Synergisms of stem cell mobilisation and toll-like receptor 4 inhibition to treat the acute-on-chronic liver failure

Background: The acute-on-chronic liver failure (ACLF) is a complex disease with high mortality without effective therapeutic opportunities. A greater understanding of disease mechanisms is necessary to identify novel targets for disease modifying therapies. Systemic inflammation is central and toll-like receptor 4 (TLR4) may be an important pathway involved in activating immune cells. Granulocyte-colony stimulating factor (G-CSF) mobilises immunomodulatory stem- and immune cells and pivotal clinical studies provided promising results in liver disease. Aims: I aimed to understand the role of TLR4 in mediating inflammation and organ injury in ACLF. I also explored how G-CSF exerts its immunomodulatory capacities in ACLF and whether combing G-CSF with TLR4 signalling inhibition by TAK-242 might act synergistically. Methods: The first experimental part comprised human samples, and septic ACLF rodent models in which I evaluated the role of TLR4 in mediating inflammation and organ injury. The second clinical part was based on a multicentre interventional trial evaluating the impact of G-CSF on the outcome of humans with ACLF. In the third experimental part G-CSF and TAK-242 were used as therapy in a septic and in a sterile ACLF mouse model to evaluate synergisms of both therapies. Results: The first part showed that TLR4 signalling prompted cytokine release and organ injury causing deaths in ACLF. TLR4 signalling inhibition by TAK-242 prevented tissue injury and fatalities. The clinical trial revealed that G-CSF was ineffective in ACLF. In the septic mouse model of ACLF GCSF alone exaggerated the inflammatory response. The combinatory therapy G-CSF plus TAK-242 acted synergistically by reducing organ injury and promoting tissue repair. Conclusion: This study highlighted the complexity of ACLF and introduced TLR4 mediated inflammation as a central pathomechanistic element. It also emphasized that combinatory therapies may be necessary to address the whole spectrum of pathomechanisms involved in ACLF

The Potential Role of Cellular Senescence in Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) represents an increasing global health burden. Cellular senescence develops in response to cellular injury, leading not only to cell cycle arrest but also to alterations of the cellular phenotype and metabolic functions. In this review, we critically discuss the currently existing evidence for the involvement of cellular senescence in NAFLD in order to identify areas requiring further exploration. Hepatocyte senescence can be a central pathomechanism as it may foster intracellular fat accumulation, fibrosis and inflammation, also due to secretion of senescence-associated inflammatory mediators. However, in some non-parenchymal liver cell types, such as hepatic stellate cells, senescence may be beneficial by reducing the extracellular matrix deposition and thereby reducing fibrosis. Deciphering the detailed interaction between NAFLD and cellular senescence will be essential to discover novel therapeutic targets halting disease progression

Prognose bestimmende inflammatorische Marker der Leberzirrhose

Die Leberzirrhose ist eine Erkrankung mit häufig progredientem Verlauf. Patienten entwickeln vermehrt Zirrhose-assoziierte Komplikationen (dekompensierte Leberzirrhose) und sterben letztendlich häufig am sogenannten akut-auf-chronischen Leberversagen mit Multiorganversagen. Bakterielle Infektionen und eine systemische Inflammationreaktion wind wesentliche Faktoren, welche den Verlauf der Erkrankung bestimmen. Allerdings sind die diagnostischen und therapeutischen Optionen bisher unzureichend, um die hohe Letalität der Zirrhose im Endstadium zu verbessern. Ziel der hier gezeigten Habilitationsarbeit war es daher, prognostische Parameter für Patienten mit fortgeschrittener Leberzirrhose zu evaluieren und damit eine Grundlage für die Anpassung von Management-Strategien für Patienten zu liefern. Über den Prognosescore CLIF-C ACLF Score war es möglich Patienten mit besonders hohem Sterberisiko und Dringlichkeit zur Anpassung der Behandlung zu identifizieren. Es zeigte sich, dass eine Dysfunktion der peritonealen neutrophilen Granulozyten für die hohe Rate an infektiösen Komplikation, insbesondere im Peritoneum, verantwortlich sein können und dass eine übermäßige Aktivierung von Neutrophilen und Monozyten mit Sequestration von Mikropartikeln im Aszites Ausdruck einer allgemeinen Immunaktivierung und schlechter Prognose sind. Zudem konnten neue PCR (polymerase chain reaction) basierte Methoden etabliert werden, über die eine rasche Identifikation und Charakterisierung von bakteriellen und fungalen Infektionen möglich ist. Auf Grundlage dieser Ergebnisse sollte nun in Zukunft evaluiert werden, ob gezielte Anpassung der Diagnostik und Therapie die Prognose von Patienten mit fortgeschrittener Leberzirrhose verbessern kann

