Carine Chagneau

Aiming at minimal invasiveness as a therapeutic strategy for Budd‐Chiari syndrome

The 1‐year spontaneous mortality rate in patients with Budd‐Chiari syndrome (BCS) approaches 70%. No prospective assessment of indications and impact on survival of current therapeutic procedures has been performed. We evaluated a therapeutic strategy uniformly applied during the last 8 years in a single referral center. Fifty‐one consecutive patients first received anticoagulation and were treated for associated diseases. Symptomatic patients were considered for hepatic vein recanalization; then for transjugular intrahepatic portosystemic shunt (TIPS), and finally for liver transplantation. The absence of a complete response led to the next procedure. Assessment was according to the strategy, whether procedures were technically applicable and successful. At entry, median (range) Child‐Pugh score and Clichy prognostic index were 8 (5–12), and 5.4 (3.1–7.7), respectively. A complete response was achieved on medical therapy alone in 9 patients; after recanalization in 6, TIPS in 20, liver transplantation in 9, and retransplantation in 1. Of the 41 patients considered for recanalization, the procedure was not feasible in 27 and technically unsuccessful in 3. Of the 34 patients considered for TIPS, the procedure was considered not feasible in 9 and technically unsuccessful in 4. At 1 year of follow‐up, a complete response to TIPS was achieved in 84%. One‐ and 5‐year survival from starting anticoagulation were 96% (95% CI, 90–100) and 89% (95% CI, 79–100), respectively. In conclusion, excellent survival can be achieved in BCS patients when therapeutic procedures are introduced by order of increasing invasiveness, based on the response to previous therapy rather than on the severity of the patients condition. (HEPATOLOGY 2006;44:1308–1316.)

Paroxysmal nocturnal hemoglobinuria in Budd-Chiari Syndrome: Findings from a cohort study

BACKGROUND/AIMS A well recognized cause of Budd-Chiari syndrome (BCS) is paroxysmal nocturnal hemoglobinuria (PNH). PNH is an acquired disorder of hematopoietic stem cells, characterized by intravascular hemolysis and venous thrombosis. Testing for this hematological disorder should be considered in all BCS patients. METHODS Using data from the EN-Vie study, a multi-center study of 163 patients with BCS, we investigated the relationship between BCS and PNH in 15 patients with combined disease and compared the results to 62 BCS patients in whom PNH was excluded. RESULTS Median follow-up for the study group (n=77) was 20 months (range 0-44 months). BCS patients with PNH presented with a significantly higher percentage of additional splanchnic vein thrombosis (SVT) as compared to BCS patients without PNH (47% vs. 10%, p=0.002). During follow-up, type and frequency of interventions for BCS was similar between both groups. Six patients with BCS and PNH were successfully treated with a transjugular intrahepatic portosystemic shunt (TIPS). Of 15 patients with PNH, six underwent allogenic stem cell transplantation after diagnosis of BCS. PNH was successfully cured in five cases. There was no significant difference in survival between BCS patients with and without PNH. CONCLUSIONS This study shows that despite a higher frequency of additional SVT, short-term prognosis of BCS patients with PNH does not differ from BCS patients without PNH. Treatment with TIPS can be safely performed in patients with PNH. Stem cell transplantation appears to be a feasible treatment option for PNH in BCS patients.

Fluorescent activated cell sorting (FACS): a rapid and reliable method to estimate the number of neurons in a mixed population.

Cells derived from the central nervous system (CNS) are usually characterised by manual counting on slides after specific immunolabelling. In this study, we investigated the possibility of using flow cytometry to determine the proportion of neurons, astrocytes or microglial cells in primary cultures. We show that parameters other than physical features are necessary to discriminate between these different cell types because of some overlap in their size and granulosity. We then used specific antibodies against intracellular markers such as Tuj-1 or GFAP to discriminate neurons from astrocytes by flow cytometry. The labelling was specific and reliable, allowing quantitative studies. Indeed, we did not find any significant difference in the number of Tuj-1 and GFAP-positive cells in primary cultures of neuronal and glial cells as determined by manual counting on slides or flow cytometry. More importantly, similar data were obtained in mixed populations, indicating that flow cytometry can be used for quantitative studies of heterogeneous cultures. The flow cytometry therefore appears to be a reliable method for the phenotypic characterisation of CNS-derived cells. This technique which enables a rapid analysis of numerous samples, might be particularly interesting for the study of neural stem cell differentiation.

Rapid failure of pig islet transplantation in non human primates

Abstract: We have previously demonstrated that adult pig islets of Langerhans are not destroyed in vitro by primate sera. Whether these islets can function when placed into the liver of non‐human primates is not known. We now report on the outcome of pig islet xenotransplantation into five non diabetic primates (four baboons and one macacus fascicularis) receiving intraportally purified adult pig islets. The average number of islet‐equivalent per graft was 110000 (60–180000). All animals received associations of ATG, cyclosporine or LF 195 (a deoxyspergualin analog), mycophenolate mofetil and corticosteroids. A specific porcine C‐peptide (C‐pep) RIA test was used to monitor insulin secretion. Two hours after grafting, porcine C‐peptide was positive (from 0.37 to 4.25 ng/ml) in all monkeys except one. Primate C‐pep was normal in all cases. Only two monkeys had detectable levels of porcine C‐pep on day 1 or 2 with undetectable levels thereafter, even after glucagon challenge between days 6 and 10. Several normal islets with moderate inflammatory infiltration were observed in one animal liver on day 2 (the time of necropsy) as well as islets with IgM and complement deposition. Among animals sacrificed on days 14, 16 and 38, some residual islet cells could be identified only in livers collected on day 14. Partial glycaemic control was achieved in some rats receiving islets from the same preparations. In conclusion, adult pig islets are not able to maintain insulin secretion for more than 24 h when injected intraportally into non diabetic immunosuppressed monkeys, suggesting immediate islet xenograft destruction.

CTLA4Ig adenoviral gene transfer induces long-term islet rat allograft survival, without tolerance, after systemic but not local intragraft expression

Genetic engineering using recombinant adenoviruses offers an opportunity to modify islet grafts in order to prevent allograft rejection. We have used an adenovirus coding for CTLA4Ig to compare its efficacy in preventing islet rejection depending on local or systemic production after gene transfer either into the islets or intramuscularly, respectively. Islet allograft survival was also evaluated using recombinant CTLA4Ig administered intraperitoneally or incubated ex vivo with islets prior to transplantation. Transduction of islets with 10(3) or 10(4) plaque-forming units (pfu) per islets of AdCTLA4Ig prolonged islet survival (mean +/- standard deviation [SD] days = 19.5 +/- 5.8 and 19.5 +/- 5.6, respectively, vs. 10.6 +/- 2.4 in control islets, p < 0.001), with low levels of circulating CTLA4Ig. In contrast, long-term survival (>60 days) was obtained after intramuscular injection of AdCTLA4Ig that resulted in sustained high levels of circulating CTLA4Ig. Islets incubated in vitro with CTLA4Ig did not show prolonged survival (10.3 +/- 2.5 days). Graft rejection was delayed after one injection of CTLA4Ig (23 +/- 7.6 days, p < 0.003 vs. control). Recipients of long-term surviving grafts after intramuscular AdCTLA4Ig gene transfer were not tolerant because second islet grafts of donor origin were rejected. These recipients also had a strong inhibition of humoral responses against nominal antigens, whereas animals receiving transduced islets showed normal responses. These data demonstrate that local production of CTLA4Ig after gene transfer was as efficient as a single injection of CTLA4Ig in preventing graft rejection. Furthermore, intramuscular gene transfer of CTLA4Ig was the most efficient way to induce long-term islet graft survival but no donor-specific tolerance was induced.

The role of nitric oxide in the reduction of protein kinase C-induced contractile response in aortae from rats with portal hypertension.

BACKGROUND/AIMS Protein kinase C plays a role in the regulation of vascular cell contraction but its activity may be reduced by nitric oxide. In portal hypertension, the exact mechanism by which nitric oxide induces vascular hyporeactivity to vasoconstrictors is unclear. The aim of this study was to investigate the role of the interaction of nitric oxide and protein kinase C in the vascular reactivity in isolated aortae from portal vein-stenosed rats. METHODS/RESULTS The contractile response to phorbol 12,13-dibutyrate, a protein kinase C activator, was significantly reduced in portal vein-stenosed aortae compared to sham-operated aortae. Preincubation with N-nitro-L-arginine or endothelium removal enhanced the response to phorbol 12,13-dibutyrate. The hyporesponsiveness to phorbol 12,13-dibutyrate in portal vein-stenosed rat aortae was only corrected after endothelium removal. The time course of contractions induced by phorbol 12,13-dibutyrate showed that the contraction was maintained for 2 h in sham-operated aortae and decreased to baseline in portal vein-stenosed rat aortae. This decrease was inhibited by N-nitro-L-arginine preincubation or endothelium removal. Protein kinase C downregulation caused a more marked reduction of phenylephrine-induced contraction in portal vein-stenosed aortae than in sham-operated aortae. The time course of total nitric oxide synthase activity in the presence of phorbol 12,13-dibutyrate showed a decrease in nitric oxide synthase activity after 30 min in both groups. Nitric oxide synthase activity remained stable for 120 min in sham-operated aortae but returned to basal level in portal vein-stenosed aortae. CONCLUSIONS Hyporeactivity to vasoconstrictors in portal vein-stenosed rat aortae may be due, in part, to a decrease in protein kinase C activation caused by nitric oxide overproduction.

Relationships between haemodynamic alterations and the development of ascites or refractory ascites in patients with cirrhosis

Objective In patients with cirrhosis, the relationships between haemodynamic alterations and the development of ascites or the occurrence of refractory ascites are unknown. The aim of the present study was to compare haemodynamic measurements obtained in patients with non-refractory ascites to haemodynamic measurements obtained in patients without ascites and in patients with refractory ascites. Methods A cohort of 121 patients was prospectively studied, of whom 29 patients did not have ascites, 45 had non-refractory ascites and 47 had refractory ascites. Splanchnic, renal and systemic haemodynamics were measured in all patients. Results The hepatic venous pressure gradient was significantly higher in patients with non-refractory ascites than in patients without ascites (18.5 ± 0.8 mmHg versus 15.8 ± 0.7 mmHg). Renal and systemic haemodynamics did not significantly differ between patients with non-refractory ascites and patients without ascites. The glomerular filtration rate and renal blood flow were significantly lower in patients with refractory ascites than in patients with non-refractory ascites (77 ± 4 versus 107 ± 5 ml/min and 867 ± 62 versus 1008 ± 68 ml/min, respectively). Splanchnic and systemic haemodynamics did not significantly differ between patients with refractory ascites and patients with non-refractory ascites. Conclusions In patients with cirrhosis, an increase in portal hypertension was the sole haemodynamic alteration related to the development of ascites. Renal vasoconstriction (and subsequent renal hypoperfusion and hypofiltration) was the only haemodynamic alteration related to the occurrence of refractory ascites. The development of ascites or refractory ascites was not associated with any alteration in systemic haemodynamics.

Endothelium-dependent blunted membrane potential responses to ATP-sensitive K+ channel modulators in aortae from rats with cirrhosis.

BACKGROUND/AIMS In vivo studies have shown that arterial vasodilation induced by synthetic openers of ATP-sensitive K+ (K(ATP)) channels is decreased in rats with cirrhosis. Since vasodilation induced by these substances is mediated by membrane potential hyperpolarization in arterial smooth muscle cells, membrane potential hyperpolarization in response to K(ATP) channel openers may be altered in cirrhotic smooth muscle cells. The aim of the present study was to investigate the effects of K(ATP) channel modulators (i.e. openers and blockers of these channels) on the membrane potential in smooth muscle cells in isolated aortae from cirrhotic and normal rats. The influence of endothelin-1 production by endothelial cells on smooth muscle cells membrane potential responses to K(ATP) channel modulators was also studied. METHODS Cells were impaled in situ (in intact and endothelium-denuded aortae) with a microelectrode that was used to measure membrane potentials. K(ATP) channel openers were diazoxide or cromakalim; blockers were glibenclamide or tolbutamide. Bosentan (a mixed endothelin receptor antagonist) and exogenous endothelin-1 were also used. Preproendothelin-1 mRNA was assayed in aortae by RNase protection assay. Aortic wall endothelin-1 concentration was measured by double antibody radioimmunoassay technique. RESULTS As expected, in smooth muscle cells in intact normal aortae, K(ATP) channel openers induced membrane potential hyperpolarization and K(ATP) channel blockers membrane potential depolarization. In smooth muscle cells in intact cirrhotic aortae, K(ATP) channel openers and blockers did not significantly change the membrane potential. Endothelium removal or exposure of intact aortae to bosentan restored normal membrane potential responses to K(ATP) channel modulators in cirrhotic smooth muscle cells and did not alter the effects of these substances in normal smooth muscle cells. In endothelium-denuded aortae, exposure to exogenous endothelin-1 suppressed membrane potential responses to K(ATP) channel modulators. In intact aortae, the abundance of preproendothelin-1 mRNA and endothelin-1 did not significantly differ between normal and cirrhotic rats. CONCLUSIONS K(ATP) channel opener-induced membrane hyperpolarization and K(ATP) channel blocker-elicited membrane depolarization are blunted in smooth muscle cells in intact cirrhotic aortae. This blunting is due to the activation of the endothelin-1 pathway in the aortic wall, downstream to the endothelial production of endothelin-1.