Research ArticleDetection of Human Cytomegalovirus in DifferentHistopathological Types of Glioma in Iraqi Patients

Haidar A Shamran1 Haider S Kadhim2 Aws R Hussain3

Abdulameer Kareem4 Dennis D Taub5 Robert L Price6 Mitzi Nagarkatti7

Prakash S Nagarkatti7 and Udai P Singh7

1Medical Research Unit School of Medicine University of Al-Nahrain Baghdad 10066 Iraq2Microbiology Department School of Medicine University of Al-Nahrain Baghdad 10066 Iraq3Department of Pathology School of Medicine University of Al-Qadisiya Dewaniya 58011 Iraq4Department of Medical Science School of Nursing University of Al-Qadisiya Dewaniya 58011 Iraq5Center for Translational Studies Medical Services VAMedical Center Department of Veteran Affairs Washington DC 20422 USA6Department of Cell Biology and Anatomy School of Medicine University of South Carolina SC 29208 USA7Department of Pathology Microbiology and Immunology School of Medicine University of South Carolina SC 29208 USA

Correspondence should be addressed to Udai P Singh udaisinghuscmedscedu

Received 5 August 2014 Revised 14 November 2014 Accepted 23 November 2014

Academic Editor William Rawlinson

Copyright copy 2015 Haidar A Shamran et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Human Cytomegalovirus (HCMV) is an endemic herpes virus that reemerges in cancer patients enhancing oncogenic potentialHCMV infection is associated with certain types of cancer morbidity such as glioblastomas HCMV like all other herpes viruseshas the ability to remain latent within the body of the host and can contribute in chronic inflammation To determine the role ofHCMV in glioma pathogenesis paraffin-embedded blocks from glioma patients (119899 = 50) and from benign meningioma patients(119899 = 30) were obtained and evaluated by immunohistochemistry and polymerase chain reaction for the evidence of HCMV antigenexpression and the presence of viral DNA We detected HCMV antigen and DNA for IEI-72 pp65 and late antigen in 33362836 and 2636 in glioblastomamultiforme patients whereas 1214 1014 and 914 in anaplastic astrocytoma patients respectivelyFurthermore 84 of glioma patients were positive for immunoglobulin G (IgG) compared to 725 among control samples(119875 = 004) These data indicate the presence of the HCMV virus in a high percentage of glioma samples demonstrating distincthistopathological grades and support previous reports showing the presence of HCMV infection in glioma tissue These studiesdemonstrate that detection of low-levels of latent viral infections may play an active role in glioma development and pathogenesis

1 Introduction

Tumors of the central nervous system (CNS) represent about2 of all cancers [1] and gliomas are the most commontumors of this system In adults gliomas account for almost80 of primary malignant brain tumors [2] Glioblastomamultiforme (GBM) is themost common type of glioma and isassociatedwith amedian survival of only 12ndash15months [3] InIraq CNS tumors are the fifth most common tumor in adultsand the secondmost common in children [4] However thesetumors are the most difficult to cure For example the totalsurgical removal of the affected part of the organ which is

used with other cancers cannot be applied to cure braintumors because each region of the brain has a vital function[5]

Human Cytomegalovirus (HCMV) a widespread beta-herpes virus persistently infects over 70 of the population[6] It has been shown that viral infections are responsiblefor approximately 15 of all cancers [7] HMCV is associatedwith brain breast and colorectal cancer [8ndash10]

Geder et al [11] demonstrated that genital isolates ofHCMV could result in oncogenic transformation of humanembryo lung fibroblasts A group led by Cinatl Jr et al[12] demonstrated three oncomodulatory aspects of HCMV

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 642652 7 pageshttpdxdoiorg1011552015642652

2 BioMed Research International

which were the expression of oncogenes transcriptional acti-vation of growth factor and interleukin synthesis Howeverthe role of this virus in brain tumor development has been amatter of debate for many years A role for HCMV in braintumors can be deduced from the association of anti-HCMVIgG and IgM Abs with the incidence of gliomas This wasconfirmed by Amirian et al [13] who found that anti-HCMVAbs level was associated with glioma risk especially amongIgM-positive individuals In contrast Sjostrom et al [14] didnot find significant association between glioma or GBM andanti-HCMV IgG levels

The presence of viral DNA or protein inside tumor versusnontumor cells can give more accurate and direct causalassociation than estimation of anti-HCMVantibodies Cobbset al [15] shows that HCMV proteins and nucleic acidsproducts are expressed in virtually all GBMs but not innormal brain or other benign tumors suggest a possibilitythat HCMV may play an active role in glioma pathogenesisFurther 92 of primary GBM tumors express viral imme-diate early protein while 73 of them express late proteins[16] In addition HCMV in tumors and the peripheral bloodof patients diagnosed with GBM shows a high percentage(gt90) of this tumor associatedwithHCMVnucleic acid andproteins and 80 of patients had detectable HCMVDNA intheir peripheral blood [17] Recent studies have also reportedthe presence of HCMV proteins in 99 of brain tumor tissuesections isolated from GBM patients [18]

HCMV in glioma and other tumors is a major concernin Iraq and until now no published information has beenavailable on the expression and association of this virus withthese tumors Studying HCMV expression and associationwith brain tumors might be beneficial in development ofpossible diagnostic and interventional strategies in thesepatients To this end in the present study we investigated theexpression represented by early (IEI-72) mid (pp65) and lateHCMV antigens in different grades of GBM and anaplasticastrocytoma in Iraqi patients

2 Materials and Methods

Patients from the Neurosurgery Hospital Neuroscience Hos-pital AL-Kadimiya Teaching Hospital AL-Fallujah Hospitaland Private Nursing Home Hospital undergoing surgicalresection of brain tumors from January 2012 to March 2013were included in this study Ethical approval to conductthe research was obtained from each hospital InstitutionalReview Board (IRB) Fifty paraffin-embedded brain tissuesfrom the patients were used (mean age 3185 plusmn 195 yearsrange 1ndash72 years) Personal data were collected through directinterview with the patient and by seeking hisher hospitalrecord and medical records Patient claims were followed asan alternative source of information when hisher previousmedical reports were not available Collected data includedage sex smoking and first relative family history of braintumors All brain tumors were graded by a histopathologistAmong the 50 specimens 36wereGBMs and 14were anaplas-tic astrocytomaThirty paraffin-embedded blocks from aged-matched benign meningioma patients were used as controls

21 Immunohistochemistry (IHC) All paraffin-embeddedsections (6 120583m thick for patients and control) were deparaf-finized by heating slides in a vertical position in an oven for 1hour followed by washing in xylene (20min) and an ethanolseries (100 95 75 and 50 for 5min each) Sectionswere blocked for endogenous peroxidase 3H

2O2for 20min

(BioGenex CA USA) Antigen retrieval was performedusing Citra Plus antigen retrieval solution (BioGenex CAUSA) for 2min at 97∘C in a microwave Protein blocks(EMDMillipore IL USA) were applied to the sections priorto application of primary antibodies (against the HCMVantigen IE1 pp65 and late antigen (1 100 for all antigens)EMD Millipore IL USA) Human lung with known HCMVinfection was used as a positive control (Chemicon Temec-ula CA USA) and negative control tumor sections that werenot treated with an antibody stained were also included theexperiments All slides were incubated overnight at 4∘C Theslides were then stainedwith secondary antibody biotinylatedgoat anti-mouse (1 175 IHC select HRP kit from EMDMilli-pore IL USA) peroxidase-labeled streptavidin (EMDMilli-pore IL USA) and 331015840-diaminobenzidine (DAB) (InnovexBiosciences USA) Hematoxylin was used as a counterstainHuman lung sections with known HCMV infection wereused as positive controls for staining (Chemicon TemeculaCA USA) Negative controls consisted of tumor sections thatwere not treated with the primary antibody andor secondaryantibody All samples were stained more than once and theresult was highly reproducible

22 Immunohistochemical Scoring Immunostained sectionswere evaluated by two independent histopathologists whowere blinded to the patient information and disease severityCases with disagreement were discussed using amultiheadedmicroscope until agreement was achieved For HCMV pro-teins (IE1-72 pp65 and late antigen) the brown staining ofthe nucleus with or without a diffuse pattern was consideredpositive The intensity of protein expression was scored asreported by Rahbar et al [18] by examining 1000 cells in 10high power fields According to the estimated percentage ofpositive cells four grades were identified grade 1 lt25 grade2 ge25ndash50 grade 3 ge51ndash75 grade 4 ge76

23 DNA Extraction A ready kit (DNeasy Blood and tissuekitQiagen Valencia) was used for viral DNA extraction fromtumor specimens according the manufacturerrsquos instructions

24 Polymerase Chain Reaction In order to reduce con-tamination and prevent false positive results preparationfor PCR was carried out in a laboratory not previouslyused for handling HCMV A nested PCR method was usedto detect viral DNA External primer sequences were theforward primer 5-CCGAAATACGCGTTTTGAGATand thereverse primer 5-CCAAGCCAAAAACAGTATAGC whichamplified a 1300 bp of the promoter region of the immediateearly (IE) gene A readymastermix (Bioneer Korea) was usedfor PCR reaction preparationThe PCR conditions were 95∘Cfor 5min followed by 45 cycles of 94∘C for 30 s 56∘C for30 s and 72∘C for 1min with a final extension at 72∘C for

BioMed Research International 3

Table 1 The percentage and grading of HCMV protein in GBM

HCMV proteinsNumber = 36

PositivityNumber()

Grading of positive sectionsGrade 1Number()

Grade 2Number()

Grade 3Number()

Grade 4Number()

IE1-72 33 (9167) 7 (2121) 6 (1818) 7 (2121) 13 (3939)pp65 28 (7778) 7 (25) 9 (3214) 7 (25) 5 (1758)Late antigen 26 (7222) 7 (2692) 10 (3846) 8 (3077) 3 (1154)Percentage changes and grading in early mid and late antigen HCMV protein in glioblastoma multiforme patients The grade number and percentage ()were calculated in a blinded fashion The number and of positive patients are indicated for each antigen

Table 2 The percentage and grading of HCMV protein in anaplastic astrocytoma

HCMV proteinsNumber = 14

PositivityNumber()

Grading of positive sectionsGrade 1Number()

Grade 2Number()

Grade 3Number()

Grade 4Number()

IE1-72 12 (8571) 2 (1667) 2 (1667) 2 (1667) 6 (50)pp65 10 (7142) 2 (20) 5 (50) 2 (20) 1 (10)Late antigen 9 (6428) 3 (3333) 3 (3333) 2 (2222) 1 (1111)Percentage changes and grading in early mid and late antigen HCMV protein in anaplastic astrocytoma patients The grade number and percentage () werecalculated in a blinded fashion The number and of positive patients are indicated for each antigen

10min Internal primer sequences were the forward primer5-GGCGGAGTT (GA) TTACGACATTT and the reverseprimer 5-ATGCGGTTTTGGCAGTACATwhich amplified a144 bp region within the 1300 bp amplicon generated by theexternal primer setThe PCR conditions were 95∘C for 5minfollowed by 40 cycles of 94∘C for 20 s 56∘C for 20 s and 72∘Cfor 20 s with a final extension at 72∘C for 5min

25 HCMV ELISA IgG Anti-CMV IgG ELISA test kits(Biokit Barcelona Spain) were used for qualitative detectionof serum anti-CMV IgG antibodies according to the manu-facturerrsquos manual

26 Statistical Analysis The Statistical Package for the SocialSciences (SPSS version 14) was used for statistical analysisThe logistic regression test was used to test the associationof HCMV immune positivity with incidence of gliomas Thechi-square test was used to compare immune positivity toCytomegalovirus in different age classes and in evaluation ofimmunostainingThe correlation test was used to calculate 119903-values for HCMV protein expression A 119875 value lt 005 wasconsidered statistically significant

3 Results

We performed IHC on paraffin sections from 50 gliomasthat included 36 GBM and 14 anaplastic astrocytomas usingmAbs specific for HCMV proteins (IE1-72 pp65 and lateantigen) Out of 36 GBM samples 33 (9167) 28 (7778)and 26 (7222) were positive for IE1-72 pp65 and lateantigen respectively (Table 1) We noticed no significantdifference in the expression of these proteins betweenGBMs and anaplastic astrocytomas Control tissue sections

(meningioma biopsy specimens) and the surrounding brainarea of malignant gliomas were devoid of immunoreactivity(Figure 1)

IE1-72 protein demonstrated stronger expression thanother proteins as 13 out of 33 positive GBM samples (3939)were grade 4 compared to 5 (1758) and 3 (1154) for pp65and late antigen respectively (1205942 = 7052 119875 = 0029)(Table 1) Furthermore IE1-72 had the lowest percentage ofsamples that were expressed as grade 1 (2121) compared to25 for pp65 and 2992 for late antigenThe correlation testrevealed highly significant positive correlation between IE1-72 late antigen (119903 = 0769 119875 lt 0001) and pp65 (119903 = 0714119875 lt 0001)

Among 14 anaplastic astrocytoma samples 12 (8571) 10(7142) and 9 (6482)were positive for IE1-72 pp65 and lateantigen respectively (Table 2) Out of 14 positive anaplasticastrocytoma samples 6 (50) 1 (10) and 1 (1111) weregrade 4 for IE1-72 pp65 and late antigen respectively witha significant difference (1205942 = 5988 119875 = 005) FurthermoreIE1-72 protein had the lowest percentage of samples that wereexpressed as grade 1 (1667) compared to pp65 (20) andlate antigen (3333) A strong significant positive correlationappeared between pp65 and late antigen (119903 = 0949 119875 lt0001) and moderate correlation between IE1-72 and pp65(119903 = 0677 119875 = 0032) No correlation between IE1-72 andlate antigen (119903 = 0567 119875 = 0111) was found

31 Polymerase Chain Reaction To determine the HCMVnucleic acids present we selected cases that were positive forHCMVby IHC and extractedDNA from the original paraffinblocks for use in PCR reactions A nested PCR approach wasutilized to detect viral DNA We detected HCMV nucleicacids in tumor cells but not in control nontumor brain cells(Figure 2)

4 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 1 Immunohistochemical staining of HCMV in glioma (a) Immunohistochemical stain of glioma section with IE1-72 protein (b)Control without secondary antibody (c) Immunohistochemical stains of glioma section with pp65 protein (d) Control without secondaryantibody (e) Immunohistochemical staining of glioma with late antigen antibody (f) Control without secondary antibody (g) Lung knownpositive control stain for HCMV

32 Seropositivity for Anti-HCMV IgG Antibodies From 50glioma patients 42 (84) were positive for anti-HCMV IgGantibodies compared to 2940 (725) among the controlgroup (Table 3) Logistic regression revealed a significant

association between seropositivity for anti-HCMV IgG anti-bodies and glioma (OR = 2779 95 CI = 1049ndash7358 119875 =004) which means that individuals who had a previousinfection of HCMV are 2779-fold more likely to get glioma

BioMed Research International 5

Table 3 The distribution of seropositivity for anti-HCMV IgG antibodies among different age classes in glioma patients and control group

Age class (years)Glioma patients Control

Number Seropositive Seronegative Number Seropositive SeronegativeNumber Number Number Number

le20 11 9 8181 2 1818 9 6 6667 3 333321ndash30 10 8 80 2 20 12 11 9167 1 83331ndash40 9 8 8889 1 1111 10 7 70 3 3041ndash50 11 9 8181 2 1818 7 4 5714 3 4286gt50 9 8 8889 1 1111 2 1 50 1 50

Figure 2 Detection of HCMV DNA in malignant glioma tissue(nested PCR products) visualized with UV light M 1000 bp DNAmarker Lanes 1ndash10 viral DNA amplified with nested PCR Lane 11negative result The size of the PCR product is 144 bp

compared with individuals with no such infection Overallthere were no significant differences in the distributionof these antibodies among different age classes of gliomapatients (Table 3) However age classes of gt50 and 31ndash40years had the highest percentage (8889) while the lowestpercentage (80) was among the youngest age class (21ndash30years) Among the control group 21ndash30-year-old individualshad a higher seropositive percentage (9167) than otherclasses with significant differences (119875 = 0037) Comparisonof the age classes between glioma patients and controlsrevealed significant differences in the seropositivity whichwas higher in glioma patients in all classes except the 21ndash30years class which was higher in the control group (9167)than that of glioma patients (80)

4 Discussion

Studying viral proteins in malignant glioma patients mayprovide more details about the mechanism that these virusesuse to modulate the cancer To the best of our knowledge thisis the first study from Iraqi patients which showed HCMVprotein and DNA in glioma specimens The results of thecurrent study indicate that 7222 and 9167 of GBM casesand 6428 to 8571 of anaplastic astrocytoma are positivefor one or more HCMV proteins and nucleic acids Theseresults corroborate previous studies (16 17 and 19) whereHCMV had an obvious association with the occurrence ofgliomas despite disparities in the percentage of tumor cellsthat gave positive results for the viral products

In immunocytochemical studies disparities in results areunavoidable because staining patterns vary by histology celltype and procedures followed during processing and stainingof paraffin-embedded tissues In this study we followedall crucial steps during detection of HCMV infection inbrain tumor tissue We used brain tumor tissue of 6 120583mbecause thicker sections do not allow proper staining andthinner sections allow excessive nonspecific binding Thiswas corroborated by the study from Scheurer et al [19]Another crucial factor in the detection of HCMV in humangliomas is the dilution of antibodies used (1 100) Our resultsare similar to other studies done by Sabatier et al [20] andPoltermann et al [21] using a lower concentration of 1 200The deparaffinization of slides may also play a role In thisstudy we deparaffinized the slides by heating them in themicrowave and treating with xylene for 20 minutes Resultsshow similarities with the study by Scheurer et al [19] butdisagree with another study [22] that used either microwaveor steam heating for much longer periods which may resultin damage to IE1-72 epitopes

The high prevalence of viral DNA and proteins in bothGBM and astrocytoma and absence of these products inbenign tumors and surrounding nontumor tissues indicatea vital role for this virus in carcinogenesis However tumorcells in general have a higher propensity to display false-positive immune-reactivity due to either nonspecific bindingtomAbs or higher levels of endogenous peroxidases that reactwith the detection substrate [17] Furthermore HCMV wasfound to use epidermal growth factor receptors (EGFR) as acellular binding and incorporation site for its entry into thecell [23] It has been shown that GMB cells uniformly ampli-fied EGFR expression while normal brain cells are largelynegative [24] Particularly with HCMV which has lifetimelatency occurrence of the cancer and subsequent treatmentinduced immunosuppression cause reactivation of the latentvirus and increase the chance for infection of the tumor cellas well as many other normal cells Presence of the viralproducts inside the tumor cells is significant and importantin the downregulation of immunogenicity of infected cellsthrough multiple mechanisms some of which are exhibitedby the tumor cells themselves These mechanisms involveinhibition of antigen presentation downregulation of surfaceMHC expression elaboration of transforming growth factor-beta (TGF-120573) from infected cells and secretion of viral IL-10 homologue (vIL-10) [25] The special features acquired bythe glioma cells might increase the tendency of HCMV to

6 BioMed Research International

enter these cells and this entry might confer protection forthe HCMV-infected cells from immune-surveillance

The nonsignificant differences in expression of the threeHCMV proteins between GBM and anaplastic astrocytomaare an expected result Cancer cells are exposed to almost thesame conditions that facilitate the entrance of the virus intothese cells Of course not all tumor cells are simultaneouslyinfected with the virus because the viral life cycle is markedlyaltered when it infects the cells However in one study theauthors reported 100 GBM positive cells for viral IE1-72protein compared to 75 of cells being positive for the sameprotein in anaplastic astrocytoma [19]

The primary HCMV antigens which induce cellularimmune response to the virus include immediate earlyprotein (IE) virion envelope glycoprotein B (late antigen55 kDa) and internal matrix protein (pp65) [26] Accumulat-ing evidence indicates that HCMV IE1 products can interactwith TP53 and Rb proteins (tumor suppressor proteins) andthereby it can induce cell cycle progression and block apop-tosis Indeed IE1 expression in a glioma cell line significantlyreduces P53 expression [27] Another study indicated thatthis protein interacts with the P13KAKT pathway one of themost important signaling pathways in glioma and sustainsactivation of AKT signaling [28] The viral protein pp65(also called pUL83) is abundantly synthesized during lyticinfection [29] It is an important target of CD8+ T-cells inthe course of immune response to HCMV infection [30]Among the many functions it has this protein is an immune-modulator and has been shown to block interferon activity[31] anddownregulate the interferon-response gene primarilythrough modulation of interferon response factor-3 IRF-3[32]

Amajor late antigen of HCMV is glycoprotein B (gB) It isa type 1 trans-membrane protein that exists as a dimer on thesurface of the viral envelope [33] This antigen is implicatedin the virus entry cell-to-cell spread [34] and fusion of theinfected cells [35]There is no evidence from available reportsthat this antigen has any role in cell transformation andthe positive result for the presence of the antigen inside thetumor cells but not the nontumor ones may be related to thefeatures of tumor cells that facilitate the entrance of HCMVcompared to the normal cells One of the most importantfeatures of IE1-72 protein is early production of this protein inthe nucleus of the infected cell It is because of this propertythat IE1-72 has stronger expression than pp65 which in turnhas stronger expression than the late antigen The last gene isknown to encode for an envelope glycoprotein and in manyinstances it scatters on the cell membrane of the infected celland cannot be easily detected These results are in part inaccordance with a previous study by Mitchell et al [17] whostated that pp65 reactivity is less ubiquitous than IE1-72 intumor cells

A significant correlation between the level of pp65 and gBantigens in the MRC-5 cell line experimentally infected withHCMV has been reported by Jun et al [36] It is not unusualto find such correlation among viral proteins as the infectionoccurs in defined sequences chronologically accompanied byexpression of immediate early early and late viral antigensHence HCMV proteins are expected to follow the pattern of

elevation and decline shown by immediate early proteins butto a lesser degree

Further support for the role ofHCMV in the developmentof this tumor results from the use of anti-HCMVdrugs Somestudies have shown that valganciclovir significantly reducedthe growth of HCMV-positive glioma tumors [37] RecentlyBaryawno et al [38] demonstrated an increase in overallsurvival of glioblastoma patients receiving valganciclovircompared with those patients who did not receive suchtreatment

The results from the present study show the high percent-age of HCMV virus expression in different histopathologicalgrades of glioma patients which clearly suggest its role ingliomapathogenesisThedetection of low-level latentHCMVinfections might suggest a role in glioma pathogenesisHowever amore detailed study ofHCMV infection in gliomapatients is required for a definitive conclusion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Ahmad Adnan in the NeuroscienceHospital for help in collection of patient samples and DrQasim Sharhan for technical assistance and scientific com-ments

References

[1] G J Tortora and B Derrickson Principles of Anatomy andPhysiology John Wiley amp Sons New York NY USA 14thedition 2014

[2] A P Kyritsis M L Bondy J S Rao and C Sioka ldquoInheritedpredisposition to gliomardquo Neuro-Oncology vol 12 no 1 pp104ndash113 2010

[3] G Prasad and D A Haas-Kogan ldquoRadiation-induced gliomasrdquoExpert Review of Neurotherapeutics vol 9 no 10 pp 1511ndash15172009

[4] Iraqi Cancer Registry 2005[5] H Ohgaki and P Kleihues ldquoEpidemiology and etiology of

gliomasrdquo Acta Neuropathologica vol 109 no 1 pp 93ndash1082005

[6] L Soroceanu and C S Cobbs ldquoIs HCMV a tumor promoterrdquoVirus Research vol 157 no 2 pp 193ndash203 2011

[7] L S YoungViruses andHuman Cancer Bios Scientific OxfordUK 1998

[8] C Hawkins and S Croul ldquoViruses and human brain tumorscytomegalovirus enters the frayrdquoThe Journal of Clinical Investi-gation vol 121 no 10 pp 3831ndash3833 2011

[9] C Taher J de Boniface A A Mohammad et al ldquoHighprevalence of human cytomegalovirus proteins and nucleicacids in primary breast cancer and metastatic sentinel lymphnodesrdquo PLoS ONE vol 8 no 2 Article ID e56795 2013

[10] F Tafvizi and Z T Fard ldquoDetection of human cytomegalovirusin patients with colorectal cancer by nested-PCRrdquo Asian PacificJournal of Cancer Prevention vol 15 no 3 pp 1453ndash1457 2014

BioMed Research International 7

[11] L Geder R Lausch F OrsquoNeill and F Rapp ldquoOncogenictransformation of human embryo lung cells by humancytomegalovirusrdquo Science vol 192 no 4244 pp 1134ndash1137 1976

[12] J Cinatl Jr J-U Vogel H Rabenau B Kornhuber and H WDoerr ldquoModulatory effects of human cytomegalovirus infectionon malignant properties of cancer cellsrdquo Intervirology vol 39no 4 pp 259ndash269 1996

[13] E S Amirian DMarquez-DoM L Bondy andM E ScheurerldquoAntihistamine use and immunoglobulin E levels in glioma riskand prognosisrdquoCancer Epidemiology vol 37 no 6 pp 908ndash9122013

[14] S Sjostrom U Hjalmars P Juto et al ldquoHuman immunoglobu-linG levels of viruses and associated glioma riskrdquoCancer Causesand Control vol 22 no 9 pp 1259ndash1266 2011

[15] C S Cobbs LHarkinsM Samanta et al ldquoHuman cytomegalo-virus infection and expression in human malignant gliomardquoCancer Research vol 62 no 12 pp 3347ndash3350 2002

[16] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas reveals apotential therapeutic targetrdquo The Journal of Clinical Investiga-tion vol 121 no 10 pp 4043ndash4055 2011

[17] D A Mitchell W Xie R Schmittling et al ldquoSensitive detectionof human cytomegalovirus in tumors and peripheral blood ofpatients diagnosed with glioblastomardquo Neuro-Oncology vol 10no 1 pp 10ndash18 2008

[18] A Rahbar G Stragliotto A Orrego et al ldquoLow levels of humancytomegalovirus infection in glioblastoma multiforme asso-ciates with patient survival a case-control studyrdquoHerpesviridaevol 3 pp 3ndash10 2012

[19] M E Scheurer M L Bondy K D Aldape T Albrecht andR El-Zein ldquoDetection of human cytomegalovirus in differenthistological types of gliomasrdquo Acta Neuropathologica vol 116no 1 pp 79ndash86 2008

[20] J Sabatier E Uro-Coste I Pommepuy et al ldquoDetection ofhuman cytomegalovirus genome and gene products in centralnervous system tumoursrdquo British Journal of Cancer vol 92 no4 pp 747ndash750 2005

[21] S Poltermann B Schlehofer K Steindorf P Schnitzler KGeletneky and J R Schlehofer ldquoLack of association of her-pesviruses with brain tumorsrdquo Journal of NeuroVirology vol 12no 2 pp 90ndash99 2006

[22] S K Lau Y-Y Chen W-G Chen et al ldquoLack of association ofcytomegalovirus with human brain tumorsrdquoModern Pathologyvol 18 no 6 pp 838ndash843 2005

[23] X Wang S-M Huong M L Chiu N Raab-Traub and E-SHuang ldquoEpidermal growth factor receptor is a cellular receptorfor human cytomegalovirusrdquoNature vol 424 no 6947 pp 456ndash461 2003

[24] T F Liu S B Tatter M C Willingham M Yang J J Hu andA E Frankel ldquoGrowth factor receptor expression varies amonghigh-grade gliomas and normal brain epidermal growth factorreceptor has excellent properties for interstitial fusion proteintherapyrdquo Molecular Cancer Therapeutics vol 2 no 8 pp 783ndash787 2003

[25] T Kossmann M C Morganti-Kossmann J M OrensteinW J Britt S M Wahl and P D Smith ldquoCytomegalovirusproduction by infected astrocytes correlates with transforminggrowth factor-120573 releaserdquoThe Journal of Infectious Diseases vol187 no 4 pp 534ndash541 2003

[26] S Saikali T Avril B Collet et al ldquoExpression of nine tumourantigens in a series of human glioblastoma multiforme interest

of EGFRvIII IL-13R1205722 gp100 and TRP-2 for immunotherapyrdquoJournal of Neuro-Oncology vol 81 no 2 pp 139ndash148 2007

[27] C S Cobbs L Soroceanu S Denham W Zhang and M HKraus ldquoModulation of oncogenic phenotype in human gliomacells by cytomegalovirus IE1-mediated mitogenicityrdquo CancerResearch vol 68 no 3 pp 724ndash730 2008

[28] R A Johnson X Wang X-L Ma S-M Huong and E-S Huang ldquoHuman cytomegalovirus up-regulates the phos-phatidylinositol 3-kinase (PI3-K) pathway inhibition of PI3-Kactivity inhibits viral replication and virus-induced signalingrdquoJournal of Virology vol 75 no 13 pp 6022ndash6032 2001

[29] S Schmolke H F Kern P Drescher G Jahn and BPlachter ldquoThe dominant phosphoprotein pp65 (UL83) ofhuman cytomegalovirus is dispensable for growth in cell cul-turerdquo Journal of Virology vol 69 no 10 pp 5959ndash5968 1995

[30] M R Wills A J Carmichael K Mynard et al ldquoThe humancytotoxic T-lymphocyte (CTL) response to cytomegalovirus isdominated by structural protein pp65 frequency specificityand T-cell receptor usage of pp65-specific CTLrdquo Journal ofVirology vol 70 no 11 pp 7569ndash7579 1996

[31] E P Browne B Wing D Coleman and T Shenk ldquoAltered cel-lular mRNA levels in human cytomegalovirus-infected fibrob-lasts viral block to the accumulation of antiviral mRNAsrdquoJournal of Virology vol 75 no 24 pp 12319ndash12330 2001

[32] D A Abate S Watanabe and E S Mocarski ldquoMajor humancytomegalovirus structural protein pp65 (ppUL83) preventsinterferon response factor 3 activation in the interferonresponserdquo Journal of Virology vol 78 no 20 pp 10995ndash110062004

[33] E R Kinzler and T Compton ldquoCharacterization of humancytomegalovirus glycoprotein-induced cell-cell fusionrdquo Journalof Virology vol 79 no 12 pp 7827ndash7837 2005

[34] M K Isaacson and T Compton ldquoHuman cytomegalovirusglycoprotein B is required for virus entry and cell-to-cell spreadbut not for virion attachment assembly or egressrdquo Journal ofVirology vol 83 no 8 pp 3891ndash3903 2009

[35] D Navarro P Paz S Tugizov K Topp J La Vail and L PereiraldquoGlycoprotein B of human cytomegalovirus promotes virionpenetration into cells transmission of infection from cell to celland fusion of infected cellsrdquoVirology vol 197 no 1 pp 143ndash1581993

[36] F Jun L Minhuan M Yadan et al ldquoDevelopment of twopotential diagnostic monoclonal antibodies against humancytomegalovirus glycoprotein Brdquo Brazilian Journal of Medicaland Biological Research vol 45 no 7 pp 583ndash590 2012

[37] G Stragliotto A Rahbar NW Solberg et al ldquoEffects of valgan-ciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma a randomized double-blind hypothesis-generating studyrdquo International Journal of Cancer vol 133 no5 pp 1204ndash1213 2013

[38] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas revealsa potential therapeutic targetrdquo Journal of Clinical Investigationvol 121 no 10 pp 4043ndash4055 2011

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 BioMed Research International

which were the expression of oncogenes transcriptional acti-vation of growth factor and interleukin synthesis Howeverthe role of this virus in brain tumor development has been amatter of debate for many years A role for HCMV in braintumors can be deduced from the association of anti-HCMVIgG and IgM Abs with the incidence of gliomas This wasconfirmed by Amirian et al [13] who found that anti-HCMVAbs level was associated with glioma risk especially amongIgM-positive individuals In contrast Sjostrom et al [14] didnot find significant association between glioma or GBM andanti-HCMV IgG levels

The presence of viral DNA or protein inside tumor versusnontumor cells can give more accurate and direct causalassociation than estimation of anti-HCMVantibodies Cobbset al [15] shows that HCMV proteins and nucleic acidsproducts are expressed in virtually all GBMs but not innormal brain or other benign tumors suggest a possibilitythat HCMV may play an active role in glioma pathogenesisFurther 92 of primary GBM tumors express viral imme-diate early protein while 73 of them express late proteins[16] In addition HCMV in tumors and the peripheral bloodof patients diagnosed with GBM shows a high percentage(gt90) of this tumor associatedwithHCMVnucleic acid andproteins and 80 of patients had detectable HCMVDNA intheir peripheral blood [17] Recent studies have also reportedthe presence of HCMV proteins in 99 of brain tumor tissuesections isolated from GBM patients [18]

HCMV in glioma and other tumors is a major concernin Iraq and until now no published information has beenavailable on the expression and association of this virus withthese tumors Studying HCMV expression and associationwith brain tumors might be beneficial in development ofpossible diagnostic and interventional strategies in thesepatients To this end in the present study we investigated theexpression represented by early (IEI-72) mid (pp65) and lateHCMV antigens in different grades of GBM and anaplasticastrocytoma in Iraqi patients

2 Materials and Methods

Patients from the Neurosurgery Hospital Neuroscience Hos-pital AL-Kadimiya Teaching Hospital AL-Fallujah Hospitaland Private Nursing Home Hospital undergoing surgicalresection of brain tumors from January 2012 to March 2013were included in this study Ethical approval to conductthe research was obtained from each hospital InstitutionalReview Board (IRB) Fifty paraffin-embedded brain tissuesfrom the patients were used (mean age 3185 plusmn 195 yearsrange 1ndash72 years) Personal data were collected through directinterview with the patient and by seeking hisher hospitalrecord and medical records Patient claims were followed asan alternative source of information when hisher previousmedical reports were not available Collected data includedage sex smoking and first relative family history of braintumors All brain tumors were graded by a histopathologistAmong the 50 specimens 36wereGBMs and 14were anaplas-tic astrocytomaThirty paraffin-embedded blocks from aged-matched benign meningioma patients were used as controls

