Functional conservation of suppressors of cytokine signaling proteins between teleosts and mammals: Atlantic salmon SOCS1 binds to JAK/STAT family members and suppresses type I and II IFN signaling

Suppressor of cytokine signaling (SOCS) proteins are crucially involved in the control of inflammatory responses through their impact on various signaling pathways including the JAK/STAT pathway. Although all SOCS protein family members are identified in teleost fish, their functional properties in non-mammalian vertebrates have not been extensively studied. To gain further insight into SOCS functions in bony fish, we have identified and characterized the Atlantic salmon (Salmo salar) SOCS1, SOCS2 and CISH genes. These genes exhibited sequence conservation with their mammalian counterparts and they were ubiquitously expressed. SOCS1 in mammalian species has been recognized as a key negative regulator of interferon (IFN) signaling and recent data for the two model fish Tetraodon (Tetraodon nigroviridis) and zebrafish (Danio rerio) suggest that these functions are conserved from teleost to mammals. In agreement with this we here demonstrate a strong negative regulatory activity of salmon SOCS1 on type I and type II IFN signaling, while SOCS2a and b and CISH only moderately affected IFN responses. SOCS1 also inhibited IFNγ-induced nuclear localization of STAT1 and a direct interaction between SOCS1 and STAT1 and between SOCS1 and the Tyk2 kinase was found. Using SOCS1 mutants lacking either the KIR domain or the ESS, SH2 and SOCS box domains showed that all domains affected the ability of SOCS1 to inhibit IFN-mediated signaling. These results are the first to demonstrate that SOCS1 is a potent inhibitor of IFN-mediated JAK-STAT signaling in teleost fish.     

诱骗寡核苷酸下调MKN-45细胞OP18基因表达及对细胞增殖的影响

目的：探讨转录因子E2F哑铃形诱骗寡核苷酸（Decoy ODNs）对MKN-45细胞中opl8基因转录调控的影响及对细胞增殖的抑制作用．方法：设计合成针对opl8启动子上E2F的结合位点序列的哑铃形DecoyODNs，然后将合成的序列进行退火、连接，使其形成哑铃形的结构，再应用阳离子脂质体Lipofectamine^TM2000将哑铃形Decoy ODNs转染MKN-45细胞中．TRIzol法提取细胞总RNA，用RT-PCR方法检测细胞中opl8 mRNA表达水平的变化．通过MTT实验监测细胞的生长增殖状况并绘制生长曲线，最后用TUNEL法观察细胞的凋亡的情况．结果：哑铃形Decoy ODNs被成功转染MKN-45细胞，并成功提取了细胞总RNA．RT-PCR检测发现哑铃形Decoy ODNs转染后的MKN-45细胞中opl8 mRNA表达水平明显低于空白对照组，MTT实验所作的生长曲线显示转染细胞增殖速度与空白对照组相比较明显减慢，TUNEL凋亡染色可见凋亡细胞．结论：哑铃形Decoy ODNs能特异性抑制E2F转录因子对op18基因的转录调控，进而抑制opl8基因表达冠MKN45缃晌增碚．     

E2F陷阱DNA对人肺癌细胞中stathmin mRNA表达的影响及促进肿瘤细胞凋亡的作用

目的：观察转录因子E2F陷阱DNA对stathmin的表达影响及抑制肿瘤细胞增生情况.方法：采用脂质体LipofectamineTM2000将E2F Decoy ODNs转染入人肺癌A549细胞中,用RT-PCR检测转染细胞中stathmin基因mRNA表达水平变化,通过细胞生长曲线观察转染细胞的增殖能力,Tunel法观察转染细胞凋亡情况.结果：E2F Decoy ODNs被成功转染入肿瘤细胞中,RT-PCR检测结果显示实验组stathmin mRNA表达水平明显低于对照组,而且显著抑制了A549细胞的增殖,空白对照组无明显变化,Tunel凋亡染色可见凋亡细胞.结论：E2F Decoy ODNs能特异性下调stathmin mRNA表达,明显抑制A549细胞的增殖,为肿瘤的基因治疗提供了新的手段.     

Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protection against the murine model of leishmaniasis

First generation Leishmania vaccines consisting of whole killed parasites with or without adjuvants have reached phase 3 trial and failed to show enough efficacy mainly due to the lack of an appropriate adjuvant. In this study, the nuclease-resistant phosphorothioate CpG oligodeoxynucleotides (PS CpG) or nuclease-sensitive phosphodiester CpG ODNs (PO CpG) were used as adjuvants to enhance immunogenicity and rate of protection against leishmaniasis. Due to the susceptibility of PO CpG to nuclease degradation, an efficient liposomal delivery system was developed to protect them from degradation. 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid was used because of its unique adjuvanticity and electrostatic interaction with negatively charged CpG ODNs. To evaluate the role of liposomal formulation in protection rate and enhanced immune response, BALB/c mice were immunized subcutaneously with liposomal soluble Leishmania antigens (SLA) co-incorporated with PO CpG (Lip-SLA-PO CpG), Lip-SLA-PS CpG, SLA+PO CpG, SLA+PS CpG, SLA or buffer. As criteria for protection, footpad swelling at the site of challenge, parasite loads, the levels of IFN-γ and IL-4, and the IgG subtypes were evaluated. The groups of mice receiving Lip-SLA-PO CpG or Lip-SLA-PS CpG showed a high protection rate compared with the control groups. In addition, there was no significant difference in immune response generation between mice immunized with PS CpG and the group receiving PO CpG when incorporated into the liposomes. The results suggested that liposomal form of PO CpG might be used instead of PS CpG in future vaccine formulations as an efficient adjuvant.     

Design and synthesis of novel galactosylated polymers for liposomes as gene drug carriers targeting the hepatic asialoglycoprotein receptor

The 18-mer oligodeoxynucleotides (ODNs) that can inhibit survivin gene expression were selected as a model gene drug to study hepatic-targeting drug delivery system. Novel galactosylated polymers (cholesteryloxycarbonylamino) ethylamine-α,β-polyasparthydrazied (CHE-PAHy-Lacs), which target asialoglycoprotein receptor on hepatic parenchymal cells (PC), were designed and synthesized as non-toxic, non-antigenic and non-teratogenic ligands for liposomes. The liposomes incorporating different CHE-PAHy-Lacs were prepared and characterized by zeta potential and particle size analyzer. The drug encapsulation efficiency was measured by gel filtration method. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate was used as a marker for all the liposome preparations in the in vivo experiments. The CHE-PAHy-Lac liposomes produced a significant improvement in the encapsulation efficiency of ODNs (28.73–51.37%) compared with conventional liposomes (9.88%). The in vivo results showed that the liposomes incorporating CHE-PAHy-Lac, which contained about 30% (w/w) galactosyl residues, exhibited marked accumulation in the liver and hepatic PC. These results suggest that the novel galactosylated polymers used for liposomes have a great potential as a gene delivery system for hepatic targeting.     

Generation 4 polyamidoamine dendrimers is a novel candidate of nano-carrier for gene delivery agents in breast cancer treatment

Polyamidoamine dendrimer (PAMAM-D) is a new gene vector developed in recent years. In this study, we successfully prepared G4PAMAM and detected its unique structure by NMR, FITR and TEM. We revealed that G4PAMAM could bind to human erythrocytes and BSA through electrostatic interaction respectively, and caused haemolysis and reduced bioavailability. However, G4PAMAM-VEGF-ASODN (antisense oligodeoxynucleotides) complex could prevent G4PAMAM from binding to the erythrocytes and BSA and remained stable as a conjugate, therefore the toxicity of the complex was reduced. Meanwhile, we showed that G4PAMAM could be used as a gene vector to deliver AODNs into breast cancer MDA-MB-231 cells without significant cell toxicity, and it enhanced cellular uptake of ODNs. In vivo experiment of human breast tumor xenograft mice model, G4PAMAM also showed more efficiency of accumulating VEGF-ASODN to inhibit the tumor vascularization of breast tumor tissue than naked AODN. Furthermore, G4PAMAM could protect DNA in cytoplasm from digestion of restriction enzymes, which was important to become an effective tool in gene research and therapy.     

CpG寡聚脱氧核苷酸抑制人膀胱癌细胞在裸鼠体内的生长

目的:观察不同剂量的CpG ODN 1668对裸鼠皮下接种人膀胱移行细胞癌T24细胞生长的抑制作用,并与卡介苗的作用进行比较.方法:通过皮下接种T24细胞,建立裸鼠皮下接种人膀胱癌细胞的动物模型.将裸鼠随机分为5组,(1)对照组;(2)BCG组;(3)小剂量CpG ODN 1668组;(4)中等剂量CpG ODN 1668组;(5)大剂量CpG ODN 1668组.自肿瘤细胞接种后第1天起给药,共4次(第1、8、15、22天),在肿瘤周围多点注射相应药物(早期给药);同样方式另设5组动物,自肿瘤细胞接种后第7天起(第7、14、21、28天)于肿瘤周围多点注射相应药物(延迟给药).观察肿瘤体积和裸鼠的生存率.结果:早期给药时,在CpG ODN 1668各剂量组和BCG组,肿瘤生长均被抑制,裸鼠的生存期得到延长.中等剂量和大剂量CpG ODN 1668组对肿瘤细胞生长的抑制作用强于BCG组.延迟给药时,CpG ODN 1668各剂量组肿瘤体积和裸鼠的生存率,与对照组和BCG组的差异无显著统计学意义.结论:CpGODN 1668的早期应用,可抑制人膀胱癌细胞T24在裸鼠体内的生长,并表现为剂量依赖性;其抗肿瘤效应是非T细胞依赖的.当加大使用剂量时,CpG ODN 1668对膀胱癌细胞生长的抑制作用强于BCG.而当肿瘤负荷较大时,CpG ODN 1668和BCG的延迟使用对膀胱肿瘤细胞在裸鼠体内的生长无抑制作用.     

CpG oligodeoxynucleotides augment the murine immune response to the Yersinia pestis F1-V vaccine in bubonic and pneumonic models of plague

The current U.S. Department of Defense candidate plague vaccine is a fusion between two Yersinia pestis proteins: the F1 capsular protein, and the low calcium response (Lcr) V-protein. We hypothesized that an immunomodulator, such as CpG oligodeoxynucleotide (ODN)s, could augment the immune response to the plague F1-V vaccine in a mouse model for plague. CpG ODNs significantly augmented the antibody response and efficacy of a single dose of the plague vaccine in murine bubonic and pneumonic models of plague. In the latter study, we also found an overall significant augmentation the immune response to the individual subunits of the plague vaccine by CpG ODN 2006. In a long-term, prime-boost study, CpG ODN induced a significant early augmentation of the IgG response to the vaccine. The presence of CpG ODN induced a significant increase in the IgG2a subclass response to the vaccine up to 5 months after the boost. Our studies showed that CpG ODNs significantly augmented the IgG antibody response to the plague vaccine, which increased the probability of survival in murine models of plague (P < 0.0001).     

The role of glutamate transporter-1a in the induction of brain ischemic tolerance in rats

This study was undertaken to determine the role of glutamate transporter-1a (GLT-1a), one of the splice variants of glutamate transporter-1, in the induction of brain ischemic tolerance by cerebral ischemic preconditioning (CIP). We used a rat global cerebral ischemic model and assessed changes by neuropathological evaluation, Western blot analysis, immunohistochemistry, real-time PCR, in vivo brain microdialysis, and high performance liquid chromatography. We found that CIP induced a significant upregulation of GLT-1a expression in the CA1 hippocampus in a time course corresponding to that of neuroprotection of CIP against brain ischemia. Severe brain ischemia for 8 min induced delayed downregulation of GLT-1a, an obvious increase in glutamate concentration and delayed neuronal death of the pyramidal neurons in the CA1 hippocampus. When the animals were pretreated with CIP before the severe ischemia, the above changes normally induced by the severe ischemia were effectively prevented. Importantly, such a preventive effect of CIP on these changes was significantly inhibited by intracerebroventricular administration of GLT-1a antisense oligodeoxynucleotides, which have been proven to specifically inhibit the expression of GLT-1a protein and mRNA, and had no effect on the expression of GLT-1b. In addition, the concentration of aspartate was also elevated after severe brain ischemic insult. However, CIP had no effect on the elevated aspartate concentrations. These results indicate that GLT-1a participated in the brain ischemic tolerance induced by CIP in rats.     

New vistas on TLR9 activation

Together with other reports, evidence published in this issue of the European Journal of Immunology by Avalos and Ploegh (Eur. J. Immunol. 2011. 41: 2820-2827) implies that trafficking of TLR9 from the ER to endolysosomal compartments, which is aided by the transmembrane UNC93B1 ER protein, is followed by proteolytic cleavage of the TLR9 ectodomain (TLR9ecto). Furthermore, Avalos and Ploegh elegantly show that RAW 264.7 macrophages stably expressing tagged TLR9 display significant amounts of cleaved TLR9 already when at rest. It is of note that inhibitory oligonucleotides (IN-ODNs) do not affect TLR9 cleavage but competitively prevent CpG-ligand binding to the C-terminal TLR9 fragment. Compared with phosphorothioated (PS) CpG-oligodeoxynucleotides (ODNs), natural phosphorodiester (PD) CpG-ODNs differ in their TLR9 activation efficiency. In this Commentary, a model is proposed that accounts for the differences in PS- and PD-ODNs with respect to TLR binding and activation.