Safety of conversion from twice-daily tacrolimus (Prograf) to once-daily prolonged-release tacrolimus (Advagraf) in stable liver transplant recipients.

Nonadherence to immunosuppressive regimens among solid organ transplantation to range has been estimated from 15% to 55%. This problem has been identified as a leading cause of preventable graft loss. Tacrolimus once daily Advagraf has been developed to provide a more convenient dosing regimen to improve adherence. The aim of this study was to analyze the safety of a 1:1 dose conversion from twice-daily tacrolimus (Prograf) to Advagraf in 36 stable liver transplant recipients. The tacrolimus whole blood trough level at T0 was 6.7 +/- 2.9 ng/mL with a daily dose of 3.7 +/- 1.8 mg. The mean tacrolimus blood trough levels at T1 (7 days) and T2 (14 days) were 5.8 +/- 2.5 and 5.8 +/- 1.8 ng/mL with mean daily doses of 3.9 +/- 1.9 and 4.1 +/- 1.8 mg, respectively. There was no significant difference between T0, T1, and T2, either for tacrolimus blood trough levels or for tacrolimus daily dosages. Liver and renal function tests remained stable; no episodes of acute rejection were encountered after the conversion. A switching policy using a dose ratio of 1:1 from twice-daily tacrolimus to once-daily prolonged-release tacrolimus was safely applied to stable liver transplant recipients.

Effect of cyanidin 3-O-beta-glucopyranoside on hepatic stellate cell proliferation and collagen synthesis induced by oxidative stress.

BACKGROUND: Reactive oxygen species play a role in the pathogenesis of hepatic fibrosis, mainly through the activation of hepatic stellate cells. Cyanidin-3-O-beta-glucopyranoside is a natural antioxidant compound distributed in several fruits and vegetables. AIM: To evaluate the effect of cyanidin-3-O-beta-glucopyranoside on hepatic stellate cells proliferation and collagen synthesis induced by a pro-oxidant agent. METHODS/RESULTS: Oxidative stress was induced by incubation of hepatic stellate cells with a ferric nitrilotriacetate complex (100 micromol/L). Incubation with ferric nitrilotriacetate induced an increased intracellular hydroperoxide formation, which was completely inhibited by cyanidin-3-O-beta-glucopyranoside at a concentration of 50mumol/L. Similarly, cyanidin-3-O-beta-glucopyranoside was able to inhibit ferric nitrilotriacetate-induced hepatic stellate cells proliferation, evaluated by an ELISA method, with a maximal effect at 50mumol/L. Incubation of hepatic stellate cells with cyanidin-3-O-beta-glucopyranoside inhibited ferric nitrilotriacetate-induced extracellular signal-regulated kinase 1/2 activation, evaluated by western blot, whereas it did not affect p70S6 kinase and AKT expression. Finally, cyanidin-3-O-beta-glucopyranoside reduced ferric nitrilotriacetate-induced Na(+)/H(+) exchange activation, evaluated by a spectrofluorimetric method, and collagen type I synthesis, evaluated by northern blot. CONCLUSION: Cyanidin-3-O-beta-glucopyranoside is able to modulate hepatic stellate cells proliferation and type I collagen synthesis induced by a pro-oxidant agent, thus suggesting a potential role for this antioxidant compound in the prevention of fibrosis in chronic liver diseases.

Hepatic stellate cell activation and liver fibrosis are associated with necroinflammatory injury and Th1-like response in chronic hepatitis C.

BACKGROUND/AIMS: The involvement of a direct viral cytopathic effect or an immune-mediated mechanism in the progression of hepatic damage in chronic hepatitis C is controversial. The type of immune response is itself a matter of controversy, and histological data are lacking. The aim of this study was to identify the factors associated with the progression of liver injury in 30 HCV/RNA-positive untreated patients with chronic hepatitis. METHODS: Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for alpha-smooth muscle actin (alphaSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT-PCR method. To study the type of immune response involved in the progression of liver injury, interferon gamma (IFNgamma)-positive cells (as expression of a Th1-like response) were evaluated by immunohistochemistry and quantitated by morphometry. RESULTS: HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The alphaSMA- and Sirius Red-positive parenchyma correlated significantly with necroinflammatory and architectural scores. IFNgamma-positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. No relationship was found between the histological features of liver injury and viral load. CONCLUSIONS: HSC activation and progression of liver injury are unrelated to viral load but associated with a Th1-like response, a plausible target for the treatment of chronic hepatitis C.

Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells.

