RNA Editing Alterations Define Disease Manifestations in the Progression of Experimental Autoimmune Encephalomyelitis (EAE).

RNA editing is an epitranscriptomic modification, leading to targeted changes in RNA transcripts. It is mediated by the action of ADAR (adenosine deaminases acting on double-stranded (ds) RNA and APOBEC (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) deaminases and appears to play a major role in the pathogenesis of many diseases. Here, we assessed its role in experimental autoimmune encephalomyelitis (EAE), a widely used non-clinical model of autoimmune inflammatory diseases of the central nervous system (CNS), which resembles many aspects of human multiple sclerosis (MS). We have analyzed in silico data from microglia isolated at different timepoints through disease progression to identify the global editing events and validated the selected targets in murine tissue samples. To further evaluate the functional role of RNA editing, we induced EAE in transgenic animals lacking expression of APOBEC-1. We found that RNA-editing events, mediated by the APOBEC and ADAR deaminases, are significantly reduced throughout the course of disease, possibly affecting the protein expression necessary for normal neurological function. Moreover, the severity of the EAE model was significantly higher in APOBEC-1 knock-out mice, compared to wild-type controls. Our results implicate regulatory epitranscriptomic mechanisms in EAE pathogenesis that could be extrapolated to MS and other neurodegenerative disorders (NDs) with common clinical and molecular features.

Changes in peripheral monocyte populations 48-72 hours after subcutaneous denosumab administration in women with osteoporosis.

OBJECTIVES: To examine the effect of denosumab administration in the peripheral blood white cell population, to further elucidate a plausible pathophysiological link between denosumab and osteonecrosis of the jaw. METHODS: Thirty women with osteoporosis, after denosumab treatment were included. Peripheral blood samples were obtained prior to and 48-72 hours following denosumab administration. Flow cytometry gated at the monocyte population for CD14/CD23/CD123/CD16 stainings were performed. RESULTS: We were able to record a number of changes in the monocyte populations between baseline and after denosumab administration. Most importantly, in the monocyte populations we were able to detect statistically significant increased populations of CD14+/CD23+ (p=0.044), CD14-/CD23+ (p=0.044), CD14+/CD123+ (p=0.011), CD14+/CD123- (p=0.011) and CD14-/CD16+ (p=0.028). In contrast, statistically significant decreased populations of CD14-/CD123+ (p=0.034), CD14+/CD16+ (p=0.037) and CD14+/CD16- (p=0.014) were detected. CONCLUSIONS: Our results provide evidence supporting the hypothesis that denosumab administration modifies the monocyte mediated immune response in a manner similar to that of bisphosphonates. This may partly explain the trivial immunity changes recorded with denosumab.

Application of antibody phage display to identify potential antigenic neural precursor cell proteins.

BACKGROUND: The discovery of neural precursor cells (NPCs) and the concomitant intensive research in the field offer regenerative medicine novel approaches, enabling it to tackle conditions, such as neurodegenerative diseases. Transplantation of NPCs is nowadays considered a cutting-edge treatment for these conditions and many related clinical trials have been already completed or are still ongoing. However, little is known about the antigenicity of NPCs, with most studies addressing the question whether their antigenicity could lead to rejection of the transplanted cells. RESULTS: In this study we investigated the antigenic potential of syngeneic NPCs emulsion, upon subcutaneous (s.c.) administration to wild type C57BL/6 mice, following a standard immunization protocol. The whole IgG repertoire expressed upon immunization was cloned into a Fab phage display vector. From the created phage display library, Fab expressing clones interacting with NPCs lysate proteins were selected with the biopanning technique. The IgG Fab fragment from clone 65 proved to be reactive against antigens originating from NPCs lysates and/or whole brain lysate in diverse immunological assays. CONCLUSIONS: Using a standard immunization protocol to administer NPCs antigens, and applying the Fab fragment phage display technique, we were able to isolate at least a monoclonal IgG Fab fragment, which interacts with different mouse brain proteins. It is not clear whether such antibodies are produced in the host organisms, following NPCs transplantation.

Tauopathy in the young autistic brain: novel biomarker and therapeutic target.

