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To date, there are no efficacious translational solutions for end-stage urinary bladder dysfunction. Current surgical strategies, including urinary diversion and bladder augmentation enterocystoplasty (BAE), utilize autologous intestinal segments (e.g. ileum) to increase bladder capacity to protect renal function. Considered the standard of care, BAE is fraught with numerous short- and long-term clinical complications. Previous clinical trials employing tissue engineering approaches for bladder tissue regeneration have also been unable to translate bench-top findings into clinical practice. Major obstacles still persist that need to be overcome in order to advance tissue-engineered products into the clinical arena. These include scaffold/bladder incongruencies, the acquisition and utility of appropriate cells for anatomic and physiologic tissue recapitulation, and the choice of an appropriate animal model for testing. In this study, we demonstrate that the elastomeric, bladder biomechanocompatible poly(1,8-octamethylene-citrate-co-octanol) (PRS; synthetic) scaffold coseeded with autologous bone marrow-derived mesenchymal stem cells and CD34+ hematopoietic stem/progenitor cells support robust long-term, functional bladder tissue regeneration within the context of a clinically relevant baboon bladder augmentation model simulating bladder trauma. Partially cystectomized baboons were independently augmented with either autologous ileum or stem-cell-seeded small-intestinal submucosa (SIS; a commercially available biological scaffold) or PRS grafts. Stem-cell synergism promoted functional trilayer bladder tissue regeneration, including whole-graft neurovascularization, in both cell-seeded grafts. However, PRS-augmented animals demonstrated fewer clinical complications and more advantageous tissue characterization metrics compared to ileum and SIS-augmented animals. Two-year study data demonstrate that PRS/stem-cell-seeded grafts drive bladder tissue regeneration and are a suitable alternative to BAE.
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The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.
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Movimento Celular , Pulmão , Mecanotransdução Celular , Neutrófilos , Animais , Camundongos , Membrana Celular , Canais Iônicos/genética , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Atividade Bactericida do Sangue/genética , Mecanotransdução Celular/genéticaRESUMO
Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.
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Macrófagos Alveolares , Imunidade Treinada , Animais , Camundongos , Transferência Adotiva , c-Mer Tirosina Quinase/genética , FagocitoseRESUMO
Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.
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Antineoplásicos Imunológicos , Neoplasias da Mama , Ligante CD27 , Resistencia a Medicamentos Antineoplásicos , MAP Quinase Quinase Quinases , Nanopartículas , Trastuzumab , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ligante CD27/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Ceramidas/química , Feminino , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/análise , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
The observation that diabetic retinopathy (DR) typically takes decades to develop suggests the existence of an endogenous system that protects from diabetes-induced damage. To investigate the existance of such a system, primary human retinal endothelial cells were cultured in either normal glucose (5 mmol/L) or high glucose (30 mmol/L; HG). Prolonged exposure to HG was beneficial instead of detrimental. Although tumor necrosis factor-α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 was unaffected after 1 day of HG, it waned as the exposure to HG was extended. Similarly, oxidative stress-induced death decreased with prolonged exposure to HG. Furthermore, mitochondrial functionality, which was compromised by 1 day of HG, was improved by 10 days of HG, and this change required increased clearance of damaged mitochondria (mitophagy). Finally, antagonizing mitochondrial dynamics compromised the cells' ability to endure HG: susceptibility to cell death increased, and basal barrier function and responsiveness to vascular endothelial growth factor deteriorated. These observations indicate the existence of an endogenous system that protects human retinal endothelial cells from the deleterious effects of HG. Hyperglycemia-induced mitochondrial adaptation is a plausible contributor to the mechanism responsible for the delayed onset of DR; loss of hyperglycemia-induced mitochondrial adaptation may set the stage for the development of DR.
