Browsing by Author "Bugarski, Diana (35616659100)"

We investigated the role of AMP-activated protein kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and bone morphogenetic protein 2 mRNA and/or protein levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-II, upregulation of proautophagic beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3β, as well as the pharmacological inhibitors of AMPK (compound C), Akt (10-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Akt inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis. © 2012 Elsevier Inc.

We investigated the role of AMP-activated protein kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and bone morphogenetic protein 2 mRNA and/or protein levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-II, upregulation of proautophagic beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3β, as well as the pharmacological inhibitors of AMPK (compound C), Akt (10-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Akt inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis. © 2012 Elsevier Inc.

Background Recent studies point to important roles for IL-17 and Th17 cells in sustaining chronic inflammation and articular destruction in rheumatoid arthritis (RA). We investigated the effects of TNF inhibitor on innate inflammatory and Th17 cytokines production by ex vivo lipopolysaccharide (LPS)-stimulated whole blood in patients with RA and the associations of cytokine levels in whole blood cultures with autoantibodies and markers of disease activity. Materials and methods Whole blood cultures from 18 healthy volunteers and 19 RA patients on etanercept therapy were stimulated with LPS and the production of IL-6, TNF-a, IL-23, IL-17A and IL-21 was measured by ELISA. Results After stimulation with LPS, the interleukin (IL)-17A (p = 0.020) and IL-21 (p = 0.0001) secretions were significantly higher in patients with RA than in controls, while the TNF-a (p = 0.002) was significantly lower at baseline. Etanercept significantly decreased IL-21 production (p = 0.007), while IL-6 production (p = 0.005) significantly increased after 6 months of therapy. IL-21 significantly correlated with RF (r = 0.917, p\0.01) and antimutated citrullinated vimentin antibodies (r = 0.770, p\0.01) at baseline. Logistic regression analysis revealed that baseline IL-21 levels (p = 0.004) were significant predictors of DAS28-ESR at 6 months follow-up. Discussion Stimulation with LPS increased production of Th17 cytokines in whole blood cultures in patients with RA. Etanercept therapy decreased IL-21 secretion, while the capacity of whole blood cells to produce IL-6 increased. IL-21 production is strongly associated with the levels of autoantibodies. Our findings suggest that IL-21 production in LPS-stimulated whole blood cultures may be predictive of clinical response to etanercept treatment in patients with RA. © 2012 Springer Basel AG.

Background Recent studies point to important roles for IL-17 and Th17 cells in sustaining chronic inflammation and articular destruction in rheumatoid arthritis (RA). We investigated the effects of TNF inhibitor on innate inflammatory and Th17 cytokines production by ex vivo lipopolysaccharide (LPS)-stimulated whole blood in patients with RA and the associations of cytokine levels in whole blood cultures with autoantibodies and markers of disease activity. Materials and methods Whole blood cultures from 18 healthy volunteers and 19 RA patients on etanercept therapy were stimulated with LPS and the production of IL-6, TNF-a, IL-23, IL-17A and IL-21 was measured by ELISA. Results After stimulation with LPS, the interleukin (IL)-17A (p = 0.020) and IL-21 (p = 0.0001) secretions were significantly higher in patients with RA than in controls, while the TNF-a (p = 0.002) was significantly lower at baseline. Etanercept significantly decreased IL-21 production (p = 0.007), while IL-6 production (p = 0.005) significantly increased after 6 months of therapy. IL-21 significantly correlated with RF (r = 0.917, p\0.01) and antimutated citrullinated vimentin antibodies (r = 0.770, p\0.01) at baseline. Logistic regression analysis revealed that baseline IL-21 levels (p = 0.004) were significant predictors of DAS28-ESR at 6 months follow-up. Discussion Stimulation with LPS increased production of Th17 cytokines in whole blood cultures in patients with RA. Etanercept therapy decreased IL-21 secretion, while the capacity of whole blood cells to produce IL-6 increased. IL-21 production is strongly associated with the levels of autoantibodies. Our findings suggest that IL-21 production in LPS-stimulated whole blood cultures may be predictive of clinical response to etanercept treatment in patients with RA. © 2012 Springer Basel AG.

