K2P2.1 is a regulator of inflammatory cell responses in idiopathic inflammatory myopathies.

K2P2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K2P2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.

Periostin as a blood biomarker of muscle cell fibrosis, cardiomyopathy and disease severity in myotonic dystrophy type 1.

BACKGROUND AND PURPOSE: Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy and is caused by an repeat expansion [r(CUG)exp] located in the 3' untranslated region of the DMPK gene. Symptoms include skeletal and cardiac muscle dysfunction and fibrosis. In DM1, there is a lack of established biomarkers in routine clinical practice. Thus, we aimed to identify a blood biomarker with relevance for DM1-pathophysiology and clinical presentation. METHODS: We collected fibroblasts from 11, skeletal muscles from 27, and blood samples from 158 DM1 patients. Moreover, serum, cardiac, and skeletal muscle samples from DMSXL mice were included. We employed proteomics, immunostaining, qPCR and ELISA. Periostin level were correlated with CMRI-data available for some patients. RESULTS: Our studies identified Periostin, a modulator of fibrosis, as a novel biomarker candidate for DM1: proteomic profiling of human fibroblasts and murine skeletal muscles showed significant dysregulation of Periostin. Immunostaining on skeletal and cardiac muscles from DM1 patients and DMSXL mice showed an extracellular increase of Periostin, indicating fibrosis. qPCR studies indicated increased POSTN expression in fibroblasts and muscle. Quantification of Periostin in blood samples from DMSXL mice and two large validation cohorts of DM1 patients showed decreased levels in animals and diseased individuals correlating with repeat expansion and disease severity and presence of cardiac symptoms identified by MRI. Analyses of longitudinal blood samples revealed no correlation with disease progression. CONCLUSIONS: Periostin might serve as a novel stratification biomarker for DM1 correlating with disease severity, presence of cardiac malfunction and fibrosis.

Validation of TREK1 ion channel activators as an immunomodulatory and neuroprotective strategy in neuroinflammation.

Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.

Neuron-oligodendrocyte potassium shuttling at nodes of Ranvier protects against inflammatory demyelination.

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS. Increasing evidence suggests that vulnerable neurons in MS exhibit fatal metabolic exhaustion over time, a phenomenon hypothesized to be caused by chronic hyperexcitability. Axonal Kv7 (outward-rectifying) and oligodendroglial Kir4.1 (inward-rectifying) potassium channels have important roles in regulating neuronal excitability at and around the nodes of Ranvier. Here, we studied the spatial and functional relationship between neuronal Kv7 and oligodendroglial Kir4.1 channels and assessed the transcriptional and functional signatures of cortical and retinal projection neurons under physiological and inflammatory demyelinating conditions. We found that both channels became dysregulated in MS and experimental autoimmune encephalomyelitis (EAE), with Kir4.1 channels being chronically downregulated and Kv7 channel subunits being transiently upregulated during inflammatory demyelination. Further, we observed that pharmacological Kv7 channel opening with retigabine reduced neuronal hyperexcitability in human and EAE neurons, improved clinical EAE signs, and rescued neuronal pathology in oligodendrocyte-Kir4.1-deficient (OL-Kir4.1-deficient) mice. In summary, our findings indicate that neuron-OL compensatory interactions promoted resilience through Kv7 and Kir4.1 channels and identify pharmacological activation of nodal Kv7 channels as a neuroprotective strategy against inflammatory demyelination.

Cladribine treatment improves cortical network functionality in a mouse model of autoimmune encephalomyelitis.

BACKGROUND: Cladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice. METHODS: In the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo. RESULTS: Oral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient. CONCLUSIONS: Our results indicate that cladribine exerts a neuroprotective effect after crossing the blood-brain barrier independently of its peripheral immunosuppressant action.

Efficient co-isolation of microvascular endothelial cells and satellite cell-derived myoblasts from human skeletal muscle.

