The association of selected multiple sclerosis symptoms with disability and quality of life: a large Danish self-report survey.

BACKGROUND: People with multiple sclerosis (MS) experience a wide range of unpredictable and variable symptoms. The symptomatology of MS has previously been reported in large sample registry studies; however, some symptoms may be underreported in registries based on clinician-reported outcomes and how the symptoms are associated with quality of life (QoL) are often not addressed. The aim of this study was to comprehensively evaluate the frequency of selected MS related symptoms and their associations with disability and QoL in a large self-report study. METHODS: We conducted a cross-sectional questionnaire survey among all patients at the Danish Multiple Sclerosis Center, Copenhagen University Hospital, Denmark. The questionnaire included information on clinical and sociodemographic characteristics, descriptors of QoL and disability, as well as prevalence and severity of the following MS symptoms: impaired ambulation, spasticity, chronic pain, fatigue, bowel and bladder dysfunction, and sleep disturbances. RESULTS: Questionnaires were returned by 2244/3606 (62%). Participants without MS diagnosis or incomplete questionnaires were excluded, n = 235. A total of 2009 questionnaires were included for analysis (mean age 49.4 years; mean disease duration 11.7 years; and 69% were women). The most frequently reported symptoms were bowel and bladder dysfunction (74%), fatigue (66%), sleep disturbances (59%), spasticity (51%) and impaired ambulation (38%). With exception of fatigue and sleep disturbances, all other symptoms increased in severity with higher disability level. Invisible symptoms (also referred to as hidden symptoms) such as fatigue, pain and sleep disturbances had the strongest associations with the overall QoL. CONCLUSION: We found invisible symptoms highly prevalent, even at mild disability levels. Fatigue, pain and sleep disturbances had the strongest associations with the overall QoL and were more frequently reported in our study compared with previous registry-based studies. These symptoms may be underreported in registries based on clinician reported outcomes, which emphasizes the importance of including standardized patient reported outcomes in nationwide registries to better understand the impact of the symptom burden in MS.

Genome-wide gene expression in a pharmacological hormonal transition model and its relation to depressive symptoms.

OBJECTIVES: Sensitivity to sex-steroid hormone fluctuations may increase risk for perinatal depression. We aimed to identify genome-wide biological profiles in women demonstrating sensitivity to pharmacological sex-hormone manipulation with gonadotrophin-releasing hormone agonist (GnRHa). METHODS: Longitudinal gene expression (Illumina Human HT12.v4) and DNA methylation data (Infinium HumanMethylation450K BeadChip) from 60 women (30 GnRHa, 30 placebo) were generated (Trial ID: NCT02661789). Differences between baseline and two follow-up points (initial stimulation- and subsequent early suppression phase) in the biphasic ovarian hormone response to GnRHa were assessed using linear mixed effects models. RESULTS: Genome-wide analysis revealed 588 probes differentially expressed from GnRHa intervention to first stimulatory phase follow-up (intervention group × time) after 10% fdr multiple testing correction. Of these, 54% genes were also significantly associated with estradiol changes over time (proxy for GnRHa response magnitude), 9.5% were associated with changes in depressive symptoms, and 38% were associated with changes in neocortical serotonin transporter binding. The genes were implicated in TGF beta signaling, adipogenesis, regulation of actin cytoskeleton, and focal adhesion pathways and enriched for DNA methylation changes (P = 0.006). CONCLUSIONS: These findings point toward an altered peripheral blood transcriptomic landscape in a pharmacological model of sex-hormone-induced depressive symptoms.

