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Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis (CF), resulting in recurrent respiratory infections and exacerbations. Strategies to rehydrate airway mucus including inhibition of the epithelial sodium channel (ENaC) have the potential to improve mucosal defense by enhancing mucociliary clearance (MCC) and reducing the risk of progressive decline in lung function. In the current work, we evaluated the effects of AZD5634, an ENaC inhibitor that shows extended lung retention and safety profile as compared with previously evaluated candidate drugs, in healthy and CF preclinical model systems. We found that AZD5634 elicited a potent inhibition of amiloride-sensitive current in non-CF airway cells and airway cells derived from F508del-homozygous individuals with CF that effectively increased airway surface liquid volume and improved mucociliary transport (MCT) rate. AZD5634 also demonstrated efficacious inhibition of ENaC in sheep bronchial epithelial cells, translating to dose-dependent improvement of mucus clearance in healthy sheep in vivo. Conversely, nebulization of AZD5634 did not notably improve airway hydration or MCT in CF rats that exhibit an MCC defect, consistent with findings from a first single-dose evaluation of AZD5634 on MCC in people with CF. Overall, these findings suggest that CF animal models demonstrating impaired mucus clearance translatable to the human situation may help to successfully predict and promote the successful translation of ENaC-directed therapies to the clinic.
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Fibrose Cística , Canais Epiteliais de Sódio , Humanos , Ratos , Ovinos , Animais , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Amilorida/farmacologia , Depuração Mucociliar/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística/tratamento farmacológico , Mucosa RespiratóriaRESUMO
A series of isoindolinone compounds have been developed showing good in vitro potency on the Kv1.5 ion channel. By modification of two side chains on the isoindolinone scaffold, metabolically stable compounds with good in vivo PK profile could be obtained leaving the core structure unsubstituted. In this way, low microsomal intrinsic clearance (CLint) could be achieved despite a relatively high logD. The compounds were synthesized using the Ugi reaction, in some cases followed by Suzuki and Diels-Alder reactions, giving a diverse set of compounds in a small number of reaction steps.
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Isoindóis/farmacologia , Canal de Potássio Kv1.5/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Relação Dose-Resposta a Droga , Humanos , Isoindóis/síntese química , Isoindóis/química , Camundongos , Modelos Animais , Estrutura Molecular , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Relação Estrutura-AtividadeRESUMO
Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis and chronic bronchitis (CB). Rehydration by hypertonic saline is efficacious but suffers from a short duration of action. We tested whether epithelial sodium channel (ENaC) inhibition would rehydrate normal and dehydrated airways to increase mucociliary clearance (MCC) over a significant time frame. For this, we used a tool compound (Compound A), which displays nanomolar ENaC affinity and retention in the airway surface liquid (ASL). Using normal human bronchial epithelial cultures (HBECs) grown at an air-liquid interface, we evaluated in vitro potency and efficacy using short-circuit current (I(sc)) and ASL height measurements where it inhibited I(sc) and increased ASL height by â¼ 50% (0.052 µM at 6 h), respectively. The in vivo efficacy was investigated in a modified guinea pig tracheal potential difference model, where we observed an effective dose (ED50) of 5 µg/kg (i.t.), and by MCC measures in rats and sheep, where we demonstrated max clearance rates at 100 µg/kg (i.t.) and 75 µg/kg (i.t.), respectively. Acute cigarette smoke-induced ASL height depletion in HBECs was used to mimic the situation in patients with CB, and pretreatment prevented both cigarette smoke-induced ASL dehydration and lessened the decrease in ciliary beat frequency. Furthermore, when added after cigarette smoke exposure, Compound A increased the rate of ASL rehydration. In conclusion, Compound A demonstrated significant effects and a link between increased airway hydration, ciliary function, and MCC. These data support the hypothesis that ENaC inhibition may be efficacious in the restoration of mucus hydration and transport in patients with CB.
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Canais Epiteliais de Sódio/metabolismo , Muco/metabolismo , Mucosa Respiratória/metabolismo , Fumar/metabolismo , Animais , Transporte Biológico Ativo , Células Cultivadas , Cílios/metabolismo , Cílios/patologia , Feminino , Cobaias , Humanos , Ratos , Ratos Wistar , Mucosa Respiratória/patologia , Ovinos , Fumar/efeitos adversos , Fumar/patologiaRESUMO
A series of lactam sulfonamides has been discovered and optimized as inhibitors of the Kv1.5 potassium ion channel for treatment of atrial fibrillation. In vitro structure-activity relationships from lead structure C to optimized structure 3y are described. Compound 3y was evaluated in a rabbit PD-model and was found to selectively prolong the atrial effective refractory period at submicromolar concentrations.