21 Immunohistochemistry (IHC) All paraffin-embeddedsections (6 120583m thick for patients and control) were deparaf-finized by heating slides in a vertical position in an oven for 1hour followed by washing in xylene (20min) and an ethanolseries (100 95 75 and 50 for 5min each) Sectionswere blocked for endogenous peroxidase 3H

2O2for 20min

(BioGenex CA USA) Antigen retrieval was performedusing Citra Plus antigen retrieval solution (BioGenex CAUSA) for 2min at 97∘C in a microwave Protein blocks(EMDMillipore IL USA) were applied to the sections priorto application of primary antibodies (against the HCMVantigen IE1 pp65 and late antigen (1 100 for all antigens)EMD Millipore IL USA) Human lung with known HCMVinfection was used as a positive control (Chemicon Temec-ula CA USA) and negative control tumor sections that werenot treated with an antibody stained were also included theexperiments All slides were incubated overnight at 4∘C Theslides were then stainedwith secondary antibody biotinylatedgoat anti-mouse (1 175 IHC select HRP kit from EMDMilli-pore IL USA) peroxidase-labeled streptavidin (EMDMilli-pore IL USA) and 331015840-diaminobenzidine (DAB) (InnovexBiosciences USA) Hematoxylin was used as a counterstainHuman lung sections with known HCMV infection wereused as positive controls for staining (Chemicon TemeculaCA USA) Negative controls consisted of tumor sections thatwere not treated with the primary antibody andor secondaryantibody All samples were stained more than once and theresult was highly reproducible

22 Immunohistochemical Scoring Immunostained sectionswere evaluated by two independent histopathologists whowere blinded to the patient information and disease severityCases with disagreement were discussed using amultiheadedmicroscope until agreement was achieved For HCMV pro-teins (IE1-72 pp65 and late antigen) the brown staining ofthe nucleus with or without a diffuse pattern was consideredpositive The intensity of protein expression was scored asreported by Rahbar et al [18] by examining 1000 cells in 10high power fields According to the estimated percentage ofpositive cells four grades were identified grade 1 lt25 grade2 ge25ndash50 grade 3 ge51ndash75 grade 4 ge76

23 DNA Extraction A ready kit (DNeasy Blood and tissuekitQiagen Valencia) was used for viral DNA extraction fromtumor specimens according the manufacturerrsquos instructions

24 Polymerase Chain Reaction In order to reduce con-tamination and prevent false positive results preparationfor PCR was carried out in a laboratory not previouslyused for handling HCMV A nested PCR method was usedto detect viral DNA External primer sequences were theforward primer 5-CCGAAATACGCGTTTTGAGATand thereverse primer 5-CCAAGCCAAAAACAGTATAGC whichamplified a 1300 bp of the promoter region of the immediateearly (IE) gene A readymastermix (Bioneer Korea) was usedfor PCR reaction preparationThe PCR conditions were 95∘Cfor 5min followed by 45 cycles of 94∘C for 30 s 56∘C for30 s and 72∘C for 1min with a final extension at 72∘C for

BioMed Research International 3

Table 1 The percentage and grading of HCMV protein in GBM

HCMV proteinsNumber = 36

PositivityNumber()

Grading of positive sectionsGrade 1Number()

Grade 2Number()

Grade 3Number()

Grade 4Number()

IE1-72 33 (9167) 7 (2121) 6 (1818) 7 (2121) 13 (3939)pp65 28 (7778) 7 (25) 9 (3214) 7 (25) 5 (1758)Late antigen 26 (7222) 7 (2692) 10 (3846) 8 (3077) 3 (1154)Percentage changes and grading in early mid and late antigen HCMV protein in glioblastoma multiforme patients The grade number and percentage ()were calculated in a blinded fashion The number and of positive patients are indicated for each antigen

Table 2 The percentage and grading of HCMV protein in anaplastic astrocytoma

HCMV proteinsNumber = 14

PositivityNumber()

Grading of positive sectionsGrade 1Number()

Grade 2Number()

Grade 3Number()

Grade 4Number()

IE1-72 12 (8571) 2 (1667) 2 (1667) 2 (1667) 6 (50)pp65 10 (7142) 2 (20) 5 (50) 2 (20) 1 (10)Late antigen 9 (6428) 3 (3333) 3 (3333) 2 (2222) 1 (1111)Percentage changes and grading in early mid and late antigen HCMV protein in anaplastic astrocytoma patients The grade number and percentage () werecalculated in a blinded fashion The number and of positive patients are indicated for each antigen

10min Internal primer sequences were the forward primer5-GGCGGAGTT (GA) TTACGACATTT and the reverseprimer 5-ATGCGGTTTTGGCAGTACATwhich amplified a144 bp region within the 1300 bp amplicon generated by theexternal primer setThe PCR conditions were 95∘C for 5minfollowed by 40 cycles of 94∘C for 20 s 56∘C for 20 s and 72∘Cfor 20 s with a final extension at 72∘C for 5min

25 HCMV ELISA IgG Anti-CMV IgG ELISA test kits(Biokit Barcelona Spain) were used for qualitative detectionof serum anti-CMV IgG antibodies according to the manu-facturerrsquos manual

26 Statistical Analysis The Statistical Package for the SocialSciences (SPSS version 14) was used for statistical analysisThe logistic regression test was used to test the associationof HCMV immune positivity with incidence of gliomas Thechi-square test was used to compare immune positivity toCytomegalovirus in different age classes and in evaluation ofimmunostainingThe correlation test was used to calculate 119903-values for HCMV protein expression A 119875 value lt 005 wasconsidered statistically significant

3 Results

We performed IHC on paraffin sections from 50 gliomasthat included 36 GBM and 14 anaplastic astrocytomas usingmAbs specific for HCMV proteins (IE1-72 pp65 and lateantigen) Out of 36 GBM samples 33 (9167) 28 (7778)and 26 (7222) were positive for IE1-72 pp65 and lateantigen respectively (Table 1) We noticed no significantdifference in the expression of these proteins betweenGBMs and anaplastic astrocytomas Control tissue sections

(meningioma biopsy specimens) and the surrounding brainarea of malignant gliomas were devoid of immunoreactivity(Figure 1)

IE1-72 protein demonstrated stronger expression thanother proteins as 13 out of 33 positive GBM samples (3939)were grade 4 compared to 5 (1758) and 3 (1154) for pp65and late antigen respectively (1205942 = 7052 119875 = 0029)(Table 1) Furthermore IE1-72 had the lowest percentage ofsamples that were expressed as grade 1 (2121) compared to25 for pp65 and 2992 for late antigenThe correlation testrevealed highly significant positive correlation between IE1-72 late antigen (119903 = 0769 119875 lt 0001) and pp65 (119903 = 0714119875 lt 0001)

Among 14 anaplastic astrocytoma samples 12 (8571) 10(7142) and 9 (6482)were positive for IE1-72 pp65 and lateantigen respectively (Table 2) Out of 14 positive anaplasticastrocytoma samples 6 (50) 1 (10) and 1 (1111) weregrade 4 for IE1-72 pp65 and late antigen respectively witha significant difference (1205942 = 5988 119875 = 005) FurthermoreIE1-72 protein had the lowest percentage of samples that wereexpressed as grade 1 (1667) compared to pp65 (20) andlate antigen (3333) A strong significant positive correlationappeared between pp65 and late antigen (119903 = 0949 119875 lt0001) and moderate correlation between IE1-72 and pp65(119903 = 0677 119875 = 0032) No correlation between IE1-72 andlate antigen (119903 = 0567 119875 = 0111) was found

31 Polymerase Chain Reaction To determine the HCMVnucleic acids present we selected cases that were positive forHCMVby IHC and extractedDNA from the original paraffinblocks for use in PCR reactions A nested PCR approach wasutilized to detect viral DNA We detected HCMV nucleicacids in tumor cells but not in control nontumor brain cells(Figure 2)

4 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 1 Immunohistochemical staining of HCMV in glioma (a) Immunohistochemical stain of glioma section with IE1-72 protein (b)Control without secondary antibody (c) Immunohistochemical stains of glioma section with pp65 protein (d) Control without secondaryantibody (e) Immunohistochemical staining of glioma with late antigen antibody (f) Control without secondary antibody (g) Lung knownpositive control stain for HCMV

32 Seropositivity for Anti-HCMV IgG Antibodies From 50glioma patients 42 (84) were positive for anti-HCMV IgGantibodies compared to 2940 (725) among the controlgroup (Table 3) Logistic regression revealed a significant

association between seropositivity for anti-HCMV IgG anti-bodies and glioma (OR = 2779 95 CI = 1049ndash7358 119875 =004) which means that individuals who had a previousinfection of HCMV are 2779-fold more likely to get glioma

BioMed Research International 5

Table 3 The distribution of seropositivity for anti-HCMV IgG antibodies among different age classes in glioma patients and control group

Age class (years)Glioma patients Control

Number Seropositive Seronegative Number Seropositive SeronegativeNumber Number Number Number

le20 11 9 8181 2 1818 9 6 6667 3 333321ndash30 10 8 80 2 20 12 11 9167 1 83331ndash40 9 8 8889 1 1111 10 7 70 3 3041ndash50 11 9 8181 2 1818 7 4 5714 3 4286gt50 9 8 8889 1 1111 2 1 50 1 50

Figure 2 Detection of HCMV DNA in malignant glioma tissue(nested PCR products) visualized with UV light M 1000 bp DNAmarker Lanes 1ndash10 viral DNA amplified with nested PCR Lane 11negative result The size of the PCR product is 144 bp

compared with individuals with no such infection Overallthere were no significant differences in the distributionof these antibodies among different age classes of gliomapatients (Table 3) However age classes of gt50 and 31ndash40years had the highest percentage (8889) while the lowestpercentage (80) was among the youngest age class (21ndash30years) Among the control group 21ndash30-year-old individualshad a higher seropositive percentage (9167) than otherclasses with significant differences (119875 = 0037) Comparisonof the age classes between glioma patients and controlsrevealed significant differences in the seropositivity whichwas higher in glioma patients in all classes except the 21ndash30years class which was higher in the control group (9167)than that of glioma patients (80)

4 Discussion

Studying viral proteins in malignant glioma patients mayprovide more details about the mechanism that these virusesuse to modulate the cancer To the best of our knowledge thisis the first study from Iraqi patients which showed HCMVprotein and DNA in glioma specimens The results of thecurrent study indicate that 7222 and 9167 of GBM casesand 6428 to 8571 of anaplastic astrocytoma are positivefor one or more HCMV proteins and nucleic acids Theseresults corroborate previous studies (16 17 and 19) whereHCMV had an obvious association with the occurrence ofgliomas despite disparities in the percentage of tumor cellsthat gave positive results for the viral products

In immunocytochemical studies disparities in results areunavoidable because staining patterns vary by histology celltype and procedures followed during processing and stainingof paraffin-embedded tissues In this study we followedall crucial steps during detection of HCMV infection inbrain tumor tissue We used brain tumor tissue of 6 120583mbecause thicker sections do not allow proper staining andthinner sections allow excessive nonspecific binding Thiswas corroborated by the study from Scheurer et al [19]Another crucial factor in the detection of HCMV in humangliomas is the dilution of antibodies used (1 100) Our resultsare similar to other studies done by Sabatier et al [20] andPoltermann et al [21] using a lower concentration of 1 200The deparaffinization of slides may also play a role In thisstudy we deparaffinized the slides by heating them in themicrowave and treating with xylene for 20 minutes Resultsshow similarities with the study by Scheurer et al [19] butdisagree with another study [22] that used either microwaveor steam heating for much longer periods which may resultin damage to IE1-72 epitopes

The high prevalence of viral DNA and proteins in bothGBM and astrocytoma and absence of these products inbenign tumors and surrounding nontumor tissues indicatea vital role for this virus in carcinogenesis However tumorcells in general have a higher propensity to display false-positive immune-reactivity due to either nonspecific bindingtomAbs or higher levels of endogenous peroxidases that reactwith the detection substrate [17] Furthermore HCMV wasfound to use epidermal growth factor receptors (EGFR) as acellular binding and incorporation site for its entry into thecell [23] It has been shown that GMB cells uniformly ampli-fied EGFR expression while normal brain cells are largelynegative [24] Particularly with HCMV which has lifetimelatency occurrence of the cancer and subsequent treatmentinduced immunosuppression cause reactivation of the latentvirus and increase the chance for infection of the tumor cellas well as many other normal cells Presence of the viralproducts inside the tumor cells is significant and importantin the downregulation of immunogenicity of infected cellsthrough multiple mechanisms some of which are exhibitedby the tumor cells themselves These mechanisms involveinhibition of antigen presentation downregulation of surfaceMHC expression elaboration of transforming growth factor-beta (TGF-120573) from infected cells and secretion of viral IL-10 homologue (vIL-10) [25] The special features acquired bythe glioma cells might increase the tendency of HCMV to

6 BioMed Research International

enter these cells and this entry might confer protection forthe HCMV-infected cells from immune-surveillance

The nonsignificant differences in expression of the threeHCMV proteins between GBM and anaplastic astrocytomaare an expected result Cancer cells are exposed to almost thesame conditions that facilitate the entrance of the virus intothese cells Of course not all tumor cells are simultaneouslyinfected with the virus because the viral life cycle is markedlyaltered when it infects the cells However in one study theauthors reported 100 GBM positive cells for viral IE1-72protein compared to 75 of cells being positive for the sameprotein in anaplastic astrocytoma [19]

The primary HCMV antigens which induce cellularimmune response to the virus include immediate earlyprotein (IE) virion envelope glycoprotein B (late antigen55 kDa) and internal matrix protein (pp65) [26] Accumulat-ing evidence indicates that HCMV IE1 products can interactwith TP53 and Rb proteins (tumor suppressor proteins) andthereby it can induce cell cycle progression and block apop-tosis Indeed IE1 expression in a glioma cell line significantlyreduces P53 expression [27] Another study indicated thatthis protein interacts with the P13KAKT pathway one of themost important signaling pathways in glioma and sustainsactivation of AKT signaling [28] The viral protein pp65(also called pUL83) is abundantly synthesized during lyticinfection [29] It is an important target of CD8+ T-cells inthe course of immune response to HCMV infection [30]Among the many functions it has this protein is an immune-modulator and has been shown to block interferon activity[31] anddownregulate the interferon-response gene primarilythrough modulation of interferon response factor-3 IRF-3[32]

Amajor late antigen of HCMV is glycoprotein B (gB) It isa type 1 trans-membrane protein that exists as a dimer on thesurface of the viral envelope [33] This antigen is implicatedin the virus entry cell-to-cell spread [34] and fusion of theinfected cells [35]There is no evidence from available reportsthat this antigen has any role in cell transformation andthe positive result for the presence of the antigen inside thetumor cells but not the nontumor ones may be related to thefeatures of tumor cells that facilitate the entrance of HCMVcompared to the normal cells One of the most importantfeatures of IE1-72 protein is early production of this protein inthe nucleus of the infected cell It is because of this propertythat IE1-72 has stronger expression than pp65 which in turnhas stronger expression than the late antigen The last gene isknown to encode for an envelope glycoprotein and in manyinstances it scatters on the cell membrane of the infected celland cannot be easily detected These results are in part inaccordance with a previous study by Mitchell et al [17] whostated that pp65 reactivity is less ubiquitous than IE1-72 intumor cells

A significant correlation between the level of pp65 and gBantigens in the MRC-5 cell line experimentally infected withHCMV has been reported by Jun et al [36] It is not unusualto find such correlation among viral proteins as the infectionoccurs in defined sequences chronologically accompanied byexpression of immediate early early and late viral antigensHence HCMV proteins are expected to follow the pattern of

elevation and decline shown by immediate early proteins butto a lesser degree

Further support for the role ofHCMV in the developmentof this tumor results from the use of anti-HCMVdrugs Somestudies have shown that valganciclovir significantly reducedthe growth of HCMV-positive glioma tumors [37] RecentlyBaryawno et al [38] demonstrated an increase in overallsurvival of glioblastoma patients receiving valganciclovircompared with those patients who did not receive suchtreatment

The results from the present study show the high percent-age of HCMV virus expression in different histopathologicalgrades of glioma patients which clearly suggest its role ingliomapathogenesisThedetection of low-level latentHCMVinfections might suggest a role in glioma pathogenesisHowever amore detailed study ofHCMV infection in gliomapatients is required for a definitive conclusion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Ahmad Adnan in the NeuroscienceHospital for help in collection of patient samples and DrQasim Sharhan for technical assistance and scientific com-ments

References

[1] G J Tortora and B Derrickson Principles of Anatomy andPhysiology John Wiley amp Sons New York NY USA 14thedition 2014

