Infusible platelet membrane microvesicles: a potential transfusion substitute for platelets

BACKGROUND: Several substitutes for intact, viable platelets have been used for transfusion, both to people and in animal models, with varied success. Infusible platelet membrane (IPM) is prepared from human platelets. IPM retains the glycoprotein (GP)lb receptor and has platelet factor 3 activity (procoagulant activity). However, factor V, serotonin, a cytoplasmic marker enzyme (purine nucleotide phosphorylase), GPIIb/IIIa complex, and HLA class I and II antigens are all absent in IPM. STUDY DESIGN AND METHODS: IPM is prepared from outdated platelets. The platelets were disrupted by freezing and thawing; they were washed and heated to inactivate possible viral contaminants, and then the sonicated membrane microvesicle fraction was separated and lyophilized. The hemostatic activity of IPM was measured by its ability to reduce the prolonged bleeding time in thrombocytopenic rabbits. RESULTS: Administration of IPM at a dose of 2 mg per kg results in a substantial reduction in the bleeding time. In a series of 23 experiments, a median preinjection bleeding time of 15 minutes was reduced to 6 minutes within 4 hours after IPM administration. Administration of IPM did show a mild enhancement in the thrombogenicity index, as measured in the Wessler rabbit model. This enhancement is, however, not significant, as a thrombogenicity index value of up to 0.6 is clinically acceptable. CONCLUSION: IPM may have clinical potential as a substitute for platelets in the treatment of bleeding due to thrombocytopenia.     

Fluorescent nanodiamond – hyaluronate conjugates for target-specific molecular imaging

Despite wide investigation on molecular imaging contrast agents, there are still strong unmet medical needs to enhance their signal-to background ratio, brightness, photostability, and biocompatibility with multimodal imaging capability. Here, we assessed the feasibility of fluorescent nanodiamonds (FNDs) as carbon based photostable and biocompatible materials for molecular imaging applications. Because FNDs have negatively charged nitrogen vacancy (NV) centers, they can emit bright red light. FNDs were conjugated to hyaluronate (HA) for target-specific molecular imaging. HA is a biocompatible, biodegradable, and linear polysaccharide with abundant HA receptors in the liver, enabling liver targeted molecular imaging. In vitro cell viability tests revealed the biocompatibility of HA–FND conjugates and the competitive cellular uptake test confirmed their target-specific intracellular delivery to HepG2 cells with HA receptors. In addition, in vivo fluorescence lifetime (FLT) assessment revealed the imaging capability of FNDs and HA–FND conjugates. After that, we could confirm the statistically significant liver-targeted delivery of HA–FND conjugates by in vivo imaging system (IVIS) analysis and ex vivo biodistribution tests in various organs. The renal clearance test and histological analysis corroborated the in vivo biocompatibility and safety of HA–FND conjugates. All these results demonstrated the feasibility of HA–FND conjugates for further molecular imaging applications.

Fluorescent nanodiamond conjugated with hyaluronate is developed as a carbon based photostable and biocompatible material for liver-targeted molecular imaging applications.     

下坡运动对大鼠骨骼肌肌浆网功能的影响

目的：观测研究下坡(离心)运动对大鼠骨骼肌肌浆网Ca2+-ATP酶活性,Ca2+摄取与释放在量与时程上的影响。此外,测定离子载体的刺激作用,即测定在含与不含(Ca2+离子载体)A23187时Ca2+-ATP酶活性的比值,用以评定囊泡的完整性。方法：成年雄性SD大鼠随机分为对照与离心运动组, 离心运动的大鼠分别于运动后即刻, 4, 24, 48, 72 和144h后取样 (n=7). 离心运动方式采用90min持续跑台下坡运动(-16°;15m/min)。取大鼠红股肌制备组织匀浆, 测定肌浆网Ca2+-ATP酶活性,Ca2+摄取与释放。结果：与对照组[19.25±1.38 nmol ·min-1·(mg protein)-1]相比, 肌浆网Ca2+摄取分别于运动后即刻和4h下降了29% and 36% (P<0.05), 24h依然降低(P<0.05). 肌浆网Ca2+释放与对照组[31.06±2.36 nmol·min-1·(mg protein)-1] 相比,也分别于运动后即刻和4h下降了37% and 39% (P<0.05), 24h持续降低(P<0.05). 用含离子载体测定的肌浆网Ca2+-ATP酶活性运动后4h降低了31%(P<0.05), 并于运动后24h仍然降低 (P<0.05)。运动后, 含与不含A23187时测定的Ca2+-ATP酶活性的比值未见显著性改变, 表明该运动没有明显改变肌浆网膜的完整性。结论：一次性低强度，长时间下坡运动导致肌浆网功能长时间降低, 运动后恢复期两天尚未完全恢复, 亦可构成离心运动诱导的骨骼肌某些功能降低的基础。提示这些变化可能产生于离心收缩时肌节长度不匀一性所造成的张力应激。     

体外制成复合软质再生颅骨修复材料填充犬颅骨缺损

目的:目前颅骨修补材料有很多种,但都为异源性无机骨替代物,并且应用该方法又要给患者再次行开颅手术,实验拟开展新型颅骨再生材料的研究。方法:实验于2006-05/11在解放军第一五七医院动物中心及中山大学附属第三医院动物实验室完成。①实验动物:30只犬随机分为实验组20只,对照组10只。②实验方法:应用纳米级羟基磷灰石为支架和成骨细胞培养,加入脱矿的犬类骨基质为载体的重组人类骨形成蛋白2,制成复合软质再生颅骨。实验组犬在右侧颅骨缺损中填补藻酸钙凝胶、成骨细胞、纳米级骨粉的复合材料,左侧颅骨缺损中填补藻酸钙凝胶、成骨细胞、纳米级骨粉和重组人类骨形成蛋白2的复合材料。对照组犬在右侧为单纯颅骨缺损,左侧颅骨缺损中填补藻酸钙凝胶、成骨细胞、纳米级骨粉和重组人类骨形成蛋白2复合材料。实验过程中对动物处置符合动物伦理学标准。③实验评估:手术后1,2,3,6个月X射线片检查颅骨缺损修复情况,对再生的颅骨组织标本进行茜素红S染色,观察成骨能力及再生材料骨膜组织细胞体外培养情况。结果:实验动物均进入结果分析。术后1个月,成骨活跃,骨端新生骨小梁基本覆盖骨断端,缺损区可见较多新生骨小梁形成,骨端新生骨小梁向缺损区长入;术后2个月可见较多散在骨岛形成;术后3个月可见成熟骨,并有髓腔形成,缺损区大量新骨形成。而各对照组骨断端处有散在骨岛,或被增生的纤维结缔组织占据,可见大量纤维组织及毛细血管长入,植入的基质材料基本被吸收,无新骨生成。结论:应用纳米级羟基磷灰石为支架和成骨细胞培养,加入脱矿骨基质为载体的重组人类骨形成蛋白2,制成的复合软质再生颅骨能自身代谢并逐渐骨化,形成新的颅骨。     

Low-frequency Sonophoresis: Pathologic and Thermal Effects in Dogs

Objective: Low-frequency sonophoresis has recently been shown to significantly facilitate transdermal permeability of various substances (e.g., insulin) in animal models, thus eliminating the need to inject such agents. Prior to human trials, the authors studied the safety profile of low-frequency sonophoresis in dogs by evaluating microscopic and temperature changes in the skin after sonophoresis. Methods: An evaluator-blinded canine study of sonophoresis using different energy intensities and probe diameters was performed. Low-frequency ultrasound was applied for 60 seconds to the clipped abdominal skin of 3 anesthetized adult mongrel dogs using a sonicator operating at a frequency of 20 KHz with a maximal energy output of 400 W. The sonicator was immersed in normal saline, and intensities of 4%, 10%, 20%, 30%, and 50% were applied during 600 msec of every second (pulsed mode). Three probes, 1-cm cylindrical, 5-cm cylindrical, and 10-cm disc-shaped, were evaluated. Each experimental condition was performed twice. Subcutaneous temperatures were measured by temperature probe before and after sonophoresis. At 30 minutes post-sonophoresis, full-thickness skin biopsies were taken for blinded histopathologic evaluation. Results: Minimal urticarial reactions were noted with the 1-cm probe at intensities of ≤20% and with the 5-cm probe at 4% intensity. With higher intensity, thermal injuries were observed grossly with erythema and vesicles. The microscopic correlates were papillary and dermal edema with neutrophils and telangiectasia. The conditions producing vesicles grossly had foci of epidermal necrosis, subepidermal vesicles, and degeneration of papillary dermal collagen. With still higher intensities, confluent epidermal necrosis became apparent. Use of the 10-cm probe did not result in any injury. Conclusions: Low-frequency ultrasound at low intensities appears safe for use to enhance the topical delivery of medications, producing only minimal urticarial reactions. Higher-intensity conditions resulted in second-degree burns, most likely attributable to localized heating.     

Validation of 3D multimodality roadmapping in interventional neuroradiology

Three-dimensional multimodality roadmapping is entering clinical routine utilization for neuro-vascular treatment. Its purpose is to navigate intra-arterial and intra-venous endovascular devices through complex vascular anatomy by fusing pre-operative computed tomography (CT) or magnetic resonance (MR) with the live fluoroscopy image. The fused image presents the real-time position of the intra-vascular devices together with the patient's 3D vascular morphology and its soft-tissue context. This paper investigates the effectiveness, accuracy, robustness and computation times of the described methods in order to assess their suitability for the intended clinical purpose: accurate interventional navigation. The mutual information-based 3D-3D registration proved to be of sub-voxel accuracy and yielded an average registration error of 0.515 mm and the live machine-based 2D-3D registration delivered an average error of less than 0.2 mm. The capture range of the image-based 3D-3D registration was investigated to characterize its robustness, and yielded an extent of 35 mm and 25° for >80% of the datasets for registration of 3D rotational angiography (3DRA) with CT, and 15 mm and 20° for >80% of the datasets for registration of 3DRA with MR data. The image-based 3D-3D registration could be computed within 8 s, while applying the machine-based 2D-3D registration only took 1.5 μs, which makes them very suitable for interventional use.     

Registration of 2D x-ray images to 3D MRI by generating pseudo-CT data

Spatial and soft tissue information provided by magnetic resonance imaging can be very valuable during image-guided procedures, where usually only real-time two-dimensional (2D) x-ray images are available. Registration of 2D x-ray images to three-dimensional (3D) magnetic resonance imaging (MRI) data, acquired prior to the procedure, can provide optimal information to guide the procedure. However, registering x-ray images to MRI data is not a trivial task because of their fundamental difference in tissue contrast. This paper presents a technique that generates pseudo-computed tomography (CT) data from multi-spectral MRI acquisitions which is sufficiently similar to real CT data to enable registration of x-ray to MRI with comparable accuracy as registration of x-ray to CT. The method is based on a k-nearest-neighbors (kNN)-regression strategy which labels voxels of MRI data with CT Hounsfield Units. The regression method uses multi-spectral MRI intensities and intensity gradients as features to discriminate between various tissue types. The efficacy of using pseudo-CT data for registration of x-ray to MRI was tested on ex vivo animal data. 2D-3D registration experiments using CT and pseudo-CT data of multiple subjects were performed with a commonly used 2D-3D registration algorithm. On average, the median target registration error for registration of two x-ray images to MRI data was approximately 1 mm larger than for x-ray to CT registration. The authors have shown that pseudo-CT data generated from multi-spectral MRI facilitate registration of MRI to x-ray images. From the experiments it could be concluded that the accuracy achieved was comparable to that of registering x-ray images to CT data.     

Use of risk stratification to target therapies in patients with recent onset arthritis; design of a prospective randomized multicenter controlled trial

Background  

Early and intensive treatment is important to inducing remission and preventing joint damage in patients with rheumatoid arthritis. While intensive combination therapy (Disease Modifying Anti-rheumatic Drugs and/or biologicals) is the most effective, rheumatologists in daily clinical practice prefer to start with monotherapy methotrexate and bridging corticosteroids. Intensive treatment should be started as soon as the first symptoms manifest, but at this early stage, ACR criteria may not be fulfilled, and there is a danger of over-treatment. We will therefore determine which induction therapy is most effective in the very early stage of persistent arthritis. To overcome over-treatment and under-treatment, the intensity of induction therapy will be based on a prediction model that predicts patients' propensity for persistent arthritis.