In vivo functional localization of the human visual cortex using positron emission tomography and magnetic resonance imaging.

Positron emission tomography (PET) and magnetic resonance imaging (MRI) are two recently developed methods for imaging the human brain in vivo. One application of PET measures stimulus-evoked changes in cerebral blood flow while MRI provides a detailed anatomical map of the brain. Here we report the combined application of these two techniques in the same human subject. Subtracted PET scans of a brain receiving visual stimulation were superimposed upon MRI images of the same brain. The PET scans were converted into the MRI coordinate space before superposition, which allowed for a more precise correlation between MRI anatomical data and PET physiological data. Responses were localized in striate and extrastriate visual areas as well as in the posterior thalamus.

Reinterventions after repair of common arterial trunk in neonates and young infants.

OBJECTIVES: To determine rates of reintervention after repair of common arterial trunk in the neonatal and early infant periods. BACKGROUND: With improving success in the early treatment of common arterial trunk, the need for reinterventional procedures in older children, adolescents and adults will become an increasingly widespread concern in the treatment of these patients. METHODS: We reviewed our experience with 159 infants younger than four months of age who underwent complete primary repair of common arterial trunk at our institution from 1975 to 1998, with a focus on postoperative reinterventions. RESULTS: Of 128 early survivors, 40 underwent early reinterventions for persistent mediastinal bleeding or other reasons. During a median follow-up of 98 months (range, 2 to 235 months), 121 reinterventions were performed in 81 patients. Actuarial freedom from reintervention was 50% at four years, and freedom from a second reintervention was 75% at 11 years. A total of 92 conduit reinterventions were performed in 75 patients, with a single reintervention in 61 patients, 2 reinterventions in 11 patients and 3 reinterventions in 3 patients. Freedom from a first conduit reintervention was 45% at five years. The only independent variable predictive of a longer time to first conduit replacement was use of an allograft conduit at the original repair (p = 0.05), despite the significantly younger age of patients receiving an allograft conduit (p < 0.001). Reintervention on the truncal valve was performed on 22 occasions in 19 patients, including 21 valve replacements in 18 patients and repair in 1, with a freedom from truncal valve reintervention of 83% at 10 years. Surgical (n = 29) or balloon (n = 12) reintervention for pulmonary artery stenosis was performed 41 times in 32 patients. Closure of a residual ventricular septal defect was required in 13 patients, all of whom underwent closure originally with a continuous suture technique. Eight of 16 late deaths were related to reintervention. CONCLUSIONS: The burden of reintervention after repair of common arterial trunk in early infancy is high. Although conduit reintervention is inevitable, efforts should be made at the time of the initial repair to minimize factors leading to reintervention, including prevention of branch pulmonary artery stenosis and residual interventricular communications.

Toxicity of cationic liposome-DNA complex in lung isografts.

BACKGROUND: Cationic lipids have been successfully employed as vectors for gene transfer in lung grafts, yet those lipid vectors have potential toxicity. Furthermore, the optimal concentration of cationic lipids for gene transfection to lung grafts has not been determined. We evaluated liposome concentration/toxicity relationships in an in vivo rat lung transplantation model. METHODS: Left lungs were harvested and infused via the pulmonary artery with chloramphenicol acetyl-transferase (CAT)-DNA/lipid 67 (cationic lipid)/dioleoylphosphatidylethanolamine complex (4:1:2 in a final concentration ratio). Donor lungs were allocated into six groups according to lipid 67 concentration: group 1, 0 microM (control); group 2, 10 microM; group 3, 50 microM; group 4, 100 microM; group 5, 250 microM; group 6, 500 microM. Forty-eight hours after orthotopic transplantation, the recipient contralateral right main pulmonary artery and bronchus were ligated. The graft was ventilated with 100% oxygen for 5 min. Arterial blood gas analysis (PaO2, PaCO2), peak airway pressure (PAP), and CAT activity of the grafts were measured. RESULTS: Recipient survival, and PaO2, PAP, and CAT levels correlated with the lipid-DNA complex concentration. The grafts in groups 4-6 were more injured as evidenced by decreased PaO2 and increased PAP levels in comparison to the control group. CAT level was significantly lower in group 2 than in groups 3-6. CONCLUSIONS: The pulmonary toxicity of cationic lipid is dose-dependent. The balance between lung graft function and transgene expression is optimal at a lipid 67 concentration of 50 microM.

Long-term follow-up of truncus arteriosus repaired in infancy: a twenty-year experience.

BACKGROUND: There have been few reports of long-term follow-up after truncus arteriosus repair in infancy. METHODS: A retrospective review was performed to assess long-term outcomes among 165 patients who survived the initial hospital stay after complete repair of truncus arteriosus since 1975. The median age at truncus repair over this 20-year experience was 3.5 months (range 2 days to 36 years), and 81% of patients were less than 1 year of age. Previous pulmonary artery banding had been performed in 15 patients, and two patients had undergone prior repair of interrupted aortic arch. Significant procedures performed along with truncus repair included truncal valve replacement (n = 10) or repair (n = 5) and repair of interrupted aortic arch (n = 4). RESULTS: Patients were followed up for up to 20.4 years (median 10.5 years). Twenty-five patients were lost at cross-sectional follow-up, with a total of 67 patient-years of follow-up available on these patients. There have been 23 late deaths, eight of which occurred within 6 months of repair and 13 of which occurred within 1 year. Ten of the late deaths were related to reoperations. Actuarial survival among all hospital survivors was 90% at 5 years, 85% at 10 years, and 83% at 15 years and was essentially identical for infants alone. A significant independent risk factor for poorer long-term survival was truncus with moderate to severe truncal valve insufficiency before repair. During the follow-up period, 107 patients underwent 133 conduit reoperations. Median time to conduit reoperation was 5.5 years, and the only factor significantly associated with shorter time to conduit replacement was smaller conduit size at initial repair. In addition, 26 patients underwent 30 truncal valve replacements. Six patients required truncal valve replacement before any conduit-related reintervention, with two associated deaths. Actuarial freedom from truncal valve replacement among patients with no prerepair truncal valve insufficiency was 95% at 10 years. Actuarial freedom from truncal valve replacement was significantly lower among patients with truncal insufficiency before initial repair (63% at 10 years). At follow-up, all patients except three were in New York Heart Association functional class I. CONCLUSIONS: Ten- to 20-year survival and functional status are excellent among infants undergoing complete repair of truncus arteriosus. Conduit replacement or revision is almost inevitably necessary in this group of patients.

In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury.

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.

Lipid-mediated ex vivo gene transfer of viral interleukin 10 in rat lung allotransplantation.

BACKGROUND: Recent studies suggest that viral interleukin 10 suppresses alloimmune response in transplantation and that cationic lipids are one of the most promising nonviral vehicles for gene therapy. The aim of this study was to examine the effect of ex vivo lipid-mediated viral IL10 gene transfer into rat lung allografts on subsequent rejection. METHODS: Male F344 rats (RT1lvl) underwent left lung transplantation with allografts from Brown Norway rats (RT1n). Allografts were transvascularly transfected 15 minutes after harvest with 5 mL of 1:20-diluted (group 1, n = 7) or 1:40-diluted (group 2, n = 6) GL67-pCMVievIL-10 complex. Group 3 (n = 7), serving as the control group, received 1:40-diluted GL67-pCF1-chloramphenicol acetyltransferase complex. All allografts were preserved for 3 hours at 10 degrees C before transplantation. In all groups recipients were killed on postoperative day 5. Transgene expression of viral interleukin 10 was assessed by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Histologic rejection score, allograft gas exchange, exhaled nitric oxide level, and allograft cytokine mRNA expression were also assessed. RESULTS: Dose-dependent transgene expression of viral interleukin 10 was detected by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Allograft gas exchange (PaO2) in groups 1 (114.06 +/- 61.1 mm Hg) and 2 (108.58 +/- 35.7 mm Hg) was significantly better than that in group 3 (66.4 +/- 8.22 mm Hg; P =.020 and P =.023, respectively). The vascular rejection score in group 1 was significantly lower than that in group 3 (P =.032, Kruskal-Wallis test). Exhaled nitric oxide levels in group 2 (5.150 +/- 6.38 ppb) were significantly lower than those in group 3 (13.517 +/- 10.4 ppb; P =.039). Allograft interleukin 2 mRNA expression levels in group 1 (1.123 +/- 0.23 relative units) were significantly lower than those in group 3 (1.753 +/- 0.71 relative units; P =.038 vs group 3). CONCLUSIONS: Lipid-mediated ex vivo viral IL10 gene transfer into rat lung allografts improved graft gas exchange, reduced histologic rejection scores, downregulated graft interleukin 2 mRNA expression, and reduced exhaled nitric oxide levels by postoperative day 5. These results suggest a therapeutic potential of graft viral IL10 gene transfer as an effective immunosuppressive strategy against lung allograft rejection.

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.

Ex vivo transfection of transforming growth factor-beta1 gene to pulmonary artery segments in lung grafts.

OBJECTIVE: Proximal pulmonary artery segment transfection may provide beneficial downstream effects on the whole-lung graft. In this study, transforming growth factor-beta1 was transfected to proximal pulmonary artery segments, and the efficacy of transforming growth factor-beta1 transfection was examined in ischemia-reperfusion injury and acute rejection models of rat lung transplantation. METHODS: In the ischemia-reperfusion injury model, orthotopic left lung transplantation was performed in F344 rats. In group I, the PPAS was isolated and injected with saline solution. In 2 other groups, lipid67:DOPE:sense (group II) or antisense transforming growth factor-beta1pDNA construct (group III) was injected instead of saline solution. After cold preservation at 4 degrees C for 18 hours, lung grafts were implanted. Graft function was assessed 24 hours later. In the acute rejection model, donor lung grafts were harvested. Proximal pulmonary artery segments were injected with saline solution (group I) or sense (group II) or antisense lipid gene construct (group III) and then implanted. Graft function was assessed on postoperative day 5. RESULTS: In the ischemia-reperfusion injury study, there were no significant differences in oxygenation, wet-to-dry weight ratios, graft myeloperoxidase activity, or transforming growth factor-beta1 levels in platelet-poor serum or proximal pulmonary artery segment homogenates. In the acute rejection study, oxygenation was significantly improved in group II receiving transforming growth factor-beta1 (group II vs I and III, 136.0 +/- 32.5 vs 54.0 +/- 9.6 mm Hg and 53.8 +/- 14.8 mm Hg; P =.016 and.016). There were no significant pathologic differences. Transforming growth factor-beta1 concentrations from proximal pulmonary artery segment homogenates in group II were significantly higher compared with controls. CONCLUSIONS: Ex vivo transfection of transforming growth factor-beta1 to proximal pulmonary artery segments did not affect reperfusion injury of lung isografts. In acute rejection, however, ex vivo transfection of transforming growth factor-beta 1 to proximal pulmonary artery segments improved allograft function. This suggests that transfection to proximal pulmonary artery segments exerts beneficial downstream effects on the whole-lung allograft.

Transforming growth factor-beta1 gene transfer ameliorates acute lung allograft rejection.

BACKGROUND: The aim of the current work was to study the feasibility of functional gene transfer using the gene encoding for transforming growth factor-beta1, a known immunosuppressive cytokine, on rat lung allograft function in the setting of acute rejection. METHODS: The rat left lung transplant technique was used in all experiments, with Brown Norway donor rats and Fischer recipient rats. After harvest, left lungs were transfected ex vivo with either sense or antisense transforming growth factor-beta1 constructs complexed to cationic lipids, then implanted into recipients. On postoperative days 2, 5, and 7, animals were put to death, arterial oxygenation measured, and acute rejection graded histologically. RESULTS: On postoperative day 2, there were no differences in acute rejection or lung function between animals treated with transforming growth factor-beta1 and control animals. On postoperative day 5, oxygenation was significantly improved in grafts transfected with the transforming growth factor-beta1 sense construct compared with antisense controls (arterial oxygen tension = 411 +/- 198 vs 103 +/- 85 mm Hg, respectively; P =.002). Acute rejection scores from lung allografts were also significantly improved, corresponding to decreases in both vascular and airway rejection (vascular rejection scores: 2.0 +/- 0. 5 vs 2.8 +/- 0.6; P =.04; airway rejection scores: 1.3 +/- 0.7 vs 2. 3 +/- 0.8, respectively; P =.02). The amelioration of acute rejection was temporary and decreased by postoperative day 7. CONCLUSIONS: The feasibility of using gene transfer techniques to introduce novel functional genes in the setting of lung transplantation is demonstrated. In this model of rat lung allograft rejection, gene transfer of transforming growth factor-beta1 resulted in temporary but significant improvements in lung allograft function and acute rejection pathology.

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.

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.

Heat shock pretreatment protects pulmonary isografts from subsequent ischemia-reperfusion injury.

BACKGROUND: Heat shock has been associated with the acquisition of tolerance to a wide variety of stressful conditions, including ischemia. This is partly mediated by the production of various heat shock proteins (HSP), including HSP70. One novel approach to the reduction of ischemia-reperfusion injury after lung transplantation is the induction of HSP70 by heat pretreatment of the donor. The purpose of this study was to investigate the feasibility of this approach in an animal model of lung transplantation. METHODS: Animals were divided into six main groups, with groups I to III representing transplanted animals: In groups I and II, donor animals were anesthetized and then underwent heat stress 6 and 12 hours before organ harvest, respectively. Control animals underwent general anesthesia but no heat stress. After harvest, left lungs from groups I to III were preserved for 18 hours at 40 degrees C and then implanted into isogeneic recipients, which were killed 24 hours after reperfusion to assess graft function. Group IV and V animals underwent heat stress followed by a recovery period of 6 and 12 hours, respectively. Lungs were collected both at the time of harvest (right lungs) and after 18 hours of cold preservation (left lungs). Group VI served as nontransplanted controls. Groups IV to VI did not undergo lung transplantation. RESULTS: At the time of harvest but before implantation, HSP70 was significantly increased in heat-shocked nontransplanted donor lungs (groups IV and V) compared with group VI controls. After 18 hours of cold preservation, HSP70 levels were higher in group IV compared with group V and group VI controls. At 24 hours after reperfusion, mean arterial oxygenation was significantly higher in group I compared with group II and group III controls (290.25+/-24.5 vs 154.5+/-23.9 and 119.6+/-11.3 mm Hg, respectively; P < .001). Myeloperoxidase activity was improved in group I compared with group III controls (0.048+/-0.018 vs 0.137+/-0.036 deltaOD/mg/min, respectively; P < .05). The wet/dry weight ratio was also improved in group I compared with group III controls (6.2+/-0.3 vs. 7.8+/-0.4, respectively; P < .05). CONCLUSIONS: Heat pretreatment of the donor 6 hours before harvest results in increased synthesis of HSP70, which offers a dramatic protective effect against subsequent ischemia-reperfusion injury in the lung isograft.

Ex vivo transfection of pulmonary artery segments in lung isografts.

