Multiple sclerosis (MS) is a multifocal demyelinating disease with progressive neurodegeneration caused by an autoimmune response to self-antigens in a genetically susceptible individual. While the formation and persistence of meningeal lymphoid follicles suggest persistence of antigens to drive the continuing inflammatory and humoral response, the identity of an antigen or infectious agent leading to the oligoclonal expansion of B and T cells is unknown. In this review we examine new paradigms for understanding the immunopathology of MS, present recent data defining the common genetic variants underlying disease susceptibility, and explore how improved understanding of immune pathway disruption can inform MS prognosis and treatment decisions.

Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease of the central nervous system, and numerous studies have shown that MS has a strong genetic component. Independent studies to identify MS-associated genes have often indicated multiple signals in physically close genomic regions, although by their proximity it is not always clear if these data indicate redundant or truly independent genetic signals. Recently, three MS study samples were genotyped in parallel using an Illumina Custom BeadChip. These revealed multiple significantly associated single-nucleotide polymorphisms within a 600 kb stretch on chromosome 16p13. Here we present a detailed analysis of variants in this region that clarifies the independent nature of these signals. The linkage disequilibrium patterns in the region and logistic regression analysis of the associations suggest that this region likely harbors three independent MS disease loci. Further, we examined cis-expression QTLs, histone modifications and CCCTC-binding factor (CTCF) binding data in the region. We also tested for correlated expression of the genes from the region using whole-genome expression array data from lymphoblastoid cell lines. Three of the genes show expression correlations across loci. Furthermore, in the GM12878 lymphoblastoid cell line, these three genes are in a continuous region devoid of H3K27 methylation, suggesting an open chromatin configuration. This region likely only contributes minimal risk to MS; however, investigation of this region will undoubtedly provide insight into the functional mechanisms of these genes. These data highlight the importance of taking a closer look at the expression and function of chromosome 16p13 in the pathogenesis of MS.

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.

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the central nervous system resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin reactive T cells in peripheral blood was observed. Using T cell cloning techniques, a modest increase in the frequency of myelin reactive T cells in patients as compared to control subjects. Here, we investigated whether MOG-specific T cells could be detected and their frequency measured using DRB1*0401/MOG97–109(107E-S) tetramers in MS subjects and healthy controls expressing HLA Class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG reactive T cells upon MOG peptide stimulation of PMBC. MOG97–109 reactive CD4+ T cells, isolated with DRB1*0401/MOG97–109 tetramers, after a short-term culture of PMBC with MOG97–109 peptides were detected more frequently from patients with MS as compared to healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG97–109(107E-S) tetramer positive cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects utilizing Class II tetramers and the frequency of MOG97–109 reactive T cells is greater in patients with MS as compared to healthy controls.

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.

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.

Human CD4+ T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4+ T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.

Gene-gene interactions are proposed as one important component of the genetic architecture of complex diseases, and are just beginning to be evaluated in the context of genome wide association studies (GWAS). In addition to detecting epistasis, a benefit to interaction analysis is that it also increases power to detect weak main effects. We conducted a knowledge-driven interaction analysis of a GWAS of 931 multiple sclerosis trios to discover gene-gene interactions within established biological contexts. We identify heterogeneous signals, including a gene-gene interaction between CHRM3 and MYLK (joint p = 0.0002), an interaction between two phospholipase-β isoforms, PLCβ1 & PLCβ4 (joint p = 0.0098), and a modest interaction between ACTN1 and MYH9 (joint p = 0.0326), all localized to calcium-signaled cytoskeletal regulation. Furthermore, we discover a main effect (joint p = 5.2E-5) previously unidentified by single-locus analysis within another related gene, SCIN, a calcium-binding cytoskeleton regulatory protein. This work illustrates that knowledge-driven interaction analysis of GWAS data is a feasible approach to identify new genetic effects. The results of this study are among the first gene-gene interactions and non-immune susceptibility loci for multiple sclerosis. Further, the implicated genes cluster within inter-related biological mechanisms that suggest a neurodegenerative component to multiple sclerosis.

