From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

A Mixture of Endocrine Disrupting Chemicals Associated with Lower Birth Weight in Children Induces Adipogenesis and DNA Methylation Changes in Human Mesenchymal Stem Cells.

Endocrine Disrupting Chemicals (EDCs) are man-made compounds that alter functions of the endocrine system. Environmental mixtures of EDCs might have adverse effects on human health, even though their individual concentrations are below regulatory levels of concerns. However, studies identifying and experimentally testing adverse effects of real-life mixtures are scarce. In this study, we aimed at evaluating an epidemiologically identified EDC mixture in an experimental setting to delineate its cellular and epigenetic effects. The mixture was established using data from the Swedish Environmental Longitudinal Mother and child Asthma and allergy (SELMA) study where it was associated with lower birth weight, an early marker for prenatal metabolic programming. This mixture was then tested for its ability to change metabolic programming of human mesenchymal stem cells. In these cells, we assessed if the mixture induced adipogenesis and genome-wide DNA methylation changes. The mixture increased lipid droplet accumulation already at concentrations corresponding to levels measured in the pregnant women of the SELMA study. Furthermore, we identified differentially methylated regions in genes important for adipogenesis and thermogenesis. This study shows that a mixture reflecting human real-life exposure can induce molecular and cellular changes during development that could underlie adverse outcomes.

High antibody levels against human herpesvirus-6A interact with lifestyle factors in multiple sclerosis development.

BACKGROUND: Infection with human herpesvirus 6A (HHV-6A) has been suggested to increase multiple sclerosis (MS) risk. However, potential interactions between HHV-6A and environmental/lifestyle risk factors for MS have not previously been studied. METHODS: We used two Swedish population-based case-control studies comprising 5993 cases and 5995 controls. Using logistic regression models, subjects with different HHV-6A antibody levels, environmental exposures, and lifestyle habits were compared regarding MS risk, by calculating odds ratios (ORs) with 95% confidence intervals (CIs). Potential interactions between high HHV-6A antibody levels and common environmental exposures and lifestyle factors were evaluated on the additive scale. RESULTS: High HHV-6A antibody levels were associated with increased risk of developing MS (OR = 1.5, 95% CI = 1.4-1.6). Regarding MS risk, significant interactions were observed between high HHV-6A antibody levels and both smoking (attributable proportion (AP) = 0.2, 95% CI = 0.1-0.3), low ultraviolet radiation (UVR) exposure (AP = 0.3, 95% CI = 0.1-0.4), and low vitamin D levels (AP = 0.3, 95% CI = 0.0-0.6). CONCLUSION: High HHV-6A antibody levels are associated with increased MS risk and act synergistically with common environmental/lifestyle risk factors for MS. Further research is needed to investigate potential mechanisms underlying the interactions presented in this study.

Bisphenol A Inhibits the Transporter Function of the Blood-Brain Barrier by Directly Interacting with the ABC Transporter Breast Cancer Resistance Protein (BCRP).

The breast cancer resistance protein (BCRP) is an important efflux transporter in the blood-brain barrier (BBB), protecting the brain from a wide range of substances. In this study, we investigated if BCRP function is affected by bisphenol A (BPA), a high production volume chemical used in common consumer products, as well as by bisphenol F (BPF) and bisphenol S (BPS), which are used to substitute BPA. We employed a transwell-based in vitro cell model of iPSC-derived brain microvascular endothelial cells, where BCRP function was assessed by measuring the intracellular accumulation of its substrate Hoechst 33342. Additionally, we used in silico modelling to predict if the bisphenols could directly interact with BCRP. Our results showed that BPA significantly inhibits the transport function of BCRP. Additionally, BPA was predicted to bind to the cavity that is targeted by known BCRP inhibitors. Taken together, our findings demonstrate that BPA inhibits BCRP function in vitro, probably by direct interaction with the transporter. This effect might contribute to BPA's known impact on neurodevelopment.

DNA methylation at GRIN2B partially mediates the association between prenatal bisphenol F exposure and cognitive functions in 7-year-old children in the SELMA study.

BACKGROUND: Accumulating evidence suggests that prenatal chemical exposure triggers epigenetic modifications that could influence health outcomes later in life. In this study, we investigated whether DNA methylation (DNAm) levels at the glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B) gene underlies the association between prenatal exposure to an endocrine disrupting chemical (EDC), bisphenol F (BPF), and lower cognitive functions in 7-year-old children. METHODS: Data from 799 children participating in the Swedish Environmental Longitudinal Mother and child Asthma and allergy (SELMA) pregnancy cohort was analyzed. Prenatal BPF exposure was assessed by measuring BPF levels in maternal urine. At age 7, DNAm of three CpG sites in a regulatory region of the GRIN2B gene was analyzed from buccal swabs using bisulfite-Pyrosequencing. Cognitive functions, including full-scale IQ and four subscales, were evaluated using the Wechsler Intelligence Scale for Children (WISC-IV). Associations between prenatal BPF exposure and GRIN2B DNAm, as well as between GRIN2B DNAm and cognitive functions, were determined using regression models adjusted for potential confounders. Generalized structural equation models (gSEM) were used to evaluate if GRIN2B DNAm mediates the association between prenatal BPF exposure and cognitive functions at 7 years of age. RESULTS: Prenatal BPF exposure was positively associated with GRIN2B DNAm levels at the third CpG site (CpG3), while CpG3 methylation was inversely associated with cognitive test scores. Mediation analyses showed that CpG3 methylation exerted 6-9% of the association between BPF exposure and full-scale IQ, as well as verbal comprehension and perceptual reasoning in boys, while not significant in girls. CONCLUSIONS: This study is the first to identify locus-specific DNAm as a mediating factor underlying an epidemiological association between prenatal EDC exposure and cognitive functions in childhood. It also confirms previous findings, that GRIN2B DNAm is responsive to environmental exposures.

Prenatal exposure to bisphenols and cognitive function in children at 7 years of age in the Swedish SELMA study.

BACKGROUND: Experimental evidence demonstrates that exposure to bisphenol A (BPA), and the recently introduced alternatives bisphenol S (BPS) and bisphenol F (BPF) alter normal neurodevelopment. More research is needed to evaluate the associations between exposure to individual BPA alternatives and neurodevelopmental outcomes in humans. OBJECTIVE: The present study aimed at examining the individual associations between prenatal BPA, BPS and BPF exposure and cognitive outcomes in children at age 7 years. METHOD: Women were enrolled in the Swedish Environmental Longitudinal Mother and Child, Asthma and Allergy (SELMA) study, at gestational median week 10.0, and their children were examined for cognitive function at 7 years of age (N = 803). Maternal urinary BPA, BPS, and BPF concentrations were measured at enrollment and childrens cognitive function at the age of 7 years was measured using the Wechsler Intelligence Scale for Children IV (WISC-IV). RESULTS: All three bisphenols were detected in over 90% of the women, where BPA had the highest geometric mean concentrations (1.55 ng/mL), followed by BPF (0.16 ng/mL) and BPS (0.07 ng/mL). Prenatal BPF exposure was associated with decreased full scale IQ (ß = -1.96, 95%CI; -3.12; -0.80), as well as with a decrease in all four sub scales covering verbal comprehension, perceptual reasoning, working memory and processing speed. This association corresponded to a 1.6-point lower IQ score for an inter-quartile-range (IQR) change in prenatal BPF exposure (IQR = 0.054-0.350 ng/mL). In sex-stratified analyses, significant associations with full scale IQ were found for boys (ß = -2.86, 95%CI; -4.54; -1.18), while the associations for girls did not reach significance (ß = -1.38, 95%CI; -2.97; 0.22). No significant associations between BPA nor BPS and cognition were found. DISCUSSION: Prenatal exposure to BPF was significantly associated with childrens cognitive function at 7 years. Since BPF is replacing BPA in numerous consumer products globally, this finding urgently call for further studies.

Childhood adversity increases methylation in the GRIN2B gene.

