L’ostéopétrose, de la souris à l’homme

Les ostéoclastes sont des acteurs essentiels du remodelage osseux, et des anomalies de leur différenciation ou de leur activité conduisent à l’apparition de maladies osseuses, dont des défauts de résorption osseuse qui se traduisent par l’apparition d’une ostéopétrose. Différents modèles murins développant une ostéopétrose secondaire à l’apparition de mutations spontanées ou à l’invalidation de gènes ont permis de décrypter, au moins en partie, les mécanismes impliqués dans la différenciation et l’activité des ostéoclastes. Chez l’être humain, en revanche, seules des anomalies d’activité de l’ostéoclaste ont été décrites. Trois modèles murins, les souris oc/oc, gl/gl et Clcn7-/-, présentent un phénotype proche de celui de patients atteints d’ostéopétrose maligne infantile, la forme d’ostéopétrose la plus sévère chez l’homme. Des mutations dans les gènes TCIRG1, GL et CLCN7 ont donc été recherchées chez des patients ostéopétrotiques, et retrouvées majoritairement dans l’ostéopétrose maligne infantile et dans l’ostéopétrose de type II. Une telle correspondance phénotypique et génétique fait de ces trois mutants de souris des modèles particulièrement adaptés à l’étude de l’ostéopétrose humaine.The osteoclast is the main effector of bone resorption. Failure in osteoclast differentiation or function leads to osteopetrosis, a bone disease characterized by an impaired bone resorption. Analysis of mouse models developing osteopetrosis as a consequence of naturally occuring mutations or gene knockouts allowed to establish the osteoclast differentiation pathway. Among these models, the oc/oc, the gl/gl and the Clcn7–/– mice present a phenotype similar to the one displayed by patients with infantile malignant osteopetrosis, the most severe form of osteopetrosis in human. Analysis of these models led to the identification of different mutations in the corresponding human genes TCIRG1, GL and CLCN7, in osteopetrotic patients. Mutations in the TCIRG1 gene seem the most frequent cause of malignant osteopetrosis and mutations in the CLCN7 gene seem the most frequent cause of type II osteopetrosis. Therefore, these three mouse models appear to be particularly well suited for the study of the osteoclast function in order to provide new insights in the therapy of osteopetrosis

Interleukin-7 partially rescues B-lymphopoiesis in osteopetrotic oc/oc mice through the engagement of B220+ CD11b+ progenitors.

OBJECTIVE: We recently identified in the mouse bone marrow a B-lymphoid/myeloid B220+ CD11b+ progenitor population. This population is accumulated in the osteopetrotic oc/oc mouse, which suggests that it could be controlled by bone marrow factors whose expression varies in this pathologic bone environment. Among the possible factors, interleukin (IL)-7 is involved in the control of B lymphopoiesis and osteoclastogenesis. Therefore, we hypothesized that IL-7 could regulate the accumulation of the B220+ CD11b+ population in oc/oc mice. METHODS: B220+ CD11b+ cells sorted from oc/oc mice were treated with IL-7 and their phenotype was analyzed by flow cytometry and real-time reverse transcriptase polymerase chain reaction (RT-PCR). In vivo, IL-7 was injected in oc/oc mice, and B220+ CD11b+ and B cells, as well as B-cell proliferation and apoptosis, were analyzed by flow cytometry. The expression of B lymphopoiesis and myelopoiesis markers was analyzed by real-time RT-PCR. RESULTS: In vitro, IL-7 induced the differentiation of B220+ CD11b+ cells into B lymphocytes through the induction of Pax5 and the inhibition of myeloid markers. In vivo, IL-7 injections in oc/oc mice induced a decrease of the B220+ CD11b+ population and the partial restoration of B-cell population, which was reduced in oc/oc mice. In parallel, upon IL-7 injections, Pax5 expression was induced in B220+ cells and B-cell apoptosis was reduced. CONCLUSIONS: Our results demonstrate that IL-7 injection can partially rescue B lymphopoiesis in oc/oc mice through the engagement of the B220+ CD11b+ population in the B-lymphoid pathway. Therefore, IL-7 delivery could represent a new therapeutic perspective to circumvent the lymphopenia observed in infantile malignant osteopetrosis patients

An alternative procedure for extraction of DNA from ancient and weathered bone fragments

International audienceBone is the most challenging tissue for DNA extraction and purification. Expensive commercial kits and specific equipments are often used in forensic and anthropology laboratories towards that goal. We present here an integrated procedure that gives satisfactory results for DNA preparation from fresh, ancient or weathered bones. Extraction is performed under simple but efficient vacuum-controlled conditions that greatly limit the risks of cross-contaminations. The whole process has been designed to minimize the need for expensive equipment and chemicals, and to be compatible with any molecular biology laboratory. In addition, no toxic reagents are necessary and the procedure is straightforward. Combined with quantitative polymerase chain reaction (qPCR), this method allows species identification and sex determination from subcellular amount of DNA (1-5 pg). In addition, enough DNA is generally obtained for human DNA profiling if necessary. The whole procedure from bone treatment to the final qPCR results takes less than 48 hours. This procedure should allow any laboratory with standard molecular biology equipment and expertise to perform bone DNA characterization whenever necessary

Characterization of IL-10-secreting T cells derived from regulatory CD4+CD25+ cells by the TIRC7 surface marker.

Natural CD25(+)CD4(+) regulatory T cells (Treg) are essential for self-tolerance and for the control of T cell-mediated immune pathologies. However, the identification of Tregs in an ongoing immune response or in inflamed tissues remains elusive. Our experiments indicate that TIRC7, T cell immune response cDNA 7, a novel membrane molecule involved in the regulation of T lymphocyte activation, identifies two Treg subsets (CD25(low)TIRC7(+) and CD25(high)TIRC7(-)) that are characterized by the expression of Foxp3 and a suppressive activity in vitro and in vivo. We also showed that the CD25(low)TIRC7(+) subset represents IL-10-secreting Tregs in steady state, which is accumulated intratumorally in a tumor-bearing mice model. Blockade of the effect of IL-10 reversed the suppression imposed by the CD25(low)TIRC7(+) subset. Interestingly, these IL-10-secreting cells derived from the CD25(high)TIRC7(-) subset, both in vitro and in vivo, in response to tumoral Ags. Our present results strongly support the notion that, in the pool of natural Tregs, some cells can recognize foreign Ags and that this recognition is an essential step in their expansion and suppressive activity in vivo

Antenne miniature implantée pour applications RFID UHF

National audienceDans cet article, la conception d’une antenne miniature implantée dans un petit animal et destinée à fonctionner dans la bande RFID UHF européenne est présentée. Un des objectifs consiste à miniaturiser l’élément rayonnant tout en préservant son efficacité afin de permettre une communication fiable entre un dispositif d’identification externe et le petit animal. L’élément rayonnant est un dipôle de taille réduite combiné avec une petite antenne boucle rectangulaire. Cette antenne de dimensions 2,4x25,4x0,44mm3 intègre une puce Impinj Monza® 4 présentant à 868MHz une impédance de (5,5-j74) Ohms. La conception et l’optimisation de l’antenne ont été réalisées à l’aide du logiciel HFSS d’ANSYS. Les résultats obtenus donnent une efficacité de rayonnement de 0,7% et un gain total de -17,5dBi

Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect.

International audienceThe incidence of oral tumors is increasing around the world and despite recent advances in early detection and diagnosis, current treatments are still unsatisfactory. Recent data suggest that tumor persistence and recurrence could be due to the presence of a rare cell population called cancer stem cells (CSCs), which are generally spared by traditional treatments. Therefore, identification and characterization of CSCs are extremely important to develop novel and effective treatment strategies for cancer. The aim of this study was to identify and isolate CSCs in an established murine head and neck squamous cell carcinoma (HNSCC) cell line and to investigate the influence of hypoxic conditions on the isolated cell popul-ation. Using the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity, which is now recognized as a CSC marker in various tumors, we isolated a cell population expressing high levels of ALDH1 (ALDH1high) representing 1±0.6% in the murine SCC-VII cell line. These cells were injected subcutaneously in syngeneic animals to evaluate their tumorigenic properties. For the lowest injected cell dose (250 injected cells), tumor occurrence and median tumor size were higher in ALDH1high injected mice than in ALDH1low injected mice. Following an in vivo passage and culture in serum-free medium, the percentage of ALDH1high cells increased by 3‑fold in SCC-VII CSCs (oral spheres) compared to the SCC-VII cell line. This percentage was further increased when oral spheres were cultured under hypoxic conditions. In conclusion, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population

Wireless interrogation of small animal phantoms with a miniature implanted UHF RFID tag

International audienceIn this article, the RSSI measurement with a miniature implanted tag into a small animal phantom, intended to work in the European UHF RFID band is presented. The miniaturization of the radiating element while preserving its efficiency allows the reliable communication between an external interrogation device and the identification tag aimed to be implanted into a small animal. The paper first presents the design of the miniature radiating element. Then, the detailed process to calculate the link budget allowing the estimation of the theoretical power received by the reader is described, showing that the gain of the proposed antenna is adapted to the application. Finally, the measurement of the Received Signal Strength Indication (RSSI) level received by the reader antenna for different positions of a mouse phantom model in a cage is presented. Results show that the proposed antenna can allow the identification of the small animal whatever its position in the cage

Identification sans fil de fantômes de petits animaux intégrant un tag RFID UHF implanté miniature

National audienceDans ce papier, la conception d'une antenne miniature implantée dans un petit modèle de fantôme représentant une souris et fonctionnant dans la bande RFID UHF européenne est présentée. Le RSSI, qui est l'indicateur de l'intensité du signal reçu par l'antenne du lecteur est mesuré pour différentes positions du modèle de fantôme dans une cage. Les résultats montrent que l'antenne proposée peut permettre l'identification du petit animal quelle que soit sa position dans la cage