Is human cytomegalovirus a target in cancer therapy?
PDF | HTML | How to cite
Metrics: PDF 2932 views | HTML 2978 views | ?
1 Childhood Cancer Research Unit, Department of Women´s and Children´s Health, Karolinska Institute, 171 76 Stockholm, Sweden
2 Karolinska Institute, Department of Medicine, Center for Molecular Medicine, Karolinska University Hospital in Solna, 171 76 Stockholm, Sweden
Received: December 12, 2011; Accepted: December 15, 2011; Published: December 31, 2011;
Keywords: cancer, human cytomegalovirus
John Inge Johnsen, email:
Human cytomegalovirus (HCMV) is a herpesvirus that is prevalent in the human population. HCMV has recently been implicated in different cancer forms where it may provide mechanisms for oncogenic transformation, oncomodulation and tumour cell immune evasion. Moreover, antiviral treatment against HCMV has been shown to inhibit tumour growth in preclinical models. Here we describe the possible involvement of HCMV in cancer and discuss the potential molecular impact expression of HCMV proteins have on tumour cells and the surrounding tumour microenvironment.
The interplay between cancer cells and the surrounding microenvironment is essential for the growth and spread of a tumour. The development of malignant tumours requires a microenvironment that supports the uncontrolled proliferation and spread of cancer cells but also conditions that avoid destruction from the various arms of the immune system must be present. The immune system represents an important tool for the destruction of the majority of cancer cells and precancerous conditions in the human body. However, malignant growing tumours have in most, if not all, cases developed immune evasion strategies to avoid destruction by immune cells. One essential immune evasion strategy that can be induced or applied by tumour cells is the formation of an inflammatory microenvironment. Tumour cells can induce inflammation directly through oncogenes that induce transcriptional programs responsible for the production of pro-inflammatory eicosanoids, cytokines and chemokines that attract different cells of the immune system to the microenvironment. Also chronic inflammation caused by viral or microbial infections, autoimmune diseases, dietary products or inflammatory conditions caused by unknown reasons can create an inflammatory microenvironment that support tumour growth . Immune cells that are recruited to the tumour are generally disabled to eliminate tumour cells. Indeed, tumour-related inflammation is regarded as one enabling characteristic crucial for the tumour cell to sustain a proliferative state, evade apoptosis, increase angiogenesis, invasion, metastasis and suppression of immune responses .
Although it has been both experimentally difficult and heavily debated, it is today well accepted that approximately 20% of the global cancer burden can be linked to infectious agents including viruses, bacteria and parasites . Recent studies indicate that the list of infectious agents linked to certain cancer forms will increase in the future.
Human cytomegalovirus (HCMV) is a beta-herpesvirus that is common in the human population. Although HCMV is not currently causally implicated in human cancer, a number of recent evidence suggests that HCMV may be specifically associated with some human malignancies. HCMV nucleic acids and proteins have been detected in 90-100% of glioblastomas and medulloblastomas, prostate, breast and colon cancers and in mucoepidermoid carcinomas of salivary glands [4-12]. Consistently, HCMV proteins are not detected in healthy tissues surrounding HCMV positive tumors. HCMV protein expression is restricted to the tumour; mainly in tumour cells, but virus proteins are sometimes found in endothelial cells and inflammatory cells within the tumour. However, infectious virus is not recovered from primary tumours. There is also a discrepancy between the number of protein positive cells and DNA positive cells within the tumour.
We have consistently observed that HCMV proteins are widespread and easily detected in a majority of tumour samples, whereas viral DNA is detected only in few cells within the tumour ( and unpublished observations). Recently, Ranganathan et. al. sequenced viral DNA from 20 different HCMV gene regions in samples obtained from glioblastoma patients and also found that only a minority of the cells in the tumour harbour the virus genome . The authors suggested that HCMV may enhance the growth or survival of a tumour through mechanisms that are distinctly different compared to classic tumour viruses that express transforming viral oncoproteins in the tumour cells. Thus, it is not likely that HCMV is an opportunistic virus capable of reactivating in the tumour and then only infects cells within in the tumour. Instead, HCMV proteins, rather than a productive infection may aid the development of HCMV positive tumours through yet undiscovered mechanisms.
HCMV; a promoter of cellular transformation or an oncogenic virus?
As of today, HCMV is not considered to have direct oncogenic properties; its potential role in cancer seems to be oncomodulatory, which imply that expression of HCMV gene products in cancer cells may promote tumour growth by enabling different hallmarks of cancer [2, 14, 15]. However, numerous recent data also indicate that several HCMV encoded proteins have biological properties that are directly related to cellular transformation and tumour development.
The US28 chemokine receptor encoded by HCMV has several characteristics resembling a viral oncoprotein [16-19]. Expression of US28 in NIH3T3 cells render these cells tumourigenic when injected into nude mice and transgenic mice with targeted expression of US28 to intestinal epithelial cells results in the development of intestinal neoplasia, which can be enhanced by inflammation . US28 targeted expression in intestinal cells inhibits glycogen synthase-3β (GSK-3β) function resulting in increased β-catenin activity and induced expression of Wnt target genes, including cyclin D, survivin and c-myc, that are involved in the control of cell proliferation  . These findings provide a direct molecular link between the expression of US28 and oncogenesis. In addition, US28 has also been shown to activate the transcription factor nuclear factor κB (NF-κb) that is a critical regulator of immunity, stress responses, apoptosis and differentiation [19, 20].
In glioblastoma cells, we found that the HCMV IE72 protein directly interacts with the hTERT promoter at SP1 binding sites to induce telomerase activity and telomere lengthening . We also found that HCMV-IE72 and hTERT were co-expressed in primary glioblastoma samples . Enhanced telomerase activity is necessary for tumour cells to divide indefinitely and is commonly induced by oncogenic viruses . Recently, Melnick et al. suggested that HCMV fulfils the criteria of Koch´s Postulates as revised for viruses and cancer, and that HCMV therefore should be designated as an “oncovirus” . They demonstrated cell specific localization of HCMV in 97% of mucoepidermoid carcinomas of salivary glands. HCMV IE and pp65 were expressed in tumour cells, but not in non-tumour cells and positively correlated with severity. HCMV protein expression correlated with activation of known oncogenic pathways such as epidermal growth factor receptor (EGFR), cyclooxygenase-2 (COX-2), Erk and amphiregulin. They also used a mouse salivary gland organ culture model and showed that murine CMV infection induces dysplasia through an upregulation of Erk phosphorylation. Phosphorylation of the ErbB receptor family members and downstream signalling may therefore be relevant targets for drug discovery also of HCMV positive tumours [9, 22].
The interaction of HCMV with its cellular receptor ligands, like integrins, during infection results in the activation of the PI3K/Akt signalling pathway and expression of IE72 protein in glioblastoma cells induces constitutive activation of Akt [23, 24]. HCMV has been shown to also activate the PI3K/Akt signalling cascade via binding of HCMV proteins to platelet-derived growth factor receptor alpha (PDGFR) and by selective phosphorylation of the cellular focal adhesion kinase (FAK) in glioblastoma and prostate cancer cells [25-27] . Furthermore, HCMV UL38 was shown to interact with tuberous sclerosis complex resulting in dysregulation of the mammalian target of rapamycin complex 1 .
HCMV encodes several proteins that interfere with the cellular apoptotic machinery. Direct anti-apoptotic activity of HCMV proteins has been located to transcripts encoded by the HCMV UL36-UL38 genes . CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis that suppresses apoptosis induced by diverse stimuli. The HCMV UL37 gene product inhibits Fas-mediated apoptosis downstream of caspase-8 activation and Bid cleavage in the mitochondria through inhibition of the pro-apoptotic Bcl-2 family members Bax and Bak [30, 31]. The HCMV UL36 gene product inhibits Fas-mediated apoptosis by binding to and inhibiting the function of caspase-8. . HCMV infection has also been shown to inhibit apoptosis and induce drug resistance by induction of the p53 tumour suppressor homologue gene product ΔN-p73α, resulting in abnormal neural cell survival . The HCMV IE86 protein binds to p53 and inhibits its transactivating function and suppresses p53-mediated apoptosis after DNA damage [26, 34-37]. The HCMV UL97 protein is a viral homologue of cellular cyclin-dependent kinases (CDK) that phosphorylates and inactivates the retinoblastoma (Rb) tumour suppressor protein resulting in cell cycle progression and inhibition of apoptosis in mammalian cells .
The functional inhibition of the p53 and Rb families of tumour suppressor proteins by HCMV encoded proteins implicates that HCMV is able to promote cell cycle progression, increase DNA synthesis and block apoptosis resulting in increased chromosomal instability [39-43]. In neuroblastoma cells HCMV induces expression of Bcl-2 resulting in inhibition of apoptosis and chemoresistance, a process that can be reversed by treatment of neuroblastoma cells with the antiviral drug ganciclovir . Interestingly, case reports of neuroblastoma patients have shown increased HCMV antibody titers and detection of HCMV in urine of small children with neuroblastoma. HCMV DNA also has been detected in neuroblastoma tissue sample [45-47]. Unpublished results from our laboratory demonstrate HCMV DNA, RNA and proteins in the majority of neuroblastoma tissue samples and in neuroblastoma cell lines. Treatment of neuroblastoma cells with the anti-viral drug ganciclovir in vitro or in vivo inhibits tumour growth (Wolmer-Solberg 2011, submitted).
Hence, HCMV encodes for a number of different proteins that have profound effects on cellular processes leading to increased proliferation, inhibition of apoptosis, stimulation of cellular migration, the release stimulatory factors, induction chemotherapeutic resistance and increased telomerase activity.
Human cytomegalovirus; an enhancer of inflammation and inducer of immune evasion in the tumour microenvironment
Symptoms of a primary HCMV infection are usually mild or asymptomatic in immunocompetent individuals but can cause severe disease in fetuses and immunocompromised patients such as transplant recipients and AIDS patients. The virus is spread through all bodily fluids and establishes a life-long latent/persistent infection. Reactivation from latency appears to be triggered by inflammation, which the virus can initiate by inducing cytokine and chemokine production and by enhancing the synthesis of pro-inflammatory eicosanoids. Indeed, the biological responses elicited by HCMV reactivation mimic those seen in leukocyte dysfunction, wound healing and chronic inflammation . HCMV reactivation has also been shown to stimulate the expression of VEGF that can induce angiogenesis [17, 18] and inhibit the expression of the potent anti-angiogenic protein thrombospondin-1 .
During evolution HCMV has coevolved with the human host and the virus has developed several immune evasion strategies to allow persistent infection and viral spread without harming its host. HCMV contains a 250 kb ds DNA genome that has 252 open reading frames and encodes approximately 200 proteins, of which only about 50 are essential for viral replication . Hence, the majority of HCMV encoded proteins have other functions in the viral lifecycle and many of these proteins are involved in immune evasion. For instance, the US11, US2 and US3 gene products prevent host cell MHC class I antigen expression that is required for CD8+ cytotoxic tumour killing. HCMV also induces a specific block in presentation of peptides of the HCMV encoded IE1 protein; one of the earliest immunodominant HCMV epitopes [50-52]. US3 and US8 inhibit presentation of MHC class II molecules on the cell surface and thereby inhibit CD4 + T cell responses [53, 54]. The HCMV pp65 protein encoded by the UL83 gene redirect HLA class II molecules to lysosomes where the alpha chain of the HLADR molecule is degraded . HCMV inhibits NK mediated lysis by several different strategies; the virus encodes for an MHC class I homologue that prevents NK cells to become activated through the missing self-hypothesis. The viral protein UL16 retains the NKG2D ligands ULBP1, 2 and MIC-B in the ER that are essential to activate an NK cell response (reviewed in ). UL16 also protects the cells from lysis mediated by cytotoxic peptides . Thus, cancer cells expressing UL16 would be protected against the action of both NK cells and T cells. Interestingly, the HCMV encoded UL83 protein pp65 and IE1/IE2 are frequently detected in both gliomas and medulloblastomas [11, 12].