Given our recent discovery of somatic mutations in autism spectrum disorder (ASD)/intellectual disability (ID) genes in postmortem aged Alzheimer's disease brains correlating with increasing tauopathy, it is important to decipher if tauopathy is underlying brain imaging results of atrophy in ASD/ID children. We concentrated on activity-dependent neuroprotective protein (ADNP), a prevalent autism gene. The unique availability of multiple postmortem brain sections of a 7-year-old male, heterozygous for ADNP de novo mutation c.2244Adup/p.His559Glnfs*3 allowed exploration of tauopathy, reflecting on a general unexplored mechanism. The tested subject exhibited autism, fine motor delays, severe intellectual disability and seizures. The patient died after multiple organ failure following liver transplantation. To compare to other ADNP syndrome mutations, immortalized lymphoblastoid cell lines from three different patients (including ADNP p.Arg216*, p.Lys408Valfs*31, and p.Tyr719* heterozygous dominant mutations) and a control were subjected to RNA-seq. Immunohistochemistry, high-throughput gene expression profiles in numerous postmortem tissues followed. Comparisons to a control brain and to extensive datasets were used. Live cell imaging investigated Tau-microtubule interaction, protecting against tauopathy. Extensive child brain tauopathy paralleled by multiple gene expression changes was discovered. Tauopathy was explained by direct mutation effects on Tau-microtubule interaction and correction by the ADNP active snippet NAP. Significant pathway changes (empirical P value < 0.05) included over 100 genes encompassing neuroactive ligand-receptor and cytokine-cytokine receptor interaction, MAPK and calcium signaling, axon guidance and Wnt signaling pathways. Changes were also seen in steroid biosynthesis genes, suggesting sex differences. Selecting the most affected genes by the ADNP mutations for gene expression analysis, in multiple postmortem tissues, identified Tau (MAPT)-gene-related expression changes compared with extensive normal gene expression (RNA-seq) databases. ADNP showed relatively reduced expression in the ADNP syndrome cerebellum, which was also observed for 25 additional genes (representing >50% of the tested genes), including NLGN1, NLGN2, PAX6, SMARCA4, and SNAP25, converging on nervous system development and tauopathy. NAP provided protection against mutated ADNP disrupted Tau-microtubule association. In conclusion, tauopathy may explain brain-imaging findings in ADNP syndrome children and may provide a new direction for the development of tauopathy protecting drug candidates like NAP in ASD/ID.

Reduced expression of L-selectin in T-cells correlates with relative lymphocyte increase in patients with RRMS treated with natalizumab - functional implication towards PML risk.

Objectives: Natalizumab (NTZ), a treatment indicated for patients with highly active Relapsing - Remitting Multiple Sclerosis (RRMS), is known to induce increased relative frequency of lymphocytes. Progressive Multifocal Leukoencephalitis (PML) is a rare but serious adverse event related to NTZ. Moreover, reduced L-selectin (CD62L) expression in T-cells in cryopreserved samples of patients with RRMS under NTZ has been proposed as a biomarker of pre-PML state. We explore the association between L-selectin expression in T-cells and hematological parameters in freshly processed samples of patients with RRMS under NTZ.Methods: We studied L-selectin expression in patients with: RRMS under NTZ (n=34), fingolimod (FTY, n=14), interferon-beta (IFNß, n=22), glatiramer acetate (GA, N=17); in 9 patients with secondary progressive (SP) MS and in 6 healthy controls. Twenty-two patients under NTZ and 6 patients under FTY were followed for 18 months. One NTZ-treated patient developed PML during the study.Results: Patients under NTZ exhibited increased relative frequency of lymphocytes (40.02±1.45) compared to patients under first-line treatment (30.57±1.68, p<0.001) and to patients with SPMS (29±1.56, p=0.02), and a lower mean L-selectin expression in (69.39±1.73) compared to patients under first-line treatment (79.1±1.17, p=0.003). A negative correlation between the relative frequency of CD4+CD62L+ T-cells and the absolute lymphocyte counts (Pearson's r=0.367, p=0.033) was observed.Discussion: We hereby provide mechanistic insight in a possible pathway implicated in NTZ-related PML risk. These results further underline the need for thorough validation of L-selectin expression in T-cells as a potential pre-PML biomarker.

Spatio-temporal expression profile of NGF and the two-receptor system, TrkA and p75NTR, in experimental autoimmune encephalomyelitis.

