异硫氰酸荧光素标记动态观察天花粉蛋白对黑色素瘤B16细胞的损伤

目的:应用异硫氰酸荧光素标记天花粉蛋白,在激光共聚焦显微镜下直接观察天花粉蛋白进入黑色素瘤B16细胞的动态过程并分析其对黑色瘤B16细胞的损伤作用。方法:实验于2003-08/2004-08在南通大学神经再生重点实验室完成。将常规传代培养的黑色素瘤B16细胞用胰酶消化,以5×109个/L的浓度种植于24孔培养板。将天花粉蛋白、异硫氰酸荧光素-天花粉蛋白、异硫氰酸荧光素-牛血清白蛋白分别加入培养细胞中,终浓度为50mg/L,同时加入等量生理盐水作为正常对照组,每组再分别设3,6,12h不同孵育时间组。异硫氰酸荧光素标记天花粉蛋白后,利用激光共聚焦显微镜观察异硫氰酸荧光素-天花粉蛋白进入细胞的过程及其荧光强度,CCK-8细胞毒性实验评估各组细胞存活率、并用单细胞凝胶电泳及hoechst33258染核观察天花粉蛋白对黑色素瘤B16细胞致DNA损伤作用。结果:①终浓度50mg/L的异硫氰酸荧光素-天花粉蛋白孵育3h时已经进入黑色素瘤细胞,6h时进入细胞量达到最高,12h时已轻度下降,各时间点比较差异有统计学意义(P<0.01)。异硫氰酸荧光素-天花粉蛋白组与异硫氰酸荧光素-牛血清白蛋白组荧光强度差异有显著性(P<0.01)。②终浓度50mg/L异硫氰酸荧光素-天花粉蛋白、天花粉蛋白孵育黑色素瘤细胞3,6h后利用单细胞电泳和Hoechst33258染色未观察到核形态的变化;但孵育6h后CCK-8细胞毒性实验显示活细胞数量明显下降;孵育12h后出现DNA损伤的特征性彗星尾现象,并观察到明显的核浓缩和边集现象,出现特征性凋亡小体。结论:孵育6h时天花粉蛋白进入细胞量最多,但并没有明显的细胞DNA损伤的作用,而孵育12h时产生明显的细胞毒作用和凋亡的发生。     

亲属活体肾移植18例报告

目的：总结亲属活体肾移植的临床经验，以期提高该技术的安全性及疗效。方法：选择2005—11／2006—12解放军第一七五医院收治的亲属活体肾移植患者18例，均知情同意。受体均为慢性肾小球肾炎；供体年龄24-74岁，17例有血缘关系，另一例为妻子供肾。均行经腰开放手术取肾，左侧供肾15例，右侧供肾3例。术后受体采用环孢素A＋霉酚酸酯＋激素三联低剂量免疫抑制剂方案。结果：①所有供体在术后10d左右出院，均无严重并发症且肾功能正常。②16例受体均在术后1-3d内移植肾功能恢复正常，另2例因供肾迷走血管损伤在术后5d左右血肌酐下降到200μmol／L，1个月左右稳定在160μmol／L。1例他克莫司仅服0．5-0．75mg／d，于术后5个月出现急性排斥反应。1例术后2个月出现环孢素A急性肾毒性（血药浓度谷值为343μg，L）。2例出现药物性肝炎。结论：亲属活体肾移植术较为安全，排斥反应少，结果满意。免疫抑制剂宜采用低剂量方案。     

