Mitochondrial dysfunctions in circulating T lymphocytes from human immunodeficiency virus-1 carriers [see comments]

In several models of lymphocyte apoptosis, two alterations of mitochondrial function precede advanced DNA fragmentation: (1) a reduction of mitochondrial transmembrane potential (delta psi m) and (2) an increase in mitochondrial generation of superoxide anion. Here we show that two fluorochromes allow for the identification of analogous mitochondrial perturbations in circulating T lymphocytes from human immunodeficiency virus (HIV)-1+ donors. The first among these fluorochromes, the cationic lipophilic dye DiOC6(3), measures delta psi m; the second marker, hydroethidine (HE), is nonfluorescent, unless it is oxidized by superoxide anions to the product ethidium (Eth). CD4+ or CD8+ cells from clinically asymptomatic HIV-1 carriers contain a significantly elevated percentage of cells endowed with enhanced HE --> Eth conversion and/or reduced DiOC6(3) uptake as compared with normal controls. Phenotypic characterization of (HE --> Eth)high cells from HIV+ donors shows that these cells possess a low delta psi m, thus demonstrating a functional alteration of mitochondria. In addition, (HE --> Eth)high cells display a reduced incorporation of the cardiolipin- specific dye nonyl-acridine orange (NAO), showing a structural defect of the cardiolipin-containing inner mitochondrial membrane. Control experiments involving rotenone, an inhibitor of the respiratory chain complex I, indicate that the reactive oxygen species responsible for HE --> Eth conversion is generated during mitochondrial electron transport. In synthesis, it appears that mitochondrial alterations occur in a significant percentage of circulating T lymphocytes from HIV- 1 carriers. The extent of delta psi m reduction, as determined ex vivo, correlates with the frequency of cells undergoing DNA fragmentation after overnight in vitro culture. These observations may be important for the understanding and for the direct ex vivo quantitation of HIV- triggered lymphocyte destruction.     

Effect of layered double hydroxide intercalated with fluoride ions on the physical,biological and release properties of a dental composite resin

Objectives

The aim of this work was the preparation of a new fluoride-releasing dental material characterized by a release of fluoride relatively constant over time without any initial toxic burst effect. This type of delivery is obtained by a matrix controlled elution and elicits the beneficial effect of a low amount of fluoride on human dental pulp stem cells (hDPSCs) towards mature phenotype.

Methods

The modified hydrotalcite intercalated with fluoride ions (LDH-F), used as filler, was prepared via ion exchange procedure and characterized by X-ray diffraction and FT-IR spectroscopy. The LDH-F inorganic particles (0.7, 5, 10, 20 wt.%) were mixed with a photo-activated Bis-GMA/TEGDMA (45/55 wt/wt) matrix and novel visible-light cured composites were prepared. The dynamic thermo-mechanical properties were determined by dynamic mechanical analyzer. The release of fluoride ions in physiological solution was determined using a ionometer. Total DNA content was measured by a PicoGreen dsDNA quantification kit to assess the proliferation rate of hDPSCs. Alkaline phosphatase activity (ALP) was measured in presence of fluoride resins.

Results

Incorporation of even small mass fractions (e.g. 0.7 and 5 wt.%) of the fluoride LDH in Bis-GMA/TEGDMA dental resin significantly improved the mechanical properties of the pristine resin, in particular at 37 °C. The observed reinforcement increases on increasing the filler concentration. The release of fluoride ions resulted very slow, lasting months. ALP activity gradually increased for 28 days in hDPSCs cell grown, demonstrating that low concentrations of fluoride contributed to the cell differentiation.

Conclusions

The prepared composites containing different amount of hydrotalcite filler showed improved mechanical properties, slow fluoride release and promoted hDPSCs cell proliferation and cell differentiation.     

