Publications by authors named "Lars Komorowski"

74 Publications

Pathogenic Activation and Therapeutic Blockage of Fc Alpha Receptor-Expressing Polymorphonuclear Leukocytes in IgA Pemphigus.

J Invest Dermatol 2021 Jul 8. Epub 2021 Jul 8.

Luebeck Inst. of Experimental Dermatology (LIED), University of Luebeck, Luebeck, Germany; Dept. of Dermatology, University of Luebeck, Luebeck, Germany. Electronic address:

Pathomechanisms in IgA pemphigus are assumed to rely on Fc-dependent cellular activation by antigen-specific IgA autoantibodies, however, models for disease and more detailed pathophysiologic data are lacking. We here aimed to establish in vitro models of disease for IgA pemphigus, allowing to study effects of the interaction of anti-keratinocyte IgA with cell-surface Fc alpha receptors. Employing multiple in vitro assays, such as a skin cryosection assay and a human skin organ culture model, we here present mechanistic data for the pathogenesis of IgA pemphigus, mediated by anti-desmoglein 3 IgA autoantibodies. Our results reveal that this disease is dependent on Fc alpha receptor-mediated activation of leukocytes in the epidermis. Importantly, this cell-dependent pathology can be dose-dependently abrogated by peptide-mediated inhibition of Fc alpha receptor:IgA interaction, as confirmed in an additional model for IgA-dependent disease, i.e., IgA vasculitis. These data suggest that IgA pemphigus can be modeled in vitro, and IgA pemphigus and IgA vasculitis are Fc alpha receptor-dependent disease entities that can be specifically targeted in these experimental systems.
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http://dx.doi.org/10.1016/j.jid.2021.06.007DOI Listing
July 2021

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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http://dx.doi.org/10.1212/NXI.0000000000000987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009278PMC
May 2021

Cross-reactivity of a pathogenic autoantibody to a tumor antigen in GABA receptor encephalitis.

Proc Natl Acad Sci U S A 2021 03;118(9)

Institute of Clinical Neuroimmunology, Biomedical Center and Hospital of the Ludwig-Maximilians-Universität München, D-82152 Martinsried, Germany;

Encephalitis associated with antibodies against the neuronal gamma-aminobutyric acid A receptor (GABA-R) is a rare form of autoimmune encephalitis. The pathogenesis is still unknown but autoimmune mechanisms were surmised. Here we identified a strongly expanded B cell clone in the cerebrospinal fluid of a patient with GABA-R encephalitis. We expressed the antibody produced by it and showed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry that it recognizes the GABA-R. Patch-clamp recordings revealed that it tones down inhibitory synaptic transmission and causes increased excitability of hippocampal CA1 pyramidal neurons. Thus, the antibody likely contributed to clinical disease symptoms. Hybridization to a protein array revealed the cross-reactive protein LIM-domain-only protein 5 (LMO5), which is related to cell-cycle regulation and tumor growth. We confirmed LMO5 recognition by immunoprecipitation and ELISA and showed that cerebrospinal fluid samples from two other patients with GABA-R encephalitis also recognized LMO5. This suggests that cross-reactivity between GABA-R and LMO5 is frequent in GABA-R encephalitis and supports the hypothesis of a paraneoplastic etiology.
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http://dx.doi.org/10.1073/pnas.1916337118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936355PMC
March 2021

GTPase Regulator Associated with Focal Adhesion Kinase 1 (GRAF1) Immunoglobulin-Associated Ataxia and Neuropathy.

Mov Disord Clin Pract 2020 Nov 14;7(8):904-909. Epub 2020 Sep 14.

Department of Neurology Mayo Clinic Rochester Minnesota USA.

Background: To date, 10 patients with GTPase Regulator Associated with Focal Adhesion Kinase 1/Rho GTPase Activating Protein 26-Immunoglobulin (GRAF1/ARHGAP26-IgG) associated neurological disorders have been described, most with ataxia.

Objective: To report the clinical, oncological, and radiological associations of GRAF1 autoantibodies.

Methods: We identified 17 patients whose serum and/or cerebrospinal fluid IgG was confirmed to target GRAF1/ARHGAP26-IgG by both tissue-based immunofluorescence and transfected cell-based assay. Clinical information was available on 14 patients.

Results: The median age at neurological symptom onset was 51 years, and 8 (47%) were men. The predominant clinical features were subacute progressive cerebellar ataxia (13) or peripheral neuropathy (2). Magnetic resonance imaging brain (7 available) showed cerebellar atrophy (4, 1 also cerebrum and brainstem atrophy). Of 7 cerebrospinal fluids available for testing, 5 showed pleocytosis with oligoclonal bands in 3. Squamous cell carcinoma was observed in 3 patients (head and neck [2], lung [1]).

