Cornelius Engelmann

Selection and use of immunosuppressive therapies after liver transplantation: current German practice

In recent years, immunosuppression (IS) after liver transplantation (LT) has become increasingly diversified as the choice of agents has expanded and clinicians seek to optimize the balance of immunosuppressive potency with the risk of adverse events in individual patients. Calcineurin inhibitors (CNIs) are the primary agents used for patients undergoing liver transplantation. Other therapeutic agents like interleukin‐2 receptor antagonists are not universally administered, but can be considered for the delay or reduction in CNI exposure. An early addition of mycophenolate mofetil (MMF) or the mTOR inhibitor everolimus also allows for the reduction in the CNI dose. To reduce the risk of malignancy, in particular of skin tumors, as well as to prevent the deterioration of renal function, everolimus‐based therapy may be advantageous. Apart from patients with autoimmune hepatitis, steroids are withdrawn within 3–6 months after transplantation. Overall, immunosuppression can only be standardized in a limited proportion of patients due to specific clinical requirements and risk factors. Future studies should attempt to refine accurate individualization of the immunosuppressive regimen in specific difficult‐to‐treat patient subpopulations.

Application of Qualitative and Quantitative Real-Time PCR, Direct Sequencing, and Terminal Restriction Fragment Length Polymorphism Analysis for Detection and Identification of Polymicrobial 16S rRNA Genes in Ascites

ABSTRACT Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct sequencing were applied for rapid detection and identification of bacterial DNA (bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a mean of 3.24 log copies ml−1. Direct sequencing of PCR products revealed 62% mixed chromatograms predominantly belonging to Gram-positive bacteria. Terminal restriction fragment length polymorphism (T-RFLP) results of a sample subset confirmed sequence data showing polymicrobial DNA contents in 67% of bactDNA-positive ascites samples.

Low sphingosine-1-phosphate plasma levels are predictive for increased mortality in patients with liver cirrhosis

Background & aim The association of circulating sphingosine-1-phosphate (S1P), a bioactive lipid involved in various cellular processes, and related metabolites such as sphinganine-1-phosphate (SA1P) and sphingosine (SPH) with mortality in patients with end-stage liver disease is investigated in the presented study. S1P as a bioactive lipid mediator, is involved in several cellular processes, however, in end-stage liver disease its role is not understood. Methods The study cohort consisted of 95 patients with end-stage liver disease and available information on one-year outcome. The median MELD (Model for end-stage liver disease) score was 12.41 (Range 6.43–39.63). The quantification of sphingolipids in citrated plasma specimen was performed after methanolic protein precipitation followed by hydrophilic interaction liquid chromatography and tandem mass spectrometric detection. Results S1P and SA1P displayed significant correlations with the MELD score. Patients with circulating S1P levels below the lowest tertile (110.68 ng/ml) showed the poorest one-year survival rate of only 57.1%, whereas one-year survival rate in patients with S1P plasma levels above 165.67 ng/ml was 93.8%. In a multivariate cox regression analysis including platelet counts, concentrations of hemoglobin and MELD score, S1P remained a significant predictor for three-month and one-year mortality. Conclusions Low plasma S1P concentrations are highly significantly associated with prognosis in end-stage liver disease. This association is independent of the stage of liver disease. Further studies should be performed to investigate S1P, its role in the pathophysiology of liver diseases and its potential for therapeutic interventions.

Detection of molecular bacterascites in decompensated cirrhosis defines a risk with decreased survival.

Background The prognostic relevance of bacterial DNA (bactDNA) detection in ascitic fluid of patients with cirrhosis is still under debate. Using quantitative real-time PCR with broad-range primers targeting the V3 and V4 variable region of the 16S rRNA gene, we measured bactDNA concentrations in patients with and without leukocytic ascites and evaluated the impact on short-term survival. Patients and methods Ascites samples from 173 patients with decompensated cirrhosis were consecutively collected between February 2011 and December 2012. BactDNA-positive ascites samples were sequenced and chromatograms were identified using RipSeq. Clinical data collection and survival analyses were carried out retrospectively and correlated with ascites bactDNA levels. Results BactDNA was detected qualitatively with a similar frequency in both nonleukocytic and leukocytic ascites [40% (57/144) and 43.5% (10/23), respectively; P=0.724]. However, the median bactDNA level was significantly higher in leukocytic ascites than in nonleukocytic ascites (1.2×104 vs. 5.7×102 copies/ml; P=0.008). Patients’ survival was associated significantly with bactDNA level. The 30-day and 180-day survival was reduced if bactDNA was above the quantification limit of 520 copies/ml (84 and 63% vs. 72 and 43%, respectively; P<0.05) and worst if bactDNA was above 5000 copies/ml. The bacterial spectrum was dominated by Gram-positive strains as shown by direct sequencing. Conclusion BactDNA quantification in ascitic fluid samples using culture-independent 16S rRNA gene-based methods seems to be an interesting approach to identify patients at risk of reduced survival. Our study warrants further evaluation of antibiotic treatment in patients with molecular bacterascites.

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.

Acute-on-Chronic Liver Failure

a Department of Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany; b Department of General and Visceral Surgery, Goethe-University Hospital Frankfurt, Frankfurt/M., Germany; c Department of Gastroenterology, Rheumatology and Hepatology, University Hospital Leipzig, Leipzig, Germany; d Department of Internal Medicine IV, University Hospital Jena, Jena, Germany; e Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Magdeburg A.ö.R., Magdeburg, Germany; f Centre de Recherche sur l’Inflammation (CRI), Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris-Diderot, Paris, France; g European Foundation for the Study of Chronic Liver Failure, Barcelona, Spain; h Department of Internal Medicine I, University of Bonn, Bonn, Germany; i Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark; j Institute for Bioengineering of Catalonia, Barcelona, Spain

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication—with implications in organ regeneration.

Molecular quantification and differentiation of Candida species in biological specimens of patients with liver cirrhosis

Patients with liver cirrhosis are susceptible to fungal infections. Due to low sensitivity of culture-based methods, we applied a real-time PCR assay targeting the 18S rRNA gene in combination with direct sequencing and terminal-restriction fragment length polymorphism (T-RFLP) in order to establish a novel tool to detect fungal DNA and to quantify and differentiate Candida DNA, also in polyfungal specimens. In total, 281 samples (blood n = 135, ascites n = 92, duodenal fluid n = 54) from 135 patients with liver cirrhosis and 52 samples (blood n = 26, duodenal fluid n = 26) from 26 control patients were collected prospectively. Candida DNA was quantified in all samples. Standard microbiological culture was performed for comparison. Blood and ascites samples, irrespective of the patient cohort, showed a method-independent low fungal detection rate of approximately 1%, and the Candida DNA content level did not exceed 3.0x101 copies ml-1 in any sample. In contrast, in duodenal fluid of patients with liver cirrhosis high fungal detection rates were discovered by using both PCR- and culture-based techniques (81.5% vs. 66.7%; p = 0.123) and the median level of Candida DNA was 3.8x105 copies ml-1 (2.3x102-6.3x109). In cirrhosis and controls, fungal positive culture results were confirmed by PCR in 96% and an additional amount of 44% of culture negative duodenal samples were PCR positive. Using T-RFLP analysis in duodenal samples, overall 85% of results from microbial culture were confirmed and in 75% of culture-negative but PCR-positive samples additional Candida species could be identified. In conclusion, PCR-based methods and subsequent differentiation of Candida DNA might offer a quick approach to identifying Candida species without prior cultivation.

Successful direct acting antiviral (DAA) treatment of HCV/HIV-coinfected patients before and after liver transplantation

OBJECTIVES The aim of this multicenter retrospective study was to investigate safety and efficacy of direct acting antiviral (DAA) treatment in the rare subgroup of patients with HCV/HIV-coinfection and advanced liver cirrhosis on the liver transplant waiting list or after liver transplantation, respectively. METHODS When contacting 54 German liver centers (including all 23 German liver transplant centers), 12 HCV/HIV-coinfected patients on antiretroviral combination therapy were reported having received additional DAA therapy while being on the waiting list for liver transplantation (patient characteristics: Child-Pugh A (n = 6), B (n = 5), C (n = 1); MELD range 7-21; HCC (n = 2); HCV genotype 1a (n = 8), 1b (n = 2), 4 (n = 2)). Furthermore, 2 HCV/HIV-coinfected patients were denoted having received DAA therapy after liver transplantation (characteristics: HCV genotype 1a (n = 1), 4 (n = 1)). RESULTS Applied DAA regimens were SOF/DAC (n = 7), SOF/LDV/RBV (n = 3), SOF/RBV (n = 3), PTV/r/OBV/DSV (n = 1), or PTV/r/OBV/DSV/RBV (n = 1), respectively. All patients achieved SVR 12, in the end. In one patient, HCV relapse occurred after 24 weeks of SOF/DAC therapy; subsequent treatment with 12 weeks PTV/r/OBV/DSV achieved SVR 12. One patient underwent liver transplantation while on DAA treatment. Analysis of liver function revealed either stable parameters or even significant improvement during DAA therapy and in follow-up. MELD scores were found to improve in 9/13 therapies in patients on the waiting list for liver transplantation; in only 2 patients a moderate increase of MELD scores persisted at the end of follow-up. CONCLUSION DAA treatment was safe and highly effective in this nation-wide cohort of patients with HCV/HIV-coinfection awaiting liver transplantation or being transplanted.

Modulation der Immunreaktion kann Organdysfunktionen bei ACLF reduzieren

Das akut-auf-chronische Leberversagen (ACLF) ist ein relativ neu definiertes Erkrankungsbild mit ungünstiger Prognose und wenigen Behandlungsoptionen. Eine Immundysbalance mit überschießender systemischer Inflammationsreaktion und eine Immunparalyse sind wesentlich für die Entwicklung und den Verlauf des ACLF. Die aktuelle Forschung konzentriert sich darauf, die zugrunde liegenden Mechanismen zu verstehen, und durch Modulation der Immunreaktion das Ausmaß der Organdysfunktionen zu reduzieren.