Research Article Autoantibody Response to Murine...

Research ArticleAutoantibody Response to Murine Double Minute 2 Protein inImmunodiagnosis of Hepatocellular Carcinoma

Mei Liu12 Su-jun Zheng1 Yu Chen1 Ning Li1 Peng-fei Ren2 Li-ping Dai2

Zhong-ping Duan1 and Jian-Ying Zhang2

1 Beijing Yoursquoan Hospital Capital Medical University Beijing 10069 China2Department of Biological Sciences The University of Texas at El Paso El Paso TX 79968 USA

Correspondence should be addressed to Zhong-ping Duan duan2517163com and Jian-Ying Zhang jzhangutepedu

Received 20 January 2014 Accepted 17 April 2014 Published 14 May 2014

Academic Editor Takami Sato

Copyright copy 2014 Mei Liu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hepatocellular carcinoma (HCC) is the fifthmost commonmalignancy worldwide Although new therapeutic strategies have beencontinuously developed and applied to clinical treatment for HCC the prognosis is still very poor Thus early detection of HCCmay enhance effective and curative management In this study autoantibody responses to MDM2 protein in HCC patientrsquos serumwere evaluated by enzyme-linked immunosorbent assay (ELISA) and part sera were evaluated by Western blotting and indirectimmunofluorescence assay Immunohistochemistry (IHC) over tissue array slides was also performed to analyze protein expressionof MDM2 in HCC and control tissues The prevalence of autoantibodies against MDM2 was significantly higher than that in livercirrhosis (LC) chronic hepatitis (CH) and normal human sera (NHS) The average titer of autoantibodies against MDM2 in HCCserumwas higher compared to that in LC CH andNHS A high titer of autoantibodies againstMDM2 in ELISA could be observedin the serum in 6 to 9months before the clinical diagnosis ofHCC in the serumof severalHCCpatients with serial bleeding samplesOur preliminary data indicate that MDM2 and anti-MDM2 system may be a potential biomarker for early stage HCC screeningand immunodiagnosis

1 Introduction

Hepatocellular carcinoma (HCC) is the fifth most commonmalignancy worldwide It represents the fifth most prevalentcancer worldwide and accounts for 500 000 deaths each year[1 2] HCC is associated with a poor prognosis due to alack of effective treatment options Although new therapeuticstrategies have been continuously developed and applied toclinical treatment of HCC the prognosis is still very poor Ithas a survival rate of less than 5 and an average survival ofless than one year after diagnosis [3]There is still no effectivetherapy for most patients with advanced or metastatic HCC[4] Early detection of HCC enhances effective and curativemanagement The sensitivity and specificity of serum alpha-fetoprotein (AFP) in HCC diagnosis are not optimal Inrecent years tumor-associated antigens (TAAs) were studiedby researchers in order to find better early stage biomarker ofHCC

The MDM2 oncogene biochemically as E3 ubiquitinprotein ligase was originally identified by virtue of itsamplification in a spontaneously transformed derivative ofmouse BALBc cells and theMDM2protein subsequentlywasshown to bind to p53 in rat cells transfectedwith p53 genes Inhumans MDM2 protein is encoded by the MDM2 gene andlocalized in chromosome 12q13-14 [5] MDM2 is a nuclearphosphoprotein that binds and inhibits transactivation bytumor protein p53 as part of an autoregulatory negative feed-back loop [6] It binds to p53 via an N-terminal hydrophobicpocket and this domain contains the highest identity atthe amino acid level The MDM2 p53-binding domainsocclude an N-terminal alpha-helix of p53 This preventsthe recruitment of transcriptional coactivators and therebyinhibits p53 transactivation function This transcriptionalantagonism can take place within the nucleus as MDM2has been detected at p53-responsive promoter elementsin chromatin [7] However MDM2 is most abundant in

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 906532 10 pageshttpdxdoiorg1011552014906532

2 Journal of Immunology Research

the cytosol in many cell lines suggesting that cytoplasmiclocalization is important for their function [8 9] MDM2both functions as an E3 ubiquitin ligase that recognizes theN-terminal transactivation domain (TAD) of the p53 and caninhibit p53 transcriptional activation [10] This protein alsoaffects the cell cycle apoptosis and tumorigenesis throughinteractions with other proteins including retinoblastoma1 and ribosomal protein L5 Overexpression of this genecan result in excessive inactivation of tumor protein p53diminishing its tumor suppressor function The MDM2proteins are deregulated in many human cancers and exerttheir oncogenic activity predominantly by inhibiting the p53tumor suppressor [11] Several human tumor types have beenshown to have increased levels of MDM2 including softtissue sarcomas bladder cancers and osteosarcomas as wellas breast tumors [12ndash15]

Diagnosis of HCC was considered as a terminal situationand the leading cause of death in cirrhotic patients [16]However when diagnosis is achieved at an early stageeffective therapies that improve long term survival are morelikely to be achieved [17] In such sense the early diagnosisof HCC is crucial for the treatment of patients In this studyMDM2 was evaluated by immunoassay as a potential TAA inHCC and autoantibody to this protein was also validated tobe an early stage biomarker in immunodiagnosis of HCC

2 Materials and Methods

21 Sera and General Information All sera used in this studyincluding 244 sera from patients with HCC 30 sera frompatients with liver cirrhosis (LC) 30 sera from patients withchronic hepatitis (CH) and 89 normal human sera (NHS) aswell as some sera from fourHCCpatients with serial bleedingsamples were obtained from the serum bank of CancerAutoimmunity and Epidemiology Research Laboratory atUTEP (University of Texas at El Paso) This study wasapproved by the Institutional Review Board of UTEP andcollaborating institutions

AllHCCpatients were diagnosed according to the criteriadescribed in a previous study [18] and had not receivedtreatment with any chemotherapy or radiotherapy Patientswith CH and LC were followed up at least 18 months aftercollecting blood to exclude individuals with primary biliarycirrhosis and asymptomatic or clinically undetectable HCCNormal human sera were collected from individuals at thesame locality during annual health examinations who had noobvious evidence of malignancy

22 Recombinant Proteins and Antibodies Used in This StudyMDM2 construct pGEX-4TMDM2WT (plasmid ID 16237)purchased from Addgenecom was subcloned into pET28avector producing a fusion protein with NH-terminal 6x histi-dine and T7 epitope tagsThe recombinant protein expressedin Escherichia coli BL21 (DE3) was purified using nickelcolumn chromatography Polyclonal anti-MDM2 rabbit anti-body and monoclonal anti-120573-actin mouse antibody werepurchased (Cell Signaling Technology Inc Danvers MA)Horseradish peroxidase- (HRP-) conjugated goat anti-human

IgG HRP-conjugated goat anti-rabbit IgG HRP-conjugatedgoat anti-mouse IgG and FITC-conjugated goat anti-humanIgG were purchased (Santa Cruz Biotechnology Inc SantaCruz CA) Anti-rabbit IgG Fab2 (Alexa Fluor 488) waspurchased (Cell Signaling Technology Inc Danvers MA)

23 Enzyme-Linked Immunosorbent Assay (ELISA) Stan-dard protocol for ELISA was used as described in ourprevious study [19 20] In brief a 96-well microtiter plate(ImmunoChemistry Technologies LLC Bloomington MN)was coated overnight at 4∘C with recombinant MDM2protein at a final concentration of 05120583gmL in phosphate-buffered saline (PBS) The antigen-coated wells were blockedwith gelatin postcoating solution at room temperature for2 h Human sera diluted at 1 100 with serum diluent wereincubated for 2 h at room temperature in the antigen-coatedwells followed by HRP-conjugated goat anti-human IgG(Caltag Laboratories San Francisco CA) at 1 4000 dilu-tion The substrate 221015840-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS Sigma-Aldrich St Louis MO) wasused as detecting reagent The average optical density (OD)value at a wavelength of 405 nm was used for data analysisThe cutoff value designating positive reaction was the meanoptical density (OD) of 89 normal human sera plus 3 standarddeviations (SD)

24 Western Blotting Denatured recombinant MDM2 pro-tein and cancer cell lysates were electrophoresed on 10 SDS-PAGE and transferred to nitrocellulose papers respectivelyAfter blocking in PBSwith 5 nonfatmilk and 005Tween-20 for 1 h at room temperature the nitrocellulose paperswere incubated overnight at 4∘C with 1 200 dilution ofhuman sera 1 500 dilution of polyclonal anti-MDM2 anti-body and 1 500 dilution of monoclonal anti-120573-actin mouseantibody separately HRP-conjugated goat anti-human IgGHRP-conjugated goat anti-rabbit IgG and HRP-conjugatedgoat anti-mouse IgG were respectively applied as secondaryantibody at a 1 10000 dilution The ECL-kit was used todetect immunoreactive bands according to the manufac-turerrsquos instructions (Thermo Scientific Waltham MA)

25 Indirect Immunofluorescence Assay (IIFA) and ConfocalMicroscopy Indirect immunofluorescence assay was per-formed on Hep2 antinuclear antigen tissue slides (BionEnterprises Des Plaines IL) The sera were diluted at1 80 in PBS and pH 74 and incubated with the slidesfor 30min at room temperature After extensive washingthe slides were incubated with fluorescein isothiocyanate-(FITC-) conjugated goat anti-human IgG secondary antibody(Santa Cruz Biotechnology CA) or anti-rabbit IgG Fab2(Alexa Fluor 488) as secondary antibody diluted 1 100 inPBS for 1 h at room temperature The slides were washedtwo times with PBS before adding a drop of mountingmedia containing 15 120583gmL 4101584061015840-diamidino-2-phenylindole(DAPI) (Vector Laboratories Inc Burlingame CA) to pre-vent photobleaching The slides were then examined underfluorescence microscopy LSM 700 Confocal Microscopy

Journal of Immunology Research 3

Table 1 Frequency of autoantibody against MDM2 in human seraby ELISA

Type of sera Number tested Autoantibody to MDM2 ()HCC 244 47 (193)LC 30 0lowastlowast

CH 30 2 (66)lowastlowast

NHS 89 0lowastlowast

The cutoff value designating positive reaction was the mean optical density(OD) of 89 normal human sera plus 3 standard deviations (SD) P valuerelative to NHS (119875 = 0000) CH (119875 = 0002) and LC (119875 = 0000)lowastlowast

119875 lt 001 HCC hepatocellular carcinoma LC liver cirrhosis CH chronichepatitis and NHS normal human sera

(Zeiss) at 400x magnification Zen 2009 software was usedfor images capture and analysis

26 Absorption of Antibodies with Recombinant Protein Thediluted human sera (1 80) were incubated with recombinantprotein (final concentration of recombinant protein in thediluted human sera was 003120583g120583L) overnight at 4∘C andthen centrifuged at 10000timesg for 10minThe supernatant wasused for immunofluorescence assay

27 Immunohistochemistry (IHC) with Tissue Array SlidesLiver cancer tissue array slide with normal tissue controls(38 cases80 cores including pathological diagnosis andpathological grades two normal liver tissues as controls) waspurchased (US Biomax Inc Rockville MD) and used todetect the expression of the MDM2 protein Tissue arrayslides were deparaffinized with xylene and dehydrated withethanol Antigen retrieval was performed by microwave-heatingmethods in Trilogy pretreatment solution for 20minAvidinbiotin blocking solution was used to prevent non-specific binding of antibodies The sections were incubatedwith polyclonal anti-MDM2 antibody (1 50 dilution) forovernight at 4∘C HRP Detection System (HRP streptavidinlabel and polyvalent biotinylated link) andDAB Substrate Kitwere used as detecting reagents After counterstaining withhematoxylin the sectionswere dehydrated andmountedTheslides were observed by light microscopy (Leica DM1000Germany)

28 Statistical Analysis The mean OD value of each groupof patientsrsquo sera was compared using the Mann-Whitney Utest the frequency of autoantibody to TAAs in each groupof patientsrsquo sera and the expression profile of MDM2 inliver cancer and normal tissue groups were compared usingthe Chi-square (1205942) test with Fisherrsquos exact test and twosignificant levels (005 and 001) were used

3 Results

31 Frequency and Titer of Autoantibodies against MDM2 inHCC The full-length recombinant MDM2 protein was usedas coating antigen in ELISA to screen autoantibodies againstMDM2 in sera from patients with HCC LC CH and NHSIn total 244 sera from patients with HCC 30 from LC 30

08

06

04

02

00

Opt

ical

den

sity

(OD

)

