Publications by authors named "Volker Siffrin"

42 Publications

Epigallocatechin Gallate in Relapsing-Remitting Multiple Sclerosis: A Randomized, Placebo-Controlled Trial.

Neurol Neuroimmunol Neuroinflamm 2021 05 24;8(3). Epub 2021 Mar 24.

From the NeuroCure Clinical Research Center (J.B.-S., F.P., J.D., A.B., V.S.), Charité-Universitätsmedizin Berlin; Medical Image Analysis Center (J.W.), University Basel; Institut for Medical Immunology (C.I.-D., E.H.), Charité-Universitätsmedizin Berlin; Department of Neurology and Neuroimaging Center (B.K.), Johannes Gutenberg University, Mainz; Charité-Universitätsmedizin Berlin (C.P.); NeuroCure Clinical Research Center (H.R., R.R.), Charité-Universitätsmedizin Berlin, Germany; Department of Neurology (O.A.), Medical Faculty, Heinrich Heine University Düsseldorf; Institut für Neuroimmunologie und Multiple Sklerose (C.H.), Universitätsklinikum Hamburg-Eppendorf, Hamburg; Klinik für Neurologie (J.F.), Asklepios Klinik Lübben/Teupitz; Department of Neurology (F.H.), Krankenhaus Martha-Maria Halle-Dölau, Halle/Saale; Medizinische Klinik für Kardiologie und Angiologie (M.L.), Campus Mitte, Charité-Universitätsmedizin Berlin; Institute of Nutritional and Food Sciences (B.Z.), University of Bonn; Department of Neurology and Neuroimaging Center (NIC) (S.G., F.Z.), Focus Program Translational Neuroscience (FTN), University Medical Center of the Johannes Gutenberg University, Mainz; and Charité-Universitätsmedizin Berlin and SOSTANA GmbH (K.-D.W.), Berlin.

Objective: To assess the safety and efficacy of epigallocatechin-3-gallate (EGCG) add-on to glatiramer acetate (GA) in patients with relapsing-remitting multiple sclerosis (RRMS).

Methods: We enrolled patients with RRMS (aged 18-60 years, Expanded Disability Status Scale [EDSS] score 0-6.5), receiving stable GA treatment in a multicenter, prospective, double-blind, phase II, randomized controlled trial. Participants received up to 800 mg oral EGCG daily over a period of 18 months. The primary outcome was the proportion of patients without new hyperintense lesions on T2-weighted (T2w) brain MRI within 18 months. Secondary end points included additional MRI and clinical parameters. Immunologic effects of EGCG were investigated in exploratory experiments.

Results: A total of 122 patients on GA were randomly assigned to EGCG treatment (n = 62) or placebo (n = 60). We could not demonstrate a difference between groups after 18 months for the primary outcome or other radiologic (T2w lesion volume, T1w hypointense lesion number or volume, number of cumulative contrast-enhancing lesions, percent brain volume change), or clinical (EDSS, MS functional composite, and annualized relapse rate) parameter. EGCG treatment did not affect immune response to GA. Pharmacologic analysis revealed wide ranging EGCG plasma levels. The treatment was well tolerated with a similar incidence of mostly mild adverse events similar in both groups.

Conclusion: In RRMS, oral EGCG add-on to GA was not superior to placebo in influencing MRI and clinical disease activity over 18 months. The treatment was safe at a daily dosage up to 800 mg EGCG. It did not influence immune parameters, despite indication of EGCG being bioavailable in patients.

Classification Of Evidence: This study provides Class II evidence that for patients with RRMS, EGCG added to GA did not significantly affect the development of new hyperintense lesions on T2-weighted brain MRI.

Trial Registration Information: Clinical trial registration number: NCT00525668.
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http://dx.doi.org/10.1212/NXI.0000000000000981DOI Listing
May 2021

Neutrophil Gelatinase-Associated Lipocalin Protects from ANCA-Induced GN by Inhibiting T17 Immunity.

J Am Soc Nephrol 2020 07 2;31(7):1569-1584. Epub 2020 Jun 2.

Experimental and Clinical Research Center, Max Delbrück Center for Molecular Medicine and Charité - Berlin University of Medicine, Corporate Member of Free University of Berlin, Humboldt University of Berlin, Berlin Institute of Health, Berlin, Germany.

Background: Neutrophil gelatinase-associated lipocalin (NGAL) is a diagnostic marker of intrinsic kidney injury produced by damaged renal cells and by neutrophils. ANCA-associated vasculitis features necrotizing crescentic GN (NCGN), and ANCA-activated neutrophils contribute to NCGN. Whether NGAL plays a mechanistic role in ANCA-associated vasculitis is unknown.

Methods: We measured NGAL in patients with ANCA-associated vasculitis and mice with anti-myeloperoxidase (anti-MPO) antibody-induced NCGN. We compared kidney histology, neutrophil functions, T cell proliferation and polarization, renal infiltrating cells, and cytokines in wild-type and NGAL-deficient chimeric mice with anti-MPO antibody-induced NCGN. To assess the role of T17 immunity, we transplanted irradiated MPO-immunized MPO-deficient mice with bone marrow from either wild-type or NGAL-deficient mice; we also transplanted irradiated MPO-immunized MPO/IL-17A double-deficient mice with bone marrow from either IL-17A-deficient or NGAL/IL-17A double-deficient mice.

Results: Mice and patients with active ANCA-associated vasculitis demonstrated strongly increased serum and urinary NGAL levels. ANCA-stimulated neutrophils released NGAL. Mice with NGAL-deficient bone marrow developed worsened MPO-ANCA-induced NCGN. Intrinsic neutrophil functions were similar in NGAL-deficient and wild-type neutrophils, whereas T cell immunity was increased in chimeric mice with NGAL-deficient neutrophils with more renal infiltrating T17 cells. NGAL-expressing neutrophils and CD3 T cells were in close proximity in kidney and spleen. CD4 T cells showed no intrinsic difference in proliferation and polarization , whereas iron siderophore-loaded NGAL suppressed T17 polarization. We found significantly attenuated NCGN in IL-17A-deficient chimeras compared with MPO-deficient mice receiving wild-type bone marrow, as well as in NGAL/IL-17A-deficient chimeras compared with NGAL-deficient chimeras.

