ORIGINAL ARTICLE

Killer Cell Immunoglobulin-Like ReceptorGenotype and Killer Cell Immunoglobulin-LikeReceptor–Human Leukocyte Antigen C LigandCompatibility Affect the Severity of Hepatitis CVirus Recurrence After Liver TransplantationAlejandro Espadas de Arias,1 Simone Elizabeth Haworth,1 Luca Saverio Belli,2 Patrizia Burra,4

Giovambattista Pinzello,2 Marcello Vangeli,2 Ernesto Minola,3 Maria Guido,5 Patrizia Boccagni,4

Tullia Maria De Feo,1 Rosanna Torelli,1 Massimo Cardillo,1 Mario Scalamogna,1 and Francesca Poli11Department of Regenerative Medicine, Organ and Tissue Transplantation Immunology, OspedaleMaggiore Policlinico, Mangiagalli, Regina Elena, Istituto di Ricovero e Cura a Carattere Scientifico, Milan,Italy; 2Hepatology and Abdominal Organ Transplantation Unit and 3Department of Histopathology,Niguarda Hospital, Milan, Italy; and Departments of 4Surgical and Gastroenterological Sciences and5Pathology, University of Padua, Padua, Italy

In 20% to 30% of infected individuals, hepatitis C virus (HCV) can cause cirrhosis and hepatocellular carcinoma, for which livertransplantation is the best treatment available. HCV re-infection is universal, and hepatitis disease recurrence occurs in mostcases with a 30% probability of progression to graft cirrhosis at 5 years post-transplant. The immunological response to HCVinvolves natural killer (NK) cells and killer cell immunoglobulin-like receptors (KIRs), which specifically recognize humanleukocyte antigen (HLA) class I antigens present on target cells. The effector functions of NK cells are influenced by inhibitoryKIR interaction with self-HLA class I ligands, with HLA-C being the most predominant. This study examines the roles of KIRgenotypes and their HLA ligands in both HCV disease recurrence and its progression. A total of 151 patients were included inthe cohort, and their clinical details were recorded. Liver biopsies were used to define the absence/presence of recurrenthepatitis, the degree of fibrosis, and the progression to cirrhosis over a 10-year period. Mismatching of KIR–HLA-C ligandsbetween donor-recipient pairs was associated with the recurrence of hepatitis (P � 0.008). The presence of KIR2DL3 in therecipient correlated with progression to liver fibrosis (P � 0.04). The mismatching of HLA-KIR ligands favored the progressionof the recurrent hepatitis to fibrosis only in the presence of KIR2DL3 (P � 0.04). These preliminary results indicate that the KIRgenotype and KIR–HLA-C ligand compatibility play roles in the recurrence and progression of hepatitis C disease in livertransplant recipients. Liver Transpl 15:390-399, 2009. © 2009 AASLD.

Received June 5, 2008; accepted September 15, 2008.

See Editorial on Page 357

The most recent World Health Organization estimate ofthe prevalence of hepatitis C virus (HCV) infection is2%,1 and 20% to 30% of HCV-positive subjects developcomplications such as cirrhosis and hepatocellular car-

cinoma (HCC), for which liver transplantation is thebest treatment available.2 Following liver transplanta-tion, HCV re-infection is universal, and hepatitis occursin most patients with a 30% cumulative probability ofprogression to graft cirrhosis at 5 years post-trans-plant.2-4

Several studies have demonstrated that the immuno-

Abbreviations: CI, confidence interval; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HLA, human leukocyte antigen; HR,hazard ratio; IFN, interferon; KIR, killer cell immunoglobulin-like receptor; IL, interleukin; NK, natural killer.This work was supported by Ricerce Corrente 2007 (Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena, Istituto di Ricovero e Cura aCarattere Scientifico, Milan, Italy).Address reprint requests to Francesca Poli, Department of Regenerative Medicine, Organ and Tissue Transplantation Immunology, OspedaleMaggiore Policlinico, Mangiagalli, Regina Elena, Istituto di Ricovero e Cura a Carattere Scientifico, Via Francesco Sforza 35, 20122 Milan, Italy.Telephone: �39-02-55034239; FAX; �39-02-55012573; E-mail: france.poli@policlinico.mi.it

DOI 10.1002/lt.21673Published online in Wiley InterScience (www.interscience.wiley.com).

LIVER TRANSPLANTATION 15:390-399, 2009

© 2009 American Association for the Study of Liver Diseases.

logical response to HCV infection involves both the in-nate and adaptive immune systems and that naturalkiller (NK) cells play a key role in the innate antiviralimmune response.5,6 NK cells express killer cell immu-noglobulin-like receptors (KIR) on their surface, whichspecifically recognize the human leukocyte antigen(HLA) class I antigens of the major histocompatibilitycomplex present on the target cells. In vivo, the effectorfunctions of NK cells are influenced by inhibitory recep-tors for self-HLA class I ligands HLA-A, HLA-B, andHLA-C, with HLA-C and Bw4 being the predominantligands for most KIR receptors. The dimorphism inamino acids present in the alpha 1 helix of the HLA-Cmolecules is characterized by Ser77/Asn80 andAsn77/Lys80, which define distinct allotypes of HLA-C(C1 and C2, respectively). The alleles corresponding tothe C1 allotype include HLA-Cw*01, HLA-Cw *03, HLA-Cw*07, and HLA-Cw*08, which interact with the KIRreceptors 2DL2/3 and 2DS2. The alleles correspondingto the C2 allotype include HLA-Cw*02, HLA-Cw*04,HLA-Cw*05, and HLA-Cw*06, which interact with the2DL1 and 2DS1 receptors.7,8 KIRs 3DL1 and 3DS1interact with HLA-Bw4, and KIR 3DL2 recognizesHLA-A3 and HLA-A11.9

