三维立体培养人胚肺成纤维母细胞前列腺素E_2释放过程中蛋白酶激活受体的调节作用

背景：前列腺素E2是蛋白酶激活受体信号转导通路中关键递质之一,在人胚肺成纤维母细胞的收缩、增殖和趋化运动中发挥重要的调节作用。但成纤维细胞三维立体培养过程中,蛋白酶激活受体对前列腺素E2释放的调节作用尚不清楚。目的：观察在三维立体培养的人胚肺成纤维母细胞过程中,蛋白酶激活受体1和蛋白酶激活受体2对前列腺素E2释放的调节作用。方法：自大鼠尾腱提取Ⅰ型胶原。体外培养人胚肺成纤维母细胞,应用免疫印迹法测定蛋白酶激活受体1、蛋白酶激活受体2的表达。将人胚肺成纤维母细胞包裹在含培养基的Ⅰ型胶原中漂浮培养作为体外组织重构模型,即人胚肺成纤维母细胞三维立体培养,分别应用蛋白酶激活受体1及蛋白酶激活受体2受体激动剂凝血酶、胰蛋白酶、SLIGKV-NH2、TFLLR-NH2刺激人胚肺成纤维母细胞,ELISA法测定培养上清液中前列腺素E2的含量。结果与结论：蛋白酶激活受体1和蛋白酶激活受体2表达于三维立体培养的人胚肺成纤维母细胞。基础状态下三维立体培养的人胚肺成纤维母细胞可释放前列腺素E2;蛋白酶激活受体1非选择性受体激动剂凝血酶、蛋白酶激活受体2非选择性受体激动剂胰蛋白酶和蛋白酶激活受体1选择性受体激动剂SLIGKV-NH2均显著刺激前列腺素E2产生（P〈0.01）,而蛋白酶激活受体2选择性受体激动剂TFLLR-NH2呈剂量依赖地抑制前列腺素E2的释放（P〈0.01）。结果提示蛋白酶激活受体1和蛋白酶激活受体2参与调节三维立体培养的人胚肺成纤维母细胞前列腺素E2的释放。     

Fas及FasL和Caspase-3在青蒿琥酯诱导人胚肺成纤维细胞凋亡中的表达

背景：青蒿琥酯具有减轻肺纤维化的作用,但相关机制的研究罕见报道。目的：探讨青蒿琥酯对人胚肺成纤维细胞凋亡的作用及其与Fas,FasL,Caspase-3表达的关系。方法：用1,10,100mg/L青蒿琥酯分别干预体外培养的人胚肺成纤维细胞。采用CCK-8法检测青蒿琥酯对人胚肺成纤维细胞增殖的影响,流式细胞术测定细胞凋亡率,RT-PCR法测定Fas,FasL,Caspase-3的mRNA的表达。结果与结论：青蒿琥酯呈浓度依赖性抑制人胚肺成纤维细胞增殖,细胞经青蒿琥酯作用后凋亡率明显增加（P〈0.05或P〈0.01）,Fas,FasL,Caspase-3mRNA的表达显著高于对照组（P〈0.05）。结果证实,青蒿琥酯可通过上调Fas,FasL,Caspase-3mRNA的表达抑制人胚肺成纤维细胞增殖、并促进细胞凋亡,发挥抗肺纤维化作用。     

