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1.
Crit Care Med ; 45(5): e516-e523, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28252538

RESUMO

OBJECTIVES: Links between microbial alterations and systemic inflammation have been demonstrated in chronic disease, but little is known about these interactions during acute inflammation. This study investigates the effect of dietary supplementation with cellulose, a nonfermentable fiber, on the gut microbiota, inflammatory markers, and survival in two murine models of sepsis. DESIGN: Prospective experimental study. SETTING: University laboratory. SUBJECTS: Six-week-old male C57BL/6 wild-type mice. INTERVENTIONS: Mice were assigned to low-fiber, normal-fiber, or high-fiber diets with or without antibiotics for 2 weeks and then subjected to sepsis by cecal ligation and puncture or endotoxin injection. Fecal samples were collected for microbiota analyses before and after dietary interventions. MEASUREMENTS AND MAIN RESULTS: Mice that received a high-fiber diet demonstrated increased survival after cecal ligation and puncture relative to mice receiving low-fiber or normal-fiber diets. The survival benefit was associated with decreased serum concentration of pro-inflammatory cytokines, reduced neutrophil infiltration in the lungs, and diminished hepatic inflammation. The high-fiber diet also increased survival after endotoxin injection. Bacterial 16S ribosomal RNA gene sequences from each sample were amplified, sequenced, and analyzed. Fiber supplementation yielded an increase in relative abundance of the genera Akkermansia and Lachnospiraceae, taxa commonly associated with metabolic health. Administration of antibiotics to mice on the high-fiber diet negated the enrichment of Akkermansia species and the survival benefit after cecal ligation and puncture. CONCLUSION: Dietary supplementation with cellulose offers a microbe-mediated survival advantage in murine models of sepsis. Improved understanding of the link between diet, the microbiota, and systemic illness may yield new therapeutic strategies for patients with sepsis.


Assuntos
Fibras na Dieta/farmacologia , Suplementos Nutricionais , Microbioma Gastrointestinal/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Sepse/tratamento farmacológico , Animais , Antibacterianos , Biomarcadores , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Estudos Prospectivos , RNA Ribossômico 16S/genética , Análise de Sobrevida
2.
Pediatr Crit Care Med ; 18(2): 191-192, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28157795
5.
Shock ; 54(2): 256-264, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31513049

RESUMO

Sepsis-induced brain injury is associated with an acute deterioration of mental status resulting in cognitive impairment and acquisition of new functional limitations in sepsis survivors. However, the exact nature of brain injury in this setting is often subtle and remains to be fully characterized both in preclinical studies and at the bedside. Given the translation potential for the use of magnetic resonance imaging (MRI) to define sepsis-induced brain injury, we sought to determine and correlate the cellular changes with neuroradiographic presentations in a classic murine model of sepsis induced by cecal ligation and puncture (CLP). Sepsis was induced in 6-10-week-old male C57/BL6 mice by CLP. We used immunohistochemistry (IHC) to define neuropathology in a mouse model of sepsis along with parallel studies using MRI, focusing on cerebral edema, blood-brain barrier (BBB) disruption, and microglial activation on days 1 and 4 days after CLP. We demonstrate that septic mice had evidence of early axonal injury, inflammation, and robust microglial activation on day 1 followed by cytotoxic edema on day 4 in the cortex, thalamus, and hippocampus in the absence of BBB disruption. We note the superiority of the MRI to detect subtle brain injury and cytotoxic cerebral edema in comparison with the traditional gold standard assessment, i.e., percent brain water (wet-dry weight method). We conclude that inflammatory changes in the septic brain can be detected in real time, and further studies are needed to understand axonal injury and the impact of inhibition of microglial activation on the development of cerebral edema.


