Flow cytometric detection of platelet-reactive antibodies and application in platelet crossmatching

Background: Alloimmunization against HLA or platelet antigens can cause refractoriness to platelet transfusions in multiply transfused patients. Crossmatching of platelet concentrates is effective in overcoming this problem. Study Design and Methods: A flow cytometric assay was used for simultaneous detection of lymphocyte-reactive and platelet-reactive antibodies in a single sample using fluorescein isothiocyanate-labeled anti-IgG. This assay was compared with the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay in selected sera containing HLA and platelet antibodies. In a further study, this assay was compared with lymphocytotoxicity test results from thrombocytopenic patients, for whom platelet concentrates were ordered. The results of both assays were then correlated with the 1-hour corrected count increment, with a corrected count increment greater then 7500 considered as an adequate transfusion response. Results: The results of the MAIPA and flow cytometric assay in detecting platelet-reactive antibodies correlated well (p<0.0001, r = 0.84). The sensitivity and specificity of the flow cytometric assay in detecting platelet-reactive antibodies were 94.7 and 96.3 percent, when the MAIPA assay was taken as a reference. In unselected sera from patients, the sensitivity and specificity of the flow cytometric assays were, respectively, 72.7 and 91.7 percent in detecting lymphocyte- reactive antibodies and 70.6 and 77.7 percent in detecting platelet- reactive antibodies, when the lymphocytotoxicity test was used as a reference. With regard to an adequate transfusion response, the sensitivities and efficiencies were 20.0 and 82.1 percent, 33.3 and 84.3 percent, and 70.0 and 88.6 percent for the lymphocytotoxicity test and the lymphocyte-reactive and platelet-reactive flow cytometric assays, respectively. Conclusion: Flow cytometric crossmatching appears to be an effective method of detecting platelet-reactive antibodies that may affect the success of platelet transfusions. This procedure is well-suited for routine conditions and can be performed within 2 hours.     

Immunohistochemical demonstration of serotonin neurons in autonomic regions of the rat spinal cord

The immunohistochemical distribution of serotonin neurons in normal and transected spinal cords of rats was examined. Intraspinal serotonin neurons were immunostained as far rostral and caudal as T3 and Co1, respectively. All serotonin neurons were located in lamina VII and X, and most were located in spinal autonomic areas. Both bipolar and multipolar neurons were observed with many of the neurons oriented longitudinally to the long axis of the cord. Spinal neurons immunostained for serotonin were visible with and without L-tryptophan and monoamine oxidase inhibitor pretreatment.     

Segmental resection spares pulmonary function in patients with stage I lung cancer

Background

Segmental resection for stage I non-small cell lung cancer remains controversial. Reports suggest that segmentectomy confers no advantage in preserving lung function and compromises survival. This study was undertaken to assess the validity of those assertions.

Methods

We retrospectively analyzed patients undergoing lobectomy (n = 147) or segmentectomy (n = 54) for stage I non-small cell lung cancer between March 1996 and June 2001. All patients were included in the survival analysis. Pulmonary function testing was obtained preoperatively and at 1 year and included forced vital capacity, forced expiratory volume in 1 second, maximum voluntary ventilation, diffusing capacity, and stair-stepper exercise. Patients with recurrent disease (lobectomy, n = 32; segmentectomy, n = 10) were excluded in the pulmonary function testing analysis to avoid the confounding variables of tumor or treatments.

Results

Preoperative pulmonary function tests in segmentectomy patients were significantly reduced compared with lobectomy (forced expiratory volume in 1 second, 75.1% versus 55.3%; p < 0.001). At 1 year, lobectomy patients experienced significant declines in forced vital capacity (85.5% to 81.1%), forced expiratory volume in 1 second (75.1% to 66.7%), maximum voluntary ventilation (72.8% to 65.2%), and diffusing capacity (79.3% to 69.6%). In contrast, a decline in diffusing capacity was the only significant change seen after segmental resection. Oxygen saturations at rest and with exercise were maintained in both groups. Actuarial survival in both groups was similar (p = 0.406) with a 1-year survival of 95% for lobectomy and 92% for segmentectomy. Four-year survivals were 67% and 62%, respectively.

Conclusions

For patients with stage I non-small cell lung cancer, segmental resection offers preservation of pulmonary function compared with lobectomy and does not compromise survival. Segmentectomy should be considered whenever permitted by anatomic location.     

Efficient conditional mutation of the vertebrate CENP-C gene

We have used gene targeting in the DT40 cell line to create a cell line which expresses a fusion between CENP-C and a mouse steroid receptor and which behaves as a conditional loss of function mutant of CENP-C. Under restrictive conditions these cells arrest at the metaphase/anaphase junction and after a delay of approximately 2.5 h die by apoptosis. These results indicate that CENP-C is either necessary for anaphase chromosome movement or for mediating a signal which triggers centromere function during anaphase. Our approach is simple and applicable to a wide range of proteins with general cell autonomous functions in vertebrates.      

卵胞质移植、克隆与逆向克隆

目的:最近人类卵细胞间的胞质移植被作为人类辅助生殖生物技术的手段并成为研究热点。本文回顾了该领域的研究进展和存在的问题,并以此为基础提出“逆向克隆技术”这一新概念。资料来源:应用计算机检索PUBMED 1998-01/2006-12期间的相关文章,检索词为“ooplasmic transfer,mitochondria heteroplasmy,animal cloning”,并限定文章语言种类为English。同时计算机检索万方数据库1998-01/2006-12期间的相关文章,检索词为“胞质转移,线粒体异质性,动物克隆”,并限定文章语言种类为中文。资料选择:对资料进行初审,并查看每篇文献后的引文。纳入标准:文章所述内容应与卵胞质移植、克隆或逆向克隆研究相关。排除标准:重复研究或Meta分析类文章。资料提炼:共收集到126篇相关文献,31篇文献符合纳入标准,排除的95篇文献为内容陈旧或重复。符合纳入标准的31篇文献中,17篇涉及卵胞质移植,13篇涉及动物克隆,1篇涉及逆克隆。资料综合:人类的生殖技术领域发展迅速,卵胞质移植技术得到广泛应用。研究表明,卵胞质对受精和胚胎发育具有重要作用,其中线粒体与受精和胚胎发育关系最为密切。大量实验对细胞质与细胞核的相互作用、基因组的重编程机制上进一步研究,同时也在技术上不断改进。克隆技术有广阔的应用前景,同时也存在着不少问题,基于此提出以胞质转移为基础的“逆向克隆技术”。结论:线粒体对卵母细胞的受精和胚胎发育有显著影响,但卵胞质移植可能导致线粒体的异质性及其潜在的问题还需要进一步研究。以卵胞质转移为基础的“逆向克隆技术”是否能达到克隆动物的结果前景喜人。     

Screening Emergency Admissions at Risk of Chronic Hepatitis C (SEARCH) to diagnose or ‘re‐diagnose’ infections is effective in Australia

The World Health Organization has set ambitious viral hepatitis elimination targets; however, difficulties in identifying and engaging patients remain. The emergency visit is an opportunity for enhanced linkage to care (LTC). We assessed the effectiveness of an automated Emergency Department (ED) screening service in identifying patients with hepatitis C (HCV) and achieving LTC. A retrospective evaluation was undertaken, analysing the first 5000 patients screened through an automatic Australian service termed ‘Screening Emergency Admissions at Risk of Chronic Hepatitis’ (SEARCH). Screening was performed for those recommended in the Australian national testing policy, specifically overseas born (OB) and Aboriginal or Torres Strait Islanders (ATSI). Healthcare worker education, patient information materials and opt‐out informed consent were used to test sera already collected for biochemistry assays. 5000 of 5801 (86.2%) consecutive eligible patients were screened (OB: 4778, ATSI: 222) from 14 093 ED presentations. HCV antibody was positive in 181 patients (3.6%); 51 (1.0%) were HCV RNA positive. Of 51 HCV RNA–positive patients, 12 were new diagnoses, 32 were ‘re‐diagnoses’ (aware but lost to follow‐up [LTFU]), and 7 were previously known but treatment contraindicated. LTC was successful in 38 viraemic patients (7 deceased, 4 LTFU, 1 treatment ineligible and 1 declined). Of RNA‐negative patients, 75 were previously treated and 49 had presumed spontaneous clearance. Opt‐out consent was acceptable to all patients and staff involved. ED screening can lead to additional diagnosing and ‘re‐diagnosing’ of HCV, with high rates of LTC. Opt‐out consent and automation removed major obstacles to testing.     

Antioxidant enriched enteral nutrition and oxidative stress after major gastrointestinal tract surgery

AIM： To investigate the effects of an enteral supplement containing antioxidants on circulating levels of antioxidants and indicators of oxidative stress after major gastrointestinal surgery. METHODS： Twenty-one patients undergoing major upper gastrointestinal tract surgery were randomised in a single centre, open label study on the effect of postoperative enteral nutrition supplemented with antioxidants. The effect on circulating levels of antioxidants and indicators of oxidative stress, such as F2-isoprostane, was studied. RESULTS： The antioxidant enteral supplement showed no adverse effects and was well tolerated. After surgery a decrease in the circulating levels of antioxidant parameters was observed. Only selenium and glutamine levels were restored to pre-operative values one week after surgery. F2-isoprostane increased in the first three postoperative days only in the antioxidant supplemented group. Lipopolysaccharide binding protein （LBP） levels decreased faster in the antioxidant group after surgery. CONCLUSION： Despite lower antioxidant levels there was no increase in the circulating markers of oxidative stress on the first day after major abdominal surgery. The rise in F2-isoprostane in patients receiving the antioxidant supplement may be related to the conversion of antioxidants to oxidants which raises questions on antioxidant supplementation. Module AOX restored the postoperative decrease in selenium levels. The rapid decrease in LBP levels in the antioxidant group suggests a possible protective effect on gut wall integrity. Further studies are needed on the role of oxidative stress on outcome and the use of antioxidants in patients undergoing major abdominal surgery.     

The insulin receptor substrate-1-related 4PS substrate but not the interleukin-2R gamma chain is involved in interleukin-13-mediated signal transduction

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3- dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin- like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL- 13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.     

银汞和玻璃多聚链烯盐开放三明治技术修复Ⅱ类洞抗折强度的研究

修复根面龋要较猞面龋复杂,比如根面龋的清理、成型片与楔子的放入、与邻牙的接触恢复和修复后悬突的处理等。尽管玻璃多聚链烯盐和复合树脂三明治技术已被广泛应用,但玻璃多聚链烯盐和银汞合金三明治技术尚未得以深入的研究。本研究旨在通过玻璃多聚链烯盐垫底,观察银汞合金充填修复Ⅱ类洞型后的洞型抗折强度。