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Sequencing of the human genome and numerous advances in molecular techniques have launched the era of genetic medicine. Increasingly precise technologies for genetic modification, manufacturing, and administration of pharmaceutical-grade biologics have proved the viability of in vivo gene therapy (GTx) as a therapeutic modality as shown in several thousand clinical trials and recent approval of several GTx products for treating rare diseases and cancers. In recognition of the rapidly advancing knowledge in this field, the regulatory landscape has evolved considerably to maintain appropriate monitoring of safety concerns associated with this modality. Nonetheless, GTx safety assessment remains complex and is designed on a case-by-case basis that is determined by the disease indication and product attributes. This article describes our current understanding of fundamental biological principles and possible procedures (emphasizing those related to toxicology and toxicologic pathology) needed to support research and development of in vivo GTx products. This article is not intended to provide comprehensive guidance on all GTx modalities but instead provides an overview relevant to in vivo GTx generally by utilizing recombinant adeno-associated virus-based GTx-the most common in vivo GTx platform-to exemplify the main points to be considered in nonclinical research and development of GTx products.
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Dependovirus , Terapia Genética , Dependovirus/genética , Terapia Genética/métodos , Humanos , Políticas , InvestigaciónRESUMEN
The inbred Fischer 344 rat is being evaluated for testing novel vaccines and therapeutics against pneumonic tularemia. Although primary pneumonic tularemia in humans typically occurs by inhalation of aerosolized bacteria, the rat model has relied on intratracheal inoculation of organisms because of safety and equipment issues. We now report the natural history of pneumonic tularemia in female Fischer 344 rats after nose-only inhalational exposure to lethal doses of aerosolized Francisella tularensis subspecies tularensis, strain SCHU S4. Our results are consistent with initial uptake of aerosolized SCHU S4 from the nasal cavity, lungs, and possibly the gastrointestinal tract. Bacteremia with hematogenous dissemination was first detected 2 days after exposure. Shortly thereafter, the infected rats exhibited fever, tachypnea, and hypertension that persisted for 24 to 36 hours and then rapidly decreased as animals succumbed to infection between days 5 and 8 after exposure. Tachycardia was observed briefly, but only after the core body temperature and blood pressure began to decrease as the animals were near death. Initial neutrophilic and histiocytic inflammation in affected tissues became progressively more fibrinous and necrotizing over time. At death, as many as 1010 colony-forming units were found in the lungs, spleen, and liver. Death was attributed to sepsis and disseminated intravascular coagulation. Overall, the pathogenesis of pneumonic tularemia in the female F344 rat model appears to replicate the disease in humans.
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Modelos Animales de Enfermedad , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Tularemia/patología , Animales , Femenino , Francisella tularensis , Ratas , Ratas Endogámicas F344RESUMEN
Inhalational anthrax is caused by inhalation of Bacillus anthracis spores. The ability of B. anthracis to cause anthrax is attributed to the plasmid-encoded A/B-type toxins, edema toxin (edema factor and protective antigen) and lethal toxin (lethal factor and protective antigen), and a poly-d-glutamic acid capsule. To better understand the contribution of these toxins to the disease pathophysiology in vivo, we used B. anthracis Ames strain and isogenic toxin deletion mutants derived from the Ames strain to examine the role of lethal toxin and edema toxin after pulmonary spore challenge of cynomolgus macaques. Lethal toxin, but not edema toxin, was required to induce sustained bacteremia and death after pulmonary challenge with spores delivered via bronchoscopy. After intravenous challenge with bacilli to model the systemic phase of infection, lethal toxin contributed to bacterial proliferation and subsequent host death to a greater extent than edema toxin. Deletion of protective antigen resulted in greater loss of virulence after intravenous challenge with bacilli than deletion of lethal toxin or edema toxin alone. These findings are consistent with the ability of anti-protective antigen antibodies to prevent anthrax and suggest that lethal factor is the dominant toxin that contributes to the escape of significant numbers of bacilli from the thoracic cavity to cause anthrax after inhalation challenge with spores.
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Carbunco/microbiología , Antígenos Bacterianos/metabolismo , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/metabolismo , Pulmón/microbiología , Infecciones del Sistema Respiratorio/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Femenino , Macaca , Masculino , Esporas Bacterianas/patogenicidad , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.
