Mitchell D. Botney

Care of patients with deep venous thrombosis in an academic medical center : limitations and lessons

PURPOSE The primary goal of our study was to review the quality of care in patients with deep vein thrombosis,, with emphasis on identifying recurrent and remedial problems. Secondary goals were (1) to evaluate the use of the vascular laboratory and (2) to characterize our patient population with deep vein thrombosis and to identify a subset of patients with uncomplicated deep vein thrombosis who might be candidates for outpatient therapy in the future. METHODS A retrospective review was performed for all patients with deep vein thrombosis diagnosed with duplex scanning who were treated as inpatients from January 1993 through March 1993. RESULTS Fifty-four (16%) of 306 duplex scans were positive; 50 patients were treated as inpatients. Forty percent of patients had uncomplicated deep vein thrombosis that was potentially treatable on an outpatient basis. Mean time to obtain a therapeutic partial thromboplastin time was 22 hours (range 4 to 54 hours). Ten (20%) patients had inferior vena cava filters placed. The in-hospital mortality rate was 4%. Management problems occurred in 18 (36%) patients and included difficulty titrating anticoagulation, (10) physician failure to provide treatment after diagnosis, (five) and inappropriate use or complication of inferior vena cava filter placement (three). CONCLUSIONS Venous duplex examination is liberally but appropriately used. The primary remediable problem resulting in suboptimal management is difficulty titrating anticoagulation; inappropriate placement of inferior vena cava filters and physician failure to provide treatment also occur. In the future a substantial number of patients may be suitable for outpatient therapy.

Inhibition of tropoelastin expression by 1,25-dihydroxyvitamin D3

Elastin production is modulated by steroid hormones and is dependent on calcium. Because vitamin D3 is involved in the regulation of calcium metabolism and influences the expression of various extracellular matrix proteins, we investigated whether vitamin D3 influences tropoelastin expression. Three elastin-producing, bovine cell types, auricular chondroblasts, nuchal ligament fibroblasts and arterial smooth muscle cells, were treated with the principal active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), and with 24,25 dihydroxyvitamin D3 (24,25[OH]2D3). Tropoelastin levels in culture media and cell layers, as measured by an enzyme-linked immunoassay, decreased in a dose and exposure dependent manner after treatment with 1,25(OH)2D3; 24,25(OH)2D3 had no effect on tropoelastin production relative to solvent-treated controls. The maximal effective dose of 1,25(OH)2D3 was 10(-7) M for 48 hr, which resulted in a severalfold reduction of tropoelastin production, and decreased tropoelastin levels were detected at 8 hr after treatment. Reduction of tropoelastin protein production was paralleled by a decrease of equal magnitude in the steady-state levels of tropoelastin mRNA. Vitamin D3 metabolites had no effect on DNA or total protein synthesis. These results suggest that vitamin D3 may be an important modulator of elastin expression.

Liposome-mediated gene transfer to lung isografts

OBJECTIVES Our objective were to determine the feasibility, efficacy, and safety of in vivo and ex vivo liposome-mediated gene transfer to lung isografts. METHODS Fischer rats were divided into three main groups: (1) Nontransplant setting: Liposome-chloramphenicol acetyl transferase cDNA was intravenously injected, and lungs were harvested at different time points: 2, 6, 12, and 24 hours; 2, 5, 8, and 21 days (n = 3). Chloramphenicol acetyl transferase activity was determined in lungs, hearts, livers, and kidneys. The distribution and type of transfected cells were evaluated by in situ hybridization. Lung toxicity was assessed by arterial oxygen tension, histology, and tumor necrosis factor-alpha levels. (2) In vivo graft transfection: Left lungs were transplanted 6 hours, 4 hours, and 15 minutes after intravenous injection and were assessed for chloramphenicol acetyl transferase activity and arterial oxygen tension on postoperative day 2. (3) Ex vivo graft transfection: Grafts were infused ex vivo with either 660 micrograms (n = 3) or 330 micrograms (n = 3) of DNA complexed to liposomes and stored at 10 degrees C for 4 hours. Chloramphenicol acetyl transferase activity was assessed 44 hours after transplantation. RESULTS Transgene expression was detected in endothelial cells, macrophages, and interstitial cells. Chloramphenicol acetyl transferase activity was present as early as 2 hours, increased significantly between 6 hours and 8 days, and then decreased to minimal levels by 21 days. Chloramphenicol acetyl transferase activity was greatest in donor lungs and hearts and minimal in livers and kidneys. Arterial oxygen tension was normal in treated animals. Inflammation was minimal, and tumor necrosis factor-alpha levels increased only sevenfold in treated animals. CONCLUSION In vivo and ex vivo liposome-mediated gene transfer to lung isografts allows significant transgene expression with minimal effects on graft function.

