RESUMO
Mucormycoses are deadly invasive infections caused by several fungal species belonging to the subphylum Mucoromycotina, order Mucorales. Hallmarks of disease progression include angioinvasion and tissue necrosis that aid in fungal dissemination through the blood stream, causing deeper infections and resulting in poor penetration of antifungal agents to the site of infection. In the absence of surgical removal of the infected focus, antifungal therapy alone is rarely curative. Even when surgical debridement is combined with high-dose antifungal therapy, the mortality associated with mucormycoses is >50%. The unacceptably high mortality rate, limited options for therapy and the extreme morbidity of highly disfiguring surgical therapy provide a clear mandate to understand the molecular mechanisms that govern pathogenesis with the hopes of developing alternative strategies to treat and prevent mucormycoses. In the absence of robust forward and reverse genetic systems available for this taxonomic group of fungi, unbiased next generation sequence (NGS)-based approaches have provided much needed insights into our understanding of many aspects of Mucormycoses, including genome structure, drug resistance, diagnostic development, and fungus-host interactions. Here, we will discuss the specific contributions that NGS-based approaches have made to the field and discuss open questions that can be addressed using similar approaches.
RESUMO
Mucormycosis is a life-threatening, invasive fungal infection that is caused by various species belonging to the order Mucorales. Rhizopus species are the most common cause of the disease, responsible for approximately 70% of all cases of mucormycosis. During pulmonary mucormycosis, inhaled Rhizopus spores must adhere to and invade airway epithelial cells in order to establish infection. The molecular mechanisms that govern this interaction are poorly understood. We performed an unbiased survey of the host transcriptional response during early stages of Rhizopus arrhizus var. delemar (R. delemar) infection in a murine model of pulmonary mucormycosis using transcriptome sequencing (RNA-seq). Network analysis revealed activation of the host's epidermal growth factor receptor (EGFR) signaling. Consistent with the RNA-seq results, EGFR became phosphorylated upon in vitro infection of human alveolar epithelial cells with several members of the Mucorales, and this phosphorylated, activated form of EGFR colocalized with R. delemar spores. Inhibition of EGFR signaling with cetuximab or gefitinib, specific FDA-approved inhibitors of EGFR, significantly reduced the ability of R. delemar to invade and damage airway epithelial cells. Furthermore, gefitinib treatment significantly prolonged survival of mice with pulmonary mucormycosis, reduced tissue fungal burden, and attenuated the activation of EGFR in response to pulmonary mucormycosis. These results indicate EGFR represents a novel host target to block invasion of alveolar epithelial cells by R. delemar, and inhibition of EGFR signaling provides a novel approach for treating mucormycosis by repurposing an FDA-approved drug.IMPORTANCE Mucormycosis is an increasingly common, highly lethal fungal infection with very limited treatment options. Using a combination of in vivo animal models, transcriptomics, cell biology, and pharmacological approaches, we have demonstrated that Mucorales fungi activate EGFR signaling to induce fungal uptake into airway epithelial cells. Inhibition of EGFR signaling with existing FDA-approved drugs significantly increased survival following R. arrhizus var. delemar infection in mice. This study enhances our understanding of how Mucorales fungi invade host cells during the establishment of pulmonary mucormycosis and provides a proof-of-concept for the repurposing of FDA-approved drugs that target EGFR function.
Assuntos
Receptores ErbB/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Pulmão/microbiologia , Mucormicose/prevenção & controle , Células A549 , Animais , Cetuximab/farmacologia , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Redes Reguladoras de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mucormicose/microbiologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Rhizopus/efeitos dos fármacos , Rhizopus/patogenicidade , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacosRESUMO
Aspergillus fumigatus is an opportunistic, ubiquitous, saprophytic mould that can cause severe allergic responses in atopic individuals as well as life-threatening infections in immunocompromised patients. A critical step in the establishment of infection is the invasion of airway epithelial cells by the inhaled fungi. Understanding how A. fumigatus senses and responds to airway cells is important to understand the pathogenesis of invasive pulmonary aspergillosis. Here, we analysed the transcriptomes of two commonly used clinical isolates, Af293 and CEA10, during infection of the A549 type II pneumocyte cell line in vitro. We focused our RNA-seq analysis on the core set of genes that are present in the genomes of the two strains. Our results suggest that: (a) A. fumigatus does not mount a conserved transcriptional response to airway epithelial cells in our in vitro model and (b) strain background and time spent in the tissue culture media have a greater impact on the transcriptome than the presence of host cells. Our analyses reveal both common and strain-specific transcriptional programmes that allow for the generation of hypotheses about gene function as it pertains to pathogenesis and the significant phenotypic heterogeneity that is observed among A. fumigatus isolates.
Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Células Epiteliais/microbiologia , Sistema Respiratório/microbiologia , Transcriptoma , Células A549 , Células Epiteliais Alveolares/microbiologia , Aspergillus fumigatus/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Fenótipo , Aspergilose Pulmonar/microbiologia , Análise de Sequência , Esporos FúngicosRESUMO
M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 (MMP1) expression. MMP1 expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced MMP1 expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time- and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFR-independent and blocked by inhibiting protein kinase C-α (PKC-α). Inhibiting activation of PKC-α, EGFR, ERK1/2, or p38-α/ß alone attenuated, but did not abolish ACh-induced MMP1 expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced MMP1 expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-α/ß inhibitor. Activating PKC-α and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated MMP1 gene and protein expression, and cell invasion. PMA- and ACh-induced MMP1 expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-α/ß and Src, indicating that Src mediates the cross-talk between PKC-α and EGFR signaling. Using siRNA knockdown, we identified p38-α as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment MMP1 expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.
Assuntos
Regulação Neoplásica da Expressão Gênica , Metaloproteinase 1 da Matriz/metabolismo , Receptor Muscarínico M3/genética , Transdução de Sinais/genética , Acetilcolina/farmacologia , Células CACO-2 , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HT29 , Humanos , Metaloproteinase 1 da Matriz/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Muscarínico M3/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
We report an aggressive fungal keratitis caused by a putatively novel species of Lophotrichus in a patient with traumatic injury to the cornea from a dog paw. The organism was isolated from the patient's necrotic cornea, which perforated despite coverage with hourly fortified broad-spectrum topical antibiotic therapy. This report represents the first case of human infection caused by this species.
Assuntos
Ascomicetos/isolamento & purificação , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/patologia , Micoses/diagnóstico , Micoses/patologia , Animais , Lesões da Córnea/complicações , Úlcera da Córnea/microbiologia , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Cães , Feminino , Histocitoquímica , Humanos , Técnicas Microbiológicas , Microscopia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Micoses/microbiologia , Análise de Sequência de DNARESUMO
Candida species are common causes of bloodstream infections (BSI), with high mortality. Four species cause >90% of Candida BSI: C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. Differentiation of Candida spp. is important because of differences in virulence and antimicrobial susceptibility. Candida QuickFISH BC, a multicolor, qualitative nucleic acid hybridization assay for the identification of C. albicans (green fluorescence), C. glabrata (red fluorescence), and C. parapsilosis (yellow fluorescence), was tested on Bactec and BacT/Alert blood culture bottles which signaled positive on automated blood culture devices and were positive for yeast by Gram stain at seven study sites. The results were compared to conventional identification. A total of 419 yeast-positive blood culture bottles were studied, consisting of 258 clinical samples (89 C. glabrata, 79 C. albicans, 23 C. parapsilosis, 18 C. tropicalis, and 49 other species) and 161 contrived samples inoculated with clinical isolates (40 C. glabrata, 46 C. albicans, 36 C. parapsilosis, 19 C. tropicalis, and 20 other species). A total of 415 samples contained a single fungal species, with C. glabrata (n = 129; 30.8%) being the most common isolate, followed by C. albicans (n = 125; 29.8%), C. parapsilosis (n = 59; 14.1%), C. tropicalis (n = 37; 8.8%), and C. krusei (n = 17; 4.1%). The overall agreement (with range for the three major Candida species) between the two methods was 99.3% (98.3 to 100%), with a sensitivity of 99.7% (98.3 to 100%) and a specificity of 98.0% (99.4 to 100%). This study showed that Candida QuickFISH BC is a rapid and accurate method for identifying C. albicans, C. glabrata, and C. parapsilosis, the three most common Candida species causing BSI, directly from blood culture bottles.
