19 research outputs found
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 CrohnÂŽs 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 Parkinsonâs 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 fĂŒr 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 hÀufig vorkommende co- und
posttranslationale Modifikation der Glykoproteine. Glykane sind von wichtiger
biologischer Relevanz und beeinflussen die strukturellen Eigenschaften sowie
die intrazellulÀren Funktionen von Proteinen. Maligne Transformationen gehen
mit quantitativen und qualitativen StrukturverÀnderungen der Glykane von
zellulÀren Glykoproteinen und Glykolipiden einher. Diese VerÀnderungen konnten
in Zellen und Gewebe aller bislang untersuchten experimentell induzierten oder
natĂŒrlich vorkommenden Tumore ohne RĂŒcksicht 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 AnsÀtze zur Diagnostik bösartiger Tumore
und EntzĂŒndungskrankheiten. Serum ist eine biologische FlĂŒssigkeit, welche man
nach Zentrifugation einer geronnenen Blutprobe erhÀlt. Im Labor hat sich das
Serum vor allem wegen dessen StabilitÀt unter diversen Bedingungen und der
hohen Proteinkonzentration als eine geeignete Quelle fĂŒr die Erforschung
neuartiger Biomarker bewÀhrt. Das Ziel dieser Arbeit war es, das Serum eines
Patientinnenkollektivs mit epithelialen Ovarialkarzinomen zu untersuchen,
wobei die Proben alle Stadien der Krankheit umfasst. AnschlieĂend 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 routinemĂ€Ăig eingesetzte Tumormarker fĂŒr die Diagnose von
Ovarialkarzinomen. FĂŒr 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 bestÀtigt. Die statistische Evaluierung des potentiellen
Biomarkers wurde mit Hilfe von SPSS-Software durchgefĂŒhrt. Die beobachteten
quantitativen und qualitativen VerÀnderungen des Serum-N-Glykoms von
Patientinnen mit epithelialen Ovarialkarzinom wurden gegenĂŒber gesunden
Kontrollen in Form eines Quotienten ausgedrĂŒckt. Trotz der Tatsache, dass die
Serum-Glykoproteine ĂŒberwiegend in der Leber synthetisiert werden wĂ€hrend
CA125 vom Tumor exprimiert wird, konnte der CA125 unabhÀngige Quotient als
potentieller Tumormarker fĂŒr die Diagnose von epithelialen Ovarialkarzinomen
eingesetzt werden. Der als GLYCOV bezeichnete Biomarker konnte fĂŒr jedes
Stadium der Krankheit und fĂŒr 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 RĂŒckfall Aszites. Die Gegenwart von Aszites korreliert mit einer
schlechten Prognose. Aufgrund der Tatsache, dass Aszites ĂŒberwiegend in
SpĂ€tstadien auftritt, ist es als biologische FlĂŒssigkeit nicht fĂŒr 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 fĂŒr das Ansprechen auf die
Behandlung des Ovarialkarzinoms zu identifizieren. In dieser Arbeit konnte
erstmalig das Aszites-N-Glykom-Profil, welches Patientinnen mit primÀren,
serösen, epithelialen Ovarialkarzinomen entnommen wurde, dem Serum-N-Glykom-
Profil gegenĂŒbergestellt werden. Die Vergleichsseren stammten von denselben
Probandinnen, um die individuumspezifischen Unterschiede beim Vergleich beider
N-Glykan-Profile auszuschlieĂen. Den Beobachtungen zufolge konnten im Aszites
dieselben N-Glykan-Modulationen nachgewiesen werden, welche zuvor in Serum von
Ovarialkarzinompatientinnen beobachtet wurden. ZusÀtzlich konnten statistisch
relevante Unterschiede hinsichtlich der AntennaritÀt, Fukosylierung und
Sialylierung herausgestellt werden
Inorganic Phosphate-Induced Extracellular Vesicles from Vascular Smooth Muscle Cells Contain Elevated Levels of Hyaluronic Acid, Which Enhance Their Interaction with Very Small Superparamagnetic Iron Oxide Particles
