RESUMEN
Human linkage and association studies suggest a gene(s) for nonsyndromic cleft lip with or without cleft palate (CL/P) on chromosome 4q31-q32 at or near the platelet-derived growth factor-C (PDGF-C) locus. The mouse Pdgfc(-/-) knockout shows that PDGF-C is essential for palatogenesis. To evaluate the role of PDGF-C in human clefting, we performed sequence analysis and SNP genotyping using 1048 multiplex CL/P families and 1000 case-control samples from multiple geographic origins. No coding region mutations were identified, but a novel -986 C>T SNP (rs28999109) was significantly associated with CL/P (P=0.01) in cases from Chinese families yielding evidence of linkage to 4q31-q32. Significant or near-significant association was also seen for this and several other PDGF-C SNPs in families from the United States, Spain, India, Turkey, China, and Colombia, whereas no association was seen in families from the Philippines, and Guatemala, and case-controls from Brazil. The -986T allele abolished six overlapping potential transcription regulatory motifs. Transfection assays of PDGF-C promoter reporter constructs show that the -986T allele is associated with a significant decrease (up to 80%) of PDGF-C gene promoter activity. This functional polymorphism acting on a susceptible genetic background may represent a component of human CL/P etiology.
Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Linfocinas/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Alelos , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , HumanosRESUMEN
BACKGROUND: Elevated levels of the macrophage inflammatory protein-1alpha (MIP-1alpha) are reported in inflammatory bone diseases including periodontitis. We evaluated the ability of interleukin-1beta (IL-1beta) and bacterial lipopolysaccharides (LPSs) to modulate MIP-1alpha expression in epithelial cells, fibroblasts, and polymorphonuclear leukocytes (PMNs). We also evaluated the effect of MIP-1alpha as an osteoclast activating factor. METHODS: Human gingival epithelial cells and fibroblasts were obtained by primary cell culture. PMNs were isolated from healthy controls. Human MG63 osteosarcoma cells were used as osteoblastic cells. After incubation of each cell type with IL-1beta, Porphyromonas gingivalis LPS, and Actinobacillus actinomycetemcomitans LPS, MIP-1alpha mRNA and secreted protein levels were quantified by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. The ability of recombinant MIP-1alpha to induce osteoclast formation was determined by tartrate resistant acid phosphatase assay. RESULTS: MIP-1alpha expression in PMNs and gingival epithelial cells was induced by IL-1beta and LPS, but neither induced MIP-1alpha expression in gingival fibroblasts or osteoblastic cells. MIP-1alpha was highly expressed in the basal epithelial layer of inflamed gingiva but not in healthy gingiva. MIP-1alpha induced osteoclast formation at an optimal concentration of 0.05 to 2 ng/ml. CONCLUSIONS: MIP-1alpha expression by gingival epithelial cells may be important in initiating inflammation by facilitating accumulation and activation of leukocytes. The ability of MIP-1alpha to facilitate formation of multinuclear bone cells indicates a possible role in periodontitis-associated bone destruction. These findings indicate MIP-1alpha may play an important role in early and later stages of inflammatory-related periodontitis.
Asunto(s)
Quimiocina CCL3/inmunología , Encía/inmunología , Interleucina-1beta/farmacología , Lipopolisacáridos/farmacología , Aggregatibacter actinomycetemcomitans , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Encía/citología , Encía/efectos de los fármacos , Gingivitis/inmunología , Gingivitis/patología , Humanos , Linfocinas/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Osteoblastos/efectos de los fármacos , Osteoblastos/inmunología , Osteoclastos/inmunología , Osteoclastos/patología , Osteosarcoma/inmunología , Osteosarcoma/patología , Porphyromonas gingivalis , Células Tumorales CultivadasRESUMEN
BACKGROUND: Elevated levels of the macrophage inflammatory protein-1α (MIP-1α) are reported in inflammatory bone diseases including periodontitis. We evaluated the ability of interleukin-1ß (IL-1ß) and bacterial lipopolysaccharides (LPSs) to modulate MIP-1α expression in epithelial cells, fibroblasts, and polymorphonuclear leukocytes (PMNs). We also evaluated the effect of MIP-1α as an osteoclast activating factor. METHODS: Human gingival epithelial cells and fibroblasts were obtained by primary cell culture. PMNs were isolated from healthy controls. Human MG63 osteosarcoma cells were used as osteoblastic cells. After incubation of each cell type with IL-1ß, Porphyromonas gingivalis LPS, and Actinobacillus actinomycetemcomitans LPS, MIP-1α mRNA and secreted protein levels were quantified by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. The ability of recombinant MIP-1α to induce osteoclast formation was determined by tartrate resistant acid phosphatase assay. RESULTS: MIP-1α expression in PMNs and gingival epithelial cells was induced by IL-1ß and LPS, but neither induced MIP-1α expression in gingival fibroblasts or osteoblastic cells. MIP-1α was highly expressed in the basal epithelial layer of inflamed gingiva but not in healthy gingiva. MIP-1α induced osteoclast formation at an optimal concentration of 0.05 to 2 ng/ml. CONCLUSIONS: MIP-1α expression by gingival epithelial cells may be important in initiating inflammation by facilitating accumulation and activation of leukocytes. The ability of MIP-1α to facilitate formation of multinuclear bone cells indicates a possible role in periodontitis-associated bone destruction. These findings indicate MIP-1α may play an important role in early and later stages of inflammatory-related periodontitis.
RESUMEN
In this study, the process of manufacturing and sterilizing an orthopedic implant constructed from poly(L-lactic acid) (PLLA) was closely simulated. The hydrogen peroxide gas plasma (HPGP) sterilization process was comparatively investigated against ethylene oxide (EtO). Characterization of the physical, thermal, mechanical, morphological, and chemical properties was monitored. The results indicate that the HPGP sterilization process did not have a significant influence on M(n) or M(w) initially or through 12 weeks of in vitro conditioning when compared with EtO. Only indications of physical aging were evident in the analysis of the thermal and mechanical properties by differential scanning calorimeter and tensile testing for each sterilization processes. Using wide angle X-ray diffraction to determine morphology characteristics, it was determined that no changes were observed between the as molded, HPGP, and EtO specimens initially or through the 12 week in vitro conditioning period. Contact angle measurements revealed a significant reduction in the surface energy following treatment by the HPGP process, suggesting the formation of polar groups. However, surface chemistry analysis by ATR-FTIR indicated no significant chemical modification from either sterilization method. PLLA showed intermediate levels of residual hydrogen peroxide absorption following processing by HPGP.
Asunto(s)
Materiales Biocompatibles , Óxido de Etileno/química , Peróxido de Hidrógeno/química , Ácido Láctico , Ensayo de Materiales , Polímeros , Prótesis e Implantes , Esterilización , Materiales Biocompatibles/química , Óxido de Etileno/análisis , Peróxido de Hidrógeno/análisis , Ácido Láctico/química , Equipo Ortopédico , Poliésteres , Polímeros/química , Propiedades de Superficie , Factores de TiempoRESUMEN
The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.
Asunto(s)
Médula Ósea , Adhesión Celular/fisiología , Proliferación Celular , Indoles/metabolismo , Mieloma Múltiple , Osteoclastos/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Médula Ósea/química , Médula Ósea/metabolismo , Proteínas Portadoras/metabolismo , Quimiocinas/metabolismo , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Humanos , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoclastos/citología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Células del Estroma/citología , Células del Estroma/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Macrophage inflammatory protein-1alpha (MIP-1alpha) is produced in high concentration by multiple myeloma (MM) cells in about 70% of patients, and MIP-1alpha levels correlate with their disease activity. Patients who have high levels of MIP-1alpha have a poor prognosis. Furthermore, blocking MIP-1alpha expression in an in vivo model of human MM profoundly decreases both tumor burden and bone destruction, suggesting that MIP-1alpha is an important mediator of MM bone disease. Therefore, to analyze the regulation of MIP-1alpha production in MM, we cloned the human MIP-1alpha promoter and characterized the transcription factor (TF) motifs that control MIP-1alpha expression in MM cells. The proximal region of MIP-1alpha promoter was composed of 2 sets of identical transcription regulatory regions consisting of GATA-2(+) AML-1(+) C/EBPalpha motifs. Since 2 alternatively spliced variants of the acute myeloid leukemia-1 (AML-1) class of TFs can bind the AML-1 region, AML-1A and AML-1B, the relationship between the expression levels of AML-1A or AML-1B in MM cells and their capacity to express MIP-1alpha was examined. AML-1A mRNA was relatively overexpressed compared with AML-1B in MM cell lines that produced high levels of MIP-1alpha (> 1 ng/mL per 10(6) cells per 72 hours), but AML-1A was not increased in MM cell lines that expressed less than 200 pg/mL MIP-1alpha. More importantly, the ratio of AML-1A to AML-1B mRNA levels was also increased in 3 of 3 highly purified myeloma cells from patients with MM who expressed increased amounts of MIP-1alpha. The ratio of AML-1A to AML-1B mRNA in patients with MM was 8-fold higher than in healthy controls. Transduction of AML-1B into the MM-derived MM.1S and ARH-77 cells totally blocked MIP-1alpha production, while AML-1A did not further increase the already high levels of MIP-1alpha produced by these cells. Taken together, these data demonstrate that in patients with MM who produce increased concentrations of MIP-1alpha, the relative level of AML-1B is significantly decreased compared with healthy controls. The data suggest that strategies that enhance AML-1B expression or decrease AML-1A in MM cells may be beneficial therapeutically.