Antibody cross-reactivity between casein and myelin-associated glycoprotein results in central nervous system demyelination.

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease of the central nervous system (CNS) with a high socioeconomic relevance. The pathophysiology of MS, which is both complex and incompletely understood, is believed to be influenced by various environmental determinants, including diet. Since the 1990s, a correlation between the consumption of bovine milk products and MS prevalence has been debated. Here, we show that C57BL/6 mice immunized with bovine casein developed severe spinal cord pathology, in particular, demyelination, which was associated with the deposition of immunoglobulin G. Furthermore, we observed binding of serum from casein-immunized mice to mouse oligodendrocytes in CNS tissue sections and in culture where casein-specific antibodies induced complement-dependent pathology. We subsequently identified myelin-associated glycoprotein (MAG) as a cross-reactive antigenic target. The results obtained from the mouse model were complemented by clinical data showing that serum samples from patients with MS contained significantly higher B cell and antibody reactivity to bovine casein than those from patients with other neurologic diseases. This reactivity correlated with the B cell response to a mixture of CNS antigens and could again be attributed to MAG reactivity. While we acknowledge disease heterogeneity among individuals with MS, we believe that consumption of cow's milk in a subset of patients with MS who have experienced a previous loss of tolerance to bovine casein may aggravate the disease. Our data suggest that patients with antibodies to bovine casein might benefit from restricting dairy products from their diet.

The presence of cerebellar B cell aggregates is associated with a specific chemokine profile in the cerebrospinal fluid in a mouse model of multiple sclerosis.

BACKGROUND: The presence of meningeal ectopic lymphoid structures (ELS) in a subgroup of patients diagnosed with secondary progressive multiple sclerosis (SPMS) corresponds to a pronounced cortical inflammation and an aggravated disease course. In MP4-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), B cell aggregates develop in the central nervous system (CNS) in the chronic stage of the disease. Therefore, the model is suitable for studying key molecules of ELS development and maintenance. Here, we investigated whether there is a specific cytokine and chemokine signature in paired cerebrospinal fluid (CSF) and serum samples associated with the presence of cerebellar B cell and T cell pathology and B cell aggregates of MP4-immunized mice. METHODS: Paired CSF and serum samples were collected from the cisterna magna and periphery of MP4-immunized mice at the chronic stage of disease. A control group with mice immunized only with the adjuvant (vehicle) was included in the study. A selected panel of 34 cytokines and chemokines were measured by MAGPIX® for both cohorts. For the assessment of B cell and T cell infiltration, immunohistochemical staining was performed and analyzed using light microscopy. To detect specific chemokine receptors additional staining was conducted. RESULTS: While we detected several upregulated cytokines and chemokines in the CSF of MP4-immunized mice independent of the extent of B cell and T cell pathology compared to vehicle-immunized mice, C-C motif chemokine ligand (CCL)-1 was associated with high B cell and T cell infiltration. Furthermore, the level of certain chemokines, including CCL1, CCL5, CCL7, CCL12, CCL22 and C-X-C motif chemokine ligand (CXCL)-13, was significantly increased (p < 0.05) in MP4-immunized mice showing a high number of B cell aggregates. While C-C motif chemokine receptor (CCR)5 had a ubiquitous expression independent of the extent of B cell and T cell pathology, C-X-C motif chemokine receptor (CXCR)-5 and CXCR6 expression was specifically associated with high B cell and T cell pathology. CONCLUSION: Our data suggest that multiple cytokines and chemokines are involved in the pathophysiology of MP4-induced EAE. Furthermore, the presence of B cell aggregates was associated with a specific chemokine profile in the CSF, which might be useful for predicting the presence of these aggregates without the necessity to histologically screen the CNS tissue.

Autoantibodies against central nervous system antigens in a subset of B cell-dominant multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), with characteristic inflammatory lesions and demyelination. The clinical benefit of cell-depleting therapies targeting CD20 has emphasized the role of B cells and autoantibodies in MS pathogenesis. We previously introduced an enzyme-linked immunospot spot (ELISpot)-based assay to measure CNS antigen-specific B cells in the blood of MS patients and demonstrated its usefulness as a predictive biomarker for disease activity in measuring the successful outcome of disease-modifying therapies (DMTs). Here we used a planar protein array to investigate CNS-reactive antibodies in the serum of MS patients as well as in B cell culture supernatants after polyclonal stimulation. Anti-CNS antibody reactivity was evident in the sera of the MS cohort, and the antibodies bound a heterogeneous set of molecules, including myelin, axonal cytoskeleton, and ion channel antigens, in individual patients. Immunoglobulin reactivity in supernatants of stimulated B cells was directed against a broad range of CNS antigens. A group of MS patients with a highly active B cell component was identified by the ELISpot assay. Those antibody reactivities remained stable over time. These assays with protein arrays identify MS patients with a highly active B cell population with antibodies directed against a swathe of CNS proteins.

