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In multiple sclerosis (MS), persisting disability can occur independent of relapse activity or development of new central nervous system (CNS) inflammatory lesions, termed chronic progression. This process occurs early and it is mostly driven by cells within the CNS. One promising strategy to control progression of MS is the inhibition of the enzyme Bruton's tyrosine kinase (BTK), which is centrally involved in the activation of both B cells and myeloid cells, such as macrophages and microglia. The benefit of BTK inhibition by evobrutinib was shown as we observed reduced pro-inflammatory activation of microglia when treating chronic experimental autoimmune encephalomyelitis (EAE) or following the adoptive transfer of activated T cells. Additionally, in a model of toxic demyelination, evobrutinib-mediated BTK inhibition promoted the clearance of myelin debris by microglia, leading to an accelerated remyelination. These findings highlight that BTK inhibition has the potential to counteract underlying chronic progression of MS., (© 2024. The Author(s).)

Introduction: Little is known about the molecular profiling associated with the effect of cladribine in patients with multiple sclerosis (MS). Here, we aimed first to characterize the transcriptomic and proteomic profiles induced by cladribine in blood cells, and second to identify potential treatment response biomarkers to cladribine in patients with MS., Methods: Gene, protein and microRNA (miRNA) expression profiles were determined by microarrays (genes, miRNAs) and mass spectrometry (proteins) in peripheral blood mononuclear cells (PBMCs) from MS patients after in vitro treatment with cladribine in its active and inactive forms. Two bioinformatics approaches to integrate the three obtained datasets were applied: (i) a multiomics discriminant analysis (DIABLO - Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies); and (ii) a multi-stage integration of features selected in differential expression analysis on each dataset and then merged. Selected molecules from the in vitro study were quantified by qPCR ex vivo in PBMCs from MS patients receiving cladribine., Results: PBMCs treated in vitro with cladribine were characterized by a major downregulation of gene, protein, and miRNA expression compared with the untreated cells. An intermediate pattern between the cladribine-treated and untreated conditions was observed in PBMCs treated with cladribine in its inactive form. The differential expression analysis of each dataset led to the identification of four genes and their encoded proteins, and twenty-two miRNAs regulating their expression, that were associated with cladribine treatment. Two of these genes (PPIF and NHLRC2), and three miRNAs (miR-21-5p, miR-30b-5p, and miR-30e-5p) were validated ex vivo in MS patients treated with cladribine., Discussion: By using a combination of omics data and bioinformatics approaches we were able to identify a multiomics molecular profile induced by cladribine in vitro in PBMCs. We also identified a number of biomarkers that were validated ex vivo in PBMCs from patients with MS treated with cladribine that have the potential to become treatment response biomarkers to this drug., Competing Interests: UB is an employee of Ares Trading SA, Eysins, Switzerland, an affiliate of Merck KGaA, Darmstadt, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fissolo, Calvo-Barreiro, Eixarch, Boschert, Villar, Costa-Frossard, Ferrer, Sanchez, Borràs, Sabidó, Espejo, Montalban and Comabella.)

Immune Cell Deconvolution methods utilizing gene expression profiling to quantify immune cells in tissues and blood are an appealing alternative to flow cytometry. Our objective was to investigate the applicability of deconvolution approaches in clinical trial settings to better investigate the mode of action of drugs for autoimmune diseases. Popular deconvolution methods CIBERSORT and xCell were validated using gene expression from the publicly available GSE93777 dataset that has comprehensive matching flow cytometry. As shown in the online tool, ~ 50% of signatures show strong correlation (r > 0.5) with the remainder showing moderate correlation, or in a few cases, no correlation. Deconvolution methods were then applied to gene expression data from the phase III CLARITY study (NCT00213135) to evaluate the immune cell profile of relapsing multiple sclerosis patients treated with cladribine tablets. At 96 weeks after treatment, deconvolution scores showed the following changes vs placebo: naïve, mature, memory CD4 + and CD8 + T cells, non-class switched, and class switched memory B cells and plasmablasts were significantly reduced, naïve B cells and M2 macrophages were more abundant. Results confirm previously described changes in immune cell composition following cladribine tablets treatment and reveal immune homeostasis of pro- vs anti-inflammatory immune cell subtypes, potentially supporting long-term efficacy., (© 2023. The Author(s).)