We recently showed that HCMV nucleic acids and proteins are present in the majority of medulloblastoma primary tumours and cell lines. We also found that US28 (the HCMV encoded chemokine receptor homologue with potential oncogenic functions) was expressed in medulloblastoma and induced expression of COX-2 in these tumours . Microarray analysis of US28 transfected cells and HCMV infected cells showed that the expression of COX-2 is highly up-regulated in these cells as compared to mock-transfected or HCMV negative cells [17, 39]. Moreover, transgenic mice with targeted expression of US28 to intestinal epithelial cells exhibit a hyperplastic intestinal epithelium resulting in tumour development, indicating that US28 is involved in tumour initiation and progression .
COX-2 is over- expressed in a number of different adult cancers of epithelial origin as well as in gliomas where high expression often is correlated with poor prognosis (reviewed in [58-60]). In paediatric solid tumours high expression of COX-2 has been found in neuroblastoma [61, 62] , medulloblastoma [63, 64] and sarcomas . COX-2 is one of the major enzymes responsible for the conversion of arachidonic acid to the pro-inflammatory eicosanoid, prostaglandin E2 (PGE2). Increased levels of prostaglandin E2 (PGE2) are perceived in malignancies of different origin, including brain tumors [66-68]. PGE2 exerts its physiological effects by interacting with a subfamily of four distinct G-protein– coupled receptors designated EP1, EP2, EP3, and EP4. PGE2 promotes tumour growth in an autocrine and/or paracrine manner by stimulating EP receptor signalling with subsequent enhancement of cellular proliferation, promotion of angiogenesis, inhibition of apoptosis and stimulation of invasion . In addition, PGE2 is an important mediator for the interaction between tumour cells and cells in the tumor microenvironment where PGE2 contributes to the generation of a tumor promoting inflammatory microenvironment that suppress the activities from cells in the immune system .
Different nonsteroidal anti-inflammatory drugs (NSAIDs) which inhibit the enzymatic function of cyclooxygenases and the production of prostaglandins and other inflammatory mediators has been shown to be promising agents for the prevention and treatment of various cancers . Elevated levels of PGE2 are required for efficient replication of HCMV by facilitating the production of the HCMV immediate-early 2 protein . Daily aspirin reduce both the risk of development of cancer and cancer deaths ; the benefit increased with duration of treatment . Interestingly, NSAIDs abrogate virus-mediated production of PGE2 and reduce the virus burden in HCMV infected cells [70, 73]; thus acting as an anti-viral agent against HCMV. Moreover, the COX-2 specific NSAID celecoxib reduces the levels of PGE2 and the expression of HCMV proteins in medulloblastoma, as well as tumour growth in vitro and in vivo .
US28 that induces the expression of COX-2 in HCMV infected cells can bind different chemokines, including CCL2, CCL5, and CX3CL1 , and suppress the host immune responses . Moreover, US28 activates NF- κB resulting in activation of the IL-6–JAK1–STAT3 signalling axis and increased interleukin-6 (IL-6), VEGF and endothelial nitric oxide synthase (e-NOS) production [14, 19]. Analysis of clinical glioblastoma samples in situ showed co-localization of US28 with phosphorylated STAT3, COX-2, VEGF and e-NOS, suggesting that US28 in addition to promoting an inflammatory microenvironment also contribute to tumour invasiveness and angiogenesis [14, 19]. Taken together US28 could provide a target for therapy in HCMV-positive tumours.
HCMV establishes latency in myeloid lineage cells, and reactivation is dependent on inflammation and differentiation of monocytes into macrophages of dendritic cells. HCMV can also persistently infect monocyte/macrophage lineage cells and induce a strong inflammatory response in these cells . In human breast and colon cancer HCMV protein expression has been detected in infiltrating inflammatory cells in the tumour microenvironment and in gliomas, macrophages and microglia cells as well as tumor cells exhibit positive HCMV protein staining [77, 78]. HCMV infection of moncyte/macrophages is associated with an induction of IL-1, IL-6, IL-10, TNF-α and TGF-β that are potent cytokines with both immune stimulating and immunosuppressive effects on the host anti-tumour response [1, 79]. In particular, CMVIL-10 and TGF-β would provide an immunosuppressive microenvironment in HCMV positive tumours [80, 81]. These evidences raise the prospect that a persistent HCMV infection could induce the same kind of “smoldering” inflammation at the same time as it creates an immunosuppressive environment, which is frequently observed in the tumour microenvironment [1, 78].
HCMV as a guardian of cancer stem cells
HCMV is a neurotropic virus that can persistently infect neural precursor cells. As a consequence HCMV is the major infectious cause of birth defects in infants, including sensori-neural hearing loss or neuronal migration disturbances during brain development, and in the most severe cases, microcephaly or anencephaly. We have demonstrated that HCMV can block the ability of neural progenitor cells to differentiate into neurons or astrocytes [82, 83]. HCMV DNA and gene products have repeatedly been detected by several laboratories in preneoplastic and neoplastic tumour cells in human glioblastoma tissue samples and the fractions of tumour cells infected with HCMV correlate significantly with tumour staging and patient survival [5, 84]. We recently reported that the majority of primary human medulloblastoma and cell lines propagated for years in laboratories contain HCMV DNA, RNA and express HCMV IE and late proteins . Our unpublished data also demonstrate that HCMV is present in the majority of childhood primary neuroblastoma and cell lines, an observation which is consistent with other reports [15, 45].
Medulloblastoma and neuroblastoma are embryonal tumours of the central and peripheral nervous systems, respectively. Compared to adult tumours, paediatric tumours generally have a dramatically shortened latency period and harbour fewer genetic aberrations causing oncogene activation or loss of apoptotic regulators. The reason for these differences is that these malignancies probably arise from stem or progenitor cells which already possess proliferative capacity as a part of the normal developmental process . Medulloblastoma and neuroblastoma are linked to dysfunctional pathways that are operative during normal development . The clinical presentation and treatment response also suggests that a tumour initiating cell population exist in these tumours [86-91].
Although the cellular origin of gliomas still is contended, recent evidence suggests that multipotent neural stem or progenitors of the subventricular zone (SVZ) are cells with the potential to form gliomas . Subpopulations of CD133+ and/or CD15+ cells in both medulloblastomas and glioblastomas have been recognized as potential cancer stem cells [89, 93]. In neuroblastoma, on the other hand, no true marker for potential cancer stem cells have been found, although CD133 and CD44 are implied as potential markers . We have detected HCMV DNA, RNA and proteins in medulloblastoma, glioblastoma and neuroblastoma cell lines used world-wide for decades in laboratories, which may indicate that the virus in condemned in a stem cell that is maintained in culture and gives rise to tumours . We observed that the expression of HCMV proteins in both medulloblastoma and neuroblastoma cell lines varied considerably between different sampling occasions over a one year period, and that protein expression increased when the cells were engrafted in nude mice. We therefore hypothesize that HCMV DNA and proteins are maintained in a stem-cell like phenotype. Indeed we observed HCMV protein expression in the majority of CD133+ medulloblastoma cells whereas in neuroblastoma this number varied between 4-34% depending on cell line and sampling time (, and unpublished observations). Likewise, in glioblastoma tissue samples 40-60% of the CD133+ cell population expressed HCMV IE1  and our own unpublished observations). These data indicate that HCMV is present in tumour cells that express stem cell markers, and that the virus is maintained in cell lines over long periods of time. The fact that HCMV is able to inhibit the differentiation of neural progenitor cells raises the possibility that HCMV encoded proteins are involved in the maintenance of a cancer stem cell population within neural tumours.
Anti-HCMV therapy as a treatment option for certain cancers
The findings that several cancer forms are HCMV positive, including those with a neural origin that usually have a dismal prognosis, opens up the possibility to treat these cancers with anti-viral drugs against HCMV. In nude mice engrafted with human medulloblastoma cells, the antiviral drug valganciclovir, significantly inhibited tumour growth. Interestingly the treatment effect was extensively enhanced when valganciclovir was combined with the COX-2 specific inhibitor celecoxib , which is known to also inhibit HCMV infection. Importantly, the inhibition of tumour growth clearly corresponded with reduction in the expression of late HCMV proteins in these tumours. However, neither valganciclovir by itself or in combination with celecoxib was able to completely eliminate the HCMV presence. In sharp contrast, valganciclovir had no effect neither on the clonogenic capacity or tumour growth of two HCMV-negative cell lines derived from prostate and pancreas adenocarcinomas . This strongly suggests that the inhibitory effect of valganciclovir on medulloblastoma growth is HCMV specific and not mediated by potential non-specific drug effects inhibiting cellular proliferation.
Medulloblastoma, neuroblastoma and glioblastoma tumors express high levels of COX-2 and NSAIDs, inhibitors of COX-2 and PGE2 production, have profound effects on the growth of these tumours [64, 94-96]. These inhibitors also efficiently prevent HCMV replication and reduce the growth of US28-expressing tumour cells [17, 18, 70, 73]. Hence, the beneficial effects seen with aspirin and other NSAIDs in cancer prevention studies could partly be due to inhibition of HCMV replication in pre-malignant lesions. Compared to conventional chemotherapeutic drugs currently used for the treatment of these tumours, both antiviral drugs for HCMV and NSAIDs are well tolerated. Hence, these drugs should undergo clinical testing in combination with conventional therapies in patients carrying HCMV-infected tumours.
In a randomized double-blinded phase II study we are currently evaluating antiviral drugs against HCMV as an adjuvant therapy for glioblastoma. Results from this study are expected to be ready soon. Also a phase I/II immunotherapy clinical trial of autologous HCMV pp65 RNA loaded dendritic cells has been initiated in which 13 patients with newly diagnosed glioblastoma multiforme were enrolled. Initial results from this study are promising. Patients exhibited a median progression-free survival of 15.4 months and overall survival of 20.6 months, numbers which are highly significant compared to historical controls .
The promising preclinical and clinical results obtained using antiviral drugs against HCMV to treat tumours carrying HCMV should be extended to include larger controlled clinical trials. Also, developing drugs that specifically inhibit the functions of HCMV encoded US28 may be of future benefit in cancer treatment since the US28 protein may possess important functions in tumour initiation through the activation of intracellular signalling pathways, angiogenesis and effects on the tumour microenvironment.
Conclusions and perspectives
The presence and functions of HCMV in cancer is still debated and scepticism vestiges regarding the relationship between HCMV and cancer. This mainly originates from conflicting results regarding the detection of HCMV in tumour samples and since HCMV by itself not has been shown to transform normal cells into cancer cells . The last statement has recently been challenged since the HCMV encoded chemokine receptor homologue US28 renders NIH3T3 cells tumorigenic when injected into nude mice and transgenic mice with targeted expression to intestinal epithelial cells develop intestinal neoplasia [16, 18, 19]. Compared to the high degree of HCMV replication and protein expression seen in primary HCMV infections and in HCMV reactivation in immunocompromised individuals, the expression of viral proteins in cancer cells is very low. The term “microinfection” has been used to describe the low levels of HCMV infection found in cancer . Clearly, the infection is different in cells that replicate the virus and produce infectious virus compared to tumour cells in vivo; in spite of the fact that several HCMV proteins are expressed, infectious virus are not isolated from tumour cells of primary tumors, primary tumour cell cultures or established tumour cell lines. Therefore, as detection of HCMV in cancer cells using standard protocols developed for the detection of active HCMV infection associated with a high HCMV replication rate and high-level expression of HCMV proteins is usually insufficient in these cases , it is believed that low levels of HCMV exists in tumours [13, 15]. However, using flow cytometry examining fresh tumour cells or indirect immunofluorescence examining frozen tumor biopsy specimens, we demonstrated the feasibility of detecting HCMV proteins in primary tumour cells from medulloblastoma, glioblastoma and neuroblastoma patients (Wolmer-solberg, submitted, [12, 98]). Research laboratories that have shown a high prevalence of HCMV nucleic acids and proteins in tumour samples have used highly sensitive immunohistochemical and molecular methods in order to detect the presence of HCMV.
As of today HCMV has been detected in glioma, medulloblastoma, neuroblastoma, breast, prostate and colon cancer and mucoepidermoid tumors of the salivary gland. Although the exact molecular functions of HCMV in these tumours still need to be further investigated, the findings that antiviral HCMV treatment inhibit the growth of certain tumours (, Wolmer-Solberg, unpublished) is exciting and future studies will elucidate whether these antiviral therapies should be included as an adjuvant treatment for patients having HCMV-positive tumours.
We apologize to our colleagues whose work we were unable to cite due to space limitations and to the specific focus of this review. The authors have no conflicting financial interests (although CS-N holds an independent grant support from Roche supporting the clinical trial evaluating the efficacy and safety of valganciclovir treatment in glioblastoma patients). This work was supported by grants from Torsten and Ragnar Söderbergs Stiftelse, Ragnar Söderbergs Foundation, The Swedish Children’s Cancer Foundation, The Swedish Cancer Society, The Swedish Research Council, the Märta and Gunnar V Philipson Foundation, The Mary Bevé Foundation, The Hans and Märit Rausing Charitable Fund, The Dämman Foundation, Swedish Society for Medical Research (SLS), Goljes Memory Foundation, Magnus Bergvalls Foundation, Swedish Society for Medical Research (SSMF) and Tore Nilsons Foundation.
1. Mantovani A, Allavena P, Sica A, Balkwill F: Cancer-related inflammation. Nature 2008, 454(7203):436-444.
2. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 2011, 144(5):646-674.
3. Zur Hausen H: The search for infectious causes of human cancers: where and why. Virology 2009, 392(1):1-10.
4. Straat K, Liu C, Rahbar A, Zhu Q, Liu L, Wolmer-Solberg N, Lou F, Liu Z, Shen J, Jia J et al: Activation of telomerase by human cytomegalovirus. J Natl Cancer Inst 2009, 101(7):488-497.
5. Scheurer ME, Bondy ML, Aldape KD, Albrecht T, El-Zein R: Detection of human cytomegalovirus in different histological types of gliomas. Acta Neuropathol 2008, 116(1):79-86.
6. Samanta M, Harkins L, Klemm K, Britt WJ, Cobbs CS: High prevalence of human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma. J Urol 2003, 170(3):998-1002.
7. Prins RM, Cloughesy TF, Liau LM: Cytomegalovirus immunity after vaccination with autologous glioblastoma lysate. N Engl J Med 2008, 359(5):539-541.
8. Mitchell DA, Xie W, Schmittling R, Learn C, Friedman A, McLendon RE, Sampson JH: Sensitive detection of human cytomegalovirus in tumors and peripheral blood of patients diagnosed with glioblastoma. Neuro Oncol 2008, 10(1):10-18.
9. Melnick M, Sedghizadeh PP, Allen CM, Jaskoll T: Human cytomegalovirus and mucoepidermoid carcinoma of salivary glands: Cell-specific localization of active viral and oncogenic signaling proteins is confirmatory of a causal relationship. Experimental and molecular pathology 2011, 92(1):118-125.
10. Harkins L, Volk AL, Samanta M, Mikolaenko I, Britt WJ, Bland KI, Cobbs CS: Specific localisation of human cytomegalovirus nucleic acids and proteins in human colorectal cancer. Lancet 2002, 360(9345):1557-1563.
11. Cobbs CS, Harkins L, Samanta M, Gillespie GY, Bharara S, King PH, Nabors LB, Cobbs CG, Britt WJ: Human cytomegalovirus infection and expression in human malignant glioma. Cancer Res 2002, 62(12):3347-3350.
12. Baryawno N, Rahbar A, Wolmer-Solberg N, Taher C, Odeberg J, Darabi A, Khan Z, Sveinbjornsson B, Fuskevag OM, Segerstrom L et al: Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target. J Clin Invest 2011, 121(10):4043-4055.
13. Ranganathan P, Clark PA, Kuo JS, Salamat MS, Kalejta RF: Significant Association of Multiple Human Cytomegalovirus Genomic Loci with Glioblastoma Multiforme Samples. J Virol 2011.
14. Soroceanu L, Cobbs CS: Is HCMV a tumor promoter? Virus Res 2011, 157(2):193-203.
15. Michaelis M, Doerr HW, Cinatl J, Jr.: Oncomodulation by human cytomegalovirus: evidence becomes stronger. Med Microbiol Immunol 2009, 198(2):79-81.
16. Bongers G, Maussang D, Muniz LR, Noriega VM, Fraile-Ramos A, Barker N, Marchesi F, Thirunarayanan N, Vischer HF, Qin L et al: The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice. J Clin Invest 2010, 120(11):3969-3978.
17. Maussang D, Langemeijer E, Fitzsimons CP, Stigter-van Walsum M, Dijkman R, Borg MK, Slinger E, Schreiber A, Michel D, Tensen CP et al: The human cytomegalovirus-encoded chemokine receptor US28 promotes angiogenesis and tumor formation via cyclooxygenase-2. Cancer Res 2009, 69(7):2861-2869.
18. Maussang D, Verzijl D, van Walsum M, Leurs R, Holl J, Pleskoff O, Michel D, van Dongen GA, Smit MJ: Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis. Proc Natl Acad Sci U S A 2006, 103(35):13068-13073.
19. Slinger E, Maussang D, Schreiber A, Siderius M, Rahbar A, Fraile-Ramos A, Lira SA, Soderberg-Naucler C, Smit MJ: HCMV-encoded chemokine receptor US28 mediates proliferative signaling through the IL-6-STAT3 axis. Sci Signal 2010, 3(133):ra58.
20. Oeckinghaus A, Hayden MS, Ghosh S: Crosstalk in NF-kappaB signaling pathways. Nat Immunol 2011, 12(8):695-708.
21. Bellon M, Nicot C: Regulation of telomerase and telomeres: human tumor viruses take control. J Natl Cancer Inst 2008, 100(2):98-108.
22. Melnick M, Abichaker G, Htet K, Sedghizadeh P, Jaskoll T: Small molecule inhibitors of the host cell COX/AREG/EGFR/ERK pathway attenuate cytomegalovirus-induced pathogenesis. Experimental and molecular pathology 2011, 91(1):400-410.
23. Cinatl J, Jr., Vogel JU, Kotchetkov R, Wilhelm Doerr H: Oncomodulatory signals by regulatory proteins encoded by human cytomegalovirus: a novel role for viral infection in tumor progression. FEMS Microbiol Rev 2004, 28(1):59-77.
24. Luo MH, Fortunato EA: Long-term infection and shedding of human cytomegalovirus in T98G glioblastoma cells. J Virol 2007, 81(19):10424-10436.
25. Blaheta RA, Beecken WD, Engl T, Jonas D, Oppermann E, Hundemer M, Doerr HW, Scholz M, Cinatl J: Human cytomegalovirus infection of tumor cells downregulates NCAM (CD56): a novel mechanism for virus-induced tumor invasiveness. Neoplasia 2004, 6(4):323-331.
26. Cobbs CS, Soroceanu L, Denham S, Zhang W, Britt WJ, Pieper R, Kraus MH: Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness. J Neurooncol 2007, 85(3):271-280.
27. Soroceanu L, Akhavan A, Cobbs CS: Platelet-derived growth factor-alpha receptor activation is required for human cytomegalovirus infection. Nature 2008, 455(7211):391-395.
28. Moorman NJ, Cristea IM, Terhune SS, Rout MP, Chait BT, Shenk T: Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex. Cell Host Microbe 2008, 3(4):253-262.
29. McCormick AL, Roback L, Mocarski ES: HtrA2/Omi terminates cytomegalovirus infection and is controlled by the viral mitochondrial inhibitor of apoptosis (vMIA). PLoS Pathog 2008, 4(5):e1000063.
30. Goldmacher VS, Bartle LM, Skaletskaya A, Dionne CA, Kedersha NL, Vater CA, Han JW, Lutz RJ, Watanabe S, Cahir McFarland ED et al: A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc Natl Acad Sci U S A 1999, 96(22):12536-12541.
31. Norris PS, Jepsen K, Haas M: High-titer MSCV-based retrovirus generated in the pCL acute virus packaging system confers sustained gene expression in vivo. J Virol Methods 1998, 75(2):161-167.
32. Skaletskaya A, Bartle LM, Chittenden T, McCormick AL, Mocarski ES, Goldmacher VS: A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc Natl Acad Sci U S A 2001, 98(14):7829-7834.
33. Allart S, Martin H, Detraves C, Terrasson J, Caput D, Davrinche C: Human cytomegalovirus induces drug resistance and alteration of programmed cell death by accumulation of deltaN-p73alpha. J Biol Chem 2002, 277(32):29063-29068.
34. Tsai HL, Kou GH, Chen SC, Wu CW, Lin YS: Human cytomegalovirus immediate-early protein IE2 tethers a transcriptional repression domain to p53. J Biol Chem 1996, 271(7):3534-3540.
35. Tanaka K, Zou JP, Takeda K, Ferrans VJ, Sandford GR, Johnson TM, Finkel T, Epstein SE: Effects of human cytomegalovirus immediate-early proteins on p53-mediated apoptosis in coronary artery smooth muscle cells. Circulation 1999, 99(13):1656-1659.
36. Speir E, Modali R, Huang ES, Leon MB, Shawl F, Finkel T, Epstein SE: Potential role of human cytomegalovirus and p53 interaction in coronary restenosis [see comments]. Science 1994, 265(5170):391-394.
37. Lukac DM, Alwine JC: Effects of human cytomegalovirus major immediate-early proteins in controlling the cell cycle and inhibiting apoptosis: studies with ts13 cells. J Virol 1999, 73(4):2825-2831.
38. Hume AJ, Finkel JS, Kamil JP, Coen DM, Culbertson MR, Kalejta RF: Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function. Science 2008, 320(5877):797-799.
39. Zhu H, Shen Y, Shenk T: Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol 1995, 69(12):7960-7970.
40. Yu Y, Alwine JC: Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 3’-OH kinase pathway and the cellular kinase Akt. J Virol 2002, 76(8):3731-3738.
41. Poma EE, Kowalik TF, Zhu L, Sinclair JH, Huang ES: The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter. J Virol 1996, 70(11):7867-7877.
42. Fortunato EA, Dell’Aquila ML, Spector DH: Specific chromosome 1 breaks induced by human cytomegalovirus. Proc Natl Acad Sci U S A 2000, 97(2):853-858.
43. Castillo JP, Kowalik TF: Human cytomegalovirus immediate early proteins and cell growth control. Gene 2002, 290(1-2):19-34.
44. Cinatl J, Jr., Cinatl J, Vogel JU, Kotchetkov R, Driever PH, Kabickova H, Kornhuber B, Schwabe D, Doerr HW: Persistent human cytomegalovirus infection induces drug resistance and alteration of programmed cell death in human neuroblastoma cells. Cancer Res 1998, 58(2):367-372.
45. Nigro G, Schiavetti A, Booth JC, Clerico A, Dominici C, Krzysztofiak A, Castello M: Cytomegalovirus-associated stage 4S neuroblastoma relapsed stage 4. Med Pediatr Oncol 1995, 24(3):200-203.
46. Wertheim P, Voute PA: Neuroblastoma, Wilms’ tumor, and cytomegalovirus. J Natl Cancer Inst 1976, 57(3):701-703.
47. Michaelis M, Doerr HW, Cinatl J, Jr.: Oncomodulation by human cytomegalovirus: evidence becomes stronger. Med Microbiol Immunol 2009, 198(2):79-81.
48. Cinatl J, Jr., Kotchetkov R, Scholz M, Cinatl J, Vogel JU, Driever PH, Doerr HW: Human cytomegalovirus infection decreases expression of thrombospondin-1 independent of the tumor suppressor protein p53. Am J Pathol 1999, 155(1):285-292.
49. Murphy E, Yu D, Grimwood J, Schmutz J, Dickson M, Jarvis MA, Hahn G, Nelson JA, Myers RM, Shenk TE: Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc Natl Acad Sci U S A 2003, 100(25):14976-14981.
50. Greijer AE, Verschuuren EA, Dekkers CA, Adriaanse HM, van der Bij W, The TH, Middeldorp JM: Expression dynamics of human cytomegalovirus immune evasion genes US3, US6, and US11 in the blood of lung transplant recipients. J Infect Dis 2001, 184(3):247-255.
51. Besold K, Wills M, Plachter B: Immune evasion proteins gpUS2 and gpUS11 of human cytomegalovirus incompletely protect infected cells from CD8 T cell recognition. Virology 2009, 391(1):5-19.
52. Benz C, Reusch U, Muranyi W, Brune W, Atalay R, Hengel H: Efficient downregulation of major histocompatibility complex class I molecules in human epithelial cells infected with cytomegalovirus. Journal of General Virology 2001, 82(Pt 9):2061-2070.
53. Tomazin R, Boname J, Hegde NR, Lewinsohn DM, Altschuler Y, Jones TR, Cresswell P, Nelson JA, Riddell SR, Johnson DC: Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells. Nat Med 1999, 5(9):1039-1043.
54. Hegde NR, Tomazin RA, Wisner TW, Dunn C, Boname JM, Lewinsohn DM, Johnson DC: Inhibition of HLA-DR assembly, transport, and loading by human cytomegalovirus glycoprotein US3: a novel mechanism for evading major histocompatibility complex class II antigen presentation. J Virol 2002, 76(21):10929-10941.
55. Odeberg J, Plachter B, Branden L, Soderberg-Naucler C: Human cytomegalovirus protein pp65 mediates accumulation of HLA-DR in lysosomes and destruction of the HLA-DR alpha-chain. Blood 2003, 101(12):4870-4877.
56. Soderberg-Naucler C: Human cytomegalovirus persists in its host and attacks and avoids elimination by the immune system. Crit Rev Immunol 2006, 26(3):231-264.
57. Odeberg J, Browne H, Metkar S, Froelich CJ, Branden L, Cosman D, Soderberg-Naucler C: The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins. J Virol 2003, 77(8):4539-4545.
58. Wang D, Dubois RN: Eicosanoids and cancer. Nat Rev Cancer 2010, 10(3):181-193.
59. Shono T, Tofilon PJ, Bruner JM, Owolabi O, Lang FF: Cyclooxygenase-2 expression in human gliomas: prognostic significance and molecular correlations. Cancer Res 2001, 61(11):4375-4381.
60. Joki T, Heese O, Nikas DC, Bello L, Zhang J, Kraeft SK, Seyfried NT, Abe T, Chen LB, Carroll RS et al: Expression of cyclooxygenase 2 (COX-2) in human glioma and in vitro inhibition by a specific COX-2 inhibitor, NS-398. Cancer Res 2000, 60(17):4926-4931.
61. Johnsen JI, Lindskog M, Ponthan F, Pettersen I, Elfman L, Orrego A, Sveinbjornsson B, Kogner P: NSAIDs in neuroblastoma therapy. Cancer Lett 2005, 228(1-2):195-201.
62. Johnsen JI, Lindskog M, Ponthan F, Pettersen I, Elfman L, Orrego A, Sveinbjornsson B, Kogner P: Cyclooxygenase-2 is expressed in neuroblastoma, and nonsteroidal anti-inflammatory drugs induce apoptosis and inhibit tumor growth in vivo. Cancer Res 2004, 64(20):7210-7215.
63. Patti R, Gumired K, Reddanna P, Sutton LN, Phillips PC, Reddy CD: Overexpression of cyclooxygenase-2 (COX-2) in human primitive neuroectodermal tumors: effect of celecoxib and rofecoxib. Cancer Lett 2002, 180(1):13-21.
64. Baryawno N, Sveinbjornsson B, Eksborg S, Orrego A, Segerstrom L, Oqvist CO, Holm S, Gustavsson B, Kagedal B, Kogner P et al: Tumor-growth-promoting cyclooxygenase-2 prostaglandin E2 pathway provides medulloblastoma therapeutic targets. Neuro Oncol 2008, 10(5):661-674.
65. Dickens DS, Kozielski R, Khan J, Forus A, Cripe TP: Cyclooxygenase-2 expression in pediatric sarcomas. Pediatr Dev Pathol 2002, 5(4):356-364.
66. Kokoglu E, Tuter Y, Yazici Z, Sandikci KS, Sonmez H, Ulakoglu EZ, Ozyurt E: Profiles of the fatty acids in the plasma membrane of human brain tumors. Cancer Biochem Biophys 1998, 16(4):301-312.
67. Loh JK, Hwang SL, Lieu AS, Huang TY, Howng SL: The alteration of prostaglandin E2 levels in patients with brain tumors before and after tumor removal. J Neurooncol 2002, 57(2):147-150.
68. Nathoo N, Barnett GH, Golubic M: The eicosanoid cascade: possible role in gliomas and meningiomas. J Clin Pathol 2004, 57(1):6-13.
69. Burn J, Gerdes AM, Macrae F, Mecklin JP, Moeslein G, Olschwang S, Eccles D, Evans DG, Maher ER, Bertario L et al: Long-term effect of aspirin on cancer risk in carriers of hereditary colorectal cancer: an analysis from the CAPP2 randomised controlled trial. Lancet 2011, Oct 27. [Epub ahead of print].
70. Zhu H, Cong JP, Yu D, Bresnahan WA, Shenk TE: Inhibition of cyclooxygenase 2 blocks human cytomegalovirus replication. Proc Natl Acad Sci U S A 2002, 99(6):3932-3937.
71. Din FV, Theodoratou E, Farrington SM, Tenesa A, Barnetson RA, Cetnarskyj R, Stark L, Porteous ME, Campbell H, Dunlop MG: Effect of aspirin and NSAIDs on risk and survival from colorectal cancer. Gut 2010, 59(12):1670-1679.