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Pulmón/efectos de los fármacos , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos/toxicidad , Receptores de Superficie Celular/genética , Animales , Femenino , Pulmón/metabolismo , Pulmón/patología , Pulmón/fisiología , Macaca , Masculino , Ratones , Oligonucleótidos/sangre , Oligonucleótidos/farmacocinética , Oligonucleótidos Antisentido/sangre , Oligonucleótidos Antisentido/farmacocinética , ARN Mensajero/metabolismoRESUMEN
Post-operative atrial fibrillation (POAF) is the most common complication after cardiac surgery. Despite implementation of several pharmacological strategies, incidence of POAF remains at approximately 30%. An adenovirus vector encoding KCNH2-G628S has proven efficacious in a porcine model of AF. In this preclinical study, 1.5 × 1010 or 1.5 × 1012 Ad-KCNH2-G628S vector particles (vp) were applied to the atrial epicardium or 1.5 × 1012 vp were applied to the whole epicardial surface of New Zealand White rabbits. Saline and vector vehicle served as procedure controls. Animals were followed for up to 42 days. Vector genomes persisted in the atria up to 42 days, with no distribution to extra-thoracic organs. There were no adverse effects attributable to test article on standard toxicological endpoints or on blood pressure, left atrial or ventricular ejection fractions, electrocardiographic parameters, or serum IL-6 or troponin concentrations. Mononuclear infiltration of the myocardium of the atrial free walls of low-dose, but not high-dose animals was observed at 7 and 21 days, but these changes did not persist or affect cardiac function. After scaling for heart size, results indicate the test article is safe at doses up to 25 times the maximum proposed for the human clinical trial.
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Fibrilación Atrial , Procedimientos Quirúrgicos Cardíacos , Conejos , Humanos , Animales , Porcinos , Distribución Tisular , Atrios Cardíacos , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Miocardio , Complicaciones Posoperatorias/etiología , Canal de Potasio ERG1RESUMEN
The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis. We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA- mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA- mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.
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Carbunco/microbiología , Carbunco/patología , Antígenos Bacterianos/biosíntesis , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/biosíntesis , Factores de Virulencia/biosíntesis , Animales , Carbunco/mortalidad , Antígenos Bacterianos/genética , Cápsulas Bacterianas/genética , Toxinas Bacterianas/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Histocitoquímica , Conejos , Análisis de Supervivencia , VirulenciaRESUMEN
Pneumonic tularemia is a life-threatening disease caused by inhalation of the highly infectious intracellular bacterium Francisella tularensis. The most serious form of the disease associated with the type A strains can be prevented in experimental animals through vaccination with the attenuated live vaccine strain (LVS). The protection is largely cell mediated, but the contribution of antibodies remains controversial. We addressed this issue in a series of passive immunization studies in Fischer 344 (F344) rats. Subcutaneous LVS vaccination induced a robust serum antibody response dominated by IgM, IgG2a, and IgG2b antibodies. Prophylactic administration of LVS immune serum or purified immune IgG reduced the severity and duration of disease in naïve rats challenged intratracheally with a lethal dose of the virulent type A strain SCHU S4. The level of resistance increased with the volume of immune serum given, but the maximum survivable SCHU S4 challenge dose was at least 100-fold lower than that shown for LVS-vaccinated rats. Protection correlated with reduced systemic bacterial growth, less severe histopathology in the liver and spleen during the early phase of infection, and bacterial clearance by a T cell-dependent mechanism. Our results suggest that treatment with immune serum limited the sequelae associated with infection, thereby enabling a sterilizing T cell response to develop and resolve the infection. Thus, antibodies induced by LVS vaccination may contribute to the defense of F344 rats against respiratory infection by type A strains of F. tularensis.
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Anticuerpos Antibacterianos/inmunología , Vacunas Bacterianas/inmunología , Francisella tularensis/inmunología , Inmunización Pasiva , Infecciones del Sistema Respiratorio/inmunología , Tularemia/inmunología , Tularemia/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/uso terapéutico , Separación Celular , Femenino , Citometría de Flujo , Ratas , Ratas Endogámicas F344 , Ratas Desnudas , Infecciones del Sistema Respiratorio/prevención & control , Vacunación , Vacunas Atenuadas/inmunologíaRESUMEN
Pneumonic tularemia is a highly debilitating and potentially fatal disease caused by inhalation of Francisella tularensis. Most of our current understanding of its pathogenesis is based on the highly virulent F. tularensis subsp. tularensis strain SCHU S4. However, multiple sources of SCHU S4 have been maintained and propagated independently over the years, potentially generating genetic variants with altered virulence. In this study, the virulence of four SCHU S4 stocks (NR-10492, NR-28534, NR-643 from BEI Resources and FTS-635 from Battelle Memorial Institute) along with another virulent subsp. tularensis strain, MA00-2987, were assessed in parallel. In the Fischer 344 rat model of pneumonic tularemia, NR-643 and FTS-635 were found to be highly attenuated compared to NR-10492, NR-28534, and MA00-2987. In the NZW rabbit model of pneumonic tularemia, NR-643 caused morbidity but not mortality even at a dose equivalent to 500x the LD50 for NR-10492. Genetic analyses revealed that NR-10492 and NR-28534 were identical to each other, and nearly identical to the reference SCHU S4 sequence. NR-643 and FTS-635 were identical to each other but were found to have nine regions of difference in the genomic sequence when compared to the published reference SCHU S4 sequence. Given the genetic differences and decreased virulence, NR-643/FTS-635 should be clearly designated as a separate SCHU S4 substrain and no longer utilized in efficacy studies to evaluate potential vaccines and therapeutics against tularemia.
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BACKGROUND: Trimethylation of lysine 27 and dimethylation of lysine 9 of histone-H3 catalyzed by the histone methyltransferases EZH2 and G9a impede gene transcription in cancer. Our human bronchial epithelial (HBEC) pre-malignancy model studied the role of these histone modifications in transformation. Tobacco carcinogen transformed HBEC lines were characterized for cytosine DNA methylation, transcriptome reprogramming, and the effect of inhibiting EZH2 and G9a on the transformed phenotype. The effects of targeting EZH2 and G9a on lung cancer prevention was assessed in the A/J mouse lung tumor model. RESULTS: Carcinogen exposure induced transformation and DNA methylation of 12-96 genes in the four HBEC transformed (T) lines that was perpetuated in malignant tumors. In contrast, 506 unmethylated genes showed reduced expression in one or more HBECTs with many becoming methylated in tumors. ChIP-on-chip for HBEC2T identified 327 and 143 genes enriched for H3K27me3 and H3K9me2. Treatment of HBEC2T and HBEC13T with DZNep, a lysine methyltransferase inhibitor depleted EZH2, reversed transformation, and induced transcriptional reprogramming. The EZH2 small molecule inhibitor EPZ6438 also affected transformation and expression in HBEC2T, while a G9a inhibitor, UNC0642 was ineffective. Genetic knock down of EZH2 dramatically reduced carcinogen-induced transformation of HBEC2. Only DZNep treatment prevented progression of hyperplasia to adenomas in the NNK mouse lung tumor model through reducing EZH2 and affecting the expression of genes regulating cell growth and invasion. CONCLUSION: These studies demonstrate a critical role for EZH2 catalyzed histone modifications for premalignancy and its potential as a target for chemoprevention of lung carcinogenesis.
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Ensamble y Desensamble de Cromatina/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Código de Histonas/efectos de los fármacos , Neoplasias/prevención & control , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosilhomocisteinasa/antagonistas & inhibidores , Animales , Benzamidas/farmacología , Compuestos de Bifenilo/farmacología , Proliferación Celular/efectos de los fármacos , Islas de CpG , Metilación de ADN/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2/farmacología , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Femenino , Código de Histonas/genética , Histona Metiltransferasas/antagonistas & inhibidores , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/farmacología , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/patología , Ratones , Morfolinas/farmacología , Fenotipo , Piridonas/farmacología , Transcriptoma/efectos de los fármacosRESUMEN
Chronic human silicosis results primarily from continued occupational exposure to silica and exhibits a long asymptomatic latency. Similarly, continued exposure of Lewis rats to low doses of silica is known to cause delayed granuloma formation with limited lung inflammation and injury. On the other hand, intratracheal exposure to large doses of silica induces acute silicosis characterized by granuloma-like formations in the lung associated with apoptosis, severe alveolitis, and alveolar lipoproteinosis. To ascertain similarities/differences between acute and chronic silicosis, in this communication, we compared cellular and molecular changes in established rat models of acute and chronic silicosis. In Lewis rats, acute silicosis was induced by intratracheal instillation of 35 mg silica, and chronic silicosis through inhalation of aerosolized silica (6.2 mg/m(3), 5 d/wk for 6 wk). Animals exposed to acute high-dose silica were sacrificed at 14 d after silica instillation while chronically silica-treated animals were sacrificed between 4 d and 28 wk after silica exposure. The lung granulomas formation in acute silicosis was associated with strong inflammation, presence of TUNEL-positive cells, and increases in caspase-3 activity and other molecular markers of apoptosis. On the other hand, lungs from chronically silica-exposed animals exhibited limited inflammation and increased expression of anti-apoptotic markers, including dramatic increases in Bcl-2 and procaspase-3, and lower caspase-3 activity. Moreover, chronic silicotic lungs were TUNEL-negative and overexpressed Bcl-3 and NF-kappaB-p50 but not NF-kappaB-p65 subunits. These results suggest that, unlike acute silicosis, chronic exposures to occupationally relevant doses of silica cause significantly lower lung inflammation and elevated expression of anti-apoptotic rather than proapoptotic markers in the lung that might result from interaction between NF-kappaB-p50 and Bcl-3.
