Secondary Logo

Journal Logo

Donor Graft Steatosis Influences Immunity to Hepatitis C Virus and Allograft Outcome After Liver Transplantation

Subramanian, Vijay1,4; Seetharam, Anil B.2; Vachharajani, Neeta1; Tiriveedhi, Venkataswarup1; Angaswamy, Nataraju1; Ramachandran, Sabarinathan1; Crippin, Jeffrey S.2; Shenoy, Surendra1; Chapman, William C.1; Mohanakumar, Thalachallour1,3; Anderson, Christopher D.1

doi: 10.1097/TP.0b013e318235a1ab
Clinical and Translational Research
Free

Background. Hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) is universal, often with accelerated allograft fibrosis. Donor liver steatosis is frequently encountered and often associated with poor early postoperative outcome. The aim of this study was to test the hypothesis that allograft steatosis alters immune responses to HCV and self-antigens promoting allograft fibrosis.

Methods. Forty-eight HCV OLT recipients (OLTr) were enrolled and classified based on amount of allograft macrovesicular steatosis at time of OLT. Group 1: no steatosis (0%–5% steatosis, n=21), group 2: mild (5%–35%, n=16), and group 3: moderate (>35%, n=11). Cells secreting interleukin (IL)-17, IL-10, and interferon gamma (IFN-γ) in response to HCV antigens were enumerated by Enzyme Linked Immunospot Assay. Serum cytokines were measured by Luminex, antibodies to Collagen I, II, III, IV, and V by ELISA.

Results. OLTr of moderate steatotic grafts had the highest incidence of advanced fibrosis in protocol 1 year post-OLT biopsy (10.8% vs. 15.8% vs. 36.6%, r=0.157, P<0.05). OLTr from groups 2 and 3 had increased HCV-specific IL-17 (P<0.05) and IL-10 (P<0.05) with reduced IFN-γ (P<0.05) secreting cells when compared with group 1. This was associated with increase in serum IL-17, IL-10, IL-1β, IL-6, IL-5, and decreased IFN-γ. In addition, there was development of antibodies to Collagen I, II, III and V in OLTr with increased steatosis (P<0.05).

Conclusion. The results demonstrate that allograft steatosis influences post-OLT HCV-specific immune responses leading to an IL-17 T-helper response and activation of humoral immune responses to liver-associated self-antigens that may contribute to allograft fibrosis and poor outcome.

1 Department of Surgery, Washington University, St. Louis, MO.

2 Department of Gastroenterology, Washington University, St. Louis, MO.

3 Department of Pathology and Immunology, Washington University, St. Louis, MO.

This work was supported by NIH grant DK065982 (T.M.) and 5-T32-DK07301-35 (A.S.), the BJC Foundation (T.M.), and in part, by the ASTS-Astellas Faculty Development Award and NIH grants P30 DK056341 and L30 DK082350 (C.D.A.).

The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

The authors have no conflicts of interests to disclose.

4Address correspondence to: Vijay Subramanian, M.D., Department of Surgery, Washington University School of Medicine, Box 8109, 3328 CSRB, 660 S. Euclid Avenue, St. Louis, MO 63110.

E-mail: subramanianv@wustl.edu

V.S. participated in research design, performing experiments, data analysis, and writing the manuscript. A.B.S. participated in research design, data analysis, and writing the manuscript. N.V. participated in data analysis and writing the manuscript. V.T. participated in data analysis and writing the manuscript. N.A. participated in data analysis and writing the manuscript. S.R. participated in data analysis and writing the manuscript. J.S.C. participated in research design and writing the manuscript. S.S. participated in research design and writing the manuscript. W.C.C. participated in research design, data analysis, and writing the manuscript. T.M. participated in research design, data analysis, and writing the manuscript. C.D.A. participated in research design, data analysis, and writing the manuscript.

Received 9 June 2011. Revision requested 14 July 2011.

Accepted 31 August 2011.

Hepatitis C virus (HCV) liver disease is the leading indication for orthotopic liver transplantation (OLT) in United States (1, 2). In 2010, among 16,904 United Network for Organ Sharing registrants, only 5763 OLTs were performed (3). To meet the demand, “extended criteria” donors after cardiac death and “steatotic” livers are often used for OLT. Steatotic allografts are cautiously used due to early postoperative complications (4–6). Because of high prevalence (25%–50%) of potential donors with significant liver steatosis (7, 8), its effect on outcome in HCV recipients requires further investigation.

HCV recurrence in the allograft is near universal often leading to accelerated fibrosis compared with native liver (9–11). Immunologic factors including T-cell responses to HCV (12–15) and immunity to extracellular matrix (ECM) antigens (Collagens [Col]) (16) have been implicated in progression of allograft fibrosis. Donor factors including graft quality can influence HCV recurrence (17). Briceño et al. (18) demonstrated that allografts with greater than 30% steatosis were associated with increased fibrosis. However, Burra et al. (19) found no impact of steatosis on fibrosis and outcome. Steatotic allografts have an increased susceptibility to ischemia-reperfusion injury (20, 21) and have poorer functional recovery (5, 22). In this context, it is interesting to note the influence of duration and degree of ischemia-reperfusion injury on HCV recurrence (23, 24).

The aim of this study was to evaluate the effect of allograft steatosis on post-OLT HCV immunity. We hypothesized that steatotic allografts increase susceptibility to HCV-mediated injury, the development of immunity against ECM antigens (Col), and thus promoting fibrosis. The results presented demonstrate that OLT recipients (OLTr) of steatotic allografts have increased Th17 and Th2 responses to HCV and suppression of Th1. This was also associated with the development of antibodies (Abs) to self-antigens (Col).

Back to Top | Article Outline

RESULTS

Patient Demographics

Eighty-five subjects were included—48 HCV OLTr, 27 non-HCV OLTr, and 10 healthy subjects. OLTr were classified by allograft macrovesicular steatosis at the time of OLT: groups 1 to 3, HCV OLTr; groups 4 to 6, non-HCV OLTr. Group 1 (n=21) and group 4 (n=11)—no steatosis; group 2 (n=16) and group 5 (n=10)—mild steatosis; and group 3 (n=11) and group 6 (n=6)—moderate/severe steatosis. Among the HCV OLTr, time from OLT for blood and biopsy was similar in all groups (312±10 days vs. 340±24 days vs. 306±22 days). No differences were noted in clinical demographics (Table 1) including pretransplant model for end-stage liver disease and donor characteristics. Peak transaminase levels after OLT were significantly higher in group 3 OLTr compared with groups 1 and 2 (aspartate aminotransferase [AST]: 1905 IU/mL vs. 2809 IU/mL vs. 3883 IU/mL, P=0.026; alanine aminotransferase [ALT]: 1236 IU/mL vs. 1359 IU/mL vs. 1776 IU/mL, P=0.039).

TABLE 1

TABLE 1

In 15 subjects (group 1—6, group 2—5, and group 3—4) biopsy due to clinical suspicion (rejection/obstruction) was performed during first year post-OLT. Biopsy- confirmed acute rejection (Banff schema [25]) was similar (25%, 27%, and 22%) including severity (mild or moderate, no severe rejection). One patient (group 1) developed biliary obstruction and underwent stent placement. Acute rejection was treated with bolus steroids as first-line therapy and in two nonresponders (group 1) with thymoglobulin/OKT3. None of the subjects received antiviral therapy post-OLT.

Clinical demographics of the non-HCV OLTr were similar (Table 2). In them also, peak transaminase levels were significantly more in moderate/severe steatotic grafts (AST: 743 vs. 1309 vs. 2708 IU/mL, P=0.03; ALT: 934 vs. 1405 vs. 2017 IU/mL, P=0.027).

TABLE 2

TABLE 2

Ten healthy subjects (no preexisting liver or autoimmune disease; age 35±8 years, male:female ratio 6:4) were included to standardize ELISA for detection of Abs to Col. Control subjects were HIV, Hepatitis B, and HCV negative.

Back to Top | Article Outline

Higher Prevalence of Advanced Fibrosis Post-OLT in Patients With Higher Donor Graft Steatosis at OLT

Allograft fibrosis was determined in biopsy by modified Batts Ludwig score (26). Group 3 HCV OLTr whose grafts had moderate/severe steatosis at OLT had the highest incidence of fibrosis (stage 3–4) when compared with groups 2 and 1—36.6% vs. 15.8% vs. 10.8%, P<0.05. Fibrosis post-OLT had a significant positive correlation with extent of steatosis at time of OLT with a spearman rho of 0.157, P less than 0.05.

