18 research outputs found

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

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    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

    Clavicle fractures: a comparison of five classification systems and their relationship to treatment outcomes

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    We compared five classification systems for clavicle fractures. The aim of this study was to evaluate the prognostic value of each system. Over a two-year period we reviewed all new radiographs of the shoulder region and identified 487 clavicle fractures. Each radiograph was classified using five classification systems. We reviewed all subsequent X-rays and clinical records until the patient was discharged. We assessed each classification system's prognostic value in predicting delayed/non-union. Our data show that 79.3% of clavicle fractures occur in the middle third, 19.3% in the lateral third and 1.4% in the medial third. The overall prevalence of delayed/non-union was 7.3%, with 3.2% requiring operative management and 4.1% developing asymptomatic non-union. The incidence of non-union in the lateral third was 9.6%, but only 0.4% required operative management. Craig's classification had the greatest prognostic value for lateral third fractures, and Robinson's classification had the greatest prognostic value for middle third fractures. Fractures of the clavicle are common injuries but non-union is an uncommon occurrence. Non-union is more common in the lateral third, but we found these to be mostly asymptomatic. Middle third fractures are more likely to require operative fixation. Middle third fractures should be classified according to Robinson's classification system and lateral third fractures according to Craig's classification. We did not assess sufficient medial third fractures for the data to be significant
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