绿色荧光蛋白转基因干细胞在癫痫大鼠脑内的存活及对脑电的影响

目的:观察神经干细胞和骨髓基质干细胞移植至癫痫大鼠海马后的存活、迁移与周围组织的整合、修复和对癫痫大鼠脑电的影响。方法:实验于2005-03/2006-02在昆明医学院神经科学研究所完成。①实验材料:6～8周龄绿色荧光蛋白转基因小鼠,雌雄不限,体质量40～60g,由新加坡国立大学提供。健康雄性SD大鼠88只,体质量(300±20)g,由昆明医学院动物科提供。将88只SD大鼠随机分为对照组(n=8)、癫痫未移植组(n=8)、生理盐水组(n=24)、神经干细胞移植组(n=24)、骨髓基质干细胞移植组(n=24)。②实验方法:剖取绿色荧光蛋白小鼠孕鼠脑,分离获得整个海马进行神经干细胞的分离,并采用免疫化学方法鉴定。取绿色荧光蛋白转基因小鼠股骨,进行骨髓基质干细胞的分离,并采用免疫组织化学方法鉴定。癫痫未移植组、生理盐水组、神经干细胞移植组、骨髓基质干细胞移植组大鼠注射300×105U/kg,浓度80万U/mL青霉素制作癫痫大鼠模型,连续注射7d,每次癫痫发作按Racine评分标准评分,对照组大鼠腹腔注射相同剂量的生理盐水作为对照。将分离、培养绿色荧光蛋白转基因小鼠神经干细胞和骨髓基质干细胞,移植至青霉素致癫痫大鼠的右侧海马内。③实验评估:移植后1,2,4周观察移植干细胞在大鼠脑内存活和迁移情况,并检测大鼠脑电改变。结果:88只大鼠全部进入结果分析,中途无脱落。①大鼠行为学改变:癫痫未移植组、生理盐水组、神经干细胞移植组、骨髓基质干细胞移植组大鼠均达到Racine分级Ⅳ～Ⅴ级,癫痫模型制备成功。②各组大鼠的脑电改变:移植干细胞可减少癫痫大鼠脑电的痫性发放,降低癫痫波的波幅,具有明显的抗痫效应。③干细胞移植后在海马内的存活和迁移:移植神经干细胞可在青霉素致痫鼠脑内存活和迁移,但随时间的延长,存活细胞数目减少。结论:干细胞移植于青霉素诱发的癫痫大鼠脑内能够存活、迁移,能够改善癫痫鼠的脑电生理功能,提示干细胞移植可能成为一种有效的癫痫治疗手段。     

Imaging early stage osteogenic differentiation of mesenchymal stem cells

Stem cells, such as mesenchymal stem cells (MSCs), contribute to bone fracture repair if they are delivered to the injury site. However, it is difficult to assess the retention and differentiation of these cells after implantation. Current options for non‐invasively tracking the transplanted stem cells are limited. Cell‐based therapies using MSCs would benefit greatly through the use of an imaging methodology that allows cells to be tracked in vivo and in a timely fashion. In this study, we implemented an in vivo imaging methodology to specifically track early events such as differentiation of implanted human MSCs (hMSCs). This system uses the collagen type 1 (Col1α1) promoter to drive expression of firefly luciferase (luc) in addition to a constitutively active promoter to drive the expression of green fluorescent protein (GFP). The resulting dual‐promoter reporter gene system provides the opportunity for osteogenic differentiation‐specific luc expression for in vivo imaging and constitutive expression of GFP for cell sorting. The function of this dual‐promoter reporter gene was validated both in vitro and in vivo. In addition, the ability of this dual‐promoter reporter system to image an early event of osteogenic differentiation of hMSCs was demonstrated in a murine segmental bone defect model in which reporter‐labeled hMSCs were seeded into an alginate hydrogel scaffold and implanted directly into the defect. Bioluminescence imaging (BLI) was performed to visualize the turn‐on of Col1α1 upon osteogenic differentiation and followed by X‐ray imaging to assess the healing process for correlation with histological analyses. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res XX:XXX–XXX, 2013 © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 871–879, 2013     

Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling

Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin’s conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin–deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.     

