- Email: [email protected]
Remnant Epitope Autoimmunity in Human Abdominal Aortic Aneurysm: A Pilot Study with Elastin Peptides
Accepted Manuscript Remnant epitope Autoimmunity in human abdominal aortic aneurysm: a pilot study with elastin PEPTIDEs Jelle Verhoeven, Alix Lambrecht, Peter Verbrugghe, Paul Herijgers, Inge Fourneau PII:
S0890-5096(17)30730-6
DOI:
10.1016/j.avsg.2017.05.036
Reference:
AVSG 3420
To appear in:
Annals of Vascular Surgery
Received Date: 19 March 2017 Accepted Date: 28 May 2017
Please cite this article as: Verhoeven J, Lambrecht A, Verbrugghe P, Herijgers P, Fourneau I, Remnant epitope Autoimmunity in human abdominal aortic aneurysm: a pilot study with elastin PEPTIDEs, Annals of Vascular Surgery (2017), doi: 10.1016/j.avsg.2017.05.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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REMNANT EPITOPE AUTOIMMUNITY IN HUMAN ABDOMINAL
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AORTIC ANEURYSM: A PILOT STUDY WITH ELASTIN PEPTIDES
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Jelle Verhoeven1
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Alix Lambrecht1
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Peter Verbrugghe1
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Paul Herijgers1
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Inge Fourneau1
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Authors:
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Herestraat 49 – bus 911, 3000 Leuven – Belgium
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[email protected], [email protected];
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[email protected]; [email protected];
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[email protected]
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Department of Cardiovascular Sciences, KU Leuven - University of Leuven, O&N1
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Corresponding Author:
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Jelle Verhoeven
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Department of cardiovascular sciences
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O&N1 Herestraat 49 - bus 911
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3000 Leuven, Belgium
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Tel: +32 495 14 33 25
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Email: [email protected]
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REMNANT EPITOPE AUTOIMMUNITY IN HUMAN ABDOMINAL
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AORTIC ANEURYSM: A PILOT STUDY WITH ELASTIN PEPTIDES
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Jelle Verhoeven, Alix Lambrecht, Peter Verbrugghe M.D., Paul Herijgers M.D. PhD, Inge Fourneau M.D. PhD Department of Cardiovascular Sciences - KU Leuven - University of Leuven Herestraat 49 – bus 911 – 3000 Leuven – Belgium Corresponding author: [email protected]
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Abstract
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Introduction
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Abdominal aortic aneurysm (AAA) is a prevalent disease affecting around 5% of the
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population over 65 years of age. The exact etiology and physiopathology of AAA still
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raises questions and elective surgery is currently the only treatment option for this
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often progressive disease. In this study we hypothesized and tested a
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pathophysiological model that depicts AAA as an inflammation triggered
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autoimmune disease with remnant vessel wall peptide fragments as the antigen.
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Material and methods
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A pilot study with male AAA patients (n=14) and male controls (n=8) was conducted.
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In both study groups peripheral blood monocytes and plasma were separated from
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whole blood by centrifugation. An ELISpot test was performed on cultured white
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blood cells for the presence of elastin specific T-lymphocytes. An ELISA test was
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performed on plasma for the presence of elastin specific IgG molecules.
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Results
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ELISpot interferon-gamma secretion in AAA (7.7±9.5%) and control (4.6±3.5%) and
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ELISA anti-elastin IgG titer in AAA (77.5±17.8%) and control (78.2±31.5%) were
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not significantly different (p= 0.94 resp. p= 0.55). Both results are expressed as a
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percentage relative to the respective positive and negative control.
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Conclusion
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ACCEPTED MANUSCRIPT The results of our pilot study did not indicate a clear and invariable autoimmune
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process directed against remnant elastin peptide fragments. Further research into the
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model mechanics and a possible antigen is still necessary. In the mean time, the model
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as presented here already offers a pathophysiological framework to further research
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into the possible remnant epitope driven AAA etiology.
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Keywords
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abdominal aortic aneurysm, autoimmunity, elastin, remnant epitope
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62 1. Introduction
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Abdominal aortic aneurysm (AAA) is defined as an abnormal balloon- or sac-like
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dilatation in the wall of the abdominal aorta. AAA is a prevalent disease affecting on
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average 5% of the population over 65 years of age and is more prevalent in men than
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in women [1, 2]. The exact etiology and physiopathology of AAA still raises
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questions and elective surgery is currently the only treatment option for this often
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progressive disease to prevent fatal rupture [3]. A better understanding of the
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physiopathology of AAA could result in a future medicinal therapy to slow down or
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even halt AAA progression. This would be a valuable future treatment alternative
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allowing to postpone or alleviate the need for surgery, especially since AAA is a
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condition of the elderly population.
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The possible autoimmune etiology of AAA has already been suggested [4, 5]. In this
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study we hypothesized a pathophysiological model that depicts AAA as an
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inflammation triggered autoimmune disease with remnant vessel wall peptide
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fragments as the antigen (Figure 1: proposed pathophysiological model of AAA). We
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ACCEPTED MANUSCRIPT then tested a part of this model in a small clinical pilot trial with remnant aortic elastin
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peptides as the candidate antigen.
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In the proposed model a chronic process like atherosclerosis could create an
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inflammatory environment inside the vessel wall. Proteases secreted by non-specific
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immune cells such as neutrophils could degrade structural proteins like elastin into
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remnant elastin peptides. These remnant elastin peptides are chemotactic and attract
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more specific and non-specific immune cells to the site [6,7]. Unlike intact elastin,
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remnant elastin peptides could be endocytosed and processed in dendritic cells. Next,
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presentation of these processed remnant elastin peptides to cells of the specific
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immune system via major histocompatibility class II (MHCII) proteins on the
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dendritic cell surface is possible. This could create a specific immune response to
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remnant elastin peptides.
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Both chemotaxis and the elastin specific immune response create a vicious circle of
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localized elastin breakdown in the atherosclerotic environment. The macroscopic
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effect of this would be AAA progression.
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The motivation for this model and the choice for elastin as a candidate peptide in the
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clinical pilot trial were based on three key elements. Firstly, the model is based on the
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remnant-epitope-generates-autoimmunity (REGA) model used to describe the
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physiopathology of other autoimmune diseases like rheumatoid arthritis [8] and
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multiple sclerosis [9]. Key elements from the REGA model such as the presence of
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specific and non-specific immune cells [10], upregulation of pro-inflammatory
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cytokines [11], secretion of proteases [12, 13], altered MHC class 2 expression and
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peptide fragmentation [14, 15] have long been shown in AAA. Secondly, remnant
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elastin peptides are auto-immunogenic in COPD patients with emphysema in a
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specific stage of the disease [16,17]. Auto-reactive T-cells and IgG molecules, both
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ACCEPTED MANUSCRIPT specific to elastin were found in peripheral blood of these patients. Thirdly, a rodent
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model of AAA can be created using elastase to digest vessel wall elastin peptides [18].
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We tested a part of our hypothetical model in a small clinical pilot trial. We assessed
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the presence of elastin specific T-cells and elastin specific IgG molecules in
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peripheral blood of AAA patients in comparison with healthy controls.
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108 2. Materials and methods
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2.1 Study design
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We conducted a clinical pilot trial with two study groups consisting of male AAA
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patients (n=14) and male controls (n=8). The ethical committee of our center
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approved this trial and an informed consent was obtained from every
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participant. Patients and controls were matched by sex but separated based on
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other risk factors for AAA development/progression to make the study more
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sensitive for subtle differences in antibody titer or immune response. Controls
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were younger, had no diagnosed AAA, no clinical signs of atherosclerosis and
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were never-smokers compared to the AAA patient group. Aneurysm diameter
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was determined with ultrasound. Smoking status was determined in pack-years.
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(Table I: study group demographics). Of each participant, 18 ml of peripheral
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blood was obtained in citrate containing blood collection tubes (BD Vacutainer®).
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2.2 Sample cryopreservation
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Because the blood sampling of all participants was not performed at the same moment,
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the blood samples of all patients were cryopreserved before being further assayed.
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The blood samples were processed in our lab within one hour, always by the same
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Falcon tube (BD®) and diluted 1:1 with DPBS (Life Technologies®). Four milliliters
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of diluted blood was carefully layered on top of 3 ml Ficoll-Paque (GE Healthcare®)
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solution into a 15 ml Falcon tube at 19 °C. This step was repeated until all the diluted
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blood was layered into Ficoll-Paque containing tubes. The layered blood tubes were
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centrifuged for 35 minutes at 400g at 19 °C. This resulted in a separate layering of
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plasma, peripheral blood monocytes (PBMC) and red blood cells. The PBMC layers
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were collected and pooled into 15 ml Falcon tubes using a 1 ml pipette. This resulted
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in 3 ml of PBMC suspension in each tube. PBMC were washed by diluting each tube
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to 14 ml with DPBS and centrifuging for 10 minutes at 640g. The resulting
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supernatant was discarded and the PBMC pellet was re-suspended in 10 ml of PBS.
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At this moment a cell-count and cell-viability test was performed using an improved
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Neubauer counting chamber by filling it with a mixture of 10 µl of the cell suspension
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and 10 µl of trypan blue (Gibco®). The tubes were then centrifuged for 10 minutes at
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470g. The resulting supernatant was discarded and cells were re-suspended in freshly
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prepared freezing medium containing 70% 1640-RPMI, 20% fetal calf serum and
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10% DMSO to a cell concentration of 8x10^6 per ml. To minimize cell death, DMSO
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was added just before freezing. Cells were pipetted into 2 ml cryovials, with a
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maximum of 1 ml of cell-suspension in each vial. The vials were placed into a Mr.
