Determination of protein turnover rates in the JAK/STAT pathway using a radioactive pulse-chase approach.

The turnover rate of different protein species in a signal transduction network strongly affects the impact of the given species on the outcome of a stimulus. Whereas stable, long-lived proteins mainly account for the transmission of a signal, unstable short-lived species often comprise regulatory functions. Here, we describe a method to determine the half-lives of proteins of the JAK/STAT pathway by a pulse-chase approach in cell culture. First, radioactive labeling with (35)S-methionine is carried out to label newly synthesized proteins (pulse). Subsequently, the dynamics of the decay of these proteins is monitored in the absence of labeled amino acids over a defined time period (chase). For this purpose the protein of interest is isolated by immunoprecipitation from total cell lysates, separated on an SDS-polyacrylamide gel, and subsequently visualized by autoradiography.

Cytokine blockade inhibits hepatic tissue inhibitor of metalloproteinase-1 expression and up-regulates matrix metalloproteinase-9 in toxic liver injury.

BACKGROUND: Tissue inhibitor of metalloproteinases (TIMP)-1, the most important endogenous inhibitor of matrix metalloproteinases, plays a pivotal role in the pathogenesis of liver fibrosis and may represent an effective therapeutic target in the design of antifibrotic strategies for chronic liver diseases. METHODS: Intraperitoneal application of a single dose of either tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta in mice led to an enhanced expression of hepatic TIMP-1 after 4-16 h. Male Sprague-Dawley rats were treated with carbon tetrachloride (CCl4) in the presence and absence of specific TNF-alpha and IL-1beta inhibitors. RESULTS: Real-time PCR revealed a significant increase of TIMP-1 mRNA in total rat liver 24 h after CCl4 injection. Repetitive injection of both, etanercept and anakinra, before and after CCl4 injection effectively inactivated TNF-alpha and IL-1beta. Anticytokine pretreatment reduced the increase of TIMP-1 expression after a single CCl4 injection by 50% and 75%, respectively. In contrast to CCl4-treated rats with and without TNF-alpha blockade, IL-1beta inactivation caused a sevenfold increase in matrix metalloproteinases-9 mRNA levels. CONCLUSIONS: In conclusion, TIMP-1 expression is up-regulated in the early phase of toxic liver injury by proinflammatory cytokines such as TNF-alpha and IL-1beta in rodents. Pharmacological inactivation of these cytokines significantly reduces TIMP-1 gene expression. Our data provide a potential new antifibrotic approach.

Recurrent thrombotic occlusion of a transjugular intrahepatic portosystemic stent-shunt due to activated protein C resistance.

The transjugular intrahepatic portosystemic stent-shunt (TIPS) has successfully been used in the management of refractory variceal bleeding and ascites in patients with portal hypertension. Major drawbacks are the induction of hepatic encephalopathy and shunt dysfunction. We present a 59-year-old woman with alcoholic liver cirrhosis who received a TIPS because of recurrent bleeding from esophageal varices. Stent occlusion occurred 4 mo after placement of the TIPS. Laboratory testing revealed resistance to activated protein C (APC). Combination therapy with low-dose enoxaparin and clopidogrel could not prevent her recurrent stent occlusion. Finally, therapy with high-dose enoxaparin was sufficient to prevent further shunt complications up to now (follow-up period of 1 year). In conclusion, early occlusion of a TIPS warrants testing for thrombophilia. If risk factors are confirmed, anticoagulation should be intensified. There are currently no evidence-based recommendations regarding the best available anticoagulant therapy and surveillance protocol for patients with TIPS.

Interleukin-6 plays a crucial role in the hepatic expression of SOCS3 during acute inflammatory processes in vivo.

BACKGROUND/AIMS: Interleukin-6 is mandatory for liver regeneration after injury and for the hepatic expression of acute phase proteins and cytochrome P450 enzymes during inflammation. Due to its crucial contribution to the maintenance of homeostasis IL-6 signaling is tightly controlled. Suppressor of cytokine signaling (SOCS) 3 is a potent IL-6-induced feedback inhibitor terminating IL-6 signal transduction. However, several signaling pathways converge on SOCS3: SOCS3 can be induced by other mediators in vitro, and it does not exclusively inhibit IL-6 signaling. The individual contribution of each cytokine to the induction of SOCS3 in vivo is unknown. METHODS: Using IL-6-deficient mice we analyzed the role of interleukin-6 for the hepatic SOCS3 expression in response to turpentine and LPS as models of aseptic and bacterial inflammation, respectively. RESULTS: In wild-type animals, turpentine and LPS elicited strong induction of SOCS3. IL-6-deficient mice, by contrast, showed severely impaired SOCS3 expression in response to both stimuli: turpentine failed to induce SOCS3 mRNA; in LPS-induced inflammation, the early inductive response 60min after LPS injection was absent, and the delayed expression of SOCS3 was markedly reduced. The residual delayed SOCS3 expression in IL-6-deficient mice was abolished in IL-6/TNFR-1 knockout mice. CONCLUSIONS: Our data strongly argue for a crucial role of IL-6 in the hepatic expression of SOCS3 during acute inflammatory processes in vivo. Although other cytokines are capable of inducing SOCS3 their contribution seems to be minor.

Patients with acute on chronic liver failure display "sepsis-like" immune paralysis.

BACKGROUND/AIMS: Cellular immune depression is linked to high mortality in sepsis, but has yet to be systematically analysed in liver cirrhosis. The aim of the present study was to directly compare functional immune parameters in patients with acute on chronic liver failure (ACLF), severe sepsis, and non-decompensated cirrhosis. METHODS: Patients with ACLF (n=27) were investigated at admission to a medical ICU. Patients with stable liver cirrhosis (n=24) and severe sepsis (n=31) served as control groups. In all subjects, serum levels of IL-6 and IL-10, ex vivo production of TNF-alpha in a whole blood assay, and monocyte surface HLA-DR expression were determined. RESULTS: In patients with ACLF or sepsis, ex vivo TNF-alpha production and HLA-DR expression were severely decreased compared to subjects with stable cirrhosis (both P<0.001). Contrary, IL-6 levels were highest in septic patients, followed by subjects with ACLF and cirrhotic patients (both P<0.05). Immune dysfunction in ACLF was independent of aetiology of liver cirrhosis and associated with high mortality. CONCLUSIONS: Patients with ACLF and severe sepsis show a similar degree of cellular immune depression. The reduced cellular immune function in subjects with ACLF might contribute to the increased infectious morbidity of these patients and provide a rational basis for prevention strategies.

Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C.

BACKGROUND: Chronic hepatitis C is frequently associated with increased hepatic iron stores. It remains controversial whether heterozygous mutations of hemochromatosis genes affect fibrosis progression. Therefore our aim was to assess associations between HFE mutations and hepatic inflammation and stage of fibrosis in German hepatitis C patients. METHODS: Liver biopsies from 166 patients were scored for inflammatory activity (A0-4) and hepatic fibrosis (F0-4). Gene mutations were determined by LightCycler, restriction fragment length polymorphism analysis, or direct sequencing. RESULTS: The frequencies of common HFE mutations C282Y and H63D are 4.2% and 21.3%, whereas the recently described S65C substitution and the Y250X mutation in the transferrin receptor 2 gene are very rare. In regression analysis, heterozygous carriers of C282Y or H63D mutations display significantly (P < 0.05) higher inflammatory activities and more advanced fibrosis than patients without mutations. For C282Y heterozygous patients, the odds ratios for marked inflammatory activity (A2-4) and advanced liver fibrosis or cirrhosis (F2-4) are 4.9 and 4.6, respectively, compared with patients carrying homozygous wild-type alleles. C282Y mutations are associated with significantly (P < 0.05) increased serum iron and aminotransferase levels, whereas H63D heterozygotes display higher transferrin saturation, serum iron, and ferritin concentrations compared to wild-type (P < 0.01). CONCLUSIONS: Common heterozygous hemochromatosis mutations are associated with higher grades of inflammation and more severe hepatic fibrosis. Our findings support a role of HFE mutations as primary risk factors for fibrogenesis and disease progression in chronic hepatitis C.

Interleukin-6 regulates hepatic transporters during acute-phase response.

Cholestasis develops during inflammatory conditions characterized by the release of cytokines like interleukin-6 (IL-6), which is the major player in the hepatic acute-phase response. However, the exact contribution of IL-6 to transporter down-regulation is unclear. Therefore, we compared wild-type and IL-6-deficient mice after IL-6-injection and induction of an aseptic (turpentine-injection) or septic (LPS-injection) acute-phase response. Down-regulation of basolateral (Ntcp, Oatp1, and Mrp3) and canalicular (Mrp2, Bsep) transporter mRNA occurred after treatment with IL-6, turpentine, and LPS. In IL-6-deficient mice, turpentine failed to decrease mRNA-levels of basolateral and canalicular transporters, whereas LPS-mediated down-regulation of Ntcp, Mrp3, and Mrp2 was abolished at later time points (24 h). In conclusion, induction of an aseptic and septic acute-phase response leads to the down-regulation of basolateral and canalicular organic anion transporters. IL-6 is required for transporter down-regulation during aseptic inflammation. Furthermore, IL-6 also contributes to transporter regulation during LPS-induced cholestasis at more delayed time points.

[Acute infectious mononucleosis-like syndrome due to infection with cytomegalovirus in immunocompetence]. / Akutes mononukleoseähnliches Krankheitsbild durch Infektion mit dem Zytomegalievirus bei Immunkompetenz.

BACKGROUND: Infections by cytomegalovirus (CMV) cause substantial morbidity and mortality in immunocompromised patients, whereas immunocompetent individuals generally experience mild disease or remain asymptomatic. CASE REPORT: A 54-year-old immunocompetent man undergoing CMV mononucleosis and hepatitis after primary infection is reported. Symptoms were pronounced with considerably reduced general condition and sustained highly febrile temperatures. Only therapy with intravenous ganciclovir led to rapid resolution of the disease. A second case describes a 31-year-old woman who, having had contact with a CMV-infected infant, developed a mononucleosis-like illness during her pregnancy. Due to the risk of congenital CMV infection, the exclusion of CMV-caused infection or the discrimination between primary and secondary infection is of particular importance, and the second case illustrates the relevant diagnostic difficulties and pitfalls. CONCLUSION: As demonstrated by the two cases, even in immunocompetent patients and pregnant women CMV should be considered in the differential diagnosis of symptomatic viral infections. Particularly during pregnancy and after organ transplantation, early diagnosis and prognostic markers are necessary because of possible severe complications such as congenital CMV infection and organ transplant failure.