RESUMO
Cystic fibrosis liver disease (CFLD) is a rapidly progressive biliary fibrosis, resembling primary sclerosing cholangitis that develops in 5-10% of patients with cystic fibrosis. Further research and evaluation of therapies are hampered by the lack of a mouse model for CFLD. Although primary sclerosing cholangitis is linked to both ulcerative colitis and loss of cystic fibrosis transmembrane conductance regulator (CFTR) ion channel function, induction of colitis with dextran sodium sulfate (DSS) in cftr(-/-) mice causes bile duct injury but no fibrosis. Since profibrogenic modifier genes are linked to CFLD, we examined whether subthreshhold doses of the profibrogenic xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), along with DSS-induced colitis, lead to bile duct injury and liver fibrosis in mice that harbor loss of CFTR function. Exon 10 heterozygous (cftr(+/-)) and homozygous (cftr(-/-)) mice treated with DDC demonstrated extensive mononuclear cell inflammation, bile duct proliferation, and periductular fibrosis. In contrast, wild-type (cftr(+/+)) littermates did not develop bile duct injury or fibrosis. Histological changes corresponded to increased levels of alkaline phosphatase, hydroxyproline, and expression of profibrogenic transcripts for transforming growth factor-ß(1), transforming growth factor-ß(2), procollagen α(1)(I), and tissue inhibitor of matrix metaloproteinase-1. Immunohistochemistry demonstrated fibrosis and activation of periductal fibrogenic cells based on positive staining for lysyl oxidase-like-2, α-smooth muscle actin, and collagen I. These data demonstrate that subthreshold doses of DDC, in conjunction with DSS-induced colitis, results in bile duct injury and periductal fibrosis in mice with partial or complete loss of CFTR function and may represent a useful model to study the pathogenic mechanisms by which CFTR dysfunction predisposes to fibrotic liver disease and potential therapies.
Assuntos
Ductos Biliares/patologia , Colite/complicações , Fibrose Cística/complicações , Cirrose Hepática/etiologia , Fígado/patologia , Animais , Ductos Biliares/metabolismo , Biomarcadores/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colite/patologia , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Regulação da Expressão Gênica , Imuno-Histoquímica , Fígado/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos CFTR , Piridinas , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Transcrição GênicaRESUMO
OBJECTIVE: To measure the changes in whole blood fatty acid levels in premature infants and evaluate associations between these changes and neonatal morbidities. STUDY DESIGN: This was a retrospective cohort study of 88 infants born at <30 weeks' gestation. Serial fatty acid profiles during the first postnatal month and infant outcomes, including chronic lung disease (CLD), retinopathy of prematurity, and late-onset sepsis, were analyzed. Regression modeling was applied to determine the association between fatty acid levels and neonatal morbidities. RESULTS: Docosahexaenoic acid (DHA) and arachidonic acid levels declined rapidly in the first postnatal week, with a concomitant increase in linoleic acid levels. Decreased DHA level was associated with an increased risk of CLD (OR, 2.5; 95% CI, 1.3-5.0). Decreased arachidonic acid level was associated with an increased risk of late-onset sepsis (hazard ratio, 1.4; 95% CI, 1.1-1.7). The balance of fatty acids was also a predictor of CLD and late-onset sepsis. An increased linoleic acid:DHA ratio was associated with an increased risk of CLD (OR, 8.6; 95% CI, 1.4-53.1) and late-onset sepsis (hazard ratio, 4.6; 95% CI, 1.5-14.1). CONCLUSION: Altered postnatal fatty acid levels in premature infants are associated with an increased risk of CLD and late-onset sepsis.
Assuntos
Ácido Araquidônico/sangue , Ácidos Docosa-Hexaenoicos/sangue , Recém-Nascido Prematuro/sangue , Pneumopatias/sangue , Doença Crônica , Estudos de Coortes , Ácidos Graxos/sangue , Feminino , Humanos , Recém-Nascido , Pneumopatias/epidemiologia , Masculino , Oxigenoterapia , Modelos de Riscos Proporcionais , Retinopatia da Prematuridade/sangue , Retinopatia da Prematuridade/epidemiologia , Estudos Retrospectivos , Sepse/sangue , Sepse/epidemiologiaRESUMO
Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. We hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidonate-derived proinflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, we determined the effect of linoleic acid supplementation on downstream proinflammatory biomarkers in two CF models: 1) in vitro cell culture model using 16HBE14o(-) sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using cftr(-/-) transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, PGE(2), and PGF(2α) secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation. cftr(-/-) Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and proinflammatory mediators.
