Sections from control and MIA male offspring were investigated for related mRNAs. Riboprobes (**Table 2**) were generated with 35S-UTP (PerkinElmer) using an *in vitro* transcription kit (Promega, Madison, WI, USA). *In situ* hybridization was performed as previously described (44), using 5 ng/mL radiolabeled riboprobes in hybridization buffer, and 35S-UTP labeled sense riboprobes as a negative control. Slides were exposed to BioMax MR (Kodak, Rochester, NY, USA) autoradiographic film (details in **Table 2**) alongside a 14C standard slide (American Radiolabeled Chemicals, St. Louis, MO, USA). Quantification of mRNAs was completed as mentioned above (see Quantification of Autoradiographic Images) with the standard Rodbard curve from the 14C standard slide.

# Statistical Analysis

Analysis was performed with IBM SPSS statistics (v23). Graphs were plotted using GraphPad Prism (v6). Data for each measure in each region (cortex, hippocampus, striatum) were analyzed using repeated measures two-way analysis of variance (RM-ANOVA) separately. Within-subject factors were the three subregions. Between-subject factors were prenatal treatment (polyI:C or vehicle), offspring sex (male or female), and gestational timing (GD10 or GD19) for the binding, and prenatal and gestational timing for the mRNAs (in male offspring only). Fisher's least significant differences were used for pairwise comparisons by treatment

TABLE 2 | *In situ* hybridization riboprobe details.

1http://rsb.info.nih.gov/nih-image. 2https://imagej.nih.gov/ij/.

when ANOVA analyses were significant. The Greenhouse Geisser correction was used if Mauchley's sphericity test was violated for within-subjects interaction effects. Overall treatment effects or effects of sex, region, or GD of exposure are presented graphically only when a significant effect or interaction was identified. In all cases, data are expressed as the mean ± SEM and *p* ≤ 0.05 was deemed statistically significant. NR2A:NR2B ratio for hippocampus was calculated by division of [3 H]CGP39653 binding values (NR2A subunit) with [3 H]Ifenprodil binding values (NR2B subunit) in each subregion and level for each animal.

# RESULTS

# Effect of Maternal PolyI:C Treatment and Timing on Ligand Binding in Adult Male and Female Offspring

Representative autoradiographs of binding assays are shown in **Figure 2** and show the expected pattern of NMDAR channel, NR2A subunit, and NR2B subunit (**Figures 2A–C**, respectively) binding and distribution in rodent brain (45–47). Briefly, signals are evenly distributed across the cortex and striatum. White matter tracts (i.e., corpus callosum) are not labeled and hippocampal architecture including neuropil areas surrounding ammon's horns region are labeled. Overall, autoradiographic signals were less in cortex and striatum (a,b) in comparison to hippocampus (c,d).

#### [ 3 H]MK-801 Binding (NMDAR Channel)

Although there was no overall effect of polyI:C on NMDAR channel binding ([3 H]MK-801 binding) in the cortex [Treatment effect: *F*(1,45) = 1.86, *p* > 0.10], polyI:C offspring had ~13% more NMDAR channel expression than control offspring in Cg [Treatment × Subregion effect: *F*(2,90) = 3.27, *p* < 0.05; Control versus polyI:C in cingulate: *F*(1,45) = 5.34, *p* < 0.05; **Figures 2A** and **3A**]. PolyI:C did not affect NMDAR channel binding in the M1 or Aud (**Figure 3A**). PolyI:C did not affect NMDAR channel binding in the hippocampus [Treatment effect: *F*(1,45) = 1.13, *p* > 0.10; **Figure 2A**; **Table 3**]. There was no effect of offspring sex or GD of exposure on NMDAR channel binding in either the cortex or the hippocampus [both, *F*(1,42) < 2.65, *p* > 0.10].

In the striatum, polyI:C offspring had ~20% more NMDAR channel binding than control offspring [Treatment effect: *F*(1,45) = 17.63, *p* < 0.001; **Table 3**; **Figure 2A**]. In addition, male polyI:C offspring had ~34% more NMDAR channel binding than male control offspring [Treatment × Sex effect: *F*(1,45) = 6.68, *p* < 0.05; Pairwise contrast: *F*(1,45) = 22.68, *p* < 0.001; **Figure 3B**]. No change was observed in polyI:C-exposed compared to control females [*F*(1,45) = 1.322, *p* > 0.05; **Figure 3B**]. There was no effect of GD of exposure on NMDAR channel binding in the striatum [*F*(1,45) = 3.473, *p* < 0.10].

#### [ 3 H]CGP39653 Binding (NR2A Subunit)

In total hippocampus, polyI:C offspring had ~30% more NR2A binding than control offspring [Treatment effect: *F*(1,44) = 6.22, *p* < 0.05; **Table 3**; **Figure 2B**], the magnitude of this increase was moderate to strong (31–44% increases) in all subregions [Treatment × Subregion × Level effect: *F*(1.7,45) = 3.68, *p* < 0.05;

Pairwise contrasts: *F*(1,44) > 5.41, *p* < 0.05; **Figure 4A**] except the molecular layer of the DG [Pairwise contrast: *F*(1,44) = 2.74, *p* > 0.10; **Figure 4A**]. There was no effect of gestation day of exposure on NR2A binding in the hippocampus, cortex, or striatum [*F*(1,45–47) < 0.178, *p* > 0.10].

Overall, polyI:C offspring had ~57% more NR2A binding than control offspring in the cortex [Treatment effect: *F*(1,45) = 11.004, *p* < 0.01; **Table 3**; **Figure 2B**]. In addition, we detected a sex by treatment interaction effect, where male polyI:C offspring had

FIGURE 3 | *N*-methyl-d-aspartate receptor (NMDAR) channel binding alterations in cortex and striatum from adult offspring exposed to vehicle (control; black) or polyI:C (gray) during early (GD10) or late (GD19) gestation. Tritiated MK-801 binding was used to quantify NMDAR channel levels. Significantly increased NMDAR binding was found in the cingulate subregion of polyI:C offspring compared to controls (A). Male, but not female, polyI:C offspring specifically exhibited significantly increased NMDAR channel binding in the striatum (B). Bars represent mean ± SEM (*n* = 15–28 rats per group). Cg, cingulate cortex; M1, motor cortex; Aud, auditory cortex (\**p* < 0.05, \*\*\**p* < 0.001).

TABLE 3 | Overall effect of gestational polyI:C exposure on NMDA receptor channel and subunit binding.

*\*p* < *0.05*

~132% more NR2A binding than male control offspring in cortical tissue [Treatment × Sex effect: *F*(1,45)= 4.794, *p*< 0.05; Pairwise contrast: *F*(1,42) = 24.61, *p* < 0.001; **Figure 4B**], an effect that was not observed for females [*F*(1,45) = 0.483, *p* > 0.10; **Figure 4B**].

Overall, polyI:C offspring had ~43% more NR2A binding than control offspring in the striatum [Treatment effect: *F*(1,47) = 6.62, *p* < 0.05; **Table 3**; **Figure 2B**]. Similar to the cortex, polyI:Cexposed male offspring had ~86% more NR2A binding than male control offspring [Treatment × Sex effect: *F*(1,47) = 5.38, *p* < 0.05; Pairwise comparison: *F*(1,47) = 11.79, *p* < 0.01; **Figure 4C**], an effect which was not observed in females [*F*(1,45) = 0.03, *p* > 0.10; **Figure 4C**].

#### [ 3 H]Ifenprodil Binding (NR2B Subunit)

There was a consistent lack of change in [3 H]Ifenprodil expression across all regions in polyI:C offspring compared to control offspring (**Table 3**; **Figure 2C**). There was no effect of treatment, or any interaction of treatment with offspring sex, GD of exposure, or anatomical region in the cortex [all *F*(1,50) and *F*(2,100)< 0.50, *p*> 0.10; **Table 3**; **Figure 2C**], hippocampus [all *F*(1,48) < 0.40, *F*(1.4,62.8) < 1.50, *F*(2,96) < 2.21, *p* > 0.10; **Table 3**; **Figure 2C**], or striatum [all *F*(1,47) and *F*(2,94) < 0.50, *p* > 0.05; **Table 3**; **Figure 2C**] on NR2B binding.

### NR2A:NR2B Ratio in Hippocampus

PolyI:C offspring had ~31% increased NR2A:NR2B ratio compared to control offspring [Treatment effect: *F*(1,46) = 5.40, *p* < 0.05; **Figure 5A**]. In addition, compared to control offspring, polyI:C offspring had ~25% higher NR2A:NR2B ratio in hippocampal afferent levels [*F*(1,46) = 4.35, *p* < 0.05] and ~38% higher NR2A:NR2B ratio in hippocampal efferent levels [*F*(1,46) = 6.37, *p* < 0.05] than control offspring [Treatment × Level effect: *F*(1,46) = 5.09, *p* < 0.05; **Figure 5B**].

# Effect of Maternal PolyI:C Treatment on NR1 and NR2A mRNA in Adult Male Offspring

Representative *in situ* autoradiographs are shown in **Figure 6** and the expected pattern of NR1 mRNA (GRIN1, **Figure 6A**)

*\*\*p* < *0.01 \*\*\*p* < *0.001*

and striatum (C). Bars represent mean ± SEM (*n* = 15–28 rats per group). DGmo, molecular layer of dentate gyrus; CA3sr, stratum radiatum of cornu ammonis 3; CA1sr, stratum radiatum of cornu ammonis 1; DGpo, polymorph layer of dentate gyrus; CA3so, stratum oriens of cornu ammonis 3; CA1so, stratum oriens of cornu ammonis 1 (\**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001).

FIGURE 5 | NR2A:NR2B binding ratio in hippocampus of adult offspring exposed to vehicle (control; black) or polyI:C (gray) during early (GD10) or late (GD19) gestation. Tritiated CGP39653 and Ifenprodil were used to calculate the ratio of NR2A:NR2B subunit binding. (A) The ratio of NR2A:NR2B ratio binding in the hippocampus was significantly increased in polyI:C offspring. (B) This increased ratio was elevated at both afferent and efferent levels of each hippocampal subregion. Bars represent mean ± SEM (*n* = 25–28 rats per group) (\**p* < 0.05).

and NR2A mRNA (GRIN2A, **Figure 6B**) distribution was identified in rodent brain (37). Briefly, signals were evenly distributed across the cortex and striatum (a,b). In the hippocampus, the granular layer of the DG and the pyramidal neuronal layers within ammons-horn (CA3–CA1) were darkly labeled for both NMDAR1 and NMDAR2A mRNAs (c,d), whereas the hilar region of CA4 had intermediate levels of both mRNAs. NR2A mRNA (**Figure 6B**) was less in the striatum relative to the cortex (a,b). White matter tracts were clearly identifiable (corpus callosum and anterior commissure) with no mRNA signal. Overall, all autoradiographic signals were less intense and more homogenous in cortex and striatum (a,b) in comparison to the hippocampus (c,d).

Maternal immune activation did not affect NR1 mRNA levels in cortical tissue [all Treatment effects/interactions: *F*(1,22) < 2.88, *p*> 0.05; **Figures 6A** and **7A**]. Similarly, no treatment main effects or interactions were observed for NR1 mRNA in hippocampus [all *F*(1,24) < 2.64, all *F*(1.4,24) < 1.0, *p* > 0.05; **Figure 7A**]. In the striatum, polyI:C offspring had a small, but statistically significant, increase (~11%) in NR1 mRNA in the AS compared to controls [Treatment × Subregion: *F*(2,44) = 6.08, *p* < 0.01; Pairwise contrast: *F*(1,22) = 8.11, *p* < 0.01; **Figures 6B** and **7B**].

In contrast to the overall lack of large changes in NR1 mRNA, polyI:C offspring had widespread increases in NR2A mRNA. Compared to controls, polyI:C offspring had ~25% more NR2A mRNA in cortex [Treatment effect: *F*(1,24) = 4.20, *p* < 0.05] and ~22% more NR2A mRNA in hippocampus [Treatment effect: *F*(1,24) = 9.02, *p* < 0.01; **Figures 6B** and **7C**]. In addition, polyI:C offspring had ~18% more NR2A mRNA in DG [*F*(1,24) = 4.53, *p* < 0.05], and ~29% more NR2A mRNA in CA1 [*F*(1,49) = 16.61,

*p* < 0.01; Subregion × Treatment effect: *F*(2,48) = 3.28, *p* < 0.05; **Figures 6B** and **7D**]. The increase of NR2A mRNA in CA3 of polyI:C offspring did not reach significance [*F*(1,24) = 3.32, *p* < 0.10] (**Figure 7D**). In striatal tissue, there was no significant treatment or between-groups interaction effect [*F*(1,23) < 3.15, *p* > 0.05; **Figures 6B** and **7C**].

(red arrowheads) and (c,d) hippocampal regions. Scale bars represent

# DISCUSSION

500 µm.

We found NMDAR-related molecular alterations in multiple brain regions from polyI:C offspring that potentially could contribute to early and late MIA behavioral changes previously reported in this model. However, contrary to our expectations, we did not find any interaction effects between the impact of MIA and GD. Additionally, while we would have expected decreased NMDAR binding overall, we found *increased* NMDAR channel binding, specifically in the Cg of polyI:C offspring. We also found novel and widespread increases in NR2A binding supported by significant increases in NR2A mRNA, along with no changes in NR2B binding, in polyI:C offspring. As hypothesized, we found more exaggerated molecular alterations in male polyI:C offspring, compared to female polyI:C offspring. Thus, we found that MIA elicited coordinated NMDAR alterations that were primarily in male offspring, in line with the exaggerated behavioral change found in males after MIA in our previous investigation (30).

