General Strategy for Decoration of Enveloped Viruses with Functionally Active Lipid-Modified Cytokines▿

Viral particles preferentially incorporate extra- and intracellular constituents of host cell lipid rafts, a phenomenon central to pseudotyping. Based on this mechanism, we have developed a system for the predictable decoration of enveloped viruses with functionally active cytokines that circumvents the need to modify viral proteins themselves. Human interleukin-2 (hIL-2), hIL-4, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), and murine IL-2 (mIL-2) were used as model cytokines and fused at their C terminus to the glycosylphosphatidylinositol (GPI) acceptor sequence of human Fcγ receptor III (CD16b). We show here that genetically modified cytokines are all well expressed on 293 producer cells. However, only molecules equipped with GPI anchors but not those linked to transmembrane/intracellular regions of type I membrane proteins are efficiently targeted to lipid rafts and consequently to virus-like particles (VLP) induced by Moloney murine leukemia virus Gag-Pol. hIL-4::GPI and hGM-CSF::GPI coexpressed on VLP were found to differentiate monocytes towards dendritic cells. Apart from myeloid-committed cell types, VLP-bound cytokines also act efficiently on lymphocytes. hIL-2::GPI strongly costimulated T-cell receptor (TCR)/CD3 dependent T-cell activation in vitro and mIL-2::GPI-coactivated antigen-specific T cells in vivo. On a molar basis, the functional activity of VLP-bound hIL-2::GPI was found to be comparable to that of soluble hIL-2. VLP decorated with hIL-2::GPI and coexpressing a TCR/CD3 ligand have an IL-2-specific activity of 5 × 104 units/mg protein. Virus particles decorated with lipid-modified cytokines might help to improve viral strains for vaccination purposes, the propagation of factor-dependent cell types, as well as gene transfer by viral systems in the future

Tunelização esofágica na fístula esofagotraqueobrônquica

Foram estudados 121 casos de câncer avançado do esôfago e da cárdia submetidos a tunelização esofágica por via endoscópica ou cirúrgica. Os doentes foram avaliados, tratados e seguidos segundo protocolo que constou de avaliação clínica, exames subsidiários, estadiamento, tratamento, avaliação intra e pós-operatória imediata. Verificaram-se a taxa de mortalidade, período de internação, complicações tardias, sobrevida e causa de óbito. Foram submetidos a tunelização cirúrgica 69 (53%) doentes, e 52 (47%) a tunelização endoscópica. Nos 17 casos de fístula esofagotraqueobrônquica houve sua oclusão eficiente pela tunelização endoscópica em 80% dos casos, com mortalidade de 13,3%, credenciando o método para tratamento dessa complicação tumoral. Os resultados obtidos com a tunelização endoscópica e cirúrgica recomendam seu uso em doentes com câncer avançado do esôfago e da cárdia

Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions

We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP+ HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R+) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2+ target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context.—Kueng, H. J., Manta, C., Haiderer, D., Leb, V. M., Schmetterer, K. G., Neunkirchner, A., Byrne, R. A., Scheinecker, C., Steinberger, P., Seed, B., Pickl, W. F. Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions