Human cytomegalovirus infection enhances 5­lipoxygenase and cycloxygenase­2 expression in colorectal cancer.

Colorectal cancer (CRC) is one of the most common and fatal types of cancer. Inflammation promotes CRC development, however, the underlying etiological factors are unknown. Human cytomegalovirus (HCMV), a virus that induces inflammation and other cancer hallmarks, has been detected in several types of malignancy, including CRC. The present study investigated whether HCMV infection was associated with expression of the pro­inflammatory enzymes 5­lipoxygenase (5­LO) and cyclooxygenase­2 (COX­2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146 individual paraffin­embedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki­67 index and EGFR by immunohistochemistry (IHC). The cores were further analyzed by IHC for the expression of two HCMV proteins (Immediate Early, IE and pp65) and the inflammatory markers 5­LO and COX­2. The CRC cell lines Caco­2 and LS­174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or anti­inflammatory drug celecoxib (CCX) and analyzed by reverse transcription­quantitative PCR and immunofluorescence for 5­LO, COX­2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in ~90% of the CRC cases tested; this was correlated with COX­2, 5­LO and KI­67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. In vitro, HCMV infection upregulated 5­LO and COX­2 transcript and proteins in both Caco­2 and LS­174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX­2, 5­LO, HCMV IE and pp65 in infected cells. HCMV was widely expressed in CRC and may promote inflammation and serve as a potential new target for CRC therapy.

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.

Enhanced neutrophil activity is associated with shorter time to tumor progression in glioblastoma patients.

Glioblastoma multiforme (GBM) is a highly malignant tumor with a poor outcome that is often positive for human cytomegalovirus (HCMV). GBM patients often have excessive numbers of neutrophils and macrophages near and within the tumor. Here, we characterized the cytokine patterns in the blood of GBM patients with and without Valganciclovir treatment. Furthermore, we determined whether neutrophil activation is related to HCMV status and patient outcome. Blood samples for analyses of cytokines and growth factors were collected from 42 GBM patients at the time of diagnosis (n = 42) and at weeks 12 and 24 after surgery. Blood neutrophils of 28 GBM patients were examined for CD11b expression. The levels of pro- and anti-inflammatory cytokines and chemokines-including interleukin (IL)-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, transforming growth factor (TGF)-ß1, interferon-γ, interferon-α, tumor necrosis factor α, and monocyte chemoattractant protein (MCP)-1were analyzed with a bead-based flow cytometry assay. During the first six months after surgery, neutrophil activity was increased in 12 patients and was unchanged or decreased in 16. Patients with increased neutrophil activity had enhanced IL-12p70, high grade HCMV and a shorter time to tumor progression (TTP) than patients without or decreased neutrophil activity (median TTP; 5.4 vs. 12 months, 95% confidence interval; 1.6-10 vs. 0.1-0.6, hazard ratio = 3 vs. 0.4, p = 0.004). The levels of IL-12p70 were significantly decreased in Valganciclovir treated patients (n = 22, T 12W vs. T 24W, p = 0.03). In conclusion, our findings suggest that neutrophil activation is an early sign of tumor progression in GBM patients.

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.

Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

Background. Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation. Methods. Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays. Results. Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs. Conclusions. Human CMV may contribute to CVD via ETBR induction.

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 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.

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

BACKGROUND: Human 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. METHODS: HCMV 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. RESULTS: The 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. CONCLUSIONS: In 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.

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103(+) dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40(+) cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Frequent detection of human cytomegalovirus in neuroblastoma: a novel therapeutic target?

Neuroblastoma is the most common and deadly tumor of childhood, where new therapy options for patients with high-risk disease are highly warranted. Human cytomegalovirus (HCMV) is prevalent in the human population and has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumor cell immune evasion. Here we show that the majority of primary neuroblastomas and neuroblastoma cell lines are infected with HCMV. Our analysis show that HCMV immediate-early protein was expressed in 100% of 36 primary neuroblastoma samples, and HCMV late protein was expressed in 92%. However, no infectious virus was detected in primary neuroblastoma tissue extracts. Remarkably, all six human neuroblastoma cell lines investigated contained CMV DNA and expressed HCMV proteins. HCMV proteins were expressed in neuroblastoma cells expressing the proposed stem cell markers CD133 and CD44. When engrafted into NMRI nu/nu mice, human neuroblastoma cells expressed HCMV DNA, RNA and proteins but did not produce infectious virus. The HCMV-specific antiviral drug valganciclovir significantly reduced viral protein expression and cell growth both in vitro and in vivo. These findings indicate that HCMV is important for the pathogenesis of neuroblastoma and that anti-viral therapy may be a novel adjuvant treatment option for children with neuroblastoma.

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.

Effects of valganciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma: a randomized, double-blind, hypothesis-generating study.

