Ex vivo 3D osteocyte network construction with primary murine bone cells.

Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models.

Inhibition of the Immunoproteasome Subunit LMP7 with ONX 0914 Ameliorates Graft-versus-Host Disease in an MHC-Matched Minor Histocompatibility Antigen-Disparate Murine Model.

In the current study we evaluated the effects of immunoproteasome inhibition using ONX 0914 (formerly PR-957) to ameliorate graft-versus-host disease (GVHD). ONX 0914, an LMP7-selective epoxyketone inhibitor of the immunoproteasome, has been shown to reduce cytokine production in activated monocytes and T cells and attenuate disease progression in mouse models of rheumatoid arthritis, colitis, systemic lupus erythematosus, and, more recently, encephalomyelitis. Inhibition of LMP7 with ONX 0914 in the B10.BR→CBA MHC-matched/minor histocompatibility antigen (miHA)-disparate murine blood and marrow transplant (BMT) model caused a modest but significant improvement in the survival of mice experiencing GVHD. Concomitant with these results, in vitro mixed lymphocyte cultures revealed that stimulator splenocytes, but not responder T cells, treated with ONX 0914 resulted in decreased IFN-γ production by allogeneic T cells in both MHC-disparate (B10.BR anti-B6) and miHA-mismatched (B10.BR anti-CBA) settings. In addition, a reduction in the expression of the MHC class I-restricted SIINFEKL peptide was observed in splenocytes from transgenic C57BL/6-Tg(CAG-OVA)916Jen/J mice exposed to ONX 0914. Taken together, these data support that LMP7 inhibition in the context of BMT modulates allogeneic responses by decreasing endogenous miHA presentation and that the consequential reduction in allogeneic stimulation and cytokine production reduces GVHD development.

Morphine-conditioned cue alters c-Fos protein expression in the brain of crayfish.

With a highly organized stereotypic behavior and a simplified neuronal system that is characterized by cellular modularity, crayfish (Orconectes rusticus) represents an excellent model that we used in this study to explore how a drug-conditioned-cue alters c-Fos protein expression in the brain of an invertebrate species. The first set of experiments revealed that a single injection of different doses of morphine (3.0 µg/g, 6.0 µg/g and 12.0 µg/g) into the circulatory system of crayfish significantly increased locomotor activity. Repeated injections of morphine increased locomotion at lower doses (3.0 µg/g and 6.0 µg/g), and decreased locomotion at a higher dose of 12.0 µg/g. The second experiment revealed that a repeated or single injection of morphine serves as reward when paired with a distinct visual environment. In the third experiment, we found that the c-Fos profile of morphine treated crayfish in an unconditioned environment did not show a significant increase from the basal level comparable to saline treated crayfish. The brains of crayfish were more active during exposure to the cue-elicited drug conditioned environment than the unconditioned environment. These results indicate that chronic morphine treatment alone is not sufficient to induce changes in the expression of c-Fos; instead, morphine-environment pairing in a specific context contributes to the expression of alterations in c-Fos regulation. The enhancement of c-Fos expression in the brain of crayfish seems to reflect the sensory or anticipatory facets of conditioning that suggests that potential and even unanticipated hypotheses in drug addiction can emerge from studies of addiction in crayfish.