Comparative Hepatology - Latest articles

Non invasive in vivo investigation of hepatobiliary structure and function in STII medaka (Oryzias latipes): Methodology and applications

Ron C Hardman, Seth W Kullman and David E Hinton — 2008-10-06

Background: A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions. Results: Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (<1 um), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 um), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated. Conclusions: The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant.

A new parameter using serum lactate dehydrogenase and alanine aminotransferase level is useful for predicting the prognosis of patients at an early stage of acute liver injury: A retrospective study

2008-08-14

Background: Although most patients with severe acute hepatitis are conservatively cured, some progress to acute liver failure (ALF) with a high rate of mortality. Based on the evidence that over-activation of macrophages, followed by disturbance of the hepatic microcirculation, plays a key role in ALF, we hypothesized that the production of serum lactate dehydrogenase (LDH) might increase in the liver under hypoxic conditions and could be an indicator to discriminate between conservative survivors and fatal patients at an early stage. Results: To confirm this hypothesis, we developed a new parameter with serum alanine aminotransferase (ALT) and LDH: the ALT-LDH index = serum ALT/(serum LDH - median of normal LDH range). We analyzed retrospectively 33 patients suffering acute liver injury (serum ALT more than 1000 U/L or prothrombin time expressed as international normalized ratio over 1.5 at admission) and evaluated the prognostic value of the ALT-LDH index, comparing data from the first 5 days of hospitalization with the Model for End-Stage Liver Disease (MELD) score. Patients whose symptoms had appeared more than 10 days before admission were excluded from this study. Among those included, 17 were conservative survivors, 9 underwent liver transplantation (LT) and 7 died waiting for LT. We found a rapid increase in the ALT-LDH index in conservative survivors but not in fatal patients. While the prognostic sensitivity and specificity of the ALT-LDH index was low on admission, at day 3 they were superior to the results of MELD. Conclusion: ALT-LDH index was useful to predict the prognosis of the patients with acute liver injury and should be helpful to begin preparation for LT soon after admission.

Relation between lipogranuloma formation and fibrosis, and the origin of brown pigments in lipogranuloma of the canine liver

Kaori Isobe, Hiroyuki Nakayama and Koji Uetsuka — 2008-05-12

Background: In a previous study we confirmed that canine hepatic lipogranuloma, defined as lesions consisting of small round cells which contain lipid vacuoles and brown pigments in their cytoplasm, was an assembly of Kupffer cells and/or macrophages, and that the cytoplasmic brown pigments in the lesions were hemosiderin and ceroid. However, the pathogenesis of the lesion remains unclear. Kupffer cells (resident macrophages) play a key role in hepatic fibrogenesis due to the production of cytokines including TGF-β. In the present study, we have examined 52 canine liver samples (age: newborn – 14 years; 25 males and 27 females) and investigated the correlation between lipogranuloma formation and fibrosis as well as the origin of brown pigments of lipogranulomas. Results: Lipogranulomas were detected histopathologically in 23 (44.2%) of the 52 liver samples. No significant correlation was found between the density of lipogranulomas and distribution of collagen type I/III in the liver. Pigmentation of lipogranulomas showed significant correlations with that on both hepatocytes and sinusoidal cells, indicating that pigments of lipogranuloma (hemosiderin and ceroid) might be derived from hepatocytes and Kupffer cells. Conclusion: Lipogranulomas are not a contributing factor in hepatic fibrosis, but might be a potential indicator of the accumulation of iron and lipid inside the liver.

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells

2008-05-05

Background: Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered in the hepatic sinusoids is in general considerably lower than the oxygen tension in the air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions with regard to oxygen would expect to improve cell survival, structure and function. In this study LSECs were isolated from rats and cultured under either 5% (normoxic) or 20% (hyperoxic) oxygen tensions, and several morpho-functional features were compared. Results: Cultivation of LSECs under normoxia, as opposed to hyperoxia improved the survival of LSECs and scavenger receptor-mediated endocytic activity, reduced the production of the pro-inflammatory mediator, interleukin-6 and increased the production of the anti-inflammatory cytokine, interleukin-10. On the other hand, fenestration, a characteristic feature of LSECs disappeared gradually at the same rate regardless of the oxygen tension. Expression of the cell-adhesion molecule, ICAM-1 at the cell surface was slightly more elevated in cells maintained at hyperoxia. Under normoxia, endogenous generation of hydrogen peroxide was drastically reduced whereas the production of nitric oxide was unaltered. Culture decline in high oxygen-treated cultures was abrogated by administration of catalase, indicating that the toxic effects observed in high oxygen environments is largely caused by endogenous production of hydrogen peroxide. Conclusion: Viability, structure and many of the essential functional characteristics of isolated LSECs are clearly better preserved when the cultures are maintained under more physiologic oxygen levels. Endogenous production of hydrogen peroxide is to a large extent responsible for the toxic effects observed in high oxygen environments.