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Apoptosis , Granuloma del Sistema Respiratorio/patología , Pulmón/patología , Dióxido de Silicio/toxicidad , Silicosis/patología , Enfermedad Aguda , Animales , Proteínas del Linfoma 3 de Células B , Biomarcadores/análisis , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Caspasa 3/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Granuloma del Sistema Respiratorio/inducido químicamente , Granuloma del Sistema Respiratorio/metabolismo , Etiquetado Corte-Fin in Situ , Exposición por Inhalación , Intubación Intratraqueal , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Endogámicas Lew , Silicosis/etiología , Silicosis/metabolismo , Organismos Libres de Patógenos Específicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The mouse is a good model for evaluating the efficacy of chemopreventive agents for lung cancer. Gene silencing by promoter hypermethylation is a critical component for the development and progression of lung cancer and an emerging target for preventive intervention by demethylating agents. Genes methylated in mouse lung tumors could serve as biomarkers to evaluate the effectiveness of demethylating agents for preventing lung cancer and causing gene reexpression in vivo. The purpose of the current study was to evaluate a panel of genes inactivated by promoter hypermethylation in human lung cancer for silencing by this epigenetic mechanism in murine lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), cigarette smoke, or arising spontaneously. Cadherin-13, estrogen receptor-alpha, progesterone receptor, and runt-related transcription factor-3 were frequently methylated in mouse lung tumor-derived cell lines, whereas cadherin-1 and suppressor of cytokine signaling-1 were not. Methylation within these four genes was associated with lack of expression that could be restored after treatment with 5-aza-2'-deoxycytidine and with methylation within the CpG island of each gene. Methylation-specific PCR revealed that methylation of these four genes occurred at prevalences of 24% to 69% in primary lung tumors arising spontaneously or induced by exposure to cigarette smoke or NNK. Estrogen receptor-alpha methylation was more frequent in spontaneously occurring lung cancer than cigarette smoke-induced or NNK-induced lung cancer, whereas runt-related transcription factor-3 showed the opposite relationship. Thus, genes can be targeted for inactivation by methylation, depending on exposure history. This study indicates that methylation events frequently observed in human lung cancer are recapitulated in the mouse model and identifies four potential biomarkers for assessing intervention approaches for reversing epigenetically mediated gene silencing.