A previous study concluded that these differentKIR–HLA-C genotypes confer either susceptibility orresistance to infectious diseases10 and that homozy-gosity for HLA-C1 and KIR2DL3 may be advantageousfor viral infections.11 Another study identified aprotective effect of HLA-Bw4I80 and the KIR3DS1gene against the development of HCC,12 whichin association with cirrhosis is a common indicationfor liver transplantation. Weaker ligand binding withKIR2DL3 has been demonstrated, indicating weakerinhibition through this receptor,13 and a recentlypublished study found that NK cell responsivenesscorresponded with the KIR-HLA genotype.14 Previousstudies have demonstrated an association withHLA-C antigens and clinical outcome in liver trans-plant recipients.15,16 Moya-Quiles et al.15 found thata decrease in the number of rejection episodes corre-lated with a decrease in the number of HLA-C allelemismatches, and Bishara et al.16 concluded that re-cipients transplanted with organs from donors withno C epitope disparity had fewer rejection episodes.The number of rejection episodes does not seem tohave an impact on HCV disease progression as indi-cated by earlier research.17 Less is known, however,about the role of NK cells and KIR-HLA interaction inliver transplant outcome with respect to HCV recur-rence. This lack of data and the considerable pres-sure of organ shortages have prompted us to examinethe possible role of KIR–HLA class I combinations inthe recurrence of HCV and disease progression fol-lowing liver transplantation. More specifically, we in-vestigated the effects of KIR genes and HLA-C allo-types on the severity of the recurrent disease in acohort of liver transplant recipients, taking advan-tage of the large sample size and the long follow-uptime.

PATIENTS AND METHODS

Study Cohort

A total of 151 consecutive donor/recipient pairs wereincluded in the cohort for this retrospective study. Thepatients were a subset of a previously described co-hort18 derived from 2 centers belonging to the NorthItaly Transplant Program19 who were transplanted be-tween 1991 and 2001; 99 patients were transplanted inMilan, and 52 were transplanted in Padua. All patientsfrom the previous cohort with either DNA or biopsymaterial available were included in this current study,and all patients involved gave informed consent. Ethicalapproval for the study was granted on the conditionthat the collected samples were screened for the pur-pose of assessing patients for transplantation.

All potential confounding factors such as other liverdiseases and transplant-related disorders were ex-cluded so that a well-defined homogeneous cohort re-mained, as described in our previous publication.1 Allconsecutive HCV-positive liver transplant recipientswithout B or delta coinfections and surviving at least 3months were considered. The main clinical features ofthe patients from the 2 centers were recorded and areshown in Table 1. All patients were of Caucasoid Italiandescent. Indications for transplantation were eitherend-stage cirrhosis (n � 109) or cirrhosis with HCC (n �42). The number of patients with HCC post-transplantwas also recorded (n � 4). In order to study the influ-ence of acute rejection on HCV disease progression, thenumber of individuals who experienced acute rejectionepisodes was noted (n � 45). Significant alcohol con-sumption (�40 g/day for women, �80 g/day for men)was seen in 21 (14%) patients prior to transplantation,but only 3 patients (2%) resumed these levels of con-sumption post-transplant.

Histological Evaluation

A minimum 1.5-fold increase in aspartate and/or ala-nine aminotransferase levels was the usual indicatorfor performing a liver biopsy examination. In addition,protocol biopsies were taken at 1, 3, 5, 7, and 10 yearspost-transplant when clinical indications of HCV recur-rence were observed in the patient. The series of biop-sies for each patient was reviewed over the study timeperiod by 2 liver pathologists and scored according tothe criteria of Ishak et al.20 in order to define the ab-sence/presence of recurrent hepatitis, the degree offibrosis, and the progression to cirrhosis. Recurrenthepatitis C disease was defined histologically from bi-opsy examinations performed because of clinical indi-cations or in protocol biopsy specimens. Only patientsshowing a necroinflammatory grade above 5, with orwithout fibrosis, throughout the follow-up period wereconsidered to have recurrent hepatitis. Patients pro-gressing beyond stage 3 were considered to have severerecurrence.

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Immunosuppression

Immunosuppressive regimens were different in the 2centers (Table 1). In Milan, quadruple drug induction(rabbit antithymocyte globulins for 5 days, azathioprinefor 1 month, corticosteroids for 1-6 months, and cyclo-sporine A) was used, followed by maintenance withlong-term cyclosporine monotherapy, which wasachieved within 6 months post-transplant in 85% ofpatients. Steroid withdrawal was anticipated between 1and 3 months in more recent years. In Padua, doubledrug induction (cyclosporine A or tacrolimus and cor-ticosteroids) was the standard protocol, whereas aza-thioprine or mycophenolate acid was added in cases ofrenal impairment with the aim of reducing the cal-cineurin-inhibitor dosage. Steroids were withdrawnwithin 6 months post-transplant in 93% of the patients.

Antiviral Therapy

A total of 72 patients with recurrent hepatitis under-went a treatment regimen of interferon-alpha 2b plusribavirin for a minimum of 6 months. A histologic find-ing of a grading score higher than 6 and a stage scorehigher than 2 with or without raised aminotransferaseswere the usual criteria for considering combined anti-viral therapy.

HCV Status

Patients were tested for anti-HCV antibodies with asecond-generation enzyme-linked immunosorbent as-say (ELISA II, Ortho Diagnostic System, Raritan, NJ),and HCV RNA was detected by nested reverse-tran-scription polymerase chain reaction (Amplicor HCV,Roche, Nutley, NJ) to determine the HCV genotype.HCV specificity was tested to determine with whichviral strain individuals were infected and if differences

in clinical outcome were influenced by differences in theviral strain.

HLA and KIR Genotyping

Genomic DNA was isolated from peripheral blood withQiagen columns (QIAmp 96 spin blood kit, QiagenGmbH, Hilden, Germany). Donor and recipient HLA-Ctyping was performed with sequence-specific oligonu-cleotide typing kits (Lipa, Innogenetics NV, Murex Bio-tech, Ltd., Dartford, UK). KIR allele genotyping wasperformed with Luminex-based reverse SSO typing(One Lambda, Los Angeles, CA). KIR haplotypes wereassumed as family data were not available and weredetermined to be haplotype AA or B/x according to thepresence of specific KIR genotypes as previously de-scribed.21

HLA-KIR ligand donor/recipient matching was di-vided into 3 groups based on the definitions of Tran etal.22 as follows: group 1 was a C1/2-Bw4 matchedgroup in which the recipient and donor shared thesame C1 (HLA-Casn80), C2 (HLA-Clys80), and Bw4epitopes; group 2 was a C1/2-Bw4 mismatched com-patible group in which the recipient and donor weremismatched for C1, C2, and Bw4 epitopes but the re-cipient’s KIR epitope was not missing in the donor’sHLA phenotype (eg, recipient with C2/C2, Bw4/Bw6;donor with C1/C2, Bw4/Bw4); and group 3 was a KIRC1/2-Bw4 mismatched incompatible group in whichthe donor’s HLA phenotype did not include a C1, C2, orBw4 epitope that was present in the recipient (eg, re-cipient with C1/C1, Bw6/Bw6; donor with C2/C2,Bw4/Bw4). KIR study groups 2 and 3, as describedhere, were also classified into a composite mismatchedgroup (Gx) for additional analysis. The recipients werealso classified into 2 groups with respect to the pres-ence/absence of the KIR2DL3 receptor in the recipientand also in terms of matching for the HLA-C1/C2

