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1.
Thorax ; 78(8): 799-807, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36261273

RESUMO

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is rare, poorly understood, with heterogeneous characteristics resulting in difficult diagnosis. We aimed to systematically review evidence of soluble markers in peripheral blood or bronchoalveolar lavage fluid (BALF) as biomarkers in SSc-ILD. METHOD: Five databases were screened for observational or interventional, peer-reviewed studies in adults published between January 2000 and September 2021 that assessed levels of biomarkers in peripheral blood or BALF of SSc-ILD patients compared with healthy controls. Qualitative assessment was performed using Critical Appraisal Skills Programme (CASP) checklists. Standardised mean difference (SMD) in biomarkers were combined in random-effects meta-analyses where multiple independent studies reported quantitative data. RESULTS: 768 published studies were identified; 38 articles were included in the qualitative synthesis. Thirteen studies were included in the meta-analyses representing three biomarkers: KL6, SP-D and IL-8. Greater IL-8 levels were associated with SSc-ILD in both peripheral blood and BALF, overall SMD 0.88 (95% CI 0.61 to 1.15; I2=1%). Greater levels of SP-D and KL-6 were both estimated in SSc-ILD peripheral blood compared with healthy controls, at an SMD of 1.78 (95% CI 1.50 to 2.17; I2=8%) and 1.66 (95% CI 1.17 to 2.14; I2=76%), respectively. CONCLUSION: We provide robust evidence that KL-6, SP-D and IL-8 have the potential to serve as reliable biomarkers in blood/BALF for supporting the diagnosis of SSc-ILD. However, while several other biomarkers have been proposed, the evidence of their independent value in diagnosis and prognosis is currently lacking and needs further investigation. PROSPERO REGISTRATION NUMBER: CRD42021282452.


Assuntos
Doenças Pulmonares Intersticiais , Escleroderma Sistêmico , Adulto , Humanos , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/etiologia , Interleucina-8 , Proteína D Associada a Surfactante Pulmonar , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/diagnóstico , Biomarcadores , Pulmão
2.
Proc Natl Acad Sci U S A ; 113(26): E3725-34, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27286825

RESUMO

Cystic fibrosis (CF) lung disease is characterized by chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in the cystic fibrosis transmembrane conductance regulator, there is still an unmet need to also normalize the inflammatory response. The prolonged and heightened inflammatory response in CF is, in part, mediated by a lack of intrinsic down-regulation of the proinflammatory NF-κB pathway. We have previously identified reduced expression of the NF-κB down-regulator A20 in CF as a key target to normalize the inflammatory response. Here, we have used publicly available gene array expression data together with a statistically significant connections' map (sscMap) to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NF-κB(p65) expression (mRNA) as well as proinflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o-, CFBE41o-) and in primary nasal epithelial cells from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with tnfαip3 (A20) knockdown (siRNA). We also show that the A20-inducing effect of ikarugamycin and quercetin is lower in CF-derived airway epithelial cells than in non-CF cells.


Assuntos
Anti-Inflamatórios/farmacologia , Fibrose Cística/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/imunologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Lactamas/farmacologia , NF-kappa B/genética , NF-kappa B/imunologia , Quercetina/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Transcriptoma , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia
3.
Med Princ Pract ; 24(4): 301-10, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25925366

RESUMO

Cystic fibrosis (CF) is a lifelong, inflammatory multi-organ disease and the most common lethal, genetic condition in Caucasian populations, with a median survival rate of 41.5 years. Pulmonary disease, characterized by infective exacerbations, bronchiectasis and increasing airway insufficiency is the most serious manifestation of this disease process, currently responsible for over 80% of CF deaths. Chronic dysregulation of the innate immune and host inflammatory response has been proposed as a mechanism central to this genetic condition, primarily driven by the nuclear factor κB (NF-κB) pathway. Chronic activation of this transcription factor complex leads to the production of pro-inflammatory cytokines and mediators such as IL-6, IL-8 and TNF-α. A20 has been described as a central and inducible negative regulator of NF-κB. This intracellular molecule negatively regulates NF-κB-driven pro-inflammatory signalling upon toll-like receptor activation at the level of TRAF6 activation. Silencing of A20 increases cellular levels of p65 and induces a pro-inflammatory state. We have previously shown that A20 expression positively correlates with lung function (FEV1%) in CF. Despite improvement in survival rates in recent years, advancements in available therapies have been incremental. We demonstrate that the experimental use of naturally occurring plant diterpenes such as gibberellin on lipopolysaccharide-stimulated cell lines reduces IL-8 release in an A20-dependent manner. We discuss how the use of a novel bio-informatics gene expression connectivity-mapping technique to identify small molecule compounds that similarly mimic the action of A20 may lead to the development of new therapeutic approaches capable of reducing chronic airway inflammation in CF.


