鹦鹉热嗜衣原体临床株的分离与鉴定及IFN-γ抗感染作用初步研究
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摘要
目的:优化鹦鹉热嗜衣原体(Chlamydophila psittaci,C.psittaci,Cps)临床株分离培养技术,建立C.psittaci感染小鼠的动物模型,为C.psittaci流行病学调查、临床诊断及致病性和致病机制研究奠定基础。初步探讨IFN-γ对C.psittaci A血清型菌株的抗感染作用,为进一步阐明机体抗衣原体免疫机制提供参考数据。
方法:通过分子生物学技术,提取禽鸟肝肺组织DNA,PCR扩增特异性C.psittaci ompA基因,进一步酶切、测序鉴定。同时将PCR阳性标本肝组织匀浆液接种到单层敏感细胞株HeLa细胞或Vero细胞中培养,Giemsa和免疫荧光染色法鉴定衣原体包涵体。将临床株扩大培养后,用2×104、2×105、2×106 IFU三个剂量鼻饲攻击C57BL/6小鼠,分别于感染后5d和10d处死小鼠,取心、肝、脾、肺、肾进行苏木素-伊红染色(HE染色法)和免疫组织化学染色,显微镜观察受染小鼠各脏器病理变化。用不同浓度的rhIFN-γ(5ng/mL、25ng/mL、50ng/mL)作用于感染C. psittaci 6BC的HeLa细胞或Vero细胞48h,计数包涵体数目,并观察包涵体形态的改变。2×106 IFU C. psittaci 6BC鼻内感染C57BL/6小鼠,于感染前、后24h内腹腔注射rmIFN-γ10μg,观察小鼠食欲情况、活动状态、生存率等一般指标的变化,于感染后5d和10d,处死小鼠,取肝、肺组织进行HE和免疫组化染色比较IFN-γ处理组与对照组的肝、肺组织的病理变化;并取感染后5d的肺组织进行培养,计数各组包涵体数目。
结果:
1)应用PCR、酶切及测序的方法从100只禽鸟标本中检测到6株C.psittaci菌株(6%),并成功地在HeLa及Vero细胞中培养出3株(3%)。
2)比较了C.psittaci在Vero细胞与HeLa细胞培养生长情况,结果显示Vero内的衣原体包涵体积大,结构致密,对衣原体感染引起的宿主细胞溶解耐受性较HeLa强,更适合用于C.psittaci的分离培养及体外研究。
3)成功建立C.psittaci的鼠呼吸道感染模型。应用2×106、2×105、2×104 IFU经呼吸道感染C57BL/6小鼠,均可引起相应的急性临床及病理变化,且呈现明显的剂量依赖性,高浓度感染组小鼠的临床表现及病理变化均较低浓度感染组严重。各剂量组小鼠感染后2天体重明显下降,分别为4.33±1.03g、2.83±1.17 g、2.67±0.82 g,而PBS对照组体重下降仅为0.17±0.75 g。感染后10天,各剂量组生存率分别为0%、33.3%、100%,PBS对照组生存率为100%。
4) IFN-γ在C.psittaci感染HeLa细胞的体外实验研究显示,IFN-γ具有明显的抗C.psittaci感染作用,呈剂量依赖性。5ng/mL、25ng/mL、50ng/mL rhIFN-γ处理组在C.psittaci 6BC感染48h后,包涵体数目明显低于对照组,各剂量rhIFN-γ处理组包涵体数分别为(23.8±5.1)×106,(10±3.58)×106,(8.0±2.22)×106),未处理组为(43.3±11.05)×106;且经rhIFN-γ处理的衣原体包涵体形状不规则,体积约为对照组1/4~1/5。
5) C.psittaci感染的小鼠体内实验也显示,IFN-γ有明显的抗C.psittaci感染作用,可明显减轻小鼠急性临床表现及脏器病理变化,且rmIFN-γ处理组生存率明显高于未处理组,感染前、后24h内腹腔注射rmIFN-γ组及未处理组生存率分别为75%、43.75%、12.5%。
结论:优化了C.psittaci临床株的分离培养技术,成功建立了C.psittaci感染小鼠的动物感染模型,并初步证明IFN-γ对宿主抗C.psittaci A血清型菌株具有明显的保护作用,且呈剂量依赖性。
Objective:
To optimize the isolation and culture technique of Chlamydophila psittaci clinical strains and establish an animal model infected with C.psittaci, which can be applied to the clincal diagnosis of C.psittaci and epidemiological or pathogenetic study. To preliminary study whether IFN-γcan resist C.psittaci serovar A infection, providing experimental evidence for further illustrating the pathogenetic or immune mechanism of Chlamydia infection. .
