Efficient Human Cytomegalovirus Replication in Primary Endothelial Cells Is SOCS3 Dependent

Background: In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. Methods: All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. Results: The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. Conclusion: These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses

Entry of human cytomegalovirus into porcine endothelial cells depends on both the cellular vascular origin and the viral strain

BACKGROUND Primary infection and reactivation of human cytomegalovirus (HCMV) is associated with allograft rejection. Pig-to-human xenotransplantation is regarded as an alternative to circumvent donor organ shortage and inevitably, porcine endothelial cells (pEC) will be exposed to human pathogens, among them HCMV. Infection of pEC with HCMV induces apoptosis and entry is sufficient to induce phenotypic alterations, which have the potential to result in rejection and vasculopathy. We investigated the mechanisms used by HCMV to enter pEC from different anatomical origins and compared them with the entry mechanisms used to enter human endothelial cells (hEC). METHODS Immortalized porcine aortic (PEDSV.15) and porcine microvascular bone marrow derived EC (2A2) as well as primary human aortic (HAEC) and microvascular EC (HMVEC) were inoculated with the endotheliotropic (TB40/E) or the fibroblast propagated (TB40/F) HCMV strains at multiplicity of infection (MOI) ranging from 0.3 to 5. EC were analyzed for receptor expression and their involvement in HCMV entry. The role of endocytosis was evaluated by treating EC with specific inhibitors, and the involvement of the endolysosomal pathway was investigated by confocal microscopy. RESULTS Silencing of platelet-derived growth factor receptor alpha resulted in a reduced expression of viral immediate early (IE) antigen only in pEC infected with either TB40/E or TB40/F whereas silencing of β1 integrins reduced expression of IE proteins in all EC except for TB40/F-infected microvascular pEC. TB40/E enters hEC and pEC by a similar mechanism dependent on dynamin-2, lipid rafts, actin and pH, whereas entry of TB40/F in pEC occurs mainly by a dynamin-2-dependent, clathrin-, lipid rafts-independent mechanism and in a pH-dispensable manner. When actin polymerization was prevented, TB40/F could enter pEC in an actin-independent fashion. Disturbance of the microtubule cytoskeleton resulted in an inhibition of infection of TB40/E-infected EC, whereas infection of TB40/F-infected pEC was not modified. Finally, viral particles located in vesicles of the endolysosomal pathway, suggesting that HCMV uses this pathway for intracellular trafficking following entry. CONCLUSIONS Our findings demonstrate that HCMV uses a variety of entry mechanisms that are dependent on the strain and on the vascular origin of the cells. Given the profound effect of pEC infection with HCMV, prevention of such an infection will be crucial for clinical application of xenotransplantation. A potential avenue is to render porcine grafts resistant to HCMV infection by blocking viral entry and propagation

Statins demonstrate a broad anti-cytomegalovirus activity in vitro in ganciclovir-susceptible and resistant strains

Vasculoprotective and cholesterol-lowering properties are hallmarks of statins. Recently, statins have been found to exhibit antiviral activity. Little is known about the potential of statins against human cytomegalovirus (HCMV), a risk factor in the pathogenesis of atherosclerosis. In this study, the in vitro anti-CMV activity of four statins (atorva-, fluva-, prava-, and simvastatin) was explored in human aortic endothelial cells (HAEC) and fibroblasts. All statins dose-dependently reduced HCMV titers in both cell types. Whereas atorva-, fluva-, and simvastatin showed comparable EC50 and EC90 within a low micromolar range in HAEC, pravastatin exhibited only limited effects. In metabolite rescue experiments, mevalonate almost completely abrogated the anti-CMV activity of all statins, whereas cholesterol failed to counteract the effects. Geranylgeranyl-pyrophosphate partially reversed the anti-CMV activity of most statins, suggesting an involvement of the non-sterol isoprenoid arm of the mevalonate pathway as the mode-of-action. The accumulation of immediate early viral antigens was blocked after 1 dpi onwards, and early and late antigen expression was completely abolished in HAEC. The antiviral activity of statins was comparable to ganciclovir and was retained in a ganciclovir-resistant HCMV strain. These findings provide new insight into the beneficial effects of statins, adding antiviral activity against HCMV to their list of pleïotropic properties, and support further clinical investigations on combined therapy for the management of active HCMV disease. J. Med. Virol. 87: 141-153, 2015. © 2014 Wiley Periodicals, Inc

The microtubule stabilizer patupilone counteracts ionizing radiation-induced matrix metalloproteinase activity and tumor cell invasion

BACKGROUND: Ionizing radiation (IR) in combination with microtubule stabilizing agents (MSA) is a promising combined treatment modality. Supra-additive treatment responses might result from direct tumor cell killing and cooperative indirect, tumor cell-mediated effects on the tumor microenvironment. Here we investigated deregulation of matrix metalloproteinase (MMP) activity, as an important component of the tumor microenvironment, by the combined treatment modality of IR with the clinically relevant MSA patupilone. METHODS: Expression, secretion and activity of MMPs and related tissue inhibitors of metalloproteinases (TIMPs) were determined in cell extracts and conditioned media derived from human fibrosarcoma HT1080 and human glioblastoma U251 tumor cells in response to treatment with IR and the MSA patupilone. Treatment-dependent changes of the invasive capacities of these tumor cell lines were analysed using a Transwell invasion assay. Control experiments were performed using TIMP-directed siRNA and TIMP-directed inhibitory antibodies. RESULTS: Enzymatic activity of secreted MMPs was determined after treatment with patupilone and irradiation in the human fibrosarcoma HT1080 and the human glioblastoma U251 tumor cell line. IR enhanced the activity of secreted MMPs up to 2-fold and cellular pretreatment with low dose patupilone (0.05-0.2 nM) counteracted specifically the IR-induced MMP activity. The cell invasive capacity of HT1080 and U251 cells was increased after irradiation with 2 Gy by 30% and 50%, respectively, and patupilone treatment completely abrogated IR-induced cell invasion. Patupilone did not alter the level of MMP expression, but interestingly, the protein level of secreted TIMP-1 and TIMP-2 was lower after combined treatment than after irradiation treatment alone. Furthermore, siRNA depletion of TIMP-1 or TIMP-2 prevented IR-mediated induction of MMP activity and cell invasion. CONCLUSIONS: These results indicate that patupilone counteracts an IR-induced MMP activation process by the reduction of secreted TIMP-1 and TIMP-2 proteins, which are required for activation of MMPs. Since IR-induced MMP activity could contribute to tumor progression, treatment combination of IR with patupilone might be of great clinical benefit for tumor therapy

Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs

BACKGROUND: Anti-Galα1,3Galβ-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb₃) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. METHODS: The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb₃ synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. RESULTS: Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb₃ on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H-type sugar structures present in GalT knockout cells as compared to wild-type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb₃ was also totally absent, whereas a fucosylated form of iGb₃ was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild-type or GalT knockout animals. CONCLUSIONS: These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Galα1,3Gal antibodies and cellular responses

Human CMV infection of porcine endothelial cells increases adhesion receptor expression and human leukocyte recruitment

BACKGROUND: Potential xenozoonosis is a concern for the clinical application of xenotransplantation. Human cytomegalovirus (HCMV) is one of the most important pathogens in allotransplantation, but the consequences of HCMV cross-species infection of porcine xenografts are unknown. Therefore, we investigated the effects of HCMV infection of porcine endothelial cells (pEC) on cell surface molecule expression and human leukocyte recruitment. METHODS: Infection of pEC inoculated with untreated, UV-inactivated, or heparin-treated HCMV at a multiplicity of infection (MOI) of 1 was analyzed by immediate early (IE) antigen expression. Cell surface receptor expression was studied by flow cytometry on pEC bulk cultures and differentially on IE-positive and -negative pEC. Adhesion of human leukocytes was tested on pEC monolayers. pEC supernatants were analyzed for cytokine content, chemotactic activity, and stimulatory effect on resting secondary pEC cultures. RESULTS: At day 2 postinfection, IE staining was evident in 10% to 20% of HCMV-infected cells. Cell-surface expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) was upregulated in both IE-negative and -positive fractions of HCMV-infected pEC. In contrast, porcine major histocompatibility complex class I expression was upregulated in IE-negative cells, but reduced in IE-positive cells. The receptor alterations in the IE-negative fraction were mediated by pEC-derived soluble factors. The increased adhesion receptor expression was paralleled by enhanced human leukocyte chemotaxis and adhesion to infected pEC cultures. Pretreatment of HCMV with heparin, but not UV-inactivation, prevented adhesion-receptor modulation and reversed the increased adhesion and chemotaxis. CONCLUSIONS: After pig-to-human solid organ transplantation HCMV may infect and activate the porcine endothelium, rendering the xenograft more susceptible to human leukocyte recruitment and rejection