TABLE 1. Clinical Characteristics of Patients from the North Italy Transplant Program (Milan/Padua)

Variable

All Patients

(n � 151)

Milan

(n � 99)

Padua

(n � 52)

Milan Versus

Padua P

Value

Gender (male/female) 113/38 73/26 40/12 0.66Mean follow-up period, months 68 � 35 64 � 37 76 � 30 0.06Mean age at transplant, years 51 � 7.7 51.6 � 7.3 50.4 � 8.5 0.37HCV genotype 1 90 (70%) 61 (64%) 29 (88%) 0.014Antilymphocyte antibody 100 (66%) 99 (100%) 1 (2%) �0.0001Azathioprine/mycophenolate 112 (74%) 99 (100%) 13 (25%) �0.0001Cyclosporine 131 (87%) 97 (98%) 34 (65%) �0.0001Tacrolimus 20 (14%) 2 (2%) 18 (35%) �0.0001Duration of steroids, months (range) 4 (0-52) 4.3 (0-52) 4.5 (1-28) 0.89Steroid pulses 45 (30%) 27 (27%) 18 (35%) 0.0010Antiviral therapy (pre-transplant) 72 (48%) 51 (52%) 21 (40%) 0.0004Recurrent hepatitis 127 (84%) 79 (80%) 48 (92%) 0.46Severe fibrosis (stages 4-6) 30 (20%) 17 (17%) 13 (25%) 0.25

NOTE: Significant P values are bolded.Abbreviation: HCV, hepatitis C virus.

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group. Individuals in whom KIR2DL3 was absent or inwhom KIR2DL3 was present but who were matched forthe HLA-C group were classified as KIR2DL3 low reac-tive; that is, they had in theory greater NK cell inhibitionand thus less NK cell activation. Individuals in whomKIR2DL3 was absent and who were mismatched for theC epitope were classified as KIR2DL3 reactive; that is,they had in theory less NK cell inhibition leading togreater NK cell activation.6,14

Data Analysis

Allele frequencies were determined for the study popu-lations for HLA-C and KIR and compared to the fre-quency of HLA-C and KIR genes in the North Italianpopulation (http://www.allelefrequencies.net).23,24 Ge-notype and allele frequencies were contrasted betweenthe patients with recurrent hepatitis C and those withoutit with the chi-square test with the level of significance setat P � 0.05 and 95% confidence intervals. For small num-bers, Fisher’s exact test was used. All tests were 2-sided.The measure of association used to determine the extentof risk associated with a particular genotype or phenotypewas the odds ratio. P values were corrected for the num-ber of comparisons according to Bonferroni.25 The pa-tients with recurrent hepatitis were stratified into types ofrecurrence throughout the follow-up period (ie, the cumu-lative incidence of fibrosis stages at years 1, 3, 5, and 10),and univariate analysis was performed to test for associ-ations between HLA class I KIR ligands, KIR genes, anddisease progression. The following clinical variables wereanalyzed for a potential association with HCV progressionand liver fibrosis outcome: donor age, recipient age attransplantation, donor gender, recipient gender, coldischemia time, transplant center, incidence of HCC pre-transplant and post-transplant, presence of rejection ep-isodes, HCV genotype, antiviral therapy, immunosup-pressive therapy, and steroid treatment. Clinical factorand allele frequency analyses were performed with theStatistical Analysis System package, version 9.1.

All variables with a P value � 0.2 at univariate anal-ysis were also analyzed with the multivariate logisticregression model.

RESULTS

Clinical Characteristics of the Patient Groups

Significant differences were observed between Milanand Padua for antiviral and immunosuppressive ther-apies, as shown in Table 1; however, there were nodifferences observed between the 2 centers in terms ofclinical outcome, that is, recurrent hepatitis and severefibrosis (P � 0.46 and P � 0.25, respectively).

HLA-C Allele and KIR Gene Distribution inLiver Transplant Recipients

The distribution of HLA-C alleles and KIR genes in ourseries of patients was not significantly different from

that of a bone marrow donor population from NorthernItaly (PHLA-C � 0.11 and PKIR genes � 0.83). Thefrequencies of the HLA and KIR alleles are shown inTable 2.

Histological Evaluation of Patient LiverBiopsy Samples

Of the 136 patients that showed clinical indications ofHCV recurrence, 11 were not available for biopsy. His-tologically proven recurrent hepatitis C [a necroinflam-matory grade greater than 5 (G � 5)] was observed in all125 biopsied individuals at a median of 9 months(range, 1-36) post-transplantation.

TABLE 2. HLA-Cw and KIR Phenotype Frequencies in

HCV Liver Recipients and Population Controls

HLA-Cw Allele Frequencies

Cw Allele HCV Liver Recipients North Pavia, Italy*

Cw 01 0.0264 0.056Cw 02 0.0625 0.068Cw 03 0.0525 0.062Cw 04 0.1720 0.204Cw 05 0.0500 0.037Cw 06 0.1355 0.112Cw 07 0.2580 0.266Cw 08 0.0525 0.031Cw 12 0.0660 0.068Cw 14 0.0265 0.031Cw 15 0.0365 0.012Cw 16 0.0430 0.044Cw 17 0.0165 0.012P � 0.107 Pz � 151 Pz � 81

KIR Phenotype Frequency (%)

KIR

Gene HCV Liver Recipients Italy*

2DL1 96 952DL2 54 532DL3 87 882DL4 100 1002DL5 51 612DS1 37 362DS2 55 532DS3 36 332DS4 94 892DS5 31 283DL1 94 963DL2 100 1003DL3 100 1003DS1 38 36P � 0.83 Pz � 151 Pz � 217

Abbreviations: HCV, hepatitis C virus; HLA, humanleukocyte antigen; KIR, killer cell immunoglobulin-likereceptor.*http://www.allelefrequencies.net.

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HLA-C Allele and KIR Gene Distribution inPatients with Recurrent Hepatitis

The analysis of HLA-C allele frequency and the pres-ence of recurrent hepatitis in this study group showedthat the allele HLA-Cw*02 was statistically significantlyincreased in HCV-recurrent patients in comparisonwith nonrecurrent patients (34% versus 10%, P � 0.03).There were no significant differences in frequency ob-served for any other HLA-C alleles.

Donor-recipient matching for HLA-KIR ligands wasexamined by a comparison of the group matched forHLA-C1 or HLA-C2 alleles and HLA-Bw (G1, n � 34)with the mismatched compatible group (G2, n � 49)and also with the mismatched incompatible group (G3,n � 68) with the development of recurrent disease (G �5; G1, 75.76% versus G2, 91.23%, P � 0.06, and G1,75.76% versus G3, 93.65%, P � 0.02). These statisti-cally significant differences in clinical outcome indicatethat the development of recurrent hepatitis is linked tothe mismatching of donor/recipient KIR ligands as wellas the absence of the recipient’s KIR ligand in the donorliver (P � 0.02). Therefore, we created a composite mis-matched group (Gx; ie, the combined group of donor-recipient pairs with HLA-KIR ligand mismatches: group2 � group 3) and found that the frequency of patientswith recurrent hepatitis (G � 5) was also increased inthe Gx group in comparison with group 1 (Gx, 93.46%versus G1, 75.76%, P � 0.008). Figure 1 shows thedistribution of HLA-KIR ligand groups G1 and Gx for 2outcomes with respect to liver inflammation (no recur-rence versus inflammation, G � 5). The analysis of theHLA-ligand mismatched group demonstrated that hep-atitis recurrence correlated with HLA-C1 or HLA-C2donor/recipient mismatch (P � 0.03) but not with HLA-Bw4 mismatch (P � 0.16).

A mismatch for the HLA-KIR ligand together with thepresence of KIR2DL3 (KIR2DL3 reactive) was signifi-cantly associated with recurrent hepatitis in compari-son with the KIR2DL3 low reactive group as definedpreviously (57.14% versus 32.14%, P � 0.02).

No association between the presence of KIR genes2DL1 (P � 0.3), 2DL2 (P � 0.8), 3DL1 (P � 0.58), 2DS1(P � 0.53), 2DS2 (P � 0.48), 2DS4 (P � 0.58), and 3DS1(P � 0.9) and hepatitis recurrence was observed.

The presence of HCC before transplantation (P �0.68) was not statistically significant. The number ofpatients with HCC post-transplantation (n � 4) was toosmall to test associations adequately. In patients withHCC pre-transplant, a protective effect of HLA-Bw4I80-receptor 3DS1 against HCV recurrence (P � 0.3) wasnot detected. The treatment of recipients with steroidshad no significant effect on HCV disease progression(P � 0.37).

The following clinical variables were statistically sig-nificant following the univariate analysis and were in-cluded in the multivariate analysis: age of recipient attransplantation, female sex of the recipient, HCV geno-type, KIR ligand mismatch, antiviral therapy, and im-munosuppression (Table 3). The donor age (P � 0.7,median age � 37 � 14.0), donor gender (P � 0.3), andcold ischemic time (P � 0.3, median time � 7 � 4.0hours) were not significant following univariate analy-sis. The multivariate analysis, however, showed only asignificant difference between the matched (group 1)and the KIR ligand mismatched recipients (Gx) in rela-tion to the recurrence of HCV in liver-transplanted pa-tients (P � 0.003).

HLA-C Allele and KIR Gene Distribution andSeverity of Fibrosis

Recurrent hepatitis progressing beyond stage 3 wasobserved in 30 of 151 patients (20%). The 1-, 3-, 5-, and10-year cumulative incidences of stage 4-6 fibrosiswere 3%, 15%, 22%, and 36%, respectively. HLA-C al-lele frequencies were stratified into those that pro-gressed from recurrent hepatitis to mild fibrosis (stages0-3) and severe fibrosis (stages 4-6). A significant asso-ciation between HLA-Cw*08 and the progression to se-vere fibrosis was observed (23% in recipients with se-vere fibrosis versus 7.4% in recipients with mildfibrosis, P � 0.02).

The frequency of patients with severe fibrosis washigher in the group of females versus the group of males(32% versus 16%, P � 0.03).

HCV type 1 was significantly associated with recur-rent hepatitis progressing beyond stage 3 (88% of pa-tients with stages 4-6 with HCV genotype 1 versus 66%of patients with stages 4-6 with the other non-1 HCVgenotypes, P � 0.02).

No association between the HLA class I alleles A3,A11, C1, C2, and Bw (previously described as being themost influential9,22) and the progression to fibrosis wasobserved. The analysis of KIR genotype distribution

Figure 1. Liver transplant recipients were classified into 2groups: those with no recurrence of liver inflammation(grade < 5) and those with inflammation (grade > 5). Theywere analyzed for recipient-donor killer cell immunoglobulin-like receptor ligand matching, that is, matched (G1) versusmismatched (Gx). P � 0.008. Abbreviation: HCV, hepatitis Cvirus.

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evidenced an association between the presence ofKIR2DL3 in the recipient and progression to liver fibro-sis (P � 0.04).

The mismatching of HLA-KIR ligands favored the pro-gression of the disease to fibrosis (ie, stages 4-6) only inthe presence of KIR2DL3 (P � 0.04). Because of thesmall number of patients who were HLA-C1–homozy-gous, the analysis of HLA-C1 homozygosity with thepresence of KIR2DL3 and the development of fibrosiswas not possible.

The study of KIR haplotypes indicated that the effectof KIR2DL3 was not associated with the presence of theAA KIR haplotype (P � 0.3).

Recipient gender, HCV genotype (1 versus others),KIR ligand mismatch, presence of KIR2DL3, donor age,and immunosuppression were included in the multi-variate analysis (Table 4).

The multivariate analysis demonstrated that HCV ge-notype 1, donor age greater than 50 years, and thepresence of the KIR2DL3 gene in the recipient wereassociated with more aggressive recurrent liver disease,that is, progression to severe fibrosis (P � 0.009, P �0.03, and P � 0.04, respectively).

There was no association between viral persistenceand clinical outcome nor any evidence of a protectiveeffect with HLA-Cw*04 (P � 0.89), HLA-Cw*06 (P �

0.60), or HLA-Cw*07 (P � 0.39), as previously report-ed.5,15,26,27

Analysis of Influence of Acute Rejection onHCV Disease Progression

The influence of acute rejection on HCV recurrence andthe development of liver fibrosis was tested but found tohave no significant effect (P � 0.7 and P � 0.36, respec-tively). The presence of KIR2DL3, presence of HLA-Cw*0727 in either the donor or recipient, and KIR hap-lotype (AA or B/x) in association with the occurrence ofacute rejection were also analyzed and again were notstatistically significant (P � 0.6, P � 0.3/P � 0.2, andP � 0.4, respectively).

DISCUSSION

Most liver allograft transplant losses are caused by re-currence of the primary disease rather than by rejec-tion,28,29 and in this respect, many contributing factorsfor progressive fibrosis have already been recognized.30

Nevertheless, the influence of the innate immunologicalsystem and the elements of this system on genetic sus-ceptibility to both HCV recurrence and disease progres-

TABLE 3. Multivariate Analysis of Clinical and Immunological Variables in 151 HCV-Positive Recipients Without

Recurrence (G � 5) or With Recurrence of Liver Inflammation (G � 5)

Variable

No Recurrence

(n � 15) Recurrence (n � 136)

P

Value Cox Analysis

Transplant center (MI/PD) 4/11 48/88 0.35 HR, 3.16; 95% CI, 0.27-36.4Age at transplantation 53.3 � 7.3 50.94 � 7.13 0.72 HR, 0.98; 95% CI, 0.89-1.09Recipient sex (male/female) 10/5 103/33 0.28 HR, 2.19; 95% CI, 0.52-9.27HCV genotype 1 71% 71% 0.24 HR, 0.39; 95% CI, 0.08-1.89KIR ligand mismatch 47% 81% 0.003 HR, 8.02; 95% CI, 2.06-31.29Antiviral therapy 33.3% 52.8% 0.34 HR, 1.89; 95% CI, 0.51-7.08Cyclosporine/tacrolimus 80% 86% 0.6 HR, 0.47; 95% CI, 0.02-11.32Steroid pulses 40% 28.8% 0.11 HR, 0.28; 95% CI, 0.06-1.38

NOTE: The significant P value is bolded.Abbreviations: CI, confidence interval; HCV, hepatitis C virus; HR, hazard ratio; KIR, killer cell immunoglobulin-like receptor.

TABLE 4. Multivariate Analysis of Clinical and Immunological Variables for HCV-Positive Patients with Mild (Stages

0-3) or Severe (Stages 4-6) Fibrosis

Variable

Severe Fibrosis

(n � 30)

Mild Fibrosis

(n � 121)

P

Value Cox Analysis

Recipient sex (male/female) 18/12 95/26 0.07 HR, 0.39; 95% CI, 0.14-1.09HCV genotype 1 88% 70% 0.009 HR, 6.39; 95% CI, 1.6-25.74KIR ligand mismatch 83% 77% 0.34 HR, 1.85; 95% CI, 0.53-6.27Presence of gene KIR2DL3 in recipient 100% 76% 0.04 HR, 5.10; 95% CI, 1.06-24.53Cyclosporine/tacrolimus 90% 86% 0.96 HR, 1.03; 95% CI, 0.23-4.51Donor age � 50 11/19 27/94 0.03 HR, 3.10; 95% CI, 1.12-8.53

NOTE: Significant P values are bolded.Abbreviations: CI, confidence interval; HCV, hepatitis C virus; HR, hazard ratio; KIR, killer cell immunoglobulin-like receptor.

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sion to cirrhosis in liver transplant recipients has neverbeen investigated.

Recurrent HCV infection may begin in a manner sim-ilar to acute HCV infection,31 and the clearance andcontrol of the virus may therefore depend on an effectiveand long-lasting NK, CD4�T, and CD8�T response.Although HCV virus per se is not cytopathic and itslevels do not correlate with the degree of liver fibrosis, ithas been demonstrated that it does induce the produc-tion of cytokines [interferon gamma (IFN-�), interleukin12 (IL-12), IL-18, and IL-15] from hepatocytes that ac-tivate the NK cells,32 which may result in liver damage.The active NK cells in turn secrete IFN-�, which inducesmonokine-induced IFN-� and IFN-�–inducible protein10, culminating in the activation and development ofCD4� and CD8� T cells. In contrast, a virus-specificCD4�T cell response is often undetectable in patientswith chronic liver disease.33

Several studies have been published in which theeffects of KIR ligand incompatibility and NK cell re-sponses in hematopoietic transplants were investigat-ed,34,35 but less is known about this in relation to solidorgan transplantation.22 To our knowledge, this is thefirst study to evaluate the role of NK receptors and theirligands in HCV-infected transplant patients, and it has4 major strengths: (1) it is based on a large unselectedcohort of consecutive Caucasoid patients with long fol-low-up times, (2) HLA typing was performed by molec-ular techniques, (3) the hepatitis recurrence is definedby objective histologic criteria, and (4) protocol liverbiopsies were available in order to assess disease pro-gression.

Our data indicate that mismatching of KIR-HLA li-gands between donor-recipient pairs in KIR2DL3-posi-tive recipients is associated with the histologic recur-rence of severe hepatitis and expand a previous studythat demonstrated the importance of HLA-Cs on livertransplantation outcome.16 Our study, however, doesnot confirm a protective effect of HLA-Bw4I80-receptor3DS1 against HCV recurrence. The highly polymorphicnature of KIR3DS1 and the peptide-specific manner ofpresentation by the molecule Bw4 might explain thedifferent findings.36 Our study did not find an associa-tion between the occurrence of acute rejection and dis-ease progression in accordance with a previousstudy.17 Our analysis of KIR2DL3, KIR haplotype, andHLA-Cw*07 as potential contributing factors for theoccurrence of acute rejection showed no significant ef-fect. Moya-Quiles et al.27 previously reported that HLA-Cw*07 has a protective effect against the occurrence ofacute rejection. It is possible that the discrepant find-ings are due to the fact that Moya-Quiles et al.’s patientgroup consisted of liver transplantees as a whole,whereas our transplant recipient group consisted spe-cifically of individuals with HCV pre-transplantation;also, the number of individuals who experienced acuterejection in our study group was small (n � 45). Ourlack of a significant finding for the role of the use ofsteroids was again possibly due to the small number ofindividuals and also to the fact that in 93% of therecipients, steroids were withdrawn within 6 months.

