Increasing incidence of multiple sclerosis in women in Northern Finland.

BACKGROUND: The geographical distribution of multiple sclerosis (MS) means that prevalence rates increase with latitude north or south of the equator. Temporally, a tendency for increased incidences of MS has been observed over the past two decades. OBJECTIVES: Since epidemiological studies of MS in areas close to the Arctic Circle are rare, we evaluated the incidence and prevalence of MS in Northern Ostrobothnia by means of a retrospective cohort study covering the period 1992-2007. METHODS: Patients with a definite clinical diagnosis of MS based on the Poser criteria and the early McDonald criteria of 2001 were identified in the region of Northern Ostrobothnia (population 386,972) and the incidence was calculated at 1-year time intervals, both overall and by gender. RESULTS: The overall prevalence was 103/100,000 (95% CI, 93-113), with a female/male ratio of 2.17. The mean overall incidence was 6.3/100,000 (95% CI, 5.2-7.2). The incidence shows a tendency to increase over the 16-year period due to a pronounced rise in the female incidence. CONCLUSIONS: Our results show a high prevalence of MS in Northern Ostrobothnia and a disproportional increase in the female MS incidence. These recent epidemiological features may be associated with environmental risk factors such as a vitamin D deficit, low life-long UV radiation and the high-latitude geographical location.

DARS2 mutations in mitochondrial leucoencephalopathy and multiple sclerosis.

BACKGROUND: Leucoencephalopathy with brain stem and spinal cord involvement and high brain lactate (LBSL) was first defined by characteristic magnetic resonance imaging and spectroscopic findings. The clinical features include childhood or juvenile onset slowly progressive ataxia, spasticity, and dorsal column dysfunction, occasionally accompanied by learning difficulties. Mutations in DARS2, encoding mitochondrial aspartyl-tRNA synthetase, were recently shown to cause LBSL. The signs and symptoms show some overlap with the most common leucoencephalopathy of young adults, multiple sclerosis (MS). OBJECTIVE: To clarify the molecular background of LBSL patients in Finland, and to look for DARS2 mutations in a group of MS patients. METHODS: Clinical evaluation of LBSL patients, DARS2 sequencing and haplotype analysis, and carrier frequency determination in Finland. RESULTS: All eight LBSL patients were compound heterozygotes for DARS2 mutations: all carried R76SfsX5 change, seven had M134_K165del, and one had C152F change. Axonal neuropathy was found in five of the eight patients. The carrier frequencies of the R76SfsX5 and M134_K165del mutations were 1:95 and 1:380, respectively. All patients shared common European haplotypes, suggestive of common European LBSL ancestors. No enrichment of the two common DARS2 mutations was found in 321 MS patients. CONCLUSION: All LBSL patients were compound heterozygotes, which suggests that DARS2 mutation homozygosity may be lethal or manifest as a different phenotype. The authors show here that despite identical mutations the clinical picture was quite variable in the patients. Axonal neuropathy was an important feature of LBSL. DARS2 mutations cause childhood-to-adolescence onset leucoencephalopathy, but they do not seem to be associated with MS.

Interferon regulatory factor 5 (IRF5) gene variants are associated with multiple sclerosis in three distinct populations.

BACKGROUND: IRF5 is a transcription factor involved both in the type I interferon and the toll-like receptor signalling pathways. Previously, IRF5 has been found to be associated with systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel diseases. Here we investigated whether polymorphisms in the IRF5 gene would be associated with yet another disease with features of autoimmunity, multiple sclerosis (MS). METHODS: We genotyped nine single nucleotide polymorphisms and one insertion-deletion polymorphism in the IRF5 gene in a collection of 2337 patients with MS and 2813 controls from three populations: two case-control cohorts from Spain and Sweden, and a set of MS trio families from Finland. RESULTS: Two single nucleotide polymorphism (SNPs) (rs4728142, rs3807306), and a 5 bp insertion-deletion polymorphism located in the promoter and first intron of the IRF5 gene, showed association signals with values of p<0.001 when the data from all cohorts were combined. The predisposing alleles were present on the same common haplotype in all populations. Using electrophoretic mobility shift assays we observed allele specific differences in protein binding for the SNP rs4728142 and the 5 bp indel, and by a proximity ligation assay we demonstrated increased binding of the transcription factor SP1 to the risk allele of the 5 bp indel. CONCLUSION: These findings add IRF5 to the short list of genes shown to be associated with MS in more than one population. Our study adds to the evidence that there might be genes or pathways that are common in multiple autoimmune diseases, and that the type I interferon system is likely to be involved in the development of these diseases.

