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1.
Thorax ; 79(8): 778-787, 2024 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-38508718

RESUMEN

INTRODUCTION: Novel therapeutic strategies are urgently needed for Mycobacterium avium complex pulmonary disease (MAC-PD). Human mesenchymal stromal cells (MSCs) can directly inhibit MAC growth, but their effect on intracellular bacilli is unknown. We investigated the ability of human MSCs to reduce bacterial replication and inflammation in MAC-infected macrophages and in a murine model of MAC-PD. METHODS: Human monocyte-derived macrophages (MDMs) were infected with M. avium Chester strain and treated with human bone marrow-derived MSCs. Intracellular and extracellular colony-forming units (CFUs) were counted at 72 hours. Six-week-old female balb/c mice were infected by nebulisation of M. avium Chester. Mice were treated with 1×106 intravenous human MSCs or saline control at 21 and 28 days post-infection. Lungs, liver and spleen were harvested 42 days post-infection for bacterial counts. Cytokines were quantified by ELISA. RESULTS: MSCs reduced intracellular bacteria in MDMs over 72 hours (median 35% reduction, p=0.027). MSC treatment increased extracellular concentrations of prostaglandin E2 (PGE2) (median 10.1-fold rise, p=0.002) and reduced tumour necrosis factor-α (median 28% reduction, p=0.025). Blocking MSC PGE2 production by cyclo-oxygenase-2 (COX-2) inhibition with celecoxib abrogated the antimicrobial effect, while this was restored by adding exogenous PGE2. MSC-treated mice had lower pulmonary CFUs (median 18% reduction, p=0.012), but no significant change in spleen or liver CFUs compared with controls. CONCLUSION: MSCs can modulate inflammation and reduce intracellular M. avium growth in human macrophages via COX-2/PGE2 signalling and inhibit pulmonary bacterial replication in a murine model of chronic MAC-PD.


Asunto(s)
Modelos Animales de Enfermedad , Células Madre Mesenquimatosas , Ratones Endogámicos BALB C , Infección por Mycobacterium avium-intracellulare , Animales , Ratones , Femenino , Humanos , Infección por Mycobacterium avium-intracellulare/microbiología , Complejo Mycobacterium avium , Trasplante de Células Madre Mesenquimatosas/métodos , Macrófagos/microbiología , Dinoprostona/metabolismo , Sulfonamidas/farmacología , Mycobacterium avium
2.
Stem Cells ; 41(5): 431-443, 2023 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-36749355

RESUMEN

Mesenchymal stromal/stem cells are multipotent adult cells that can be extracted from numerous tissues, including the lungs. Lung-resident MSCs (LR-MSCs) are localized to perivascular spaces where they act as important regulators of pulmonary homeostasis, mediating the balance between lung injury/damage and repair processes. LR-MSCs support the integrity of the lung tissue via modulation of the immune response and release of trophic factors. However, in the context of chronic lung diseases, the ability of LR-MSCs to maintain pulmonary homeostasis and facilitate repair is diminished. In this setting, LR-MSC can contribute to the pathogenesis of disease, through their altered secretory and immunomodulatory properties. In addition, they are capable of differentiating into myofibroblasts, thereby contributing to the fibrotic aspects of numerous lung diseases. For example, in idiopathic pulmonary fibrosis, a variety of factors can stimulate their differentiation into myofibroblasts including tumor necrosis factor-α (TNF-(α), transforming growth factor-ß1 (TGF-ß1), endoplasmic reticulum (ER) stress, Hedgehog (HH), and Wingless/integrated (Wnt) signaling. Here, we review the current literature on the characterization of LR-MSCs and describe their roles in pulmonary homeostasis/repair and in the pathogenesis of chronic lung disease.


Asunto(s)
Fibrosis Pulmonar Idiopática , Células Madre Mesenquimatosas , Humanos , Proteínas Hedgehog , Pulmón/patología , Fibrosis Pulmonar Idiopática/patología , Diferenciación Celular
3.
Thorax ; 78(6): 617-630, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-35948417

RESUMEN

RATIONALE: A better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory distress syndrome (ARDS). Macrophages are important mediators of ARDS inflammatory response. Suppressor of cytokine signalling (SOCS) proteins are key regulators of the macrophage phenotype switch. We therefore investigated whether SOCS proteins are involved in mediation of the MSC effect on human macrophage reprogramming. METHODS: Human monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS) or plasma samples from patients with ARDS (these samples were previously classified into hypo-inflammatory and hyper-inflammatory phenotype) and treated with MSC conditioned medium (CM) or EVs. Protein expression was measured by Western blot. EV micro RNA (miRNA) content was determined by miRNA sequencing. In vivo: LPS-injured C57BL/6 mice were given EVs isolated from MSCs in which miR-181a had been silenced by miRNA inhibitor or overexpressed using miRNA mimic. RESULTS: EVs were the key component of MSC CM responsible for anti-inflammatory modulation of human macrophages. EVs significantly reduced secretion of tumour necrosis factor-α and interleukin-8 by LPS-stimulated or ARDS plasma-stimulated MDMs and this was dependent on SOCS1. Transfer of miR-181a in EVs downregulated phosphatase and tensin homolog (PTEN) and subsequently activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) leading to upregulation of SOCS1 in macrophages. In vivo, EVs alleviated lung injury and upregulated pSTAT5 and SOCS1 expression in alveolar macrophages in a miR181-dependent manner. Overexpression of miR-181a in MSCs significantly enhanced therapeutic efficacy of EVs in this model. CONCLUSION: miR-181a-PTEN-pSTAT5-SOCS1 axis is a novel pathway responsible for immunomodulatory effect of MSC EVs in ARDS.


Asunto(s)
Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Síndrome de Dificultad Respiratoria , Animales , Ratones , Humanos , Lipopolisacáridos , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Macrófagos/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/terapia , Síndrome de Dificultad Respiratoria/metabolismo , Vesículas Extracelulares/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo
4.
Am J Respir Crit Care Med ; 205(7): 769-782, 2022 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-35073247

RESUMEN

Rationale: Although the cysteine protease cathepsin S has been implicated in the pathogenesis of several inflammatory lung diseases, its role has not been examined in the context of acute respiratory distress syndrome, a condition that still lacks specific and effective pharmacological treatments. Objectives: To characterize the status of cathepsin S in acute lung inflammation and examine the role of cathepsin S in disease pathogenesis. Methods: Human and mouse model BAL fluid samples were analyzed for the presence and activity of cathepsin S and its endogenous inhibitors. Recombinant cathepsin S was instilled directly into the lungs of mice. The effects of cathepsin S knockout and pharmacological inhibition were examined in two models of acute lung injury. Protease-activated receptor-1 antagonism was used to test a possible mechanism for cathepsin S-mediated inflammation. Measurements and Main Results: Pulmonary cathepsin S concentrations and activity were elevated in acute respiratory distress syndrome, a phenotype possibly exacerbated by the loss of the endogenous antiprotease cystatin SN. Direct cathepsin S instillation into the lungs induced key pathologies of acute respiratory distress syndrome, including neutrophilia and alveolar leakage. Conversely, in murine models of acute lung injury, genetic knockdown and prophylactic or therapeutic inhibition of cathepsin S reduced neutrophil recruitment and protein leakage. Cathepsin S may partly mediate its pathogenic effects via protease-activated receptor-1, because antagonism of this receptor abrogated cathepsin S-induced airway inflammation. Conclusions: Cathepsin S contributes to acute lung injury and may represent a novel therapeutic target for acute respiratory distress syndrome.


