RESUMO
During competitive freestyle swimming, the change of direction requires a turn followed by â¼15 m of underwater kicking at various intensities that require a â¼5 s breath-hold (BH). Upon surfacing, breathing must be regulated, as head rotation is necessary to facilitate the breath while completing the length of the pool (â¼25 s). This study compared the respiratory and muscle deoxygenation responses of regulated breathing vs. free breathing, during these 25-5 s cycles. It was hypothesized that with the addition of a BH and sprint during heavy-intensity (HVY) exercise, oxygen uptake (VO2) and oxygen saturation (SatO2) would decrease, and muscle deoxygenation ([HHb]) and total hemoglobin ([Hbtot]) would increase. Ten healthy male participants (24 ± 3 years) performed 4-6 min trials of HVY cycling in the following conditions: (1) continuous free breathing (CONLD); (2) continuous with 5 s BH every 25 s (CONLD-BH); (3) Fartlek (FLK), a 5 s sprint followed by 25 s of HVY; and (4) a combined Fartlek and BH (FLK-BH). Continuous collection of VO2 and SatO2, [Hbtot], and [HHb] via breath-by-breath gas analysis and near-infrared spectroscopy (normalized to baseline) was performed. Breathing frequency and tidal volumes were matched between CONLD and CONLD-BH and between FLK and FLK-BH. As a result, VO2 was unchanged between CONLD (2.12 ± 0.35 L/min) and CONLD-BH (2.15 ± 0.42 L/min; p = 0.116) and between FLK (2.24 ± 0.40 L/min) and FLK-BH (2.20 ± 0.45 L/min; p = 0.861). SatO2 was higher in CONLD (63 ± 1.9%) than CONLD-BH (59 ± 3.3%; p < 0.001), but was unchanged between FLK (61 ± 2.2%) and FLK-BH (62 ± 3.1%; p = 0.462). Δ[Hbtot] is higher in CONLD (3.3 ± 1.6 µM) than CONLD-BH (-2.5 ± 1.2 µM; Δ177%; p < 0.001), but was unchanged between FLK (2.0 ± 1.6 µM) and FLK-BH (0.82 ± 1.4 µM; p = 0.979). Δ[HHb] was higher in CONLD (7.3 ± 1.8µM) than CONLD-BH (7.0 ± 2.0µM; Δ4%; p = 0.011) and lower in FLK (6.7 ± 1.8µM) compared to FLK-BH (8.7 ± 2.4 µM; p < 0.001). It is suggested that the unchanged VO2 between CONLD and CONLD-BH was supported by increased deoxygenation as reflected by decreased Δ[Hbtot] and blunted Δ[HHb], via apneic-driven redistribution of blood flow away from working muscles, which was reflected by the decreased SatO2. However, the preserved VO2 during FLK-BH vs. FLK has been underpinned by an increase in [HHb].
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OBJECTIVES: Inflammation is crucial for the pathogenesis of acquired sensorineural hearing loss, but the precise mechanism involved remains elusive. Among a number of inflammatory mediators, tumor necrosis factor-alpha (TNF-α) plays a pivotal role in cisplatin ototoxicity. However, TNF-α alone is cytotoxic to cochlear sensory cells only at the extremely high concentrations, suggesting the involvement of other factors that may sensitize cells to TNF-α cytotoxicity. Since interferon gamma (IFN-γ) importantly contributes to the cochlear inflammatory processes, we aim to determine whether and how IFN-γ affects TNF-α cytotoxicity to cochlear sensory cells. METHODS: TNF-α expression was determined with western blotting in RSL cells and immunolabeling of mouse temporal bone sections. HEI-OC1 cell viability was determined with MTT assays, cytotoxicity assays, and cytometric analysis with methylene blue staining. Cochlear sensory cell injury was determined in the organotypic culture of the mouse organ of Corti. RESULTS: Spiral ligament fibrocytes were shown to upregulate TNF-α in response to pro-inflammatory stimulants. We demonstrated IFN-γ increases the susceptibility of HEI-OC1 cells to TNF-α cytotoxicity via JAK1/2-STAT1 signaling. TNFR1-mediated Caspase-1 activation was found to mediate the sensitization effect of IFN-γ on TNF-α cytotoxicity. The combination of IFN-γ and TNF-α appeared to augment cisplatin cytotoxicity to cochlear sensory cells ex vivo. CONCLUSIONS: Taken together, these findings suggest the involvement of IFN-γ in the sensitization of cochlear cells to TNF-α cytotoxicity, which would enable us to better understand the complex mechanisms underlying inflammation-mediated cochlear injury.
