| 1. |
- Abu Hamdeh, Sami, et al.
(författare)
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Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.
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| 2. |
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| 3. |
- Almandoz-Gil, Leire, et al.
(författare)
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Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways
- 2017
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Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 110, s. 421-431
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Tidskriftsartikel (refereegranskat)abstract
- Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.
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| 4. |
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| 5. |
- Emami Khoonsari, Payam, et al.
(författare)
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Chitinase-3-like protein 1 (CH3L1) and Neurosecretory protein VGF (VGF) as two novel CSF biomarker candidates for improved diagnostics in Alzheimer’s disease
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by amyloid-β (Aβ) plaque deposition and accumulation of intracellular neurofibrillary tangles. This pathology is mirrored in the cerebrospinal fluid (CSF), where decreased Aβ42 together with increased total (t-tau) and phospho-tau (p-tau) today is used as a diagnostic marker. Although these biomarkers have a fairly good sensitivity and specificity, additional biomarkers are needed to further improve the accuracy for early disease detection and to monitor disease development. In this study, we used mass spectrometry-based shotgun proteomics to investigate the CSF proteome of patients with AD and mild cognitive impairment (MCI) as well as of non-demented controls. By combining the diagnostic markers (Aβ42, total t-tau, and p-tau) with a selection of proteomics biomarkers, the accuracy of predicting MCI to AD conversion increased from 83% to 92% with a specificity of 1.0 and sensitivity of 0.86. Among these markers, the levels of protein chitinase-3-like protein 1 (CH3L1) were significantly higher in AD and MCI converters compared to controls. In addition to Aβ42, t-tau, and p-tau the protein CH3L1 contributed mostly to the prediction accuracy. We also found statistically significant lower CSF levels of the neurosecretory protein VGF (VGF) in AD compared to controls. Taken together, our findings suggest that incorporating new CSF biomarkers can further enhance early diagnosis of AD.
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| 6. |
- Karademir, Betul, et al.
(författare)
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Proteomic approach for understanding milder neurotoxicity of Carfilzomib against Bortezomib
- 2018
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The proteasomal system is responsible for the turnover of damaged proteins. Because of its important functions in oncogenesis, inhibiting the proteasomal system is a promising therapeutic approach for cancer treatment. Bortezomib (BTZ) is the first proteasome inhibitor approved by FDA for clinical applications. However neuropathic side effects are dose limiting for BTZ as many other chemotherapeutic agents. Therefore second-generation proteasome inhibitors have been developed including carfilzomib (CFZ). Aim of the present work was investigating the mechanisms of peripheral neuropathy triggered by the proteasome inhibitor BTZ and comparing the pathways affected by BTZ and CFZ, respectively. Neural stem cells, isolated from the cortex of E14 mouse embryos, were treated with BTZ and CFZ and mass spectrometry was used to compare the global protein pool of treated cells. BTZ was shown to cause more severe cytoskeletal damage, which is crucial in neural cell integrity. Excessive protein carbonylation and actin filament destabilization were also detected following BTZ treatment that was lower following CFZ treatment. Our data on cytoskeletal proteins, chaperone system, and protein oxidation may explain the milder neurotoxic effects of CFZ in clinical applications.
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| 7. |
- Musunuri, Sravani
(författare)
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Mass Spectrometry-based Neuroproteomics : Deciphering the Human Brain Proteome
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mammalian brain is challenging to study due to its heterogeneity and complexity. However, recent advances in molecular imaging, genomics and proteomics have contributed significantly to achieve insights into molecular basis of brain function and pathogenesis of neurological disorders. Efficient sample preparation is an integral part of a successful mass spectrometry (MS)-based proteomics. Apart from the identification, quantification of proteins is needed to investigate the alterations between proteome profiles from different sample sets. Therefore, this thesis investigates optimizing and application of the MS compatible sample preparation techniques for the identification and quantification of proteins from brain tissue.The central objective of this thesis was (i) to improve the extraction of proteins as well as membrane proteins (MPs) from the brain tissue and (ii) to apply the optimized method along with the stable isotope dimethyl labeling (DML) and label-free (LF) MS approaches for the relative quantification of the brain proteome profiles during neurological conditions such as Alzheimer’s disease (AD) and traumatic brain injury (TBI). First study described in this thesis is focused on the qualitative aspects for the brain tissue sample preparation. The optimized extraction buffers from first study containing n-octyl-β-glucopyranside or triton X-114 were used in the further quantitative studies to extract the proteins from patient (AD or TBI) and control human brain samples. Triton X-114 has additional advantage of separating MPs into a micellar phase. Therefore we also investigated the possibility to apply this in combination with DML quantitation approach for enrichment of low abundant MPs from AD brains.AD and TBI causes severe socio-economic burden on the society and therefore there is a need to develop diagnostic markers to detect the early changes in the pathology of the disease. Analytical tools and techniques applied and discussed in this thesis for neuroproteomics applications proved to be powerful and reliable for analyzing complex biological samples to generate high-throughput screening and unbiased identification and quantitation of disease-specific proteins that are of great importance in understanding the disease pathology.
