Publications by authors named "Bianca Teegen"

27 Publications

  • Page 1 of 1

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

Association of thyroid peroxidase antibodies with anti-neuronal surface antibodies in health, depression and schizophrenia - Complementary linkage with somatic symptoms of major depression.

Brain Behav Immun 2020 11 1;90:47-54. Epub 2020 Aug 1.

Laboratory of Translational Psychiatry, University of Magdeburg, Magdeburg, Germany; Department of Psychiatry and Psychotherapy, University of Magdeburg, Magdeburg, Germany.

Hashimoto's thyroiditis has been associated with major depression (MD) and schizophrenia (Sz) in epidemiological studies. However, diagnostically relevant antibodies (Abs) against thyroid peroxidase (TPO) and thyroglobulin (Tg) do not act directly on neurons. We hypothesized that an increased prevalence of anti-brain-Abs in thyroid-Ab-carriers could be linked with MD and Sz even without clinically manifest Hashimoto's thyroiditis. Serum samples from 638 acutely-ill patients with MD, Sz or matched controls were systematically screened for TPO- and Tg-Abs, other endocrine-Abs and a spectrum of specific anti-brain-Abs (directed against neuronal cell surface, synaptic, other neuronal or glial proteins). Analyses were based on indirect immunofluorescence in biochip mosaics of frozen tissue sections and transfected HEK293 cells expressing respective recombinant target antigens. Psychopathology was assessed on admission and after 6 weeks treatment by HAMD-21 (in MD) or PANSS (in Sz). Seroprevalence of TPO- and/or Tg-Abs was comparable in ill and healthy individuals (MD ~10%, Sz ~7%, controls ~9%) but thyroid-Abs were associated with neuronal cell surface/synaptic-Abs (p = 0.005), particularly in schizophrenia. Thyroid Ab-positive MD patients showed higher HAMD-21 scores (particularly somatic symptoms) at baseline (p = 0.026) and better reduction of symptoms after 6 weeks (p = 0.049) than thyroid-Ab-negative patients. This was unrelated to antidepressant drug dosage, thyroid hormonal-, inflammation- and anti-brain-Ab-status. No link with PANSS scores was observed in Sz. In conclusion, the co-occurrence of thyroid-Abs and neuronal surface/synaptic-Abs may be associated with Sz. Future cerebrospinal fluid research may be promising to clarify if thyroid-Ab-associated neuronal-Abs reach the brain in Sz patients. Thyroid-Ab-related differences regarding disease-severity and -course in MD are currently unexplained, but may be caused by un-identified anti-brain-Abs or a direct action of TPO-Abs on astrocytes.
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http://dx.doi.org/10.1016/j.bbi.2020.07.039DOI Listing
November 2020

International multicenter examination of MOG antibody assays.

Neurol Neuroimmunol Neuroinflamm 2020 03 5;7(2). Epub 2020 Feb 5.

From the Clinical Department of Neurology (M. Reindl, K.S., M. Ramberger, H.H.), Medical University of Innsbruck, Innsbruck, Austria; Oxford Autoimmune Neurology Group (M.W., M. Ramberger, M.I.L., J.P., S.R.I., P.W.), Nuffield Department of Clinical Neurosciences, University of Oxford, United Kingdom; Brain Autoimmunity Group (F.T., S.R., R.C.D., F.B.), Kids Neuroscience Centre at Kids Research at the Children's Hospital at Westmead, Brain and Mind Centre, University of Sydney, New South Wales, Australia; Department of Neurology (J.S., J.P.F., J.M., E.P.F., S.J.P.), Mayo Clinic, Rochester, MN; Euroimmun Medizinische Labordiagnostika AG (B.T., S.M., N.R., U.K., W.S., C.P.), Lübeck, Germany; Institute for Quality Assurance (ifQ) affiliated to Euroimmun (J.E., M.P.), Lübeck, Germany; Paediatric Neurology (K.R.), Witten/Herdecke University, Children's Hospital Datteln, Datteln, Germany; and Department of Neurology (T.B.), Medical University of Vienna, Austria.

Objective: To compare the reproducibility of 11 antibody assays for immunoglobulin (Ig) G and IgM myelin oligodendrocyte glycoprotein antibodies (MOG-IgG and MOG-IgM) from 5 international centers.

