RESUMEN
Administration of active vitamin D sterols to treat secondary hyperparathyroidism in patients with chronic kidney disease receiving dialysis has been associated with elevated serum calcium and phosphorus levels, which may lead to increased risk of vascular calcification. However, calcimimetics, by binding to the parathyroid gland calcium-sensing receptors, reduce serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Using cultured bovine aorta vascular smooth muscle cells (BASMCs), an in vitro model of vascular calcification, we compared calcification levels and gene expression profiles after exposure to the phosphate source ss-glycerolphosphate (BGP), the active vitamin D sterols calcitriol and paricalcitol, the calcimimetic R-568, or BGP with the active vitamin D sterols or R-568. Cells exposed to BGP (10 mM) alone or with calcitriol or paricalcitol showed dose-dependent BASMC calcification. No change in calcification was observed in cultures exposed to BGP with R-568, consistent with the observed lack of calcium-sensing receptor expression. Microarray analysis using total cellular RNA from cultures exposed to vehicle or BGP in the absence and presence of 10(-8) M calcitriol or paricalcitol for 7 days showed that cells exposed to BGP with calcitriol or BGP with paricalcitol had virtually identical gene expression profiles, which differed from those of cells treated with BGP or vehicle alone. Several osteoblast- and chondrocyte-associated genes were modulated by BGP and vitamin D exposure. In this study, exposure of BASMCs to phosphate and active vitamin D sterols induced calcification and changes in expression of genes associated with mineralized tissue.
Asunto(s)
Compuestos de Anilina/farmacología , Calcinosis/prevención & control , Calcitriol/farmacología , Ergocalciferoles/farmacología , Glicerofosfatos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Proteínas Wnt/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Calcinosis/inducido químicamente , Calcinosis/metabolismo , Calcio/agonistas , Calcio/metabolismo , Calcio/farmacología , Bovinos , Células Cultivadas , Combinación de Medicamentos , Expresión Génica/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenetilaminas , Fósforo/metabolismo , Fósforo/farmacología , Propilaminas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores Sensibles al Calcio/efectos de los fármacos , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND AND AIMS: Transfer of CD4+CD45RBHi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. METHODS: CD4+CD45RBHi or CD4+CD45RBLo T cells (4 x 10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. RESULTS: Mice that received CD4+CD45RBHi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4+CD45RBLo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4+CD45RBHi or CD4+CD45RBLo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4+CD45RBHi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4+CD45RBHi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG. CONCLUSIONS: CD4+CD45RBHi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.
Asunto(s)
Enfermedades Óseas Metabólicas/prevención & control , Glicoproteínas/uso terapéutico , Enfermedades Inflamatorias del Intestino/complicaciones , Transfusión de Linfocitos/efectos adversos , Receptores Citoplasmáticos y Nucleares/uso terapéutico , Animales , Densidad Ósea/efectos de los fármacos , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Enfermedades Óseas Metabólicas/etiología , Linfocitos T CD4-Positivos/trasplante , Femenino , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Intestino Grueso/patología , Ratones , Ratones SCID , Osteoblastos/patología , Osteoclastos/patología , Osteoprotegerina , Hormona Paratiroidea/sangre , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes/uso terapéutico , Proteína Amiloide A Sérica/metabolismo , Subgrupos de Linfocitos T/trasplante , Pérdida de PesoRESUMEN
The epithelium of the oral cavity and small intestine of the gastrointestinal tract have a high rate of cell renewal and as such, are sensitive to cytotoxic therapies that kill rapidly dividing cells. Mucositis is a complication of cancer therapy where impairment of the regenerative capacity of the epithelium leads to atrophy, ulceration and a loss of barrier function. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth and differentiation factor that is trophic for the mucosal epithelium of the gastrointestinal tract. In this study, KGF in normal animals caused epithelial thickening in the squamous epithelium of the oral cavity and increased crypt depth and villus height of the small intestine. It also appeared to regulate gene expression in these tissues including that of some antioxidant enzymes and intestinal trefoil protein. KGF has been shown to be efficacious in several preclinical models of mucositis where KGF pretreatment reduced weight loss typically seen during and after the course of therapy and significantly improved survival. At a tissue level KGF reduced atrophy, accelerated regrowth, and decreased ulcer formation of the oral epithelium after irradiation, and improved crypt survival and prevented villus atrophy in the small intestine of irradiated or chemotherapy-treated mice. Preliminary studies suggest that its efficacy may be partly a consequence of the growth and differentiation effect, and also partly due to regulation of the expression of genes that play a role in mucosal protection. These data suggest that KGF may be useful for the prevention or treatment of mucositis in patients treated with regimens of cancer therapy that have gastrointestinal toxicity.
