Lack of Association between Antimyelin Antibodies and Progression to Multiple Sclerosis
Jens Kuhle, Cristoph Pohl, Matthias Mehling, Gilles Edan, Mark S. Freedman, Hans-Peter Hartung, Chris H. Polman, David H. Miller, Xavier Montalban, Frederik Barkhof, Lars Bauer, Susanne Dahms, Raija Lindberg, Ludwig Kappos, and Rupert Sandbrink.
Summary
Serum anti-MOG and anti-MBP IgG and IgM antibodies were measured in 462 MS patients. No associations were found with progression to clinically definite MS or a diagnosis of MS as outlined by the McDonald criteria.
To read the full article please refer to NEJM 2007, 56: 371-8
B-cell activating factor (BAFF)
TNF superamily member and plays a role in the survival and maturation of B cells. Has been found to be elevated in the serum of SLE and Sjogren's patients.
Clusterin and Chromogranin A
Elevated in the CSF of MS patients. Clusterin regulates complement activation and is involved in apoptosis, whilst chromogranin A has been found in GM along the meneigeal border and may play a role in the maintenance of neuronal integrity.
Vitamin D
Children with lower levels of vitamin D seem to be at a greater risk of being diagnosed with MS. Vitamin D has been shown to regulate the immune system.
Apolipoprotein E (Apo-E)
Apo-E ε4 allele is assocated with MS and may predispose carriers to faster disease progression.
Ferritin
Ferritin is an acute phase reactant and increased levels have been found in MS patients. Serum ferritin levels have been associated with male gender and relapsing-progressive MS.
Genomics
Genome-Wide Association Study Using High-Density SNP Arrays on Pooled Genomic DNA from Relapsing-Remitting Multiple Sclerosis Patients and Healthy Controls
Comabella M., Craig D., Camina M., Montalban, X., Martin, R.
Background
A large body of evidence supports the idea that multiple sclerosis (MS) is a complex disorder resulting from an interaction between an inherent genetic susceptibility and environmental exposures such as viral infections. Regarding the genetic background, only genes of the HLA-class II region have consistently been associated with MS. Genome-wide case-control association studies are a powerful approach to identify genes that predispose to genetically complex disorders.
Objective
To identify genes that confer susceptibility to MS by means of a genome-wide case control association study using high-density single nucleotide polymorphisms (SNPs) arrays.
Methods
DNA from 250 relapsing-remitting (RR) and secondary progressive (SP) MS patients and 250 sex-matched healthy donors was pooled and nine pooling replicates for cases and nine pooling replicates for controls were hybridized to high-density SNP arrays (Affymetrix GeneChip Mappign 500K). SNPs were ranked based on statistical tests that incorporated both relative allele signals (RAS)-1 and RAS-2, SNP variability between pools, and allelic frequencies.
Results and Conclusions
Genome-wide association study using SNP arrays in MS patients and healthy controls lead to a number of canidate genes that conferred susceptibility for the disease. The use of high-density genotyping technologies in association studies may prove to be a powerful tool to uncover the genetic component of complex disorders such as MS
Integration of whole genome SNP and gene expression profiling studies in multiple sclerosis patient responders and non-responders to treatment with interferon-b
Comabella M, Craig D, Camina M, Rio J, Sanchez A, Lopez C, Montalban X, Martin R
Background
Treatment with interferon-beta (IFN-b) in patients with RR-MS has been shown to decrease clinical relapses, reduce brain MRI activity, and possibly slow progression of disability. Although several studies have described the effects of IFN-b on gene expression, no definite treatment-response profile has been identified in MS. In addition, the cost of IFN-b is significant, the drug is associated with a number of adverse reactions, and there is a relatively large proportion of patients that don not respond to therapy.
Objective
To identify biomarkers (single nucleotide polymorphisms, SNPs; differentially expressed genes) associated with the responder and non-responder status in relapsing-remitting MS (RR-MS) patients.
Methods
Two independent cohorts of RR-MS patients were classified by stringent clinical criteria as IFN-b responders or non-responders after 2 years follow-up. DNA and RNA samples were isolated from peripheral blood mononuclear cells before and after treatment. SNP polymorphism mapping was performed on pooled DNA samples using high-density SNP arrays (Affymetrix GeneChip Mapping 500K). Differential gene expression was examined from individual RNA samples using oligonucleotide microarray (Affymetrix Human Genome U133 Plus 2.0). In the first round of analysis 53 non-responders and 53 responders were compared, and in a second confirmatory experiment 25 responders with 25 non-reponders. Several biostatistical methods were applied to compare data between groups at the level of SNP mapping and gene expression profiling, and subsequently the two data sets were cross-related.