Use of bacterial DNA concentration in ascites as a marker for spontaneous bacterial peritonitis

Background & Aims Spontaneous bacterial peritonitis (SBP) is a common and serious complication in patients with decompensated cirrhosis. Precise quantification of bacterial DNA (bactDNA) and the related inflammatory response might add further information on the course of disease. The aim of the study was to evaluate the association between bactDNA, cytokine levels and clinical outcome. Methods Ascites and serum samples of 98 patients with decompensated liver cirrhosis (42 with SBP and 56 without SBP) as well as serum samples of 21 healthy controls were collected. BactDNA in ascites and serum was detected and quantified by 16S rRNA PCR. Concentrations of IL-1β, TNF-α, IL-6, IL-8 and IL-10 were measured by a LEGENDplexTM multi-analyte flow assay. Clinical data were collected and analyzed retrospectively. Results BactDNA was detected more frequently in ascites of patients with SBP (n=24/42; 57.1%) than in ascites of patients without SBP (n=5/56; 8.9%; p < 0.001). Additionally, IL-6 levels in both ascites and serum were significantly higher in patients with SBP (ascites p < 0.001, serum p = 0.036). The quantity of bactDNA in ascites was strongly correlated with polymorphonuclear neutrophil count in ascites (r = 0.755; p < 0.001) as well as ascites IL-6 levels (r = 0.399; p < 0.001). Receiver operating characteristic (ROC) curve analysis to diagnose SBP provided an AUC of 0.764 (95% CI: 0.661 – 0.867) for serum IL-6 levels, an AUC of 0.810 (95% CI: 0.714 - 0.905) for ascites IL-6 levels, and an AUC of 0.755 (95% CI: 0.651 – 0.858) for bactDNA levels in ascites. Conclusions The correlation between the amount of bactDNA and IL-6 confirms the pathophysiological relevance of bactDNA and IL-6 as potential biomarkers for the diagnosis of SBP

Ascites’ neutrophil function is significantly impaired in patients with decompensated cirrhosis but can be restored by autologous plasma incubation

Systemic immune cell dysfunction is a typical feature of liver diseases and increases the risk of bacterial infection, especially spontaneous bacterial peritonitis. We evaluated functional properties of neutrophil granulocytes in blood and ascites of patients both with and without decompensated cirrhosis. We collected blood and ascites samples from 63 patients with cirrhosis and eight without cirrhosis. Phagocytosis activity (PA) and oxidative burst activity (OBA) were evaluated after ex vivo stimulation with E. coli, while fluorescence signals were measured by flow cytometry. Ascites’ neutrophil function tests were repeated after incubation with autologous plasma. Ascites’ neutrophils showed an impaired PA and OBA (median blood PA 98.1% (86.8–99.8) vs. ascites’ PA 50.5% (0.4–97.3), p < 0.0001; median blood OBA 98.7% (27.5–100) vs. ascites’ OBA 27.5% (0.3–96.7), p < 0.0001). Patients with non- cirrhotic ascites showed higher PA but equally suppressed OBA. Ascites’ neutrophil function could be partially restored after incubation with autologous plasma (median increase PA: 22.5% (−49.7 – +93.2), p = 0.002; OBA: 22.8% (−10.4 – +48.8), p = 0.002). Ascites’ neutrophils of patients with cirrhosis are functionally impaired, but could be partially restored after incubation with plasma. Further investigations are needed to identify the factors in ascites that are associated with neutrophils’ function

Body fat composition determines outcomes before and after liver transplantation in patients with cirrhosis

Cachexia occurs in late stages of liver cirrhosis, and a low-fat mass is potentially associated with poor outcome. This study compared different computed tomography (CT)–derived fat parameters with respect to its prognostic impact on the development of complications and death before and after liver transplantation. Between 2001 and 2014, 612 patients with liver cirrhosis without hepatocellular carcinoma listed for liver transplantation met the inclusion criteria, including abdominal CT scan (±200 days to listing). A total of 109 patients without cirrhosis served as controls. The subcutaneous fat index (SCFI), the paraspinal muscle fat index, and the visceral fat index were assessed at L3/L4 level and normalized to the height (cm2/m2). Data were collected and analyzed retrospectively. Low SCFI was associated with a higher rate of ascites and increased C-reactive protein levels (p < 0.001). In addition, multivariate Cox regression analysis adjusting for sex, age, body mass index (BMI), and Model for End-Stage Liver Disease showed that decreasing SCFI was also associated with an increased risk of cirrhosis-related complications (p = 0.003) and death on the transplant wait list (p = 0.013). Increased paraspinal and visceral fat were not only positively correlated with creatinine levels (p < 0.001), BMI, and metabolic comorbidities (all p < 0.001) before transplantation, but also predictive for 1-year mortality after transplantation. Conclusion: The distribution of body fat is a major determinant for complications and outcome in cirrhosis before and after liver transplantation

