Proteins and their peptide motifs in acellular apatite mineralization of scaffolds for tissue engineering

Many proteins in the inorganic=organic matrix of bone induce or modulate or inhibit mineralization of apatite in vivo. Many attempts have been made to mimic and understand this mechanism as part of bone formation, and ectopic mineralization and control thereof. Many attempts have also been made to use such proteins or protein fragments to harness their potential for improved mineralization. Such proteins and peptide motifs have also been the inspiration for attempts of making mimics of their structures and motifs using chemical or biological synthesis. The aim of this review is to highlight how proteins and (poly)peptides themselves impact mineralization in the human body, and how those could be used and have been used for improving apatite mineralization, for example, on or in materials that by themselves do not induce apatite mineralization but otherwise have interesting properties for use as bone tissue engineering scaffolds.J. Benesch wishes to acknowledge the financial support from FCT, postdoctoral fellowship scholarship SFRH/BPD/17584/2004. This work was carried out under the scope of the European Union NoE EXPERTISSUES (NMP3-CT-2004500283) and partially funded by the European Union FP6 STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and FCT project ProteoLight (PTDC/FIS/68517/2006)

Clinical impact of radiolabeled anti-CD4 antibodies in the diagnosis of rheumatoid arthritis

Human rheumatoid arthritis (RA) is characterized by severe chronic synovitis with abundance of CD4-positive T-cells and macrophages in the inflamed synovial tissue. These cells likely play a central pathogenetic role in RA and experimental models of arthritis. CD4 is a surface molecule present on the helper/inducer subset of T lymphocytes and macrophages, although with a lower density on the latter. CD4+ T-cells/macrophages and their cytokine products, therefore, represent potential therapeutic and diagnostic targets in RA. CD4, a 55 kDa monomeric glycoprotein, binds as a T-cell coreceptor to conserved areas of the major histocompatibility complex II on antigen-presenting cells, and thereby participates in the formation of the immunological synapse and the provision of the so-called “second signal” required for full activation of T-helper cells. A specific diagnostic or therapeutic approach is the direct targeting of CD4+ T-cells by anti-CD4 monoclonal antibodies (mAbs). In addition to therapeutic clinical trials with anti-CD4 mAbs in RA, which have yielded only ambiguous results, anti-CD4 mAbs have also been developed and applied for diagnostic purposes. The studies thus far conducted in RA have focused on the following aspects: 1) comparison of anti-CD4 mAb imaging to the established early methylene diphosphonate (MDP) scan; 2) biodistribution/ pharmacokinetics studies; and 3) specificity of joint imaging with anti-CD4 mAbs in comparison to control immunoglobulins with irrelevant specificity. The available results in RA and arthritis models show that 99mTc-anti-CD4 mAbs are well-suited to actively image diseased joints, and clearly allow more specific imaging than 99mTc-MDP or control immunoglobulins. Because effective treatment is known to reduce the density of CD4+ cells in the inflamed synovial membrane, diagnostic methods targeted to CD4 warrant further attention, also for early diagnosis of clinically silent joints, precise description of the cellular infiltrates, and monitoring of anti-rheumatic therapy

ADAM15 in Apoptosis Resistance of Synovial Fibroblasts: Converting Fas/CD95 Death Signals Into the Activation of Prosurvival Pathways by Calmodulin Recruitment

Objective: To investigate mechanisms underlying the capability of ADAM15 to transform FasL‐mediated death‐inducing signals into prosurvival activation of Src and focal adhesion kinase (FAK) in rheumatoid arthritis synovial fibroblasts (RASFs). Methods: Caspase 3/7 activity and apoptosis rate were determined in RASFs and ADAM15‐transfected T/C28a4 cells upon Fas/CD95 triggering using enzyme assays and annexin V staining. Phosphorylated Src and FAK were analyzed by immunoblotting. Interactions of ADAM15 and CD95 with calmodulin (CaM), Src, or FAK were analyzed by pull‐downs using CaM–Sepharose and coimmunoprecipitations with specific antibodies. Protein binding assays were performed using recombinant CaM and ADAM15. Immunofluorescence was performed to investigate subcellular colocalization of ADAM15, Fas/CD95, and CaM. Results: The antiapoptotic effect of ADAM15 in FasL‐stimulated cells was demonstrated either by increased apoptosis of cells transfected with an ADAM15 construct lacking the cytoplasmic domain compared to cells transfected with full‐length ADAM15 or by reduced apoptosis resistance of RASFs upon RNA interference silencing of ADAM15. Fas ligation triggered a Ca2+ release‐activated Ca2+/calcium release‐activated calcium channel protein 1 (CRAC/Orai1) channel–dependent CaM recruitment to Fas/CD95 and ADAM15 in the cell membrane. Simultaneously, Src associated with CaM was shown to become engaged in the ADAM15 complex also containing cytoplasmic‐bound FAK. Accordingly, Fas ligation in RASFs led to ADAM15‐dependent phosphorylation of Src and FAK, which was associated with increased survival. Pharmacologic interference with either the CaM inhibitor trifluoperazine or the CRAC/Orai inhibitor BTP‐2 simultaneously applied with FasL synergistically enhanced Fas‐mediated apoptosis in RASFs. Conclusion: ADAM15 provides a scaffold for formation of CaM‐dependent prosurvival signaling complexes upon CRAC/Orai coactivation by FasL‐induced death signals and a potential therapeutic target to break apoptosis resistance in RASFs

A novel Saa3-promoter reporter distinguishes inflammatory subtypes in experimental arthritis and human synovial fibroblasts

Item does not contain fulltextOBJECTIVE: To evaluate the applicability of a lentiviral (LV) serum amyloid A3 (Saa3)-promoter luciferase (Luc) reporter for assessing inflammation in experimental arthritis, synovial fibroblasts (SF) from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. METHODS: In mice, synovium was transduced in vivo by cholesterol optimised LV, and two flares of acute joint inflammation were induced by injection of streptococcal cell wall (SCW) material into the knee-joint cavity. The time course of synovial inflammation was assessed using ex vivo luciferase assays, and histology. Uptake of (99m)technetium (Tc) was used to assess oedema. SF (n=12) of RA and OA patients were stratified by hierarchical clustering of whole genome expression profiles. Relative Saa3-promoter responses were determined in cytokine- or toll-like receptor (TLR)-stimulated SF subgroups. RESULTS: In vivo, the Saa3-promoter reporter activity was strongly upregulated at 1 and 2 days after the first and second SCW challenge. The Saa3-promoter activities during acute inflammation correlated with Tc uptake measurements but were more sensitive and able to respond to the ongoing synovitis in the chronic phase of SCW arthritis. Molecular stratification defined two inflammatory SF subtypes, unrelated to disease classification. Relative Saa3-promoter responses to interleukin 1beta, tumour necrosis factor alpha and TLR4 agonist were significantly increased in OA/RA SF with a high compared to a low inflammatory profile subtype. Serum stimulation of the Saa3-promoter reporter cell-line could distinguish between healthy and RA patients. CONCLUSION: The Saa3-promoter reporter demonstrates a robust and feasible tool for assessing the course and severity of experimental arthritis and for distinguishing molecularly distinct inflammatory SF subtypes from a heterogeneous patient population