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Circulating cell-free DNA (ccfDNA) of mitochondrial origin (ccf-mtDNA) consists of a minor fraction of total ccfDNA in blood or in other biological fluids. Aberrant levels of ccf-mtDNA have been observed in many pathologies. Here, we introduce a simple and effective standardized Taqman probe-based dual-qPCR assay for the simultaneous detection and relative quantification of nuclear and mitochondrial fragments of ccfDNA. Three pathologies of major burden, one malignancy (Breast Cancer, BrCa), one inflammatory (Osteoarthritis, OA) and one metabolic (Type 2 Diabetes, T2D), were studied. Higher levels of ccf-mtDNA were detected both in BrCa and T2D in relation to health, but not in OA. In BrCa, hormonal receptor status was associated with ccf-mtDNA levels. Machine learning analysis of ccf-mtDNA datasets was used to build biosignatures of clinical relevance. (A) a three-feature biosignature discriminating between health and BrCa (AUC: 0.887) and a five-feature biosignature for predicting the overall survival of BrCa patients (Concordance Index: 0.756). (B) a five-feature biosignature stratifying among T2D, prediabetes and health (AUC: 0.772); a five-feature biosignature discriminating between T2D and health (AUC: 0.797); and a four-feature biosignature identifying prediabetes from health (AUC: 0.795). (C) a biosignature including total plasma ccfDNA with very high performance in discriminating OA from health (AUC: 0.934). Aberrant ccf-mtDNA levels could have diagnostic/prognostic potential in BrCa and Diabetes, while the developed multiparameter biosignatures can add value to their clinical management.
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Neoplasias da Mama , Ácidos Nucleicos Livres , DNA Mitocondrial , Diabetes Mellitus Tipo 2 , Humanos , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , Feminino , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Pessoa de Meia-Idade , Masculino , Idoso , Aprendizado de MáquinaRESUMO
PURPOSE: Miscarriage is one of the most common complications of pregnancy. Although chromosomal abnormalities of the embryo is a well-known cause of miscarriage, a lot of cases remain unexplained, with immunologic and vascular growth alterations being considered as probable causes. Chemokines are produced by a variety of cells and exhibit several functions including both pro and anti-angiogenic properties. In this study, we investigated the role of the angiogenic and angiostatic chemokines in placenta and decidua tissues from spontaneous and induced abortions. METHODS: Total RNA was extracted from the placenta and decidua tissues, which was then purified and converted into cDNA. Real-time PCR was then performed for the expression of the angiogenic CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 and CXCL4, and the angiostatic CXCL9, CXCL10, CXCL11, CXCL12 and CXCL14 and results were then statistically analyzed. RESULTS: Regarding the placenta, CXCL7 (2.29-fold, 2.16-2.38, p < 0.05), CXCL4 (1.01-fold, 0.74-4.447, p < 0.05), CXCL9 (0.87-fold, 0.43-1.34, p < 0.05) and CXCL11 (0.31-fold, 0.22-0.45, p < 0.05) were altered in spontaneous abortions. CCL2, CCL5, CXCL2-3, CXCL8, CXCL10, CXCL12 and CXCL14 were not statistically significant altered. Regarding the decidua, CXCL7 (7.13-fold, 6.32-7.54, p < 0.01), CXCL8 (11.02-fold, 8.58-13.45, p < 0.05), CCL20 (1.21-fold, 0.29-1.89, p < 0.05) and CXCL9 (5.49-fold, 3.67-6.39, p < 0.05) were overexpressed in spontaneous abortions. CXCL2-4, CCL2, CCL5, CXCL10-12 and CXCL14 did not show any differences. The expression of the chemokines CXCL1, CXCL5-6 was absent in either tissue or group. CONCLUSION: Our results show that the overexpression of angiostatic and diminished expression of angiogenic chemokines takes place in the placenta and decidua of spontaneous abortions, suggesting that dysregulation of angiogenesis could be a contributive factor to the pathogenesis of miscarriage.