It is well known that the major histocompatibilitycomplex class I molecules bind and present endoge-nous antigens to CD8� cytotoxic T cell molecules andare directly involved in the activation and inhibition ofNK cells. The level of HLA-C expression regulates NKcell activity37 and thus represents a potential target forvirally induced modulation of NK responses.

HLA-C matching, as shown in our study, recalls theconcept of the dualistic effect of HLA matching, whichon the one hand reduces the risk of rejection but on theother hand enhances immune-mediated injury.38 Wehypothesize that HLA-C matching prevents the activa-tion of the T lymphocytes, and this explains its impor-tance in the development of HCV recurrence.

The importance of KIR2DL3 and NK cells in the pro-gression to graft cirrhosis is evidenced by our study. Inrelation to this, Khakoo et al.6 demonstrated that thepresence of the KIR2DL3 receptor in association withHLA C1C1 favors HCV clearance only in patients with alow viral load.

We postulate that in immunocompetent individuals,NK cells with receptor KIR2DL3 would have a protectiveeffect during the first stage of the infection but not laterwhen the viral load increases and the innate immuneresponse is possibly overwhelmed.39-41

It has been shown in animal models that the deple-tion of NK cells before a hepatotropic viral infectionleads to inhibition of a virus-specific T cell response aswell as inhibition of liver injury.42 In 1 human study, itwas demonstrated that during a persistent HCV infec-tion, the virus faces diminished immune pressure overtime, either from mutation to an immune-resistant se-quence or from immunological exhaustion. This dimin-ished immune response may be reflected by diminishedinflammatory activity.43 When the HCV virus infects apatient, a weaker inhibitory receptor ligand such asKIR2DL3–HLA-C1 will be protective because it canmore easily prevail by activating signals than a strongerinhibitory interaction such as KIR2DL2–HLAC1 orKIR2DL1–HLA-C2.13 This activation is important forvirus clearance only during the infective phase; if theactivation persists, it damages the liver because ofNKR�CD8�T cell accumulation in the liver parenchy-ma.44,45

With this hypothesis in mind, the absence of theKIR2DL3 receptor and matching for the HLA-KIR li-gands in HCV-positive liver transplant recipients mighthave a protective effect for the evolution of HCV recur-rence. Conversely, the NK cell receptor KIR2DL3 maymodify cell activity in the presence of the mismatch,facilitating the progression to fibrosis. A recent studyprovides support for this hypothesis as it demonstrateda differential NK cell responsiveness to viral infectionsdepending on the KIR-HLA genotype and showed thatKIR2DL3-positive NK cells secreted more IFN-� at anearlier time point following infection.14 Our preliminarydata indicate that KIR2DL3-positive recipients wouldbe better assigned a matched donor for the HLA-KIRs inorder to reduce the risk of developing severe fibrosisafter liver transplantation. Tran et al.22 showed a sim-ilar trend, in that renal recipient survival was better in

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matched individuals than in mismatched individualsand in incompatible individuals, although these findingwere not statistically significant. Therefore, it would bepotentially beneficial to the HCV-positive patients if theKIR genotype were determined pre-transplantation. Weare currently undertaking a prospective study includ-ing functional cellular assays to test the previously dis-cussed findings.

As far as the effect of posttransplant antiviral therapyis concerned, there are discrepant opinions at present.Several studies have demonstrated an increase in re-jection episodes in recipients treated with IFN,46

whereas others deny the association.47 Our results in-dicate, however, that the presence of acute rejection isnot associated with HCV progression. On the basis ofour results, its appears that posttransplant antiviraltherapy is effective only in the early stages of re-infec-tion when, as previously reported, an increase in NKcells and higher NK activity can be observed.48 In theevent of HCV recurrence, the activation of the defensiveinnate system produces an increase in inflammationwith a possible evolution to severe fibrosis. Our data,however, confirm several studies that indicate that ge-notype 1b is harder to eradicate than genotypes 2 and 3and that subjects with severe fibrosis are less respon-sive to the therapy.49-51 This is likely to be a result of agreater enhancement of the T cell response due to IFNtreatment in genotype 2 and 3–infected patients versusgenotype 1b–infected patients.52 In agreement with aprevious study,4 our data show an association betweendonor age greater than 50 years and the development ofsevere fibrosis in the multivariate analysis. Anotherstudy reported an association between cold ischemictime greater than 10 hours and the development of HCVrecurrence,53 but this was not confirmed by our group;however, it must be noted that for our liver transplants,the cold ischemic time was rarely greater than 10hours.54

In terms of HLA matching alone, in a previous studywe demonstrated that HLA-DRB1 mismatching is asso-ciated with the recurrence of histologic hepatitis and itsprogression beyond stage 3.18 Based on our previousresults and current results, in our opinion, HCV-posi-tive recipients should ideally be matched for HLA-C andDRB1 with their donors.

The presence of HLA-Cw*02 is significantly higher inpatients without recurrence and shows a negative trendin patients with severe fibrosis. It is possible that HLA-Cw*02 presents HCV peptides that lead an effectiveimmune response or that HLA-Cw*02 interacts with thevirus or another host protein that favors viral clearance.

On the contrary, the frequency of HLA-Cw*08 is sig-nificantly higher in recipients who develop severe fibro-sis. It can be postulated that subtle structural changesin HLA-Cw*08 molecules differentiate them from otherHLA-C molecules, leading to greater specificity for pep-tide engagement and causing increased alloreactivityand risk for fibrosis.

In summary, the results presented in this study showthat a disparity in HLA-C allotypes between the recipi-ent and donor may increase the risk of recurrence of

hepatic inflammation and support the importance ofthe KIR2DL3 receptor in the development of the dis-ease. These are potentially important factors for theselection of the most appropriate HCV-positive recipi-ent of liver transplantation and thus the optimum useof the limited organs available.

An extension of this study and confirmation of ourfindings will help to elucidate the role of HLA–KIR ge-notype combinations in the recurrence and progressionof hepatitis C in liver transplant patients.

REFERENCES

1. Shepard CW, Finelli L, Alter MJ. Global epidemiology ofhepatitis C virus infection. Lancet Infect Dis 2005;5:558-567.

2. Berenguer M, Lopez-Labrador FX, Wright TL. Hepatitis Cand liver transplantation. J Hepatol 2001;35:666-678.

3. Feray C, Caccamo L, Alexander GJ, Ducot B, GugenheimJ, Casanovas, et al. European collaborative study on fac-tors influencing outcome after liver transplantation forhepatitis C. European Concerted Action on Viral Hepatitis(EUROHEP) Group. Gastroenterology 1999;117:619-625.

4. Charlton M, Seaberg E, Wiesner R, Everhart J, ZettermanR, Lake J, et al. Predictors of patient and graft survivalfollowing liver transplantation for hepatitis C. Hepatology1998;28:823-830.

5. Thio CL, Gao X, Goedert JJ, Vlahov D, Nelson KE, Hilgar-tner MW, et al. HLA-Cw*04 and hepatitis C virus persis-tence. J Virol 2002;76:4792-4797.

6. Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astem-borski J, et al. HLA and NK cell inhibitory receptor genes inresolving hepatitis C virus infection. Science 2004;305:872-874.

7. Mandelboim O, Reyburn HT, Vales-Gomez M, Pazmany L,Colonna M, Borsellino G, et al. Protection from lysis bynatural killer cells of group 1 and 2 specificity is mediatedby residue 80 in human histocompatibility leukocyte an-tigen C alleles and also occurs with empty major histocom-patibility complex molecules. J Exp Med 1996;184:913-922.

8. Falk CS, Schendel DJ. HLA-C revisited. Ten years ofchange. Immunol Res 1997;16:203-214.

9. Pende D, Biassoni R, Cantoni C, Verdiani S, Falco M, diDonato C, et al. The natural killer cell receptor specific forHLA-A allotypes: a novel member of the p58/p70 family ofinhibitory receptors that is characterized by three immu-noglobulin-like domains and is expressed as a 140-kDdisulphide-linked dimer. J Exp Med 1996;184:505-518.

10. Carrington M, Martin MP. The impact of variation at theKIR gene cluster on human disease. Curr Top MicrobiolImmunol 2006;298:225-257.

11. Hawes SE, Kiviat NB. Are genital infections and inflam-mation cofactors in the pathogenesis of invasive cervicalcancer? J Natl Cancer Inst 2002;94:1592-1593.

12. Lopez-Vazquez A, Rodrigo L, Martınez-Borra J, Perez R,Rodrıguez M, Fdez-Morera JL, et al. Protective effect of theHLA-Bw4I80 epitope and the killer cell immunoglobulin-like receptor 3DS1 gene against the development of hep-atocellular carcinoma in patients with hepatitis C virusinfection. J Infect Dis 2005;192:162-165.

13. Winter CC, Gumperz JE, Parham P, Long EO, WagtmannN. Direct binding and functional transfer of NK cell inhib-itory receptors reveal novel patterns of HLA-C allotyperecognition. J Immunol 1998;161:571-577.

14. Ahlensteil G, Martin MP, Gao X, Carrington M, Reher-mann B. Distinct KIR/HLA compound genotypes affectthe kinetics of human antiviral natural killer cell re-sponses. J Clin Invest 2008;118:1017-1026.

SEVERITY OF HEPATITIS C VIRUS RECURRENCE 397

LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

15. Moya-Quiles MR, Muro M, Torıo A, Sanchez-Bueno F,Miras M, Marın L, et al. Human leukocyte antigen-C inshort- and long-term liver graft acceptance. Liver Transpl2003;9:218-227.

16. Bishara A, Brautbar C, Zamir G, Eid A, Safadi R. Impact ofHLA-C and Bw epitopes disparity on liver transplantationoutcome. Hum Immunol 2005;66:1099-1105.

17. Yilmaz N, Shiffman ML, Stravitz RT, Sterling RK, Luketic VA,Sanyal AJ, et al. A prospective evaluation of fibrosis progres-sion in patients with recurrent hepatitis C virus followingliver transplantation. Liver Transpl 2007;13:975-983.

18. Belli LS, Burra P, Poli F, Battista Alberti A, Silini E, Zava-glia C, et al. HLA-DRB1 donor-recipient mismatch affectsthe outcome of hepatitis C disease recurrence after livertransplantation. Gastroenterology 2006;130:695-702.

19. Sirchia G, Poli F, Cardillo M, Scalamogna M, Rebulla P,Taioli E, et al. A cadaver kidney allocation in the NorthItaly transplant program on the eve of the new millen-nium. Clin Transpl 1998:133-145.

20. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J,Gudat F, et al. Histological grading and staging of chronichepatitis. J Hepatol 1995;22:696-699.

21. Stern M, Elsasser H, Honger G, Steiger J, Schaub S, HessC. The number of activating KIR genes inversely correlateswith the rate of CMV infection/reactivation in kidneytransplant recipients. Am J Transplant 2008;8:1312-1317.

22. Tran TH, Mytilineos J, Scherer S, Laux G, Middleton D,Opelz G. Analysis of KIR ligand incompatibility in humanrenal transplantation. Transplantation 2005;80:1121-1123.

23. Bontadini A, Testi M, Cuccia MC, Martinetti M, CarcassiC, Chiesa A, et al. Distribution of killer cell immunoglob-ulin-like receptors genes in the Italian Caucasian popula-tion. J Transl Med 2006;4:44.

24. Martinetti M, Degioanni A, D’Aronzo AM, Benazzi E, Car-panelli R, Castellani L, et al. An immunogenetic map ofLombardy (Northern Italy). Ann Hum Genet 2002;66:37-48.

25. Svejgaard A, Ryder LP. HLA and disease associations: de-tecting the strongest association. Tissue Antigens 1994;43:18-27.

26. Alric L, Fort M, Izopet J, Vinel JP, Bureau C, Sandre K, etal. Study of host- and virus-related factors associated withspontaneous hepatitis C virus clearance. Tissue Antigens2000;56:154-158.

27. Moya-Quiles MR, Alvarez R, Miras M, Gomez-Mateo J,Lopez-Alvarez MR, Marin-Moreno I, et al., for the SpanishGroup of Transplant Immunology and Immunotolerance(G03/104). Impact of recipient HLA-C in liver transplant:a protective effect of HLA-Cw*07 on acute rejection. HumImmunol 2007;68:51-58.

28. Garcia RF, Garcia CE, McMaster P. Chronic rejection ofthe liver: the role of immunosuppression. BioDrugs 2000;14:283-297.

29. Ghobrial RM, Farmer DG, Baquerizo A, Colquhoun S,Rosen HR, Yersiz H, et al. Orthotopic liver transplantationfor hepatitis C: outcome, effect of immunosuppression,and causes of retransplantation during an 8-year single-center experience. Ann Surg 1999;229:824-831.

30. Piscaglia F, Cucchetti A, Benlloch S, Vivarelli M, Beren-guer J, Bolondi L, et al. Prediction of significant fibrosis inhepatitis C virus infected liver transplant recipients byartificial neural network analysis of clinical factors. Eur JGastroenterol Hepatol 2006;18:1255-1261.

31. McCaughan GW, Zekry A. Pathogenesis of hepatitis C vi-rus recurrence in the liver allograft. Liver Transpl 2002;8(suppl 1):S7–S13.

32. Ahmad A, Alvarez F. Role of NK and NKT cells in the

immunopathogenesis of HCV-induced hepatitis. J LeukocBiol 2004;76:743-759.

33. Elliot LN, Lloyd AR, Ziegler JB, French RA. Protective im-munity against hepatitis C virus infection. Immunol CellBiol 2006;84:239-249.

34. Ruggeri L, Capanni M, Urbani E, Perruccio K, ShlomchikWD, Tosti A, et al. Effectiveness of donor natural killer cellalloreactivity in mismatched hematopoietic transplants.Science 2002 15;295:2097-2100.

35. Giebel S, Locatelli F, Lamparelli T, Velardi A, Davies S,Frumento G, et al. Survival advantage with KIR ligandincompatibility in hematopoietic stem cell transplantationfrom unrelated donors. Blood 2003;102:814-819.

36. O’Connor GM, Guinan KJ, Cunningham RT, Middleton D,Parham P, Gardiner CM. Functional polymorphism of theKIR3DL1/S1 receptor on human NK cells. J Immunol2007;178:235-241.

37. Liao NS, Bix M, Zijlstra M, Jaenisch R, Raulet D. MHCclass I deficiency: susceptibility to natural killer (NK) cellsand impaired NK activity. Science 1991;253:199-202.

38. Donaldson P, Underhill J, Doherty D, Hayllar K, Calne R,Tan KC, et al. Influence of human leukocyte antigenmatching on liver allograft survival and rejection: “thedualistic effect”. Hepatology 1993;17:1008-1015.

39. Ciccorossi P, Maina AM, Oliveri F, Petruccelli S, LeandroG, Colombatto P, et al. Viral load 1 week after liver trans-plantation, donor age and rejections correlate with theoutcome of recurrent hepatitis C. Liver Int 2007;27:612-619.

40. Chazouilleres O, Kim M, Combs C, Ferrell L, Bacchetti P,Roberts J, et al. Quantitation of hepatitis C virus RNA in livertransplant recipients. Gastroenterology 1994;106:994-999.

41. Gane EJ, Naoumov NV. A longitudinal analysis of hepati-tis C virus replication following liver transplantation. Gas-troenterology 1996;110:167-177.

42. Liu ZX, Govindarajan S, Okamoto S, Dennert G. NK cellscause liver injury and facilitate the induction of T cell-mediated immunity to a viral liver infection. J Immunol2000;164:6480-6486.

43. Hanada K, Tanaka Y, Mizokami M, Gojobori T, Alter HJ. Areduction in selective immune pressure during the courseof chronic hepatitis C correlates with diminished biochem-ical evidence of hepatic inflammation. Virology 2007;361:27-33.

44. Bonorino P, Leroy V, Dufeu-Duchesne T, Tongiani-Das-han S, Sturm N, Pernollet M, et al. Features and distribu-tion of CD8 T cells with human leukocyte antigen classI-specific receptor expression in chronic hepatitis C.Hepatology 2007;46:1375-1386.

45. Paladino N, Flores AC, Marcos CY, Fainboim H, Theiler G,Arruvito L, et al. Increased frequencies of activating nat-ural killer receptors are associated with liver injury inindividuals who do not eliminate hepatitis C virus. TissueAntigens 2007;69(suppl 1):109-111.

46. Berenguer M, Prieto M, Palau A, Rayon JM, Carrasco D,Juan FS, et al. Severe recurrent hepatitis C after liverretransplantation for hepatitis C virus-related graft cir-rhosis. Liver Transpl 2003;9:228-235.

47. Wright HI, Gavaler JS, Van Theil DH. Preliminary experiencewith alpha-2b-interferon therapy of viral hepatitis in liverallograft recipients. Transplantation 1992;53:121-124.

48. Oertel M, Kohlhaw K, Diepolder HM, Schroder S, SchwarzR, Tannapfel A, et al. Alloreactivity of natural killer cells inallogeneic liver transplantation. Transplantation 2001;72:116-122.

49. Bruno S, Crosignani A, Maisonneuve P, Rossi S, Silini E,Mondelli MU. Hepatitis C virus genotype 1b as a majorrisk factor associated with hepatocellular carcinoma inpatients with cirrhosis: a seventeen-year prospective co-hort study. Hepatology 2007;46:1350-1356.

398 ESPADAS DE ARIAS ET AL.

LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

50. Bruno S, Silini E, Crosignani A, Borzio F, Leandro G, BonoF, et al. Hepatitis C virus genotypes and risk of hepatocel-lular carcinoma in cirrhosis: a prospective study. Hepa-tology 1997;25:754-758.

51. Silini E, Bottelli R, Asti M, Bruno S, Candusso ME, Bram-billa S, et al. Hepatitis C virus genotypes and risk of hep-atocellular carcinoma in cirrhosis: a case-control study.Gastroenterology 1996;111:199-205.

52. Missale G, Cariani E, Lamonaca V, Ravaggi A, Rossini A,Bertoni R, et al. Effects of interferon treatment on theantiviral T-cell response in hepatitis C virus genotype 1b-

and genotype 2c-infected patients. Hepatology 1997;26:792-797.

53. Iacob S, Cicinnati VR, Hilgard P, Iacob RA, Gheorghe LS,Popescu I, et al. Predictors of graft and patient survival inhepatitis C virus (HCV) recipients: model to predict HCVcirrhosis after liver transplantation. Transplantation2007;84:56-63.

54. Botha JF, Thompson E, Gilroy R, Grant WJ, Mukherjee S,Lyden ER, et al. Mild donor liver steatosis has no impacton hepatitis C virus fibrosis progression following livertransplantation. Liver Int 2007;27:758-763.

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