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1.
IBJ-Iranian Biomedical Journal. 2017; 21 (1): 24-31
in English | IMEMR | ID: emr-185664

ABSTRACT

Background: Mesenchymal stem cells [MSCs] are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, the study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells [ESCs] and bone marrow cells after hypoxia and normoxia preconditioning


Methods: ESCs differentiated into MSCs and characterized by flow cytometry as well as by differentiation into adipocytes and osteoblasts. The experimental groups were consisted of individual groups of ESC-MSCs and BM-MSCs [bone marrow-derived mesenchymal stromal cells], which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell [PBMC] assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs


Results: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium


Conclusions: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BMMSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor alpha, which needs further investigation


Subject(s)
Humans , Human Embryonic Stem Cells , Culture Media, Conditioned/pharmacology , Cell Hypoxia/physiology , Blood Cells , Leukocytes, Mononuclear/physiology , Interleukin-10/metabolism
2.
Article in English | WPRIM | ID: wpr-61560

ABSTRACT

Obesity is recognized as a chronic low-grade inflammatory state due to adipose tissue expansion being accompanied by an increase in the production of proinflammatory adipokines. Our group is the first to report that B-cell-activating factor (BAFF) is produced from adipocytes and functions as a proinflammatory adipokine. Here, we investigated how loss of BAFF influenced diet-induced obesity in mice by challenging BAFF-/- mice with a high-fat diet for 10 weeks. The results demonstrated that weight gain in BAFF-/- mice was >30% than in control mice, with a specific increase in the fat mass of the subcutaneous region rather than the abdominal region. Expression of lipogenic genes was examined by quantitative real-time PCR, and increased lipogenesis was observed in the subcutaneous adipose tissue (SAT), whereas lipogenesis in the epididymal adipose tissue (EAT) was reduced. A significant decrease in EAT mass resulted in the downregulation of inflammatory gene expression in EAT, and more importantly, overall levels of inflammatory cytokines in the circulation were reduced in obese BAFF-/- mice. We also observed that the macrophages recruited in the enlarged SAT were predominantly M2 macrophages. 3T3-L1 adipocytes were cultured with adipose tissue conditioned media (ATCM), demonstrating that EAT ATCM from BAFF-/- mice contains antilipogenic and anti-inflammatory properties. Taken together, BAFF-/- improved systemic inflammation by redistributing adipose tissue into subcutaneous regions. Understanding the mechanisms by which BAFF regulates obesity in a tissue-specific manner would provide therapeutic opportunities to target obesity-related chronic diseases.


Subject(s)
3T3-L1 Cells , Adipocytes/drug effects , Adiposity/genetics , Animals , B-Cell Activating Factor/genetics , Cells, Cultured , Culture Media, Conditioned/pharmacology , Diet, High-Fat , Disease Models, Animal , Gene Knockout Techniques , Inflammation/genetics , Lipogenesis/genetics , Macrophages/metabolism , Male , Mice , Mice, Knockout , Obesity/etiology
3.
Indian J Exp Biol ; 2014 Nov; 52(11): 1138-1146
Article in English | IMSEAR | ID: sea-153804

ABSTRACT

Realization of hazardious effects of chemical fungicides has led to an interest in the usage of biocontrol agents. The present study, therefore, evaluates the biocontrol efficacy of Western Ghats (India) soil bacterial isolates. A potential strain NII 1006 was evaluated for its antagonistic property against a diverse range of moulds and yeasts. The strain was characterized morphologically, biochemically and molecularly, which revealed the isolate belonged to Streptomyces genus. Organic solvent extracts of NII 1006 culture filtrates inhibited the growth of the test pathogens indicating that growth suppression was due to extracellular anti-fungal metabolites present in the culture filtrates. The strain produced extracellular chitinase enzyme in addition to some stable partially purified anti-fungal compounds. Morphological changes such as hyphae degradation into debris and abnormal shapes were observed in test fungi and yeast grown on potato dextrose broth that contained the NII 1006 culture filtrate. The cell free supernatant has a tolerance to wide range of pH, temperature and enzymes such as lipase and protease. The biocontrol potential of NII 1006 strain may be correlated significantly with their ability to produce antibiotics as well as extracellular hydrolytic enzymes particularly chitinolytic enzyme.


Subject(s)
Acetates , Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Carbon/metabolism , Chitinases/isolation & purification , Chitinases/pharmacology , Chloroform , Culture Media, Conditioned/pharmacology , Drug Evaluation, Preclinical , Fungi/drug effects , Glucans/metabolism , Glycoside Hydrolases/isolation & purification , Glycoside Hydrolases/pharmacology , Hexanes , Hydrogen-Ion Concentration , Hyphae/drug effects , India , Nitrogen/metabolism , Plant Extracts/pharmacology , Plant Proteins/isolation & purification , Plant Proteins/pharmacology , Soil Microbiology , Solvents , Streptomyces/chemistry , Streptomyces/enzymology , Streptomyces/isolation & purification , Yeasts/drug effects
4.
Braz. dent. j ; 25(5): 435-441, Sep-Oct/2014. tab, graf
Article in English | LILACS | ID: lil-731060

ABSTRACT

This study compared the physicochemical properties and interfacial adaptation to canal walls of Endo-CPM-Sealer, Sealapex and Activ GP with the well-established AH Plus sealer. The following analyses were performed: radiopacity, pH variation and solubility using samples of each material and scanning electron microscopy of root-filled bovine incisors to evaluate the interfacial adaptation. Data were analyzed by the parametric and no-parametric tests (α=0.05). All materials were in accordance with the ANSI/ADA requirements for radiopacity. Endo-CPM-Sealer presented the lowest radiopacity values and AH Plus was the most radiopaque sealer (p=0.0001). Except for ActiV GP, which was acidic, all other sealers had basic chemical nature and released hydroxyl ions. Regarding solubility, all materials met the ANSI/ADA recommendations, with no statistically significant difference between the sealers (p=0.0834). AH Plus presented the best adaptation to canal walls in the middle (p=0.0023) and apical (p=0.0012) thirds, while the sealers Activ GP and Endo-CPM-Sealer had poor adaptation to the canal walls. All sealers, except for ActiV GP, were alkaline and all of them fulfilled the ANSI/ADA requirements for radiopacity and solubility. Regarding the interfacial adaptation, AH Plus was superior to the others considering the adaptation to the bovine root canal walls.


Este estudo comparou as propriedades físico-químicas e a adaptação interfacial às paredes do canal dos cimentos Endo-CPM-Sealer, Sealapex e Activ GP com o bem estabelecido cimento AH Plus. As seguintes análises foram realizadas: radiopacidade, variação de pH e de solubilidade utilizando amostras de cada material, e microscopia eletrônica de varredura utilizando incisivos bovinos obturados para avaliar a adaptação interfacial. Os dados foram analisados utilizando testes paramétricos e não-paramétricos (α=0,05). Todos os materiais estavam de acordo com os requerimentos da ANSI/ADA para radiopacidade, sendo que o Endo-CPM-Sealer apresentou os menores valores de radiopacidade e o AH Plus foi o cimento mais radiopaco (p=0,0001). Exceto o Activ GP, que foi ácido, todos os outros cimentos apresentaram natureza química básica e liberaram íons hidroxila. Com relação à solubilidade, todos os materiais estavam de acordo com as recomendações da ANSI /ADA, sem diferença significante entre os cimentos (p=0,0834). O AH Plus apresentou a melhor adaptação às paredes do canal nos terços médio (p=0,0023) e apical (p=0,0012), enquanto que os cimentos Activ GP e Endo-CPM-Sealer apresentaram uma pobre adaptação às paredes do canal. Em conclusão, todos os cimentos, exceto o Activ GP, foram alcalinos e todos preencheram os requerimentos da ANSI/ADA para radiopacidade e solubilidade. Com relação à adaptação interfacial, o AH Plus foi superior aos demais para adaptação às paredes do canal radicular de incisivos bovinos.


Subject(s)
Animals , Female , Humans , Mice , Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Dextrans/pharmacology , Growth Inhibitors/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/pathology , Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents/therapeutic use , Culture Media, Conditioned/pharmacology , Dose-Response Relationship, Drug , Dextrans/chemistry , Dextrans/therapeutic use , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Growth Inhibitors/therapeutic use , Mice, Inbred BALB C , Mice, Nude , Necrosis , Phenylacetates/pharmacology , Phenylacetates/therapeutic use , Xenograft Model Antitumor Assays/statistics & numerical data
5.
Article in English | WPRIM | ID: wpr-36639

ABSTRACT

Mesenchymal stem cells (MSCs) have been widely studied for their applications in stem cell-based regeneration. During myocardial infarction (MI), infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. We hypothesized that MSCs may modulate the immunologic environment to accelerate regeneration. This study was designed to assess the functional relationship between the macrophage phenotype and MSCs. MSCs isolated from bone marrow and bone marrow-derived macrophages (BMDMs) underwent differentiation induced by macrophage colony-stimulating factor. To determine the macrophage phenotype, classical M1 markers and alternative M2 markers were analyzed with or without co-culturing with MSCs in a transwell system. For animal studies, MI was induced by the ligation of the rat coronary artery. MSCs were injected within the infarct myocardium, and we analyzed the phenotype of the infiltrated macrophages by immunostaining. In the MSC-injected myocardium, the macrophages adjacent to the MSCs showed strong expression of arginase-1 (Arg1), an M2 marker. In BMDMs co-cultured with MSCs, the M1 markers such as interleukin-6 (IL-6), IL-1beta, monocyte chemoattractant protein-1 and inducible nitric oxide synthase (iNOS) were significantly reduced. In contrast, the M2 markers such as IL-10, IL-4, CD206 and Arg1 were markedly increased by co-culturing with MSCs. Specifically, the ratio of iNOS to Arg1 in BMDMs was notably downregulated by co-culturing with MSCs. These results suggest that the preferential shift of the macrophage phenotype from M1 to M2 may be related to the immune-modulating characteristics of MSCs that contribute to cardiac repair.


Subject(s)
Animals , Biomarkers/metabolism , Cell Differentiation , Cells, Cultured , Coculture Techniques , Culture Media, Conditioned/pharmacology , Humans , Macrophage Activation , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/drug effects , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Myocardial Infarction/surgery , Rats
6.
Article in English | WPRIM | ID: wpr-17803

ABSTRACT

Chronic neuroinflammation is an integral pathological feature of major neurodegenerative diseases. The recruitment of microglia to affected brain regions and the activation of these cells are the major events leading to disease-associated neuroinflammation. In a previous study, we showed that neuron-released alpha-synuclein can activate microglia through activating the Toll-like receptor 2 (TLR2) pathway, resulting in proinflammatory responses. However, it is not clear whether other signaling pathways are involved in the migration and activation of microglia in response to neuron-released alpha-synuclein. In the current study, we demonstrated that TLR2 activation is not sufficient for all of the changes manifested by microglia in response to neuron-released alpha-synuclein. Specifically, the migration of and morphological changes in microglia, triggered by neuron-released alpha-synuclein, did not require the activation of TLR2, whereas increased proliferation and production of cytokines were strictly under the control of TLR2. Construction of a hypothetical signaling network using computational tools and experimental validation with various peptide inhibitors showed that beta1-integrin was necessary for both the morphological changes and the migration. However, neither proliferation nor cytokine production by microglia was dependent on the activation of beta1-integrin. These results suggest that beta1-integrin signaling is specifically responsible for the recruitment of microglia to the disease-affected brain regions, where neurons most likely release relatively high levels of alpha-synuclein.


Subject(s)
Animals , Integrin beta1/genetics , Cell Line, Tumor , Cell Movement , Cells, Cultured , Culture Media, Conditioned/pharmacology , Gene Regulatory Networks , Humans , Mice , Mice, Inbred C57BL , Microglia/drug effects , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction , Toll-Like Receptor 2/metabolism , alpha-Synuclein/pharmacology
7.
Article in English | WPRIM | ID: wpr-219417

ABSTRACT

This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits.


Subject(s)
Alkalies/toxicity , Amnion/cytology , Animals , Cornea/injuries , Corneal Diseases/chemically induced , Culture Media, Conditioned/pharmacology , Epithelial Cells/physiology , Humans , Male , Rabbits
8.
Article in English | WPRIM | ID: wpr-119450

ABSTRACT

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.


Subject(s)
Activating Transcription Factor 2/metabolism , Animals , Cell Separation , Chemokines/biosynthesis , Chemotaxis/drug effects , Culture Media, Conditioned/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , GTP-Binding Proteins/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Mice , NF-kappa B/metabolism , Neutrophils/cytology , Pertussis Toxin/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Wnt/metabolism , Type C Phospholipases/metabolism , Wnt Proteins/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism
9.
Braz. j. med. biol. res ; 42(6): 506-514, June 2009. ilus, tab, graf
Article in English | LILACS | ID: lil-512771

ABSTRACT

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.


Subject(s)
Animals , Male , Mice , Calcium/metabolism , Culture Media, Conditioned/pharmacology , Cytochalasin B/pharmacology , Mesenchymal Stem Cells , Oocytes/drug effects , Parthenogenesis/drug effects , Microscopy, Confocal , Oocytes/physiology , Parthenogenesis/physiology
10.
Article in English | WPRIM | ID: wpr-98677

ABSTRACT

Aldosterone has been shown to stimulate renal TGF-beta1 expression. However, the mechanisms for aldosterone-induced TGF-beta1 expression have not been clearly determined in mesangial cells. We examined the role of extracellular-signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and activator protein- 1 (AP-1) in the aldosterone-induced TGF-beta1 expression in rat mesangial cells. TGF-beta1 protein in the conditioned medium released from rat mesangial cells was measured by sandwich ELISA, TGF-beta1 mRNA expression was analyzed by Northern blotting, AP-1 DNA binding activity was measured by EMSA and the ERK1/2, JNK activity was analyzed by western blotting. Aldosterone significantly stimulated TGF-beta1 protein production and TGF-beta1 mRNA expression in mesangial cells in a dose-dependent manner. Aldosterone significantly increased AP-1 DNA binding activity in mesangial cells. Pre-treatment of cells with AP-1 inhibitor, curcumin, blocked aldosterone-induced AP-1 DNA binding activity as well as aldosterone-induced TGF-beta1 production. Aldosterone increased phosphorylation of ERK1/2 and JNK in mesangial cells. Pre-treatment of cells with ERK1/2 inhibitor, PD98059, or JNK inhibitor, SP600125 significantly inhibited aldosterone-induced ERK1/2 and JNK activity and subsequently TGF-beta1 production, respectively. We conclude that aldosteroneinduced TGF-beta1 expression in mesangial cells is regulated by the ERK1/ 2, JNK and AP-1 intracellular signaling pathways.


Subject(s)
Aldosterone/pharmacology , Animals , Culture Media, Conditioned/pharmacology , DNA/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Enzymologic , Humans , MAP Kinase Signaling System , Mesangial Cells/metabolism , Models, Biological , Phosphorylation , Protein Binding , Rats , Transcription Factor AP-1/metabolism , Transforming Growth Factor beta1/biosynthesis
11.
Braz. j. med. biol. res ; 41(11): 978-985, Nov. 2008. ilus, graf
Article in English | LILACS | ID: lil-500368

ABSTRACT

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), alpha-minimum essential medium (alpha-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), alpha-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with alpha-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.


Subject(s)
Animals , Female , Mice , Pregnancy , Culture Media, Conditioned/pharmacology , Mesenchymal Stem Cells , Oocytes/growth & development , Cumulus Cells/cytology , Embryo, Mammalian/embryology , Embryonic Development/physiology , Fertilization in Vitro , Meiosis/physiology , Ovarian Follicle/growth & development
12.
Biocell ; 31(2): 237-245, ago. 2007. graf
Article in English | LILACS | ID: lil-491530

ABSTRACT

Interaction between parenchyma and stroma is essential for organogenesis, morphogenesis, and differentiation. Mammary gland has being the chosen model for developmental biologist because the most striking changes in morphology and function take place after birth. We have demonstrated a regulation of triglyceride accumulation by protein factors synthesized by normal mouse mammary gland epithelial cells (NMMG), acting on a cell line, 3T3-L1, long used as a model for adipogenesis. In this paper, we demonstrate that this inhibitory effect seems to be shared by other cells of epithelial origin but not by other cell types. We found a regulation of cell proliferation when NMMG cells are cultured in the presence of conditioned media from Swiss 3T3 or 3T3-L1 cells. We found a possible point of regulation for the mammary factor on a key enzyme of the lipid metabolic pathway, the glycerol-3-phosphate dehydrogenase. The inhibitory factor seems to have an effect on this enzyme's activity and reduces it. The results presented herein contribute to the understanding of cell-cell communication in a model of a normal mammary gland.


Subject(s)
Humans , Animals , Rats , Adipocytes, White/cytology , Adipocytes, White/metabolism , Epithelial Cells/cytology , Mammary Glands, Animal/cytology , Culture Media, Conditioned/pharmacology , Triglycerides/metabolism , Cell Differentiation , Cells, Cultured , Cell Communication/physiology , HeLa Cells , Cell Proliferation
13.
J Vector Borne Dis ; 2006 Sep; 43(3): 95-103
Article in English | IMSEAR | ID: sea-117874

ABSTRACT

BACKGROUND & OBJECTIVES: Plasmodium falciparum, the causative agent of the most serious form of malaria, infects about 5-10% of the world human population per year. It is well established that the erythrocytic stages of the malaria parasite rely mainly on glycolysis for their energy supply. In the present study, the glucose utilisation of erythrocyte population with parasitaemia levels similar to that of malaria patients was measured. The results allowed us to assess the effect of the parasites on the glucose utilisation of the vast majority of uninfected erythrocytes. METHODS: Using [2-13C]glucose and nuclear magnetic resonance (NMR) technique, the glucose utilisation in normal red blood cell (RBC) and P. falciparum infected red blood cell (IRBC) populations was measured. The IRBC population consisted of > 96% RBC and < 4% of parasite infected red blood cells (PRBC). The glycolytic enzymes were assayed to assess the effect of infected red cells on the enzymatic activities of uninfected ones. RESULTS: The rate of glucose utilisation by IRBC was considerably higher than that of RBC. Upon addition of 25% v/v conditioned culture medium (CM) of IRBC, RBCs exhibited a significant decrease in glucose utilisation. The CM could directly inhibit the activities of RBC glycolytic enzymes-phosphofructokinase (PFK) and pyruvate kinase (PK), without interfering with the activity of the pentose phosphate pathway enzyme-glucose-6-phosphate dehydrogenase (G-6-PD). INTERPRETATION & CONCLUSION: The present study showed that the clinical level of P. falciparum infected RBCs (< 4% parasitaemia) significantly enhance the glycolytic flux as well as down-regulate the glucose utilisation rate in the majority of uninfected RBC population. The mechanism of inhibition seems to be direct inhibition of the regulatory glycolytic enzymes-PFK and PK.


Subject(s)
Adult , Animals , Coculture Techniques , Culture Media, Conditioned/pharmacology , Down-Regulation , Erythrocytes/metabolism , Glucose/metabolism , Glucosephosphate Dehydrogenase/metabolism , Glycolysis , Host-Parasite Interactions , Humans , Male , Mice , Mice, Inbred BALB C , Phosphofructokinases/antagonists & inhibitors , Plasmodium falciparum/physiology , Pyruvate Kinase/antagonists & inhibitors , Time Factors
14.
Indian J Exp Biol ; 2006 Mar; 44(3): 189-92
Article in English | IMSEAR | ID: sea-55719

ABSTRACT

Although numerous investigations have demonstrated the beneficial effects of co-culture system of different somatic cells on in vitro development of embryos, the effects of conditioned-media of co-culture cells have not been well documented. The objective of this study was to compare the effects of human granulosa cells co-culture system and its conditioned medium on the developmental rate of mouse embryos in vitro. Two sets of experiments were undertaken: in the first one 317 mouse one-cell embryos were cultured in human granulosa cell co-culture system (GC). Ham's F10 medium conditioned with granulosa cells (CM) and non-conditioned Ham's F10 for 120 h. In the second experiment. 391 late two-cell embryos were cultured in the 3 fore-mentioned culture treatments for 72 h. Embryos were obtained from NMRI mice. Granulosa cells were collected from patients undergoing an IVF program during oocyte pickup. In the first set of experiments, 23.6, 14.5 and 11.1% of one-cell embryos passed two-cell block and continued growing to 4-cell in GC, CM and HF, respectively. This index in GC was significantly different from two other treatments. Also significantly more embryos reached blastocyst stage in GC compared with two other treatments. The blastocyst rate was not significantly different between CM and HF. In the second set of experiments the proportion of blastocyst stage was significantly higher in CM than that in HF and lower than that in GC. In conclusion, although human granulosa cell-conditioned medium has beneficial effects on mouse embryo development, it was not as effective as co-culture of these cells.


Subject(s)
Animals , Cell Proliferation/drug effects , Coculture Techniques/methods , Culture Media, Conditioned/pharmacology , Embryo, Mammalian/cytology , Female , Granulosa Cells/cytology , Humans , Ice , Mice
15.
Article in English | WPRIM | ID: wpr-53154

ABSTRACT

Heat shock protein 70 (Hsp70) release and its effects on pro-inflammatory cytokine production have been controversial. In this study, we investigated whether Hsp70 could be released from monocytes and activates matrix metalloproteinase-9 (MMP-9) gene expression. Hsp70 overexpression in human monocytic cell line U937 was found to increase PMA- induced MMP-9 expression and enhance cell motility. Hsp70 cDNA transfectants released Hsp70 protein into culture supernatants, and a part of released Hsp70 subsequently was bound to the surface of U937 cells. Addition of culture medium containing the extracelluar Hsp70 led to an increase not only in proMMP-9 secretion, but also the invasiveness of U937 cells through Matrigel or human umbilical vascular endothelial cells (HUVEC) in vitro. Immunodepletion of Hsp70 abolished its effect on MMP-9 expression. The released Hsp70 activated nuclear factor kappa B (NF-kappa B) and activating protein-1 (AP-1), which led to the activation of MMP-9 transcription. Taken together, these results suggest that extracellular Hsp70 induces the expression of MMP-9 gene through activation of NF-kappa B and AP-1.


Subject(s)
U937 Cells , Transfection , Transcription Factor AP-1/metabolism , NF-kappa B/metabolism , Matrix Metalloproteinase 9/metabolism , Humans , HSP70 Heat-Shock Proteins/metabolism , Gene Expression Regulation , Culture Media, Conditioned/pharmacology , Cell Movement/drug effects
16.
Article in English | WPRIM | ID: wpr-201940

ABSTRACT

Autocrine stimulation via coexpression of hepatocyte growth factor (HGF) and its receptor (Met) has been reported in many human sarcomas, but few in carcinomas. In this report, we found that one gastric cancer cell line, SNU-484, among 11 gastric cell lines tested has an autocrine HGF- Met stimulation. RT-PCR, ELISA and scattering assay using MDCK cells revealed that SNU-484 cells secreted a significant amount of active HGF (about 1.25 +/- 0.41 ng/24 h/106 cells) into conditioned medium. Resultantly, Met in this cell line was constitutively phosphorylated. Neutralizing antibodies against HGF reduced the tyrosine phosphorylation of Met, resulting in the inhibition of cell proliferation and migration (P <0.005). To the best of our knowledge, this is the first report on autocrine HGF-Met signaling in a gastric cancer cell line. Our observations with SNU-484 cells suggest that HGF is involved in the development and/or progression of some gastric carcinoma through an autocrine mechanism.


Subject(s)
Animals , Antibodies, Neoplasm/immunology , Autocrine Communication , Cell Movement , Cell Proliferation , Culture Media, Conditioned/pharmacology , Dogs , Enzyme-Linked Immunosorbent Assay , Hepatocyte Growth Factor/immunology , Neutralization Tests , Phosphorylation , Proto-Oncogene Proteins c-met/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stomach Neoplasms/immunology , Tumor Cells, Cultured , Tyrosine/metabolism
17.
Article in English | WPRIM | ID: wpr-156006

ABSTRACT

Bone destruction is primarily mediated by osteoclastic bone resorption, and cancer cells stimulate the formation and activation of osteoclasts next to metastatic foci. Accumulating evidences indicate that receptor activator of NF-kB ligand (RANKL) is the ultimate extracellular mediator that stimulates osteoclast differentiation into mature osteoclasts. In contrast, osteoprotegerin (OPG) inhibits osteoclast development. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MDA-MB-231, were directly co-cultured with ST2, MC3T3-E1, or with primary mouse calvarial cells. Osteoclast-like cells and tartarate resistant acid phosphatase (TRAP) activities were then quantitated. We examined these cell lines and samples from breast cancer by RT-PCR for the expressions of OPG and RANKL mRNA. Compared to controls, co-culture of MDA-MB-231 cells with stromal or osteoblastic cells induced an increase in number of osteoclasts and TRAP activities. MDA-MB-231 cells alone or breast cancer samples did not express RANKL mRNA. However, co-culture of these cancer cells with stromal or osteoblastic cells induced RANKL mRNA expression and decreased OPG mRNA expression. These experiments demonstrate that direct interactions between breast cancer and stromal or osteoblastic cells induce osteoclastogenesis in vitro through modulating RANKL expression.


Subject(s)
3T3 Cells , Acid Phosphatase/metabolism , Animals , Bone Neoplasms/metabolism , Breast Neoplasms/pathology , Carrier Proteins/biosynthesis , Cell Differentiation , Cell Line, Tumor , Cells, Cultured , Coculture Techniques , Culture Media, Conditioned/pharmacology , Glycoproteins/biosynthesis , Humans , Isoenzymes/metabolism , Male , Membrane Glycoproteins/biosynthesis , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , Osteoblasts/metabolism , Osteoclasts/metabolism , Protein Binding , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
18.
Article in English | WPRIM | ID: wpr-221856

ABSTRACT

This study investigated the production of stromal cell-derived factor-1 (SDF-1) and the expression of CXCR4 in human bone marrow endothelial cells (BMECs). Human BMEC cell line BMEC-1 cells expressed SDF-1 mRNA, and conditioned medium induced chemoattraction of CD34+ cells. Migration was not inhibited by pretreating the input cells with pertussis toxin, indicating that the chemoattractive activity was not dependent on SDF-1. Three-day culture of BMEC-1 and primary human BMEC cells produced 1,710+/-204 and 1,050+/-153 pg/mL SDF-1alpha, respectively, which was much less than primary human BM stromal cells (29,536+/-532 pg/ mL). By immuno-histochemistry, CXCR4 was detected in the endothelial cells lining sinusoids, arterioles, and venules in the bone marrow. However, cultured BMECs and BMEC-1 cells did not express CXCR4 on their surfaces. These results indicate that BMECs produce and release small amounts of SDF-1 and express CXCR4 in vivo only.


Subject(s)
Antigens, CD34/biosynthesis , Bone Marrow Cells/metabolism , Cell Movement , Cells, Cultured , Chemokines, CXC/biosynthesis , Chemotaxis , Culture Media, Conditioned/pharmacology , Endothelial Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Hematopoietic Stem Cells/metabolism , Humans , Immunohistochemistry , Pertussis Toxin/pharmacology , RNA, Messenger/metabolism , Receptors, CXCR4/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Umbilical Veins/cytology
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