Polymorphisms of the estrogen receptor alpha (ESR1) gene and the risk of Alzheimer's disease in a southern Chinese community

Background: Alzheimer's disease (AD) is a neurodegenerative disease with a higher prevalence in women. Expression of estrogen receptor 1 (ESR1) gene has been identified throughout the brain. Owing to the putative neuroprotective effects of estrogen, estro

Presence of estrogenic activity from emission of fossil fuel combustion as detected by a recombinant yeast bioassay

Estrogenic activities of emission samples generated by fossil fuel combustion were investigated with human estrogen receptor (ER) recombinant yeast bioassay. The results showed that there were weak but clear estrogenic activities in combustion emissions of fossil fuels including coal, petroleum, and diesel. The estrogenic relative potency (RP) of fossil fuel combustion was the highest in petroleum-fired car, followed by coal-fired stove, diesel-fired agrimotor, coal-fired electric power station. On the other hand, the estrogenic relative inductive efficiency (RIE) was the highest in coal-fired stove and coal-fired electric power station, followed by petroleum-fired car and diesel-fired agrimotor. The estrogenic activities in the sub-fractions from chromatographic separation of emitted materials were also determined. The results indicated that different chemical fractions in these complex systems have different estrogenic potencies. The GC/MS analysis of the emission showed that there were many aromatic carbonyls, big molecular alcohol, PAHs and derivatives, and substituted phenolic compounds and derivatives which have been reported as environmental estrogens. The existence of estrogenic substances in fossil fuel combustion demands further investigation of their potential adverse effects on human and on the ecosystem. The magnitude of pollution due to global usage of fossil fuels makes it imperative to understand the issue of fossil fuel-derived endocrine activities and the associated health risks, particularly the aggregated risks stemmed from exposure to toxicants of multiple sources. (C) 2003 Elsevier Science Ltd. All rights reserved.

Environmental signals: Synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS 1500 was applied. The in vivo results showed that HS 1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSH beta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals. (c) 2005 Elsevier Ltd. All rights reserved.

Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials

Numerous environmental pollutants have been detected for estrogenic activity by interacting with the estrogen receptor, but little information is available about their interactions with the progesterone receptor. In this study, emission samples generated by fossil fuel combustion (FFC) and air particulate material (APM) collected from an urban location near a traffic line in a big city of China were evaluated to interact with the human progesterone receptor (hPR) signaling pathway by examining their ability to interact with the activity of hPR expressed in yeast. The results showed that the soot of a petroleum-fired vehicle possessed the most potent anti-progesteronic activity, that of coal-fired stove and diesel fired agrimotor emissions took the second place, and soot samples of coal-fired heating work and electric power station had lesser progesterone inhibition activity. The anti-progesteronic activity of APM was between that of soot from petroleum-fired vehicle and soot from coal-fired establishments and diesel fired agrimotor. Since there was no other large pollution source near the APM sampling sites, the endocrine disrupters were most likely from vehicle emissions, tire attrition and house heating sources. The correlation analysis showed that a strong relationship existed between estrogenic activity and anti-progesteronic activity in emissions of fossil fuel combustion. The discoveries that some environmental pollutants with estrogenic activity can also inhibit OR activity indicate that further studies are required to investigate potential mechanisms for the reported estrogenic activities of these pollutants. (c) 2005 Elsevier B.V. All rights reserved.

Differing estrogen activities in the organic phase of air particulate matter collected during sunny and foggy weather in a Chinese city detected by a recombinant yeast bioassay