Summary of Recent Advances

Human regulatory T cells (Tregs) play a critical role in preventing autoimmunity, and their failure contributes to autoimmune diseases. In recent years, our understanding of human Tregs has been greatly enhanced by improvements in the definition and isolation of pure human Tregs, as well as by the discovery of phenotypically and functionally distinct human Treg subsets. This progress has also yielded a better understanding of the mechanisms of human Treg suppression and the role of human Tregs in autoimmune diseases. An unexpected discovery is that human Tregs have considerable plasticity that allows them to produce the pro-inflammatory cytokine IL-17 under certain conditions. These recent advances highlight the importance of studying the roles of both mouse and human Tregs in autoimmunity.

The primary genetic risk factor in multiple sclerosis (MS) is the HLA-DRB1*1501 allele; however, much of the remaining genetic contribution to MS has yet to be elucidated. Several lines of evidence support a role for neuroendocrine system involvement in autoimmunity which may, in part, be genetically determined. Here, we comprehensively investigated variation within eight candidate hypothalamic–pituitary–adrenal (HPA) axis genes and susceptibility to MS. A total of 326 SNPs were investigated in a discovery dataset of 1343 MS cases and 1379 healthy controls of European ancestry using a multi-analytical strategy. Random Forests, a supervised machine-learning algorithm, identified eight intronic SNPs within the corticotrophin-releasing hormone receptor 1 or CRHR1 locus on 17q21.31 as important predictors of MS. On the basis of univariate analyses, six CRHR1 variants were associated with decreased risk for disease following a conservative correction for multiple tests. Independent replication was observed for CRHR1 in a large meta-analysis comprising 2624 MS cases and 7220 healthy controls of European ancestry. Results from a combined meta-analysis of all 3967 MS cases and 8599 controls provide strong evidence for the involvement of CRHR1 in MS. The strongest association was observed for rs242936 (OR = 0.82, 95% CI = 0.74–0.90, P = 9.7 × 10−5). Replicated CRHR1 variants appear to exist on a single associated haplotype. Further investigation of mechanisms involved in HPA axis regulation and response to stress in MS pathogenesis is warranted.

Background

Postoperative ventricular dysfunction (VnD) occurs in 9–20% of coronary artery bypass graft (CABG) surgical patients and is associated with increased postoperative morbidity and mortality. Understanding genetic causes of postoperative VnD should enhance patient risk stratification and improve treatment and prevention strategies. We aimed to determine if genetic variants associate with occurrence of in-hospital VnD after CABG surgery.

Methods

A genome-wide association study identified single nucleotide polymorphisms (SNPs) associated with postoperative VnD in male subjects of European ancestry undergoing isolated primary CABG surgery with cardiopulmonary bypass. VnD was defined as the need for ≥2 inotropes or mechanical ventricular support after CABG surgery. Validated SNPs were assessed further in two replication CABG cohorts and meta-analysis was performed.

Results

Over 100 SNPs were associated with VnD (P<10−4), with one SNP (rs17691914) encoded at 3p22.3 reaching genome-wide significance (Padditive model = 2.14×10−8). Meta-analysis of validation and replication study data for 17 SNPs identified three SNPs associated with increased risk for developing postoperative VnD after adjusting for clinical risk factors. These SNPs are located at 3p22.3 (rs17691914, ORadditive model = 2.01, P = 0.0002), 3p14.2 (rs17061085, ORadditive model = 1.70, P = 0.0001) and 11q23.2 (rs12279572, ORrecessive model = 2.19, P = 0.001).

Conclusions

No SNPs were consistently associated with strong risk (ORadditive model>2.1) of developing in-hospital VnD after CABG surgery. However, three genetic loci identified by meta-analysis were more modestly associated with development of postoperative VnD. Studies of larger cohorts to assess these loci as well as to define other genetic mechanisms and related biology that link genetic variants to postoperative ventricular dysfunction are warranted.

