Detection of human cytomegalovirus in synovial neutrophils obtained from patients with rheumatoid arthritis.

Objectives: To examine whether signs of an active human cytomegalovirus (HCMV) infection are present in affected joints of patients with rheumatoid arthritis (RA).Method: Polymorphonuclear leucocytes (PMNLs) were obtained from synovial fluid (SF) of 17 RA patients and were analysed for HCMV-pp65 and HCMV-immediate early (IE) proteins using the antigenemia assay. Peripheral blood (PB) and SF obtained from these 17 patients and from 17 additional RA patients (n = 34) were tested for HCMV-IE and pp150 DNA with Taqman polymerase chain reaction. Plasma samples from the patients were analysed for HCMV-immunoglobulin M (IgM) and immunoglobulin G (IgG) by enzyme-linked immunosorbent assay and compared to 71 healthy gender-matched blood donors.Results: HCMV-pp65 protein was detected in 65% of synovial PMNL samples, but in only 18% of PMNLs from PB. In contrast, HCMV IE protein was not found in any of the analysed PMNL samples. On the DNA level, HCMV-IE and pp150 DNA was detected in SF of 13/32 (41%) and 14/23 (61%) of RA patients, respectively. HCMV-IE and pp150 DNA was also found in 24/33 (73%) and in 16/24 (67%) of PB samples obtained from RA patients, respectively. HCMV IgG seroprevalence was 76% in RA patients as well as in healthy controls, while only one RA patient was positive for specific IgM.Conclusions: HCMV pp65 antigen was found in PMNLs from SF of RA patients, indicating an active infection in the affected joint. Future studies are needed to determine whether HCMV infection can aggravate the inflammatory process in these patients.

Human cytomegalovirus replication induces endothelial cell interleukin-11.

Endothelial cells (EC) are critical sites of human cytomegalovirus (hCMV) infection in vivo. Infection can induce the production of various EC cytokines, such as interleukin (IL-)6, which can have autocrine and/or paracrine effector functions. Here, we report that hCMV induces the production of EC IL-11, a relatively understudied member of the IL-6-type cytokine family. We detail temporal EC IL-11 translation and protein secretion dynamics in response to hCMV infection, and reveal distinct differences compared to EC IL-6. Viral replication had markedly opposing effects on the regulation of these closely related cytokines, representing a major driving force behind IL-11 production, whilst concurrently suppressing IL-6 expression. This is the first report of any biological agent that stimulates EC IL-11 production.

Locally Transplanted CD34+ Bone Marrow-Derived Cells Contribute to Vascular Healing After Vascular Injury.

INTRODUCTION: Vascular progenitor cells contribute to repair of injured vasculature. In this study, we aimed to investigate the role of bone marrow-derived cells in the intimal formation after arterial injury. METHODS AND RESULTS: Balloon injury of the femoral artery of wild-type mice was followed by local delivery of bone marrow-derived cells from GFP transgenic mice. The arteries were collected 1, 4, 7, and 14 days after injury and studied for morphology, localization, and phenotypes of delivered cells. Bone marrow-derived cells were present in the intima only at the early stages of arterial injury and expressed endothelial progenitor cell markers (CD31, CD34, and VEGFR-2). In the areas where intima was thicker, bone marrow-derived cells differentiated to intimal smooth muscle cells but they did not fuse with intimal cells. Delivery of CD34+ cells contributed to a 1.5-fold inhibition of intimal hyperplasia. CONCLUSION: Bone marrow-derived endothelial cells differentiated but did not fuse with vascular smooth muscle cells at the early stages of intimal formation and contributed to intimal hyperplasia.

Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells: prognostic significance and biological impact.

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor.

Apolipoprotein B100 danger-associated signal 1 (ApoBDS-1) triggers platelet activation and boosts platelet-leukocyte proinflammatory responses.

