Multiplatform metabolome and proteome profiling identifies serum metabolite and protein signatures as prospective biomarkers for schizophrenia.

Schizophrenia is a severe mental illness with a biological basis. However, the search for reliable biomarkers suitable for clinical routine has been futile so far. Accordingly, there is a need for innovative approaches such as genomics and proteomics to achieve this goal. In the present study, we compared metabolomic and proteomic data from 26 schizophrenia patients as well as from unaffected controls carefully matched for age and gender in a multi-platform approach. The combined analysis identified many signatures with initially good biomarker characteristics. After statistical analysis and comparison of these identified serum metabolites (analysed by Gas Chromatography Mass Spectrometry) and hydrophobic serum proteins (analysed by matrix-assisted laser desorption ionisation mass spectrometry), several markers (e.g., 2-piperidinec carboxylic acid, 6-deoxy-mannofuranose, galactoseoxime and a serum peptide of m/z 3177) were determined as having the best discriminating value between the groups. Our findings represent a proof of principle indicating that metabolomic and proteomic approaches can be successfully used in psychiatric biomarker research, even though the results should be regarded as preliminary with a need for replication in larger samples.

C3-dependent mechanism of microglial priming relevant to multiple sclerosis.

Microglial priming predisposes the brain to neurodegeneration and affects disease progression. The signal to switch from the quiescent to the primed state is unknown. We show that deleting the C3 convertase regulator complement receptor 1-related protein y (Crry) induces microglial priming. Mice that were double-knockout for Crry and either C3 or factor B did not show priming, demonstrating dependence on alternative pathway activation. Colocalization of C3b/iC3b and CR3 implicated the CR3/iC3b interaction in priming. Systemic lipopolysaccharide challenge overactivated primed microglia with florid expression of proinflammatory molecules, which were blocked by complement inhibition. Relevance for neurodegenerative disease is exemplified by human multiple sclerosis (MS) and by experimental autoimmune encephalomyelitis (EAE), a model of MS. In human MS, microglial priming was evident in perilesional white matter, in close proximity to C3b/iC3b deposits. EAE was accelerated and exacerbated in Crry-deficient mice, and was dependent on C activation. In summary, C3-dependent microglial priming confers susceptibility to other challenges. Our observations are relevant to progression in MS and other neurological diseases exacerbated by acute insults.

ZNF804A genotype modulates neural activity during working memory for faces.

BACKGROUND: Genetic susceptibility to schizophrenia (SZ) has been suggested to influence the cortical systems supporting working memory (WM) and face processing. Genetic imaging studies link the SZ risk variant rs1344706 on the ZNF804A gene to psychosis via alterations in functional brain connectivity during WM, but no work has looked at the effects of ZNF804A on WM with face-processing components. METHODS: We therefore investigated healthy controls that were genotyped for rs1344706 with a face WM task during functional magnetic resonance imaging. We suggested that variation at the rs1344706 locus would be associated with similar alterations as patients previously tested using the same WM task for faces. RESULTS: The rs1344706 risk allele was indeed associated with altered activation in the right dorsolateral prefrontal (rDLPFC) cortex. We established that the rDLPFC was activated in a task-dependent manner, suggesting that the differences in activation between rs1344706 genotype groups reflected alterations in task processing. Furthermore, we demonstrated that the rDLPFC region showed significant volumetric overlap with the rDLPFC which had previously been reported to be altered during task processing for patients with SZ. CONCLUSIONS: The findings support an association between rs1344706 and alterations in DLPFC activity during WM for faces. We further suggest that WM for faces may be a useful intermediate phenotype in the investigation of genetic susceptibility to psychosis.

Protein and peptide profiling as a tool for biomarker discovery in depression.

This study sought to determine whether protein and/or peptide profiles from serum were able to distinguish patients suffering from depression from healthy control subjects and thereby act as biomarker candidates with potential diagnostic value. Serum samples were collected from patients (n = 39) and controls (n = 30). A C8 magnetic bead protocol was used to prepare serum proteins, while a microextraction C18 packed tip was used to isolate serum peptides. Both protein and peptide profiles were recorded by MALDI-MS and the data were exported for further analysis. No protein signals differentiated patients from controls and principle component analysis of the entire peptide profile did not allow for distinct clustering of the two groups. Further analysis of individual peptides however identified three peptide signals whose intensities were significantly different between patients and control subjects. The efficacy of these potential biomarker candidates to identify the patients was therefore studied using a receiver operating characteristic (ROC) curve and the combined use of all three candidates together offered the most specific and sensitive identification of true positive cases of depression.

Targeting neural-restrictive silencer factor sensitizes tumor cells to antibody-based cancer immunotherapy in vitro via multiple mechanisms.

Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Recently, we designed a peptide derived from the neural-restrictive silencer factor (REST), REST68, which we showed to inhibit expression of CD59 in tumors lacking the full-length REST and proposed a detailed model for regulation of CD59 expression via interplay between REST and nucleolin (NCL) transcription factors. In this paper, we study in detail the mechanisms for sensitization of malignant cells to Ab-based cancer immunotherapy by the REST68 peptide and the implications of the REST/NCL model for the design of treatment resulting in higher tumor susceptibility. REST68 inhibited CD59 expression in malignant cells expressing either truncated or full-length REST, but not in nonmalignant cells. However, activation of protein kinase C (PKC) in nonmalignant cells, a process that contributes to cellular transformation, phosphorylated NCL and enabled suppression of CD59 expression by the REST68. Combined treatment of different tumor types with REST68 and PKC inhibitor synergized to further suppress CD59 expression and reduce resistance to complement lysis. The combined treatment also increased susceptibility of tumors expressing either of the REST isoforms to PBMC-mediated killing, which, at least in part, accounted for the strong promotion of apoptosis by the REST68/PKC inhibitor. These data demonstrate that REST68 sensitizes tumors to Ab-based cancer immunotherapy via multiple mechanisms. Furthermore, the REST/NCL interplay model for regulation of expression of cd59 and other genes involved in cell survival enables the design of treatments for different tumor types to achieve more efficient tumor clearance.

Adult attention deficit hyperactivity disorder and other psychiatric symptoms in recreational polydrug users.

AIMS: Previous research has shown that recreational drug use is associated with more psychiatric symptoms and psychobiological distress. This study investigated whether symptoms of adult attention deficit hyperactivity disorder (ADHD) were also raised in polydrug users. METHODS: We assessed a non-clinical sample of 84 unpaid volunteers (mean age 27.5 years): n = 17 light-novice polydrug users; n = 29 moderate polydrug users; and n = 38 non-user controls (14 non-drug users, 24 alcohol/tobacco users). They completed the Symptom Checklist 90 (SCL-90) self-rating inventory for psychiatric symptoms, the Adult ADHD Self-report Scale symptom checklist for adult ADHD, and also the questions on positive moods and sociability. Saliva samples provided a neuroendocrine cortisol measure. RESULTS: Moderate polydrug users reported significantly higher adult ADHD symptoms and SCL-90 psychiatric symptoms and lower sociability than non-user controls and light polydrug users. Novice-light polydrug users did not differ from control groups on any measure. There were no significant group differences in cortisol. These findings are debated using the interactive diathesis-distress model. Psychoactive drugs can affect both mood and cognition. When taken regularly, the drug-induced psychobiological vacillation may exacerbate prior problems with mood stability and attentional-cognitive control. CONCLUSIONS: It is not polydrug usage per se, but rather their regular-repeated usage, that is associated with increased signs of psychiatric and attentional-hyperactivity distress.

Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation.

Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases.

Crry deficiency in complement sufficient mice: C3 consumption occurs without associated renal injury.

The rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry) is critical for complement homeostasis. Gene deletion is 100% embryonically lethal; Crry-deficient (Crry(-/-)) mice were rescued by back-crossing onto C3 deficiency, confirming that embryo loss was complement mediated. In order to rescue viable Crry(-/-) mice without deleting C3, we have tested inhibition of C5 during gestation. Crry(+/-) females were given neutralizing anti-C5 mAb immediately prior to mating with Crry(+/-) males and C5 inhibition maintained through pregnancy. A single, healthy Crry(-/-) female was obtained and mating with Crry(+/-) males yielded healthy litters containing equal numbers of Crry(+/-) and Crry(-/-) pups. Inter-crossing Crry(-/-) mice yielded healthy litters of expected size. Although the mice were not anemic, exposure of Crry(-/-) erythrocytes to normal mouse serum caused C3 deposition and lysis, while transfusion into normal or C6(-/-) mice resulted in rapid clearance. Complement activity and C3 levels in Crry(-/-) mice were markedly reduced. Comparison with factor H deficient (CfH(-/-)) mice revealed similar levels of residual C3; however, unlike the CfH(-/-) mice, Crry(-/-) mice showed no evidence of renal injury, demonstrating distinct roles for these regulators in protecting the kidney.

A Novel, Orally Delivered Antibody Therapy and Its Potential to Prevent Clostridioides difficile Infection in Pre-clinical Models.