72. Rothwell PM, Fowkes FG, Belch JF, Ogawa H, Warlow CP, Meade TW: Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials. Lancet 2011, 377(9759):31-41.
73. Speir E, Yu ZX, Ferrans VJ, Huang ES, Epstein SE: Aspirin attenuates cytomegalovirus infectivity and gene expression mediated by cyclooxygenase-2 in coronary artery smooth muscle cells. Circ Res 1998, 83(2):210-216.
74. Bodaghi B, Jones TR, Zipeto D, Vita C, Sun L, Laurent L, Arenzana-Seisdedos F, Virelizier JL, Michelson S: Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. Journal of Experimental Medicine 1998, 188(5):855-866.
75. Randolph-Habecker JR, Rahill B, Torok-Storb B, Vieira J, Kolattukudy PE, Rovin BH, Sedmak DD: The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production. Cytokine 2002, 19(1):37-46.
76. Sinclair J, Sissons P: Latent and Persistent Infections Of Monocytes and Macrophages. Intervirology 1996, 39(5-6):293-301.
77. Harkins LE, Matlaf LA, Soroceanu L, Klemm K, Britt WJ, Wang W, Bland KI, Cobbs CS: Detection of human cytomegalovirus in normal and neoplastic breast epithelium. Herpesviridae 2010, 1(1):8.
78. Soroceanu L, Matlaf L, Bezrookove V, Harkins L, Martinez R, Greene M, Soteropoulos P, Cobbs CS: Human Cytomegalovirus US28 Found in Glioblastoma Promotes an Invasive and Angiogenic Phenotype. Cancer Res 2011, 71(21):6643-6653.
79. Chan G, Bivins-Smith ER, Smith MS, Smith PM, Yurochko AD: Transcriptome analysis reveals human cytomegalovirus reprograms monocyte differentiation toward an M1 macrophage. J Immunol 2008, 181(1):698-711.
80. Kotenko SV, Saccani S, Izotova LS, Mirochnitchenko OV, Pestka S: Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10). Proc Natl Acad Sci U S A 2000, 97(4):1695-1700.
81. Michelson S, Alcami J, Kim SJ, Danielpour D, Bachelerie F, Picard L, Bessia C, Paya C, Virelizier JL: Human cytomegalovirus infection induces transcription and secretion of transforming growth factor beta 1. J Virol 1994, 68(9):5730-5737.
82. Odeberg J, Wolmer N, Falci S, Westgren M, Seiger A, Soderberg-Naucler C: Human cytomegalovirus inhibits neuronal differentiation and induces apoptosis in human neural precursor cells. J Virol 2006, 80(18):8929-8939.
83. Odeberg J, Wolmer N, Falci S, Westgren M, Sundtrom E, Seiger A, Soderberg-Naucler C: Late human cytomegalovirus (HCMV) proteins inhibit differentiation of human neural precursor cells into astrocytes. J Neurosci Res 2007, 85(3):583-593.
84. Soderberg-Naucler C: HCMV microinfections in inflammatory diseases and cancer. J Clin Virol 2008, 41(3):218-223.
85. Scotting PJ, Walker DA, Perilongo G: Childhood solid tumours: a developmental disorder. Nat Rev Cancer 2005, 5(6):481-488.
86. Baryawno N, Sveinbjornsson B, Kogner P, Johnsen JI: Medulloblastoma: a disease with disorganized developmental signaling cascades. Cell Cycle 2010, 9(13):2548-2554.
87. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC: PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev 2008, 22(4):436-448.
88. Hansford LM, McKee AE, Zhang L, George RE, Gerstle JT, Thorner PS, Smith KM, Look AT, Yeger H, Miller FD et al: Neuroblastoma cells isolated from bone marrow metastases contain a naturally enriched tumor-initiating cell. Cancer Res 2007, 67(23):11234-11243.
89. Read TA, Fogarty MP, Markant SL, McLendon RE, Wei Z, Ellison DW, Febbo PG, Wechsler-Reya RJ: Identification of CD15 as a marker for tumor-propagating cells in a mouse model of medulloblastoma. Cancer Cell 2009, 15(2):135-147.
90. Schuller U, Heine VM, Mao J, Kho AT, Dillon AK, Han YG, Huillard E, Sun T, Ligon AH, Qian Y et al: Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma. Cancer Cell 2008, 14(2):123-134.
91. Yang ZJ, Ellis T, Markant SL, Read TA, Kessler JD, Bourboulas M, Schuller U, Machold R, Fishell G, Rowitch DH et al: Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells. Cancer Cell 2008, 14(2):135-145.
92. Gilbertson RJ, Rich JN: Making a tumour’s bed: glioblastoma stem cells and the vascular niche. Nat Rev Cancer 2007, 7(10):733-736.
93. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB: Identification of human brain tumour initiating cells. Nature 2004, 432(7015):396-401.
94. Ma HI, Chiou SH, Hueng DY, Tai LK, Huang PI, Kao CL, Chen YW, Sytwu HK: Celecoxib and radioresistant glioblastoma-derived CD133+ cells: improvement in radiotherapeutic effects. Laboratory investigation. J Neurosurg 2011, 114(3):651-662.
95. Sareddy GR, Geeviman K, Ramulu C, Babu PP: The nonsteroidal anti-inflammatory drug celecoxib suppresses the growth and induces apoptosis of human glioblastoma cells via the NF-kappaB pathway. J Neurooncol 2012, 106(1):99-109.
96. Sharma V, Dixit D, Ghosh S, Sen E: COX-2 regulates the proliferation of glioma stem like cells. Neurochem Int 2011, 59(5):567-571.
97. Cobbs CS: Evolving evidence implicates cytomegalovirus as a promoter of malignant glioma pathogenesis. Herpesviridae 2011, 2(1):10.
98. Dziurzynski K, Wei J, Qiao W, Hatiboglu MA, Kong LY, Wu A, Wang Y, Cahill D, Levine N, Prabhu S et al: Glioma-associated cytomegalovirus mediates subversion of the monocyte lineage to a tumor propagating phenotype. Clin Cancer Res 2011, 17(14):4642-4649.
All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.