Effect of Hemorrhage on Medial Collateral Ligament Healing in a Mouse Model

Background Medial collateral ligament injuries heal by a scar response. Hypothesis increased hemorrhage at the site of medial collateral ligament injury improves healing. Study Design Controlled laboratory study. Methods Ninety-six mice were divided into two groups. Group 1 mice underwent knee medial collateral ligament transection with the opposite knee as a sham-operated control and group 2 animals additionally had 0.25 ml of tail cut blood pipetted to the medial collateral ligament transection site and sham-operated opposite knee. Ligament specimens were harvested at 3, 7, 21, and 28 days. Results Immunohistochemical analysis demonstrated peak macrophage counts at day 7 in all transected specimens. Macrophage counts were higher in group 2 than in group 1 at all time points, with a statistically significant increase of macrophages noted at day 7. In situ hybridization demonstrated increased collagen gene expression, with peaks at 7 and 28 days after transection. Group 2 animals showed increased gene expression at all time points as compared with group 1, with a statistically significant increase noted at 7 and 28 days. Biomechanical testing demonstrated progressive healing at each time point. At 28 days, the load to failure was 67% that of the sham-operated knee. Conclusions This study suggests there is an increased healing response with bleeding at the ligament injury site. Clinical Relevance Identification of the factors involved with increased healing may allow manipulation of the healing response in the clinical setting.

Ex vivo liposome-mediated gene transfer to lung isografts

OBJECTIVE Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts. METHODS Left lungs were harvested and perfused via the pulmonary vein with chloramphenicol acetyltransferase complementary deoxyribonucleic acid complexed with cationic liposomes. Several variables were examined: (1) Influence of temperature: In group I (n = 4), grafts were stored for 4 hours at 23 degrees C and transplanted. Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. In groups II and III (n = 4), grafts were stored at 10 degrees and 4 degrees C, respectively. Arterial oxygen tension and inflammatory infiltrate were also determined. (2) Influence of storage time: Grafts were preserved at 10 degrees C for 1, 2, 3, 4 (n = 4), and 10 hours (n = 5). chloramphenicol acetyltransferase activity was assessed on postoperative day 2. (3) Rapidity and duration of transgene expression: Grafts were preserved at 10 degrees C for 1 hour and then transplanted. Chloramphenicol acetyltransferase activity was assessed 2, 4, 6, 12, and 24 hours and 2, 7, 14, 21, and 28 days after implantation. RESULTS Chloramphenicol acetyltransferase expression was apparently less in lungs transfected at 4 degrees C than in those transfected at 10 degrees and 23 degrees C. Storage for 1 hour at 10 degrees C was sufficient to yield significant expression. Increasing the exposure time to 10 hours did not increase toxicity. There were no differences in arterial oxygen tension between transfected and nontransfected lungs. Chloramphenicol acetyltransferase expression was detected for at least 28 days. CONCLUSION Ex vivo liposome-mediated transfection of lung isografts can be achieved after a short time of cold storage, with minimal toxicity.

Time course and cellular localization of inducible nitric oxide synthases expression during cardiac allograft rejection

