Sex-driven variability in TSPO-expressing microglia in MS patients and healthy individuals.

Background: Males with multiple sclerosis (MS) have a higher risk for disability progression than females, but the reasons for this are unclear. Objective: We hypothesized that potential differences in TSPO-expressing microglia between female and male MS patients could contribute to sex differences in clinical disease progression. Methods: The study cohort consisted of 102 MS patients (mean (SD) age 45.3 (9.7) years, median (IQR) disease duration 12.1 (7.0-17.2) years, 72% females, 74% relapsing-remitting MS) and 76 age- and sex-matched healthy controls. TSPO-expressing microglia were measured using the TSPO-binding radioligand [11C](R)-PK11195 and brain positron emission tomography (PET). TSPO-binding was quantified as distribution volume ratio (DVR) in normal-appearing white matter (NAWM), thalamus, whole brain and cortical gray matter (cGM). Results: Male MS patients had higher DVRs compared to female patients in the whole brain [1.22 (0.04) vs. 1.20 (0.02), p = 0.002], NAWM [1.24 (0.06) vs. 1.21 (0.05), p = 0.006], thalamus [1.37 (0.08) vs. 1.32 (0.02), p = 0.008] and cGM [1.25 (0.04) vs. 1.23 (0.04), p = 0.028]. Similarly, healthy men had higher DVRs compared to healthy women except for cGM. Of the studied subgroups, secondary progressive male MS patients had the highest DVRs in all regions, while female controls had the lowest DVRs. Conclusion: We observed higher TSPO-binding in males compared to females among people with MS and in healthy individuals. This sex-driven inherent variability in TSPO-expressing microglia may predispose male MS patients to greater likelihood of disease progression.

Association of serum neurofilament light with microglial activation in multiple sclerosis.

BACKGROUND: Translocator protein (TSPO)-PET and neurofilament light (NfL) both report on brain pathology, but their potential association has not yet been studied in multiple sclerosis (MS) in vivo. We aimed to evaluate the association between serum NfL (sNfL) and TSPO-PET-measurable microglial activation in the brain of patients with MS. METHODS: Microglial activation was detected using PET and the TSPO-binding radioligand [11C]PK11195. Distribution volume ratio (DVR) was used to evaluate specific [11C]PK11195-binding. sNfL levels were measured using single molecule array (Simoa). The associations between [11C]PK11195 DVR and sNfL were evaluated using correlation analyses and false discovery rate (FDR) corrected linear regression modelling. RESULTS: 44 patients with MS (40 relapsing-remitting and 4 secondary progressive) and 24 age-matched and sex-matched healthy controls were included. In the patient group with elevated brain [11C]PK11195 DVR (n=19), increased sNfL associated with higher DVR in the lesion rim (estimate (95% CI) 0.49 (0.15 to 0.83), p(FDR)=0.04) and perilesional normal appearing white matter (0.48 (0.14 to 0.83), p(FDR)=0.04), and with a higher number and larger volume of TSPO-PET-detectable rim-active lesions defined by microglial activation at the plaque edge (0.46 (0.10 to 0.81), p(FDR)=0.04 and 0.50 (0.17 to 0.84), p(FDR)=0.04, respectively). Based on the multivariate stepwise linear regression model, the volume of rim-active lesions was the most relevant factor affecting sNfL. CONCLUSIONS: Our demonstration of an association between microglial activation as measured by increased TSPO-PET signal, and elevated sNfL emphasises the significance of smouldering inflammation for progression-promoting pathology in MS and highlights the role of rim-active lesions in promoting neuroaxonal damage.

Association between microglial activation and serum kynurenine pathway metabolites in multiple sclerosis patients.

BACKGROUND: Microglial activation associates with MS progression but it is unclear what drives their persistent pro-inflammatory state. Metabolites of the kynurenine pathway (KP), the main metabolism route of tryptophan, can influence the function of brain innate immune cells. OBJECTIVE: To investigate whether tryptophan metabolites in blood associate with TSPO-PET measurable microglial activation in MS brain. METHODS: Microglial activation was detected using PET imaging and the TSPO-binding radioligand [11C]PK11195. Distribution volume ratios (DVR) for specific [11C]PK11195-binding in the normal appearing white matter (NAWM), lesions, and thalamus were calculated. Ultrahigh performance liquid chromatography-tandem mass spectrometry was used to measure serum levels of tryptophan and kynurenine pathway metabolites. RESULTS: The study cohort consisted of 48 MS patients. Increased DVR in the NAWM and thalamus correlated with decreased serum 3-hydroxykynurenine level (R = -0.31, p = 0.031 and R = -0.32, p = 0.028). Increased EDSS correlated with decreased 3-hydroxykynurenine and xanthurenic acid (R = -0.36, p = 0.012 and R = -0.31, p = 0.034) and increased DVR in the NAWM and thalamus (R = 0.33, p = 0.023 and R = 0.34, p = 0.020, respectively). CONCLUSIONS: This clinical study demonstrates an association between low serum 3-hydroxykynurenine and high microglial activation in MS. Further investigations are warranted for elucidation of the biological mechanisms behind this association.

Increased serum glial fibrillary acidic protein associates with microstructural white matter damage in multiple sclerosis: GFAP and DTI.

BACKGROUND: Astrocytes and microglial cells are now recognized as active players in contributing to the diffuse neuroaxonal damage associated with disease progression of multiple sclerosis (MS). The serum level of glial fibrillary acidic protein (GFAP), a biomarker for astrocytic activation, is increased in MS and associates with disease progression and disability. Similarly, diffusion tensor imaging (DTI) parameters for microstructural changes in brain, including demyelination and axonal loss, associate with disability. The association between brain DTI parameters and serum GFAP has not been previously explored in MS. The objective of the study was to get insights into DTI-measurable pathological correlates of elevated serum GFAP in the normal appearing white matter (NAWM) of MS. METHODS: A total of 62 MS patients with median age of 49.2 years were included in the study. Study patients underwent DTI-MRI and blood sampling for GFAP determination by single molecule array (Simoa). Mean fractional anisotropy (FA) and mean (MD), axial (AD) and radial (RD) diffusivities were calculated within the entire NAWM and six segmented NAWM regions. The associations between the DTI parameters and GFAP levels were analysed using Spearman correlation analysis and multiple regression model with sex and disease modifying treatment (no, 1st line or 2nd line) as adjustments. RESULTS: Elevated serum GFAP levels correlated significantly with decreased FA values within the entire (ρ = -0.39, p = 0.03), frontal (ρ = -0.42, p = 0.02), temporal (ρ = -0.37; p = 0.04) and cingulate (ρ = -0.38, p = 0.034) NAWM, and increased MD and RD within the frontal NAWM (ρ = 0.36, p = 0.046 for both). Similarly, higher GFAP associated with lower FA in frontal and cingulate NAWM in the multiple regression model corrected for confounding variables (standardised regression coefficient ß = -0.29, p = 0.045 and ß = -0.33, p = 0.025). CONCLUSIONS: Our results give evidence that increased serum GFAP levels associate with DTI-measurable micro-damage in the NAWM in MS. Our work supports the use of serum GFAP as a biomarker for MS pathology-related astrocytopathy and related diffuse white matter damage.

Fingolimod treatment reverses signs of diffuse white matter damage in multiple sclerosis: A pilot study.

BACKGROUND: In multiple sclerosis (MS) diffuse normal appearing white matter (NAWM) damage may drive chronic worsening independent of relapse activity. Diffusion tensor imaging (DTI) is a nonconventional MRI technique that can be used to assess microstructural alterations in myelin and axons. The aim of our study was to investigate the effect of six months fingolimod treatment on the integrity of entire and segmented NAWM in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: Ten RRMS patients initiating fingolimod treatment were included in the study. Patients underwent 3 T MRI including diffusion tensor sequences at baseline before the initiation of treatment and at six months. The mean values for fractional anisotropy (FA), and mean, radial and axial diffusivities (MD, RD and AD) were calculated within the whole NAWM and in six segmented sub-regions of NAWM (frontal, parietal, temporal, occipital, cingulate and deep NAWM). Clinical characteristics, Expanded Disability Status Scale (EDSS) and volumetric MRI data were also evaluated. RESULTS: In the cingulate NAWM FA was increased and RD was decreased significantly at six months compared to baseline (0.462 vs. 0.472, P = 0.027 and 0.000646 vs. 0.000634, P = 0.041, respectively), indicating improvements in myelin and axonal integrity following fingolimod treatment, whereas there were no alterations in cingulate MD or AD. Cingulate and temporal FA and RD correlated with T2 lesion volume percentage of cingulate and temporal areas. EDSS change correlated with change of the whole NAWM AD. CONCLUSIONS: Increased FA and decreased RD in the cingulate NAWM might suggest microstructural fingolimod-induced improvements in the normal appearing cingulate white matter. Our results support the concept that DTI can be used as a marker of diffuse neuronal damage also in interventional settings.

High serum neurofilament associates with diffuse white matter damage in MS.

OBJECTIVE: To evaluate to which extent serum neurofilament light chain (NfL) increase is related to diffusion tensor imaging-MRI measurable diffuse normal-appearing white matter (NAWM) damage in MS. METHODS: Seventy-nine patients with MS and 10 healthy controls underwent MRI including diffusion tensor sequences and serum NfL determination by single molecule array (Simoa). Fractional anisotropy and mean, axial, and radial diffusivities were calculated within the whole and segmented (frontal, parietal, temporal, occipital, cingulate, and deep) NAWM. Spearman correlations and multiple regression models were used to assess the associations between diffusion tensor imaging, volumetric MRI data, and NfL. RESULTS: Elevated NfL correlated with decreased fractional anisotropy and increased mean, axial, and radial diffusivities in the entire and segmented NAWM (for entire NAWM ρ = -0.49, p = 0.005; ρ = 0.49, p = 0.005; ρ = 0.43, p = 0.018; and ρ = 0.48, p = 0.006, respectively). A multiple regression model examining the effect of diffusion tensor indices on NfL showed significant associations when adjusted for sex, age, disease type, the expanded disability status scale, treatment, and presence of relapses. In the same model, T2 lesion volume was similarly associated with NfL. CONCLUSIONS: Our findings suggest that elevated serum NfL in MS results from neuroaxonal damage both within the NAWM and focal T2 lesions. This pathologic heterogeneity ought to be taken into account when interpreting NfL findings at the individual patient level.

Natalizumab treatment leads to an increase in circulating CXCR3-expressing B cells.

OBJECTIVE: To study the effects of natalizumab treatment on subgroups of circulating peripheral blood B cell populations. METHODS: We studied the proportions and absolute numbers of CD19+CD20+, CD10+, and CD5+ B cell populations, and determined very late activation antigen-4 and chemokine receptor CXCR3, CCR5, and CCR6 expression on B cells in the peripheral blood of 14 natalizumab-treated patients with relapsing-remitting multiple sclerosis. Five blood samples per patient were obtained longitudinally before and during the first year of treatment. Blood samples were analyzed by 6-color flow cytometry. RESULTS: Proportions of B cells and CD10+ pre-B cells were significantly increased, and very late activation antigen-4 expression on the B cell surface was significantly decreased already after 1 week of natalizumab treatment. Natalizumab-induced sustained increase in the proportion and absolute number of CXCR3-expressing B cells was statistically significant after 1 month of treatment. There were no changes in the proportions of CCR5- or CCR6-expressing B cells. CONCLUSIONS: The rapid and persistent increase in circulating CXCR3-expressing B cells in response to natalizumab treatment possibly reflects the relevance of this chemokine receptor in controlling migration of B cells into the CNS in humans in vivo.

CCR7 expression on peripheral blood lymphocytes is up-regulated following treatment of multiple sclerosis with interferon-beta.

A prospective study of the expression of the chemokine receptor CCR7 was performed in 11 relapsing-remitting multiple sclerosis (MS) patients during 12 months of interferon-beta (IFNbeta) treatment. The results show increased expression of CCR7 on peripheral blood lymphocytes (PBL) of MS patients receiving IFNbeta treatment as well as lymphocytes from healthy subjects treated with IFNbeta in vitro. Our results suggest that in addition to modulating the expression of adhesion molecules, the mode of action of IFNbeta also involves the control of the chemokine receptor CCR7. The net effect is a key change in the control of lymphocyte traffic between immune organs and the central nervous system (CNS) and a shift from CCR7 negative effector T cells to CCR7 positive central memory T cells.

Clinical and immunologic evaluation of women with multiple sclerosis during and after pregnancy.

BACKGROUND: Multiple sclerosis (MS) is a disabling autoimmune disease of the central nervous system, typically affecting women of childbearing years. Although the disease course of MS is highly unpredictable, disease activity is almost invariably halted during pregnancy. After delivery, however, the relapse rate increases. Despite early recognition of this pattern of disease activity, its explanation remains a mystery. OBJECTIVE: The aim of this study was to elucidate the underlying mechanisms responsible for the amelioration of MS during pregnancy and for its reactivation after delivery. METHODS: This Finnish prospective study included clinical and immunologic follow-up of patients with MS during pregnancy and 6 months into the postpartum period. Groups of patients with MS who were not pregnant, along with pregnant and nonpregnant healthy women, served as controls. Laboratory investigations included subtype analysis of T, B, and natural killer (NK) cells during and after pregnancy, using immunofluorescence staining and fluorescence-activated cell sorting analysis RESULTS: The clinical and immunologic follow-up data from 42 pregnant patients with MS indicated that the percentage of circulating NK cells decreases during the last trimester of pregnancy and increases again soon after the delivery. This correlates with disease activity as measured by annualized relapse rate. Early postpartum treatment with interferon-0 was effective in preventing relapses, and good response to postpartum treatment coincided with a reduction in the circulating NK cell levels. CONCLUSIONS: Our findings have implications for the treatment and follow-up of pregnant women with MS. To prevent postpartum relapses, disease-modifying treatment should be initiated as early as possible.

Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level.

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides. Current analytical methods for sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the functionality of one mutant in some real sample matrices.