ICAP-1对脐静脉内皮细胞PI3K表达及细胞增殖的影响

目的 研究整合素胞浆结构域关联蛋白-1(ICAP-1)对人脐静脉内皮细胞(HUVEC)增殖及PI3K信号通路表达的影响,以进一步研究其生物学作用及其在血管形成中的作用机制.方法 通过构建PCDNA3.1-ICAP-1质粒并转染HUVEC,采用MTT法测定HUVEC增殖化,Western Blot检测ICAP-1及PI3K表达.结果 PCDNA3.1-ICAP-1转染组ICAP-1和PI3K表达明显增加,PCDNA3.1-ICAP-1转染组与正常对照组、PCDNA3.1-GFP转染组比较表达差异有统计学意义(P<0.05).PCDNA3.1-ICAP-1转染组较PCDNA3.1-GFP转染组和正常对照组细胞增殖能力增强,差异有统计学意义(P<0.05).结论 ICAP-1具有促进内皮细胞增殖的作用,ICAP-1可能通过上调PI3K表达,促进内皮细胞增殖达到促进血管新生的作用.     

同型半胱氨酸对人脐静脉内皮细胞增殖活性的影响及瑞舒伐他汀干预研究

目的血浆同型半胱氨酸(homocysteine,Hcy)水平升高被认为是动脉粥样硬化的独立危险因子;瑞舒伐他汀(rosuvastatin,RSV)是目前降低LDL-C最强的调脂药物兼有调脂以外的作用。本研究观察Hcy对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)增殖的影响以及RSV的保护作用。方法将HUVEC分为3组进行培养包括正常对照组、Hcy不同浓度刺激组和RSV干预组。通过MTT法测定各组细胞的增殖活力,realvtimePCR检测内皮细胞血管细胞间粘附因子1(VCAM-1)mRNA和细胞间粘附因子1(ICAM-1)mRNA的表达。结果 (1)用不同浓度的Hcy与HUVECs共孵育24 h后观察细胞增殖情况,从0.1～1 mmol/L随着Hcy浓度逐渐增大,细胞活性逐渐减少,0.4～1 mmol/L各组细胞活性均明显小于正常对照组(P<0.05);(2)RSV 5、2、1、0.5、0.1μmol/L浓度干预组VCAM-1mRNA和ICAM-1 mRNA表达较Hcy组明显下降(P>0.05)。结论 Hcy对HUVEC有明显抑制细胞增殖的作用;RSV能有效抑制Hcy对HUVEC的损伤,促进HUVEC增殖;RSV可浓度依赖性抑制Hcy诱导的内皮细胞ICAM-1mRNA和VCAM-1mRNA表达的上调。提示RSV的保护作用可能与抗炎作用有关。     

银杏叶提取物对内皮细胞的保护作用

目的 探讨银杏叶提取物(GBE)对血管紧张素Ⅱ(Ang Ⅱ)致体外培养人脐静脉内皮细胞(HUVEC)损伤的保护作用.方法 将处于对数生长期的HUVEC分为正常对照组、AngⅡ组及高、中、低3个GBE处理组,其中正常对照组用含0.5%小牛血清的DMEM培养基培养,Ang Ⅱ组在正常对照组培养基基础上含Ang Ⅱ 10-8mol/L,GBE处理组在AngⅡ组基础上分别加GBE 50、100、200 mg/L做为GBE低、中、高组,采用MTT法测定培养细胞增殖能力.并应用Western印迹法检测各组细胞中磷酸化Akt表达.结果 MTT法检测结果表明,Ang Ⅱ组细胞增殖能力明显高于正常对照组(P＜0.05),除GBE低剂量组外,GBE高、中剂量组A值均明显高于Ang Ⅱ组(P＜0.05);Western 印迹法检测各组培养细胞磷酸化Akt表达结果显示,与正常对照组比较,Ang Ⅱ组磷酸化Akt蛋白表达量明显降低(P＜0.05),GBE高剂量组磷酸化Akt表达明显高于Ang Ⅱ组(P＜0.05),且与正常对照组比较,差异无统计学意义,GBE中、低剂量组磷酸化Akt蛋白表达量明显降低(P＜0.05),但仍显著高于Ang Ⅱ组(P＜0.05).结论 Ang Ⅱ导致血管内皮细胞增殖能力下降可能与其抑制磷酸化Akt表达量有关,GBE可通过上调Akt表达抵抗Ang Ⅱ引起的细胞损伤.     

miR-206对血管内皮细胞增殖的影响及其机制

目的研究miR-206对血管内皮细胞增殖的影响及其机制,为临床动脉硬化疾病提供新的诊疗方向。方法收集临床病理首次确诊为糖尿病下肢动脉粥样硬化病变的患者病变处动脉内皮组织和正常内皮组织,qPCR检测miR-206的表达;而后通过基础实验研究将HUVEC分为未转染组、miR-206-mimics组和miR-206-inhibitor组,观察转染效率;三组细胞均采用CCK8检测细胞增殖情况,Transwell进行迁移能力评估,流式细胞仪检测细胞周期情况,Western blot检测Cx43表达情况。结果糖尿病动脉硬化患者下肢动脉(均为胫腓动脉)病变处内皮细胞miR-206的mRNA表达较正常组织内皮细胞明显增加(P0.05);HUVEC转染miR-206-mimics后,miR-206表达显著升高(P0.05),转染miR-206-inhibitor后,miR-206表达明显降低(P0.05);在HUVEC转染miR-206-mimics 24 h后,细胞增殖率相比于未转染组和miR-206-inhibitor组要明显降低(P0.05);在HUVEC转染miR-206-mimics后,细胞迁移能力明显降低(P0.05),细胞周期在G2期发生阻滞,而HUVEC转染miR-206-inhibitor后细胞迁移能力提高(P0.05);miR-206-mimics组细胞Cx43表达明显降低,而miR-206-inhibitor组细胞Cx43表达明显增加。结论 miR-206可以抑制血管内皮细胞的增殖,将内皮细胞阻滞在有丝分裂G2期,并且抑制内皮细胞迁移,其对内皮细胞增殖的影响可能是通过调节Cx43表达来实现的。     

激酶非催化的C-叶域蛋白质1对人脐静脉内皮细胞衰老作用的影响及机制

目的:探讨激酶非催化的C-叶域蛋白质1(KNDC1)对人脐静脉内皮细胞(HUVEC)衰老作用的分子机制。方法:体外分离培养HUVEC,选择第6代HUVEC转染KNDC1腺病毒为实验组,另设空白对照组与阴性对照组。显微镜下观察细胞形态变化,通过q PCR检测内皮细胞的KNDC1的mRNA的表达,用β-半乳糖苷酶染色检测细胞衰老情况,Westem blot检测衰老相关蛋白,分光光度法检测内皮细胞抗氧化酶活性。结果:第6代的HUVEC呈现出有别于前期传代细胞的衰老特征,细胞变得扁平,体积增大,增殖能力降低;实验组KNDC1的mRNA水平较对照组增高(239%~344%,P<0.05)及蛋白质表达也显著增高(249%~441%,P<0.05),实验组HUVEC的SA-β-gal染色阳性率显著上升(P<0.01),与KNDC1腺病毒转染剂量呈剂量依赖性。实验组磷酸化p53较对照组增加[(244.3±19.5)vs.(113.5±9.0),P<0.05]及抗氧化酶SOD及GPX含量(0.84±0.079);(0.82±0.062)较对照组下降,差异有统计学意义(P<0.05)。结论:KNDC1过度表达可能通过启动p53信号通路增加ROS表达,进而加速细胞衰老的进程。     

