須田 隆

INTRODUCTION: The purpose of this study was to identify independent unfavorable prognostic factors for patients who underwent video-assisted thoracoscopic surgery for resection of pulmonary metastases from colorectal cancer (CRC). METHODS: Between January 2004 and December 2013, 131 patients with pulmonary metastases from CRC underwent the aforementioned procedure for the first time at our institution. Kaplan-Meier survival curves and log-rank tests were used to analyze the survival rates. Multivariate analyses were performed using the Cox proportional hazards regression model. RESULTS: The 5-year disease-free survival (DFS) rate of these 131 patients was 34 %. Multivariate analyses showed two variables to be independent significant unfavorable prognostic factors for DFS: preoperative high serum carcinoembryonic antigen (CEA) level and a greater number of pulmonary metastases. According to subgroup analyses that combined these two risk factors, the 5-year DFS rates were 58, 25, and 12 % for patients with 0, 1, or 2 risk factors, respectively. CONCLUSION: In patients who underwent video-assisted thoracoscopic surgery for pulmonary metastases from CRC, we identified two independent unfavorable prognostic factors for DFS: a high CEA level before metastasectomy and a greater number of pulmonary metastases. These factors can be used to identify higher- and lower-risk subgroups, which may help with selecting patients who would benefit the most from video-assisted thoracoscopic pulmonary metastasectomy.

OBJECTIVES: The purpose of this study was to investigate the prognostic factors for repeat lung metastasectomy in patients with colorectal cancer, which may be clinically helpful in defining a subset of patients who are most likely to benefit from repeat lung metastasectomy. METHODS: In total, 138 patients underwent complete lung resection for the first time due to metastases of colorectal cancer between January 2004 and December 2013 at Fujita Health University School of Medicine. Among them, 33 underwent repeat pulmonary metastasectomy for lung tumour recurrence. Kaplan-Meier survival curves and log-rank tests were used to analyse the survival rates. RESULTS: No patient died as a direct result of surgery, and all patients were discharged after the repeat pulmonary metastasectomy. The 5-year survival rate after the initial pulmonary resection of the 33 patients who underwent repeat lung resection was 64%, which was not significantly different from that of the 105 patients who did not undergo repeat lung resection (5-year survival rate, 61%; P = 0.779). Univariate analysis identified only one significant prognostic factor: preoperative serum carcinoembryonic antigen (CEA) level (P = 0.002). The 5-year survival rates of patients with high preoperative CEA levels and normal CEA levels after repeat metastasectomy were significantly different at 47 and 90%, respectively. CONCLUSIONS: Prethoracotomy serum CEA levels affect survival rates after repeat pulmonary resection. The preoperative assessment of serum CEA levels before repeat metastasectomy is important when considering repeat pulmonary resection, and prethoracotomy CEA levels should be taken into account when selecting patients for repeat lung resection.

BACKGROUND: We have previously reported on single-port thymectomy (SPT), which involves performing thymectomy via a single subxiphoid incision, and trans-subxiphoid robotic thymectomy (TRT), which is performed using the da Vinci surgical system. The aim of this study was to investigate the early surgical outcomes of thymectomy using the SPT and TRT subxiphoid approaches and to discuss their appropriate uses. METHODS: The subjects included 80 patients who underwent thymectomy via a subxiphoid approach. These patients were selected from among 99 surgical cases of myasthenia gravis or anterior mediastinal tumors at Fujita Health University Hospital between March 2011 and November 2015. The patients were divided into a SPT group (n=72) and a TRT group (n=8). RESULTS: The operative time was shorter in the SPT group compared with that in the TRT group (135±48 and 20±40 min, respectively; P=0.0004). There were no significant differences between the groups in terms of blood loss volume (5.9±16.8 and 5.4±4.6 mL, respectively; P=0.48), postoperative hospital stay duration (4.0±2.0 and 4.3±3.6 days, respectively; P=0.21), or the period of postoperative oral analgesic use (10.7±5.4 and 10.1±3.4 days, respectively; P=0.89). There were no intraoperative complications, such as intraoperative bleeding, in either group. In the SPT group, there was one case (1.4%) of postoperative left phrenic nerve paralysis and one case (1.4%) of transient paroxysmal atrial fibrillation. No one died during or after the surgery. CONCLUSIONS: TRT may be as equally minimally invasive as SPT. In cases where the thymoma has infiltrated the surrounding organs, the extent of the infiltration should be used to determine whether to select TRT, or median sternotomy.

