Prospects and Pitfalls of Plasma Complement C4 in Schizophrenia: Building a Better Biomarker.

Complex brain disorders like schizophrenia may have multifactorial origins related to mis-timed heritable and environmental factors interacting during neurodevelopment. Infections, inflammation, and autoimmune diseases are over-represented in schizophrenia leading to immune system-centered hypotheses. Complement component C4 is genetically and neurobiologically associated with schizophrenia, and its dual activity peripherally and in the brain makes it an exceptional target for biomarker development. Studies to evaluate the biomarker potential of plasma or serum C4 in schizophrenia do so to understand how peripheral C4 might reflect central nervous system-derived neuroinflammation, synapse pruning, and other mechanisms. This effort, however, has produced mostly conflicting results, with peripheral C4 sometimes elevated, reduced, or unchanged between comparison groups. We undertook a pilot biomarker development study to systematically identify sociodemographic, genetic, and immune-related variables (autoimmune, infection-related, gastrointestinal, inflammatory), which may be associated with plasma C4 levels in schizophrenia (SCH; n = 335) and/or in nonpsychiatric comparison subjects (NCs; n = 233). As with previously inconclusive studies, we detected no differences in plasma C4 levels between SCH and NCs. In contrast, levels of general inflammation, C-reactive protein (CRP), were significantly elevated in SCH compared to NCs (ANOVA, F = 20.74, p < 0.0001), suggestive that plasma C4 and CRP may reflect different sources or causes of inflammation. In multivariate regressions of C4 gene copy number variants, plasma C4 levels were correlated only for C4A (not C4B, C4L, C4S) and only in NCs (R Coeff = 0.39, CI = 0.01-0.77, R2 = 0.18, p < 0.01; not SCH). Other variables associated with plasma C4 levels only in NCs included sex, double-stranded DNA IgG, tissue-transglutaminase (TTG) IgG, and cytomegalovirus IgG. Toxoplasma gondii IgG was the only variable significantly correlated with plasma C4 in SCH but not in NCs. Many variables were associated with plasma C4 in both groups (body mass index, race, CRP, N-methyl-D-aspartate receptor (NMDAR) NR2 subunit IgG, TTG IgA, lipopolysaccharide-binding protein (LBP), and soluble CD14 (sCD14). While the direction of most C4 associations was positive, autoimmune markers tended to be inverse, and associated with reduced plasma C4 levels. When NMDAR-NR2 autoantibody-positive individuals were removed, plasma C4 was elevated in SCH versus NCs (ANOVA, F = 5.16, p < 0.02). Our study was exploratory and confirmation of the many variables associated with peripheral C4 requires replication. Our preliminary results point toward autoimmune factors and exposure to the pathogen, T. gondii, as possibly significant contributors to variability of total C4 protein levels in plasma of individuals with schizophrenia.

Complement C4 associations with altered microbial biomarkers exemplify gene-by-environment interactions in schizophrenia.

Schizophrenia is a complex brain disorder with genetic and environmental factors contributing to its etiology. Complement C4 genes are schizophrenia susceptibility loci and are activated in response to infections and gut microbiome imbalances. We hypothesize that C4 genetic susceptibility predisposes individuals to neuropathological effects from pathogen exposures or a microbiome in dysbiosis. In 214 individuals with schizophrenia and 123 non-psychiatric controls, we examined C4 gene copy number and haplotype groups for associations with schizophrenia and microbial plasma biomarkers. C4A copy number and haplotypes containing HERV-K insertions (C4A-long; C4AL-C4AL) conferred elevated odds ratios for schizophrenia diagnoses (OR 1.58-2.56, p < 0.0001), while C4B-short (C4BS) haplogroups conferred decreased odds (OR 0.43, p < 0.0001). Haplogroup-microbe combinations showed extensive associations with schizophrenia including C4AL with Candida albicans IgG (OR 2.16, p < 0.0005), C4AL-C4BL with cytomegalovirus (CMV) IgG (OR 1.79, p < 0.008), C4BS with lipopolysaccharide-binding protein (LBP) (OR 1.18, p < 0.0001), and C4AL-C4AL with Toxoplasma gondii IgG (OR = 17.67, p < 0.0001). In controls, only one haplogroup-microbe combination was significant: C4BS with CMV IgG (OR 0.52, p < 0.02). In schizophrenia only, LBP and CMV IgG levels were inversely correlated with C4A and C4S copy numbers, respectively (R2 = 0.13-0.16, p < 0.0001). C4 haplogroups were associated with altered scores of cognitive functioning in both cases and controls and with psychiatric symptom scores in schizophrenia. Our findings link complement C4 genes with a susceptibility to infections and a dysbiotic microbiome in schizophrenia. These results support immune system mechanisms by which gene-environmental interactions may be operative in schizophrenia.