Dynamic study of the finger interphalangeal joint volar plate-motion analysis with magnetic resonance cinematography and histologic comparison.

OBJECTIVE: We aimed to further improve knowledge about volar plate (VP) motion of the finger proximal interphalangeal joint (PIP), by analyzing the dynamic VP shape during a full range of finger flexion using magnetic resonance cinematography of the fingers (MRCF), and to compare the results with anatomical cross sections from cadaver specimens. MATERIALS AND METHODS: The dynamic sagittal VP shape was visualized with MRCF in a total number of 23 healthy volunteers. The length, angle, and thickness as well as the contact length of the VP to the PIP joint base were measured. Statistical analysis included t-test or rank-sum testing. Anatomical cross sections with differing degrees of PIP joint flexion were obtained from 12 cadaver specimens (fingers) for comparison. RESULTS: Significant positive correlations between PIP joint flexion angle and VP area, length, depth and the VP contact length were found. This matched histologically to fiber rearrangements especially within the loose third VP layer. CONCLUSION: Our study analyzed the full range of motion dynamic VP shape of the PIP joint using MRCF. This contributes to a more precise understanding of the complex interaction of the VP with the PIP joint and may facilitate evaluation of clinical cases such as VP avulsion or pulley rupture.

Impact of the Voltage-Gated Calcium Channel Antagonist Nimodipine on the Development of Oligodendrocyte Precursor Cells.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While most of the current treatment strategies focus on immune cell regulation, except for the drug siponimod, there is no therapeutic intervention that primarily aims at neuroprotection and remyelination. Recently, nimodipine showed a beneficial and remyelinating effect in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Nimodipine also positively affected astrocytes, neurons, and mature oligodendrocytes. Here we investigated the effects of nimodipine, an L-type voltage-gated calcium channel antagonist, on the expression profile of myelin genes and proteins in the oligodendrocyte precursor cell (OPC) line Oli-Neu and in primary OPCs. Our data indicate that nimodipine does not have any effect on myelin-related gene and protein expression. Furthermore, nimodipine treatment did not result in any morphological changes in these cells. However, RNA sequencing and bioinformatic analyses identified potential micro (mi)RNA that could support myelination after nimodipine treatment compared to a dimethyl sulfoxide (DMSO) control. Additionally, we treated zebrafish with nimodipine and observed a significant increase in the number of mature oligodendrocytes (* p≤ 0.05). Taken together, nimodipine seems to have different positive effects on OPCs and mature oligodendrocytes.

Bone marrow-derived myeloid progenitors in the leptomeninges of adult mice.

Although the bone marrow contains most hematopoietic activity during adulthood, hematopoietic stem and progenitor cells can be recovered from various extramedullary sites. Cells with hematopoietic progenitor properties have even been reported in the adult brain under steady-state conditions, but their nature and localization remain insufficiently defined. Here, we describe a heterogeneous population of myeloid progenitors in the leptomeninges of adult C57BL/6 mice. This cell pool included common myeloid, granulocyte/macrophage, and megakaryocyte/erythrocyte progenitors. Accordingly, it gave rise to all major myelo-erythroid lineages in clonogenic culture assays. Brain-associated progenitors persisted after tissue perfusion and were partially inaccessible to intravenous antibodies, suggesting their localization behind continuous blood vessel endothelium such as the blood-arachnoid barrier. Flt3Cre lineage tracing and bone marrow transplantation showed that the precursors were derived from adult hematopoietic stem cells and were most likely continuously replaced via cell trafficking. Importantly, their occurrence was tied to the immunologic state of the central nervous system (CNS) and was diminished in the context of neuroinflammation and ischemic stroke. Our findings confirm the presence of myeloid progenitors at the meningeal border of the brain and lay the foundation to unravel their possible functions in CNS surveillance and local immune cell production.