应用细胞培养、流式细胞技术和Western Blot检测氮杂糖化合物SZL在体外抑制人宫颈癌HeLa细胞增殖与诱导凋亡的效应

目的:存在于细胞表面的糖复合物参与细胞的通讯、增殖、迁移、分化等生理过程,在机体中具有复杂的生物学调控作用。探讨氮杂糖化合物SZL在体外对人宫颈癌HeLa细胞生长、DNA损伤、线粒体膜电位以及细胞凋亡的干预。方法:实验于2004-12/2006-12在北京大学医学部细胞生物学系完成。①实验材料:人子宫颈癌HeLa细胞株由北京大学医学部细胞生物学系杜晓娟副教授提供。SZL由北京大学天然药物及仿生药物重点实验室叶新山教授合成。②实验方法:取对数生长期HeLa细胞,消化后制成细胞悬液,按1.5×103/孔的密度接种,培养24h细胞完全贴壁后,SZL组加入5,10,25,50,100μmol/L的SZL,空白对照组加入DMEM培养液。③实验评估:SZL作用24,48,72h后,采用酸性磷酸酶法检测细胞生长抑制情况,瑞氏染色镜下观察细胞形态;AnnexinV-PI染色后,流式细胞仪检测细胞凋亡率;Rhodamine123标记活细胞后,流式细胞仪检测线粒体膜电位的变化;WesternBlot法检测SZL作用后凋亡相关蛋白的表达;单细胞凝胶电泳检测SZL作用后细胞DNA损伤情况。结果:①细胞生长抑制:5,10,25,50,100μmol/LSZL作用24,48,72h后的半数抑制浓度分别为73.6,43.2,33.6μmol/L,显著抑制Hela细胞的增殖。镜下空白对照组HeLa细胞分布均匀,胞质透明清亮,呈铺展状态生长;50μmol/LSZL作用24h后,HeLa细胞密度变稀,体积变小,胞核染色质呈聚集状态。②细胞凋亡:50,100μmol/LSZL作用24h后细胞凋亡率分别为(3.51±0.41)%,(58.46±8.45)%;在24～48h过程中,凋亡的细胞逐渐坏死,SZL作用48h后细胞凋亡率分别为(10.51±2.20)%,(17.29±7.52)%。空白对照组24,48h后细胞凋亡率均为1%。③线粒体跨膜电位的变化:50μmol/LSZL作用24,48h后,HeLa细胞的线粒体膜电位平均荧光强度均明显低于空白对照组(P<0.01)。④凋亡相关蛋白的表达:25,50,100μmol/LSZL作用HeLa细胞48h后,凋亡相关蛋白pro-caspase3、Bcl-2表达降低,细胞色素c含量升高。⑤细胞DNA损伤:50μmol/LSZL作用24h后,受损细胞DNA在电场中迁出,呈现彗星状拖尾。结论:氮杂糖类化合物SZL能够抑制HeLa细胞的增殖,诱导细胞发生DNA损伤,通过干预线粒体途径实现细胞凋亡。     

Loss of volunteer blood donors because of unconfirmed enzyme immunoassay screening results. Retrovirus Epidemiology Donor Study

BACKGROUND: Blood donors who test repeatably reactive on enzyme immunoassay (EIA) and are not confirmed as positive are a continuing problem for blood banks. Units are discarded and donors are deferred, in spite of multiple studies indicating that such donors are very rarely infected with the transmissible agents. Few data are available, however, with which to evaluate whether the discarded units are more likely to come from particular demographic subgroups. STUDY DESIGN AND METHODS: The Retrovirus Epidemiology Donor Study database of over 2 million allogeneic whole-blood donations collected in the years 1991 through 1993 was utilized. The prevalence of false-positive and indeterminate test results within demographic subgroups was computed for antibodies to human immunodeficiency virus, hepatitis C virus, and human T-lymphotropic virus (anti-HIV, anti-HCV, anti-HTLV, respectively) and hepatitis B surface antigen (false-positive only) as the proportion of donations that were repeatably reactive on EIA but negative or indeterminate on the confirmatory or supplemental test. RESULTS: Several demographic groups with increased prevalence of false- positive and indeterminate anti-HIV results were the same females, younger age groups, blacks, and first-time donors. Likewise, many of the demographic subgroups with increased prevalence of false-positive and indeterminate anti-HCV results were similar: older age groups, non- whites, lower education levels, first-time donors, donors making directed donations, and donors who had received transfusions. For anti- HTLV, by contrast, the oldest group had the highest prevalence of false- positive results but the lowest prevalence of indeterminate results: blacks had the lowest prevalence of false positive results but the highest prevalence of indeterminate results. CONCLUSION: If units that test repeatably reactive on EIA but that are not confirmed as positive are almost always from individuals not infected with the virus in question, then these results indicate that there may be sex-, race-, and/or age-linked proteins cross-reacting with the test kit materials. Elucidation of these antigenic determinates and their subsequent removal should be a priority.     