Asunto(s)
Factores de Crecimiento de Fibroblastos/farmacología , Mucosa Bucal/patología , Estomatitis/tratamiento farmacológico , Estomatitis/patología , Animales , Modelos Animales de Enfermedad , Factor 7 de Crecimiento de Fibroblastos , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Traumatismos Experimentales por Radiación/patologíaRESUMEN
SALL1 is a mammalian homolog of the Drosophila region-specific homeotic gene spalt (sal); heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We have isolated a mouse homolog of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1-deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Riñón/embriología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Uréter/embriología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cruzamientos Genéticos , Regulación hacia Abajo , Marcadores Genéticos/genética , Heterocigoto , Humanos , Hibridación in Situ , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Reactive oxygen species (ROS) and glutathione (GSH) depletion contribute to organ injury after bone marrow transplantation (BMT). Keratinocyte growth factor (KGF) ameliorates graft-versus-host disease (GVHD)-associated organ injury in murine BMT models. METHODS: B10.BR mice received total body irradiation (TBI; day -1) +/- cyclophosphamide (Cy; 120 mg/kg/day i.p., days -3 and -2), then were transplanted on day 0 with C57BL/6 bone marrow + spleen cells as a source of GVHD-causing T cells. KGF (5 mg/kg/day subcutaneously [s.c.]) or saline was given on days -6, -5, and -4. Lung and liver GSH and oxidized GSH disulfide (GSSG) levels were measured on days 0 and 5 and glutathione redox potential (Eh) calculated. Organ malondialdehyde (MDA) was determined on day 5 as an index of ROS-mediated lipid peroxidation. RESULTS: In lung, TBI+BMT oxidized GSH Eh and increased MDA. Cy further oxidized lung GSH Eh. In liver, neither BMT regimen altered GSH redox status or MDA. KGF prevented the decrease in lung GSH after TBI+Cy and decreased lung MDA after both TBI and TBI+Cy. KGF increased liver GSH levels and GSH Eh after TBI and GSH Eh after TBI+Cy. CONCLUSIONS: In murine allogeneic BMT, TBI oxidizes the lung GSH redox pool and Cy exacerbates this response by 5 days post-BMT. In contrast, liver GSH redox status is maintained under these experimental conditions. KGF treatment attenuates the Cy-induced decrease in lung GSH, decreases post-BMT lung lipid peroxidation, and improves liver GSH redox indices. KGF may have a therapeutic role to prevent or attenuate GSH depletion and ROS-mediated organ injury in BMT.
