Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

28Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically 29 administered drugs demand high doses and are accompanied by poor selectivity and side 30 effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is 31 based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled 32 release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous 33 deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating 34 electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron 35 oxide nanoparticles (SPIONs). Here, we show proof-of-principle of controlled release by the 36 simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, 37 which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that 38 had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we 39 demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 40 (SPION-loaded macrophage : tumour cells). Overall, our results demonstrate that AMF 41 induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may 42 be an attractive novel strategy for targeted drug release. 43 Keywords: 44 Cell based drug delivery; macrophages; magnetic silica nanoparticles; hyperthermia; 45 controlled drug delivery, 3D tumor model 46 47 48 102 tumor size was observed, indicating that the therapeutic potential of hyperthermia induced by 103such an approach might be limited [42]. 104 In the last years, macrophages emerged as another/an interesting cell type for take up of 105 nanoparticles [43, 44]. However, controlled drug release from macrophage entrapped 106 nanoparticles remains to be explored. Here, we explore macrophages as carriers for SPIONs 107 carrying a silica shell. We show that macrophages efficiently take up the silica-coated SPION 108 nanoparticles. AMF-induced hyperthermia can induce death of loaded macrophages in a cell 109 type specific manner. Using silica-coated SPION nanoparticles functionalized with a toxin via 110 a thermosensitive linker (T-SPIONs) we demonstrate that AMF is sufficient to liberate the 111 toxin from T-SPION loaded cells, with negligible spontaneous release. The release of the 112 toxin could specifically induce the death of Kaposi's sarcoma cells in 3D spheroid co-113 cultures, thereby potentiating the efficiency of heat-induced killing of tumour cells.114 5 Materials and Methods 115 Cell culture 116 The mouse macrophage cell lines IC21, J774a.1, RAW264.7 as well as the human breast 117 carcinoma cell line MCF-7 and the human monocyte cell line U937 were obtained from 118 DSMZ (Braunschweig, Germany). IC21 and U937 cells were cultured in RPMI media 119 supplemented with 10% fetal calf serum, 60 µg/mL penicillin and 100 µg/mL streptomycin. 120 J774a.1 and RAW264.7 cells were cultured in DMEM media supplemented w...

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies: Kaposi’s sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. Previous research has shown that several cellular tyrosine kinases play crucial roles during several steps in the virus replication cycle. Two KSHV proteins also have protein kinase function: open reading frame (ORF) 36 encodes a serine-threonine kinase, while ORF21 encodes a thymidine kinase (TK), which has recently been found to be an efficient tyrosine kinase. In this study, we explore the role of the ORF21 tyrosine kinase function in KSHV lytic replication. By generating a recombinant KSHV mutant with an enzymatically inactive ORF21 protein, we show that the tyrosine kinase function of ORF21/TK is not required for the progression of the lytic replication in tissue culture but that it is essential for the phosphorylation and activation to toxic moieties of the antiviral drugs zidovudine and brivudine. In addition, we identify several tyrosine kinase inhibitors, already in clinical use against human malignancies, which potently inhibit not only ORF21 TK kinase function but also viral lytic reactivation and the development of KSHV-infected endothelial tumors in mice. Since they target both cellular tyrosine kinases and a viral kinase, some of these compounds might find a use in the treatment of KSHV-associated malignancies. IMPORTANCE Our findings address the role of KSHV ORF21 as a tyrosine kinase during lytic replication and the activation of prodrugs in KSHV-infected cells. We also show the potential of selected clinically approved tyrosine kinase inhibitors to inhibit KSHV TK, KSHV lytic replication, infectious virion release, and the development of an endothelial tumor. Since they target both cellular tyrosine kinases supporting productive viral replication and a viral kinase, these drugs, which are already approved for clinical use, may be suitable for repurposing for the treatment of KSHV-related tumors in AIDS patients or transplant recipients.

An endothelial cell line infected by Kaposi's sarcoma associated herpes virus (KSHV) allows the investigation of Kaposi's sarcoma and the validation of novel viral inhibitors in vitro and in vivo

Pseudotyped viruses bearing the glycoprotein(s) of a donor virus over the nucleocapsid core of a surrogate virus are widely used as safe substitutes for infectious virus in virology studies. Retroviral particles pseudotyped with influenza A virus glycoproteins have been used recently for the study of influenza hemagglutinin and neuraminidase-dependent processes. Here, we report the development of vesicular-stomatitis-virus-based pseudotypes bearing the glycoproteins of influenza A virus. We show that pseudotypes bearing the hemagglutinin and neuraminidase of H5N1 influenza A virus mimic the wild-type virus in neutralization assays and sensitivity to entry inhibitors. We demonstrate the requirement of NA for the infectivity of pseudotypes and show that viruses obtained with different NA proteins are significantly different in their transduction activities. Inhibition studies with oseltamivir carboxylate show that neuraminidase activity is required for pseudovirus production, but not for the infection of target cells with H5N1-VSV pseudovirus. The HA-NA-VSV pseudoviruses have high transduction titers and better stability than the previously reported retroviral pseudotypes and can replace live influenza virus in the development of neutralization assays, screening of potential antivirals, and the study of different HA/NA reassortants.