Low-density lipoproteins (LDL), occurring in vivo in both their native and oxidative form, modulate platelet function and thereby contribute to atherothrombosis. We recently identified and demonstrated that 'ApoB100 danger-associated signal 1' (ApoBDS-1), a native peptide derived from Apolipoprotein B-100 (ApoB100) of LDL, induces inflammatory responses in innate immune cells. Platelets are critically involved in the development as well as in the lethal consequences of atherothrombotic diseases, but whether ApoBDS-1 has also an impact on platelet function is unknown. In this study we examined the effect of ApoBDS-1 on human platelet function and platelet-leukocyte interactions in vitro. Stimulation with ApoBDS-1 induced platelet activation, degranulation, adhesion and release of proinflammatory cytokines. ApoBDS-1-stimulated platelets triggered innate immune responses by augmenting leukocyte activation, adhesion and transmigration to/through activated HUVEC monolayers, under flow conditions. These platelet-activating effects were sequence-specific, and stimulation of platelets with ApoBDS-1 activated intracellular signalling pathways, including Ca2+, PI3K/Akt, PLC, and p38- and ERK-MAPK. Moreover, our data indicates that ApoBDS-1-induced platelet activation is partially dependent of positive feedback from ADP on P2Y1 and P2Y12, and TxA2. In conclusion, we demonstrate that ApoBDS-1 is an effective platelet agonist, boosting platelet-leukocyte's proinflammatory responses, and potentially contributing to the multifaceted inflammatory-promoting effects of LDL in the pathogenesis of atherothrombosis.

Human cytomegalovirus infection induces leukotriene B4 and 5-lipoxygenase expression in human placentae and umbilical vein endothelial cells.

INTRODUCTION: Human cytomegalovirus (HCMV) can cause congenital infection with risk of neurological disability. Maternal-fetal transmission is associated with placental inflammation. 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of Leukotrienes (LTs), which are proinflammatory mediators. This study investigated the effect of HCMV infection on 5-LO expression and Leukotriene-B4 (LTB4) induction in human placentae and umbilical vein endothelial cells (HUVEC). METHODS: Seven placentae from fetuses with congenital HCMV infection and brain damage and six controls were stained with HCMV-immediate-early-antigen (HCMV-IEA) and 5-LO by immunohistochemistry. 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4 were measured in culture supernatant from ex vivo HCMV-infected placental histocultures by liquid chromatography. In vitro, HCMV infected HUVEC cells were analyzed for 5-LO mRNA and protein expression by real time PCR and immunofluorescence staining. RESULTS: HCMV-IEA was abundant in all HCMV infected placentae but absent in control placentae. 5-LO expression was higher in endothelial and smooth muscle cells of HCMV-infected placentae, compared to control placentae. HCMV infection induced an up-regulation of LTB4 in ex vivo placental explants with higher levels of LTB4 at 72 h compared to controls (p = 0.002). In vitro, 5-LO transcript and protein expression were significantly induced in HCMV-infected HUVEC, compared to the control cultures (p = 0.036). CONCLUSION: The presence of HCMV coincided with high 5-LO expression in cells of in vivo HCMV infected placentae. HCMV induced up-regulation of 5-LO in both ex vivo HCMV-infected placental explants and HUVEC. HCMV induced LT-biosynthesis in congenitally infected placentae may have a role in pathogenesis of congenital HCMV disease.

Human cytomegalovirus induces a biphasic inflammatory response in primary endothelial cells.

Contradictory studies report either pro- or anti-inflammatory endothelial cell (EC) responses to human cytomegalovirus (hCMV) infection, hindering the validation of a potential link between this virus and associated inflammatory pathologies. Clarifying this issue, we report that hCMV induces a biphasic response. Early after inoculation, hCMV promoted lymphocyte and, to a lesser extent, neutrophil capture under in vivo relevant shear stresses. In contrast, later stages of infection rendered EC refractory to basal, or cytokine-induced, leukocyte recruitment.

Significance of cytomegalovirus infection in the failure of native arteriovenous fistula.

High cytomegalovirus (CMV) IgG levels have been identified as a risk factor for arteriovenous fistula (AVF) failure. None of the 68 patents in our study were CMV IgM positive, although 96% were CMV IgG positive. CMV antigens were detected in the radial artery or cephalic vein of 46% of patients who received an AVF. The presence of CMV antigens or high serum CMV IgG levels had no prognostic value for AVF failure.

Detection of cytotoxic CD13-specific autoantibodies in sera from patients with ulcerative colitis and Crohn's disease.

Recent evidence suggests an association between inflammatory bowel disease (IBD) and human cytomegalovirus (HCMV) infection, but the exact pathogenic role of HCMV in this disease remains unclear. HCMV infection has for a long time been known to be associated with various autoimmune manifestations and the formation of autoantibodies. Previous studies from our group have shown that HCMV is associated with a human protein, CD13 (aminopeptidase N) and that autoantibodies against this protein are frequently found in HCMV infected bone marrow transplant patients with chronic graft versus host disease. We have recently observed that 90% of IBD patients have an active HCMV infection. In this study, we examined the presence and cytotoxicity of CD13-specific autoantibodies in sera obtained from 28 patients with ulcerative colitis and 26 patients with Crohn's disease, and in sera obtained from healthy blood donors by using flow cytometric assays against mouse cells transfected with human CD13 or a microcytotoxicity assay against different CD13 positive human cells. Cytotoxic CD13-specific autoantibodies were identified in 66% of the sera obtained from HCMV-IgG positive patients with ulcerative colitis and in 58% of the sera obtained from HCMV-IgG positive patients with Crohn's disease, but not in control individuals. These cytotoxic autoantibodies may interfere with biological cell functions and could thereby contribute to the chronic inflammation in patients with IBD.

Does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer?

Human cytomegalovirus (HCMV) is a herpes virus that infects and is carried by 70-100% of the world's population. During its evolution, this virus has developed mechanisms that allow it to survive in an immunocompetent host. For many years, HCMV was not considered to be a major human pathogen, as it appeared to cause only rare cases of HCMV inclusion disease in neonates. However, HCMV is poorly adapted for survival in the immunosuppressed host and has emerged as an important human pathogen in AIDS patients and in patients undergoing immunosuppressive therapy following organ or bone marrow transplantation. HCMV-mediated disease in such patients has highlighted the possible role of this virus in the development of other diseases, in particular inflammatory diseases such as vascular diseases, autoimmune diseases and, more recently, with certain forms of cancers. Current research is focused on determining whether HCMV plays a causative role in these diseases or is merely an epiphenomenon of inflammation. Inflammation plays a central role in the pathogenesis of HCMV. This virus has developed a number of mechanisms that enable it to hide from the cells of the immune system and, at the same time, reactivation of a latent infection requires immune activation. Numerous products of the HCMV genome are devoted to control central functions of the innate and adaptive immune responses. By influencing the regulation of various cellular processes including the cell cycle, apoptosis and migration as well as tumour invasiveness and angiogenesis, HCMV may participate in disease development. Thus, the various drugs now available for treatment of HCMV disease (e.g. ganciclovir, acyclovir and foscarnet), may also prove to be useful in the treatment of other, more widespread diseases.

The antiviral cytomegalovirus inducible gene 5/viperin is expressed in atherosclerosis and regulated by proinflammatory agents.

OBJECTIVE: Inflammatory processes play an important role in atherosclerosis, and increasing evidence implies that microbial pathogens and proinflammatory cytokines are involved in the development and activation of atherosclerotic lesions. To find new inflammatory genes, we explored the vascular transcriptional response to an activator of innate immunity bacterial lipopolysaccharides (LPSs). METHODS AND RESULTS: Gene arrays identified the cytomegalovirus-inducible gene 5 (cig5)/viperin among the genes most potently induced by LPS in human vascular biopsies. Viperin was expressed by endothelial cells in atherosclerotic arteries and significantly elevated in atherosclerotic compared with normal arteries. In culture, cytomegalovirus infection, interferon-gamma, and LPS induced viperin expression. CONCLUSIONS: Viperin is expressed in atherosclerosis and induced in vascular cells by inflammatory stimuli and cytomegalovirus infection. The putative functions of viperin in atherosclerosis may relate to disease-associated microbes.

Recognition of cytomegalovirus clinical isolate antigens by sera from cytomegalovirus-negative blood donors.