Assuntos
Fibrose Cística/fisiopatologia , Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , NF-kappa B/metabolismo , Técnicas de Cultura de Células , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Humanos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fenótipo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/biossíntese
5.
Am J Physiol Lung Cell Mol Physiol ; 304(5): L371-82, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23316065

RESUMO

The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF.


Assuntos
Brônquios/imunologia , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/imunologia , Receptor 4 Toll-Like/metabolismo , Brônquios/citologia , Linhagem Celular , Fibrose Cística/patologia , Endossomos/metabolismo , Humanos , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Infecções por Pseudomonas/imunologia , Mucosa Respiratória/citologia , Proteínas de Transporte Vesicular/biossíntese , Proteínas rab de Ligação ao GTP/biossíntese , proteínas de unión al GTP Rab7
6.
Eur Respir J ; 41(6): 1315-23, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23018911

RESUMO

A20 is a lipopolysaccharide (LPS)-inducible, cytoplasmic zinc finger protein, which inhibits Toll-like receptor-activated nuclear factor (NF)-κB signalling by deubiquitinating tumour necrosis factor receptor-associated factor (TRAF)-6. The action of A20 is facilitated by complex formation with ring finger protein (RNF)-11, Itch and TAX-1 binding protein-1 (TAX1BP1). This study investigated whether the expression of A20 is altered in the chronically inflamed cystic fibrosis (CF) airway epithelium. Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-κB subunits were analysed. Formation of the A20 ubiquitin editing complex was also investigated. In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-. The expression of A20 is significantly altered in CF, and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-κB-driven inflammation.


Assuntos
Fibrose Cística/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Brônquios/citologia , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Citometria de Fluxo , Humanos , Inflamação , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
7.
Mol Neurobiol ; 60(7): 3963-3978, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37004607

RESUMO

Post-traumatic stress disorder (PTSD), gaining increasing attention, is a multifaceted psychiatric disorder that occurs following a stressful or traumatic event or series of events. Recently, several studies showed a close relationship between PTSD and neuroinflammation. Neuroinflammation, a defense response of the nervous system, is associated with the activation of neuroimmune cells such as microglia and astrocytes and with changes in inflammatory markers. In this review, we first analyzed the relationship between neuroinflammation and PTSD: the effect of stress-derived activation of the hypothalamic-pituitary-adrenal (HPA) axis on the main immune cells in the brain and the effect of stimulated immune cells in the brain on the HPA axis. We then summarize the alteration of inflammatory markers in brain regions related to PTSD. Astrocytes are neural parenchymal cells that protect neurons by regulating the ionic microenvironment around neurons. Microglia are macrophages of the brain that coordinate the immunological response. Recent studies on these two cell types provided new insight into neuroinflammation in PTSD. These contribute to promoting comprehension of neuroinflammation, which plays a pivotal role in the pathogenesis of PTSD.


Assuntos
Transtornos de Estresse Pós-Traumáticos , Humanos , Transtornos de Estresse Pós-Traumáticos/metabolismo , Doenças Neuroinflamatórias , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Encéfalo/metabolismo
8.
Eur Respir J ; 39(6): 1377-84, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22088966

RESUMO

Epidemiological evidence supports a positive relationship between fruit and vegetable (FV) intake, lung function and chronic obstructive pulmonary disease (COPD). Increasing FV intake may attenuate the oxidative stress and inflammation associated with COPD. An exploratory randomised controlled trial to examine the effect of increased consumption of FV on oxidative stress and inflammation in moderate-to-severe COPD was conducted. 81 symptomatically stable patients with a habitually low FV intake (two or fewer portions of FV per day) were randomised to the intervention group (five or more portions of FV per day) or the control group (two or fewer portions of FV per day). Each participant received self-selected weekly home deliveries of FV for 12 weeks. 75 participants completed the intervention. There was a significant between-group change in self-reported FV intake and biomarkers of FV intake (zeaxanthin (p = 0.034) and ß-cryptoxanthin (p = 0.015)), indicating good compliance; post-intervention intakes in intervention and control groups were 6.1 and 1.9 portions of FV per day, respectively. There were no significant changes in biomarkers of airway inflammation (interleukin-8 and myeloperoxidase) and systemic inflammation (C-reactive protein) or airway and systemic oxidative stress (8-isoprostane). This exploratory study demonstrated that patients with moderate-to-severe COPD were able to comply with an intervention to increase FV intake; however, this had no significant effect on airway or systemic oxidative stress and inflammation.


Assuntos
Dieta , Comportamento Alimentar , Frutas , Inflamação/reabilitação , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/reabilitação , Verduras , Idoso , Idoso de 80 Anos ou mais , Anticarcinógenos/sangue , Biomarcadores/sangue , Proteína C-Reativa/análise , Criptoxantinas , Dinoprosta/análogos & derivados , Dinoprosta/urina , Feminino , Humanos , Inflamação/fisiopatologia , Interleucina-8/sangue , Masculino , Pessoa de Meia-Idade , Cooperação do Paciente , Peroxidase/sangue , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Índice de Gravidade de Doença , Escarro/química , Xantofilas/sangue , Zeaxantinas
9.
Nat Commun ; 13(1): 6358, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289219

RESUMO

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-ß induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.


Assuntos
Receptores de Adiponectina , Escleroderma Sistêmico , Animais , Camundongos , Bleomicina , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose , Camundongos Knockout , Receptores de Adiponectina/metabolismo , Escleroderma Sistêmico/metabolismo , Transdução de Sinais/genética , Pele/patologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinas/metabolismo
10.
Am J Respir Cell Mol Biol ; 44(6): 743-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21239605

RESUMO

Persistent activation of NF-κB is central to the pathogenesis of many inflammatory lung disorders, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in autoimmune and inflammatory disorders, including diabetes and Crohn's disease, but which has received little attention in terms of chronic lung disorders. This review examines the existing body of research on A20 regulation of NF-κB signaling and details the mechanism and regulation of A20 action focusing, where possible, on pulmonary inflammation. A20 and its associated signaling molecules are highlighted as being of potential therapeutic interest for the treatment of inflammatory disorders, and a proposed model of A20 activity in inflammatory lung disease is provided.


Assuntos
Quimiocina CCL20/metabolismo , Regulação da Expressão Gênica , Pneumopatias/metabolismo , NF-kappa B/metabolismo , Animais , Doença Crônica , Células Endoteliais/citologia , Humanos , Inflamação , Pulmão/metabolismo , Pneumopatias/microbiologia , Camundongos , Modelos Biológicos , Estrutura Terciária de Proteína , Transdução de Sinais
11.
J Cyst Fibros ; 20(4): 682-691, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34112603

RESUMO

BACKGROUND: In Cystic Fibrosis (CF) airways, the dehydrated, thick mucus promotes the establishment of persistent polymicrobial infections and drives chronic airways inflammation. This also predisposes the airways to further infections, the vicious, self-perpetuating cycle causing lung damage and progressive lung function decline. The airways are a poly-microbial environment, containing both aerobic and anaerobic bacterial species. Pseudomonas aeruginosa (P. aeruginosa) infections contribute to the excessive inflammatory response in CF, but the role of anaerobic Prevotella spp., frequently found in CF airways, is not known. MATERIALS: We assessed innate immune signalling in CF airway epithelial cells in response to clinical strains of P. histicola, P. nigresens and P. aeruginosa. CFBE41o- cells were infected with P. aeruginosa (MOI 100, 2h) followed by infection with P. histicola or P. nigrescens (MOI 100, 2h). Cells were incubated under anaerobic conditions for the duration of the experiments. RESULTS: Our study shows that P. histicola and P. nigresens can reduce the growth of P. aeruginosa and dampen the inflammatory response in airway epithelial cells. We specifically illustrate that the presence of the investigated Prevotella spp. reduces Toll-like-receptor (TLR)-4, MAPK, NF-κB(p65) signalling and cytokine release (Interleukin (IL)-6, IL-8) in mixed infections. CONCLUSION: Our work, for the first time, strongly indicates a relationship between P. aeruginosa and anaerobic Prevotella spp.. The observed modified NF-κB and MAPK signalling indicates some mechanisms underlying this interaction that could offer a novel therapeutic approach to combat chronic P. aeruginosa infection in people with CF.


Assuntos
Brônquios/citologia , Brônquios/microbiologia , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Células Epiteliais/imunologia , Inflamação/etiologia , Inflamação/microbiologia , Prevotella/fisiologia , Infecções por Pseudomonas/etiologia , Pseudomonas aeruginosa/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/microbiologia , Células Cultivadas , Fibrose Cística/imunologia , Humanos
12.
PLoS One ; 15(10): e0235803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031374

RESUMO

Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.


Assuntos
Brônquios/imunologia , Fibrose Cística/imunologia , NF-kappa B/metabolismo , Prevotella/imunologia , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/imunologia , Receptores Toll-Like/metabolismo , Brônquios/metabolismo , Brônquios/microbiologia , Brônquios/patologia , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Interleucina-8/metabolismo , NF-kappa B/genética , Prevotella/isolamento & purificação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Transdução de Sinais , Receptores Toll-Like/imunologia
13.
FASEB J ; 21(3): 766-76, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17209128

RESUMO

Alpha-tocopherol (aT), the predominant form of vitamin E in mammals, is thought to prevent oxidation of polyunsaturated fatty acids. In the lung, aT is perceived to be accumulated in alveolar type II cells and secreted together with surfactant into the epithelial lining fluid. Conventionally, determination of aT and related compounds requires extraction with organic solvents. This study describes a new method to determine and image the distribution of aT and related compounds within cells and tissue sections using the light-scattering technique of Raman microscopy to enable high spatial as well as spectral resolution. This study compared the nondestructive analysis by Raman microscopy of vitamin E, in particular aT, in biological samples with data obtained using conventional HPLC analysis. Raman spectra were acquired at spatial resolutions of 2-0.8 microm. Multivariate analysis techniques were used for analyses and construction of corresponding maps showing the distribution of aT, alpha-tocopherol quinone (aTQ), and other constituents (hemes, proteins, DNA, and surfactant lipids). A combination of images enabled identification of colocalized constituents (heme/aTQ and aT/surfactant lipids). Our data demonstrate the ability of Raman microscopy to discriminate between different tocopherols and oxidation products in biological specimens without sample destruction. By enabling the visualization of lipid-protein interactions, Raman microscopy offers a novel method of investigating biological characterization of lipid-soluble compounds, including those that may be embedded in biological membranes such as aT.


Assuntos
Antioxidantes/análise , Pulmão/metabolismo , alfa-Tocoferol/análise , Antioxidantes/metabolismo , Antioxidantes/farmacocinética , Oxirredução , Análise Espectral Raman , Distribuição Tecidual , alfa-Tocoferol/metabolismo , alfa-Tocoferol/farmacocinética
15.
Lipids ; 41(2): 105-12, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17707975

RESUMO

The alpha-tocopherol transfer protein (TTP) plays an important role in the regulation of plasma alpha-tocopherol concentrations. We hypothesized that hepatic TTP levels would be modulated by dietary vitamin E supplementation and/or by oxidative stress. Mice were fed either a High E (1150 mg RRR-alpha-tocopheryl acetate/kg diet) or a Low E (11.5 mg/kg diet) diet for 2 wk. High E increased plasma and liver alpha-tocopherol concentrations approximately 8- and 40-fold, respectively, compared with Low E-fed mice, whereas hepatic TTP increased approximately 20%. Hepatic TTP concentrations were unaffected by fasting (24 h) in mice fed either diet. To induce oxidative stress, chow-fed mice were exposed for 3 d to environmental tobacco smoke (ETS) for 6 h/d (total suspended particulate, 57.4 +/- 1.8 mg/m3). ETS exposure, while resulting in pulmonary and systemic oxidative stress, had no effect on hepatic alpha-tocopherol concentrations or hepatic TTP. Overall, changes in hepatic TTP concentrations were minimal in response to dietary vitamin E levels or ETS-related oxidative stress. Thus, hepatic TTP concentrations may be at sufficient levels such that they are unaffected by either modulations of dietary vitamin E or by the conditions of environmentally related oxidative stress used in the present studies.


Assuntos
Proteínas de Transporte/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Vitamina E/administração & dosagem , Animais , Peso Corporal/efeitos dos fármacos , Dieta , Ingestão de Alimentos/efeitos dos fármacos , Jejum/fisiologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poluição por Fumaça de Tabaco , Vitamina E/sangue
16.
Antioxid Redox Signal ; 7(1-2): 25-31, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15650393

RESUMO

Lipid oxidation and environmental pollutants are major sources of alpha,beta-unsaturated aldehydes such as acrolein and 4-hydroxynonenal. Acrolein (2-propenal), a major product of organic combustion such as tobacco smoke, represents the most reactive alpha,beta-unsaturated aldehyde, with high reactivity toward nucleophilic targets such as sulfhydryl groups. To investigate how acrolein affects respiratory tract cell activation, we exposed either primary (NHBE) or immortalized human bronchial epithelial cells (HBE1) to 0-25 microM acrolein, and determined effects on basal and tumor necrosis factor-alpha (TNFalpha)-induced production of the chemokine interleukin (IL)-8. Cell exposure to acrolein dose-dependently suppressed IL-8 mRNA levels in HBE1 cells (26, 40, and 79% at 5, 10, and 25 microM acrolein concentrations, respectively) and resulted in corresponding decreases in IL-8 production. Studies of nuclear factor-kappaB (NFkappaB) activation, an essential event in IL-8 production, showed decreased TNFalpha-induced NFkappaB activation by acrolein, illustrated by inhibition of nuclear translocation of NFkappaB and reduced IkappaBalpha degradation. Immunochemical analysis of IkappaB kinase (IKK), a redox-sensitive regulator of NFkappaB activation, indicated direct modification of the IKK beta-subunit by acrolein, suggesting that acrolein may act directly on IKK. In summary, our results demonstrate that acrolein can suppress inflammatory processes in the airways by inhibiting epithelial IL-8 production through direct or indirect inhibitory effects on NFkappaB activation.


Assuntos
Acroleína/farmacologia , Brônquios/citologia , Células Epiteliais/citologia , Interleucina-8/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Western Blotting , Brônquios/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Humanos , Quinase I-kappa B , Imuno-Histoquímica , Imunoprecipitação , Inflamação , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Sistema Respiratório/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/metabolismo
18.
Free Radic Biol Med ; 35(11): 1343-54, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-14642382

RESUMO

Alpha-tocopherol transfer protein (TTP) regulates the retention and secretion of alpha-tocopherol (alpha-T) by the liver. Deletion of the TTP gene (Ttpa) in mice results in systemic deficiency of alpha-T and neurological dysfunctions described in patients with mutated Ttpa. We have explored genome-wide changes in mRNAs from brain cortex and liver of Ttpa-deficient (Ttpa(-/-)) mice and wild-type (Ttpa(+/+)) mice. Selective inductions of genes regulated by antioxidant response elements were detected in Ttpa(-/-) livers compared to Ttpa(+/+) livers, suggesting increased oxidant stress in Ttpa(-/-) livers. The activation of cell proliferation pathways in Ttpa(-/-) livers was indicated by the induction of genes that encode growth factor-binding proteins, mitogen-activated protein kinase kinase 3, and apoptosis inhibitor 6. The induction of synuclein-alpha and repression of synuclein-beta genes was detected in Ttpa(-/-) cortex. This may predispose Ttpa(-/-) cortex to increased formation of synuclein-alpha aggregates and Lewy body, often associated with oxidant stress. Cortex of Ttpa(-/-) mice revealed repression of genes encoding synaptic proteins, protein kinase C family members, and myelin proteins. A 13-fold decrease in the expression of retinoic acid receptor-related orphan receptor-alpha mRNA predicts staggerer-like phenotype (ataxia and deficits of motor coordination) of Ttpa(-/-) mice. The repression of specific genes that determine synaptic plasticity and neuronal development may account for suppressed electrophysiological activities of cortex and impaired behavior in Ttpa(-/-) mice.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Doenças Neurodegenerativas/metabolismo , Oxidantes , Animais , Encéfalo/metabolismo , Divisão Celular , DNA/metabolismo , Feminino , Deleção de Genes , Fígado/metabolismo , MAP Quinase Quinase 3 , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Bainha de Mielina/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/metabolismo , Transativadores , Fatores de Transcrição
19.
Free Radic Biol Med ; 37(9): 1393-401, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15454278

RESUMO

Ascorbic acid (AA) is thought to be an important antioxidant in the respiratory tract, whose regulation is yet to be fully characterized. We investigated whether AA in respiratory tract lining fluids (RTLFs) can be augmented by oral supplementation with AA. Plasma, nasal lavage fluids (NLFs), induced sputum (IS), and saliva were analyzed for AA immediately before and 2 h after ingestion of 2 g of AA in 13 healthy subjects. Concentrations of AA (median and range) were 52.5 (16.0-88.5), 2.4 (0.18-4.66), 2.4 (0.18-6.00), and 0.55 (0.18-18.90) micromol/l, respectively. Two hours after ingestion of AA, plasma AA increased 2-fold (p = .004), NLF AA increased 3-fold (p = .039), but IS and saliva AA did not increase. As AA concentrations in saliva and tracheobronchial secretions were low compared with other common extracellular components (such as urate), we evaluated the fate of AA in these fluids. Addition of AA to freshly obtained saliva or IS resulted in rapid depletion, which could be largely prevented or reversed by sodium azide or dithiothreitol. These findings suggest that oxidant-producing systems in saliva and airway secretions, such as heme peroxidases and other oxidizing substances, rapidly consume AA. Whereas oral supplementation resulted in detectable increases of AA in NLFs, its levels in tracheobronchial lining fluid, as measured by IS, were unaffected and remained relatively low, suggesting that AA may play a less significant antioxidant role in this compartment as compared with most other extracellular compartments.


Assuntos
Ácido Ascórbico/análise , Mucosa Nasal/química , Saliva/química , Escarro/química , Administração Oral , Adulto , Idoso , Antioxidantes/análise , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/sangue , Ácido Ascórbico/farmacologia , Brônquios/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Irrigação Terapêutica , Traqueia/química
20.
Free Radic Biol Med ; 35(12): 1560-7, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-14680679

RESUMO

We hypothesized that the high concentrations of reactive nitrogen species in cigarette smoke and the known stimulatory effects of cigarette smoke on the inflammatory immune systems would lead to the formation of 5-nitro-gamma-tocopherol (NGT). In order to assess gamma-tocopherol nitration, human plasma was exposed in vitro to gas phase cigarette smoke (GPCS) or air for up to 6 h. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantitate NGT. Detector response was linear from 0.1 to 3 pmol NGT, with a detection limit of 20 fmol. After a 1 h lag time, 6 h plasma exposure to GPCS depleted approximately 75% of alpha-T, approximately 60% of gamma-T and increased NGT from 3 to 134 nmol/l. The increase in NGT accounted for approximately 20% of the gamma-T decrease. NGT also correlated (R2 = 0.9043) with nitrate concentrations in GPCS-exposed plasma. The physiologic relevance of NGT was evaluated in a group of healthy humans. Smokers (n = 15) had plasma NGT concentrations double those of nonsmokers (n = 19), regardless of corrections using lipids or gamma-T; plasma alpha-T and gamma-T concentrations were similar between the groups. Our results show that LC-MS can be successfully used for NGT quantitation in biologic samples. Importantly, NGT in smokers' plasma suggests that cigarette smoking causes increased nitrosative stress.


Assuntos
Fumar/sangue , gama-Tocoferol/análogos & derivados , gama-Tocoferol/sangue , Humanos , Técnicas In Vitro , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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