A-H, Representative results of rat-brain immunohistochemistry testing using human sera. Autoantibody reactivity was shown by using a biotinylated antihuman IgG-specific secondary antibody, streptavidin-conjugated peroxidase and diaminobenzidine. The grading of rat-brain immunohistochemistry was based on 4 staining intensities of the hippocampus (A, grade 0, from a healthy control participant; B, grade 1, and C, grade 2, from the sera of the affected cohort; and D, grade 3, from the serum of an patient with autoimmune encephalitis and anti–contactin-associated protein-like 2 [Caspr2] antibodies). E-H, Samples that were also reactive on cell-based assays or neuronal staining. Scale bar = 500 μm (D). I-T, Results from live cell-based assays for Caspr2. Human sera were incubated on live human embryonic kidney 293 cells transfected with Caspr2 (with mCherry tag [red], I-L) and stained with antihuman IgG Alexa-488 (green). The presented results include a positive result from a participant with anti-Caspr2 encephalitis (M and Q; the same serum as in D) and individuals with psychosis (N and R) or from the control group (O and S) and a negative sample from a control participant (P and T). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Q-T, Images show mCherry, DAPI, and positive IgG staining. Scale bar = 50 μm (T). U-X, Staining on rat hippocampal primary live neurons. Neurons were incubated with human serum samples and subsequently with antihuman IgG Alexa-488 and DAPI. Each image represents 1 or 2 neurons (scale bar = 50 μm [X]), and underneath, the zoomed-in image of the area identified with a white outline (scale bar = 10 μm [AB]). U, A positive result is represented by serum from a patient with encephalitis and autoantibodies against dipeptidyl-peptidase-like protein-6 (DPPX); V, sera of a patient with schizophrenia (the same individual as in G) and an individual from the control cohort (the same individual as in H), both tested with low reactivity on neurons, and the control participant with a negative result was represented by serum from a healthy individual. All sera were diluted 1:50. Nuclei were stained with DAPI.
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Hoffmann C, Zong S, Mané-Damas M, et al. Absence of Autoantibodies Against Neuronal Surface Antigens in Sera of Patients With Psychotic Disorders. JAMA Psychiatry. 2020;77(3):322–325. doi:10.1001/jamapsychiatry.2019.3679
Patients with antibodies against neuronal surface antigens (NSAbs), including the N-methyl-d-aspartate receptor (NMDAR), typically develop autoimmune encephalitis with characteristic neurological and psychiatric symptoms.1 In 4% of individuals with NMDAR encephalitis, isolated psychotic episodes occur without simultaneous neurological involvement.2 This raises the question of whether NSAbs are underdiagnosed in patients with psychotic disorders. Results of studies testing this, however, are contradictory,3-5 possibly because of differences in the methods of antibody detection. Previously, in a cohort of 475 patients with schizophrenia, we found no one with NMDAR antibodies after excluding 2 false-positive results by combined immunohistochemistry (IHC) tests and cell-based assays (CBAs) across laboratories.6 In this analysis, we extend these studies to other common NSAbs known to cause encephalitis, aiming to expand the clinical spectrum of psychosis potentially caused by autoantibodies.
The cohort included individuals with psychotic disorders, who were further divided into those with schizophrenia, schizoaffective disorder, brief psychotic disorder, and first-episode psychosis diagnoses (as defined by the DSM-IV), and control participants (Table). Samples from all participants were screened by IHC testing for immunoreactivity to rat hippocampus, and if results were questionable or positive (grades 1-3), the samples were analyzed by live and fixed CBAs. If samples were graded 3 on IHC testing, they were also tested on live primary rat hippocampal neurons (Figure, A).
Approval for this study was obtained from the review boards of Erasmus Medical Centre, University Medical Centre Utrecht, and Katholieke Universiteit Leuven. Written consent was obtained from patients and control participants. Images of rat-brain IHC were taken by the iScan HT slide scanner (Ventana; ×20 objective) and visually graded 0 to 3 (Ventana Image Viewer) for the hippocampal immunoreactivity of sera based on the intensity and contrast of the staining. Statistical analyses were done in SPSS version 23.0 for Windows (IBM). Data were collected from February 2016 to February 2018.
A total of 621 affected individuals (mean [SD] age, 34.4 [11.9] years; 248 female participants [39.9%]) and 257 control participants (mean [SD] age, 44.1 [16.6] years; 112 female participants [47.5%]) were included. Overall, 40 of 878 serum samples (4.6%) were IHC positive (grades 2-3), with no difference between groups (Table). Representative stainings with grade 0 to 3 are shown in the Figure, as well as staining patterns of 4 cases that also tested positive on live CBA or neurons. Further analyses of the IHC-reactive sera for known NSAbs revealed that 6 of 94 samples (6.4%) had contactin-associated protein-like 2 (Caspr2) autoantibodies detected by a live CBA but not a fixed CBA; 3 were from individuals with psychotic disorders and 3 from control participants (Figure). However, of these, only 3 had patterns similar to Caspr2 on IHC testing (1 from an individual with schizophrenia and 2 from control participants). One serum sample from a person with schizophrenia and 1 from a control participant (at 1:50 dilution) had weakly positive results on live neurons (Figure). Sera graded 3 by IHC testing or found to be positive by any CBA (n = 25) were retested in a reference diagnostic laboratory according to established diagnostic procedures for autoimmune encephalitis. No known autoantibodies by IHC testing, fixed CBA, or staining on live neurons (at 1:200 dilution) were detected there.