OBJECTIVES: Compared with conventional median sternotomy, approaches used in thymectomy for myasthenia gravis and anterior mediastinal tumours have become much less invasive in recent years. We previously developed a surgical technique called single-port thymectomy (SPT) to excise the thymus through a single opening made below the xiphoid process. In this study, to show the utility of SPT, we compared factors contributing to low surgical invasiveness between SPT and conventional video-assisted thoracoscopic surgery (VATS) thymectomy. METHODS: Between January 2005 and December 2014, 146 patients underwent surgery for anterior mediastinal tumour or myasthenia gravis at our hospital. After excluding patients diagnosed with tumour invasion of nearby organs or those who had undergone concurrent removal of nearby organs, 81 patients were enrolled in this retrospective study as subjects. Patients were divided into the VATS thymectomy group (VATS group, n = 35) and the SPT group (n = 46). Surgical duration, blood loss, duration of hospital stay and the amount and duration of postoperative oral analgesics were compared between the groups. RESULTS: Operating time did not vary significantly between the VATS and SPT groups (P = 0.0853). The amount of blood loss was higher in the VATS group than in the SPT group (P < 0.0001). The duration of hospital stay was longer in the VATS group than in the SPT group (P = 0.0008). The amount of postoperative oral analgesics was significantly higher in the VATS group than in the SPT group (P = 0.0092). Similarly, the duration of postoperative oral analgesics was significantly longer in the VATS group than in the SPT group (P = 0.0312). CONCLUSIONS: Compared with VATS thymectomy, SPT required a similar operating time, was associated with less blood loss and enabled postoperative analgesics to be discontinued earlier. Therefore, it could be considered a less invasive surgical approach.

When endoscopic surgery is indicated for myasthenia gravis and thymomas, most institutions use a lateral thoracic approach that includes robot-assisted surgery. However, with the unilateral thoracic approach, it can be difficult to ensure the operative field in the neck and difficult to identify the location of the contralateral phrenic nerve. In 2015, we reported on a robotic subxiphoid thymectomy (RST) in which the camera is inserted from the subxiphoid incision and robotic forceps are inserted from the bilateral intercostal spaces. With this approach, a camera is inserted into a subxiphoid incision which is the midline of the body and a surgical field comparable to that in a median sternotomy can be achieved. This makes it easier to identify the location of the bilateral phrenic nerves and offer the good visualization in the neck area. Here we report on our RST techniques. For a thymectomy without suturing, a subxiphoid, single-port thymectomy is performed because it is minimally invasive. In patients who require suturing, such as with a pericardial patch closure, RST is selected. The RST has excellent operability when performed with a robot, making it suitable for more difficult procedures. In the future, we believe that a robot-assisted thymectomy might become the standard method.

With a uniportal subxiphoid video-assisted thoracoscopic surgery (VATS) thymectomy, the thymus is not accessed through the ribs, which avoids intercostal nerve damage. Furthermore, compared to a VATS thymectomy via the lateral chest, decreased use of postoperative analgesics (lower doses and shorter duration), decreased blood loss, and shorter surgical duration and hospital stay have been reported. Here we report our surgical method and solutions for a uniportal subxiphoid VATS thymectomy. A uniportal subxiphoid VATS thymectomy is indicated for patients with myasthenia gravis and anterior mediastinal tumors who do not require surgical suturing. The operator stands between the patient's legs and the scopist stands to the right of the patient to operate the camera scope. To begin with, a 3-cm transverse incision was made along Langer's lines 1 cm caudal below the xiphoid process. The CO2 insufflation is performed in the mediastinum at 8 mmHg. The pericardial adipose tissue and thymus are detached from the pericardium in an anterior manner from the bilateral phrenic nerves. The resected thymus is placed in a pouch in the mediastinum and removed from the body through the subxiphoid incision. The subxiphoid approach is highly beneficial for the patient in that it results in superior esthetic outcomes considering that there is no sternotomy and no intercostal nerve damage; thus, it is a surgical procedure that surgeons should learn.

