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1.
Sci Total Environ ; 659: 1307-1321, 2019 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-31096342

RESUMEN

Tritium dating requires a good understanding of the tritium and water inputs into hydrologic systems, including their main trends due to latitudinal, seasonal and altitudinal effects. Although tritium reached ambient levels at the end of the 20th century, tritium released from nuclear facilities and bomb tests since then has the potential to confound use of tritium for age dating. We therefore collected precipitation and snowpack samples for tritium analysis to confirm that tritium levels in Japanese precipitation had not exceeded ambient levels following the North Korean nuclear tests in January 6th 2016 and September 3rd 2017. As the result, the highest tritium concentration was 5.52(±0.27)TU at samples collected from January 8 to 11th at one Honshu and four Hokkaido locations and samples collected at six Honshu locations had 8.01(±1.5)TU from September 6 to 19th 2017. Confirming ambient tritium concentrations after both events we investigated the latitude tritium effect at selected coastal stations in Asia, indicating a break of latitude trend around Tokyo area, and established the latitude scaling factors to the north and south of the Tokyo area data. The seasonal trend was investigated during the winter-spring 2016 in precipitation samples confirming the higher spring tritium compared with winter continental tritium values. The altitude effect on tritium and stable (18O and 2H) isotopes was observed in Hokkaido snowpack, which had tritium concentrations ranging between 4.08 and 5.93 TU during March-April, and demonstrated two trends for western and central Hokkaido mountain ranges. Using established latitude and altitude scaling factors with the long-term continuous time-series of monthly Tokyo area tritium we estimated the annual weighted tritium at 110 meteorological stations in Japan with monthly precipitation demonstrating the applicability of this approach for future tritium-tracer studies across Asia.

2.
J Periodontal Res ; 53(5): 870-882, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29900548

RESUMEN

OBJECTIVE AND BACKGROUND: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. MATERIAL AND METHODS: Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids. RESULTS: The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate "stemness" were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation. CONCLUSION: Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Osteogénesis/fisiología , Ligamento Periodontal/citología , Esferoides Celulares , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Células Cultivadas , Medios de Cultivo , Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Osteogénesis/genética , Ligamento Periodontal/metabolismo , Transducción de Señal/fisiología
3.
J Dairy Sci ; 101(7): 5789-5798, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29680655

RESUMEN

The activation of phagocytosis is one important approach to clearing pathogenic cells in a host. This study evaluated the ability of probiotic lactobacilli to induce phagocytic activity as well as the clearance of a periodontal pathogen, Aggregatibacter actinomycetemcomitans. First, the activation of phagocytosis was found by using lyophilized dead cells. Probiotic Lactobacillus strains significantly enhanced the phagocytic activity of macrophage cells, indicating that the probiotic lactobacilli have a remarkable ability to stimulate the macrophages. Essentially, 3 Lactobacillus strains tested did not have any critical toxic effect on the murine macrophage, and Lactobacillus johnsonii NBRC 13952 showed the least cytotoxic effect on the RAW264.7 macrophages. The expression of classically activated macrophage markers, IL-1ß, and cluster of differentiation 80 increased by L. johnsonii NBRC 13952; however, there was no significant difference for IL-18. The highest phagocytic activity by macrophages was found in a condition in which the macrophage activated by L. johnsonii NBRC 13952 functions to kill the cells of A. actinomycetemcomitans. Correlating with the result, a high amount of hypodiploid DNA (SubG1) was detected from the macrophage cells stimulated by L. johnsonii NBRC 13952. Taken together, the results suggest that macrophages activated by the Lactobacillus strain can facilitate the phagocytosis of A. actinomycetemcomitans cells by linking with enhanced apoptotic activities. In conclusion, L. johnsonii NBRC 13952 has a certain role in activating the RAW264.7 macrophages, thereby counteracting the infection of A. actinomycetemcomitans.