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Biomarcadores de Tumor/genética , Metilación de ADN , Silenciador del Gen , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Cadherinas/genética , Islas de CpG/efectos de los fármacos , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Decitabina , Receptor alfa de Estrógeno/genética , Genes Relacionados con las Neoplasias , Humanos , Neoplasias Pulmonares/inducido químicamente , Ratones , Regiones Promotoras Genéticas , Receptores de Progesterona/genética , Factores de Transcripción TCF/genética , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
BACKGROUND: Postoperative atrial fibrillation (POAF) is the most common complication occurring after cardiac surgery. Multiple studies have shown significantly increased risks of stroke, myocardial infarction, and death associated with POAF. Current prophylaxis strategies are inadequate to eliminate this problem. We examined the preclinical efficacy and safety of KCNH2-G628S gene transfer to prevent POAF. METHODS: Domestic pigs received AdKCNH2-G628S by epicardial atrial gene painting and atrial pacemaker implantation for continuous-burst pacing to induce atrial fibrillation. In an initial dose-ranging evaluation, 3 pigs received 5 × 1010 to 5 × 1011 virus particles. In the formal study, 16 pigs were randomized to 3 groups: 5 × 1011 virus particles of AdKCNH2-G628S with 20% Pluronic P407 in saline, 20% Pluronic P407 in saline with no virus, and saline alone. Animals were followed with daily efficacy and safety evaluations through the period of peak adenovirus-mediated transgene expression. After 14 days, pacing was discontinued, and the animals were followed in sinus rhythm for an additional 14 days to assess any longer-term toxicity. RESULTS: In the primary efficacy analysis, the G628S animals exhibited a significant increase in the average time in sinus rhythm compared with the Pluronic control group (59 ± 7% vs 14 ± 6%; P = .009). There was no significant difference between the Pluronic and saline controls (14 ± 6% vs 32 ± 12%; P = .16). Safety assessment showed improved left ventricular function in the G628S animals; otherwise there were no significant differences among the groups in any safety measure. CONCLUSIONS: These data indicate that KCNH2-G628S gene therapy can successfully and safely reduce the risk of AF.
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Fibrilación Atrial , Procedimientos Quirúrgicos Cardíacos , Animales , Canal de Potasio ERG1 , Terapia Genética , Atrios Cardíacos , Humanos , Periodo Posoperatorio , PorcinosRESUMEN
Loss of expression of the death-associated protein (DAP)-kinase gene by aberrant promoter methylation may play an important role in cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the DAP-kinase gene in adenocarcinomas induced in mice by chronic exposure to mainstream cigarette smoke, the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate, and the occupational carcinogen methylene chloride. The timing for inactivation was also determined in alveolar hyperplasias that arise in lung cancer induced in the A/J mouse by NNK. The DAP-kinase gene was not expressed in three of five NNK-induced lung tumor-derived cell lines or in a spontaneously arising lung tumor-derived cell line. Treatment with 5-aza-2'-deoxycytidine restored expression; dense methylation throughout the DAP-kinase CpG island detected by bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the DAP-kinase gene in 43% of tumors associated with cigarette smoke, a frequency similar to those reported in human non-small cell lung cancer. In addition, DAP-kinase methylation was detected in 52%, 60%, and 50% of tumors associated with NNK, vinyl carbamate, and methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced hyperplasias and adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for tumor development in the mouse lung. Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of adenocarcinoma development. This study is the first to use a murine model of cigarette smoke-induced lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development of this disease in humans.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Carcinógenos/toxicidad , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/genética , Nitrosaminas/toxicidad , Fumar/efectos adversos , Uretano/análogos & derivados , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Islas de CpG , Metilación de ADN/efectos de los fármacos , Proteínas Quinasas Asociadas a Muerte Celular , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen/efectos de los fármacos , Hiperplasia , Pulmón/patología , Neoplasias Pulmonares/inducido químicamente , Cloruro de Metileno/toxicidad , Ratones , Ratones Endogámicos A , Regiones Promotoras Genéticas/efectos de los fármacos , Uretano/toxicidadRESUMEN
Interleukin-1 (IL-1) plays an important role in the pathophysiology of osteoarthritis (OA), and gene transfer of IL-1 receptor antagonist (IL-1Ra) holds promise for OA treatment. A preclinical safety and biodistribution study evaluated a self-complementary adeno-associated viral vector carrying rat IL-1Ra transgene (sc-rAAV2.5rIL-1Ra) at 5 × 10(8), 5 × 10(9), or 5 × 10(10) vg/knee, or human IL-1Ra transgene (sc-rAAV2.5hIL-1Ra) at 5 × 10(10) vg/knee, in Wistar rats with mono-iodoacetate (MIA)-induced OA at days 7, 26, 91, 180, and 364 following intra-articular injection. The MIA-induced OA lesions were consistent with the published data on this model. The vector genomes persisted in the injected knees for up to a year with only limited vector leakage to systemic circulation and uptake in tissues outside the knee. Low levels of IL-1Ra expression and mitigation of OA lesions were observed in the vector-injected knees, albeit inconsistently. Neutralizing antibodies against the vector capsid developed in a dose-dependent manner, but only the human vector induced a small splenic T-cell immune response to the vector capsid. No local or systemic toxicity attributable to vector administration was identified in the rats as indicated by clinical signs, body weight, feed consumption, clinical pathology, and gross and microscopic pathology through day 364. Taken together, the gene therapy vector demonstrated a favorable safety profile.
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Microcystins, a family of cyclic heptapeptides produced by the cyanobacteria, Microcystis aeruginosa, have documented hepatotoxic and tumor promoting activities. The purpose of this study was to evaluate the toxicity of inhaled microcystin LR (microcystin). Male BALB/c mice were exposed by nose-only inhalation to 260-265 microg microcystin/m(3) for 7 days. The low-, mid- and high-dose groups were exposed for 0.5, 1, and 2h, respectively. Control animals were sham exposed to aerosolized vehicle. Treatment-related microscopic lesions were observed only in the nasal cavity of the mid- and high-dose groups. These lesions consisted of minimal to moderate multifocal degeneration and necrosis of the respiratory epithelium, with variable neutrophilic inflammation and minimal to marked degeneration, necrosis, and atrophy of the olfactory epithelium. The no-adverse-effect dose for the nasal lesions was approximately 3 microg/kg body weight, or 20 ng/cm(2) of nasal epithelium. In serum, only two protein peaks, occurring at m/zs of 11,688 and 11,829 Da, exhibited decreases in intensity that were microcystin dose-dependent. While these proteins have not been positively identified, they may be useful in the future as biomarkers of microcystin exposure in humans.
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Mucosa Olfatoria/efectos de los fármacos , Péptidos Cíclicos/toxicidad , Mucosa Respiratoria/efectos de los fármacos , Administración por Inhalación , Análisis de Varianza , Animales , Proteínas Sanguíneas , Relación Dosis-Respuesta a Droga , Técnicas Histológicas , Masculino , Toxinas Marinas , Ratones , Ratones Endogámicos BALB C , Microcistinas , Necrosis , Nivel sin Efectos Adversos Observados , Mucosa Olfatoria/patología , Péptidos Cíclicos/administración & dosificación , Mucosa Respiratoria/patología , Factores de TiempoRESUMEN
Oncostatin M (OSM), an interleukin 6-type cytokine, induces sustained up-regulation of CCAAT/enhancer-binding protein (C/EBP) delta mRNA and protein in nonneoplastic HC11 mouse mammary epithelial cells. This up-regulation is dependent on signaling by phospho-Stat3 (signal transducers and activators of transcription). The same signaling pathway is activated in two human breast cancer cell lines, a neoplastic mouse mammary epithelial cell line and a second nonneoplastic mouse mammary epithelial cell line. [3H]Thymidine incorporation and flow cytometry demonstrate that OSM inhibits the growth of HC11 cells by reducing the number of S-phase cells. These phenotypic changes are accompanied by reduced expression of S-phase genes with a corresponding increased expression of G0 genes in HC11 cells. Reduction of C/EBPdelta protein in HC11 cells by expression of a C/EBPdelta antisense construct inhibits OSM-mediated growth arrest. These data demonstrate that OSM induces up-regulation of C/EBPdelta via a Stat3-dependent pathway in mammary epithelial cells and that the growth inhibition induced by OSM depends on the presence of C/EBPdelta.