Assuntos
Sangue/microbiologia , Candida/classificação , Candida/isolamento & purificação , Candidemia/diagnóstico , Candidemia/microbiologia , Hibridização in Situ Fluorescente/métodos , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Candida/genética , Humanos , Sensibilidade e EspecificidadeRESUMO
(1-3)-ß-d-Glucan (BDG) from cerebrospinal fluid (CSF) is a promising marker for diagnostic and prognostic aid of central nervous system (CNS) fungal infection, but its relationship to serum values has not been studied. Herein, we detected BDG from CSF at levels 2-fold lower than those in serum in patients without evidence of fungal disease but 25-fold higher than those in in serum in noncryptococcal CNS fungal infections. CSF BDG may be a useful biomarker in the evaluation of fungal CNS disease.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Infecções Fúngicas do Sistema Nervoso Central/líquido cefalorraquidiano , Infecções Fúngicas do Sistema Nervoso Central/diagnóstico , Infecções Fúngicas do Sistema Nervoso Central/epidemiologia , beta-Glucanas/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteoglicanas , Adulto JovemRESUMO
Angioinvasive fungal infections (AFIs) are an important cause of morbidity and mortality among immunocompromised patients. However, clinicomicrobiological characteristics and treatment of many AFI agents remain poorly defined. We report the first human case of infection with Westerdykella dispersa, an emergent cause of AFI, which was successfully treated in a neutropenic pediatric patient.
Assuntos
Ascomicetos/isolamento & purificação , Micoses/diagnóstico , Micoses/patologia , Neutropenia/complicações , Vasculite/diagnóstico , Vasculite/patologia , Ascomicetos/classificação , Ascomicetos/genética , Criança , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Histocitoquímica , Humanos , Hospedeiro Imunocomprometido , Injeções/efeitos adversos , Masculino , Técnicas Microbiológicas , Microscopia , Dados de Sequência Molecular , Micoses/microbiologia , Radiografia Torácica , Análise de Sequência de DNA , Tomografia Computadorizada por Raios X , Vasculite/microbiologiaAssuntos
Candida/classificação , Candida/isolamento & purificação , Meios de Cultura/química , Reações Falso-Positivas , Técnicas Microbiológicas/métodos , Azul de Bromotimol/metabolismo , Canavanina/metabolismo , Pré-Escolar , Dermatomicoses/diagnóstico , Dermatomicoses/microbiologia , Glicina/metabolismo , Humanos , Masculino , Dermatoses do Couro Cabeludo/diagnóstico , Dermatoses do Couro Cabeludo/microbiologiaRESUMO
BACKGROUND: Gene expression profiling of peripheral blood mononuclear cells (PBMCs) is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells. METHODOLOGY/PRINCIPAL FINDINGS: The transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH), 19 patients with systemic sclerosis without pulmonary hypertension (SSc), 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH), and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD) were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease. CONCLUSIONS/SIGNIFICANCE: In PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression.
Assuntos
Células Eritroides/metabolismo , Hipertensão Pulmonar/genética , Leucócitos Mononucleares/metabolismo , Transcriptoma , Adulto , Idoso , Feminino , Regulação da Expressão Gênica , Hemodinâmica , Humanos , Hipertensão Pulmonar/fisiopatologia , Leucócitos Mononucleares/patologia , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
CONTEXT: The overwhelming majority of benign lesions of the adrenal cortex leading to Cushing syndrome are linked to one or another abnormality of the cAMP or protein kinase pathway. PRKAR1A-inactivating mutations are responsible for primary pigmented nodular adrenocortical disease, whereas somatic GNAS activating mutations cause macronodular disease in the context of McCune-Albright syndrome, ACTH-independent macronodular hyperplasia, and, rarely, cortisol-producing adenomas. OBJECTIVE AND DESIGN: The whole-genome expression profile (WGEP) of normal (pooled) adrenals, PRKAR1A- (3) and GNAS-mutant (3) was studied. Quantitative RT-PCR and Western blot were used to validate WGEP findings. RESULTS: MAPK and p53 signaling pathways were highly overexpressed in all lesions against normal tissue. GNAS-mutant tissues were significantly enriched for extracellular matrix receptor interaction and focal adhesion pathways when compared with PRKAR1A-mutant (fold enrichment 3.5, P < 0.0001 and 2.1, P < 0.002, respectively). NFKB, NFKBIA, and TNFRSF1A were higher in GNAS-mutant tumors (P < 0.05). Genes related to the Wnt signaling pathway (CCND1, CTNNB1, LEF1, LRP5, WISP1, and WNT3) were overexpressed in PRKAR1A-mutant lesions. CONCLUSION: WGEP analysis revealed that not all cAMP activation is the same: adrenal lesions harboring PRKAR1A or GNAS mutations share the downstream activation of certain oncogenic signals (such as MAPK and some cell cycle genes) but differ substantially in their effects on others.
Assuntos
Hiperfunção Adrenocortical/genética , Códon sem Sentido , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Mutação em Linhagem Germinativa , Sistemas do Segundo Mensageiro , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Insuficiência Adrenal/genética , Insuficiência Adrenal/metabolismo , Insuficiência Adrenal/patologia , Hiperfunção Adrenocortical/metabolismo , Hiperfunção Adrenocortical/patologia , Ciclo Celular , Cromograninas , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Via de Sinalização WntRESUMO
Although T cells have been shown to play a direct role in kidney ischemia-reperfusion injury (IRI), little is known about the underlying mechanisms. We hypothesized that studying the transcriptional responses in kidney-infiltrating T cells would help elucidate novel therapeutic targets for kidney IRI. Unilateral renal pedicle clamping for 45 min was performed in male C57BL/6 mice, and CD3(+) T cells were isolated from the kidney and purified. Transcriptional activities of T cell were measured by array-based PCR compared between ischemic kidneys and contralateral nonischemic kidneys. Among total of 89 genes analyzed, 24, 22, 24, and 37 genes were significantly changed at 6 h, day 3, day 10, and day 28 after IRI. Genes associated with cytokines, chemokines, and costimulatory molecules were upregulated. Pathway analysis identified CC motif chemokine receptor 5 (CCR5) as a candidate pathophysiological pathway. CCR5 upregulation was validated at the protein level, and CCR5 blockade improved renal function after kidney IRI. Using discovery techniques to identify transcriptional responses in purified kidney-infiltrating cells enabled the elucidation of novel mechanisms and therapeutic targets for IRI.
Assuntos
Regulação da Expressão Gênica/fisiologia , Rim/lesões , Rim/patologia , Receptores CCR5/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Linfócitos T/metabolismo , Animais , Anticorpos , Complexo CD3/genética , Complexo CD3/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Rim/citologia , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Masculino , Camundongos , Receptores CCR5/genética , Traumatismo por Reperfusão/metabolismo , Organismos Livres de Patógenos EspecíficosRESUMO
There is extensive evidence that posttranscriptional mechanisms of gene regulation, such as mRNA turnover, critically affect the patterns of expressed mRNAs. Conventional microarray analysis measures steady-state messenger RNA (mRNA) levels, which represents the dynamic balance between new transcription and mRNA degradation. Accordingly, only de novo transcription can accurately reflect the temporal and spatial events of transcriptional regulation. In this chapter, we describe a recently reported method to study transcription systematically. It involves the genome-wide labeling of nascent transcripts using nonradioactive modified nucleotides, their isolation for amplification, and their hybridization and analysis using commercial microarrays.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Biotina , Linhagem Celular , Humanos , Camundongos , Estabilidade de RNA/genética , RNA Mensageiro/genéticaRESUMO
CONTEXT: Massive macronodular adrenocortical disease or ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a clinically and genetically heterogeneous disorder. OBJECTIVE AND DESIGN: Whole-genome expression profiling and oligonucleotide array comparative genomic hybridization changes were analyzed in samples of different nodules from the same patients with AIMAH. Quantitative RT-PCR and staining were employed to validate the mRNA array data. RESULTS: Chromosomal gains were more frequent in larger nodules when compared with smaller nodules from the same patients. Among the 50 most overexpressed genes, 50% had a chromosomal locus that was amplified in the comparative genomic hybridization data. Although the list of most over- and underexpressed genes was similar between the nodules of different size, the gene set enrichment analysis identified different pathways associated with AIMAH that corresponded to the size; the smaller nodules were mainly enriched for metabolic pathways, whereas p53 signaling and cancer genes were enriched in larger nodules. Confirmatory studies demonstrated that BCL2, E2F1, EGF, c-KIT, MYB, PRKCA, and CTNNB1 were overexpressed in the larger nodules at messenger and/or protein levels. Chromosomal enrichment analysis showed that chromosomes 20q13 and 14q23 might be involved in progression of AIMAH from smaller to larger tumors. CONCLUSION: Integrated transcriptomic and genomic data for AIMAH provides supporting evidence to the hypothesis that larger adrenal lesions, in the context of this chronic, polyclonal hyperplasia, accumulate an increased number of genomic and, subsequently, transcript abnormalities. The latter shows that the disease appears to start with mainly tissue metabolic derangements, as suggested by the study of the smaller nodules, but larger lesions showed aberrant expression of oncogenic pathways.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Córtex Suprarrenal/patologia , Carcinoma/genética , Transformação Celular Neoplásica/genética , Neoplasias Primárias Múltiplas/genética , Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Adulto , Carcinoma/patologia , Transformação Celular Neoplásica/patologia , Hibridização Genômica Comparativa , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Genômica/métodos , Humanos , Hiperplasia , Masculino , Neoplasias Primárias Múltiplas/patologia , Integração de Sistemas , Carga TumoralRESUMO
Patients with genetic defects of the cyclic (c) adenosine-monophosphate (AMP)-signaling pathway and those with neonatal-onset multisystem inflammatory disease (NOMID) develop tumor-like lesions of the long bones. The molecular basis of this similarity is unknown. NOMID is caused by inappropriate caspase-1 activity, which in turn activates the inflammasome. The present study demonstrates that NOMID bone lesions are derived from the same osteoblast progenitor cells that form fibroblastoid tumors in mice and humans with defects that lead to increased cAMP-dependent protein kinase A (PKA) signaling. NOMID tumor cells showed high PKA activity, and an increase in their cAMP signaling led to PKA-specific activation of caspase-1. Increased PKA led to inflammation-independent activation of caspase-1 via over-expression of the proto-oncogene (and early osteoblast factor) Ets-1. In NOMID tumor cells, as in cells with defective PKA regulation, increased prostaglandin E2 (PGE2) led to increased cAMP levels and activation of Wnt signaling, like in other states of inappropriate PKA activity. Caspase-1 and PGE2 inhibition led to a decrease in cell proliferation of both NOMID and cells with abnormal PKA. These data reveal a previously unsuspected link between abnormal cAMP signaling and defective regulation of the inflammasome and suggest that caspase-1 and PGE2 inhibition may be therapeutic targets in bone lesions associated with defects of these two pathways.
Assuntos
Síndromes Periódicas Associadas à Criopirina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Osteoblastos/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Células-Tronco/metabolismo , Animais , Osso e Ossos/patologia , Caspase 1/genética , Células Cultivadas , Síndromes Periódicas Associadas à Criopirina/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Dinoprostona/genética , Dinoprostona/metabolismo , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Camundongos , Proto-Oncogene Mas , Proteína Proto-Oncogênica c-ets-1/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Células Estromais/metabolismo , Ativação Transcricional/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
INTRODUCTION: Molecular biomarkers validated previously in animal models are increasingly being studied in conjunction with traditional clinical endpoints in therapeutic trials. PATIENT AND METHODS: We hypothesized that human kidneys would exhibit a brisk, gene-specific inflammatory response during ischemia reperfusion injury (IRI), which would be modified by anti-adhesive therapy. Forty deceased-donor kidneys were biopsied prior to implantation and â¼1 h after reperfusion during an intervention trial with the selectin antagonist YSPSL (recombinant P-selectin glycoprotein ligand Ig). Ten inflammatory genes were measured by RT-PCR and normalized to three housekeeping genes. RESULTS: Pre-implantation kidney biopsies were already significantly inflamed relative to healthy tissue, with transcripts encoding IL-6, IL-8, and CD25 > 10-fold elevated. After reperfusion, IL-6 and IL-8 increased additional 60- and 120-fold (p < 0.05), while already elevated CD25-levels remained stable. Furthermore, transcripts encoding MCP-1, E-selectin, and TNFα were also induced significantly upon reperfusion (p < 0.0005). Systemic treatment of the recipient with YSPSL pre-reperfusion, with or without pre-implantation YSPSL flush of the donor organ, attenuated the post-reperfusion increase in MCP-1 and TGFß (p < 0.05), E-selectin and hemoxygenase 1 transcripts (p < 0.1). CONCLUSIONS: Our data in humans demonstrate a robust increase in inflammatory gene transcript levels during kidney transplantation IRI and reduction thereof by inhibition of leukocyte adhesion.
Assuntos
Biomarcadores/análise , Citocinas/genética , Rim/fisiopatologia , Glicoproteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Traumatismo por Reperfusão/diagnóstico , Traumatismo por Reperfusão/prevenção & controle , Doadores de Tecidos , Adulto , Idoso , Cadáver , Estudos de Coortes , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Selectina-P/antagonistas & inibidores , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Reperfusão , Traumatismo por Reperfusão/genética , Transplante HomólogoRESUMO
A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation. Mice heterozygous for a null allele of prkar1a (Prkar1a(+/-)), the primary receptor for cyclic adenosine monophosphate (cAMP) and regulator of protein kinase A (PKA) activity, developed bone lesions that were derived from cAMP-responsive osteogenic cells and resembled fibrous dysplasia (FD). Prkar1a(+/-) mice were crossed with mice that were heterozygous for catalytic subunit Calpha (Prkaca(+/-)), the main PKA activity-mediating molecule, to generate a mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a(+/-)Prkaca(+/-)). Unexpectedly, Prkar1a(+/-)Prkaca(+/-) mice developed a greater number of osseous lesions starting at 3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions originated from an area proximal to the growth plate, expressed osteogenic cell markers, and showed higher PKA activity that was mostly type II (PKA-II) mediated by an alternate pattern of catalytic subunit expression. Gene expression profiling confirmed a preosteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial transition and increased Wnt signaling. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by alternate PKA and catalytic subunit activity; abnormal proliferation of these cells leads to skeletal lesions that have similarities to human FD and bone tumors.
Assuntos
Envelhecimento/patologia , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Animais , Osso e Ossos/diagnóstico por imagem , Calcificação Fisiológica , Domínio Catalítico , Heterozigoto , Mesoderma/metabolismo , Camundongos , Diester Fosfórico Hidrolases/metabolismo , Análise Espectral Raman , Células Estromais/enzimologia , Células Estromais/patologia , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: The definition of transcriptional networks through measurements of changes in gene expression profiles and mapping of transcription factor binding sites is limited by the moderate overlap between binding and gene expression changes and the inability to directly measure global nuclear transcription (coined "transactome"). METHODOLOGY/PRINCIPAL FINDINGS: We developed a method to measure nascent nuclear gene transcription with an Array-based Nuclear Run-On (ANRO) assay using commercial microarray platforms. This strategy provides the missing component, the transactome, to fully map transcriptional networks. ANRO measurements in an inducible c-Myc expressing human P493-6 B cell model reveals time-dependent waves of transcription, with a transactome early after c-Myc induction that does not persist at a late, steady-state phase, when genes that are regulated by c-Myc and E2F predominate. Gene set matrix analysis further uncovers functionally related groups of genes putatively regulated by waves of transcription factor motifs following Myc induction, starting with AP1 and CREB that are followed by EGR1, NFkB and STAT, and ending with E2F, Myc and ARNT/HIF motifs. CONCLUSIONS/SIGNIFICANCE: By coupling ANRO with previous global mapping of c-Myc binding sites by chromatin immunoprecipitation (ChIP) in P493-6 cells, we define a set of transcriptionally regulated direct c-Myc target genes and pave the way for the use of ANRO to comprehensively map any transcriptional network.
