Promoter region methylation does not account for the frequent loss of expression of the Fas gene in colorectal carcinoma

Expression of the apoptosis-promoting Fas gene is frequently reduced or lost during the development of colorectal carcinoma. However, loss of heterozygosity at the Fas locus or Fas gene rearrangements do not account for the loss of expression of Fas, raising the possibility that methylation of the Fas promoter may inhibit gene expression in colorectal carcinomas. We have examined the Fas promoter region CpG island for evidence of hypermethylation in colorectal tumours. Forty-seven specimens of colorectal adenoma and carcinoma, as well as six samples of normal colonic mucosa, were examined by Southern blotting for methylation at HpaII and Cfol sites in this region. No methylation was detected in any of the specimens, suggesting that hypermethylation is not primarily responsible for the loss of expression of the Fas gene during colorectal tumorigenesis.     

Impact of Site-Specific Nucleobase Deletions on the Arthritogenicity of DNA

Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN) potently stimulate the innate and acquired immune system. We have compared the in vivo and in vitro inflammatogenic properties of CpG ODNs containing a specific nucleobase deletion either 5'-upstream (ODN-2) or 3'-downstream (ODN-3) of the CpG motif, comparing with a prototype CpG ODN (ODN-1). The frequency of arthritis was similar after intra-articular (i.a.) injections of ODN-1 or ODN-3, but was significantly lower (p < 0.02) after i.a. injections of ODN-2. In vitro production of the pro-inflammatory cytokine TNF-alpha was higher in mouse spleen cell cultures exposed to ODN-2 in comparison to ODN-1. In addition, the level of IL-10 induced by ODN-2 was higher than that induced by ODN-1. On the other hand, TNF-alpha, IL-10, and MCP-1 levels, as well as splenocyte proliferative responses were all significantly lower for ODN-3 than for ODN-1. These results suggest that a 5'-upstream nucleobase deletion reduces arthritogenicity, while maintaining or increasing the production of pro- and anti-inflammatory factors. In contrast, a 3'-downstream nucleobase deletion has no effect on arthritogenicity, despite significantly lower levels of proliferation and pro- and anti-inflammatory cytokines, compared with ODN-1. This study indicates that specific structural elements within the ODN sequence but outside the CpG motif, modulate the immunostimulatory properties of CpG ODNs.     

Immunoregulatory activity of CpG oligonucleotides in humans and nonhuman primates

Oligodeoxynucleotides (ODN) containing CpG motifs mimic the ability of microbial DNA to activate the innate immune system. The resultant response limits the early spread of infectious organisms while promoting the development of adaptive immunity. CpG ODN show promise as vaccine adjuvants and in the treatment of asthma, allergy, infection, and cancer. Due to evolutionary divergence in CpG recognition between species, CpG ODN that are most active in rodents are poorly immunostimulatory in primates. Thus, evidence that CpG ODN have therapeutic activity in mice must be confirmed in primates. Two distinct types of CpG ODN were identified that stimulate primate PBMC. D-type ODN trigger plasmacytoid DC to secrete IFNalpha, monocytes to mature into functionally active DC, and NK cells to secrete IFNgamma. K-type ODN stimulate B cells and monocytes to proliferate and secrete IgM, IL-10, and/or IL-6. In vivo studies in nonhuman primates indicate that proinflammatory or humoral immune responses can be selectively facilitated by judicious use of these distinct types of ODN.     

CpG island methylation status and mutation analysis of the RB1 gene essential promoter region and protein-binding pocket domain in nervous system tumours

A series of 136 nervous system tumours were studied to determine the methylation status of the CpG island contained within the promoter region of the RB1 gene, as well as mutation analysis of the essential promoter region and exons 20-24 (and surrounding intronic regions) coding for the protein-binding pocket domain. Methylation of the RB1 CpG island was detected in 26 samples corresponding to nine glioblastomas, three anaplastic astrocytomas, one mixed oligo-astrocytoma, one ependymoma, two medulloblastomas, two primary central nervous system lymphomas, two neurofibrosarcomas, and six brain metastasis from solid tumours. No inactivating mutations were found within the RB1 promoter region, whereas one glioblastoma and one oligodendroglioma displayed similar sequence variations consisting of 12 and 8 base pair deletions at intron 21. These results suggest that RB1 CpG island hypermethylation is a common epigenetic event that is associated with the development of malignant nervous system tumours.     

Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients.

Hypermethylation of exon 1 of p16INK4a was examined in tumour and plasma DNA of a series of breast cancer patients. De novo methylation was observed in the tumours of eight patients (23%), and in plasma DNA in five (14%) of these eight patients. Our data show that de novo methylation of exon 1 of p16INK4a can be demonstrated in plasma DNA of breast cancer patients, a fact that provides additional evidence of the tumour-related origin of free plasma DNA in cancer patients.     

TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner

Purpose

Invariant natural killer T (iNKT) cells may play an important role in the pathogenesis of asthma in mice and humans. Thus, an agent that modulates the function of iNKT cells may have therapeutic potential to control asthma. We hypothesized that lipopolysaccharide (LPS)-, flagellin-, or CpG-induced changes in the cytokine milieu may modify and even inhibit the function of airway iNKT cells in asthma.

Methods

Because increased α-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR) reflects the presence of airway iNKT cells, α-GalCer-induced AHR, as well as inflammatory cells and cytokines in bronchoalveolar lavage (BAL) fluid, were determined 24 hours after in vivo treatment with LPS, flagellin, or CpG in naïve BALB/c mice. Intracellular IL-4 and IFN-γ were measured in spleen iNKT cells after in vitro treatment with LPS, flagellin, or CpG. A role for IL-12 following the treatments was determined.

Results

Intranasal administration of LPS, flagellin, or CpG reduced development of α-GalCer-induced AHR, eosinophilic airway inflammation, and Th1 and Th2 cytokine responses in BAL fluid, while producing IL-12 in BAL fluid. Intraperitoneal administration of IL-12 mAb blocked the suppressive effect of LPS, flagellin, or CpG. In vitro treatment with LPS, flagellin, or CpG reduced production of IL-4 and IFN-γ from α-GalCer-stimulated spleen iNKT cells; these effects were ameliorated by addition of anti-IL-12 mAb.

Conclusions

TLR4, 5, and 9 agonists may suppress the function of airway and spleen iNKT cells via IL-12-dependent mechanisms. Anergy of iNKT cells by IL-12 might play a role in suppression by these TLR agonists.     

不同剂量CpG ODN联合STAg鼻内免疫BALB／c小鼠诱导的免疫应答

目的 观察弓形虫可溶性速殖子抗原（STAg）与不同剂量的CpG ODN联合滴鼻免疫小鼠的免疫效果，探索CpG ODN佐剂最佳剂量。方法 5-6周龄BALB／c小鼠50只随机分为5组，每组10只，实验组以20μg STAg联合不同剂量CpG ODN佐剂滴鼻免疫小鼠，佐剂剂量分别为0、1、5、10μg，对照组以PBS滴鼻，间隔2wk免疫2次。末次免疫后30d断颈处死全部小鼠，分离脾、Peyer＇s patch（PP结）、肠系膜淋巴结（MLN）和IEL淋巴细胞，观察淋巴细胞增殖情况。用ELISA的方法检测血清IgG和粪便IgA含量。结果 与PBS组和0剂量佐剂组相比，10μg CpG ODN联合STAg免疫后，小鼠肠粘膜淋巴组织和脾淋巴细胞增殖明显（P〈0．05）。10μg组血清IgG远高于对照组（P〈0．05），粪便IgA高于对照组（P〉0．05）。结论 CpG ODN可作为STAg的粘膜佐剂，10μg CpG ODN与STAg联合具有最佳的免疫效果。     

MGMT、hMLH1和hMSH2基因启动子甲基化状态对脑胶质瘤预后的影响

目的探讨脑胶质瘤DNA修复基因O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）和错配修复基因（MMR）（hMLH1、hMSH2）启动子甲基化状态及其对患者预后和烷化剂化疗敏感性的影响。方法采用甲基化特异性PCR（MSP）方法检测39例脑胶质瘤和6例正常脑组织MGMT、hMLH1和hMSH2基因启动子区的甲基化状态,免疫组化方法测定其蛋白表达。绘制Kaplan-merier生存曲线。结果脑胶质瘤组织MGMT、hMLH1和hMSH2基因启动子区甲基化发生率分别为46.2%、10.3%和20.5%,而正常脑组织相应基因启动子区未发生甲基化;三种基因启动子未甲基化模式与其对应蛋白表达模式相似。MGMT基因甲基化的脑胶质瘤患者存活率显著高于未甲基化者（P〈0.05）;MMR基因甲基化患者中MGMT基因甲基化与未甲基化者的生存期无统计学差异（P〉0.05）。结论hMLH1、hMSH2及MGMT甲基化是脑胶质瘤发生过程中常见的分子事件;联合检测MGMT、hMLH1和hM-SH2基因启动子甲基化状态可判断脑胶质瘤患者的预后及其对烷化剂化疗的敏感性。