Severe infection in a lung transplant recipient caused by donor-transmitted carbapenem-resistant Acinetobacter baumannii.

We describe a case of proven donor transmission of carbapenem-resistant Acinetobacter baumannii, which resulted in severe infectious complications after lung transplantation. A single bla(OXA-23) positive strain, belonging to a new multilocus sequence type (ST231), was isolated from donor and recipient, who died 65 days after transplantation. This report highlights the current challenges associated with the potential transmission of multidrug-resistant infections through organ transplantation.

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.

Inhibition of inducible nitric oxide synthase ameliorates functional and histological changes of acute lung allograft rejection.

BACKGROUND: We recently demonstrated that inhibition of inducible nitric oxide synthase (iNOS) ameliorated severe acute lung allograft rejection. This study used a rat lung transplant model to determine (1) the time course and cellular localization of iNOS expression during the histological progression of unmodified acute rejection and (2) whether inhibition of iNOS prevented impaired gas exchange function of the allograft lung and/or ameliorated the histological changes of acute rejection. METHODS AND RESULTS: iNOS mRNA and enzyme activity were expressed in allograft lungs during mild, moderate, and severe acute rejection, but not in normal, isograft, or allograft lungs before histological changes of mild acute rejection. iNOS expression in allografts resulted in elevated serum nitrite/nitrate levels, indicative of increased in vivo nitric oxide (NO) production. In situ hybridization demonstrated iNOS mRNA expression in infiltrating inflammatory cells, but not in allograft parenchymal cells. Allografts had significantly impaired gas exchange, which was prevented with the selective iNOS inhibitor aminoguanidine (PaO2 of 566+/-19, 76+/-22, and 504+/-105 mmHg for isograft, allograft, and aminoguanidine-treated allograft, respectively; P<0.0002). Aminoguanidine also significantly improved the histological rejection scores. CONCLUSIONS: (1) iNOS expression and increased NO 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; (2) aminoguanidine ameliorated the histological and functional changes of acute rejection; and (3) increased NO production, detected by the presence of iNOS mRNA, protein, or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft 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.

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.

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.

Vasoactive intestinal peptide ameliorates reperfusion injury in rat lung transplantation.

BACKGROUND: Vasoactive intestinal peptide (VIP) has been reported to have some properties that provide protection from lung injury. Furthermore, its protective effect in cold storage of donor lungs has been confirmed. We examined its effect and the timing of administration in an in vivo rat lung transplantation model. METHODS: All lungs were flushed with low-potassium dextran-1% glucose solution, and orthotopic left lung transplantations were performed. Rats were divided into four groups (n = 6). Group I received no preservation or storage. Groups II, III, and IV grafts were stored for 18 hours at 4 degrees C. Group II received no VIP. Group III received VIP (0.1 g/ml) via the flush solution. Group IV recipients received VIP (3 microg/kg) intravenously just after reperfusion. Twenty-four hours after transplantation, the right main pulmonary artery and right main bronchus were ligated, and the rats were ventilated with 100% O2 for 5 minutes. Mean pulmonary arterial pressure, peak airway pressure, blood gas analysis, serum lipid peroxide level, tissue myeloperoxidase activity, and wet-dry weight ratio were measured. RESULTS: The partial O2 tension values of groups III and IV were better than group II (groups II, III, and IV: 147.4 +/- 71.4, 402.1 +/- 64.8, 373.4 +/- 81.0 mm Hg; p < 0.05). Peak airway pressure was lower in groups III and IV than in group II (groups II, III, and IV: 19.7 +/- 0.8, 16.7 +/- 0.9. and 16.3 +/- 1.0 mm Hg; p < 0.05). Mean pulmonary arterial pressure in group III was lower than group II (groups II and III: 36.3 +/- 3.0 and 22.1 +/- 2.2 mm Hg; p < 0.01). Wet-dry weight ratio in group III was lower than in groups II and IV (group II, III, and IV: 5.2 +/- 0.2, 4.4 +/- 0.2, and 5.2 +/- 0.3; II vs III; p < 0.05, III vs IV; p < 0.01). Serum lipid peroxide levels in groups III and IV were significantly lower (groups II, III, and IV: 2.643 +/- 0.913, 0.455 +/- 0.147, and 0.325 +/- 0.124 nmol/ml; p < 0.01). CONCLUSION: VIP ameliorates reperfusion injury in an in vivo rat lung transplantation model. Either administration of VIP via the flush solution or systemically just after reperfusion was associated with improved pulmonary function.

