An improved method for assessing the incorporation of labeled precursors into DNA by human mononuclear cells

The blastogenic responsiveness of activated lymphoid cells is usually assessed in vitro by measuring the incorporation of radioactive thymidine or iododeoxyuridine, a thymidine analog, into DNA. The accuracy of this method is compromised by the presence in activated and unactivated lymphocytes and in some of the substances used to activate them, of degradative enzymes which compete with DNA synthetase, the incorporation efficiency of exogenous precursor is inherently low. We have done studies aimed at improving both the efficiency and the accuracy of the assay system by selectively inhibiting the enzymes responsible for thymidylate synthesis de novo and DNA precursor degradation. Culture conditions were investigated and potential inhibitors were tested using human peripheral blood mononuclear cells activated with phytohemagglutinin. Nucleoside-degrading activity of mammalian and bacterial cells is due largely to nucleoside phosphorylases, enzymes that require orthophosphate for activity. We partly inhibited DNA precursor degradation by lowering the phosphate concentration in the culture medium and lowering the pH, thereby reducing the orthophosphate concentration. To reduce precursor degradation further, we tested several potential nucleoside phosphorylase and thymidylate synthetase inhibitors at various concentrations. Our data show that the addition of 1 mM fluorouracil and 1 mM deoxyuridine to the culture medium largely prevents degradation of radioactive thymidine and iododeoxyuridine without unduly compromising the DNA-labeling efficiency of cells activated with mitogens or bacterial homogenates. Under these conditions, label incorporation increases linearly as the number of blast cells or the labeling time increases.     

硫芥中毒对小鼠小肠DNA合成的影响

用[~3H]TdR参入法看到1LD(30mg/kg)硫芥中毒后4h,小肠DNA生物合成受到明显抑制;8h降至最低值,尔后逐渐恢复。中毒后3d超过正常水平,5d达峰值。胸腺、脾脏和小肠对硫芥的敏感度和恢复速度不同。胸腺最敏感;小肠恢复最快。体重在中毒后呈渐进性下降。     

益气活血冲剂对创伤骨折致肾脏损伤的保护作用

目的：探讨益气活血冲剂对创伤骨折所致肾损伤的保护作用。方法：将大鼠或小鼠分为空白组、对照组和实验组。分别于术后不同时期处死动物,测定三组的肾指数、肾中柠檬酸、DNA、RNA含量及^3H-TdR ^3H-UR参入率。结果：创伤骨折引起大鼠肾指数明显下降,^3H-TdR 参入受抑制,DNA含量降低,^3H-UR参入率和RNA含量升高;应用益气活血冲剂可以改善这些变化、促进DNA和RNA的合成。结论：创伤骨折后应用益气活血冲剂可以改善创伤引起的肾脏的变化。     

Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells

Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis. The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P), which can be converted to dR-5-P, glyceraldehyde-3-phosphate (G3P) or deoxyribose (dR). However, it is unclear which sugar accumulates in this reaction. Therefore, in the TP overexpressing Colo320 TP1 and RT112/TP cells we determined by LC-MS/MS which sugars accumulated, their subcellular localization (using ³H-TdR) and whether dR was secreted from the cells. In both TP-overexpressing cell lines, dR-1-P and dR-5-P accumulated intracellularly at high levels and dR was secreted extensively by the cells. A specific inhibitor of TP completely blocked TdR conversion, and thus no sugars were formed. To examine whether these sugars may be used for the production of angiogenic factors or other products, we determined with ³H-TdR in which subcellular location these sugars accumulated. TdR-derived sugars accumulated in the cytoskeleton and to some extent in the cell membrane, while incorporation into the DNA was responsible for trapping in the nucleus. In conclusion, various metabolic routes were entered, of which the TdR-derived sugars accumulated in the cytoskeleton and membrane. Future studies should focus on which exact metabolic pathway is involved in the induction of angiogenesis.     

Autoreactive T-cell responses in primary biliary cirrhosis are proinflammatory whereas those of controls are regulatory

BACKGROUND & AIMS: Autoreactive T cells that proliferate in response to autoantigens are found in both autoimmune disease and controls but have important qualitative differences in relative activation states, costimulation signal requirements, and pathogenetic significance. Understanding the mechanism for activation of autoreactive T cells will be critical in the treatment of autoimmune diseases. METHODS: To understand the differences between autoreactive T cells in primary biliary cirrhosis (PBC) versus controls, we have developed autoreactive T-cell clones (TCCs) from patients with PBC and healthy controls and have used a peptide corresponding to the CD4 major autoepitope to define the relative proliferative and cytokine response. RESULTS: Using an enzyme-linked immunosorbent spot assay, peripheral blood mononuclear cells (PBMCs) from PBC, but not from controls, produce interferon (IFN)-gamma regardless of whether costimulation-competent or -incompetent antigen-presenting cells (APC) were used. In contrast, a significant number of IFN-gamma-producing cells were found in PBMCs from controls but only if costimulation-competent PBMCs presented an autoantigenic peptide. In addition, costimulation-dependent autoreactive TCCs became anergic after a single round of stimulation in the presence of APC that did not provide a costimulatory signal, whereas some costimulation-independent autoreactive TCCs required repeated stimulation to become anergic and the others did not become anergic. Finally, anergic TCCs produced interleukin-10, but no IFN-gamma, and exhibited regulatory functions in an antigen-dependent, cell contact-independent, and partially interleukin-10-mediated manner. CONCLUSIONS: These data relate specifically to the functional characteristics of autoreactive T cells in PBC but are also generically important for understanding the mechanisms for generating pathogenetic autoreactive T cells.     