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Frosty freezing container (Nalgene®) and put into a -80°C freezer for 24 hours before
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being transferred to liquid nitrogen before being assayed. The plasma after ficoll
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paque centrifugation was pipetted into cryovials and stored at -80 °C.
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2.3 Thawing of PBMC and plasma
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When all participants' samples were obtained and underwent cryopreserving, all
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PBMC vials were thawed simultaneously. PBMC containing vials were transferred
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ACCEPTED MANUSCRIPT from liquid nitrogen into a -80°C freezer for one hour. Vials were then placed into a
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37°C preheated water bath until the PBMC solution was almost completely thawed. A
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37°C solution of complete RPMI medium with 20% fetal calf serum was added
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dropwise to each cyrovial until the total volume equaled 2 ml. The content of each
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cryovial was added to a 8ml containing RPMI/FCS 15ml falcon tube and centrifuged
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at 330g for 10 minutes. Supernatant was discarded and cells were resuspended in 6ml
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of RPMI/FCS mixture. A cell-count and cell-viability test was performed as described
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in the sample cryopreservation section above. Tubes were centrifuged for 10 minutes
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at 640 g, supernatant was discarded and a solution of RPMI/FCS containing 50 units
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of penicillin per ml were added to obtain a cell concentration of 4*10^6 cells per ml.
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Plasma containing vials were thawed completely in a 37°C water bath.
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2.4 ELISpot
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The presence of elastin specific T-cells in each participant PBMC collection was
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assayed using a human interferon-gamma ELISpot Plus kit with 4 pre-coated plates
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(Mabtech®). For each participant we used 9 wells of a 96-well plate. In each well,
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200.000 live cells were seeded by pipetting 50 µl of the thawed PBMC. Three wells
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were used as negative control by pipetting 50µl of cell medium into the corresponding
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wells. Three wells were used as a positive control by pipetting 50µl of the kit CD-3
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antibody solution into the corresponding wells. CD-3 stimulation results in a maximal
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activation of T-cells, maximizing the interferon-gamma secretion. The remaining
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three wells were stimulated with human aortic elastin peptide solution (RY53, Elastin
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Products Company®) by pipetting 50 µl of a 30 µg/ml solution into the corresponding
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wells. Plates were incubated for 12 hours into a 37°C humidified incubator containing
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5% CO2. After incubation, plates were developed to visualize the amount of
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interferon-gamma secretion according to the kit manual. All wells were photographed
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ACCEPTED MANUSCRIPT using a surgical microscope (Zeiss®). The area of IFN-gamma secretion was
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automatically measured and counted with the Colony Blob Count tool macro plugin
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for ImageJ.
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2.5 ELISA
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The presence of elastin specific IgG molecules in the plasma of each participant was
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assayed using a human IgG ELISA development kit (3850-1AD-6, Mabtech®). Two
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wells were used for the plasma of each participant. Two wells were used for plasma of
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a systemic sclerosis patient with specific elastin IgG as positive control [19]. Two
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wells were used for the negative control, these would be filled with DPBS instead of
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plasma. Sixteen wells were used for making a standard curve to monitor the
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correctness of the ELISA development process. A protein binding Immulon 4HBX
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plate (Thermo Fisher Scientific®) was coated with human aortic elastin peptide (RY53,
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Elastin Products Company®) by pipetting 60 µl of DPBS containing a 25 µg/ml elastin
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into the wells of each participant, positive and negative control. The plate was
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incubated overnight at 4-8°C. After washing twice with 200 µl of DPBS, the plate was
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blocked with DPBS containing 0,05% Tween 20 and 0,1% FCS to prevent further
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protein binding. The plate was incubated for one hour at room temperature before
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washing each well five times with 200 µl DPBS containing 0,05% Tween 20. One
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hundred microliters of plasma, DPBS and IgG standard solution were added to their
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respective wells and incubated for two hours at room temperature. All wells were
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washed five times with 200 µl DPBS containing 0,05% Tween 20. 100 µl of IgG
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antibody conjugated with alkaline phosphatase was added to each well and incubated
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for one hour at room temperature. All wells were washed five times with 200 µl
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DPBS containing 0,05% Tween 20. One hundred microliters of para-
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nitrophenolphosphate solution (Sigma-Aldrich®) was added to each well. After thirty
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ACCEPTED MANUSCRIPT 203
minutes the plate was read at 405 nm using a Multiskan EX ELISA reader (Thermo
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Fisher Scientific®).
205 3. Results
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3.1 ELISpot
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The amount of interferon-gamma secretion of the PBMC cell cultures after human
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aortic elastin peptide stimulation was a measure for the amount of elastin specific T-
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lymphocytes. The mean interferon-gamma secretion of the AAA patient group and
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control group was assayed. Both results were expressed as a relative percentage with a
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secretion level of zero percent corresponding to the interferon-gamma secretion of the
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negative control wells. A secretion level of hundred percent corresponded to the
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interferon-gamma secretion of the positive control wells. The mean interferon-gamma
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secretion levels of the control group (7.7±9.5%) and the AAA patient group
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(4.6±3.5%) were not significantly different (p = 0,80), using a Mann Whitney U test
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in SPSS (Figure 2: PBMC culture IFN-γ secretion).
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3.2 ELISA
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The mean elastin specific antibody amount of the AAA patient group and the control
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group was assayed. Both results were expressed as a relative percentage with a level
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of zero percent corresponding to the antibody concentration of the negative control
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wells. A level of hundred percent corresponded to the antibody concentration of the
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positive control. The mean elastin specific antibody level of the control group
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(77,5±17,8%) and the AAA patient group (78.2±31.5%) were not significantly
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different (p = 0,43), using a Mann Whitney U test in SPSS (Figure 3: anti-elastin IgG
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titer).
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ACCEPTED MANUSCRIPT 228 4. Discussion
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Based on the results of our pilot study, the validity of the proposed model could not
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be shown. There was no indication for an invariable and specific autoimmune
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response against elastin peptides in atherosclerotic AAA. However, we used a small
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number of participants and we only tested a specific part of the model in our pilot
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study. Repeating the pilot study in larger patient groups with other inclusion criteria
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and/or using tissue samples instead of peripheral blood and other candidate peptides is
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necessary before being able to (partially) accept or reject the proposed model.
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The clinical relevance of this study was not only to identify a specific antigen as a
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possible therapeutic target, but also to propose a usable model itself. Indeed, proving
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the (partial) validity of the model offers other therapeutic targets to slow or stop the
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autoimmune response by intervening with the various mechanisms of the model [9].
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The model places atherosclerosis and AAA as points on a continuous spectrum.
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However, we do not state a one-to-one relationship between atherosclerosis and AAA
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[20]. We hypothesized that atherosclerosis is the origin of the aspecific chronic
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inflammatory reaction that is necessary to cause AAA initiation via the creation of
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remnant peptide fragments. It is possible that atherosclerosis stimulates a pro-
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inflammatory cytokine activating protein complex like the inflammasome [21].
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Inflammasome activation could result in AAA initiation [22, 23] because the delicate
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balance between pro- and anti-inflammatory cytokines would change. The disruption
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of this balance is necessary in our model to initiate the vicious process of structural
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vessel wall peptide degradation. Indeed, pro-inflammatory cytokines such as
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interleukin-1β and interferon-gamma are also key elements of the REGA model [9].
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All of this can only happen if the necessary genetic and environmental factors are
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ACCEPTED MANUSCRIPT present. Therefore, it would be possible to have severe atherosclerotic lesions without
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the occurrence of AAA initiation. It has been shown that there is an altered expression
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pattern of MHCII alleles in AAA compared with controls [14, 24]. It has also been
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shown that in AAA there is an imbalance between proteases such as matrix
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metalloproteinase and their inhibitors, favoring activation of these proteases [12].
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Further research is necessary to further elucidate all of the genetic and environmental
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factors necessary for AAA initiation and progression.
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5. Conclusion
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The results of our pilot study did not indicate a clear and invariable autoimmune
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process directed against remnant elastin peptide fragments. However, the goal of the
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study was not only to find a specific antigen but also to propose a pathophysiological
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AAA model. Further research into the model mechanics and a possible antigen is still
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necessary. In the mean time, the model as presented here already offers a
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pathophysiological framework to further research into the possible remnant epitope
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driven AAA etiology.
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6. Acknowledgement
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This study was funded from our own lab resources. We would like to thank our
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student colleagues for their assistance with the sample processing: Leontine Grozema,
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Sofie Ordies, Isabelle Sreeram, Laurens Van Melkebeke and Lucas Van Hoof.
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7. Conflicts of interest
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None
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8. Funding
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This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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et al. Human leukocyte antigen class II immune response genes, female gender, and
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cigarette smoking as risk and modulating factors in abdominal aortic aneurysms. J
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Vasc Surg. 2002 May;35(5):988-93.