Methods: The following samples were analyzed: MOG-IgG clearly positive sera (n = 39), MOG-IgG low positive sera (n = 39), borderline negative sera (n = 13), clearly negative sera (n = 40), and healthy blood donors (n = 30). As technical controls, 18 replicates (9 MOG-IgG positive and 9 negative) were included. All samples and controls were recoded, aliquoted, and distributed to the 5 testing centers, which performed the following antibody assays: 5 live and 1 fixed immunofluorescence cell-based assays (CBA-IF, 5 MOG-IgG, and 1 MOG-IgM), 3 live flow cytometry cell-based assays (CBA-FACS, all MOG-IgG), and 2 ELISAs (both MOG-IgG).

Results: We found excellent agreement (96%) between the live CBAs for MOG-IgG for samples previously identified as clearly positive or negative from 4 different national testing centers. The agreement was lower with fixed CBA-IF (90%), and the ELISA showed no concordance with CBAs for detection of human MOG-IgG. All CBAs showed excellent interassay reproducibility. The agreement of MOG-IgG CBAs for borderline negative (77%) and particularly low positive (33%) samples was less good. Finally, most samples from healthy blood donors (97%) were negative for MOG-IgG in all CBAs.

Conclusions: Live MOG-IgG CBAs showed excellent agreement for high positive and negative samples at 3 international testing centers. Low positive samples were more frequently discordant than in a similar comparison of aquaporin-4 antibody assays. Further research is needed to improve international standardization for clinical care.
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http://dx.doi.org/10.1212/NXI.0000000000000674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051197PMC
March 2020

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 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

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

Onconeural Antibodies in Acute Psychiatric Inpatient Care.

J Neuropsychiatry Clin Neurosci 2017 31;29(1):74-76. Epub 2016 Aug 31.

From the Dept. of Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway (SGS, MS, AV, DK, SKR); the Dept. of Psychiatry, St. Olav's University Hospital, Trondheim, Norway (SGS, MS, AV, SKR); the Institute for Experimental Immunology, Euroimmun AG, Lübeck, Germany (WS, BT, KB); and the Dept. of Neurology, Rigshospitalet, Copenhagen, Denmark (DK).

Paraneoplastic neurological disorders associated with onconeural antibodies often appear with neuropsychiatric symptoms. To study the prevalence of onconeural antibodies in patients admitted to acute psychiatric inpatient care, the serum of 585 such patients was tested for antibodies targeting MOG, GLRA1B, DPPX, GRM1, GRM5, DNER, Yo, ZIC4, GAD67, amphiphysin, CV2, Hu, Ri, Ma2, and recoverin. Only one sample was positive (antirecoverin IgG). The present findings suggest that serum onconeural antibody positivity is rare among patients acutely admitted for inpatient psychiatric care. The clinical implications of this finding are discussed.
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http://dx.doi.org/10.1176/appi.neuropsych.16050110DOI Listing
April 2017

Multiparametric serological testing in autoimmune encephalitis using computer-aided immunofluorescence microscopy (CAIFM).

Autoimmun Rev 2016 Oct 1;15(10):937-42. Epub 2016 Aug 1.

Institute for Experimental Immunology, Euroimmun AG, Seekamp 31, 23560 Lübeck, Germany. Electronic address:

Autoantibodies against neuronal cell surface antigens are tightly associated with immunotherapy-responsive autoimmune encephalitis, and a considerable number of corresponding autoantigens has been identified in recent years. Most patients initially present with overlapping symptoms, and a broad range of autoantibodies has to be considered to establish the correct diagnosis and initiate treatment as soon as possible to prevent irreversible and sometimes even life-threatening damage to the brain. Recombinant cell-based immunofluorescence allows to authentically present fragile membrane-associated surface antigens and, in combination with multiparametric analysis in the form of biochip mosaics, has turned out to be highly beneficial for parallel and prompt determination of anti-neuronal autoantibodies and comprehensive differential diagnostics. For the evaluation of recombinant cell-based IIFT, a semi-automated novel function was introduced into an established platform for computer-aided immunofluorescence microscopy. The system facilitates the microscopic analysis of the tests and supports the laboratory personnel in the rapid issuance of diagnostic findings, which is of major importance for autoimmune encephalitis patients since timely initiation of treatment may lead to their full recovery.
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http://dx.doi.org/10.1016/j.autrev.2016.07.024DOI Listing
October 2016

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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http://dx.doi.org/10.1212/NXI.0000000000000255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946772PMC
August 2016

Anti-GP2 IgA autoantibodies are associated with poor survival and cholangiocarcinoma in primary sclerosing cholangitis.

