Bianca Teegen

Seroprevalence of autoantibodies against brain antigens in health and disease.

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.

Herpes simplex virus–1 encephalitis can trigger anti-NMDA receptor encephalitis: Case report

Relapsing symptoms post herpes simplex virus-1 (HSV) encephalitis (HSVE) usually occur a few weeks after viral therapy and represent either 1) a true viral relapse of HSVE (CSF PCR positive for HSV, new necrotic lesions on brain MRI, and response to acyclovir therapy) or 2) a disorder postulated to be immune-mediated (CSF negative for HSV, no new necrotic lesions, and no response to acyclovir).1,2 It has been suggested that this immune-mediated disorder may be related to NMDA receptor (NMDAR) antibodies,3 and we recently reported a child in whom relapsing symptoms post HSVE were the presentation of anti-NMDAR encephalitis.4 We report an adult with this disorder, demonstrate that synthesis of NMDAR antibodies began after HSVE, and show that relapsing symptoms were due to steroid-responsive anti-NMDAR encephalitis.

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

BACKGROUND Autoantibodies against exocrine pancreas (PAb) have been reported to be pathognomonic markers of Crohns 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.

Prevalence of N-Methyl-D-Aspartate Receptor Autoantibodies in the Peripheral Blood: Healthy Control Samples Revisited

Prevalence of N-Methyl-D-Aspartate Receptor Autoantibodies in the Peripheral Blood: Healthy Control Samples Revisited In March 2013, we published a study in JAMA Psychiatry assessing the prevalence of anti–N-methyl-D-aspartate glutamate receptor (NMDAR) serum autoantibodies in unmedicated acutely ill patients with schizophrenia, major depression (MD), borderline personality disorder (BLPD), and healthy control individuals without neuropsychiatric disorder.1 The laboratory analyses of this set of samples were done in 2010. N-methyl-Daspartate glutamate receptor antibodies were identified in 15 patients, primarily in those with a clinical diagnosis of schizophrenia (12 of 121, 9.9%) as opposed to those with MD (2 of 70, 2.9%), those with BLPD (0 of 38, 0%), and control individuals (1 of 230, 0.4%). Two patients were initially misdiagnosed as having catatonic or disorganized schizophrenia. Retrospectively, these cases were reclassified as cases with NMDAR encephalitis (presence of specific IgG anti-NR1a autoantibodies in both serum and cerebrospinal fluid).

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

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

DNA‐Bound Lactoferrin Is the Major Target for Antineutrophil Perinuclear Cytoplasmic Antibodies in Ulcerative Colitis

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.

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

Abstract 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.

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

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 Crohns 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 Crohns 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.

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

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: Patients 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 patients 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.

Prevalence of serum anti-neuronal autoantibodies in patients admitted to acute psychiatric care.

BACKGROUND Autoimmune encephalitis associated with anti-neuronal antibodies may be challenging to distinguish from primary psychiatric disorders. The significance of anti-neuronal antibodies in psychiatric patients without clear evidence of autoimmune encephalitis is unknown. We investigated the serum prevalence of six anti-neuronal autoantibodies in a cohort of unselected patients admitted to acute psychiatric care. METHOD Serum was drawn from 925 patients admitted to acute psychiatric in-patient care. Psychiatric diagnoses were set according to International Classification of Diseases (ICD)-10 criteria. Antibody analysis was performed with an indirect immunofluorescence test for N-methyl d-aspartate receptor (NMDAR) antibodies and five other anti-neuronal autoantibodies of the immunoglobulin (Ig) classes IgA, IgG and IgM isotype. RESULTS Anti-neuronal autoantibodies were found in 11.6% of patients: NMDAR antibodies in 7.6%, contactin-associated protein-like 2 (CASPR2) antibodies in 2.5%, glutamic acid decarboxylase-65 (GAD65) antibodies in 1.9%, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antibodies in 0.1%. Leucine-rich glioma-inactivated protein-1 (LGI1) and γ-aminobutyric acid B (GABAB) receptor antibodies were not detected. NMDAR antibodies of class IgG were present in five patients only (0.5%). NMDAR antibodies of all Ig classes were equally prevalent in patients with and without psychosis. There were no significant differences in antibody prevalence in the different diagnostic categories, except for a higher odds ratio of being NMDAR antibody positive for patients without a specific psychiatric diagnosis. CONCLUSIONS NMDAR IgG autoantibodies, which are known to be strongly associated with anti-NMDAR encephalitis, were rarely found. CASPR2 and GAD65 antibodies were more frequently encountered in the present study than previously reported. Further research on the clinical significance of anti-neuronal autoantibodies in patients with acute psychiatric symptoms is needed.