Roberto De Masi

Background/Objectives: Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS characterized by immune-mediated inflammation and neurodegeneration. Based on its immunoablative effect targeting the CD20-positive B lymphocytes, ocrelizumab has emerged as a valid therapeutic option for MS. Although the on-treatment dynamics of leukocyte subpopulations are crucial for optimizing therapeutic strategies, little data are available in this field. Here, we aim to investigate how pre- and post-infusion immune-cell subsets change and correlate with each other, and how CD19+ B cell repopulation evolves over time. Methods: In an observational, retrospective, and prospective real-world study, we studied 51 relapsing-remitting MS ocrelizumab-treated patients who underwent flow cytometric immunophenotyping before and after each infusion and at three-month intervals, up to the eighth (4 years), for a total of 408 cycles. Results: After an initial decrease, post-infusion lymphocyte count returned to normal, while total white blood cells showed a sawtooth pattern driven by post-infusion increases. Granulocytes and natural killer cell counts remained substantially stable, while monocytes showed a significant cyclic pattern: increasing post-infusion, decreasing at the intermediate time point, with a more pronounced reduction pre-infusion, and rising again after re-administration. CD3+ and CD4+ T cells exhibited the same cyclic sawtooth pattern observed for monocytes. The CD4+/CD8+ ratio progressively increased, reflecting a significant elevation of CD4+, CD3+ and, to a lesser extent, CD8+ T cells. Pre-infusion CD19+ B cell counts progressively decreased, approaching zero by the fourth treatment year. Pre-treatment CD3+, CD4+, CD8+, lymphocyte, monocyte, and granulocyte counts differed significantly from on-treatment values. Correlation analyses showed that the major relationships among leukocyte subsets remained preserved throughout treatment. Conclusions: This study details leukocyte subpopulation dynamics during ocrelizumab therapy, unveiling previously unrecognized immunomodulatory properties beyond B-cell depletion. Ocrelizumab predominantly affects adaptive immunity, with potential indirect effects on innate immune compartments, and is associated with changes across immune-cell populations. These findings could have implications for individualized treatment strategies, but their direct application in this field still requires confirmatory studies on immunophenotyping and peripheral B-cell repopulation dynamics.

Background and Objectives: Ocrelizumab significantly reduces inflammatory activity in relapsing-remitting multiple sclerosis (RRMS), but treatment-induced brain volume change and the specific contributions of white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF) compartments to global atrophy and pseudoatrophy remain unclear. We aim to characterize the longitudinal and infusion-related dynamics of brain compartments in ocrelizumab-treated RRMS patients and identify the clinical and time-dependent predictors of these changes. Methods: Fifty-one RRMS patients were enrolled in a four-year prospective study. Brain volumes, including WM, GM, peripheral GM (pGRAY), CSF, ventricular CSF (vCSF), total brain volume (TBV) and their respective fractions (WMF, GMF, BPF) were evaluated by absolute time points (baseline to 4 years) and infusion-based intervals (baseline to 8th infusion), before and after each ocrelizumab cycle. Correlations between brain volume measures and the Expanded Disability Status Scale (EDSS), as well as time-dependent variables such as age, age at onset and disease duration (DD) were examined. Results: Mean age was 41.62 ± 9.76 years, mean age at onset 28.17 ± 7.85, mean DD 12.89 ± 8.55 years and mean EDSS 3.26 ± 1.5, indicating moderate disability. During the study, vCSF and CSF significantly increased, whereas WM, WMF, TBV and BPF significantly decreased. Notably, these measures exhibited marked, non-linear, and transient changes during the first year of ocrelizumab treatment, consistent with pseudoatrophy, likely reflecting early resolution of inflammation rather than irreversible tissue loss. GM and pGRAY remained relatively stable, with minor early increases. Correlation analyses revealed that higher EDSS scores, older age, later age at onset and longer DD were associated with lower GM, pGRAY and TBV, emphasizing the interplay between disease progression and development of brain volumetric patterns. Conclusions: Ocrelizumab-induced pseudoatrophy is predominantly WM-driven and age-dependent, with WM shrinkage being the main contributor to parenchymal loss and secondary widening of CSF spaces. Age remains a powerful predictor of brain atrophy and neurological impairment. These findings provide mechanistic insights into the biological basis of this modulation in RRMS.

Background: The role of age and time-dependent variables in determining the response to disease-modifying therapies (DMTs) has aroused growing interest in the Multiple Sclerosis (MS) field. Although it is a very hot topic, related literature on the subject is considerably lacking. Objectives: The aim of this study is to deepen the understanding of how time-dependent variables influence disability accumulation and drug response in an MS population, assuming DMF as the first-line treatment, and to expand our knowledge of the risk–benefit evaluation of DMF. Methods: We investigated, in a real-world setting, the efficacy of Dimethyl Fumarate (DMF) in naive versus switcher MS patients, correlated with age, in preventing disability accumulation. Starting from an initial population of 234 DMF-treated patients, we selected 169 of them based on their similar time in therapy (TinT) with DMF of 5.9 ± 2.3 year and sex ratio. Of these, 74 were naive and 95 were lateral switchers at the start of treatment. The mean Expanded Disability Status Scale (EDSS), Disease Duration (DD), age and age at onset were compared between groups. Results: The switcher group showed higher EDSS and age compared to the naive group (2.7 vs. 1.8, p < 0.001; 40.2 vs. 35.5, p = 0.005, respectively). Age correlated with DD, EDSS and age at onset in both naive (r = 0.39, p = 0.007; r = 0.53, p = 0.000; r = 0.63, p = 0.000, respectively) and switcher (r = 0.46, p = 0.002; r = 0.49, p = 0.000; r = 0.61, p = 0.000, respectively) groups. Kaplan–Meier curves, adjusted for age, also indicated that the naive group retained an EDSS score status of 0.5–3.5 more frequently (p < 0.001) and reached elevated disability less frequently (p = 0.002) than switchers. The mean EDSS percentage ratio between paired naive and switcher patients, representing the differential neurological impairment (DNI), was 69%, inversely correlating with age in both naive (r = −0.52, p < 0.001) and switcher patients (r = −0.47, p < 0.001). Finally, logistic regression analysis indicated age as an independent and predictive variable with respect to EDSS. Conclusions: We conclude that age is the main contributor to disability progression and the primary predictive factor for treatment effectiveness for DMF in both naive and switcher MS patients.

Growing evidence identifies extracellular vesicles (EVs) as important cell-to-cell signal transducers in autoimmune disorders, including multiple sclerosis (MS). If the etiology of MS still remains unknown, its molecular physiology has been well studied, indicating peripheral blood mononuclear cells (PBMCs) as the main pathologically relevant contributors to the disease and to neuroinflammation. Recently, several studies have suggested the involvement of EVs as key mediators of neuroimmune crosstalk in central nervous system (CNS) autoimmunity. To assess the role of EVs in MS, we applied electron microscopy (EM) techniques and Western blot analysis to study the morphology and content of plasma-derived EVs as well as the ultrastructure of PBMCs, considering four MS patients and four healthy controls. Through its exploratory nature, our study was able to detect significant differences between groups. Pseudopods and large vesicles were more numerous at the plasmalemma interface of cases, as were endoplasmic vesicles, resulting in an activated aspect of the PBMCs. Moreover, PBMCs from MS patients also showed an increased number of multivesicular bodies within the cytoplasm and amorphous material around the vesicles. In addition, we observed a high number of plasma-membrane-covered extensions, with multiple associated large vesicles and numerous autophagosomal vacuoles containing undigested cytoplasmic material. Finally, the study of EV cargo evidenced a number of dysregulated molecules in MS patients, including GANAB, IFI35, Cortactin, Septin 2, Cofilin 1, and ARHGDIA, that serve as inflammatory signals in a context of altered vesicular dynamics. We concluded that EM coupled with Western blot analysis applied to PBMCs and vesiculation can enhance our knowledge in the physiopathology of MS.