Our most robust finding was increased NR2A binding in the brain of adult male polyI:C offspring. NR2A-rich NMDARs have relatively shorter channel opening duration (38), and faster decay kinetics (37, 48). We did not detect corresponding altered NMDAR channel binding in the hippocampus of polyI:C offspring, suggesting that more NMDARs would be of the NR1– NR2A containing type but may not be dramatically changed in overall numbers. This concurs with our finding of increased NR2A mRNA, and no change in NR1 mRNA, in several brain regions. The lack of interaction between treatment and gestational timing indicates that hippocampal NMDARs are potentially vulnerable to MIA at either gestational stage and suggests that increases in NMDAR2A could be a common brain response to developmental activation of the immune system. Previous studies report increased NR2A mRNA in the hippocampus of adult rats (7–8 weeks of age) exposed postnatally (at 2 weeks of age) to polyI:C (49) or the bacterial mimic LPS (50), suggesting that this NMDAR2A response to immune activation can even occur if the inflammation occurred after birth. This demonstrates that the mechanisms that govern NR2A expression in adulthood are potentially vulnerable to long-lasting effects of inflammation, regardless of the precise timing of inflammatory stress. In contrast to our lack of change in NR1 mRNA in the hippocampus, previous studies have reported ~20% reduced NR1 mRNA in the hippocampus of polyI:C mouse offspring exposed to late MIA, but not early MIA (13). Our data in rats suggest that polyI:C treatment during gestation may not affect NR1 gene expression to the same extent or as consistently as it does NR2A gene expression in the hippocampus. These variable findings could be due to subtle differences in the timing of vulnerable neurodevelopmental epochs, or species differences.

Our results show that the CA1 subregion exhibited increased NR2A mRNA and increased NR2A binding and NR2A:NR2B ratio in the apical dendritic region (afferent) in polyI:C offspring, regardless of gestational timing of MIA and offspring sex. This suggests that synaptic input coming from CA3, which is critically dependant on NMDAR function for learning and memory (Schaffer collaterals), is significantly changed after developmental immune activation. Indeed, increases in NR2A:NR2B ratio reduces both long-term potentiation and long-term depression in the CA3-to-CA1 circuitry [see Ref. (38) for details], limiting plasticity and making it more difficult to learn and remember. Our present findings therefore suggest that polyI:C offspring have associated molecular alterations in CA1 that perhaps would produce longer term memory retrieval deficits, consistent with long-term memory deficits found in adult polyI:C rat offspring (26).

We find localized increases in NMDAR binding in the Cg of polyI:C offspring, suggesting that MIA can elicit long-lasting changes in NMDAR expression within cortical regions of adult offspring, regardless of sex or gestational timing of MIA. Although systemic MK-801 did not alter locomotion in polyI:C offspring in our previous studies (30), other studies have reported

both increased (18) and decreased (19–21) locomotion in polyI:C offspring in response to systemic MK-801. The mechanism by which MK-801 elicits behavioral changes, and the contribution of other neurotransmitter systems, is unclear (51–54). Although the increased NMDARs in the cingulate may not be sufficient to elicit consistent MK-801 locomotion alterations in all cohorts, it may impact cognition dependent on Cg. Indeed, previous studies report increased glutamatergic processes (55) and NMDAR channel binding in anterior (42) and posterior (56) Cg of people with schizophrenia.

cornu ammonis 1; DS, dorsal striatum; AC, nucleus accumbens core; AS, nucleus accumbens shell (\**p* < 0.05, \*\**p* < 0.01).

We found some interesting changes within the striatal brain areas examined. Our current investigation of exaggerated molecular changes in striatal NMDAR of males appears to align with our previous behavioral investigation that showed that male, but not female, polyI:C rat offspring exhibit sensorimotor gating deficits (30). Our current molecular data shows that male, but not female, polyI:C offspring had increased NMDAR channel binding in the striatum, and elevated NR2A binding in both the cortex and striatum. Given that these molecular and behavioral alterations were exaggerated in male polyI:C offspring, one interpretation would be that these cortical and striatal changes contribute to the sensorimotor gating deficits that male polyI:C offspring exhibit at adulthood. However, further research is needed to elucidate how MIA-induced changes in NR2A-rich NMDARs are modified by sex and how NMDARs may contribute to sensorimotor gating function. In the present study, the coordinated increase in striatal NR2A and NMDAR channel binding in male polyI:C offspring indicates they potentially have increased NR2A-rich NMDARs in the striatum. In normal rodents, the "indirect" striatopallidal pathway is primarily mediated by NR1–NR2A-rich NMDARs, while the direct "striatonigral–entopeduncular" pathway is mediated by NR1–NR2B rich NMDARs (57–59). The changes in NMDARs in the MIA model could suggest more activity in the indirect striatopallidal pathway, which is also known to be enriched in D2 receptors. However, how the striatal changes found here map onto these distinct striatal pathways needs to be determined. PolyI:C offspring show increased dopamine turnover and reduced D2-like receptor levels in the striatum (60), supporting that the indirect striatal pathway may have more pathology in response to MIA, especially in male offspring. However, our previous investigation found no change in D2 receptor mRNA in the striatum of polyI:C offspring (30). This suggests that the NMDAR changes may precede or be independent of D2 receptor changes. Previously, we found increased D1 dopamine receptor mRNA in the nucleus accumbens of male GD10 polyI:C offspring (30). In this study, we found that male polyI:C offspring had increased NR1 mRNA in the nucleus accumbens shell only, regardless of gestational timing but with overall increases in striatal NMDAR channel binding. This suggests male polyI:C offspring may have elevated NMDAR synthesis and activity in the striatum, particularly in the ventral portion. Taken together, the results on striatal abnormalities suggest dopaminergic and glutamatergic alterations found in early and/or late MIA rat offspring may interact and perhaps be rescued by pharmacotherapies that target either dopamine [as evidenced in MIA rat offspring treated with antipsychotics (18, 27)] or NMDAR, particularly NMDAR2A.

The primary finding of this study is that immune challenge *in utero* irrespective of timing of the challenge has an effect on glutamatergic signaling in adult offspring, an effect that seems to be largely restricted, or more extreme, in male offspring. In terms of the validity of the MIA model of schizophrenia, the sex differences are consistent with epidemiological and clinical evidence of a small increased incidence of schizophrenia in males and a more chronic course of the disorder in males (61). The long-term changes in NMDAR, particularly the consistent, anatomically widespread and quite robust increase in the NMDAR2A subunit may have deleterious effects on synaptic plasticity, learning and memory. We suggest that even transient developmental immune activation mediates neural and possibly behavioral changes, through adult changes in the synthesis and function of NMDAR. The clear and robust increases in NMDAR2A, whose gene has been recently linked to the increased risk of schizophrenia (62), suggest a possible point of convergence between genetic and environmental risk (maternal infection) factors in schizophrenia. Our study that identifies increased NMDAR2A after MIA suggests that future efforts could focus on determining when this NMDAR2A change occurs developmentally and could test if and when reversing the NMDAR2A increase could restore normal behavior.

# REFERENCES

# ETHICS STATEMENT

Experiments were performed in accordance with the National Health and Medical Research Council's *Australian code for the care and use of animals for scientific purposes*. The current study was approved by the University of Newcastle's Animal Care and Ethics Committee (Approval number A-2009-108).

# AVAILABILITY OF DATA AND MATERIAL

The data analyzed during the current study are available from the corresponding author on reasonable request.

# AUTHOR CONTRIBUTIONS

KZ, US, JT, DH, and PM conceived and designed the experiments. CW assisted in the design of the experiments. LH and CM undertook the PolyI:C treatments and provided the rat brain tissue. KZ undertook the binding experiments, GRIN1 in situ experiment, contributed to the analysis of the hippocampus data, and provided a first draft based on hippocampus data only. TR undertook the GRIN2A experiment and analyzed the data. TR, TP-T, and CW wrote the final draft. TP-T and LH checked the data and edited the manuscript. All authors contributed to the interpretation and approval of the final manuscript.

# FUNDING

This study was supported by a National Health and Medical Research Council (NHMRC) project grant (APP1026070: PM, DH, KZ, US, and JT). CM is a recipient of an Australian Postgraduate Award (APA). US is supported by the Schizophrenia Research Institute (utilizing infrastructure funding from the NSW Ministry of Health and NSW Ministry of Trade and Investment, Australia). CSW is supported by the Schizophrenia Research Institute (utilising infrastructure funding from the NSW Ministry of Health and the Macquarie Group Foundation), the University of New South Wales, and Neuroscience Research Australia and is a recipient of a National Health and Medical Research Council (Australia) Principal Research Fellowship (#1117079).

throughout development. *Brain Behav Immun* (2013) 31:54–68. doi:10.1016/j. bbi.2012.07.008

**Conflict of Interest Statement:** CW is a member of an Advisory Board for Lundbeck Australia Pty Ltd. The other authors declare that they have no competing interests.

*Copyright © 2017 Rahman, Zavitsanou, Purves-Tyson, Harms, Meehan, Schall, Todd, Hodgson, Michie and Weickert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Hydrogen Sulfide Affects Radical Formation in the Hippocampus of LPS Treated Rats and the Effect of Antipsychotics on Hydrogen Sulfide Forming Enzymes in Human Cell Lines

# Edited by:

Karl Bechter, Universität Ulm, Germany

### Reviewed by:

Dasiel Oscar Borroto-Escuela, Karolinska Institute (KI), Sweden Xu Cao, Memorial Sloan Kettering Cancer Center, United States

### \*Correspondence:

Hans-Willi Clement<EMAIL_ADDRESS>
> †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry

Received: 14 December 2016 Accepted: 24 September 2018 Published: 16 October 2018

#### Citation:

Sommer O, Aug RL, Schmidt AJ, Heiser P, Schulz E, Vedder H and Clement H-W (2018) Hydrogen Sulfide Affects Radical Formation in the Hippocampus of LPS Treated Rats and the Effect of Antipsychotics on Hydrogen Sulfide Forming Enzymes in Human Cell Lines. Front. Psychiatry 9:501. doi: 10.3389/fpsyt.2018.00501 Olaf Sommer 1†, Rosana L. Aug1†, Andreas J. Schmidt 2 , Philip Heiser 1 , Eberhard Schulz 1 , Helmut Vedder 2 and Hans-Willi Clement 1 \*

1 Department of Child and Adolescent Psychiatry, Psychotherapy, and Psychosomatics, Albert-Ludwigs-University Freiburg, Freiburg, Germany, 2 Department of Psychiatry and Psychotherapy, Philipps-University, Marburg, Germany

Objectives: Psychiatric disorders, such as schizophrenia and other neuroinflammatory diseases are accompanied by an increase in the oxidative stress and changes in the immune system and in the metabolic, hormonal and neurological components of the central nervous system (CNS). Hydrogen sulfide (H2S) is a gaseous molecule that is endogenously produced in the peripheral and central nervous system through cysteine by the following major H2S producing enzymes in the brain: cystathionine-γlyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST). The physiological effects of H2S are broad, with antioxidative properties being a major role in the body. The aims of our investigation were to analyze the central nervous antioxidant, metabolic and neuronal effects in the hippocampus of the rat after inflammatory peripheral lipopolysaccharide (LPS) treatment; and to examine the effects of antipsychotics on the expression of these enzymes in human cell lines.

Material and Methods: Male Lewis rats (250 g) received an i.p. LPS injection (1 mg/kg) 24 h before microdialysis experiments. Conscious rats were infused via these probes (1.5 µl/min) with a radical scavenger 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) in Krebs-Ringer solution. Sodiumhydrogensulfide (NaHS, 10 µg/min) was infused after a 2- h baseline for 1 h. Corticosterone, glutamate, glucose and lactate were measured by Elisa. Reactive oxygen species (ROS) were detected by electron spin resonance spectroscopy (ESR). The impact of the antipsychotics haloperidol, clozapine, olanzapine and risperidone on the expression of genes encoding the key enzymes of H2S synthesis was studied at the human neuroblastoma SH-SY5Y and monocytic U-937 cell lines. The cells were incubated for 24 h with 30µM antipsychotic following which mRNA levels were measured by polymerase chain reaction. Results: Microdialysate glucose and lactate levels dramatically increased in the hippocampus of LPS untreated rats by local application of NaHS. By contrast, in the LPS pretreated rats, there was no effect of NaHS infusion on glucose but a further significant increase in microdialysate lactate was found. It was LPS pretreatment alone that particularly enhanced lactate levels. There was a marked increase in hippocampal microdialysate glutamate levels after local NaHS infusion in LPS untreated animals. In LPS treated rats, no change was observed by NaHS, but LPS itself had the strongest effect on microdialysate glutamate levels. Microdialysate corticosterone levels were reduced by NaHS in both LPS pretreated and untreated rats. The formation of free radicals in the hippocampus significantly reduced in LPS pretreated rats, while in LPS untreated rats a significant increase was observed after NaHS infusion. In human SH-SY5Y and U-937 cells, all three major enzymes of H2S-Synthesis, namely cystathionine-γ-lyase, cystathione ß-synthase and 3-mercaptopyruvate sulfurtransferase, could be detected by PCR. The antipsychotics haloperidol, clozapine, olanzapine and risperidone affected all three enzymes in different ways; with haloperidol and risperidone showing major effects that led to reductions in CBS or CSE expression.

Discussion: The local application of NaHS in the hippocampus of the rat strongly affected glucose, lactate and glutamate release. Contrastingly, in LPS pretreated rats, a decreased radical formation was the only effect found. H2S synthetizing enzymes may be involved in antipsychotic mechanisms, although no clear common mechanism could be found.

Keywords: schizophrenia, electron spin resonance spectroscopy (ESR), reactive oxygen species (ROS), hydrogensulfide (H2S), lipopolysaccharide (LPS)

# INTRODUCTION

Psychiatric disorders, such as schizophrenia and other neuroinflammatory diseases are accompanied by an increased oxidative stress, changes in the immune system and in metabolic, hormonal and neurological components of the central nervous system (1–22). More and more evidence suggests various dysregulations of the hypothalamus-pituitary-adrenal (HPA)- Axis in the course of numerous mental disorders, such as affective disorders (23–25), and schizophrenia (18–21).

The response to antipsychotics in schizophrenia shows a high variability. There are several patient cases that are resistant to clozapine, which is called "ultra-resistance to treatments in schizophrenia" (UTRS). Peripheral inflammations are associated with UTRS (26).