Conclusion: GTPase Regulator Associated with Focal Adhesion Kinase 1 autoimmunity manifests commonly with subacute ataxia and cerebellar degeneration with a potential association with squamous cell carcinoma. Peripheral neuropathy may also be encountered. Cases in this series responded poorly to immunotherapy.
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http://dx.doi.org/10.1002/mdc3.13036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604666PMC
November 2020

Contactin-1 autoimmunity: Serologic, neurologic, and pathologic correlates.

Neurol Neuroimmunol Neuroinflamm 2020 07 27;7(4). Epub 2020 May 27.

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

Objective: To determine serologic characteristics, frequency, phenotype, paraneoplastic associations, and electrodiagnostic and histopathologic features accompanying contactin-1 autoimmunity.

Methods: Archived sera known to produce synaptic tissue-based immunofluorescence patterns were reevaluated, and contactin-1 specificity was confirmed by recombinant protein assays. Screening of 233 chronic/relapsing demyelinating neuropathies for additional cases was performed.

Results: We identified 10 contactin-1 IgG seropositive cases. Frequency of contactin-1 immunoglobulin (Ig) G among tested Mayo Clinic chronic/relapsing demyelinating neuropathies was 2%. Sensory predominant presentations (n = 9, 90%), neuropathic pain (n = 6, 60%), and subacute progression (n = 5, 50%) were commonly encountered among contactin-1 neuropathies. Two patients had chronic immune sensory polyradiculopathy-like phenotype at presentation. Electrodiagnostic studies were consistent with demyelination (slowed conduction velocities and/or prolonged distal latencies) without conduction block. Markedly elevated CSF protein (median 222 mg/dL, range 69-960 mg/dL), thickening/gadolinium enhancement of nerve roots (4/5), and subperineural edema on nerve biopsy (4/4) were other characteristic features. Three cases were diagnosed with paraneoplastic demyelinating neuropathies (thymoma, n = 1; breast cancer, n = 1; plasmacytoma, n = 1). Four of the 9 patients treated with IV immunoglobulin demonstrated initial clinical improvement, but the favorable response was sustained in only 1 case (median follow-up, 60 months). Sustained clinical stabilization or improvement was observed among 3 of the 6 cases in whom second-line therapies (rituximab, cyclophosphamide, and azathioprine) were used.

Conclusion: Contactin-1 IgG has a distinct sensory predominant presentation commonly associated with neuropathic pain, with demyelinating changes on electrophysiologic studies. A paraneoplastic cause should be considered. Testing of contactin-1 IgG among cases with similar presentations may guide immunotherapy selection, especially second-line immunotherapy consideration.
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http://dx.doi.org/10.1212/NXI.0000000000000771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286654PMC
July 2020

Multicenter prospective study on multivariant diagnostics of autoimmune bullous dermatoses using the BIOCHIP technology.

J Am Acad Dermatol 2020 Nov 28;83(5):1315-1322. Epub 2020 Jan 28.

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

Background: The current standard in the serologic diagnosis of autoimmune bullous diseases (AIBD) is a multistep procedure sequentially applying different assays. In contrast, the BIOCHIP Mosaic technology combines multiple substrates for parallel analysis by indirect immunofluorescence.

Methods: Sera from 749 consecutive, prospectively recruited patients with direct immunofluorescence-positive AIBD from 13 international study centers were analyzed independently and blinded by using (1) a BIOCHIP Mosaic including primate esophagus, salt-split skin, rat bladder, monkey liver, monkey liver with serosa, recombinant BP180 NC16A, and gliadin GAF3X, as well as HEK293 cells expressing recombinant desmoglein 1, desmoglein 3, type VII collagen, and BP230 C-terminus and (2) the conventional multistep approach of the Department of Dermatology, University of Lübeck.

Results: In 731 of 749 sera (97.6%), specific autoantibodies could be detected with the BIOCHIP Mosaic, similar to the conventional procedure (725 cases, 96.8%). The Cohen κ for both serologic approaches ranged from 0.84 to 1.00. In 6.5% of sera, differences between the 2 approaches occurred and were mainly attributed to autoantigen fragments not present on the BIOCHIP Mosaic.

Limitations: Laminin 332 and laminin γ1 are not represented on the BIOCHIP Mosaic.

Conclusions: The BIOCHIP Mosaic is a standardized time- and serum-saving approach that further facilitates the serologic diagnosis of AIBD.
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http://dx.doi.org/10.1016/j.jaad.2020.01.049DOI Listing
November 2020

Effects of IVIg treatment on autoantibody testing in neurological patients: marked reduction in sensitivity but reliable specificity.

J Neurol 2020 Mar 14;267(3):715-720. Epub 2019 Nov 14.

Department of Neurology, St. Josef Hospital, Ruhr-University Bochum, Gudrunstr. 56, 44791, Bochum, Germany.

Background: Therapy of autoimmune diseases of the central and peripheral nervous system with intravenous IgG immunoglobulin (IVIg) is well established. Since IVIg is produced from pooled human plasma, autoantibodies can be found in IVIg products and, accordingly, in patient sera after transfusion. The de novo evidence or disappearance of anti-neural autoantibodies after IVIg treatment has so far not been systematically examined.

