Toll-like receptors 2 polymorphism is associated with psoriasis: A case-control study in the northern Chinese population

Background:Psoriasis is a disease caused by genetics and immune system dysfunction,affecting the skin and joints.Toll-like receptors(TLRs)play an important role in triggering the innate immune response and controlling adaptive immunity.The role of TLR2 in the progression of psoriasis is not well understood.Methods:A case-control study was conducted on a northern Chinese Han population,consisting of psoriasis patients and healthy control subjects.Genotyping was performed using the tetra-primer amplification refractory mutation system-polymerase chain reaction(ARMS-PCR),and allele and genotype frequencies of four SNPs in TLR2 were analyzed in 270 psoriasis patients and 246 healthy controls.Results:Four TLR2 SNPs(rs11938228,rs4696480,rs3804099,rs5743699)were genotyped and found to be in linkage disequilibrium.The genotype distributions of rs11938228 and rs4696480 in two groups were in Hardy-Weinberg equilibrium and statistically significant except for the overdominance model.The haplotypes ATTC and ATCC were found to be protective against psoriasis.Conclusion:Our study found a correlation between TLR2 genetic variations and the likelihood of psoriasis in northern China.

Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

The noble gas argon has the potential to protect neuronal cells from cell death.So far,this effect has been studied in treatment after acute damage.Preconditioning using argon has not yet been investigated.In this study,human neuroblastoma SH-SY5Y cells were treated with different concentrations of argon(25%,50%,and 74%;21%O_(2),5%CO_(2),balance nitrogen)at different time intervals before inflicting damage with rotenone(20μM,4 hours).Apoptosis was determined by flow cytometry after annexin V and propidium iodide staining.Surface expressions of Toll-like receptors 2 and 4 were also examined.Cells were also processed for analysis by western blot and qPCR to determine the expression of apoptotic and inflammatory proteins,such as extracellular-signal regulated kinase(ERK1/2),nuclear transcription factor-κB(NF-κB),protein kinase B(Akt),caspase-3,Bax,Bcl-2,interleukin-8,and heat shock proteins.Immunohistochemical staining was performed for TLR2 and 4 and interleukin-8.Cells were also pretreated with OxPAPC,an antagonist of TLR2 and 4 to elucidate the molecular mechanism.Results showed that argon preconditioning before rotenone application caused a dose-dependent but not a time-dependent reduction in the number of apoptotic cells.Preconditioning with 74%argon for 2 hours was used for further experiments showing the most promising results.Argon decreased the surface expression of TLR2 and 4,whereas OxPAPC treatment partially abolished the protective effect of argon.Argon increased phosphorylation of ERK1/2 but decreased NF-κB and Akt.Preconditioning inhibited mitochondrial apoptosis and the heat shock response.Argon also suppressed the expression of the pro-inflammatory cytokine interleukin-8.Immunohistochemistry confirmed the alteration of TLRs and interleukin-8.OxPAPC reversed the argon effect on ERK1/2,Bax,Bcl-2,caspase-3,and interleukin-8 expression,but not on NF-κB and the heat shock proteins.Taken together,argon preconditioning protects against apoptosis of neuronal cells and mediates its action via Toll-like receptors.Argon may represent a promising therapeutic alternative in various clinical settings,such as the treatment of stroke.

Inetetamab combined with tegafur as second-line treatment for human epidermal growth factor receptor-2-positive gastric cancer: A case report

BACKGROUND Human epidermal growth factor receptor-2(HER-2)plays a vital role in tumor cell proliferation and metastasis.However,the prognosis of HER2-positive gastric cancer is poor.Inetetamab,a novel anti-HER2 targeting drug independently developed in China,exhibits more potent antibody-dependent cell-mediated cytotoxicity than trastuzumab,which is administered as the first-line treatment for HER2-positive gastric cancer in combination with chemotherapy.In this case,the efficacy and safety of inetetamab combined with tegafur was investigated as a second-line treatment for HER2-positive gastric cancer.CASE SUMMARY A 52-year-old male patient with HER2-positive gastric cancer presented with abdominal distension,poor appetite,and fatigue two years after receiving six cycles of oxaliplatin combined with tegafur as first-line treatment after surgery,followed by tegafur monotherapy for six months.The patient was diagnosed with postoperative recurrence of gastric adenocarcinoma.He received 17 cycles of a combination of inetetamab,an innovative domestically developed anti-HER2 monoclonal antibody,and tegafur chemotherapy as the second-line treatment(inetetamab 200 mg on day 1,every 3 wk combined with tegafur twice daily on days 1–14,every 3 wk).Evaluation of the efficacy of the second-line treatment revealed that the patient achieved a stable condition and progression-free survival of 17 months.He tolerated the treatment well without exhibiting any grade 3-4 adverse events.CONCLUSION Inetetamab combined with chemotherapy for the treatment of metastatic HER2-positive gastric cancer demonstrates significant survival benefits and acceptable safety.

