research article demonstration of hepatitis c virus rna...

Hindawi Publishing CorporationInternational Journal of HepatologyVolume 2013 Article ID 249535 7 pageshttpdxdoiorg1011552013249535

Research ArticleDemonstration of Hepatitis C Virus RNA with In SituHybridization Employing a Locked Nucleic Acid Probe inHumanized Liver of Infected Chimeric Mice and inNeedle-Biopsied Human Liver

Kazuya Shiogama1 Ken-ichi Inada1 Michinori Kohara2 Hidemi Teramoto3

Yasuyoshi Mizutani1 Takanori Onouchi1 and Yutaka Tsutsumi1

1 Department of Pathology Fujita Health University School of Medicine Toyoake Aichi 470-1192 Japan2Department of Microbiology and Cell Biology Tokyo Metropolitan Institute of Medical Science Tokyo 156-8506 Japan3Department of Internal Medicine Kojin Hospital Nagoya 463-8530 Japan

Correspondence should be addressed to Yutaka Tsutsumi tsutsumifujita-huacjp

Received 30 December 2012 Revised 21 May 2013 Accepted 23 May 2013

Academic Editor Herve Lerat

Copyright copy 2013 Kazuya Shiogama 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

Background In situ hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA informalin-fixed paraffin-embedded (FFPE) sections of the liver Methods ISH employing a locked-nucleic-acid- (LNA-)modifiedoligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in theliver tissue Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using totalRNA extracted from FFPE sectionsThe target tissues included FFPE tissue sections of humanized livers in HCV-infected chimericmice (HCVgenotypes 1a 1b and 2a and noninfected) and of needle-biopsied livers fromHCV-infected patientsResults HCV-RNAwas demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82) of 11 patients withHCV infection The HCV signals were sensitive to RNase Nested RT-PCR confirmed the genotype in 8 (73) of 11 livers (type 1b6 lesions and type 2a 2 lesions) HCV-RNA was not identified in chronic hepatitis B lesions fatty liver autoimmune hepatitis andhepatocellular carcinoma Conclusion ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detectingHCV-RNA in routinely prepared FFPE liver specimens

1 Introduction

Hepatitis C virus (HCV) is a single-stranded RNA virus amember of the Flaviviridae family Since the first identifica-tion of the HCV genome by Choo et al [1] HCV study hasprogressed mainly in the field of HCV functional analysisand therapeutic implications Because of low viral levels inthe serum the diagnosis of HCV infection has been madewith a branched chain DNA signal amplification assay andreverse-transcription polymerase chain reaction (RT-PCR)[2ndash4] The sensitivity of HCV detection in the serum thusbecame reproducible and clinically relevant

Pathologists are often requested to detect viral pathogenswithin the routinely prepared biopsy tissue samples and

therefore simple and reliable histochemical techniques areneeded Techniques for visualizing HCV localization withindiseased hepatocytes have also been developed Reports havedescribed the detection ofHCV inhuman liver tissue by usingimmunohistochemistry and in situ hybridization (ISH) [4ndash14] as well as by in situ RT-PCR [4 6 15ndash20] At the momentHCV detection with immunohistochemistry and ISH is notyet reproducible enough we believe In situ RT-PCR maydemonstrate highly sensitive signals but with concomitantincrease of false positivity Reliable histochemical techniquesfor detecting HCV in formalin-fixed paraffin-embedded(FFPE) liver tissues are needed particularly for routinediagnostic purpose

2 International Journal of Hepatology

In the present study we established an ISH techniqueusing a locked nucleic acid (LNA) probe and biotin-freetyramide amplification system (CSAII) for detecting HCV-RNA in FFPE tissue sections of the liver

2 Materials and Methods

21 Samples As positive controls we sampled the humanizedliver of HCV-infected chimericmice [21 22] and FFPE tissuesections were prepared The chimeric mice were maintainedbyM Kohara Department ofMicrobiology and Cell BiologyTokyo Metropolitan Institute of Medical Science Tokyo Atotal of 13 humanized liver specimens from HCV-infectedchimeric mice were used (noninfected 3 genotype 1a-infect-ed 3 genotype 1b-infected 4 and genotype 2a-infected 3)

A total of 11 needle biopsy liver specimens of chronichepatitis Cwere culled from the computer file at theDiagnos-tic Pathology Division of Fujita Health University HospitalToyoake Japan and FFPE sections were cut Needle biopsyspecimens of hepatitis-B-virus- (HBV-)infected chronic hep-atitis virus-negative fatty liver and autoimmune hepatitis(119899 = 1 resp) as well as two surgically removed HCC lesionscaused by HCV or HBV infection were also examinedThe nonneoplastic part of the liver tissue (chronic hepatitisB) in the HBV-related HCC case was also evaluated Non-neoplastic liver tissue was scarcely included in the HCV-related HCC case Hematoxylin and eosin (HE) staining wasperformed for demonstrating histological featuresThe activ-ity of inflammation and the degree of fibrosis were evaluatedaccording to the New Inuyama Classification (1996) [23]SerumHCV-RNA levels in the patients were measured usingan RT-PCR assay the Amplicor HCVMonitor Assay (RocheDiagnostics) and serum HCV subtypes were determinedwith nested RT-PCR (Roche Diagnostics)

22 LNA-Modified Oligonucleotide Probe The sequence ofan HCV-common probe was designed according to theprevious description [15] The target was within the HCV51015840-untranslated region (HCV-51015840UTR) which is a highlyconserved portion in the HCV genome An LNA-modifiedoligonucleotide probe labeled with digoxygenin at the 31015840-end was prepared in Gene Design Inc Ibaraki OsakaJapan The nucleotide sequence of the 45-mer probe was51015840-ALtTTLgGGLcGTLgCCLcCCLgCGLaGALcTGLcTALg-CCLgAGLtAGLtGTLtGGLgT-31015840 in which La Lt Lc and Lgrepresent LNA monomers corresponding to the bases A TC and G respectively

23 ISH FFPE sections were dewaxed in xylene and rehy-drated in graded ethanol Endogenous peroxidase activitywas quenched with 03 hydrogen peroxide in methanolfor 60 minutes at room temperature (RT) After rinsingthrice in diethyl-pyrocarbonate- (DEPC-)treated water sec-tions were digested with 40 120583gmL proteinase K (RocheDiagnostics Tokyo Japan) at 37∘C for 15 minutes washedthrice in DEPC-treated water submerged in 95 ethanolfor 1 minute and air-dried completely Then sections wereheat-treated for 5 minutes at 95∘C on a hot plate and

hybridized with 001120583molL LNA-modified oligonucleotideprobe diluted with in situ Hybridization Buffer (Enzo LifeScience Bulter Pike PA USA) in an incubation chamberovernight at 37∘C After rinsing in 1x saline sodium citrate(SSC) for 30 minutes at 50∘C sections were rinsed twicein 50mM Tris-HCl-buffered saline (TBS) pH 76 at RTA horseradish-peroxidase-labeled antidigoxygenin antibody(Roche Diagnostics) diluted at 1 100 with 1 bovine serumalbumin in TBS was applied for 60 minutes at RT For ampli-fying signals fluorescein-isothiocyanate- (FITC-)conjugatedtyramide enclosed in CSAII (Dako Glostrup Denmark) wasapplied to the sections for 15 minutes at RT Finally a horse-radish-peroxidase-labeled anti-FITC antibody equipped inthe CSAII kit was reacted for 30 minutes at RT Reactionproducts were visualized in 50mM Tris-HCl buffer pH 76containing 20mgdL diaminobenzidine tetrahydrochlorideand 0006 hydrogen peroxidase The nuclei were lightlycounterstained with Mayerrsquos hematoxylin

In order to verify the specificity of the ISH techniquefor HCV-RNA detection we carried out the treatment withDNase and RNase before the hybridization with the LNAprobe on consecutive sections Namely either 10 IUmLDNase I (Wako Osaka Japan) or 100 120583gmL RNase (Sigma-Aldrich St Louis MO USA) was incubated for 30 minutesat 37∘C

24 Immunostaining Using a Human Specific Cytokeratin 818(CK818) Antibody In order to distinguish mouse hepato-cytes from chimeric human hepatocytes in HCV-infectedchimeric mice we utilized a human-specific CK818 mono-clonal antibody (clone NCL 5D3 MP Biochemicals SantaAna CA USA) After blocking endogenous peroxidase activ-ity hydrated heat-assisted epitope retrieval was employedusing a pressure pan cooker (Delicio 6L T-FAL ClichyFrance) for 10 minutes Preliminary study revealed that asa soaking solution 1mM ethylenediamine tetraacetic acidsolution pH 80 was optimal to retrieve the antigenicityAfter pressure cooking sectionswere left for 30minutes at RTfor cooling In order for avoiding nonspecific signals inmousehepatocytes a Mouse Stain kit (Nichirei Bioscience Tokyo)was applied before and after the primary antibody incubationThe human-specific CK818 monoclonal antibody at a 1 100dilution was incubated overnight at RT After rinsing in10mM phosphate-buffered saline (PBS) pH 72 the sectionswere reacted with a secondary polymer reagent Novolink(Novocastra Newcastle UK) Reaction products were visu-alized in the diaminobenzidine-hydrogen peroxide solutionNuclear counterstaining with hematoxylin followed