三维立体培养人胚肺成纤维母细胞前列腺素E2释放过程中蛋白酶激活受体的调节作用

背景:前列腺素E2是蛋白酶激活受体信号转导通路中关键递质之一,在人胚肺成纤维母细胞的收缩、增殖和趋化运动中发挥重要的调节作用.但成纤维细胞三维立体培养过程中,蛋白酶激活受体对前列腺素E2释放的调节作用尚不清楚.目的:观察在三维立体培养的人胚肺成纤维母细胞过程中,蛋白酶激活受体1和蛋白酶激活受体2对前列腺素E2释放的调节作用.方法:自大鼠尾腱提取Ⅰ型胶原.体外培养人胚肺成纤维母细胞,应用免疫印迹法测定蛋白酶激活受体1、蛋白酶激活受体2的表达.将人胚肺成纤维母细胞包裹在含培养基的Ⅰ型胶原中漂浮培养作为体外组织重构模型,即人胚肺成纤维母细胞三维立体培养,分别应用蛋白酶激活受体1及蛋白酶激活受体2受体激动剂凝血酶、胰蛋白酶、SLIGKV-NH2、TFLLR-NH2刺激人胚肺成纤维母细胞,ELISA法测定培养上清液中前列腺素E2的含量.结果与结论:蛋白酶激活受体1和蛋白酶激活受体2表达于三维立体培养的人胚肺成纤维母细胞.基础状态下三维立体培养的人胚肺成纤维母细胞可释放前列腺素E2;蛋白酶激活受体1非选择性受体激动剂凝血酶、蛋白酶激活受体2非选择性受体激动剂胰蛋白酶和蛋白酶激活受体1选择性受体激动剂SLIGKV-NH2均显著刺激前列腺素E2产生(P<0.01),而蛋白酶激活受体2选择性受体激动剂TFLLR-NH2呈剂量依赖地抑制前列腺素E2的释放(P<0.01).结果提示蛋白酶激活受体1和蛋白酶激活受体2参与调节三维立体培养的人胚肺成纤维母细胞前列腺素E2的释放.     

Ⅶ因子活化蛋白酶对肺纤维母细胞功能的影响及其临床意义

目的 探讨Ⅶ因子活化蛋白酶（FSAP）对肺纤维母细胞增殖和迁移的影响及其在人类急性肺损伤和肺纤维化过程中的可能作用.方法 体外培养人肺纤维母细胞,分别用CCK8法和Transwell小室法测定FSAP对人肺纤维母细胞增殖和迁移的影响;PCR法检测FSAP对PDGF-BB介导的人肺纤维母细胞胶原Ⅲ mRNA表达的影响.结果 FSAP能抑制人肺纤维母细胞的增殖、迁移.并能抑制PDGF-BB引起的人肺纤维母细胞胶原Ⅲ合成.结论 FSAP能抑制人肺纤维母细胞活化,提示FSAP可能与肺损伤时肺纤维化形成有关.     

苦参碱对AngⅡ诱导的大鼠心肌成纤维细胞的影响及其机制研究

目的：研究苦参碱对血管紧张素Ⅱ（AngⅡ）诱导大鼠心肌成纤维细胞增殖和胶原合成的影响,探讨其抑制纤维化的机制。方法以AngⅡ诱导大鼠心肌成纤维细胞为研究对象,0.25～1.0 mmol/L浓度的Mat作用24 h后,采用四氮唑盐（MTT）法检测Mat对心肌成纤维细胞增殖的影响;羟脯氨酸测定胶原合成量;ELISA 法测定血管紧张素Ⅱ1型受体（AT1-R）、血管紧张素Ⅱ2型受体（AT2-R）、肿瘤坏死因子α（TNF-α）、转化生长因子β1（TGF-β1）和结缔组织生长因子（CTGF）的含量变化。结果 Mat以浓度依赖性方式抑制AngⅡ诱导的心肌成纤维细胞增殖和胶原合成（P＜0.01）,并显著抑制AT1-R和CTGF的表达（P＜0.01）。结论 Mat能够显著降低AngⅡ诱导的心肌成纤维细胞中AT1-R和CTGF的含量,从而抑制细胞增殖和胶原合成增加,发挥出抗心肌纤维化的作用。     

辛伐他汀和胰岛素样生长因子-Ⅰ对人肺成纤维细胞增殖的影响

目的研究辛伐他汀和胰岛素样生长因子-Ⅰ（IGF-Ⅰ）共同对人肺成纤维细胞（HFL-1）增殖的影响。方法培养人肺成纤维细胞,采用MTT法检测在辛伐他汀和IGF-Ⅰ单独／共同作用下对人肺成纤维细胞增殖的影响。结果辛伐他汀和IGF-Ⅰ共同作用于人肺成纤维细胞时,IGF-Ⅰ的促细胞增殖作用受到抑制。结论辛伐他汀可抑制IGF-Ⅰ对人肺成纤维细胞的促增殖作用。     

纤维连接蛋白诱导人胚肺成纤维细胞增殖信号转导机制探讨

【目的】探讨纤维连接蛋白（FN）诱导人胚肺成纤维细胞（HFL1）增殖的细胞外信号调节激酶（ERK）信号转导通路。【方法】用四甲基偶氮唑蓝（MTT）法检测各组细胞增殖情况。采用不同浓度FN刺激HFL1并观察不同浓度下细胞ERK信号转导通路阻断剂PD98059对FN诱导HFL1增殖作用的影响;并用蛋白免疫印记法（Western Blot）观察FN浓度为50ng／mL时,PD98059对HFL1中磷酸化细胞外信号调节激酶（p-ERK）表达的影响。[结果]FN对HFL1诱导增殖作用呈剂量依赖性升高趋势,在50ng／mL时作用显著;ERK抑制剂PD98059可抑制FN诱导的HFL1细胞增殖。【结论】FN诱导HFL1的细胞增殖可以通过ERK信号转导途径实现。     

干扰素γ与甲强龙联合应用对人胚肺成纤维细胞增殖的影响

目的:目前间质性肺疾病的发病机制尚不完全清楚.实验拟进一步验证干扰素γ与甲强龙联合应用对人胚肺成纤维细胞增殖的影响,探究可否成为间质性肺疾病的有效治疗方法之一.方法:实验于2005-07/2006-05在河北医科大学细胞研究室完成.①实验分组:选用第8代人胚肺成纤维细胞进行体外培养.参考两药人体常用药物剂量范围,选取细胞单独用药浓度将实验分为对照组、甲强龙组(15 mg/L)、干扰素γ(250,500,750,1000.1250 U/mL)组及甲强龙与γ干扰素联合应用组.②实验方法:不同浓度的干扰素γ与甲强龙单独或联合应用于人胚肺成纤维细胞48 h.③实验评估:应用四甲基偶氮唑盐法观察人胚肺成纤维细胞的抑制率;应用流式细胞技术观察人胚肺成纤维细胞的细胞周期变化并计算增殖指数;应用免疫细胞化学方法检测增殖细胞核抗原的表达.结果:①甲强龙和干扰素γ对人胚肺成纤维细胞均有抑制作用(P＜0.05),两药联合应用具有相加乃至协同作用(Q＞0.85).②干扰素γ主要作用于G1和G2M期,甲强龙作用于S期,两药联合应用后细胞的增殖指数较两药单独应用降低(P＜0.05).③甲强龙与不同浓度干扰素γ单独或联合应用均具有抑制人胚肺成纤维细胞增殖细胞核抗原表达的作用(P＜0.05),两药联合应用抑制增殖细胞核抗原表达作用增强(P＜0.05).结论:甲强龙及干扰素γ均可抑制人胚肺成纤维细胞增殖,两药联合应用具有协同作用.干扰素γ与甲强龙联合应用的抗纤维化效果可能优于两药单独应用.     

Fas及FasL和Caspase-3在青蒿琥酯诱导人胚肺成纤维细胞凋亡中的表达

背景:青蒿琥酯具有减轻肺纤维化的作用,但相关机制的研究罕见报道.目的:探讨青蒿琥酯对人胚肺成纤维细胞凋亡的作用及其与Fas,FasL,Caspase-3表达的关系.方法:用1,10,100 mg/L青蒿琥酯分别干预体外培养的人胚肺成纤维细胞.采用CCK-8法检测青蒿琥酯对人胚肺成纤维细胞增殖的影响,流式细胞术测定细胞凋亡率,RT-PCR法测定Fas,FasL,Caspase-3 的mRNA的表达.结果与结论:青蒿琥酯呈浓度依赖性抑制人胚肺成纤维细胞增殖,细胞经青蒿琥酯作用后凋亡率明显增加(P < 0.05或P < 0.01),Fas,FasL,Caspase-3 mRNA的表达显著高于对照组(P < 0.05).结果证实,青蒿琥酯可通过上调Fas,FasL,Caspase-3 mRNA的表达抑制人胚肺成纤维细胞增殖、并促进细胞凋亡,发挥抗肺纤维化作用.     

矽肺纤维化发生发展过程中CXCL11功能的实验研究

目的构建CXC趋化因子11(CXCL11)基因小干扰RNA片段的表达质粒,进而探讨CXCL11在矽肺纤维化发生发展中的作用。方法采用RNA干扰技术,将CXCL11-siRNA表达质粒转染人胚肺成纤维细胞,筛选出最佳干扰质粒序列。实验分为4组:空白对照组(C),常规10%胎牛血清MEM培养基培养;刺激组(S),加入经SiO2粉尘刺激的肺泡巨噬细胞培养上清;转染组(T),将筛选的最佳干扰质粒转染FB后,再加入经SiO2粉尘刺激的肺泡巨噬细胞培养上清;阴性质粒转染组(N),将阴性质粒转染FB后,再加入经SiO2粉尘刺激的肺泡巨噬细胞培养上清。用Western blot法检测各组Ⅰ、Ⅲ型胶原蛋白的表达。结果经肺泡巨噬细胞上清刺激后,S组与C组比较,Ⅰ、Ⅲ型胶原表达量增加(P<0.05),T组与S组比较,Ⅰ、Ⅲ型胶原表达量减少(P<0.05)。结论抑制CXCL11表达可以下调SiO2引起的人胚肺成纤维细胞的Ⅰ型和Ⅲ型胶原表达,提示CXCL11表达与矽肺纤维化的发生发展有关。     

CpG oligodeoxynucleotides inhibit the proliferation and osteoclastic differentiation of RAW264.7 cells

Clinical prevention and treatment of periodontitis-induced bone absorption remains a challenge. The anti-infection role of CpG oligodeoxynucleotides (CpG ODNs) is well known; however, their effect on osteoclasts is still unclear. Here, we show that some CpG ODNs can regulate osteoclastogenesis of RAW264.7 cells. The phosphorothioate CpG ODN was efficiently taken up by the cells within 1 h and distributed in the cytoplasm. BW006, YW001, YW002, and FC004 CpG ODNs significantly repressed cell proliferation by targeting several cyclin proteins to arrest the cells in the G2 phase and to further initiate cell apoptosis. Regarding differentiation, we selected six CpG ODNs (FC002, BW006, YW002, YW001, FC004, and MT01) that markedly inhibited the gene expression levels of Nfatc, c-fos, RANK, and MMP9. TRAP staining showed that only YW002, YW001, and FC004 suppressed osteoclast generation and maturation. These three CpG ODNs dramatically declined the protein levels of osteoclastogenic proteins by elevating the ratio of OPG/RANKL and also downregulating the inflammatory factors (TNF-α, IL-1β, IL-6, and IL-17) at different stages. Thus, the selected CpG ODNs may be a potential molecular therapy for the prevention and treatment of periodontitis-mediated bone resorption.

Clinical prevention and treatment of periodontitis-induced bone absorption remains a challenge.     

CpG ODN增强树突状细胞对胃癌细胞增殖周期和凋亡影响的实验研究

目的探讨CpG ODN1826增强树突状细胞(DC)抗胃癌效应的作用。方法分离正常人外周血DC,用GM-CSF和IL-4培养,于第5天加入TNF-α并分成Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ、Ⅷ组继续培养。第6天各组均加入胃癌细胞冻融抗原50μl,Ⅱ、Ⅳ、Ⅵ、Ⅷ组再分别加入CpG ODN182610μg/ml,第10天ELISA检测IL-12和IFN-γ的水平,MTT法检测CTL对MKN45、MKN28、SGC7901和A549细胞的体外杀伤效应。流式细胞术检测CpG ODN1826增强DC对胃癌细胞增殖周期、凋亡的影响。结果体外培养第10天,加入CpG ODN1826后各组IL-12、IFN-γ的分泌量明显提高;CpG ODN1826协助DC对同种不同分化类型的胃癌细胞株MKN45、MKN28、SGC7901均有强烈的杀伤效应(P<0.01),杀伤活性显著高于A549(P<0.01)。体外应用CpG ODN1826对肿瘤细胞周期比例:G0/G1间期(MKN4568.35%、MKN2869.23%、SGC790169.80%),S期(MKN4539.45%、MKN2839.75%、SGC790139.55%),G2/M期(MKN456.50%、MKN286.30%、SGC79016.42%);与A549(G0/G1间期57.68%,S期25.13%,G2/M期18.46%)比较,差异有统计学意义(P<0.01);同时,不同肿瘤细胞株的凋亡率分别为MKN4575.20%、MKN2373.87%、SGC790172.43%、A54951.43%,表明CpG ODN1826能显著增强DC对胃癌细胞周期的抑制作用,促进胃癌细胞的早期凋亡(P<0.01)。结论 CpG ODN1826体外可显著增强DC诱导出高效而特异的抗胃癌效应,显著抑制胃癌细胞分裂增殖、促进凋亡,可作为胃癌免疫治疗的一种有效方法。     

Heterogeneity in the human response to immunostimulatory CpG oligodeoxynucleotides

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs trigger an immune response characterized by proliferation, B-cell activation, and cytokine production. CpG ODNs show promise in animal models as vaccine adjuvants and anti-allergens and for the treatment of infectious diseases and cancer. Several different CpG motifs capable of stimulating human cells have been identified, and several CpG-containing ODNs are now in various stages of clinical trials. However, little is known of the comparative magnitude or breadth of the immune responses these CpG ODNs elicit. This work compares the proliferative, IgM, and IL-6 response of PBMCs from 100 normal donors to a panel of 11 ODNs selected as highly active from over 100 phosphorothioate ODNs. While PBMCs from every donor responded to at least one member of the ODN panel, no single ODN was optimally stimulatory in all subjects or for all responses. A mixture of ODNs with complementary activities induced significantly broader stimulation of donor PBMCs than any single ODN, providing a rational basis for proceeding into clinical trials with such a mixture.     

Phosphorothioate-modified oligodeoxynucleotides inhibit human cytomegalovirus replication by blocking virus entry

Studies in animal models have provided evidence that Toll-like receptor 9 (TLR9) agonists, such as synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory deoxycytidyl-deoxyguanosine (CpG) motifs (CpG ODNs), protect against a wide range of viral pathogens. This antiviral activity has been suggested to be indirect and secondary to CpG-induced cytokines and inflammatory responses triggered through TLR9 activation. However, few studies have addressed the potential of CpG ODNs as direct antiviral agents. Here, we report on the ability of some CpG ODNs to directly suppress, almost completely, human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells. Murine CMV replication was inhibited as well, whereas no inhibition was observed for herpes simplex virus type 1, adenovirus, or vesicular stomatitis virus. The antiviral activity of these ODNs was significantly reduced when they were added after virus adsorption, indicating that their action may be primarily targeted to the very early phases of the HCMV cycle. In fact, the B-class prototype CpG ODN 2006 effectively prevented the nuclear localization of pp65 and input viral DNA, which suggests that it inhibits HCMV entry. Moreover, a CpG 2006 control, ODN 2137 without CpG motifs, also showed a potent inhibitory activity on the HCMV entry phase, indicating that the anticytomegaloviral activity is independent of the CpG motif. In contrast, a phosphodiester version of CpG 2006 showed reduced antiviral activity, indicating that the inhibitory activity is dependent on the phosphorothioate backbone of the ODN. These results suggest that this yet-unrecognized activity of CpG ODNs may be of interest in the development of novel anticytomegaloviral molecules.     

CpG oligonucleotide therapy cures subcutaneous and orthotopic tumors and evokes protective immunity in murine bladder cancer

Bacillus Calmette-Guerin (BCG) instillation is standard immunotherapy for superficial bladder carcinoma. However, many patients become refractory to BCG, giving impetus to the development of alternative therapies. CpG oligodeoxynucleotide (ODN) therapy has been shown to promote T(H)1-oriented antitumor responses in various tumor models. To investigate its therapeutic effect in bladder cancer, we used different CpG ODNs to treat C57BL/6 mice bearing the subcutaneous murine bladder tumor MB49. CpG type B ODN 1668 was superior at inhibiting tumor growth, leading to complete regression of large tumors. More importantly, CpG ODN 1668 also regressed orthotopically growing MB49 tumors for the first time. Rechallenge of CpG ODN-cured mice with MB49 showed that a majority of the mice were protected long term, demonstrating that CpG ODN therapy evokes a memory response. Adenoviral vectors (Ad) encoding CD40L, tumor necrosis factor-related activation-induced cytokine, lymphotactin, interleukin (IL) 2, and IL-15 were also investigated. AdCD40L and AdIL-15 transduction could abolish MB49 tumorigenicity, and these vectors were combined with CpG ODN 1668 to investigate any enhanced effects. No such effects were seen. All groups of mice treated with CpG ODNs, alone or in combination with adenoviral vector, exhibited increased serum concentrations of IL-12, indicative of a T(H)1 response. Our results show that CpG ODN therapy cures established subcutaneous and orthotopic bladder cancer via a T(H)1-mediated response and provides long-lasting protective immunity.     

CpGODN2216对白血病缓解期患者外周血单个核细胞的活化能力及活化的细胞对K562细胞的杀伤作用

本研究探讨CpGODN2216对白血病缓解期患者外周血单个核细胞（peripheral blood mononuclear cells,PBMNC）的活化能力以及CpGODN2216活化的PBMNC对白血病细胞K562的杀伤作用。取白血病缓解期患者外周血,以淋巴细胞分离液（Ficoll）分离PBMNC,用含10％胎牛血清的RPMI1640培养液调整PBMNC浓度为2．0×10^6／ml。对照组加入生理盐水（NS）,实验组加入CpGODN2216,培养1周,提取上清,用ELISA检测上清中IFN-γ、IL-12、IL-4、IL-10的水平。取白血病缓解期患者外周血,分离PBMNC,对照组加入生理盐水,实验组加入CpGODN2216,培养48小时,用流式细胞仪检测PBMNC中淋巴细胞的Th1／Th2,Tc1／Tc2细胞比例;流式细胞术检测活化的PBMNC对肿瘤细胞K562的杀伤能力。结果表明：实验组上清中IFN-γ、IL-12水平与对照纽相比有显著差异（P〈0．01）;IL-4、IL-10水平与对照组相比无明显差异（P〉0．05）。CpGODN2216能刺激白血病缓解期患者PBMNC向Th1／Tc1转化,与对照组相比有显著差异（P〈0．01）,但对Th2／Tc2无明显的刺激作用（P〉0．05）。CpGODN2216活化的PBMNC对K562细胞的杀伤能力明显高于对照组（P〈0．01）。结论：CpGODN2216可在体外诱导白血病缓解期患者PBMNC分泌Th1型细胞因子,诱导PBMNC向Th1方向转化,并强烈地活化CD8^＋T细胞应答。CpGODN2216活化的PBMNC对白血病细胞K562具有较强的杀伤作用.     

Immunotherapeutic utility of stimulatory and suppressive oligodeoxynucleotides

Bacterial DNA contains immunostimulatory CpG motifs that interact with toll-like receptor 9 on immune cells to stimulate the production of cytokines, chemokines and immunoglobulins. Synthetic oligodeoxynucleotides (ODNs) containing CpG motifs mimic the activity of bacterial DNA. Recently, several structurally distinct types of CpG ODN were identified that differentially activate human immune cells. These ODNs may be useful as vaccine adjuvants, anti-allergens and in the treatment of infectious diseases and cancer. Yet CpG-driven immune activation can have deleterious consequences, such as increasing the host's susceptibility to autoimmune disease. The immunomodulatory activity of CpG DNA can be blocked by DNA containing G-rich 'suppressive' motifs. The therapeutic potential of these immunostimulatory and immunosuppressive ODNs are discussed in this review.     

Selection of oligonucleotide aptamers with enhanced uptake and activation of human leukemia B cells

The clinical use of oligonucleotide (ODN) therapeutics has been hampered by their limited ability to penetrate intact cells. To identify ODN properties that would facilitate cellular uptake, we developed a repetitive selection procedure using an ODN library containing at least 10(14) different molecules and human B lymphoma cells as a target. Natural phosphodiester single-stranded DNA ODNs (R-aptamers) were obtained after 10 rounds of selection. A common feature in the R-aptamers was guanine-rich 3' terminal sequences, and many also contained potential immunostimulatory (ISS) CpG sequence motifs. Two R-aptamers (R10-60 and D-R15-8) with the predominant shared characteristics were selected for further study on primary human chronic lymphocytic leukemia (CLL) B cells, which are well known to be difficult to transfect and activate. Flow cytometry analysis of the CLL cells demonstrated that the fluorochrome-labeled R-aptamers were internalized much more efficiently than nonselected random sequence ODN. Studies on sequence modifications indicated that efficient uptake required ODN multimerization, that was promoted by guanine-rich sequences at the 3' terminus. In addition, CLL cells that were exposed to the aggregating R-aptamers containing CpG motifs were strongly activated, as indicated by upregulation of CD40 levels as compared to cells treated with nonaggregating CpG R-aptamers. Together, these findings suggest that the sequence compositions in R-aptamers that promote multimerization and contain optimal ISS CpG motifs facilitate the delivery of ISS-ODN to CLL cells and enhance the activation of these cells.     

Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.