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ACCEPTED MANUSCRIPT AAA
Controls
Sex
Male
Number (n=)
14
8
Mean age (years)
70,8 ± 6,5
29,4 ± 3,6
Pack-years (years)
49,5 ± 17,19
0
Aneurysm diameter (mm)
42,3 ± 8,84
% with hypertension (%)
90
% with hypercholesterolemia (%)
91
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Parameter
/
0 0
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Table I: study group demographics
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S0890-5096(17)30730-6
DOI:
10.1016/j.avsg.2017.05.036
Reference:
AVSG 3420
To appear in:
Annals of Vascular Surgery
Received Date: 19 March 2017 Accepted Date: 28 May 2017
Please cite this article as: Verhoeven J, Lambrecht A, Verbrugghe P, Herijgers P, Fourneau I, Remnant epitope Autoimmunity in human abdominal aortic aneurysm: a pilot study with elastin PEPTIDEs, Annals of Vascular Surgery (2017), doi: 10.1016/j.avsg.2017.05.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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REMNANT EPITOPE AUTOIMMUNITY IN HUMAN ABDOMINAL
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AORTIC ANEURYSM: A PILOT STUDY WITH ELASTIN PEPTIDES
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Jelle Verhoeven1
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Alix Lambrecht1
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Peter Verbrugghe1
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Paul Herijgers1
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Inge Fourneau1
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Authors:
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Herestraat 49 – bus 911, 3000 Leuven – Belgium
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[email protected], [email protected];
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[email protected]; [email protected];
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[email protected]
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Department of Cardiovascular Sciences, KU Leuven - University of Leuven, O&N1
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Corresponding Author:
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Jelle Verhoeven
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Department of cardiovascular sciences
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O&N1 Herestraat 49 - bus 911
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3000 Leuven, Belgium
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Tel: +32 495 14 33 25
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Email: [email protected]
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REMNANT EPITOPE AUTOIMMUNITY IN HUMAN ABDOMINAL
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AORTIC ANEURYSM: A PILOT STUDY WITH ELASTIN PEPTIDES
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Jelle Verhoeven, Alix Lambrecht, Peter Verbrugghe M.D., Paul Herijgers M.D. PhD, Inge Fourneau M.D. PhD Department of Cardiovascular Sciences - KU Leuven - University of Leuven Herestraat 49 – bus 911 – 3000 Leuven – Belgium Corresponding author: [email protected]
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Abstract
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Introduction
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Abdominal aortic aneurysm (AAA) is a prevalent disease affecting around 5% of the
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population over 65 years of age. The exact etiology and physiopathology of AAA still
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raises questions and elective surgery is currently the only treatment option for this
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often progressive disease. In this study we hypothesized and tested a
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pathophysiological model that depicts AAA as an inflammation triggered
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autoimmune disease with remnant vessel wall peptide fragments as the antigen.
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Material and methods
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A pilot study with male AAA patients (n=14) and male controls (n=8) was conducted.
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In both study groups peripheral blood monocytes and plasma were separated from
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whole blood by centrifugation. An ELISpot test was performed on cultured white
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blood cells for the presence of elastin specific T-lymphocytes. An ELISA test was
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performed on plasma for the presence of elastin specific IgG molecules.
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Results
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ELISpot interferon-gamma secretion in AAA (7.7±9.5%) and control (4.6±3.5%) and
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ELISA anti-elastin IgG titer in AAA (77.5±17.8%) and control (78.2±31.5%) were
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not significantly different (p= 0.94 resp. p= 0.55). Both results are expressed as a
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percentage relative to the respective positive and negative control.
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Conclusion
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ACCEPTED MANUSCRIPT The results of our pilot study did not indicate a clear and invariable autoimmune
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process directed against remnant elastin peptide fragments. Further research into the
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model mechanics and a possible antigen is still necessary. In the mean time, the model
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as presented here already offers a pathophysiological framework to further research
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into the possible remnant epitope driven AAA etiology.
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Keywords
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abdominal aortic aneurysm, autoimmunity, elastin, remnant epitope
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62 1. Introduction
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Abdominal aortic aneurysm (AAA) is defined as an abnormal balloon- or sac-like
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dilatation in the wall of the abdominal aorta. AAA is a prevalent disease affecting on
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average 5% of the population over 65 years of age and is more prevalent in men than
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in women [1, 2]. The exact etiology and physiopathology of AAA still raises
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questions and elective surgery is currently the only treatment option for this often
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progressive disease to prevent fatal rupture [3]. A better understanding of the
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physiopathology of AAA could result in a future medicinal therapy to slow down or
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even halt AAA progression. This would be a valuable future treatment alternative
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allowing to postpone or alleviate the need for surgery, especially since AAA is a
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condition of the elderly population.
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The possible autoimmune etiology of AAA has already been suggested [4, 5]. In this
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study we hypothesized a pathophysiological model that depicts AAA as an
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inflammation triggered autoimmune disease with remnant vessel wall peptide
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fragments as the antigen (Figure 1: proposed pathophysiological model of AAA). We
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ACCEPTED MANUSCRIPT then tested a part of this model in a small clinical pilot trial with remnant aortic elastin
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peptides as the candidate antigen.
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In the proposed model a chronic process like atherosclerosis could create an
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inflammatory environment inside the vessel wall. Proteases secreted by non-specific
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immune cells such as neutrophils could degrade structural proteins like elastin into
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remnant elastin peptides. These remnant elastin peptides are chemotactic and attract
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more specific and non-specific immune cells to the site [6,7]. Unlike intact elastin,
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remnant elastin peptides could be endocytosed and processed in dendritic cells. Next,
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presentation of these processed remnant elastin peptides to cells of the specific
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immune system via major histocompatibility class II (MHCII) proteins on the
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dendritic cell surface is possible. This could create a specific immune response to
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remnant elastin peptides.
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Both chemotaxis and the elastin specific immune response create a vicious circle of
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localized elastin breakdown in the atherosclerotic environment. The macroscopic
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effect of this would be AAA progression.
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The motivation for this model and the choice for elastin as a candidate peptide in the
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clinical pilot trial were based on three key elements. Firstly, the model is based on the
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remnant-epitope-generates-autoimmunity (REGA) model used to describe the
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physiopathology of other autoimmune diseases like rheumatoid arthritis [8] and
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multiple sclerosis [9]. Key elements from the REGA model such as the presence of
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specific and non-specific immune cells [10], upregulation of pro-inflammatory
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cytokines [11], secretion of proteases [12, 13], altered MHC class 2 expression and
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peptide fragmentation [14, 15] have long been shown in AAA. Secondly, remnant
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elastin peptides are auto-immunogenic in COPD patients with emphysema in a
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specific stage of the disease [16,17]. Auto-reactive T-cells and IgG molecules, both
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ACCEPTED MANUSCRIPT specific to elastin were found in peripheral blood of these patients. Thirdly, a rodent
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model of AAA can be created using elastase to digest vessel wall elastin peptides [18].
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We tested a part of our hypothetical model in a small clinical pilot trial. We assessed
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the presence of elastin specific T-cells and elastin specific IgG molecules in
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peripheral blood of AAA patients in comparison with healthy controls.
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108 2. Materials and methods
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2.1 Study design
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We conducted a clinical pilot trial with two study groups consisting of male AAA
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patients (n=14) and male controls (n=8). The ethical committee of our center
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approved this trial and an informed consent was obtained from every
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participant. Patients and controls were matched by sex but separated based on
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other risk factors for AAA development/progression to make the study more
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sensitive for subtle differences in antibody titer or immune response. Controls
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were younger, had no diagnosed AAA, no clinical signs of atherosclerosis and
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were never-smokers compared to the AAA patient group. Aneurysm diameter
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was determined with ultrasound. Smoking status was determined in pack-years.
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(Table I: study group demographics). Of each participant, 18 ml of peripheral
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blood was obtained in citrate containing blood collection tubes (BD Vacutainer®).
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2.2 Sample cryopreservation
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Because the blood sampling of all participants was not performed at the same moment,
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the blood samples of all patients were cryopreserved before being further assayed.
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The blood samples were processed in our lab within one hour, always by the same
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Falcon tube (BD®) and diluted 1:1 with DPBS (Life Technologies®). Four milliliters
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of diluted blood was carefully layered on top of 3 ml Ficoll-Paque (GE Healthcare®)
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solution into a 15 ml Falcon tube at 19 °C. This step was repeated until all the diluted
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blood was layered into Ficoll-Paque containing tubes. The layered blood tubes were
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centrifuged for 35 minutes at 400g at 19 °C. This resulted in a separate layering of
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plasma, peripheral blood monocytes (PBMC) and red blood cells. The PBMC layers
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were collected and pooled into 15 ml Falcon tubes using a 1 ml pipette. This resulted
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in 3 ml of PBMC suspension in each tube. PBMC were washed by diluting each tube
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to 14 ml with DPBS and centrifuging for 10 minutes at 640g. The resulting
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supernatant was discarded and the PBMC pellet was re-suspended in 10 ml of PBS.
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At this moment a cell-count and cell-viability test was performed using an improved
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Neubauer counting chamber by filling it with a mixture of 10 µl of the cell suspension
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and 10 µl of trypan blue (Gibco®). The tubes were then centrifuged for 10 minutes at
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470g. The resulting supernatant was discarded and cells were re-suspended in freshly
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prepared freezing medium containing 70% 1640-RPMI, 20% fetal calf serum and
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10% DMSO to a cell concentration of 8x10^6 per ml. To minimize cell death, DMSO
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was added just before freezing. Cells were pipetted into 2 ml cryovials, with a
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maximum of 1 ml of cell-suspension in each vial. The vials were placed into a Mr.
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Frosty freezing container (Nalgene®) and put into a -80°C freezer for 24 hours before
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being transferred to liquid nitrogen before being assayed. The plasma after ficoll
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paque centrifugation was pipetted into cryovials and stored at -80 °C.
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2.3 Thawing of PBMC and plasma
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When all participants' samples were obtained and underwent cryopreserving, all
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PBMC vials were thawed simultaneously. PBMC containing vials were transferred
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ACCEPTED MANUSCRIPT from liquid nitrogen into a -80°C freezer for one hour. Vials were then placed into a
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37°C preheated water bath until the PBMC solution was almost completely thawed. A
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37°C solution of complete RPMI medium with 20% fetal calf serum was added
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dropwise to each cyrovial until the total volume equaled 2 ml. The content of each
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cryovial was added to a 8ml containing RPMI/FCS 15ml falcon tube and centrifuged
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at 330g for 10 minutes. Supernatant was discarded and cells were resuspended in 6ml
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of RPMI/FCS mixture. A cell-count and cell-viability test was performed as described
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in the sample cryopreservation section above. Tubes were centrifuged for 10 minutes
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at 640 g, supernatant was discarded and a solution of RPMI/FCS containing 50 units
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of penicillin per ml were added to obtain a cell concentration of 4*10^6 cells per ml.
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Plasma containing vials were thawed completely in a 37°C water bath.
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2.4 ELISpot
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The presence of elastin specific T-cells in each participant PBMC collection was
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assayed using a human interferon-gamma ELISpot Plus kit with 4 pre-coated plates
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(Mabtech®). For each participant we used 9 wells of a 96-well plate. In each well,
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200.000 live cells were seeded by pipetting 50 µl of the thawed PBMC. Three wells
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were used as negative control by pipetting 50µl of cell medium into the corresponding
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wells. Three wells were used as a positive control by pipetting 50µl of the kit CD-3
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antibody solution into the corresponding wells. CD-3 stimulation results in a maximal
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activation of T-cells, maximizing the interferon-gamma secretion. The remaining
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three wells were stimulated with human aortic elastin peptide solution (RY53, Elastin
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Products Company®) by pipetting 50 µl of a 30 µg/ml solution into the corresponding
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wells. Plates were incubated for 12 hours into a 37°C humidified incubator containing
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5% CO2. After incubation, plates were developed to visualize the amount of
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interferon-gamma secretion according to the kit manual. All wells were photographed
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ACCEPTED MANUSCRIPT using a surgical microscope (Zeiss®). The area of IFN-gamma secretion was
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automatically measured and counted with the Colony Blob Count tool macro plugin
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for ImageJ.
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2.5 ELISA
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The presence of elastin specific IgG molecules in the plasma of each participant was
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assayed using a human IgG ELISA development kit (3850-1AD-6, Mabtech®). Two
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wells were used for the plasma of each participant. Two wells were used for plasma of
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a systemic sclerosis patient with specific elastin IgG as positive control [19]. Two
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wells were used for the negative control, these would be filled with DPBS instead of
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plasma. Sixteen wells were used for making a standard curve to monitor the
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correctness of the ELISA development process. A protein binding Immulon 4HBX
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plate (Thermo Fisher Scientific®) was coated with human aortic elastin peptide (RY53,
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Elastin Products Company®) by pipetting 60 µl of DPBS containing a 25 µg/ml elastin
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into the wells of each participant, positive and negative control. The plate was
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incubated overnight at 4-8°C. After washing twice with 200 µl of DPBS, the plate was
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blocked with DPBS containing 0,05% Tween 20 and 0,1% FCS to prevent further
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protein binding. The plate was incubated for one hour at room temperature before
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washing each well five times with 200 µl DPBS containing 0,05% Tween 20. One
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hundred microliters of plasma, DPBS and IgG standard solution were added to their
197
respective wells and incubated for two hours at room temperature. All wells were
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washed five times with 200 µl DPBS containing 0,05% Tween 20. 100 µl of IgG
199
antibody conjugated with alkaline phosphatase was added to each well and incubated
200
for one hour at room temperature. All wells were washed five times with 200 µl
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DPBS containing 0,05% Tween 20. One hundred microliters of para-
202
nitrophenolphosphate solution (Sigma-Aldrich®) was added to each well. After thirty
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ACCEPTED MANUSCRIPT 203
minutes the plate was read at 405 nm using a Multiskan EX ELISA reader (Thermo
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Fisher Scientific®).
205 3. Results
207
3.1 ELISpot
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The amount of interferon-gamma secretion of the PBMC cell cultures after human
209
aortic elastin peptide stimulation was a measure for the amount of elastin specific T-
210
lymphocytes. The mean interferon-gamma secretion of the AAA patient group and
211
control group was assayed. Both results were expressed as a relative percentage with a
212
secretion level of zero percent corresponding to the interferon-gamma secretion of the
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negative control wells. A secretion level of hundred percent corresponded to the
214
interferon-gamma secretion of the positive control wells. The mean interferon-gamma
215
secretion levels of the control group (7.7±9.5%) and the AAA patient group
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(4.6±3.5%) were not significantly different (p = 0,80), using a Mann Whitney U test
217
in SPSS (Figure 2: PBMC culture IFN-γ secretion).
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3.2 ELISA
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The mean elastin specific antibody amount of the AAA patient group and the control
221
group was assayed. Both results were expressed as a relative percentage with a level
222
of zero percent corresponding to the antibody concentration of the negative control
223
wells. A level of hundred percent corresponded to the antibody concentration of the
224
positive control. The mean elastin specific antibody level of the control group
225
(77,5±17,8%) and the AAA patient group (78.2±31.5%) were not significantly
226
different (p = 0,43), using a Mann Whitney U test in SPSS (Figure 3: anti-elastin IgG
227
titer).
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ACCEPTED MANUSCRIPT 228 4. Discussion
230
Based on the results of our pilot study, the validity of the proposed model could not
231
be shown. There was no indication for an invariable and specific autoimmune
232
response against elastin peptides in atherosclerotic AAA. However, we used a small
233
number of participants and we only tested a specific part of the model in our pilot
234
study. Repeating the pilot study in larger patient groups with other inclusion criteria
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and/or using tissue samples instead of peripheral blood and other candidate peptides is
236
necessary before being able to (partially) accept or reject the proposed model.
237
The clinical relevance of this study was not only to identify a specific antigen as a
238
possible therapeutic target, but also to propose a usable model itself. Indeed, proving
239
the (partial) validity of the model offers other therapeutic targets to slow or stop the
240
autoimmune response by intervening with the various mechanisms of the model [9].
241
The model places atherosclerosis and AAA as points on a continuous spectrum.
242
However, we do not state a one-to-one relationship between atherosclerosis and AAA
243
[20]. We hypothesized that atherosclerosis is the origin of the aspecific chronic
244
inflammatory reaction that is necessary to cause AAA initiation via the creation of
245
remnant peptide fragments. It is possible that atherosclerosis stimulates a pro-
246
inflammatory cytokine activating protein complex like the inflammasome [21].
247
Inflammasome activation could result in AAA initiation [22, 23] because the delicate
248
balance between pro- and anti-inflammatory cytokines would change. The disruption
249
of this balance is necessary in our model to initiate the vicious process of structural
250
vessel wall peptide degradation. Indeed, pro-inflammatory cytokines such as
251
interleukin-1β and interferon-gamma are also key elements of the REGA model [9].
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All of this can only happen if the necessary genetic and environmental factors are
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ACCEPTED MANUSCRIPT present. Therefore, it would be possible to have severe atherosclerotic lesions without
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the occurrence of AAA initiation. It has been shown that there is an altered expression
255
pattern of MHCII alleles in AAA compared with controls [14, 24]. It has also been
256
shown that in AAA there is an imbalance between proteases such as matrix
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metalloproteinase and their inhibitors, favoring activation of these proteases [12].
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Further research is necessary to further elucidate all of the genetic and environmental
259
factors necessary for AAA initiation and progression.
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5. Conclusion
262
The results of our pilot study did not indicate a clear and invariable autoimmune
263
process directed against remnant elastin peptide fragments. However, the goal of the
264
study was not only to find a specific antigen but also to propose a pathophysiological
265
AAA model. Further research into the model mechanics and a possible antigen is still
266
necessary. In the mean time, the model as presented here already offers a
267
pathophysiological framework to further research into the possible remnant epitope
268
driven AAA etiology.
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6. Acknowledgement
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This study was funded from our own lab resources. We would like to thank our
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student colleagues for their assistance with the sample processing: Leontine Grozema,
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Sofie Ordies, Isabelle Sreeram, Laurens Van Melkebeke and Lucas Van Hoof.
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7. Conflicts of interest
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None
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8. Funding
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This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors.
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Inflammasome activation by mitochondrial oxidative stress in macrophages leads to
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the development of angiotensin II-induced aortic aneurysm. Arterioscler Thromb
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Vasc Biol. 2015 Jan;35(1):127-36. http://dx.doi.org/10.1161/ATVBAHA.114.303763.
ACCEPTED MANUSCRIPT 377 [23] Warnatsch A, Ioannou M, Wang Q, Papayannopoulos V. Inflammation.
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Neutrophil extracellular traps license macrophages for cytokine production in
380
atherosclerosis. Science. 2015 Jul 17;349(6245):316-20.
381
http://dx.doi.org/10.1126/science.aaa8064.
382
RI PT
378
[24] Rasmussen TE, Hallett JW Jr, Tazelaar HD, Miller VM, Schulte S, O'Fallon WM
384
et al. Human leukocyte antigen class II immune response genes, female gender, and
385
cigarette smoking as risk and modulating factors in abdominal aortic aneurysms. J
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Vasc Surg. 2002 May;35(5):988-93.
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Controls
Sex
Male
Number (n=)
14
8
Mean age (years)
70,8 ± 6,5
29,4 ± 3,6
Pack-years (years)
49,5 ± 17,19
0
Aneurysm diameter (mm)
42,3 ± 8,84
% with hypertension (%)
90
% with hypercholesterolemia (%)
91
RI PT
Parameter
/
0 0
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SC
Table I: study group demographics
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