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Role of cytokine gene polymorphism and hepatic transforming growth factor β1 expression in recurrent hepatitis C after liver transplantation
www.elsevier.com/locate/jnlabr/ycyto Cytokine 27 (2004) 7e14
Role of cytokine gene polymorphism and hepatic transforming growth factor b1 expression in recurrent hepatitis C after liver transplantation Ziv Ben-Aria,), Orit Pappob, Tamarah Druzdb, Jaqueline Sulkesc, Tirza Kleind, Zmira Samrae, Rahamim Gadbae, Anat R. Tamburf, Ran Tur-Kaspaa, Eytan Morg a
The Liver Institute and Department of Medicine D, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel b Department of Pathology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel c Epidemiology Unit, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel d Tissue Typing Laboratory, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel e Department of Microbiology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel f Tissue Typing Laboratory, Rush Medical Center, Chicago, IL, USA g Department of Transplantation, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel Received 11 December 2003; received in revised form 8 March 2004; accepted 15 March 2004
Abstract Recurrent hepatitis C virus (HCV) infection after orthotopic liver transplantation (OLT) is nearly universal. Cytokines play an important role in the immune response to viral infection, and cytokine gene polymorphism affects the overall expression and secretion of cytokines. The objective of this study was to define the relationship between cytokine polymorphism and recurrent hepatitis C after OLT. Blood samples were collected from 36 patients at a mean of 44:6 G 30:4 months after OLT for chronic HCV infection. DNA was extracted from peripheral blood mononuclear cells, and polymerase chain reaction-sequence specific primers (PCR-SSP) analysis was performed on promoter sequences of transforming growth factor b1 (TGF-b1), interleukin 6 (IL-6) interleukin 10 (IL-10), tumor necrosis factor a (TNF-a) and interferon g (INF-g). Liver biopsies performed at diagnosis of recurrent disease were graded with the Knodell score, and hepatic TGF-b1 expression was determined semiquantitatively by immunohistochemistry. The gene polymorphism of TGF-b1 was correlated with its expression on hepatocytes and sinusoids. Polymorphism in all studied cytokine genes was correlated with recurrence, and interval to recurrence (O12 or %12 months post-OLT), and clinical (ascites, ChildePugh score and death), biochemical parameters of recurrent HCV (serum alanine aminotransferase (ALT)), INR, albumin, bilirubin), and virological parameters (HCV genotype and load). Biopsies revealed recurrent HCV in 31 patients (86.1%); in 21 (67.7%), the interval to recurrence was 12 months. There was a statistically significant correlation between TGF-b1 gene polymorphism, i.e., the genetic ability to produce high levels of TGF-b1, and the intensity of TGF-b1 staining on hepatocytes (p ¼ 0:003) and sinusoids (p ¼ 0:003), and the degree of fibrosis (p ¼ 0:02). A borderline correlation was found with the presence of ascites (p ¼ 0:07), but not with ChildePugh score, synthetic liver function tests or HCV genotype and load. The genetic ability to produce low levels of IFN-g was correlated with recurrent disease (p ¼ 0:015). No such correlation was found for TGF-b1 gene polymorphism. In conclusion, polymorphism in the TGF-b1 gene correlates with its in situ hepatic expression in patients with recurrent HCV after liver transplantation. INF-g, but not TGF-b1 gene polymorphism, correlates with early recurrent hepatitis C after transplantation. These findings might help to design preemptive prevention therapy in selected patients at risk. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cytokine gene polymorphism; Hepatitis C; Liver transplantation
) Corresponding author. Tel.: C972-3-9377253; fax: C972-39377277. E-mail address: [email protected] (Z. Ben-Ari). 1043-4666/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2004.03.009
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Z. Ben-Ari et al. / Cytokine 27 (2004) 7e14
1. Introduction
2. Patients and methods
Hepatic C virus (HCV)-related liver disease is the leading indication for orthotopic liver transplantation (OLT). Recurrent HCV infection, defined as viremia after OLT, is nearly universal, with histological indices in the majority of patients [1]. Although patients with HCV after OLT have a similar short-term survival to patients who undergo OLT for other causes of liver failure, 20%e30% develop allograft cirrhosis within 5 years [2,3]. The recurrence of HCV is associated with several interrelated viral and host factors, including pretransplant and early post-OLT HCV viral load, genotype 1b, multiple episodes of rejection, and level of immunosuppression [4]. Cytokines play an important role in the generation of the immune response to viral infections, both directly, by inhibition of viral replication, and indirectly, through determination of the predominant pattern of the host response [5]. The in vitro maximal capacity to produce different cytokines in response to mitogen stimulation varies among individuals. These differences are attributable to certain molecular mechanisms, including variations in transcription, translation and secretion pathways [6,7], and conservative mutations within cytokine coding regions, as well as nucleotide variations within more pronounced regulatory regions (i.e., promoter sequences). These genetic polymorphisms have been shown to affect the overall expression and secretion of cytokines, both in in vitro and, sporadically, in in vivo systems [8e13]. A potential association with allelic variations in certain cytokine genes have also been reported in allograft rejection and allograft fibrosis [14e16]. We have recently reported an association between cytokine gene polymorphism and recurrence of hepatitis C in OLT recipients [17]. The inherent ability to produce higher transforming growth factor (TGF)-b1 levels, low interleukin (IL)-10 levels and high interferon (IFN)-g levels was less pronounced in patients who had a disease recurrence than in those who did not [17]. These results were surprising in light of findings that levels of hepatic TGF-b1, a profibrogenic cytokine [18,19], were increased in patients with chronic HCV infection [20], and that serum and tissue levels of TGF-b1 could serve as predictors of progressive liver fibrosis in hepatitis C infection [20]. The objectives of the present study were to further identify and validate, in a larger cohort, potential genetic markers of susceptibility to recurrence of hepatitis C in liver transplant recipients. The gene polymorphism of five cytokines (tumor necrosis factor (TNF)-a, TGF-b1, IFN-g, IL-6, and IL-10) was analyzed and compared between the recurrence and non-recurrence groups, and the findings for the TGF-b1 cytokine gene polymorphism were correlated with its hepatic expression by semiquantitative immunostaining of liver biopsy sections.
2.1. Patients The initial sample included all 42 liver transplant recipients who underwent OLT for chronic HVC infection at Rabin Medical Center between 1992 and 2000. Six patients were excluded because of concomitant alcoholic liver disease (n ¼ 2), a history of hepatitis B (n ¼ 2), or unavailability of patients slides for review (n ¼ 2), leaving 36 patients for study. All had more than one year of post-OLT follow-up (mean, 44:6 G 30:4 months). The diagnosis of recurrent HCV infection was based on the presence of viremia by quantitative PCR assay, increased serum transaminase levels, and histologic findings at diagnosis of lobular hepatitis on liver biopsy in association with hepatocyte necrosis and midzonal macrovesicular steatosis [21]. The immunosuppressive regimen included cyclosporine, azathioprine, or cellcept, and corticosteroids in 11 patients and tacrolimus and corticosteroids in 25 patients. Histologically proven acute cellular rejection episodes were treated with three consecutive boluses of IV solomedrol 1.0 g/day and steroid-resistant episodes were treated with OKT3. Patient files were reviewed for demographic characteristics, pretransplant HCV RNA load and genotype, immunosuppressive regimen, rejection episodes, interval to recurrence, clinical progression at follow-up based on levels of serum bilirubin, alanine aminotransferase (ALT), serum albumin and INR, presence of ascites, ChildePugh score [22], and death. 2.2. Genetic polymorphism assessment The genetic profile of five cytokines was analyzed in all patients. 2.2.1. DNA extraction Genomic DNA was isolated by proteinase K digestion of fresh peripheral blood mononuclear cells, followed by phenol extraction and ethanol precipitation. DNA samples were quantified and subjected to specific PCR reactions as described. 2.2.2. Cytokine gene polymorphism Single nucleotide mutations were analyzed in five cytokines, for genotype and phenotype assignment. Specifically, for TNF-a, we examined the presence of a G or A nucleotide in position 308 of the promoter region, which generates three potential genotypes corresponding to two different phenotypes: the A/A and G/A genotypes represent the potential to produce high levels of TNF-a; and the G/G genotype represents the potential to produce low levels [8]. For IL-6 promoter, we examined the presence of a single nucleotide
Z. Ben-Ari et al. / Cytokine 27 (2004) 7e14
