Der Einfluss von 12/15-Lipoxygenase-Hemmung und Tacrolimus-Behandlung auf die Glutathionperoxidase-4-abhängige Signalkaskade im hepatischen Ischämie-Reperfusionsschaden

Hintergrund In Zeiten des Organmangels, in denen vermehrt marginale Spenderorgane zur Transplantation akzeptiert werden, steht die Erforschung potentieller Mechanismen der Verbesserung von Organqualität und Verlängerung von Transplantatüberleben im Vordergrund. Der hepatische Ischämie-Reperfusionsschaden (IRI) stellt hierbei eine der zentralen Komplikationen nach Lebertransplantationen dar, weil er das Organüberleben signifikant einschränkt. Der hepatische IRI entsteht durch die Ausbildung von oxidativem Stress nach der Gewebsreperfusion, ist charakterisiert durch ein komplexes Zusammenspiel aus Mikrozirkulationsstörung, zellulärer Immunantwort, sowie proinflammatorischen Mediatoren und mündet in unterschiedlichen Formen des Zelltods. In den letzten Jahren konnte die Relevanz der Glutathion-peroxidase-4 (Gpx4) -abhängigen Signalkaskade, in welcher die 12/15-Lipoxygenase (12/15-LOX) als Lipidperoxidquelle einen oxidativen Zelltodinitiator darstellt, im IRI anderer Organsysteme nachgewiesen werden. Jedoch konnten die genauen Wirkmechanismen und Zelltodmediatoren der Gpx4-abhängigen Signalkaskade bisher noch nicht vollständig geklärt werden. Zudem wurde das etablierte Immunsuppressivum und Calcineurininhibitor Tacrolimus in den vergangenen Jahren immer wieder mit einer Wirkung auf den Glutathionmetabolismus in Verbindung gebracht, wenngleich auch hier die genaue Interaktion nicht endgültig beschrieben werden konnte. Ziel dieser Studie war daher, die Relevanz der Gpx4-abhängigen Signalkaskade für den hepatischen IRI zu erörtern, neue Erkenntnisse über potentielle Mediatormechanismen zu erlangen und die Interaktion dieser Signalkaskade mit Tacrolimus zu beleuchten. Methoden An Lebern von C57BL/6-Mäusen wurde unter peritoneal applizierter Narkose operativ ein hepatischer IRI induziert, indem der gemeinsame Pedikel des rechten anterioren, linken an-terioren und linken posterioren Lebersegments für 60 Minuten abgeklemmt wurde, was eine 65%ige Leberischämie zur Folge hatte. In der weiteren Folge erfolgte eine 90-minütige Reperfusionsphase. Die verschiedenen Versuchstiere wurden entsprechend ihrer Experimentalgruppe ohne medikamentöse Vorbehandlung untersucht, beziehungsweise nach 30-minütiger Vorbehandlung mit Baicalein, einem potenten Hemmstoff der 12/15-LOX, und der Trägerlösung von Baicalein, DMSO. Zudem wurde eine Versuchsgruppe 24 Stunden vor Laparotomie mit Tacrolimus vorbehandelt, sowie eine Versuchsgruppe kombiniert mit Tacrolimus und Baicalein. Das Gewebe der postischämischen Lebersegmente wurden hin-sichtlich des Zelltodausmaßes mittels TUNEL-Assay quantifiziert, bezüglich der proapoptotischen Proteine ERK1/2, JNK, PARP und Caspase-3 mithilfe von Western Blot-Analysen analysiert und für die Bestimmung der Glutathionextinktion spektralphotometrisch untersucht. Ferner erfolgten eine laborchemische Auswertung der Mausseren zum Ende des Experiments in Bezug auf Leber- und Nierenparameter, sowie eine kontinuierliche, intraarterielle Kreislaufüberwachung. Für sämtliche Versuchsgruppen wurden zudem scheinoperierte Kontrollgruppen anhand derselben Verfahren untersucht. Ergebnisse Die Analyse der TUNEL-Assays ergab eine signifikante Abnahme abgestorbener Zellen nach IRI-Induktion und Vorbehandlung mit Baicalein (-74,8%; p<0,001), Tacrolimus (-61,8%; p<0,01) und der kombinierten Vortherapie mit beiden Medikamenten (-62,3%; p<0,001). Nach alleiniger DMSO-Therapie konnte zumindest ein negativer Regulationstrend hinsichtlich der Zelltodabnahme festgestellt werden (-23,2%). Die Western Blot-Analysen ergaben eine tendenzielle Minderexpression von ERK1/2 nach Baicalein- (-36,8%), Tacrolimus- (-19,1%) und Kombinationstherapie (-28,1%), sowie eine signifikante Abnahme der Aktivität von JNK durch Baicalein- (-83,0%; p<0,01), DMSO- (-69,7%; p<0,05) und Tacrolimusgabe (-84,6%; p<0,05), von PARP durch Baicalein- (-73,8%; p<0,05), DMSO- (-68,9%; p<0,05) und Kombinationstherapie (-80,1%; p<0,05), wie auch von Caspase-3 nach Baicalein- (-45,0%; p<0,001), Tacrolimus- (-66,9%; p<0,001) und kombinierter Applikation (-59,2%; p<0,001). Ein negativer Regulationstrend der PARP-Aktivität konnte auch nach Tacrolimusgabe verzeichnet werden (-59,9%). Zudem war eine signifikante Steigerung des oxidativen Glutathionmetabolismus nach Baicalein- (+45,7%; p<0,05), Tacrolimus- (+75,1%; p<0,05) und kombinierter Therapie (+43,7%; p<0,05) festzustellen. Während allein die Gabe von Baicalein, DMSO oder der Kombinationstherapie zu einer signifikant gesteigerten Ausschüttung der Transaminasen und GLDH ins Blut führte, konnte eine relative Abnahme der postischäm erfassten Laborwerte nach Baicalein- (GOT -56,4%; p<0,001; GPT: -16,8%; GLDH -62,1%; p<0,001), DMSO- (GOT -36,5%; p<0,01; GPT -43,7%; p<0,001; GLDH -40,0%; p<0,001), Tacrolimus- (GOT -28,8%; p<0,05; GPT -53,4%; p<0,001; GLDH -3,4%) und der Kombinationstherapie (GOT -64,4%; p<0,001; GPT -10,4%; GLDH -47,1%; p<0,001), größtenteils signifikant, gemessen werden. Schlussfolgerung Die Vorbehandlung mit Baicalein führt zu einer signifikanten Zelltodabnahme nach hepatischem IRI, sowie zu einer signifikanten Steigerung der Glutathionoxidierung. Daraus kann geschlussfolgert werden, dass die Gpx4-abhängige Signalkaskade eine relevante Rolle im hepatischen IRI zu spielen scheint. Diese hepatoprotektiven Effekte werden durch die signifikante Reduktion der JNK-, PARP- und Caspase-3-Aktivität, sowie durch eine tendenzielle Abnahme der ERK1/2-Aktivität bewirkt, welche somit relevante Zelltodmediatoren in der Gpx4-abhängigen Signalkaskade zu sein scheinen. Auch eine Tacrolimustherapie hat ähnliche Auswirkungen auf die Zelltodentwicklung und den Glutathionmetabolismus zur Folge, während sie dieselben Zelltodmediatoren in vergleichbarer Weise affektiert. Somit ist zu mutmaßen, dass auch Tacrolimus seine postischäm hepatoprotektiven Eigenschaften durch die Interaktion mit dem Gpx4-Signalweg erhält. Ebenso scheint die als Antioxidans bekannte Trägerlösung DMSO in weitaus diskreterem Ausmaß als die anderen beschriebenen Substanzen Teile der dargestellten Signalketten des oxidativen Glutathionstoffwechsels positiv zu beeinflussen. Eine Kombinationstherapie aus Tacrolimus und Baicalein konnte jedoch keine Steigerung der jeweiligen Therapieeffekte erbringen. Dies ist am ehesten auf die nachgewiesenen, relevanten toxischen Effekte der Einzelsubstanzen auf Zelltod, Leberwerte im Serum und Kreislaufparameter zurückzuführen, welche im postischämischen Umfeld jedoch durch die protektiven Effekte amortisiert zu sein schienen. Baicalein und Tacrolimus stellen somit potentielle Therapieansätze zur Reduktion des hepatischen IRI dar, sofern ein sinnvolles Maß an Protektivität im Vergleich zur Toxizität erlangt werden kann