The etiology of Multiple Sclerosis (MS) remains undetermined. Its pathogenic risk factors are thought to play a negligible role individually in the development of the disease, instead assuming a pathogenic role when they interact with each other. Unfortunately, the statistical weighting of this pathogenic role in predicting MS risk is currently elusive, preventing clinical and health insurance applications. Here, we aim to develop a population-based multi-criterial model for weighting biological risk factors in MS; also, to calculate the individual MS risk value useful for health insurance application. Accordingly, among 596 MS patients retrospectively assessed at the time of diagnosis, the value of vitamin D < 10 nm/L, BMI (Body Mass Index) < 15 Kg/m² and >30 Kg/m², female sex, degree of family kinship, and the range of age at onset of 20–45 years were considered as biological risk factors for MS. As a result, in a 30-year-old representative patient having a BMI of 15 and second degree of family kinship for MS, the major developmental contributor for disease is the low vitamin D serum level of 10 nm/L, resulting in an MS risk of 0.110 and 0.106 for female and male, respectively. Furthermore, the Choquet integral applied to uncertain variables, such as biological risk factors, evidenced the family kinship as the main contributor, especially if coincident with the others, to the MS risk. This model allows, for the first time, for the risk stratification of getting sick and the application of the health insurance in people at risk for MS.

We report the singular case of a 31-year-old woman who developed very serious Fluphenazine-induced parkinsonism over a few days due to a doubly incongruent drug prescription by indication and dosage having been applied to a healthy subject over one week instead of seven months. Unlike gradual drug-induced parkinsonism, our patient experienced acute extrapyramidal syndrome (EPS), reaching significant motor and sphincter disability in just a few days, followed by a gradual incomplete recovery over more than six months. In fact, after drug discontinuation, hypomimia and slight left hemi-somatic rigidity with bradykinesia remained, as well as stable non-progressive memory disturbances. Despite bio-humoral and instrumental investigations and DaTScan were negative, MRI post-analysis evidenced a 6.5% loss in brain volume. Specifically, irreversible cortical and sub-cortical grey matter reduction and cerebrospinal fluid space enlargement with spared white matter were found. Our observations suggest that the sudden availability of Fluphenazine results in a kind of plateau effect of parkinsonism presentation, partially reversible due to the neurotoxic drug effect on the cortical and sub-cortical grey matter, resulting in asymmetric EPS and stable memory loss, respectively. Our report confirms the debated neurotoxicity of first-generation neuroleptics and the postulated theory of differential susceptibility to the cytotoxic stressors on the central nervous system.

This is a case report concerning a Natalizumab-associated Progressive Multifocal Leukoencephalopathy (PML) with cerebellar localization and wakefulness disturbances. Awakening and clinical improvement dramatically occurred as soon as the immune reconstitution inflammatory syndrome (IRIS) took place, being it mild in nature and colocalizing with the PML lesion. In these ideal experimental conditions, we applied brain magnetic resonance imaging post-analysis in order to know changes in brain volumes underlying the pathological process over the infection period. White matter volume increased with a decrease in grey matter during IRIS. Conversely, we found a constant increase in cerebrospinal fluid volume throughout the duration of PML, suggesting a widespread abiotrophic effect, far from the lesion. Furthermore, brain parenchymal fraction significantly decreased as expected while the total brain volume remained stable at all times. Neurodegeneration is the main contributor to the steady disability in Natalizumab-associated PML. This process is thought to be widespread and inflammatory in nature as well as sustained by IRIS and humoral factors derived from the PML lesion.