Results and Conclusions
Broad screening for biomarkers associated with the clinical response to IFN-b lead to a number of SNPs and differentially expressed genes that are related to and predicitive of the responder status. The use of genomics and gene expression platforms at the level of the entire genome offers unique opportunities for biomarker research. Cross-validation of genomic data by information from gene expression profiling strengthens the search for candidate biomarkers.
24S-Hydroxycholesterol
Crucial for maintaining lipid neuronal membranes. Higher CSF 24-OH-chol levels were demonstrated in patients with GAD-enhancing lesions indicating release from damaged cells during inflammation.
Interferon-beta
Binding properties of antibodies against interferon-beta mutants
C.Gneiss, B.Kuenz, R.Ehling, A. Lutterotti, R.Egg, I.Mayringer, M.Reindl, T.Berger and F.Deisenhammer
Background
Interferon-beta (IFN-b) therapy in multiple sclerosis is associated with the occurrence of anti-IFN-b antibodies. The total population of these antibodies is referred to as binding antibodies (BAB). A subset of BAB is capable of interfering with the IFN-receptor activation and is therefore called neutralizing antibodies (NAB). There is some evidence in literature that the N-terminus of IFN-b molecule is a specific domain for the binding of neutralizing antibodies (NAB). Substitutions or deletions of amino acids on the N-terminus of IFN-beta might reduce the immunogenicity of IFN-b.
Aim
To evaluate the binding behaviour of IFN-b antibodies to commercially available IFN-b preparations, an IFN-b wildtype and two different IFN-b mutants with substitutions on the N-terminus
Methods
Six different IFN-b antigens were used in the test-systems: three commercially available IFN-b preparations, IFN-b-1a s.c. (Rebif®), IFN-b-1a i.m. (Avonex®) and IFN-b-1b s.c. (Betaferon®), an unformulated IFN-b-1a wildtype and two mutants of IFN-b (A1,A2). Mutant A1 had 5 substitutions to alanine in residues 1 to 11 of the molecule and mutant A2 had 6 substitutions to alanine in residues 15-23 of the molecule. To get comparable results the same protein weight of antigens was used (femtogram), as reference we used the protein weight of Betaferon®. NAB titres were determined by an MxA-bioassay. BAB titers of 30 and NAB titers of 6 serum samples were measured against the six different antigens.
Results
BAB titres to the 6 antigens, which were determined in 30 samples of BAB positive IFN-b treated patients, differed significantly, with higher titres against the commercially available IFN-b preparations compared to IFN-b mutants and the unformulated wildetype (p<0.0001). In a subset of 6 samples, NAB titres were higher when tested against mutants and the unformulated wildtype as compared to the commercial IFN-b preparations (p=0.02), non of the subtests showed a significant difference.
Conclusions
This preliminary study shows a higher antibody binding capacity of the commercial IFN-b preparations compared to the mutants and unformulated IFN-b. This indicates an influence of the formulation of IFN-b on the antibody binding strength. The elevated NAB titers also support this observation because - owing to the properties of Kawade's NAB titre calculation - low binding capacity result in high titres.
c-Jun
c-Jun has been found on microglia and oligodendrocytes in chronic active MS lesions, comapared to silent lesions and OND/non-neurological controls. Inductions of c-Jun has been shown to correlate with apoptosis.
Granzyme K
Granzyme K plays plays a role in immunoregulation of adaptive immunity. Gene silencing of granzyme K inhibited the ability of natural killer (NK)-92 cells to kill activated syngeneic T cells.
Kallikreins
KLK-1 and KLK-6 may serve as biomarkers of progressive MS; highest levels are found in secondary progressive MS.
Lactate
CSF Lactate levels have been found to reduced in MS compared to controls and may be related to altererd astrocytic metabolism during disease. This has diagnostic potential in MR spectroscopy.
Mannan-binding Lectin
Mannan-binding lectin related to CSF and MRI findings in optic neuritis and multiple sclerosis.
V.veyhe, A. Tsakiri, I. Laursen, J.L. Frederiksen
Background
Mannan-binding lectin (MBL) plays an important role in the innate immune system. We examined the concentration of serum MBL in patients with multiple sclerosis (MS) and acute optic neuritis (ON). We studied if MBL was influenced by the number of days from onset to venous puncture and the disease severity.
