No abstract

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIIIdeficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A. IntroductionHemophilia A is a severe, X-linked bleeding disorder caused by a deficiency of blood coagulation factor VIII (FVIII). Hemophilia A has an incidence approaching 1 in 4000 males in all populations, 1 and in its severe form, is a life-threatening, crippling disease. Infusion of plasma-derived or recombinant FVIII protein in response to bleeding crises is currently the most widely accepted therapy 1 and has dramatically increased the life expectancy and quality of life for many patients with hemophilia. However, the high cost and short supply of FVIII replacement products has resulted in their availability being limited to less than 10% of the world's hemophilic population.Gene therapy for hemophilia A would provide prophylactic expression of FVIII and correction of the coagulation defect. Considerable progress has been made recently in the development of adenoviral vector-mediated gene therapy for hemophilia A. 2,3 Potent adenoviral vectors encoding a human FVIII complementary DNA (cDNA) have been developed that mediated expression of physiologic levels of FVIII in mice, 4-7 monkeys, 8 and dogs, 9 and sustained human FVIII expression in normal 5 and hemophilic mice. 7 Treatment of hemophilic mice and dogs resulted in human FVIII expression and complete phenotypic correction, verifying the feasibility of adenoviral vector administration for the treatment of hemophilia A. 7,[9][10][11] Expression in the hemophilic mice was sustained for at least 1 year, 7,11 whereas the duration of expression in the hemophilic dogs was short-term, limited by a rapid antibody response to the human FVIII protein. 9 Canine hemophilia A was first described 50 years ago, 12,13 and FVIII-deficient dogs have been used to support the development of FVIII pharmaceutical products. [14][15][16][17][18][19] However, human FVIII is highly immunogenic in dogs when the protein is delivered intravenously 20 or v...

Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 jzM and a pH optimum of 7.0-75. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylhlanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.

The addition of 0.1 mug/ml of phorbol myristate acetate (PMA) to a suspension of resting human neutrophils causes a marked stimulation of all aspects of cellular oxidative metabolism normally associated with phagocytosis. PMA induces a greatly increased rate of glucose oxidation via the hexose monophosphate shunt, increased production of superoxide anion and of hydrogen peroxide, increased cellular chemiluminescence, and increased iodination of protein material. The time course of hexose monophosphate shunt activation and of chemiluminescence are similar to those observed following phagocytosis of opsonized zymosan; the levels of activation achieved in all cases approximate those seen following phagocytosis. These phenomena are not simply reflections of altered cellular permeability, since PMA actually inhibits the uptake of radioactive 2-deoxyglucose and of uniformly labeled amino acids. The addition of PMA similarly inhibits the uptake of 14C-labeled bacteria, suggesting a competition between the effect of the chemical and the process of phagocytosis. These results suggest that PMA activates the cell in the same manner as does phagocytosis. This compound should provide a useful tool for elucidating the metabolic events underlying the phenomena of phagocytosis and bacterial killing by polymorphonuclear leukocytes.