Difference in prevalence of congenital cryptorchidism in infants between two Nordic countries.

BACKGROUND: Several investigators have shown striking differences in semen quality and testicular cancer rate between Denmark and Finland. Since maldescent of the testis is a shared risk factor for these conditions we undertook a joint prospective study for the prevalence of congenital cryptorchidism. METHODS: 1068 Danish (1997-2001) and 1494 Finnish boys (1997-99) were consecutively recruited prenatally. We also established prevalence data for all newborns at Turku University Central Hospital, Finland (1997-99, n=5798). Testicular position was assessed by a standardised technique. All subtypes of congenital cryptorchidism were included, but retractile testes were considered normal. FINDINGS: Prevalence of cryptorchidism at birth was 9.0% (95% CI 7.3-10.8) in Denmark and 2.4% (1.7-3.3) in Finland. At 3 months of age, prevalence rates were 1.9% (1.2-3.0) and 1.0% (0.5-1.7), respectively. Significant geographic differences were still present after adjustment for confounding factors (birthweight, gestational age, being small for gestational age, maternal age, parity, mode of delivery); odds ratio (Denmark vs Finland) was 4.4 (2.9-6.7, p<0.0001) at birth and 2.2 (1.0-4.5, p=0.039) at three months. The rate in Denmark was significantly higher than that reported 40 years ago. INTERPRETATION: Our findings of increasing and much higher prevalence of congenital cryptorchidism in Denmark than in Finland contribute evidence to the pattern of high frequency of reproductive problems such as testicular cancer and impaired semen quality in Danish men. Although genetic factors could account for the geographic difference, the increase in reproductive health problems in Denmark is more likely explained by environmental factors, including endocrine disrupters and lifestyle.

Measles and rubella virus antibodies in patients with multiple sclerosis. A longitudinal study of serum and CSF specimens by radioimmunoassay.

A longitudinal study on rubella, measles, and respiratory syncytial virus antibodies in serial serum and CSF specimens from 20 multiple sclerosis (MS) patients was performed, using solid-phase radioimmunoassay. Albumin and immunoglobulin G (IgG) levels were also measured to check the integrity of the blood-brain barrier and the intrathecal IgG production. All the patients had local IgG production in their CNA. A local antibody production against one or more of the viruses studied was evident in 15 patients. Fluctuations in the intrathecal viral antibody synthesis were evident in eight patients. No correlation was found between these changes and the clinical course of the disease. The results suggest that the intrathecal antibody synthesis in MS is only partially against any given virus, and in most patients the bulk of the oligoclonal CSF antibodies is against antigens other than those studied here.

Antibodies to coronaviruses OC43 and 229E in multiple sclerosis patients.

Multiple sclerosis (MS) and matched control sera had similar antibody titers to coronaviruses OC43 and 229E when tested by a radioimmunoassay method. In contrast, cerebrospinal fluid from MS patients contained coronavirus antibodies more frequently and in higher titers than matched controls. Intrathecal antibody synthesis to OC43 and 229E viruses was detected in 41% (9/22) and 26% (7/27) of MS patients, respectively, but was not found in any of the neurologic control patients. This intrathecal antibody synthesis may mean that coronaviruses play an etiologic or pathogenic role in MS. Alternatively, intrathecal synthesis of coronavirus antibodies may be but part of a generalized and variable intrathecal antibody synthesis that is typical for MS patients.

T-cell subpopulations in the cerebrospinal fluid and peripheral blood of patients with multiple sclerosis: a follow-up study.

We analyzed the relative and absolute numbers of CD4+ cells positive and negative for CD45RA marker as well as the numbers of CD8+ cells positive and negative for CD11b in the cerebrospinal fluid (CSF) and peripheral blood (PB) of 17 patients with multiple sclerosis (MS) during and 60 days after an exacerbation. We also studied samples from 27 control patients, 17 of whom had a noninflammatory neurologic disease and 10 of whom had an inflammatory neurologic disease other than MS. The results confirmed the small number of CD45RA-positive (naive or suppressor inducer) CD4+ cells as well as CD11b-positive (suppressor) CD8+ cells in the CSF compared with the PB for all paired CSF-blood comparisons. The relative numbers of these cells did not significantly differ between samples taken during an exacerbation and those taken 2 months later. In PB, the percentage of CD8+ CD11b-positive lymphocytes was lower in patients with MS or in patients with an inflammatory neurologic disease than in patients with a noninflammatory neurologic disease. The results indicate that there are fewer cells responsible for suppression-related phenomena in CSF than in PB but that this phenomenon is not specific to MS.

