Adenoviruses (Ad) are commonly used both experimentally and clinically, including oncolytic virotherapy applications. In the clinical area, efficacy is frequently hampered by the high rates of neutralizing immunity, estimated as high as 90% in some populations that promote vector clearance and limit bioavailability for tumor targeting following systemic delivery. Active tumor targeting is also hampered by the ubiquitous nature of the Ad5 receptor, hCAR, as well as the lack of highly tumor-selective targeting ligands and suitable targeting strategies. Furthermore, significant off-target interactions between the viral vector and cellular and proteinaceous components of the bloodstream have been documented that promote uptake into non-target cells and determine dose-limiting toxicities. Novel strategies are therefore needed to overcome the obstacles that prevent efficacious Ad deployment for wider clinical applications. The use of less seroprevalent Ad serotypes, non-human serotypes, capsid pseudotyping, chemical shielding and genetic masking by heterologous peptide incorporation are all potential strategies to achieve efficient vector escape from humoral immune recognition. Conversely, selective vector arming with immunostimulatory agents can be utilized to enhance their oncolytic potential by activation of cancer-specific immune responses against the malignant tissues. This review presents recent advantages and pitfalls occurring in the field of adenoviral oncolytic therapies.
Encouraging results from recent clinical trials are revitalizing the field of oncolytic virotherapies. Human adenovirus type 5 (HAdV-C5/Ad5) is a common vector for its ease of manipulation, high production titers and capacity to transduce multiple cell types. However, effective clinical applications are hindered by poor tumor-selectivity and vector neutralization. We generated Ad5/kn48 by pseudotyping Ad5 with the fiber knob domain from the less seroprevalent HAdV-D48 (Ad48). The vector was shown to utilize coxsackie and adenovirus receptor (CAR) but not CD46 for cell entry. A 20-amino acid peptide NAVPNLRGDLQVLAQKVART (A20) was inserted into the Ad5. Luc HI loop (Ad5.HI.A20) and Ad5/kn48 DG loop (Ad5/kn48.DG.A20) to target a prognostic cancer cell marker, αvβ6 integrin. Relative to the Ad5.Luc parent vector, Ad5.HI.A20, Ad5.KO1.HI.A20 (KO1, ablated CAR-binding) and Ad5/kn48.DG.A20 showed ∼ 160-, 270- and 180-fold increased transduction in BT-20 breast carcinoma cells (αvβ6high). Primary human epithelial ovarian cancer (EOC) cultures derived from clinical ascites provided a useful ex vivo model for intraperitoneal virotherapy. Ad5.HI.A20, Ad5.KO1.HI.A20 and Ad5/kn48.DG.A20 transduction was ∼ 70-, 60- and 16-fold increased relative to Ad5.Luc in EOC cells (αvβ6high), respectively. A20 vectors transduced EOC cells at up to ∼ 950-fold higher efficiency in the presence of neutralizing ovarian ascites, as compared to Ad5.Luc. Efficient transduction and enhanced cancer-selectivity via a non-native αvβ6-mediated route was demonstrated, even in the presence of pre-existing anti-Ad5 immunity. Consequently, αvβ6-targeted Ad vectors may represent a promising platform for local intraperitoneal treatment of ovarian cancer metastases.
Objective-Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. Approach and Results-Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs.Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via β-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. was not translated into protein, and recombinant Wnt2 (rWnt2) protein did not increase VSMC proliferation in vitro. 5 The Wnt pathway has also been shown to have a role in cell migration, with Wnt3a involvement in both migration and adhesion of VSMCs through integrin linked kinase regulation of β1-integrin.10 However, on initiation of this study, it was unclear which Wnt proteins modulated VSMC growth factor-induced migration and whether Wntinduced VSMC migration promoted intimal thickening in vivo. Many genes are upregulated by the Wnt pathway, some of which are known to modulate VSMC migration, including Wnt-1-inducible signaling pathway protein-1 (WISP-1/CCN4 Materials and MethodsMaterials and Methods are available in the online-only Data supplement. Nonstandard Abbreviations and Acronyms Results Wnt2 Was Upregulated in Migrating VSMCs In VitroWnt mRNA levels during VSMC migration were assessed using an in vitro scratch wound assay with multiple wounds to stimulate migration of the VSMCs. mRNA was extracted from VSMCs and applied to a focussed Wnt pathway microarray to assess whether the level of expression changed during migration. A significant increase was only observed in Wnt2 mRNA ( Figure 1A), and this change was confirmed using quantitative polymerase chain reaction ( Figure 1A). No significant change was seen in the mRNA levels of any other Wnts (see Table III in the online-only Data Supplement). It was observed by Western blotting ( Figure 1B) and immunocytochemistry ( Figure I in the online-only Data Supplement) that the increase in Wnt2 mRNA was translated into augmented Wnt2 protein levels in migrating VSMCs. Migrating VSMCs on the wound edge could be seen to express higher levels of Wnt2 protein than nonmigratory VSMCs further away from the wound edge ( Figure I in the online-only Data Supplement). Wnt2 Promoted VSMC Migration In VitroAddition of rWnt2 protein significantly increased VSMC migration in vitro, whereas knockdown of Wnt2 using silencing RNA (siRNA) inhibited migration ( Figure 1C). When rWnt2 was ad...
COVID-19 is characterised by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterised by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients’ long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2 specific T cells to elucidate markers which may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that whilst some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation.
Background: Roles of the multifunctional kinase PKCα in bone are unknown.Results: Female Prkca−/− mice form bone in their medullary cavities associated with higher osteoblastic differentiation. Bone and spleen changes in Prkca−/− mice resemble features of Gaucher disease.Conclusion: PKCα regulates osteoblast differentiation and bone architecture.Significance: PKCα-targeting therapies may benefit low bone mass conditions, including Gaucher disease and osteoporosis.
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