25 HCV Genotyping by Nested RT-PCR Five slices of 5120583mthickness sections were collected in Eppendorf tubes TotalRNA was extracted from dewaxed sections using RecoverAllTotal Nucleic Acid Isolation kit (Applied Biosytems AustinTX USA) according to the manufacturerrsquos protocol andstored at minus80∘C until use Nested RT-PCR for HCV genotyp-ing was performed as described previously [24]

International Journal of Hepatology 3

Table 1 HCV-RNA demonstration with LNA-based ISH and nested RT-PCR in FFPE human liver tissues

Patient Biopsy orsurgery

DiagnosisNew Inuyama Classification

Serum FFPE liver tissueHCV-RNA(KIUmL)

NestedRT-PCR

NestedRT-PCR ISH

Case 1 B Chronic hepatitis C A2F2 51 times 102 ≦ 1b 1b + FocalpartialCase 2 B Chronic hepatitis C A1F1 28 times 102 1b 1b + FocalpartialCase 3 B Chronic hepatitis C A2F1 24 times 102 2a 2a + DiffuseCase 4 B Chronic hepatitis C A1F0 19 times 102 2a 2a minus

Case 5 B Chronic hepatitis C A1F1 25 times 102 1b 1b + FocalpartialCase 6 B Chronic hepatitis C A2F3 27 times 102 1b minus minus

Case 7 B Chronic hepatitis C A2F1 22 times 102 2a minus + FocalpartialCase 8 B Chronic hepatitis C A3F3 51 times 102 1b 1b + FocalpartialCase 9 B Chronic hepatitis C A2F1 29 times 102 1b 1b + DiffuseCase 10 B Chronic hepatitis C A2F2 31 times 102 2a minus + FocalpartialCase 11 B Chronic hepatitis C A2F3 33 times 102 1b 1b + DiffuseCase 12 B Chronic hepatitis B A3F3 Not detected Not done minus minus

Case 13 B Fatty liver A0F0 Not detected Not done minus minus

Case 14 B Autoimmune hepatitis A3F3 Not detected Not done minus minus

Case 15 S Hepatitis-B-related HCCChronic hepatitis B A2F2 Not detected Not done minus

minus

minus

Case 16 S Hepatitis-C-related HCC 84 Not done minus minus

New Inuyama Classification (1996)Activity of inflammation A1 mild A2 moderate and A3 severeFibrosis F0 none F1 portal widening F2 bridging fibrosis and F3 bridging fibrosis with lobular distortion

26 Ethical Issue The present study was approved by theinstitutional ethical review board for clinical and epidemi-ological investigations at Fujita Health University ToyoakeThe approval number is 12-193Written informed consentwasobtained from each patient

3 Results

31 HCV-RNA Localization in Humanized Liver of HCV-Infected Chimeric Mice In HE-stained sections mousehepatocytes showed densely eosinophilic cytoplasm whilechimeric human hepatocytes were weakly eosinophilic withfrequent deposition of fat droplets Immunoreactivity ofhuman CK818 was seen only in human hepatocytes In allthe 10 HCV-infected livers but not the livers of three HCVnoninfected mice ISH demonstrated diffuse cytoplasmicsignals in the human hepatocytes In Figure 1 employingconsecutive sections HCV-RNA was visualized with the ISHtechnique in human CK818-positive chimeric hepatocytesfrom chimeric mice infected HCV genotypes 1a 1b and 2a(in Figure 1 dotted lines encircle the mouse hepatocyte area)No signals were detected in the noninfected chimeric livertissue It is of note that HCV-RNA was demonstrated inthe cytoplasm of almost all chimeric human hepatocytesThe positive signals remained after DNase treatment butwere completely abolished after RNase treatment Figure 2illustrates HCV genotype 1b-infected chimeric mouse livershowing HE histology and positive cytoplasmic signalswhich were resistant to DNase but sensitive to RNase

