RESUMO
Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflammation, chemotaxis of progenitor cells, mitosis, differentiation of cartilage, and replacement with bone. This multistep cascade is orchestrated by cytokines and morphogens. Members of the interleukin (IL)-17 family, including IL-17B, have been identified in cartilage, but their expression during fracture healing is unknown. In this study, we determined the immunolocalization of cytokines IL-17A and IL-17B, along with the IL-17 receptor (IL-17R) and IL-17 receptor-like protein (IL-17RL), during the sequence of fracture repair in a standard model. The results were extended to developmental changes in the epiphyseal growth plate of long bones. Members of the IL-17 family were localized in chondrocytes in the fracture callus. Moreover, we found significant parallels to the localization of these cytokines and their receptors in chondrocytes during an endochondral differentiation program in the epiphyseal growth plate.
Assuntos
Condrócitos/metabolismo , Consolidação da Fratura , Interleucina-17/biossíntese , Receptores de Interleucina-17/biossíntese , Animais , Animais Recém-Nascidos , Calo Ósseo/metabolismo , Diferenciação Celular , Condrócitos/citologia , Fraturas do Fêmur/metabolismo , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Imuno-Histoquímica , Masculino , Ratos , Ratos Long-Evans , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismoRESUMO
To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.
Assuntos
Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Técnicas Biossensoriais/métodos , Antígeno Prostático Específico/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/normas , Humanos , Cinética , Ligantes , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/imunologia , Ligação Proteica , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Ressonância de Plasmônio de Superfície/normas , Fatores de TempoRESUMO
Members of the interleukin-17 cytokine family are present in a variety of tissues (1-3), although the founding member, interleukin-17, is expressed exclusively in T cells and B cells (4-8). The cloning and characterization of a novel single-pass transmembrane protein with limited homology to the interleukin-17 receptor is reported. High mRNA levels were detected in prostate, cartilage, kidney, liver, heart, and muscle, whereas transcripts were barely detected in thymus and leukocytes. At least 11 RNA splice variants were found, transcribed from 19 exons on human chromosome 3p25.3-3p24.1. Differential exon usage was found in different tissues by quantitative reverse transcriptase-PCR. Predicted proteins range from 186 to 720 amino acids. Soluble secreted proteins lacking transmembrane and intracellular domains are predicted from several splice isoforms and may function as extracellular antagonists to cytokine signaling by functioning as soluble decoy receptors. Using antibodies directed at the cytoplasmic and extracellular domains of this protein, we investigated its localization and found that it was expressed in a variety of normal human tissues including prostate and in prostate cancer.