modification in position 174. Both the G/G and G/C genotypes are known to correlate with a high-production phenotype, whereas C/C is associated with low IL-6 production [9]. An additional coding sequence mutation was analyzed for IFN-g at position C874 (T versus A): the homozygous T produces high levels of IFN-g; the heterozygous T/A is an intermediate producer; and the homozygous A genotype represents the potential to generate only low amounts of IFN-g [10]. For TGF-b1, two single nucleotide mutations in the coding region were analyzed: codon C10 can be either T or C, and codon C25, either C or G. Potentially, there are nine different combinations of these two isolated mutations which give rise to three different secretion phenotypes: high, intermediate, and low [11]. For the IL-10 promoter, three polymorphisms were studied: position 1082 (G versus A); position 819 (C versus T); and position 592 (A versus C) [12,13]. 2.2.3. PCR-SSP PCR amplification was carried out according to the manufacturer’s instructions (One Lambda Inc., Canoga Park, CA). Briefly, after the addition of the appropriate primer pairs, salts, buffer and Taq polymerase, the samples were subjected to 30 cycles of PCR as follows: one cycle of 130 s at 96 (C, dropping to 63 (C for an additional 60 s; 9 cycles of 10 s at 96 (C, 60 s at 63 (C; and the final 20 cycles which included a three-temperature rampdannealing for 10 s at 96 (C, hybridization for 50 s at 59 (C, and an extension step of 30 s at 72 (C. PCR products were then loaded onto an agarose gel and photographed with an ultraviolet transilluminator. 2.3. Histological assessment One pathologist (O.P.) blindly reviewed all hepatic specimens of the studied patients for overall necroinflammatory activity (grade 0e12) and fibrosis (stage 0e4) according to Knodell’s score [23]. Intercurrent disease processes, such as acute cellular rejection, cytomegalovirus infection, biliary obstruction, and ischemia were excluded using serologic, immunohistochemical, radiological and endoscopic studies. 2.4. TGF-b1 immunohistochemistry staining and scoring Immunohistology was performed on deparaffinized liver biopsy sections. Briefly, mouse monoclonal antihuman TGF-b1 antibody (Chemicon Int. Inc., Temecula, CA) was used in a dilution of 1:300. Antigen retrieval was performed before application of the primary antibody. The tissue sections were placed in a bath with citrate buffer (pH = 6.0) and microwaved in a pressure cooker at high power (900e1000 W) for 13 min until the cooker was fully pressurized. Thereafter, the
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microwave level was reduced by 40% (380 W) for another 5 min. Endogenous peroxidase reactions were blocked by adding 3% H2O2 for 20 min. Endogenous avidin and biotin were blocked using Dako’s blocking kit (Dako Corp., Carpinteria, CA). Sections were then incubated with the primary antibody for 45 min, followed by the Dako LSABC Kit (Dako Corp., Carpinteria, CA) peroxidase, which consists of labelled streptavidin biotin reagents. Reactive sites were revealed by incubation with DAB (3,3-diaminobenzidine). Sections were counterstained with hematoxylin. Sections of reactive lymph nodes and kidney served as positive controls, and liver biopsies run concurrently without the primary antibody, as negative controls. The evaluation was done semiquantitatively according to the percentage of cells that stained positively. For sinusoidal cells: 0, staining similar to the negative controls for sinusoidal cells; 1, positive staining in 25% of sinusoidal cells; 2, 50% positive staining; 3, 75% positive staining; 4, 100% positive staining. For hepatocytes: 1, positive staining in 10% of hepatocytes; 2, 20% positive staining; 3, 30% positive staining; 4, 40% positive staining. The hepatic expression of TGF-b1 was correlated with its gene polymorphism.
2.5. Virological assays HCV RNA was tested by nested reverse-transcription polymerase chain reaction assay (Cobas Amplicor HCV Monitor Test, Roche Diagnostic Systems Inc., Branchburg, NJ, USA). Analytical sensitivity of the assay was 600 IU/ml. HCV genotypes were determined by the means of a line-probe hybridization assay (INNO-LiPa Innogenetics, Ghent, Belgium) directed to the 5# untranslated regions of the different HCV genotypes.
2.6. Statistical analysis Continuous variables are given as means G standard deviations. Pearson correlation coefficients (r) and their significance ( p) were calculated between the variables. Chi-squared test or Fisher exact test were used, as appropriate, to analyze statistically significant relationships in the distribution of categorical data. Analysis of variance with the Duncan multiple comparison option was used to analyze statistically significant differences in mean continuous parameters (age, bilirubin, albumin, etc.) between TGF-b1 groups (low, intermediate and high production). Student’s t-test was used to analyze statistically significant differences in mean continuous parameters between two groups of patients (recurrence vs. non-recurrence, etc.). A correlation factor was used for multiple comparisons. A p value of less than or equal to 0.05 was considered statistically significant.
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follow-up was 44:6 G 30:4 months. Five patients did not develop histological recurrence of HCV infection during a follow-up period of 40:5 G 9:6 months (all of them underwent, at least once, a liver biopsy during the follow-up period due to increased serum ALT level, which was found to be related to other causes and resolved).
Table 1 Baseline characteristics of patient (n ¼ 36) Age (years)
53.4 G 8.2
Sex (M/F) (%) HCV genotype 1 HCV load (IU/ml) Rejection (%) Rate Gradea 1 2 3 Cyclosporine/tacrolimus (%) Recurrent HCV infection (%) Interval to recurrence (months) ALT (U/l)b Bilirubin (mg/dl)b Necroinflammatory scoreb Fibrosis scoreb Cirrhosis (%) Follow-up (months)
58.3/41.7 84% 231,770.9 G 541,787.7 12 (33.3%) 6 (50%) 5 (41.6%) 1 (8.4%) 30.5/69.5 31 (86.1%) 14.5 G 15.9 128.6 G 79.9 1.3 G 1.7 5.4 G 2.5 1.5 G 1.1 6 (19.3%) 44.6 G 30.4
3.1. Phenotypic expression Table 2 summarizes the phenotypic expression deduced from the genetic polymorphism in the five selected cytokines in the patients with and without recurrent HCV infection after transplantation. The allelic polymorphism of IFN-g translated directly into three phenotypic levels of expression. A highly statistically significant difference in the distribution of the IFN-g gene polymorphism was observed between the groups (p ¼ 0:001) (Fig. 1). The majority (80%) of the patients without recurrence exhibited the potential to produce high levels of IFN-g (T/T genotype), and 20% exhibited the potential to produce intermediate levels; none had the genetic ability to produce low levels of IFN-g (A/A genotype). By contrast, among the patients with recurrent HCV infection, only 3.3% had the potential to produce high levels of IFN-g, whereas the majority exhibited the potential to produce either low levels of IFN-g (41.9%) or intermediate levels (54.8%). There was also a borderline statistical difference ( probably due to the small sample size) in the distribution of the IFN-g gene polymorphism between patients who developed a recurrent HCV infection early (within 12 months) after transplantation (n ¼ 21) or later (n ¼ 10) (p ¼ 0:07) (Fig. 1). In the first subgroup, 10 patients (47.6%) exhibited the genetic ability to produce low and 11 intermediate levels of IFN-g, and none of the patients had the ability to produce high levels. Among the patients in whom a recurrent infection developed more than 12 months after transplantation, three (30%) had the genetic ability to produce low levels of IFN-g, whereas six (60%) exhibited the potential to produce intermediate levels and one (10%), high levels. There was no statistically significant difference in the ability to produce TNF-a, IL-6, TGF-b1 and IL-10 between patients with and without a recurrent HCV infection (Table 2).
ALT Z alanine aminotransferase. Note: values are mean G SD unless otherwise indicated. a1 Z mild; 2 Z moderate; 3 Z severe. bAt diagnosis of recurrent HCV after transplantation.
3. Results The baseline characteristics of the patients included in this study are presented in Table 1. Mean age was 53:4 G 8:2 years; 58.3% were male. HCV genotypes were available for 25 patients. Twenty-two patients (84%) were genotype 1, and three patients were genotype non-1 (two patients 2a, one patient 3a). The mean pretransplant HCV RNA load was 231; 770:9 G 541; 787:7 IU=ml. The immunosuppressive regimen was based on cyclosporine in 30.5% of the patients and on tacralimus in 69.5%. The rejection episode rate was 33.3%. Only one patient was treated with OKT3. Recurrent HCV infection developed in 31 patients (86.1%) after a mean interval of 14:5 G 15:9 months. The interval to recurrence was %12 months in 21 (67.7%) patients and O12 months in 10 (32.3%). At recurrence, mean serum alanine aminotransferase (ALT) level was 128:6 G 79:9 U=l, and mean serum bilirubin, 1:3 G 1:7 mg=dl. Mean necroinflammatory score at recurrence was 5:4 G 2:5, and mean fibrosis score, 1:5 G 1:1. Six patients (19.3%) developed histologically proven cirrhosis during the follow-up period. Mean duration of
Table 2 Phenotypic expression of five cytokines in patients who underwent OLT due to hepatitis C infection Posttransplantation HCV recurrence
TNF-a (%)
IL-6 (%)
IFN-g (%)
Low
Low
Low
High
High
Interm.
TGF-b1 High
Low
IL-10 Interm.
High
Low
Interm. High
Recurrence (n ¼ 31) 26 (83.8) 5 (16.2) 3 (9.7) 28 (90.3) 13 (41.9) 17 (54.8) 1 (3.3) 2 (6.5) 10 (32.2) 19 (61.3) 18 (58.0) 8 (25.8) 5 (16.1) No recurrence (n ¼ 5) 3 (60.0) 2 (40.0) 0 (0) 5 (100) 0 (0) 1 (20) 4 (80) 0 (0) 3 (60) 2 (40) 3 (60) 1 (20) 1 (20) p Value N.S. N.S. 0.001 N.S. N.S. Note: all values are n (%).
