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Background: Uveal melanoma is a poor prognosis cancer. Ergolide, a sesquiterpene lactone isolated from Inula Brittanica, exerts anti-cancer properties. The objective of this study was to 1) evaluate whether ergolide reduced metastatic uveal melanoma (MUM) cell survival/viability in vitro and in vivo; and 2) to understand the molecular mechanism of ergolide action. Methods: Ergolide bioactivity was screened via long-term proliferation assay in UM/MUM cells and in zebrafish MUM xenograft models. Mass spectrometry profiled proteins modulated by ergolide within whole cell or extracellular vesicle (EVs) lysates of the OMM2.5 MUM cell line. Protein expression was analyzed by immunoblots and correlation analyses to UM patient survival used The Cancer Genome Atlas (TCGA) data. Results: Ergolide treatment resulted in significant, dose-dependent reductions (48.5 to 99.9%; p<0.0001) in OMM2.5 cell survival in vitro and of normalized primary zebrafish xenograft fluorescence (56%; p<0.0001) in vivo, compared to vehicle controls. Proteome-profiling of ergolide-treated OMM2.5 cells, identified 5023 proteins, with 52 and 55 proteins significantly altered at 4 and 24 hours, respectively ( p<0.05; fold-change >1.2). Immunoblotting of heme oxygenase 1 (HMOX1) and growth/differentiation factor 15 (GDF15) corroborated the proteomic data. Additional proteomics of EVs isolated from OMM2.5 cells treated with ergolide, detected 2931 proteins. There was a large overlap with EV proteins annotated within the Vesiclepedia compendium. Within the differentially expressed proteins, the proteasomal pathway was primarily altered. Interestingly, BRCA2 and CDKN1A Interacting Protein (BCCIP) and Chitinase Domain Containing 1 (CHID1), were the only proteins significantly differentially expressed by ergolide in both the OMM2.5 cellular and EV isolates and they displayed inverse differential expression in the cells versus the EVs. Conclusions: Ergolide is a novel, promising anti-proliferative agent for UM/MUM. Proteomic profiling of OMM2.5 cellular/EV lysates identified candidate pathways elucidating the action of ergolide and putative biomarkers of UM, that require further examination.
The most common form of adult eye cancer is uveal melanoma (UM). Once UM cancer cells spread to organs in the rest of the body, metastatic UM (MUM), there is a poor prognosis for patients with only one approved drug treatment. Hence, it is vital to better understand the cellular and extracellular proteins that regulate UM pathology in order to uncover biomarkers of disease and therapeutic targets. In this original study, we demonstrate a compound called ergolide is capable of severely reducing the metabolic activity and growth of UM cancer cells, grown as isolated monolayers. Ergolide was also able to reduce the growth of human MUM cells growing as tumors in transplanted zebrafish larvae. We identify that ergolide alters specific proteins found in the human UM cells. These proteins once analyzed in detail offer opportunities to understand how new treatment strategies can be developed for UM.
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BACKGROUND: The phase II neo-adjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy in combination with trastuzumab, lapatinib or both in patients with HER2+ breast cancer. We studied circulating immune cells looking for alterations in phenotype, genotype and cytotoxic capacity (direct and antibody-dependent cell-mediated cytotoxicity (ADCC)) in the context of treatment response. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from pre- (n = 41) and post- (n = 25) neo-adjuvant treatment blood samples. Direct/trastuzumab-ADCC cytotoxicity of patient-derived PBMCs against K562/SKBR3 cell lines was determined ex vivo. Pembrolizumab was interrogated in 21 pre-treatment PBMC ADCC assays. Thirty-nine pre-treatment and 21 post-treatment PBMC samples were immunophenotyped. Fc receptor genotype, tumour infiltrating lymphocyte (TIL) levels and oestrogen receptor (ER) status were quantified. RESULTS: Treatment attenuated the cytotoxicity/ADCC of PBMCs. CD3+/CD4+/CD8+ T cells increased following therapy, while CD56+ NK cells/CD14+ monocytes/CD19+ B cells decreased with significant post-treatment immune cell changes confined to patients with residual disease. Pembrolizumab-augmented ex vivo PBMC ADCC activity was associated with residual disease, but not pathological complete response. Pembrolizumab-responsive PBMCs were associated with lower baseline TIL levels and ER+ tumours. CONCLUSIONS: PBMCs display altered phenotype and function following completion of neo-adjuvant treatment. Anti-PD-1-responsive PBMCs in ex vivo ADCC assays may be a biomarker of treatment response.
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Antineoplásicos , Neoplasias , Humanos , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Leucócitos Mononucleares/metabolismo , Terapia Neoadjuvante , Neoplasias/tratamento farmacológico , Fenótipo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologiaRESUMO
Kinase Suppressor of RAS 1 (KSR1) is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS-mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E-transformed melanoma cell line. KSR1 loss produced a complex phenotype characterised by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.
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Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/genética , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas ras/metabolismoRESUMO
CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.
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Dióxido de Carbono , Anidrase Carbônica II , Dióxido de Carbono/metabolismo , Anidrase Carbônica II/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hipercapnia/enzimologia , Hipercapnia/metabolismo , IsoenzimasRESUMO
BACKGROUND: Mesenchymal stromal cells (MSC) have demonstrated ability to improve diabetic nephropathy (DN) in experimental models, as well as by improving kidney endogenous progenitor cells proliferation and differentiation. Many studies have demonstrated the effect of hypoxia on MSC improving their functionality but the potential enhancement of the nephroprotective properties of MSC cultured under low oxygen concentration has been explored in few studies, none of them in the context of DN. On the other hand, diabetes is associated with abnormalities in MSCs functionality. These findings related to the hypoxia preconditioning ability to enhance adipose-tissue derived-MSC (ASC) performance have led us to wonder if hypoxia could increase the known beneficial effect of normal ASC in DN and if it could correct the expected inability of diabetic rat-derived ASC to exert this effect in vivo. To answer these questions, in the present study we have used ASC from healthy and diabetic-induced rats, cultured under standard conditions or hypoxia preconditioned, in a DN rat model induced by streptozotocin (STZ). METHODS: Diabetes was induced in Wistar-rats by 60 mg/kg streptozotocin (STZ) intraperitoneal injection. Fifteen days thereafter, five diabetic-induced rats and five healthy, previously injected with saline, were sacrificed and used as ASC donors . Both healthy and diabetic rat-derived ASC (cASC and dASC, respectively) were cultured under standard conditions (21%O2)(N) or were subjected to a 48 h conditioning period in hypoxia (3%O2)(H). Thus, four types of cells were generated depending on their origin (healthy or diabetic-induced rats) and the culture conditions(N or H):cASC-N, cASC-H, dASC-N and dASC-H. DN experimental study were carried out fifteen days after STZ induction of diabetes in fifty-two healthy rats. DN-induced-animals were randomly assigned to be injected with 200 µL saline as placebo or with 3 × 106 cASC-N, cASC-H, dASC-N or dASC-H, according to the study group. Serum glucose, urea and creatinine, and urine albumin levels were measured at 2-weeks intervals until day+ 45 after ND-induction.Animals were sacrificed and kidneys extracted for histopathological and transmission electron microcopy analysis RESULTS: None of the four study groups that received cell treatment showed significant changes in serum glucose, urea and creatinine levels, urine albumin concentration and body weight compared to placebo ND-induced group. Interestingly, only the group that received cASC-H showed a reduction in glucose and creatinine levels although it did not reach statistical significance.All DN-induced groups treated with ASC reduced significantly renal lesions such as mesangial expansion, mesangiolysis, microaneurysms and acute tubular necrosis compared to ND-induced placebo group (p ≤ 0.05). Renal injuries such as clear tubular cell changes, thickening of tubular basement membrane, tubular cysts and interstitial fibrosis significantly showed reduction in ND-induced rats treated with cASC-H regarding to their received cASCN (p ≤ 0.05). Non statistical differences were observed in the improvement capacity of cASC and dASC culture under standard condition.However, hypoxia preconditioning reduces the presence of tubular cysts (p ≤ 0.01). CONCLUSIONS: Hypoxia preconditioning enhances the ability of healthy rat-derived ASC to improve kidney injury in a rat model of DN. Moreover, diabetic-derived ASC exhibits a similar ability to healthy ASC which is clearly more than expected, but it is not significantly modified by hypoxia preconditioning.
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Diabetes Mellitus Experimental/cirurgia , Nefropatias Diabéticas/cirurgia , Rim/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/citologia , Albuminúria/induzido quimicamente , Albuminúria/cirurgia , Albuminúria/urina , Animais , Glicemia/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Creatinina/sangue , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/patologia , Fibrose , Rim/metabolismo , Masculino , Ratos Wistar , Estreptozocina , Ureia/sangueRESUMO
Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD deficiency (Phd1-/-, Phd2+/-, and Phd3-/-) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2 haplodeficiency (Phd2+/-) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/- (but not PHI or Phd1-/-) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/- induced immunomodulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/--mediated M2 polarization of macrophages, conferring a favorable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.
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Anastomose Cirúrgica , Colo/metabolismo , Macrófagos/metabolismo , Prolil Hidroxilases/metabolismo , Abdome/cirurgia , Aminoácidos Dicarboxílicos , Anastomose Cirúrgica/efeitos adversos , Fístula Anastomótica , Animais , Células CACO-2 , Colágeno/metabolismo , Colo/patologia , Colo/cirurgia , Feminino , Humanos , Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Isquemia , Masculino , Camundongos , RNA Mensageiro/metabolismo , Sepse , CicatrizaçãoRESUMO
Articular cartilage and synovial tissue from patients with osteoarthritis (OA) show an overactivity of connexin43 (Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration and disease progression. The aim of this study was to determine the effect of oleuropein on Cx43 and cellular senescence for tissue engineering and regenerative medicine strategies for OA treatment. Oleuropein regulates Cx43 promoter activity and enhances the propensity of hMSCs to differentiate into chondrocytes and bone cells, reducing adipogenesis. This small molecule reduce Cx43 levels and decrease Twist-1 activity in osteoarthritic chondrocytes (OACs), leading to redifferentiation, restoring the synthesis of cartilage ECM components (Col2A1 and proteoglycans), and reducing the inflammatory and catabolic factors mediated by NF-kB (IL-1ß, IL-6, COX-2 and MMP-3), in addition to lowering cellular senescence in OACs, synovial and bone cells. Our in vitro results demonstrate the use of olive-derived polyphenols, such as oleuropein, as potentially effective therapeutic agents to improve chondrogenesis of hMSCs, to induce chondrocyte re-differentiation in OACs and clearing out senescent cells in joint tissues in order to prevent or stop the progression of the disease.
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Antirreumáticos/farmacologia , Cartilagem Articular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Iridoides/farmacologia , Olea , Osteoartrite/tratamento farmacológico , Polifenóis/farmacologia , Regeneração/efeitos dos fármacos , Idoso , Antirreumáticos/isolamento & purificação , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Linhagem Celular , Microambiente Celular , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno Tipo II/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Frutas , Humanos , Glucosídeos Iridoides , Iridoides/isolamento & purificação , Masculino , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Olea/química , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteogênese/efeitos dos fármacos , Polifenóis/isolamento & purificação , Transdução de Sinais , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
BACKGROUND: Fasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties. OBJECTIVES: Given the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co-infection with F. hepatica might have on the course of Johne's disease (JD). METHODS: This study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products. RESULTS: We found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre-stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake. CONCLUSIONS: Overall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co-stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.
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Antígenos de Bactérias/imunologia , Doenças dos Bovinos/imunologia , Fasciola hepatica/imunologia , Imunidade , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/imunologia , Animais , Apoptose , Bovinos , Doenças dos Bovinos/parasitologia , Proliferação de Células , Coinfecção , Citocinas/metabolismo , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Paratuberculose/parasitologia , Células Th1/imunologiaRESUMO
The prostate is the only male accessory gland in cetaceans. However, little is known about this organ in these species. Anatomical and histological characteristics of the prostate have been described in only a few cetacean species, further, one study reported a high incidence of prostatic pathologies in cetaceans that may impair reproduction. The objective of this work was to describe and compare the morphological, histological, and cytological characteristics of the prostate in different odontocete cetaceans. To this end, the prostate glands of 47 animals from nine different species of cetaceans were macroscopically and microscopically studied. Members of the families Delphinidae, Ziphiidae, and Physeteridae were included. In general, the prostate appeared as a musculo-glandular organ with two distinct parts-the Corpus prostatae and the Pars disseminata prostatae. In the pygmy sperm whale (Kogia breviceps) and the Cuvier's beaked whale (Ziphius cavirostris), the prostate was a discrete gland with a small Corpus prostatae. Microscopically, the prostates of different delphinids species shared similarities; however, the prostate of the pygmy sperm whale revealed significant histological differences compared to those of the delphinids. Immunohistochemical analysis was performed using low- and high-molecular-weight cytokeratin, vimentin, and prostatic specific antigen commercial antibodies. Electron microscopy analysis was performed on the prostate of a bottlenose dolphin and the cytomorphological differences among the major epithelial components of the prostatic epithelium were described. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:2036-2053, 2020. © 2019 American Association for Anatomy.
