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Increased prevalence of Chlamydophila DNA in post-mortem brain frontal cortex from patients with schizophrenia
Schizophrenia Research 129 (2011) 191–195
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Schizophrenia Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c h r e s
Increased prevalence of Chlamydophila DNA in post-mortem brain frontal cortex from patients with schizophrenia Barbara Fellerhoff a,b,⁎, Rudolf Wank b,⁎ a b
Institute of Immunology, University of Munich, Goethestrasse 31, D-80336 Munich, Germany Immunotherapy Research Center, Immunis e.V., Pettenkoferstrasse 8, D-80336 Munich, Germany
a r t i c l e
i n f o
Article history: Received 21 May 2010 Received in revised form 5 April 2011 Accepted 14 April 2011 Available online 5 May 2011 Keywords: Chlamydiaceae Chlamydophila psittaci Chlamydophila pneumoniae CNS Infection Schizophrenia Stanley Medical Research Institute (SMRI)
a b s t r a c t Infection can initiate symptoms of mental illness. It has been shown previously that Chlamydophila DNA is present six times more often in the blood of patients with schizophrenia than in the blood of control individuals. Monocytes, the main targets of Chlamydiaceae infection, are microglia precursors. We identified Chlamydiaceae infection using blinded brain DNA samples derived from the frontal cortex. Using PCR and sequence analysis, we found Chlamydophila DNA to be four times greater in patients with schizophrenia than in controls (schizophrenia: N = 34, microbial DNA frequency 23.5%; controls: N = 35, microbial DNA frequency 5.7%; P = 0.045, OR = 5.08). Persistent Chlamydophila-infected microglia or neuronal cells may impair neuronal circuits and thus be a mechanism for causing psychiatric illness in these patients. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Microbial infections can evoke symptoms of mental illness. Unrecognized Treponema pallidum infection, for example, causes dementia in approximately 10% of infected individuals (Luxon et al., 1979); hepatitis B virus may cause depression (Martini and Strohmeyer, 1974), and some HIV-infected individuals may develop dementia (Wolcott et al., 1985). Usually, symptoms of acute infection disappear with the resolution of the infection. However, some microbes are able to escape surveillance by the immune system, and after the acute illness, symptoms may flare up during infection by other microbes. These new infections, which stimulate inflammatory cytokines, can help to “resuscitate” chronic infections. Persistent infection, as well as new infection by different microbes, can stimulate a polarization of immune cell subpopulations (Wucherpfennig and Strominger, 1995). As a consequence, the polarized and amplified immune cell populations will produce enhanced levels of the same cytokine. Which cytokines are produced and how they may affect mental status depends on the individual's immunogenetic constellation and on the type and number of microbial pathogens encountered. Abnormal
⁎ Corresponding authors at: Immunotherapy Research Center, Immunis e.V., Pettenkoferstrasse 8, D-80336 Munich, Germany. Tel.: + 49 89 3090737 40; fax: + 49 89 3090737 49. E-mail addresses: [email protected] (B. Fellerhoff), [email protected] (R. Wank). 0920-9964/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2011.04.015
levels of several cytokines have been shown to be associated with schizophrenia (Drexhage et al., 2010). The present investigation was prompted by the history of two children who presented with serious cases of pneumonia. The pneumonia was followed by mental deterioration within months that did not improve, leading to autism in one child and schizophrenia in the other (Wank, 2002). Unusually high antibody titers against Chlamydophila were observed in both patients. In addition, it was noted that the patient with schizophrenia had extensive contact with dogs and the autistic child, with pigeons. Chlamydia trachomatis (C. trachomatis) and Chlamydophila pneumoniae (C. pneumoniae) are common human pathogens and can persist in infected monocytes. Less is known about Chlamydophila psittaci (C. psittaci.), but human-to-human and nosocomial transmission seem to be rare. C. pneumoniae and C. psittaci can affect the function of various organs and exhibit neurotropism. They can cause encephalitis (Reis et al., 1985) and meningitis (Pozniak et al., 2002). C. pneumoniae is probably spread via respiratory secretions (usually human-to-human transmission) (Falsey and Walsh, 1993; Saikku, 1992). To date, C. pneumoniae has not been found in pets but has been isolated from a horse, clawed frogs, koalas, and amphibians (Bodetti et al., 2002). Infection with C. psittaci is transmitted by aerosols from infected birds and other domestic animals, for example, from cats, but does not often affect humans (Peeling and Brunham, 1996). Most infected individuals are able to clear the Chlamydophila infection. If symptoms of mental illness were present, they disappear. We propose that in few infected individuals, a persistent infection
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may permanently disturb brain function and result in the complex symptoms of schizophrenia. It is important to note that antibody titers prove only that encounter with the infectious agent has occurred, but these titers do not prove that the infectious agent is still present in the body. Only a PCR assay with specific DNA probes can verify a persistent infection with a microbe. Previously, we identified C. psittaci and/or C. pneumoniae DNA by PCR in blood samples of 40% of patients with schizophrenia from Germany, compared to 6% of control individuals (Fellerhoff et al., 2007). We hypothesized that Chlamydophila (or other Chlamydiaceae) can persist not only in monocytes but also in the brain cells of patients with schizophrenia. To test our hypothesis, we analyzed blinded DNA samples from the Stanley Medical Research Institute (SMRI) brain collection, taken from the frontal cortex of American Caucasian patients with schizophrenia, with bipolar disorder and from unaffected individuals using nested PCR and sequence analyses.
PCR) was used as a positive control and was included in every PCR run. Any sample with a visible band in the subsequent agarose gel electrophoresis analysis was considered to be positive (Fig. 1). To confirm results after the first PCR analysis, the PCR was repeated at least once. All samples were verified as positive by the second PCR. To obtain PCR amplification for sequencing, individual rather than multiplex PCRs were used. The Sequencing Service (SeqService) of the Department of Biology, University of Munich, Germany, sequenced all amplification products of positive samples (defined as prevalence of detectable DNA amounts) to verify the specificity of the PCR products. The primers used for cycle sequencing were CPS101, CPN91 and CTR71 (Madico et al., 2000). The sequenced genes were 16S rRNA for C. trachomatis and C. pneumoniae and 16S and 16S–23S spacer rRNA for C. psittaci. As expected, all sequences matched the PCR amplification products for each organism without any polymorphisms, as verified by a BLASTN search (http://www.ncbi.nlm.nih.gov).
