Luc Delriviere

Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo

Abstract. Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non-dividing cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes –E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field.

Is there disparity between risk and incidence of cardiovascular disease after liver transplant

Background. Hypertension and hypercholesterolemia are recognized complications of liver transplantation, but whether they contribute to the development of cardiovascular disease is uncertain. We aimed first to determine the prevalence of risk factors for coronary heart disease (CHD) after liver transplantation and second to study the effect of liver transplantation on the predicted 10-year risk of developing CHD and the incidence of cardiovascular events in comparison with a matched local population. Methods. Data on blood pressure, serum lipids, weight, diabetes mellitus, smoking, and incidence of myocardial infarction (MI) and stroke were obtained retrospectively from the case notes of 181 consecutive adult liver transplant recipients (median follow-up 54 months). The Framingham coronary risk equations were used to calculate the 10-year probability of developing CHD. Results. The prevalences of hypertension and hypercholesterolemia after transplantation were 77% and 62%, respectively. The predicted 10-year risk of CHD increased from 6.9% before transplantation to 11.5% at 1 year after transplantation, whereas that of a matched local population was 7%. Compared with a matched nontransplant population, the incidence ratios for MI and stroke were 0.55 (95% confidence interval, 0.01–3.06 ) and 1.45 (95% confidence interval, 0.18–5.22), respectively. No patients died from MI or stroke. Conclusions. Liver transplant recipients have a high prevalence of risk factors for cardiovascular disease, exceeding that of the general population, and have a higher predicted risk of developing CHD. Despite this, there were no deaths from CHD or stroke during the study period.

Monitoring systemic donor lymphocyte macrochimerism to aid the diagnosis of graft-versus-host disease after liver transplantation.

Background. The diagnosis of graft-versus-host disease (GvHD) after liver transplantation can be difficult because early symptoms are often nonspecific. In this study, the presence of donor lymphocyte macrochimerism in recipient peripheral blood was examined as a diagnostic aid for GvHD after cadaveric donor liver transplantation. Methods. Between 1996 and 2002, 33 liver transplant recipients with a clinical suspicion of GvHD (skin rash, diarrhea, pyrexia, pancytopenia, or anemia, without an obvious alternative cause) were investigated for peripheral blood donor lymphocyte macrochimerism. Donor macrochimerism was determined at the time of first clinical presentation by a low-sensitivity polymerase chain reaction (PCR) to detect donor human leukocyte antigen (HLA) alleles using genomic DNA extracted from recipient peripheral blood. Where donor HLA alleles were detected, the percentage of donor T cells was quantified by two-color flow cytometric analysis using antibodies specific for mismatched donor and recipient HLA alleles. The relationship between the presence or absence of donor lymphocyte macrochimerism and final diagnoses based on clinical and histological criteria was examined. Results. Seven of the 33 patients were PCR positive for donor HLA alleles. All had macrochimerism, with donor T lymphocyte levels ranging from 4% to 50% of circulating lymphocytes. All seven patients had normal liver function tests, skin rash, and diagnosis of GvHD histologically confirmed by skin or gut biopsies. Twenty-six patients were PCR negative, and, in 23, an alternative diagnosis was eventually established. The remaining three patients made a rapid and spontaneous recovery with no further symptoms suggestive of GvHD. Conclusions. Donor lymphocyte macrochimerism was present in all patients in whom the diagnosis of GvHD was confirmed. In patients with symptoms consistent with GvHD and a negative PCR for donor HLA, an alternative diagnosis was eventually established or the patients recovered spontaneously. Detection of donor HLA alleles in recipient peripheral blood by PCR is a useful diagnostic tool for GvHD after liver transplantation.

Technical details for safer venous and biliary anastomoses for liver transplantation in the rat

Although orthotopic liver transplantation in the rat is a well‐established experimental model, no detailed illustrated description of the procedure is available in the literature. As a result, achieving success in the technique without prolonged learning time requires training in specialized centers where the step‐by‐step details essential to success in the technique can be learnt. The aim of this paper is to provide beginners with the culminative experience of 15 years of improvements made to Kamadas original model, clearly illustrating the necessary steps and fine detail of each anastomosis to help beginners avoid unnecessary complications. We hope that this complete description of orthotopic liver transplantation in the rat, which remains the most difficult rodent transplant model to master, will greatly help microsurgeons acquire this demanding procedure.

Size reduction of small bowels from adult cadaveric donors to alleviate the scarcity of pediatric size-matched organs an anatomical and feasibility study

BACKGROUND Small bowel transplantation in children weighing less than 10 kg is hindered by the lack of size-matched donors. The ability to create reduced size small bowel grafts from adult cadaveric donors suitable for use in young children has been studied. METHODS Volumetric assessment of computed tomography scans were used to evaluate abdominal cavity and small bowel volumes in children. Small bowels were retrieved from adult cadaveric donors and reduced in size. RESULTS Computed tomography studies of the abdominal cavity showed that the mean volume available for a small bowel graft was 260 ml in children less than 5 kg (n = 5) and 460 ml in children weighing 5-10 kg (n = 5). Fifteen small bowels were successfully reduced to provide an ileal graft of one meter while keeping the whole length of the superior mesenteric artery and vein after their dissection in the proximal part of the mesentery. The mean volume of the grafts created was 270 ml in seven thin patients (body mass index [BMI] <25), 390 ml in five preobese patients (25< BMI<30), and 490 ml in three obese patients (BMI>30). Mesenteric transillumination in thin donors allowed safe dissection and complete hemostasis. No diameter reduction was required. Technical modifications permitted the creation of two grafts, one ileal and the other jejunal from a single donor. Volumetric and surgical data show that implantation of up to two meters of ileum from a thin adult weighing up to 80 kg is feasible in children weighing less than 10 kg. CONCLUSION Size reduction of adult cadaveric small bowels can provide suitable grafts for children of less than 10 kg and could expand the potential pool of donors for these patients.

Basic anatomical and physiological differences between species should be considered when choosing combinations for use in models of hepatic xenotransplantation: an investigation of the guinea pig-to-rat combination.

Published data on the guinea pig-to-rat hepatic xenotransplant model describe problems concerning poor graft reperfusion. To further investigate this phenomenon, orthotopic liver xenotransplantation between weight-matched guinea pigs and rats were performed using Kamadas technique. On reperfusion, all cases had portal venous inflow block with hypoperfusion of the hepatic parenchyma. Histological examination showed no evidence of hyperacute rejection, although deposits of IgG2a and C3 but not IgM were identified within the central area of the liver. To increase blood inflow, arterialized partial liver grafts were performed without changing the outcome. We hypothesize that the hypoperfusion may be related to anatomical and physiological differences between the species. Guinea pig portal vein branches were found to have muscular walls susceptible to spasm, and portal blood flow is four times greater in the guinea pig than in the rat because the guinea pig intestine is both longer (two times as long) and of greater diameter. The combination of reperfusion injury, early immunological events, and the rats lower portal blood flow induces spasm of the intrahepatic portal system resulting in hypoperfusion. These findings demonstrate the importance of recognizing basic anatomical and physiological differences between species when selecting xenotransplantation models.

Allograft and Patient Outcomes of a Tumourectomized Kidney Transplant Program: The Western Australia Experience

Background There is an increased utilization of marginal donor kidneys for transplantation due to the shortage of donor kidneys. Patients with a small renal cell cancer occasionally undergo radical nephrectomy, which represents an opportunity to consider the utilization of these kidneys for transplantation that would otherwise be discarded. We report the long-term outcome of kidney transplant recipients who have received kidneys from live-unrelated donors with resected small renal cell cancers of less than 3 cm. Materials & Methods The potential recipients were informed and consent was obtained before they were listed for kidney transplantation by using a kidney graft, in which a small tumor was excised. The referral was sent from urologists to transplant team when a radical nephrectomy was decided as an option for treating a renal tumor. As such, the patient was offered an option of kidney donation after radical nephrectomy and the informed consent was signed. In our structured program, the decision for partial or radical nephrectomy for management of a small renal mass was purely made between the discussion of a patient and his or her treating urologist. The selection criteria for the potential donor and recipient have been established prior to implementation. The frozen section was performed after tumor excision on bench table to ensure the margin was clear prior to transplantation. The recipients were selected as age over 60 years or age 55 with urgent need for transplantation. Results From Feb 2007 to Aug 2017, 28 patients with a small renal mass chose radical nephrectomy and were considered suitable for kidney donation after tumor excision. The tumor size was from 1 cm to 4 cm, average 2.7 cm. There were 24 malignant tumors; with most of them were Fuhrman grade I-II; and 4 benign tumors. The recipients age were from 52-80 years old; average 63 year old at the time for kidney transplantation M:F=15:13. The average warm ischemic time was 5 minutes; cold ischemic time was from 155 minutes to 340 minutes (average 275). In our cohort the follow up period was from 2-10 years, median 7 years. There was no tumor recurrence. Of recipients, 68% are alive with average age 70 years old at follow up. The average Cr level was136 mmol/L. At early phase 3 patients developed urine leakage from the tumor excision area and one patient developed pseudoaneurysm, but all recoved well after interventional management. Conclusion Transplantation of tumourectomized kidneys from patients with small renal cell cancer is associated with comparable allograft and patient outcomes. The decision of utilizing these kidneys for transplantation needs careful informed consent process, balancing the benefit of kidney transplantation and the small but potential risk of tumour recurrence. The absence of tumor recurrence in this cohort may be associated with cold perfusion, which inhibits tumor cell proliferation and clear excision of tumor on bench table.