Conclusions: Our findings support that bone marrow-derived, presumably neutrophil, NGAL protects from ANCA-induced NCGN by downregulating T17 immunity.
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http://dx.doi.org/10.1681/ASN.2019090879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350985PMC
July 2020

Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes.

J Neurosci Methods 2020 03 9;333:108545. Epub 2019 Dec 9.

Experimental and Clinical Research Center (ECRC), Charité - Universitätsmedizin Berlin und Max Delbrück Center or Molecular Medicine in the Helmholtz Association, Berlin Germany. Electronic address:

Background: Molecular analyses of cell populations and single cells have been instrumental in the advancement of our understanding of the physiology and pathologic processes of the nervous system. However, the limitation of these methods is the dependence on a gentle, efficient and specific enrichment procedure for the target cell population. In particular, this has been challenging for tightly interconnected cells, for example central nervous system (CNS) endogenous cells such as astrocytes.

New Method: Here we adopted one of the most common methods of cell extraction, namely, enzymatic tissue digestion followed by fluorescence-activated cell sorting (FACS) of individual cells. We evaluated different enzymatic/mechanical tissue dissociation procedures and analyzed different astrocyte lineage transgenic models. Furthermore, we compared the cell extraction efficiency from spinal cord vs. brain.

Results: Enzymatic digestion of CNS tissue of Glast-Crex tdTomato or Aldh1l1-Crex tdTomato followed by FACS resulted in highly purified astrocytes. Automated tissue digestion strongly improved the isolated cell numbers. Aldh1l1-Cre identified more astrocytes than Glast-Cre; isolation from brain yields higher numbers than from spinal cord.

Comparison With Existing Methods: We compared the efficiency and purity of the enzymatic dissociation/FACS approach with a more modern procedure consisting of tissue homogenization followed by translating ribosome affinity purification (TRAP).

Conclusion: We found that both methods result in highly enriched astrocytic RNA. However, only TRAP isolation resulted in reliably detectable RNA concentrations from spinal cord tissue on a single animal level. Depending on the aim of the study both methods have advantages and disadvantages but both are acceptable for astrocytic RNA analysis.
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http://dx.doi.org/10.1016/j.jneumeth.2019.108545DOI Listing
March 2020

Disease Modification in Multiple Sclerosis by Flupirtine-Results of a Randomized Placebo Controlled Phase II Trial.

Front Neurol 2018 9;9:842. Epub 2018 Oct 9.

NeuroCure Clinical Research Center, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Berlin, Germany.

Central nervous system inflammation and neurodegeneration are the pathophysiological hallmarks of multiple sclerosis (MS). While inflammation can readily be targeted by current disease modifying drugs, neurodegeneration is by far less accessible to treatment. Based on suggested additional neuroprotective capacities of the orally available non-opioid and centrally acting analgesic drug flupirtine maleate we hypothesized that treatment with flupirtine maleate might be beneficial in MS patients. The upirtine as al treatment n ultiple clerosis (FLORIMS) study was a multi-center, randomized and stratified, placebo-controlled double-blind phase II trial to investigate safety and efficacy in terms of clinical and radiographical activity of flupirtine maleate (300 mg per day) given orally for 12 months, add-on to interferon beta 1b subcutaneously in patients with relapsing remitting MS. Due to a substantial delay in recruitment, enrolment of patients was prematurely terminated after randomization of only 30 of the originally planned 80 patients. Of these, 24 regularly terminated study after 12 months of treatment. Data were analyzed as originally planned. Treatment with flupirtine maleate was overall well tolerated. We observed moderate and asymptomatic elevations of liver enzymes in several cases but no overt hepatotoxicity. Neither the intention to treat nor the per protocol analysis revealed any significant treatment effects of flupirtine maleate with respect to occurrence of MS relapses, disability progression, or development of new lesions on cranial MRI. However, substantial methodological limitations need to be considered when interpreting these results. In conclusion, the results of the FLORIMS study neither add further evidence to nor argue against the hypothesized neuroprotective or disease modifying effects of flupirtine maleate in MS.
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http://dx.doi.org/10.3389/fneur.2018.00842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190842PMC
October 2018

CCR7 on CD4 T Cells Plays a Crucial Role in the Induction of Experimental Autoimmune Encephalomyelitis.

J Immunol 2018 04 16;200(8):2554-2562. Epub 2018 Mar 16.

Department of Neurology, Focus Program Translational Neuroscience and Immunotherapy, Rhine Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany.

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the CNS. Myelin-specific CD4 Th lymphocytes are known to play a major role in both MS and its animal model experimental autoimmune encephalomyelitis (EAE). CCR7 is a critical element for immune cell trafficking and recirculation, that is, lymph node homing, under homeostatic conditions; blocking CCR7 central memory cells from egress of lymph nodes is a therapeutic approach in MS. To define the effect of CD4 T cell-specific constitutive deletion of CCR7 in the priming and effector phase in EAE, we used an active EAE approach in T cell reconstituted Rag1 mice, as well as adoptive transfer EAE, in which mice received in vitro-primed CCR7 or CCR7 myelin Ag TCR-transgenic 2d2 Th17 cells. Two-photon laser scanning microscopy was applied in living anesthetized mice to monitor the trafficking of CCR7-deficient and wild-type CD4 T cells in inflammatory lesions within the CNS. We demonstrate that CD4 T cell-specific constitutive deletion of CCR7 led to impaired induction of active EAE. In adoptive transfer EAE, mice receiving in vitro-primed CCR7 2d2 Th17 cells showed similar disease onset as mice adoptively transferred with CCR7 2d2 Th17 cells. Using two-photon laser scanning microscopy CCR7 and CCR7 CD4 T cells did not reveal differences in motility in either animal model of MS. These findings indicate a crucial role of CCR7 in neuroinflammation during the priming of autoimmune CD4 T cells but not in the CNS.
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http://dx.doi.org/10.4049/jimmunol.1701419DOI Listing
April 2018

Phenotype of Antigen Unexperienced T Cells in the Inflamed Central Nervous System in Experimental Autoimmune Encephalomyelitis.