BACKGROUND: Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection. Proximal pulmonary artery endothelial transfection may provide beneficial downstream effects on the whole lung graft. We have already demonstrated the feasibility of in vivo and ex vivo transfection in proximal pulmonary artery segments of rat lung grafts. The aim of this study was to determine the optimal conditions for and duration of transfection. METHODS: Orthotopic left lung transplantation was performed in F344 rats after donor lung proximal pulmonary artery segments were isolated and injected with lipid 67/DOPE-chloramphenicol acetyl transferase (CAT) complementary deoxyribonucleic acid construct. Effect of exposure time was studied by exposing donor pulmonary artery segments to the construct for 0, 30, and 60 minutes prior to transplantation. In another series of experiments, pulmonary artery segments were exposed to the construct for 60 minutes prior to transplantation. Onset and duration of gene expression were determined after sacrificing animals at 3, 6, 12, and 24 hours and 3 days as well as 1 week, 2, 4, and 8 weeks after transplantation. Effect of exposure temperature was studied by exposing pulmonary artery segments to the construct for 60 minutes at 4 degrees, 10 degrees, and 23 degrees C. These recipients were sacrificed on postoperative day 3. Effect of exposure pressure was studied by using two volumes of the construct (0.01 and 0.03 mL). These recipients were sacrificed on postoperative day 3. Transgene expression was assessed by chloramphenicol acetyl transferase activity assay. RESULTS: Transgene expression was similar after 30- and 60-minute exposure. Transgene expression was evident within 3 to 6 hours after operation and persisted at 8 weeks after operation. Expression was detected at all temperatures and was equivalent at both exposure pressures. CONCLUSIONS: Gene transfection into graft pulmonary artery segments is possible under a range of conditions applicable to clinical lung transplantation.

Transfection of pulmonary artery segments in lung isografts during storage.

BACKGROUND: Proximal pulmonary artery segment (PPAS) endothelial transfection of lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection and may provide beneficial downstream effects on the whole lung graft. Transfection immediately after lung transplantation may be efficacious in ameliorating allograft dysfunction after transplantation. METHODS: In F344 rats, the PPAS was isolated and injected with 0.03 mL of GL-67/DOPE-chloramphenicol acetyl transferase (CAT) plasmid DNA. The PPASs were exposed for 60 minutes at several temperatures. The lung grafts were stored in saline solution (group 1, n = 24) or LPDG solution (group 2, n = 27) for 12 or 24 hours at 4 degrees to 37 degrees C. In group 3 (n = 42), PPASs were stored in endothelial cell culture medium and incubated at 10 degrees or 37 degrees C in a carbon dioxide incubator for 3 to 72 hours. Group 4 (n = 18) served as transplanted controls; after 3 to 24 hours' preservation at 4 degrees C in LPDG solution, lung grafts were transplanted. Transgene expression of PPASs was assessed with two CAT activity assays, thin-layer chromatography enzyme-linked immunosorbent assay and immediately after the preservation period (groups 1 to 3) or 24 hours after transplantation (group 4). RESULTS: In group 1, transgene expression did not appear. In groups 2 and 3, transgene expression was apparent after any storage duration at 37 degrees C. Transgene expression increased successively with longer storage periods. In group 4, transgene expression was detected after any storage duration. The enzyme-linked immunosorbent assay is able to quantify the expression of CAT activity, but thin-layer chromatography is more sensitive. CONCLUSIONS: Transgene expression did not occur during conventional cold storage. Transgene expression in rat PPASs during storage is possible with warm storage (37 degrees C) and appropriate storage solution.

Gene transfer of heat shock protein 70 protects lung grafts from ischemia-reperfusion injury.

BACKGROUND: We recently demonstrated that heat stress induction of heat shock protein 70 (HSP70) in donor animals before harvest decreases posttransplant ischemia-reperfusion injury in preserved rat lung isografts. The purpose of this study was to investigate the feasibility of HSP70 gene transfection into rat lung isografts using an adenoviral vector, and to study the effects of gene expression on subsequent ischemia-reperfusion injury. METHODS: In preliminary studies to determine the optimal titer, animals were injected with various titers of adenovirus-HSP70 (saline, 5 x 10(9), 1 x 10(10), and 2 x 10(10) plaque forming units [pfu]) and sacrificed 5 days after injection. To determine the optimal exposure time, animals were sacrificed at different times (0, 6, 24, and 72 hours) after intravenous injection of adenovirus-HSP70. In a subsequent series of transplant experiments, donors were allocated to three groups according to transfection strategy. Group 1 (n = 8) donors received 5 x 10(9) pfu adenovirus-HSP70 intravenously, group 2 (n = 7) donors received 5 x 10(9) pfu adenovirus-beta-galactosidase (as a virus control), and group 3 (n = 7) donors received saline and served as a negative control. Twenty-four hours after treatment all grafts were harvested and stored for 18 hours before orthotopic left lung transplantation. Twenty-four hours after implantation animals were sacrificed for assessment. The expression of HSP70 was assessed by Western blot analysis. RESULTS: In preliminary studies, HSP70 was detectable even at low titers (5 x 10(9) pfu) of adenovirus-HSP70, and was detectable at low levels as early as 6 hours after intravenous administration. Heat shock protein 70 expression was maximal at 24 hours. In transplant experiments, Western blot analysis showed that overexpression of HSP70 occurred in the HSP70-transfected lungs. The mean arterial oxygenation 24 hours after reperfusion in group 1 was superior in comparison with other groups (p < 0.05). Wet to dry weight ratio (p < 0.05) and myeloperoxidase activity (p < 0.05) were also significantly less in group 1 grafts compared with the other groups. CONCLUSIONS: This study demonstrates that in vivo, donor adenovirus-mediated gene transfer of HSP70 decreases subsequent ischemia-reperfusion injury in rat lung isografts.