Asunto(s)
Trasplante de Médula Ósea , Factores de Crecimiento de Fibroblastos/farmacología , Glutatión/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Acondicionamiento Pretrasplante , Animales , Ciclofosfamida/farmacología , Femenino , Factor 7 de Crecimiento de Fibroblastos , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Oxidación-Reducción , Especies Reactivas de Oxígeno , Trasplante Homólogo , Irradiación Corporal TotalRESUMEN
PTH is a potent bone anabolic factor, and its combination with antiresorptive agents has been proposed as a therapy for osteoporosis. We tested the effects of PTH, alone and in combination with the novel antiresorptive agent OPG, in a rat model of severe osteopenia. Sprague Dawley rats were sham-operated or ovariectomized at 3 months of age. Rats were untreated for 15 months, at which time ovariectomy had caused significant decreases in bone mineral density in the lumbar vertebrae and femur. Rats were then treated for 5.5 months with vehicle (PBS), human PTH-(1-34) (80 microg/kg), rat OPG (10 mg/kg), or OPG plus PTH (all three times per wk, sc). Treatment of ovariectomized rats with OPG or PTH alone increased bone mineral density in the lumbar vertebrae and femur, whereas PTH plus OPG caused significantly greater and more rapid increases than either therapy alone (P < 0.05). OPG significantly reduced osteoclast surface in the lumbar vertebrae and femur (P < 0.05 vs. sham or ovariectomized), but had no effect on osteoblast surface at either site. Ovariectomy significantly decreased the mechanical strength of the lumbar vertebrae and femur. In the lumbar vertebrae, OPG plus PTH was significantly more effective than PTH alone at reversing ovariectomy-induced deficits in stiffness and elastic modulus. These data suggest that OPG plus PTH represent a potentially useful therapeutic option for patients with severe osteoporosis.
Asunto(s)
Enfermedades Óseas Metabólicas/tratamiento farmacológico , Glicoproteínas/farmacología , Fragmentos de Péptidos/farmacología , Teriparatido/farmacología , Animales , Densidad Ósea/efectos de los fármacos , Enfermedades Óseas Metabólicas/fisiopatología , Interacciones Farmacológicas , Quimioterapia Combinada , Femenino , Glicoproteínas/uso terapéutico , Osteoprotegerina , Ovariectomía , Fragmentos de Péptidos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/uso terapéutico , Receptores del Factor de Necrosis Tumoral , Teriparatido/análogos & derivados , Teriparatido/uso terapéuticoRESUMEN
This study examines the ability of osteoprotegerin (OPG) to prevent the local bone resorption caused by sciatic nerve damage. Sixty-five 18-week-old male mice were assigned to one of six groups (n = 10-11/group). A baseline control group was sacrificed on day zero of the 10-day study. The remaining groups were placebo sham operated, placebo nerve crush (Plac NC) operated, 0.1 mg/kg/day OPG + nerve crush (LOW), 0.3 mg/kg/day OPG + nerve crush (MED), and 1.0 mg/kg/day OPG + nerve crush (HI). Nerve crush or sham operations were performed on the right leg. The left leg served as a contralateral control to the nerve crushed (ipsilateral) leg. The difference in mass between the right and left femur and tibia was examined. Additionally, quantitative histomorphometry was performed on the right and left femur and tibia diaphyses. Nerve crush resulted in a significant loss of bone mass in the ipsilateral side compared to the contralateral side. Bone mass for the ipsilateral bones of the Plac NC group were significantly reduced by 3.8% in the femur and 3.5% in the tibia compared to the contralateral limb. The percent diminution was reduced for OPG treated mice compared to the Plac NC group for both the femur and tibia. In the femur, the percent reduction of ipsilateral bone mass was reduced to 1.0% (LOW), 1.3% (MED) and 1.6% (HI) compared to the contralateral limb. In the tibia, loss of bone mass in the ipsilateral limb was reduced to 1.4% (LOW), 1.4% (MED), and 2.4% (HI) compared to the contralateral. OPG also decreased the amount of tibial endocortical resorption compared to the Plac NC group. In summary, OPG mitigated bone loss caused by damage to the sciatic nerve.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Glicoproteínas/farmacología , Nervio Ciático/fisiología , Animales , Resorción Ósea/patología , Diáfisis/patología , Modelos Animales de Enfermedad , Fémur/inervación , Fémur/patología , Húmero/anatomía & histología , Masculino , Ratones , Ratones Endogámicos C57BL , Compresión Nerviosa , Tamaño de los Órganos , Osteoprotegerina , Receptores Citoplasmáticos y Nucleares , Receptores del Factor de Necrosis Tumoral , Tibia/inervación , Tibia/patologíaRESUMEN
Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/secundario , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Glicoproteínas/farmacología , Osteólisis/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Neoplasias Óseas/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Transformada , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones Desnudos , Osteoprotegerina , Receptores Citoplasmáticos y Nucleares , Receptores del Factor de Necrosis Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Osteoprotegerin (OPG) regulates bone resorption by inhibiting osteoclast formation, function, and survival. The current studies employed a mouse ovariectomy (OVX) model of estrogen deficiency to investigate gene therapy with OPG as a means of preventing osteoporosis. Young adult females injected with a recombinant adenoviral (Ad) vector carrying cDNA of either full-length OPG or a fusion protein combining the hOPG ligand-binding domain with the human immunoglobulin constant domain (Ad-hOPG-Fc) developed serum OPG concentrations exceeding the threshold needed for efficacy. However, elevated circulating OPG levels were sustained for up to 18 months only in mice given Ad-hOPG-Fc. Administration of Ad-hOPG-Fc titers between 10(7) and 10(9) pfu yielded dose-dependent increases in serum OPG. Mice subjected to OVX or sham surgery followed by immediate treatment with Ad-hOPG-Fc had significantly more bone volume with reduced osteoclast numbers in axial and appendicular bones after 4 weeks. In contrast, animals given OVX and either a control vector or vehicle had significantly less bone than did comparably treated sham-operated mice. This study demonstrates that a single adenoviral gene transfer can produce persistent high-level OPG expression and shows that gene therapy to provide sustained delivery of OPG may prove useful in treating osteoporosis.
Asunto(s)
Adenoviridae/genética , Glicoproteínas/genética , Osteoporosis/terapia , Receptores Citoplasmáticos y Nucleares/genética , Animales , Bioensayo , Southern Blotting , Western Blotting , Densidad Ósea/efectos de los fármacos , Resorción Ósea , ADN Complementario/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Osteoprotegerina , Ovariectomía , Ovario/fisiología , Pelvis/diagnóstico por imagen , Radiografía , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
Osteoprotegerin (OPG) is a naturally secreted protein that decreases bone resorption by inhibiting osteoclast differentiation and activation while promoting osteoclast apoptosis [8]. In this study, the effects of osteoprotegerin injections on long bone mechanical and material properties were investigated in young male Sprague-Dawley rats. OPG increased fracture strength at the femur mid-diaphysis in three-point bending by 30%, without affecting the elastic or maximum strength. At the femoral neck, OPG significantly increased the elastic (45%), maximum (15%), and fracture (35%) strengths. There was not a difference in microhardness at the femur mid-diaphysis in comparing the placebo and OPG groups. There were, however, significant increases in whole bone dry mass (25%), mineral mass (30%), organic mass (17%), and percent mineralization (4%); percent mineralization at the mid-diaphysis (3%); and percent mineralization at the distal epiphysis (6%) due to the OPG treatment. While OPG decreased endocortical bone formation (52%), total bone area, endocortical bone area, and periosteal bone formation were maintained with OPG treatment. A 30% increase in the X-ray opacity of the bone at the proximal metaphysis of the right tibiae was observed. Overall, OPG increased mineralization and strength indices in the rat femur. Its effects on strength were more pronounced in the femoral neck than at the mid-diaphysis.
