内皮源性吲哚胺2,3-双加氧酶抑制周细胞迁移及收缩蛋白表达

 目的: 探讨内皮源性吲哚胺2,3-双加氧酶(IDO)对周细胞迁移及收缩蛋白表达的影响。方法: 体外培养人肺动脉内皮细胞(HPAECs)及大鼠脑微血管周细胞。建立过表达IDO的HPAECs模型(IDO-HPAECs)。实验分为3组:对照组,以HPAECs条件培养基干预周细胞;处理组,以IDO-HPAECs条件培养基干预周细胞;抑制组,以含1-甲基色氨酸(1-mT)的IDO-HPAECs条件培养基干预周细胞。测定共培养体系一氧化氮(NO)、色氨酸及犬尿氨酸浓度。观察周细胞活性、迁移及收缩蛋白表达情况。结果: IDO-HPAECs条件培养基干预周细胞6～48 h对其活性无显著影响。处理组的周细胞迁移能力显著低于对照组(P < 0.01),抑制组显著高于处理组(P < 0.01)。共培养体系的NO浓度在各组间差异无统计学意义(P > 0.05)。处理组的色氨酸浓度显著低于对照组(P < 0.01),而抑制组显著高于处理组(P < 0.01)。处理组的犬尿氨酸浓度显著高于对照组(P < 0.01),而抑制组显著低于处理组(P < 0.01)。处理组的α-平滑肌肌动蛋白与结蛋白表达水平显著低于对照组(P < 0.01),抑制组显著高于处理组(P < 0.01)。结论: 内皮源性IDO抑制周细胞迁移及收缩蛋白表达,可能参与机体微血管功能调控。     

Cell-autonomous control of interferon type I expression by indoleamine 2,3-dioxygenase in regulatory CD19+ dendritic cells

Following CD80/86 (B7) and TLR9 ligation, small subsets of splenic dendritic cells expressing CD19 (CD19(+) DC) acquire potent T cell regulatory functions due to induced expression of the intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan. In CD19(+) DC, IFN type I (IFN-alpha) is the obligate inducer of IDO. We now report that IFN-alpha production needed to stimulate high-level expression of IDO following B7 ligation is itself dependent on basal levels of IDO activity. Genetic and pharmacologic ablation of IDO completely abrogated IFN-alpha production by CD19(+) DC after B7 ligation. In contrast, IDO ablation did not block IFN-alpha production by CD19(+) DC after TLR9 ligation. IDO-mediated control of IFN-alpha production depended on tryptophan depletion as adding excess tryptophan also blocked IFN-alpha expression after B7 ligation. Consistent with this, DC from mice deficient in general control of non-derepressible-2 (GCN2)-kinase, a component of the cellular stress response to amino acid withdrawal, did not produce IFN-alpha following B7 ligation, but produced IFN-alpha after TLR9 ligation. Thus, B7 and TLR9 ligands stimulate IFN-alpha expression in CD19(+) DC via distinct signaling pathways. In the case of B7 ligation, IDO activates cell-autonomous signals essential for IFN-alpha production, most likely by activating the GCN2-kinase-dependent stress response.     

Expression and function analysis of indoleamine 2 and 3-dioxygenase in bladder urothelial carcinoma

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for tryptophan metabolism inducing immune tolerance of tumors. The purpose of this study is to investigate IDO expression and its prognostic significance in bladder urothelial carcinoma (BUC). In this study, immunohistochemical staining for IDO expression in BUC tissues (n = 84) and normal bladder tissues (n = 22) was performed. The mRNA expression levels of IDO in BUC and normal bladder were analyzed by quantitative RT-PCR. Survival analysis was performed for the correlation of IDO expression and clinicopathological factors with disease-free survival. Positive expression of IDO was found in 48 of 84 cases in BUC tissues and was significantly correlated with histological classification, histological grade and TNM stage. While IDO expression in normal bladder tissues was expressed in only 4 of 22 (18.2%) cases. Moreover, IDO mRNA levels of BUC were significantly higher than that of normal bladder. We also found that IDO, histological grade and TNM stage were closely associated with DFS. These results indicated that IDO was related to the progression of BUC and might be one of the crucial prognostic factors for BUC.     