Gut 2017 01 12;66(1):137-144. Epub 2016 Jul 12.

Molecular Gastroenterology, Medical Department 1, University of Lübeck, Lübeck, Germany.

Objective: Pancreatic autoantibodies (PABs), comprising antibodies against glycoprotein 2 (anti-GP2), are typically associated with complicated phenotypes in Crohn's disease, but have also been observed with variable frequencies in patients with UC. In a previous study, we observed a high frequency of primary sclerosing cholangitis (PSC) in patients with anti-GP2-positive UC. We therefore aimed to characterise the role of anti-GP2 in PSC.

Design: In an evaluation phase, sera from 138 well-characterised Norwegian patients with PSC were compared with healthy controls (n=52), and patients with UC without PSC (n=62) for the presence of PABs by indirect immunofluorescence. Further, 180 German patients with PSC served as a validation cohort together with 56 cases of cholangiocarcinoma without PSC, 20 of secondary sclerosing cholangitis (SSC) and 18 of autoimmune hepatitis.

Results: Anti-GP2 IgA specifically occurred at considerable rates in large bile duct diseases (cholangiocarcinoma=36%, PSC and SSC about 50%). In PSC, anti-GP2 IgA consistently identified patients with poor survival during follow-up (Norwegian/German cohort: p Log Rank=0.016/0.018). Anti-GP2 IgA was associated with the development of cholangiocarcinoma in both PSC cohorts, yielding an overall OR of cholangiocarcinoma in patients with anti-GP2 IgA-positive PSC of 5.0 (p=0.001). Importantly, this association remained independent of disease duration, bilirubin level and age.

Conclusions: Anti-GP2 IgA can be hypothesised as a novel marker in large bile duct diseases. In particular, in PSC, anti-GP2 IgA identified a subgroup of patients with severe phenotype and poor survival due to cholangiocarcinoma. Anti-GP2 IgA may therefore be a clinically valuable tool for risk stratification in PSC.
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http://dx.doi.org/10.1136/gutjnl-2016-311739DOI Listing
January 2017

Diagnostic and clinical significance of Crohn's disease-specific pancreatic anti-GP2 and anti-CUZD1 antibodies.

Clin Chem Lab Med 2016 Feb;54(2):249-56

Background: Pancreatic autoantibodies (PAB) targeting GP2 and CUZD1 are Crohn's disease (CrD)-markers. The clinical significance of anti-GP2 antibodies has been assessed, but that of anti-CUZD1 remains elusive. The aim of the study was to assess the clinical utility of anti-CUZD1/anti-GP2 by novel cell-based indirect immunofluorescence (IIF) assays in CrD.

Methods: A total of 212 CrD and 249 UC patients followed up at a London IBD centre were investigated to simultaneously detect PABs, anti-GP2 and anti-CUZD1 by IIF using primate pancreatic tissue, and HEK293 over-expressing CUZD1 or GP2.

Results: Overall, 88 (41.5%) CrDs compared to 26 (10.4%) UCs (p<0.001) tested positive for IgA and/or IgG anti-GP2 and/or anti-CUZD1 antibodies, while ASCA were found in 67.5% CrDs versus 19.2% UCs (p<0.0001); ASCA and/or PAB (anti-GP2 or anti-CUZD1) were detected in 76% CrD versus 34% UC patients. IgG anti-GP2 antibodies were less prevalent in L2 phenotype (p=0.002) and more prevalent in patients with stricturing disease (p=0.0418), even when a higher cut-off (≥1000 RU) was used (p=0.0396). Also, anti-GP2 IgG positive CrD patients had younger age of disease onset. IgA and/or IgG ASCA and anti-GP2 IgG antibody positive CrDs had younger onset of disease (p<0.0001), were more likely to have both ileal and colonic disease (p<0.0001) and had more stricturing (p<0.0001) than seronegative patients. Clinical correlates were not found for anti-CUZD1 positivity.

