Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether that response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18α-glycyrrhetinic acid (αGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MK to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation.

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes (MK) can induce osteoblast (OB) proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of MKs with another cell-type of mesenchymal origin -the fibroblast (FB). Our findings implicate the involvement of fibronectin/RGD-binding integrins including α 3 β 1 (VLA-3) and α 5 β 1 (VLA-5) as well as glycoprotein IIb (CD41), all of which are known to be expressed on MK membranes. Furthermore, we demonstrate that interleukin (IL)-3 can enhance MK-induced OB activation in vitro, as demonstrated in the MK-FB model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous in several ways. KeywordsMegakaryocytes; Osteoblasts; Integrins; CD41; IL-3 Skeletal fragility has emerged as a major limitation to quality of life as we age. Osteoporosis and the ensuing hip, wrist, and vertebral fractures are significant sources of morbidity and pain among the elderly: such a fracture can be the sentinel event that transforms a relatively healthy, independent senior citizen into a person requiring significant assistance for daily living. This downward spiral is evidenced by a one-year post-hip fracture mortality of 24 percent (National Osteoporosis Foundation). As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. (Thiede et al., 1994;Kelm et al., 1992;Breton-Gorius et al., 1992;Chenu and Delmas, 1992;Frank et al., 1993;Sipe et al., 2004;Kacena et al., 2004;Ciovacco et al., 2009;Miao et al., 2004;Bord et al., 2005; Ciovacco et al., in press). Simultaneously, MKs may regulate bone resorption by expressing/secreting several factors known to be involved in osteoclastogenesis, and recent studies demonstrate that MKs can inhibit osteoclast (OC) formation in vitro (Ciovacco et al., in press;Bord...

IntroductionWe sought to develop evidence-based recommendations for the prehospital evaluation and treatment of adult and pediatric patients with a seizure and to compare these recommendations against the current protocol used by the 33 emergency medical services (EMS) agencies in California.MethodsWe performed a review of the evidence in the prehospital treatment of patients with a seizure, and then compared the seizure protocols of each of the 33 EMS agencies for consistency with these recommendations. We analyzed the type and route of medication administered, number of additional rescue doses permitted, and requirements for glucose testing prior to medication. The treatment for eclampsia and seizures in pediatric patients were analyzed separately.ResultsProtocols across EMS Agencies in California varied widely. We identified multiple drugs, dosages, routes of administration, re-dosing instructions, and requirement for blood glucose testing prior to medication delivery. Blood glucose testing prior to benzodiazepine administration is required by 61% (20/33) of agencies for adult patients and 76% (25/33) for pediatric patients. All agencies have protocols for giving intramuscular benzodiazepines and 76% (25/33) have protocols for intranasal benzodiazepines. Intramuscular midazolam dosages ranged from 2 to 10 mg per single adult dose, 2 to 8 mg per single pediatric dose, and 0.1 to 0.2 mg/kg as a weight-based dose. Intranasal midazolam dosages ranged from 2 to 10 mg per single adult or pediatric dose, and 0.1 to 0.2 mg/kg as a weight-based dose. Intravenous/intrasosseous midazolam dosages ranged from 1 to 6 mg per single adult dose, 1 to 5 mg per single pediatric dose, and 0.05 to 0.1 mg/kg as a weight-based dose. Eclampsia is specifically addressed by 85% (28/33) of agencies. Forty-two percent (14/33) have a protocol for administering magnesium sulfate, with intravenous dosages ranging from 2 to 6 mg, and 58% (19/33) allow benzodiazepines to be administered.ConclusionProtocols for a patient with a seizure, including eclampsia and febrile seizures, vary widely across California. These recommendations for the prehospital diagnosis and treatment of seizures may be useful for EMS medical directors tasked with creating and revising these protocols.

Dimethyl sulfoxide (DMSO) is routinely used in the laboratory as a solvent and vehicle for organic molecules. Although it has been used in previous studies involving myeloid cells and macrophages, we are unaware of data demonstrating the effects of DMSO alone on osteoclast development. Recently, we were using DMSO as a vehicle and included a non-vehicle control. Surprisingly, we observed a marked change in osteoclast development, and therefore designed this study to examine the effects of DMSO on osteoclast development. Osteoclasts were generated from two sources: bone marrow macrophages and an osteoclast progenitor cell line. Cells were cultured with DMSO for various durations and at differing concentrations and mature, multinucleated (>3 nuclei) TRAP+ cells were assessed in terms of cell number, cell surface area, and number of nuclei/cell. Osteoclast surface area increased in 5 μM DMSO to a mean of 156,422 pixels from a mean of 38,510 pixels in control culture, and subsequently decreased in 10 μM DMSO to a mean of 18,994 pixels. With serial addition of DMSO over 5 d, a significant increase in mean surface area, and number of nuclei/cell was also observed, while the opposite was true when DMSO was serially removed from culture. These findings show that DMSO exerts a marked effect on osteoclast differentiation. Since many investigators use DMSO to solubilize compounds for treatment of osteoclasts, caution is warranted as altering DMSO concentrations may have a profound effect on the final data, especially if osteoclast differentiation is being assessed.

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