In the biopsy of 15 subjects who underwent biopsy within 6 months post-OLT for clinical indications, there was no significant fibrosis. There was no correlation of other factors including rejection (r=0.024, P=0.45), donor age, and so forth, with post-OLT fibrosis. Non-HCV OLTr did not demonstrate significant fibrosis post-OLT.

Back to Top | Article Outline

Increased Interleukin-17, Interleukin-10, and Decreased Interferon Gamma Secreting Cells to HCV Antigens in OLTr of Higher Grades of Graft Steatosis

Immune responses to HCV antigens (NS3, NS4, NS5, Core) were determined by Enzyme Linked Immunospot Assay (ELISpot). Group 3 HCV OLTr had significantly higher frequency of HCV-specific interleukin (IL)-17 cells when compared with groups 2 and 1 (in mean spots per million cells [spm] ±SE; groups 3, 2, and 1: Core: 23.3±3.4 vs. 12.2±5.2 vs. 10±2.1, P=0.043; NS3 P=0.03; NS4 P=0.051; NS5 P=0.049; Fig. 1A). There was no difference in the cells secreting IL-17 in response to HIV and phytohemagglutinin (PHA) among the groups.

FIGURE 1.

FIGURE 1.

Groups 2 and 3 HCV OLTr demonstrated significantly increased frequency of HCV specific IL-10 cells when compared with group 1 (groups 2, 3, and 1; Core: 259.6±30.5 vs. 402.3±37.2 vs. 107.2±23.5 spm, P=0.01; NS3, P<0.01; NS4, P=0.02; NS5, P<0.01) (Fig. 1B).

When compared with group 1, both groups 2 and 3 HCV OLTr had significantly lower frequency of cells secreting interferon gamma (IFN-γ) in response to HCV (groups 2, 3, and 1; core: 34.3±14.1 vs. 9.1±2.4 vs. 133.6±42.7, P=0.01; NS3, P=0.032; NS4, P=0.02; NS5, P=0.045) (Fig. 1C). These results indicate that with increasing steatosis, there was an increase in IL-17 and IL-10 in response to HCV antigens with a decrease in IFN-γ.

Back to Top | Article Outline

Increased Serum Cytokines (IL-1β, IL-4, IL-17, IL-10, IL5, IL-6, and IL-8) and Chemokines (IP10, MIP, Eotaxin, and MCP1) in HCV OLTr of Grafts With Moderate-to-Severe Steatosis

Serum IL-17, IL-6, IL-1β, IL-4, IL-5, and IL-10, and chemokines IL-8, IP10, MIP, Eotaxin, and MCP-1 were increased in groups 2 and 3 (Table 3). Ten subjects were analyzed 6 months post-OLT groups 1—4, and groups 2 and 3—3 each. Recipients in group 3 demonstrated a significant increase in these cytokines and chemokines when compared with groups 1 and 2. In addition, IFN-γ in group 3 OLTr sera were significantly lower than groups 1 and 2 (at 1 year: 23.9±1.1 pg/mL vs. 27.9±2.3 pg/mL vs. 25.8±1.8 pg/mL; and at 6 months: 22.74±4.57 pg/mL vs. 63.29±3.69 pg/mL vs. 34.69±6.53 pg/mL, P=0.046; Table 3). There were no differences in the levels of other cytokines and chemokines (data not shown). Thus, there was an increase in IL-17, pro Th-17 (IL-6, IL-1β), Th2 (IL-10, IL-4) cytokines, and pro-inflammatory chemokines in OLTr of higher steatotic grafts with a suppression of Th1 (IFN-γ) cytokines and which was significant in the early post-OLT period.

TABLE 3

TABLE 3

Back to Top | Article Outline

HCV OLTr of Mild-to-Severe Steatotic Grafts Develop Increased Abs to Self-Antigens (Col)

All HCV OLTr developed Abs to Col I, II, III, and V when compared with controls. Additionally, both groups 2 and 3 had significantly higher levels of Abs to Col I (P=0.031), Col II (P=0.029), Col III (P=0.047), and Col V (P=0.048) (Fig. 2A-C,E). There was no significant difference in the Abs to Col IV among the groups (P=0.23) (Fig. 2D). The Abs titers in non-HCV OLTr in the various steatotic groups did not significantly differ from healthy controls (Fig. 2A-E).

FIGURE 2.

FIGURE 2.

