Wellcome Trust (grant number 098051).

The heterogeneity of the molecular pathology of HCC poses a formidable obstacle to the development of non-cytotoxic therapies. Several pro-tumorigenic signaling pathways can be aberrantly activated in HCC, including those triggered by Wnts. Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan that is overexpressed in most HCCs, promotes the growth of these tumors by stimulating Wnt signaling. Because GPC3 binds with high affinity to Wnts, and its growthpromoting activity requires attachment to the cell membrane, we have hypothesized that a mutated GPC3 lacking the GPI anchoring domain (sGPC3) will block Wnt signaling and inhibit the growth of Wnt-dependent tumors. In addition, because sGPC3 displays heparan sulfate chains, this secreted glypican could also inhibit HCC growth by blocking the activity of other heparin-binding growth factors. To test this hypothesis, HCC cell lines were infected with an sGPC3-expressing lentivirus or virus control, and the effect of sGPC3 on the in vitro and in vivo growth was investigated. In addition, the signaling pathways targeted by sGPC3 were identified. We observed that sGPC3-expressing cells had lower proliferation rate. In addition, sGPC3 significantly inhibited the in vivo growth of the Huh6, HepG2 and Huh7 HCC cell lines. sGPC3 blocked Wnt signaling in Huh6-and Huh7-derived tumors and Erk1/2 and Akt phosphorylation in tumors generated by Huh7 and HepG2 cells, respectively. An anti-angiogenic effect in Huh7 and HepG2-derived tumors was also observed. We conclude that sGPC3 can inhibit HCC tumorigenicity by blocking the activity of several pro-tumorigenic growth factors.

Basic fibroblast growth factor (bFGF, FGF-2) is a potent angiogenic factor and endothelial cell mitogen. Although bFGF levels are increased in chronically inflamed tissue, its role in inflammation is unclear. We investigated the effect of bFGF on acute dermal inflammation and the recruitment of monocytes, T cells, and neutrophils. Leukocyte recruitment to inflamed sites was quantified with radiolabeled leukocytes. Intradermal injection of bFGF in rats did not induce leukocyte recruitment or inflammation. However, the recruitment of leukocytes to inflammation induced by tumor necrosis factor-alpha, interferon-gamma, C5a, or a delayed hypersensitivity reaction was enhanced by bFGF by 55 to 132% (P < 0.05). Either acute or prolonged bFGF treatment of dermal sites had this effect. The potentiating effect of bFGF on leukocyte recruitment was also seen in joints. There was no associated modulation of vascular permeability, blood flow, or angiogenesis in the sites by bFGF. However, the expression of the endothelial cell adhesion molecules (CAMs) for leukocytes, P-selectin, E-selectin, and ICAM-1, was significantly up-regulated in the inflamed tissue by bFGF, as quantified by radiolabeled anti-CAM antibody binding in vivo. Thus, although not directly proinflammatory, bFGF synergistically potentiates inflammatory mediator-induced leukocyte recruitment, at least in part, by enhancing CAM up-regulation on endothelium.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are produced at sites of inflammation. Previously, we demonstrated that bFGF enhances leukocyte recruitment and endothelial cell adhesion molecule (CAM) expression during inflammation. Here, we investigated the influence of VEGF during acute inflammation and whether VEGF and bFGF cooperate to modulate leukocyte recruitment. Inflammation was induced in skin of rats by intradermal injection of inflammatory stimuli +/- VEGF +/- bFGF. Migration of 51Cr-monocytes and 111In-polymorphonuclear leukocytes (PMN) to the dermal lesions and 125I-anti-CAM monoclonal antibody binding to the dermal vasculature were quantitated after 2 h. VEGF significantly enhanced tumor necrosis factor alpha (TNF-alpha)-induced monocyte recruitment by 39 +/- 16% and increased P-selectin, E-selectin, and intercellular CAM-1 expression by two- to threefold over TNF-alpha alone. However, recruitment of monocytes to TNF-alpha + interferon-gamma (IFN-gamma) and of PMN to all stimuli tested was not affected by VEGF. In contrast, bFGF enhanced recruitment of both leukocyte types to all stimuli tested. With the potent TNF-alpha + IFN-gamma stimulus, in contrast to bFGF, VEGF did not enhance E-selectin or ICAM-1 expression. bFGF, but not VEGF, increased the chemotactic activity for PMN in TNF-alpha + IFN-gamma-inflamed sites by 54%. The limited effect of VEGF on these mechanisms likely contributed to the differential effect of VEGF and bFGF on leukocyte recruitment. However, VEGF + bFGF increased PMN recruitment more than did either factor alone. Thus, bFGF and VEGF differentially but synergistically enhance leukocyte recruitment to inflammatory stimuli and individually as well as jointly function as positive regulators of inflammatory cell recruitment.

Highlights Third-generation cephalosporins are common first line agents to empirically treat typhoid fever. An outbreak of extensively-drug resistant (XDR) typhoid has emerged in Pakistan that is resistant to ceftriaxone. This is the first Canadian case of XDR typhoid in a child who travelled to Pakistan and was successfully treated with 2 weeks of meropenem. Clinicians should consider adapting their empiric management of typhoid for travellers returning from XDR regions.

Summary Dendritic cells (DCs) are the most potent antigen‐presenting cells and populate many tissues where they may participate in inflammatory reactions. The infiltration of polymorphonuclear leucocytes (PMNLs) into tissues is a prominent feature of inflammation. The mechanisms of PMNL recruitment depend on chemotactic factors and adhesion molecules expressed on endothelial cells. The aim of the present study was to determine whether DCs participate in the early recruitment of PMNLs. Dendritic cells derived from peripheral blood monocytes were used for this study. PMNLs incubated with culture supernatant (CS) from untreated or from tumour necrosis factor‐α (TNF‐α)‐treated (1 hr, 100 U/ml, 37°) monocyte‐derived DCs (moDCs) had increased surface expression of both CD11b and CD18. Moreover, both untreated and TNF‐α‐treated moDCs induced PMNL chemotaxis. By blocking CXCL8, CXCL5, CXCL7 and Pan GRO (CXCL1, CXCL2, CXCL3), we observed that CXCL8/interleukin‐8 might be the chemokine that induced the PMNL chemotactic activity in the CS of untreated and TNF‐α‐treated moDC. Furthermore, we investigated the regulation of CXCL8 production in moDCs by adhesion molecule engagement. Our data demonstrated that CD31, CD18, CD29 and CD49d participated in the adhesion of immature moDCs to endothelium. Moreover, engagement of domains 1–3 of CD31, but not of CD29 or CD18, decreased the production of CXCL8 by immature but not mature moDCs (which display lower CD31 levels than immature moDCs). Overall, these results suggest that DCs not only trigger a specific immune response, but also the innate immune response by recruiting PMNLs. Furthermore, our results also suggest that CXCL8 production by immature DCs might be regulated by signalling through CD31 during their migration through the vascular endothelium.

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These results indicate that alloimmunization may be useful in the treatment of RSA.