Liposome-mediated gene transfer in rat lung transplantation: A comparison between the in vivo and ex vivo approaches

OBJECTIVE: We compared the efficacy of in vivo and ex vivo liposome transfection in rat lung transplantation. METHODS: (1) Chloramphenicol acetyltransferase group: Fischer rats underwent isogeneic transplantation (n = 4 per group). Recipients were put to death on postoperative day 2 for chloramphenicol acetyltransferase activity. Ex vivo setting: Grafts received cDNA complexed or not with liposomes and were transplanted after 1.5 or 10 hours at 10 degreesC. In vivo setting: Donors were intravenously injected with cDNA complexed or not with liposomes. Lungs were harvested after 1.5 or 10 hours, preserved at 10 degreesC, and transplanted. (2) Transforming growth factor-beta1 group: Brown-Norway rats served as donors and Fischer rats as recipients. All grafts were preserved for 3 hours at 10 degreesC. On postoperative day 5, arterial oxygenation and histologic rejection scores were assessed. Ex vivo setting: Grafts received transforming growth factor-beta1 sense (n = 8) or antisense (n = 7) complexed with liposomes or cDNA alone (n = 5). In vivo setting: Donors were intravenously injected with liposome:transforming growth factor-beta1 sense cDNA (n = 7). Exposure time was 3 hours. RESULTS: (1) Chloramphenicol acetyltransferase-transfection was superior in the ex vivo group but was not statistically different for longer exposure times. (2) Transforming growth factor-beta1-arterial oxygenation was superior in the ex vivo liposome:sense group. cDNA alone was inefficient. Rejection scores were not statistically different between ex vivo and in vivo liposome:sense groups but were better when the ex vivo liposome:sense group was compared with the cDNA alone or the antisense groups. CONCLUSIONS: (1) With current liposome technology, the ex vivo route is superior to the in vivo approach; (2) cDNA alone does not provide transgene expression at levels to produce a functional effect.     

Therapeutic implications of thymic uptake of radioiodine in thyroid carcinoma

BACKGROUND: NPC18915, a member of new antiinflammatory agent called nactins (neutrophil activation inhibitors), has been shown to reduce reperfusion injury in rat lung transplantation at high dosage. In vitro studies have demonstrated effectiveness of this compound even at low dosage. We hypothesized that this compound ameliorates lung ischemia reperfusion injury even at low dosage levels if administration is optimally timed. The aim of this study was to determine the efficacy and the best timing for administration of low-dose NPC18915. METHODS: Forty syngeneic rat left lung transplantations were performed. All isografts were flushed with low-potassium dextran-1% glucose solution 20 ml and preserved for 18 hours at 4 degrees C. Animals were divided into four groups. Group I animals (n = 10) served as control subjects. In groups II (n = 10), III (n = 10), and IV (n = 10), NPC18915 (0.04 mg) was added to the flush solution and was administered intravenously (0.4 mg/kg) immediately before reperfusion (group II) and 60 minutes (group III) and 120 minutes (group IV) after reperfusion. Pulmonary function was assessed 24 hours after reperfusion. RESULTS: In group III, oxygenation improved in comparison to group I (247.2 +/- 59.8 versus 76.6 +/- 16.0 mm Hg, p < 0.002). Wet-to-dry weight ratio and graft myeloperoxidase activity were significantly improved (group III versus group I, 6.02 +/- 0.21 versus 7.19 +/- 0.41, p = 0.013) (group III versus group I, 0.093 +/- 0.019 versus 0.207 +/- 0.023 delta optical density/min/mg, p < 0.002). There were no significant differences in CD11b expression. CONCLUSION: These data suggest that delayed administration of NPC18915, 60 minutes after reperfusion, dramatically improves pulmonary graft function.     

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.     

Chloramphenicol acetylation in Streptomyces

Twenty-one strains of actinomycetes were screened for the presence of chloramphenicol acetyltransferase, the enzyme responsible for chloramphenicol resistance in many species of bacteria. Only five strains, belonging to three species, yielded mycelial lysates which catalysed the formation of chloramphenicol acetates in the presence of acetyl-coenzyme A: Streptomyces coelicolor Müller, S. acrimycini, and S. griseus. A mutant of S. acrimycini selected for an increase in resistance to chloramphenicol had a higher specific activity for chloramphenicol acetyltransferase than that found in the parental strain; the enzyme was not inducible in the mutant, the parental strain, or any other strain tested. Chloramphenicol was not acetylated by lysates of a strain of S. venezuelae, the organism known to produce it.     

Expression of endothelin-1 and effects of an endothelin receptor antagonist, TAK-044, at reperfusion after cold preservation in a canine lung transplantation model

BACKGROUND: Rapid increase of pulmonary vascular resistance (PVR) early after reperfusion remains a major issue in clinical lung transplantation. A potent vasoconstrictor peptide, endothelin- plays an important role in various pulmonary pathophysiologic conditions and might induce increased PVR. We investigated the expression and influence of endothelin-1, and the effects of an ETA and ETB nonselective endothelin receptor antagonist, TAK-044, at reperfusion after cold preservation in a canine lung transplantation model. METHODS: Left single lung allotransplantation procedures were performed in three groups of animals. In group I (n=5) lungs were preserved for 12 hours; in group II (n=5) lungs were preserved for 18 hours; and in group III (n=6) lungs were also preserved for 18 hours, and TAK-044 (5 mg/kg) was administered just before reperfusion. All donor lungs were flushed and preserved with low-potassium dextran glucose solution at 4 degrees C. RESULTS: Six hours after reperfusion, arterial oxygen tension (mm Hg, inspired oxygen fraction=1.0) was 512.9+/-34.7 in group I, 152.4+/-46.7 in group II, and 509.6+/-29.0 in group III; PVR index (dyne x sec x cm(-5) x m2) was 1130+/-142 in group I, 1820+/-142 in group II, and 1287+/-191 in group III. Plasma endothelin-1 level was elevated significantly, and endothelin-1-like immunoreactivity was found in a variety of pulmonary vascular tissue and was seen less with immunohistochemical evaluation in group II in bronchial tissue. Conclusions: These results suggest that endothelin-1 is expressed as a result of ischemia-reperfusion injury and may worsen early graft function. TAK-044 is beneficial in protecting the graft from high pulmonary vascular resistance and pulmonary edema during the early posttransplantation stage.     

Mild posttraumatic hypothermia reduces mortality after severe controlled cortical impact in rats

The effect of posttraumatic hypothermia (brain temperature controlled at 32 degrees C for 4 h) on mortality after severe controlled cortical impact (CCI) was studied in rats. Four posttraumatic brain temperatures were compared: 37 degrees C (n = 10), 36 degrees C (n = 4), 32 degrees C (n = 10), and uncontrolled (UC; n = 6). Rats were anesthetized and subjected to severe CCI (4.0-m/s velocity, 3.0-mm depth) to the exposed left parietal cortex. At 10 min posttrauma the rats were cooled or maintained at their target brain temperature, using external cooling or warming. Brain temperature in the UC group was recorded but not regulated, and rectal temperature was maintained at 37 +/- 0.5 degrees C. After 4 h, rats were rewarmed over a 1-h period to 37 degrees C, extubated, and observed for 24 h. In the 37 and 36 degree C groups, 24-h mortality was 50% (37 degrees C = 5/10, 36 degrees C = 2/4). In the 32 degree C group, 24-h mortality was 10% (1/10). In the UC group, brain temperature was 35.4 +/- 0.6 degrees C during the 4-h treatment period and 24-h mortality was 0% (0/6). Mortality was higher in groups with brain temperatures > or = 36 degrees C versus those with brain temperatures < 36 degrees C (50 vs. 6%, respectively; p < 0.05). Additionally, electroencephalograms (EEG) were recorded in subsets of each temperature group and the percentage of time that the EEG was suppressed (isoelectric) was determined. Percentage of EEG suppression was greater in the hypothermic (32 degrees C, n = 6; UC, n = 4) groups than in the normothermic (36 degrees C, n = 3; 37 degrees C, n = 6) groups (23.3 +/- 14.3 vs. 1.2 +/- 3.1%, respectively; p < 0.05). Posttraumatic hypothermia suppressed EEG during treatment and reduced mortality after severe CCI. The threshold for this protective effect appears to be a brain temperature < 36 degrees C. Thus, even mild hypothermia may be beneficial after severe brain trauma.     

The effect of triiodothyronine on myocardial contractile performance after epinephrine exposure: implications for donor heart management

BACKGROUND: This study analyzes in the experimental model of isolated human atrial myocardium whether the myocardial contractile depression occurring after high-dose/long-term catecholamine exposure (as typically occurring in brain-dead organ donors) can be reversed by thyroid hormone administration. METHODS: Isolated trabeculae were prepared from atrial myocardium from patients undergoing coronary artery bypass (n = 15). Initial measurements of isometric force were carried out (measurement conditions of 37 degrees C, Krebs Henseleit solution, supramaximal electrical stimulation, 1 Hz, at optimal length). Then the trabeculae were incubated for 6 hours at 26 degrees C in a Krebs Henseleit solution containing epinephrine 10(-7) mol/L and the fluorescent dye FURA-2/AM for calcium measurements. At the end of the incubation period, isometric force, isotonic shortening, and intracellular calcium transient (FURA-2 "ratio method") were measured. After 30 minutes administration of triiodothyronine (5 x 10(-9) mol/L), the measurements were repeated. Control groups included 6 hours incubation in 4 degrees C Krebs Henseleit solution (n = 5); 6 hours incubation in 26 degrees C FURA-2/AM (n = 5); and 6 hours incubation in epinephrine 10(-7) mol/L (n = 5). RESULTS: After 6 hours catecholamine exposure isometric force declined significantly to 56.8% (p < .0001) and isotonic shortening to 54% of its initial value (p < .01). Administration of triiodothyronine was associated with a significant recovery of the isotonic shortening amplitude (p < .005), of isometric force development (p < .01), an increased velocity of force development (p < .0001), and of diastolic force decay (p < .005). At the same time the shape of the intracellular calcium transient became smaller as a result of an accelerated diastolic decay. The amplitude of the calcium transient remained unaltered, whereas the calcium time integral was reduced (p < .05). CONCLUSION: In the model of isolated human myocardium, experimental depression of the contractile performance resulting from long-term catecholamine exposure could be reversed by a 30-minute triiodothyronine incubation. The experimental data showing increased force amplitudes at unaltered amplitudes of the intracellular calcium transient and an even-reduced calcium time integral provide strong evidence for a sensitization of the contractile apparatus for calcium by triiodothyronine. The data provide additional knowledge to explain the successful administration of triiodothyronine in donor heart management.     

Preservation with 8-bromo-cyclic GMP improves pulmonary function after prolonged ischemia

BACKGROUND: Cyclic guanosine monophosphate (cGMP) is a potent second messenger for the nitric oxide pathway in the pulmonary vasculature. Increased cytosolic cGMP levels elicit pulmonary vasodilatation resulting in decreased pulmonary vascular resistance and maximized pulmonary function after ischemia-reperfusion injury. We hypothesized that the addition of a membrane-permeable cGMP analogue (8-bromo-cGMP) to a Euro-Collins (EC) preservation solution would ameliorate pulmonary reperfusion injury better than prostaglandin E1 injection alone after prolonged hypothermic ischemia. METHODS: All lungs from New Zealand White rabbits (weight, 3 to 3.5 kg) were harvested en bloc, flushed with EC solution, and reperfused with whole blood for 30 minutes. Group 1 lungs (immediate control) were immediately reperfused. Group 2 lungs (control) were stored inflated at 4 degrees C for 18 hours before reperfusion. Groups 3 and 4 lungs were flushed with EC solution containing 200 micromol/L 8-bromo-cGMP and stored at 4 degrees C for 18 and 30 hours, respectively. Fresh, nonrecirculated venous blood was used to determine single-pass pulmonary venous-arterial oxygen gradients at 10-minute intervals. Assays for cGMP, cyclic adenosine monophosphate, nitric oxide synthase activity, and myeloperoxidase were performed on all lung tissue samples. Wet to dry weight ratios were determined after 2 weeks of passive desiccation. RESULTS: Oxygenation (venous-arterial oxygen gradient), pulmonary artery pressure, pulmonary vascular resistance, and edema formation were significantly improved in groups 3 and 4 (addition of 8-bromo-cGMP to EC plus 18 or 30 hours of hypothermic ischemia). Hypothermic storage (groups 2, 3, and 4) decreased both nitric oxide synthase activity and myeloperoxidase levels compared with immediate reperfusion (group 1). CONCLUSIONS: These results suggest that the addition of a membrane-permeable cGMP analogue to an EC pulmonary flush solution improves pulmonary function after prolonged storage compared with EC and prostaglandin (E1) preservation alone. The finding of myeloperoxidase reduced levels after hypothermic storage and subsequent reperfusion may suggest a more important role for pulmonary hemodynamic control in mitigating pulmonary reperfusion injury.     

Myocardial preservation by continuous perfusion of Krebs-Henseleit solution--the temperature dependency of the optimal perfusion pressure

The aim of this study was to determine the temperature dependency of the optimal pressure in myocardial preservation by continuous perfusion with Krebs-Henseleit bicarbonate buffer (KHBB) solution. Hearts from Wistar male rats were perfused with KHBB solution and cardiac function (aortic flow) was measured using an isolated working rat heart preparation. In the preliminary experiment, hearts were then preserved using Langendorff perfusion with KHBB solution of 37, 20 or 4 degrees C for 2 hours at a perfusion pressure of 100 cmH2O. This was followed by 15 min of Langendorff perfusion (37 degrees C, 100 cmH2O) and 20 min working perfusion. The 37 degrees C group and 20 degrees C group exhibited better functional recoveries of aortic flow (%AF) in the post-preservation period compared to the 4 degrees C group. In the test experiment, hearts were preserved using Langendorff perfusion for 4 hours at 37 degrees C or for 8 hours at 20 degrees C at various perfusion pressures. At 37 degrees C, %AF after 4 hours of the preservation were 64.7 +/- 2.6, 69.0 +/- 3.2, 81.9 +/- 3.1, 94.7 +/- 3.3 and 63.5 +/- 4.0% (p < 0.05 vs the 100 cmH2O group) at the perfusion pressure of 100, 60, 20, 15 and 10 cmH2O, respectively. %AF after 8 hours of the preservation at 15 cmH2O was 56.3 +/- 2.5%. At 20 degrees C, %AF after 8 hours of the preservatin was 78.9 +/- 3.3, 81.9 +/- 2.3, 67.4 +/- 1.9 and 65.9 +/- 2.2% (p < 0.05 vs the 60 cmH2O group) at the perfusion pressure of 60, 30, 10 and 15 cmH2O, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid reperfusion causes stress failure in ischemic rat lungs

OBJECTIVE: Rapid reperfusion may be injurious to the ischemic lung. Our aim was to confirm that slow reperfusion improves postischemic pulmonary function and to elucidate the ultrastructural changes associated with slow versus rapid reperfusion. METHODS. We used an ex vivo perfused rat lung transplant model to study the effect of slow versus rapid reperfusion on subsequent lung function and morphologic conditional. Functional assessment was performed in (1) fresh lung, slowly reperfused; (2) fresh lung, rapidly reperfused; (3) ischemic lung (4 hours at 22 degrees C), slowly reperfused; and (4) ischemic lung, rapidly reperfused. RESULTS: In group 4, the shunt fraction (P=.001), airway pressure (P=.001), and wet/dry ratio (P=.01) were significantly higher than in groups 1 through 3. Light and electron microscopy of slowly reperfused ischemic lungs (n=4) appeared normal. Rapidly reperfused ischemic lungs (n=4) demonstrated massive alveolar edema hemorrhage, and epithelial "blebbing" by light microscopy. Electron microscopy identified the blebbing as separation of the epithelial layer from an intact basement membrane by edema fluid. The epithelial layer was disrupted in numerous locations. Complete disruption of all layers of the blood-gas barrier was occasionally present. CONCLUSION: Rapid reperfusion of the ischemic lung is an important contributing factor to reperfusion lung injury resulting in mechanical stress failure of the alveolar/capillary barrier. Gradual reintroduction of blood flow to the ischemic lung improves oxygenation.     

High-performance liquid chromatographic analysis of chlorhexidine in saliva after mouthrinsing

