CpG寡核苷酸联合热固化瘤苗对肝癌细胞Hca-F荷瘤小鼠的免疫治疗研究

目的探讨CpG寡核苷酸(ODN)联合热固化瘤苗的抗肿瘤作用。方法剥取肿瘤．称重并计算抑瘤率;MTT法测免疫小鼠CTL、Mψ和CTL细胞的体外杀伤活性;ELISA法测免疫小鼠血清中IL-10、IL-12水平。结果联合使用CpG ODN和热固化瘤苗可以诱导荷瘤小鼠的抑瘤作用,其抑瘤率与单独使用瘤苗或CpG ODN比较差异有显著性;联合使用CpG ODN和热固化瘤苗可以提高荷瘤小鼠NK、Mψ和CTL细胞的体外杀伤活性,与单独使用瘤苗组相比较,差异具有显著性,但与单独使用CpG ODN组相比较,NK、Mψ细胞的体外杀伤活性差异无显著性;联合使用CpG ODN和热固化瘤苗可以提高荷瘤小鼠血清中IL-12水平并降低血清中IL-10水平,与二者单独使用组比较差异都具有显著性。结论联合使用CpG ODN和热固化瘤苗能显著提高机体的免疫功能,尤其是特异性细胞免疫功能。     

CpG 寡核苷酸的安全性实验研究

目的研究CpG 寡核苷酸(CpG ODN)作为免疫佐剂应用可行性.方法按要求配制CpG ODN检测内毒素,采用昆明小鼠和豚鼠进行异常毒性试验和超敏试验;分别以10 μg /只、100 μg /只、300 μg /只3个剂量组CpG ODN进行小鼠骨髓嗜多染红细胞微核试验;以10 μg /只、30 μg/只、90 μg/只3个剂量组的CpG ODN分别交替于双侧后肢胫前肌肌肉注射Balb/c小鼠,每周2次,连续4周,进行血液学、血生化、病理组织学检查及抗核抗体、抗双链DNA抗体和肿瘤坏死因子检测.结果内毒素检测阴性,实验期内所有动物均未出现异常毒性反应和超敏反应,实验结束后所有动物全部健存且体重增加. CpG ODN小鼠骨髓嗜多染红细胞微核试验为阴性结果;实验期间各组动物未见异常表现,体重增加各组间差异无显著性,血液学、血生化和病理组织学检查未见明显异常.未检测到ANA、ds-DNA.各剂量组肿瘤坏死因子含量在与对照组之间比较差异均有显著性.结论所选用CpG ODN作为生物制品使用是安全的;在受试剂量下微核试验阴性;在小鼠亚急性毒性实验中未引起毒性损害,安全性良好.     

Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.     

Complex roles of CpG in liposomal delivery of DNA and oligonucleotides

Unmethylated CpG in bacterial DNA has recently been recognized as a danger signal to the mammalian immune system. This CpG signal can be greatly amplified when DNA is delivered via a lipidic vector. The CpG effects are affected by the administration route, and can be either beneficial or harmful. In this review, we will summarize our current understanding about the mechanism of action of the immunostimulatory motifs. Emphasis will be placed on the discussion of the complicated roles of CpG when CpG DNA or oligonucleotides are administered in vivo using liposomes as a delivery vehicle.     

Synthesis and anti-tumor activity study of water-soluble PEG-celastrol coupling derivatives as self-assembled nanoparticles

To improve the drug-ability of celastrol, a series of PEGylation celastrol (PEGC) were designed and synthesized by conjugation with different kinds of polyethylene glycols (PEGs) with celastrol. Most of PEGCs could easily dissolve in water. In particular, one of them (DC1000) could be dispersed in water to form nanoparticles by self-assembly. The cytotoxic evaluation of PEGCs revealed that some of PEGCs showed more potent cytotoxicity than celastrol, and the molecular weight of PEG parts in PEGCs had apparent influence on their cytotoxic activity. Anti-tumor evaluation in vivo showed DC1000 had higher tumor inhibition rate and better safety than celastrol by intravenous administration with equivalent molar weight. These results revealed PEGylation might be an efficient and economical method to improve the water solubility and safety of celastrol and similar natural products.     