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Canal de Potássio Kv1.5/antagonistas & inibidores , Lactamas/química , Bloqueadores dos Canais de Potássio/química , Pirrolidinonas/química , Sulfonamidas/química , Animais , Cães , Meia-Vida , Humanos , Canal de Potássio Kv1.5/metabolismo , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/farmacocinética , Pirrolidinonas/síntese química , Pirrolidinonas/farmacocinética , Coelhos , Ratos , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacocinéticaRESUMO
Rationale: Bronchiectasis is a chronic, progressive disease of bronchial dilation, inflammation, and scarring leading to impaired mucociliary clearance and increased susceptibility to infection. Identified causes include previous severe respiratory infections. A small, single-center UK study demonstrated a reduction in bronchiectasis exacerbations during the first year of the coronavirus disease (COVID-19) pandemic. No studies have been conducted in a U.S. (commercially insured) cohort to date. Objectives: To explore the impact of the COVID-19 pandemic on the frequency of exacerbations in a large cohort of commercially insured U.S. patients with bronchiectasis by testing the hypothesis that U.S. patients with bronchiectasis had fewer exacerbations during the pandemic. Methods: This retrospective observational cohort study used health insurance claims data from Optum's deidentified Clinformatics Data Mart database, which included U.S. patients and their covered dependents. Eligible patients were ⩾18 years of age with bronchiectasis; patients with other respiratory conditions were excluded. The main study cohort excluded patients with frequent asthma and/or chronic obstructive pulmonary disease diagnoses. The primary objective was to compare the bronchiectasis exacerbation rates before and during the COVID-19 pandemic. Results: The median number of exacerbations per patient per year decreased significantly from the year before the COVID-19 pandemic to the first year of the pandemic (1 vs. 0; P < 0.01). More patients had zero exacerbations during the first year of the pandemic than the year prior (57% vs. 24%; McNemar's chi-square = 122.56; P < 0.01). Conclusions: In a U.S. population-based study of patients with International Classification of Diseases codes for bronchiectasis, the rate of exacerbations during Year 1 of the COVID-19 pandemic was reduced compared with the 2-year time period preceding the pandemic.
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Bronquiectasia , COVID-19 , Seguro , Humanos , Estudos Retrospectivos , Pandemias , COVID-19/epidemiologia , Bronquiectasia/epidemiologia , Progressão da DoençaRESUMO
Differential white blood cell counts are frequently used in diagnosis, patient stratification, and treatment selection to optimize therapy responses. Referral laboratories are often used but challenged with use of different hematology platforms, variable blood shipping times and storage conditions, and the different sensitivities of specific cell types. To extend the scientific literature and knowledge on the temporal commutability of blood samples between hematology analyzers, we performed a comparative ex-vivo study using four of the most utilized commercial platforms, focusing on the assessment of eosinophils given its importance in asthma management. Whole blood from healthy volunteers with and without atopy (n = 6+6) and participants with eosinophilic asthma (n = 6) were stored under different conditions (at 4, 20, 30, and 37°C, with or without agitation) and analyzed at different time points (3, 6, 24, 48 and 72h post-sampling) in parallel on the Abbott CELL-DYN Sapphire, Beckman Coulter DxH900, Siemens ADVIA 2120i and Sysmex XN-1000V. In the same blood samples, eosinophil-derived neurotoxin (EDN), eosinophil activation and death markers were analyzed. All platforms gave comparable measurements of cell differentials on fresh blood within the same day of sampling. However, by 24 hours, significant temporal and temperature-dependent differences were observed, most markedly for eosinophils. None of the platforms performed perfectly across all temperatures tested during the 72 hours, showing that handling conditions should be optimized depending on the cell type of interest and the hematology analyzer. Neither disease status (healthy vs. asthma) nor agitation of the sample affected the cell quantification result or EDN release. The eosinophil activation markers measured by flow cytometry increased with time, were influenced by temperature, and were higher in those with asthma versus healthy participants. In conclusion, hematology analyzer, time window from sampling until analysis, and temperature conditions must be considered when analyzing blood cell differentials, particularly for eosinophils, via central labs to obtain counts comparable to the values obtained in freshly sampled blood.
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Asma , Eosinófilos , Humanos , Asma/sangue , Asma/diagnóstico , Eosinófilos/citologia , Feminino , Masculino , Adulto , Contagem de Células Sanguíneas/instrumentação , Contagem de Células Sanguíneas/métodos , Contagem de Leucócitos/instrumentação , Contagem de Leucócitos/métodos , Pessoa de Meia-Idade , Hematologia/instrumentação , Hematologia/métodosRESUMO
BACKGROUND: Benralizumab is indicated as add-on therapy in patients with uncontrolled, severe eosinophilic asthma; it has not yet been evaluated in a large Asian population with asthma in a clinical trial. OBJECTIVE: To evaluate the efficacy and safety of benralizumab in patients with severe asthma in Asia. METHODS: MIRACLE (NCT03186209) was a randomized, Phase 3 study in China, South Korea, and the Philippines. Patients aged 12-75 years with severe asthma receiving medium-to-high-dose inhaled corticosteroid/long-acting ß2-agonists, stratified (2:1) by baseline blood eosinophil count (bEOS) (≥300/µL; <300/µL), were randomized (1:1) to benralizumab 30 mg or placebo. Endpoints included annual asthma exacerbation rate (AAER; primary endpoint), change from baseline at Week 48 in pre-bronchodilator (BD) forced expiratory volume in 1 second (pre-BD FEV1) and total asthma symptom score (TASS). Safety was evaluatedâ¯≤â¯Week 56. RESULTS: Of 695 patients randomized, 473 had baseline bEOS ≥300/µL (benralizumab nâ¯=â¯236; placebo nâ¯=â¯237). In this population, benralizumab significantly reduced AAER by 74% (rate ratio 0.26 [95% CI 0.19, 0.36], pâ¯<â¯0.0001) and significantly improved pre-BD FEV1 (least squares difference [LSD] 0.25 L [95% CI 0.17, 0.34], pâ¯<â¯0.0001) and TASS (LSD -0.25 [-0.45, -0.05], pâ¯=â¯0.0126) versus placebo. In patients with baseline bEOS <300/µL, there were numerical improvements in AAER, pre-BD FEV1, and TASS with benralizumab versus placebo. The frequency of adverse events was similar for benralizumab (76%) and placebo (80%) in the overall population. CONCLUSIONS: MIRACLE data reinforces the efficacy and safety of benralizumab for severe eosinophilic asthma in an Asian population, consistent with the global Phase 3 results.
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The T-type calcium channel inhibitor Mibefradil was reported to protect the heart from atrial remodeling, a key process involved in the development of atrial fibrillation and arrhythmias. Mibefradil is not a selective T-type calcium channel inhibitor and also affects the function of different ion channels. Our aim was to develop a selective T-type calcium channel inhibitor to validate the importance of T-type-related pharmacology in atrial fibrillation. Structural optimisation of a previously disclosed hit series focussed on minimising exposure to the central nervous system and improving pharmacokinetic properties, while maintain adequate potency and selectivity. This resulted in the design of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide, a novel, selective, peripherally restricted chemical probe to verify the role of T-type calcium channel inhibition on atrial fibrillation protection.