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Retinopatia Diabética , Hiperglicemia , Humanos , Mitofagia , Células Endoteliais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Glucose/metabolismo , Hiperglicemia/patologia , Retinopatia Diabética/patologiaRESUMO
MicroRNAs (miRNA, miR-) play important roles in disease development. In this study, we identified an anti-proliferative miRNA, miR-212-5p, that is induced in pulmonary artery smooth muscle cells (PASMCs) and lungs of pulmonary hypertension (PH) patients and rodents with experimental PH. We found that smooth muscle cell (SMC)-specific knockout of miR-212-5p exacerbated hypoxia-induced pulmonary vascular remodeling and PH in mice, suggesting that miR-212-5p may be upregulated in PASMCs to act as an endogenous inhibitor of PH, possibly by suppressing PASMC proliferation. Extracellular vesicles (EVs) have been shown recently to be promising drug delivery tools for disease treatment. We generated endothelium-derived EVs with an enriched miR-212-5p load, 212-eEVs, and found that they significantly attenuated hypoxia-induced PH in mice and Sugen/hypoxia-induced severe PH in rats, providing proof of concept that engineered endothelium-derived EVs can be used to deliver miRNA into lungs for treatment of severe PH.
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In the lung, communication between pulmonary vascular endothelial cells (PVEC) and pulmonary artery smooth muscle cells (PASMC) is essential for the maintenance of vascular homeostasis. In pulmonary hypertension (PH), the derangement in their cell-cell communication plays a major role in the pathogenesis of pulmonary vascular remodeling. In this study, we focused on the role of PVEC-derived extracellular vesicles (EV), specifically their microRNA (miRNA, miR-) cargo, in the regulation of PASMC proliferation and vascular remodeling in PH. We found that the amount of pro-proliferative miR-210-3p was increased in PVEC-derived EV in hypoxia (H-EV), which contributes to the H-EV-induced proliferation of PASMC and the development of PH.
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Implant therapy is a treatment option to ensure prosthesis survival rate and it is also done as a fixed dental prosthesis for replacing single and multiunit gaps. Posterior maxilla often has insufficient bone quality and quantity; for this reason it makes implant placement challenging in the site. Posterior edentulous maxilla presents special challenges to implant surgeons that are unique to this region compared to other regions of the maxilla. Thus, the aim of this study is to determine the common implant dimensions used in posterior maxilla. Completed case sheets were collected from a private dental hospital software system. Case sheets were taken from June 2019 to March 2020. Data was retrieved and evaluated by two reviewers. The parameters taken were patients, age groups, gender, teeth indicated for implants (maxillary premolars and molars), implant height, and implant width. Two-hundred fifty-four implants have been placed on the posterior maxilla of which 139 were premolars and 115 were molars. There was no statistical significance between the implants placed in both males and females (p value: 0.274). Between the age groups, the highest number of implants was seen in 41-60 years (n = 146) followed by 17-40 years (n = 78) and finally > 61 years (n = 30). The p value was 0.000, which was statistically significant. Various implant sizes for posterior maxilla have been introduced due to its challenging site. Thus in our study, we can see there is a difference in sizes for premolars and molars. Implant dimensions with increased height are used in the premolars compared to the molars. Implant dimensions with increased width are used in the molars compared to the premolars. In general, implant width and implant height can range from 3.6 to 4.5 mm and implant height ranging from 9.50 to 12.00 mm.
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Maxila , Próteses e Implantes , Adulto , Feminino , Humanos , Masculino , Maxila/cirurgia , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
The complex involvement of neutrophils in inflammatory diseases makes them intriguing but challenging targets for therapeutic intervention. Here, we tested the hypothesis that varying endocytosis capacities would delineate functionally distinct neutrophil subpopulations that could be specifically targeted for therapeutic purposes. By using uniformly sized (â¼120 nm in diameter) albumin nanoparticles (ANP) to characterize mouse neutrophils in vivo, we found two subsets of neutrophils, one that readily endocytosed ANP (ANPhigh neutrophils) and another that failed to endocytose ANP (ANPlow population). These ANPhigh and ANPlow subsets existed side by side simultaneously in bone marrow, peripheral blood, spleen, and lungs, both under basal conditions and after inflammatory challenge. Human peripheral blood neutrophils showed a similar duality. ANPhigh and ANPlow neutrophils had distinct cell surface marker expression and transcriptomic profiles, both in naive mice and in mice after endotoxemic challenge. ANPhigh and ANPlow neutrophils were functionally distinct in their capacities to kill bacteria and to produce inflammatory mediators. ANPhigh neutrophils produced inordinate amounts of reactive oxygen species and inflammatory chemokines and cytokines. Targeting this subset with ANP loaded with the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, mitigated the effects of polymicrobial sepsis by reducing tissue inflammation while fully preserving neutrophilic host-defense function.
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Nanopartículas , Neutrófilos , Albuminas/metabolismo , Animais , Endocitose , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Neutrófilos/metabolismoRESUMO
TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.
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Antígenos/imunologia , Ativação Linfocitária/imunologia , Ligante OX40/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Biomarcadores , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Imunofenotipagem , Ligantes , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Família Multigênica , Fatores de Necrose Tumoral/genéticaRESUMO
Macrophages demonstrate remarkable plasticity that is essential for host defense and tissue repair. The tissue niche imprints macrophage identity, phenotype and function. The role of vascular endothelial signals in tailoring the phenotype and function of tissue macrophages remains unknown. The lung is a highly vascularized organ and replete with a large population of resident macrophages. We found that, in response to inflammatory injury, lung endothelial cells release the Wnt signaling modulator Rspondin3, which activates ß-catenin signaling in lung interstitial macrophages and increases mitochondrial respiration by glutaminolysis. The generated tricarboxylic acid cycle intermediate α-ketoglutarate, in turn, serves as the cofactor for the epigenetic regulator TET2 to catalyze DNA hydroxymethylation. Notably, endothelial-specific deletion of Rspondin3 prevented the formation of anti-inflammatory interstitial macrophages in endotoxemic mice and induced unchecked severe inflammatory injury. Thus, the angiocrine-metabolic-epigenetic signaling axis specified by the endothelium is essential for reprogramming interstitial macrophages and dampening inflammatory injury.
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Reprogramação Celular , Metabolismo Energético , Epigênese Genética , Inflamação/etiologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Trombospondinas/genética , Animais , Biomarcadores , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunofluorescência , Inflamação/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Trombospondinas/metabolismoRESUMO
INTRODUCTION: The management of gingival recession associated with esthetic concerns and root hypersensitivity is challenging, and its sequelae is based on the assessment of etiological factors and the degree of tissue involvement. Procedures using pedicle flaps, free soft tissue grafts, combination of pedicle flaps with grafts, barrier membranes, and the use of platelet concentrates are all effective for this purpose. The use of the third-generation platelet concentrate, advanced platelet-rich fibrin (A-PRF), has evolved as a promising regenerative material for root coverage procedure wherein it acts as a scaffold and also accelerates wound healing due to its dense fibrin meshwork. CASE PRESENTATION: This case report, discusses treating an isolated maxillary Miller Class I recession in a 25-year-old male patient by a periosteal inversion method along with the A-PRF membrane. A partial thickness flap was reflected; periosteum was inverted; and an A-PRF membrane was placed over the denuded root surface which aided in enhanced regeneration; 100% root coverage was obtained as seen in follow-up visits. CONCLUSION: The periosteal inversion technique along with an A-PRF membrane seems to be a novel approach in managing an isolated Miller Class I maxillary gingival recession.
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Retração Gengival , Fibrina Rica em Plaquetas , Adulto , Estética Dentária , Fibrina , Retração Gengival/cirurgia , Humanos , Masculino , Resultado do TratamentoRESUMO
The sex-biased disease pulmonary arterial hypertension (PAH) is characterized by the proliferation and overgrowth of dysfunctional pulmonary artery endothelial cells (PAECs). During inflammation associated with PAH, granzyme B cleaves intersectin-1 to produce N-terminal (EHITSN) and C-terminal (SH3A-EITSN) protein fragments. In a murine model of PAH, EHITSN triggers plexiform arteriopathy via p38-ELK1-c-Fos signaling. The SH3A-EITSN fragment also influences signaling, having dominant-negative effects on ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively). Using PAECs engineered to express tagged versions of EHITSN and SH3A-EITSN, we demonstrate that the two ITSN fragments increase both p38-ELK1 activation and the ratio of p38 to ERK1 and ERK2 activity, leading to PAEC proliferation, with female cells being more responsive than male cells. Furthermore, expression of EHITSN substantially upregulates the expression and activity of the long non-coding RNA Xist in female PAECs, which in turn upregulates the X-linked gene ELK1 and represses expression of krüppel-like factor 2 (KLF2). These events are recapitulated by the PAECs of female idiopathic PAH patients, and may account for their proliferative phenotype. Thus, upregulation of Xist could be an important factor in explaining sexual dimorphism in the proliferative response of PAECs and the imbalanced sex ratio of PAH.
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Hipertensão Pulmonar , Artéria Pulmonar , Animais , Proliferação de Células , Células Cultivadas , Células Endoteliais , Feminino , Humanos , Masculino , Camundongos , Caracteres SexuaisRESUMO
It is of interest to assess the association of age and gender of patients undergoing class V tooth colored restoration in maxillary teeth. Records were collected by reviewing the data of 86,000 patients of which 1580 patients had undergone class V tooth colored restoration in maxillary teeth. Patients were divided into age groups 18-30, 31-40,41-50,71-80 years. The most common age group who had undergone class V restoration was in the age group 41-50 years (30.8%). Cervical abrasions were the most common (79%). Most of the patients underwent direct restoration (87.7%). Direct restoration was found to be more prevalent in the age group 41-50 years due to cervical abrasion.
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INTRODUCTION: Treating the patients with excessive gingival display to provide a pleasant smile is a challenge to the periodontist. Gummy smile can be due to excessive vertical bone growth, dentoalveolar extrusion, short upper lip, upper lip hyperactivity, or altered passive eruption. Gummy smile associated with hyperactivity of smile elevator muscles can be treated by surgical techniques like lip repositioning, botulinum toxin injection, lip elongation with rhinoplasty, detachment of the lip muscles, and myectomy. Regardless of the technique used, to achieve a predictable result with long-term stability limiting upper lip movement when the patient smiles, firm muscle containment is imperative. CASE PRESENTATION: The case report describes the excessive gingival display having a multifactorial etiology in a 25-year-old female patient. Altered passive eruption in upper anterior teeth was treated by crown lengthening followed by management of hyperactive lip using a diode laser-assisted lip repositioning along with traction and muscle containment. Excellent and predictable results were obtained after a 1-year follow-up without the relapse of gummy smile. CONCLUSIONS: The case report showed an excellent result when treated by a combined approach of an innovative procedure with laser-assisted lip repositioning aimed at maintaining the traction and containment of the smile elevator muscles along with crown lengthening procedure by gingivectomy.
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Aumento da Coroa Clínica , Estética Dentária , Gengivectomia , Lábio , Adulto , Feminino , Humanos , Lábio/anatomia & histologia , Lábio/cirurgia , SorrisoRESUMO
BACKGROUND AND PURPOSE: Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets. RESULTS: TrBs were defined by surface marker expression as CD19+CD24hiCD38hi. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+CD38+), early memory (CD24hiCD38-), and late memory (CD24-CD38-) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation. CONCLUSIONS: We optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies.