Purification,characterization and immunostimulatory activity of polysaccharide from Cipangopaludina chinensis

In this study, we investigated the purification, preliminary characterization and immunostimulatory activity in vivo of polysaccharide from Cipangopaludina chinensis (CCPS). Firstly, crude CCPS was prepared by hot water extraction. And the crude CCPS was sequentially purified by chromatography of DEAE-52 and Sephadex G-100, resulting in two purified fractions of CCPS-1 and CCPS-2. We found the two fractions were homogeneous heteropolysaccharides mainly composed of rhamnose and glucose with the average molecular weight of 226 and 235 kDa, respectively. CCPS-2 was quite different from CCPS-1. It had much higher content of uronic acid and sulfuric radical. For immunostimulatory activity in vivo, crude CCPS could significantly increase the thymus and spleen indices, enhance the macrophage function, and increase the level of serum hemolysin in cyclophosphamide-treated mice, suggesting CCPS had a potent immunostimulatory activity and could be explored as a potential natural immunomodulatory agent     

Structure,molecular modification and anti-tumor activity of melanin from Lachnum singerianum

Intracellular melanin (LIM) was extracted from Lachnum singerianum YM296 mycelium. LIM-a, LIM-b and LIM-c were resolved from LIM by Sephadex G-15 column chromatography, in which LIM-a was the main homogeneous component with a molecular weight of 530 Da. Based on the elemental analysis, mass spectrometry, infrared spectroscopy and NMR analysis, the molecular formula (C₂₈H₂₀N₂O₇S₂) and possible structural formula of LIM-a were proposed. In order to increase its water solubility, non-water-soluble LIM-a was modified by histidine, lysine and arginine, and histidine-melanin (HLIM-a) had the highest water solubility, being 47.7 mg mL⁻¹ in distilled water at room temperature. Infrared spectroscopy, mass spectroscopy and ¹H NMR analysis of HLIM-a indicated that histidine-melanin was formed by conjugating LIM-a molecule with histidine molecule. In vivo test showed that both LIM-a and HLIM-a had significant anti-tumor activity, in which HLIM-a showed better efficacy. Immunohistochemistry analysis and cytokines detection suggested that LIM-a and HLIM-a may repress tumor growth through activating the immune response.     

Synthesis and in vitro anti-tumor activity of carboranyl levodopa

Reactions of closo-1-Me-2-Iodobutyl-1,2-closo-dicarborane, 1-Me-2-I(CH₂)₄-C₂B₁₀H₁₀, with l-dopa methyl ester can produce carboranyl l-dopa methyl esters in 54% yield in the presence of sodium hydroxide. The appended closo-carboranes can be decapitated with sodium hydroxide in a mixed solvent of ethanol and deionized water to produce highly water-soluble carboranyl levodopa in 64% yield. All the new compounds were characterized by ¹H, ¹³C, ¹¹B NMR, FT-IR spectroscopy and elemental analysis. The highly water soluble carboranyl levodopa 4 shows promising efficacy of anti-tumors in vitro in the presence of slow neutron beams.     

Cytotoxicity, qualitative structure-activity relationship (QSAR), and anti-tumor activity of bismuth dithiocarbamate complexes

Bismuth dithiocarbamate complexes of general formula Bi(S(2)CNR(2))(3) demonstrate potent in vitro cytotoxicity against a panel of seven human cancer cell lines; a structure-activity relationship has been established. Potency exhibited by the R=Et (2) derivative, for example, is unrivalled by standard cancer drugs with the exception of paclitaxel. In vivo studies indicate a significant anti-tumor effect exerted by (2) against both OVCAR-3, an ovarian cancer cell line, and HT-29, a colon carcinoma cell line.     