Assuntos
Células Ciliadas Auditivas/efeitos dos fármacos , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Sobrevivência Celular , Cisplatino/farmacologia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Inflamação , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The original version of this article unfortunately contained a mistake.
RESUMO
PURPOSE: The purpose was to compare the singular and combined effects of 5 s breath holds (BH) and 5 s sprints, every 30 s, during continuous high-intensity exercise, on ventilation ([Formula: see text]), oxygen uptake ([Formula: see text]O2) and associated kinetics (τ), carbon dioxide production ([Formula: see text]CO2), and arterialized-capillary lactate concentration ([La-]). METHODS: Ten men (24 ± 3 years) performed 4-6 min ergometer protocols that included a step-transition from 20 W to a power output of 50% of the difference between lactate threshold and [Formula: see text]O2 peak (Δ50%) including: (1) a continuous protocol (CONT) with free breathing, (2) an intermittent BH protocol (CONT-BH); repeated cycles of 5 s BH: 25 s free breathing, (3) a Fartlek protocol (Fartlek); repeated 5 s at peak aerobic power output: 25 s at Δ50%; (4) combining the 5 s Fartlek and CONT-BH protocol (Fartlek-BH). Breath-by-breath gas exchange, measured by mass spectrometry and turbine, was recorded. RESULTS: [Formula: see text] E (L min-1) was greater (p < 0.05) than CONT (90 ± 7) in all conditions CONT-BH (98 ± 16), Fartlek (105 ± 10), and Fartlek-BH (101 ± 19). [Formula: see text]O2 and [Formula: see text]CO2 (L min-1) were unchanged in CONT-BH (2.73 ± 0.14 and 3.16 ± 0.38) and greater in Fartlek (2.85 ± 0.12 and 3.43 ± 0.16), compared to CONT (2.71 ± 0.12 and 3.12 ± 0.13). Whereas, [Formula: see text]CO2 during Fartlek-BH was higher (3.28 ± 0.35) and [Formula: see text]O2 was unchanged (2.73 ± 0.14). Fartlek-BH resulted in slower [Formula: see text]O2 kinetics (62.2 ± 19 s) and greater blood lactate concentrations (11.5 ± 2.7 mM), compared to CONT (48.8 ± 12 s; 9.0 ± 2.3 mM, respectively). CONCLUSIONS: It was demonstrated that the CONT-BH resulted in increased ventilation that sustained [Formula: see text]O2. However, [Formula: see text]O2 was restricted when an additional work was combined with the BH condition.
Assuntos
Suspensão da Respiração , Treinamento Intervalado de Alta Intensidade/métodos , Consumo de Oxigênio , Adulto , Dióxido de Carbono/metabolismo , Humanos , Ácido Láctico/sangue , Masculino , Troca Gasosa Pulmonar , Ventilação Pulmonar , Distribuição Aleatória , Natação/fisiologiaRESUMO
Persistent müllerian duct syndrome (PMDS) is a form of disordered sex development in which rudimentary müllerian structures are identified in phenotypically and genotypically normal males. It is caused by defects in the anti-müllerian hormone (AMH) system. Since patients with PMDS present with undescended testes, testosterone production by Leydig cells later in life is often decreased. The role of androgens in prostate cancerogenesis is well known. Cryptorchid testes and diminished testosterone levels in post-pubertal life in patients with PMDS play a protective role against prostate cancer, and hence, prostate cancer is a rare event in patients with PMDS. Herein, we present a patient who underwent prostatectomy for high-grade prostatic adenocarcinoma with persistent müllerian structures (such as rudimentary uterus, fallopian tubes, and cervix) identified during surgery. To our knowledge, this is the second case reported in the English language literature where PMDS was associated with prostate cancer.
Assuntos
Adenocarcinoma/complicações , Transtorno 46,XY do Desenvolvimento Sexual/complicações , Neoplasias da Próstata/complicações , Adenocarcinoma/patologia , Transtorno 46,XY do Desenvolvimento Sexual/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/patologiaRESUMO
Inflammatory reaction plays a crucial role in the pathophysiology of acquired hearing loss such as ototoxicity and labyrinthitis. In our earlier work, we showed the pivotal role of otic fibrocytes in cochlear inflammation and the critical involvement of proinflammatory cytokines in cisplatin ototoxicity. We also demonstrated that otic fibrocytes inhibit monocyte chemoattractant protein 1 (CCL2) upregulation in response to interleukin-10 (IL-10) via heme oxygenase 1 (HMOX1) signaling, resulting in suppression of cochlear inflammation. However, it is still unclear how IL-10 affects inflammation-mediated cochlear injury. Here we aim to determine how hypochlorous acid, a model inflammation mediator affects cochlear cell viability and how IL-10 affects hypochlorous acid-mediated cochlear cell injury. NaOCl, a sodium salt of hypochlorous acid (HOCl) was found to induce cytotoxicity of HEI-OC1 cells in a dose-dependent manner. Combination of hydrogen peroxide and myeloperoxidase augmented cisplatin cytotoxicity, and this synergism was inhibited by N-Acetyl-L-cysteine and ML-171. The rat spiral ligament cell line (RSL) appeared to upregulate the antioxidant response element (ARE) activities upon exposure to IL-10. RSL cells upregulated the expression of NRF2 (an ARE ligand) and NR0B2 in response to CoPP (a HMOX1 inducer), but not to ZnPP (a HMOX1 inhibitor). Adenovirus-mediated overexpression of NR0B2 was found to suppress CCL2 upregulation. IL-10-positive cells appeared in the mouse stria vascularis 1 day after intraperitoneal injection of lipopolysaccharide (LPS). Five days after injection, IL-10-positive cells were observed in the spiral ligament, spiral limbus, spiral ganglia, and suprastrial area, but not in the stria vascularis. IL-10R1 appeared to be expressed in the mouse organ of Corti as well as HEI-OC1 cells. HEI-OC1 cells upregulated Bcl-xL expression in response to IL-10, and IL-10 was shown to attenuate NaOCl-induced cytotoxicity. In addition, HEI-OC1 cells upregulated IL-22RA upon exposure to cisplatin, and NaOCl cytotoxicity was inhibited by IL-22. Taken together, our findings suggest that hypochlorous acid is involved in cochlear injury and that IL-10 potentially reduces cochlear injury through not only inhibition of inflammation but also enhancement of cochlear cell viability. Further studies are needed to determine immunological characteristics of intracochlear IL-10-positive cells and elucidate molecular mechanisms involved in the otoprotective activity of IL-10.
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Otitis media (OM) in children is clinically important because of its detrimental effects on the development of language and motor coordination and is the most common reason for prescription of antibiotics. A recent bacteriological change in OM pathogens such as emergence of antibiotic resistance and vaccination-mediated pathogenic shift urges us to develop a new non-antibiotic strategy. The middle ear epithelium abundantly secretes a variety of antimicrobial molecules suppressing the viability of the common OM pathogens. Recently, we have demonstrated that the adenoviral vector is able to deliver the ß-defensin 2 gene to the middle ear epithelial cells in vitro and in vivo, and adenovirus-mediated overexpression of ß-defensin 2 is protective for experimental OM. There are many hurdles limiting successful clinical application of gene delivery to the respiratory epithelium of the tubotympanum; however, intratympanic gene therapy with ß-defensin 2 is a promising alternative or adjuvant strategy for the management of OM.
Assuntos
Anti-Infecciosos/uso terapêutico , Terapia Genética , Otite Média/terapia , Animais , Antibacterianos/uso terapêutico , Orelha Média , Epitélio , Humanos , Otite Média/genéticaRESUMO
Cisplatin has many adverse effects, which are a major limitation to its use, including ototoxicity, neurotoxicity, and nephrotoxicity. This study aims to elucidate the protective mechanisms of erdosteine against cisplatin in HEI-OC1 cells. Pretreatment with erdosteine protects HEI-OC1 cells from cisplatin-medicated apoptosis, which is characterized by increase in nuclear fragmentation, DNA laddering, sub-G0/G1 phase, H2AX phosphorylation, PARP cleavage, and caspase-3 activity. Erdosteine significantly suppressed the production of reactive nitrogen/oxygen species and pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in cisplatin-treated cells. Studies using pharmacologic inhibitors demonstrated that phosphatidylinositol-3-kinases (PI3K) and protein kinase B (Akt) have protective roles in the action of erdosteine against cisplatin in HEI-OC1 cells. In addition, pretreatment with erdosteine clearly suppressed the phosphorylation of p53 (Ser15) and expression of p53-upregulated modulator of apoptosis. Erdosteine markedly induces expression of NF-E2-related factor 2 (Nrf2), which may contribute to the increase in expression of glutathione redox genes γ-l-glutamate-l-cysteine-ligase catalytic and γ-l-glutamate-l-cysteine-ligase modifier subunits, as well as in the antioxidant genes HO-1 and SOD2 in cisplatin-treated HEI-OC1 cells. Furthermore, the increase in expression of phosphorylated p53 induced by cisplatin is markedly attenuated by pretreatment with erdosteine in the mitochondrial fraction. This increased expression may inhibit the cytosolic expression of the apoptosis-inducing factor, cytochrome c, and Bax/Bcl-xL ratio. Thus, our results suggest that treatment with erdosteine is significantly attenuated cisplatin-induced damage through the activation of Nrf2-dependent antioxidant genes, inhibition of pro-inflammatory cytokines, activation of the PI3K/Akt signaling, and mitochondrial-related inhibition of pro-apoptotic protein expression in HEI-OC1 auditory cells.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Cisplatino/toxicidade , Citocinas/metabolismo , Orelha Interna/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Tioglicolatos/farmacologia , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Citocinas/imunologia , Citoproteção , Relação Dose-Resposta a Droga , Orelha Interna/imunologia , Orelha Interna/metabolismo , Orelha Interna/patologia , Regulação da Expressão Gênica , Mediadores da Inflamação/imunologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cochlear inflammatory diseases, such as tympanogenic labyrinthitis, are associated with acquired sensorineural hearing loss. Although otitis media is extremely frequent in children, tympanogenic labyrinthitis is not commonly observed, which suggests the existence of a potent anti-inflammatory mechanism modulating cochlear inflammation. In this study, we aimed to determine the molecular mechanism involved in cochlear protection from inflammation-mediated tissue damage, focusing on IL-10 and hemoxygenase-1 (HMOX1) signaling. We demonstrated that IL-10Rs are expressed in the cochlear lateral wall of mice and rats, particularly in the spiral ligament fibrocytes (SLFs). The rat SLF cell line was found to inhibit nontypeable Haemophilus influenzae (NTHi)-induced upregulation of monocyte chemotactic protein-1 (MCP-1; CCL2) in response to IL-10. This inhibition was suppressed by silencing IL-10R1 and was mimicked by cobalt Protoporphyrin IX and CO-releasing molecule-2. In addition, IL-10 appeared to suppress monocyte recruitment through reduction of NTHi-induced rat SLF cell line-derived chemoattractants. Silencing of HMOX1 was found to attenuate the inhibitory effect of IL-10 on NTHi-induced MCP-1/CCL2 upregulation. Chromatin immunoprecipitation assays showed that IL-10 inhibits NTHi-induced binding of p65 NF-κB to the distal motif in the promoter region of MCP-1/CCL2, resulting in suppression of NTHi-induced NF-κB activation. Furthermore, IL-10 deficiency appeared to significantly affect cochlear inflammation induced by intratympanic injections of NTHi. Taken together, our results suggest that IL-10/HMOX1 signaling is involved in modulation of cochlear inflammation through inhibition of MCP-1/CCL2 regulation in SLFs, implying a therapeutic potential for a CO-based approach for inflammation-associated cochlear diseases.
Assuntos
Quimiocina CCL2/imunologia , Cóclea/imunologia , Doenças Cocleares/imunologia , Regulação da Expressão Gênica/imunologia , Heme Oxigenase (Desciclizante)/imunologia , Heme Oxigenase-1/imunologia , Interleucina-10/imunologia , Proteínas de Membrana/imunologia , Animais , Linhagem Celular , Cóclea/patologia , Doenças Cocleares/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Ratos , Ratos Wistar , Elementos de Resposta/imunologia , Fator de Transcrição RelA/imunologiaRESUMO
Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.
Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Cisplatino/toxicidade , Cisteína/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular , Cisteína/farmacologia , Técnicas de Silenciamento de Genes , Heme Oxigenase-1/genética , Espaço Intracelular/metabolismo , Masculino , Desintoxicação Metabólica Fase II/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , Óxido Nítrico/biossíntese , Interferência de RNA , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genéticaRESUMO
Otitis media (OM), one of the most prevalent diseases in young children, is clinically important owing to its high incidence in children and its potential impact on language development and motor coordination. OM is the most common reason for the prescription of antibiotics (accounting for 25% of prescriptions) due to its extremely high incidence. A recent increase in antibiotic resistance among OM pathogens is emerging as a major public health concern globally, which led us to consider non-antibiotic approaches for the management of OM. In this study, we evaluated gene transfer of an antimicrobial peptide, human ß-defensin 2 (DEFB4), using an adenoviral vector (Ad5 with deletions of E1/E3/E4) as a potential therapeutic approach. We demonstrated that the transduction of human ß-defensin 2 induces the production of human ß-defensin 2 and suppresses non-typeable Haemophilus influenzae (NTHi) adhesion to human middle ear epithelial cells. Moreover, intratympanic inoculation of Ad-DEFB4 was found to attenuate NTHi-induced middle ear effusions without eliciting a significant immune response. Most importantly, intratympanic inoculation of Ad-DEFB4 appeared to significantly augment clearance of NTHi from middle ear cavity. Collectively, our results suggest that intratympanic gene delivery of antimicrobial molecules may serve as an alternative/adjuvant approach for the management of OM.
Assuntos
Orelha Média/efeitos dos fármacos , Células Epiteliais/fisiologia , Terapia Genética , Infecções por Haemophilus/terapia , Haemophilus influenzae/patogenicidade , Otite Média/prevenção & controle , beta-Defensinas/administração & dosagem , Adenoviridae/genética , Animais , Aderência Bacteriana/genética , Carga Bacteriana/efeitos dos fármacos , Células Cultivadas , Criança , Orelha Média/microbiologia , Orelha Média/patologia , Células Epiteliais/microbiologia , Vetores Genéticos , Infecções por Haemophilus/complicações , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Otite Média/etiologia , Deleção de Sequência/genética , Transgenes/genética , beta-Defensinas/genéticaRESUMO
Middle ear infection, otitis media (OM), is clinically important due to the high incidence in children and its impact on the development of language and motor coordination. Previously, we have demonstrated that the human middle ear epithelial cells up-regulate ß-defensin 2, a model innate immune molecule, in response to nontypeable Haemophilus influenzae (NTHi), the most common OM pathogen, via TLR2 signaling. NTHi does internalize into the epithelial cells, but its intracellular trafficking and host responses to the internalized NTHi are poorly understood. Here we aimed to determine a role of cytoplasmic pathogen recognition receptors in NTHi-induced ß-defensin 2 regulation and NTHi clearance from the middle ear. Notably, we observed that the internalized NTHi is able to exist freely in the cytoplasm of the human epithelial cells after rupturing the surrounding membrane. The human middle ear epithelial cells inhibited NTHi-induced ß-defensin 2 production by NOD2 silencing but augmented it by NOD2 over-expression. NTHi-induced ß-defensin 2 up-regulation was attenuated by cytochalasin D, an inhibitor of actin polymerization and was enhanced by α-hemolysin, a pore-forming toxin. NOD2 silencing was found to block α-hemolysin-mediated enhancement of NTHi-induced ß-defensin 2 up-regulation. NOD2 deficiency appeared to reduce inflammatory reactions in response to intratympanic inoculation of NTHi and inhibit NTHi clearance from the middle ear. Taken together, our findings suggest that a cytoplasmic release of internalized NTHi is involved in the pathogenesis of NTHi infections, and NOD2-mediated ß-defensin 2 regulation contributes to the protection against NTHi-induced otitis media.
Assuntos
Infecções por Haemophilus/imunologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Otite Média/imunologia , beta-Defensinas/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Orelha Média/metabolismo , Endocitose , Regulação da Expressão Gênica , Inativação Gênica , Gentamicinas/química , Infecções por Haemophilus/microbiologia , Humanos , Imunidade Inata , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Otite Média/microbiologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismoRESUMO
Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5'-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/ß-IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.
Assuntos
Elementos Facilitadores Genéticos/genética , Células Epiteliais/metabolismo , Haemophilus influenzae/fisiologia , NF-kappa B/metabolismo , beta-Defensinas/genética , Adulto , Técnicas de Tipagem Bacteriana , Sequência de Bases , Sítios de Ligação/genética , Regulação da Expressão Gênica , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , Fosforilação , Ligação Proteica/genética , Fator de Transcrição RelA/metabolismo , beta-Defensinas/metabolismoRESUMO
BACKGROUND: Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Currently, there is an urgent need for developing novel therapeutic agents for treating OM based on full understanding of molecular pathogenesis in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. OBJECTIVE: To provide a state-of-the-art review concerning recent advances in OM in the areas of molecular biology, biochemistry, genetics, and animal model studies and to discuss the future directions of OM studies in these areas. DATA SOURCES AND REVIEW METHODS: A structured search of the current literature (since June 2007). The authors searched PubMed for published literature in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. RESULTS: Over the past 4 years, significant progress has been made in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. These studies brought new insights into our understanding of the molecular and biochemical mechanisms underlying the molecular pathogenesis of OM and helped identify novel therapeutic targets for OM. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Our understanding of the molecular pathogenesis of OM has been significantly advanced, particularly in the areas of inflammation, innate immunity, mucus overproduction, mucosal hyperplasia, middle ear and inner ear interaction, genetics, genome sequencing, and animal model studies. Although these studies are still in their experimental stages, they help identify new potential therapeutic targets. Future preclinical and clinical studies will help to translate these exciting experimental research findings into clinical applications.
Assuntos
Otite Média , Animais , Biomarcadores/sangue , Quimiocinas/sangue , Criança , Citocinas/sangue , Modelos Animais de Doenças , Orelha Interna/imunologia , Orelha Média/imunologia , Medicina Baseada em Evidências , Expressão Gênica , Predisposição Genética para Doença , Perda Auditiva Condutiva/etiologia , Humanos , Imunidade Inata/imunologia , Otite Média/sangue , Otite Média/complicações , Otite Média/genética , Otite Média/imunologia , Otite Média/microbiologia , Otite Média/terapiaRESUMO
The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.
Assuntos
Quimiocina CXCL2/fisiologia , Haemophilus influenzae/imunologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Ligamento Espiral da Cóclea/citologia , Animais , Sítios de Ligação , Linhagem Celular/metabolismo , Linhagem Celular/microbiologia , Movimento Celular , Células Cultivadas/metabolismo , Células Cultivadas/microbiologia , Quimiocina CXCL2/biossíntese , Quimiocina CXCL2/genética , Regulação da Expressão Gênica , MAP Quinase Quinase 1/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Otite Média/imunologia , Ratos , Proteínas Recombinantes de Fusão , Transdução de Sinais , Especificidade da Espécie , Fator de Transcrição AP-1/metabolismo , Transfecção , Regulação para CimaRESUMO
The middle ear infection is the most common childhood infection. In order to elucidate the cell and molecular mechanisms involved in bacterial recognition and innate immune response, we have established a stable human middle ear cell line, which has contributed to the current knowledge concerning the molecular pathogenesis of the middle ear infection. The inner ear, a sensory organ responsible for hearing and balance, is filled with inner ear fluid, and disturbance of the fluid homeostasis results in dizziness and hearing impairment. It has been suggested that the endolymphatic sac (ES) may play a critical role in the fluid homeostasis of the inner ear. We have established a stable human ES cell line and are undertaking cell and molecular characterization of this cell line.
Assuntos
Orelha Média/citologia , Saco Endolinfático/citologia , Linhagem Celular , Orelha Média/ultraestrutura , Saco Endolinfático/ultraestrutura , Células Epiteliais/fisiologia , HumanosRESUMO
There is no licensed vaccine available against Moraxella catarrhalis, an exclusive human pathogen responsible for otitis media in children and respiratory infections in adults. We previously developed conjugate vaccine candidates based on lipooligosaccharides (LOSs) of M. catarrhalis serotypes A, B, and C, each of which was shown to cover a portion of the clinical strains. To generate conserved LOS antigens and eliminate a potential autoimmune response to a similar epitope between M. catarrhalis LOS moiety Galα1-4Galß1-4Glc and human P(k) antigen, two LOS mutants from strain O35E were constructed. Mutant O35Elgt5 or O35EgalE revealed a deletion of one or two terminal galactose residues of wild type O35E LOS. Each LOS molecule was purified, characterized, detoxified, and coupled to tetanus toxoid (TT) to form conjugates, namely dLOS-TT. Three subcutaneous immunizations using dLOS-TT from O35Elgt5 or O35EgalE elicited significant increases (a 729- or 1263-fold above the preimmune serum levels) of serum immunoglobulin (Ig)G against O35E LOS in rabbits with an adjuvant or without an adjuvant (an 140- or 140-fold above the preimmune serum levels). Rabbit antisera demonstrated elevated complement-mediated bactericidal activities against the wild type strain O35E. The rabbit sera elicited by O35Elgt5 dLOS-TT were further examined and showed cross bactericidal activity against all additional 19 M. catarrhalis strains and clinical isolates studied. Moreover, the rabbit sera displayed cross-reactivity not only among three serotype strains but also clinical isolates in a whole-cell enzyme-linked immunosorbent assay (ELISA), which was further confirmed under transmission electron microscopy. In conclusion, O35Elgt5 dLOS-TT may act as a vaccine against most M. catarrhalis strains and therefore can be used for further in vivo efficacy studies.
Assuntos
Vacinas Bacterianas/imunologia , Lipopolissacarídeos/imunologia , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Atividade Bactericida do Sangue , Proteínas do Sistema Complemento/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização Secundária/métodos , Imunoglobulina G/sangue , Injeções Subcutâneas , Lipopolissacarídeos/genética , Moraxella catarrhalis/genética , Infecções por Moraxellaceae/imunologia , Coelhos , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/imunologia , Vacinação/métodos , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologiaRESUMO
We herein investigated the role of the STAT signaling cascade in the production of pro-inflammatory cytokines and cisplatin ototoxicity. A significant hearing impairment caused by cisplatin injection was observed in Balb/c (wild type, WT) and STAT4(-/-), but not in STAT6(-/-) mice. Moreover, the expression levels of the protein and mRNA of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, were markedly increased in the serum and cochlea of WT and STAT4(-/-), but not STAT6(-/-) mice. Organotypic culture revealed that the shape of stereocilia bundles and arrays of sensory hair cell layers in the organ of Corti from STAT6(-/-) mice were intact after treatment with cisplatin, whereas those from WT and STAT4(-/-) mice were highly distorted and disarrayed after the treatment. Cisplatin induced the phosphorylation of STAT6 in HEI-OC1 auditory cells, and the knockdown of STAT6 by STAT6-specific siRNA significantly protected HEI-OC1 auditory cells from cisplatin-induced cell death and inhibited pro-inflammatory cytokine production. We further demonstrated that IL-4 and IL-13 induced by cisplatin modulated the phosphorylation of STAT6 by binding with IL-4 receptor alpha and IL-13Rα1. These findings suggest that STAT6 signaling plays a pivotal role in cisplatin-mediated pro-inflammatory cytokine production and ototoxicity.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Citocinas/metabolismo , Citotoxinas/toxicidade , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/patologia , Citocinas/genética , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Genes Reporter , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Perda Auditiva/induzido quimicamente , Perda Auditiva/patologia , Perda Auditiva/fisiopatologia , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/metabolismo , Fator de Transcrição STAT6/genética , Transcrição GênicaRESUMO
BACKGROUND: There are no licensed vaccines available against Moraxella catarrhalis, a significant human respiratory pathogen. Lipooligosaccharide (LOS) based conjugate vaccines derived from individual serotype M. catarrhalis only showed partial protection coverage. A vaccine combining LOS conjugates of two or three serotypes might provide a broader protection. METHODS: Mice were immunized intranasally with the combined conjugates consisting of LOS from serotype A and B or serotype A, B, and C followed by challenge with different M. catarrhalis strains of three serotypes. Mouse lungs, nasal washes, and sera were collected after each challenge for bacterial counts, histological evaluation, cytokine profiles, antibody level and binding activity determinations. RESULTS: Intranasal administration of the combined LOS conjugates not only enhanced pulmonary bacterial clearance of all three serotypes of M. catarrhalis strains in vaccinated mice, but also elevated serotype-specific anti-LOS immunoglobulin (Ig)A and IgG titers in nasal wash and serum respectively. Mice vaccinated with the combined LOS conjugates also showed increased interferon (IFN)-γ, interleukin (IL)-12, and IL-4 in the lungs after challenges. Compared to the control group, mice immunized with the combined LOS conjugates also showed reduced lung inflammation after M. catarrhalis infections. The hyperimmune sera induced by the combined conjugates exhibited a broad cross-reactivity toward all three serotypes of M. catarrhalis under transmission electron microscopy. CONCLUSIONS: The combined vaccine of serotype A and B LOS conjugates provides protection against most M. catarrhalis strains by eliciting humoral and cellular immune responses.