Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer

Fabry disease is an X-linked recessive inborn metabolic disorder characterized by systemic and vascular accumulation of globotriaosylceramide (Gb(3)) caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). The condition is associated with an increased morbidity and mortality due to renal failure, cardiac disease, and early onset of stroke. Hemizygous males are primarily affected clinically with variable expression in heterozygous females. Gene-therapy trials have been initiated recently in alpha-gal A knockout mouse models of Fabry disease by using a variety of viral vectors. In the present investigation we administered single i.v. injections of 1 x 10(10) genomes of recombinant adeno-associated virus (rAAV) encoding the human alpha-gal A gene driven by a modified chicken beta-actin (CAG) promoter to alpha-gal A knockout (Fabry) mice. Transgenic mice were analyzed for expression of alpha-gal A activity and Gb(3) levels in liver, kidney, heart, spleen, small intestine, lung, and brain. Administration of the rAAV-CAG-hAGA vector resulted in stable expression of alpha-gal A in organs of the Fabry mice for >6 months. alpha-Gal A activity in the organs became equal to or higher than that of wild-type mice. Accumulated Gb(3) in the liver, heart, and spleen was reduced to that of wild-type mice with lesser but significant reductions in kidney, lung, and small intestine. Injection of the rAAV-CAG-hAGA construct into skeletal muscle did not result in expression of alpha-gal A in it or in other tissues. This study provides a basis for a simple and efficient gene-therapy approach for patients with Fabry disease and is indicative of its potential for the treatment of other lysosomal storage disorders.     

Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells

Fabry disease is a compelling target for gene therapy as a treatment strategy. A deficiency in the lysosomal hydrolase alpha-galactosidase A (alpha-gal A; EC ) leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop vascular occlusions that cause cardiovascular, cerebrovascular, and renal disease. Unlike for some lysosomal storage disorders, there is limited primary nervous system involvement in Fabry disease. The enzyme defect can be corrected by gene transfer. Overexpression of alpha-gal A by transduced cells results in secretion of this enzyme. Secreted enzyme is available for uptake by nontransduced cells presumably by receptor-mediated endocytosis. Correction of bystander cells may occur locally or systemically after circulation of the enzyme in the blood. In this paper we report studies on long-term genetic correction in an alpha-gal A-deficient mouse model of Fabry disease. alpha-gal A-deficient bone marrow mononuclear cells (BMMCs) were transduced with a retrovirus encoding alpha-gal A and transplanted into sublethally and lethally irradiated alpha-gal A-deficient mice. alpha-gal A activity and Gb3 levels were analyzed in plasma, peripheral blood mononuclear cells, BMMCs, liver, spleen, heart, lung, kidney, and brain. Primary recipient animals were followed for up to 26 weeks. BMMCs were then transplanted into secondary recipients. Increased alpha-gal A activity and decreased Gb3 storage were observed in all recipient groups in all organs and tissues except the brain. These effects occurred even with a low percentage of transduced cells. The findings indicate that genetic correction of bone marrow cells derived from patients with Fabry disease may have utility for phenotypic correction of patients with this disorder.     

Mandibular division trigeminal nerve injuries following primary endodontic treatment. A case series

The aim of this study is to report a series of patients with mandibular division trigeminal nerve (V₃) injuries secondary to endodontic treatment, evaluate presentation characteristics and identify prevention strategies. This article describes a retrospective review of patients referred to a tertiary clinic 2007–2015 with V₃ injury following endodontic treatment. The sample included 12 male and 16 female patients with a mean age of 41.5 years. Sixteen cases presented following endodontic treatment of the first and second molar, premolar teeth in eight cases and canine in two cases. Fifteen patients reported immediate post‐operative symptoms, in eleven cases there was a 24–48 h asymptomatic period. The average referral delay was 23.1 months. Twenty patients had permanent neuropathy. Four patients experienced resolution of symptoms within 8 weeks. V₃ injury following endodontic treatment is rare but can result in permanent neuropathy and functional impairment. This can be avoided through comprehensive pre‐operative radiographic examination, identification and referral of high‐risk cases.     

Pegaptanib for choroidal neovascularization in treatment-na?ve exudative age-related macular degeneration.

BACKGROUND AND OBJECTIVE: To evaluate the use of intravitreal pegaptanib in the treatment of choroidal neovascularization secondary to age-related macular degeneration (AMD) in treatment-naive patients. PATIENTS AND METHODS: In a consecu-tive, retrospective case series, treatment-na?ve patients with exudative AMD were treated with intravitreal pegaptanib. Intravitreal injections were typically given every 6 weeks at the discretion of the treating physician. Snellen visual acuity (VA), clinical course, and adverse events were monitored. A minimum of three pegaptanib injections were given. Retreatment criteria included persistent submacular fluid, macular edema, new macular hemorrhage, and loss of vision. RESULTS: The average change in VA for all lesions was a loss of 2.9 lines. Fifteen (14%) patients gained more than 3 lines of VA. The average number of in-jections was 4.8. Ninety-two of 111 lesions were able to be categorized by size. Sixty-six patients had small lesions (< 4 disc areas) with an average change of -2.0 lines, and 26 had large lesions (> or = 4 disc areas) with an average change of -5.4 lines (P < .02). Patients with larger lesions were at greater risk for severe visual loss (P < .01). The average follow-up was approximately 31 weeks (range: 12 to 82 weeks) after the first injection. CONCLUSIONS: Pegaptanib therapy resulted in a 2.9 average line loss in patients when all lesions were considered. Small lesions responded favorably, with 15% of patients gaining more than 3 lines of VA. Larger lesions had an increased risk of progression and poor visual outcome.     

Dimere Austauschprodukte aromatisch substituierter α-Aminonitrile VII. Mitt. über Umsetzungen von α-Aminonitrilen

Dimer Products of aromatic substituted Aminonitriles The dimer-products which in addition to hydrogen-exchange products are produced during the reaction of aromatic substituted aminonitriles with metallic sodium in toluene are unstable. The initially produced diphenyldiamine derivatives decompose during isolation under elimination of amine and then hydrolyse to form desoxybenzoin derivatives.     

Synthesis of Simian Virus 40 DNA in Isolated Nuclei

Nuclei isolated from African green monkey kidney cells infected with simian virus 40 at different times after infection maintain in vitro the same temporal sequence of host and viral DNA synthesis as that seen in intact cells. The viral DNA synthesized by the nuclei of cells previously infected for 32-35 hr was characterized by centrifugation through neutral and alkaline sucrose gradients, and by isopycnic banding in a propidium iodide-cesium chloride gradient. DNA synthesis in this system is maintained for only 4-5 min. Neutral sucrose gradient analysis showed that most of the radioactivity is associated with the replicative intermediate of simian virus 40 DNA and the rest sediments at 5-7 S. Alkaline gradient analysis showed that 50-60% of the radioactivity sediments as 3-7S fragments, and the rest between 7 and 16 S. Pulsechase experiments showed that in this system 3-7S fragments do not mature into long chains. A model is presented to explain the failure of these fragments to join into long chains in this in vitro nuclear system.     

Adduktbildung und Dimerisierung von α-Piperidinoacetonitril bei Umsetzung mit n-Butylmagnesiumchlorid. V. Mitt. über Umsetzungen von α-Aminonitrilen

Dimerisation of α-Piperidinoacetonitrile in the Reaction with n-Butylmagnesium chloride The reaction mechanisms which determine the course of the reaction between t- and n-butyl-magnesium chloride and α-piperidinoacetonitrile, have been studied. The formation of different quantities of the reaction products 3-amino-2,4-dipiperidinocrotononitrile and butyl-α-piperidinomethylketone can be explained on the basis of steric hindrace and also through the difference of nucleophilic activity of t- and n-butyl groups.