Amplification and molecular cloning of HTLV-I sequences from DNA of multiple sclerosis patients.

Techniques of gene amplification, molecular cloning, and sequence analysis were used to test for the presence of sequences related to human T-lymphotropic virus type I (HTLV-I) in peripheral blood mononuclear cells of six patients with multiple sclerosis (MS) and 20 normal individuals. HTLV-I sequences were detected in all six MS patients and in one individual from the control group by DNA blot analysis and molecular cloning of amplified DNAs. The viral sequence in MS patients were associated with adherent cell populations consisting predominantly of monocytes and macrophages. Molecular cloning and nucleotide sequence analysis indicated that these amplified viral sequences were related to the HTLV-I proviral genome.

Pathogenesis of autoimmune diseases: antibodies against transglutaminase, peptidylarginine deiminase and protein-bound citrulline in primary Sjögren's syndrome, multiple sclerosis and Alzheimer's disease.

Coeliac disease (CD) is becoming a model for understanding the pathogenesis of autoimmune disorders. In CD, antibodies against transglutaminase 2 (TG2) and specific residues of gliadins have been identified. A similar situation is seen in rheumatoid arthritis (RA) with both anti-citrullinated protein antibodies (ACPA) and auto-antibodies against the citrullinating enzyme, peptidylarginine deiminase (PAD). Previously, we have suggested that a complex between an enzyme and its modified substrate constitutes the neoantigen in autoimmune diseases. Our hypothesis is challenged by findings in patients of primary Sjögren's syndrome (pSS) who do not express ACPA, but who have been reported to carry anti-PAD. The aims of our investigation were to reproduce the study claiming the presence of anti-PAD in pSS and screen for ACPA and antibodies against TG2 and PAD in pSS (n = 78), multiple sclerosis (MS) (n = 85) and Alzheimer's disease (AD) (n = 79) using ELISA. With blood donors (n = 100) as controls, no increased occurrence of autoantibodies was found among the patient groups tested. Contrary to what has been published previously, patients with pSS do not express anti-PAD. The hypothesis of a complex between an enzyme and its modified substrate constituting the neoantigen in autoimmune diseases is still valid. The prevalence of anti-PAD, anti-TG2 and ACPA is comparatively restricted. PAD and TG2 do not seem to be involved directly in autoimmune mechanisms in pSS, MS or AD.

The role of MRI of the brain and spinal cord, and CSF examination for the diagnosis of primary progressive multiple sclerosis.

The clinical applicability of the revised McDonald diagnostic criteria of primary progressive multiple sclerosis (PPMS) was assessed in 17 patients with a longstanding PPMS diagnosis (mean 15 years). All patients were re-evaluated with clinical examinations, magnetic resonance imaging (MRI) of the brain and the spinal cord, extensive laboratory tests, and 12 patients underwent cerebrospinal fluid (CSF) examination. No diagnosis more likely than PPMS was disclosed. All patients had brain and spinal cord lesions on MRI. In 15 patients the brain lesions and in 14 the spinal cord lesions fulfilled the revised McDonald criteria for positive scans. No contrast-enhancing lesion was observed despite administration of triple doses of gadolinium. In total, 12 patients fulfilled the revised McDonald MRI criteria for PPMS. Of the remaining five patients who incompletely fulfilled the revised MRI criteria, all had CSF findings supporting the diagnosis PPMS. Thus, CSF analysis was required in addition to MRI in about one-third of the patients to establish the diagnosis of PPMS.

Comparative tolerance of IFN beta-1a regimens in patients with relapsing multiple sclerosis. The EVIDENCE study.

The EVIDENCE study was a direct comparative study of two dose regimens of interferon (IFN) beta-1a used in the treatment of relapsing-remitting multiple sclerosis (RRMS): 30 mcg intramuscularly once weekly (qw; n=338) and 44 mcg subcutaneously three times weekly (tiw; n=339). The study continued for an average of 64 weeks. The safety population consisted of all patients receiving at least one dose of study drug. Clinical assessments occurred every 4 weeks for 24 weeks and then every 12 weeks. Blood tests for safety were taken at baseline and at weeks 4 and 12, and every 12 weeks thereafter. Overall adverse events were more common with the 44 mcg tiw regimen (p=0.007), and were due predominantly to differences in injection-site reactions. The majority of adverse events were rated mild by investigators. Hepatic and haematological adverse events and asymptomatic laboratory abnormalities were more common with 44 mcg tiw (p<0.001),with no difference seen for severe events. Flu-like symptoms were more common with 30 mcg qw (p=0.031), were more severe and persisted for longer. Serious adverse events were comparable for both groups, as were drug discontinuations. In conclusion, although adverse events were more common with high-dose, high-frequency IFN therapy, differences were primarily for mild events and did not affect treatment adherence. Based on superior clinical and magnetic resonance imaging outcomes over an average of 64 weeks, coupled with modest safety differences, the risk-benefit ratio for IFN therapy in RRMS favours the 44 mcg tiw regimen over this period of time.

