RESUMO
As electronic cigarette (E-cig) use, also known as "vaping", has rapidly increased in popularity, data regarding potential pathologic effects are recently emerging. Recent associations between vaping and lung pathology have led to an increased need to scrutinize E-cigs for adverse health impacts. Our previous work (and others) has associated vaping with Ca2+-dependent cytotoxicity in cultured human airway epithelial cells. Herein, we develop a vaped e-liquid pulmonary exposure mouse model to evaluate vaping effects in vivo. Using this model, we demonstrate lung pathology through the use of preclinical measures, that is, the lung wet: dry ratio and lung histology/H&E staining. Further, we demonstrate that acute vaping increases macrophage chemotaxis, which was ascertained using flow cytometry-based techniques, and inflammatory cytokine production, via Luminex analysis, through a Ca2+-dependent mechanism. This increase in macrophage activation appears to exacerbate pulmonary pathology resulting from microbial infection. Importantly, modulating Ca2+ signaling may present a therapeutic direction for treatment against vaping-associated pulmonary inflammation.
Assuntos
Cálcio/metabolismo , Misturas Complexas/efeitos adversos , Infecções por Klebsiella/etiologia , Klebsiella pneumoniae/patogenicidade , Pneumonia Bacteriana/etiologia , Vaping/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Quimiotaxia/imunologia , Sistemas Eletrônicos de Liberação de Nicotina , Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/fisiologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
RATIONALE: The popularity of new and emerging tobacco products such as E-cigarettes (E-cigs) is rapidly expanding worldwide. However, uncertainties surrounding the potential health consequences due to the use of such products exist and warrant further study. METHODS: Cultured A549 and Calu-3 airway epithelia were exposed to three out of the eight types of JUUL brand e-liquids ("Mint", "Virginia Tobacco" and "Menthol", all containing 3% nicotine at 1% and 3% (vol/vol) dilutions) and assessed for viability using a resazurin-based assay. Intracellular Ca2+ levels were measured using fluorescent indicators and pro-inflammatory cytokine levels were monitored by quantitative PCR (qPCR). Cultures were also analyzed by flow cytometry to evaluate apoptotic markers and cell viability. RESULTS: Exposing the airway epithelial cells to the flavored JUUL e-liquids led to significant cytotoxicity, with the "Mint" flavor being the overall most cytotoxic. The "Mint" flavored e-liquid also led to significant elevations in intracellular Ca2+ and upregulation of the pro-inflammatory cytokine IL-6 and early apoptotic marker Annexin V. CONCLUSIONS: JUUL e-liquid challenge resulted in a loss of airway epithelial cell viability, induced pro-inflammatory responses and eventually caused apoptosis.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Sistemas Eletrônicos de Liberação de Nicotina , Células Epiteliais/efeitos dos fármacos , Mucosa Respiratória/citologia , Células A549 , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Citocinas/análise , Citocinas/metabolismo , Aromatizantes/toxicidade , Humanos , Mentha , Nicotina/análise , Mucosa Respiratória/efeitos dos fármacosRESUMO
4-Anilinoquinolines were identified as potent and narrow-spectrum inhibitors of the cyclinâ G associated kinase (GAK), an important regulator of viral and bacterial entry into host cells. Optimization of the 4-anilino group and the 6,7-quinoline substituents produced GAK inhibitors with nanomolar activity, over 50 000-fold selectivity relative to other members of the numb-associated kinase (NAK) subfamily, and a compound (6,7-dimethoxy-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine; 49) with a narrow-spectrum kinome profile. These compounds may be useful tools to explore the therapeutic potential of GAK in prevention of a broad range of infectious and systemic diseases.
Assuntos
Compostos de Anilina/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Quinazolinas/química , Compostos de Anilina/metabolismo , Compostos de Anilina/farmacologia , Sítios de Ligação , Domínio Catalítico , Desenho de Fármacos , Endocitose/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Relação Estrutura-Atividade , Vírus/patogenicidadeRESUMO
BACKGROUND AIMS: Evaluation of the BD Stem Cell Enumeration Kit was conducted at four clinical sites with flow cytometry CD34(+) enumeration to assess agreement between two investigational methods: (i) the BD FACSCanto II and BD FACSCalibur systems and (ii) the predicate method (Beckman Coulter StemKit and StemTrol, Immunotech SAS, Beckman Coulter, Marseille Cedex 9, France). METHODS: Leftover and delinked specimens (n = 1032) from clinical flow cytometry testing were analyzed on the BD FACSCanto II (n = 918) and BD FACSCalibur (n = 905) in normal and mobilized blood, frozen and thawed bone marrow and leucopheresis and cord blood anticoagulated with citrate phosphate dextrose, anticoagulant citrate dextrose-solution A, heparin and ethylenediaminetetraacetate, alone or in combination. Fresh leucopheresis analysis addressed site equivalency for sample preparation, testing and analysis. RESULTS: The mean relative bias showed agreement within predefined parameters for the BD FACSCanto II (-2.81 to 4.31 ±7.1) and BD FACSCalibur (-2.69 to 5.2 ±7.9). Results are reported as absolute and relative differences compared with the predicate for viable CD34(+), percentage of CD34(+) in CD45(+) and viable CD45(+) populations (or gates). Bias analyses of the distribution of the predicate low, mid and high bin values were done using BD FACSCanto II optimal gating and BD FACSCalibur manual gating for viable CD34(+), percentage of CD34(+) in CD45(+) and viable CD45(+). Bias results from both investigational methods show agreement. Deming regression analyses showed a linear relationship with R(2) > 0.92 for both investigational methods. DISCUSSION: In conclusion, the results from both investigational methods demonstrated agreement and equivalence with the predicate method for enumeration of absolute viable CD34(+), percentage of viable CD34(+) in CD45(+) and absolute viable CD45(+) populations.
Assuntos
Antígenos CD34/metabolismo , Citometria de Fluxo/métodos , Transplante de Células-Tronco , Células-Tronco/citologia , Antígenos CD34/imunologia , Contagem de Células , Linhagem da Célula/genética , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo/instrumentação , Humanos , Células-Tronco/imunologiaRESUMO
Benzimidazole nucleosides have been shown to be potent inhibitors of human cytomegalovirus (HCMV) replication in vitro. As part of the exploration of structure-activity relationships within this series, we synthesized the 2-isopropylamino derivative (3322W93) of 1H-beta-D-ribofuranoside-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and the biologically unnatural L-sugars corresponding to both compounds. One of the L derivatives, 1H-beta-L-ribofuranoside-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94), showed significant antiviral potency in vitro against both laboratory HCMV strains and clinical HCMV isolates, including those resistant to ganciclovir (GCV), foscarnet, and BDCRB. 1263W94 inhibited viral replication in a dose-dependent manner, with a mean 50% inhibitory concentration (IC(50)) of 0.12 +/- 0.01 microM compared to a mean IC(50) for GCV of 0.53 +/- 0.04 microM, as measured by a multicycle DNA hybridization assay. In a single replication cycle, 1263W94 treatment reduced viral DNA synthesis, as well as overall virus yield. HCMV mutants resistant to 1263W94 were isolated, establishing that the target of 1263W94 was a viral gene product. The resistance mutation was mapped to the UL97 open reading frame. The pUL97 protein kinase was strongly inhibited by 1263W94, with 50% inhibition occurring at 3 nM. Although HCMV DNA synthesis was inhibited by 1263W94, the inhibition was not mediated by the inhibition of viral DNA polymerase. The parent benzimidazole D-riboside BDCRB inhibits viral DNA maturation and processing, whereas 1263W94 does not. The mechanism of the antiviral effect of L-riboside 1263W94 is thus distinct from those of GCV and of BDCRB. In summary, 1263W94 inhibits viral replication by a novel mechanism that is not yet completely understood.