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Subchondral bone loss is a characteristic feature of inflammatory arthritis. Recently, estrogen receptor-related receptor-alpha (ERR-alpha), an orphan nuclear receptor, has been found to be involved in activation of macrophages. We hypothesized that ERR-alpha which is expressed and also functional in articular chondrocytes, osteoblasts and osteoclasts, may be involved in rodent models of inflammatory arthritis.Erosive arthritis was induced in DBA/1 mice by injection of type II collagen in Freund's complete adjuvant. RNA was isolated from the bone and joints and expression of ERR-alpha and cartilage (GDF5 and Col2a1) and bone [bone sialoprotein (BSP) and osteocalcin (OCN)] markers was analysed by semi-quantitative PCR.We report for the first time that the expression of ERR-alpha is dysregulated in bones and joints in a mouse model of inflammatory arthritis. Specifically, we show that ERR-alpha expression is down-regulated early in bone and later in joints of mice with type II CIA. Concomitantly, temporal changes were observed in GDF-5 and Col2a1 expression in joints following both initial injection and booster injection of type II collagen. Similarly, down-regulation of ERR-alpha mRNA expression in subchondral bone in mice with induced joint inflammation was also paralleled by down-regulation of markers of bone formation (BSP, OCN).These data suggest that dysregulation of ERR-alpha expression may precede and contribute to the destruction of cartilage and bone accompanying inflammatory arthritis.

Objectifs Le Fibroblast Growth Factor 23 (FGF23), hormone cle de l’axe « rein/parathyroides/os », est secrete par les osteocytes. Aucune donnee n’a ete publiee sur l’effet local du FGF23 sur les osteoclastes (OC), cellules geantes multinucleees capables de resorber la matrice osseuse. Objectif Etudier l’effet du FGF23 sur la physiologie de l’OC, en comparaison a la 1,25(OH) 2 vitamine D (1,25D). Materiels et methodes Les OC, obtenus a partir de monocytes humains purifies, ont ete exposes au FGF23 et a la 1,25D. L’osteoclastogenese a ete analysee grâce au comptage des cellules colorees (TRAP) de plus de 3 noyaux. L’activite de resorption a ete etudiee grâce a la quantification des surfaces resorbees par OC. Le FGF recepteur inhibiteur (FGFRi) et l’etude de la voie de signalisation Akt ont ete utilises pour confirmer les resultats. Resultats principaux La 1,25D et le FGF23 inhibaient l’osteoclastogenese, respectivement de 91% et de 23% (p

Macrophages and osteoclasts develop unique contact sites with the extracellular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their function is lacking. We have used chicken and human monocytes differentiating in vitro into osteoclast-like cells in the presence of RANKL-ODF to study these cytoskeletal structures. During the differentiation process, podosomes are redistributed from the cell body in early macrophages to the cell periphery in increasingly spread and multinucleated cells expressing high levels of integrin alphaVbeta3. Immunofluorescence with anti-phosphotyrosine antibodies revealed increased tyrosine-phosphorylation at the basal tips of these podosomes. RANKL-ODF treatment reinforced the peripheral location of podosomes and initiated their partial fusion to larger F-actin-containing structures that displayed reduced levels of tyrosine phosphorylation. Paxillin and the FAK-related kinase Pyk2 colocalized with integrin alphaVbeta3 in the juxtamembrane region surrounding individual podosomes. In lysates of macrophages and differentiated osteoclasts both paxillin and Pyk2 associated with synthetic and recombinant polypeptides containing the C-terminal region of the integrin beta3 cytoplasmic domain. These in vitro interactions were direct and they were abolished by substitutions in the beta3 integrin peptides known to disrupt integrin function in vivo. The marked adhesion-dependent tyrosinephosphorylation of Pyk2 and paxillin however did not detectably alter their interaction with beta3 tail peptides in cell lysates. Our results provide novel insight into the molecular architecture and the phosphorylation dynamics in podosomes. Moreover, they outline a novel potential mechanism for the recruitment of paxillin and Pyk2 to beta3 integrin-dependent cell contacts.

Background Autoantibodies against citrullinated proteins (ACPA) are amongst the strongest risk factors for bone destruction in rheumatoid arthritis (RA) Objectives We therefore hypothesized that these autoantibodies directly influence bone metabolism. Methods A strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption was found in the serum of RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination such as PAD2 and revealed specific inducible N-terminal citrullination of vimentin during their differentiation process. Affinity purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to the osteoclast surface, but also lead to a robust induction of osteoclastogenesis and bone resorptive activity. Adoptive transfer of human MCV antibodies into RAG1-/- mice was followed by induction of osteopenia and increased osteoclastogenesis in vivo. This effect was based on the inducible release of TNFa from osteoclast precursors and the subsequent increase of CD11b+ osteoclast precursor cell numbers with enhanced expression of RANK and CSF1 receptors in vivo . Conclusions Our data thus suggest that autoantibody formation to citrullinated vimentin directly induces bone loss and provides a novel link between the adaptive immune system and bone. Disclosure of Interest None Declared

To study the contribution of v-Jun homodimers to oncogenesis, we constructed artificial v-Jun derivatives in which the natural dimerization domain of v-Jun was replaced by an heterologous homodimerization domain from either the viral EB1 or the yeast GCN4 transcription factor. The resulting v-Jun chimeric proteins, called v-Juneb1 and v-Jungcn4, which can no longer dimerize with Jun or Fos, should only form homodimers in the cell. Helper-independent retroviruses expressing v-Jun, v-Juneb1 and v-Jungcn4 were generated. All three viruses transformed primary cultures of chick embryo cells with the same high efficiency and promoted local tumor growth after subcutaneous injection of infected cells in young animals. In contrast, after intravenous injection of viral suspensions into chick embryos, only the chimeric proteins produced internal tumors that were lethal. These tumors were leiomyosarcomas located within the liver and along the digestive tract. Thus, in vivo, v-Juneb1 and v-Jungcn4 are more potent oncoproteins than v-Jun. These data demonstrate that when forced to accumulate, v-Jun homodimers can induce tumors efficiently. They also show that the oncogenic potential of v-Jun can be regulated through the properties of its dimerization domain.

Transforming growth factor beta 1 (TGF-beta 1) inhibits growth of primary cultures of chick embryo fibroblasts by affecting G1 and strongly increasing the generation time. This inhibition is reversed by the nuclear oncogenes v-jun, v-fos, v-myc, but not v-erbA and v-ets. It is also reversed by v-myb from either avian myeloblastosis virus or avian E26 retrovirus. Taken together, these results strongly suggest that independent, functional interferences may take place between the TGF-beta 1-induced growth inhibitory pathway and the oncogen-driven stimulatory pathway(s) at the level of the AP-1, Myc, and Myb transcription factors.

The v-erbA oncogene encodes an altered form of the nuclear receptor of the thyroid hormone triiodothyronine (T3). This altered receptor is unable to bind T3, and blocks the differentiation of chicken erythrocyte progenitor cells. To identify the cellular target genes of v-ErbA in the transformed cells, we analysed the expression of several genes in normal erythrocytic cells exposed to T3, and found that the gene encoding carbonic anhydrase II is transcriptionally activated by the hormone. In contrast, this gene is repressed in erythroleukemic cells transformed by the v-erbA product. To investigate in more details the effects of v-ErbA, we constructed a mutant of v-ErbA in which we restored the ability to bind T3. This mutant developed its oncogenicity only in the absence of T3. Upon binding of T3, the transformed cells differentiated and immediately expressed the carbonic anhydrase II gene. These data show that v-ErbA directly inhibits the transcription of the carbonic anhydrase II gene, presumably by competing with normal T3 receptors. The carbonic anhydrase II gene is the first identified target gene of the v-ErbA oncoprotein in erythroleukemic cells.

The enumeration of erythropoietic colony-forming cells in vitro has allowed us to complete previous data on changes in the various erythroid cell populations during chick embryogenesis. Erythrocytic colony-forming units in culture (CFU-cE) which are sensitive to avian erythropoietin appear in the blastoderm as soon as the 24th hour of development. They represent most likely precursors of the megalocytic erythropoiesis, and do not seem to derive from stem cells common with normocytic erythropoiesis. Data concerning vitelline normocytic erythropoiesis were analysed in a kinetic model based on stochastic change of the stem cells. From this model it appears that 17−20 cell divisions are required for differentiation of erythrocytes from stem cells.

The response to infection of chicken hemopoietic cells derived from the early stages of embryogenesis by avian myeloblastosis virus (AMV) and avian erythroblastosis virus (AEV) was investigated. It was found that erythroid progenitor cells were present in the blastoderm at a higher frequency than that of myeloid progenitor cells. These results correlate with the observation that target cells for AEV were found to be more numerous than those for AMV. Therefore, blastoderm cells are of potential value in understanding the mechanisms of oncogenesis at the level of the target cells.

Embryonic chick cells from the primitive streak stage to later stages of the developing embryo were infected with avian erythroblastosis virus (AEV). The data indicate that the greatest number of target cells for AEV was observed in the 12-somite blastoderm and gradually decreased in hemopoietic tissues with the development of the embryo. The target cell for AEV is not in the BFU-E compartment, as it is in the adult bone marrow, but is probably recruited within the CFU-M compartment which precedes the BFU-E compartment. Our studies also show that a significant number of transformed colonies derived from embryonic hemopoietic tissues undergo hemoglobinization in contrast with what is observed in transformed colonies of bone marrow. A complete characterization of the embryonic and adult hemoglobin is at present under study.

Young chick blastoderms contain erythropoietic stem cells able to produce normocytic clones when grafted to irradiated chickens. These stem cells are present as early as the 36th hour of incubation, long before the occurrence of the first normoblasts. Later, they exhibit exponential growth.

The dual leukemogenic response, involving both the erythroid and myeloid hemopoietic systems in chickens infected with E26 virus, has previously been described ( C. Moscovici, J. Samarut, L. Gazzolo, and M. G. Moscovici, 1981 . Virology 113, 765–768; K. Radke, H. Beug, S. Kornfeld, and T. Graf, 1982. Cell 31, 643–653 ). Similarly, the in vitro response of the two lineages resulted in the concomitant transformation and proliferation of erythroblast and myeloblast leukemic cells. The present study, using embryonic tissues at very early stages of development, was valuable in implying that E26 target cells are recruited among uncommitted erythroid-myeloid stem cells as well as myeloid- or erythroid-committed progenitor cells. Therefore, E26 may be the first avian retrovirus capable of interacting with uncommitted hemopoietic precursor cells.

The enumeration of erythropoietic colony-forming cells in vitro has allowed us to complete previous data on changes in the various erythroid cell populations during chick embryo-genesis. Erythrocytic colony-forming units in culture (CFU-cE) which are sensitive to avian erythropoietin appear in the blastoderm as soon as the 24th hour of development. They represent most likely precursors of the megalocytic erythropoiesis, and do not seem to derive from stem cells common with normocytic erythropoiesis. Data concerning vitelline normocytic erythropoiesis were analysed in a kinetic model based on stochastic change of the stem cells. From this model it appears that 17-20 cell divisions are required for differentiation of erythrocytes from stem cells.

It has recently been suggested that the endogenous retroviruses present in many different species might be involved during stimulation of the immune system of their hosts. We have now studied the expression of two avian retroviral antigens p27 and gp85 in chicken lymphoid cells by indirect immunofluorescence (IIF) and by complement-dependent microcytotoxicity (CDM). We have now found that these viral antigens are expressed in peripheral blood leukocytes of adults and embryos and in splenic and bursal lymphocytes of Spafas gs- chf- chickens but they are not expressed in fibroblasts cultured from the feather follicles of the same individual adult birds nor in fibroblasts cultured from embryos of the same flock. The differential expression of viral antigens in leukocytes may be related to a specific property or function of these cells.

The hemoglobin patterns of 293 cord bloods from Northern Algeria were examined by electrophoresis on cellulose-acetate strips. A fast-moving component, identified as Hb Bart's, was found in about 10% of the cases. The levels of Hb Bart's ranged from 0.1 to 10% of the total hemoglobin. There was a significant correlation between the Hb Bart's levels and the decrease in MCV. The relative rates of globin chain synthesis measured by 3H-leucine incorporation was estimated in 15 cord bloods. It was found imbalanced in the 5 cord bloods which contained more than 0.5% Hb Bart's. These findings suggest that elevated Hb Bart's levels in the Algerian population are due to the presence of alpha-thalassemia.