Prevalence of elevated serum anti-N-methyl-D-aspartate receptor antibody titers in patients presenting exclusively with psychiatric symptoms: a comparative follow-up study

The present study included inpatients that developed new-onset or exacerbations of schizophrenia-like symptoms and were from neurology and psychiatry departments at institutions from June 2012 to March 2014. Target cases were chosen by the study partaker of each institution. When agreement was not provided by the patients themselves, we explained the purpose of the present study to a family member and obtained their consent. We obtained written consent from patients and/or their families (caregivers) when patients were unable to comprehend because of psychiatric symptoms. Furthermore, written consent to publication of more detailed clinical information such as age, sex, and symptoms was included.

The present study was conducted with the approval of the Bioethics Committee for Clinical Research, Jichi Medical University Hospital. Any collaborating institution without its own ethical review board was also represented by the Bioethics Committee for Clinical Research, as requested in writing.

A total of 59 cases (23 male, 36 female) were enrolled. The mean age was 42.0 ± 13.7 (SD) years. Twenty-one patients had a first episode of symptoms, 37 had recurrent episodes (second, third, fourth, and fifth recurrences in 19, five, three, and ten patients, respectively), and one case was unclear.

Patient information was collected using a questionnaire completed by participants. The initial questionnaire at the time of the first examination included questions regarding age, sex, blood pressure, pulse rate, body temperature, and Japan coma scale (JCS). Further sections were as follows: clinical diagnosis according to Diagnostic and Statistical Manual of Mental Disorders IV text revision (DSM-IV TR); history of preceding infection; characteristics of onset or recurrence; number of recurrences and disease duration; psychiatric symptoms (anxiety/depression, mania, or schizophrenia-like symptoms); and neurological symptoms. Furthermore, we included items related to psychiatric symptoms and neurological symptoms (Table 1). The JCS is the original Japanese consciousness scale and is more widely used than the Glasgow coma scale (GCS) in Japan. However, the accurate comparison of scores between the GCS and JCS is challenging as there is no item corresponding to the “Verbal” score in the JCS. A consciousness score of 0 indicates the patient is lucid, and scores of 1–3 are equivalent to a GCS of E4, indicating spontaneous eye-opening. Scores of 10–30 indicate eye-opening in response to stimulation, with a score of 10 being equivalent to a GCS score of E3 and scores of 20–30 being equivalent to E2. Scores >100 are equivalent to E1, and a score of 300 is equivalent to a score of M1 on the GCS.

Patients were divided into two groups on the basis of the presence or absence of anti-NMDA receptor antibodies, and answers to individual questionnaire items were compared. Welch’s t-test was performed for comparison of continuous variables, including age, blood pressure, pulse rate, and body temperature. For categorical data such as sex and items assessing the presence or absence of symptoms, Fisher’s direct exact test was used because of the small number of antibody-positive patients. JCS at the time of the hospitalization was divided into seven classes: 0, 1, 5, 3, 10, 30, and 100. The number of recurrences was divided into five classes: 1, 2, 3, 4, and >5. Disease duration was divided into seven classes: first, 1 month, 3 month, 1 year, 5 years, 10 years, and >10 years, which were assessed using the Mann–Whitney U test. All statistics were performed using the software “Stat Flex ver.6”(Artech, Osaka, Japan). All tests were two-tailed, and significance was set at P < 0.05.

Blood and/or cerebrospinal fluid was collected at the time of hospitalization at each institution. Serum was extracted, frozen, and stored until further use. All samples were anonymized and sent to the Department of Neurology, Jichi Medical University. A frozen serum specimen for each patient was subsequently sent to the Department of Neurology, Kanazawa Medical University.

All serum obtained at the first study visit was measured using the following methods.

Human embryonic kidney (HEK) 293 cells transfected with plasmid DNAs encoding the NMDA receptor subunits NR1 and NR2B with cell membrane co-expression were used [3]. Cell culture was maintained in Dulbecco’s modified Eagle's medium with 10 % fetal calf serum supplemented with 10 μM MK-801 (Wako, Tokyo, Japan) for neuroprotection. At 12 h after transfection, HEK cells were fixed in 4 % paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 20 min. Non-specific binding was blocked with 10 % goat serum/PBS, and cells were incubated with patient sera (1:10–400) in 0.02 % Triton X-100 and 10 % goat serum in PBS overnight at 4 °C and then with fluorescein isothiaocyanate (FITC)-conjugated anti-human IgG (DAKO, Glostrup, Denmark; 1:50) and phycoerythrin (PE)-conjugated anti-rabbit IgG secondary antibodies (DAKO, Glostrup, Denmark; 1:50) for 1 h. SlowFade Gold anti-fade reagent (Invitrogen Japan, Tokyo) was then applied to the slides, and staining was observed under a fluorescence microscope (Axiovision, Zeiss). To confirm the localization of NMDAR antibody-binding sites, double staining was performed with both patient samples and a mixture of rabbit anti-NR1 (PhosphoSolutions, CO, USA; 1:10) and anti-NR2B (Frontier Science, Sapporo, Japan; 1:10) antibodies. Then, primary antibodies were visualized by FITC-anti-human IgG and R-PE-anti-rabbit IgG and observed under a laser scanning microscope (Zeiss LSM 710). IgG binding was confirmed as IgG1. No staining was observed in HEK cells expressing homomeric NR2B subunits or transfected with an empty vector. Serial dilutions were used, and where co-localization was observed, antibody titers were measured (Fig. 1). Serum from subsequent follow-up visits was analyzed using the same methodology.

Paraffin-embedded sections of mouse brain (Genostaff Co. Ltd. Tokyo, Japan) were stained with rabbit anti-GluN1 antibody and anti-GluN2B antibody (Frontier Institute Co. Ltd. Hokkaido, Japan) at a 1:200 dilution or human serum at a 1:1000 dilution that included the serum of an antibody-positive 59-year-old male patient. Staining was detected using biotinylated anti-rabbit or anti-human Ig secondary antibodies (Vector Laboratories, Inc., Burlingame, CA, USA). For histochemical evaluation, hematoxylin staining was used to visualize the overall structure of each section.

Animal experiments were conducted in a humane manner after receiving approval from the Institutional Animal Experiment Committee of the GENOSTAFF CO.LTD. and in accordance with the Institutional Regulation for Animal Experiments and Fundamental Guideline for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the justification of the Ministry of Education, Culture, Sports, Science and Technology.