C Trautwein

CCR2 Mediates Homeostatic and Inflammatory Release of Gr1 high Monocytes from the Bone Marrow, but Is Dispensable for Bladder Infiltration in Bacterial Urinary Tract Infection

CCR2 is thought to recruit monocytes to sites of infection. Two subpopulations of murine blood monocytes differing in Gr1 and CCR2 expression have been described. The exact role of CCR2 in migration of CCR2lowGr1low and CCR2highGr1high monocytes into nonlymphoid tissue is controversial. In this study, we have addressed this question in a murine model of bacterial urinary tract infection. Only Gr1high monocytes were recruited into the infected bladder. CCR2 deficiency reduced their frequency in this organ, indicating a requirement of this chemokine receptor. Importantly, CCR2-deficient mice also showed reduced Gr1high monocyte numbers in the blood, but not in the bone marrow (BM), indicating that CCR2 acted at the step of monocyte release into the circulation. The same was found also in noninfected mice, indicating a further involvement of CCR2 in steady-state BM egress. An additional requirement of CCR2 in monocyte recruitment from the blood into the bladder was excluded by tracking particle-labeled endogenous monocytes and by adoptive transfer of BM-derived monocyte subsets. These findings demonstrate that CCR2 governs homeostatic and infection-triggered release of Gr1high monocytes from the BM into the blood but is dispensable for recruitment into a nonlymphoid tissue.

Circulating microRNA-150 serum levels predict survival in patients with critical illness and sepsis.

Background and Aims Down-regulation of miR-150 was recently linked to inflammation and bacterial infection. Furthermore, reduced serum levels of miR-150 were reported from a small cohort of patients with sepsis. We thus aimed at evaluating the diagnostic and prognostic value of miR-150 serum levels in patients with critically illness and sepsis. Methods miR-150 serum levels were analyzed in a cohort of 223 critically ill patients of which 138 fulfilled sepsis criteria and compared to 76 healthy controls. Results were correlated with clinical data and extensive sets of routine and experimental biomarkers. Results Measurements of miR-150 serum concentrations revealed only slightly reduced miR-150 serum levels in critically ill patients compared to healthy controls. Furthermore miR-150 levels did not significantly differ in critically ill patients with our without sepsis, indicating that miR-150 serum levels are not suitable for diagnostic establishment of sepsis. However, serum levels of miR-150 correlated with hepatic or renal dysfunction. Low miR-150 serum levels were associated with an unfavorable prognosis of patients, since low miR-150 serum levels predicted mortality with high diagnostic accuracy compared with established clinical scores and biomarkers. Conclusion Reduced miR-150 serum concentrations are associated with an unfavorable outcome in patients with critical illness, independent of the presence of sepsis. Besides a possible pathogenic role of miR-150 in critical illness, our study indicates a potential use of circulating miRNAs as a prognostic rather than diagnostic marker in critically ill patients.

Gp130-Dependent Astrocytic Survival Is Critical for the Control of Autoimmune Central Nervous System Inflammation

Astrocytes are activated in experimental autoimmune encephalomyelitis (EAE) and have been suggested to either aggravate or ameliorate EAE. However, the mechanisms leading to an adverse or protective effect of astrocytes on the course of EAE are incompletely understood. To gain insight into the astrocyte-specific function of gp130 in EAE, we immunized mice lacking cell surface expression of gp130, the signal-transducing receptor for cytokines of the IL-6 family, with myelin oligodendrocyte glycoprotein35–55 peptide. These glial fibrillary acid protein (GFAP)-Cre gp130fl/fl mice developed clinically a significantly more severe EAE than control mice and succumbed to chronic EAE. Loss of astrocytic gp130 expression resulted in apoptosis of astrocytes in inflammatory lesions of GFAP-Cre gp130fl/fl mice, whereas gp130fl/fl control mice developed astrogliosis. Astrocyte loss of GFAP-Cre gp130fl/fl mice was paralleled by significantly larger areas of demyelination and significantly increased numbers of CD4 T cells in the CNS. Additionally, loss of astrocytes in GFAP-Cre gp130fl/fl mice resulted in a reduction of CNS regulatory Foxp3+ CD4 T cells and an increase of IL-17–, IFN-γ–, and TNF-producing CD4 as well as IFN-γ– and TNF-producing CD8 T cells, illustrating that astrocytes regulate the phenotypic composition of T cells. An analysis of mice deficient in either astrocytic gp130– Src homology region 2 domain-containing phosphatase 2/Ras/ERK or gp130–STAT1/3 signaling revealed that prevention of astrocyte apoptosis, restriction of demyelination, and T cell infiltration were dependent on the astrocytic gp130–Src homology region 2 domain-containing phosphatase 2/Ras/ERK, but not on the gp130–STAT1/3 pathway, further demonstrating that gp130-dependent astrocyte activation is crucial to ameliorate EAE.

