Objective
Gray matter (GM) damage and meningeal inflammation have been associated with early disease onset and a more aggressive disease course in multiple sclerosis (MS), but can these changes be ...identified in the patient early in the disease course?
Methods
To identify possible biomarkers linking meningeal inflammation, GM damage, and disease severity, gene and protein expression were analyzed in meninges and cerebrospinal fluid (CSF) from 27 postmortem secondary progressive MS and 14 control cases. Combined cytokine/chemokine CSF profiling and 3T magnetic resonance imaging (MRI) were performed at diagnosis in 2 independent cohorts of MS patients (35 and 38 subjects) and in 26 non‐MS patients.
Results
Increased expression of proinflammatory cytokines (IFNγ, TNF, IL2, and IL22) and molecules related to sustained B‐cell activity and lymphoid‐neogenesis (CXCL13, CXCL10, LTα, IL6, and IL10) was detected in the meninges and CSF of postmortem MS cases with high levels of meningeal inflammation and GM demyelination. Similar proinflammatory patterns, including increased levels of CXCL13, TNF, IFNγ, CXCL12, IL6, IL8, and IL10, together with high levels of BAFF, APRIL, LIGHT, TWEAK, sTNFR1, sCD163, MMP2, and pentraxin III, were detected in the CSF of MS patients with higher levels of GM damage at diagnosis.
Interpretation
A common pattern of intrathecal (meninges and CSF) inflammatory profile strongly correlates with increased cortical pathology, both at the time of diagnosis and at death. These results suggest a role for detailed CSF analysis combined with MRI as a prognostic marker for more aggressive MS. Ann Neurol 2018 Ann Neurol 2018;83:739–755
Recent studies of cortical pathology in secondary progressive multiple sclerosis have shown that a more severe clinical course and the presence of extended subpial grey matter lesions with ...significant neuronal/glial loss and microglial activation are associated with meningeal inflammation, including the presence of lymphoid-like structures in the subarachnoid space in a proportion of cases.
To investigate the molecular consequences of pro-inflammatory and cytotoxic molecules diffusing from the meninges into the underlying grey matter, we carried out gene expression profiling analysis of the motor cortex from 20 post-mortem multiple sclerosis brains with and without substantial meningeal inflammation and 10 non-neurological controls.
Gene expression profiling of grey matter lesions and normal appearing grey matter not only confirmed the substantial pathological cell changes, which were greatest in multiple sclerosis cases with increased meningeal inflammation, but also demonstrated the upregulation of multiple genes/pathways associated with the inflammatory response. In particular, genes involved in tumour necrosis factor (TNF) signalling were significantly deregulated in MS cases compared with controls. Increased meningeal inflammation was found to be associated with a shift in the balance of TNF signalling away from TNFR1/TNFR2 and NFkB-mediated anti-apoptotic pathways towards TNFR1- and RIPK3-mediated pro-apoptotic/pro-necroptotic signalling in the grey matter, which was confirmed by RT-PCR analysis. TNFR1 was found expressed preferentially on neurons and oligodendrocytes in MS cortical grey matter, whereas TNFR2 was predominantly expressed by astrocytes and microglia.
We suggest that the inflammatory milieu generated in the subarachnoid space of the multiple sclerosis meninges by infiltrating immune cells leads to increased demyelinating and neurodegenerative pathology in the underlying grey matter due to changes in the balance of TNF signalling.
Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification ...(PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4
T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples.
Antigen-induced T-cell exhaustion and T-cell senescence are peripheral regulatory mechanisms that control effector T-cell responses. Markers of exhaustion and senescence on T Cells indicate the ...previous activation by repetitive stimulation with specific antigens. Malignant tumors are accompanied by enhanced T-cell exhaustion and T-cell senescence resulting in immune evasion, while these control mechanisms might be diminished in autoimmune diseases including multiple sclerosis (MS). To better understand the involvement of antigen-induced T-cell senescence in controlling CD4+ T-cell-mediated autoimmune responses in MS, we have analyzed the re-expression of CD45RA and the downregulation of CD28 and CD27 molecules as markers of antigen-induced T-cell senescence in fresh cerebrospinal fluid (CSF)-infiltrating and paired circulating T cells from patients with MS. Patients with different levels of CD4+ T-cell senescence were identified and characterized regarding demographical and clinical features as well as intrathecal markers of neurodegeneration. CD4+ T-cell senescence was also analyzed in control patients to explore a putative deficit of this regulatory mechanism in MS. This study shows heterogeneity of markers of CD4+ T-cell senescence in patients with MS. Patients with high levels of CD4+ T-cell senescence in peripheral blood showed increased frequencies of CSF-infiltrating CD28+ CD27-EM CD4+ T cells with a proinflammatory Th1 functional phenotype. The correlation of these cells with the intrathecal levels of neurofilament light chain, a marker of neurodegeneration, suggests their relevance in disease pathogenesis and the involvement of T-cell senescence in their regulation. Markers of antigen-induced T-senescence, therefore, show promise as a tool to identify pathogenic CD4+ T cells in patients with MS.
The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as ...antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.
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•HLA-DR15 present abundant HLA-DR-derived self-peptides on B cells•Autoreactive T cells in MS recognize HLA-DR-derived self-peptides/DR15 complexes•Foreign peptides/DR15 complexes trigger potential autoreactive T cells in MS•HLA-DR15 shape an autoreactive T cell repertoire by cross-reactivity/restriction
The immunopeptidome presented by HLA-DR15 molecules links the most important genetic and environmental risk factors for multiple sclerosis, the HLA-DR15 haplotype and Epstein-Barr virus, by shaping a cross-reactive CD4+ T cell repertoire.
Encouraged by the enormous progress that the identification of specific autoantigens added to the understanding of neurologic autoimmune diseases, we undertook here an in-depth study of T-cell ...specificities in the autoimmune disease multiple sclerosis (MS), for which the spectrum of responsible autoantigens is not fully defined yet. The identification of target antigens in MS is crucial for therapeutic strategies aimed to induce antigen-specific tolerance. In addition, knowledge of relevant T-cell targets can improve our understanding of disease heterogeneity, a hallmark of MS that complicates clinical management.
The proliferative response and interferon gamma (IFN-γ) release of CSF-infiltrating CD4
T cells from patients with MS against several autoantigens was used to identify patients with different intrathecal T-cell specificities. Fresh CSF-infiltrating and paired circulating lymphocytes in these patients were characterized in depth by ex vivo immunophenotyping and transcriptome analysis of relevant T-cell subsets. Further examination of these patients included CSF markers of inflammation and neurodegeneration and a detailed characterization with respect to demographic, clinical, and MRI features.
By testing CSF-infiltrating CD4
T cells from 105 patients with MS against seven long-known myelin and five recently described GDP-l-fucose synthase peptides, we identified GDP-l-fucose synthase and myelin oligodendrocyte glycoprotein (35-55) responder patients. Immunophenotyping of CSF and paired blood samples in these patients revealed a significant expansion of an effector memory (CCR7
CD45RA
) CD27
Th1 CD4
cell subset in GDP-l-fucose synthase responders. Subsequent transcriptome analysis of this subset demonstrated expression of Th1 and cytotoxicity-associated genes. Patients with different intrathecal T-cell specificities also differ regarding inflammation- and neurodegeneration-associated biomarkers, imaging findings, expression of HLA class II alleles, and seasonal distribution of the time of the lumbar puncture.
Our observations reveal an association between autoantigen reactivity and features of disease heterogeneity that strongly supports an important role of T-cell specificity in MS pathogenesis. These data have the potential to improve patient classification in clinical practice and to guide the development of antigen-specific tolerization strategies.