Bone microarchitecture and bone mineral density in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) patients are at increased risk of reduced bone mineral density (BMD) and fractures. The aetiology of bone loss in MS is unclear. Trabecular bone score (TBS) is a novel analytical tool that provides a measurement of the bone microarchitecture. Decreased TBS predicts increased fracture risk independently of BMD. To date, no studies have investigated TBS in MS patients. OBJECTIVES: To assess bone quality in MS patients by TBS and to evaluate potential risk factors that may affect BMD and TBS in patients with MS. METHODS: Two hundred sixty MS patients were included. TBS was calculated using TBS iNsight software (MediMaps® ). Multivariable regression analyses were performed with information on smoking, alcohol, glucocorticoid (GC) treatment, sun exposure, physical activity, vitamin D and BMI. RESULTS: Trabecular bone score was not significantly different from an age-matched reference population. Low TBS was associated with high age (P = .014) and smoking (P = .03). Smoking and physical inactivity were associated with low BMD in spine (P = .034, P = .032). GC treatment was not associated with TBS. CONCLUSION: We could not find altered TBS values among MS patients, suggesting that BMD alone, and not the bone microarchitecture, is affected in MS. However, larger studies are needed to verify these findings and to establish the role of TBS in MS. As in the background population, physical activity and non-smoking habits are associated with better bone health in MS.

Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis.

Quantitative real-time PCR (qPCR) involves the need of a proper standard for normalizing the gene expression data. Different studies have shown the validity of reference genes to vary greatly depending on tissue, cell subsets and experimental context. This study aimed at the identification of suitable reference genes for qPCR studies using different peripheral blood cell subsets (whole blood (WB) cells, peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD4(+) T cells, CD8(+) T cells, NK cells, monocytes, B cells and dendritic cells) from healthy controls (HC), patients with relapsing-remitting multiple sclerosis (RRMS) and interferon-ß-treated patients with RRMS (RRMS-IFN-ß). Eight candidate reference genes (CASC3, EEF1A1, GAPDH, HPRT1, RPLP0, UBC, UBE2D2 and YWHAZ) were analysed using normfinder and genorm algorithms to identify the most stably expressed genes. We found reference gene expression varied most across cell subsets, and less variation between the donor groups. UBE2D2 was the most stably expressed gene across both HC and RRMS patients and across cell subsets, while UBC was the most stably expressed gene between HC, RRMS and RRMS-IFN-ß patients. UBE2D2 and HPRT1 was the most stable combination for analyses of cell subsets between HC and RRMS patients, while the combination of UBC and YWHAZ was superior for analysis of cell subsets between HC, RRMS and RRMS-IFN-ß groups. GAPDH was generally unsuitable for blood cell subset studies in multiple sclerosis. In conclusion, we found that blood cell subsets result in marked variation in reference gene expression, and we identified suitable reference genes for studies involving PBMC subsets, RRMS patients and interferon-ß treatment.

Joint bleeds increase the inhibitor response to human factor VIII in a rat model of severe haemophilia A.

INTRODUCTION: The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII in on-demand treatment, increasing the inhibitor risk. AIM: To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. METHOD: HA rats were divided into two groups: one group (n = 10) receiving three needle induced knee joint bleeds 14 days apart and a control group (n = 9) receiving three sham procedures. Three hours after each injury/sham 50 IU kg(-1) rhFVIII was administrated intravenously. Subsequently, both groups continued rhFVIII treatment for another 9 weeks. Binding antibodies were analysed using an enzyme-linked immunosorbent assay and neutralizing antibodies using a Bethesda-like assay. RESULTS: Rats in the knee-bleed group developed a significantly faster inhibitor response and reached significantly higher inhibitor levels. In the knee-bleed group, 80% developed inhibitors vs. 33% in the control group, demonstrating a 2.4 times higher inhibitor risk when treating concurrent with bleeds. CONCLUSION: FVIII treatment in relation to a bleed potentiates inhibitor development compared to FVIII treatment alone in this HA rat, indicating that bleeding is a potential danger signal. Our results support the theory that FVIII replacement therapy concurrent with a bleeding episode increases the inhibitor risk, which to the best of our knowledge, has not been confirmed in an animal model before.

Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models.

In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed.

Trichuris suis ova therapy in relapsing multiple sclerosis is safe but without signals of beneficial effect.

