肌腱玻璃化法冷冻保存的体外实验

目的:目前临床上常用低温冷冻法来保存同种异体肌腱,但操作较复杂费时,并且所保存的肌腱活性较低而限制其应用。采用已筛选的玻璃化法冷冻保存鸡屈趾深肌腱,并将复温后的玻璃化肌腱进行体外检测,探索其作为肌腱移植材料的可行性。方法:实验于2003-11/2005-02在解放军总医院骨科研究所完成。①实验材料及分组:来亨鸡16只,雄性,体质量2.5kg左右,随机分为2组,玻璃化组为玻璃化肌腱,新鲜肌腱组为新鲜肌腱,每组8只。②实验过程:切取来亨鸡屈趾深肌腱,置入玻璃化液内快速投入液氮保存2周制备玻璃化肌腱。③实验评估:将2组肌腱在体外进行大体、组织学及超微结构观察,羟脯氨酸含量测定,生物力学性能检测,并对两组肌腱进行细胞培养与鉴定。结果:①玻璃化组肌腱的大体、组织学及超微结构与新鲜肌腱组相似,其细胞及细胞外结构得以良好保存。②应用碱解法测定玻璃化肌腱内的羟脯氨酸含量为69.27mg/g,与新鲜肌腱组间差异无统计学意义。③玻璃化组肌腱破裂强度为165.58MPa,弹性模量1.41GPa,与新鲜肌腱组的力学性能差异无统计学意义。④将玻璃化组肌腱进行细胞培养,细胞第8天自组织块长出,第21天后传代,培养3代后出现明显的退化现象,其生物学特性与新鲜肌腱组相似。⑤将两组肌腱培养的细胞分别进行免疫组织化学染色,经鉴定均为肌腱细胞。结论:玻璃化法保存的肌腱具有良好的细胞活性、细胞外结构及力学性能,其生物学及生物力学特性无明显变异。     

EUROPEAN STROKE PREVENTION STUDY 2: DIPYRIDAMOLE AND ACETYLSALICYLIC ACID IN THE SECONDARY PREVENTION OF STROKE

In 1988, an optimal antiplatelet regimen for secondary stroke prevention remained to be defined. We undertook a randomised, placebo-controlled, double-blind trial to investigate the safety and efficacy of low-dose acetylsalicylic acid (ASA), modified-release dipyridamole, and the two agents in combination. Patients with prior stroke or transient ischaemic attack (TIA) were randomised to treatment with ASA alone (50 mg daily), modified-release dipyridamole alone (400 mg daily), the two agents in a combined formulation, or placebo. Primary endpoints were stroke, death, and stroke or death. TIA and other vascular events were secondary endpoints. Patients were followed on treatment for two years. We concluded that dipyridamole, in a modified-release form, at a dose of 200 mg b.d. and ASA 25 mg b.d., have been shown to be equally effective in the secondary prevention of ischaemic stroke and TIA; that when co-prescribed, the protective effects are additive, the combination being significantly more effective than each agent prescribed singly; and that low-dose ASA does not eliminate the propensity for induced bleeding.     

A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a K_D of 0.35 μM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC₅₀ of 3.9 μM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a K_D of 0.08 μM and inhibited it with an EC₅₀ of 0.65 μM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 μM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.     

Prior CSF shunting increases the risk of endoscopic third ventriculostomy failure in the treatment of obstructive hydrocephalus in adults

INTRODUCTION: Endoscopic third ventriculostomy (ETV) is accepted as an effective treatment for obstructive hydrocephalus (OHC); however, its benefit in patients previously treated with cerebrospinal fluid (CSF) shunting remains unclear. The value of concurrent ETV and ventriculoperitoneal (VP) shunting in patients with frequent shunt failure remains unstudied. METHODS: Outcomes were compared between OHC patients receiving ETV as initial CSF diversion treatment (n= 19) versus OHC patients receiving ETV for shunt failure (n= 11) by log-rank analysis and Kaplan-Meier plots of recurrence-free periods. To determine if the performance of ETV with concurrent shunt revision decreased the incidence of catastrophic treatment failure in patients experiencing frequent and emergent shunt failures (n = 8), the time to treatment failure after ETV and shunt revision was compared with the mean duration of their previous CSF shunts. RESULTS: ETV after shunt failure was 2.5-fold more likely to fail [risk ratio (RR): 2.48, p<0.05] versus ETV as initial CSF diversion treatment for OHC. Following ETV as initial CSF diversion treatment, 17 patients (89%) experienced immediate improvement and 65% remained recurrence-free at year 2. Following ETV after shunt failure, 16 patients (71%) experienced immediate improvement, but only 25% remained recurrence-free at year 2. In patients with a history of multiple shunt revisions and complications, concurrent use of ETV and VP shunt did not significantly decrease treatment failure. However, the incidence of catastrophic shunt failure requiring acute intervention decreased (43% versus 17%). CONCLUSION: In our experience with ETV for OHC, prior CSF shunting in patients with obstructive hydrocephalus was associated with the decreased time to treatment failure following conversion to ETV. ETV may be less effective for the treatment of OHC in previously shunted patients. ETV combined with concurrent CSF shunting may be an important strategy to prevent catastrophic treatment failure in OHC patients with a history of multiple shunt revisions and complications.     

The 2005 Italian Quality Control Study for the evaluation of CD4 cells in centers involved in the treatment of HIV type 1 patients

We report the results of an external quality control program, including 17 Italian centers involved in the care of patients infected by HIV, to evaluate CD4 T cell count proficiency and reproducibility. The centers received two commercial stabilized blood preparations, one with "normal" and one with "low" CD4 T cell content. The centers were asked to process the samples two times, 1 week apart, with the same procedure used for samples from HIV patients. Most centers showed a good performance of CD4 frequency and absolute count determinations. In particular, the "low" sample was correctly analyzed by all centers; only two underestimated the "normal" sample CD4 frequency, and only one underestimated the CD4 absolute count by >100 CD4 cells/microl. Overall, our data suggest that most Italian laboratories provide reliable and reproducible results in evaluating CD4 T cells in HIV(+) samples.