Back to Top | Article Outline

Patient and Graft Survival in HCV OLTr

Patient and graft outcome in all OLTr (HCV=131; non-HCV=178) between January 2002 and December 2008 was analyzed (Fig. 3A-D). Mean patient survival was similar in the groups (HCV OLTr: 5.4±0.2 years, 6.3±0.4 years, 4.9±0.8 years; non-HCV OLTr: 6.4±0.3 years, 6.8±0.6 years, 4.7±0.7 years). Mean graft survival among HCV OLTr was 5.3±0.25 years, 6.3±0.4 years, 4.9±0.8 years and among non-HCV OLTr was 6.3±0.4 years, 6.7±0.6 years, 4.6±0.7 years. Patient survival at 3 months in HCV OLTr was 97%, 100%, 91%, and at 1 year: 93% vs. 94% vs. 82% (P=0.24). In non-HCV OLTr, 3-month survival was 95%, 88%, and 86% and 1-year survival was 90% vs. 85% vs. 71% (P=0.47). There was no difference in survival between HCV and non-HCV OLTr (Fig. 3A, C).

FIGURE 3.

FIGURE 3.

Back to Top | Article Outline

DISCUSSION

Many factors both in the donor and recipient including immune responses affect prognosis in HCV OLTr (14, 17, 23, 27–32). This study evaluated the impact of donor graft steatosis on changes in post-OLT HCV immunity. In HCV OLTr, increasing grade of allograft steatosis positively correlated with fibrosis 1 year post-OLT. These OLTr had increased HCV-specific IL-17 and IL-10 with decreased IFN-γ secreting cells (Fig. 1). This was associated with an increase in serum IL-17, pro Th-17 (IL-6, IL-1β), and Th2 (IL-4, IL-10) cytokines (Table 3). Additionally, HCV OLTr also developed Abs to self-antigens (Col I, II, III, and V) whose titer was more in OLTr of moderate/severe steatotic grafts. These results support the contention of donor steatosis being an important factor in viral recurrence and fibrosis (18) and further suggest a role for allograft steatosis in influencing post-OLT HCV immune responses development of immunity to self-antigens.

Earlier studies have associated graft steatosis with poor function (28, 29), but these grafts when appropriately selected can have functional recovery (5). The degree of steatosis decreases soon after OLT (33), and in this study also there was lack of any significant steatosis in the grafts 1 year post-OLT (data not shown) with no difference in overall survival. Similar to previous findings (5–7, 20, 22), this report demonstrates that OLTr of moderate/severe steatotic grafts had higher transaminases (AST and ALT, P<0.05) in the first week after OLT (Table 1) signifying increased reperfusion injury. OLTr of moderate-to-severe steatotic grafts have increased coagulopathy and a stormier postoperative period (5, 6). Several studies including by Verran et al. (6) and Chui et al. (34) report increased early poor function and survival in moderate/severe steatotic grafts. These could serve as a possible explanation for the poor early survival of steatotic allografts; however, this was not statistically significant (Fig. 3).

HCV recurrence is universal (35, 36) and in peri-OLT period early preservation and reperfusion injuries have been associated with HCV recurrence and poor outcome (23, 24). Therefore, it is likely that postoperative stress sets up an inflammatory process that can alter the host immunological response to HCV. This in turn can promote HCV pathologic injury and recurrence resulting in development of allograft fibrosis. Although events immediately post-OLT could not be analyzed due to nonavailability of serial samples, the significant differences in cytokine levels 6 months post-OLT even with a smaller number of samples (Table 3) support the hypothesis that early inflammatory events may determine the course and progression of HCV infection and fibrosis.

The immune response to HCV is a critical determinant of allograft fibrosis, and a predominant Th2 (13, 14, 37) and Th17 (15) immunity is associated with increased fibrosis and poor outcome. Cytokines including IL-1β, IL-6, and IL-8 have been shown to promote fibrosis (38–41). These cytokines also favor Th17 responses and suppress IFN-γ (15). This study demonstrated that such HCV immune responses are more prominent in OLTr of grafts with higher grade of steatosis (Fig. 1, Table 3). We propose that this prevents viral clearance and enhances liver damage, increased ECM turnover, and the development of fibrosis.