Assuntos
Ácido Araquidônico/biossíntese , Fibrose Cística/dietoterapia , Suplementos Nutricionais , Eicosanoides/biossíntese , Ácido Linoleico/administração & dosagem , Mucosa Respiratória/citologia , Animais , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/fisiopatologia , Modelos Animais de Doenças , Eicosanoides/metabolismo , Ácidos Graxos/metabolismo , Humanos , Inflamação/fisiopatologia , Interleucina-8/imunologia , Interleucina-8/metabolismo , Ácido Linoleico/uso terapêutico , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Camundongos Transgênicos , Pseudomonas aeruginosa/imunologiaRESUMO
Ovarian cancer (OvCa), while accounting for only 3% of all women's cancer, is the fifth leading cause of cancer death among women. One of the most significant obstacles to successful OvCa treatment is chemoresistance. The current lack of understanding of the driving mechanisms underlying chemoresistance hinders the development of effective therapeutics against this obstacle. Adipocytes are key components of the OvCa microenvironment and have been shown to be involved in OvCa cell proliferation, however, little is known about their impact on OvCa chemoresistance. In the current study, we found that adipocytes, of both subcutaneous and visceral origin, secrete factors that enhance the resistance of OvCa cells against chemotherapeutic drugs by activating the Akt pathway. Importantly, we have demonstrated that secreted lipids mediate adipocyte-induced chemoresistance. Through a comprehensive lipidomic analysis, we have identified this chemo-protective lipid mediator as arachidonic acid (AA). AA acts on OvCa cells directly, not through its downstream derivatives such as prostaglandins, to activate Akt and inhibit cisplatin-induced apoptosis. Taken together, our study has identified adipocytes and their secreted AA as important mediators of OvCa chemoresistance. Strategies that block the production of AA from adipocytes or block its anti-apoptotic function may potentially inhibit chemoresistance in OvCa patients.
Assuntos
Adipócitos/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Ovarianas/metabolismo , Adipócitos/fisiologia , Ácido Araquidônico/metabolismo , Ácido Araquidônico/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Lipidômica/métodos , Lipídeos/fisiologia , Neoplasias Ovarianas/fisiopatologia , Ovário/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologiaRESUMO
BACKGROUND: Cystic fibrosis (CF) is characterized by an excessive inflammatory response in epithelial cells and macrophages. In CF mice, lung inflammation can be abrogated by oral treatment with docosahexaenoic acid (DHA). Since PPARs and LXRs are important regulators of inflammation and fatty acid metabolism in macrophages, we hypothesized that these pathways are dysregulated in CF macrophages and are corrected with DHA treatment. METHODS: Peritoneal macrophages were obtained from wild type and cftr(-/-) mice. LPS induced cytokine secretion and NFkappaB activity were analyzed with and without oral DHA treatment. The expression and activity of PPARalpha,gamma, delta and LXRalpha were analyzed by RT-PCR and EMSA. RESULTS: LPS induced TNFalpha and IL-6 secretion and NFkappaB p65 activity were increased in CF macrophages. This was associated with low basal PPARgamma expression and attenuated LPS induced induction of PPARdelta, LXRalpha and ABCA1. DHA pretreatment in vivo decreased TNFalpha secretion and p65 activity, and increased PPARalpha and gamma expression and function. The effects of DHA could be reproduced by PPAR agonists and blocked by a PPARalpha antagonist. CONCLUSION: Impaired regulation of nuclear receptors may contribute to the abnormal LPS induced signaling in CF macrophages and is reversed by DHA.
Assuntos
Fibrose Cística/imunologia , Proteínas de Ligação a DNA/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Imunidade Inata/imunologia , Macrófagos Peritoneais/imunologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/farmacologia , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/imunologia , Receptores X do Fígado , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Receptores Nucleares Órfãos , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: Patients with cystic fibrosis have altered levels of plasma fatty acids. We previously demonstrated that arachidonic acid levels are increased and docosahexaenoic acid levels are decreased in affected tissues from cystic fibrosis-knockout mice. In this study we determined whether humans with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have a similar fatty acid defect in tissues expressing CFTR. METHODS: Fatty acids from nasal- and rectal-biopsy specimens, nasal epithelial scrapings, and plasma were analyzed from 38 subjects with cystic fibrosis and compared with results in 13 obligate heterozygotes, 24 healthy controls, 11 subjects with inflammatory bowel disease, 9 subjects with upper respiratory tract infection, and 16 subjects with asthma. RESULTS: The ratio of arachidonic to docosahexaenoic acid was increased in mucosal and submucosal nasal-biopsy specimens (P<0.001) and rectal-biopsy specimens (P=0.009) from subjects with cystic fibrosis and pancreatic sufficiency and subjects with cystic fibrosis and pancreatic insufficiency, as compared with values in healthy control subjects. In nasal tissue, this change reflected an increase in arachidonic acid levels and a decrease in docosahexaenoic acid levels. In cells from nasal mucosa, the ratio of arachidonic to docosahexaenoic acid was increased in subjects with cystic fibrosis (P<0.001), as compared with healthy controls, with values in obligate heterozygotes intermediate between these two groups (P<0.001). The ratio was not increased in subjects with inflammatory bowel disease. Subjects with asthma and those with upper respiratory tract infection had values intermediate between those in subjects with cystic fibrosis and those in healthy control subjects. CONCLUSIONS: These data indicate that alterations in fatty acids similar to those in cystic fibrosis-knockout mice are present in CFTR-expressing tissue from subjects with cystic fibrosis.
Assuntos
Ácido Araquidônico/metabolismo , Fibrose Cística/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Asma/metabolismo , Biópsia , Estudos de Casos e Controles , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ácidos Graxos/sangue , Ácidos Graxos/metabolismo , Heterozigoto , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Mutação , Mucosa Nasal/metabolismo , Reto/metabolismo , Valores de Referência , Infecções Respiratórias/metabolismoRESUMO
Infants with short bowel syndrome (SBS) are at high risk for malabsorption, malnutrition, and failure to thrive. The objective of this study was to evaluate in a porcine model of SBS, the systemic absorption of a novel enteral Docosahexaenoic acid (DHA) formulation that forms micelles independent of bile salts (DHA-ALT®). We hypothesized that enteral delivery of DHA-ALT® would result in higher blood levels of DHA compared to a control DHA preparation due to improved intestinal absorption. SBS was induced in term piglets through a 75% mid-jejunoileal resection and the piglets randomized to either DHA-ALT® or control DHA formulation (N=5 per group) for 4 postoperative days. The median±IQR difference in final vs starting weight was 696±425 g in the DHA-ALT® group compared to 132±278 g in the controls (P=.08). Within 12 hours, median±IQR DHA and eicosapentaenoic acid plasma levels (mol%) were significantly higher in the DHA-ALT® vs control group (4.1±0.3 vs 2.5±0.5, P=.009; 0.7±0.3 vs 0.2±0.005, P=.009, respectively). There were lower fecal losses of DHA and greater ileal tissue incorporation with DHA-ALT® vs the control. Morphometric analyses demonstrated an increase in proximal jejunum and distal ileum villus height in the DHA-ALT® group compared to controls (P=.01). In a neonatal porcine model of SBS, enteral administration of a novel DHA preparation that forms micelles independent of bile salts resulted in increased fatty acid absorption, increased ileal tissue incorporation, and increased systemic levels of DHA.
Assuntos
Gorduras na Dieta/sangue , Ácidos Docosa-Hexaenoicos/sangue , Íleo/metabolismo , Absorção Intestinal/efeitos dos fármacos , Síndrome do Intestino Curto/terapia , Animais , Animais Recém-Nascidos , Ácidos e Sais Biliares , Peso Corporal , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/sangue , Nutrição Enteral , Micelas , Distribuição Aleatória , SuínosRESUMO
An association has been reported between alterations in fatty acid metabolism and cystic fibrosis (CF). We hypothesized that these alterations are specific for a particular lipid component(s) and are the result of a specific metabolic defect. The different lipid classes were examined for fatty acid changes by using pancreatic homogenates and primary cultures of pancreatic acini from cftr(-/-) (CF) and wild-type mice. Lipid classes and phospholipids were separated by aminopropyl column chromatography and high-performance liquid chromatography, and fatty acid methyl esters were analyzed. The results indicate that in CF mice (1) linoleate was decreased in phospholipids but not in neutral lipids; (2) there was an increase in dihomo-gamma-linolenate and in docosapentaenoate, the terminal fatty acid of the n-6 pathway, in total lipids and total phospholipids, but not in the neutral lipid class; and (3) the docosapentaenoate (n-6)/docosahexaenoate (n-3) ratio was significantly elevated in neutral phospholipids. This suggests an enhanced flux through the n-6 pathway beyond arachidonate. This study provides a more in-depth understanding of the fatty acid alterations found in CF, as reflected by the cftr(-/-) mouse model.
Assuntos
Ácidos Graxos Ômega-6/metabolismo , Ácidos Graxos Insaturados/metabolismo , Pâncreas/metabolismo , Fosfolipídeos/análise , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Ácido Linoleico/análise , Lipídeos/análise , Camundongos , Camundongos Endogâmicos CFTR , Fosfolipídeos/químicaRESUMO
BACKGROUND: Morbidities of impaired immunity and dysregulated inflammation are common in preterm infants. Postnatal Intestinal development plays a critical role in the maturation of the immune system and is, in part, driven by exposure to an enteral diet. OBJECTIVE: The aim of this study was to evaluate the influence of the timing of the first enteral feeding on intestinal inflammation and risk of disease. METHODS: 130 infants <33 weeks' gestation were studied. Maternal and infant data were abstracted from the medical record. Single and multiplex ELISA assays quantified cytokines from fecal and serum samples at two weeks postnatal age. RESULTS: A delay in enteral feedings after the third postnatal day is associated with a 4.5 (95% CI 1.8-11.5, p=0.002) fold increase in chronic lung disease, 2.9 (1.1-7.8, p=0.03) fold increase in retinopathy of prematurity, and 3.4 (1.2-9.8, p=0.02) fold increase in multiple comorbidities compared to infants fed on or before the third day. Additionally, a delay in the initiation of feedings is associated with increased fecal IL-8 levels and a decreased IL-10:IL-8 ratio. CONCLUSIONS: A delay in enteral feeding is associated with intestinal inflammation and increased risks of morbidities. To improve neonatal outcomes, early nutritional practices need to be reevaluated.
Assuntos
Doenças do Prematuro/etiologia , Recém-Nascido Prematuro/fisiologia , Inflamação/etiologia , Intestinos/patologia , Adulto , Nutrição Enteral/métodos , Idade Gestacional , Humanos , Recém-Nascido , Recém-Nascido Prematuro/metabolismo , Doenças do Prematuro/metabolismo , Doenças do Prematuro/patologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-10/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Cuidado Pós-Natal/métodos , Fatores de TempoRESUMO
BACKGROUND: The critical fatty acids Docosahexaenoic Acid (DHA) and Arachidonic Acid (AA) decline in preterm infants within the first postnatal week and are associated with neonatal morbidities, including bronchopulmonary dysplasia (BPD). DHA and AA are precursors to downstream metabolites that terminate the inflammatory response. We hypothesized that treatment with Resolvin D1 and/or Lipoxin A4 would prevent lung injury in a murine model of BPD. OBJECTIVE: To determine the effect of Resolvin D1 and/or Lipoxin A4 on hyperoxia-induced lung injury. METHODS: C57/BL6 pups were randomized at birth to Room Air, Hyperoxia (>90% oxygen), Hyperoxia + Resolvin D1, Hyperoxia + Lipoxin A4, or Hyperoxia + Resolvin D1/Lipoxin A4. Resolvin D1 and/or Lipoxin A4 (2 ng/g) were given IP on days 0, 3, 6, and 9. On day 10, mice were sacrificed and lungs collected for morphometric analyses including Mean Linear Intercept (MLI), Radial Alveolar Count (RAC), and Septal Thickness (ST); RT-PCR analyses of biomarkers of lung development and inflammation; and ELISA for TGFß1 and TGFß2. RESULT: The increased ST observed with hyperoxia exposure was normalized by both Resolvin D1 and Lipoxin A4; while, hyperoxia-induced alveolar simplification was attenuated by Lipoxin A4. Relative to hyperoxia, Resolvin D1 reduced the gene expression of CXCL2 (2.9 fold), TIMP1 (6.7 fold), and PPARγ (4.8 fold). Treatment with Lipoxin A4 also led to a reduction of CXCL2 (2.4 fold) while selectively increasing TGFß2 (2.1 fold) and Smad3 (1.58 fold). CONCLUSION: The histologic and biochemical changes seen in hyperoxia-induced lung injury in this murine model can be reversed by the addition of DHA and AA fatty acid downstream metabolites that terminate the inflammatory pathways and modulate growth factors. These fatty acids or their metabolites may be novel therapies to prevent or treat lung injury in preterm infants.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Hiperóxia/complicações , Lipoxinas/uso terapêutico , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/etiologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologiaRESUMO
Cystic fibrosis (CF) is associated with fatty acid alterations characterized by low linoleic and docosahexaenoic acid. It is not clear whether these fatty acid alterations are directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction or result from nutrient malabsorption. We hypothesized that if fatty acid alterations are a result of CFTR dysfunction, those alterations should be demonstrable in CF cell culture models. Two CF airway epithelial cell lines were used: 16HBE, sense and antisense CFTR cells, and C38/IB3-1 cells. Wild-type (WT) and CF cells were cultured in 10% fetal bovine serum (FBS) or 10% horse serum. Fatty acid levels were analyzed by GC-MS. Culture of both WT and CF cells in FBS resulted in very low linoleic acid levels. When cells were cultured in horse serum containing concentrations of linoleic acid matching those found in human plasma, physiological levels of linoleic acid were obtained and fatty acid alterations characteristic of CF tissues were then evident in CF compared with WT cells. Kinetic studies with radiolabeled linoleic acid demonstrated in CF cells increased conversion to longer and more-desaturated fatty acids such as arachidonic acid. In conclusion, these data demonstrate that CFTR dysfunction is associated with altered fatty acid metabolism in cultured airway epithelial cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Ácidos Graxos/metabolismo , Animais , Elementos Antissenso (Genética) , Brônquios/citologia , Contagem de Células , Células Cultivadas , Meios de Cultura , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Humanos , Ácido Linoleico/metabolismo , Ácido Linoleico/farmacologia , CamundongosRESUMO
This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.
Assuntos
Microscopia/métodos , Organelas , Linhagem Celular , Sobrevivência Celular , HumanosRESUMO
We have developed a novel optical method for observing submicrometer intracellular structures in living cells, which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light-scattering spectroscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales of the order of 100 nm.
