The acute and residual effects of escalating, analgesic-range doses of ketamine on driving performance: A simulator study.

Ketamine hydrochloride elicits potent psychotomimetic and neurobehavioural effects which make it incompatible with driving; however, the direct effect on driving performance is yet to be assessed. Using an open label, within-subjects protocol, 15 males and 5 females (mean age = 30.8 years) were administered three fixed, stepwise increasing sub-anaesthetic doses of intravenous (IV) ketamine solution [(i) 8 mg/h IV infusion plus 30 mg bolus, (ii) 12 mg/h IV infusion and (iii) 20 mg/h infusion]. Whole blood ketamine and norketamine concentrations were determined at each treatment step and at 2 h post-infusion. Driving performance was assessed at baseline, at each treatment step and at 2 h post-treatment using a validated computerised driving simulator. Standard Deviation of Lateral Position (SDLP) and Steering Variability (SV) were assessed. Linear Fixed Effect Modelling indicated a main effect for time (dose) for SDLP (F[4,72] = 33.22, p < 0.0001) and SV (F[4,72] = 4.65, p < 0.002). Post-hoc analyses revealed significant differences from baseline at each treatment step for SDLP (all p < 0.001), and for 12 mg/h treatment step for SV (p = 0.049). Post-treatment driving performance returned to baseline levels. Weak positive linear associations were observed between SDLP and whole blood ketamine concentrations (R2 = 0.11, ß = 29.96, p = 0.001) and norketamine (R2 = 0.09, ß = 28.87, p = 0.003). These findings suggest that even under highly controlled conditions, ketamine intoxication significantly alters simulated driving performance. At the highest dose, ketamine produced changes to SDLP considered incompatible with safe driving, highlighting how ketamine consumption may translate to an increased risk of road trauma.

Neurocognitive and behavioural performance of healthy volunteers receiving an increasing analgesic-range infusion of ketamine.

BACKGROUND: The acute and delayed effect of analgesic-range doses of ketamine on neurocognitive and behavioural outcomes is understudied. Using a non-controlled open-labelled design, three (1-h duration) increasing intravenous (IV) ketamine infusions comprising (i) 30 mg bolus of ketamine + 8 mg/h IV infusion, (ii) 12 mg/h IV infusion and (iii) 20 mg/h infusion were administered to 20 participants (15 male, 5 female, mean age = 30.8 years). Whole-blood ketamine and norketamine concentrations were determined at each treatment step and post-infusion. METHODS: The Cambridge Neuropsychological Test Automated Battery (CANTAB) was used to assess reaction/movement time (RTI, Simple and 5-Choice), visuospatial working memory (SWM), spatial planning (SOC) and subjective effects (visual analogue scale; VAS) during treatment and at post-treatment. RESULTS: Significant main effects were reported for time (dose) on CANTAB RTI 5-Choice reaction (F(4,18) = 3.41, p = 0.029) and movement time (F(4,18) = 4.42, p = 0.011), SWM (F(4,18) = 4.19, p = 0.014) and SOC (F(4,18) = 4.13, p = 0.015), but not RTI Simple reaction or movement time. Post hoc analyses revealed dose-dependent effects for both RTI 5-Choice reaction and movement time (all p < 0.05). Post-treatment performance on all neurocognitive and behavioural tasks returned to baseline levels. Regression analyses revealed a weak positive linear association between SWM 'strategy' score (R2 = 0.103, p < 0.001), all performance-based CANTAB VAS items (R2 range 0.005-0.137, all p < 0.05) and ketamine blood concentrations. DISCUSSION: The open-label, non-controlled trial design somewhat precludes the ability to adequately account for random treatment effects. Notwithstanding, these results suggest that analgesic doses of ketamine produce acute, selective, dose-dependent deficits in higher-order neurocognitive and behavioural domains.

Long-term imatinib therapy promotes bone formation in CML patients.

