Trichrome staining of heart sections 4, 7, 14, 31, 40 and 94 days post infarction and 4 days post sham. Concurrently, functional restoration of cardiac output was monitored using echocardiography and MRI. Protein profiles of myocardial tissue 4, 14 and 30 days post infarction and 4 days post sham was produced using LC-MS/MS.
Results and discussion: The axolotl restores complete myocardial structure and functionality within 2-3 months after a 25% myocardial infarction.
Interestingly, the initial response to injury is fibrosis forming a scar as found in mammals. However, this response is replaced within a few weeks when cardiomyocytes are deposited in the fibrotic scaffold aiding a gradual recovery of cardiac output.
This infarction model has the potential to improve our understanding of regeneration in the heart, and we hope to be able to identify signalling events at the protein level using the proteomics data.
O03.03 Dennis Kjølhede Jeppesen
EXOSOME ISOLATION BY DIFFERENTIAL CENTRIFUGATION: ANALYSIS OF DISCRETE FRACTIONS
D.K. Jeppesen1, M.L. Hvam2, B. Primdal-Bengtson1, B. Whitehead2, K.A.
Howard2, M.S. Ostenfeld1
1Department of Molecular Medicine, Aarhus University Hospital, 2The Interdisciplinary Nanoscience Center (iNANO), Aarhus University Cells release a mixture of extracellular vesicles (EV) that differ in size,
composition and cellular origin. Exosomes, the most studied of the EVs, are small membrane vesicles (40-120 nm) of endocytic origin. The most basic and standard method for obtaining samples of exosomes from fluid
samples is ultracentrifugation at 100,000 × g. The rapidly expanding field of exosome research necessitates a better understanding of the proper steps needed to ensure efficient generation of pure exosome samples. Here we have used the recently developed Nanoparticle Tracking Analysis (NTA) technique to examine the size and concentration of EVs present in samples from FL3 bladder cancer cells as well as HEK293 cells before and after centrifugation at a range of speeds from 33,000 × g to 200,000 × g.
Analyses revealed that efficient sedimentation of extracellular material occurs at lower speeds for HEK293 cell compared to FL3 cells. Western Blot analysis for the exosomal marker proteins CD81, Syntenin and TSG101 revealed that exosomes can be sedimented already at 33,000 × g and confirmed that HEK293 exosomes are rapidly purified at lower speeds compared to FL3 exosomes. Mitochondrial contamination of samples could be removed by a pre-clearing centrifugation step at 2,000 × g while contaminating microsomes persisted even after pre-clearing at 15,000 × g.
NTA analysis indicated that there is significant, persistent aggregation of exosomes after pelleting by ultracentrifugation. In conclusion, our results indicate and highlight the need to examine and analyze each cell type or biofluid individually to determine the optimal preparations of exosome samples free from contaminants.
O03.04 Tue Wenzel Kragstrup
DECREASED PLASMA LEVELS OF SOLUBLE CD18 LINK LEUKOCYTE MIGRATION WITH DISEASE ACTIVITY IN SPONDYLOARTHRITIS
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T.W. Kragstrup1, 2, B. Jalilian1, M. Hvid1, A.G. Kjærgaard1, R. Østgård1, B.
Schiøttz-Christensen3, A.G. Jurik4, W.H. Robinson2, T. Vorup-Jensen1, B.
Deleuran1, 3
1Department of Biomedicine, Aarhus University, 2Division of Immunology and Rheumatology, Stanford University, Palo Alto, CA, USA, 3Department of Rheumatology, Aarhus University Hospital, 4Department of Radiology, Aarhus University Hospital
Introduction: Spondyloarthritis (SpA) is an autoimmune disease
characterized by spinal joint inflammation causing pain and disability. The inflammatory process in SpA is poorly understood, and tools for monitoring and treating the disease are needed. The CD18 integrins on leukocytes are pivotal for the generation of inflammatory responses. Recently, we
demonstrated a soluble form of CD18 (sCD18) in the blood with the potential to attenuate inflammation. Here, we study the role of sCD18 in SpA.
Methods: Plasma levels of sCD18 in a study population with 84 SpA patients and matched healthy controls were analyzed with a time resolved
immunoflourometric assay (TRIFMA). Binding of sCD18 to endothelial cells was studied with confocal microscopy and shedding of CD18 from peripheral blood mononuclear cells (PBMC) was studied with flow cytometry and TRIFMA.
