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Necroptosis is a form of programmed cell death executed by receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL). Platelets are circulating cells that play central roles in haemostasis and pathological thrombosis. In this study we demonstrate seminal contribution of MLKL in transformation of agonist-stimulated platelets to active haemostatic units progressing eventually to necrotic death on a temporal scale, thus attributing a yet unrecognized fundamental role to MLKL in platelet biology. Physiological agonists like thrombin instigated phosphorylation and subsequent oligomerization of MLKL in platelets in a RIPK3-independent but phosphoinositide 3-kinase (PI3K)/AKT-dependent manner. Inhibition of MLKL significantly curbed agonist-induced haemostatic responses in platelets that included platelet aggregation, integrin activation, granule secretion, procoagulant surface generation, rise in intracellular calcium, shedding of extracellular vesicles, platelet-leukocyte interactions and thrombus formation under arterial shear. MLKL inhibition, too, prompted impairment in mitochondrial oxidative phosphorylation and aerobic glycolysis in stimulated platelets, accompanied with disruption in mitochondrial transmembrane potential, augmented proton leak and drop in both mitochondrial calcium as well as ROS. These findings underscore the key role of MLKL in sustaining OXPHOS and aerobic glycolysis that underlie energy-intensive platelet activation responses. Prolonged exposure to thrombin provoked oligomerization and translocation of MLKL to plasma membranes forming focal clusters that led to progressive membrane permeabilization and decline in platelet viability, which was prevented by inhibitors of PI3K/MLKL. In summary, MLKL plays vital role in transitioning of stimulated platelets from relatively quiescent cells to functionally/metabolically active prothrombotic units and their ensuing progression to necroptotic death.
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Plaquetas , Proteínas Quinasas , Proteínas Quinasas/metabolismo , Plaquetas/metabolismo , Necroptosis , Calcio/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Trombina/farmacología , Trombina/metabolismo , Muerte Celular , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
BACKGROUND: There is an urgent need to understand the key events driving pathogenesis of severe COVID-19 disease, so that precise treatment can be instituted. In this respect NETosis is gaining increased attention in the scientific community, as an important pathological process contributing to mortality. We sought to test if indeed there exists robust evidence of NETosis in multiple transcriptomic data sets from human subjects with severe COVID-19 disease. Gene set enrichment analysis was performed to test for up-regulation of gene set functional in NETosis in the blood of patients with COVID-19 illness. RESULTS: Blood gene expression functional in NETosis increased with severity of illness, showed negative correlation with blood oxygen saturation, and was validated in the lung of COVID-19 non-survivors. Temporal expression of IL-6 was compared between severe and moderate illness with COVID-19. Unsupervised clustering was performed to reveal co-expression of IL-6 with complement genes. In severe COVID-19 illness, there is transcriptional evidence of activation of NETosis, complement and coagulation cascade, and negative correlation between NETosis and respiratory function (oxygen saturation). An early spike in IL-6 is observed in severe COVID-19 illness that is correlated with complement activation. CONCLUSIONS: Based on the transcriptional dynamics of IL-6 expression and its downstream effect on complement activation, we constructed a model that links early spike in IL-6 level with persistent and self-perpetuating complement activation, NETosis, immunothrombosis and respiratory dysfunction. Our model supports the early initiation of anti-IL6 therapy in severe COVID-19 disease before the life-threatening complications of the disease can perpetuate themselves autonomously.
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COVID-19/inmunología , Trampas Extracelulares , Interleucina-6 , Trombosis/virología , Transcriptoma , COVID-19/patología , Proteínas del Sistema Complemento/genética , Humanos , Interleucina-6/genética , OxígenoRESUMEN
BACKGROUND AND AIM: Alcohol exerts its effects on organs in multiple ways. Alcoholic chronic pancreatitis (ACP) is a disease in which alcohol triggers the pathological changes in pancreas, leading to chronic inflammation and fibrosis. The molecular mechanism behind these changes is not clear. Identification of key circulating miRNA changes in ACP patients and determination of the fraction that is secreted from diseased pancreas not only could serve as potential biomarker for assessing disease severity, but also could help identifying the molecular alterations prevailing in the organ precipitating the disease, to some extent. METHODS: We performed microRNA microarray using the Affymetrix miRNA 4.0 platform to identify differentially expressed miRNAs in serum of ACP patients as compared to alcoholic control individuals and then found out how many of them could be pancreas-specific and exosomally secreted. We further analyzed a pancreatitis-specific gene expression data set to find out the differentially expressed genes in diseased pancreas and explored the possible role of those selected miRNAs in regulation of gene expression in ACP. RESULTS: We identified 14 miRNAs differentially expressed in both serum and pancreas and also identified their experimentally validated targets. Transcription factors modulating the miRNA expression in an alcohol-dependent manner were also identified and characterized to derive the miRNA-gene-TF interaction network responsible for progression of the disease. CONCLUSIONS: Differentially expressed miRNA signature demonstrated significant changes in both pro- and anti-inflammatory pathways probably balancing the chronic inflammation in the pancreas. Our findings also suggested possible involvement of pancreatic stellate cells in disease progression.
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BACKGROUND: Most often, the patients with pancreatic diseases are presented with a mass in pancreatic head region and existing methods of diagnosis fail to confirm whether the head mass is malignant or benign. As subsequent management of the disease hugely depends on the correct diagnosis, we wanted to explore possible biomarkers which could distinguish benign and malignant pancreatic head masses. METHODS: In order to address that gap, we performed a case-control study to identify genome-wide differentially expressed coding and noncoding genes between pancreatic tissues collected from benign and malignant head masses. These genes were next shortlisted using stringent criteria followed by selection of top malignancy specific genes. They subsequently got validated by quantitative RT-PCR and also in other patient cohorts. Survival analysis and ROC analysis were also performed. RESULTS: We identified 55 coding and 13 noncoding genes specific for malignant pancreatic head masses. Further shortlisting and validation, however, resulted in 5 coding genes as part of malignancy specific multi-gene signature, which was validated in three independent patient cohorts of 145 normal and 153 PDAC patients. We also found that overexpression of these genes resulted in survival disadvantage in the patients and ROC analysis identified that combination of 5 coding genes had the AUROC of 0.94, making them potential biomarker. CONCLUSIONS: Our study identified a multi-gene signature comprising of 5 coding genes (CDCA7, DLGAP5, FOXM1, TPX2 and OSBPL3) to distinguish malignant head masses from benign ones.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Estudios de Casos y Controles , Perfilación de la Expresión Génica , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Transcriptoma/genéticaRESUMEN
BACKGROUND: Sepsis and cancer are both leading causes of death, and occurrence of any one, increases the likelihood of the other. While cancer patients are susceptible to sepsis, survivors of sepsis are also susceptible to develop certain cancers. This mutual dependence for susceptibility suggests shared biology between the two disease categories. Earlier analysis had revealed a cancer-related pathway to be up-regulated in Septic Shock (SS), an advanced stage of sepsis. This has motivated a more comprehensive comparison of the transcriptomes of SS and cancer. METHODS: Gene Set Enrichment Analysis was performed to detect the pathways enriched in SS and cancer. Thereafter, hierarchical clustering was applied to identify relative segregation of 17 cancer types into two groups vis-a-vis SS. Biological significance of the selected pathways was explored by network analysis. Clinical significance of the pathways was tested by survival analysis. A robust classifier of cancer groups was developed based on machine learning. RESULTS: A total of 66 pathways were observed to be enriched in both SS and cancer. However, clustering segregated cancer types into two categories based on the direction of transcriptomic change. In general, there was up-regulation in SS and one group of cancer (termed Sepsis-Like Cancer, or SLC), but not in other cancers (termed Cancer Alone, or CA). The SLC group mainly consisted of malignancies of the gastrointestinal tract (head and neck, oesophagus, stomach, liver and biliary system) often associated with infection. Machine learning classifier successfully segregated the two cancer groups with high accuracy (> 98%). Additionally, pathway up-regulation was observed to be associated with survival in the SLC group of cancers. CONCLUSION: Transcriptome-based systems biology approach segregates cancer into two groups (SLC and CA) based on similarity with SS. Host response to infection plays a key role in pathogenesis of SS and SLC. However, we hypothesize that some component of the host response is protective in both SS and SLC.
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Redes Reguladoras de Genes , Neoplasias/genética , Choque Séptico/genética , Biología de Sistemas/métodos , Análisis por Conglomerados , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Aprendizaje Automático , Masculino , Neoplasias/clasificación , Neoplasias/mortalidad , Choque Séptico/mortalidad , Análisis de SupervivenciaRESUMEN
Septic shock is a major medical problem with high morbidity and mortality and incompletely understood biology. Integration of multiple data sets into a single analysis framework empowers discovery of new knowledge about the condition that may have been missed by individual analysis of each of these datasets. Electronic search was performed on medical literature and gene expression databases for selection of transcriptomic studies done in circulating leukocytes from human subjects suffering from septic shock. Gene-level meta-analysis was conducted on the six selected studies to identify the genes consistently differentially expressed in septic shock. This was followed by pathway-level analysis using three different algorithms (ORA, GSEA, SPIA). The identified up-regulated pathway, Osteoclast differentiation pathway (hsa04380) was validated in two independent cohorts. Of the pathway, 25 key genes were selected that serve as an expression signature of Septic Shock.
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Diferenciación Celular/genética , Osteoclastos/citología , Choque Séptico/genética , Transcriptoma , Regulación hacia Arriba , Algoritmos , Bases de Datos Factuales , Humanos , Leucocitos/citología , Leucocitos/metabolismo , Modelos Biológicos , Osteoclastos/metabolismo , Análisis de Componente Principal , Choque Séptico/fisiopatologíaRESUMEN
Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver-specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV-HCC and HBV-control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR-126 and miR-142-3p showed increased expression in plasma of HBV-HCC compared to HBV-non-HCC patients. Subsequently, ROC curve analysis revealed that neither miR-126 nor miR-142-3p performed better than AFP in discriminating HCC from non-HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR-126 + miR-142-3p + AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR-126 was noticed significantly higher in HBV-HCC patients with low-AFP [<250 ng/ml] compared to either non-HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir-126 in HCV-HCC or non-viral-HCC revealed that miR-126 + AFP might be specific to HBV-HCC. To understand the physiological role of these two miRNAs in hepato-carcinogenesis, target genes related to cancer pathways (APAF1, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR-126 + AFP is a promising noninvasive diagnostic biomarker for HBV-HCC and may be useful in the management of HCC patients.
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Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Neoplasias Hepáticas/sangre , MicroARNs/sangre , alfa-Fetoproteínas/genética , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/patogenicidad , Hepatitis B Crónica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , MasculinoRESUMEN
Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor-clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures.
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Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Glioblastoma/diagnóstico , Glioblastoma/genética , Receptores Notch/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Hipoxia de la Célula , Análisis por Conglomerados , Glioblastoma/mortalidad , Glioblastoma/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Estimación de Kaplan-Meier , Osteopontina/genética , Osteopontina/metabolismo , Fosfoglicerato Quinasa/genética , Fosfoglicerato Quinasa/metabolismo , Análisis de Componente Principal , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/metabolismo , Transducción de Señal , Factor de Transcripción HES-1 , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
BACKGROUND: There is an inverse secular trend between the incidence of obesity and gastric colonization with Helicobacter pylori, a bacterium that can affect the secretion of gastric hormones that relate to energy homeostasis. H. pylori strains that carry the cag pathogenicity island (PAI) interact more intimately with gastric epithelial cells and trigger more extensive host responses than cag(-) strains. We hypothesized that gastric colonization with H. pylori strains differing in cag PAI status exert distinct effects on metabolic and inflammatory phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we examined metabolic and inflammatory markers in db/db mice and mice with diet-induced obesity experimentally infected with isogenic forms of H. pylori strain 26695: the cag PAI wild-type and its cag PAI mutant strain 99-305. H. pylori colonization decreased fasting blood glucose levels, increased levels of leptin, improved glucose tolerance, and suppressed weight gain. A response found in both wild-type and mutant H. pylori strain-infected mice included decreased white adipose tissue macrophages (ATM) and increased adipose tissue regulatory T cells (Treg) cells. Gene expression analyses demonstrated upregulation of gastric PPAR γ-responsive genes (i.e., CD36 and FABP4) in H. pylori-infected mice. The loss of PPAR γ in immune and epithelial cells in mice impaired the ability of H. pylori to favorably modulate glucose homeostasis and ATM infiltration during high fat feeding. CONCLUSIONS/SIGNIFICANCE: Gastric infection with some commensal strains of H. pylori ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism and modulates macrophage and Treg cell infiltration into the abdominal white adipose tissue.