Minimally invasive surgery has replaced median sternotomy for resectable anterior mediastinal masses and is performed by various approaches. We developed a new minimally invasive surgical procedure by combining the subxiphoid approach performed through a midline camera port with the use of a robotic surgery system (Intuitive Surgical, Sunnyvale, CA, USA). A 3-cm transverse incision was made 1 cm below the xiphoid process. Then, a port designed for single-port surgery was inserted. Through this port, CO2 gas was injected at 8 mmHg. The thymus was then detached from the back of the sternum. A 1-cm skin incision was made bilaterally in the sixth intercostal space, followed by insertion of a port for the robotic system. A camera port was inserted into the subxiphoid port, to which the camera scope was mounted, and thymectomy was performed. We have performed the operation in 3 patients. In our experience, this procedure provides a good operative view in the neck region and makes verification of the phrenic nerve easy. Furthermore, with the da Vinci surgical system, which enables surgical manipulation from a correct angle due to the multijoint robotic arms, trans-subxiphoid robotic thymectomy may be a promising new thymectomy procedure.

OBJECTIVES: As surgical robots have become increasingly used, verification of their usefulness in the general thoracic surgery field is required. Initial results of robot-assisted thoracoscopic surgery in Japan were investigated. METHODS: A questionnaire survey was performed to retrospectively examine the current status of robotic surgery for general thoracic disease in Japan. The subjects were 112 cases performed by the end of September 2012 at 9 institutions. RESULTS: There were 60 cases of primary lung cancer, 38 cases of anterior-middle mediastinal disease, and 14 cases of posterior mediastinal disease. In lung cancer cases, the operative time was 284.7 min, the blood loss was 129 mL, the drainage period was 3.3 days, and the conversion rate was 3.3 %. The incidence of postoperative complications was 6.7 %. The postoperative hospital stay was 8.2 days. In cases of anterior-middle mediastinal disease, the operative time was 184.3 min, the blood loss was 43.8 mL, the drainage period was 2.3 days, and there was no conversion. The incidence of postoperative complications was 7.9 %. The postoperative hospital stay was 7.1 days. In cases of posterior mediastinal disease, the operative time was 142.6 min, the blood loss was 61.4 mL, the drainage period was 1.6 days, and there was no conversion. No postoperative complication developed in any case. The postoperative hospital stay was 5 days. In all cases underwent robotic surgery, there was no operation related mortality. CONCLUSIONS: Robotic surgery was safely introduced, and the incidence of postoperative complications tended to be low, although the operative time was long. Preparations for its employment in advanced medical care and coverage by national health insurance are urgent issue.

We have previously reported single-port thymectomy (SPT) through an infrasternal approach, a procedure in which the thymus is removed through a single port. The dual-port thymectomy procedure developed by adding another port to the single-port procedure has eliminated the risk of interference between forceps operated by both hands of the surgeon and has thereby significantly simplified operative procedures. This procedure provides the same operative field as that obtained by median sternotomy and has excellent maneuverability of devices. Therefore, the dual-port procedure can be used by surgeons who have not been sufficiently trained for SPT, as an alternative procedure in the event of experiencing technical difficulty during SPT, or as a new approach for thymectomy.

In recent years, tumors producing granulocyte-colony-stimulating factor have been reported in an increasing number of patients, the majority of which have lung cancer. We experience a case of lung carcinoma producing granulocyte-colony-stimulating factor treated by resection and chemotherapy. He remains well 2 years and 10 months after surgery, with no recurrence of the carcinoma.

Hepatic resection for colorectal metastases was performed for 188 patients. Overall survival rates after the first hepatectomy are 41.4% and 32.7% for 5 and 10 years, respectively. The survival rate of 116 cases with unilobar hepatic metastases (H1) is significantly higher than those of 48 cases with two to four bilobar metastases (H2) and 24 cases with more than four (H3), respectively. However, the differences between the survival rates from H1 with multiple metastases, H2, and H3 are not significant, even though the H3 group has no 10-year survivors. The 5-year survival rates after the second hepatectomy (30 patients) and the resection of the lung (26 patients) are 30.3% and 35.2%, respectively, in this series. In those patients, the 5-year survival rates from the first metastasectomy are 43.4% and 50.3%, respectively. There are 14 5-year survivors with multiple metastases and 8 of those patients underwent multiple surgeries. There are 13 patients with three or more repeat resections of the liver and/or lung. The 5-year survival rates of the patients from the first and third metastasectomy are 53.9% and 22.5%, respectively. Repeat operations for the liver and the lung contribute to the improving prognosis.

