Recurrence of Hepatocellular Carcinoma After Liver Transplantation is Associated with Episodes of Acute Rejections.

Purpose The impact of acute rejection (AR) after liver transplantation (LT) for hepatocellular carcinoma (HCC) on patient outcome is uncertain. This aim of this study is to investigate whether AR is associated with HCC relapse and overall survival. Patients and Methods Patients undergoing LT for HCC between 2001 and 2015 were retrospectively analyzed with regard to histopathological proven AR within the median time until recurrence. Cox's regression analysis was conducted revealing risk factors for HCC recurrence. Results HCC recurred in 47 of 252 analyzed patients with a median time to recurrence of 20 months. Patients with AR (28.6%) had a significantly higher frequency of recurrence compared to patients without AR (13.0%, p=0.002). Multiple Cox regression analyses identified AR within 20 months to be an independent risk factor for HCC recurrence both as dichotomized (HR=2.91, 95%CI: 1.30-6.53; p=0.009) and as a continuous variable (HR=1.81, 95%CI: 1.28-2.54; p=0.001). HCC recurrence and AR were associated with higher grades of liver fibrosis one year after LT, when compared to patients without AR (p=0.019). Conclusion Our results demonstrate an association of AR with HCC recurrence after LT with implications for intervals of monitoring in tumor surveillance. Graft fibrosis and immune mechanisms are potentially related and causal interactions are worth further investigation

Gestational Weight Gain and Body Mass Index in Children: Results from Three German Cohort Studies

Previous studies suggested potential priming effects of gestational weight gain (GWG) on offspring's body composition in later life. However, consistency of these effects in normal weight, overweight and obese mothers is less clear. We combined the individual data of three German cohorts and assessed associations of total and excessive GWG (as defined by criteria of the Institute of Medicine) with offspring's mean body mass index (BMI) standard deviation scores (SDS) and overweight at the age of 5-6 years (total: n = 6,254). Quantile regression was used to examine potentially different effects on different parts of the BMI SDS distribution. All models were adjusted for birth weight, maternal age and maternal smoking during pregnancy and stratified by maternal pre-pregnancy weight status. In adjusted models, positive associations of total and excessive GWG with mean BMI SDS and overweight were observed only in children of non- overweight mothers. For example, excessive GWG was associated with a mean increase of 0.08 (95% CI: 0.01, 0.15) units of BMI SDS (0.13 (0.02, 0.24) kg/m(2) of 'real' BMI) in children of normal-weight mothers. The effects of total and excessive GWG on BMI SDS increased for higher- BMI children of normal-weight mothers. Increased GWG is likely to be associated with overweight in offspring of non-overweight mothers

The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice

<div><h3>Background</h3><p>Recent studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes (T1D) support the notion that tyrosine kinase inhibitors have the potential for modulating disease development. However, the therapeutic effects of AG490 on the development of T1D are unknown.</p> <h3>Materials and Methods</h3><p>Female NOD mice were treated with AG490 (i.p, 1 mg/mouse) or DMSO starting at either 4 or 8 week of age, for five consecutive week, then once per week for 5 additional week. Analyses for the development and/or reversal of diabetes, insulitis, adoptive transfer, and other mechanistic studies were performed.</p> <h3>Results</h3><p>AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points). Monotherapy of newly diagnosed diabetic NOD mice with AG490 markedly resulted in disease remission in treated animals (n = 23) in comparision to the absolute inability (0%; 0/10, p = 0.003, Log-rank test) of DMSO and sustained eugluycemia was maintained for several months following drug withdrawal. Interestingly, adoptive transfer of splenocytes from AG490 treated NOD mice failed to transfer diabetes to recipient NOD.<em>Scid</em> mice. CD4 T-cells as well as bone marrow derived dendritic cells (BMDCs) from AG490 treated mice, showed higher expression of Foxp3 (p<0.004) and lower expression of co-stimulatory molecules, respectively. Screening of the mouse immune response gene arrary indicates that expression of costimulaotry molecule Ctla4 was upregulated in CD4+ T-cell in NOD mice treated with AG490, suggesting that AG490 is not a negative regulator of the immune system.</p> <h3>Conclusion</h3><p>The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.</p> </div

Genome-wide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes.

OBJECTIVE: Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. RESEARCH DESIGN AND METHODS: We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. RESULTS: Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. CONCLUSIONS: We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis

Improving perioperative safety after liver resection

Einleitung Die Leberteilresektion stellt für die meisten Tumore der Leber die einzige kurative Therapiealternative dar. Neben der richtigen Indikationsstellung ist eine Abschätzung des perioperativen Risikos essentiell. Insbesondere Resektionen von mehr als drei Segmenten sind immer noch mit einer hohen Rate an Leberinsuffizienz und assoziierter Morbidität vergesellschaftet. Nach Etablierung onkologischer Prinzipien in vergangenen Dekaden sind derzeitige Bestrebungen davon geprägt, die perioperative Sicherheit zu erhöhen und die Lebensqualität nach Leberteilresektion zu verbessern. Methoden Es erfolgte zunächst eine retrospektive Auswertung aller Patienten, bei denen im Zeitraum von 2010 bis 2017 an der Chirurgischen Klinik ein minimalinvasiver leberchirurgischer Eingriff durchgeführt wurde. Die Iwate-Kriterien zur Einschätzung des Schwierigkeitsgrads einer laparoskopischen Leberteilresektion wurden bei Patienten mit hepatozellulärem Karzinom (HCC) zwischen 01/2010 und 11/2017 hinsichtlich der Vorhersagekraft der perioperativen Sicherheit evaluiert. Zudem erfolgte eine Befragung aller Patienten nach laparoskopischer und offener Leberteilresektion zwischen 06/2014 und 10/2016 hinsichtlich verschiedener Dimensionen der postoperativen Lebensqualität. Um die Bedeutung extrahepatischer Stammzellen zur Leberregeneration und damit Erhöhung der postoperativen Sicherheit zu untersuchen, erfolgten zudem experimentelle Studien. Balb/c nu-nu Knock-out Mäusen wurden humane mesenchymale Stammzellen (MSC) 24h nach 70% Leberteilresektion intraperitoneal (i.p.) appliziert. Zwei, 3 und 5 Tage nach der Leberteilresektion wurden die Tiere finalisiert und immunhistochemische Färbungen, Hämatoxylin-Eosin-Färbungen sowie Serumanalysen nach MSC Therapie durchgeführt. Ergebnisse Die Iwate-Kriterien zeigten eine Korrelation mit der Operationszeit, dem Aufenthalt und der postoperativen Komplikationsrate. Die Operationszeit verlängerte sich dabei analog zu den Schwierigkeitsgraden (p<0,001). Entgegen der eigentlichen Intention der Iwate-Kriterien korrelierte auch die postoperative Komplikationsrate mit der Komplexität der Eingriffe (Grad 4: 50%; Grad 1: 0%). Entsprechend war Schwierigkeitsgrad 4 auch mit einem signifikant längeren Gesamtaufenthalt assoziiert (p=0,008). Der Vergleich der Health related quality of Life (HRQoL) zeigte trotz einer signifikant höheren Rate von Nebenerkrankungen (p=0,044) und vorbestehender Leberzirrhose (p=0,016) in der Gruppe nach laparoskopischer Leberteilresektion eine zur Gruppe der konventionellen, offenen Leberchirurgie vergleichbare HRQoL. Eine MSC-Therapie nach experimenteller Leberteilresektion im Mausmodellführte zu einer signifikant höheren Proliferationsrate von Hepatozyten (p=0,0096) und sinusoidalen Endothelzellen (p=0,0019). Es zeigte sich zudem eine inverse Korrelation zwischen der hepatozellulären Proliferationsrate und der hepatozytären Verfettung (r2= 0,4064; p=0,0079), wobei keine mit MSC behandelte Maus den schwersten Steatosegrad aufwies. Schlussfolgerung Die Iwate-Kriterien haben neben der Einteilung der intraoperativen Komplexität bei laparoskopischen Leberteilresektionen auch einen Stellenwert zur Vorhersage der postoperativen Morbidität. Patienten mit Komorbiditäten profitieren möglicherweise aufgrund der schnelleren Rekonvaleszenz von einem minimalinvasiven Vorgehen. Die Stammzelltherapie scheint ein vielversprechender Ansatz zur Unterstützung der postoperativen Leberregeneration und könnte helfen, die postoperative Sicherheit zu verbessern.Introduction Liver resection represents the only curative therapy for tumors in the liver. Beside the correct indication for resection, an adequate perioperative risk assessment is essential. After resections of more than three liver segments, postoperative liver failure is associated with high mortality and morbidity. Moreover, several studies have indicated minimal-invasive procedures to be linked to faster recovery. Improving perioperative safety after liver resections is a topic of considerable recent scientific efforts and is of paramount importance. Methods A retrospective analysis of all patients who underwent a laparoscopic liver resection (LLR) between 2010 and 2017 at the Department of Surgery was performed. To evaluate the predictionof the Iwate criteria, all patients with hepatocellular carcinoma (HCC) who underwent a LLR between 01/2010 und 11/2017 were included, graded according to the Iwate criteria (level 1-4) and analyzed retrospectively. In addition, we performed a survey withregard to dimensions of postoperative life quality of among all patients who underwent a LLR or open liver resection (OLR) between 06/2014 and 10/2016. The pro-regenerative role of human mesenchymal stem cell (MSC) treatment after experimental hepatectomy was evaluated in Balb/c nu-nu knock-out mice. MSC were administrated i.p. 24h after 70% hepatectomy and animals were sacrificed 2d, 3d and 4d after surgery. We performed haematoxylin and eosin staining and immunostaining as well as serum analyses. Results The Iwate criteria showed a good correlation to operation time, hospital stay and postoperative complications. The operation time significantly correlated with increasing difficulty levels (p=<0.001). Likewise, postoperative complication rate correlated with the complexity of the procedures. Patients undergoing procedures for level 4 lesions showed a significant longer hospital stay (p=0.008). When comparing the health related quality of life (HRQoL), we observed despite a higher rate of comorbidities (p=0.044) and liver cirrhosis (p=0.016) in the OLR group, no differences regarding the HRQoL. Mice of the MSC treatment group showed a significant higher hepatocellular (p=0.0096) and liver sinusoidal endothelial cells (LSEC) proliferation rate (p=0.0019). Additionally, there was an inverse correlation of steatosis and hepatocellular proliferation (r2=0.4064; p=0.0079) with no severe steatosis in the treatment group. Conclusion Postoperative complications after laparoscopic liver resections can be predicted by Iwate criteria. Particularly, patients with liver cirrhosis and comorbidities may profit from the minimally invasive approach because of a faster recovery. MSC treatment is an encouraging approach to support postoperative liver regeneration and may improve the postoperative safety after major partial liver resections

Evaluating the impact of hydroxychloroquine on mouse lymphocyte proliferation and cytokine production in vivo and in vitro

Summary: Immunomodulatory drugs can alter lymphocyte function. Hydroxychloroquine (HCQ) is prescribed for many autoimmune diseases and is under investigation as an anti-tumor autophagy inhibitor. Here, we describe a protocol to evaluate the influence of HCQ on lymphocyte function by measuring the in vitro and ex vivo proliferation and cytokine production. The protocol can provide insights into potential immunomodulatory effects of HCQ and can be used for assessing other medications' effects on lymphocyte functions.For complete details on the use and execution of this protocol, please refer to Wabitsch et al. (2021)

Establishment of lipofection for studying miRNA function in human adipocytes.

miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000

Lysophosphatidic acid inhibits adipocyte differentiation via lysophosphatidic acid 1 receptor-dependent down-regulation of peroxisome proliferator-activated receptor gamma2.

Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue

APOB-LIPOPROTEINS AND PCSK9 AS MODULATORS OF HUMAN WHITE ADIPOSE TISSUE FUNCTION AND NLRP3 INFLAMMASOME ACTIVITY

International Symposium on Atherosclerosis (ISA), Toronto, CANADA, JUN 09-12, 2018International audienc