Immunotargeting of glucose oxidase: intracellular production of H(2)O(2) and endothelial oxidative stress.

Extracellular and intracellular reactive oxygen species attack different targets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellular flux of a defined reactive oxygen species in endothelium, we used immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-(125)I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-(125)I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H(2)O(2) from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H(2)O(2) was generated intracellularly, whereas 30% of H(2)O(2) was detected in the cell medium. Catalase added to the cells eliminated H(2)O(2) in the medium but had little effect on the intracellular generation of H(2)O(2) by anti-PECAM-GOX. Both H(2)O(2) added exogenously to the cell medium (extracellular H(2)O(2)) and that generated by anti-PECAM-GOX caused oxidative stress manifested by time- and dose-dependent irreversible plasma membrane damage. Inactivation of cellular catalase by aminotriazole treatment augmented damage caused by either extracellular H(2)O(2) or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H(2)O(2), yet failed to protect cells against injury induced by anti-PECAM-GOX. Therefore, treatment of PECAM-positive cells with anti-PECAM-GOX leads to conjugate internalization, predominantly intracellular H(2)O(2) generation and intracellular oxidative stress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxidative stress in PECAM-positive cells.

Lack of reactivation of shigellosis in naturally infected enrofloxacin-treated cynomolgus monkeys after exogenous immunosuppression.

Four cynomolgus macaques housed at our facility became acutely ill with dysenteric symptoms. Enteric isolates established an etiologic diagnosis of Shigella flexneri. Enrofloxacin antimicrobial therapy cleared the infection with no perceptible bacterial shedding or clinical signs of disease. High-dose methyl-prednisolone therapy was administered to the four monkeys for 5 weeks. The animals were monitored for signs of shigellosis and bacterial shedding weekly throughout the study, for a total of 7 weeks. Although methylprednisolone therapy induced marked cellular immunosuppression in all four animals, as measured by in vitro assays, no animal had evidence of clinical shigellosis or bacterial shedding. These results suggest that cynomolgus macaques naturally infected with S. flexneri and appropriately treated with enrofloxacin are unlikely to have reactivation of shigellosis and shedding of bacteria in the feces during periods of stress or profound immunosuppression.

Diaphragmatic hernia in a llama.

A 7-year-old castrated male llama was admitted for evaluation of mild colic. Exploratory celiotomy revealed a diaphragmatic hernia that was entrapping the proximal portion of the ascending colon. The hernia was reduced, and the devitalized ileum, cecum, and 45 cm of the proximal portion of the ascending colon were resected. Surgical repair of the defect in the diaphragm could not be accomplished from the ventral celiotomy, and the owners declined definitive repair via left thoracotomy. The llama recovered and did well until 9 months after surgery, when the hernia recurred. Necropsy revealed a defect, with characteristics consistent with a congenital origin, in the dorsal part of the left hemidiaphragm. On the basis of the findings in this llama, repair of such defects is strongly advocated.