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Fig. 2. The genetic potential to produce high levels of TGF-b1 correlated significantly with the fibrosis score (p ¼ 0:02). Fig. 1. The genetic ability to produce low levels of IFN-g was associated with recurrence of HCV after transplantation.
3.2. TGF-b1 polymorphism and histological findings (Table 3) There was a statistically significant correlation between the fibrosis score and TGF-b1 gene polymorphism. The genetic potential to produce high levels of TGF-b1 correlated significantly with the fibrosis score (r ¼ 0:43, p ¼ 0:02) (Fig. 2), but not with the necroinflammatory score (r ¼ 0:23, p ¼ 0:23). On immunohistology, a high statistically significant correlation was found between the genetic ability to produce high levels of TGF-b1 and the hepatic expression of TGF-b1 in hepatocytes (r ¼ 0:52, p ¼ 0:003) and sinusoidal cells (r ¼ 0:52, p ¼ 0:003). The close correlation between the fibrosis score and the TGF-b1 polymorphism suggests that liver-derived TGF-b1 is a relevant mediator of fibrosis. All biopsies were scored at least 1. Biopsies with a low score exhibited TGF-b1 staining only in sinusoidal cells (Fig. 3a), whereas biopsies with a high score displayed positive staining of sinusoidal cells and of hepatocyte cytoplasm with focal distribution (Fig. 3b) and association with the fibrosis stage (Fig. 4). An inverse, statistically significant difference was noted between IL-10 polymorphism and fibrosis, with a low fibrosis score associated with the genetic ability to produce high levels of IL-10 (r ¼ 0:38, p ¼ 0:04).
3.3. Correlation between the five selected cytokine gene polymorphism and clinical parameters A borderline statistically significant association was noted between the genetic ability to produce TGF-b1 and the presence of ascites at follow-up (r ¼ 0:33, p ¼ 0:07). However, there was no significant correlation
Table 3 TGF-b1 gene polymorphism and histological findings (mean G SD) TGF-b1 polymorphism
Fibrosis score
Inflammation score
TGF-b1 hepatocyte staining score
TGF-b1 sinusoid staining score
Low Intermediate High p Value
0 1.1 G 1.1 1.8 G 0.52 0.02
3.1 G 1.4 5.5 G 2.5 5.8 G 2.7 0.22
0 0.8 G 0.58 1.4 G 0.7 0.003
0.75 G 0.35 1.2 G 0.71 1.8 G 0.52 0.003
Fig. 3. TGF-b1 immunohistochemistry in liver biopsy. (a) A low score indicated TGF-b1 staining only in sinusoidal cells. (b) A high score indicated positive staining of sinusoidal cells and cytoplasm of hepatocytes with focal distribution.
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Fig. 4. Portal fibrosis and septal formation, positive staining for TGF-b1 is noted in scattered hepatocytes.
between TGF-b1 gene polymorphism and HCV genotype and load, ChildePugh score, synthetic liver function tests, ALT at follow-up, or death. An inverse, statistically significant difference was noted between the genetic ability to produce IL-10 and the presence of ascites (r ¼ 0:35, p ¼ 0:05). No correlation was found between TNF-a, IL-6 and IFN-g gene polymorphisms and the clinical parameters studied.
4. Discussion Liver transplant recipients have an accelerated course of HCV infection compared to immunocompetent patients [24], with a significantly higher rate of fibrosis progression to graft cirrhosis and higher subsequent risk of decompensation than before transplantation [24e26]. The risk factors associated with poor outcome are as yet not clearly defined [4] and include treatment of acute rejection with steroid boluses, or OKT3 for steroidresistant rejection, high viral load either pretransplantation or early after transplantation and genotype 1b [21]. In the present study, we observe a highly statistically significant difference in the IFN-g polymorphism patients who did and did not have a recurrent HCV infection after transplantation, validating our previous findings in a smaller cohort [17]. The patients with recurrent disease had the genetic ability to produce low levels of IFN-g, whereas those without recurrence had the ability to produce high levels of IFN-g (p ¼ 0:001). Moreover, within the recurrence group, those in whom the recurrence developed early (%12 months posttransplantation) exhibited the potential to produce low and intermediate levels of IFN-g compared to those in
whom recurrent disease developed later, who exhibited the potential to produce intermediate and high levels of IFN-g. When comparing the distribution of IFN-g gene polymorphism in our study with previously published studies that included normal controls, the frequency of low levels of IFN-g is almost double, 42% in our patients with recurrent HCV vs. 25%e27% in the normal population [27,28]. Several researchers have suggested that an adequate T-helper 1 (Th1) response (i.e., high IFN-g secretion by peripheral blood mononuclear cells) may be associated with a protective antiviral immune response [29], while insufficient systemic Th1 cytokine secretion may be associated with increased viral load and disease progression [29,30]. Indeed, serum samples from HCV patients contain significantly lower levels of soluble IFN-g compared with controls [31]. The cytokine profile of immunocompromised patients with recurrent HCV infection was also studied recently. Schirren et al. [32] reported that this profile does not differ principally from that of immunocompetent patients with HCV. Thus, our findings, if confirmed in other large-scale studies, might have implications for the pretransplantation identification of a subset of patients who are at risk of early, recurrent HCV infection posttransplantation and who might benefit from preemptive antiviral therapy immediately after transplantation. No significant statistical correlation was found between the TNF-a gene polymorphism at position 308 and recurrent HCV infection. Ho¨hler et al. [33] reported an association of general susceptibility to HCV infection with TNF-a promoter polymorphism, but it involved only the polymorphism at position 238 (p ! 0:009); as in our study, no such correlation was found at position 308. The distribution of TNF-a gene polymorphism in our study was comparable with the previously reported studies of the distribution in normal controls [27]. The reports on the role of IL-6 in chronic HCV infection are contradictory. McGuinness et al. [34] found that IL-6 mRNA is significantly downregulated in HCV-positive liver tissue, whereas others noted high serum IL-6 levels in patients with chronic HCV infection [35]. We did not find any significant statistical correlation between IL-6 gene polymorphism and recurrent HCV infection. The distribution of IL-6 gene polymorphism was comparable with the previously reported studies of the distribution in normal controls [28]. In our previous report [17], a borderline statistically significant difference in IL-10 polymorphism was found between patients with and without recurrent HCV posttransplantation (p ¼ 0:07). In the present study, which included a larger number of patients, no difference in the genetic ability to produce IL-10 was found between the groups. However, when comparing the distribution of IL-10 gene polymorphism in our study to previously published studies that included
Z. Ben-Ari et al. / Cytokine 27 (2004) 7e14
normal controls, the frequency of low level IL-10 was doubled, 58% in our patients with recurrent HCV vs. 27%e28% in the normal population [27,28]. In a recent study, Edwards-Smith et al. [36] recently noted that the heterogeneity in the promoter region of the IL-10 gene played a role in the initial response to IFN-a therapy in patients with HCV infection: those who were genetically predisposed to high IL-10 production had a poor response to IFN-a. We did not find any significant statistical correlation between TGF-b1 gene polymorphism and recurrent HCV infection. The distribution of TGF-b1 gene polymorphism was comparable with the previously reported studies of the distribution in normal controls [27,28]. Regarding TGF-b1, our study showed a statistically significant correlation between the genetic ability to produce high levels of TGF-b1 and the fibrosis score in patients with untreated chronic HCV infection. The fibrosis score in the initial posttransplantation liver biopsies [24,26,37] has been found to be a useful predictor of the development of severe chronic HCV. Accordingly, the TGF-b1 polymorphism may have a prognostic significance in patients with recurrent HCV infection, thereby possibly directing more aggressive therapy towards those patients with a genetic ability to produce high levels of TGF-b1. Furthermore, in the complex multivariate setting of the posttransplantation period, the patient’s ability to produce low levels of TGF-b1 could also serve as an additional guide to physicians to decide against antiviral therapy because of minimal disease activity. These assumptions are supported by our findings of a correlation of both TGF-b1 expression within the liver and TGF-b1 polymorphism with the progression of liver disease, and the association of progressive fibrosis with a typical pattern of TGF-b1 expression in both hepatocytes and sinusoidal cells in the liver. By contrast, in patients with stable disease, hepatic TGF-b1 expression was mostly limited to existing fibrous septa. Apart from their prognostic significance, these findings provide further proof that hepatocytes are a potential source of TGF-b1 under pathological conditions, as demonstrated in patients with autoimmune hepatitis [38] and HCV infection [20]. Thus, the source of TGF-b1 in the liver is not only from the recipient graft infiltrating cells but also from the donor hepatocytes. TGF-b1 has been implicated as a mediating factor in hepatic fibrogenesis in chronic HCV infection [39]. Host genetic factors that influence fibrogenesis may account for some of the variability in the progression of the disease. Powell et al. [40] recently reported a significant relationship between inheritance of the high TGF-b1producing genotype and the development of hepatic fibrosis. In addition, TGF-b1 inhibits proliferation and function of T and B lymphocytes, also its high expression could lead to suppression of anti-HCV cytotoxic T-cell function [41].
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In our study, the TGF-b1 gene polymorphism correlated with the presence of ascites but not with the results of synthetic liver function tests. These findings were not unexpected, as TGF-b1 is a fibrogenic cytokine. Therefore, the genetic ability to produce high levels of TGF-b1 would affect the stage of fibrosis and thereby portal hypertension, but not hepatocellular function. In conclusion, we found that TGF-b1 gene polymorphism correlates with its in situ hepatic expression and degree of hepatic fibrosis in patients with recurrent HCV after liver transplantation. In addition, the IFN-g gene polymorphism correlates with early recurrent hepatitis C after transplantation. These findings might help to design preemptive aggressive antiviral therapy in selected patients at risk.
References [1] Fukumoto T, Berg T, Ku Y, Bechstein WO, Hopf U, Neuhaus P. Viral dynamics of hepatitis C early after orthotopic liver transplantation: evidence for rapid turnover of serum virions. Hepatology 1996;24:1351e4. [2] Forman LM, Lucey MR. Orthotopic liver transplantation for hepatitis C: analysis of allograft survival using the UNOS database. Am J Transpl 2001;1(Suppl 1):S156A. [3] Gane EJ, Portmann BC, Naoumov NV, Smith HM, Underhill JA, Donaldson PT, et al. Long-term outcome of hepatitis C infection after liver transplantation. N Engl J Med 1996;334:821e7. [4] Keeffe EB. Liver transplantation: current status and novel approaches to liver replacement. Gastroenterology 2001;120: 749e62. [5] Kosiel MJ. Cytokines in viral hepatitis. Semin Liver Dis 1999;19: 157e69. [6] Bidwell JL, Wood NA, Morse HR, Olomolaiye OO, Laundy GJ. Human cytokine gene nucleotide sequence alignments. Eur J Immunogenet 1998;25:83e265. [7] Bidwell JL, Wood NA, Morse HR, Olomolaiye OO, Keen LJ, Laundy GJ. Human cytokine gene nucleotide sequence alignments. Eur J Immunogenet 1999;26:135e223. [8] Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A 1997;94:3195e9. [9] Fishman D, Faulds G, Jeffrey R, Jeffery R, Mohamed-Ali V, Yudkin JS, et al. The effect of novel polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic onset juvenile chronic arthritis. J Clin Invest 1998;102:1369e76. [10] Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ, Hutchinson IV. In vitro production of IFN-g gene correlates with CA repeat polymorphism in the human IFN-g gene. Eur J Immunogenet 1999;26:1e3. [11] He B, Xu C, Yang B, Landtblom AM, Fredrikson S, Hillert J. Linkage and association analysis of gene encoding cytokine and myelin proteins in multiple sclerosis. J Neuroimmunol 1998;86: 13e9. [12] Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet 1997;24:1e8. [13] Tagore A, Gonsalkorale WM, Pravica V, Hajeer AH, McMahon R, Whorwell PJ, et al. Interleukin-10 (IL-10) genotype in inflammatory bowel disease. Tissue Antigens 1999;54:389e90.
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[14] Turner D, Grant SC, Yonan N, Sheldon S, Dyer PA, Sinnott PJ, et al. Cytokine gene polymorphism and heart transplant rejection. Transplantation 1997;64:776e9. [15] Awad M, Pravica V, Perrey C, El Gamel A, Yonan N, Sinnott PJ, et al. CA repeat allele polymorphism in the first intron of the human interferon-gamma gene is associated with lung allograft fibrosis. Hum Immunol 1999;60:343e6. [16] Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV. Genotype variation in the transforming growth factor-beta 1 gene-association with transforming growth factor-pi production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation 1998;66:1014e20. [17] Tambur AR, Ortegel JW, Ben-Ari Z, Shabtai E, Klein T, Michoviz R, et al. Role of cytokine gene polymorphism in hepatitis C recurrence and allograft rejection among liver transplant recipients. Transplantation 2001;71:1475e80. [18] Czaja MJ, Weiner FR, Flanders KC, Giambrone MA, Wind R, Biempica L, et al. In vitro and in vivo association of transforming growth factor B1 with hepatic fibrosis. J Cell Biol 1989;108: 2477e8224. [19] Nakatsukasa H, Nagy P, Evarts RP, Hsia CC, Marsden E, Thorgeitsson SS. Cellular distribution of transforming growth factor-beta 1 and procollagen types I, III and IV transcripts in carbon tetrachloride-induced rate liver fibrosis. J Clin Invest 1990; 85:1833e4318. [20] Kanzler S, Baumann M, Schirmacher P, Dries V, Bayer E, Gerken G, et al. Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levels of transforming growth factor-b. J Viral Hepat 2001;8:430e7. [21] Berenguer M, Lopez-Labrador FX, Wright TL. Hepatitis C and liver transplantation. J Hepatol 2001;35:666e78. [22] Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973;60:646e9. [23] Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981;1:431e5. [24] Berenguer M, Ferrell L, Watson J, Prieto M, Kim M, Rayon M, et al. HCV-related fibrosis progression following liver transplantation: increase in recent years. J Hepatol 2000;32:673e84. [25] Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet 1997;349: 825e32. [26] Testa G, Crippin JS, Netto GJ, Goldstein RM, Jennings LW, Brkic BS, et al. Liver transplantation for hepatitis C: recurrence and disease progression in 300 patients. Liver Transpl 2000;6:553e61. [27] Perey C, Pravica V, Sinnott P, Hutchinson IV. Genotyping for polymorphisms in interferon-g, interleukin-10, transforming growth factor-b1 and tumour necrosis factor-a genes: a technical report. Transpl Immunol 1998;6:193e7. [28] Hoffman SC, Stanley EM, Cox ED, DiMercurio BS, Koziol DE, Harlan DM, et al. Ethnicity greatly influences cytokine gene polymorphism distribution. Am J Transpl 2002;2:560e7.
[29] Koziel MJ, Dudley D, Afdhal N, Grakoui A, Rice CM, Choo QL, et al. HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release. J Clin Invest 1995;96: 2311e21. [30] Lechmann M, Woitas RP, Langhans B, Kaiser R, Ihlenfeldt HG, Jung G, et al. Decreased frequency of HCV core-specific peripheral blood mononuclear cells with type 1 cytokine secretion in chronic hepatitis C. J Hepatol 1999;31:971e8. [31] Cribier B, Schmitt C, Rey D, Lang JM, Kirn A, Stoll-Keller F. Production of cytokines in patients infected by hepatitis C virus. J Med Virol 1998;55:89e91. [32] Schirren CA, Jung M, Worzfeld T, Marmin M, Baretton GB, Gruener NH, et al. Cytokine profile of liver- and blood derived nonspecific T cells after liver transplantation: T helper cell type 1/0 lymphokines dominate in recurrent hepatitis C virus infection and rejection. Liver Transpl 2000;6:222e6228. [33] Ho¨hler T, Kruger A, Gerken G, Schneider PM, Meyer zum Buschenfelde KH, Rittner C. Tumor necrosis factor alpha promoter polymorphism at position 238 is associated with chronic active hepatitis C infection. J Med Virol 1998;54: 173e7. [34] McGuinness PH, Painter D, Davies S, McCaughan GW. Increased intrahepatic CD68 positive cells, MAC 387 positive cells, and proinflammatory cytokines in chronic hepatitis C infection. Gut 2000;46:260e9. [35] Malaguarnera M, Trovato BA, Laurino A, Di Fazio I, Romeo MA, Motta M. Interleukin-6 in hepatitis C cirrhosis. Panminerva 1996;38:207e10. [36] Edwards-Smith CJ, Jonsson JR, Purdie DM, Bansal A, Shorthouse C, Powell EE. Interleukein-10 promoter polymorphism predicts initial response of chronic hepatitis C to interferon alpha. Hepatology 1999;30:526e30. [37] Rosen HR, Gretch DR, Oehlke M, Flora KD, Benner KG, Rabkin JM, et al. Timing and severity of initial hepatitis C recurrence as predictors of long-term liver allograft injury. Transplantation 1998;65:1178e82. [38] Bayer EM, Herr W, Kanzler S, Waldmann C, Meyer zum Buschenfelde KH, Dienes HP, et al. Transforming growth factor-b1 in autoimmune hepatitis. Correlation of liver tissue expression and serum levels with disease activity. J Hepatol 1998; 28:803e11. [39] Nelson DR, Gonzalez-Peralta RP, Qian K, Xu Y, Marousis CG, Davis GL, et al. Transforming growth factor-beta 1 in chronic hepatitis C. J Viral Hepatol 1997;4:29e35. [40] Powell EE, Edwards-Smith CJ, Hay JI, Clouston AD, Crawford DH, Shorthouse C, et al. Host genetic factors influence disease progression in chronic hepatitis C. Hepatology 2000;31: 828e33. [41] Kanto T, Takehara T, Katayama K, Ito A, Mochizuki K, Kuzushita N, et al. Neutralization of transforming growth factor beta 1 augments hepatitis C virus-specific cytotoxic T lymphocytes induction in vitro. J Clin Immunol 1997;17: 462e71.