Conclusions: PAB testing increases ASCA's serological sensitivity for CrD. Anti-GP2 detection, in isolation or in combination with ASCA, stratify CrD patients who phenotypically are characterised by a much younger onset of disease, extensive and stricturing behaviour.
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http://dx.doi.org/10.1515/cclm-2015-0376DOI Listing
February 2016

Pancreatic Autoantibodies Against CUZD1 and GP2 Are Associated with Distinct Clinical Phenotypes of Crohn's Disease.

Inflamm Bowel Dis 2015 Dec;21(12):2864-72

*Molecular Gastroenterology/Medical Department 1, University Hospital Schleswig Holstein, Campus Lübeck, Germany; †Institute for Anatomy, University of Lübeck, Campus Lübeck, Germany; ‡Department of Experimental Immunology, EUROIMMUN AG, Campus Lübeck, Germany; and §Medical Department 1, Gastroenterology, Institute for Systemic Inflammation Research, University of Lübeck, Campus Lübeck, Germany.

Background: Inflammatory bowel disease (IBD) is characterized by a broad spectrum of clinical phenotypes with different outcomes. In the last decades, several IBD-associated autoantibodies have been identified and investigated for their diagnostic relevance. Autoantibodies against the pancreatic glycoproteins (PAB) CUB and zona pellucida-like domains-containing protein 1 (CUZD1), and glycoprotein 2 (GP2) have been demonstrated to possess high specificity for the diagnosis of IBD. Although several studies have shown significant interrelations of anti-GP2 positivity with disease phenotype, associations of clinical phenotypes with anti-CUZD1 are still unknown. The aim was to identify the association of clinical phenotypes with anti-CUZD1 and anti-GP2 in a well-defined German IBD cohort.

Methods: Patients with IBD (224 patients with Crohn's disease and 136 patients with ulcerative colitis), who were tested for anti-GP2 and anti-CUZD1 immunoglobulin G and immunoglobulin A by indirect immunofluorescence on transfected cells between 2005 and 2013, were included. Serotype and specified phenotypic data were collected in retrospect and statistically analyzed.

Results: Both anti-GP2 (P < 0.001) and anti-CUZD1 (P < 0.001) were significantly more prevalent in patients with Crohn's disease than in ulcerative colitis. PAB positivity was associated with ileocolonic disease (P = 0.002), perianal disease (P = 0.011), immunosuppressive treatment (P = 0.036), and ASCA positivity (P = 0.036). Anti-CUZD1 positivity was associated with ileocolonic (P = 0.016) and perianal disease (P = 0.002), whereas anti-GP2 positivity was positively associated with stricturing behavior (P = 0.016).

Conclusions: We found distinct clinical phenotypes to be associated with PAB positivity. Therefore, determination of PABs and their subgroup analysis might identify patients with complicated disease behavior. However, the clinical relevance of our findings should be further evaluated in prospective cohorts.
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http://dx.doi.org/10.1097/MIB.0000000000000564DOI Listing
December 2015

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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http://dx.doi.org/10.1212/WNL.0000000000001493DOI Listing
April 2015

High prevalence of NMDA receptor IgA/IgM antibodies in different dementia types.

Ann Clin Transl Neurol 2014 Oct 18;1(10):822-32. Epub 2014 Oct 18.

Department of Neurology, Charité - Universitätsmedizin Berlin Berlin, Germany ; German Center for Neurodegenerative Diseases (DZNE) Berlin Berlin, Germany ; Center of Stroke Research Berlin, Germany.

Objective: To retrospectively determine the frequency of N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) autoantibodies in patients with different forms of dementia.

Methods: Clinical characterization of 660 patients with dementia, neurodegenerative disease without dementia, other neurological disorders and age-matched healthy controls combined with retrospective analysis of serum or cerebrospinal fluid (CSF) for the presence of NMDAR antibodies. Antibody binding to receptor mutants and the effect of immunotherapy were determined in a subgroup of patients.