Carbohydrate selectin inhibitor CY-1503 reduces neutrophil migration and reperfusion injury in canine pulmonary allografts.

BACKGROUND: Neutrophil adhesion is initiated by the interaction of rapidly expressed endothelial selectins with oligosaccharide structures (sialyl Lewis(x) on polymorphonuclear neutrophils (PMN). The carbohydrate sialyl Lewis X analogue CY-1503 blocks selectin receptors, thereby inhibiting PMN rolling and subsequent firm adhesion and migration. METHODS: We evaluated the inhibitory effect of CY-1503 on PMN migration and reperfusion injury in canine left lung allografts. Donor lungs were flushed with modified Euro-Collins solution (1500 ml, 4 degrees C) and preserved for 21 hours at 1 degree C. Left lung allotransplantation was subsequently performed in 14 mongrel dogs. Immediately after transplantation and allograft reperfusion, the recipient contralateral right pulmonary artery and bronchus were ligated to permit assessment of isolated allograft function during a 6-hour postreperfusion period (FIO2 = 1.0). Allograft gas exchange (q 15 minutes) and hemodynamics (q 60 minutes) were assessed. After sacrifice, allograft bronchoalveolar lavage fluid (BALF) PMN count and allograft tissue myeloperoxidase (MPO) activity were measured. Two groups were studied: In group I (n = 7) CY-1503 was added to the donor lung flush (20 mg/L) and given to the recipient (35 mg/kg intravenous bolus) before reperfusion, followed by a continuous infusion (5.25 mg/kg/h intravenously) during the 6-hour assessment period. Group II animals (n = 7) received no CY-1503. RESULTS: Gas exchange in group I was superior throughout the assessment period (p < 0.01 at 6 hours after reperfusion). BALF PMN count in group I was reduced to 0.57 +/- 0.3 x 10(6) PMN/ml compared with 3.9 +/- 1.3 x 10(6) PMN/ml in group II (p < 0.05). Group I allograft MPO activity was 0.21 +/- 0.06 compared with 0.40 +/- 0.02 delta OD/mg/ min in controls (p < 0.02). Two animals in each group died early after reperfusion as a result of graft failure and were excluded from analysis. CONCLUSIONS: Our observations indicate that selectin inhibition effectively reduces PMN adhesion, migration, and subsequent reperfusion injury in preserved canine lung allografts.

Inducible nitric oxide synthase is expressed during experimental acute lung allograft rejection.

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.

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.

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.

Isolated lung liposome-mediated gene transfer produces organ-specific transgenic expression.

BACKGROUND: Gene therapy is a promising strategy for the treatment of inoperable pulmonary tumors and rejection after lung transplantation. However, unlike ex vivo administration, intravenous in vivo transfection lacks organ specificity and has a limited duration of expression. The objectives of this study were to limit transfection to a single lung and to increase the duration of gene expression in vivo. METHODS: Sixteen male Fisher rats were anesthetized and divided into two groups. Animals in group I (n = 7) received an intrajugular administration of 1,320 microg of chloramphenicol acetyl transferase (CAT) complementary DNA complexed with cationic liposomes. Animals in group II (n = 9) received 660 microg of CAT complementary DNA complexed with cationic liposomes into the pulmonary artery of an isolated left lung over 10 minutes. After 40 minutes of incubation, the lung was flushed with 10 mL of normal saline solution, and the perfusate was suctioned through a left pulmonary venotomy. The circulation to the left lung was then restored. After 48 hours, the animals were divided into subgroups (a and b) and CAT activity was assessed in the lungs, hearts, livers, and kidneys of groups Ia (n = 3) and IIa (n = 5). After 21 days, CAT activity was assessed in the left lungs of groups Ib (n = 4) and IIb (n = 4). RESULTS: After 48 hours, animals that had received intravenous administration of CAT cDNA showed strong expression in the lungs and hearts and negligible expression in the livers and kidneys. In contrast, animals in group IIa, which had received isolated left lung perfusion of CAT cDNA showed expression only in the left lung. After 21 days, the left lungs of animals in group Ib, which had received intravenous administration of CAT complementary DNA, showed no CAT expression, but the left lungs of animals in group IIb, which had received isolated left lung perfusion of CAT complementary DNA, exhibited strong CAT expression. CONCLUSIONS: Compared with intravenous administration, isolated lung liposome-mediated gene transfer provides prolonged organ-specific gene expression. This provides a useful model to study the effects of gene therapy on pulmonary tumors, which may have further application when gene therapy is used in clinical practice.

Recombinant Kunitz protease inhibitor ameliorates reperfusion injury in rat lung transplantation.

BACKGROUND: Recombinant Kunitz protease inhibitor (rKPI-BG022) is more homologous to human Kunitz protease inhibitor than is aprotinin. Because aprotinin has been reported to inhibit free radicals, we hypothesized that rKPI would ameliorate reperfusion injury caused by free radicals. We examined its effect and the timing of administration in an in vivo rat lung transplantation model. METHODS: All lungs were flushed with low-potassium dextran-1% glucose solution and stored for 24 hours at 4 degrees C, then orthotopic left lung transplantations were performed. Rats were divided into 4 groups (n=6) as follows: group 1 served as control; in Group 2, rKPI was added to the flush solution (10 micromol/L); in group 3, rKPI (5 mg/kg) was administered intravenously to the recipient just after reperfusion; and in group 4, rKPI was added to the flush solution (10 micromol/L) and rKPI (5 mg/kg) was administered intravenously to the recipient just after reperfusion. Twenty-four hours after transplantation, the right main pulmonary artery and right main bronchus were ligated, and the rats were ventilated with 100% O2 for 5 minutes. Peak airway pressure, blood gas analysis, serum lipid peroxide level, tissue myeloperoxidase activity, and wet-dry weight ratio were measured. RESULTS: The partial oxygen tension values of group 2 were higher than those of groups 1 and 4 (groups 1, 2, and 4: 104.8+/-15.8, 245.1+/-49.0, 101.4+/-4.5 mm Hg, respectively; p < 0.01). The partial carbon dioxide tension values of groups 3 and 4 were lower than those of group 1 (groups 1, 3, and 4: 74.5+/-5.7, 42.0+/-11.0, 46.0+/-8.4 mm Hg, respectively; p < 0.05). Peak airway pressures were lower in groups 2 and 3 than in groups 1 and 4 (groups 1, 2, 3, and 4: 22.5+/-0.5, 18.2+/-0.5, 19.2+/-0.8, 22.5+/-1.1 mm Hg; p < 0.01). Serum lipid peroxide levels in groups 2 and 3 were lower than those of groups 1 and 4 (groups 1, 2, 3, and 4: 0.793+/-0.037, 0.577+/-0.069, 0.560+/-0.029, and 0.785+/-0.053 nmol/mL, respectively; groups 2 and 3 vs group 1, and group 3 vs group 4: p < 0.01; group 2 vs group 4: p < 0.05). There were no differences in wet-dry weight ratio and tissue myeloperoxidase activity between the groups. CONCLUSION: Recombinant Kunitz protease inhibitor ameliorates reperfusion injury caused by free radicals in an in vivo rat lung transplantation model. Administration of rKPI through the flush solution and intravenous injection after reperfusion were effective separately, but the combination of the two administrations was not effective.

Inhaled nitric oxide at the time of harvest improves early lung allograft function.