The aim of this study was to evaluate intracellular pH (pHi) regulation in nonactivated and activated rat hepatic stellate cells (HSC). The fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was used to measure pHi in the presence and absence of HCO3-. In the absence of HCO3-, baseline pHi was significantly higher (P < 0.001) in activated than in nonactivated HSC (7.1 +/- 0.1 vs. 6.9 +/- 0.2) and decreased, in both groups, after amiloride administration and after Na+ removal. After an acid-loading maneuver, pHi recovery was significantly higher (P < 0.03) in activated than in nonactivated HSC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pHi 6.6) and was inhibited by amiloride and Na+ removal. In the presence of HCO3-, baseline pHi was higher in both groups and decreased after amiloride administration. Amiloride and Na+ removal inhibited pHi recovery after an intracellular acid load by 77 and 93%, respectively, in nonactivated and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited pHi recovery by only 27%. Acute Cl- removal increased pHi by 0.07 +/- 0.01 pH unit/min in the absence but not in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid in nonactivated and activated HSC in an Na(+)-independent manner. In activated HSC, 24 h of incubation with 25 ng/ml platelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pHi (7.07 +/- 0.1 vs. 7.08 +/- 0.1 in HSC cultured in 0.5% serum only) but significantly (P < 0.02) increased, with respect to controls, pHi recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed as percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-induced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0.001). Our results show that 1) the Na+/H+ exchanger is the main pHi regulator in rat HSC, 2) activation of HSC is associated with an increase in pHi and in the activity of the Na+/H+ exchanger, 3) PDGF increases the activity of this exchanger, and 4) amiloride is able to inhibit HSC proliferation induced by PDGF.

Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis.

Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin-positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha-smooth-muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation.

Regulation of intracellular pH in periportal and perivenular hepatocytes isolated from ethanol-treated rats.

The aim of this study was to gain information on intracellular pH (pHi) regulation in periportal (PP) and perivenular (PV) hepatocytes isolated from rats pair-fed liquid diets with either ethanol (T rats) or isocaloric carbohydrates (C rats). pHi was analyzed by the pH-sensitive dye BCECF in perfused subconfluent hepatocyte monolayers. Cells were acid-loaded by pulse exposure to NH4Cl and were alkali-loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHout. In cells from C rats: (a) steady-state pHi was higher in PP than in PV hepatocytes in the presence, but not in the absence, of bicarbonate; (b) pHi recovery from an acid load was 35% higher in PP than in PV cells in the presence of HCO3-, whereas it was similar in HCO3(-)-free experiments; and, on the contrary, (c) pHi recovery from an alkaline load was 30% higher in PV than in PP cells. In cells from T rats: (a) steady-state pHi was always lower than in cells isolated from pair-fed animals; (b) steady-state pHi was similar in PP and PV hepatocytes either in the presence or absence of bicarbonate in the perfusate; (c) pHi recovery from an acid load was not significantly different in PP and PV cells either in the presence of HCO3- or in HCO3(-)-free experiments; and (d) pHi recovery from an alkaline load was similar in PP and PV cells. Our data suggest that chronic ethanol treatment selectively modifies pHi by affecting the activity of ion transport mechanisms regulating pHi in PP and PV hepatocytes isolated from rat liver.

Chronic ethanol feeding increases apoptosis and cell proliferation in rat liver.

The present study was conducted to evaluate if the increased rate of apoptosis previously reported in the liver of ethanol-treated rats was accompanied by increased cell renewal. A quantitative analysis of apoptosis was performed in rats fed an ethanol-containing liquid diet for 5 weeks. S-phase cells were demonstrated by immunohistochemistry, using the Bromodeoxyuridine/anti-Bromodeoxyuridine method. In ethanol-fed rats apoptosis was five times greater than in pair-fed controls. Bromodeoxyuridine-labelled hepatocytes increased from 0.07 +/- 0.009% in controls to 0.17 +/- 0.013% (p < 0.001) and Bromodeoxyuridine-labelled lipocytes (desmin-positive sinusoidal cells) increased from 3.43 +/- 0.28% to 6.60 +/- 1.04% (p < 0.001). The lobular distribution of labelled cells was modified with a shift towards the perivenular areas. The results of this study suggest that the replacement of liver cells lost by ethanol-induced apoptosis is not impaired in intact (non-operated) animals. The impaired regeneration following partial hepatectomy reported in ethanol-fed rats is possibly due to differences in the extent of parenchymal loss, to altered relationships between hepatocytes and blood supply and to the modalities of regeneration involved.

Cell proliferation following extrahepatic biliary obstruction. Evaluation by immunohistochemical methods.

The aim of the present investigation was to conduct an immunohistochemical study using bromodeoxyuridine (BrdU) incorporation as a marker of S-phase cells and cytokeratins as markers of biliary epithelial cells, in bile-duct-ligated rats at intervals of 1, 3, 7, 14 and 21 days after total biliary obstruction. Data obtained using only BrdU incorporation by S-phase nuclei were compared with those obtained by the simultaneous demonstration of S-phase nuclei and cytoplasmic cytokeratins. The labelling index of parenchymal liver cells and of biliary epithelial cells was evaluated as an index of the cellular growth pattern after total biliary obstruction. Our data show that following total biliary obstruction: (a) cell proliferation follows a similar pattern for biliary epithelial cells hepatocytes with a peak on the 3rd day; (b) the labelling index is significantly higher in biliary epithelial cells than in hepatocytes; and (c) sequential immunohistochemical staining using cytokeratin as a marker allows better identification of biliary epithelial cells, especially when the ductular lumen is not clearly outlined, or in isolated biliary cells which appear as components of the wall of the ducts of Hering.