Visualization of Inflammation in Experimental Colitis by Magnetic Resonance Imaging Using Very Small Superparamagnetic Iron Oxide Particles

Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, co-localization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content

ARHGEF7 (BETA-PIX) Acts as Guanine Nucleotide Exchange Factor for Leucine-Rich Repeat Kinase 2

Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson’s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. Methodology/Principal Findings: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. Conclusions/Significance: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPas

Serum-basierte N-Glykan Biomarker für Diagnose von epithelialen Ovarialkarzinomen. Das Aszites-N-Glykom von epithelialen Ovarialkarzinomen.

Glycosylation is an important co- and posttranslational modification that frequently occurs in proteins. Glycans are of biological relevance as they affect many fundamental functions and properties of proteins including the structural, intracellular and immunological aspects. Malignant transformations are associated with qualitative and quantitative changes of glycan structures of all cellular glycoproteins and glycolipids. These changes could be confirmed on cells and in tissues of all experimentally-induced or naturally occurring malignancies that were analysed so far regardless of type, stage and cause of tumors. As glycans can reflect the normal or abnormal status of an individual, glycan-based biomarkers are of great interest for the diagnosis of multiple diseases including cancer and inflammation. Serum is the liquid fraction that is obtained after whole blood has been allowed to clot. It is considered to be a biological fluid suitable for biomarker discovery as it is relatively stable under various conditions as well as it is rich in glycoproteins. In the present work, whole serum from patients suffering from epithelial ovarian cancer (EOC) in all disease stages was analyzed and compared with age-matched healthy subjects and women suffering from benign ovarian masses. Further, the diagnostic performance of the proposed biomarker candidate was compared with CA125, the routine diagnostic marker, to assess whether total serum glycome can be used as a biomarker for early-stage diagnosis. For this purpose, N-glycans were digested from total serum glycoproteins, previously denatured, using the PNGase F enzyme. Samples were subsequently purified and permethylated. The characterization of total N-glycan profile was performed using MALDI-TOF mass spectrometry and glycan structural isomericity was confirmed using MALDI-TOF/-TOF-MS. The statistical evaluation of potential biomarker candidates was done using the SPSS software. Quantitative and qualitative changes in the serum N-glycome from EOC patients were expressed for the first time as a ratio that can be used as a tumor marker, which is independent from CA125 as serum glycoproteins stem from the liver whereas CA125 is expressed by the tumor itself. The proposed biomarker candidate, termed as GLYCOV, has proved its worth at any disease stage and allows discrimination between ovarian cancer and the control group consisting of patients with benign ovarian masses. More than one third of ovarian cancer patients develop ascites in the course of the disease, and almost all of them in case of cancer recurrence. The presence of ascites is usually correlated with a bad diagnosis. As this event occurs at late stages of EOC, ascites cannot be used for early-stage biomarker discovery. The exploration of ascites glycome is rather thought to complete integrated bioinformatics data and to identify usable putative biomarkers that assess treatment response of ovarian cancer. In this thesis are reported for the first time the N-glycome profiles of ascitic fluid from primary serous EOC patients that are compared with the serum N-glycome of the same patients to overcome individual-specific glycosylation pattern. For the first time, it could be demonstrated that the glycome modulations previously reported in ovarian cancer serum were also present in ascites. In addition, statistical differences between ascites and serum samples were observed for glycan antennarity, sialylation and fucosylation.Glykosylierung ist eine wichtige und häufig vorkommende co- und posttranslationale Modifikation der Glykoproteine. Glykane sind von wichtiger biologischer Relevanz und beeinflussen die strukturellen Eigenschaften sowie die intrazellulären Funktionen von Proteinen. Maligne Transformationen gehen mit quantitativen und qualitativen Strukturveränderungen der Glykane von zellulären Glykoproteinen und Glykolipiden einher. Diese Veränderungen konnten in Zellen und Gewebe aller bislang untersuchten experimentell induzierten oder natürlich vorkommenden Tumore ohne Rücksicht auf den Typ, das Stadium oder die Ursache der Entstehung nachgewiesen werden. Da die Glykane den physiologischen und pathologischen Status eines Individuums reflektieren können, bieten glykanbasierte Serumtumormarker neue Ansätze zur Diagnostik bösartiger Tumore und Entzündungskrankheiten. Serum ist eine biologische Flüssigkeit, welche man nach Zentrifugation einer geronnenen Blutprobe erhält. Im Labor hat sich das Serum vor allem wegen dessen Stabilität unter diversen Bedingungen und der hohen Proteinkonzentration als eine geeignete Quelle für die Erforschung neuartiger Biomarker bewährt. Das Ziel dieser Arbeit war es, das Serum eines Patientinnenkollektivs mit epithelialen Ovarialkarzinomen zu untersuchen, wobei die Proben alle Stadien der Krankheit umfasst. Anschließend erfolgte ein Vergleich mit alterskorrelierten Kontrollseren von gesunden Probandinnen und Patientinnen mit gutartigen Ovarialtumoren. Im Folgenden, sollte die diagnostische Leistung des vorgeschlagenen, potentiellen Biomarkers mit der von CA125 verglichen werden. CA125 ist aktuell der einzige, klinisch erprobte und routinemäßig eingesetzte Tumormarker für die Diagnose von Ovarialkarzinomen. Für diesen Zweck wurden die N-Glykane von zuvor denaturierten Serum- Glykoproteinen unter Verwendung von PNGase F enzymatisch abgespaltet, aufgereinigt und permethyliert. Das N-Glykanprofil wurde mittels MALDI-TOFMassenspektroskopie charakterisiert und die Strukturisomere mit MALDI-TOF/-TOFMS bestätigt. Die statistische Evaluierung des potentiellen Biomarkers wurde mit Hilfe von SPSS-Software durchgeführt. Die beobachteten quantitativen und qualitativen Veränderungen des Serum-N-Glykoms von Patientinnen mit epithelialen Ovarialkarzinom wurden gegenüber gesunden Kontrollen in Form eines Quotienten ausgedrückt. Trotz der Tatsache, dass die Serum-Glykoproteine überwiegend in der Leber synthetisiert werden während CA125 vom Tumor exprimiert wird, konnte der CA125 unabhängige Quotient als potentieller Tumormarker für die Diagnose von epithelialen Ovarialkarzinomen eingesetzt werden. Der als GLYCOV bezeichnete Biomarker konnte für jedes Stadium der Krankheit und für die Diskriminierung zwischen Ovarialkarzinomen und gutartigen Ovarialtumoren validiert werden. Mehr als ein Drittel der Frauen mit Ovarialmalignomen entwickeln im Verlauf der Krankheit und bei fast jedem Rückfall Aszites. Die Gegenwart von Aszites korreliert mit einer schlechten Prognose. Aufgrund der Tatsache, dass Aszites überwiegend in Spätstadien auftritt, ist es als biologische Flüssigkeit nicht für die Erforschung neuer Biomarker geeignet. Die Erforschung des Aszites-N-Glykoms könnte aber dennoch existierende Daten aus der Bioinformatik erweitern und dazu beitragen einen geeigneten Biomarker für das Ansprechen auf die Behandlung des Ovarialkarzinoms zu identifizieren. In dieser Arbeit konnte erstmalig das Aszites-N-Glykom-Profil, welches Patientinnen mit primären, serösen, epithelialen Ovarialkarzinomen entnommen wurde, dem Serum-N-Glykom- Profil gegenübergestellt werden. Die Vergleichsseren stammten von denselben Probandinnen, um die individuumspezifischen Unterschiede beim Vergleich beider N-Glykan-Profile auszuschließen. Den Beobachtungen zufolge konnten im Aszites dieselben N-Glykan-Modulationen nachgewiesen werden, welche zuvor in Serum von Ovarialkarzinompatientinnen beobachtet wurden. Zusätzlich konnten statistisch relevante Unterschiede hinsichtlich der Antennarität, Fukosylierung und Sialylierung herausgestellt werden

Serum Glycome Profiling: A Biomarker for Diagnosis of Ovarian Cancer

During the development of cancer, changes in cellular glycosylation are observed, indicating that alterations of the glycome occur in extracellular fluids as well as in serum and could therefore serve as tumor biomarkers. In the case of epithelial ovarian cancer (EOC), common tumor markers such as CA125 are known to have poor specificity; therefore, better biomarkers are needed. The aim of this work was to identify new potential glycan biomarkers in EOC-patients. N-Glycans were cleaved from serum glycoproteins from 63 preoperative primary EOC-patients along with 33 age-matched healthy women, permethylated, and analyzed using MALDI-TOF mass spectrometry. A value named GLYCOV was calculated from the relative areas of the 11 N-glycan biomarkers revealed by SPSS statistical analyses, namely four high-mannose and seven complex-type fucosylated N-glycans. GLYCOV diagnosed primary EOC with a sensitivity of 97% and a specificity of 98.4% whereas CA-125 showed a sensitivity of 97% and a specificity of 88.9%. Our study is the first one to compare glycan values with the established tumor marker CA125 and to give better results. Therefore, the N-glycome could potentially be used as a biomarker

No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

<div><p>Mutations within the <i>LRRK2</i> gene have been identified in Parkinson’s disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.</p></div

Neurite outgrowth parameters analyzed with the BD AttoVision^TM V1.6 Software from BD Bioscience.

<p>Neurite outgrowth parameters analyzed with the BD AttoVision^TM V1.6 Software from BD Bioscience.</p

Analysis of neurite outgrowth and branching complexity of primary hippocampal neurons.

<p><b>A-B:</b> Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; <b>C-H:</b> Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. <b>C1-H2:</b> Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.</p

Generation of human wild type and G2019S mutant LRRK2 transgenic mice.

<p><b>A:</b> Human full length wild type or G2019S mutant LRRK2 cDNA were cloned into the murine Thy-1.2 promoter element driving neuronal-specific transgene expression. <b>B:</b> Western blot analysis of LRRK2 protein expression in brain lysates from LRRK2, GS-LRRK2 line 1, GS-LRRK2 line 2 and non-tg littermates using MID antibody which recognizes human and murine LRRK2 protein. <b>C:</b> Densitometry quantification revealed approximately twice the amount of total LRRK2 protein in all transgenic lines compared to endogenous Lrrk2 levels in non-tg controls. Data represent means ± SEM; n = 2 for each transgenic line.</p

LRRK2 mRNA and protein expression in brain regions in the three transgenic mouse lines.

<p><b>A:</b> RT-PCR semi-quantification of LRRK2 expression in whole brains at different embryonic and postnatal stages indicates robust transgene expression at postnatal day 2 (P2) in all three lines. Data represents means ± SEM; n = 3–5 animals per group. <b>B:</b> In-situ hybridization of coronal brain sections at the level of posterior hippocampus and midbrain with two human specific LRRK2 probes showed comparable transgene expression levels in hippocampus and cortex of 11-month-old LRRK2 and GS-LRRK2 lines 1 and 2. <b>C:</b> Western blot analysis of LRRK2 protein showed robust expression levels of LRRK2 in hippocampus (HC) and cortex (CTX) of 10-month-old animals with the human-specific LRRK2 antibody MJFF5; n = 3 animals per genotype.</p