Vascularization of tissue-engineered constructs remains a key challenge in the field of skeletal muscle tissue engineering. One strategy for vascularizing organoids is in vitro pre-vascularization, relying on de novo assembly of undifferentiated endothelial cells into capillaries, a process termed vasculogenesis. In most endothelial cell research to date, human umbilical vein endothelial cells have been used primarily because of their availability. Nevertheless, this endothelial cell type is naturally not occurring in skeletal muscle tissue. Since endothelial cells display a tissue-specific phenotype, it is of interest to use muscle-specific microvascular endothelial cells to study pre-vascularization in skeletal muscle tissue engineering research. Thus far, tissue biopsies had to be processed in two separate protocols to obtain cells from the myogenic and the endothelial compartment. Here, we describe a novel, detailed protocol for the co-isolation of human skeletal muscle microvascular endothelial cells and satellite cell-derived myoblasts. It incorporates an automated mechanical and enzymatic tissue dissociation followed by magnetically activated cell sorting based on a combination of endothelial and skeletal muscle cell markers. Qualitative, quantitative, and functional characterization of the obtained cells is described and demonstrated by representative results. The simultaneous isolation of both cell types from the same donor is advantageous in terms of time efficiency. In addition, it may be the only possible method to isolate both cell types as the amount of tissue biopsy is often limited. The isolation of the two cell types is crucial for further studies to elucidate cell crosstalk in health and disease. Furthermore, the use of muscle-specific microvascular endothelial cells allows a shift towards engineering more physiologically relevant functional tissue, with downstream applications including drug screening and regenerative medicine.

Deficiency of the Two-Pore Potassium Channel KCNK9 Impairs Intestinal Epithelial Cell Survival and Aggravates Dextran Sodium Sulfate-Induced Colitis.

BACKGROUND & AIMS: The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells. METHODS: Kcnk9-/- mice were challenged with 3% dextran sulfate sodium (DSS) to induce experimental acute colitis. Primary cultures of intestinal epithelial cells were generated, and expression of potassium channels as well as cytosolic calcium levels and susceptibility to apoptosis were evaluated. Furthermore, we evaluated whether KCNK9 deficiency was compensated by the closely related 2-pore potassium channel KCNK3 in vivo or in vitro. RESULTS: Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9-dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk9-/- mice but did not prevent apoptosis after DSS exposure. Conversely, increased levels of KCNK9 in Kcnk3-/- mice were associated with an ameliorated course of DSS-induced colitis. CONCLUSIONS: KCNK9 enhances mitochondrial stability, reduces apoptosis, und thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3-/- resulted in less mitochondrial damage and apoptosis and was associated with beneficial outcomes in DSS-induced colitis.

Alemtuzumab-induced immune phenotype and repertoire changes: implications for secondary autoimmunity.

Alemtuzumab is a monoclonal antibody that causes rapid depletion of CD52-expressing immune cells. It has proven to be highly efficacious in active relapsing-remitting multiple sclerosis; however, the high risk of secondary autoimmune disorders has greatly complicated its use. Thus, deeper insight into the pathophysiology of secondary autoimmunity and potential biomarkers is urgently needed. The most critical time points in the decision-making process for alemtuzumab therapy are before or at Month 12, where the ability to identify secondary autoimmunity risk would be instrumental. Therefore, we investigated components of blood and CSF of up to 106 multiple sclerosis patients before and after alemtuzumab treatment focusing on those critical time points. Consistent with previous reports, deep flow cytometric immune-cell profiling (n = 30) demonstrated major effects on adaptive rather than innate immunity, which favoured regulatory immune cell subsets within the repopulation. The longitudinally studied CSF compartment (n = 18) mainly mirrored the immunological effects observed in the periphery. Alemtuzumab-induced changes including increased numbers of naïve CD4+ T cells and B cells as well as a clonal renewal of CD4+ T- and B-cell repertoires were partly reminiscent of haematopoietic stem cell transplantation; in contrast, thymopoiesis was reduced and clonal renewal of T-cell repertoires after alemtuzumab was incomplete. Stratification for secondary autoimmunity did not show clear immununological cellular or proteomic traits or signatures associated with secondary autoimmunity. However, a restricted T-cell repertoire with hyperexpanded T-cell clones at baseline, which persisted and demonstrated further expansion at Month 12 by homeostatic proliferation, identified patients developing secondary autoimmune disorders (n = 7 without secondary autoimmunity versus n = 5 with secondary autoimmunity). Those processes were followed by an expansion of memory B-cell clones irrespective of persistence, which we detected shortly after the diagnosis of secondary autoimmune disease. In conclusion, our data demonstrate that (i) peripheral immunological alterations following alemtuzumab are mirrored by longitudinal changes in the CSF; (ii) incomplete T-cell repertoire renewal and reduced thymopoiesis contribute to a proautoimmune state after alemtuzumab; (iii) proteomics and surface immunological phenotyping do not identify patients at risk for secondary autoimmune disorders; (iv) homeostatic proliferation with disparate dynamics of clonal T- and B-cell expansions are associated with secondary autoimmunity; and (v) hyperexpanded T-cell clones at baseline and Month 12 may be used as a biomarker for the risk of alemtuzumab-induced autoimmunity.

Skeletal muscle provides the immunological micro-milieu for specific plasma cells in anti-synthetase syndrome-associated myositis.

Anti-synthetase syndrome (ASyS)-associated myositis is a major subgroup of the idiopathic inflammatory myopathies (IIM) and is characterized by disease chronicity with musculoskeletal, dermatological and pulmonary manifestations. One of eight autoantibodies against the aminoacyl-transferase RNA synthetases (ARS) is detectable in the serum of affected patients. However, disease-specific therapeutic approaches have not yet been established.To obtain a deeper understanding of the underlying pathogenesis and to identify putative therapeutic targets, we comparatively investigated the most common forms of ASyS associated with anti-PL-7, anti-PL-12 and anti-Jo-1. Our cohort consisted of 80 ASyS patients as well as healthy controls (n = 40), diseased controls (n = 40) and non-diseased controls (n = 20). We detected a reduced extent of necrosis and regeneration in muscle biopsies from PL-12+ patients compared to Jo-1+ patients, while PL-7+ patients had higher capillary dropout in biopsies of skeletal muscle. Aside from these subtle alterations, no significant differences between ASyS subgroups were observed. Interestingly, a tissue-specific subpopulation of CD138+ plasma cells and CXCL12+/CXCL13+CD20+ B cells common to ASyS myositis were identified. These cells were localized in the endomysium associated with alkaline phosphatase+ activated mesenchymal fibroblasts and CD68+MHC-II+CD169+ macrophages. An MHC-I+ and MHC-II+ MxA negative type II interferon-driven milieu of myofiber activation, topographically restricted to the perifascicular area and the adjacent perimysium, as well as perimysial clusters of T follicular helper cells defined an extra-medullary immunological niche for plasma cells and activated B cells. Consistent with this, proteomic analyses of muscle tissues from ASyS patients demonstrated alterations in antigen processing and presentation. In-depth immunological analyses of peripheral blood supported a B-cell/plasma-cell-driven pathology with a shift towards immature B cells, an increase of B-cell-related cytokines and chemokines, and activation of the complement system. We hypothesize that a B-cell-driven pathology with the presence and persistence of a specific subtype of plasma cells in the skeletal muscle is crucially involved in the self-perpetuating chronicity of ASyS myositis. This work provides the conceptual framework for the application of plasma-cell-targeting therapies in ASyS myositis.

Immune response to SARS-CoV-2 vaccination in relation to peripheral immune cell profiles among patients with multiple sclerosis receiving ocrelizumab.

BACKGROUND: Vaccination has proven to be effective in preventing SARS-CoV-2 transmission and severe disease courses. However, immunocompromised patients have not been included in clinical trials and real-world clinical data point to an attenuated immune response to SARS-CoV-2 vaccines among patients with multiple sclerosis (MS) receiving immunomodulatory therapies. METHODS: We performed a retrospective study including 59 ocrelizumab (OCR)-treated patients with MS who received SARS-CoV-2 vaccination. Anti-SARS-CoV-2-antibody titres, routine blood parameters and peripheral immune cell profiles were measured prior to the first (baseline) and at a median of 4 weeks after the second vaccine dose (follow-up). Moreover, the SARS-CoV-2-specific T cell response and peripheral B cell subsets were analysed at follow-up. Finally, vaccination-related adverse events were assessed. RESULTS: After vaccination, we found anti-SARS-CoV-2(S) antibodies in 27.1% and a SARS-CoV-2-specific T cell response in 92.7% of MS cases. T cell-mediated interferon (IFN)-γ release was more pronounced in patients without anti-SARS-CoV-2(S) antibodies. Antibody titres positively correlated with peripheral B cell counts, time since last infusion and total IgM levels. They negatively correlated with the number of previous infusion cycles. Peripheral plasma cells were increased in antibody-positive patients. A positive correlation between T cell response and peripheral lymphocyte counts was observed. Moreover, IFN-γ release was negatively correlated with the time since the last infusion. CONCLUSION: In OCR-treated patients with MS, the humoral immune response to SARS-CoV-2 vaccination is attenuated while the T cell response is preserved. However, it is still unclear whether T or B cell-mediated immunity is required for effective clinical protection. Nonetheless, given the long-lasting clinical effects of OCR, monitoring of peripheral B cell counts could facilitate individualised treatment regimens and might be used to identify the optimal time to vaccinate.

Translational value of choroid plexus imaging for tracking neuroinflammation in mice and humans.

Neuroinflammation is a pathophysiological hallmark of multiple sclerosis and has a close mechanistic link to neurodegeneration. Although this link is potentially targetable, robust translatable models to reliably quantify and track neuroinflammation in both mice and humans are lacking. The choroid plexus (ChP) plays a pivotal role in regulating the trafficking of immune cells from the brain parenchyma into the cerebrospinal fluid (CSF) and has recently attracted attention as a key structure in the initiation of inflammatory brain responses. In a translational framework, we here address the integrity and multidimensional characteristics of the ChP under inflammatory conditions and question whether ChP volumes could act as an interspecies marker of neuroinflammation that closely interrelates with functional impairment. Therefore, we explore ChP characteristics in neuroinflammation in patients with multiple sclerosis and in two experimental mouse models, cuprizone diet-related demyelination and experimental autoimmune encephalomyelitis. We demonstrate that ChP enlargement-reconstructed from MRI-is highly associated with acute disease activity, both in the studied mouse models and in humans. A close dependency of ChP integrity and molecular signatures of neuroinflammation is shown in the performed transcriptomic analyses. Moreover, pharmacological modulation of the blood-CSF barrier with natalizumab prevents an increase of the ChP volume. ChP enlargement is strongly linked to emerging functional impairment as depicted in the mouse models and in multiple sclerosis patients. Our findings identify ChP characteristics as robust and translatable hallmarks of acute and ongoing neuroinflammatory activity in mice and humans that could serve as a promising interspecies marker for translational and reverse-translational approaches.

Pathophysiological Role of K2P Channels in Human Diseases.

The family of two-pore domain potassium (K2P) channels is critically involved in central cellular functions such as ion homeostasis, cell development, and excitability. K2P channels are widely expressed in different human cell types and organs. It is therefore not surprising that aberrant expression and function of K2P channels are related to a spectrum of human diseases, including cancer, autoimmune, CNS, cardiovascular, and urinary tract disorders. Despite homologies in structure, expression, and stimulus, the functional diversity of K2P channels leads to heterogeneous influences on human diseases. The role of individual K2P channels in different disorders depends on expression patterns and modulation in cellular functions. However, an imbalance of potassium homeostasis and action potentials contributes to most disease pathologies. In this review, we provide an overview of current knowledge on the role of K2P channels in human diseases. We look at altered channel expression and function, the potential underlying molecular mechanisms, and prospective research directions in the field of K2P channels.

Production of glycosylphosphatidylinositol-anchored proteins for vaccines and directed binding of immunoliposomes to specific cell types.

BACKGROUND: Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. Furthermore, the introduction of a universal immunoglobulin-binding domain can make liposomes targetable to virtually any desired receptor for which antibodies exist. METHODS: We developed a system for the production of recombinant proteins with GPI anchors and histidine tags and Strep-tags for simplified purification from cells. This system was applied to i) the green fluorescent protein (GFP) as a reporter, ii) the promising Plasmodium falciparum vaccine antigen PfRH5 and iii) a doubled immunoglobulin Fc-binding domain termed ZZ from protein A of Staphylococcus aureus. As the GPI-attachment domain, the C-terminus of murine CD14 was used. After the recovery of these three recombinant proteins from Chinese hamster ovary (CHO) cells and association with liposomes, their vaccine potential and ability to target the CD4 receptor on lymphocytes in ex vivo conditions were tested. RESULTS: Upon immunization in mice, the PfRH5-GPI-loaded liposomes generated antibody titers of 103 to 104, and showed a 45% inhibitory effect on in vitro growth at an IgG concentration of 600 µg/mL in P. falciparum cultures. Using GPI-anchored ZZ to couple anti-CD4 antibodies to liposomes, we created immunoliposomes with a binding efficiency of 75% to CD4+ cells in splenocytes and minimal off-target binding. CONCLUSIONS: Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes. Importantly, the CHO-cell and GPI-tagged produced PfRH5 elicited invasion-blocking antibodies qualitatively comparable to other approaches.

Production of glycosylphosphatidylinositol-anchored proteins for vaccines and directed binding of immunoliposomes to specific cell types

Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. Furthermore, the introduction of a universal immunoglobulin-binding domain can make liposomes targetable to virtually any desired receptor for which antibodies exist. Methods: We developed a system for the production of recombinant proteins with GPI anchors and histidine tags and Strep-tags for simplified purification from cells. This system was applied to i) the green fluorescent protein (GFP) as a reporter, ii) the promising Plasmodium falciparum vaccine antigen PfRH5 and iii) a doubled immunoglobulin Fc-binding domain termed ZZ from protein A of Staphylococcus aureus. As the GPI-attachment domain, the C-terminus of murine CD14 was used. After the recovery of these three recombinant proteins from Chinese hamster ovary (CHO) cells and association with liposomes, their vaccine potential and ability to target the CD4 receptor on lymphocytes in ex vivo conditions were tested. Results: Upon immunization in mice, the PfRH5-GPI-loaded liposomes generated antibody titers of 103 to 104, and showed a 45% inhibitory effect on in vitro growth at an IgG concentration of 600 µg/mL in P. falciparum cultures. Using GPI-anchored ZZ to couple anti-CD4 antibodies to liposomes, we created immunoliposomes with a binding efficiency of 75% to CD4+ cells in splenocytes and minimal off-target binding. Conclusions: Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes. Importantly, the CHO-cell and GPI-tagged produced PfRH5 elicited invasion-blocking antibodies qualitatively comparable to other approaches.(AU)

A multilamellar nanoliposome stabilized by interlayer hydrogen bonds increases antimalarial drug efficacy.

Lipid particles for drug delivery can be modified to create multilayer vesicles with higher stability and improved cargo interaction. Here, we used lipids capable of forming hydrogen bonds instead of covalent bonds and designed stable vesicles-inside-vesicles with a high capacity of entrapping antimalarial drugs such as chloroquine (hydrophilic) and Artemisinin (lipophilic). In vitro treatment of the drug-sensitive P. falciparum strain NF54 showed that encapsulated drugs resulted in 72% and 60% lower IC50 values for each drug, respectively. Fluorochrome-labeling of a cargo-peptide or of membrane-resident lipids indicated that vesicles interacted more specifically with parasite-infected erythrocytes than with normal red blood cells. Accordingly, vesicle-confined chloroquine was able to elicit a stronger antiparasitic effect than free chloroquine in a lethal murine model of infection. Being permissive not only to small molecules but also to larger peptides, hydrogen-bond linked multilamellar liposomes are a very promising approach for enhanced drug delivery.

The next-generation sphingosine-1 receptor modulator BAF312 (siponimod) improves cortical network functionality in focal autoimmune encephalomyelitis.

Autoimmune diseases of the central nervous system (CNS) like multiple sclerosis (MS) are characterized by inflammation and demyelinated lesions in white and grey matter regions. While inflammation is present at all stages of MS, it is more pronounced in the relapsing forms of the disease, whereas progressive MS (PMS) shows significant neuroaxonal damage and grey and white matter atrophy. Hence, disease-modifying treatments beneficial in patients with relapsing MS have limited success in PMS. BAF312 (siponimod) is a novel sphingosine-1-phosphate receptor modulator shown to delay progression in PMS. Besides reducing inflammation by sequestering lymphocytes in lymphoid tissues, BAF312 crosses the blood-brain barrier and binds its receptors on neurons, astrocytes and oligodendrocytes. To evaluate potential direct neuroprotective effects, BAF312 was systemically or locally administered in the CNS of experimental autoimmune encephalomyelitis mice with distinct grey- and white-matter lesions (focal experimental autoimmune encephalomyelitis using an osmotic mini-pump). Ex-vivo flow cytometry revealed that systemic but not local BAF312 administration lowered immune cell infiltration in animals with both grey and white matter lesions. Ex-vivo voltage-sensitive dye imaging of acute brain slices revealed an altered spatio-temporal pattern of activation in the lesioned cortex compared to controls in response to electrical stimulation of incoming white-matter fiber tracts. Here, BAF312 administration showed partial restore of cortical neuronal circuit function. The data suggest that BAF312 exerts a neuroprotective effect after crossing the blood-brain barrier independently of peripheral effects on immune cells. Experiments were carried out in accordance with German and EU animal protection law and approved by local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen; 87-51.04.2010.A331) on December 28, 2010.

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional "myovascular unit" (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes. This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Targeting Voltage-Dependent Calcium Channels with Pregabalin Exerts a Direct Neuroprotective Effect in an Animal Model of Multiple Sclerosis.

BACKGROUND/AIMS: Multiple sclerosis (MS) is a prototypical autoimmune central nervous system (CNS) disease. Particularly progressive forms of MS (PMS) show significant neuroaxonal damage as consequence of demyelination and neuronal hyperexcitation. Immuno-modulatory treatment strategies are beneficial in relapsing MS (RMS), but mostly fail in PMS. Pregabalin (Lyrica®) is prescribed to MS patients to treat neuropathic pain. Mechanistically, it targets voltage-dependent Ca2+ channels and reduces harmful neuronal hyperexcitation in mouse epilepsy models. Studies suggest that GABA analogues like pregabalin exert neuroprotective effects in animal models of ischemia and trauma. METHODS: We tested the impact of pregabalin in a mouse model of MS (experimental autoimmune encephalomyelitis, EAE) and performed histological and immunological evaluations as well as intravital two-photon-microscopy of brainstem EAE lesions. RESULTS: Both prophylactic and therapeutic treatments ameliorated the clinical symptoms of EAE and reduced immune cell infiltration into the CNS. On neuronal level, pregabalin reduced long-term potentiation in hippocampal brain slices indicating an impact on mechanisms of learning and memory. In contrast, T cells, microglia and brain endothelial cells were unaffected by pregabalin. However, we found a direct impact of pregabalin on neurons during CNS inflammation as it reversed the pathological elevation of neuronal intracellular Ca2+ levels in EAE lesions. CONCLUSION: The presented data suggest that pregabalin primarily acts on neuronal Ca2+ channel trafficking thereby reducing Ca2+-mediated cytotoxicity and neuronal damage in an animal model of MS. Future clinical trials need to assess the benefit for neuronal survival by expanding the indication for pregabalin administration to MS patients in further disease phases.

Skeletal muscle cells actively shape (auto)immune responses.

Histopathological analyses of muscle specimens from myositis patients indicate that skeletal muscle cells play an active role in the interaction with immune cells. Research over the last few decades has shown that skeletal muscle cells exhibit immunobiological properties that perfectly define them as non-professional antigen presenting cells. They are able to present antigens via major histocompatibility complex molecules, exhibit costimulatory molecules and secrete soluble molecules that actively shape the immune response in an either pro- or anti-inflammatory manner. Skeletal muscle cells regulate both innate and adaptive immune responses and are essentially involved in the pathophysiological processes of idiopathic inflammatory myopathies. Understanding the role of skeletal muscle cells might help to identify new therapeutic targets for these devastating diseases. This review summarizes the immunobiological features of skeletal muscle cells, especially in the context of idiopathic inflammatory myopathies, and discusses shortcomings and limitations in skeletal muscle related research providing potential perspectives to overcome them in the future.

The role of ion channels in malignant brain tumors.

Malignant gliomas are the most common primary brain tumors and have poor clinical prognosis, despite multimodal therapeutic strategies. In recent years, ion channels have emerged as major players in tumor pathophysiology regarding all hallmarks of cancer. Since ion channels are easily accessible structures, they may prove to be effective targets for canner therapy, although their broad expression pattern and role in physiological processes should be taken into consideration. This review summarizes the current knowledge on the role of ion channels in the pathophysiology of malignant gliomas, especially glioblastoma, and evaluates their potential role in targeted antiglioma therapy.