BACKGROUND: The most common way to prevent transmission of CMV by blood transfusion is to use blood products from seronegative donors. Screening of blood donors for CMV infection is usually based on detection of antigens obtained from the CMV laboratory strain AD 169. Recent evidence suggests that approximately up to 20 percent of CMV-negative blood donors may in fact be CMV-DNA positive by PCR analyses. STUDY DESIGN AND METHODS: In this study, sera from CMV-seronegative, CMV-seropositive, and CMV-DNA-positive/seronegative individuals, and from patients with acute and convalescent CMV infection for detection of CMV antibodies were analyzed. CMV antigens prepared from cells infected with CMV clinical isolates or the CMV laboratory strain AD 169 in ELISA and Western blot assays were used. RESULTS: All CMV-positive sera from blood donors were seropositive for the CMV antigens prepared from AD 169 (A2) or from a CMV clinical isolate (C6). Interestingly, whereas all CMV-negative blood donors were negative in tests for the CMV antigen A2, 36 percent were CMV seropositive using the CMV antigen C6 in ELISA. CONCLUSION: The data suggest that a substantial number of CMV-seronegative/CMV-DNA-positive serum samples contain antibodies that recognize CMV clinical isolate antigens.

Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-golgi network 46, and mannosidase II.

Although considerable progress has been made towards characterizing virus assembly processes, assignment of the site of tegumentation and envelopment for human cytomegalovirus (HCMV) is still not clear. In this study, we examined the envelopment of HCMV particles in human lung fibroblasts (HF) HL 411 and HL 19, human umbilical vein endothelial cells, human pulmonary arterial endothelial cells, and arterial smooth muscle cells at different time points after infection by electron microscopy (EM), immunohistochemistry, and confocal microscopy analysis. Double-immunofluorescence labeling experiments demonstrated colocalization of the HCMV glycoprotein B (gB) with the Golgi resident enzyme mannosidase II, the Golgi marker TGN (trans-Golgi network) 46, and the secretory vacuole marker Rab 3 in all cell types investigated. Final envelopment of tegumented capsids was observed at 5 days postinfection by EM, when tegumented capsids budded into subcellular compartments located in the cytoplasm, in close proximity to the Golgi apparatus. Immunogold labeling and EM analysis confirmed staining of the budding compartment with HCMV gB, Rab 3, and mannosidase II in HL 411 cells. However, the markers Rab 1, Rab 2, Rab 7, Lamp 1 (late endosomes and lysosomes), and Lamp 2 (lysosomes) neither showed specific staining of the budding compartment in the immunogold labeling experiments nor colocalized with gB in the immunofluorescent colocalization experiments in any cell type studied. Together, these results suggest that the final envelopment of HCMV particles takes place mainly into a Golgi-derived secretory vacuole destined for the plasma membrane, which may release new infectious virus particles by fusion with the plasma membrane.

Human cytomegalovirus (HCMV)-infected endothelial cells and macrophages are less susceptible to natural killer lysis independent of the downregulation of classical HLA class I molecules or expression of the HCMV class I homologue, UL18.

A number of reports have suggested that human cytomegalovirus (HCMV)-infected fibroblasts are resistant to natural killer (NK) lysis, and that the HCMV-encoded human leucocyte antigen (HLA) class I homologue UL18 may be responsible for this effect. While fibroblasts are easy to infect in vitro, their role in HCMV pathogenesis in vivo is unclear. Here, we have established systems to address NK recognition of infected endothelial cells and macrophages, two important HCMV cellular reservoirs in vivo. The HCMV-infected endothelial cells exhibited increased resistance to NK killing, and, in most experiments, infected macrophages demonstrated a decreased susceptibility to NK lysis. Infection with the mutant HCMV strain RV670, lacking the genes US1-9 and US11 that are responsible for downregulation of HLA class I molecules, also led to decreased NK susceptibility. Furthermore, reduced NK susceptibility was independent of the expression of the HLA class I homologue UL18, since cells infected with the UL18Delta HCMV strain were also less susceptible to NK killing. These results suggest that HCMV-induced resistance to NK cytotoxicity in endothelial cells and macrophages is independent of known pathways that interfere with the expression of cellular HLA class I A, B and C surface antigens and the HCMV encoded class I homologue UL18.

Multicenter evaluation of PCR methods for detecting CMV DNA in blood donors.

BACKGROUND: CMV DNA screening may be a useful adjunct to serologic tests in distinguishing potentially infectious blood donations from those that are "CMV-safe." However, there is currently no consensus on the optimal assay method for accurate detection of CMV DNA in donors. STUDY DESIGN AND METHODS: A blinded multicenter evaluation of seven CMV PCR assays was performed by five laboratories by using coded sets of analytical controls and donor blood samples. RESULTS: Five assays displayed sufficient sensitivity for donor screening, as judged by consistent detection of a minimum of 25 CMV genome equivalents (geq) in analytical controls constructed to contain from 1 to 100 CMV geq in background DNA from 250,000 cells, while the other two assays displayed inadequate sensitivity. Three sensitive assays, two based on nested PCR directed at the UL93 and UL32 regions of the CMV genome and another test (Monitor Assay, Roche), did not detect CMV DNA in samples from any of 20 pedigreed CMV-seronegative, Western blot-negative (S-/WB-) donors. Two other assays based on nested PCR occasionally detected CMV DNA in S-WB- samples, and one sensitive nested PCR assay directed at UL123 detected CMV DNA in a large proportion (85%) of S-WB- samples. CONCLUSION: Seven CMV PCR assays currently used for research and/or diagnostic applications displayed marked variations in sensitivity, specificity, and reproducibility when applied to coded analytical and clinical control samples containing cellular DNA from the equivalent of 250,000 WBCs. These results will be useful in the selection of assays with performance characteristics appropriate to donor screening objectives. They may also help explain discrepant findings from previous studies that used PCR to determine CMV DNA prevalence in seronegative and seropositive blood donors.

Viral infections and their impact on chronic renal allograft dysfunction.

Viral infections, particularly those involving HCMV, are an important complication of renal transplantation. Transplantation protocols and treatment regimens that increase HCMV infection and disease may promote the development of CRAD and impair long-term renal allograft survival. Investigators are beginning to illuminate the mechanisms by which HCMV infection may cause chronic rejection in general and transplant vascular sclerosis in particular. Migration and proliferation of SMCs within the intimal layer of blood vessels is an important component of transplant vascular sclerosis, and HCMV appears to facilitate both of these processes. Current management strategies for HCMV focus on prevention, either using a focal preemptive therapeutic approach or by administering antiviral therapies to all or at-risk patients.

Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent.

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4(+) and CD8(+) T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14(+) monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-gamma) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-gamma was crucial for reactivation of latent HCMV, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-gamma in the reactivation of HCMV.

Reduced expression of HLA class II molecules and Iinterleukin-10- and transforming growth factor beta1-independent suppression of T-cell proliferation in human cytomegalovirus-infected macrophage cultures.

After a primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in myeloid lineage cells, and the virus has developed several mechanisms to avoid immune recognition and destruction of infected cells. In this study, we show that HCMV utilizes two different strategies to reduce the constitutive expression of HLA-DR, -DP, and -DQ on infected macrophages and that infected macrophages are unable to stimulate a specific CD4+ T-cell response. Downregulation of the HLA class II molecules was observed in 90% of the donor samples and occurred in two phases: at an early (1 day postinfection [dpi]) time point postinfection and at a late (4 dpi) time point postinfection. The early inhibition of HLA class II expression and antigen presentation was not dependent on active virus replication, since UV-inactivated virus induced downregulation of HLA-DR and inhibition of T-cell proliferation at 1 dpi. In contrast, the late effect required virus replication and was dependent on the expression of the HCMV unique short (US) genes US1 to -9 or US11 in 77% of the samples. HCMV-treated macrophages were completely devoid of T-cell stimulation capacity at 1 and 4 dpi. However, while downregulation of HLA class II expression was rather mild, a 66 to 90% reduction in proliferative T-cell response was observed. This discrepancy was due to undefined soluble factors produced in HCMV-infected cell cultures, which did not include interleukin-10 and transforming growth factor beta1. These results suggest that HCMV reduces expression of HLA class II molecules on HCMV-infected macrophages and inhibits T-cell proliferation by different distinct pathways.

The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration.

Human cytomegalovirus (HCMV) infection of smooth muscle cells (SMCs) in vivo has been linked to a viral etiology of vascular disease. In this report, we demonstrate that HCMV infection of primary arterial SMCs results in significant cellular migration. Ablation of the chemokine receptor, US28, abrogates SMC migration, which is rescued only by expression of the viral homolog and not a cellular G protein-coupled receptor (GPCR). Expression of US28 in the presence of CC chemokines including RANTES or MCP-1 was sufficient to promote SMC migration by both chemokinesis and chemotaxis, which was inhibited by protein tyrosine kinase inhibitors. US28-mediated SMC migration provides a molecular basis for the correlative evidence that links HCMV to the acceleration of vascular disease.