Pierre Balladur

Induction of Cytochrome P450 2B6 and 3A4 Expression by Phenobarbital and Cyclophosphamide in Cultured Human Liver Slices

AbstractPurpose. To examine the potential of cultured human liver slices to predict cytochrome P450 (CYP) inducibility, regarding global and zonal CYP expression, together with drug-induced histologic changes.nMethods. We first assessed whether CYP2B6, 3A4, and 2C9 expression was maintained in cultured liver slices. Cultured hepatocytes were used as the reference culture system. Then we tested the effects of phenobarbital and cyclophosphamide on CYP expression in both models.nResults. Morphologic features are preserved in slices. Basal CYP expression declines with time in culture in both models. Slices display the same region specificity of CYP2B6, 2C9, and 3A4 expression as intact liver. CYP2B6 and 3A4 mRNA, apoprotein, and enzyme-related activities were induced by phenobarbital and cyclophosphamide, whereas CYP2C9 apoprotein was not. Their immunoreactivities were also increased, while their zonal distribution was preserved on slice tissue sections. Microsomal enzyme induction was confirmed by histology.nConclusions. Cultured human liver slices are an attractive alternative to hepatocyte culture for the prediction of human CYP isoenzyme induction by xenobiotics.

Permeability and biocompatibility of a new hydrogel used for encapsulation of hepatocytes

A new high-water-content (83%) and highly permeable anionic polyelectrolyte hydrogel was obtained by phase inversion of a polymer solution containing 6% polyacrylonitrile-sodium methallylsulphonate, 91% dimethylsulphoxide and 3% physiological saline solution. Hydrogel-based hollow fibres (HFs) were fabricated with a co-extrusion apparatus in collaboration with Hospal (France). HFs have an internal diameter of 800 microns and a wall thickness of 100 microns. Experimental results demonstrated that hydrogel-based HFs were permeable to albumin (mol. wt 69,000) and human immunoglobulin G (150,000), but were impermeable to immunoglobulins A (170,000) and M (900,000) after 24 h of diffusion. In vitro, the viability of isolated rat hepatocytes injected into the HFs was 64 +/- 6% after 10 d versus 30 +/- 5% for hepatocytes cultured in Petri dishes (P = 0.0001). Under these conditions, the amount of albumin released by encapsulated hepatocytes was 12 +/- 3 micrograms/24 h/10(6) cells at day 10, whereas at that time no albumin was released by hepatocytes cultured in Petri dishes. In vivo, histological study of hydrogel HFs implanted up to 6 wk in the peritoneum of rats revealed a low inflammatory tissue reaction without giant multinucleate cells in the foreign tissue, which decreased after the third week. The survival rate of encapsulated hepatocytes was over 85% 45 d after transplantation in the peritoneum of syngeneic Lewis rats. Therefore, this hydrogel demonstrates highly favourable properties for encapsulation of hepatocytes with regard to its biocompatibility, permeability and ability to maintain hepatocytes in a functional state for prolonged periods.

Effect of liver transplantation on sex-hormone disorders in male patients with alcohol-induced or post-viral hepatitis advanced liver disease

The effects of liver transplantation on the pituitary-gonadal axis and sex-hormone metabolism were evaluated by studying hormonal status (androgens, oestrogens, and gonadotropins) and sex-hormone-binding globulin levels in men with advanced liver disease of both alcoholic and viral origins. Comparison of the results prior to and 6 months after liver transplantation showed that successful liver transplantation in men induced significant differences in sex-hormone levels and in pituitary-gonadal function in both alcoholic and post-hepatitis patients. Plasma testosterone and dihydrotestosterone levels increased, oestrogen (oestrone and oestradiol) and androstenedione levels fell while gonadotropin (FSH and LH) levels increased. There was also a fall in plasma prolactin levels. Sex-hormone binding globulin plasma levels were elevated prior to transplantation and decreased thereafter. These data show that male patients with advanced liver disease have biological hypogonadism and feminization, irrespective of the aetiology, and that these abnormalities rapidly improve after successful liver transplantation. Therefore in men with advanced liver disease the biochemical signs of sex hormone disturbance are reversible and may be largely related to the liver disease.

Transplantation of allogeneic hepatocytes without immunosuppression: long-term survival.

UNLABELLEDnHepatocyte transplantation could be an alternative to whole liver transplantation. Allogeneic hepatocytes are rejected if transplanted without immunosuppression. The aim of this study was to transplant allogeneic hepatocytes in the peritoneum and to protect them from rejection by encapsulation in a new semi-permeable membrane.nnnMETHODSnRats hepatocytes were encapsulated in hydrogel-based hollow fibers, obtained from AN69 polymer, before being transplanted into the peritoneum of rats. Outcome of allogeneic hepatocytes encapsulated in hollow fibers was compared to that of free allogeneic hepatocytes. Cell viability was assessed by erythrosine exclusion, morphology by electronic microscopy and function by albumin release.nnnRESULTSnUp to 90 days, viability of allogenic hepatocytes in hollow fibers was above 80%. The morphology remained normal at electronic microscopy. Albumin release was 16.5 +/- 0.3 (day 15), 14.2 +/- 2.0 (day 30), 8.8 +/- 0.1 (day 60) and 11.4 +/- 0.3 micrograms/24 h/10(6) hepatocytes (day 90). Free hepatocytes did not survive at day 15.nnnCONCLUSIONnViability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppression.

Morphological and Biochemical Integrity of Human Liver Slices in Long-Term Culture: Effects of Oxygen Tension

We tested the effects of low 20% O2) and high (70% O2) oxygen tension on the morphological and biochemical integrity of human liver slices incubated for up to 72 h in supplemented Williams E medium in a dynamic rotating culture system. High oxygen tension was more effective than low oxygen tension for preserving morphological integrity in long-term culture 48–72 h). After 72 h of culture with 70% O2, the lobular pattern was well preserved, and the survival of hepatocytes approximately 80%) and other cell types was good. Immunohistochemical studies showed good preservation of the region-specific expression of CYP2E1 and CYP3A4 isoenzymes for up to 72 h of incubation in 70% O2. As compared to 20% O2, the oxidized glutathione content and reactive oxygen species production were slightly increased in 70% O2, suggesting that minimal oxidative stress occurred with the high oxygen tension. In conclusion, despite slight oxidative stress associated with high oxygen tension, 70% O2 appeared more appropriate than 20% O2 for preserving the morphological and biochemical integrity of human liver slices cultured in a dynamic organ culture system for up to 72 h.

Semiautomatic macroencapsulation of fresh or cryopreserved porcine hepatocytes maintain their ability for treatment of acute liver failure.

We have previously demonstrated that fresh or cryopreserved xenogeneic hepatocytes manually macroencapsulated in AN69 polymer and transplanted intraperitoneally in rats were able to improve the survival rate after 95% hepatectomy without immunosuppression. In addition, we developed a semiautomatic device where porcine hepatocytes were coextruded with AN69 hydrogel in order to macroencapsulate large amounts of cells. The purpose of the present study was to 1) test whether transplanted porcine hepatocytes macroencapsulated in this device remained functional as evaluated by their ability to prevent death from acute liver failure, and 2) compare the efficiency of cryopreserved or freshly isolated hepatocytes. Fresh or cryopreserved porcine hepatocytes were macroencapsulated in the semiautomatic device by coextrusion in AN69 polymer in 2-m minitubes containing 6 × 107 cells. Acute liver failure was induced in rats by two-step 95% hepatectomy. At the time of completion of liver resection, rats were either not transplanted with minitubes (control group I, n = 13), or were implanted with two minitubes containing culture medium (control group II, n = 11), hepatocytes killed by heat treatment (control group III, n = 10), coextruded fresh hepatocytes (group IV, n = 11), or coextruded cryopreserved hepatocytes (group V, n = 11), without immunosuppression. The survival rate at day 7 was between 0% and 31% in the three control groups. By contrast, coextruded fresh hepatocytes significantly improved the survival rate (group IV, 82%) as did cryopreserved cells (group V, 91% survival). In surviving rats, minitubes were explanted after 20 days: either fresh or cryopreserved hepatocytes appeared morphologically viable and their ultrastructure was preserved. Their detoxification capacities evaluated by the activity of the cyt P450 CYP3A4 were partly maintained. In conclusion, porcine hepatocytes macroencapsulated by coextrusion using a semiautomatic device and transplanted without immunosuppression were able to prevent death from acute liver failure in rats. Cryopreserved cells were as efficient as fresh hepatocytes.

