RESUMO
Skin represents the main barrier against the external environment, but also plays a role in human relations, as one of the prime determinants of beauty, resulting in a high consumer demand for skincare-related pharmaceutical products. Given the importance of skin aging in both medical and social spheres, the present research aims to characterize microscopic changes in human skin composition due to intrinsic aging (as opposed to aging influenced by external factors) via histological analysis of a photoprotected body region. Samples from 25 autopsies were taken from the periumbilical area and classified into four age groups: group 1 (0-12 years), group 2 (13-25 years), group 3 (26-54 years), and group 4 (≥ 55 years). Different traditional histological (hematoxylin-eosin, Masson's trichrome, orcein, toluidine, Alcian blue, and Feulgen reaction) and immunohistochemical (CK20, CD1a, Ki67, and CD31) stains were performed. A total of 1879 images photographed with a Leica DM3000 optical microscope were morphometrically analyzed using Image ProPlus 7.0 for further statistical analysis with GraphPad 9.0. Our results showed a reduction in epidermis thickness, interdigitation and mitotic indexes, while melanocyte count was raised. Papillary but not reticular dermis showed increased thickness with aging. Specifically, in the papillary layer mast cells and glycosaminoglycans were expanded, whereas the reticular dermis displayed a diminution in glycosaminoglycans and elastic fibers. Moreover, total cellularity and vascularization of both dermises were diminished with aging. This morphometric analysis of photoprotected areas reveals that intrinsic aging significantly influences human skin composition. This study paves the way for further research into the molecular basis underpinning these alterations, and into potential antiaging strategies.
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Carbon black nanomaterial (CB-NM), as an industrial product with a large number of applications, poses a high risk of exposure, and its impact on health needs to be assessed. The most common testing platform for engineered (E)NMs is in vitro toxicity assessment, which requires prior ENM dispersion, stabilization, and characterization in cell culture media. Here, asymmetric flow field-flow fractionation (AF4) coupled to UV-Vis and dynamic light scattering (DLS) detectors in series was used for the study of CB dispersions in cell culture media, optimizing instrumental variables and working conditions. It was possible to disperse CB in a non-ionic surfactant aqueous solution due to the steric effect provided by surfactant molecules attached on the CB surface which prevented agglomeration. The protection provided by the surfactant or by culture media alone was insufficient to ensure good dispersion stability needed for carrying out in vitro toxicity studies. On the other hand, cell culture media in combination with the surfactant improved dispersion stability considerably, enabling the generation of shorter particles and a more favourable zeta potential magnitude, leading to greater stability due to electrostatic repulsion. It was demonstrated that the presence of amino acids in the culture media improved the monodisperse nature and stability of the CB dispersions, and resulted in a turn towards more negative zeta potential values when the pH was above the amino acid isoelectric point (IEP). Culture media used in real cell culture scenarios were also tested, and in vitro toxicity assays were developed optimizing the compatible amount of surfactant.
Assuntos
Fracionamento por Campo e Fluxo , Nanoestruturas , Surfactantes Pulmonares , Técnicas de Cultura de Células , Meios de Cultura , Fracionamento por Campo e Fluxo/métodos , Nanoestruturas/toxicidade , Nanoestruturas/química , Tamanho da Partícula , Fuligem/toxicidade , Tensoativos/toxicidade , Ponto IsoelétricoRESUMO
Myocardial fibrosis is a pathological hallmark of cardiac dysfunction. Oncostatin M (OSM) is a pleiotropic cytokine that can promote fibrosis in different organs after sustained exposure. However, OSM released by macrophages during cardiac fibrosis suppresses cardiac fibroblast activation by modulating transforming growth factor beta 1 (TGF-ß1) expression and extracellular matrix deposition. Small extracellular vesicles (SEVs) from mesenchymal stromal cells (MSCs) are being investigated to treat myocardial infarction, using different strategies to bolster their therapeutic ability. Here, we generated TERT-immortalized human MSC cell lines (MSC-T) engineered to overexpress two forms of cleavage-resistant OSM fused to CD81TM (OSM-SEVs), which allows the display of the cytokine at the surface of secreted SEVs. The therapeutic potential of OSM-SEVs was assessed in vitro using human cardiac ventricular fibroblasts (HCF-Vs) activated by TGF-ß1. Compared with control SEVs, OSM-loaded SEVs reduced proliferation in HCF-V and blunted telo-collagen expression. When injected intraperitoneally into mice treated with isoproterenol, OSM-loaded SEVs reduced fibrosis, prevented cardiac hypertrophy, and increased angiogenesis. Overall, we demonstrate that the enrichment of functional OSM on the surface of MSC-T-SEVs increases their potency in terms of anti-fibrotic and pro-angiogenic properties, which opens new perspectives for this novel biological product in cell-free-based therapies.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Humanos , Animais , Camundongos , Oncostatina M/farmacologia , Oncostatina M/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Isoproterenol , Fibrose , Células-Tronco Mesenquimais/metabolismo , Vesículas Extracelulares/metabolismoRESUMO
