Joszef Dudas

IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF).

Several lines of evidence suggest a role of insulin-like growth factor I (IGF-I) in the regulation of apoptosis. Up to now its impact on many specific cells is unknown. We therefore studied the effect of IGF-I on two similar mesenchymal matrix-producing cell types of the liver, the hepatic stellate cells (HSC) and the myofibroblasts (rMF). The present study aimed to reveal the influence of IGF-I on cell cycle and apoptosis of HSC and rMF and to elucidate responsible signaling. While IGF-I significantly increased DNA synthesis in HSC, cell number decreased and apoptosis increased. In rMF IGF-I also increased DNA synthesis, which is, however, followed by proliferation. Blocking extracellular signal regulating kinase (ERK) revealed that in HSC, bcl-2 upregulation and bax downregulation are effected downstream of ERK, whereas downregulation of NFκB and consecutive of bcl-xL is mediated upstream. In the rMF upregulation of both, the antiapoptotic bcl-2 and bcl-xL is mediated upstream of ERK. The expression of the proapoptotic bax is not regulated by IGF-I in rMF. The studies demonstrate a completely different effect and signaling of IGF-I in two morphologically and functionally similar matrix-producing cells of the liver.

Atorvastatin induces apoptosis by a caspase‐9‐dependent pathway: an in vitro study on activated rat hepatic stellate cells

Background: Statins are shown to have cholesterol‐independent properties such as anti‐inflammation and immunomodulation. Activated hepatic stellate cells (HSCs) acquire the capacity to synthesize matrix proteins in damaged liver. We tested the hypothesis that atorvastatin may be capable of inducing apoptosis in HSCs.

Effect of Radiation on Gene Expression of Rat Liver Chemokines: In Vivo and In Vitro Studies

Abstract Moriconi, F., Christiansen, H., Raddatz, D., Dudas, J., Hermann, R. M., Rave-Fränk, M., Sheikh, N., Saile, B., Hess, C. F. and Ramadori, G. Effect of Radiation on Gene Expression of Rat Liver Chemokines: In Vivo and In Vitro Studies. Radiat. Res. 169, 162–169 (2008). The aim of the study was to analyze the effect of a single irradiation on chemokine gene expression in the rat liver and in isolated rat hepatocytes. RNA extracted from livers and from hepatocytes within the first 48 h after irradiation was analyzed by real-time PCR and the Northern blot assay. The chemokine concentrations in the serum of irradiated rats were measured quantitatively by ELISA. A significant radiation-induced increase of CINC1, IP10, MCP1, MIP3α, MIP3β, MIG and ITAC gene expression could be detected at the RNA level in the liver. CINC1, MCP1 and IP10 serum levels were significantly increased. In rat hepatocytes in vitro, only MIP3α showed a radiation-induced increase in expression, while CINC1, IP10, MIP3β, MIG, MIP1α, ITAC and SDF1 RNA levels were significantly down-regulated. However, incubation of irradiated hepatocytes in vitro with either TNF-α, IL1β, or IL6 plus TNF-α led to up-regulation of MCP1, IP10 and MCP1 or CINC1 and MIP3β, respectively. Irradiation of the liver induces up-regulation of the genes of the main proinflammatory chemokines, probably through the action of locally synthesized proinflammatory cytokines. The reason for the lack of liver inflammation in this model has still to be clarified.

Immune cells in primary gastrointestinal stromal tumors

Introduction Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. They are regarded as having relatively uniform histology, although their potential for malignant behavior varies. Despite a strong promoting role of tumor-infiltrating innate immune cells in neoplastic progression, the presence of immune cells in GISTs has not yet been studied. Methods A total of 47 untreated, c-kit-positive primary GISTs were immunohistochemically analyzed to distinguish histiocytic and dendritic cells (DCs) (KIM-1P, fascin, and CD68) from cells of lymphoplasmacellular origin (CD3, CD20, and CD56). Furthermore, the gene expression of proinflammatory cytokines was characterized by real-time, reverse transcription-PCR analysis of total RNA extracted from frozen tissue samples. Results KIM-1P+ cells were the dominant immune cells (851±295 cells/mm2) and were scattered among the tumor cells. Most of the KIM-1P+ cells showed cellular projections characteristic of DCs. Fascin positivity identified a subgroup of DCs. In comparison to KIM-1P+ cells, there were significantly fewer CD68+ macrophages (196±217 cells/mm2). CD3+ T cells were the dominant lymphocytes (201±331 cells/mm2), whereas B cells (60±126 cells/mm2) were few. On transcriptional level, a concomitant gene expression of cytokines for the classical acute phase cytokines TNF-&agr; and IL-6 was missing, thus supporting the rather innate status of immune cells. Conclusion GISTs contain, beside T lymphocytes, a high number of monocyte-derived cells, which we suggest are, at least in part, immature DCs. Together with the lack of gene expression of inflammatory cytokines in tumor tissue our results point to a possible ‘symbiotic relationship’ between the tumor and the local immune cells.

Effect of irradiation on gene expression of rat liver adhesion molecules: in vivo and in vitro studies.

Background and Purpose:Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro.Material and Methods:Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology.Results:Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), β1-integrin, β2-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, β1-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), β2-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β, or IL-6 plus TNF-α led to an upregulation of P-selectin, ICAM-1 and VCAM-1.Conclusion:The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver.Hintergrund und Ziel:Tierexperimentelle Studien haben gezeigt, dass es in der Akutphase nach Einzeitbestrahlung der Leber mit 25 Gy im Gegensatz zu anderen toxischen Leberschädigungen trotz strahleninduzierter Expression von Chemokinen nicht zu einer inflammatorischen Reaktion mit Einwanderung von Entzündungszellen kommt. Ziel der vorliegenden experimentellen Studie war die Messung der Auswirkungen ionisierender Strahlung auf die Expression der wichtigsten Adhäsionsmoleküle nach Leberbestrahlung in vivo und Bestrahlung von Hepatozyten in vitro im etablierten Modell.Material und Methodik:Innerhalb von 48 h nach selektiver Leberbestrahlung in vivo (25 Gy) sowie Bestrahlung von Hepatozyten in vitro (8 Gy) wurde RNA extrahiert und mittels Real-Time-PCR (Polymerase-Kettenreaktion) und Northern-Blot analysiert. Neben alleinig bestrahlten Hepatozyten wurden dabei in vitro auch Zellen untersucht, die zusätzlich zur Bestrahlung mit Tumor-Nekrose-Faktor-(TNF-)α, Interleukin-(IL-)1β oder einer Kombination aus IL-6/TNF-α inkubiert wurden. Adhäsionsmolekülkonzentrationen im Serum wurden mittels ELISA („enzyme-linked immunosorbent assay“) gemessen, Lebergewebe auch mittels Immunhistochemie untersucht.Ergebnisse:ICAM-1 („intercellular adhesion molecule-1“), VCAM-1 („vascular cell adhesion molecule-1“), JAM-1, („junctional adhesion molecule-1“), β1-Integrin, β2-Integrin, E-Cadherin und P-Selectin waren in vivo nach Bestrahlung vermehrt exprimiert, die PECAM-1-Expression („platelet-endothelial cell adhesion molecule-1“) blieb jedoch unverändert. In vitro kam es zu einer vermehrten Expression von β1-Integrin, JAM-1 und ICAM-2 und einer verminderten Expression von P-Selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 („mucosal addressin cell adhesion molecule 1“), β2-Integrin und E-Cadherin. Nach zusätzlicher Inkubation der Hepatozyten mit TNF-α, IL-1β oder IL-6 und TNF-α kam es auch in vitro zu einer vermehrten Expression von P-Selectin, ICAM-1 und VCAM-1.Schlussfolgerung:Leberbestrahlung führt zu einer vermehrten Expression der wichtigsten Adhäsionsmoleküle in vivo und in durch Zytokine aktivierten Hepatozyten. Die PECAM-1-Expression wird allerdings nicht beeinflusst. Dies könnte einer der Gründe für die fehlende Inflammation in diesem Modell sein.

Effect of Irradiation on Gene Expression of Rat Liver Adhesion Molecules

Background and Purpose:Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro.Material and Methods:Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology.Results:Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), β1-integrin, β2-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, β1-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), β2-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β, or IL-6 plus TNF-α led to an upregulation of P-selectin, ICAM-1 and VCAM-1.Conclusion:The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver.Hintergrund und Ziel:Tierexperimentelle Studien haben gezeigt, dass es in der Akutphase nach Einzeitbestrahlung der Leber mit 25 Gy im Gegensatz zu anderen toxischen Leberschädigungen trotz strahleninduzierter Expression von Chemokinen nicht zu einer inflammatorischen Reaktion mit Einwanderung von Entzündungszellen kommt. Ziel der vorliegenden experimentellen Studie war die Messung der Auswirkungen ionisierender Strahlung auf die Expression der wichtigsten Adhäsionsmoleküle nach Leberbestrahlung in vivo und Bestrahlung von Hepatozyten in vitro im etablierten Modell.Material und Methodik:Innerhalb von 48 h nach selektiver Leberbestrahlung in vivo (25 Gy) sowie Bestrahlung von Hepatozyten in vitro (8 Gy) wurde RNA extrahiert und mittels Real-Time-PCR (Polymerase-Kettenreaktion) und Northern-Blot analysiert. Neben alleinig bestrahlten Hepatozyten wurden dabei in vitro auch Zellen untersucht, die zusätzlich zur Bestrahlung mit Tumor-Nekrose-Faktor-(TNF-)α, Interleukin-(IL-)1β oder einer Kombination aus IL-6/TNF-α inkubiert wurden. Adhäsionsmolekülkonzentrationen im Serum wurden mittels ELISA („enzyme-linked immunosorbent assay“) gemessen, Lebergewebe auch mittels Immunhistochemie untersucht.Ergebnisse:ICAM-1 („intercellular adhesion molecule-1“), VCAM-1 („vascular cell adhesion molecule-1“), JAM-1, („junctional adhesion molecule-1“), β1-Integrin, β2-Integrin, E-Cadherin und P-Selectin waren in vivo nach Bestrahlung vermehrt exprimiert, die PECAM-1-Expression („platelet-endothelial cell adhesion molecule-1“) blieb jedoch unverändert. In vitro kam es zu einer vermehrten Expression von β1-Integrin, JAM-1 und ICAM-2 und einer verminderten Expression von P-Selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 („mucosal addressin cell adhesion molecule 1“), β2-Integrin und E-Cadherin. Nach zusätzlicher Inkubation der Hepatozyten mit TNF-α, IL-1β oder IL-6 und TNF-α kam es auch in vitro zu einer vermehrten Expression von P-Selectin, ICAM-1 und VCAM-1.Schlussfolgerung:Leberbestrahlung führt zu einer vermehrten Expression der wichtigsten Adhäsionsmoleküle in vivo und in durch Zytokine aktivierten Hepatozyten. Die PECAM-1-Expression wird allerdings nicht beeinflusst. Dies könnte einer der Gründe für die fehlende Inflammation in diesem Modell sein.

Phagocytosis of gadolinium chloride or zymosan induces simultaneous upregulation of hepcidin- and downregulation of hemojuvelin- and Fpn-1-gene expression in murine liver.

The liver and the spleen are the organs in which cellular material and aged erythrocytes are eliminated from the blood. Within the liver, Kupffer cells (KCs) are mainly responsible for this task, as such KCs have a pivotal role in iron metabolism. The aim of this study is to investigate the changes of hepatic gene expression in two models of KC phagocytosis. Gadolinium chloride (GD) or zymosan was injected intraperitoneally into rats and to endotoxin-resistant mice (C3H/HeJ). The animals were killed at different time points and their livers were immediately frozen in liquid nitrogen for RNA isolation and immunohistological studies. RNA was analyzed by real-time PCR and northern blot. Sera were used to measure transaminases, hepcidin and iron levels. The expression of iron metabolism genes, hepcidin, hemojuvelin (Hjv), ferroportin-1 (Fpn-1) and of the inflammatory cytokines IL-6, IL-1β, TNF-α and IFN-γ was determined. Although phagocytosed material was detected in ED-1- and C1q-positive cells, no inflammatory cells were identified within the liver parenchyma. Serum levels of hepcidin, iron and transaminases did not differ from those of control animals. Both GD and zymosan induced an upregulation of hepcidin-gene expression in rat liver as early as 3 h, reaching a maximum 6 h after treatment. Hjv- and Fpn-1-gene expression was downregulated at the same time. IL-6 was by far the most induced acute-phase-cytokine in GD- and zymosan-treated livers, although IL-1β and TNF-α were also strongly upregulated by zymosan and to a lesser extent by GD. Similar results were obtained in the C3H/HeJ mouse strain excluding the possible role of contaminating endotoxin. This study shows that phagocytosis upregulates hepcidin-gene expression and downregulates Hjv- and Fpn-1-gene expression within the liver. These changes in iron-regulating-gene expression may be mediated by the locally produced acute-phase-cytokines.

Irradiation leads to sensitization of hepatocytes to TNF-α-mediated apoptosis by upregulation of IκB expression

This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis. IκB was upregulated in irradiated hepatocytes. Administration of IκB antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-α administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by IκB antisense oligonucleotides was mediated by suppression of caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF-α-administration depend on radiation-induced upregulation of IκB, thus demonstrating an apoptosis permitting effect of IκB.

Effect of Irradiation on Gene Expression of Rat Liver Adhesion Molecules@@@Einfluss ionisierender Strahlung auf die Expression von Adhäsionsmolekülen in der Leber. In-vivo- und In-vitro-Studien: In Vivo and In Vitro Studies

Background and Purpose:Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro.Material and Methods:Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology.Results:Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), β1-integrin, β2-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, β1-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), β2-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-)α, interleukin-(IL-)1β, or IL-6 plus TNF-α led to an upregulation of P-selectin, ICAM-1 and VCAM-1.Conclusion:The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of inflammation in the irradiated rat liver.Hintergrund und Ziel:Tierexperimentelle Studien haben gezeigt, dass es in der Akutphase nach Einzeitbestrahlung der Leber mit 25 Gy im Gegensatz zu anderen toxischen Leberschädigungen trotz strahleninduzierter Expression von Chemokinen nicht zu einer inflammatorischen Reaktion mit Einwanderung von Entzündungszellen kommt. Ziel der vorliegenden experimentellen Studie war die Messung der Auswirkungen ionisierender Strahlung auf die Expression der wichtigsten Adhäsionsmoleküle nach Leberbestrahlung in vivo und Bestrahlung von Hepatozyten in vitro im etablierten Modell.Material und Methodik:Innerhalb von 48 h nach selektiver Leberbestrahlung in vivo (25 Gy) sowie Bestrahlung von Hepatozyten in vitro (8 Gy) wurde RNA extrahiert und mittels Real-Time-PCR (Polymerase-Kettenreaktion) und Northern-Blot analysiert. Neben alleinig bestrahlten Hepatozyten wurden dabei in vitro auch Zellen untersucht, die zusätzlich zur Bestrahlung mit Tumor-Nekrose-Faktor-(TNF-)α, Interleukin-(IL-)1β oder einer Kombination aus IL-6/TNF-α inkubiert wurden. Adhäsionsmolekülkonzentrationen im Serum wurden mittels ELISA („enzyme-linked immunosorbent assay“) gemessen, Lebergewebe auch mittels Immunhistochemie untersucht.Ergebnisse:ICAM-1 („intercellular adhesion molecule-1“), VCAM-1 („vascular cell adhesion molecule-1“), JAM-1, („junctional adhesion molecule-1“), β1-Integrin, β2-Integrin, E-Cadherin und P-Selectin waren in vivo nach Bestrahlung vermehrt exprimiert, die PECAM-1-Expression („platelet-endothelial cell adhesion molecule-1“) blieb jedoch unverändert. In vitro kam es zu einer vermehrten Expression von β1-Integrin, JAM-1 und ICAM-2 und einer verminderten Expression von P-Selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 („mucosal addressin cell adhesion molecule 1“), β2-Integrin und E-Cadherin. Nach zusätzlicher Inkubation der Hepatozyten mit TNF-α, IL-1β oder IL-6 und TNF-α kam es auch in vitro zu einer vermehrten Expression von P-Selectin, ICAM-1 und VCAM-1.Schlussfolgerung:Leberbestrahlung führt zu einer vermehrten Expression der wichtigsten Adhäsionsmoleküle in vivo und in durch Zytokine aktivierten Hepatozyten. Die PECAM-1-Expression wird allerdings nicht beeinflusst. Dies könnte einer der Gründe für die fehlende Inflammation in diesem Modell sein.

Effect of tumour necrosis factor-α and irradiation alone or in combination on the viability of hepatocellular and biliary adenocarcinoma cell lines in vitro

Background: Tumour necrosis factor α (TNF‐α) may exhibit antitumoral activity and can influence the reaction of both tumour and normal tissue to radiation.