Methods
The concentration of MBL in 138 adult patients with MS or ON was measured by a sandwich ELISA method and was compared to the concentration in 87 healthy donors.The patients were divided into 3 groups: monosymptomatic acute ON, acute ON as part of MS, and clinically definite MS.
Results
The mean concentration of MBL in patients (1.22 ug/ml, 95% confidence interval 1.03-1.42 ug/ml) did not differ significantly from healthy controls (0.97 ug/ml, 95% confidence interval 0.79-1.17). The results within subgroups did neither differ from each other nor from the donors. There were neither significant correlations between the concentration of MBL and interval from onset, nor with CSF and MRI findings. Nineteen (13.8 %) of patients had a very low (< 0.05 ug/ml) and 24 (17.4%) of patients a low (< 0.1 ug/ml) concentration of MBL.
Conclusions
The mean serum concentration of MBL did not differ from healthy controls, but 13.8 % of patients had a very low and 17.4% of low value of MBL. The value of MBL was neither influenced by the number of days from onset to venous puncture nor to the disease severity judged by MRI and CSF findings.
N-acetylaspartate
N-acetylaspartate as a CSF marker for axonal damage in MS patients and controls
aCE. Teunissen, bE. Jacobeaus, bM. Khademi, bL. Brundin, cNanda Verhoeven, cC. Jakobs and aCD. Dijkstra
aNUBIN, Department of Molecular Cell Biology and Immunology, VUmc, Amsterdam; bCentre of Molecular Medicine, Neuroimmunology Unit Karolinska Institutet Stockholm and cDepartment of Clinical Chemistry VUmc, Amsterdam
Background:
N-acetylaspartate (NAA) has been known as a MRI marker for axonal loss in MS. In a previous study, we investigated CSF levels of NAA. We observed lower levels in SP MS patients compared to RR MS patients in the Amsterdam MS cohort. Furthermore, the correlation of CSF NAA levels with EDSS scores and atrophy suggested that CSF NAA levels could be a marker for disease progression in MS. So far, it is not known whether CSF NAA levels are changed early during the disease course of MS.
Objectives:
To investigate whether CSF NAA levels are changed in early stages of MS compared to the levels in well defined control groups. The second objective was to repeat the previous findings in a different and larger population of MS patients (Swedish cohort).
Methods: CSF NAA levels were determined by a previously described gas-chromatography/mass spectrometry method. The following groups were included: patients with clinically isolated syndrome (n=40); RR MS (n=42) and SP MS (n=29); non-inflammatory neurological controls (n=41) and inflammatory neurological controls (n=40).
Results:
In agreement with our previous findings, CSF NAA levels were decreased in SP MS patients compared to RR MS patients. The median CSF levels in CIS patients were similar to those in RR MS patients. The median CSF NAA levels in non-inflammatory neurological controls were lower compared to the inflammatory controls or RR MS patients. NAA levels in three patients with headache complaints were similar to levels in CIS and RR MS patients. A negative correlation with EDSS was observed and disease duration in MS patients.
Conclusions:
The data suggest that CSF NAA levels decrease during the MS disease course. Decreased CSF NAA levels likely are a reflection of axonal loss in various neurological diseases.
Acknowledgements:
Dutch Brain Foundation
Neurofilaments
Cannabinoid-receptor 1 null mice are susceptible to neurofilament damage and caspase 3 activation.
Jackson SJ, Pryce G, Diemel LT, Cuzner ML, Baker D.
Administered cannabinoids have been shown to ameliorate signs of CNS inflammatory disease in a number of animal models, including allergic encephalomyelitis. More recently, neuroprotective actions have been attributed to activation of the cannabinoid 1 receptor in a number of in vitro and in vivo models. One of these, chronic relapsing experimental allergic encephalomyelitis, is considered a robust analog of multiple sclerosis.
In this study, spinal cord tissue from cannabinoid receptor 1 knockout mice was analyzed for neurofilament H and myelin basic protein content, as markers of neurons/axons and myelin respectively, during the course of chronic relapsing experimental allergic encephalomyelitis.
Dephosphorylation of a neurofilament H epitope, immunoreactive to the
SMI32 antibody, was assessed as a marker of axonal damage and levels of the endpoint cell death mediator caspase 3 were evaluated.
It was found that both neurofilament and myelin basic protein levels decrease over the course of disease, indicating concomitant neuronal/axonal loss and demyelination. Loss of each marker was more severe in cannabinoid receptor 1 knockout animals. Increased SMI32 reactivity was observed as disease progressed. SMI32 reactivity was significantly increased in knockout animals over wildtype counterparts, an indication of greater axonal dephosphorylation and injury. Active caspase 3 levels were increased in all animals during disease, with knockout animals displaying highest levels, even in knockout animals prior to disease induction.
