The use of a recombinant immunoblot assay in the interpretation of anti- hepatitis C virus reactivity among prospectively followed patients, implicated donors, and random donors

Samples from prospectively followed recipients, their respective donors, and a cohort of random donors were used to evaluate the specificity and efficacy of a recombinant immunoblot assay (RIBA) as an adjunct to anti-hepatitis C virus (HCV) testing by enzyme immunoassay (EIA). RIBA reacted (RIBA+) in 100 percent of patients who developed hepatitis associated with anti-HCV seroconversion documented by EIA and in 100 percent of the EIA-positive (EIA+) donors implicated in these cases. In contrast, RIBA reacted in none of 10 recipients who were EIA+ but did not develop hepatitis, in none of 7 EIA+ patients with hepatitis B or cytomegalovirus infection, in 33 percent of EIA+ donors who were not implicated in hepatitis transmission, and in 37 percent of EIA+ random donors. Hence, the vast majority of EIA+ individuals who have ancillary evidence of HCV infection react on RIBA, whereas the majority of EIA+ individuals in low-risk settings do not react (RIBA-negative, or RIBA-). There was a strong association between RIBA reactivity and the presence of a surrogate marker (elevated alanine aminotransferase [ALT] and/or antibody to hepatitis B core antigen); 43 percent of RIBA+ implicated donors had a surrogate marker as compared to none of 14 EIA+, RIBA- donors. Among EIA+ random donors, 77 percent of those with a surrogate marker were RIBA+, as compared with 29 percent of those without a surrogate marker. In addition, in EIA+ donors, RIBA reactivity correlated with the extent of ALT elevation; 86 percent of those with an ALT greater than 135 IU per L were RIBA+ compared with 18 percent of those with an ALT less than 30 IU per L.(ABSTRACT TRUNCATED AT 250 WORDS)     

人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中构建组织工程软骨

目的:观察人脂肪干细胞复合脱细胞软骨基质支架在生物反应器中初步构建组织工程软骨的可行性。方法:实验于2005-04/2006-05在解放军总医院骨科研究所完成。脂肪组织和关节软骨均来自膝关节置换术中切除的组织,并经患者知情同意。关节软骨冻干后经粉碎机粉碎,过筛,选取25～38μm大小的软骨微粒。在样品中先加入2.5g/L胰蛋白酶,37℃消化24h,再加入1%Triton X-100震荡72h。将软骨微粒和蒸馏水按1∶3的比例混合后滴加在模板中,置入冷冻干燥机冻干后行紫外线交联。紫外线照射8h完成。最后经25kGy 60Co辐照灭菌完成支架制备。取膝关节置换术中切除的髌下脂肪垫,酶消法获得脂肪干细胞,扩增后复合于脱细胞软骨基质制成圆柱状三维支架上(细胞密度5×1010L-1),置于生物反应器中进行诱导培养,同时设静态培养组作为对照,3周后观测大体形态和组织学形态变化,同时进行组织化学(包括番红花O,阿利新蓝染色)和Ⅱ型胶原免疫组织化学分析。结果:生物反应器组诱导培养3周苏木精-伊红染色显示支架结构消失,只有中心区域残存少量支架结构;静态培养组支架结构尚存在,有少量基质分泌。番红花O染色显示生物反应器组细胞外有大量蛋白聚糖沉积,阿利新蓝染色表明有软骨特异性蛋白多糖的聚集;而静态培养组只有部分区域染色且淡于生物反应器组。Ⅰ型胶原免疫组化的结果显示,在生物反应器组细胞能够合成大量软骨细胞特异性胶原成分,而静态培养组呈弱阳性。结论:生物反应器培养明显促进了脂肪干细胞的增殖与软骨分化,是体外构建组织工程软骨的良好方法。     

应用遥测肌电技术实测分析人体助行带的生物力学作用

目的:人体助行带为一种随身佩带的动力装置,应用遥测肌电技术测定和分析自行研制的人体助行带的生物力学作用机制。方法:实验于2005-12/2006-06在天津体育大学运动生物力学实验室进行。选取天津市天津医院30名受试对象,均对本实验知情同意。按照生物力学的测试要求,首先对受试者进行5次以上行走状态下的遥测肌电测定,然后在同等条件下对受试者佩带助行带实施对照研究。实验采用的助行带为自行研制的,由腰至足以双层弹力带作为动力装置的弹性结构。结果:行走摆动相大腿肌肌电:佩带助行带后肌内侧肌、肌二头肌、半腱肌肌电高于佩带前(P<0.01)。支撑相小腿肌肌电:佩带助行带后胫前肌、腓骨长肌、腓肠肌肌电高于佩带前(P<0.01)。结论:通过弹力带施加于下肢的外部载荷,能够起到增强下肢肌电以及产生助行的生物力学功效。     

蒿甲醚对大鼠肺泡Ⅱ型上皮细胞DNA的影响

目的:蒿甲醚除了具有良好的临床治疗作用外,对正常组织有极大的副作用。实验拟观察蒿甲醚对大鼠肺泡Ⅱ型上皮细胞的影响。方法:实验于2005-09-20/2007-03-10在山东省泰山医学院生命科学研究所、中心实验室和机能实验室完成。①实验材料:蒿甲醚购自昆明制药厂,分析纯,使用前先进行预处理,处理方法:研磨后用溶液配成1g/L的母液,超声助溶解后供实验用。Wistar大鼠70只,体质量(70±15)g,雌雄不拘,由山东大学医学院实验动物中心提供。实验过程中对动物处置符合动物伦理学标准。②实验方法:将大鼠随机分为普通饲料对照组(n=10)和实验组(n=60),实验组分6个亚组,分别在饲料中加入0.1,0.2,0.4,0.8,1.6,3.2mg/kg的蒿甲醚。采用单细胞凝胶电泳技术观察大鼠肺泡Ⅱ型上皮细胞DNA的改变。结果:70只大鼠,饲养过程中实验组死亡6只,死亡原因为饲料添加蒿甲醚造成。当饲料中的蒿甲醚浓度为0.1mg/kg时,大鼠肺泡Ⅱ型上皮细胞DNA损伤率与对照组相比,差异无统计学意义;饲料中的蒿甲醚浓度超过0.2mg/kg时,大鼠肺泡Ⅱ型上皮细胞DNA损伤率明显增加,与对照组相比,差异有统计学意义。且损伤程度与饲料中蒿甲醚含量呈正相关。结论:当饲料中的蒿甲醚浓度超过0.2mg/kg时,蒿甲醚可在一定程度上改变肺泡Ⅱ型上皮细胞的DNA生物学特性。且随着蒿甲醚浓度的升高,这种损害更为明显。     

Observations on the effect of BMP-2 on rat bone marrow cells cultured on titanium substrates of different roughness

The objective of this study was to examine the osteoinductive capacity of different concentrations of BMP-2 on bone marrow stromal cells in vitro. Further, we intended to determine whether titanium provided with an increased surface roughness is more efficient in osteoblast differentiation than machined titanium. Therefore, 20,000 cells/ml were seeded and cultured on machined and grit-blasted titanium discs for 4, 8 and 16 days. Different concentrations of rhBMP-2 (0, 10, 100, 1000 ng/ml) were supplemented to the medium for 8 days of culturing. To evaluate cellular proliferation and differentiation, specimens were examined for DNA, alkaline phosphatase activity, and calcium content. Morphological appearance of the specimens at 8 and 16 days of incubation was evaluated using scanning electron microscopy. Two separate experimental runs were performed.Evaluation of the DNA and alkaline phosphatase data revealed that a significant difference existed for these data between both experimental runs. Further analysis of the DNA figures learned that roughening of the titanium surface and addition of BMP-2 had no effect on cell proliferation. The alkaline phosphatase analysis and calcium measurements revealed that BMP-2 stimulated the early differentiation of osteogenic cells on machined titanium substrates in a dose-dependent manner. After 16 days of culture, no significant differences in calcium content could be observed anymore between machined and roughened titanium surfaces. Further, the data revealed that the machined surfaces showed a significant increase in calcium deposition when 100 and 1000 ng/ml BMP-2 were supplemented to the medium. However, the roughened surfaces showed this significant enhancement in calcium content only with 1000 ng/ml BMP-2. In addition, SEM evaluation revealed a dose-dependent response to BMP-2. Increasing BMP-2 concentrations resulted in more calcified globular accretions on bone surfaces than when no BMP-2 was added.On the basis of our results, we conclude that (1) due to the heterogeneous nature of bone marrow, experimental results with primary rat bone marrow cells are difficult to reproduce from one experiment to the other, and (2) addition of rhBMP-2 in the medium stimulates the early differentiation and matrix mineralization of osteogenic cells on machined titanium surfaces in a dose-responsive manner. Further, we concluded that our roughened titanium surfaces had no effect on proliferation and differentiation of primary derived rate bone marrow cells.     

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Differences in the global methylation pattern, ie hyper‐ as well as hypo‐methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5‐ cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam‐2 before and after demethylation using 5‐azacytidine. Exposure to 5‐azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell‐specific marker VASA, showed increased expression. Following treatment with 5‐azacytidine, TCam‐2 cells were analysed using a high‐throughput methylation screen for changes in the methylation sites of 14 000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation

Yoo E‐H, Woo H, Ki C‐S, Lee HJ, Kim D‐K, Kang I‐S, Park P, Sung K, Lee CS, Chung T‐Y, Moon JR, Han H, Lee S‐T, Kim J‐W. Clinical and genetic analysis of Korean patients with Marfan syndrome: possible ethnic differences in clinical manifestation. Marfan syndrome (MFS) is an autosomal dominant disorder of the fibrous connective tissue caused by mutations in the fibrillin‐1 (FBN1) gene. Although clinical and genetic analyses have been performed in various populations, there have been few studies in Korea. The aim of this study was to investigate the clinical characteristics and genetic background of Korean patients with MFS. In 39 Korean patients with MFS who met the Ghent criteria, the most common clinical finding was aortic dilatation and/or dissection (94.9%), whereas only 35.9% of patients had ectopia lentis. The majority of MFS patients had fewer than four of the skeletal findings required to fulfill the major skeletal Ghent criterion for MFS. Only 21% of Korean patients had major skeletal abnormalities and most cases showed only minor skeletal involvement. FBN1 gene mutations were detected in 35 out of 39 patients (89.7%), which is similar to rates presented in the previous reports. These results suggest that some clinical features in Korean patients with MFS differed from those reported in Western MFS patients.     

Bone formation in CaP-coated and noncoated titanium fiber mesh

The osteogenic activity of calcium phosphate (CaP)-coated and noncoated porous titanium (Ti) fiber mesh loaded with cultured syngeneic osteogenic cells after prolonged in situ culturing was compared in a syngeneic rat ectopic assay model. Rat bone marrow (RBM) cells were loaded onto the CaP-coated and noncoated Ti scaffolds using either a droplet or a suspension loading method. After loading, the RBM cells were cultured for 8 days in vitro. Thereafter, implants were subcutaneously placed in 39 syngeneic rats. The rats were euthanized and the implants retrieved at 2, 4, and 8 weeks postoperatively. Further, in the 8 week group fluorochrome bone markers were injected at 2, 4, and 6 weeks. Histological analysis demonstrated that only the CaP-coated meshes supported bone formation. The amount of newly formed bone varied between single and multiple spheres to filling a significant part of the mesh porosity. In the newly formed bone, osteocytes embedded in a mineralized matrix could be observed clearly. On the other hand, in the noncoated titanium implants, abundant deposition of calcium-containing material was seen. This deposit lacked a bonelike tissue organization. Further analysis revealed that the cell-loading method did not influence the final amount of bone formation. In CaP-coated implants the accumulation sequence of the fluorochrome markers showed that bone formation started on the mesh fibers. In conclusion, our results prove that the combination of a thin CaP coating, Ti-mesh, and RBM cells can indeed generate ectopic bone formation after prolonged in vitro culturing. No effect of the loading method was observed on the final amount of bone.