Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, "outrunning" the host's immune response in demyelinating plaques, thus continuously eliciting new lesions.

Tumefactive demyelinating lesions during etanercept treatment requiring decompressive hemicraniectomy

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory and immunoregulatory cytokine involved in the pathogenesis of several autoimmune disorders. Etanercept, a TNF-α antagonist (anti-TNF-α) acting as a soluble TNF-α receptor, has been associated with neurological demyelinating disorders. This paper aims to report an unusual case showing tumefactive central nervous system (CNS) inflammatory demyelination in a patient in the course of TNF -α antagonist therapy, requiring decompressive hemicraniectomy. This report is based on magnetic resonance imaging (MRI) findings and histology. A biopsy confirmed the inflammatory demyelinating nature of the lesions. The clinical presentation is unusual due to the severity of the disease process, requiring decompressive hemicraniotomy with a clinically favorable outcome.

Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

Canine microglial cells: stereotypy in immunophenotype and specificity in function?

Microglial cells represent the endogenous immune system of the central nervous system (CNS). Upon pathological insults they reveal their immunological potential aimed at regaining homeostasis. These reactions have long been believed to follow a uniform and unspecific pattern which is irrespective to the underlying disease entity. Evidence is growing that this view seriously underrates microglial competence as the defenders of the CNS. In the present study, microglial cells of 47 dogs were examined ex vivo by means of flow cytometry. Ex vivo examination included immunophenotypic characterization using eight different surface markers and functional studies such as phagocytosis assay and the reactive oxygen species (ROS) generation test. The dogs were classified according to their histopathological diagnoses in disease categories (controls, canine distemper virus (CDV) induced demyelination, other diseases of the CNS) and results of microglial reaction profiles were compared. Immunophenotypic characterization generally revealed relative high conformity in the microglial disease response among the different groups, however the functional response was shown to be more specific. Dogs with intracranial inflammation and dogs with demyelination showed an enhanced phagocytosis, whereas a significant up-regulation of ROS generation was found in dogs with demyelination due to CDV infection. This strongly suggests a specific response of microglia to infection with CDV in the settings of our study and underlines the pivotal role of microglial ROS generation in the pathogenesis of demyelinating diseases, such as canine distemper.

Inhibition of L-type amino acid transport with non-physiological amino acids in the Pahenu2 mouse model of phenylketonuria

Phenylketonuria, an autosomal recessive Mendelian disorder, is one of the most common inborn errors of metabolism. Although currently treated by diet, many suboptimal outcomes occur for patients. Neuropathological outcomes include cognitive loss, white matter abnormalities, and hypo- or demyelination, resulting from high concentrations and/or fluctuating levels of phenylalanine. High phenylalanine can also result in competitive exclusion of other large neutral amino acids from the brain, including tyrosine and tryptophan (essential precursors of dopamine and serotonin). This competition occurs at the blood brain barrier, where the L-type amino acid transporter, LAT1, selectively facilitates entry of large neutral amino acids. The hypothesis of these studies is that certain non-physiological amino acids (NPAA; DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), α-aminoisobutyrate (AIB), and α-methyl-aminoisobutyrate (MAIB)) would competitively inhibit LAT1 transport of phenylalanine (Phe) at the blood-brain barrier interface. To test this hypothesis, Pah-/- mice (n=5, mixed gender; Pah+/-(n=5) as controls) were fed either 5% NL, 0.5% NB, 5% AIB or 3% MAIB (w/w 18% protein mouse chow) for 3 weeks. Outcome measurements included food intake, body weight, brain LNAAs, and brain monoamines measured via LCMS/MS or HPLC. Brain Phe values at sacrifice were significantly reduced for NL, NB, and MAIB, verifying the hypothesis that these NPAAs could inhibit Phe trafficking into the brain. However, concomitant reductions in tyrosine and methionine occurred at the concentrations employed. Blood Phe levels were not altered indicating no effect of NPAA competitors in the gut. Brain NL and NB levels, measured with HPLC, verified both uptake and transport of NPAAs. Although believed predominantly unmetabolized, NL feeding significantly increased blood urea nitrogen. Pah-/-disturbances of monoamine metabolism were exacerbated by NPAA intervention, primarily with NB (the prototypical LAT inhibitor). To achieve the overarching goal of using NPAAs to stabilize Phe transport levels into the brain, a specific Phe-reducing combination and concentration of NPAAs must be found. Our studies represent the first in vivo use of NL, NB and MAIB in Pah-/- mice, and provide proof-of-principle for further characterization of these LAT inhibitors. Our data is the first to document an effect of MAIB, a specific system A transport inhibitor, on large neutral amino acid transport.

Antigen Processing and Presentation in Multiple Sclerosis

CD4(+) T cells play a central role in the pathogenesis of multiple sclerosis (MS). Generation, activation and effector function of these cells crucially depends on their interaction with MHC II-peptide complexes displayed by antigen presenting cells (APC). Processing and presentation of self antigens by different APC therefore influences the disease course at all stages. Selection by thymic APC leads to the generation of autoreactive T cells, which can be activated by peripheral APC. Reactivation by central nervous system APC leads to the initiation of the inflammatory response resulting in demyelination. In this review we will focus on how MHC class II antigenic epitopes are created by different APC from the thymus, the periphery and from the brain, and will discuss the relevance of the balance between creation and destruction of such epitopes in the context of MS. A solid understanding of these processes offers the possibility for designing future therapeutic strategies.

Polyganglioradiculoneuritis in a young cat: clinical and histopathological findings

An 18-month-old European shorthair cat was presented with a two week history of progressive decrease in consciousness, ambulatory tetraparesis, moderate ataxia and generalised decreased-to-absent postural reactions. Bilateral facial and nasal hypalgesia, absent menace response and anisocoria were found, and segmental spinal reflexes were normal. Neurological signs progressed to nonambulatory tetraparesis, tremor and spinal hyperalgesia. Histopathological examination revealed a mild-to-moderate lymphoplasmacytic and histiocytic infiltration, predominantly in the dorsal spinal roots, cranial nerves and ganglia in association with marked demyelination and proliferation of Schwann cells. Neurons and axons were preserved. Lesions were multi-focal and varied in severity. A predominantly sensory polyganglioradiculoneuritis was diagnosed. This lesion has not been reported previously in cats. Rabies, herpesviruses, feline infectious peritonitis, feline immunodeficiency virus, Toxoplasma gondii and feline leukaemia virus were excluded as possible aetiologies. Infections by other viruses or an autoimmune disease are discussed.

Emergence of Canine Distemper Virus Strains With Modified Molecular Signature and Enhanced Neuronal Tropism Leading to High Mortality in Wild Carnivores

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination-the classic presentation of CDV infection-was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Multiple Sclerosis: Current Knowledge and Future Outlook

Background: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal loss. The etiology of MS is unknown; however, environmental and genetic factors play a key role in the development of MS. Diagnostic criteria have been adapted to facilitate earlier diagnosis with increased sensitivity and specificity. Our understanding of the pathophysiology of MS has deepened considerably in recent years, resulting in different therapies to modify the disease course. Furthermore, several drugs have lately shown efficacy in phase III studies and their approval is expected in the near future. As treatment options expand, a future challenge will be to find the optimal treatment for the individual patient. Summary: This mini-review gives an overview of the current knowledge of MS with emphasis on the latest diagnostic criteria and both current and upcoming treatment options. Key Messages: Treatment of MS changes rapidly as the knowledge and therapeutic options in MS expand. Clinical Impact: Diagnosis of MS is based on McDonald criteria. MS therapy can be divided into relapse, disease-modifying and symptomatic treatment. Relapses are commonly treated with intravenous methylprednisolone. First-line therapy consists of either interferon-β, glatiramer acetate or teriflunomide. In general, agents used as escalation therapies (natalizumab, fingolimod and mitoxantrone) are more potent than the agents used for first-line therapy; however, these have potentially serious side effects and should be used with care.