Childhood adversity is an early life stressor associated with increased risk of several psychiatric disorders such as depression. Epigenetic changes, primarily DNA methylation, can be affected by early life stress, which in turn might contribute to altered disease susceptibility later in life. One plausible biomarker of early life stress is methylation of the ionotropic glutamate receptor NMDA type subunit 2B (GRIN2B) gene, which has been previously shown to be epigenetically affected by prenatal environmental stressors. Here, we set out to investigate if stress-inducing adversity during childhood is associated with changes in methylation of GRIN2B in adulthood. We studied 186 individuals from a Swedish naturalistic population-based cohort who had provided saliva samples (DNA) as well as information regarding both childhood adversity (CA) and depressive symptoms (dep) (nCA,dep = 41, nCA,no-dep = 56, nno-CA,dep = 40, Nno-CA,no-dep = 49). Methylation at four CpG sites in a regulatory region of GRIN2B was analysed using bisulfite pyrosequencing. Associations for methylation status to childhood adversity and to depression status were investigated using linear regression models. Our study shows that childhood adversity is associated with increased methylation levels of GRIN2B in adulthood, for three of the measured CpGs (p = 0.007, 0.006 and 5 × 10-14). This indicates that GRIN2B methylation is susceptible to early life stress, and that methylation at this gene is persistent over time. No association was found between GRIN2B methylation and depression status. Yet, this does not rule out a role for alterations in GRIN2B methylation for other neuropsychological outcomes not studied here.

Increased Serological Response Against Human Herpesvirus 6A Is Associated With Risk for Multiple Sclerosis.

Human herpesvirus (HHV)-6A or HHV-6B involvement in multiple sclerosis (MS) etiology has remained controversial mainly due to the lack of serological methods that can distinguish the two viruses. A novel multiplex serological assay measuring IgG reactivity against the immediate-early protein 1 from HHV-6A (IE1A) and HHV-6B (IE1B) was used in a MS cohort (8,742 persons with MS and 7,215 matched controls), and a pre-MS cohort (478 individuals and 476 matched controls) to investigate this further. The IgG response against IE1A was positively associated with MS (OR = 1.55, p = 9 × 10-22), and increased risk of future MS (OR = 2.22, p = 2 × 10-5). An interaction was observed between IE1A and Epstein-Barr virus (EBV) antibody responses for MS risk (attributable proportion = 0.24, p = 6 × 10-6). In contrast, the IgG response against IE1B was negatively associated with MS (OR = 0.74, p = 6 × 10-11). The association did not differ between MS subtypes or vary with severity of disease. The genetic control of HHV-6A/B antibody responses were located to the Human Leukocyte Antigen (HLA) region and the strongest association for IE1A was the DRB1*13:01-DQA1*01:03-DQB1*06:03 haplotype while the main association for IE1B was DRB1*13:02-DQA1*01:02-DQB1*06:04. In conclusion a role for HHV-6A in MS etiology is supported by an increased serological response against HHV-6A IE1 protein, an interaction with EBV, and an association to HLA genes.

The effect of human herpesvirus 6B infection on the MAPK pathway.

BACKGROUND: Human herpesvirus 6B (HHV-6B) is a neurotropic virus that has been repeatedly associated with mesial temporal lobe epilepsy (MTLE). However, the mechanism behind this suggested association is not known. Therefore, the aim of this study was to investigate what genes were affected by HHV-6B, possibly revealing HHV-6B induced disease causing mechanisms. MATERIAL AND METHOD: First, gene expression in MTLE tissue positive for HHV-6B DNA (n = 10) and negative for HHV-6B DNA (n = 14) was compared using the Affymetrix® Human Gene 2.1 ST Array. Secondly, in vitro experiments were conducted where Molt-3 T cells were infected with HHV-6B and gene expression of MAP2K4 (MKK4) and 89 other genes in the MAPK signaling pathway was investigated using qPCR. In addition, phosphorylated MKK4 was assessed using IFA and the DNA methylation investigated with Illumina Infinium HumanMethylation450 BeadChip array. RESULTS: MAP2K4 was one of the most differently expressed genes in the Affymetrix array, suggesting an upregulation by HHV-6B infection in MTLE tissue. No gene reached statistical significance but MAP2K4 was selected for further investigation in vitro, where it was clearly upregulated by HHV-6B infection both on gene expression and protein expression level. Further investigating expression of genes in the MAPK pathways in vitro revealed that several genes were affected by HHV-6B infection, but none of these genes displayed viral induced changes in DNA methylation. CONCLUSIONS: As the MAPK pathways are involved in transforming different stimuli (like stress) into a cellular responses (like apoptosis or inflammation), it may not be surprising that genes in these pathways are affected by virus infection. This is the first report of HHV-6B's effect on these signaling cascades and given that both dysregulation of the MAPK pathways and an association with HHV-6B have been previously observed in epilepsy, a possible link of infection induced dysregulation of MAPK in epilepsy warrant further investigation.

Human Herpesvirus 6B Induces Hypomethylation on Chromosome 17p13.3, Correlating with Increased Gene Expression and Virus Integration.

Human herpesvirus 6B (HHV-6B) is a neurotropic betaherpesvirus that achieves latency by integrating its genome into host cell chromosomes. Several viruses can induce epigenetic modifications in their host cells, but no study has investigated the epigenetic modifications induced by HHV-6B. This study analyzed methylation with an Illumina 450K array, comparing HHV-6B-infected and uninfected Molt-3 T cells 3 days postinfection. Bisulfite pyrosequencing was used to validate the Illumina results and to investigate methylation over time in vitro Expression of genes was investigated using quantitative PCR (qPCR), and virus integration was investigated with PCR. A total of 406 CpG sites showed a significant HHV-6B-induced change in methylation in vitro Remarkably, 86% (351/406) of these CpGs were located <1 Mb from chromosomal ends and were all hypomethylated in virus-infected cells. This was most evident at chromosome 17p13.3, where HHV-6B had induced CpG hypomethylation after 2 days of infection, possibly through TET2, which was found to be upregulated by the virus. In addition, virus-induced cytosine hydroxymethylation was observed. Genes located in the hypomethylated region at 17p13.3 showed significantly upregulated expression in HHV-6B-infected cells. A temporal experiment revealed HHV-6B integration in Molt-3 cell DNA 3 days after infection. The telomere at 17p has repeatedly been described as an integration site for HHV-6B, and we show for the first time that HHV-6B induces hypomethylation in this region during acute infection, which may play a role in the integration process, possibly by making the DNA more accessible.IMPORTANCE The ability to establish latency in the host is a hallmark of herpesviruses, but the mechanisms differ. Human herpesvirus 6B (HHV-6B) is known to establish latency through integration of its genome into the telomeric regions of host cells, with the ability to reactivate. Our study is the first to show that HHV-6B specifically induces hypomethylated regions close to the telomeres and that integrating viruses may use the host methylation machinery to facilitate their integration process. The results from this study contribute to knowledge of HHV-6B biology and virus-host interaction. This in turn will lead to further progress in our understanding of the underlying mechanisms by which HHV-6B contributes to pathological processes and may have important implications in both disease prevention and treatment.

Investigation of reference gene expression during human herpesvirus 6B infection indicates peptidylprolyl isomerase A as a stable reference gene and TATA box binding protein as a gene up-regulated by this virus.

When using relative gene expression for quantification of RNA it is crucial that the reference genes used for normalization do not change with the experimental condition. We aimed at investigating the expressional stability of commonly used reference genes during Human herpesvirus 6B (HHV-6B) infection. Expression of eight commonly used reference genes were investigated with quantitative PCR in a T-cell line infected with HHV-6B. The stability of genes was investigated using the 2(-ΔΔCT) method and the algorithms BestKeeper, GeNorm and NormFinder. Our results indicate that peptidylprolyl isomerase A (PPIA) is the most stably expressed gene while TATA box binding protein (TBP) is the least stably expressed gene during HHV-6B infection. In a confirmatory experiment, TBP was demonstrated to be dose and time dependently upregulated by HHV-6B. The stability of PPIA is in line with other studies investigating different herpesvirus infections whereas the finding that HHV-6B significantly upregulates TBP is novel and most likely specific to HHV-6B.

The structures of CyMe4-BTBP complexes of americium(iii) and europium(iii) in solvents used in solvent extraction, explaining their separation properties.

Separation of trivalent actinoid (An(iii)) and lanthanoid (Ln(iii)) ions is extremely challenging due to their similar ionic radii and chemical properties. Poly-aromatic nitrogen compounds acting as tetradentate chelating ligands to the metal ions in the extraction, have the ability to sufficiently separate An(iii) from Ln(iii). One of these compounds, 6,6'-bis(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-benzol[1,2,4]triazin-3-yl)[2,2]bipyridine, CyMe4-BTBP, has proven to be resistant towards acidic environments and strong radiation from radioactive decomposition. EXAFS studies of the dicomplexes of CyMe4-BTBP with americium(iii) and europium(iii) in nitrobenzene, cyclohexanone, 1-hexanol, 1-octanol and malonamide (DMDOHEMA) in 1-octanol have been carried out to get a deeper understanding of the parameters responsible for the separation. The predominating complexes independent of solvent used are [Am(CyMe4-BTBP)2(NO3)](2+) and [Eu(CyMe4-BTBP)2](3+), respectively, which are present as outer-sphere ion-pairs with nitrate ions in the studied solvents with low relative permittivity. The presence of a nitrate ion in the first coordination sphere of the americium(iii) complex compensates the charge density of the complex considerably in comparison when only outer-sphere ion-pairs are formed as for the [Eu(CyMe4-BTBP)2](3+) complex. The stability and solubility of a complex in a solvent with low relative permittivity increase with decreasing charge density. The [Am(CyMe4-BTBP)2(NO3)](2+) complex will therefore be increasingly soluble and stabilized over the [Eu(CyMe4-BTBP)2](3+) complex in solvents with decreasing relative permittivity of the solvent. The separation of americium(iii) from europium(iii) with CyMe4-BTBP as extraction agent will increase with decreasing relative permittivity of the solvent, and thereby also with decreasing solubility of CyMe4-BTBP. The choice of solvent is therefore a balance of a high separation factor and sufficient solubility of the CyMe4-BTBP ligand.

HLA-A(∗)02, gender and tobacco smoking, but not multiple sclerosis, affects the IgG antibody response against human herpesvirus 6.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system. Both genetic and environmental factors contribute to disease susceptibility and two viruses associated with MS are human herpesvirus (HHV)-6A and HHV-6B, together referred to as HHV-6. This study characterized the plasma IgG antibody response against HHV-6 in MS patients (n=446) and healthy controls (n=487), and the relationship between MS susceptibility factors and the anti-HHV-6 response was investigated. In addition, 134 samples were further investigated for IgG against the early HHV-6 antigen p41. Antibody levels were measured with ELISA. The overall seroprevalence against HHV-6 was 90%, with no significant difference in positivity or levels between MS patients and controls. Interestingly, carriership of HLA-A(∗)02 and tobacco smoking was associated with lower anti-HHV-6 IgG levels (p=0.0017 and p=0.026 respectively), whereas females sex was associated with higher levels (p=0.0090). No difference in IgG titers against p41 was observed between MS patients and controls. In conclusion, the IgG response against HHV-6 was associated with several factors that have previously been associated with MS susceptibility, possibly reflecting a relation between autoimmunity and how the immune system handles viral infections.

Anti-interferon beta antibody titers strongly correlate between two bioassays and in vivo biomarker expression, and indicates that a titer of 150 TRU/mL is a biologically functional cut-point.

Interferon beta (IFNß) is used as a first-line treatment in relapsing-remitting multiple sclerosis (MS). The occurrence of neutralizing antidrug antibodies (NAbs) against IFNß may reduce treatment response. Therefore, clinical monitoring of NAbs is currently executed using bioassays, but several bioassays are available and it is unclear how well their readouts correlate. We made a comparison between 2 bioassays; myxovirus resistance protein A (MxA) gene expression assay (MGA) and iLite™ anti-Human IFNß bioassay, to measure IFNß-specific NAb titers in 44 MS patients. We further studied how NAb titers affected in vivo transcription of IFN-induced genes myxovirus resistant 1 (MX1) and C-X-C motif chemokine 10 (CXCL10), in addition to serum CXCL10 protein levels. There were significant correlations between NAb titer levels measured with MGA and iLite (Spearman r=0.9368). MX1 and CXCL10 gene expression was strongly induced by IFNß and NAb positivity significantly reduced this expression. A NAb titer of 150 TRU/mL was observed to be a biological cut-point applicable to both assays, since MX1 and CXCL10 expression was greatly reduced or blocked in patients above this titer level. In conclusion, NAb titers measured with the MGA and iLite bioassays are comparable, but the threshold for positivity in both assays does not correspond to the biologically functional cut-point.

First therapeutic use of Artesunate in treatment of human herpesvirus 6B myocarditis in a child.

BACKGROUND: Human herpesvirus 6 (HHV-6) is an important cause of fulminant or acute viral myocarditis. However, insufficiency of standard antiviral treatment against HHV-6 is an emerging problem. OBJECTIVES: To describe the case of child with HHV-6 myocarditis who was treated by unloading with a left ventricular assist device and Artesunate. STUDY DESIGN: Case report supported by histological and viral diagnoses via a combination of histology/immunohistochemistry and polymerase chain reaction techniques performed on cardiac tissues before and after treatment. RESULTS: Following therapeutic intervention, the clinical status and heart function improved. Endomyocardial biopsies revealed decreased levels of HHV-6B DNA in the myocardium and the disappearance of histological findings in support of lymphocytic myocarditis. Left ventricular assist device could be explanted. No adverse effects of Artesunate were noted. CONCLUSIONS: In addition to existing heart failure treatments, Artesunate can be considered as an effective candidate for clinical use in cases of HHV-6B associated myocarditis.

Human herpesvirus 6A partially suppresses functional properties of DC without viral replication.

Human herpesvirus 6A (HHV-6A) is a common virus with a worldwide distribution that has been associated with multiple sclerosis. Whether HHV-6A can replicate in dendritic cells (DC) and how the infection might modulate the functional properties of the cell are currently not well known and need further investigations. Here, we show that a non-productive infection of HHV-6A in DC leads to the up-regulation of HLA-ABC, via autocrine IFN-α signaling, as well as the up-regulation of HLA-DR and CD86. However, HHV-6A exposure reduces IL-8 secretion by DC and their capacity to stimulate allogenic T cell proliferation. The ability to suppress DC functions important for activation of innate and adaptive immune responses might be one successful strategy by which HHV-6A avoids the induction of appropriate host defense mechanisms, and thus facilitating persistent infection.

Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6.

BACKGROUND: For titer assessment of human herpesvirus 6 (HHV-6), IFA targeting viral proteins or a TCID(50) method with ocular inspection for CPE can be used. These methods rely on the subjective decision of the assessor, obstructing the ability to obtain unanimous results. FINDINGS: We have developed and validated an alternative TCID(50) read-out approach where infection in the titration culture plate is assessed by viral DNA load change by quantitative PCR. A ten time increase in viral DNA load was determined as cut point for infection since that yielded a maximum correlation with viral protein expression (93%). The average intra-assay CV was 9% for quantitative PCR read-out of TCID(50) compared to 45% for ocular inspection read-out of TCID(50) , 14% for IFA read-out of TCID(50), and 43% for an infectious units approach using IFA. The average inter-assay CV for quantitative PCR read-out of TCID(50) was 73%, compared to 66%, 25% and 77% for the ocular inspection read-out for TCID(50), IFA read-out of TCID(50)and infectious unit approaches respectively. CONCLUSIONS: The quantitative PCR based read-out of TCID(50)proved to be more robust and easier to interpret than traditional TCID(50)assessment approaches for HHV-6, and therefore it might be considered as an alternative method.

Prevalence of anti-drug antibodies against interferon beta has decreased since routine analysis of neutralizing antibodies became clinical practice.

BACKGROUND: Neutralizing antibodies (NAbs) against interferon beta (IFNß) lead to loss of treatment efficacy in multiple sclerosis patients. The seroprevalence of NAbs in multiple sclerosis patients treated with IFNß during 2003-2004 was 32% in a cross-sectional analysis of routine data. OBJECTIVES: The aim of this study was to investigate whether the seroprevalence of NAbs, the levels of NAb titres and the IFNß preparations used for treatment of multiple sclerosis patients had changed in 2009-2010. METHODS: This study included 1296 patients, analysed for NAbs with the myxovirus resistance protein A gene expression assay in 2009-2010. RESULTS: The seroprevalence of NAbs had decreased to 19% in 2009-2010, which is significantly lower compared with the previous study in 2003-2004 (p<0.0001). This decrease was attributed to the IFNß-1a preparations only, not to IFNß-1b. The frequency of patients with high positive titres decreased the most, from 16% to 7% (p<0.0001). CONCLUSIONS: NAb seroprevalence has decreased since NAb monitoring became clinical practice in 2003, especially for patients with high NAb titres. This might be due to the stricter monitoring of NAb titres that prompt NAb positive patients to stop treatment, to preferential use of less immunogenic drugs and to alteration of drug formulations.