Hydrogen sulfide (H2S) is a gaseous molecule that is endogenously and enzymatically produced in the peripheral and central nervous system by three major H2S producing enzymes: cystathionine-γ-lyase (CSE) (27), cystathionine ß-synthase (CBS) (28) and 3-mercaptopyruvate sulfurtransferase (MPST) (29, 30); besides other mechanisms (31). High endogenous concentrations of H2S were found in the hippocampus and cerebellum which are parts of the human brain where CBS seems to be the most important enzyme for the synthesis (32, 33).

H2S is known to regulate a multitude of physiological and pathophysiological functions in the vascular, immune and nervous system [for review see (34, 35)]. There is evidence for a role of H2S in neurodegeneration (36), but also radical scavenging effects regarding nitric oxide (37) or glutamate mediated oxidative stress (38). Other authors report a role for H2S in Alzheimer's disease (39) or Parkinson's disease (40).

Promising studies have demonstrated the therapeutic effect of H2S donation in various disease models of cancer, inflammation or neuroinflammation (41, 42). Additionally, the pharmacological inhibition of H2S production (43–47) or the genetic deficiency of H2S producing enzymes (48–50) results in beneficial effects.

Xiong et al. could show that the H2S levels of patients with schizophrenia are significantly low. H2S is a regulator for the N-methyl-D-aspartate receptor (NMDAR) function and low H2S levels can cause a hypofunction of NMDAR. Given that a hypofunction of the NMDAR receptor is related to the pathogenesis of this disorder, H2S levels in the liquor are also likely to have an effect on the pathophysiology of this disorder (51).

The aims of our investigation were to analyze antioxidant, metabolic, hormonal and neuronal changes of NaHS application in the hippocampus in naive and LPS pretreated rats. Furthermore, we aim to show the effects of different antipsychotics on the expression of several enzymes related with the H2S synthesis in a neuronal and an immunological human cell lines. Especially the role of exogenous H2S as a possible therapeutic mediator in inflammatory and neurodegenerative diseases will be analyzed.

# MATERIALS AND METHODS

# Animals

Animal experiments were carried out at the Department of Surgical Research, University Hospital, Freiburg, Germany. All procedures were performed in accordance with the German animal protection law, FELASA, the national animal welfare body, GV-SOLAS and the NIH guide for the care and use of laboratory animals; and were approved by the animal welfare committee of the University of Freiburg (AZ: G-10/44). Male Lewis rats (250–350 g; Charles River, Germany) were housed at a temperature of 21◦ C in plastic cages with lights turned on from 06:00 to 19:00 h and with free access to food and water. Experiments were conducted during the light phase after at least 1 week of adaptation.

# In vivo Microdialysis Experimental Design

CMA/12 microdialysis probes were implanted under isoflurane anesthesia (3%) using stereotaxic coordinates according to (52) A: +5.2 mm; L: +2.0 mm; V: −4.4 mm from Cortex top (53). The microdialysis experiments started after awakening using the CMA freely moving system in groups of 8 animals. Microdialysis samples were collected every 30 min for a period of 4 h at a constant flow rate of 1.5 µL/min (Krebs-Ringer). Microdialysis samples were collected and stored at −20◦C until analysis. Electron spin resonance (ESR) measurements were performed immediately. The same effluents were used to measure Corticosterone (Enzyme immunoassay, IBL, Hamburg, Germany), Glucose, Lactate and Glutamate (Colorimetric Assays, BioCat, Heidelberg, Germany).

To detect central nervous protective effects of hydrogen sulfide, a series of rats received an intraperitoneal injection of 1 mg/kg body weight Lipopolysaccharide from E. coli (LPS; Sigma Aldrich Chemicals, Steinheim, Germany). The injections were made 24 h before microdialysis probes were implanted. NaHS was infused intrahippocampal via the microdialysis cannula at a flowrate and dose of 10 µg/min 2 h after beginning of sampling. ROS, Glutamate, Corticosterone, Lactate and Glucose-Uptake (difference between glucose inflow concentration and outflow concentration) were measured.

# Histology

At the end of the experiment, one group of animals was euthanized with CO2. To verify probe placement, the brain was removed and stored at −20◦C.

# Chemicals

Krebs-Ringer's solution was obtained from Delta-Select, Pfullingen, Germany; LPS (Lipopolysaccharides from Escherichia coli, Serotype 0127:B8 purified by trichloroacetic acid extraction) from Sigma, Steinheim, Germany; 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) from Noxygen, Elzach, Germany; Isofluorane from Abbott, Wiesbaden, Germany; Primers were obtained from MWG Biotech, Ebersberg, Germany.

# ROS Measurements

Hippocampal detection of reactive oxygen species is based on the reaction of CMH and ROS as reactant in dialysates. CMH solution (1 mg/ml Krebs-Ringer) was prepared fresh every day. The spin probe CMH was applied by infusion via a microdialysis cannula at a flow rate of 1.5 µl/min. The oxidation of spin probe CMH by reactive oxygen species generates stable 3 methoxycarbonyl-proxyl radicals (CM) as shown by Dikalov et al. (54, 55). Autoxidation was found to be in the range of 1– 2%. The amount of CM. radical is equivalent to the formation of reactive oxygen species in vivo. The amount of reacted ROS was determined from the ESR amplitude according to a calibration curve using standard CM. solutions. ESR measurements were performed at room temperature using an EMX ESR spectrometer (MiniScope MS 200, Magnettech, Berlin, Germany). The ESR had the following settings: center field g = 2.001, sweep wide 60 G, sweep time 5 ms over 10 scans, modulation amplitude 2.4 G, microwave power 20 mW. The total spin probe concentration was measured to determine the concentration of free radicals (56).

# Cells and Culture Conditions

U-937 cells were maintained from the Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heatinactivated fetal calf serum (FCS) (Gibco/BRL and Seromed, Berlin, Germany), 0.5% glutamine, and 1% gentamycine at 37◦C in a 5% CO2 atmosphere. For the studies, cells were plated at a concentration of 225,000 cells in 3 ml medium per well into six-well culture plates (Greiner, Frickenhausen, Germany).

Cells were preincubated for 24 h and then treated with the antipsychotics, dissolved in ethanol at a concentration of 30µM. These were added to the plates at a quantity of 300 µl to each well. The same procedure was performed with the ethanol controls without antipsychotics. Antipsychotics were obtained from Sigma, Deisenhofen, Germany.

Neuroblastoma SH-SY5Y cells were cultured in heatinactivated Roswell Park Memorial Institute medium (RPMI) (Gibco/BRL, Eggenstein, Germany) supplemented with 15% fetal calf serum (FCS) (Biochrom, Berlin, Germany), 1% penicillinstreptomycin and 1% glutamine in a 5% CO2 atmosphere. For further studies, cells were plated at a number of 225.000 cells/dish in 10 mm culture dishes. Antipsychotics and ethanol-control treatments were performed at a density of 450.000 cells/dish. The antipsychotics were dissolved in ethanol and were further diluted in culture medium. The cells were exposed to antipsychotics at 30µM for 24 h at 37◦C.

# RNA Extraction

After incubation, the cells were collected from the culture dishes and total RNA was extracted by the use of Trizol R reagent in accordance with the manufacturer's instructions. The amount of extracted total RNA was quantified by established optical methods at A260/A280 (Genequant II, Pharmacia Biotech, Freiburg, Germany) and structural integrity checked by agarosegel electrophoresis [1.5% agarose (Gibco/BRL)].

# Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

RT-PCR was used to analyse the transcription of the GRs and the house-keeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 1 mg of cellular total RNA was reverse-transcribed with 40 U of Superscript II (Gibco/BRL) and 1 mg oligo-(dT) in a volume of 20 ml following the manufacturer's protocol.

The following primer pairs were used to amplify the cDNA's:

CBS: Sense 5′ -CGATGGGTACCATATGCAGAAAAGACG CCTCCTCACAAGG-3′ , Antisense 5′ -CGGTACCTCGAGTTA CTACTGTGATTCCACTTGGAGGGTGTGCTGCC-3′ . CSE: Sense 5′ -GGCCTGAAGTGTGAGCTCTT-3′ , Antisense 5′ -TTG GGGATTTCGTTCTTCAG-3′ . MPST: Sense 5′ -GACCCC GCCTTCATCAAG-3′ , Antisense 5′ -CATGTACCACTCCAC CCA-3′ . GAPDH: Sense 5′ -CGTCTTCACCACCATGGAGA-3′ , Antisense 5′ -CGGCCATCACGCCACAGTTT-3′ .

Aliquots of 1 ml cDNA were amplified with a PCR cycler (Biometra Trio, Göttingen, Germany) for the enzymes and GAPDH using the primers described above with the following cycling program: denaturation for 45 s at 95◦C, annealing for 60 s at 59◦C, and extension for 60 s at 72◦C. PCR products were analyzed for all enzymes after amplification with 28 cycles and gel electrophoresis in 1.5% agarose gels. Semi-quantitative determination was achieved by digitization of gels with a Polaroid video system (Rothaar & Schroeder, Heidelberg, Germany) and further densitometric evaluation achieved with the Gelscan 4.0 Professional Program (LTF/BioSciTec, Landau/Frankfurt, Germany).

# Quantification of mRNA by RT-PCR and Densitometry

Different approaches were used to minimize variations and to ensure the reliability of the quantification procedure: first, the purity of mRNA probes was determined by measuring the optical density at A260/A280. This revealed a ratio of 1.56 ± 0.05 for all extractable probes. Second, integrity and amounts of mRNA measured were checked by gel electrophoresis. Third, relevant impurities of DNA were routinely excluded by PCR-amplification of extracted mRNA probes after omission of the RT reaction. The reliability of the further steps of quantification (PCR reaction, gel densitometry and evaluation of results) including nonsaturating conditions of the PCR was determined using different amounts of RT products. This yielded a near-linear dose-product relationship in the gel electrophoresis.

# Statistical Analyses

The statistical analysis of the microdialysis data was performed using Microsoft Excel 8.0 and SPSS version 9.0 (SPSS Inc., Chicago, USA). The given data comprises mean ± standard deviation (SD). For microdialysis experiments significance was assessed by unpaired Student t-test, one-tailed (since we had a clear hypothesis from the previous in-vitro studies). An αlevel of p < 0.05 was considered significant (57). Data are presented in percent of the mean of the respective control samples and are shown as mean ± SD values. The results from at least three independent different experiments were pooled and analyzed by Mann-Whitney Rank Sum tests. A p < 0.05 (∗ ) was accepted as a statistically significant difference. For statistical evaluations, Sigmastat (Jandel Scientific, Kerpenich, Germany) was applied (58).

# RESULTS

The local intrahippocampal application of NaHS via microdialysis cannula (10 µg/min. at 1.5 ml/min. for 1 h) affected different neuronal parameters. In LPS untreated rats, NaHS infusion exerted massive increases in both microdialysis glucose and lactate release in the hippocampus. These effects were not seen in LPS treated rats (1 mg/kg i.p. 24 h before microdialysis experiments), **Figure 1**.

Microdialysis monitoring of Glutamate and Corticosterone showed a highly significant increase in Glutamate and a diminished formation of Corticosterone after 10 µg/min NaHS compared to the previous control periods. In the LPS treated rats no further increase in Glutamate could be observed. The decreasing effect of NaHS on Corticosterone was also observed in LPS treated animals (**Figure 2**).

ROS are also affected significantly by NaHS (**Figure 3**). The infusion of NaHS, 10 µg/min, led to a significant increase in free radical formation in LPS-untreated animals. In contrary, in LPS treated animals we saw a free radical decrease of NaHS.

Our present study demonstrated effects of typical (haloperidol) as well as atypical antipsychotics (clozapine, olanzapine, risperidone) on the main H2S synthesizing enzymes CBS, CBE, and MPST in SH-SY5Y cells, and U-937 cells (**Figure 4**). In essence, all measured significant effects of the used antipsychotics in the employed concentration were reductions in the expression of both enzymes CBS and CSE in the neuronal cell line. Haloperidol significantly reduced the expression of CBS and CSE in SH-SY5Y cells, but the effect of haloperidol was not significant in U-937 cells. In U-937 cells the antipsychotics olanzapine and risperidone reduced the m-RNA of CSE significantly. The expression of MPST was only affected in U-937 cells and only by olanzapine. Clozapine, the most important antipsychotic did not show any effect in both cell lines.

# DISCUSSION

Our data indicate that H2S affects parameters involved in the pathophysiology of inflammatory psychiatric illnesses, such as depression and schizophrenia. Especially in normal Lewis rats, extracellular glucose, lactate release and glutamate release are highly increased in the hippocampus after local application of NaHS via microdialysis cannula. Hippocampal corticosterone levels are found to be significantly reduced under these conditions. At this concentration, an increase in free radical formation could also be observed, whereas after

LPS pretreatment of animals a significant reduction could be observed. Under the conditions of LPS pretreatment the effects of NaHS on glutamate, corticosterone, glucose, and lactate release were missing. Antipsychotics seem to have individual effects on the expression of the major H2S-synthetizing enzymes with haloperidol exerting mostly reducing effects, while clozapine was ineffective in both SH-SY5Y and U-937 cell lines.

Hayden et al. (59) have also shown elevated blood glucose levels after exposure with H2S. Pichette and Gagnon summarize the literature on the effects of H2S and glucose regulation, indicating that mechanisms besides insulin regulation, such as glucagon-like-peptide (GLP)-1 or peptide YY are involved (60). Our data confirm findings of Lin et al. (61) that exogenous H2S protects cells by its glucose reducing activity.

The increase in lactate by NaHS-Application may be supported by the observation that in fish exposed to H2S, an increase in whole blood lactate could be observed (62). Without LPS pretreatment, our results suggest that the local application of NaHS leads to a general metabolic activation of neurons and glial cells in the hippocampus.

Our findings of the significant release of glutamate in the hippocampus of the rat induced by local NaHS application might be due to postsynaptic effects of H2S on the membrane potential, and thus excitability of CNS neurons, as it has been reported in dorsal raphe (63), paraventricular nucleus (64), subfornical organs (65) and dorsal root ganglion neurons (66). The effects in the dorsal raphe have been suggested to be the result of direct modulatory actions of H2S on calcium dependent potassium channels (63).

Significant alterations in the HPA axis activity occur in course of the schizophrenia, regarding basal cortisol secretion, probably in response to a decrease in the amount of glucocorticoid receptors (25, 67, 68). Walder et al. (69) revealed a significant positive correlation between salivary cortisol concentrations and symptoms severity. A study of Walker et al. (70) revealed that increased cortisol levels in patients who developed psychosis. Therefore, the hypothesis that HPA axis distortion is closely bound with symptoms and pathophysiology of the schizophrenia is becoming increasingly recognized. Antipsychotic medication leads to the reduction of cortisol concentrations in patients as well as in healthy controls (71, 72). Flores et al. (73) suggested that this might be responsible for their effectivity (17). Wang et al. (74) showed that inhibitors of the H2S producing enzymes, such as CBS or CSE, or the application of small interfering RNAs lead to mitochondrial oxidative stress and dysfunction, resulting in an even blunted corticosterone response to ACTH in adrenal glands. These effects were significantly attenuated by the treatment of H2S donor GYY4137. As shown by Navarra et al. (75), NaHS application is associated with the inhibition of the stimulated release of corticotropin-releasing hormone from rat hypothalamic explants.

In our experiments we found a significant reduction on free radical production in the hippocampus induced by the

local NaHS application in the LPS pretreated rats. These findings support the role of H2S as an important antioxidative anti-inflammatory mediator. H2S was found to increase the antioxidative properties of cells in different ways (76). It decreases lipid peroxidation induced by homocysteine (77). H2S is able to directly react with reactive oxygen but its endogenous concentrations are too low to act as an important endogenous antioxidant. On the other hand H2S has a cytoprotective effect in brain cells by elevating the reduced glutathione (GSH) production via activation of cystine/cysteine transporters and redistribution of GSH to mitochondria (38, 78, 79). On the molecular level, several mechanisms seem to be involved in the antioxidant and neuroprotective role of H2S, such as the nuclear factor (NF)-κB pathway (80). H2S significantly reduced levels of malondialdehyde and 4-hydroxynonenal and elevated levels of superoxide dismutase and reduced glutathione in the hippocampus of streptozotocin (STZ)-induced diabetic rats (81). They also found that H2S alleviated depressive-like behaviors of STZ-induced diabetic rats in the forced swimming and tail suspension tests and reduced their anxiety-like behaviors in the elevated plus maze test. The results provide evidence for antidepressant-like and anxiolytic-like effects of H2S in STZinduced diabetic rats and suggest that the therapeutic effects may result from inhibition of hippocampal oxidative stress (81). In HT22 neuronal cells, Kimura et al. (82) observed a cytoprotective role of H2S by activating ATP-dependent K+ (KATP) and Clchannels, in addition to increasing the levels of glutathione. In LPS-treated mice the findings of (48) indicated that a deficiency in MPST does not significantly affect endotoxemia but a deficiency in CBS or CSE slightly ameliorates the outcome of LPS-induced endotoxemia in vivo.

In summary our data support Huang and Moore's (83) notion, that H2S is not only a toxic agent but also a gasotransmitter with growing therapeutical potential. Here we could demonstrate that it might be a double sided sword and that the body condition is important for the effects of H2S.

We here observed that the neuroleptics haloperidol, clozapine, olanzapine and risperidone have different effects on human SH-SY5Y and U-937 cell lines. Several drugs or even vitamins, such as

vitamin D3 (84) are able to change the concentrations of H2S and these effects seem to be tissue dependent (35). The mechanisms through which these effects occur are still unclear. The regulation of gene expression seems to be different in respect to tissue and cell type since antipsychotics, such as haloperidol and quetiapine seem to reduce genes encoding antioxidant enzyme expression (58), the reduction of H2S-forming enzymes, CBS and CSE, especially by haloperidol in neuronal cell line. It cannot be excluded that the inhibition of CSE in U-937 cells by olanzapine and risperidone is combined not only with beneficial effects of these drugs but also with shared side effects. Furthermore, Fond et al. (26) recently found that ultra-resistance to treatment in schizophrenia (URTS) is independently associated with peripheral low-grade inflammation in schizophrenia patients (26).

H2S does not seem to be a classical neurotransmitter as specific receptors are not reported until now. Changes in cytokine production, such as IL-1 or TNF-a in the LPS model are reported (85). Although several mechanisms report that H2S can exert its physiological effects, such as sulfhydration or hemeprotein interactions [for review see (31)], other mechanisms, such as the influence on receptor heterocomplexes that are important in the pathophysiology of schizophrenia cannot be excluded from consideration (86, 87).

Schizophrenic Patients have olfactory impairments as was shown by Turetsky et al. (88) and show higher depolarization responses after stimulation with H2S indicating a special role for H2S in schizophrenia. Nevertheless, it is still unclear whether there is avoidance or an even beneficial effect (88).

Taken together our data confirm that hydrogen sulfide affects mechanisms involved in the pathophysiology of

# REFERENCES

neuroinflammatory diseases, such as schizophrenia and depression and antipsychotic treatment might alter H2Srelated mechanisms. We could show that H2S has effects on several brain metabolites and hormones, and our data confirm the idea that H2S can have toxic and protective properties depending on the state the body is in, here demonstrated for the case of inflammation. Nevertheless, the collected results of the study have not adequately clarified the therapeutic benefits H2S has for neurodegenerative diseases in brain, and the conditions and concentrations under which the beneficial properties H2S exceed the toxicity of this gasotransmitter. Further beneficial effects of H2S and related compounds might be studied in animal models of schizophrenia.

# AUTHOR CONTRIBUTIONS

All the authors meet the following criteria: made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; and drafted the work or revised it critically for important intellectual content; and approved the final the version to be published; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. In addition OS performed the animal experiments and AS performed the cell culture experiments.

# ACKNOWLEDGMENTS

We thank S. Fischer for her technical assistance.

patients versus healthy controls. BMC Neurosci. (2011) 12:13. doi: 10.1186/1471-2202-12-13

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Sommer, Aug, Schmidt, Heiser, Schulz, Vedder and Clement. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Schizophrenia or Atypical Lupus Erythematosus with Predominant Psychiatric Manifestations over 25 Years: Case Analysis and Review

*Axel Mack1 \*, Christiane Pfeiffer 2 , E. Marion Schneider 3 and Karl Bechter1 \**

*1Department of Psychiatry and Psychotherapy II, University Ulm/Bezirkskrankenhaus Guenzburg, Guenzburg, Germany, 2Department of Dermatology and Allergology, University Hospital Ulm, Ulm, Germany, 3Sektion Experimentelle Anaesthesiologie, University Hospital Ulm, Ulm, Germany*

We observed a case over 25 years of relapsing–remitting schizophrenic spectrum disorder, varying regarding the main symptomatology between more depressive or more schizoaffective or rather typical schizophrenic syndrome. Diseased phases were repeatedly accompanied by minor skin lesions, which were initially classified as mixed tissue disorder. Psychotic phases were waxing–waning over years. During one later relapse, skin involvement was severe, classified to likely represent an allergic reaction to psychopharmaca; this generalized exanthema remitted rapidly with cortisone treatment and azathioprine. Under continued azathioprine and low dose neuroleptics, the patient remitted completely, appearing psychiatrically healthy for 16 years. When azathioprine was set off due to pregnancy, an extraordinary severe relapse of schizophrenia like psychosis accompanied by most severe skin lesions developed within a few weeks, then requiring 2 years of psychiatric inpatient treatment. Finally, a diagnosis of systemic lupus erythematodes plus neuropsychiatric lupus was made. A single CSF sample in 2013 showed suspicious biomarkers, matching with CSF cytokine profiling in schizophrenic and affective spectrum disorder patients and indicated mild neuroinflammation. Complex immune suppressive treatment was reinitiated short after relapse, but was only partially successful. However, surprisingly the psychosis and skin lesions remitted (in parallel) when belimumab was given (add-on). The very details of this complicated, long-term disease course are discussed also with regard to general ideas, in particular with respect to the question if this case of seemingly comorbid schizophrenia with minor autoimmunity signs represented a case of one emerging autoimmune disorder with variant manifestations systemically and within the CNS, though atypically with predominant appearance as a schizophrenia spectrum disorder.

Keywords: mild encephalitis, neuroinflammation, chronic schizophrenia, autoimmune encephalitis, autoimmune diseases, neuropsychiatric lupus erythematosus, lupus erythematosus, systemic

# INTRODUCTION

Schizophrenia is understood as a partly heritable brain disease, recent interesting findings showing alleles of the complement component C4 genes play a role in both in the CNS and the immune system (1). Autoimmune diseases and severe infections are associated with a later increased risk of schizophrenia and other psychiatric disorders (2, 3). The relative risk of schizophrenia for an

### *Edited by:*

*Gretchen Hermes, Yale University, United States*

#### *Reviewed by:*

*Bernhard J. Mitterauer, Volitronics-Institute for Basic Research Psychopathology and Brain Philosophy, Austria Armando D'Agostino, Università degli Studi di Milano, Italy*

#### *\*Correspondence:*

*Axel Mack<EMAIL_ADDRESS>Karl Bechter<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry*

*Received: 12 December 2016 Accepted: 07 July 2017 Published: 27 July 2017*

### *Citation:*

*Mack A, Pfeiffer C, Schneider EM and Bechter K (2017) Schizophrenia or Atypical Lupus Erythematosus with Predominant Psychiatric Manifestations over 25 Years: Case Analysis and Review. Front. Psychiatry 8:131. doi: 10.3389/fpsyt.2017.00131*

**159**

individual with a history of autoimmune disease in themselves or in their family is elevated by about 45%. Inversely schizophrenia is associated with a nearly 50% elevated lifetime prevalence of autoimmune diseases (4).

We observed a case of a female patient with a 25-year psychiatric history, her disease beginning at the age of 24. As the medical records show, the diagnosis was changing several times, from depression (bipolar disorder hypothesized) to schizoaffective to schizophrenic disorder, this was the predominating diagnosis over disease cause. Besides psychiatric process, the disease course in this patient was atypical, as her psychotic phases were often accompanied by appearance of minor non-specific skin lesions on the trunk and limbs. It has been suggested that there might be an autoimmune process; however, she did not fulfill any criteria for a defined autoimmune disorder. During one particular psychotic relapse she developed severe skin efflorescences, which were treated with cortisone and later azathioprine. Under this regimen skin improved rapidly and surprisingly after this particular inpatient treatment, the patient remained psychiatrically well with few exceptions over many years. Sixteen years later, this patient became pregnant and azathioprine had been set off because of possible teratogenic side effects. Soon, after discontinuing azathioprine, she rapidly relapsed and for the first time the criteria of an established autoimmune disorder, systemic lupus erythematosus (SLE), were fulfilled and could be even extended to neuropsychiatric lupus erythematosus (NPSLE).

The question discussed in this single case, is whether the patient suffered from the two unrelated disorders of schizophrenia and SLE, or an atypical course of a not clearly defined autoimmune disorder with early predominant psychiatric and late neuropsychiatric manifestations, with minor systemic manifestations and late fulfilling criteria of SLE/NPSLE.

# BACKGROUND

In previous CSF studies, we accumulated evidence of minor neuroinflammation and immune activation in large subgroups of both schizophrenic and affective disorders (5). These findings were recently confirmed by others (6). In addition we recently found high CSF cytokines, especially at IL-8 in each patient (7), also increased CSF neopterin (8). All these findings supported the recently updated mild encephalitis (ME) hypothesis (9). Also epidemiological studies are well compatible with ME hypothesis, in that infections and autoimmune disorders are additive risk factors for a spectrum of severe psychiatric disorders (3). Discrete features of neuroinflammation are seen in a variety of CNS disorders, including degenerative diseases like Alzheimer's disease, where neuroinflammation seems to represent a disease escalating factor (10). Most interesting is that in single case studies of acute psychosis, despite normal magnetic resonance imaging scans of the brain and normal CSF and without detection of CNS autoantibodies, there was nevertheless proof of mild neuroinflammation in the cortex biopsy (11). These findings point to the difficulties in detecting mild inflammatory processes and show the limitations of available diagnostic methods including CSF diagnostics (12).

The study was approved by the ethics committee of the University of Ulm (the patient gave written informed consent to publication) was to gain a better insight into possible relationships between a seemingly primary psychiatric disorder and a poorly defined autoimmune process. By using a careful retrospective analysis of an unusual case with a long-standing disease course and considerable available clinical material, the possibility of a unifying diagnosis over the disease course, against the established assumption of two separate disorders (schizophrenia and autoimmune disorder) was tested.

# FAMILY HISTORY

Within the framework of the patient's sixth hospitalization, the suspicious family history for both psychiatric and autoimmune disorders became apparent: both parents and the maternal grandmother suffered from a longtime depression with several psychiatric hospitalizations; the grandmother committed suicide. The patient's only sibling has a schizophrenic disorder as well as Crohn's disease.

# RETROSPECTIVE CASE ANALYSIS OF 25 YEARS

In 1989, a 24-year-old woman suffering from severe depression (ICD-10 F32.3) with predominant loss of interest, hypersomnia, feelings of guilt, and worthlessness and diminished ability to concentrate was hospitalized in our clinic for the first time. The physical findings were unremarkable except slight anemia, increased blood sedimentation rate and positive rheumatoid factor. Besides the psychiatric symptomatology no signs of autoimmunity were registered. The patient was treated successfully with a combination of pimozide, flupentixol, and amitriptyline and released after 3 months inpatient stay in good mental condition.

During 1990, now 25 years old, the patient again demonstrated emerging anxiety, thought disorder (derailment and thought blocking), disorganized speech and for the first time acoustic hallucinations (commenting voices) and initially catatonic stupor. In the required second psychiatric hospital stay she showed slight leukopenia and hypochromic microcytic anemia. One month after hospitalization some non-specific skin manifestations on her chest and back appeared. Internal and dermatological consultancies happened, tissue samples were made, but neither serological nor histopathological results could name the disorder. Now the differential diagnosis of collagenosis was considered and further blood testing was undertaken. For the first time, antinuclear antibodies (ANA) tested positive.  There was no evidence of infection, electrolyte disturbances or metabolic derangements. Established criteria of a defined autoimmune disorder were not fulfilled and therefore no immune suppressive therapy was started. With a combination of bromperidol and clorazepate, she distanced from productive psychotic content. During the cause, more depressive symptoms developed, tranylcypromine were added. The patient was dismissed in acceptable mental status.