BACKGROUND: Neutrophils contribute to the host defense mechanism, but they can cause remote organ injury in peritonitis. The purpose of this study was to examine neutrophil adhesion to the peritoneum and remote organs simultaneously in peritonitis using a fluorescence microscopic method. STUDY DESIGN: Experiment 1: Sprague-Dawley rats (n = 16) were injected intraperitoneally (ip) with saline solution or 10(5), 10(7), or 10(9) Escherichia coli. Five hours after challenge, 1 x 10(6) fluorescein-labeled neutrophils were infused. Two minutes after neutrophil injection, five peritoneal samples (the greater omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy. Lung myeloperoxidase (MPO) activity was also determined. Experiment 2: Rats (n = 23) were given 10(9) E. coli ip. Before challenge (0 h) or at 1, 5, or 10 h after challenge, labeled neutrophils were infused. Then, the labeled neutrophil numbers in organs and lung MPO activities were assessed as described for Experiment 1. Hemodynamic and arterial blood gas data were also obtained in another set of rats before and at 1, 5, 8 and 10 h after 10(9) E. coli ip challenge. RESULTS: Experiment 1: The labeled neutrophil numbers in the peritoneum, lungs, and kidney showed significant positive correlations with the injected bacterial numbers. Lung MPO also positively correlated with E. coli number and labeled neutrophil number in the lungs. Experiment 2: Labeled neutrophil numbers in the peritoneum and kidney peaked at 5 h. The pulmonary labeled neutrophil number rose, reaching a plateau at 5 h. No remarkable change was observed in the hepatic labeled neutrophil number. There was a positive correlation between lung MPO activity and pulmonary labeled neutrophil number. Hemodynamic and blood gas data reflected a hyperdynamic state. CONCLUSIONS: Concomitant dose-dependent increases in neutrophil adhesion in the peritoneum, lungs, and kidney were observed in this peritonitis model. Increased neutrophil adhesion was transient in the peritoneum and kidney but persistent in the lungs. Strategies modulating neutrophil adhesion in organs are anticipated to be useful for the treatment of peritonitis.     

Impaired endothelin-1 release in tilt-induced syncope

BACKGROUND: Nitric oxide (NO) is considered to be one of the endogenous inhibitory factors of ischemic reperfusion injury. In this study, the NO-producing ability of the preserved lung, flushed at various pulmonary artery pressures (flushing pressure), was studied during reperfusion using an ex vivo rabbit lung perfusion model. METHODS: The lungs were flushed with 200 ml of preservation solution with flushing pressures adjusted to 15, 15, 20, and 25 mmHg for groups 1, 2, 3, and 4, respectively (n=5 in each group). In the control group (group 1), the heart-lung block was harvested after flushing and the lungs were assessed without preservation. In the other groups, the harvested blocks were preserved at 8 degrees C for 24 hr and reperfused with homologous blood for pulmonary functional assessment. Pulmonary function was assessed by measuring mean airway pressure, mean pulmonary arterial pressure, partial oxygen tension of pulmonary venous effluent blood, and pulmonary wet-dry weight ratio. The sequential changes in the concentration of NO-related substances (NO-RS) in the serum of reperfused blood were also measured by chemiluminescence. RESULTS: During reperfusion, biphasic increases in NO-RS were observed in all groups. In groups 3 and 4, the increases in NO-RS were significantly lower than those of groups 1 and 2, and pulmonary function deteriorated. CONCLUSION: These data suggest that in order to maintain the endogenous NO-producing ability of preserved lung, the flushing pressure must be less than 20 mmHg.     

Interactions between hypothermia and the latency to ischemic depolarization: implications for neuroprotection

BACKGROUND: The authors postulated that hypothermic neuroprotection can be attributed to a delayed onset of ischemic depolarization. METHODS: Halothane-anesthetized rats were prepared for near-complete forebrain ischemia. Direct current (DC) potential microelectrodes were placed in hippocampal CA1. The pericranial temperature was maintained at 31 degrees C, 33 degrees C, 35 degrees C, or 37 degrees C (n = 6 per group). Bilateral carotid occlusion with systemic hypotension was initiated for 10 min. The time to onset of the DC shift was recorded. In a second experiment, rats were assigned to 37 degrees C or 31 degrees C for 10 min of ischemia, or to 31 degrees C for 14 min of ischemia (n = 8 per group). These durations of ischemia were defined to allow 9 min of ischemic depolarization in the 37 degrees C-10 min and 31 degrees C-14 min groups. Neurologic and histologic outcomes were examined 7 days later. RESULTS: Hippocampal CA1 time to depolarization increased with decreasing temperature (P < 0.0001). Time to depolarization was increased by approximately 4 min in the rats maintained at 31 degrees C compared with those at 37 degrees C. Time to repolarization during reperfusion was not affected by temperature. Increasing the duration of ischemia from 10 min to 14 min with the pericranial temperature maintained at 31 degrees C resulted in a duration of depolarization that was equivalent in the 37 degrees C-10 min and 31 degrees C-14 min groups. However, hippocampal CA1 damage was not increased (31 degrees C-10 min = 4 +/- 1% dead neurons; 31 degrees C-14 min = 6 +/- 1% dead neurons, 95% CI, -1% to 3%; 37 degrees C-10 min = 90 +/- 17% dead neurons). CONCLUSIONS: Despite similar durations of DC depolarization, outcome in hypothermic rats was markedly improved compared with normothermic rats. This indicates that hypothermic neuroprotection can be attributed to mechanisms other than the delay in time to onset of ischemic depolarization.     

Controlled reperfusion and pentoxifylline modulate reperfusion injury after single lung transplantation

OBJECTIVE: Rodent models have suggested that initial low-pressure reperfusion of transplanted lungs reduces injury after ischemia. We investigated this phenomenon and the use of pentoxifylline in a porcine model of left single lung transplantation. METHODS: Donor lungs were preserved with Euro-Collins solution for a mean ischemic time of 18.4 hours. Neutrophil trapping in the graft, pulmonary artery pressure, and gas exchange were assessed over a 12-hour period. Partial occlusion of the contralateral pulmonary artery allowed manipulation of the pulmonary artery pressure in the transplanted lung. Group A (n = 5) was perfused at a mean pulmonary artery pressure of 20 mm Hg, group B was reperfused at a mean pulmonary artery pressure of 45 mm Hg for 10 minutes before reducing the pressure to the same as group A, and group C was reperfused at a mean pressure of 20 mm Hg for 10 minutes, then increased to a mean of 45 mm Hg for the remainder of the experiment. Group D was reperfused as in group A with the addition of intravenous pentoxifylline. RESULTS: Leukocyte sequestration was observed in the first 10 minutes after reperfusion in groups A, B, and C, with maximal sequestration at 2 minutes. Significantly more sequestration was observed in the first 6 minutes in group B than in groups A and C, which were similar. Pentoxifylline significantly reduced leukocyte sequestration. Pulmonary venous oxygen tension in the allograft lung was worst in group B. Groups A and C were similar, but group D was superior to all other groups (p < 0.001). CONCLUSIONS: Low-pressure reperfusion, even when limited to the first 10 minutes, modulates reperfusion injury possibly through a leukocyte-dependent mechanism. The addition of pentoxifylline in the recipient confers significant additional benefit.     

The co-culture of dermal fibroblasts with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts

BACKGROUND: We recently demonstrated that inhibition of nitric oxide (NO) production ameliorated acute pulmonary allograft rejection. This study examined whether inducible NO synthase (iNOS) was expressed in the transplanted lung during acute rejection. METHODS: With a rat left lung transplant model, tissue from syngeneic (Fischer 344 to Fischer 344) and allogeneic (Brown Norway to Fischer 344) transplants were harvested on postoperative day 4 and analyzed for iNOS mRNA expression (ribonuclease protection assay), iNOS enzyme activity (conversion of L-[3H]-arginine to NO and L-[3H]-citrulline), and serum nitrite/nitrate levels. RESULTS: The iNOS mRNA was expressed in allograft lungs but was not detected in isografts or controls. The iNOS protein was present in allograft lungs, as demonstrated by high levels of L-[3H]-citrulline production compared with minimal iNOS enzyme activity in isograft and control lungs (10.1 +/- 2.4 vs 0.6 +/- 0.2 and 0.7 +/- 0.2 pmol L-[3H]-citrulline.mg-1.min-1, respectively; n = 6, p < 0.001). Allografts had significantly elevated systemic serum nitrite/nitrate levels compared with isografts and controls (38 +/- 6 vs 18 +/- 2 and 16 +/- 1 mumol/L, respectively; n = 6; p < 0.005). CONCLUSIONS: These results, together with our previous demonstration that iNOS inhibition ameliorated lung allograft rejection, suggest that (1) iNOS expression and increased NO production contributed to acute rejection of the transplanted lung, (2) iNOS inhibition may offer an alternative in management of acute lung allograft rejection, and (3) increased NO production, detected by the presence of iNOS mRNA or protein or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.     

Biodistribution of radiolabeled lipid-DNA complexes and DNA in mice

The tissue biodistribution and expression of [33P]DNA-1-[2-[9-(Z)-octadecenoyloxy]ethyl]]-2-[8](Z)-heptadece nyl]-3 -[hydroxyethyl]imidazolinium chloride (DOTIM):cholesterol complexes and 33P-radiolabeled DNA expressing chloramphenicol acetyl transferase (CAT; 4.7 kB) were studied after intravenous (iv) injection in ICR mice. Mice were injected with 200 microL of complex containing DNA at 3 mg/kg or DNA alone. One group received 8 microCi of radioactivity and were sacrificed at 5 and 20 min, and 1, 2, 4 and 24 h post-dose (n = 4/time point). A second group received the equivalent of 3.9 microCi of radioactivity and were sacrificed at 20 min, and 2 and 24 h for subsequent whole body autoradiographic analysis (WBA; n = 2/time point). The tissue distribution of intact DNA was assessed by Southern blot at 24 h post-dose, whereas the integrity of complexes and DNA incubated in heparinized whole blood was studied separately. In further studies, the time course of expression in lung tissue over a 48-h period was examined, and the relative lung-expression of purified open circular (OC) versus supercoiled (SC) DNA at 24 h was evaluated. Approximately 42% of the radioactivity was found in the lungs 5 min after injection and about half this percentage was found in the liver. By 2 h, only 5% remained in the lungs, but 48% was present in the liver. No other tissue accumulated >5% of the dose throughout the duration of the study. WBA radiograms confirmed the tissue distribution results and highlighted significant accumulation of radioactivity in bone over time. Southern Blot analysis demonstrated intact DNA in many tissues 24 h after dosing. In contrast, the majority of DNA incubated in blood was degraded within 2 h, although the complexes afforded some protection relative to DNA alone. The OC DNA expressed equivalently to SC DNA in lung tissue (OC = 1035 +/- 183 pg; SC = 856 +/- 257 pg/mg soluble protein, n = 6, mean +/- SEM) at 24 h, and detectable levels of CAT were present within 2 h of dosing (21.3 +/- 7.2 pg, n >/= 8, mean +/- SD). The results confirm that DNA-DOTIM:cholesterol complexes are initially deposited in the lungs after iv administration.     

Temperature-dependent cardioprotection of exogenous substrates in long-term heart preservation with continuous perfusion: twenty-four-hour preservation of isolated rat heart with St. Thomas' Hospital solution containing glucose, insulin, and aspartate

BACKGROUND AND METHODS: Using an isolated working rat heart model, we determined the effects of glucose, insulin, and aspartate on recovery of cardiac function when used as components of preservation solution at different temperatures. After measurement of baseline cardiac function, hearts (n = 6 per group) were perfused with oxygenated St. Thomas' Hospital solution containing (1) vehicle, glucose (9 mmol/L) or aspartate (20 mmol/L) for 12 hours at either 20 degrees or 4 degrees C; (2) glucose or glucose + insulin (10 U/L) at 20 degrees C for 20 hours; and (3) glucose + insulin at 20 degrees C or glucose + insulin + aspartate at either 20 degrees or 4 degrees C for 24 hours. Cardiac function was measured after preservation and expressed as a percentage of baseline values. RESULTS: At 20 degrees C, both glucose and aspartate increased recovery of cardiac output (vehicle, 57.7% +/- 3.8%; glucose, 76.5% +/- 2.4% [p < 0.05 versus vehicle]; aspartate, 79.9% +/- 1.4% [p < 0.05 versus vehicle]). At 4 degrees C, glucose decreased recovery of cardiac output, whereas aspartate did not change the value (vehicle, 74.4% +/- 2.2%; glucose, 61.4% +/- 2.8% [p < 0.05 versus vehicle]; aspartate, 80.5% +/- 1.7%). The addition of insulin to glucose increased recovery of cardiac output (glucose, 24.6% +/- 4.0%; insulin + glucose, 69.2% +/- 2.0%: p < 0.05). The combined use of these three agents showed an additive effect in improvement of recovery of cardiac output at 20 degrees C (glucose + insulin, 64.2% +/- 2.2%; glucose + insulin + aspartate, 76.0% +/- 1.1%; p < 0.05), but the recovery at 4 degrees C (63.1% +/- 1.8%) was significantly lower than at 20 degrees C. CONCLUSIONS: These results suggest that glucose and aspartate afford differential cardioprotective effects depending on the temperature of the preservation solution and that combined use of glucose, insulin, and aspartate at the optimal temperature may extend graft preservation time.     

Cryopreservation and culture of human corneal keratocytes

PURPOSE: To assess the effects of two different concentrations of albumin in a cryoprotective solution and two freezing methods on human corneal keratocyte ctyopreservation. METHODS: Isolated keratocytes were used for cryopreservation. Solutions of 10% dimethylsulfoxide with either 2% or 10% human albumin were used as cryoprotective agents. Cells either were transferred directly into a -80 degrees C freezer (freezing rate, 2 degrees C/min) or were cooled in a programmed freezer (1 degrees C/min until -40 degrees C and then 10 degrees C/min), which resulted in four different cryopreservation protocols. Cells were stored at -80 degrees C, then were thawed at 37 degrees C, and subsequently were cultured. Keratocytes were studied by means of trypan blue staining, growth assay, apoptosis assays, transmission electron microscopy, and immunochemistry. RESULTS: The percentage of cells that were alive after thawing ranged from 80% to 99% by trypan blue staining and from 45% to 60% by flow cytometry. The ratio of the number of living cells at the end of primary culture after cryopreservation to that before cryopreservation was significantly (P=0.04) higher after direct transfer into the -80 degrees C freezer than after controlled-rate freezing, whereas the albumin concentration had no significant influence on this ratio (P=0.45). The percentage of apoptotic cells was significantly higher after cryopreservation than in the control group of noncryopreserved cells; more than 5% 24 hours after thawing. Cryopreservation did not modify the keratocyte ultrastructure. Fibroblast growth factor dramatically decreased the serum-induced cell expression of alpha smooth muscle actin, whereas cryopreservation had no influence on this cell expression. CONCLUSIONS: A freeze-thaw trauma, which was related to cryopreservation-induced cell apoptosis, was revealed during primary culture after thawing. Direct transfer into the -80 degrees C freezer resulted in better postcryopreservation growth in the culture than controlled-rate freezing. A change in albumin concentration from 2% to 10% did not affect the results.     

Retinal cholinergic system: characterization of rat retinal acetyltransferases using specific inhibitors of choline- and carnitine-acetyltransferases

Choline acetyltransferase catalyzes the synthesis of acetylcholine from choline and acetylcoenzyme A (ACoA) in both nervous and non-nervous tissues. Carnitine acetyltransferase occurs in several tissues and transfers acetyl groups from ACoA to carnitine forming acetylcarnitine and exhibits weak choline acetyltransferase activity. Several haloacetylcholines and haloacetylcarnitines were synthesized to develop selective inhibitors of choline acetyltransferase and carnitine acetyltransferase. Acetylcholine is a transmitter for some presynaptic neurons and/or amacrine cells in retina. Selective inhibitors of choline acetyltransferase and carnitine acetyltransferase were used in the evaluation of choline acetyltransferase and carnitine acetyltransferase activities in the rat retina. Choline acetyltransferase and carnitine acetyltransferase activities were assayed by transferring of [14C]acetyl group from [14C]ACoA to choline or carnitine and estimating [14C]-acetylcholine or [14C]acetylcarnitine. This study gave the following results: (a) Bromoacetylcholine (BrACh) was a selective inhibitor of purified choline acetyltransferase (I50, 2.2 microM); (b) (R)-bromoacetylcarnitine [(R)-BrACa] was more potent for inhibiting purified carnitine acetyltransferase (I50, 4 microM) than purified choline acetyltransferase (I50, 46 microM); (c) Rat retinal sonicate gave choline acetyltransferase activity of 98 +/- 6 nmol of ACh formed/mg/10 min. When the carnitine acetyltransferase was completely inhibited by (R)-BrACa, the activity for choline acetyltransferase decreased to 47 +/- 1 nmol, and this decrease was possibly due to the formation of some [14C]acetylcholine by carnitine acetyltransferase. The net retinal choline acetyltransferase activity was 51 nmol acetylcholine/mg protein/10 min; (d) Rat retinal sonicate contained carnitine acetyltransferase activity of 102 +/- 7 nmol acetylcarnitine formed/mg protein/10 min. This was not altered by inhibition of choline acetyltransferase with BrACh. This means that choline acetyltransferase did not use carnitine as a substrate. Choline acetyltransferase and carnitine acetyltransferase activities did not change after dialysis of retinal sonicates at 4 degrees C for 24 hrs. These observations suggest that BrACh and (R)-BrACa are useful for assessing the correct values for choline acetyltransferase and carnitine acetyltransferase activities in retinal tissues.