Regional hyperthermia with cisplatin added to gemcitabine versus gemcitabine in patients with resected pancreatic ductal adenocarcinoma: The HEAT randomised clinical trial.

BACKGROUND: Regional hyperthermia (RHT) with cisplatin added to gemcitabine showed efficacy in gemcitabine-pre-treated patients with advanced pancreatic ductal adenocarcinoma. We conducted a randomised clinical trial to investigate RHT with cisplatin added to gemcitabine (GPH) compared with gemcitabine (G) in the adjuvant setting of resected pancreatic ductal adenocarcinoma. METHODS: This randomised, multicentre, open-label trial randomly assigned patients to either GPH (gemcitabine 1000 mg/m2 on day 1, 15 and cisplatin 25 mg/m2 with RHT on day 2, 3 and 15,16) or to G (gemcitabine 1000 mg/m2 on day 1,8,15), four-weekly over six cycles. Disease-free survival (DFS) was the primary end-point. Secondary end-points included overall survival (OS) and safety. RESULTS: A total of 117 eligible patients (median age, 63 years) were randomly allocated to treatment (57 GPH; 60 G). With a follow-up time of 56.6 months, the median DFS was 12.7 compared to 11.2 months for GPH and G, respectively (p = 0.394). Median post-recurrence survival was significantly prolonged in the GPH-group (15.3 versus 9.8 months; p = 0.031). Median OS reached 33.2 versus 25.2 months (p = 0.099) with 5-year survival rates of 28.4% versus 18.7%. Excluding eight patients who received additional capecitabine in the G-arm (investigators choice), median OS favoured GPH (p = 0.052). Adverse events CTCAE (Common Terminology Criteria for Adverse Events) grade ≥3 occurred in 61.5% (GPH) versus 63.6% (G) of patients. Two patients in the G-group died because of treatment-related toxic effects. CONCLUSIONS: The randomised controlled Hyperthermia European Adjuvant Trial study failed to demonstrate a significant difference in DFS. However, it suggests a difference in post-recurrence survival and a trend for improved OS. CLINICALTRIALS: gov, number NCT01077427.

[Standards and recent advancements in esophageal cancer treatment]. / Aktuelle Standards und Ausblicke in der Behandlung des Ösophaguskarzinoms.

Das Ösophaguskarzinom macht ca. 1 % aller malignen Erkrankungen aus und besitzt trotz aller Fortschritte in der Diagnostik und Therapie eine sehr schlechte Prognose. In der Therapie sind multimodale Konzepte fest verankert und es bedarf der interdisziplinären Zusammenarbeit der Gastroenterologen, Onkologen, Chirurgen und Strahlentherapeuten.Bei mukosalen Karzinomen stellt die endoskopische Resektion die Therapie der Wahl da. Im Falle von cT1b- oder cT2- Tumoren erfolgt die primäre chirurgische Resektion, demgegenüber wird bei lokal fortgeschrittenen Karzinomen cT3- oder N+-Tumoren erst nach neoadjuvanter Vorbehandlung mittels Radiochemotherapie oder alleiniger Chemotherapie die operative Resektion durchgeführt. In der metastasierten Situation stehen nur palliative Radio- bzw. Chemotherapien als Behandlungskonzepte zur Verfügung, wobei die Effizienz dieser Therapien eingeschränkt ist. Dieser Übersichtsartikel fasst die aktuellen multimodalen Therapiekonzepte zusammen.

Anti-inflammatory consequences of bile acid accumulation in virus-infected bile duct ligated mice.

Cholestatic patients exhibiting high bile acid serum levels were reported to be more susceptible to bacterial and viral infections. Animal studies in bile duct ligated (BDL) mice suggest that cholestasis leads to an aggravation of hepatic bacterial infections. We have investigated the impact of cholestasis on mouse cytomegalovirus (MCMV)-induced immune responses and viral replication. While MCMV did not aggravate BDL-induced liver damage, BDL markedly reduced MCMV-triggered chemokine expression and immune cell recruitment to the liver. MCMV-infected BDL mice showed diminished trafficking of Ly6C+/F4/80+ myeloid cells and NK1.1+ NK cells to the liver compared to MCMV infected control mice. Moreover, virus-driven expression of CCL7, CCL12, CXCL9 and CXCL10 was clearly impaired in BDL- compared to sham-operated mice. Furthermore, production of the anti-inflammatory cytokine IL-10 was massively augmented in infected BDL mice. In contrast, intra- and extrahepatic virus replication was unaltered in BDL-MCMV mice when compared to sham-MCMV mice. Cholestasis in the BDL model severely impaired pathogen-induced chemokine expression in the liver affecting CCR2- and CXCR3-dependent cell trafficking. Cholestasis resulted in reduced recruitment of inflammatory monocytes and NK cells to the liver.

Reprogramming of pro-inflammatory human macrophages to an anti-inflammatory phenotype by bile acids.

Cholestasis is caused by autoimmune reactions, drug-induced hepatotoxicity, viral infections of the liver and the obstruction of bile ducts by tumours or gallstones. Cholestatic conditions are associated with impaired innate and adaptive immunity, including alterations of the cellular functions of monocytes, macrophages, NK cells and T-cells. Bile acids act as signalling molecules, affecting lipopolysaccharide (LPS)-induced cytokine expression in primary human macrophages. The present manuscript investigates the impact of bile acids, such as taurolithocholic acid (TLC), on the transcriptome of human macrophages in the presence or absence of LPS. While TLC itself has almost no effect on gene expression under control conditions, this compound modulates the expression of 202 out of 865 transcripts in the presence of LPS. Interestingly, pathway analysis revealed that TLC specifically supressed the expression of genes involved in mediating pro-inflammatory effects, phagocytosis, interactions with pathogens and autophagy as well as the recruitment of immune cells, such as NK cells, neutrophils and T cells. These data indicate a broad influence of bile acids on inflammatory responses and immune functions in macrophages. These findings may contribute to the clinical observation that patients with cholestasis present a lack of response to bacterial or viral infections.

Multimodal and sequential treatment improves survival in patients with hepatocellular carcinoma.

Background and aims Therapy of hepatocellular carcinoma (HCC) mainly depends on tumor stage and liver function. The aim of this study was to identify additional predictors of overall survival in HCC patients with a particular attention to multimodal therapies. Methods Six hundred and seven consecutive HCC-patients treated in a tertiary center between 1988 and 2011 were retrospectively analyzed. Multivariate analysis was performed by logistic and Cox-regression, overall survival was analyzed by Kaplan Meier statistics. Results In comparison to unimodal therapies, multimodal treatment increased overall survival in BCLC-A patients from 16 to 26 months (p < 0.001), in patients with BCLC-B stage from 9.5 to 16 months (p < 0.001), in BCLC-C patients from 6 to 18 months (p < 0.001), and in stage BCLC-D from 2 to 8 months (not significant). Survival increased throughout all Child Pugh scores, and patients experienced benefits from multimodal therapy irrespective of alfa-fetoprotein levels. Comparing the time span 1988 - 1999 with 2000 - 2011, the rate of multimodal/sequential treatment increased from 12.3 % to 30 % (p < 0.001), and the overall survival of all (treated and non-treated) patients increased from 7 months (1988 - 1999) to 10 months (2000 - 2011, p < 0.001). In multivariate analysis, multimodal treatment was shown to be an independent predictor for overall survival besides elevated alfa-fetoprotein, Child Pugh score, and BCLC stage. Conclusion Multimodal therapies increase overall survival in HCC patients and should be considered in patients with HCC if practicable.

Bile Acids Act as Soluble Host Restriction Factors Limiting Cytomegalovirus Replication in Hepatocytes.

UNLABELLED: The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete, and recycle a chemically diverse set of bile acids, with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC), glycochenodeoxycholic acid, and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anticytomegalovirus activity required active import of bile acids via the sodium-taurocholate-cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which alpha interferon (IFN-α) lacks antiviral activity, physiological TCDC concentrations were similarly effective as IFN-γ. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells. IMPORTANCE: Cytomegaloviruses are members of the Betaherpesvirinae subfamily. Primary infection leads to latency, from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations, including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds.

MAPKAP kinase 2 regulates IL-10 expression and prevents formation of intrahepatic myeloid cell aggregates during cytomegalovirus infections.

BACKGROUND & AIMS: The kinase p38(MAPK) and its downstream target MAPKAP kinase (MK) 2 are critical regulators of inflammatory responses towards pathogens. To date, the relevance of MK2 for regulating IL-10 expression and other cytokine responses towards cytomegalovirus (CMV) infection and the impact of this pathway on viral replication in vitro and in vivo is unknown and the subject of this study. METHODS: The effect of MK2, interferon-α receptor (IFNAR)1, tristetraprolin (TTP) and IL-10 on mouse (M)CMV virus titres, cytokine expression, signal transduction, transcript stability, liver enzymes release, immune cell recruitment and aggregation in response to MCMV infection were studied ex vivo in hepatocytes and macrophages, as well as in vivo. RESULTS: MK2 is critical for MCMV-induced production of IL-10, IFN-α2 and 4, IFN-ß, IL-6, and TNF-α but not for IFN-γ. The MCMV-induced IL-10 production requires activation of IFNAR1 and is further regulated by MK2 and TTP-dependent stabilization of IL-10 transcripts. MK2(-/-) mice are able to control acute MCMV replication, despite deregulated cytokine production. This may be related to the observation that MCMV-infected MK2(-/-) mice show enhanced formation of focal intrahepatic lymphocyte infiltrates resembling intrahepatic myeloid cell aggregates of T cell expansion (iMATEs), which were also observed in MCMV-infected IL-10(-/-) mice but are almost absent in MCMV-infected wild-type controls. CONCLUSIONS: The data suggest that MK2 is critical for regulating cytokine responses towards acute MCMV infection, including that of IL-10 via IFNARI-mediated circuits. MCMV stimulates expression of MK2-dependent cytokines, in particular IL-10 and thereby prevents enhanced formation of intrahepatic iMATE-like cellular aggregates.

Transient Elastography for the Detection of Liver Damage in Patients with HIV.

INTRODUCTION: Highly active antiretroviral therapy (HAART) is effective and well tolerated, but hepatotoxicity is relatively common. Different non-invasive methods are available for detecting liver fibrosis in patients with chronic liver disease. METHODS: Patients who were HIV positive and who had given their informed consent were included in this cross-sectional study. Transient elastography [FibroScan(®) (FS); Echosens], serum hyaluronic acid (HA), Hepascore (HS), Fibrosis-4 (FIB-4), and aspartate aminotransferase to platelet ratio index (APRI) were used to detect liver fibrosis in the patients. The agreement between FS and the other methods was evaluated. To observe the hepatotoxicity of HAART, patients with chronic viral hepatitis B or C were excluded by detection of hepatitis B surface antigens and hepatitis C virus antibodies. Patients with chronic alcohol intake were excluded by measuring carbohydrate-deficient transferrin (CDT). FS correlation with the duration of therapy with protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI) was evaluated. RESULTS: Overall, 203 patients were included in the study. The agreement between the different tests ranged from 64% to 77%: FS vs. HA, 72%; FS vs. APRI, 74%; FS vs. HS, 77%; and FS vs. FIB-4, 64%. After excluding patients with chronic hepatitis B or C and elevated CDT, 153 patients remained for studying the hepatotoxicity of HAART. A significant correlation of FS with the duration of medication intake was observed for PIs (P = 0.026; r = 0.18). NRTI and NNRTI therapy duration did not correlate with FS. CONCLUSIONS: The agreement between FS and other tests ranged from 64% to 77%. A significant correlation was found between liver stiffness and the duration of therapy with PIs, which underlines the known hepatotoxicity of this substance group. FUNDING: Heinz-Ansmann Foundation.

Metastasized pancreatic carcinoma with neoadjuvant FOLFIRINOX therapy and R0 resection.

Patients with metastasized carcinoma of the pancreas have a very poor prognosis, and long-term survival cannot be expected. This case report describes two patients with an initial diagnosis of metastatic pancreatic cancer, both with hepatic metastases and one with an additional peritoneal carcinomatosis. Initially, both patients were treated intravenously with the FOLFIRINOX chemotherapy regimen, consisting of 5-FU, folinic acid, irinotecan and oxaliplatin. Surprisingly, the FOLFIRINOX treatment resulted in complete resolution of the hepatic metastases in both patients, with no lesions detectable by computed tomography scan. Furthermore, treatment response included decreased diameter of the primary tumor in the tail of the pancreas and disappearance of the additional peritoneal carcinomatosis. Both patients were discussed by our multidisciplinary tumor board, which recommended surgical resections of the carcinoma. The R0 resection of the primary tumor was successful in both cases and, interestingly, the resected tissues showed no evidence of the hepatic metastases intraoperatively. In the first case, the patient received a postoperative 6-mo course of adjuvant chemotherapy with gemcitabine. In the second case, the patient continued to receive the FOLFIRINOX regimen for an additional 6 mo postoperatively. At 12 mo after the operation, a nonresectable retroperitoneal lymph node metastasis was detected in the first patient, whereas the second patient remained in complete remission at the time of this report (5 mo after the adjuvant therapy was discontinued). This case report is the first of its kind to describe two cases of hepatic metastatic pancreatic carcinoma that were resectable following treatment with FOLFIRINOX. Further studies are required to examine the role of FOLFIRINOX as a neoadjuvant treatment option in subgroups of patients with initially metastasized pancreatic carcinoma.

Multimodal treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) represents the most common liver cancer with an increasing incidence and it accounts for the third most common cause of cancer-related death worldwide. Even though the clinical diagnosis and management of HCC improved significantly in the last decades, this malignant disease is still associated with a poor prognosis. It has to be distinguished between patients with HCCs, which developed from liver cirrhosis, and patients without underlying liver cirrhosis as classification systems, prognosis estimation and therapy recommendations differ in-between. In case of HCC in patients with liver cirrhosis in Europe, treatment allocation and prognosis estimation are mainly based on the Barcelona-Clinic Liver Cancer (BCLC) staging system. Based on this staging system different surgical, interventional radiological/sonographical and non-interventional procedures have been established for the multimodal treatment of HCC. The BCLC classification system represents a decision guidance; however because of its limitations in selected patients treatment allocation should be determined on an individualized rather than a guideline-based medicine by a multidisciplinary board in order to offer the best treatment option for each patient. This review summarizes the current management of HCC and illustrates controversial areas of therapeutic strategies.

Bile acids PKA-dependently induce a switch of the IL-10/IL-12 ratio and reduce proinflammatory capability of human macrophages.

That cholestatic conditions are accompanied by an enhanced susceptibility to bacterial infection in human and animal models is a known phenomenon. This correlates with the observation that bile acids have suppressive effects on cells of innate and adaptive immunity. The present study provides evidence that in human macrophages, bile acids inhibit the LPS-induced expression of proinflammatory cytokines without affecting the expression of the anti-inflammatory cytokine IL-10. This results in a macrophage phenotype that is characterized by an increased IL-10/IL-12 ratio. Correspondingly, bile acids suppress basal phagocytic activity of human macrophages. These effects of bile acids can be mimicked by cAMP, which is presumably induced TGR5-dependently. The data provided further suggest that in primary human macrophages, modulation of the macrophage response toward LPS by bile acids involves activation of CREB, disturbed nuclear translocation of NF-κB, and PKA-dependent enhancement of LPS-induced cFos expression. The increase in cFos expression is paralleled by an enhanced formation of a protein complex comprising cFos and the p65 subunit of NF-κB. In summary, the data provided suggest that in human macrophages, bile acids induce an anti-inflammatory phenotype characterized by an increased IL-10/IL-12 ratio via activation of PKA and thereby, prevent their activation as classically activated macrophages. This bile acid-induced modulation of macrophage function may also be responsible for the experimentally and clinically observed anti-inflammatory and immunosuppressive effects of bile acids.

Inhibition of interferon-α-induced signaling by hyperosmolarity and hydrophobic bile acids.

Apart from viral conditions, host factors such as elevated bile acid concentrations are determinants of successful interferon-α (IFN-α) treatment in patients with chronic hepatitis C or B. The present study demonstrates that hydrophobic bile acids inhibit Jak1- and Tyk2-phosphorylation, which lead to blockade of STAT1-mediated IFN-α-signaling in the sodium-taurocholate cotransporting peptide (NTCP)-transfected human hepatoma cell line HepG2, resulting in a decreased mRNA and protein expression of IFN-stimulated genes such as myxovirus resistance protein A (MxA) or dsRNA-activated protein kinase (PKR). In addition, hyperosmotic stress leads to an inhibition of IFN-α-induced Jak1- and Tyk2-phosphorylation, and STAT1/STAT2-phosphorylation and gene expression. This inhibitory effect of hydrophobic bile acids or hyperosmolarity is not due to caspase-mediated cleavage or lysosomal degradation of the cognate receptors or to the generation of oxidative stress, activation of p38- or Erk-mediated MAPK pathways or phosphatase activity. Preincubation with the organic osmolyte betaine blocked the inhibitory effect of bile acids or hyperosmolarity on MxA protein expression, but had no effect on transcript levels or activation of STAT1, suggesting that betaine mediates its effects on MxA expression at a translational or post-translational level. Our findings could provide a rationale for betaine use in cholestatic HBV/HCV patients undergoing interferon therapy.

Enhanced insulin sensitivity of gene-targeted mice lacking functional KCNQ1.

The pore-forming K+-channel alpha-subunit KCNQ1 is expressed in a wide variety of tissues including heart, skeletal muscle, liver, and epithelia. Most recent evidence revealed an association of the KCNQ1 gene with the susceptibility to type 2 diabetes. KCNQ1 participates in the regulation of cell volume, which is, in turn, critically important for the regulation of metabolism by insulin. The present study explored the influence of KCNQ1 on insulin-induced cellular K+ uptake and glucose metabolism. Insulin (100 nM)-induced K+ uptake was determined in isolated perfused livers from KCNQ1-deficient mice (kcnq1(-/-)) and their wild-type littermates (kcnq1(+/+)). Moreover, plasma glucose and insulin levels, intraperitoneal glucose (3 g/kg) tolerance, insulin (0.15 U/kg)-induced hypoglycemia, and peripheral uptake of radiolabeled 3H-deoxy-glucose were determined in both genotypes. Insulin-stimulated hepatocellular K+ uptake was significantly more sustained in isolated perfused livers from kcnq1(-/-) mice than from kcnq1(+/+)mice. The decline of plasma glucose concentration following an intraperitoneal injection of insulin was again significantly more sustained in kcnq1(-/-) than in kcnq1(+/+) mice. Both fasted and nonfasted plasma glucose and insulin concentrations were significantly lower in kcnq1(-/-) than in kcnq1(+/+)mice. Following an intraperitoneal glucose injection, the peak plasma glucose concentration was significantly lower in kcnq1(-/-) than in kcnq1(+/+)mice. Uptake of 3H-deoxy-glucose into skeletal muscle, liver, kidney and lung tissue was significantly higher in kcnq1(-/-) than in kcnq1(+/+)mice. In conclusion, KCNQ1 counteracts the stimulation of cellular K+ uptake by insulin and thereby influences K+-dependent insulin signaling on glucose metabolism. The observations indicate that KCNQ1 is a novel molecule affecting insulin sensitivity of glucose metabolism.

SGK1 dependence of insulin induced hypokalemia.

Insulin stimulates cellular K+ uptake leading to hypokalemia. Cellular K+ uptake is accomplished by parallel stimulation of Na+/H+ exchange, Na+,K+,2Cl- co-transport, and Na+/K+ ATPase and leads to cell swelling, a prerequisite for several metabolic effects of the hormone. Little is known about underlying signaling. Insulin is known to activate the serum and glucocorticoid-inducible kinase SGK1, which in turn enhances the activity of all three transport proteins. The present study thus explored the contribution of SGK1 to insulin-induced hypokalemia. To this end, gene-targeted mice lacking SGK1 (sgk1-/-) and their wild-type littermates (sgk1+/+) have been infused with insulin (2 mU kg(-1) min(-1)) and glucose at rates leaving the plasma glucose concentration constant. Moreover, isolated liver perfusion experiments have been performed to determine stimulation of cellular K+ uptake by insulin (100 nM). As a result, combined glucose and insulin infusion significantly decreased plasma K+ concentration despite a significant decrease of urinary K+ excretion in sgk1+/+ but not in sgk1-/- mice. Accordingly, the plasma K+ concentration was within 60 min significantly lower in sgk1+/+ than in sgk1-/- mice. In isolated liver perfusion experiments, cellular K+ uptake was stimulated by insulin (100 nM), an effect blunted by 72% in sgk1-/- mice as compared to sgk1+/+ mice. Accordingly, insulin-induced cell hydration was 63% lower in sgk1-/- mice than in sgk1+/+ mice. Moreover, volume regulatory K+ release was 31% smaller in sgk1-/- mice than in sgk1+/+ mice. In conclusion, the serum and glucocorticoid-inducible kinase SGK1 participates in the signaling mediating the hypokalemic effect of insulin.

Caspase-mediated cleavage of the signal-transducing IL-6 receptor subunit gp130.

The present study characterizes the molecular mechanisms of CD95L-induced inhibition of IL-6 signaling, which is known to mediate hepatoprotective effects in response to various toxins. CD95L-induced caspase activation leads to degradation of gp130, thereby suppressing IL-6-induced phosphorylation of STAT3 (Tyr(705)) and of tyrosine phosphatase SHP2 (Tyr(580)). Degradation of gp130 protein in response to CD95L was largely prevented after inhibition of caspase 3 or 8. Introduction of a point mutation into a newly identified caspase cleavage site located within position 800-806 (DHVDGGD) of the cytoplasmic tail of gp130 leads to cleavage resistance of the respective receptor in an in vitro assay with recombinant active caspase 3. Correspondingly, the release of a C-terminal gp130-cleavage product of approximately 18kDa was also inhibited after mutagenesis of this cleavage motif. In conclusion, this study demonstrates that caspase activation by CD95L antagonizes IL-6 signaling by a caspase-mediated cleavage of gp130 thereby probably counteracting hepatoprotective effects of IL-6.

Taurolithocholic acid-3 sulfate impairs insulin signaling in cultured rat hepatocytes and perfused rat liver.

BACKGROUND/AIMS: The role of bile acids for insulin resistance in cholestatic liver disease is unknown. METHODS: The effect of taurolithocholic acid-3 sulfate (TLCS) on insulin signaling was studied in cultured rat hepatocytes and perfused rat liver. RESULTS: TLCS induced insulin resistance at the level of insulin receptor (IR) beta Tyr(1158) phosphorylation, phosphoinositide (PI) 3-kinase activity and protein kinase (PK)B Ser(473) phosphorylation in cultured hepatocytes. Consistently, the insulin stimulation of the PI 3-kinase-dependent K(+) uptake, hepatocyte swelling and proteolysis inhibition was blunted by TLCS in perfused rat liver. The PKC inhibitor Go6850 and tauroursodeoxycholate (TUDC) counteracted the suppression of insulin-induced IRbeta and PKB phosphorylation by TLCS. Rapamycin and dibutyryl-cAMP, which inhibited basal signaling via mammalian target of rapamycin (mTOR), restored insulin-induced PKB- but not IRbeta phosphorylation. In livers from 7 day bile duct-ligated rats PKB Ser(473) phosphorylation was decreased by about 50%. CONCLUSION: TLCS induces insulin resistance by a PKC-dependent suppression of insulin-induced IRbeta phosphorylation and the PI 3-kinase/PKB path. This can in part be compensated by a decrease of mTOR activity, which may release insulin-sensitive components downstream of the insulin receptor from tonic inhibition. The data suggest that retention of hydrophobic bile acids confers insulin resistance on the cholestatic liver.

Caspases and receptor cleavage.

In addition to their established functions in programmed cell death, there is increasing evidence that caspases contribute to several other cellular processes beside of apoptosis. So-called "dependence receptors" represent a group of receptors, which derive from different protein families, but are functionally linked by their capability to regulate cell survival in presence of their respective ligands thereby preserving cellular homeostasis. In the absence of their ligands these receptors are cleaved by caspases thereby releasing pro-apoptotic receptor fragments (e.g. rearranged during transfection [RET]) or permitting the exposure of death domains, which were masked before through other receptor domains (e.g. deleted in colorectal carcinoma [DCC]). Apart from these, there are other plasma membrane receptors such as the epidermal growth factor receptor, which have been identified as substrates of caspases. In terms of signal-transduction, caspase-mediated cleavage of these receptors blocks ligand-induced activation of their intracellular signalling. It is hypothesized that this might be another mechanism, whereby caspases trigger cell toxicity through shut-down of survival signals.

Hydrophobic bile salts induce hepatocyte shrinkage via NADPH oxidase activation.

Hydrophobic bile salts activate NADPH oxidase through a ceramide and protein kinase Czeta-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. As shown in the present study, hydrophobic bile salts such as glycochenodeoxycholate, taurochenodeoxycholate or taurolithocholylsulfate (TLCS) also induce within 30 min hepatocyte shrinkage in perfused rat liver. TLCS-induced hepatocyte shrinkage was strongly blunted in presence of desipramine, apocynin, bafilomycin and DIDS, i.e. maneuvres previously shown to inhibit TLCS-induced NADPH oxidase activation and the subsequent oxidative stress response. The antioxidant N-acetylcysteine inhibited TLCS-induced hepatocyte shrinkage. N-acetylcysteine by itself increased hepatocyte hydration, suggesting that a basal production of reactive oxygen intermediates is involved in the regulation of liver cell hydration. TLCS failed to induce shrinkage of hepatocytes from p47(phox) knock-out, but not control mice. Likewise, hepatocytes from p47(phox) knock-out mice were resistant towards TLCS-induced apoptosis and failed to activate the CD95 system. No cell shrinkage was observed in response to taurocholate and tauroursodesoxycholate, i.e. bile salts which do not induce an oxidative stress signal and apoptosis. NADPH oxidase activation also counteracts volume recovery in response to hyperosmotic hepatocyte shrinkage. The findings indicate that hydrophobic, proapoptotic bile salts induce hepatocyte shrinkage largely through NADPH oxidase-derived oxidative stress. Because cell shrinkage in turn activates NADPH oxidase, which blunts cell volume recovery, a vicious cycle ensues between oxidative stress and cell shrinkage, which propagates CD95 activation and may finally lead to apoptosis. In addition, cell shrinkage induced by proapoptotic bile salts may augment apoptosis by increasing protein breakdown and induction of cholestasis.

Bile acids inhibit interleukin-6 signaling via gp130 receptor-dependent and -independent pathways in rat liver.

Interleukin-6 (IL-6) is a major regulator of the acute phase reaction in the liver and is thought to mediate protective effects in response to hepatotoxins. In this study, the influence of bile acids on IL-6 signal transduction was analyzed. It was shown that hydrophobic bile acids such as glycochenodeoxycholate (GCDC) inhibited IL-6-induced tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 3 in hepatocytes and in perfused rat liver. This inhibition was accompanied by GCDC-mediated downregulation of glycoprotein (gp) 130 expression, whereas gp130 and suppressor of cytokine signaling 3 messenger RNA and gp80 protein levels remained unaffected. The GCDC-induced downregulation of gp130 protein expression was insensitive to inhibition of proteasomal or lysosomal protein degradation but turned out to be sensitive to inhibition of caspase-3 or caspase-8 activity. Accordingly, treatment of cell extracts with active recombinant caspase-3 led to a decay of immunoreactive gp130. Moreover, activation of caspases by CD95 ligand or hyperosmotic stress also resulted in a downregulation of gp130 levels. This indicates that caspase activation antagonizes IL-6 signaling by decay of gp130 levels. However, caspase inhibition did not prevent GCDC-dependent inhibition of IL-6-induced STAT3 activation, which turned out to be at least partially sensitive to suppression of p38(MAPK) activation. In conclusion, hydrophobic bile acids compromise IL-6 signaling through both a caspase-mediated downregulation of gp130 and a p38(MAPK)-dependent inhibition of STAT3 phosphorylation. This may contribute to bile acid-induced hepatotoxicity in cholestasis through counteracting the known hepatoprotective effects of IL-6.