B cell-specific knockout of AID protects against atherosclerosis.

Antigen-naive IgM-producing B cells are atheroprotective, whereas mature B cells producing class-switched antibodies promote atherosclerosis. Activation-induced cytidine deaminase (AID), which mediates class switch recombination (CSR), would thus be expected to foster atherosclerosis. Yet, AID also plays a major role in the establishment of B cell tolerance. We sought to define whether AID affects atherosclerotic plaque formation. We generated Ldlr-/- chimeras transplanted with bone marrow from Aicda-/- or wild-type (WT) mice, fed a HFD for 14 weeks. Decreased B cell maturation in Ldlr-/-Aicda-/- mice was demonstrated by 50% reduction in splenic and aortic BAFFR expression, a key signaling component of B2 cell maturation. This was associated with increased plasma IgM in Ldlr-/-Aicda-/- compared with Ldlr-/-WT animals. Importantly, Ldlr-/-Aicda-/- mice had reduced atherosclerotic lesion area (0.20 ± 0.03mm2) compared with Ldlr-/-WT (0.30 ± 0.04mm2, P < 0.05), although no differences in plaque composition were noted between groups. In addition, immunofluorescence analysis revealed increased splenic B and T cell areas independent of cell number. AID depletion directly inhibits atherosclerotic plaque formation.

Assessment of the effect of systemic delivery of sclerostin antibodies on Wnt signaling in distraction osteogenesis.

Sclerostin is a known inhibitor of the Wnt signaling pathway which is involved in osteogenesis and, when inactivated, stimulates bone formation. To our knowledge, this effect has not been studied in the context of distraction osteogenesis (DO). Tibial DO was conducted on a total of 24 wild-type mice, which were then divided into 2 groups-a saline injection group (control) and an anti-sclerostin (Scl-Ab) injection group (treatment). The mice in the treatment group received 100 mg/kg intravenous injections of the antibody weekly until killing. The 12 mice in each group were subdivided into four time points according to post-osteotomy time of killing-11 days (mid-distraction), 17 days (late distraction), 34 days (mid-consolidation) and 51 days (late consolidation), with 3 mice per subgroup. After killing, the tibia specimens were collected for immunohistochemical analysis. Our results show that the group injected with anti-sclerostin had an earlier peak (day 11) in the distraction phase of the osteogenic molecules involved in the Wnt signaling pathway in comparison to the placebo group. In addition, downregulation of the inhibitors of this pathway was noted in the treatment group when compared with the placebo group. Furthermore, LRP-5 showed a significant increase in expression in the treatment group. Sclerostin inhibition has a significant effect on the DO process through its effect on the Wnt pathway. This effect was evident through the decreased effect of sclerostin on LRP-5 and earlier upregulation of the osteogenic molecules involved in this pathway.

OP-1 injection increases VEGF expression but not angiogenesis in a rabbit model of distraction osteogenesis.

We have previously shown that a single injection of rhBMP-7 (OP-1) applied to the regenerate early during distraction accelerates bone consolidation in a rabbit model of distraction osteogenesis. In the present study, we hypothesised that the injection of OP-1 improves bone consolidation by increasing blood flow to the distracted site. Blood flow into the regenerate of a rabbit model was measured and vascular endothelial growth factor (VEGF) expression was tested using semi-quantitative PCR. Immunohistochemistry was used for assessing the temporal and spatial expression of platelet endothelial cell adhesion molecule (PECAM), VEGF and its receptors following OP-1 injection. We observed a higher expression of VEGF and its receptors in the regenerate with OP-1 treatment. However, there was no difference in the increase in bone blood flow nor PECAM expression between the treated and control groups of animals. Interestingly, the increased expression of VEGF and its receptors was associated with chondrocyte and fibroblast-like cells, but not with endothelial cells. These results suggest that accelerated ossification by OP-1 may depend on a non-vascular mechanism, possibly involving a non-angiogenic function of VEGF signalling.

Characterizing the BMP pathway in a wild type mouse model of distraction osteogenesis.

Distraction osteogenesis (DO) is a well established surgical technique for limb lengthening and replacement of bone loss due to trauma, infection or malignancies. Although the technique is widely used, one of its limitations is the long period of time required for the newly formed bone to consolidate. We have previously shown that exogenous application of bone morphogenetic proteins (BMPs) can increase bone formation during DO, however, exogenous BMPs have many drawbacks. An alternative method for accelerating the rate of bone formation may be to modulate the intrinsic BMP signaling pathway. The aim of the current study was to analyze the expression of various genes involved in the BMP pathway at various time periods during DO in order to identify potential targets for therapeutic manipulation. DO was applied to the right tibia of 80 adult wild type mice. Distraction began after a latency period of 5 days at a rate of 0.2 mm/12 h for 2 weeks. Mice were sacrificed in groups of 12 at the following times post surgery: day 5 (latency), days 11 and 17 (distraction) and days 34 and 51 (consolidation). Specimens were examined using radiology, microCT, histology, RT(2)PCR, immunohistochemistry and Western analysis. Genes involved in the BMP pathway including the BMP ligands, receptors, antagonists and downstream effectors were examined. A significant upregulation of BMPs 2, 4 and 6 was observed using both PCR and immunohistochemistry during the distraction phase. The expression of BMP7 remained constant throughout the distraction and consolidation process. Surprisingly, the only receptors which were upregulated significantly were the Activin Receptor Type 1 (ActR1) during distraction and Activin Receptor Type 2b (ActR2b) during consolidation. Most interestingly, simultaneously with the ligands, an increase in the expression of the antagonists, Noggin, Chordin, Inhibin and BMP3 was observed. This study provides a clearer understanding of expression patterns during DO, which is a valuable resource for finding therapeutic options to stimulate bone formation. The results suggest that blocking BMP inhibitors may be a possible method for increasing the function of intrinsic growth factors involved in bone regeneration.

Early injection of OP-1 during distraction osteogenesis accelerates new bone formation in rabbits.

Distraction osteogenesis (DO) is a surgical technique for generating new bone by applying controlled distraction of two bony segments post osteotomy. A limitation of the technique is the long time required for the new bone to consolidate. We investigated the effect of injecting osteogenic protein 1 (OP-1) at the beginning of distraction in a rabbit model of DO. Regenerate bone was evaluated using radiology, densitometry, micro-computed tomography (microCT) and histomorphometry. Immunohistochemsitry was used to evaluate changes in expression of various ligands, growth factors and receptors following OP-1 treatment. Compared to the control, a two-fold increase in bone volume was apparent for treated groups at 3 weeks post injection. An upregulation of almost all of the 41 genes examined was observed. Results suggested that applying OP-1 early during distraction can accelerate bone formation by the activation of numerous pathways. This study provides further insights on strategies to improve bone regeneration rate in DO.

Heat shock protein 90 expression in Epstein-Barr virus-infected B cells promotes gammadelta T-cell proliferation in vitro.

The aim of this study was to elucidate the in vitro response of gammadelta T cells to Epstein-Barr virus (EBV)-infected B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as gammadelta T-cell stimulants. Cytofluorometric analysis revealed HSP90 cell surface expression in 12% of the EBV-immortalized B-cell population in all four of the B-cell lines tested. HSP27, HSP60, and HSP70 were not detected on the cell surface by cytofluorometry in these same B-cell lines. HSP90 and HSP60, but not HSP70 or HSP27, were detected on the cell surface after 125I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal antibodies. In vitro kinetic studies indicated that gammadelta T cells increased at least twofold by day 11 postinfection in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of alphabeta T cells in these same cultures either decreased slightly or remained relatively unchanged in response to EBV infection. Addition of anti-human HSP90 monoclonal antibody to the EBV-infected lymphocyte cultures inhibited gammadelta T-cell expansion by 92%. The inhibition of gammadelta T-cell expansion by anti-HSP90 antibody was reversed upon treatment with exogenous HSP90. Taken together, these results indicate that HSP90 played an important role in the stimulation of gammadelta T cells during EBV infection of B cells in vitro and may serve as an important immunomodulator of gammadelta T cells during acute EBV infection.