RESUMEN
BACKGROUND: Rhinoviruses (RVs) are the most common trigger for asthma exacerbations, and there are currently no targeted therapies for viral-induced asthma exacerbations. RV infection causes neutrophilic inflammation, which is often resistant to effects of glucocorticoids. IL-1 receptor antagonist (IL-1RA) treatment reduces neutrophilic inflammation in humans challenged with inhaled endotoxin and thus may have therapeutic potential for RV-induced asthma exacerbations. OBJECTIVE: We sought to test the hypothesis that IL-1RA treatment of airway epithelium reduces RV-mediated proinflammatory cytokine production, which is important for neutrophil recruitment. METHODS: Human bronchial epithelial cells from deceased donors without prior pulmonary disease were cultured at air-liquid interface and treated with IL-13 to approximate an asthmatic inflammatory milieu. Human bronchial epithelial cells were infected with human RV-16 with or without IL-1RA treatment. RESULTS: RV infection promoted the release of IL-1α and the neutrophil-attractant cytokines IL-6, IL-8, and CXCL10. Proinflammatory cytokine secretion was significantly reduced by IL-1RA treatment without significant change in IFN-ß release or RV titer. In addition, IL-1RA reduced MUC5B expression after RV infection without impacting MUC5AC. CONCLUSIONS: These data suggest that IL-1RA treatment significantly reduced proinflammatory cytokines while preserving the antiviral response. These results provide evidence for further investigation of IL-1RA as a novel targeted therapy against neutrophil-attractant cytokine release in RV-induced airway inflammatory responses.
Asunto(s)
Asma , Infecciones por Enterovirus , Infecciones por Picornaviridae , Humanos , Rhinovirus/fisiología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Receptores de Interleucina-1 , Asma/tratamiento farmacológico , Citocinas/metabolismo , Epitelio/metabolismo , Células Epiteliales/metabolismo , Inflamación/tratamiento farmacológico , Infecciones por Picornaviridae/tratamiento farmacológicoRESUMEN
The steady increase in global temperatures, resulting from the combustion of fossil fuels and the accumulation of greenhouse gases (GHGs), continues to destabilize all ecosystems worldwide. Although annual emissions must be halved by 2030 and reach net zero by 2050 to limit some of the most catastrophic impacts associated with a warming planet, the world's efforts to curb GHG emissions fall short of the commitments made in the 2015 Paris Agreement. To this effect, July 2021 was recently declared the hottest month ever recorded in 142 years. The ramifications of these changes for global temperatures are complex and further promote outdoor air pollution, pollen exposure, and extreme weather events. Besides worsening respiratory health, air pollution promotes atopy and susceptibility to infections. The effects of GHGs on pollen affect the frequency and severity of asthma and allergic rhinitis. Changes in temperature, air pollution, and extreme weather events exert adverse multisystemic health effects and disproportionally affect disadvantaged and vulnerable populations. This review article is an update for allergists and immunologists about the health impacts of climate change that are already evident in our daily practices. It is also a call to action and advocacy, including to integrate climate change-related mitigation, education, and adaptation measures to protect our patients and avert further injury to our planet.
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Alergia e Inmunología , Asma/inmunología , Rinitis Alérgica/inmunología , Contaminación del Aire , Animales , Asma/epidemiología , Cambio Climático , Ecosistema , Combustibles Fósiles , Salud Global , Calentamiento Global , Gases de Efecto Invernadero/efectos adversos , Humanos , Rinitis Alérgica/epidemiologíaRESUMEN
Electronic cigarettes (ECs) have been growing rapidly in popularity among youth and adults in the United States over the last decade. This increasing prevalence is driven partially by the ability to customize devices, flavors, and nicotine content and the general notion that ECs are harmless, particularly in comparison with conventional cigarettes. In vitro and in vivo murine models have demonstrated a number of harmful biological effects of e-liquids and their aerosols. However, limited clinical data exist on whether these effects translate into detrimental long-term outcomes in human subjects. The short-term harmful respiratory effects of EC use demonstrated in nonsmokers argue against their use. However, slightly more favorable data exist for the respiratory benefits of substituting conventional cigarettes with ECs and the short-term efficacy of ECs as smoking cessation tools. Nonetheless, available research is severely limited in regard to long-term outcomes and by study designs fraught with bias, pointing to the need for additional research efforts with well-designed longitudinal studies to guide US Food and Drug Administration regulatory efforts. The hurdle presented by diverse device designs and e-liquid permutations, which contribute to the inconsistency of available data, also highlights the need for legislative standardization of ECs.
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Sistemas Electrónicos de Liberación de Nicotina , Vapeo/efectos adversos , Animales , Humanos , Ratones , Prevalencia , Vapeo/epidemiologíaAsunto(s)
Contaminantes Atmosféricos , Contaminación del Aire , Asma , Contaminantes Atmosféricos/efectos adversos , Contaminantes Atmosféricos/análisis , Contaminación del Aire/efectos adversos , Contaminación del Aire/análisis , Asma/epidemiología , Granjas , Humanos , Material Particulado , Emisiones de VehículosRESUMEN
Allergic disease prevalence has increased significantly in recent decades. Primary prevention efforts are being guided by study of the exposome (or collective environmental exposures beginning during the prenatal period) to identify modifiable factors that affect allergic disease risk. In this review we explore the evidence supporting a relationship between key components of the external exposome in the prenatal and early-life periods and their effect on atopy development focused on microbial, allergen, and air pollution exposures. The abundance and diversity of microbial exposures during the first months and years of life have been linked with risk of allergic sensitization and disease. Indoor environmental allergen exposure during early life can also affect disease development, depending on the allergen type, dose, and timing of exposure. Recent evidence supports the role of ambient air pollution in allergic disease inception. The lack of clarity in the literature surrounding the relationship between environment and atopy reflects the complex interplay between cumulative environmental factors and genetic susceptibility, such that no one factor dictates disease development in all subjects. Understanding the effect of the summation of environmental exposures throughout a child's development is needed to identify cost-effective interventions that reduce atopy risk in children.
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Exposición a Riesgos Ambientales , Hipersensibilidad/epidemiología , Contaminación del Aire , Alérgenos , Animales , Humanos , Microbiota , Factores de RiesgoRESUMEN
UNLABELLED: Sulforaphane (SFN), a naturally occurring isothiocyanate found in cruciferous vegetables, is implicated as a possible therapy for airway inflammation via induction of the transcription factor NF-E2-related factor 2 (NRF2). In this proof-of-concept clinical study, we show that supplementation of SFN with broccoli sprout homogenate in healthy human subjects did not induce expression of antioxidant genes or protect against neutrophilic airway inflammation in an ozone-exposure model. Therefore, dietary sulforaphane supplementation is not a promising candidate for larger scale clinical trials targeting airway inflammation. TRIAL REGISTRATION: NCT01625130 . Registered 19 June, 2012.
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Antiinflamatorios/uso terapéutico , Isotiocianatos/uso terapéutico , Trastornos Leucocíticos/prevención & control , Pulmón/efectos de los fármacos , Factor 2 Relacionado con NF-E2/agonistas , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neumonía/prevención & control , Adolescente , Adulto , Antiinflamatorios/aislamiento & purificación , Brassica/química , Estudios Cruzados , Femenino , Voluntarios Sanos , Humanos , Isotiocianatos/aislamiento & purificación , Trastornos Leucocíticos/inducido químicamente , Trastornos Leucocíticos/inmunología , Trastornos Leucocíticos/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Factor 2 Relacionado con NF-E2/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Ozono , Fitoterapia , Plantas Medicinales , Neumonía/inducido químicamente , Neumonía/inmunología , Neumonía/metabolismo , Sulfóxidos , Adulto JovenRESUMEN
The biological effect of an inorganic particle (i.e., silica) can be associated with a disruption in cell iron homeostasis. Organic compounds included in particles originating from combustion processes can also complex sources of host cell iron to disrupt metal homeostasis. We tested the postulate that (1) wood smoke particle (WSP) sequesters host cell iron resulting in a disruption of metal homeostasis, (2) this loss of essential metal results in both an oxidative stress and biological effect in respiratory epithelial cells, and (3) humic-like substances (HULIS), a component of WSP, have a capacity to appropriate cell iron and initiate a biological effect. BEAS-2B cells exposed to WSP resulted in diminished concentrations of mitochondrial (57)Fe, whereas preincubation with ferric ammonium citrate (FAC) prevented significant mitochondrial iron loss after such exposure. Cellular oxidant generation was increased after WSP exposure, but this signal was diminished by coincubation with FAC. Similarly, exposure of BEAS-2B cells to 100 µg/mL WSP activated mitogen-activated protein (MAP) kinases, elevated NF-E2-related factor 2/antioxidant responsive element (Nrf2 ARE) expression, and provoked interleukin (IL)-6 and IL-8 release, but all these changes were diminished by coincubation with FAC. The biological response to WSP was reproduced by exposure to 100 µg/mL humic acid, a polyphenol comparable to HULIS included in the WSP that complexes iron. We conclude that (1) the biological response following exposure to WSP is associated with sequestration of cell iron by the particle, (2) increasing available iron in the cell diminished the biological effects after particle exposure, and (3) HULIS included in WSP can sequester the metal initiating the cell response.
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Hierro/metabolismo , Humo/efectos adversos , Madera , Línea Celular , Núcleo Celular/metabolismo , Humanos , Sustancias Húmicas , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Transcripción/genéticaRESUMEN
Inhalation of particulate matter has presented a challenge to human health for thousands of years. The underlying mechanism for biological effect following particle exposure is incompletely understood. We tested the postulate that particle sequestration of cell and mitochondrial iron is a pivotal event mediating oxidant generation and biological effect. In vitro exposure of human bronchial epithelial cells to silica reduced intracellular iron, which resulted in increases in both the importer divalent metal transporter 1 expression and metal uptake. Diminished mitochondrial (57)Fe concentrations following silica exposure confirmed particle sequestration of cell iron. Preincubation of cells with excess ferric ammonium citrate increased cell, nuclear, and mitochondrial metal concentrations and prevented significant iron loss from mitochondria following silica exposure. Cell and mitochondrial oxidant generation increased after silica incubation, but pretreatment with iron diminished this generation of reactive oxygen species. Silica exposure activated MAP kinases (ERK and p38) and altered the expression of transcription factors (nF-κB and NF-E2-related factor 2), proinflammatory cytokines (interleukin-8 and -6), and apoptotic proteins. All of these changes in indexes of biological effect were either diminished or inhibited by cell pretreatment with iron. Finally, percentage of neutrophils and total protein concentrations in an animal model instilled with silica were decreased by concurrent exposure to iron. We conclude that an initiating event in the response to particulate matter is a sequestration of cell and mitochondrial iron by endocytosed particle. The resultant oxidative stress and biological response after particle exposure are either diminished or inhibited by increasing the cell iron concentration.
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Bronquios/efectos de los fármacos , Hierro/metabolismo , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Material Particulado/farmacología , Dióxido de Silicio/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Bronquios/citología , Bronquios/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Ferritinas/metabolismo , Citometría de Flujo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Oxidantes/farmacología , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
RATIONALE: Diesel exhaust enhances allergic inflammation, and pollutants are associated with heightened susceptibility to viral respiratory infections. The effects of combined diesel and virus exposure in humans are unknown. OBJECTIVES: Test whether acute exposure to diesel modifies inflammatory responses to influenza virus in normal humans and those with allergies. METHODS: We conducted a double-blind, randomized, placebo-controlled study of nasal responses to live attenuated influenza virus in normal volunteers and those with allergic rhinitis exposed to diesel (100 µg/m(3)) or clean air for 2 hours, followed by standard dose of virus and serial nasal lavages. Endpoints were inflammatory mediators (ELISA) and virus quantity (quantitative reverse-transcriptase polymerase chain reaction). To test for exposure effect, we used multiple regression with exposure group (diesel vs. air) as the main explanatory variable and allergic status as an additional factor. MEASUREMENTS AND MAIN RESULTS: Baseline levels of mediators did not differ among groups. For most postvirus nasal cytokine responses, there was no significant diesel effect, and no significant interaction with allergy. However, diesel was associated with significantly increased IFN-γ responses (P = 0.02), with no interaction with allergy in the regression model. Eotaxin-1 (P = 0.01), eosinophil cationic protein (P < 0.01), and influenza RNA sequences in nasal cells (P = 0.03) were significantly increased with diesel exposure, linked to allergy. CONCLUSIONS: Short-term exposure to diesel exhaust leads to increased eosinophil activation and increased virus quantity after virus inoculation in those with allergic rhinitis. This is consistent with previous literature suggesting a diesel "adjuvant" effect promoting allergic inflammation, and our data further suggest this change may be associated with reduced virus clearance.Clinical trial registered with www.clinicaltrials.gov (NCT00617110).
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Contaminantes Atmosféricos/efectos adversos , Vacunas contra la Influenza/administración & dosificación , Mucosa Nasal/inmunología , Orthomyxoviridae/aislamiento & purificación , Rinitis Alérgica Perenne/inmunología , Emisiones de Vehículos , Administración Intranasal , Adulto , Antivirales/inmunología , Biomarcadores/sangre , Quimiocina CCL11/inmunología , Método Doble Ciego , Proteína Catiónica del Eosinófilo/inmunología , Femenino , Humanos , Factores Inmunológicos/inmunología , Inflamación/inmunología , Interferón gamma/inmunología , Masculino , Lavado Nasal (Proceso) , Estudios Prospectivos , Análisis de Regresión , Vacunas Atenuadas/administración & dosificaciónRESUMEN
Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.
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Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T del Adulto/virología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas del Núcleo Viral/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , División Celular/fisiología , Supervivencia Celular/fisiología , Daño del ADN/fisiología , Fase G2/fisiología , Células HEK293 , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/patología , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Humanos , Células Jurkat , Leucemia-Linfoma de Células T del Adulto/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Complejos Multiproteicos/metabolismo , Unión Proteica/fisiología , Proteínas del Núcleo Viral/genéticaRESUMEN
Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA) protein binds to sialic acid residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host cell membrane, which is mediated by host trypsin-like serine protease. We show data here demonstrating that the protease:antiprotease ratio is increased in the nasal mucosa of asthmatics and that these changes were associated with increased proteolytic activation of influenza. These data suggest that disruption of the protease balance in asthmatics enhances activation and infection of influenza virus.
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Asma/enzimología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Hipersensibilidad Inmediata/enzimología , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Nariz/enzimología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Serina Endopeptidasas/metabolismo , Replicación Viral , Adulto , Animales , Estudios de Casos y Controles , Perros , Femenino , Humanos , Células de Riñón Canino Madin Darby , Masculino , Líquido del Lavado Nasal/química , Adulto JovenRESUMEN
Ozone (O3) is a ubiquitous outdoor air pollutant, which may be derived from various primary pollutants such as nitrates, hydrocarbons, and volatile organ compounds through ultraviolet radiation exposure, and has been shown to negatively impact respiratory health. O3 is the most common noninfectious environmental cause of asthma exacerbations among children and adults. Its effects on pediatric respiratory health could be due to multiple physiologic factors that may contribute to enhanced O3 exposure seen in children compared with adults, including differences in lung surface area per unit of body weight and ventilation rates. O3 can reach the distal regions of human lungs due to its low water solubility, resulting in either injury or activation of airway epithelial cells and macrophages. Multiple epidemiologic studies have highlighted a link between exposure to air pollution and the development of asthma. This review article specifically focuses on examining the impact of early life O3 exposure on lung development, lung function, and the risk of developing atopic diseases including asthma, allergic rhinitis, and atopic dermatitis among children.
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Contaminantes Atmosféricos , Asma , Ozono , Niño , Humanos , Contaminantes Atmosféricos/efectos adversos , Asma/epidemiología , Nitratos , Ozono/efectos adversos , Rayos Ultravioleta , AguaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) infection causes adult T-cell leukemia and is associated with a variety of lymphocyte-mediated disorders. It has been hypothesized that a highly regulated pattern of HTLV-1 gene expression is critical for virus survival and disease pathogenesis. In this study, real-time reverse transcriptase PCR was used to determine the kinetics of viral gene expression in cells transiently transfected with an HTLV-1 proviral plasmid, in newly infected human peripheral blood mononuclear cells (PBMCs), and in PBMCs from newly infected rabbits. The HTLV-1 gene expression profiles in transiently transfected and infected cells were similar; over time, all transcripts increased and then maintained stable levels. gag/pol, tax/rex, and env mRNA were detected first and at the highest levels, whereas the expression levels of the accessory genes, including the antisense Hbz, were significantly lower than the tax/rex levels (ranging from 1 to 4 logs depending on the specific mRNA). In infected rabbits, tax/rex and gag/pol mRNA levels peaked early after inoculation and progressively decreased, which correlated inversely with the proviral load and host antibody response against viral proteins. Interestingly, Hbz mRNA was detectable at 1 week postinfection and increased and stabilized. The expression levels of all other HTLV-1 genes in infected rabbit PBMCs were at or below our limit of detection. This analysis provides insight into viral gene expression under various in vitro and in vivo experimental conditions. Our in vivo data indicate that in infected rabbits, Hbz mRNA expression over time directly correlates with the proviral load, which provides the first evidence linking Hbz expression to proviral load and the survival of the virus-infected cell in the host.
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Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/fisiología , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Animales , Línea Celular , Células Cultivadas , Humanos , Leucocitos Mononucleares/virología , Provirus/crecimiento & desarrollo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Human T-cell leukemia virus type 2 (HTLV-2) Rex is a transacting regulatory protein required for efficient cytoplasmic expression of the unspliced and incompletely spliced viral mRNA transcripts encoding the structural and enzymatic proteins. Previously, it was demonstrated that phosphorylation of Rex-2, predominantly on serine residues, is correlated with an altered conformation, as observed by a gel mobility shift and the detection of two related protein species (p24(Rex) and p26(Rex)). Rex-2 phosphorylation is required for specific binding to its viral-mRNA target sequence and inhibition of mRNA splicing and may be linked to subcellular compartmentalization. Thus, the phosphorylation-induced structural state of Rex in the infected cell may be a switch that determines whether HTLV exists in a latent or productive state. We conducted a phosphoryl and functional mapping of both structural forms of mammalian-cell-expressed Rex 2 using affinity purification, liquid chromatography-tandem mass spectrometry, and site-directed substitutional mutational analysis. We identified two phosphorylation sites in p24(Rex) at Ser-117 and Thr-164. We also identified six phosphorylation sites in p26(Rex) at Thr-19, Ser-117, Ser-125, Ser-151, Ser-153, and Thr-164. We evaluated the functional significance of these phosphorylation events and found that phosphorylation on Thr-164, Ser-151, and Ser-153 is critical for Rex-2 function in vivo and that phosphorylation of Ser-151 is correlated with nuclear/nucleolar subcellular localization. Overall, this work is the first to completely map the phosphorylation sites in Rex-2 and provides important insight into the phosphorylation continuum that tightly regulates Rex-2 structure, cellular localization, and function.
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Regulación Viral de la Expresión Génica , Productos del Gen rex/metabolismo , Virus Linfotrópico T Tipo 2 Humano/fisiología , Secuencia de Aminoácidos , Núcleo Celular/química , Cromatografía de Afinidad , Citoplasma/química , Productos del Gen rex/genética , Productos del Gen rex/aislamiento & purificación , Humanos , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Serina/metabolismo , Espectrometría de Masas en Tándem , Treonina/metabolismoRESUMEN
Human T-cell leukemia virus (HTLV) regulatory protein, Rex, functions to increase the expression of the viral structural and enzymatic gene products. The phosphorylation of two serine residues (S151 and S153) at the C terminus is important for the function of HTLV-2 Rex (Rex-2). The Rex-2 phosphomimetic double mutant (S151D, S153D) is locked in a functionally active conformation. Since rex and tax genes overlap, Rex S151D and S153D mutants were found to alter the Tax oncoprotein coding sequence and transactivation activities. Therefore, additional Rex-2 mutants including P152D, A157D, S151Term, and S158Term were generated and characterized ("Term" indicates termination codon). All Rex-2 mutants and wild-type (wt) Rex-2 localized predominantly to the nucleus/nucleolus, but in contrast to the detection of phosphorylated and unphosphorylated forms of wt Rex-2 (p26 and p24), mutant proteins were detected as a single phosphoprotein species. We found that Rex P152D, A157D, and S158Term mutants are more functionally active than wt Rex-2 and that the Rex-2 C terminus and its specific phosphorylation state are required for stability and optimal expression. In the context of the provirus, the more active Rex mutants (A157D or S158Term) promoted increased viral protein production, increased viral infectious spread, and enhanced HTLV-2-mediated cellular proliferation. Moreover, these Rex mutant viruses replicated and persisted in inoculated rabbits despite higher antiviral antibody responses. Thus, we identified in Rex-2 a novel C-terminal inhibitory domain that regulates functional activity and is positively regulated through phosphorylation. The ability of this domain to modulate viral replication likely plays a key role in the infectious spread of the virus and in virus-induced cellular proliferation.
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Productos del Gen rex/metabolismo , Virus Linfotrópico T Tipo 2 Humano/fisiología , Replicación Viral , Animales , Regulación Viral de la Expresión Génica , Productos del Gen rex/genética , Productos del Gen tax/metabolismo , Infecciones por HTLV-II/virología , Células HeLa , Virus Linfotrópico T Tipo 2 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/metabolismo , Humanos , Células Jurkat , Fosforilación , Estabilidad Proteica , Conejos , Eliminación de Secuencia , Linfocitos T/virologíaRESUMEN
BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1) is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. RESULTS: We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. CONCLUSION: We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into the regulation of Rex-1 function.
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Productos del Gen rex/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Secuencia de Aminoácidos , Línea Celular , Regulación Viral de la Expresión Génica , Productos del Gen rex/química , Productos del Gen rex/genética , Productos del Gen rex/aislamiento & purificación , Virus Linfotrópico T Tipo 1 Humano/química , Humanos , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Fosfoserina/metabolismo , Fosfotreonina/metabolismoRESUMEN
BACKGROUND: Human T-cell leukemia virus (HTLV) type 1 and type 2 are related but distinct pathogenic complex retroviruses. HTLV-1 is associated with adult T-cell leukemia and a variety of immune-mediated disorders including the chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraparesis. In contrast, HTLV-2 displays distinct biological differences and is much less pathogenic, with only a few reported cases of leukemia and neurological disease associated with infection. In addition to the structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory proteins. Tax and Rex positively regulate virus production and are critical for efficient viral replication and pathogenesis. Using an over-expression system approach, we recently reported that the accessory gene product of the HTLV-1 and HTLV-2 open reading frame (ORF) II (p30 and p28, respectively) acts as a negative regulator of both Tax and Rex by binding to and retaining their mRNA in the nucleus, leading to reduced protein expression and virion production. Further characterization revealed that p28 was distinct from p30 in that it was devoid of major transcriptional modulating activity, suggesting potentially divergent functions that may be responsible for the distinct pathobiologies of HTLV-1 and HTLV-2. RESULTS: In this study, we investigated the functional significance of p28 in HTLV-2 infection, proliferation, and immortaliztion of primary T-cells in culture, and viral survival in an infectious rabbit animal model. An HTLV-2 p28 knockout virus (HTLV-2Deltap28) was generated and evaluated. Infectivity and immortalization capacity of HTLV-2Deltap28 in vitro was indistinguishable from wild type HTLV-2. In contrast, we showed that viral replication was severely attenuated in rabbits inoculated with HTLV-2Deltap28 and the mutant virus failed to establish persistent infection. CONCLUSION: We provide direct evidence that p28 is dispensable for viral replication and cellular immortalization of primary T-lymphocytes in cell culture. However, our data indicate that p28 function is critical for viral survival in vivo. Our results are consistent with the hypothesis that p28 repression of Tax and Rex-mediated viral gene expression may facilitate survival of these cells by down-modulating overall viral gene expression.
Asunto(s)
Virus Linfotrópico T Tipo 2 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/patogenicidad , Proteínas de los Retroviridae/metabolismo , Replicación Viral , Animales , Anticuerpos Antivirales/sangre , Línea Celular , Proliferación Celular , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Genes Reporteros , Genoma Viral , Infecciones por HTLV-II/inmunología , Infecciones por HTLV-II/virología , Virus Linfotrópico T Tipo 2 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/aislamiento & purificación , Humanos , Leucocitos Mononucleares/fisiología , Leucocitos Mononucleares/virología , Conejos , Proteínas de los Retroviridae/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Mechanistic pathways underlying inflammatory injury following exposures to vanadium-containing compounds are not defined. We tested the postulate that the in vitro biological effect of vanadium results from its impact on iron homeostasis. Human bronchial epithelial (HBE) cells exposed to vanadyl sulfate (VOSO4) showed a time- and dose-dependent increase in vanadium relative to PBS. HBE cells exposed to VOSO4 and then exposed to ferric ammonium citrate (FAC) significantly increased intracellular iron import supporting an interaction between the two metals. Following exposure to VOSO4, there was an increase (336±73%) in RNA for divalent metal transporter 1 (DMT1), a major iron importer. With inclusion of VOSO4 in the incubation, vanadium could be measured in the nuclear and mitochondrial fractions and the supernatant. Non-heme iron in the nuclear and mitochondrial fractions were decreased immediately following VOSO4 exposure while there was an increased concentration of non-heme iron in the supernatant. Provision of excess iron inhibited changes in the concentration of this metal provoked by VOSO4 exposures. Using Amplex Red, VOSO4 was shown to significantly increase oxidant generation by HBE cells in a time- and dose-dependent manner. HBE cells pre-treated with FAC and then exposed to VOSO4 demonstrated a decreased generation of oxidants. Similarly, activation of the transcription factor NF-ĸB promoter and release of interleukin-6 and -8 were increased following VOSO4 exposure and these effects were diminished by pre-treatment with FAC. We conclude that an initiating event in biological effect after exposure to vanadyl sulfate is a loss of requisite cell iron.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Compuestos Férricos/farmacología , Compuestos de Amonio Cuaternario/farmacología , Compuestos de Vanadio/farmacología , Células Cultivadas , HumanosRESUMEN
Cells lining the respiratory tract are equipped with mechanisms that dampen the effects of oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a mediator involved in regulating oxidative stress. Recent data indicate Nrf2 also controls expression of secretory leukocyte protease inhibitor (SLPI). Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, enhances Nrf2 activity. Therefore, we hypothesized that SFN supplementation induces SLPI secretion in the nasal mucosa in an Nrf2 dependent manner. Healthy nonsmoking adults ingested SFN-containing broccoli shake homogenate (BSH) for 3 consecutive days. Nasal lavage fluid (NLF) was collected before and after BSH ingestion and analyzed for SLPI protein levels. In follow up in vitro experiments, differentiated primary nasal epithelial cells were used to evaluate the relationship between SFN, Nrf2, and SLPI. Epithelial cells were transduced with Nrf2-specific shRNA to examine the regulatory role of Nrf2 on SLPI expression. Supplementation with BSH significantly increased SLPI levels in NLF. SFN supplementation in vitro significantly enhanced SLPI secretion and these effects were significantly decreased in cells transduced with Nrf2-specific shRNA. Our data support a relationship between nutritional supplementation, Nrf2 activation, and SLPI secretion. Therefore, ingestion of SFN-containing foods has therapeutic potential to augment SLPI expression in the nasal mucosa.