Sorting of Phagocytic Cells Infected with Legionella pneumophila

International audienceThe ability of Legionella pneumophila to colonize host cells and to form a replicative vacuole depends on its ability to counteract the host cell response by secreting more than 300 effectors. The host cell responds to this bacterial invasion with extensive intracellular signaling to counteract the replication of the pathogen. When studying L. pneumophila infection in vitro, only a small proportion of the cell lines or primary cells used to analyze the host response are infected; the study of such a mixed cell population leads to unprecise results. In order to study the multitude of pathogen-induced phenotypic changes occurring in the host cell, the separation of infected from uninfected cells is a top priority. Here we describe a highly efficient FACS-derived protocol to separate cells infected with a L. pneumophila strain encoding a fluorescent protein. Indeed, the highly infected, homogenous cell population obtained after sorting is the best possible starting point for the studies of infection-induced effects

Osteoanabolic effect of alendronate and zoledronate on bone marrow stromal cells (BMSCs) isolated from aged female osteoporotic patients and its implications for their mode of action in the treatment of age-related bone loss

In the present study, we evaluated the potential for aminobisphosphonates to enhance the development of bone-forming osteoblasts from progenitor cells isolated from aged female osteoporotic patients. The aminobisphosphonates tested significantly enhanced osteoblast formation and thus lend further insights into their possible mode of action in the treatment of osteoporosis. INTRODUCTION: The primary aim of this study was to evaluate the influence of aminobisphosphonates on the osteogenesis of human bone marrow stromal cells (hBMSCs) and mineralization of differentiating bone-forming cells isolated from osteoporotic patients. METHODS: The influence of aminobisphosphonate treatment on hBMSC osteogenesis was assessed by the quantitative measurement of alkaline phosphatase (ALP) activity, in addition to quantitative reverse transcription polymerase chain reaction and Western blot analysis of known osteogenic markers. Mineralized matrix formation by hBMSC-derived osteoblasts was visualized and quantified using Alizarin red staining. RESULTS: hBMSC cultures treated with osteogenic medium supplemented with zoledronate demonstrated a significant increase in Alizarin red staining after 3 weeks as compared to cells cultured in osteogenic medium alone. Similarly, cultures of differentiating hBMSCs isolated from patients receiving alendronate treatment also demonstrated an increased propensity for mineralization, even in the absence of further in vitro stimulation by zoledronate. The stimulatory effects of aminobisphosphonate treatment on hBMSC-derived osteoblast-mediated mineralization were independent of any alterations in ALP activity, although significant decreases in the expression levels of osteopontin (SPP1) were evident in hBMSCs following exposure to aminobisphosphonates. Further analysis including Western blotting and loss-of-function studies revealed osteopontin as having a negative influence on the mineralization of differentiating osteoporotic bone-forming cells. CONCLUSIONS: The results presented here demonstrate for the first time that aminobisphosphonate treatment of osteoporotic hBMSCs enhances their capacity for osteoblast formation and subsequent mineral deposition, thus supporting the concept of aminobisphosphonates as having an osteoanabolic effect in osteoporosis