长效干扰素α-2a治疗慢性乙型肝炎过程中外周血浆样树突状细胞的变化及其与疗效的关系

目的 观察长效干扰素(PEG-IFN α-2a)治疗慢性乙型肝炎过程中外周血浆样树突状细胞(pDC)亚群的变化及其与临床疗效的关系. 方法 41例慢性乙型肝炎患者接受PEG-IFN α-2a(180 μg)每周皮下注射一次,治疗48周;治疗过程中检测肝功能、血清HBV病毒学标志物和HBVDNA,在治疗前及治疗开始后2、12、24、36、48周分别检测外周血pDC数量和功能及Toll样受体(TLR)9的表达水平、血清肿瘤坏死因子(TNF)α及IFN γ水平.对数据进行成组设计t检验、非参数检验、重复测量资料的方差分析. 结果 应答组与无应答组比较,TLR9平均荧光强度、pDC数量及INF α分泌能力在治疗2周时均明显下降,应答组在12周时TLR9平均荧光强度恢复(66.25±13.10),无应答组仍处于低水平(51.47±16.85),差异有统计学意义(t=2.478,P＜0.05);12周时应答组pDC数量恢复(5.24±1.61),无应答组为(3.74±1.25),差异有统计学意义(t=2.644,P＜0.05);12周时应答组IFN α分泌能力明显升高(459.94±200.27) pg/ml,显著高于无应答组[(237.18± 123.57)pg/ml],差异有统计学意义(t=2.942,P＜0.05).治疗24周时,应答组血清IFN γ水平明显升高[(67.81±16.64) pg/ml],显著高于无应答组[(43.73± 15.97) pg/ml],差异有统计学意义(t=3.396,P＜ 0.05);TNFα水平为[(268.94±64.32)pg/ml],也显著高于无应答组[(206.45±78.28) pg/ml],差异有统计学意义(t=2.22,P＜0.05).结论 pDC在PEG-IFNα-2a治疗诱发的早期免疫应答中发挥重要作用,抗病毒治疗过程中pDC数量和功能的恢复可能是机体抗病毒治疗应答的重要因素.     

Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells

Modulation of macrophage/dendritic cell (DC) cytokine production by the filarial nematode phosphorylcholine (PC)-containing product, ES-62, is mediated by Toll-like receptor (TLR) 4 and signal transduction depends on the TLR adaptor MyD88. Intriguingly, comparison of TLR4 knock-out (ko) mice with TLR4 mutant C3H/HeJ mice indicates that ES-62 cytokine responses are not dependent on the Pro712 residue of TLR4, which is crucial for the response to bacterial lipopolysaccharide (LPS). Because other immunomodulatory effects of ES-62 have been attributed to PC we have now investigated, using PC conjugated to ovalbumin (PC-Ova), whether PC is responsible for the interaction of ES-62 with TLR4. PC-Ova mimicked the modulation of interleukin (IL)-12 production by ES-62 in a TLR4- and MyD88-dependent manner and as with native ES-62, PC-Ova effects were not dependent on Pro712. Furthermore, both native ES-62 and PC-Ova suppressed Akt phosphorylation, whereas neither altered the activation of p38 or Erk MAP kinases. To rule out any role for the ES-62 protein component, we tested a PC-free recombinant ES-62 (rES-62) generated in the yeast Pichia pastoris. Surprisingly, rES-62 also modulated IL-12 production, but in a TLR4/MyD88-independent manner. Furthermore, rES-62 strongly activated both the p38 and Erk MAP kinases and Akt. However, recent biophysical analysis suggests there are differences in folding/shape between native and rES-62 and hence data obtained with the latter should be treated with caution. Nevertheless, although our study indicates that PC is likely to be primarily responsible for the modulation of cytokine production observed with native ES-62, an immunomodulatory role for the protein component cannot be ruled out.     

TLRs在溃疡性结肠炎小鼠结肠中的表达研究

目的 观察溃疡性结肠炎（UC）小鼠模型结肠组织中TLR2、TLR4和TLR9的表达情况,为UC发病机制的研究提供新思路.方法 将6～8周龄健康雄性BALB/c小鼠随机分为两组：正常对照组（n=10）和UC模型组（n=10）,正常对照组小鼠蒸馏水自由饮用7d.UC模型组小鼠5％ DSS溶液自由饮用7d造模.7d后处死小鼠,采用实时荧光定量PCR方法检测各组小鼠结肠黏膜组织中TLR2、TLR4和TLR9 mRNA的表达情况.结果 正常对照组结肠组织中无TLR2、TLR4和TLR9 mRNA的表达,而UC组结肠组织中TLR2、TLR4和TLR9 mRNA表达明显,两组比较差异有统计学意义（P＜0.05）.结论 TLR2、TLR4、TLR9可能参与了UC的发病过程.     

Toll样受体7及9在系统性红斑狼疮患者外周血B淋巴细胞内的表达及意义

目的 了解Toll样受体(TLR)7及9在系统性红斑狼疮(SLE)患者外周血B淋巴细胞内的表达情况及其意义.方法 流式细胞术检测50例SLE患者及30名健康人外周血B淋巴细胞内TLR7及9的表达水平.并将其与有关临床及实验窜指标进行相关性分析.结果 SLE患者B淋巴细胞内TLR7+及9+细胞所占比例均高于对照组.TLR7与患者红细胞沉降率(ESR)、SLE疾病活动指数(SLEDAI)呈正相关,与C3呈负相关.TLR9与患者尿蛋白、SLEDAI呈正相关.结论 TLR7及9在SLE患者B淋巴细胞内表达上调,且与病情有一定的相关性.     

Loss of CD103~+ intestinal dendritic cells during colonic inflammation

AIM:To investigate possible differences in dendritic cells(DC)within intestinal tissue of mice before and after induction of colitis. METHODS:Mucosal DC derived from intestinal tissue,as well as from mesenteric lymph nodes and spleen,were analyzed by fluorescence activated cell sorting(FACS) analysis.Supernatants of these cells were analyzed for secretion of different pro-and anti-inflammatory cytokines. Immunohistochemistry and immunofluorescence were performed on cryosections of mucosal tissue derived fro...     

Loss of CD103~+ intestinal dendritic cells during colonic inflammation

AIM:To investigate possible differences in dendritic cells(DC)within intestinal tissue of mice before and after induction of colitis. METHODS:Mucosal DC derived from intestinal tissue,as well as from mesenteric lymph nodes and spleen,were analyzed by fluorescence activated cell sorting(FACS) analysis.Supernatants of these cells were analyzed for secretion of different pro-and anti-inflammatory cytokines. Immunohistochemistry and immunofluorescence were performed on cryosections of mucosal tissue derived fro...     

神经激肽-1受体在溃疡性结肠炎黏膜中的表达

目的：了解神经激肽-1受体(NK-1R)在溃疡性结肠炎(UC)病人结肠活检黏膜中的表达，探讨该受体的表达与UC严重程度的关系。方法：38个UC黏膜标本取自因该病而行结肠镜检查的病人，男23例，女15例；对照组结肠黏膜取自15例肠易激综合征(IBS)病人，男8例，女7例。应用逆转录聚合酶链反应(RT-PCR)检测对照组和UC肠黏膜NK-1R的mRNA表达水平，应用Western blot技术检测NK-1R的蛋白水平，以免疫组化方法进行NK-1R的组织学定位。结果：与对照组肠黏膜相比，UC黏膜中NK-1R mRNA和蛋白都过度表达，NK-1R mRNA的表达与疾病的严重程度相关。免疫组化检查显示，NK-1R的表达主要位于UC的肠黏膜表面、黏膜固有层的单核细胞、黏膜下层的动静脉等处。结论：UC黏膜组织中NK-1R的表达水平明显上调，扰乱了神经激肽的作用环节，加剧了肠黏膜的病理改变。     

肠易激综合征腹泻型患者结肠黏膜Toll样受体2和4的表达

目的:研究Toll样受体(TLR)2、TLR4在肠易激综合征(IBS)患者和正常人结肠黏膜的表达.方法:研究30例IBS腹泻型患者及12名健康志愿者结肠黏膜TLR2和TLR4的表达,半定量分析TLR2及TLR4平均吸光度值.结果:与健康对照者相比,IBS患者结肠黏膜固有层水肿疏松.有大量的炎性细胞浸润.健康对照组和IBS组肠腔肠上皮细胞(IEC)的TLR2表达差异无统计学意义,(P<0.05).IBS患者的隐窝IEC的TLR2弱表达者较健康对照组少(6.7%比50.0%),而均匀表达增强者多(40.0%比0,P<0.05).86.7%的IBS患者基底侧及肠腔侧IEC均表达TLR4,较健康对照组增高(50.0%,P<0.05).细胞计数表明,IBS患者固有层TLR4阳性细胞数大于健康对照组(70.084±21.887比20.577±4.546,P<0.01),IBS患者的结肠黏膜TLR2及TLR4的吸光度值均增高单位分别为0.3079±0.0283比0.3886±0.0510和0.3044±0.0481比0.3971±0 0996(P<0 01).结论:IBS患者结肠有炎症表现,TLR2、TLR4存IBS发病中可能起一定作用.     

Clinical efficacy of the Toll-like receptor 9 agonist cobitolimod using patient-reported-outcomes defined clinical endpoints in patients with ulcerative colitis

BackgroundThe Toll-like-receptor 9 (TLR-9) agonist cobitolimod (DIMS0150, Kappaproct^®) is a promising therapeutic option for ulcerative colitis (UC) patients.AimsThe objectives of this post-hoc analysis using the COLLECT study data was to investigate the clinical effects of cobitolimod using patient-reported-outcomes (PRO) defined endpoints.MethodsDual topical administration of cobitolimod was studied in a randomised, multicentre clinical trial named COLLECT in moderate-to-severe UC patients. Symptomatic remission (SR) was studied in 104 patients based on their e-diary records and was defined as absence of blood in stool and a mean daily stool frequency (SF) < 4.ResultsSR was achieved at week 4 in 17.1% of cobitolimod vs. 5.9% of placebo treated patients (p = 0.13), at week 8 in 35.7% vs. 17.6% (p = 0.07), and at week 12 in 38.6% vs. 17.6% (p = 0.04) of the patients, respectively.SR rates with cobitolimod and placebo in anti-TNFα experienced patients were smaller but with a broadly similar relative effect-size to anti-TNFα naïve patients. Clinical efficacy was higher in patients with moderate compared to severe disease.ConclusionsApplication of the Toll-like-receptor 9 (TLR-9) agonist cobitolimod is able to induce remission as assessed by PRO measures in UC patients with moderate-to-severe activity as well as in anti-TNFα experienced and naïve patients supporting the overall efficacy of the substance.     

B-lymphoid cells with attributes of dendritic cells regulate T cells via indoleamine 2,3-dioxygenase

A discrete population of splenocytes with attributes of dendritic cells (DCs) and coexpressing the B-cell marker CD19 is uniquely competent to express the T-cell regulatory enzyme indoleamine 2,3-dioxygenase (IDO) in mice treated with TLR9 ligands (CpGs). Here we show that IDO-competent cells express the B-lineage commitment factor Pax5 and surface immunoglobulins. CD19 ablation abrogated IDO-dependent T-cell suppression by DCs, even though cells with phenotypic attributes matching IDO-competent cells developed normally and expressed IDO in response to interferon γ. Consequently, DCs and regulatory T cells (Tregs) did not acquire T-cell regulatory functions after TLR9 ligation, providing an alternative perspective on the known T-cell regulatory defects of CD19-deficient mice. DCs from B-cell–deficient mice expressed IDO and mediated T-cell suppression after TLR9 ligation, indicating that B-cell attributes were not essential for B-lymphoid IDO-competent cells to regulate T cells. Thus, IDO-competent cells constitute a distinctive B-lymphoid cell type with quintessential T-cell regulatory attributes and phenotypic features of both B cells and DCs.     

TLR3与TLR9在溃疡性结肠炎中表达状态及临床意义的研究

目的观察TLR3和TLR9在溃疡性结肠炎(UC)患者病变组织和正常大肠组织中的表达情况,探讨其在UC发病机制中的作用。方法收集UC病例及正常对照结肠镜活检标本各30例。采用免疫组化和实时荧光定量PCR技术,检测UC患者及正常对照组肠黏膜中TLR3、TLR9的表达情况。结果 UC病变组织、正常结肠组织中均有TLR3 mRNA及TLR9 mRNA的表达,但UC组织中TLR9 mRNA表达显著高于正常结肠组织,而TLR3 mRNA与正常结肠组织相比差异无统计学意义。在免疫组化染色图片上,发现TLR3、TLR9主要在细胞的胞浆中表达,在UC病变组织中TLR9阳性表达率明显高于正常结肠组织(P0.05)。结论TLR9在UC患者中表达高度上调,推测它可能参与了UC的发病过程。     

Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen

Human cancer vaccines are often prepared with altered "analog" or "heteroclitic" antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity.     

Conventional type 2 lung dendritic cells are potent inducers of follicular helper T cells in the asthmatic lung

BackgroundFollicular helper T (Tfh) cells represent a unique subset of helper CD4⁺ T cells in lymphoid follicles. Recently, Tfh cells were shown to play an important role in asthma through B cell differentiation. Conventional lung DCs are classified into two major subsets: conventional type 1 (cDC1) and type 2 (cDC2). Although the two subsets are different in driving particular T cell responses, the subset that induces Tfh cells in the asthmatic lung primarily has yet to be fully elucidated.MethodsWe evaluated Tfh cells, defined by the expression of CD4 and CXCR5, in HDM-challenged mice. Next, we characterized cDC1 and cDC2 purified from antigen-primed lung and examined their Tfh cell-inducing capacity. Additionally, the ability of lung DC-induced Tfh cells to cause germinal center B (GCB) cells to produce antigen-specific IgE was assessed.ResultsIn HDM-challenged mice, Bcl-6-expressing Tfh cells were significantly increased in the mediastinal lymph nodes. Lung cDC2, but not lung cDC1, increased after HDM priming, and cDC2 secreted larger amounts of IL-6 with higher ICOS-L expression than cDC1. In the co-cultures with OVA-specific naïve CD4⁺ T cells, cDC2 from OVA-primed lung induced Bcl-6-expressing Tfh cells more efficiently, together with larger amounts of IL-6 and IL-21, than cDC1. Blockage of IL-6 or ICOS-L significantly reduced Tfh cell induction. Finally, cDC2-induced Tfh cells enabled GCB cells to produce OVA-specific IgE.ConclusionsIn asthmatic lung, cDC2 is the primary DC subset responsible for Tfh cell differentiation and plays an important role in humoral immunity in asthma by inducing Tfh cells.     

Toll样受体9和低氧诱导因子1α在胰腺癌中的表达及其意义

目的 研究Toll样受体9(TLR9)和低氧诱导因子1α(HIF-1 α)在胰腺癌组织中的表达,探讨其临床意义.方法 采用实时定量PCR法、蛋白质印迹法及免疫组织化学法检测30例胰腺癌组织及其相邻癌旁组织、10例正常胰腺组织中TLR9和HIF-1α的表达,分析两者表达的相关性、与肿瘤临床病理特征的关系以及对患者生存时间的影响.结果 胰腺癌TLR9 mRNA及HIF-1αmRNA表达量分别为正常胰腺组织的2.32(1.41～3.22)倍和2.26(1.62～2.89)倍,癌旁组织表达量分别为正常胰腺组织的1.23(1.18 ～1.28)倍和1.36(1.17～1.55)倍,胰腺癌的表达量显著高于癌旁组织(t=2.642,P=0.023;t=4.076,P=0.001).胰腺癌组织TLR9和HIF-1α蛋白阳性表达率分别为73.3％(22/30)和70.0％(21/30),癌旁组织分别为33.3％ (10/30)和36.7％ (11/30),正常胰腺组织分别为20％(2/10)和10％(1/10),呈递减趋势(x2=13.99,P=0.001;x2=13.15,P=0.001).胰腺癌组织TLR9 mRNA与HIF-1 αmRNA表达呈正相关(r=0.537,P=0.003),TLR9和HIF-1α蛋白阳性表达率亦呈正相关(r =0.511,P=0.001).胰腺癌组织TLR9和HIF-1α表达与肿瘤分化程度、TNM分期、淋巴结转移呈正相关,与患者的生存时间呈负相关.结论 胰腺癌组织TLR9和HIF-1α基因均高表达,且与肿瘤细胞的恶性生物学行为及患者预后差有关.     

Double-negative T cells regulate the progression of liver fibrosis through Th9 cells differentiation

Our previous study found that double negative T cells (DNTs) could promote the NLRP3 activation through high expression of TNF-α, thereby leading to hepatic fibrosis progression. We focused on investigating the role and mechanism of DNTs in regulating the Th9 cells differentiation in liver fibrosis. In our results, among patients with liver fibrosis, the proportions of peripheral blood DNTs and Th9 cells were up-regulated and positively correlated. While promoting the progression of liver fibrosis in mice, DNTs could elevate the proportion of Th9 cells and activate the TNFR2-STAT5-NF-κB pathway. The use of IL-9 and TNF-α monoclonal antibodies (mAbs) inhibited the effect of DNTs and lowered the proportion of Th9 cells in tissues. In vitro experiments showed that DNTs could promote the Th9 cells differentiation of Naive T cells, while TNF-α mAbs could inhibit such effect of DNTs to lower the proportion of Th9 cells. We found that DNTs can activate TNFR2-STAT5-NF-κB pathway by secreting TNF-α, thereby promoting the Th9 Cells differentiation to facilitate the progression of liver fibrosis. There is interaction between DNTs and Th9 cells.     

CpG的信号传递机制及其在支气管哮喘等过敏性疾病的应用

支气管哮喘在全球范围特别是发达国家的发病率迅速增加.这被认为与患者幼年时期没有充分暴露在细菌或细菌产物环境下而导致过敏易感性增加有关.CpG-脱氧核苷酸是模拟细菌DNA的合成核苷酸链,可以通过Toll样受体(TLR)被细胞识别,并通过TLR/核因子κB(NF-κB)途径和MAPK途径等诱导Th1型细胞反应,下调Th2型细胞反应,调节免疫应答类型.动物实验证实其可以有效抑制哮喘模型的气道炎症、气道高反应性和气道重塑等,在临床实验中亦已开展诸多研究,因此有关CpG及其作用机制的研究可能成为支气管哮喘治疗的新突破口.     

CpG寡脱氧核苷酸促进小鼠清除体内结核分枝杆菌的机制

目的 探讨CpG寡脱氧核苷酸(CpG-ODN)增强小鼠体内结核分枝杆菌清除能力的可能机制.方法 将雌性BALB/c小鼠随机分为免疫组36只和对照组24只,并分别腹腔注射CpG-ODN 30 μg(溶于200 μl生理盐水)和200 μl生理盐水.2周后每只小鼠经尾静脉注射结核分枝杆菌H37Rv 1×106 CFU.感染后3周和4周每组分别处死12只小鼠,免疫组小鼠饲养至感染后6周处死.肺和脾组织进行菌落计数,观察肺和脾组织的病理学变化.用实时定量聚合酶链反应方法检测肺和脾组织γ-干扰素、白细胞介素(IL)-4、IL-10、IL-12、IL-18和诱导型一氧化氮合酶(iNOS)mRNA表达的相对含量.组间比较采用t检验.结果 感染后3周,免疫组小鼠的肺和脾组织经培养后无结核分枝杆菌生长.感染后4周,免疫组小鼠的肺和脾组织培养后有结核分枝杆菌生长,但明显少于对照组;肺组织中IL-18、γ-干扰素和iNOS的mRNA表达的相对含量分别为(3.6±0.5、0.32±0.14和23.2±4.7),均明显高于对照组的(1.6±1.1、0.20±0.10和16.2±5.1),IL-12p40 mRNA表达的相对含量(5.7±0.6)明显低于对照组(14.5±1.9),IL-4和IL-10 mRNA表达的相对含量(0.30±0.09和0.28±0.05)与对照组(0.26±0.05和0.29±0.08)无明显差别;脾组织中IL-18、γ-干扰素和iNOS mRNA表达的相对含量(5.5±1.3、0.52±0.07和9.1±1.8)明显高于对照组(0.8±0.4、0.21±0.06和6.0±1.4),IL-12p40、IL-4和IL-10 mRNA表达的相对含量(2.1±0.3、0.23 ±0.10和0.10±0.04)明显低于对照组(5.1±0.4、1.21±0.26和0.57±0.13).与感染后4周比较,免疫组小鼠感染后6周,肺组织γ-干扰素mRNA表达的相对含量(0.95±0.27)明显增高,IL-18、IL-4和IL-10 mRNA表达的相对含量(3.51±0.86、0.45±0.35和0.24±0.21)无明显变化,IL-12p40和iNOS mRNA表达的相对含量(1.72±1.41和1.1±0.5)明显降低;脾组织IL-12p40和IL-18 mRNA表达的相对含量(0.08±0.02)和(0.11±0.03)明显降低,IL-10 mRNA表达的相对含量(0.39±0.11)明显增高,γ-干扰素、IL-4和iNOS mRNA表达的相对含量(0.63±0.32、0.30±0.16和8.4±2.7)无明显变化.结论 CpG-ODN增强小鼠清除体内结核分枝杆菌的能力与IL-18、γ-干扰素和iNOS的表达增强及IL-4和IL-10的表达抑制密切相关.