Methods:
C.psittaci ompA gene was amplified from DNA extracted from bird livers or lungs by polymerase chain reactions (PCR). The PCR products were identified by enzymes cleavage or sequencing. For the PCR positive clinical samples, the liver tissues were homogenized and used to incubated with HeLa or Vero cell monolayers for 24 h in different dilutions,and chlamydia inclusion bodies were detected by immunofluorescence or Giemsa staining. Different dose of the clinical strains(2×104, 2×105, 2×106 IFUs) were used to attack C57BL/6 mice by intranasal injection,mice were sacrificed on day 5 or day 10 after infection, and the histopathology changes were analyzed by H&E and immunohistochemistry staining in different organs. To study the role of IFN-γ, C. psittaci 6BC-infected HeLa cells were treated with different concentrations of rhIFN-γ(5ng/mL, 25ng/mL, 50ng/mL). Forty-eight hours later, inclusion bodies were calculated and morphology was compared in different groups. For the in vivo experiment, C57BL/6 mice were inoculated intranasal with 2×106 IFU of live C. psittaci 6BC in 50μL SPG, and 10ug rmIFN-γor PBS were injected intraperitoneally at 24 hours prior or after infection. The body weight, activity and survival rate were recorded, and histopathology changes were analyzed by H&E and immunohistochemistry staining in mice livers and lungs after sacrificed. The lung tissues were homogenized, followed by sonication on ice, and the released organisms were titrated on Vero cell monolayers.
Results:
1) Six of one hundred clinical samples were positive by C.psittaci ompA gene amplification, and three were positive by cell culture.
2) Vero cells showed stronger tolerance of cytolysis than HeLa cells. And chlamydia inclusion bodies were larger and more dense in Vero cells than in HeLa cells.
3) C.psittaci infected mouse model was successfully established. After intranasally infected with 2×106, 2×105 or 2×104 IFUs of C.psittaci, corresponding clinical symptoms and pathology changes could be seen in all mice group in a dose-dependent manner. The body weight was decreased 4.33±1.03g, 2.83±1.17g, 2.67±0.82g respectively, but only decreased a little bit(0.17±0.75g) in control group. The survival rate was 0%, 33.3% or 100%, and no mice died in control group till day10 after infection.
4) The in vitro experiment showed that IFN-γcould resist C.psittaci infection in a dose dependent manner. After incobulated 48h with the same amount of chlamydia and different dose (5ng/m, 25ng/mL, 50ng/mL) of rhIFN-γ, and the inclusion body numbers were significantly lower than those of control groups ((23.8±5.1)×106, (10±3.58)×106, (8.0±2.22)×106, (43.3±11.05)×106, respectively). The inclusion body was out-of-shape, and the size is only one fourth or one fifth of the control group.
5) The results in the animal model of mouse infected with C.psittaci also showed that IFN-γcan resist C.psittaci infection and significantly alleviate the symptoms and pathology. And the survival rate was much higher than that of untreated group (75%, 43.75%, 12.5%, respectively) on day 10 after infection.
Conclusion:
The isolation and culture condition was optimized for C.psittaci clinical strains, and animal model of respiratory tract infection by C.psittaci was successfully established. IFN-γwas found to mediate strong protection against C.psittaci serovar A strain in a dose-dependent manner.
方法:通过分子生物学技术,提取禽鸟肝肺组织DNA,PCR扩增特异性C.psittaci ompA基因,进一步酶切、测序鉴定。同时将PCR阳性标本肝组织匀浆液接种到单层敏感细胞株HeLa细胞或Vero细胞中培养,Giemsa和免疫荧光染色法鉴定衣原体包涵体。将临床株扩大培养后,用2×104、2×105、2×106 IFU三个剂量鼻饲攻击C57BL/6小鼠,分别于感染后5d和10d处死小鼠,取心、肝、脾、肺、肾进行苏木素-伊红染色(HE染色法)和免疫组织化学染色,显微镜观察受染小鼠各脏器病理变化。用不同浓度的rhIFN-γ(5ng/mL、25ng/mL、50ng/mL)作用于感染C. psittaci 6BC的HeLa细胞或Vero细胞48h,计数包涵体数目,并观察包涵体形态的改变。2×106 IFU C. psittaci 6BC鼻内感染C57BL/6小鼠,于感染前、后24h内腹腔注射rmIFN-γ10μg,观察小鼠食欲情况、活动状态、生存率等一般指标的变化,于感染后5d和10d,处死小鼠,取肝、肺组织进行HE和免疫组化染色比较IFN-γ处理组与对照组的肝、肺组织的病理变化;并取感染后5d的肺组织进行培养,计数各组包涵体数目。
结果:
1)应用PCR、酶切及测序的方法从100只禽鸟标本中检测到6株C.psittaci菌株(6%),并成功地在HeLa及Vero细胞中培养出3株(3%)。
2)比较了C.psittaci在Vero细胞与HeLa细胞培养生长情况,结果显示Vero内的衣原体包涵体积大,结构致密,对衣原体感染引起的宿主细胞溶解耐受性较HeLa强,更适合用于C.psittaci的分离培养及体外研究。
3)成功建立C.psittaci的鼠呼吸道感染模型。应用2×106、2×105、2×104 IFU经呼吸道感染C57BL/6小鼠,均可引起相应的急性临床及病理变化,且呈现明显的剂量依赖性,高浓度感染组小鼠的临床表现及病理变化均较低浓度感染组严重。各剂量组小鼠感染后2天体重明显下降,分别为4.33±1.03g、2.83±1.17 g、2.67±0.82 g,而PBS对照组体重下降仅为0.17±0.75 g。感染后10天,各剂量组生存率分别为0%、33.3%、100%,PBS对照组生存率为100%。
4) IFN-γ在C.psittaci感染HeLa细胞的体外实验研究显示,IFN-γ具有明显的抗C.psittaci感染作用,呈剂量依赖性。5ng/mL、25ng/mL、50ng/mL rhIFN-γ处理组在C.psittaci 6BC感染48h后,包涵体数目明显低于对照组,各剂量rhIFN-γ处理组包涵体数分别为(23.8±5.1)×106,(10±3.58)×106,(8.0±2.22)×106),未处理组为(43.3±11.05)×106;且经rhIFN-γ处理的衣原体包涵体形状不规则,体积约为对照组1/4~1/5。
5) C.psittaci感染的小鼠体内实验也显示,IFN-γ有明显的抗C.psittaci感染作用,可明显减轻小鼠急性临床表现及脏器病理变化,且rmIFN-γ处理组生存率明显高于未处理组,感染前、后24h内腹腔注射rmIFN-γ组及未处理组生存率分别为75%、43.75%、12.5%。
结论:优化了C.psittaci临床株的分离培养技术,成功建立了C.psittaci感染小鼠的动物感染模型,并初步证明IFN-γ对宿主抗C.psittaci A血清型菌株具有明显的保护作用,且呈剂量依赖性。
Objective:
To optimize the isolation and culture technique of Chlamydophila psittaci clinical strains and establish an animal model infected with C.psittaci, which can be applied to the clincal diagnosis of C.psittaci and epidemiological or pathogenetic study. To preliminary study whether IFN-γcan resist C.psittaci serovar A infection, providing experimental evidence for further illustrating the pathogenetic or immune mechanism of Chlamydia infection. .
Methods:
C.psittaci ompA gene was amplified from DNA extracted from bird livers or lungs by polymerase chain reactions (PCR). The PCR products were identified by enzymes cleavage or sequencing. For the PCR positive clinical samples, the liver tissues were homogenized and used to incubated with HeLa or Vero cell monolayers for 24 h in different dilutions,and chlamydia inclusion bodies were detected by immunofluorescence or Giemsa staining. Different dose of the clinical strains(2×104, 2×105, 2×106 IFUs) were used to attack C57BL/6 mice by intranasal injection,mice were sacrificed on day 5 or day 10 after infection, and the histopathology changes were analyzed by H&E and immunohistochemistry staining in different organs. To study the role of IFN-γ, C. psittaci 6BC-infected HeLa cells were treated with different concentrations of rhIFN-γ(5ng/mL, 25ng/mL, 50ng/mL). Forty-eight hours later, inclusion bodies were calculated and morphology was compared in different groups. For the in vivo experiment, C57BL/6 mice were inoculated intranasal with 2×106 IFU of live C. psittaci 6BC in 50μL SPG, and 10ug rmIFN-γor PBS were injected intraperitoneally at 24 hours prior or after infection. The body weight, activity and survival rate were recorded, and histopathology changes were analyzed by H&E and immunohistochemistry staining in mice livers and lungs after sacrificed. The lung tissues were homogenized, followed by sonication on ice, and the released organisms were titrated on Vero cell monolayers.
