Effects of disodium ascorbyl phytostanol phosphates (FM-VP4) on cholesterol accumulation within rat intestinal cells

The objective of this study was to determine whether FM-VP4, a novel compound derived from plant sterols, can effectively reduce cholesterol accumulation within rat intestinal epithelial crypt (IEC-6) cells. IEC-6 cells were cultured in Dulbeccos minimal essential medium (DMEM) containing 5% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.1 units/mL insulin at 37°C under a humidified 5% CO₂ atmosphere and seeded at 6.4×10⁴ cells/well in 48-well plates. Experiments were initiated 14 days postconfluence. IEC-6 cells were exposed to [³H]cholesterol micelles (containing oleic and taurcholic acids), co-incubated with FM-VP4 (0, 10, 50, and 100 μM) in Hepes Buffered Sterile Saline (HBSS). Cells were also preincubated with FM-VP4 prior to [³H]cholesterol micelle incubation to determine whether its effects are elicited intracellularly. The cellular localization of cholesterol was determined using digitonin. To determine the effects of cholesterol on the extent of FM-VP4 accumulation within IEC-6 cells, [³H]FM-VP4 was incubated with IEC-6 cells in the presence of unlabeled cholesterol micelles (0, 10, and 50 μM). The extent of [³H]cholesterol or [³H]FM-VP4 associated with cell monolayers was determined after cell lysis using liquid scintillation counting in a Beckman LS6500 Scintillation Counter. Dose-response and time course studies were performed in which control (no FM-VP4 treatment) and FM-VP4 (10–100 μM) were co-incubated with 50-μM [³H]cholesterol micelles from 1 minute to 24 hours. Incubation with only 50-μM FM-VP4 for less than 24 hours resulted in a 50% to 60% reduction (n=6, P<.05) in [³H]cholesterol associated with the monolayer compared with control (n=6). Preincubation of FM-VP4 did not elicit a significant reduction in cholesterol accumulation compared with control (n=6). Approximately 25% of the total [³H]cholesterol associated with the cells was determined to be cytosolic, while 75% was noncytosolic in the presence and/or absence of FM-VP4. [³H]FM-VP4 was also shown to associate with IEC-6 cells at similar concentrations to cholesterol with the most pronounced inhibition of FM-VP4 accumulation occurring at a cholesterol concentration of 50 μM. However, cholesterol-induced inhibition was detectable only after 1 hour of incubation. FM-VP4 inhibits cholesterol accumulation within IEC-6 cells and is most effective at equimolar concentrations with cholesterol. Our findings further suggest that the action of FM-VP4 is likely at the cell surface and not elicited intracellularly.     

PHB1 Efficient Strategies for Osteoporosis Testing

There are multiple screening and testing tools for osteoporosis. We need to understand the most cost-efficient way to utilize these tools to identify postmenopausal women with osteoporosis. The objective of this study was to identify efficient strategies for detecting low bone mass in postmenopausal women and estimate the incremental cost per case found.
METHODS: The study sample consists of 392 women age >50. Each participant completed the Simple Calculated Osteoporosis Risk Estimation (SCORE ^* ) (a prescreening questionnaire), and bone mineral density (BMD) levels were collected at different skeletal sites. Assumed costs were: $5 for SCORE, $35 for peripheral site (pDXA) testing at the forearm, $120 for single central (DXA) site testing at hip or spine, and $200 for multiple site DXA. An osteoporotic woman was defined as a woman with BMD < −2 SD below peak adult mean at any site.
RESULTS: The cost, efficient frontier consisted of 7 strategies ranging in cost from $33 to $189 per patient, with corresponding sensitivity of 53% to 100%. The incremental cost per case found ranged from $62.30 to $1,100. Most importantly, the current gold standard (testing all women at the hip and spine) is not on the efficient frontier.
CONCLUSION: The efficiency of osteoporosis testing can be greatly increased through the appropriate use of sequential instruments to identify postmenopausal women with osteoporosis.     

Microparticles of human atherosclerotic plaques enhance the shedding of the tumor necrosis factor-alpha converting enzyme/ADAM17 substrates, tumor necrosis factor and tumor necrosis factor receptor-1

Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 +/- 2007 TACE/ADAM17(+) MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304(TNF)), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304(TNF) cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17(+) MPs could regulate the inflammatory balance in the culprit lesion.     

小鼠胎盘间充质干细胞的生物学特性

目的:胎盘中包含大量能够自我更新、具有多向分化潜能的细胞,其低免疫原性几乎不引起移植物抗宿主反应疾病。观察体外培养的小鼠胎盘来源间充质干细胞的生物学特性。方法:实验于2006-01/11在首都医科大学组织胚胎学教研室实验室(国家中医药管理局三级实验室)完成。①实验方法:孕10dICR小鼠100只,取出子宫收集胎盘实体组织,经0.1%IV型胶原酶消化获得细胞,以3×106接种于25cm2培养瓶中,加入含体积分数0.1胎牛血清、200U/mL青链霉素的高糖DMEM培养基进行体外培养。②实验评估:每天在倒置显微镜下观察细胞形态;MTT检测细胞生长状况,记录生长曲线;流式细胞术分析细胞周期;免疫细胞化学方法分析胎盘间充质干细胞的免疫表型表达。结果:①小鼠胎盘间充质干细胞的形态学特征:原代培养初期仅有少量细胞贴壁,3d后贴壁细胞逐渐增多,培养过程中细胞呈梭形,散在、集落式分布,细胞集落随着增殖而逐渐融合。②小鼠胎盘间充质干细胞的生长特性:接种3d后细胞增殖速度加快,随后保持稳定增殖速度,第9天速度大幅增长达峰值,10d后进入平台期。G0/G1期、S期、G2期的比例分别为64.4%,25.2%,10.4%。③小鼠胎盘间充质干细胞免疫表型检测:原代培养的小鼠胎盘间充质干细胞CD105、CD44、波形蛋白均呈阳性表达。结论:小鼠胎盘来源的间充质干细胞可成功在体外培养并大量扩增,生物学性状稳定,是组织工程优良的种子细胞来源之一。     

Regulation of macrophage and granulocyte proliferation. Specificities of prostaglandin E and lactoferrin

Hemopoietic colony-forming cells committed to macrophage differentiation (M-CFC) are selectively and differentially inhibited by prostaglandin E (PGE). A hierarchy of sensitivity was observed among murine CFC stimulated by colony-stimulating factors (CSF) which differ in their ability to initiate proliferation of morphologically distinct colony types, or stimulated by CSF provided by macrophage feeder layers. Inhibition of macrophage colony formation to 50 percent levels occurred with PGE concentrations between 10(-8) and 10(-9) M, and was still evident at 10(-10) -10(-11) M PGE concentrations. The growth of mixed colonies containing both macrophages and neutrophils was less sensitive to the inhibitory effects of PGE, however, the monocytoid component of these colonies was reduced in the presence of PGE. Neutrophil progenitor cell proliferation was not influenced by PGE concentrations below 10(-6) M, regardless of time of addition of PGE, whereas clonal macrophage expansion, as well as clone size, was sensitive to inhibition by PGE when added as late as 3 d after culture initiation. Prostaglandin F(2α), was not inhibitory to colony formation. Experimental evidence for a selective role of macrophage PGE in the regulation of macrophage colony formation was directly provided by utilizing resident peritoneal macrophages as a source of CSF for bone marrow target cell overlays. Simultaneous morphological analysis of colonies proliferating in bilayer culture in response to increasing concentrations of macrophages, and direct measurements of PGE synthesized by an identical number of macrophages maintained in liquid culture demonstrate that a specific decline in macrophage colony formation occurs coincident with a linear increase in macrophage PGE synthesis. Inhibition of macrophage PGE synthesis by indomethacin results in the specific enhancement of macrophage colony formation. Furthermore, macrophage PGE synthesis is induced by CSF preparations with the selective capacity to differentially stimulate macrophage proliferation, but not by those which preferentially stimulate granulocyte colony formation. In comparison to the effects of PGE on M-CFC, polymorphonuclear granulocyte-derived lactoferrin (LF) reduces macrophage production of colony-stimulating activities for macrophage, mixed macrophage- neutrophil and neutrophil colony formation. The ability of LF to reduce macrophage PGE synthesis, presumably by decreasing CSF production, suggests that LF and PGE can interact in the control of macrophage and granulocyte proliferation.     