What Is This Summary About?: This is a summary of an article originally published in the Multiple Sclerosis Journal . Cladribine tablets (MAVENCLAD ® ) are an oral (taken by mouth) medication, approved for the treatment of people with relapsing forms of multiple sclerosis (MS, with episodes of new or worsening symptoms). They are administered for a maximum of 10 days per year, over a period of 2 years. Cladribine tablets work by temporarily reducing the number of lymphocytes, which are immune cells that help to fight off infections. Because of this, people with MS (also called PwMS) may have concerns about the effect of cladribine tablets on vaccines, as these work via immune cells to build protection against infection., What Happened in the Magnify-Ms Study?: A study called MAGNIFY-MS investigated how long it takes for cladribine tablets to begin to work in people with a type of MS called highly active relapsing MS. During the study, some participants received their usual vaccinations against flu (influenza) and against the chickenpox virus (also called varicella zoster virus) as part of their routine medical care. The MAGNIFY-MS study gave the researchers an opportunity to look at how cladribine tablets affect the way the flu and chickenpox virus vaccines work in the body., What Were the Results?: Cladribine tablets do not affect how well the body responds to flu and chickenpox vaccines., What Do the Results Mean?: PwMS taking cladribine tablets who are vaccinated against chickenpox, flu or both can be protected against these diseases.

Background and Objectives: Cladribine tablets cause a reduction in lymphocytes with a predominant effect on B-cell and T-cell counts. The MAGNIFY-MS substudy reports the dynamic changes on multiple peripheral blood mononuclear cell (PBMC) subtypes and immunoglobulin (Ig) levels over 12 months after the first course of cladribine tablets in patients with highly active relapsing multiple sclerosis (MS)., Methods: Immunophenotyping was performed at baseline (predose) and at the end of months 1, 2, 3, 6, and 12 after initiating treatment with cladribine tablets. Assessments included lymphocyte subtype counts of CD19 + B cells, CD4 + and CD8 + T cells, CD16 + natural killer cells, plasmablasts, and Igs. Immune cell subtypes were analyzed by flow cytometry, and serum IgG and IgM were analyzed by nephelometric assay. Absolute cell counts and percentage change from baseline were assessed., Results: The full analysis set included 57 patients. Rapid reductions in median CD19 + , CD20 + , memory, activated, and naive B-cell counts were detected, reaching nadir by month 2. Thereafter, total CD19 + , CD20 + , and naive B-cell counts subsequently reconstituted, but memory B cells remained reduced by 93%-87% for the remainder of the study. The decrease in plasmablasts was slower, reaching nadir at month 3. Decrease in T-cell subtypes was also slower and more moderate compared with B-cell subtypes, reaching nadir between months 3 and 6. IgG and IgM levels remained within the normal range over the 12-month study period., Discussion: Cladribine tablets induce a specific pattern of early and sustained PBMC subtype dynamics in the absence of relevant Ig changes: While total B cells were reduced dramatically, T cells were affected significantly less. Naive B cells recovered toward baseline, naive CD4 and CD8 T cells did not, and memory B cells remained reduced. The results help to explain the unique immune depletion and repopulation architecture regarding onset of action and durability of effects of cladribine tablets while largely maintaining immune competence., Trial Registration Information: ClinicalTrials.gov Identifier: NCT03364036. Date registered: December 06, 2017., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Recent clinical trials have shown promising results for the next-generation Bruton's tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti-VLA-4 antibody) treatment. Under in vitro T follicular helper-like conditions, BTK phosphorylation was enhanced by T-bet-inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet-associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