In 1993, a third severe relapse occurred requiring hospitalization again. Main symptoms were restlessness, anxiety and extensive productive psychotic symptoms. One month after hospitalization, additional symptoms of severe maculopapular exanthema on the trunk and limbs appeared. This was diagnosed as a Stevens–Johnson syndrome and thought to be caused by the psychopharmacological drugs, propyphenazone ergotamine or acetylsalicylic acid (her own headache medication). The patient was transferred to the internal medicine department, where high-dose cortisone (100 mg/day) was initiated, 11 days later the exanthema had remitted, and patient was transferred back to the psychiatric ward. Cortisone was gradually phased out until the patient was discharged from the hospital. A few months later, in outpatient treatment the patient attempted suicide during a psychotic episode and was readmitted to the hospital. Clinical examination now demonstrated speckled exanthema on her chest, which was rapidly progressing, then confluating and spreading over the limbs (see **Figure 1**). Blood testing showed high ANA Titer and for the first time autoantibodies against Ro/SSA and La/SSB (see **Table 1**). In October 1993, an atypical collagenosis was hypothesized. Under this assumption, prednisolone was

FIGURE 1 | Skin manifestations of the patient in April 1993, hypothesized as Stevens–Johnson Syndrome.

administered for over 6 months. During this period, skin and psychosis improved, though some symptoms continued to fluctuate.

# SURPRISING REMISSION UNDER AZATHIOPRINE TREATMENT FOR ALMOST 16 YEARS

In the time period between 1994 and 1996 no severe problems were stated, according to the patient's own evaluation "skin and mind came to rest." However, in June 1996 another relapse of psychosis occurred: she presented initially symptoms of thought disorder, delusional ideas, paranoia, depersonalization and later acoustic hallucinations. Thioridazine, fluphenazine, and diazepam were added. In the course, fluphenazine was reduced and because of emerging comorbid depressive symptoms and affective instability, amitriptyline and later lithium was added. Because of suspected drug-induced leukopenia, fluphenazine was replaced by olanzapine. Skin eruptions on the trunk followed psychiatric symptoms about 5 months later. Another therapy trial with prednisolone was initiated, with worsening of her skin and mental status, so that treatment on a protected psychiatric ward became necessary. CSF examination ruled out any brain infection, however, did show unspecific inflammation. A diagnosis of mixed connective tissue disorder was then made. Azathioprine (Imurek®) was prescribed, starting in January 1997 and continued for nearly 16 years. About 2 weeks after the start of azathioprine, the exanthema regressed and prednisolone was tapered out. As the psychiatric symptoms gradually improved, with a complex psychopharmacological treatment including lithium, olanzapine, amitriptyline, the patient was discharged from hospital. Azathioprine was reduced from 100 to 50 mg future years. Because of her good mental health, olanzapine and amitriptyline were gradually reduced but maintained until 2004. Lithium was phased out in subsequent 2 years. Under this treatment the patient remained stable with regard to mental status and skin status for nearly 16 years (except for one exception in 2004). After release she was in psychiatric and sporadic rheumatologic supervision. Interim, she married a second time. She chose early retirement but was able to work part-time in a fashion store. The ambulatory protocols in this time noted occasionally social anxieties, but predominant an "emotionally and psychologically stable" status.

In 2004, a brief hospitalization became necessary, because of restlessness and affective tension, probably as a consequence of life events (suicide attempt of her mother and sister and the sisters diagnosis schizophrenic disorder). During inpatient stay, acoustic hallucinations were noted and quetiapine was added next to amitriptyline, olanzapine, and azathioprine. She was released after 2 months in good condition and was mentally stable for another 8 years.

# SEVERE RELAPSE AFTER DISCONTINUANCE OF AZATHIOPRINE

In 2012, because of pregnancy, the patient decided together with the gynecologist to stop azathioprine medication immediately,

Mack et al.

July 2017 | Volume 8 | Article 131

*Treatment: ST, stationary treatment (number of hospitalization in brackets); AMB, ambulatory treatment.*

*antiphospolipid (antibody); ANCA, antineutrophil cytoplasmic antibodies.*

*NDA, no data available; green, pathologically elevated; red, pathologically elevated.*

*Laboratory marker and reference range/unit: AB, antibody; ANA, antinuclear antibody; CCP, (anti) citrullinated protein antibodies; DsDNA, double stranded deoxyribonucleic acid; ESR, erythrocyte sedimentation rate; aPL,*

balancing the teratogenic potential versus the need for continuing azathioprine. Shortly after, the pregnancy was terminated at the request of the patient, but azathioprine was still left out. As a result, an unexpected and dramatic change in the overall health status of the patient reoccurred. Within 4 weeks the patient developed signs of restlessness, insomnia and mania, all symptoms rapidly deteriorating further to a full blown psychosis, and again requiring hospitalization in May 2012. She soon developed progressive disorganized behavior, thought disorder overall incoherence and paranoid ideation. The initial therapy was undergoing a continual adjustment, including benperidol (stopped because of side effects), later fluphenazine, quetiapine, asenapine, and because of depressive symptoms trimipramine and amitriptyline. Finally, a combination of risperidone, olanzapine, chlorprothixene, and valproate was given. Electroconvulsive therapy was tried. Besides psychotropic medication, cortisone and azathioprine were restarted from the beginning. Exactly 19 days after admission, an exanthema appeared on her cleavage and upper back. Simultaneously, the patient reported to suffer from light sensitivity and arthralgia. ANA titer and antibodies against SSA–Ro were rising, C3 complement was low (compare **Table 1**). Severe skin eruptions were constantly treated in the department of Dermatology, at Ulm University.

In January 2013, the disease fulfilled the criteria of subacute cutaneous lupus erythematosus (psychosis, discoid lesions, photosensitivity, leukopenia, and ANA titer). Despite immediate resumption of immune suppression, the clinical situation was unchanged if not worsening. Because of self injurious behavior she was taken to the protected ward for weeks, partly medical restraint become necessary. Subsequently, instead of azathioprine, mycophenolate mofetil (CellCept® 3 g/day) was prescribed. Nevertheless, there was little therapeutic success to be observed. In March 2013, additionally even the scalp and face were affected by skin lesions. After 10 months of inpatient stay, the patient agreed to another CSF examination, which showed oligoclonal IgG bands (compare **Tables 2** and **3**). There were no neuronal antibodies. Also levels of tumor necrosis factor (TNF)-α and SCDs 25 were increased in the blood and elevated biomarkers in CSF were found (compare **Table 2**). The results of CSF examination now justified a diagnosis of NPSLE in accordance with established criteria (12–15).

# OVERCOMING THERAPY RESISTANCE WITH MONOCLONAL ANTIBODY THERAPY

Because of therapy resistance for more than 1 year and the initial diagnoses of SCLE with NPSLE, actual immune suppressive therapy was reconsidered, and intravenous therapy with the monoclonal antibody belimumab (Benlysta®) was started as an add-on treatment to the previous therapy (see **Figure 2**). Gradually, the patient improved over the next 2 months in every aspect and was discharged in December 2013. However, 2 weeks later she again attempted suicide by drug intoxication, requiring treatment in the intensive care unit, where all psychiatric and immune suppressive treatments were stopped. Eight weeks later, severe skin eruptions again emerged. In spite of hydroxychloroquine (Quensyl® 400 mg/day) being prescribed now, the skin eruptions worsened further, and psychiatric hospitalization was again required because of her severe psychosis relapse. Subsequently belimumab was restarted again. With the combination of mycophenolate mofetil (1,000 mg/day), hydroxychloroquine (400 mg/day), urbason (2 mg/day), and belimumab (10 mg kg/bodyweight per cycle) a rapid remission was achieved. Both, psychotic symptoms and skin manifestations improved in parallel. An attempt at pausing hydroxychloroquine was discarded because of repeated erupting skin manifestations. The patient was discharged from hospital treatment in an acceptable clinical status. During the next continuous outpatient treatment months she improved to full remission in psychosis and skin manifestations.

Following dismissal, she remained clinically stable with a complex of psychopharmacological and immune suppressive medications (for details see **Figure 2**).

# DISCUSSION

We conducted a detailed analysis case study of a patient with schizophrenia in regard to the question of whether autoimmunity and schizophrenia were unrelated comorbid disorders, or whether both disorders could be interpreted as a unified disorder, representing in fact the varied manifestations of an atypical

TABLE 2 | Plasma cytokines and CSF cytokines (red framed) of the patient in 2011–2015 based on research done by Prof. Dr. E. Marion Schneider, Experimental Anesthesiology Section, University Clinic Ulm, 89075 Ulm, Germany.

*Normal range: erythropoietin (EPO)* <*30 mU/ml; IL-10* <*3–15 pg/ml; IL-1β* <*1–3 pg/ml; IL-6* <*6–10 pg/ml; IL-8* <*70.0 pg/l; tumor necrosis factor (TNF)-α* <*3–15 pg/ml; sCD25* = *200–500 U/ml (adult); plasma ferritin* <*50 ng/ml; lipopolysaccharide binding protein (LBP)* <*15 μg.*

course of SLE, predominantly with psychiatric symptoms and accompanying skin manifestations most over the time. The latter hypothesis was plausible from a first look because of concurrent minor autoimmune signs and findings with the psychotic phases. To assume indeed a unifying disease hypothesis, we need, however, more arguments. The following points support the unifying perspective:

Lactate 1.4 mmol/l

FIGURE 2 | Timeline of the development of the disease, the severity of the skin lesons and immunotherapeutic treatments over time. Abbreviations and explanations: 1 well documentated, specific cutaneous, and mucosal lesions; 2 except for topical application; 3 / 4 methylprednisolone/mycophenolate mofetil was paused due to a suicide attempt. (A–G) period of stationary treatment: (A) 14.04.–23.06.89; (B) 15.02.–11.07.90; (C) 11.03.–27.07.90; (D) 20.08.93–30.03.94; (E) 11.06.96–12.06.97; (F) 19.07.–25.09.2004; (G) 29.05.12–03.05.13; (H) 29.05.–13.12.1; (I) 03.01.–30.04.14 and 01.05.–06.05.14.

depression and paranoid schizophrenia (ICD-10 F20.0, F32.2)] and autoimmune disorders [sister with Crohn's disease (ICD-10 K50.1)].

Because our patient rejected repeatedly CSF diagnostics, there is only a single CSF sample with cytokine profiling. The sampling was done in June 2013, during a phase of severe psychotic symptoms. In this CSF sample, we found suspicious biomarkers (compare **Table 2**): chemokine IL-8 was selectively higher than in the corresponding serum samples, other CSF cytokines were marginally elevated, TNF-α was not increased. These results match with CSF and serum cytokine profiling in schizophrenic and affective spectrum disorder patients (7). The presence of oligoclonal bands in CSF, few of them also in serum (compare **Table 3**), indicates in agreement with established criteria (13, 14) neuroinflammation, here overall mild and such was found in small groups of patients with affective and schizophrenic spectrum disorders in several studies (5, 6).

We conclude from our case analysis of 25 years disease course, that our case represented an atypical case of autoimmune disorder, lately diagnosed as LE, beginning with a focus on the CNS, explaining the variant psychiatric Syndrome from variant autoimmune-inflammatory disease activity. We could only partially define the apparently variant disease activity and only partially detect the immune inflammatory pathomechanisms involved. Overall the case represents a prototype of what was preliminary defined and hypothesized as ME. Cases with incomplete diagnostic autoimmune disorders over years, which later may develop a classified autoimmune disorder, are well-known, for example, presenting with mixed tissue disorder years before (17–20). Comparable cases of NPSLE were not described according to our knowledge. Our many arguments to assume an atypical case of remitting relapsing, or chronic when without immune suppressive treatment, NPSLE may be strong, though proof is missing: there were no definite, in part from circumstantial reasons, signs of neuroinflammation by imaging or CSF analysis in the early course. However, such was not unexpected from many arguments (9) and in rare single cases of schizophrenic syndromes with normal or quite normal CSF, nevertheless brain biopsy demonstrated mild inflammation (11, 21). We did not find neuronal antibodies, but this does not argue against our conclusion, cases with brain biopsy proven ME described by Najjar et al. (11) did also not demonstrate neuronal autoantibodies. We had not the opportunity to take a brain biopsy. On the other hand, we had the advantage of a long-term observation including several time periods of immune suppressive treatment, given from somewhat only partially established diagnostic criteria, detecting in retrospect a rather plausible if not clear evidence of positive treatment effects from immune suppression, which continues up to the time point of writing this paper. In sum, our arguments appear to represent strong arguments in total, to interpret our case as an atypical course of NPSLE from the beginning focused on the CNS.

With this case several difficult scientific and ethical questions had to be considered: After remission of the debated severe skin disease, thought to represent an allergic reaction to pharmaca or an undefined autoimmune disorder, also the possibility of a unified autoimmune disorder, as discussed in this paper, was considered by author Karl Bechter and discussed with the patient, though by majority of psychiatric doctors in our hospital rather held implausible. The patient was clearly informed about these scientific uncertainties and the novelty of the hypothesis of mild neuroinflammation to possibly causally underlie the psychotic disorder, held by Karl Bechter. The question of a relative indication of long-term treatment with azathioprine was evaluated independently by specialist in internal medicine (Dr. Peter Müller), with the result that long time azathioprine was justified (defined as relative indication) by the case history and because of continued mild leukopenia. With this informed knowledge the patient preferred to be treated with azathioprine as documented and the psychopharmacological medication to be reduced. The patient continued to visit Drs. Karl Bechter and Peter Müller, all over the respective years reported, though visiting Karl Bechter only sporadically but continuous over many years with remitted psychiatric syndrome but immediately with incipient relapse after set off of azathioprine.

# DIFFICULTIES IN DIAGNOSING AUTOIMMUNE DISORDERS IN PSYCHIATRIC PATIENTS

Psychiatric symptoms are rarely reported as an initial and isolated feature of SLE (22), in spite of many patients having the feeling that psychiatric symptoms occurred before they were diagnosed with SLE. However, central inflammation does not always coincide with systemic signs. Tests on mice suggest that NPSLE is not always a complication of SLE and can occur in absence of systemic autoimmunity (23). This supports the importance that early CSF diagnostics are done in longtime psychiatric patients. The European League Against Rheumatism emphasizes the increased risk of neuropsychiatric manifestations in SLE and requests early diagnostics including CSF examination in neuropsychiatric patients (even if only to exclude CNS infections) (24).

In our case, there was a delay of 4 years between initial psychiatric symptoms and first noticed mild skin manifestations. Accompanying systemic signs are maybe misinterpreted or remain undiagnosed, in our case even the established criteria of lupus were fulfilled only late in the course. This case shows the difficulties in establishing a diagnosis of common autoimmune disorders such as SLE, by regarding criteria like those of the American College of Rheumatology (ACR)-97 or Systemic Lupus International Collaborating Criteria (SLICC)-12. Our patient never had any further neuropsychiatric symptoms like seizures or neuropathy. No cerebrovascular accidents were noticed. Missing manifestations or incomplete laboratory markers create major uncertainties for clinicians. The general problem of incomplete criteria is known in autoimmune disorders, the diagnosis of mixed tissue disorder, a respective phenomenon, number of cases later being diagnosed with classical autoimmune disorders (19). Also atypical courses in other organ systems, e.g., with visceral focus have been described (25).

# CONCLUSION

Psychosis as the initial or predominant manifestation of SLE over years is an unusual course of the disease presented here*.* If our conclusion is correct, that indeed we observed one disorder with manifestations in different organ systems, there is an apparent need for improved diagnostic instruments and methods. For better detection of unnoticed clinical signs we recently developed a questionnaire called "FAISF" [(16), in review]. Another problem is, as our case strikingly demonstrates, that even known signs and minor findings of autoimmunity do not usually allow a definitive diagnosis of the defined autoimmune disorder, as established for example by the ACR or the SLICC. This problem of sub diagnostic cases has also been recognized in rheumatology.

A careful analysis of our case with 25 years disease including actual and previous material, clearly suggests that this case of seemingly comorbid schizophrenia with minor autoimmunity signs represented a case of one emerging autoimmune disorder

# REFERENCES

with variant manifestations systemically and within the CNS, though atypically with predominant appearance as a schizophrenia spectrum disorder. We do not exclude, that more such cases exist, but were certainly inappropriately understood. This case demonstrates the therapeutic potential if detected in an early phase and treated appropriately including with immunosuppressive options. The consequences of our perspective would likely include psychoimmunological aspects in psychiatric routine examinations. Recommendations should be developed including clinical signs and findings.

# AUTHOR CONTRIBUTIONS

AM—collecting data and summary of the patient. CP and KB treatment of the patient. EMS—CSF analysis, serum analysis, and cytokine profiling.

# ACKNOWLEDGMENTS

The authors thank Dr. Peter Müller, Kreiskliniken Günzburg, for his continued cooperation. The authors also thank the Margarete-Ammon-Stiftung, München, for continued financial support for research on mild neuroinflammation.

# FUNDING

Research on such cases including the one described was over years generously supported by the Margarete-Ammon-Stiftung, München.

connective tissue disease: successful treatment with plasmapheresis and rituximab. *Case Rep Rheumatol* (2013) 2013:4. doi:10.1155/2013/857694

of systemic disease in MRL/lpr mice. *J Neuroinflammation* (2015) 12:205. doi:10.1186/s12974-015-0423-4

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Mack, Pfeiffer, Schneider and Bechter. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Dominique Endres1 , Evgeniy Perlov 2 , Anne Nicole Riering1 , Viktoria Maier 1 , Oliver Stich3 , Rick Dersch3 , Nils Venhoff 4 , Daniel Erny 5,6, Irina Mader 7 and Ludger Tebartz van Elst1 \**

*1 Faculty of Medicine, Department of Psychiatry, Section for Experimental Neuropsychiatry, University of Freiburg, Freiburg, Germany, 2Clinic for Psychiatry Luzern, Luzern, Switzerland, 3 Faculty of Medicine, Department of Neurology, University of Freiburg, Freiburg, Germany, 4 Faculty of Medicine, Department of Rheumatology and Clinical Immunology, University of Freiburg, Freiburg, Germany, 5 Faculty of Medicine, Institute of Neuropathology, University of Freiburg, Freiburg, Germany, 6Berta-Ottenstein-Programme, Faculty of Medicine, University of Freiburg, Freiburg, Germany, 7 Faculty of Medicine, Department of Neuroradiology, University of Freiburg, Freiburg, Germany*

### *Edited by:*

*Karl Bechter, University of Ulm, Germany*

### *Reviewed by:*

*Harald Prüss, German Center for Neurodegenerative Diseases (DZNE) Berlin, Germany Assen Veniaminov Jablensky, University of Western Australia, Australia*

*\*Correspondence:*

*Ludger Tebartz van Elst<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry*

*Received: 28 January 2017 Accepted: 06 April 2017 Published: 21 April 2017*

#### *Citation:*

*Endres D, Perlov E, Riering AN, Maier V, Stich O, Dersch R, Venhoff N, Erny D, Mader I and Tebartz van Elst L (2017) Steroid-Responsive Chronic Schizophreniform Syndrome in the Context of Mildly Increased Antithyroid Peroxidase Antibodies. Front. Psychiatry 8:64. doi: 10.3389/fpsyt.2017.00064*

Background: Schizophreniform syndromes can be divided into primary forms from polygenic causes or secondary forms due to immunological, epileptiform, monogenic, or degenerative causes. Steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT) is a secondary immunological form associated with increased thyroid antibodies, such as antithyroid peroxidase antibodies and shows a good response to corticosteroids.

Case presentation: We present the case of a 41-year-old woman suffering from a schizophreniform syndrome. Starting at the age of 35, she developed psychotic exacerbations with formal thought disorder, acoustic hallucinations, cenesthopathic experiences, and loss of ego boundaries. At the same time, she began to suffer from chronic sexual delusions and olfactory hallucinations, which did not respond to neuroleptic medication. Her levels of antithyroid peroxidase antibodies were slightly increased, and the blood–brain barrier was disturbed. An electroencephalogram (EEG) showed intermittent generalized slowing, and cerebral magnetic resonance imaging (cMRI) depicted mild temporolateral atrophy. High-dose corticosteroid treatment led to convincing improvement of attentional performance and the disappearance of delusions and olfactory hallucinations.

Conclusion: SREAT can mimic typical symptoms of schizophreniform syndromes. The increased titer of antithyroid peroxidase antibodies in combination with the EEG slowing, blood–brain barrier dysfunction, and the cMRI alterations were the basis for suspecting an immunological cause in our patient. Chronic delusions, olfactory hallucinations, and cognitive deficits were successfully treated with corticosteroids. The occurrence of secondary immunological forms of schizophreniform syndromes demonstrates the need for innovative immunosuppressive treatment options.

Keywords: Hashimoto encephalopathy, steroid-responsive encephalopathy associated with autoimmune thyroiditis, schizophrenia, thyroiditis, thyroid peroxidase, corticosteroids

**168**

# BACKGROUND

Schizophreniform syndromes are common severe disorders that are characterized by delusions, hallucinations, loss of ego boundaries, cognitive deficits, impaired motivation, and social withdrawal (1). Primary schizophrenia, which has polygenic causes, can be distinguished from various secondary forms of schizophreniform syndromes caused by immunological, epileptiform, monogenic, or degenerative factors. Immunological encephalopathies can be associated with antibodies against thyroid tissue, such as steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT) with antithyroid peroxidase antibodies, intracellular onconeural or synaptic antigens, such as limbic encephalitis with anti-Hu antibodies, or neuronal cell surface antigens, such as autoimmune encephalitis due to anti-*N*-methyld-aspartate receptor (NMDAR) antibodies (2, 3). SREAT should be considered in the context of neuropsychiatric symptoms, autoimmune thyroiditis, increased thyroid antibodies, such as antithyroid peroxidase or anti-thyroglobulin antibodies, and other organic alterations [e.g., blood–brain barrier dysfunction in the cerebrospinal fluid, abnormal encephalopathic patterns as identified by an electroencephalogram (EEG), and non-specific white matter lesions as identified by cerebral magnetic resonance imaging (cMRI) (4–6)]. SREAT can mimic schizophreniform and other psychiatric syndromes (3, 7).

# CASE PRESENTATION

We present the case of a single 41-year-old female teacher and sports therapist suffering from a schizophreniform syndrome. In her third decade, she developed recurrent reactive depressive episodes. In her fourth decade, she developed a sexualized delusional system, which chronified. During this time, she suffered from repeated episodes with hallucinations.

The patient suffered reactive depressive episodes at 22, 28, and 32 years of age. At age 32, she experienced delusions for the first time. At age 35, she experienced her first schizophreniform episode with delusions, hallucinations, and promiscuity. During this episode, her mood was hypomanic. Since that time, she has suffered from a complex system of chronic delusions and olfactory hallucinations. At 40 and 41 years of age, she developed exacerbations with hallucinations. During these episodes, she suffered from formal thought disorder (long, incoherent train of thought) and auditory hallucinations. During the auditory hallucinations, she heard the voices of her neighbors ask her to have sexual intercourse and masturbate, the voice of a man who talked about the sexual lives of her friends, and a computer voice that told her which clothes to wear and that she was a "porn queen." She also had cenesthopathic experiences in which she felt that she was being irradiated, her breasts were tense, and her intestines were pulsating, and felt that she was being externally controlled. In the latter case, she thought that she was being irradiated by the neighbors, her thoughts were being tracked, and she could feel the bodily sensations of her neighbors. These symptoms disappeared after she received neuroleptic treatment.

However, simultaneously at age 35, she developed a complex system of chronic delusions in which she attributed sexual body parts to everyday gestures. In this sexualized delusional system, the nostrils represented the buttocks, the mouth was the vagina, the tongue was the penis, and the ears were a combination of the bottom and the genitals. If a person scratched at their nose, our patient identified this gesture as proof that the person would like to have or that they had anal intercourse. The precise area of the nose that the person scratched was attributed to different positions of sexual intercourse. She had similar convictions if a person touched the mouth, tongue, or ears. Wrinkles at different locations in the face were assigned specific meanings with respect to sexual preferences (**Figure 1**). The patient was absolutely convinced of her interpretations.  In addition, she had olfactory hallucinations in which she smelled urine, feces, and vaginal secretions in different situations. She took care not to wear provocative clothes to avoid attracting the sexual attention of men. All these symptoms persisted under treatment with neuroleptic medication.

# Developmental, Somatic, and Family History

The patient reported no history of *in utero* or birth complications, febrile convulsions, inflammatory brain diseases, or cerebral contusions. Her childhood development was normal, and there was no evidence of any neurodevelopmental disorder, such as attention-deficit hyperactivity disorder or autism. There was also no evidence of a personality disorder. Her somatic history was unremarkable, except that she had been diagnosed with Hashimoto's thyroiditis. She also had no history of alcohol or drug abuse.

# Investigations

from behind; lower right: oral sex.

A neurological examination of the patient was normal. Serum analysis showed increased antithyroid peroxidase antibodies (35–55 IU/mL over the course of the disease and before steroid treatment; normal range <34 IU/mL). Levels of antithyroglobulin and antithyroid-stimulating hormone receptor antibodies were unremarkable. Thyroid-stimulating hormone levels were in the upper range; triiodothyronine and thyroxine

levels were normal. Screening for rheumatoid factors, antinuclear antibodies, and antineutrophil cytoplasmic antibodies, as well as infectious diseases, such as *Borrelia*, lues, and HIV, showed no relevant abnormalities. In the cerebrospinal fluid analyses, we detected mild blood–brain barrier impairment and increased age-dependent albumin quotient (7.5; age-dependent reference <6.7 × 10−3 ). The white cell count (2/μL) and IgG index (0.43) were normal. No cerebrospinal fluid-specific oligoclonal bands were found. Antibodies against neuronal cell surface antigens [NMDAR, AMPA-R, GABA-B-R, and VGKC-complex (LGI1, Caspr2)] were negative in the cerebrospinal fluid. No antibodies against intracellular onconeural or synaptic antigens (Yo, Hu, CV2/CRMP5, Ri, Ma1/2, SOX1, GAD, and amphiphysin) were found in the serum. The cMRI showed a few right-accentuated hippocampus cysts in the cortical region. No white matter lesions were found. Mild temporolateral atrophy was identified. Repeated EEGs showed intermittent rhythmic delta activity (**Figure 2**). The neuropsychological test of attentional performance (TAP) revealed severe deficits in alertness and working memory (**Figure 3**).

# Differential Diagnosis

The symptoms met the ICD-10 criteria for paranoid hallucinatory schizophrenia (F20.0). Because the patient had Hashimoto's thyroiditis and increased antithyroid peroxidase antibodies, we also considered SREAT.

# Treatment

Different neuroleptics (amisulpride, aripiprazole, clozapine, flupentixol, olanzapine, quetiapine, and risperidone) were used to successfully treat our patient's acoustic and cenesthopathic experiences and loss of ego boundaries. However, average doses were not tolerated by the patient, and the neuroleptics were not able to treat the chronic delusional system and olfactory hallucinations. The same was true for additional treatment with lithium and valproic acid. The patient's hypothyroidism was successfully treated with 100 µg thyroxine daily. Because of treatment resistance and organic abnormalities due to potential SREAT, we started treatment with 500 mg methylprednisolone administered intravenously once per day for five consecutive days. Methylprednisolone treatment was continued with 100 mg administered orally each day, which was then gradually tapered over nearly 3 months. Treatment with amisulpride (400 mg), lithium (450 mg), pipamperone (20 mg), thyroxine (100 µg), selenium (0.1 mg), and cholecalciferol (0.025 mg) was continued.

## Outcome and Follow-up

Following the high-dose methylprednisolone treatment, the patient experienced full remission of all psychotic symptoms, including the delusional system and olfactory hallucinations. After realizing that the former system was a delusion, our patient developed depression. She felt sad and embarrassed about her previous misinterpretations and the "wasted years" of the disease. TAP at that time showed deterioration (t1, **Figure 3**).

Approximately 3 weeks later, her mood normalized. A TAP was performed again 2 months after starting high-dose methylprednisolone treatment and showed that the patient's alertness, divided attention, and working memory had all improved. However, the cMRI and EEG results were unchanged. Analysis of the cerebrospinal fluid showed that it normalized after steroid treatment; the blood–brain barrier dysfunction was no longer detected. Levels of antithyroid peroxidase antibodies were no longer elevated. Because of the positive response to the immunosuppressive treatment, we diagnosed SREAT encoded as an organic schizophreniform syndrome (ICD-10: F06.2). Six months later, the patient was still free of delusions, hallucinations, and loss of ego boundaries. Amisulpride treatment was reduced to 50 mg, pipamperone was stopped, and lithium, thyroxine, selenium, and cholecalciferol treatment remained unchanged. However, a TAP showed decreased results for divided attention and working memory (**Figure 3**). Antithyroid peroxidase antibody titers were still normal.

# DISCUSSION

We present the case of a female patient with a chronic schizophreniform syndrome, who had side effects and insufficient responsiveness to neuroleptic medication, had mild organic alterations with increased levels of antithyroid peroxidase antibodies, and responded to treatment with corticosteroids. Therefore, we diagnosed the patient with SREAT, which is typically characterized by seizures (47%), confusion (46%), memory disturbances (43%), disordered speech (37%), gait disturbance (27%), delusions (25%), myoclonic jerks (22%), and depression (12%) (6). Clinical manifestations of isolated schizophreniform syndromes have been described for individual cases in the literature (8–12). Most case studies reported acute onset or repetitive episodes of SREAT (9–12).

The distinctive features of our case were chronic delusions and olfactory hallucinations, which were successfully treated with high doses of corticosteroids. The EEG slowing and blood–brain barrier dysfunction identified in our patient are frequent, but nonspecific alterations that are found in over 80% of SREAT patients on a review level (6). We suggested in earlier studies that the EEG slowing might lead to clinical symptoms *via* local area network inhibition (3, 13). Blood–brain barrier dysfunction might allow potentially pathogenic autoantibodies to enter the central nervous system (CNS), thereby causing subtle CNS inflammation. A similar cause has been proposed for anti-NMDAR encephalitis (14). One earlier study showed cross reactivity between antithyroid peroxidase antibodies and cerebellar astrocytes (15). The role of the cerebellum in psychiatric symptoms has also been described (16, 17). Therefore, anti-thyroideal antibodies might have a direct pathophysiological role in the development of neuropsychiatric symptoms. However, Blanchin et al. (15) only showed antibody binding, but no neuronal damage. Therefore, the thyroid antibodies might alternatively function as an epiphenomenon, similar to the MRZ reaction in patients with multiple sclerosis (18), along with increased susceptibility to autoimmune conditions.

Most researchers favor the idea that thyroid antibodies do not play a relevant role in the development of neuropsychiatric symptoms (19, 20). Thyroid antibodies have been found in the serum of 13% of healthy individuals (21, 22). Isolated elevated levels of antithyroid peroxidase antibodies were found in 34% of SREAT cases (6). In our patient, autoantibody titers were only slightly increased; however, earlier studies have shown that antibody titers are not correlated with clinical severity (4–6). Our patient had no white matter lesions, which have been described in up to 52% of SREAT patients (6). However, our patient did have mild temporolateral atrophy; atrophy has been described in earlier SREAT cases (7). Following the current diagnostic recommendations, SREAT can only be diagnosed by exclusion (22). Therefore, the presence of other well-characterized neuronal antibodies was excluded in our patient. However, it is possible that our patient's symptoms might also be due to new or unknown antineuronal antibodies. An unbiased search on rodent brain sections could be an additional tool in future cases.

We treated our patient in a probatory manner with high-dose corticosteroids. According to prior research, in cases of autoimmune encephalitis, first-line therapy should include corticosteroids, intravenous immunoglobulins, or plasmapheresis, whereas second-line therapy should include rituximab or cyclophosphamide (23). In our patient, the blood–brain barrier disturbance and antithyroid peroxidase antibody titer normalized following corticosteroid therapy. Cognitive testing showed substantial improvement within 2 months, and the psychotic symptoms disappeared. If the autoantibodies played a direct pathophysiological role, closure of the blood–brain barrier would have helped avoid direct autoantibody-mediated effects in the CNS. Furthermore, one could speculate about alternative mechanisms. For example, the corticosteroid treatment could have led to epigenetic effects that modulated the genome functionality of different neural, glial, and immunological cell populations.

The worsening of neurocognitive testing after 5 months raises a question regarding the need for repeated or long-term immunosuppressive treatment. Patients with SREAT that have an insufficient response to corticosteroids are mostly treated with a second course of corticosteroids alone or combined with other immunosuppressive agents, such as azathioprine, intravenous immunoglobulins, or even plasmapheresis (6). For long-term immunosuppressive maintenance therapy, lowdose corticosteroids, azathioprine, methotrexate, cyclosporine A, mycophenolate mofetil, cyclophosphamide, rituximab, and intravenous immunoglobulins are available (23). Presently, there are no clear recommendations regarding long-term treatment for patients with SREAT. Therefore, further research is necessary in this field. Independent from such considerations, we found that neuroleptic treatment was initially sufficient for treating our patient's acoustic hallucinations, cenesthopathic experiences, and loss of ego boundaries. Therefore, an (initial) combination therapy with neuroleptics and corticosteroids seems a reasonable treatment for patients with SREAT.

# CONCLUSION

Single cases of chronic schizophreniform symptoms in the context of autoimmune thyroiditis and slightly increased antithyroid peroxidase antibodies can be successfully treated with corticosteroids. However, these effects were only shown for a short-term follow-up in our patient; a diagnosis of

# REFERENCES

cenesthopathic schizophrenia in our patient cannot be fully ruled out. We suggest that clinical screening for autoimmune conditions should become a routine procedure among patients with schizophreniform syndromes. The decision to administer immunosuppressive therapy in the presence of organ-specific autoantibodies should take into account other organic alterations, such as EEG and cMRI findings, or blood–brain barrier dysfunction. Therefore, cerebrospinal fluid analyses should also have a greater importance in patients with schizophreniform syndromes (24).

# ETHICS STATEMENT

The patient has given her signed written informed consent for this case report, including the presented images, to be published.

# AUTHOR CONTRIBUTIONS

DEndres and LTvE treated the patient and performed the data research. VM supported the data research. DEndres wrote the paper. IM performed the cMRI analyses. RD performed the EEG analyses. OS performed the CSF analyses. NV performed the rheumatological analyses. ANR performed the neuropsychological testing. EP and DErny critically reviewed the diagnostic results and contributed to the manuscript preparation. All authors were critically involved in the theoretical discussion and composition of the manuscript; read and approved the final version of the manuscript.

multiple sclerosis in a tertiary hospital cohort. *Fluids Barriers CNS* (2015) 12:27. doi:10.1186/s12987-015-0024-8

24. Endres D, Perlov E, Baumgartner A, Hottenrott T, Dersch R, Stich O, et al. Immunological findings in psychotic syndromes: a tertiary care hospital's CSF sample of 180 patients. *Front Hum Neurosci* (2015) 9:476. doi:10.3389/ fnhum.2015.00476

**Conflict of Interest Statement:** DEndres, EP, ANR, VM, RD, and DErny: None. OS: consulting and lecture fees, grant and research support from Bayer Vital GmbH, Biogen Idec, Genzyme, Merck Serono, Novartis, Sanofi-Aventis, and Teva. NV: advisory boards, lectures, research or travel grants within the last 3 years: Janssen-Cilag, Roche, Novartis, AbbVie, GSK, Medac, and Pfizer. IM: lecture fees from UCB Pharma GmbH, Germany. LTvE: advisory boards, lectures, or travel grants within the last 3 years: Eli Lilly, Janssen-Cilag, Novartis, Shire, UCB, GSK, Servier, Janssen, and Cyberonics.

*Copyright © 2017 Endres, Perlov, Riering, Maier, Stich, Dersch, Venhoff, Erny, Mader and Tebartz van Elst. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Dominique Endres1 , Evgeniy Perlov1 , Bernd Feige1 , Dirk-Matthias Altenmüller2 , Nils Venhoff 3 and Ludger Tebartz van Elst1 \**

*1Section for Experimental Neuropsychiatry, Department of Psychiatry, Faculty of Medicine, University of Freiburg, Freiburg, Germany, 2Freiburg Epilepsy Center, Department of Neurosurgery, Faculty of Medicine, University of Freiburg, Freiburg, Germany, 3Department of Rheumatology and Clinical Immunology, Faculty of Medicine, University of Freiburg, Freiburg, Germany*

Background: Schizophrenia-like disorders can be divided into endogenic or primary, idiopathic, polygenetic forms, and different secondary, organic subgroups [e.g., (para)epileptic, immunological, degenerative]. Epileptic and paraepileptic explanatory approaches have a long tradition due to the high rate of electroencephalography (EEG) alterations in patients with schizophrenia.

### *Edited by:*

*Karl Bechter, University of Ulm, Germany*

### *Reviewed by:*

*Sabina Berretta, Harvard Medical School, USA Mark Cunningham, Newcastle University, UK*

*\*Correspondence:*

*Ludger Tebartz van Elst<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry*

*Received: 27 October 2016 Accepted: 17 January 2017 Published: 08 February 2017*

### *Citation:*

*Endres D, Perlov E, Feige B, Altenmüller D-M, Venhoff N and Tebartz van Elst L (2017) Schizophrenia Associated with Epileptiform Discharges without Seizures Successfully Treated with Levetiracetam. Front. Psychiatry 8:12. doi: 10.3389/fpsyt.2017.00012*

Case presentation: We present the case of a 23-year-old female patient suffering, since the age of 14 years, from a fluctuating paranoid-hallucinatory syndrome with formal thought disorder, fear, delusions of persecution, auditory, visual, and tactile hallucinations, as well as negative and cognitive symptoms. Laboratory measurements showed increased titers of antinuclear antibodies (ANAs) in the context of ulcerative colitis. While there was no clear history or evidence of epileptic seizures, the EEG showed generalized 3 Hz polyspike wave complexes. Under treatment with levetiracetam, the symptoms disappeared and the patient was able to complete vocational training.

Conclusion: The schizophrenia-like symptoms associated with epileptiform discharges but not overt seizures and the good response to antiepileptic treatment could be interpreted in the context of a (para)epileptic pathomechanism. The EEG alterations might be due to a polygenetic effect due to different genes. Mild immunological mechanisms in the framework of ulcerative colitis and increased ANA titers might have supported the network instability. This case report illustrates (1) the importance of EEG screenings in schizophrenia, (2) a potential pathogenetic role of epileptiform discharges in a subgroup of patients with schizophrenia-like symptoms, and (3) that antiepileptic medication with levetiracetam could be a successful treatment alternative in schizophrenia-like disorders with EEG alterations.

Keywords: epilepsy, schizophrenia, epileptiform discharges, levetiracetam, paraepileptic, LANI hypothesis

**Abbreviations:** ACR, American College of Rheumatology; ANA, antinuclear antibody; CSF, cerebrospinal fluid; EEG, electroencephalography; FIRDA, frontal intermittent rhythmic delta activity; GABA, γ-aminobutyric acid; HV, hyperventilation; ICA, independent component analyses; IRDA; intermittent rhythmic delta activity; LANI, local area network inhibition; SLE, systemic lupus erythematosus; SV2A, synaptic vesicle glycoprotein 2A.

# BACKGROUND

Schizophrenia-like disorders are characterized by delusional perception and delusions of control, hallucinations (e.g., commenting or discussing voices), thought insertion or withdrawal, cognitive impairment, thought disorders, or social withdrawal.1 In addition to primary, endogenic or idiopathic, polygenetic forms, different secondary pathophysiological mechanisms [e.g., (para)epileptic, immunological, degenerative] can be assumed. Because of the high rates of electroencephalography (EEG) alterations, ranging from 7 to 60% in patients with schizophreniform syndromes, epileptic and paraepileptic explanatory approaches have a long tradition (1–3). In line with this assumption, we reported the first case of a young patient with a schizophrenialike disorder, generalized spike-and-slow-wave complexes without epileptic seizures but with remission under treatment with valproate (4, 5). Immunological reasons might be due to autoantibody-associated autoimmune encephalitis, cerebral vasculitis, or collagenosis [e.g., systemic lupus erythematosus (SLE)] (6). Immunological effects might lead to network instability and therefore cause (para)epileptic phenomena (7). The detection of a (para)epileptic or immunological mechanism opens new treatment perspectives, in that antiepileptics or immunomodulators may be helpful (4, 5, 7–10).

# CASE PRESENTATION

# Clinical Presentation

We present the case of a 23-year-old female office clerk suffering from fluctuating paranoid-hallucinatory symptoms since the age

1http://apps.who.int/classifications/icd10/browse/2010/en#/F20-F29.

of 14 years (2007). Therefore, the diagnosis of paranoid schizophrenia was made by different psychiatrists. Although taking neuroleptics, in the course of the disease, the patient developed five episodes (for several weeks) with paranoid-hallucinatory exacerbation. In these episodes, the patient suffered from formal thought disorder, fear, delusions of persecution, auditory hallucinations with commenting, discussing, and commanding voices, visual hallucinations with seeing maggots in her room, and tactile hallucinations with the feeling of being touched from behind. In parallel to these exacerbations, the patient developed severe negative and cognitive symptoms including attention and memory deficits, fatigue, depressive mood, and sleep disturbances thus completing the psychopathological features of comprehensive schizophrenia. Neurological and medical examinations were normal.

# Family History

There was a positive family history for unipolar depression, which was diagnosed earlier in two sisters, both parents, and both grandmothers. There was no history for schizophrenia-like psychopathology, bipolar disorder, or epilepsy.

