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1.
Sci Rep ; 9(1): 12855, 2019 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-31492934

RESUMEN

Cedrus libani is a majestic evergreen tree native to the Mediterranean mountains of Lebanon, Syria and Turkey. In this study, the tree heart wood was extracted using hexane to produce C. libani oil extract (CLOE) as a dark oil. GCMS analysis of CLOE identified up to 30 compounds whereby 2-himachalen-7-ol (7-HC) was the most abundant (40%). 7-HC was isolated using column chromatography and the identity of the white crystalline solid was confirmed via NMR spectroscopy and X-Ray Crystallography. 7-HC demonstrated potent cytotoxic activity against several human cancer cell lines including brain (SF-268, IC50 8.1 µg/mL) and colon (HT-29, IC50 10.1 µg/mL; Caco-2, IC50 9.9 µg/mL) with ovarian (Sk-OV-3, IC50 > 50 µg/mL) cells being the most resistant. However, while HT-29 displayed resistance to Cisplatin, 7-HC was 8-10 folds more potent. Co-treatment with 7-HC and Cisplatin showed a significant synergistic anti-proliferative effect against SF-268, HT-29 and Caco-2 cells. 7-HC also exhibited significant anti-inflammatory effect in formalin-induced paw edema in rats. Western blot analysis revealed that 7-HC displayed dose dependent inhibition of LPS-induced COX-2 protein expression in isolated rat monocytes. The present study demonstrates that 7-HC possesses promising anticancer and anti-inflammatory activities, and may serve as a lead molecule in cancer therapy.

2.
ACS Appl Mater Interfaces ; 11(36): 32623-32632, 2019 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-31424195

RESUMEN

Chronic kidney disease is characterized by a gradual decline in renal function that progresses toward end-stage renal disease. Podocytes are highly specialized glomerular epithelial cells which form with the glomerular basement membrane (GBM) and capillary endothelium the glomerular filtration barrier. GBM is an extracellular matrix (ECM) that acts as a mechanical support and provides biophysical signals that control normal podocytes behavior in the process of glomerular filtration. Thus, the ECM stiffness represents an essential characteristic that controls podocyte function. Hydrolyzed Polyacrylamide (PAAm) hydrogels are smart polyelectrolyte materials. Their biophysical properties can be tuned as desired to mimic the natural ECM. Therefore, these hydrogels are investigated as new ECM-like constructs to engineer a podocyte-like basement membrane that forms with cultured human podocytes a functional glomerular-like filtration barrier. Such ECM-like PAAm hydrogel construct will provide unique opportunity to reveal podocyte cell biological responses in an in vivo-like setting by controlling the physical properties of the PAAm membranes. In this work, Hydrolyzed PAAm scaffolds having different stiffness ranging between 0.6-44 kPa are prepared. The correlation between the hydrogel structural and mechanical properties and Podocyte morphology, elasticity, cytoskeleton reorganization, and podocin expression is evaluated. Results show that hydrolyzed PAAm hydrogels promote good cell adhesion and growth and are suitable materials for the development of future 3D smart scaffolds. In addition, the hydrogel properties can be easily modulated over a wide physiological range by controlling the cross-linker concentration. Finally, tuning the hydrogel properties is an effective strategy to control the cells function. This work addressed the complexity of podocytes behavior which will further enhance our knowledge to develop a kidney-on-chip model much needed in kidney function studies in both healthy and diseased states.

3.
J Cell Physiol ; 234(11): 21145-21152, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31041809

RESUMEN

Adipose tissue-derived mesenchymal stromal cells (ASCs) hold the promise of achieving successful immunotherapeutic results due to their ability to regulate different T-cell fate. ASCs also show significant adaptability to environmental stresses by modulating their immunologic profile. Cell-based therapy for inflammatory diseases requires a detailed understanding of the molecular relation between ASCs and Th17 lymphocytes taking into account the influence of inflammation and cell ratio on such interaction. Accordingly, a dose-dependent increase in Th17 generation was only observed in high MSC:T-cell ratio with no significant impact of inflammatory priming. IL-23 receptor (IL-23R) expression by T cells was not modulated by ASCs when compared to levels in activated T cells, while ROR-γt expression was significantly increased reaching a maximum in high (1:5) unprimed ASC:T-cell ratio. Finally, multiplex immunoassay showed substantial changes in the secretory profile of 15 cytokines involved in the Th17 immune response (IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-22, IL-21, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40, and TNF-α), which was modulated by both cell ratio and inflammatory priming. These findings suggest that Th17 lymphocyte pathway is significantly modulated by ASCs that may lead to immunological changes. Therefore, future ASC-based immunotherapy should take into account the complex and detailed molecular interactions that depend on several factors including inflammatory priming and cell ratio.