通络药物对血管内皮细胞与单核细胞黏附作用的影响

目的:观察氧化低密度脂蛋白(ox-LDL)损伤的血管内皮细胞(HUVEC)与人白血病单核细胞(THP-1)的黏附作用,以及通络药物(通心络超微粉溶液和人参皂苷Rb1)干预的影响。方法:采用ox-LDL建立血管内皮细胞损伤模型,将细胞分为正常对照组、模型组、通心络组和人参皂苷Rb1组。实验采用MTS比色法检测ox-LDL损伤的HUVEC的生存活性,用活细胞染色方法观察不同组HUVEC与THP-1细胞的黏附率,用酶联免疫吸附测定(ELISA)方法检测HUVEC培养上清中单核细胞趋化因子(MCP-1)、可溶性血管内皮细胞间黏附分子(s VCAM-1)、可溶性内皮细胞间黏附分子(sICAM-1)、E-选择素(E-selectin)的水平,用蛋白免疫印迹法(Western Blot)检测各组HUVEC条件培养基培养的单核细胞趋化因子受体2(CCR2)、极迟抗原4(VLA-4)、巨噬细胞分化抗原-1(Mac-1)的表达。结果:与正常对照组(100.00±1.31)%比较,模型组HUVEC生存活性降低为正常对照组的(75.57±1.02)%,通心络组和人参皂苷Rb1组HUVEC生存活性明显分别提高了(99.25±1.40)%、(99.48±2.15)%。与正常对照组比较,模型组黏附于HUVEC的THP-1细胞数量明显增多,与模型组比较,通心络组和人参皂苷Rb1组黏附于HUVEC细胞的THP-1细胞数量减少。通心络组和人参皂苷Rb1组HUVEC细胞培养上清中MCP-1、sVCAM-1、sICAM-1、E-selectin的水平和THP-1细胞上相应受体CCR2、VLA-4、Mac-1的表达较模型组降低。上述比较差异均有统计学意义(P0.05)。结论:通心络和人参皂苷Rb1能够保护HUVEC,减少ox-LDL损伤的血管内皮细胞趋化、黏附分子的分泌及单核细胞上相应受体的表达,从而抑制单核细胞向受损血管内皮的趋化黏附。     

芎芍胶囊含药血清对人脐静脉内皮细胞增殖的影响

目的研究芎芍胶囊含药血清对人脐静脉内皮细胞(HUVEC)的增殖作用。方法采用血清药理学和体外培养HUVEC的方法,通过倒置显微镜观察、MTT法和流式细胞术(FCM)检测芎芍胶囊高、中、低3个浓度含药血清对HUVEC增殖的影响。结果MTT法检测结果显示高、中、低剂量的芎芍胶囊含药血清均有促进HUVEC增殖的作用,增殖率分别为54.14%、54.98%、33.77%;FCM法检测发现高、中、低剂量芎芍胶囊含药血清均可增加S期细胞比例,分别为52.14%、42.65%、37.08%;显微镜观察结果也显示各含药血清具有促进HUVEC增殖的作用。结论芎芍胶囊能促进HUVEC的增殖,其促进细胞增殖的作用与浓度有一定的关系;提示芎芍胶囊的促血管新生作用可能是通过促进血管内皮细胞的增殖。     

NF-κB参与银杏叶提取物保护血管内皮细胞的作用

目的阐明银杏叶提取物(GBE)对H2O2介导的人脐静脉内皮细胞(HUVEC)损伤的保护作用。方法 HUVEC分为对照组、H2O2处理组、50 mg/L及100 mg/L GBE处理组。用四甲基偶氮唑蓝(MTT)法检测各组细胞增殖能力。Western印迹分析各组细胞(NF-κB P65)表达。结果H2O2处理组细胞增殖率明显低于对照组(P0.01);GBE高、低剂量组细胞增殖能力明显高于H2O2处理组(P0.01,P0.05)。各组细胞细胞核NF-κB P65表达量差异有显著性,与H2O2处理组比较,GBE高、低剂量组NF-κB P65核/浆比明显升高(P0.01)。结论 GBE通过NF-κB通路影响血管内皮细胞的增殖能力。     

沙利度胺对系统性红斑狼疮患者外周血T淋巴细胞的免疫调节作用

目的 探讨沙利度胺(Thd)对系统性红斑狼疮(SLE)患者外周血T淋巴细胞的免疫凋节作用.方法 用四甲基偶氮唑蓝(MTT)比色法检测Thd对SLE患者外周血T淋巴细胞增殖的影响,用流式细胞术检测T淋巴细胞早期凋亡及CD3~+CD28~+和CD8~+CD152~+的表达,用实时定量聚合酶链反应(PCR)检测T淋巴细胞白细胞介素(IL)-6、IL-10、肿瘤坏死因子(TNF)-a mRNA的表达,采用单因素方差分析进行统计学处理.结果 在体外,500 μg/ml Thd组CD3~+CD28~+表达为(48±9)%显著低于对照组(57±9)%(P<0.05)、500μg/ml Thd组T淋巴细胞凋亡率为(36±8)%显著高于对照组(23±5)%(P<0.05);100、300、500μg/ml Thd组A_(570nm)值分别为:0.39±0.05、0.34±0.04、0.30±0.03较对照组(0.51±0.07)均有明显下降(P<0.05);100、500 μg/ml Thd组CD8~+CD152~+表达分别为(5.0±0.6)%、(7.8±0.7)%,明显高于对照组(4.2±0.6)%(P<0.05);500μg/ml Thd能抑制IL-6 mRNA的表达,各剂量组均抑制IL-10、TNF-a mRNA表达.结论 Thd可能通过抑制T淋巴细胞增殖、CD28的表达、IL-6、IL-10、TNF-a mRNA的表达和促进T淋巴细胞凋亡、CD152表达来下调SLE患者的免疫反应.     

高糖影响内皮细胞功能不良机制的初步探究

目的探讨高糖对人脐静脉内皮细胞(HUVEC)功能不良的机制。方法分离并培养HUVEC,取第3代细胞分为正常对照组、甘露醇对照组(33mmol/L)和高糖对照组(33mmol/L),处理36h后,用RT-PCR和Western blot测哺乳动物雷帕霉素靶蛋白雷帕霉素不敏感配体(RICTOR)表达情况。转染RICTOR过表达腺病毒为高糖腺病毒组,转染空白病毒AD-GFP作为高糖空病毒组,检测蛋白激酶B(Akt)和内皮型一氧化氮合酶(eNOS)的磷酸化水平,用硝酸还原酶法测定NO释放量。结果高糖对照组RICTOR蛋白表达量较正常对照组显著降低(1.00±0.16 vs 2.69±0.07,P<0.01)。与高糖空病毒组比较,高糖腺病毒组RICTOR表达增加(0.57±0.03 vs0.29±0.02,P<0.01),磷酸化Akt-Ser473(0.95±0.05 vs 0.56±0.04,P<0.01)及磷酸化eNOS-Ser1177增加(0.97±0.05 vs 0.55±0.07,P<0.01),内皮细胞NO释放增多(0.85±0.06 vs 0.56±0.04,P<0.05)。结论纠正RICTOR表达能改善高糖诱发的血管内皮细胞功能不良。     

Degradation of human calcitonin in human plasma

The degradation in vitro of human calcitonin (HCT) in plasma from normal subjects and patients with disorders of calcium metabolism was studied. As measured by radioimmunoassay, the hormone was stable at 37°C in phosphate buffer for 24 hr but progressively disappeared when incubated in normal human plasma. The loss was temperature and pH dependent, being maximal at 37°C between pH 6.0 and 7.0. Under these conditions ¹²⁵I-HCT appeared to be degraded to at least one labeled fragment that behaved on polyacrylamide gels as if it were smaller than 1000 daltons. The rate of formation of this fragment correlated well with the rate of loss of immunoreactive HCT. Compared to plasma from normal individuals, plasma from patients with hypercalcemia due to causes other than hyperparathyroidism degraded HCT significantly more rapidly. The mean loss of HCT in 5 hr in plasma from nine such patients was 54% ± 17% (± 2 SEM), compared to 22% ± 5% in plasma from 22 healthy volunteers (p < 0.001). Those patients whose plasma most rapidly degraded HCT had milkalkali syndrome, metastatic carcinoma of the colon, metastatic oat cell carcinoma of the lung, and metastatic breast carcinoma. Rates of HCT degradation in plasma from eight hypercalcemic patients with hyperparathyroidism and seventeen normocalcemic patients with malignancy were within the normal range. From these findings we conclude that human plasma contains one or more enzymes that degrade HCT and that the hormone is degraded more rapidly in plasma from some patients with hypercalcemia.     

Interaction of human chorionic gonadotropin and human luteinizing hormone with human thyroid membranes

In an attempt to identify a possible pathogenetic role for the hCG molecule in the mechanism of the hyperthyroidism which occurs in choriocarcinoma, we have looked for evidence that the hCG molecule has a thyrotropic action on the human thyroid. The thyrotropic activity of various hCG preparations on the human thyroid was assessed by measuring the stimulation of adenylate cyclase activity in human thyroid plasma membranes purified by sucrose density gradient centrifugation. The highly purified hCG CR119 preparation stimulated human thyroid adenylate cyclase activity. Its activity was more than 654 times greater than could be accounted for by human TSH (hTSH) contamination of the preparation, as determined by RIA. The thyrotropic activity intrinsic to 1.0 IU hCG was equivalent to roughly 0.27 microU hTSH. Significant saturable binding of the 125I-labeled highly purified hCG preparation to human thyroid membranes was demonstrated, and the bound component was characterized. Its apparent molecular size, subunit composition, and testis receptor-binding characteristics were those of the hCG molecule. Examination of a crude urinary hCG preparation in adenylate cyclase and TSH radioligand assays using human thyroid membranes showed no evidence of any molecule other than hCG with a thyrotropic action on the human thyroid. Given that hCG binds to and stimulates adenylate cyclase activity in human thyroid tissue, as the above data indicate, then human LH (hLH) would be expected to do the same, since hLH and hCG have such strong structural and functional similarities. As anticipated, a highly purified hLH preparation exhibited TSH binding inhibition and adenylate cyclase stimulation. Its activity was more than 1030 times greater than could be accounted for by hTSH contamination of the preparation. The thyrotropic activity intrinsic to 1.0 IU hLH was equivalent to roughly 44 microU hTSH. Thus, in addition to other shared properties, the hLH molecule and the hCG molecule share the ability to interact with human thyroid tissue. These results strongly indicate that the hCG molecule has a thyrotropic action on the human thyroid and support the hypothesis that hCG is the thyrotropic factor that mediates the hyperthyroidism which occurs in patients with hCG-secreting neoplasms.     

Degradation of human calcitonin in human plasmas.

The degradation in vitro of human calcitonin (HCT) in plasma from normal subjects and patients with disorders of calcium metabolism was studied. As measured by radioimmunoassay, the hormone was stable at 37°C in phosphate buffer for 24 hr but progressively disappeared when incubated in normal human plasma. The loss was temperature and pH dependent, being maximal at 37°C between pH 6.0 and 7.0. Under these conditions ¹²⁵I-HCT appeared to be degraded to at least one labeled fragment that behaved on polyacrylamide gels as if it were smaller than 1000 daltons. The rate of formation of this fragment correlated well with the rate of loss of immunoreactive HCT. Compared to plasma from normal individuals, plasma from patients with hypercalcemia due to causes other than hyperparathyroidism degraded HCT significantly more rapidly. The mean loss of HCT in 5 hr in plasma from nine such patients was 54% ± 17% (± 2 SEM), compared to 22% ± 5% in plasma from 22 healthy volunteers (p < 0.001). Those patients whose plasma most rapidly degraded HCT had milkalkali syndrome, metastatic carcinoma of the colon, metastatic oat cell carcinoma of the lung, and metastatic breast carcinoma. Rates of HCT degradation in plasma from eight hypercalcemic patients with hyperparathyroidism and seventeen normocalcemic patients with malignancy were within the normal range. From these findings we conclude that human plasma contains one or more enzymes that degrade HCT and that the hormone is degraded more rapidly in plasma from some patients with hypercalcemia.     

Effect of human recombinant Endostatin protein on human angiogenesis

Tumor growth and metastasis are dependent on the development of new blood vessels. Inhibitors of new vessel growth have been widely investigated as anti-tumor agents. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII inhibits endothelial cell proliferation, induces endothelial cell apoptosis, and can both inhibit and reverse tumor growth in mice. However, human recombinant endostatin has had limited testing against human tissue targets. To investigate the effect of human endostatin on a human vessel target over a broad range of concentrations (10^–12–10^–4 M), human placental vein disks were grown for a period of 2 weeks in a 0.3% fibrin clot overlayed with growth medium. Disks from five individual placentas were tested. For each placenta utilized, a control (medium and 20% fetal bovine serum [FBS]) group and a group treated with heparin (300 g/ml) and hydrocortisone 21-phosphate (350 g/ml) (heparin-steroid) at a dose known to inhibit angiogenesis were included. Endostatin was tested at concentrations of 10^–12–10^–4 M in medium containing 20% FBS. The rate of initiation and the angiogenic growth index (on a visually graded semi-quantitative scale of 0–16) were determined for all experimental conditions. Endostatin inhibited angiogenesis in our model only in high concentrations. At 10^–5 M, endostatin did not alter the percent of wells that initiated an angiogenic response, but significantly inhibited subsequent vessel growth. At 10^–4 M, endostatin was able to inhibit both initiation and subsequent new vessel growth. Human endostatin can inhibit the initiation of a human angiogenic response and inhibit the subsequent proliferation of human neovessels when used at high doses in a continuous exposure model.     

Absence of a directly causative role for human herpesvirus 7 in human lymphoma and a review of human herpesvirus 6 in human malignancy

 In search of a (new) viral etiological agent, we screened 64 lymph node samples from Hodgkin's disease (HD) and 43 samples (32 lymph node and 11 skin biopsies) from non-Hodgkin's lymphoma (NHL) for human herpesvirus 7 (HHV-7). Twenty-nine control samples were tested as well, including 17 with benign lymphadenopathy. None of the samples tested positive by Southern blot hybridization using HHV-7-specific probes. We conclude that there is no major HHV-7 load in human lymphoma and that HHV-7 is not likely to be directly involved in its etiology. This is in contrast to a small minority of human lymphoproliferative diseases in which HHV-6 can be found at high copy number, but in which an etiological role is still uncertain. Received: September 8, 1998 / Accepted: November 2, 1998     

Comparison of recombinant human thrombin and plasma-derived human alpha-thrombin

Bleeding can be a serious complication of surgery, and topical thrombin is widely used as an adjunct to hemostasis in diverse surgical settings. The potent hemostatic properties of thrombin derive from its ability to activate platelets directly to aggregate and adhere to damaged vessels and to catalyze the formation simultaneously of a fibrin matrix. Application of exogenous thrombin bypasses the physiological process of generating a thrombin burst by directly initiating the terminal reactions of blood clot formation. Currently, thrombin used to control surgical bleeding is primarily from bovine plasma, with a small percentage from human plasma. Human thrombin isolated from pooled plasma carries the risk of transmitting plasma-borne pathogens or prion diseases. The bovine preparations have been associated with protein and preparative contaminants that pose potential risks of developing cross-reacting antibodies. There is a need for a pure therapeutic preparation of human thrombin. Recombinant human thrombin (rhThrombin) has been efficiently produced from a prethrombin-1 precursor obtained from Chinese hamster ovary cell culture. This rhThrombin is substantially free of process-derived contaminants and has been characterized extensively in terms of composition, primary, secondary, and tertiary structure, enzymatic activity; and in vivo pharmacology. In vivo studies of topically applied rhThrombin have shown it is effective in achieving hemostasis in a rabbit liver excisional wound model. Clinical studies are ongoing to evaluate the safety and efficacy of rhThrombin as an adjunct to hemostasis in patients undergoing surgery.     

Effects of zymosan-activated human granulocytes on isolated human airways

In asthma a temporal association exists between the late allergic reaction (LAR), the influx of granulocytes into the airway wall, and an increase in bronchial responsiveness. We therefore tested the hypothesis that activated human granulocytes constrict isolated human airways and increase their sensitivity to cholinergic stimuli. Bronchial rings were dissected from 23 lung tissue specimens collected at thoracotomy and studied isotonically in organ baths. Airways were incubated with 1, 2, 5, 10, or 20 x 10(6) granulocytes from normal or atopic donors. Activation of the cells with serum-treated zymosan (STZ, 0.2 mg/ml), which itself did not alter baseline airway caliber, resulted in a bronchoconstriction proportional to the number of zymosan-activated granulocytes (ZAG) present (rs = 0.79, p less than 0.001). This contraction was reduced by about 70% with the leukotriene C4/D4 receptor antagonist FPL 55712 (11.5 microM; p less than 0.001) or with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM; p less than 0.001). The scavengers of activated oxygen molecules superoxide dismutase (300 U/ml) and bovine catalase (5,000 U/ml), the cyclooxygenase inhibitor indomethacin (10 microM), or the histamine (H1) receptor antagonist mepyramine (2.8 microM) had no effect. Granulocyte suspensions from atopic donors contained more eosinophils (p less than 0.001), and the magnitude of the contraction to 10 x 10(6) ZAG was related to the proportion of eosinophils (rs = 0.66, p less than 0.01). The sensitivity of the airways to methacholine was unchanged in the presence of 1, 2, or 5 x 10(6) ZAG and decreased with 10 or 20 x 10(6) ZAG (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)