Methods: We screened 50 neurological patients before and after IVIg treatment for classical onconeural and the most common neurological surface autoantibodies as well as for ganglioside autoantibodies and 23 different antinuclear autoantibodies using immunoblot or cell-based indirect immunofluorescence assays. Furthermore, we screened 31 neurological patients with previously known seropositivity for disappearance of the corresponding antibody after treatment.

Results: After IVIg treatment, 90% of all sera were de novo positive for antinuclear antibodies, especially for Ro-52. In contrast, 94% of all sera did not show any de novo-positive anti-neural antibodies. In the remaining three cases, titers were very low. Importantly, 12.9% of all tested sera of patients with known antibody positivity turned false negative after IVIg treatment and titers were falsely low in 37% of the remaining sera.

Conclusions: Here, we present for the first time results of a broad screening for clinically relevant autoantibodies before and after IVIg treatment in neurological patients. We identified a high specificity but reduced sensitivity for anti-neural antibody testing after IVIg transfusion. In contrast, antinuclear antibody testing is not reliable after IVIg treatment. These results are of high practical importance for diagnostic of neuroimmunological diseases.
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http://dx.doi.org/10.1007/s00415-019-09614-4DOI Listing
March 2020

Serological Diagnosis of Autoimmune Bullous Skin Diseases.

Front Immunol 2019 20;10:1974. Epub 2019 Aug 20.

Institute for Experimental Immunology, Euroimmun AG, Lübeck, Germany.

Autoimmune bullous dermatoses (AIBD) encompass a variety of organ-specific autoimmune diseases that manifest with cutaneous and/or mucosal blisters and erosions. They are characterized by autoantibodies targeting structural proteins of the skin, which are responsible for the intercellular contact between epidermal keratinocytes and for adhesion of the basal keratinocytes to the dermis. The autoantibodies disrupt the adhesive functions, leading to splitting and blister formation. In pemphigus diseases, blisters form intraepidermally, whereas in all other disease types they occur subepidermally. Early identification of autoimmune bullous dermatoses is crucial for both treatment and prognosis, particularly as regards tumor-associated disease entities. The diagnosis is based on clinical symptoms, histopathology, direct immunofluorescence to detect antibody/complement deposits, and the determination of circulating autoantibodies. The identification of various target antigens has paved the way for the recent development of numerous specific autoantibody tests. In particular, optimized designer antigens and multiplex test formats for indirect immunofluorescence and ELISA have enhanced and refined the laboratory analysis, enabling highly efficient serodiagnosis and follow-up. This review elaborates on the current standards in the serological diagnostics for autoimmune bullous dermatoses.
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http://dx.doi.org/10.3389/fimmu.2019.01974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736620PMC
October 2020

Neurochondrin neurological autoimmunity.

Neurol Neuroimmunol Neuroinflamm 2019 11 11;6(6). Epub 2019 Sep 11.

Department of Laboratory Medicine and Pathology (S.S., T.J.K., E.P.F., S.R.H., V.A.L., S.J.P., A.M.), Department of Neurology (E.P.F., V.A.L., S.J.P., A.M.), and Department of Immunology (V.A.L.), College of Medicine, Mayo Clinic; Euroimmun AG (L.K., R.M.), Lubeck, Germany; and Department of Neurology (M.D.A.), University of Mississippi Medical Center, Jackson, MS.

Objectives: To describe the neurologic spectrum and treatment outcomes for neurochondrin-IgG positive cases identified serologically in the Mayo Clinic Neuroimmunology Laboratory.

Methods: Archived serum and CSF specimens previously scored positive for IgGs that stained mouse hippocampal tissue in a nonuniform synaptic pattern by immunofluorescence assay (89 among 616,025 screened, 1993-2019) were reevaluated. Antibody characterization experiments revealed specificity for neurochondrin, confirmed by recombinant protein assays.

Results: IgG in serum (9) or CSF (4) from 8 patients yielded identical neuron-restricted CNS patterns, most pronounced in hippocampus (stratum lucidum in particular), cerebellum (Purkinje cells and molecular layer), and amygdala. All were neurochondrin-IgG positive. Five were women; median symptom onset age was 43 years (range, 30-69). Of 7 with clinical data, 6 presented with rapidly progressive cerebellar ataxia, brainstem signs, or both; 1 had isolated unexplained psychosis 1 year prior. Five of 6 had cerebellar signs, 4 with additional brainstem symptoms or signs (eye movement abnormalities, 3; dysphagia, 2; nausea and vomiting, 1). One patient with brainstem signs (vocal cord paralysis and VII nerve palsy) had accompanying myelopathy (longitudinally extensive abnormality on MRI; aquaporin-4-IgG and myelin oligodendrocyte glycoprotein-IgG negative). The 7th patient had small fiber neuropathy only. Just 1 of 7 had contemporaneous cancer (uterine). Six patients with ataxia or brainstem signs received immunotherapy, but just 1 remained ambulatory. At last follow-up, 5 had MRI evidence of severe cerebellar atrophy.

Conclusion: In our series, neurochondrin autoimmunity was usually accompanied by a nonparaneoplastic rapidly progressive rhombencephalitis with poor neurologic outcomes. Other phenotypes and occasional paraneoplastic causes may occur.
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http://dx.doi.org/10.1212/NXI.0000000000000612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745726PMC
November 2019

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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http://dx.doi.org/10.1212/WNL.0000000000008061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745733PMC
September 2019

GABA receptor autoimmunity: A multicenter experience.

Neurol Neuroimmunol Neuroinflamm 2019 05 4;6(3):e552. Epub 2019 Apr 4.

Department of Laboratory Medicine and Pathology (K.O'.C., A.Z., V.L., S.J.P., A.M.), Department of Neurology (A.Z., V.L., S.J.P., A.M.), and Department of Immunology (V.L.), Mayo Clinic, Rochester, MN; Oxford Autoimmune Neurology Group (P.J.W., V.C.M.), Nuffield Department of Clinical Neurosciences, UK; Institute for Experimental Immunology (L.K., C.P., S.M., B.T.), Affiliated to Euroimmun AG, Luebeck, Germany; and the Department of Neurology (C.Y.G., J.M.G., M.D.G.), University California, San Francisco.

Objective: We sought to validate methods for detection and confirmation of GABA receptor (R)-IgG and clinically characterize seropositive cases.

Methods: Archived serum and CSF specimens (185 total) suspected to harbor GABAR-IgG were evaluated by indirect immunofluorescence assay (IFA). Twenty-six specimens from 19 patients appeared suspicious for GABAR-IgG positivity by IFA, based on prior reports and comparison with commercial GABAR antibody staining. Aliquots of those specimens were tested at the University of Oxford, United Kingdom, and Euroimmun, Lubeck, Germany, for GABAR-IgG by cell-based assays (CBAs) using HEK293-indicator cells transfected with plasmids encoding different GABAR subunits.

Results: Eight specimens (of 26 tested; 4 serums, 4 CSFs) from 5 patients were confirmed by CBA to be GABAR-IgG positive. Patient IgGs were always reactive with α1β3 GABAR subunits. One more patient was identified clinically after this validation study. Median age for the 6 patients at serologic diagnosis was 44 years (range, 1-71 years), and 4 of them were male. Among the 4 for whom clinical information was available (2 treated by the authors), all had encephalitis and antiepileptic drug refractory seizures. Three out of 4 patients treated with a combination of immunotherapies had good outcomes. The fourth, recognized to have an autoimmune cause late in the clinical course, had severe permanent neurologic sequelae and brain atrophy.

Conclusions: Though not as common as NMDA-R encephalitis, GABAR encephalitis generally has a characteristic clinical-radiologic presentation and is treatable, making accurate laboratory diagnosis critical.
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http://dx.doi.org/10.1212/NXI.0000000000000552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501640PMC
May 2019

A multicenter comparison of MOG-IgG cell-based assays.

Neurology 2019 03 6;92(11):e1250-e1255. Epub 2019 Feb 6.

From the Oxford Autoimmune Neurology Group (P.J.W., M.W., S.R.I.), Nuffield Department of Clinical Neurosciences, UK; Institute for Experimental Immunology (L.K., S.L.), Affiliated to Euroimmun AG, Luebeck, Germany; and Departments of Neurology (M.M., E.P.F., A.C.K., A.M., S.J.P.) and Laboratory Medicine and Pathology (J.F., J.M., E.P.F., A.C.K., A.M., S.J.P.), Mayo Clinic, College of Medicine, Rochester, MN.

Objectives: To compares 3 different myelin oligodendrocyte glycoprotein-immunoglobulin G (IgG) cell-based assays (CBAs) from 3 international centers.

Methods: Serum samples from 394 patients were as follows: acute disseminated encephalomyelitis (28), seronegative neuromyelitis optica (27), optic neuritis (21 single, 2 relapsing), and longitudinally extensive (10 single, 3 recurrent). The control samples were from patients with multiple sclerosis (244), hypergammaglobulinemia (42), and other (17). Seropositivity was determined by visual observation on a fluorescence microscope (Euroimmun fixed CBA, Oxford live cell CBA) or flow cytometry (Mayo live cell fluorescence-activated cell sorting assay).

Results: Of 25 samples positive by any methodology, 21 were concordant on all 3 assays, 2 were positive at Oxford and Euroimmun, and 2 were positive only at Oxford. Euroimmun, Mayo, and Oxford results were as follows: clinical specificity 98.1%, 99.6%, and 100%; positive predictive values (PPVs) 82.1%, 95.5%, and 100%; and negative predictive values 79.0%, 78.8%, and 79.8%. Of 5 false-positives, 1 was positive at both Euroimmun and Mayo and 4 were positive at Euroimmun alone.