Antigen processing gene polymorphisms in HLA-DR2 multiple sclerosis.

The association between multiple sclerosis (MS) and alleles of the HLA class II genes indicates that at least one MS susceptibility gene is linked to the HLA class II region. However, the actual locus responsible has not been precisely identified. The recent cloning of new genes involved in antigen processing that map within the HLA class II region led us to investigate--using the restriction fragment length polymorphism (RFLP) technique and sequence-specific oligonucleotide analysis--whether these genes might play a role in conferring susceptibility to MS. We studied large multifunctional protease (LMP) 2 and 7 and transporter associated with antigen processing (TAP) 1 and 2 gene polymorphisms in 60 HLA-DR2 MS patients and 60 HLA-DR2 healthy subjects and found no specific or preferential RFLP patterns or coding sequence variants in the patient group. Our data do not support a role for these genes in MS susceptibility.

Primary Sjögren's syndrome in patients with multiple sclerosis.

The relationship between multiple sclerosis (MS) and primary Sjögren's syndrome (PSS) is ambiguous; it was suggested that some patients diagnosed with MS may instead have PSS. In a recent epidemiologic study, the prevalence of PSS was 2.7% in southern Sweden. We randomly selected 30 patients with definite MS from our patient population and investigated them for evidence of PSS according to the Copenhagen criteria. One patient had clinical symptoms compatible with PSS and also fulfilled the Copenhagen criteria. In addition, five patients had keratoconjunctivitis sicca, one had xerostomia, and three had histopathologic evidence of sialadenitis in the lower lip salivary glands. However, one of these findings alone is not sufficient to support the diagnosis of PSS. We conclude that MS and PSS may coexist in the same individual, but PSS is not more common among MS patients than expected in the general population.

A randomized, double-blind, dose-comparison study of weekly interferon beta-1a in relapsing MS.

BACKGROUND: Interferon beta-1a (IFNbeta-1a; Avonex) is effective for the treatment of relapsing MS; however, the optimal dose of IFNbeta-1a is not known. OBJECTIVE: To determine whether IFNbeta-1a 60 micro g IM once weekly is more effective than IFNbeta-1a 30 micro g IM once weekly in reducing disability progression in relapsing MS. METHODS: In a double-blind, parallel-group, dose-comparison study, 802 patients with relapsing MS from 38 centers in Europe were randomized to IFNbeta-1a 30 micro g (n = 402) or 60 micro g (n = 400) IM once weekly for >/=36 months. The primary endpoint was disability progression, defined as time to a sustained increase of >/=1.0 point on the Expanded Disability Status Scale (EDSS) persisting for 6 months. Additional endpoints included relapses, MRI, safety, immunogenicity, and subgroup analyses of disability progression. RESULTS: Both groups showed equal rates of disability progression (hazard ratio, 0.96; 95% CI, 0.77 to 1.20; p = 0.73). In both groups the proportion of subjects with progression of disability by 36 months estimated from Kaplan-Meier curves was 37%. No dose effects were observed on any of the secondary clinical endpoints. Only one MRI measure at one time point, number of new or enlarging T2 lesions at month 36 compared with month 24, showed a difference favoring the 60- micro g dose. Both doses were well tolerated; however, slightly higher incidences of flulike symptoms and muscle weakness were observed in the 60- micro g group. The incidences of neutralizing antibodies (titers >/= 20) were 2.3% in the 30- micro g group and 5.8% in the 60- micro g group. CONCLUSION: There was no difference between IFNbeta-1a 30 micro g and 60 micro g IM in clinical or MRI measures.

Analysis of B-cell activation of cerebrospinal fluid lymphocytes in multiple sclerosis.

We have developed a microculture system to study pokeweed mitogen (PWM)-induced B-cell differentiation responses of CSF lymphocytes from patients with multiple sclerosis (MS) and other neurologic diseases (OND). B-cell differentiation was assessed by (1) enumeration of immunoglobulin-secreting cells (IgSC) by a protein A reverse hemolytic plaque assay; and (2) quantitation of supernatant IgG by ELISA. Cultures of MS CSF cells and OND CSF cells responded to PWM with a similar frequency, with responses in CSF cell cultures exceeding responses in corresponding blood cell cultures in several instances in both groups of patients. Numbers of IgSC in unstimulated cultures of MS CSF cells exceeded numbers in cultures of autologous peripheral blood mononuclear cells (PBM). Results suggest that CSF cells may be a particularly reactive population compared with PBM.

Humoral immune responses within the human central nervous system following systemic immunization.

We investigated sequential humoral immune responses in the CSF and blood of 6 stable multiple sclerosis (MS) patients without decreases in the blood-brain barrier. Anti-tetanus toxoid antibodies (anti-TT Ab) increased to a similar relative degree within the CSF and blood starting within 2 weeks after subcutaneous booster injection of TT. In 3 of 4 subjects, CSF lymphocytes obtained at 2 weeks secreted anti-TT Ab to the same degree as autologous blood lymphocytes when cultured with pokeweed mitogen. These findings suggest a prompt antibody response within the CSF to systemically administered antigen, not due to diffusion from the serum, with active trafficking of TT-sensitized lymphocytes into the central nervous system.

No genetic linkage between multiple sclerosis and the interferon alpha/beta locus.

Multiple sclerosis (MS) is probably caused by an interaction of genetic and environmental factors. The genetic component is reflected by a ten-fold higher concordance rate in monozygotic (27%) compared to dizygotic (3%) twin pairs. Treatment with interferon was recently reported to have a favorable effect in patients with relapsing-remitting MS. In the present familial study, we have investigated the possibility of a genetic association between the highly polymorphic Interferon alpha beta locus and the development of MS. Based on our data, we conclude that there is no linkage between the Interferon alpha beta locus and MS.

Tumor necrosis factor alpha gene polymorphism in multiple sclerosis and optic neuritis.

The NcoI tumor necrosis factor (TNF alpha) polymorphism was studied in relapsing/remitting multiple sclerosis and monosymptomatic optic neuritis. The frequency of the NcoI marker phenotypes did not differ between healthy controls and the two disease groups. No extra or missing DNA fragments were observed in the disease groups when compared with controls.

Golli-MBP gene in multiple sclerosis susceptibility.

Multiple sclerosis (MS) is an oligo- or polygenic disease but no specific susceptibility genes have been identified so far. In the Finnish population we have previously found evidence for linkage between MS and the myelin basic protein gene (here called Golli-MBP gene) suggesting that either Golli-MBP or another gene in its vicinity contributes to MS suceptibility. Here we have screened the Golli-MBP gene for nucleotide variations and carried out multipoint association analyses in a Finnish case-control data-set as well as in an independent data-set composed of 151 MS families from Finland and Sweden. In both data-sets we found association between MS and alleles in the 1.27 kilobase (kb) range at a tetranucleotide repeat element (TGGA)n which is located 1 kb upstream of the MBP exon 1. Haplotype analyses suggested that the MS-associated 1.27 kb alleles can be split into predisposing and non-predisposing variants and provided evidence that the candidate DNA region contributing to MS susceptibility should be located at the Golli-MBP gene within a 20-25 kb segment that was conserved in the predisposing haplotypes. These findings suggest a role for the Golli-MBP locus in MS susceptibility, at least in a subset of patients, and serve as a basis for highly focused attempts to identify predisposing mutation(s).

No linkage or association of the nitric oxide synthase genes to multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology. Nitric oxide (NO) is a free radical that participates in a variety of biological processes. It is an important mediator in the immune response. Several studies indicate involvement of NO in the pathogenesis of MS. We studied five markers within the three NO synthase genes with regards to susceptibility and disease course in 156 affected sib-pairs and in 96 "benign" and 96 "severe" definite MS patients and 148 controls. We found no significant association or evidence for linkage in our data sets.

Association of susceptibility to multiple sclerosis in Sweden with HLA class II DRB1 and DQB1 alleles.

The association of MS with HLA class II alleles was studied by PCR-based typing of the DQA1, DQB1, DRB1, and DPB1 loci in 94 Swedish patients with relapses and remissions of the disease. The haplotype DRB1*1501-DQA1*0102-DQB1*0602 was found to be positively associated and three haplotypes were found to be negatively associated with MS. Linkage disequilibrium makes it difficult to assess whether DRB1 or DQB1 plays the primary role in the disease association, while the association with DPB1 and DQA1 appears to be secondary to that of DQB1 and DRB1. Two of the three haplotypes negatively associated with MS carry the DQB1*0301 allele. Also, the negatively associated DRB1*0401-DQA1*0301-DQB1*0301 haplotype differs from those with nonassociated DRB1*0401-DQA1*0301-DQB1*0302 haplotype only at DQB1. These results suggest that DQB1 alleles, as well as some DRB1 alleles, are involved in susceptibility and protection to MS. In searching for sequence motifs in the DR beta chain associated with MS susceptibility, all DRB1 alleles on haplotypes positively associated with MS, including the DRB1*1501, were found to encode a Val at position 86 of the DR beta chain. Also, DRB1 alleles that are negatively associated with MS all encode a Gly at position 86, suggesting that the residue at position 86 may be critical in conferring susceptibility and protection to MS. Finally, when the effect of the DRB1*1501 haplotype was removed there was no support for the hypothesis that MS is associated with a putative DQ-alpha beta heterodimer, encoded for by certain DQA1 and DQB1 alleles.

Monocyte phenotype in blood and cerebrospinal fluid: compartment-specific pattern is unrelated to neurological disease.

An immunocytochemical study of surface and cytoplasmic markers of the monocyte/macrophage lineage was undertaken on peripheral blood (PB) and cerebrospinal fluid (CSF) cells in patients with multiple sclerosis (MS) or other neurological diseases (OND) and in three healthy controls. Size and granularity were assessed by means of cytofluorimetric analysis. Fewer monocytes were positive for complement receptors 1 and 3, myeloperoxidase, KiM6, IOM-3 and CD14 in CSF than in PB. Only DR-positive monocytes were present with the same frequency in the two compartments. There was no difference in the monocyte phenotype between patients and healthy controls. Furthermore, there was no difference between patients with MS or OND. Cytofluorimetric analysis showed that in CSF, regardless of clinical status, few cells displayed the physical features of blood monocytes. A population of granular and large granular cells that was not normally present among PB mononuclear cells was recognized in the CSF. A large proportion of these cells was able to adhere to plastic and--when sorted--displayed the morphology and surface markers of monocyte lineage. The results of our study demonstrate compartment-specific differences in the monocyte phenotype between CSF and PB but suggest that these changes may be unrelated to the investigated neurological diseases.

Increased frequency of chromosome aberrations in long-term cultured cerebrospinal fluid lymphocytes of patients with multiple sclerosis.

A method employing long-term lymphocyte culturing was developed to study chromosome aberrations in samples with very few cells. It was used to examine lymphocytes from the cerebrospinal fluid (CSF) and peripheral blood (PB) in 23 patients with clinically definite multiple sclerosis (MS), nine patients with other neurological diseases (OND), and eight healthy individuals. MS patients had significantly more aberrations in CSF lymphocytes than in PB lymphocytes (6.4 vs 4.1; P = 0.003). In contrast, no such difference was noted among patients with OND (3.8 vs. 3.7; P = 0.89) or healthy controls (3.6 vs 3.5; P = 0.90). CSF lymphocytes from MS patients had more aberrations than CSF lymphocytes from healthy controls (P = 0.012), but there was no difference between PB lymphocytes from MS patients and controls (P = 0.58). The patients with OND were similar to healthy controls both in CSF (3.8 vs 3.6; P = 0.91) and PB lymphocytes (3.7 vs 3.5; P = 0.90).

The intrathecal immune response in the early stage of multiple sclerosis.

Sequential pairs of cerebrospinal fluid (CSF) and serum samples from 10 patients followed for 2.5-12 years after onset of unilateral optic neuritis (ON) were studied. Eight patients developed definite multiple sclerosis (MS) during the observation period. All patients had normal CSF protein patterns on agar or agarose gel electrophoresis at onset. Six patients developed oligoclonal immunoglobulin (Ig) bands in the CSF during the observation period. Imprint immunofixation of electrofocused specimens disclosed intrathecal synthesis of oligoclonal IgG antibodies to 1 or more of 6 viruses (measles, herpes simplex type 1, varicella-zoster, cytomegalo, mumps, rota) during the observation period in 8 patients. Changes in patterns of intrathecally synthesized viral antibodies, characterized by the appearance of "new" antibody populations and the waxing or waning of others were observed in 6 patients. The results suggest that the early stage of MS in some patients is associated with transient as well as permanent recruitment of B cell clones producing viral antibodies of different specificities.