Imatinib inhibits tyrosine kinases important in osteoclast (c-Fms) and osteoblast (platelet-derived growth factor receptor [PDGF-R], c-Abl) function, suggesting that long-term therapy may alter bone homeostasis. To investigate this question, we measured the trabecular bone volume (TBV) in iliac crest bone biopsies taken from chronic myeloid leukemia (CML) patients at diagnosis and again after 2 to 4 years of imatinib therapy. Half the patients (8 of 17) showed a substantive increase in TBV (> 2-fold), after imatinib therapy, with the TBV in the posttreatment biopsy typically surpassing the normal upper limit for the patient's age group. Imatinib-treated patients exhibited reduced serum calcium and phosphate levels with hypophosphatemia evident in 53% (9 of 17) of patients. In vitro, imatinib suppressed osteoblast proliferation and stimulated osteogenic gene expression and mineralized-matrix production by inhibiting PDGF receptor function. In PDGF-stimulated cultures, imatinib dose-dependently inhibited activation of Akt and Crk-L. Using pharmacologic inhibitors, inhibition of PI3-kinase/Akt activation promoted mineral formation, suggesting a possible molecular mechanism for the imatinib-mediated increase in TBV in vivo. Further investigation is required to determine whether the increase in TBV associated with imatinib therapy may represent a novel therapeutic avenue for the treatment of diseases that are characterized by generalized bone loss.

RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.

UNLABELLED: RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool. INTRODUCTION: The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs. MATERIALS AND METHODS: Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions. RESULTS: RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK-RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45(+)CD14(+)alphaVbeta3(+)c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45(+)alphaVbeta3(+)TRACP(+) OCs. Importantly, sorted CD14(+)RANK(high) PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANK(mid) or RANK(low) cells. CONCLUSIONS: These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity.

Human trabecular bone-derived osteoblasts support human osteoclast formation in vitro in a defined, serum-free medium.

While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking. We tested the ability of normal human trabecular bone-derived osteoblasts (NHBCs) to support osteoclast formation from human peripheral blood mononuclear cells (PBMC) in response to treatment with either 1alpha,25-dihydroxyvitamin D3 (1,25D) or parathyroid hormone (PTH), using a serum-replete medium previously used to support human osteoclast formation on a stroma of murine ST-2 cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC phenotype in these media. We next tested a defined, serum-free medium (SDM) on NHBC phenotype, their expression of RANKL and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic maturation of NHBC, based on the expression of STRO-1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of supporting osteoclast formation. Consistent with this, co-culture of NHBC with CD14+ PBMC, or bone marrow mononuclear cell (BMMC), or CD34+ BMMC precursors in SDM + 1,25D + DEX, resulted in functional osteoclast formation. Osteoclast formation also occurred in PTH + DEX stimulated co-cultures. Interestingly, SDM supplemented with recombinant RANKL (25-100 ng/ml) and M-CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal-free cultures, unlike serum-replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this process.

Isolation of a human homolog of osteoclast inhibitory lectin that inhibits the formation and function of osteoclasts.

UNLABELLED: Osteoclast inhibitory lectin (OCIL) is a newly recognized inhibitor of osteoclast formation. We identified a human homolog of OCIL and its gene, determined its regulation in human osteoblast cell lines, and established that it can inhibit murine and human osteoclast formation and resorption. OCIL shows promise as a new antiresorptive. INTRODUCTION: Murine and rat osteoclast inhibitory lectins (mOCIL and rOCIL, respectively) are type II membrane C-type lectins expressed by osteoblasts and other extraskeletal tissues, with the extracellular domain of each, expressed as a recombinant protein, able to inhibit in vitro osteoclast formation. MATERIALS AND METHODS: We isolated the human homolog of OCIL (hOCIL) from a human fetal cDNA library that predicts a 191 amino acid type II membrane protein, with the 112 amino acid C-type lectin region in the extracellular domain having 53% identity with the C-type lectin sequences of rOCIL and mOCIL. The extracellular domain of hOCIL was expressed as a soluble recombinant protein in E. coli, and its biological effects were determined. RESULTS AND CONCLUSIONS: The hOCIL gene is 25 kb in length, comprised of five exons, and is a member of a superfamily of natural killer (NK) cell receptors encoded by the NK gene complex located on chromosome 12. Human OCIL mRNA expression is upregulated by interleukin (IL)-1alpha and prostaglandin E2 (PGE2) in a time-dependent manner in human osteogenic sarcoma MG63 cells, but not by dexamethasone or 1,25 dihydroxyvitamin D3. Soluble recombinant hOCIL had biological effects comparable with recombinant mOCIL on human and murine osteoclastogenesis. In addition to its capacity to limit osteoclast formation, OCIL was also able to inhibit bone resorption by mature, giant-cell tumor-derived osteoclasts. Thus, a human homolog of OCIL exists that is highly conserved with mOCIL in its primary amino acid sequence (C-lectin domain), genomic structure, and activity to inhibit osteoclastogenesis.

Receptor activator of nuclear factor-kappaB ligand expression by human myeloma cells mediates osteoclast formation in vitro and correlates with bone destruction in vivo.

Multiple myeloma (MM) is an incurable B-cell malignancy able to mediate massive destruction of the axial skeleton. The aim of this study was to examine the involvement of the tumor necrosis factor-ligand family member, receptor activator of nuclear factor-kappaB ligand (RANKL), and its naturally occurring antagonist, osteoprotegerin (OPG), in MM biology. Using flow cytometry and two independent anti-RANKL antibodies, we demonstrate RANKL expression in CD38(+++)CD45(+) and CD38(+++)CD45(-) myeloma plasma cell (MPC) subpopulations derived from patients with osteolytic MM. In addition, highly purified subpopulations of MPC express mRNA for both transmembrane and soluble RANKL isoforms but lack expression of OPG mRNA and protein. We also show that RANKL expressed by MPC is functional as in vitro coculture of CD38(+++)CD45(+) and CD38(+++)CD45(-) MPC subpopulations with peripheral blood mononuclear cells resulted in the formation of multinucleate, tartrate-resistant acid phosphatase-positive osteoclasts-like cells capable of forming typical resorption pits. Furthermore, high expression of membrane-associated RANKL by CD38(+++) MPC correlated with the presence of multiple radiological bone lesions in individuals with MM. Together, our data strongly suggest that RANKL expression by MPC confers on them the ability to participate directly in the formation of osteoclast in vivo and extends our knowledge of the involvement of RANKL and OPG in the osteolysis characteristic of this disease.

RANKL expression is related to the differentiation state of human osteoblasts.

Human osteoblast phenotypes that support osteoclast differentiation and bone formation are not well characterized. Osteoblast differentiation markers were examined in relation to RANKL expression. RANKL expression was induced preferentially in immature cells. These results support an important link between diverse osteoblast functions. Cells of the osteoblast lineage support two apparently distinct functions: bone formation and promotion of osteoclast formation. The aim of this study was to examine the relationship between these phenotypes in human osteoblasts (NHBC), in terms of the pre-osteoblast marker, STRO-1, and the mature osteoblast marker, alkaline phosphatase (AP), and the expression of genes involved in osteoclast formation, RANKL and OPG. The osteotropic stimuli, 1alpha,25(OH)2vitamin D3 (vitD3) and dexamethasone, were found to have profound proliferative and phenotypic effects on NHBCs. VitD3 inhibited NHBC proliferation and increased the percentage of cells expressing STRO-1 over an extended culture period, implying that vitD3 promotes and maintains an immature osteogenic phenotype. Concomitantly, RANKL mRNA expression was upregulated and maintained in NHBC in response to vitD3. Dexamethasone progressively promoted the proliferation of AP-expressing cells, resulting in the overall maturation of the cultures. Dexamethasone had little effect on RANKL mRNA expression and downregulated OPG mRNA expression in a donor-dependent manner. Regression analysis showed that RANKL mRNA expression was associated negatively with the percentage of cells expressing AP (p < 0.01) in vitD3- and dexamethasone-treated NHBCs. In contrast, RANKL mRNA expression was associated positively with the percentage of STRO-1+ cells (p < 0.01). In NHBCs sorted by FACS based on STRO-1 expression (STRO-1bright and STRO-1dim populations), it was found that vitD3 upregulated the expression of RANKL mRNA preferentially in STRO-1bright cells. The results suggest that immature osteoblasts respond to osteotropic factors in a potentially pro-osteoclastogenic manner. Additionally, the dual roles of osteoblasts, in supporting osteoclastogenesis or forming bone, may be performed by the same lineage of cells at different stages of their maturation.