沉默吲哚胺2,3-双加氧酶2基因对黑色素瘤细胞B16-BL6增殖、迁移和侵袭的影响

目的:探讨沉默吲哚胺2,3-双加氧酶2(IDO2)基因对小鼠黑色素瘤B16-BL6细胞增殖、迁移及侵袭等生物学行为的影响。方法:IDO2-siRNA转染体外培养的黑色素瘤细胞B16-BL6,应用real-time PCR和Western blot检测IDO2和IDO1基因的表达;平板集落形成实验检测IDO2基因沉默对肿瘤细胞增殖的影响;细胞划痕实验和Transwell小室细胞迁移实验观察IDO2对肿瘤细胞迁移的影响;Transwell小室侵袭实验观察肿瘤细胞侵袭能力。结果:沉默B16-BL6细胞中IDO2基因能使细胞单集落形成密度降低,划痕迁移变慢,Transwell小室细胞迁移数减少,侵袭细胞数减少。结论:沉默IDO2可以影响黑色素瘤细胞B16-BL6的增殖、迁移和侵袭能力。     

IDO/TTS色氨酸代谢途径对类风湿关节炎滑膜T细胞增殖的影响

自身反应性T细胞在类风湿关节炎(RA)疾病的发生发展中发挥着极其重要的作用,抑制这群细胞是RA治疗的关键.表达吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)的树突状细胞(DC)能够通过此代谢通路剥夺环境中的色氨酸,并生成促凋亡因子犬尿氨酸等,使T细胞的增殖和功能受抑制,最终诱导...     

人外周血单个核细胞和肿瘤细胞中吲哚胺2,3双加氧酶mRNA水平的实时定量RT-PCR分析

目的 定量分析HIV-1感染者及高危人群外周血单个核细胞(PBMC)和人源肿瘤细胞在Toll样受体(TLR)配体刺激前后吲哚胺2,3双加氧酶(indoleamine 2,3-dioxygenase,IDO)的表达水平.方法 建立人IDO mRNA的TaqMan探针实时荧光定量RT-PCR检测方法;检测HIV-1阳性个体和高危人群PBMC中IDO mRNA水平;检测TLR4、TLR7/8、TLR9配体刺激前后人黏膜(T84、Caco2、Hela)和白细胞(THP-1、MT-4)来源肿瘤细胞中IDO mRNA的水平.结果 HIV-1阳性个体PBMC中IDO mRNA水平较高危人群高(103.42拷贝IDO mRNA/106拷贝GAPDH mRNA);但也有部分高危人群个体的PBMC含有较高水平的IDO mRNA;肿瘤细胞在TLR配体刺激后IDO mRNA的水平可以显著升高,因细胞系和TLR配体种类不同而异.结论 本研究表明IDO可能在我国HIV-1感染者体内病毒免疫逃逸和肿瘤发生中起作用,值得深入研究.     

干扰素调节因子3 rs2304204野生型及突变型重组质粒的构建及活性分析

目的构建干扰素调节因子3（IRF-3）rs2304204野生型及突变型重组质粒并比较二者启动子活性。为进一步研究IRF-3对HBV感染的影响打下基础。方法以人全血DNA为模板,扩增含rs2304204位点的IRF-3启动子区1355bp目的序列,插入pGL3-Basic载体中,构建rs2304204野生型的重组质粒（wIRF-3）。以wIRF-3为模板,利用基因定点突变试剂盒构建rs2304204突变型重组质粒（mIRF-3）。wIRF．3、mIRF-3和pGL3-Basic质粒分别转染HEK293细胞,采用双荧光素酶报告基因检测试剂盒检测荧光素酶活性,并计算荧光素酶相对活性单位（RLU）。结果成功构建IRF-3rs2304204野生型及突变型重组质粒,且wIRF-3质粒转染组RLU明显高于mIRF-3质粒转染组（P〈0．005）。结论野生型重组质粒的启动子活性明显高于突变型重组质粒,启动子活性的不同可能进而影响机体抗HBV感染的临床结局。     