Genome-wide association (GWA) studies have identified numerous, replicable, genetic associations between common single nucleotide polymorphisms (SNPs) and risk of common autoimmune and inflammatory (immune-mediated) diseases, some of which are shared between two diseases. Along with epidemiological and clinical evidence, this suggests that some genetic risk factors may be shared across diseases—as is the case with alleles in the Major Histocompatibility Locus. In this work we evaluate the extent of this sharing for 107 immune disease-risk SNPs in seven diseases: celiac disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes. We have developed a novel statistic for Cross Phenotype Meta-Analysis (CPMA) which detects association of a SNP to multiple, but not necessarily all, phenotypes. With it, we find evidence that 47/107 (44%) immune-mediated disease risk SNPs are associated to multiple—but not all—immune-mediated diseases (SNP-wise PCPMA<0.01). We also show that distinct groups of interacting proteins are encoded near SNPs which predispose to the same subsets of diseases; we propose these as the mechanistic basis of shared disease risk. We are thus able to leverage genetic data across diseases to construct biological hypotheses about the underlying mechanism of pathogenesis.

Author Summary

Over the last five years we have found over 100 genetic variants predisposing to common diseases affecting the immune system. In this study we analyze 107 such variants across seven diseases and find that almost half are shared across diseases. We also find that the patterns of sharing across diseases cluster these variants into groups; proteins encoded near variants in the same group tend to interact. This suggests that genetic variation may influence entire pathways to create risk to multiple diseases.

Here we present a microfluidic method for the analysis of single cell secretions. The method co-encapsulates cells with microspheres conjugated with capture antibodies and detection fluorescence-labeled antibodies. The secreted substance captured on the microsphere surface and detected via detection antibodies generating a localized fluorescent signal on a microsphere surface. Using this method, CD4+CD25+ regulatory T cells were encapsulated and assayed to detect IL-10 secreting cell in population.

Summary

The large diversity of cells that comprise the human immune system requires methods that can resolve the individual contributions of specific subsets to an immunological response. Microengraving is process that uses a dense, elastomeric array of microwells to generate microarrays of proteins secreted from large numbers of individual live cells (~104–105 cells/assay). In this paper, we describe an approach based on this technology to quantify the rates of secretion from single immune cells. Numerical simulations of the microengraving process indicated an operating regime between 30 min-4 h that permits quantitative analysis of the rates of secretion. Through experimental validation, we demonstrate that microengraving can provide quantitative measurements of both the frequencies and the distribution in rates of secretion for up to four cytokines simultaneously released from individual viable primary immune cells. The experimental limits of detection ranged from 0.5 to 4 molecules/s for IL-6, IL-17, IFNγ, IL-2, and TNFα. These multidimensional measures resolve the number and intensities of responses by cells exposed to stimuli with greater sensitivity than single-parameter assays for cytokine release. We show that cells from different donors exhibit distinct responses based on both the frequency and magnitude of cytokine secretion when stimulated under different activating conditions. Primary T cells with specific profiles of secretion can also be recovered after microengraving for subsequent expansion in vitro. These examples demonstrate the utility of quantitative, multidimensional profiles of single cells for analyzing the diversity and dynamics of immune responses in vitro and for identifying rare cells from clinical samples.

The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-β and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-β during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-β to the Th17-inducing cytokines (IL-1β, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-β–dependent coexpression of IL-9 and IL-17 was identified to be IL-1β. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1β. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9+IL-17+ cells. These data demonstrate the presence of IL-17+IL-9+ CD4 cells induced by IL-1β that may play a role in human autoimmune disease.

The MHC class II transactivator gene (CIITA) is an important transcription factor regulating gene required for HLA class II MHC-restricted antigen presentation. Association with HLA class II variation, particularly HLA-DRB1*1501, has been well-established for multiple sclerosis (MS). In addition, the −168A/G CIITA promoter variant (rs3087456) has been reported to be associated with MS. Thus, a multi-stage investigation of variation within CIITA, DRB1*1501 and MS was undertaken in 6108 individuals. In stage 1, 24 SNPs within CIITA were genotyped in 1320 cases and 1363 controls (n = 2683). Rs4774 (missense +1614G/C; G500A) was associated with MS (P = 4.9 × 10−3), particularly in DRB1*1501 +individuals (P = 1 × 10−4). No association was observed for the −168A/G promoter variant. In stage 2, rs4774 was genotyped in 973 extended families; rs4774*C was also associated with increased risk for MS in DRB1*1501+ families (P = 2.3 × 10−2). In a third analysis, rs4774 was tested in cases and controls (stage 1) combined with one case per family (stage 2) for increased power. Rs4774*C was associated with MS (P = 1 × 10−3), particularly in DRB1*1501+ cases and controls (P = 1 × 10−4). Results obtained from logistic regression analysis showed evidence for interaction between rs4774*C and DRB1*1501 associated with risk for MS (ratio of ORs = 1.72, 95% CI 1.28–2.32, P = 3 × 10−4). Furthermore, rs4774*C was associated with DRB1*1501+ MS when conditioned on the presence (OR = 1.67, 95% CI = 1.19–2.37, P = 1.9 × 10−3) and absence (OR = 1.49, 95% CI = 1.15–1.95, P = 2.3 × 10−3) of CLEC16A rs6498169*G, a putative MS risk allele adjacent to CIITA. Our results provide strong evidence supporting a role for CIITA variation in MS risk, which appears to depend on the presence of DRB1*1501.

Several single nucleotide polymorphism (SNP) genome-wide association studies (GWAS) have been completed in multiple sclerosis. Follow-up studies of the variants with the most promising rankings, especially when supplemented by informed candidate gene selection, have proven to be extremely successful. We report here the results of a multi-stage replication analysis of the putatively associated SNPs identified in the Wellcome Trust Case-Control Consortium non-synonymous SNP screen. In total the replication sample consisted of 3444 patients and 2595 controls. A combined analysis of the nsSNP screen and replication data provides evidence implicating a novel additional locus, rs3748816 in MMEL1 (OR=1.16, p=3.54×10−6) in multiple sclerosis susceptibility.

Background

Predicting susceptibility to multiple sclerosis may have important clinical applications either as part of a diagnostic algorithm or as a tool with which to identify high-risk individuals for prospective studies. Here, we examine the utility of an aggregate measure of risk of multiple sclerosis (MS) based on genetic susceptibility loci. Secondarily, we assess the added effect of environmental risk factors that have been associated with susceptibility for MS.

Methods

We created a weighted genetic risk score (wGRS) that includes 16 MS susceptibility loci. We tested our model using data from (1) 2215 MS cases and 2189 controls (derivation samples), (2) a validation set of 1340 cases and 1109 controls taken from several MS therapeutic trials (TT samples), and (3) a second validation set of 143 cases and 281 controls from the U.S. Nurses’ Health Studies I and II (NHS) for whom we also have information regarding exposure to smoking and Epstein-Barr Virus (EBV).

Findings

. Patients with wGRS > 1.25 standard deviations from the mean had a significantly higher odds ratio for MS in all datasets. The area under the curve for a purely genetic model was 0.70 and for a gender + genetic model was 0.74 in the derivation samples (P <0.0001), 0.64 and 0.72 in the TT cohort (P <0.0001). Similarly, consideration of smoking and immune response to EBV enhanced the AUC of 0.64 for the genetic model to 0.68 in the NHS cohort (P =0.02). The wGRS does not appear to be correlated with conversion of a clinically isolated syndrome to MS.

Interpretation

The current combination of 16 susceptibility alleles into a wGRS modestly predicts MS risk and shows consistent discriminatory ability in independent subject samples and is enhanced by considering non-genetic risk factors.

Multiple sclerosis (MS) is characterized as an autoimmune demyelinating disease. Numerous family studies have confirmed a strong genetic component underlying its etiology. After several decades of frustrating research, the advent and application of affordable genotyping of dense SNP maps in large datasets has ushered in a new era in which rapid progress is being made in our understanding of the genetics underlying many complex traits. For MS, one of the first discoveries to emerge in this new era was the association with rs6897932[T244I] in the interleukin-7 receptor alpha chain (IL7RA) gene (Gregory et al. 2007; International Multiple Sclerosis Genetics Consortium 2007; Lundmark 2007), a discovery that was accompanied by functional data that suggest this variant is likely to be causative rather than a surrogate proxy (Gregory et al. 2007). We hypothesized that variations in other genes functionally related to IL7RA might also influence MS. We investigated this hypothesis by examining genes in the extended biological pathway related to IL7RA to identify novel associations. We identified 73 genes with putative functional relationships to IL7RA and subsequently genotyped 7,865 SNPs in and around these genes using an Illumina Infinium BeadChip assay. Using 2,961 case-control dataset, two of the gene regions examined, IL7 and SOCS1, had significantly associated single-nucleotide polymorphisms (SNPs) that further replicated in an independent case-control dataset (4,831 samples) with joint p-values as high as 8.29×10-6 and 3.48×10-7, respectively, exceeding the threshold for experiment-wise significance. Our results also implicate two additional novel gene regions that are likely to be associated with MS: PRKCE with p-values reaching 3.47×10-4 and BCL2 with p-values reaching 4.32×10-4. The TYK2 gene, which also emerged in our analysis, has recently been associated with MS (Ban et al. 2009). These results help to further delineate the genetic architecture of MS and validate our pathway approach as an effective method to identify novel associations in a complex disease.

Background

Multiple sclerosis (MS) is the most common cause of chronic neurologic disability beginning in early to middle adult life. Results from recent genome-wide association studies (GWAS) have substantially lengthened the list of disease loci and provide convincing evidence supporting a multifactorial and polygenic model of inheritance. Nevertheless, the knowledge of MS genetics remains incomplete, with many risk alleles still to be revealed.

Methods

We used a discovery GWAS dataset (8,844 samples, 2,124 cases and 6,720 controls) and a multi-step logistic regression protocol to identify novel genetic associations. The emerging genetic profile included 350 independent markers and was used to calculate and estimate the cumulative genetic risk in an independent validation dataset (3,606 samples). Analysis of covariance (ANCOVA) was implemented to compare clinical characteristics of individuals with various degrees of genetic risk. Gene ontology and pathway enrichment analysis was done using the DAVID functional annotation tool, the GO Tree Machine, and the Pathway-Express profiling tool.

Results

In the discovery dataset, the median cumulative genetic risk (P-Hat) was 0.903 and 0.007 in the case and control groups, respectively, together with 79.9% classification sensitivity and 95.8% specificity. The identified profile shows a significant enrichment of genes involved in the immune response, cell adhesion, cell communication/signaling, nervous system development, and neuronal signaling, including ionotropic glutamate receptors, which have been implicated in the pathological mechanism driving neurodegeneration. In the validation dataset, the median cumulative genetic risk was 0.59 and 0.32 in the case and control groups, respectively, with classification sensitivity 62.3% and specificity 75.9%. No differences in disease progression or T2-lesion volumes were observed among four levels of predicted genetic risk groups (high, medium, low, misclassified). On the other hand, a significant difference (F = 2.75, P = 0.04) was detected for age of disease onset between the affected misclassified as controls (mean = 36 years) and the other three groups (high, 33.5 years; medium, 33.4 years; low, 33.1 years).

Conclusions

The results are consistent with the polygenic model of inheritance. The cumulative genetic risk established using currently available genome-wide association data provides important insights into disease heterogeneity and completeness of current knowledge in MS genetics.

The frequencies of antigen-specific CD4+ T cells in samples of human tissue has been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or Type 1 diabetes. Conventional approaches involve the expansion of primary T cells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide-loaded MHC Class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNγ and IL-17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical samples.

Previous functional studies have proposed that solution-phase loading of human insulin A-chain peptides into cell surface Class II molecules may be limited by the redox state of intrinsic cysteine residues within the A-chain peptide. T cell functional studies of a human insulin A-chain analogue (KR A1-15) comprised of residues 1-15 of the A-chain peptide as well as an amino-terminal lysine-arginine extension have been carried out in a reducing environment. These data suggest that free thiol moieties within this peptide may participate in major histocompatibility complex (MHC) II/peptide interactions. Two-dimensional 1H NMR spectroscopy data partnered with quantum chemical calculations identified that KR A1-15 exists in a conformational flux sampling heterogeneous redox dependent conformations including: one reduced and two oxidized states. These findings were further supported by mass spectrometry analysis of this peptide that confirmed the presence of a redox state dependent conformational equilibrium. Interestingly, the presence of a free thiol (1Hγ) resonance for cysteine 8 in the oxidized state supports the existence of the third redox dependent conformation represented as a mixed disulfide conformation. We believe these data support the presence of a redox-dependent mechanism for reg ulating the a ctivity of huma n insulin and provides a better understanding of redox chemistry that may be extended to other protein systems.

Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signaling and is required for the maintenance of immunological tolerance in mice1, 2. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 Caucasian subjects with relatively common autoimmune disorders and in 2/648 Caucasian controls. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in 8 autoimmune subjects but in no control subjects. The Odds Ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.

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.

Background

Interleukin 12 (IL-12), a cytokine that promotes generation of helper T cells subtype 1, is increased in multiple sclerosis. Albuterol sulfate, a β2-adrenergic agonist, reduces IL-12 expression, so we tested the effect of albuterol as an add-on treatment to glatiramer acetate therapy.

Objectives

To investigate the clinical and immunologic effects of albuterol treatment as an add-on therapy in patients starting glatiramer acetate treatment.

Design

Single-center double-masked clinical trial.

Setting

Academic research.

Patients

Subjects with relapsing-remitting multiple sclerosis.

Main Outcome Measures

In this single-center double-masked clinical trial, subjects with relapsing-remitting multiple sclerosis were randomized to receive a subcutaneous injection of glatiramer acetate (20 mg) plus an oral dose of placebo daily for 2 years or a subcutaneous injection of glatiramer acetate (20 mg) plus an oral dose of albuterol daily for 2 years. The primary clinical efficacy measurement was the change in Multiple Sclerosis Functional Composite at 2 years, and the primary immunologic end point was the change in expression of IL-13 and interferon γ at each study time point. The classification level of evidence from this trial is C for each question, as this is the first class II clinical trial addressing the efficacy of glatiramer acetate plus albuterol.

Results

Forty-four subjects were randomized to receive glatiramer acetate plus albuterol or glatiramer acetate plus placebo, and 39 subjects contributed to the analysis. Improvement in the Multiple Sclerosis Functional Composite was observed in the glatiramer acetate plus albuterol group at the 6-month (P = .005) and 12-month (P = .04) time points but not at the 24-month time point. A delay in the time to first relapse was also observed in the glatiramer acetate plus albuterol group (P = .03). Immunologically, IL-13 and interferon-γ production decreased in both treatment groups, and a treatment effect on IL-13 production was observed at the 12-month time point (P < .05). Adverse events were generally mild, and only 3 moderate or severe events were considered related to the treatment.

Conclusion

Treatment with glatiramer acetate plus albuterol is well tolerated and improves clinical outcomes in patients with multiple sclerosis.