Eiden, et al. Immunostimulatory sequence DNA linked to the Amb a 1 allergen promotes TH1 cytokine expression while downregulating TH2 cytokine expression in PBMCs from human patients with ragweed allergy[J]. Allergy Clin Immunol, 2001, 108: 191–7.
[35]. Kazuhisa Asai, Susan C Foley, et al. Amb a 1-immunostimulatory oligodeoxynucleotide conjugate immunotherapy increases CD4+CD25+ T cells in the nasal mucosa of subjects with allergic rhinitis[J]. Allergol Int, 2008, 57(4): 377–81
[36]. Kenji Takabayashi, Lev Libet, Dugald Chisholm, et al. Intranasal immunotherapy is more effective than intradermal immunotherapy for the induction of airway allergen tolerance in Th2-sensitized mice[J]. The Journal of Immunology, 2003, 170: 3898–3905.
[37]. K. Kitagaki, T. R. Businga and J. N. Kline. Oral administration of CpG-ODNs suppresses antigen-induced asthma in mice[J]. Clinical and Experimental Immunology, 2005;143: 249–259.
[38]. Kline JN, Krieg AM, Waldschmidt TJ, et al. CpG oligodeoxynucleotides do not require Th1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma[J]. Allergy Clin Immunol, 1999, 104(6): 1258–64.
[39]. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM. CpG motifs in bacterial DNA trigger direct B-cell activation[J]. Nature, 1995, 374: 546–549.
[40]. Kumar RK, Foster PS: Modeling allergic asthma in mice-pitfalls and opportunities[J]. Am JRespir Cell Mol Biol, 2002, 27: 267–272.
[41]. Kunihiko Kitagaki, Vipul V. Jain, Thomas R. Businga, et al. Immunomodulatory effects of CpG Oligodeoxynucleotides on established Th2 responses[J]. Clinical and Diagnostic Laboratory Immunology, 2002, p. 1260–1269.
[42]. Michelle V. Fanucchi, Edward S. Schelegle, Gregory L. Baker, et al. Immunostimulatory oligonucleotides attenuate airways remodeling in allergic monkeys[J]. Am J Respir Crit Care Med, 2004, 170: 1153–1157.
[43]. Peter S. Creticos, John T. Schroeder, et al. Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis[J]. N Engl J Med, 2006, 355(14): 1445–55.
[44]. Reid K. Ikeda, Jyothi Nayar, Jae Youn Cho, et al. Resolution of airway inflammation following ovalbumin inhalation comparison of ISS DNA and Corticosteroids[J]. Am. J. Respir. Cell Mol. Biol, 2003, 28: 655–663.
[45]. Repa, A. et al. In?uence of the route of sensitization on local and systemic immune responses in a murine model of type I allergy[J]. Clin. Exp.Immunol, 2004, 137: 12–18.
[46]. Sanjiv Sur, James S. Wild, Barun K. Choudhury, et al. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG Oligodeoxynucleotides[J]. The Journal of Immunology, 1999;162: 6284–6293.
[47]. Schelegle ES, Gershwin LJ, Miller LA, Fanucchi MV, et al. Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae) [J]. Am J Pathol, 2001, 158: 333–341.
[48]. Sel S, Wegmann M, Dicke T, et al. Effective prevention and therapy of experimental allergic asthma using a GATA-3 specific DNAzyme[J]. Allergy Clin Immunol, 2008, 121(4): 910–916.
[49]. Shigeru Ashino1, Daiko Wakita1, Yue Zhang, et al. CpG-ODN inhibits airway inflammation at effector phase through down-regulation of antigen-specific Th2-cell migration into lung[J]. International Immunology, 2007, 20(2): 259–266.
[50]. Sook Young Lee, Jae Youn Cho, Marina Miller, et al. Immunostimulatory DNA inhibits allergen induced peribronchial angiogenesis in mice [J]. Allergy Clin Immunol, 2006, 117: 597–603.
[51]. S. Underwood, M. Foster, D. Raeburn, et al. Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness[J]. Eur Respir J, 1995, 8: 2104–2113.
[52]. Takeahita F, Gursel I , Ishii KJ, et al. Signal transduction pathways mediated by the interaction of CpG DNA with Toll - like receptor 9 [J]. Sem in Immnol, 2004, 16(1): 17–21.
[53]. Takeda K, Hamelmann E, Joetham A, Shultz LD, Larsen GL, Irvin CG, Gelfand EW. Development of eosinophilic airway infammation and airway hyperresponsiveness in mast cell-defcient mice[J]. Exp Med, 1997, 186: 449–54.
[54]. Tahereh Mousavi, Nader Tajik, Maziar Moradi, et al. CpG Immunotherapy in Chenopodium album sensitized mice: The comparison of IFN-gamma, IL-10 and IgE responses in intranasal and subcutaneous administrations[J]. Clinical and Molecular Allergy, 2008, 6: 10–15.
[55]. Tulic MK, Fiset PO, Christodoulopoulos P, et al. Amb a 1-immunostimulatory oligodeoxynucleotide conjugate immunotherapy decreases the nasal in?ammatory response[J]. Allergy Clin Immunol, 2004, 113: 235–41.
[56]. Victor Matheu, Alexandra Treschow, Ingrid Teige, et al. Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-βknock-out mice[J]. Respiratory Research, 2005, 6: 25–37.
[57]. Vipul V. Jain, Kunihiko Kitagaki, Thomas Businga, et al. CpG-oligodeoxy nucleotides inhibit airway remodeling in a murine model of chronic asthma[J]. Allergy Clin Immunol, 2002, 110:867–72.
[58]. Vipul V. Jain, Thomas R. Businga, Kunihiko Kitagaki, et al. Mucosal immunotherapy with CpG oligodeoxynucleotides reverses a murine model of chronic asthma induced by repeated antigen exposure[J]. Am J Physiol Lung Cell Mol Physiol, 2003, 285: 1137–1146.
[59]. Vollmer J, Weeratna R, Payette P, Jurk M, Schetter C, Laucht M, Wader T, Tluk S, Liu M, Davis HL, Krieg AM. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities[J]. Eur. J. Immunol, 2004, 34: 251–262
[60]. Wen Xu, Toshiki Tamura, Kiyoshi Takatsu, et al. CpG ODN mediated prevention from ovalbumin-induced anaphylaxis in mouse through B cell pathway[J]. InternationalImmunopharmacology, 2008, 8: 351–361.
[61]. Wilson DR, Nouri Aria KT, Walker SM, et al. Grass pollen immunotherapy: symptomatic improvement correlates with reductions in eosinophils and IL -5 mRNA expression in the nasal mucosa during the pollen season[J]. Allergy Clin Immunol, 2001, 107(6): 971–976.
[62]. Zosky GR, Sly PD. Animal models of asthma[J]. Clin Exp Allergy, 2007, 37: 973–988.
[63]. Zu-qun WU, Yi-ping XU, Hua XIANG, Hua-hao SHEN. Effects of CpG oligodeoxynucleotide on transcription factors GATA-3 and T-bet mRNA expression in asthmatic mice[J]. Acta Pharmacologica Sinica, 2005, 26(9): 1117–1122.
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