Ethical approval

All parts of this study were approved by the Lund University Medical Ethics Commitee. All subjects gave verbal informed consent to participate in the study. Written informed consent was not required per the ethical approval. Consent was registered in the patients’ chart. Subjects who, in the investigator's judgment, lacked the ability to make an informed decision regarding study participation were not included in the study.

Subject recruitment

Between 1987 and 2001, 29 subjects were enrolled in the study on admission to Lund University after a suicide attempt, as defined by Beck et al. [31]. Suicide attempters were evaluated by a certified psychiatrist for DSM axis I and II diagnoses [32]. Depressive symptoms were rated using the Montgomery-Åsberg Depression Rating Scale [33]. The 29 subjects included in this pilot study were randomly selected from a larger cohort of suicide attempters, that has been described in more detail elsewhere [7]. All subjects underwent a general physical examination before the lumbar puncture, and they were all somatically healthy except for two subjects with migraines, and two subjects with gastritis. Although some of the subjects had previously been treated with psychoactive medications, they did not receive any antidepressants or antipsychotics during a washout-period before the lumbar puncture. Occasional doses of benzodiazepines were allowed during the washout. Lumbar punctures were performed in the morning between 8 am and 9 am, after a night of fasting and bed rest. CSF was collected from the L4-L5 interspace using a standardized protocol, and immediately stored at -80°C. Twenty of the 29 CSF samples had been previously thawed/frozen at least once before the analyses.

Assays

Six different autoantibodies previously implicated in autoimmune encephalitis [20] were analyzed; NMDAR, the α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid receptor (AMPAR), the γ-amino-butyric acid B-receptor (GABAbR), the leucine-rich, glioma-inactivated 1 (LGI1), the contactin-associated protein-like 2 (Caspr2), and the dipeptidyl-peptidase-like protein-6 (DPPX). These autoantibodies, associated clinical syndromes and frequent symptoms are summarized in Table 1 (adapted from Wandinger et al 2011 [34]).

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Table 1. Autoantibodies tested in the present study, associated clinical syndromes and frequent symptoms (adapted from Wandinger et al 2011).

https://doi.org/10.1371/journal.pone.0176358.t001

Antibody detection was done using the Autoimmune Encephalitis 6 Biochip mosaics (EUROIMMUN, Lübeck, Germany) by trained laboratory personnel at a specialized diagnostic laboratory affiliated with Lund University Hospital (Wieslab, Malmö, Sweden). Each biochip contains transfected cells expressing a specific antigen (NMDAR, AMPAR, GABAbR, LGI1, Caspr2, and DPPX). Biochip mosaics were incubated each with 30 μl of un-diluted CSF for 30 minutes at room temperature, washed with PBS-Tween, and immersed in PBS-Tween for 5 minutes. Bound antibodies were stained with fluorescein isothiocyanate–labeled goat anti-human IgG, (Euroimmun) for 30 minutes at room temperature. Slides were washed again with a flush of PBS-Tween and then immersed in PBS-Tween for 5 minutes. Drops of PBS-buffered glycerol were placed onto a cover glass, and the biochip slides were embedded in this mounting medium simultaneously and examined by fluorescence microscopy. Positive and negative controls were included with every test procedure. Autoantibodies against the corresponding antigen react specifically with the corresponding transfected cells and results are given as positive or negative. NMDA receptor autoantibodies react specifically with the corresponding transfected cells and induce a fine granular cytoplasmic fluorescence, while the cell nuclei are only slightly stained. Antibodies against AMPA receptors, CASPR2, LGI1, and GABAB1/B2 receptors react specifically with the cytoplasm of the corresponding transfected cells inducing a cytoplasmic fluorescence, with some fluorescence of the cell membrane, while the cell nuclei are only slightly stained. Antibodies against DPPX react with the transfected cells of the test substrate. They produce a spread, smooth to fine-speckled cytoplasmic fluorescence, partly with fluorescence of the cell membrane. The cell nuclei are only slightly stained.

Samples with no autoantibodies or negative control cells results in no fluorescent staining. The test results were interpreted manually by extensively trained personnel by reading the fluorescence of fixated cells in a microscopy and results are reported as positive or negative. There are no predetermined cut-off values, since a negative result will show no fluorescence.

Albumin was determined in serum and CSF as previously described [29]. CSF/serum albumin ratio was calculated as CSF albumin (g/l)/serum albumin (mg/l).

Demographics

Demographic and clinical characteristics of the study participants are summarized in Table 2. Approximately two-thirds of the subjects had Axis II co-morbidity, cluster B being the most frequent specifier (n = 10).