Infection and inflammation in schizophrenia and bipolar disorder.

The present study investigated the relationship between exposure to infectious agents and inflammation markers in individuals with schizophrenia (SZ), bipolar disorder (BP), and controls without a psychiatric disorder. We measured plasma levels of antibodies and innate immune markers and correlated them with clinical symptoms and cognitive function. In both SZ and BP, we found an increase in soluble CD14, and in BP an increase in C-reactive protein, IgM class antibodies against cytomegalovirus (CMV), and IgG class antibodies against herpes simplex virus 2. Furthermore in BP, we observed a negative relationship between IgG antibodies against CMV and scores for cognitive function.

Seroreactive marker for inflammatory bowel disease and associations with antibodies to dietary proteins in bipolar disorder.

OBJECTIVES: Immune sensitivity to wheat glutens and bovine milk caseins may affect a subset of individuals with bipolar disorder. Digested byproducts of these foods are exorphins that have the potential to impact brain physiology through action at opioid receptors. Inflammation in the gastrointestinal (GI) tract might accelerate exposure of food antigens to systemic circulation and help explain elevated gluten and casein antibody levels in individuals with bipolar disorder. METHODS: We measured a marker of GI inflammation, anti-Saccharomyces cerevisiae antibodies (ASCA), in non-psychiatric controls (n = 207), in patients with bipolar disorder without a recent onset of psychosis (n = 226), and in patients with bipolar disorder with a recent onset of psychosis (n = 38). We compared ASCA levels to antibodies against gluten, casein, Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), influenza A, influenza B, measles, and Toxoplasma gondii. RESULTS: Elevated ASCA conferred a 3.5-4.4-fold increased odds ratio of disease association (age-, race-, and gender-corrected multinomial logistic regressions, p ≤ 0.00001) that was independent of type of medication received. ASCA correlated with food antibodies in both bipolar disorder groups (R(2)  = 0.29-0.59, p ≤ 0.0005), and with measles and T. gondii immunoglobulin G (IgG) in the recent onset psychosis bipolar disorder group (R(2)  = 0.31-0.36, p ≤ 0.004-0.01). CONCLUSIONS: Elevated seropositivity of a GI-related marker and its association with antibodies to food-derived proteins and self-reported GI symptoms suggest a GI comorbidity in at least a subgroup of individuals with bipolar disorder. Marker seroreactivity may also represent part of an overall heightened activated immune state inherent to this mood disorder.

C-reactive protein is elevated in schizophrenia.

BACKGROUND: Increased levels of inflammatory markers have been reported in schizophrenia, but few studies have examined levels of high sensitivity C-reactive protein (CRP), a non-specific inflammatory marker. METHODS: Levels of high sensitivity CRP were measured in individuals with schizophrenia, bipolar disorder, and non-psychiatric controls. Linear regression analyses were used to compare the CRP levels among the three groups adjusting for demographic and clinical variables. Logistic regression analyses were used to determine the odds ratios associated with elevated levels of CRP, defined as >=75th and 90th percentile in the controls. RESULTS: The sample consisted of 715 individuals: 295 with schizophrenia, 192 with bipolar disorder, and 228 without a psychiatric disorder. The levels of CRP in the schizophrenia group, but not in the bipolar disorder group, were significantly increased compared to controls adjusting for age, gender, race, maternal education, smoking status, and Body Mass Index (BMI) (t=3.78, p=<.001). The individuals with schizophrenia had significantly increased odds of having elevated levels of CRP relative to both the 75th and 90th percentile levels of the controls adjusting for the same covariates (OR 1.79, 95% CI 1.14, 2.82; p=.012; OR 2.76, 95% CI 1.58, 4.83, p=<.001). In the multivariate linear and logistic regression analyses, levels of CRP were also associated with BMI and female gender. CONCLUSIONS: Individuals with schizophrenia may be at risk for the adverse health consequences associated with elevated CRP in the overall population. Trials of interventions directed at lowering the level of CRP and other inflammatory markers are indicated.

Maternal antibodies to infectious agents and risk for non-affective psychoses in the offspring--a matched case-control study.

BACKGROUND: An increasing number of studies suggest that certain maternal infections are associated with non-affective psychoses in the offspring. Here we investigated if maternal exposure to Toxoplasma gondii, cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) prior to delivery was associated with future diagnosis of schizophrenia or other non-affective psychoses in the offspring. METHODS: This case-control study included 198 individuals born in Sweden 1975-85, diagnosed with schizophrenia (ICD-10, F20) and other non-affective psychoses (ICD-10, F21-29) as in- or outpatients, and 524 matched controls. Specific immunoglobulin G (IgG) levels in archived neonatal dried blood samples from these individuals were determined by immunoassays. Reference levels were determined by prevalences among pregnant women in Sweden 1975-85. Odds ratios (OR) for schizophrenia and other non-affective psychoses were calculated, considering maternal and gestational factors as covariates. RESULTS: Levels of IgG directed at T. gondii corresponding to maternal exposure was associated with subsequent schizophrenia (OR=2.1, 95% CI 1.0-4.5) as were levels of IgG directed at CMV (OR=2.2, 95% CI 1.0-5.1) but not at HSV-1 or -2. There were even stronger associations with higher levels of T. gondii or CMV antibodies. There were no associations between any of the infectious agents and other non-affective psychoses. CONCLUSIONS: This study supports findings of maternal exposure to T. gondii and schizophrenia risk in offspring, and extends the risk to also include maternal exposure to CMV. Future studies should confirm the association with CMV exposure and identify mechanisms underlying these associations.

Maternal antibodies to dietary antigens and risk for nonaffective psychosis in offspring.

OBJECTIVE: The authors analyzed archival dried blood spots obtained from newborns to assess whether levels of immunoglobulin G (IgG) directed at dietary antigens were associated with a later diagnosis of a nonaffective psychotic disorder. METHOD: The study population consisted of individuals born in Sweden between 1975 and 1985 with verified register-based diagnoses of nonaffective psychoses made between 1987 and 2003 and comparison subjects matched on sex, date of birth, birth hospital, and municipality. A total of 211 case subjects and 553 comparison subjects consented to participate in the study. Data on factors associated with maternal status, pregnancy, and delivery were extracted from the Swedish Medical Birth Register. Levels of IgG directed at gliadin (a component of gluten) and casein (a milk protein) were analyzed in eluates from dried blood spots by enzyme-linked immunosorbent assay. Odds ratios were calculated for levels of IgG directed at gliadin or casein for nonaffective psychosis. RESULTS: Levels of anti-gliadin IgG (but not anti-casein IgG) above the 90th percentile of levels observed among comparison subjects were associated with nonaffective psychosis (odds ratio=1.7, 95% CI=1.1-2.8). This association was not confounded by differences in maternal age, immigrant status, or mode of delivery. Similarly, gestational age at birth, ponderal index, and birth weight were not related to maternal levels of anti-gliadin IgG. CONCLUSIONS: High levels of anti-gliadin IgG in the maternal circulation are associated with an elevated risk for the development of a nonaffective psychosis in offspring. Research is needed to identify the mechanisms underlying this association in order to develop preventive strategies.

Gastrointestinal inflammation and associated immune activation in schizophrenia.

Immune factors are implicated in normal brain development and in brain disorder pathogenesis. Pathogen infection and food antigen penetration across gastrointestinal barriers are means by which environmental factors might affect immune-related neurodevelopment. Here, we test if gastrointestinal inflammation is associated with schizophrenia and therefore, might contribute to bloodstream entry of potentially neurotropic milk and gluten exorphins and/or immune activation by food antigens. IgG antibodies to Saccharomyces cerevisiae (ASCA, a marker of intestinal inflammation), bovine milk casein, wheat-derived gluten, and 6 infectious agents were assayed. Cohort 1 included 193 with non-recent onset schizophrenia, 67 with recent onset schizophrenia and 207 non-psychiatric controls. Cohort 2 included 103 with first episode schizophrenia, 40 of whom were antipsychotic-naïve. ASCA markers were significantly elevated and correlated with food antigen antibodies in recent onset and non-recent onset schizophrenia compared to controls (p≤0.00001-0.004) and in unmedicated individuals with first episode schizophrenia compared to those receiving antipsychotics (p≤0.05-0.01). Elevated ASCA levels were especially evident in non-recent onset females (p≤0.009), recent onset males (p≤0.01) and in antipsychotic-naïve males (p≤0.03). Anti-food antigen antibodies were correlated to antibodies against Toxoplasma gondii, an intestinally-infectious pathogen, particularly in males with recent onset schizophrenia (p≤0.002). In conclusion, gastrointestinal inflammation is a relevant pathology in schizophrenia, appears to occur in the absence of but may be modified by antipsychotics, and may link food antigen sensitivity and microbial infection as sources of immune activation in mental illness.

Serological evidence of exposure to Herpes Simplex Virus type 1 is associated with cognitive deficits in the CATIE schizophrenia sample.

Cognitive impairment is a core feature of schizophrenia. Previous studies have indicated that exposure to neurotropic infectious agents such as Herpes Simplex Virus type 1 may contribute to cognitive deficits and neuroanatomical abnormalities in individuals with schizophrenia. We examined the association between exposure to neurotropic infectious agents and cognitive function in 1308 participants in the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) trial. This sample included all of the individuals in the CATIE trial for whom baseline blood samples were available. Cognition was evaluated at baseline by a test battery which yielded composite scores in the domains of processing speed, verbal memory, vigilance, reasoning, and working memory as well as a summary neurocognitive score. Solid phase immunoassay techniques were used to measure IgG class antibodies to Herpes Simplex Virus type 1 (HSV-1), Herpes Simplex Virus type 2 (HSV-2), Cytomegalovirus (CMV), and to Toxoplasma gondii (T gondii) in the sera of the study individuals. We found a significant association between the neurocognitive summary score and antibodies to HSV-1 but not to HSV-2, CMV, or T. gondii. There was also a significant association between HSV-1 exposure and the Verbal Memory, Vigilance, and Processing Speed composite scores. HSV-1 may modulate the neurocognitive function of individuals with schizophrenia through its ability to establish latency in the central nervous system and undergo periodic reactivation. A better understanding of the role of HSV-1 may lead to better methods of treatment for the cognitive impairments associated with schizophrenia.

Immune activation by casein dietary antigens in bipolar disorder.

OBJECTIVES: Inflammation and other immune processes are increasingly linked to psychiatric diseases. Antigenic triggers specific to bipolar disorder are not yet defined. We tested whether antibodies to bovine milk caseins were associated with bipolar disorder, and whether patients recognized different epitopes of the casein protein than control individuals. METHODS: Anti-bovine casein immunoglobulin G (IgG) levels were measured with solid-phase immunoassays in 75 individuals with bipolar disorder and 65 controls. Epitope recognition was evaluated in immunoassays by cross neutralization with anti-bovine casein polyclonal antibodies of defined reactivity. Group-specific reactivity and associations with symptom severity scores were detected with age-, gender-, and race-controlled regression models. RESULTS: Individuals with bipolar disorder had significantly elevated anti-casein IgG (t-test, p ≤0.001) compared to controls. Casein IgG seropositivity conferred odds ratios of 3.97 for bipolar disorder [n=75, 95% confidence interval (CI): 1.31-12.08, p ≤0.015], 5.26 for the bipolar I subtype (n=56, 95% CI: 1.66-16.64, p ≤0.005), and 3.98 for bipolar disorder with psychosis (n=54, 95% CI: 1.32-12.00, p ≤0.014). Lithium and/or antipsychotic medication did not significantly affect anti-casein IgG levels. Casein IgG measures correlated with severity of manic (R(2) =0.15, 95% CI: 0.05-0.24, p ≤0.02) but not depressive symptoms. Unlike controls, sera from individuals with bipolar disorder did not inhibit binding of casein-reactive animal sera (t-test/χ(2) , p ≤0.0001). CONCLUSIONS: Anti-casein IgG associations with bipolar I diagnoses, psychotic symptom history, and mania severity scores suggest that casein-related immune activation may relate to the psychosis and mania components of this mood disorder. Case-control differences in epitope recognition implicate disease-related alterations in how the casein molecule is digested and/or how resulting casein-derived structures are rendered immunogenic.

Markers of gluten sensitivity and celiac disease in recent-onset psychosis and multi-episode schizophrenia.

BACKGROUND: Increased immune sensitivity to gluten has been reported in schizophrenia. However, studies are inconsistent about this association. METHODS: The sample of 471 individuals included 129 with recent-onset psychosis, 191 with multi-episode schizophrenia, and 151 controls. Immunoglobulin (Ig)G and IgA antibodies to gliadin and to tissue transglutaminase, and IgG antibodies to deamidated gliadin were measured. Quantitative levels of antibodies in the psychiatric groups were compared with controls. All participants were categorized as to whether their levels of antibodies met standardized cutoffs for celiac disease. HLA DQ2 and HLA DQ8 alleles were detected by real-time polymerase chain reaction. RESULTS: Individuals with recent-onset psychosis had increased levels of IgG (odds ratio [OR] 5.50; 95% confidence interval [CI] 2.65-11.42) and IgA (OR 2.75; 95% CI 1.31-5.75) antibodies to gliadin compared with control subjects. Individuals with multi-episode schizophrenia also had significantly increased levels of IgG antibodies to gliadin (OR 6.19; 95% CI 2.70-14.16). IgG antibodies to deamidated gliadin and IgA antibodies to tissue transglutaminase were not elevated in either psychiatric group, and fewer than 1% of individuals in each of the groups had levels of these antibodies predictive of celiac disease. There were no significant differences in the distribution of the HLA DQ2/8 alleles among the groups. CONCLUSIONS: Individuals with recent-onset psychosis and with multi-episode schizophrenia who have increased antibodies to gliadin may share some immunologic features of celiac disease, but their immune response to gliadin differs from that of celiac disease.

Subunit and whole molecule specificity of the anti-bovine casein immune response in recent onset psychosis and schizophrenia.

Previous studies show increased antibody levels to bovine casein in some individuals with schizophrenia. The immunogenicity of specific domains of bovine casein varies among people with milk sensitivities and thus could vary among different neuropsychiatric disorders. Using ELISAs and immunoblotting, we characterized IgG class antibody specificity to whole bovine casein and to the alpha(s), beta, and kappa subunits in individuals with recent onset psychosis (n=95), long-term schizophrenia (n=103), and non-psychiatric controls (n=65). In both patient groups, we found elevated IgG to casein proteins, particularly to whole casein and the alpha(s) subunit (p

Polymorphisms in human endogenous retrovirus K-18 and risk of type 2 diabetes in individuals with schizophrenia.

Type 2 diabetes is a major health problem in individuals with schizophrenia. The genetic basis of diabetes risk in individuals with schizophrenia has not been previously defined. We measured polymorphisms in a human endogenous retrovirus, Herv K-18, which is located in the CD48 signaling lymphocyte activating (SLAM) gene on chromosome 1. The study population consisted of 229 individuals with schizophrenia, 29 of whom had a history of type 2 diabetes, as well as 136 control individuals without a history of a psychiatric disorder or type 2 diabetes. We found that a haplotype defined by 2 polymorphisms in the envelope region of Herv K-18 is highly associated with type 2 diabetes in a population of 229 individuals with schizophrenia, with an odds ratio of 9.0 (95% confidence limits 2.3-34.7, p<.001) adjusted for race, gender and type of antipsychotic medication. Lower levels of association were found in other polymorphisms located in the 3'untranslated region of Herv K-18 and in adjacent loci in CD48. Polymorphisms in endogenous retroviruses which are located near immunomodulatory genes may constitute risk factors for diabetes in individuals with schizophrenia.