J Neuroimmune Pharmacol 2017 06 10;12(2):305-313. Epub 2016 Nov 10.

Department of Neurology, Focus Program Translational Neuroscience (FTN) and Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn2), University Medical Center of the Johannes Gutenberg University, Langenbeckstrasse 1, Building 708, 55131, Mainz, Germany.

Multiple sclerosis is a chronic, disseminated inflammation of the central nervous system which is thought to be driven by autoimmune T cells. Genetic association studies in multiple sclerosis and a large number of studies in the animal model of the disease support a role for effector/memory T helper cells. However, the mechanisms underlying relapses, remission and chronic progression in multiple sclerosis or the animal model experimental autoimmune encephalomyelitis, are not clear. In particular, there is only scarce information on the role of central nervous system-invading naive T helper cells in these processes. By applying two-photon laser scanning microscopy we could show in vivo that antigen unexperienced T helper cells migrated into the deep parenchyma of the inflamed central nervous system in experimental autoimmune encephalomyelitis, independent of their antigen specificity. Using flow cytometric analyses of central nervous system-derived lymphocytes we found that only antigen-specific, formerly naive T helper cells became activated during inflammation of the central nervous system encountering their corresponding antigen.
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http://dx.doi.org/10.1007/s11481-016-9718-1DOI Listing
June 2017

Dendritic cells tip the balance towards induction of regulatory T cells upon priming in experimental autoimmune encephalomyelitis.

J Autoimmun 2017 01 2;76:108-114. Epub 2016 Oct 2.

Department of Neurology, Focus Program Translational Neuroscience (FTN) and Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn(2)), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany. Electronic address:

Counter-balancing regulatory mechanisms, such as the induction of regulatory T cells (Treg), limit the effects of autoimmune attack in neuroinflammation. However, the role of dendritic cells (DCs) as the most powerful antigen-presenting cells, which are intriguing therapeutic targets in this context, is not fully understood. Here, we demonstrate that conditional ablation of DCs during the priming phase of myelin-specific T cells in experimental autoimmune encephalomyelitis (EAE) selectively aborts inducible Treg (iTreg) induction, whereas generation of T helper (Th)1/17 cells is unaltered. DCs facilitate iTreg induction by creating a milieu with high levels of interleukin (IL)-2 due to a strong proliferative response. In the absence of DCs, B220 B cells take over priming of Th17 cells in the place of antigen-presenting cells (APCs), but not the induction of iTreg, thus leading to unregulated, severe autoimmunity.
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http://dx.doi.org/10.1016/j.jaut.2016.09.008DOI Listing
January 2017

Role of IL-17-producing lymphocytes in severity of multiple sclerosis upon natalizumab treatment.

Mult Scler 2017 Apr 21;23(4):567-576. Epub 2016 Jul 21.

Focus Program Translational Neurosciences (FTN), Rhine-Main Neuroscience Network (rmn2), Department of Neurology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.

Objective: Natalizumab is known to prevent T-helper cells entering the central nervous system (CNS). We hypothesize that more pathogenic T-helper cells are present outside the CNS and a possible relationship to disease severity.

Methods: Characterization and enrichment of human CD4+IL-17+ cells were performed ex vivo using peripheral blood mononuclear cells from natalizumab-treated relapsing-remitting multiple sclerosis (RRMS) patients ( n = 33), untreated RRMS patients ( n = 13), and healthy controls ( n = 33). Magnetic resonance imaging (MRI) scans were performed routinely for patients.

Results: Lymphocytes were elevated in peripheral blood of natalizumab-treated patients compared to untreated patients and healthy controls. Whereas group comparison for CD4+IL-17+ numbers also differed, CD4+IFN-γ+ and CD4+IL-22+ counts were not increased. CD4+IL-17+ cells not only expressed but also secreted IL-17. In natalizumab-treated patients, IL-17+ cell frequency was found to correlate with T1-hypointense lesions, but was not an indicator for rebound activity after treatment discontinuation, except in one patient who experienced a fulminant rebound, and interestingly, in whom the highest IL-17+ cell levels were observed.

Conclusion: Increased lymphocytes and CD4+IL-17+ cells in the blood of RRMS patients receiving natalizumab corroborate the drug's mechanism of action, that is, blocking transmigration to CNS. Correlation between IL-17-expressing lymphocytes and T1-hypointense lesions underlines the important role of these cells in the disease pathology.
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http://dx.doi.org/10.1177/1352458516658559DOI Listing
April 2017

In vivo and in vitro effects of multiple sclerosis immunomodulatory therapeutics on glutamatergic excitotoxicity.

J Neurochem 2016 Mar 11;136(5):971-80. Epub 2016 Jan 11.

Department of Neurology, Focus Program Translational Neuroscience (FTN), Rhine Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.

In multiple sclerosis (MS), a candidate downstream mechanism for neuronal injury is glutamate (Glu)-induced excitotoxicity, leading to toxic increases in intraneuronal Ca(2+) . Here, we used in vivo two-photon imaging in the brain of TN-XXL transgenic Ca(2+) reporter mice to test whether promising oral MS therapeutics, namely fingolimod, dimethyl fumarate, and their respective metabolites fingolimod-phosphate and monomethyl fumarate, can protect neurons against acute glutamatergic excitotoxic damage. We also assessed whether these drugs can protect against excitotoxicity in vitro using primary cortical neurons, and whether they can directly inhibit Glu release from pathogenic T-helper 17 lymphocytes. In vivo, direct and acute (1 h) administration of 100 mM Glu to the brainstem resulted in a rapid and significant up-regulation in neuronal Ca(2+) signaling as well as morphological excitotoxic changes that were attenuated by the NMDA-receptor antagonist MK801. Direct CNS administration of MS drugs prior to Glu significantly delayed or reduced, but did not prevent the neuronal Ca(2+) increase or morphological changes. In vitro, prolonged (24 h) treatment of primary neurons with the fumarates significantly protected against neurotoxicity induced by Glu as well as NMDA, similar to MK801. Furthermore, monomethyl fumerate significantly reduced Glu release from pathogenic T-helper 17 lymphocytes. Overall, these data suggest that MS drugs may mediate neuroprotection via excitotoxicity modulating effects. Evidence suggests MS pathogenesis may involve neuronal excitotoxicity, induced by local release of glutamate. However, current MS drugs, including dimethyl fumerate (DMF) and fingolimod (FTY720) are largely anti-inflammatory and not yet fully tested for their neuroprotective potential. Here, we show that the drugs, in particular DMF metabolite monomethyl fumerate (MMF), protect neurons by excitotoxicity modulating effects. Th17, T-helper 17.
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http://dx.doi.org/10.1111/jnc.13456DOI Listing
March 2016

Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation.

EMBO J 2016 Jan 26;35(1):89-101. Epub 2015 Nov 26.

Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²) University Medical Center of the Johannes Gutenberg University, Mainz, Germany

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c(+) cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c(+) cells. These CD11c(+) cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c(+) cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c(+) cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS.
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http://dx.doi.org/10.15252/embj.201591488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718005PMC
January 2016

Role of Sortilin in Models of Autoimmune Neuroinflammation.

J Immunol 2015 Dec 13;195(12):5762-9. Epub 2015 Nov 13.

Department of Neurology, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany;

The proneurotrophin receptor sortilin is a protein with dual functions, being involved in intracellular protein transport, as well as cellular signal transduction. The relevance of the receptor for various neuronal disorders, such as dementia, seizures, and brain injury, is well established. In contrast, little is known about the role of sortilin in immune cells and inflammatory diseases. The aim of our study was to elucidate the distribution of sortilin in different immune cell types in mice and humans and to analyze its function in autoimmune CNS inflammation. Sortilin was expressed most profoundly in murine and human macrophages and dendritic cells and to a much lesser extent in B and T cells. In dendritic cells, sortilin had an impact on Ag processing. Accordingly, sortilin was highly expressed by infiltrated perivascular myeloid cells, mainly in vessel cuffs, in the CNS of patients suffering from multiple sclerosis, the most common inflammatory autoimmune disease of the CNS. Yet, sortilin gene-targeted mice (Sort1(-/-)) and chimeras deficient in sortilin in the immune system were as susceptible as wild-type littermates to T cell-dependent experimental autoimmune encephalomyelitis. Considering our results and recent data from other investigators, we conclude that the proneurotrophin receptor sortilin plays a role in innate, rather than in adaptive, immune processes and, thus, not in autoimmune neuroinflammation.
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http://dx.doi.org/10.4049/jimmunol.1403156DOI Listing
December 2015

PML risk stratification using anti-JCV antibody index and L-selectin.

Mult Scler 2016 07 2;22(8):1048-60. Epub 2015 Oct 2.

Department of Neurology, University of Münster, Germany.

Background: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters.

Objective: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification.

Methods: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients).

Results: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor "CD62L low" increasing a patient's relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group.

Conclusions: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence.
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http://dx.doi.org/10.1177/1352458515607651DOI Listing
July 2016

Flow cytometric analysis of T cell/monocyte ratio in clinically isolated syndrome identifies patients at risk of rapid disease progression.

Mult Scler 2016 Apr 10;22(4):483-93. Epub 2015 Jul 10.

Neurology Department, Johannes Gutenberg University Mainz, Germany/Charité - Universitätsmedizin Berlin, Germany

Background: Multiple sclerosis is a chronic inflammatory central nervous system disease diagnosed by clinical presentation and characteristic magnetic resonance imaging findings. The role of cerebrospinal fluid (CSF) analysis has been emphasized in particular in the context of differential diagnosis in patients with a first episode suggestive of multiple sclerosis.

Objective: We investigated here the potential additional value of analysis of CSF cellularity by fluorescence activated cell sorting (FACS) in the setting of a routine diagnostic work-up in our inpatient clinic.

Methods: CSF cells from back-up samples from patients with suspected chronic inflammatory central nervous system disorder were analyzed by FACS and correlated with clinical data, magnetic resonance imaging findings and oligoclonal band status.

Results: We found distinct changes of T cell/monocyte (CD4/CD14) and B cell/monocyte (CD20/CD14) ratios between clinically isolated syndrome (CIS)/multiple sclerosis and other neurologic diseases or other inflammatory neurologic diseases. In particular, patients with a rapid transition from CIS to multiple sclerosis had an elevated CD4/CD14 ratio. A subgroup analysis showed diagnostic value of CD4/CD8 ratio in the differential diagnosis of CIS/multiple sclerosis to neurosarcoidosis.

Conclusion: The diagnostic and prognostic accuracy of autoimmune neuroinflammatory diseases can be improved by FACS analysis of CSF cells.
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http://dx.doi.org/10.1177/1352458515593821DOI Listing
April 2016

The impact of isolated lesions on white-matter fiber tracts in multiple sclerosis patients.

Neuroimage Clin 2015 10;8:110-6. Epub 2015 Apr 10.

Department of Neurology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany ; Neuroimage Center (NIC) of the Focus Program Translational Neuroscience (FTN), Johannes Gutenberg University, Mainz, Germany.