2. Materials and methods 2.1. Tissues, PCR and sequencing methods
2.2. Patients and control individuals
DNA from the frontal cortex (4 ng/μl, SMRI brain collection) was analyzed using the published nested PCR method to identify current Chlamydiaceae infections (Fellerhoff et al., 2007). No information about the composition/ratio of gray and white matter was available. We established a nested PCR with a specific primer set designed for each of the three Chlamydiaceae species based on the DNA sequences of the 16S rRNA and 16S–23S spacer rRNA genes in the first step. In brief, in a 50 μl PCR reaction mixture 50 ng of DNA, 200 mM of each dNTP (Pharmacia, Freiburg Germany), 25 pM of each primer, 3 mM MgCl2, 1× PCR-Puffer (Invitrogen, Karlsruhe, Germany), and 2.5 μl ELONGASE DNA polymerase (Invitrogen, Karlsruhe, Germany) were used. Cycling times were 2 min at 94 °C, followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and extension at 68 °C for 90 s. A final extension step of 10 min at 68 °C was added (Fellerhoff et al., 2007). In the second step, we followed the end point PCR method of Madico et al., 2000 to detect less than 1 colony-forming unit of C. psittaci, C. pneumoniae, and C. trachomatis. 1 μl of the PCR reaction of the first step was used for the specific PCR reaction of Madico et al., 2000 and followed by the published analysis. Steps to minimize cross-contamination included physical separation of the PCR steps in three rooms and interspersed negative control individuals. A low concentration of DNA (from 10 inclusion-forming units of each Chlamydiaceae species per
35 samples originated from unaffected Caucasian Americans, 32 from Caucasian Americans with bipolar disorder, and 34 from Caucasian American patients with schizophrenia. Diagnoses (performed according to DMS-IV) and demographic data of patients and of unaffected control individuals (free of psychiatric deficits) are summarized in Table 1. Subcategories of bipolar disorder were not provided. Tables 2A and 2B include previously published results obtained by the analysis of blood samples of German Caucasian patients and control individuals (Fellerhoff et al., 2007).
2.3. Statistics SPSS 10.1.3 was used for all statistical analyses (SPSS GmbH Software, Munich, Germany). Two-tailed Fisher's exact tests were used to determine frequency differences between infected and non-infected samples and were considered significant when P b 0.05. Infected samples were counted only one time, regardless of whether there was a single or double infection in the respective sample. The results of the current study of the frequencies of Chlamydophila infections in the frontal cortex were also compared to the previously published frequencies of Chlamydophila infections in peripheral blood mononuclear cells (PBMCs) (Tables 2A and 2B).
Fig. 1. Nested PCR products obtained from Chlamydiaceae species and sample DNA. Lanes 1 and 10 show the molecular length standard (L) (lengths in base pairs as indicated on the right side of the image); lanes 2–4 show products obtained from sample DNA: lanes 2–4, patients (P) 26, 5, and 35, respectively; lanes 5–7 show positive controls for C. psittaci (111 bp), C. pneumoniae (197 bp), and C. trachomatis (315 bp), respectively; lane 8 shows a positive control with all three Chlamydiaceae species combined (comb.); and lane 9 shows the no template control (ntc).
B. Fellerhoff, R. Wank / Schizophrenia Research 129 (2011) 191–195 Table 1 Demographic details and Chlamydiaceae infections in brain samples from patients with schizophrenia and bipolar disorder and from unaffected control individuals (SMRI, American Caucasians).
Mean age and range Sex Diagnoses
Unaffected control individuals
Patients with schizophrenia
Patients with bipolar disorder
N = 35
N = 34
N = 32
45.5 (19–64)
44.2 (31–60)
42.7 (19–59)
26M, 9F No axis I
25M, 9F 25 undifferentiated 8 paranoid 1 disorganized
14M, 18F 21 BP I
Psychotic features
0
34
C. C. C. C.
1 (2.86%) 1 (2.86%) 0 0
4 (11.76%) 3 (8.82%) 0 1 (2.94%)
4 BP II 6 BP NOS 1 BP SA 20+, 10−, 2 unclear 1 (3.13%) 4 (12.5%) 1 (3.13%) 0
2 (5.71%)
8 (23.53%)a
6 (18.75%)b
psittaci pneumoniae trachomatis psittaci + C. pneumoniae Sum of Chlamydiaceaeinfected individuals
Additional information can be viewed at http://www.stanleyresearch.org. Abbreviations are as follows: bipolar disorder I (BP I), bipolar disorder II (BP II), bipolar disorder not otherwise specified (BP NOS), and bipolar schizoaffective type (BP SA), respectively. a Samples from the frontal cortex: patients with schizophrenia vs. unaffected control individuals, P = 0.045 (Fisher's exact test, two-tailed); Chi 2 = 4.42; OR = 5.08 (confidence interval [CI] = 1.00–25.98). b Samples from the frontal cortex: patients with bipolar disorder vs. unaffected control individuals, P = 0.139 (Fisher's exact test, two-tailed); Chi2 = 2.70; OR = 3.81 (CI = 0.71–20.45).
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Table 2B Comparison of Chlamydiaceae infections of samples from PBMCs (German Caucasians) with brain samples (SMRI, American Caucasians), respectively. B. Comparison of brain samples with PBMC samples from patients with bipolar disorder/depression. Frontal cortex
PBMCs
Unaffected Patients with Unaffected Patients control bipolar control with individualsa depression individuals disorder
C. psittaci C. pneumoniae C. trachomatis C. psittaci + C. pneumoniae C. pneumoniae + C. trachomatis Sum of Chlamydiaceae infected individuals
N = 35
N = 32
1 (2.86%) 1 (2.86%) 0 (0%) 0 (0%) 0 (0%)
1 (3.13%) 4 (12.5%) 1 (3.13%) 0 (0%) 0 (0%)
2 (5.71%)
6 (18.75%)b
N = 225 2 (0.9%) 8 (3.6%) 4 (1.8%) 1 (0.4%) 0 (0%) 15 (6.7%)
N = 16 1 (6.3%) 1 (6.3%) 1 (6.3%) 0 (0%) 0 (0%) 3 (18.8%)c
a For additional information regarding age and sex, see published data (Fellerhoff et al., 2007). b Samples from the frontal cortex: patients with bipolar disorder vs. unaffected control individuals (see also Table 1), P = 0.139 (Fisher's exact test, two-tailed); 2 Chi = 2.70; OR = 3.81 (CI = 0.71–20.45). c Samples from PBMCs: patients with depression vs. unaffected control individuals, P = 0.11 (Fisher's exact test, two-tailed); Chi2 = 3.16; OR = 3.23 (CI = 0.83–12.59).
amplification products for each organism without any polymorphisms, as verified by a BLASTN search. Infected samples were counted only one time, regardless of whether there was a single or double infection in the respective sample. 3.2. Detection of Chlamydiaceae DNA in brain samples of American Caucasian patients with bipolar disorder
3. Results 3.1. Detection of Chlamydiaceae DNA in brain samples of American Caucasian patients with schizophrenia and controls DNA of C. psittaci and C. pneumoniae was identified in blinded DNA samples taken from the frontal cortex of patients with schizophrenia four times more often than in brain samples of unaffected individuals (P = 0.045, Table 1). All sequences matched the expected PCR
Table 2A Comparison of Chlamydiaceae infections of samples from PBMCs (German Caucasians) with brain samples (SMRI, American Caucasians), respectively. A. Comparison of brain samples with PBMC samples from schizophrenia patients. PBMCsa
Frontal cortex
C. C. C. C.
psittaci pneumoniae trachomatis psittaci + C. pneumoniae C. pneumoniae + C. trachomatis Sum of Chlamydiaceae infected individuals
Unaffected control individuals
Patients with schizophrenia
Unaffected control individuals
Patients with schizophrenia
N = 35
N = 34
N = 225
N = 72
1 (2.86%) 1 (2.86%) 0 (0%) 0 (0%)
4 (11.76%) 3 (8.82%) 0 (0%) 1 (2.94%)
0 (0%)
0 (0%)
2 (5.71%)
8 (23.53%)
2 (0.9%) 8 (3.6%) 4 (1.8%) 1 (0.4%)
b
13 (18.1%) 11 (15.3%) 0 (0%) 3 (4.2%)
0 (0%)
2 (2.8%)
15 (6.7%)
29 (40.3%)c
a For additional information regarding age and sex, see published data (Fellerhoff et al., 2007). b Samples from the frontal cortex: patients with schizophrenia vs. unaffected control individuals (see also Table 1), P = 0.045 (Fisher's exact test, two-tailed); Chi2 = 4.42; OR = 5.08 (CI = 1.00–25.98). c Samples from PBMCs: patients with schizophrenia vs. unaffected control individuals, P = 1.3 × 10−10 (Fisher's exact test, two-tailed); Chi2 = 48.83; OR= 9.43 (CI = 4.67– 19.23).
Chlamydiaceae DNA was detected in brain samples of American Caucasian patients with bipolar disorder three times more often than in control samples (P = 0.139, Table 1). While not significant, this trend indicates the need for further studies with a larger subject pool. Infected samples were counted only one time, regardless of whether there was a single or double infection in the respective sample. 4. Discussion Infections have been previously proposed to be causal or contributing pathogenic factors in schizophrenia (Torrey et al., 2006; Torrey and Yolken, 1995; Xavier et al., 2005; Yilmaz et al., 2008; Yolken et al., 2000). Many infections have been shown to induce symptoms of mental illness, but these symptoms generally disappear after recovery from the acute illness. However, some symptoms may not disappear if an acute infection becomes chronic. One could raise the question as to whether some psychiatric diseases represent a pool of unrecognized chronic infections. Microbes, which have the ability to permanently reside in the body, could permanently disturb brain functions. Today, the nested PCR technique allows successful amplification of only a few copies of the DNA of a specific pathogen. This technique allows the search for known pathogenic microbes. However, a combination of known and unknown microbes could also chronically infect individuals, leading to symptoms. Investigators have previously defined conditions that suggest the participation of unknown microbes in the diseases, for example, by comparing the levels of immune cytokines of healthy and affected individuals (Barak et al., 1995; Licinio et al., 1993; McAllister et al., 1995). Based on serological observations in a few patients (Wank, 2002) and by later analyzing DNA from peripheral blood, we hypothesized that C. psittaci and C. pneumoniae are important pathogenic factors in schizophrenia (Fellerhoff et al., 2005). We confirmed these observations using blood samples from a large German Caucasian panel, which revealed a 6-fold higher frequency of persistent infections with
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C. psittaci and/or C. pneumoniae in patients with schizophrenia (Fellerhoff et al., 2007). Although chronic infections in the peripheral blood can induce immune cells to produce certain cytokines in excess and thus affect brain functions (Barak et al., 1995; Licinio et al., 1993; McAllister et al., 1995), the disruption of brain circuits would be a more plausible explanation for the mental illness symptoms, if cells of the CNS were targeted by the infection. Therefore, we analyzed DNA from brain samples of patients with schizophrenia. To our knowledge, this represents the first time that the brains of patients with schizophrenia have been investigated for the presence of Chlamydiaceae DNA. In this study, blinded DNA samples were isolated from the frontal cortex of North American Caucasians with schizophrenia and bipolar disorder and from healthy control individuals. The increased rate of infection in the brains of American Caucasian patients with schizophrenia was significant (P= 0.045, odds ratio [OR]= 5.08), but this rate was lower than that previously observed in the peripheral blood of German Caucasian patients, i.e., 4-fold compared to 6-fold (Table 2A). This difference may reflect the small dataset used or differences in the rates of infection between the two populations. This difference in the observed infection rate may also reflect the distinct distribution of microglia in the brain for the following reasons. The primary targets of Chlamydophila infection in the blood are monocytes, which represent about 20–40% of PBMCs. The primary targets of Chlamydophila infection in the brain are probably microglia cells, which arise from monocyte subpopulations (Kaur et al., 2007). Microglia participate in neuronal signal transduction and are not equally distributed throughout the brain. The reported frequency of microglia in the gray matter is 0.5%, compared to 13% in the white matter, representing a 26-fold difference (Mittelbronn et al., 2001). Furthermore, microglia in the white matter are only half as frequent as monocytes in the peripheral blood. Microglia-poor samples from the gray matter may not have reached the threshold required to detect infected cells by PCR. It remains to be seen whether infectious particles are evenly distributed in the brain or are more frequent in certain brain areas. It will be also interesting to see whether certain symptoms are correlated with infection of certain brain areas. It would have been interesting to compare the PCR results with antibody titers. Unfortunately, there was no material available to perform serological investigations. However, it is assumed that nearly all individuals become infected at some time with C. pneumoniae, as the frequency of positive titers increases with age (Grayston, 1992). The high infection rate in the elderly is often interpreted as a “boost” resulting from frequent re-infections. The low seropositivity for C. pneumoniae in children aged less than 5 years increases to nearly 40% at 10 years of age. Seropositivity is greater than 75% in the elderly (5242 investigated individuals in Seattle, USA) (Grayston, 1992). A recent epidemiological study in Finland used both ELISA and immunofluorescence techniques and found similar infection rates for children: 55% for children 10–14 years of age and 70% for children 15–19 years of age (742 investigated individuals in Finland) (Tuuminen et al., 2000). The onset of schizophrenia typically occurs between the late teens and the mid-30s; onset prior to adolescence is rare. These statistical studies suggest that C. pneumoniae infects individuals before the onset of schizophrenia. Whereas C. pneumoniae is transferred from person to person via inhalation, the route of C. psittaci infection is not known. In addition to infected birds and poultry, cats may serve as a natural reservoir because of the high rates of chlamydial infection in household cats and asymptomatic C. psittaci infection in cats from breeding facilities (Peeling and Brunham, 1996). Using PCR to identify Chlamydophila DNA, we and others found that approximately 4% of the population is persistently infected with C. pneumoniae (Fellerhoff et al., 2007; Fukano, 2004; Sessa et al., 2001). Other investigators using the same species-specific detection method with PBMC-derived DNA failed to detect C. psittaci in smaller control panels of 38 healthy individuals (Ferreri et al., 2004). However, only about 1% of the population succumbs to schizophrenia. If we accept the supposition based on the high infection rate of C. psittaci
and C. pneumoniae in patients with schizophrenia that these infections are major factors in the development of schizophrenia, then we can assume that the immunogenetic constellation protects most infected individuals from serious negative sequelae and most of the persistently infected individuals from serious mental illness. Immunogenetics can explain the individual reactions against infections in the general population (Blackwell et al., 2009; Carter, 2009). Vulnerability genes for schizophrenia have been shown to be located in the HLA region (Thaker and Carpenter, 2001). We found that not only certain variants of classical HLA antigens but also variants of genes in the HLA complex, which select and transport microbial peptides, are more frequent in patients with schizophrenia than in control individuals (Fellerhoff and Wank, 2009). These genetic variants may predispose carriers to chronic Chlamydophila infections. We found a dramatic influence for a persistent infection with C. psittaci for carriers of HLA-A10. For these individuals, infection with C. psittaci represents a 50-fold increased relative risk for schizophrenia (Fellerhoff et al., 2007). The higher incidence of Chlamydophila infection in patients with schizophrenia may result in part from poor hygiene, a characteristic of many of these patients. However, based on serological data from the USA and in Finland, there is a good chance that Chlamydophila infection occurred in these patients before the onset of schizophrenia. Other indirect arguments for infection with Chlamydophila before the onset of schizophrenia derive from the infection rates seen with the non-neurotropic Chlamydia trachomatis, which are comparable in patients with schizophrenia and control subjects (Buka et al., 2001; Fellerhoff et al., 2007). Additionally, persistent Bartonella henselae infection (defined as the presence of detectable DNA using PCR), which infects household pets, showed no increased frequency among patients with schizophrenia; none of the tested brain samples was positive for this pathogen (Fellerhoff and Wank, unpublished results). Furthermore, using blood samples from our previous study, we found that 5.6% of German Caucasian patients with schizophrenia (N = 90) were positive for Bartonella henselae, as compared to 6.2% positive samples in the control group (N = 113) (Fellerhoff and Wank, unpublished results). This result argues against a generalized status of chronic infection induced by poor hygiene in patients with schizophrenia. It instead suggests a specific immunogenetic susceptibility to persistent infection with Chlamydiaceae pathogens, as shown for HLA-A10 (Fellerhoff et al., 2005; Laumbacher et al., 2003). Chlamydophila infections were three times more frequent in the brains of patients with bipolar depression than in control individuals, but this difference did not reach significance after correction for the number of comparisons (P = 0.139, Table 1). A three-fold increase of chlamydial DNA (18.75%) was also observed in samples from the peripheral blood of German Caucasian patients with depressive disorder (P = 0.11, Table 2B). These results suggest that further studies should be performed to evaluate Chlamydophila infections in the blood and brain of patients with bipolar depression. Whether Chlamydophila infections require co-infections to disturb CNS synaptic activities of patients with schizophrenia, for example with herpes virus or Toxoplasma gondii (Buka et al., 2001; Carter, 2009), requires further investigation. The neurotrophins BDNF and NT-3, which have been linked to psychiatric disorders, are produced by immune cells and by microglia (Besser and Wank, 1999; Roy et al., 2007), and the infection of microglia may affect brain functions. We have observed that genetic variants involved in transporting and chaperoning cellular and microbial proteins are also associated with schizophrenia (Fellerhoff and Wank, 2009). Could our results support other therapeutic modalities? We observed significant but short-lived improvements in symptoms with one time treatment in two of three azithromycin-treated patients with schizophrenia (Fellerhoff et al., 2005; Wank, 2002). These findings are also supported by others (Frykholm, 2009). In birds, azithromycin is given for 6 weeks to eradicate infections with C. psittaci (Smith et al., 2005), but it is premature to suggest such
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prolonged treatment for patients with schizophrenia. Nevertheless, given the higher prevalence of persistent Chlamydophila infections in patients with schizophrenia, it would be worthwhile to control the success of antibiotic treatment to further evaluate the role of Chlamydophila infections in schizophrenia. Based on our present knowledge, antibiotic treatment should be restricted to patients with Chlamydophilae-positive PCR results. Because Chlamydophilae spp. were not present in all patients with schizophrenia, one could consider Chlamydophilae spp.-infected patients as a subgroup. In other subgroups, other microbial infections may be the main pathogenic factor in the development of schizophrenia. The number of investigated brain samples and the sampling from the microglia-poor gray matter has limited the present study. Although C. psittaci is not known to commonly affect humans, surprising findings in the white matter cannot be excluded. Most recently, C. psittaci was found in a patient with a marginal zone B-cell lymphoma in the CNS (primary central nervous system lymphoma) (Ponzoni et al., 2011). C. psittaci could be only identified in the lymphomatous tissue, not in immune cells of the peripheral blood (Ponzoni et al., 2011). In neurological abnormalities such as transverse myelitis, confusion, meningitis, and encephalitis, the results of the examination of the cerebrospinal fluid are usually normal. Therefore, only meticulous investigation of all brain areas, of gray and white matter, of patients with schizophrenia and perhaps of patients with depressive disorders with nested PCR and sequencing will tell us the frequency and impact of Chlamydophila infections in these psychiatric patients. Role of funding source Funding for this study was provided by Verein zur Förderung der adoptiven Immunzelltherapie, and Immunis e.V; neither of them had any further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Contributors All authors contributed equally to and have approved the final manuscript. Conflict of interest All other authors declare that they have no conflicts of interest. Acknowledgements We thank the Verein zur Förderung der adoptiven Immunzelltherapie, Immunis e.V, and Annemarie and Max Gansbühler for generous support. Specimens were donated by The Stanley Medical Research Institute Brain Collection courtesy of Drs. Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken.
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Schizophrenia Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c h r e s
Increased prevalence of Chlamydophila DNA in post-mortem brain frontal cortex from patients with schizophrenia Barbara Fellerhoff a,b,⁎, Rudolf Wank b,⁎ a b
Institute of Immunology, University of Munich, Goethestrasse 31, D-80336 Munich, Germany Immunotherapy Research Center, Immunis e.V., Pettenkoferstrasse 8, D-80336 Munich, Germany
a r t i c l e
i n f o
Article history: Received 21 May 2010 Received in revised form 5 April 2011 Accepted 14 April 2011 Available online 5 May 2011 Keywords: Chlamydiaceae Chlamydophila psittaci Chlamydophila pneumoniae CNS Infection Schizophrenia Stanley Medical Research Institute (SMRI)
a b s t r a c t Infection can initiate symptoms of mental illness. It has been shown previously that Chlamydophila DNA is present six times more often in the blood of patients with schizophrenia than in the blood of control individuals. Monocytes, the main targets of Chlamydiaceae infection, are microglia precursors. We identified Chlamydiaceae infection using blinded brain DNA samples derived from the frontal cortex. Using PCR and sequence analysis, we found Chlamydophila DNA to be four times greater in patients with schizophrenia than in controls (schizophrenia: N = 34, microbial DNA frequency 23.5%; controls: N = 35, microbial DNA frequency 5.7%; P = 0.045, OR = 5.08). Persistent Chlamydophila-infected microglia or neuronal cells may impair neuronal circuits and thus be a mechanism for causing psychiatric illness in these patients. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Microbial infections can evoke symptoms of mental illness. Unrecognized Treponema pallidum infection, for example, causes dementia in approximately 10% of infected individuals (Luxon et al., 1979); hepatitis B virus may cause depression (Martini and Strohmeyer, 1974), and some HIV-infected individuals may develop dementia (Wolcott et al., 1985). Usually, symptoms of acute infection disappear with the resolution of the infection. However, some microbes are able to escape surveillance by the immune system, and after the acute illness, symptoms may flare up during infection by other microbes. These new infections, which stimulate inflammatory cytokines, can help to “resuscitate” chronic infections. Persistent infection, as well as new infection by different microbes, can stimulate a polarization of immune cell subpopulations (Wucherpfennig and Strominger, 1995). As a consequence, the polarized and amplified immune cell populations will produce enhanced levels of the same cytokine. Which cytokines are produced and how they may affect mental status depends on the individual's immunogenetic constellation and on the type and number of microbial pathogens encountered. Abnormal
⁎ Corresponding authors at: Immunotherapy Research Center, Immunis e.V., Pettenkoferstrasse 8, D-80336 Munich, Germany. Tel.: + 49 89 3090737 40; fax: + 49 89 3090737 49. E-mail addresses: [email protected] (B. Fellerhoff), [email protected] (R. Wank). 0920-9964/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2011.04.015
levels of several cytokines have been shown to be associated with schizophrenia (Drexhage et al., 2010). The present investigation was prompted by the history of two children who presented with serious cases of pneumonia. The pneumonia was followed by mental deterioration within months that did not improve, leading to autism in one child and schizophrenia in the other (Wank, 2002). Unusually high antibody titers against Chlamydophila were observed in both patients. In addition, it was noted that the patient with schizophrenia had extensive contact with dogs and the autistic child, with pigeons. Chlamydia trachomatis (C. trachomatis) and Chlamydophila pneumoniae (C. pneumoniae) are common human pathogens and can persist in infected monocytes. Less is known about Chlamydophila psittaci (C. psittaci.), but human-to-human and nosocomial transmission seem to be rare. C. pneumoniae and C. psittaci can affect the function of various organs and exhibit neurotropism. They can cause encephalitis (Reis et al., 1985) and meningitis (Pozniak et al., 2002). C. pneumoniae is probably spread via respiratory secretions (usually human-to-human transmission) (Falsey and Walsh, 1993; Saikku, 1992). To date, C. pneumoniae has not been found in pets but has been isolated from a horse, clawed frogs, koalas, and amphibians (Bodetti et al., 2002). Infection with C. psittaci is transmitted by aerosols from infected birds and other domestic animals, for example, from cats, but does not often affect humans (Peeling and Brunham, 1996). Most infected individuals are able to clear the Chlamydophila infection. If symptoms of mental illness were present, they disappear. We propose that in few infected individuals, a persistent infection
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may permanently disturb brain function and result in the complex symptoms of schizophrenia. It is important to note that antibody titers prove only that encounter with the infectious agent has occurred, but these titers do not prove that the infectious agent is still present in the body. Only a PCR assay with specific DNA probes can verify a persistent infection with a microbe. Previously, we identified C. psittaci and/or C. pneumoniae DNA by PCR in blood samples of 40% of patients with schizophrenia from Germany, compared to 6% of control individuals (Fellerhoff et al., 2007). We hypothesized that Chlamydophila (or other Chlamydiaceae) can persist not only in monocytes but also in the brain cells of patients with schizophrenia. To test our hypothesis, we analyzed blinded DNA samples from the Stanley Medical Research Institute (SMRI) brain collection, taken from the frontal cortex of American Caucasian patients with schizophrenia, with bipolar disorder and from unaffected individuals using nested PCR and sequence analyses.
PCR) was used as a positive control and was included in every PCR run. Any sample with a visible band in the subsequent agarose gel electrophoresis analysis was considered to be positive (Fig. 1). To confirm results after the first PCR analysis, the PCR was repeated at least once. All samples were verified as positive by the second PCR. To obtain PCR amplification for sequencing, individual rather than multiplex PCRs were used. The Sequencing Service (SeqService) of the Department of Biology, University of Munich, Germany, sequenced all amplification products of positive samples (defined as prevalence of detectable DNA amounts) to verify the specificity of the PCR products. The primers used for cycle sequencing were CPS101, CPN91 and CTR71 (Madico et al., 2000). The sequenced genes were 16S rRNA for C. trachomatis and C. pneumoniae and 16S and 16S–23S spacer rRNA for C. psittaci. As expected, all sequences matched the PCR amplification products for each organism without any polymorphisms, as verified by a BLASTN search (http://www.ncbi.nlm.nih.gov).