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Antígenos CD/imunologia , Subpopulações de Linfócitos B , Citometria de Fluxo/métodos , Memória Imunológica , Sistema de Sinalização das MAP Quinases/imunologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: Pulmonary endothelial cells' (ECs) injury and apoptotic death are necessary and sufficient for the pathogenesis of the acute respiratory distress syndrome (ARDS), regardless of epithelial damage. Interaction of dysfunctional ECs with circulatory extracellular vesicles (EVs) holds therapeutic promise in ARDS. However, the presence in the blood of long-term ARDS survivors of EVs with a distinct phenotype compared to the EVs of non-surviving patients is not reported. With a multidisciplinary translational approach, we studied EVs from the blood of 33 patients with moderate-to-severe ARDS. RESULTS: The EVs were isolated from the blood of ARDS and control subjects. Immunoblotting and magnetic beads immunoisolation complemented by standardized flow cytometry and nanoparticles tracking analyses identified in the ARDS patients a subset of EVs with mesenchymal stem cell (MSC) origin (CD73+CD105+Cd34-CD45-). These EVs have 4.7-fold greater counts compared to controls and comprise the transforming growth factor-beta receptor I (TßRI)/Alk5 and the Runx1 transcription factor. Time course analyses showed that the expression pattern of two Runx1 isoforms is critical for ARDS outcome: the p52 isoform shows a continuous expression, while the p66 is short-lived. A high ratio Runx1p66/p52 provided a survival advantage, regardless of age, sex, disease severity or length of stay in the intensive care unit. Moreover, the Runx1p66 isoform is transiently expressed by cultured human bone marrow-derived MSCs, it is released in the EVs recoverable from the conditioned media and stimulates the proliferation of lipopolysaccharide (LPS)-treated ECs. The findings are consistent with a causal effect of Runx1p66 expression on EC proliferation. Furthermore, morphological and functional assays showed that the EVs bearing the Runx1p66 enhanced junctional integrity of LPS-injured ECs and decreased lung histological severity in the LPS-treated mice. CONCLUSIONS: The expression pattern of Runx1 isoforms might be a reliable circulatory biomarker of ARDS activity and a novel determinant of the molecular mechanism for lung vascular/tissue repair and recovery after severe injury.
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Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders hemoglobin susceptible to polymerization when deoxygenated, affects millions of people worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors eliminate their mitochondria during the terminal differentiation process. Strikingly, we observed an increased percentage of RBCs retaining mitochondria in SCD patient blood samples compared with healthy individuals. In addition, using an experimental SCD mouse model, we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mitochondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1 showed increased expression of mitophagy genes. Our findings suggest that reduction of mitochondria-retaining RBCs may provide a new therapeutic approach to preventing excessive ROS in SCD.
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Anemia Falciforme/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Histona Desmetilases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Biológicos , Rodaminas/farmacologia , Sirolimo/farmacologia , Compostos de Espiro/farmacologia , Tiofenos/farmacologiaRESUMO
Antigen uptake, processing and presentation by antigen presenting cells (APCs) are tightly coupled processes which consequently lead to the activation of innate and adaptive immune responses. However, the regulatory role of microRNA (miRNAs) in these critical pathways is poorly understood. In this study, we show that overexpression of miR-24, miR-30b and miR-142-3p attenuates uptake and processing of soluble antigen ovalbumin (Ova) in primary human macrophages and dendritic cells. MiRNA mimic transfected APCs exhibit defects in antigen presentation (Ova and CMV antigen) to CD4+ T-cells leading to reduced cell proliferation. Using transgenic OT-II mice we demonstrated that this impairment in T-cell proliferation is specific to antigen provided i.e., Ova. Further, human T-cells co-cultured with miRNA transfected dendritic cells secrete low levels of T helper (Th)-1 polarization associated cytokines. Analysis of molecules regulating APC and T-cell receptor interaction shows miRNA-mediated induced expression of Programmed Death-Ligand 1 (PD-L1) which inhibits T-cell proliferation. Blocking PD-L1 with antibodies rescues miRNA-mediated inhibition of T cell priming by DCs. These results uncover regulatory functions of miR-24, miR-30b and miR-142-3p in pairing innate and adaptive components of immunity.