Conclusions: Overall, a high degree of agreement was observed across 3 different MOG-IgG CBAs. Both live cell-based methodologies had superior PPVs to the fixed cell assays, indicating that positive results in these assays are more reliable indicators of MOG autoimmune spectrum disorders.
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http://dx.doi.org/10.1212/WNL.0000000000007096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511109PMC
March 2019

Intracellular Localization of Microbial Transglutaminase and Its Influence on the Transport of Gliadin in Enterocytes.

J Pediatr Gastroenterol Nutr 2019 03;68(3):e43-e50

Department of Paediatrics.

Objective: Celiac disease (CD) is a systemic inflammatory disorder, characterized by the destruction of duodenal epithelium. The CD8 T cells involved are associated with cross-presentation. In addition to other factors, the rising prevalence of CD might be induced by microbial transglutaminase (mTG) an enzyme frequently used in food production that shares enzymatic and antigenic properties of tissue transglutaminase (TG2), the autoantigen in CD. We hypothesized that mTG and gliadin are transported into the endoplasmic reticulum (ER), indicating cross-presentation of both antigens.

Methods: Apical incubation of duodenal biopsies from CD and control patients was performed with mTG alone or with mTG and simultaneously with Frazer's fraction. Evaluation was carried out by immunofluorescence and electron microscopy.

Results: Approximately 6% to 9% of the intracellular mTG and gliadin were transported to the ER of enterocytes. RACE cells (Rapid uptake of Antigen into the Cytosol of Enterocytes) displayed an enhanced antigen uptake into a dilated ER. mTG strongly localized at the basolateral membrane and the lamina propria.

Conclusions: mTG and gliadin are transported to the ER of enterocytes and to a greater extent to the ER of RACE cells, suggesting cross-presentation of exogenous antigens. The strong localization of mTG at the basolateral membrane and the lamina propria may also indicate a potential antigenic interaction with cells of the immune system. Since mTG may not only been taken up with food stuffs but could also be released by bacteria within the intestinal microbiota, further investigations are needed regarding the role of mTG in CD pathogenesis.
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http://dx.doi.org/10.1097/MPG.0000000000002171DOI Listing
March 2019

Immunoadsorption of Desmoglein-3-Specific IgG Abolishes the Blister-Inducing Capacity of Pemphigus Vulgaris IgG in Neonatal Mice.

Front Immunol 2018 3;9:1935. Epub 2018 Sep 3.

Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany.

Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease which is associated with autoantibodies directed against two desmosomal proteins, desmoglein (Dsg) 3 and 1. Treatment of PV is rather challenging and relies on the long-term use of systemic corticosteroids and additional immunosuppressants. More recently, autoantibody-depleting therapies such as rituximab, high-dose intravenous immunoglobulins, and immunoadsorption were shown to be valuable treatment options in PV. Specific removal of pathogenic autoantibodies would further increase efficacy and usability of immunoadsorption. Here, we tested the capacity of our recently developed prototypic Dsg1- and Dsg3-specific adsorbers to remove circulating pathogenic autoantibodies from three different PV patients. The pathogenic potential of the Dsg3/1-depleted IgG fractions and the anti-Dsg3-specific IgG was explored in two different assays based on cultured human keratinocytes, the desmosome degradation assay and the dispase-based dissociation assay. In addition, the neonatal mouse model of PV was used. In both assays, no difference between the pathogenic effect of total PV IgG and anti-Dsg3-specific IgG was seen, while Dsg3/1-depleted and control IgG were not pathogenic. For the samples of all 3 PV patients, depletion of anti-Dsg3/1 IgG resulted in a complete loss of pathogenicity when injected into neonatal mice. In contrast, injection of anti-Dsg3-specific IgG, eluted from the column, induced gross blistering in the mice. Our data clearly show that anti-Dsg3-specific IgG alone is pathogenic and , whereas Dsg3/1-depletion results in a complete loss of pathogenicity. Furthermore, our data suggest that Dsg-specific adsorption may be a suitable therapeutic modality to efficiently reduce pathogenic autoantibodies in patients with severe PV.
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http://dx.doi.org/10.3389/fimmu.2018.01935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130267PMC
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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http://dx.doi.org/10.3389/fimmu.2018.01447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046535PMC
July 2018

Reliable Serological Testing for the Diagnosis of Emerging Infectious Diseases.

Adv Exp Med Biol 2018;1062:19-43

Institute for Experimental Immunology, Euroimmun AG, Lübeck, Germany.

Climate change, increased urbanization and international travel have facilitated the spread of mosquito vectors and the viral species they carry. Zika virus (ZIKV) is currently spreading in the Americas, while dengue virus (DENV) and chikungunya virus (CHIKV) have already become firmly established in most tropical and also many non-tropical regions. ZIKV, DENV and CHIKV overlap in their endemic areas and cause similar clinical symptoms, especially in the initial stages of infection. Infections with each of these viruses can lead to severe complications, and co-infections have been reported. Therefore, laboratory analyses play an important role in differential diagnostics. A timely and accurate diagnosis is crucial for patient management, prevention of unnecessary therapies, rapid adoption of vector control measures, and collection of epidemiological data.There are two pillars to diagnosis: direct pathogen detection and the determination of specific antibodies. Serological tests provide a longer diagnostic window than direct methods, and are suitable for diagnosing acute and past infections, for disease surveillance and for vaccination monitoring. ELISA and indirect immunofluorescence test (IIFT) systems based on optimized antigens enable sensitive and specific detection of antibodies against ZIKV, DENV and CHIKV in patient serum or plasma. In recent years, Euroimmun (Lübeck, Germany) has developed numerous test systems for the serological diagnosis of (re-)emerging diseases, including a very sensitive and specific anti-ZIKV ELISA.
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http://dx.doi.org/10.1007/978-981-10-8727-1_3DOI Listing
November 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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http://dx.doi.org/10.1212/NXI.0000000000000418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778826PMC
January 2018

Routine detection of serum antidesmocollin autoantibodies is only useful in patients with atypical pemphigus.

Exp Dermatol 2017 12 29;26(12):1267-1270. Epub 2017 Oct 29.

Lübeck Institute of Experimental Dermatology (LIED), Lübeck, Germany.

Autoantibodies against the 3 desmocollin (Dsc; Dsc1-Dsc3) isoforms have been described in different pemphigus variants. Here, we developed state-of-the-art detection systems for serum anti-Dsc1, Dsc2 and Dsc1 IgG and IgA. These assays were applied in 5 different cohorts including pemphigus vulgaris (PV) patients with compatible direct immunofluorescence (IF) microscopy but no reactivity against desmogleins 1 and 3 (n = 24) and sera from patients with autoimmune blistering diseases with positive direct IF microscopy taken at the time of diagnosis (n = 749). We found that detection of anti-Dsc serum reactivity is not helpful in the routine diagnosis of PV, pemphigus foliaceus and paraneoplastic pemphigus but may be valuable in pemphigus vegetans.
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http://dx.doi.org/10.1111/exd.13409DOI Listing
December 2017

Autoantibodies against "rods and rings"-related IMPDH2 in hepatitis C genotype 1 and DAA therapy in a "real life" cohort.

Med Microbiol Immunol 2017 Oct 16;206(5):379-382. Epub 2017 Aug 16.

Department of Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany.

Autoantibodies against inosine-5'-monophosphate-dehydrogenase-2 (IMPDH2; "rods and rings" pattern) develop in chronic hepatitis C (CHC) patients under treatment with peg-interferon (IFN) and ribavirin (RBV), an inhibitor of IMPDH2. We investigated the influence of the alternative therapy with direct-acting antivirals (DAA)/ribavirin on anti-IMPDH2 autoantibody generation and the use of anti-IMPDH2 development as a marker for therapy outcome (sustained virologic response, SVR). We analyzed a "real life" cohort of 104 unselected CHC genotype 1 (GT1) patients treated with IFN/first-generation DAA/RBV prospectively compared to a historic cohort of 59 IFN/RBV-treated CHC GT1 patients. First-generation DAA were boceprevir (BOC) or telaprevir (TPR). Serum autoantibodies were tested by indirect immunofluorescence (IFA) using recombinant IMPDH2 expressing HEK293 cells and native HEp2-cells as substrates. 64/163 (39%) CHC patients turned anti-IMPDH2 positive during therapy, but only 43/163 (26%) showed also "rods and rings" structures. 99/163 (61%) were tested as anti-IMPDH2 negative. 53/104 (51%) CHC patients undergoing IFN/DAA/RBV therapy were anti-IMPDH2 positive and 38/104 (37%) were in parallel anti-"rods and rings" positive. HCV clearance/SVR rate after IFN/DAA/RBV therapy and anti-IMPDH2 status were not significantly dependent. CHC GT1 patients treated with IFN/first-generation DAA/RBV developed anti-IMPDH2 autoantibodies comparable to previous studies including patients under IFN/RBV therapy. Anti-IMPDH2 titers show no use as a marker for therapy outcome in CHC GT1 patients.
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http://dx.doi.org/10.1007/s00430-017-0516-zDOI Listing
October 2017

IgLON5 antibody: Neurological accompaniments and outcomes in 20 patients.

Neurol Neuroimmunol Neuroinflamm 2017 Sep 18;4(5):e385. Epub 2017 Jul 18.