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Cetáceos/anatomia & histologia , Próstata/anatomia & histologia , Animais , Masculino , Próstata/citologiaRESUMO
Osteoarthritis (OA), a chronic disease characterized by articular cartilage degeneration, is a leading cause of disability and pain worldwide. In OA, chondrocytes in cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favouring disease progression. Similar to other wound-healing disorders, chondrocytes from OA patients show a chronic increase in the gap junction channel protein connexin43 (Cx43), which regulates signal transduction through the exchange of elements or recruitment/release of signalling factors. Although immature or stem-like cells are present in cartilage from OA patients, their origin and role in disease progression are unknown. In this study, we found that Cx43 acts as a positive regulator of chondrocyte-mesenchymal transition. Overactive Cx43 largely maintains the immature phenotype by increasing nuclear translocation of Twist-1 and tissue remodelling and proinflammatory agents, such as MMPs and IL-1ß, which in turn cause cellular senescence through upregulation of p53, p16INK4a and NF-κB, contributing to the senescence-associated secretory phenotype (SASP). Downregulation of either Cx43 by CRISPR/Cas9 or Cx43-mediated gap junctional intercellular communication (GJIC) by carbenoxolone treatment triggered rediferentiation of osteoarthritic chondrocytes into a more differentiated state, associated with decreased synthesis of MMPs and proinflammatory factors, and reduced senescence. We have identified causal Cx43-sensitive circuit in chondrocytes that regulates dedifferentiation, redifferentiation and senescence. We propose that chondrocytes undergo chondrocyte-mesenchymal transition where increased Cx43-mediated GJIC during OA facilitates Twist-1 nuclear translocation as a novel mechanism involved in OA progression. These findings support the use of Cx43 as an appropriate therapeutic target to halt OA progression and to promote cartilage regeneration.
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Cartilagem Articular/imunologia , Comunicação Celular/genética , Senescência Celular/genética , Condrócitos/imunologia , Conexina 43/genética , Osteoartrite/genética , Adipócitos/efeitos dos fármacos , Adipócitos/imunologia , Adipócitos/patologia , Antígenos CD/genética , Antígenos CD/imunologia , Carbenoxolona/farmacologia , Cartilagem Articular/patologia , Estudos de Casos e Controles , Comunicação Celular/imunologia , Diferenciação Celular , Senescência Celular/imunologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Conexina 43/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/imunologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/patologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Osteoartrite/imunologia , Osteoartrite/patologia , Cultura Primária de Células , Índice de Gravidade de Doença , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/imunologiaRESUMO
Mesenchymal stem cells (MSC) are osteoblasts progenitors and a variety of studies suggest that they may play an important role for the health in the field of bone regeneration. Magnesium supplementation is gaining importance as adjuvant treatment to improve osteogenesis, although the mechanisms involving this process are not well understood. The objective of this study was to investigate the effects of magnesium on MSC differentiation. Here we show that in rat bone marrow MSC, magnesium chloride increases MSC proliferation in a dose-dependent manner promoting osteogenic differentiation and mineralization. These effects are reduced by 2-APB administration, an inhibitor of magnesium channel TRPM7. Of note, magnesium supplementation did not increase the canonical Wnt/ß-catenin pathway, although it promoted the activation of Notch1 signaling, which was also decreased by addition of 2-APB. Electron microscopy showed higher proliferation, organization and maturation of osteoblasts in bone decellularized scaffolds after magnesium addition. In summary, our results demonstrate that magnesium chloride enhances MSC proliferation by Notch1 signaling activation and induces osteogenic differentiation, shedding light on the understanding of the role of magnesium during bone regeneration.
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Diferenciação Celular/efeitos dos fármacos , Cloreto de Magnésio/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Osteogênese/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Osso e Ossos/citologia , Compostos de Boro/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/metabolismo , Microscopia Eletrônica , Ratos , Canais de Cátion TRPM/antagonistas & inibidoresRESUMO
BACKGROUND: Effects of cell therapy on dilated cardiomyopathy (DCM) have been investigated in pre-clinical models using distinct cellular types in each study. A single study that compares the effectiveness of different cells is lacking. METHODS: We have compared the effects of intramyocardial injection (IMI) of bone marrow (BM)-derived mononuclear cells (MNCs), BM and adipose tissue (AT) mesenchymal stromal cells (BM-MSCs and AT-MSCs) on heart function, histological changes and myocardial ultrastructure in a rat model of DCM. Isogenic Wistar rats were used to isolate the different cell types and to induce DCM by autoimmune myocarditis. Animals were randomly assigned to receive BM-MNCs, BM-MSCs, AT-MSCs or placebo at day 42 by IMI. Serial echocardiography was used to assess cardiac function and hearts obtained after sacrifice at day 70, were used for histological and ultrastructural analysis. Serum levels of type B-natriuretic peptide (BNP) and vascular endothelial growth-factor (VEGF) were determined at different time points. RESULTS: BM-MSC treatment induced significant improvement in ejection fraction (EF), fractional shortening (FS), left ventricular systolic diameter (LVESD) and systolic volume (LVESV). In contrast, changes in echocardiographic parameters with respect to pre-treatment values in animals receiving placebo, AT-MSCs or BM-MNCs were not statistically significant. EF and FS in animals receiving AT-MSCs were superior to those receiving placebo. BM-MSC transplantation induced also improvement in cardiac fibers organization and capillary density, fibrotic tissue reduction, increase in final VEGF concentration and BNP decrease. DISCUSSION: IMI of BM or AT-MSCs improves LV function and induces more angiogenesis processes than BM-MNCs. In addition, BM-MSCs showed more anti-fibrotic effects and more ability to reorganize myocardial tissue compared with the other cell types.
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Tecido Adiposo/citologia , Cardiomiopatia Dilatada/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Modelos Animais de Doenças , Ecocardiografia , Coração/fisiologia , Injeções , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Miocárdio/ultraestrutura , Ratos WistarRESUMO
BACKGROUND: The primary aim of this study is to evaluate the effectiveness of different doses (intensity) of supervised exercise training - concomitant with lifestyle counselling - as a primary care intervention tool for the management of metabolic syndrome risk factors in low-active adults with one or more such factors (programme name in Catalan: Bellugat de CAP a peus). METHODS/DESIGN: Three-arm, randomized controlled clinical trial implemented in the primary care setting, with a duration of 40 weeks (16 weeks intervention and 24-week follow-up). Adults aged 30 to 55 years with metabolic risk factors will be randomized into three intervention groups: 1) aerobic interval training (16 supervised training lessons) plus a healthy lifestyle counselling programme (6 group and 3 individual meetings); 2) low-to-moderate intensity continuous training (16 supervised training lessons) plus the same counselling programme; or 3) the counselling- programme without any supervised physical exercise. The main output variables assessed will be risk factors for metabolic syndrome (waist circumference, blood pressure, and levels of plasma triglycerides, high-density lipoproteins and glucose), systemic inflammation, cardiorespiratory fitness, physical activity and sedentary behaviour, dietary habits, health-related quality of life, self-efficacy and empowerment. Economic factors will also be analysed in order to determine the cost-effectiveness of the programme. These variables will be assessed three times during the study: at baseline, at the end of the intervention, and at follow-up. We estimate to recruit 35 participants per group. DISCUSSION: The results of this study will provide insight into the immediate and medium-term effects on metabolic risk and lifestyle of a combined approach involving aerobic interval training and a multidisciplinary behavioural intervention. If effective, the proposed intervention would provide both researchers and practitioners in this field with a platform on which to develop similar intervention programmes for tackling the repercussions of an unhealthy lifestyle. TRIAL REGISTRATION: Clinical trials.gov. NTC02832453 . Registered 6 July 2016 (retrospectively registered).
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Terapia Comportamental , Aconselhamento , Exercício Físico , Promoção da Saúde/métodos , Estilo de Vida , Síndrome Metabólica/prevenção & controle , Adulto , Análise Custo-Benefício , Comportamento Alimentar , Comportamentos Relacionados com a Saúde , Humanos , Síndrome Metabólica/etiologia , Pessoa de Meia-Idade , Atenção Primária à Saúde , Qualidade de Vida , Projetos de Pesquisa , Fatores de RiscoRESUMO
BACKGROUND: Newborn infants are endotoxin tolerant which may be responsible for their increased susceptibility to bacterial sepsis. Vitamin D has an immunomodulatory effect and newborn infants are at risk of vitamin D deficiency. We examined the in vitro effect of 1, 25-dihydroxyvitamin D (1,25OHD) on whole blood phagocytic toll-like receptor 4 (TLR4), CD11b, and reactive oxygen intermediates (ROIs) in newborn infants during sepsis. METHODS: Whole blood from preterm infants <32-wk gestation, control term neonates, and adults were sampled for phagocytic expression of ROI, TLR4, CD11b in response to lipopolysaccharide (LPS), and 1,25OHD using flow cytometer. RESULTS: ROI production from newborn phagocytes incubated with LPS alone was decreased. Pretreatment with 1,25OHD demonstrated increased (P = 0.001) phagocytic ROI production in newborns but not in adults. 1,25OHD did not have any effect on TLR4 and CD11b in both newborns and adults. Pretreatment with ROI inhibitors (apocynin (APO) and diphenyleneiodonium), phosphoinositide 3-kinase (PI3K) inhibitor, and p38 inhibitor blocked neutrophil ROI production. CONCLUSION: Neonatal phagocytic cells had diminished ROI production in the presence of LPS, however, pretreatment with 1,25OHD reversed this hyporesponsiveness. This action by 1,25OHD was mediated by activation of nicotinamide adenine dinucleotide phosphate oxidase system through PI3K signaling enzymes.
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Ácido Ascórbico/administração & dosagem , Recém-Nascido Prematuro , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Adulto , Estudos de Casos e Controles , Cromonas/farmacologia , Humanos , Imidazóis/farmacologia , Recém-Nascido , Morfolinas/farmacologia , Neutrófilos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The food packaging industry is in continuous development in order to obtain more secure and stable food and beverages. The incorporation of inorganic and organic materials with plastic polymers leads to polymer composites. Among the inorganic compounds, clays such as montmorillonite (MTT) and its derivatives are of great interest due to their advantageous properties. The Technological Institute of Packaging, Transport,and Logistics (ITENE) developed a novel nanocomposite based on a poly(lactic) acid (PLA) polymer using an MMT derivative, named Clay1, as filler, to be used in the beverage industry. The improvement of the technological properties of this new material was demonstrated, but safety issues are also of concern. In the present study, a histopathological examination by optical and electron microscopy of organs from Wistar rats exposed for 90 d to a migration extract of PLA-Clay1 nanocomposite was carried out. Moreover, different clinical biochemistry, inflammation,and oxidative stress biomarkers were determined. Results showed no apparent evidence of damage, indicating that this nanocomposite has a good profile to be used in the food packaging industry, although further research is still needed.
Assuntos
Bentonita/toxicidade , Ácido Láctico/toxicidade , Nanocompostos/química , Nanocompostos/toxicidade , Polímeros/toxicidade , Animais , Bentonita/química , Biomarcadores , Fracionamento Químico/métodos , Armazenamento de Alimentos , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/genética , Interleucina-6/metabolismo , Ácido Láctico/química , Masculino , Estresse Oxidativo/efeitos dos fármacos , Poliésteres , Polímeros/química , Ratos , Ratos WistarRESUMO
Cylindrospermopsin (CYN) is increasingly recognized as a potential threat to drinking water safety, due to its ubiquity. This cyanotoxin has been found to cause toxic effects in mammals, and although fish could be in contact with this toxin, acute toxicity studies on fish are nonexistent. This is the first study showing that single doses of CYN pure standard (200 or 400 µg CYN/kg fish bw) by oral route (gavage) generate histopathological effects in fish (Tilapia-Oreochromis niloticus) exposed to the toxin under laboratory condition. Among the morphological changes, disorganized parenchymal architecture in the liver, dilated Bowman's space in the kidney, fibrolysis in the heart, necrotic enteritis in the intestines, and hemorrhages in the gills, were observed. Moreover, some oxidative stress biomarkers in the liver and kidney of tilapias were altered. Thus, CYN exposure induced increased protein oxidation products in both organs, NADPH oxidase activity was significantly increased with the kidney being the most affected organ, and decreased GSH contents were also detected in both organs, at the higher dose assayed.
Assuntos
Estresse Oxidativo/efeitos dos fármacos , Tilápia/anatomia & histologia , Tilápia/metabolismo , Toxinas Biológicas/toxicidade , Uracila/análogos & derivados , Alcaloides , Animais , Toxinas Bacterianas , Biomarcadores/metabolismo , Toxinas de Cianobactérias , Brânquias/efeitos dos fármacos , Brânquias/patologia , Glutationa/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Miocárdio/patologia , NADPH Oxidases/metabolismo , Oxirredução , Uracila/toxicidadeRESUMO
MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP) and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.
Assuntos
Adenosina Trifosfatases/genética , Autoantígenos/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , MicroRNAs/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Quinases Associadas a Fase S/genética , Adenosina Trifosfatases/metabolismo , Animais , Autoantígenos/metabolismo , Células CHO , Cricetulus , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Estrogen receptor 1 and 2 (ESR1 and 2) mediate estrogen (E) action on gonadotrope function. While much is known about the effects of ESR1 on the gonadotrope, there is still some controversy regarding the effects of ESR2. To investigate the role of ESR2 in the gonadotrope, 45-day-old female mice of two different genotypes were used: wild type (WT) and pituitary (gonadotropes and thyrotropes)-specific Esr1 knockout (KO). All mice were ovariectomized (OVX) and 15 days later injected over 3 days with 2.5 µg 17ß-estradiol (E(2)), 0.2 mg of the selective ESR1 or 2 agonists, propylpyrazole triol and diarylpropionitrile, respectively, or 0.1 ml oil. The day after treatment, anterior pituitary glands were dissected out for evaluation of gonadotrope ultrastructural morphology and pituitary immunohistochemical expression of progesterone receptor (Pgr (Pr)). Blood was collected and serum LH levels were assessed. Activation of ESR1 in WT mice resulted in the following: i) uterine ballooning and vaginal cornification, ii) negative feedback on LH secretion, iii) increased number of homogeneous (functional) gonadotropes, and iv) pituitary Pgr expression (35.9±2.0% of pituitary cells). Activation of ESR1 in KO mice induced normal uterine, vaginal, and LH secretion responses, but failed to increase the number of functional gonadotropes, and induced significantly lower Pgr expression (21.0±3.0% of pituitary cells) than in WT mice. Whilst activation of ESR2 had no significant effects in WT mice, it doubled the number of functional gonadotropes exhibited by KO mice injected with oil. It is concluded that E(2) exerted its action in KO mouse gonadotropes via ESR2.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Gonadotrofos/metabolismo , Receptores de Progesterona/metabolismo , Animais , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Feminino , Gonadotrofos/ultraestrutura , Imuno-Histoquímica , Hormônio Luteinizante/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Útero/fisiologia , Vagina/citologiaRESUMO
BACKGROUND: Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. METHODS: Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. RESULTS: Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumorigenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. CONCLUSIONS: Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.
Assuntos
Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/patologia , Actinas/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/ultraestrutura , Técnicas de Cultura de Células , Proliferação de Células , Forma Celular , Senescência Celular , Feminino , Humanos , Queratinas Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/ultraestrutura , Células Tumorais Cultivadas , Vimentina/metabolismoRESUMO
To study numerical changes in intestinal macrophages and variations in cytokine production by immune cells in the intestine, conventional C57BL/6J mice were orally infected with the Rocky Mountain Laboratory strain of scrapie. Animals were sacrificed at different timepoints, and samples were taken and processed by routine methods for morphological and immunohistochemical analysis. The results point to a possible role for macrophages in the uptake and transport of the infective agent to Peyer's patches. The observed increase in macrophage numbers in subepithelial sites, taken in conjunction with a drop in tumour necrosis factor-alpha production at these sites, suggests a possible secretory inhibition that could be induced by the disease-associated prion protein (PrPd). On the other hand, cytokine dynamics indicated the presence of an impaired Th1-Th2 cell mediated response, which could facilitate the spread of PrPd to the central nervous system. Further research is required to confirm these hypotheses.