32 HCV-RNA Localization in Needle-Biopsied or SurgicallyRemoved Human Liver Tissues Results of the human mate-rial are summarized in Table 1 Most of the HCV-positivepatients showed high levels of HCV-RNA in the serumExceptionally a surgical case of HCV-related HCC showeda low level of HCV-RNA in the serum Nested RT-PCRgenotyping for HCV using total RNA extracted from FFPEsections was successful in 8 (73) of 11 biopsied chronichepatitis C lesions and in all the 8 cases the genotypeswere comparable with the serum analysis LNA-based ISHdetected signals in 9 (82) of 11 chronic hepatitis C lesionsincluding three showing diffuse cytoplasmic positivity andsix showing focal or partial positivity In three nested RT-PCR negative lesions ISH revealed negativity (1 lesion) orfocalpartial positivity (2 lesions) One lesion was nested RT-PCR-positive and ISH-negative No HCV-RNA was detectedin HBV-related lesions fatty liver autoimmune hepatitisand in HCC lesions Figure 3 demonstrates representativehistochemical findings Nonspecific binding of the CSAIIreagent to Kupffer cells or lymphoid cells was occasionallyobserved in both the HCV-infected and HCV-unrelatedlesions as arrows indicate

4 Discussion

We showed herein that LNA-based ISH for HCV-RNAyielded clear cytoplasmic reactivity in infected hepatocytesin FFPE sections The LNA-modified oligonucleotide probewe employed represents one of the most sensitive probes for


lowast

lowastlowast

(a) (b) (c)

lowastlowastlowast

(d) (e) (f)

lowast

lowastlowast

(g) (h) (i)

lowast

lowastlowast

(j) (k) (l)

Figure 1 HCV-RNA localization with LNA-based ISH in the liver of chimeric mice infected with HCV of different genotypes (a)ndash(c) HCVnoninfected (d)ndash(f) HCV genotype 1a-infected (g)ndash(i) HCV genotype 1b-infected (j)ndash(l) HCV genotype 2a-infected ((a) (d) (g) (j)) HEstain ((b) (e) (h) (k)) immunostaining for human-specificCK818 ((c) (f) (i) (l)) LNA-based ISH (lowast)mouse hepatocytes and (lowastlowast) humanhepatocytes HCV-RNA is demonstrated diffusely in the cytoplasm of human hepatocytes in the chimeric livers No signals are seen in thenoninfected liver

ISH [25ndash29] LNA-based ISH has been applied to detectingcellular microRNA in FFPE tumor tissues [30ndash32] The 45-mer LNA probe was targeted at the HCV-51015840UTR containinga sequence common to the HCV subtypes Positive signalsfor HCV-RNA were clearly demonstrated in HCV 1a- 1b-and 2a-infected humanized livers of chimeric mice HCV-infected chimeric mice with humanized liver thus functioned

as a usefulmodel for histochemically demonstrating theHCVgenome particularly when immunostaining for human-specific CK818 was combined

In needle-biopsied human liver specimens HCV-RNAwas detected in 8 of 11 HCV-infected samples with the nestedRT-PCR analysis and in 9 of 11 with the ISH technique Innested RT-PCR negative cases ISH was negative or partially


lowast

lowastlowast

(a) (b) (c)

Figure 2 Confirmation of the specificity of LNA-based ISH (a) HE stain (b) ISH after DNase treatment (c) ISH after RNase treatment (lowast)mouse hepatocytes and (lowastlowast) human hepatocytesThemouse hepatocytes are densely eosinophilic while the human hepatocytes appear palewith deposition of fat droplets HCV-RNA is observed only in the human hepatocytes in the chimeric liver infected with HCV genotype 1bThe HCV-RNA signals are resistant to DNase treatment but disappear after RNase treatment

(a) (b) (c) (d)

(e) (f) (g) (h) (i) (j)

Figure 3 HCV-RNA localization with LNA-based ISH in FFPE biopsy or surgical specimens of the liver ((a) (b)) Case 10 with chronichepatitis C ((c) (d)) case 11 with chronic hepatitis C ((e) (f)) case 15 with chronic hepatitis B (noncancerous portion of a surgical case forHCC) ((g) (h)) case 13 with fatty liver ((i) (j)) case 16 with surgically removed hepatitis-C-related HCC ((a) (c) (e) (g) (i)) HE stain and((b) (d) (f) (h) (j)) LNA-based ISH In HCV-infected human livers the HCV-RNA signals are demonstrated in a focalpartial pattern (b)or in a diffuse pattern (d) Signals are not demonstrated in the hepatocytes of HBV-infected liver (f) fatty liver (h) and hepatitis-C-relatedHCC (j) Nonspecific binding of the reagent to Kupffer cells and lymphoid cells is occasionally observed (arrows in the panels (b) (d) (f)and (h))

positive suggesting that the detection threshold may dependon the virus load Diffuse HCV expression pattern in thehepatocytes was already reported by Nuriya et al [15] Lianget al [33] and Li et al [34]