Asunto(s)
Neumonía , Síndrome de Dificultad Respiratoria , Animales , Líquido del Lavado Bronquioalveolar , Catepsinas , Modelos Animales de Enfermedad , Humanos , Pulmón/patología , Ratones
5.
Mediators Inflamm ; 2021: 6682657, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33828414

RESUMEN

BACKGROUND: Elevated levels of the cysteine protease cathepsin S (CatS) are associated with chronic mucoobstructive lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). We have previously demonstrated that prophylactic treatment with a CatS inhibitor from birth reduces inflammation, mucus plugging, and lung tissue damage in juvenile ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice with chronic inflammatory mucoobstructive lung disease. In this study, we build upon this work to examine the effects of therapeutic intervention with a CatS inhibitor in adult ßENaC-Tg mice with established disease. METHODS: ßENaC-Tg mice and wild-type (WT) littermates were treated with a CatS inhibitor from 4 to 6 weeks of age, and CatS-/- ßENaC-Tg mice were analysed at 6 weeks of age. Bronchoalveolar lavage (BAL) fluid inflammatory cell counts were quantified, and lung tissue destruction and mucus obstruction were analysed histologically. RESULTS: At 6 weeks of age, ßENaC-Tg mice developed significant airway inflammation, lung tissue damage, and mucus plugging when compared to WT mice. CatS-/- ßENaC-Tg mice and ßENaC-Tg mice receiving inhibitor had significantly reduced airway mononuclear and polymorphonuclear (PMN) cell counts as well as mucus plugging. However, in contrast to CatS-/- ßENaC-Tg mice, therapeutic inhibition of CatS in ßENaC-Tg mice had no effect on established emphysema-like lung tissue damage. CONCLUSIONS: These results suggest that while early CatS targeting may be required to prevent the onset and progression of lung tissue damage, therapeutic CatS targeting effectively inhibited airway inflammation and mucus obstruction. These results indicate the important role CatS may play in the pathogenesis and progression of mucoobstructive lung disease.


Asunto(s)
Catepsinas/antagonistas & inhibidores , Fibrosis Quística , Canales Epiteliales de Sodio , Animales , Fibrosis Quística/patología , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológico , Inflamación/patología , Pulmón/patología , Ratones , Ratones Transgénicos , Moco
6.
Am J Respir Cell Mol Biol ; 62(3): 300-309, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31499011

RESUMEN

Previous studies demonstrated spontaneous type 2 airway inflammation with eosinophilia in juvenile Scnn1b (sodium channel, non-voltage-gated 1, ß-subunit)-transgenic (Scnn1b-Tg) mice with muco-obstructive lung disease. IL-1 receptor (IL-1R) signaling has been implicated in allergen-driven airway disease; however, its role in eosinophilic inflammation in muco-obstructive lung disease remains unknown. In this study, we examined the role of IL-1R signaling in the development of airway eosinophilia and type 2 inflammation in juvenile Scnn1b-Tg mice. We determined effects of genetic deletion of Il1r1 (IL-1 receptor type I) on eosinophil counts, transcript levels of key type 2 cytokines, markers of eosinophil activation and apoptosis, and tissue morphology in lungs of Scnn1b-Tg mice at different time points during neonatal development. Furthermore, we measured endothelial surface expression of intercellular adhesion molecule 1 (ICAM-1), an integrin involved in eosinophil transendothelial migration, and determined effects of eosinophil depletion using an anti-IL-5 antibody on lung morphology. Lack of IL-1R reduced airway eosinophilia and structural lung damage, but it did not reduce concentrations of type 2 cytokines and associated eosinophil activation in Scnn1b-Tg mice. Structural lung damage in Scnn1b-Tg mice was also reduced by eosinophil depletion. Lack of IL-1R was associated with reduced expression of ICAM-1 on lung endothelial cells and reduced eosinophil counts in lungs from Scnn1b-Tg mice. We conclude that IL-1R signaling is implicated in airway eosinophilia independent of type 2 cytokines in juvenile Scnn1b-Tg mice. Our data suggest that IL-1R signaling may be relevant in the pathogenesis of eosinophilic airway inflammation in muco-obstructive lung diseases, which may be mediated in part by ICAM-1-dependent transmigration of eosinophils into the lungs.


Asunto(s)
Enfermedades Pulmonares Obstructivas/fisiopatología , Moco/metabolismo , Eosinofilia Pulmonar/fisiopatología , Receptores Tipo I de Interleucina-1/deficiencia , Envejecimiento/inmunología , Animales , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Apoptosis , Líquido del Lavado Bronquioalveolar/citología , Quimiotaxis de Leucocito , Citocinas/sangre , Citocinas/fisiología , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/ultraestructura , Células Endoteliales/metabolismo , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Eosinófilos/patología , Molécula 1 de Adhesión Intercelular/fisiología , Interleucina-5/inmunología , Enfermedades Pulmonares Obstructivas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Eosinofilia Pulmonar/tratamiento farmacológico , Eosinofilia Pulmonar/prevención & control , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/fisiología , Transducción de Señal , Organismos Libres de Patógenos Específicos
7.
Am J Respir Crit Care Med ; 200(1): 51-62, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-30641028

RESUMEN

Rationale: CTSS (cathepsin S) is a cysteine protease that is observed at higher concentrations in BAL fluid and plasma of subjects with chronic obstructive pulmonary disease (COPD). Objectives: To investigate whether CTSS is involved in the pathogenesis of cigarette smoke-induced COPD and determine whether targeting upstream signaling could prevent the disease. Methods: CTSS expression was investigated in animal and human tissue and cell models of COPD. Ctss-/- mice were exposed to long-term cigarette smoke and forced oscillation and expiratory measurements were recorded. Animals were administered chemical modulators of PP2A (protein phosphatase 2A) activity. Measurements and Main Results: Here we observed enhanced CTSS expression and activity in mouse lungs after exposure to cigarette smoke. Ctss-/- mice were resistant to cigarette smoke-induced inflammation, airway hyperresponsiveness, airspace enlargements, and loss of lung function. CTSS expression was negatively regulated by PP2A in human bronchial epithelial cells isolated from healthy nonsmokers and COPD donors and in monocyte-derived macrophages. Modulating PP2A expression or activity, with silencer siRNA or a chemical inhibitor or activator, during acute smoke exposure in mice altered inflammatory responses and CTSS expression and activity in the lung. Enhancement of PP2A activity prevented chronic smoke-induced COPD in mice. Conclusions: Our study indicates that the decrease in PP2A activity that occurs in COPD contributes to elevated CTSS expression in the lungs and results in impaired lung function. Enhancing PP2A activity represents a feasible therapeutic approach to reduce CTSS activity and counter smoke-induced lung disease.


Asunto(s)
Catepsinas/metabolismo , Fumar Cigarrillos/metabolismo , Pulmón/metabolismo , Nicotiana , Proteína Fosfatasa 2/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Humo/efectos adversos , Animales , Bronquios/citología , Estudios de Casos y Controles , Fumar Cigarrillos/efectos adversos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Silenciador del Gen , Humanos , Pulmón/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Ratones , Ratones Noqueados , Ácido Ocadaico/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Mucosa Respiratoria/citología
8.
Int J Mol Sci ; 21(17)2020 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-32887484

RESUMEN

The arrival of cystic fibrosis transmembrane conductance regulator (CFTR) modulators as a new class of treatment for cystic fibrosis (CF) in 2012 represented a pivotal advance in disease management, as these small molecules directly target the upstream underlying protein defect. Further advancements in the development and scope of these genotype-specific therapies have been transformative for an increasing number of people with CF (PWCF). Despite clear improvements in CFTR function and clinical endpoints such as lung function, body mass index (BMI), and frequency of pulmonary exacerbations, current evidence suggests that CFTR modulators do not prevent continued decline in lung function, halt disease progression, or ameliorate pathogenic organisms in those with established lung disease. Furthermore, it remains unknown whether their restorative effects extend to dysfunctional CFTR expressed in phagocytes and other immune cells, which could modulate airway inflammation. In this review, we explore the effects of CFTR modulators on airway inflammation, infection, and their influence on the impaired pulmonary host defences associated with CF lung disease. We also consider the role of inflammation-directed therapies in light of the widespread clinical use of CFTR modulators and identify key areas for future research.


Asunto(s)
Antiinflamatorios/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Terapia Molecular Dirigida , Mucosa Respiratoria/efectos de los fármacos , Animales , Fibrosis Quística/inmunología , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Humanos , Inflamación/inmunología , Inflamación/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología
9.
Eur Respir J ; 53(3)2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30655278

RESUMEN

Cathepsin S (CatS) is upregulated in the lungs of patients with cystic fibrosis (CF). However, its role in CF lung disease pathogenesis remains unclear.In this study, ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice, a model of CF-like lung disease, were crossed with CatS null (CatS-/-) mice or treated with the CatS inhibitor VBY-999.Levels of active CatS were elevated in the lungs of ßENaC-Tg mice compared with wild-type (WT) littermates. CatS-/-ßENaC-Tg mice exhibited decreased pulmonary inflammation, mucus obstruction and structural lung damage compared with ßENaC-Tg mice. Pharmacological inhibition of CatS resulted in a significant decrease in pulmonary inflammation, lung damage and mucus plugging in the lungs of ßENaC-Tg mice. In addition, instillation of CatS into the lungs of WT mice resulted in inflammation, lung remodelling and upregulation of mucin expression. Inhibition of the CatS target, protease-activated receptor 2 (PAR2), in ßENaC-Tg mice resulted in a reduction in airway inflammation and mucin expression, indicating a role for this receptor in CatS-induced lung pathology.Our data indicate an important role for CatS in the pathogenesis of CF-like lung disease mediated in part by PAR2 and highlight CatS as a therapeutic target.


Asunto(s)
Catepsinas/metabolismo , Fibrosis Quística/metabolismo , Moco/metabolismo , Neumonía/metabolismo , Receptor PAR-2/metabolismo , Obstrucción de las Vías Aéreas/metabolismo , Animales , Catepsinas/genética , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/genética , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/etiología
10.
Eur Respir J ; 50(1)2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28705940

RESUMEN

Eppin is a serine protease inhibitor expressed in male reproductive tissues.The aim of this study was to investigate the localisation and regulation of eppin expression in myeloid and epithelial cell lines, and explore its potential role as a multifunctional host defence protein.Using immunohistochemistry and Western blotting, eppin was detected in the lungs of patients with acute respiratory distress syndrome and cystic fibrosis lung disease. Expression of eppin in monocytic cells was unaffected by stimulation with Toll-like receptor agonists, cytokines and hormone receptor agonists. However, upregulated expression and secretion of eppin was observed following treatment of monocytes with epidermal growth factor. Incubation of recombinant eppin with monocytic cells resulted in significant inhibition of lipopolysaccharide-induced chemokine production. Furthermore, eppin inhibited lipopolysaccharide-induced NF-κB activation by a mechanism which involved accumulation of phosphorylated IκBα. In an in vivo model of lung inflammation induced by lipopolysaccharide, eppin administration resulted in decreased recruitment of neutrophils to the lung with a concomitant reduction in the levels of the neutrophil chemokine macrophage inflammatory protein-2.Overall, these results suggest a role for eppin outside of the reproductive tract and that eppin may have a role in the innate immune response in the lung.


Asunto(s)
Fibrosis Quística/metabolismo , Citocinas/metabolismo , Pulmón/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , Líquido del Lavado Bronquioalveolar/química , Línea Celular Tumoral , Humanos , Inmunidad Innata , Masculino , Síndrome de Dificultad Respiratoria/genética , Transducción de Señal , Esputo/química , Receptores Toll-Like/metabolismo
11.
Biol Chem ; 398(4): 425-440, 2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-27930359

RESUMEN

Members of the whey acidic protein (WAP) or WAP four-disulfide-core (WFDC) family of proteins are a relatively under-explored family of low molecular weight proteins. The two most prominent WFDC proteins, secretory leukocyte protease inhibitor (SLPI) and elafin (or the precursor, trappin-2), have been shown to possess multiple functions including anti-protease, anti-bacterial, anti-viral and anti-inflammatory properties. It is therefore of no surprise that both SLPI and elafin/trappin-2 have been developed as potential therapeutics. Given the abundance of SLPI and elafin/trappin-2 in the human lung, most work in the area of WFDC research has focused on the role of WFDC proteins in protecting the lung from proteolytic attack. In this review, we will outline the current evidence regarding the expanding role of WFDC protein function with a focus on WFDC activity in lung disease as well as emerging data regarding the function of some of the more recently described WFDC proteins.


Asunto(s)
Enfermedades Pulmonares/fisiopatología , Proteínas de la Leche/metabolismo , Fenómenos Fisiológicos Respiratorios , Humanos , Enfermedades Pulmonares/prevención & control , Proteínas de la Leche/clasificación , Proteínas/metabolismo , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP
12.
Stem Cells ; 34(8): 2210-23, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27059413

RESUMEN

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte-derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210-2223.


Asunto(s)
Macrófagos/patología , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Nanotubos/química , Fagocitosis , Síndrome de Dificultad Respiratoria/patología , Animales , Antiinfecciosos/metabolismo , Comunicación Celular , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Escherichia coli/fisiología , Humanos , Macrófagos Alveolares/metabolismo , Ratones , Neutrófilos/metabolismo , Neumonía/microbiología , Neumonía/patología
13.
Prostate ; 74(3): 273-85, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24242708

RESUMEN

BACKGROUND: A contributing factor to the emergence of castrate resistant prostate cancer (CRPC) is the ability of the tumor to circumvent low circulating levels of testosterone during androgen deprivation therapy (ADT), through the production of "intracrine" tumoral androgens from precursors including cholesterol and dehydroepiandrosterone (DHEA). As these processes promote AR signaling and prostate cancer progression their modulation is required for disease prevention and treatment. METHODS: We evaluated the involvement of the vitamin D receptor ligand EB1089 in the regulation of genes with a role in androgen metabolism using the androgen dependent cell lines LNCaP and LAPC-4. EB1089 regulation of androgen metabolism was assessed using QRT-PCR, luciferase promoter assays, western blotting, enzyme activity assays, and LC-MS analyses. RESULTS: EB1089 induced significant expression of genes involved in androgen metabolism in prostate cancer cells. Real-Time PCR analysis revealed that VDR mediated significant regulation of CYP3A4, CYP3A5, CYP3A43, AKR1C1-3, UGT2B15/17, and HSD17B2. Data revealed potent regulation of CYP3A4 at the level of mRNA, protein expression and enzymatic activity, with VDR identified as the predominant regulator. Inhibition of CYP3A activity using the specific inhibitor ritonavir resulted in alleviation of the anti-proliferative response of VDR ligands in prostate cancer cells. Mass spectrometry revealed that overexpression of CYP3A protein in prostate cancer cells resulted in a significant increase in the oxidative inactivation of testosterone and DHEA to their 6-ß-hydroxy-testosterone and 16-α-hydroxy-DHEA metabolites, respectively. CONCLUSIONS: These data highlight a potential application of VDR-based therapies for the reduction of growth-promoting androgens within the tumor micro-environment.


Asunto(s)
Andrógenos/metabolismo , Calcitriol/análogos & derivados , Próstata/efectos de los fármacos , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores de Calcitriol/fisiología , Andrógenos/genética , Calcitriol/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP3A/análisis , Citocromo P-450 CYP3A/genética , Inhibidores del Citocromo P-450 CYP3A , Deshidroepiandrosterona/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/prevención & control , ARN Mensajero/análisis , Receptores de Calcitriol/agonistas , Ritonavir/farmacología , Testosterona/metabolismo
14.
Mol Cancer ; 10: 147, 2011 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-22168338

RESUMEN

BACKGROUND: Proteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting. RESULTS: Here we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect. CONCLUSIONS: This data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Catepsinas/inmunología , Citotoxicidad Inmunológica , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Catepsinas/química , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Femenino , Citometría de Flujo , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Masculino , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Ratas , Ratas Sprague-Dawley
15.
PLoS One ; 9(2): e89506, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24586832

RESUMEN

Vitamin D receptor (VDR) is a substrate for modification with small ubiquitin-like modifier (SUMO). To further assess the role of reversible SUMOylation within the vitamin D hormonal response, we evaluated the effects of sentrin/SUMO-specific proteases (SENPs) that can function to remove small ubiquitin-like modifier (SUMO) from target proteins upon the activities of VDR and related receptors. We report that SENP1 and SENP2 strikingly potentiate ligand-mediated transactivation of VDR and also its heterodimeric partner, retinoid X receptor (RXRα) with depletion of cellular SENP1 significantly diminishing the hormonal responsiveness of the endogenous vitamin D target gene CYP24A1. We find that SENP-directed modulation of VDR activity is cell line-dependent, achieving potent modulatory effects in Caco-2 and HEK-293 cells, while in MCF-7 cells the vitamin D signal is unaffected by any tested SENP. In support of their function as novel modulators of the vitamin D hormonal pathway we demonstrate that both SENP1 and SENP2 can interact with VDR and reverse its modification with SUMO2. In a preliminary analysis we identify lysine 91, a residue known to be critical for formation and DNA binding of the VDR-RXR heterodimer, as a minor SUMO acceptor site within VDR. In combination, our results support a repressor function for SUMOylation of VDR and reveal SENPs as a novel class of VDR/RXR co-regulatory protein that significantly modulate the vitamin D response and which could also have important impact upon the functionality of both RXR-containing homo and heterodimers.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Receptores de Calcitriol/genética , Receptores X Retinoide/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Animales , Western Blotting , Células CHO , Cricetulus , Cisteína Endopeptidasas/genética , Endopeptidasas/genética , Células HEK293 , Humanos , Células MCF-7 , Mutagénesis Sitio-Dirigida , Mutación/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Calcitriol/metabolismo , Receptores X Retinoide/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Transcripción Genética
16.
J Steroid Biochem Mol Biol ; 132(1-2): 24-31, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22564762

RESUMEN

The present study investigated the potential for members of the protein inhibitors of activated STAT (PIAS) family to function as co-regulators of the vitamin D signal pathway. Among the PIAS proteins evaluated, we establish PIAS4 as a potent inhibitor of the transcriptional responses of the CYP3A4 and CYP24A1 target genes to the active hormonal form of vitamin D, a repression that was observed to be dependent upon an intact SUMO-ligase function of PIAS4. We report that PIAS4 represents a direct binding partner for vitamin D receptor (VDR) and also facilitates its modification with SUMO2, a process that preferentially occurs on the apo-form of VDR and which is reversed upon binding of ligand. Our results implicate PIAS4 and the process of SUMOylation as important modulators of VDR-mediated signaling which may both represent flexible mechanistic components as to how vitamin D achieves its pleiotropic effects.


Asunto(s)
Proteínas Inhibidoras de STAT Activados/metabolismo , Receptores de Calcitriol/metabolismo , Citocromo P-450 CYP3A/genética , Células HEK293 , Células HeLa , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Esteroide Hidroxilasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Vitamina D3 24-Hidroxilasa
17.
Mol Cell Endocrinol ; 364(1-2): 54-64, 2012 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-22939842

RESUMEN

We investigated the capacity for vitamin D receptor (VDR) to modulate the expression of CYP3A4 and other genes that may facilitate the oxidative inactivation of androgens such as testosterone and androstanediol within prostate cells. We report that exposure to the active hormonal form of vitamin D markedly increased gene expression of CYP3A4 and CYP3A5 and ultimately achieved levels of intracellular CYP3A enzyme activity within LNCaP prostate cancer cells that were comparable to that observed for Caco2 cells, an established model of CYP3A induction, and resulted in the increased turnover of testosterone to its inactive 6ß-OH metabolite. We demonstrate that VDR directs CYP3A4 and CYP3A5 expression through binding to distinct regulatory motifs located within the 5' promoter regions of both genes. The current data highlight the potential application of VDR-based treatment regimes as a means to limit the bioavailability of growth-promoting androgens within the tumor microenvironment.


Asunto(s)
Androstenodiol/metabolismo , Calcitriol/farmacología , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP4A/genética , Neoplasias de la Próstata/genética , Receptores de Calcitriol/genética , Testosterona/metabolismo , Células CACO-2 , Calcitriol/metabolismo , Línea Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP4A/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Masculino , Regiones Promotoras Genéticas , Próstata/efectos de los fármacos , Próstata/enzimología , Próstata/patología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Receptores de Calcitriol/metabolismo , Transfección
18.
Patient Saf Surg ; 2: 24, 2008 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-18817559

RESUMEN

BACKGROUND: The implementation of the European Working Time Directive has meant the introduction of shift patterns of working for junior doctors. Patient handover between shifts has become a necessary part of practice in order to reduce the risk of medical errors. Data handed over between shifts are used to prioritise clinical jobs outstanding, and to create theatre lists. We present a closed-loop audit of handover practice to assess whether standardised proformas improve clinical data transfer between shifts during handover in our Orthopaedic Unit. METHODS: We collected data handed over between shifts for a period of one week at our department. The data were in the form of hand written data on plain paper used to assist verbal handover. Data were analysed and a standardised handover sheet was trialled. After feedback from juniors the sheet was revised and implemented. A re-audit, of handover data, was then undertaken using the revised standardised proforma during a period of 1 week. RESULTS: Forty-eight patients were handed over in week 1 while 55 patients were handed over during re-audit. The standardised proformas encouraged use of pre-printed patient labels which contained legible patient identifiers, use of labels increased from 72.9% to 93.4%. Handover of outstanding jobs increased from 31.25% to 100%. Overall data handed over increased from 72.6% to 93.2%. Handover of relevant blood results showed little improvement from 18.8% to 20.7% CONCLUSION: This audit highlights the issue of data transfer between shifts. Standardised proformas encourage filling of relevant fields and increases the data transferred between shifts thereby reducing the potential for clinical error cause by shift patterns.

19.
J Foot Ankle Surg ; 46(4): 314-6, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17586449

RESUMEN

Arterial pseudoaneurysm formation as a complication of ankle arthroscopy is extremely rare. We present a case of anterior tibial artery pseudoaneurysm identified 10 days after ankle arthroscopy in a patient with hemophilia. The diagnosis was confirmed with a duplex ultrasound scan. The patient was referred to the vascular surgeon and underwent evacuation of the hematoma, resection of the damaged segment of the artery, and reconstruction with a reversed long saphenous vein interposition graft. The patient had an uneventful recovery after the second surgery. The prevention of this complication in patients with hemophilia is discussed, as well as diagnosis and management. Preventative measures include careful dissection while making the portals, preoperative mapping of the artery with a duplex or a handheld Doppler in patients with coagulopathy, and performance of open rather than arthroscopic surgery to excise large osteophytes.


Asunto(s)
Aneurisma Falso/etiología , Articulación del Tobillo/cirugía , Artroscopía/efectos adversos , Hemofilia A/complicaciones , Arterias Tibiales , Adulto , Humanos , Masculino
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