Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 33
Filtrar
1.
PLoS One ; 16(1): e0244173, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33411744

RESUMEN

The novel coronavirus (COVID-19) is an emergent disease that initially had no historical data to guide scientists on predicting/ forecasting its global or national impact over time. The ability to predict the progress of this pandemic has been crucial for decision making aimed at fighting this pandemic and controlling its spread. In this work we considered four different statistical/time series models that are readily available from the 'forecast' package in R. We performed novel applications with these models, forecasting the number of infected cases (confirmed cases and similarly the number of deaths and recovery) along with the corresponding 90% prediction interval to estimate uncertainty around pointwise forecasts. Since the future may not repeat the past for this pandemic, no prediction model is certain. However, any prediction tool with acceptable prediction performance (or prediction error) could still be very useful for public-health planning to handle spread of the pandemic, and could policy decision-making and facilitate transition to normality. These four models were applied to publicly available data of the COVID-19 pandemic for both the USA and Italy. We observed that all models reasonably predicted the future numbers of confirmed cases, deaths, and recoveries of COVID-19. However, for the majority of the analyses, the time series model with autoregressive integrated moving average (ARIMA) and cubic smoothing spline models both had smaller prediction errors and narrower prediction intervals, compared to the Holt and Trigonometric Exponential smoothing state space model with Box-Cox transformation (TBATS) models. Therefore, the former two models were preferable to the latter models. Given similarities in performance of the models in the USA and Italy, the corresponding prediction tools can be applied to other countries grappling with the COVID-19 pandemic, and to any pandemics that can occur in future.


Asunto(s)
/epidemiología , Predicción/métodos , Modelos Biológicos , /mortalidad , Control de Enfermedades Transmisibles , Simulación por Computador , Toma de Decisiones , Humanos , Italia/epidemiología , Estados Unidos/epidemiología
2.
JMIR Med Inform ; 8(12): e23530, 2020 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-33325834

RESUMEN

BACKGROUND: Despite steady gains in life expectancy, individuals with cystic fibrosis (CF) lung disease still experience rapid pulmonary decline throughout their clinical course, which can ultimately end in respiratory failure. Point-of-care tools for accurate and timely information regarding the risk of rapid decline is essential for clinical decision support. OBJECTIVE: This study aims to translate a novel algorithm for earlier, more accurate prediction of rapid lung function decline in patients with CF into an interactive web-based application that can be integrated within electronic health record systems, via collaborative development with clinicians. METHODS: Longitudinal clinical history, lung function measurements, and time-invariant characteristics were obtained for 30,879 patients with CF who were followed in the US Cystic Fibrosis Foundation Patient Registry (2003-2015). We iteratively developed the application using the R Shiny framework and by conducting a qualitative study with care provider focus groups (N=17). RESULTS: A clinical conceptual model and 4 themes were identified through coded feedback from application users: (1) ambiguity in rapid decline, (2) clinical utility, (3) clinical significance, and (4) specific suggested revisions. These themes were used to revise our application to the currently released version, available online for exploration. This study has advanced the application's potential prognostic utility for monitoring individuals with CF lung disease. Further application development will incorporate additional clinical characteristics requested by the users and also a more modular layout that can be useful for care provider and family interactions. CONCLUSIONS: Our framework for creating an interactive and visual analytics platform enables generalized development of applications to synthesize, model, and translate electronic health data, thereby enhancing clinical decision support and improving care and health outcomes for chronic diseases and disorders. A prospective implementation study is necessary to evaluate this tool's effectiveness regarding increased communication, enhanced shared decision-making, and improved clinical outcomes for patients with CF.

