Human hepatocellular carcinomas with a periportal phenotype have the lowest potential for early recurrence after curative resection.

Hepatocellular carcinomas (HCCs) exhibit a diversity of molecular phenotypes, raising major challenges in clinical management. HCCs detected by surveillance programs at an early stage are candidates for potentially curative therapies (local ablation, resection, or transplantation). In the long term, transplantation provides the lowest recurrence rates. Treatment allocation is based on tumor number, size, vascular invasion, performance status, functional liver reserve, and the prediction of early (<2 years) recurrence, which reflects the intrinsic aggressiveness of the tumor. Well-differentiated, potentially low-aggressiveness tumors form the heterogeneous molecular class of nonproliferative HCCs, characterized by an approximate 50% ß-catenin mutation rate. To define the clinical, pathological, and molecular features and the outcome of nonproliferative HCCs, we constructed a 1,133-HCC transcriptomic metadata set and validated findings in a publically available 210-HCC RNA sequencing set. We show that nonproliferative HCCs preserve the zonation program that distributes metabolic functions along the portocentral axis in normal liver. More precisely, we identified two well-differentiated, nonproliferation subclasses, namely periportal-type (wild-type ß-catenin) and perivenous-type (mutant ß-catenin), which expressed negatively correlated gene networks. The new periportal-type subclass represented 29% of all HCCs; expressed a hepatocyte nuclear factor 4A-driven gene network, which was down-regulated in mouse hepatocyte nuclear factor 4A knockout mice; were early-stage tumors by Barcelona Clinic Liver Cancer, Cancer of the Liver Italian Program, and tumor-node-metastasis staging systems; had no macrovascular invasion; and showed the lowest metastasis-specific gene expression levels and TP53 mutation rates. Also, we identified an eight-gene periportal-type HCC signature, which was independently associated with the highest 2-year recurrence-free survival by multivariate analyses in two independent cohorts of 247 and 210 patients. CONCLUSION: Well-differentiated HCCs display mutually exclusive periportal or perivenous zonation programs. Among all HCCs, periportal-type tumors have the lowest intrinsic potential for early recurrence after curative resection. (Hepatology 2017;66:1502-1518).

Molecular profiling of stroma identifies osteopontin as an independent predictor of poor prognosis in intrahepatic cholangiocarcinoma.

UNLABELLED: Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary cancer in the liver. ICC is an aggressive cancer with poor prognosis and limited therapeutic strategies. The identification of new drug targets and prognostic biomarkers is an important clinical challenge for ICC. The presence of an abundant stroma is a histological hallmark of ICC. Given the well-established role of the stromal compartment in the progression of cancer diseases, we hypothesized that relevant biomarkers could be identified by analyzing the stroma of ICC. By combining laser capture microdissection and gene expression profiling, we demonstrate that ICC stromal cells exhibit dramatic genomic changes. We identified a signature of 1,073 nonredundant genes that significantly discriminate the tumor stroma from nontumor fibrous tissue. Functional analysis of differentially expressed genes demonstrated that up-regulated genes in the stroma of ICC were related to cell cycle, extracellular matrix, and transforming growth factor beta (TGFß) pathways. Tissue microarray analysis using an independent cohort of 40 ICC patients validated at a protein level the increased expression of collagen 4A1/COL4A1, laminin gamma 2/LAMC2, osteopontin/SPP1, KIAA0101, and TGFß2 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of osteopontin, TGFß2, and laminin in the stroma of ICC was significantly correlated with overall patient survival. More important, multivariate analysis demonstrated that the stromal expression of osteopontin was an independent prognostic marker for overall and disease-free survival. CONCLUSION: The study identifies clinically relevant genomic alterations in the stroma of ICC, including candidate biomarkers for prognosis, supporting the idea that tumor stroma is an important factor for ICC onset and progression.

COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration.

BACKGROUND: The orphan receptors COUP-TF (chicken ovalbumin upstream promoter transcription factor) I and II are members of the nuclear receptor superfamily that play distinct and critical roles in vertebrate organogenesis. The involvement of COUP-TFs in cancer development has recently been suggested by several studies but remains poorly understood. METHODS: MCF-7 breast cancer cells overexpressing COUP-TFI and human breast tumors were used to investigate the role of COUP-TFI in the regulation of CXCL12/CXCR4 signaling axis in relation to cell growth and migration. We used Immunofluorescence, western-blot, RT-PCR, Formaldehyde-assisted Isolation of Regulatory Elements (FAIRE) assays, as well as cell proliferation and migration assays. RESULTS: Previously, we showed that COUP-TFI expression is enhanced in breast cancer compared to normal tissue. Here, we report that the CXCL12/CXCR4 signaling pathway, a crucial pathway in cell growth and migration, is an endogenous target of COUP-TFI in breast cancer cells. The overexpression of COUP-TFI in MCF-7 cells inhibits the expression of the chemokine CXCL12 and markedly enhances the expression of its receptor, CXCR4. Our results demonstrate that the modification of CXCL12/CXCR4 expression by COUP-TFI is mediated by the activation of epithelial growth factor (EGF) and the EGF receptor. Furthermore, we provide evidence that these effects of COUP-TFI increase the growth and motility of MCF-7 cells in response to CXCL12. Cell migration toward a CXCL12 gradient was inhibited by AMD3100, a specific antagonist of CXCR4, or in the presence of excess CXCL12 in the cell culture medium. The expression profiles of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA in 82 breast tumors and control non-tumor samples were measured using real-time PCR. CXCR4 expression was found to be significantly increased in the tumors and correlated with the tumor grade, whereas the expression of CXCL12 was significantly decreased in the tumors compared with the healthy samples. Significantly higher COUP-TFI mRNA expression was also detected in grade 1 tumors. CONCLUSIONS: Together, our mechanistic in vitro assays and in vivo results suggest that a reduction in chemokine CXCL12 expression, with an enhancement of CXCR4 expression, provoked by COUP-TFI, could be associated with an increase in the invasive potential of breast cancer cells.

Epithelial cell adhesion molecule is a prognosis marker for intrahepatic cholangiocarcinoma.

BACKGROUND: Recently, we identified a gene signature of intrahepatic cholangiocarcinoma (ICC) stroma and demonstrated its clinical relevance for prognosis. The most upregulated genes included epithelial cell adhesion molecule (EpCAM), a biomarker of cancer stem cells (CSC). We hypothesized that CSC biomarkers could predict recurrence of resected ICC. METHODS: Both functional analysis of the stroma signature previously obtained and immunohistochemistry of 40 resected ICC were performed. The relationships between the expression of CSC markers and clinicopathologic factors including survival were assessed by univariate and multivariable analyzes. RESULTS: Gene expression profile of the stroma of ICC highlighted embryonic stem cells signature. Immunohistochemistry on tissue microarray showed at a protein level the increased expression of CSC biomarkers in the stroma of ICC compared with nontumor fibrous liver tissue. The overexpression of EpCAM in the stroma of ICC is an independent risk factor for overall (hazard ratio = 2.6; 95% confidence interval, 1.3-5.1; P = 0.005) and disease-free survival (hazard ratio = 2.2; 95% confidence interval, 1.2-4.2; P = 0.012). In addition, the overexpression of EpCAM in nontumor fibrous liver tissue is closely correlated with a worst disease-free survival (P = 0.035). CONCLUSIONS: Our findings provide new arguments for a potential role of CSC on ICC progression supporting the idea that targeting CSC biomarkers might represent a promise personalized treatment.

DNAX accessory molecule-1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vgamma9Vdelta2 T cells.

Human Vgamma9Vdelta2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway-derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate. They display a strong cytotoxic activity against several tumor types, including hepatocellular carcinoma (HCC). Little is known about the mechanisms underlying Vgamma9Vdelta2 T-cell recognition of tumor cells, but there is strong evidence that activating NK receptors play a role in gammadelta T-cell cytotoxicity. In this study, we showed that the two NK receptors DNAX accessory molecule-1 (DNAM-1) and CD96 were expressed by Vgamma9Vdelta2 T cells. The ligands Nectin-like-5 specific of both DNAM-1 and CD96, and also Nectin-2, an additional ligand of DNAM-1, were present on all HCC cell lines analyzed. Furthermore, we demonstrated by mAb-mediated masking experiments that cytotoxicity against HCC cells as well as IFN-gamma production in gammadelta T cells were dependent on DNAM-1. Our experiments indicated that Nectin-like-5 but not Nectin-2 was involved in DNAM-1-dependent gammadelta T-cell functions. We did not reveal a role for CD96 in the killing of HCC cells. Finally, we showed by combined mAb-mediated blockade that DNAM-1 and NKG2D could cooperate in the cell lysis of HCC.

[Tgammadelta lymphocytes in oncology: unconventional killer lymphocytes]. / Lymphocytes Tgd en cancérologie: des lymphocytes tueurs non conventionnels.

Gamma delta T cells are a distinct subset of CD3+ T cells featuring both T cells receptors that are encoded by Vgamma- and Vdelta- gene segments and characteristics of innate immunity. In human blood, 80% of those express Vgamma9Vdelta2-TCRs that are specific for conserved non peptidic compound, phosphoantigens (PAgs). Vgamma9Vdelta2 T cells recognize in vitro a wide array of transformed cells and are activated in vivo in various tumor. Owing to their ability to directly kill tumor cells and produce inflammatory cytokines (such as IFN-gamma) boosting antitumor properties of other immune effector cells, gamma delta T cells contribute to protective immunity against cancers. These observations, and the recent availability of synthetic clinical grade PAg or pharmacological inducers of PAg (e.g. aminobisphosphonates) able to trigger Vgamma9Vdelta2 T cell, have fostered development of new gammadelta T cell-based therapeutic strategies, which are depicted in this review.

Integrating Biobank Data into a Clinical Data Research Network: The IBCB Project.

Development of biobanks is still hampered by difficulty to collect high quality sample annotations using patient clinical information. The IBCB project evaluated the feasibility of a nationwide clinical data research network for this purpose. METHOD: the infrastructure, based on eHOP and I2B2 technologies, was interfaced with the legacy IT components of 3 hospitals. The evaluation focused on the data management process and tested 5 expert queries in Hepatocarcinoma. RESULTS: the integration of biobank data was comprehensive and easy. Five out of 5 queries were successfully performed and shown consistent results with the data sources excepted one query which required to search in unstructured data. The platform was designed to be scalable and showed that with few effort biobank data and clinical data can be integrated and leveraged between hospitals. Clinical or phenotyping concepts extraction techniques from free text could significantly improve the samples annotation with fine granularity information.

Assessment of in vitro applicability of reversibly immortalized NKNT-3 cells and clonal derivatives.

In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4-20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.

Gene expression profiling of the tumor microenvironment in human intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common type of malignant primary tumors in the liver. ICC is an aggressive cancer with a poor survival and limited therapeutic options. At the histological level, ICC is characterized by an abundant stroma (i.e. the tumor microenvironment that notably includes components of the extracellular matrix, stromal cells and soluble factors). Tumor microenvironment is known to play a key role in tumor onset and progression but it is poorly characterized at the molecular level. Thus, this study was specifically designed to identify genes that are significantly deregulated in the tumor microenvironment of human ICC. Here we provide a detailed description of the experimental design and methods used to acquire the genomic data deposited into Gene Expression Omnibus (GEO) under the accession number GSE45001. Our genomic dataset provides insights on the molecular pathways altered in the microenvironment of ICC and allows the identification of novel ICC biomarkers, as exemplified previously in Hepatology (PMID: 23775819).

"Fibrous nests" in human hepatocellular carcinoma express a Wnt-induced gene signature associated with poor clinical outcome.

Hepatocellular carcinoma (HCC) is the 3rd cause of cancer-related death worldwide. Most cases arise in a background of chronic inflammation, extracellular matrix (ECM) remodeling, severe fibrosis and stem/progenitor cell amplification. Although HCCs are soft cellular tumors, they may contain fibrous nests within the tumor mass. Thus, the aim of this study was to explore cancer cell phenotypes in fibrous nests. Combined anatomic pathology, tissue microarray and real-time PCR analyses revealed that HCCs (n=82) containing fibrous nests were poorly differentiated, expressed Wnt pathway components and target genes, as well as markers of stem/progenitor cells, such as CD44, LGR5 and SOX9. Consistently, in severe liver fibroses (n=66) and in HCCs containing fibrous nests, weighted correlation analysis revealed a gene network including the myofibroblast marker ACTA2, the basement membrane components COL4A1 and LAMC1, the Wnt pathway members FZD1; FZD7; WNT2; LEF1; DKK1 and the Secreted Frizzled Related Proteins (SFRPs) 1; 2 and 5. Moreover, unbiased random survival forest analysis of a transcriptomic dataset of 247 HCC patients revealed high DKK1, COL4A1, SFRP1 and LAMC1 to be associated with advanced tumor staging as well as with bad overall and disease-free survival. In vitro, these genes were upregulated in liver cancer stem/progenitor cells upon Wnt-induced mesenchymal commitment and myofibroblast differentiation. In conclusion, fibrous nests express Wnt target genes, as well as markers of cancer stem cells and mesenchymal commitment. Fibrous nests embody the specific microenvironment of the cancer stem cell niche and can be detected by routine anatomic pathology analyses.

De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas.

About 20% hepatocellular carcinomas (HCCs) display wild-type ß-catenin, enhanced Wnt signaling, hepatocyte dedifferentiation and bad outcome, suggesting a specific impact of Wnt signals on HCC stem/progenitor cells. To study Wnt-specific molecular pathways, cell fates and clinical outcome, we fine-tuned Wnt/ß-catenin signaling in liver progenitor cells, using the prototypical Wnt ligand Wnt3a. Cell biology assays and transcriptomic profiling were performed in HepaRG hepatic progenitors exposed to Wnt3a after ß-catenin knockdown or Wnt inhibition with FZD8_CRD. Gene expression network, molecular pathology and survival analyses were performed on HCCs and matching non-tumor livers from 70 patients by real-time PCR and tissue micro-array-based immunohistochemistry. Wnt3a reprogrammed liver progenitors to replicating fibrogenic myofibroblast-like cells displaying stem and invasive features. Invasion was inhibited by 30 nM FZD7 and FZD8 CRDs. Translation of these data to human HCCs revealed two tight gene networks associating cell surface Wnt signaling, stem/progenitor markers and mesenchymal commitment. Both networks were linked by Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1), that appeared de novo in aggressive HCCs expressing cytoplasmic ß-catenin and stem cell markers. HAPLN1 was independently associated with bad overall and disease-free outcome. In vitro, HAPLN1 was expressed de novo in EPCAM¯/NCAM+ mesoderm-committed progenitors, upon spontaneous epithelial-mesenchymal transition and de-differentiation of hepatocyte-like cells to liver progenitors. In these cells, HAPLN1 knockdown downregulated key markers of mesenchymal cells, such as Snail, LGR5, collagen IV and α-SMA. In conclusion, HAPLN1 reflects a signaling network leading to stemness, mesenchymal commitment and HCC progression.

Inhibition of carcinogen-bioactivating cytochrome P450 1 isoforms by amiloride derivatives.

We examined the effects of amiloride derivatives, especially 5-(N-ethyl-N-isopropyl)amiloride (EIPA), on the activity of cytochrome P450 (CYP) 1 isoforms, known to metabolize carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BP), into mutagenic metabolites and whose cellular expression can be induced through interaction of PAHs with the arylhydrocarbon receptor. EIPA was found to cause a potent and dose-dependent inhibition of CYP1-related ethoxyresorufine O-deethylase (EROD) activity in both liver cells and microsomes. It also markedly reduced activity of human recombinant CYP1A1 enzyme through a competitive mechanism; activities of other human CYP1 isoforms, i.e. CYP1A2 and CYP1B1, were also decreased. However, EIPA did not affect BP-mediated induction of CYP1A1 mRNA and protein levels in rat liver cells, likely indicating that EIPA does not block activation of the arylhydrocarbon receptor by PAHs. Inhibition of CYP1 activity by EIPA was associated with a decreased metabolism of BP, a reduced formation of BP-derived DNA adducts and a diminished BP-induced apoptosis in liver cells. The present data suggest that amiloride derivatives, such as EIPA, may be useful for preventing toxicity of chemical carcinogens, such as PAHs, through inhibition of CYP1 enzyme activity.

Hypothermic storage and cryopreservation of hepatocytes: the protective effect of alginate gel against cell damages.

Hepatocyte-based therapy has been proposed as an alternative to organ transplantation in the treatment of liver disorders. In the clinical context, a major issue is the constant supply of quality assurance-controlled hepatocytes, thereby requiring their cold storage in good conditions. We have analyzed the protective effects of alginate entrapment of rat hepatocytes after either 24 or 48 h of hypothermic storage or cryopreservation on the cell viability, cell yield, both mitochondrial and other cytoplasmic functional activities, and apoptosis. Decrease in viability, as evaluated by the MTT inclusion test, was 4% and 13% (24 h at 4 degrees C), 15% and 33% (48 h at 4 degrees C), and 9% and 19% (liquid nitrogen) for entrapped and free suspended hepatocytes, respectively. Viable cell yields were 86 +/- 8% and 51 +/- 6% for cryopreserved entrapped and free suspended hepatocytes, respectively. The mitochondrial (MTS assay), 7-ethoxyresorufin O-deethylase (EROD), and glutathione-S-transferase (GST) activities were better preserved in entrapped than in free suspended hepatocytes. Both hypothermic storage and cryopreservation were found to induce early caspase-3-like activities, being always much lower in entrapped hepatocytes, particularly after cryopreservation (98.4 +/- 42.4 vs. 6.4 +/- 4.0 fluorescence arbitrary units/hours/microg protein). Thus, cold-induced apoptosis in hepatocytes can be significantly reduced following their entrapment within alginate gel beads and this is associated with an improvement of both their viability and function.

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection.

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.

HLA-A*0201-restricted CEA-derived peptide CAP1 is not a suitable target for T-cell-based immunotherapy.

Carcinoembryonic antigen (CEA) is a potential target for antigen-specific immunotherapy, as it is frequently overexpressed in human carcinomas. Moreover, an epitope derived from CEA, designated CAP1 (YLSGANLNL), has been proposed as naturally processed and presented by tumors in the human leukocyte antigen (HLA)-A*0201 context. Our aim was to fully characterize and assess the clinical relevance of the HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response against CEA. Stable and potent artificial antigen presenting cells (AAPCs) were used to evaluate T-cell response against CEA. These cells efficiently activate CTLs against tumor-derived epitopes after transduction with the antigenic peptides or full-length proteins. We found that AAPCs genetically modified to express CAP1, the agonist peptide CAP1-6D, or the whole CEA protein were not able to activate CAP1-specific CTLs from HLA-A*0201+ healthy donors or patients with colorectal carcinoma, even after multiple stimulations. In addition, we showed that a CAP1-specific T-cell clone, obtained after multiple stimulations of T cells of a HLA-A*0201+ healthy donor in vitro with autologous antigen presenting cells, recognized CEA(-) HLA-A*0201+ tumors transduced with a minigene encoding CAP1 but failed to react against HLA-A*0201+ tumor cells expressing CEA. Finally, AAPCs expressing the whole CEA protein did not induce any specific CTL response against CEA+ HLA-A*0201+ tumor cells highlighting the potential difficulty of mounting an efficacious T-cell response against this autoantigen. Altogether, our data indicate that CAP1 is not efficiently processed and presented by CEA+ tumor cells, and therefore, is not an appropriate target for T-cell-based immunotherapy.

Assessment of in Vitro Applicability of Reversibly Immortalized NKNT-3 Cells and Clonal Derivatives.

In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4-20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.

Reduced encephalopathy in pigs with ischemia-induced acute hepatic failure treated with a bioartificial liver containing alginate-entrapped hepatocytes.

OBJECTIVE: To analyze the effects of an extracorporeal bioartificial liver containing alginate bead-entrapped hepatocytes on pigs with ischemia-induced acute hepatic failure. DESIGN: Prospective animal study. SETTING: University and INSERM laboratory. SUBJECTS: Fifteen Large White/Pietrin female pigs weighing 20-30 kg. INTERVENTIONS: Acute hepatic failure was induced by end-to-side portocaval shunt and ligature of the whole porta hepatitis. The bioartificial liver was in a thermostabilized column, containing a fluidized bed of alginate beads that embedded porcine hepatocytes, connected to a plasmapheresis system. Intracranial pressure; survival; ammonia, total bilirubin, aminotransferases, alkaline phosphatase, and lactate concentrations; and clotting factors were studied. The groups were pigs with acute hepatic failure (group 1, n = 4), pigs with acute hepatic failure treated with bioartificial liver containing empty beads (group 2, n = 4), or porcine hepatocytes (group 3, n = 5). MEASUREMENTS AND MAIN RESULTS: In group 1, survival of pigs averaged 10.9 +/- 1.0 hrs; intracranial pressure reached 32.3 +/- 3.8 mm Hg and was associated with coma and cerebral edema. After connection to the bioartificial liver, the survival of acute hepatic failure pigs was 12.1 +/- 1.4 hrs in group 2 and 14.8 +/- 2.5 hrs in group 3. In group 3, intracranial pressure and bilirubin concentrations were reduced significantly compared with both group 1 and group 2. Neither signs of encephalopathy nor cerebral edema was observed in any animal of group 3. In all animals, plasma ammonium, aminotransferases, alkaline phosphatase, and lactate concentrations increased and clotting factors decreased with no significant differences between the three groups. Autopsy revealed a total necrosis of the liver, which was histologically confirmed. CONCLUSIONS: The ischemia-induced model of acute hepatic failure in pigs is reproducible and provides measurable clinical and biological features. A bioartificial liver containing alginate bead-entrapped hepatocytes improves the signs of encephalopathy in pigs with ischemia-induced acute hepatic failure, suggesting that the bioartificial liver can clear out toxic compounds that are released from necrotic livers.

Follow-up by one- and two-dimensional NMR of plasma from pigs with ischemia-induced acute liver failure treated with a bioartificial liver.

Hepatic encephalopathy may occur following acute hepatic failure (AHF), which results in the release of toxic compounds from the injured liver. These compounds, which induce cerebral edema, are not well characterized, yet. The aim of this study was to evaluate the potential interest of NMR spectroscopy in the follow-up of different plasma compounds in pigs with ischemia-induced fulminant hepatic failure treated or not with a bioartificial liver (BAL), which has been previously shown to improve the neurological status of the animals. Qualitative analysis of pig plasma was achieved by one-dimensional-(1)H CPMG, two-dimensional homonuclear (1)H-(1)H TOCSY CPMG and heteronuclear (1)H-(13)C HSQC sequences. Semi-quantitative analysis of selected plasma metabolites along the disease evolution was carried out on pigs with ischemia-induced AHF treated with the BAL containing alginate beads with or without hepatocytes. A quantitative longitudinal follow-up was performed on characteristic metabolites via a one-dimensional CPMG sequence, including choline, glutamine, N-acetyl-glucosamine (NAG), pyruvate and trimethylamine-N-oxide (TMAO). The concentrations of choline and TMAO increased from the beginning to the end in animals treated with the BAL containing alginate beads without hepatocytes. Treatment of pigs with BAL containing hepatocytes resulted in an improvement of survival, the plasma concentrations of choline and TMAO being decreased in three out of five animals. Thus, NMR spectroscopy is a useful approach for the identification of toxic compounds which are involved in hepatic encephalopathy associated with AHF. These compounds can be cleared by a BAL resulting in the improvement of survival and neurological parameters of the animals.