Spontaneous proliferation of cerebrospinal fluid mononuclear cells in multiple sclerosis. A longitudinal study.

Cerebrospinal fluid (CSF) and peripheral blood (PB) specimens from 32 MS patients longitudinally followed for up to 24 months and from a group of control patients without intrathecal inflammation were studied for the occurrence of activated lymphocytes with an autoradiography method. MS patients had higher numbers of proliferating mononuclear cells in CSF than did the controls, both during remission and exacerbation phases, whereas this difference was not found in the PB. ACTH treatment decreased the number of proliferating cells in CSF but had no effect on those of the PB of MS patients. A large variation in spontaneous proliferation of CSF cells was evident during the follow-up of individual patients, and there seemed to be no uniform correlation to the clinical fluctuations.

Intrathecal synthesis of antibodies to diphtheria and tetanus toxoids in multiple sclerosis patients.

Antibodies to diphtheria and tetanus toxoids were measured in 30 multiple sclerosis (MS) patients and 30 neurological control patients by enzyme-immunoassays. The frequency of antibody-positive patients and the titer distribution were similar in both groups. In spite of this, some MS patients had intrathecal antibody synthesis to these toxoids. The histories of these patents did not reveal any diseases caused by diphtheria of tetanus bacteria but they had been vaccinated 2-24 years before the first clinical symptoms of MS. The findings suggest that lymphocytes earlier committed to specific antibody synthesis outside the brain compartment could enter the brain before or after the clinical onset of MS.

HLA-Dw1 and BfF as protective markers in multiple sclerosis.

The frequencies of Properdin factor B allotypes were studied in 54 multiple sclerosis patients and 58 healthy control subjects, and the association of various phenotypes with HLA-Dw1 and Dw2 antigens (found with decreased and increased frequency in MS patients, respectively) was further studied. SS genotype was found in 76% of MS patients and 63% of control subjects (n.s.) F types (FS + FF) were found to be strongly associated with HLA-Dw1 in control subjects (P less than 0.0014), whereas neither SS nor F containing types were associated with Dw2. The findings support the concept of a hypothetical MS resistance factor in the HLA gene area.

Characterization of immune complexes in multiple sclerosis by an antigen-specific immune complex radioimmunoassay.

Serum and cerebrospinal fluid samples from multiple sclerosis (MS) patients containing various levels of Clq-reactive immune complexes (IC) and samples from age- and sex-matched controls were tested by an antigen-specific IC radioimmunoassay which detects IC containing myelin membrane-related antigens. Positive reactivity in the assay was significantly associated with IC-containing MS sera (P less than 0.005) but such an association was not observed with MS cerebrospinal fluid. Analysis of longitudinal specimens revealed that the levels of the antigen-specific serum IC fluctuated with time. Significant correlation between serum levels of Clq-reactive IC and serum levels of IC containing myelin membrane-related antigens was observed (r = 0.62; P less than 0.001). Sucrose gradient centrifugation of sera from 1 patient showed that the IC had a peak density of 1.075 g/ml, indicating the presence of lipid material. The results suggest that serum IC of MS patients frequently contain myelin membrane-related antigens and that these antigens may be lipids or lipid-associated.

Antibodies in cerebrospinal fluid to white matter glycoproteins in multiple sclerosis patients.

Cerebrospinal fluid (CSF) specimens from 45 patients with multiple sclerosis (MS) and 45 age- and sex-matched controls with other neurological diseases (OND) were tested for antibodies to white matter (WM) membrane glycoprotein (GP) fractions prepared from MS and control WM membranes by lentil lectin chromatography. The binding of the CSF IgG to the 125I-labeled GP fractions was determined by immunoprecipitation using Protein A-Sepharose. CSF from patients with MS bound highly significantly more strongly to the GP fraction prepared from MS WM than did the OND CSF specimens (P less than 0.001). There was no such difference when control GP fraction was used as an antigen. No highly significant differences were observed when 20 paired serum specimens were tested. Electrophoresis of the immunoprecipitates showed that components with molecular weights (MWs) of 157,300, 135,600, 111,100, 93,000, 75,700, 63,300, 50,100, 24,300, 20,300 and 17,000 daltons were precipitated from the MS GP fraction by CSF specimens of both MS and OND groups, whereas components with MWs of 50,100, 24,300, 20,300 and 17,000 daltons were precipitated from the control GP fraction.