Dementia-associated compulsive singing (DACS) is a neurotransmettitorial-based behavioral disturbance, characterized by an unabating melodic expression, occurring in patients that suffer from evolved dementia. Previously described only as a “punding” aspect of the dopamine dysregulation syndrome (DDS) in the Parkinson’s disease (PD), compulsive singing has now been described, for the first time, in four non-PD patients effectively treated with Haloperidol or Quetiapine. Unlike the DDS-associated conditions, in our cases DACS is not pharmacologically induced, being that all patients were L-dopa-free. We detected a diffuse hyperintensity of the white matter and brain atrophy, with insular shrinkage as well as ventricular system and/or sub-arachnoid space enlargement in our DACS patients. Furthermore, similarly to the other behavioral symptoms of dementia, DACS also seems to be correlated to the degree of cognitive and functional impairment, rather than its subtype. In conclusion, DACS is a non-cognitive, unpublished clinical aspect of evolved dementia, which is interesting due to the involvement of the extra-nigral dopaminergic system, resulting in an unabating altered behavior, but also to the enrichment of our knowledge in the involutional diseases of the central nervous system and their physiopathological manifestations.

Glycans are one of the four fundamental macromolecular components of living matter, and they are highly regulated in the cell. Their functions are metabolic, structural and modulatory. In particular, ER resident N-glycans participate with the Glc₃Man₉GlcNAc₂ highly conserved sequence, in protein folding process, where the physiological balance between glycosylation/deglycosylation on the innermost glucose residue takes place, according GANAB/UGGT concentration ratio. However, under abnormal conditions, the cell adapts to the glucose availability by adopting an aerobic or anaerobic regimen of glycolysis, or to external stimuli through internal or external recognition patterns, so it responds to pathogenic noxa with unfolded protein response (UPR). UPR can affect Multiple Sclerosis (MS) and several neurological and metabolic diseases via the BiP stress sensor, resulting in ATF6, PERK and IRE1 activation. Furthermore, the abnormal GANAB expression has been observed in MS, systemic lupus erythematous, male germinal epithelium and predisposed highly replicating cells of the kidney tubules and bile ducts. The latter is the case of Polycystic Liver Disease (PCLD) and Polycystic Kidney Disease (PCKD), where genetically induced GANAB loss affects polycystin-1 (PC1) and polycystin-2 (PC2), resulting in altered protein quality control and cyst formation phenomenon. Our topics resume the role of glycans in cell physiology, highlighting the N-glycans one, as a substrate of GANAB, which is an emerging key molecule in MS and other human pathologies.

Discovered in 1993 by Bange et al., the 35-kDa interferon-induced protein (IFP35) is a highly conserved cytosolic interferon-induced leucine zipper protein with a 17q12-21 coding gene and unknown function. Belonging to interferon stimulated genes (ISG), the IFP35 reflects the type I interferon (IFN) activity induced through the JAK-STAT phosphorylation, and it can homodimerize with N-myc-interactor (NMI) and basic leucine zipper transcription factor (BATF), resulting in nuclear translocation and a functional expression. Casein kinase 2-interacting protein-1 (CKIP-1), retinoic acid-inducible gene I (RIG-I), and laboratory of genetics and physiology 2 Epinephelus coioides (EcLGP2) are thought to regulate IFP35, via the innate immunity pathway. Several in vitro and in vivo studies on fish and mammals have confirmed the IFP35 as an ISG factor with antiviral and antiproliferative functions. However, in a mice model of sepsis, IFP35 was found working as a damage associated molecular pattern (DAMP) molecule, which enhances inflammation by acting in the innate immune-mediated way. In human pathology, the IFP35 expression level predicts disease outcome and response to therapy in Multiple Sclerosis (MS), reflecting IFN activity. Specifically, IFP35 was upregulated in Lupus Nephritis (LN), Rheumatoid Arthritis (RA), and untreated MS. However, it normalized in the MS patients undergoing therapy. The considered data indicate IFP35 as a pleiotropic factor, suggesting it as biologically relevant in the innate immunity, general pathology, and human demyelinating diseases of the central nervous system.