Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting.

BACKGROUND: Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. METHODS: The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. RESULTS: Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. CONCLUSIONS: Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases.

Immunomodulatory Effects of Endoscopic Ultrasound-Guided Thermal Ablation in Patients with Pancreatic Ductal Adenocarcinoma.

Immunological consequences of endoscopic ultrasound (EUS)-local thermal ablation (LTA) for pancreatic ductal adenocarcinoma (PDAC) have not been extensively assessed. We aimed to explore EUS-LTA effects on the systemic immune response in PDAC. Peripheral blood was collected from 10 treatment-naïve patients with borderline resectable and locally advanced PDAC, randomly allocated to Nab-paclitaxel plus Gemcitabine chemotherapy (CT-arm, n = 5) or EUS-LTA with HybridTherm Probe plus CT (HTP + CT-arm, n = 5). Twenty healthy donors were included as controls. Flow-cytometry and multiplex assays were used to profile immune cell subsets and measure serum cytokines/chemokines, respectively. At baseline, PDAC patients showed increased circulating monocytes and lower circulating lymphocytes and CD19+ B cells counts compared to healthy controls. After 4 months, CT induced decrease of B regulatory cells, CD4+ cytotoxic T cells and IL-1ß. The addition of EUS-HTP to CT selectively decreased the serum levels of APRIL/TNFSF13 as well as T regulatory cells, total, classic and inflammatory monocytes. Serum levels of APRIL/TNFSF13 and total, classic and inflammatory monocytes counts at baseline were associated with worse overall survival. EUS-HTP has the potential to selectively impact on immune cells and cytokines associated with poor outcomes in PDAC.

Ceruloplasmin oxidized and deamidated by Parkinson's disease cerebrospinal fluid induces epithelial cells proliferation arrest and apoptosis.

In Parkinson's disease, the ferroxidase ceruloplasmin (Cp) is oxidized and deamidated by the pathological cerebrospinal fluid (CSF) environment. These modifications promote the gain of integrin binding properties, fostered by the deamidation of two NGR-motifs present in the Cp sequence that convert into the isoDGR-motif. Through isoDGR/integrin binding, the oxidized/deamidated-Cp (Cp-ox/de) mediates cell adhesion and transduces an intracellular signal in epithelial cells that seems to be addressed to regulate cell cycle, proliferation and cytoskeletal re-arrangement. However, the effect fostered on cells by integrins engagement via Cp-ox/de is not known. We found that in HaCaT epithelial cells, the incubation with Cp-ox/de resulted in proliferation inhibition mediated by isoDGR, cell cycle arrest and apoptosis induction. Similar proliferation inhibition was induced by treatment with purified Cp previously incubated in the CSF from Parkinson's disease patients, but not by Cp incubated in the CSF from healthy subjects. In human primary choroid plexus epithelial cells, a possible in vivo target of Cp-ox/de generated in pathological CSFs, we found that Cp-ox/de mediated cell adhesion via isoDGR/integrins binding and transduced an intracellular signal, which resulted in cell proliferation inhibition. Thus, the generation of Cp-ox/de in pathological CSFs and the consequent apoptosis induction of epithelial cells facing the liquor, might represent a novel mechanism that contributes to neurodegeneration.

Release of Prometastatic Platelet-Derived Microparticles Induced by Breast Cancer Cells: A Novel Positive Feedback Mechanism for Metastasis.

Circulating platelets and platelet-derived microparticles are regulators of cancer metastasis. In this study, we show that breast cancer cells induce platelet aggregation and lead to the release of platelet-derived microparticles. Although able to cause comparable aggregation, the highly aggressive MDA-MB-231 cells were more potent than the poorly aggressive MCF7 cells in inducing platelet-derived microparticles release, which was comparable to that promoted by thrombin. MDA-MB-231 cells were able to bind and internalize both MCF7- and MDA-MB-231-induced platelet-derived microparticles with comparable efficiency. By contrast, MCF7 cells did not interact with either type of platelet-derived microparticles. Upon internalization, only platelet-derived microparticles released by platelet stimulation with MDA-MB-231 cells, but not those released upon stimulation with MCF7 cells, caused activation of MDA-MB-231 cells and promoted the phosphorylation of selected signaling proteins, including p38MAPK and myosin light chain. Accordingly, MDA-MB-231-induced, but not MCF7-induced, platelet-derived microparticles dose-dependently stimulated migration and invasion of targeted MDA-MB-231 cells. These results identify a novel paracrine positive feedback mechanism initiated by aggressive breast cancer cell types to potentiate their invasive phenotype through the release of platelet-derived microparticles.

Expression of IFNγR2 mutated in a dileucine internalization motif reinstates IFNγ signaling and apoptosis in human T lymphocytes.

In T lymphocytes, the internalization of the R2 chain of the IFN-γ receptor (IFN-γR2) prevents the switching-on of pro-apoptotic and anti-proliferative genes induced by the IFN-γ/STAT1 pathway. In fibroblasts, a critical role of controlling the IFN-γR2 internalization is played by the LI(255-256) intracellular motif. Here we show that, in human malignant T cells, the expression of a mutated IFN-γR2 chain in which the LI(255-256) internalization motif is replaced by two alanines (LI(255-256)AA) induces cell surface accumulation of the receptor and reinstates the cell sensitivity to IFN-γ. In comparison with T cells that expressed wild-type IFN-γR2, cells that expressed the mutated receptor displayed, in response to IFN-γ a sustained activation of STAT1. The activation of this signaling pathway leads to higher induction of MHC class I and FasL expression and triggered apoptosis. Malignant ST4 cells transduced with either wild-type or mutated receptor were able to grow in SCID mice, but only the proliferation of T cells expressing the mutated receptor was inhibited by IFN-γ. Finally, lentiviral-mediated transduction of the mutated receptor in T lymphoblasts from healthy donors reinstated their IFN-γ-dependent apoptosis. As a whole, these data indicate that perturbation of IFN-γR2 internalization by mutating the LI(255-256) motif induces a timely coordinated activation of IFN-γ/STAT1 signaling pathways that leads to the apoptosis of T cells.

Ups and downs: the STAT1:STAT3 seesaw of Interferon and gp130 receptor signalling.

Downstream of cytokine or growth factor receptors, STAT3 counteracts inflammation and promotes cell survival/proliferation and immune tolerance while STAT1 inhibits proliferation and favours innate and adaptive immune responses. STAT1 and STAT3 activation are reciprocally regulated and perturbation in their balanced expression or phosphorylation levels may re-direct cytokine/growth factor signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory. Here we review the functional canonical and non-canonical effects of STAT1/3 activation and discuss the hypothesis that perturbation of their expression and/or activation levels may provide novel therapeutic strategies in different clinical settings and particularly in cancer.

IFN-gamma regulates Fas ligand expression in human CD4+ T lymphocytes and controls their anti-mycobacterial cytotoxic functions.

Fas and Fas Ligand (FasL) expression, activation-induced cell death (AICD) and mycobacterial antigen-specific cytotoxicity of peripheral T cells from patients with complete inherited IFN-gamma receptor 1 binding chain deficiency (IFN-gammaR1-/-) were investigated. Fas was equally expressed in both normal and deficient T lymphoblasts and they underwent apoptosis when stimulated with agonist anti-Fas mAb. By contrast, T lymphoblasts and CD4+ T cell clones (TCC) from deficient patients displayed a reduced surface FasL expression and resistance to AICD. CD8+ TCC from healthy and deficient patients displayed similar high level of FasL and susceptibility to AICD. In Jurkat CD4+ T cells competent to transduce IFN-gamma signaling, IFN-gamma induced surface FasL export and their Fas-dependent apoptosis. Effector T cells generated from a patient with a dominant negative mutation of IFN-gammaR1 (IFN-gammaR1DN) following stimulation with mycobacterial antigens were unable to kill MHC class II-matched, mycobacterial antigen-pulsed macrophages. Normal Fas expression in T cells and FasL in CD8+ cells may account for the absence of autoimmune disorders in these patients. Conversely, defective FasL expression on IFN-gammaR1DN CD4+ T cells impairs their cytotoxic functions and highlights a novel role for IFN-gamma signaling in the control of mycobacterial infection in humans.

Iron regulates T-lymphocyte sensitivity to the IFN-gamma/STAT1 signaling pathway in vitro and in vivo.

The refractoriness of T cells to the interferon-gamma (IFN-gamma)/signal transducer and activator of transcription 1 (STAT1) pathway, which shields them from the antiproliferative effect of IFN-gamma, is attributed mainly to down-regulation of the IFN-gammaR2 signaling chain. However, the mechanisms responsible for this down-regulation are unclear. Here we show that iron uptake mediated by the transferrin receptor (TfR) delivers a signal that leads to IFN-gammaR2 internalization and thus plays an essential role in attenuating activation of the IFN-gamma/STAT1 pathway in human T lymphocytes. The effect of iron on IFN-gammaR2 internalization was specific as it did not affect expression of the IFN-gammaR1 binding chain. Deferoxamine (DFO), an iron-chelating agent, up-regulated IFN-gammaR2 surface expression and reinstated IFN-gamma/STAT1 activation in proliferating T lymphocytes. Resistance of malignant T cells to the antiproliferative effect of IFN-gamma in vitro was abrogated by addition of DFO. Conversely, iron inhibited IFN-gamma-induced apoptosis in malignant T cells in serum-free conditions. In combination but not individually, DFO and IFN-gamma strongly inhibited growth of human malignant T cells in an in vivo severe combined immunodeficient (SCID) mouse model. These data provide valuable insights for novel therapeutic approaches aimed at reinstating the IFN-gamma/STAT1 apoptotic signaling pathway in autoreactive or neoplastic T cells by means of iron chelation.