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1.  Antibodies from Inflamed Central Nervous System Tissue Recognize Myelin Oligodendrocyte Glycoprotein1 
Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) can induce demyelination and oligodendrocyte loss in models of multiple sclerosis (MS). Whether anti-MOG Abs play a similar role in patients with MS or inflammatory CNS diseases by epitope spreading is unclear. We have therefore examined whether autoantibodies that bind properly folded MOG protein are present in the CNS parenchyma of MS patients. IgG was purified from CNS tissue of 14 postmortem cases of MS and 8 control cases, including cases of encephalitis. Binding was assessed using two independent assays, a fluorescence-based solid-phase assay and a solution-phase RIA. MOG autoantibodies were identified in IgG purified from CNS tissue by solid-phase immunoassay in 7 of 14 cases with MS and 1 case of subacute sclerosing panencephalitis, but not in IgG from noninflamed control tissue. This finding was confirmed with a solution-phase RIA, which measures higher affinity autoantibodies. These data demonstrate that autoantibodies recognizing MOG are present in substantially higher concentrations in the CNS parenchyma compared with cerebrospinal fluid and serum in subjects with MS, indicating that local production/accumulation is an important aspect of autoantibody-mediated pathology in demyelinating CNS diseases. Moreover, chronic inflammatory CNS disease may induce autoantibodies by virtue of epitope spreading.
PMCID: PMC4515951  PMID: 16034142
2.  The immune cell infiltrate populating meningiomas is composed of mature, antigen-experienced T and B cells 
Neuro-Oncology  2013;15(11):1479-1490.
Background
Meningiomas often harbor an immune cell infiltrate that can include substantial numbers of T and B cells. However, their phenotype and characteristics remain undefined. To gain a deeper understanding of the T and B cell repertoire in this tumor, we characterized the immune infiltrate of 28 resected meningiomas representing all grades.
Methods
Immunohistochemistry was used to grossly characterize and enumerate infiltrating lymphocytes. A molecular analysis of the immunoglobulin variable region of tumor-infiltrating B cells was used to characterize their antigen experience. Flow cytometry of fresh tissue homogenate and paired peripheral blood lymphocytes was used to identify T cell phenotypes and characterize the T cell repertoire.
Results
A conspicuous B and T cell infiltrate, primarily clustered in perivascular spaces, was present in the microenvironment of most tumors examined. Characterization of 294 tumor-infiltrating B cells revealed clear evidence of antigen experience, in that the cardinal features of an antigen-driven B cell response were present. Meningiomas harbored populations of antigen-experienced CD4+ and CD8+ memory/effector T cells, regulatory T cells, and T cells expressing the immune checkpoint molecules PD-1 and Tim-3, indicative of exhaustion. All of these phenotypes were considerably enriched relative to their frequency in the circulation. The T cell repertoire in the tumor microenvironment included populations that were not reflected in paired peripheral blood.
Conclusion
The tumor microenvironment of meningiomas often includes postgerminal center B cell populations. These tumors invariably include a selected, antigen-experienced, effector T cell population enriched by those that express markers of an exhausted phenotype.
doi:10.1093/neuonc/not110
PMCID: PMC3813416  PMID: 23978377
B cell; meningioma; T cell; tumor-infiltrating lymphocytes
3.  The neuroinflammation marker translocator protein is not elevated in individuals with mild-to-moderate depression: A [11C]PBR28 PET study 
Brain, behavior, and immunity  2013;33:131-138.
Depression is associated with systemic inflammation. In animals, systemic inflammation can induce neuroinflammation and activation of microglia; however, postmortem studies have not convincingly shown that there is neuroinflammation in depression. The purpose of this study was to use positron emission tomography (PET) with [11C]PBR28, which binds to the neuroinflammation marker translocator protein 18 kDa (TSPO), to compare the level of TSPO between individuals with depression and control subjects. Ten individuals who were in an acute episode of major depression and 10 control subjects matched for TSPO genotype and other characteristics had a PET scan with arterial input function to quantify levels of TSPO in brain regions of interest (ROIs). Total volume of distribution (VT) of [11C]PBR28 was used as a measure of total ligand binding. The primary outcome was the difference in VT between the two groups; this was assessed using a linear mixed model with group as a between-subject factor and region as a within-subject factor. There was no statistically significant difference in [11C]PBR28 binding (VT) between the two groups. In fact, 7 of 10 individuals with depression had lower [11C]PBR28 binding in all ROIs compared to their respective genotype-matched control subjects. Future studies are needed to determine whether individuals with mild-to-moderate depression have lower TSPO levels and to assess whether individuals with severe depression and/or with elevated levels of systemic inflammation might have higher TSPO levels than control subjects.
doi:10.1016/j.bbi.2013.06.010
PMCID: PMC3899398  PMID: 23850810
Depression; Neuroinflammation; Microglia; Translocator protein 18 kDa; PET
4.  pRESTO: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires 
Bioinformatics  2014;30(13):1930-1932.
Summary: Driven by dramatic technological improvements, large-scale characterization of lymphocyte receptor repertoires via high-throughput sequencing is now feasible. Although promising, the high germline and somatic diversity, especially of B-cell immunoglobulin repertoires, presents challenges for analysis requiring the development of specialized computational pipelines. We developed the REpertoire Sequencing TOolkit (pRESTO) for processing reads from high-throughput lymphocyte receptor studies. pRESTO processes raw sequences to produce error-corrected, sorted and annotated sequence sets, along with a wealth of metrics at each step. The toolkit supports multiplexed primer pools, single- or paired-end reads and emerging technologies that use single-molecule identifiers. pRESTO has been tested on data generated from Roche and Illumina platforms. It has a built-in capacity to parallelize the work between available processors and is able to efficiently process millions of sequences generated by typical high-throughput projects.
Availability and implementation: pRESTO is freely available for academic use. The software package and detailed tutorials may be downloaded from http://clip.med.yale.edu/presto.
Contact: steven.kleinstein@yale.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu138
PMCID: PMC4071206  PMID: 24618469
5.  B lymphocytes in neuromyelitis optica 
Neuromyelitis optica (NMO) is an inflammatory autoimmune disorder of the CNS that predominantly affects the spinal cord and optic nerves. A majority (approximately 75%) of patients with NMO are seropositive for autoantibodies against the astrocyte water channel aquaporin-4 (AQP4). These autoantibodies are predominantly IgG1, and considerable evidence supports their pathogenicity, presumably by binding to AQP4 on CNS astrocytes, resulting in astrocyte injury and inflammation. Convergent clinical and laboratory-based investigations have indicated that B cells play a fundamental role in NMO immunopathology. Multiple mechanisms have been hypothesized: AQP4 autoantibody production, enhanced proinflammatory B cell and plasmablast activity, aberrant B cell tolerance checkpoints, diminished B cell regulatory function, and loss of B cell anergy. Accordingly, many current off-label therapies for NMO deplete B cells or modulate their activity. Understanding the role and mechanisms whereby B cells contribute to initiation, maintenance, and propagation of disease activity is important to advancing our understanding of NMO pathogenesis and developing effective disease-specific therapies.
doi:10.1212/NXI.0000000000000104
PMCID: PMC4426682  PMID: 25977932
6.  Serum autoantibodies to myelin peptides distinguish acute disseminated encephalomyelitis from relapsing-remitting multiple sclerosis 
Background and objective
Acute disseminated encephalomyelitis (ADEM) and relapsing remitting multiple sclerosis share overlapping clinical, radiologic, and laboratory features at onset. Because autoantibodies may contribute to the pathogenesis of both diseases, we sought to identify autoantibody biomarkers capable of distinguishing them.
Methods
We used custom antigen arrays to profile anti-myelin-peptide autoantibodies in sera derived from individuals with pediatric ADEM (n = 15), pediatric multiple sclerosis (n = 11), and adult multiple sclerosis (n = 15). Using isotype-specific secondary antibodies,we profiled both IgG and IgM reactivities. We used Statistical Analysis of Microarrays to confirm differences in autoantibody reactivity profiles between ADEM and multiple sclerosis samples. We used Prediction Analysis of Microarrays to generate and validate prediction algorithms based on the autoantibody reactivity profiles.
Results
ADEM was characterized by IgG autoantibodies targeting epitopes derived from myelin basic protein, proteolipid protein, myelin-associated oligodendrocyte basic glycoprotein, and alpha-B-crystallin. In contrast, multiple sclerosis was characterized by IgM autoantibodies targeting myelin basic protein, proteolipid protein, myelin-associated oligodendrocyte basic glycoprotein, and oligodendrocyte specific protein. We generated and validated prediction algorithms that distinguish ADEM serum (sensitivity 62–86%; specificity 56–79%) from multiple sclerosis serum (sensitivity 40–87%; specificity 62–86%) on the basis of combined IgG and IgM anti-myelin autoantibody reactivity to a small number of myelin peptides.
Conclusions
Combined profiles of serum IgG and IgM autoantibodies identify myelin antigens that may be useful for distinguishing multiple sclerosis from ADEM. Further studies are required to establish clinical utility. Further biological assays are required to delineate the pathogenic potential of these antibodies.
doi:10.1177/1352458513485653
PMCID: PMC4411183  PMID: 23612879
pediatric; multiple sclerosis; acute disseminated encephalomyelitis; myelin; autoantibody; antigen; array; diagnosis; biomarker; immunoglobulin; IgG; IgM
7.  Related B cell clones populate the meninges and parenchyma of patients with multiple sclerosis 
Brain  2011;134(2):534-541.
In the central nervous system of patients with multiple sclerosis, B cell aggregates populate the meninges, raising the central question as to whether these structures relate to the B cell infiltrates found in parenchymal lesions or instead, represent a separate central nervous system immune compartment. We characterized the repertoires derived from meningeal B cell aggregates and the corresponding parenchymal infiltrates from brain tissue derived primarily from patients with progressive multiple sclerosis. The majority of expanded antigen-experienced B cell clones derived from meningeal aggregates were also present in the parenchyma. We extended this investigation to include 20 grey matter specimens containing meninges, 26 inflammatory plaques, 19 areas of normal appearing white matter and cerebral spinal fluid. Analysis of 1833 B cell receptor heavy chain variable region sequences demonstrated that antigen-experienced clones were consistently shared among these distinct compartments. This study establishes a relationship between extraparenchymal lymphoid tissue and parenchymal infiltrates and defines the arrangement of B cell clones that populate the central nervous system of patients with multiple sclerosis.
doi:10.1093/brain/awq350
PMCID: PMC3030766  PMID: 21216828
multiple sclerosis; B cells; clonal expansion; antigen experience; central nervous system
8.  B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes 
Science translational medicine  2014;6(248):248ra107.
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF) and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS resident B cells encounter antigen and experience affinity maturation. In this study, paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clonal families were more often found in the draining CLNs. More mature clonal family members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or impact the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS.
doi:10.1126/scitranslmed.3008879
PMCID: PMC4388137  PMID: 25100741
9.  MOG cell-based assay detects non-MS patients with inflammatory neurologic disease 
Objective:
To optimize sensitivity and disease specificity of a myelin oligodendrocyte glycoprotein (MOG) antibody assay.
Methods:
Consecutive sera (n = 1,109) sent for aquaporin-4 (AQP4) antibody testing were screened for MOG antibodies (Abs) by cell-based assays using either full-length human MOG (FL-MOG) or the short-length form (SL-MOG). The Abs were initially detected by Alexa Fluor goat anti-human IgG (H + L) and subsequently by Alexa Fluor mouse antibodies to human IgG1.
Results:
When tested at 1:20 dilution, 40/1,109 sera were positive for AQP4-Abs, 21 for SL-MOG, and 180 for FL-MOG. Only one of the 40 AQP4-Ab–positive sera was positive for SL-MOG-Abs, but 10 (25%) were positive for FL-MOG-Abs (p = 0.0069). Of equal concern, 48% (42/88) of sera from controls (patients with epilepsy) were positive by FL-MOG assay. However, using an IgG1-specific secondary antibody, only 65/1,109 (5.8%) sera were positive on FL-MOG, and AQP4-Ab– positive and control sera were negative. IgM reactivity accounted for the remaining anti-human IgG (H + L) positivity toward FL-MOG. The clinical diagnoses were obtained in 33 FL-MOG–positive patients, blinded to the antibody data. IgG1-Abs to FL-MOG were associated with optic neuritis (n = 11), AQP4-seronegative neuromyelitis optica spectrum disorder (n = 4), and acute disseminated encephalomyelitis (n = 1). All 7 patients with probable multiple sclerosis (MS) were MOG-IgG1 negative.
Conclusions:
The limited disease specificity of FL-MOG-Abs identified using Alexa Fluor goat anti-human IgG (H + L) is due in part to detection of IgM-Abs. Use of the FL-MOG and restricting to IgG1-Abs substantially improves specificity for non-MS demyelinating diseases.
Classification of evidence:
This study provides Class II evidence that the presence of serum IgG1- MOG-Abs in AQP4-Ab–negative patients distinguishes non-MS CNS demyelinating disorders from MS (sensitivity 24%, 95% confidence interval [CI] 9%–45%; specificity 100%, 95% CI 88%–100%).
doi:10.1212/NXI.0000000000000089
PMCID: PMC4370386  PMID: 25821844
10.  Epstein–Barr virus infection is not a characteristic feature of multiple sclerosis brain 
Brain  2009;132(12):3318-3328.
Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system (CNS) that is thought to be caused by a combination of genetic and environmental factors. To date, considerable evidence has associated Epstein–Barr virus (EBV) infection with disease development. However, it remains controversial whether EBV infects multiple sclerosis brain and contributes directly to CNS immunopathology. To assess whether EBV infection is a characteristic feature of multiple sclerosis brain, a large cohort of multiple sclerosis specimens containing white matter lesions (nine adult and three paediatric cases) with a heterogeneous B cell infiltrate and a second cohort of multiple sclerosis specimens (12 cases) that included B cell infiltration within the meninges and parenchymal B cell aggregates, were examined for EBV infection using multiple methodologies including in situ hybridization, immunohistochemistry and two independent real-time polymerase chain reaction (PCR) methodologies that detect genomic EBV or the abundant EBV encoded RNA (EBER) 1, respectively. We report that EBV could not be detected in any of the multiple sclerosis specimens containing white matter lesions by any of the methods employed, yet EBV was readily detectable in multiple Epstein–Barr virus-positive control tissues including several CNS lymphomas. Furthermore, EBV was not detected in our second cohort of multiple sclerosis specimens by in situ hybridization. However, our real-time PCR methodologies, which were capable of detecting very few EBV infected cells, detected EBV at low levels in only 2 of the 12 multiple sclerosis meningeal specimens examined. Our finding that CNS EBV infection was rare in multiple sclerosis brain indicates that EBV infection is unlikely to contribute directly to multiple sclerosis brain pathology in the vast majority of cases.
doi:10.1093/brain/awp200
PMCID: PMC2792367  PMID: 19638446
B cells; Epstein–Barr virus; multiple sclerosis brain
11.  Comprehensive Phenotyping in Multiple Sclerosis: Discovery Based Proteomics and the Current Understanding of Putative Biomarkers 
Disease Markers  2006;22(4):213-225.
Currently, there is no single test for multiple sclerosis (MS). Diagnosis is confirmed through clinical evaluation, abnormalities revealed by magnetic resonance imaging (MRI), and analysis of cerebrospinal fluid (CSF) chemistry. The early and accurate diagnosis of the disease, monitoring of progression, and gauging of therapeutic intervention are important but elusive elements of patient care. Moreover, a deeper understanding of the disease pathology is needed, including discovery of accurate biomarkers for MS. Herein we review putative biomarkers of MS relating to neurodegeneration and contributions to neuropathology, with particular focus on autoimmunity. In addition, novel assessments of biomarkers not driven by hypotheses are discussed, featuring our application of advanced proteomics and metabolomics for comprehensive phenotyping of CSF and blood. This strategy allows comparison of component expression levels in CSF and serum between MS and control groups. Examination of these preliminary data suggests that several CSF proteins in MS are differentially expressed, and thus, represent putative biomarkers deserving of further evaluation.
doi:10.1155/2006/670439
PMCID: PMC3851054  PMID: 17124343
12.  PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis 
Journal of autoimmunity  2013;43:1-9.
Autoimmune disease results from a loss of tolerance to self-antigens in genetically susceptible individuals. Completely understanding this process requires that targeted antigens be identified, and so a number of techniques have been developed to determine immune receptor specificities. We previously reported the construction of a phage-displayed synthetic human peptidome and a proof-of-principle analysis of antibodies from three patients with neurological autoimmunity. Here we present data from a large-scale screen of 298 independent antibody repertoires, including those from 73 healthy sera, using phage immunoprecipitation sequencing. The resulting database of peptide-antibody interactions characterizes each individual’s unique autoantibody fingerprint, and includes specificities found to occur frequently in the general population as well as those associated with disease. Screening type 1 diabetes (T1D) patients revealed a prematurely polyautoreactive phenotype compared with their matched controls. A collection of cerebrospinal fluids and sera from 63 multiple sclerosis patients uncovered novel, as well as previously reported antibody-peptide interactions. Finally, a screen of synovial fluids and sera from 64 rheumatoid arthritis patients revealed novel disease-associated antibody specificities that were independent of seropositivity status. This work demonstrates the utility of performing PhIP-Seq screens on large numbers of individuals and is another step toward defining the full complement of autoimmunoreactivities in health and disease.
doi:10.1016/j.jaut.2013.01.013
PMCID: PMC3677742  PMID: 23497938
autoantigen discovery; high throughput screening; PhIP-Seq; proteomics
13.  Specific peripheral B cell tolerance defects in patients with multiple sclerosis 
The Journal of Clinical Investigation  2013;123(6):2737-2741.
Multiple sclerosis (MS) is a genetically mediated autoimmune disease of the central nervous system. B cells have recently emerged as major contributors to disease pathogenesis, but the mechanisms responsible for the loss of B cell tolerance in patients with MS are largely unknown. In healthy individuals, developing autoreactive B cells are removed from the repertoire at 2 tolerance checkpoints during early B cell development. Both of these central and peripheral B cell tolerance checkpoints are defective in patients with rheumatoid arthritis (RA) and type 1 diabetes (T1D). Here, we found that only the peripheral, but not the central, B cell tolerance checkpoint is defective in patients with MS. We show that this specific defect is accompanied by increased activation and homeostatic proliferation of mature naive B cells. Interestingly, all of these MS features parallel defects observed in FOXP3-deficient IPEX patients, who harbor nonfunctional Tregs. We demonstrate that in contrast to patients with RA or T1D, bone marrow central B cell selection in MS appears normal in most patients. In contrast, patients with MS suffer from a specific peripheral B cell tolerance defect that is potentially attributable to impaired Treg function and that leads to the accumulation of autoreactive B cell clones in their blood.
doi:10.1172/JCI68775
PMCID: PMC3668812  PMID: 23676463
14.  Models of Somatic Hypermutation Targeting and Substitution Based on Synonymous Mutations from High-Throughput Immunoglobulin Sequencing Data 
Analyses of somatic hypermutation (SHM) patterns in B cell immunoglobulin (Ig) sequences contribute to our basic understanding of adaptive immunity, and have broad applications not only for understanding the immune response to pathogens, but also to determining the role of SHM in autoimmunity and B cell cancers. Although stochastic, SHM displays intrinsic biases that can confound statistical analysis, especially when combined with the particular codon usage and base composition in Ig sequences. Analysis of B cell clonal expansion, diversification, and selection processes thus critically depends on an accurate background model for SHM micro-sequence targeting (i.e., hot/cold-spots) and nucleotide substitution. Existing models are based on small numbers of sequences/mutations, in part because they depend on data from non-coding regions or non-functional sequences to remove the confounding influences of selection. Here, we combine high-throughput Ig sequencing with new computational analysis methods to produce improved models of SHM targeting and substitution that are based only on synonymous mutations, and are thus independent of selection. The resulting “S5F” models are based on 806,860 Synonymous mutations in 5-mer motifs from 1,145,182 Functional sequences and account for dependencies on the adjacent four nucleotides (two bases upstream and downstream of the mutation). The estimated profiles can explain almost half of the variance in observed mutation patterns, and clearly show that both mutation targeting and substitution are significantly influenced by neighboring bases. While mutability and substitution profiles were highly conserved across individuals, the variability across motifs was found to be much larger than previously estimated. The model and method source code are made available at http://clip.med.yale.edu/SHM
doi:10.3389/fimmu.2013.00358
PMCID: PMC3828525  PMID: 24298272
immunoglobulin; B cell; somatic hypermutation; mutability; substitution; targeting; AID; affinity maturation
15.  Autoantibodies Produced at the Site of Tissue Damage Provide Evidence of Humoral Autoimmunity in Inclusion Body Myositis 
PLoS ONE  2012;7(10):e46709.
Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens.
doi:10.1371/journal.pone.0046709
PMCID: PMC3465259  PMID: 23071619
16.  Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein 
Nature medicine  2007;13(2):211-217.
The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.
doi:10.1038/nm1488
PMCID: PMC3429369  PMID: 17237795
17.  Related B cell clones that populate the CSF and CNS of patients with multiple sclerosis produce CSF immunoglobulin 
Journal of neuroimmunology  2011;233(1-2):245-248.
We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.
doi:10.1016/j.jneuroim.2011.01.010
PMCID: PMC3090654  PMID: 21353315
multiple sclerosis; B cells; cerebrospinal fluid; central nervous system; oligoclonal bands
18.  Cortical injury in multiple sclerosis; the role of the immune system 
BMC Neurology  2011;11:152.
The easily identifiable, ubiquitous demyelination and neuronal damage that occurs within the cerebral white matter of patients with multiple sclerosis (MS) has been the subject of extensive study. Accordingly, MS has historically been described as a disease of the white matter. Recently, the cerebral cortex (gray matter) of patients with MS has been recognized as an additional and major site of disease pathogenesis. This acknowledgement of cortical tissue damage is due, in part, to more powerful MRI that allows detection of such injury and to focused neuropathology-based investigations. Cortical tissue damage has been associated with inflammation that is less pronounced to that which is associated with damage in the white matter. There is, however, emerging evidence that suggests cortical damage can be closely associated with robust inflammation not only in the parenchyma, but also in the neighboring meninges. This manuscript will highlight the current knowledge of inflammation associated with cortical tissue injury. Historical literature along with contemporary work that focuses on both the absence and presence of inflammation in the cerebral cortex and in the cerebral meninges will be reviewed.
doi:10.1186/1471-2377-11-152
PMCID: PMC3266198  PMID: 22145746
20.  Acute Demyelinating Disease after Oral Therapy with Herbal Extracts 
Case Reports in Neurology  2011;3(2):141-146.
Central nervous system demyelinating processes such as multiple sclerosis and acute disseminated encephalomyelitis constitute a group of diseases not completely understood in their physiopathology. Environmental and toxic insults are thought to play a role in priming autoimmunity. The aim of the present report is to describe a case of acute demyelinating disease with fatal outcome occurring 15 days after oral exposure to herbal extracts.
doi:10.1159/000329734
PMCID: PMC3130893  PMID: 21738505
Phytomedicine; Demyelinating disease; Immunostimulant
21.  Intrathecal Anti-MOG Antibody Production is Elevated in Multiple Sclerosis 
Archives of neurology  2010;67(9):1102-1108.
Objective
Evaluate antibodies (Ab) to myelin oligodendrocyte glycoprotein (MOG) in the serum and cerebrospinal fluid (CSF) of multiple sclerosis (MS) subjects and controls.
Design
Prospective case control series.
Setting
Academic referral center.
Subjects
Twenty-six controls with non-inflammatory neurologic disease (NIND) and 35 MS subjects donated serum and CSF for rMOG Ab determination.
Main Outcome Measures
Serum and CSF rMOG Ab and albumin levels were used to calculate an “rMOG index”. Clinical disability, CSF markers, and magnetic resonance (MR) metrics were correlated to rMOG index.
Results
rMOG index was elevated in MS subjects compared to controls (p=0.012). Progressive MS subjects exhibited elevated rMOG indices compared to relapsing remitting MS (RRMS) (p=0.041). rMOG index was inferior to IgG index in differentiating MS subjects from controls. However, 7 of 16 MS subjects with normal IgG indices had an elevated rMOG index. rMOG index did not correlate with clinical disability, other CSF markers, or radiographic outcome measures.
Conclusions
rMOG index, a marker of intrathecal MOG Ab production, may provide complementary information to routine CSF testing in the diagnosis of MS. Furthermore, intrathecal anti-MOG Ab production may be more pronounced in progressive than relapsing forms of MS.
doi:10.1001/archneurol.2010.197
PMCID: PMC3051403  PMID: 20837855
22.  Age-dependent B cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis 
Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface antigens in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. While antibodies to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum antibodies that brightly labeled the MOG transfectant. Antibodies to two other myelin surface antigens were largely absent. Affinity purification of MOG antibodies as well as competition of binding with soluble MOG documented their binding specificity. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG antibodies, compared to 14.7% of patients in the 10–18 year age group. B cell autoimmunity to this myelin surface antigen is therefore most common in patients with a very early onset of MS.
doi:10.4049/jimmunol.0801888
PMCID: PMC2795331  PMID: 19687098
Human; Multiple Sclerosis; Autoantibodies; MOG
23.  Antibodies Produced by Clonally Expanded Plasma Cells in Multiple Sclerosis Cerebrospinal Fluid 
Annals of neurology  2009;65(6):639-649.
Objective
Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic.
Methods
We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy-and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections.
Results
Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immuno-reactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells.
Interpretation
The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.
doi:10.1002/ana.21641
PMCID: PMC2843543  PMID: 19557869
24.  Paradoxical dysregulation of the neural stem cell pathway Sonic hedgehog-Gli1 in autoimmune encephalomyelitis and multiple sclerosis 
Annals of neurology  2008;64(4):417-427.
Objective
Neurovascular niches have been proposed as critical components of the neural stem cell (NSC) response to acute central nervous system (CNS) injury, however, it is unclear whether these potential reparative niches remain functional during chronic injury. Here we asked how CNS inflammatory injury regulates the intrinsic properties of NSCs and their niches.
Methods
We investigated the sonic hedgehog (Shh)-Gli1 pathway, an important signaling pathway for NSCs, in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) and its regulation of by inflammatory cytokines.
Results
We show that Shh is markedly up-regulated by reactive and perivascular astroglia in areas of injury in MS lesions and during EAE. Astroglia outside the subventricular zone (SVZ) niche can support NSC differentiation towards neurons and oligodendrocytes and Shh is a critical mediator of this effect. Shh induces differential upregulation of the transcription factor Gli1, which mediates Shh-induced NSC differentiation. However, despite the increase in Shh and the fact that Gli1 was initially increased during early inflammation of EAE and active lesions of MS, Gli1 was significantly decreased in spinal cord oligodendrocyte precursor cells (OPCs) after onset of EAE and in chronic active and inactive lesions from MS brain. The Th1 cytokine IFN-γ was unique in inducing Shh expression in astroglia and NSCs, while paradoxically suppressing Gli1 expression in NSCs and inhibiting Shh-mediated NSC differentiation.
Interpretation
Our data suggest that endogenous repair potential during chronic injury appears to be limited by inflammation-induced alterations in intrinsic NSC molecular pathways such as Gli1.
doi:10.1002/ana.21457
PMCID: PMC2757750  PMID: 18991353
25.  Antigen Specificity of Clonally Expanded and Receptor Edited Cerebrospinal Fluid B cells from Patients with Relapsing Remitting MS 
Journal of neuroimmunology  2007;186(1-2):164-176.
We re-engineered the immunoglobulin rearrangements from clonally expanded CSF B cells of three Multiple Sclerosis patients as Fab fragments, and used three methods to test for their Ag-specificity. Nine out of ten Fab fragments were reactive to Myelin Basic Protein (MBP). The one Fab that did not react to MBP was a product of receptor editing. Two of the nine MBP-reactive Fabs were also reactive to GFAP and CNPase, indicating that these clones were polyreactive. Targeting the mechanisms that allows these self-reactive B cells to reside in the CSF of MS patients may prove to be a potent immunotherapeutic strategy.
doi:10.1016/j.jneuroim.2007.03.002
PMCID: PMC2709235  PMID: 17451814
Multiple Sclerosis; B lymphocytes; antigen specificity; antibodies; gene rearrangement

Results 1-25 (27)