Aged hind-limb clasping experimental autoimmune encephalomyelitis models aspects of the neurodegenerative process seen in multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is the most common model of multiple sclerosis (MS). This model has been instrumental in understanding the events that lead to the initiation of central nervous system (CNS) autoimmunity. Though EAE has been an effective screening tool for identifying novel therapies for relapsing-remitting MS, it has proven to be less successful in identifying therapies for progressive forms of this disease. Though axon injury occurs in EAE, it is rapid and acute, making it difficult to intervene for the purpose of evaluating neuroprotective therapies. Here, we describe a variant of spontaneous EAE in the 2D2 T cell receptor transgenic mouse (2D2+ mouse) that presents with hind-limb clasping upon tail suspension and is associated with T cell-mediated inflammation in the posterior spinal cord and spinal nerve roots. Due to the mild nature of clinical signs in this model, we were able to maintain cohorts of mice into middle age. Over 9 mo, these mice exhibited a relapsing-remitting course of hind-limb clasping with the development of progressive motor deficits. Using a combined approach of ex vivo magnetic resonance (MR) imaging and histopathological analysis, we observed neurological progression to associate with spinal cord atrophy, synapse degradation, and neuron loss in the gray matter, as well as ongoing axon injury in the white matter of the spinal cord. These findings suggest that mild EAE coupled with natural aging may be a solution to better modeling the neurodegenerative processes seen in MS.

Effects of a Fully Humanized Type II Anti-CD20 Monoclonal Antibody on Peripheral and CNS B Cells in a Transgenic Mouse Model of Multiple Sclerosis.

Successful therapy with anti-CD20 monoclonal antibodies (mAbs) has reinforced the key role of B cells in the immunopathology of multiple sclerosis (MS). This study aimed to determine the effects of a novel class of anti-CD20 mAbs on vascular and extravascular central nervous system (CNS)-infiltrating B cells in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male hCD20xhIgR3 mice and wild-type C57BL/6 (B6) mice were immunized with human myelin oligodendrocyte glycoprotein (MOG)1-125 to induce EAE. While hCD20xhIgR3 mice were injected intravenously with an anti-human CD20 mAb (5 mg/kg) (rituximab (a type I anti-CD20 mAb) or obinutuzumab (a type II anti-CD20 mAb), B6 mice received the anti-mouse CD20 antibody 18B12. Neither mAb affected clinical disease or serum antibody levels. Obinutuzumab and rituximab had an impact on splenic and CNS-infiltrated B cells with slightly differential depletion efficacy. Additionally, obinutuzumab had beneficial effects on spinal cord myelination. B cell depletion rates in the 18B12/B6 model were comparable with those observed in obinutuzumab-treated hCD20xhIgR3 mice. Our results demonstrate the usefulness of anti-CD20 mAbs for the modulation of B cell-driven peripheral immune response and CNS pathology, with type II antibodies potentially being superior to type I in the depletion of tissue-infiltrating B cells.

Impact of Siponimod on Enteric and Central Nervous System Pathology in Late-Stage Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Although immune modulation and suppression are effective during relapsing-remitting MS, secondary progressive MS (SPMS) requires neuroregenerative therapeutic options that act on the CNS. The sphingosine-1-phosphate receptor modulator siponimod is the only approved drug for SPMS. In the pivotal trial, siponimod reduced disease progression and brain atrophy compared with placebo. The enteric nervous system (ENS) was recently identified as an additional autoimmune target in MS. We investigated the effects of siponimod on the ENS and CNS in the experimental autoimmune encephalomyelitis model of MS. Mice with late-stage disease were treated with siponimod, fingolimod, or sham. The clinical disease was monitored daily, and treatment success was verified using mass spectrometry and flow cytometry, which revealed peripheral lymphopenia in siponimod- and fingolimod-treated mice. We evaluated the mRNA expression, ultrastructure, and histopathology of the ENS and CNS. Single-cell RNA sequencing revealed an upregulation of proinflammatory genes in spinal cord astrocytes and ependymal cells in siponimod-treated mice. However, differences in CNS and ENS histopathology and ultrastructural pathology between the treatment groups were absent. Thus, our data suggest that siponimod and fingolimod act on the peripheral immune system and do not have pronounced direct neuroprotective effects.

Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens.