BACKGROUND: Inhalation of nitric oxide (NO) has been shown to have beneficial effects on a variety of acute lung injuries, including lung allograft reperfusion injury. The purpose of the present study was to investigate the effects of inhaled NO at the time of harvest on function of canine left lung allografts after transplantation. METHODS: Ten dogs underwent left lung allotransplantation. Donor lungs were flushed with modified Euro-Collins solution and stored for 21 hours at 1 degree C. Immediately after transplantation, the contralateral main pulmonary artery and bronchus were ligated to assess isolated allograft function. Hemodynamics and arterial blood gases (inspired oxygen fraction, 1.0) were assessed intermittently for 6 hours prior to sacrifice. Allograft myeloperoxidase activity and wet to dry weight ratio were assessed. Donor animals were divided into two groups. Group I animals (n = 5) received no NO. In group II (n = 5), donors received inhaled NO (60 ppm) at the time of harvest. RESULTS: Pulmonary vascular resistance decreased to 79.6% of baseline because of inhalation of 60 ppm NO in group II donor animals. Thiobarbituric acid-reactive materials were reduced during the storage period in group II, a finding suggesting less oxidant injury during storage in donor lungs treated with NO. Throughout the 6-hour assessment, oxygenation in group II was superior to that in group I (p < 0.05). At 360 minutes of assessment, mean arterial oxygen tension in groups I and II was 88.9 +/- 11.4 mm Hg and 169.1 +/- 33.0 mm Hg, respectively. Myeloperoxidase activity was significantly decreased in group II (p < 0.05), data indicating reduced neutrophil sequestration. Wet to dry weight ratio was significantly lower in group II. CONCLUSIONS: These data suggest that inhaled NO at the time of harvest improves early function of preserved lung allografts by attenuating oxidant injury during storage and subsequent neutrophil sequestration.

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.

Fas ligand gene transfer combined with low dose cyclosporine A reduces acute lung allograft rejection.

The interaction between Fas and its ligand (FasL) induces apoptosis in the Fas-expressing cell. We hypothesized that liposome-mediated FasL gene transduction to the lung allograft, in addition to low-dose immunosuppression, might reduce acute rejection. Orthotopic left lung allotransplantation was performed in male rats (Brown Norway to Fischer F344). FasL gene transfer was performed by use of the plasmid pBCMGSNeo carrying the gene coding for murine FasL and the cationic liposome GL#67:DOPE. Six hundred and sixty micrograms of DNA in 250 microl H2O and 0.5 micromol GL#67 in 250 microl H2O were diluted to 5 ml with saline solution. This emulsion (20 degrees C) was instilled retrogradely through the left pulmonary vein after flushing with LPD solution (20 ml, at 4 degrees C). Subsequently, the graft was stored at 10 degrees C for 3 h. A single dose of cyclosporine A (CsA; 2.5 mg/kg i.m.) was given to all groups 48 h after the transplantation. In group 1 (n = 6), FasL/GL#67 was instilled as described. In group 2 (n = 5), GL#67 was given without DNA. Group 3 (n = 5) animals received CsA only. Five days after transplantation, gas exchange was assessed after exclusion of the contralateral native lung (FiO2 = 1.0). Grafts were flushed with saline solution and fixed in formaldehyde for histological evaluation. No statistical difference in gas exchange (PaO2) between the two control groups 2 (6.4 +/- 0.4 kPa) and 3 (7.4 +/- 0.4 kPa) could be detected 5 days postoperatively (P = 0.9). In contrast, grafts transduced with FasL (group 1) had significantly better gas exchange on postoperative day 5 (PaO2: group 1 37.0 +/- 10.6 kPa vs group 2 6.4 +/- 0.41 kPa; P = 0.002). Two animals in group 1 revealed no or only minimal improvement in gas exchange. Histologically, all lung specimen of all groups showed signs of acute rejection (A2). Leukocyte infiltrates, rated by two independent observers, were less severe in all group 1 animals. Liposome-mediated FasL gene transfer at the time of harvest in combination with low-dose CsA reduces acute rejection in four out of six animals in this model of rat lung allotransplantation.