Clostridioides difficile infection (CDI) is a toxin-mediated infection in the gut and a major burden on healthcare facilities worldwide. We rationalized that it would be beneficial to design an antibody therapy that is delivered to, and is active at the site of toxin production, rather than neutralizing the circulating and luminal toxins after significant damage of the layers of the intestines has occurred. Here we describe a highly potent therapeutic, OraCAb, with high antibody titers and a formulation that protects the antibodies from digestion/inactivation in the gastrointestinal tract. The potential of OraCAb to prevent CDI in an in vivo hamster model and an in vitro human colon model was assessed. In the hamster model we optimized the ratio of the antibodies against each of the toxins produced by C. difficile (Toxins A and B). The concentration of immunoglobulins that is effective in a hamster model of CDI was determined. A highly significant difference in animal survival for those given an optimized OraCAb formulation versus an untreated control group was observed. This is the first study testing the effect of oral antibodies for treatment of CDI in an in vitro gut model seeded with a human fecal inoculum. Treatment with OraCAb successfully neutralized toxin production and did not interfere with the colonic microbiota in this model. Also, treatment with a combination of vancomycin and OraCAb prevented simulated CDI recurrence, unlike vancomycin therapy alone. These data demonstrate the efficacy of OraCAb formulation for the treatment of CDI in pre-clinical models.

Neuronal death in Alzheimer's disease and therapeutic opportunities.

Alzheimer's disease (AD) is an age-related neurodegenerative disease that affects approximately 24 million people worldwide. A number of different risk factors have been implicated in AD; however, neuritic (amyloid) plaques are considered as one of the defining risk factors and pathological hallmarks of the disease. In the past decade, enormous efforts have been devoted to understand the genetics and molecular pathogenesis leading to neuronal death in AD, which has been transferred into extensive experimental approaches aimed at reversing disease progression. Modern medicine is facing an increasing number of treatments available for vascular and neurodegenerative brain diseases, but no causal or neuroprotective treatment has yet been established. Almost all neurological conditions are characterized by progressive neuronal dysfunction, which, regardless of the pathogenetic mechanism, finally leads to neuronal death. The particular emphasis of this review is on risk factors and mechanisms resulting in neuronal loss in AD and current and prospective opportunities for therapeutic interventions. This review discusses these issues with a view to inspiring the development of new agents that could be useful for the treatment of AD.

Implication of complement system and its regulators in Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease that affects approximately 24 million people worldwide. A number of different risk factors have been implicated in AD, however, neuritic (amyloid) plaques are considered as one of the defining risk factors and pathological hallmarks of the disease. Complement proteins are integral components of amyloid plaques and cerebral vascular amyloid in Alzheimer brains. They can be found at the earliest stages of amyloid deposition and their activation coincides with the clinical expression of Alzheimer's dementia. This review emphasizes on the dual key roles of complement system and complement regulators (CRegs) in disease pathology and progression. The particular focus of this review is on currently evolving strategies for design of complement inhibitors that might aid therapy by restoring the fine balance between activated components of complement system, thus improving the cognitive performance of patients. This review discusses these issues with a view to inspiring the development of new agents that could be useful for the treatment of AD.

The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility.

In mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function.

The role of membrane complement regulatory proteins in cancer immunotherapy.

Anti-tumor monoclonal antibody therapy represents one of the earliest targeted therapies in clinical cancer care and has achieved great clinical promise. Complement activation mediated by anti-tumor mAbs can result in direct tumor lysis or enhancement of antibody-dependent cellular cytotoxicy. Chemotaxis of phagocytic cells by complement activation products C5a is also required for certain cancer immunotherapy such as combined beta-glucan with anti-tumor mAb therapy. However, high expression levels of membrane-bound complement regulatory proteins (mCRPs) such as CD46, CD55 and CD59 on tumors significantly limit the anti-tumor mAb therapeutic efficacy. In addition, mCRPs have been shown to directly or indirectly down-regulate adaptive T cell responses. Therefore, it is desirable to combine anti-tumor mAb therapy or tumor vaccines with the blockade of mCRPs. Such strategies so far include the utilization of neutralizing mAbs for mCRPs, small interfering RNAs or anti-sense oligos for mCRPs, and chemotherapeutic drugs or cytokines. In vitro studies have demonstrated the feasibility and efficacy of such methods, although concerns have been raised about the utilization of neutralizing mAbs in vivo due to widespread expression of mCRPs on normal cells and tissues. Strategies have been developed to address these issues and more in vivo studies are needed to further validate these combination approaches.

CD55 in rat male reproductive tissue: differential expression in testis and expression of a unique truncated isoform on spermatozoa.

CD55 is a key regulator of complement activation, expressed on most tissues and cells in man and other mammals. In the rat, alternative splicing in the gene encoding CD55 yields GPI-anchored (GPI-CD55) and transmembrane (TM-CD55) forms. Published Northern blot analysis indicated that while GPI-CD55 was broadly expressed, TM-CD55 was primarily expressed in the testis, although the precise site of expression was not identified. To clarify the distribution of CD55 isoforms in rat reproductive tissues, we first performed immunohistochemistry and Western blot analysis with an anti-rat CD55 mAb that recognized all reported CD55 isoforms, and a polyclonal immunoglobulin specific for TM-CD55. CD55 was absent in testis prior to puberty. Post-puberty, CD55 was expressed at high levels on all spermiogenic cells from step 6 spermatid onward, and on mature spermatozoa focussed on the acrosome, but was absent from support cells and early progenitors. Enzymatic digestion revealed that GPI-CD55 was predominant in testis and spermatozoa. Staining for TM-CD55 with specific immunoglobulin confirmed its absence from mature sperm and expression on spermatids only between steps 11 and 14 of development. GPI-CD55 on spermatozoa was of lower molecular weight than that in testis and other tissues; sequencing from spermatozoal mRNA identified a unique isoform of GPI-CD55 missing short consensus repeat 4. The predominant acrosome expression and presence of a unique, truncated isoform of CD55 on spermatozoa provides further support for the hypothesis that the acrosome is a highly specialized region in which closely regulated complement activation may contribute to reproductive function.

CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens.

CD59a is the primary regulator of membrane attack complex in mice. Recently, we have shown that CD59a-deficient (Cd59a-/-) mice exhibit enhanced CD4+ T cell responses. Here, we explored the effects of CD59a on B cell function and antibody production. Contrary to our expectations, Cd59a-/- mice showed a decreased humoral immune response to a T cell dependent antigen, sheep red blood cells. We found that the decreased humoral immune response was associated with a reduction in plasma cell number in vivo and reduced ability to respond to stimuli during in vitro culture experiments. Using MLR studies in which purified wild type or Cd59a-/- CD4+ T cells were mixed with purified B cells from each source, we found that the reduced B cell activation was largely due to the absence of CD59a on CD4+ T cells. Furthermore, a CD59a fusion protein bound specifically to mouse B cells, and enhanced B cell proliferation in a MLR, demonstrating that B cells express an as yet unidentified ligand for CD59a that aids in B cell activation.

p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

It has been recently hypothesized that the CD59 gene has two putative p53-responsive elements that may be involved in defense of host cells from damage by the complement system in inflammation. Here we have examined the roles of these putative p53-binding sequences within the CD59 gene in regulation of CD59 expression. We have shown that both of these potential responsive elements bind p53 in vitro. Knocking down expression of p53 using small interfering RNA led to a 6-fold decrease in CD59 protein expression in HeLa cells. We have previously observed a decrease of CD59 in camptothecin-induced apoptotic IMR32 cells, whereas expression was increased in the surviving fraction compared with untreated cells. Here, we have shown that these changes are associated with altered expression levels and acetylation status of p53. We have also shown that acetylation status of p53 regulates CD59 expression on cells exposed to inflammatory cytokines to model inflammation. Our data suggest that p53 and in vivo positive/negative regulators of p53 could be used to modulate susceptibility of tumor cells to complement lysis in chemotherapy.

A quantitative method for comparison of expression of alternatively spliced genes using different primer pairs.

In this paper we describe a novel two-step method for comparison of expression of alternatively spliced genes by quantitative PCR (QPCR) applying different primer pairs. As a model system we used rat decay accelerating factor (DAF; CD55) mRNA, which comprises three different isoforms: soluble (sDAF), transmembrane (tmDAF) and glycosyl-phosphatidylinositol (GPI) anchored (gpiDAF) forms. The first step was to prepare solid phase specific for each mRNA isoform and purify the three DAF-forms from total RNA. We then assessed amplification efficiency of primer pairs designed to recognise each of the isoforms using equimolar amounts of the three purified DAF mRNAs. The final step in our assay was to compare expression of the three DAF-isoforms in testis by QPCR taking into account the efficiency of their amplification to enable quantification. The RNA capture/QPCR method we described here can be used for quantifying the expression ratios of alternatively spliced mRNAs from a single gene or for direct comparison of expression of different genes.

Alterations in Gene Expression in Depression: Prospects for Personalize Patient Treatment.

The number of people around the world suffering from depression has dramatically increased in last few decades. It has been predicted that by 2020 depression will become the second most common cause of disability. Furthermore, depression is often misdiagnosed and confused with other psychiatric disorders showing similar symptoms, i.e., anxiety and bipolar disorder, due to the fact that diagnosing is often carried out by medical workers who are not psychiatrically trained. These facts prompt us to prepare this review which focuses on alterations in gene expression in depression. We believe that an in-depth knowledge of molecular bases of behavior in depression and other mood disorders would be of a great benefit for the correct diagnosing of these disorders, as well as for prescribing a treatment that best suits each individual depending on expression alterations in depression-related genes. Therefore, the main aim of this review is to promote further translational research on the biochemistry of mood disorders and take the results further for the design of new targeted therapeutics that can be used for personalized treatment with minimal adverse effects.