Consensus care recommendations for alfapump® in cirrhotic patients with refractory or recurrent ascites

BACKGROUND: The alfapump® is an implantable class III medical device that pumps ascitic fluid from the peritoneal space to the urinary bladder from where it is excreted. The pump reduces or abrogates the need for repeated paracentesis in patients with recurrent or refractory ascites. AIMS: To improve outcomes for alfapump® implantation and pre- and post-implant patient management in both clinical trial and real-world settings by development of consensus recommendations. METHODS: The alfapump® working group consisting of hepatologists and surgeons with extensive experience in implantation of the alfapump® and patient management met on two occasions: (1) to determine the key areas where recommendations should be made; and (2) to discuss the experiences of the working group within those areas and formulate draft statements. Developed statements were submitted to the group and consensus sought on relevance and wording through a collaborative iterative approach in order to consolidate the recommendations into consensus statements. Only recommendations agreed upon unanimously were included. RESULTS: Twenty-three consensus recommendations were developed in the areas of pre-implantation procedure, (three statements), surgical implant procedure (11 statements), immediate post-implant care (three statements) and long-term management (six statements). CONCLUSIONS: The consensus statements are a valuable reference resource for physicians managing patients with the alfapump® and for those considering management strategies for patients with refractory ascites

Consensus care recommendations for alfapump® in cirrhotic patients with refractory or recurrent ascites.

BACKGROUND The alfapump® is an implantable class III medical device that pumps ascitic fluid from the peritoneal space to the urinary bladder from where it is excreted. The pump reduces or abrogates the need for repeated paracentesis in patients with recurrent or refractory ascites. AIMS To improve outcomes for alfapump® implantation and pre- and post-implant patient management in both clinical trial and real-world settings by development of consensus recommendations. METHODS The alfapump® working group consisting of hepatologists and surgeons with extensive experience in implantation of the alfapump® and patient management met on two occasions: (1) to determine the key areas where recommendations should be made; and (2) to discuss the experiences of the working group within those areas and formulate draft statements. Developed statements were submitted to the group and consensus sought on relevance and wording through a collaborative iterative approach in order to consolidate the recommendations into consensus statements. Only recommendations agreed upon unanimously were included. RESULTS Twenty-three consensus recommendations were developed in the areas of pre-implantation procedure, (three statements), surgical implant procedure (11 statements), immediate post-implant care (three statements) and long-term management (six statements). CONCLUSIONS The consensus statements are a valuable reference resource for physicians managing patients with the alfapump® and for those considering management strategies for patients with refractory ascites

Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is an unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with Granulocyte-Colony Stimulating Factor (G-CSF) targets inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride followed by either lipopolysaccharide (LPS) or galactosamine (GalN) as extrahepatic or hepatic insults, respectively. G-CSF and/or TLR4-antagonist, TAK-242, were administered daily. The treatment duration was 24h and 5d in the LPS model and 48h in the GalN model, respectively. RESULTS: In a LPS-induced ACLF mouse model treatment with G-CSF was associated with a significant mortality of 66% after 48 hours compared with 0% without G-CSF. Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterized by p21 over-expression suggesting hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase of markers of hepatocyte regeneration. CONCLUSION: TLR4 inhibition with TAK-242 rescued G-CSF-driven cell death, inflammation, enhanced tissue repair, and significantly induced regeneration thus suggesting that the combination of G-CSF and TAK-242 is a novel approach for the treatment of ACLF. LAY SUMMARY: The combinatorial therapy of Granulocyte-Colony Stimulating Factor and TAK-242, a Toll-like Receptor-4 inhibitor, achieves the dual aim of reducing hepatic inflammation and inducing liver regeneration for the treatment of acute-on-chronic liver failure

Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study

Background: Microparticles (MPs) are small (488.4 MP/mu L 94.7%, log rank p = 0.001) and such patients had a higher relative amount of ascites microparticles derived from neutrophils and lymphocytes. Low levels of ascites MPs, high MELD score and antibiotic treatment were independent risk factors for death within 30 days. Conclusions: Ascites MP levels predict short-term survival along with the liver function in patients with decompensated cirrhosis. Further studies which evaluate ascites MPs as disease specific biomarker with a validation cohort and which investigate its underlying mechanisms are needed. Neutrophils and lymphocytes contributed more frequently to the release of microparticles in patients with low ascites levels, possibly indicating an immune activation in this cohort