Higher Human Cytomegalovirus (HCMV) Specific IgG Antibody Levels in Plasma Samples from Patients with Metastatic Brain Tumors Are Associated with Longer Survival.

Background: Human cytomegalovirus (HCMV) has been detected in tissue samples from patients with glioblastoma but little is known about the systemic immunological response to HCMV in these patients. Objectives: To investigate the presence and clinical significance of HCMV antibodies levels in plasma samples obtained from patients with brain tumors. Materials and Methods: HCMV-specific IgG and IgM antibody levels were determined in 59 plasma samples collected from brain tumor patients included in a prospective study and in 114 healthy individuals. We examined if the levels of HCMV specific antibodies varied in patients with different brain tumor diagnoses compared to healthy individuals, and if antibody levels were predictive for survival time. Results: HCMV specific IgG antibodies were detected by ELISA in 80% and 89% of patients with GBM and astrocytoma grades II-III, respectively, in all samples (100%) from patients with secondary GBM and brain metastases, as well as in 80% of healthy donors (n = 114). All plasma samples were negative for HCMV-IgM. Patients with brain metastases who had higher plasma HCMV-IgG titers had longer survival times (p = 0.03). Conclusions: HCMV specific IgG titers were higher among all brain tumor patient groups compared with healthy donors, except for patients with secondary GBM. Higher HCMV specific IgG levels in patients with brain metastases but not in patients with primary brain tumors were associated with prolonged survival time.

Overexpression of endothelin B receptor in glioblastoma: a prognostic marker and therapeutic target?

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with median survival of 12-15 months. Owing to uncertainty in clinical outcome, additional prognostic marker(s) apart from existing markers are needed. Since overexpression of endothelin B receptor (ETBR) has been demonstrated in gliomas, we aimed to test whether ETBR is a useful prognostic marker in GBM and examine if the clinically available endothelin receptor antagonists (ERA) could be useful in the disease treatment. METHODS: Data from The Cancer Genome Atlas and the Gene Expression Omnibus database were analyzed to assess ETBR expression. For survival analysis, glioblastoma samples from 25 Swedish patients were immunostained for ETBR, and the findings were correlated with clinical history. The druggability of ETBR was assessed by protein-protein interaction network analysis. ERAs were analyzed for toxicity in in vitro assays with GBM and breast cancer cells. RESULTS: By bioinformatics analysis, ETBR was found to be upregulated in glioblastoma patients, and its expression levels were correlated with reduced survival. ETBR interacts with key proteins involved in cancer pathogenesis, suggesting it as a druggable target. In vitro viability assays showed that ERAs may hold promise to treat glioblastoma and breast cancer. CONCLUSIONS: ETBR is overexpressed in glioblastoma and other cancers and may be a prognostic marker in glioblastoma. ERAs may be useful for treating cancer patients.

Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells.

Human cytomegalovirus (HCMV) infection results in the production of virions, dense bodies (DBs) and non-infectious enveloped particles, all of which incorporate proteins and RNAs that can be transferred to host cells. Here, we investigated whether virions and DBs also carry microRNAs (miRNAs) and assessed their delivery and functionality in cells. Human lung fibroblasts (MRC-5) were infected with the HCMV strain AD169, and conditioned cell culture medium was collected and centrifuged. The pellets were treated with RNase-ONE, and the virions and DBs were purified with a potassium tartrate-glycerol gradient and dialysed. The virions and DBs were incubated with micrococcal nuclease, DNA and RNA were extracted and then analysed with TaqMan PCR assays, while the proteins were examined with Western blots. To assess the delivery of miRNAs to cells and their functionality, virions and DBs were irradiated with UV light. The purity of the virions and DBs was confirmed by typical morphology, the presence of the structural protein pp65 and the HCMV genome, the ability to infect MRC-5 cells and the absence of the host genome. RNA analysis revealed the presence of 14 HCMV-encoded miRNAs (UL22A-5p, US25-1-5p, UL22A-3p, US5-2-3p, UL112-3p, US25-2-3p, US25-2-5p, US33-3p, US5-1, UL36-5p, US4-5p, UL36-3p, UL70-5p and US25-1-3p), HCMV immediate-early mRNA and long non-coding RNA2.7, moreover, two host-encoded miRNAs (hsa-miR-218-5p and hsa-miR-21-5p) and beta-2-microglobulin RNA. UV-irradiated virions and DBs delivered viral miRNAs (US25-1-5p and UL112-3p) to the host cells, and miR-US25-1-5p was functional in a luciferase reporter assay. We conclude that virions and DBs carry miRNAs that are biologically functional and can be delivered to cells, which may affect cellular processes.