IgM-class rheumatoid factor in serum and cerebrospinal fluid of multiple sclerosis and matched neurological control patients.

Paired serum and cerebrospinal fluid (CSF) specimens from 30 multiple sclerosis (MS) patients and 30 matched neurological control (NC) patients were quantitatively tested for IgM-class rheumatoid factor (RF). Significantly elevated RF levels were found in serum from 6 MS patients and 12 NC patients. Seven of the latter 12 patients had a diagnosis of recurrent head pain. RF was detected in CSF from 2 MS patients and 2 NC patients. In 3 of the 4 cases, this could be explained by blood-brain barrier damage or normal diffusion of RF into CSF. Intrathecal RF synthesis was found in the remaining MS patient. These results suggest that RF production may be related to underlying pathological mechanisms in at least some MS and NC patients. They also confirm that RF must be carefully accounted for in etiological studies designed to demonstrate pathogen-specific IgM antibodies in, for example, MS patients.

Lymphocyte blast transformation responses and viral antibodies in relation to HLA antigens in multiple sclerosis.

Fifty four clinically stable multiple sclerosis (MS) patients and 54 age- and sex-matched control subjects were HLA-typed, and their responses to herpes simplex, measles, mumps and rubella antigens were examined by the lymphocyte blast transformation test and by serum antibody titrations. Blast transformation response to purified tuberculin (PPD), mitogen phytohaemagglutinin (PHA), pokeweed mitogen (PWN) and concanavalin A (Con A) and spontaneous proliferation of lymphocytes were also studied. MS patients differed from controls by higher antibody levels to measles and rubella viruses and by lower specific blast transformation responses to rubella and measles antigens. When the relative strength of transformation responses was measured, mumps and herpes simplex responses were also lower in MS patients than in controls. In addition, spontaneous lymphocyte proliferation of MS patients in 6-day cultures was lower than that of control lymphocytes. In mitogen stimulations there were no differences between whole groups, but the oldest patients had lower responses to PHA and Con A than their matched controls. The frequency of HLA-Dw2 was 56.6% in MS patients and 32.1% in controls. The patients with and without Dw2 differed from each other only by a lower specific response to PPD in the Dw2-positive group. The immunological response of Dw2-positive controls resembled that of MS patients: low transformation response to viral antigens, low spontaneous proliferation and elevated measles antibodies. This finding supports the function of a genetically determined type of immune responsiveness with low cell-mediated immunity and high levels of certain viral antibodies as one susceptibility factor in multiple sclerosis.

Defective production of interferon-alpha associated with HLA-DW2 antigen in stable multiple sclerosis.

Interferon (IFN) production by peripheral blood mononuclear cells after induction with one purified and three crude viral antigens was studied in 29 patients with stable multiple sclerosis (MS) and 29 healthy controls. Antiviral substance produced was characterized as interferon-alpha. MS patients produced significantly less IFN-alpha after induction with mumps and purified measles virus antigens and the same tendency was seen after induction with rubella virus antigen. However, when herpes simplex virus antigen was used as the stimulating agent, no difference was seen between MS patients and controls. The decreased ability to produce IFN-alpha was associated with the histocompatibility antigen Dw2. Control subjects positive for Dw2 also produced less IFN-alpha than Dw2-negative controls. In conclusion, we suggest that the observed impaired interferon-alpha production in MS is at least partially due to a high prevalence of Dw2 antigen in this disease.

NK activity and NK-like non-specific cytolysis after PPD, rubella and measles antigen stimulation in multiple sclerosis.

Blood samples were collected from 14 pairs of multiple sclerosis (MS) patients and from age- and sex-matched healthy controls. The cytotoxic activity of peripheral blood mononuclear cells was tested against the K562 cell line on the day of collection and again after 3 days in vitro culture with medium or with purified protein derivative of tuberculin (PPD), inactivated rubella virus or inactivated measles virus antigen. Lymphocytes from MS patients had a lower spontaneous cytotoxic activity compared to the controls both on the day of collection (P less than 0.025) and after 3 days in culture with medium alone (P less than 0.025). The activity decreased during in vitro culture in both groups, but the decrease was greater among MS patients (P less than 0.05). In cultures with antigens, a strong increase of NK-like cell-mediated cytolysis (NK-like CMC) was noted especially in cultures stimulated with PPD. There were no significant differences in the increase of the activity among MS patients and control subjects. A significant correlation between the increase of NK-like CMC and the lymphocyte blast transformation response induced by each antigen was found.

Production of viral antibodies in vitro by CSF cells from mumps meningitis and multiple sclerosis patients.

Cerebrospinal fluid (CSF) cells from 4 mumps meningitis and 11 multiple sclerosis (MS) patients were cultured in vitro for 7 days with and without pokeweed mitogen (PWM) stimulation. The cells produced varying amounts of IgG without stimulation and no significant increase of IgG synthesis was observed after PWM stimulation. Antibodies against mumps, measles, rubella, herpes simplex, and adeno viruses were measured in the supernatants of the cultures by a sensitive enzyme immunoassay. In the mumps meningitis patients, the largest amount of antibody was against mumps virus but low amounts of antibodies with other specificities were also synthesized by CSF cells of one patient. The most commonly detected specificities in MS patients were against measles and rubella viruses, whereas antibodies against adeno and mumps viruses were detected in only one CSF cell supernatant. No antibodies produced against herpes simplex virus in vitro were detected in any of the supernatants. The amounts of viral antibodies produced in vitro and intrathecally were only partially correlated.

Mitogen and antigen stimulation of multiple sclerosis cerebrospinal fluid lymphocytes in vitro.

Responses of cerebrospinal fluid (CSF) and peripheral blood (PB) lymphocytes from 20 MS patients to phytohemagglutinin (PHA), measles, rubella, mumps and herpes simplex virus antigens were followed during periods of from 6 to 13 months. Up to 6 examinations, each with 1-5 stimulants, were performed with a lymphocyte blast transformation test. Most of the patients responded with their CSF cells to PHA (14/19) and at least to some of the viral antigens tested (15/20) during the follow-up. Although the maximal responses of CSF and PB cells to PHA and measles virus antigen were of the same magnitude, non-reactive or weakly responding lymphocytes were more common in CSF than in PB. In 7 of 15 patients having viral antigen responsive CSF cells simultaneous reactivity to several antigens could be shown. The stimulation results of CSF lymphocytes did not correlate with the numbers of CSF leukocytes or the intrathecal IgG synthesis. A negative correlation was observed between the strength of the CSF cellular response to PHA or measles virus antigen and the rate of intrathecal antibody synthesis to measles virus antigen, suggesting that the stimulated cells may at least partially represent suppressor cells.

Circulating immune complexes in patients with multiple sclerosis. A longitudinal study of serum and CSF by C1q and platelet binding tests.

Two hundred and twenty-eight paired serum and CSF samples collected from 31 patients with MS during a 2-3-year follow-up were analyzed for the presence of immune complexes (IC) by C1q RIA and PIPA (platelet [125I]protein A) techniques. One hundred and forty-four sera from 11 healthy individuals were analyzed as controls. In almost all patients (29/31) IC were detectable during some period of the disease, as tested by either of the techniques. The results obtained by C1q RIA and PIPA correlated positively with each other in MS when mean serum values of each patient were compared. The mean CSF IC levels detected by C1q RIA appeared to correlate to the mean IgG indexes, an indicator of the intrathecal rate of IgG synthesis. The amount of IC in serum and CSF fluctuated independently in MS. The results of the PIPA test for MS serum IC correlated significantly to the duration of the disease. The PIPA test results also showed that patients in stable or chronic phases of MS displayed IC in serum and CSF more often than patients with a relapsing/remitting course of disease but there was no clear correlation between fluctuations in IC levels in individual patients measured by C1q RIA or PIPA techniques and the disease course. Because of the lack of a clear correlation between the presence, quantity and fluctuation of IC and the clinical picture we suggest that those IC detected in the present study are probably not a precipitating factor in the pathogenesis of multiple sclerosis.

Volatile halocarbons in tap water as a problem in haemodialysis therapy.

The concentrations of volatile halocarbons in the tap water of Turku and of Turku University Central Hospital are quite high and are reduced but not eliminated during water treatment at the hospital. Before haemodialysis is started, only trichloromethane is found in the blood of the patients. Two hours later dichlorobromomethane and dibromochloromethane could also be found. These substances are absorbed, and possibly accumulate, in the body or are metabolised and excreted because all their concentrations are lower at the end of dialysis therapy. Therefore, maximum levels for volatile halocarbons in drinking water should be sufficiently low to prevent these substances being detected in body fluids and special care should be taken with hospital water.