Expression of CYP24A1 and other multiple sclerosis risk genes in peripheral blood indicates response to vitamin D in homeostatic and inflammatory conditions.

Although genetic and epidemiological evidence indicates vitamin D insufficiency contributes to multiple sclerosis (MS), and serum levels of vitamin D increase on treatment with cholecalciferol, recent metanalyses indicate that this vitamin D form does not ameliorate disease. Genetic variation in genes regulating vitamin D, and regulated by vitamin D, affect MS risk. We evaluated if the expression of vitamin D responsive MS risk genes could be used to assess vitamin D response in immune cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls and people with MS treated with dimethyl fumarate. We assayed changes in expression of vitamin D responsive MS risk (VDRMS) genes in response to treatment with 25 hydroxy vitamin D in the presence or absence of inflammatory stimuli. Expression of CYP24A1 and other VDRMS genes was significantly altered in PBMCs treated with vitamin D in the homeostatic and inflammatory models. Gene expression in MS samples had similar responses to controls, but lower initial expression of the risk genes. Vitamin D treatment abrogated these differences. Expression of CYP24A1 and other MS risk genes in blood immune cells indicate vitamin D response and could enable assessment of immunological response to vitamin D in clinical trials and on therapy.

The latitude-dependent autoimmune disease risk genes ZMIZ1 and IRF8 regulate mononuclear phagocytic cell differentiation in response to vitamin D.

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.

The Interaction of Human and Epstein-Barr Virus miRNAs with Multiple Sclerosis Risk Loci.

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein-Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

The CYP27B1 variant associated with an increased risk of autoimmune disease is underexpressed in tolerizing dendritic cells.

Genome-wide association studies have identified a linkage disequilibrium (LD) block on chromosome 12 associated with multiple sclerosis (MS), type 1 diabetes and other autoimmune diseases. This block contains CYP27B1, which catalyzes the conversion of 25 vitamin D3 (VitD3) to 1,25VitD3. Fine-mapping analysis has failed to identify which of the 17 genes in this block is most associated with MS. We have previously used a functional approach to identify the causal gene. We showed that the expression of several genes in this block in whole blood is highly associated with the MS risk allele, but not CYP27B1. Here, we show that CYP27B1 is predominantly expressed in dendritic cells (DCs). Its expression in these cells is necessary for their response to VitD, which is known to upregulate pathways involved in generating a tolerogenic DC phenotype. Here, we utilize a differentiation protocol to generate inflammatory (DC1) and tolerogenic (DC2) DCs, and show that for the MS risk allele CYP27B1 is underexpressed in DCs, especially DC2s. Of the other Chr12 LD block genes expressed in these cells, only METT21B expression was as affected by the genotype. Another gene associated with autoimmune diseases, CYP24A1, catabolizes 1,25 VitD3, and is predominantly expressed in DCs, but equally between DC1s and DC2s. Overall, these data are consistent with the hypothesis that reduced VitD pathway gene upregulation in DC2s of carriers of the risk haplotype of CYP27B1 contributes to autoimmune diseases. These data support therapeutic approaches aimed at targeting VitD effects on DCs.

The low EOMES/TBX21 molecular phenotype in multiple sclerosis reflects CD56+ cell dysregulation and is affected by immunomodulatory therapies.

Multiple Sclerosis (MS) is an autoimmune disease treated by therapies targeting peripheral blood cells. We previously identified that expression of two MS-risk genes, the transcription factors EOMES and TBX21 (ET), was low in blood from MS and stable over time. Here we replicated the low ET expression in a new MS cohort (p<0.0007 for EOMES, p<0.028 for TBX21) and demonstrate longitudinal stability (p<10(-4)) and high heritability (h(2)=0.48 for EOMES) for this molecular phenotype. Genes whose expression correlated with ET, especially those controlling cell migration, further defined the phenotype. CD56+ cells and other subsets expressed lower levels of Eomes or T-bet protein and/or were under-represented in MS. EOMES and TBX21 risk SNP genotypes, and serum EBNA-1 titres were not correlated with ET expression, but HLA-DRB1*1501 genotype was. ET expression was normalised to healthy control levels with natalizumab, and was highly variable for glatiramer acetate, fingolimod, interferon-beta, dimethyl fumarate.