Deoxycytidine kinase (dCK) and 5' deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by mediating phosphorylation (activation) or phosphorolysis (deactivation) of 2CdA, respectively, these enzymes promote or prevent its accumulation in the cell, which leads to cell death. In particular, lymphocytes which present with a high intracellular dCK/NT5C2 ratio are more sensitive to 2CdA than other immune cells. We aim at determining if the expression of these enzymes and/or their activity differ in specific progenitor and mature immune cells and are influenced by cellular activation and/or exposure to 2CdA. Flow cytometry analysis showed no difference in dCK/NT5C2 ratio in progenitor and mature immune cells. 2CdA induced apoptosis in stimulated T and B cells and unstimulated B cells. dCK expression was enhanced by 2CdA at mRNA and protein levels in activated T cells and mRNA level in activated B cells. dCK activity, measured through an in-house luminescence release enzyme assay was higher in activated T and B cells, and such an increase was abrogated in activated B cells, but not T cells, upon exposure to 2CdA. These results reveal an important relationship between dCK activity and the effect of 2CdA on B and T cells, according to their activation status. Further study is warranted to evaluate whether dCK activity could, in the future, be a suitable predictive biomarker of lymphocyte response to 2CdA., (© 2021. The Author(s).)

Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.

Leptomeningeal inflammation in multiple sclerosis is associated with worse clinical outcomes and greater cortical pathology. Despite progress in identifying this process in multiple sclerosis patients using post-contrast fluid-attenuated inversion recovery imaging, early trials attempting to target meningeal inflammation have been unsuccessful. There is a lack of appropriate model systems to screen potential therapeutic agents targeting meningeal inflammation. We utilized ultra-high field (11.7 T) MRI to perform post-contrast imaging in SJL/J mice with experimental autoimmune encephalomyelitis induced via immunization with proteolipid protein peptide (PLP139-151) and complete Freund's adjuvant. Imaging was performed in both a cross-sectional and longitudinal fashion at time points ranging from 2 to 14 weeks post-immunization. Following imaging, we euthanized animals and collected tissue for pathological evaluation, which revealed dense cellular infiltrates corresponding to areas of contrast enhancement involving the leptomeninges. These areas of meningeal inflammation contained B cells (B220+), T cells (CD3+) and myeloid cells (Mac2+). We also noted features consistent with tertiary lymphoid tissue within these areas, namely the presence of peripheral node addressin-positive structures, C-X-C motif chemokine ligand-13 (CXCL13)-producing cells and FDC-M1+ follicular dendritic cells. In the cortex adjacent to areas of meningeal inflammation we identified astrocytosis, microgliosis, demyelination and evidence of axonal stress/damage. Since areas of meningeal contrast enhancement persisted over several weeks in longitudinal experiments, we utilized this model to test the effects of a therapeutic intervention on established meningeal inflammation. We randomized mice with evidence of meningeal contrast enhancement on MRI scans performed at 6 weeks post-immunization, to treatment with either vehicle or evobrutinib [a Bruton tyrosine kinase (BTK) inhibitor] for a period of 4 weeks. These mice underwent serial imaging; we examined the effect of treatment on the areas of meningeal contrast enhancement and noted a significant reduction in the evobrutinib group compared to vehicle (30% reduction versus 5% increase; P = 0.003). We used ultra-high field MRI to identify areas of meningeal inflammation and to track them over time in SJL/J mice with experimental autoimmune encephalomyelitis, and then used this model to identify BTK inhibition as a novel therapeutic approach to target meningeal inflammation. The results of this study provide support for future studies in multiple sclerosis patients with imaging evidence of meningeal inflammation., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Anti-CD20-mediated B-cell depletion effectively reduces acute multiple sclerosis (MS) flares. Recent data shows that antibody-mediated extinction of B cells as a lasting immune suppression, harbors the risk of developing humoral deficiencies over time. Accordingly, more selective, durable and reversible B-cell-directed MS therapies are needed. We here tested inhibition of Bruton's tyrosine kinase (BTK), an enzyme centrally involved in B-cell receptor signaling, as the most promising approach in this direction. Using mouse models of MS, we determined that evobrutinib, the first BTK inhibiting molecule being developed, dose-dependently inhibited antigen-triggered activation and maturation of B cells as well as their release of pro-inflammatory cytokines. Most importantly, evobrutinib treatment functionally impaired the capacity of B cells to act as antigen-presenting cells for the development of encephalitogenic T cells, resulting in a significantly reduced disease severity in mice. In contrast to anti-CD20, BTK inhibition silenced this key property of B cells in MS without impairing their frequency or functional integrity. In conjunction with a recent phase II trial reporting that evobrutinib is safe and effective in MS, our mechanistic data highlight therapeutic BTK inhibition as a landmark towards selectively interfering with MS-driving B-cell properties.

Background: Microglia are the resident macrophages of the central nervous system (CNS). In multiple sclerosis (MS) and related experimental models, microglia have either a pro-inflammatory or a pro-regenerative/pro-remyelinating function. Inhibition of Bruton's tyrosine kinase (BTK), a member of the Tec family of kinases, has been shown to block differentiation of pro-inflammatory macrophages in response to granulocyte-macrophage colony-stimulating factor in vitro . However, the role of BTK in the CNS is unknown., Methods: Our aim was to investigate the effect of BTK inhibition on myelin repair in ex vivo and in vivo experimental models of demyelination and remyelination. The remyelination effect of a BTK inhibitor (BTKi; BTKi-1) was then investigated in LPC-induced demyelinated cerebellar organotypic slice cultures and metronidazole-induced demyelinated Xenopus MBP-GFP-NTR transgenic tadpoles., Results: Cellular detection of BTK and its activated form BTK-phospho-Y223 (p-BTK) was determined by immunohistochemistry in organotypic cerebellar slice cultures, before and after lysophosphatidylcholine (LPC)-induced demyelination. A low BTK signal detected by immunolabeling under normal conditions in cerebellar slices was in sharp contrast to an 8.5-fold increase in the number of BTK-positive cells observed in LPC-demyelinated slice cultures. Under both conditions, approximately 75% of cells expressing BTK and p-BTK were microglia and 25% were astrocytes. Compared with spontaneous recovery, treatment of demyelinated slice cultures and MTZ-demyelinated transgenic tadpoles with BTKi resulted in at least a 1.7-fold improvement of remyelination., Conclusion: Our data demonstrate that BTK inhibition is a promising therapeutic strategy for myelin repair., Competing Interests: R. Grenningloh is an employee of EMD Serono, Billerica, MA, USA (a business of Merck KGaA, Darmstadt, Germany). U. Boschert is an employee of Merck Serono S.A., Eysin, Switzerland., (© 2020 – IOS Press and the authors. All rights reserved.)

Background: Cladribine tablets 3.5 mg/kg cumulative over 2 years (CT3.5) had significant clinical/imaging effects in patients with clinically isolated syndrome (CIS; ORACLE-MS) or relapsing-remitting MS (RRMS; CLARITY and CLARITY Extension). This analysis compared the effect of cladribine tablets on the dynamics of immune cell reduction and reconstitution in ORACLE-MS, CLARITY, and CLARITY Extension during the first year of treatment (i.e. the first course of CT1.75) in patients randomized to CT3.5., Methods: Lymphocyte subtypes were analyzed using multiparameter flow cytometry. Changes in cell counts and relative proportions of lymphocytes were evaluated at weeks 5, 13, 24, and 48., Results: Across studies, consistent and comparable selective kinetics of immune cell populations occurred following the first treatment year with CT. A rapid reduction in CD16 + /CD56 + cells (week 5 nadir), a more marked reduction in CD19 + B cells (week 13 nadir), and a less-pronounced effect on CD4 + (week 13 nadir) and CD8 + T cells (week 24 nadir) was shown. There was little effect on neutrophils or monocytes. Lymphocyte recovery began after treatment with CT3.5. Regarding relative proportions of naïve and memory T-cell subtypes in ORACLE-MS, the proportion of naïve-like naturally occurring T-regulatory cells (nTregs) decreased, and the proportion of memory-like nTregs increased, relative to total CD4 + T cells., Conclusions: CT3.5 has comparable effects on the immune systems of patients with CIS or RRMS. The pronounced reduction and recovery dynamics of CD19 + B cells and relative changes in the proportion of some immune cell subtypes may underlie the clinical effects of CT3.5., Competing Interests: Conflict of interest statement: OS serves on the editorial boards of the Multiple Sclerosis Journal, and Therapeutic Advances in Neurological Disorders. He has served on data monitoring committees for Genentech-Roche, Pfizer, and TG Therapeutics without monetary compensation. He has advised EMD Serono, Celgene, Genzyme, and Serono. He currently receives grant support from Sanofi Genzyme. He received travel support from Shire PSS has served on advisory boards for Biogen, Merck KGaA, Novartis, Teva, MedDay Pharmaceuticals, and GSK; on steering committees or independent data monitoring boards in trials sponsored by Merck KGaA, Teva, GSK, and Novartis; has received speaker honoraria from Biogen Idec, Merck KGaA, Teva, Sanofi-Aventis, Genzyme, and Novartis. His department has received research support from Biogen, Merck KGaA, Teva, Novartis, Roche, and Genzyme. TL has received consultancy fees or clinical research grants from Acorda, Bayer, Biogen, Daiichi, EMD Serono, Novartis, ONO, Pfizer, and Teva Neuroscience. GG has received speaker honoraria and consulting fees from Abbvie, Actelion, Atara Bio, Almirall, Bayer Schering Pharma, Biogen Idec, FivePrime, GlaxoSmithKline, GW Pharma, Merck & Co., Merck KGaA, Pfizer Inc, Protein Discovery Laboratories, Teva Pharmaceutical Industries Ltd, Sanofi-Genzyme, UCB, Vertex Pharmaceuticals, Ironwood, and Novartis; and has received research support unrelated to this study from Biogen Idec, Merck & Co, Novartis, and Ironwood. YH, DD, FD, and UB are employees of EMD Serono Research & Development Institute Inc., a business of Merck KGaA, Darmstadt, Germany.

Objective: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood-onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking., Methods: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole-cell patch-clamp recordings and K + -sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC., Results: An early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K + dynamics and neuronal network activity are abnormal in MLC mice., Interpretation: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636-649., (© 2018 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.)

Objective: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic infantile-onset disease characterized by macrocephaly and white matter edema due to loss of MLC1 function. Recessive mutations in either MLC1 or GLIALCAM cause the disease. MLC1 is involved in astrocytic volume regulation; GlialCAM ensures the correct membrane localization of MLC1. Their exact role in brain ion-water homeostasis is only partly defined. We characterized Glialcam -null mice for further studies., Methods: We investigated the consequences of loss of GlialCAM in Glialcam -null mice and compared GlialCAM developmental expression in mice and men., Results: Glialcam -null mice had early-onset megalencephaly and increased brain water content. From 3 weeks, astrocytes were abnormal with swollen processes abutting blood vessels. Concomitantly, progressive white matter vacuolization developed due to intramyelinic edema. Glialcam -null astrocytes showed abolished expression of MLC1, reduced expression of the chloride channel ClC-2 and increased expression and redistribution of the water channel aquaporin4. Expression of other MLC1-interacting proteins and the volume regulated anion channel LRRC8A was unchanged. In mice, GlialCAM expression increased until 3 weeks and then stabilized. In humans, GlialCAM expression was highest in the first 3 years to then decrease and stabilize from approximately 5 years., Interpretation: Glialcam -null mice replicate the early stages of the human disease with early-onset intramyelinic edema. The earliest change is astrocytic swelling, further substantiating that a defect in astrocytic volume regulation is the primary cellular defect in MLC. GlialCAM expression affects expression of MLC1, ClC-2 and aquaporin4, indicating that abnormal interplay between these proteins is a disease mechanism in megalencephalic leukoencephalopathy with cysts.

This randomized, double-blind, placebo-controlled, 6-arm, parallel-design study investigated cardiac and hematological pharmacodynamic effects of ceralifimod (ONO-4641), a selective sphingosine-1-phosphate (S1P) receptor modulator, over a broad dose range in direct comparison with the nonselective S1P modulator fingolimod. Healthy subjects were assigned to ceralifimod (0.01, 0.025, 0.05, or 0.10 mg), fingolimod (0.5 mg), or placebo once daily for 14 days (n = 24 per group). After 14 days of treatment, mean absolute lymphocyte count percentage change from baseline was greatest in the fingolimod (-62%) and ceralifimod 0.10 mg (-56%) groups. On treatment cessation, lymphocyte recovery was faster in the ceralifimod versus the fingolimod group. Ceralifimod showed dose- and concentration-dependent chronotropic effect. Cardiac effects in the fingolimod group were dependent on fingolimod-P concentrations. Maximum mean heart rate (HR) effect on day 1 was larger with fingolimod (placebo-adjusted change from time-matched baseline HR [ΔΔHR], -14.9 beats per minute [bpm]) versus ceralifimod (ΔΔHR, -6.2 and -12.0 bpm for the 0.05- and 0.10-mg doses, respectively). Ceralifimod's effect on the PR interval was minor. Safety biomarker results suggest that potential therapeutic doses of ceralifimod, in particular the 0.05-mg dose, might result in reduced occurrence of bradycardia, atrioventricular block absolute lymphocyte count and grade 3/4 lymphopenia compared with fingolimod 0.5 mg., (© 2015, The American College of Clinical Pharmacology.)

Sphingosine-1-phosphate (S1P) receptor agonists have shown promise as therapeutic agents for multiple sclerosis (MS) due to their regulatory roles within the immune, central nervous system, and cardiovascular system. Here, the design and optimization of novel [1,2,4]oxadiazole derivatives as selective S1P receptor agonists are described. The structure-activity relationship exploration was carried out on the three dominant segments of the series: modification of the polar head group (P), replacement of the oxadiazole linker (L) with different five-membered heterocycles, and the use of diverse 2,2'-disubstituted biphenyl moieties as the hydrophobic tail (H). All three segments have a significant impact on potency, S1P receptor subtype selectivity, physicochemical properties, and in vitro absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of the compounds. From these optimization studies, a selective S1P1 agonist, N-methyl-N-(4-{5-[2-methyl-2'-(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)glycine (45), and a dual S1P1,5 agonist, N-methyl-N-(3-{5-[2'-methyl-2-(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)glycine (49), emerged as frontrunners. These compounds distribute predominantly in lymph nodes and brain over plasma and induce long lasting decreases in lymphocyte count after oral administration. When evaluated head-to-head in an experimental autoimmune encephalomyelitis mouse model, together with the marketed drug fingolimod, a pan-S1P receptor agonist, S1P1,5 agonist 49 demonstrated comparable efficacy while S1P1 -selective agonist 45 was less potent. Compound 49 is not a prodrug, and its improved property profile should translate into a safer treatment of relapsing forms of MS., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: Sphingosine-1-phosphate receptor (S1PR) modulating therapies are currently in the clinic or undergoing investigation for multiple sclerosis (MS) treatment. However, the expression of S1PRs is still unclear in the central nervous system under normal conditions and during neuroinflammation., Methods: Using immunohistochemistry we examined tissues from both grey and white matter MS lesions for sphingosine-1-phosphate receptor 1 (S1P1 ) and 5 (S1P5 ) expression. Tissues from Alzheimer's disease (AD) cases were also examined., Results: S1P1 expression was restricted to astrocytes and endothelial cells in control tissues and a decrease in endothelial cell expression was found in white matter MS lesions. In grey matter MS lesions, astrocyte expression was lost in active lesions, while in quiescent lesions it was restored to normal expression levels. CNPase colocalization studies demonstrated S1P5 expression on myelin and both were reduced in demyelinated lesions. In AD tissues we found no difference in S1P1 expression., Conclusion: These data demonstrate a differential modulation of S1PRs in MS lesions, which may have an impact on S1PR-directed therapies., (© 2013 British Neuropathological Society.)

CXCL12α has been shown to be selectively processed at the N- and C-termini in blood and plasma in vitro. In order to study the processing in vivo, several versions of CXCL12α were expressed and purified. The protein was administered either iv or sc to mice, and at different time points postadministration plasma was collected and analyzed. To detect modifications of the CXCL12α molecule in crude plasma a SELDI TOF-MS-based method was developed. Anti-CXCL12 antibodies were immobilized on the SELDI chip and CXCL12α binding to the antibodies was detected by SELDI-TOF-MS. The protein was found to be processed both at the C- and N-termini. The same processed CXCL12α forms as detected in vitro were found; however, in addition further processing was detected at the N-terminus, where altogether seven amino acids were removed. At the C-terminus the lysine was removed as has been seen in vitro, and no further processing was detected. The full-length CXCL12α disappeared within minutes after administration, whereas the processed forms of the protein were detectable for up to 6-8 h postadministration. The same processed forms appeared after iv and sc administration, only the kinetics was different. When the shortest processed form detected in plasma, 7ΔN1ΔC-CXCL12α, was administered directly, no further processed forms were detected. Interestingly, a version of CXCL12α containing a N-terminal methionine was protected against N-terminal processing in plasma in vitro; however, in vivo no protection was seen, the protein was processed in the same way as full-length CXCL12α., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using structure based genome mining targeting vascular endothelial and platelet derived growth factor immunoglobulin (Ig) like folds, we have identified a sequence corresponding to a single transmembrane protein with two Ig domains, which we cloned from a human brain cDNA library. The cDNA is identical to hepatocyte cell adhesion molecule (hepaCAM), which was originally described as a tumor suppressor gene in liver. Here, we show that the protein is predominantly expressed in the mouse and human nervous system. In liver, the expression is very low in humans, and is not detected in mice. To identify the central nervous system (CNS) regions and cell types expressing the protein, we performed a LacZ reporter gene assay on heterozygous mice in which one copy of the gene encoding the novel protein had been replaced with beta-galactosidase. beta-galactosidase expression was prominent in white matter tracts of the CNS. Furthermore, expression was detected in ependymal cells of the brain ventricular zones and the central canal of the spinal cord. Double labeling experiments showed expression mainly in CNPase positive oligodendrocytes (OL). Since the protein is predominantly expressed in the CNS glial cells, we named the molecule glial cell adhesion molecule (GlialCAM). A potential role for GlialCAM in myelination was supported by its up-regulation during postnatal mouse brain development, where it was concomitantly expressed with myelin basic protein (MBP). In addition, in vitro, GlialCAM was observed in various developmental stages of OL and in astrocytes in processes and at cell contact sites. In A2B5 positive OL, GlialCAM colocalizes with GAP43 in OL growth cone like structures. Overall, the data presented here indicate a potential function for GlialCAM in glial cell biology., ((c) 2008 Wiley-Liss, Inc.)

In order to fully understand biological processes it is essential to identify interactions in protein complexes. There are several techniques available to study this type of interactions, such as yeast two-hybrid screens, affinity chromatography, and coimmunoprecipitation. We propose a novel strategy to identify protein-protein interactions, comprised of first detecting the interactions using ProteinChips and SELDI-TOF MS, followed by the isolation of the interacting proteins through affinity beads and RP-HPLC and finally identifying the proteins using nano-LC MS/MS. The advantages of this new strategy are that the primary high-throughput screening of samples can be performed with small amounts of sample, no specific antibody is needed and the proteins represented on the SELDI-TOF MS spectra can be identified with high confidence. Furthermore, the method is faster and less labor-intensive than other current approaches. Using this novel method, we isolated and identified the interactions of two mouse plasma proteins, mannose binding lectin C and properdin, with GlialCAM, a type 1 transmembrane glycoprotein that belongs to the Ig superfamily.

Chemokines have recently been postulated to have important functions in the central nervous system (CNS) in addition to their principal role of directional migration of leukocytes. In particular, it has been shown that chemokines may play a role in the regulation of oligodendrocyte biology. Here, we have chosen to study the role of certain chemokines in regulating myelination. We have used the murine oligodendrocyte precursor-like cell line, Oli-neu, and primary mixed cortical cultures as experimental systems to assess their activities on oligodendrocyte precursor proliferation and developmental in vitro myelination. GRO-alpha, IL-8, SDF-1alpha and RANTES dose-dependently increased proliferation of this mouse A2B5 precursor-like cell line, while MCP-1 did not. Furthermore, the CXC chemokines GRO-alpha, IL-8 and SDF-1alpha stimulated myelin basic protein synthesis in a dose-dependent manner in primary myelinating cultures and enhanced myelin segment formation in this system, while the CC chemokines MCP-1 and RANTES did not. We also demonstrate that the receptor for SDF-1alpha, CXCR4, is expressed in mixed cortical cultures by PDGFalphaR positive oligodendrocyte precursors (OLPs) as well as by Oli-neu cells. SDF-1alpha induced proliferation in primary mixed cultures and the Oli-neu cell line was mediated through this receptor. We propose, therefore, that CXC chemokines and in particular SDF-1alpha regulates CNS myelination via their effects on cells of the oligodendrocyte lineage, specifically stimulation of OLP proliferation.

Osteopontin (OPN) has been defined as a key cytokine promoting the release of IL-12 and hence inducing the development of protective cell-mediated immunity to viruses and intracellular pathogens. To further characterize the role of OPN in antiviral immunity, OPN-deficient (OPN-/-) mice were analyzed after infection with influenza virus and vaccinia virus. Surprisingly, we found that viral clearance, lung inflammation, and recruitment of effector T cells to the lung were unaffected in OPN-/- mice after influenza infection. Furthermore, effector status of T cells was normal as demonstrated by normal IFN-gamma production and CTL lytic activity. Moreover, activation and Th1 differentiation of naive TCR transgenic CD4+ T cells by dendritic cells and cognate Ag was normal in the absence of OPN in vitro. Contrary to a previous report, we found that OPN-/- mice mounted a normal immune response to Listeria monocytogenes. In conclusion, OPN is dispensable for antiviral immune responses against influenza virus and vaccinia virus.

Multiple sclerosis, a primary autoimmune disease of the central nervous system has been characterized by the presence of the demyelinating lesions (plaques) in the CNS. To further understand the gene transcription status of the two most common lesions, chronic active and chronic inactive, we have performed a cDNA microarray analysis of these two lesion type. Comparative analysis of differential gene expression of chronic active and inactive lesions have confirmed the existence of a significant difference in the transcriptional profiles of these two lesion types in both marginal and central areas. Different sets of genes were highlighted, including genes of inflammatory characteristics, apoptosis related and stress-induced, indicating their potential role in MS pathogenesis.

We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN(-/-) mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.