[2] A P Kyritsis M L Bondy J S Rao and C Sioka ldquoInheritedpredisposition to gliomardquo Neuro-Oncology vol 12 no 1 pp104ndash113 2010

[3] G Prasad and D A Haas-Kogan ldquoRadiation-induced gliomasrdquoExpert Review of Neurotherapeutics vol 9 no 10 pp 1511ndash15172009

[4] Iraqi Cancer Registry 2005[5] H Ohgaki and P Kleihues ldquoEpidemiology and etiology of

gliomasrdquo Acta Neuropathologica vol 109 no 1 pp 93ndash1082005

[6] L Soroceanu and C S Cobbs ldquoIs HCMV a tumor promoterrdquoVirus Research vol 157 no 2 pp 193ndash203 2011

[7] L S YoungViruses andHuman Cancer Bios Scientific OxfordUK 1998

[8] C Hawkins and S Croul ldquoViruses and human brain tumorscytomegalovirus enters the frayrdquoThe Journal of Clinical Investi-gation vol 121 no 10 pp 3831ndash3833 2011

[9] C Taher J de Boniface A A Mohammad et al ldquoHighprevalence of human cytomegalovirus proteins and nucleicacids in primary breast cancer and metastatic sentinel lymphnodesrdquo PLoS ONE vol 8 no 2 Article ID e56795 2013

[10] F Tafvizi and Z T Fard ldquoDetection of human cytomegalovirusin patients with colorectal cancer by nested-PCRrdquo Asian PacificJournal of Cancer Prevention vol 15 no 3 pp 1453ndash1457 2014

BioMed Research International 7

[11] L Geder R Lausch F OrsquoNeill and F Rapp ldquoOncogenictransformation of human embryo lung cells by humancytomegalovirusrdquo Science vol 192 no 4244 pp 1134ndash1137 1976

[12] J Cinatl Jr J-U Vogel H Rabenau B Kornhuber and H WDoerr ldquoModulatory effects of human cytomegalovirus infectionon malignant properties of cancer cellsrdquo Intervirology vol 39no 4 pp 259ndash269 1996

[13] E S Amirian DMarquez-DoM L Bondy andM E ScheurerldquoAntihistamine use and immunoglobulin E levels in glioma riskand prognosisrdquoCancer Epidemiology vol 37 no 6 pp 908ndash9122013

[14] S Sjostrom U Hjalmars P Juto et al ldquoHuman immunoglobu-linG levels of viruses and associated glioma riskrdquoCancer Causesand Control vol 22 no 9 pp 1259ndash1266 2011

[15] C S Cobbs LHarkinsM Samanta et al ldquoHuman cytomegalo-virus infection and expression in human malignant gliomardquoCancer Research vol 62 no 12 pp 3347ndash3350 2002

[16] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas reveals apotential therapeutic targetrdquo The Journal of Clinical Investiga-tion vol 121 no 10 pp 4043ndash4055 2011

[17] D A Mitchell W Xie R Schmittling et al ldquoSensitive detectionof human cytomegalovirus in tumors and peripheral blood ofpatients diagnosed with glioblastomardquo Neuro-Oncology vol 10no 1 pp 10ndash18 2008

[18] A Rahbar G Stragliotto A Orrego et al ldquoLow levels of humancytomegalovirus infection in glioblastoma multiforme asso-ciates with patient survival a case-control studyrdquoHerpesviridaevol 3 pp 3ndash10 2012

[19] M E Scheurer M L Bondy K D Aldape T Albrecht andR El-Zein ldquoDetection of human cytomegalovirus in differenthistological types of gliomasrdquo Acta Neuropathologica vol 116no 1 pp 79ndash86 2008

[20] J Sabatier E Uro-Coste I Pommepuy et al ldquoDetection ofhuman cytomegalovirus genome and gene products in centralnervous system tumoursrdquo British Journal of Cancer vol 92 no4 pp 747ndash750 2005

[21] S Poltermann B Schlehofer K Steindorf P Schnitzler KGeletneky and J R Schlehofer ldquoLack of association of her-pesviruses with brain tumorsrdquo Journal of NeuroVirology vol 12no 2 pp 90ndash99 2006

[22] S K Lau Y-Y Chen W-G Chen et al ldquoLack of association ofcytomegalovirus with human brain tumorsrdquoModern Pathologyvol 18 no 6 pp 838ndash843 2005

[23] X Wang S-M Huong M L Chiu N Raab-Traub and E-SHuang ldquoEpidermal growth factor receptor is a cellular receptorfor human cytomegalovirusrdquoNature vol 424 no 6947 pp 456ndash461 2003

[24] T F Liu S B Tatter M C Willingham M Yang J J Hu andA E Frankel ldquoGrowth factor receptor expression varies amonghigh-grade gliomas and normal brain epidermal growth factorreceptor has excellent properties for interstitial fusion proteintherapyrdquo Molecular Cancer Therapeutics vol 2 no 8 pp 783ndash787 2003

[25] T Kossmann M C Morganti-Kossmann J M OrensteinW J Britt S M Wahl and P D Smith ldquoCytomegalovirusproduction by infected astrocytes correlates with transforminggrowth factor-120573 releaserdquoThe Journal of Infectious Diseases vol187 no 4 pp 534ndash541 2003

[26] S Saikali T Avril B Collet et al ldquoExpression of nine tumourantigens in a series of human glioblastoma multiforme interest

of EGFRvIII IL-13R1205722 gp100 and TRP-2 for immunotherapyrdquoJournal of Neuro-Oncology vol 81 no 2 pp 139ndash148 2007

[27] C S Cobbs L Soroceanu S Denham W Zhang and M HKraus ldquoModulation of oncogenic phenotype in human gliomacells by cytomegalovirus IE1-mediated mitogenicityrdquo CancerResearch vol 68 no 3 pp 724ndash730 2008

[28] R A Johnson X Wang X-L Ma S-M Huong and E-S Huang ldquoHuman cytomegalovirus up-regulates the phos-phatidylinositol 3-kinase (PI3-K) pathway inhibition of PI3-Kactivity inhibits viral replication and virus-induced signalingrdquoJournal of Virology vol 75 no 13 pp 6022ndash6032 2001

[29] S Schmolke H F Kern P Drescher G Jahn and BPlachter ldquoThe dominant phosphoprotein pp65 (UL83) ofhuman cytomegalovirus is dispensable for growth in cell cul-turerdquo Journal of Virology vol 69 no 10 pp 5959ndash5968 1995

[30] M R Wills A J Carmichael K Mynard et al ldquoThe humancytotoxic T-lymphocyte (CTL) response to cytomegalovirus isdominated by structural protein pp65 frequency specificityand T-cell receptor usage of pp65-specific CTLrdquo Journal ofVirology vol 70 no 11 pp 7569ndash7579 1996

[31] E P Browne B Wing D Coleman and T Shenk ldquoAltered cel-lular mRNA levels in human cytomegalovirus-infected fibrob-lasts viral block to the accumulation of antiviral mRNAsrdquoJournal of Virology vol 75 no 24 pp 12319ndash12330 2001

[32] D A Abate S Watanabe and E S Mocarski ldquoMajor humancytomegalovirus structural protein pp65 (ppUL83) preventsinterferon response factor 3 activation in the interferonresponserdquo Journal of Virology vol 78 no 20 pp 10995ndash110062004

[33] E R Kinzler and T Compton ldquoCharacterization of humancytomegalovirus glycoprotein-induced cell-cell fusionrdquo Journalof Virology vol 79 no 12 pp 7827ndash7837 2005

[34] M K Isaacson and T Compton ldquoHuman cytomegalovirusglycoprotein B is required for virus entry and cell-to-cell spreadbut not for virion attachment assembly or egressrdquo Journal ofVirology vol 83 no 8 pp 3891ndash3903 2009

[35] D Navarro P Paz S Tugizov K Topp J La Vail and L PereiraldquoGlycoprotein B of human cytomegalovirus promotes virionpenetration into cells transmission of infection from cell to celland fusion of infected cellsrdquoVirology vol 197 no 1 pp 143ndash1581993

[36] F Jun L Minhuan M Yadan et al ldquoDevelopment of twopotential diagnostic monoclonal antibodies against humancytomegalovirus glycoprotein Brdquo Brazilian Journal of Medicaland Biological Research vol 45 no 7 pp 583ndash590 2012

[37] G Stragliotto A Rahbar NW Solberg et al ldquoEffects of valgan-ciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma a randomized double-blind hypothesis-generating studyrdquo International Journal of Cancer vol 133 no5 pp 1204ndash1213 2013

[38] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas revealsa potential therapeutic targetrdquo Journal of Clinical Investigationvol 121 no 10 pp 4043ndash4055 2011

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 3

Table 1 The percentage and grading of HCMV protein in GBM

HCMV proteinsNumber = 36

PositivityNumber()

Grading of positive sectionsGrade 1Number()

Grade 2Number()

Grade 3Number()

Grade 4Number()

IE1-72 33 (9167) 7 (2121) 6 (1818) 7 (2121) 13 (3939)pp65 28 (7778) 7 (25) 9 (3214) 7 (25) 5 (1758)Late antigen 26 (7222) 7 (2692) 10 (3846) 8 (3077) 3 (1154)Percentage changes and grading in early mid and late antigen HCMV protein in glioblastoma multiforme patients The grade number and percentage ()were calculated in a blinded fashion The number and of positive patients are indicated for each antigen

Table 2 The percentage and grading of HCMV protein in anaplastic astrocytoma

HCMV proteinsNumber = 14

PositivityNumber()

Grading of positive sectionsGrade 1Number()

Grade 2Number()

Grade 3Number()

Grade 4Number()

IE1-72 12 (8571) 2 (1667) 2 (1667) 2 (1667) 6 (50)pp65 10 (7142) 2 (20) 5 (50) 2 (20) 1 (10)Late antigen 9 (6428) 3 (3333) 3 (3333) 2 (2222) 1 (1111)Percentage changes and grading in early mid and late antigen HCMV protein in anaplastic astrocytoma patients The grade number and percentage () werecalculated in a blinded fashion The number and of positive patients are indicated for each antigen

10min Internal primer sequences were the forward primer5-GGCGGAGTT (GA) TTACGACATTT and the reverseprimer 5-ATGCGGTTTTGGCAGTACATwhich amplified a144 bp region within the 1300 bp amplicon generated by theexternal primer setThe PCR conditions were 95∘C for 5minfollowed by 40 cycles of 94∘C for 20 s 56∘C for 20 s and 72∘Cfor 20 s with a final extension at 72∘C for 5min

25 HCMV ELISA IgG Anti-CMV IgG ELISA test kits(Biokit Barcelona Spain) were used for qualitative detectionof serum anti-CMV IgG antibodies according to the manu-facturerrsquos manual

26 Statistical Analysis The Statistical Package for the SocialSciences (SPSS version 14) was used for statistical analysisThe logistic regression test was used to test the associationof HCMV immune positivity with incidence of gliomas Thechi-square test was used to compare immune positivity toCytomegalovirus in different age classes and in evaluation ofimmunostainingThe correlation test was used to calculate 119903-values for HCMV protein expression A 119875 value lt 005 wasconsidered statistically significant

3 Results

We performed IHC on paraffin sections from 50 gliomasthat included 36 GBM and 14 anaplastic astrocytomas usingmAbs specific for HCMV proteins (IE1-72 pp65 and lateantigen) Out of 36 GBM samples 33 (9167) 28 (7778)and 26 (7222) were positive for IE1-72 pp65 and lateantigen respectively (Table 1) We noticed no significantdifference in the expression of these proteins betweenGBMs and anaplastic astrocytomas Control tissue sections

(meningioma biopsy specimens) and the surrounding brainarea of malignant gliomas were devoid of immunoreactivity(Figure 1)

IE1-72 protein demonstrated stronger expression thanother proteins as 13 out of 33 positive GBM samples (3939)were grade 4 compared to 5 (1758) and 3 (1154) for pp65and late antigen respectively (1205942 = 7052 119875 = 0029)(Table 1) Furthermore IE1-72 had the lowest percentage ofsamples that were expressed as grade 1 (2121) compared to25 for pp65 and 2992 for late antigenThe correlation testrevealed highly significant positive correlation between IE1-72 late antigen (119903 = 0769 119875 lt 0001) and pp65 (119903 = 0714119875 lt 0001)

Among 14 anaplastic astrocytoma samples 12 (8571) 10(7142) and 9 (6482)were positive for IE1-72 pp65 and lateantigen respectively (Table 2) Out of 14 positive anaplasticastrocytoma samples 6 (50) 1 (10) and 1 (1111) weregrade 4 for IE1-72 pp65 and late antigen respectively witha significant difference (1205942 = 5988 119875 = 005) FurthermoreIE1-72 protein had the lowest percentage of samples that wereexpressed as grade 1 (1667) compared to pp65 (20) andlate antigen (3333) A strong significant positive correlationappeared between pp65 and late antigen (119903 = 0949 119875 lt0001) and moderate correlation between IE1-72 and pp65(119903 = 0677 119875 = 0032) No correlation between IE1-72 andlate antigen (119903 = 0567 119875 = 0111) was found

31 Polymerase Chain Reaction To determine the HCMVnucleic acids present we selected cases that were positive forHCMVby IHC and extractedDNA from the original paraffinblocks for use in PCR reactions A nested PCR approach wasutilized to detect viral DNA We detected HCMV nucleicacids in tumor cells but not in control nontumor brain cells(Figure 2)

4 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 1 Immunohistochemical staining of HCMV in glioma (a) Immunohistochemical stain of glioma section with IE1-72 protein (b)Control without secondary antibody (c) Immunohistochemical stains of glioma section with pp65 protein (d) Control without secondaryantibody (e) Immunohistochemical staining of glioma with late antigen antibody (f) Control without secondary antibody (g) Lung knownpositive control stain for HCMV

32 Seropositivity for Anti-HCMV IgG Antibodies From 50glioma patients 42 (84) were positive for anti-HCMV IgGantibodies compared to 2940 (725) among the controlgroup (Table 3) Logistic regression revealed a significant

association between seropositivity for anti-HCMV IgG anti-bodies and glioma (OR = 2779 95 CI = 1049ndash7358 119875 =004) which means that individuals who had a previousinfection of HCMV are 2779-fold more likely to get glioma

BioMed Research International 5

Table 3 The distribution of seropositivity for anti-HCMV IgG antibodies among different age classes in glioma patients and control group

Age class (years)Glioma patients Control

Number Seropositive Seronegative Number Seropositive SeronegativeNumber Number Number Number

le20 11 9 8181 2 1818 9 6 6667 3 333321ndash30 10 8 80 2 20 12 11 9167 1 83331ndash40 9 8 8889 1 1111 10 7 70 3 3041ndash50 11 9 8181 2 1818 7 4 5714 3 4286gt50 9 8 8889 1 1111 2 1 50 1 50

Figure 2 Detection of HCMV DNA in malignant glioma tissue(nested PCR products) visualized with UV light M 1000 bp DNAmarker Lanes 1ndash10 viral DNA amplified with nested PCR Lane 11negative result The size of the PCR product is 144 bp

compared with individuals with no such infection Overallthere were no significant differences in the distributionof these antibodies among different age classes of gliomapatients (Table 3) However age classes of gt50 and 31ndash40years had the highest percentage (8889) while the lowestpercentage (80) was among the youngest age class (21ndash30years) Among the control group 21ndash30-year-old individualshad a higher seropositive percentage (9167) than otherclasses with significant differences (119875 = 0037) Comparisonof the age classes between glioma patients and controlsrevealed significant differences in the seropositivity whichwas higher in glioma patients in all classes except the 21ndash30years class which was higher in the control group (9167)than that of glioma patients (80)

4 Discussion

Studying viral proteins in malignant glioma patients mayprovide more details about the mechanism that these virusesuse to modulate the cancer To the best of our knowledge thisis the first study from Iraqi patients which showed HCMVprotein and DNA in glioma specimens The results of thecurrent study indicate that 7222 and 9167 of GBM casesand 6428 to 8571 of anaplastic astrocytoma are positivefor one or more HCMV proteins and nucleic acids Theseresults corroborate previous studies (16 17 and 19) whereHCMV had an obvious association with the occurrence ofgliomas despite disparities in the percentage of tumor cellsthat gave positive results for the viral products

In immunocytochemical studies disparities in results areunavoidable because staining patterns vary by histology celltype and procedures followed during processing and stainingof paraffin-embedded tissues In this study we followedall crucial steps during detection of HCMV infection inbrain tumor tissue We used brain tumor tissue of 6 120583mbecause thicker sections do not allow proper staining andthinner sections allow excessive nonspecific binding Thiswas corroborated by the study from Scheurer et al [19]Another crucial factor in the detection of HCMV in humangliomas is the dilution of antibodies used (1 100) Our resultsare similar to other studies done by Sabatier et al [20] andPoltermann et al [21] using a lower concentration of 1 200The deparaffinization of slides may also play a role In thisstudy we deparaffinized the slides by heating them in themicrowave and treating with xylene for 20 minutes Resultsshow similarities with the study by Scheurer et al [19] butdisagree with another study [22] that used either microwaveor steam heating for much longer periods which may resultin damage to IE1-72 epitopes

