Dysfunctional Cori and Krebs cycle and inhibition of lactate transporters constitute a mechanism of primary nonfunction of fatty liver allografts.

Orthotopic liver transplantation (OLT) is a lifesaving procedure. However, grafts may fail due to primary nonfunction (PNF). In the past, we demonstrated PNFs to be mainly associated with fatty allografts, and given its unpredictable nature, the development of a disease model is urgently needed. In an effort to investigate mechanism of fatty allograft-associated PNFs, we induced fatty liver disease in donor animals by feeding rats a diet deficient in methionine and choline (MCD). We performed OLT with allografts of different grades of hepatic steatosis and compared the results to healthy ones. We assessed liver function by considering serum biochemistries, and investigated genome wide responses following OLT of healthy and fatty allograft-associated PNFs. Furthermore, we performed immunohistochemistry to evaluate markers of oxidative stress and reperfusion injury, inflammation, glycolysis and gluconeogenesis, lactate transport, and its utilization as part of the Cori cycle. Strikingly, PNFs are strictly lipid content dependent. Nonetheless, a fat content of ≤17% and an increase in the size of hepatocytes of ≤11% (ballooning) greatly improved outcome of OLTs and the hepatic microcirculation. Mechanistically, PNFs arise from a dysfunctional Cori cycle with complete ablation of the lactate transporter SLC16A1. Thus, lipid-laden hepatocytes fail to perform gluconeogenesis via lactate reutilization, and the resultant hyperlactatemia and lactic acidosis causes cardiac arrhythmogenicity and death. Furthermore, the genomic and immunohistochemistry investigations underscore a dysfunctional Krebs cycle with impaired energy metabolism in lipid-burdened mitochondria. Together, we show fatty allografts to be highly vulnerable towards ischemia/reperfusion-injury, and stabilizing the Cori cycle is of critical importance to avert PNFs.

The pathogenesis of diclofenac induced immunoallergic hepatitis in a canine model of liver injury.

Hypersensitivity to non-steroidal anti-inflammatory drugs is a common adverse drug reaction and may result in serious inflammatory reactions of the liver. To investigate mechanism of immunoallergic hepatitis beagle dogs were given 1 or 3 mg/kg/day (HD) oral diclofenac for 28 days. HD diclofenac treatment caused liver function test abnormalities, reduced haematocrit and haemoglobin but induced reticulocyte, WBC, platelet, neutrophil and eosinophil counts. Histopathology evidenced hepatic steatosis and glycogen depletion, apoptosis, acute lobular hepatitis, granulomas and mastocytosis. Whole genome scans revealed 663 significantly regulated genes of which 82, 47 and 25 code for stress, immune response and inflammation. Immunopathology confirmed strong induction of IgM, the complement factors C3&B, SAA, SERPING1 and others of the classical and alternate pathway. Alike, marked expression of CD205 and CD74 in Kupffer cells and lymphocytes facilitate antigen presentation and B-cell differentiation. The highly induced HIF1A and KLF6 protein expression in mast cells and macrophages sustain inflammation. Furthermore, immunogenomics discovered 24, 17, 6 and 11 significantly regulated marker genes to hallmark M1/M2 polarized macrophages, lymphocytic and granulocytic infiltrates; note, the latter was confirmed by CAE staining. Other highly regulated genes included alpha-2-macroglobulin, CRP, hepcidin, IL1R1, S100A8 and CCL20. Diclofenac treatment caused unprecedented induction of myeloperoxidase in macrophages and oxidative stress as shown by SOD1/SOD2 immunohistochemistry. Lastly, bioinformatics defined molecular circuits of inflammation and consisted of 161 regulated genes. Altogether, the mechanism of diclofenac induced liver hypersensitivity reactions involved oxidative stress, macrophage polarization, mastocytosis, complement activation and an erroneous programming of the innate and adaptive immune system.

Immune-mediated liver injury of the cancer therapeutic antibody catumaxomab targeting EpCAM, CD3 and Fcγ receptors.

The immunotherapeutic catumaxomab targets EpCAM positive cancers and is approved for the treatment of peritoneal carcinomatosis. To assess the safety of intravenous applications a phase 1 clinical trial was initiated. Treatment of EpCAM positive tumor patients with catumaxomab caused dose dependent hepatitis as evidenced by significant elevations in serum alanine- and aspartate aminotransferases, bilirubin, γGT and induction of the acute phase C-reactive protein (CRP) and the cytokines IL6 and IL8. The first patient receiving 10µg catumaxomab experienced fatal acute liver failure which led to the termination of the study. Immmunopathology revealed catumaxomab to bind via its Fc-fragment to FcγR-positive Kupffer cells to stimulate CRP, chemokine and cytokine release. The observed CD3+T-cell margination at activated hepatic macrophages exacerbated T-cell mediated cytotoxicity. Strikingly, the combined Kupffer/T-cell responses against liver cells did not require hepatocytes to be EpCAM-positive. Catumaxomab's off-target activity involved T-cell mediated lysis of the granzyme B cell death pathway and the molecular interaction of hepatic sinusoidal macrophages with T-cells induced cytolytic hepatitis. Although the bile ducts were surrounded by densely packed lymphocytes these rarely infiltrated the ducts to suggest an intrahepatic cholestasis as the cause of hyperbilirubinaemia. Lastly, evidence for the programming of memory T-cells was observed with one patient that succumbed to his cancer six weeks after the last catumaxomab infusion. In conclusion, our study exemplifies off-target hepatotoxicity with molecularly targeted therapy and highlights the complexities in the clinical development of immunotherapeutic antibodies.

Immunogenomics reveal molecular circuits of diclofenac induced liver injury in mice.

Diclofenac is a non-steroidal anti-inflammatory drug and its use can be associated with severe adverse reactions, notably myocardial infarction, stroke and drug-induced liver injury (DILI). In pursue of immune-mediated DILI mechanisms an immunogenomic study was carried out. Diclofenac treatment of mice at 30 mg/kg for 3 days caused significant serum ALT and AST elevations, hepatomegaly and degenerative changes including hepatic glycogen depletion, hydropic swelling, cholesterolosis and eosinophilic hepatocytes with one animal presenting subsegmental infarction due to portal vein thrombosis. Furthermore, portal/periportal induction of the rate limiting enzyme in ammonia detoxification, i.e. carbamoyl phosphate synthetase 1 was observed. The performed microarray studies informed on > 600 differential expressed genes of which 35, 37 and 50 coded for inflammation, 51, 44 and 61 for immune and 116, 129 and 169 for stress response, respectively after single and repeated dosing for 3 and 14 days. Bioinformatic analysis defined molecular circuits of hepatic inflammation with the growth hormone (Ghr)- and leptin receptor, the protein-tyrosine-phosphatase, selectin and the suppressor-of-cytokine-signaling (Socs) to function as key nodes in gene regulatory networks. Western blotting confirmed induction of fibronectin and M-CSF to hallmark tissue repair and differentiation of monocytes and macrophages. Transcript expression of the macrophage receptor with collagenous structure increased > 7-fold and immunohistochemistry of CD68 evidenced activation of tissue-resident macrophages. Importantly, diclofenac treatment prompted strong expression of phosphorylated Stat3 amongst individual animals and the associated 8- and 4-fold Soc3 and Il-6 induction reinforced Ghr degradation as evidenced by immunoblotting. Moreover, immunohistochemistry confirmed regulation of master regulatory proteins of diclofenac treated mice to suggest complex pro-and anti-inflammatory reactions in immune-mediated hepatic injury. The findings encourage translational research.

Decoding c-Myc networks of cell cycle and apoptosis regulated genes in a transgenic mouse model of papillary lung adenocarcinomas.

The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays.Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies.

MYC-regulated genes involved in liver cell dysplasia identified in a transgenic model of liver cancer.

Foci of liver cell dysplasia (LCD) are distinct morphological entities and may evolve into hepatocellular carcinomas (HCCs). While most HCCs overexpress c-Myc, its role in LCD remains uncertain. Therefore, a c-Myc transgenic model of HCC was investigated to understand the genetic events forcing liver cells into dysplasia and subsequent malignant transformation. Specifically, whole genome scans enabled fingerprinting of genes at different stages of disease, ie LCD and HCC, while laser microdissected LCD lesions were used to validate regulation of candidate genes by quantitative real-time RT-PCR, ie Mybbp1a, Rps7, Rps19, Rpl10a, Skp1a, Tfdp1, Nhp2, and Bola2. EMSA band shift assays confirmed c-Myc DNA binding at regulatory sequences of candidate gene-specific promoters. Additionally, published ChIP-seq data helped to define the candidate genes as c-Myc bona fide targets. Treatment of the human hepatoma cell line HepG2 with hepatic growth factor (Hgf) caused c-Myc protein induction and transcriptional up-regulation of candidate genes, albeit at different levels when individual genes were compared. A significant increase of HepG2 entering the G1-phase was associated with up-regulation of the candidate genes in an Hgf concentration-dependent matter. Finally, we confirmed regulation of candidate genes in patients' samples with low- and high-grade dysplasia and HCC staged T1 to T3, while their expression was unchanged in focal nodular hyperplasia and hepatic adenoma, therefore asserting the diagnostic value and clinical significance of these candidate genes. Overall, novel c-Myc targeted genes were identified and may contribute to hepatocyte transformation by altering cell cycle control, thereby contributing to c-Myc's oncogenic activity.

Combined microPET/CT for imaging of hepatocellular carcinoma in mice.

The EGF-transgenic mouse is a genetic model of hepatocellular carcinoma that allows for a comprehensive study of signal pathways, molecular interactions and the evaluation of novel therapeutic concepts. In this regard, non-invasive imaging tools for serial in-vivo monitoring of tumor load and growth are highly desirable. This study therefore aimed at demonstrating the feasibility of non-invasive in-vivo imaging of primary liver malignancies in mice using combined contrast-enhanced microCT and F-18 FDG microPET. In our murine disease model, microCT enabled imaging of primary liver tumors down to a lesional diameter of 0.9 mm. F-18 FDG tumor-to-non-tumor ratio of HCCs was observed to be dependent on lesion size and linked to overpression of glucose transporters and hexokinase isoenzymes as determined by gene expression studies. Histopathologic analyses indicated an increased cellular dedifferentiation with increase lesion size, as well.

Epidermal growth factor-induced hepatocellular carcinoma: gene expression profiles in precursor lesions, early stage and solitary tumours.

Epidermal growth factor is an important mitogen for hepatocytes. Its overexpression promotes hepatocellular carcinogenesis. To identify the network of genes regulated through EGF, we investigated the liver transcriptome during various stages of hepatocarcinogenesis in EGF2B transgenic mice. Targeted overexpression of IgEGF induced distinct hepatocellular lesions and eventually solid tumours at the age of 6-8 months, as evidenced by histopathology. We used the murine MG U74Av2 oligonucleotide microarrays to identify transcript signatures in 12 tumours of small (n=5, pooled), medium (n=4) and large sizes (n=3), and compared the findings with three nontumorous transgenic livers and four control livers. Global gene expression analysis at successive stages of carcinogenesis revealed hallmarks linked to tumour size. A comparison of gene expression profiles of nontumorous transgenic liver versus control liver provided insight into the initial events predisposing liver cells to malignant transformation, and we found overexpression of c-fos, eps-15, TGIF, IGFBP1, Alcam, ets-2 and repression of Gas-1 as distinct events. Further, when gene expression profiles of small manifested tumours were compared with nontumorous transgenic liver, additional changes were obvious and included overexpression of junB, Id-1, minopontin, villin, claudin-7, RR M2, p34cdc2, cyclinD1 and cyclinB1 among others. These genes are therefore strongly associated with tumour formation. Our study provided new information on the tumour stage-dependent network of EGF-regulated genes, and we identified candidate genes linked to tumorigenes and progression of disease.