Sustained virological and biochemical responses to lamivudine and adefovir dipivoxil combination in a chronic hepatitis B infection despite mutations conferring resistance to both drugs

2008-03-12

Background: Sequential monotherapies of nucleotide analogs used in chronic hepatitis B treatment can lead to the selection of a resistance mutation to each antiviral drug.Case presentationA patient with chronic hepatitis B was successively treated with lamivudine monotherapy, lamivudine-adefovir dual therapy, adefovir monotherapy and again with an adefovir-lamivudine dual therapy. Lamivudine-associated mutations (rtL180M and rtM204V/I) followed by adefovir-associated mutations (rtN236T and rtA181V) emerged during the two monotherapy regimens. Despite the presence of rtM204V/I, rtA181V, and rtN236T mutations at the beginning of the second dual therapy, sustained biochemical and virological responses have been observed thus far after 23 months. Conclusion: This case illustrates that rtM204V/I, rtA181V, and rtN236T resistance mutations can coexist in a patient but do not preclude the recycling of lamivudine and adefovir in combination therapy, when no other therapeutic choices are available.

Over-expression of glutamine synthase in focal nodular hyperplasia (part 1): Early stages in the formation support the hypothesis of a focal hyper-arterialisation with venous (portal and hepatic) and biliary damage

2008-02-29

Background: Most focal nodular hyperplasia (FNH) cases are diagnosed by chance. We studied a case of pre-FNH. We used glutamine synthase as an immunohistochemical marker for perivenous zones. Results: Neither fibrotic scars nor hepatocytic nodules surrounded by fibrosis with a ductular reaction were observed in the sections studied. Most sections generally displayed preserved architecture. The glutamine synthase-positive hepatocyte areas were wider than those observed in non-tumoural surrounding liver, and they tended to extend outwards. Portal tracts bordering the nodule were more fibrotic, with an absence of portal veins and ducts and with arterial proliferation often in proximity with large draining veins; isolated arteries were present and hepatic veins were rare in the nodule. These features appeared prior to the identification of other major criteria characteristics of FNH, thus supporting the "hypothesis of Wanless". Conclusion: The findings confirm that in FNH there is a portal tract injury leading to local portal vein injury. This leads to a cascade of events, including arterial venous shunts, ductular reaction, and scar formation.

Plasma redox status is impaired in the portacaval shunted rat – the risk of the reduced antioxidant ability

2008-02-05

Background: Portacaval shunting in rats produces a reduction of hepatic oxidant scavenging ability. Since this imbalance in hepatic oxidant/antioxidant homeostasis could coexist with systemic changes of oxidant stress/antioxidant status, plasma oxidants and antioxidant redox status in plasma of portacaval shunted-rats were determined. Results: Male Wistar male: Control (n = 11) and with portacaval shunt (PCS; n = 11) were used. Plasma levels of the oxidant serum advanced oxidation protein products (AOPP), lipid hydroperoxides (LOOH), the antioxidant total thiol (GSH) and total antioxidant status (TAX) were measured. Albumin, ammonia, Aspartate-aminotransferase (AST), Alanine-aminotransferase (ALT), thiostatin and alpha-1-acid glycoprotein (α1-AGP) were also assayed 4 weeks after the operation. AOPPs were significantly higher (50.51 ± 17.87 vs. 36.25 ± 7.21 μM; p = 0.02) and TAX was significantly lower (0.65 ± 0.03 vs. 0.73 ± 0.06 mM; p = 0.007) in PCS compared to control rats. Also, there was hypoalbuminemia (2.54 ± 0.08 vs. 2.89 ± 0.18 g/dl; p = 0.0001) and hyperammonemia (274.00 ± 92.25 vs. 104.00 ± 48.05 μM; p = 0.0001) and an increase of thiostatin (0.23 ± 0.04 vs. 0.09 ± 0.01 mg/ml; p = 0.001) in rats with a portacaval shunt. The serum concentration of ammonia is correlated with albumin levels (r = 0.624; p = 0.04) and TAX correlates with liver weight (r = 0.729; p = 0.017) and albumin levels (r = 0.79; p = 0.007) Conclusion: These findings suggest that in rats with a portacaval shunt a systemic reduction of oxidant scavenging ability, correlated with hyperammonemia, is principally produced. It could be hypothesized, therefore, that the reduced antioxidant defences would mediate a systemic inflammation.

Establishment of an early liver fibrosis model by the hydrodynamics-based transfer of TGF-β1 gene

Kun-Lin Yang, Kuo-Chen Hung, Wen-Teng Chang and Eric IC Li — 2007-10-19

Background: Liver fibrosis represents a significant and severe health care problem and there are no efficient drugs for therapy so far. Preventing the progression of fibrogenesis and revival endogenous repair activities is an important strategy for both current and future therapies. Many studies of liver fibrosis consist of animal testing with various hepatotoxins. Although this method is often used, the model at which cirrhosis or extensive fibrosis becomes irreversible has not been well defined and is not representative of early-stage fibrogenesis. We here report the establishment of a transient and reversible liver fibrosis animal model which may better represent an early and natural fibrotic event. We used a high-speed intravenous injection of naked plasmid DNA of transforming growth factor-β1 (TGF-β1) gene which is under the control of a metallothionein-regulated gene in a pPK9A expression vector into the tail vein (the hydrodynamics-based transfer) and fed the mouse with zinc sulfate (ZnSO4)-containing water simultaneously. Results: Using our hydrodynamics-based gene transfer model we found that upon induction by ZnSO4, the serum TGF-β1 level in Balb/c mice and Sp1 transcription factor binding activity peaked at 48 h and declined thereafter to a normal level on the 5th day. In addition, mRNA and protein levels of TGF-β1 in the liver were also upregulated at 48 h. Furthermore, induction of TGF-β1 increased the α-smooth muscle actin (α-SMA), p-Smad2/3, hydroxyproline and collagen 1A2 (Col 1A2) levels in the liver, suggesting a significant liver fibrosis. Conclusion: Our results show that TGF-β1 in pPK9a-transferred mice liver with ZnSO4 feeding can achieve a high expression level with significant fibrosis. However, since TGF-β1 induction is transient in our model, the fibrotic level does not reach a large scale (panlobular fibrosis) as seen in the CCl4-treated liver. Our model hence represents a dynamic and reversible liver fibrosis and could be a useful tool for studying early molecular mechanism of fibrogenesis or screening of antifibrotic drugs for clinical use.

Major HGF-mediated regenerative pathways are similarly affected in human and canine cirrhosis

2007-07-31

Background: The availability of non-rodent animal models for human cirrhosis is limited. We investigated whether privately-owned dogs (Canis familiaris) are potential model animals for liver disease focusing on regenerative pathways. Several forms of canine hepatitis were examined: Acute Hepatitis (AH), Chronic Hepatitis (CH), Lobular Dissecting Hepatitis (LDH, a specific form of micronodulair cirrhosis), and Cirrhosis (CIRR). Canine cirrhotic samples were compared to human liver samples from cirrhotic stages of alcoholic liver disease (hALC) and chronic hepatitis C infection (hHC). Results: Canine specific mRNA expression of the regenerative hepatocyte growth factor (HGF) signaling pathway and relevant down-stream pathways were measured by semi-quantitative PCR and Western blot (STAT3, PKB, ERK1/2, and p38-MAPK). In all canine groups, levels of c-MET mRNA (proto-oncogenic receptor for HGF) were significantly decreased (p < 0.05). Surprisingly, ERK1/2 and p38-MAPK were increased in CH and LDH. In the human liver samples Western blotting indicated a high homology of down-stream pathways between different etiologies (hALC and hHC). Similarly activated pathways were found in CIRR, hALC, and hHC. Conclusion: In canine hepatitis and cirrhosis the major regenerative downstream pathways were activated. Signaling pathways are similarly activated in human cirrhotic liver samples, irrespective of the differences in etiology in the human samples (alcohol abuse and HCV-infection). Therefore, canine hepatitis and cirrhosis could be an important clinical model to evaluate novel interventions prior to human clinical trials.

Evolving concepts of liver fibrogenesis provide new diagnostic and therapeutic options

Olav A Gressner, Ralf Weiskirchen and Axel M Gressner — 2007-07-30

Despite intensive studies, the clinical opportunities for patients with fibrosing liver diseases have not improved. This will be changed by increasing knowledge of new pathogenetic mechanisms, which complement the "canonical principle" of fibrogenesis. The latter is based on the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts induced by hepatocellular injury and consecutive inflammatory mediators such as TGF-β. Stellate cells express a broad spectrum of matrix components. New mechanisms indicate that the heterogeneous pool of (myo-)fibroblasts can be supplemented by epithelial-mesenchymal transition (EMT) from cholangiocytes and potentially also from hepatocytes to fibroblasts, by influx of bone marrow-derived fibrocytes in the damaged liver tissue and by differentiation of a subgroup of monocytes to fibroblasts after homing in the damaged tissue. These processes are regulated by the cytokines TGF-β and BMP-7, chemokines, colony-stimulating factors, metalloproteinases and numerous trapping proteins. They offer innovative diagnostic and therapeutic options. As an example, modulation of TGF-β/BMP-7 ratio changes the rate of EMT, and so the simultaneous determination of these parameters and of connective tissue growth factor (CTGF) in serum might provide information on fibrogenic activity. The extension of pathogenetic concepts of fibrosis will provide new therapeutic possibilities of interference with the fibrogenic mechanism in liver and other organs.