Recent findings with live CBA tests showed that NMDAR autoantibodies were present in 3% of patients with first-episode psychosis but not in control participants; other NSAbs (including anti-Caspr2) were not disease specific.5 In contrast, we did not find any individuals who were anti-NMDAR positive in the subcohort with first-episode psychosis. The Caspr2 antibodies detected by live CBA were not confirmed by other methods. Antibodies that are only detectable by live CBA are thus unlikely to be relevant, highlighting the importance of careful laboratory procedures and standardization regarding autoantibody measurements.
Although brain-reactive autoantibodies were found by IHC testing in both the group with disorder and the control group, no individual sample showed a known NSAbs pattern, as confirmed by the fixed CBA results and the reference laboratory results. Only 1 individual with a disorder and 1 control participant had unidentified NSAbs by live-neuron staining. These results indicate despite extensive laboratory testing that the levels of autoantibodies did not differ between affected individuals and control participants within the study population.
Limitations of this study are that cerebrospinal fluid samples were unavailable and positive samples could have been missed by IHC testing (eg, because of species differences). In light of the cross-sectional nature of the study and the relatively small number of individuals in each diagnostic group, it is conceivable that autoantibodies might be found in studies with larger sample sizes and/or longitudinal samples collected at different times during the course of the illness.
Corresponding Author: Pilar Martinez-Martinez, PhD, School for Mental Health and Neuroscience, Maastricht University, Universiteitssingel 50, 6229 ER, Maastricht, the Netherlands (firstname.lastname@example.org).
Published Online: November 20, 2019. doi:10.1001/jamapsychiatry.2019.3679
Author Contributions: Dr Martinez-Martinez had access to the data in the study and takes responsibility for the integrity and data analysis. Ms Hoffmann and Mr Zong contributed equally to this work.
Concept and design: Hoffmann, Zong, Losen, Martinez.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Hoffmann, Zong, Molenaar, Martinez.
Critical revision of the manuscript for important intellectual content: All authors.
Statistical analysis: Zong, Martinez.
Obtained funding: Zong, Martinez.
Administrative, technical, or material support: Hoffmann, Zong, Mané-Damas, Losen, Titulaer, Martinez.
Supervision: Losen, Martinez.
Conflict of Interest Disclosures: Dr Titulaer reported grants from the Dutch Epilepsy Foundation, Netherlands Organisation for Scientific Research, Netherlands Organisation for Health Research and Development/ZonMW, and an Erasmus Medical Centre fellowship and received unrestricted research funding from Euroimmun AG, MedImmune LLC, Guidepoint Global LLC, and Novartis outside the submitted work. In addition, Dr Titulaer’s institution, the Erasmus Medical Center, has a patent for methods for typing neurological disorders and cancer and devices for use therein pending. No other disclosures were reported.
Funding/Support: This study was supported by the Netherlands Organization for Scientific Research (grant 022005019), the Brain Foundation of the Netherlands (grant KS2012(1)-157), and the ZonMW Netherlands Organisation for Health Research and Development Program Translationeel Onderzoek (grant 40-41200-98-9257); a fellowship of the Brain Foundation of the Netherlands (grant FS2008(1)-28); and the China Scholarship Council (grant 201507720015). The European Network of National Schizophrenia Networks Studying Gene-Environment Interactions Project was funded by the European Community's Seventh Framework Programme (grant HEALTH-F2-2010-241909), and the collection of samples in Turkey was supported by the Research Fund of the University of Istanbul (grant 23979).
Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Group Information: We acknowledge the collaborators for this article of the European Consortium of Autoimmune Mental Disorders (CAIMED): Maastricht University, Maastricht, Netherlands: Jo Stevens, PhD, Gisela Nogales-Gadea, PhD, Jan Damoiseaux, PhD, Ashna Silas, MSc, Marc De Baets, MD, PhD, Wim Buurman, PhD, Bart Rutten, MD, PhD, Jim van Os, MD, PhD; Erasmus University Medical Center Rotterdam Netherlands: Nico van Beveren, MD, PhD, Marco Schreurs, PhD; IMMCO Diagnostics, Buffalo, New York: Kishore Malyavantham, PhD, Vincent Ramsperger, MSc, Lakshmanan Suresh, PhD; Istanbul University, Istanbul, Turkey: Cem İsmail Küçükali, MD, PhD, Erdem Tüzün, MD; Hôpitaux Universitaires Créteil, Créteil, France: Andrei Szoke, MD, PhD; Universiteit Leuven, Leuven, Belgium: Marc De Hert, MD, PhD; Universidad Complutense Madrid Spain: Emiliano González-Vioque, PhD; Hospital General Universitario Madrid Spain: Celso Arango, MD, PhD
Additional Contributions: We thank Josep Dalmau, MD, PhD, Hospital Clinic, University of Barcelona, and Frank Leypoldt, MD, PhD, University Hospital Schleswig-Holstein, for their scientific advice. We also thank Fabienne Brilot, PhD, University of Sydney, Patrick Waters, PhD, Oxford University, Catherine Faivre-Sarrailh, PhD, Aix-Marseille Université, and Francesc Graus, MD, PhD, Hospital Clinic, University of Barcelona, for generous gifts of plasmid DNA for antigen expression, and Christiane Hampe, PhD, University of Washington, for a gift of GAD antibodies. No compensation was received from a funding sponsor for such contributions.
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