Assessment of different transfection parameters in efficiency optimization.

Achieving optimal transfection efficiency is the most critical step in overcoming the primary obstacle to success in nonviral-mediated gene therapy. Several transfection parameters were being examined including the effects of different types of transfection media, glucose concentration, reporter DNA concentration, and incubation time in lipotransfectant. Efficiency of transfection obtained was highest for Opti-MEM I (29 +/- 2.28%; p = 0.001) followed by M199 (24 +/- 1.54%; p = 0.009), both of which performed significantly better than DMEM (14 +/- 0.28%) as a transfection medium. The rate of transfection was affected by glucose levels in only DMEM with higher efficiency achieved using low glucose containing DMEM (17 +/- 0.38%) than its counterpart. Furthermore, transfection rate and cell viability were severely hampered by lengthened exposure to transfection complexes, leading to an overall mean efficiency of 5 +/- 0.87%. However, doubling the DNA content in the transfection mixture did not significantly change the mean rate of transfection in M199 medium (24 +/- 1.54% to 27 +/- 1.54%; p = 0.273). The overall range of mean efficiency acquired with our protocol under different transfection conditions was between 14% and 29%. Hopefully results from this study will further potential success in nonviral-mediated gene transfer.

Assessment of Different Transfection Parameters in Efficiency Optimization.

Achieving optimal transfection efficiency is the most critical step in overcoming the primary obstacle to success in nonviral-mediated gene therapy. Several transfection parameters were being examined including the effects of different types of transfection media, glucose concentration, reporter DNA concentration, and incubation time in lipotransfectant. Efficiency of transfection obtained was highest for Opti-MEM I (29 ± 2.28%; p = 0.001) followed by M199 (24 ± 1.54%; p = 0.009), both of which performed significantly better than DMEM (14 ± 0.28%) as a transfection medium. The rate of transfection was affected by glucose levels in only DMEM with higher efficiency achieved using low glucose containing DMEM (17 ± 0.38%) than its counterpart. Furthermore, transfection rate and cell viability were severely hampered by lengthened exposure to transfection complexes, leading to an overall mean efficiency of 5 ± 0.87%. However, doubling the DNA content in the transfection mixture did not significantly change the mean rate of transfection in M199 medium (24 ± 1.54% to 27 ± 1.54%; p = 0.273). The overall range of mean efficiency acquired with our protocol under different transfection conditions was between 14% and 29%. Hopefully results from this study will further potential success in nonviral-mediated gene transfer.

In vitro senescence occurring in normal human endothelial cells can be rescued by ectopic telomerase activity.

Telomerase activation is a means to delay in vitro replicative senescence in human cells via telomere maintainence; however, this enzymatic activity is virtually absent in almost all normal somatic cells. As a result, cell senesce, leading to an eventual loss of graft function. Aging allografts, either due to cell injury related to transplantation and/or the use of organs from older donors, pose a threat to the long-term survival of a graft as constitutive cells of an aging organ have a much reduced ability to thrive after transplantation. In our study, human endothelial cells were found to undergo replicative senescence in culture with an increase in the percentage of senescent cells (beta-gal staining at pH 6) and a decrease in both the fraction of S-phase cycling cells and the proliferative index measured using CFDA-SE dye. Aging endothelial cells also demonstrated slow rates of proliferation and migration compared to younger cells. Unlike control cells that were transfected with an irrelevant gene vector, telomerase-transfected endothelial cells recovered rapidly after media replacement in cultures that had been serum starved for 2 weeks. Telomerase-transfected cells also retained a high proliferative index comparable to young cells as opposed to untransfected control cells. This young phenotype provided by telomerase expression through restoration of the telomeres may help to increase the longevity of organ transplants.

Troponin T and I are not reliable markers of cardiac transplant rejection.