Assuntos
Edema Encefálico/patologia , Sepse/patologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Ceco/lesões , Corpo Caloso/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Sepse/metabolismo , Tálamo/metabolismo
6.
J Pediatr Intensive Care ; 8(1): 25-31, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31073505

RESUMO

The incidence of multiple organ dysfunction syndrome (MODS) in sepsis varies from 17 to 73% and furthermore, increases the risk of death by 60% when controlled for the number of dysfunctional organs. Several MODS phenotypes exist, each unique in presentation and pathophysiology. Common to the phenotypes is the stimulation of the immune response by pathogen-associated molecular patterns (PAMPs), or danger-associated molecular patterns (DAMPs) causing an unremitting inflammation. Two of the MODS phenotypes are discussed in detail, thrombocytopenia-associated multiple organ failure (TAMOF) and the hyperinflammatory phenotype-macrophage activating syndrome (MAS) and hemophagocytic lymphohistiocytosis (HLH). In the end, we will briefly review the role of mitochondrial dysfunction as a significant contributor to the pathogenesis of MODS.

7.
Cancer Biol Ther ; 5(8): 933-40, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16775428

RESUMO

Glucocorticoid receptor (GR) activation has recently been implicated in the initiation of anti-apoptotic signaling pathways in epithelial cell lines grown in culture. However, the evidence that GR-mediated inhibition of tumor cell apoptosis is the mechanism that diminishes chemotherapy effectiveness in vivo is limited. We therefore initiated a breast cancer xenograft study to examine whether or not pretreatment with glucocorticoids (GCs) decreases tumor response to chemotherapy by inhibiting tumor cell apoptosis. Here we report a significant decrease in paclitaxel-induced apoptosis in xenografts from mice pretreated with dexamethasone (Dex). A significant difference in apoptosis in xenografts from Dex/paclitaxel versus paclitaxel treated animals was seen eight days following initiation of chemotherapy. Nine days later, mice treated with Dex/paclitaxel had significantly larger tumors compared with those that received paclitaxel alone (p = 0.032). Dex pretreatment did not significantly affect tumor cell proliferation rates. Taken together, these results demonstrate that systemic Dex administration results in significantly reduced breast cancer xenograft apoptosis in the context of chemotherapy treatment. We also found that systemic Dex treatment results in upregulation of the anti-apoptotic gene MKP-1 and downregulation of pro-apoptotic Bid and TRAIL genes in tumor cells six hours following Dex treatment. These in vivo gene expression changes correlated with significant inhibition of chemotherapy-induced apoptosis. Interestingly, the decreased chemotherapeutic response of Dex-pretreated tumors persisted for several weeks following treatment. These data suggest that GR-mediated transcriptional regulation of pro- and anti-apoptotic genes contributes to the mechanism through which GCs decrease paclitaxel-induced apoptosis.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Paclitaxel/uso terapêutico , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/metabolismo , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos SCID , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transplante Heterólogo , Células Tumorais Cultivadas
8.
Cancer Res ; 64(5): 1757-64, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-14996737

RESUMO

Activation of the glucocorticoid receptor (GR) results in diverse physiological effects depending on cell type. For example, glucocorticoids (GC) cause apoptosis in lymphocytes but can rescue mammary epithelial cells from growth factor withdrawal-induced death. However, the molecular mechanisms of GR-mediated survival remain poorly understood. In this study, a large-scale oligonucleotide screen of GR-regulated genes was performed. Several of the genes that were found to be induced 30 min after GR activation encode proteins that function in cell survival signaling pathways. We also demonstrate that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced apoptosis in a GR-dependent manner and is associated with the transcriptional induction of at least two genes identified in our screen, serum and GC-inducible protein kinase-1 (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1). Furthermore, GC treatment alone or GC treatment followed by chemotherapy increases both SGK-1 and MKP-1 steady-state protein levels. In the absence of GC treatment, ectopic expression of SGK-1 or MKP-1 inhibits chemotherapy-induced apoptosis, suggesting a possible role for these proteins in GR-mediated survival. Moreover, specific inhibition of SGK-1 or MKP-1 induction by the introduction of SGK-1- or MKP-1-small interfering RNA reversed the anti-apoptotic effects of GC treatment. Taken together, these data suggest that GR activation in breast cancer cells regulates survival signaling through direct transactivation of genes that encode proteins that decrease susceptibility to apoptosis. Given the widespread clinical administration of dexamethasone before chemotherapy, understanding GR-induced survival mechanisms is essential for achieving optimal therapeutic responses.


Assuntos
Apoptose , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Glucocorticoides/farmacologia , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas Fosfatases , Células Cultivadas , Dexametasona/farmacologia , Fosfatase 1 de Especificidade Dupla , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/análise , Proteínas Imediatamente Precoces/fisiologia , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/fisiologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais
9.
Shock ; 45(5): 506-11, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26682947

RESUMO

Mitochondrial DNA (mtDNA) is a novel danger-associated molecular pattern that on its release into the extracellular milieu acts via toll-like receptor-9, a pattern recognition receptor of the immune system. We hypothesized that plasma mtDNA concentrations will be elevated in septic children, and these elevations are associated with an increase in the severity of illness. In a separate set of in vitro experiments, we test the hypothesis that exposing peripheral blood mononuclear cells (PBMC) to mtDNA activates the immune response and induces tumor necrosis factor (TNF) release. Children with sepsis/systemic inflammatory response syndrome or control groups were enrolled within 24  h of admission to the pediatric intensive care unit. Mitochondrial gene cytochrome c oxidase 1 (COX1) concentrations were measured by real-time quantitative PCR in the DNA extracted from plasma. PBMCs were treated with mtDNA (10  µg/mL) and supernatant TNF levels were measured. The median plasma mtDNA concentrations were significantly elevated in the septic patients as compared with the critically ill non-septic and healthy control patients [1.75E+05 (IQR 6.64E+04-3.67E+05) versus 5.73E+03 (IQR 3.90E+03-1.28E+04) and 6.64E+03 (IQR 5.22E+03-1.63E+04) copies/µL respectively]. The median concentrations of plasma mtDNA were significantly greater in patients with MOF as compared with patients without MOF (3.2E+05 (IQR 1.41E+05-1.08E+06) vs. 2.9E+04 (IQR 2.47E+04-5.43E+04) copies/µL). PBMCs treated with mtDNA demonstrated higher supernatant TNF levels as compared with control cells (6.5 ±â€Š1.8 vs. 3.5 ±â€Š0.5  pg/mL, P > 0.05). Our data suggest that plasma mtDNA is a novel danger-associated molecular pattern in pediatric sepsis and appears to be associated with MOF.


Assuntos
Alarminas/sangue , DNA Mitocondrial/sangue , Plasma/metabolismo , Sepse/sangue , Sepse/genética , Adolescente , Criança , Pré-Escolar , Estado Terminal , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/metabolismo
10.
Curr Treat Options Pediatr ; 1(1): 38-47, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26042193

RESUMO

Acute central nervous system conditions due to hypoxic-ischemic encephalopathy, traumatic brain injury (TBI), status epilepticus, and central nervous system infection/inflammation, are a leading cause of death and disability in childhood. There is a critical need for effective neuroprotective therapies to improve outcome targeting distinct disease pathology. Fever, defined as patient temperature > 38°C, has been clearly shown to exacerbate brain injury. Therapeutic hypothermia (HT) is an intervention using targeted temperature management that has multiple mechanisms of action and robust evidence of efficacy in multiple experimental models of brain injury. Prospective clinical evidence for its neuroprotective efficacy exists in narrowly-defined populations with hypoxic-ischemic injury outside of the pediatric age range while trials comparing hypothermia to normothermia after TBI have failed to demonstrate a benefit on outcome but consistently demonstrate potential use in decreasing refractory intracranial pressure. Data in children from prospective, randomized controlled trials using different strategies of targeted temperature management for various outcomes are few but a large study examining HT versus controlled normothermia to improve neurological outcome in cardiac arrest is underway.

11.
Cancer Prev Res (Phila) ; 2(10): 850-861, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19789294

RESUMO

Clinical studies have revealed that social support improves the outcome of cancer patients, whereas epidemiologic studies suggest that social isolation increases the risk of death associated with several chronic diseases. However, the precise molecular consequences of an unfavorable social environment have not been defined. To do so, robust, reproducible preclinical models are needed to study the mechanisms whereby an adverse environment affects gene expression and cancer biology. Because random assignment of inbred laboratory mice to well-defined social environments allows accurate and repeated measurements of behavioral and endocrine parameters, transgenic mice provide a preclinical framework with which to begin to determine gene-environment mechanisms. In this study, we found that female C3(1)/SV40 T-antigen mice deprived of social interaction from weaning exhibited increased expression of genes encoding key metabolic pathway enzymes in the premalignant mammary gland. Chronic social isolation was associated with up-regulated lipid synthesis and glycolytic pathway gene expression-both pathways are known to contribute to increased breast cancer growth. Consistent with the expression of metabolic genes in premalignant mammary tissue, isolated mice subsequently developed a significantly larger mammary gland tumors burden compared with group-housed mice. Endocrine evaluation confirmed that isolated mice developed a heightened corticosterone stress response compared with group-housed mice. Together, these transdisciplinary studies show for the first time that an adverse social environment is associated with altered mammary gland gene expression and tumor growth. Moreover, the identification of specific alterations in metabolic pathways gene expression favoring tumor growth suggests potential molecular biomarkers and/or targets (e.g., fatty acid synthesis) for preventive intervention in breast cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/psicologia , Isolamento Social , Animais , Western Blotting , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Imuno-Histoquímica , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/fisiopatologia , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
J Biol Chem ; 280(6): 4117-24, 2005 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-15590693

RESUMO

Glucocorticoid receptor (GR) activation has recently been shown to inhibit apoptosis in breast epithelial cells. We have previously described a group of genes that is rapidly up-regulated in these cells following dexamethasone (Dex) treatment. In an effort to dissect the mechanisms of GR-mediated breast epithelial cell survival, we now examine the molecular events downstream of GR activation. Here we show that GR activation leads to both the rapid induction of MAPK phosphatase-1 (MKP-1) mRNA and its sustained expression. Induction of the MKP-1 protein in the MCF10A-Myc and MDA-MB-231 breast epithelial cell lines was also seen. Paclitaxel treatment resulted in MAPK activation and apoptosis of MDA-MB-231 breast cancer cells, and both processes were inhibited by Dex pretreatment. Furthermore, induction of MKP-1 correlated with the inhibition of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) activity, whereas p38 activity was minimally affected. Blocking Dex-induced MKP-1 induction using small interfering RNA increased ERK1/2 and JNK phosphorylation and decreased cell survival. ERK1/2 and JNK inactivation was associated with Ets-like transcription factor-1 (ELK-1) dephosphorylation. To explore the gene expression changes that occur downstream of ELK-1 dephosphorylation, we used a combination of temporal gene expression data and promoter element analyses. This approach revealed a previously unrecognized transcriptional target of ELK-1, the human tissue plasminogen activator (tPA). We verified the predicted ELK-1--> tPA transcriptional regulatory relationship using a luciferase reporter assay. We conclude that GR-mediated MAPK inactivation contributes to cell survival and that the potential transcriptional targets of this inhibition can be identified from large scale gene array analysis.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Paclitaxel/farmacologia , Fosfoproteínas Fosfatases/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Glucocorticoides/metabolismo , Motivos de Aminoácidos , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Northern Blotting , Western Blotting , Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA/biossíntese , Regulação para Baixo , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Luciferases/metabolismo , MAP Quinase Quinase 4 , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ativador de Plasminogênio Tecidual/metabolismo , Fatores de Transcrição/biossíntese , Transcrição Gênica , Transfecção , Proteínas Elk-1 do Domínio ets , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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