Quantification of neurodegeneration by measurement of brain-specific proteins

Quantification of neurodegeneration in animal models is typically assessed by time-consuming and observer-dependent immunocytochemistry. This study aimed to investigate if newly developed ELISA techniques could provide an observer-independent, cost-effective and time-saving tool for this purpose. Neurofilament heavy chain (NfH(SM135)), astrocytic glial fibrillary acidic protein (GFAP), S100B and ferritin, markers of axonal loss, gliosis, astrocyte activation and microglial activation, respectively, were quantified in the spinal cord homogenates of mice with chronic relapsing experimental allergic encephalomyelitis (CREAE, n=8) and controls (n=7). Levels of GFAP were found to be threefold elevated in CREAE (13 ng/mg protein) when compared to control animals (4.5 ng/mg protein, p<0.001). The inverse was observed for NfH(SM135) (21 ng/mg protein vs. 63 ng/mg protein, p<0.001), ferritin (542 ng/mg protein vs. 858 ng/mg protein, p<0.001) and S100B (786 ng/mg protein vs. 2080 ng/mg protein, N.S.). These findings were confirmed by immunocytochemistry, which demonstrated intense staining for GFAP and decreased staining for NfH(SM135) in CREAE compared to control animals. These findings indicate that axonal loss and gliosis can be estimated biochemically using the newly developed ELISA assays for NfH(SM135) and GFAP. These assays may facilitate the quantification of pathological features involved in neurodegeneration

Memory B Cells are Major Targets for Effective Immunotherapy in Relapsing Multiple Sclerosis

Although multiple sclerosis (MS) is considered to be a CD4, Th17-mediated autoimmune disease, supportive evidence is perhaps circumstantial, often based on animal studies, and is questioned by the perceived failure of CD4-depleting antibodies to control relapsing MS. Therefore, it was interestingly to find that current MS-treatments, believed to act via T cell inhibition, including: beta-interferons, glatiramer acetate, cytostatic agents, dimethyl fumarate, fingolimod, cladribine, daclizumab, rituximab/ocrelizumab physically, or functionally in the case of natalizumab, also depleted CD19+, CD27+ memory B cells. This depletion was substantial and long-term following CD52 and CD20-depletion, and both also induced long-term inhibition of MS with few treatment cycles, indicating induction-therapy activity. Importantly, memory B cells were augmented by B cell activating factor (atacicept) and tumor necrosis factor (infliximab) blockade that are known to worsen MS. This creates a unifying concept centered on memory B cells that is consistent with therapeutic, histopathological and etiological aspects of MS

The impact of sphingosine-1-phosphate receptor modulators on COVID-19 and SARS-CoV-2 vaccination.

BACKGROUND: Sphingosine-one phosphate receptor (S1PR) modulation inhibits S1PR1-mediated lymphocyte migration, lesion formation and positively-impacts on active multiple sclerosis (MS). These S1PR modulatory drugs have different: European Union use restrictions, pharmacokinetics, metabolic profiles and S1PR receptor affinities that may impact MS-management. Importantly, these confer useful properties in dealing with COVID-19, anti-viral drug responses and generating SARS-CoV-2 vaccine responses. OBJECTIVE: To examine the biology and emerging data that potentially underpins immunity to the SARS-CoV-2 virus following natural infection and vaccination and determine how this impinges on the use of current sphingosine-one-phosphate modulators used in the treatment of MS. METHODS: A literature review was performed, and data on infection, vaccination responses; S1PR distribution and functional activity was extracted from regulatory and academic information within the public domain. OBSERVATIONS: Most COVID-19 related information relates to the use of fingolimod. This indicates that continuous S1PR1, S1PR3, S1PR4 and S1PR5 modulation is not associated with a worse prognosis following SARS-CoV-2 infection. Whilst fingolimod use is associated with blunted seroconversion and reduced peripheral T-cell vaccine responses, it appears that people on siponimod, ozanimod and ponesimod exhibit stronger vaccine-responses, which could be related notably to a limited impact on S1PR4 activity. Whilst it is thought that S1PR3 controls B cell function in addition to actions by S1PR1 and S1PR2, this may be species-related effect in rodents that is not yet substantiated in humans, as seen with bradycardia issues. Blunted antibody responses can be related to actions on B and T-cell subsets, germinal centre function and innate-immune biology. Although S1P1R-related functions are seeming central to control of MS and the generation of a fully functional vaccination response; the relative lack of influence on S1PR4-mediated actions on dendritic cells may increase the rate of vaccine-induced seroconversion with the newer generation of S1PR modulators and improve the risk-benefit balance IMPLICATIONS: Although fingolimod is a useful asset in controlling MS, recently-approved S1PR modulators may have beneficial biology related to pharmacokinetics, metabolism and more-restricted targeting that make it easier to generate infection-control and effective anti-viral responses to SARS-COV-2 and other pathogens. Further studies are warranted