Multiple sclerosis (MS) has been considered to specifically affect the central nervous system (CNS) for a long time. As autonomic dysfunction including dysphagia can occur as accompanying phenomena in patients, the enteric nervous system has been attracting increasing attention over the past years. The aim of this study was to identify glial and myelin markers as potential target structures for autoimmune processes in the esophagus. RT-PCR analysis revealed glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), and myelin basic protein (MBP) expression, but an absence of myelin oligodendrocyte glycoprotein (MOG) in the murine esophagus. Selected immunohistochemistry for GFAP, PLP, and MBP including transgenic mice with cell-type specific expression of PLP and GFAP supported these results by detection of (1) GFAP, PLP, and MBP in Schwann cells in skeletal muscle and esophagus; (2) GFAP, PLP, but no MBP in perisynaptic Schwann cells of skeletal and esophageal motor endplates; (3) GFAP and PLP, but no MBP in glial cells surrounding esophageal myenteric neurons; and (4) PLP, but no GFAP and MBP in enteric glial cells forming a network in the esophagus. Our results pave the way for further investigations regarding the involvement of esophageal glial cells in the pathogenesis of dysphagia in MS.

Same same but different: A Web-based deep learning application revealed classifying features for the histopathologic distinction of cortical malformations.

OBJECTIVE: The microscopic review of hematoxylin-eosin-stained images of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex remains challenging. Both entities are distinct subtypes of human malformations of cortical development that share histopathological features consisting of neuronal dyslamination with dysmorphic neurons and balloon cells. We trained a convolutional neural network (CNN) to classify both entities and visualize the results. Additionally, we propose a new Web-based deep learning application as proof of concept of how deep learning could enter the pathologic routine. METHODS: A digital processing pipeline was developed for a series of 56 cases of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex to obtain 4000 regions of interest and 200 000 subsamples with different zoom and rotation angles to train a neural network. Guided gradient-weighted class activation maps (Guided Grad-CAMs) were generated to visualize morphological features used by the CNN to distinguish both entities. RESULTS: Our best-performing network achieved 91% accuracy and 0.88 area under the receiver operating characteristic curve at the tile level for an unseen test set. Novel histopathologic patterns were found through the visualized Guided Grad-CAMs. These patterns were assembled into a classification score to augment decision-making in routine histopathology workup. This score was successfully validated by 11 expert neuropathologists and 12 nonexperts, boosting nonexperts to expert level performance. SIGNIFICANCE: Our newly developed Web application combines the visualization of whole slide images with the possibility of deep learning-aided classification between focal cortical dysplasia IIb and tuberous sclerosis complex. This approach will help to introduce deep learning applications and visualization for the histopathologic diagnosis of rare and difficult-to-classify brain lesions.

Generation of Cardiomyocytes From Vascular Adventitia-Resident Stem Cells.

RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.

Nimodipine fosters remyelination in a mouse model of multiple sclerosis and induces microglia-specific apoptosis.

Despite continuous interest in multiple sclerosis (MS) research, there is still a lack of neuroprotective strategies, because the main focus has remained on modulating the immune response. Here we performed in-depth analysis of neurodegeneration in experimental autoimmune encephalomyelitis (EAE) and in in vitro studies regarding the effect of the well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated clinical EAE and spinal cord degeneration and promoted remyelination. Surprisingly, we observed calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell-type specific and irrespective of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS and reactive oxygen species levels in EAE. In addition, increased numbers of Olig2+APC+ oligodendrocytes were detected. Overall, nimodipine application seems to generate a favorable environment for regenerative processes and therefore could be a treatment option for MS, because it combines features of immunomodulation with beneficial effects on neuroregeneration.

Strategies for Neuroprotection in Multiple Sclerosis and the Role of Calcium.

Calcium ions are vital for maintaining the physiological and biochemical processes inside cells. The central nervous system (CNS) is particularly dependent on calcium homeostasis and its dysregulation has been associated with several neurodegenerative disorders including Parkinson's disease (PD), Alzheimer's disease (AD) and Huntington's disease (HD), as well as with multiple sclerosis (MS). Hence, the modulation of calcium influx into the cells and the targeting of calcium-mediated signaling pathways may present a promising therapeutic approach for these diseases. This review provides an overview on calcium channels in neurons and glial cells. Special emphasis is put on MS, a chronic autoimmune disease of the CNS. While the initial relapsing-remitting stage of MS can be treated effectively with immune modulatory and immunosuppressive drugs, the subsequent progressive stage has remained largely untreatable. Here we summarize several approaches that have been and are currently being tested for their neuroprotective capacities in MS and we discuss which role calcium could play in this regard.

Obinutuzumab-Induced B Cell Depletion Reduces Spinal Cord Pathology in a CD20 Double Transgenic Mouse Model of Multiple Sclerosis.

B cell-depleting therapies have recently proven to be clinically highly successful in the treatment of multiple sclerosis (MS). This study aimed to determine the effects of the novel type II anti-human CD20 (huCD20) monoclonal antibody (mAb) obinutuzumab (OBZ) on spinal cord degeneration in a B cell-dependent mouse model of MS. Double transgenic huCD20xHIGR3 (CD20dbtg) mice, which express human CD20, were immunised with the myelin fusion protein MP4 to induce experimental autoimmune encephalomyelitis (EAE). Both light and electron microscopy were used to assess myelination and axonal pathology in mice treated with OBZ during chronic EAE. Furthermore, the effects of the already established murine anti-CD20 antibody 18B12 were assessed in C57BL/6 wild-type (wt) mice. In both models (18B12/wt and OBZ/CD20dbtg) anti-CD20 treatment significantly diminished the extent of spinal cord pathology. While 18B12 treatment mainly reduced the extent of axonal pathology, a significant decrease in demyelination and increase in remyelination were additionally observed in OBZ-treated mice. Hence, the data suggest that OBZ could have neuroprotective effects on the CNS, setting the drug apart from the currently available type I anti-CD20 antibodies.

Contribution of LTi and TH17 cells to B cell aggregate formation in the central nervous system in a mouse model of multiple sclerosis.

BACKGROUND: In a subgroup of patients suffering from progressive multiple sclerosis (MS), which is an inflammation-mediated neurodegenerative disease of the central nervous system (CNS), B cell aggregates were discovered within the meninges. Occurrence of these structures was associated with a more severe disease course and cortical histopathology. We have developed the B cell-dependent MP4-induced experimental autoimmune encephalomyelitis (EAE) as a mouse model to mimic this trait of the human disease. The aim of this study was to determine a potential role of lymphoid tissue inducer (LTi) and TH17 cells in the process of B cell aggregate formation in the MP4 model. METHODS: We performed flow cytometry of cerebellar and splenic tissue of MP4-immunized mice in the acute and chronic stage of the disease to analyze the presence of CD3-CD5-CD4+RORγt+ LTi and CD3+CD5+CD4+RORγt+ TH17 cells. Myelin oligodendrocyte glycoprotein (MOG):35-55-induced EAE was used as B cell-independent control model. We further determined the gene expression profile of B cell aggregates using laser capture microdissection, followed by RNA sequencing. RESULTS: While we were able to detect LTi cells in the embryonic spleen and adult intestine, which served as positive controls, there was no evidence for the existence of such a population in acute or chronic EAE in neither of the two models. Yet, we detected CD3-CD5-CD4-RORγt+ innate lymphoid cells (ILCs) and TH17 cells in the CNS, the latter especially in the chronic stage of MP4-induced EAE. Moreover, we observed a unique gene signature in CNS B cell aggregates compared to draining lymph nodes of MP4-immunized mice and to cerebellum as well as draining lymph nodes of mice with MOG:35-55-induced EAE. CONCLUSION: The absence of LTi cells in the cerebellum suggests that other cells might take over the function as an initiator of lymphoid tissue formation in the CNS. Overall, the development of ectopic lymphoid organs is a complex process based on an interplay between several molecules and signals. Here, we propose some potential candidates, which might be involved in the formation of B cell aggregates in the CNS of MP4-immunized mice.