Symptomatic hepatitis B virus (HBV) reactivation despite reduced viral fitness is associated with HBV test and immune escape mutations in an HIV-coinfected patient.

Two sequential hepatitis B virus (HBV) strains obtained before and during an icteric flare-up of an occult HBV infection in a patient coinfected with human immunodeficiency virus revealed HBV surface antigen (HBsAg) test escape mutations, although the patient had never received hepatitis B-specific immunoglobulin. In contrast to the high HBV DNA loads, recurrence of HBsAg, and resulting icteric hepatitis, phenotypic analysis of the mutated HBV strains revealed significantly reduced replication efficacies in vitro, compared with wild-type HBV. Therefore, immune escape in the transiently anti-HBs-positive patient appeared to be crucial for persistence and reactivation. Immune escape mutants evolved even without exogenous selective pressure, hampered detection in HBsAg screening, and might be transmitted during reactivation with high HBV loads.

Adipocytokines in Severe Sepsis and Septic Shock

Among the different conditions of critical illness leading to admission to an Intensive Care Unit (ICU), sepsis remains the leading cause of death at the non-coronary medical ICU [1]. Even with optimal therapy, mortality rates of severe sepsis and septic shock are about 40 to 50% [2, 3]. Although there are numerous studies with varying methods from different countries, the incidence of severe sepsis is constantly approximately one out of ten admissions to all ICUs worldwide [4]. With millions of individuals concerned every year, worldwide sepsis is one of the major healthcare problems today. The proportion of severe sepsis, and case fatal outcomes increased during the last years [2]. It is crucial to establish the diagnosis sepsis as early as possible and to identify its origin, in order to initiate an appropriate therapy permitting to achieve the best possible outcome [5]. Sepsis is defined as a systemic inflammatory response syndrome (SIRS) caused by an infection. The association with organ dysfunction or sepsis-induced hypotension is termed severe sepsis. Septic shock, as a subset of severe sepsis, is characterized by sepsis-induced hypotension, persisting despite adequate fluid resuscitation [6]. Early differentiation between sepsis and SIRS is a considerable problem in the treatment of patients on ICU. Due to the early systemic release of inflammatory cytokines as compared with synthesis of acutephase-proteins, cytokines have been widely investigated for their diagnostic potential in predicting sepsis [7]. Nevertheless, until now the perfect biomarker for differentiation of sepsis and SIRS has not been found yet and ongoing research focuses on identification of appropriate diagnostic biomarkers for sepsis [8]. Despite a growing number of studies, the physiopathology of sepsis is not satisfyingly understood. Data show that physiopathology is characterized by a large number of proand anti-inflammatory cytokines and mediators of inflammation with complex interactions [9]. For instance, the application of a single bolus i.v. infusion of endotoxin to a healthy individual leads to the expression of 3147 genes, (> 10% of the human genome) [10]. These mediators and cytokines lead to endothelial dysfunction and activation of inflammatory and coagulation pathways as reaction to the invasion of a pathogen [11]. In order to reduce the sepsis-related high mortality, a better understanding of common pathogenic mechanisms of sepsis and other critical diseases is needed, potentially resulting in more effective treatment options.

Mechanisms of Acute Liver Failure

Acute liver failure is characterized by the sudden onset of liver failure in a patient without evidence of chronic liver disease. This definition is important, as it differentiates patients with acute liver failure from patients who suffer from liver failure owing to end-stage chronic liver disease.

Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma

Significance The two E-type cyclins E1 and E2 are known to interact with Cdk2 and are thought to trigger cell cycle activity in carcinogenesis. However, the individual contributions of cyclin E1, cyclin E2, and Cdk2 for initiation and progression of hepatocellular carcinoma (HCC) are unknown. In the present study, we discovered that only cyclin E1—but not cyclin E2—is essential for initiation of liver cancer and requires Cdk2. Unexpectedly, advanced liver cancer progression can be mediated in presence of any E-cyclin, but in a Cdk2-independent manner. We identified the specific expression profiles of cyclin E1-dependent and cyclin E1-independent hepatoma cells. These signatures are useful for predicting patient prognosis and for developing novel cyclin E-based HCC therapies. E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

Regulation of Cell Cycle During Liver Regeneration

Abstract In the last few years, several studies have revisited long-held paradigm and postulated the fundamentally new vision on the mechanisms of cell-cycle regulation during liver regeneration. The numerous cyclins and cyclin-dependent kinases that are involved in regulating the cell cycle have being successfully dissected using molecular and genetic methods. These approaches have opened new avenues in this field of research, namely, the hypothesis of redundancy and cell-type specificity. A better understanding of the cellular processes which govern hepatic proliferation will provide important insights into disease-derived compensatory mechanism of liver regeneration or into aberrant cellular proliferation in liver cancer. The chapter will cover the actual knowledge about the role of cell cycle during liver regeneration and addresses questions which remain open.

Sorafenib steuert differentielle Signalwege in murinen Hepatozyten und Hepatomzellen und inhibiert Leberregeneration durch Zellzyklusarrest und nicht-apoptotische Leberschädigung

Hintergrund: Der Multikinase-Inhibitor Sorafenib erhoht signifikant das Uberleben in Patienten mit fortgeschrittenem hepatozellularem Karzinom (HCC). Bisherige Daten zeigten, dass Sorafenib mitogen-aktivierte Proteinkinasen (MAPK) inhibiert und Apoptose induziert. Allerdings wurden diese Sorafenib-bedingten Effekte bislang weitestgehend in Tumorzelllinien untersucht. In dieser Studie sollte die Wirkung von Sorafenib in isogenen murinen Hepatomzellen und gesunden Hepatozyten miteinander verglichen werden, um protektive oder schadigende Einflusse in der regenerierenden Leber zu identifizieren. Methoden: Sorafenib Tosylat wurde von Bayer Pharmazeutika bereitgestellt. Fur in vitro Studien wurden murine Hepa1–6 Hepatomzellen sowie primare Hepatozyten aus C57/BL6Mausen verwendet. C57/BL6 Wildtypmause wurden vor und nach partieller Hepatektomie (PH) einmal taglich mit Sorafenib (100mg/kg, p.o.) behandelt und zu verschiedenen Zeitpunkten auf Hepatozytenproliferation, Leberschadigung und Regeneration bis zu einem Zeitpunkt von 96h nach PH analysiert. Ergebnisse: In vitro inhibierte Sorafenib gleichermasen die Zellzyklusprogression in Hepatomzellen und Hepatozyten, unter anderem durch Inhibition der Cycline D, E und A uber transkriptionelle und post-translationale Mechanismen. Zusatzlich bewirkt Sorafenib in beiden Zellspezies die Herabregulation der anti-apoptotischen Proteine Akt, Bcl-2 und Mcl-1. Interessanterweise induzierte Sorafenib Apoptose ausschlieslich in Hepatomzellen, aber nicht in primaren Hepatozyten. Diese Ergebnisse zeigen, dass die pro-apoptotische Wirkung von Sorafenib nicht allein auf einer Reduktion anti-apoptotischer Signale beruht, sondern eine maligne Zelltransformation erfordert. In vivo fuhrte die Applikation von Sorafenib uber einen Zeitraum von bis zu 4 Tagen zu einer transienten Inhibition der DNA-Synthese und Zellzyklusprogression nach PH. Allerdings wurde die Rekonstitution der Lebermasse nach PH durch Sorafenib nicht beeinflusst. In Ubereinstimmung mit den in vitro erhobenen Daten induzierte Sorafenib keine Apoptose in hepatektomierten Lebern. Allerdings erhohte sich durch die Applikation von Sorafenib die nicht-apoptotische Leberschadigung nach PH, was anhand erhohter Serumtransaminasenaktivitat sowie dem vermehrten Auftreten von Hepatozyten mit irregularen Mitosen und freier (cytoplasmatischer) kondensierter DNA nachgewiesen wurde Schlussfolgerung: Sorafenib induziert selektiv Apoptose in maligne transformierten Hepatomzellen, jedoch nicht in proliferierenden Hepatozyten in vitro oder in vivo. Weiterhin deuten unsere Daten darauf hin, dass Sorafenib in regenerierenden Lebern eine nicht-apoptotische Schadigung der Hepatozyten hervorrufen kann.

135 Jnk1 IN HEPATIC STELLATE CELLS MODULATES LIVER FIBROGENESIS IN VIVO AND IN VITRO

Chronic liver damage may eventually progress to end-stage liver cirrhosis and hepatocellular carcinoma. Cytoglobin (Cygb) is a 21 kDa globin expressed in hepatic stellate cells and functions as a hypoxia sensor and a gas carrier. It serves as local peroxidase by degrading H2O2. However, its pathophysiological role in vivo remains undetermined. Here, we report the promotion of liver cancer development in Cygb-deficient (KO) mice administrated with either diethylnitrosamine (DEN) or choline-deficient amino acid-defined (CDAA) diet that induces hepatosteatosis. Methods: Cygb KO mice and corresponding wild-type (WT) mice at 8 week-old were treated with either DEN, CDAA, or CSAA diet for 8–32 weeks. Macroscopic and microscopic observations were performed. Gene expressions and intracellular signaling pathways were analyzed. Oxidative stress was determined by the formation of 8-OHdG, DHE, and nitrotyrosine. Results: Model 1; 25 or 0.05 ppm DEN treatment for 25 or 36 weeks induced liver tumor formation in 100% or 44%, respectively, in Cygb KO mice compared to 44% or 0%, respectively, in WT mice. Background liver developed fibrosis together with the augmented expression of mRNA of TGF-beta 3, collagen 1a1, and TIMP-1. Inflammatory gene expressions and augmented oxidative stress formation were evident in KO mice. Model 2; as early as 8 weeks of CDAA treatment, Cygb KO mice exhibited dominant steatohepatitis, which resulted in advanced fibrosis at 16 week-point, compared with WT as assessed by pathological NASH scores, collagen deposition, alpha-smooth muscle actin expression, and hepatic hydroxyproline content. Surprisingly, after 32 weeks under CDAA administration, 100% of both male and female Cygb KO mice developed liver cancers, compared to 0% in corresponding WT mice. Analyses at 32 week-point showed histologically severe inflammatory reactions concomitant with increased mRNA expression of Tnfa, Tgfb1, Il-1b and Il-6 in Cygb KO mice compared with WT mice. Cygb KO mice showed increased hepatocyte proliferation (Ki67 staining) and expression of AFP. Oxidative stress and antioxidant defense PCR array identified altered expression of 31 genes involved in the metabolism of reactive oxygen species in Cygb KO mice. Conclusion: Deficiency of Cygb promotes liver cancer development through activating inflammatory reaction and oxidative stress pathway.