Exhaled nitric oxide correlates with experimental lung transplant rejection.

BACKGROUND: Increased nitric oxide production accompanies acute lung allograft rejection. Transforming growth factor-beta1 is an immunosuppressive cytokine capable of ameliorating acute rejection. The purpose of this study was to determine whether exhaled nitric oxide (eNO) concentrations correlated with the degree of acute rejection. METHODS: A model of acute lung transplant rejection in the rat was developed, and concentrations of eNO were measured at the time of animal sacrifice. In group 1 (partial immunosuppression), donor lungs were pretreated with transforming growth factor-beta1 before implantation. In group 2 (fulminant acute rejection), no immunosuppression was used. In group 3 (full immunosuppression), recipients received cyclosporine. Group 4 were normal rats. RESULTS: When measured from both lungs, eNO concentrations were 4.97+/-0.68 versus 6.73+/-2.90 ppb for groups 1 and 2, respectively (p = 0.58). When measured selectively from transplanted left lungs, eNO concentrations were 8.61+/-0.97 versus 42.14+/-7.27 ppb, respectively (p<0.001). In groups 3 and 4, eNO concentrations were 1.02+/-0.21 and 1.51+/-0.74 ppb, respectively. CONCLUSIONS: Exhaled nitric oxide is elevated in fulminant acute rejection, is reduced after partial immunosuppression using transforming growth factor-beta1 gene therapy, and is in the normal range in cyclosporine-treated animals. The measurement of eNO correlates with the degree of acute lung allograft rejection and may serve as a noninvasive measure of acute lung transplant rejection in the clinical setting.

Options for repair of a bicuspid aortic valve and ascending aortic aneurysm.

BACKGROUND: Intrinsic abnormality of the aortic wall may explain the association of bicuspid aortic valves with ascending aortic aneurysms. Separate valve and graft repair of such lesions, rather than composite valve graft replacement, is more straightforward but leaves potentially abnormal sinuses behind. METHODS: Between January 1985 and January 1998, 45 patients underwent separate valve and graft (n = 27) or composite valve graft (n = 18) for an ascending aortic aneurysm and bicuspid aortic valve. Perioperative events and late results were compared. RESULTS: Patients undergoing separate valve and graft were older (mean age, 60 +/- 13 vs 42 +/- 12 years, p < 0.001) and were more likely to have purely stenotic (48% vs 6%, p = 0.003) than purely regurgitant (11% vs 72%, p < 0.001) disease. They were also more likely to require concomitant coronary artery bypass grafting (56% vs 6%, p = 0.001). There were no significant differences in operative risk and no known late complications related to recurrent aneurysms. CONCLUSIONS: Root replacement with a composite valve graft can be accomplished with low operative risk and is the first choice for repair of this lesion. Separate valve and graft repair, however, yields satisfactory early and late results and remains an acceptable option, especially when the coronary ostea are not displaced or when concomitant procedures must be performed.

Trends in the management of truncal valve insufficiency.