3.
J Cyst Fibros ; 19(4): 632-640, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-31870630

RESUMEN

BACKGROUND: Circulating biomarkers reflective of lung disease activity and severity have the potential to improve patient care and accelerate drug development in CF. The objective of this study was to leverage banked specimens to test the hypothesis that blood-based biomarkers discriminate CF children segregated by lung disease severity. METHODS: Banked serum samples were selected from children who were categorized into two extremes of phenotype associated with lung function ('mild' or 'severe') based on CF-specific data and were matched on age, gender, CFTR genotype, and P. aeruginosa infection status. Targeted inflammatory proteins, lipids, and discovery metabolite profiles were measured in these serum samples. RESULTS: The severe cohort, characterized by a lower CF-specific FEV1 percentile, had significantly higher circulating concentrations of high sensitivity C-reactive protein, serum amyloid A, granulocyte colony stimulating factor, and calprotectin compared to the mild cohort. The mild cohort tended to have higher serum linoleic acid concentrations. The metabolite arabitol was lower in the severe cohort while other CF relevant metabolic pathways showed non-significant differences after adjusting for multiple comparisons. A sensitivity analysis to correct for biased estimates that may result from selecting subjects using an extremes of phenotype approach confirmed the protein biomarker findings. CONCLUSIONS: Circulating inflammatory proteins differ in CF children segregated by lung function. These findings serve to demonstrate the value of maintaining centralized, high quality patient derived samples for future research, with linkage to clinical information to answer testable hypotheses in biomarker development.

4.
J Clin Invest ; 129(8): 3448-3463, 2019 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-31145101

RESUMEN

Cystic Fibrosis (CF) is a multi-organ progressive genetic disease caused by loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. Previously, we identified a significant dysfunction in CF cells and model mice of the transcription factor nuclear-factor-E2-related factor-2 (Nrf2), a major regulator of redox balance and inflammatory signaling. Here we report that approved F508del CFTR correctors VX809/VX661 recover diminished Nrf2 function and colocalization with CFTR in CF human primary bronchial epithelia by proximity ligation assay, immunoprecipitation, and immunofluorescence, concordant with CFTR correction. F508del CFTR correctors induced Nrf2 nuclear translocation, Nrf2-dependent luciferase activity, and transcriptional activation of target genes. Rescue of Nrf2 function by VX809/VX661 was dependent on significant correction of F508del and was blocked by inhibition of corrected channel function, or high-level shRNA knockdown of CFTR or F508del-CFTR. Mechanistically, F508del-CFTR modulation restored Nrf2 phosphorylation and its interaction with the coactivator CBP. Our findings demonstrate that sufficient modulation of F508del CFTR function corrects Nrf2 dysfunction in CF.


Asunto(s)
Núcleo Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Mucosa Respiratoria/metabolismo , Transporte Activo de Núcleo Celular/genética , Animales , Línea Celular , Núcleo Celular/genética , Núcleo Celular/patología , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/patología , Humanos , Ratones , Ratones Transgénicos , Mutación , Factor 2 Relacionado con NF-E2/genética , Fosforilación/genética , Mucosa Respiratoria/patología
5.
Neuroscience ; 329: 264-74, 2016 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-27180285

RESUMEN

Evidence suggests that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, may reduce the risk of Alzheimer's disease (AD). Statin action in patients with AD, as in those with heart disease, is likely to be at least partly independent of the effects of statins on cholesterol. Statins can alter cellular signaling and protein trafficking through inhibition of isoprenylation of Rho, Cdc42, and Rab family GTPases. The effects of statins on protein isoprenylation in vivo, particularly in the central nervous system, are poorly studied. We utilized two-dimensional gel electrophoresis approaches to directly monitor the levels of isoprenylated and non-isoprenylated forms of Rho and Rab family GTPases. We report that simvastatin significantly inhibits RhoA and Rab4, and Rab6 isoprenylation at doses as low as 50nM in vitro. We also provide the first in vivo evidence that statins inhibit the isoprenylation of RhoA in the brains of rats and RhoA, Cdc42, and H-Ras in the brains of mice treated with clinically relevant doses of simvastatin.


Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Fármacos del Sistema Nervioso Central/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Prenilación de Proteína/efectos de los fármacos , Simvastatina/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Electroforesis en Gel Bidimensional , Focalización Isoeléctrica , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas Endogámicas SHR , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rhoA/metabolismo
6.
J Cyst Fibros ; 15(6): 714-723, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-28215711

RESUMEN

PURPOSE: To provide a review of the status of biomarkers in cystic fibrosis drug development, including regulatory definitions and considerations, a summary of biomarkers in current use with supportive data, current gaps, and future needs. METHODS: Biomarkers are considered across several areas of CF drug development, including cystic fibrosis transmembrane conductance regulator modulation, infection, and inflammation. RESULTS: Sweat chloride, nasal potential difference, and intestinal current measurements have been standardized and examined in the context of multicenter trials to quantify CFTR function. Detection and quantification of pathogenic bacteria in CF respiratory cultures (e.g.: Pseudomonas aeruginosa) are commonly used in early phase antimicrobial clinical trials, and to monitor safety of therapeutic interventions. Sputum (e.g.: neutrophil elastase, myeloperoxidase, calprotectin) and blood biomarkers (e.g.: C reactive protein, calprotectin, serum amyloid A) have had variable success in detecting response to inflammatory treatments. CONCLUSIONS: Biomarkers are used throughout the drug development process in CF, and many have been used in early phase clinical trials to provide proof of concept, detect drug bioactivity, and inform dosing for later-phase studies. Advances in the precision of current biomarkers, and the identification of new biomarkers with 'omics-based technologies, are needed to accelerate CF drug development.


Asunto(s)
Biomarcadores/análisis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística , Descubrimiento de Drogas/métodos , Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Humanos
7.
J Gen Virol ; 96(9): 2543-2556, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26297201

RESUMEN

Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.


Asunto(s)
Células Epiteliales/metabolismo , Receptores de Quimiocina/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Secuencias de Aminoácidos , Receptor 1 de Quimiocinas CX3C , Línea Celular , Células Epiteliales/virología , Humanos , Unión Proteica , Receptores de Quimiocina/química , Receptores de Quimiocina/genética , Infecciones por Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Sistema Respiratorio/citología , Sistema Respiratorio/metabolismo , Sistema Respiratorio/virología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo
8.
Chembiochem ; 16(14): 2017-22, 2015 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-26227551

RESUMEN

Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX-809 has been reported to facilitate the folding and trafficking of F508del-CFTR and augment its channel function. The mechanism of action of VX-809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX-809: does it bind CFTR directly in order to exert its action? We synthesized two VX-809 derivatives, ALK-809 and SUL-809, that possess an alkyne group and retain the rescue capacity of VX-809. By using Cu(I) -catalyzed click chemistry, we provide evidence that the VX-809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.


Asunto(s)
Aminopiridinas/química , Aminopiridinas/farmacología , Benzodioxoles/química , Benzodioxoles/farmacología , Química Clic , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Aminopiridinas/síntesis química , Benzodioxoles/síntesis química , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Descubrimiento de Drogas , Células HEK293 , Humanos , Mutación , Unión Proteica
9.
Cell Signal ; 27(7): 1345-55, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25841995

RESUMEN

Multidrug resistance protein 4 (MRP4), a member of the ATP binding cassette transporter family, functions as a plasma membrane exporter of cyclic nucleotides. Recently, we demonstrated that fibroblasts lacking the Mrp4 gene migrate faster and contain higher cyclic-nucleotide levels. Here, we show that cAMP accumulation and protein kinase A (PKA) activity are higher and polarized in Mrp4(-/-) fibroblasts, versus Mrp4(+/+) cells. MRP4-containing macromolecular complexes isolated from these fibroblasts contained several proteins, including actin, which play important roles in cell migration. We found that actin interacts with MRP4, predominantly at the plasma membrane, and an intact actin cytoskeleton is required to restrict MRP4 to specific microdomains of the plasma membrane. Our data further indicated that the enhanced accumulation of cAMP in Mrp4(-/-) fibroblasts facilitates cortical actin polymerization in a PKA-dependent manner at the leading edge, which in turn increases the overall rate of cell migration to accelerate the process of wound healing. Disruption of actin polymerization or inhibition of PKA activity abolished the effect of MRP4 on cell migration. Together, our findings suggest a novel cAMP-dependent mechanism for MRP4-mediated regulation of fibroblast migration whereby PKA and actin play critical roles as downstream effectors.


Asunto(s)
Actinas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/química , Animales , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Microscopía Fluorescente , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Células 3T3 NIH , Propionatos/toxicidad , Unión Proteica , Quinolinas/toxicidad
10.
Int J Biochem Cell Biol ; 52: 113-23, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24657650

RESUMEN

The homeostatic balance between oxidants and antioxidants in biological systems is known as redox balance, and is regulated by complex processes. Redox balance regulates many of the known cellular pathways and disease processes. The dysregulation of redox balance can lead to acute or long-term oxidative or reductive stresses that are associated with many of the abnormalities observed in cystic fibrosis (CF). Over the past 5 decades researchers have examined contributors to redox dysregulation, their molecular products, and their impact on ion transport, cell proliferation, inflammation, bacterial killing, and the metabolism of nucleic acids, proteins, and lipids in CF. CF patients exhibit elevated markers of oxidative stress when compared to non-CF healthy controls; however, whether the reported redox imbalance is sufficient to produce pathology has been controversial. In addition, comparisons between CF and non-CF disease controls have been lacking. To better understand the mechanisms which mediate the generation of oxidants and antioxidants in CF and the importance of their balance in effecting oxidative or reductive stress, we will review the determinants of redox balance in the blood, lumen, and cellular compartments. From the perspective of methodological application, we will focus on the approaches most often used to study oxidant and antioxidants in CF, including biochemical, proteomic, metabolomic, and lipidomic studies, with a discussion of the few transcriptomic analyses that predict changes in the expression of regulators of redox. Finally, we will discuss the utility of oxidants and antioxidants as biomarkers of disease and the use of antioxidant therapy in CF.


Asunto(s)
Fibrosis Quística/metabolismo , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Hemostasis , Humanos , Oxidación-Reducción , Estrés Oxidativo/fisiología
11.
Small ; 10(9): 1799-804, 2014 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-24515950

RESUMEN

A photoprecursor Pc 227 is covalently bound onto gold nanoparticles (Au NPs) to produce the known photodynamic therapy (PDT) drug Pc 4 upon 660 nm photoirradiation. The photochemical formation of the photoproduct Pc 4 is identified by spectroscopy, chromatography, and mass spectrometry and its PDT efficacy is equal to Pc 4 when administered non-covalently by Au NPs, with the added benefit of improved covalent delivery and targeted NIR-triggered release from the covalent Pc 227-Au NP conjugate, while during transport the attached Pc 227 is quenched by the Au NP and PDT inactivated.


Asunto(s)
Portadores de Fármacos/química , Oro/química , Indoles/farmacología , Rayos Infrarrojos , Nanopartículas del Metal/química , Fotoquimioterapia , Células HeLa , Humanos , Nanopartículas del Metal/ultraestructura , Preparaciones Farmacéuticas , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray
12.
Am J Physiol Lung Cell Mol Physiol ; 302(11): L1221-31, 2012 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-22467641

RESUMEN

Cystic fibrosis (CF) is characterized by inflammatory lung disease that significantly contributes to morbidity and mortality. Airway epithelial cells play a role in the inflammatory signaling in CF and have been reported to exhibit a number of dysfunctions in signaling cascades that modulate inflammation. Previously, we reported that the activity of nuclear factor erythroid-derived-like 2 (Nrf2), a transcription factor that regulates antioxidant and cytoprotective protein expression, is diminished in CF epithelia (7). In this report, we examined the mechanism of Nrf2 dysregulation in vitro in human airway epithelial cell lines and primary cells and in vivo in nasal epithelia excised from ΔF508 CF mutant mice. We found that cAMP-mediated signaling markedly reduces Nrf2 activity in CF vs. non-CF cells. Rp-cAMPS, a cAMP competitor, significantly corrected Nrf2 activity in CF cells, predominantly by increasing the nuclear accumulation of the transcription factor. Furthermore, we found that Rp-cAMPS significantly decreased NF-κB activation following inflammatory stimulation of CF cells. Further investigation revealed that Nrf2 and NF-κB compete for the transcriptional coactivator cAMP responsive element-binding protein (CREB) binding protein (CBP) and that Rp-cAMPS shifts CBP association in favor of Nrf2. Thus our findings provide a link between feedback to CF transmembrane regulator dysfunction and dysregulation of an inflammatory signaling pathway that modulates the coordinated activities of Nrf2 and NF-κB. Furthermore, our studies suggest that strategies that shift CBP association away from NF-κB and toward Nrf2 could have potential therapeutic efficacy for reducing inflammation in patients with CF.