BACKGROUND We have demonstrated that inhibition of inducible nitric oxide synthase (NOS) ameliorated acute cardiac allograft rejection. This study determined the time course and cellular localization of inducible NOS expression during the histologic progression of unmodified acute rat cardiac allograft rejection. METHODS Tissue from syngeneic (ACI to ACI) and allogeneic (Lewis to ACI) transplants were harvested on postoperative days 3 through 10 and analyzed for inducible NOS mRNA expression (ribonuclease protection assay), inducible NOS enzyme activity (conversion of L-[3H]arginine to nitric oxide and L-[3H]citrulline), and nitric oxide production (serum nitrite/nitrate levels). Inducible NOS mRNA and protein expression were localized using in situ hybridization and immunohistochemistry. RESULTS Inducible NOS mRNA and enzyme activity were expressed in allografts during mild, moderate, and severe acute rejection (postoperative days 4 through 10), but were not detected in normals, isografts, or allografts before histologic changes of mild acute rejection (postoperative day 3). Inducible NOS expression resulted in increased serum nitrite/nitrate levels during mild and moderate rejection (postoperative days 4 through 6). Inducible NOS mRNA and protein expression localized to infiltrating mononuclear inflammatory cells in allograft tissue sections during all stages of rejection but were not detected in allograft parenchymal cells or in normals or isografts. CONCLUSIONS Inducible NOS expression and increased nitric oxide production occurred during the early stages of acute rejection, persisted throughout the unmodified rejection process, and localized to infiltrating inflammatory cells but not allograft parenchymal cells during all stages of acute rejection.

Successful in vivo and ex vivo transfection of pulmonary artery segments in lung isografts

OBJECTIVE Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection. Efficient gene transfection to the whole organ may prove problematic. Proximal pulmonary artery endothelial transfection might provide beneficial downstream effects on the whole graft. The aim of this study was to determine the feasibility of transfecting proximal pulmonary artery segments in lung isografts. METHODS Male Fischer rats were divided into six groups. In vivo transfection: In group I (n = 7), a proximal segment of the left pulmonary artery was isolated and injected with saline solution by means of a catheter inserted through the right ventricle. After an exposure period of 20 minutes, clamps were removed and blood flow was restored. In group II (n = 7), the isolated arterial segments were injected with adenovirus carrying the Escherichia coli LacZ gene encoding for beta-galactosidase. Ex vivo transfection: In group III (n = 5), arterial segments were injected ex vivo with saline solution and in group IV (n = 5) with the adenovirus construct. In group V (n = 6), arteries were injected with saline solution and in group VI (n = 11) with liposome chloramphenicol acetyl transferase cDNA. In groups I to IV, animals were killed on postoperative day 3 and transgene expression was assessed by Bluo-Gal staining. In groups V and VI, animals were killed on postoperative day 2 and transgene expression was assessed by chloramphenicol acetyl transferase activity assay. RESULTS Transgene expression was detected grossly and microscopically in endothelial and smooth muscle cells of pulmonary artery segments from all surviving animals of groups II and IV. In group VI, chloramphenicol acetyl transferase activity was significant in all assessed arterial segments. CONCLUSION Significant transgene expression is observed in proximal pulmonary artery segments after both in vivo and ex vivo exposure.

Adenovirus mediated gene transfer into rat lung grafts at the time of harvest.

OBJECTIVE New methods to introduce genetic material into cells in vivo may revolutionize current treatment modalities. Expression of functional genes in lung allografts could be used as a prophylactic strategy for reperfusion injury and rejection. We studied the feasibility of ex vivo adenovirus mediated transfection of rat lung allografts. METHODS In group I (n = 3) donor rat lungs (Fisher) were flushed with Low Potassium Dextran Glucose (LPDG) solution (20 ml, 20 degrees C). 4 x 10(11) viral particles of adenovirus 5 containing the E. coli lacZ reporter gene coding for beta-galactosidase (AdCMV-beta-Gal) were added to the last milliliter of the flush solution. Lung grafts were stored for 3.5 h at room temperature followed by syngenic orthotopic transplantation (Fisher to Fisher) using a microsurgical cuff technique. On postoperative day 5 the heart lung block was extracted and flushed with x-Gal (beta-Gal substrate) and kept in x-Gal for 3 h at 37 degrees C. Color development was observed macroscopically and plastic embedded sections were used for histologic examination. Group II grafts (n = 3) served as controls and were flushed without adenovirus. RESULTS X-Gal stained the transfected lung grafts blue, indicating high reporter gene expression. Control lungs did not stain with x-Gal. In group I histological examination demonstrated transfection predominantly in type II pneumocytes. Surprisingly endothelial cells showed no beta-Gal activity. CONCLUSION This study demonstrates that ex vivo transfection of lung grafts at the time of harvest is a feasible method of gene transfer and results in gene expression after transplantation.