Assuntos
Linfócitos B/citologia , Núcleo Celular/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , DNA Complementar/metabolismo , Redes Reguladoras de Genes , Humanos , Células Jurkat , Modelos Biológicos , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Acute kidney injury (AKI) is being increasingly shown to be a risk factor for chronic kidney disease (CKD), but little is known about the possible mechanistic links. We hypothesized that analysis of the genomic signature in the repair stage after AKI would reveal pathways that could link AKI and CKD. Unilateral renal pedicle clamping for 45 min was performed in male C57BL/6J mice. Mice were euthanized at 3, 10, and 28 days after ischemia-reperfusion injury (IRI). Total RNA was isolated from kidney and analyzed using an Illumina mouse array. Among 24,600 tested genes, 242, 146, and 46 genes were upregulated at days 3, 10, and 28 after IRI, and 85, 35, and 0 genes were downregulated, respectively. Gene ontology analysis showed that gene expression changes were primarily related to immune and inflammatory pathways both early and late after AKI. The most highly upregulated genes late after AKI were hepatitis A virus cellular receptor 1 (Havcr1) and lipocalin 2 (Lcn2), which code for kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), respectively. This was unexpected since they are both primarily potential biomarkers of the early stage of AKI. Furthermore, increases observed in gene expression in amiloride binding protein 1, vascular cell adhesion molecule-1, and endothelin 1 could explain the salt-sensitive hypertension that can follow AKI. These data suggested that 1) persistent inflammation and immune responses late after AKI could contribute to the pathogenesis of CKD, 2) late upregulation of KIM-1 and NGAL could be a useful marker for sustained renal injury after AKI, and 3) hypertension-related gene changes could underlie mechanisms for persistent renal and vascular injury after AKI.
Assuntos
Proteínas de Fase Aguda/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Nefropatias/genética , Rim/enzimologia , Lipocalinas/genética , Proteínas de Membrana/genética , Proteínas Oncogênicas/genética , Traumatismo por Reperfusão/genética , Transcrição Gênica , Doença Aguda , Proteínas de Fase Aguda/metabolismo , Animais , Biomarcadores/metabolismo , Doença Crônica , Modelos Animais de Doenças , Fibrose , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Receptor Celular 1 do Vírus da Hepatite A , Inflamação/genética , Inflamação/imunologia , Rim/imunologia , Rim/patologia , Nefropatias/enzimologia , Nefropatias/imunologia , Nefropatias/patologia , Lipocalina-2 , Lipocalinas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para CimaRESUMO
OBJECTIVE: Wegener's granulomatosis (WG) is a systemic inflammatory disease that is associated with substantial morbidity. The aim of this study was to understand the biology underlying WG and to discover markers of disease activity that would be useful for prognosis and treatment guidance. METHODS: Gene expression profiling was performed using total RNA from peripheral blood mononuclear cells (PBMCs) and granulocyte fractions from 41 patients with WG and 23 healthy control subjects. Gene set enrichment analysis (GSEA) was performed to search for candidate WG-associated molecular pathways and disease activity biomarkers. Principal components analysis was used to visualize relationships between subgroups of WG patients and controls. Longitudinal changes in proteinase 3 (PR3) gene expression were evaluated using reverse transcription-polymerase chain reaction, and clinical outcomes, including remission status and disease activity, were determined using the Birmingham Vasculitis Activity Score for WG (BVAS-WG). RESULTS: Eighty-six genes in WG PBMCs and 40 in WG polymorphonuclear neutrophils (PMNs) were significantly up-regulated relative to controls. Genes up-regulated in WG PBMCs were involved in myeloid differentiation, and these included the WG autoantigen PR3. The coordinated regulation of myeloid differentiation genes was confirmed by GSEA. The median expression values of the 86 up-regulated genes in WG PBMCs were associated with disease activity (P = 1.3 x 10(-4)), and WG patients with low-level expression of the WG signature genes showed expression profiles that were only modestly different from that in healthy controls (P = 0.07). PR3 transcription was significantly up-regulated in WG PBMCs (P = 1.3 x 10(-5), false discovery rate [FDR] 0.002), but not in WG PMNs (P = 0.03, FDR 0.28), and a preliminary longitudinal analysis showed that the fold change in PR3 RNA levels in WG PBMCs corresponded to changes in the BVAS-WG score over time. CONCLUSION: Transcription of PR3 and related myeloid differentiation genes in PBMCs may represent novel markers of disease activity in WG.