Results:
1) Six of one hundred clinical samples were positive by C.psittaci ompA gene amplification, and three were positive by cell culture.
2) Vero cells showed stronger tolerance of cytolysis than HeLa cells. And chlamydia inclusion bodies were larger and more dense in Vero cells than in HeLa cells.
3) C.psittaci infected mouse model was successfully established. After intranasally infected with 2×106, 2×105 or 2×104 IFUs of C.psittaci, corresponding clinical symptoms and pathology changes could be seen in all mice group in a dose-dependent manner. The body weight was decreased 4.33±1.03g, 2.83±1.17g, 2.67±0.82g respectively, but only decreased a little bit(0.17±0.75g) in control group. The survival rate was 0%, 33.3% or 100%, and no mice died in control group till day10 after infection.
4) The in vitro experiment showed that IFN-γcould resist C.psittaci infection in a dose dependent manner. After incobulated 48h with the same amount of chlamydia and different dose (5ng/m, 25ng/mL, 50ng/mL) of rhIFN-γ, and the inclusion body numbers were significantly lower than those of control groups ((23.8±5.1)×106, (10±3.58)×106, (8.0±2.22)×106, (43.3±11.05)×106, respectively). The inclusion body was out-of-shape, and the size is only one fourth or one fifth of the control group.
5) The results in the animal model of mouse infected with C.psittaci also showed that IFN-γcan resist C.psittaci infection and significantly alleviate the symptoms and pathology. And the survival rate was much higher than that of untreated group (75%, 43.75%, 12.5%, respectively) on day 10 after infection.
Conclusion:
The isolation and culture condition was optimized for C.psittaci clinical strains, and animal model of respiratory tract infection by C.psittaci was successfully established. IFN-γwas found to mediate strong protection against C.psittaci serovar A strain in a dose-dependent manner.
引文
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[5] Wira CR, Fahey JV, Sentman CL et al. Innate and adaptive immunity in female genital tract: cellular responses and interactions[J]. Immunol Rev,2005, 206:306-335
[6] Puolakkainen M. Innate immunity and vaccines in chlamydial infection with special emphasis on Chlamydia pneumoniae[J]. FEMS Immunol Med Microbiol , 2009, 55 (2):167-177
[7] Hafner L, Beagley K, Timms P. Chlamydia trachomatis infection: host immune responses and potential vaccines [J]. Mucosal Immunol ,2008, 1(2):116-130
[8] Rottenberg ME, Gigliotti Rothfuchs A, Gigliotti D et al. Regulation and role of IFN-gamma in the innate resistance to infection with Chlamydia pneumoniae [J]. J Immunol, 2000,164 (9) :4812-4818
[9] Tseng CT, Rank RG. Role of NK cells in early host response to chlamydial genital infection[J]. Infect Immun ,1998,66(12):5867-5875
[10] Rottenberg ME, Gigliotti-Rothfuchs A, Wigzell H. The role of IFN-gamma in the outcome of chlamydial infection[J]. Curr Opin Immunol, 2002,14 (4):444-451
[11] Joyee AG, Qiu H, Wang S et al. Distinct NKT cell subsets are induced by different Chlamydia species leading to differential adaptive immunity and host resistance to the infections[J]. J Immunol, 2007, 178 (2):1048-1058
[12] Bilenki L, Wang S, Yang J et al. NK T cell activation promotes Chlamydia trachomatis infection in vivo[J]. J Immunol, 2005, 175 (5):3197-3206
[13] Rothfuchs AG, Gigliotti D, Palmblad K et al. IFN-alpha beta-dependent, IFN-gamma secretion by bone marrow-derived macrophages controls an intracellular bacterial infection[J]. J Immunol, 2001,167 (11):6453-6461
[14] Rothfuchs AG, Kreuger MR, Wigzell H, Rottenberg ME. Macrophages, CD4+ or CD8+ cells are each sufficient for protection against Chlamydia pneumoniae infection through their ability to secrete IFN-gamma[J]. J Immunol, 2004, 172(4):2407-2415
[15] Roan NR, Gierahn TM, Higgins DE, Starnbach MN. Monitoring the T cell response to genital tract infection[J]. Proc Natl Acad Sci U S A, 2006, 103(32):12069-12074.