In the present ISH study HCV-RNA was undetectablein the hepatocytes in HBV-infected livers nonviral liverdisorders (fatty liver and autoimmune hepatitis) and inHCV-related HCC With ISH using a digoxygenin-labeledcDNA probe Tang et al reported positivity in 8 (67) of 12HCV-related HCC [12] It has been documented with an insitu RT-PCR technique that HCV-RNA decreases along withthe progression from liver cirrhosis to HCC [35] More cases

of HCC need to be analyzed in relation to the distribution ofthe HCV genome in cancerous lesions

Revie and Salahuddin reviewed that HCV replicated inmacrophages B and T lymphocytes and other nonhepa-tocellular components [36] However we judged the non-hepatocellular bindings as nonspecific reactions primarilybecause of the staining in non-HCV cases Under our presentcondition nonspecific binding of the reagents particularlyCSAII to Kupffer cells and lymphoid cells was observedFurther technical improvement is requested for reliable andreproducible HCV-RNA detection in FFPE specimens

In conclusion the ISH technique using the LNA-modifiedoligonucleotide probe and CSAII can be applied to detecting


HCV-RNA in liver biopsy specimens the sensitivity probablybeing comparable to in situ RT-PCR For detecting the HCVgenome in routinely prepared liver biopsy specimens ourISH sequence is relatively simple and requires no specialequipment to perform At the moment this method seems tobe suitable for demonstrating HCV genome in FFPE sectionsand will become a valuable tool for routine histopathologicaldiagnosis of HCV infection using FFPE liver biopsy speci-mens

Conflict of Interests

The authors have no conflict of interests

Acknowledgments

The authors thank Mr Yutaka Hirasawa and Ms Satomi ItoDivision of Diagnostic Pathology Fujita Health UniversityHospital for their cooperation in culling the specimensProfessor Kentaro Yoshioka Department of Liver BiliaryTract and Pancreas Diseases and Professor Atsushi SugiokaDepartment of Surgery Fujita Health University School ofMedicine Toyoake kindly allowed the authors to use thebiopsy specimens The skillful technical assistance by MsMika Maeshima and Ms Sayaka Takeuchi Department ofPathology Fujita Health University School of Medicine isalso cordially acknowledgedThis workwas in part supportedby the Research Grant-in-Aid from the Japanese Ministryof Education Culture Sports Science and Technology (no24790367)

References

[1] Q-L Choo G Kuo A J Weiner L R Overby D W Bradleyand M Houghton ldquoIsolation of a cDNA clone derived from ablood-borne non-A non-B viral hepatitis genomerdquo Science vol244 no 4902 pp 359ndash362 1989

[2] A M Majid and D R Gretch ldquoCurrent and future hepatitis Cvirus diagnostic testing problems and advancementsrdquoMicrobesand Infection vol 4 no 12 pp 1227ndash1236 2002

[3] L Muratori ldquoIn situ reverse transcriptase-polymerase chainreaction an innovative tool for hepatitis C virus RNA detectionand localisation and for quantification of infected cellsrdquo Euro-pean Journal of Histochemistry vol 42 no 2 pp 133ndash136 1998

[4] P Komminoth V Adams A A Long et al ldquoEvaluationof methods for hepatitis C virus detection in archival liverbiopsies Comparison of histology immunohistochemistry in-situ hybridization reverse transcriptase polymerase chain reac-tion (RT-PCR) and in-situ RT-PCRrdquo Pathology Research andPractice vol 190 no 11 pp 1017ndash1025 1994

[5] H S Carvalho M L Baptista M A Pinto et al ldquoDetection ofhepatitis C virus RNA by in situ hybridization in paraformalde-hyde fixed biopsiesrdquo Memorias do Instituto Oswaldo Cruz vol100 no 3 pp 269ndash272 2005

[6] X Qian R B Guerrero T B Plummer V F Alves and R VLloyd ldquoDetection of hepatitis C virus RNA in formalin-fixedparaffin-embedded sections with digoxigenin-labeled cRNAprobesrdquo Diagnostic Molecular Pathology vol 13 no 1 pp 9ndash142004

[7] S de Lucas J Bartolome E Rodrıguez-Inigo et al ldquoDistribu-tion of hepatitis C virus infection in liver biopsies from childrenand adults with chronic hepatitis Crdquo Journal ofMedical Virologyvol 64 no 1 pp 1ndash5 2001

[8] M Chang A P Marquardt B L Wood et al ldquoin situ distribu-tion of hepatitis C virus replicative-intermediate RNA in hep-atic tissue and its correlation with liver diseaserdquo Journal ofVirology vol 74 no 2 pp 944ndash955 2000

[9] V Agnello G Abel G B Knight and EMuchmore ldquoDetectionof widespread hepatocyte infection in chronic hepatitis CrdquoHepatology vol 28 no 2 pp 573ndash584 1998

[10] D Sansonno V Cornacchiulo V Racanelli and F DammaccoldquoIn situ simultaneous detection of hepatitis C virus RNA andhepatitis C virus-related antigens in hepatocellular carcinomardquoCancer vol 80 no 1 pp 22ndash33 1997

[11] S W Cho S G Hwang D C Han et al ldquoIn situ detection ofhepatitis C virus RNA in liver tissue using a digoxigenin-labeledprobe created during a polymerase chain reactionrdquo Journal ofMedical Virology vol 48 no 3 pp 227ndash233 1996

[12] L Tang Y Tanaka N Enomoto F Marumo and C SatoldquoDetection of hepatitis C virus RNA in hepatocellular carci-noma by in situ hybridizationrdquo Cancer vol 76 no 11 pp 2211ndash2216 1995

[13] M Gastaldi A Massacrier R Planells et al ldquoDetection by insituhybridization of hepatitis C virus positive andnegativeRNAstrands using digoxigenin-labeled cRNA probes in human livercellsrdquo Journal of Hepatology vol 23 no 5 pp 509ndash518 1995

[14] Y Tanaka N Enomoto S Kojima et al ldquoDetection of hepatitisC virus RNA in the liver by in situ hybridizationrdquo Liver vol 13no 4 pp 203ndash208 1993

[15] H Nuriya K Inoue T Tanaka et al ldquoDetection of hepatitis Band C viruses in almost all hepatocytes by modified PCR-basedin situ hybridizationrdquo Journal of Clinical Microbiology vol 48no 11 pp 3843ndash3851 2010

[16] A J Alzahrani P J Vallely and R F T McMahon ldquoDevelop-ment of a novel nested in situ PCR-ISH method for detectionof hepatitis C virus RNA in liver tissuerdquo Journal of VirologicalMethods vol 99 no 1-2 pp 53ndash61 2002

[17] P Biagini L Benkoel F Dodero et al ldquoHepatitis C virusRNA detection by in situ RT-PCR in formalin-fixed paraffin-embedded liver tissue Comparison with serum and tissueresultsrdquo Cellular and Molecular Biology vol 47 pp OL167ndashOL171 2001

[18] D Bettinger CMougin B Fouque B Kantelip J PMiguet andM Lab ldquoDirect in situ reverse transcriptase-linked polymerasechain reaction with biotinylated primers for the detection ofhepatitis C virus RNA in liver biopsiesrdquo Journal of ClinicalVirology vol 12 no 3 pp 233ndash241 1999

[19] F M Walker M Dazza M Dauge et al ldquoDetection and local-ization by in situmolecular biology techniques and immunohis-tochemistry of hepatitis C virus in livers of chronically infectedpatientsrdquo Journal of Histochemistry and Cytochemistry vol 46no 5 pp 653ndash660 1998

[20] G K K Lau J W S Fang P C Wu G L Davis and J Y NLau ldquoDetection of hepatitis C virus genome in formalin-fixedparaffin-embedded liver tissue by in situ reverse transcriptionpolymerase chain reactionrdquo Journal of Medical Virology vol 44no 4 pp 406ndash409 1994

[21] C Tateno Y YoshizaneN Saito et al ldquoNear completely human-ized liver in mice shows human-type metabolic responses todrugsrdquo American Journal of Pathology vol 165 no 3 pp 901ndash912 2004


[22] D F Mercer D E Schiller J F Elliott et al ldquoHepatitis Cvirus replication in mice with chimeric human liversrdquo NatureMedicine vol 7 no 8 pp 927ndash933 2001

[23] F Ichida ldquoHistological grading criteria of chronic hepatitis newInuyama classificationrdquo in Proceedings of the 19th Inuyama Sym-posium pp 183ndash188 Chugai Igaku 1996

[24] K Shiogama H Teramoto Y Morita et al ldquoHepatitis C virusinfection in a Japanese leprosy sanatorium for the past 67 yearsrdquoJournal of Medical Virology vol 82 no 4 pp 556ndash561 2010

[25] L A Neely S Patel J Garver et al ldquoA single-molecule methodfor the quantitation of microRNA gene expressionrdquo NatureMethods vol 3 no 1 pp 41ndash46 2006

[26] W P Kloosterman E Wienholds E de Bruijn S Kauppinenand R H A Plasterk ldquoIn situ detection of miRNAs in animalembryos using LNA-modified oligonucleotide probesrdquo NatureMethods vol 3 no 1 pp 27ndash29 2006

[27] K Kubota A Ohashi H Imachi and H Harada ldquoImprovedin situ hybridization efficiency with locked-nucleic-acid- incor-poratedDNAprobesrdquoApplied and EnvironmentalMicrobiologyvol 72 no 8 pp 5311ndash5317 2006

[28] R Thomsen P S Nielsen and T H Jensen ldquoDramaticallyimproved RNA in situ hybridization signals using LNA-modi-fied probesrdquo RNA vol 11 no 11 pp 1745ndash1748 2005

[29] A Valoczi C Hornyik N Varga J Burgyan S Kauppinenand Z Havelda ldquoSensitive and specific detection of microRNAsby northern blot analysis using LNA-modified oligonucleotideprobesrdquoNucleic Acids Research vol 32 no 22 article e175 2004

[30] K Sakurai C Furukawa T Haraguchi et al ldquoMicroRNAsmiR-199a-5p and -3p target the Brm subunit of SWISNF to generatea double-negative feedback loop in a variety of human cancersrdquoCancer Research vol 71 no 5 pp 1680ndash1689 2011

[31] N Yamamichi R Shimomura K Inada et al ldquoLocked nucleicacid in situ hybridization analysis of miR-21 expression duringcolorectal cancer developmentrdquo Clinical Cancer Research vol15 no 12 pp 4009ndash4016 2009

[32] P T Nelson D A BaldwinW P Kloosterman S Kauppinen RH A Plasterk and Z Mourelatos ldquoRAKE and LNA-ISH revealmicroRNA expression and localization in archival humanbrainrdquo RNA vol 12 no 2 pp 187ndash191 2006

[33] Y Liang T Shilagard S Xiao et al ldquoVisualizing hepatitis Cvirus infections in human liver by two-photon microscopyrdquoGastroenterology vol 137 no 4 pp 1448ndash1458 2009

[34] G Li K Li A S Lea et al ldquoIn situ hybridization for thedetection of hepatitis C virus RNA in human liver tissuerdquoJournal of Viral Hepatitis vol 20 no 3 pp 183ndash192 2013

[35] T Tanaka K Inoue Y Hayashi et al ldquoVirological significanceof low-level hepatitis B virus infection in patients with hepatitisC virus associated liver diseaserdquo Journal ofMedical Virology vol72 no 2 pp 223ndash229 2004

[36] D Revie and S Z Salahuddin ldquoHuman cell types important forHepatitis C Virus replication in vivo and in vitro Old assertionsand current evidencerdquo Virology Journal vol 8 pp 346ndash3702011

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


In the present study we established an ISH techniqueusing a locked nucleic acid (LNA) probe and biotin-freetyramide amplification system (CSAII) for detecting HCV-RNA in FFPE tissue sections of the liver

2 Materials and Methods

21 Samples As positive controls we sampled the humanizedliver of HCV-infected chimericmice [21 22] and FFPE tissuesections were prepared The chimeric mice were maintainedbyM Kohara Department ofMicrobiology and Cell BiologyTokyo Metropolitan Institute of Medical Science Tokyo Atotal of 13 humanized liver specimens from HCV-infectedchimeric mice were used (noninfected 3 genotype 1a-infect-ed 3 genotype 1b-infected 4 and genotype 2a-infected 3)

A total of 11 needle biopsy liver specimens of chronichepatitis Cwere culled from the computer file at theDiagnos-tic Pathology Division of Fujita Health University HospitalToyoake Japan and FFPE sections were cut Needle biopsyspecimens of hepatitis-B-virus- (HBV-)infected chronic hep-atitis virus-negative fatty liver and autoimmune hepatitis(119899 = 1 resp) as well as two surgically removed HCC lesionscaused by HCV or HBV infection were also examinedThe nonneoplastic part of the liver tissue (chronic hepatitisB) in the HBV-related HCC case was also evaluated Non-neoplastic liver tissue was scarcely included in the HCV-related HCC case Hematoxylin and eosin (HE) staining wasperformed for demonstrating histological featuresThe activ-ity of inflammation and the degree of fibrosis were evaluatedaccording to the New Inuyama Classification (1996) [23]SerumHCV-RNA levels in the patients were measured usingan RT-PCR assay the Amplicor HCVMonitor Assay (RocheDiagnostics) and serum HCV subtypes were determinedwith nested RT-PCR (Roche Diagnostics)

22 LNA-Modified Oligonucleotide Probe The sequence ofan HCV-common probe was designed according to theprevious description [15] The target was within the HCV51015840-untranslated region (HCV-51015840UTR) which is a highlyconserved portion in the HCV genome An LNA-modifiedoligonucleotide probe labeled with digoxygenin at the 31015840-end was prepared in Gene Design Inc Ibaraki OsakaJapan The nucleotide sequence of the 45-mer probe was51015840-ALtTTLgGGLcGTLgCCLcCCLgCGLaGALcTGLcTALg-CCLgAGLtAGLtGTLtGGLgT-31015840 in which La Lt Lc and Lgrepresent LNA monomers corresponding to the bases A TC and G respectively

23 ISH FFPE sections were dewaxed in xylene and rehy-drated in graded ethanol Endogenous peroxidase activitywas quenched with 03 hydrogen peroxide in methanolfor 60 minutes at room temperature (RT) After rinsingthrice in diethyl-pyrocarbonate- (DEPC-)treated water sec-tions were digested with 40 120583gmL proteinase K (RocheDiagnostics Tokyo Japan) at 37∘C for 15 minutes washedthrice in DEPC-treated water submerged in 95 ethanolfor 1 minute and air-dried completely Then sections wereheat-treated for 5 minutes at 95∘C on a hot plate and

hybridized with 001120583molL LNA-modified oligonucleotideprobe diluted with in situ Hybridization Buffer (Enzo LifeScience Bulter Pike PA USA) in an incubation chamberovernight at 37∘C After rinsing in 1x saline sodium citrate(SSC) for 30 minutes at 50∘C sections were rinsed twicein 50mM Tris-HCl-buffered saline (TBS) pH 76 at RTA horseradish-peroxidase-labeled antidigoxygenin antibody(Roche Diagnostics) diluted at 1 100 with 1 bovine serumalbumin in TBS was applied for 60 minutes at RT For ampli-fying signals fluorescein-isothiocyanate- (FITC-)conjugatedtyramide enclosed in CSAII (Dako Glostrup Denmark) wasapplied to the sections for 15 minutes at RT Finally a horse-radish-peroxidase-labeled anti-FITC antibody equipped inthe CSAII kit was reacted for 30 minutes at RT Reactionproducts were visualized in 50mM Tris-HCl buffer pH 76containing 20mgdL diaminobenzidine tetrahydrochlorideand 0006 hydrogen peroxidase The nuclei were lightlycounterstained with Mayerrsquos hematoxylin

In order to verify the specificity of the ISH techniquefor HCV-RNA detection we carried out the treatment withDNase and RNase before the hybridization with the LNAprobe on consecutive sections Namely either 10 IUmLDNase I (Wako Osaka Japan) or 100 120583gmL RNase (Sigma-Aldrich St Louis MO USA) was incubated for 30 minutesat 37∘C

24 Immunostaining Using a Human Specific Cytokeratin 818(CK818) Antibody In order to distinguish mouse hepato-cytes from chimeric human hepatocytes in HCV-infectedchimeric mice we utilized a human-specific CK818 mono-clonal antibody (clone NCL 5D3 MP Biochemicals SantaAna CA USA) After blocking endogenous peroxidase activ-ity hydrated heat-assisted epitope retrieval was employedusing a pressure pan cooker (Delicio 6L T-FAL ClichyFrance) for 10 minutes Preliminary study revealed that asa soaking solution 1mM ethylenediamine tetraacetic acidsolution pH 80 was optimal to retrieve the antigenicityAfter pressure cooking sectionswere left for 30minutes at RTfor cooling In order for avoiding nonspecific signals inmousehepatocytes a Mouse Stain kit (Nichirei Bioscience Tokyo)was applied before and after the primary antibody incubationThe human-specific CK818 monoclonal antibody at a 1 100dilution was incubated overnight at RT After rinsing in10mM phosphate-buffered saline (PBS) pH 72 the sectionswere reacted with a secondary polymer reagent Novolink(Novocastra Newcastle UK) Reaction products were visu-alized in the diaminobenzidine-hydrogen peroxide solutionNuclear counterstaining with hematoxylin followed

25 HCV Genotyping by Nested RT-PCR Five slices of 5120583mthickness sections were collected in Eppendorf tubes TotalRNA was extracted from dewaxed sections using RecoverAllTotal Nucleic Acid Isolation kit (Applied Biosytems AustinTX USA) according to the manufacturerrsquos protocol andstored at minus80∘C until use Nested RT-PCR for HCV genotyp-ing was performed as described previously [24]






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