OBJECTIVE: Heart transplant recipients undergo a number of invasive endomyocardial biopsies to screen for rejection. Serum assays of troponin T and/or I may provide a less invasive alternative. The purpose of this study was to evaluate troponin T and I as markers of cardiac transplant rejection. METHODS: We conducted a prospective analysis comparing troponin T and I levels to biopsy results in heart transplant recipients. Plasma was assayed for troponin T and I preoperatively, on the first 3 postoperative days, and with each subsequent biopsy. RESULTS: Twenty-nine patients entered the study. A total of 173 biopsies were performed at a mean follow-up of 129+/-9 days (range: 12-564 days). There were two rejection episodes (> or = grade 3), one in each of two patients. There were no significant relationships between troponin T or I and biopsy-proven rejection (> or = grade 3; P=0.59 and 0.54, respectively). There were also no correlations between troponin T or I levels and biopsy grade (P=0.40 and 0.92, respectively). Troponin T and I levels peaked on postoperative day 1 and fell to baseline over long-term follow-up with no peak in serum markers associated with rejection episodes. Donor ischemic time was significantly correlated to troponin T on postoperative days 1-3 (r=0.58, P=0.005; r=0.61, P=0.004; and r=0.61, P=0.003, respectively). CONCLUSIONS: Troponin T and I are not useful indicators of cardiac rejection, but do correlate with donor heart ischemic injury.

Resection of renal cell carcinomas with inferior vena caval extension using deep hypothermic circulatory arrest.

Renal cell carcinoma with tumour thrombus extension into the inferior vena cava presents a difficult surgical challenge. The conventional surgical approach, which involves isolating the inferior vena cava, incising its wall and removing the thrombus, can have high incidences of perioperative mortality and embolization of the tumour thrombus compounded by severe hemorrhage. Four patients with renal cell carcinomas extending into the inferior vena cava were supported with cardiopulmonary bypass and deep hypothermic circulatory arrest during tumour excision. All of the operations were successfully performed with no mortality and minimal morbidity. The technique allowed the surgeon to operate in a bloodless field, thereby improving visibility and allowing complete tumour excision without significantly prolonging operative time. It is believed that this technique has improved the safety and technical feasibility of what had previously been a complicated and risky surgical procedure.

Extended donor ischemic times and recipient outcome after orthotopic cardiac transplantation.

BACKGROUND: The effect of extended donor ischemic times on mortality following heart transplantation is a matter of considerable debate. PATIENTS AND METHODS: A retrospective study of the 261 consecutive heart transplantations performed at the centre (University of Alberta, Edmonton, Alberta) between July 1985 and June 1999 was conducted. Patients were divided into the following two groups based on donor ischemic time: 4 h or less and longer than 4 h. Donor and recipient factors were analyzed for their effects on 30-day and 90-day survival. RESULTS: Thirty-day mortality was not significantly greater with prolonged donor ischemic times (13%) than with shorter ischemic times (7%, P=0.14). There was also no significant increase in 90-day mortality with longer ischemic times (16%) than with shorter ischemic times (10%, P=0.27). Actuarial survival (10 years) was similar between the groups (P=0.33). Predictors of 30-day and 90-day mortality were cardiopulmonary bypass time (P<0.001 and P<0.001, respectively) and lower donor weight (P=0.008 and P=0.02, respectively). CONCLUSIONS: Longer donor ischemic times were not significantly related to decreased 30-day, 90-day or 10-year actuarial survival.

Successful cardiac transplantation with methanol or carbon monoxide-poisoned donors.

BACKGROUND: Patients succumbing to methanol or carbon monoxide poisoning are usually rejected for heart donation. Increasing demand for donors has lead to the expansion of acceptance criteria and increased use of the marginal donor. METHODS: We transplanted hearts from donors who had had methanol intoxication in three cases and carbon monoxide exposure in two cases. Standard donor evaluation criteria and transplantation techniques were used. RESULTS: All of the transplants were successful. Three of the recipients required significant inotropic support for a few days postoperatively; however, all of the hearts functioned well over the intermediate and long term. Two recipients (1 from each group) died of complications other than heart failure (1.5 and 2 years postoperatively). CONCLUSIONS: Successful heart transplantation can be achieved using the hearts from patients succumbing to methanol or carbon monoxide poisoning. Routine evaluation of cardiac function and myocardial damage is adequate for screening these donors. Hearts from methanol-poisoning victims may require longer inotropic support postoperatively before complete recovery, but can provide excellent long-term function and results.

Anomalous right coronary artery originating from the left main coronary artery.

A 54-year-old man with no cardiac history presented with exertional angina. Cardiac catheterization revealed an anomalous right coronary artery originating from the left main coronary artery with external compression during its course between the aorta and the pulmonary artery. He was successfully managed with surgical reimplantation of the right coronary artery into the aorta.

One-stage repair of a massive aortic arch aneurysm.

A 61-year-old woman with a massive aortic aneurysm extending from the aortic root to the proximal descending thoracic aorta required urgent surgical intervention. She underwent successful replacement of her ascending aorta, transverse arch and descending aorta in a single operation.

Reduction of leg wound infections following coronary artery bypass surgery.

OBJECTIVE: To reduce the rate of infection at the saphenous vein harvest site after coronary artery bypass surgery, to identify predictors of infection and to determine the best method for leg wound closure. DESIGN: A randomized clinical trial was undertaken to determine the best technique for reducing the postoperative leg wound infection rate. Patients were allocated to one of four leg wound closure methods: staples, close immediately; staples, close after protamine administration; subcuticular sutures, close immediately; and subcuticular sutures, close after protamine. Risk factors evaluated were age, sex, diabetes, obesity, peripheral vascular disease, reoperation, time in surgery, wound length, wound depth, time that the wound was open, wound quality and harvest site. SETTING: The Walter C Mackenzie Health Sciences Centre, University of Alberta, Edmonton, Alberta. PATIENTS: All consenting patients undergoing elective coronary artery bypass surgery involving saphenous vein harvesting were considered for the study. Exclusion criteria were insertion of a drain, insertion of an intra-aortic balloon pump in the index limb and inability to complete follow-up at the authors' centre. Eighty patients were initially enrolled, with 77 completing the study. INTERVENTIONS: Patients underwent standard saphenous vein harvesting followed by wound closure as indicated by the study group. MAIN RESULTS: The major infection rate was reduced from 13% to 3% (P = 0.02). Each closure method was equally effective, and wound depth was the only factor related to infection. CONCLUSIONS: Leg wound infections continue to be a major source of morbidity after coronary bypass surgery.

Biatrial myxoma: a rare cardiac tumor.

A previously healthy 48-year-old man presented to the hospital with a transient ischemic attack. Echocardiography revealed a large left atrial tumor with a second tumor in the right atrium. Surgical excision revealed a large left atrial myxoma with extension through the interatrial septum into the right atrium.

Coarctation of the aorta: tailoring the surgical approach.

OBJECTIVE: To employ a flexible approach for repairing coarctation of the aorta in an attempt to minimize residual coarctation and avoid the use of synthetic material. DESIGN: Retrospective study of consecutive children undergoing surgical repair of coarctation of the aorta. SETTING: Walter C Mackenzie Health Sciences Centre, University of Alberta, Edmonton, Alberta. PATIENTS: Children presenting with coarctation of the aorta between June 1993 and October 1996 (n = 42), treated by one surgeon. INTERVENTIONS: Children had repair by one of three methods: subclavian flap angioplasty for discrete juxtaductal coarctation, 17 (40%); resection and end-to-end anastomosis, 13 (31%); and resection with extended transverse arch repair, 12 (29%). MAIN RESULTS: Follow-up was 22 +/- 2 months. The preoperative mean arm-leg gradient was 23 +/- 3 mmHg and postoperatively was 4 +/- 2 mmHg (P < 0.001). In late follow-up, five children developed a significant gradient (end-to-end anastomosis, one; transverse arch repair, two; subclavian flap angioplasty, two) necessitating balloon dilation, one of whom (subclavian flap angioplasty) eventually required end-to-end repair. Another child, who had a subclavian flap angioplasty, underwent transverse arch repair at the time of complete cardiac repair. There was one perioperative death in a child who was in extremis preoperatively and three late deaths in children with additional complex intracardiac anomalies. CONCLUSIONS: A flexible surgical approach with avoidance of synthetic material and low threshold for extended repair has yielded good early and intermediate term results.

Acute aortic dissection and ventricular septal defect repair.

A 37-year-old male with long-standing heart murmur and ventricular septal defect developed acute chest pain and was diagnosed with an aortic dissection and aortic insufficiency. The ventricular septal defect and aortic dissection were repaired successfully as a combined procedure. At late follow-up (three years), he continued to enjoy an excellent result.

Papillary fibroelastoma of the tricuspid valve.

A 50-year-old woman with a long-standing history of palpitations was found by echocardiography to have a 1.5 cm mass on the atrial surface of the anterior leaflet of the tricuspid valve. Surgical excision included a portion of surrounding leaflet tissue. The tumour was a papillary fibroelastoma. She remained asymptomatic with no recurrence in follow-up.

Bilateral partial anomalous pulmonary venous return.

Two unusual cases of atrial septal defects with bilateral partial anomalous pulmonary venous return are described. Total repair was achieved in both children by direct connection of the left upper lobe vein to the left atrium and baffle redirection of the right upper lobe vein.

Recurrent aortic dissection after orthotopic heart transplantation.

A patient with Marfan's syndrome and previous Bentall repair for aortic dissection required orthotopic cardiac transplantation for end-stage cardiomyopathy. Postoperatively he suffered recurrent aortic dissection involving the transverse and descending aorta leading to tracheal and esophageal compression. He underwent successful surgical replacement of his ascending aorta, transverse arch, and descending aorta.

Perimembranous ventricular septal defect repair: keeping it simple.

OBJECTIVE: A simplified method of repairing perimembranous ventricular septal defects (VSDs) was employed to reduce the incidence of complete heart block and significant residual defects. PATIENTS AND INTERVENTIONS: Twenty-three children (mean age 1.2 +/- 0.3 years, range 0.1 to 5.8; mean weight 8.2 +/- 0.9 kg, range 3.7 to 20), one adolescent (16 years old) and one adult (30 years old) with perimembranous VSDs were operated on using the simplified method to correct the defect. The repairs were done from a right atrial approach by sewing the patch directly to the rim of the defect, by using very small bites in the area of the conduction tissue. In 36% of cases, the tricuspid valve was temporarily detached close to the annulus to improve operative exposure. Concomitant procedures were required in 91% of children. MAIN RESULTS: Early postoperative echocardiography showed a tiny residual ventricular septal defect in 28% (seven of 25) of patients, none requiring reoperation. There were no perioperative deaths. At follow-up there were no significant residual VSDs and no patient required reoperation. Postoperative electrocardiography revealed no evidence of heart block, but 64% showed a right bundle branch block pattern. There were no problems with tricuspid valve function postoperatively. At late follow-up (22 +/- 2 months) there were no significant problems related to the VSD repair. CONCLUSIONS: This simplified method of VSD repair produces excellent results avoiding significant leaks and the need for reoperation. This method has shown freedom from complete heart block and the need for pacemaker implantation. Temporary detachment of the tricuspid valve improves visualization in many children and is a safe practice.

Modified Blalock-Taussig shunts: to heparinize or not to heparinize?

OBJECTIVE: To demonstrate shunt function of modified Blalock-Taussig Shunts (MBTSs) when no heparin is administered postoperatively. PATIENTS AND INTERVENTIONS: Twenty-four MBTSs were performed on 23 children with various congenital heart problems needing palliation before the primary repair could be completed. A single low dose of heparin was given intraoperatively and no heparin was given postoperatively. Patients were evaluated for shunt patency, bleeding problems and the incidence of significant seromas at the graft site. MAIN RESULTS: There was one perioperative death and no incidence of early shunt failure. Two children died from causes unrelated to shunt function. There were no postoperative bleeding difficulties (no reoperations), and no seromas surrounding the grafts. Late follow-up (mean 18 +/- 1 months) revealed no shunt failure. CONCLUSIONS: Avoidance of heparin after an MBTS procedure is a safe practice and may reduce bleeding problems and the incidence of significant seromas surrounding the graft. Postoperative shunt thrombosis is more likely related to intraoperative technical difficulty or extremely small pulmonary artery size.

Splenorenal arterial bypass in a child with Takayasu's disease: a case report.

In adults splenorenal arterial bypass is a highly effective treatment for renovascular hypertension, but in children the procedure has been less successful because of the small size of the splenic artery. However, with the improvement in microvascular techniques the procedure is now possible in children. A 2-year-old child with Takayasu's arteritis, previous right nephrectomy and severe renovascular hypertension required revascularization to salvage his remaining left kidney. A splenorenal arterial bypass was performed through a left retroperitoneal flank incision. Postoperatively his creatinine level returned to normal and his requirements for antihypertensive medication were markedly diminished. The advantages of a splenorenal arterial bypass for left renal revascularization in a developing child are discussed.

Traumatic rupture of the thoracic aorta during the second trimester of pregnancy.

A 36-year-old woman in the second trimester of pregnancy underwent emergent operative repair of a traumatic aortic disruption caused by a motor vehicle accident. Left atrial-to-femoral artery bypass was used to maintain fetal circulation during the cross-clamp period. Her healthy, full-term child was subsequently delivered 3 months later by normal vaginal delivery.