[16] Dong-Ji Z, Yang X, Shen C et al. Priming with Chlamydia trachomatis major outer membrane protein (MOMP) DNA followed by MOMP ISCOM boosting enhances protection and is associated with increased immunoglobulin A and Th1 cellular immune responses[J]. Infect Immun, 2000, 68(6):3074-3078
[17] Svanholm C, Bandholtz L, Castanos-Velez E et al. Protective DNA immunization against Chlamydia pneumoniae[J]. Scand J Immunol, 2000,51 (4):345-353
[18] Nelson DE, Virok DP, Wood H et al. Chlamydial IFN-gamma immune evasion is linked to host infection tropism[J]. Proc Natl Acad Sci U S A, 2005,102 (30):10658-10663
[19] Murthy AK, Chambers JP, Meier PA et al. Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous gamma interferon production[J]. Infect Immun,2007, 75(2):666-676
[20] Li W, Murthy AK, Guentzel MN et al. Antigen-specific CD4+ T cells produce sufficient IFN-gamma to mediate robust protective immunity against genital Chlamydia muridarum infection[J]. J Immuno,l 2008,180 (5):3375-3382
[21] Wizel B, Nystrom-Asklin J, Cortes C, Tvinnereim A. Role of CD8(+)T cells in the host response to Chlamydia[J]. Microbes Infect ,2008,10(14-15):1420-1430
[22] Starnbach MN, Loomis WP, Ovendale P et al. An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+ T cell response[J]. J Immunol, 2003,171 (9):4742-4749
[23] Gervassi AL, Grabstein KH, Probst P et al. Human CD8+ T cells recognize the 60-kDa cysteine-rich outer membrane protein from Chlamydia trachomatis[J]. J Immunol, 2004,173(11):6905-6913
[24] Tvinnereim A, Wizel B. CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent[J]. J Immunol, 2007,179 (6):3947-3957
[25] Roshick C, Wood H, Caldwell HD, McClarty G. Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mousecells[J]. Infect Immun ,2006,74 (1):225-238
[26] Taylor GA. IRG proteins: key mediators of interferon-regulated host resistance to intracellular pathogens[J]. Cell Microbiol, 2007,9 (5):1099-1107
[27] Ong ST, Ho JZ, Ho B, Ding JL. Iron-withholding strategy in innate immunity[J]. Immunobiology ,2006, 211(4):295-314
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[30] Coers J, Bernstein-Hanley I, Grotsky D et al. Chlamydia muridarum evades growth restriction by the IFN-gamma-inducible host resistance factor Irgb10[J]. J Immunol, 2008, 180(9):6237-6245
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[32] Zhong G, Fan T, Liu L. Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1[J]. J Exp Med ,1999,189 (12):1931-1938
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[1] Roan NR, Starnbach MN. Immune-mediated control of Chlamydia infection [J]. Cell Microbiol, 2008, 10(1):9-19
[2] Brunham RC, Rey-Ladino J. Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine[J]. Nat Rev Immunol , 2005, 5 (2):149-161
[3] Jupelli M, Guentzel MN, Meier PA et al. Endogenous IFN-gamma production is induced and required for protective immunity against pulmonary chlamydial infection in neonatal mice[J]. J Immunol , 2008, 180 (6):4148-4155
[4] Morrison RP, Caldwell HD. Immunity to murine chlamydial genital infection[J].Infect Immun, 2002, 70(6):2741-2751
[5] Wira CR, Fahey JV, Sentman CL et al. Innate and adaptive immunity in female genital tract: cellular responses and interactions[J]. Immunol Rev,2005, 206:306-335
[6] Puolakkainen M. Innate immunity and vaccines in chlamydial infection with special emphasis on Chlamydia pneumoniae[J]. FEMS Immunol Med Microbiol , 2009, 55 (2):167-177
[7] Hafner L, Beagley K, Timms P. Chlamydia trachomatis infection: host immune responses and potential vaccines [J]. Mucosal Immunol ,2008, 1(2):116-130
[8] Rottenberg ME, Gigliotti Rothfuchs A, Gigliotti D et al. Regulation and role of IFN-gamma in the innate resistance to infection with Chlamydia pneumoniae [J]. J Immunol, 2000,164 (9) :4812-4818
[9] Tseng CT, Rank RG. Role of NK cells in early host response to chlamydial genital infection[J]. Infect Immun ,1998,66(12):5867-5875
[10] Rottenberg ME, Gigliotti-Rothfuchs A, Wigzell H. The role of IFN-gamma in the outcome of chlamydial infection[J]. Curr Opin Immunol, 2002,14 (4):444-451
[11] Joyee AG, Qiu H, Wang S et al. Distinct NKT cell subsets are induced by different Chlamydia species leading to differential adaptive immunity and host resistance to the infections[J]. J Immunol, 2007, 178 (2):1048-1058
[12] Bilenki L, Wang S, Yang J et al. NK T cell activation promotes Chlamydia trachomatis infection in vivo[J]. J Immunol, 2005, 175 (5):3197-3206
[13] Rothfuchs AG, Gigliotti D, Palmblad K et al. IFN-alpha beta-dependent, IFN-gamma secretion by bone marrow-derived macrophages controls an intracellular bacterial infection[J]. J Immunol, 2001,167 (11):6453-6461
[14] Rothfuchs AG, Kreuger MR, Wigzell H, Rottenberg ME. Macrophages, CD4+ or CD8+ cells are each sufficient for protection against Chlamydia pneumoniae infection through their ability to secrete IFN-gamma[J]. J Immunol, 2004, 172(4):2407-2415
[15] Roan NR, Gierahn TM, Higgins DE, Starnbach MN. Monitoring the T cell response to genital tract infection[J]. Proc Natl Acad Sci U S A, 2006, 103(32):12069-12074.
[16] Dong-Ji Z, Yang X, Shen C et al. Priming with Chlamydia trachomatis major outer membrane protein (MOMP) DNA followed by MOMP ISCOM boosting enhances protection and is associated with increased immunoglobulin A and Th1 cellular immune responses[J]. Infect Immun, 2000, 68(6):3074-3078
[17] Svanholm C, Bandholtz L, Castanos-Velez E et al. Protective DNA immunization against Chlamydia pneumoniae[J]. Scand J Immunol, 2000,51 (4):345-353
[18] Nelson DE, Virok DP, Wood H et al. Chlamydial IFN-gamma immune evasion is linked to host infection tropism[J]. Proc Natl Acad Sci U S A, 2005,102 (30):10658-10663
[19] Murthy AK, Chambers JP, Meier PA et al. Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous gamma interferon production[J]. Infect Immun,2007, 75(2):666-676
[20] Li W, Murthy AK, Guentzel MN et al. Antigen-specific CD4+ T cells produce sufficient IFN-gamma to mediate robust protective immunity against genital Chlamydia muridarum infection[J]. J Immuno,l 2008,180 (5):3375-3382
[21] Wizel B, Nystrom-Asklin J, Cortes C, Tvinnereim A. Role of CD8(+)T cells in the host response to Chlamydia[J]. Microbes Infect ,2008,10(14-15):1420-1430
[22] Starnbach MN, Loomis WP, Ovendale P et al. An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+ T cell response[J]. J Immunol, 2003,171 (9):4742-4749
[23] Gervassi AL, Grabstein KH, Probst P et al. Human CD8+ T cells recognize the 60-kDa cysteine-rich outer membrane protein from Chlamydia trachomatis[J]. J Immunol, 2004,173(11):6905-6913
[24] Tvinnereim A, Wizel B. CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent[J]. J Immunol, 2007,179 (6):3947-3957
[25] Roshick C, Wood H, Caldwell HD, McClarty G. Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mousecells[J]. Infect Immun ,2006,74 (1):225-238
[26] Taylor GA. IRG proteins: key mediators of interferon-regulated host resistance to intracellular pathogens[J]. Cell Microbiol, 2007,9 (5):1099-1107
[27] Ong ST, Ho JZ, Ho B, Ding JL. Iron-withholding strategy in innate immunity[J]. Immunobiology ,2006, 211(4):295-314
[28] Ludwiczek S, Aigner E, Theurl I, Weiss G. Cytokine-mediated regulation of iron transport in human monocytic cells[J]. Blood, 2003,101(10):4148-4154
[29] Paradkar PN, De Domenico I, Durchfort N et al. Iron depletion limits intracellular bacterial growth in macrophages[J]. Blood, 2008,112 (3):866-874
[30] Coers J, Bernstein-Hanley I, Grotsky D et al. Chlamydia muridarum evades growth restriction by the IFN-gamma-inducible host resistance factor Irgb10[J]. J Immunol, 2008, 180(9):6237-6245
[31] Bernstein-Hanley I, Coers J, Balsara ZR et al. The p47 GTPases Igtp and Irgb10 map to the Chlamydia trachomatis susceptibility locus Ctrq-3 and mediate cellular resistance in mice[J]. Proc Natl Acad Sci U S A, 2006, 103(38):14092-14097
[32] Zhong G, Fan T, Liu L. Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1[J]. J Exp Med ,1999,189 (12):1931-1938