To establish a set of variables that define a predictive profile of events of maxillofacial trauma resulting from interpersonal violence, we analyzed sociodemographic variables and clinical characteristics of injuries recorded in three tertiary care hospital centers in Chile. To assess the relation between categories, we applied a multiple correspondence analysis. We identified 567 cases. Two dimensions explained 53.4% of the model. The first dimension was composed of variables related to the severity of the injury: medical-legal prognosis (.574), type of trauma (.511), and the destination of the patient (.332); the second dimension was composed of variables related to the typology of interpersonal violence: type of violence (.398) and sex of the patient (.370). Two profiles were recognized: women, victims of domestic violence, with lesions affecting mainly soft tissues and not requiring hospitalization and men, victims of community violence, with lesions involving fractures associated with greater severity and requiring hospitalization. There are two key dimensions in the diagnosis of maxillofacial trauma resulting from interpersonal violence: severity of the injury and typology of the interpersonal violence. Exploring these predictive profiles can be a useful complement to the current screening tools of violence in clinical practice.
Assuntos
Violência Doméstica , Traumatismos Maxilofaciais , Masculino , Humanos , Feminino , Conduta Expectante , Serviço Hospitalar de Emergência , Traumatismos Maxilofaciais/epidemiologia , Estudos RetrospectivosRESUMO
Many anticancer agents used in clinics induce premature senescence in healthy tissues generating accelerated aging processes and adverse side-effects in patients. Cardiotoxicity is a well-known limiting factor of anticancer treatment with doxorubicin (DOX), a very effective anthracycline widely used as antitumoral therapy in clinical practice, that leads to long-term morbidity and mortality. DOX exposure severely affects the population of cardiac cells in both mice and human hearts by inducing premature senescence, which may represent the molecular basis of DOX-induced cardiomyopathy. Here, we demonstrate that senescence induction in the heart contributes to impaired cardiac function in mice upon DOX treatment. Concomitant elimination of senescent cells with the senolytic Navitoclax in different formulations produces a significant decrease in senescence and cardiotoxicity markers together with the restoration of the cardiac function in mice followed by echocardiography. These results evidence the potential clinical use of senolytic therapies to alleviate cardiotoxicities induced in chemotherapy-treated patients.
Assuntos
Cardiomiopatias , Cardiotoxicidade , Animais , Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/prevenção & controle , Cardiotoxicidade/tratamento farmacológico , Doxorrubicina/efeitos adversos , Humanos , Camundongos , Miócitos Cardíacos , SenoterapiaRESUMO
Impaired wound healing in patients with type 2 diabetes (DM2) is characterized by chronic inflammation, which delays wound closure. Specialized pro-resolving lipid mediators (SPMs) are bioactive molecules produced from essential polyunsaturated fatty acids (PUFAs), principally omega-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). SPMs are potent regulators of inflammation and have been used to suppress chronic inflammation in peripheral artery disease, non-alcoholic fatty liver disease, and central nervous system syndromes. LIPINOVA® is a commercially available safe-grade nutritional supplement made from a fractionated marine lipid concentrate derived from anchovy and sardine oil that is rich in SPMs and EPA, as well as DHA precursors. Here, we assessed the effect of LIPINOVA® in wound dressing applications. LIPINOVA® showed biocompatibility with keratinocytes and fibroblasts, reduced the abundance of pro-inflammatory macrophages (Mφ1), and promoted in vitro wound closure. Daily application of the marine oil to open wounds made by punch biopsy in db/db mice promoted wound closure by accelerating the resolution of inflammation, inducing neoangiogenesis and Mφ1/Mφ2 macrophage polarization. In conclusion, LIPINOVA® displays pro-resolutive properties and could be exploited as a therapeutic agent for the treatment of diabetic ulcers.
Assuntos
Diabetes Mellitus Tipo 2 , Ácidos Graxos Ômega-3 , Administração Tópica , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-3/uso terapêutico , Inflamação/tratamento farmacológico , Macrófagos , Camundongos , CicatrizaçãoRESUMO
Marfan syndrome (MFS) is a systemic connective tissue disorder caused by mutations in the fibrillin-1 (FBN1) gene, and cardiovascular involvement is the leading cause of mortality. We sought to examine the genotype/phenotype realtionship in 61 consecutive patients with a phenotype and genotype compatible with MFS. The FBN1 gene was analyzed by massive sequencing using a hybridization capture-based target enrichment custom panel. Forty-three different variants of FBN1 were identified, of which 17 have not been previously reported. The causal variants of MFS were grouped into mutations resulting in haploinsufficiency (HI group; 23 patients) and mutations producing a dominant-negative effect (DN group; 38 patients). Patient information was collected from electronic medical records and clinical evaluation. While no significant differences were found between the two groups, the HI group included more cases with aortic dissection and occurring at a younger age that the DN group (34.7% vs. 15.8%; p = 0.160). Irrespective of the mutation group, males presented with a higher probability of aortic involvement (4-fold higher risk than females) and aortic dissections events occurred at younger ages. Patients with DN variants carrying a cysteine substitution had a higher incidence of ectopia lentis.
Assuntos
Fibrilina-1/genética , Síndrome de Marfan/genética , Adolescente , Adulto , Doenças da Aorta/genética , Doenças Cardiovasculares/genética , Criança , Estudos de Coortes , Estudos Transversais , Feminino , Estudos de Associação Genética , Genótipo , Haploinsuficiência , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Adulto JovemRESUMO
Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared the results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and a Rapid Barcoding Kit (RBK) vs a Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in thymine and adenine homopolymeric regions than the R9, at the expenses of a larger rate (~10%) of mismatches in these regions. The LSK had a larger yield than the RBK, and provided longer reads than the RBK. It also resulted in a larger percentage of aligned reads (99 vs 93%) and also in a complete consensus genome. The results from this study suggest that the LSK preparation library provided longer DNA fragments which contributed to a better assembly of the SARS-CoV-2, despite an impaired detection of variants in a R10 flow cell. Nanopore sequencing could be used in epidemiological surveillance of SARS-CoV-2. KEY POINTS: ⢠Sequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance. ⢠Nanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome. ⢠Ligation sequencing is preferred rather than the rapid kit using transposases.
Assuntos
Genoma Viral , Nanoporos , SARS-CoV-2/genética , Análise de Sequência de RNA/métodosRESUMO
The dextro-transposition of the great arteries (d-TGA) is one of the most common congenital heart diseases. To identify biological processes that could be related to the development of d-TGA, we established induced pluripotent stem cell (iPSC) lines from two patients with d-TGA and from two healthy subjects (as controls) and differentiated them into endothelial cells (iPSC-ECs). iPSC-EC transcriptome profiling and bioinformatics analysis revealed differences in the expression level of genes involved in circulatory system and animal organ development. iPSC-ECs from patients with d-TGA showed impaired ability to develop tubular structures in an in vitro capillary-like tube formation assay, and interactome studies revealed downregulation of biological processes related to Notch signaling, circulatory system development and angiogenesis, pointing to alterations in vascular structure development. Our study provides an iPSC-based cellular model to investigate the etiology of d-TGA.
Assuntos
Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Receptores Notch/genética , Transposição dos Grandes Vasos/patologia , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Análise de Sequência de RNA , Transdução de Sinais , Transposição dos Grandes Vasos/genéticaRESUMO
Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EVMSC-T-HIFc). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EVMSC-T-HIFc suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.
Assuntos
Vesículas Extracelulares/transplante , Hipersensibilidade Tardia/terapia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imunomodulação , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Linfócitos T/imunologia , Animais , Linhagem Celular , Proliferação de Células , Células Cultivadas , Citocinas/farmacologia , Polpa Dentária/citologia , Vesículas Extracelulares/imunologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lentivirus/genética , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfócitos T/fisiologia , Telomerase/genética , Telomerase/metabolismo , Adulto JovemRESUMO
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have therapeutic potential in the treatment of several immune disorders, including ulcerative colitis, owing to their regenerative and immunosuppressive properties. We recently showed that MSCs engineered to overexpress hypoxia-inducible factor 1-alpha and telomerase (MSC-T-HIF) and conditioned with pro-inflammatory stimuli release EVs (EVMSC-T-HIFC) with potent immunomodulatory activity. We tested the efficacy of EVMSC-T-HIFC to repolarize M1 macrophages (Mφ1) to M2-like macrophages (Mφ2-like) by analyzing surface markers and cytokines and performing functional assays in co-culture, including efferocytosis and T-cell proliferation. We also studied the capacity of EVMSC-T-HIFC to dampen the inflammatory response of activated endothelium and modulate fibrosis. Finally, we tested the therapeutic capacity of EVMSC-T-HIFC in an acute colitis model. EVMSC-T-HIFc induced the repolarization of monocytes from Mφ1 to an Mφ2-like phenotype, which was accompanied by reduced inflammatory cytokine release. EVMSC-T-HIFc-treated Mφ1 had similar effects of immunosuppression on activated peripheral blood mononuclear cells (PBMC) as Mφ2, and reduced the adhesion of PBMCs to activated endothelium. EVMSC-T-HIFc also prevented myofibroblast differentiation of TGF-ß-treated fibroblasts. Finally, administration of EVMSC-T-HIFc promoted healing in a TNBS-induced mouse colitis model in terms of preserving colon length and intestinal mucosa architecture and altering the ratio of Mφ1/ Mφ2 infiltration. In conclusion, EVMSC-T-HIFC have effective anti-inflammatory properties, making them potential therapeutic agents in cell free-based therapies for the treatment of Crohn's disease and likely other immune-mediated inflammatory diseases.
Assuntos
Doença de Crohn/terapia , Vesículas Extracelulares/transplante , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Adesão Celular , Polaridade Celular , Doença de Crohn/induzido quimicamente , Doença de Crohn/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Telomerase/metabolismo , Ácido Trinitrobenzenossulfônico/efeitos adversos , Adulto JovemRESUMO
Late-onset heart failure (HF) is a known side effect of doxorubicin chemotherapy. Typically, patients are diagnosed when already at an irreversible stage of HF, which allows few or no treatment options. Identifying the causes of compromised cardiac function in this patient group may improve early patient diagnosis and support treatment selection. To link doxorubicin-induced changes in cardiac cellular and tissue mechanical properties to overall cardiac function, we apply a multiscale biophysical biomechanics model of the heart to measure the plausibility of changes in model parameters representing the passive, active, or anatomical properties of the left ventricle for reproducing measured patient phenotypes. We create representative models of healthy controls (N = 10) and patients with HF induced by (N = 22) or unrelated to (N = 25) doxorubicin therapy. The model predicts that HF in the absence of doxorubicin is characterized by a 2- to 3-fold stiffness increase, decreased tension (0-20%), and ventricular dilation (of order 10-30%). HF due to doxorubicin was similar but showed stronger bias toward reduced active contraction (10-30%) and less dilation (0-20%). We find that changes in active, passive, and anatomical properties all play a role in doxorubicin-induced cardiotoxicity phenotypes. Differences in parameter changes between patient groups are consistent with doxorubicin cardiotoxicity having a greater dependence on reduced cellular contraction and less anatomical remodeling than HF not caused by doxorubicin.