Removal of ABO-incompatible red cells from lymphocytapheresis and granulocytapheresis components before transfusion

BACKGROUND: The collection of allogeneic lymphocytapheresis and granulocytapheresis components containing significant volumes of ABO- incompatible red cells is sometimes necessary. STUDY DESIGN AND METHODS: Twenty-nine ABO-incompatible lymphocytapheresis components collected for transfusion to three patients and 11 ABO-incompatible granulocytapheresis components collected for transfusion to five patients were depleted of red cells by gravity sedimentation aided by the addition of hetastarch solution. The efficacy of red cell depletion and white cell retention and the complications of transfusion were analyzed. RESULTS: Lymphocytapheresis components contained 82 +/− 13 percent of the original white cells and 5 +/− 3 mL of red cells after depletion; however, for those components containing < 70 mL of red cells before depletion (n = 12), white cell recovery was 92 +/− 5 percent. After depletion, granulocytapheresis components contained 96 +/− 3 percent of the original white cells and 6 +/− 2 mL of red cells. No clinical or laboratory evidence of hemolysis was observed after the transfusion of any leukapheresis component. CONCLUSION: Red cells can be effectively removed from leukacytapheresis components by a simple gravity sedimentation technique with added hetastarch. This allows safe transfusion of ABO-incompatible components.     

额叶皮质灌注不足的纯构音障碍

目的:观察纯构音障碍患者病变部位及脑血流量的变化。方法:选择1988-06/1997-02在日本Hyogo医科大学第十五医院收治的12例右利手纯构音障碍患者。纳入标准:均符合构音障碍主要特点,包括发音不清、慢,低音韵律,且没有其他神经受累,如伴随其他神经症状也为轻度,如面部肌肉受累、舌肌受累,无口语听理解和书写能力障碍。年龄49～81岁,平均(65±13)岁,其中男10例,女2例。选择11名CT或MRI显示无脑损伤同期入院健康体检健康者作为对照组,平均(68±11)岁。所有纳入受试对象均对检测项目知情同意。①对病例组患者进行头部MRI检查,确定脑梗死部位。②对两组受试对象进行局部脑血流量检测,局部脑血流量半量值按以下方法计算:选取4×4象素的兴趣区(ROI),定量分析脑区摄取的123I-IMP。参考脑功能定位图谱,检查15mm×15mm×7.5mm大小的脑体积所对应的28个标准化位置,包括前额叶2个(中、侧面)、前盖部1个、前环部1个、前中运动区1个,感觉运动区2个(上部、下部)、顶叶2个(上、后)、颞叶3个(上、中、下)、枕叶1个、小脑1个,均选择左右两侧。每1区域的吸收由此区的每象素的平均数来确定。测量每个区域的吸收率[吸收率=2×(垂直棒)左-(垂直棒)右/L R]。结果:纳入12例患者和11名健康对照者全部进入结果分析。①病例组患者MRI检查结果:11例患者为两侧多发腔隙梗死,1例为单侧内囊-放射冠梗死。所有患者病变部位均在内囊或放射冠(3例为单侧、9例为双侧)受累。8例为内囊梗死,其中6例为后肢损伤、3例为膝部、1例在前肢。9例有放射冠梗死(3例在前部、2例在后部、4例前后部均受累),3例为桥脑梗死,1例为丘脑梗死,1例为基底节梗死。10例在T2加权MRI上显示脑室周围高信号。②病例组患者脑血流量检测结果:8例患者前额叶皮质呈低灌注状态,其中1例患者主要在旁矢状面和前盖区,其他区域皮质未检测到灌注不足。局部脑血流量半定量检测显示,病例组患者前额叶前部内侧、外侧、前环区、前盖及前运动区的脑血流量分别为(0.800±0.065),(0.821±0.064),(0.746±0.092),(0.803±0.093),(0.851±0.065),明显少于对照组[(0.875±0.081),(0.874±0.054),(0.835±0.073),(0.881±0.067),(0.932±0.055),t=-2.447,-2.125,-2.560,-2.970,-2.449,P<0.05-0.01]。结论:纯构音障碍多见于多发腔隙梗死患者,额叶皮质低灌注,尤其是前盖和额叶中部低灌注,可能是引起纯构音障碍的重要原因。