Asunto(s)
Proteína de Unión a CREB/metabolismo , Fibrosis Quística/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Mucosa Respiratoria/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Línea Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Inflamación/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Tionucleótidos/farmacología
13.
Annu Rev Pathol ; 7: 267-82, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22017581

RESUMEN

Cystic fibrosis (CF), a lethal genetic disease, is characterized by substantial clinical heterogeneity. Work over the past decade has established that much of the variation is genetically conferred, and recent studies have begun to identify chromosomal locations that identify specific genes as contributing to this variation. Transcriptomic and proteomic data, sampling hundreds and thousands of genes and their products, point to pathways that are altered in the cells and tissues of CF patients. Genetic studies have examined more than half a million polymorphic sites and have identified regions, and probably genes, that contribute to the clinical heterogeneity. The combination of these approaches has great potential because genetic profiling identifies putative disease-modifying processes, and transcript and protein profiling is shedding light on the biology involved. Such studies are providing new insights into the disease, such as altered apoptotic responses, oxidative stress dysregulation, and neuronal involvement, all of which may open new therapeutic avenues to exploration.


Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos
14.
J Leukoc Biol ; 91(2): 311-20, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22158781

RESUMEN

Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5ß1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.


Asunto(s)
Proteínas Bacterianas/fisiología , Linfocitos T CD4-Positivos/inmunología , Integrina alfa5beta1/fisiología , Activación de Linfocitos/inmunología , Mycobacterium tuberculosis/fisiología , Proteínas Bacterianas/química , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Humanos , Memoria Inmunológica , Integrina alfa5/química , Integrina alfa5beta1/química , Interferón gamma/biosíntesis , Interferón gamma/genética , Interferón gamma/metabolismo , Oligopéptidos , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba
15.
Methods Mol Biol ; 742: 51-76, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21547726

RESUMEN

Cystic fibrosis is characterized by excessive pulmonary inflammation, which presents early in life and becomes self-sustaining, eventually leading to the destruction of the lung. Treating inflammation is one of the most pressing needs in CF therapy and has been shown to slow lung function deterioration. However, it remains unclear whether excessive inflammation is a direct result of CFTR dysfunction, and thus innate, or develops in response to early stimulation of inflammatory pathways. Here, we will discuss clinically relevant studies and the methods employed by them. We will focus on investigations in cell and animal models as well as patients. Our discussion will describe the character of pulmonary inflammation in CF and present potential therapeutic approaches that can ameliorate excessive responses and improve disease prognosis.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/fisiopatología , Inflamación/fisiopatología , Animales , Antiinflamatorios/uso terapéutico , Western Blotting , Técnicas de Cultivo de Célula , Fibrosis Quística/complicaciones , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/inmunología , Fibrosis Quística/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/metabolismo , Lipopolisacáridos/inmunología , Pulmón/metabolismo , Pulmón/fisiopatología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Pseudomonas aeruginosa/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Mol Imaging ; 10(5): 327-39, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21521549

RESUMEN

In this study, we used bioluminescence imaging (BLI) to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA) combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol) form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs) into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.


Asunto(s)
Encéfalo/fisiología , ADN/administración & dosificación , Técnicas de Transferencia de Gen , Mediciones Luminiscentes/métodos , Nanopartículas/administración & dosificación , Análisis de Varianza , Animales , Encéfalo/metabolismo , Química Encefálica , Vectores Genéticos , Histocitoquímica/métodos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Microinyecciones , Ratas , Ratas Sprague-Dawley , Transgenes
17.
Infect Immun ; 79(2): 663-73, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21078852

RESUMEN

The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4(+) T cells. In the absence of APCs, activation of memory CD4(+) T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4(+) T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4(+) T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Lipoproteínas/metabolismo , Activación de Linfocitos/fisiología , Mycobacterium tuberculosis/metabolismo , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Acilación , Adulto , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Memoria Inmunológica/fisiología , Lipoproteínas/genética , Lipoproteínas/inmunología , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Receptor Toll-Like 1/genética , Receptor Toll-Like 2/genética , Adulto Joven
18.
Mol Ther ; 19(1): 93-102, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20959809

RESUMEN

DNA nanoparticles (DNPs) are nonviral gene transfer vectors with excellent in vivo potential. Previously, we reported that cell surface nucleolin directly binds DNPs, and functions as an important receptor for DNPs. However, the fate of the nucleolin-DNP complex following cellular uptake remains elusive. In this study, we examined the role of lipid rafts in the uptake of DNPs, and found that both nucleolin and DNPs are recovered from the low-density raft fractions of the sucrose gradient. Furthermore, nucleolin colocalizes with, and coimmunoprecipitates with a raft protein, flotillin. Disruption of lipid rafts by depleting membrane cholesterol significantly inhibited DNP transfection, while inhibition of other endocytic pathways had little effect. Following the uptake, the nuclear import of the DNPs required microtubules but not F-actin. By coimmunoprecipitation in conjunction with tandem mass spectrometry, we identified glucocorticoid receptor (GCR) as a nucleolin-associated protein, and confirmed this result by western blot. Cortisone or dexamethasone increased nucleolin's association with GCR, and transfection by DNPs. Finally, we detected the expression of nucleolin on the surface of airway epithelia in vivo. Taken together, our findings shed light on important determinants of DNP trafficking in cells and support the notion that nucleolin is a good target for nonviral gene delivery.


Asunto(s)
ADN/administración & dosificación , Terapia Genética/métodos , Vectores Genéticos/metabolismo , Microdominios de Membrana/metabolismo , Microtúbulos/metabolismo , Nanopartículas/química , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Actinas/metabolismo , Animales , Western Blotting/métodos , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Cortisona/metabolismo , ADN/genética , ADN/metabolismo , Dexametasona/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Endocitosis/genética , Vectores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación/métodos , Microdominios de Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microtúbulos/genética , Unión Proteica , Receptores de Glucocorticoides/metabolismo , Sacarosa/metabolismo , Espectrometría de Masas en Tándem/métodos , Transfección/métodos , Células Tumorales Cultivadas
19.
Microsc Res Tech ; 73(9): 918-28, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20306536

RESUMEN

DNA nanoparticles (DNPs) formed by compacting DNA with polyethyleneglycolylated poly-L-lysine are a nonviral vector shown to be safe and efficacious in animals and humans. To extend our capabilities of assessing the efficacy and duration of expression achieved by DNPs, we tested the utility of bioluminescent imaging (BLI) of transgene expression in wildtype and cystic fibrosis (CF) mouse models. We tested the effect of route of administration, mouse coat color, anesthesia, dose, and promoter sequence on the level and duration of expression. Furthermore, we investigated the correlation between imaging and direct analysis of luciferase expression in lung homogenates. We found that intratracheal instillation, and the use of deep and prolonged anesthesia with avertin produced significantly higher expression compared with intranasal administration, and the use of lighter anesthesia with isoflurane. Although similar expression was observed for both dark and light coat animals, imaging signal intensity was attenuated in mice with dark fur. Furthermore, good correlation between imaging and direct homogenate analysis was observed for single dose (r = 0.96), and dose response studies in wildtype (r = 0.82) and CF mice (r = 0.87). Finally, we used imaging to track gene expression over a 56-day time course. We found that the human ubiquitin B promoter gives stable transgene expression up to 49 days following nanoparticle administration, while expression with the cytomegalovirus promoter diminished after 2 days and returned to background levels by day 14. Taken together, our results demonstrate that BLI is an effective and useful modality for measuring gene expression conferred by DNPs in the lung.


Asunto(s)
Expresión Génica , Técnicas de Transferencia de Gen , Genes Reporteros , Luciferasas/metabolismo , Pulmón/química , Nanopartículas/química , Polietilenglicoles/química , Animales , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , ADN/administración & dosificación , ADN/genética , ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen/instrumentación , Humanos , Luciferasas/administración & dosificación , Luciferasas/análisis , Luciferasas/genética , Sustancias Luminiscentes/química , Sustancias Luminiscentes/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos
20.
Am J Physiol Lung Cell Mol Physiol ; 297(5): L828-36, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19700644

RESUMEN

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-kappaB activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.


Asunto(s)
Fibrosis Quística/complicaciones , Fibrosis Quística/prevención & control , Inflamación/complicaciones , Inflamación/prevención & control , Ácido Oleanólico/análogos & derivados , Triterpenos/farmacología , Animales , Antioxidantes/metabolismo , Bronquios/citología , Lavado Broncoalveolar , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Flagelina/administración & dosificación , Flagelina/farmacología , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Ácido Oleanólico/administración & dosificación , Ácido Oleanólico/farmacología , Oxidación-Reducción/efectos de los fármacos , Proteómica , Tráquea/citología , Triterpenos/administración & dosificación
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA