Diffusion-weighted magnetic resonance imaging using a preclinical 1 T PET/MRI in healthy and tumor-bearing rats

Background: Hybrid positron emission tomography and magnetic resonance imaging (PET/MRI) scanners are increasingly used for both clinical and preclinical imaging. Especially functional MRI sequences such as diffusionweighted imaging (DWI) are of great interest as they provide information on a molecular level, thus, can be used as surrogate biomarkers. Due to technical restrictions, MR sequences need to be adapted for each system to perform reliable imaging. There is, to our knowledge, no suitable DWI protocol for 1 Tesla PET/MRI scanners. We aimed to establish such DWI protocol with focus on the choice of b values, suitable for longitudinal monitoring of tumor characteristics in a rat liver tumor model. Material and methods: DWI was first performed in 18 healthy rat livers using the scanner-dependent maximum of 4 b values (0, 100, 200, 300 s/mm2). Apparent diffusion coefficients (ADC) were calculated from different b value combinations and compared to the reference measurement with four b values. T2-weighted MRI and optimized DWI with best agreement between accuracy, scanning time, and system performance stability were used to monitor orthotopic hepatocellular carcinomas (HCC) in five rats of which three underwent additional 2-deoxy-2-(18F)fluoro-D-glucose(FDG)-PET imaging. ADCs were calculated for the tumor and the surrounding liver parenchyma and verified by histopathological analysis. Results: Compared to the reference measurements, the combination b = 0, 200, 300 s/mm2 showed the highest correlation coefficient (rs = 0.92) and agreement while reducing the acquisition time. However, measurements with less than four b values yielded significantly higher ADCs (p < 0.001). When monitoring the HCC, an expected drop of the ADC was observed over time. These findings were paralleled by FDG-PET showing both an increase in tumor size and uptake heterogeneity. Interestingly, surrounding liver parenchyma also showed a change in ADC values revealing varying levels of inflammation by immunohistochemistry. Conclusion: We established a respiratory-gated DWI protocol for a preclinical 1 T PET/MRI scanner allowing to monitor growth-related changes in ADC values of orthotopic HCC liver tumors. By monitoring the changes in tumor ADCs over time, different cellular stages were described. However, each study needs to adapt the protocol further according to their question to generate best possible results

Independent effects of sham laparotomy and anesthesia on hepatic microRNA expression in rats

Background: Studies on liver regeneration following partial hepatectomy (PH) have identified several microRNAs (miRNAs) that show a regulated expression pattern. These studies involve major surgery to access the liver, which is known to have intrinsic effects on hepatic gene expression and may also affect miRNA screening results. We performed two-third PH or sham laparotomy (SL) in Wistar rats to investigate the effect of both procedures on miRNA expression in liver tissue and corresponding plasma samples by microarray and qRT-PCR analyses. As control groups, non-treated rats and rats undergoing anesthesia only were used. Results: We found that 49 out of 323 miRNAs (15%) were significantly deregulated after PH in liver tissue 12 to 48 hours postoperatively (>20% change), while 45 miRNAs (14%) were deregulated following SL. Out of these miRNAs, 10 miRNAs were similarly deregulated after PH and SL, while one miRNA showed opposite regulation. In plasma, miRNA upregulation was observed for miR-133a and miR-133b following PH and SL, whereas miR-100 and miR-466c were similarly downregulated following anesthesia and surgery. Conclusions: We show that miRNAs are indeed regulated by sham laparotomy and anesthesia in rats. These findings illustrate the critical need for finding appropriate control groups in experimental surgery

Diagnostic Biomarkers to Diagnose Acute Allograft Rejection After Liver Transplantation: Systematic Review and Meta-Analysis of Diagnostic Accuracy Studies

Objective: A systematic review and meta-analysis of diagnostic biomarkers for noninvasive diagnosis of acute allograft rejection following liver transplantation.Background: Noninvasive blood and urine markers have been widely explored in recent decades for diagnosing acute rejection after liver transplantation. However, none have been translated into routine clinical use so far due to uncertain diagnostic accuracy, and liver biopsy remains the gold standard.Methods: Systematic literature searches of Medline, Cochrane and Embase were conducted up to February 2019 to identify studies evaluating the use of noninvasive markers in diagnosing allograft rejection following liver transplantation. Meta-analysis was performed using a random effects model with DerSimonian–Laird weighting and the hierarchical summary receiver operating curve.Results: Of 560 identified studies, 15 studies (1,445 patients) met the inclusion criteria. The following markers were tested: acid labile nitroso-compounds (NOx), serum amyloid A protein, procalcitonin, peripheral blood eosinophil count, peripheral blood T-cell activation and interleukin 2 (IL-2) receptor, guanylate-binding protein-2 mRNA, graft-derived cell-free DNA, pi-glutathione S-transferase, alpha-glutathione S-transferase and serum HLA class I soluble antigens. Only eosinophil count was tested in multiple studies, and they demonstrated high heterogeneity (I2 = 72% [95% CI: 0.5–0.99]). IL-2 receptor demonstrated the highest sensitivity (89% [95% CI: 0.78–0.96]) and specificity (81% [95% CI: 0.69–0.89]).Conclusion: IL-2 receptor expression demonstrated the highest diagnostic accuracy, while the peripheral eosinophil count was the only marker tested in more than one study. Presently, liver biopsy remains superior to noninvasive diagnostic biomarkers as most studies exhibited inferior designs, hindering possible translation into clinical application

Development and evaluation of micron-sized, oligonucleotide-labeled iron oxide particles for the stimulation of cultivated primary human hepatocytes

Die Leberzelltransplantation ist eine erfolgversprechende Therapie zur Behandlung angeborener Stoffwechseldefekte der Leber sowie des akuten Leberversagens. Im Rahmen der Leberzelltransplantation bieten Partikel- basierte Transfektionssysteme (engl. Particle based delivery systems; PDS), die auf der Inkorporation von Eisenoxidpartikeln in Leberzellen beruhen, perspektivisch die Möglichkeit der Kombination von Diagnostik und Therapie. Ziel der vorliegenden Dissertation war die Entwicklung und Evaluierung eines multifunktionalen oligonukleotid-gekoppelten biokompatiblen mikroskaligen Eisenoxidpartikels (sMPIO-α-LNA) zur Verfolgung transplantierter Leberzellen im MRT und dessen therapeutische Manipulation mittels RNA Interferenz. Der sMPIO bestand aus nanoskaligen Magnetitpartikeln, welche homogen in einer SiO2-basierten Polymermatrix eingebettet waren. Die Oberfläche der Partikel wurde mit Carboxylgruppen konjugiert, was die Anbindung funktioneller Gruppen und therapeutischer Wirkstoffe ermöglichte. In dieser Arbeit wurden antisense Locked Nucleic Acids (α-LNAs) an die Oberfläche des sMPIOs gekoppelt. Die zentrale Funktion des sMPIOs bestand in der Erzeugung eines reproduzierbaren hypointensen MRT-Signals. Hierfür wurden zwei Oberflächenmodifikationen (Poly-L-Lysin, Streptavidin) hinsichtlich ihrer Inkorporationsfähigkeit in Leberzellen getestet. Es konnte eine signifikante Signalauslöschung ab einer Zellzahl von 1.000 primären humanen Hepatozyten bzw. von 5.000 Zellen der Zelllinie Huh-7 in der T2*-gewichteten Sequenz erzielt werden. Für die Verwendung des Partikels als intrazellulären Stimulus der zu transplantierenden Hepatozyten wurde die Let7g-spezifischeα-LNA an den Partikel kovalent gebunden. Die Depletion von über 80% der endogenen microRNA (miRNA) Let7g induzierte 24 Stunden nach Partikelinkubation eine Hochregulation der Zellzyklus-assoziierten Proteine Cyclin D1 und c-Myc. Nachfolgende Proteomanalysen offenbarten weitere Zielproteine, von denen vier als Interaktionspartner der Let7g bekannt sind. Die Transplantation von Partikel-markierten Zellen zeigte nach sieben Tagen eine verstärkte Signalauslöschung im MRT. Das Engraftment der Hepatozyten sowie die Eisenoxidpartikel konnten histologisch nachgewiesen werden. Die Entwicklung des neuartigen Partikels ermöglicht die theragnostische Anwendung in der Leberzelltransplantation. Darüber hinaus ist eine Nutzung des Partikels in der Diagnostik und Therapie von Tumorerkrankungen denkbar.The liver cell transplantation is a promising therapy for the treatment of inborn metabolic liver disease and acute liver failure. In the course of liver cell transplantation particle based delivery systems (PDS) consisting of iron oxide particles incorporated in liver cells might provide a tool for combination of diagnosis and therapy. The aim of this study was to design a multifunctional oligonucleotide-labeled biocompatible micron-sized iron oxide particle (sMPIO-α-LNA), which combines MRI cell tracking and therapeutic manipulation of primary human hepatocytes by miRNA targeting. The sMPIO consisted of nanometer-scaled magnetite-particles, which were distributed homogenously in a silica-based polymer matrix. The polymeric shell was covered with carboxyl groups allowing the binding of functional groups and therapeutic agents. In this study antisense locked nucleic acids (α-LNA) were covalently bound to the surface of the sMPIO. The main purpose of the sMPIO was to enable a reproducible hypointense MRI signal. Therefore two different surface modifications (Poly-L-Lysin, Streptavidin), which were coupled onto particle surfaces, were tested regarding the incorporation capacity into liver cells. The results showed that 1,000 primary hepatocytes and 5,000 cells of the cell line Huh-7 could be quantified in a T2* weighted imaging. In order to cause an intracellular stimulus of transplanted hepatocytes, a α-LNA specific for the depletion of the microRNA (miRNA) Let7g was covalently bound to the particle surface. The Let7g depletion (80%) resulted in up-regulation of the cell cycle associated target proteins Cyclin-D1 and c-Myc. In further proteome analyses, additional target proteins, which were regulated by sMPIO-α-LNA treatment, were detected. Four of them were identified as interaction partners of Let7g. The transplantation of sMPIO-labeled cells caused an enhanced signal loss in MRI after seven days. The engraftment of the liver cells as well as the iron oxide particles could be visualized by histological staining. The new particle might be used as a theragnostic tool for cell transplantation and additionally for diagnosis and therapy of malignoma

The nanomolar sensing of nicotinamide adenine dinucleotide in human plasma using a cycling assay in albumin modified simulated body fluids

Abstract Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies

Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

Acute cellular rejection (ACR) after liver transplantation (LT) goes along with allograft dysfunction, which is diagnosed by liver biopsy and concomitant histological analysis, representing the gold standard in clinical practice. Yet, liver biopsies are invasive, costly, time-intensive and require expert knowledge. Herein we present substantial evidence that blood plasma residing peripheral liver-derived extracellular particles (EP) could be employed to diagnose ACR non-invasively. In vitro experiments showed organ-specific EP release from primary human hepatocytes under immunological stress. Secondly, analysis of consecutive LT patients (n=11) revealed significant heightened EP concentrations days before ACR. By conducting a diagnostic accuracy study (n = 69, DRKS00011631), we explored the viability of using EP as a liquid biopsy for diagnosing ACR following LT. Consequently, novel EP populations in samples were identified using visualization of t-distributed stochastic neighbor embedding (viSNE) and self-organizing maps (FlowSOM) algorithms. As a result, the ASGR1(+)CD130(+)Annexin V+ EP subpopulation exhibited the highest accuracy for predicting ACR (area under the curve: 0.80, 95% confidence interval [CI], 0.70-0.90), with diagnostic sensitivity and specificity of 100% (95% CI, 81.67-100.0%) and 68.5% (95% CI, 55.3-79.3%), respectively. In summary, this new EP subpopulation presented the highest diagnostic accuracy for detecting ACR in LT patients