Department of Laboratory Medicine and Pathology (J.A.H., S.R.H., V.A.L., S.J.P., A.M.), Department of Neurology (K.A.J., E.K.S.L., N.K., A.G., V.A.L., S.J.P., A.M.), Department of Medicine (E.K.S.L.), Department of Immunology (V.A.L.), and Center for Sleep Medicine (E.K.S.L.), College of Medicine, Mayo Clinic, Rochester, MN; and Institute of Experimental Immunology (L.K., K.F., S.L.), Euroimmun AG, Lubeck, Germany.

Objective: To describe the phenotypes, treatment response, and outcome of IgLON5 autoimmunity.

Methods: Archived serum and CSF specimens from 367 patients known to harbor unclassified antibodies which stained neural synapses diffusely (mimicking amphiphysin-IgG) were reevaluated by indirect immunofluorescence assay (IFA) using a composite of mouse tissues and recombinant IgLON5-transfected cell-based assay (CBA, Euroimmun).

Results: Available specimens (serum, 25; CSF, 9) from 26/367 patients (7%) had identical IFA appearance and robust IgLON5 CBA positivity. Clinical information was available for 20/26 patients; 13 were women. Median disease-onset age was 62 years (range, 46-75 years). Most patients had insidious onset and progression of neurological symptoms affecting movement and sleep predominantly. Sleep disorders were sleep-disordered breathing (11) and parasomnias (3). Brainstem disorders were gait instability (14), dysphagia (10), abnormal eye movements (7), respiratory dysfunction (6), ataxia (5), craniocervical dystonia (3), and dysarthria (3). Findings compatible with hyperexcitability included myoclonus (3), cramps (3), fasciculations (2), and exaggerated startle (2). Neuropsychiatric disorders included cognitive dysfunction (6), psychiatric symptoms (5), and seizures (1). Dysautonomia, in 9, affected bladder function (7), gastrointestinal motility (3), thermoregulation (3), and orthostatic tolerance (1). Just 2 patients had coexisting autoimmune disease. Brain MRI findings were nonspecific and CSF was noninflammatory in all tested. Seven of 9 immunotherapy-treated patients improved: 6 of those 7 were stable at last follow-up. Three untreated patients died. Each IgLON5-IgG subclass (1-4) was readily detectable in ≥80% of specimens using CBA.

Conclusions: IgLON5-IgG is diagnostic of a potentially treatable neurological disorder, where autoimmune clues are otherwise lacking.
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http://dx.doi.org/10.1212/NXI.0000000000000385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515599PMC
September 2017

Synapsin-antibodies in psychiatric and neurological disorders: Prevalence and clinical findings.

Brain Behav Immun 2017 Nov 18;66:125-134. Epub 2017 Jul 18.

Department of Neurology, Charité - Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

Objective: To study the prevalence of autoantibodies to synapsin in patients with psychiatric and neurological disorders and to describe clinical findings in synapsin antibody positive patients.

Methods: Sera of 375 patients with different psychiatric and neurological disorders and sera of 97 healthy controls were screened (dilution 1:320) for anti-synapsin IgG using HEK293 cells transfected with rat synapsin Ia. Positive sera were further analyzed by immunoblots with brain tissue from wild type and synapsin knock out mice and with HEK293 cells transfected with human synapsin Ia and Ib. Binding of synapsin IgG positive sera to primary neurons was studied using murine hippocampal neurons.

Results: IgG in serum from 23 (6.1%) of 375 patients, but from none of the 97 healthy controls (p=0.007), bound to rat synapsin Ia transfected cells with a median (range) titer of 1:1000 (1:320-1:100,000). Twelve of the 23 positive sera reacted with a protein of the molecular size of synapsin I in immunoblots of wild type but not of synapsin knock out mouse brain tissue. Out of 19/23 positive sera available for testing, 13 bound to human synapsin Ia and 16 to human synapsin Ib transfected cells. Synapsin IgG positive sera stained fixed and permeabilized murine hippocampal neurons. Synapsin IgG positive patients had various psychiatric and neurological disorders. Tumors were documented in 2 patients (melanoma, small cell lung carcinoma); concomitant anti-neuronal or other autoantibodies were present in 8 patients.

Conclusions: Autoantibodies to human synapsin Ia and Ib are detectable in a proportion of sera from patients with different psychiatric and neurological disorders, warranting further investigation into the potential pathophysiological relevance of these antibodies.
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http://dx.doi.org/10.1016/j.bbi.2017.07.011DOI Listing
November 2017

Mechanisms of Autoantibody-Induced Pathology.

Front Immunol 2017 31;8:603. Epub 2017 May 31.

Department of Biology, Institute of Genetics, University of Erlangen-Nuremberg, Erlangen, Germany.

Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves' disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein.
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http://dx.doi.org/10.3389/fimmu.2017.00603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449453PMC
May 2017

Rho-associated protein kinase 2 (ROCK2): a new target of autoimmunity in paraneoplastic encephalitis.

Acta Neuropathol Commun 2017 05 29;5(1):40. Epub 2017 May 29.

Department of Neurology, St. Josef Hospital Bochum, Ruhr University Bochum, Bochum, Germany.

Onconeural antibodies are associated with cancer and paraneoplastic encephalitis. While their pathogenic role is still largely unknown, their high diagnostic value is undisputed. In this study we describe the discovery of a novel target of autoimmunity in an index case of paraneoplastic encephalitis associated with urogenital cancer.A 75-year-old man with a history of invasive bladder carcinoma 6 years ago with multiple recurrences and a newly discovered renal cell carcinoma presented with seizures and progressive cognitive decline followed by super-refractory status epilepticus. Clinical and ancillary findings including brain biopsy suggested paraneoplastic encephalitis. Immunohistochemistry of the brain biopsy was used to characterize the inflammatory response. Indirect immunofluorescence assay (IFA) was used for autoantibody screening. The autoantigen was identified by histo-immunoprecipitation and mass spectrometry and was validated by expressing the recombinant antigen in HEK293 cells and neutralization tests. Sera from 125 control patients were screened using IFA to test for the novel autoantibodies.IFA analysis of serum revealed a novel autoantibody against brain tissue. An intracellular enzyme, Rho-associated protein kinase 2 (ROCK2), was identified as target-antigen. ROCK2 was expressed in affected brain tissue and archival bladder tumor samples of this patient. Brain histopathology revealed appositions of cytotoxic CD8 T cells on ROCK2-positive neurons. ROCK2 antibodies were not found in the sera of 20 patients with bladder cancer and 17 with renal cancer, both without neurological symptoms, 49 healthy controls, and 39 patients with other antineuronal autoantibodies. In conclusion, novel onconeural antibodies targeting ROCK2 are associated with paraneoplastic encephalitis and should be screened for when paraneoplastic neurological syndromes, especially in patients with urogenital cancers, occur.
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http://dx.doi.org/10.1186/s40478-017-0447-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448146PMC
May 2017

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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http://dx.doi.org/10.1212/NXI.0000000000000326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292928PMC
March 2017

Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies.

J Neurol Neurosurg Psychiatry 2017 04 23;88(4):353-361. Epub 2017 Jan 23.

Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.

Objectives: Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined.

Methods: Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin (I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of I-αDTX and I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2.

Results: VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical-serological correlations and a limited immunotherapy response.

Conclusions: Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against non-mammalian αDTX. These antibodies should no longer be classified as neuronal-surface antibodies. They consequently lack pathogenic potential and do not in themselves support the use of immunotherapies.
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http://dx.doi.org/10.1136/jnnp-2016-314758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5644714PMC
April 2017

Proteomic Analysis of Pemphigus Autoantibodies Indicates a Larger, More Diverse, and More Dynamic Repertoire than Determined by B Cell Genetics.

Cell Rep 2017 01;18(1):237-247

Department of Dermatology, 1008 BRB, 421 Curie Boulevard, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity.
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http://dx.doi.org/10.1016/j.celrep.2016.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221611PMC
January 2017

Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016.

Euro Surveill 2016 Dec;21(50)

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

Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0-78.4) for IgM, 88.2% (95% CI: 64.4-98.0) for IgG, and 100% (95% CI: 78.4-100) for IgM/IgG, at 99.8% (95% CI: 99.2-100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0-3.0) and 0.4% (95% CI: 0-2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas.
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http://dx.doi.org/10.2807/1560-7917.ES.2016.21.50.30426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291135PMC
December 2016

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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http://dx.doi.org/10.1212/NXI.0000000000000307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141526PMC
January 2017

Anti-Hu antibodies activate enteric and sensory neurons.

Sci Rep 2016 12 1;6:38216. Epub 2016 Dec 1.

Human Biology, Technical University of Munich, Freising, Germany.

IgG of type 1 anti-neuronal nuclear antibody (ANNA-1, anti-Hu) specificity is a serological marker of paraneoplastic neurological autoimmunity (including enteric/autonomic) usually related to small-cell lung carcinoma. We show here that IgG isolated from such sera and also affinity-purified anti-HuD label enteric neurons and cause an immediate spike discharge in enteric and visceral sensory neurons. Both labelling and activation of enteric neurons was prevented by preincubation with the HuD antigen. Activation of enteric neurons was inhibited by the nicotinic receptor antagonists hexamethonium and dihydro-β-erythroidine and reduced by the P2X antagonist pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid (PPADS) but not by the 5-HT antagonist tropisetron or the N-type Ca-channel blocker ω-Conotoxin GVIA. Ca imaging experiments confirmed activation of enteric neurons but not enteric glia. These findings demonstrate a direct excitatory action of ANNA-1, in particular anti-HuD, on visceral sensory and enteric neurons, which involves nicotinic and P2X receptors. The results provide evidence for a novel link between nerve activation and symptom generation in patients with antibody-mediated gut dysfunction.
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http://dx.doi.org/10.1038/srep38216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131267PMC
December 2016
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