RESUMEN
Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1ß levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1ß induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.
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Infecciones por Acinetobacter , Acinetobacter , Ortopedia , Acinetobacter/fisiología , Infecciones por Acinetobacter/microbiología , Animales , Proteínas Bacterianas/farmacología , Humanos , Inflamación , Ratones , Oseointegración , Percepción de QuorumRESUMEN
PURPOSE: The classic fracture model, described by Bonnarens and Einhorn in 1984, enlists a blunt guillotine to generate a closed fracture in a pre-stabilized rodent femur. However, in less experienced hands, this technique yields considerable variability in fracture pattern and requires highly-specialized equipment. This study describes a reproducible and low-cost model of mouse fracture healing using an open femoral osteotomy. METHODS: Femur fractures were produced in skeletally mature male and female mice using an open femoral osteotomy after intramedullary stabilization. Mice were recovered for up to 28 days prior to analysis with microradiographs, histomorphometry, a novel µCT methodology, and biomechanical torsion testing at weekly intervals. RESULTS: Eight mice were excluded due to complications (8/193, 4.1%), including unacceptable fracture pattern (2/193, 1.0%). Microradiographs showed progression of the fracture site to mineralized callus by 14 days and remodelling 28 days after surgery. Histomorphometry from 14 to 28 days revealed decreased cartilage area and maintained bone area. µCT analysis demonstrated a reduction in mineral surface from 14 to 28 days, stable mineral volume, decreased strut number, and increased strut thickness. Torsion testing at 21 days showed that fractured femurs had 61% of the ultimate torque, 63% of the stiffness, and similar twist to failure when compared to unfractured contralateral femurs. CONCLUSIONS: The fracture model described herein, an open femoral osteotomy, demonstrated healing comparable to that reported using closed techniques. This simple model could be used in future research with improved reliability and reduced costs compared to the current options.
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The survival rate for osteosarcoma patients with localized disease is 70% and only 25% for patients with metastases. Therefore, novel therapeutic and prognostic tools are needed. In this study, extensive screening and validation strategies identified Axl, EphB2, FGFR2, IGF-1R and Ret as specific receptor tyrosine kinases (RTKs) that are activated and promote the in vitro phenotype of two genetically different metastatic osteosarcoma cell lines. Initial phosphoproteomic screening identified twelve RTKs that were phosphorylated in 143B and/or LM7 metastatic human osteosarcoma cells. A small interfering RNA (siRNA) screen demonstrated that siRNA pools targeting ten of the twelve RTKS inhibited the in vitro phenotype of one or both cell lines. To validate the results, we individually tested the four siRNA duplexes that comprised each of the effective siRNA pools from the initial screen. The pattern of phenotype inhibition replicated the pattern of mRNA knockdown by the individual duplexes for seven of the ten RTKs, indicating the effects are consistent with on-target silencing. Five of those seven RTKs were further validated using independent approaches including neutralizing antibodies (IGF-1R), antisense-mediated knockdown (EphB2, FGFR2, and Ret) or small molecule inhibitors (Axl), indicating that those specific RTKs promote the in vitro behavior of metastatic osteosarcoma cell lines and are potential therapeutic targets for osteosarcoma. Immunohistochemistry demonstrated that Axl is frequently activated in osteosarcoma patient biopsy samples, further supporting our screening and validation methods to identify RTKs that may be valuable targets for novel therapies for osteosarcoma patients.
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Aseptic loosening of orthopedic implants is thought to be caused primarily by osteoclast differentiation induced by bone resorptive cytokines produced in response to phagocytosis of implant-derived wear particles. This study examined whether adherent endotoxin on the wear particles is responsible for inducing osteoclast differentiation as well as production of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor a (TNF-alpha). Removal of adherent endotoxin almost completely inhibited the responses to titanium (Ti) particles by both murine marrow cells and human peripheral blood monocytes. In vivo experiments showed that endotoxin removal reduced particle-induced osteolysis by 50-70%. Addition of lipopolysaccharide (LPS) to the "endotoxin-free" particles restored their ability to induce cytokine production and osteoclast differentiation in vitro. Moreover, marrow cells from mice that are hyporesponsive to endotoxin because of mutation of Toll-like receptor 4 induced significantly less cytokine production and osteoclast differentiation in response to Ti particles with adherent endotoxin than did marrow cells from normoresponsive mice. This mutation also resulted in significantly less particle-induced osteolysis in vivo. Taken together, these results show that adherent endotoxin is involved in many of the biological responses induced by orthopedic wear particles and should stimulate development of new approaches designed to reduce the activity of adherent endotoxin in patients with orthopedic implants.
Asunto(s)
Citocinas/biosíntesis , Proteínas de Drosophila , Endotoxinas/toxicidad , Osteoclastos/citología , Falla de Prótesis , Adhesividad , Animales , Resorción Ósea/etiología , Diferenciación Celular , Humanos , Técnicas In Vitro , Lipopolisacáridos/toxicidad , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Monocitos/fisiología , Mutación , Osteólisis/etiología , Receptores de Superficie Celular/genética , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Aseptic loosening is the most common cause of orthopaedic implant failure. This process is thought to be due to osteolysis induced by implant-derived wear particles. Teitelbaum and colleagues have recently developed a promising murine calvarial model of wear particle-induced osteolysis. However, prior to this study, this model had only been assessed qualitatively. We now report a reproducible, quantitative version of the calvarial model of wear particle-induced osteolysis, in which the extent of osteolysis (and repair) of entire parietal bones is assessed by histomorphometry of contact microradiographs. Using this model, we found that the osteolytic response is transient and rapidly repaired in one month old mice. The extent of osteolysis peaks 7 days after particle implantation and returns to baseline levels by 13 days. A similar amount of osteolysis and even more extensive repair is observed when particles are implanted repeatedly. In contrast, aged mice develop progressive osteolysis with no detectable repair. As a result, 26 month old mice have approximately 17-fold more osteolysis than one month old mice 21 days after particle implantation. Skeletally mature, adult mice (4-16 months old) show an intermediate pattern of response. Osteolysis in these mice peaks at 7 days after particle implantation but it is repaired more slowly than in the one month old mice. Taken together, these results underscore the role of an imbalance between bone resorption and bone formation in the development of aseptic loosening and suggest that agents that stimulate bone formation maybe useful in prevention or treatment of aseptic loosening.
Asunto(s)
Envejecimiento/fisiología , Osteólisis/fisiopatología , Hueso Parietal/efectos de los fármacos , Hueso Parietal/fisiopatología , Titanio/efectos adversos , Cicatrización de Heridas , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Osteólisis/patología , Hueso Parietal/patología , Factores de TiempoRESUMEN
BACKGROUND: Loosening of orthopaedic implants is mediated by cytokines that elicit bone resorption and are produced in response to phagocytosis of implant-derived wear particles. This accelerated bone resorption could be due to increased osteoclastic activity, survival, or differentiation. Although a number of in vitro studies have shown that wear particles increase osteoclastic activity, the increase was less than twofold in all cases. The objective of the current study was to test the hypothesis that wear particles stimulate bone resorption by inducing osteoclast differentiation. METHODS: Conditioned media were prepared from murine marrow cells or human peripheral blood monocytes incubated in the presence or absence of titanium particles. The effects of conditioned media on osteoclast differentiation were examined with use of a recently developed assay in which osteoclast precursors are co-cultured with mesenchymal support cells. RESULTS: The present study showed that titanium particles induced both murine marrow cells and human peripheral blood monocytes to produce factors that stimulated osteoclast differentiation. The mean increase in osteoclast differentiation was 29.3+/-9.4-fold. The stimulation of osteoclast differentiation led to a parallel increase in bone resorption. The amount of stimulation was regulated in a dose-dependent manner by the concentration of both titanium particles and conditioned media. The stimulation of osteoclast differentiation required interactions between the cells and the particles themselves and, therefore, was not due to metal ions, soluble contaminants released from the particles, or submicrometer particles. In contrast, conditioned media from control cells incubated in the absence of titanium particles had no detectable effect on any of the examined parameters. CONCLUSIONS: The present study showed that titanium particles stimulate in vitro bone resorption primarily by inducing osteoclast differentiation. In contrast, the titanium particles had only small effects on osteoclast activity or survival.
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Resorción Ósea , Osteoclastos/citología , Titanio/farmacología , Adulto , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Diferenciación Celular , Medios de Cultivo Condicionados , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/fisiología , Ratones , Osteoclastos/efectos de los fármacos , Falla de PrótesisRESUMEN
Neural networks with asymmetric synaptic connections (w(ij) not equal to w(ji)) display a broad range of dynamical behavior including fixed point, periodic, and "chaotic" trajectories. Previous work has shown that such networks undergo an order-chaos phase transition as various network parameters, such as the connectivity or the degree of asymmetry, are changed. Here, using an information theoretic approach, we present results which suggest that neurons are able to communicate information to each other most effectively in networks that are near the order-chaos transition. We then extend the model to incorporate some biologically relevant features.
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Biofisica/métodos , Red Nerviosa , Dinámicas no Lineales , Sinapsis , Animales , Modelos Estadísticos , Factores de TiempoRESUMEN
Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.
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Antígenos de Superficie/inmunología , Antígeno B7-1 , Proteínas Sanguíneas , Activación de Linfocitos , Péptidos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD , Apoptosis , Proteínas Reguladoras de la Apoptosis , Antígeno B7-H1 , Antígenos CD28/inmunología , Células CHO , Células Cultivadas , Cricetinae , Citocinas/biosíntesis , Humanos , Péptidos y Proteínas de Señalización Intercelular , Células Jurkat , Ligandos , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Proteína 2 Ligando de Muerte Celular Programada 1 , Receptor de Muerte Celular Programada 1 , Receptores de Antígenos de Linfocitos T/inmunología , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.
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Activación de Linfocitos/inmunología , Proteínas de la Membrana/análisis , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/análisis , Antígenos CD/inmunología , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Humanos , Receptores de Hialuranos/análisis , Receptores de Hialuranos/inmunología , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Glicoproteínas de Membrana Plaquetaria/análisis , Glicoproteínas de Membrana Plaquetaria/inmunología , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/inmunología , Receptores de Complemento/análisis , Receptores de Complemento/inmunología , Receptores de Complemento 3b , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Linfocitos T/citología , Tetraspanina 30RESUMEN
Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.
Asunto(s)
Antígenos CD28/biosíntesis , Antígenos CD28/genética , Molécula 1 de Adhesión Intercelular/fisiología , Activación de Linfocitos , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígeno B7-1/biosíntesis , Antígeno B7-1/genética , Antígeno B7-2 , Antígenos CD28/fisiología , Complejo CD3/inmunología , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Sueros Inmunes/farmacología , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Interleucina-2/biosíntesis , Interleucina-2/metabolismo , Activación de Linfocitos/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Receptores de IgG/biosíntesis , Receptores de IgG/genética , Subgrupos de Linfocitos T/inmunología , TransfecciónRESUMEN
The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos CD28/fisiología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Diferenciación de Linfocitos T/fisiología , Unión Competitiva/genética , Unión Competitiva/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Citocinas/biosíntesis , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Inmunoglobulinas/farmacología , Ligando Coestimulador de Linfocitos T Inducibles , Proteína Coestimuladora de Linfocitos T Inducibles , Ligandos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Proteínas/genética , Proteínas/metabolismo , Proteínas/farmacología , Proteínas/fisiología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
The BCR/ABL oncogene causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and myeloid cells. It is shown here that transformation of the hematopoietic cell lines Ba/F3, 32Dcl3, and MO7e with BCR/ABL results in an increase in reactive oxygen species (ROS) compared with quiescent, untransformed cells. The increase in ROS was directly due to BCR/ABL because it was blocked by the ABL-specific tyrosine kinase inhibitor STI571. Oxidative stress through ROS is believed to have many biochemical effects, including the potential ability to inhibit protein-tyrosine phosphatases (PTPases). To understand the significance of increased production of ROS, a model system was established in which hydrogen peroxide (H(2)O(2)) was added to untransformed cells to mimic the increase in ROS induced constitutively by BCR/ABL. H(2)O(2) substantially reduced total cellular PTPase activity to a degree approximately equivalent to that of pervanadate, a well known PTPase inhibitor. Further, stimulation of untransformed cells with H(2)O(2) or pervanadate increased tyrosine phosphorylation of each of the most prominent known substrates of BCR/ABL, including c-ABL, c-CBL, SHC, and SHP-2. Treatment of the BCR/ABL-expressing cell line MO7/p210 with the reducing agents pyrrolidine dithiocarbamate or N-acetylcysteine reduced the accumulation of ROS and also decreased tyrosine phosphorylation of cellular proteins. Further, treatment of MO7e cells with H(2)O(2) or pervanadate increased the tyrosine kinase activity of c-ABL. Drugs that alter ROS metabolism or reactivate PTPases may antagonize BCR/ABL transformation.
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Proteínas de Fusión bcr-abl/metabolismo , Células Madre Hematopoyéticas/metabolismo , Megacariocitos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Transformación Celular Neoplásica , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Proteínas Tirosina Fosfatasas/metabolismo , Rotenona/farmacología , Transducción de Señal , Vanadatos/farmacologíaRESUMEN
Osteoclasts are the primary cell type responsible for bone resorption. This paper reviews many of the known regulators of osteoclast activity, including hormones, cytokines, ions, and arachidonic acid metabolites. Most of the hormones and cytokines that inhibit osteoclast activity act directly on the osteoclasts. In contrast, most of the hormones and cytokines that stimulate osteoclast activity act indirectly through osteoblasts. Particularly interesting in this regard are agents that directly inhibit activity of highly purified osteoclasts yet stimulate activity of osteoclasts that are co-cultured with osteoblasts. Recent studies have demonstrated that the primary mechanism by which bone resorptive agents stimulate osteoclast activity indirectly is likely to be up-regulation of production of osteoclast differentiation factor/osteoprotegerin ligand (ODF/OPGL) by the osteoblasts. In addition to discussing regulators of osteoclast activity per se, this paper also reviews the role of osteoclast apoptosis to limit the extent of bone resorption.
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Osteoclastos/fisiología , Animales , Apoptosis , Resorción Ósea/etiología , Calcio/farmacología , Proteínas Portadoras/fisiología , Estrógenos/farmacología , Humanos , Interleucina-6/farmacología , Glicoproteínas de Membrana/fisiología , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Ligando RANK , Receptor Activador del Factor Nuclear kappa-BRESUMEN
For T cells to become functionally activated they require at least two signals. The B7 costimulatory molecules B7-1 and B7-2 provide the "second signal" pivotal for T cell activation. In this report, we studied the relative roles of B7-1 and B7-2 molecules in the induction of antitumor immunity to the T cell thymoma, EL4. We generated EL4 tumor cells that expressed B7-1, B7-2, and B7-1+B7-2 by transfecting murine cDNAs. Our results demonstrate that EL4-B7-1 cells are completely rejected in syngeneic mice. Unlike EL4-B7-1 cells, we find that EL4-B7-2 cells are not rejected but progressively grow in the mice. A B7-1- and B7-2-EL4 double transfectant was generated by introducing B7-2 cDNA into the EL4-B7-1 tumor line that regressed in vivo. The EL4-B7-1+B7-2 double transfectant was not rejected when implanted into syngeneic mice but progressively grew to produce tumors. The double transfectant EL4 cells could costimulate T cell proliferation that could be blocked by anti-B7-1 antibodies, anti-B7-2 antibodies, or hCTLA4 immunoglobulin, showing that the B7-1 and B7-2 molecules expressed on the EL4 cells were functional. In vivo, treatment of mice implanted with double-transfected EL4 cells with anti-B7-2 monoclonal antibody resulted in tumor rejection. Furthermore, the EL4-B7-2 and EL4-B7-1+B7-2 cells, but not the wild-type EL4 cells, were rejected in interleukin 4 (IL-4) knockout mice. Our data suggests that B7-2 expressed on some T cell tumors inhibits development of antitumor immunity, and IL-4 appears to play a critical role in abrogation of the antitumor immune response.
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Antígenos CD/fisiología , Tolerancia Inmunológica , Interleucina-4/fisiología , Glicoproteínas de Membrana/fisiología , Timoma/inmunología , Animales , Antígeno B7-1/fisiología , Antígeno B7-2 , Femenino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Transfección , Células Tumorales CultivadasRESUMEN
Cytokines that stimulate bone resorption are produced by cells found in bone marrow. However, marrow cells produce multiple factors, some of which may be inhibitors of osteoclast differentiation or activity. Thus, it is not possible to predict a priori whether the mixture of factors produced by marrow cells will have a net stimulatory or inhibitory effect on bone resorption. In this study, we showed that the net effect of whole marrow is to inhibit osteoclast activity induced by parathyroid hormone. Fractionation of the marrow revealed that the inhibitory activity was in the marrow fluid. However, conditioned media obtained from marrow cell cultures also inhibited osteoclast activity. Thus, it is likely that the inhibitory factors are produced in vivo by cells residing in the marrow. These inhibitory factors may represent a physiological regulatory process that plays an important role in maintaining the balance between bone resorption and formation. Because we have previously shown that interleukin-6 is one of the cytokines that parathyroid hormone induces in osteoblastic cells to stimulate osteoclast activity, one potential mechanism by which the marrow-derived inhibitory factors might act is by preventing this production of interleukin-6. However, we found that the marrow cell-conditioned media do not inhibit the production or activity of interleukin-6. Thus, the inhibitory factors appear to block osteoclast activity through a mechanism that does not involve interleukin-6. Taken together, these results demonstrate the importance of factors that inhibit bone resorption and emphasize that the presence of cytokines that stimulate bone resorption in conditions such as osteoporosis and orthopaedic implant loosening should be interpreted with caution unless evidence exists demonstrating their functional importance.
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Factores Biológicos/fisiología , Células de la Médula Ósea/metabolismo , Osteoclastos/metabolismo , Animales , Animales Recién Nacidos , Células de la Médula Ósea/efectos de los fármacos , Resorción Ósea/metabolismo , Fraccionamiento Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Interleucina-6/biosíntesis , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
We examined a patient who was clinically much better at reporting tactile stimulation when he could see his stimulated hand. Experimentally, we found that he had difficulty detecting taps accompanied by a salient (but not predictive) light located directly above his concealed hand. However, his performance was dramatically improved if the light was attached to a rubber hand situated in line with the patient's hidden hand. Previous studies have suggested that tactile sensitivity can be improved by nearby visual stimulation. However, our effect shows that crossmodal sensory facilitation does not only depend upon simple spatial proximity alone. Rather, a simultaneous visual event dramatically improves perception of touch specifically when it is attributed to the perceiver's stimulated limb.
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Mano , Goma , Tacto/fisiología , Lateralidad Funcional/fisiología , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Estimulación LuminosaRESUMEN
The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production. In contrast, Ox40L-deficient mice do not have defects in T cell homing or humoral immune responses. In vitro, Ox40L-deficient dendritic cells are defective in costimulating T cell cytokine production. Thus, Ox40L has a critical costimulatory function in vitro and in vivo for dendritic cell:T cell interactions.
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Células Dendríticas/inmunología , Glicoproteínas de Membrana , Receptores del Factor de Necrosis Tumoral/inmunología , Linfocitos T/inmunología , Células 3T3 , Animales , Antígenos T-Independientes/inmunología , Dermatitis por Contacto/inmunología , Haptenos/inmunología , Hemocianinas/inmunología , Hipersensibilidad Tardía/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ligando OX40 , Ovalbúmina/inmunología , Receptores del Factor de Necrosis Tumoral/genética , Factores de Necrosis TumoralRESUMEN
Aseptic loosening is thought to be due primarily to osteolysis induced by cytokines and prostaglandins that are produced in response to implant-derived wear particles. Because endotoxin has many of the same effects as have been reported for wear particles, we hypothesized that adherent endotoxin may be responsible for the biological responses induced by wear particles. We demonstrated the presence of significant levels of adherent endotoxin on commonly used preparations of titanium particles as well as on titanium and titanium-alloy implant surfaces. In contrast, supernatants obtained by centrifugation of particle suspensions contained approximately 1% as much endotoxin as did the particles. Therefore, it is erroneous to assume that particles do not contain endotoxin on the basis of data that it cannot be detected in their supernatants or filtrates. These results emphasize the importance of considering the potential role of adherent endotoxin when examining the in vitro effects of wear particles and the in vivo performance of orthopaedic implants. We also developed a protocol that removed more than 99.94% of the adherent endotoxin from the titanium particles without detectably affecting their size or shape. The removal of adherent endotoxin will allow comparison of the biological responses induced by particles with or without adherent endotoxin.
Asunto(s)
Endotoxinas/análisis , Procedimientos Ortopédicos , Prótesis e Implantes , Titanio , Endotoxinas/aislamiento & purificaciónRESUMEN
The current model of T cell activation requires two signals. The first signal is specific, requiring T cell receptor recognition and binding to MHC/Antigen presented by an antigen-presenting cell. The second signal is nonspecific, resulting from the binding of B7 ligand on the antigen-presenting cell with its receptor, CD28, on the T cell. If both signals are provided, the T cell will proliferate and secrete cytokines. Recently, it has been shown that CTLA4, another receptor for B7 that is upregulated following T cell after activation, can deliver an inhibitory signal, downregulating T cell proliferation. The B7 family of ligands has two family members, B7-1 and B7-2. They both bind to CD28 and CTLA4, but they differ in their binding affinity, structure, and temporal expression. Considerable research has been done on the CD28/B7 costimulatory pathway. Different ways of manipulating this pathway could provide insights into the mechanism and treatment of opposing pathological states. Blocking the CD28/B7 pathway could result in immunosuppression, with implications for the treatment of autoimmune diseases, organ transplantation, and graft vs. host disease. Activating the CD28/B7 pathway could be useful for including the immune system to recognize and eliminate tumors that evade the immune system. Finally, the CD28/B7 pathway could be involved with maintaining immune tolerance, as recent studies suggest the preferential binding of the B7-CTLA4 pathway results in the down-regulation of the responding T cells. Thus, the B7/CD28/CTLA4 pathway has the ability to both positively and negatively regulate immune responses.