Highly efficient redirected anti-tumor activity of human lymphocytes transduced with a completely human chimeric immune receptor

BACKGROUND: Novel antibody-based immunotherapeutic strategies exploit chimeric immune receptors (CIR), expressed on the surface of transduced human peripheral blood mononuclear cells (PBMC), to redirect potent non-MHC-dependent cytotoxicity to tumor cells expressing a tumor-associated antigen. However, clinical application of the strategy has been hampered by the potential side effects associated with immunogenicity and by low transduction efficiency. METHODS: A fully human CIR was constructed that triggers immune activation through the zeta chain of CD3 and contains a human single-chain antibody fragment specific for an extracellular epitope of HER2. PBMC were transduced with the CIR using gibbon-ape leukemia virus envelope pseudotyped retroviruses. In vitro cytotoxicity and inhibition assays were carried out using normal and tumor cell lines expressing different levels of HER2. RESULTS: Bulk populations of CIR-transduced PBMC could express high levels of the construct and subcloning ensured stable expression. CIR-mediated killing and growth inhibition of targets expressing high HER2 levels were very efficient at low effector-to-target ratios. Under the same experimental conditions, CIR-mediated activity against normal cells expressing low HER2 levels was marginal. The CIR-mediated recognition of target cells induced the release of soluble factors able to inhibit growth of both HER-positive and HER2-negative bystander tumor cells. CONCLUSIONS: Human CIR-transduced PBMC exert a potent and dose-dependent anti-tumor activity. Target antigen level appeared to be a critical determinant of specificity and delivery of signals leading to redirected effector functions. Soluble factors, released by redirected effectors at the site of antigen-driven activation, mediate potent bystander killing.     

细菌感染对家蚕DNA甲基化与组蛋白乙酰化相关基因表达的影响

【目的】探讨DNA甲基化及组蛋白乙酰化是否参与家蚕 Bombyx mori 免疫反应的调控。【方法】对家蚕与其他生物的DNA甲基转移酶 (DNMT)、组蛋白去乙酰化酶（HDAC）与组蛋白乙酰转移酶（HAT）的蛋白序列进行系统进化分析;利用定量PCR检测家蚕5龄第3天幼虫感染病原菌绿脓杆菌 Pseudomonas aeruginosa 和金黄色葡萄球菌 Staphylococcus aureus 后 BmDNMT 1, BmHDACI-1, BmHDACI-2和 BmHAT 1在家蚕脂肪体组织中的表达变化;给家蚕5龄第2天幼虫注射DNMT, HDAC和HAT抑制剂,观察它们对家蚕感染细菌后的存活率的影响。【结果】系统进化分析显示,BmDNMT1在进化上呈现特殊性,独立于其他昆虫DNMT1的进化,而BmHDACs和BmHAT在进化上相对保守。定量PCR检测表明,在两种细菌感染后,BmDNMT1, BmHDACs 和 BmHAT1 在家蚕幼虫脂肪体中的表达水平均有不同程度的上升。然而,DNMT, HDAC和HAT抑制剂对家蚕幼虫感染细菌后的存活率并无明显影响。【结论】本研究发现感染绿脓杆菌和金黄色葡萄球菌后,家蚕幼虫脂肪体中 BmDNMT1, BmHDACs 和 BmHAT1 的表达水平有不同程度的上调,推测DNA甲基化和组蛋白乙酰化/去乙酰化可能参与家蚕免疫反应的调控。     

Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX⁺ foci), enhanced senescence-associated β-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the 'distended' morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.     

Improved cellular immune response elicited by a ubiquitin-fused ESAT-6 DNA vaccine against Mycobacterium tuberculosis

The present study evaluated the immune response elicited by a ubiquitin-fused ESAT-6 DNA vaccine against Mycobacterium tuberculosis. BALB/c mice were vaccinated with plasmid DNA encoding ESAT-6 protein, ubiquitin-fused ESAT-6 DNA vaccine (UbGR-ESAT-6), pcDNA3-ubiquitin and blank vector, respectively. ESAT-6 DNA vaccine immunization induced a Thl-polarized immune response. The production of Thl-type cytokine (IFN-γ) and proliferative T-cell responses was enhanced significantly in mice immunized with UbGR-ESAT-6 fusion DNA vaccine, compared to non-fusion DNA vaccine. This fusion DNA vaccine also resulted in an increased relative ratio of IgG_2a to IgG_l and the cytotoxicity of T cells. Thus, the present study demonstrated that the UbGR-ESAT-6 fusion DNA vaccine inoculation improved antigen-specific cellular immune responses, which is helpful for protection against tuberculosis infection.     

Adjuvant effect of CIA07, a combination of Escherichia coli DNA fragments and modified lipopolysaccharides, on the immune response to hepatitis B virus surface antigen

CIA07 is an immunostimulatory agent composed of bacterial DNA fragments and modified lipopolysaccharide, which has antitumor activity against bladder cancer in mice. In this study, the adjuvant activity of CIA07 was evaluated using hepatitis B virus surface antigen (HBsAg) as the immunogen. Mice were immunized intramuscularly three times at 1-week intervals with HBsAg alone or in combination with alum, bacterial DNA fragments, modified lipopolysaccharide, CIA07 or CpG1826, and immune responses were assessed. At 1 week after the final injection, the HBsAg-specific total serum IgG antibody titer in CIA07-treated mice was 14 times higher than that in animals administered antigen alone, six times higher than in mice given alum or bacterial DNA fragments and twice as high as those treated with modified lipopolysaccharide or CpG1826, and remained maximal until 8 weeks postimmunization. Animals receiving antigen alone or plus alum displayed barely detectable HBsAg-specific serum IgG2a antibody responses. However, coadministration of CIA07 with antigen led to markedly enhanced serum IgG2a antibody titer and IFN-gamma(+) production in splenocytes, indicating that CIA07 effectively induces Th1-type immune responses. In addition, the number of HBsAg-specific CD8(+) T cells in peripheral blood mononuclear cells was elevated in CIA07-treated mice. These data clearly demonstrate that CIA07 is able to induce both cellular and humoral immune responses to HBsAg, and confirm its potential as an adjuvant in therapeutic vaccines for hepatitis B virus infections.     

Steelhead trout Oncorhynchus mykiss metabolic rate is affected by dietary Aloe vera inclusion but not by mounting an immune response against formalin‐killed Aeromonas salmonicida

The oxygen consumption of two groups of 10° C acclimated steelhead trout Oncorhynchus mykiss was measured for 72 h after they were given a 100 µl kg^?1 intraperitoneal injection of formalin‐killed Aeromonas salmonicida (ASAL) or phosphate‐buffered saline (PBS). In addition, plasma cortisol levels were measured in fish from both groups prior to, and 1 and 3 h after, they were given a 30 s net stress. The first group was fed an unaltered commercial diet for 4 weeks, whereas the second group was fed the same diet but with 0·5% (5 g kg^?1) Aloe vera powder added; A. vera has potential as an immunostimulant for use in aquaculture, but its effects on basal and acute phase response (APR)‐related metabolic expenditures and stress physiology, are unknown. Injection of ASAL v. PBS had no measurable effect on the of O. mykiss indicating that the APR in this species is not associated with any net increase in energy expenditure. In contrast, incorporating 0·5% A. vera powder into the feed decreased routine metabolic rate by c. 8% in both injection groups and standard metabolic rate in the ASAL‐injected group (by c. 4 mg O₂ kg^?1 h^?1; 5%). Aloe vera fed fish had resting cortisol levels that were approximately half of those in fish on the commercial diet (c. 2·5 v. 5·0 ng ml^?1), but neither this difference nor those post‐stress reached statistical significance (P > 0·05).     

果蝇免疫研究进展及其感染分枝杆菌的免疫特征

耐药性、持续感染以及与HIV病毒的共感染等诸多因素导致一度得到控制的结核病死灰复燃, 有效控制日益严峻的结核病迫切需要深入认识其致病菌——结核分枝杆菌Mycobacterium tuberculosis的基础生物学特性, 以及宿主相应的免疫控制机理。目前尚无一个动物模型能够同时回答这些关键问题, 而利用多种动物模型有望从不同角度回答上述问题, 普遍认为果蝇Drosophila 是比较理想的研究结核病天然免疫的简易模式动物之一。本文综述了果蝇免疫研究的最新进展, 包括免疫途径及其新成员与负调控子, 重点总结了用海分枝菌杆菌M. marinum、偶发分枝杆菌M. fortuitum和耻垢分枝杆菌M. smegmatis等分枝杆菌感染果蝇的新发现, 其中包括感染期间不诱导抗菌肽表达, 多个宿主因子(如CD36家族成员和ESCRT)参与了应答, 鉴定出具有杀灭分支杆菌作用的β-己糖酰胺酶, 感染期间能量代谢相关基因差异表达等。这些工作为利用果蝇模型快速筛选治疗结核病的新药物靶标和药物先导物提供了思路。     

质粒拯救法克隆细菌基因组大片段

[目的]细菌基因组大片段尤其是基因簇的克隆与操作,是细菌基因功能分析的一个难点.基因组测序工作的不断完成和序列信息的大量积累,为细菌基因组:DNA的操作提供了方便.本文报道了利用细菌的全基因组信息和质粒拯救法的原理建立的一种克隆细菌基因组大片段的方法.[方法]首先,根据基因组序列信息,在待克隆片段的一侧扩增一段DNA,并将其克隆到自杀载体上构建打靶质粒,然后,将打靶质粒整合到细菌的基因组中构建重组菌,提取重组菌的基因组DNA,酶切,自连,转化,将自杀质粒与待克隆的目的片段一起拯救出来.最后,根据需要将拯救的DNA片段亚克隆到新的载体中.[结果]我们利用该方法克隆了布鲁氏菌中长度为11kb的virB操纵子,并构建了互补质粒.将该质粒导入到virB的突变株中后使virB操纵子的转录活性得到了恢复,表明该策略切实可行.[结论]这种重组克隆策略给我们提供了一种新的对细菌基因组大片段进行操作的方法.     

病毒感染与宿主细胞DNA损伤应答

病毒感染宿主细胞后,利用细胞内的营养物质和原料进行复制和增殖,同时能引起宿主细胞启动抗病毒免疫应答的防御机制。此外,近年来的研究还表明病毒感染能够引起宿主细胞的DNA损伤应答,该反应是细胞另一种防止病毒入侵的自我保护机制。同时发现,病毒在长期进化过程中形成了不同的机制来对抗宿主细胞的DNA损伤应答,从而消除细胞对其复制和繁殖产生的不利影响。因此,研究和阐述病毒感染后引起宿主细胞DNA损伤应答途径的机制,可使我们采取相应对策选择新的抗病毒靶点,从而有利于新型抗病毒药物的开发。     

阿尔茨海默病核酸疫苗的构建及诱导体液免疫反应的初步研究

近年,内源性蛋白主动免疫预防和治疗阿尔茨海默病(AD)的策略成为AD研究领域的热点,并获得迅猛发展。构建了以β-片层结构的淀粉样蛋白(AB)及其可溶性与病理性突变体为抗原基因的DNA疫苗用于免疫小鼠,经过对免疫方案的探索和调整,结果能够打破其自身耐受,在外周血中诱发出较高滴度的抗-AB抗体。初步探讨了诱导体液免疫学效应的规律。并在原代培养的海马神经元AB毒性模型中验证了免疫后血清的生物学活性。     

Activation of immune receptor Rx1 triggers distinct immune responses culminating in cell death after 4 hours

Intracellular nucleotide-binding leucine-rich repeat (NLR)-type immune receptors are a fundamental part of plant immune systems. As infection occurs at foci, activation of immune responses is typically non-uniform and non-synchronized, hampering the systematic dissection of their cellular effects and determining their phasing. We investigated the potato NLR Rx1 using the CESSNA (Controlled Expression of effectors for Synchronized and Systemic NLR Activation) platform. CESSNA-mediated Potato virus X coat protein (CP) expression allowed the monitoring of Rx1-mediated immune responses in a quantitative and reproducible manner. Rx1 was found to trigger a reactive oxygen species (ROS) burst and ion leakage within 1 h and a change in autofluorescence within 2 h after the induction of CP production. After 2 h, HIN1 expression was increased and single-stranded DNA (ssDNA) damage and loss of cellular integrity became apparent, followed by double-stranded DNA (dsDNA) damage after 3 h and increased PR-1a, LOX, ERF1 and AOX1B expression and cell death at 4 h. Nuclear exclusion of Rx1 resulted in increased basal levels of ROS and permitted Rx1 activation by an Rx1-breaking CP variant. In contrast, nuclear-targeted Rx1 showed diminished basal ROS levels, and only avirulent CP could trigger a compromised ROS production. Both nuclear-excluded and nuclear-targeted Rx1 triggered a delayed ion leakage compared with non-modified Rx1, suggesting that ion leakage and ROS production originate from distinct signalling pathways. This work offers novel insights into the influence of Rx1 localization on its activity, and the interplay between Rx1-triggered processes.     

Bacterial DNA containing methylated CpG motifs retains immunostimulatory activity in synergy with modified lipopolysaccharides

We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF-kappaB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs.     

磁小体对小鼠免疫应答的影响

【目的】研究细菌磁小体对小鼠免疫应答的影响,为磁小体的体内应用提供参考依据。【方法】以卵清白蛋白(OVA)作为抗原分别与弗氏完全佐剂(阳性对照)、磁小体(BMP)悬液、PBS缓冲液(阴性对照)混合免疫小鼠。两周后检测血清中抗OVA特异性抗体及亚型、细胞因子表达水平以及淋巴细胞增殖能力。【结果】与阴性对照(PBS组)相比,BMP组在IL-2、IFN-γ和IL-4、IL-10的表达水平、淋巴细胞增殖能力、特异性抗体的产生没有明显差异。【结论】磁小体对小鼠免疫应答无显著的影响。     

IL-12增强HBV融合抗原DNA疫苗在小鼠诱导的细胞免疫应答

乙型肝炎病毒(hepatitis B virus,HBV)极易形成慢性感染,主要机制在于感染者不能产生强有力的细胞免疫应答以清除病毒[1].慢性HBV感染者体内虽然存在HBV抗原特异性T淋巴细胞,但对HBV抗原的反应性较低.研究发现,增强这类T淋巴细胞的反应性,可以促进HBV的清除[2].     

土壤细菌DNA提取及多样性分析的T-RFLP方法

获得高质量的土壤总DNA是土壤细菌生态学的关键步骤之一.实验通过综合应用两个试剂盒(Soilmaster kit和DNA IQTM系统)的优点进行土壤样品总DNA的提取,结果证明该方法是一种快速、有效、灵敏、稳定的土壤DNA提取方法.另外尝试将16S rDNA序列和T-RFLP(Terminal restriction fragment 1ength polymorphism)技术引入土壤细菌DNA群落多样性的研究中,证明T-RFLP是一种有力的土壤细菌多样性分析工具.     

Chemokines and chemokine receptors: Insights from human disease and experimental models of helminthiasis

Infection with helminth parasites affects more than 1.5 billion people and is concentrated in global areas of extreme poverty, having a significant impact on public health, social life and the economy. Upon entry into the host, helminth parasites often migrate through specific tissues triggering host immunity. The immune response triggered by helminth infections is complex and depends on parasite load, site of infection, acuteness/chronicity of the infection and is species-dependent. In general, susceptibility or resistance to the infection involves the participation of the innate immune response and then the balance between several effector CD4⁺ T cells subsets, such as Th1, Th2, Th9, Th17, Tfh and Treg, coordinated by immune mediators such as cytokines and chemokines. Chemokines guide the recruitment and activation of leukocytes under inflammatory and homeostatic states. The chemokine system has been associated with several diseases and experimental models with a significant inflammatory component, including infection with helminth parasites. Therefore, this critical review will highlight the main findings concerning chemokine responses elicited by the interaction between helminth parasites and the hosts’ immune system, hence contributing to the understanding of the relevance of chemokine synthesis and biology in the immunological response to infection by parasitic helminths.     

Detection of mycoplasmas infecting cell cultures by DNA hybridization

Summary Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of theEco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes ofMycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants,Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, andAcholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10⁵ mycoplasmas. The study was supported by the U.S. Public Health Service Grant GM25286 awarded to G. G. and by a grant from the United States-Israel Binational Agricultural Research Development Fund (BARD) awarded to S. R.