Endoscopic vacuum therapy for in- and outpatient treatment of colorectal defects

Background. Evidence for endoscopic vacuum therapy (EVT) for colorectal defects is still based on small patient series from various institutions, employing different treatment algorithms and methods. As EVT was invented at our institution 20 years ago, the aim was to report the efficacy and safety of EVT for colorectal defects as well as to analyze factors associated with efficacy, therapy duration, and outpatient treatment. Methods. Cohort study with analysis of prospectively collected data of patients receiving EVT for colorectal defects at a tertiary referral center in Germany (n=281). Results. The majority of patients had malignant disease (83%) and an American Society of Anesthesiologists classification of III/IV (81%). Most frequent indications for EVT were anastomotic leakage after sigmoid or rectal resection (67%) followed by rectal stump leakage (20%). EVT was successful in 256 out of 281 patients (91%). EVT following multi-visceral resection (P = 0.037) and recent surgical revision after primary surgery (P = 0.009) were risk factors for EVT failure. EVT-associated adverse events occurred in 27 patients (10%). Median treatment duration was 25 days. Previous chemo-radiation (P = 0.006) was associated with a significant longer duration of EVT. Outpatient treatment was conducted in 49% of patients with a median hospital stay reduction of 15 days and 98% treatment success. Younger patient age (P = 0.044) was associated with the possibility of outpatient treatment. Restoration of intestinal continuity was achieved in 60% of patients where technically possible with a 12-month rate of 52%. Conclusions. In patients with colorectal defects, EVT appears to be a safe and effective, minimally invasive option for in- and outpatient treatment

Infections after kidney transplantation: A comparison of mTOR‐Is and CNIs as basic immunosuppressants. A systematic review and meta‐analysis

Background Side effects of the immunosuppressive therapy after solid organ transplantation are well known. Recently, significant benefits were shown for mTOR‐Is with respect to certain viral infections in comparison with CNIs. However, reported total incidences of infections under mTOR‐Is vs CNIs are usually not different. This raises the question to additional differences between these immunosuppressants regarding development and incidence of infections. Methods The current literature was searched for prospective randomized controlled trials in renal transplantation. There were 954 trials screened of which 19 could be included (9861 pts.). The 1‐year incidence of infections, patient and graft survival were assessed in meta‐analyses. Results Meta‐analysis on 1‐year incidence of infections showed a significant benefit of an mTOR‐I based therapy when combined with a CNI vs CNI‐based therapy alone (OR 0.76). There was no difference between mTOR‐I w/o CNI and CNI therapy (OR 0.97). For pneumonia, a significant disadvantage was seen only for mTOR‐I monotherapy compared to CNI's (OR 2.09). The incidence of CMV infections was significantly reduced under mTOR‐I therapy (combination with CNI: OR 0.30; mTOR w/o CNI: OR: 0.46). There was no significant difference between mTOR‐I and CNI therapy with respect to patient survival (mTOR‐I w/o CNI vs CNI: OR 1.22; mTOR‐I with CNI vs CNI: OR 0.86). Graft survival was negatively affected by mTOR‐I monotherapy (OR 1.52) but not when combined with a CNI (OR 0.97). Conclusion Following renal transplantation the incidence of infections is lower when mTOR‐Is are combined with a CNI compared to a standard CNI therapy. Pneumonia occurs more often under mTOR‐I w/o CNI

Differential significance of early surgical complications for acute and long-term recurrence-free survival following surgical resection of hepatocellular carcinoma: do comorbidities play a role?

Background Postoperative complications of Clavien-Dindo grade 3 or more are of prognostic significance in patients who undergo liver resection for hepatocellular carcinoma (HCC). However, perioperative mortality and patient comorbidities represent relevant factors that interfere with postoperative long-term survival. To clarify this, a retrospective single-center study was carried out. Patients and methods Patient data were prospectively collected in a continuously updated liver resection database. Overall, 184 consecutive patients who underwent liver resection for HCC with a curative intent between March 2003 and December 2013 were selected for the study. The patients were assigned to two groups according to the presence or absence of postoperative complications. Pre-existing comorbidities, perioperative mortality, surgical outcome, and long-term survival data were analyzed. Results Postoperative complications requiring revision surgery were identified in 17.4% of the patients. The in-house mortality rate was 4.8%. Compared with patients without complications, patients with complications were older and had significantly more pre-existing comorbidities, more advanced tumors, more intrahepatic metastasis, longer operation times, greater blood loss, and more extensive resections. The overall 5-year survival rates were 40.1 and 52.5% in patients with or without postoperative complications, respectively. The corresponding 5-year recurrence-free survival rates were 46.3 and 46.7% (perioperative mortality excluded). Multivariate analysis showed that elevation of the Charlson Comorbidity Index was associated independently with decreased overall and recurrence-free survival. Conclusion In patients with HCC, posthepatectomy complications are confirmed to have predictive value. However, closer analysis and exclusion of perioperative mortality effects show an independent impact of pre-existing comorbidities on long-term overall und recurrence-free survival

Hypothermic Oxygenated Machine Perfusion (HOPE) Prior to Liver Transplantation Mitigates Post-Reperfusion Syndrome and Perioperative Electrolyte Shifts

(1) Background: Post-reperfusion syndrome (PRS) and electrolyte shifts (ES) represent considerable challenges during liver transplantation (LT) being associated with significant morbidity. We aimed to investigate the impact of hypothermic oxygenated machine perfusion (HOPE) on PRS and ES in LT. (2) Methods: In this retrospective study, we compared intraoperative parameters of 100 LTs, with 50 HOPE preconditioned liver grafts and 50 grafts stored in static cold storage (SCS). During reperfusion phase, prospectively registered serum parameters and vasopressor administration were analyzed. (3) Results: Twelve percent of patients developed PRS in the HOPE cohort vs. 42% in the SCS group (p = 0.0013). Total vasopressor demand in the first hour after reperfusion was lower after HOPE pretreatment, with reduced usage of norepinephrine (-26%;p = 0.122) and significant reduction of epinephrine consumption (-52%;p = 0.018). Serum potassium concentration dropped by a mean of 14.1% in transplantations after HOPE, compared to a slight decrease of 1% (p < 0.001) after SCS. The overall incidence of early allograft dysfunction (EAD) was reduced by 44% in the HOPE group (p = 0.04). (4) Conclusions: Pre-transplant graft preconditioning with HOPE results in higher hemodynamic stability during reperfusion and lower incidence of PRS and EAD. HOPE has the potential to mitigate ES by preventing hyperpotassemic complications that need to be addressed in LT with HOPE-pre-treated grafts

The Role of Gut-Derived Lipopolysaccharides and the Intestinal Barrier in Fatty Liver Diseases

BACKGROUND Hepatosteatosis is the earliest stage in the pathogenesis of nonalcoholic fatty (NAFLD) and alcoholic liver disease (ALD). As NAFLD is affecting 10-24% of the general population and approximately 70% of obese patients, it carries a large economic burden and is becoming a major reason for liver transplantation worldwide. ALD is a major cause of morbidity and mortality causing 50% of liver cirrhosis and 10% of liver cancer related death. Increasing evidence has accumulated that gut-derived factors play a crucial role in the development and progression of chronic liver diseases. METHODS A selective literature search was conducted in Medline and PubMed, using the terms \textquotedblnonalcoholic fatty liver disease,\textquotedbl \textquotedblalcoholic liver disease,\textquotedbl \textquotedbllipopolysaccharide,\textquotedbl \textquotedblgut barrier,\textquotedbl and \textquotedblmicrobiome.\textquotedbl RESULTS Gut dysbiosis and gut barrier dysfunction both contribute to chronic liver disease by abnormal regulation of the gut-liver axis. Thereby, gut-derived lipopolysaccharides (LPS) are a key factor in inducing the inflammatory response of liver tissue. The review further underlines that endotoxemia is observed in both NAFLD and ALD patients. LPS plays an important role in conducting liver damage through the LPS-TLR4 signaling pathway. Treatments targeting the gut microbiome and the gut barrier such as fecal microbiota transplantation (FMT), probiotics, prebiotics, synbiotics, and intestinal alkaline phosphatase (IAP) represent potential treatment modalities for NAFLD and ALD. CONCLUSIONS The gut-liver axis plays an important role in the development of liver disease. Treatments targeting the gut microbiome and the gut barrier have shown beneficial effects in attenuating liver inflammation and need to be further investigated