RESUMEN
We have previously shown that pretreatment of mice with keratinocyte growth factor (KGF), an epithelial tissue repair factor, can ameliorate graft-versus-host disease (GVHD) after intensive chemoradiotherapeutic conditioning and allogeneic bone marrow transplantation (BMT). To determine whether this effect was dependent on a KGF-mediated mechanism affecting repair of conditioning-induced epithelial cell injury, we studied GVHD in the absence of conditioning using BALB/c severe combined immune-deficient (SCID) recipients given C57BL/6 T cells. KGF (5 mg/kg per day, subcutaneously) given either before or after T-cell transfer enhanced body weights and extended survival. KGF-treated recipients had elevated serum levels of the Th2 cytokine interleukin 13 (IL-13) on day 6 after T-cell transfer concomitant with reduced levels of the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma). A 3-day KGF pretreatment also depressed the secondary in vitro mixed lymphocyte response (MLR) of C57BL/6 splenocytes taken 7 days after in vivo alloimmunization with irradiated BALB/c spleen cells. To determine whether KGF would inhibit host-antidonor-mediated BM rejection, pan-T-cell-depleted BALB/c BM cells were infused into sublethally irradiated C57BL/6 mice and administered KGF either before or before and after BMT. Surprisingly, all KGF schedules tested actually resulted in enhanced alloengraftment. The presence of KGF receptor on donor antihost alloreactive T cells could not be detected by binding studies with radiolabeled KGF, reverse transcriptase-polymerase chain reaction, and Western blotting. Therefore, the mechanism of action of KGF on inhibiting T-cell-mediated immune effects may not be due to a direct effect of KGF on T cells. These studies demonstrate that KGF, by mechanisms independent of repair of conditioning-induced injury, has great potential as an anti-GVHD therapeutic agent with the added benefit of inhibiting the rejection of pan-T-cell-depleted donor BM allografts. (Blood. 2000;96:4350-4356)
Asunto(s)
Trasplante de Médula Ósea , Factores de Crecimiento de Fibroblastos , Supervivencia de Injerto/efectos de los fármacos , Enfermedad Injerto contra Huésped/prevención & control , Sustancias de Crecimiento/uso terapéutico , Acondicionamiento Pretrasplante/efectos adversos , Animales , Evaluación Preclínica de Medicamentos , Células Epiteliales/efectos de la radiación , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Inmunización , Interferón gamma/sangre , Interleucina-13/sangre , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Quimera por Radiación , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Células Th2/metabolismo , Factor de Necrosis Tumoral alfa/análisis , Irradiación Corporal Total/efectos adversosRESUMEN
Osteoclast precursors (OCPs) circulate in the mononuclear fraction of peripheral blood (PB), but their abundance and surface characteristics are unknown. Previous studies suggest that the receptor activator for NF-kappaB (RANK) on cytokine-treated OCPs in mouse bone marrow interacts with osteoprotegerin ligand (OPGL/TRANCE/RANKL/ODF) to initiate osteoclast differentiation. Hence, we used a fluorescent form of human OPGL (Hu-OPGL-F) to identify possible RANK-expressing OCPs in untreated peripheral blood mononuclear cells (PBMCs) using fluorescence-activated cell sorting analysis. Monocytes [CD14-phycoerythrin (PE) antibody (Ab) positive (+) cells, 10-15% of PBMCs] all (98-100%) co-labelled with Hu-OPGL-F (n > 18). T lymphocytes (CD3-PE Ab+ cells, 66% of PBMCs) did not bind Hu-OPGL-F; however, B cells (CD19-PE Ab+ cells, 9% of PBMCs) were also positive for Hu-OPGL-F. All Hu-OPGL-F+ monocytes also co-labelled with CD33, CD61, CD11b, CD38, CD45 and CD54 Abs, but not CD34 or CD56 Abs. Hu-OPGL-F binding was dose dependent and competed with excess Hu-OPGL. When Hu-OPGL-F+, CD14-PE Ab+, CD33-PE Ab+, Hu-OPGL-F+/CD14-PE Ab+ or Hu-OPGL-F+/CD33-PE Ab+ cells were cultured with OPGL (20 ng/ml) and colony-stimulating factor (CSF)-1 (25 ng/ml), OC-like cells readily developed. Thus, all freshly isolated monocytes demonstrate displaceable Hu-OPGL-F binding, suggesting the presence of RANK on OCPs in PB; also, OCPs within a purified PB monocyte population form osteoclast-like cells in the complete absence of other cell types in OPGL and CSF-1 containing medium.
Asunto(s)
Proteínas Portadoras , Glicoproteínas/metabolismo , Leucocitos Mononucleares/metabolismo , Glicoproteínas de Membrana , Osteoclastos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Linfocitos B/metabolismo , Diferenciación Celular , Células Cultivadas , Factores Estimulantes de Colonias/farmacología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Glicoproteínas/farmacología , Humanos , Leucocitos Mononucleares/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Rastreo , Monocitos/metabolismo , Osteoclastos/ultraestructura , Osteoprotegerina , Unión Proteica , Ligando RANK , Receptor Activador del Factor Nuclear kappa-BRESUMEN
We reported that systemic keratinocyte growth factor (KGF) given before bone marrow transplantation (BMT) prevents allogeneic T cell-dependent lung inflammation assessed on Day 7 post-BMT, but the antiinflammatory effects of KGF were impaired in mice injected with both T cells and conditioning regimen of cyclophosphamide (Cy). Intratracheal KGF is known to stimulate the expression of surfactant protein A (SP-A), an oxidant-sensitive T cell immunomodulator produced by alveolar type II cells. We hypothesized that systemic KGF up-regulates SP-A after allogeneic BMT, and the addition of Cy may interfere with the ability of KGF to enhance SP-A production. The subcutaneous administration of recombinant human KGF (5 mg/kg on Days -6, -5, and -4 pre-BMT) increased SP-A protein and mRNA in allogeneic T cell-recipient irradiated mice measured on Day 7 post-BMT. In contrast, the same KGF treatment in irradiated mice given T cells and Cy failed to up-regulate SP-A mRNA and protein expression. In mixed lymphocyte reaction experiments designed to simulate the in vivo model, the addition of human SP-A (5-50 microg) to alloactivated T cells suppressed the production of interleukin-2 in a dose-dependent fashion. We conclude that the systemic pre-BMT injection of KGF in recipients of allogeneic T cells up-regulates SP-A, which may contribute to the early antiinflammatory effects of KGF. The protective KGF-mediated SP-A production is abolished in mice given alloreactive T cells plus Cy.
Asunto(s)
Trasplante de Médula Ósea , Ciclofosfamida/farmacología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Inmunosupresores/farmacología , Enfermedades Pulmonares/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Acondicionamiento Pretrasplante , Tirosina/análogos & derivados , Animales , Trasplante de Médula Ósea/efectos adversos , Líquido del Lavado Bronquioalveolar/química , Femenino , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Interleucina-2/metabolismo , Queratinocitos , Enfermedades Pulmonares/etiología , Prueba de Cultivo Mixto de Linfocitos , Depleción Linfocítica , Ratones , Ratones Endogámicos , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Linfocitos T/trasplante , Tirosina/análisis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Osteoprotegerin-ligand (OPGL) is a key osteoclast differentiation/activation factor essential for bone remodeling. We report that mice lacking OPGL or its receptor RANK fail to form lobulo-alveolar mammary structures during pregnancy, resulting in death of newborns. Transplantation and OPGL-rescue experiments in opgl-/- and rank-/- pregnant females showed that OPGL acts directly on RANK-expressing mammary epithelial cells. The effects of OPGL are autonomous to epithelial cells. The mammary gland defect in female opgl-/- mice is characterized by enhanced apoptosis and failures in proliferation and PKB activation in lobulo-alveolar buds that can be reversed by recombinant OPGL treatment. These data provide a novel paradigm in mammary gland development and an evolutionary rationale for hormonal regulation and gender bias of osteoporosis in females.
Asunto(s)
Proteínas Portadoras/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas , Animales , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/fisiología , Proteínas Portadoras/genética , División Celular/fisiología , Supervivencia Celular/fisiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/trasplante , Femenino , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Hormonas Esteroides Gonadales/farmacología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Osteoporosis/etiología , Osteoporosis/fisiopatología , Osteoprotegerina , Fenotipo , Fosforilación , Embarazo/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores del Factor de Necrosis TumoralRESUMEN
Osteoprotegerin ligand (OPGL) targets osteoclast precursors and osteoclasts to enhance differentiation and activation, however, little is known about OPGL effects on osteoclast survival. In vitro, the combination of OPGL + colony-stimulating factor-1 (CSF-1) is required for optimal osteoclast survival. Ultrastructurally, apoptotic changes were observed in detached cells and culture lysates exhibited elevated caspase 3 activity, particularly in cultures lacking CSF-1. DEVD-FMK (caspase 3 inhibitor) partially protected cells when combined with OPGL, but not when used alone or in combination with CSF-1. CSF-1 maintained NF-kappaB activation and increased the expression of bcl-2 and bcl-X(L) mRNA, but had no effect on JNK activation. In contrast, OPGL enhanced both NF-kappaB and JNK kinase activation and increased the expression of c-src, but not bcl-2 and bcl-X(L) mRNA. These data suggest that aspects of both OPGL's and CSF-1's signaling/survival pathways are required for optimal osteoclast survival. In mice, a single dose of OPG, the OPGL decoy receptor, led to a >90% loss of osteoclasts because of apoptosis within 48 hours of exposure without impacting osteoclast precursor cells. Therefore, OPGL is essential, but not sufficient, for osteoclast survival and endogenous CSF-1 levels are insufficient to maintain osteoclast viability in the absence of OPGL.
Asunto(s)
Proteínas Portadoras/farmacología , Supervivencia Celular/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Osteoclastos/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/farmacología , Inyecciones Subcutáneas , Factor Estimulante de Colonias de Macrófagos/farmacología , Masculino , Ratones , Ratones Endogámicos C3H , FN-kappa B/metabolismo , Osteoclastos/citología , Osteoclastos/ultraestructura , Osteoprotegerina , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ligando RANK , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B , Receptores del Factor de Necrosis Tumoral , Factores de Tiempo , Proteína bcl-XRESUMEN
High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG(-/-) mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG(-/-) mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG(-/-) mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-kappaB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG(-/-) mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.
Asunto(s)
Densidad Ósea/fisiología , Calcinosis/fisiopatología , Glicoproteínas/metabolismo , Osteoclastos/metabolismo , Osteopetrosis/metabolismo , Osteoporosis/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Aorta/patología , Western Blotting , Células CHO , Catepsina K , Catepsinas/metabolismo , Cricetinae , Fémur/anatomía & histología , Fémur/diagnóstico por imagen , Fémur/metabolismo , Glicoproteínas/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/metabolismo , Osteoclastos/ultraestructura , Osteopetrosis/genética , Osteoporosis/genética , Osteoprotegerina , Radiografía , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Fosfatasa Ácida TartratorresistenteRESUMEN
Idiopathic pneumonia syndrome (IPS) is a significant complication following bone marrow transplantation (BMT). We have developed a murine model in which severe IPS is induced by pre-BMT conditioning and allogeneic T cells and is characterized by the recruitment of host monocytes and donor T cells into the lung by day 7 post-BMT. Chemokines regulate cellular recruitment and the migration of cells into inflammatory lesions. In this study, we examined the profiles of chemokines produced locally in the lung (parenchyma and bronchoalveolar lavage fluid) and systemically (serum) during the generation of IPS in the peri-BMT period. Protein and messenger RNA (mRNA) levels of CC chemokines (monocyte/lymphocyte attractants), especially monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, RANTES (regulated upon activation normal T-cell expressed and secreted), and C10, were preferentially induced in the lung by day 7 postallogeneic BMT. In addition, there was an increase in mRNA for IP-10 (a monocyte and Th1-cell chemoattractant). The CXC chemokines MIP-2 and KC, known neutrophil attractants, were moderately elevated. For the most part, these increases in chemokines were dependent on the coinfusion of allogeneic T cells with the BM inoculum. Ribonuclease protection assay and in situ hybridization analyses post-BMT showed that the lung was a major producer of MCP-1, a potent inducer of monocyte chemotaxis. Increases in MCP-1 levels in the lung preceded host APC influx whereas MIP-1alpha levels accompanied donor T-cell infiltration. In summary, we have shown that monocyte- and T-cell-attracting chemokines are associated with monocyte and T-cell recruitment during IPS.
Asunto(s)
Trasplante de Médula Ósea , Quimiocinas/inmunología , Monocitos/inmunología , Neumonía/inmunología , Linfocitos T/inmunología , Inmunología del Trasplante , Animales , Trasplante de Médula Ósea/efectos adversos , Movimiento Celular/inmunología , Quimiocinas/biosíntesis , Factores Quimiotácticos/biosíntesis , Factores Quimiotácticos/inmunología , Ratones , Monocitos/patología , Neumonía/etiología , Neumonía/patología , Síndrome , Linfocitos T/patología , Trasplante HomólogoRESUMEN
Osteoprotegerin (OPG) is a recently discovered protein related to the tumor necrosis factor receptor family. It has been shown to inhibit ovariectomy (ovx)-induced resorption in rats and increase bone mineral density in young mice. Tail suspension is a procedure that inhibits bone formation in maturing rodents. This study was designed to quantify OPG's effect on cortical bone formation. Fifty-four mice were assigned to one of five groups (n = 10-11/group). A baseline control group was killed on day 0 of the 10 day study. The remaining groups were: vivarium housed (nonsuspended) control mice receiving 0.3 mg/kg per day OPG; vivarium control mice receiving daily placebo injections; tail-suspended mice receiving 0. 3 mg/kg per day OPG; and tail-suspended mice receiving placebo injections. Tetracycline was administered on days 0 and 8. OPG treatment of tail-suspended mice produced mechanical properties similar to those of placebo-treated, vivarium-housed mice: structural stiffness (8.5%, 20.7%) and elastic (13.9%, 10.1%) and maximum (4.7%, 8.1%) force were increased compared with placebo controls (vivarium, suspended groups). Percent mineral composition was highly significantly greater (p < 0.001 for all comparisons) for OPG-treated mice in the femur, tibia, and humerus, relative to placebo treatment. Matrix mass was also significantly increased in the femur, although not to the same degree as mineral mass. OPG decreased the amount of femoral endocortical resorption compared with the placebo-treated groups for both vivarium (27%) and suspended (24%) mice. Administration of OPG significantly decreased endocortical formation of the tibia. Periosteal bone formation rates were not altered by OPG. OPG-mitigated tail suspension induced osteopenia not by returning bone formation to normal levels, but by inhibiting resorption and increasing percent mineral composition.
Asunto(s)
Enfermedades Óseas Metabólicas/tratamiento farmacológico , Glicoproteínas/farmacología , Suspensión Trasera , Receptores Citoplasmáticos y Nucleares , Animales , Enfermedades Óseas Metabólicas/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoprotegerina , Placebos , Ratas , Receptores del Factor de Necrosis TumoralRESUMEN
We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.
Asunto(s)
Antígeno B7-1/genética , Trasplante de Médula Ósea/inmunología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Alveolos Pulmonares/inmunología , Serina Endopeptidasas/genética , Animales , Antígenos CD/genética , Antígeno B7-2 , Trasplante de Médula Ósea/efectos adversos , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Expresión Génica/inmunología , Granzimas , Hipersensibilidad/inmunología , Hibridación in Situ , Interleucina-13/sangre , Interleucina-13/genética , Interleucina-13/inmunología , Interleucina-4/sangre , Interleucina-4/genética , Interleucina-4/inmunología , Interleucina-6/sangre , Interleucina-6/inmunología , Macrófagos Alveolares/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Monocitos/inmunología , Neumonía/etiología , Neumonía/inmunología , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/ultraestructura , ARN Mensajero/análisis , Células Th2/inmunología , Acondicionamiento PretrasplanteRESUMEN
We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.