Administration of adenoviral vectors induces gangrene in acutely ischemic rat hindlimbs: role of capsid protein-induced inflammation.

PURPOSE: The initial purpose of this study was to determine the effects of intravascular adenoviral vector-mediated gene transfer of endothelial nitric oxide synthase (AdeNOS) on experimental hindlimb ischemia in the rat. Unexpectedly, administration of AdeNOS immediately after induction of acute limb ischemia led to limb gangrene. We subsequently sought to define the molecular mechanisms responsible for this unusual effect and to devise adenoviral gene transfer strategies to prevent the development of gangrene in acutely ischemic limbs. METHODS: Phosphate-buffered saline or adenoviral vectors containing the bovine endothelial nitric oxide synthase gene (AdeNOS) or no transgene (Ad-E1) were injected intra-arterially into the hindlimb of a rat under vascular isolation immediately after surgical induction of severe ischemia. Hematoxylin and eosin staining was performed on muscle sections to evaluate inflammation. A separate group of animals was injected with an adenovirus containing a nontranscribable genome, treated with cyclosporine, or received delayed administration of the adenoviral vector. Gene expression after delayed adenoviral gene transfer was assessed with immunohistochemistry, Western blotting, and nitric oxide synthase (NOS) activity assay. RESULTS: Both AdeNOS and Ad-E1 caused gangrene of the entire hindlimb within 12 days in a dose-dependent manner, at a threshold concentration of 1 x 10(9) plaque-forming unit/mL. Adenoviral delivery was associated with more inflammation and edema compared with phosphate-buffered saline histologically. Inactivation of adenoviral DNA transcription did not affect induction of gangrene. However, gangrene was prevented by concurrent immunosuppression with cyclosporine or delayed administration of the vector. Delayed administration allowed adenoviral gene expression as determined by immunohistochemistry, NOS protein levels, and an assay of NOS enzyme activity. CONCLUSION: Intra-arterial administration of adenoviral vectors, under vascular isolation, immediately after induction of acute ischemia causes inflammation and subsequent limb gangrene. The inflammatory response is unrelated to the expression of the recombinant transgene or the adenoviral genome and is likely due to the adenoviral capsid proteins. However, administration of cyclosporine or delayed injection of the adenoviral vector is a method that can be used for adenoviral mediated gene transfer in limb ischemia.

Exercise-induced hyperemia unmasks regional blood flow deficit in experimental hindlimb ischemia.

UNLABELLED: Many experimental models of hindlimb ischemia are characterized by spontaneous and rapid normalization of resting muscle blood flow (BF) rates which complicates the long-term evaluation of angiogenic therapies to reverse limb ischemia. We tested the hypothesis that peroneal nerve stimulation in an ischemic hindlimb would increase the oxygen (O(2)) demand and BF rate, thereby unmasking a severe blood flow deficit that is not apparent at rest. METHODS: Ischemia was induced in adult rats by ligation of the left common iliac, femoral arteries, and their branches. Peroneal nerves were stimulated to allow measurement of exercise-induced regional BF rates with fluorescent microspheres. Hemodynamics were monitored. Fluorescent microspheres were injected before and after 5 min of nerve stimulation 3, 10, and 24 days postischemia. The tibialis anterior (TA) and gastrocnemius (GC) muscles and skin were harvested and weighed, and fluorescence was measured. BF rate was calculated as milliters per minute per gram of tissue and compared to normal muscle and skin of unoperated control rats. In order to determine the accuracy of BF rate measurements in ischemic muscle when <400 microspheres was delivered per specimen, 3 rats were studied by simultaneous injection of 4 x 10(5) blue and 1 x 10(5) yellow-green fluorescent microspheres. The correlation coefficient between the number of different colored microspheres delivered was measured. RESULTS: The ischemia caused atrophy of the TA and GC muscles. The mean muscle mass of the ischemic TA and GC as a percentage of total body weight decreased over time vs control [TA 0.13 +/- 0.05% vs 0.25 +/- 0.03%, P < 0.05; GC 0.51 +/- 0.27% vs 0.70 +/- 0.07%, P = 0.07 at 24 days (24D)]. Despite clinical evidence of severe hindlimb ischemia in experimental groups, i.e., pressure sores, muscle atrophy, and weakness, resting BF rates were not significantly different from those of control. The BF rate of the TA was of 0.11 ml/min/g after 3D of ischemia, 0.14 ml/min/g after 10D, and 0.13 ml/min/g after 24D. The mean BF rate in normal muscle of unoperated controls was 0.16 ml/min/g (P > 0.05). However, the exercise-induced hyperemia in the skeletal muscle was significantly blunted in all of the ischemic groups. The unoperated control TA had a greater than 10-fold increase in BF to 1.95 ml/min/g in response to exercise while the ischemic TA had no increase in BF at 3D, 2-fold increase at 10D, and a 5-fold increase at 24D. Parallel findings were noted in the GC muscles. There was no significant difference in the BF rate in the skin. The accuracy of this microsphere technique in measuring very low BF rates found in ischemic muscle was supported by the significant correlation coefficient (r = 0.99) comparing two quantities of microspheres injected simultaneously. CONCLUSION: Despite clinical signs of severe hindlimb ischemia, resting BF rates in the ischemic groups were not significantly decreased. Peroneal nerve stimulation resulted in up to 10-fold increase in BF rate and unmasked a severe deficit in vascular reserve in the ischemic groups. Resting BF rate is not always an accurate reflection of the flow deficit in models of critical limb ischemia, and this model of exercise-induced hindlimb hyperemia may allow better long-term evaluation of angiogenic therapies designed to reverse critical limb ischemia.

