Poul Erik Hyldgaard Jensen

Anti-JC virus antibody prevalence in a multinational multiple sclerosis cohort

JC virus (JCV) is an opportunistic virus known to cause progressive multifocal leukoencephalopathy. Anti-JC virus (Anti-JCV) antibody prevalence in a large, geographically diverse, multi-national multiple sclerosis (MS) cohort was compared in a cross-sectional study. Overall, anti-JCV antibody prevalence was 57.6%. Anti-JCV antibody prevalence in MS patients ranged from approximately 47% to 68% across these countries: Norway, 47.4%; Denmark, 52.6%; Israel, 56.6%; France, 57.6%; Italy, 58.3%; Sweden, 59.0%; Germany, 59.1%; Austria, 66.7% and Turkey, 67.7%. Prevalence increased with age (from 49.5% in patients < 30 years of age to 66.5% in patients ≥ 60 years of age; p < 0.0001 comparing all age categories), was lower in females than in males (55.8% versus 61.9%; p < 0.0001) and was not affected by prior immunosuppressant or natalizumab use.

CSF inflammation and axonal damage are increased and correlate in progressive multiple sclerosis.

Background: The mechanism underlying disease progression in progressive multiple sclerosis (MS) is uncertain. Pathological studies found widespread inflammation in progressive MS brains correlating with disease progression and axonal damage. Objectives: To study cerebrospinal fluid (CSF) biomarkers and clarify whether inflammation and axonal damage are associated in progressive MS. Methods: Using enzyme-linked immunosorbent assay (ELISA), we analysed CSF from 40 secondary progressive (SPMS), 21 primary progressive (PPMS), and 36 relapsing–remitting (RRMS) and 20 non-inflammatory neurological disease (NIND) patients. Twenty-two of the SPMS patients participated in an MBP8298 peptide clinical trial and had CSF follow-up after one year. Results: Compared to NIND patients, inflammatory biomarkers osteopontin and matrix metalloproteinase-9 (MMP9) were increased in all MS patients while CXCL13 was increased in RRMS and SPMS patients. Biomarkers of axonal damage (NFL) and demyelination (MBP) were increased in all MS patients. In progressive MS patients CSF levels of osteopontin and CXCL13 correlated with NFL while osteopontin and MMP9 correlated with MBP. MBP8298 treatment did not affect the levels of the biomarkers after one year of treatment. All biomarkers were continuously increased after one year of follow-up except MBP, which decreased. Conclusion: CSF biomarkers of inflammation, axonal damage and demyelination are continuously increased in progressive MS patients and correlate. These findings parallel pathology studies, emphasise a relationship between inflammation, axonal damage and demyelination and support the use of CSF biomarkers in progressive MS clinical trials.

Oligoclonal band status in Scandinavian multiple sclerosis patients is associated with specific genetic risk alleles.

The presence of oligoclonal bands (OCB) in cerebrospinal fluid (CSF) is a typical finding in multiple sclerosis (MS). We applied data from Norwegian, Swedish and Danish (i.e. Scandinavian) MS patients from a genome-wide association study (GWAS) to search for genetic differences in MS relating to OCB status. GWAS data was compared in 1367 OCB positive and 161 OCB negative Scandinavian MS patients, and nine of the most associated SNPs were genotyped for replication in 3403 Scandinavian MS patients. HLA-DRB1 genotypes were analyzed in a subset of the OCB positive (n = 2781) and OCB negative (n = 292) MS patients and compared to 890 healthy controls. Results from the genome-wide analyses showed that single nucleotide polymorphisms (SNPs) from the HLA complex and six other loci were associated to OCB status. In SNPs selected for replication, combined analyses showed genome-wide significant association for two SNPs in the HLA complex; rs3129871 (p = 5.7×10−15) and rs3817963 (p = 5.7×10−10) correlating with the HLA-DRB1*15 and the HLA-DRB1*04 alleles, respectively. We also found suggestive association to one SNP in the Calsyntenin-2 gene (p = 8.83×10−7). In HLA-DRB1 analyses HLA-DRB1*15∶01 was a stronger risk factor for OCB positive than OCB negative MS, whereas HLA-DRB1*04∶04 was associated with increased risk of OCB negative MS and reduced risk of OCB positive MS. Protective effects of HLA-DRB1*01∶01 and HLA-DRB1*07∶01 were detected in both groups. The groups were different with regard to age at onset (AAO), MS outcome measures and gender. This study confirms both shared and distinct genetic risk for MS subtypes in the Scandinavian population defined by OCB status and indicates different clinical characteristics between the groups. This suggests differences in disease mechanisms between OCB negative and OCB positive MS with implications for patient management, which need to be further studied.

Intravenous immunoglobulin ameliorates experimental autoimmune encephalomyelitis and reduces neuropathological abnormalities when administered prophylactically.

Abstract Background and methods: Immunomodulation with intravenous immunoglobulin (IVIG) represents a way of interfering with the disease process in multiple sclerosis (MS). In this study, the effects of IVIG on neurological symptoms and central nervous system (CNS) pathology were evaluated in experimental autoimmune encephalomyelitis (EAE), an MS animal model. EAE was induced in susceptible Dark Agouti rats by active immunization with a spinal cord homogenate, and infusions of 1 g/kg IVIG were given prophylactically or therapeutically. Results: The administration of IVIG at the time of immunization significantly suppressed the development of neurological symptoms compared with infusions of placebo (mean EAE score 0.6±0.3 versus 2.3±0.4). Moreover, the prophylactic IVIG administration resulted in a significant inhibition of the inflammatory response in CNS tissue (inflammation score 1.1±0.2 versus 1.8±0.2 after placebo). No beneficial effects were obtained by therapeutic IVIG infusions as the EAE disease course and the degree of inflammation and demyelination in the CNS were not different from animals receiving treatment with placebo. Conclusions: The present study indicates that IVIG reduces the symptoms of EAE by suppression of the CNS inflammation that characterizes CNS pathology in these animals. Taking into account data from clinical trials of IVIG in MS, the results further suggest that IVIG acts primarily during the induction phase of the immune response thus preventing the development of relapses in MS.

IVIG enters the central nervous system during treatment of experimental autoimmune encephalomyelitis and is localised to inflammatory lesions.

Intravenous immunoglobulin (IVIG) treatment reduces the relapse rate in relapsing–remitting multiple sclerosis (MS) and may interfere with MS pathology through its various anti-inflammatory and immunomodulatory properties. It is presently unknown whether IVIG enters the central nervous system (CNS) in sufficient amounts to influence the local immune response within the brain and spinal cord, or if the treatment effects are entirely due to peripheral actions of IVIG. The purpose of the present study was to evaluate if IVIG radiolabeled with 99mTc enters the CNS during treatment of experimental autoimmune encephalomyelitis (EAE) in the susceptible rat strain Dark Agouti. After in vivo administration of 99mTc-IVIG we observed significantly increased accumulation in the brain and spinal cord from rats with EAE. Accumulation of 99mTc-IVIG was not detectable in CNS tissue from control animals. In peripheral tissue samples minor increases in 99mTc-IVIG organ binding were observed in the liver and kidney during EAE. Localisation of 99mTc-IVIG in the brain tissue was visualised by autoradiography and revealed significant accumulation of IVIG only in areas also affected by perivascular inflammation and leakage of serum proteins. In conclusion, the results indicate that significant extravasation of IVIG to the CNS only occurs when blood–brain barrier function is compromised during EAE.

Prediction of antibody persistency from antibody titres to natalizumab

Background:In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients. Objective:To use titres of anti-natalizumab antibodies to predict persistency of antibodies. Patients and methods:In 525 consecutive natalizumab treated patients tested for anti-natalizumab antibodies 43 (8.2%) were antibody-positive. Thirty of the antibody-positive patients, who were tested both at three and at six months after treatment start, had antibody titres in blood measured using an extended ELISA method. Results:Samples from persistently positive patients ( N =18) had higher titre values than samples from transiently positive patients ( N =12). A cut-off value for high titre values was generated, above which patients may discontinue natalizumab therapy after three months. The method had a sensitivity of 0.83, a specificity of 1.00 and a diagnostic accuracy of 0.90. Conclusion:An extended ELISA method for measuring anti-natalizumab antibody titres in multiple sclerosis patients on natalizumab therapy may be used for evaluation of antibody persistence. A test at three months may identify patients with high titres, who should discontinue natalizumab therapy, and patients with transient low-titre antibodies, who may continue natalizumab therapy despite development of antibodies.

Identifying Patients with Myasthenia for Epidemiological Research by Linkage of Automated Registers

Background: We validated a new method of identifying patients with incident myasthenia in automated Danish registers for the purpose of conducting epidemiological studies of the disorder. Methods: For residents of a Danish county (population 484,862) in 1993–2008, we identified any hospital contacts coded for myasthenia in a nationwide patient register and any prescriptions for pyridostigmine in the county prescription register. Results from an acetylcholine receptor antibody register were linked to the data. We verified the diagnosis by a review of medical records. Results: Subjects identified in the Patient Register (n = 83) were comparable with individuals found in the Prescription Register (n = 89) with regard to age and gender, but were more often seropositive (83.1 vs. 74.2%). Seropositivity increased to 91.6% by restricting the data to individuals recorded in both Patient and Prescription Registers (n = 71). We found that for subjects identified in both Patient and Prescription Registers the positive predictive value of the register diagnosis was 92.9% (95% confidence interval, CI, 84.3–97.7), the false-positive rate was low (2.8%), and the sensitivity was acceptable (81.2%; 95% CI 71.2–88.8). Conclusions: Our data indicate that this novel approach of combining diagnosis register and prescription register information provides a feasible and valid method to trace incident myasthenia patients for population-based epidemiological studies.

Aberrant forms of α2-macroglobulin purified from patients with multiple sclerosis

Abstract The biochemical properties of α2-macroglobulin were investigated in four patients with multiple sclerosis and compared to α2-macroglobulin from healthy controls. An impaired stability of α2-macroglobulin from the multiple sclerosis patients was demonstrated as a spontaneous conversion to an electrophoretic‘‘fast” form of α2-macroglobulin upon purification and storage, with a concomitant decrease in functional capacity to inhibit proteinases. The ability to form complexes with proteinases was significantly reduced in α2-macroglobulin purified from the multiple sclerosis patients. The aberrant molecular arrangements of the protein were not due to proteinase cleavages in the bait regions of α2-macroglobulin, as demonstrated by gel electrophoresis and protein sequencing. The number of functional thiol esters, however, was reduced in α2-macroglobulin purified from the multiple sclerosis patients, an observation compatible with the impaired proteinase binding property. Furthermore, differences in isoelectric points were observed between α2-macroglobulin from the multiple sclerosis patients and α2-macroglobulin from healthy controls. The results suggest that aberrant forms of α2-macroglobulin may be present in patients with multiple sclerosis.

Native and transformed α2‐macroglobulin in plasma from patients with multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease with unknown etiology. Various proteinases have been observed in increased levels in the central nervous system of patients with MS, which may contribute to the release of immunogenic myelin components. α2‐Macroglobulin (α2M) inhibits a broad spectrum of proteinases sterically, undergoing major conformational changes induced by the proteinases themselves. Moreover, α2M acts as a carrier of several cytokines in the systemic circulation. By use of radial immunodiffusion, we determined the total α2M levels in plasma from 28 MS patients and 15 control subjects [14 patients with other neurologic diseases (OND) and one healthy individual]. No significant differences in total α2M concentration were observed between the MS patients and the control subjects. A comparison of the degree of α2M transformation in MS patients with different disease courses and controls was performed, using monoclonal antibodies (mAbs) specific for binding to native and transformed α2M, respectively. The fractions of transformed α2M were significantly increased in patients with secondary or primary progressive disease course compared with the controls. No significant differences were obtained using a native‐specific mAb. At least a major proportion of α2M from the MS patients was able to change conformation from its native to its transformed state, as demonstrated by a shift in mAb reactivity, following methylamine treatment of the plasma samples. In conclusion, the results indicate that plasma α2M may be inactivated at a higher degree in patients with chronic progressive MS compared with patients with OND. This may influence the levels of proteinases and cytokines in the systemic circulation and may furthermore have diagnostic implications.

Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein α2-macroglobulin

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.