Results: Serum NMDAR antibodies of IgM, IgA, or IgG subtypes were detected in 16.1% of 286 dementia patients (9.5% IgM, 4.9% IgA, and 1.7% IgG) and in 2.8% of 217 cognitively healthy controls (1.9% IgM and 0.9% IgA). Antibodies were rarely found in CSF. The highest prevalence of serum antibodies was detected in patients with "unclassified dementia" followed by progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease-related dementia, and primary progressive aphasia. Among the unclassified dementia group, 60% of 20 patients had NMDAR antibodies, accompanied by higher frequency of CSF abnormalities, and subacute or fluctuating disease progression. Immunotherapy in selected prospective cases resulted in clinical stabilization, loss of antibodies, and improvement of functional imaging parameters. Epitope mapping showed varied determinants in patients with NMDAR IgA-associated cognitive decline.

Interpretation: Serum IgA/IgM NMDAR antibodies occur in a significant number of patients with dementia. Whether these antibodies result from or contribute to the neurodegenerative disorder remains unknown, but our findings reveal a subgroup of patients with high antibody levels who can potentially benefit from immunotherapy.
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http://dx.doi.org/10.1002/acn3.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241809PMC
October 2014

Absence of dopamine receptor serum autoantibodies in schizophrenia patients with an acute disease episode.

Schizophr Res 2014 Sep 5;158(1-3):272-4. Epub 2014 Jul 5.

Department of Psychiatry, University of Magdeburg, Magdeburg, Germany; Center for Behavioral Brain Sciences, Magdeburg, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.schres.2014.06.025DOI Listing
September 2014

Prevalence of N-methyl-D-aspartate receptor autoantibodies in the peripheral blood: healthy control samples revisited.

JAMA Psychiatry 2014 Jul;71(7):838-9

Department of Psychiatry, University of Magdeburg, Magdeburg, Germany2Center for Behavioral Brain Sciences, Magdeburg, Germany.

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http://dx.doi.org/10.1001/jamapsychiatry.2014.469DOI Listing
July 2014

Seroprevalence of autoantibodies against brain antigens in health and disease.

Ann Neurol 2014 Jul 23;76(1):82-94. Epub 2014 Jun 23.

Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

Objective: We previously reported an unexpectedly high seroprevalence (~10%) of N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1) autoantibodies (AB) in healthy and neuropsychiatrically ill subjects (N = 2,817). This finding challenges an unambiguous causal relationship of serum AB with brain disease. To test whether similar results would be obtained for other brain antigen-directed AB previously connected with pathological conditions, we systematically screened serum samples of 4,236 individuals.

Methods: Serum samples of healthy (n = 1,703) versus neuropsychiatrically ill subjects (schizophrenia, affective disorders, stroke, Parkinson disease, amyotrophic lateral sclerosis, personality disorder; total n = 2,533) were tested. For analysis based on indirect immunofluorescence, we used biochip mosaics of frozen brain sections (rat, monkey) and transfected HEK293 cells expressing respective recombinant target antigens.

Results: Seroprevalence of all screened AB was comparable in healthy and ill individuals. None of them, however, reached the abundance of NMDAR1 AB (again ~10%; immunoglobulin [Ig] G ~1%). Appreciable frequency was noted for AB against amphiphysin (2.0%), ARHGAP26 (1.3%), CASPR2 (0.9%), MOG (0.8%), GAD65 (0.5%), Ma2 (0.5%), Yo (0.4%), and Ma1 (0.4%), with titers and Ig class distribution similar among groups. All other AB were found in ≤0.1% of individuals (anti-AMPAR-1/2, AQP4, CV2, Tr/DNER, DPPX-IF1, GABAR-B1/B2, GAD67, GLRA1b, GRM1, GRM5, Hu, LGl1, recoverin, Ri, ZIC4). The predominant Ig class depended on antigen location, with intracellular epitopes predisposing to IgG (chi-square = 218.91, p = 2.8 × 10(-48) ).

Interpretation: To conclude, the brain antigen-directed AB tested here are comparably detectable in healthy subjects and the disease groups studied here, thus questioning an upfront pathological role of these serum AB.
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http://dx.doi.org/10.1002/ana.24189DOI Listing
July 2014

Activated CD4+ T cells enter the splenic T-cell zone and induce autoantibody-producing germinal centers through bystander activation.

