Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model.

INTRODUCTION: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. METHODS: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. RESULTS: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. CONCLUSIONS: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Use of preoperative haemostasis and ABO blood typing tests in children: a retrospective observational study using a nationwide claims database in Japan.

OBJECTIVES: To describe the prevalence and factors associated with preoperative haemostasis and ABO blood typing tests for children because these tests might represent low-value care. DESIGN: A retrospective observational study. SETTING: Nationwide insurance claims database in Japan. PARTICIPANTS: Patients aged 1-17 years who underwent common non-cardiac surgeries between April 2012 and March 2018 were included. Patients with high-risk comorbidities for bleeding (n=175) and those with multiple eligible surgeries were excluded (n=2121). MAIN OUTCOME MEASURES: We described the proportions of each preoperative test performed within 60 days before an index surgery, including platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT) and ABO blood typing tests. We also explored the associations between patient-level and institutional-level factors and any preoperative tests, using multilevel logistic regression analysis. RESULTS: We included 13 018 patients (median (IQR) age, 5.2 (2.9-7.7) years; 8276 (63.6%) boys) from 1499 institutions. The overall proportion of each test was as follows: platelet count, 78.6%; PT, 54.4%; aPTT, 56.4% and ABO blood typing tests, 50.4%. The proportion of patients undergoing any preoperative tests in the overall sample was 79.3%. Multilevel logistic regression analysis indicated that preoperative tests were associated with type of anaesthesia (general anaesthesia: adjusted OR 7.06; 95% CI 4.94 to 10.11), type of surgery (tonsillectomy: adjusted OR 3.45; 95% CI 2.75 to 4.33) and surgical setting (inpatient procedure: adjusted OR 5.41; 95% CI 3.83 to 7.66). There was one postoperative transfusion event (0.008%) in the entire cohort and 37 postoperative reoperation events for surgical bleeding after tonsillectomy (0.90%). CONCLUSIONS: In the largest Japanese cohort reported to date, preoperative haemostasis and ABO blood typing tests were performed in a majority of children prior to common paediatric surgeries. Preoperative tests were associated with anaesthesia, surgical type and surgical setting.

Prevalence and associated factors of preoperative functional disability in elective surgical patients over 55 years old: a prospective cohort study.

PURPOSE: In preoperative settings, patients may have functional disabilities due to the disease for which surgery is being performed or comorbidities, but the associated and predictive factors remain unknown. This study examined the prevalence of preoperative functional disability and clarified the associated factors. METHODS: Individuals aged ≥ 55 years who were scheduled to undergo surgery in a tertiary-care hospital in Japan between April 2016 and September 2016 were eligible for enrolment in the study. Patients with the diseases requiring psychiatric treatment and patients unable to complete the questionnaire without help were excluded. After obtaining informed consent, each patient was asked to complete the 12-item World Health Organization Disability Assessment Schedule-2.0, which is a standardized evaluation tool for assessing comprehensive living function. Data from these questionnaires and the patients' characteristics were evaluated. Multiple logistic regression analysis was conducted to determine independent factors associated with preoperative functional disability. RESULTS: Of 1201 recruited patients, 912 (75.9%) were included in our analysis. The prevalence of preoperative functional disability was 29.2%. Regression analysis identified six independent associated factors for preoperative functional disability: body mass index ≥ 30 kg m-2, mixed lung disease, serum albumin values, malnutrition, risk of malnutrition, and preoperative use of corticosteroids. CONCLUSIONS: In total, 29.2% of preoperative patients had functional disability. Obesity, nutritional deficiency, respiratory complications, and low serum albumin values were determined as potentially modifiable factors.

Feasibility, reliability, and validity of the Japanese version of the 12-item World Health Organization Disability Assessment Schedule-2 in preoperative patients.

PURPOSE: The avoidance of postoperative functional disability is one of the most important concerns of patients facing surgery, but methods to evaluate disability have not been definitively established. The aim of our study was to evaluate the feasibility, reliability, and validity of the Japanese version of the 12-item World Health Organization Disability Assessment Schedule-2 (WHODAS 2.0-J) in preoperative patients. METHODS: Individuals aged ≥55 years who were scheduled to undergo surgery in a tertiary-care hospital in Japan between April 2016 and September 2016 were eligible for enrolment in the study. All patients were assessed preoperatively using the WHODAS 2.0-J, the 8-Item Short Form (SF-8) questionnaire, and the Tokyo Metropolitan Institute of Gerontology Index (TMIG Index). The feasibility, reliability, and validity of WHODAS2.0-J were evaluated using response rate, Cronbach's alpha (a measure of reliability), and the correlation between the WHODAS 2.0-J and the SF-8 questionnaire and TMIG Index, respectively. RESULTS: A total of 934 patients were enrolled in the study during the study period, of whom 930 completed the WHODAS 2.0-J (response rate 99.5%) preoperatively. Reliability and validity were assessed in the 898 patients who completed all three assessment tools (WHODAS 2.0-J, SF-8 questionnaire, and TMIG Index) and for whom all demographic data were available. Cronbach's alpha was 0.92. The total score of the WHODAS 2.0-J showed a mild or moderate correlation with the SF-8 questionnaire and TMIG Index (r = -0.63 to -0.34). CONCLUSION: The WHODAS 2.0-J is a feasible, reliable, and valid instrument for evaluating preoperative functional disability in surgical patients.