Infratentorial lesions have been assigned an equivalent weighting to supratentorial plaques in the new McDonald criteria for diagnosing multiple sclerosis. Moreover, their presence has been shown to have prognostic value for disability. However, their spatial distribution and impact on network damage is not well understood. As a preliminary step in this study, we mapped the overall infratentorial lesion pattern in relapsing-remitting multiple sclerosis patients (N = 317) using MRI, finding the pons (lesion density, 14.25/cm(3)) and peduncles (13.38/cm(3)) to be predilection sites for infratentorial lesions. Based on these results, 118 fiber bundles from 15 healthy controls and a subgroup of 23 patients showing lesions unilaterally at the predilection sites were compared using diffusion tensor imaging to analyze the impact of an isolated infratentorial lesion on the affected fiber tracts. Fractional anisotropy, mean diffusion as well as axial and radial diffusivity were investigated at the lesion site and along the entire fiber tract. Infratentorial lesions were found to have an impact on the fractional anisotropy and radial diffusivity not only at the lesion site itself but also along the entire affected fiber tract. As previously found in animal experiments, inflammatory attack in the posterior fossa in multiple sclerosis impacts the whole affected fiber tract. Here, this damaging effect, reflected by changes in diffusivity measures, was detected in vivo in multiple sclerosis patients in early stages of the disease, thus demonstrating the influence of a focal immune attack on more distant networks, and emphasizing the pathophysiological role of Wallerian degeneration in multiple sclerosis.
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http://dx.doi.org/10.1016/j.nicl.2015.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473264PMC
March 2016

FRET based ratiometric Ca(2+) imaging to investigate immune-mediated neuronal and axonal damage processes in experimental autoimmune encephalomyelitis.

J Neurosci Methods 2015 Jul 10;249:8-15. Epub 2015 Apr 10.

Neurology Department, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany.

Background: Irreversible axonal and neuronal damage are the correlate of disability in patients suffering from multiple sclerosis (MS). A sustained increase of cytoplasmic free [Ca(2+)] is a common upstream event of many neuronal and axonal damage processes and could represent an early and potentially reversible step.

New Method: We propose a method to specifically analyze the neurodegenerative aspects of experimental autoimmune encephalomyelitis by Förster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca(2+) dynamics by two-photon laser scanning microscopy (TPLSM).

Results: Using the genetically encoded Ca(2+) sensor TN-XXL expressed in neurons and their corresponding axons, we confirm the increase of cytoplasmic free [Ca(2+)] in axons and neurons of autoimmune inflammatory lesions compared to those in non-inflamed brains. We show that these relative [Ca(2+)] increases were associated with immune-neuronal interactions.

Comparison With Existing Methods: In contrast to Ca(2+)-sensitive dyes the use of a genetically encoded Ca(2+) sensor allows reliable intraaxonal free [Ca(2+)] measurements in living anesthetized mice in health and disease. This method detects early axonal damage processes in contrast to e.g. cell/axon morphology analysis, that rather detects late signs of neurodegeneration.

Conclusions: Thus, we describe a method to analyze and monitor early neuronal damage processes in the brain in vivo.
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http://dx.doi.org/10.1016/j.jneumeth.2015.04.005DOI Listing
July 2015

Cross-recognition of a myelin peptide by CD8+ T cells in the CNS is not sufficient to promote neuronal damage.

J Neurosci 2015 Mar;35(12):4837-50

Department of Neurology, Research Center Translational Neurosciences, Rhine Main Neuroscience Network (rmn), and Molecular Neurology Group, Max Delbrueck Center for Molecular Medicine Berlin-Buch, 13125 Berlin, Germany, Charité University Medicine Berlin, ECRC, 13125 Berlin, Germany, and

Multiple sclerosis (MS) is an inflammatory disease of the CNS thought to be driven by CNS-specific T lymphocytes. Although CD8(+) T cells are frequently found in multiple sclerosis lesions, their distinct role remains controversial because direct signs of cytotoxicity have not been confirmed in vivo. In the present work, we determined that murine ovalbumin-transgenic (OT-1) CD8(+) T cells recognize the myelin peptide myelin oligodendrocyte glycoprotein 40-54 (MOG40-54) both in vitro and in vivo. The aim of this study was to investigate whether such cross-recognizing CD8(+) T cells are capable of inducing CNS damage in vivo. Using intravital two-photon microscopy in the mouse model of multiple sclerosis, we detected antigen recognition motility of the OT-1 CD8(+) T cells within the CNS leading to a selective enrichment in inflammatory lesions. However, this cross-reactivity of OT-1 CD8(+) T cells with MOG peptide in the CNS did not result in clinically or subclinically significant damage, which is different from myelin-specific CD4(+) Th17-mediated autoimmune pathology. Therefore, intravital imaging demonstrates that local myelin recognition by autoreactive CD8(+) T cells in inflammatory CNS lesions alone is not sufficient to induce disability or increase axonal injury.
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http://dx.doi.org/10.1523/JNEUROSCI.3380-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705380PMC
March 2015

New candidates for CD4 T cell pathogenicity in experimental neuroinflammation and multiple sclerosis.

Brain 2015 Apr 9;138(Pt 4):902-17. Epub 2015 Feb 9.

1 Department of Neurology, Focus Program Translational Neuroscience (FTN) and Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn), University Medical Centre of the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany

Multiple sclerosis is a chronic autoimmune demyelinating disease of the central nervous system, which is thought to be triggered by environmental factors in genetically susceptible individuals leading to activation of autoreactive T lymphocytes. Large multi-centre genome-wide association studies have identified multiple genetic risk loci in multiple sclerosis. In this study, we investigated T cell transcriptomic changes in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We correlated these findings with the multiple sclerosis risk genes postulated by the most recent Immunochip analysis and found that multiple sclerosis susceptibility genes were significantly regulated in experimental autoimmune encephalomyelitis. Our data indicate that nine distinct genes associated with multiple sclerosis risk, Bach2, Il2ra, Irf8, Mertk, Odf3b, Plek, Rgs1, Slc30a7 and Thada, can be confirmed to be differentially regulated in pathogenic CD4(+) T cells. During the effector phase within the inflamed CNS, CD4(+) T cells undergo comprehensive transformation and we identified key transcription factors and signalling networks involved in this process. The transformation was linked to metabolic changes with the involvement of liver X receptor/retinoid X receptor signalling and cholesterol biosynthesis, which might control the T cell effector function in the central nervous system. Thus, our study confirms the involvement of multiple sclerosis risk genes in the pathophysiology of the animal model and sheds light on additional disease-relevant inflammatory networks.
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http://dx.doi.org/10.1093/brain/awu408DOI Listing
April 2015

MHCII-independent CD4+ T cells protect injured CNS neurons via IL-4.

J Clin Invest 2015 Feb 20;125(2):699-714. Epub 2015 Jan 20.

A body of experimental evidence suggests that T cells mediate neuroprotection following CNS injury; however, the antigen specificity of these T cells and how they mediate neuroprotection are unknown. Here, we have provided evidence that T cell-mediated neuroprotection after CNS injury can occur independently of major histocompatibility class II (MHCII) signaling to T cell receptors (TCRs). Using two murine models of CNS injury, we determined that damage-associated molecular mediators that originate from injured CNS tissue induce a population of neuroprotective, IL-4-producing T cells in an antigen-independent fashion. Compared with wild-type mice, IL-4-deficient animals had decreased functional recovery following CNS injury; however, transfer of CD4+ T cells from wild-type mice, but not from IL-4-deficient mice, enhanced neuronal survival. Using a culture-based system, we determined that T cell-derived IL-4 protects and induces recovery of injured neurons by activation of neuronal IL-4 receptors, which potentiated neurotrophin signaling via the AKT and MAPK pathways. Together, these findings demonstrate that damage-associated molecules from the injured CNS induce a neuroprotective T cell response that is independent of MHCII/TCR interactions and is MyD88 dependent. Moreover, our results indicate that IL-4 mediates neuroprotection and recovery of the injured CNS and suggest that strategies to enhance IL-4-producing CD4+ T cells have potential to attenuate axonal damage in the course of CNS injury in trauma, inflammation, or neurodegeneration.
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http://dx.doi.org/10.1172/JCI76210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319416PMC
February 2015

Cardiotoxicity of mitoxantrone treatment in a german cohort of 639 multiple sclerosis patients.

J Clin Neurol 2014 Oct 6;10(4):289-95. Epub 2014 Oct 6.

Department of Neurology, Focus Program Translational Neuroscience, Rhine Main Neuroscience Network, University Medical Center, Johannes Gutenberg University of Mainz, Mainz, Germany.

Background And Purpose: The aim of this study was to elucidate the role of therapy-related cardiotoxicity in multiple sclerosis (MS) patients treated with mitoxantrone and to identify potential predictors for individual risk assessment.

Methods: Within a multicenter retrospective cohort design, cardiac side effects attributed to mitoxantrone were analyzed in 639 MS patients at 2 MS centers in Germany. Demographic, disease, treatment, and follow-up data were collected from hospital records. Patients regularly received cardiac monitoring during the treatment phase.

Results: None of the patients developed symptomatic congestive heart failure. However, the frequency of patients experiencing cardiac dysfunction of milder forms after mitoxantrone therapy was 4.1% (26 patients) among all patients. Analyses of the risk for cardiotoxicity revealed that cumulative dose exposure was the only statistically relevant risk factor associated with cardiac dysfunction.

Conclusions: The number of patients developing subclinical cardiac dysfunction below the maximum recommended cumulative dose is higher than was initially assumed. Interestingly, a subgroup of patients was identified who experienced cardiac dysfunction shortly after initiation of mitoxantrone and who received a low cumulative dose. Therefore, each administration of mitoxantrone should include monitoring of cardiac function to enhance the treatment safety for patients and to allow for early detection of any side effects, especially in potential high-risk subgroups (as determined genetically).
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http://dx.doi.org/10.3988/jcn.2014.10.4.289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198709PMC
October 2014

Modulation of dendritic cell immunobiology via inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase.

PLoS One 2014 11;9(7):e100871. Epub 2014 Jul 11.

Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine, Berlin, Germany.

The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100871PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094470PMC
March 2015

Experimental applications of TNF-reporter mice with far-red fluorescent label.

Methods Mol Biol 2014 ;1155:151-62

German Rheumatism Research Center, a Leibniz Institute, Berlin, Germany.

This chapter provides protocols for in vitro and in vivo analysis of TNF-producing cells from a novel TNF reporter mouse. In these transgenic mice, genetic sequence encoding far-red reporter protein Katyushka (FRFPK) was placed under control of the same regulatory elements as TNF, thus providing the basis for detection, isolation, and visualization of TNF-producing cells.
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http://dx.doi.org/10.1007/978-1-4939-0669-7_13DOI Listing
December 2014

How to treat tumefactive demyelinating disease?

Mult Scler 2014 Apr 17;20(5):631-3. Epub 2013 Dec 17.

Department of Neurology, Johannes Gutenberg University Mainz, Germany.

Glioma-like inflammatory demyelinating lesions can be found in patients with pre-diagnosed multiple sclerosis, but they have also been described as an isolated disease entity. The initial diagnostic work-up usually includes a biopsy for histopathological analysis. However, even after unambiguous histopathologic classification, tumefactive lesions pose a therapeutic challenge. Until now, there have been no guidelines on how to treat patients with these rare and extreme lesion phenotypes. Here we report a patient with a relapsing unifocal tumefactive demyelinating lesion. The patient initially showed a good response to steroid treatment, with full clinical recovery. However, after relapse of the same lesion, recovery was incomplete. Although immunosuppression was initiated, the patient presented with subsequent further deterioration. Only maximal escalation of immunosuppression was able to stop the inflammatory activity. Due to the length of time of the step-wise escalation treatment however, the lengthy lesion activity led to irreversible tissue destruction and residual non-remitting disability. Early aggressive treatment with an induction therapy regimen might be more appropriate for these rare and often strongly disabling lesion subtypes.
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http://dx.doi.org/10.1177/1352458513516891DOI Listing
April 2014

Membrane-type 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases.