Nav1.6 promotes inflammation and neuronal degeneration in a mouse model of multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) model of MS, the Nav1.6 voltage-gated sodium (Nav) channel isoform has been implicated as a primary contributor to axonal degeneration. Following demyelination Nav1.6, which is normally co-localized with the Na+/Ca2+ exchanger (NCX) at the nodes of Ranvier, associates with ß-APP, a marker of neural injury. The persistent influx of sodium through Nav1.6 is believed to reverse the function of NCX, resulting in an increased influx of damaging Ca2+ ions. However, direct evidence for the role of Nav1.6 in axonal degeneration is lacking. METHODS: In mice floxed for Scn8a, the gene that encodes the α subunit of Nav1.6, subjected to EAE we examined the effect of eliminating Nav1.6 from retinal ganglion cells (RGC) in one eye using an AAV vector harboring Cre and GFP, while using the contralateral either injected with AAV vector harboring GFP alone or non-targeted eye as control. RESULTS: In retinas, the expression of Rbpms, a marker for retinal ganglion cells, was found to be inversely correlated to the expression of Scn8a. Furthermore, the gene expression of the pro-inflammatory cytokines Il6 (IL-6) and Ifng (IFN-γ), and of the reactive gliosis marker Gfap (GFAP) were found to be reduced in targeted retinas. Optic nerves from targeted eyes were shown to have reduced macrophage infiltration and improved axonal health. CONCLUSION: Taken together, our results are consistent with Nav1.6 promoting inflammation and contributing to axonal degeneration following demyelination.

Characterization of blood-brain barrier integrity in a B-cell-dependent mouse model of multiple sclerosis.

Recent studies with B-cell-depleting antibodies have demonstrated clinical success in the treatment of multiple sclerosis (MS) patients. While these antibodies efficiently target B cells in the blood, it is unclear how effective they are in the central nervous system (CNS), especially in the context of limited blood-brain barrier (BBB) permeability and the ongoing discussion on the relevance of B-cell aggregate formation in the brains of SP-MS patients. The aim of this study was to evaluate BBB integrity in the context of B-cell-dependent neuroinflammation in a mouse model of MS. C57BL/6 mice were actively immunized with either myelin oligodendrocyte glycoprotein peptide 35-55 to induce T-cell-dependent experimental autoimmune encephalomyelitis (EAE), or with the myelin basic protein-proteolipid protein fusion protein MP4 for additional B-cell dependence. BBB integrity was assessed using Evans Blue or fluorescein isothiocyanate-dextran injection, respectively, in combination with immunofluorescence staining for key components of the BBB. In both EAE models, tracer leakage into the CNS parenchyma was observed indicating BBB leakiness. Yet, intensity and distribution patterns of leakage differed between the two models. There was no difference in the severity of BBB damage comparing acute and chronic MP4-induced EAE, but the formation of B-cell aggregates was associated with local BBB impairment in this model. This study underscores that a leaky BBB is a characteristic feature of EAE, but it also suggests that extent and region specificity of BBB damage differs between individual EAE models that vary in the underlying immunopathology.

Digital pathology imaging and computer-aided diagnostics as a novel tool for standardization of evaluation of aganglionic megacolon (Hirschsprung disease) histopathology.

Based on a recently introduced immunohistochemical panel (Bachmann et al. 2015) for aganglionic megacolon (AM), also known as Hirschsprung disease, histopathological diagnosis, we evaluated whether the use of digital pathology and 'machine learning' could help to obtain a reliable diagnosis. Slides were obtained from 31 specimens of 27 patients immunohistochemically stained for MAP2, calretinin, S100ß and GLUT1. Slides were digitized by whole slide scanning. We used a Definiens Developer Tissue Studios as software for analysis. We configured necessary parameters in combination with 'machine learning' to identify pathological aberrations. A significant difference between AM- and non-AM-affected tissues was found for calretinin (AM 0.55% vs. non-AM 1.44%) and MAP2 (AM 0.004% vs. non-AM 0.07%) staining measurements and software-based evaluations. In contrast, S100ß and GLUT1 staining measurements and software-based evaluations showed no significant differences between AM- and non-AM-affected tissues. However, no difference was found in comparison of suction biopsies with resections. Applying machine learning via an ensemble voting classifier, we achieved an accuracy of 87.5% on the test set. Automated diagnosis of AM by applying digital pathology on immunohistochemical panels was successful for calretinin and MAP2, whereas S100ß and GLUT1 were not effective in diagnosis. Our method suggests that software-based approaches are capable of diagnosing AM. Our future challenge will be the improvement of efficiency by reduction of the time-consuming need for large pre-labelled training data. With increasing technical improvement, especially in unsupervised training procedures, this method could be helpful in the future.

Anti-CD52 antibody treatment depletes B cell aggregates in the central nervous system in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable. METHODS: We used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 µg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA. RESULTS: Treatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment. CONCLUSION: Anti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.