4.
Cell Biochem Funct ; 37(4): 245-255, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31017709

RESUMEN

Damage to podocytes is a key event in glomerulopathies. While energy dense food can contribute to kidney damage, the role of the orixegenic hormone "ghrelin" in podocyte biology is still unknown. In the present study, we investigated the effect of ghrelin on podocyte survival as well as the signalling pathways mediating ghrelin effect in immortalized cultured rat podocytes. RT-PCR analysis revealed that GHS-R1 is expressed in rat podocytes. Western blot analysis showed that ghrelin upregulated COX-2 protein expression in a time and dose-dependent manner. Additionally, ghrelin activated P38 MAPK, AKT, and ERK1/2 pathways and also induced P38 MAPK phosphorylation in high glucose conditions. Ghrelin induced ROS release and dose dependently reduced podocyte survival. Ghrelin mediated podocyte cell death was partially reversed by pharmacologically inhibiting P38 MAPK or phospholipase C (PLC). Furthermore, PLC inhibitor (U73122) inhibited ghrelin induced P38 MAPK activation. While PI3K inhibitor (LY294002) was without effect on cell survival or P38 MAPK activation, it inhibited ghrelin induced ERK1/2 phosphorylation. Finally, ghrelin induced TAU phosphorylation was reversed by pharmacologic inhibitors of either P38 MAPK or PKA. In conclusion, ghrelin activated harmful molecular pathways in podocytes that can be damaging to the glomerular filtration barrier SIGNIFICANCE OF THE STUDY: Endocrine derangements secondary to obesity are major players in the aetiology of renal injuries. Furthermore, energy dense diet is thought to be the major element in developing obesity. Appetite and increase in energy intake are regulated by complex hormonal pathways which mainly include the orexigenic hormone "ghrelin" in addition to leptin. To date no study have highlighted a significant role for ghrelin in kidney biology, and therefore, it is thought that its endocrine effect is mostly limited to adipose tissue metabolism and appetite regulation. In this study, we first showed that ghrelin receptor is expressed on glomerular podocytes. Also, ghrelin showed negative impact on podocyte survival through modulating signalling pathways such as P38 MAPK and AKT known to play a key role in podocyte health. Moreover, the negative effects of ghrelin on podocytes were further exacerbated in hyperglycemic conditions. Of note, podocytes contribute to the formation and the maintenance of the glomerular filtration barrier and thus are important for normal renal function. Therefore, ghrelin secretion in the context of obesity could be involved in the aetiology of kidney injury, a well-known hallmark found in obese patients.


Asunto(s)
Ghrelina/farmacología , Podocitos/citología , Podocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Calcio/análisis , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , Ratones , Podocitos/metabolismo
5.
Curr Pharm Biotechnol ; 20(1): 84-96, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30727882

RESUMEN

BACKGROUND: Propolis is a resinous substance produced by bees and known to possess antioxidant, antimicrobial, antiproliferative and anti-inflammatory activities. OBJECTIVE: This study is aimed at evaluating the in vivo and in vitro anti-inflammatory potential of the Crude Ethanolic Extract (CE) of Lebanese propolis and its Ethyl Acetate Fraction (EAF). METHOD: Chemical content of propolis was characterized using high-performance liquid chromatography and LC-MS/MS. COX-2 and iNOS protein expression, nitric oxide (NO) and prostaglandin (PGE2) release in LPS-activated RAW monocytes were achieved respectively by western blot and spectrophotometry. Antioxidant activity was evaluated by DPPH free radical scavenging assay. Measurement of paw thickness in carrageenan-induced paw edema in mice and pathologic assessment of inflammation in paw sections were used to judge the anti-inflammatory properties of propolis. RESULTS: Pathology analysis revealed in the treated group significant reduction of immune cell infiltration and edema. Both extract and ethyl acetate fraction showed significant anti-inflammatory and antioxidant effects in LPS-treated RAW cells characterized by the inhibition of COX-2 and iNOS protein expression, as well as PGE2 and NO release. Chemical analysis of the crude extract and its ethyl acetate fraction identified 28 different compounds of which two phenolic acids and nine other flavonoids were also quantified. Ferulic acid, caffeic acid, chrysin, galangin, quercetin, and pinocembrin were among the most representative compounds. CONCLUSION: Lebanese propolis is rich in a various amount of flavonoids which showed promising antiinflammatory and antioxidant properties. Additionally, chemical analysis showed unique chemical compositions with the potential of identifying ingredients with interesting anti-inflammatory activities.


Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Antioxidantes/química , Antioxidantes/uso terapéutico , Própolis/química , Própolis/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Abejas , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cromatografía Liquida/métodos , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Edema/tratamiento farmacológico , Edema/patología , Líbano , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Espectrometría de Masas en Tándem/métodos
6.
Inflamm Res ; 68(3): 203-213, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30506263

RESUMEN

OBJECTIVE AND DESIGN: The objective of the study is to uncover the influence of human bone marrow-derived mesenchymal stem cells (BM-MSCs) on the generation of Th17 lymphocytes in co-cultures of both BM-MSCs and T cells. MATERIALS AND METHODS: BM-MSCs, characterized according to the international society for cellular therapy (ISCT) criteria, were co-cultured with T cells isolated from peripheral blood. The expression levels of IL-17 receptor, RORγt and IL-23 receptor were evaluated using flow cytometry. The levels of cytokines involved in Th17 immunomodulation were measured using multiplex assay. TREATMENT: Inflammatory primed and non-primed BM-MSCs were co-cultured with either activated or non-activated T cells either at (1/80) and (1/5) ratio respectively. RESULTS: MSC/T-cell ratio and inflammation significantly influenced the effect of BM-MSCs on the generation of Th17 lymphocytes. Cocultures of either primed or non-primed BM-MSCs with activated T cells significantly induced IL-17A-expressing lymphocytes. Interestingly, the expression of the transcription factor RORγt was significantly increased when compared to levels in activated T cells. Finally, both cell ratio and priming of BM-MSCs with cytokines substantially influenced the cytokine profile of BM-MSCs and T cells. CONCLUSION: Our findings suggest that BM-MSCs significantly modulate the Th17 lymphocyte pathway in a complex manner.


Asunto(s)
Células Madre Mesenquimatosas/inmunología , Células Th17/inmunología , Células de la Médula Ósea/citología , Técnicas de Cocultivo , Citocinas/inmunología , Humanos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Receptores de Interleucina/inmunología
7.
J Cell Physiol ; 234(4): 4825-4839, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30207376

RESUMEN

Vascular calcification (VC) is the pathological accumulation of calcium phosphate crystals in one of the layers of blood vessels, leading to loss of elasticity and causing severe calcification in vessels. Medial calcification is mostly seen in patients with chronic kidney disease (CKD) and diabetes. Identification of key enzymes and their actions during calcification will contribute to understand the onset of pathological calcification. Phospholipase D (PLD1, PLD2) is active at the earlier steps of mineralization in osteoblasts and chondrocytes. In this study, we aimed to determine their effects during high-phosphate treatment in mouse vascular smooth muscle cell line MOVAS, in the ex vivo model of the rat aorta, and in the in vivo model of adenine-induced CKD. We observed an early increase in PLD1 gene and protein expression along with the increase in the PLD activity in vascular muscle cell line, during calcification induced by ascorbic acid and ß-glycerophosphate. Inhibition of PLD1 by the selective inhibitor VU0155069, or the pan-PLD inhibitor, halopemide, prevented calcification. The mechanism of PLD activation is likely to be protein kinase C (PKC)-independent since bisindolylmaleimide X hydrochloride, a pan-PKC inhibitor, did not affect the PLD activity. In agreement, we found an increase in Pld1 gene expression and PLD activity in aortic explant cultures treated with high phosphate, whereas PLD inhibition by halopemide decreased calcification. Finally, an increase in both Pld1 and Pld2 expression occurred simultaneously with the appearance of VC in a rat model of CKD. Thus, PLD, especially PLD1, promotes VC in the context of CKD and could be an important target for preventing onset or progression of VC.