2. Materials and methods 2.1. Tissues, PCR and sequencing methods
2.2. Patients and control individuals
DNA from the frontal cortex (4 ng/μl, SMRI brain collection) was analyzed using the published nested PCR method to identify current Chlamydiaceae infections (Fellerhoff et al., 2007). No information about the composition/ratio of gray and white matter was available. We established a nested PCR with a specific primer set designed for each of the three Chlamydiaceae species based on the DNA sequences of the 16S rRNA and 16S–23S spacer rRNA genes in the first step. In brief, in a 50 μl PCR reaction mixture 50 ng of DNA, 200 mM of each dNTP (Pharmacia, Freiburg Germany), 25 pM of each primer, 3 mM MgCl2, 1× PCR-Puffer (Invitrogen, Karlsruhe, Germany), and 2.5 μl ELONGASE DNA polymerase (Invitrogen, Karlsruhe, Germany) were used. Cycling times were 2 min at 94 °C, followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and extension at 68 °C for 90 s. A final extension step of 10 min at 68 °C was added (Fellerhoff et al., 2007). In the second step, we followed the end point PCR method of Madico et al., 2000 to detect less than 1 colony-forming unit of C. psittaci, C. pneumoniae, and C. trachomatis. 1 μl of the PCR reaction of the first step was used for the specific PCR reaction of Madico et al., 2000 and followed by the published analysis. Steps to minimize cross-contamination included physical separation of the PCR steps in three rooms and interspersed negative control individuals. A low concentration of DNA (from 10 inclusion-forming units of each Chlamydiaceae species per
35 samples originated from unaffected Caucasian Americans, 32 from Caucasian Americans with bipolar disorder, and 34 from Caucasian American patients with schizophrenia. Diagnoses (performed according to DMS-IV) and demographic data of patients and of unaffected control individuals (free of psychiatric deficits) are summarized in Table 1. Subcategories of bipolar disorder were not provided. Tables 2A and 2B include previously published results obtained by the analysis of blood samples of German Caucasian patients and control individuals (Fellerhoff et al., 2007).
2.3. Statistics SPSS 10.1.3 was used for all statistical analyses (SPSS GmbH Software, Munich, Germany). Two-tailed Fisher's exact tests were used to determine frequency differences between infected and non-infected samples and were considered significant when P b 0.05. Infected samples were counted only one time, regardless of whether there was a single or double infection in the respective sample. The results of the current study of the frequencies of Chlamydophila infections in the frontal cortex were also compared to the previously published frequencies of Chlamydophila infections in peripheral blood mononuclear cells (PBMCs) (Tables 2A and 2B).
Fig. 1. Nested PCR products obtained from Chlamydiaceae species and sample DNA. Lanes 1 and 10 show the molecular length standard (L) (lengths in base pairs as indicated on the right side of the image); lanes 2–4 show products obtained from sample DNA: lanes 2–4, patients (P) 26, 5, and 35, respectively; lanes 5–7 show positive controls for C. psittaci (111 bp), C. pneumoniae (197 bp), and C. trachomatis (315 bp), respectively; lane 8 shows a positive control with all three Chlamydiaceae species combined (comb.); and lane 9 shows the no template control (ntc).
B. Fellerhoff, R. Wank / Schizophrenia Research 129 (2011) 191–195 Table 1 Demographic details and Chlamydiaceae infections in brain samples from patients with schizophrenia and bipolar disorder and from unaffected control individuals (SMRI, American Caucasians).
Mean age and range Sex Diagnoses
Unaffected control individuals
Patients with schizophrenia
Patients with bipolar disorder
N = 35
N = 34
N = 32
45.5 (19–64)
44.2 (31–60)
42.7 (19–59)
26M, 9F No axis I
25M, 9F 25 undifferentiated 8 paranoid 1 disorganized
14M, 18F 21 BP I
Psychotic features
0
34
C. C. C. C.
1 (2.86%) 1 (2.86%) 0 0
4 (11.76%) 3 (8.82%) 0 1 (2.94%)
4 BP II 6 BP NOS 1 BP SA 20+, 10−, 2 unclear 1 (3.13%) 4 (12.5%) 1 (3.13%) 0
2 (5.71%)
8 (23.53%)a
6 (18.75%)b
psittaci pneumoniae trachomatis psittaci + C. pneumoniae Sum of Chlamydiaceaeinfected individuals
Additional information can be viewed at http://www.stanleyresearch.org. Abbreviations are as follows: bipolar disorder I (BP I), bipolar disorder II (BP II), bipolar disorder not otherwise specified (BP NOS), and bipolar schizoaffective type (BP SA), respectively. a Samples from the frontal cortex: patients with schizophrenia vs. unaffected control individuals, P = 0.045 (Fisher's exact test, two-tailed); Chi 2 = 4.42; OR = 5.08 (confidence interval [CI] = 1.00–25.98). b Samples from the frontal cortex: patients with bipolar disorder vs. unaffected control individuals, P = 0.139 (Fisher's exact test, two-tailed); Chi2 = 2.70; OR = 3.81 (CI = 0.71–20.45).
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Table 2B Comparison of Chlamydiaceae infections of samples from PBMCs (German Caucasians) with brain samples (SMRI, American Caucasians), respectively. B. Comparison of brain samples with PBMC samples from patients with bipolar disorder/depression. Frontal cortex
PBMCs
Unaffected Patients with Unaffected Patients control bipolar control with individualsa depression individuals disorder
C. psittaci C. pneumoniae C. trachomatis C. psittaci + C. pneumoniae C. pneumoniae + C. trachomatis Sum of Chlamydiaceae infected individuals
N = 35
N = 32
1 (2.86%) 1 (2.86%) 0 (0%) 0 (0%) 0 (0%)
1 (3.13%) 4 (12.5%) 1 (3.13%) 0 (0%) 0 (0%)
2 (5.71%)
6 (18.75%)b
N = 225 2 (0.9%) 8 (3.6%) 4 (1.8%) 1 (0.4%) 0 (0%) 15 (6.7%)
N = 16 1 (6.3%) 1 (6.3%) 1 (6.3%) 0 (0%) 0 (0%) 3 (18.8%)c
a For additional information regarding age and sex, see published data (Fellerhoff et al., 2007). b Samples from the frontal cortex: patients with bipolar disorder vs. unaffected control individuals (see also Table 1), P = 0.139 (Fisher's exact test, two-tailed); 2 Chi = 2.70; OR = 3.81 (CI = 0.71–20.45). c Samples from PBMCs: patients with depression vs. unaffected control individuals, P = 0.11 (Fisher's exact test, two-tailed); Chi2 = 3.16; OR = 3.23 (CI = 0.83–12.59).
amplification products for each organism without any polymorphisms, as verified by a BLASTN search. Infected samples were counted only one time, regardless of whether there was a single or double infection in the respective sample. 3.2. Detection of Chlamydiaceae DNA in brain samples of American Caucasian patients with bipolar disorder
3. Results 3.1. Detection of Chlamydiaceae DNA in brain samples of American Caucasian patients with schizophrenia and controls DNA of C. psittaci and C. pneumoniae was identified in blinded DNA samples taken from the frontal cortex of patients with schizophrenia four times more often than in brain samples of unaffected individuals (P = 0.045, Table 1). All sequences matched the expected PCR
Table 2A Comparison of Chlamydiaceae infections of samples from PBMCs (German Caucasians) with brain samples (SMRI, American Caucasians), respectively. A. Comparison of brain samples with PBMC samples from schizophrenia patients. PBMCsa
Frontal cortex
C. C. C. C.
psittaci pneumoniae trachomatis psittaci + C. pneumoniae C. pneumoniae + C. trachomatis Sum of Chlamydiaceae infected individuals
Unaffected control individuals
Patients with schizophrenia
Unaffected control individuals
Patients with schizophrenia
N = 35
N = 34
N = 225
N = 72
1 (2.86%) 1 (2.86%) 0 (0%) 0 (0%)
4 (11.76%) 3 (8.82%) 0 (0%) 1 (2.94%)
0 (0%)
0 (0%)
2 (5.71%)
8 (23.53%)
2 (0.9%) 8 (3.6%) 4 (1.8%) 1 (0.4%)
b
13 (18.1%) 11 (15.3%) 0 (0%) 3 (4.2%)
0 (0%)
2 (2.8%)
15 (6.7%)
29 (40.3%)c
a For additional information regarding age and sex, see published data (Fellerhoff et al., 2007). b Samples from the frontal cortex: patients with schizophrenia vs. unaffected control individuals (see also Table 1), P = 0.045 (Fisher's exact test, two-tailed); Chi2 = 4.42; OR = 5.08 (CI = 1.00–25.98). c Samples from PBMCs: patients with schizophrenia vs. unaffected control individuals, P = 1.3 × 10−10 (Fisher's exact test, two-tailed); Chi2 = 48.83; OR= 9.43 (CI = 4.67– 19.23).
Chlamydiaceae DNA was detected in brain samples of American Caucasian patients with bipolar disorder three times more often than in control samples (P = 0.139, Table 1). While not significant, this trend indicates the need for further studies with a larger subject pool. Infected samples were counted only one time, regardless of whether there was a single or double infection in the respective sample. 4. Discussion Infections have been previously proposed to be causal or contributing pathogenic factors in schizophrenia (Torrey et al., 2006; Torrey and Yolken, 1995; Xavier et al., 2005; Yilmaz et al., 2008; Yolken et al., 2000). Many infections have been shown to induce symptoms of mental illness, but these symptoms generally disappear after recovery from the acute illness. However, some symptoms may not disappear if an acute infection becomes chronic. One could raise the question as to whether some psychiatric diseases represent a pool of unrecognized chronic infections. Microbes, which have the ability to permanently reside in the body, could permanently disturb brain functions. Today, the nested PCR technique allows successful amplification of only a few copies of the DNA of a specific pathogen. This technique allows the search for known pathogenic microbes. However, a combination of known and unknown microbes could also chronically infect individuals, leading to symptoms. Investigators have previously defined conditions that suggest the participation of unknown microbes in the diseases, for example, by comparing the levels of immune cytokines of healthy and affected individuals (Barak et al., 1995; Licinio et al., 1993; McAllister et al., 1995). Based on serological observations in a few patients (Wank, 2002) and by later analyzing DNA from peripheral blood, we hypothesized that C. psittaci and C. pneumoniae are important pathogenic factors in schizophrenia (Fellerhoff et al., 2005). We confirmed these observations using blood samples from a large German Caucasian panel, which revealed a 6-fold higher frequency of persistent infections with
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C. psittaci and/or C. pneumoniae in patients with schizophrenia (Fellerhoff et al., 2007). Although chronic infections in the peripheral blood can induce immune cells to produce certain cytokines in excess and thus affect brain functions (Barak et al., 1995; Licinio et al., 1993; McAllister et al., 1995), the disruption of brain circuits would be a more plausible explanation for the mental illness symptoms, if cells of the CNS were targeted by the infection. Therefore, we analyzed DNA from brain samples of patients with schizophrenia. To our knowledge, this represents the first time that the brains of patients with schizophrenia have been investigated for the presence of Chlamydiaceae DNA. In this study, blinded DNA samples were isolated from the frontal cortex of North American Caucasians with schizophrenia and bipolar disorder and from healthy control individuals. The increased rate of infection in the brains of American Caucasian patients with schizophrenia was significant (P= 0.045, odds ratio [OR]= 5.08), but this rate was lower than that previously observed in the peripheral blood of German Caucasian patients, i.e., 4-fold compared to 6-fold (Table 2A). This difference may reflect the small dataset used or differences in the rates of infection between the two populations. This difference in the observed infection rate may also reflect the distinct distribution of microglia in the brain for the following reasons. The primary targets of Chlamydophila infection in the blood are monocytes, which represent about 20–40% of PBMCs. The primary targets of Chlamydophila infection in the brain are probably microglia cells, which arise from monocyte subpopulations (Kaur et al., 2007). Microglia participate in neuronal signal transduction and are not equally distributed throughout the brain. The reported frequency of microglia in the gray matter is 0.5%, compared to 13% in the white matter, representing a 26-fold difference (Mittelbronn et al., 2001). Furthermore, microglia in the white matter are only half as frequent as monocytes in the peripheral blood. Microglia-poor samples from the gray matter may not have reached the threshold required to detect infected cells by PCR. It remains to be seen whether infectious particles are evenly distributed in the brain or are more frequent in certain brain areas. It will be also interesting to see whether certain symptoms are correlated with infection of certain brain areas. It would have been interesting to compare the PCR results with antibody titers. Unfortunately, there was no material available to perform serological investigations. However, it is assumed that nearly all individuals become infected at some time with C. pneumoniae, as the frequency of positive titers increases with age (Grayston, 1992). The high infection rate in the elderly is often interpreted as a “boost” resulting from frequent re-infections. The low seropositivity for C. pneumoniae in children aged less than 5 years increases to nearly 40% at 10 years of age. Seropositivity is greater than 75% in the elderly (5242 investigated individuals in Seattle, USA) (Grayston, 1992). A recent epidemiological study in Finland used both ELISA and immunofluorescence techniques and found similar infection rates for children: 55% for children 10–14 years of age and 70% for children 15–19 years of age (742 investigated individuals in Finland) (Tuuminen et al., 2000). The onset of schizophrenia typically occurs between the late teens and the mid-30s; onset prior to adolescence is rare. These statistical studies suggest that C. pneumoniae infects individuals before the onset of schizophrenia. Whereas C. pneumoniae is transferred from person to person via inhalation, the route of C. psittaci infection is not known. In addition to infected birds and poultry, cats may serve as a natural reservoir because of the high rates of chlamydial infection in household cats and asymptomatic C. psittaci infection in cats from breeding facilities (Peeling and Brunham, 1996). Using PCR to identify Chlamydophila DNA, we and others found that approximately 4% of the population is persistently infected with C. pneumoniae (Fellerhoff et al., 2007; Fukano, 2004; Sessa et al., 2001). Other investigators using the same species-specific detection method with PBMC-derived DNA failed to detect C. psittaci in smaller control panels of 38 healthy individuals (Ferreri et al., 2004). However, only about 1% of the population succumbs to schizophrenia. If we accept the supposition based on the high infection rate of C. psittaci
and C. pneumoniae in patients with schizophrenia that these infections are major factors in the development of schizophrenia, then we can assume that the immunogenetic constellation protects most infected individuals from serious negative sequelae and most of the persistently infected individuals from serious mental illness. Immunogenetics can explain the individual reactions against infections in the general population (Blackwell et al., 2009; Carter, 2009). Vulnerability genes for schizophrenia have been shown to be located in the HLA region (Thaker and Carpenter, 2001). We found that not only certain variants of classical HLA antigens but also variants of genes in the HLA complex, which select and transport microbial peptides, are more frequent in patients with schizophrenia than in control individuals (Fellerhoff and Wank, 2009). These genetic variants may predispose carriers to chronic Chlamydophila infections. We found a dramatic influence for a persistent infection with C. psittaci for carriers of HLA-A10. For these individuals, infection with C. psittaci represents a 50-fold increased relative risk for schizophrenia (Fellerhoff et al., 2007). The higher incidence of Chlamydophila infection in patients with schizophrenia may result in part from poor hygiene, a characteristic of many of these patients. However, based on serological data from the USA and in Finland, there is a good chance that Chlamydophila infection occurred in these patients before the onset of schizophrenia. Other indirect arguments for infection with Chlamydophila before the onset of schizophrenia derive from the infection rates seen with the non-neurotropic Chlamydia trachomatis, which are comparable in patients with schizophrenia and control subjects (Buka et al., 2001; Fellerhoff et al., 2007). Additionally, persistent Bartonella henselae infection (defined as the presence of detectable DNA using PCR), which infects household pets, showed no increased frequency among patients with schizophrenia; none of the tested brain samples was positive for this pathogen (Fellerhoff and Wank, unpublished results). Furthermore, using blood samples from our previous study, we found that 5.6% of German Caucasian patients with schizophrenia (N = 90) were positive for Bartonella henselae, as compared to 6.2% positive samples in the control group (N = 113) (Fellerhoff and Wank, unpublished results). This result argues against a generalized status of chronic infection induced by poor hygiene in patients with schizophrenia. It instead suggests a specific immunogenetic susceptibility to persistent infection with Chlamydiaceae pathogens, as shown for HLA-A10 (Fellerhoff et al., 2005; Laumbacher et al., 2003). Chlamydophila infections were three times more frequent in the brains of patients with bipolar depression than in control individuals, but this difference did not reach significance after correction for the number of comparisons (P = 0.139, Table 1). A three-fold increase of chlamydial DNA (18.75%) was also observed in samples from the peripheral blood of German Caucasian patients with depressive disorder (P = 0.11, Table 2B). These results suggest that further studies should be performed to evaluate Chlamydophila infections in the blood and brain of patients with bipolar depression. Whether Chlamydophila infections require co-infections to disturb CNS synaptic activities of patients with schizophrenia, for example with herpes virus or Toxoplasma gondii (Buka et al., 2001; Carter, 2009), requires further investigation. The neurotrophins BDNF and NT-3, which have been linked to psychiatric disorders, are produced by immune cells and by microglia (Besser and Wank, 1999; Roy et al., 2007), and the infection of microglia may affect brain functions. We have observed that genetic variants involved in transporting and chaperoning cellular and microbial proteins are also associated with schizophrenia (Fellerhoff and Wank, 2009). Could our results support other therapeutic modalities? We observed significant but short-lived improvements in symptoms with one time treatment in two of three azithromycin-treated patients with schizophrenia (Fellerhoff et al., 2005; Wank, 2002). These findings are also supported by others (Frykholm, 2009). In birds, azithromycin is given for 6 weeks to eradicate infections with C. psittaci (Smith et al., 2005), but it is premature to suggest such
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prolonged treatment for patients with schizophrenia. Nevertheless, given the higher prevalence of persistent Chlamydophila infections in patients with schizophrenia, it would be worthwhile to control the success of antibiotic treatment to further evaluate the role of Chlamydophila infections in schizophrenia. Based on our present knowledge, antibiotic treatment should be restricted to patients with Chlamydophilae-positive PCR results. Because Chlamydophilae spp. were not present in all patients with schizophrenia, one could consider Chlamydophilae spp.-infected patients as a subgroup. In other subgroups, other microbial infections may be the main pathogenic factor in the development of schizophrenia. The number of investigated brain samples and the sampling from the microglia-poor gray matter has limited the present study. Although C. psittaci is not known to commonly affect humans, surprising findings in the white matter cannot be excluded. Most recently, C. psittaci was found in a patient with a marginal zone B-cell lymphoma in the CNS (primary central nervous system lymphoma) (Ponzoni et al., 2011). C. psittaci could be only identified in the lymphomatous tissue, not in immune cells of the peripheral blood (Ponzoni et al., 2011). In neurological abnormalities such as transverse myelitis, confusion, meningitis, and encephalitis, the results of the examination of the cerebrospinal fluid are usually normal. Therefore, only meticulous investigation of all brain areas, of gray and white matter, of patients with schizophrenia and perhaps of patients with depressive disorders with nested PCR and sequencing will tell us the frequency and impact of Chlamydophila infections in these psychiatric patients. Role of funding source Funding for this study was provided by Verein zur Förderung der adoptiven Immunzelltherapie, and Immunis e.V; neither of them had any further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Contributors All authors contributed equally to and have approved the final manuscript. Conflict of interest All other authors declare that they have no conflicts of interest. Acknowledgements We thank the Verein zur Förderung der adoptiven Immunzelltherapie, Immunis e.V, and Annemarie and Max Gansbühler for generous support. Specimens were donated by The Stanley Medical Research Institute Brain Collection courtesy of Drs. Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken.
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