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Aborto Espontâneo , Gravidez , Feminino , Humanos , Placenta/metabolismo , Decídua/metabolismoRESUMO
BACKGROUND: The aim of this study was to investigate bacterial translocation and its possible role in the development of post-resuscitation inflammatory response following Cardio-Pulmonary Resuscitation (CPR) after cardiac arrest. METHODS: Munich female swine were employed for a model of cardiac arrest via application of electrical current. After 7 min, CPR was initiated, and animals were either successfully return to spontaneous circulation (ROSC) within 40 min or not (no-ROSC). At the end of experimental period and prior to sacrifice, samples from the intestine, mesenteric lymph nodes (MLN), liver and portal vein blood were obtained. Evaluation of inflammation and gut permeability was performed; MLN, liver and portal vein samples were analyzed for 16 s rRNA detection and cytokine mRNA expression. RESULTS: A decreased expression of the tight junction protein Occludin, with higher levels of inflammation, greater epithelial disintegration, ulceration, loss of crypts and villi height were found in the intestines of the ROSC swine in comparison to no-ROSC. The macrophage surface antigen CD-14 staining was relatively more intense in the ROSC than in no-ROSC. Higher levels of TNF-α mRNA expression were present in the liver of the ROSC group. Finally, despite the inflammatory response and the gut mucosal alterations in ROSC group, no bacterial translocation was detected in liver, MLN and portal vein. CONCLUSIONS: We show that resuscitation from cardiac arrest induces inflammatory response and intestinal permeability in swine 4h after resuscitation, but not a bacterial translocation. Bacterial translocation is not an early phase phenomenon but probably part of the pathophysiologic sequelae.
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Reanimação Cardiopulmonar , Parada Cardíaca , Síndrome Pós-Parada Cardíaca , Animais , Translocação Bacteriana , Feminino , Parada Cardíaca/complicações , Parada Cardíaca/terapia , Inflamação , RNA Mensageiro , SuínosRESUMO
Idiopathic pulmonary fibrosis (IPF) is caused by progressive lung tissue impairment due to extended chronic fibrosis, and it has no known effective treatment. The use of conditioned media (CM) from an immortalized human adipose mesenchymal stem cell line could be a promising therapeutic strategy, as it can reduce both fibrotic and inflammatory responses. We aimed to investigate the anti-inflammatory and anti-fibrotic effect of CM on human pulmonary subepithelial myofibroblasts (hPSM) and on A549 pulmonary epithelial cells, treated with pro-inflammatory or pro-fibrotic mediators. CM inhibited the proinflammatory cytokine-induced mRNA and protein production of various chemokines in both hPSMs and A549 cells. It also downregulated the mRNA expression of IL-1α, but upregulated IL-1ß and IL-6 mRNA production in both cell types. CM downregulated the pro-fibrotic-induced mRNA expression of collagen Type III and the migration rate of hPSMs, but upregulated fibronectin mRNA production and the total protein collagen secretion. CM's direct effect on the chemotaxis and cell recruitment of immune-associated cells, and its indirect effect on fibrosis through the significant decrease in the migration capacity of hPSMs, makes it a plausible candidate for further development towards a therapeutic treatment for IPF.
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Fibrose Pulmonar Idiopática , Células-Tronco Mesenquimais , Anti-Inflamatórios/farmacologia , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/metabolismo , Fibrose , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Células-Tronco Mesenquimais/metabolismo , Miofibroblastos/metabolismo , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: To measure plasma glutamine (GLN) levels in systemic and portal circulation after combined enteral and parenteral administration in early endotoxemic swine. We hypothesized that this combination will be more efficient than intravenous administration alone in restoring plasma levels during the course of endotoxemia. MATERIALS AND METHODS: Endotoxemia was induced with Escherichia coli O111:B4 lipopolysaccharide (LPS) (250 µg/kg body weight) in 16 anes-thetized, fasted swine and maintained by constant infusion (2 µg/kg/h) over 180 min. Another 16 swine served as controls. After infusion with LPS or placebo, GLN was administered intravenously, enterally or in combination (0.5 g/kg i.v. plus 0.5 g/kg enterally) over 30 min. At 0, 15, 30, 45, 60, 120 and 180 min, blood was drawn from the systemic and portal circulation for colorimetric assessment of GLN. RESULTS: In healthy, placebo-alone swine, GLN levels remained stable throughout the study. Intravenous and combined infusion increased systemic levels (p = 0.001), but after enteral administration alone, a smaller effect was observed (p = 0.026). Portal levels were increased after combined, enteral and intravenous administration (p = 0.001). In endotoxemia, systemic and portal levels decreased significantly. Intravenous and, to a greater extent, combined administration increased systemic levels (p = 0.001), while enteral administration only had a small effect (p = 0.001). In the portal vein, intravenous and combined treatment increased plasma levels (p = 0.001), whereas enteral supplementation alone had again a small, yet significant effect (p = 0.001). CONCLUSIONS: The findings indicate that combined GLN supplementation is superior to intravenous treatment alone, in terms of enhanced availability in systemic and portal circulations. Thus, combined treatment at the onset of endotoxemia is a beneficial practice, ensuring adequate GLN to compensate for the resulting intracellular shortage.
