Advancing personalised medicine in multiple sclerosis: Exploring biomarkers and point-of-care testing

Multiple sclerosis (MS) is a chronic, immune-mediated disorder of the central nervous system characterized by inflammation, demyelination, and neurodegeneration. Despite major advances in disease-modifying therapies (DMTs), early diagnosis and reliable monitoring remain challenging, as current approaches are often invasive, expensive, time-consuming, and sometimes insufficiently sensitive. This thesis investigated molecular, functional, and technological strategies to improve diagnosis and monitoring in MS, with a particular focus on saliva as a non-invasive biofluid that offers faster and more cost-effective biomarker detection. The principal aim was to evaluate neuronal and immunological biomarkers in saliva, with neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and κ free light chains (κFLC) as primary targets. To the best of our knowledge, this is the first study to successfully measure both NfL and κFLC in saliva of people with MS (pwMS) simultaneously, demonstrating detectable levels that differentiated pwMS from healthy controls. Receiver operating characteristic (ROC) curve analysis revealed moderate diagnostic accuracy for κFLC alone, and improved performance when combined with GFAP, NfL, or λFLC. Furthermore, normalization strategies inspired by the CSF κFLC index were applied for the first time in saliva, enhancing diagnostic evaluation. Collectively, these findings establish saliva as a feasible, non-invasive source of both neuronal (NfL) and immunological (κFLC) biomarkers. A second innovative aspect of this thesis was the evaluation of liver enzymes (AST and ALT) in saliva of pwMS. While serum AST was elevated in relapsing-remitting MS (RRMS), salivary measurements did not yield clear diagnostic value. Nonetheless, this proof-of-concept study introduces a novel avenue for non-invasive monitoring of treatment-related hepatotoxicity. In parallel, this thesis presents the first investigation of endoplasmic reticulum (ER) stress-related gene expression in peripheral blood mononuclear cells (PBMCs) of pwMS. Significant downregulation of PERK, XBP1, GRP94, and calreticulin was observed compared to healthy controls, while BiP, CHOP, and ATF6 remained unchanged. These results contrast with findings from experimental autoimmune encephalomyelitis (EAE) models and other autoimmune diseases, suggesting compartment- and context-specific regulation of ER stress in MS. Functional markers were also explored using the University of Pennsylvania Smell Identification Test (UPSIT) in two independent cohorts. Olfactory impairment was frequent among pwMS and associated with cognitive deficits, depressive symptoms, and motor dysfunction, underscoring its potential as a non-invasive functional biomarker. Finally, a wax-printed, paper-based ELISA (p-ELISA) platform was designed and optimized for multiplex biomarker detection, with salivary GFAP employed as a model analyte. Incorporating magnetic bead-based capture and smartphone-based readout, the assay enabled sensitive detection within one hour, offering a rapid and resource-efficient point-of-care tool. In summary, this thesis demonstrates the feasibility of saliva as a clinically relevant biofluid for biomarker measurement in MS, while introducing novel approaches including salivary NfL quantification, salivary liver enzyme assessment, and ER stress gene profiling in PBMCs. By integrating molecular, functional, and technological perspectives, this work advances the development of non-invasive strategies to improve patient stratification and disease management in MS.

Funder

Research Ireland

Publisher

University of Galway

Rights

CC BY-NC-ND

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