Results: Plasma levels of sCD18 were decreased in SpA patients compared with healthy volunteers and exhibited an inverse correlation with clinical scores of disease activity in a multiple regression model. The mechanisms for these changes could be simulated in vitro. First, sCD18 in plasma adhered to endothelial cells, indicating increased consumption. Second, CD18 shedding from SpA PBMC correlated inversely with disease activity, suggesting insufficient generation.
Conclusions: Taken together, SpA patients fail to maintain normal sCD18 levels in the blood, and this facilitates leukocyte migration to the spinal joints and aggregates disease activity. In the future, our findings could be utilized by using the level of sCD18 as a marker of inflammatory activity or a compound resembling sCD18 as a therapeutic drug.
O03.05 Shivani Joshi IDENTIFICATION OF DISEASE-RELATED GENES IN FAMILIAL STEROID SENSITIVE NEPHROTIC SYNDROME
S. Joshi1, R. Andersen1, K. Moeller2, T. Seeman3, L. Podracká4, H. Eiberg5, S.
Rittig1
1Pediatric Research Lab-A, Department of Clinical Medicine, Aarhus University Hospital, Brendstrupgårdsvej 100, 8200, Aarhus N, Denmark.,
2Department of Pediatric and Adolescent Medicine, Klinikum Links der Weser, Senator-Wessling-Str.1, 28277 Bremen, 3Department of Pediatrics, Charles University in Prague - 2 Faculty of Medicine, The Úvalu 84, Praha 5, 150 06., 4I. Department of Children and Adolescents UPJŠ LF and DFN, Class SNP 1, 040 11 Košice, Slovak Republic, 5Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, The Panum Institute, 3B Blegdamsvej, 2200 Copenhagen N, Denmark.
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Nephrotic syndrome (NS) is defined by heavy proteinuria, edema and hypoalbuminemia. Most pediatric patients respond well to steroid therapy and have good prognosis. These patients have steroid sensitive NS (SSNS).
Familial aggregates and kindred have been described in patients with SSNS, suggesting a potential genetic origin. However, very little data on molecular genetics of familial SSNS is available in literature. Fuchshuber et al reported in 2001 that familial SSNS was found to be genetically different from changes in NPHS2 gene. Till date, only one locus for SSNS has been mapped on chromosome 2p12-13.212, but no gene(s) have been ascertained for SSNS. Family studies could help to further identify genes that are altered in this patient group and will contribute to a better understanding of underlying molecular mechanism in familial SSNS. The purpose of this study is to identify new candidate genes in 9 families with siblings with SSNS, using SNP 6.0 microarray whole genome linkage analysis in all participants, followed by whole exome sequencing in selected patients. Our findings of whole genome linkage analysis and exome sequencing will be discussed.
Key words: nephrotic syndrome, SNP 6.0 microarray whole genome linkage analysis, whole exome sequencing
O03.06 Kristian Ravlo EFFECT OF REMOTE ISCHEMIC CONDITIONING ON DENDRITIC CELLS AFTER KIDNEY TRANSPLANTATION FROM DECEASED DONORS
K. Ravlo1,2, N. Krogstrup1, M. Steen Petersen2, M. Bue Svendsen1, C. Abdel-Halim1, B. Møller2, B. Jespersen1
1Department of Clinical Medicine – Medicine and Nephrology, Aarhus University, 2Department of Clinical Immunology, Aarhus University Hospital In kidney transplantation, remote ischemic conditioning (rIC) protects the
graft against ischemia-reperfusion injury and reduces the risk of rejection. In essence, rIC consists of repeated, brief, non-damaging periods of ischemia in a limb. But why this procedure protects against rejection is unknown.
During graft rejection, the main antigen-presenting cells are probably dendritic cells (DCs). Because dendritic cells are attracted to ischemic endothelium, we hypothesise that rIC protects the organ by 'trapping' circulating DCs in the ischemic limb. As a result, fewer DCs are available to infiltrate the graft and present foreign antigens. To test this hypothesis, we shall measure the quantity of dendritic cells in circulation before and after rIC.
The CONTEXT study is a randomised, controlled clinical study of 200 patients, who are randomised to rIC or non-rIC. Blood samples will be obtained at baseline, day 1, day 3, and day 5; and 1 month and 3 months after transplantation. In these samples, myeloid and plasmacytoid DCs will be identified and quantified by flow cytometry after staining with
antibodies against CD3, CD14, CD19, CD56, ILT3, HLA-DR, CD123, CD11c, and CD86.
Preliminary data are presented.
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