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Vias de Administração de Medicamentos , Endotoxemia/tratamento farmacológico , Infecções por Escherichia coli/tratamento farmacológico , Glutamina/administração & dosagem , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologia , Administração Intravenosa , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Endotoxemia/sangue , Escherichia coli , Infecções por Escherichia coli/sangue , Feminino , Glutamina/análise , Grécia , Sistema Porta/efeitos dos fármacos , Suínos , Doenças dos Suínos/sangueRESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by infiltration of inflammatory cells, excessive collagen production and accumulation of myofibroblasts. We explored the possible role of subepithelial lung myofibroblasts (SELMs) in the development of fibrosis in IPF. SELMs, isolated from surgical specimens of healthy lung tissue, were cultured with pro-inflammatory factors or bronchoalveolar lavage fluid (BALF) from patients with IPF or idiopathic non-specific interstitial pneumonia (iNSIP) and their fibrotic activity was assessed. Stimulation of SELMs with pro-inflammatory factors induced a significant increase of Tissue Factor (TF) and Tumor necrosis factor-Like cytokine 1 A (TL1A) expression and collagen production in culture supernatants. Stimulation with BALF from IPF patients with mild to moderate, but not severe disease, and from iNSIP patients induced a significant increase of TF expression. BALF from all IPF patients induced a significant increase of TL1A expression and collagen production, while BALF from iNSIP patients induced a significant increase of TL1A, but not of collagen production. Interestingly, TGF-ß1 and BALF from all IPF, but not iNSIP patients, induced a significant increase in SELMs migration. In conclusion, BALF from IPF patients induces fibrotic activity in lung myofibroblasts, similar to mediators associated with lung fibrosis, indicating a key role of SELMs in IPF.
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Líquido da Lavagem Broncoalveolar , Pneumonias Intersticiais Idiopáticas/fisiopatologia , Fibrose Pulmonar Idiopática/fisiopatologia , Miofibroblastos/metabolismo , Colágeno/metabolismo , Humanos , Índice de Gravidade de Doença , Tromboplastina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismoRESUMO
BACKGROUND: Peritoneal adhesions, organized as fibrous bands after abdominal surgery, are related with considerable morbidity and repeated hospitalization. Phospholipids, natural constituents of the peritoneal fluid, seem to display excellent antiadhesive properties. The aim of this study was to investigate whether intraperitoneal application of phospholipids is capable of reducing postoperative adhesions and the possible underlying mechanisms. MATERIALS AND METHODS: Twenty male Wistar rats were subjected to a midline laparotomy and a standard peritoneal and cecum abrasion trauma. Before laparotomy closure, a bolus of 3 mL of phospholipids (12 mg/mL) or NaCl (placebo) was given intraperitoneally. Seven days later, the quality and the quantity of adhesions, as well as serum proinflammatory and/or profibrotic mediators, were blindly assessed. Human colonic subepithelial myofibroblasts were isolated from normal controls and cultured with transforming growth factor-ß1 (TGFß1, 5 ng/mL) in the presence of phospholipids (30-300 µg/mL). Collagen production in culture supernatants and migratory activity of myofibroblasts were also assessed. RESULTS: Phospholipids reduced intra-abdominal adhesions (P < 0.001), with respect to their intensity and area, and serum levels of cytokines (interleukin 1ß, interleukin 6, platelet-derived growth factor-1, and TGFß1) compared with placebo-treated rats. Stimulation of myofibroblasts with TGFß1 significantly increased (P < 0.001) the basic collagen production. The presence of phospholipids significantly reduced (P < 0.001) both the TGFß1 induced and the basic collagen production. Using a wound healing assay, phospholipids were found to reduce the basic and the TGFß1-induced migration of myofibroblasts in a concentration-dependent manner. CONCLUSIONS: Intraperitoneal phospholipids might be involved in the prevention of postoperative adhesions formation via the reduction of proinflammatory and/or profibrotic mediators and by inhibiting fibrogenic properties of mesenchymal cells.
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Miofibroblastos/efeitos dos fármacos , Fosfolipídeos/uso terapêutico , Complicações Pós-Operatórias/prevenção & controle , Aderências Teciduais/prevenção & controle , Animais , Biomarcadores/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Humanos , Injeções Intraperitoneais , Laparotomia , Masculino , Miofibroblastos/metabolismo , Peritônio/cirurgia , Fosfolipídeos/farmacologia , Complicações Pós-Operatórias/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Aderências Teciduais/etiologia , Aderências Teciduais/metabolismoRESUMO
INTRODUCTION: Th17 cells play a crucial role in neutrophilic inflammation and tissue injury in non-cystic fibrosis (non-CF) bronchiectasis. Clarithromycin demonstrates anti-inflammatory and immunomodulatory properties but the effect of long-term clarithromycin prophylaxis on the Th17 response in non-CF bronchiectasis is still unexplored. METHODS: Th17 response was studied in 22 patients with stable non-CF bronchiectasis receiving daily 500-mg clarithromycin for 12 weeks. We analysed IL-17 concentrations in exhaled breath condensate (EBC) and peripheral blood Th17 cells, whereas functional parameters and clinical data were recorded in parallel. RESULTS: Both, post-treatment absolute counts of CD4+IL17+ cells in peripheral blood and IL-17 levels in EBC decreased significantly (post-treatment CD4+IL17+ mean 2.418 ± 0.414 cells/µl versus pre-treatment 3.202 ± 0.507 cells/µl, p = 0.036 and post-treatment IL-17 mean levels 7.16 ± 0.47 pg/ml versus pre-treatment 9.32 ± 0.47 pg/ml, p < 0.001, respectively). Post-treatment EBC IL-17 levels decreased significantly in both patients who exhibited exacerbations and those who remained stable during the study period (mean 6.72 ± 0.37 versus 9.12 ± 0.64 pg/ml, p = 0.01 and 7.69 ± 0.9 versus 9.53 ± 0.72 pg/ml, p = 0.042, respectively), while pre-treatment and post-treatment levels did not differ between the two groups (p = 0.665 and p = 0.465, respectively). PaO(2) improved significantly (post-treatment mean 77.73 ± 2.23 mmHg versus pre-treatment 73.18 ± 2.22 mmHg, p = 0.025), while PaCO(2), post-bronchodilation FEV1, and post-bronchodilation FVC remained unaltered. CONCLUSIONS: Our results argue for a reduction of both systemic and local Th17 response after prophylactic, low-dose clarithromycin administration in patients with non-CF bronchiectasis, suggestive of a potential anti-inflammatory and/or immunomodulatory action.
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Bronquiectasia/tratamento farmacológico , Bronquiectasia/imunologia , Claritromicina/administração & dosagem , Células Th17/efeitos dos fármacos , Adulto , Idoso , Gasometria , Testes Respiratórios , Bronquiectasia/patologia , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Coortes , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , Humanos , Interleucina-17/metabolismo , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória , Medição de Risco , Estudos de Amostragem , Índice de Gravidade de Doença , Células Th17/imunologia , Resultado do TratamentoRESUMO
Metabolites produced by dysbiotic intestinal microbiota can influence disease pathophysiology by participating in ligand-receptor interactions. Our aim was to investigate the differential expression of metabolite receptor (MR) genes between inflammatory bowel disease (IBD), healthy individuals (HIs), and disease controls in order to identify possible interactions with inflammatory and fibrotic pathways in the intestine. RNA-sequencing datasets containing 643 Crohn's disease (CD) patients, 467 ulcerative colitis (UC) patients and 295 HIs, and 4 Campylobacter jejuni-infected individuals were retrieved from the Sequence Read Archive, and differential expression was performed using the RaNA-seq online platform. The identified differentially expressed MR genes were used for correlation analysis with up- and downregulated genes in IBD, as well as functional enrichment analysis using a R based pipeline. Overall, 15 MR genes exhibited dysregulated expression in IBD. In inflamed CD, the hydroxycarboxylic acid receptors 2 and 3 (HCAR2, HCAR3) were upregulated and were associated with the recruitment of innate immune cells, while, in the non-inflamed CD ileum, the cannabinoid receptor 1 (CNR1) and the sphingosine-1-phospate receptor 4 (S1PR4) were downregulated and were involved in the regulation of B-cell activation. In inflamed UC, the upregulated receptors HCAR2 and HCAR3 were more closely associated with the process of TH-17 cell differentiation, while the pregnane X receptor (NR1I2) and the transient receptor potential vanilloid 1 (TRPV1) were downregulated and were involved in epithelial barrier maintenance. Our results elucidate the landscape of metabolite receptor expression in IBD, highlighting associations with disease-related functions that could guide the development of new targeted therapies.
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BACKGROUND: Oncostatin-M (OSM) is associated with antitumor necrosis factor (anti-TNF)-α resistance in inflammatory bowel disease (IBD) and fibrosis in inflammatory diseases. We studied the expression of OSM and its receptors (OSMR, gp130) on intestinal subepithelial myofibroblasts (SEMFs) and the effect of OSM stimulation on SEMFs. METHODS: The mRNA and protein expression of OSM, OSMR, gp130, and several fibrotic and chemotactic factors were studied in mucosal biopsies and isolated human intestinal SEMFs of patients with IBD and healthy controls (HCs) and in a model of human intestinal organoids (HIOs). Subepithelial myofibroblasts and HIOs were stimulated with OSM and interleukin (IL)-1α/TNF-α. RNAseq data of mucosal biopsies were also analyzed. RESULTS: Oncostatin-M receptors and gp130 were overexpressed in mucosal biopsies of patients with IBD (Pâ <â .05), especially in inflamed segments (Pâ <â .05). The expression of OSM, OSMR, and gp130 in SEMFs from HCs was increased after stimulation with IL-1α/TNF-α (Pâ <â .001; Pâ <â .01; Pâ <â .01). The expression of CCL2, CXCL9, CXCL10, and CXCL11 was increased in SEMFs from patients with IBD and HCs after stimulation with OSM in a dose-dependent manner (Pâ <â .001; Pâ <â .05; Pâ <â .001; Pâ <â .001) and was further increased after prestimulation with IL-1α/TNF-α (Pâ <â .01 vs OSM-alone). Similar results were yielded after stimulation of HIOs (Pâ <â .01). Oncostatin-M did not induce the expression of collagen I, III, and fibronectin. Oncostatin-M receptor expression was positively correlated with CCL2, CXCL9, CXCL10, and CXCL11 expression in mucosal biopsies (Pâ <â .001; Pâ <â .001; Pâ =â .045; Pâ =â .033). CONCLUSIONS: Human SEMFs overexpress OSMR in an inflammatory microenvironment. Oncostatin-M may promote inflammation in IBD via its stimulatory effects on SEMFs, which primarily involve chemoattraction of immune cells to the intestinal mucosa.
Oncostatin-M/OSMR show elevated expression on intestinal fibroblasts that is regulated by IBD-relevant pro-inflammatory stimuli. In turn, OSM induces a pro-inflammatory phenotype on primary intestinal fibroblasts, with prominent overexpression of chemotactic factors, without demonstrating a substantial profibrotic effect.
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Pluripotent stem cells are key players in regenerative medicine. Embryonic pluripotent stem cells, despite their significant advantages, are associated with limitations such as their inadequate availability and the ethical dilemmas in their isolation and clinical use. The discovery of very small embryonic-like (VSEL) stem cells addressed the aforementioned limitations, but their isolation technique remains a challenge due to their small cell size and their efficiency in isolation. Here, we report a simplified and effective approach for the isolation of small pluripotent stem cells derived from human peripheral blood. Our approach results in a high yield of small blood stem cell (SBSC) population, which expresses pluripotent embryonic markers (e.g., Nanog, SSEA-3) and the Yamanaka factors. Further, a fraction of SBSCs also co-express hematopoietic markers (e.g., CD45 and CD90) and/or mesenchymal markers (e.g., CD29, CD105 and PTH1R), suggesting a mixed stem cell population. Finally, quantitative proteomic profiling reveals that SBSCs contain various stem cell markers (CD9, ITGA6, MAPK1, MTHFD1, STAT3, HSPB1, HSPA4), and Transcription reg complex factors (e.g., STAT5B, PDLIM1, ANXA2, ATF6, CAMK1). In conclusion, we present a novel, simplified and effective isolating process that yields an abundant population of small-sized cells with characteristics of pluripotency from human peripheral blood.
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Therapeutic drug monitoring (TDM) is the clinical practice of measuring drug concentrations. TDM can be used to determine treatment efficacy and to prevent the occurrence or reduce the risk of drug-induced side effects, being, thus, a tool of personalized medicine. Drugs for which TDM is applied should have a narrow therapeutic range and exhibit both significant pharmacokinetic variability and a predefined target concentration range. The aim of our study was to assess the current status of TDM in Greek public hospitals and estimate its progress over the last 20 years. All Greek public hospitals were contacted to provide data and details on the clinical uptake of TDM in Greece for the years 2003 and 2021 through a structured questionnaire. Data from 113 out of 132 Greek hospitals were collected in 2003, whereas for 2021, we have collected data from 98 out of 122 hospitals. Among these, in 2003 and 2021, 64 and 51 hospitals, respectively, performed TDM. Antiepileptics and antibiotics were the most common drug categories monitored in both years. The total number of drug measurement assays decreased from 2003 to 2021 (153,313 ± 7794 vs. 90,065 ± 5698; p = 0.043). In direct comparisons between hospitals where TDM was performed both in 2003 and 2021 (n = 35), the mean number of measurements was found to decrease for most drugs, including carbamazepine (198.8 ± 46.6 vs. 46.6 ± 10.1, p < 0.001), phenytoin (253.6 ± 59 vs. 120 ± 34.3; p = 0.001), amikacin (147.3 ± 65.2 vs. 91.1 ± 71.4; p = 0.033), digoxin (783.2 ± 226.70 vs. 165.9 ± 28.9; p < 0.001), and theophylline (71.5 ± 28.7 vs. 11.9 ± 6.4; p = 0.004). Only for vancomycin, a significant increase in measurements was recorded (206.1 ± 96.1 vs. 789.1 ± 282.8; p = 0.012). In conclusion, our findings show that TDM clinical implementation is losing ground in Greek hospitals. Efforts and initiatives to reverse this trend are urgently needed.
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Niclosamide is a commonly used helminthicidic drug for the treatment of human parasitosis by helminths. Recently, efforts have been focusing on repurposing this drug for the treatment of other diseases, such as idiopathic pulmonary fibrosis. Subepithelial lung myofibroblasts (SELMs) isolated from tissue biopsies of patients undergoing surgery for lung cancer were stimulated with TNF-α (50 ng/mL), IL-1α (5 ng/mL), added alone or in combination, and TGF-ß1 (5 ng/mL). After treatment with niclosamide at 30 nM and 100 nM concentrations, expression of collagen type I, collagen type III, and fibronectin was studied by total RNA isolation and qRT-PCR and protein collagen secretion with the use of Sircol collagen assay. The migration of SELMs was assessed by a wound-healing assay. Niclosamide had no effect on baseline SELM fibrotic factor expression. When stimulated with TGF-ß1, IL-1α, and/or TNF-α, SELM expression of collagen type I, type III, and fibronectin were upregulated, as was the secretion of total collagen in the culture medium. Treatment with niclosamide attenuated the effects of cytokine stimulation leading to a notable decrease in the mRNA expression of collagen type I, type III, and fibronectin in a concentration-dependent manner. SELM collagen secretion was also reduced by niclosamide at 100 nM concentration when examined at the protein level. Migration of both TGF-ß1 stimulated and unstimulated SELMs was also inhibited by niclosamide. In this study, we highlight the anti-fibrotic properties of niclosamide on SELMs under stimulation with pro-fibrotic and pro-inflammatory cytokines, thus proposing this compound as a possible new therapeutic agent against lung fibrosis.
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The probiotics Lactiplantibacillus plantarum UBLP-40, Lactobacillus rhamnosus UBLR-58 and Bifidobacterium longum UBBL-64 seem to promote wound healing when applied topically. Our aim was to investigate their effect on the mRNA expression of pro-inflammatory, healing and angiogenetic factors during the healing process of a standardized excisional wound model in rats. Rats subjected to six dorsal skin wounds were allocated to Control; L. plantarum; combined formula of L. rhamnosus plus B. longum; L. rhamnosus; and B. longum treatments, applied every two days, along with tissue collection. The pro-inflammatory, wound-healing, and angiogenetic factors of mRNA expression were assessed by qRT-PCR. We found that L. plantarum exerts a strong anti-inflammatory effect in relation to L. rhamnosus-B. longum, given alone or in combination; the combined regime of L. rhamnosus-B. longum, works better, greatly promoting the expression of healing and angiogenic factors than L. plantarum. When separately tested, L. rhamnosus was found to work better than B. longum in promoting the expression of healing factors, while B. longum seems stronger than L. rhamnosus in the expression of angiogenic factors. We, therefore, suggest that an ideal probiotic treatment should definitively contain more than one probiotic strain to speed up all three healing phases.
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Bifidobacterium longum , Lacticaseibacillus rhamnosus , Probióticos , Ratos , Animais , Cicatrização , RNA MensageiroRESUMO
OBJECTIVE: The aim of this study was to examine the role of growth factors associated with angiogenesis and oxidative stress in the pathogenesis of spontaneous miscarriage. METHODS: We performed a comparative mRNA expression analysis of VEGF, PlGF, Flt-1, Angiogenin and Endoglin using Real-Time PCR, in the placenta and decidua collected from 12 patients presenting with spontaneous abortion and from 14 women undergoing induced abortion, during the first and second trimester of pregnancy. RESULTS: The mRNA expression of Flt-1 was significantly upregulated in the placenta of spontaneous abortions (5.17-fold, IQR: 2.72-9.11, p < 0.01). The placental expression of the soluble isoforms of Flt-1, sFlt-1 e15a and sFlt-1 i13, was also significantly upregulated in spontaneous abortions (sFlt-1 e15a: 2.35-fold, IQR: 0.98-2.83, p < 0.01; sFlt-1 i13: 3.47-fold, IQR: 2.37-5.08, p < 0,05). Placental tmFlt-1, PlGF and Endoglin showed a tendency of higher expression levels in spontaneous abortions, although they did not reach statistical significance (tmFlt-1: 7.42-fold, IQR: 3.58-14.32; PlGF: 2.36-fold, IQR: 0.90-4.12; Endoglin: 1.97-fold, IQR: 1.18-2.43). VEGF and Angiogenin mRNA expression in induced, as well as in spontaneous abortions, did not convey any statistically significant difference. In the decidua, the expression levels of Flt-1 and its splice variants sFlt-1 e15a, sFlt-1 i13 and tmFlt-1 did not show any statistically significant differences, as was the case for the rest of the herein examined growth factors. CONCLUSIONS: In this study, we observed higher levels of sFlt-1 mRNA expression in the placenta of spontaneous abortions, while expression of other growth factors in placenta and decidua remained constant. This suggests that an imbalance of sFlt-1 expression in the placenta might contribute to the pathogenesis of spontaneous abortion, probably via oxidative stress, providing a possible biomarker for prompt identification of this condition.
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Aborto Espontâneo , Pré-Eclâmpsia , Feminino , Humanos , Gravidez , Aborto Espontâneo/genética , Aborto Espontâneo/metabolismo , Endoglina/genética , Endoglina/metabolismo , Placenta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator de Crescimento Placentário/metabolismo , Estresse Oxidativo , RNA Mensageiro/metabolismo , Pré-Eclâmpsia/metabolismoRESUMO
Introduction: Extracellular matrix turnover, a ubiquitous dynamic biological process, can be diverted to fibrosis. The latter can affect the intestine as a serious complication of Inflammatory Bowel Diseases (IBD) and is resistant to current pharmacological interventions. It embosses the need for out-of-the-box approaches to identify and target molecular mechanisms of fibrosis. Methods and results: In this study, a novel mRNA sequencing dataset of 22 pairs of intestinal biopsies from the terminal ileum (TI) and the sigmoid of 7 patients with Crohn's disease, 6 with ulcerative colitis and 9 control individuals (CI) served as a validation cohort of a core fibrotic transcriptomic signature (FIBSig), This signature, which was identified in publicly available data (839 samples from patients and healthy individuals) of 5 fibrotic disorders affecting different organs (GI tract, lung, skin, liver, kidney), encompasses 241 genes and the functional pathways which derive from their interactome. These genes were used in further bioinformatics co-expression analyses to elucidate the site-specific molecular background of intestinal fibrosis highlighting their involvement, particularly in the terminal ileum. We also confirmed different transcriptomic profiles of the sigmoid and terminal ileum in our validation cohort. Combining the results of these analyses we highlight 21 core hub genes within a larger single co-expression module, highly enriched in the terminal ileum of CD patients. Further pathway analysis revealed known and novel inflammation-regulated, fibrogenic pathways operating in the TI, such as IL-13 signaling and pyroptosis, respectively. Discussion: These findings provide a rationale for the increased incidence of fibrosis at the terminal ileum of CD patients and highlight operating pathways in intestinal fibrosis for future evaluation with mechanistic and translational studies.
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Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Doença de Crohn/metabolismo , Colite Ulcerativa/patologia , Colo Sigmoide/patologia , FibroseRESUMO
Bifidobacterium lactis, Lactobacillus acidophilus, Lactiplantibacillus plantarum and Saccharomyces boulardii are common probiotic supplements. Colonic subepithelial myofibroblasts (cSEMFs) are actively involved in mucosal wound healing and inflammation. cSEMFs, isolated from healthy individuals, were stimulated with 102 or 104 cfu/mL of these probiotic strains alone and in combination, and their effect on chemokine and wound healing factor expression was assessed by qRT-PCR, ELISA and Sircol Assay, and on cSEMFs migration, by Wound Healing Assay. These strains remained viable and altered cSEMFs' inflammatory and wound healing behavior, depending on the strain and concentration. cSEMFs treated with a combination of the four probiotics had a moderate, but statistically significant, increase in the mRNA and/or protein expression of chemokines CXCL1, CXCL2, CXCL4, CXCL8, CXCL10, CCL2 and CCL5, and healing factors, collagen type I and III, fibronectin and tissue factor. In contrast, when each strain was administered alone, different effects were observed, with greater increase or decrease in chemokine and healing factor expression, which was balanced by the mixture. Overall, this study highlights that the use of multiple probiotic strains can potentially alert the gut mucosal immune system and promote wound healing, having a better effect on mucosal immunity than the use of single probiotics.
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BACKGROUND: Self-transfusion has been proven as an effective management of blood loss after total knee arthroplasty (TKA). Considering that the high local concentration of antibiotic from bone cement is delivered intravenously through the self-transfusion process, systematic toxicity has never been evaluated. In addition, the effectiveness of self-transfusion with the routine concomitant use of other modern blood-salvage strategies, like tranexamic acid, should also be assessed. Therefore, we performed a randomised study to assess: 1) the safety of self-transfusion in TKA by comparing the gentamicin concentrations resulting from the use or not of autologous blood transfusion; 2) the efficacy of self-transfusion in TKA, with the concomitant administration of tranexamic acid. HYPOTHESIS: Self-transfusion in TKA elevates the serum gentamicin concentration and the potential risk of nephrotoxicity. METHODS: The serum concentration of aminoglycosides was measured in two groups of 20 patients each, after TKA, according to the use of self-transfusion. Hemoglobin, renal function and calculated blood loss were compared at several points in time between groups. RESULTS: The only time where there was a statistically significant difference in serum gentamicin, was at 48h postoperatively between groups [0.3 ug/mL±0.21, range: 0.15 to 0.72 vs. 0.14ug/mL±0.1, range: 0 to 0.35 (p=0.02)]. There were no significant differences in total blood loss [1341mL±501, range: 830 to 2230 vs. 1263mL±459 range: 840 to 2480 (p=0.67)] and need of allogeneic blood transfusion [3 units vs. 2 units] between groups. CONCLUSION: The use of autologous blood transfusion was found to be safe, in terms of nephrotoxicity of aminoglycosides after TKA, but it seemed to be ineffective as a blood salvage strategy, when used concomitantly with the administration of tranexamic acid. LEVEL OF EVIDENCE: II; low-powered randomised study. CLINICALTRIALS. GOV REGISTRATION NUMBER: NCT04505748.
Assuntos
Antifibrinolíticos , Artroplastia do Joelho , Ácido Tranexâmico , Antifibrinolíticos/uso terapêutico , Artroplastia do Joelho/efeitos adversos , Perda Sanguínea Cirúrgica , Transfusão de Sangue , Transfusão de Sangue Autóloga , Gentamicinas , Humanos , Hemorragia Pós-Operatória , Ácido Tranexâmico/uso terapêuticoRESUMO
Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells' fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.
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The endocannabinoid system has been shown to be a putative therapeutic target for retinal disease. Here, we aimed to investigate the ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) and novel inhibitors of its metabolic enzymes, α/ß-hydrolase domain-containing 6 (ABHD6) and monoacylglycerol lipase (MAGL), a) to protect the retina against excitotoxicity and b) the mechanisms involved in the neuroprotection. Sprague-Dawley rats, wild type and Akt2-/- C57BL/6 mice were intravitreally administered with phosphate-buffered saline or (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA). 2-AG was intravitreally co-administered with AMPA in the absence and presence of AM251 or AM630 (cannabinoid 1 and 2 receptor antagonists, respectively) or Wortmannin [Phosphoinositide 3-Kinase (PI3K)/Akt inhibitor]. Inhibitors of ABHD6 and dual ABHD6/MAGL (AM12100 and AM11920, respectively) were co-administered with AMPA intravitreally in rats. Immunohistochemistry was performed using antibodies raised against retinal neuronal markers (bNOS), microglia (Iba1) and macroglia (GFAP). TUNEL assay and real-time PCR were also employed. The CB2 receptor was expressed in rat retina (approx. 62% of CB1 expression). 2-AG attenuated the AMPA-induced increase in TUNEL+ cells. 2-AG activation of both CB1 and CB2 receptors and the PI3K/Akt downstream signaling pathway, as substantiated by the use of Akt2-/- mice, afforded neuroprotection against AMPA excitotoxicity. AM12100 and AM11920 attenuated the AMPA-induced glia activation and produced a dose-dependent partial neuroprotection, with the dual inhibitor AM11920 being more efficacious. These results show that 2-AG has the pharmacological profile of a putative therapeutic for retinal diseases characterized by neurodegeneration and neuroinflammation, when administered exogenously or by the inhibition of its metabolic enzymes.