CNS damage can increase the susceptibility of the blood-brain barrier (BBB) to changes induced by systemic inflammation. The aim of this study is to better understand BBB permeability in patients ...with MS and to examine whether compromised BBB integrity in some of these patients is associated with CNS damage and systemic inflammation.
Routine CSF measurements of 121 patients with MS were analyzed including number and type of infiltrating cells, total protein, lactate, and oligoclonal bands, as well as intrathecal production of immunoglobulins and CSF/serum quotients for albumin, immunoglobulins, and glucose. In addition, in a subcohort of these patients, we performed ex vivo immunophenotyping of CSF-infiltrating and paired circulating lymphocytes using a panel of 13 monoclonal antibodies, we quantified intrathecal neurofilament light chain (NF-L) and chitinase 3-like 1 (CHI3L1), and we performed intrathecal lipidomic analysis.
Patients with MS with abnormal high levels of albumin in the CSF showed a distinct CSF cell infiltrate and markers of CNS damage such as increased intrathecal levels of NF-L and CHI3L1 as well as a distinct CSF lipidomic profile. In addition, these patients showed higher numbers of circulating proinflammatory Th1 and Th1* cells compatible with systemic inflammation. Of interest, the abnormally high levels of albumin in the CSF of those patients were preserved over time.
Our results support the hypothesis that CNS damage may increase BBB vulnerability to systemic inflammation in a subset of patients and thus contribute to disease heterogeneity.
•A method for 33 PFASs was developed in liver using LC-Q-Orbitrap.•Satisfactory performance characteristics were obtained.•LOQs were generally lower than 100 pg g−1.•Method was employed for the ...analysis of farm animal liver samples.•Chicken was the less contaminated species.
An analytical method for the quantification of thirty-three perfluoroalkyl and polyfluoroalkyl substances (PFASs) in animal liver was developed applying the isotopic dilution methodology with twenty-one labelled isotopologues of native compounds. The proposed protocol involved the determination of short and long aliphatic chain PFASs (C4C18) extracting liver with acetonitrile followed by two clean-up steps. The instrumental analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. The acquisition method combined full MS/dd-MS2, t-SIM/dd-MS2 and SIM experiments with variable resolution in order to maximize in one chromatographic run accuracy, sensitivity and selectivity. An eight-level validation study was performed evaluating linearity, trueness, precision, quantification and detection limits. Trueness was from 94 to 126% with intra-laboratory reproducibility lower than 20%. Limits of quantification were in the range 2–100 pg g−1, except for 2,3,3,3-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)-propanoic acid, HFPO-DA (500 pg g−1). The analysis of a certified reference material (IRMM-427) and participation in a proficiency test scheme (FAPAS – 0687) confirmed these satisfactory performances. Finally, the application of the developed procedure to detect PFASs in sixteen liver samples of farm animals revealed that chicken was the less contaminated species.
Inonotus hispidus (Bull.) P. Karst. has been used as traditional medicine for the treatment of dyspepsia, cancer, and diabetes. Numerous studies have confirmed the antimicrobial, antiviral, ...antioxidant, anti-inflammatory, immunomodulatory, antiproliferative and cytotoxic biological activities of extracts from this species. The purpose of this study was a comparative analysis of the antioxidant and the antimicrobial activities of methanol extracts from fruit and liquid-cultured mycelia. Four compounds (N-butylbenzenesulfonamide, lauramidopropyl betaine, 3,5-di-tert-butyl-4-hydroxybenzaldehyde, and uplandicine), determined by hybrid HRMS, were found only in mycelia culture extracts. Free radical scavenging, measured by DPPH assay on methanol extracts, showed an activity of about 17.2% and 22.1% of Trolox in fruiting bodies and mycelia, respectively. The I. hispidus methanol extracts from fruit and mycelia culture were found to have varying degrees of antibacterial and antifungal effects against the pathogenic microorganisms tested (minimum inhibitory concentration from 0.17 to 2.56 μg mL
−1
).
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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