Total air suspended particles (PM 100) collected from an urban location near a traffic line in Wuhan, China, were examined for estrogen using a recombinant yeast bioassay. Wuhan, located at the central part of China, is the fourth biggest city in China with 7 million populations. Today, Wuhan has developed into the biggest city and the largest traveling center of central China, becoming one of the important bases of industry, education and research. Wuhan is right at the confluent point of Yangzi River, the third longest river in the world, and its largest distributary Hanjiang, with mountains and more than 100 takes in downtown area. Therefore, by its unique landscape, Wuhan has formed clear four seasons with relatively long winter and summer and short spring and autumn. Foggy weather usually happen in early spring. The yeast line used in this assay stably expresses human estrogen receptor-alpha. Weak but clear estrogenic activities were detected in the organic phase of crude extracts of air particle materials (APM) in both sunny and foggy weather by 0.19-0.79 mug E2/gPM(100) which were statistically significantly elevated relative to the blank control responding from 20% to 50% of the maximum E2 response, and the estrogenic activity was much higher in foggy weather than in sunny weather. The estrogenic activities in the sub-fractions from chromatographic separation of APM sampled in foggy days were also determined. The results indicated that the responses of the fractions were obviously higher than the crude extracts. Since there is no other large pollution source nearby, the estrogenic material was most likely from vehicle emissions, house heating sources and oil fumes of house cooking. The GC/MS analysis of the PM100 collected under foggy weather showed that there were many phenol derivatives, oxy-PAHs and resin acids which have been reported as environmental estrogens. These results of the analysis of estrogenic potency in sunny and foggy weather in a subtropical city of China indicate that further studies are required to investigate the actual risks for the associated health and atmospheric system. (C) 2004 Elsevier Ltd. All rights reserved.

瓦山安息香中苯并呋喃促雌激素合成的机理研究

雌激素是人体内重要的激素之一，具有广泛的生理功能。雌激素缺乏与许多疾病相关，如卵巢功能低下，更年期综合征以及骨质疏松等；雌激素过剩也将导致某些疾病，如乳腺癌、卵巢癌、子宫内膜癌等。目前，如何降低肿瘤组织中的雌激素水平而达到治疗肿瘤的目的，已经得到广泛的研究，但促雌激素生成或调节卵巢功能药物或其相关研究则很少。 本实验室前期的研究发现，瓦山安息香属植物果实中的乙醇提取物具有促雌激素生成作用，通过活性追踪和结构鉴定，确认促E2 生成的主要成分为苯并呋喃类化合物。苯并呋喃类化合物的作用与芳香酶有关，但其确切的作用机理有待证实和深入研究。 为了探讨安息香苯并呋喃类化合物的促雌激素合成的作用机理，拟采用如下的实验方案： 1、细胞学方面，对小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、IGROV1 等细胞株，采用RT-PCR 和ELISA 方法研究芳香酶Aro基因的表达和雌二醇E2 的生成，芳香酶抑制剂Formestane 作为阳性对照，研究时效曲线和量效曲线，确定安息香苯并呋喃类化合物SP25 的有效浓度和作用时间。 2、RNAi 方面，设计合成了针对人芳香酶Aro基因的3 对RNAi 序列，转染入细胞，芳香酶促进剂Forskolin 和地塞米松、芳香酶抑制剂Formestane 作为阳性对照，采用实时定量PCR 技术，研究RNA 干扰后，安息香苯并呋喃类化合物SP25 对人芳香酶Aro基因表达水平瓦山安息香苯并呋喃促雌激素合成的机理研究的影响。 3、雌激素受体方面，设计一段ERE 的雌激素调控元件，构建重组荧光素酶报告基因载体，瞬时转染人乳腺癌细胞株MDA-MB-231，建立针对雌激素受体的报告基因筛选模型，观察安息香苯并呋喃类化合物SP25 对雌激素受体的选择性和亲和力，从受体水平考察安息香苯并呋喃类化合物SP25 促进雌激素生成的药理学机理。 实验结果显示： 1、分化后的小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7 、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-8 等细胞株具有芳香酶基因的表达。睾酮向雌二醇的转化能够被芳香酶抑制剂Formestane 所阻断，其中OVCAR-3 最适合进行下一步的RNAi研究。 2、RNAi 实验结果显示，设计的3 对RNAi 序列中R2 的干扰效果最强，相应的阴性对照C2 与R2 的表达量相差118 倍（24 小时）和19 倍（48 小时），显示R2/C2 这组序列可用于进一步的RNAi 试验。以R2 干扰OVCAR-3 细胞株，药物作用24、48 小时后，芳香酶抑制剂Formestane 与R2 相对表达量相比分别为0.83 倍和0.04 倍；芳香酶促进剂Forskolin 与R2 相对表达量相比分别为3.61 和1.84 倍；芳香酶促进剂地塞米松与R2 相对表达量相比分别为5.76 倍和3.49倍；苯并呋喃类化合物SP25 与R2 相对表达量相比分别为8.13 倍和4.59 倍。实验证实安息香苯并呋喃类化合物SP25 能够促进因RNAi 而发生基因沉默的人芳香酶Aro表达水平的上调。 3、雌激素受体实验结果显示，构建成功重组pERE-pGL3-promoter 荧光素酶报告基因载体和基于报告基因系统的雌激素受体激动剂或拮抗剂的细胞筛选模型。实验结果表明安息香苯并呋喃类化合物SP25 与雌激素受体ERα和ERβ亲和力选择性之比约为3：1 ，SP25通过与雌激素受体ERα结合作用其受体，刺激芳香酶的表达。 本课题通过RNA 干扰、ELISA、荧光实时定量PCR、报告基因筛选模型等技术手段，从细胞水平、蛋白酶水平和基因表达水平、雌激素受体水平等方面系统地研究了从瓦山安息香属植物果实中提取的苯并呋喃SP25 促进促雌激素生成的机理研究。试验结果显示苯并呋喃类化合物SP25 促雌激素生成的主要作用机制是直接促进芳香酶基因表达水平，以及与雌激素受体a 结合，刺激芳香酶活性。 Estrogen is an important hormone that has versatile physiologicalfunctions. Lack of estrogen will lead to many diseases such as lower ovarianfunction, climacteric syndrome and osteoporosis. Excessive estrogen alsoinduces breast carcinoma, oophoroma and endometrial carcinoma and otherdiseases. To depress the estrogen level in tumor tissue to cure carcinomawas widely studied, but there is only few studies reported on the induction ofestrogen and on the regulation of ovary function. We found that the extracts from seeds of Styrax perkinsiae couldpromote the synthesis of estrogen. The active compounds benzofurans wereidentified. Effect of benzofurans may be related to aromatase, but the mechanism was not clear. To reveal the mechanism of these benzofurans to promote estrogensynthesis, the following protocols were adopted: 1 Cytology: 3T3-L1 preadipocytes,human ovary carcinoma celllines OVCAR-3,OVCAR-4,OVCAR-5,OVCAR-8,IGROV1 andbreast carcinoma cell lines MCF-7 and MDA-MB-231 were usedto determine Aro gene expression and estrogen production withRT-PCR AND ELISA methods. Formestane, an aromataseinhibitor, was used as positive control. And dose-curve,time-curve and the effective concentration of SP25 were also studied. 2 Designed 3 pairs of RNAi for human aromatase gene, andtransfected into cell. Aromatase inducer Forskolin andDexamethasone, and aromatase inhibitor Formestane were usedas positive controls. We studied the change of Aro expressionlevel with SP25 by using real-time PCR after RNA interfering. 3 Estrogen Receptor: We constructed the recombined Luciferasereport vector and establish a screening system for estrogenagonist and antagon. With this system, we studied the affinity ofSP25 and estrogen receptor. Results: 1 Differentiated 3T3-L1 preadipocytes¡¢human ovary carcinomacell lines:OVCAR-3, OVCAR-4, OVCAR-8 and breast carcinomacell lines MCF-7, MDA-MB-231 had detected aromatase geneexpression.And OVCAR-3 is more suitable for further aromatasegene function research. 2 In RNAi assay, R2 has a strong interfering effcet in OVCAR-3 cellline, and ratio of C2 (the negative control) to R2 were 118 times(24 hours) and 19 times (48 hours). This means sucessful inRNA interfering. After R2 acted on OVCAR-3 cell line, the ratiosof formestane to R2 were 0.83 and 0.04 times, 5.76 and 3.49times (Dex), 3.61 and 1.84 times (forskolin) and 8.13 and 4.59times (sp25) after drug treated 24 or 48 hours respectively.These results indicated that SP25 can directly induce aromatasegene up-regulation. 3 We had constructed pERE-pGL3-promoter recombined vectorand the Luciferase report gene screening system. Luciferasereport gene assay showed that sp25 had a higher affinity with strogen receptor alpha than estrogen receptor beta, this indicated that SP25 can act on estrogen receptor and induce aromatase. Our results revealed that the mechanisms of benzofuran to promoteestrogen were the upregulation aromatase gene expression and promotion ofaromatase activity and have partially elective affinity with estrogen receptoralpha.

水稻中OsERD2和OsRAA1基因的功能分析

内质网中一些可溶性蛋白含有Lys-Asp-Glu-Leu（KDEL）基序作为内质网滞留的信号，这些内质网滞留蛋白可以离开内质网进入高尔基体进行糖基化修饰。目前的研究表明，KDEL基序可以被滞留蛋白受体识别，通过反向运输途径将其运回内质网。ERD2是第一个在酵母中被鉴定的内质网滞留蛋白受体。在人、拟南芥、弓形虫等生物中也鉴定出类似的内质网滞留蛋白受体。ERD2在拟南芥中的同源基因aERD2受内质网胁迫信号的诱导，在水稻中还未见该类受体的报道。 本工作从水稻中克隆到ERD2的同源基因OsERD2。OsERD2的cDNA全长为1081bp，编码一含215个氨基酸的蛋白。OsERD2与酵母、拟南芥、人中的内质网滞留蛋白受体的同源性分别为43.38%、72.56%、54.42%。疏水性分析显示该蛋白具有7个跨膜区；OsERD2呈组成型表达模式；亚细胞定位显示OsERD2主要分布于高尔基体中；利用酵母互补实验证明OsERD2可以恢复酵母erd2缺失突变体的表型。这些结果表明，OsERD2是水稻中的内质网滞留蛋白受体。 借助农杆菌介导的转化将OsERD2在水稻中超表达，分析转基因水稻对二硫苏糖醇（DTT）处理的响应。结果显示DTT处理抑制水稻幼苗生长，超表达OsERD2株系受抑制程度更为明显。表明OsERD2转基因水稻对内质网胁迫更加敏感。因此，OsERD2可能参与了水稻中的未折叠蛋白响应。 本论文还比较分析了OsRAA1/AtFPF1的一些新功能。OsRAA1（Oryza sativa root architecture associated 1）是拟南芥AtFPF1在水稻中的同源基因，参与水稻根发育的调控。我们将OsRAA1在拟南芥中异源超表达，发现OsRAA1的积累使转基因拟南芥的开花时间提前，同时发现在白光条件下转基因拟南芥的下胚轴长度增加。进一步分析表明，在蓝光、远红光和黑暗条件下转OsRAA1拟南芥下胚轴长度和野生型没有明显区别，但在红光条件下，转基因拟南芥的下胚轴长度是野生型的两倍。AtFPF1转基因拟南芥也表现出类似的表型，说明RAA1/FPF1蛋白不但可以调控拟南芥开花时间而且参与红光对下胚轴生长的光抑制过程，它们在进化过程中保留了这两个方面的功能。

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes

Perfluorochemicals (PFCs) are emerging persistent organic pollutants (POPs) and are widely present in the environment, wildlife and humans. Recently, reports have suggested that PFCs may have endocrine-disrupting activities. In the present study, we have developed a non-competitive enzyme-linked immunosorbent assay (ELISA) method to investigate estrogenic activities of selected PFCs using vitellogenin (VTG) induction in primary cultured hepatocytes of freshwater male tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to various concentrations of perfluorooctanyl sulfonate (PFOS), pentadecafluorooctanoic acid (PFOA), 1H, 1H, 2H, 2H-nonafluoro-1-hexanol (4:2 FTOH), 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH) and 1H, 1H, 2H, 2H-perfluoro-1-decanol (8:2 FTOH) for 48h, while 17 beta-estradiol (E2) and 4-nonylphenol (4-NP) were used as positive controls. A dose-dependent induction of VTG was observed in E2-, 4-NP-, PFOS-, PFOA- and 6:2 FrOH-treated cells, whereas VTG levels remained unchanged in the 4:2 FTOH and 8:2 FTOH exposure groups at the concentrations tested. The estimated 48-h EC50 values for E2,4-NP, PFOS, PFOA and 6:2 FTOH were 4.7 x 10(-7), 7.1 x 10(-6), 1.5 x 10(-5), 2.9 x 10(-5) and 2.8 x 10(-5) M, respectively. In the time-course study, significant VTG induction took place at 24 h (E2), 6 It (4-NP), 48 It (PFOS), 48 It (PFOA), 72 It (4:2 FTOH), 12 h (6:2 FTOH), 72 h (8:2 FTOH), and increased further after 96 It of exposure. Co-exposure to binary mixtures of individual PFCs and E2 for 48 It significantly inhibited E2-induced hepatocellular VTG production in a dose-dependent manner except for 4:2 FTOH. The estimated 48-h IC50 (concentration of a compound that elicits 50% inhibition of maximally E2-induced VTG) values for PFOS, PFOA, 6:2 FTOH and 8:2 FTOH were 3.1 x 10(-7), 5.1 X 10(-7), 1.1 X 10(-6) and 7.5 x 10(-7) M, respectively. In order to further investigate the estrogenic mechanism of PFCs, the hepatocytes were co-exposed to binary mixtures of individual chemicals (E2,4-NP, PFOS, PFOA and 6:2 FTOH) and the known estrogen receptor inhibitor tamoxifen for 48 h; tamoxifen significantly inhibited the ability of these chemicals to stimulate vitellogenesis. The overall results demonstrated that PFOS, PFOA and FTOHs have estrogenic activities and that exposure to a combination of E2 and PFCs produced anti-estrogenic effects. The results of the estrogen receptor inhibition assay further suggested that the estrogenic effect of PFCs may be mediated by the estrogen receptor pathway in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp (Carassius auratus) hepatocytes

Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in vitro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estrogenic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL) seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1 mumol/L), and P-naphtho-flavone (beta-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with beta-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for beta-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.

Estrogenic effects from household stoves

With the application of a genetically modified yeast, estrogen receptor-activating compounds were detected in the soot and emission gas of a wood-burning household stove. The EC50 value of 17beta-estradiol was divided by the EC50 value of soot, and the obtained relative estrogenic value for raw soot was 2.37E-5, indicating that soot was about 100,000 times less estrogenic than 17beta-estradiol. Chemical analysis revealed that alkyl phenol, benzonic acid, and PAHs represented the major constituents in the most potent fractions of the soot. Along with PAHs, other constituents might also contribute to the estrogenicity of soot. (C) 2002 Elsevier Science (USA).

Measurement of estrogen activity in two Chinese lakes

Estrogenic activities of samples from two Chinese lakes, Ya-Er Lake and Donghu Lake, were measured by in vitro recombinant yeast assay and found in both lakes polluted with industrial and domestic wastewater. In methanol extracts of lake water samples the estrogen-like activity was higher than in toluene extracts. These results have been inverted for solid samples. Furthermore, the EC50 value of the water samples was close to the original concentration in the lake.

Measuring Receptor/Ligand Interaction at the Single-Bond Level: Experimental and Interpretative Issues

There is increased interest in measuring kinetic rates, lifetimes, and rupture forces of single receptor/ligand bonds. Valuable insights have been obtained from previous experiments attempting such measurements. However, it remains difficult to know with sufficient certainty that single bonds were indeed measured. Using exemplifying data, evidence supporting single-bond observation is examined and caveats in the experimental design and data interpretation are identified. Critical issues preventing definitive proof and disproof of single-bond observation include complex binding schemes, multimeric interactions, clustering, and heterogeneous surfaces. It is concluded that no single criterion is sufficient to ensure that single bonds are actually observed. However, a cumulative body of evidence may provide reasonable confidence. 0 2002 Biomedical Engineering Society.