PURPOSE: To determine if Noguchi's classification can be evaluated accurately by frozen-section diagnosis before limited surgery. METHODS: We performed frozen-section diagnosis in 31 of 343 patients who underwent excision of primary lung cancer at our hospital between 1993 and 2004. All 31 patients had pulmonary adenocarcinoma, with a tumor diameter of < or = 20 mm. There were 20 men and 11 women, ranging in age from 42 to 79 years (mean, 63.2 years). We assessed the rate of correct Noguchi's classification by categorizing all lesions into the following three groups on the basis of tumor diameter: < or = 10 mm, 11-15 mm, and 16-20 mm. RESULTS: The overall rate of correct frozen-section diagnosis during surgery was 67.7%; being 100%, 41.7%, and 70% in the < or = 10 mm, 11-15 mm, and 16-20 mm groups, respectively. CONCLUSION: Limited surgery for primary lung cancer can be performed when the tumor diameter is < or = 10 mm, by confirming it as either type A or B according to Noguchi's classification, using frozen-section diagnosis. Thus, examination of frozen sections might be an important diagnostic procedure before intentional limited surgery for lung cancer.

OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in acute lung graft rejection. We investigated whether gene cotransfection into the recipient reduces acute lung graft rejection. METHODS: Brown Norway rats were used as donors, and F344 rats were used as recipients. Recipient animals were injected with saline (groups I/VI) or 1 x 10(10) pfu of adenovirus encoding beta-galactosidase (groups II/VII), transforming growth factor beta1 (groups III/VIII), interleukin 10 (groups IV/IX), or both transforming growth factor beta1 and interleukin 10 (groups V/X) into both leg muscles 2 days before transplantation (groups I-V) or at the time of harvest (groups VI-X). The Kruskal-Wallis test for rejection score and 1-way analysis of variance were used to compare groups. RESULTS: Oxygenation was significantly improved in the cotransfected groups treated 2 days before transplantation and at the time of harvest. Rejection scores were also reduced in the cotransfected groups. In group V cotransfection suppressed endogenous interleukin 2 but not interferon gamma and tumor necrosis factor alpha. CONCLUSION: Recipient intramuscular cotransfection of transforming growth factor beta1 and interleukin 10 suppressed interleukin 2 expression and provided a synergistic effect that reduced acute lung graft rejection. This approach might be applied to the clinical setting because transplant recipients could be treated at the time of implantation.

BACKGROUND: Soluble type I interleukin-1 receptor is a competitive inhibitor of interleukin-1 and may reduce its proinflammatory actions. The objective of this experiment was to demonstrate that endobronchial gene transfer of soluble type I interleukin-1 receptor IgG to donor lung grafts reduces posttransplant ischemia-reperfusion injury. METHODS: All experiments utilized an orthotopic left lung isograft transplant model. Donors were divided into three groups (n = 6 each) for endobronchial transfection: group I received 2 x 10(7) plaque-forming units of adenovirus encoding soluble type I interleukin-1 receptor IgG; group II received 2 x 10(7) plaque-forming units of nonfunctional control adenovirus encoding beta-galactosidase; and group III received 0.1 mL of saline. Left lungs were harvested 24 hours after transfection and stored for 18 hours before transplantation. Graft function was assessed 24 hours after reperfusion using three measurements: isolated graft oxygenation, wet-to-dry lung weight ratio, and tissue myeloperoxidase activity. Transgene expression of soluble type I interleukin-1 receptor IgG was also evaluated using enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Isolated graft arterial oxygenation was significantly improved in group I compared with groups II and III (281.8 +/- 134.8 versus 115.7 +/- 121.5 and 88.0 +/- 58.9 mm Hg, p = 0.0197 and p = 0.0081, respectively). Myeloperoxidase activity was also significantly reduced in group I compared with groups II and III (0.083 +/- 0.044 versus 0.155 +/- 0.043 and 0.212 +/- 0.079 optical density units per minute per milligram protein, p = 0.0485 and p = 0.0016, respectively). Expression of soluble type I interleukin-1 receptor IgG was detected only in lungs from group I. CONCLUSIONS: Endobronchial gene transfer of soluble type I interleukin-1 receptor IgG to donor lung grafts subjected to prolonged cold ischemia ameliorates ischemia-reperfusion injury by improving graft oxygenation and reducing lung edema and neutrophil sequestration.

BACKGROUND: Gene therapy may be an effective strategy for modulating lung graft ischemia-reperfusion injury. We investigated whether recipient intramuscular (IM) naked plasmid gene transfer of transforming growth factor beta1-active (TGF-beta1-active) ameliorates lung graft ischemia-reperfusion injury. METHODS: Preliminary studies in F344 rats demonstrated that gastrocnemius muscle transfection of TGF-beta1-active produced muscle and plasma protein expression at 24 and 48 hours after transfection. Recipients (n = 8) received IM injection of naked plasmid-encoding chloramphenicol acetyl transferase (CAT), TGF-beta1-latent or TGF-beta1-active, respectively, at 24 or at 48 hours before left lung transplantation. We did not treat the control group before transplantation (18-hour cold ischemia). Donor lungs were flushed with low-potassium dextran-1% glucose and stored for 18 hours at 4 degrees C. All groups were killed at 24 hours after transplantation. Immediately before killing the animals, we clamped the contralateral right hilum and assessed graft function. We measured wet-to-dry ratio (W/D), myeloperoxidase, pro-inflammatory cytokines (interleukin 1 [IL-1], tumor necrosis factor alpha [TNF-alpha], interferon-gamma [INF-gamma], and IL-2) and performed immunohistochemistry. RESULTS: Arterial oxygenation was greatest in the recipient group transfected with TGF-beta1-active at 24 hours before transplantation compared with CAT, TGF-beta1-latent, and 18-hour cold ischemia groups (p < 0.01). The W/D ratio and myeloperoxidase decreased in both 24- and 48-hour groups, with TGF-beta1-active compared with CAT, and 18-hour cold ischemia groups (W/D, p < 0.02 and p < 0.004, respectively; myeloperoxidase, p < 0.05 and p < 0.01, respectively). All pro-inflammatory cytokines decreased in the 24-hour TGF-beta1-active group compared with CAT, TGF-beta1-latent, 18-hour and 1-hour cold ischemia, and non-treated lung groups (IL-1beta, p < 0.03; TNF-alpha, p < 0.02; IFN-gamma, p < 0.001; IL-2, p < 0.0001). In 24- and 48-hour groups with TGF-beta1-active, immunohistochemistry showed marked staining of Type I and Type II alveolar cells and of macrophages from the apical to the caudal sections of the lung grafts. CONCLUSIONS: Recipient IM administration of naked plasmid encoding TGF-beta1-active before transplantation ameliorates lung isograft reperfusion injury after prolonged ischemia.

OBJECTIVE: Tumor necrosis factor is an important mediator of lung transplant ischemia-reperfusion injury, and soluble type I tumor necrosis factor receptor binds to tumor necrosis factor and works as a tumor necrosis factor inhibitor. The objectives of this study were to demonstrate that gene transfer of type I tumor necrosis factor receptor-IgG fusion protein reduces lung isograft ischemia-reperfusion injury and to compare donor endobronchial versus recipient intramuscular transfection strategies. METHODS: Three donor groups of Fischer rats (n = 6/group) underwent endobronchial transfection with either saline, 2 x 10(7) plaque-forming units of control adenovirus encoding beta-galactosidase, or 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein. Left lungs were harvested 24 hours later. Two recipient groups (n = 6/group) underwent intramuscular transfection with 2 x 10(7) plaque-forming units or 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein 24 hours before transplantation. All donor lung grafts were stored for 18 hours before orthotopic lung transplantation. Graft function was assessed 24 hours after reperfusion. Transgene expression was evaluated by means of enzyme-linked immunosorbent assay and immunohistochemistry of type I tumor necrosis factor receptor. RESULTS: Endobronchial transfection of donor lung grafts with 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein significantly improved arterial oxygenation compared with the saline and beta-galactosidase donor groups (366.6 +/- 137.9 vs 138.8 +/- 159.9 and 140.6 +/- 131.4 mm Hg, P =.009 and.010, respectively). Recipient intramuscular transfection with 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein improved lung graft oxygenation compared with that seen in the low-dose intramuscular group (2 x 10(7); 320.3 +/- 188.6 vs 143.6 +/- 20.2 mm Hg, P =.038). Type I tumor necrosis factor receptor-IgG fusion protein was expressed in endobronchial transfected grafts. In addition, intramuscular type I tumor necrosis factor receptor-IgG fusion protein expression was dose dependent. CONCLUSIONS: Donor endobronchial and recipient intramuscular adenovirus-mediated gene transfer of type I tumor necrosis factor receptor-IgG fusion protein improved experimental lung graft oxygenation after prolonged ischemia. However, donor endobronchial transfection required 500-fold less vector. Furthermore, at low vector doses, it does not create significant graft inflammation.