Role of cytokine gene polymorphism and hepatic transforming growth factor b1 expression in recurrent hepatitis C after liver transplantation Ziv Ben-Aria,), Orit Pappob, Tamarah Druzdb, Jaqueline Sulkesc, Tirza Kleind, Zmira Samrae, Rahamim Gadbae, Anat R. Tamburf, Ran Tur-Kaspaa, Eytan Morg a
The Liver Institute and Department of Medicine D, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel b Department of Pathology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel c Epidemiology Unit, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel d Tissue Typing Laboratory, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel e Department of Microbiology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel f Tissue Typing Laboratory, Rush Medical Center, Chicago, IL, USA g Department of Transplantation, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel Received 11 December 2003; received in revised form 8 March 2004; accepted 15 March 2004
Abstract Recurrent hepatitis C virus (HCV) infection after orthotopic liver transplantation (OLT) is nearly universal. Cytokines play an important role in the immune response to viral infection, and cytokine gene polymorphism affects the overall expression and secretion of cytokines. The objective of this study was to define the relationship between cytokine polymorphism and recurrent hepatitis C after OLT. Blood samples were collected from 36 patients at a mean of 44:6 G 30:4 months after OLT for chronic HCV infection. DNA was extracted from peripheral blood mononuclear cells, and polymerase chain reaction-sequence specific primers (PCR-SSP) analysis was performed on promoter sequences of transforming growth factor b1 (TGF-b1), interleukin 6 (IL-6) interleukin 10 (IL-10), tumor necrosis factor a (TNF-a) and interferon g (INF-g). Liver biopsies performed at diagnosis of recurrent disease were graded with the Knodell score, and hepatic TGF-b1 expression was determined semiquantitatively by immunohistochemistry. The gene polymorphism of TGF-b1 was correlated with its expression on hepatocytes and sinusoids. Polymorphism in all studied cytokine genes was correlated with recurrence, and interval to recurrence (O12 or %12 months post-OLT), and clinical (ascites, ChildePugh score and death), biochemical parameters of recurrent HCV (serum alanine aminotransferase (ALT)), INR, albumin, bilirubin), and virological parameters (HCV genotype and load). Biopsies revealed recurrent HCV in 31 patients (86.1%); in 21 (67.7%), the interval to recurrence was 12 months. There was a statistically significant correlation between TGF-b1 gene polymorphism, i.e., the genetic ability to produce high levels of TGF-b1, and the intensity of TGF-b1 staining on hepatocytes (p ¼ 0:003) and sinusoids (p ¼ 0:003), and the degree of fibrosis (p ¼ 0:02). A borderline correlation was found with the presence of ascites (p ¼ 0:07), but not with ChildePugh score, synthetic liver function tests or HCV genotype and load. The genetic ability to produce low levels of IFN-g was correlated with recurrent disease (p ¼ 0:015). No such correlation was found for TGF-b1 gene polymorphism. In conclusion, polymorphism in the TGF-b1 gene correlates with its in situ hepatic expression in patients with recurrent HCV after liver transplantation. INF-g, but not TGF-b1 gene polymorphism, correlates with early recurrent hepatitis C after transplantation. These findings might help to design preemptive prevention therapy in selected patients at risk. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cytokine gene polymorphism; Hepatitis C; Liver transplantation
) Corresponding author. Tel.: C972-3-9377253; fax: C972-39377277. E-mail address: [email protected] (Z. Ben-Ari). 1043-4666/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2004.03.009
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1. Introduction
2. Patients and methods
Hepatic C virus (HCV)-related liver disease is the leading indication for orthotopic liver transplantation (OLT). Recurrent HCV infection, defined as viremia after OLT, is nearly universal, with histological indices in the majority of patients [1]. Although patients with HCV after OLT have a similar short-term survival to patients who undergo OLT for other causes of liver failure, 20%e30% develop allograft cirrhosis within 5 years [2,3]. The recurrence of HCV is associated with several interrelated viral and host factors, including pretransplant and early post-OLT HCV viral load, genotype 1b, multiple episodes of rejection, and level of immunosuppression [4]. Cytokines play an important role in the generation of the immune response to viral infections, both directly, by inhibition of viral replication, and indirectly, through determination of the predominant pattern of the host response [5]. The in vitro maximal capacity to produce different cytokines in response to mitogen stimulation varies among individuals. These differences are attributable to certain molecular mechanisms, including variations in transcription, translation and secretion pathways [6,7], and conservative mutations within cytokine coding regions, as well as nucleotide variations within more pronounced regulatory regions (i.e., promoter sequences). These genetic polymorphisms have been shown to affect the overall expression and secretion of cytokines, both in in vitro and, sporadically, in in vivo systems [8e13]. A potential association with allelic variations in certain cytokine genes have also been reported in allograft rejection and allograft fibrosis [14e16]. We have recently reported an association between cytokine gene polymorphism and recurrence of hepatitis C in OLT recipients [17]. The inherent ability to produce higher transforming growth factor (TGF)-b1 levels, low interleukin (IL)-10 levels and high interferon (IFN)-g levels was less pronounced in patients who had a disease recurrence than in those who did not [17]. These results were surprising in light of findings that levels of hepatic TGF-b1, a profibrogenic cytokine [18,19], were increased in patients with chronic HCV infection [20], and that serum and tissue levels of TGF-b1 could serve as predictors of progressive liver fibrosis in hepatitis C infection [20]. The objectives of the present study were to further identify and validate, in a larger cohort, potential genetic markers of susceptibility to recurrence of hepatitis C in liver transplant recipients. The gene polymorphism of five cytokines (tumor necrosis factor (TNF)-a, TGF-b1, IFN-g, IL-6, and IL-10) was analyzed and compared between the recurrence and non-recurrence groups, and the findings for the TGF-b1 cytokine gene polymorphism were correlated with its hepatic expression by semiquantitative immunostaining of liver biopsy sections.
2.1. Patients The initial sample included all 42 liver transplant recipients who underwent OLT for chronic HVC infection at Rabin Medical Center between 1992 and 2000. Six patients were excluded because of concomitant alcoholic liver disease (n ¼ 2), a history of hepatitis B (n ¼ 2), or unavailability of patients slides for review (n ¼ 2), leaving 36 patients for study. All had more than one year of post-OLT follow-up (mean, 44:6 G 30:4 months). The diagnosis of recurrent HCV infection was based on the presence of viremia by quantitative PCR assay, increased serum transaminase levels, and histologic findings at diagnosis of lobular hepatitis on liver biopsy in association with hepatocyte necrosis and midzonal macrovesicular steatosis [21]. The immunosuppressive regimen included cyclosporine, azathioprine, or cellcept, and corticosteroids in 11 patients and tacrolimus and corticosteroids in 25 patients. Histologically proven acute cellular rejection episodes were treated with three consecutive boluses of IV solomedrol 1.0 g/day and steroid-resistant episodes were treated with OKT3. Patient files were reviewed for demographic characteristics, pretransplant HCV RNA load and genotype, immunosuppressive regimen, rejection episodes, interval to recurrence, clinical progression at follow-up based on levels of serum bilirubin, alanine aminotransferase (ALT), serum albumin and INR, presence of ascites, ChildePugh score [22], and death. 2.2. Genetic polymorphism assessment The genetic profile of five cytokines was analyzed in all patients. 2.2.1. DNA extraction Genomic DNA was isolated by proteinase K digestion of fresh peripheral blood mononuclear cells, followed by phenol extraction and ethanol precipitation. DNA samples were quantified and subjected to specific PCR reactions as described. 2.2.2. Cytokine gene polymorphism Single nucleotide mutations were analyzed in five cytokines, for genotype and phenotype assignment. Specifically, for TNF-a, we examined the presence of a G or A nucleotide in position 308 of the promoter region, which generates three potential genotypes corresponding to two different phenotypes: the A/A and G/A genotypes represent the potential to produce high levels of TNF-a; and the G/G genotype represents the potential to produce low levels [8]. For IL-6 promoter, we examined the presence of a single nucleotide
Z. Ben-Ari et al. / Cytokine 27 (2004) 7e14
modification in position 174. Both the G/G and G/C genotypes are known to correlate with a high-production phenotype, whereas C/C is associated with low IL-6 production [9]. An additional coding sequence mutation was analyzed for IFN-g at position C874 (T versus A): the homozygous T produces high levels of IFN-g; the heterozygous T/A is an intermediate producer; and the homozygous A genotype represents the potential to generate only low amounts of IFN-g [10]. For TGF-b1, two single nucleotide mutations in the coding region were analyzed: codon C10 can be either T or C, and codon C25, either C or G. Potentially, there are nine different combinations of these two isolated mutations which give rise to three different secretion phenotypes: high, intermediate, and low [11]. For the IL-10 promoter, three polymorphisms were studied: position 1082 (G versus A); position 819 (C versus T); and position 592 (A versus C) [12,13]. 2.2.3. PCR-SSP PCR amplification was carried out according to the manufacturer’s instructions (One Lambda Inc., Canoga Park, CA). Briefly, after the addition of the appropriate primer pairs, salts, buffer and Taq polymerase, the samples were subjected to 30 cycles of PCR as follows: one cycle of 130 s at 96 (C, dropping to 63 (C for an additional 60 s; 9 cycles of 10 s at 96 (C, 60 s at 63 (C; and the final 20 cycles which included a three-temperature rampdannealing for 10 s at 96 (C, hybridization for 50 s at 59 (C, and an extension step of 30 s at 72 (C. PCR products were then loaded onto an agarose gel and photographed with an ultraviolet transilluminator. 2.3. Histological assessment One pathologist (O.P.) blindly reviewed all hepatic specimens of the studied patients for overall necroinflammatory activity (grade 0e12) and fibrosis (stage 0e4) according to Knodell’s score [23]. Intercurrent disease processes, such as acute cellular rejection, cytomegalovirus infection, biliary obstruction, and ischemia were excluded using serologic, immunohistochemical, radiological and endoscopic studies. 2.4. TGF-b1 immunohistochemistry staining and scoring Immunohistology was performed on deparaffinized liver biopsy sections. Briefly, mouse monoclonal antihuman TGF-b1 antibody (Chemicon Int. Inc., Temecula, CA) was used in a dilution of 1:300. Antigen retrieval was performed before application of the primary antibody. The tissue sections were placed in a bath with citrate buffer (pH = 6.0) and microwaved in a pressure cooker at high power (900e1000 W) for 13 min until the cooker was fully pressurized. Thereafter, the
9
microwave level was reduced by 40% (380 W) for another 5 min. Endogenous peroxidase reactions were blocked by adding 3% H2O2 for 20 min. Endogenous avidin and biotin were blocked using Dako’s blocking kit (Dako Corp., Carpinteria, CA). Sections were then incubated with the primary antibody for 45 min, followed by the Dako LSABC Kit (Dako Corp., Carpinteria, CA) peroxidase, which consists of labelled streptavidin biotin reagents. Reactive sites were revealed by incubation with DAB (3,3-diaminobenzidine). Sections were counterstained with hematoxylin. Sections of reactive lymph nodes and kidney served as positive controls, and liver biopsies run concurrently without the primary antibody, as negative controls. The evaluation was done semiquantitatively according to the percentage of cells that stained positively. For sinusoidal cells: 0, staining similar to the negative controls for sinusoidal cells; 1, positive staining in 25% of sinusoidal cells; 2, 50% positive staining; 3, 75% positive staining; 4, 100% positive staining. For hepatocytes: 1, positive staining in 10% of hepatocytes; 2, 20% positive staining; 3, 30% positive staining; 4, 40% positive staining. The hepatic expression of TGF-b1 was correlated with its gene polymorphism.
2.5. Virological assays HCV RNA was tested by nested reverse-transcription polymerase chain reaction assay (Cobas Amplicor HCV Monitor Test, Roche Diagnostic Systems Inc., Branchburg, NJ, USA). Analytical sensitivity of the assay was 600 IU/ml. HCV genotypes were determined by the means of a line-probe hybridization assay (INNO-LiPa Innogenetics, Ghent, Belgium) directed to the 5# untranslated regions of the different HCV genotypes.
2.6. Statistical analysis Continuous variables are given as means G standard deviations. Pearson correlation coefficients (r) and their significance ( p) were calculated between the variables. Chi-squared test or Fisher exact test were used, as appropriate, to analyze statistically significant relationships in the distribution of categorical data. Analysis of variance with the Duncan multiple comparison option was used to analyze statistically significant differences in mean continuous parameters (age, bilirubin, albumin, etc.) between TGF-b1 groups (low, intermediate and high production). Student’s t-test was used to analyze statistically significant differences in mean continuous parameters between two groups of patients (recurrence vs. non-recurrence, etc.). A correlation factor was used for multiple comparisons. A p value of less than or equal to 0.05 was considered statistically significant.
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follow-up was 44:6 G 30:4 months. Five patients did not develop histological recurrence of HCV infection during a follow-up period of 40:5 G 9:6 months (all of them underwent, at least once, a liver biopsy during the follow-up period due to increased serum ALT level, which was found to be related to other causes and resolved).
Table 1 Baseline characteristics of patient (n ¼ 36) Age (years)
53.4 G 8.2
Sex (M/F) (%) HCV genotype 1 HCV load (IU/ml) Rejection (%) Rate Gradea 1 2 3 Cyclosporine/tacrolimus (%) Recurrent HCV infection (%) Interval to recurrence (months) ALT (U/l)b Bilirubin (mg/dl)b Necroinflammatory scoreb Fibrosis scoreb Cirrhosis (%) Follow-up (months)
58.3/41.7 84% 231,770.9 G 541,787.7 12 (33.3%) 6 (50%) 5 (41.6%) 1 (8.4%) 30.5/69.5 31 (86.1%) 14.5 G 15.9 128.6 G 79.9 1.3 G 1.7 5.4 G 2.5 1.5 G 1.1 6 (19.3%) 44.6 G 30.4
3.1. Phenotypic expression Table 2 summarizes the phenotypic expression deduced from the genetic polymorphism in the five selected cytokines in the patients with and without recurrent HCV infection after transplantation. The allelic polymorphism of IFN-g translated directly into three phenotypic levels of expression. A highly statistically significant difference in the distribution of the IFN-g gene polymorphism was observed between the groups (p ¼ 0:001) (Fig. 1). The majority (80%) of the patients without recurrence exhibited the potential to produce high levels of IFN-g (T/T genotype), and 20% exhibited the potential to produce intermediate levels; none had the genetic ability to produce low levels of IFN-g (A/A genotype). By contrast, among the patients with recurrent HCV infection, only 3.3% had the potential to produce high levels of IFN-g, whereas the majority exhibited the potential to produce either low levels of IFN-g (41.9%) or intermediate levels (54.8%). There was also a borderline statistical difference ( probably due to the small sample size) in the distribution of the IFN-g gene polymorphism between patients who developed a recurrent HCV infection early (within 12 months) after transplantation (n ¼ 21) or later (n ¼ 10) (p ¼ 0:07) (Fig. 1). In the first subgroup, 10 patients (47.6%) exhibited the genetic ability to produce low and 11 intermediate levels of IFN-g, and none of the patients had the ability to produce high levels. Among the patients in whom a recurrent infection developed more than 12 months after transplantation, three (30%) had the genetic ability to produce low levels of IFN-g, whereas six (60%) exhibited the potential to produce intermediate levels and one (10%), high levels. There was no statistically significant difference in the ability to produce TNF-a, IL-6, TGF-b1 and IL-10 between patients with and without a recurrent HCV infection (Table 2).
ALT Z alanine aminotransferase. Note: values are mean G SD unless otherwise indicated. a1 Z mild; 2 Z moderate; 3 Z severe. bAt diagnosis of recurrent HCV after transplantation.
3. Results The baseline characteristics of the patients included in this study are presented in Table 1. Mean age was 53:4 G 8:2 years; 58.3% were male. HCV genotypes were available for 25 patients. Twenty-two patients (84%) were genotype 1, and three patients were genotype non-1 (two patients 2a, one patient 3a). The mean pretransplant HCV RNA load was 231; 770:9 G 541; 787:7 IU=ml. The immunosuppressive regimen was based on cyclosporine in 30.5% of the patients and on tacralimus in 69.5%. The rejection episode rate was 33.3%. Only one patient was treated with OKT3. Recurrent HCV infection developed in 31 patients (86.1%) after a mean interval of 14:5 G 15:9 months. The interval to recurrence was %12 months in 21 (67.7%) patients and O12 months in 10 (32.3%). At recurrence, mean serum alanine aminotransferase (ALT) level was 128:6 G 79:9 U=l, and mean serum bilirubin, 1:3 G 1:7 mg=dl. Mean necroinflammatory score at recurrence was 5:4 G 2:5, and mean fibrosis score, 1:5 G 1:1. Six patients (19.3%) developed histologically proven cirrhosis during the follow-up period. Mean duration of
Table 2 Phenotypic expression of five cytokines in patients who underwent OLT due to hepatitis C infection Posttransplantation HCV recurrence
TNF-a (%)
IL-6 (%)
IFN-g (%)
Low
Low
Low
High
High
Interm.
TGF-b1 High
Low
IL-10 Interm.
High
Low
Interm. High
Recurrence (n ¼ 31) 26 (83.8) 5 (16.2) 3 (9.7) 28 (90.3) 13 (41.9) 17 (54.8) 1 (3.3) 2 (6.5) 10 (32.2) 19 (61.3) 18 (58.0) 8 (25.8) 5 (16.1) No recurrence (n ¼ 5) 3 (60.0) 2 (40.0) 0 (0) 5 (100) 0 (0) 1 (20) 4 (80) 0 (0) 3 (60) 2 (40) 3 (60) 1 (20) 1 (20) p Value N.S. N.S. 0.001 N.S. N.S. Note: all values are n (%).
Z. Ben-Ari et al. / Cytokine 27 (2004) 7e14
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Fig. 2. The genetic potential to produce high levels of TGF-b1 correlated significantly with the fibrosis score (p ¼ 0:02). Fig. 1. The genetic ability to produce low levels of IFN-g was associated with recurrence of HCV after transplantation.
3.2. TGF-b1 polymorphism and histological findings (Table 3) There was a statistically significant correlation between the fibrosis score and TGF-b1 gene polymorphism. The genetic potential to produce high levels of TGF-b1 correlated significantly with the fibrosis score (r ¼ 0:43, p ¼ 0:02) (Fig. 2), but not with the necroinflammatory score (r ¼ 0:23, p ¼ 0:23). On immunohistology, a high statistically significant correlation was found between the genetic ability to produce high levels of TGF-b1 and the hepatic expression of TGF-b1 in hepatocytes (r ¼ 0:52, p ¼ 0:003) and sinusoidal cells (r ¼ 0:52, p ¼ 0:003). The close correlation between the fibrosis score and the TGF-b1 polymorphism suggests that liver-derived TGF-b1 is a relevant mediator of fibrosis. All biopsies were scored at least 1. Biopsies with a low score exhibited TGF-b1 staining only in sinusoidal cells (Fig. 3a), whereas biopsies with a high score displayed positive staining of sinusoidal cells and of hepatocyte cytoplasm with focal distribution (Fig. 3b) and association with the fibrosis stage (Fig. 4). An inverse, statistically significant difference was noted between IL-10 polymorphism and fibrosis, with a low fibrosis score associated with the genetic ability to produce high levels of IL-10 (r ¼ 0:38, p ¼ 0:04).
3.3. Correlation between the five selected cytokine gene polymorphism and clinical parameters A borderline statistically significant association was noted between the genetic ability to produce TGF-b1 and the presence of ascites at follow-up (r ¼ 0:33, p ¼ 0:07). However, there was no significant correlation
Table 3 TGF-b1 gene polymorphism and histological findings (mean G SD) TGF-b1 polymorphism
Fibrosis score
Inflammation score
TGF-b1 hepatocyte staining score
TGF-b1 sinusoid staining score
Low Intermediate High p Value
0 1.1 G 1.1 1.8 G 0.52 0.02
3.1 G 1.4 5.5 G 2.5 5.8 G 2.7 0.22
0 0.8 G 0.58 1.4 G 0.7 0.003
0.75 G 0.35 1.2 G 0.71 1.8 G 0.52 0.003
Fig. 3. TGF-b1 immunohistochemistry in liver biopsy. (a) A low score indicated TGF-b1 staining only in sinusoidal cells. (b) A high score indicated positive staining of sinusoidal cells and cytoplasm of hepatocytes with focal distribution.
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Fig. 4. Portal fibrosis and septal formation, positive staining for TGF-b1 is noted in scattered hepatocytes.
between TGF-b1 gene polymorphism and HCV genotype and load, ChildePugh score, synthetic liver function tests, ALT at follow-up, or death. An inverse, statistically significant difference was noted between the genetic ability to produce IL-10 and the presence of ascites (r ¼ 0:35, p ¼ 0:05). No correlation was found between TNF-a, IL-6 and IFN-g gene polymorphisms and the clinical parameters studied.
4. Discussion Liver transplant recipients have an accelerated course of HCV infection compared to immunocompetent patients [24], with a significantly higher rate of fibrosis progression to graft cirrhosis and higher subsequent risk of decompensation than before transplantation [24e26]. The risk factors associated with poor outcome are as yet not clearly defined [4] and include treatment of acute rejection with steroid boluses, or OKT3 for steroidresistant rejection, high viral load either pretransplantation or early after transplantation and genotype 1b [21]. In the present study, we observe a highly statistically significant difference in the IFN-g polymorphism patients who did and did not have a recurrent HCV infection after transplantation, validating our previous findings in a smaller cohort [17]. The patients with recurrent disease had the genetic ability to produce low levels of IFN-g, whereas those without recurrence had the ability to produce high levels of IFN-g (p ¼ 0:001). Moreover, within the recurrence group, those in whom the recurrence developed early (%12 months posttransplantation) exhibited the potential to produce low and intermediate levels of IFN-g compared to those in
whom recurrent disease developed later, who exhibited the potential to produce intermediate and high levels of IFN-g. When comparing the distribution of IFN-g gene polymorphism in our study with previously published studies that included normal controls, the frequency of low levels of IFN-g is almost double, 42% in our patients with recurrent HCV vs. 25%e27% in the normal population [27,28]. Several researchers have suggested that an adequate T-helper 1 (Th1) response (i.e., high IFN-g secretion by peripheral blood mononuclear cells) may be associated with a protective antiviral immune response [29], while insufficient systemic Th1 cytokine secretion may be associated with increased viral load and disease progression [29,30]. Indeed, serum samples from HCV patients contain significantly lower levels of soluble IFN-g compared with controls [31]. The cytokine profile of immunocompromised patients with recurrent HCV infection was also studied recently. Schirren et al. [32] reported that this profile does not differ principally from that of immunocompetent patients with HCV. Thus, our findings, if confirmed in other large-scale studies, might have implications for the pretransplantation identification of a subset of patients who are at risk of early, recurrent HCV infection posttransplantation and who might benefit from preemptive antiviral therapy immediately after transplantation. No significant statistical correlation was found between the TNF-a gene polymorphism at position 308 and recurrent HCV infection. Ho¨hler et al. [33] reported an association of general susceptibility to HCV infection with TNF-a promoter polymorphism, but it involved only the polymorphism at position 238 (p ! 0:009); as in our study, no such correlation was found at position 308. The distribution of TNF-a gene polymorphism in our study was comparable with the previously reported studies of the distribution in normal controls [27]. The reports on the role of IL-6 in chronic HCV infection are contradictory. McGuinness et al. [34] found that IL-6 mRNA is significantly downregulated in HCV-positive liver tissue, whereas others noted high serum IL-6 levels in patients with chronic HCV infection [35]. We did not find any significant statistical correlation between IL-6 gene polymorphism and recurrent HCV infection. The distribution of IL-6 gene polymorphism was comparable with the previously reported studies of the distribution in normal controls [28]. In our previous report [17], a borderline statistically significant difference in IL-10 polymorphism was found between patients with and without recurrent HCV posttransplantation (p ¼ 0:07). In the present study, which included a larger number of patients, no difference in the genetic ability to produce IL-10 was found between the groups. However, when comparing the distribution of IL-10 gene polymorphism in our study to previously published studies that included
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normal controls, the frequency of low level IL-10 was doubled, 58% in our patients with recurrent HCV vs. 27%e28% in the normal population [27,28]. In a recent study, Edwards-Smith et al. [36] recently noted that the heterogeneity in the promoter region of the IL-10 gene played a role in the initial response to IFN-a therapy in patients with HCV infection: those who were genetically predisposed to high IL-10 production had a poor response to IFN-a. We did not find any significant statistical correlation between TGF-b1 gene polymorphism and recurrent HCV infection. The distribution of TGF-b1 gene polymorphism was comparable with the previously reported studies of the distribution in normal controls [27,28]. Regarding TGF-b1, our study showed a statistically significant correlation between the genetic ability to produce high levels of TGF-b1 and the fibrosis score in patients with untreated chronic HCV infection. The fibrosis score in the initial posttransplantation liver biopsies [24,26,37] has been found to be a useful predictor of the development of severe chronic HCV. Accordingly, the TGF-b1 polymorphism may have a prognostic significance in patients with recurrent HCV infection, thereby possibly directing more aggressive therapy towards those patients with a genetic ability to produce high levels of TGF-b1. Furthermore, in the complex multivariate setting of the posttransplantation period, the patient’s ability to produce low levels of TGF-b1 could also serve as an additional guide to physicians to decide against antiviral therapy because of minimal disease activity. These assumptions are supported by our findings of a correlation of both TGF-b1 expression within the liver and TGF-b1 polymorphism with the progression of liver disease, and the association of progressive fibrosis with a typical pattern of TGF-b1 expression in both hepatocytes and sinusoidal cells in the liver. By contrast, in patients with stable disease, hepatic TGF-b1 expression was mostly limited to existing fibrous septa. Apart from their prognostic significance, these findings provide further proof that hepatocytes are a potential source of TGF-b1 under pathological conditions, as demonstrated in patients with autoimmune hepatitis [38] and HCV infection [20]. Thus, the source of TGF-b1 in the liver is not only from the recipient graft infiltrating cells but also from the donor hepatocytes. TGF-b1 has been implicated as a mediating factor in hepatic fibrogenesis in chronic HCV infection [39]. Host genetic factors that influence fibrogenesis may account for some of the variability in the progression of the disease. Powell et al. [40] recently reported a significant relationship between inheritance of the high TGF-b1producing genotype and the development of hepatic fibrosis. In addition, TGF-b1 inhibits proliferation and function of T and B lymphocytes, also its high expression could lead to suppression of anti-HCV cytotoxic T-cell function [41].
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In our study, the TGF-b1 gene polymorphism correlated with the presence of ascites but not with the results of synthetic liver function tests. These findings were not unexpected, as TGF-b1 is a fibrogenic cytokine. Therefore, the genetic ability to produce high levels of TGF-b1 would affect the stage of fibrosis and thereby portal hypertension, but not hepatocellular function. In conclusion, we found that TGF-b1 gene polymorphism correlates with its in situ hepatic expression and degree of hepatic fibrosis in patients with recurrent HCV after liver transplantation. In addition, the IFN-g gene polymorphism correlates with early recurrent hepatitis C after transplantation. These findings might help to design preemptive aggressive antiviral therapy in selected patients at risk.
References [1] Fukumoto T, Berg T, Ku Y, Bechstein WO, Hopf U, Neuhaus P. Viral dynamics of hepatitis C early after orthotopic liver transplantation: evidence for rapid turnover of serum virions. Hepatology 1996;24:1351e4. [2] Forman LM, Lucey MR. Orthotopic liver transplantation for hepatitis C: analysis of allograft survival using the UNOS database. Am J Transpl 2001;1(Suppl 1):S156A. [3] Gane EJ, Portmann BC, Naoumov NV, Smith HM, Underhill JA, Donaldson PT, et al. Long-term outcome of hepatitis C infection after liver transplantation. N Engl J Med 1996;334:821e7. [4] Keeffe EB. Liver transplantation: current status and novel approaches to liver replacement. Gastroenterology 2001;120: 749e62. [5] Kosiel MJ. Cytokines in viral hepatitis. Semin Liver Dis 1999;19: 157e69. [6] Bidwell JL, Wood NA, Morse HR, Olomolaiye OO, Laundy GJ. Human cytokine gene nucleotide sequence alignments. Eur J Immunogenet 1998;25:83e265. [7] Bidwell JL, Wood NA, Morse HR, Olomolaiye OO, Keen LJ, Laundy GJ. Human cytokine gene nucleotide sequence alignments. Eur J Immunogenet 1999;26:135e223. [8] Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A 1997;94:3195e9. [9] Fishman D, Faulds G, Jeffrey R, Jeffery R, Mohamed-Ali V, Yudkin JS, et al. The effect of novel polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic onset juvenile chronic arthritis. J Clin Invest 1998;102:1369e76. [10] Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ, Hutchinson IV. In vitro production of IFN-g gene correlates with CA repeat polymorphism in the human IFN-g gene. Eur J Immunogenet 1999;26:1e3. [11] He B, Xu C, Yang B, Landtblom AM, Fredrikson S, Hillert J. Linkage and association analysis of gene encoding cytokine and myelin proteins in multiple sclerosis. J Neuroimmunol 1998;86: 13e9. [12] Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet 1997;24:1e8. [13] Tagore A, Gonsalkorale WM, Pravica V, Hajeer AH, McMahon R, Whorwell PJ, et al. Interleukin-10 (IL-10) genotype in inflammatory bowel disease. Tissue Antigens 1999;54:389e90.
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Z. Ben-Ari et al. / Cytokine 27 (2004) 7e14
[14] Turner D, Grant SC, Yonan N, Sheldon S, Dyer PA, Sinnott PJ, et al. Cytokine gene polymorphism and heart transplant rejection. Transplantation 1997;64:776e9. [15] Awad M, Pravica V, Perrey C, El Gamel A, Yonan N, Sinnott PJ, et al. CA repeat allele polymorphism in the first intron of the human interferon-gamma gene is associated with lung allograft fibrosis. Hum Immunol 1999;60:343e6. [16] Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV. Genotype variation in the transforming growth factor-beta 1 gene-association with transforming growth factor-pi production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation 1998;66:1014e20. [17] Tambur AR, Ortegel JW, Ben-Ari Z, Shabtai E, Klein T, Michoviz R, et al. Role of cytokine gene polymorphism in hepatitis C recurrence and allograft rejection among liver transplant recipients. Transplantation 2001;71:1475e80. [18] Czaja MJ, Weiner FR, Flanders KC, Giambrone MA, Wind R, Biempica L, et al. In vitro and in vivo association of transforming growth factor B1 with hepatic fibrosis. J Cell Biol 1989;108: 2477e8224. [19] Nakatsukasa H, Nagy P, Evarts RP, Hsia CC, Marsden E, Thorgeitsson SS. Cellular distribution of transforming growth factor-beta 1 and procollagen types I, III and IV transcripts in carbon tetrachloride-induced rate liver fibrosis. J Clin Invest 1990; 85:1833e4318. [20] Kanzler S, Baumann M, Schirmacher P, Dries V, Bayer E, Gerken G, et al. Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levels of transforming growth factor-b. J Viral Hepat 2001;8:430e7. [21] Berenguer M, Lopez-Labrador FX, Wright TL. Hepatitis C and liver transplantation. J Hepatol 2001;35:666e78. [22] Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973;60:646e9. [23] Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981;1:431e5. [24] Berenguer M, Ferrell L, Watson J, Prieto M, Kim M, Rayon M, et al. HCV-related fibrosis progression following liver transplantation: increase in recent years. J Hepatol 2000;32:673e84. [25] Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet 1997;349: 825e32. [26] Testa G, Crippin JS, Netto GJ, Goldstein RM, Jennings LW, Brkic BS, et al. Liver transplantation for hepatitis C: recurrence and disease progression in 300 patients. Liver Transpl 2000;6:553e61. [27] Perey C, Pravica V, Sinnott P, Hutchinson IV. Genotyping for polymorphisms in interferon-g, interleukin-10, transforming growth factor-b1 and tumour necrosis factor-a genes: a technical report. Transpl Immunol 1998;6:193e7. [28] Hoffman SC, Stanley EM, Cox ED, DiMercurio BS, Koziol DE, Harlan DM, et al. Ethnicity greatly influences cytokine gene polymorphism distribution. Am J Transpl 2002;2:560e7.
[29] Koziel MJ, Dudley D, Afdhal N, Grakoui A, Rice CM, Choo QL, et al. HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release. J Clin Invest 1995;96: 2311e21. [30] Lechmann M, Woitas RP, Langhans B, Kaiser R, Ihlenfeldt HG, Jung G, et al. Decreased frequency of HCV core-specific peripheral blood mononuclear cells with type 1 cytokine secretion in chronic hepatitis C. J Hepatol 1999;31:971e8. [31] Cribier B, Schmitt C, Rey D, Lang JM, Kirn A, Stoll-Keller F. Production of cytokines in patients infected by hepatitis C virus. J Med Virol 1998;55:89e91. [32] Schirren CA, Jung M, Worzfeld T, Marmin M, Baretton GB, Gruener NH, et al. Cytokine profile of liver- and blood derived nonspecific T cells after liver transplantation: T helper cell type 1/0 lymphokines dominate in recurrent hepatitis C virus infection and rejection. Liver Transpl 2000;6:222e6228. [33] Ho¨hler T, Kruger A, Gerken G, Schneider PM, Meyer zum Buschenfelde KH, Rittner C. Tumor necrosis factor alpha promoter polymorphism at position 238 is associated with chronic active hepatitis C infection. J Med Virol 1998;54: 173e7. [34] McGuinness PH, Painter D, Davies S, McCaughan GW. Increased intrahepatic CD68 positive cells, MAC 387 positive cells, and proinflammatory cytokines in chronic hepatitis C infection. Gut 2000;46:260e9. [35] Malaguarnera M, Trovato BA, Laurino A, Di Fazio I, Romeo MA, Motta M. Interleukin-6 in hepatitis C cirrhosis. Panminerva 1996;38:207e10. [36] Edwards-Smith CJ, Jonsson JR, Purdie DM, Bansal A, Shorthouse C, Powell EE. Interleukein-10 promoter polymorphism predicts initial response of chronic hepatitis C to interferon alpha. Hepatology 1999;30:526e30. [37] Rosen HR, Gretch DR, Oehlke M, Flora KD, Benner KG, Rabkin JM, et al. Timing and severity of initial hepatitis C recurrence as predictors of long-term liver allograft injury. Transplantation 1998;65:1178e82. [38] Bayer EM, Herr W, Kanzler S, Waldmann C, Meyer zum Buschenfelde KH, Dienes HP, et al. Transforming growth factor-b1 in autoimmune hepatitis. Correlation of liver tissue expression and serum levels with disease activity. J Hepatol 1998; 28:803e11. [39] Nelson DR, Gonzalez-Peralta RP, Qian K, Xu Y, Marousis CG, Davis GL, et al. Transforming growth factor-beta 1 in chronic hepatitis C. J Viral Hepatol 1997;4:29e35. [40] Powell EE, Edwards-Smith CJ, Hay JI, Clouston AD, Crawford DH, Shorthouse C, et al. Host genetic factors influence disease progression in chronic hepatitis C. Hepatology 2000;31: 828e33. [41] Kanto T, Takehara T, Katayama K, Ito A, Mochizuki K, Kuzushita N, et al. Neutralization of transforming growth factor beta 1 augments hepatitis C virus-specific cytotoxic T lymphocytes induction in vitro. J Clin Immunol 1997;17: 462e71.