A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus into Dendritic Cells

Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation

Nonoperative treatment of closed displaced midshaft clavicle fractures.

none7BACKGROUND: closed displaced midshaft clavicle fractures used to be treated nonoperatively, and many studies have reported that nonoperative treatment gave good results. However, more recent studies have reported poorer results following nonoperative treatment, whereas the results of operative treatment have improved considerably. The aim of this paper was to report the results of treating closed displaced midshaft clavicle fractures nonoperatively. MATERIALS AND METHODS: one hundred Edinburgh type 2B clavicle fractures (69 type 2B1 and 31 type 2B2) in 100 patients (78 males and 22 females) aged between 18 and 67 ears (mean 32 years) were treated. All patients were treated using a figure-of-eight bandage. Clinical and radiographic assessment was performed at the time of trauma, 1, 2 and 3 months after the trauma, and then at an average follow-up of 3 years (range 1-5 years). The outcome was rated at the last follow-up using the DASH score. RESULTS: ninety-seven of the 100 fractures healed. Three nonunions were observed. Average healing time was 9 weeks (range 8-12 weeks). No statistically significant correlation between the type of fracture and the healing time was observed. The average DASH score was 24 (range 0-78) and, based on this score, 81 patients presented excellent results, 12 good, 5 fair, and 2 poor. No statistically significant correlation between the type of the fracture and the score was observed. CONCLUSIONS: we believe that nonoperative treatment is still appropriate in most cases, as it yields good results without incurring the potential complications of surgery.noneFaldini C.; Nanni M.; Leonetti D.; Acri F.; Galante C.; Luciani D.; Giannini S.Faldini C.; Nanni M.; Leonetti D.; Acri F.; Galante C.; Luciani D.; Giannini S