These results indicate that lack of the cannabinoid receptor
1 is associated with increased caspase activation and greater loss and/or compromise of myelin and axonal/neuronal proteins. The increase of caspase 3 in knockout mice prior to disease induction indicates a latent physiological effect of the missing receptor. The data presented further strengthen the hypothesis of neuroprotection elicited via cannabinoid receptor 1 signaling.
Cannabinoid-mediated neuroprotection following interferon-gamma treatment in a three-dimensional mouse brain aggregate cell culture.
Jackson SJ, Baker D, Cuzner ML, Diemel LT.
Multiple sclerosis is increasingly recognized as a neurodegenerative disease which is triggered by inflammation in the central nervous system (CNS). Demyelination-associated axonal or neuronal damage is a primary cause of disability and has thus far not been successfully targeted by available drug therapies. The neuroprotective properties of both endogenous and administered cannabinoids have been shown in in vivo and in vitro models of CNS damage following excitotoxic, oxidative, traumatic and ischaemic insults, with a predominantly apoptotic effector mechanism.
In this study a foetal mouse telencephalon aggregate cell culture system was developed to compare tissue from cannabinoid receptor
1 knockout mice with wildtype counterparts. Aggregate formation and neurofilament/myelin basic protein accumulation were dependent on the age of foetal dissection and species used. Following treatment with interferon-gamma, levels of myelin basic protein, neurofilament, neuronal dephosphorylation and caspase 3 activation were assessed in telencephalon tissue in vitro.
Cytokine treatment resulted in significant loss of the neuronal marker neurofilament-H in cannabinoid receptor 1 knockout cultures but not in wildtypes, indicating that presence of the cannabinoid receptor 1 gene can be neuroprotective.
Caspase 3 activation was higher in cultures from knockout animals, indicating an apoptotic mechanism of cell death. Dephosphorylated neurofilament levels were significantly elevated in knockout mice, lending support to the premise that neurofilament dephosphorylation is a marker for neuronal damage. Taken together, these results indicate that neuroprotection could be elicited through the cannabinoid receptor 1, and point towards a potential therapeutic role for cannabinoid compounds in demyelinating conditions such as multiple sclerosis.
Osteopontin
Osteopontin is a pro-inflammatory cytokine and elevated levels have been demonstrated during relapses in RRMS. It has been also found to be highly expressed in MS lesions.
Proteasome autoantibodies
Retrospectively proteasome autoantibodies to human proteasomes predict multiple sclerosis in monosymptomatic optic neuritis.
J. Milthers, N.H. Beyer, G. Houen, J.L. Frederiksen
Introduction
Mayo et al [1] suggested the human proteasomes as a major autoantigen in MS. We have previously shown that proteasome autoantibodies (PAB) in serum to human purified proteasomes were present in 28 % of optic neuritis (ON) patients and 47 % of multiple sclerosis (MS) patients and 8 % of a control group using population based data.
Objective
We wanted to confirm that our ELISA method of PAB detection is valid. Additionally, we wanted to investigate whether the presence of serum PAB in acute monosymptomatic ON (AMON) is predicative to MS.
Method
All patient samples were collected at the time of ON and samples were taken and 79 patient samples (33 AMON and 46 MS patients) were included. ON was diagnosed using Celesia [2] criteria and MS was diagnosed using Poser criteria’s and information on MS development after AMON was sought in all available patient files.
A quantitative immunoassay, ELISA, was used for the detection of autoantibodies against human proteasome in all patient sera using a pool of purified human proteasome for patient sample investigation.
Results
Six out of 33 (18.2 %) AMON patients were PAB positive and all 6 patient developed MS, confirmed by patient files. From the 26 PAB negative AMON patients, 11 developed MS and 13 did not. No information was available on the remaining 2 patients. The distribution is significant using chi-square testing.
These findings corroborate with our previous results using ELISA for the determination of proteasome autoantibodies in MS-patients.
Discussion
We have used population based data prospectively and patient material from a well described cohort of ON and MS. We can confirm that serum PABs are predicative in AMON for development of MS. Our ELISA method of serum PAB detection was reproducible and validated. Our numbers of PAB positive AMON patients may be too small and more detailed prospective sample studies are necessary.
[1] Mayo et al
[2] Celesia et al 1990
Proteomic Profiles
Cerebrospinal fluid proteome profile in patients with multiple sclerosis
H. Tumani, S. Süssmuth, G. Tauscher, J. Brettschneider, S. Felk, F. Gillardon, V. Lehmensiek (Ulm, D)
Background
Cerebrospinal fluid (CSF) proteins may provide important information about the pathomechanisms present in multiple sclerosis (MS). Although diagnostic criteria for early MS are available, there is still a need for biomarkers predicting disease subtype and progression. Therefore, we intended to analyze the CSF proteome profile of MS patients.
Methods
CSF samples from 12 patients with relapsing remitting MS (RRMS, mean age 40.4 years; median disease duration 24 months) and from 12 patients with clinically isolated syndrome (CIS, mean age 33.8 years; median disease duration 3 weeks) were analyzed and compared to age matched normal controls; all MS and CIS patients received a lumbar puncture during an acute relapse. CSF samples were analyzed by 2-dimensional difference in-gel electrophoresis (2-D-DIGE) technology. Only those protein spots that showed more than 2-fold difference between both groups were selected for further analysis with MALDI-TOF mass spectrometry.
Results
In RRMS, 11 different spots were detected. Only one of them was up-regulated which was identified as Ig kappa chain NIG precursor protein; on the other hand, 10 out of 11 proteins were down-regulated which were identified as transferrin, some serine proteinase inhibitors, alpha-2-HS-glycoprotein, apolipoprotein E and transthyretin. In contrast, 14 different spots were detected in the CSF of CIS patients. Two of the different proteins were up-regulated which were identified as IgG kappa chain and proapo-A-1-protein. Down-regulated proteins included serum albumine precursor, serum albumine, complement factor 3, some serine proteinase inhibitors, vitamin D-binding protein, translation-initiation factor elF-4-gamma, apolipoprotein E precursor and transthyretin. Some of these proteins, serine proteinase inhibitors, alpha-2-HS-glycoprotein and translation-initiation factor eIF-4-gamma, have not been reported in CSF of MS patients yet.
Conclusion
Our preliminary results show clear differences in the proteome profile of patients with RRMS and CIS as compared to normal controls. Though the pathopysiological role of these proteins still remain to be elucidated in detail and further validation of the findings is needed, this non-hypothesis driven approach may have a relevant impact on the identification of disease-specific markers.
CCL5/RANTES
RANTES (regulated upon activation, normal T-cell expressed and secreted), a CC chemokine, enhances inflammatory response. Inteferon-beta-1b treatment reduces RANTES production.
S100B
S100B is a biochemical of neurodegeneration. CSF and serum levels of S100B are increased in MS patients.
TNF-a
Tumour necrosis factor is pro-inflammatory cytokine and is involved in the pathophysiology of MS.
Uric Acid
Serum uric acid levels are decreased in patients with optic neuritis and multiple sclerosis.
Tsakiri A 1, Frederiksen Jette 1, Department of neurology 1 Glostrup Hospital University of Copenhagen Denmark
Background
A few small series have reported low serum levels of the endogenous antioxidant uric acid (UA) in patients with multiple sclerosis (MS) and patients with optic neuritis (ON). We aimed to confirm this in a population-based greater material and to correlate the values to results of CSF examination and MRI of the brain.
Methods
The mean age of patients was 38 years. The first group comprised 76 consecutive untreated patients with acute ON: fifty-three patients (70%) had clinically isolated ON. The remaining 23 patients (30%) had ON as part of MS. The second group comprised 33 consecutively examined patients with relapsing remitting (RR) MS (mean disease duration 1.6 years). The reference interval of UA was 0.20-0.45 mM.
Results
Twelve patients (15%) with ON had decreased UA, of whom 9 had isolated ON. In RRMS 5 patients (15%) had decreased UA. In ON the frequency of decreased UA was significantly higher in women than in men (p=0.03).
Conclusions
Decreased serum values of UA was present in 15% of patients with ON or RRMS, equally distributed between these two groups. Only in ON was the frequency of decreased UA significantly higher in women than in men. The relationship of UA levels and the results of CSF and MRI findings will be presented at the congress.
VEGF-A
VEGF-A (vascular endothelial growth factor-A) expression is reduced CSF cells in MS patients compared to controls and SPMS cases have reduced VEGF-A mRNA expression in PBMC compared to RRMS and controls.
YKL-40
YKL-40 (chitinase 3-like protein) is expressed in inflammatory conditions and has been found to be increased in lentiviral encephalitis and MS.
Zinc
Zinc levels in erythroctes have been found to be elevated in MS patients, however serum levels have been found to be low.