参芪健胃颗粒抗炎效应

目的：观察参芪健胃颗粒对急性炎症模型的抗炎作用。方法：实验于2006—02／05在河南中医学院动物实验中心完成。①将50只昆明小鼠随机均分为5组：大、中、小剂量的参芪健胃颗粒组（2．4，1．2，0．6g/kg），三九胃泰颗粒组（10g／kg）及生理盐水组。给药体积0．02mL/g。灌服给药1次/d，连续给药5d，于最后1次给药后1h，每鼠右耳涂二甲苯，4h后处死小鼠，沿耳廓基线解剖位置仔细剪下全耳，对齐双耳，修剪，使每耳大小一致，迅速用分析天平称重，并计算肿胀度（肿胀度=右耳重-左耳重）。②将40只SD大鼠随机均分为5组：大、中、小剂量的参芪健胃颗粒组（16，8，4g／kg），三九胃泰颗粒组（6．7g／kg）及同体积生理盐水组。给药体积2mL/kg。灌服给药1次/d，连续给药5d，于最后1次给药后立即在大鼠足趾肿胀仪上测大鼠正常左后足跖体积，给药后30min立即给每鼠左后足跖皮下注射0．1mL新配制的10％新鲜鸡蛋清液，分别于给蛋清后30，60，120，240，360min再次在大鼠足趾肿胀仪上测大鼠左后足跖体积，并计算肿胀率[足跖肿胀率=（给药后不同时问足跖体积-同侧正常足跖体积），同侧正常足跖体积]。结果：50只小鼠和40只大鼠均进入结果分析。①大、中剂量的参芪健胃颗粒组和三九胃泰颗粒组耳壳肿胀度显著小于生理盐水组（P〈0．01），小剂量参芪健胃颗粒组小于生理盐水组（P〈0．05）。②与生理盐水组比，大剂量参芪健胃颗粒组和三九胃泰颗粒组在给药后30～60min均可明显抑制大鼠蛋清性足跖肿胀，使足跖肿胀率明显减小（P〈0．05），在给药后120-360min均可显著抑制大鼠蛋清性足跖肿胀，使足跖肿胀度明显减小（P〈0．01）；中剂量参芪健胃颗粒组在给药后30-120min可明显抑制大鼠蛋清性足跖肿胀，使足跖肿胀率明显减少（P〈0．05），在给药后240-360min可显著抑制大鼠蛋清性足跖肿胀，使足跖肿胀率显著减少（P〈0．01）；小剂量参芪健胃颗粒在给药后30～360min仅有抑制大鼠蛋清性足跖肿胀的趋势。结论：参芪健胃颗粒可显著抑制小鼠耳壳肿胀和大鼠足趾肿胀程度，有较好的抗炎作用。     

共病致大肠癌漏诊情况分析55例

目的:总结共病状态下大肠癌漏诊的具体原因.方法:回顾性分析55例漏诊大肠癌的临床资料,对率的比较采用卡方检验.结果:被漏诊的疾病中,大肠癌合并痔疮18例,合并消化性溃疡10例,合并结直肠息肉7例,合并急性阑尾炎5例,合并缺铁性贫血5例,合并慢性胆囊炎4例,合并溃疡性结肠炎3例,合并慢性盆腔炎2例,合并阑尾周围脓肿1例.结论:不少临床医师拘泥于“一因论”的思维模式而忽视了共病的存在,这是导致共病状态下大肠癌漏诊的主要原因.     

Red blood cell size is important for adherence of blood platelets to artery subendothelium

The hematocrit is one of the main factors influencing platelet adherence to the vessel wall. Raising the hematocrit causes an increase of platelet accumulation of about an order of magnitude. Our studies concern the role of red cell size. We have studied this effect using an annular perfusion chamber, according to Baumgartner, with human umbilical arteries and a steady-flow system. Normal human red blood cells (MCV 95 cu mu) increased platelet adherence sevenfold, as the hematocrit increases from 0 to 0.6. Small erythrocytes from goats (MCV 25 cu mu) caused no increment in adherence in the same hematocrit range. Rabbit erythrocytes (MCV 70 cu mu) caused an intermediate increase in adherence. Red blood cells from newborns (MCV 110-130 cu mu) caused a larger increase in platelet adherence than normal red cells at hematocrit 0.4. These results were further confirmed with large red blood cells from two patients. Experiments with small red cells (MCV 70 cu mu) of patients with iron deficiency showed that platelet adherence was similar to normal red cells, provided the red cell diameter was normal. Small red blood cells of a patient with sideroblastic anemia caused decreased adherence. These data indicate that red cell size is of major importance for platelet adherence. Red cell diameter is more important than average volume. However, for size differences in the human range, the hematocrit remains the dominant parameter.