Publications by authors named "Madeleine Scharf"

10 Publications

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Autoantibodies Against the Purkinje Cell Protein RGS8 in Paraneoplastic Cerebellar Syndrome.

Neurol Neuroimmunol Neuroinflamm 2021 05 29;8(3). Epub 2021 Mar 29.

From the Institute for Experimental Immunology (R.M., M.S., A.O., Y.D., N.R., C.P., L.K.), Affiliated to EUROIMMUN Medizinische Labordiagnostika AG, Luebeck; Department of Neurology (P.S., H.D.), Krankenhaus St. Elisabeth, Damme; Laboratory Krone (C.I.B.), Bad Salzuflen; Department of Neurology (C.B., P.K.), Nordwest-Krankenhaus Sanderbusch, Sande; Clinical Immunological Laboratory Prof. Dr. Med. Winfried Stöcker (K.B., B.T.), Luebeck; and Department of Neurology (C.F.), Charité - Universitätsmedizin Berlin, Berlin, Germany.

Objective: To describe the identification of regulator of G-protein signaling 8 (RGS8) as an autoantibody target in patients with cerebellar syndrome associated with lymphoma.

Methods: Sera of 4 patients with a very similar unclassified reactivity against cerebellar Purkinje cells were used in antigen identification experiments. Immunoprecipitations with cerebellar lysates followed by mass spectrometry identified the autoantigen, which was verified by recombinant immunofluorescence assay, immunoblot, and ELISA with the recombinant protein.

Results: The sera and CSF of 4 patients stained the Purkinje cells and molecular layer of the cerebellum. RGS8 was identified as the target antigen in all 4 sera. In a neutralization experiment, recombinant human RGS8 was able to neutralize the autoantibodies' tissue reaction. Patient sera and CSF showed a specific reactivity against recombinant RGS8 in ELISA and immunoblot, whereas no such reactivity was detectable in the controls. Clinical data were available for 2 of the 4 patients, remarkably both presented with cerebellar syndrome accompanied by B-cell lymphoma of the stomach (patient 1, 53 years) or Hodgkin lymphoma (patient 2, 74 years).

Conclusion: Our results indicate that autoantibodies against the intracellular Purkinje cell protein RGS8 represent new markers for paraneoplastic cerebellar syndrome associated with lymphoma.

Classification Of Evidence: This study provided Class IV evidence that autoantibodies against the intracellular Purkinje cell protein RGS8 are associated with paraneoplastic cerebellar syndrome in lymphoma.
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May 2021

Autoimmune gait disturbance accompanying adaptor protein-3B2-IgG.

Neurology 2019 09 1;93(10):e954-e963. Epub 2019 Aug 1.

From the Departments of Laboratory Medicine and Pathology (J.A.H., T.J.K., S.R.H., V.A.L., S.J.P., C.J.K., A.M.), Neurology (A.S.L.-C., V.A.L., S.J.P., C.J.K., A.M.), and Immunology (V.A.L.), College of Medicine, Mayo Clinic, Rochester, MN; and Euroimmun, AG (L.K., M.S.), Lubeck, Germany.

Objective: To describe phenotypes, treatment response, and outcomes of autoimmunity targeting a synaptic vesicle coat protein, the neuronal (B2) form of adaptor protein-3 (AP3).

Methods: Archived serum and CSF specimens (from 616,025 screened) harboring unclassified synaptic antibodies mimicking amphiphysin-immunoglobulin G (IgG) on tissue-based indirect immunofluorescence assay (IFA) were re-evaluated for novel IgG staining patterns. Autoantigens were identified by western blot and mass spectrometry. Recombinant western blot and cell-binding assay (CBA) were used to confirm antigen specificity. Clinical data were obtained retrospectively.

Results: Serum (10) and CSF (6) specimens of 10 patients produced identical IFA staining patterns throughout mouse nervous system tissues, most prominently in cerebellum (Purkinje neuronal perikarya, granular layer synapses, and dentate regions), spinal cord gray matter, dorsal root ganglia, and sympathetic ganglia. The antigen revealed by mass spectrometry analysis and confirmed by recombinant assays (western blot and CBA) was AP3B2 in all. Of 10 seropositive patients, 6 were women; median symptom onset age was 42 years (range 24-58). Clinical information was available for 9 patients, all with subacute onset and rapidly progressive gait ataxia. Neurologic manifestations were myeloneuropathy (3), peripheral sensory neuropathy (2), cerebellar ataxia (2), and spinocerebellar ataxia (2). Five patients received immunotherapy; none improved, but they did not worsen over the follow-up period (median 36 months; range 3-94). Two patients (both with cancer) died. One of 50 control sera was positive by western blot only (but not by IFA or CBA).

Conclusion: AP3B2 (previously named β-neuronal adaptin-like protein) autoimmunity appears rare, is accompanied by ataxia (sensory or cerebellar), and is potentially treatable.
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September 2019

A Spectrum of Neural Autoantigens, Newly Identified by Histo-Immunoprecipitation, Mass Spectrometry, and Recombinant Cell-Based Indirect Immunofluorescence.

Front Immunol 2018 9;9:1447. Epub 2018 Jul 9.

Institute of Experimental Immunology, EUROIMMUN AG, Lübeck, Germany.

Background: A plurality of neurological syndromes is associated with autoantibodies against neural antigens relevant for diagnosis and therapy. Identification of these antigens is crucial to understand the pathogenesis and to develop specific immunoassays. Using an indirect immunofluorescence assay (IFA)-based approach and applying different immunoprecipitation (IP), chromatographic and mass spectrometric protocols was possible to isolate and identify a spectrum of autoantigens from brain tissue.

Methods: Sera and CSF of 320 patients suspected of suffering from an autoimmune neurological syndrome were comprehensively investigated for the presence of anti-neural IgG autoantibodies by IFA using mosaics of biochips with brain tissue cryosections and established cell-based recombinant antigen substrates as well as immunoblots. Samples containing unknown brain tissue-specific autoantibodies were subjected to IP with cryosections of cerebellum and hippocampus (rat, pig, and monkey) immobilized to glass slides or with lysates produced from homogenized tissue, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tryptic digestion, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Identifications were confirmed by IFA with recombinant HEK293 cells and by neutralizing the patients' autoantibodies with the respective recombinantly expressed antigens in the tissue-based immunofluorescence test.

Results: Most samples used in this study produced speckled, granular, or homogenous stainings of the hippocampal and cerebellar molecular and/or granular layers. Others exclusively stained the Purkinje cells. Up to now, more than 20 different autoantigens could be identified by this approach, among them ATP1A3, CPT1C, Flotillin1/2, ITPR1, NBCe1, NCDN, RGS8, ROCK2, and Syntaxin-1B as novel autoantigens.

Discussion: The presented antigen identification strategy offers an opportunity for identifying up to now unknown neural autoantigens. Recombinant cell substrates containing the newly identified antigens can be used in serology and the clinical relevance of the autoantibodies can be rapidly evaluated in cohort studies.
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July 2018

ITPR1 autoimmunity: Frequency, neurologic phenotype, and cancer association.

Neurol Neuroimmunol Neuroinflamm 2018 Jan 8;5(1):e418. Epub 2017 Dec 8.

Department of Laboratory Medicine and Pathology (N.A., A.G., V.A.L., S.H., A.M., S.J.P.), Department of Neurology (V.A.L., A.M., S.J.P.), and Department of Immunology (V.A.L.), Mayo Clinic, Rochester, MN; and Institute for Experimental Immunology (L.K., M.S.), Affiliated to Euroimmun AG, Luebeck, Germany.

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January 2018

Paraneoplastic cerebellar degeneration associated with anti-ITPR1 antibodies.

Neurol Neuroimmunol Neuroinflamm 2017 Mar 3;4(2):e326. Epub 2017 Feb 3.

AP-HP Pitié-Salpêtrière (G.B., C.D., J.-Y.D., D.P.), Service de Neurologie Mazarin, Paris, France; Neuroscience Consortium (G.B.), University of Pavia, Monza Policlinico and Pavia Mondino, Italy; Nuffield Department of Clinical Neurosciences (Y.H.), John Radcliffe Hospital, University of Oxford, United Kingdom; Institute of Experimental Immunology (L.K., M.S.), affiliated to Euroimmun AG, Lübeck, Germany; AP-HP Pitié-Salpêtrière (D.L.), Service de Neuroradiologie; Département de Chirurgie Oncologique (V.F.); Département de Biologie des Tumeurs (B.B.), Service Génétique, Institut Curie, Paris; Centre National de Référence pour les Syndromes Neurologiques Paranéoplasiques (J.H.), Hospices Civils de Lyon, Institut NeuroMyoGene INSERM U1217/CNRS UMR 5310, Université de Lyon, France; and Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) (F.G.), Service of Neurology, Hospital Clinic, Barcelona, Spain.

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March 2017

Neurochondrin is a neuronal target antigen in autoimmune cerebellar degeneration.

Neurol Neuroimmunol Neuroinflamm 2017 Jan 5;4(1):e307. Epub 2016 Dec 5.

Institute of Experimental Immunology (R.M., M. Scharf, I.M.D., S.M., Y.D., B.T., C.P., S.B., W.S., L.K.), Euroimmun AG, Lübeck; Department of Neurology (C.C.G., K.S.G., M.H., U.B., A.S.-M., K.B., C.S., H.L., M.D., T.W., H.W., S.G.M., N.M.), University of Münster; Centre for Neurology and Hertie-Institute for Clinical Brain Research (L.S., M. Synofzik), Tübingen; German Center for Neurodegenerative Diseases (DZNE) (L.S., M. Synofzik), Tübingen; and Institute of Clinical Chemistry and Department of Neurology (K.-P.W.), University Hospital of Schleswig-Holstein, Lübeck, Germany.

Objective: To report on a novel neuronal target antigen in 3 patients with autoimmune cerebellar degeneration.

Methods: Three patients with subacute to chronic cerebellar ataxia and controls underwent detailed clinical and neuropsychological assessment together with quantitative high-resolution structural MRI. Sera and CSF were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IFA) and immunoblot. Immunoprecipitation with lysates of hippocampus and cerebellum combined with mass spectrometric analysis was used to identify the autoantigen, which was verified by recombinant expression in HEK293 cells and use in several immunoassays. Multiparameter flow cytometry was performed on peripheral blood and CSF, and peripheral blood was subjected to T-cell receptor spectratyping.

Results: Patients presented with a subacute to chronic cerebellar and brainstem syndrome. MRI was consistent with cortical and cerebellar gray matter atrophy associated with subsequent neuroaxonal degeneration. IFA screening revealed strong immunoglobulin G1 reactivity in sera and CSF with hippocampal and cerebellar molecular and granular layers, but not with a panel of 30 recombinantly expressed established neural autoantigens. Neurochondrin was subsequently identified as the target antigen, verified by IFA and immunoblot with HEK293 cells expressing human neurochondrin as well as the ability of recombinant neurochondrin to neutralize the autoantibodies' tissue reaction. Immune phenotyping revealed intrathecal accumulation and activation of B and T cells during the acute but not chronic phase of the disease. T-cell receptor spectratyping suggested an antigen-specific T-cell response accompanying the formation of antineurochondrin autoantibodies. No such neurochondrin reactivity was found in control cohorts of various neural autoantibody-associated neurologic syndromes, relapsing-remitting multiple sclerosis, cerebellar type of multiple system atrophy, hereditary cerebellar ataxias, other neurologic disorders, or healthy donors.

Conclusion: Neurochondrin is a neuronal target antigen in autoimmune cerebellar degeneration.
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January 2017

Autoantibodies against glutamate receptor δ2 after allogenic stem cell transplantation.