Functional characterization of tissue factor in von Willebrand factor-dependent thrombus formation under whole blood flow conditions.

Von Willebrand factor (VWF) plays an important role in mediating platelet adhesion and aggregation under high shear rate conditions. Such platelet aggregates are strengthened by fibrin-network formation triggered by tissue factor (TF). However, little is known about the role of TF in VWF-dependent thrombus formation under blood flow conditions. We evaluated TF in thrombus formation on immobilized VWF under whole blood flow conditions in an in vitro perfusion chamber system. Surface-immobilized TF amplified intra-thrombus fibrin generation significantly under both low and high shear flow conditions, while TF in sample blood showed no appreciable effects. Furthermore, immobilized TF enhanced VWF-dependent platelet adhesion and aggregation significantly under high shear rates. Neutrophil cathepsin G and elastase increased significantly intra-thrombus fibrin deposition on immobilized VWF-TF complex, suggesting the involvement of leukocyte inflammatory responses in VWF/TF-dependent mural thrombogenesis under these flow conditions. These results reveal a functional link between VWF and TF under whole blood flow conditions, in which surface-immobilized TF and VWF mutually contribute to mural thrombus formation, which is essential for normal hemostasis. By contrast, TF circulating in blood may be involved in systemic hypercoagulability, as seen in sepsis caused by severe microbial infection, in which neutrophil inflammatory responses may be active.

Coagulation potential of immobilised factor VIII in flow-dependent fibrin generation on platelet surfaces.

Coagulation factor VIII (FVIII) plays an essential role in haemostasis. To date, physiologic activity of FVIII circulating in the bloodstream (S-FVIII) is evaluated by classic coagulation assays. However, the functional relevance of FVIII (-von Willebrand factor complex) immobilised on thrombogenic surfaces (I-FVIII) remains unclear. We used an in vitro perfusion chamber system to evaluate the function of I-FVIII in the process of mural thrombus formation under whole blood flow conditions. In perfusion of either control or synthetic haemophilic blood, the intra-thrombus fibrin generation on platelet surfaces significantly increased as a function of I-FVIII, independent of S-FVIII, under high shear rate conditions. This I-FVIII effect was unvarying regardless of anti-FVIII inhibitor levels in synthetic haemophilic blood. Thus, our results illustrate coagulation potentials of immobilised clotting factors, distinct from those in the bloodstream, under physiologic flow conditions and may give a clue for novel therapeutic approaches for haemophilic patients with anti-FVIII inhibitors.

[Comparison of thoracoscopic and open repair of esophageal atresia with tracheoesophageal fistula].

BACKGROUND: With the increasing use of endoscopic surgery in children, several papers report the comparison between the thoracoscopic and open repair of the neonatal esophageal atresia with tracheoesophageal fistula (EA/TEF). Most of them focus on the duration and outcome of the surgery with few focusing on the neonatal tolerance to the thoracoscopic procedure and intraoperative anesthetic management. METHODS: We retrospectively reviewed the repair surgery of EA/TEF performed during 2001 and 2006 in our institution and compared thoracoscopic repair (thoracoscopy group, n=5) with open repair (open group, n=7). The right main bronchus was blocked with Fogarty catheter in thoracoscopic repair, but not in open repair. Thoracoscopic repair was performed with insufflation of carbon dioxide (3-5 mmHg). RESULTS: The thoracoscopy group had a higher incidence of intraoperative hypercapnia and acidosis and required higher inspired oxygen fraction. On admission to ICU Pa(CO2) was in the normal range in both groups and there was no difference in the duration of mechanical ventilation and ICU stay. CONCLUSIONS: Hypercapnia and acidosis were severer in thoracoscopy group. Careful perioperative adjustment of inspired oxygen fraction and ventilator setting is required.

[Epidural blood patch for postdural puncture headache in a five-year-old child].

