Broader anti-EBV TCR repertoire in multiple sclerosis: disease specificity and treatment modulation.

Epstein-Barr virus (EBV) infection has long been associated with the development of multiple sclerosis (MS). MS patients have elevated titers of EBV-specific antibodies in serum and show signs of CNS damage only after EBV infection. Regarding CD8+ T-cells, an elevated but ineffective response to EBV was suggested in MS patients, who present with a broader MHC-I-restricted EBV-specific T-cell receptor beta chain (TRB) repertoire compared to controls. It is not known whether this altered EBV response could be subject to dynamic changes, e.g., by approved MS therapies, and whether it is specific for MS. 1317 peripheral blood TRB repertoire samples of healthy donors (n=409), patients with MS (n=710) before and after treatment, patients with neuromyelitis optica spectrum disorder (n=87), myelin-oligodendrocyte-glycoprotein antibody-associated disease (n=64) and Susac's syndrome (n=47) were analyzed. Apart from MS, none of the evaluated diseases presented with a broader anti-EBV TRB repertoire. In MS patients undergoing autologous hematopoietic stem-cell transplantation, EBV reactivation coincided with elevated MHC-I-restricted EBV-specific TRB sequence matches. Therapy with ocrelizumab, teriflunomide or dimethyl fumarate reduced EBV-specific, but not CMV-specific MHC-I-restricted TRB sequence matches. Together, this data suggests that the aberrant MHC-I-restricted T-cell response directed against EBV is specific to MS with regard to NMO, MOGAD and Susac's Syndrome and that it is specifically modified by MS treatments interfering with EBV host cells or activated lymphocytes.

Understanding sexual distress related to sexual function (SDRSF): A preliminary framework based on a qualitative study with clinical sexologists.

Introduction: Sexual distress related to sexual function (SDRSF) is pivotal in diagnosing sexual dysfunction. However, there is a lack of theoretical models for its comprehension and of knowledge concerning how to address it in clinical practice. Aim: To contribute to theory building and clinical practice about SDRSF by collecting clinicians' accounts, aiming to inform a preliminary framework to study and intervene in SDRSF. Method: Reflexive thematic analysis was used to analyze the data from 16 semi-structured interviews with clinical sexologists. Results: Three main themes were created: (1) Burning from the inside, (2) Wicked games, and (3) Running up that hill. Participants revealed a multidimensional understanding of SDRSF in clinical settings that integrates individual, sociocultural, interpersonal and situational factors. This underscores the interconnected nature of SDRSF, revealing its links to different facets of overall distress in clinical settings. We present a preliminary framework that may be analytically generalized to enhance the comprehension of the specificities of SDRSF. Conclusion: These insights frame a comprehensive conceptualization of SDRSF in clinical settings that goes beyond sexual activity and implies that interpersonal and societal factors need to be considered in research and intervention in this field.

Systems Analysis of Immune Changes after B-cell Depletion in Autoimmune Multiple Sclerosis.

Multiple sclerosis (MS) is a complex genetically mediated autoimmune disease of the central nervous system where anti-CD20-mediated B cell depletion is remarkably effective in the treatment of early disease. While previous studies investigated the effect of B cell depletion on select immune cell subsets using flow cytometry-based methods, the therapeutic impact on patient immune landscape is unknown. In this study, we explored how a therapy-driven " in vivo perturbation " modulates the diverse immune landscape by measuring transcriptomic granularity with single-cell RNA sequencing (scRNAseq). We demonstrate that B cell depletion leads to cell type-specific changes in the abundance and function of CSF macrophages and peripheral blood monocytes. Specifically, a CSF-specific macrophage population with an anti-inflammatory transcriptomic signature and peripheral CD16 + monocytes increased in frequency post-B cell depletion. In addition, we observed increases in TNFα messenger RNA and protein in monocytes post-B cell depletion, consistent with the finding that anti-TNFα treatment exacerbates autoimmune activity in MS. In parallel, B cell depletion also induced changes in peripheral CD4 + T cell populations, including increases in the frequency of TIGIT + regulatory T cells and marked decreases in the frequency of myelin peptide loaded-tetramer binding CD4 + T cells. Collectively, this study provides an exhaustive transcriptomic map of immunological changes, revealing different mechanisms of action contributing to the high efficacy in B cell depletion treatment of MS.

The effect of emotional regulation difficulties on sexual and psychological distress using repetitive negative thinking as a mediator.

BACKGROUND: Sexual distress, a determinant factor in diagnosing sexual dysfunction, plays a significant role in individuals' sexual well-being, yet it has been overlooked in research. AIM: This exploratory study adopted a transdiagnostic approach to sexual distress and sought to examine the association between emotional regulation difficulties and sexual and psychological distress, with repetitive negative thinking as a potential mediator. METHODS: We used a quantitative cross-sectional design with a sample of 509 partnered individuals. OUTCOMES: The survey included a sociodemographic questionnaire, the Difficulties in Emotion Regulation Scale-Short Form, the Kessler Psychological Distress Scale, the Persistent and Intrusive Negative Thoughts Scale, and the Female Sexual Distress Scale-Revised. RESULTS: Correlational analysis revealed significant associations among emotional regulation difficulties, repetitive negative thinking, psychological distress, and sexual distress. Furthermore, a mediation model demonstrated that repetitive negative thinking significantly mediated emotion dysregulation and psychological and sexual distress. CLINICAL IMPLICATIONS: These findings underscore the importance of considering emotion regulation difficulties and repetitive negative thinking as a maladaptive coping strategy when evaluating sexual distress and suggest that therapeutic interventions targeting such difficulties and thoughts may yield beneficial outcomes. STRENGTHS AND LIMITATIONS: These findings reinforce the importance of considering the role of emotional regulation difficulties and, consequently, repetitive negative thinking as a dysfunctional coping strategy, when studying and intervening in sexual distress. Future research with clinical samples should be developed to establish better the significance of considering these two dimensions in assessment and therapeutic intervention. CONCLUSION: Future research should corroborate and expand upon these findings to advance our understanding of sexual distress and optimize interventions in this domain.

Anti-SARS-CoV-2 monoclonal antibodies for the treatment of mild-to-moderate COVID-19 in multiple sclerosis: A retrospective cohort study.

BACKGROUND: The use and potential benefit of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs) for the treatment of mild-to-moderate coronavirus disease 2019 (COVID-19) in people living with multiple sclerosis (pwMS) remains poorly studied. The objective of this study is to describe the therapeutic use of anti-SARS-CoV-2 mAbs among pwMS. METHODS: This retrospective cohort study used electronic medical records data from the TriNetX Dataworks USA Network and included adult pwMS, diagnosed with COVID-19, who received anti-SARS-CoV-2 mAbs in the outpatient setting between November 2020 and April 2022. We analyzed COVID-19 severity at anti-SARS-CoV-2 mAb initiation and up to 30 days, stratified by before/after emergence of Omicron variant and by disease-modifying therapy (DMT). RESULTS: The study included 434 pwMS treated with anti-SARS-CoV-2 mAbs for mild-to-moderate COVID-19, including 270 patients before and 174 after Omicron emergence. Most pwMS were female (80.2%), mean age (SD) was 51.5 (12.5) years. Two-hundred-and-five patients were on DMTs, 51% of whom received anti-CD20s. One patient with moderate COVID-19 was hospitalized whilst receiving glatiramer acetate. No patients required intensive care and there were no deaths. COVID-19 outcomes were comparable following anti-SARS-CoV-2 mAb therapy in patients receiving different DMTs. CONCLUSION: Anti-SARS-CoV-2 mAb treatment for pwMS with mild-to-moderate COVID-19 may reduce the risk of COVID-19-related hospitalization and death.

Long-Term Immune Response Profiles to SARS-CoV-2 Vaccination and Infection in People with Multiple Sclerosis on Anti-CD20 Therapy.

Our objective was to analyze longitudinal cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in people with multiple sclerosis (pwMS) on B-cell depleting treatment (BCDT) compared to pwMS without immunotherapy. We further evaluated the impact of COVID-19 infection and vaccination timing. PwMS (n = 439) on BCDT (ocrelizumab, rituximab, ofatumumab) or without immunotherapy were recruited for this prospective cohort study between June 2021 and June 2022. SARS-CoV-2 spike-specific antibodies and interferon-γ release of CD4 and CD8 T-cells upon stimulation with spike protein peptide pools were analyzed at different timepoints (after primary vaccination, 3 and 6 months after primary vaccination, after booster vaccination, 3 months after booster). Humoral response to SARS-CoV-2 was consistently lower whereas T-cell response was higher in patients with BCDT compared to controls. Cellular and humoral responses decreased over time after primary vaccination and increased again upon booster vaccination, with significantly higher antibody titers after booster than after primary vaccination in both untreated and B-cell-depleted pwMS. COVID-19 infection further led to a significant increase in SARS-CoV-2-specific responses. Despite attenuated B-cell responses, a third vaccination for patients with BCDT seems recommendable, since at least partial protection can be expected from the strong T-cell response. Moreover, our data show that an assessment of T-cell responses may be helpful in B-cell-depleted patients to evaluate the efficacy of SARS-CoV-2 vaccination.

Phenotypic Characterization of Oral Mucor Species from Eurasian Vultures: Pathogenic Potential and Antimicrobial Ability.

Due to poisoning and decline in the food resources of Eurasian vultures, there has been a rise in the number of Griffon (Gyps fulvus) and Cinereous vultures (Aegypius monachus) needing veterinary care. In captivity, vultures often develop oral and other infectious diseases which can affect their survival and the probability of reintroduction in the wild. Therefore, it is important to characterize relevant microbial species present in the oral cavity of vultures, such as Mucor spp. In this work, seven Mucor spp. isolates previously obtained from Gyps fulvus and Aegypius monachus oral swabs collected at two rehabilitation centers in Portugal were characterized regarding their pathogenic enzymatic profile and antimicrobial activity. Isolates were identified by macro and microscopic observation, and PCR and ITS sequencing. Their antimicrobial activity was determined using a collection of pathogenic bacteria and two yeast species. Results showed that 86% of the isolates produced α-hemolysis, 71% expressed DNase, 57% produce lecithinase and lipase, 29% expressed gelatinase, and 29% were biofilm producers. Four isolates showed inhibitory activity against relevant human and veterinary clinical isolates, including Escherichia coli, Enterococcus faecium, Neisseria zoodegmatis, and Staphylococcus aureus. In conclusion, accurate management programs should consider the benefits and disadvantages of Mucor spp. presence in the oral mucosa.

A structured evaluation of cryopreservation in generating single-cell transcriptomes from cerebrospinal fluid.

Single-cell transcriptomics allows characterization of cerebrospinal fluid (CSF) cells at an unprecedented level. Here, we report a robust cryopreservation protocol adapted for the characterization of fragile CSF cells by single-cell RNA sequencing (RNA-seq) in moderate- to large-scale studies. Fresh CSF was collected from twenty-one participants at two independent sites. Each CSF sample was split into two fractions: one was processed fresh, while the second was cryopreserved for months and profiled after thawing. B and T cell receptor sequencing was also performed. Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. We find no significant difference in cell type proportions and cellular transcriptomes between fresh and cryopreserved cells. Results were comparable at both sites and with different single-cell sequencing chemistries. Cryopreservation did not affect recovery of T and B cell clonotype diversity. Our CSF cell cryopreservation protocol provides an important alternative to fresh processing of fragile CSF cells.

Isolated Peptide from Spider Venom Modulates Dendritic Cells In Vitro: A Possible Application in Oncoimmunotherapy for Glioblastoma.

Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1ß, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.

Immune Profiling Reveals the T-Cell Effect of Ocrelizumab in Early Relapsing-Remitting Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Ocrelizumab (OCR), a humanized anti-CD20 monoclonal antibody, is highly efficient in patients with relapsing-remitting multiple sclerosis (RR-MS). We assessed early cellular immune profiles and their association with disease activity at treatment start and under therapy, which may provide new clues on the mechanisms of action of OCR and on the disease pathophysiology. METHODS: A first group of 42 patients with an early RR-MS, never exposed to disease-modifying therapy, was included in 11 centers participating to an ancillary study of the ENSEMBLE trial (NCT03085810) to evaluate the effectiveness and safety of OCR. The phenotypic immune profile was comprehensively assessed by multiparametric spectral flow cytometry at baseline and after 24 and 48 weeks of OCR treatment on cryopreserved peripheral blood mononuclear cells and analyzed in relation to disease clinical activity. A second group of 13 untreated patients with RR-MS was included for comparative analysis of peripheral blood and CSF. The transcriptomic profile was assessed by single-cell qPCRs of 96 genes of immunologic interest. RESULTS: Using an unbiased analysis, we found that OCR as an effect on 4 clusters of CD4+ T cells: one corresponding to naive CD4+ T cells was increased, the other clusters corresponded to effector memory (EM) CD4+CCR6- T cells expressing homing and migration markers, 2 of them also expressing CCR5 and were decreased by the treatment. Of interest, one CD8+ T-cell cluster was decreased by OCR corresponding to EM CCR5-expressing T cells with high expression of the brain homing markers CD49d and CD11a and correlated with the time elapsed since the last relapse. These EM CD8+CCR5+ T cells were enriched in the CSF of patients with RR-MS and corresponded to activated and cytotoxic cells. DISCUSSION: Our study provides novel insights into the mode of action of anti-CD20, pointing toward the role of EM T cells, particularly a subset of CD8 T cells expressing CCR5.

CD171 Multi-epitope peptide design based on immuno-informatics approach as a cancer vaccine candidate for glioblastoma.

Glioblastoma (GB) is a common primary malignancy of the central nervous system, and one of the highly lethal brain tumors. GB cells can promote therapeutic resistance and tumor angiogenesis. The CD171 is an adhesion molecule in neuronal cells that is expressed in glioma cells as a regulator of brain development during the embryonic period. CD171 is one of the immunoglobulin-like CAMs (cell adhesion molecules) families that can be associated with prognosis in a variety of human tumors. The multi-epitope peptide vaccines are based on synthetic peptides with a combination of both B-cell epitopes and T-cell epitopes, which can induce specific humoral or cellular immune responses. Moreover, Cholera toxin subunit B (CTB), a novel TLR agonist was utilized in the final construct to polarize CD4+ T cells toward T-helper 1 to induce strong cytotoxic T lymphocytes (CTL) responses. In the present study, several immune-informatics tools were used for analyzing the CD171 sequence and studying the important characteristics of a designed vaccine. The results included molecular docking, molecular dynamics simulation, immune response simulation, prediction and validation of the secondary and tertiary structure, physicochemical properties, solubility, conservancy, toxicity as well as antigenicity and allergenicity of the promising candidate for a vaccine against CD171. The immuno-informatic analyze suggested 12 predicted multi-epitope peptides, whose construction consists of 582 residues long. Therewith, cloning adaptation of the designed vaccine was performed, and eventually sequence was inserted into pET30a (+) vector for the application of the anti-glioblastoma vaccine development.Communicated by Ramaswamy H. Sarma.

Timing of SARS-CoV-2 Vaccination Matters in People With Multiple Sclerosis on Pulsed Anti-CD20 Treatment.

BACKGROUND AND OBJECTIVES: Our objective was to investigate cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in a cohort of people with multiple sclerosis (pwMS) on pulsed B-cell-depleting treatment (BCDT). In particular, we intended to evaluate a possible association between immune responses and the timing of vaccination under BCDT. METHODS: We conducted a cross-sectional study among pwMS on pulsed BCDT or without disease-modifying treatment after completed SARS-CoV-2 vaccination. Samples were collected during routine clinical visits at the Multiple Sclerosis Center Dresden, Germany, between June 2021 and September 2021. Blood was analyzed for SARS-CoV-2 spike protein-specific antibodies and interferon-γ release of CD4 and CD8 T cells on stimulation with spike protein peptide pools. Lymphocyte subpopulations and total immunoglobulin levels in the blood were measured as part of clinical routine. RESULTS: We included 160 pwMS in our analysis, comprising 133 pwMS on BCDT (n = 132 on ocrelizumab and n = 1 on rituximab) and 27 without disease-modifying treatment. Humoral and cellular anti-SARS-CoV-2 responses were reciprocally regulated by the time between the last BCDT cycle and vaccination. Although antibody responses increased with prolonged intervals between the last BCDT cycle and vaccination, CD4 and CD8 T-cell responses were higher in pwMS vaccinated at early time points after the last BCDT cycle compared with untreated pwMS. T-cellular vaccination responses correlated with total, CD3 CD4, and partly with CD3 CD8 lymphocyte counts. Humoral responses correlated with CD19 lymphocyte counts. Status post coronavirus disease 2019 infection led to significantly increased SARS-CoV-2-specific T-cell and antibody responses. DISCUSSION: Delaying BCDT is currently discussed as a strategy to optimize humoral responses to SARS-CoV-2 vaccination. However, T cells represent an important line of defense against SARS-CoV-2 infection as well, especially in light of emerging variants of concern. We observed enhanced CD4 and CD8 T-cellular responses in pwMS receiving vaccination at early time points after their last BCDT cycle. These data may influence clinical decision making with respect to vaccination strategies in patients receiving BCDT.

Hybrid and vaccine-induced immunity against SAR-CoV-2 in MS patients on different disease-modifying therapies.

OBJECTIVE: To compare "hybrid immunity" (prior COVID-19 infection plus vaccination) and post-vaccination immunity to SARS CoV-2 in MS patients on different disease-modifying therapies (DMTs) and to assess the impact of vaccine product and race/ethnicity on post-vaccination immune responses. METHODS: Consecutive MS patients from NYU MS Care Center (New York, NY), aged 18-60, who completed primary COVID-19 vaccination series ≥6 weeks previously were evaluated for SARS CoV-2-specific antibody responses with electro-chemiluminescence and multiepitope bead-based immunoassays and, in a subset, live virus immunofluorescence-based microneutralization assay. SARS CoV-2-specific cellular responses were assessed with cellular stimulation TruCulture IFNγ and IL-2 assay and, in a subset, with IFNγ and IL-2 ELISpot assays. Multivariate analyses examined associations between immunologic responses and prior COVID-19 infection while controlling for age, sex, DMT at vaccination, time-to-vaccine, and vaccine product. RESULTS: Between 6/01/2021 and 11/11/2021, 370 MS patients were recruited (mean age 40.6 years; 76% female; 53% non-White; 22% with prior infection; common DMT classes: ocrelizumab 40%; natalizumab 15%, sphingosine-1-phosphate receptor modulators 13%; and no DMT 8%). Vaccine-to-collection time was 18.7 (±7.7) weeks and 95% of patients received mRNA vaccines. In multivariate analyses, patients with laboratory-confirmed prior COVID-19 infection had significantly increased antibody and cellular post-vaccination responses compared to those without prior infection. Vaccine product and DMT class were independent predictors of antibody and cellular responses, while race/ethnicity was not. INTERPRETATION: Prior COVID-19 infection is associated with enhanced antibody and cellular post-vaccine responses independent of DMT class and vaccine type. There were no differences in immune responses across race/ethnic groups.

Broader Epstein-Barr virus-specific T cell receptor repertoire in patients with multiple sclerosis.

Epstein-Barr virus (EBV) infection precedes multiple sclerosis (MS) pathology and cross-reactive antibodies might link EBV infection to CNS autoimmunity. As an altered anti-EBV T cell reaction was suggested in MS, we queried peripheral blood T cell receptor ß chain (TCRß) repertoires of 1,395 MS patients, 887 controls, and 35 monozygotic, MS-discordant twin pairs for multimer-confirmed, viral antigen-specific TCRß sequences. We detected more MHC-I-restricted EBV-specific TCRß sequences in MS patients. Differences in genetics or upbringing could be excluded by validation in monozygotic twin pairs discordant for MS. Anti-VLA-4 treatment amplified this observation, while interferon ß- or anti-CD20 treatment did not modulate EBV-specific T cell occurrence. In healthy individuals, EBV-specific CD8+ T cells were of an effector-memory phenotype in peripheral blood and cerebrospinal fluid. In MS patients, cerebrospinal fluid also contained EBV-specific central-memory CD8+ T cells, suggesting recent priming. Therefore, MS is not only preceded by EBV infection, but also associated with broader EBV-specific TCR repertoires, consistent with an ongoing anti-EBV immune reaction in MS.

The CELLO trial: Protocol of a planned phase 4 study to assess the efficacy of Ocrelizumab in patients with radiologically isolated syndrome.

BACKGROUND: Patients with radiologically isolated syndrome (RIS) exhibit CNS lesions suggestive of multiple sclerosis (MS) in the absence of overt neurological symptoms characteristic of the disease. They may have concurrent brain atrophy, subtle cognitive impairment, and intrathecal inflammation. At least half ultimately develop MS, cementing RIS as preclinical MS for many. However, high-quality data, including immunologic biomarkers, to guide treatment decisions in this population are lacking. Early intervention with ocrelizumab, a humanized monoclonal antibody approved for relapsing and primary progressive MS that targets CD20+ B-cells, may affect disease course and improve long-term outcomes. The objective of this study is to describe the protocol for CELLO, a clinical trial assessing the effect of ocrelizumab on RIS. METHODS: The CELLO clinical trial, a phase 4, multicenter, randomized, double-blind, placebo-controlled study conducted as an academic-industry collaboration, aims to (1) assess the efficacy of ocrelizumab in patients with RIS and (2) identify biomarkers indicative of emerging autoimmunity as well as immune recovery after transient B-cell depletion. The study will enroll 100 participants across ≥15 sites. Participants will be aged 18 to 40 years, have RIS (defined as meeting 2017 revised McDonald criteria for dissemination in space), and have either been diagnosed with RIS within the last 5 years or have had new brain lesions identified within 5 years of study entry. A screening program of first-degree relatives of patients with MS will be used to boost recruitment. Eligible patients will be randomized 1:1 to receive 3 courses of ocrelizumab or placebo at baseline, week 24, and week 48. Patients will subsequently be followed up for ≥3 years. The primary outcome is time to development of new radiological or clinical evidence of MS. Secondary and exploratory objectives will investigate neuroimaging, serological and immunologic biomarkers, cognitive function, and patient-reported outcomes. A substudy using single-cell RNA sequencing to characterize blood and CSF immune cells will assess markers associated with conversion to clinical MS. CONCLUSION: The CELLO study will improve the understanding of B-cell biology in early MS disease pathophysiology, characterize the emergence of CNS autoimmunity, and provide evidence to inform treatment decision-making for individuals with RIS. CLINICALTRIALS: GOV: NCT04877457.

Lateralization in hemi-parkinsonian rats is affected by deep brain stimulation or glutamatergic neurotransmission in the inferior colliculus.

After unilateral lesion of the medial forebrain bundle (MFB) by 6-OHDA rats exhibit lateralized deficits in spontaneous behavior or apomorphine-induced rotations. We investigated whether such lateralization is attenuated by either deep brain stimulation (DBS) or glutamatergic neurotransmission in the inferior colliculus (IC) of Wistar rats. Intracollicular DBS did not affect spontaneous lateralization but attenuated apomorphine-induced rotations. Spontaneous lateralization disappeared after both glutamatergic antagonist MK-801 or the agonist NMDA microinjected in the IC. Apomorphine-induced rotations were potentiated by MK-801 but were not affected by NMDA intracollicular microinjection. After injecting a bidirectional neural tract tracer into the IC, cell bodies and/or axonal fibers were found in the periaqueductal gray, superior colliculus, substantia nigra, cuneiform nucleus and pedunculo-pontine tegmental nucleus, suggesting the involvement of these structures in the motor improvement after IC manipulation. Importantly, the side of the IC microinjection regarding the lesion (ipsi- or contralateral) is particularly important and this effect may not involve the neostriatum directly.Significance StatementThe inferior colliculus, usually viewed as an auditory structure, when properly manipulated may counteract motor deficits in Parkinsonian rats. Indeed, the present study showed that 30 Hz deep brain stimulation or glutamatergic neural network in the inferior colliculus reduced body asymmetry induced by medial forebrain bundle unilateral 6-OHDA lesion in rats, an animal model of Parkinsonism. Understanding how glutamatergic mechanisms in the inferior colliculus influence motor control, classically attributed to the basal nuclei circuitry, could be useful in the development of new therapeutics to treat Parkinson's disease and other motor disorders.

Cannabinoid CB2 Receptors Modulate Microglia Function and Amyloid Dynamics in a Mouse Model of Alzheimer's Disease.

The distribution and roles of the cannabinoid CB2 receptor in the CNS are still a matter of debate. Recent data suggest that, in addition to its presence in microglial cells, the CB2 receptor may be also expressed at low levels, yet biologically relevant, in other cell types such as neurons. It is accepted that the expression of CB2 receptors in the CNS is low under physiological conditions and is significantly elevated in chronic neuroinflammatory states associated with neurodegenerative diseases such as Alzheimer's disease. By using a novel mouse model (CB2 EGFP/f/f), we studied the distribution of cannabinoid CB2 receptors in the 5xFAD mouse model of Alzheimer's disease (by generating 5xFAD/CB2 EGFP/f/f mice) and explored the roles of CB2 receptors in microglial function. We used a novel selective and brain penetrant CB2 receptor agonist (RO6866945) as well as mice lacking the CB2 receptor (5xFAD/CB2 -/-) for these studies. We found that CB2 receptors are expressed in dystrophic neurite-associated microglia and that their modulation modifies the number and activity of microglial cells as well as the metabolism of the insoluble form of the amyloid peptide. These results support microglial CB2 receptors as potential targets for the development of amyloid-modulating therapies.

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.

Anti-SARS-CoV-2 T-stem cell memory persists in ocrelizumab-treated MS patients.

BACKGROUND: Development of long-lasting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) T-cell responses in persons with multiple sclerosis (pwMS) treated with ocrelizumab is questioned. OBJECTIVE: Investigate antiviral T-cell responses after infection with SARS-CoV-2 in ocrelizumab-treated pwMS. Control groups included ocrelizumab-treated pwMS without SARS-CoV-2 infection, and non-MS individuals with and without SARS-CoV-2 infection. METHODS: Peripheral blood mononuclear cells were stimulated with SARS-CoV-2 peptide pools and T-cell reactivity was assessed by ELISPOT for interferon (IFN)-γ detection, and by multiparametric fluorescence-activated cell sorting (FACS) analyses for assessment and characterization of T-cell activation. RESULTS: ELISPOT assay against the spike and the N protein of SARS-CoV-2 displayed specific T-cell reactivity in 28/29 (96%) pwMS treated with ocrelizumab and infected by SARS-CoV-2, similar to infected persons without MS. This reactivity was present 1 year after infection and independent from the time of ocrelizumab infusion. FACS analysis following stimulation with SARS-CoV-2 peptide pools showed the presence of activation-induced markers (AIMs) in both CD4+ and CD8+ T-cell subsets in 96% and 92% of these individuals, respectively. Within naïve AIM+ CD4+ and CD8+ T-cells, we detected T memory stem cells, suggesting the acquisition of long-term memory. CONCLUSIONS: B-cell depletion using ocrelizumab does not impair the development of long-lasting anti-SARS-CoV-2 T-cell responses.