Investigation of leptomeningeal enhancement in MS: A postcontrast FLAIR MRI study

OBJECTIVE To investigate possible leptomeningeal contrast enhancement using postcontrast fluid-attenuated inversion recovery (FLAIR) MRI as an additional marker of inflammation in patients with multiple sclerosis (MS). METHODS A cohort of 112 patients (73 women) with clinically definitive MS or a clinically isolated syndrome suggestive of CNS demyelination were included. A pathologic control group of 5 stroke patients was also examined. MRI was performed on a 3T system including FLAIR, T2-weighted, T1-weighted-contrast injection, followed by T1-weighted and FLAIR. RESULTS Of the 112 patients, 39 had an acute relapse at the time of MRI. In total, 96 contrast-enhancing lesions were identified on postcontrast T1-weighted images. The pathologic control group demonstrated the sensitivity of postcontrast FLAIR images demonstrating leptomeningeal enhancement in all cases. In contrast, only 1 out of 112 examined patients with MS showed a single area of abnormal leptomeningeal contrast enhancement. CONCLUSIONS In contrast to intraparenchymal blood-brain barrier (BBB) dysfunction that is frequently seen in patients with MS, BBB dysfunction of leptomeningeal vessels is usually not detectable in patients with early MS.

Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke

Each year about 650,000 Europeans die from stroke and a similar number lives with the sequelae of multiple sclerosis (MS). Stroke and MS differ in their etiology. Although cause and likewise clinical presentation set the two diseases apart, they share common downstream mechanisms that lead to damage and recovery. Demyelination and axonal injury are characteristics of MS but are also observed in stroke. Conversely, hallmarks of stroke, such as vascular impairment and neurodegeneration, are found in MS. However, the most conspicuous common feature is the marked neuroinflammatory response, marked by glia cell activation and immune cell influx. In MS and stroke the blood-brain barrier is disrupted allowing bone marrow-derived macrophages to invade the brain in support of the resident microglia. In addition, there is a massive invasion of auto-reactive T-cells into the brain of patients with MS. Though less pronounced a similar phenomenon is also found in ischemic lesions. Not surprisingly, the two diseases also resemble each other at the level of gene expression and the biosynthesis of other proinflammatory mediators. While MS has traditionally been considered to be an autoimmune neuroinflammatory disorder, the role of inflammation for cerebral ischemia has only been recognized later. In the case of MS the long track record as neuroinflammatory disease has paid off with respect to treatment options. There are now about a dozen of approved drugs for the treatment of MS that specifically target neuroinflammation by modulating the immune system. Interestingly, experimental work demonstrated that drugs that are in routine use to mitigate neuroinflammation in MS may also work in stroke models. Examples include Fingolimod, glatiramer acetate, and antibodies blocking the leukocyte integrin VLA-4. Moreover, therapeutic strategies that were discovered in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, turned out to be also effective in experimental stroke models. This suggests that previous achievements in MS research may be relevant for stroke. Interestingly, the converse is equally true. Concepts on the neurovascular unit that were developed in a stroke context turned out to be applicable to neuroinflammatory research in MS. Examples include work on the important role of the vascular basement membrane and the BBB for the invasion of immune cells into the brain. Furthermore, tissue plasminogen activator (tPA), the only established drug treatment in acute stroke, modulates the pathogenesis of MS. Endogenous tPA is released from endothelium and astroglia and acts on the BBB, microglia and other neuroinflammatory cells. Thus, the vascular perspective of stroke research provides important input into the mechanisms on how endothelial cells and the BBB regulate inflammation in MS, particularly the invasion of immune cells into the CNS. In the current review we will first discuss pathogenesis of both diseases and current treatment regimens and will provide a detailed overview on pathways of immune cell migration across the barriers of the CNS and the role of activated astrocytes in this process. This article is part of a Special Issue entitled: Neuro inflammation: A common denominator for stroke, multiple sclerosis and Alzheimer's disease, guest edited by Helga de Vries and Markus Swaninger.