J Neurosci Res 2014 Mar 9;92(3):275-86. Epub 2013 Dec 9.

Cellular Neurosciences, Max Delbrück Centre for Molecular Medicine, Berlin, Germany; Department of Neurology, Charité, Universitätsmedizin Berlin, Charité Campus Virchow, Berlin, Germany.

We previously reported that glioma cells induce the expression of membrane-type 1 metalloproteinase (MT1-MMP or MMP-14) in tumor-associated microglia/macrophages and promote tumor growth, whereas MMP-14 expression in microglia under physiological conditions is very low. Here, we show that the increase in MMP-14 expression is also found in microglia/macrophages associated with neurodegenerative and neuroinflammatory pathologies in mouse models as well as in human biopsies or post-mortem tissue. We found that microglial/macrophage MMP-14 expression was upregulated in Alzheimer's disease tissue, in active lesions of multiple sclerosis, and in tissue from stage II stroke as well as in the corresponding mouse models for the human diseases. In contrast, we observed no upregulation for MMP-14 in microglia/macrophages in the early phase of stroke or in the corresponding mouse model, in human amyotrophic lateral sclerosis (ALS) tissue or in a mouse model of ALS as well as in human cases of acute brain trauma. These data indicate that MMP-14 expression is not a general marker for activated microglia/macrophages but is upregulated in defined stages of neuroinflammatory and neurodegenerative diseases and that there is generally a good match between mouse models and human brain pathologies.
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http://dx.doi.org/10.1002/jnr.23288DOI Listing
March 2014

Microglial activation milieu controls regulatory T cell responses.

J Immunol 2013 Dec 21;191(11):5594-602. Epub 2013 Oct 21.

Institute for Cell Biology and Neurobiology, Charité-University Medicine Berlin, 10117 Berlin, Germany;

Although mechanisms leading to brain-specific inflammation and T cell activation have been widely investigated, regulatory mechanisms of local innate immune cells in the brain are only poorly understood. In this study, to our knowledge we show for the first time that MHC class II(+)CD40(dim)CD86(dim)IL-10(+) microglia are potent inducers of Ag-specific CD4(+)Foxp3(+) regulatory T cells (Tregs) in vitro. Microglia differentially regulated MHC class II expression, costimulatory molecules, and IL-10 depending on the amount of IFN-γ challenge and Ag dose, promoting either effector T cell or Treg induction. Microglia-induced Tregs were functionally active in vitro by inhibiting Ag-specific proliferation of effector T cells, and in vivo by attenuating experimental autoimmune encephalomyelitis disease course after adoptive transfer. These results indicate that MHC class II(+)CD40(dim)CD86(dim)IL-10(+) microglia have regulatory properties potentially influencing local immune responses in the CNS.
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http://dx.doi.org/10.4049/jimmunol.1203331DOI Listing
December 2013

The role of CD8+ T cells and their local interaction with CD4+ T cells in myelin oligodendrocyte glycoprotein35-55-induced experimental autoimmune encephalomyelitis.

J Immunol 2013 Nov 11;191(10):4960-8. Epub 2013 Oct 11.

Department of Neurology, Focus Program Translational Neurosciences, Rhine Main Neuroscience Network, Johannes Gutenberg University Mainz, 55131 Mainz, Germany;

T cells have an essential role in the induction of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Although for CD4(+) T cells it is well established that they contribute to the disease, less is known about the role of CD8(+) T cells. Our aim was to determine the individual contribution of CD4(+) and CD8(+) T cells in myelin oligodendrocyte glycoprotein (MOG)35-55-induced EAE. We investigated MOG35-55-activated CD8(+) T cells to clarify their potential to induce or attenuate EAE. We monitored the behavior of CD8(+) T cells and their interaction with CD4(+) T cells directly at the site of inflammation in the CNS using intravital imaging of the brainstem of EAE-affected living anesthetized mice. We found that mice without CD4(+) T cells did not develop relevant clinical signs of disease, although CD8(+) T cells were present in the CNS of these mice. These CD8(+) T cells displayed reduced motility compared with those in the presence of CD4(+) T cells. In mice that harbored CD4(+) and CD8(+) T cells, we saw a similar extent of clinical signs of EAE as in mice with only CD4(+) T cells. Furthermore, the dynamic motility and viability of CD4(+) T cells were not disturbed by CD8(+) T cells in the lesions of these mice. Therefore, we conclude that in MOG35-55-induced EAE, CD8(+) T cell accumulation in the CNS represents instead an epiphenomenon with no impact on clinical disease or on the effects of CD4(+) T cells, the latter being the true inducers of the disease.
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http://dx.doi.org/10.4049/jimmunol.1300822DOI Listing
November 2013

Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis.

Authors:
Ashley H Beecham Nikolaos A Patsopoulos Dionysia K Xifara Mary F Davis Anu Kemppinen Chris Cotsapas Tejas S Shah Chris Spencer David Booth An Goris Annette Oturai Janna Saarela Bertrand Fontaine Bernhard Hemmer Claes Martin Frauke Zipp Sandra D'Alfonso Filippo Martinelli-Boneschi Bruce Taylor Hanne F Harbo Ingrid Kockum Jan Hillert Tomas Olsson Maria Ban Jorge R Oksenberg Rogier Hintzen Lisa F Barcellos Cristina Agliardi Lars Alfredsson Mehdi Alizadeh Carl Anderson Robert Andrews Helle Bach Søndergaard Amie Baker Gavin Band Sergio E Baranzini Nadia Barizzone Jeffrey Barrett Céline Bellenguez Laura Bergamaschi Luisa Bernardinelli Achim Berthele Viola Biberacher Thomas M C Binder Hannah Blackburn Izaura L Bomfim Paola Brambilla Simon Broadley Bruno Brochet Lou Brundin Dorothea Buck Helmut Butzkueven Stacy J Caillier William Camu Wassila Carpentier Paola Cavalla Elisabeth G Celius Irène Coman Giancarlo Comi Lucia Corrado Leentje Cosemans Isabelle Cournu-Rebeix Bruce A C Cree Daniele Cusi Vincent Damotte Gilles Defer Silvia R Delgado Panos Deloukas Alessia di Sapio Alexander T Dilthey Peter Donnelly Bénédicte Dubois Martin Duddy Sarah Edkins Irina Elovaara Federica Esposito Nikos Evangelou Barnaby Fiddes Judith Field Andre Franke Colin Freeman Irene Y Frohlich Daniela Galimberti Christian Gieger Pierre-Antoine Gourraud Christiane Graetz Andrew Graham Verena Grummel Clara Guaschino Athena Hadjixenofontos Hakon Hakonarson Christopher Halfpenny Gillian Hall Per Hall Anders Hamsten James Harley Timothy Harrower Clive Hawkins Garrett Hellenthal Charles Hillier Jeremy Hobart Muni Hoshi Sarah E Hunt Maja Jagodic Ilijas Jelčić Angela Jochim Brian Kendall Allan Kermode Trevor Kilpatrick Keijo Koivisto Ioanna Konidari Thomas Korn Helena Kronsbein Cordelia Langford Malin Larsson Mark Lathrop Christine Lebrun-Frenay Jeannette Lechner-Scott Michelle H Lee Maurizio A Leone Virpi Leppä Giuseppe Liberatore Benedicte A Lie Christina M Lill Magdalena Lindén Jenny Link Felix Luessi Jan Lycke Fabio Macciardi Satu Männistö Clara P Manrique Roland Martin Vittorio Martinelli Deborah Mason Gordon Mazibrada Cristin McCabe Inger-Lise Mero Julia Mescheriakova Loukas Moutsianas Kjell-Morten Myhr Guy Nagels Richard Nicholas Petra Nilsson Fredrik Piehl Matti Pirinen Siân E Price Hong Quach Mauri Reunanen Wim Robberecht Neil P Robertson Mariaemma Rodegher David Rog Marco Salvetti Nathalie C Schnetz-Boutaud Finn Sellebjerg Rebecca C Selter Catherine Schaefer Sandip Shaunak Ling Shen Simon Shields Volker Siffrin Mark Slee Per Soelberg Sorensen Melissa Sorosina Mireia Sospedra Anne Spurkland Amy Strange Emilie Sundqvist Vincent Thijs John Thorpe Anna Ticca Pentti Tienari Cornelia van Duijn Elizabeth M Visser Steve Vucic Helga Westerlind James S Wiley Alastair Wilkins James F Wilson Juliane Winkelmann John Zajicek Eva Zindler Jonathan L Haines Margaret A Pericak-Vance Adrian J Ivinson Graeme Stewart David Hafler Stephen L Hauser Alastair Compston Gil McVean Philip De Jager Stephen J Sawcer Jacob L McCauley

Nat Genet 2013 Nov 29;45(11):1353-60. Epub 2013 Sep 29.

1] John P. Hussman Institute for Human Genomics, University of Miami, Miller School of Medicine, Miami, Florida, USA. [2].

Using the ImmunoChip custom genotyping array, we analyzed 14,498 subjects with multiple sclerosis and 24,091 healthy controls for 161,311 autosomal variants and identified 135 potentially associated regions (P < 1.0 × 10(-4)). In a replication phase, we combined these data with previous genome-wide association study (GWAS) data from an independent 14,802 subjects with multiple sclerosis and 26,703 healthy controls. In these 80,094 individuals of European ancestry, we identified 48 new susceptibility variants (P < 5.0 × 10(-8)), 3 of which we found after conditioning on previously identified variants. Thus, there are now 110 established multiple sclerosis risk variants at 103 discrete loci outside of the major histocompatibility complex. With high-resolution Bayesian fine mapping, we identified five regions where one variant accounted for more than 50% of the posterior probability of association. This study enhances the catalog of multiple sclerosis risk variants and illustrates the value of fine mapping in the resolution of GWAS signals.
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http://dx.doi.org/10.1038/ng.2770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832895PMC
November 2013

Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.

PLoS One 2013 16;8(4):e60100. Epub 2013 Apr 16.

German Rheumatism Research Center, Berlin, Germany.

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060100PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629055PMC
November 2013

Modulation of dendritic cell properties by laquinimod as a mechanism for modulating multiple sclerosis.

Brain 2013 Apr 20;136(Pt 4):1048-66. Epub 2013 Mar 20.

Focus Program Translational Neuroscience, Rhine Main Neuroscience Network, University Medical Centre of the Johannes Gutenberg-University of Mainz, 55131 Mainz, Germany.

Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.
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http://dx.doi.org/10.1093/brain/awt023DOI Listing
April 2013

Two-photon imaging of immune cells in neural tissue.

Cold Spring Harb Protoc 2013 Mar 1;2013(3). Epub 2013 Mar 1.

To develop new therapeutic strategies for many central nervous system (CNS) diseases, it is essential to observe the motility and function of immune cells within neural tissue. Two-photon laser-scanning microscopy is an outstanding technique for imaging these phenomena under in vivo-like conditions. To gain deeper insight into the pathological phenomena that occur during chronic neuroinflammation of the CNS, we use it to view acute murine hippocampal slices cocultured with different subpopulations of immune cells and to view in vivo the brain stem of anesthetized transgenic mice affected by experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. This protocol describes the preparation of cocultures of acute hippocampal slices with antigen-specific T helper 17 (Th17) cells migrating into the parenchyma, and the preparation of anesthetized mice for imaging the brain stem. We also discuss technical aspects of dual-color, two-photon laser-scanning microscopy that is used to image these samples and that allows for greater flexibility in the choice of fluorophores.
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http://dx.doi.org/10.1101/pdb.prot073528DOI Listing
March 2013