Didier Houssin

Cytochromes P-450 in human hepatocyte plasma membrane: Recognition by several autoantibodies

BACKGROUND Anti-cytochrome P-450 autoantibodies are present in several forms of autoimmune hepatitis. The possibility that cytochromes P-450 are present in the plasma membrane of human hepatocytes was examined. METHODS (1) Plasma membranes with microsomal contamination < 1%, as judged from the activities of glucose-6-phosphatase and NADH-cytochrome c reductase, were prepared. (2) After exposure of uncut, fixed hepatocytes to antibodies, immunofluorescence and immunoperoxidase studies were performed. RESULTS (1) The specific content of cytochrome P-450 in plasma membrane was 9% of that in microsomes. Plasma membranes showed NADPH-cytochrome c reductase and mono-oxygenase activities; immunoblots showed the presence of cytochromes P-450 1A2, 2C, 2D6, 2E1, and 3A4; cytochromes P-450 1A2, 2D6, and 2C were also recognized by anti-liver microsome and anti-liver/kidney microsome type 1 and type 2 autoantibodies, respectively. (2) Immunofluorescence and immunoperoxidase labeling of the plasma membrane was observed with the three auto-antibodies and with anti-cytochrome P-450 1A2, 2C, 2E1, or 3A4. CONCLUSIONS It is concluded that cytochromes P-450 are present and functional in the plasma membrane of human hepatocytes and that anti-cytochrome P-450 autoantibodies recognize epitopes expressed on the outer surface.

Immunosuppressive properties of chenodeoxycholic and ursodeoxycholic acids in the mouse

Cell-mediated immunity is impaired during cholestasis, and there is evidence that bile acids play a role in this immune defect. Ursodeoxycholic acid (UDCA), which corrects the immunological abnormalities observed in primary biliary cirrhosis, could counter the detrimental effects of the endogenous bile acids. Accordingly, we assessed the respective effects of cholestasis, chenodeoxycholic acid (CDCA), and UDCA, using mixed lymphocyte culture as a model of allogeneic immune response. CDCA induced a dose-dependent inhibition of the proliferative response (0-150 mumol/L). Mononuclear cells obtained from bile duct-ligated mice had a normal immunostimulatory effect, whereas responder cells obtained from such animals showed a profoundly impaired proliferative response, suggesting that responder T cels are the main target of the cholestasis-induced immune defect. Supplementation of cultures with exogenous interleukins partially compensated for the inhibitory effect of 25 mumol/L CDCA, but not for that of 50 mumol/L CDCA, suggesting that impaired secretion of interleukins is not the only factor involved in the effect of bile acids. In contrast to CDCA, UDCA had no inhibitory effect on the allogenic immune response at concentrations of up to 50 mumol/L.

Orthotopic liver transplantation in children with chronic liver disease and severe hypoxemia

Liver transplantation has been considered until recently as an absolute contraindication in hypoxemic patients. We report our experience in nine patients who had orthotopic liver transplantation between June 1986 and June 1992. These patients had cirrhosis-related hypoxemia with intrapulmonary shunting (IPS). The arterial oxygen pressure (PaO2) on room air ranged from 47 to 78 mmHg. OLT resulted in resolution of hypoxemia and closure of IPS in five patients whose hypoxemia was higher than 60 mmHg, and in death for the remaining four patients who had severe hypoxemia (PaO2 < 60 mmHg). We conclude that hypoxemia is no longer a contraindication to liver transplantation. Patients having PaO2 levels higher than 60 mmHg should have OLT as soon as possible before reaching lower levels of PaO2, and combined lung-liver transplantation or heart-lung-liver transplantation should be discussed in patients with severe hypoxemia (PaO2 < 60 mmHg).

Hyperacute xenograft rejection in the swine-to-human donor-recipient combination : in vitro analysis of complement activation

Complement activation is central to the rejection of discordant xenografts. In order to assess the respective roles of direct and alternative pathways, an in vitro model of hyperacute rejection in the swine-to-human donor-recipient combination was designed, using a complement-dependent cytotoxicity test with swine endothelial cells in culture as targets, and fresh human serum as the source of xenogeneic antibodies and complement. The cytotoxic activity of the sera was evaluated by a colorimetric assay using (3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Pure human serum lysed 58 +/- 5% of swine endothelial cells. Selective inhibition of the direct pathway by adding EGTA to the serum reduced cytolysis to 51 +/- 2% (P < 0.01 versus normal serum). Similarly, when using C1q-deficient human sera, only 37 +/- 7% of swine endothelial cells were killed (P < 0.001 versus normal serum). When the alternative pathway was selectively inhibited by heating for 20 min at 50 degrees C, the lytic activity of human serum dropped to 42 +/- 5% (P < 0.001 versus normal serum). Factor B-deficient human serum could only lyse 42 +/- 10% of porcine endothelial cells (P < 0.001 versus normal serum). Syngeneic normal swine serum and heat-inactivated serum were not cytotoxic. Mixing serum with deficient direct pathway and serum with deficient alternative pathway restored the cytotoxicity to normal levels. Similarly, the cytotoxic activity of deficient serum supplemented with purified C1q or factor B at physiological concentrations reached that of normal human serum. In this model of in vitro hyperacute rejection, both pathways of complement activation are involved, suggesting that regimens designed to inhibit hyperacute rejection of swine xenografts into humans should take into account the dual activation of complement in this donor-recipient combination.

ORTHOTOPIC LIVER TRANSPLANTATION FOR BYLER DISEASE

Byler disease is a rare form of familial intrahepatic cholestasis that is fatal before puberty. This retrospective study reviewed the results of orthotopic liver transplantation in 14 children with Byler disease using 12 whole-liver grafts and 2 reduced-size grafts. One post-operative death occurred after retransplantation for arterial thrombosis. In the other patients, infectious problems and rejection episodes were the most frequent complications during the postoperative period. In the 13 patients alive, graft function, growth, and quality of life were good after an average follow-up of 17 months without any sign of disease recurrence.

Surgical management of gallstones in cirrhotic patients.

Among the cirrhotic patients admitted to our department, 64 (17 percent) were found to have cholelithiasis. In 14 patients (22 percent), cholelithiasis caused cholecystitis, obstructive jaundice, or biliary pain. These 14 patients were operated on and underwent cholecystectomy. There was one postoperative complication (gastrointestinal bleeding from esophageal varices) and one death (due to acute respiratory failure). In 50 patients (78 percent) cholelithiasis was asymptomatic. Ten of the 50 patients died from liver failure and the stones were discovered at necropsy. Seven of the patients had radiographically demonstrated stones that were not operated on. They are alive at the present time, more than 2 years later. In the remaining 33 patients, the stones were discovered during portasystemic shunt procedures. In these patients, cholelithiasis was systematically treated by cholecystectomy (8 patients) or cholecystolithotomy (25 patients). Postoperative mortality and morbidity rates were not different in these 33 patients when compared with the rates in 170 patients who underwent portal surgery alone during the same period. Our results confirm the high incidence of cholelithiasis in cirrhotic patients. Complications of gallstones are not frequent but require an emergency operation that carries a high risk in these patients. On the other hand, elective surgical treatment of asymptomatic cholelithiasis at the time of portal diversion does not bear any peculiar risk. In such a situation, cholecystolithotomy is easier and probably safer than cholecystectomy.

The influence of cold ischemia time on biliary complications following liver transplantation

Biliary complications are a continuing source of morbidity and mortality following orthotopic liver transplantation. The results of 100 whole-liver allografts performed in 92 adult patients were reviewed to determine whether cold ischemia time and preservation injury influenced both the incidence and type of biliary complications. Mean cold ischemia time was 10.2 +/- 0.5 h (range 3.6-19). Eighteen patients (19.6%) developed 25 biliary complications: there were eight anastomotic leaks, eight anastomotic strictures, six non-anastomotic strictures, two cystic duct mucoceles, and one biliary fistula following T-tube removal. Despite the high rate of reoperative surgery (68%), no death was attributable to biliary complications. Neither cold ischemia time nor early graft function influenced the rate of biliary complications or strictures of either type. Furthermore, an analysis of different factors revealed no predisposing effect of the pre-operative status of the recipient, type of biliary reconstruction, blood requirement, vascular complications, rejection or cytomegalovirus infection on the incidence of biliary complications or strictures. Only chronic rejection could be singled out as a risk factor for non-anastomotic strictures (p = 0.05). These results suggest that prolonged cold ischemia time does not seem to affect the rate or type of biliary complications following orthotopic liver transplantation. In view of these data, there is no clear reason to reconsider prolonged cold ischemia up to 15 h in University of Wisconsin solution, as it has transformed liver transplantation from an emergency operation to a semi-elective procedure and allows longer back-table preparation for graft reduction of splitting.

Cyclosporine-induced stimulation of the renin-angiotensin system after liver and heart transplantation

To analyze the status of the renin-angiotensin system in hypertensive transplant recipients on cyclosporine, we prospectively explored 21 cardiac (CTR: 52 ± 8.2 yr) and 12 liver (LTR: 45 ± 10 yr) transplant recipients on a normal salt diet with 19 normotensive controls in the same age range. Systolic and diastolic blood pressure was measured in the supine and standing positions. Renal function was assessed by serum creatinine values, and 24-hr urinary sodium and potassium excretion were recorded. Plasma renin activity (PRA), active renin, total renin, angiotensinogen, aldosterone, and cortisol plasma levels were simultaneously determined. Results were expressed as mean ± SD, and between-group differences were compared using variance analysis. Supine blood pressure (± SD) was 158 ± 15/103 ± 8.4 in CTR and 155 ± 21.4/102 ± 11.7 mmHg in LTR. Serum creatinine was higher in CTR (159 ± 52 μmol/L) than in LTR (117 ± 24.7, P<0.05) and values in both groups were above controls (83 ± 14.1, P<0.05). Urinary sodium excretion tended to be lower in transplant recipients (59 ± 42 mmol/L) for CTR and 44 ± 36.7 in LTR than in healthy controls (117 ± 24.7 mmol/L). Supine and upright PRA values tended to be higher in hypertensive transplant recipients than in healthy volunteers, although not significantly. Supine active renin was significantly higher in CTR (47 ± 42 pg/ml) and in LTR (44 ± 29.8 pg/ml) than in normal subjects (17 ± 4.8 pg/ml, P<0.05). Total renin levels in CTR (supine: 716 ± 357 pg/ml) and in LTR (supine: 647 ± 365 pg/ml) were 3− to 4-fold higher than in controls (supine: 207 ± 69 pg/ml) (P<0.05), as were inactive renin levels (P<0.01). Active renin was effectively correlated with PRA (P<0.001) and with total renin (P<0.001) in the supine and in the upright position. Plasma aldosterone was almost within the normal range in CTR and in LTR, and it did not correlate with PRA values. Plasma angiotensinogen levels were normal in LTR (1032 ± 226 ng/ml) but were significantly lower in CTR (938 ± 216 ng/ml, P<0.05). Cortisol plasma levels were lower in both CTR (7 ± 4.4 μg/L) and LTR (6 ± 1.9 μg/L) than in healthy controls (11 ± 4 μg/L, P<0.01). We conclude that an increase in plasma active renin and in inactive renin demonstrates the presence of unexpected stimulation of the renin angiotensin system among hypertensive CTR and LTR on cyclosporine. These findings support the hypothesis that direct and indirect cyclosporine toxic effects on the renal vascular wall and on the juxtaglomerular apparatus, associated with cyclosporine-induced sympathetic stimulation, contribute to the pathophysiology of cyclosporine-induced hypertension.

Endothelin-1 induces liver vasoconstriction through both ETA and ETB receptors

BACKGROUND/AIMS We investigated which endothelin receptors mediate the vasoconstrictive effects of endothelin-1 on liver circulation. METHODS An isolated perfused rat liver model in recirculation was used. RESULTS The perfusion of 10(-10) M endothelin-1 had no significant influence on the liver flow, whereas 10(-9) M endothelin-1 induced significant vasoconstriction, with flow dropping from 3.20 +/- 0.34 to 1.48 +/- 0.28 ml. min-1.g-1 liver tissue (p < 0.01 vs controls). The liver flow was interrupted following the perfusion of 10(-8) M endothelin-1. Sarafatoxin C and BQ 3020, two agonists of ETB receptor, had vasoconstrictive effects in this model. Sarafatoxin C decreased the liver flow in a dose-dependent manner, from 3.32 +/- 0.21 to 2.18 +/- 0.20, 1.60 +/- 0.09, and 1.01 +/- 0.06 ml.min-1. g-1, respectively, with 10(-9) M, 10(-8) M, and 10(-7) M. While BQ 123, an antagonist of ETA receptor, or BQ 788, an antagonist of ETB receptor, partially reversed the effect of 10(-9) M endothelin-1, the simultaneous administration of BQ 123 and BQ 788 completely reversed these effects. CONCLUSIONS These results indicate that the vasoconstrictive effects of endothelin-1 on the liver circulation are mediated through both ETA and ETB receptors.

HETEROTOPIC LIVER TRANSPLANTATION IN END-STAGE HBsAg-POSITIVE CIRRHOSIS

A patient with end-stage cirrhosis complicating HBsAg-positive chronic active hepatitis given a heterotopic liver transplant is alive and well 28 months after transplantation; liver function is normal, and there are no hepatitis-B related lesions on the liver graft despite positivity of HBsAg in the serum. This observation confirms that a heterotopic liver graft can offer long-term survival in man and suggests that this technique may be a more suitable procedure than orthotopic transplantation in end-stage cirrhotics.