[Methods for Recovering Residual Anticancer Drugs in Medical Settings to Improve Working Environments].

We examined the methods for recovering residual anticancer drugs in medical settings to prevent health hazards caused by exposure to anticancer drugs. Presently, the lactose hydrate recovery rates(Lac, an alternative sample for an anticancer drug)were determined using 2 drug recovery methods that are based on a procedure manual(procedure manual method) and smart remote support(remote support method). Using the procedure manual method, 5 healthcare workers recovered Lac after receiving a detailed face-to-face methodological explanation. Using the remote support method, 3 healthcare workers recovered Lac regarded by an instructor waiting at a remote site without using a procedure manual. As a result, the Lac recovery rates were>80% for both methods; however, they showed the need for improvement. Eventually, the issues found presently will be resolved to improve the working environments of healthcare workers, caregivers, and medical service providers.

Intra-cisterna magna delivery of an AAV vector with the GLUT1 promoter in a pig recapitulates the physiological expression of SLC2A1.

Glucose transporter 1 deficiency syndrome (GLUT1DS) is caused by haplo-insufficiency of SLC2A1, which encodes GLUT1, resulting in impaired hexose transport into the brain. Previously, we generated a tyrosine-mutant AAV9/3 vector in which SLC2A1 was expressed under the control of the endogenous GLUT1 promoter (AAV-GLUT1), and confirmed the improved motor function and cerebrospinal fluid glucose levels of Glut1-deficient mice after cerebroventricular injection of AAV-GLUT1. In preparation for clinical application, we examined the expression of transgenes after intra-cisterna magna injection of AAV-GFP (tyrosine-mutant AAV9/3-GFP with the CMV promoter) and AAV-GLUT1. We injected AAV-GFP or AAV-GLUT1 (1.63 × 1012 vector genomes/kg) into the cisterna magna of pigs to compare differential promoter activity. After AAV-GFP injection, exogenous GFP was expressed in broad areas of the brain and peripheral organs. After AAV-GLUT1 injection, exogenous GLUT1 was expressed predominantly in the brain. At the cellular level, exogenous GLUT1 was mainly expressed in the endothelium, followed by glia and neurons, which was contrasted with the neuronal-predominant expression of GFP by the CMV promotor. We consider intra-cisterna magna injection of AAV-GLUT1 to be a feasible approach for gene therapy of GLUT1DS.

Intestinal mucosa staple line integrity and anastomotic leak pressure after healing in a porcine model.

PURPOSE: The aim of this study was to evaluate both the intestinal mucosa staple line integrity and anastomotic leak pressure after healing in a porcine survival model. METHODS: We used two suture models using two different size staples (incomplete mucosal closure model: group G [staple height 0.75 mm], complete mucosal closure model: group B [staple height 1.5 mm]) in the porcine ileum. Five staple lines were created in each group made in the ileum for each model, and the staple sites harvested on days 0, 2, and 7. The leak pressure at the staple site was measured at each time point. RESULTS: On day 0, the leak pressure for group G (79.5 mmHg) was significantly lower than that for group B (182.3 mmHg) (p < 0.01). On days 2 and 7, there was no significant difference between groups G and B (171 mmHg and 175.5 mmHg on day 2, 175.5 mmHg and 175.5 mmHg on day 7, p > 0.05). The histological findings in both groups showed similar healing at postoperative days 2 and 7. CONCLUSION: The integrity of the mucosal staple lines was associated with the postoperative leak pressure on day 0. However, there was no association with the leak pressure at two days or more postoperatively in a porcine model.

Utility of microminipigs for evaluating liver-mediated gene expression in the presence of neutralizing antibody against vector capsid.

Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.

Dorsal approach plus branch patch technique is the preferred method for liver transplanting small babies with monosegmental grafts.

PURPOSE: When living donor liver transplantation (LDLT) is performed on small infant patients, the incidence of hepatic artery complications (HACs) is high. Here, we present a retrospective analysis that focuses on our surgical procedure for hepatic arterial reconstruction and the outcomes of monosegmental LDLT. METHODS: Of the 275 patients who underwent LDLT between May 2001 and December 2015, 13 patients (4.7 %) underwent monosegmental LDLT. Hepatic artery reconstruction was performed under a microscope. The size discrepancy between the graft and the recipient's abdominal cavity was defined as the graft to recipient distance ratio (GRDR) between the left hepatic vein and the portal vein (PV) bifurcation on a preoperative computed tomography scan. HACs were defined as hepatic arterial hypoperfusion. RESULTS: Recipient hepatic arteries were selected for the branch patch technique in five cases (38.5 %), and the diameter was 2.2 ± 0.6 mm. The anastomotic approaches selected were the dorsal position of the PV in seven cases (53.8 %) and the ventral position in six, and the GRDRs were 2.8 ± 0.4 and 1.9 ± 0.5, respectively (p = 0.012). The incidence rate of HACs caused by external factors, such as compression or inflammation around the anastomotic site, was significantly higher in monosegmental than in non-monosegmental graft recipients (15.4 vs. 1.1 %, p < 0.001). CONCLUSION: Although monosegmental graft recipients experienced HACs caused by external factors around the anastomotic field, hepatic arterial reconstruction could be safely performed. Important components of successful hepatic arterial reconstructions include the employment of the branch patch technique and the selection of the dorsal approach.

Minimizing the inhibitory effect of neutralizing antibody for efficient gene expression in the liver with adeno-associated virus 8 vectors.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.

Laparoscopic cholecystectomy and laparoscopic common bile duct exploration for cholecystolithiasis and choledocholithiasis in a patient with situs inversus totalis: A case report.

Situs inversus complicates diagnosis and treatment due to the mirrored organ placement in relation to normal anatomy. This report describes a 78-year-old female patient with situs inversus totalis who underwent laparoscopic cholecystectomy and laparoscopic common bile duct exploration for cholecystolithiasis and choledocholithiasis. Utilizing the "French mirror technique" for port placement, the surgeon adeptly mirrored standard maneuvers with a 2-mm needle forceps in the left hand and a 5-mm forceps in the right in a reversed anatomical setting. This technique maintained familiar hand movements, despite the patient's unique anatomy. The surgeon applied transcystic ductal bile duct exploration, using choledochoscopy for duct exploration and a basket catheter for stone removal. Laparoscopic cholecystectomy and common bile duct exploration through the transcystic ductal route are viable and effective for patients with situs inversus.

Efficient gene transduction in pigs and macaques with the engineered AAV vector AAV.GT5 for hemophilia B gene therapy.

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination.

Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs.

BACKGROUND AIMS: Transplantation of synovial mesenchymal stromal cells (MSCs) may induce repair of cartilage defects. We transplanted synovial MSCs into cartilage defects using a simple method and investigated its usefulness and repair process in a pig model. METHODS: The chondrogenic potential of the porcine MSCs was compared in vitro. Cartilage defects were created in both knees of seven pigs, and divided into MSCs treated and non-treated control knees. Synovial MSCs were injected into the defect, and the knee was kept immobilized for 10 min before wound closure. To visualize the actual delivery and adhesion of the cells, fluorescence-labeled synovial MSCs from transgenic green fluorescent protein (GFP) pig were injected into the defect in a subgroup of two pigs. In these two animals, the wounds were closed before MSCs were injected and observed for 10 min under arthroscopic control. The defects were analyzed sequentially arthroscopically, histologically and by magnetic resonance imaging (MRI) for 3 months. RESULTS: Synovial MSCs had a higher chondrogenic potential in vitro than the other MSCs examined. Arthroscopic observations showed adhesion of synovial MSCs and membrane formation on the cartilage defects before cartilage repair. Quantification analyses for arthroscopy, histology and MRI revealed a better outcome in the MSC-treated knees than in the non-treated control knees. CONCLUSIONS: Leaving a synovial MSC suspension in cartilage defects for 10 min made it possible for cells to adhere in the defect in a porcine cartilage defect model. The cartilage defect was first covered with membrane, then the cartilage matrix emerged after transplantation of synovial MSCs.

Hepatocellular telomere length in biliary atresia measured by Q-FISH.

BACKGROUND: Liver transplantation for biliary atresia is indicated whenever a Kasai portoenterostomy is considered unfeasible. However, the timing of liver transplantation in biliary atresia has not been precisely defined. Excessive shortening of hepatocellular telomeres may occur in patients with biliary atresia, and therefore, telomere length could be a predictor of hepatocellular reserve capacity. METHODS: Hepatic tissues were obtained from 20 patients with biliary atresia who underwent LT and 10 age-matched autopsied individuals (mean age, 1.7 and 1.2 years, respectively). Telomere lengths were measured by Southern blotting and quantitative fluorescence in situ hybridization using the normalized telomere-centromere ratio. The correlation between the normalized telomere-centromere ratio for the hepatocytes in biliary atresia and the pediatric end-stage liver disease score was analyzed. RESULTS: The median terminal restriction fragment length of the hepatic tissues in biliary atresia was not significantly different from that of the control (p = 0.425), whereas the median normalized telomere-centromere ratio of hepatocytes in biliary atresia was significantly smaller than that of the control (p < 0.001). Regression analysis demonstrated a negative correlation of the normalized telomere-centromere ratio with the pediatric end-stage liver disease score in biliary atresia (p < 0.001). CONCLUSIONS: Telomere length analysis using quantitative fluorescence in situ hybridization could be an objective indicator of hepatocellular reserve capacity in patients with biliary atresia, and excessive telomere shortening supports the early implementation of liver transplantation.

Organ perfusion during partial REBOA in haemorrhagic shock: dynamic 4D-CT analyses in swine.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) increases proximal blood pressure while inducing distal ischemia of visceral organs. The evaluation of distal ischemia severity during REBOA is a prerequisite for safe resuscitation of haemorrhagic shock patients with REBOA. We evaluated changes in blood flow and organ perfusion due to the degree of occlusion using dynamic 4D-computed tomography (CT). We compared the results with those of a previous study on euvolemic status. Delayed enhancement of the inferior vena cava (IVC) without retrograde flow was observed in the 4D-volume rendering images in the high-degree occlusion. The time-density curve (TDC) of the liver parenchyma (liver perfusion) and superior mesenteric vein (SMV) demonstrated a decreased peak density and a delayed peak in high-degree occlusion. The change rate of the area under the TDC of the liver and SMV decreased linearly as the degree of occlusion increased (PV, Y = -1.071*X + 106.8, r2 = 0.972, P = 0.0003; liver, Y = -1.050*X + 101.8, r2 = 0.933, P = 0.0017; SMV, Y = -0.985*X + 100.3, r2 = 0.952, P = 0.0009). Dynamic 4D-CT revealed less severe IVC congestion during P-REBOA in haemorrhagic shock than in euvolemia. Analyses of TDC of the liver and SMV revealed a linear change in organ perfusion, regardless of intravascular volume.

Bicuspidalization of the Native Tricuspid Aortic Valve: A Porcine in Vivo Model of Bicuspid Aortopathy.

Objective: To examine early histologic changes in the aorta exposed to bicuspid flow. Material and Methods: A porcine bicuspid aortopathy model was developed by suturing aortic cusps. Of nine pigs, eight underwent sham surgery (n=3) or bicuspidalization (n=5); one was used as an intact control. Wall shear stress (WSS) was assessed by computational fluid dynamics (CFD). Animals were exposed to normal or bicuspid flow for 48 h and were then euthanized for histologic examinations. Results: No animal died intraoperatively. One animal subjected to bicuspidalization died of respiratory failure during postoperative imaging studies. Echocardiography showed the aortic valve area decreased from 2.52±1.15 to 1.21±0.48 cm2 after bicuspidalization, CFD revealed increased maximum WSS (10.0±5.2 vs. 54.0±25.7 Pa; P=0.036) and percentage area of increased WSS (>5 Pa) in the ascending aorta (30.3%±24.1% vs. 81.3%±13.4%; P=0.015) after bicuspidalization. Hematoxylin-eosin staining and transmission electron microscopy showed subintimal edema and detached or degenerated endothelial cells following both sham surgery and bicuspidalization, regardless of WSS distribution. Conclusion: A bicuspid aortic valve appears to increase aortic WSS. The endothelial damage observed might have been related to non-pulsatile flow (cardiopulmonary bypass). Chronic experiments are needed to clarify the relationship between hemodynamic stress and development of bicuspid aortopathy.

Endovascular interventions for hepatic artery complications immediately after pediatric liver transplantation.

Hepatic artery complications after living donor liver transplantation (LDLT) can directly affect both graft and recipient outcomes. For this reason, early diagnosis and treatment are essential. In the past, relaparotomy was generally employed to treat them. Following recent advances in interventional radiology, favorable outcomes have been reported with endovascular treatment. However, there is ongoing discussion regarding the best and safe time for definitive endovascular interventions. We herein report a retrospective analysis for six children with early hepatic artery complication after pediatric LDLT who underwent endovascular treatment as primary therapy at our institution. We evaluate the usefulness of endovascular treatment for hepatic artery complication and its optimal timing. The mean patient age was 11.9 months and mean body weight at LDLT was 6.7 kg. The mean duration between the transplantation and first endovascular treatment was 5.3 days. Five of the six patients were technically successful treated by only endovascular treatment. Of these five patients, two developed biliary complications. Endovascular procedures were performed 10 times in six patients without any complications and nine of the 10 procedures were successful. By selecting optimal devices, our findings suggest that endovascular treatment can be feasible and safe in the earliest time period after pediatric LDLT.

Double-balloon enteroscopy for bilioenteric anastomotic stricture after pediatric living donor liver transplantation.

Bilioenteric anastomotic stricture after liver transplantation is still frequent and early detection and treatment is important. We established the management using double-balloon enteroscopy (DBE) and evaluated the intractability for bilioenteric anastomotic stricture after pediatric living donor liver transplantation (LDLT). We underwent DBE at Jichi Medical University from May 2003 to July 2009 for 25 patients who developed bilioenteric anastomotic stricture after pediatric LDLT. The patients were divided into two types according to the degree of dilatation of the anastomotic sites before and after interventional radiology (IVR) using DBE. Type I is an anastomotic site macroscopically dilated to five times or more, and Type II is an anastomotic site dilated to less than five times. The rate of DBE reaching the bilioenteric anastomotic sites was 68.0% (17/25), and the success rate of IVR was 88.2% (15/17). There were three cases of Type I and 12 cases of Type II. Type II had a significantly longer cold ischemic time and higher recurrence rate than Type I (P = 0.005 and P = 0.006). In conclusion, DBE is a less invasive and safe treatment method that is capable of reaching the bilioenteric anastomotic site after pediatric LDLT and enables IVR to be performed on strictures, and its treatment outcomes are improving. Type II and long cold ischemic time are risk factors for intractable bilioenteric anastomotic stricture.

Pediatric living donor liver transplantation for biliary atresia with hepatopulmonary syndrome: the gift of a second wind.

PURPOSE: Hepatopulmonary syndrome (HPS) is a progressive, deteriorating complication of end-stage liver disease (ESLD) that occurs in 13-47% of liver transplant candidates. Although LT is the only therapeutic option for HPS, it has a high morbidity and mortality, especially in patients with severe hypoxemia before transplantation, but the course of HPS after living donor liver transplantation (LDLT), especially for biliary atresia (BA) patients is not well established. PATIENTS AND METHODS: The present study evaluated 122 patients who received an LDLT for BA and of these, 3 patients had HPS at the time of LDLT in a single-center series. RESULTS: Two patients of the HPS patients them had biliary and/or vascular complications, but they recovered uneventfully with interventional treatment. None of the patients required supplemental oxygen and had no residual cardiopulmonary abnormalities at a follow-up of more than 24 months. CONCLUSION: Although a series of three patients is too small for definitive conclusion and further investigations must be conducted, pediatric LDLT can be a favorable therapeutic option for HPS.

Bowel perforation after pediatric living donor liver transplantation.

PURPOSE: Bowel perforation after liver transplantation (LT) is a rare, but highly lethal complication with a poor prognosis. Here, we report the outcome of cases of bowel perforation after pediatric LT in our department. PATIENTS AND METHODS: The study subjects were 148 patients who underwent pediatric living donor liver transplantation. The 114 with biliary atresia (BA) were divided into two groups: those with associated bowel perforation (Group A) and those without (Group B). RESULTS: Four patients in all (2.5%) suffered bowel perforation. Their original disease was BA and emergency surgery was performed in all cases, with a mortality rate of 50.0%. Comparison of Groups A and B revealed significant differences in the patient age, body weight, duration of surgery, cold ischemic time, and blood loss volume. The survival rates in Groups A and B were 50.0 and 99.1%, respectively (p < 0.01). Duration of surgery was an independent risk factor (p = 0.05). CONCLUSION: Bowel perforation after LT is a potentially fatal complication. LT is a procedure that requires care and precision, and the possibility of bowel perforation should always be borne in mind during post-operative management, when the duration of surgery has been long.

[Draft of guidelines for human body dissection for clinical anatomy education and research and commentary].

This article analyses the Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research drawn by the Study Group for Future Training Systems of Surgical Skills and Procedures established by the Fiscal Year 2010 research program of the Ministry of Health, Labor and Welfare. The purpose of the Draft of Guidelines is: First, to lay out the required basic guidelines for human cadaver usage to allow medical and dental faculty to conduct clinical education and research in accordance with existing regulations. Second, the guidelines are expected to give physicians a regulatory framework to carry out cadaver training in accordance with the current legal framework. This article explains the Draft of Guidelines in detail, outlines the future of cadaver training, and describes issues which must still be solved.

[Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research and commentary].

This article analyses the Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research drawn by the Study Group for Future Training Systems of Surgical Skills and Procedures established by the Fiscal Year 2010 research program of the Ministry of Health, Labor and Welfare. The purpose of the Draft of Guidelines is: First, to lay out the required basic guidelines for human cadaver usage to allow medical and dental faculty to conduct clinical education and research in accordance with existing regulations. Second, the guidelines are expected to give physicians a regulatory framework to carry out cadaver training in accordance with the current legal framework. This article explains the Draft of Guidelines in detail, outlines the future of cadaver training, and describes issues which must still be solved.

Distal pressure monitoring and titration with percent balloon volume: feasible management of partial resuscitative endovascular balloon occlusion of the aorta (P-REBOA).

INTRODUCTION: Resuscitative endovascular balloon occlusion of the aorta (REBOA) increases proximal arterial pressure, but may also induce life-threatening distal ischemia. Partial REBOA (P-REBOA) is thought to mitigate distal ischemia during aortic occlusion. However, feasible indicators of the degree of P-REBOA remain inconsistent. We hypothesised percent balloon volume could be a substitute for pressure measurements of gradients during P- REBOA. This study aimed to compare balloon volume and arterial pressure gradient, and analysed with intra-balloon pressure and balloon shape. METHODS: Proximal (carotid) and distal (femoral) arterial pressures were recorded and a 7-Fr REBOA catheter was placed in four swine. Total REBOA was defined as a cessation of distal pulse pressure and maximum balloon volume was documented. The balloon volume was titrated by 20% increments of maximum capacity to adjust the degree of P-REBOA. The distal/proximal arterial pressure gradient and the intra-balloon pressures were also recorded. The changes in shape and the cross-sectional area of the balloon were evaluated with computed tomography (CT) images. RESULTS: The proximal mean arterial pressure (MAP) plateaued after 60% balloon volume; meanwhile, distal pulse pressure was still left. The balloon pressure was traced with proximal MAP before contact with aortic wall. The balloon shape changed unevenly from "cone" to "spindle" shape, although the balloon cross-sectional area of the mid-segment linearly increased. CONCLUSION: Monitoring distal pressure and titrating percent balloon volume is feasible to manage P-REBOA. In this experiment, 60% balloon volume was enough inflation to elevate central pressure allowing distal perfusion. The intra-balloon pressure was not reliable due to the strong influence of proximal MAP and uneven change of the balloon shape.