8.
Inflamm Res ; 67(8): 711-722, 2018 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-29922854

RESUMEN

OBJECTIVE AND DESIGN: The objective of this study is to uncover the signal transduction pathways of N-formyl methionyl-leucyl-phenylalanine (fMLP) in monocyte. MATERIALS OR SUBJECTS: Freshly isolated human peripheral blood monocytes (PBMC) were used for in vitro assessment of signal transduction pathways activated by fMLP. TREATMENT: Time-course and dose-response experiments were used to evaluate the effect of fMLP along with the specific inhibitors/stimulators on the activation of downstream signaling kinases. METHODS: Freshly isolated human PBMC were stimulated with fMLP for the desired time. Western blot and siRNA analysis were used to evaluate the activated intracellular signaling kinases, and flow analysis was performed to assess the levels of CD11b. Furthermore, luminescence spectrometry was performed to measure the levels of released hydrogen peroxide in the media. RESULTS: fMLP strongly stimulated the activation of AKT and ERK1/2 through a RhoA-GTPase-dependent manner and also induced H2O2 release by monocytes. Furthermore, fMLP mediated its effects through restricted activation of formylpeptide receptor-like 1 (FPRL1/FPR2), but independently of either EGFR transactivation or intracellular calcium release. In addition, NAC reversed fMLP- and H2O2-induced activation of Akt and RhoA-GTPase. CONCLUSION: Collectively, these data suggested that fMLP-activated ERK1/2 and Akt pathways through specific activation of the FPRL1/ROS/RoA-GTPase pathway.


Asunto(s)
Peróxido de Hidrógeno/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Células Cultivadas , Humanos , Leucocitos Mononucleares/metabolismo , Ratas
9.
Inflamm Res ; 67(2): 191-201, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29085960

RESUMEN

OBJECTIVE AND DESIGN: The aim of this study is to elucidate TGF-ß1 signaling pathways involved in COX-2 protein induction and modulation of TAU protein phosphorylation in cultured podocytes. MATERIALS, TREATMENT AND METHODS: In vitro cultured immortalized podocytes were stimulated with TGF-ß1 in presence and absence of pharmacologic inhibitors for various signaling pathways and phosphatases. Then, COX-2 protein expression, as well as P38MAPK, AKT and TAU phosphorylation levels were evaluated by western blot analysis. RESULTS: TGF-ß1 induction of COX-2 protein levels was completely blocked by pharmacologic inhibitors of phosphatases, P38 MAPK, or NF-қB pathways. Time course experiments showed that TGF-ß1 activated p38 MAPK after 5 min of stimulation. Interestingly, podocyte co-incubated with TGF-ß1, high glucose and/or PGE2 showed strong increase in p38 MAPK and AKT phosphorylation as well as COX- 2 protein expression levels. Levels of phosphorylated AKT were further reduced and levels of phosphorylated p38 were increased when PGE2 was added to the culture media. Interestingly, selective phosphatases inhibitors completely abrogated PGE2-induced P38 MAPK and TAU phosphorylation. Also, inhibition of phosphatases reversed TGF-ß1-induced COX-2 protein expression either alone or when incubated with high glucose or PGE2. CONCLUSION: These data suggest TGF-ß1 mediates its effect in podocyte through novel signaling mechanisms including phosphatases and TAU protein phosphorylation.


Asunto(s)
Ciclooxigenasa 2/biosíntesis , Monoéster Fosfórico Hidrolasas/farmacología , Podocitos/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Proteínas tau/metabolismo , Animales , Células Cultivadas , Glucosa/farmacología , Ratones , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Podocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
10.
Biomed Pharmacother ; 98: 52-62, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29245066

RESUMEN

Human hepatic stellate cells (HSCs) demonstrated great immunological plasticity with important consequences for liver cell therapy. Activated HSCs (aHSCs) are in vitro reverted (rHSCs) to a quiescent-like phenotype with potential benefit to reduce liver fibrosis. The goal of this study is to establish and compare the immunological profile of activated and in vitro reverted HSCs and to investigate the impact of inflammatory priming on the immunobiology of both HSCs populations. The distribution of inflammatory primed activated and reverted HSCs across the different phases of the cell cycle is assessed by flow cytometry. In addition, Flow analysis was done to assess the expression level of neuronal, endothelial and stromal markers, cell adhesion molecules, human leucocyte antigens, co-stimulatory molecules, immunoregulatory molecules and natural killer ligands. Our results showed that the cell cycle distribution of both HSCs populations is significantly modulated by inflammation. Accordingly, activated HSC that were in G1 phase switch to S- and G2 phases when exposed to inflammation, while reverted HSCs mostly redistribute into sub-G0 phase. In a HSC state dependent manner, inflammatory priming modulated the expression of the stromal marker CD90, biological receptors (CD95 and CD200R), cell adhesion molecules (CD29, CD54, CD58, CD106 and CD166), human leucocyte antigen HLA-G, co-stimulatory molecules (CD40 and CD252), as well as the immunoregulatory molecules (CD200 and CD274). In conclusion, the immunologic profile of HSCs is significantly modulated by their activation state and inflammation and is important for the development of novel HSC liver cell-based therapy.


Asunto(s)
Células Estrelladas Hepáticas/inmunología , Hígado/inmunología , Biomarcadores/metabolismo , Moléculas de Adhesión Celular/metabolismo , Ciclo Celular/inmunología , Línea Celular , Antígenos HLA-G/inmunología , Células Estrelladas Hepáticas/metabolismo , Humanos , Factores Inmunológicos/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Hígado/metabolismo , Cirrosis Hepática/inmunología , Cirrosis Hepática/metabolismo
11.
Biomed Pharmacother ; 95: 298-307, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28850929

RESUMEN

Chemical composition, anti-proliferative and proapoptotic activity as well as the effect of various fractions of Lebanese propolis on the cell cycle distribution were evaluated on Jurkat leukemic T-cells, glioblastoma U251 cells, and breast adenocarcinoma MDA-MB-231 cells using cytotoxic assays, flow cytometry as well as western blot analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that ferulic acid, chrysin, pinocembrin, galangin are major constituents of the ethanolic crude extract of the Lebanese propolis, while the hexane fraction mostly contains chrysin, pinocembrin, galangin but at similar levels. Furthermore chemical analysis was performed using gas chromatography-mass spectrometry (GC-MS) to identify major compounds in the hexane fraction. Reduction of cell viability was observed in Jurkat cells exposed to the ethanolic crude extract and the hexane fraction, while viability of U251 and MDA-MB-231 cells was only affected upon exposure to the hexane fraction; the other fractions (aqueous phase, methylene chloride, and ethyl acetate) were without effect. Maximum toxic effect was obtained when Jurkat cells were cultivated with 90µg/ml of both the crude extract and hexane faction. Toxicity started early after 24h of incubation and remained till 72h. Interestingly, the decrease in cell viability was accompanied by a significant increase in p53 protein expression levels and PARP cleavage. Cell cycle distribution showed an increase in the SubG0 fraction in Jurkat, U251 and MDA-MB-231 cells after 24h incubation with the hexane fraction. This increase in SubG0 was further investigated in Jurkat cells by annexinV/PI and showed an increase in the percentage of cells in early and late apoptosis as well as necrosis. In conclusion, Lebanese propolis exhibited significant cytotoxicity and anti-proliferative activity promising enough that warrant further investigations on the molecular targets and mechanisms of action of Lebanese propolis.


Asunto(s)
Citotoxinas/química , Citotoxinas/farmacología , Própolis/química , Própolis/farmacología , Espectrometría de Masas en Tándem/métodos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cromatografía Liquida/métodos , Citotoxinas/análisis , Relación Dosis-Respuesta a Droga , Humanos , Células Jurkat , Líbano , Própolis/análisis
12.
Histol Histopathol ; 32(3): 307-313, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27264533

RESUMEN

Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and ß-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and ß-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and ß-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.


Asunto(s)
Biomarcadores de Tumor/análisis , Western Blotting/métodos , Técnicas de Preparación Histocitológica , Proteínas/aislamiento & purificación , Humanos , Adhesión en Parafina , Fijación del Tejido , Xilenos
13.
Inflamm Res ; 66(2): 129-139, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27783097

RESUMEN

OBJECTIVE: The role of direct cell-cell interactions mediating selective bone metastasis by breast cancer cells (BCCs) niche is still mostly unknown. MATERIALS AND METHODS: Conditioned medium and direct cell-cell contacts experiments were used to investigate the effect of bone marrow-derived mesenchymal stromal cells (MSCs), osteoprogenitor-like cells (MG-63) and osteosarcoma cells (SaOS-2) on luminal-like (MCF-7) and basal-like (MDA-MB-231) BCCs flow cytometry was used to assess the purity of isolated BCCs and osteoblasts. Expression of osteoblastic markers was investigated by semi-quantitative RT-PCR. RANKL and OPG levels were measured by ELISA. RESULTS: Conditioned medium from MSCs and osteoblasts induced the expression of osteoblastic markers in BCCs. While co-culture assays with SaOS-2 increased the expression of osteoblastic markers in MCF-7 cells, SaOS-2 cell conditioned medium increased the expression of RANKL, PTHrP, VEGF and NOGGIN in MCF-7 cells. Co-cultures with either MG-63 cells or MSCs induced OPG and MMP-2 in both tumor cell lines. Interestingly, conditioned medium from co-cultures of MSCs and MDA-MB-231 cells significantly decreased the proliferation of activated T lymphocytes which was reversed by addition of anti-OPG antibodies to the co-cultures. CONCLUSION: Our data suggest that MSCs strongly contribute to the adaptation and invasiveness of breast cancer cells in skeletal tissues.


Asunto(s)
Neoplasias de la Mama/inmunología , Células Madre Mesenquimatosas/inmunología , Células de la Médula Ósea/citología , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Femenino , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/inmunología , Osteoblastos/metabolismo , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología
14.
Chem Biol Drug Des ; 90(1): 83-96, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28032452

RESUMEN

This study reports the synthesis of two series of new purine bioisosteres comprising a pyrazolo[3,4-d]pyrimidine scaffold linked to piperazine moiety through different amide linkages. The newly synthesized compounds were evaluated for anticancer activity against four cell lines (MDA-MB-231, MCF-7, SF-268, B16F-10) and cyclooxygenase (COX-2) protein expression inhibition in lipopolysaccharide (LPS)-activated rat monocytes. The results revealed that most of the synthesized compounds showed moderate-to-high cytotoxic activity against at least one cell line, with compound 10b being the most active against all used cell lines (IC50 values 5.5-11 µg/ml) comparable to cisplatin. In addition, six of these compounds (7b, 10a-d, and 12c) demonstrated inhibition of LPS-induced COX-2 protein expression at low concentration (25 µg/ml) as compared to the control non-stimulated cells and showed a COX-2 selectivity index range comparable to diclofenac sodium. The overall results indicate that many of these pyrazolopyrimidine derivatives possess in vitro anti-inflammatory and anticancer activities at varying doses, and the most active compounds will be subjected to in vivo pharmacological evaluation.


Asunto(s)
Antiinflamatorios/síntesis química , Antineoplásicos/síntesis química , Inhibidores de la Ciclooxigenasa/síntesis química , Pirazoles/química , Pirazoles/síntesis química , Pirimidinas/química , Pirimidinas/síntesis química , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 1/química , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/química , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Diclofenaco/farmacología , Diseño de Drogas , Humanos , Concentración 50 Inhibidora , Lipopolisacáridos/farmacología , Células MCF-7 , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Pirazoles/metabolismo , Pirazoles/farmacología , Pirimidinas/metabolismo , Pirimidinas/farmacología , Ratas
15.
Cytokine ; 90: 130-134, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27865205

RESUMEN

AIM: Uncertainty about the safety of cell therapy continues to be a major challenge to the medical community. Inflammation and the associated immune response represent a major safety concern hampering the development of long-term clinical therapy. In vivo interactions between the cell graft and the host immune system are mediated by functional environmental sensors and stressors that play significant roles in the immunobiology of the graft. Within this context, human liver stellate cells (HSC) demonstrated marked immunological plasticity that has main importance for future liver cell therapy application. METHODS: By using qPCR technique, we established the cytokine gene expression profile of HSCs and investigated the effect of an inflammatory environment on the immunobiology of HSCs. RESULTS AND DISCUSSION: HSCs present a specific immunological profile as demonstrated by the expression and modulation of major immunological cytokines. Under constitutive conditions, the cytokine pattern expressed by HSCs was characterized by the high expression of IL-6. Inflammation critically modulated the expression of major immunological cytokines. As evidenced by the induction of the expression of several inflammatory genes, HSCs acquire a pro-inflammatory profile that ultimately might have critical implications for their immunological shape. CONCLUSION: These new observations have to be taken into account in any future liver cell therapy application based on the use of HSCs.


Asunto(s)
Células Estrelladas Hepáticas/inmunología , Hepatitis/inmunología , Interleucina-6/inmunología , Células Cultivadas , Células Estrelladas Hepáticas/patología , Hepatitis/patología , Humanos , Inflamación/inmunología , Inflamación/patología
16.
BMC Pharmacol Toxicol ; 17(1): 51, 2016 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-27817746

RESUMEN

BACKGROUND: Antidotes stocking is a critical component of hospital care for poisoned patients in emergency. Antidote stocking represents a major health challenge worldwide and in Lebanon. Systematic data monitoring of antidote stocking in Lebanese hospitals is lacking. The objective of this study is to assess the adequacy of antidotes stocking in Lebanese hospitals according to type and quantity and explore the characteristics associated with their differential availability. METHODS: Data collection to assess antidote availability and its correlate was undertaken through a self-administered questionnaire. The questionnaires were distributed by the unit of surveillance at the Ministry of Public Health to eligible hospitals providing emergency care services. The list of essential antidotes was adapted from the World Health Organization (WHO) list and the British Columbia Drug and Poison Information Centre. RESULTS: Among the 85 Lebanese hospitals surveyed none had in stock all the 35 essential antidotes required. The frequency of stocking by type of antidote varied from a minimum of 1.2 % of the hospitals having a (cyanide kit) to 100 % availability of (atropine and calcium gluconate). Teaching hospitals and those with a large bed-capacity reported a higher number of available antidotes for both immediate and non-immediate use than non-teaching hospitals while controlling for the hospital geographical region and public vs private sector. CONCLUSION: The Lebanese hospitals have a suboptimal stock of essential antidotes supply. It is recommended that the Lebanese Ministry of Public Health monitors closely on the hospital premises the adequacy and availability of essential antidotes stock.


Asunto(s)
Antídotos/provisión & distribución , Servicios Médicos de Urgencia/provisión & distribución , Servicio de Urgencia en Hospital , Hospitales de Enseñanza/provisión & distribución , Servicio de Farmacia en Hospital/provisión & distribución , Antídotos/normas , Estudios Transversales , Servicios Médicos de Urgencia/normas , Servicio de Urgencia en Hospital/normas , Hospitales de Enseñanza/normas , Humanos , Líbano/epidemiología , Servicio de Farmacia en Hospital/normas
17.
Inflamm Res ; 65(6): 501-10, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26956767

RESUMEN

OBJECTIVE: Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are well known to modulate T cells. However, the molecular mechanisms that mark hBM-MSCs immunomodulation of T cells are not fully resolved. MATERIALS AND METHODS: hBM-MSCs harvested from sternum or iliac crest of five healthy donors and characterized in accordance with the International Society of Cellular Therapy (ISCT) guidelines are co-cultured with T cells. Additionally, modulatory effects of MSCs on T-cell viability, proliferation, cytokine profile, co-stimulatory pathway, activation and immunomodulation are also determined. RESULTS: hBM-MSCs significantly reduced the expression of T-cell activation marker CD38 as well as co-stimulatory markers CD134 and CD154, whilst that of CD27 remained unchanged. BrdU, CFSE and Ki67 proliferation assays showed that hBM-MSCs reduced T-cell proliferation. Moreover, viability of T cells remained unchanged when co-cultured with hBM-MSCs. Finally, T cells when co-cultured with hBM-MSCs showed increased secretion of IL-10 and IL-11. CONCLUSION: Collectively, hBM-MSCs are able to modulate the main steps involved in T-cell response toward a tolerogenic state. Thus, establishing immunobiological criteria defining the immunosuppressive effect of hBM-MSCs is of importance to reach efficient immunotherapeutic intervention.


Asunto(s)
Células Madre Mesenquimatosas/inmunología , Linfocitos T/inmunología , Apoptosis , Médula Ósea , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Técnicas de Cocultivo , Humanos , Inmunomodulación , Interleucina-10/inmunología , Interleucina-11/inmunología , Activación de Linfocitos
18.
Data Brief ; 6: 974-9, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26949729

RESUMEN

The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels) and reduced into smaller fat pieces of around 1-3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in "Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent" [1].

19.
J Enzyme Inhib Med Chem ; 31(6): 1079-94, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26482802

RESUMEN

Four series of new bipyrazoles comprising the N-phenylpyrazole scaffold linked to polysubstituted pyrazoles or to antipyrine moiety through different amide linkages were synthesized. The synthesized compounds were evaluated for their anti-inflammatory and analgesic activities. In vitro COX-1/COX-2 inhibition study revealed that compound 16b possessed the lowest IC50 value against both COX-1 and COX-2. Moreover, the effect of the most promising compounds on inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) protein expression in lipopolysaccharide (LPS)-activated rat monocytes was also investigated. The results revealed that some of the synthesized compounds showed anti-inflammatory and/or analgesic activity with less ulcerogenic potential than the reference drug diclofenac sodium and are well tolerated by experimental animals. Moreover, they significantly inhibited iNOS and COX-2 protein expression induced by LPS stimulation. Compounds 16b and 18 were proved to display anti-inflammatory activity superior to diclofenac sodium and analgesic activity equivalent to it with minimal ulcerogenic potential.


Asunto(s)
Amidas/química , Analgésicos/farmacología , Antiinflamatorios/farmacología , Pirazoles/química , Evaluación Preclínica de Medicamentos , Análisis Espectral/métodos
20.
Inflamm Res ; 64(7): 501-12, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25966976

RESUMEN

OBJECTIVE: This study is aimed at evaluating the effects of a cafeteria diet (obesity) mouse model on early multi-organ functional, structural, endocrine and biochemical alterations. MATERIALS AND METHODS: Multi-organ damage is assessed using clinical, biochemical, pathological, and inflammatory parameters in 30 mice fed one of the three diets for 15 weeks: standard chow diet (SC), high fat (HF), or "Cafeteria diet" (CAF) (standard SC and a choice of highly palatable human cafeteria foods: chocolate, biscuits, and peanut butter). RESULTS: CAF diet was associated with an increase in body weight, energy intake, and serum cholesterol levels compared to the other diets, as well as higher insulin levels and lower glucose tolerance. Additionally, consumption of the CAF diet was associated with significantly higher weight gain, abdominal fat, and serum IL-6 levels, as well as more damage in the heart (coronary perivascular fibrosis and steatosis), kidney (chronic interstitial inflammation and glomerular sclerosis), and liver (liver weight, portal fibrosis, apoptosis, and steatosis) compared to the HF diet. CONCLUSION: Functional and structural damage in CAF were higher than HF of similar macronutrient composition. This study provides a novel dietary model in mice that mimics multi-organ physiologic alterations in humans secondary to obesity.


Asunto(s)
Dieta , Inflamación/patología , Obesidad/patología , Grasa Abdominal/efectos de los fármacos , Animales , Composición Corporal/efectos de los fármacos , Colesterol/sangre , Dieta Alta en Grasa/efectos adversos , Sistema Endocrino/patología , Ingestión de Energía/efectos de los fármacos , Preferencias Alimentarias , Intolerancia a la Glucosa , Inflamación/metabolismo , Insulina/sangre , Interleucina-6/metabolismo , Riñón/patología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Miocardio/patología , Obesidad/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Aumento de Peso/efectos de los fármacos
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