Yusuke Kita

Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene.

BACKGROUND CTLA4Ig, a soluble recombinant fusion protein that contains the extracellular domain of the CTLA4 and Fc portion of IgG1, strongly adheres to the B7 molecule to block CD28-mediated costimulatory signals and inhibits in vitro and in vivo immune responses. In vivo gene transfer using adenovirus vector achieves a high transfection rate into organ cells that usually contain adenoviral receptors. In this study, we investigated expression levels of the transfected gene and the survival times of the allografts in cardiac recipients systemically administered adenoviral vectors containing CTLA4Ig. METHODS Hearts from DA rats (RT-1a) were transplanted into a cervical location in LEW recipients (RT1(1)). The adenoviral vectors containing CTLA4Ig was injected via a recipient vein immediately after grafting. RESULTS The serum level of CTLA4Ig reached to maximum at 51-93 microg/ml 3 to 7 days after gene-transfection and declined after 14 days, although detectable levels were observed up to 49 days. The median survival time of the allografts in the gene-transfected group were significantly prolonged (27 days) in compared to the control group (6 days). In addition, down-regulation of IL-2 and IFN-gamma mRNAs and persistence of IL-4 and IL-10 transcripts were observed in the graft infiltrating cells. CONCLUSION The adenovirous-mediated CTLA4Ig gene transfer into a recipient liver by systemic administration resulted in remarkable prolongation of cardiac allograft survival. Its action mechanisms may be mediated by inhibition of CD28-associated signal transduction, reduction of Th1-type cytokine production, and continuous expression of Th2-type cytokines in the activating lymphocytes.

Fas-mediated apoptosis is involved in the elimination of gene-transduced hepatocytes with E1/E3-deleted adenoviral vectors.

Gene‐transduced hepatocytes with E1/E3‐deleted adenoviral vectors are eliminated immediately and the expression of transduced genes disappears rapidly following the vector administration. In this report, we analysed the involvement of apoptotic cell death in the elimination of hepatocytes infected with adenoviral vectors. An E1/E3‐deleted adenoviral vector expressing Escherichia coli β‐galactosidase (LacZ) was injected via the portal vein into congenitally Fas‐deficient mice (lpr), Fas ligand‐deficient mice (gld) and their control mice, MRL and C3H. 5‐Bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactoside (X‐gal) staining of the liver specimens showed that 80–100% of hepatocytes were LacZ positive at 7 days after virus administration, suggesting that most of the hepatocytes received the injected adenoviral vectors. In normal mice, the number of LacZ‐positive cells decreased dramatically at 14 and 21 days after transduction and few positive cells were observed at day 28. β‐Galactosidase activity, quantified by the O‐nitrophenyl‐beta‐D‐galactopyranoside assay, gave comparable results to X‐gal staining. At days 14 or 21, many apoptotic hepatocytes and apoptotic infiltrating cells were detected with the terminal deoxyribonucleotidyl transferase‐mediated dUTP‐digoxigenin nick end‐labelling (TUNEL) in situ apoptosis detection method. This observation suggested that the apoptotic process was associated with the elimination of adenovirus‐infected hepatocytes. To test the involvement of the Fas—Fas ligand interaction in this apoptotic process, the period of transgene expression was measured in 1pr and gld mice, which had received the same amount of AxCALacZ. X‐Gal histochemical analysis detected many LacZ‐positive cells in 1pr or gld mice liver even at 21 or 28 days after AxCALacZ injection. There were significant differences in the reduction rates of β‐galactosidase activity of liver homogenates between lpr and MRL, or gld and C3H mice. Based on these observations, we conclude that the Fas‐mediated apoptotic process is involved in the elimination of hepatocytes infected with E1/E3‐deleted adenoviral vectors.

Long-term Graft Acceptance in Rat Heart Transplantation by CTLA4Ig Gene Transfection Combined with FTY720 Treatment

CTLA4Ig strongly adheres to B7 molecules on antigen-presenting cells to block intracellular signal transduction via CD28 on helper T cells, which eventually inhibits immune responses. We have demonstrated that the administration to recipient animals of adenoviral vectors containing CTLA4Ig gene (adCTLA4Ig) prolonged graft survival, although the gene expression diminished in a time-dependent manner and the grafts were finally rejected. In addition, recipient animals treated with FTY720, a new immunosuppressant, exhibited a decrease in the number of peripheral lymphocytes due to apoptosis. In this study, we performed adCTLA4Ig transfection combined with FTY720 treatment in heart-grafted rats to determine if the combination could induce a mutual effect on graft survival. The recipient animals were given injections of 1 × 109 plaque-forming units of adCTLA4Ig via the tail vein immediately after grafting. On the day before transplantation we administered FTY720 orally to some of these animals at a dosage of 5 mg/kg and again on the day of transplantation. The median graft survival period in the adCTLA4Ig-only group was 27 days, whereas that in the combination group was markedly prolonged to 56 days. Of 15 grafts, 5 survived indefinitely. In these groups we observed detectable levels of CTLA4Ig in the sera 49 days after grafting; the levels were always higher in the combination group than in the adCTLA4Ig-only group. As a result, this study revealed that FTY720 and adCTLA4Ig have a potent mutual effect on graft survival during rat heart transplantation. Furthermore, it is highly possible that FTY720 enhances gene expression of adCTLA4Ig, which may be related to the long-term acceptance of grafts.

CTLA4Ig-gene transfection inhibits obliterative airway disease in rats.

BACKGROUND Obliterative airway disease (OAD) is a major cause of long-term morbidity following lung transplantation. Its pathologic characteristics are small-airway inflammation and occlusion by fibrous tissue. However, the pathogenesis is uncertain and therapy is ineffective. This study presents the effects of CTLA4Ig-gene therapy on OAD in heterotopically transplanted rat tracheal allografts. METHODS Dark Agouti (DA, RT1a) allografts and Lewis (LEW, RT1l) isografts were transplanted into Lewis recipients. The tracheal graft was transplanted heterotopically into the subcutaneous pocket into the back. Adenoviral vectors (1.0x10(9) pfu) containing the CTLA4Ig-gene (AdCTLA4Ig) or the LacZ-gene (AdLacZ) were injected into the tail vein immediately after grafting. Grafts were harvested and examined after more than 35 days for mononuclear cell infiltration development and lumen occlusion with fibrosis. RESULTS Fully allogenic DA tracheas, treated with AdCTLA4Ig had significantly lower pathologic scores and infiltrating scores than the control allografts. The pathologic findings of the grafts, treated with AdCTLA4Ig, were very similar to those of the syngeneic grafts. The animals experienced no adverse events during follow-up. No evidence of vector-mediated tissue damage was seen in any graft. CONCLUSIONS Adenoviral vectors containing the CTLA4Ig-gene markedly inhibited the obliteration of the airway lumen. OAD may be associated with T-cell responses against graft tissue and alloimmune injury.

Spontaneous parathyroid adenoma hemorrhage.

We report a case of spontaneous parathyroid adenoma hemorrhage. A 50-year-old man with a sore throat, and swelling and ecchymosis of the entire anterior neck was found in cervical and chest computed tomography revealed to have a low-density area extending from the parapharyngeal region to below the carina, Suspecting descending necrotizing mediastinitis secondary to a peritonsillar abscess, we conducted mediastinal and cervical drainage, but found no abscess. No evidence was found, either, in bacteriological culture of sputum and pleural effusion. After the hematoma disappeared, cervical ultrasonography indicated parathyroid adenoma. Serum calcium was marginally increased, indicating that serum calcium should be determined if cervical or mediastinal hematoma develops without an obvious cause.

Resected early lung cancer with pulmonary aspergilloma

Pulmonary aspergillosis and lung cancer rarely occur simultaneously. We report a 66-year-old man with a round shadow in the thin-wall cavity of the right upper lobe. Radiological findings and transbronchial biopsy revealed squamous cell carcinoma complicated by aspergilloma at the site. Right upper lobectomy suggested that early lung cancer arose from preexisting lung scars containing an aspergilloma.

Intraoperative computed tomography after tumor marking with metal clips for non-palpable lung tumors

Background Locating small, non-palpable lung tumors during video-assisted thoracoscopic surgery (VATS) is difficult. In this paper, we report a simple method to identify such tumors during VATS, using intraoperative computed tomography (IO-CT). Methods From 2015 to 2017, we performed IO-CT scans for patients who preoperatively seemed to have non-palpable lung tumors. We initially tried to locate these tumors by finger palpation through the thoracoscopic ports. IO-CT scans were performed after marking tumors with metal clips. However, difficult-to-palpate tumors were marked by initially locating the intercostal muscle from preoperative CT. Metal clips were applied just under the intercostal muscle, and IO-CT scans were performed. After locating the tumor in relationship to the marking clips, patients would undergo wedge resections during VATS, using surgical staplers. Results We used this procedure on 21 tumors in 18 patients, including 9 non-palpable tumors and 12 palpable tumors (mean tumor size: 7.3 mm; mean distance from pleura: 6.8 mm). All tumors were identified intraoperatively, and all patients successfully underwent wedge resections during VATS, with no intra-postoperative complications. Conclusion IO-CT scans after tumor marking with metal clips during VATS can accurately locate non-palpable small sized lung tumors. IO-CT scans should be indicated for tumors that are preoperatively considered to be non-palpable.

Intrathoracic Suture Abscess After Lobectomy for Early Lung Cancer

Intrathoracic suture abscess may occur around sutures on the pleura or in the lung parenchyma, although it is rare to encounter such cases clinically. We report on a 68-year-old woman with an intrathoracic (extrapulmonary) suture abscess, which was discovered on a chest X-ray film one year after right-middle lobectomy for early lung cancer. The abscess was removed surgically, and the postoperative course was uneventful. Pathological examination showed that it was caused by braided polyester sutures.