Transscaphoid transcapitate transtriquetral perilunate fracture-dislocation: a case report.

We present a rare stage III greater arc fracture-dislocation of the carpus including transscaphoid, transcapitate, and transtriquetral dorsal perilunate fracture-dislocation.

Acute dislocation of the elbow caused by a solitary osteochondroma of the proximal radius.

A young man presented with acute dislocation of the left elbow at the radio-capitellar articulation caused by trapping of the biceps tendon at the stalk of a solitary osteochondroma. There was no deformity of the ulna and radius shaft suggestive of a developmental growth disturbance of the forearm bones. Good reduction could be achieved by simple relocation of the biceps tendon. The osteochondroma was excised.

A new method of functional tendon transfer for the dysfunction of extensor hallucis longus.

A surgical technique of functional tendon transfer for the treatment of extensor hallucis longus (EHL) rupture is described. By using the extensor digitorum longus tendon of the second toe, the patient regains active dorsiflexion of the big toe and the deformity of the toe is corrected.

Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient 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.

In vivo evaluation of a novel epitope-tagged human factor VIII-encoding adenoviral vector.

Haemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and is an attractive target for gene therapy. Adenoviral vectors encoding a human B-domain deleted (BDD) FVIII cDNA have been shown previously to mediate expression of high levels of human FVIII and correct the bleeding defect in haemophiliac mice and dogs. While vector assessment in a non-human primate model would have a significant preclinical benefit, a haemophiliac non-human primate model is not available, and assays that distinguish human FVIII from monkey FVIII have not been developed successfully. As a first step to enable vector evaluation in non-human primates, we have constructed an epitope-tagged FVIII molecule by the addition of 16 amino-acids to the carboxy terminus of the BDD protein (BDD-E). Following vector administration to normal mice, therapeutic levels of BDD-E FVIII were expressed for at least 20 weeks. Treatment of haemophiliac mice revealed that the BDD-E protein was biologically active in vivo. To distinguish the BDD-E protein from non-human primate FVIII, a sensitive immunoprecipitation/Western assay was developed that reproducibly detected 1 ng mL-1 of the epitope-tagged human FVIII in the presence of monkey plasma. These data demonstrate that the addition of an epitope tag had no effect on FVIII function or immunogenicity, and suggest that the BDD-E vector will be an effective reagent for non-human primate studies.

In vivo evaluation of an adenoviral vector encoding canine factor VIII: high-level, sustained expression in hemophiliac mice.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Circumvention of immunity to the adenovirus major coat protein hexon.

Immunity to adenoviruses is an important hurdle to be overcome for successful gene therapy. The presence of antibodies to the capsid proteins prevents efficacious adenovirus vector administration in vivo. We tested whether immunity to a particular serotype of adenovirus (Ad5) may be overcome with a vector that encodes the hexon sequences from a different adenovirus serotype (Ad12). We successfully constructed an adenovirus vector with a chimeric Ad5-Ad12 hexon which was not neutralized by plasma from C57BL/6 mice immunized with Ad5. The vector was also capable of transducing the livers of C57BL/6 mice previously immunized with Ad5.

Superoxide generation by the human polymorphonuclear leukocyte in response to latex beads.

In this study, superoxide formation was not immediately detected when polymorphonuclear leukocytes were treated with linoleyl alcohol-coated, carboxy-modified latex beads. However, all other measures of neutrophil activation were present. Superoxide was not detected until 30 min after the initial exposure to beads. However, an O2(-)-producing, NADPH-dependent oxidase is active 15 min after exposure. When polymorphonuclear leukocytes are pretreated with cytochalasin B, superoxide production is detected immediately after exposure to the beads. Superoxide secretion after treatment with linoleyl alcohol-coated latex beads is compared with the response to other latex beads. The results imply that neutrophils form a phagolysosome around linoleyl alcohol-coated latex beads that is tightly sealed and does not allow superoxide to escape into the medium where it could be detected by the reduction of ferricytochrome c.

Role of free radical processes in stimulated human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes produce large quantities of superoxide when they attack and kill bacteria. However, superoxide is a weak oxidizing and reducing agent, and other more reactive oxygen species derived from reactions of superoxide are suggested to participate in the killing processes. To test the hypothesis that a reactive free radical or singlet oxygen is involved in bactericidal activity, human polymorphonuclear leukocytes were exposed to phagocytozable particles containing lipids that contain the easily autoxidized 1,4-diene moiety. After incubation the preparations were extracted and the extracts reduced with NaBH4 to convert hydroperoxides to stable alcohols. Using gas chromatography/mass spectrometry to analyze the extracts, we were unable to detect products unless iron salts were added to the medium. The products obtained by extraction are those that would be expected if both free radical chain autoxidation and 1O2 oxidation were taking place. In summary, we find that polymorphonuclear leukocytes do not cause peroxidation, implying that formation of strongly oxidizing free radicals is not an intrinsic property of the leukocyte. Added iron catalyzes peroxidation by activated leukocytes yielding an unusual distribution of hydroxylated products.

Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.

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 microM and a pH optimum of 7.0-7.5. 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-phenylalanine 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.

Effects of asbestos on the random migration of rabbit alveolar macrophages.

The toxicity of sized and characterized chrysotile, crocidolite, and amosite preparations obtained from Dr. K. R. Spurny have been evaluated using alveolar macrophage (AM) migration inhibition assays and viability tests. These results have been compared with asbestos samples obtained from the National Institute of Environmental Health Sciences (NIEHS). These latter samples are designated chrysotile A (RT), crocidolite (RT), and amosite (RT). In addition, filter-isolated preparations of chrysotile A (RT) that consisted mainly of large nonphagocytosable fibers were also tested. Chrysotile (Spurny) and sonicated chrysotile A (RT) produced 50% migration inhibition at about 115 micrograms/mL. Spurny crocidolite produced 50% migration inhibition at about 340 micrograms/mL, whereas RT crocidolite produced 50% migration inhibition at about 230 micrograms/mL. RT amosite caused 50% migration inhibition at about 180 micrograms/mL, whereas Spurny amosite was inactive up to 500 micrograms/mL. The large nonphagocytosable chrysotile A (RT) fibers produced 50% migration inhibition at about 66 micrograms/mL. This indicates that fibers can be toxic for AM through extracellular membrane contact. In general the results from the viability studies paralleled the migration inhibition observations. None of the asbestos preparations induced a burst in the hexose monophosphate shunt of BCG-immune AM at 1 mg/mL. BCG-immune AM were more susceptible to cell death than normal AM when incubated with chrysotile A (RT), amosite (RT) and zymosan. Migration inhibition induced by asbestos fibers probably reflects toxicity of the asbestos preparations and could play an important role in blocking normal alveolar clearance of inhaled particles.

Co-localization of superoxide generation and NADP formation in plasma membrane fractions from human neutrophils.

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

The use of acetylated cytochrome c in detecting superoxide anion production in rabbit alveolar macrophages.

Polymorphonuclear phagocytes have been shown to undergo marked alteration in oxidative metabolism during phagocytosis. These alterations, collectively known as the "respiratory burst", include increased glucose oxidation through the hexose monophosphate shunt (1), increased oxygen consumption (1), and increased superoxide (O-2)3 (2) and H2O2 production (3). Similar metabolic events have also been shown to occur in the rabbit alveolar macrophage (AM). There is consistent evidence that the macrophage undergoes increased oxygen consumption (4-6) and hexose monophosphate shunt activity (4-9) upon phagocytosis. There are conflicting data, however, concerning the ability of the macrophage to produce O-2. Some studies suggest that macrophages are incapable of producing measurable amounts of O-2 upon phagocytosis (7, 10-12). Other studies, however, suggest that macrophages are indeed capable of producing substantial amounts of O-2 during phagocytosis (8, 13-15). This study was designed to resolve the discrepancies in the literature concerning O-2 production in macrophages.

Further evaluation of luminol-enhanced luminescence in the diagnosis of disorders of leukocyte oxidative metabolism: role of myeloperoxidase.

Chemiluminescence can be used to identify defects in the oxidative metabolism of granulocytes. This procedure has recently been adopted for use with microliter quantities of whole blood, appropriate for prenatal or neonatal study. Although the contribution of myeloperoxidase to the chemiluminescence assay has been noted, the possible diagnostic confusion between chronic granulomatous disease of childhood (which is rare and severe) and myeloperoxidase deficiency (which is common and of little clinical consequence) has not been stressed. We report a father and his infant daughter whose cells emitted no light in the luminol-enhanced luminescence assay; both patients are totally peroxidase deficient. These results emphasize the hereditary nature of myeloperoxidase deficiency, and the possibility for erroneous diagnosis of chronic granulomatous disease of childhood based on the luminol-enhanced luminescence test.

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils.

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.

Comparison of the calcium ionophore and phorbol myristate acetate on the initiation of the respiratory burst in human neutrophils.

We investigated the ability of the calcium ionophore A23187 and phorbol myristate acetate to elicit a respiratory burst in human neutrophils. In general, the ionophore was considerably more potent than phorbol myristate acetate in generating a chemiluminescent response and slightly less active in generating superoxide anion. In contrast, the ionophore caused a much smaller stimulation of glucose oxidation via the hexose monophosphate shunt. This relative inability of the ionophore to stimulate the shunt could not be ascribed to an effect on glucose transport or to a direct inhibition of any of the enzymes involved in glucose metabolism. These data suggest that different stimuli may have markedly different effects on various activities associated with the respiratory burst and emphasize the necessity for measurement of more than one parameter to assess the oxidative metabolism of the neutrophil.