Longitudinal micro-computed tomography monitoring of progressive liver regeneration in a mouse model of partial hepatectomy.

The partial hepatectomy (PH) model is widely used to study liver regeneration. Currently, the extent of regeneration is analyzed by measuring the weight of the liver post-mortem or by magnetic resonance imaging. In this study we aimed to determine whether liver volume gain can be accurately measured using micro-computed tomography (microCT). Approximately 42% of the liver was removed by ligation in C57BL/6 N mice. Mice were divided into two study groups. In group 1 conventional characterization of liver hyperplasia was performed by weighing the liver post-mortem. In group 2, liver volume gain was determined by microCT volume estimation. MicroCT results showed equivalent regeneration rates compared with the conventional method without the need to mathematically determine initial liver weights before PH. This parameter is strongly influenced by the age, strain and sex of the mice. In addition non-invasive microCT determination of volume gain over multiple time-points using the same animal reduces the number of animals needing to be used (in line with the 3R principle of replacement, reduction and refinement).

Early hepatocyte DNA synthetic response posthepatectomy is modulated by IL-6 trans-signaling and PI3K/AKT activation.

BACKGROUND & AIMS: Interleukin-6 (IL-6) is a crucial factor in liver regeneration following partial hepatectomy (PH); however, the role of IL-6 and IL-6 trans-signaling in particular, in hepatocyte mitosis remains controversial. IL-6 trans-signaling relies upon the release of the soluble IL-6R (sIL-6R), which binds IL-6 to form an agonistic IL-6/sIL-6R complex. Herein we have examined the hypothesis that IL-6 trans-signaling plays a crucial and distinct role in liver regeneration following PH. METHODS: The specific IL-6/sIL-6R antagonist, sgp130Fc, was expressed in mice and analyzed for its effect on hepatocyte mitosis following PH. Alternatively, we examined the effect of the IL-6/sIL-6R super-agonist, Hyper-IL-6, or IL-6 expressed either alone or in combination with hepatocyte growth factor (HGF) on hepatocyte mitosis in the absence of PH. RESULTS: Following PH, the dramatic rise of circulating IL-6 levels is accompanied by a concurrent ∼2-fold increase in circulating sIL-6R levels. Ectopic expression of sgp130Fc reduced hepatocyte mitosis by about 40% at early times following PH, while substantially reducing AKT, but not STAT3, activation. But, ectopic Hyper-IL-6 expression in mice without PH was not mitogenic to hepatocytes in vivo. Rather, Hyper-IL-6, but not IL-6, markedly increased HGF-induced hepatocyte mitosis. This cooperative effect correlated with greater resistance of HIL-6 than IL-6 to HGF-mediated reduction of AKT activation, rather than changes in STAT3 or MAPK signaling, and was completely blocked by PI3K inhibition. CONCLUSIONS: Following PH, IL-6/sIL-6R cooperates with growth factors, through a PI3K/AKT-dependent mechanism to promote entry of hepatocytes into the cell cycle.

Role of IL-6 trans-signaling in CCl4induced liver damage.

Interleukin-6 (IL-6) plays an important role in liver regeneration and protection against liver damage. In addition to IL-6 classic signaling via membrane bound receptor (mIL-6R), IL-6 signaling can also be mediated by soluble IL-6R (sIL-6R) thereby activating cells that do not express membrane bound IL-6R. This process has been named trans-signaling. IL-6 trans-signaling has been demonstrated to operate during liver regeneration. We have developed methods to specifically block or mimic IL-6 trans-signaling. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling whereas an IL-6/sIL-6R fusion protein (Hyper-IL-6) mimics IL-6 trans-signaling. Using these tools we investigate the role of IL-6 trans-signaling in CCl4 induced liver damage. Blockade of IL-6 trans-signaling during CCl4 induced liver damage led to higher liver damage, although induction of Cyp4502E1 and thus bioactivation of CCl4 was unchanged. Depletion of neutrophils resulted in reduced liver transaminase levels irrespective of IL-6 trans-signaling blockade. Furthermore, IL-6 trans-signaling was important for refilling of hepatocyte glycogen stores, which were depleted 24 h after CCl4 treatment. We conclude that IL-6 trans-signaling via the soluble IL-6R is important for the physiologic response of the liver to CCl4 induced chemical damage.

Impact of interleukin-6 classic- and trans-signaling on liver damage and regeneration.

Interleukin-6 (IL-6) has been suggested to play a pivotal role in liver regeneration. IL-6 on target cells activates a receptor complex consisting of the IL-6 receptor (IL-6R) and the signal transducing receptor subunit gp130. Not all cells in the body express the IL-6R on the cell surface. IL-6 can signal via two different pathways: classical signaling via the membrane bound IL-6R and IL-6 trans-signaling via a naturally occurring soluble IL-6R (sIL-6R). This second pathway widens the scope of IL-6 signaling since also cells expressing no membrane bound IL-6R can be stimulated by the trans-signal pathway. Mimicking IL-6 trans-signaling via a designer molecule, Hyper-IL-6 has been shown to accelerate liver regeneration. Another designer molecule, sgp130Fc, specifically blocks IL-6 trans-signaling. Using these proteins we investigated the contribution of IL-6 classic- and trans-signaling in the liver. Here we review the role of IL-6 signaling in response to liver damage and during liver regeneration.

Interleukin-6 trans-signaling regulates glycogen consumption after D-galactosamine-induced liver damage.

The cytokine interleukin-6 (IL-6) is important for liver regeneration. IL-6 can stimulate target cells either by binding to the membrane-bound IL-6 receptor (IL-6R) leading to dimerization and activation of gp130 or by binding to a soluble IL-6R that results in an activation of gp130 independently of membrane-bound IL-6R, a process called trans-signaling. We have established a transgenic mouse line, in which only trans-signaling is abrogated whereas signaling via the membrane-bound IL-6R is intact. In the present study we employed this mouse model to ask whether the activity of IL-6 during repair of mild liver damage acts via classic or trans-signaling. We analyzed liver regeneration and showed that intracellular signaling, proliferation, and glycogenolysis are reduced in the transgenic mice and thus are regulated by IL-6 trans-signaling. Taken together our results show that upon liver damage, activation of the gp130 pathway depends on the sIL-6R.

Tarde demais para os deuses”: Três características de uma perspectiva ser-historial sobre a religião / To late for the “gods”: Three characteristics of a historial perspective be on religion

Heidegger é mais lembrado hoje como o pensador da técnica e da longa noite indigente do niilismo, com boas razões. Uma visão sobre a religião pode ser derivada daí. Mais difícil de coadunar com a pergunta sobre a técnica, e com a perspectiva ser-historial sobre a religião que daí deriva, é a importância crucial da obra de Hölderlin para Heidegger. Nem Hölderlin é apenas um poeta que interdita a presença dos deuses entre nós, nem Heidegger é só o pensador da indigência técnica quando o interpreta.

Nowadays, Heidegger is most often remembered as the thinker of technique and of the long precarious night of nihilism, and with good reason. One might derive a view on religion from this standpoint, although hard to reconcile with the crucial role played by Hölderlin in Heidegger’s thinking. Hölderlin is not just a poet who denies the presence of gods among us, and Heidegger is not only the thinker of technical poverty when he reads him.

HB-EGF is a paracrine growth stimulator for early tumor prestages in inflammation-associated hepatocarcinogenesis.

BACKGROUND/AIMS: We studied the impact of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on inflammation-driven hepatocarcinogenesis. METHODS: HB-EGF expression was determined by qRT-PCR and immunodetection in hepatocellular adenoma and carcinoma and in mesenchymal (MC) and parenchymal liver cells obtained from different models of inflammation. The functions of HB-EGF in early hepatocarcinogenesis were assessed in co-cultures of unaltered and initiated/premalignant hepatocytes. RESULTS: In human and rat (pre)malignant liver lesions, HB-EGF levels were comparable to that of the surrounding tissue. In inflamed livers HB-EGF was expressed predominantly in MC and was further increased by pro-inflammatory lipopolysaccharide (LPS) or linoleic acid hydroperoxide (LOOH). In culture, DNA-replication occurred rather in initiated/premalignant than unaltered hepatocytes and was further elevated by LOOH- or LPS-stimulated MC-supernatants. The supernatant effects were abrogated by pre-incubation with HB-EGF-neutralizing antisera. HB-EGF itself induced DNA-replication and mitosis preferentially in the initiated/premalignant cells. When transducing hepatocytes with a dominant-negative ErbB1-construct, HB-EGF-induced DNA-replications were blocked completely in unaltered hepatocytes but incompletely in initiated/premalignant cells, which suggests elevated ErbB-mediated signal transduction in first stages of hepatocarcinogenesis. CONCLUSIONS: Pro-inflammatory stimuli induce the release of HB-EGF from MC, which stimulates DNA-replication in initiated/premalignant hepatocytes. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

Deregulation of the activin/follistatin system in hepatocarcinogenesis.

BACKGROUND/AIMS: Activins A and E negatively regulate hepatic cell number by inhibiting cell replication and inducing apoptosis. Follistatin and follistatin-like 3 bind activins and antagonise their biological activities. Aim of our study was to investigate, whether activins and follistatins may play a role in hepatocarcinogenesis. METHODS: Expression levels of follistatin, follistatin-like 3, and activin subunits beta(A) as well as beta(E) were investigated in chemically induced rat and human liver tumours by real-time PCR and immunohistochemistry. In addition, the effects of follistatin and activin A on DNA synthesis of normal as well as preneoplastic hepatocytes and hepatoma cells were analysed. RESULTS: Follistatin was overexpressed while both activin subunits were downregulated in the majority of rat and human liver tumours. Follistatin-like 3 expression was low in normal but enhanced in malignant rat liver. In human normal liver, in contrast, it was abundantly expressed but downregulated in liver cancer. Administration of follistatin to normal and preneoplastic hepatocytes stimulated DNA synthesis preferentially in preneoplastic rat hepatocytes, whereas activin A repressed it. CONCLUSIONS: The balanced expression of follistatins and activins becomes deregulated during hepatocarcinogenesis. The sensitivity of preneoplastic hepatocytes to activin signals suggests the activin/follistatin system as promising target for therapeutic intervention.

Non-parenchymal liver cells support the growth advantage in the first stages of hepatocarcinogenesis.

Hepatocellular carcinoma almost always arises in chronically inflamed livers. We developed a culture model to study the role of non-parenchymal cells (NPCs) for inflammation-driven hepatocarcinogenesis. Rats were treated with the carcinogen N-nitrosomorpholine, which induced initiated hepatocytes expressing the marker placental glutathione-S-transferase (GSTp). After 21 days two preparations of hepatocytes were made: (i) conventional ones (Hep-conv) containing NPCs and (ii) hepatocytes purified of NPCs (Hep-pur). Initiated hepatocytes, being positive for GSTp (GSTp-pos) were present in both preparations and were cultured along with normal hepatocytes, being negative for GSTp (GSTp-neg). Under any culture condition DNA synthesis was approximately 4-fold higher in GSTp-pos than in GSTp-neg hepatocytes demonstrating the inherent growth advantage of the first stages of hepatocarcinogenesis. Hepatocytes showed approximately 3-fold lower rates of DNA synthesis in Hep-pur than in Hep-conv, which was elevated above Hep-conv levels by addition of NPC or NPC-supernatant. Pretreatment of NPCs with proinflammatory lipopolysaccharide (LPS) further increased DNA synthesis. Thus, NPCs release soluble growth stimulators. Next we investigated the effect of specific cytokines produced by NPCs. Tumour necrosis factor alpha and interleukin 6 barely altered DNA synthesis, whereas hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and the heparin-binding epidermal growth factor-like growth factor (HB-EGF) were potent inducers of DNA replication in both, GSTp-neg and GSTp-pos cells. In conclusion, DNA synthesis of hepatocytes is increased by factors released from NPCs, an effect augmented by LPS-stimulation. NPC-derived cytokines, such as KGF, HGF and HB-EGF, stimulate DNA synthesis preferentially in initiated hepatocytes, presumably resulting in tumour promotion. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

Expression of activins C and E induces apoptosis in human and rat hepatoma cells.

Activins C and E (homodimers of the betaC and betaE subunits), which are almost exclusively expressed in the liver, are members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. We examined their expression in three different hepatoma cell lines and found that, compared with normal liver or primary hepatocytes, human hepatoblastoma (HepG2), human hepatocellular carcinoma (Hep3B) and rat hepatoma (H4IIEC3) cells have either completely lost or drastically reduced the expression of activins C and E. In order to elucidate the biological function of these proteins we transiently transfected HepG2, Hep3B and H4IIEC3 cell lines with rat activin betaC or betaE cDNA to study the consequences of restoring activin expression in hepatoma cells. Transfection with activin betaA, a known inhibitor of hepatic DNA synthesis and inducer of apoptosis, served as a positive control. We found that transfection of the three cell lines with activin betaC or betaE, as well as with activin betaA, reduced the increase in cell number by up to 40% compared with cells transfected with a control plasmid. Co-culture with a CHO cell clone secreting activin C also inhibited HepG2 cell multiplication. Furthermore, the three hepatoma cell lines studied showed an enhanced rate of apoptosis and elevated levels of active caspases in response to activin transfection. These results indicate that activins C and E share the potential to induce apoptosis in liver derived cell lines with activin A and TGFbeta1.

Experiência nacional e interpretação: a recepção americana de Heidegger / National experience and interpretation: the american reception of Heidegger

Uma comparação entre dois interpretes americanos de Heidegger, que destaca as premissas e os objetivos comuns a ambos. Dreyfus e Rorty usam o pensamento de Heidegger seletivamente. A autora aponta as dificuldades a que tal leitura leva, e faz uma distinção entre as respostas de Drefuys e de Rorty. Dreyfus atribui a Heideggger uma má compreensão do seu próprio projeto. Rorty reconhece que suas premissas e objetivos não são heideggerianos, e defende que é legítimo usar uma teoria a serviços do pragmatismo e da democracia. A filosofia, e, por conseguinte,a interpretação, é um modo de esclarecer e legitimar as preferências da comunidade nacional a qual pertencemos. A conclusão desse estudo é que Rorty está certo em parte, na medida em que não se pode evitar uma apropriação nacional, seletiva de uma obra. Mas pode-se e deve colocar em questão as premisssas e os objetivos que motivam e orientam nossa interpretação(AU)