Direct infection of primary endothelial cells with human cytomegalovirus prevents angiogenesis and migration.

Human cytomegalovirus (hCMV) is a beta herpesvirus that establishes lifelong infection. Although the virus does not usually cause overt clinical symptoms in immunocompetent individuals it can have deleterious effects in immunocompromised patients, such as those on post-transplant medication or with HIV infection. hCMV is the most common congenital infection and can lead to serious fetal sequelae. Endothelial cells (ECs) are natural hosts for hCMV in vivo, therefore, investigations of how this cell type is modulated by infection are key to understanding hCMV pathogenesis. Previous studies have examined the effect of secretomes from hCMV-infected cells on EC angiogenesis, whereas the effect of direct infection on this process has not been so well investigated. Here, we show that placental ECs are viral targets during congenital infection and that vessels in infected tissue appear morphologically abnormal. We demonstrate that the clinical hCMV strain VR1814 impaired EC tube assembly in in vitro angiogenesis assays and inhibited wound healing ability in scratch assays. Secretomes from infected cultures did not impair angiogenesis of uninfected ECs, suggesting that cell-intrinsic changes, as opposed to secreted factors, were responsible. We observed viral gene transcription dependent downregulation of the expression of angiogenesis-associated genes, including angiopoietin-2, TEK receptor and vascular endothelial growth factor receptors. An alternative clinical hCMV stain, TB40E showed similar effects on EC angiogenesis. Together, our data indicate that direct infection with hCMV can induce an anti-migratory and anti-angiogenic EC phenotype, which could have a detrimental effect on the vasculature development in infected tissues.

Platelets mediate oxidized low-density lipoprotein-induced monocyte extravasation and foam cell formation.

OBJECTIVE: A growing body of evidence indicates that platelets contribute to the onset and progression of atherosclerosis by modulating immune responses. We aimed to elucidate the effects of oxidized low-density lipoprotein (OxLDL) on platelet-monocyte interactions and the consequences of these interactions on platelet phagocytosis, chemokine release, monocyte extravasation, and foam cell formation. APPROACH AND RESULTS: Confocal microscopy and flow cytometric analysis revealed that in vitro and in vivo stimulation with OxLDL resulted in rapid formation of platelet-monocyte aggregates, with a preference for CD16+ monocyte subsets. This platelet-monocyte interaction facilitated OxLDL uptake by monocytes, in a process that involved platelet CD36-OxLDL interaction, release of chemokines, such as CXC motif ligand 4, direct platelet-monocyte interaction, and phagocytosis of platelets. Inhibition of cyclooxygenase with acetylsalicylic acid and antagonists of ADP receptors, P2Y1 and P2Y12, partly abrogated OxLDL-induced platelet-monocyte aggregates and platelet-mediated lipid uptake in monocytes. Platelets also enhanced OxLDL-induced monocyte transmigration across an endothelial monolayer via direct interaction with monocytes in a transwell assay. Importantly, in LDLR(-/-) mice, platelet depletion resulted in a significant decrease of peritoneal macrophage recruitment and foam cell formation in a thioglycollate-elicited peritonitis model. In platelet-depleted wild-type mice, transfusion of ex vivo OxLDL-stimulated platelets induced monocyte extravasation to a higher extent when compared with resting platelets. CONCLUSIONS: Our results on OxLDL-mediated platelet-monocyte aggregate formation, which promoted phenotypic changes in monocytes, monocyte extravasation and enhanced foam cell formation in vitro and in vivo, provide a novel mechanism for how platelets potentiate key steps of atherosclerotic plaque development and plaque destabilization.

Human cytomegalovirus-platelet interaction triggers toll-like receptor 2-dependent proinflammatory and proangiogenic responses.

OBJECTIVE: Human cytomegalovirus (HCMV) is a widespread pathogen that correlates with various clinical complications, including atherosclerosis. HCMV is released into the circulation during primary infection and periodic viral reactivation, allowing virus-platelet interactions. Platelets are important in the onset and development of atherosclerosis, but the consequences of platelet-HCMV interactions are unclear. APPROACH AND RESULTS: We studied the effects of HCMV-platelet interactions in blood from healthy donors using the purified clinical HCMV isolate VR1814. We demonstrated that HCMV bound to a Toll-like receptor (TLR) 2-positive platelet subpopulation, which resulted in signal transduction, degranulation, and release of proinflammatory CD40L and interleukin-1ß and proangiogenic vascular endothelial-derived growth factor. In mice, murine CMV activated wild-type but not TLR2-deficient platelets. However, supernatant from murine CMV-stimulated wild-type platelets also activated TLR2-deficient platelets, indicating that activated platelets generated soluble mediators that triggered further platelet activation, independent of TLR2 expression. Inhibitor studies, using ADP receptor antagonists and apyrase, revealed that ADP release is important to trigger secondary platelet activation in response to HCMV. HCMV-activated platelets rapidly bound to and activated neutrophils, supporting their adhesion and transmigration through endothelial monolayers. In an in vivo model, murine CMV induced systemic upregulation of platelet-leukocyte aggregates and plasma vascular endothelial-derived growth factor in mice and showed a tendency to enhance neutrophil extravasation in a TLR2-dependent fashion. CONCLUSIONS: HCMV is a well-adapted pathogen that does not induce immediate thrombotic events. However, HCMV-platelet interactions lead to proinflammatory and proangiogenic responses, which exacerbate tissue damage and contribute to atherogenesis. Therefore, platelets might contribute to the effects of HCMV in accelerating atherosclerosis.

Human cytomegalovirus inhibits erythropoietin production.

Anemia is a feature of CKD and a complication of renal transplantation, often caused by impaired production of erythropoietin. The kidney is a target organ for human cytomegalovirus (hCMV) in such patients, but it is not known whether hCMV effects erythropoietin production. We found that kidneys from patients with CKD were positive for hCMV protein and that blood levels of hCMV IgG inversely correlated with red blood cell count. In mice, systemic murine cytomegalovirus infection decreased serum erythropoietin levels. In human erythropoietin-producing cells, hCMV inhibited hypoxia-induced expression of erythropoietin mRNA and protein. hCMV early gene expression was responsible, as ultraviolet-inactivated virus had no effect and valganciclovir treatment showed that late gene expression was nonessential. Hypoxia-induced gene transcription is controlled by the transcription factors hypoxia-inducible transcription factor (HIF)-1α and HIF2α, which are constitutively produced but stable only under low oxygen conditions. We found that hCMV inhibited constitutive production of HIF2α mRNA. HIF2α is thought to be the master regulator of erythropoietin transcription. Single-cell analysis revealed that nuclear accumulation of HIF2α was inhibited in hCMV-infected cells, and the extent of inhibition correlated with hCMV protein expression. Our findings suggest that renal hCMV infection could induce or exacerbate anemia in patients.

Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis.

A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMV's role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

Human cytomegalovirus induces upregulation of arginase II: possible implications for vasculopathies.

Both human cytomegalovirus (HCMV) and arginase II (ARG II) have been implicated in the pathogenesis of cardiovascular diseases. The effects of HCMV on ARG II are unknown. The aim of this study was to investigate the effects of HCMV on ARG II expression in endothelial and vascular smooth muscle cells (SMC) both in vitro and ex vivo. Endothelial and SMC were infected with either HCMV or UV-irradiated HCMV. Expression of ARG II, endothelial or inducible nitric oxide synthase (eNOS and iNOS, respectively) and viral immediate early (IE) was quantified using quantitative PCR. Ganciclovir and short interfering RNA were used to determine the viral gene mediating the effects on ARG II. Detection of viral antigens and ARG II expression was performed by immunofluorescence or immunohistochemistry. HCMV infection increased both ARG II mRNA and protein levels in the examined cells; this effect was mediated by the HCMV IE2-p86 protein. The upregulation of ARG II was accompanied by a downregulation of eNOS but an induction of iNOS in HCMV-infected endothelial cells. Both eNOS and iNOS expressions were induced in HCMV-infected SMC. ARG II was abundantly expressed in endothelial cells, foam cells and SMC and was importantly significantly upregulated in HCMV-immunoreactive human carotid atherosclerotic plaques. HCMV IE2-p86 mediates ARG II upregulation in vitro and ARG II is co-expressed with HCMV antigens in human carotid atherosclerotic plaques. We speculate that HCMV may contribute to endothelial dysfunction via ARG II induction and reduced eNOS production.

Human cytomegalovirus (HCMV) induces human endogenous retrovirus (HERV) transcription.

BACKGROUND: Emerging evidence suggests that human cytomegalovirus (HCMV) is highly prevalent in tumours of different origin. This virus is implied to have oncogenic and oncomodulatory functions, through its ability to control host gene expression. Human endogenous retroviruses (HERV) are also frequently active in tumours of different origin, and are supposed to contribute as cofactors to cancer development. Due to the high prevalence of HCMV in several different tumours, and its ability to control host cell gene expression, we sought to define whether HCMV may affect HERV transcription. FINDINGS: Infection of 3 established cancer cell lines, 2 primary glioblastoma cells, endothelial cells from 3 donors and monocytes from 4 donors with HCMV (strains VR 1814 or TB40/F) induced reverse transcriptase (RT) activity in all cells tested, but the response varied between donors. Both, gammaretrovirus-related class I elements HERV-T, HERV-W, HERV-F and ERV-9, and betaretrovirus-related class II elements HML-2 - 4 and HML-7 - 8, as well as spuma-virus related class III elements of the HERV-L group were up-regulated in response to HCMV infection in GliNS1 cells. Up-regulation of HERV activity was more pronounced in cells harbouring active HCMV infection, but was also induced by UV-inactivated virus. The effect was only slightly affected by ganciclovir treatment and was not controlled by the IE72 or IE86 HCMV genes. CONCLUSIONS: Within this brief report we show that HCMV infection induces HERV transcriptional activity in different cell types.

Envelope-chimeric entry-targeted measles virus escapes neutralization and achieves oncolysis.

Measles virus (MV) is a promising vector for cancer therapy and multivalent vaccination, but high prevalence of pre-existing neutralizing antibodies may reduce therapeutic efficacy, particularly following systemic administration. MV has only one serotype, but here we show that its envelope glycoproteins can be exchanged with those of the closely related canine distemper virus (CDV), generating a chimeric virus capable of escaping neutralization. To target its entry, we displayed on the CDV attachment protein a single-chain antibody specific for a designated receptor. To enhance oncolytic efficacy we armed the virus with a prodrug convertase gene capable of locally activating chemotherapeutic prodrugs. The new virus achieved high titers, was genetically stable, and was resistant to neutralization by sera from both MV-immunized mice and MV-immune humans. The new virus targeted syngeneic murine tumor cells expressing the designated receptor implanted in immunocompetent mice, and synergized with a chemotherapeutic prodrug in a model of oncolysis. Importantly, the chimeric MV remained oncolytic when administered systemically even in the presence of anti-MV antibodies capable of abrogating the therapeutic efficacy of the parental, nonshielded MV. This work shows that targeting, arming, and shielding can be combined to generate a tumor-specific, neutralization-resistant virus that can synergize with chemotherapeutics.

Intragraft cytomegalovirus protein expression is associated with reduced renal allograft survival.

BACKGROUND: Cytomegalovirus (CMV) infection is a risk factor for acute and chronic rejection of transplanted organs and is thought to mediate rejection indirectly. METHODS: In this retrospective observational cohort study, early- and end-stage biopsies from renal allografts lost because of chronic allograft dysfunction (n = 29) were examined for CMV antigens and DNA using immunohistochemistry, in situ hybridization, and real-time polymerase chain reaction. RESULTS: CMV immediate-early and late proteins were present in 27 (93%) of 29 of the end-stage chronic allograft dysfunction biopsies and in 64% of the corresponding early biopsies but not in pretransplant biopsies from CMV-seronegative donors (n = 3). Graft survival time was reduced in patients with moderate or high CMV levels in the graft soon after transplantation compared with that in patients with no or low CMV levels in the graft. No significant difference was observed in serum creatinine obtained at the time of early biopsies. CONCLUSIONS: We provide evidence that intragraft CMV protein expression is associated with end-stage chronic renal allograft dysfunction, that intragraft CMV levels increase as graft function deteriorates, and that CMV protein expression in the grafts soon after transplant is associated with reduced graft survival. Thus, CMV may have a pathological role in chronic renal allograft dysfunction.

The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

Human Cytomegalovirus Reduces Endothelin-1 Expression in Both Endothelial and Vascular Smooth Muscle Cells.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

Tioman virus, a paramyxovirus of bat origin, causes mild disease in pigs and has a predilection for lymphoid tissues.

Disease manifestation, pathology, and tissue tropism following infection with Tioman virus (TioPV), a newly isolated, bat-derived paramyxovirus, was investigated in subcutaneously (n = 12) and oronasally (n = 4) inoculated pigs. Pigs were either asymptomatic or developed pyrexia, but all of the animals produced neutralizing antibodies. The virus (viral antigen and/or genome) was detected in lymphocytes of the thymus, tonsils, spleen, lymph nodes and Peyer's patches (ileum), tonsillar epithelium, and thymic epithelioreticular cells. Virus was isolated from oral swabs but not from urine. Our findings suggest that the pig could act as an intermediate or amplifying host for TioPV and that oral secretion is a possible means of viral transmission.

Tioman virus infection in experimentally infected mouse brain and its association with apoptosis.

Tioman virus is a newly described bat-urine derived paramyxovirus isolated in Tioman Island, Malaysia in 2001. Hitherto, neither human nor animal infection by this virus has been reported. Nonetheless, its close relationship to another paramyxovirus, the Menangle virus which had caused diseases in humans and pigs [Philbey, A.W., Kirkland, P.D., Ross, A.D., Davis, R.J., Gleeson, A.B., Love, R.J., Daniels, P.W., Gould, A.R., Hyatt, A.D., 1998. An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4, 269-271], raises the possibility that it may be potentially pathogenic. In this study, mice were experimentally infected with Tioman virus by intraperitoneal and intracerebral routes, and the cellular targets and topographical distribution of viral genome and antigens were examined using in situ hybridization and immunohistochemistry, respectively. The possible association between viral infection and apoptosis was also investigated using the TUNEL assay and immunohistochemistry to FasL, Caspase-3, Caspase-8, Caspase-9 and bcl-2. The results showed that Tioman virus inoculated intracerebrally was neurotropic causing plaque-like necrotic areas, and appeared to preferentially replicate in the neocortex and limbic system. Viral infection of inflammatory cells was also demonstrated. TUNEL and Caspase-3 positivity was found in inflammatory cells but not in neurons, while FasL, Caspase-8 and Caspase-9 were consistently negative. This suggests that neuronal infection was associated with necrosis rather than apoptosis. Moreover, the data suggest that there may be an association between viral infection and apoptosis in inflammatory cells, and that it could, at least in part, involve Caspase-independent pathways. Bcl-2 was expressed in some neurons and inflammatory cells indicating its possible role in anti-apoptosis. There was no evidence of central nervous system infection via the intraperitoneal route.

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Human cytomegalovirus may promote tumour progression by upregulating arginase-2.

BACKGROUND: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.

Discordant humoral and cellular immune responses to Cytomegalovirus (CMV) in glioblastoma patients whose tumors are positive for CMV.

Background. Glioblastoma (GBM) is the most common malignant brain tumor in adults and is nearly always fatal. Emerging evidence suggests that human Cytomegalovirus (HCMV) is present in 90-100% of GBMs and that add-on antiviral treatment for HCMV show promise to improve survival. Methods. In a randomized, placebo-controlled trial of valganciclovir in 42 GBM patients, blood samples were collected for analyses of HCMV DNA, RNA, reactivity against HCMV peptides, IgG, and IgM at baseline and at 3, 12, and 24 weeks of treatment. Results. All 42 tumors were positive for HCMV protein. All patients examined had at least one blood sample positive for HCMV DNA, 63% were HCMV RNA positive, and 21% were IgM positive. However, 29% of GBM patients were IgG negative for HCMV. Five of these samples were positive in an enzyme-linked immunosorbent assay (ELISA) that used antigens derived from a clinical isolate. Blood T cells from 11 of 13 (85%) HCMV IgG-negative GBM patients reacted against HCMV peptides. Valganciclovir did not affect IgG titers, DNA, or RNA levels of the HCMV immediate early (HCMV IE) gene in blood. Conclusion. In GBM patients, HCMV activity is higher than in healthy controls and serology is a poor test to define previous or active HCMV infection in these patients.