Neurol Neuroimmunol Neuroinflamm 2016 Aug 14;3(4):e255. Epub 2016 Jul 14.

Institute of Experimental Immunology (R.M., S.H., I.M.D., S.M., Y.D., S.B., B.T., C.P., W.S., L.K.), Euroimmun AG, Lübeck; Department of Neurology (T.R., M.M., C.T.), Asklepios Klinik St. Georg, Hamburg; Department of Neurology (N.M.), University of Münster; and Department of Neurology (H.-M.M.), University of Heidelberg, Germany.

Objective: To report on a Caucasian patient who developed steroid-responsive transverse myelitis, graft vs host disease of the gut, and anti-GluRδ2 after allogenic stem cell transplantation.

Methods: Histoimmunoprecipitation (HIP) with the patient's serum and cryosections of rat and porcine cerebellum followed by mass spectrometry was used to identify the autoantigen. Correct identification was verified by indirect immunofluorescence using recombinant GluRδ2 expressed in HEK293 cells.

Results: The patient's serum produced a granular staining of the cerebellar molecular layer (immunoglobulin G1 and immunoglobulin G3; endpoint titer: 1:1,000) but did not react with other CNS tissues or 28 established recombinant neural autoantigens. HIP revealed a unique protein band at ∼110 kDa that was identified as GluRδ2. The patient's serum also stained GluRδ2 transfected but not mock-transfected HEK293 cells. Control sera from 38 patients with multiple sclerosis, 85 patients with other neural autoantibodies, and 205 healthy blood donors were negative for anti-GluRδ2. Preadsorption with lysate from HEK293-GluRδ2 neutralized the patient's tissue reaction whereas control lysate had no effect. In addition to anti-GluRδ2, the patient's serum contained immunoglobulin G autoantibodies against the pancreatic glycoprotein CUZD1, which are known to be markers of Crohn disease.

Conclusions: In the present case, the development of anti-GluRδ2 was associated with transverse myelitis, which was supposedly triggered by the stem cell transplantation. Similar to encephalitis in conjunction with anti-GluRδ2 reported in a few Japanese patients, the patient's neurologic symptoms ameliorated after steroid therapy.
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August 2016

Neuronal Na+/K+ ATPase is an autoantibody target in paraneoplastic neurologic syndrome.

Neurology 2015 Apr 25;84(16):1673-9. Epub 2015 Mar 25.

From the Institute of Experimental Immunology (M.S., R.M., I.-M.B., N.B., Y.D., S.T., C.D., W. Schlumberger, M.U., B.T., W. Stöcker, C.P., L.K.), EUROIMMUN AG, Lübeck; and Departments of Neurology and Clinical Neurophysiology (F.H., R.G.) and Diagnostic and Interventional Radiology (P.L.), Hospital of the Henriettenstiftung, Hannover, Germany.

Objectives: To identify an autoreactivity in a 66-year-old woman who presented with combined brainstem and cerebellar syndrome including vertical gaze palsy, severe progressive ataxia, and spastic tetraparesis, an acute deterioration of vision, dysarthria, and dysphagia with concurrent diagnosis of a colon adenocarcinoma.

Methods: Patient's serum and CSF underwent comprehensive autoantibody screening by indirect immunofluorescence assay and immunoblot. For autoantigen purification, a histo-immunoprecipitation technique was developed followed by mass spectrometrical analysis. Recombinant candidate antigens were expressed in HEK293 and used to verify the identification.

Results: Indirect immunofluorescence assay screening revealed strong immunoglobulin G reactivity with neural tissues in serum and CSF, but not with a panel of 28 recombinantly expressed established neural autoantigens. The hitherto unknown target antigen was identified as the neuronal Na(+)/K(+) ATPase. Epitope mapping and competitive inhibition experiments showed that the autoantibodies were directed against the membrane-spanning alpha 3 subunit (ATP1A3) of the enzyme but did not bind to extracellular epitopes. Immunohistochemical analysis revealed overexpression of this subunit in the patient's tumor.

Conclusions: We describe a case of an anti-ATP1A3-associated neurologic disorder. Mutations in the gene encoding this neuronal surface protein have already been recognized as the cause of infantile alternating hemiplegia, rapid-onset dystonia parkinsonism, and CAPOS syndrome. Although the autoantibodies are unlikely to be pathogenic, they are likely to be rare biomarkers for the apparently paraneoplastic neurologic syndrome or for the tumor itself.
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April 2015

Antibodies to the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) in cerebellar ataxia.

J Neuroinflammation 2014 Dec 11;11:206. Epub 2014 Dec 11.

Institute of Experimental Immunology, affiliated to Euroimmun AG, Seekamp 31, 23560, Lübeck, Germany.

We report on a serum autoantibody associated with cerebellar ataxia. Immunohistochemical studies of sera from four patients referred for autoantibody testing revealed binding of high-titer (up to 1:5,000) IgG antibodies, mainly IgG1, to the molecular layer, Purkinje cell layer, and white matter on mouse, rat, porcine, and monkey cerebellum sections. The antibody bound to PC somata, dendrites, and axons, resulting in a binding pattern similar to that reported for anti-Ca/anti-ARHGAP26, but did not react with recombinant ARHGAP26. Extensive control studies were performed to rule out a broad panel of previously described paraneoplastic and non-paraneoplastic anti-neural autoantibodies. The characteristic binding pattern as well as double staining experiments suggested inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) as the target antigen. Verification of the antigen included specific neutralization of the tissue reaction following preadsorption with ITPR1 (but not ARHGAP26) and a dot-blot assay with purified ITPR1 protein. By contrast, anti-ARHGAP26-positive sera did not bind to ITPR1. In a parallel approach, a combination of histoimmunoprecipitation and mass spectrometry also identified ITPR1 as the target antigen. Finally, a recombinant cell-based immunofluorescence assay using HEK293 cells expressing ITPR1 and ARHGAP26, respectively, confirmed the identification of ITPR1. Mutations of ITPR1 have previously been implicated in spinocerebellar ataxia with and without cognitive decline. Our findings suggest a role of autoimmunity against ITPR1 in the pathogenesis of autoimmune cerebellitis and extend the panel of diagnostic markers for this disease.
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December 2014

Mitogen-activated protein kinase-activated protein kinases 2 and 3 regulate SERCA2a expression and fiber type composition to modulate skeletal muscle and cardiomyocyte function.

Mol Cell Biol 2013 Jul 22;33(13):2586-602. Epub 2013 Apr 22.

Department of Biochemistry, Hannover Medical School, Hannover, Germany.

The mitogen-activated protein kinase (MAPK)-activated protein kinases 2 and 3 (MK2/3) represent protein kinases downstream of the p38 MAPK. Using MK2/3 double-knockout (MK2/3(-/-)) mice, we analyzed the role of MK2/3 in cross-striated muscle by transcriptome and proteome analyses and by histology. We demonstrated enhanced expression of the slow oxidative skeletal muscle myofiber gene program, including the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α). Using reporter gene and electrophoretic gel mobility shift assays, we demonstrated that MK2 catalytic activity directly regulated the promoters of the fast fiber-specific myosin heavy-chain IId/x and the slow fiber-specific sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) gene. Elevated SERCA2a gene expression caused by a decreased ratio of transcription factor Egr-1 to Sp1 was associated with accelerated relaxation and enhanced contractility in MK2/3(-/-) cardiomyocytes, concomitant with improved force parameters in MK2/3(-/-) soleus muscle. These results link MK2/3 to the regulation of calcium dynamics and identify enzymatic activity of MK2/3 as a critical factor for modulating cross-striated muscle function by generating a unique muscle phenotype exhibiting both reduced fatigability and enhanced force in MK2/3(-/-) mice. Hence, the p38-MK2/3 axis may represent a novel target for the design of therapeutic strategies for diseases related to fiber type changes or impaired SERCA2 function.
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July 2013