Endoplasmic reticulum stress response and inflammatory cytokines in type 2 diabetic nephropathy: Role of indoleamine 2,3-dioxygenase and programmed death-1

We tested the hypotheses that a) type 2 diabetes increases endoplasmic reticulum (ER) stress response, production of pro-inflammatory cytokines and kidney cell death and b) downregulations of renal indoleamine 2,3-dioxygenase (IDO) and programmed death-1 (PD-1) contribute to exacerbated inflammation and tissue injury. The growth arrest and DNA damage-inducible protein 153 (GADD153; a marker of ER stress response), inflammatory cytokines and cell death were determined in the context of assessment of IDO and PD-1 in an animal model of type 2 diabetic nephropathy (i.e., db/db mouse). Peripheral blood of 4-month-old db/db mice manifested significantly greater percents of interleukin (IL)-17 and IL-23 positive cells in association with greater percents of cells that were positive for PD-1 or GADD153. Compared to kidneys of db/m controls, renal cells prepared from kidneys of db/db mice displayed a) increased percent of cells that were positive for IL-17, IL-23, PD-1 and GADD153, b) decreased JC-1 aggregates but increased JC-1 monomers suggestive of disruption of mitochondrial membrane potential and c) increased apoptotic and necrotic cell death. Immunohistochemical analyses also revealed increased staining of renal tissue of db/db mice for IL-17, IL23, GADD153, Annexin V, caspase 3, PD-1 and IDO compared to db/m kidneys; these changes were generally more prominent in the glomeruli. In conclusion, type 2 diabetes upregulates systemic and local ER stress response and pro-inflammatory mechanisms thereby contributing to renal injury. However, the accompanying upregulations of PD-1 and IDO likely reflect activation of compensatory mechanisms to curtail inflammation and cell injury.     

Apoptosis is induced in Chlamydia trachomatis-infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Citation Huston WM, Gloeckl S, de Boer L, Beagley KW, Timms P. Apoptosis is induced in Chlamydia trachomatis‐infected HEp‐2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone. Am J Reprod Immunol 2011; 65: 460–465 Problem Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. Method of study Innate immune activation of HEp‐2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. Results The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp‐2 cells under these conditions. Conclusion This is the first time that induction of apoptosis has been reported in C. trachomatis‐infected cells when treated with a combination of innate immune activators and wedelolactone.     

Abatement of cyclophosphamide-induced splenic immunosuppressive indoleamine 2, 3-dioxygenase and altered hematological indices in Wister rats by dietary quercetin

The immunotoxicity mediated by cyclophosphamide (CYP) was earlier reported. Quercetin, due to its anti-oxidative/inflammatory properties elicits a plethora of health benefits. However, the influence of quercetin on the splenic/immunotoxicity linked with CYP-induced indoleamine 2,3-dioxygenase (IDO) is unavailable in the literature. We investigated the effects of quercetin on the splenic immunosuppressive IDO and hematological indices of immune response in rats. Animals were treated with CYP (100 mg/kg) alone or co-treated (CYP + quercetin [100 mg/kg + 50 mg/kg respectively]) at days 1, 3, 5, and 7. Results revealed that CYP treatment alone significantly provoked an oxidative-inflammatory response, increased serum kynurenine concentration, and concomitantly increased immunosuppressive IDO and tryptophan 2, 3-dioxygenase (TDO), in the spleen as well as altering hematological indices. Quercetin co-treatment enhanced activities of antioxidant enzymes, inhibited myeloperoxidase (MPO) activity, lowered levels of nitric oxide, interferon-Υ (IFN-Υ), and interleukin-6 (IL-6), and reduced kynurenine concentration as well as IDO/TDO activities. Quercetin co-treatment augmented white blood cell (WBC), CD4-T cells, and other hematological indices of the immune response. In conclusion, quercetin prevented CYP-induced alterations in immune response in rats by lowering the activities of immunosuppressive IDO and TDO, inhibiting oxidative-inflammatory stress, diminution of kynurenine concentrations, and augmenting hematological parameters.     

Inhibition of indoleamine 2,3-dioxygenase activity enhances the anti-tumour effects of a Toll-like receptor 7 agonist in an established cancer model

Toll-like receptor (TLR) agonists have been shown to have anti-tumour activity in basic research and clinical studies. However, TLR agonist monotherapy does not sufficiently eliminate tumours. Activation of the innate immune response by TLR agonists is effective at driving adaptive immunity via interleukin-12 (IL-12) or IL-1, but is counteracted by the simultaneous induction of immunosuppressive cytokines and other molecules, including IL-10, transforming growth factor-β, and indoleamine 2,3-dioxygenase (IDO). In the present study, we evaluated the anti-cancer effect of the TLR7 agonist, imiquimod (IMQ), in the absence of IDO activity. The administration of IMQ in IDO knockout (KO) mice inoculated with tumour cells significantly suppressed tumour progression compared with that in wild-type (WT) mice, and improved the survival rate. Moreover, injection with IMQ enhanced the tumour antigen-specific T helper type 1 response in IDO-KO mice with tumours. Combination therapy with IMQ and an IDO inhibitor also significantly inhibited tumour growth. Our results indicated that the enhancement of IDO expression with TLR agonists in cancer treatment might impair host anti-tumour immunity while the inhibition of IDO could enhance the therapeutic efficacy of TLR agonists via the increase of T helper type 1 immune response.     

Expression of TLR 2, TLR 4 and iNOS in cervical monocytes of Chlamydia trachomatis-infected women and their role in host immune response

Citation Agrawal T, Bhengraj AR, Vats V, Salhan S, Mittal A. Expression of Toll‐like receptors (TLR) 2, TLR 4 and inducible nitric oxide synthase (iNOS) in cervical monocytes of Chlamydia trachomatis‐infected women and their role in host immune response. Am J Reprod Immunol 2011; 66: 534–543 Problem To study the innate immune response ‐TLR2 TLR 4 and iNOS expression in female genital Chlamydia trachomatis infection. Method TLR 2, TLR 4, and iNOS expression was evaluated by real‐time PCR in C. trachomatis‐infected asymptomatic, mucopurulent cervicitis (MPC), and fertility disorders (FD) women. Expression of TLR signaling pathway genes was checked in vivo in C. trachomatis‐infected cervical monocytes. Further, inos gene expression and nitric oxide release was assessed in vitro in THP‐1 cell line upon chlamydial infection. Results TLR2, TLR4, and iNOS expression was significantly (P < 0.05) higher in C. trachomatis‐positive women with FD, MPC, and asymptomatic women, respectively, than in control. Chlamydial infection significantly upregulates CD86, TLR4, MyD88, IRAK2, nF‐κB, IL‐1,β and IL‐12 genes. Expression of iNOS gene was found to be significantly (P < 0.05) high 12 hrs post‐infection. Conclusions Chlamydia trachomatis stimulates innate immune cells by activation of TLR2/TLR 4. Overall data indicate that recognition by TLR4 helps in initiation of immune response while recognition by TLR2 leads to secretion of inflammatory cytokines while iNOS‐induced nitric oxide production helps in clearing Chlamydia. These results are first to provide initial insights into how innate immune response operates in human cervical monocytes upon chlamydial infection.     

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:C) and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TTTC motifs and five γ-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type II IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN.     

Indoleamine 2,3-dioxygenase and regulatory dendritic cells contribute to the allograft protection induced by infusion of donor-specific splenic stromal cells

It has been reported that splenic stromal cells (SSCs) are capable of directly supporting the development of CD11c(lo)CD45RB(+ )IL-10-producing dendritic cells (DCs) from lineage-negative c-kit(+) progenitor cells in the absence of exogenous cytokines. In vitro, DCs that differentiate on stromal cells suppress mixed leukocyte reaction responses and induce primary alloreactive CD4(+) T cells to differentiate into IL-10-producing Tr1 cells. However, the precise mechanisms by which these SSCs exert their regulatory functions in vivo remain undefined. Furthermore, their possible contribution to the development of allograft transplantation tolerance has yet to be examined. Here, we have used both murine skin and cardiac allograft transplantation models to explore whether in vivo alloresponses can be regulated by infusion with donor-derived SSCs and to investigate the possible mechanisms by which SSCs exert regulatory effects to prevent allograft rejection. We show that intravenous SSC infusion prolonged murine skin allograft survival. The prolonged graft survival is associated with augmentation of the generation of regulatory DC subsets and CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), as well as upregulation of the production of suppressive cytokines IL-10 and transforming growth factor (TGF)-β. Moreover, we found that indoleamine 2,3-dioxygenase and SSC-derived regulatory DCs contribute to allograft protection by infusion of donor-specific SSCs. Our data suggest that donor-derived SSCs could be used as a therapeutic target to promote transplantation tolerance.     

沙眼衣原体主要外膜蛋白在减毒鼠伤寒杆菌中的表达及 …

目的 采用作为疫苗载体的减毒鼠伤寒杆菌致死性平衡系统,构建能异源表达沙眼衣原体（Ｃｔ）主要外膜蛋白（ＭＯＭＰ）的重组菌株。方法 以Ｄ型Ｃｔ的ＤＮＡ为模板,用所设计的特异性引物扩增编码Ｃｔ ＭＯＭＰ高变区（ＶＤＩ～ＶＤＩＶ）基因,并将扩增产物定向克隆至质粒ｐＵＣ１９中。序列分析后,再亚克隆至与减毒鼠伤寒杆菌Ｘ４５５０互补的质粒ｐＹＡ３３４１中,以重组质粒转化减毒鼠伤寒杆菌Ｘ４５５０。结果 对构建的重     

Association of interferon-γ and interferon regulatory factor 1 polymorphisms with asthma in a family-based association study in Taiwan

BACKGROUND: Asthma is a multi-factorial disorder caused by complex interactions between genetic and environmental factors. IFN-gamma and IFN regulatory factor 1 (IRF-1) affect Th1/Th2 cytokine balance, and influence the differentiation of Th2 cells, which influence the development of asthma. OBJECTIVE: This study investigated CA repeats polymorphism of the IFN-gamma gene and GT repeats polymorphism of the IRF-1 gene, which may predispose individuals to asthma pathogenesis. METHODS: In the present study, we used the transmission/disequilibrium test (TDT) to investigate the relationship between asthma and the IFN-gamma and IRF-1 polymorphisms by studying 348 subjects composed of 232 parents and 116 asthmatic children. RESULTS: For global TDT test, IFN-gamma CA repeats and IRF-1 GT repeat polymorphisms showed a significant association with asthma in children (P=0.009 and 0.017, respectively). We demonstrated that 13 CA repeats (138 bp) of IFN-gamma gene and 11 GT repeats (306 bp) of IRF-1 gene are significantly preferentially transmitted to asthmatic children (T/NT=89/61, chi2=8.43, P<0.005 and T/NT=75/49, chi2=8.18, P<0.005, respectively). The offspring will have an increased risk of asthma when their parents transmit IFN-gamma 13 CA repeats (OR=1.83, P=0.009) and IRF1 11 GT repeats (OR=1.88, P=0.007) to them. But we observed that the IFN-gamma and IRF-1 polymorphisms are not associated with IgE concentrations. CONCLUSION: These findings provide strong evidence of which IFN-gamma CA repeat and IRF-1 GT repeat polymorphisms influence the risk of asthma for children in Taiwan.