Asunto(s)
Aggregatibacter actinomycetemcomitans/metabolismo , Lactobacillus/fisiología , Fagocitosis , Probióticos , Animales , Macrófagos , Ratones
4.
Int Endod J ; 49(3): 271-8, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25702817

RESUMEN

AIM: To investigate the effects of PRP on odontoblastic differentiation using dental pulp progenitor cells derived from the dental papilla of rat incisors. METHODOLOGY: Monolayer cultures of odontoblastic lineage KN-3 cells were incubated with PRP for various time periods. The expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1) was determined using real-time reverse transcription-polymerase chain reaction and Western blot analyses. To further clarify the role of PRP in odontogenesis, KN-3 cells were stimulated with PRP in the presence of ascorbic acid and ß-glycerophosphate. The cells were stained for alkaline phosphatase (ALP), and ALP activity was quantified in cell lysates. The formation of mineralized nodules was assessed by alizarin red staining. Statistical analysis was performed by one-way analysis of variance. RESULTS: PRP increased the mRNA and protein expressions of odontoblastic markers, such as DSPP and DMP-1. Furthermore, PRP stimulated the ALP activity and mineralized nodule formation induced by ascorbic acid and ß-glycerophosphate in a time-dependent manner. CONCLUSIONS: PRP enhances odontoblastic differentiation of KN-3 cells. These results indicate that PRP could be a potential candidate for use in the regeneration of dentine-pulp complex.


Asunto(s)
Pulpa Dental/citología , Odontoblastos/efectos de los fármacos , Plasma Rico en Plaquetas , Fosfatasa Alcalina/metabolismo , Animales , Ácido Ascórbico/farmacología , Western Blotting , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/metabolismo , Glicerofosfatos/metabolismo , Humanos , Fosfoproteínas/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Sialoglicoproteínas/metabolismo
5.
Oral Dis ; 21(8): 969-76, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26332604

RESUMEN

OBJECTIVE: To develop a model of osteonecrosis using oral bisphosphonate in ovariectomy-induced osteoporotic rats. MATERIALS AND METHODS: Thirty-six rats were subjected to ovariectomy or sham surgery. After 8 weeks, rats received oral alendronate (1.0 mg kg(-1) ) or saline once weekly for 4 weeks; then, serum C-telopeptide cross-linked collagen type I levels were measured to evaluate bone metabolism. Twelve rats from each group were injected with either lipopolysaccharide or saline into the bone marrow of the mandibles and femurs, and the areas of osteonecrosis were evaluated by histomorphometry. RESULTS: Serum C-telopeptide cross-linked collagen type I levels were significantly increased in the ovariectomy group (105.1 ± 2.1 ng ml(-1) ) compared with the sham group (78.9 ± 12.5 ng ml(-1) ); they were significantly reduced following oral alendronate administration in the ovariectomy group (91.0 ± 4.4 ng ml(-1) ). Following alendronate and lipopolysaccharide administration, extensive osteonecrosis was observed in the mandibles and femurs of ovariectomy (0.45 ± 0.08 mm(2) , 1.69 ± 0.72 mm(2) , respectively) and sham (1.12 ± 0.45 mm(2) , 1.84 ± 0.66 mm(2) , respectively) groups. Significantly wider osteonecrosis occurred in the mandibles of sham-operated rats than ovariectomy rats following alendronate or lipopolysaccharide treatment. CONCLUSIONS: We successfully developed a model of osteonecrosis in ovariectomised rats following oral bisphosphonate administration.


Asunto(s)
Alendronato/efectos adversos , Conservadores de la Densidad Ósea/efectos adversos , Modelos Animales de Enfermedad , Osteonecrosis/inducido químicamente , Administración Oral , Alendronato/administración & dosificación , Animales , Conservadores de la Densidad Ósea/administración & dosificación , Femenino , Inyecciones , Lipopolisacáridos/administración & dosificación , Mandíbula , Ovariectomía , Ratas , Ratas Wistar
6.
Int J Oral Maxillofac Surg ; 44(4): 528-34, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25442743

RESUMEN

The pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ) is highly controversial. We have previously reported the development of osteonecrosis by periodontal pathogenic stimulation in the jaw and femur of rats treated with bisphosphonate. Since the major toxicity factor of Gram-negative bacteria is lipopolysaccharide (LPS), the present study aimed to evaluate the relationship between osteonecrosis and LPS in a rat model of BRON-like lesions. Seventeen male rats were injected subcutaneously with zoledronic acid weekly for 4 weeks and divided into three groups: LPS (LPS administered into the bone marrow of the mandible and femur) and LPS plus polymyxin B (PMB) and saline groups (given neutralized LPS with PMB or saline, respectively, using the same protocol). At 4 weeks after the procedure, harvested specimens were analyzed using histomorphology (n=5 from each group) and histochemistry (n=1 each from LPS and LPS plus PMB groups). There was a significantly wider area of osteonecrosis in the LPS group as compared to the saline and LPS plus PMB groups in both the mandible (P=0.030 and P=0.009, respectively) and femur (P=0.002 and P=0.020, respectively). Our results indicate that LPS stimulation is deeply involved in the development and promotion of BRON.


Asunto(s)
Osteonecrosis de los Maxilares Asociada a Difosfonatos/etiología , Difosfonatos/toxicidad , Imidazoles/toxicidad , Lipopolisacáridos/toxicidad , Animales , Masculino , Ratas , Ratas Wistar , Ácido Zoledrónico
7.
Infect Immun ; 82(5): 1904-13, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24566623

RESUMEN

The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.


Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Enfermedades Óseas Metabólicas/etiología , Resorción Ósea/etiología , Inflamación/etiología , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Infecciones por Pasteurellaceae/complicaciones , Periodontitis/complicaciones , Animales , Regulación de la Expresión Génica , Proteína Antagonista del Receptor de Interleucina 1/genética , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Ratones Noqueados , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Infecciones por Pasteurellaceae/microbiología , Periodontitis/microbiología , Ligando RANK/genética , Ligando RANK/metabolismo
8.
Theriogenology ; 81(5): 683-8, 2014 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-24411494

RESUMEN

The ATP-binding cassette subfamily B member 1 (ABCB1) is an efflux transporter that excretes xenobiotics and waste matter. High expression of ABCB1 induced by forskolin (FSK) and rifampicin (RIF) in the bovine blastocysts reportedly improves the cellular quality. In the present study, interferon-α, similar to FSK and RIF, was highly potent in inducing the expression of ABCB1 in the bovine blastocysts but did not exhibit an additive effect with FSK and RIF. Bovine blastocysts stimulated by the combined treatment with FSK, RIF, and interferon-α to express high levels of ABCB1 displayed better freezing resistance as indicated by higher cell numbers in post thawing cultures. On transfer to recipients, such embryos established pregnancies with significantly higher frequencies in repeat breeder cows rather than normal ones.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Bovinos/embriología , Implantación del Embrión/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Blastocisto/química , Blastocisto/citología , Blastocisto/fisiología , Recuento de Células , Colforsina/farmacología , Criopreservación/veterinaria , Interacciones Farmacológicas , Femenino , Expresión Génica/efectos de los fármacos , Interferón-alfa/farmacología , Embarazo , Rifampin/farmacología
9.
Osteoarthritis Cartilage ; 22(1): 111-20, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24185105

RESUMEN

OBJECTIVE: To determine the effects of high molecular weight hyaluronic acid (HMW-HA) on osteoclast differentiation by monocytes co-cultured with stromal cells. METHODS: Mouse bone marrow stromal cell line ST2 cells were incubated with HMW-HA or 4-methylunbeliferone (4-MU) for various times. In some experiments, cells were pre-treated with the anti-CD44 monoclonal antibody (CD44 mAb) or Rho kinase pathway inhibitors (simvastatin or Y27632), then treated with HMW-HA. The expression of receptor activator of NF-κB ligand (RANKL) was determined using real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence microscopy, while the amount of active RhoA was measured by a pull-down assay. To further clarify the role of HMW-HA in osteoclastogenesis, mouse monocyte RAW 264.7 cells were co-cultured with ST2 cells pre-stimulated with 1,25(OH)2D3. Osteoclast-like cells were detected by staining with tartrate-resistant acid phosphatase (TRAP). RESULTS: HMW-HA decreased RANKL mRNA and protein expressions, whereas inhibition of hyaluronic acid (HA) synthesis by 4-MU enhanced RANKL expression. Blockage of HA-CD44 binding by CD44 mAb suppressed HMW-HA-mediated inhibition of RANKL. Pull-down assay findings also revealed that HMW-HA transiently activated RhoA in ST2 cells and pre-treatment with CD44 mAb inhibited the activation of RhoA protein mediated by HMW-HA. Moreover pre-treatment with Rho kinase pathway inhibitors also blocked the inhibition of RANKL by HMW-HA. Co-culture system results showed that HMW-HA down-regulated differentiation into osteoclast-like cells by RAW 264.7 cells induced by 1,25(OH)2D3-stimulated ST2 cells. CONCLUSIONS: These results indicated that HA-CD44 interactions down-regulate RANKL expression and osteoclastogenesis via activation of the Rho kinase pathway.


Asunto(s)
Ácido Hialurónico/farmacología , Osteoclastos/efectos de los fármacos , Ligando RANK/antagonistas & inhibidores , Quinasas Asociadas a rho/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/administración & dosificación , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Ratones , Peso Molecular , Monocitos/citología , Monocitos/efectos de los fármacos , Osteoclastos/citología , Ligando RANK/genética , Ligando RANK/metabolismo , Ligando RANK/fisiología , ARN Mensajero/genética , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo
10.
J Periodontal Res ; 48(6): 748-56, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23586622

RESUMEN

BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.


Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Citocinas/efectos de los fármacos , Dinoprostona/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Lipopolisacáridos/farmacología , Osteoclastos/efectos de los fármacos , Regulación hacia Arriba , Fosfatasa Ácida/análisis , Animales , Células de la Médula Ósea/efectos de los fármacos , Resorción Ósea/inmunología , Técnicas de Cultivo de Célula , Ciclooxigenasa 2/efectos de los fármacos , Células Gigantes/efectos de los fármacos , Proteína Antagonista del Receptor de Interleucina 1/genética , Interleucina-1alfa/análisis , Interleucina-1beta/efectos de los fármacos , Interleucina-6/análisis , Isoenzimas/análisis , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones Noqueados , Receptor Activador del Factor Nuclear kappa-B/efectos de los fármacos , Subtipo EP4 de Receptores de Prostaglandina E/efectos de los fármacos , Cráneo/inmunología , Fosfatasa Ácida Tartratorresistente , Factor de Necrosis Tumoral alfa/efectos de los fármacos
11.
J Dent Res ; 92(3): 241-6, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23318766

RESUMEN

The objective of this study was to examine whether native low-density lipoprotein (LDL) induces foam cell formation by macrophages and to examine the effect of lipopolysaccharide (LPS) on native LDL-induced foam cell formation by macrophages in vitro. RAW 264.7 cells were cultured with LDL or high-density lipoprotein (HDL) in the presence of LPS derived from Aggregatibacter actinomycetemcomitans. Foam cell formation was determined by staining with Oil-red-O to visualize cytoplasmic lipid droplet accumulation. The expression of LDL-receptor and the degree of internalization of FITC-conjugated LDL in RAW 264.7 cells were examined by immunofluorescence microscopy. The images were digitally recorded and analyzed with Image J software. Statistical analysis was performed by JMP software. Foam cell formation was induced by the addition of native LDL in dose- and time-dependent manners, whereas HDL showed no effect. LPS enhanced the foam cell formation induced by native LDL. In addition, LPS stimulated the expression of LDL-receptor protein on RAW 264.7 cells and enhanced the internalization of LDL. The enhancement of foam cell formation induced by LPS and LDL was inhibited by the depolymerizing agent nocodazole and amiloride analog 5-(N-ethyl-N-isoprophyl) amiloride (EIPA). Our findings indicate that LPS plays an important role in foam cell formation by LDL-stimulated macrophages.


Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Células Espumosas/metabolismo , Lipoproteínas LDL/farmacología , Pinocitosis/efectos de los fármacos , Receptores de LDL/biosíntesis , Moduladores de Tubulina/farmacología , Bloqueadores del Canal Iónico Sensible al Ácido/farmacología , Aggregatibacter actinomycetemcomitans/química , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Línea Celular , Sinergismo Farmacológico , Células Espumosas/citología , Células Espumosas/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Lipoproteínas LDL/fisiología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Microtúbulos/efectos de los fármacos , Nocodazol/farmacología , Periodontitis/microbiología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores
12.
J Periodontal Res ; 45(4): 550-6, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20412415

RESUMEN

BACKGROUND AND OBJECTIVE: Inflammatory agents, such as lipopolysaccharide (LPS), in periodontal pockets may promote atherogenesis by activating leukocytes. In our previous study, we developed a microchannel chip to observe the cell adhesion process in a fluid system. The objective of this investigation was to examine the mechanism by which periodontopathic bacterial LPS enhances plaque-like formation on a microchannel chip. MATERIAL AND METHODS: To evaluate the effect of Aggregatibacter actinomycetemcomitans LPS on the expression of adhesion molecules, e.g. intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen 1 (LFA-1) and L-selectin, on the surface of murine macrophage RAW264.7 cells, the expression of each adhesion molecule was examined by flow cytometry and western blot analysis. Moreover, a flow test on the microchannel chip involving anti-adhesion molecule antibodies was conducted to clarify which adhesion molecule is related to plaque-like formation of RAW264.7 cells. RESULTS: The expressions of ICAM-1 and LFA-1 on the surface of RAW 264.7 cells increased following 12 h culture with LPS; L-selectin expression was unaffected. An increase in ICAM-1 expression was also confirmed by western blot analysis. The flow test revealed that anti-ICAM-1 antibody inhibited plaque-like formation of LPS-stimulated macrophages on the micropillars of the microchannel chip. CONCLUSION: These findings indicate that ICAM-1 plays an important role in plaque-like formation of LPS-stimulated macrophages. Our microchannel chip is a suitable tool for the investigation of etiological factors of atherosclerosis, including periodontitis, in vitro.


Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Selectina L/efectos de los fármacos , Lipopolisacáridos/farmacología , Antígeno-1 Asociado a Función de Linfocito/efectos de los fármacos , Macrófagos/efectos de los fármacos , Animales , Anticuerpos , Aterosclerosis/patología , Western Blotting , Adhesión Celular/efectos de los fármacos , Agregación Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Citometría de Flujo , Molécula 1 de Adhesión Intercelular/análisis , Selectina L/análisis , Dispositivos Laboratorio en un Chip , Antígeno-1 Asociado a Función de Linfocito/análisis , Ratones
13.
Oral Dis ; 16(3): 305-9, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20374514

RESUMEN

OBJECTIVES: Infection of murine macrophage cell line J774.1 with the periodontopathic bacterium Aggregatibacter actinomycetemcomitans induces apoptotic cell death. The infection induces cell cycle arrest in the G1 phase prior to the appearance of apoptotic cells. This study determined the involvement of various cell cycle-related signal molecules in A. actinomycetemcomitans-induced G1 cell cycle arrest. MATERIALS AND METHODS: Cell cycle in J774.1 cells infected with A. actinomycetemcomitans was analyzed with a flow cytometer. Immunoblot analysis was also employed to determine the expression levels of intracellular signal molecules. RESULTS: Flow cytometric analysis revealed that the percentage of cells in the G1 phase increased to 77.2% at 12 h after A. actinomycetemcomitans infection. Additionally, according to immunoblot analysis, expression levels of hyperphosphorylated forms of retinoblastoma protein (ppRb) declined in J774.1 cells following A. actinomycetemcomitans infection, whereas hypophosphorylated Rb (pRb) expression levels were elevated slightly. Expression levels of cyclin D1 and D2 in the cells decreased gradually postinfection; CDK2, CDK4, CDK6 and cyclin E levels were not changed. Furthermore, postinfection, p21(CIP1/WAF1) expression increased at 6 h, followed by a subsequent decrease. CONCLUSION: These findings suggest that cyclin D1 and D2 and p21(CIP1/WAF1) participate in G1 cell cycle arrest in A. actinomycetemcomitans-infected J774.1 cells.


Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Apoptosis/fisiología , Fase G1/fisiología , Macrófagos/microbiología , Animales , Línea Celular , Ciclina D1/biosíntesis , Ciclina D2/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Macrófagos/metabolismo , Ratones , Fosforilación , Proteína de Retinoblastoma/metabolismo
14.
J Oral Rehabil ; 36(11): 808-13, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19744262

RESUMEN

Although the number of sound or decayed teeth has been reported to be associated with cognitive function in elderly populations with dementia, little is known about this association in elderly populations without dementia. We evaluated this relationship, with adjustment for confounding factors, in Japanese populations of 60-year-old (n = 270; 120 males and 150 females) and 65-year-old (n = 123; 57 males and 66 females) individuals residing in Fukuoka Prefecture of Japan. Dental examinations were performed in all subjects, along with the Mini-mental state examination (MMSE) for assessing cognitive function. Among the total of 393 subjects, the mean MMSE score was 27.9 +/- 1.9, and 391 subjects scored 24 or higher. The mean numbers of sound and decayed teeth were 12.0 +/- 6.3 and 0.5 +/- 1.2, respectively. Associations were found between the numbers of sound and decayed teeth and MMSE in total subjects and males, but not in females, by multiple regression analysis adjusted for gender, age, level of education, marital status, smoking, alcohol drinking, working status, systolic blood pressure and blood glucose. An association was also found between MMSE and the number of sound teeth in a logistic regression analysis. In conclusion, associations were found between normal-range cognitive function and the numbers of sound and decayed teeth, after adjustment for various confounding factors, in an elderly Japanese population.


Asunto(s)
Trastornos del Conocimiento/epidemiología , Caries Dental/epidemiología , Higiene Bucal/normas , Enfermedades Periodontales/epidemiología , Autocuidado/normas , Anciano , Presión Sanguínea/fisiología , Trastornos del Conocimiento/complicaciones , Encuestas de Salud Bucal , Femenino , Indicadores de Salud , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Enfermedades Periodontales/etiología , Características de la Residencia
15.
J Periodontal Res ; 44(5): 609-15, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19453861

RESUMEN

BACKGROUND AND OBJECTIVE: In the present study, micro-channel arrays were fabricated on the surface of plastic-based disposable chips. The cell adhesion process and the detection of plaque-forming macrophages were observed. Further, we evaluated cell adhesion in a fluid system in vitro. MATERIAL AND METHODS: Features of the micro-channel (1.4 mm wide and 10 mm long) included twenty micro-pillars (with a projection of 200 microm diameter and 250 microm high) coated in a 50 microm thick silicon rubber layer, which were regularly arranged at the bottom of each channel. The efficiency of cell capture was expected to increase by arrangement of micro-pillars in a micro-channel. Mouse macrophage RAW264.7 cells, stimulated for 24 h with lipopolysaccharide (LPS) derived from periodontopathic bacteria, were circulated continuously for 2 h at room temperature by the pump in a chip. RESULTS: Control cells had not formed plaques on micro-pillars 20 min into the experiment. By contrast, LPS-activated macrophages produced plaques at the side walls of micro-pillars after 20 min. The plaques grew during the flow test, and image shading became clearer with increasing flow time for 120 min. The maximal adhesion rate per unit area appeared at 20% for control cells, whereas the peak was shifted to 30% for LPS-activated macrophages (n = 20). The average adhesion rate was 3.0 +/- 2.0% for control cells and 5.0 +/- 3.9% for LPS-activated macrophages (n = 100). CONCLUSION: These findings indicate that LPS-activated macrophages accumulate in micro-channel arrays, and suggest that macrophage plaque formation is a two-step procedure: (1) LPS-activated macrophages adhere physically to the silicon rubber layer on micro-pillars; and (2) consequently, the cells adhere to the activated macrophage layer.


Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Algoritmos , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular , Diseño de Equipo , Procesamiento de Imagen Asistido por Computador , Dispositivos Laboratorio en un Chip , Ratones , Reología , Elastómeros de Silicona , Propiedades de Superficie , Factores de Tiempo
16.
Gut ; 57(10): 1431-40, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18579666

RESUMEN

BACKGROUND: Obesity is a risk factor for acute pancreatitis (AP), but the molecular mechanism remains unclear. Adiponectin, an adipose tissue-derived secretory factor, has anti-inflammatory properties in addition to various biological functions, and its plasma concentrations are reduced in obese subjects. However, the role of adiponectin in AP has not been investigated. AIM: To determine the effects of adiponectin on AP. METHODS: We investigated the effects of adiponectin on experimental AP by using adiponectin-knockout (APN-KO) mice and adenovirus-mediated adiponectin over-expression. AP was induced by 10 hourly intraperitoneal injections of low-dose caerulein (10 microg/kg) after 2 week feeding of normal chow or a high-fat diet (HFD) in wild-type (WT) and APN-KO mice. We evaluated the severity of AP biochemically and morphologically. RESULTS: Low-dose caerulein treatment did not induce pancreatic damage in either WT or APN-KO mice under normal chow feeding. APN-KO mice, but not WT mice, fed a HFD and then treated with caerulein developed pancreatic damage and inflammation, accompanied by increased macrophage/neutrophil infiltration and upregulation of pro-inflammatory mediators such as tumour necrosis factor alpha in the pancreas. Adenovirus-mediated over-expression of adiponectin attenuated the severity of HFD/caerulein-induced AP in APN-KO mice. CONCLUSIONS: Adiponectin plays a protective role in caerulein-induced AP in HFD-fed mice.


Asunto(s)
Adiponectina/fisiología , Pancreatitis/prevención & control , Enfermedad Aguda , Adiponectina/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Ceruletida , Grasas de la Dieta/administración & dosificación , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/complicaciones , Pancreatitis/inducido químicamente , Pancreatitis/metabolismo , Pancreatitis/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba
17.
Oral Microbiol Immunol ; 23(3): 259-64, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18402614

RESUMEN

INTRODUCTION: Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms. METHODS: LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization. RESULTS: The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors. CONCLUSION: These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.


Asunto(s)
Aggregatibacter actinomycetemcomitans , Colágeno/metabolismo , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Lipopolisacáridos/farmacología , Fagocitosis/efectos de los fármacos , Actinas/efectos de los fármacos , Proteínas de Fase Aguda/farmacología , Adulto , Androstadienos/farmacología , Proteínas Portadoras/farmacología , Células Cultivadas , Cromonas/farmacología , Citocalasina D/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli , Fibroblastos/fisiología , Encía/citología , Humanos , Receptores de Lipopolisacáridos/farmacología , Masculino , Glicoproteínas de Membrana/farmacología , Morfolinas/farmacología , Fagocitosis/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Wortmanina
18.
Cancer Gene Ther ; 15(3): 140-53, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18157144

RESUMEN

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.


Asunto(s)
Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/genética , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Neoplasias del Cuello Uterino/terapia , Animales , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular/genética , Senescencia Celular/fisiología , Femenino , Terapia Genética/métodos , Células HeLa , Papillomavirus Humano 16/crecimiento & desarrollo , Humanos , Immunoblotting , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas E7 de Papillomavirus , ARN Interferente Pequeño/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección , Carga Tumoral , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Ensayos Antitumor por Modelo de Xenoinjerto
19.
Biofouling ; 23(1-2): 73-7, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17453731

RESUMEN

An isothiazolone biocide, 5-chloro-2-methyl-4-isothiazolin-3-one (CMI), was degraded in the presence of iron. According to the Fe-dependent degradation of CMI, stoichiometric production of chloride was observed. Copper and stainless steel did not enhance the physico-chemical degradation of CMI, whilst phosphate inhibited the Fe-dependent degradation. Neither aerobic nor anaerobic conditions influenced the Fe-dependent CMI degradation. Furthermore, FeO(OH)-powder and Fe(3)O(4)-powder did not lead to the physico-chemical degradation of CMI. Rapid disappearance of CMI was observed in an operating cooling water plant. CMI added to the cooling tower declined from 1.4 mg l(-1) to < 0.1 mg l(-1) in 2 d. This finding is important in optimising the use of CMI and combating resistance if encountered.


Asunto(s)
Desinfectantes , Tiazoles , Aerobiosis , Anaerobiosis , Biodegradación Ambiental , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Fenómenos Químicos , Química Física , Desinfectantes/metabolismo , Desinfectantes/farmacología , Hierro , Tiazoles/metabolismo , Tiazoles/farmacología , Microbiología del Agua
20.
Cancer Gene Ther ; 14(4): 354-63, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17273182

RESUMEN

Recently, ultrasound-targeting microbubble destruction has been employed in molecular gene therapy, and a new potent nonviral gene transfer method known as 'sonoporation' has been developed. We investigated the efficiency of sonoporation toward growth inhibition of human gingival squamous carcinoma cell line, Ca9-22, in vitro and in vivo. The cytotoxicity of bleomycin (BLM) was investigated using flow-cytometric analysis and Hoechst's staining in vitro assay systems. We found that the delivery of BLM by sonoporation induced cytotoxic effect toward Ca9-22 cells in vitro. Our in vivo results showed that tumors nearly disappeared in Ca9-22 cell-implanted nude KSN/slc mice treated with a low dose of BLM followed by sonoporation during the 4-week experimental period. Histological analysis revealed that the cytotoxic effect was mainly apoptosis. We previously reported that the cytolethal distending toxin B (cdtB) from Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, is responsible for cell cycle arrest and apoptosis in vitro. Thus, we used sonoporation to transfect a cdtB-expressing plasmid into Ca9-22 cells and examined cell viability in vitro and in vivo. We found that an administration of cdtB-expressing plasmid followed by sonoporation-induced marked growth inhibition of Ca9-22 cells and apoptotic cells were also observed in vitro and in vivo. These findings suggest that local administration of cytotoxic agents with sonoporation is a useful method for molecular cancer therapy.


Asunto(s)
Antineoplásicos/administración & dosificación , Toxinas Bacterianas/genética , Bleomicina/administración & dosificación , Carcinoma de Células Escamosas/terapia , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Neoplasias Gingivales/terapia , Ultrasonido , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Terapia Combinada , Citotoxinas/administración & dosificación , Neoplasias Gingivales/tratamiento farmacológico , Neoplasias Gingivales/patología , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto
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