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Mucosa Gástrica/microbiología , Islas Genómicas/genética , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/crecimiento & desarrollo , Homeostasis/fisiología , Obesidad/microbiología , PPAR gamma/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/inmunología , Animales , Glucemia , Peso Corporal , Antígenos CD36/metabolismo , Ensayo de Inmunoadsorción Enzimática , Proteínas de Unión a Ácidos Grasos/metabolismo , Citometría de Flujo , Mucosa Gástrica/inmunología , Mucosa Gástrica/metabolismo , Perfilación de la Expresión Génica , Ghrelina/sangre , Infecciones por Helicobacter/inmunología , Helicobacter pylori/genética , Insulina/sangre , Leptina/sangre , Macrófagos/inmunología , Ratones , Linfocitos T Reguladores/inmunologíaRESUMEN
Gene expression profiling has revolutionized functional genomics research by providing a quick handle on all the transcriptional changes that occur in the cell in response to internal or external perturbations or developmental programs. Microarrays have become the most popular technology for recording gene expression profiles. This chapter describes all the necessary steps for analyzing Affymetrix microarray data using the open-source statistical tools (R and bioconductor). The reader is walked through all the basic steps of data analysis: reading raw data, assessing quality, preprocessing/normalization, discovery of differentially expressed genes, comparison of gene lists, functional enrichment analysis, and saving results to files for future reference. Some familiarity with computer is assumed. This chapter is self-contained with installation instructions for R and bioconductor packages along with links to downloadable data and code for reproducing the examples.
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Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Programas InformáticosRESUMEN
BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor whose activation has been shown to modulate macrophage and T cell-mediated inflammation. The objective of this study was to investigate the mechanisms by which the deletion of PPAR gamma in T cells modulates immune cell distribution and colonic gene expression and the severity of experimental IBD. METHODS: PPAR gamma flfl; CD4 Cre+ (CD4cre) or Cre- (WT) mice were challenged with 2.5% dextran sodium sulfate in their drinking water for 0, 2, or 7 days. Mice were scored on disease severity both clinically and histopathologically. Flow cytometry was used to assess lymphocyte and macrophage populations in the blood, spleen, and mesenteric lymph nodes (MLN). Global gene expression in colonic mucosa was profiled using Affymetrix microarrays. RESULTS: The deficiency of PPAR gamma in T cells accelerated the onset of disease and body weight loss. Examination of colon histopathology revealed significantly greater epithelial erosion, leukocyte infiltration, and mucosal thickening in the CD4cre mice on day 7. CD4cre mice had more CD8+ T cells than WT mice and fewer CD4+ FoxP3+ regulatory T cells (Treg) and IL10+ CD4+ T cells in blood and MLN, respectively. Transcriptomic profiling revealed around 3000 genes being transcriptionally altered as a result of DSS challenge in CD4cre mice. These included up-regulated mRNA expression of adhesion molecules, proinflammatory cytokines interleukin-6 (IL-6) and IL-1beta, and suppressor of cytokine signaling 3 (SOCS-3) on day 7. Gene set enrichment analysis (GSEA) showed that the ribosome and Krebs cycle pathways were downregulated while the apoptosis pathway was upregulated in colons of mice lacking PPAR gamma in T cells. CONCLUSIONS: The expression of PPAR gamma in T cells is involved in preventing gut inflammation by regulating colonic expression of adhesion molecules and inflammatory mediators at later stages of disease while favoring the recruitment of Treg to the mucosal inductive sites.
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Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , PPAR gamma/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patología , Animales , Apoptosis , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Colon/metabolismo , Colon/patología , Citocinas/metabolismo , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Enfermedades Inflamatorias del Intestino/inducido químicamente , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR gamma/genética , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patologíaRESUMEN
BACKGROUND: Peroxisome proliferator-activated receptors are nuclear receptors highly expressed in intestinal epithelial cells (IEC) and immune cells within the gut mucosa and are implicated in modulating inflammation and immune responses. The objective of this study was to investigate the effect of targeted deletion of PPAR gamma in IEC on progression of experimental inflammatory bowel disease (IBD). METHODOLOGY/PRINCIPAL FINDINGS: In the first phase, PPAR gamma flfl; Villin Cre- (VC-) and PPAR gamma flfl; Villin Cre+ (VC+) mice in a mixed FVB/C57BL/6 background were challenged with 2.5% dextran sodium sulfate (DSS) in drinking water for 0, 2, or 7 days. VC+ mice express a transgenic recombinase under the control of the Villin-Cre promoter that causes an IEC-specific deletion of PPAR gamma. In the second phase, we generated VC- and VC+ mice in a C57BL/6 background that were challenged with 2.5% DSS. Mice were scored on disease severity both clinically and histopathologically. Flow cytometry was used to phenotypically characterize lymphocyte and macrophage populations in blood, spleen and mesenteric lymph nodes. Global gene expression analysis was profiled using Affymetrix microarrays. The IEC-specific deficiency of PPAR gamma in mice with a mixed background worsened colonic inflammatory lesions, but had no effect on disease activity (DAI) or weight loss. In contrast, the IEC-specific PPAR gamma null mice in C57BL/6 background exhibited more severe inflammatory lesions, DAI and weight loss in comparison to their littermates expressing PPAR gamma in IEC. Global gene expression profiling revealed significantly down-regulated expression of lysosomal pathway genes and flow cytometry results demonstrated suppressed production of IL-10 by CD4+ T cells in mesenteric lymph nodes (MLN) of IEC-specific PPAR gamma null mice. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that adequate expression of PPAR gamma in IEC is required for the regulation of mucosal immune responses and prevention of experimental IBD, possibly by modulation of lysosomal and antigen presentation pathways.
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Colon/patología , Células Epiteliales/patología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/patología , PPAR gamma/inmunología , Animales , Células Epiteliales/inmunología , Perfilación de la Expresión Génica , Inmunidad , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: It has been shown previously that administration of Francisella tularensis (Ft) Live Vaccine Strain (LVS) lipopolysaccharide (LPS) protects mice against subsequent challenge with Ft LVS and blunts the pro-inflammatory cytokine response. METHODS: To further investigate the molecular mechanisms that underlie Ft LVS LPS-mediated protection, we profiled global hepatic gene expression following Ft LVS LPS or saline pre-treatment and subsequent Ft LVS challenge using Affymetrix arrays. RESULTS: A large number of genes (> 3,000) were differentially expressed at 48 hours post-infection. The degree of modulation of inflammatory genes by infection was clearly attenuated by pre-treatment with Ft LVS LPS in the surviving mice. However, Ft LVS LPS alone had a subtle effect on the gene expression profile of the uninfected mice. By employing gene set enrichment analysis, we discovered significant up-regulation of the fatty acid metabolism pathway, which is regulated by peroxisome proliferator activated receptors (PPARs). CONCLUSIONS: We hypothesize that the LPS-induced blunting of pro-inflammatory response in mouse is, in part, mediated by PPARs (alpha and gamma).
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Vacunas Bacterianas/inmunología , Hígado/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Tularemia/genética , Animales , Ácidos Grasos/metabolismo , Femenino , Francisella tularensis , Perfilación de la Expresión Génica , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Tularemia/inmunología , Vacunas Atenuadas/inmunologíaRESUMEN
Type I IFNs were discovered as the primary antiviral cytokines and are now known to serve critical functions in host defense against bacterial pathogens. Accordingly, established mediators of IFN antiviral activity may mediate previously unrecognized antibacterial functions. RNase-L is the terminal component of an RNA decay pathway that is an important mediator of IFN-induced antiviral activity. Here, we identify a role for RNase-L in the host antibacterial response. RNase-L(-/-) mice exhibited a dramatic increase in mortality after challenge with Bacillus anthracis and Escherichia coli; this increased susceptibility was due to a compromised immune response resulting in increased bacterial load. Investigation of the mechanisms of RNase-L antibacterial activity indicated that RNase-L is required for the optimal induction of proinflammatory cytokines that play essential roles in host defense from bacterial pathogens. RNase-L also regulated the expression of the endolysosomal protease, cathepsin-E, and endosome-associated activities, that function to eliminate internalized bacteria and may contribute to RNase-L antimicrobial action. Our results reveal a unique role for RNase-L in the antibacterial response that is mediated through multiple mechanisms. As a regulator of fundamental components of the innate immune response, RNase-L represents a viable therapeutic target to augment host defense against diverse microbial pathogens.