Cytomegalovirus is highly prevalent in glioblastomas. In 2006, we initiated a randomized, double-blind, placebo-controlled, hypothesis-generating study to examine the safety and potential efficacy of Valganciclovir as an add-on therapy for glioblastoma. Forty-two glioblastoma patients were randomized in double-blind fashion to receive Valganciclovir or placebo in addition to standard therapy for 6 months. Magnetic resonance images were obtained before and immediately and 3 and 6 months after surgery to evaluate treatment efficacy by measuring contrast enhancing tumor volume (primary end point). Survival data were analyzed for patients and controls in explorative analyses to aid the design of future randomized trials. Trends but no significant differences were observed in tumor volumes in Valganciclovir and placebo patients at 3 (3.58 vs. 7.44 cm3, respectively, p = 0.2881) and 6 (3.31 vs. 13.75 cm3, p = 0.2120) months. Median overall survival (OS) was similar in both groups (17.9 vs. 17.4 months, p = 0.430). Patients could take Valganciclovir for compassionate use after the study phase. Explorative analyses showed an OS of 24.1 months (95% CI, 17.4-40.3) in patients receiving >6 months of Valganciclovir (Val > 6M) versus 13.1 months (95% CI, 7.9-17.7, p < 0.0001) in patients receiving Valganciclovir for 0 or <6 months, and 13.7 months (95% CI, 6.9-17.3, p = 0.0031) in contemporary controls. OS at 4 years was 27.3% in Val>6M patients versus 5.9% in controls (p = 0.0466). Prolonged OS in Val>6M patients suggest that future randomized trials are warranted and should evaluate whether continuous antiviral treatment can improve outcome in glioblastoma patients.

Low levels of Human Cytomegalovirus Infection in Glioblastoma multiforme associates with patient survival; -a case-control study.

BACKGROUND: Glioblastoma multiforme (GBM) represent the most aggressive brain tumor with a median overall survival of about 12-15 months. Over 90% of GBM tumors have recently been shown to be infected with human cytomegalovirus (HCMV). In this case-control study, we evaluated whether there was an association between the grade of HCMV infection and long-term survival (> 18 months) in GBM patients. MATERIAL AND METHODS: Brain tumor tissue sections from consecutive GBMs patients who survived more than 18 months (n = 40), and an equal number of GBM patients, matched to date of diagnosis and surgery, operated at Karolinska University Hospital in 2000-2005 were selected. HCMV infection grade was determined by estimation of the number of HCMV positive cells (scored negative or grade 1-4) in tumor tissue specimens. Using Chi-Square test and logistic regression analysis, we analyzed whether there was an association between long-term survival and HCMV low-grade infection or other clinical parameters known to be associated with prolonged survival of GBM patients; age under 50 years, radical surgery or low recursive partition analysis (RPA) subclass. RESULTS: HCMV infection was detected in tumor samples from 79 of 80 patients (99%). Among patients surviving > 18 months, HCMV infection grade 1 in the GBM tumor was predominant. A low grade HCMV infection was found in 19 patients, of these 16 survived > 18 months. Thus, 16 of 40 (40%) GBM patients who lived > 18 months had low-grade HCMV infection while only 3 of 40 (8%) GBM patients who lived < 18 months did (p .0006, Chi-Square test). Multiple logistic regression analyses yielded an odds ratio estimate of 6.604 with 95% confidence interval (1.36-32.1) (p .019) for low grade HCMV after adjustment for RPA class III and IV, radical surgery, age and gamma knife treatment. CONCLUSION: In conclusion, we found that low-grade HCMV infection was strongly associated with long-term survival in GBM patients.

Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target.

Medulloblastomas are the most common malignant brain tumors in children. They express high levels of COX-2 and produce PGE2, which stimulates tumor cell proliferation. Human cytomegalovirus (HCMV) is prevalent in the human population and encodes proteins that provide immune evasion strategies and promote oncogenic transformation and oncomodulation. In particular, HCMV induces COX-2 expression; STAT3 phosphorylation; production of PGE2, vascular endothelial growth factor, and IL-6; and tumor formation in vivo. Here, we show that a large proportion of primary medulloblastomas and medulloblastoma cell lines are infected with HCMV and that COX-2 expression, along with PGE2 levels, in tumors is directly modulated by the virus. Our analysis indicated that both HCMV immediate-early proteins and late proteins are expressed in the majority of primary medulloblastomas. Remarkably, all of the human medulloblastoma cell lines that we analyzed contained HCMV DNA and RNA and expressed HCMV proteins at various levels in vitro. When engrafted into immunocompromised mice, human medulloblastoma cells induced expression of HCMV proteins. HCMV and COX-2 expression correlated in primary tumors, cell lines, and medulloblastoma xenografts. The antiviral drug valganciclovir and the specific COX-2 inhibitor celecoxib prevented HCMV replication in vitro and inhibited PGE2 production and reduced medulloblastoma tumor cell growth both in vitro and in vivo. Ganciclovir did not affect the growth of HCMV-negative tumor cell lines. These findings imply an important role for HCMV in medulloblastoma and suggest HCMV as a novel therapeutic target for this tumor.