OBJECTIVE: Because almost all pulmonary diseases are not caused by one gene, multiple gene transfection is required for current gene therapy. Adenovirus is an important gene therapy vector, but a short duration and the inability of repeated administration remain limitations. The aims of this study were to evaluate whether adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs gene expression and facilitates repeated vector administration to investigate the feasibility of a cotransfection strategy. METHODS: F344 rats received intratracheal administration of 1 x 10(9) plaque-forming units of adenoviral vector encoding beta-galactosidase or both adenoviral vector encoding beta-galactosidase and adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin. In the expression study beta-galactosidase gene expression in the lung was examined by means of enzyme-linked immunosorbent assay on days 2, 7, 14, 28, and 56 (n = 4/day). In the repeated transfection study, soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase were readministered once (7 days after the first adenovirus administration) or twice (on days 7 and 14; n = 4/day). A 2-way factorial analysis of variance was used for statistical analysis. RESULTS: Soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolonged the duration of beta-galactosidase expression. However, antiadenovirus antibody production was significantly increased in the cotransfection group. In addition, there was no increase in beta-galactosidase expression after readministration of soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase. CONCLUSION: Adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs beta-galactosidase expression but does not increase beta-galactosidase expression after repeated administration. These results suggest that tumor necrosis factor alpha is one of the most important factors in regulating the duration of gene expression. The cotransfection approach is feasible, but the increase of antiadenovirus antibodies might make repeated cotransfection unfeasible.

OBJECTIVE: Gene transfer to experimental lung grafts has been shown to reduce ischemia-reperfusion injury and acute rejection. The optimal delivery route should produce high lung expression with no inflammation and minimal systemic expression. The goal of this study was to determine the optimal gene transfer route for use in experimental lung transplantation. METHODS: F344 rats were injected with 2.9 x 10(10) plaque-forming units of adenovirus vector encoding beta-galactosidase through intratracheal, intravenous, intraperitoneal, or intramuscular delivery routes and killed 48 hours later. Gene expression was measured by means of enzyme-linked immunosorbent assay. RESULTS: Intratracheal delivery produces significantly greater gene expression in the lung (75,350 +/- 47,288 pg/100 microg of protein, P <.001 vs intravenous, intraperitoneal, and intramuscular routes) and minimal systemic expression (nonsignificant in serum, kidney, liver, spleen, and muscle vs that seen in control animals, P =.016 for heart). Immunohistochemistry staining showed beta-galactosidase expression in the bronchial epithelium of lungs transfected through the intratracheal route with mild inflammation. CONCLUSIONS: Intratracheal gene transfer provides significant expression in the lung with mild to no inflammation and minimal systemic expression. This delivery strategy has tremendous potential in experimental lung transplant models to reduce ischemia-reperfusion injury and acute allograft rejection and should be investigated further.

Interleukin-10 (IL-10) has potent anti-inflammatory properties but its direct effects on neutrophil trafficking in lung transplant ischemia-reperfusion (I/R) injury are unknown. This study was performed to determine if recipient intramuscular IL-10 gene transfer reduces neutrophil infiltration in lung isografts and ameliorates I/R injury. Twenty-four hours before transplantation, recipient rodents received intramuscular injection with 1 x 10(10) plaque-forming units (pfu) adenovirus encoding human IL-10 (hIL-10), 1 x 10(10) pfu adenovirus control encoding p-galactosidase, or saline. Gene expression in muscle and plasma was confirmed. Lung grafts were harvested, stored at 4 degrees C for 18h, and assessed 24 h after transplantation. Peak muscle and plasma expression of hIL-10 was achieved 24h after gene transfer and returned to baseline by 7 days (p < 0.05 vs. controls). Gene transfer of hIL-10 reduced neutrophil sequestration and emigration in lung grafts as measured by morphometry and myeloperoxidase activity (p < 0.03 vs. controls). Furthermore, hIL-10 improved graft oxygenation and reduced lung edema (p <0.01 vs. controls). Intramuscular gene transfer of hIL-10 releases hIL-10 protein into plasma and reduces neutrophil sequestration and emigration in lung isografts. This is associated with a reduction in I/R injury with improved isograft oxygenation and reduced tissue edema. Intramuscular gene transfer may be a useful strategy to reduce clinical l/R injury.

OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in lung graft ischemia-reperfusion injury. In this study we analyzed whether recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 would result in amelioration of lung graft ischemia-reperfusion injury. METHODS: Forty-eight hours before transplantation, 6 groups (n = 6) of F344 rats received intramuscular injection of naked plasmid encoding chloramphenicol acetyltransferase, chloramphenicol acetyltransferase plus beta-galactosidase, transforming growth factor beta(1), interleukin 10, or transforming growth factor beta(1) plus interleukin 10 or were not treated. Donor lungs were flushed and stored for 18 hours at 4 degrees C before transplantation. Twenty-four hours later, grafts were assessed immediately before the animals were killed. Arterial oxygenation, wet/dry ratio, myeloperoxidase, and proinflammatory cytokines (interleukin 1, tumor necrosis factor alpha, interferon gamma, and interleukin 2) were measured, and immunohistochemistry was performed. RESULTS: For lung graft function, the arterial oxygenation was considerably higher in the cotransfected group receiving transforming growth factor beta(1) plus interleukin 10 compared with that in all other groups (P < or =.03). The wet/dry ratio, reflecting lung edema, was reduced in the cotransfected group compared with that in control animals (nontreated, P <.02; chloramphenicol acetyltransferase, P <.03; chloramphenicol acetyltransferase plus beta-galactosidase, P <.01). Myeloperoxidase, which measures neutrophil sequestration, was also reduced with cotransfection compared with that seen in control animals (P < or =.03). All proinflammatory cytokines were decreased in the cotransfected group compared with those in all other groups (interleukin 1beta, P <.04; tumor necrosis factor alpha, P <.002; interferon gamma, P <.0001; interleukin 2, P <.03). These results indicate that cotransfection provides a synergistic benefit in graft function versus either cytokine alone, neutrophil sequestration, or inflammatory cytokine expression. Immunohistochemistry showed positive staining of transforming growth factor beta(1) plus interleukin 10 in type I and II pneumocytes and localized edema fluid. CONCLUSIONS: Recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 provides a synergistic effect in ameliorating lung reperfusion injury after prolonged ischemia.

OBJECTIVE: This study was undertaken to determine whether low-dose endobronchial transfer to the donor of the gene for human interleukin 10 would decrease ischemia-reperfusion injury in lung transplantation. METHODS: Experiments used male Fischer rats. Donor animals underwent right thoracotomy. A catheter was introduced into the left main bronchus, and vector was instilled. Group I (n = 6) received 2 x 10(7) plaque-forming units of adenovirus encoding human interleukin 10, group II (n = 6) received an adenovirus control encoding beta-galactosidase, and group III (n = 6) received saline solution. After instillation the left main bronchus was clamped for 60 minutes. Lungs were removed 24 hours later and stored in low-potassium dextran glucose solution for 18 hours before left lung transplantation. Graft function was assessed at 24 hours immediately before the animals were killed. Ratio of wet to dry weight and tissue myeloperoxidase activity were measured. Transgenic expression of human interleukin 10 was evaluated by means of enzyme-linked immunosorbent assay and immunohistochemical assay. RESULTS: Arterial oxygenation was significantly improved in group I relative to groups II and III (257.6 +/- 59.7 mm Hg vs 114.6 +/- 66.9 mm Hg and 118.6 +/- 91.1 mm Hg, P =.008 and P =.007, respectively). Neutrophil sequestration, as measured by myeloperoxidase activity, was also significantly reduced in group I relative to groups II and III (0.141 +/- 0.025 vs 0.304 +/- 0.130 and 0.367 +/- 0.153 Delta optical density units/[min. mg protein], P =.029 and P =.004, respectively). Enzyme-linked immunosorbent assay and immunohistochemical assay demonstrated the expression of human interleukin 10 in transfected lungs only. CONCLUSIONS: Low-dose endobronchial transfer to the donor of the gene for human interleukin 10 ameliorated ischemia-reperfusion injury in rodent lung transplantation by improving graft oxygenation and reducing neutrophil sequestration. Only 2 x 10(7) plaque-forming units of adenoviral vector were required for functional transgenic expression. Endobronchial gene transfer to lung grafts may be a useful delivery route even at low doses.