A Novel Deep Learning Model as a Donor-Recipient Matching Tool to Predict Survival after Liver Transplantation

Background: The digital era in the field of medicine is the new here and now. Artificial intelligence has entered many fields of medicine and is recently emerging in the field of organ transplantation. Solid organs remain a scarce resource. Being able to predict the outcome after liver transplantation promises to solve one of the long-standing problems within organ transplantation. What is the perfect donor recipient match? Within this work we developed and validated a novel deep-learning-based donor-recipient allocation system for liver transplantation. Method: In this study we used data collected from all liver transplant patients between 2004 and 2019 at the university transplantation centre in Munich. We aimed to design a transparent and interpretable deep learning framework to predict the outcome after liver transplantation. An individually designed neural network was developed to meet the unique requirements of transplantation data. The metrics used to determine the model quality and its level of performance are accuracy, cross-entropy loss, and F1 score as well as AUC score. Results: A total of 529 transplantations with a total of 1058 matching donor and recipient observations were added into the database. The combined prediction of all outcome parameters was 95.8% accurate (cross-entropy loss of 0.042). The prediction of death within the hospital was 94.3% accurate (cross-entropy loss of 0.057). The overall F1 score was 0.899 on average, whereas the overall AUC score was 0.940. Conclusion: With the achieved results, the network serves as a reliable tool to predict survival. It adds new insight into the potential of deep learning to assist medical decisions. Especially in the field of transplantation, an AUC Score of 94% is very valuable. This neuronal network is unique as it utilizes transparent and easily interpretable data to predict the outcome after liver transplantation. Further validation must be performed prior to utilization in a clinical context

Modulation of Glutathione Hemostasis by Inhibition of 12/15-Lipoxygenase Prevents ROS-Mediated Cell Death after Hepatic Ischemia and Reperfusion

Background. Reactive oxygen species-(ROS-) mediated ischemia-reperfusion injury (IRI) detrimentally impacts liver transplantation and resection. 12/15-Lipoxygenase (12/15-LOX), an antagonistic protein of the glutathione peroxidase 4 (GPX4) signaling cascade, was proven to mediate cell death in postischemic cerebral and myocardial tissue. The aim of this study was to investigate the impact of 12/15-LOX inhibition on hepatic IRI. Methods. Livers of C57BL/6 mice were exposed to 60 minutes of partial warm ischemia and 90 minutes of reperfusion after previous Baicalein administration, an inhibitor of 12/15-LOX. Tissue samples were analyzed by TUNEL assay, Western blot, and spectral photometry. Results. TUNEL labeling showed a significant reduction of hepatic cell death following baicalein pretreatment. Western Blot analysis revealed a significant downregulation of Jun-amino-terminal-kinase (JNK), caspase-3, and poly-ADP-ribose-polymerase (PARP), besides considerably lowered p44/42MAP- kinase (ERK1/2) expression after Baicalein administration. A significant elevation of glutathione oxidation was measured in Baicalein pretreated livers. Conclusion. Our data show that inhibition of 12/15-lipoxygenase causes significant cell death reduction after hepatic ischemia and reperfusion by enhancing glutathione metabolism. We conclude that GPX4-dependent cell death signaling cascade might play a major role in development of hepatic IRI, in which the investigated proteins JNK, caspase3, ERK1/2, and PARP might contribute to tissue damage