Characterization of anti -sulfatide autoantibodies in demyelinating diseases

Multiple sclerosis (MS) is the most common autoimmune disease of the central nerve system and Guillain Barré Syndrome (GBS) is an inflammatory neuropathy involving the peripheral nerves. Anti-myelin immunoglobins may play a role in the demyelination processes of the both diseases. Sulfatide is an abundant glycolipid on myelin and is a candidate target antigen for disease related autoantibodies. The objective of this study was to characterize anti-sulfatide antibodies and compare antibodies from GBS and MS patients with fetal antibodies. Our hypothesis is that some B cells producing disease-associated autoantibodies are derived from or related to B cells of the fetal repertoire. Here we report that reactivity of plasma IgM against sulfatide was elevated in twelve MS patients compared with twelve normal subjects. This result implies that anti-sulfatide antibodies are disease-related. A total of sixteen human B lymphocyte clones producing anti-sulfatide autoantibodies were isolated from MS patients, GBS patients and a human fetus. Seven of the clones were from three MS patients, four of the clones were from three GBS patients and five were from the spleen of a twenty-week human fetus. Sequences have been obtained for the heavy and light chain variable regions (VDJ and VJ regions) of all of the anti-sulfatide immunoglobulins. Seven of the sixteen antibodies used VH3 for the variable region gene of the heavy chain consistent with the rate of VH3 usage in randomly selected B cells. Somatic mutations were significantly more frequent in the patient antibodies than in the fetus and somatic mutations in CDR's (Complementarity Determining Region) were significantly more frequent than in framework regions. No significant difference was found between patients and fetus in length of VH CDRIII. However, it is reported that antibodies from randomly selected normal adult B cells have longer CDRIII lengths than those of the fetus (Sanz I, 1991 Journal of Immunology Sep 1;147(5):1720-9). Our results are consistent with derivation of the precursors of B cells producing these autoantibodies from B cells related to those of the fetal repertoire. These findings are consistent with a model in which quiescent B cells from clones produced early in development undergo proliferation in dysregulated disease states, accumulating somatic mutations and increasing in reactivity toward self-antigens. ^ Epitope mapping and molecular modeling were done to elucidate the relationships between antibody structure and binding characteristics. The autoantibodies were tested for binding activity to three different antigens: sulfatide, galactoceramide and ceramide. Molecular modeling suggests that antibodies with positive charge surrounded by or adjacent to hydrophobic groups in the binding pocket bind to the head of sulfatide via the sulfate group through electrostatic interactions. However, the antibodies with hydrophobic groups separated from positive charges appear to bind to the hydrophobic tail of sulfatide. This observation was supported by a study of the effect of NaCl concentration on antigen binding. The result suggested that electrostatic interactions played a major role in sulfate group binding and that hydrophobic interactions were of greater importance for binding to the ceramide group. Our three-dimensional structure data indicated that epitope specificity of these antibodies is more predictable at the level of tertiary than primary structure and suggested positive selection based on structure occurred in the. formation of those autoantibodies. ^

Neurotransplantation of magnetically labeled oligodendrocyte progenitors: Magnetic resonance tracking of cell migration and myelination

Demyelination is a common pathological finding in human neurological diseases and frequently persists as a result of failure of endogenous repair. Transplanted oligodendrocytes and their precursor cells can (re)myelinate axons, raising the possibility of therapeutic intervention. The migratory capacity of transplanted cells is of key importance in determining the extent of (re)myelination and can, at present, be evaluated only by using invasive and irreversible procedures. We have exploited the transferrin receptor as an efficient intracellular delivery device for magnetic nanoparticles, and transplanted tagged oligodendrocyte progenitor cells into the spinal cord of myelin-deficient rats. Cell migration could be easily detected by using three-dimensional magnetic resonance microscopy, with a close correlation between the areas of contrast enhancement and the achieved extent of myelination. The present results demonstrate that magnetic resonance tracking of transplanted oligodendrocyte progenitors is feasible; this technique has the potential to be easily extended to other neurotransplantation studies involving different precursor cell types.

Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood–brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood–brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

Phenotype of arylsulfatase A-deficient mice: Relationship to human metachromatic leukodystrophy

Metachromatic leukodystrophy is a lysosomal sphingolipid storage disorder caused by the deficiency of arylsulfatase A. The disease is characterized by progressive demyelination, causing various neurologic symptoms. Since no naturally occurring animal model of the disease is available, we have generated arylsulfatase A-deficient mice. Deficient animals store the sphingolipid cerebroside-3-sulfate in various neuronal and nonneuronal tissues. The storage pattern is comparable to that of affected humans, but gross defects of white matter were not observed up to the age of 2 years. A reduction of axonal cross-sectional area and an astrogliosis were observed in 1-year-old mice; activation of microglia started at 1 year and was generalized at 2 years. Purkinje cell dendrites show an altered morphology. In the acoustic ganglion numbers of neurons and myelinated fibers are severely decreased, which is accompanied by a loss of brainstem auditory-evoked potentials. Neurologic examination reveals significant impairment of neuromotor coordination.