An Inhibitor of the F1 Subunit of ATP Synthase (IF1) Modulates the Activity of Angiostatin on the Endothelial Cell Surface

Angiostatin binds to endothelial cell (EC)-surface F1-F0 ATP synthase, leading to inhibition of EC3 migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin-mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is Inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F1 ATP synthase and inhibited F1-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. While exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared to angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin-mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase, and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Monitoring the Initiation and Kinetics of Human Dendritic Cell-Induced Polarization of Autologous Naive CD4⁺ T Cells

<div><p>A crucial step in generating <i>de novo</i> immune responses is the polarization of naive cognate CD4⁺ T cells by pathogen-triggered dendritic cells (DC). In the human setting, standardized DC-dependent systems are lacking to study molecular events during the initiation of a naive CD4⁺ T cell response. We developed a TCR-restricted assay to compare different pathogen-triggered human DC for their capacities to instruct functional differentiation of autologous, naive CD4⁺ T cells. We demonstrated that this methodology can be applied to compare differently matured DC in terms of kinetics, direction, and magnitude of the naive CD4⁺ T cell response. Furthermore, we showed the applicability of this assay to study the T cell polarizing capacity of low-frequency blood-derived DC populations directly isolated <i>ex vivo</i>. This methodology for addressing APC-dependent instruction of naive CD4⁺ T cells in a human autologous setting will provide researchers with a valuable tool to gain more insight into molecular mechanisms occurring in the early phase of T cell polarization. In addition, it may also allow the study of pharmacological agents on DC-dependent T cell polarization in the human system.</p></div