# Somatic and Developmental History

Symptoms started 6 weeks after pain of the large joints. Therefore, a rheumatological disease was discussed. During an external work-up of repeated diarrhea, a chronic inflammation gut disease (ulcerative colitis) was diagnosed in 2014 and treated with mesalazine. No birth complications or *in utero* abnormalities were remembered; the birth was performed by cesarean section. The early childhood development was normal. No febrile convulsions or inflammatory brain diseases were remembered. The patient suffered mild cerebral contusions at the age of 4 and 12 years.

# Diagnostic Findings

The diagnostic findings are summarized in **Table 1**. Taken together, the immunological alterations were compatible with the previously known ulcerative colitis (11). The electrophysiological findings (**Figure 1**) would be compatible with primary (idiopathic) generalized epilepsy; however, the history for epileptic seizures including absences and myoclonic jerks was negative.

# Differential Diagnosis

The schizophreniform symptoms fulfilled the criteria of paranoidhallucinatory schizophrenia (see footnote 1). Therefore, the most important differential diagnoses would be schizophrenia plus coincidental epileptiform discharges. Although the findings of the investigation led us to our consideration of neuropsychiatric SLE, the American College of Rheumatology classification criteria for SLE were not fulfilled.2

# Therapy and Outcome

External neuroleptic treatment with risperidone (4 mg), amisulpride (600 mg), perazine (100 mg), aripiprazole (15 mg), and clozapine (275 mg) did not lead to long-term stabilization. Additional treatment with fluoxetine (20 mg) and citalopram (20 mg) for affective, negative, and cognitive symptoms did not successfully improve these symptoms. During the first visit in our clinic, in 2013, we detected the abovementioned epileptiform discharges. Assuming a (para)epileptic pathomechanism, we added antiepileptic treatment with valproate (1,500 mg) to the neuroleptic medication with clozapine and aripiprazole. At this point, the cognitive deficits improved significantly. Also, the EEG improved except for the (F)IRDAs. Another paranoid-hallucinatory episode in 2014 was treated successfully with a dose increase of clozapine and valproate. Because of a strong weight gain, the therapy with valproate was changed to topiramate (200 mg) in 2014. Assuming a (para)epileptic pathomechanism, clozapine was reduced and stopped in 2014. Aripiprazole was reduced in January 2014 and stopped in 2015. Normal results were found in both the routine EEG and in the EEG after sleep deprivation (2014). In the further course, topiramate led to a severe loss of appetite and was therefore changed to levetiracetam (1,500 mg) in 2014. The mental condition stabilized with the short-term antiepileptic treatment with topiramate and the subsequent antiepileptic treatment with levetiracetam (since 2014, and since 2015 as monotherapy). There were no more paranoid-hallucinatory episodes, the negative symptoms declined, the patient became a mother (in the spring of 2016); she lived alone, took care of her daughter, and simultaneously finished her vocational training (in the summer of 2016). She was able to suspend the mesalazine therapy and therefore only took levetiracetam (1,500 mg) at the time of stabilization.

# DISCUSSION

We present the case of a patient with a schizophrenia-like disorder and, following our judgment, a (para)epileptic pathomechanism, because of the distinct epileptiform discharges without seizures and remission under the anticonvulsive treatment with topiramate and subsequently levetiracetam monotherapy.

# Reason and Potential Pathophysiology of Network Instability

The EEG alterations might be due to a polygenetic effect caused by different genes (12). The immunological mechanisms in the framework of ulcerative colitis and increased antinuclear antibody titers might have supported the network instability by mild inflammatory processes (7, 13–15). Medication might also disclose underlying polygenetic or immunological network instability (16). The local area network inhibition (LANI) hypothesis might explain the causal relationship between epileptiform EEG discharges and schizophrenia-like symptoms. Excitatory network activity, as represented by the 3 Hz polyspike wave complexes, might lead to consecutive inhibitory processes in a physiological attempt of the central nervous system to stabilize the excitatory– inhibitory equilibrium of local cerebral networks. The repetitive

2www.rheumatology.org/Portals/0/Files/1997%20Update%20of%201982%20 Revised.pdf.

excitatory activity, as documented by consecutive EEGs in our patient, could have exceeded a critical threshold, leading to the successive hyperinhibition of cerebral networks. Following the LANI hypothesis, the symptoms are due to the secondary induced processes of hyperinhibition (e.g., temporal hyperinhibition might have led to auditory hallucinations or memory deficits) (2–5, 17).

# Treatment Considerations

Following the LANI hypothesis, the cognitive improvement after the addition of valproate to the neuroleptic treatment would be explained by the reduced epileptic activity and therefore the subsequent amelioration of inhibitory processes. In line with this assumption, comprehensive long-term stabilization was not achieved by several attempts of neuroleptic medication alone in spite of clear and very convincing effects of the treatment with clozapine in particular on positive symptoms. However, such a comprehensive improvement and even full remission was achieved with topiramate and later levetiracetam monotherapy. Thus, clozapine with its wellknown proconvulsive properties might well have counteracted inhibitory processes, while it is at the same time most likely unable to improve causative excitatory neuronal activity. By contrast, by reducing the epileptiform activity, topiramate and levitiracetam monotherapy might have resulted in a more causal and therefore more comprehensive improvement of relevant pathophysiology. Earlier, we published a case of a (para) epileptic schizophrenia-like disorder successfully treated with valproate (4). Valproate, and likewise lamotrigine, is already established as an augmentative treatment strategy in schizophrenia (18). One might hypothesize that patients with (para) epileptic pathomechanisms will benefit significantly more from antiepileptic treatment than other subgroups. To our knowledge, this is the first published case study that describes a patient with a schizophrenia-like disorder who was successfully treated with levetiracetam. Levetiracetam is rarely used off-label in psychiatry probably because of its potential side effects, such as agitation, aggression, fear, and psychosis (2). The advantage of levetiracetam is that it can be rapidly dosed up to effective concentrations. Therefore, on a single case basis, the working hypothesis of a (para)epileptic pathomechanism could be tested quickly. In comparison, valproate effects could be due to combined γ-aminobutyric acid (GABAergic) and antiglutamatergic effects, and lamotrigine effects might be due to potential antiglutamatergic effects. However, the mechanism of levetiracetam cannot be explained by such direct transmitter effects. The effects of levetiracetam seem to be associated with

# REFERENCES

the binding of the synaptic vesicle glycoprotein 2A (SV2A) (2). SV2A can be found in presynaptic membranes; it controls the calcium-dependent exocytosis of different neurotransmitters into the synaptic gap (19, 20). Therefore, it might also influence GABAergic and glutamatergic transmission (21).

# Limitations

Epileptiform discharges are found in less than 1% of healthy adults (1, 22–24) and as a rare consequence of clozapine treatment (16). Therefore, the EEG alterations could be interpreted either as an incidental finding in a patient with schizophrenia or as a clozapine side effect. However, the clinical course—with improvement under antiepileptic treatment in parallel with EEG normalization—speaks against the assumption. The pathophysiological processes might be explained by the LANI hypothesis; however, this is only an unproven, theoretical framework that needs further investigation.

# Conclusion

This case report illustrates the idea of a possible (para)epileptic pathomechanism in a patient with a schizophrenia-like disorder. Regarding diagnostic procedure, our case shows the importance of EEG examinations in typical schizophrenia-like disorders. Regarding pathophysiology, the case illustrates a potential pathogenetic role of epileptiform discharges in a subgroup of patients with schizophrenia-like symptoms. Regarding treatment, the case demonstrates that anticonvulsive medication with levetiracetam and also topiramate or valproate could be a successful treatment alternative in schizophrenia with EEG alterations.

# ETHICS STATEMENT

The patient has given her informed and written consent for this case report, including the presented images, to be published.

# AUTHOR CONTRIBUTIONS

LTvE treated the patient. DE wrote the paper and performed the data collection. DE and LTvE performed the interpretation of the diagnostic findings and therapy effects. BF performed and interpreted the EEG analysis. NV performed and interpreted the immunological analyses. EP and D-MA reviewed the diagnostic results and contributed to the manuscript preparation. All the authors were significantly involved in the theoretical discussion and the preparation of the manuscript, and they read and approved the final version of the manuscript.

3. Endres D, Perlov E, Feige B, Fleck M, Bartels S, Altenmüller D, et al. Electroencephalographic findings in schizophreniform and affective disorders. *Int J Psychiatry Clin Pract* (2016) 16:1–8. doi:10.1080/13651501.2016.1 181184

4. van Elst LT, Schulze-Bonhage A, Altenmüller D, Ebert D. Generalised spikeand-slow-wave complexes without seizures in schizophrenia. *Br J Psychiatry* (2011) 199:253–4. doi:10.1192/bjp.199.3.253a

5. Tebartz van Elst L, Krishnamoorthy ES, Schulze-Bonhage A, Altenmüller D, Richter H, Ebert D, et al. Local area network inhibition: a model of a potentially important paraepileptic pathomechanism in neuropsychiatric disorders. *Epilepsy Behav* (2011) 22:231–9. doi:10.1016/j.yebeh.2011. 06.016

**Conflict of Interest Statement:** DE, EP, and BF: none; D-MA: lecture fees from UCB Pharma; NV: advisory boards, lectures, research, or travel grants within the last 3 years: Janssen-Cilag, Roche, Novartis, AbbVie, GSK, and Medac. LTvE: lectures, work shops, or travel grants within the last 3 years: Eli Lilly, Medice, Shire, UCB, Servier, and Cyberonics.

*Copyright © 2017 Endres, Perlov, Feige, Altenmüller, Venhoff and Tebartz van Elst. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Lawrence T. C. Ong1 \*, Gary Galambos2 and David A. Brown1,3*

*1Centre for Immunology, Westmead Institute for Medical Research, University of Sydney, Sydney, NSW, Australia, 2St Vincent's Private Hospital, Sydney, NSW, Australia, 3Westmead Hospital, Sydney, NSW, Australia*

There is an increasing awareness that autoimmune diseases can present with neuropsychiatric manifestations. We present the case of a 17-year-old female requiring psychiatric hospitalization for obsessive–compulsive disorder and major depressive disorder with mixed affective features, who was subsequently diagnosed with primary Sjogren's syndrome. Treatment with potent immunosuppression resulted in remission of psychiatric illness. Due to a lack of awareness and/or the lack of specific biomarkers, clinicians may not associate psychiatric symptoms with autoimmune disease, including primary Sjogren's syndrome. This case demonstrates that Sjogren's syndrome may be a causative or aggravating factor in mental disorders and that autoimmune diseases should be carefully considered in the differential diagnosis of psychiatric illness especially in cases of concurrent physical symptomatology and severity or treatment resistance of psychiatric disease.

### *Edited by:*

*Michael Noll-Hussong, University of Ulm, Germany*

### *Reviewed by:*

*Elham Hossny, Ain Shams University, Egypt Danielle Cath, Utrecht University, Netherlands*

### *\*Correspondence:*

*Lawrence T. C. Ong<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Mood and Anxiety Disorders, a section of the journal Frontiers in Psychiatry*

*Received: 06 September 2016 Accepted: 28 June 2017 Published: 11 July 2017*

#### *Citation:*

*Ong LTC, Galambos G and Brown DA (2017) Primary Sjogren's Syndrome Associated With Treatment-Resistant Obsessive– Compulsive Disorder. Front. Psychiatry 8:124. doi: 10.3389/fpsyt.2017.00124*

Keywords: Sjogren's syndrome, obsessive–compulsive disorder, major depressive disorder, treatment resistance, autoimmune serology

Although there is increasing awareness that a subset of psychiatric presentations may result from underlying autoimmune disease, the evidence for an association between autoimmune disease and obsessive–compulsive disorder (OCD) specifically, appears modest, and is perhaps strongest in conditions such as rheumatic fever (1). Here, we present a case of OCD, which appears to be associated with underlying autoimmune disease, most likely primary Sjogren's syndrome (pSS) and discuss the need for a high degree of suspicion in diagnosis and timely management of such presentations.

Written, informed consent was obtained for the publication of the following case report. A 17-year-old female was admitted to a private psychiatric unit with features consistent with severe OCD. The patient suffered from recurrent obsessions regarding contamination, hoarding and symmetry, and compulsions including hand washing, showering, cleaning, and checking. Her symptoms had caused significant distress and interference with academic and social functioning, to the extent that she had become isolated from her peers and was unable to attend school. There was no drug use or diagnosed medical illnesses to account for her symptoms. Therefore, her symptomatology satisfied DSM-IV criteria for diagnosis of OCD. A score of 36 out of 40 on the Yale-Brown Obsessive–Compulsiveness score (YBOCS) indicated that symptoms were extreme in severity.

In addition to satisfying DSM-IV criteria for OCD, she also satisfied criteria for major depressive disorder, with melancholic features—in particular psychomotor agitation. She had been referred from a local general hospital after presenting there with suicidal ideation. A psychologist in the community had been managing her with psychotherapy for a number of months but her mental

**179**

state had continued to deteriorate. She had been commenced on amitriptyline 10 mg daily by her family physician 2 weeks prior to admission, which was replaced fluoxetine 20 mg daily 1 week prior to admission.

There was no history of previously diagnosed mental disorder, although frequent nocturnal awakenings had been a problem for most of her life. Obsessive–compulsive symptoms were noted during primary school years, including checking the alignment of objects on a wall and checking that power points had been switched off. As a child, the patient demonstrated age-appropriate developmental milestones. The patient's academic performance was considered to be superior throughout her school career. This was, however, interposed with a history of bullying and later depressive symptoms following a relationship breakup.

Her past medical history included facial lacerations from a dog attack in early childhood, subsequently requiring plastic surgery, tonsillectomy, adenoidectomy, and grommet insertion. There was a family history of anorexia nervosa, systemic lupus erythematosus (without cerebral lupus), but not OCD.

The patient was hospitalized and fluoxetine was increased to 80 mg daily (for her OCD and depressive features). In addition, quetiapine 350 mg daily (for her agitation) and prazosin 2 mg daily (for her nightmares) were commenced, in conjunction with group therapy and individual psychotherapy with her psychiatrist. There was a modest partial improvement in her condition.