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1.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-36232826

RESUMEN

A small library of new angelicin derivatives was designed and synthesized with the aim of bypassing the side effects of trimethylangelicin (TMA), a promising agent for the treatment of cystic fibrosis. To prevent photoreactions with DNA, hindered substituents were inserted at the 4 and/or 6 positions. Unlike the parent TMA, none of the new derivatives exhibited significant cytotoxicity or mutagenic effects. Among the synthesized compounds, the 4-phenylderivative 12 and the 6-phenylderivative 25 exerted a promising F508del CFTR rescue ability. On these compounds, preliminary in vivo pharmacokinetic (PK) studies were carried out, evidencing a favorable PK profile per se or after incorporation into lipid formulations. Therefore, the selected compounds are good candidates for future extensive investigation to evaluate and develop novel CFTR correctors based on the angelicin structure.


Asunto(s)
Fibrosis Quística , Furocumarinas , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , ADN/uso terapéutico , Furocumarinas/química , Furocumarinas/farmacología , Furocumarinas/uso terapéutico , Humanos , Lípidos/uso terapéutico , Mutación
2.
Int J Mol Sci ; 23(22)2022 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-36430961

RESUMEN

A series of new-generation TMA (4,6,4'-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4'-dimethyl-angelicin) and GY964 (4-phenyl-6,4'-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, these molecules were found to have no pro-apoptotic, mutagenic and phototoxic effects, facilitating our decision to test the efficacy in vivo by using a mouse model of acute P. aeruginosa lung infection. The anti-inflammatory effect of GY971a was confirmed in vivo; this derivative was able to deeply decrease the total number of inflammatory cells, the neutrophil count and the cytokine/chemokine profile in the P. aeruginosa acute infection model, without evident toxicity. Considering all the obtained and reported in vitro and in vivo pre-clinical results, GY971a seems to have interesting anti-inflammatory effects, modulating the NF-κB pathway, as well as the starting lead compound TMA, but without side effects.


Asunto(s)
Fibrosis Quística , Quistes , Furocumarinas , Humanos , Fibrosis Quística/genética , FN-kappa B/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Furocumarinas/farmacología , Quistes/tratamiento farmacológico , Pseudomonas aeruginosa/metabolismo
3.
Glycoconj J ; 37(5): 623-633, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32666337

RESUMEN

Human primary bronchial epithelial cells differentiated in vitro represent a valuable tool to study lung diseases such as cystic fibrosis (CF), an inherited disorder caused by mutations in the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator. In CF, sphingolipids, a ubiquitous class of bioactive lipids mainly associated with the outer layer of the plasma membrane, seem to play a crucial role in the establishment of the severe lung complications. Nevertheless, no information on the involvement of sphingolipids and their metabolism in the differentiation of primary bronchial epithelial cells are available so far. Here we show that ceramide and globotriaosylceramide increased during cell differentiation, whereas glucosylceramide and gangliosides content decreased. In addition, we found that apical plasma membrane of differentiated bronchial cells is characterized by a higher content of sphingolipids in comparison to the other cell membranes and that activity of sphingolipids catabolic enzymes associated with this membrane results altered with respect to the total cell activities. In particular, the apical membrane of CF cells was characterized by high levels of ceramide and glucosylceramide, known to have proinflammatory activity. On this basis, our data further support the role of sphingolipids in the onset of CF lung pathology.


Asunto(s)
Diferenciación Celular/genética , Fibrosis Quística/genética , Hidrolasas/genética , Esfingolípidos/genética , Bronquios/enzimología , Membrana Celular/enzimología , Membrana Celular/genética , Ceramidas/genética , Fibrosis Quística/enzimología , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Glucosilceramidas/genética , Humanos , Hidrolasas/química , Cultivo Primario de Células , Esfingolípidos/metabolismo
4.
Int J Mol Sci ; 21(9)2020 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-32397443

RESUMEN

Iminosugars are sugar analogues endowed with a high pharmacological potential. The wide range of biological activities exhibited by these glycomimetics associated with their excellent drug profile make them attractive therapeutic candidates for several medical interventions. The ability of iminosugars to act as inhibitors or enhancers of carbohydrate-processing enzymes suggests their potential use as therapeutics for the treatment of cystic fibrosis (CF). Herein we review the most relevant advances in the field, paying attention to both the chemical synthesis of the iminosugars and their biological evaluations, resulting from in vitro and in vivo assays. Starting from the example of the marketed drug NBDNJ (N-butyl deoxynojirimycin), a variety of iminosugars have exhibited the capacity to rescue the trafficking of F508del-CFTR (deletion of F508 residue in the CF transmembrane conductance regulator), either alone or in combination with other correctors. Interesting results have also been obtained when iminosugars were considered as anti-inflammatory agents in CF lung disease. The data herein reported demonstrate that iminosugars hold considerable potential to be applied for both therapeutic purposes.


Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Compuestos Heterocíclicos con 1 Anillo/uso terapéutico , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/uso terapéutico , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Glicósido Hidrolasas/antagonistas & inhibidores , Glicosiltransferasas/antagonistas & inhibidores , Compuestos Heterocíclicos con 1 Anillo/síntesis química , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Iminopiranosas/química , Iminopiranosas/uso terapéutico , Inflamación , Estructura Molecular , Mutación , Eliminación de Secuencia , Tartratos/química , Tartratos/uso terapéutico
5.
Int J Mol Sci ; 21(12)2020 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-32599772

RESUMEN

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.


Asunto(s)
Adyuvantes Inmunológicos/farmacología , Aminofenoles/farmacología , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Fibrosis Quística/tratamiento farmacológico , Gangliósido G(M1)/farmacología , Quinolonas/farmacología , Adyuvantes Inmunológicos/química , Aminofenoles/química , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Bronquios/patología , Agonistas de los Canales de Cloruro/química , Agonistas de los Canales de Cloruro/farmacología , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Gangliósido G(M1)/química , Humanos , Mutación , Quinolonas/química , Terapias en Investigación
6.
Am J Respir Cell Mol Biol ; 59(4): 428-436, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29668297

RESUMEN

The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-ß3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca2+ release from endoplasmic reticulum and rise of Ca2+ concentration, 2) activation of conventional protein kinase C isoform ß, and 3) induction of IL-8 release. These results, besides identifying S845L as a loss-of-function variant, strengthen the importance of targeting PLCB3 to mitigate the CF inflammatory response in bronchial epithelial cells without blunting the immune response.


Asunto(s)
Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Interleucina-8/metabolismo , Fosfolipasa C beta/deficiencia , Pseudomonas aeruginosa/fisiología , Bronquios/patología , Señalización del Calcio , Línea Celular , Simulación por Computador , Humanos , Moco/metabolismo , Mutación/genética , Fosfolipasa C beta/química , Fosfolipasa C beta/genética , Fosfolipasa C beta/metabolismo , Serina/metabolismo , Relación Estructura-Actividad
7.
J Neurooncol ; 136(1): 51-62, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29076001

RESUMEN

Malignant gliomas, the most frequent primary brain tumors, are characterized by a dismal prognosis. Reliable biomarkers complementary to neuroradiology in the differential diagnosis of gliomas and monitoring for post-surgical progression are unmet needs. Altered expression of several microRNAs in tumour tissues from patients with gliomas compared to normal brain tissue have been described, thus supporting the rationale of using microRNA-based biomarkers. Although different circulating microRNAs were proposed in association with gliomas, they have not been introduced into clinical practice so far. Blood samples were collected from patients with high and low grade gliomas, both before and after surgical resection, and the expression of miR-21, miR-222 and miR-124-3p was measured in exosomes isolated from serum. The expression levels of miR-21, miR-222 and miR-124-3p in serum exosomes of patients with high grade gliomas were significantly higher than those of low grade gliomas and healthy controls and were sharply decreased in samples obtained after surgery. The analysis of miR-21, miR-222 and miR-124-3p in serum exosomes of patients affected by gliomas can provide a minimally invasive and innovative tool to help the differential diagnosis of gliomas at their onset in the brain and predict glioma grading and non glial metastases before surgery.


Asunto(s)
Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/diagnóstico , Exosomas/metabolismo , Glioma/sangre , Glioma/diagnóstico , MicroARNs/sangre , Adulto , Anciano , Biomarcadores de Tumor , Neoplasias Encefálicas/cirugía , Diagnóstico Diferencial , Femenino , Glioma/cirugía , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad
8.
Mediators Inflamm ; 2017: 1730245, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29333001

RESUMEN

Cystic fibrosis (CF) is the most common autosomal genetic recessive disease caused by mutations of gene encoding for the cystic fibrosis transmembrane conductance regulator. Patients with CF display a wide spectrum of symptoms, the most severe being chronic lung infection and inflammation, which lead to onset of cystic fibrosis lung disease. Several studies indicate that sphingolipids play a regulatory role in airway inflammation. The inhibition and downregulation of GBA2, the enzyme catabolizing glucosylceramide to ceramide, are associated with a significant reduction of IL-8 production in CF bronchial epithelial cells. Herein, we demonstrate that GBA2 plays a role in the proinflammatory state characterizing CF cells. We also report for the first time that Pseudomonas aeruginosa infection causes a recruitment of plasma membrane-associated glycosphingolipid hydrolases into lipid rafts of CuFi-1-infected cells. This reorganization of cell membrane may be responsible for activation of a signaling cascade, culminating in aberrant inflammatory response in CF bronchial epithelial cells upon bacterial infection. Taken together, the presented data further support the role of sphingolipids and their metabolic enzymes in controlling the inflammatory response in CF.


Asunto(s)
Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Glicósido Hidrolasas/metabolismo , Infecciones por Pseudomonas/metabolismo , Esfingolípidos/metabolismo , beta-Glucosidasa/metabolismo , Bronquios/metabolismo , Bronquios/microbiología , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/microbiología , Fibrosis Quística/complicaciones , Glucosilceramidasa , Humanos , Mediadores de Inflamación/metabolismo , Microdominios de Membrana/metabolismo , Modelos Biológicos , Infecciones por Pseudomonas/complicaciones , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Transducción de Señal
9.
Molecules ; 23(1)2017 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-29286300

RESUMEN

Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , MicroARNs/metabolismo , Ácidos Nucleicos de Péptidos/farmacología , Regiones no Traducidas 3'/genética , Apoptosis/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Evolución Molecular , Humanos , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos
10.
Am J Respir Cell Mol Biol ; 55(5): 645-656, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27281024

RESUMEN

Pseudomonas aeruginosa colonization, prominent inflammation with massive expression of the neutrophil chemokine IL-8, and luminal infiltrates of neutrophils are hallmarks of chronic lung disease in patients with cystic fibrosis (CF). The nociceptive transient receptor potential ankyrin (TRPA) 1 calcium channels have been recently found to be involved in nonneurogenic inflammation. Here, we investigate the role of TRPA1 in CF respiratory inflammatory models in vitro. Expression of TRPA1 was evaluated in CF lung tissue sections and cells by immunohistochemistry and immunofluorescence. Epithelial cell lines (A549, IB3-1, CuFi-1, CFBE41o-) and primary cells from patients with CF were used to: (1) check TRPA1 function modulation, by Fura-2 calcium imaging; (2) down-modulate TRPA1 function and expression, by pharmacological inhibitors (HC-030031 and A-967079) and small interfering RNA silencing; and (3) assess the effect of TRPA1 down-modulation on expression and release of cytokines upon exposure to proinflammatory challenges, by quantitative RT-PCR and 27-protein Bioplex assay. TRPA1 channels are expressed in the CF pseudostratified columnar epithelium facing the bronchial lumina exposed to bacteria, where IL-8 is coexpressed. Inhibition of TRPA1 expression results in a relevant reduction of release of several cytokines, including IL-8 and the proinflammatory cytokines IL-1ß and TNF-α, in CF primary bronchial epithelial cells exposed to P. aeruginosa and to the supernatant of mucopurulent material derived from the chronically infected airways of patients with CF. In conclusion, TRPA1 channels are involved in regulating the extent of airway inflammation driven by CF bronchial epithelial cells.


Asunto(s)
Canales de Calcio/metabolismo , Fibrosis Quística/complicaciones , Pulmón/patología , Proteínas del Tejido Nervioso/metabolismo , Neumonía/complicaciones , Neumonía/patología , Canales de Potencial de Receptor Transitorio/metabolismo , Células A549 , Adulto , Bronquios/patología , Fibrosis Quística/genética , Fibrosis Quística/patología , Citocinas/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Silenciador del Gen , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Modelos Biológicos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neumonía/genética , Pseudomonas aeruginosa/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Canal Catiónico TRPA1 , Donantes de Tejidos , Transcripción Genética , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Adulto Joven
11.
Genet Med ; 18(2): 145-51, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26087173

RESUMEN

PURPOSE: We evaluated the effects of cystic fibrosis (CF) carrier screening on birth prevalence trends and newborn screening (NBS) efficiency by comparing two Italian regions; carrier screening was performed in one region (eastern region (ER)) and not in the other (western region (WR)). METHODS: Annual births of infants with CF, NBS false-positive results, NBS uncertain diagnoses (borderline sweat chloride (BSC)), carrier tests performed, and carriers detected were monitored during the 1993-2013 period. MEASUREMENTS AND MAIN RESULTS: A total of 259 newborns with CF were detected. In the ER, 150 carrier couples were found. Mean annual percentage of birth prevalence decrease was 9% per 10,000 (P = 0.002) and was greater in the ER (15%, P = 0.0008; WR 1%, P = ns). The WR estimated birth prevalence was 1/3,589 in 1993 and 1/3,870 in 2013; in the ER it was 1/2,730 in 1993 and 1/14,200 in 2013. The ER birth prevalence correlated inversely with the number of carrier couples (P = 0.0032). The ratio between CF cases and NBS-positive results significantly decreased in the ER (1.6%, P = 0.0001) but not in the WR. The ratio between prevalence of BSC and of CF cases increased in the ER (P = 0.008) but not in the WR (P = 0.1). CONCLUSION: Carrier screening was connected with a decrease in birth prevalence of CF. Poorer NBS performance was observed in the carrier screening area.


Asunto(s)
Fibrosis Quística/epidemiología , Fibrosis Quística/genética , Heterocigoto , Tamizaje Neonatal , Fibrosis Quística/diagnóstico , Pruebas Genéticas , Humanos , Recién Nacido , Italia/epidemiología , Prevalencia
13.
Pflugers Arch ; 466(12): 2269-78, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24595473

RESUMEN

Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Infecciones por Pseudomonas/metabolismo , Mucosa Respiratoria/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Bronquios/citología , Bronquios/metabolismo , Bronquios/microbiología , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Pulmón/citología , Pulmón/metabolismo , Pulmón/microbiología , Ratones , Mutación , Fosfoproteínas/genética , Fosforilación , Pseudomonas aeruginosa , Mucosa Respiratoria/microbiología , Intercambiadores de Sodio-Hidrógeno/genética , Ubiquitinación
14.
ACS Pharmacol Transl Sci ; 7(6): 1807-1822, 2024 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-38898954

RESUMEN

Over the past few years, l-iminosugars have revealed attractive pharmacological properties for managing rare diseases including Cystic Fibrosis (CF). The iminosugar N-butyl-l-deoxynojirimycin (l-NBDNJ, ent-1), prepared by a carbohydrate-based route, was herein evaluated for its anti-inflammatory and anti-infective potential in models of CF lung disease infection. A significant decrease in the bacterial load in the airways was observed in the murine model of Pseudomonas aeruginosa chronic infection in the presence of l-NBDNJ, also accompanied by a modest reduction of inflammatory cells. Mechanistic insights into the observed activity revealed that l-NBDNJ interferes with the expression of proteins regulating cytoskeleton assembly and organization of the host cell, downregulates the main virulence factors of P. aeruginosa involved in the host response, and affects pathogen adhesion to human cells. These findings along with the observation of the absence of an in vitro bacteriostatic/bactericidal action of l-NBDNJ suggest the potential use of this glycomimetic as an antivirulence agent in the management of CF lung disease.

15.
J Immunol ; 187(11): 6069-81, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-22031759

RESUMEN

IL-8 released from bronchial epithelial cells infected with Pseudomonas aeruginosa plays a crucial role in the chronic lung pathology of patients affected by cystic fibrosis. Novel anti-inflammatory approaches will benefit from a thorough understanding of the regulatory mechanisms involved in the transcription of this chemokine to identify potential pharmacological targets. We addressed this issue by investigating the role of phosphoproteins and transcription factors (TFs) on transcription of IL-8 gene in the human bronchial epithelial IB3-1, CuFi-1, and Calu-3 cells. P. aeruginosa increased the basal phosphorylation of the ERK1/2 pathway components 90-kDa ribosomal S6 kinase (RSK)1/2 and mitogen- and stress-activated kinase-2 and of the p38 MAPK pathway components p38α/δ/γ and heat shock protein 27 (HSP27). The involvement of these kinases in the expression of IL-8 gene was confirmed with pharmacological inhibitors of ERK1/2, RSK, p38, and HSP27 both at transcription and secretion levels. Transfection of TF decoy oligodeoxynucleotides, designed to interfere with the interaction of the TFs NF-κB, NF-IL6, AP-1, CREB, and CHOP with the corresponding consensus sequences identified in the IL-8 promoter, reduced the P. aeruginosa-dependent transcription of IL-8, suggesting their participation in the transcriptional machinery. Stimulation of IB3-1 cells with IL-1ß led to a similar pattern of activation, whereas the pattern of phosphoproteins and of TFs modulated by TNF-α differentiated sharply. In conclusion, the results highlight a novel role for RSK1/2 and HSP27 phosphoproteins and of the cooperative role of the TFs NF-κB, NF-IL6, AP-1, CHOP, and CREB in P. aeruginosa-dependent induction of transcription of the IL-8 gene in human bronchial epithelial cells.


Asunto(s)
Regulación de la Expresión Génica/genética , Interleucina-8/genética , Fosfoproteínas/genética , Infecciones por Pseudomonas/genética , Mucosa Respiratoria/inmunología , Transducción de Señal/inmunología , Bronquios/inmunología , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Células Epiteliales/inmunología , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/inmunología , Humanos , Interleucina-8/biosíntesis , Fosfoproteínas/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/inmunología , Transcripción Genética , Transfección
16.
J Immunol ; 186(8): 4946-58, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21411730

RESUMEN

Respiratory insufficiency is the major cause of morbidity and mortality in patients affected by cystic fibrosis (CF). An excessive neutrophilic inflammation, mainly orchestrated by the release of IL-8 from bronchial epithelial cells and amplified by chronic bacterial infection with Pseudomonas aeruginosa, leads to progressive tissue destruction. The anti-inflammatory drugs presently used in CF patients have several limitations, indicating the need for identifying novel molecular targets. To address this issue, we preliminarily studied the association of 721 single nucleotide polymorphisms from 135 genes potentially involved in signal transduction implicated in neutrophil recruitment in a cohort of F508del homozygous CF patients with either severe or mild progression of lung disease. The top ranking association was found for a nonsynonymous polymorphism of the phospholipase C-ß3 (PLCB3) gene. Studies in bronchial epithelial cells exposed to P. aeruginosa revealed that PLCB3 is implicated in extracellular nucleotide-dependent intracellular calcium signaling, leading to activation of the protein kinase Cα and Cß and of the nuclear transcription factor NF-κB p65. The proinflammatory pathway regulated by PLCB3 acts by potentiating the Toll-like Receptors' signaling cascade and represents an interesting molecular target to attenuate the excessive recruitment of neutrophils without completely abolishing the inflammatory response.


Asunto(s)
Fibrosis Quística/genética , Células Epiteliales/metabolismo , Interleucina-8/genética , Fosfolipasa C beta/genética , Adenosina Trifosfato/farmacología , Calcio/metabolismo , Línea Celular Transformada , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Activación Enzimática , Células Epiteliales/microbiología , Expresión Génica/efectos de los fármacos , Frecuencia de los Genes , Genotipo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno , Humanos , Interleucina-8/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Microscopía Fluorescente , Fosfolipasa C beta/metabolismo , Polimorfismo de Nucleótido Simple , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Pseudomonas aeruginosa/fisiología , Interferencia de ARN , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo
17.
FEBS Open Bio ; 13(9): 1601-1614, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37315117

RESUMEN

Cystic fibrosis (CF) is the most common inherited, life-limiting disorder in Caucasian populations. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to an impairment of protein expression and/or function. CFTR is a chloride/bicarbonate channel expressed at the apical surface of epithelial cells of different organs. Nowadays, more than 2100 CFTR genetic variants have been described, but not all of them cause CF. However, around 80-85% of the patients worldwide are characterized by the presence, at least in one allele, of the mutation F508del. CFTR mutations cause aberrant hydration and secretion of mucus in hollow organs. In the lungs, this condition favors bacterial colonization, allowing the development of chronic infections that lead to the onset of the CF lung disease, which is the main cause of death in patients. In recent years, evidence has reported that CFTR loss of function is responsible for alterations in a particular class of bioactive lipids, called sphingolipids (SL). SL are ubiquitously present in eukaryotic cells and are mainly asymmetrically located within the external leaflet of the plasma membrane, where they organize specific platforms capable of segregating a selected number of proteins. CFTR is associated with these platforms that are fundamental for its functioning. Considering the importance of SL in CFTR homeostasis, we attempt here to provide a critical overview of the literature to determine the role of these lipids in channel stability and activity, and whether their modulation in CF could be a target for new therapeutic approaches.


Asunto(s)
Fibrosis Quística , Humanos , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mutación/genética , Membrana Celular/metabolismo , Lípidos
18.
J Cyst Fibros ; 22(1): 156-160, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-35973900

RESUMEN

We previously demonstrated that ß-sitosterol (BSS) inhibits the expression of the chemokine IL-8 in CF bronchial epithelial cells exposed to P. aeruginosa. In the mouse model of lung chronic infection, herein shown, BSS significantly reduced leukocyte recruitment in the bronchoalveolar lavage fluid and decreased bacteria recovered in the airways. Treatment with BSS decreased the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in the lung homogenate. This anti-inflammatory activity is accompanied by a beneficial protecting activity against infection and improvement of health status. Our data suggest that BSS has the potential to become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS.


Asunto(s)
Fibrosis Quística , Neumonía , Infecciones por Pseudomonas , Ratones , Animales , Pseudomonas aeruginosa , Fibrosis Quística/complicaciones , Pulmón/metabolismo , Inflamación , Modelos Animales de Enfermedad , Infecciones por Pseudomonas/tratamiento farmacológico , Neutrófilos/metabolismo
19.
Nat Commun ; 14(1): 132, 2023 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-36627352

RESUMEN

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Fibrosis Quística , SARS-CoV-2 , Internalización del Virus , Humanos , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación hacia Abajo , Receptores Virales/genética , Receptores Virales/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Replicación Viral
20.
Cancers (Basel) ; 14(14)2022 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-35884554

RESUMEN

Circulating miRNAs are increasingly studied and proposed as tumor markers with the aim of investigating their role in monitoring the response to therapy as well as the natural evolution of primary or secondary brain tumors. This study aimed to evaluate the modulation of the expression of three miRNAs, miR-21, miR-222 and miR-124-3p, in the serum exosomes of patients with high-grade gliomas (HGGs) and brain metastases (BMs) to verify their usefulness in the differential diagnosis of brain masses; then, it focused on their variations following the surgical and/or radiosurgical treatment of the BMs. A total of 105 patients with BMs from primary lung or breast cancer, or melanoma underwent neurosurgery or radiosurgery treatment, and 91 patients with HGGs were enrolled, along with 30 healthy controls. A significant increase in miR-21 expression in serum exosomes was observed in both HGGs and BMs compared with healthy controls; on the other hand, miR-124-3p was significantly decreased in BMs, and it was increased in HGGs. After the surgical or radiosurgical treatment of patients with BMs, a significant reduction in miR-21 was noted with both types of treatments. This study identified a signature of exosomal miRNAs that could be useful as a noninvasive complementary analysis both in the differential diagnosis of BMs from glial tumors and in providing information on tumor evolution over time.

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