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| 8. |
- Musunuri, Sravani, et al.
(författare)
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Micellar extraction possesses a new advantage for the analysis of Alzheimer's disease brain proteome
- 2015
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 407:4, s. 1041-1057
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Tidskriftsartikel (refereegranskat)abstract
- Integral membrane proteins (MPs), such as transporters, receptors, and ion channels, are of great interest because of their participation in various vital cellular functions including cell-cell interactions, ion transport, and signal transduction. However, studies of MPs are complicated because of their hydrophobic nature, heterogeneity, and low abundance. Cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was performed to simultaneously extract and phase separate hydrophobic and hydrophilic proteins from Alzheimer's disease (AD) and unaffected control brain tissue. Quantitative proteomics analysis of temporal neocortex samples of AD patients and controls was performed using a shotgun approach based on stable isotope dimethyl labeling (DML) quantification technique followed by nanoLC-MS/MS analysis. A total of 1096 unique proteins were identified and quantified, with 40.3 % (211/524) predicted as integral MPs with at least one transmembrane domain (TMD) found in the detergent phase, and 10 % (80/798) in the detergent-depleted phase. Among these, 62 proteins were shown to be significantly altered (p-value < 0.05), in AD versus control samples. In the detergent fraction, we found 10 hydrophobic transmembrane proteins containing up to 14 putative TMDs that were significantly up- or down-regulated in AD compared with control brains. Changes in four of these proteins, alpha-enolase (ENOA), lysosome-associated membrane glycoprotein 1 (LAMP1), 14-3-3 protein gamma (1433G), and sarcoplasmic/endoplasmic reticulum calcium ATPase2 (AT2A2) were validated by immunoblotting. Our results emphasize that separating hydrophobic MPs in CPE contributes to an increased understanding of the underlying molecular mechanisms in AD. Such knowledge can become useful for the development of novel disease biomarkers.
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| 9. |
- Musunuri, Sravani, et al.
(författare)
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Neuroproteomic profiling of human brain tissue using multidimensional separation techniques and selective enrichment of membrane proteins
- 2012
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 33:24, s. 3779-3785
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Tidskriftsartikel (refereegranskat)abstract
- Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature-induced phase fractionation with the nonionic surfactant Triton X-114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two-dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In-solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano-LC and analyzed using two different high performance mass spectrometers, linear ion trap-Fourier transform and a linear ion trap-Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap-Fourier transform and linear ion trap-Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.
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| 10. |
- Musunuri, Sravani, et al.
(författare)
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Quantification of the Brain Proteome in Alzheimer's Disease Using Multiplexed Mass Spectrometry
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:4, s. 2056-2068
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Tidskriftsartikel (refereegranskat)abstract
- We have compared the brain proteome in the temporal neocortex between Alzheimer's disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.
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| 11. |
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| 12. |
- Shevchenko, Ganna, et al.
(författare)
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Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:4, s. 2441-2451
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Tidskriftsartikel (refereegranskat)abstract
- This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ: FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-beta-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.
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| 13. |
- Shevchenko, Ganna, et al.
(författare)
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Neuroproteomics Tools in Clinical Practice
- 2015
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1854:7, s. 705-717
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Tidskriftsartikel (refereegranskat)abstract
- Abstract Neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases.11 This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.
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| 14. |
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| 15. |
- Sravani, Musunuri, 1987-, et al.
(författare)
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Increased Levels of Extracellular Microvesicle Markers and Decreased Levels of Endocytic/Exocytic Proteins in the Alzheimer's Disease Brain
- 2016
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Ingår i: Journal of Alzheimer's Disease. - : IOS Press. - 1387-2877 .- 1875-8908. ; 54:4, s. 1671-1686
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Tidskriftsartikel (refereegranskat)abstract
- Background: Alzheimer's disease (AD) is a chronic neurodegenerative disorder accounting for more than 50% of all dementia cases. AD neuropathology is characterized by the formation of extracellular plaques and intracellular neurofibrillary tangles consisting of aggregated amyloid-beta and tau, respectively. The disease mechanism has only been partially elucidated and is believed to also involve many other proteins. Objective: This study intended to perform a proteomic profiling of post mortem AD brains and compare it with control brains as well as brains from other neurological diseases to gain insight into the disease pathology. Methods: Here we used label-free shotgun mass spectrometry to analyze temporal neocortex samples from AD, other neurological disorders, and non-demented controls, in order to identify additional proteins that are altered in AD. The mass spectrometry results were verified by antibody suspension bead arrays. Results: We found 50 proteins with altered levels between AD and control brains. The majority of these proteins were found at lower levels in AD. Pathway analyses revealed that several of the decreased proteins play a role in exocytic and endocytic pathways, whereas several of the increased proteins are related to extracellular vesicles. Using antibody-based analysis, we verified the mass spectrometry results for five representative proteins from this group of proteins (CD9, HSP72, PI42A, TALDO, and VAMP2) and GFAP, a marker for neuroinflammation. Conclusions: Several proteins involved in exo-endocytic pathways and extracellular vesicle functions display altered levels in the AD brain. We hypothesize that such changes may result in disturbed cellular clearance and a perturbed cell-to-cell communication that may contribute to neuronal dysfunction and cell death in AD.
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| 16. |
- Warnecke, Andreas, et al.
(författare)
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Nitration of MOG diminishes its encephalitogenicity depending on MHC haplotype
- 2017
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Ingår i: Journal of Neuroimmunology. - : Elsevier BV. - 0165-5728 .- 1872-8421. ; 303, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- Post-translational modifications of autoantigens are hypothesized to affect their immunogenicity. We here report that nitration of tyrosine 40 in Myelin Oligodendrocyte Glycoprotein (MOG) abrogates its encephalitogenicity both at protein and peptide levels in the experimental autoimmune encephalomyelitis (EAE) model in H2(b) C57BL/6 mice. Furthermore, nitrated MOG displays inferior antigen-specific proliferation of 2D2 splenocytes in vitro. Conversely, H2(q) DBA1 mice remain fully susceptible to EAE induction using nitrated MOG as the dominant epitope of H2q mice is unaltered. Molecular modeling analysis of the MOG(35-55)/H2-lA(b) complex and bioinformatics peptide binding predictions indicate that the lack of T cell reactivity towards nitrated MOG can be attributed to the inability of murine H2-IA(b) to efficiently present the altered peptide ligand of MOG(35-55) because the nitrated tyrosine 40 cannot be accommodated in the p1 anchor pocket. In conclusion we demonstrate nitration as a relevant determinant affecting T cell recognition of carrier antigen depending on MHC haplotype. Our data have implications for understanding the role of post-translationally modified antigen in autoimmunity. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.
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| 17. |
- Warnecke, Andreas, et al.
(författare)
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Scavenger Receptor A Mediates the Clearance and Immunological Screening of MDA-Modified Antigen by M2-Type Macrophages
- 2017
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Ingår i: Neuromolecular medicine. - : Springer Science and Business Media LLC. - 1535-1084 .- 1559-1174. ; 19:4, s. 463-479
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we investigated the uptake of malondialdehyde (MDA)-modified myelin oligodendrocyte glycoprotein (MOG) in the context of lipid peroxidation and its implications in CNS autoimmunity. The use of custom-produced fluorescently labeled versions of MOG or MDA-modified MOG enabled us to study and quantify the uptake by different macrophage populations and to identify the responsible receptor, namely SRA. The SRA-mediated uptake of MDA-modified MOG is roughly tenfold more efficient compared to that of the native form. Notably, this uptake is most strongly associated with anti-inflammatory M2-type macrophages. MDA-modified MOG was demonstrated to be resistant to degradation by lysine-dependent proteases in vitro, but the overall digestion fragments appeared to be similar in cell lysates, although their relative abundance appeared to be altered as a result of faster uptake. Accordingly, MDA-modified MOG is processed for presentation by APCs, allowing maximized recall proliferation of MOG(35-55)-specific 2D2 T cells in vitro due to higher uptake. However, MDA modification of MOG did not enhance immune priming or disease course in the in vivo MOG-EAE model, but did induce antibody responses to both MOG and MDA adducts. Taken together our results indicate that MDA adducts primarily constitute clearance signals for phagocytes and promote rapid removal of antigen, which is subjected to immunological screening by previously licensed T cells.
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