Koon-Chu Yaiw

High Prevalence of Human Cytomegalovirus Proteins and Nucleic Acids in Primary Breast Cancer and Metastatic Sentinel Lymph Nodes

Background Breast cancer is a leading cause of death among women worldwide. Increasing evidence implies that human cytomegalovirus (HCMV) infection is associated with several malignancies. We aimed to examine whether HCMV is present in breast cancer and sentinel lymph node (SLN) metastases. Materials and Methods Formalin-fixed paraffin-embedded tissue specimens from breast cancer and paired sentinel lymph node (SLN) samples were obtained from patients with (n = 35) and without SLN metastasis (n = 38). HCMV immediate early (IE) and late (LA) proteins were detected using a sensitive immunohistochemistry (IHC) technique and HCMV DNA by real-time PCR. Results HCMV IE and LA proteins were abundantly expressed in 100% of breast cancer specimens. In SLN specimens, 94% of samples with metastases (n = 34) were positive for HCMV IE and LA proteins, mostly confined to neoplastic cells while some inflammatory cells were HCMV positive in 60% of lymph nodes without metastases (n = 35). The presence of HCMV DNA was confirmed in 12/12 (100%) of breast cancer and 10/11 (91%) SLN specimens from the metastatic group, but was not detected in 5/5 HCMV-negative, SLN-negative specimens. There was no statistically significant association between HCMV infection grades and progesterone receptor, estrogen receptor alpha and Elston grade status. Conclusions The role of HCMV in the pathogenesis of breast cancer is unclear. As HCMV proteins were mainly confined to neoplastic cells in primary breast cancer and SLN samples, our observations raise the question whether HCMV contributes to the tumorigenesis of breast cancer and its metastases.

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.

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.

High Prevalence of Human Cytomegalovirus in Brain Metastases of Patients with Primary Breast and Colorectal Cancers

BACKGROUND: Brain metastases (BMs) develop by largely unknown mechanisms and cause major morbidity and mortality in patients with solid tumors. Human cytomegalovirus (HCMV) is frequently detected in tumor tissue from patients with different cancers. Here, we aimed to determine the prevalence and potential prognostic role of HCMV in BMs. METHODS: We obtained archived samples of BMs from 41 patients with breast cancer and 37 with colorectal cancer and paired primary tumor tissues from 13 and 12 patients in each respective group. In addition, primary breast cancer tissues from 15 patients were included. HCMV proteins were detected with an immunohistochemical technique and Western blot. HCMV nucleic acids were detected with TaqMan polymerase chain reaction (PCR) assay. RESULTS: HCMV proteins were abundantly expressed in 99% of BM specimens, and in 12 of 13 (92%) paired primary breast cancer specimens. All 12 paired colon cancer samples were positive for HCMV proteins. Protein staining was mainly confined to neoplastic cells. Western blot analysis detected an HCMV-IE reactive protein in 53% of breast cancer specimens, and PCR detected the presence of HCMV DNA and transcripts in 92% and 80% of samples, respectively. Patients with high-level expression of HCMV-IE proteins in their tumors had a shorter time to tumor progression and shorter overall survival. CONCLUSIONS: The prevalence of HCMV proteins and nucleic acids is very high in primary and metastatic tumors and may drive the development of metastatic brain tumors; therefore, this virus may represent a potential therapeutic target in metastatic cancer.

Human Cytomegalovirus (HCMV) induces Human Endogenous Retrovirus (HERV) transcription

BackgroundEmerging 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.FindingsInfection 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.ConclusionsWithin this brief report we show that HCMV infection induces HERV transcriptional activity in different cell types.

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 HCMVs 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.

Detection of Circulating hcmv-miR-UL112-3p in Patients with Glioblastoma, Rheumatoid Arthritis, Diabetes Mellitus and Healthy Controls

Background microRNAs (miRNA) are 18–22 nucleotides long non-coding RNAs that regulate gene expression at a post-transcriptional level. Human cytomegalovirus (HCMV) encodes at least 26 known mature miRNAs. hcmv-miR-UL112-3p (miR-UL112-3p) is the most well characterized HCMV miRNA, which is suggested to play role in establishment and maintenance of viral latency. Elevated miR-UL112-3p levels have been reported to be present in plasma of patients with hypertension. Objectives In this study, we aimed to quantify miR-UL112-3p levels in the plasma/serum of patients with Diabetes Mellitus (DM; from the DIGAMI-2 cohort), Glioblastoma multiforme (GBM), Rheumatoid Arthritis (RA) and Healthy Controls (HC). Study Design Total RNA was isolated from plasma/serum samples of 87 patients and controls, a TaqMan miRNA assay was performed to detect miR-UL112-3p and the copy numbers were normalized to 10 ng of total RNA. HCMV IgG and IgM were analysed using ELISA. Results HCMV miR-UL112-3p was detected in 14/27 (52%) of DM, 5/20 (25%) of GBM, 1/20 (5%) of RA patients and in 2/20 (10%) of HC, respectively. Anti-HCMV IgG was detected in 85%, 65%, 75% of patients and 70% of HC, respectively. Anti-HCMV IgM was found only in one GBM patient of 87 examined patients and controls. Conclusions A higher prevalence of miR-UL112-3p was detected in DM and GBM patients than in RA patients and HC. Elevated levels of miR-UL112-3p and higher prevalence of HCMV IgG were observed in DM patients. Whether the presence of circulating miR-UL112-3p denotes a biomarker of HCMV latency or active replication in patients warrants further investigation.

High prevalence of human cytomegalovirus in carotid atherosclerotic plaques obtained from Russian patients undergoing carotid endarterectomy

BackgroundHuman cytomegalovirus (HCMV) infection is associated with cardiovascular disease (CVD) but the role of this virus in CVD progression remains unclear. We aimed to examine the HCMV serostatus in Russian patients (n = 90) who had undergone carotid endarterectomy (CEA) and controls (n = 82) as well as to determine the prevalence of HCMV immediate early (IE) and late (LA) antigens in carotid atherosclerotic plaques obtained from 89 patients. In addition, we sought to determine whether HCMV infection was associated with inflammatory activity in the plaque by quantifying infiltrating CD3 and CD68 positive cells and 5-LO immunoreactivity.MethodsHCMV serology was assessed with ELISA and immunohistochemistry staining was performed to detect HCMV antigens, CD3, CD68 and 5-LO reactivity. The Fisher’s exact test was used to compare i) seroprevalence of HCMV IgG between patients and controls and ii) HCMV-positive or –negative to that of CD3, CD68 and 5-LO immunoreactive cells in plaque samples. The student-t test was performed to connote the significance level of mean optical density between patients and controls.ResultsThe seroprevalence for HCMV IgG was high in both patients and controls (99% and 98%, respectively). Controls had significantly higher IgG titers for HCMV compared with patients (p = 0.0148). Strikingly, we found a high prevalence of HCMV antigens in atherosclerotic plaques; 57/89 (64%) and 47/87 (54%) were HCMV IE and LA positive, respectively. Most plaques had rather low HCMV reactivity with distinct areas of HCMV-positive cells mainly detected in shoulder regions of the plaques, but also in the area adjacent to the necrotic core and fibrous cap. In plaques, the cellular targets for HCMV infection appeared to be mainly macrophages/foam cells and smooth muscle cells. HCMV-positive plaques trended to be associated with increased numbers of CD68 positive macrophages and CD3 positive T cells, while 5-LO reactivity was high in both HCMV-positive and HCMV-negative plaques.ConclusionsIn Russian patients undergoing CEA, HCMV proteins are abundantly expressed in carotid plaques and may contribute to the inflammatory response in plaques via enhanced infiltration of CD68 and CD3 cells.

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.

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.