BACKGROUND: An observational study has suggested that relapsing-remitting multiple sclerosis patients with helminth infections have lower disease activity and progression than uninfected multiple sclerosis patients. OBJECTIVE: To evaluate the safety and efficacy on MRI activity of treatment with TSO in relapsing MS. METHODS: The study was an open-label, magnetic resonance imaging assessor-blinded, baseline-to-treatment study including ten patients with relapsing forms of multiple sclerosis. Median (range) age was 41 (24-55) years, disease duration 9 (4-34) years, Expanded Disability Status Scale score 2.5 (1-5.0), and number of relapses within the last two years 3 (2-5). Four patients received no disease modifying therapy, while six patients received IFN-ß. After an observational period of 8 weeks, patients received 2500 ova from the helminth Trichuris suis orally every second week for 12 weeks. Patients were followed with serial magnetic resonance imaging, neurological examinations, laboratory safety tests and expression of immunological biomarker genes. RESULTS: Treatment with Trichuris suis orally was well-tolerated apart from some gastrointestinal symptoms. Magnetic resonance imaging revealed 6 new or enlarged T2 lesions in the run-in period, 7 lesions in the early period and 21 lesions in the late treatment period. Two patients suffered a relapse before treatment and two during treatment. Eight patients developed eosinophilia. The expression of cytokines and transcription factors did not change. CONCLUSIONS: In a small group of relapsing multiple sclerosis patients, Trichuris suis oral therapy was well tolerated but without beneficial effect.

Central and peripheral hemodynamics in exercising humans: leg vs arm exercise.

In humans, arm exercise is known to elicit larger increases in arterial blood pressure (BP) than leg exercise. However, the precise regulation of regional vascular conductances (VC) for the distribution of cardiac output with exercise intensity remains unknown. Hemodynamic responses were assessed during incremental upright arm cranking (AC) and leg pedalling (LP) to exhaustion (Wmax) in nine males. Systemic VC, peak cardiac output (Qpeak) (indocyanine green) and stroke volume (SV) were 18%, 23%, and 20% lower during AC than LP. The mean BP, the rate-pressure product and the associated myocardial oxygen demand were 22%, 12%, and 14% higher, respectively, during maximal AC than LP. Trunk VC was reduced to similar values at Wmax. At Wmax, muscle mass-normalized VC and fractional O2 extraction were lower in the arm than the leg muscles. However, this was compensated for during AC by raising perfusion pressure to increase O2 delivery, allowing a similar peak VO2 per kg of muscle mass in both extremities. In summary, despite a lower Qpeak during arm cranking the cardiovascular strain is much higher than during leg pedalling. The adjustments of regional conductances during incremental exercise to exhaustion depend mostly on the relative intensity of exercise and are limb-specific.

Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and Caucasians in the arctic winter.

During evolution, mitochondrial DNA haplogroups of arctic populations may have been selected for lower coupling of mitochondrial respiration to ATP production in favor of higher heat production. We show that mitochondrial coupling in skeletal muscle of traditional and westernized Inuit habituating northern Greenland is identical to Danes of western Europe haplogroups. Biochemical coupling efficiency was preserved across variations in diet, muscle fiber type, and uncoupling protein-3 content. Mitochondrial phenotype displayed plasticity in relation to lifestyle and environment. Untrained Inuit and Danes had identical capacities to oxidize fat substrate in arm muscle, which increased in Danes during the 42 days of acclimation to exercise, approaching the higher level of the Inuit hunters. A common pattern emerges of mitochondrial acclimatization and evolutionary adaptation in humans at high latitude and high altitude where economy of locomotion may be optimized by preservation of biochemical coupling efficiency at modest mitochondrial density, when submaximum performance is uncoupled from VO2max and maximum capacities of oxidative phosphorylation.

Maintained peak leg and pulmonary VO2 despite substantial reduction in muscle mitochondrial capacity.

We recently reported the circulatory and muscle oxidative capacities of the arm after prolonged low-intensity skiing in the arctic (Boushel et al., 2014). In the present study, leg VO2 was measured by the Fick method during leg cycling while muscle mitochondrial capacity was examined on a biopsy of the vastus lateralis in healthy volunteers (7 male, 2 female) before and after 42 days of skiing at 60% HR max. Peak pulmonary VO2 (3.52 ± 0.18 L.min(-1) pre vs 3.52 ± 0.19 post) and VO2 across the leg (2.8 ± 0.4L.min(-1) pre vs 3.0 ± 0.2 post) were unchanged after the ski journey. Peak leg O2 delivery (3.6 ± 0.2 L.min(-1) pre vs 3.8 ± 0.4 post), O2 extraction (82 ± 1% pre vs 83 ± 1 post), and muscle capillaries per mm(2) (576 ± 17 pre vs 612 ± 28 post) were also unchanged; however, leg muscle mitochondrial OXPHOS capacity was reduced (90 ± 3 pmol.sec(-1) .mg(-1) pre vs 70 ± 2 post, P < 0.05) as was citrate synthase activity (40 ± 3 µmol.min(-1) .g(-1) pre vs 34 ± 3 vs P < 0.05). These findings indicate that peak muscle VO2 can be sustained with a substantial reduction in mitochondrial OXPHOS capacity. This is achieved at a similar O2 delivery and a higher relative ADP-stimulated mitochondrial respiration at a higher mitochondrial p50. These findings support the concept that muscle mitochondrial respiration is submaximal at VO2max , and that mitochondrial volume can be downregulated by chronic energy demand.

The chemokine receptor CCR5 Δ32 allele in natalizumab-treated multiple sclerosis.

OBJECTIVE: The chemokine receptor CCR5 may be important for the recruitment of pathogenic T cells to the CNS in multiple sclerosis (MS). We hypothesized that this chemokine receptor might still be important for T-cell migration during treatment with anti-very late antigen (VLA)-4 antibody. We therefore analysed whether natalizumab-treated MS patients carrying the CCR5 Δ32 deletion allele, which results in reduced expression of CCR5 on the cell surface, had lower disease activity. METHODS: CCR5 Δ32 was analysed in 212 natalizumab-treated MS patients. RESULTS: CCR5 Δ32 status had no significant impact on the frequency of relapses 1 year prior to natalizumab treatment or during the first 48 weeks of treatment. The multiple sclerosis severity score (MSSS) was significantly lower at baseline in patients carrying CCR5 Δ32 (P = 0.031). CONCLUSIONS: CCR5 Δ32 is not associated with lower disease activity in MS patients treated with natalizumab. We found lower MSSS scores in patients carrying CCR5 Δ32 compared with the remaining patients, which is consistent with previous studies reporting an association with a more favourable disease course. Further studies are, however, needed before the relationship between CCR5 Δ32 and disease activity in MS can be definitely established.

Prediction of response to interferon therapy in multiple sclerosis.

OBJECTIVE: Single nucleotide polymorphisms (SNPs) in the genes encoding interferon response factor (IRF)-5, IRF-8 and glypican-5 (GPC5) have been associated with disease activity in multiple sclerosis (MS) patients treated with interferon (IFN)-ß. We analysed whether SNPs in the IRF5, IRF8 and GPC5 genes are associated with clinical disease activity in MS patients beginning de novo treatment with IFN-ß. METHODS: The SNPs rs2004640, rs3807306 and rs4728142 in IRF5, rs13333054 and rs17445836 in IRF8 and rs10492503 in GPC5 were genotyped in 575 patients with relapsing-remitting MS followed prospectively after the initiation of their first treatment with IFN-ß. RESULTS: 62% of patients experienced relapses during the first 2 years of treatment, and 32% had disability progression during the first 5 years of treatment. Patients with a pretreatment annualized relapse rate >1 had an increased risk of relapse (hazard ratio 1.53, 95% confidence interval 1.24-1.90) and progression (hazard ratio 1.48, 95% confidence interval 1.10-1.99) on treatment and patients with breakthrough relapses in the form of relapses during the first 2 years of treatment had an increased risk of progression during the first 5 years of treatment (hazard ratio 2.04, 95% confidence interval 1.47-2.85).The gene variants in IRF5, IRF8 and GPC5 were not associated with risk of relapse or disease progression. CONCLUSIONS: Pretreatment relapse rate and clinical disease activity during the first 2 years of treatment may be associated with disease progression in MS patients treated with IFN-ß. Genetic analysis of the studied gene variants do not provide additional information.

Human T cells depend on functional calcineurin, tumour necrosis factor-α and CD80/CD86 for expansion and activation in mice.

Dysregulated T cells are a hallmark of several autoimmune and inflammatory diseases; thus, models to study human T cells in vivo are advantageous, but limited by lacking insight into human T cell functionality in mice. Using non-obese diabetic (NOD), severe combined immunodeficient (SCID) or recombination activating gene-1 (RAG1)(-/-) and interleukin-2 receptor gamma-chain (IL-2Rγ)(-/-) mice reconstituted with human peripheral blood mononuclear cells (PBMCs), we have studied the mechanisms of human T cell expansion and activation in mice. Injection of human PBMCs into mice caused consistent xeno-engraftment with polyclonal expansion and activation of functional human T cells and production of human cytokines. Human T cell expansion coincided with development of a graft-versus-host disease (GVHD)-like condition observed as weight loss, multi-organ immune infiltration and liver damage. CD8(+) T cells alone were sufficient for expansion and required for disease development; in contrast, CD4(+) T cells alone expanded but did not induce acute disease and, rather, exerted regulatory capacity through CD25(+)CD4(+) T cells. Using various anti-inflammatory compounds, we demonstrated that several T cell-activation pathways controlled T cell expansion and disease development, including calcineurin-, tumour necrosis factor-α and co-stimulatory signalling via the CD80/CD86 pathway, indicating the diverse modes of action used by human T cells during expansion and activation in mice as well as the pharmacological relevance of this model. Overall, these data provide insight into the mechanisms used by human T cells during expansion and activation in mice, and we speculate that PBMC-injected mice may be useful to study intrinsic human T cell functions in vivo and to test T cell-targeting compounds.

Dendritic cell, monocyte and T cell activation and response to glatiramer acetate in multiple sclerosis.

BACKGROUND: Treatment with glatiramer acetate (GA) modestly decreases disease activity in multiple sclerosis (MS). The mechanism of action is incompletely understood and differences in the response to treatment between individuals may exist. OBJECTIVE: To study the activation of CD4+ T cells, monocytes and dendritic cells (DC) in relation to disease activity in MS patients treated with GA. METHODS: Flow cytometry was used to study the activation of CD4+ T cells and T cell subsets (CD25(high) and CD26(high) cells), monocytes and DCs in a cross-sectional study of 39 untreated and 29 GA-treated MS patients, the latter followed prospectively for one year. Gd-enhanced magnetic resonance imaging (MRI) studies were conducted in all patients. Disease activity was assessed as relapses. RESULTS: The median percentage of DCs expressing CD40 was 10% in untreated MS patients and 5.9% in GA-treated patients (Bonferroni-corrected p=0.0005). The hazard ratio of relapse was 1.32 (95% confidence interval 1.05-1.64) per 1% increase in CD40+ DCs. Patients treated with GA had fewer CD4+ T cells expressing surface markers associated with T helper type 1 effector responses and more CD4+ T cells expressing surface markers associated with regulatory, naïve or central memory T cell populations, but CD4+ T cell activation was not related with relapse risk. CONCLUSIONS: MS patients treated with GA show prominent changes in circulating antigen-presenting cells and CD4+ T cells. Expression of CD40 on DCs is significantly lower and associated with relapse risk in MS patients treated with GA.

Gene expression analysis of relapsing-remitting, primary progressive and secondary progressive multiple sclerosis.

BACKGROUND: Previous studies of multiple sclerosis (MS) have indicated differences in the pathogenesis in relapsing-remitting (RRMS), secondary progressive (SPMS) and primary progressive (PPMS) disease. OBJECTIVE: We hypothesized that different MS subtypes would show differences in gene expression that could be traced to specific subsets of peripheral blood mononuclear cells (PBMCs). METHODS: Gene expression in RRMS, SPMS, PPMS and healthy control (HC) PBMCs was analyzed on Affymetrix arrays. In addition, we studied gene expression in pools of purified PBMC subsets. RESULTS: We found 380 genes that were differentially expressed in RRMS, PPMS, SPMS and HCs (false discovery rate < 5%). There were no major differences between the subtypes of MS. The genes showing most prominent expression changes in RRMS were associated with adaptive immune pathways, while genes in PPMS were associated with innate immune system pathways. SPMS patients shared pathways with RRMS and PPMS patients. Gene expression changes were most prominent in B cells, CD8+ T cells and monocytes. CONCLUSION: Differences in gene expression, which could be traced to B cells, CD8+ T cells and monocytes, were found between MS patients and HCs but only minor differences were observed between MS subgroups.