Assuntos
Doxorrubicina/efeitos adversos , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/patologia , Miocárdio/patologia , Colágeno/metabolismo , Feminino , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/efeitos dos fármacos , FenótipoRESUMO
Insufficient vessel growth associated with ischemia remains an unresolved issue in vascular medicine. Mesenchymal stem cells (MSCs) have been shown to promote angiogenesis via a mechanism that is potentiated by hypoxia. Overexpression of hypoxia inducible factor (HIF)-1α in MSCs improves their therapeutic potential by inducing angiogenesis in transplanted tissues. Here, we studied the contribution of exosomes released by HIF-1α-overexpressing donor MSCs (HIF-MSC) to angiogenesis by endothelial cells. Exosome secretion was enhanced in HIF-MSC. Omics analysis of miRNAs and proteins incorporated into exosomes pointed to the Notch pathway as a candidate mediator of exosome communication. Interestingly, we found that Jagged1 was the sole Notch ligand packaged into MSC exosomes and was more abundant in HIF-MSC than in MSC controls. The addition of Jagged1-containing exosomes from MSC and HIF-MSC cultures to endothelial cells triggered transcriptional changes in Notch target genes and induced angiogenesis in an in vitro model of capillary-like tube formation, and both processes were stimulated by HIF-1α. Finally, subcutaneous injection of Jagged 1-containing exosomes from MSC and HIF-MSC cultures in the Matrigel plug assay induced angiogenesis in vivo, which was more robust when they were derived from HIF-MSC cultures. All Jagged1-mediated effects could be blocked by prior incubation of exosomes with an anti-Jagged 1 antibody. All together, the results indicate that exosomes derived from MSCs stably overexpressing HIF-1α have an increased angiogenic capacity in part via an increase in the packaging of Jagged1, which could have potential applications for the treatment of ischemia-related disease. Stem Cells 2017;35:1747-1759.
Assuntos
Exossomos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína Jagged-1/genética , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Animais , Anticorpos Neutralizantes/farmacologia , Hipóxia Celular , Técnicas de Cocultura , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Exossomos/química , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína Jagged-1/antagonistas & inibidores , Proteína Jagged-1/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Cultura Primária de Células , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Transdução Genética , Transplante Heterólogo , Proteína Vermelha FluorescenteRESUMO
Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF-κB) in human MSCs. This pathway is activated by TNF-α that is generated following TCR stimulation of T cells. Inhibition of NF-κB through silencing of IκB kinase ß or the TNF-α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC-associated NF-κB activation primarily leads to inhibition of T-cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF-α/NF-κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF-κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.
Assuntos
Ativação Linfocitária/imunologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , NF-kappa B/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
RATIONALE: Transplantation of allogeneic cardiac stem/progenitor cells (CPC) in experimental myocardial infarction promoted cardiac regeneration and improved heart function. Although this has enhanced prospects of using allogeneic CPC for cardiac repair, the mechanisms regulating the behavior of these allogeneic cells, which are central to clinical applications, remain poorly understood. OBJECTIVE: T cells orchestrate the allogeneic adaptive immune response. Therefore, to provide insight into the mechanisms regulating the immunologic behavior of human CPC (hCPC), we investigated the allogeneic T-cell response elicited by cryopreserved c-kit-selected hCPC. METHODS AND RESULTS: By using an experimental model of allogeneic stimulation, we demonstrate that, whether under inflammatory conditions or not, hCPC do not trigger conventional allogeneic Th1 or Th2 type responses but instead induce proliferation and selective expansion of suppressive CD25(high)CD127(low)human leukocyte antigen-DR(+)FoxP3(high) effector regulatory T cells. The regulatory T-cell proliferation and amplification were dependent on the interaction with the B7 family member programmed death ligand 1 (PD-L1), which is substantially expressed on hCPC and increased under inflammatory conditions. Thus, hCPC in allogeneic settings acquire the capacity to downregulate an ongoing immune response, which was dependent on PD-L1. CONCLUSIONS: Collectively, these data reveal that hCPC in allogeneic settings have a tolerogenic immune behavior, promoting a contact PD-L1-dependent regulatory response and a PD-L1-dependent allogeneic-driven immunomodulation. Our study attributes an important role for PD-L1 in the immune behavior of allogeneic hCPC and raises the possibility of using PD-L1 expression as a marker to identify and select low-risk high-benefit allogeneic cardiac repair cells.
Assuntos
Imunidade Adaptativa , Antígeno B7-H1/metabolismo , Comunicação Celular , Tolerância Imunológica , Miócitos Cardíacos/imunologia , Células-Tronco/imunologia , Linfócitos T Reguladores/imunologia , Antígeno B7-H1/genética , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Criopreservação , Fatores de Transcrição Forkhead/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ativação Linfocitária , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Interferência de RNA , Transplante de Células-Tronco , Células-Tronco/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/metabolismo , TransfecçãoRESUMO
BACKGROUND: Small Extracellular Vesicles (sEVs) are nano-sized vesicles that are present in all biofluids including human milk (HM) playing a crucial role in cell-to-cell communication and the stimulation of the neonatal immune system. Oxylipins, which are bioactive lipids formed from polyunsaturated fatty acids, have gained considerable attention due to their potential role in mitigating disease progression and modulating the inflammatory status of breastfed infants. This study aims at an in-depth characterization of the oxylipin profiles of HM and, for the first time, of HM-derived sEVs (HMEVs) employing an ad-hoc developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. RESULTS: The UPLC-MS/MS method covered a panel of 13 oxylipins for quantitation and 93 oxylipins for semi-quantitation. In 200 µL of HM and HMEV isolates of 15 individuals, 42 out of 106 oxylipins were detected in either HM or HMEVs, with 38 oxylipins being detected in both matrices. Oxylipins presented distinct profiles in HM and HMEVs, suggesting specific mechanisms responsible for the encapsulation of target molecules in HMEVs. Ten and eight oxylipins were quantified with ranges between 0.03 - 73 nM and 0.30 pM-0.07 nM in HM and HMEVs, respectively. The most abundant oxylipins found in HMEVs were docosahexaenoic acid derivatives (17-HDHA and 14-HDHA) with known anti-inflammatory properties, and linoleic acid derivatives (9-10-DiHOME and 12,13-DiHOME) in HM samples. SIGNIFICANCE AND NOVELTY: This is the first time a selective, relative enrichment of anti-inflammatory oxylipins in HMEVs has been described. Future studies will focus on the anti-inflammatory and pro-healing capacity of oxylipins encapsulated in HMEVs, with potential clinical applications in the field of preterm infant care, specifically the prevention of severe intestinal complications including necrotizing enterocolitis.
Assuntos
Vesículas Extracelulares , Leite Humano , Oxilipinas , Espectrometria de Massas em Tandem , Humanos , Leite Humano/química , Oxilipinas/análise , Oxilipinas/química , Vesículas Extracelulares/química , Cromatografia Líquida de Alta Pressão , FemininoRESUMO
BACKGROUND/AIMS: Extracellular vesicles (EVs) derived from dental pulp mesenchymal stem cells (DP-MSCs) are a promising therapeutic option for the treatment of myocardial ischemia. The aim of this study is to determine whether MSC-EVs could promote a pro-resolving environment in the heart by modulating macrophage populations. METHODS: EVs derived from three independent biopsies of DP-MSCs (MSC-EVs) were isolated by tangential flow-filtration and size exclusion chromatography and were characterized by omics analyses. Biological processes associated with these molecules were analyzed using String and GeneCodis platforms. The immunomodulatory capacity of MSC-EVs to polarize macrophages towards a pro-resolving or M2-like phenotype was assessed by evaluating surface markers, cytokine production, and efferocytosis. The therapeutic potential of MSC-EVs was evaluated in an acute myocardial infarction (AMI) model in nude rats. Infarct size and the distribution of macrophage populations in the infarct area were evaluated 7 and 21 days after intramyocardial injection of MSC-EVs. RESULTS: Lipidomic, proteomic, and miRNA-seq analysis of MSC-EVs revealed their association with biological processes involved in tissue regeneration and regulation of the immune system, among others. MSC-EVs promoted the differentiation of pro-inflammatory macrophages towards a pro-resolving phenotype, as evidenced by increased expression of M2 markers and decreased secretion of pro-inflammatory cytokines. Administration of MSC-EVs in rats with AMI limited the extent of the infarcted area at 7 and 21 days post-infarction. MSC-EV treatment also reduced the number of pro-inflammatory macrophages within the infarct area, promoting the resolution of inflammation. CONCLUSION: EVs derived from DP-MSCs exhibited similar characteristics at the omics level irrespective of the biopsy from which they were derived. All MSC-EVs exerted effective pro-resolving responses in a rat model of AMI, indicating their potential as therapeutic agents for the treatment of inflammation associated with AMI.
RESUMO
A barn owl (Tyto alba) died with neurological signs compatible with a viral infection. After discarding other possible infections caused by circulating viruses in the area, analysis of the central nervous system using a pan-viral microarray revealed hybridization to canary bornavirus 2 (CnBV-2). Subsequent sequence analysis confirmed the presence of a virus sharing more than 83% identity with CnBV-2. Surprisingly, the new sequence corresponds to a new virus, here named Barn owl Bornavirus 1 (BoBV-1), within the Orthobornavirus serini species. Moreover, it is the first member of this species that has been detected in a non-passerine bird, indicating that Orthobornavirus serini species comprises viruses with a wider range of hosts than previously presumed. The use of this microarray has proven to be an excellent tool for viral detection in clinical samples, with capacity to detect new viral variants. This allows the diagnosis of a great range of viruses, which can cause similar disease symptoms and which identification by PCR methods might be tedious, probably unsuccessful and, in the long run, expensive. This platform is highly useful for a fast and precise viral detection, contributing to the improvement of diagnostic methods.
Assuntos
Bornaviridae , Estrigiformes , Animais , Bornaviridae/genéticaRESUMO
BACKGROUND: West Nile Virus (WNV) is a zoonotic arbovirus worldwide spread. Seasonal WNV outbreaks occur in the Mediterranean basin since the late 1990's with ever-increasing incidence. In Southern Spain WNV is endemic, as disease foci - caused by WNV lineage 1 (WNV-L1) strains - occur every year. On the contrary, WNV-L2 is the dominant lineage in Europe, so most European WNV sequences available belong to this lineage, WNV-L1 sequences being still scarce. METHODS: To fill this gap, this study reports the genetic characterisation of 27 newly described WNV-L1 strains, involved in outbreaks affecting wild birds and horses during the last decade in South-Western Spain. RESULTS: All strains except one belong to the Western Mediterranean-1 sub-cluster (WMed-1), related phylogenetically to Italian, French, Portuguese, Moroccan and, remarkably, Senegalese strains. This sub-cluster persisted, spread and evolved into three distinguishable WMed-1 phylogenetic groups that co-circulated, notably, in the same province (Cádiz). They displayed different behaviours: from long-term persistence and rapid spread to neighbouring regions within Spain, to long-distance spread to different countries, including transcontinental spread to Africa. Among the different introductions of WNV in Spain revealed in this study, some of them succeeded to get established, some extinguished from the territory shortly afterwards. Furthermore, Spain's southernmost province, Cádiz, constitutes a hotspot for virus incursion. CONCLUSION: Southern Spain seems a likely scenario for emergence of exotic pathogens of African origin. Therefore, circulation of diverse WNV-L1 variants in Spain prompts for an extensive surveillance under a One Health approach.
Assuntos
Aves , Filogenia , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/classificação , Vírus do Nilo Ocidental/isolamento & purificação , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Febre do Nilo Ocidental/transmissão , Animais , Espanha/epidemiologia , Aves/virologia , Doenças das Aves/virologia , Doenças das Aves/epidemiologia , Cavalos/virologia , Europa (Continente)/epidemiologia , Surtos de Doenças , África/epidemiologia , Doenças dos Cavalos/virologia , Doenças dos Cavalos/epidemiologia , Humanos , Animais Selvagens/virologiaRESUMO
BACKGROUND: Vector-borne diseases like West Nile virus (WNV) pose a global health challenge, with rising incidence and distribution. Culex mosquitoes are crucial WNV vectors. Avian species composition and bird community diversity, along with vector communities, influence WNV transmission patterns. However, limited knowledge exists on their impact in southwestern Spain, an area with active WNV circulation in wild birds, mosquitoes, and humans. METHODS: To address this, we conducted a comprehensive study investigating the contributions of migratory and exotic bird species to WNV transmission and the influence of mosquito community composition. RESULTS: Analysing 1194 serum samples from 44 avian species, we detected WNV antibodies in 32 samples from 11 species, four for the first time in Europe. Migratory birds had higher WNV exposure likelihood than native and exotic species, and higher phylogenetic diversity in bird communities correlated with lower exposure rates. Moreover, in 5859 female mosquitoes belonging to 12 species, we identified WNV competent vectors like Cx. pipiens s.l. and the Univittatus subgroup. Birds with WNV antibodies were positively associated with competent vector abundance, but negatively with overall mosquito species richness. CONCLUSIONS: These findings highlight the complex interactions between bird species, their phylogenetics, and mosquito vectors in WNV transmission. Understanding these dynamics will help to implement effective disease control strategies in southwestern Spain.