A case was presented of a 5-year-old girl who suffered an accidental dural puncture during placement of an epidural catheter under general anesthesia for orthopedic surgery. She complained of headache 4 days after the operation, which was relieved on supine position but became worse on sitting position. Her symptoms failed to respond to conservative management. An epidural blood patch was performed under general anesthesia and completely resolved her symptoms. The reported incidence of epidural blood patch for post dural puncture headache following accidental dural puncture in children is low. We outline this case and the consideration for management for epidural blood patch in pediatric patients.

[General anesthesia for a girl with Joubert syndrome].

Joubert syndrome is a rare autosomal recessive disorder, which is characterized by absence or underdevelopment of the cerebellar vermis and severe developmental delay. The other common features include ataxia, an abnormal breathing pattern, abnormal eye movements and hypotonia. We report the anesthetic management in a 13-year-old girl with Joubert syndrome, scheduled for cauterization of nasal mucosa under general anesthesia. She had episodes of tachypnea and apnea. Oral midazolam 10 mg and famotidine 20 mg were administered 30 min before surgery. Anesthesia was induced and maintained with sevoflurane and nitrous oxide in oxygen. Vecuronium 2 mg was used to facilitate tracheal intubation. Mechanical ventilation was performed with a low ventilation setting of respiratory rate 5 beats x min(-1) and peak inspiratory pressure 9 cm H2O to maintain normal end-tidal CO2. Flurbiprofen axetil 30 mg was administered intravenously for analgesia, because opioids are not recommended. After reversal of muscle relaxation by atropin 0.5 mg and neostigmine 1.5 mg, her trachea was extubated. She did not develop postoperative apnea. In this patient with Joubert syndrome, midazolam, sevoflurane, nitrous oxide and flurbiprofen axetil were used without any complications.

[Recovery from fatal ventricular fibrillation after immediate application of percutaneous cardiopulmonary support].

We present a patient who recovered from refractory ventricular fibrillation after immediate application of percutaneous cardiopulmonary support (PCPS). On the postoperative day (POD) 3 after the Y-grafting surgery for abdominal aortic aneurysm, circulation collapsed due to sudden onset of ventricular fibrillation. Because ventricular fibrillation had persisted in spite of medical treatment and defibrillation, we established PCPS and his circulation recovered. Although an emergent coronary angiography revealed no new lesions, we performed an emergent percutaneous catheter intervention to deny the possibility that ischemic changes had contributed to the arrhythmia. Soon after percutaneous transluminal coronary angioplasty, we successfully weaned him from PCPS, and extubated his trachea on the POD 5 without any neurological deficits. On the POD 8, ventricular fibrillation occurred again and defibrillation was effective at this time. We suspected cardiac ischemia, prolonged QT interval, and electrical remodeling due to hypertrophic heart as possible causes of refractory ventricular fibrillation. Therefore, we performed percutaneous transluminal coronary angioplasty, terminated famotidine administration, maintained normal electrolytes level, started administration of beta-blocker, and implanted an cardioverter defibrillator. On the POD 16, he was discharged from the ICU with no neurological deficits.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine pretreatment attenuates methamphetamine-induced dopamine toxicity.

The effects of pretreatment with MPTP (1-methyl4-phenyl-1,2,3,6-tetrahydropyridine) on the acute and long-term effects of methamphetamine on striatal dopamine were evaluated in BALB/c mice. Four subcutaneous injections of a non-toxic dose of MPTP (8 mg/kg, at 2 hr intervals) were followed three days later by a toxic regimen of methamphetamine (four injections of 4 mg/kg, at 2 hr intervals) and mice were sacrificed immediately or three days later. Control mice received saline in place of the MPTP or methamphetamine and mice were observed for acute changes in body temperature, self-injurious behaviour, and striatal dopamine metabolites, or long-term changes in striatal dopamine levels, tyrosine hydroxylase immunoreactivity and glial fibrillary acidic protein. It was observed that pretreatment with MPTP protected mice against the acute increase in body temperature caused by the methamphetamine but, at the same time, delayed the occurrence of self-injurious behaviour following the repeated injections of methamphetamine. Likewise, pretreatment with MPTP attenuated the long-term depletion of striatal dopamine induced by the methamphetamine as well as the large increase in glial fibrillary acidic protein and the reduction in tyrosine hydroxylase immunoreactivity. The MPTP-treatment itself did not alter any of these neurotoxic markers. Finally, the acute decrease in 3,4-dihydroxyphenyacetic acid levels and increased ratio of 3-methoxytyramine/dopamine observed 60 min. after a single injection of methamphetamine (4 mg/kg) were also attenuated in MPTP-treated mice. These results are discussed in the context of the hypothesis that the low-dose treatment with MPTP may modify exchange diffusion across the striatal cell membrane thereby altering the acute and long-lasting effects of methamphetamine.