Patients with chronic kidney disease (CKD) have a high prevalence of hyperphosphatemia, where uremic toxins like inorganic phosphate (Pi) induce a cardiovascular remodeling. Related disorders like atherosclerosis bear the risk of increased morbidity and mortality. We previously found that Pi stimulates the synthesis and sulfation of the negatively charged glycosaminoglycans (GAGs) heparan sulfate and chondroitin sulfate in vascular smooth muscle cells (VSMC). Similar GAG alterations were detected in VSMC-derived exosome-like extracellular vesicles (EV). These EV showed a strong interaction with very small superparamagnetic iron oxide particles (VSOP), which are used as imaging probes for experimental magnetic resonance imaging (MRI). Hyaluronic acid (HA) represents another negatively charged GAG which is supposed to function as binding motif for VSOP as well. We investigated the effects of Pi on the amounts of HA in cells and EV and studied the HA-dependent interaction between VSOP with cells and EV. Rat VSMC were treated with elevated concentrations of Pi. CKD in rats was induced by adenine feeding. EV were isolated from culture supernatants and rat plasma. We investigated the role of HA in binding VSOP to cells and EV via cell-binding studies, proton relaxometry, and analysis of cellular signaling, genes, proteins, and HA contents. Due to elevated HA contents, VSMC and EV showed an increased interaction with VSOP after Pi stimulation. Amongst others, Pi induced hyaluronan synthase (HAS)2 expression and activation of the Wnt pathway in VSMC. An alternative upregulation of HA by iloprost and an siRNA-mediated knockdown of HAS2 confirmed the importance of HA in cells and EV for VSOP binding. The in vitro-derived data were validated by analyses of plasma-derived EV from uremic rats. In conclusion, the inorganic uremic toxin Pi induces HA synthesis in cells and EV, which leads to an increased interaction with VSOP. HA might therefore be a potential molecular target structure for improved detection of pathologic tissue changes secondary to CKD like atherosclerosis or cardiomyopathy using EV, VSOP and MRI
The Cerebrospinal Fluid Free-Glycans Hex<sub>1</sub> and HexNAc<sub>1</sub>Hex<sub>1</sub>Neu5Ac<sub>1</sub> as Potential Biomarkers of Alzheimerâs Disease
Alzheimerâs disease (AD) is the most common neurodegenerative disorder, affecting a growing number of elderly people. In order to improve the early and differential diagnosis of AD, better biomarkers are needed. Glycosylation is a protein post-translational modification that is modulated in the course of many diseases, including neurodegeneration. Aiming to improve AD diagnosis and differential diagnosis through glycan analytics methods, we report the glycoprotein glycome of cerebrospinal fluid (CSF) isolated from a total study cohort of 262 subjects. The study cohort consisted of patients with AD, healthy controls and patients suffering from other types of dementia. CSF free-glycans were also isolated and analyzed in this study, and the results reported for the first time the presence of 19 free glycans in this body fluid. The free-glycans consisted of complete or truncated N-/O-glycans as well as free monosaccharides. The free-glycans Hex1 and HexNAc1Hex1Neu5Ac1 were able to discriminate AD from controls and from patients suffering from other types of dementia. Regarding CSF N-glycosylation, high proportions of high-mannose, biantennary bisecting core-fucosylated N-glycans were found, whereby only about 20% of the N-glycans were sialylated. O-Glycans and free-glycan fragments were less sialylated in AD patients than in controls. To conclude, this comprehensive study revealed for the first time the biomarker potential of free glycans for the differential diagnosis of AD
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 Parkinsonâs 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
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 Ă-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 Ă-tubulin III staining images (C-H) obtained from Attovision Software.</p
Neurite outgrowth parameters analyzed with the BD AttoVision<sup>TM</sup> V1.6 Software from BD Bioscience.
<p>Neurite outgrowth parameters analyzed with the BD AttoVision<sup>TM</sup> V1.6 Software from BD Bioscience.</p