Assuntos
Aumento da Imagem/instrumentação , Microscopia Confocal/instrumentação , Análise Espectral/instrumentação , Tomografia de Coerência Óptica/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Estudos de Viabilidade , Aumento da Imagem/métodos , Microscopia Confocal/métodos , Reprodutibilidade dos Testes , Espalhamento de Radiação , Sensibilidade e Especificidade , Análise Espectral/métodos , Tomografia de Coerência Óptica/métodosRESUMO
BACKGROUND: Primary sclerosing cholangitis (PSC) is associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. As proof of concept that CFTR dysfunction plays a role in PSC, induction of colitis in cftr mice results in bile duct injury that can be prevented by pretreatment with docosahexaenoic acid (DHA). OBJECTIVES: Determine whether 1) CFTR dysfunction in cftr mice through a reduction in peroxisome proliferator activated receptor (PPAR)alpha or gamma leads to bile duct injury and 2) whether DHA prevents bile duct injury through an increase in PPAR. METHODS: Cftr and wild-type (WT) mice were treated with dextran sodium sulfate (DSS) to induce colitis with or without pretreatment with oral DHA. PPARalpha and gamma as well as tumor necrosis factor (TNF)alpha were analyzed in liver tissue. PPARalpha mice were also treated with DSS and histology examined. RESULTS: PPARgamma mRNA levels were low, with DSS suppressing mRNA levels equally in WT and cftr mice. PPARalpha levels were no different between cftr and WT litter mates by reverse-transcription polymerase chain reaction. After DSS, WT mice showed a 9.3-fold increase in PPARalpha mRNA levels and increased nuclear localization compared with no DSS (P < 0.05), with no increase seen in cftr mice. This was not caused by changes in TNFalpha. DHA treatment led to 7.0-fold increase in PPARalpha mRNA levels in cftr mice (P < 0.01). PPARalpha mice treated with DSS did not develop bile duct injury, indicating that PPARalpha alone is not sufficient to cause bile duct inflammation. CONCLUSION: DSS induced bile duct injury in cftr mice is associated with a defect in PPARalpha expression, which is reversed by DHA.
Assuntos
Ductos Biliares/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Fibrose Cística/metabolismo , Ácidos Docosa-Hexaenoicos/uso terapêutico , PPAR alfa/metabolismo , Animais , Ductos Biliares/lesões , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Sulfato de Dextrana/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , PPAR gama/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Fatty acid ethyl esters (FAEE) are nonoxidative ethanol metabolites shown to produce toxic effects in the liver and pancreas in vivo and in vitro. Because alcohol-induced chronic pancreatitis is associated with mutations in the gene responsible for cystic fibrosis (CFTR), we hypothesized that CFTR dysfunction leads to increased levels of these toxic nonoxidative ethanol metabolites following alcohol administration. METHODS: Cystic fibrosis (CF) and wild-type (WT) mice were injected intraperitoneally with 1, 2, or 3 g/kg of 50% ethanol. Mice were sacrificed and the liver and pancreas removed for FAEE analysis. RESULTS: The mean FAEE concentration (pmol/g) detected in the liver of cftr mice following injection with 2 g/kg of ethanol was significantly greater than the amount detected in WT (p < 0.005). A similar trend in FAEE concentration was seen in the pancreas, but the difference was not statistically different. In both the liver and pancreas, analysis of individual FAEE species demonstrated a selective increase in ethyl oleate. CONCLUSION: These data show an association between CFTR dysfunction and qualitative and quantitative changes in FAEE in liver and pancreas upon ethanol exposure.
Assuntos
Fibrose Cística/metabolismo , Etanol/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Pâncreas/metabolismo , Animais , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Ésteres/análise , Ésteres/metabolismo , Etanol/administração & dosagem , Etanol/toxicidade , Ácidos Graxos/análise , Ácidos Graxos/biossíntese , Injeções Intraperitoneais , Fígado/química , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Mutação , Ácidos Oleicos/análise , Ácidos Oleicos/metabolismo , Pâncreas/química , Pâncreas/efeitos dos fármacos , Pancreatite Alcoólica/metabolismoRESUMO
It is unknown why some patients with inflammatory bowel disease develop primary sclerosing cholangitis. We have recently shown that patients with primary sclerosing cholangitis have an increased prevalence of mutations in the gene responsible for cystic fibrosis (CFTR) compared with individuals with inflammatory bowel disease alone. Our aim was to examine whether induction of colitis by oral dextran leads to bile duct injury in mice heterozygous or homozygous for mutations in CFTR. The effect of oral administration of docosahexaenoic acid to correct a fatty acid imbalance associated with cystic fibrosis was also examined to determine whether this would prevent bile duct inflammation. Wild-type mice and mice heterozygous and homozygous for CFTR mutations were given dextran orally for 14 days to induce colitis. Bile duct injury was quantitated by blinded histological scoring and measurement of serum alkaline phosphatase activity. The effect of pretreatment with docosahexaenoic acid for 7 days was examined. Treatment of mice with 100 mg dextran/day for 9 days followed by 85 mg/day for 5 days resulted in a significant increase in bile duct injury as determined by histological scoring in homozygous cystic fibrosis mice compared with wild-type mice (P = 0.005). The bile duct injury seen in cystic fibrosis mice was reflected in a threefold increase in serum alkaline phosphatase (P = 0.0006). Pretreatment with oral docosahexaenoic acid decreased both histological evidence of bile duct injury and serum alkaline phosphatase levels. In the setting of colitis, loss of CFTR function leads to bile duct injury.
Assuntos
Ductos Biliares/patologia , Colite/metabolismo , Colite/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Administração Oral , Fosfatase Alcalina/sangue , Animais , Ductos Biliares/efeitos dos fármacos , Colite/induzido quimicamente , Colo/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Sulfato de Dextrana/administração & dosagem , Ácidos Docosa-Hexaenoicos/administração & dosagem , Heterozigoto , Homozigoto , Camundongos , Camundongos Knockout , MutaçãoRESUMO
Patients with cystic fibrosis (CF) exhibit an excessive host inflammatory response. The aim of this study was to determine (i) whether interleukin 8 (IL-8) secretion is increased from monocytes from subjects heterozygous as well as homozygous for cystic fibrosis transmembrane conductance regulator (CFTR) mutations and (ii) whether this is due to increased cell surface lipopolysaccharide (LPS) receptors or, alternatively, increased activation of mitogen-activated protein kinases (MAPK). The basal level of IL-8 secretion was higher from monocytes from CF patients than from monocytes from healthy controls (P = 0.02) and obligate heterozygotes (parents of the CF patients). The 50% effective concentrations for LPS-induced IL-8 production for monocytes from both CF patients and obligate heterozygotes were 100-fold lower than those for monocytes from healthy controls (P < 0.05). No differences in the levels of IL-1beta production were seen between these groups. Expression of the LPS surface receptors CD14 and Toll-like receptor 4 were not different between CF patients and healthy controls. In contrast, phosphorylation of the MAPKs p38 and ERK occurred at lower doses of LPS in monocytes from patients heterozygous and homozygous for CFTR mutations. These results indicate that a single allelic CFTR mutation is sufficient to augment IL-8 secretion in response to LPS. This is not a result of increased LPS receptor expression but, rather, is associated with alterations in MAPK signaling.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Interleucina-8/metabolismo , Monócitos/metabolismo , Mutação , Adulto , Estudos de Casos e Controles , Feminino , Heterozigoto , Humanos , Receptores de Lipopolissacarídeos/análise , Sistema de Sinalização das MAP Quinases , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores de Superfície Celular/análise , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
Some of the pathological manifestations of cystic fibrosis are in accordance with an impaired expression and/or activity of PPARgamma. We hypothesized that PPARgamma expression is altered in tissues lacking the normal cystic fibrosis transmembrane regulator protein (CFTR). PPARgamma mRNA levels were measured in colonic mucosa, ileal mucosa, adipose tissue, lung, and liver from wild-type and cftr-/- mice by quantitative RT-PCR. PPARgamma expression was decreased twofold in CFTR-regulated tissues (colon, ileum, and lung) from cftr-/- mice compared to wild-type littermates. In contrast, no differences were found in fat and liver. Immunohistochemical analysis of PPARgamma in ileum and colon revealed a predominantly nuclear localization in wild-type mucosal epithelial cells while tissues from cftr-/- mice showed a more diffuse, lower intensity labeling. A significant decrease in PPARgamma expression was confirmed in nuclear extracts of colon mucosa by Western blot analysis. In addition, binding of the PPARgamma/RXR heterodimer to an oligonucletotide containing a peroxisome proliferator responsive element (PPRE) was also decreased in colonic mucosa extracts from cftr-/- mice. Treatment of cftr-/- mice with the PPARgamma ligand rosiglitazone restored both the nuclear localization and binding to DNA, but did not increase RNA levels. We conclude that PPARgamma expression in cftr-/- mice is downregulated at the RNA and protein levels and its function diminished. These changes may be related to the loss of function of CFTR and may be relevant to the pathogenesis of metabolic abnormalities associated with cystic fibrosis in humans.