Edaravone (EDA), an antioxidant drug approved for the treatment of ischemic stroke and amyotrophic lateral sclerosis, was recently proposed as a remyelinating candidate for the treatment of multiple sclerosis. Here, we synthesized twelve EDA analogues 2b–4c showing three substitution patterns A–C, searching for improved remyelinating agents and putative molecular targets responsible for their regenerative activity. We profiled them in three primary assays to determine their stimulation of oligodendrocyte progenitor cell metabolism (tetrazolium MTT assay), their antioxidant potential (2,2-diphenyl-1-picrylhydrazyl-DPPH assay) and to predict their bioavailability (virtual ADME profile). Active 4′-carboxylate 2b, 4′-ester 2c and N¹-carbamate-4′-ester 4a were further characterized, justifying their in vitro effects and selecting 4a as a putative EDA 1 prodrug suitable for in vivo testing.

Objective: Multiple sclerosis (MS) is a chronic autoimmune disease where B cells play a central pathogenic role. Cladribine, an oral therapy, provides durable benefits by reshaping lymphocyte populations, yet its specific long‐term impact on distinct B‐cell subsets is not fully understood. This study aimed to define cladribine's longitudinal effects on B‐cell subsets and characterize their inflammatory properties. Methods: We conducted a 48‐month longitudinal study of 36 persons with relapsing–remitting MS treated with cladribine who had completed 2 annual treatment cycles, using high‐parameter flow cytometry to track B‐cell subset dynamics. In a parallel cross‐sectional analysis, B cells from 16 untreated patients and 16 healthy donors were profiled for polyfunctional cytokine production granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α) and key surface markers. Results: Cladribine induced profound, sustained depletion of memory B cells, with marginal zone‐like (MZ‐like) cells showing the deepest and most persistent loss, alongside reconstitution of transitional and naïve populations. Functionally, MZ‐like B cells were the most potent polyfunctional producers of proinflammatory cytokines and immunoglobulin M (IgM), a capacity significantly enhanced in persons with MS compared to healthy donors, and expressed the highest levels of CD1c and CD1d, consistent with specialization in lipid antigen presentation. Interpretation: MZ‐like B cells are a dominant, polyfunctional inflammatory subset in MS, equipped for non‐canonical lipid antigen presentation and IgM secretion. They represent a preferential pharmacological target of cladribine. Sustained reduction of this pathogenic memory pool, followed by repopulation with less inflammatory B cells, offers a mechanistic basis for cladribine's durable clinical benefit and nominates MZ‐like cells as biomarkers of response and therapeutic targets. ANN NEUROL 2026

Innate immune responses depend on diverse cell subsets whose frequencies and functions can shift rapidly during infection or inflammation. We developed a high-parameter flow cytometry platform that uses <1 mL of fresh whole blood to identify multiple circulating innate cell subsets and to measure, in each subset, intracellular cytokines and chemokines after stimulation with lipopolysaccharide (LPS; bacterial mimic) or R848 (resiquimod; viral mimic). The platform operates on less than 1 mL of fresh whole blood, preserving mononuclear phagocytes and avoiding density-gradient cell separation, with phenotypic and functional readouts integrated into a single acquisition. Because several canonical lineage markers, most notably CD16, are rapidly downmodulated upon activation, the gating strategy relies instead on markers whose expression remains stable under stimulation, allowing nonclassical monocytes and other subsets to be tracked reliably across resting and activated conditions. Combining lineage exclusion with these stable markers, we resolve plasmacytoid and conventional dendritic cells, classical, intermediate, and nonclassical monocytes, and additional minor subsets including inflammatory dendritic cells. LPS elicits broad TNF, IL-6, and IL-1β responses with subset-specific magnitudes, whereas R848 preferentially induces type I interferon programs alongside pro-inflammatory cytokines in selected lineages. The same gating framework was further validated in PBMCs challenged with intact UV-inactivated bacterial and fungal preparations, demonstrating portability across sample matrices. This compact, scalable assay enables precise monitoring of both subset composition and effector functions from scarce clinical samples, supporting translational studies in infection, autoimmunity, and immunotherapy where blood volume and turnaround time are limited.