MR-guided laser-induced thermotherapy (LITT) of liver tumours: experimental and clinical data

MR-guided laser-induced interstitial thermotherapy (LITT) is a percutaneous, minimally invasive treatment modality for treating liver lesions/metastases, soft tissue tumours and musculoskeletal lesions. In this group, MR-guided LITT is currently performed under local anaesthesia on an out-patient basis with a specially designed saline-cooled laser application system. Nd:YAG laser (1064?nm wave length) was used for tumour ablation. Magnetic resonance imaging (MRI) using both open and closed MR units has proven clinically effective in validating the exact positioning of optical fibres. It also allows for real time-monitoring of thermal effects and the evaluation of treatment-induced coagulation necrosis. In liver tumours, percutaneous MR-guided LITT achieves a local tumour control rate of 98.7% at 3 months post-therapy and 97.3% at 6 months with metastases smaller than 5?cm in diameter. The mean survival rate for 1259 patients with 3440 metastases treated with 14 694 laser applications at the institute (calculated with the Kaplan-Meier method) was 4.4 years (95% confidence interval: 4.1–4.8?years) and median survival was 3.00 years. No statistically significant difference in survival rates was observed in patients with liver metastases from colorectal cancer vs metastases from other primary tumours. The rate of clinically relevant side effects and complications requiring secondary treatment was 2.2%. The clinical use of MR guided LITT (size<5?cm, number<5) is justified in patients with liver metastases of colorectal and/or breast cancers if the inclusion criteria are carefully observed. Further indications for MR guided LITT include recurrent cancer lesions in the head and neck, lung metastases and bone and soft tissue lesions.     

Efficacy and tolerability of 8 weeks'' treatment with terbinafine in children with tinea capitis caused by Microsporum canis: a comparison of three doses

BACKGROUND: Tinea capitis caused by Microsporum canis is the most common mycosis of the scalp in preschool and school-aged children in Greece. OBJECTIVE: To compare the efficacy, safety and tolerability of an 8-week course of oral terbinafine at different doses. METHODS: Patients received oral terbinafine at doses ranging from 3.3 to 12.5 mg/kg/day for 8 weeks, as follows: group A, terbinafine 3.3 to 6.0 to 7.0 mg/kg/day (23 patients); group C, terbinafine > 7.0 to 12.5 mg/kg/day (37 patients). Fungal microscopy and cultures were performed 4 weeks before the start of the treatment, at the end of the treatment (week 8) and at a follow-up visit at week 16. RESULTS: At week 8 mycological cure was achieved in one patient (2.7%) in group A, in 21 patients (91.3%) in group B and in 34 patients (97.1%) in group C. At week 16 mycological cure was achieved in one patient (2.7%) in group A, in 22 patients (95.7%) in group B and in 35 patients (100%) in group C. There was a statistically significant difference (P < 0.0005) between dose level and efficacy of terbinafine at the end of the treatment period and also at the follow-up visit at week 16. Five patients (three in group A and two in group C) discontinued treatment because of adverse events. CONCLUSIONS: The administration of terbinafine at a dose of either 6-7 or 7-12.5 mg/kg/day for 8 weeks is safe and effective for the treatment in children of tinea capitis caused by M. canis.     

DETECTION OF PROSTATE CANCER WITH C-METHIONINE POSITRON EMISSION TOMOGRAPHY

PurposeWe studied the detection of primary prostate cancer with positron emission tomography (PET) using ¹¹C-labeled methionine (MET) in patients with increased prostate specific antigen (PSA) levels and repeatedly negative biopsies.Materials and MethodsA total of 20 consecutive patients with increased serum PSA and negative repeat biopsies were included in the study. Patient age ranged from 52 to 75 years (average 65). PSA levels ranged from 3.49 to 28.6 ng/ml (average 9.36). Dynamic PET images were obtained from the prostate region using ¹¹C-labeled MET. Suspicious accumulations of the tracer were anatomically localized using magnetic resonance images and were used as guidance during the next biopsy.ResultsPET was positive in 15 (75%) patients, in 7 of whom (46.7%) the next repeat biopsy verified carcinoma. The overall detection rate was 35% (7 of 20) and 46.7% (7 of 15) in the whole group and in the positive PET group, respectively. All 5 of 5 patients with negative MET PET had negative biopsies.ConclusionsMET PET of the prostate with short dynamic scanning and multicore biopsy is a useful method to ensure a high detection rate of prostate cancer in patients with increased PSA and repeat negative biopsies.