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Benzamidas/química , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio Tipo T/química , Animais , Benzamidas/síntese química , Benzamidas/farmacocinética , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/farmacocinética , Canais de Cálcio Tipo T/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Frequência Cardíaca/efeitos dos fármacos , Humanos , Relação Estrutura-AtividadeRESUMO
Diphenylphosphinic amides and diphenylphosphine oxides have been synthesized and tested as inhibitors of the Kv1.5 potassium ion channel as a possible treatment for atrial fibrillation. In vitro structure-activity relationships are discussed and several compounds with Kv1.5 IC(50) values of <0.5 µM were discovered. Selectivity over the ventricular IKs current was monitored and selective compounds were found. Results from a rabbit PD-model are included.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Canal de Potássio Kv1.5/antagonistas & inibidores , Óxidos/síntese química , Óxidos/farmacologia , Fosfinas/síntese química , Fosfinas/farmacologia , Amidas/química , Animais , Compostos de Bifenilo/química , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Óxidos/química , Fosfinas/química , Ácidos Fosfínicos/química , Ligação Proteica/efeitos dos fármacos , Coelhos , Relação Estrutura-AtividadeRESUMO
Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family mainly targeting cytosolic nonhistone substrates, such as α-tubulin, cortactin, and heat shock protein 90 to regulate cell proliferation, metastasis, invasion, and mitosis in tumors. We describe the identification and characterization of a series of 2-(difluoromethyl)-1,3,4-oxadiazoles (DFMOs) as selective nonhydroxamic acid HDAC6 inhibitors. By comparing structure-activity relationships and performing quantum mechanical calculations of the HDAC6 catalytic mechanism, we show that potent oxadiazoles are electrophilic substrates of HDAC6 and propose a mechanism for the bioactivation. We also observe that the inherent electrophilicity of the oxadiazoles makes them prone to degradation in water solution and the generation of potentially toxic products cannot be ruled out, limiting the developability for chronic diseases. However, the oxadiazoles demonstrate high oral bioavailability and low in vivo clearance and are excellent tools for studying the role of HDAC6 in vitro and in vivo in rats and mice.
Assuntos
Neoplasias , Oxidiazóis , Ratos , Camundongos , Animais , Desacetilase 6 de Histona , Oxidiazóis/farmacologia , Tubulina (Proteína)/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/químicaRESUMO
Background: Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Objectives: We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. Methods: The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. Results: We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. Conclusion: This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.
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Airway epithelial damage is a common feature in respiratory diseases such as COPD and has been suggested to drive inflammation and progression of disease. These features manifest as remodeling and destruction of lung epithelial characteristics including loss of small airways which contributes to chronic airway inflammation. Histone deacetylase 6 (HDAC6) has been shown to play a role in epithelial function and dysregulation, such as in cilia disassembly, epithelial to mesenchymal transition (EMT) and oxidative stress responses, and has been implicated in several diseases. We thus used ACY-1083, an inhibitor with high selectivity for HDAC6, and characterized its effects on epithelial function including epithelial disruption, cytokine production, remodeling, mucociliary clearance and cell characteristics. Primary lung epithelial air-liquid interface cultures from COPD patients were used and the impacts of TNF, TGF-ß, cigarette smoke and bacterial challenges on epithelial function in the presence and absence of ACY-1083 were tested. Each challenge increased the permeability of the epithelial barrier whilst ACY-1083 blocked this effect and even decreased permeability in the absence of challenge. TNF was also shown to increase production of cytokines and mucins, with ACY-1083 reducing the effect. We observed that COPD-relevant stimulations created damage to the epithelium as seen on immunohistochemistry sections and that treatment with ACY-1083 maintained an intact cell layer and preserved mucociliary function. Interestingly, there was no direct effect on ciliary beat frequency or tight junction proteins indicating other mechanisms for the protected epithelium. In summary, ACY-1083 shows protection of the respiratory epithelium during COPD-relevant challenges which indicates a future potential to restore epithelial structure and function to halt disease progression in clinical practice.
Assuntos
Inibidores de Histona Desacetilases , Doença Pulmonar Obstrutiva Crônica , Citocinas/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Inflamação/metabolismo , Pulmão/metabolismo , Mucinas/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/metabolismo , Proteínas de Junções Íntimas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Epithelial sodium channel (ENaC) inhibitors may offer clinical benefit in cystic fibrosis (CF); however, data are limited. We report the outcomes of a Phase I (NCT02679729) and a Phase Ib (NCT02950805) study of AZD5634, a novel inhaled ENaC inhibitor. METHODS: A Phase I, first-in-human, single-blind, placebo-controlled, single ascending dose, sequential dose group study assessed the safety, tolerability, and pharmacokinetics of AZD5634 in healthy subjects (n=53) in part A following inhaled doses up to 1700 µg, and, in part B, following administration of single inhaled (1700 µg) and intravenous (65 µg) doses. A Phase Ib, randomized, double-blind, placebo-controlled, single-dose, 2-way cross-over study assessed the effects of a single dose (600 µg) of inhaled AZD5634 on mucociliary clearance (MCC), pharmacokinetics and safety and tolerability in patients with CF (n=11). Nasal potential difference (NPD) was assessed as an in situ target engagement exploratory biomarker. RESULTS: Absolute bioavailability of AZD5634 after inhalation was approximately 3%, indicating minimal distribution into the systemic circulation. Urinary excretion was a minor elimination pathway. Administration of inhaled AZD5634 did not improve MCC in CF patients, but AZD5634 inhibited ENaC in the nasal epithelium, as measured by NPD. AZD5634 was safe and well tolerated in both studies. CONCLUSIONS: AZD5634 showed favorable pharmacokinetics and safety in healthy subjects and patients with CF. However, despite achieving target engagement, proof of mechanism was not achieved after a single dose in patients with CF. Further evaluation into multiple dose studies is warranted to explore its therapeutic potential.
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Fibrose Cística , Administração por Inalação , Estudos Cross-Over , Fibrose Cística/diagnóstico , Fibrose Cística/tratamento farmacológico , Método Duplo-Cego , Voluntários Saudáveis , Humanos , Método Simples-CegoRESUMO
Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor (TCR) signaling. As with all protein tyrosine phosphatases, the activity of PTPN22 is redox regulated, but if or how such regulation can modulate inflammatory pathways in vivo is not known. To determine this, we created a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 (C129S), a residue proposed to alter the redox regulatory properties of PTPN22 by forming a disulfide with the catalytic C227 residue. The C129S mutant mouse showed a stronger T-cell-dependent inflammatory response and development of T-cell-dependent autoimmune arthritis due to enhanced TCR signaling and activation of T cells, an effect neutralized by a mutation in Ncf1, a component of the NOX2 complex. Activity assays with purified proteins suggest that the functional results can be explained by an increased sensitivity to oxidation of the C129S mutated PTPN22 protein. We also observed that the disulfide of native PTPN22 can be directly reduced by the thioredoxin system, while the C129S mutant lacking this disulfide was less amenable to reductive reactivation. In conclusion, we show that PTPN22 functionally interacts with Ncf1 and is regulated by oxidation via the noncatalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T-cell-dependent autoimmunity.
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Doenças Autoimunes , Linfócitos T , Animais , Dissulfetos/metabolismo , Inflamação/metabolismo , Camundongos , Oxirredução , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
Cystic fibrosis (CF) is a recessive inherited disease caused by mutations affecting anion transport by the epithelial ion channel cystic fibrosis transmembrane conductance regulator (CFTR). The disease is characterized by mucus accumulation in the airways and intestine, but the major cause of mortality in CF is airway mucus accumulation, leading to bacterial colonization, inflammation and respiratory failure. Several drug targets are under evaluation to alleviate airway mucus obstruction in CF and one of these targets is the epithelial sodium channel ENaC. To explore effects of ENaC inhibitors on mucus properties, we used two model systems to investigate mucus characteristics, mucus attachment in mouse ileum and mucus bundle transport in piglet airways. We quantified mucus attachment in explants from CFTR null (CF) mice and tracheobronchial explants from newborn CFTR null (CF) piglets to evaluate effects of ENaC or sodium/hydrogen exchanger (NHE) inhibitors on mucus attachment. ENaC inhibitors detached mucus in the CF mouse ileum, although the ileum lacks ENaC expression. This effect was mimicked by two NHE inhibitors. Airway mucus bundles were immobile in untreated newborn CF piglets but were detached by the therapeutic drug candidate AZD5634 (patent WO, 2015140527). These results suggest that the ENaC inhibitor AZD5634 causes detachment of CF mucus in the ileum and airway via NHE inhibition and that drug design should focus on NHE instead of ENaC inhibition.
Assuntos
Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/metabolismo , Pulmão/metabolismo , Muco/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Bicarbonatos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Canais Epiteliais de Sódio/efeitos dos fármacos , Feminino , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Muco/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/genética , SuínosRESUMO
We have modified the periplasmic Escherichia coli glucose/galactose binding protein (GBP) and labelled with environmentally sensitive fluorophores to further explore its potential as a sensor for the evaluation of glucose concentration in airway surface liquid (ASL). We identified E149C/A213R GBP labelled with N,N'-Dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD, emission wavelength maximum 536nm) with a Kd for D-glucose of 1.02mM and a fluorescence dynamic range of 5.8. This sensor was specific for D-glucose and exhibited fluorescence stability in experiments for several hours. The use of E149C/A213R GBP-IANBD in the ASL of airway cells grown at air-liquid-interface (ALI) detected an increase in glucose concentration 10 minutes after raising basolateral glucose from 5 to 15mM. This sensor also reported a greater change in ASL glucose concentration in response to increased basolateral glucose in H441 airway cells compared to human bronchial epithelial cells (HBEC) and there was less variability with HBEC data than that of H441 indicating that HBEC more effectively regulate glucose movement into the ASL. The sensor detected glucose in bronchoalveolar lavage fluid (BALf) from diabetic db/db mice but not normoglycaemic wildtype mice, indicating limited sensitivity of the sensor at glucose concentrations <50µM. Using nasal inhalation of the sensor and spectral unmixing to generate images, E149C/A213R GBP-IANBD fluorescence was detected in luminal regions of cryosections of the murine distal lung that was greater in db/db than wildtype mice. In conclusion, this sensor provides a useful tool for further development to measure luminal glucose concentration in models of lung/airway to explore how this may change in disease.
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Técnicas Biossensoriais , Glucose , Animais , Proteínas de Ligação ao Cálcio , Células Epiteliais , Camundongos , Proteínas de Transporte de Monossacarídeos , Proteínas Periplásmicas de LigaçãoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0210308.].
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Alopecia areata (AA) is a hair loss disorder resulting from an autoimmune reaction against hair follicles. T-helper 1 cells are a major contributor to this disorder, but little is known about the role of T-regulatory cells (Tregs) in AA. Here, we analysed the distribution of circulating Treg subsets in twenty AA patients with active hair loss and fifteen healthy subjects by flow cytometry. The Treg suppressor HLA-DR+ subpopulation was significantly reduced in the patients (P<0.001) and there were significantly fewer cells expressing CD39 among the CD4+CD25+Foxp3+ Treg subpopulation in patients (P = 0.001). FOXP3 CD39 Treg cells were also reduced in hair follicles; by 75% in non-lesional skin and 90% in lesional skin, when compared to control healthy skin. To further characterise Treg cells in AA; Tregs (CD4+CD25+FOXP3+) were investigated for their TCRß sequence. PCR products analysed by Next Generation Sequencing techniques, showed that all frequent public clonotypes in AA Tregs were also present in controls at relatively similar frequencies, excepting two public clonotypes: CATSRDEGGLDEKLFF (V15 D1 J1-4) and CASRDGTGPSNYGYTF (V2 D1 J1-2), which were exclusively present in controls. This suggests that these Treg clonotypes may have a protective effect and that they may be an exciting subject for future therapeutic applications.
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Alopecia em Áreas/imunologia , Antígenos CD/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Alopecia em Áreas/metabolismo , Feminino , Humanos , MasculinoRESUMO
INTRODUCTION: This review aims to raise the potential of the modern society's impact on gut integrity often leading to increased intestinal permeability, as a cause or driver of Alopecia Areata (AA) in genetically susceptible people. With the increasing rate of T cell-driven autoimmunity, we hypothesize that there is a common root cause of these diseases that originates from chronic inflammation, and that the gut is the most commonly exposed area with our modern lifestyle. Areas covered: We will discuss the complexity in the induction of AA and its potential link to increased intestinal permeability. Our main focus will be on the gut microbiome and mechanisms involved in the interplay with the immune system that may lead to local and/or peripheral inflammation and finally, tissue destruction. Expert opinion: We have seen a link between AA and a dysfunctional gastrointestinal system which raised the hypothesis that an underlying intestinal inflammation drives the priming and dysregulation of immune cells that lead to hair follicle destruction. While it is still important to resolve local inflammation and restore the IP around the hair follicles, we believe that the root cause needs to be eradicated by long-term interventions to extinguish the fire driving the disease.
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Alopecia em Áreas/imunologia , Microbioma Gastrointestinal/imunologia , Inflamação/imunologia , Alopecia em Áreas/terapia , Animais , Autoimunidade/imunologia , Gastroenteropatias/complicações , Gastroenteropatias/imunologia , Folículo Piloso/imunologia , Folículo Piloso/patologia , Humanos , Inflamação/complicações , Estilo de Vida , Linfócitos T/imunologiaRESUMO
The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.