Heat Shock Protein 90 Expression in Epstein-Barr Virus-Infected B Cells Promotes γδ T-Cell Proliferation In Vitro

The aim of this study was to elucidate the in vitro response of γδ T cells to Epstein-Barr virus (EBV)-infected B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as γδ 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 (125)I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal antibodies. In vitro kinetic studies indicated that γδ T cells increased at least twofold by day 11 postinfection in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of αβ 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 γδ T-cell expansion by 92%. The inhibition of γδ 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 γδ T cells during EBV infection of B cells in vitro and may serve as an important immunomodulator of γδ T cells during acute EBV infection

Activation of Wnt signaling in human fracture callus and nonunion tissues

The Wnt signaling pathway is a key molecular process during fracture repair. Although much of what we now know about the role of this pathway in bone is derived from in vitro and animal studies, the same cannot be said about humans. As such, we hypothesized that Wnt signaling will also be a key process in humans during physiological fracture healing as well as in the development of a nonunion (hypertrophic and oligotrophic). We further hypothesized that the expression of Wnt-signaling pathway genes/proteins would exhibit a differential expression pattern between physiological fracture callus and the pathological nonunion tissues. We tested these two hypotheses by examining the mRNA levels of key Wnt-signaling related genes: ligands (WNT4, WNT10a), receptors (FZD4, LRP5, LRP6), inhibitors (DKK1, SOST) and modulators (CTNNB1 and PORCN). RNA sequencing from calluses as well as from the two nonunion tissue types, revealed that all of these genes were expressed at about the same level in these three tissue types. Further, spatial expression experiments identified the cells responsible of producing these proteins. Robust expression was detected in osteoblasts for the majority of these genes except SOST which displayed low expression, but in contrast, was mostly detected in osteocytes. Many of these genes were also expressed by callus chondrocytes as well. Taken together, these results confirm that Wnt signaling is indeed active during both human physiological fracture healing as well as in pathological nonunions

Assessment of the Effect of Systemic Delivery of Sclerostin Antibodies on WNT Signaling in Distraction Osteogenesis Using Immunohistochemistry

Introduction Sclerostin is a known inhibitor of the WNT signaling pathway involved in osteogenesis, therefore when inactivated bone formation is stimulated. One of the potential ways of inactivation of this molecule is the sclerostin antibody injection. This antibody has been shown to improve fracture healing in the mouse model but to our knowledge its effect has not been studied in the context of distraction osteogenesis (DO). Objective The objective of this study was to determine the immunohistochemical effect of sclerostin antibody injection at various time points during bone formation in a wild-type mouse model of DO. Materials and Methods Tibial DO was conducted on a total of 24 wild type mice, which were then divided into 2 groups; saline injection group (control) and anti-sclerostin (Scl-Ab) injection group (treatment). The mice in the treatment group received 100mg/kg intravenous injections of the antibody weekly till sacrifice. The 12 mice in each group were subdivided into four time points according to time post-osteotomy for sacrifice, these were 11 days (mid-distraction), 17 days (late distraction), 34 days (mid-consolidation) and 51 days (late consolidation) with 3 mice per subgroup. The tibia specimens post-sacrifice were then collected for immunohistochemical analysis. Results Our results showed 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 to the noted downregulation of the inhibitors of this pathway in the treatment group when compared with the placebo group. Also LRP-5 should a significant increase in expression in the treatment group. Conclusion 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. Clinical Relevance This is the first report of the immunohistochemical effect of sclerostin antibody injection in DO, which could be a potential treatment modality to accelerate healing of the gap created during the process of DO, thus reducing the complications associated with this