BACKGROUND: The single semilunar valve in patients with truncus arteriosus frequently is dysplastic and dysfunctional. Truncal valve insufficiency has been associated with poor outcome. Although the management of truncal valve insufficiency has evolved over the years, approaches to this problem vary considerably and remain a serious dilemma in many cases. METHODS: We reviewed the records of 89 patients with unrepaired truncus arteriosus and mild (n = 37), moderate (n = 33), or severe (n = 19) truncal valve insufficiency who were admitted to our institution between 1975 and 1995. Eight patients (7 neonates) with moderate or severe insufficiency died before surgical intervention, and 4 patients underwent palliative pulmonary artery banding. The remaining 77 patients underwent repair. The median age at repair was 3.2 months (range, 2 days to 15 years; 83% infants), and it decreased from 4 months between 1975 and 1985 to 1 month between 1986 and 1995. Truncal valve replacement (mechanical = 6, allograft = 4) was performed in 10 patients, and 5 patients underwent valve repair. RESULTS: All 4 patients who underwent pulmonary artery banding died either early or late. The hospital (or 30-day) mortality rate after repair was 34% (26/77). At a median follow-up of 10 years, 11 hospital survivors had died, with overall 1- and 10-year actuarial survival rates of 56% and 48%, respectively, and poorer survival among patients with severe truncal valve insufficiency (p = 0.02). Late truncal valve replacement (n = 24) had been performed in 21 patients. Freedom from truncal valve replacement was better in patients with mild truncal insufficiency than in those with moderate or severe preoperative insufficiency (p < 0.001). Four late deaths were related directly to reoperation for truncal valve replacement or to prosthetic valve dysfunction. Three of the 4 neonates who received allograft root replacements died within 7 months of repair, and severe allograft valve insufficiency requiring replacement 1 year after operation developed in the fourth. CONCLUSIONS: The prospects for patients with truncal valve insufficiency have been improving over time. Nevertheless, the results in patients with severe insufficiency continue to be poor.

Ex vivo adenoviral-mediated gene transfer to lung isografts during cold preservation.

BACKGROUND: Although whole-organ gene transfer has been reported in heart and liver transplant models, it has not been well characterized in lung grafts. The aim of this study was to determine the feasibility of ex vivo gene transfer to rat lung isografts during cold preservation using an adenoviral vector. METHODS: F344 rats, divided into four groups, underwent orthotopic left lung transplantation. In group I, lung grafts were flushed with adenovirus carrying the beta-galactosidase gene. After storage at 10 degrees C, grafts were implanted in recipient animals. Group II underwent the same procedure but graft storage was at 4 degrees C. Groups III (10 degrees C) and IV (4 degrees C) served as controls. On postoperative day 5, recipients were sacrificed, and native and transplanted lungs were examined. RESULTS: In group I, all animals showed successful, albeit patchy, gene expression. This occurred in 2 of 4 animals in group II, the other 2 showing no expression. Transduced cells were consistent morphologically with endothelial cells and pneumocytes. A minimal mononuclear inflammatory infiltrate was present. Control groups showed no transduction. CONCLUSIONS: It is feasible to perform ex vivo adenoviral-mediated gene transfer to rat lung isografts during cold preservation.

31P magnetization transfer studies in the monkey brain.

The forward and reverse rates through the creatine-kinase (CK) catalyzed reaction, phosphocreatine + ADP+ H+ kf in equilibrium with kr creatine + ATP in the in vivo monkey brain were measured using the techniques of saturation transfer (ST) and inversion transfer (IT) 31P nuclear magnetic resonance (NMR) spectroscopy. Independent checks on the ST apparent longitudinal relaxation (tau) data could be obtained from the parameters determined from the IT analyses. At near-equilibrium it is assumed that the forward-to-reverse flux ratio lies close to 1.0. In the monkey brain the value for the forward-to-reverse flux ratio obtained is 1.37 +/- 0.26 calculated from ST with average tau values from IT initial slopes, a value which is not significantly different from unity. The present NMR data point to the CK reaction in the living monkey brain being maintained at or near equilibrium.