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Antineoplásicos/farmacología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Epitelio/metabolismo , Inhibidores de Crecimiento/farmacología , Glándulas Mamarias Animales/metabolismo , Péptidos/farmacología , Animales , Northern Blotting , Western Blotting , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Humanos , Ratones , Oncostatina M , Fosforilación , Fase S , Factor de Transcripción STAT3 , Transducción de Señal , Timidina/metabolismo , Factores de Tiempo , Transactivadores/metabolismo , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 µg.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Proteínas Recombinantes/inmunología , Vacunación/métodos , Hidróxido de Aluminio/inmunología , Animales , Carbunco/sangre , Carbunco/prevención & control , Vacunas contra el Carbunco/administración & dosificación , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Mutación , Pseudomonas fluorescens/genética , Conejos , Proteínas Recombinantes/genética , Esporas Bacterianas/inmunologíaRESUMEN
Alpha-1 antitrypsin (α1AT) deficiency is a common autosomal recessive disorder characterized by a marked reduction in serum α1AT levels, lung and liver disease. α1AT is mainly produced and secreted by hepatocytes, with its primary function to protect the lung against the proteolytic activity of neutrophil elastase. Serum α1AT levels <11 µM are associated with progressive destruction of lung parenchyma and early-onset of panacinar emphysema in the age range 35-45. The current approved treatment for α1AT deficiency is a costly protein augmentation therapy requiring weekly intravenous infusion of purified α1AT from pooled human plasma. Gene therapy offers the advantage of a single vector administration, eliminating the burden of the repeated purified protein infusions, with the consequent reduced overall drug cost and improved compliance. We have developed a novel, highly efficient gene therapy approach for α1AT deficiency based on the administration of AAVrh.10hα1AT, an adeno-associated viral vector serotype rh.10 coding for normal M-type human α1AT via the intrapleural route. On the basis of prior murine studies, this approach provides sustained α1AT proximal to the lung with a highly efficient vector. In support of a clinical trial for this approach, we carried out a study to assess the safety of intrapleural administration of AAVrh.10hα1AT to 280 mice and 36 nonhuman primates. The data demonstrate that this approach is safe, with no toxicity issues. Importantly, there was persistent expression of the human α1AT mRNA in chest cavity cells for the duration of the study (6 months in mice and 1 year in nonhuman primates). Together, these data support the initiation of a clinical trial of intrapleural human AAVrh.10hα1AT for the treatment of α1AT deficiency.
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Dependovirus/genética , Terapia Genética , Vectores Genéticos/administración & dosificación , Deficiencia de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Animales , Vectores Genéticos/efectos adversos , Humanos , Inyecciones , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Primates , alfa 1-Antitripsina/metabolismoRESUMEN
Orthopoxviruses encode multiple proteins that modulate host immune responses. We determined whether cowpox virus (CPXV), a representative orthopoxvirus, modulated innate and acquired immune functions of human primary myeloid DCs and plasmacytoid DCs and monocyte-derived DCs (MDDCs). A CPXV infection of DCs at a multiplicity of infection of 10 was nonproductive, altered cellular morphology, and failed to reduce cell viability. A CPXV infection of DCs did not stimulate cytokine or chemokine secretion directly, but suppressed toll-like receptor (TLR) agonist-induced cytokine secretion and a DC-stimulated mixed leukocyte reaction (MLR). LPS-stimulated NF-κB nuclear translocation and host cytokine gene transcription were suppressed in CPXV-infected MDDCs. Early viral immunomodulatory genes were upregulated in MDDCs, consistent with early DC immunosuppression via synthesis of intracellular viral proteins. We conclude that a nonproductive CPXV infection suppressed DC immune function by synthesizing early intracellular viral proteins that suppressed DC signaling pathways.
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Virus de la Viruela Vacuna/inmunología , Virus de la Viruela Vacuna/patogenicidad , Células Dendríticas/inmunología , Células Dendríticas/virología , Evasión Inmune , Supervivencia Celular , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Humanos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/inmunologíaRESUMEN
Recombinant adeno-associated virus (rAAV) vectors offer promise for gene therapy of alpha-1 antitrypsin (AAT) deficiency. A toxicology study in mice evaluated intramuscular injection of an rAAV vector expressing human AAT (rAAV-CB-hAAT) produced using a herpes simplex virus (HSV) complementation system or a plasmid transfection (TFX) method at doses of 3 × 10(11) vg (1.2 × 10(13) vg/kg) for both vectors and 2 × 10(12) vg (8 × 10(13) vg/kg) for the HSV-produced vector. The HSV-produced vector had favorable in vitro characteristics in terms of purity, efficiency of transduction, and hAAT expression. There were no significant differences in clinical findings or hematology and clinical chemistry values between test article and control groups and no gross pathology findings. Histopathological examination demonstrated minimal to mild changes in skeletal muscle at the injection site, consisting of focal chronic interstitial inflammation and muscle degeneration, regeneration, and vacuolization, in vector-injected animals. At the 3 × 10(11) vg dose, serum hAAT levels were higher with the HSV-produced vector than with the TFX-produced vector. With the higher dose of HSV-produced vector, the increase in serum hAAT levels was dose-proportional in females and greater than dose-proportional in males. Vector copy numbers in blood were highest 24 hr after dosing and declined thereafter, with no detectable copies present 90 days after dosing. Antibodies to hAAT were detected in almost all vector-treated animals, and antibodies to HSV were detected in most animals that received the highest vector dose. These results support continued development of rAAV-CB-hAAT for treatment of AAT deficiency.