Update on acute endovascular and surgical stroke treatment.

Emergency stroke care has become a natural part of the emerging discipline of neurocritical care and demands close cooperation between the neurologist and neurointerventionists, neurosurgeons, and anesthesiologists. Endovascular treatment (EVT), including intra-arterial thrombolysis, mechanical thrombectomy and angioplasty/stenting, is under rapid development. Although EVT has yet to be shown in randomized controlled trials to improve clinical outcome compared to intravenous thrombolysis, it is far better in achieving recanalization of occluded large cerebral vessels, which is crucial for rescuing the penumbra. Moreover, decompressive craniectomy is now a well-established treatment option for malignant middle cerebral artery infarction and cerebellar stroke. Using a case-based approach, this article reviews recent achievements in advanced treatment options for patients with acute ischemic stroke.

Glatiramer acetate antibodies, gene expression and disease activity in multiple sclerosis.

BACKGROUND: Glatiramer acetate (GA) treatment suppresses disease activity in multiple sclerosis (MS). The immunological response to treatment may differ in patients who are stable on GA therapy and patients with breakthrough disease activity, but the results of previous studies are inconsistent. OBJECTIVES: We studied the immunological response to GA and its relationship with disease activity. METHODS: Anti-GA antibodies in plasma and the expression of genes encoding cytokines and T-cell-polarizing transcription factors in blood cells were analysed by flow cytometric bead array and polymerase chain reaction (PCR) analysis in 39 untreated and 29 GA-treated relapsing-remitting MS patients. Definition of breakthrough disease was based on the occurrence of relapses, disability progression, or gadolinium (Gd)-enhanced MRI. RESULTS: The expression of T helper type 1 (Th1) and Th17 cytokines and transcription factors was reduced during long-term treatment, but there was no relationship between the expression of cytokines and transcription factors and anti-GA antibodies. High expression of mRNA encoding GATA3 and lymphotoxin-ß (LT-ß) was associated with low disease activity in Gd-enhanced MRI studies. None of the variables studied were associated with clinical disease activity. GA treatment resulted in the development of IgG and IgG4 anti-GA antibodies during the first months of treatment, persisting during long-term treatment. CONCLUSIONS: The observed relationship between the expression of mRNA encoding GATA3 and LT-ß expression and MRI disease activity deserves further analysis in future studies. The development of anti-GA antibodies was observed in all patients treated with GA, but this was not related with measures of cellular immunity, clinical or MRI disease activity.

Correlation between anti-interferon-ß binding and neutralizing antibodies in interferon-ß-treated multiple sclerosis patients.

BACKGROUND AND PURPOSE: Measurements of binding antibodies (BAbs), neutralizing antibodies (NAbs) and MX1 mRNA expression are used to analyse the immunological reactions in patients with MS treated with IFN-ß. The correlations between these are yet not fully understood. METHODS: We measured BAbs and NAbs to IFN-ß in 110 serum samples from 83 patients with MS treated with IFN-ß, and in a subgroup, antibody titre was compared with corresponding expressions of MX1 mRNA. The methods used were capture ELISA assay, luciferase reporter gene assay and mRNA RT-PCR for MX1 gene expression. RESULTS: There were significant correlations between binding, neutralizing and MX1 results. Cut-off values are suggested for the definition of samples of BAbs and NAbs as negative, positive and grey zones. Naturally occurring groups of low and high antibody titres were identified by the correlation between BAbs and NAbs, probably as a result of an immunological maturation process of antibodies. The low-titre group had lower correlations between BAbs and NAbs than the high-titre group. CONCLUSIONS: High correlation is demonstrated between the results obtained by the three methods, and we suggest the possibility of using ELISA measurements of BAbs to identify patients with high titres of anti-IFN-ß antibodies that block the biological response to IFN-ß. Ιn patients with low titres, we suggest to supplement ELISA with measurement of MX1 mRNA to establish whether the bioavailability of IFN-ß is preserved.

Disease protection and interleukin-10 induction by endogenous interferon-ß in multiple sclerosis?

OBJECTIVE: An immune activation response resembling virus or type I interferon responses has been observed in untreated multiple sclerosis (MS), but its pathogenic significance is uncertain. We studied the relationship between a type I interferon-like response in untreated patients with MS and disease activity. METHODS: Gene expression was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR) in whole blood samples and by microarray analysis of mononuclear cells from untreated patients with MS, patients with MS treated with IFN-ß, and patients with MS with anti-IFN-ß neutralizing antibodies (NAb). Disease activity was assessed by gadolinium-enhanced magnetic resonance imaging. RESULTS: Eight of 36 untreated patients with MS had spontaneously increased expression of the type I IFN-induced gene MX1. Microarray gene expression analysis demonstrated that patients with increased spontaneous MX1 expression also had increased expression of other genes induced by regular IFN-ß treatment of MS. MX1 expression correlated with FOXP3 and IL10 expression, and IL10 expression correlated negatively with disease activity on magnetic resonance imaging. Further, in vivo IL10 expression was lower in NAb-positive patients than in untreated patients with MS and healthy controls. Finally, ex vivo treatment of mononuclear blood cells with IFN-ß induced the expression of IL10, and this was blocked by the addition of serum from NAb-positive patients with MS. CONCLUSION: Our findings suggest that endogenous IFN-ß may induce the expression of immunoregulatory IL10 in MS and that this might be associated with dampening of inflammatory disease activity.

Circulating levels of tight junction proteins in multiple sclerosis: Association with inflammation and disease activity before and after disease modifying therapy.

BACKGROUND: Tight junction proteins contribute to maintenance of epithelial and endothelial barriers such as the intestinal barrier and the blood brain barrier (BBB). Increased permeability of these barriers has been linked to disease activity in MS and there is currently a lack of easily accessible biomarkers predicting disease activity in MS. AIM: To investigate whether levels of circulating tight junction proteins occludin and zonula occludens-1 (ZO-1) are associated with biomarkers of inflammation and disease activity; and to determine whether they could serve as clinical biomarkers. METHODS: We prospectively included 72 newly diagnosed patients with relapsing remitting MS or clinically isolated syndrome with no prior disease modifying therapy (DMT) use and 50 healthy controls (HCs). Patients were followed with blood samples, 3 tesla MRI, and clinical evaluation for 12 months. Occludin, ZO-1, calprotectin and soluble urokinase-type plasminogen activator receptor (suPAR) were measured by ELISA; serum neurofilament light (NfL) and IL-6 by single-molecule array (SIMOA). The mRNA expression of IFNG, IL1R1, IL10, IL1B, ARG1 and TNF was measured by quantitative real time polymerase chain reaction (qPCR) in whole blood. RESULTS: Plasma occludin levels were higher in MS patients compared with HCs. After 12 months on DMT, occludin levels were reduced by approximately 25% irrespective of 1st or 2nd line DMT (p<0.001). Furthermore, NfL and calprotectin levels were significantly reduced by 31% and 29%, respectively. Occludin and ZO-1 did not correlate with biomarkers of inflammation and did not predict disease activity at baseline or after 12 months. CONCLUSIONS: Higher levels of occludin suggest an increased permeability of the BBB and/or the intestinal barrier in MS patients. The reduction of occludin after 12 months on DMTs might reflect repair of these barriers upon treatment. However, plasma levels of ZO-1 and occludin could not predict clinical or MRI disease activity as determined by regression and ROC-curve analysis. Our results do not indicate a clear clinically relevant role for circulating tight junction proteins as biomarkers of disease activity in MS and further investigations in larger cohorts are needed to clarify this issue.