NHS CH LC HCC

Figure 1 Titer of autoantibody against MDM2 in human sera byELISA The range of antibody titers to MDM2 was expressed asoptical density (OD) obtained fromELISAThemean + 3SD of NHSwas shown in relationship to all serum samples Titer of anti-MDM2in HCCwas much higher than that in other types of sera (119875 lt 001)The cutoff value line for positive samples is indicated with red color

1 2 3 4 5 6 7

MDM2

8

82kDa

Figure 2 Western blot analysis with representative sera in ELISALane 1 the polyclonal anti-MDM2 antibody was used as positivecontrol Lanes 2ndash5 four representative HCC sera which were pos-itive in ELISA also had strong reactivity with MDM2 recombinantprotein inWestern blot analysis Lanes 6ndash8 three randomly selectedNHS had negative reactivity with MDM2 recombinant protein

from CH and 89 sera from normal human individuals wereused in this study As shown in Table 1 the prevalence ofautoantibody against MDM2 was 193 (47244) in HCCwhich was significantly higher than that in LC (119875 = 0000)CH (119875 = 0002) and NHS (119875 = 0000) Titer of anti-MDM2 antibodies in human sera is shown in Figure 1 Theaverage titer of autoantibody against MDM2 in HCC serawas higher than that in LC CH and NHS (119875 lt 001) TheELISA results were also confirmed by Western blot analysisFigure 2 shows that representative HCC sera with positivereaction to MDM2 in ELISA also have strong reactivity inWestern blotting compared to the normal sera Autoantibodyto MDM2 in serial serum samples from four HCC patients(case 1 to case 4) was also tested The ELISA results wereshown in Figure 3 In HCC case 1 anti-MDM2 autoantibodytiter was high in nine months before HCC was diagnosed InHCCcase 2 case 3 and case 4 anti-MDM2 autoantibody titerbecame higher at six months before HCC was detected

32 Intense Nuclear Staining Pattern Shown in Hep2 Cellsby Indirect Immunofluorescence Assay with RepresentativePositive HCC Sera To further confirm the reactivity ofautoantibody in HCC sera to MDM2 and the intracellularlocation of MDM2 commercially purchased Hep2 cell slideswere used in indirect immunofluorescence assay to detect


010

008

006

004

002

000

Opt

ical

den

sity

(OD

)

1994

215

1994

510

1994

89

1994

11

8

1995

27

1995

512

lowast

(a)

03

02

01

00

Opt

ical

den

sity

(OD

)

1992

317

1992

616

1992

10

20

1992

11

24

1993

32

lowast

(b)

020

015

010

005

000

Opt

ical

den

sity

(OD

)

1992

918

1993

112

1993

511

1993

824

1993

10

27

lowast

(c)

015

010

005

000

Opt

ical

den

sity

(OD

)

1991

417

1991

731

1991

11

27

1992

129

1992

47

lowast

(d)

Figure 3 Autoantibody to MDM2 in serial serum samples from four HCC patients (cases 1 2 3 and 4) (a) First HCC patient (case 1) wasdiagnosed on 02071995 with high titer of anti-MDM2 autoantibody Totally we had six serum samples within more than one year of timespan collected from this HCC patientThe titer of anti-MDM2 autoantibody became higher at month 9 before HCCwas detected (b) Total offive serum samples were acquired from the secondHCCpatient (case 2)The titer of anti-MDM2 autoantibody became high atmonth 6 beforethe date (11241992) of HCC diagnosed There was low titer of anti-MDM2 autoantibody before 03171992 (c) The third HCC patient (case3) was diagnosed at the date of 08241993 with a high titer of anti-MDM2 autoantibody During one-year period (09181992 to 10271993)six serum samples collected from this patient showed that the titer of anti-MDM2 autoantibody became higher at month 6 before HCC wasdetected (d) The fourth HCC patient (case 4) was diagnosed at the date of 01291992 and five serum samples were acquired for test From04171991 to 01291992 (the date of detection) a gradually increased titer of anti-MDM2 autoantibody can be seen It became high at month6 before HCC was detected The asterisk means the date of HCC diagnosed

HCC sera with anti-MDM2 positive in ELISA As shown inFigure 4 a representative anti-MDM2 positive HCC serumhad an intense nuclear staining pattern which was similarin fluorescent staining pattern and cellular location to thatshown by polyclonal anti-MDM2 antibody The fluorescentstainingwas significantly reducedwhen the sameHCCserumwas preabsorbed with recombinant MDM2 protein

33 Expression of MDM2 in Liver Cancer Tissues andNormal Hepatic Tissues by Immunohistochemistry In thecurrent study the expression profile of MDM2 in liver

cancer tissues and normal liver tissues was examined byimmunohistochemistry with tissue array slides Tissue arrayslides were commercially available for this study including30 HCC tissue specimens and 10 normal hepatic tissuespecimens The polyclonal anti-MDM2 antibody was usedas primary antibody to detect the expression of MDM2 inliver cancer and normal hepatic tissues As a result 24 ofthe 30 HCC tissues were positively stained (800) None ofthe 10 normal hepatic tissues were positively stained (0)The characteristics of patients andMDM2 expression in livercancer are shown in Table 2The expression ofMDM2 in livercancer and normal hepatic tissues is shown in Figure 5


DAPI FITC Merge

(a)

(b)

(c)

(d)

Figure 4 Representative immunofluorescence staining pattern of anti-MDM2 autoantibody positive HCC serum (performed on Hep2antinuclear antigen tissue slides) (a) NHS were used as negative control (b) polyclonal anti-MDM2 antibody which showed a nuclearimmunofluorescence staining pattern was used as positive control (c) a representative anti-MDM2 autoantibody positive HCC serumdemonstrated an intense nuclear staining pattern (d) the same HCC serum used in panel (c) was preabsorbed with recombinant MDM2The nuclear fluorescent staining was significantly reduced


Table 2 Characteristics of patients and MDM2 expression in livercancer

Variable Frequency

Age ge60 7 233lt60 23 767

Gender Male 20 667Female 10 333

Grade II 15 500III 15 500

Normal liver tissue Negative 10 1000Positive 0 00

Liver cancer Negative 6 200Positive 24 800lowastlowast

lowastlowastP value of liver cancer to normal tissue P lt 001

4 Discussion

Many studies demonstrated that sera from patients withcancer contain antibodies which react with a unique groupof autologous cellular antigens generally known as tumor-associated antigens (TAAs) During the progression fromchronic liver disease to HCC the novel autoantibodiesappearing with malignant transformation will be more likelyto be related to events associated with tumorigenesis andtherefore these autoantibodies can be used as reportersidentifying aberrant cellular mechanisms in tumorigenesisand also serve as immunodiagnostic markers for HCCdetection [21ndash23] As we know AFP is the most commonand traditional biomarker being used for diagnosis of HCCin current clinical screening However both its sensitivityand specificity are limited There are still about 40 ofHCC patients that cannot be identified by this approachHCC patients with small tumors or with well-to-moderatelydifferentiated tumors may not have high level of serum AFPTheultrasonic and computer tomography are useful when thetumors are big enough to be detected Usually it is too late forpatients to get effective therapy when the HCC is detected bythesemethodsTherefore identification of a better biomarkerfor the early stage HCC is crucial for reducing the HCCmortality rate in the population Anti-TAAs autoantibodieswhich have been called ldquoreportersrdquo from the immune systemcan be used as biomarkers in immunodiagnosis of earlystage HCC [24] In our previous study over ten oncoproteinshave been evaluated and validated as TAAs in HCC andautoantibodies against these TAAs have been detected insera from patients with HCC [25] But the sensitivity andspecificity of autoantibodies to single TAA as a diagnosticmarker inHCC are not high enough for the diagnosis ofHCC[26] The method to solve this problem is to combine theknown TAAs making a TAA array with multiple TAAs bywhich a higher sensitivity and specificity can be acquired butit is still not good enough till now It is necessary to work onthe validation of TAAswhich are good at early stage diagnosisin HCC patients

The MDM2 protein plays a critical role in the nega-tive regulation of p53 MDM2 can lead to nuclear exportubiquitination and nuclear and cytoplasmic proteasomal

degradation of p53 through directly binding to the p53 trans-activation domain [27 28] p53-mediated tumor suppressingactivity can be inhibited by high levels of MDM2 throughp53 inactivation [29] The MDM2 protein also interacts withseveral cell cycle regulatory proteins that may contribute toits tumorigenic ability So the expression level of MDM2 hasclose relationshipwith tumorigenesis [30]The role ofMDM2in HCC and its overexpression in HCC patients are studiedby many researchers A study on Japanese patients with HCVinfection showed that MDM2 gene was significantly associ-ated with development and recurrence of HCC [31] MDM2SNP309 plays a major role in the carcinogenesis of HCCespecially among Caucasian populations [29] The findingsfrom different areas support that MDM2 is significantlyassociated with increased risk of hepatocellular carcinoma[32ndash35] MDM2 SNP309 G allele is a susceptibility genefor the development of viral hepatitis-related hepatocellularcarcinoma [36ndash38] The combination of MDM2 SNP 309and TP53 Arg72Pro genotypes confers higher risk to developHCC [39ndash41]TheMDM2 gene silenced by shRNA effectivelyinhibits HCC tumorigenesis of subcutaneously xenograftedHepG2 cells in nude mice [42] MDM2 antagonist caninhibit tumor growth in HCC with different types of p53 invitro [43] HCC patients with a low expression of MDM2survived significantly longer as compared with patients withhigh expression [44] The expressions of MDM2 proteinand gene were also related to the high invasiveness ofHCC through inactivating the tumor-suppressor functionof the p53 gene [45 46] MDM2 was considered to be avaluable target for cancer therapy and MDM2 blockade withsuitable antagonists was shown to block tumor growth ina number of models [47] MDM2 overexpression was alsoconsidered to be a useful predictor of poor prognosis inpatientswithHCC following hepatic resection [48]Howeverthere is nothing reported about the possibility of MDM2 as abiomarker of cancer MDM2 is predominantly distributed inthe cell nucleus and some of MDM2 can be detected in thecytoplasmThe expression ofMDM2 can be increased duringthe tumorigenesis On the situation of cell injury or the highpenetration of the cell membrane the immune system canfind the exposed MDM2 protein and the autoantibodies willappear in the serum Since MDM2 is overexpressed in HCCwe try to evaluate whether the MDM2 protein is a potentialTAA in HCC and validate if autoantibody to this protein canbe used as an early stage biomarker in immunodiagnosis ofHCC

In our present study the results indicated that almosttwenty percent of HCC sera showed immune response toMDM2 recombinant protein The mean titer of autoan-tibodies against MDM2 in sera from patients with HCCwas significantly higher than that in LC CH and normalindividuals The results were also confirmed by Westernblot analysis which showed that representative HCC serawith positive reaction to MDM2 in ELISA also have strongreactivity compared to normal sera In the further study anti-MDM2 autoantibodies were detected in sera from severalHCC patients with serial bleeding samples A high titer ofautoantibodies against MDM2 in ELISA can be seen in serain 6 months to 9 months before the clinical diagnosis of


(a) (b)

(c) (d)

(e) (f)

Figure 5 Expression of MDM2 in liver cancer and normal hepatic tissues by immunohistochemistry The polyclonal anti-MDM2 antibodywas used as primary antibody to detect the expression of MDM2 in liver cancer and normal hepatic tissues (a) A normal hepatic tissue hadnegative staining (b) HCC tissue (Grade II) had positive staining (c) HCC tissue (Grade III) had positive staining (d) a normal hepatictissue with HE staining (e) HCC tissue (Grade II) with HE staining (f) HCC tissue (Grade III) with HE staining Grade II moderatelydifferentiated cells appear slightly different from normal Grade III poorly differentiated cells appear abnormal and tend to grow and spreadmore aggressively

HCC Most cases of HCC are secondary to either a chronicviral hepatitis or cirrhosis HCC develops when there is agene mutation to the cellular machinery which causes thecell to replicate at a high rate which results in the cellavoiding of apoptosis Constant cycle of damage followedby repair can lead to mistakes during DNA repair whichin turn lead to carcinogenesis During the course of HCCformation some new presenting autoantibodies maybe havethe value for diagnosis of HCC patients with an early stageof tumorigenesis Anti-MDM2 autoantibodies are potential

biomarkers for immunodiagnosis of HCC patients due tothe low positive rate in LC CH and normal individuals andhigher positive rate in HCC patients

It has been reported that MDM2 protein is overexpressedin HCC tissue In a previous report by MF Zhangrsquos groupMDM2 expression was examined in 181 pairs of HCC tissuesand the adjacent hepatic tissues by using immunohisto-chemistry through which the group found that MDM2 wasoverexpressed in all theHCC cases [49] But in another studythe result of immunohistochemistry showed that MDM2


was expressed in 26 of HCC and its expression correlatedpositively with p53 mutations [50] In our present studyeighty percent of HCC liver tissues were positively stainedwith anti-MDM2 antibody and there were no differencesbetween HCC tissues with Grade III and Grade II in usingimmunohistochemistry approach with HCC tissue arrayslide IHC study with HCC tissue array has verified that thecellular localization of MDM2 is found in the nucleus of cellWe also verified the cellular localization ofMDM2 by indirectimmunofluorescence assay and anti-MDM2 autoantibodypositive HCC serum demonstrated an intense nuclear stain-ing pattern The cellular localization of MDM2 is associatedwith its oncoprotein function as negative regulator of tumorsuppressor protein p53

MDM2 plays an important role in HCC tumorigenesisData from a Chinese population shows that the MDM2 indelpolymorphism may be a genetic modifier for developingHCC [51] So MDM2 protein maybe appeared in early stageof HCC generation and its antibodies have the possibilityof early biomarker in HCC patients Our current studyfocused on the possibility of its role as a biomarker inearly stage HCC diagnosis The data from our study hasdemonstrated that MDM2 is a potential TAA in HCC andthe anti-MDM2 autoantibodies may be used as a biomarkerin early stage HCC diagnosis because a higher titer ofautoantibodies against MDM2 in ELISA can be seen in seraat 6 months to 9 months before the clinical diagnosis ofHCC The limitation of our study is that no information oftheseHCCpatients with serial bleeding samples was acquiredafter the HCC diagnosis We did not know the reason whythe titer of autoantibodies against MDM2 decreased afterdiagnosis The affecting factors may range from the surgeryprocedures the drug used by the patients the chemotherapyor the radiotherapy Therefore in our future study morecases with complete clinical data should be included in orderto further investigate the correlation of the role of hepatictissueMDM2 protein expression and the presence of elevatedMDM2 autoantibodies in serum of patients with HCC to beevaluated as early biomarkers in immunodiagnostic of HCC

Conflict of Interests

There are no commercial affiliations or conflict of interests todisclose

Acknowledgments

This work was supported by Grants (SC1CA166016) fromthe National Institutes of Health (NIH) and also partiallysupported by a Grant of High Technical Personnel TrainingItem from Beijing Health System (2011-3-083) Yoursquoan liverdisease and AIDS funding (BJYAH-2011-047 and BJYAH-2011-045) National Science and Technology Key Projecton ldquoMajor Infectious Diseases such as HIVAIDS ViralHepatitis Prevention and Treatmentrdquo (2012ZX10002004-006 2012ZX10004904-003-001 and 2013ZX10002002-006)and Beijing Municipal Administration of Hospitals Clin-ical Medicine Development of Special Funding Support

(XM201308) The authors thank the Border BiologicalResearch Center (BBRC) Core Facilities at The University ofTexas at El Paso (UTEP) for their support whichwere fundedby NIH Grant (5G12MD007592) The authors also thank DrQing Zhursquos help to give comments on their paper

References

[1] F X Bosch J Ribes M Dıaz and R Cleries ldquoPrimary livercancer worldwide incidence and trendsrdquo Gastroenterology vol127 no 5 pp S5ndashS16 2004

[2] J A Marrero ldquoHepatocellular carcinomardquo Current Opinion inGastroenterology vol 22 no 3 pp 248ndash253 2006

[3] D M Parkin F Bray J Ferlay and P Pisani ldquoGlobal cancerstatistics 2002rdquo CA A Cancer Journal for Clinicians vol 55 no2 pp 74ndash108 2005

[4] T-J Song E W K Ip and Y Fong ldquoHepatocellular carcinomacurrent surgical managementrdquo Gastroenterology vol 127 no 5pp S248ndashS260 2004

[5] J D Oliner K W Kinzler P S Meltzer D L George and BVogelstein ldquoAmplification of a gene encoding a p53-associatedprotein in human sarcomasrdquo Nature vol 358 no 6381 pp 80ndash83 1992

[6] D A Freedman L Wu and A J Levine ldquoFunctions of theMDM2 oncoproteinrdquo Cellular and Molecular Life Sciences vol55 no 1 pp 96ndash107 1999

[7] J-P Kruse andW Gu ldquoModes of p53 Regulationrdquo Cell vol 137no 4 pp 609ndash622 2009

[8] Y V Wang M Wade E Wong Y-C Li L W Rodewald andG M Wahl ldquoQuantitative analyses reveal the importance ofregulated Hdmx degradation for P53 activationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 104 no 30 pp 12365ndash12370 2007

[9] M Wade Y V Wang and G M Wahl ldquoThe p53 orchestramdm2 and Mdmx set the tonerdquo Trends in Cell Biology vol 20no 5 pp 299ndash309 2010

[10] K Itahana HMao A Jin et al ldquoTargeted inactivation ofMdm2RING finger E3 ubiquitin ligase activity in the mouse revealsmechanistic insights into p53 regulationrdquo Cancer Cell vol 12no 4 pp 355ndash366 2007

[11] M Wade Y C Li and G M Wahl ldquoMDM2 MDMX and p53in oncogenesis and cancer therapyrdquoNature Reviews Cancer vol13 no 2 pp 83ndash96 2013

[12] V A Floslashrenes G M Moelandsmo A Forus A AndreassenO Myklebost and O Fodstad ldquoMDM2 gene amplification andtranscript levels in human sarcomas relationship to TP53 genestatusrdquo Journal of the National Cancer Institute vol 86 no 17pp 1297ndash1302 1994

[13] P Lianes I Orlow Z-F Zhang et al ldquoAltered patterns ofMDM2and TP53 expression in human bladder cancerrdquo Journal of theNational Cancer Institute vol 86 no 17 pp 1325ndash1330 1994

[14] A Yoshida T Ushiku T Motoi et al ldquoMDM2 and CDK4immunohistochemical coexpression in high-grade osteosar-coma correlation with a dedifferentiated subtyperdquo The Amer-ican Journal of Surgical Pathology vol 36 no 3 pp 423ndash4312012

[15] M Choschzick U Heilenkotter A Lebeau et al ldquoMDM2amplification is an independent prognostic feature of node-negative estrogen receptor-positive early-stage breast cancerrdquoCancer Biomarkers vol 8 no 2 pp 53ndash60 2010


[16] L Benvegnu M Gios S Boccato and A Alberti ldquoNaturalhistory of compensated viral cirrhosis a prospective study onthe incidence and hierarchy of major complicationsrdquo Gut vol53 no 5 pp 744ndash749 2004

[17] J M Llovet A Burroughs and J Bruix ldquoHepatocellularcarcinomardquoThe Lancet vol 362 no 9399 pp 1907ndash1917 2003

[18] P J Johnson N Leung P Cheng et al ldquolsquoHepatoma-specificrsquoalphafetoprotein may permit preclinical diagnosis of malignantchange in patients with chronic liver diseaserdquo British Journal ofCancer vol 75 no 2 pp 236ndash240 1997

[19] J-Y Zhang C A Casiano X-X Peng J A Koziol E K LChan and E M Tan ldquoEnhancement of antibody detection incancer using panel of recombinant tumor-associated antigensrdquoCancer Epidemiology Biomarkers amp Prevention vol 12 no 2 pp136ndash143 2003

[20] J-Y Zhang R Megliorino X-X Peng E M Tan Y Chenand E K L Chan ldquoAntibody detection using tumor-associatedantigen mini-array in immunodiagnosing human hepatocellu-lar carcinomardquo Journal of Hepatology vol 46 no 1 pp 107ndash1142007

[21] J-Y Zhang E K L Chan X-X Peng and E M Tan ldquoA novelcytoplasmic proteinwith RNA-bindingmotifs is an autoantigenin human hepatocellular carcinomardquo Journal of ExperimentalMedicine vol 189 no 7 pp 1101ndash1110 1999

[22] L S Hoo J Y Zhang and E K L Chan ldquoCloning andcharacterization of a novel 90 kDa ldquocompanionrdquo auto-antigenof p62 overexpressed in cancerrdquo Oncogene vol 21 no 32 pp5006ndash5015 2002

[23] K S Looi E S Nakayasu R A de Diaz E M Tan IC Almeida and J-Y Zhang ldquoUsing proteomic approach toidentify tumor-associated antigens as markers in hepatocellularcarcinomardquo Journal of ProteomeResearch vol 7 no 9 pp 4004ndash4012 2008

[24] J Zhang K Wang J Zhang S S Liu L Dai and J-Y ZhangldquoUsing proteomic approach to identify tumor-associated pro-teins as biomarkers in human esophageal squamous cell carci-nomardquo Journal of Proteome Research vol 10 no 6 pp 2863ndash2872 2011

[25] Y Chen Y Zhou S Qiu et al ldquoAutoantibodies to tumor-associated antigens combined with abnormal alpha-fetoproteinenhance immunodiagnosis of hepatocellular carcinomardquo Can-cer Letters vol 289 no 1 pp 32ndash39 2010

[26] J-Y Zhang ldquoMini-array of multiple tumor-associated antigensto enhance autoantibody detection for immunodiagnosis ofhepatocellular carcinomardquo Autoimmunity Reviews vol 6 no 3pp 143ndash148 2007

[27] J Chen V Marechal and A J Levine ldquoMapping of the p53 andmdm-2 interaction domainsrdquo Molecular and Cellular Biologyvol 13 no 7 pp 4107ndash4114 1993

[28] YHaupt RMayaAKazaz andMOren ldquoMdm2promotes therapid degradation of p53rdquo Nature vol 387 no 6630 pp 296ndash299 1997

[29] Q W Chen H Chen J S Cheng and Z Q Meng ldquoMDM2SNP309TgtG polymorphism and hepatocellular carcinomarisk a meta-analysisrdquo Tumor Biology 2013

[30] S P Deb ldquoFunction and dysfunction of the human oncoproteinMDM2rdquo Frontiers in Bioscience vol 7 no 1 pp d235ndashd2432002

[31] T Tomoda K Nouso A Sakai et al ldquoGenetic risk of hepatocel-lular carcinoma in patients with hepatitis C virus a case controlstudyrdquo Journal of Gastroenterology and Hepatology vol 27 no4 pp 797ndash804 2012

[32] Y Liu S Kuang J Zheng et al ldquoMurine double minute 2rs2279744 polymorphism and hepatocellular carcinoma risk inEast Asians a meta-analysisrdquo Tumor Biology vol 35 no 2 pp961ndash965 2013

[33] S Ezzikouri A E El Feydi R Afifi et al ldquoMDM2 SNP309TgtG polymorphism and risk of hepatocellular carcinoma a case-control analysis in a Moroccan populationrdquo Cancer Detectionand Prevention vol 32 no 5 pp 380ndash385 2009

[34] Y J Yoon H Y Chang S H Ahn et al ldquoMDM2 andp53 polymorphisms are associated with the development ofhepatocellular carcinoma in patients with chronic hepatitis Bvirus infectionrdquo Carcinogenesis vol 29 no 6 pp 1192ndash11962008

[35] N Dharel N Kato R Muroyama et al ldquoMDM2 promoterSNP309 is associated with the risk of hepatocellular carcinomain patients with chronic hepatitis Crdquo Clinical Cancer Researchvol 12 no 16 pp 4867ndash4871 2006

[36] V di Vuolo L Buonaguro F Izzo et al ldquoTP53 andMDM2 genepolymorphisms and risk of hepatocellular carcinoma amongItalian patientsrdquo Infectious Agents and Cancer vol 6 article 132011

[37] H Akkiz A T Sumbul S Bayram A Bekar and E AkgolluldquoMDM2 promoter polymorphism is associated with increasedsusceptibility to hepatocellular carcinoma in Turkish popula-tionrdquo Cancer Epidemiology vol 34 no 4 pp 448ndash452 2010

[38] T Acun E Terzioglu-Kara O Konu M Ozturk and M CYakicier ldquoMdm2 Snp309 G allele displays high frequency andinverse correlation with somatic P53 mutations in hepatocellu-lar carcinomardquoMutation Research vol 684 no 1-2 pp 106ndash1082010

[39] Q Peng X Lao Z Chen et al ldquoTP53 and MDM2 genepolymorphisms gene-gene interaction and hepatocellular car-cinoma risk evidence from an updated meta-analysisrdquo PLoSONE vol 8 no 12 Article ID e82773 2013

[40] Y Yang T Xia N Li et al ldquoCombined effects of p53 andMDM2polymorphisms on susceptibility and surgical prognosisin hepatitis B virus-related hepatocellular carcinomardquoProteinampCell vol 4 no 1 pp 71ndash81 2013

[41] X Wang X Zhang B Qiu et al ldquoMDM2 SNP309TgtGpolymorphism increases susceptibility to hepatitis B virus-related hepatocellular carcinoma in a northeast Han Chinesepopulationrdquo Liver International vol 32 no 7 pp 1172ndash11782012

[42] Y Y Zhao Y G Li Y J Sun et al ldquoShRNA-mediated silencingof MDM2 inhibits growth of HepG2 hepatocellular carcinomacells xenografted in nude micerdquo Zhonghua Gan Zang Bing ZaZhi vol 21 no 3 pp 213ndash217 2013

[43] JWang T Zheng X Chen et al ldquoMDM2 antagonist can inhibittumor growth in hepatocellular carcinoma with different typesof p53 in vitrordquo Journal of Gastroenterology and Hepatology vol26 no 2 pp 371ndash377 2011

[44] M Schoniger-Hekele S Hanel F Wrba and C Muller ldquoHep-atocellular carcinomamdashsurvival and clinical characteristics inrelation to various histologic molecular markers in Westernpatientsrdquo Liver International vol 25 no 1 pp 62ndash69 2005

[45] S-J Qiu S-L Ye Z-Q Wu Z-Y Tang and Y-K Liu ldquoTheexpression of the mdm2 gene may be related to the aberrationof the p53 gene in human hepatocellular carcinomardquo Journal ofCancer Research and Clinical Oncology vol 124 no 5 pp 253ndash258 1998


[46] S Qiu S Ye and Z Wu ldquoThe expression of mdm2 generelated to the invasiveness of human hepatocellular carcinomardquoZhonghua Zhong Liu Za Zhi vol 19 no 4 pp 256ndash259 1997

[47] S Shangary and S Wang ldquoSmall-molecule inhibitors of theMDM2-p53 protein-protein interaction to reactivate p53 func-tion a novel approach for cancer therapyrdquo Annual Review ofPharmacology and Toxicology vol 49 no 1 pp 223ndash241 2009

[48] K Endo T Ueda T Ohta and T Terada ldquoProtein expressionof MDM2 and its clinicopathological relationships in humanhepatocellular carcinomardquo Liver vol 20 no 3 pp 209ndash2152000

[49] M-F Zhang Z-Y Zhang J Fu Y-F Yang and J-P YunldquoCorrelation between expression of p53 p21WAF1 andMDM2proteins and their prognostic significance in primary hepatocel-lular carcinomardquo Journal of Translational Medicine vol 7 no 1article 110 2009

[50] T J Song Y Fong S J Cho et al ldquoComparison of hepatocellularcarcinoma in American and Asian patients by tissue arrayanalysisrdquo Journal of Surgical Oncology vol 106 no 1 pp 84ndash882012

[51] D Dong X Gao Z Zhu Q Yub S Biana and Y GaoldquoA 40-bp insertiondeletion polymorphism in the constitutivepromoter of MDM2 confers risk for hepatocellular carcinomain a Chinese populationrdquo Gene vol 497 no 1 pp 66ndash70 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the cytosol in many cell lines suggesting that cytoplasmiclocalization is important for their function [8 9] MDM2both functions as an E3 ubiquitin ligase that recognizes theN-terminal transactivation domain (TAD) of the p53 and caninhibit p53 transcriptional activation [10] This protein alsoaffects the cell cycle apoptosis and tumorigenesis throughinteractions with other proteins including retinoblastoma1 and ribosomal protein L5 Overexpression of this genecan result in excessive inactivation of tumor protein p53diminishing its tumor suppressor function The MDM2proteins are deregulated in many human cancers and exerttheir oncogenic activity predominantly by inhibiting the p53tumor suppressor [11] Several human tumor types have beenshown to have increased levels of MDM2 including softtissue sarcomas bladder cancers and osteosarcomas as wellas breast tumors [12ndash15]

Diagnosis of HCC was considered as a terminal situationand the leading cause of death in cirrhotic patients [16]However when diagnosis is achieved at an early stageeffective therapies that improve long term survival are morelikely to be achieved [17] In such sense the early diagnosisof HCC is crucial for the treatment of patients In this studyMDM2 was evaluated by immunoassay as a potential TAA inHCC and autoantibody to this protein was also validated tobe an early stage biomarker in immunodiagnosis of HCC

2 Materials and Methods

21 Sera and General Information All sera used in this studyincluding 244 sera from patients with HCC 30 sera frompatients with liver cirrhosis (LC) 30 sera from patients withchronic hepatitis (CH) and 89 normal human sera (NHS) aswell as some sera from fourHCCpatients with serial bleedingsamples were obtained from the serum bank of CancerAutoimmunity and Epidemiology Research Laboratory atUTEP (University of Texas at El Paso) This study wasapproved by the Institutional Review Board of UTEP andcollaborating institutions

AllHCCpatients were diagnosed according to the criteriadescribed in a previous study [18] and had not receivedtreatment with any chemotherapy or radiotherapy Patientswith CH and LC were followed up at least 18 months aftercollecting blood to exclude individuals with primary biliarycirrhosis and asymptomatic or clinically undetectable HCCNormal human sera were collected from individuals at thesame locality during annual health examinations who had noobvious evidence of malignancy

22 Recombinant Proteins and Antibodies Used in This StudyMDM2 construct pGEX-4TMDM2WT (plasmid ID 16237)purchased from Addgenecom was subcloned into pET28avector producing a fusion protein with NH-terminal 6x histi-dine and T7 epitope tagsThe recombinant protein expressedin Escherichia coli BL21 (DE3) was purified using nickelcolumn chromatography Polyclonal anti-MDM2 rabbit anti-body and monoclonal anti-120573-actin mouse antibody werepurchased (Cell Signaling Technology Inc Danvers MA)Horseradish peroxidase- (HRP-) conjugated goat anti-human

IgG HRP-conjugated goat anti-rabbit IgG HRP-conjugatedgoat anti-mouse IgG and FITC-conjugated goat anti-humanIgG were purchased (Santa Cruz Biotechnology Inc SantaCruz CA) Anti-rabbit IgG Fab2 (Alexa Fluor 488) waspurchased (Cell Signaling Technology Inc Danvers MA)

23 Enzyme-Linked Immunosorbent Assay (ELISA) Stan-dard protocol for ELISA was used as described in ourprevious study [19 20] In brief a 96-well microtiter plate(ImmunoChemistry Technologies LLC Bloomington MN)was coated overnight at 4∘C with recombinant MDM2protein at a final concentration of 05120583gmL in phosphate-buffered saline (PBS) The antigen-coated wells were blockedwith gelatin postcoating solution at room temperature for2 h Human sera diluted at 1 100 with serum diluent wereincubated for 2 h at room temperature in the antigen-coatedwells followed by HRP-conjugated goat anti-human IgG(Caltag Laboratories San Francisco CA) at 1 4000 dilu-tion The substrate 221015840-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS Sigma-Aldrich St Louis MO) wasused as detecting reagent The average optical density (OD)value at a wavelength of 405 nm was used for data analysisThe cutoff value designating positive reaction was the meanoptical density (OD) of 89 normal human sera plus 3 standarddeviations (SD)

24 Western Blotting Denatured recombinant MDM2 pro-tein and cancer cell lysates were electrophoresed on 10 SDS-PAGE and transferred to nitrocellulose papers respectivelyAfter blocking in PBSwith 5 nonfatmilk and 005Tween-20 for 1 h at room temperature the nitrocellulose paperswere incubated overnight at 4∘C with 1 200 dilution ofhuman sera 1 500 dilution of polyclonal anti-MDM2 anti-body and 1 500 dilution of monoclonal anti-120573-actin mouseantibody separately HRP-conjugated goat anti-human IgGHRP-conjugated goat anti-rabbit IgG and HRP-conjugatedgoat anti-mouse IgG were respectively applied as secondaryantibody at a 1 10000 dilution The ECL-kit was used todetect immunoreactive bands according to the manufac-turerrsquos instructions (Thermo Scientific Waltham MA)

25 Indirect Immunofluorescence Assay (IIFA) and ConfocalMicroscopy Indirect immunofluorescence assay was per-formed on Hep2 antinuclear antigen tissue slides (BionEnterprises Des Plaines IL) The sera were diluted at1 80 in PBS and pH 74 and incubated with the slidesfor 30min at room temperature After extensive washingthe slides were incubated with fluorescein isothiocyanate-(FITC-) conjugated goat anti-human IgG secondary antibody(Santa Cruz Biotechnology CA) or anti-rabbit IgG Fab2(Alexa Fluor 488) as secondary antibody diluted 1 100 inPBS for 1 h at room temperature The slides were washedtwo times with PBS before adding a drop of mountingmedia containing 15 120583gmL 4101584061015840-diamidino-2-phenylindole(DAPI) (Vector Laboratories Inc Burlingame CA) to pre-vent photobleaching The slides were then examined underfluorescence microscopy LSM 700 Confocal Microscopy












3 Results


08

06

04

02

00

Opt

ical

den

sity

(OD

)

NHS CH LC HCC


1 2 3 4 5 6 7

MDM2

8

82kDa





010

008

006

004

002

000

Opt

ical

den

sity

(OD

)

1994

215

1994

510

1994

89

1994

11

8

1995

27

1995

512

lowast

(a)

03

02

01

00

Opt

ical

den

sity

(OD

)

1992

317

1992

616

1992

10

20

1992

11

24

1993

32

lowast

(b)

020

015

010

005

000

Opt

ical

den

sity

(OD

)

1992

918

1993

112

1993

511

1993

824

1993

10

27

lowast

(c)

015

010

005

000

Opt

ical

den

sity

(OD

)

1991

417

1991

731

1991

11

27

1992

129

1992

47

lowast

(d)






DAPI FITC Merge

(a)

(b)

(c)

(d)




Variable Frequency

Age ge60 7 233lt60 23 767






4 Discussion






(a) (b)

(c) (d)

(e) (f)










Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results


08

06

04

02

00

Opt

ical

den

sity

(OD

)

NHS CH LC HCC


1 2 3 4 5 6 7

MDM2

8

82kDa





010

008

006

004

002

000

Opt

ical

den

sity

(OD

)

1994

215

1994

510

1994

89

1994

11

8

1995

27

1995

512

lowast

(a)

03

02

01

00

Opt

ical

den

sity

(OD

)

1992

317

1992

616

1992

10

20

1992

11

24

1993

32

lowast

(b)

020

015

010

005

000

Opt

ical

den

sity

(OD

)

1992

918

1993

112

1993

511

1993

824

1993

10

27

lowast

(c)

015

010

005

000

Opt

ical

den

sity

(OD

)

1991

417

1991

731

1991

11

27

1992

129

1992

47

lowast

(d)






DAPI FITC Merge

(a)

(b)

(c)

(d)




Variable Frequency

Age ge60 7 233lt60 23 767






4 Discussion






(a) (b)

(c) (d)

(e) (f)










Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















010

008

006

004

002

000

Opt

ical

den

sity

(OD

)

1994

215

1994

510

1994

89

1994

11

8

1995

27

1995

512

lowast

(a)

03

02

01

00

Opt

ical

den

sity

(OD

)

1992

317

1992

616

1992

10

20

1992

11

24

1993

32

lowast

(b)

020

015

010

005

000

Opt

ical

den

sity

(OD

)

1992

918

1993

112

1993

511

1993

824

1993

10

27

lowast

(c)

015

010

005

000

Opt

ical

den

sity

(OD

)

1991

417

1991

731

1991

11

27

1992

129

1992

47

lowast

(d)






DAPI FITC Merge

(a)

(b)

(c)

(d)




Variable Frequency

Age ge60 7 233lt60 23 767






4 Discussion






(a) (b)

(c) (d)

(e) (f)










Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DAPI FITC Merge

(a)

(b)

(c)

(d)




Variable Frequency

Age ge60 7 233lt60 23 767






4 Discussion






(a) (b)

(c) (d)

(e) (f)










Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Variable Frequency

Age ge60 7 233lt60 23 767






4 Discussion






(a) (b)

(c) (d)

(e) (f)










Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)










Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Autoantibody Response to Murine...

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