The autoimmune disease-associated transcription factors EOMES and TBX21 are dysregulated in multiple sclerosis and define a molecular subtype of disease.

We have identified a marked over-representation of transcription factors controlling differentiation of T, B, myeloid and NK cells among the 110 MS genes now known to be associated with multiple sclerosis (MS). To test if the expression of these genes might define molecular subtypes of MS, we interrogated their expression in blood in three independent cohorts of untreated MS (from Sydney and Adelaide) or clinically isolated syndrome (CIS, from San Francisco) patients. Expression of the transcription factors (TF) controlling T and NK cell differentiation, EOMES, TBX21 and other TFs was significantly lower in MS/CIS compared to healthy controls in all three cohorts. Expression was tightly correlated between these TFs, with other T/NK cell TFs, and to another downregulated gene, CCL5. Expression was stable over time, but did not predict disease phenotype. Optimal response to therapy might be indicated by normalization of expression of these genes in blood.

Functional disruption of yeast metacaspase, Mca1, leads to miltefosine resistance and inability to mediate miltefosine-induced apoptotic effects.

Miltefosine (MI) is a novel, potential antifungal agent with activity against some yeast and filamentous fungal pathogens. We previously demonstrated in the model yeast, Saccharomyces cerevisiae, that MI causes disruption of mitochondrial membrane potential and apoptosis-like cell death via interaction with the Cox9p sub-unit of cytochrome c oxidase (COX). To identify additional mechanisms of antifungal action, MI resistance was induced in S. cerevisiae by exposure to the mutagen, ethyl methanesulfonate, and gene mutation(s) responsible for resistance were investigated. An MI-resistant haploid strain (H-C101) was created. Resistance was retained in the diploid strain (D-C101) following mating, confirming dominant inheritance. Phenotypic assessment of individual D-C101 tetrads revealed that only one mutant gene contributed to the MI-resistance phenotype. To identify this gene, the genome of H-C101 was sequenced and 17 mutated genes, including metacaspase-encoding MCA1, were identified. The MCA1 mutation resulted in substitution of asparagine (N) with aspartic acid (D) at position 164 (MCA1(N164D)). MI resistance was found to be primarily due to MCA1(N164D), as single-copy episomal expression of MCA1(N164D), but not two other mutated genes (FAS1(T1417I) and BCK2(T104A)), resulted in MI resistance in the wild-type strain. Furthermore, an MCA1 deletion mutant (mca1Δ) was MI-resistant. MI treatment led to accumulation of reactive oxygen species (ROS) in MI-resistant (MCA1(N164D)-expressing and mca1Δ) strains and MI-susceptible (MCA1-expressing) strains, but failed to activate Mca1 in the MI-resistant strains, demonstrating that ROS accumulation does not contribute to the fungicidal effect of MI. In conclusion, functional disruption of Mca1, leads to MI resistance and inability to mediate MI-induced apoptotic effects. Mca1-mediated apoptosis is therefore a major mechanism of MI-induced antifungal action.

Functionally significant differences in expression of disease-associated IL-7 receptor alpha haplotypes in CD4 T cells and dendritic cells.

Common genetic variants of IL-7 receptor alpha (IL-7Ralpha) have recently been shown to affect susceptibility to multiple sclerosis (MS) and type 1 diabetes, and survival following bone marrow transplantation. Transcription of the gene produces two dominant isoforms, with or without exon 6, which code for membrane-bound or soluble IL-7Ralpha, respectively. The haplotypes produce different isoform ratios. We have tested IL-7Ralpha mRNA expression in cell subsets and in models of T cell homeostasis, activation, tolerance, and differentiation into regulatory T cell/Th1/Th2/Th17, memory, and dendritic cells (DCs) under the hypothesis that the conditions in which haplotype differences are maximal are those likely to be the basis for their association with disease pathogenesis. Maximal differences between haplotypes were found in DCs, where the ligand is mainly thymic stromal lymphopoietin (TSLP). The MS-protective haplotype produces a much lower ratio of soluble to membrane-bound receptor, and so potentially, DCs of this haplotype are more responsive to TSLP. The TSLP/IL-7Ralpha interaction on DCs is known to be critical for production of thymic regulatory T cells, and reduced production of these cells in MS susceptibility haplotypes may be a basis for its association with this disease. IL-7Ralpha mRNA expression varies greatly through cell differentiation so that it may be a useful marker for cell states. We also show that serum levels of soluble receptor are much higher for the MS susceptibility haplotype (p = 4 x 10(-13)). Because signaling through IL-7Ralpha controls T cell regulation, this haplotype difference is likely to affect the immunophenotype and disease pathogenesis.

Miltefosine induces apoptosis-like cell death in yeast via Cox9p in cytochrome c oxidase.

Miltefosine has antifungal properties and potential for development as a therapeutic for invasive fungal infections. However, its mode of action in fungi is poorly understood. We demonstrate that miltefosine is rapidly incorporated into yeast, where it penetrates the mitochondrial inner membrane, disrupting mitochondrial membrane potential and leading to an apoptosis-like cell death. COX9, which encodes subunit VIIa of the cytochrome c oxidase (COX) complex in the electron transport chain of the mitochondrial membrane, was identified as a potential target of miltefosine from a genomic library screen of the model yeast Saccharomyces cerevisiae. When overexpressed in S. cerevisiae, COX9, but not COX7 or COX8, led to a miltefosine-resistant phenotype. The effect of miltefosine on COX activity was assessed in cells expressing different levels of COX9. Miltefosine inhibited COX activity in a dose-dependent manner in Cox9p-positive cells. This inhibition most likely contributed to the miltefosine-induced apoptosis-like cell death.

Gender and the Sex Hormone Estradiol Affect Multiple Sclerosis Risk Gene Expression in Epstein-Barr Virus-Infected B Cells.

Multiple Sclerosis (MS) is a complex immune-mediated disease of the central nervous system. Treatment is based on immunomodulation, including specifically targeting B cells. B cells are the main host for the Epstein-Barr Virus (EBV), which has been described as necessary for MS development. Over 200 genetic loci have been identified as increasing susceptibility to MS. Many MS risk genes have altered expression in EBV infected B cells, dependent on the risk genotype, and are themselves regulated by the EBV transcription factor EBNA2. Females are 2-3 times more likely to develop MS than males. We investigated if MS risk loci might mediate the gender imbalance in MS. From a large public dataset, we identified gender-specific associations with EBV traits, and MS risk SNP/gene pairs with gender differences in their associations with gene expression. Some of these genes also showed gender differences in correlation of gene expression level with Estrogen Receptor 2. To test if estrogens may drive these gender specific differences, we cultured EBV infected B cells (lymphoblastoid cell lines, LCLs), in medium depleted of serum to remove the effects of sex hormones as well as the estrogenic effect of phenol red, and then supplemented with estrogen (100 nM estradiol). Estradiol treatment altered MS risk gene expression, LCL proliferation rate, EBV DNA copy number and EBNA2 expression in a sex-dependent manner. Together, these data indicate that there are estrogen-mediated gender-specific differences in MS risk gene expression and EBV functions. This may in turn contribute to gender differences in host response to EBV and to MS susceptibility.

Age-dependent VDR peak DNA methylation as a mechanism for latitude-dependent multiple sclerosis risk.

BACKGROUND: The mechanisms linking UV radiation and vitamin D exposure to the risk of acquiring the latitude and critical period-dependent autoimmune disease, multiple sclerosis, is unclear. We examined the effect of vitamin D on DNA methylation and DNA methylation at vitamin D receptor binding sites in adult and paediatric myeloid cells. This was accomplished through differentiating CD34+ haematopoietic progenitors into CD14+ mononuclear phagocytes, in the presence and absence of calcitriol. RESULTS: Few DNA methylation changes occurred in cells treated with calcitriol. However, several VDR-binding sites demonstrated increased DNA methylation in cells of adult origin when compared to cells of paediatric origin. This phenomenon was not observed at other transcription factor binding sites. Genes associated with these sites were enriched for intracellular signalling and cell activation pathways involved in myeloid cell differentiation and adaptive immune system regulation. CONCLUSION: These results suggest vitamin D exposure at critical periods during development may contribute to latitude-related differences in autoimmune disease incidence.

Evidence from genome wide association studies implicates reduced control of Epstein-Barr virus infection in multiple sclerosis susceptibility.

BACKGROUND: Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. METHODS: We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. RESULTS: These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10-4), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10-16). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype. CONCLUSIONS: These data indicate targeting EBV may be of therapeutic benefit in MS.

Association of Regulatory T-Cell Expansion With Progression of Amyotrophic Lateral Sclerosis: A Study of Humans and a Transgenic Mouse Model.

Importance: Neuroinflammation appears to be a key modulator of disease progression in amyotrophic lateral sclerosis (ALS) and thereby a promising therapeutic target. The CD4+Foxp3+ regulatory T-cells (Tregs) infiltrating into the central nervous system suppress neuroinflammation and promote the activation of neuroprotective microglia in mouse models of ALS. To our knowledge, the therapeutic association of host Treg expansion with ALS progression has not been studied in vivo. Objective: To assess the role of Tregs in regulating the pathophysiology of ALS in humans and the therapeutic outcome of increasing Treg activity in a mouse model of the disease. Design, Setting, and Participants: This prospective multicenter human and animal study was performed in hospitals, outpatient clinics, and research institutes. Clinical and function assessment, as well as immunological studies, were undertaken in 33 patients with sporadic ALS, and results were compared with 38 healthy control participants who were consecutively recruited from the multidisciplinary ALS clinic at Westmead Hospital between February 1, 2013, and December 31, 2014. All data analysis on patients with ALS was undertaken between January 2015 and December 2016. Subsequently, we implemented a novel approach to amplify the endogenous Treg population using peripheral injections of interleukin 2/interleukin 2 monoclonal antibody complexes (IL-2c) in transgenic mice that expressed mutant superoxide dismutase 1 (SOD1), a gene associated with motor neuron degeneration. Main Outcomes and Measures: In patients with ALS, Treg levels were determined and then correlated with disease progression. Circulating T-cell populations, motor neuron size, glial cell activation, and T-cell and microglial gene expression in spinal cords were determined in SOD1G93A mice, as well as the association of Treg amplification with disease onset and survival time in mice. Results: The cohort of patients with ALS included 24 male patients and 9 female patients (mean [SD] age at assessment, 58.9 [10.9] years). There was an inverse correlation between total Treg levels (including the effector CD45RO+ subset) and rate of disease progression (R = -0.40, P = .002). Expansion of the effector Treg population in the SOD1G93A mice was associated with a significant slowing of disease progression, which was accompanied by an increase in survival time (IL-2c-treated mice: mean [SD], 160.6 [10.8] days; control mice: mean [SD], 144.9 [10.6] days; P = .003). Importantly, Treg expansion was associated with preserved motor neuron soma size and marked suppression of astrocytic and microglial immunoreactivity in the spinal cords of SOD1G93A mice, as well as elevated neurotrophic factor gene expression in spinal cord and peripheral nerves. Conclusions and Relevance: These findings establish a neuroprotective effect of Tregs, possibly mediated by suppression of toxic neuroinflammation in the central nervous system. Strategies aimed at enhancing the Treg population and neuroprotective activity from the periphery may prove therapeutically useful for patients with ALS.

Fungal Inositol Pyrophosphate IP7 Is Crucial for Metabolic Adaptation to the Host Environment and Pathogenicity.

UNLABELLED: Inositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis. We investigate the cryptococcal kinases responsible for the production of PP-IPs (IP7/IP8) and the hierarchy of PP-IP importance in pathogenicity. Using gene deletion and inositol polyphosphate profiling, we identified Kcs1 as the major IP6 kinase (producing IP7) and Asp1 as an IP7 kinase (producing IP8). We show that Kcs1-derived IP7 is the most crucial PP-IP for cryptococcal drug susceptibility and the production of virulence determinants. In particular, Kcs1 kinase activity is essential for cryptococcal infection of mouse lungs, as reduced fungal burdens were observed in the absence of Kcs1 or when Kcs1 was catalytically inactive. Transcriptome and carbon source utilization analysis suggested that compromised growth of the KCS1 deletion strain (Δkcs1 mutant) in the low-glucose environment of the host lung is due to its inability to utilize alternative carbon sources. Despite this metabolic defect, the Δkcs1 mutant established persistent, low-level asymptomatic pulmonary infection but failed to elicit a strong immune response in vivo and in vitro and was not readily phagocytosed by primary or immortalized monocytes. Reduced recognition of the Δkcs1 cells by monocytes correlated with reduced exposure of mannoproteins on the Δkcs1 mutant cell surface. We conclude that IP7 is essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity. IMPORTANCE: Cryptococcus neoformans is responsible for 1 million cases of AIDS-associated meningitis and ~600,000 deaths annually. Understanding cellular pathways responsible for pathogenicity might have an impact on new drug development. We characterized the inositol polyphosphate kinases Kcs1 and Asp1, which are predicted to catalyze the production of inositol pyrophosphates containing one or two diphosphate moieties (PP-IPs). Using gene deletion analysis and inositol polyphosphate profiling, we confirmed that Kcs1 and Asp1 are major IP6 and IP7 kinases, respectively. Kcs1-derived IP7, but not Asp1-derived IP8, is crucial for pathogenicity. Global expression profiling and carbon source utilization testing suggest that IP7-deficient cryptococci cannot adapt their metabolism to allow growth in the glucose-poor environment of the host lung, and consequently, fungal burdens are significantly reduced. Persistent asymptomatic Δkcs1 mutant infection correlated with decreased mannoprotein exposure on the Δkcs1 mutant surface and reduced phagocytosis. We conclude that IP7 is crucial for the metabolic adaptation of C. neoformans to the host environment and for pathogenicity.

HIV type 1 Nef increases the association of T cell receptor (TCR)-signaling molecules with T cell rafts and promotes activation-induced raft fusion.

HIV-1 Nef (Nef) is a myristoylated protein that contributes to HIV disease pathogenesis. Nef has a modulatory effect on T cell receptor (TCR) signaling, resulting in up-regulation of interleukin-2 (IL-2) production in stimulated T cells. Recent studies have shown that efficient TCR signaling requires enhanced association of TCR-signaling molecules with plasma membrane microdomains (lipid rafts) and fusion of rafts into larger structures. We utilized Jurkat T cell lines expressing wild-type Nef (Nef(wt)) and a myristoylation-deficient form of Nef (Nef(G)2(A)), from an inducible promoter, to determine the effects of Nef on the association of TCR-signaling molecules with rafts in nonstimulated T cells. In addition, the effect of Nef on raft size, before and after TCR activation by CD3 cross-linking, was also examined. Following induction, Nef(wt) was associated with both rafts and nonrafts, while Nef(G)2(A) was almost exclusively cytosolic. Induction of Nef(wt), but not Nef(G)2(A), coincided with an increased association of the src family tyrosine kinase, Lck, and TCRzeta with rafts, but not with nonrafts. Further, rafts were found to be significantly larger in CD3-activated T cells in the presence of Nef(wt) when compared to nonexpressing cells. We propose that myristoylated, raft-localized Nef primes resting T cells for activation by increasing the levels of signaling molecules within rafts, and that TCR activation is enhanced by the capacity of Nef to promote raft fusion.

IL7Rα expression and upregulation by IFNß in dendritic cell subsets is haplotype-dependent.

The IL7Rα gene is unequivocally associated with susceptibility to multiple sclerosis (MS). Haplotype 2 (Hap 2) confers protection from MS, and T cells and dendritic cells (DCs) of Hap 2 exhibit reduced splicing of exon 6, resulting in production of relatively less soluble receptor, and potentially more response to ligand. We have previously shown in CD4 T cells that IL7Rα haplotypes 1 and 2, but not 4, respond to interferon beta (IFNß), the most commonly used immunomodulatory drug in MS, and that haplotype 4 (Hap 4) homozygotes have the highest risk of developing MS. We now show that IL7R expression increases in myeloid cells in response to IFNß, but that the response is haplotype-dependent, with cells from homozygotes for Hap 4 again showing no response. This was shown using freshly derived monocytes, in vitro cultured immature and mature monocyte-derived dendritic cells, and by comparing homozygotes for the common haplotypes, and relative expression of alleles in heterozygotes (Hap 4 vs not Hap 4). As for T cells, in all myeloid cell subsets examined, Hap 2 homozygotes showed a trend for reduced splicing of exon 6 compared to the other haplotypes, significantly so in most conditions. These data are consistent with increased signaling being protective from MS, constitutively and in response to IFNß. We also demonstrate significant regulation of immune response, chemokine activity and cytokine biosynthesis pathways by IL7Rα signaling in IFNß -treated myeloid subsets. IFNß-responsive genes are over-represented amongst genes associated with MS susceptibility. IL7Rα haplotype may contribute to MS susceptibility through reduced capacity for IL7Rα signalling in myeloid cells, especially in the presence of IFNß, and is currently under investigation as a predictor of therapeutic response.

Novel approaches to detect serum biomarkers for clinical response to interferon-beta treatment in multiple sclerosis.

Interferon beta (IFNbeta) is the most common immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS). However, some patients fail to respond to treatment. In this study, we identified putative clinical response markers in the serum and plasma of people with multiple sclerosis (MS) treated with IFNbeta. In a discovery-driven approach, we use 2D-difference gel electrophoresis (DIGE) to identify putative clinical response markers and apply power calculations to identify the sample size required to further validate those markers. In the process we have optimized a DIGE protocol for plasma to obtain cost effective and high resolution gels for effective spot comparison. APOA1, A2M, and FIBB were identified as putative clinical response markers. Power calculations showed that the current DIGE experiment requires a minimum of 10 samples from each group to be confident of 1.5 fold difference at the p<0.05 significance level. In a complementary targeted approach, Cytometric Beadarray (CBA) analysis showed no significant difference in the serum concentration of IL-6, IL-8, MIG, Eotaxin, IP-10, MCP-1, and MIP-1alpha, between clinical responders and non-responders, despite the association of these proteins with IFNbeta treatment in MS.

BAFF is a biological response marker to IFN-beta treatment in multiple sclerosis.

Multiple sclerosis (MS) is a complex autoimmune disease characterized by the destruction of the myelin sheath of neurons. Interferon beta (IFN-beta) is currently the major drug used to treat MS. Some patients fail to respond to this treatment, in some cases due to the development of neutralizing antibodies (NAb) to IFN-beta. We used microarray analysis and RT-PCR to measure gene expression in whole blood, 9-15 h postinjection, in patients with and without NAbs to IFN-beta. The canonical marker of biological response to IFN-beta, myxovirus resistance protein A, was upregulated in all NAb- patients while remaining unchanged in NAb+ patients. Genes functioning in immune response pathways were dominant in the set of differentially expressed genes: 73 immune response genes were identified as upregulated and 29 genes were identified as downregulated. B-cell activating factor (BAFF) is a strong candidate marker for biological and clinical response as well as for predisposition to NAb development. We demonstrate that it is responsive to IFN-beta in vitro and in vivo, and that its soluble form is elevated in serum from NAb- but not NAb+ patients. We conclude BAFF is a good biomarker for IFN-beta response, and requires further studies to determine its value as a marker for clinical response and NAb predisposition.

CD127 immunophenotyping suggests altered CD4+ T cell regulation in primary progressive multiple sclerosis.

Aberrant regulatory T cell populations, characterised by a wide array of CD markers, have been identified in many autoimmune diseases. CD127 has recently been identified as a specific marker for the CD4(+)CD25(Hi) (Tregs) subset. CD127 is the first non-HLA gene to have its association with multiple sclerosis widely replicated. We demonstrate that the regulatory or suppressor T cells CD4(+)CD25(Hi) (Tregs), CD8(+)CD28(-), and CD3(+)CD56(+) (NKT) all produce low levels of CD127, and so could be at a disadvantage in survival and/or proliferation where IL7 is limiting. The remissions seen in relapsing remitting multiple sclerosis (RRMS) could be driven by regulatory T cells, and the absence of remissions seen in primary progressive MS (PPMS) may point to a particularly reduced function of this cell subset. We found that the proportions of CD4(+)FoxP3(+)CD25(Hi) regulatory T cells were not aberrant in PPMS. There was, however, a trend towards reduced FoxP3 expression per cell in this fraction (p<0.083), which has been highly correlated with suppressor function. Notably, we found that the target of regulatory T cells, the CD4(+)CD25(-) cells, was in excess (p<0.009); and in PPMS a protective CD127 haplotype is correlated with higher CD127 expression (p<0.01). These data support further investigations into the regulatory T cell immunophenotype in MS.

Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.