国人全膝关节假体置换胫骨近端截骨面至腘血管距离的测量及意义

目的:测量国人全膝关节假体置换术胫骨近端截骨面后缘至腘窝血管之间的距离,以期为临床全膝关节置换术中避免损伤腘窝血管提供参考数据。方法:选择2006-06/12于解放军第二军医大学长征医院体检的50名正常成人(53膝),男29名(31膝),女21名(22膝)。所有观察对象均知情同意,且得到医院伦理道德委员会批准。对所有膝关节进行MRI扫描,在胫骨外侧平台以下10mm水平横断面上辨认腘动静脉,并测量胫骨近端截骨面后缘至腘窝动静脉的距离。结果:53膝全部进入结果分析,无脱落。①男性胫骨近端截骨面后缘至腘动脉、腘静脉平均距离为(6.7±2.5,7.3±2.3)mm,95%可信区间分别为5.8～7.6mm,6.5～8.1mm。②女性胫骨近端截骨面后缘至腘动脉、腘静脉平均距离为(6.6±1.9,7.1±2.7)mm,95%可信区间分别为:5.8～7.4mm,5.9～8.3mm。③不同性别观察对象胫骨近端截骨面后缘至腘血管的距离差异无显著性意义(P>0.05)。结论:腘窝血管紧邻全膝关节假体置换术胫骨近端截骨面后缘,不同性别间无明显差异。全膝关节假体置换术中进行胫骨近端截骨,特别是后方操作时需特别谨慎,以避免损伤腘窝血管。     

不同孔径纳米羟基磷灰石人工骨修复兔桡骨缺损效果比较

目的:纳米级的羟基磷灰石材料与人体内组织成分更为相似,具有更佳的生物性能。评价不同孔径的多孔纳米羟基磷灰石人工骨的骨缺损修复能力,从而筛选出适合的孔径以达到骨传导功能与生物力学性能的良好统一。方法:实验于2005-10/2006-10在深圳市第二人民医院中心实验室完成。①实验材料:纳米羟基磷灰石人工骨以硝酸钙和磷酸二氢铵为原料,采用溶胶-絮凝法制备粉体,运用压力成型、木模成型和浸渍成型分别制得孔隙分布均匀的孔径分别为50～150μm、100～250μm和300～500μm的多孔纳米羟基磷灰石人工骨。②实验动物:雄性新西兰大白兔60只随机分为植入50～150μm孔径材料组、植入100～250μm孔径材料组、植入300～500μm孔径材料组、空白对照组,每组15只。实验过程中对动物处置符合动物伦理学要求。③实验方法:制备双侧桡骨骨缺损动物模型,然后用3种不同孔径的纳米羟基磷灰石人工骨材料植入骨缺损处进行修复,空白对照组不植入任何材料。④实验评估:术后4,8和12周分别行大体标本观察、X射线片观察、扫描电镜观察及生物力学测试,比较各组材料修复骨缺损的能力。结果:实验动物均进入结果分析。①X射线片检查结果:术后4周、8周、12周,植入100～250μm孔径材料组X射线评分高于植入50～150μm,300～500μm孔径材料组,差异有显著性意义(P<0.05)。②生物力学检测结果:术后4周、8周、12周,植入100～250μm孔径材料组生物力学强度高于植入50～150μm,300～500μm孔径材料组,差异有显著性意义(P<0.05)。③扫描电镜观察结果:植入100～250μm孔径材料组成骨效果明显优于植入50～150μm,300～500μm孔径材料组和空白对照组。结论:纳米羟基磷灰石人工骨具有良好的成骨能力,但其骨修复能力受孔径因素的影响,孔径100～250μm的纳米羟基磷灰石人工骨材料成骨能力较好。     

上海市0～6岁小儿佝偻病的现状调查

目的:了解上海市小儿佝偻病的现状及影响因素。方法:2005年春季以整群和分层随机抽样法抽取上海市部分城区0～6岁小儿821名,采取问卷调查方法了解小儿的生活环境、饮食习惯、户外活动、营养状况、既往疾病史及母亲的妊娠情况等。佝偻病的诊断以1996年国家卫生部颁布的"婴幼儿佝偻病防治方案"为诊断标准。结果:取得完整有效资料769名,其中男童396名,女童373名;集居儿童456名,散居儿童313名。①小儿佝偻病患病率为17.3%(133/769),其中男童患病率为17.4%、女童为17.2%。②佝偻病与喂养方式(母乳喂养的患病率为13.0%、混合喂养的患病率为17.5%、人工喂养的患病率为25.2%)、鱼肝油添加(按时添加维生素D的患病率为13.5%、偶加或未加维生素D的患病率为32.5%)、居住环境(居住在市区的患病率为23.6%、居住在郊区的患病率为10.6%;集居儿童患病率为13.8%、散居儿童患病率为22.4%)、户外活动时间(经常户外活动的患病率为12.9%、偶尔户外活动的患病率为31.8%)、反复呼吸道感染(小儿有反复呼吸道感染的患病率为26.9%、无反复呼吸道感染的患病率为12.9%)、母孕期缺钙(母孕期有缺钙的患病率为33.2%、无缺钙的患病率为12.0%)等因素有关(P<0.01)。结论:上海市小儿佝偻病患病率有上升趋势。影响因素与城市环境污染、母乳喂养减少、年轻父母科学育儿知识缺乏等有关。     

A population-based survey of the epidemiology of symptom-defined gastroesophageal reflux disease: the Systematic Investigation of Gastrointestinal Diseases in China

Background

Hepatitis B immune globulins (HBIG) in combination with nucleos(t)ide analogues (NA) are effectively used for the prevention of hepatitis B virus (HBV) recurrence after liver transplantation (LT). However, associated treatment costs for HBIG are exceedingly high.

Methods

Fresh frozen plasma obtained from blood donors with high anti-HBs levels (hyperimmune plasma, HIP) containing at least 4,500 IU anti-HBs was used as alternative treatment for HBV recurrence prophylaxis post-LT.

Results

Twenty-one HBV-related LT recipients received HIP starting at transplantation, followed by long-term combination treatment with NA. Mean follow-up time was 4.5 years (range 0.5-12.6) and each patient received on average 8.2 HIP per year (range 5.8-11.4). Anti-HBs terminal elimination kinetic after HIP administration was 20.6 days (range 13.8-30.9), which is comparable to values reported for commercial HBIG products. All 21 patients remained free of HBV recurrence during follow-up and no transfusion-transmitted infection or other serious complication was observed. Seven patients developed reversible mild transfusion reactions. The cost for one HIP unit was US$140; average yearly HBIG treatment cost was US$1,148 per patient, as compared to US$25,000-100,000 for treatment with commercial HBIG.

Conclusion

The results of this study suggest that the use of HIP may be a useful and economical approach for the prevention of HBV recurrence post-LT if used in combination with NA. Additional prospective controlled studies in larger populations are needed to confirm these results.     

Modern management of chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder of phagocytic cells resulting in failure to kill a characteristic spectrum of bacteria and fungi and in defective degradation of inflammatory mediators with concomitant granuloma formation. Current prophylaxis with trimethoprim-sulfamethoxazole, itraconazole and in selected cases additional interferon gamma is efficient, but imperfect. A significant recent progress towards new antibiotic (e.g. linezolid) and antifungal (e.g. voriconazole and posaconazole) therapy will allow survival of most patients into adulthood. Adolescent and adult CGD is increasingly characterized by inflammatory complications, such as granulomatous lung and inflammatory bowel disease, requiring immunosupressive therapy. Allogeneic haematopoietic stem cell transplantation from a human leucocyte antigen identical donor is currently the only proven curative treatment for CGD and can be offered to the selected patients. Gene-replacement therapy for patients lacking a suitable stem cell donor is still experimental and faces major obstacles and risks. However, it may offer some transitory benefits and has helped in a few cases to overcome life-threatening infections.     

Allogeneic bone marrow transplantation for chronic myeloid leukemia in first chronic phase: a randomized trial of busulfan-cytoxan versus cytoxan-total body irradiation as preparative regimen: a report from the French Society of Bone Marrow Graft (SFGM)

From March 1988 to March 1991, 19 French bone marrow transplant (BMT) centers participated in a prospective randomized trial comparing two conditioning regimens for patients with chronic myeloid leukemia transplanted in first chronic phase with an HLA identical sibling donor. A total of 120 consecutive patients were randomized to receive either 120 mg/kg of cyclophosphamide followed by total body irradiation (CY-TBI; n = 55) or 16 mg/kg of busulfan followed by 120 mg/kg of CY (BU-CY; n = 65). Two different TBI regimens were used. Thirteen patients received a 10-Gy single-dose TBI (SDTBI), and 42 received a fractionated TBI (FTBI). Median time between diagnosis and BMT was 315 days. Overall 5-year actuarial survival was 62.9% (65.8% +/- 12.5% for CY-TBI and 60.6 +/- 11.7% for BU-CY; P = .5), and overall disease-free survival was 55% (51% +/- 14% for CY-TBI and 59.1% +/- 11.8% for BU-CY; P = .75). All patients conditioned with CY-TBI experienced sustained engraftment; in contrast, 4 of 65 patients conditioned with BU-CY rejected the graft (P = .18). There was no significant statistical difference between the two groups regarding transplant-related mortality (29% for CY-TBI and 38% for BU-CY; P = .44). So far, with a median follow up of 42 months, 11 patients have relapsed; 9 relapses occurred after CY-TBI, mostly after FTBI (8 of 9) and 2 after BU-CY (P = .02). The actuarial risk of relapse was 4.4% +/- 6.7% after BU-CY, 11.1% +/- 20.8% after SDTBI, and 31.3% +/- 18.1% after FTBI (P = .039). In addition, independently of the conditioning regimen, the increase of posttransplant immunosuppression in 16 patients with an anti- interleukin-2 receptor monoclonal antibody (MoAb) in addition to a short course of methotrexate and cyclosporine was shown to increase the actuarial risk of relapse (57% +/- 30% with MoAb v 9% +/- 7.3% without MoAb; P = .001). We conclude that BU is an acceptable alternative to TBI for patients with chronic myeloid leukemia in first chronic phase receiving BMT from HLA identical sibling donors. Both BU-CY and CY-TBI regimens gave similar transplant-related mortality, and the antileukemic efficiency of BU-CY regimen was either similar or even higher than that of CY-TBI.     

T-cell death by apoptosis in vertically human immunodeficiency virus- infected children coincides with expansion of CD8+/interleukin-2 receptor-/HLA-DR+ T cells: sign of a possible role for herpes viruses as cofactors?

One mechanism proposed to play a role in T-cell depletion in human immunodeficiency virus (HIV) infection is apoptosis (activation-induced cell death). We assessed whether apoptosis is related to activation of T cells in vivo and its possible triggers. DNA was extracted from peripheral blood mononuclear cells (PBMC) taken from 16 vertically HIV- infected children and 9 HIV-negative children born to HIV-positive mothers (controls) and tested by agarose gel electrophoresis for the presence of DNA fragments specific for apoptosis. Signs of apoptosis were found on in vitro culture of PBMC from 12 of 16 HIV-infected children, but not in PBMC from the nine controls. Eleven of the 12 HIV- infected children with apoptosis showed an elevated (> 15%) proportion of CD3+/HLA-DR+ cells. This was due to an increased proportion of CD8+/HLA-DR+ cells, as shown in 7 of 7 further tested patients. In none of the probands an increased (> 5%) proportion of IL-2 receptor expressing CD3+ cells was found. T cells undergoing apoptosis were preferentially of the CD8+ phenotype. Expansion of circulating CD8+/interleukin-2 receptor (IL-2R)-/HLA-DR+ T cells is known to occur during active infection with herpes viruses. To investigate the possible role of herpes viral coinfections for apoptosis in HIV infection, we focused on Epstein-Barr virus (EBV) as an example for a herpes virus usually acquired during childhood. In 10 of 12 patients with apoptosis, we found increased levels of EBV genome in PBMC and/or tissues, indicating active EBV replication. By contrast, no increased burden of EBV was found in the four HIV-infected patients without apoptosis or in the controls. Our data indicate that in children the occurrence of apoptosis in HIV infection is closely related to activation of CD8+ T cells. Furthermore, primoinfection with or reactivation of herpes viruses, such as EBV, may substantially contribute to such T-cell activation and the ensuing apoptosis. Additional studies are warranted to evaluate the contribution of herpes virus-triggered apoptosis to the T-cell loss leading to the acquired immunodeficiency syndrome.