Learning from other autoimmunities to understand targeting of B cells to control multiple sclerosis

Although many suspected autoimmune diseases are thought to be T cell-mediated, the response to therapy indicates that depletion of B cells consistently inhibits disease activity. In multiple sclerosis, it appears that disease suppression is associated with the long-term reduction of memory B cells, which serves as a biomarker for disease activity in many other CD20+ B cell depletion-sensitive, autoimmune diseases. Following B cell depletion, the rapid repopulation by transitional (immature) and naïve (mature) B cells from the bone marrow masks the marked depletion and slow repopulation of lymphoid tissue-derived, memory B cells. This can provide long-term protection from a short treatment cycle. It seems that memory B cells, possibly via T cell stimulation, drive relapsing disease. However, their sequestration in ectopic follicles and the chronic activity of B cells and plasma cells in the central nervous system may drive progressive neurodegeneration directly via antigen-specific mechanisms or indirectly via glial-dependent mechanisms. While unproven, Epstein-Barr virus may be an aetiological trigger of multiple sclerosis. This infects mature B cells, drives the production of memory B cells and possibly provides co-stimulatory signals promoting T cell-independent activation that breaks immune tolerance to generate autoreactivity. Thus, a memory B cell centric mechanism can integrate: potential aetiology, genetics, pathology and response to therapy in multiple sclerosis and other autoimmune conditions with ectopic B cell activation that are responsive to memory B cell-depleting strategies

Detecting and predicting neutralization of alemtuzumab responses in MS

Objective: To test the hypothesis that anti-drug antibodies against alemtuzumab could become relevant after repeated treatments for some individuals, possibly explaining occasional treatment resistance. Methods: Recombinant alemtuzumab single-chain variable fragment antibody with a dual tandem nanoluciferase reporter linker was made and used to detect binding anti-drug antibodies. Alemtuzumab IgG Alexa-Fluor 488 conjugate was used in a competitive-binding cell based assay to detect neutralizing anti-drug antibodies. The assays were used to retrospectively screen, blinded, banked-serum samples from people with multiple sclerosis (n=32) who had received three or more cycles of alemtuzumab. Lymphocyte depletion was measured between baseline and about 1 month post-infusion. Results: The number of individuals showing limited depletion of lymphocytes increased with the number of treatment cycles. Lack of depletion was also a poor prognostic feature for future disease activity. Anti-drug antibody responses were detected in 29/32 (90.6%) individuals. Neutralizing antibodies occurred prior to the development of limited depletion in 6/7 individuals (18.8% of the whole sample). Pre-infusion, anti-drug antibody levels predicted limited, post-infusion lymphocyte depletion. Conclusions: Although anti-drug antibodies to alemtuzumab have been portrayed as being of no clinical significance, alemtuzumab-specific antibodies appear to be clinically relevant for some individuals, although causation remains to be established. Monitoring of, lymphocyte depletion and the anti-drug response may be of practical value in patients requiring additional cycles of alemtuzumab. Anti-drug antibody detection may help to inform on re-treatment or switching to another treatment

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PMCID: PMC379391

Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity

Autoimmune encephalomyelitis in NOD mice is not initially a progressive multiple sclerosis model.

OBJECTIVE: Despite progress in treating relapsing multiple sclerosis (MS), effective inhibition of nonrelapsing progressive MS is an urgent, unmet, clinical need. Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE), provide valuable tools to examine the mechanisms contributing to disease and may be important for developing rational therapeutic approaches for treatment of progressive MS. It has been suggested that myelin oligodendrocyte glycoprotein (MOG) peptide residues 35-55 (MOG35-55 )-induced EAE in nonobese diabetic (NOD) mice resembles secondary progressive MS. The objective was to determine whether the published data merits such claims. METHODS: Induction and monitoring of EAE in NOD mice and literature review. RESULTS: It is evident that the NOD mouse model lacks validity as a progressive MS model as the individual course seems to be an asynchronous, relapsing-remitting neurodegenerative disease, characterized by increasingly poor recovery from relapse. The seemingly progressive course seen in group means of clinical score is an artifact of data handling and interpretation. INTERPRETATION: Although MOG35-55 -induced EAE in NOD mice may provide some clues about approaches to block neurodegeneration associated with the inflammatory penumbra as lesions form, it should not be used to justify trials in people with nonactive, progressive MS. This adds further support to the view that drug studies in animals should universally adopt transparent raw data deposition as part of the publication process, such that claims can adequately be interrogated. This transparency is important if animal-based science is to remain a credible part of translational research in MS.Stichting MS ResearchWellcome TrustMedical Research CouncilNational Multiple Sclerosis Society. Grant Number: RG4132A5/