Desflurane improves the throughput of patients in the PACU. A cost-effectiveness comparison with isoflurane

PurposeIn a pharmacoeconomic approach of anesthesia, postanesthesia care unit (PACU) occupancy can be chosen as a criteria of effectiveness to compare two anesthetic drugs with different rates of elimination and different costs of administration. Our objective was to develop a cost-effectiveness approach for the comparison of isoflurane (I) and desflurane (D).MethodIn this prospective observational study, 68 patients aged 18–70 received either D or I for maintenance of anesthesia for inpatient abdominal procedures. Length of stay (LOS) in PACU was collected by a blinded observer. After the relationship between duration of surgery and LOS in PACU had been established in the 68 observed patients, we estimated the PACU occupancy according to duration of surgery and time of admission in PACU using a computer model of 204 consecutive patients, based on the hypothesis of an exclusive use of either D or I. Outcome measures were direct costs of the anesthesia procedure and occupancy of the PACU.ResultsThe direct cost of the anesthetic was significantly higher with D than with I. This represents an increase of CAN

Tumor necrosis factor-α in liver transplantation and resection: No evidence for a key role in ischemia-reperfusion injury

2 708 for the 204 patients. PACU occupancy was reduced by at least one patient (out of five beds) during 26.1 % of the time with D (P < 0.01).DiscussionImproving the throughput of patients in PACU by using new halogenated anesthetic agents with faster rates of elimination may outweigh the incremental cost of this strategy. This becomes particularly meaningful in operating theatres experiencing frequent overcrowded periods.RésuméObjectifL’occupation de la salle de réveil (SDR) peut être choisie comme critère pour réaliser une étude coût-efficacité entre deux agents d’anesthésie de coûts d’administration différents. Le but du présent travail est de réaliser une comparaison coût-efficacité entre l’isoflurane (I) et le desflurane (D).MéthodeDans cette étude prospective observationelle, 68 patients de 18 à 70 ans, hospitalisés pour une intervention chirurgicale abdominale, ont reçu soit I, soit D pour le maintien de l’anesthésie. La durée de séjour (DDS) en SDR était mesurée par un observateur indépendant. Après avoir établi la relation liant la durée opératoire et la DDS en SDR pour les 68 patients observés, nous avons bâti un modèle permettant de prédire l’occupation en SDR en fonction de la durée d’opération et de l’heure d’arrivée en SDR pour 204 patients consécutifs avec l’hypothèse d’une utilisation exclusive soit de I, soit de D. Les paramètres évalués ont été le coût d’administration et l’occupation de la SDR.RésultatsPour les 204 patients du modèle, le surcoût lié à l’utilisation de D a été de 2 708

Glutamine metabolism and neuropathologic disorders in experimental hepatic encephalopathy: Effect of transplanted hepatocytes

CAN. Avec le D, l’occupation de SDR a été réduite d’au moins un patient (sur cinq postes) durant 26 % du temps d’ouverture de la SDR (P < 0,01).ConclusionLa réduction de l’occupation de la SDR peut compenser le surcoût entraîné par l’usage de D par rapport à I. Cela est particulièrement intéressant dans les SDR où il existe des périodes de saturation.

Semiautomatic macroencapsulation of large numbers of porcine hepatocytes by coextrusion with a solution of AN69 polymer

Abstract Experimental studies have shown that liver ischemia-reperfusion induces Kupffer cell activation and tumor necrosis factor- α (TNF α ) release. The aim of this work was to determine whether severe hepatic ischemia and subsequent reperfusion triggers TNF α release in man. Serum TNF α was measured before and 3, 10, 30, 60, 120 min after revascularization and postoperatively at day 1 and 2 in 11 patients with orthotopic liver transplantation (group 1 and 4 patients with liver resection with vascular occlusion (group 2). In group 1, TNF α levels decreased during the first few minutes of reperfusion, then increased slightly to peak at 120 min (40 ± 13 pg/ml). Primary non-function occurred in 1 patient in whom low peroperative levels of TNF α levels were measured. In group 2, no significant changes in TNF α levels were observed. These data, in a small number of patients: (a) show that hepatic ischemia reperfusion does not result in major TNF α production; (b) do not support a primary pathogenic role for TNF α in damage after ischemia-reperfusion in humans.