Adenoviral-mediated gene transfer of ICP47 inhibits major histocompatibility complex class I expression on vascular cells in vitro.

PURPOSE: Many viruses have evolved mechanisms to evade detection by the host immune system. The herpes simplex gene ICP47 encodes a protein that binds to the host antigen-processing transporter, inhibiting the formation of major histocompatibility complex class I (MHC-I) antigens in infected cells. MHC-I antigen expression is also important in acute allograft rejection. This study was designed to quantitate the effect of adenoviral-mediated gene transfer of ICP47 on MHC-I cell surface expression of human vascular cells. We hypothesized that the transduction of vascular cells with a replication-incompetent adenoviral vector that was expressing ICP47 (AdICP47) would inhibit constitutive and inducible MHC-I expression and thereby reduce the rate of cytolysis of ICP47-transduced vascular cells by sensitized cytotoxic T lymphocytes (CTL). METHODS: A replication-incompetent adenoviral vector, AdICP47, was created to express ICP47 driven by the cytomegalovirus immediate early promoter. Cultured human vascular endothelial and smooth muscle cells and human dermal fibroblasts were transduced with either AdICP47 or the control empty vector AddlE1. Cell surface constitutive and gamma-interferon-induced MHC-I expression were quantitated by flow cytometry. A standard 4-hour chromium release cytotoxicity assay was used to determine the percent cytolysis of transduced and nontransduced endothelial cells by sensitized CTL. Finally, to quantitate the specificity of the effect of ICP47 on MHC-I expression, adhesion molecule expression was quantitated in both transduced and nontransduced cells. RESULTS: Constitutive MHC-I expression in AdICP47-transduced endothelial cells was inhibited by a mean of 84% +/- 5% (SEM) in five experiments. After 48 hours of exposure to gamma-interferon, AdICP47-transduced cells exhibited a mean of 66% +/- 8% lower MHC-I expression than nontransduced cells. Similar inhibition in MHC-I expression was achieved in AdICP47-transduced vascular smooth muscle cells and dermal fibroblasts. Percent cytolysis of AdICP47-transduced endothelial cells by CTL was reduced by 72%. Finally, the specificity of the effect of transduction of ICP47 on vascular cell MHC-I expression was confirmed by a lack of significant change in either constitutive or tumor necrosis factor-induced vascular cell adhesion molecule/intercellular adhesion molecule expression. CONCLUSION: Transduction of vascular cells with AdICP47 strongly inhibits both constitutive and inducible MHC-I expression in human vascular cells. AdICP47-transduced cells exhibited a substantial reduction in cytolysis by CTL. Thus AdICP47 transduction holds promise as a technique to characterize the role of MHC-I expression in acute vascular allograft rejection in vivo and as a potential therapeutic intervention.

Giant tumors of the chest: preoperative embolization and resection.

Giant tumors of the chest are rare. These tumors comprise a spectrum of disease from benign lesions to highly aggressive malignant tumors with cells of origin in the pleura, pulmonary parenchyma, blood vessels, thymus, and connective tissues. We report four cases of giant tumors of the thorax treated with preoperative arterial embolization followed by complete surgical resection. Their diagnostic and treatment courses, imaging, and pathology are described.

U tubes and rare hepatobiliary complications.

U tubes have been used for a wide variety of hepatobiliary problems. We report a patient with multiple complications possibly related to the use of a U tube. These include secondary biliary cirrhosis, intrahepatic bilomas, and enterocutaneous fistula. The relatively rare entities of intrahepatic biloma and enterocutaneous fistula are reviewed.

Pyocolpos: diagnosis and treatment.

Pyocolpos is a rare complication of hydrocolpos. Hydrocolpos usually presents during adolescence and is associated with an imperforate hymen. The following is a case of a 3-month-old girl with pyocolpos. Her history was significant for a urinary tract infection (UTI) at 7 weeks of age. The authors believe that her UTI was caused by urinary retention secondary to hydrocolpos. A complete evaluation may have prevented the complication of pyocolpos.

A surgical technique that allows reuse of an existing venotomy site for multiple central venous catheterizations.

During long-term use, central venous catheters are damaged or inadvertently displaced, and replacement is required. The authors developed a technique that allows multiple recatheterizations through an existing venotomy site. The technique is very advantageous for patients who need long-term central venous access.