{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T19:17:50Z","timestamp":1787771870321,"version":"build-2784847793"},"reference-count":40,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2025,7,25]],"date-time":"2025-07-25T00:00:00Z","timestamp":1753401600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Research Council of Norway","award":["288164"],"award-info":[{"award-number":["288164"]}]},{"name":"Gerda Meyer Nyquist Gulbranson &amp; Gerdt Meyer Nyquist legacy","award":["288164"],"award-info":[{"award-number":["288164"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The effective suppression of inflammation using disease-modifying therapies is essential in the treatment of multiple sclerosis (MS). Anti-CD20 monoclonal antibodies are commonly used long-term as maintenance therapies, largely due to the lack of reliable biomarkers to guide dosing and evaluate treatment response. However, prolonged use increases the risk of infections and other immune-mediated side effects. The unique ability of brain-derived blood extracellular vesicles (EVs) to cross the blood\u2013brain barrier and reflect the central nervous system (CNS) immune status has sparked interest in their potential as biomarkers. This study aimed to assess whether blood-derived L1CAM+ EVs could serve as biomarkers of treatment response to rituximab (RTX) in patients with relapsing-remitting MS (RRMS). Serum samples (n = 25) from the baseline (month 0) and after 6 months were analyzed from the RTX arm of the ongoing randomized clinical trial OVERLORD-MS (comparing anti-CD20 therapies in RRMS patients) and were compared with serum samples from healthy controls (n = 15). Baseline cerebrospinal fluid (CSF) samples from the same study cohort were also included. EVs from both serum and CSF samples were characterized, considering morphology, size, and concentration, using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The immunophenotyping of EV surface receptors was performed using flow cytometry with the MACSPlex exosome kit, while label-free quantitative proteomics of EV protein cargo was conducted using a proximity extension assay (PEA). TEM confirmed the presence of EVs with the expected round morphology with a diameter of 50\u2013150 nm. NTA showed significantly higher concentrations of L1CAM+ EVs (p &lt; 0.0001) in serum total EVs and EBNA1+ EVs (p &lt; 0.01) in serum L1CAM+ EVs at baseline (untreated) compared to in healthy controls. After six months of RTX therapy, there was a significant reduction in L1CAM+ EV concentration (p &lt; 0.0001) and the downregulation of TNFRSF13B (p = 0.0004; FC = \u22120.49) in serum total EVs. Additionally, non-significant changes were observed in CD79B and CCL2 levels in serum L1CAM+ EVs at baseline compared to in controls and after six months of RTX therapy. In conclusion, L1CAM+ EVs in serum showed distinct immunological profiles before and after rituximab treatment, underscoring their potential as dynamic biomarkers for individualized anti-CD20 therapy in MS.<\/jats:p>","DOI":"10.3390\/ijms26157213","type":"journal-article","created":{"date-parts":[[2025,7,25]],"date-time":"2025-07-25T12:56:43Z","timestamp":1753448203000},"page":"7213","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["In-Depth Characterization of L1CAM+ Extracellular Vesicles as Potential Biomarkers for Anti-CD20 Therapy Response in Relapsing\u2013Remitting Multiple Sclerosis"],"prefix":"10.3390","volume":"26","author":[{"given":"Shamundeeswari","family":"Anandan","sequence":"first","affiliation":[{"name":"Department of Clinical Medicine, University of Bergen, 5021 Bergen, Norway"},{"name":"Neuro-SysMed, Department of Neurology, Haukeland University Hospital, 5021 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3098-1113","authenticated-orcid":false,"given":"Karina","family":"Maciak","sequence":"additional","affiliation":[{"name":"Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, 90-136 Lodz, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7172-9443","authenticated-orcid":false,"given":"Regina","family":"Breinbauer","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, Friedrich-Alexander-University Erlangen-Nuremberg (FAU), 91054 Erlangen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8765-6341","authenticated-orcid":false,"given":"Laura","family":"Otero-Ortega","sequence":"additional","affiliation":[{"name":"Neurology and Cerebrovascular Diseases Group, Neurology Department, Neurosciences Area, La Paz Hospital Institute for Health Research, 28046 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giancarlo","family":"Feliciello","sequence":"additional","affiliation":[{"name":"Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM-R), Personalized Tumor Therapy, 93053 Regensburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nata\u0161a","family":"Stojanovi\u0107 Gu\u017evi\u0107","sequence":"additional","affiliation":[{"name":"Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM-R), Personalized Tumor Therapy, 93053 Regensburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5294-2866","authenticated-orcid":false,"given":"Oivind","family":"Torkildsen","sequence":"additional","affiliation":[{"name":"Department of Clinical Medicine, University of Bergen, 5021 Bergen, Norway"},{"name":"Neuro-SysMed, Department of Neurology, Haukeland University Hospital, 5021 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0980-510X","authenticated-orcid":false,"given":"Kjell-Morten","family":"Myhr","sequence":"additional","affiliation":[{"name":"Department of Clinical Medicine, University of Bergen, 5021 Bergen, Norway"},{"name":"Neuro-SysMed, Department of Neurology, Haukeland University Hospital, 5021 Bergen, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1816","DOI":"10.1177\/1352458520970841","article-title":"Rising prevalence of multiple sclerosis worldwide: Insights from the Atlas of MS, third edition","volume":"26","author":"Walton","year":"2020","journal-title":"Mult. 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