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Carbunco/enzimología , Bacillus anthracis , Endorribonucleasas/biosíntesis , Infecciones por Escherichia coli/enzimología , Escherichia coli , Interferón Tipo I/biosíntesis , Animales , Carbunco/genética , Carbunco/inmunología , Bacillus anthracis/inmunología , Catepsina E/biosíntesis , Catepsina E/genética , Catepsina E/inmunología , Endorribonucleasas/genética , Endorribonucleasas/inmunología , Endosomas/enzimología , Endosomas/genética , Endosomas/inmunología , Escherichia coli/inmunología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/inmunología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Ratones , Ratones Noqueados , Estabilidad del ARN/genética , Estabilidad del ARN/inmunologíaRESUMEN
Currently, no effective tool exists for screening or early diagnosis of head and neck squamous cell carcinoma (HNSCC). Here, we describe an approach for cancer detection based on analysis of patterns of serum immunoreactivity against a panel of biomarkers selected using microarray-based serologic profiling and specialized bioinformatics. We biopanned phage display libraries derived from three different HNSCC tissues to generate 5,133 selectively cloned tumor antigens. Based on their differential immunoreactivity on protein microarrays against serum immunoglobulins from 39 cancer and 41 control patients, we reduced the number of clones to 1,021. The performance of a neural network model (Multilayer Perceptron) for cancer classification on a data set of 80 HNSCC and 78 control samples was assessed using 10-fold cross-validation repeated 100 times. A panel of 130 clones was found to be adequate for building a classifier with sufficient sensitivity and specificity. Using these 130 markers on a completely new and independent set of 80 samples, an accuracy of 84.9% with sensitivity of 79.8% and specificity of 90.1% was achieved. Similar performance was achieved by reshuffling of the data set and by using other classification models. The performance of this classification approach represents a significant improvement over current diagnostic accuracy (sensitivity of 37% to 46% and specificity of 24%) in the primary care setting. The results shown here are promising and show the potential use of this approach toward eventual development of diagnostic assay with sufficient sensitivity and specificity suitable for detection of early-stage HNSCC in high-risk populations.
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Autoanticuerpos/sangre , Biomarcadores de Tumor/sangre , Carcinoma de Células Escamosas/diagnóstico , Neoplasias de Cabeza y Cuello/diagnóstico , Autoanticuerpos/inmunología , Bacteriófago T7/genética , Biomarcadores de Tumor/inmunología , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/inmunología , Epítopos/inmunología , Femenino , Biblioteca de Genes , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/inmunología , Humanos , Inmunoglobulina G/inmunología , Masculino , Redes Neurales de la Computación , Análisis por Matrices de Proteínas/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Phage display technology has emerged into a powerful tool for identifying proteins with specific binding properties. This technology adds amino acid sequences to the carboxy terminus of a phage capsid protein, thus generating a fusion protein displayed on the surface of the phage. Here, we have developed a high-throughput strategy to synthesize purified protein that solves many of the problems associated with crude phage lysates. Phage DNA was used as a template for a nested PCR that added the T7 promoter, ribosome binding site, and a His6-tag. The PCR product was then used as a template for in vitro transcription/translation. The resulting His6-tagged recombinant protein was then purified by nickel affinity chromatography. The functionality of the purified protein was verified using protein microarray analysis.
Asunto(s)
Bacteriófago T7/genética , Clonación Molecular , Biblioteca de Péptidos , Análisis por Matrices de Proteínas/métodos , Proteínas Virales/biosíntesis , Western Blotting , Histidina/química , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Tinción con Nitrato de Plata , Proteínas Virales/aislamiento & purificaciónRESUMEN
A noninvasive screening test would significantly facilitate early detection of epithelial ovarian cancer. This study used a combination of high-throughput selection and array-based serologic detection of many antigens indicative of the presence of cancer, thereby using the immune system as a biosensor. This high-throughput selection involved biopanning of an ovarian cancer phage display library using serum immunoglobulins from an ovarian cancer patient as bait. Protein macroarrays containing 480 of these selected antigen clones revealed 65 clones that interacted with immunoglobulins in sera from 32 ovarian cancer patients but not with sera from 25 healthy women or 14 patients having other benign or malignant gynecologic diseases. Sequence analysis data of these 65 clones revealed 62 different antigens. Among the markers, we identified some known antigens, including RCAS1, signal recognition protein-19, AHNAK-related sequence, nuclear autoantogenic sperm protein, Nijmegen breakage syndrome 1 (Nibrin), ribosomal protein L4, Homo sapiens KIAA0419 gene product, eukaryotic initiation factor 5A, and casein kinase II, as well as many previously uncharacterized antigenic gene products. Using these 65 antigens on protein microarrays, we trained neural networks on two-color fluorescent detection of serum IgG binding and found an average sensitivity and specificity of 55% and 98%, respectively. In addition, the top 6 of the most specific clones resulted in an average sensitivity and specificity of 32% and 94%, respectively. This global approach to antigenic profiling, epitomics, has applications to cancer and autoimmune diseases for diagnostic and therapeutic studies. Further work with larger panels of antigens should provide a comprehensive set of markers with sufficient sensitivity and specificity suitable for clinical testing in high-risk populations.