Introduction: Mesenchymal stem cells from Wharton's Jelly of a human umbilical cord (WJ-MSCs) are a potential tool in regenerative medicine based on their availability, proliferative potential and differentiation capacity. Since their physiological niche contains low oxygen levels, we investigated whether cultivation of WJ-MSCs at 3% O2 affects their main features. Methods: WJ-MSCs were cultured under 21% and 3% O2. Proliferation rate was followed by short and long term proliferation assays, clonogenic capacity by CFU-F assay and cell cycle and death by flow cytometry. Differentiation capacity was investigated by histochemical staining after induced differentiation. Pluripotency and differentiation markers' expression was determined by RT-PCR. Migration capacity was followed by scratch assay and mobilization from collagen, and the activity of proteolytic enzymes by zymography. Specific inhibitors of MAPK and Wnt/β-catenin pathways were used to investigate underlying molecular mechanisms. Results: Compared to standard 21% O2, cultivation of WJ-MSCs at 3% O2 did not influence their immunophenotype, while it modulated their differentiation process and enhanced their clonogenic and expansion capacity. 3% O2 induced transient change in cell cycle and prevented cell death. The expression of NANOG, OCT4A, OCT4B and SOX2 was increased at 3% O2. Both cultivation and preculturing of WJ-MSCs at 3% O2 increased their in vitro migratory capacity and enhanced the activity of proteolytic enzymes. ERK1/2 mediated WJ-MSCs’ mobilization from collagen regardless of oxygen levels, while Wnt/β-catenin pathway was activated during migration and mobilization at standard conditions. Conclusion: Culturing of WJ-MSCs under 3% O2 should be considered a credible condition when investigating their properties and potential use. © 2019 Elsevier Ltd

Introduction: Mesenchymal stem cells from Wharton's Jelly of a human umbilical cord (WJ-MSCs) are a potential tool in regenerative medicine based on their availability, proliferative potential and differentiation capacity. Since their physiological niche contains low oxygen levels, we investigated whether cultivation of WJ-MSCs at 3% O2 affects their main features. Methods: WJ-MSCs were cultured under 21% and 3% O2. Proliferation rate was followed by short and long term proliferation assays, clonogenic capacity by CFU-F assay and cell cycle and death by flow cytometry. Differentiation capacity was investigated by histochemical staining after induced differentiation. Pluripotency and differentiation markers' expression was determined by RT-PCR. Migration capacity was followed by scratch assay and mobilization from collagen, and the activity of proteolytic enzymes by zymography. Specific inhibitors of MAPK and Wnt/β-catenin pathways were used to investigate underlying molecular mechanisms. Results: Compared to standard 21% O2, cultivation of WJ-MSCs at 3% O2 did not influence their immunophenotype, while it modulated their differentiation process and enhanced their clonogenic and expansion capacity. 3% O2 induced transient change in cell cycle and prevented cell death. The expression of NANOG, OCT4A, OCT4B and SOX2 was increased at 3% O2. Both cultivation and preculturing of WJ-MSCs at 3% O2 increased their in vitro migratory capacity and enhanced the activity of proteolytic enzymes. ERK1/2 mediated WJ-MSCs’ mobilization from collagen regardless of oxygen levels, while Wnt/β-catenin pathway was activated during migration and mobilization at standard conditions. Conclusion: Culturing of WJ-MSCs under 3% O2 should be considered a credible condition when investigating their properties and potential use. © 2019 Elsevier Ltd

Introduction Mesenchymal stem cells (MSCs) are a promising tool for regenerative medicine, but due to the heterogeneity of their populations, different sources and isolation techniques, the characteristics defining MSCs are inconsistent. Objective The aim of this study was to compare the characteristics of MSCs derived from two different human tissues: peripheral blood (PB-MSCs) and umbilical cord Wharton's Jelly (UC-MSCs). Methods The PB-MSC and UC-MSC were isolated by adherence to plastic after gradient-density separation or an explant culture method, respectively, and compared regarding their morphology, clonogenic efficiency, proliferating rates, immunophenotype and differentiation potential. Results MSCs derived from both sources exhibit similar morphology, proliferation capacity and multilineage (osteogenic, chondrogenic, adipogenic and myogenic) differentiation potential. Differences were observed in the clonogenic capacity and the immunophenotype, since UC-MSCs showed higher CFU-F (colony-forming units-fibroblastic) cloning efficiency, as well as higher embryonic markers (Nanog, Sox2, SSEA4) expression. When additional surface antigens were analyzed by flow cytometry (CD44, CD90, CD105, CD33, CD34, CD45, CD11b, CD235a) or immunofluorescent labeling (vimentin, STRO-1 and α-smooth muscle actin), most appeared to have similar epitope profiles irrespective of MSC source. Conclusion The results obtained demonstrated that both MSCs represent good alternative sources of adult MSCs that could be used in cell therapy applications.

Purpose: To evaluate the reliability of ultrasound scan (US) findings in the preoperative assessment of the nature of adnexal masses infernales. Methods: After detailed history, a preoperative US examination was performed in all women. Tumor diameter, localization, the presence of solid, cystic and multilocular components, excrescences, metastasis and free fluid were assessed. Doppler scan was done and pulsatility (PI) and resistance indices (RI) were determined. These data were compared with postoperatively obtained histopathological findings and statistically analyzed. Results: The study included 609 women out of which 20.7% had malignant, 73.7% benign, and 5.6% borderline tumors. Patients with malignant tumors were oldest (p<0.001). There were significantly more positive US parameters in malignant than in benign tumors (p<0.001). Also, there were significant differences (p<0.001) between malignant, benign and borderline tumors regarding all examined US and Doppler parameters except tumor multilocularity. RI had sensitivity 75%, specificity 61.2%, positive predictive value (PPV) 42.70% and negative predictive value (NPV) 96.16%. PI had sensitivity 50%, specificity 35.3%, PPV 8.37% and NPV 25.93%. Sensitivity of US characteristics was 94.34%, specificity 30.62%, PPV 22.27% and NPV 96.25%. Conclusions: US pattern characteristics and Doppler parameters were found to be moderately reliable in discriminating malignant, benign and borderline adnexal tumors. Tumor of solid or mixed consistency, presence of ascites and excrescences were the best predictors of malignancy.

Purpose: To evaluate the reliability of ultrasound scan (US) findings in the preoperative assessment of the nature of adnexal masses infernales. Methods: After detailed history, a preoperative US examination was performed in all women. Tumor diameter, localization, the presence of solid, cystic and multilocular components, excrescences, metastasis and free fluid were assessed. Doppler scan was done and pulsatility (PI) and resistance indices (RI) were determined. These data were compared with postoperatively obtained histopathological findings and statistically analyzed. Results: The study included 609 women out of which 20.7% had malignant, 73.7% benign, and 5.6% borderline tumors. Patients with malignant tumors were oldest (p<0.001). There were significantly more positive US parameters in malignant than in benign tumors (p<0.001). Also, there were significant differences (p<0.001) between malignant, benign and borderline tumors regarding all examined US and Doppler parameters except tumor multilocularity. RI had sensitivity 75%, specificity 61.2%, positive predictive value (PPV) 42.70% and negative predictive value (NPV) 96.16%. PI had sensitivity 50%, specificity 35.3%, PPV 8.37% and NPV 25.93%. Sensitivity of US characteristics was 94.34%, specificity 30.62%, PPV 22.27% and NPV 96.25%. Conclusions: US pattern characteristics and Doppler parameters were found to be moderately reliable in discriminating malignant, benign and borderline adnexal tumors. Tumor of solid or mixed consistency, presence of ascites and excrescences were the best predictors of malignancy.