The high prevalence of viral DNA and proteins in bothGBM and astrocytoma and absence of these products inbenign tumors and surrounding nontumor tissues indicatea vital role for this virus in carcinogenesis However tumorcells in general have a higher propensity to display false-positive immune-reactivity due to either nonspecific bindingtomAbs or higher levels of endogenous peroxidases that reactwith the detection substrate [17] Furthermore HCMV wasfound to use epidermal growth factor receptors (EGFR) as acellular binding and incorporation site for its entry into thecell [23] It has been shown that GMB cells uniformly ampli-fied EGFR expression while normal brain cells are largelynegative [24] Particularly with HCMV which has lifetimelatency occurrence of the cancer and subsequent treatmentinduced immunosuppression cause reactivation of the latentvirus and increase the chance for infection of the tumor cellas well as many other normal cells Presence of the viralproducts inside the tumor cells is significant and importantin the downregulation of immunogenicity of infected cellsthrough multiple mechanisms some of which are exhibitedby the tumor cells themselves These mechanisms involveinhibition of antigen presentation downregulation of surfaceMHC expression elaboration of transforming growth factor-beta (TGF-120573) from infected cells and secretion of viral IL-10 homologue (vIL-10) [25] The special features acquired bythe glioma cells might increase the tendency of HCMV to

6 BioMed Research International

enter these cells and this entry might confer protection forthe HCMV-infected cells from immune-surveillance

The nonsignificant differences in expression of the threeHCMV proteins between GBM and anaplastic astrocytomaare an expected result Cancer cells are exposed to almost thesame conditions that facilitate the entrance of the virus intothese cells Of course not all tumor cells are simultaneouslyinfected with the virus because the viral life cycle is markedlyaltered when it infects the cells However in one study theauthors reported 100 GBM positive cells for viral IE1-72protein compared to 75 of cells being positive for the sameprotein in anaplastic astrocytoma [19]

The primary HCMV antigens which induce cellularimmune response to the virus include immediate earlyprotein (IE) virion envelope glycoprotein B (late antigen55 kDa) and internal matrix protein (pp65) [26] Accumulat-ing evidence indicates that HCMV IE1 products can interactwith TP53 and Rb proteins (tumor suppressor proteins) andthereby it can induce cell cycle progression and block apop-tosis Indeed IE1 expression in a glioma cell line significantlyreduces P53 expression [27] Another study indicated thatthis protein interacts with the P13KAKT pathway one of themost important signaling pathways in glioma and sustainsactivation of AKT signaling [28] The viral protein pp65(also called pUL83) is abundantly synthesized during lyticinfection [29] It is an important target of CD8+ T-cells inthe course of immune response to HCMV infection [30]Among the many functions it has this protein is an immune-modulator and has been shown to block interferon activity[31] anddownregulate the interferon-response gene primarilythrough modulation of interferon response factor-3 IRF-3[32]

Amajor late antigen of HCMV is glycoprotein B (gB) It isa type 1 trans-membrane protein that exists as a dimer on thesurface of the viral envelope [33] This antigen is implicatedin the virus entry cell-to-cell spread [34] and fusion of theinfected cells [35]There is no evidence from available reportsthat this antigen has any role in cell transformation andthe positive result for the presence of the antigen inside thetumor cells but not the nontumor ones may be related to thefeatures of tumor cells that facilitate the entrance of HCMVcompared to the normal cells One of the most importantfeatures of IE1-72 protein is early production of this protein inthe nucleus of the infected cell It is because of this propertythat IE1-72 has stronger expression than pp65 which in turnhas stronger expression than the late antigen The last gene isknown to encode for an envelope glycoprotein and in manyinstances it scatters on the cell membrane of the infected celland cannot be easily detected These results are in part inaccordance with a previous study by Mitchell et al [17] whostated that pp65 reactivity is less ubiquitous than IE1-72 intumor cells

A significant correlation between the level of pp65 and gBantigens in the MRC-5 cell line experimentally infected withHCMV has been reported by Jun et al [36] It is not unusualto find such correlation among viral proteins as the infectionoccurs in defined sequences chronologically accompanied byexpression of immediate early early and late viral antigensHence HCMV proteins are expected to follow the pattern of

elevation and decline shown by immediate early proteins butto a lesser degree

Further support for the role ofHCMV in the developmentof this tumor results from the use of anti-HCMVdrugs Somestudies have shown that valganciclovir significantly reducedthe growth of HCMV-positive glioma tumors [37] RecentlyBaryawno et al [38] demonstrated an increase in overallsurvival of glioblastoma patients receiving valganciclovircompared with those patients who did not receive suchtreatment

The results from the present study show the high percent-age of HCMV virus expression in different histopathologicalgrades of glioma patients which clearly suggest its role ingliomapathogenesisThedetection of low-level latentHCMVinfections might suggest a role in glioma pathogenesisHowever amore detailed study ofHCMV infection in gliomapatients is required for a definitive conclusion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Ahmad Adnan in the NeuroscienceHospital for help in collection of patient samples and DrQasim Sharhan for technical assistance and scientific com-ments

References

[1] G J Tortora and B Derrickson Principles of Anatomy andPhysiology John Wiley amp Sons New York NY USA 14thedition 2014

[2] A P Kyritsis M L Bondy J S Rao and C Sioka ldquoInheritedpredisposition to gliomardquo Neuro-Oncology vol 12 no 1 pp104ndash113 2010

[3] G Prasad and D A Haas-Kogan ldquoRadiation-induced gliomasrdquoExpert Review of Neurotherapeutics vol 9 no 10 pp 1511ndash15172009

[4] Iraqi Cancer Registry 2005[5] H Ohgaki and P Kleihues ldquoEpidemiology and etiology of

gliomasrdquo Acta Neuropathologica vol 109 no 1 pp 93ndash1082005

[6] L Soroceanu and C S Cobbs ldquoIs HCMV a tumor promoterrdquoVirus Research vol 157 no 2 pp 193ndash203 2011

[7] L S YoungViruses andHuman Cancer Bios Scientific OxfordUK 1998

[8] C Hawkins and S Croul ldquoViruses and human brain tumorscytomegalovirus enters the frayrdquoThe Journal of Clinical Investi-gation vol 121 no 10 pp 3831ndash3833 2011

[9] C Taher J de Boniface A A Mohammad et al ldquoHighprevalence of human cytomegalovirus proteins and nucleicacids in primary breast cancer and metastatic sentinel lymphnodesrdquo PLoS ONE vol 8 no 2 Article ID e56795 2013

[10] F Tafvizi and Z T Fard ldquoDetection of human cytomegalovirusin patients with colorectal cancer by nested-PCRrdquo Asian PacificJournal of Cancer Prevention vol 15 no 3 pp 1453ndash1457 2014

BioMed Research International 7

[11] L Geder R Lausch F OrsquoNeill and F Rapp ldquoOncogenictransformation of human embryo lung cells by humancytomegalovirusrdquo Science vol 192 no 4244 pp 1134ndash1137 1976

[12] J Cinatl Jr J-U Vogel H Rabenau B Kornhuber and H WDoerr ldquoModulatory effects of human cytomegalovirus infectionon malignant properties of cancer cellsrdquo Intervirology vol 39no 4 pp 259ndash269 1996

[13] E S Amirian DMarquez-DoM L Bondy andM E ScheurerldquoAntihistamine use and immunoglobulin E levels in glioma riskand prognosisrdquoCancer Epidemiology vol 37 no 6 pp 908ndash9122013

[14] S Sjostrom U Hjalmars P Juto et al ldquoHuman immunoglobu-linG levels of viruses and associated glioma riskrdquoCancer Causesand Control vol 22 no 9 pp 1259ndash1266 2011

[15] C S Cobbs LHarkinsM Samanta et al ldquoHuman cytomegalo-virus infection and expression in human malignant gliomardquoCancer Research vol 62 no 12 pp 3347ndash3350 2002

[16] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas reveals apotential therapeutic targetrdquo The Journal of Clinical Investiga-tion vol 121 no 10 pp 4043ndash4055 2011

[17] D A Mitchell W Xie R Schmittling et al ldquoSensitive detectionof human cytomegalovirus in tumors and peripheral blood ofpatients diagnosed with glioblastomardquo Neuro-Oncology vol 10no 1 pp 10ndash18 2008

[18] A Rahbar G Stragliotto A Orrego et al ldquoLow levels of humancytomegalovirus infection in glioblastoma multiforme asso-ciates with patient survival a case-control studyrdquoHerpesviridaevol 3 pp 3ndash10 2012

[19] M E Scheurer M L Bondy K D Aldape T Albrecht andR El-Zein ldquoDetection of human cytomegalovirus in differenthistological types of gliomasrdquo Acta Neuropathologica vol 116no 1 pp 79ndash86 2008

[20] J Sabatier E Uro-Coste I Pommepuy et al ldquoDetection ofhuman cytomegalovirus genome and gene products in centralnervous system tumoursrdquo British Journal of Cancer vol 92 no4 pp 747ndash750 2005

[21] S Poltermann B Schlehofer K Steindorf P Schnitzler KGeletneky and J R Schlehofer ldquoLack of association of her-pesviruses with brain tumorsrdquo Journal of NeuroVirology vol 12no 2 pp 90ndash99 2006

[22] S K Lau Y-Y Chen W-G Chen et al ldquoLack of association ofcytomegalovirus with human brain tumorsrdquoModern Pathologyvol 18 no 6 pp 838ndash843 2005

[23] X Wang S-M Huong M L Chiu N Raab-Traub and E-SHuang ldquoEpidermal growth factor receptor is a cellular receptorfor human cytomegalovirusrdquoNature vol 424 no 6947 pp 456ndash461 2003

[24] T F Liu S B Tatter M C Willingham M Yang J J Hu andA E Frankel ldquoGrowth factor receptor expression varies amonghigh-grade gliomas and normal brain epidermal growth factorreceptor has excellent properties for interstitial fusion proteintherapyrdquo Molecular Cancer Therapeutics vol 2 no 8 pp 783ndash787 2003

[25] T Kossmann M C Morganti-Kossmann J M OrensteinW J Britt S M Wahl and P D Smith ldquoCytomegalovirusproduction by infected astrocytes correlates with transforminggrowth factor-120573 releaserdquoThe Journal of Infectious Diseases vol187 no 4 pp 534ndash541 2003

[26] S Saikali T Avril B Collet et al ldquoExpression of nine tumourantigens in a series of human glioblastoma multiforme interest

of EGFRvIII IL-13R1205722 gp100 and TRP-2 for immunotherapyrdquoJournal of Neuro-Oncology vol 81 no 2 pp 139ndash148 2007

[27] C S Cobbs L Soroceanu S Denham W Zhang and M HKraus ldquoModulation of oncogenic phenotype in human gliomacells by cytomegalovirus IE1-mediated mitogenicityrdquo CancerResearch vol 68 no 3 pp 724ndash730 2008

[28] R A Johnson X Wang X-L Ma S-M Huong and E-S Huang ldquoHuman cytomegalovirus up-regulates the phos-phatidylinositol 3-kinase (PI3-K) pathway inhibition of PI3-Kactivity inhibits viral replication and virus-induced signalingrdquoJournal of Virology vol 75 no 13 pp 6022ndash6032 2001

[29] S Schmolke H F Kern P Drescher G Jahn and BPlachter ldquoThe dominant phosphoprotein pp65 (UL83) ofhuman cytomegalovirus is dispensable for growth in cell cul-turerdquo Journal of Virology vol 69 no 10 pp 5959ndash5968 1995

[30] M R Wills A J Carmichael K Mynard et al ldquoThe humancytotoxic T-lymphocyte (CTL) response to cytomegalovirus isdominated by structural protein pp65 frequency specificityand T-cell receptor usage of pp65-specific CTLrdquo Journal ofVirology vol 70 no 11 pp 7569ndash7579 1996

[31] E P Browne B Wing D Coleman and T Shenk ldquoAltered cel-lular mRNA levels in human cytomegalovirus-infected fibrob-lasts viral block to the accumulation of antiviral mRNAsrdquoJournal of Virology vol 75 no 24 pp 12319ndash12330 2001

[32] D A Abate S Watanabe and E S Mocarski ldquoMajor humancytomegalovirus structural protein pp65 (ppUL83) preventsinterferon response factor 3 activation in the interferonresponserdquo Journal of Virology vol 78 no 20 pp 10995ndash110062004

[33] E R Kinzler and T Compton ldquoCharacterization of humancytomegalovirus glycoprotein-induced cell-cell fusionrdquo Journalof Virology vol 79 no 12 pp 7827ndash7837 2005

[34] M K Isaacson and T Compton ldquoHuman cytomegalovirusglycoprotein B is required for virus entry and cell-to-cell spreadbut not for virion attachment assembly or egressrdquo Journal ofVirology vol 83 no 8 pp 3891ndash3903 2009

[35] D Navarro P Paz S Tugizov K Topp J La Vail and L PereiraldquoGlycoprotein B of human cytomegalovirus promotes virionpenetration into cells transmission of infection from cell to celland fusion of infected cellsrdquoVirology vol 197 no 1 pp 143ndash1581993

[36] F Jun L Minhuan M Yadan et al ldquoDevelopment of twopotential diagnostic monoclonal antibodies against humancytomegalovirus glycoprotein Brdquo Brazilian Journal of Medicaland Biological Research vol 45 no 7 pp 583ndash590 2012

[37] G Stragliotto A Rahbar NW Solberg et al ldquoEffects of valgan-ciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma a randomized double-blind hypothesis-generating studyrdquo International Journal of Cancer vol 133 no5 pp 1204ndash1213 2013

[38] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas revealsa potential therapeutic targetrdquo Journal of Clinical Investigationvol 121 no 10 pp 4043ndash4055 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 BioMed Research International

(a) (b)

(c) (d)

(e) (f)

(g)

Figure 1 Immunohistochemical staining of HCMV in glioma (a) Immunohistochemical stain of glioma section with IE1-72 protein (b)Control without secondary antibody (c) Immunohistochemical stains of glioma section with pp65 protein (d) Control without secondaryantibody (e) Immunohistochemical staining of glioma with late antigen antibody (f) Control without secondary antibody (g) Lung knownpositive control stain for HCMV

32 Seropositivity for Anti-HCMV IgG Antibodies From 50glioma patients 42 (84) were positive for anti-HCMV IgGantibodies compared to 2940 (725) among the controlgroup (Table 3) Logistic regression revealed a significant

association between seropositivity for anti-HCMV IgG anti-bodies and glioma (OR = 2779 95 CI = 1049ndash7358 119875 =004) which means that individuals who had a previousinfection of HCMV are 2779-fold more likely to get glioma

BioMed Research International 5

Table 3 The distribution of seropositivity for anti-HCMV IgG antibodies among different age classes in glioma patients and control group

Age class (years)Glioma patients Control

Number Seropositive Seronegative Number Seropositive SeronegativeNumber Number Number Number

le20 11 9 8181 2 1818 9 6 6667 3 333321ndash30 10 8 80 2 20 12 11 9167 1 83331ndash40 9 8 8889 1 1111 10 7 70 3 3041ndash50 11 9 8181 2 1818 7 4 5714 3 4286gt50 9 8 8889 1 1111 2 1 50 1 50

Figure 2 Detection of HCMV DNA in malignant glioma tissue(nested PCR products) visualized with UV light M 1000 bp DNAmarker Lanes 1ndash10 viral DNA amplified with nested PCR Lane 11negative result The size of the PCR product is 144 bp

compared with individuals with no such infection Overallthere were no significant differences in the distributionof these antibodies among different age classes of gliomapatients (Table 3) However age classes of gt50 and 31ndash40years had the highest percentage (8889) while the lowestpercentage (80) was among the youngest age class (21ndash30years) Among the control group 21ndash30-year-old individualshad a higher seropositive percentage (9167) than otherclasses with significant differences (119875 = 0037) Comparisonof the age classes between glioma patients and controlsrevealed significant differences in the seropositivity whichwas higher in glioma patients in all classes except the 21ndash30years class which was higher in the control group (9167)than that of glioma patients (80)

4 Discussion

Studying viral proteins in malignant glioma patients mayprovide more details about the mechanism that these virusesuse to modulate the cancer To the best of our knowledge thisis the first study from Iraqi patients which showed HCMVprotein and DNA in glioma specimens The results of thecurrent study indicate that 7222 and 9167 of GBM casesand 6428 to 8571 of anaplastic astrocytoma are positivefor one or more HCMV proteins and nucleic acids Theseresults corroborate previous studies (16 17 and 19) whereHCMV had an obvious association with the occurrence ofgliomas despite disparities in the percentage of tumor cellsthat gave positive results for the viral products

In immunocytochemical studies disparities in results areunavoidable because staining patterns vary by histology celltype and procedures followed during processing and stainingof paraffin-embedded tissues In this study we followedall crucial steps during detection of HCMV infection inbrain tumor tissue We used brain tumor tissue of 6 120583mbecause thicker sections do not allow proper staining andthinner sections allow excessive nonspecific binding Thiswas corroborated by the study from Scheurer et al [19]Another crucial factor in the detection of HCMV in humangliomas is the dilution of antibodies used (1 100) Our resultsare similar to other studies done by Sabatier et al [20] andPoltermann et al [21] using a lower concentration of 1 200The deparaffinization of slides may also play a role In thisstudy we deparaffinized the slides by heating them in themicrowave and treating with xylene for 20 minutes Resultsshow similarities with the study by Scheurer et al [19] butdisagree with another study [22] that used either microwaveor steam heating for much longer periods which may resultin damage to IE1-72 epitopes

The high prevalence of viral DNA and proteins in bothGBM and astrocytoma and absence of these products inbenign tumors and surrounding nontumor tissues indicatea vital role for this virus in carcinogenesis However tumorcells in general have a higher propensity to display false-positive immune-reactivity due to either nonspecific bindingtomAbs or higher levels of endogenous peroxidases that reactwith the detection substrate [17] Furthermore HCMV wasfound to use epidermal growth factor receptors (EGFR) as acellular binding and incorporation site for its entry into thecell [23] It has been shown that GMB cells uniformly ampli-fied EGFR expression while normal brain cells are largelynegative [24] Particularly with HCMV which has lifetimelatency occurrence of the cancer and subsequent treatmentinduced immunosuppression cause reactivation of the latentvirus and increase the chance for infection of the tumor cellas well as many other normal cells Presence of the viralproducts inside the tumor cells is significant and importantin the downregulation of immunogenicity of infected cellsthrough multiple mechanisms some of which are exhibitedby the tumor cells themselves These mechanisms involveinhibition of antigen presentation downregulation of surfaceMHC expression elaboration of transforming growth factor-beta (TGF-120573) from infected cells and secretion of viral IL-10 homologue (vIL-10) [25] The special features acquired bythe glioma cells might increase the tendency of HCMV to

6 BioMed Research International

enter these cells and this entry might confer protection forthe HCMV-infected cells from immune-surveillance

The nonsignificant differences in expression of the threeHCMV proteins between GBM and anaplastic astrocytomaare an expected result Cancer cells are exposed to almost thesame conditions that facilitate the entrance of the virus intothese cells Of course not all tumor cells are simultaneouslyinfected with the virus because the viral life cycle is markedlyaltered when it infects the cells However in one study theauthors reported 100 GBM positive cells for viral IE1-72protein compared to 75 of cells being positive for the sameprotein in anaplastic astrocytoma [19]

The primary HCMV antigens which induce cellularimmune response to the virus include immediate earlyprotein (IE) virion envelope glycoprotein B (late antigen55 kDa) and internal matrix protein (pp65) [26] Accumulat-ing evidence indicates that HCMV IE1 products can interactwith TP53 and Rb proteins (tumor suppressor proteins) andthereby it can induce cell cycle progression and block apop-tosis Indeed IE1 expression in a glioma cell line significantlyreduces P53 expression [27] Another study indicated thatthis protein interacts with the P13KAKT pathway one of themost important signaling pathways in glioma and sustainsactivation of AKT signaling [28] The viral protein pp65(also called pUL83) is abundantly synthesized during lyticinfection [29] It is an important target of CD8+ T-cells inthe course of immune response to HCMV infection [30]Among the many functions it has this protein is an immune-modulator and has been shown to block interferon activity[31] anddownregulate the interferon-response gene primarilythrough modulation of interferon response factor-3 IRF-3[32]

Amajor late antigen of HCMV is glycoprotein B (gB) It isa type 1 trans-membrane protein that exists as a dimer on thesurface of the viral envelope [33] This antigen is implicatedin the virus entry cell-to-cell spread [34] and fusion of theinfected cells [35]There is no evidence from available reportsthat this antigen has any role in cell transformation andthe positive result for the presence of the antigen inside thetumor cells but not the nontumor ones may be related to thefeatures of tumor cells that facilitate the entrance of HCMVcompared to the normal cells One of the most importantfeatures of IE1-72 protein is early production of this protein inthe nucleus of the infected cell It is because of this propertythat IE1-72 has stronger expression than pp65 which in turnhas stronger expression than the late antigen The last gene isknown to encode for an envelope glycoprotein and in manyinstances it scatters on the cell membrane of the infected celland cannot be easily detected These results are in part inaccordance with a previous study by Mitchell et al [17] whostated that pp65 reactivity is less ubiquitous than IE1-72 intumor cells

A significant correlation between the level of pp65 and gBantigens in the MRC-5 cell line experimentally infected withHCMV has been reported by Jun et al [36] It is not unusualto find such correlation among viral proteins as the infectionoccurs in defined sequences chronologically accompanied byexpression of immediate early early and late viral antigensHence HCMV proteins are expected to follow the pattern of

elevation and decline shown by immediate early proteins butto a lesser degree

Further support for the role ofHCMV in the developmentof this tumor results from the use of anti-HCMVdrugs Somestudies have shown that valganciclovir significantly reducedthe growth of HCMV-positive glioma tumors [37] RecentlyBaryawno et al [38] demonstrated an increase in overallsurvival of glioblastoma patients receiving valganciclovircompared with those patients who did not receive suchtreatment

The results from the present study show the high percent-age of HCMV virus expression in different histopathologicalgrades of glioma patients which clearly suggest its role ingliomapathogenesisThedetection of low-level latentHCMVinfections might suggest a role in glioma pathogenesisHowever amore detailed study ofHCMV infection in gliomapatients is required for a definitive conclusion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Ahmad Adnan in the NeuroscienceHospital for help in collection of patient samples and DrQasim Sharhan for technical assistance and scientific com-ments

References

[1] G J Tortora and B Derrickson Principles of Anatomy andPhysiology John Wiley amp Sons New York NY USA 14thedition 2014

[2] A P Kyritsis M L Bondy J S Rao and C Sioka ldquoInheritedpredisposition to gliomardquo Neuro-Oncology vol 12 no 1 pp104ndash113 2010

[3] G Prasad and D A Haas-Kogan ldquoRadiation-induced gliomasrdquoExpert Review of Neurotherapeutics vol 9 no 10 pp 1511ndash15172009

[4] Iraqi Cancer Registry 2005[5] H Ohgaki and P Kleihues ldquoEpidemiology and etiology of

gliomasrdquo Acta Neuropathologica vol 109 no 1 pp 93ndash1082005

[6] L Soroceanu and C S Cobbs ldquoIs HCMV a tumor promoterrdquoVirus Research vol 157 no 2 pp 193ndash203 2011

[7] L S YoungViruses andHuman Cancer Bios Scientific OxfordUK 1998

[8] C Hawkins and S Croul ldquoViruses and human brain tumorscytomegalovirus enters the frayrdquoThe Journal of Clinical Investi-gation vol 121 no 10 pp 3831ndash3833 2011

[9] C Taher J de Boniface A A Mohammad et al ldquoHighprevalence of human cytomegalovirus proteins and nucleicacids in primary breast cancer and metastatic sentinel lymphnodesrdquo PLoS ONE vol 8 no 2 Article ID e56795 2013

[10] F Tafvizi and Z T Fard ldquoDetection of human cytomegalovirusin patients with colorectal cancer by nested-PCRrdquo Asian PacificJournal of Cancer Prevention vol 15 no 3 pp 1453ndash1457 2014

BioMed Research International 7

[11] L Geder R Lausch F OrsquoNeill and F Rapp ldquoOncogenictransformation of human embryo lung cells by humancytomegalovirusrdquo Science vol 192 no 4244 pp 1134ndash1137 1976

[12] J Cinatl Jr J-U Vogel H Rabenau B Kornhuber and H WDoerr ldquoModulatory effects of human cytomegalovirus infectionon malignant properties of cancer cellsrdquo Intervirology vol 39no 4 pp 259ndash269 1996

[13] E S Amirian DMarquez-DoM L Bondy andM E ScheurerldquoAntihistamine use and immunoglobulin E levels in glioma riskand prognosisrdquoCancer Epidemiology vol 37 no 6 pp 908ndash9122013

[14] S Sjostrom U Hjalmars P Juto et al ldquoHuman immunoglobu-linG levels of viruses and associated glioma riskrdquoCancer Causesand Control vol 22 no 9 pp 1259ndash1266 2011

[15] C S Cobbs LHarkinsM Samanta et al ldquoHuman cytomegalo-virus infection and expression in human malignant gliomardquoCancer Research vol 62 no 12 pp 3347ndash3350 2002

[16] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas reveals apotential therapeutic targetrdquo The Journal of Clinical Investiga-tion vol 121 no 10 pp 4043ndash4055 2011

[17] D A Mitchell W Xie R Schmittling et al ldquoSensitive detectionof human cytomegalovirus in tumors and peripheral blood ofpatients diagnosed with glioblastomardquo Neuro-Oncology vol 10no 1 pp 10ndash18 2008

[18] A Rahbar G Stragliotto A Orrego et al ldquoLow levels of humancytomegalovirus infection in glioblastoma multiforme asso-ciates with patient survival a case-control studyrdquoHerpesviridaevol 3 pp 3ndash10 2012

[19] M E Scheurer M L Bondy K D Aldape T Albrecht andR El-Zein ldquoDetection of human cytomegalovirus in differenthistological types of gliomasrdquo Acta Neuropathologica vol 116no 1 pp 79ndash86 2008

[20] J Sabatier E Uro-Coste I Pommepuy et al ldquoDetection ofhuman cytomegalovirus genome and gene products in centralnervous system tumoursrdquo British Journal of Cancer vol 92 no4 pp 747ndash750 2005

[21] S Poltermann B Schlehofer K Steindorf P Schnitzler KGeletneky and J R Schlehofer ldquoLack of association of her-pesviruses with brain tumorsrdquo Journal of NeuroVirology vol 12no 2 pp 90ndash99 2006

[22] S K Lau Y-Y Chen W-G Chen et al ldquoLack of association ofcytomegalovirus with human brain tumorsrdquoModern Pathologyvol 18 no 6 pp 838ndash843 2005

[23] X Wang S-M Huong M L Chiu N Raab-Traub and E-SHuang ldquoEpidermal growth factor receptor is a cellular receptorfor human cytomegalovirusrdquoNature vol 424 no 6947 pp 456ndash461 2003

[24] T F Liu S B Tatter M C Willingham M Yang J J Hu andA E Frankel ldquoGrowth factor receptor expression varies amonghigh-grade gliomas and normal brain epidermal growth factorreceptor has excellent properties for interstitial fusion proteintherapyrdquo Molecular Cancer Therapeutics vol 2 no 8 pp 783ndash787 2003

[25] T Kossmann M C Morganti-Kossmann J M OrensteinW J Britt S M Wahl and P D Smith ldquoCytomegalovirusproduction by infected astrocytes correlates with transforminggrowth factor-120573 releaserdquoThe Journal of Infectious Diseases vol187 no 4 pp 534ndash541 2003

[26] S Saikali T Avril B Collet et al ldquoExpression of nine tumourantigens in a series of human glioblastoma multiforme interest

of EGFRvIII IL-13R1205722 gp100 and TRP-2 for immunotherapyrdquoJournal of Neuro-Oncology vol 81 no 2 pp 139ndash148 2007

[27] C S Cobbs L Soroceanu S Denham W Zhang and M HKraus ldquoModulation of oncogenic phenotype in human gliomacells by cytomegalovirus IE1-mediated mitogenicityrdquo CancerResearch vol 68 no 3 pp 724ndash730 2008

[28] R A Johnson X Wang X-L Ma S-M Huong and E-S Huang ldquoHuman cytomegalovirus up-regulates the phos-phatidylinositol 3-kinase (PI3-K) pathway inhibition of PI3-Kactivity inhibits viral replication and virus-induced signalingrdquoJournal of Virology vol 75 no 13 pp 6022ndash6032 2001

[29] S Schmolke H F Kern P Drescher G Jahn and BPlachter ldquoThe dominant phosphoprotein pp65 (UL83) ofhuman cytomegalovirus is dispensable for growth in cell cul-turerdquo Journal of Virology vol 69 no 10 pp 5959ndash5968 1995

[30] M R Wills A J Carmichael K Mynard et al ldquoThe humancytotoxic T-lymphocyte (CTL) response to cytomegalovirus isdominated by structural protein pp65 frequency specificityand T-cell receptor usage of pp65-specific CTLrdquo Journal ofVirology vol 70 no 11 pp 7569ndash7579 1996

[31] E P Browne B Wing D Coleman and T Shenk ldquoAltered cel-lular mRNA levels in human cytomegalovirus-infected fibrob-lasts viral block to the accumulation of antiviral mRNAsrdquoJournal of Virology vol 75 no 24 pp 12319ndash12330 2001

[32] D A Abate S Watanabe and E S Mocarski ldquoMajor humancytomegalovirus structural protein pp65 (ppUL83) preventsinterferon response factor 3 activation in the interferonresponserdquo Journal of Virology vol 78 no 20 pp 10995ndash110062004

[33] E R Kinzler and T Compton ldquoCharacterization of humancytomegalovirus glycoprotein-induced cell-cell fusionrdquo Journalof Virology vol 79 no 12 pp 7827ndash7837 2005

[34] M K Isaacson and T Compton ldquoHuman cytomegalovirusglycoprotein B is required for virus entry and cell-to-cell spreadbut not for virion attachment assembly or egressrdquo Journal ofVirology vol 83 no 8 pp 3891ndash3903 2009

[35] D Navarro P Paz S Tugizov K Topp J La Vail and L PereiraldquoGlycoprotein B of human cytomegalovirus promotes virionpenetration into cells transmission of infection from cell to celland fusion of infected cellsrdquoVirology vol 197 no 1 pp 143ndash1581993

[36] F Jun L Minhuan M Yadan et al ldquoDevelopment of twopotential diagnostic monoclonal antibodies against humancytomegalovirus glycoprotein Brdquo Brazilian Journal of Medicaland Biological Research vol 45 no 7 pp 583ndash590 2012

[37] G Stragliotto A Rahbar NW Solberg et al ldquoEffects of valgan-ciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma a randomized double-blind hypothesis-generating studyrdquo International Journal of Cancer vol 133 no5 pp 1204ndash1213 2013

[38] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas revealsa potential therapeutic targetrdquo Journal of Clinical Investigationvol 121 no 10 pp 4043ndash4055 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 5

Table 3 The distribution of seropositivity for anti-HCMV IgG antibodies among different age classes in glioma patients and control group

Age class (years)Glioma patients Control

Number Seropositive Seronegative Number Seropositive SeronegativeNumber Number Number Number

le20 11 9 8181 2 1818 9 6 6667 3 333321ndash30 10 8 80 2 20 12 11 9167 1 83331ndash40 9 8 8889 1 1111 10 7 70 3 3041ndash50 11 9 8181 2 1818 7 4 5714 3 4286gt50 9 8 8889 1 1111 2 1 50 1 50

Figure 2 Detection of HCMV DNA in malignant glioma tissue(nested PCR products) visualized with UV light M 1000 bp DNAmarker Lanes 1ndash10 viral DNA amplified with nested PCR Lane 11negative result The size of the PCR product is 144 bp

compared with individuals with no such infection Overallthere were no significant differences in the distributionof these antibodies among different age classes of gliomapatients (Table 3) However age classes of gt50 and 31ndash40years had the highest percentage (8889) while the lowestpercentage (80) was among the youngest age class (21ndash30years) Among the control group 21ndash30-year-old individualshad a higher seropositive percentage (9167) than otherclasses with significant differences (119875 = 0037) Comparisonof the age classes between glioma patients and controlsrevealed significant differences in the seropositivity whichwas higher in glioma patients in all classes except the 21ndash30years class which was higher in the control group (9167)than that of glioma patients (80)

4 Discussion

Studying viral proteins in malignant glioma patients mayprovide more details about the mechanism that these virusesuse to modulate the cancer To the best of our knowledge thisis the first study from Iraqi patients which showed HCMVprotein and DNA in glioma specimens The results of thecurrent study indicate that 7222 and 9167 of GBM casesand 6428 to 8571 of anaplastic astrocytoma are positivefor one or more HCMV proteins and nucleic acids Theseresults corroborate previous studies (16 17 and 19) whereHCMV had an obvious association with the occurrence ofgliomas despite disparities in the percentage of tumor cellsthat gave positive results for the viral products

In immunocytochemical studies disparities in results areunavoidable because staining patterns vary by histology celltype and procedures followed during processing and stainingof paraffin-embedded tissues In this study we followedall crucial steps during detection of HCMV infection inbrain tumor tissue We used brain tumor tissue of 6 120583mbecause thicker sections do not allow proper staining andthinner sections allow excessive nonspecific binding Thiswas corroborated by the study from Scheurer et al [19]Another crucial factor in the detection of HCMV in humangliomas is the dilution of antibodies used (1 100) Our resultsare similar to other studies done by Sabatier et al [20] andPoltermann et al [21] using a lower concentration of 1 200The deparaffinization of slides may also play a role In thisstudy we deparaffinized the slides by heating them in themicrowave and treating with xylene for 20 minutes Resultsshow similarities with the study by Scheurer et al [19] butdisagree with another study [22] that used either microwaveor steam heating for much longer periods which may resultin damage to IE1-72 epitopes

The high prevalence of viral DNA and proteins in bothGBM and astrocytoma and absence of these products inbenign tumors and surrounding nontumor tissues indicatea vital role for this virus in carcinogenesis However tumorcells in general have a higher propensity to display false-positive immune-reactivity due to either nonspecific bindingtomAbs or higher levels of endogenous peroxidases that reactwith the detection substrate [17] Furthermore HCMV wasfound to use epidermal growth factor receptors (EGFR) as acellular binding and incorporation site for its entry into thecell [23] It has been shown that GMB cells uniformly ampli-fied EGFR expression while normal brain cells are largelynegative [24] Particularly with HCMV which has lifetimelatency occurrence of the cancer and subsequent treatmentinduced immunosuppression cause reactivation of the latentvirus and increase the chance for infection of the tumor cellas well as many other normal cells Presence of the viralproducts inside the tumor cells is significant and importantin the downregulation of immunogenicity of infected cellsthrough multiple mechanisms some of which are exhibitedby the tumor cells themselves These mechanisms involveinhibition of antigen presentation downregulation of surfaceMHC expression elaboration of transforming growth factor-beta (TGF-120573) from infected cells and secretion of viral IL-10 homologue (vIL-10) [25] The special features acquired bythe glioma cells might increase the tendency of HCMV to

6 BioMed Research International

enter these cells and this entry might confer protection forthe HCMV-infected cells from immune-surveillance

The nonsignificant differences in expression of the threeHCMV proteins between GBM and anaplastic astrocytomaare an expected result Cancer cells are exposed to almost thesame conditions that facilitate the entrance of the virus intothese cells Of course not all tumor cells are simultaneouslyinfected with the virus because the viral life cycle is markedlyaltered when it infects the cells However in one study theauthors reported 100 GBM positive cells for viral IE1-72protein compared to 75 of cells being positive for the sameprotein in anaplastic astrocytoma [19]

The primary HCMV antigens which induce cellularimmune response to the virus include immediate earlyprotein (IE) virion envelope glycoprotein B (late antigen55 kDa) and internal matrix protein (pp65) [26] Accumulat-ing evidence indicates that HCMV IE1 products can interactwith TP53 and Rb proteins (tumor suppressor proteins) andthereby it can induce cell cycle progression and block apop-tosis Indeed IE1 expression in a glioma cell line significantlyreduces P53 expression [27] Another study indicated thatthis protein interacts with the P13KAKT pathway one of themost important signaling pathways in glioma and sustainsactivation of AKT signaling [28] The viral protein pp65(also called pUL83) is abundantly synthesized during lyticinfection [29] It is an important target of CD8+ T-cells inthe course of immune response to HCMV infection [30]Among the many functions it has this protein is an immune-modulator and has been shown to block interferon activity[31] anddownregulate the interferon-response gene primarilythrough modulation of interferon response factor-3 IRF-3[32]

Amajor late antigen of HCMV is glycoprotein B (gB) It isa type 1 trans-membrane protein that exists as a dimer on thesurface of the viral envelope [33] This antigen is implicatedin the virus entry cell-to-cell spread [34] and fusion of theinfected cells [35]There is no evidence from available reportsthat this antigen has any role in cell transformation andthe positive result for the presence of the antigen inside thetumor cells but not the nontumor ones may be related to thefeatures of tumor cells that facilitate the entrance of HCMVcompared to the normal cells One of the most importantfeatures of IE1-72 protein is early production of this protein inthe nucleus of the infected cell It is because of this propertythat IE1-72 has stronger expression than pp65 which in turnhas stronger expression than the late antigen The last gene isknown to encode for an envelope glycoprotein and in manyinstances it scatters on the cell membrane of the infected celland cannot be easily detected These results are in part inaccordance with a previous study by Mitchell et al [17] whostated that pp65 reactivity is less ubiquitous than IE1-72 intumor cells

A significant correlation between the level of pp65 and gBantigens in the MRC-5 cell line experimentally infected withHCMV has been reported by Jun et al [36] It is not unusualto find such correlation among viral proteins as the infectionoccurs in defined sequences chronologically accompanied byexpression of immediate early early and late viral antigensHence HCMV proteins are expected to follow the pattern of

elevation and decline shown by immediate early proteins butto a lesser degree

Further support for the role ofHCMV in the developmentof this tumor results from the use of anti-HCMVdrugs Somestudies have shown that valganciclovir significantly reducedthe growth of HCMV-positive glioma tumors [37] RecentlyBaryawno et al [38] demonstrated an increase in overallsurvival of glioblastoma patients receiving valganciclovircompared with those patients who did not receive suchtreatment

The results from the present study show the high percent-age of HCMV virus expression in different histopathologicalgrades of glioma patients which clearly suggest its role ingliomapathogenesisThedetection of low-level latentHCMVinfections might suggest a role in glioma pathogenesisHowever amore detailed study ofHCMV infection in gliomapatients is required for a definitive conclusion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Ahmad Adnan in the NeuroscienceHospital for help in collection of patient samples and DrQasim Sharhan for technical assistance and scientific com-ments

References

[1] G J Tortora and B Derrickson Principles of Anatomy andPhysiology John Wiley amp Sons New York NY USA 14thedition 2014

[2] A P Kyritsis M L Bondy J S Rao and C Sioka ldquoInheritedpredisposition to gliomardquo Neuro-Oncology vol 12 no 1 pp104ndash113 2010

[3] G Prasad and D A Haas-Kogan ldquoRadiation-induced gliomasrdquoExpert Review of Neurotherapeutics vol 9 no 10 pp 1511ndash15172009

[4] Iraqi Cancer Registry 2005[5] H Ohgaki and P Kleihues ldquoEpidemiology and etiology of

gliomasrdquo Acta Neuropathologica vol 109 no 1 pp 93ndash1082005

[6] L Soroceanu and C S Cobbs ldquoIs HCMV a tumor promoterrdquoVirus Research vol 157 no 2 pp 193ndash203 2011

[7] L S YoungViruses andHuman Cancer Bios Scientific OxfordUK 1998

[8] C Hawkins and S Croul ldquoViruses and human brain tumorscytomegalovirus enters the frayrdquoThe Journal of Clinical Investi-gation vol 121 no 10 pp 3831ndash3833 2011

[9] C Taher J de Boniface A A Mohammad et al ldquoHighprevalence of human cytomegalovirus proteins and nucleicacids in primary breast cancer and metastatic sentinel lymphnodesrdquo PLoS ONE vol 8 no 2 Article ID e56795 2013

[10] F Tafvizi and Z T Fard ldquoDetection of human cytomegalovirusin patients with colorectal cancer by nested-PCRrdquo Asian PacificJournal of Cancer Prevention vol 15 no 3 pp 1453ndash1457 2014

BioMed Research International 7

[11] L Geder R Lausch F OrsquoNeill and F Rapp ldquoOncogenictransformation of human embryo lung cells by humancytomegalovirusrdquo Science vol 192 no 4244 pp 1134ndash1137 1976

[12] J Cinatl Jr J-U Vogel H Rabenau B Kornhuber and H WDoerr ldquoModulatory effects of human cytomegalovirus infectionon malignant properties of cancer cellsrdquo Intervirology vol 39no 4 pp 259ndash269 1996

[13] E S Amirian DMarquez-DoM L Bondy andM E ScheurerldquoAntihistamine use and immunoglobulin E levels in glioma riskand prognosisrdquoCancer Epidemiology vol 37 no 6 pp 908ndash9122013

[14] S Sjostrom U Hjalmars P Juto et al ldquoHuman immunoglobu-linG levels of viruses and associated glioma riskrdquoCancer Causesand Control vol 22 no 9 pp 1259ndash1266 2011

[15] C S Cobbs LHarkinsM Samanta et al ldquoHuman cytomegalo-virus infection and expression in human malignant gliomardquoCancer Research vol 62 no 12 pp 3347ndash3350 2002

[16] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas reveals apotential therapeutic targetrdquo The Journal of Clinical Investiga-tion vol 121 no 10 pp 4043ndash4055 2011

[17] D A Mitchell W Xie R Schmittling et al ldquoSensitive detectionof human cytomegalovirus in tumors and peripheral blood ofpatients diagnosed with glioblastomardquo Neuro-Oncology vol 10no 1 pp 10ndash18 2008

[18] A Rahbar G Stragliotto A Orrego et al ldquoLow levels of humancytomegalovirus infection in glioblastoma multiforme asso-ciates with patient survival a case-control studyrdquoHerpesviridaevol 3 pp 3ndash10 2012

[19] M E Scheurer M L Bondy K D Aldape T Albrecht andR El-Zein ldquoDetection of human cytomegalovirus in differenthistological types of gliomasrdquo Acta Neuropathologica vol 116no 1 pp 79ndash86 2008

[20] J Sabatier E Uro-Coste I Pommepuy et al ldquoDetection ofhuman cytomegalovirus genome and gene products in centralnervous system tumoursrdquo British Journal of Cancer vol 92 no4 pp 747ndash750 2005

[21] S Poltermann B Schlehofer K Steindorf P Schnitzler KGeletneky and J R Schlehofer ldquoLack of association of her-pesviruses with brain tumorsrdquo Journal of NeuroVirology vol 12no 2 pp 90ndash99 2006

[22] S K Lau Y-Y Chen W-G Chen et al ldquoLack of association ofcytomegalovirus with human brain tumorsrdquoModern Pathologyvol 18 no 6 pp 838ndash843 2005

[23] X Wang S-M Huong M L Chiu N Raab-Traub and E-SHuang ldquoEpidermal growth factor receptor is a cellular receptorfor human cytomegalovirusrdquoNature vol 424 no 6947 pp 456ndash461 2003

[24] T F Liu S B Tatter M C Willingham M Yang J J Hu andA E Frankel ldquoGrowth factor receptor expression varies amonghigh-grade gliomas and normal brain epidermal growth factorreceptor has excellent properties for interstitial fusion proteintherapyrdquo Molecular Cancer Therapeutics vol 2 no 8 pp 783ndash787 2003

[25] T Kossmann M C Morganti-Kossmann J M OrensteinW J Britt S M Wahl and P D Smith ldquoCytomegalovirusproduction by infected astrocytes correlates with transforminggrowth factor-120573 releaserdquoThe Journal of Infectious Diseases vol187 no 4 pp 534ndash541 2003

[26] S Saikali T Avril B Collet et al ldquoExpression of nine tumourantigens in a series of human glioblastoma multiforme interest

of EGFRvIII IL-13R1205722 gp100 and TRP-2 for immunotherapyrdquoJournal of Neuro-Oncology vol 81 no 2 pp 139ndash148 2007

[27] C S Cobbs L Soroceanu S Denham W Zhang and M HKraus ldquoModulation of oncogenic phenotype in human gliomacells by cytomegalovirus IE1-mediated mitogenicityrdquo CancerResearch vol 68 no 3 pp 724ndash730 2008

[28] R A Johnson X Wang X-L Ma S-M Huong and E-S Huang ldquoHuman cytomegalovirus up-regulates the phos-phatidylinositol 3-kinase (PI3-K) pathway inhibition of PI3-Kactivity inhibits viral replication and virus-induced signalingrdquoJournal of Virology vol 75 no 13 pp 6022ndash6032 2001

[29] S Schmolke H F Kern P Drescher G Jahn and BPlachter ldquoThe dominant phosphoprotein pp65 (UL83) ofhuman cytomegalovirus is dispensable for growth in cell cul-turerdquo Journal of Virology vol 69 no 10 pp 5959ndash5968 1995

[30] M R Wills A J Carmichael K Mynard et al ldquoThe humancytotoxic T-lymphocyte (CTL) response to cytomegalovirus isdominated by structural protein pp65 frequency specificityand T-cell receptor usage of pp65-specific CTLrdquo Journal ofVirology vol 70 no 11 pp 7569ndash7579 1996

[31] E P Browne B Wing D Coleman and T Shenk ldquoAltered cel-lular mRNA levels in human cytomegalovirus-infected fibrob-lasts viral block to the accumulation of antiviral mRNAsrdquoJournal of Virology vol 75 no 24 pp 12319ndash12330 2001

[32] D A Abate S Watanabe and E S Mocarski ldquoMajor humancytomegalovirus structural protein pp65 (ppUL83) preventsinterferon response factor 3 activation in the interferonresponserdquo Journal of Virology vol 78 no 20 pp 10995ndash110062004

[33] E R Kinzler and T Compton ldquoCharacterization of humancytomegalovirus glycoprotein-induced cell-cell fusionrdquo Journalof Virology vol 79 no 12 pp 7827ndash7837 2005

[34] M K Isaacson and T Compton ldquoHuman cytomegalovirusglycoprotein B is required for virus entry and cell-to-cell spreadbut not for virion attachment assembly or egressrdquo Journal ofVirology vol 83 no 8 pp 3891ndash3903 2009

[35] D Navarro P Paz S Tugizov K Topp J La Vail and L PereiraldquoGlycoprotein B of human cytomegalovirus promotes virionpenetration into cells transmission of infection from cell to celland fusion of infected cellsrdquoVirology vol 197 no 1 pp 143ndash1581993

[36] F Jun L Minhuan M Yadan et al ldquoDevelopment of twopotential diagnostic monoclonal antibodies against humancytomegalovirus glycoprotein Brdquo Brazilian Journal of Medicaland Biological Research vol 45 no 7 pp 583ndash590 2012

[37] G Stragliotto A Rahbar NW Solberg et al ldquoEffects of valgan-ciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma a randomized double-blind hypothesis-generating studyrdquo International Journal of Cancer vol 133 no5 pp 1204ndash1213 2013

[38] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas revealsa potential therapeutic targetrdquo Journal of Clinical Investigationvol 121 no 10 pp 4043ndash4055 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 BioMed Research International

enter these cells and this entry might confer protection forthe HCMV-infected cells from immune-surveillance

The nonsignificant differences in expression of the threeHCMV proteins between GBM and anaplastic astrocytomaare an expected result Cancer cells are exposed to almost thesame conditions that facilitate the entrance of the virus intothese cells Of course not all tumor cells are simultaneouslyinfected with the virus because the viral life cycle is markedlyaltered when it infects the cells However in one study theauthors reported 100 GBM positive cells for viral IE1-72protein compared to 75 of cells being positive for the sameprotein in anaplastic astrocytoma [19]

The primary HCMV antigens which induce cellularimmune response to the virus include immediate earlyprotein (IE) virion envelope glycoprotein B (late antigen55 kDa) and internal matrix protein (pp65) [26] Accumulat-ing evidence indicates that HCMV IE1 products can interactwith TP53 and Rb proteins (tumor suppressor proteins) andthereby it can induce cell cycle progression and block apop-tosis Indeed IE1 expression in a glioma cell line significantlyreduces P53 expression [27] Another study indicated thatthis protein interacts with the P13KAKT pathway one of themost important signaling pathways in glioma and sustainsactivation of AKT signaling [28] The viral protein pp65(also called pUL83) is abundantly synthesized during lyticinfection [29] It is an important target of CD8+ T-cells inthe course of immune response to HCMV infection [30]Among the many functions it has this protein is an immune-modulator and has been shown to block interferon activity[31] anddownregulate the interferon-response gene primarilythrough modulation of interferon response factor-3 IRF-3[32]

Amajor late antigen of HCMV is glycoprotein B (gB) It isa type 1 trans-membrane protein that exists as a dimer on thesurface of the viral envelope [33] This antigen is implicatedin the virus entry cell-to-cell spread [34] and fusion of theinfected cells [35]There is no evidence from available reportsthat this antigen has any role in cell transformation andthe positive result for the presence of the antigen inside thetumor cells but not the nontumor ones may be related to thefeatures of tumor cells that facilitate the entrance of HCMVcompared to the normal cells One of the most importantfeatures of IE1-72 protein is early production of this protein inthe nucleus of the infected cell It is because of this propertythat IE1-72 has stronger expression than pp65 which in turnhas stronger expression than the late antigen The last gene isknown to encode for an envelope glycoprotein and in manyinstances it scatters on the cell membrane of the infected celland cannot be easily detected These results are in part inaccordance with a previous study by Mitchell et al [17] whostated that pp65 reactivity is less ubiquitous than IE1-72 intumor cells

A significant correlation between the level of pp65 and gBantigens in the MRC-5 cell line experimentally infected withHCMV has been reported by Jun et al [36] It is not unusualto find such correlation among viral proteins as the infectionoccurs in defined sequences chronologically accompanied byexpression of immediate early early and late viral antigensHence HCMV proteins are expected to follow the pattern of

elevation and decline shown by immediate early proteins butto a lesser degree

Further support for the role ofHCMV in the developmentof this tumor results from the use of anti-HCMVdrugs Somestudies have shown that valganciclovir significantly reducedthe growth of HCMV-positive glioma tumors [37] RecentlyBaryawno et al [38] demonstrated an increase in overallsurvival of glioblastoma patients receiving valganciclovircompared with those patients who did not receive suchtreatment

The results from the present study show the high percent-age of HCMV virus expression in different histopathologicalgrades of glioma patients which clearly suggest its role ingliomapathogenesisThedetection of low-level latentHCMVinfections might suggest a role in glioma pathogenesisHowever amore detailed study ofHCMV infection in gliomapatients is required for a definitive conclusion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Ahmad Adnan in the NeuroscienceHospital for help in collection of patient samples and DrQasim Sharhan for technical assistance and scientific com-ments

References

[1] G J Tortora and B Derrickson Principles of Anatomy andPhysiology John Wiley amp Sons New York NY USA 14thedition 2014

[2] A P Kyritsis M L Bondy J S Rao and C Sioka ldquoInheritedpredisposition to gliomardquo Neuro-Oncology vol 12 no 1 pp104ndash113 2010

[3] G Prasad and D A Haas-Kogan ldquoRadiation-induced gliomasrdquoExpert Review of Neurotherapeutics vol 9 no 10 pp 1511ndash15172009

[4] Iraqi Cancer Registry 2005[5] H Ohgaki and P Kleihues ldquoEpidemiology and etiology of

gliomasrdquo Acta Neuropathologica vol 109 no 1 pp 93ndash1082005

[6] L Soroceanu and C S Cobbs ldquoIs HCMV a tumor promoterrdquoVirus Research vol 157 no 2 pp 193ndash203 2011

[7] L S YoungViruses andHuman Cancer Bios Scientific OxfordUK 1998

[8] C Hawkins and S Croul ldquoViruses and human brain tumorscytomegalovirus enters the frayrdquoThe Journal of Clinical Investi-gation vol 121 no 10 pp 3831ndash3833 2011

[9] C Taher J de Boniface A A Mohammad et al ldquoHighprevalence of human cytomegalovirus proteins and nucleicacids in primary breast cancer and metastatic sentinel lymphnodesrdquo PLoS ONE vol 8 no 2 Article ID e56795 2013

[10] F Tafvizi and Z T Fard ldquoDetection of human cytomegalovirusin patients with colorectal cancer by nested-PCRrdquo Asian PacificJournal of Cancer Prevention vol 15 no 3 pp 1453ndash1457 2014

BioMed Research International 7

[11] L Geder R Lausch F OrsquoNeill and F Rapp ldquoOncogenictransformation of human embryo lung cells by humancytomegalovirusrdquo Science vol 192 no 4244 pp 1134ndash1137 1976

[12] J Cinatl Jr J-U Vogel H Rabenau B Kornhuber and H WDoerr ldquoModulatory effects of human cytomegalovirus infectionon malignant properties of cancer cellsrdquo Intervirology vol 39no 4 pp 259ndash269 1996

[13] E S Amirian DMarquez-DoM L Bondy andM E ScheurerldquoAntihistamine use and immunoglobulin E levels in glioma riskand prognosisrdquoCancer Epidemiology vol 37 no 6 pp 908ndash9122013

[14] S Sjostrom U Hjalmars P Juto et al ldquoHuman immunoglobu-linG levels of viruses and associated glioma riskrdquoCancer Causesand Control vol 22 no 9 pp 1259ndash1266 2011

[15] C S Cobbs LHarkinsM Samanta et al ldquoHuman cytomegalo-virus infection and expression in human malignant gliomardquoCancer Research vol 62 no 12 pp 3347ndash3350 2002

[16] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas reveals apotential therapeutic targetrdquo The Journal of Clinical Investiga-tion vol 121 no 10 pp 4043ndash4055 2011

[17] D A Mitchell W Xie R Schmittling et al ldquoSensitive detectionof human cytomegalovirus in tumors and peripheral blood ofpatients diagnosed with glioblastomardquo Neuro-Oncology vol 10no 1 pp 10ndash18 2008

[18] A Rahbar G Stragliotto A Orrego et al ldquoLow levels of humancytomegalovirus infection in glioblastoma multiforme asso-ciates with patient survival a case-control studyrdquoHerpesviridaevol 3 pp 3ndash10 2012

[19] M E Scheurer M L Bondy K D Aldape T Albrecht andR El-Zein ldquoDetection of human cytomegalovirus in differenthistological types of gliomasrdquo Acta Neuropathologica vol 116no 1 pp 79ndash86 2008

[20] J Sabatier E Uro-Coste I Pommepuy et al ldquoDetection ofhuman cytomegalovirus genome and gene products in centralnervous system tumoursrdquo British Journal of Cancer vol 92 no4 pp 747ndash750 2005

[21] S Poltermann B Schlehofer K Steindorf P Schnitzler KGeletneky and J R Schlehofer ldquoLack of association of her-pesviruses with brain tumorsrdquo Journal of NeuroVirology vol 12no 2 pp 90ndash99 2006

[22] S K Lau Y-Y Chen W-G Chen et al ldquoLack of association ofcytomegalovirus with human brain tumorsrdquoModern Pathologyvol 18 no 6 pp 838ndash843 2005

[23] X Wang S-M Huong M L Chiu N Raab-Traub and E-SHuang ldquoEpidermal growth factor receptor is a cellular receptorfor human cytomegalovirusrdquoNature vol 424 no 6947 pp 456ndash461 2003

[24] T F Liu S B Tatter M C Willingham M Yang J J Hu andA E Frankel ldquoGrowth factor receptor expression varies amonghigh-grade gliomas and normal brain epidermal growth factorreceptor has excellent properties for interstitial fusion proteintherapyrdquo Molecular Cancer Therapeutics vol 2 no 8 pp 783ndash787 2003

[25] T Kossmann M C Morganti-Kossmann J M OrensteinW J Britt S M Wahl and P D Smith ldquoCytomegalovirusproduction by infected astrocytes correlates with transforminggrowth factor-120573 releaserdquoThe Journal of Infectious Diseases vol187 no 4 pp 534ndash541 2003

[26] S Saikali T Avril B Collet et al ldquoExpression of nine tumourantigens in a series of human glioblastoma multiforme interest

of EGFRvIII IL-13R1205722 gp100 and TRP-2 for immunotherapyrdquoJournal of Neuro-Oncology vol 81 no 2 pp 139ndash148 2007

[27] C S Cobbs L Soroceanu S Denham W Zhang and M HKraus ldquoModulation of oncogenic phenotype in human gliomacells by cytomegalovirus IE1-mediated mitogenicityrdquo CancerResearch vol 68 no 3 pp 724ndash730 2008

[28] R A Johnson X Wang X-L Ma S-M Huong and E-S Huang ldquoHuman cytomegalovirus up-regulates the phos-phatidylinositol 3-kinase (PI3-K) pathway inhibition of PI3-Kactivity inhibits viral replication and virus-induced signalingrdquoJournal of Virology vol 75 no 13 pp 6022ndash6032 2001

[29] S Schmolke H F Kern P Drescher G Jahn and BPlachter ldquoThe dominant phosphoprotein pp65 (UL83) ofhuman cytomegalovirus is dispensable for growth in cell cul-turerdquo Journal of Virology vol 69 no 10 pp 5959ndash5968 1995

[30] M R Wills A J Carmichael K Mynard et al ldquoThe humancytotoxic T-lymphocyte (CTL) response to cytomegalovirus isdominated by structural protein pp65 frequency specificityand T-cell receptor usage of pp65-specific CTLrdquo Journal ofVirology vol 70 no 11 pp 7569ndash7579 1996

[31] E P Browne B Wing D Coleman and T Shenk ldquoAltered cel-lular mRNA levels in human cytomegalovirus-infected fibrob-lasts viral block to the accumulation of antiviral mRNAsrdquoJournal of Virology vol 75 no 24 pp 12319ndash12330 2001

[32] D A Abate S Watanabe and E S Mocarski ldquoMajor humancytomegalovirus structural protein pp65 (ppUL83) preventsinterferon response factor 3 activation in the interferonresponserdquo Journal of Virology vol 78 no 20 pp 10995ndash110062004

[33] E R Kinzler and T Compton ldquoCharacterization of humancytomegalovirus glycoprotein-induced cell-cell fusionrdquo Journalof Virology vol 79 no 12 pp 7827ndash7837 2005

[34] M K Isaacson and T Compton ldquoHuman cytomegalovirusglycoprotein B is required for virus entry and cell-to-cell spreadbut not for virion attachment assembly or egressrdquo Journal ofVirology vol 83 no 8 pp 3891ndash3903 2009

[35] D Navarro P Paz S Tugizov K Topp J La Vail and L PereiraldquoGlycoprotein B of human cytomegalovirus promotes virionpenetration into cells transmission of infection from cell to celland fusion of infected cellsrdquoVirology vol 197 no 1 pp 143ndash1581993

[36] F Jun L Minhuan M Yadan et al ldquoDevelopment of twopotential diagnostic monoclonal antibodies against humancytomegalovirus glycoprotein Brdquo Brazilian Journal of Medicaland Biological Research vol 45 no 7 pp 583ndash590 2012

[37] G Stragliotto A Rahbar NW Solberg et al ldquoEffects of valgan-ciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma a randomized double-blind hypothesis-generating studyrdquo International Journal of Cancer vol 133 no5 pp 1204ndash1213 2013

[38] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas revealsa potential therapeutic targetrdquo Journal of Clinical Investigationvol 121 no 10 pp 4043ndash4055 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 7

[11] L Geder R Lausch F OrsquoNeill and F Rapp ldquoOncogenictransformation of human embryo lung cells by humancytomegalovirusrdquo Science vol 192 no 4244 pp 1134ndash1137 1976

[12] J Cinatl Jr J-U Vogel H Rabenau B Kornhuber and H WDoerr ldquoModulatory effects of human cytomegalovirus infectionon malignant properties of cancer cellsrdquo Intervirology vol 39no 4 pp 259ndash269 1996

[13] E S Amirian DMarquez-DoM L Bondy andM E ScheurerldquoAntihistamine use and immunoglobulin E levels in glioma riskand prognosisrdquoCancer Epidemiology vol 37 no 6 pp 908ndash9122013

[14] S Sjostrom U Hjalmars P Juto et al ldquoHuman immunoglobu-linG levels of viruses and associated glioma riskrdquoCancer Causesand Control vol 22 no 9 pp 1259ndash1266 2011

[15] C S Cobbs LHarkinsM Samanta et al ldquoHuman cytomegalo-virus infection and expression in human malignant gliomardquoCancer Research vol 62 no 12 pp 3347ndash3350 2002

[16] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas reveals apotential therapeutic targetrdquo The Journal of Clinical Investiga-tion vol 121 no 10 pp 4043ndash4055 2011

[17] D A Mitchell W Xie R Schmittling et al ldquoSensitive detectionof human cytomegalovirus in tumors and peripheral blood ofpatients diagnosed with glioblastomardquo Neuro-Oncology vol 10no 1 pp 10ndash18 2008

[18] A Rahbar G Stragliotto A Orrego et al ldquoLow levels of humancytomegalovirus infection in glioblastoma multiforme asso-ciates with patient survival a case-control studyrdquoHerpesviridaevol 3 pp 3ndash10 2012

[19] M E Scheurer M L Bondy K D Aldape T Albrecht andR El-Zein ldquoDetection of human cytomegalovirus in differenthistological types of gliomasrdquo Acta Neuropathologica vol 116no 1 pp 79ndash86 2008

[20] J Sabatier E Uro-Coste I Pommepuy et al ldquoDetection ofhuman cytomegalovirus genome and gene products in centralnervous system tumoursrdquo British Journal of Cancer vol 92 no4 pp 747ndash750 2005

[21] S Poltermann B Schlehofer K Steindorf P Schnitzler KGeletneky and J R Schlehofer ldquoLack of association of her-pesviruses with brain tumorsrdquo Journal of NeuroVirology vol 12no 2 pp 90ndash99 2006

[22] S K Lau Y-Y Chen W-G Chen et al ldquoLack of association ofcytomegalovirus with human brain tumorsrdquoModern Pathologyvol 18 no 6 pp 838ndash843 2005

[23] X Wang S-M Huong M L Chiu N Raab-Traub and E-SHuang ldquoEpidermal growth factor receptor is a cellular receptorfor human cytomegalovirusrdquoNature vol 424 no 6947 pp 456ndash461 2003

[24] T F Liu S B Tatter M C Willingham M Yang J J Hu andA E Frankel ldquoGrowth factor receptor expression varies amonghigh-grade gliomas and normal brain epidermal growth factorreceptor has excellent properties for interstitial fusion proteintherapyrdquo Molecular Cancer Therapeutics vol 2 no 8 pp 783ndash787 2003

[25] T Kossmann M C Morganti-Kossmann J M OrensteinW J Britt S M Wahl and P D Smith ldquoCytomegalovirusproduction by infected astrocytes correlates with transforminggrowth factor-120573 releaserdquoThe Journal of Infectious Diseases vol187 no 4 pp 534ndash541 2003

[26] S Saikali T Avril B Collet et al ldquoExpression of nine tumourantigens in a series of human glioblastoma multiforme interest

of EGFRvIII IL-13R1205722 gp100 and TRP-2 for immunotherapyrdquoJournal of Neuro-Oncology vol 81 no 2 pp 139ndash148 2007

[27] C S Cobbs L Soroceanu S Denham W Zhang and M HKraus ldquoModulation of oncogenic phenotype in human gliomacells by cytomegalovirus IE1-mediated mitogenicityrdquo CancerResearch vol 68 no 3 pp 724ndash730 2008

[28] R A Johnson X Wang X-L Ma S-M Huong and E-S Huang ldquoHuman cytomegalovirus up-regulates the phos-phatidylinositol 3-kinase (PI3-K) pathway inhibition of PI3-Kactivity inhibits viral replication and virus-induced signalingrdquoJournal of Virology vol 75 no 13 pp 6022ndash6032 2001

[29] S Schmolke H F Kern P Drescher G Jahn and BPlachter ldquoThe dominant phosphoprotein pp65 (UL83) ofhuman cytomegalovirus is dispensable for growth in cell cul-turerdquo Journal of Virology vol 69 no 10 pp 5959ndash5968 1995

[30] M R Wills A J Carmichael K Mynard et al ldquoThe humancytotoxic T-lymphocyte (CTL) response to cytomegalovirus isdominated by structural protein pp65 frequency specificityand T-cell receptor usage of pp65-specific CTLrdquo Journal ofVirology vol 70 no 11 pp 7569ndash7579 1996

[31] E P Browne B Wing D Coleman and T Shenk ldquoAltered cel-lular mRNA levels in human cytomegalovirus-infected fibrob-lasts viral block to the accumulation of antiviral mRNAsrdquoJournal of Virology vol 75 no 24 pp 12319ndash12330 2001

[32] D A Abate S Watanabe and E S Mocarski ldquoMajor humancytomegalovirus structural protein pp65 (ppUL83) preventsinterferon response factor 3 activation in the interferonresponserdquo Journal of Virology vol 78 no 20 pp 10995ndash110062004

[33] E R Kinzler and T Compton ldquoCharacterization of humancytomegalovirus glycoprotein-induced cell-cell fusionrdquo Journalof Virology vol 79 no 12 pp 7827ndash7837 2005

[34] M K Isaacson and T Compton ldquoHuman cytomegalovirusglycoprotein B is required for virus entry and cell-to-cell spreadbut not for virion attachment assembly or egressrdquo Journal ofVirology vol 83 no 8 pp 3891ndash3903 2009

[35] D Navarro P Paz S Tugizov K Topp J La Vail and L PereiraldquoGlycoprotein B of human cytomegalovirus promotes virionpenetration into cells transmission of infection from cell to celland fusion of infected cellsrdquoVirology vol 197 no 1 pp 143ndash1581993

[36] F Jun L Minhuan M Yadan et al ldquoDevelopment of twopotential diagnostic monoclonal antibodies against humancytomegalovirus glycoprotein Brdquo Brazilian Journal of Medicaland Biological Research vol 45 no 7 pp 583ndash590 2012

[37] G Stragliotto A Rahbar NW Solberg et al ldquoEffects of valgan-ciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma a randomized double-blind hypothesis-generating studyrdquo International Journal of Cancer vol 133 no5 pp 1204ndash1213 2013

[38] N Baryawno A Rahbar N Wolmer-Solberg et al ldquoDetec-tion of human cytomegalovirus in medulloblastomas revealsa potential therapeutic targetrdquo Journal of Clinical Investigationvol 121 no 10 pp 4043ndash4055 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom