Outer retinal transduction can be achieved after intravitreal delivery of adeno-associated virus serotype 2 vector with glycosidic enzymes

BackgroundGene therapies for retinal disorders, including in current clinical trials, so far have relied on subretinal delivery of adeno-associated virus (AAV) vectors carrying therapeutic DNA into outer retinal cells. Subretinal injection has many limitations over the less-invasive route of administration into the vitreal cavity. However, at present only limited retinal transduction can be achieved after intravitreal delivery of AAV vectors. We hypothesise that the inner limiting membrane and extracellular matrix proteoglycans act as a barrier to AAV vector entry into and movement across the retina. Therefore, glycosidic enzymes, which degrade these extracellular barriers, can improve retinal gene therapy. In this study we investigated the effects of enzymatic digestion of extracellular matrices on the depth of vector penetration into the retina.MethodsThe green fluorescent protein (GFP)-expressing AAV serotype 2 (AAV2) vector was co-injected intravitreally with glycosidic enzymes at their optimum concentration. Efficacy of virus transduction was assessed by visualising fluorescence in histological cross-sections with fluorescence microscopy. We also analysed safety of these treatments and retinal function using electroretinography.FindingsGlycosidic enzymes led to a significant improvement in retinal transduction after intravitreal delivery of AAV2. These enzymes markedly improved transduction of the outer retina, including photoreceptor cells. Electroretinograms were unchanged (compared with controls) even at much higher doses of enzymes than were needed for optimum retinal transduction.InterpretationAAV2-mediated retinal transduction is improved by co-injection of glycosidic enzymes into the vitreous. Improved transduction efficiency may allow intravitreal injection to become the preferred route for delivering gene therapy to the inner and outer retina in both preclinical and clinical settings.FundingUK Medical Research Council.     

Obstruction of the left hepatic duct: diagnosis and treatment by selective fine-needle cholangiography and percutaneous biliary drainage

Obstruction of the left hepatic duct due to periportal pathologic conditions was identified by fine-needle transhepatic cholangiography in 28 patients. Selective cholangiography of the left duct was performed in 30 instances and subsequent selective left-sided catheter decompression was carried out in 23 cases. Techniques for selective puncture and drainage of the left duct required procedure modifications to accommodate the specific anatomic and pathologic features of the obstructed left duct system. Ultrasonography was indispensable as an aid to delineation of the left duct anatomy for directing needle puncture. Accurate documentation and successful catheter drainage of left duct obstruction are important contributions to the total management of patients with high biliary obstruction.     

Optimal nutrition during the period of mechanical ventilation decreases mortality in critically ill, long-term acute female patients: a prospective observational cohort study

Introduction  

Optimal nutrition for intensive care patients has been proposed to be the provision of energy as determined by indirect calorimetry, and protein provision of at least 1.2 g/kg pre-admission weight per day. The evidence supporting these nutritional goals is based on surrogate outcomes and is not yet substantiated by patient oriented, clinically meaningful endpoints. In the present study we evaluated the effects of achieving optimal nutrition in ICU patients during their period of mechanical ventilation on mortality.     

美国国家骨髓库无关供者造血干细胞移植20年回顾

自美国国家骨髓库(NMDP)开展第一例无关供者移植以来,至今已有20年.NMDP目前的库容量已逾700万,已为6大洲提供了30 000多份无关供者造血干细胞.这一辉煌成就是美国国家骨髓库600多名工作人员共同努力的结果,同时也得益于广泛的国际合作,包括171个移植中心,73个供者中心,24个脐血库,97个骨髓采集中心,91个血液净化中心,26个HLA分型实验室和26个合作供者登记处.本文回顾了美国国家骨髓库的历史,阐述了20年来移植病人、移植物来源和预处理方案几方面的主要变化趋势.     

尿中睾酮与表睾酮的三甲基硅烷化及其比值的GC—MS测定

对睾酮及表睾酮的三甲基硅烷化进行了详细考察,找到了较好的抗氧剂巯基乙醇,确定了较好的衍生化条件,衍生化产物单一。并采用GC—MS法测定了尿中睾酮与表睾酮的比值。实验条件为:以氦为载气,SE—54熔融石英柔性毛细管柱、程序升温进行样品分离,多离子检测(MID),监测m/z432的离子。该法专属、灵敏、快速。睾酮与表睾酮比值在1:1～10:1(睾酮为20ng/μl)与相应峰面积比呈线性关系(r=0.998),最低检测限为1ng,最低检测尿药浓度为8ng/ml。     

Walker-Warburg Syndrome with POMT1 mutations can be associated with cleft lip and cleft palate

Walker-Warburg Syndrome (WWS) is an alpha-dystroglycan deficient congenital muscular dystrophy that is associated with brain and eye abnormalities. Patients present with hypotonia, weakness, developmental delay, mental retardation and occasional seizures. Other abnormalities were also described including cleft lip and palate. Mutations in POMT1, POMT2, fukutin, FKRP and LARGE genes are found in 20-30% of children with WWS. We report a novel mutation in POMT1 gene and provide further evidence that WWS with cleft lip and palate is associated with POMT1 mutations. We recommend POMT1 analysis in WWS cases associated with cleft lip and palate when considering which gene to sequence first.     

Elimination of Daudi lymphoblasts from human bone marrow using avidin- biotin immunoadsorption

Biotinylated antibodies and an avidin-Sepharose 6MB column were utilized in a novel approach to deplete selected cell populations from human bone marrow. Fluorescein-labeled Daudi lymphoblasts were mixed with bone marrow mononuclear cells in a model system, and removal of Daudi cells was quantitatively assessed with an inverted fluorescent microscope. Treatment using the biotinylated monoclonal antibody 2H7 reactive with Bp32 antigen (expressed on Daudi cells) followed by passage over avidin-Sepharose produced greater than two logs of Daudi cell removal from bone marrow. An alternative method was tested by treating cells successively with nonbiotinylated monoclonal antibody and biotinylated goat antimouse immunoglobulin followed by passage over avidin-Sepharose. Up to three logs of Daudi cells could be eliminated from bone marrow with quantitative recovery of hematopoietic progenitors. The use of biotinylated goat antimouse immunoglobulin eliminates the need to prepare a biotin conjugate of each individual monoclonal antibody. The clinical application of cellular immunoadsorption using the avidin-biotin system may prove useful in bone marrow transplantation.