Eur J Immunol 2014 Jan 4;44(1):93-102. Epub 2013 Nov 4.

Center for Structural and Cell Biology in Medicine, Institute of Anatomy, University of Lübeck, Lübeck, Germany.

CD4(+) T (helper) cells migrate in huge numbers through lymphoid organs. However, little is known about traffic routes and kinetics of CD4(+) T-cell subsets within different organ compartments. Such information is important because there are indications that CD4(+) T cells may influence the function of microenvironments depending on their developmental stage. Therefore, we investigated the migration of resting (naïve), activated, and recently activated (memory) CD4(+) T cells through the different compartments of the spleen. Resting and recently activated CD4(+) T cells were separated from thoracic duct lymph and activated CD4(+) T cells were generated in vitro by cross-linking the T-cell receptor and CD28. The present study shows that all three CD4(+) T-cell subsets selectively accumulate in the T-cell zone of the spleen. However, only activated T cells induce the formation of germinal centers (GCs) and autoantibodies in rats and mice. Our results suggest that in a two-step process they first activate B cells independent of the T-cell receptor repertoire and CD40 ligand (CD154) expression. The activated B cells then form GCs whereby CD154-dependent T-cell help is needed. Thus, activated T cells may contribute to the development of autoimmune diseases by activating autoreactive B cells in an Ag-independent manner.
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http://dx.doi.org/10.1002/eji.201343811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4209793PMC
January 2014

Herpes simplex virus-1 encephalitis can trigger anti-NMDA receptor encephalitis: case report.

Neurology 2013 Oct 2;81(18):1637-9. Epub 2013 Oct 2.

From August Pi i Sunyer Biomedical Research Institute (IDIBAPS) (F.L., M.J.T., E.A., J.D.), Barcelona, Spain; University Medical-Center Hamburg-Eppendorf (F.L., J.W., M.B., S.H., M.L., M.R., T.M.), Hamburg, Germany; Erasmus Medical Center (M.J.T.), Rotterdam, the Netherlands; Institute of Experimental Immunology (B.T.), Euroimmun, Lübeck, Germany; and Catalan Institution for Research and Advanced Studies (ICREA) (J.D.), Barcelona, Spain.

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http://dx.doi.org/10.1212/WNL.0b013e3182a9f531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806918PMC
October 2013

Seroprevalence of anti-N-methyl-D-aspartate receptor antibodies in women with ovarian teratoma.

J Neurol 2013 Nov 24;260(11):2831-5. Epub 2013 Aug 24.

Department of Gynecology, Charitéplatz 1, Charité Campus Mitte, 10117, Berlin, Germany,

Recently antibodies against neuronal receptors have been identified as cause of a new type of encephalitis. The anti-N-methyl-D-aspartate receptor (anti-NMDA-R) encephalitis is the prototype of these disorders. Patients have a high incidence of teratomata. Removal of teratoma is considered the essential treatment of anti-NMDA-R encephalitis. Here, we aimed to investigate whether neurologically asymptomatic individuals suffering from ovarian teratomata may have positive anti-NMDA-R antibodies to be detected by an established assay. Over a time period of 15 months, all patients suffering from ovarian teratomata without neurological symptoms were included in this prospective study. Twenty consecutive patients were pair matched to patients with other benign ovarian disease and healthy controls. Preoperatively, patients had a gynaecological examination, transvaginal ultrasound, neurological examination and determination of anti-NMDA-R antibodies. None of the patients or controls presented with neurological symptoms. All tumours could be removed completely by laparoscopy. Anti-NMDA-R antibodies were absent in the group of patients with teratomata as well as in patients with benign ovarian tumours and healthy controls. Testing for anti-NMDA-R antibodies revealed negative findings in well-characterised patients with ovarian teratomata lacking neurological symptoms. Our data support the current clinical practice that a systematic screening for anti-NMDA-R antibodies in teratoma patients is not indicated.
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http://dx.doi.org/10.1007/s00415-013-7074-0DOI Listing
November 2013

High prevalence of IgA class anti-neutrophil cytoplasmic antibodies (ANCA) is associated with increased risk of bacterial infection in patients with cirrhosis.

J Hepatol 2013 Sep 29;59(3):457-66. Epub 2013 Apr 29.

2nd Department of Medicine, Division of Gastroenterology, University of Debrecen, Debrecen, Hungary.

Background & Aims: Anti-neutrophil cytoplasmic antibodies (ANCA) are a non-uniform family of antibodies recognizing diverse components of neutrophil granulocytes. ANCA formation might be induced by protracted bacterial infections or probably reflect an abnormal immune response to commensal microorganisms. Bacterial infections are common complications in cirrhosis with high incidence of episodes caused by enteric organisms, therefore, we sought to study the presence and clinical importance of ANCA in cirrhosis.

Methods: Sera of 385 patients with cirrhosis of different etiologies were assayed for ANCA of IgG, IgA, IgA1, IgA2, and secretory IgA subtypes by indirect immunofluorescence and ELISAs. The control group comprised 202 patients with chronic liver diseases without cirrhosis and 100 healthy subjects. In cirrhosis, a 2-year follow-up, observational study was conducted to assess a possible association between the presence of ANCA and clinically significant bacterial infections.

Results: Prevalence of ANCA IgA was significantly higher in cirrhosis (52.2%) compared to chronic liver diseases (18.6%) or healthy controls (0%, p<0.001 for both). ANCA IgA subtyping assays revealed marked increase in the proportion of IgA2 subtype (46% of total ANCA IgA) and presence of the secretory component concurrently. Presence of ANCA IgA was associated with disease-specific clinical characteristics (Child-Pugh stage and presence of ascites, p<0.001). During a 2-year follow-up period, risk of infections was higher among patients with ANCA IgA compared to those without (41.8% vs. 23.4%, p<0.001). ANCA IgA positivity was associated with a shorter time to the first infectious complication (pLogRank <0.001) in Kaplan-Meier analysis and was identified as an independent predictor in multivariate Cox-regression analysis (HR:1.74, 95% CI: 1.18-2.56, p=0.006).

Conclusions: Presence of IgA type ANCA is common in cirrhosis. Involvement of gut mucosal immune system is in center of their formation and probably reflects sustained exposure to bacterial constituents.
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http://dx.doi.org/10.1016/j.jhep.2013.04.018DOI Listing
September 2013

Development of a recombinant cell-based indirect immunofluorescence assay for the determination of autoantibodies against soluble liver antigen in autoimmune hepatitis.

Clin Dev Immunol 2013 16;2013:572815. Epub 2013 Jan 16.

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

Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.
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http://dx.doi.org/10.1155/2013/572815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562650PMC
July 2013

Development of a recombinant cell-based indirect immunofluorescence assay (RC-IFA) for the determination of autoantibodies against "rings and rods"-associated inosine-5'-monophosphate dehydrogenase 2 in viral hepatitis C.

Clin Chim Acta 2013 Mar 16;418:91-6. Epub 2013 Jan 16.

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

Background And Aims: Autoantibodies against so-called "rings and rods" structures, as determined by indirect immunofluorescence assay (IFA) using the human cell line HEp-2, have been described in chronic hepatitis C virus (HCV) infected patients treated with interferon/ribavirin. Recently, cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the enzyme inhibited by ribavirin, were proposed as the target antigens. We wanted to confirm the identification and setup a robust system for autoantibody testing in routine laboratories.

Methods: CTPS and IMPDH2 were individually expressed in HEK293 cells and the recombinant cells were used in IFA (RC-IFA) to analyze sera from 33 anti-"rings and rods" antibody positive individuals with unknown diagnosis, 50 patients with chronic HCV infection, 100 with autoimmune hepatitis, 50 with primary biliary cirrhosis and 50 healthy blood donors.

Results: We found that all sera with anti-"rings and rods" reacted with recombinant IMPDH2 but none with CTPS. In western blot or ELISA, anti-IMPDH2 positive sera reacted only weakly, if at all, with Escherichia coli derived recombinant IMPDH2 indicating that the autoantibody reaction probably depends on the 3-dimensional conformation of the antigen. A rabbit hyperimmune serum raised against bacterially expressed IMPDH2 produced the ring/rods pattern in IFA using HEp-2.

Conclusions: We conclude, that IMPDH2 is indeed the main target of anti-"rings and rods" while CTPS is an unlikely target. Moreover, the novel RC-IFA test system allows a standardized semi-quantitative determination of anti-IMPDH2 basing on a defined recombinant antigen.
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http://dx.doi.org/10.1016/j.cca.2013.01.003DOI Listing
March 2013

Autoantibodies against exocrine pancreas in Crohn's disease are directed against two antigens: the glycoproteins CUZD1 and GP2.

J Crohns Colitis 2013 Nov 7;7(10):780-90. Epub 2012 Nov 7.

Institute of Experimental Immunology, Euroimmun AG, Luebeck, Germany. Electronic address:

Background: Autoantibodies against exocrine pancreas (PAb) have been reported to be pathognomonic markers of Crohn's disease (CD). Recently, the glycoprotein GP2 has been proposed as the exclusive target for PAb but two equally prevalent binding patterns can be observed in the indirect immunofluorescence test (IIFT) using cryosections of human pancreas: a reticulogranular and a droplet pattern.

Aim: To identify autoantigens corresponding to the staining patterns.

Methods: Different lectins were screened for their ability to immobilize PAb-reactive glycoproteins from cell free human pancreas. The glycoproteins were then purified via UEA-I affinity chromatography and identified by mass spectrometry. The two candidate autoantigens were separately expressed in HEK293 cells, and the recombinant cells applied as substrates in IIFT to analyze sera from 96 patients with CD, 89 controls and hybridoma supernatants during the generation of murine monoclonal antibodies.

Results: The UEA-I eluate was able to neutralize PAb reactivity of both patterns in IIFT. It contained two major constituents which were identified as the glycoproteins CUZD1 and GP2. With the recombinant cells, 35.4% of the CD patients exhibited positive reactions (CUZD1 alone 19.8%, GP2 alone 9.4%, and both antigens 6.2%). The reaction with the CUZD1 expressing cells was strictly correlated to the reticulogranular pattern, whereas the antibodies causing the droplet pattern stained the GP2 expressing cells. Antigen-capture ELISA using the newly generated monoclonal antibodies against CUZD1 and GP2 verified this relationship.

Conclusions: The concordant reactivities of the different platforms can be regarded as a proof for the authenticity of the two identified autoantigens.
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http://dx.doi.org/10.1016/j.crohns.2012.10.011DOI Listing
November 2013

DNA-bound lactoferrin is the major target for antineutrophil perinuclear cytoplasmic antibodies in ulcerative colitis.

Ann N Y Acad Sci 2009 Sep;1173:161-5

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

Lactoferrin has repeatedly been proposed to be a target for antineutrophil perinuclear cytoplasmic antibodies (P-ANCA), which are present in 67% of ulcerative colitis (UC) cases. However, this high prevalence has not been achieved with either Western blots or monospecific ELISA on the basis of purified lactoferrin bound to the solid phase. We reevaluated autoantibodies against lactoferrin by indirect immunofluorescence (IIF), using a lactoferrin-tuned granulocyte substrate. Slides with ethanol-fixed human granulocytes were stripped of their P-ANCA targets by high-salt treatment and then reconstituted with human lactoferrin (LFR granulocytes). The slides were then subjected to IIF with a panel of sera (39 UC, 10 antimyeloperoxidase-positive vasculitis, 50 healthy blood donors). The human sera were also analyzed with antilactoferrin ELISA. In 28 of 39 (71.8%) sera from UC patients, antibodies could be determined that bound exclusively to LFR granulocytes. Nuclease-treated cells failed to show this reactivity. ELISA detected antilactoferrin antibodies in only two UC sera. Lactoferrin is a major P-ANCA target in UC but requires DNA to present the epitopes relevant for the reaction with the autoantibodies. Antigen-stripped and lactoferrin-reconstituted granulocytes can be used in IIF to diagnose antilactoferrin antibodies in UC more reliably than with existing ELISA systems.
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http://dx.doi.org/10.1111/j.1749-6632.2009.04752.xDOI Listing
September 2009
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