短棒状杆菌胞壁肽聚糖的实验研究

目的提取短棒状杆菌的有效成分,观察生物学效应,为研制新型治疗制剂奠定基础。方法将短棒状杆菌77—1株用冷热外压法获得细胞壁,再经苯酚及三氯甲烷萃取肤聚糖成分。结果提纯品经紫外吸收测定、淋巴细胞转化试验、抑瘤试验、脾激活试验、毒性试验证实,具有生物学活性及安全性。其主要成分经生化检测含有多肽及聚糖类物质。结论短棒状杆菌的有效成分是细胞壁上的肽聚糖类物质;实验采用的提纯工艺具有可操作性和实际应用价值,为新产品开发提供了可靠依据。     

厌氧棒状杆菌与双歧杆菌抗小鼠腹水瘤活性的比较

厌氧棒状杆菌和双歧杆菌分别制成死菌苗,在相同实验条件下观察两菌对小鼠腹水瘤的抗瘤效果。结果显示,两种菌苗均有显著的抗瘤活性,而厌氧棒状杆菌抗瘤作用更强于双歧杆菌,特别在延长观察期尤为显著。应用两菌苗后,脾指数均有不同程度升高,未见明显毒性作用。     

厌氧棒状杆菌群抗血清的血清学分析

厌氧棒状杆菌具有刺激网状内皮系统、抗感染、调节免疫反应、抑制肿瘤等一系列生物学活性。近年来制备的厌氧棒状杆菌菌苗作为肿瘤免疫治疗剂受到了重视。这类细菌的分类较混乱,1966年Prevot将其分为12个属,但其中不少为同义名。1972年Johnson和Cummins用细胞壁分析、DNA同源性以及血清学研究分为3大群4个血清学群;[即疮疱丙酸杆菌     

乳酸菌表面蛋白对免疫细胞的诱导增殖及粘附抑制效应

目的研究表面蛋白在乳酸菌体外粘附和激活免疫细胞中的作用。方法应用5mol/L氯化锂结合盐酸胍提取植物乳杆菌LpYZU09、干酪乳杆菌LcYZU02、鼠李糖乳杆菌LrGG、发酵乳杆菌LfYZU15及戊糖片球菌PpYZU32的表面蛋白,并分析提取物对乳酸菌粘附鼠肠上皮细胞、巨噬细胞和脾细胞的抑制作用及诱导增殖效应。结果表面蛋白对3种细胞粘附菌体均具有显著抑制效应,抑制作用具有细胞和菌株差异性,其中菌株LrGG表面蛋白对巨噬细胞粘附5种菌体普遍显示了较强的抑制作用,抑制率为38.7%~76.0%。不同菌株表面蛋白对肠上皮细胞的诱导增殖指数为0.05~0.35,对巨噬细胞为0.05~0.42,对脾细胞为0.02~0.40,诱导效应具有菌株和剂量依赖性,菌株LrGG的诱导增殖指数显著高于其他四种。结论乳酸菌表面的蛋白类因子在粘附和激活免疫细胞中发挥了重要作用。     

细胞壁缺陷白喉棒状杆菌致病作用的观察及其毒力基因检测

目的比较白喉棒状杆菌亲代细菌型及其稳定L型动物致病性的差异,了解白喉棒状杆菌稳定L型变异的特点,探讨其变异的性质及其与细胞壁缺陷突变的关系。方法用氨苄青霉素在非高渗培养基内人工诱导产毒性白喉棒状杆菌为稳定L型。采用白喉棒状杆菌稳定L型纯培养物及其代谢产物皮内感染家兔,观察局部感染部位皮肤或全身的病理改变。采用微量法提取白喉棒状杆菌稳定L型的染色体DNA,用Tox基因特异性引物进行PCR扩增以检测毒素蛋白结构基因,并进行序列测定和分析。结果白喉棒状杆菌在氨苄青霉素作用下可发生细胞壁缺陷而成为L型,白喉棒状杆菌稳定L型不能引起动物局部或全身发生异常表现,该稳定L型的传代培养物可仍然保留同其亲代细菌型一致的Tox基因及其核苷酸序列。结论提示细胞壁缺失将导致白喉棒状杆菌与产生毒素蛋白有关结构基因在宿主菌细胞内的表达受到抑制,以致使白喉棒状杆菌稳定L型丧失了产生外毒素致病的作用。     

细胞壁缺陷对白喉棒状杆菌致病性影响的研究

检测白喉棒状杆菌稳定L型对动物的致病性,探讨细胞壁缺陷对白喉棒状杆菌致病性的影响及其可能的分子机制。采用氨苄青霉素在非高渗培养基内诱导并获得产毒性白喉棒状杆菌稳定L型纯培养物。收集白喉棒状杆菌稳定L型纯培养物及其代谢产物,将收集的高于细菌型10 000倍浓度的白喉棒状杆菌稳定L型纯培养物及其代谢产物皮内注射家兔,观察局部注射部位皮肤或全身的病理改变。分别采用对流免疫电泳(CIEP)和SDS-不连续聚丙烯酰胺凝胶电泳(SDS-PAGE)检测白喉棒状杆菌稳定L型可溶性代谢产物中的白喉毒素蛋白质。结果显示,白喉棒状杆菌稳定L型不能引起动物局部或全身发生异常表现,在其可溶性代谢产物中并未检测到白喉毒素蛋白质。提示细胞壁缺陷变异可影响白喉棒状杆菌产生白喉毒素蛋白质,从而使其丧失了产生外毒素致病的作用。     

国产厌气棒状杆菌菌苗小型生产工艺

本文报道一种小型、简易、廉价的方法,用以制备厌气捧状杆菌菌苗。实验证明本方法生产的菌苗质量(包括菌团大小、脾指数及抑瘤效果等)达到了较好的水平。对动物没有可见的毒性作用,对人无严重的副反应。可供实验研究和临床试用。     

通过接合作用质粒从大肠杆菌到棒状杆菌的转移

构建了可接合转移的穿梭质粒pXZ911、pBZ51和pBZ52。这些质粒中含有具转移功能的Mob片段和在棒状杆菌中复制的复制区。以大肠杆菌S17—1为供体菌,通过接合转移作用,可将这些质粒转移到谷氨酸棒杆菌ATCCl3032、谷氨酸棒杆菌ATCC21543、北京棒杆菌B3、北京棒杆菌1．299、裂氏棒杆菌B43、黄色短杆菌ATCC 14067等棒状杆菌苗株,接合转移频率分别为：9X10^-5,1X10^-4,8．5x10,2．3X10^-4×10^-5,2．9X10^-5。本文还探索了大肠杆菌和几种革兰氏阳性棒状杆菌问基因转移的方法、转移频率、影响转移频率的因素、宿主范围等问题。     

短小棒状杆菌对小鼠实体瘤及免疫功能的影响

目的研究短小棒状杆菌对H22荷瘤小鼠及其免役功能的影响。方法采用抑瘤实验、小鼠碳廓清实验、MTT法观察短小棒状杆菌对小鼠实体瘤和免疫功能的影响。结果短小棒状杆菌可使荷瘤小鼠的碳廓清指数、胸腺指数、脾脏指数升高,并由刀豆蛋白(ConA)、脂多糖(LPS)诱导体外小鼠T、B淋巴细胞增殖,能够抑制小鼠实体瘤生长。结论短小棒状杆菌能够增强小鼠机体免疫功能,并有抑瘤作用。     

应用纤维素结合域标签构建谷氨酸棒状杆菌表达系统

为优化谷氨酸棒状杆菌表达系统的纯化工艺,合成里氏木霉的CBD基因,将其与谷氨酸棒状杆菌分泌表达载体pXMJ19-sp连接,构建以CBD为纯化标签的重组载体pXMJ 19-sp-CBD.在该载体中插入GFP基因并转化至谷氨酸棒状杆菌,可获得分泌表达融合蛋白GFP-CBD的重组菌.该菌经IPTG诱导后的发酵液在紫外灯下显示强烈的绿色荧光,重组蛋白的分泌表达量达200 mg/L.利用CBD标签对纤维素柱的可逆性吸附,可直接对谷氨酸棒状杆菌分泌到培养基中的重组蛋白进行纯化,从而简化工艺和降低成本,为工业化大生产奠定基础.     

Immunogenic comparison for two different recombinant chimeric peptides (CP12 and CP22) containing one or two copies of three linear B cell epitopes from β-hCG subunit

To develop a superior chimeric peptide (CP) vaccine of human chorionic gonadotropin (hCG), two CP antigens (named CP12 and CP22) encoding one or two copies of three linear B cell epitopes from the β-hCG subunit and six foreign T cell epitopes, including two promiscuous TCEs from hepatitis B surface antigen and tetanus toxoid, were constructed and biosynthesized. The hCG CP12 and CP22 of 21 or 23 kDa, respectively, were expressed in Escherichia coli at the level of ∼1% of total cell proteins when inserted into thermo-inducible pBV221 expression vector. The purified CP12 and CP22 proteins with >95% relative homogeneity are immunogenic, and elicited antibodies against the β5, β9 and β8 BCEs of β-hCG in both rabbits and three different inbred strains of mice. A mouse uterine weight study in Balb/c mice demonstrated that the CP12 and CP22 antigens with an additional β5 neutralizing epitope enhanced the in vivo bio-neutralization capacity of the induced antibodies compared to the C-terminal immunogen of β-hCG. We propose that the biosynthesized CP22, possessing with two copies of three BCEs, represents a novel candidate antigen for an hCG contraceptive or tumor therapeutic vaccine.     

Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

Mitigation of regulatory T cell-mediated immunosuppression and elicitation of immunogenic tumor cell death are crucial events for optimal anti-tumor immune activity in vivo. This study was designed to investigate the potential synergistic activity of the combined use of cyclophosphamide (CP) and doxorubicin (DR), both of which are known to resolve these two issues. BALB/c mice were inoculated subcutaneously with CT-26 carcinoma cells in the bilateral flank and treated with an intraperitoneal injection of a low dose of CP followed by an intratumoral injection of DR into one side of the tumor. We found that, in addition to a significant suppression of growth on the DR-treated side of the tumor, combination therapy suppressed the growth of DR-untreated remote tumors in both tumor-specific and T cell-dependent manners. Mitomycin C showed no such synergistic anti-tumor activity with CP treatment. Combination therapy increased the frequency of interferon (IFN)-γ-producing T lymphocytes specific to a CT-26-associated class I-binding tumor peptide in the tumor-draining lymph nodes. Real-time PCR analysis revealed that combination therapy led to an increase in IFN-γ and tumor necrosis factor-α mRNA expression; however, levels of Foxp3 and transforming growth factor-β within the remote tumor tissues were decreased. In addition, knock down of calreticulin expression in CT-26 cells using small interfering RNA attenuated anti-tumor vaccine effects induced by DR-treated CT-26 cells. These results provide an immunological rationale for the combined use of chemotherapeutic drugs, i.e., CP and DR, and further recommend their use with current cancer vaccines.     

The anti-tumor efficacy of a GM-CSF-secreting tumor cell vaccine is not inhibited by docetaxel administration

Docetaxel has demonstrated therapeutic efficacy against breast, prostate, and ovarian cancer and other solid tumors. The tumoricidal activity of docetaxel is mainly attributed to its ability to block microtubule depolymerization, thus inducing G₂-M arrest and apoptosis. Mounting evidence indicates that docetaxel also possesses immunomodulatory activity such as augmenting macrophage and lymphokine activated killer activity and inducing pro-inflammatory cytokines, suggesting that docetaxel may be a good chemotherapeutic agent to combine with cancer immunotherapies, assuming that it does not inhibit the vaccine-induced immune response. The anti-tumor activity of the combination of docetaxel and a GM-CSF-secreting B16F10 tumor cell vaccine (B16.GM) was evaluated in the murine B16 melanoma model. Dose levels of docetaxel and the B16.GM vaccine known to be ineffective when used as single agents were selected. Three iv treatments of 6 mg/kg docetaxel per injection given on days 5, 9, and 13 after tumor challenge or a single vaccination with 2–3×10⁶ B16.GM cells on day 3 were ineffective at inhibiting tumor growth when used as single agents [median survival time (MST)=24 days in both treatment groups and in control animals]. However, combination of docetaxel and B16.GM vaccine significantly delayed tumor growth, increasing MST to 45 days. A similar improvement in anti-tumor efficacy was observed using multiple treatment cycles of the B16.GM vaccine/docetaxel combination. Administration of docetaxel every 4 days between bi-weekly B16.GM vaccinations increased the median survival of tumor-bearing mice from 31 to 52 days compared to multiple B16.GM vaccinations alone. In summary, these data demonstrate that rather than inhibiting the anti-tumor effects of a GM-CSF-secreting tumor cell vaccine, docetaxel combined with a whole cell vaccine significantly inhibits tumor growth, increases survival time and does not impede T-cell activation in the murine B16F10 melanoma tumor model. GM-secreting tumor cell vaccines in combination with docetaxel may represent a new strategy for combining chemo and immunotherapy for cancer.     

Chemoimmunotherapy of stage III breast carcinoma with BCG and a live allogeneic tumor cell vaccine

Summary Ten women with metastatic breast carcinoma were treated with CMF, BCG, and a live TCV in a phase I study to determine whether viable allogeneic tumor cells can safely be given to women receiving adjuvant chemotherapy. Side effects of fatigue, nausea, vomiting, alopecia, and myelosuppression were attributable to chemotherapy. Side effects of BCG therapy were malaise, fever, and pruritis at the injection site, as previously described. Four patients developed hepatitis B due to viral contamination of the human agamma serum in which the tumor cells were grown. This complication has now been eliminated by omitting human sera, and hence the possibility of hepatitis B virus, from tumor cell growth media. No local tumor growth was observed in any patient. Results of this study show that live TCV is safe for patients receiving chemotherapy, and could be tested in combination with BCG and chemotherapy for control of micrometastases from breast cancer following mastectomy. Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; CMF, cyclophosphamide, methotrexate, and 5-fluorouracil; DNCB, dinitrochlorobenzene; PPD, purified protein derivative; TCV, tumor cell vaccine(s).     

Anticancer effects of a plant lignan 7-hydroxymatairesinol on a prostate cancer model in vivo

Clinical intervention studies and experimental studies with lignan-rich diets suggest that lignans may have inhibitory effects on prostate cancer, but no clinical or experimental studies with purified lignans have been published. The purpose of this study was to investigate the effect of a plant lignan 7-hydroxymatairesinol (HMR) on LNCaP human prostate cancer xenografts in athymic mice. Athymic nude male mice were injected subcutaneously with LNCaP cells. Starting 3 days after tumor cell injections, a control diet or a control diet supplemented with 0.15% or 0.30% of HMR was administered to mice and the tumor take rate and growth was observed for 9 weeks. HMR diet inhibited the growth of LNCaP tumors. Mice treated with HMR had smaller tumor volume, lower tumor take rate, increased proportion of nongrowing tumors, and higher tumor cell apoptotic index compared with controls. Furthermore, the cell proliferation index was reduced in mice receiving the 0.30% HMR diet compared with mice receiving the control diet. Our results suggest that dietary HMR started at the early phase of the tumor development inhibits the growth of the LNCaP human prostate cancer xenografts in athymic male mice.     

在低蛋白质饲料中补充必需氨基酸对大口黑鲈生长、体组成和免疫指标的影响

为了研究在低蛋白质饲料中补充晶体必需氨基酸对大口黑鲈生长、体组成和免疫指标的影响,根据鱼体的必需氨基酸组成模式设计了7种等能的试验饲料。其中4种饲料（45CP、40CP、35CP和30CP）的粗蛋白质水平分别为45%、40%、35%和30%,另3种饲料（40AA、35AA和30AA）是在低蛋白质饲料（40CP、35CP和30CP）的基础上添加必需氨基酸,使它们的必需氨基酸含量与45CP（对照组）相一致。用上述饲料对初始体重为（10.13 0.01） g的大口黑鲈进行了89d的饲养试验。饲养试验在室内循环水养殖系统中进行,每种饲料设3个重复,每重复放养30尾鱼。方差分析显示:试验鱼的生长性能、饲料效率、全鱼和肌肉的粗蛋白质含量、成活率以及免疫指标均随着饲料蛋白质含量的降低而显著降低（P0.05）。添加必需氨基酸的35AA和30AA的饲料效率和蛋白质保留率分别显著高于对应的未添加必需氨基酸的35CP和30CP组（P0.05）,但仍显著低于45CP组（P0.05）。40AA的试验鱼血清溶菌酶活性和血清补体活性与45CP组差异不显著（P0.05）。35AA和30AA组的头肾白细胞呼吸爆发活性显著高于35CP和30CP组（P0.05）。30AA组的全鱼粗蛋白质含量以及肥满度显著高于30CP（P0.05）。各组试验鱼的水分和灰分均无显著差异（P0.05）。回归分析显示:在低蛋白质饲料中补充晶体必需氨基酸对大口黑鲈幼鱼的生长、饲料效率和蛋白质保留率所产生的影响与其引起增加了的饲料蛋白质水平而不是饲料的必需氨基酸水平正相关。研究表明,在低蛋白质饲料中补充的晶体必需氨基酸对大口黑鲈的生长、体组成和免疫指标产生的有益作用不及等量的以蛋白质为来源的必需氨基酸。       

Presence of Active and Inactive Molecules of a Cell Wall-Associated Proteinase in Lactobacillus helveticus CP790

Monoclonal antibodies against a cell wall-associated 45-kDa proteinase from Lactobacillus helveticus CP790 were prepared and used for an immunoblotting analysis of the cell wall extract of CP790. They were found to react with an unidentified 46-kDa protein as well as the 45-kDa proteinase. The 46-kDa protein was copurified with the 45-kDa proteinase by affinity column chromatography using antibody-fixed Sepharose and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted from the gels. An elution profile of the cyanogen bromide digest of the purified 46-kDa protein obtained by reversed-phase high-performance liquid chromatography was identical to that of the 45-kDa proteinase except for one peak. An analysis of the N-terminal 21-amino-acid sequence revealed that the 46-kDa protein possesses an extra 7 amino acids at the N terminus of the 45-kDa proteinase. The 46-kDa protein was produced at constant levels during fermentation in a skim milk medium, while the 45-kDa protein was mainly observed in the middle of the exponential phase of growth and was produced in proportion to the proteinase activity. Moreover, only the 46-kDa protein was detected in the crude extract of L. helveticus CP791, a variant strain of CP790 defective in proteinase activity. These data strongly suggest that the 46-kDa protein is a precursor, inactive form of the 45-kDa proteinase.     

小鼠白细胞介素21瘤苗的构建及其抗肿瘤效应研究

目的:建立稳定表达小鼠白细胞介素21(mIL-21)的肿瘤细胞瘤苗,观察其在小鼠体内是否能够诱导有效的抗肿瘤免疫反应及免疫记忆效应。方法:将已鉴定的重组质粒pcDNA3.1/mIL-21用脂质体法转染Sp2/0细胞制备瘤苗,RT-PCR法鉴定瘤苗中mIL-21的表达。通过流式细胞仪检测细胞周期来反映瘤苗体外增殖活性,再将其接种BALB/c小鼠,监测肿瘤生长情况,观察mIL-21瘤苗诱导的抗肿瘤效应;用ELISA法检测血清IFN-γ和IL-4含量。结果:得到稳定表达mIL-21的瘤苗Sp2/0-mIL-21。与对照组相比,体外增殖活性无差异。皮下接种BALB/c小鼠后,肿瘤生长缓慢,部分小鼠无瘤体生长并长期存活;用野生株Sp2/0瘤细胞再次攻击未长肿瘤的实验小鼠,4周后亦未见肿瘤生长。接种瘤苗小鼠血清中IFN-γ水平明显上升,IL-4无明显增高。结论:成功构建了mIL-21瘤苗Sp2/0-mIL-21,其能诱导有效的抗肿瘤免疫反应及免疫记忆效应。     

Directed evolution for improved secretion of cancer-testis antigen NY-ESO-1 from yeast

NY-ESO-1 is a highly immunogenic tumor antigen and a promising vaccine candidate in cancer immunotherapy. Access to purified protein both for vaccine formulations and for monitoring antigen-specific immune responses is vital to vaccine development. Currently available recombinant Escherichia coli-derived NY-ESO-1 is isolated from inclusion bodies as a complex protein mixture and efforts to improve the purity of this antigen are required, especially for later-stage clinical trials. Using yeast cell surface display and fluorescence activated cell sorting techniques, we have engineered an NY-ESO-1 variant (NY-ESO-L5; C(75)A C(76)A C(78)A L(153)H) with a 100x improved display level on yeast compared to the wild-type protein. This mutant can be effectively produced as an Aga2p-fusion and purified in soluble form directly from the yeast cell wall. In the process, we have identified the epitope recognized by anti-NY-ESO-1 mAb E978 (79-87, GARGPESRL). The availability of an alternative expression host for this important antigen will help avoid artifactual false positive tests of patient immune response due to reaction against expression-host-specific contaminants.     

Deoxycholate-treated, nontoxic, whole-cell vaccine protective against experimental salmonellosis of mice

A vaccine prepared from the residue after extraction of whole cells of Salmonella typhimurium with 2% sodium deoxycholate proved to be nontoxic and highly immunogenic. The material was not lethal for mice at 6.0 mg and was essentially nontoxic in rabbit skin, whereas endotoxic activity was found in the dialyzed extract. A high dosage, above 2.0 mg, was less protective than lower doses, indicating a degree of "immunologic paralysis." Three inoculations of low doses, 0.25 mg each, induced protection against death and tissue infection in animals challenged with 2,000 ld(50) of virulent homologous S. typhimurium and against death, but not against tissue infection, after heterologous challenge with S. enteritidis. Residues of purified cell walls were as effective as residues of whole cells, indicating that the immunizing antigen(s) resided in the cell wall.