瘦素与人类外周血单核细胞增殖作用相关性的探讨

目的探讨瘦素(leptin)与人外周血单核细胞(PBMC)增殖作用的相关性。方法分离并培养PBMC,采用[3H]TdR掺入试验方法,观察瘦素对PBMC增殖作用的影响。结果瘦素可以促PBMC增殖,也可协同PHA或ConA对PBMC的刺激增殖作用,并在一定浓度范围内呈现剂量依赖性。结论瘦素可以促进PBMC增殖。     

局部加温对裸小鼠部分器官~(3H)TdR掺入量影响的观察

采用~(3H)TdR掺入法观察用微波局部加温治疗裸小鼠人脑恶性胶质瘤模型后,裸小鼠脾、肠、肝、脑、皮肤、血等主要器官组织细胞DNA合成功能的影响,发现:经44℃、不同的加温时间(10min,20min,30min,40min)1次加温移植于裸小鼠的人脑恶性胶质瘤后,裸小鼠脾、肠、肝、脑、皮肤、血等主要器官组织~(3H)TdR掺入量与未加温的对照组相比无显著性变化,而加温20min,30min和40min三组移植瘤的~(3H)TdR掺入量与加温10min组及对照组相比,有显著性降低。说明局部微波加温治疗肿瘤时,裸小鼠部分主要器官组织细胞DNA合成功能无明显影响,而对肿癌细胞的DNA合成有明显的抑制作用,提示局部微波加温治疗恶性胶质瘤为一种安全而有效的方法。     

离心管技术构建的组织工程软骨细胞生物学特性的研究

目的 观察离心管技术构建的组织工程化软骨的细胞生物学特点，探讨该技术构建组织工程化软骨的可行性。方法 取3周龄兔关节软骨的表层软骨，经酶消化获得大量的软骨细胞，用离心管技术构建软骨组织，对培养3周的组织工程软骨进行组织学，免疫组织化学，透射电镜，^3H-TdR掺入量等检查。结果 组织学显示软骨细胞位于陷窝，基质异染，Ⅱ型胶原免疫组化阳性，细胞内富含高尔基体，分泌囊泡和糖原颗粒，^3H-TdR掺入量显示软骨细胞DNA较正常软骨细胞明显增强。结论 离心管内软骨细胞的增殖较正常关节软骨增强，离心管技术可用于组织工程软骨的构建。     

抗辐射菌形态及敏感性研究

目的 研究抗辐射菌（Deinococcus radiodurans,Dr)KD8301和KH3111的抗辐射特性，分离纯化DNA质粒，分析其抗辐射基因蛋白产物对哺乳动物细胞放射敏感性的影响。方法 用集落形成法及^3H-TdR掺入法绘制剂量－效应曲线，分析其辐射耐受性，并与对照菌比较，用聚丙烯酰胺凝胶电泳法检测DNA含量。结果 Dr菌是红色微球菌，多呈二联体和四迭体生长，平均致死剂量（D0）、准阈剂量（Dq)、63％细胞的死亡剂量（D37）、n值为最大，D0＝2.9kGy,Dq=3.1kGy,D37=6.0kGy,n=5，以KD8301的辐射抗性最强。结论 用集落形成法测定剂量存活效应曲线显示，经过8kGy照射后，该辐射菌仍具有很强的集落形成能力。     

Antiviral activity of 5-ethyl pyrimidine deoxynucleosides.

The antiviral activity of the α- and β-anomers of 5-ethyl-2′-deoxyuridine (EtUdR) and 5-ethyl-2′-deoxycytidine (EtCdR) has been assessed in primary rabbit kidney (PRK) cell cultures. Cytosine arabinoside (ara-C) and 5-iodo-2′-deoxyuridine (IUdR) were included as reference materials. When inhibition of vaccinia virus multiplication was measured the following order of (decreasing) activity was established: ara-C > IUdR > (β-)EtUdR > (β-)EtCdR. The α-anomers of EtUdR and EtCdR were completely inactive.In marked contrast with ara-C and IUdR which were found to inhibit PRK cell growth and thymidine incorporation into cell DNA at relatively low doses (0.08–0.4 μg/ml and 8–40 μg/ml respectively), EtUdR did not inhibit cell growth or thymidine incorporation into DNA unless very high doses were used (200–500 μg/ml). At 0.4–200 μg/ml EtUdR had a stimulatory effect on the subsequent incorporation of thymidine and deoxycytidine into DNA, most probably due to a starvation of the cells for these precursors during their contact period with EtUdR. EtCdR did not markedly alter thymidine incorporation into DNA.Antiviral indices were calculated for EtUdR, IUdR and ara-C. These were defined as the ratios of the minimum toxic doses (inhibiting cell growth or thymidine incorporation by 50 per cent) to the minimum effective doses (inhibiting vaccinia virus induced cytopathogenicity by 50 per cent). The antiviral indices of EtUdR and IUdR were almost identical but considerably greater than that of ara-C.