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Table 2. Demographic and clinical characteristics of study participants (n = 29).

https://doi.org/10.1371/journal.pone.0176358.t002

Most of the subjects (n = 21) had made a non-violent suicide attempt, e.g. intoxication, while the remainder (n = 8) had made a violent suicide attempt, e.g. carbon monoxide poisoning or jumping in front of a train [35]. Ten of the subjects had attempted suicide more than once and for 19 subjects this was the first attempt.

Autoantibodies in CSF

None of the following autoantibodies were detected in any of the CSF samples: NMDAR, AMPAR, GABAbR, LGI1, Caspr2, DPPX.

CSF/serum albumin ratio

Five subjects had increased CSF/serum albumin ratio, in relation to age-adjusted reference values previously described [36]. For individuals <45 years values above 6.8 were considered high, while the cut-off was 10.2 for those aged 45 and over.

Interpretation and discussion of results

In this pilot study we investigated, the presence of autoimmune antibodies in CSF samples from a group of recent suicide attempters. Given that neuroinflammation has been implicated in the pathophysiology of suicidality [1], this is an important research question that needs to be pursued. Although classical autoimmune encephalopathies are typically associated with aberrant neurological signs and symptoms, such as seizures, affected motor functions and speech, there is also a growing interest in autoimmune encephalopathies that may present with “pure” psychiatric symptoms. The present sample was randomly selected from a larger cohort of somatically healthy and medication-free recent suicide attempters. We tested six different autoantibodies previously implicated in clinical encephalopathies characterized by a psychiatric symptoms [20]. The findings of the present study were negative; we did not detect any of these autoantibodies in any of the CSF samples. These results, however, should be interpreted with some caution as outlined below.

Various psychiatric manifestations have been documented in subjects with anti-NMDAR encephalitis and other autoimmune encephalitis. These include psychotic symptoms, cognitive impairment, depression, irritability, and personality disturbances [37, 38]. Fewer studies have investigated the presence of autoantibodies in primary psychiatric cohorts. The available studies have detected NMDAR antibodies in blood samples from subjects diagnosed with schizophrenia [22, 23], although there have also been negative studies [24, 25]. Bergquist et al showed that suicide attempters have increased CSF levels of immunoglobin G with affinity for dopaminergic receptors (DA-IgG) compared to healthy controls [26]. Comparability between that study and the present one is, however, limited since different assays were used and Bergquist et al. measured one specific antibody (DA-IgG) while we explored six different autoantibodies implicated in autoimmune encephalitis.

In those cases autoantibodies are detected in individuals with psychiatric and neurologic manifestations, their origin is not fully understood. Several potential mechanisms have been proposed including paraneoplastic processes, past influenza infections and genetic susceptibility [28, 39]. Moreover, it has been hypothesized that a compromised BBB may facilitate transportation of autoantibodies from the periphery to the brain [28]. In our sample, approximately 17% of the subjects had high CSF/serum albumin ratio, indicative of increased BBB permeability, yet CSF autoimmune antibodies were not detectable in any of these subjects. Our findings do not support the involvement of impaired BBB integrity in autoimmune processes in psychiatric patients, although larger case series are clearly needed in order to definitely confirm or refute this hypothesis.

The present study is among the first to investigate autoantibodies in CSF samples from psychiatric subjects. An important strength of the study is the inclusion of a well-characterized sample of unmedicated recent suicide attempters. The present study, however, also comes with several caveats. Firstly, the sample size was relatively small, thus any conclusions must be considered preliminary and the results should serve primarily as pilot data for testing in future larger scale studies. Even though the test panel that was used included six different autoantibodies, thus increasing the possibility of detection, the number of subjects tested might have been insufficient. Secondly, the assays used in our study were originally designed as a diagnostic tool for targeted populations of neurology patients where autoimmune processes in the brain are suspected. Thus, we cannot rule out that a more sensitive experimental assay would have been able to detect more subtle levels of autoantibodies. Future studies might consider using experimental assays where lower concentrations of autoantibodies might be detected. Finally, this is a cross-sectional study and we cannot completely rule the possibility that autoantibodies, if present, might have been detectable immediately after the suicide attempt but not after the wash-out period at the time of the lumbar puncture. Longitudinal studies of anti-NMDA receptor encephalitis, however, have shown that although titres may decrease over time, antibodies are often detected in consecutive samples [30].

In conclusion, we were not able to detect any autoantibodies in CSF samples from recent suicide attempters. This is one of the first studies of its kind, and future larger scale studies using sensitive assays are warranted before any firm conclusions may be drawn.