BACKGROUND: Nerve growth factor (NGF) and its receptors, tropomyosin receptor kinase A (TrkA) and pan-neurotrophin receptor p75 (p75NTR), are known to play bidirectional roles between the immune and nervous system. There are only few studies with inconclusive results concerning the expression pattern and role of NGF, TrkA, and p75NTR (NGF system) under the neuroinflammatory conditions in multiple sclerosis (MS) and its mouse model, the experimental autoimmune encephalomyelitis (EAE). The aim of this study is to investigate the temporal expression in different cell types of NGF system in the central nervous system (CNS) during the EAE course. METHODS: EAE was induced in C57BL/6 mice 6-8 weeks old. CNS tissue samples were collected on specific time points: day 10 (D10), days 20-22 (acute phase), and day 50 (chronic phase), compared to controls. Real-time PCR, Western Blot, histochemistry, and immunofluorescence were performed throughout the disease course for the detection of the spatio-temporal expression of the NGF system. RESULTS: Our findings suggest that both NGF and its receptors, TrkA and p75NTR, are upregulated during acute and chronic phase of the EAE model in the inflammatory lesions in the spinal cord. NGF and its receptors were co-localized with NeuN+ cells, GAP-43+ axons, GFAP+ cells, Arginase1+ cells, and Mac3+ cells. Furthermore, TrkA and p75NTR were sparsely detected on CNPase+ cells within the inflammatory lesion. Of high importance is our observation that despite EAE being a T-mediated disease, only NGF and p75NTR were shown to be expressed by B lymphocytes (B220+ cells) and no expression on T lymphocytes was noticed. CONCLUSION: Our results indicate that the components of the NGF system are subjected to differential regulation during the EAE disease course. The expression pattern of NGF, TrkA, and p75NTR is described in detail, suggesting possible functional roles in neuroprotection, neuroregeneration, and remyelination by direct and indirect effects on the components of the immune system.

Exercise intensity-dependent immunomodulatory effects on encephalomyelitis.

BACKGROUND: Exercise training (ET) has beneficial effects on multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the intensity-dependent effects of ET on the systemic immune system in EAE remain undefined. OBJECTIVE: (1) To compare the systemic immune modulatory effects of moderate versus high-intensity ET protocols in protecting against development of EAE; (2) To investigate whether ET affects autoimmunity selectively, or causes general immunosuppression. METHODS: Healthy mice performed moderate or high-intensity treadmill running programs. Proteolipid protein (PLP)-induced transfer EAE was utilized to examine ET effects specifically on the systemic immune system. Lymph node (LN)-T cells from trained versus sedentary donor mice were transferred to naïve recipients and EAE severity was assessed, by clinical assessment and histopathological analysis. LN-T cells derived from donor trained versus sedentary PLP-immunized mice were analyzed in vitro for proliferation assays by flow cytometry analysis and cytokine and chemokine receptor gene expression using real-time PCR. T cell-dependent immune responses of trained versus sedentary mice to the nonautoantigen ovalbumin and susceptibility to Escherichia coli-induced acute peritonitis were examined. RESULTS: High-intensity training in healthy donor mice induced significantly greater inhibition than moderate-intensity training on proliferation and generation of encephalitogenic T cells in response to PLP-immunization, and on EAE severity upon their transfer into recipient mice. High-intensity training also inhibited LN-T cell proliferation in response to ovalbumin immunization. E. coli bacterial counts and dissemination were not affected by training. INTERPRETATION: High-intensity training induces superior effects in preventing autoimmunity in EAE, but does not alter immune responses to E. coli infection.

Induction of apoptosis in CD4(+) T-cells is linked with optimal treatment response in patients with relapsing-remitting multiple sclerosis treated with Glatiramer acetate.

BACKGROUND: Induction of T-cell apoptosis constitutes a mechanism of action for Glatiramer Acetate (GA). We investigated whether activation of apoptotic T-cell death may be indicative of optimal treatment response in patients with relapsing-remitting Multiple Sclerosis (RRMS), with respect to radiological activity. METHODS: We studied apoptotic markers on blood T-cells of forty patients with RRMS, 19 patients under GA and 21 patients under interferon-ß (IFNß), by flow cytometry. Patients were relapse-free and were further classified into optimal and sub-optimal responders based on radiological activity. Eighteen patients (8 patients under GA and 10 patients under IFNß were additionally evaluated at a 12-month follow-up and were relapse- and radiological activity-free. For these patients, apoptosis was also evaluated by molecular techniques. RESULTS: At inclusion, optimal responders to GA exhibited increased (23.6 ±â€¯1.976) relative % frequency of CD4(+)AnnexinV(+)7AAD(-) T-cells, compared to sub-optimal responders (14.478 ±â€¯1.204, p = 0.001). Similarly, relative % frequency of caspase-3(+) T-cells was 1.517 ±â€¯0.436 versus 0.45 ±â€¯0.149 (p = 0.041), respectively. Anti-apoptotic molecule bcl-2 showed an inverse pattern 4.532 ±â€¯1.321 versus 13.094 ±â€¯3.987, p = 0.044, respectively. These differences were not evident for IFNß-treated patients. CONCLUSIONS: T-cell apoptotic markers may be applied as a biomarker useful in evaluating optimal treatment response under GA, thus allowing for personalized treatment decisions.

The autism/neuroprotection-linked ADNP/NAP regulate the excitatory glutamatergic synapse.

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, was discovered as a leading de novo mutated gene causing the autism-like ADNP syndrome. This syndrome is phenotypically characterized by global developmental delays, intellectual disabilities, speech impediments, and motor dysfunctions. The Adnp haploinsufficient mouse mimics the human ADNP syndrome in terms of synapse density and gene expression patterns, as well as in developmental, motor, and cognitive abilities. Peripheral ADNP was also discovered as a biomarker for Alzheimer's disease and schizophrenia, with nasal administration of the ADNP snippet peptide NAP (enhancing endogenous ADNP activity) leading to partial cognitive and functional protection at the cellular, animal and clinical settings. Here, a novel formulation for effective delivery of NAP is provided with superior brain penetration capabilities. Also provided are methods for treating pertinent clinical implications such as autism, cognitive impairments, olfactory deficits, and muscle strength using the formulation in the Adnp haploinsufficient mouse. Results showed a dramatically specific increase in brain/body bioavailability with the new formulation, without breaching the blood brain barrier. Additional findings included improvements using daily intranasal treatments with NAP, at the behavioral and brain structural levels, diffusion tensor imaging (DTI), translatable to clinical practice. Significant effects on hippocampal and cerebral cortical expression of the presynaptic Slc17a7 gene encoding vesicular excitatory glutamate transporter 1 (VGLUT1) were observed at the RNA and immunohistochemical levels, explaining the DTI results. These findings tie for the first time a reduction in presynaptic glutamatergic synapses with the autism/Alzheimer's/schizophrenia-linked ADNP deficiency coupled with amelioration by NAP (CP201).

Atypical Auditory Brainstem Response and Protein Expression Aberrations Related to ASD and Hearing Loss in the Adnp Haploinsufficient Mouse Brain.

Autism is a wide spread neurodevelopmental disorder with growing morbidity rates, affecting more boys than girls worldwide. Activity-dependent neuroprotective protein (ADNP) was recently recognized as a leading gene accounted for 0.17% of autism spectrum disorder (ASD) cases globally. Respectively, mutations in the human ADNP gene (ADNP syndrome), cause multi-system body dysfunctions with apparent ASD-related traits, commencing as early as childhood. The Adnp haploinsufficient (Adnp+/-) mouse model was researched before in relations to Alzheimer's disease and autism. Adnp+/- mice suffer from deficient social memory, vocal and motor impediments, irregular tooth eruption and short stature, all of which corresponds with reported phenotypes in patients with the ADNP syndrome. Recently, a more elaborated description of the ADNP syndrome was published, presenting impediments such as hearing disabilities in > 10% of the studied children. Irregular auditory brainstem response (ABR) has been connected to ASD-related cases and has been suggested as a potential hallmark for autism, allowing diagnosis of ASD risk and early intervention. Herein, we present detriment hearing in the Adnp+/- mice with atypical ABR and significant protein expression irregularities that coincides with ASD and hearing loss studies in the brain.

Exposure to 3-Nitropropionic Acid Mitochondrial Toxin Induces Tau Pathology in Tangle-Mouse Model and in Wild Type-Mice.

Oxidative stress, particularly of mitochondrial origin, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease (AD) and other tauopathies. Controversies regarding the responses of tau phosphorylation state to various stimuli causing oxidative stress have been reported. Here we investigated the effect of 3-nitropropionic acid (3NP), a mitochondrial toxin which induces oxidative stress, on the tangle-pathology in our previously generated double mutant (E257T/P301S, DM) -Tau-tg mice and in WT-mice. We detected an increase in tangle pathology in the hippocampus and cortex of the DM-Tau-tg mice following exposure of the mice to the toxin, as well as generation of tangles in WT-mice. This increase was accompanied with alterations in the level of the glycogen synthase kinase 3ß (GSK3ß), the kinase which phosphorylates the tau protein, and in the phosphorylation state of this kinase. A response of microglial cells was noticed. These results point to the involvement of mitochondrial dysfunction in the development of the tangle-pathology and may suggest that interfering with mitochondrial dysfunction may have an anti-tangle therapeutic potential.

Cyclization of PLP139-151 peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis.

We report the novel synthesis of cyclic PLP139-151 (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H147 replaces W144 and F151-K150 replace H147 as TCR contacts, which may explain the difference on each peptide's response.

Exercise training attenuates experimental autoimmune encephalomyelitis by peripheral immunomodulation rather than direct neuroprotection.

BACKGROUND: Conflicting results exist on the effects of exercise training (ET) on Experimental Autoimmune Encephalomyelitis (EAE), nor is it known how exercise impacts on disease progression. OBJECTIVE: We examined whether ET ameliorates the development of EAE by modulating the systemic immune system or exerting direct neuroprotective effects on the CNS. METHODS: Healthy mice were subjected to 6weeks of motorized treadmill running. The Proteolipid protein (PLP)-induced transfer EAE model in mice was utilized. To assess effects of ET on systemic autoimmunity, lymph-node (LN)-T cells from trained- vs. sedentary donor mice were transferred to naïve recipients. To assess direct neuroprotective effects of ET, PLP-reactive LN-T cells were transferred into recipient mice that were trained prior to EAE transfer or to sedentary mice. EAE severity was assessed in vivo and the characteristics of encephalitogenic LN-T cells derived from PLP-immunized mice were evaluated in vitro. RESULTS: LN-T cells obtained from trained mice induced an attenuated clinical and pathological EAE in recipient mice vs. cells derived from sedentary animals. Training inhibited the activation, proliferation and cytokine gene expression of PLP-reactive T cells in response to CNS-derived autoantigen, but strongly enhanced their proliferation in response to Concanavalin A, a non-specific stimulus. However, there was no difference in EAE severity when autoreactive encephalitogenic T cells were transferred to trained vs. sedentary recipient mice. CONCLUSION: ET inhibits immune system responses to an auto-antigen to attenuate EAE, rather than generally suppressing the immune system, but does not induce a direct neuro-protective effect against EAE.

Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents.

BACKGROUND: Neural precursor cells (NPCs) located in the subventricular zone (SVZ), a well-defined NPC niche, play a crucial role in central nervous system (CNS) homeostasis. Moreover, NPCs are involved in the endogenous reparative process both in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the possibility that NPCs may be vulnerable to immune-related components may not be ruled out. Therefore, we investigated the potential affinity of myelin oligodendrocyte glycoprotein (MOG)-induced humoral response(s) to NPCs. METHODS: MOG35-55-EAE was induced in C57BL/6 mice; blood-sampling was performed on days 17-21 (acute phase) along with a naive group and corresponding antisera (AS) were collected (EAE-AS, NAIVE-AS). The presence of anti-CNS autoantibodies was examined with western blotting. Furthermore, using the collected antisera and anti-MOG antibody (as positive control), immunohistochemistry and double immunofluorescence were implemented on normal neonatal, postnatal, and adult mouse brain sections. Targeted NPCs were identified with confocal microscopy. In vitro immunoreactivity assessment on NPCs challenged with autoantibodies was evaluated for apoptotic/autophagic activity. RESULTS: Western blotting verified the existence of autoantibodies in EAE mice and demonstrated bands corresponding to yet unidentified NPC surface epitopes. A dominant selective binding of EAE-AS in the subventricular zone in all age groups compared to NAIVE-AS (p < 0.001) was observed. Additionally, anti-BrdU+/EAE-AS+ colocalization was significantly higher than anti-BrdU+/anti-MOG+, a finding suggesting that the EAE humoral response colocalized with NPCs(BrdU+), cells that do not express MOG. Well-established NPC markers (Nestin, m-Musashi-1, Sox2, DCX, GFAP, NG2) were used to identify the distinct cell types which exhibited selective binding with EAE-AS. The findings verified that EAE-AS exerts cross-reactivity with NPCs which varies throughout the neonatal to adult stage, with a preference to cells of early developmental stages. Finally, increased expressions of Caspase 3 and Beclin 1 on NPCs were detected. CONCLUSION: We provide evidence for the first time that MOG35-55 EAE induces production of antibodies with affinity to SVZ of naive mice in three different age groups. These autoantibodies target lineage-specific NPCs as brain develops and have the potential to trigger apoptotic pathways. Thus, our findings provide indication that cross-talk between immunity and NPCs may lead to functional alteration of NPCs regarding their viability and potentially oligodendrogenesis and effective remyelination.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.

The Impact of Methylprednisolone Pulses during Relapses of Multiple Sclerosis on the Kinetics of Anti-Interferon-Beta Antibodies.

BACKGROUND/AIMS: We assessed the, hitherto unknown, impact of intravenous methylprednisolone (ivMP) pulses during relapses of multiple sclerosis (MS) on the kinetics of anti-interferon-beta neutralizing antibodies (Nabs) and binding antibodies (Babs). METHODS: Babs (ELISA) and Nabs (antiviral cytopathic effect assay) titers were evaluated before, immediately after and at 1 month following ivMP in 60 MS patients. RESULTS: ivMP reduces Nabs and Babs titers for at least 1 month. Baseline titers determine Nabs and Babs seronegativity at the end of ivMP. Clinical response to ivMP tends to be better in Nabs(+) patients. CONCLUSION: Sampling for Nabs/Babs should be avoided during or shortly after ivMP to avoid transient positive or negative results that may obscure the decision to switch treatment.

Immunophenotype of mouse cerebral hemispheres-derived neural precursor cells.

Postnatally isolated neural precursor cells (piNPCs) from mouse cerebral tissue have been studied in cell-based therapeutic approaches for Experimental Autoimmune Encephalomyelitis (EAE). Transplantation experiments in EAE rodents revealed that piNPCs manage to integrate into the host tissue and ameliorate clinical symptoms. When cultured in vitro, mouse cerebral piNPCs form neurospheres consisting of immature cells positive for polysialylated neural adhesion molecule (PSA-NCAM) that differentiate mainly towards glial cells, but also neurons. Herein, we have characterized piNPCs immunophenotype, with flow cytometry. NPCs were positive for CD24, CD44, and CD133 though negative for CD15, CD184 and CD49d. This immunophenotype, determined for the first time, among cells isolated from neonates might be useful for the identification of NPC population aiming at the development of transplantation protocols.

Subcutaneous Transplantation of Neural Precursor Cells in Experimental Autoimmune Encephalomyelitis Reduces Chemotactic Signals in the Central Nervous System.

UNLABELLED: Neural precursor cell (NPC) transplantation has been proposed as a therapy for multiple sclerosis (MS) and other degenerative disorders of the central nervous system (CNS). NPCs are suggested to exert immune modulation when they are transplanted in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Herein, we explore whether the effect of NPC transplantation on the clinical course and the pathological features of EAE is combined with the modulation of chemokines levels expressed in the inflamed CNS. NPCs were isolated from brains of neonatal C57/Bl6 mice and were subcutaneously administered in female mice with myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Clinical signs of the disease and transcript analysis of the CNS in the acute phase were performed. In addition, the presence of inflammatory components in the spinal cord was evaluated and ex vivo proliferation of lymphocytes was measured. NPC recipients exhibited ameliorated clinical outcome and less pronounced pathological features in their spinal cord. Downregulation of chemokine mRNA levels throughout the CNS was correlated with diminished Mac-3-, CD3-, and CD4-positive cells and reduced expression levels of antigen-presenting molecules in the spinal cord. Moreover, NPC transplantation resulted in lymphocyte-related, although not splenocyte-related, peripheral immunosuppression. We conclude that NPCs ameliorated EAE potentially by modulating the levels of chemokines expressed in the inflamed CNS, thus resulting in the impaired recruitment of immune cells. These findings further contribute to the better understanding of NPCs' immunomodulatory properties in neuroinflammatory disorders, and may lead to faster translation into potential clinical use. SIGNIFICANCE: Endogenous neural precursor cells of the central nervous system are able to migrate and differentiate toward mature cells to repair an injury. There is increasing evidence that autologous transplantation of these cells in experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, may have a beneficial effect on the disease process. Several mechanisms have been proposed-among them, the potentiation of endogenous precursor cell differentiation of the central nervous system and the modulation of demyelinating and neurodegenerative immune-mediated processes. This article provides evidence of interference in immune signaling within the central nervous system as a potential mechanism underlying the immunomodulatory properties of transplanted neural precursor cells.

Connexin43 and connexin47 alterations after neural precursor cells transplantation in experimental autoimmune encephalomyelitis.

Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune-modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG-induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post-induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP(+) NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP(+) cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP(+) NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue.