RESUMO
Schizophrenia is a severe psychotic illness affecting 1% of the general population. There are no consistent pathological features, and the disorder is defined by a complex symptomatology, which overlaps with other psychiatric illnesses. Diagnosis is based on a clinical interview, relying on the patient meeting criteria according to diagnosis manuals, including Diagnostic and Statistical Manual of Mental Disorders, 4th Ed. and International Statistical Classification of Diseases, 10th Revision. Because of the ambiguous symptoms, the diagnostic process can take many months and often years. Rapid and effective treatment has been shown to impact positively on disease progression and outcome, and it is therefore important to identify disease-associated biomarkers allowing early diagnosis. Reliable biomarkers can be used for the development of diagnostic tests and may also help us understand the underlying pathology of this disorder. In the present study, proteins from anti-CD3 stimulated and unstimulated peripheral blood T cell lysates from 15 minimally medicated and unmedicated patients and 15 age-, sex-, race-, and smoking-matched controls were profiled on cation exchange (CM10) chips using SELDI-TOF. Partial least squares discriminate analysis was used to separate patient and control groups according to the expression of 108 detected peaks, and two peaks of 3,374 and 3,450 Da, corresponding to alpha-defensins based on masses and cationic properties, were found to contribute significantly to the separation of patient and control groups. Reduction of T cell lysates with DTT resulted in a 6-Da shift in the mass of these peaks consistent with the presence of three cysteine bonds in the structure, confirming them as alpha-defensins. Quantification of alpha-defensins in T cell lysates from six patients and 18 healthy controls was carried out by ELISA, which also showed that alpha-defensin levels were significantly increased in patient lysates when compared with matched controls (p = 0.0197). Plasma from 21 monozygotic twins discordant for schizophrenia and eight healthy unaffected twin pairs was also analyzed for the expression of alpha-defensins by ELISA. Notably both affected and unaffected twins were found to have significantly elevated alpha-defensin levels compared with healthy control twin pairs (p = 0.0014 and p = 0.0115, respectively). Increased expression of alpha-defensins in unaffected as well as affected discordant monozygotic twins is of particular interest as monozygotic twins share genes and usually environmental upbringing. The unaffected twin therefore represents the biological and environmental risk of developing schizophrenia in the absence of overt symptomatology and therapeutic medication. These findings suggest that alpha-defensins could be an important early indicator of the risk of schizophrenia.
Assuntos
Suscetibilidade a Doenças/sangue , Esquizofrenia/sangue , alfa-Defensinas/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Análise por Conglomerados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linfócitos T/química , Linfócitos T/metabolismo , Gêmeos Monozigóticos , Regulação para CimaRESUMO
Many cases of tuberculosis result from reactivation of previously acquired latent infections. Models to study such persister forms often involve gradual depletion of oxygen during culture as poor aeration is a characteristic of non-progressive TB granulomas. Anaerobically cultured bacilli develop a thickened outer-most cell wall layer. Here, we analyzed this layer from anaerobically cultured Mycobacterium tuberculosis and Mycobacterium bovis BCG. By six weeks of anaerobiosis a pigment was detected at levels > 60-fold higher in anaerobic than aerobic bacilli. This pigment was responsible for the electron-dense appearance of the thickened cell wall layer and gave an electrospray mass spectrometry peak at 409 Da (M+Na)+ or (M+H)+. We termed this pigment APP1, anaerobically produced pigment 1, the first pigment identified in M. tuberculosis.
Assuntos
Mycobacterium bovis/química , Mycobacterium tuberculosis/química , Pigmentos Biológicos/análise , Anaerobiose , Parede Celular/química , Parede Celular/ultraestrutura , Etanol/química , Peso Molecular , Mycobacterium bovis/metabolismo , Mycobacterium bovis/ultraestrutura , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/química , Espectrometria de Massas por Ionização por Electrospray , Fatores de TempoRESUMO
BACKGROUND: Previous studies of brain and peripheral tissues in schizophrenia patients have indicated impaired energy supply to the brain. A number of studies have also demonstrated dysfunction of the microvasculature in schizophrenia patients. Together these findings are consistent with a hypothesis of blood-brain barrier dysfunction in schizophrenia. In this study, we have investigated the cerebral vascular endothelium of schizophrenia patients at the level of transcriptomics. METHODOLOGY/PRINCIPAL FINDINGS: We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from schizophrenia patients and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using two independent microarray platforms, Affymetrix HG133plus2.0 GeneChips and CodeLink Whole Human Genome arrays. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. We then compared neuronal and endothelial data separately between schizophrenic subjects and controls. Analysis of the endothelial samples showed differences in gene expression between schizophrenics and controls which were reproducible in a second microarray platform. Functional profiling revealed that these changes were primarily found in genes relating to inflammatory processes. CONCLUSIONS/SIGNIFICANCE: This study provides preliminary evidence of molecular alterations of the cerebral microvasculature in schizophrenia patients, suggestive of a hypo-inflammatory state in this tissue type. Further investigation of the blood-brain barrier in schizophrenia is warranted.
Assuntos
Encéfalo/irrigação sanguínea , Terapia a Laser/métodos , Microdissecção/métodos , Microvasos/cirurgia , Esquizofrenia/cirurgia , Adulto , Algoritmos , Autopsia , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Esquizofrenia/genética , Esquizofrenia/patologia , Adulto JovemRESUMO
Despite decades of research into the aetiology and pathophysiology of schizophrenia, our understanding of this devastating disorder remains incomplete, with adverse consequences for both diagnosis and treatment. Here we investigate whether differences between patients and controls can be observed in peripheral patient tissue, with a view of establishing a means for dynamic investigations into cell function. In vitro stimulation of peripheral blood CD3+ pan T cells with anti-CD3 (clone OKT3) was used to investigate disease-associated cell responses. T cells from both medicated (n = 39), unmedicated (n = 6) and minimally medicated (n = 5) schizophrenia patients were found to have significantly lower proliferative responses to stimulation, compared to well-matched controls (n = 32). Expression of CD3 and TCR (T cell receptor) alphabeta chains was equivalent between patients and controls, ensuring equal stimulation with anti-CD3, and there was no significant difference in the proportions of CD4+ and CD8+ T cells between samples (n = 12). Lower T cell proliferation in schizophrenia patients was not found to result from deficient early tyrosine phosphorylation signalling or lower IL-2 (interleukin-2) production, as these parameters were similar between patients and controls, as was the expression of CD25, the IL-2 receptor alpha chain. Analysis of CD45 isoforms, however, revealed that patients had a significantly greater percentage of CD8+ and CD4+ CD45RA+ cells before stimulation and significantly higher fluorescence intensity of CD45RA on CD4+ and CD8+ cells before and after stimulation. There was significantly higher expression of CD45 RB on both CD4+ and CD8+ unstimulated cells, with a trend towards lower numbers of CD45RO+ T cells in patient blood. Gene expression analysis in freshly isolated T cells from six minimally treated or first onset patients and six controls was carried out using human whole-genome CodeLink microarrays to identify functional pathways that may affect the ability of patient cells to respond to stimulation. Functional profiling showed prominent transcript changes in categories pertaining to cell cycle machinery, intracellular signalling, oxidative stress and metabolism. Intriguingly, chromosomal location analysis of genes significantly altered between schizophrenia and controls revealed clusters at 1p36, 1q42 and 6p22, which have previously been identified as strong susceptibility loci for schizophrenia.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Esquizofrenia/imunologia , Esquizofrenia/fisiopatologia , Subpopulações de Linfócitos T/imunologia , Animais , Antipsicóticos/uso terapêutico , Complexo CD3/genética , Complexo CD3/imunologia , Proliferação de Células , Mapeamento Cromossômico , Feminino , Perfilação da Expressão Gênica , Humanos , Interleucina-2/genética , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Esquizofrenia/tratamento farmacológico , Transdução de Sinais/imunologiaRESUMO
Neutrophils are abundant, short-lived leukocytes with a key role in the defense against rapidly dividing bacteria. They enter apoptosis spontaneously within 24-48 h of leaving the bone marrow. However, their life span can be extended during inflammatory responses by several proinflammatory cytokines. Inappropriate survival of neutrophils contributes to chronic inflammation and tissue damage associated with diseases such as rheumatoid arthritis. We have previously reported that type I IFNs can inhibit both cytokine deprivation and Fas-induced apoptosis in activated T cells. IFN-beta locally produced by hyperplastic fibroblasts within the pannus tissue of patients with rheumatoid arthritis contributes to the inappropriately extended life span of infiltrating T cells. Type I IFNs are equally effective at delaying spontaneous apoptosis in human neutrophils. In the work presented here we investigated the signaling pathways involved in mediating this effect. The antiapoptotic actions of IFN-beta were targeted at an early stage of neutrophil apoptosis, occurring upstream of mitochondrial permeability transition, and were phosphatidylinositol 3-kinase (PI3K) dependent, as they were blocked by the PI3K inhibitor LY294002. Analysis of signaling pathways downstream of PI3K revealed that the antiapoptotic effect of type 1 IFN was inhibited by rottlerin, SN50, and cycloheximide, indicating requirements for activation of protein kinase C-delta, NF-kappaB, and de novo protein synthesis, respectively. Moreover, EMSA was used to show that the activation of NF-kappaB occurred downstream of PI3K and protein kinase C-delta activation. We conclude that type I IFNs inhibit neutrophil apoptosis in a PI3K-dependent manner, which requires activation of protein kinase C-delta and induction of NF-kappaB-regulated genes.
Assuntos
Apoptose/imunologia , Comunicação Celular/imunologia , Interferon beta/fisiologia , NF-kappa B/fisiologia , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Quinase C/fisiologia , Transdução de Sinais/imunologia , Apoptose/efeitos dos fármacos , Caspase 3 , Caspase 9 , Caspases/metabolismo , Comunicação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Cicloeximida/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Humanos , Interferon-alfa/fisiologia , Membranas Intracelulares/imunologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologia , Mitocôndrias/imunologia , NF-kappa B/antagonistas & inibidores , Neutrófilos/citologia , Neutrófilos/enzimologia , Permeabilidade , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-delta , Transdução de Sinais/efeitos dos fármacosRESUMO
In the absence of survival-inducing cytokines activated T cells and neutrophils enter apoptosis spontaneously. Phosphatidylinositol 3-kinase (PI3 K) activation and signaling through PKB/AKT have been widely linked to the inhibition of apoptosis by cytokines. Here we have investigated the role of PKB in the inhibition of spontaneous apoptosis of activated human CD4+ T cells and neutrophils. We used a range of cytokines known to induce survival and/or activation of PKB. We found activation of PKB in T cells treated with IL-2 and insulin, and neutrophils cultured with N-formyl-Met-Leu-Phe (fMLP), insulin or granulocyte-macrophage colony-stimulating factor. Insulin did not inhibit apoptosis in neutrophils or T cells and fMLP did not delay neutrophil apoptosis. Intriguingly, IFN-beta induced PI3 K-dependent survival in both cell types, but did not activate PKB. IL-2 mediated rescue of T cells from apoptosis but no induction of proliferation occurred in thepresence of LY294002, an inhibitor of PI3 K, which also blocked subsequent PKB activation. The main role of PI3 K in IL-2-mediated signaling may therefore be in the regulation of proliferation. These findings suggest that activation of PKB and inhibition of apoptosis can be dissociated in cytokine-mediated rescue of non-transformed CD4+ T cells and neutrophils.
Assuntos
Apoptose/imunologia , Apoptose/fisiologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/metabolismo , Neutrófilos/citologia , Neutrófilos/imunologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Cromonas/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Morfolinas/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt , Transdução de SinaisRESUMO
Neutrophils are abundant, short-lived leukocytes, and their death by apoptosis is central to hemostasis and the resolution of inflammation, yet the trigger for their entry into apoptosis is unknown. We show here that death receptor signaling, including CD95 death-inducing signaling complex (DISC) formation and caspase 8 activation, occurred early in neutrophil apoptosis. However, death receptor ligation was not required for apoptosis, suggesting a novel mechanism for caspase 8 activation. We detected ceramide generation and clustering of CD95 in lipid rafts early in neutrophil apoptosis, and neutrophil apoptosis and ceramide generation were both significantly inhibited in acid sphingomyelinase knockout (ASM(-/-)) mice compared to wild-type littermates. Further studies revealed that ceramide generation, CD95 clustering, and neutrophil apoptosis were dependent on reactive oxygen species (ROSs) and were preceded by a fall in reduced glutathione levels. We propose that accumulation of ROSs, as a consequence of altered redox status, initiates ligand-independent death receptor signaling via activation of ASM and clustering of preformed DISC components in lipid rafts and is therefore a primary factor limiting neutrophil life span.