人类基因组上CpG岛的定位和系统分析

为了研究CpG岛产生和消失机制以及位于基因启动子区域外的CpG岛保守性等问题,我们通过序列比对和进化保守性分析等方法,分析在人类和小鼠中保守的基因上的CpG岛。结果显示已有保守序列的突变以及序列插入删除是CpG岛产生和消失的主要原因,进一步分析发现52%的在小鼠基因组上保守序列完全缺失的CpG岛位于两个转座子之间,提示转座子所介导的序列插入是CpG岛形成和消失的重要原因。人类基因组上在启动子区域外的CpG岛中约有79%为新产生的CpG岛,显著高于启动子区域内新产生的CpG岛比例(41%)。GO分析表明与这些CpG岛相关的部分基因与神经系统发育显著相关,提示新产生的CpG岛参与神经发育过程。     

人类全基因组范围的CpG岛的预测与分析

CpG岛的甲基化是表观遗传中基因表达调控的重要机制。虽然目前已存在几个从DNA序列判别CpG岛的标准,但如何在标准中选择合适的参数仍是研究的焦点。文章通过分析比较两种经典CpG岛判定标准与三种预测方法,提出了改进的CpG岛预测方法——CpGISeeker。应用该预测方法,结合判定标准中的三个基本参数组合出的13组组合参数,在人类全基因组范围内进行了CpG岛预测,并统计分析了CpG岛的重复序列组成以及相对于基因转录起始位点的位置分布情况。分析结果表明CpGISeeker具有更精确判定CpG岛的特性;同时还提示,随着判定标准严格性的增加,CpG岛的重复序列含量降低,与基因转录起始位点的相关性提高。将CpG岛最小尺寸为500bp、GC含量为60%、CpG出现率达到0.65的组合参数作为标准,是目前预测CpG岛的最佳方式。     

Protective effect of vaccination with Toxoplasma lysate antigen and CpG as an adjuvant against Toxoplasma gondii in susceptible C57BL/6 mice

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems to both humans and livestock and of great economic impact worldwide. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote Th1 responses, an adjuvant activity that is desirable for vaccination against intracellular pathogens. We investigated the feasibility of using CpG as an adjuvant combined with Toxoplasma lysate antigen (TLA) as a vaccine against toxoplasmosis. Genetically susceptible C57BL/6 mice were vaccinated with TLA with or without CpG ODN as an adjuvant and then challenged with 85 cysts of the moderately virulent RRA (Beverley) strain of T. gondii. Prior to challenge infection, immunization with TLA plus CpG ODN directed cellular and humoral immunity toward a Th1 pattern, characterized by enhanced INF gamma production by splenic cells in response to TLA, and enhanced production of toxoplasma-specific IgG and IgG (2a) antibodies. Consequently, CpG/TLA-treated mice showed prolonged survival and 64% reduction in brain parasite burden compared to non-CpG/TLA treated group. Our results suggest that CpG ODN would provide a stable and effective adjuvant for use in vaccination against toxoplasmosis.     

Green synthesis,characterization and biological activity of Solanum trilobatum-mediated silver nanoparticles

Biologically inspired synthesis of nanoparticles was found to be more attractive in metal nanoparticle synthesis. The present study reported an in-situ biogenic synthesis of silver nanoparticles (AgNPs) using Solanum trilobatum aqueous leaf extract. On this basis, the aqueous leaf extract of S. trilobatum acted as a reducing agent and stabilizing agent to synthesize highly stable AgNPs at ambient temperature. Eventually, the synthesized and stabilized AgNPs surface plasmon resonance was near 430 nm through a UV–visible (UV–vis) spectrophotometer. Here, the stability of the silver colloids monitored through zeta potential and mean particle size was evaluated through diffraction light scattering (DLF). Further, the average particle size was found to be 27.6 nm and spherical, confirmed with transmission electron microscopy (TEM). Also, colloidal AgNPs and aqueous extract are found to be rich sources of antioxidants and exhibit higher free radical scavenging ability. Thus, efficient inhibition with COX1 and COX2 enzymes and the protective effect with human red blood cell (HRBC) membrane stability showed significant results. These features are promising, suggesting the possibility of the AgNPs to be useful to disease-modifying for treating inflammatory disorders and associated complications.     

Synthesis, characterization, solid-state photo-luminescence and anti-tumor activity of zinc(II) 4′-phenyl-terpyridine compounds

Reactions between 4′-phenyl-terpyridine (L) and several Zn(II) salts (sulfate, nitrate, chloride or acetate) led to the formation of the complexes [Zn₂(μ-O₂SO₂)₂L₂(CH₃OH)₂] (1), [Zn(NO₃)L(H₂O)]NO₃ (2), [Zn(Cl)₂L] (3) and [Zn(CH₃COO)₂L] (4) which were characterized by IR, ¹H NMR and fluorescence spectroscopies, elemental analysis and single crystal X-ray diffraction. In the dinuclear molecule of 1 the Zn atom is hexacoordinated, with a N₃O₃ coordination environment and forms an octagonal ZnOSOZnOSO metallacycle. In the remaining structures, the metal atom is envisaged as possessing highly distorted N₃X₂ (X = O or Cl) square pyramid coordination geometries. The structure of 3 presents two different packing patterns which lead to distinct π-π stackings. In both structures 2 and 4, strong intermolecular hydrogen bonds were identified. The complexes exhibit promising in vitro tumor-inhibiting activities, which are higher than that of cisplatin, against the following human tumor cell lines: promyelocyticfina leukaemia (HL-60), hepatocellular carcinoma (Bel-7402), gastric carcinoma (BGC-823) and nasopharyngeal carcinoma (KB).     

Beckmann rearrangement products of methyl 3-oxo-tirucall-8, 24-dien-21-oate from Boswellia serrata gum and their anti-tumor activity

3-Oxo-tirucall-8, 24-dien-3-one-21-oic acid is a minor natural product isolated from Boswellia serrata gum apart from β-boswellic acids. Since oxidation of 3-hydroxy group of β-boswellic acids leads to unstable beta-keto acids, Beckmann rearrangement could not be tried. Hence A-ring modified 3-oxo-tirucall-8, 24-dien-21 methyl esters (2–6) were synthesized for the first time via Beckmann rearrangement and evaluated for their anticancer potential against five human cancer cell lines (MCF-7, SW-982, HeLa, PC-3 and IMR-132) by MTT assay. While naturally occurring 3-oxo-tirucallic acid (1) and its methyl ester (2) exhibited nearly the same antiproliferative activity, A-ring modified molecules displayed improved anti-tumor activity with methyl A-homo-4-aza-3-oxo-tirucall-8, 24, dien-3-one-21-oate (4) exhibiting significant effect against prostate cancer cell lines.     

Significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides produced by submerged culture of Tuber melanosporum

The significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides was systematically studied in the submerged fermentation of Tuber melanosporum. The lowest weight-average molecular weight (M_w) (i.e., 115.3 × 10⁴ g/mol) of intracellular polysaccharides (IPS) was obtained when Mg²⁺ and K⁺ was added in the fermentation medium. The IPS with the lower M_w exhibited a higher inhibition ratio against S-180 tumor cells. The compact conformation of extracellular polysaccharides (EPS) was formed when only K⁺ was supplied in the fermentation medium. Interestingly, EPS with compact conformation exhibited a higher inhibition ratio (i.e., 59.2%) than EPS with branched polymer chain (i.e., 9.2%) against A549 tumor cells. The highest inhibition ratio for EPS with α-glycosidic linkages against the tumor cell line HepG2 reached 32.2% when Mg²⁺ or K⁺ was supplied in the fermentation medium. The addition of metal ion Mg²⁺, K⁺, and their combination to the fermentation medium is a vital factor affecting the structures of Tuber polysaccharides, which further determine their anti-tumor activities. The information obtained in this work will be useful for the efficient and directed production of polysaccharides with anti-tumor activities by the submerged fermentation of edible fungi mycelium.     

NK cells are migrated and indispensable in the anti-tumor activity induced by CCL27 gene therapy

Natural killer (NK) cells have been demonstrated could play an important role in the treatment of a number of tumors in mice. In the present study, chemokine CCL27, which be considered only selectively chemoattracts cutaneous lymphocyte antigen positive memory T cells and Langerhans cells, firstly demonstrated that it could induce the accumulation of NK cells into tumor by the intratumoral injection of CCL27-encoding fiber-mutant vector, AdRGD-CCL27. Experiments using spleen cell fractionation and RT-PCR showed CCL27 receptor, mCCR10, was strongly expressed in NK cells, suggesting the accumulation of NK cells in tumor was attributed to chemoattractant activity of CCL27 itself. Moreover, the combination of AdRGD-CCL27 and AdRGD-IL-12 induced the synergistic anti-tumor activity via NK-dependent manner and induced more NK cells infiltration into tumor nodule than that induced by AdRGD-CCL27 alone or AdRGD-IL-12 alone.     

Clinical assessment of the combination therapy with liposomal gels of tretinoin and benzoyl peroxide in acne

Conclusions  The findings of this investigation thus conclusively demonstrate the promising role of concomitant therapy of liposomal TRE and BP gels in treating acne patients. Allthough their use improved the therapeutic response, more benefits of the liposomal form of drugs were observed in terms of reduction in adverse effects of therapy, thus fostering better patient compliance. It can be concluded that this study underscores the potential utility of concomitant therapy with liposomal TRE and BP gels in the treatment of acne. However, the role of liposmal formulations may only be established after clinical evaluation of alarge number of patients, with a special focus on the adverse symptoms of therapy. Published: July 17, 2001.     

The distribution of the dinucleotide CpG and cytosine methylation in the vitellogenin gene family

Summary Sequence data from regions of five vertebrate vitellogenin genes were used to examine the frequency, distribution, and mutability of the dinucleotide CpG, the preferred modification site for eukaryotic DNA methyltransferases. The observed level of the CpG dinucleotide in all five genes was markedly lower than that expected from the known mononucleotide frequencies. CpG suppression was greater in introns than in exons. CpG-containing codons were found to be avoided in the vitellogenin genes, but not completely despite the redundancy of the genetic code. Frequency and distribution patterns of this dinucleotide varied dramatically among these otherwise closely related genes. Dense clusters of CpG dinucleotides tended to appear in regions of either functional or structural interest (e.g., in the transposon-like Vi-element ofXenopus) and these clusters contained 5-methylcytosine (5 mC). 5 mC is known to undergo deamination to form thymidine, but the extent to which this transition occurs in the heavily methylated genomes of vertebrates and its contribution to CpG suppression are still unclear. Sequence comparison of the methylated vitellogenin gene regions identified CT and GA substitutions that were found to occur at relatively high frequencies. The predicted products of CpG deamination, TpG and CpA, were elevated. These findings are consistent with the view that CpG distribution and methylation are interdependent and that deamination of 5 mC plays an important role in promoting evolutionary change at the nucleotide sequence level.     

Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells

Glioblastomas are the most malignant and most frequent brain tumors and exciting targets of gene and immunotherapy. Despite rapid development of experimental therapy little is known about the cellular behaviour of therapeutic oligodeoxynucleotides (ODNs). Here we designed uptake, cellular distribution and cellular binding proteins of immunostimulatory CpG-ODNs in glioblastoma cells by flow cytometry, fluorescence microscopy and mass spectrometry. Our data show that the phosphorothioate (PS) CpG-ODNs uptake in T98G and C6 cells is dose-, time-, temperature-dependent and independent of the CpG dinucleotides. Uptake can be inhibited by sodium azide, polyanions but not by chloroquine. After internalisation FITC labelled CpG-ODNs showed a spotted distribution in cytoplasm. Dozens of cellular binding proteins were identified using mass spectrometry. The binding of ODNs to proteins is dependent on modification and sequence but independent on CpG motif. ODNs bind to cellular proteins that are important for RNA processing and transport. Furthermore, three novel membrane proteins were identified, which might contribute to uptake of ODNs. ODNs binding to these proteins might interfere with the physiological function and thus might cause unwanted effects. Such binding also might influence the uptake efficiency or cellular distribution of therapeutic ODNs.