An ex vivo perfusion system emulating in vivo conditions in noncirrhotic and cirrhotic human liver.

Various models are used for investigating human liver diseases and testing new drugs. However, data generated in such models have only limited relevance for in vivo conditions in humans. We present here an ex vivo perfusion system using human liver samples that enables the characterization of parameters in a functionally intact tissue context. Resected samples of noncirrhotic liver (NC; n = 10) and cirrhotic liver (CL; n = 12) were perfused for 6-h periods. General and liver-specific parameters (glucose, lactate, oxygen, albumin, urea, and bile acids), liver enzymes (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, and γ-glutamyl transferase), overall (M65) and apoptotic (M30) cell-death markers, and indicators of phase-I/phase-II biotransformations were analyzed. The measurement readings closely resembled (patho)physiological characteristics in patients with NC and CL. Mean courses of glucose levels reflected the CLs' reduced glycogen storage capability. Furthermore, CL samples exhibited significantly stronger increases in lactate, bile acids, and the M30/M65 ratio than NC specimens. Likewise, NC samples exhibited more rapid phase-I transformations of phenacetin, midazolam, and diclofenac and phase-I to phase-II turnover rates of the respective intermediates than CL tissue. Collectively, these findings reveal the better hepatic functionality in NC. Perfusion of human liver tissue with this system emulates in vivo conditions and clearly discriminates between noncirrhotic and cirrhotic tissue. This highly reliable device for investigating basic hepatic functionality and testing safety/toxicity, pharmacokinetics/pharmacodynamics and efficacies of novel therapeutic modalities promises to generate superior data compared with those obtained via existing economic perfusion systems.

Major histocompatibility complex class I-related chains A and B (MIC A/B): a novel role in nonalcoholic steatohepatitis.

UNLABELLED: Stress-induced soluble major histocompatibility complex class I-related chains A/B (MIC A/B) are increased in chronic liver diseases and hepatocellular malignancy. We investigated the impact of these molecules on liver injury, apoptosis, and fibrosis in nonalcoholic steatohepatitis (NASH). Blood and liver tissue were obtained from 40 patients with NASH undergoing bariatric surgery for obesity. The control group consisted of 10 healthy individuals. We also investigated 10 patients with nonalcoholic fatty liver (NAFL). Polymerase chain reaction was used to measure messenger RNA (mRNA) transcripts of MIC A/B, natural killer cell receptor G2D (NKG2D), CD95/Fas, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-death receptor 5 (DR5). Apoptosis was quantified by way of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (intrahepatic) and M30/M65 (systemic). Liver injury was assessed histopathologically and serologically (alanine aminotransferase/aspartate aminotransferase). Fibrosis was identified by Sirius red staining, quantitative morphometry, and alpha-smooth muscle actin and collagen 1alpha transcripts. Compared with controls, patients with NASH revealed significant increases in (1) NKG2D mRNA (13.1-fold) and MIC A/B mRNA (3.6-fold and 15.8-fold, respectively); (2) TRAIL-DR5 and CD95/Fas mRNA (2.7-fold and 3.6-fold, respectively); (3) TUNEL-positive hepatocytes (4.0-fold); and (4) M30 and M65 levels (4.6-fold and 3.4-fold, respectively). We found relevant correlations between MIC protein expression rates and NAS and fibrosis stages. In contrast, NKG2D and MIC A/B transcripts were attenuated in patients with NAFL compared with NASH. Histopathologically, NASH patients revealed increased NAS scores, an accumulation of natural killer cells, and 2.7-fold increased hepatic fibrosis by quantitative morphometry. CONCLUSION: Our findings suggest an important role for MIC A/B in liver injury. Therapeutic intervention aimed at reducing MIC A/B levels may beneficially affect the progression of NASH.

Resveratrol amplifies profibrogenic effects of free fatty acids on human hepatic stellate cells.

AIM: To ascertain whether resveratrol affects the expression of free fatty acids (FFA)-induced profibrogenic genes, death receptors, and/or apoptosis-related molecules in human hepatic stellate cells, using the LX-2 cell line. METHODS: Cells were cultured in the presence of FFAs (2:1 oleate : palmitate) and subsequently treated with resveratrol. Gene expression rates were determined by quantitative real-time PCR. The 50% lethal dose (LD(50)) of resveratrol in the presence of FFAs was assessed with the MTT viability test. RESULTS: Compared to vehicle controls, incubation of LX-2 cells with 0.5 mM FFAs induced profibrogenic genes (alpha-SMA x 2.9; TGF-beta1 x 1.6; TIMP-1 x 1.4), death receptors (CD95/Fas x 3.8; TNFR-1 x 1.4), and anti-apoptotic molecules (Bcl-2 x 2.3; Mcl-1 x 1.3). Subsequent addition of 15 microM resveratrol (LD(50) = 23.2 microM) significantly (P < 0.05) upregulated further these genes (alpha-SMA x 6.5; TGF-beta1 x 1.9; TIMP-1 x 2.2; CD95/Fas x 13.1, TNFR-1 x 2.1; Bcl-2 x 3.6; Mcl-1 x 1.9). Importantly, this effect was only observed in the presence of FFAs. CONCLUSION: Resveratrol amplifies the profibrogenic activation of human hepatic LX-2 stellate cells. This finding raises the possibility that in obese patients with elevated FFAs reserveratrol could provoke hepatic fibrogenesis. In-vivo studies are necessary to further validate this conclusion.

Adiponectin inhibits steatotic CD95/Fas up-regulation by hepatocytes: therapeutic implications for hepatitis C.

BACKGROUND/AIMS: Steatosis may trigger hepatocytes to up-regulate CD95/Fas thereby increasing susceptibility to apoptosis, inflammation and fibrosis. We investigated this concept and potential roles of adiponectin and its receptors (AdipoR1; AdipoR2) in chronically HCV-infected patients. METHODS: In 98 HCV+ patients and 20 controls, sera were tested for HCV genotypes, FFAs, adiponectin and the M30 apoptosis indicator, and biopsies were evaluated for steatosis/inflammation/fibrosis, CD95/Fas (mRNA/protein), adiponectin (mRNA/protein), AdipoR1/-R2 (mRNA) and M30 (protein). We also questioned whether adiponectin protects HepG2 hepatoblastoma cells from FFA-triggered CD95/Fas up-regulation and apoptosis. RESULTS: Patients [HCV clades 1 (78%), 2 (3%) and 3 (19%)] revealed increased FFA and adiponectin serum levels (p = .005). Hepatocyte CD95/Fas up-regulation correlated with steatosis, inflammation and fibrosis (p = .004). Advanced fibrosis correlated significantly (p = .05) with serum M30. Liver adiponectin correlated with steatosis (p = .016), CD95/Fas (p < .001) and inflammation/fibrosis. Hepatocyte AdipoR2 mRNA specifically correlated with serum adiponectin and steatosis (p = .003), while hepatocyte AdipoR1 mRNA dropped in pronounced fibrosis (p = .060). Finally, adiponectin protected HepG2 cells from FFA-triggered CD95/Fas expression and induction of apoptosis (p = .0396). CONCLUSIONS: In chronic HCV infection, steatosis up-regulates hepatocyte CD95/Fas and thus increases apoptosis, which facilitates inflammation and fibrosis. The physiologic countermeasure of adiponectin up-regulation may offer clues for future therapeutic intervention.

Apoptosis versus necrosis rate as a predictor in acute liver failure following acetaminophen intoxication compared with acute-on-chronic liver failure.

Cell death is known to play a crucial role in liver diseases. We compared the clinical courses of two cases of liver failure of different origins correlated with the extent of cell death (apoptosis and/or necrosis), measured in the patient's blood. Patient 1 was admitted with acute liver failure following acetaminophen intoxication. Intravenous therapy with acetylcysteine was performed. The percentage of apoptotic cell death measured by M30 was three times higher than normal and the overall cell death measured by M65 was more than 30 x higher than normal. High levels of aminotransferases were detected, indicating high rates of necrosis. M30 and M65 values rapidly returned to normal levels, while the liver function test (LFT) levels decreased with latency. Patient 2 was admitted with acute-on-chronic liver failure with known liver fibrosis. M30 values were 16 x higher than normal. In contrast, M65 values were lower than in the first case. This time, no movement in M30 levels was seen until the M30 levels rapidly increased indicating the inevitable death of the patient, while LFTs did not change. These results indicate the role of M30 and M65 immunoexpression as markers for functioning liver cell mass, capacity for recovery and therefore as predictive markers in acute liver failure.