Relationship of Toll-Like Receptors 2 and 4 Gene Polymorphisms with Essential Hypertension in Chinese Han Population

Objective: There are numerous studies suggesting that genetic polymor-phisms of inflammation factors Toll-like receptors 2 and 4 (TLR2, TLR4) might play a role in the pathophysiological process of hypertension. In this study, we evaluated the association in a sample of members of the Chinese Han population. Method: We selected four single nucleotide polymor-phisms (SNP) of TLR2 (rs3804099, rs3804100, rs7656411) and TLR4 (rs1927906) genes, and measured the distributions of genotypic and allelic frequencies in 1063 participants, including 391 essential hypertension pa-tients and 672 controls. Result: No significant differences in the genotypic and allelic frequencies of the four SNPs were detected between cases and controls. However, three haplotypes, CCG, TTG and TTT of TLR2, were significantly associated with a decrease in the risk of essential hyperten-sion (OR: 0.512, 95% CI: 0.397 - 0.660, P P = 0.0038;OR: 0.797, 95% CI: 0.667 - 0.952, P = 0.0122, respectively). Inversely, the risk of essential hypertension increased sig-nificantly in patients with the CTG, TCG or TCT haplotypes (OR: 2.924, 95% CI: 2.157 - 3.963, P P P Conclusion: Our study suggested that haplotypes (CCG, TTG, TTT, CTG, TCG and TCT) of TLR2 might have profound effects on the development of essential hypertension in the Chinese Han population.

广谱模式识别分子Toll-like receptor 2的研究进展

TLR-2（Toll-like receptor 2,TLR-2）是哺乳动物TLRs（Toll-like receptors,TLRs）家族的一员,作为细胞表面的天然受体蛋白,主要参与病原微生物产物的识别及炎症信号传导,介导天然抗感染兔疫;最近又发现其参与机体对非感染因子所致炎性组织损伤的识别。通过对TLR-2参与的识别和细胞内信号传导机制的研究,可为深入探讨抵御微生物感染的机制、对自身正常与非正常组织的识别提供新的思路。

人Toll-like receptor 2配基模拟肽的初步研究

TLR-2(Toll-likereceptor2)是介导天然免疫的重要模式识别分子,可参与识别多种病原体及其产物.为探索被TLR-2所识别配基的结构共性,以真核细胞表达的人TLR-2胞外段蛋白(A26￣T588)为钓饵筛选噬菌体12肽库,获得一高度保守的阳性噬菌体克隆P12-1,实验发现P12-1可与不同形式的TLR-2胞外段结合,并且可刺激细胞分泌TNFα,提示P12-1可能模拟TLR-2配基的结构与生物学活性.

人Toll-like receptor 2胞外段的克隆和表达

目的 克隆、表达人Toll-like receptor2(TLR2)胞外段(A26-T588)基因,获得人TLR2胞外段蛋白。方法 RT-PCR扩增TLR2胞外段基因,以pcDNA3.1+质粒为载体在HEK293细胞中表达TLR2胞外段蛋白,同时以pcDNA3.1+/TLR2(A26-T588)重组质粒免疫昆明鼠,制备抗TLR2胞外段蛋白多抗。结果 PCR扩增及重组质粒测序结果表明成功地构建了pcDNA3.1+/TLR2(A26-T588)真核表达质粒,SDS-PAGE分析纯化产物在M_r为68000处出现明显蛋白条带。重组质粒DNA免疫小鼠3次后,血清抗体滴度可达1∶250。TLR2胞外段蛋白可与LPS结合,并在一定的质量浓度范围内呈剂量依赖性。结论 构建的pcDNA3.1+/TLR2(A26-T588)真核表达质粒可在哺乳动物细胞中表达TLR2胞外段蛋白,并与重组质粒DNA免疫小鼠抗血清及TLR2单克隆抗体TL2.1特异性反应,由此证明其表达正确。

外周血TLR2、TIPE2、NLR联合检测对机械通气新生儿呼吸机相关性肺炎的预测价值

目的探讨外周血Toll样受体2(TLR2)、肿瘤坏死因子α诱导蛋白8样因子2(TIPE2)、中性粒细胞与淋巴细胞比值(NLR)联合检测对机械通气新生儿呼吸机相关性肺炎(VAP)的预测价值。方法选取2020年6月至2023年6月河北北方学院附属第一医院进行机械通气治疗的232例新生儿作为研究对象。将发生VAP的新生儿纳入VAP组,其余纳入非VAP组。检测并比较两组外周血TLR2、TIPE2表达水平和NLR。绘制受试者工作特征(ROC)曲线评估外周血TLR2、TIPE2、NLR单独及联合预测机械通气新生儿VAP发生的价值。采用多因素Logistic回归分析机械通气新生儿发生VAP的影响因素。结果232例机械通气新生儿中,60例发生VAP,发生率为25.86%,纳入VAP组;其余172例未发生VAP,纳入非VAP组。VAP组和非VAP组机械通气时间、置管次数和吸痰次数比较,差异均有统计学意义(P<0.05)。VAP组TLR2相对表达水平和NLR高于非VAP组,TIPE2相对表达水平低于非VAP组,差异均有统计学意义(P<0.05)。多因素Logistic回归分析结果显示,机械通气时间≥3 d、置管次数≥2次、吸痰次数≥5次/天、TLR2相对表达水平升高、NLR升高是械通气新生儿发生VAP的危险因素(P<0.05),TIPE2相对表达水平升高是新生儿发生VAP的保护因素(P<0.05)。ROC曲线分析结果显示,TLR2、TIPE2和NLR单独预测机械通气新生儿发生VAP的曲线下面积(AUC)分别为0.783、0.754、0.745;且外周血TLR2、TIPE2、NLR联合检测预测机械通气新生儿发生VAP的AUC为0.823。结论外周血TLR2、TIPE2和NLR均是机械通气新生儿发生VAP的独立影响因素,且联合检测有助于预测VAP的发生。

Toll样受体2与肾脏疾病关系的研究进展

Toll样受体2(TLR2)属于固有免疫模式识别受体,其广泛分布于体内多种免疫细胞表面,使免疫细胞活化、释放细胞因子,引起免疫炎症反应。近年来,发现TLR2在肾脏固有细胞中也有表达,TLR2异常激活与急性肾损伤、慢性肾脏病、尿路感染等密切相关。TLR2配体及其下游信号因子繁多,TLR2被激活后通过复杂的转导机制在肾脏病理生理过程中起“双刃剑”的作用。未来,可对TLR2在肾脏疾病中的作用及其可能涉及的机制进行深入探索,以期为肾脏疾病的防治提供新的方法和理论依据。

儿童肺炎支原体肺炎肺泡灌洗液CARDS TX、HMGB1、TLR2表达及临床意义

目的研究肺炎支原体肺炎(MPP)患儿肺泡灌洗液中社区获得性呼吸窘迫综合征毒素(CARDS TX)、高迁移率族蛋白1(HMGB1)、Toll样受体2(TLR2)表达,探讨其在MPP发病中的临床意义。方法回顾性分析55例MPP病例组及20例对照组临床资料,同时检测两组支气管肺泡灌洗液(BALF)中CARDS TX、HMGB1、TLR2、TLR4、晚期糖基化终末产物受体(RAGE)及髓样分化因子88(MyD88)表达,体外检测不同浓度CARDS TX刺激下HMGB1、TLR2的表达并进行比较。结果与对照组相比,MPP组LYM绝对值计数、PA、CD3^(+)、CD3^(+)CD4^(+)、CD3^(+)CD8^(+)、CD3-CD19^(+)、NK cell、CD19^(+)CD23^(+)明显下降,CRP、LDH、D-二聚体、IgA、IgG、IgM、CARDS TX、HMGB1、TLR2、MyD88均升高,差异有统计学意义(P<0.05或0.001)。CARDS TX刺激后HMGB1、TLR2的表达升高,且呈正相关,差异有统计学意义(P<0.001)。结论CARDS TX可能通过HMGB1-TLR2-MyD88途径参与肺炎支原体(MP)的致病。

Toll样受体2及Toll样受体4 mRNA表达水平与前列腺癌术后尿路感染发生及转归的关系分析

目的分析Toll样受体2(TLR2)及Toll样受体4(TLR4)mRNA表达水平与前列腺癌(PC)根治术后尿路感染发生及转归的关系。方法选取2020年3月至2023年3月衡水市第二人民医院收治的80例PC根治术后尿路感染患者作为感染组,另选取同期80例PC根治术后未发生尿路感染患者作为非感染组。比较两组TLR2、TLR4 mRNA相对表达量及血清炎症因子水平,分析TLR2、TLR4 mRNA表达量与炎症因子的相关性;根据感染组治疗后转归情况分为预后良好组和预后不良组,分析TLR2、TLR4 mRNA对不良预后的预测效能。结果与非感染组比较,感染组外周血单个核细胞TLR2、TLR4 mRNA表达量及血清白介素(IL)-6、IL-8、肿瘤坏死因子-α(TNF-α)水平更高(P<0.05);Pearson分析显示,TLR2、TLR4 mRNA表达量与血清IL-6、IL-8、TNF-α水平均呈正相关(P<0.05);感染组治疗后61例预后良好(预后良好组),其外周血单个核细胞TLR2、TLR4 mRNA表达量较预后不良组更高(P<0.05);TLR2、TLR4 mRNA单独及两者联合预测尿路感染不良预后的灵敏度/特异度分别为89.50%/63.90%、73.70%/73.80%、94.70%/62.30%,曲线下面积(AUC)分别为0.808、0.790、0.890。结论PC根治术后尿路感染的发生及转归与外周血TLR2、TLR4 mRNA表达有关,其作用机制可能为上调IL-6、IL-8、TNF-α等炎症因子表达。

Toll-like receptor 2及其信号传导通路在阿片类物质诱导细胞凋亡中的作用

目的:成瘾物质(Addictive drugs)导致的中毒性脑病的中枢神经系统损害以神经元凋亡为突出表现,具体机制仍有待进一步的研究探讨。方法:Toll-like receptor 2(TLR2)不仅是激活机体自身免疫防御和炎症反应的重要受体,而且广泛参与多种细胞功能,包括细胞凋亡。我们使用体外培养的HEK293和高表达TLR2的HEK2932种细胞,以及体外培养小鼠皮层原代神经元细胞,研究经过吗啡处理后细胞存活率和凋亡的变化,以探讨TLR2信号通路在吗啡诱导的细胞凋亡中的作用。结果:TLR2高表达导致吗啡诱导的细胞存活率下降和细胞凋亡的显著增加。使用MyD88抑制体竞争性抑制MyD88并阻断TLR2信号通路后,吗啡诱导的TLR2高表达细胞的凋亡也被明显抑制。长期的吗啡处理导致正常神经元的TLR2在信使RNA(mRNA)水平和蛋白水平表达明显增高,而且在TLR2功能缺失的原代神经元细胞中,吗啡诱导的caspase-3活性增高被阻断,吗啡诱导的神经元凋亡也被明显抑制。结论:TLR2信号通路参与吗啡诱导的神经元凋亡。

黄芪甲苷通过Toll-like受体通路对HepG2高脂细胞脂质积累的抑制作用

目的探讨黄芪甲苷(AS-Ⅳ)对HepG2高脂细胞模型血脂代谢的影响以及Toll-like信号通路在其中的作用。方法使用HepG2细胞构建高脂血症模型,并随机分为六组:AS-Ⅳ低、中、高(0.05、0.1、0.2 mmol/L)剂量组,对照组,抑制剂组,AS-Ⅳ+抑制剂组。分别进行实时荧光定量聚合酶链式反应(PCR)和蛋白质免疫印迹(WB)分析,检测Toll样受体4(TLR4)、髓样分化因子88(MyD88)、肿瘤坏死因子受体相关因子6(TRAF6)mRNA和蛋白表达。结果①在AS-Ⅳ高剂量组中,甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白(LDL)的水平显著降低(在AS-Ⅳ低剂量组中降低不显著)。②在AS-Ⅳ中、高剂量组中,TLR4、MYD88和TRAF6mRNA的表达水平明显下降(在AS-Ⅳ低剂量组中降低不显著)。③在AS-Ⅳ高、中剂量组中,TLR4、MyD88、TRAF6蛋白的表达水平显著下降(在AS-Ⅳ低剂量组中降低不显著)。④TLR4抑制剂TAK-242、MyD88抑制剂ST2825和TRAF6抑制剂C25-140能够抑制AS-Ⅳ的作用。结论AS-Ⅳ在HepG2高脂细胞中有降低血脂累积作用,可能通过减轻脂肪代谢障碍实现,其机理与Toll-like信号路径有关。

单核细胞Toll样受体和核因子-κB水平与2型糖尿病患者自主神经病变的相关性

目的探讨单核细胞中Toll样受体(TLR)、核因子-κB(NF-κB)表达水平与2型糖尿病(T2DM)患者自主神经病变的相关性。方法选择2021年4月至2022年12月濮阳市人民医院收治的112例T2DM患者为观察组,另选择同期在本院进行体检的50例健康志愿者为对照组。采用反转录聚合酶链反应检测2组受试者血浆单核细胞中TLR2、TLR4、NF-κB表达水平。根据是否并发有自主神经病变将观察组患者分为自主神经病变组(n=46)和无自主神经病变组(n=66),比较2组患者的临床资料,将差异有统计学意义的指标进一步行多因素logistic回归来分析T2DM患者发生自主神经病变的危险因素;采用Spearman相关分析TLR2、TLR4、NF-κB表达水平与T2DM患者发生自主神经病变的相关性。结果观察组患者单核细胞中TLR2、TLR4、NF-κB水平显著高于对照组(P<0.05)。单因素分析结果显示,自主神经病变组与无自主神经病变组患者的年龄、单核细胞/高密度脂蛋白比值(MHR)及胱抑素C、TLR2、TLR4、NF-κB水平比较差异有统计学意义(P<0.05)。多因素logistic回归分析结果显示,MHR、TLR2、TLR4、NF-κB是T2DM患者发生自主神经病变的危险因素(P<0.05)。Spearman相关分析结果显示,TLR2、TLR4、NF-κB水平与T2DM患者发生自主神经病变呈正相关(r=0.825、0.822、0.819,P<0.05)。结论TLR2、TLR4、NF-κB在T2DM患者中表达上调,TLR2、TLR4、NF-κB表达水平与T2DM患者自主神经病变有关。

Toll样受体2对儿童肺炎支原体肺炎肺部炎症表现和肿瘤坏死因子-α的调节作用

目的 探讨Toll样受体2(TLR2)对肺炎支原体肺炎(MPP)病儿肺部炎症表现和肿瘤坏死因子-α(TNF-α)的调节作用。方法 选取2021年1月至2022年1月郑州大学附属儿童医院诊治的MPP病儿27例,其中难治性肺炎支原体肺炎(RMPP)病儿12例、MPP病儿15例。选取非感染病儿12例作为对照(NC)组。检测并比较不同病儿外周血TLR和TNF-α的表达,培养A549细胞并比较其与中性粒细胞肺炎支原体(MP)刺激的TNF-α表达,以及TLR2敲除后MP刺激的TNF-α表达。结果 MPP病儿血清TNF-α水平高于NC组,且RMPP组高于MPP组(P<0.05)。RMPP组外周血中性粒细胞TLR1和TLR2mRNA高于MPP组(P<0.05)。MPP组TLR2 mRNA高于NC组(P<0.05)。MP刺激组的TNF-α[A549细胞:(19.50±1.30)ng/L,中性粒细胞:(505.60±92.40)ng/L]和TLR2(A549细胞:3 760.17±772.06,中性粒细胞:4 224.00±711.12)表达显著高于NC组(P<0.05)。TLR2敲除后A549细胞后,与对照组相比,TLR2敲除的细胞MP刺激诱导的TNF-α表达受到显著抑制。蛋白质印迹法显示,MP刺激A549细胞中MyD88和NF-κB蛋白表达均明显增加(P<0.05)。阻断Myd88或NF-κB可明显减弱MP诱导的A549细胞TNF-α表达(P<0.05)。结论 TLR2可调节MPP通过TLR2-MyD88-NF-κB信号通路介导的肺部炎症和TNF-α释放。

Effects of Propofol on The mRNA Expression of Toll-like Receptor 4 in BV-2 Cells During Mimic Ischemia-Reperfusion Injury In Vitro

This study investigated the effects of propofol on the mRNA expression of Toll-like receptor-4 （TLR4） in BV-2 cells during mimic ischemia-reperfusion （I/R） injury in vitro. BV-2 cells, a mouse microglia line, were cultured and divided into 4 groups at random： control group （group C）, ischemia/reperfusion group （group I/R）, low-dose propofol （25 μmol/L） intervention group （group PF25） and high-dose propofol （100 μmol/L） intervention group （group PF100）. The mRNA expression of TLR4 and NF-κB was measured by means of RT-PCR. TNF-α levels in the supernatants of BV-2 cells were detected by ELISA. The results showed that the mRNA expression of TLR4 and NF-κB was significantly higher in groups I/R, PF25 and PF100 than in group C （P〈0.01）. And the TNF-α level in the supernatants was elevated in groups I/R, PF25 and PF100 as compared with that in group C （P〈0.01）. After pre-treatment with propofol, the mRNA expressions of TLR4 and NF-κB and the TNF-α level were significantly decreased in groups PF25 and PF100 in comparison to those in group I/R （P〈0.01）. And the decrease in those indicators was more significant in group PF100 than in group PF25 （P〈0.01）. It was concluded that propofol exerted brain-protecting effects during I/R injury by suppressing the mRNA expressions of TLR4 and NF-κB and deceasing the TNF-α level.

Divergent expression of bacterial wall sensing toll-like receptors 2 and 4 in colorectal cancer

To characterize the expression of toll-like receptors (TLR) 2 and 4 in colorectal cancer (CRC) and in normal colorectal mucosa. METHODSWe analysed tissue samples from a prospective series of 118 unselected surgically treated patients with CRC. Sections from formalin fixed, paraffin embedded specimens were analysed for TLR2 and TLR4 expression by immunohistochemistry. Two independent assessors evaluated separately expression at the normal mucosa, at the invasive front and the bulk of the carcinoma, and in the lymph node metastases when present. Expression levels in different locations were compared and their associations with clinicopathological features including TNM-stage and the grade of the tumour and 5-year follow-up observations were analysed. RESULTSNormal colorectal epithelium showed a gradient of expression of both TLR2 and TLR4 with low levels in the crypt bases and high levels in the surface. In CRC, expression of both TLRs was present in all cases and in the major proportion of tumour cells. Compared to normal epithelium, TLR4 expression was significantly weaker but TLR2 expression stronger in carcinoma cells. Weak TLR4 expression in the invasive front was associated with distant metastases and worse cancer-specific survival at 5 years. In tumours of the proximal colon the cancer-specific survival at 5 years was 36.9% better with strong TLR4 expression as compared with those with weak expression (P = 0.044). In contrast, TLR2 expression levels were not associated with prognosis. Tumour cells in the lymph node metastases showed higher TLR4 expression and lower TLR2 expression than cells in primary tumours. CONCLUSIONTumour cells in CRC show downregulation of TLR4 and upregulation of TLR2. Low expression of TLR4 in the invasive front predicts poor prognosis and metastatic disease.

Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects

AIM: To investigate the effects of our tumor vaccines on reversing immune tolerance and generating therapeutic response.METHODS: Vaccines were synthesized by solid phase using an Fmoc strategy,where a small molecule toll-like receptor-7 agonist(T7) was conjugated to a monoclonal gastric cancer 7 antigen mono-epitope(T7-MG1) or tri-epitope(T7-MG3).Cytokines were measured in both mouse bone marrow dendritic cells and mouse spleen lymphocytes after exposed to the vaccines.BALB/c mice were intraperitoneally immunized with the vaccines every 2 wk for a total of three times,andthen subcutaneously challenged with Ehrlich ascites carcinoma(EAC) cells.Three weeks later,the mice were killed,and the tumors were surgically removed and weighed.Serum samples were collected from the mice,and antibody titers were determined by ELISA using an alkaline phosphate-conjugated detection antibody for total Ig G.Antibody-dependent cell-mediated cytotoxicity was detected by the lactate dehydrogenase method using natural killer cells as effectors and antibody-labeled EAC cells as targets.Cytotoxic T lymphocyte activities were also detected by the lactate dehydrogenase method using lymphocytes as effectors and EAC cells as targets.RESULTS: Vaccines were successfully synthesized and validated by analytical high performance liquid chromatography and electrospray mass spectrometry,including T7,T7-MG1,and T7-MG3.Rapid inductions of tumor necrosis factor-α and interleukin-12 in bone marrow dendritic cells and interferon γ and interleukin-12 in lymphocytes occurred in vitro after T7,T7-MG1,and T7-MG3 treatment.Immunization with T7-MG3 reduced the EAC tumor burden in BALB/c mice to 62.64% ± 5.55% compared with PBS control(P < 0.01).Six or nine weeks after the first immunization,the monoclonal gastric cancer 7 antigen antibody increased significantly in the T7-MG3 group compared with the PBS control(P < 0.01).As for antibody-dependent cell-mediated cytotoxicity,antisera obtained by immunization with T7-MG3 were able to markedly enhance cell lysis compared to PBS control(31.58% ± 2.94% vs 18.02% ± 2.26%; P < 0.01).As for cytotoxic T lymphocytes,T7-MG3 exhibited obviously greater cytotoxicity compared with PBS control(40.92% ± 4.38% vs 16.29% ± 1.90%; P < 0.01).CONCLUSION: A successful method is confirmed for the design of gastric cancer vaccines by chemical conjugation of T7 and multi-repeat-epitope of monoclonal gastric cancer 7 antigen.

Protection against hepatic ischemia/reperfusion injury via downregulation of toll-like receptor 2 expression by inhibition of Kupffer cell function

AIM： To elucidate the mechanism of liver protection by inhibition of Kupffer cells （KCs） function.METHODS： All the animals were randomly divided into three groups. Blockade group （gadolinium chloride solution （GdCl3） injection plus ischemia/reperfusion （I/R） injury）：GdCl3 solution was injected once every 24 h for 2 d via the tail vein before I/R injury. Non-blockade group （saline solution injection plus I/R injury）： saline instead of GdCl3 as a control was injected as in the blockade group. Sham group： saline was injected without I/R injury. Liver samples were collected 4 h after blood inflow restoration. The blockade of the function of KCs was verified by immunostaining with an anti-CD68 mAb. Toll-like receptor2 （TLR2） was immunostained with a goat antimouse polydonal anti-TLR2 antibody. Membrane proteins were extracted from the liver samples and TLR2 protein was analyzed by Western blot. Portal vein serum and plasma were taken respectively at the same time point for further detection of the levels of tumor necrosis factor-α （TNF-α） and alanine aminotransferase （ALT）, an indicator of liver function.I/R injury via downregulation of theexpression of TLR2.RESULTS： Compared to non-blockade group, CD^68＋ cells significantly reduced in blockade group （OPTDI, optical density integral）： 32.97±10.55 vs 185.65±21.88, P〈0.01） and the liver function impairment was relieved partially （level of ALT： 435.89±178.37 U/L vs 890.21±272.91 U/L,P〈0.01）. The expression of TLR2 protein in blockade group significantly decreased compared to that in non- group（method of immunohistochemistry, OPDTI： 75.74±17.44 vs 170.58±25.14, P〈0.01; method of Western blot, A value： 125.89±15.49 vs 433.91±35.53, P〈0.02）. The latter correlated with the variation of CD68 staining （r = 0.745,P〈0.05）. Also the level of portal vein TNF-α decreased in blockade group compared to that in non-blockade group （84.45±14.73 ng/L vs 112.32±17.56 ng/L, P〈0.05）, butwas still higher than that in sham group （84.45±14.73 ng/L vs 6.07±5.33 ng/L, P〈0.01）.CONCLUSION： Inhibition of the function of KCs may protect liver against I/R injury via downregulation of the expression of TLR2.

Association between polymorphisms in the Toll-like receptor 4,CD14,and CARD15/NOD2and inflammatory bowel disease in the Greek population

AIM: Crohn's disease(CD)and ulcerative colitis(UC)are multifactorial diseases with a significant genetic background.Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp,Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC.METHODS: DNA was obtained from 120 patients with CD,85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis.RESULTS: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequendes of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026＜0.05; P = 0.0048＜0.01 and P= 0.047＜0.05 respectively). Concerning the NOD2/CARD15mutations the overall presence in CD patients was significantly higher than that in UC patients or in controls.Additionally, 51.67% of the CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one variant of the NOD2/CARD15, compared to 27% of the UC patients. It should be pointed out that both frequencies significantly increased as compared with the 10% frequency of multiple carriers found in healthy controls. A possible interaction of the NOD2/CARD15 with TLR4 and especially CD14, increased the risk of developing inflammatory bowel disease (IBD).CONCLUSION: Our results indicate that co-existence of a mutation in either the TLR4 or CD14 gene, and in NOD2/CARD15is associated with an increased susceptibility to developing CD compared to UC, and to developing either CD or UC compared to healthy individuals.