The inflammatory process that follows ischemia reperfusion injury and HCV-mediated liver damage may lead to exposure of cryptic self-antigens such as Col, thus precipitating an immune response. A recent study from our laboratory found that Abs to Col I, II, V are associated with the development of fibrosis both in non-OLT and post-OLT HCV patients (16). In this study as well, the degree of allograft steatosis demonstrated positive correlations with Abs titer and fibrosis (Fig. 2). The mechanistic role of these Abs in the development of fibrosis can be postulated from reports in lung and heart transplantation wherein humoral and cellular (especially Th17) responses to self-antigens are implicated in the immunopathogenesis of fibrosis and chronic allograft rejection (42, 43).

This is further explained by an increase in IL-17 that has been shown to play a role in the development of autoimmune B cells and liver fibrosis (15, 44). An increase in IL-17 in steatotic allografts along with a pro-Th17 cytokine milieu (IL-6, IL-1β) may facilitate Th-17 immune responses to self- antigens (Col) leading to the production of Abs. These immune responses characterized by increased IL-6 and IL-17 can also lead to increase in other pro-fibrotic growth factors (transforming growth factor beta and connective tissue growth factor), which will result in ECM turnover and allograft fibrosis (45).

In non-HCV OLTr, however, there is no significant immune response to self-antigens (Fig. 2) and there is no significant fibrosis post-OLT (data not shown). In these non-HCV OLTr, only ischemia-reperfusion injury plays a role and the additional HCV-mediated responses does not occur. Thus, both an early postoperative stress and the continued HCV-mediated liver damage may be critical for exposure of cryptic self-antigens or determinants for development of immune responses to self-antigens and subsequent allograft fibrosis.

A limitation of this study is that due to lack of serial samples post-OLT, early changes and effects of steatosis on HCV replication and immune responses and the temporal and mechanistic correlation of development of Abs to self-antigens and allograft fibrosis could not be determined.

In conclusion, this study demonstrates that extent of graft steatosis significantly influences post-OLT HCV- specific immune responses and development of allograft fibrosis. Increasing grades of steatosis favor the development of predominant Th17-type HCV-specific responses with a concomitant suppression of Th1 (IFN-γ). In addition, early inflammatory changes in the allograft due to steatosis and inflammatory cytokine milieu can perpetuate the development of Abs to self-antigens (Col). We propose that HCV-specific and immune responses to self-antigens collectively promote allograft fibrosis and lead to a poor outcome especially in HCV OLTr of grafts with moderate to severe grades of steatosis.

Back to Top | Article Outline

MATERIALS AND METHODS

Patient Population

HCV OLTr at Barnes Jewish Hospital, Washington University, St. Louis, were consecutively enrolled (January 2002 to December 2008). Among 131 HCV OLTr, a cross-sectional analysis was conducted on 48 HCV OLTr by obtaining blood and post-OLT biopsy on same day of 1-year follow-up. Twenty-seven non-HCV OLTr were also enrolled and blood collected at similar time point. HCV infection was confirmed by HCV+ RNA polymerase chain reaction (Roche Diagnostics), anti-HCV Abs (Abbot Laboratories, Chicago, IL), and liver pathology. Patients with Hepatitis B and/or HIV coinfection were excluded. In 10 HCV OLTr, additional 6 months post-OLT samples were analyzed. Clinicodemographic data were obtained retrospectively. Laboratory parameters were at time of biopsy, and peak transaminase level was their highest concentration immediately after OLT. Control healthy subjects were enrolled when donating blood at human leukocyte antigen laboratory. The study was approved by the Institutional Review Board and informed consent was obtained from all subjects.

Back to Top | Article Outline

Allograft Histology

Pathologists estimated steatosis in graft biopsies (wedge or core from left lateral segment) taken either at the time of procurement or immediately after reperfusion (5). HCV (groups 1–3) and non-HCV (groups 4–6) OLTr were stratified by macrovesicular steatosis estimated by extent of large droplet fat occupying the parenchymal area: groups 1 and 4, up to 5% steatosis; groups 2 and 5, 5% to 35% steatosis; and groups 3 and 6, greater than 35% steatosis.

Protocol 1 year post-OLT and biopsies obtained for clinical indications were graded by pathologists for fibrosis by modified Batts Ludwig score (26). Patients were dichotomized into those with advanced fibrosis (stage 3–4) and without fibrosis (stage 0–2). Acute rejection was scored by Banff schema (25).

Back to Top | Article Outline

Isolation of Mononuclear Cells and Serum Samples

Serum was stored at −70°C. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient using Ficoll-Hypaque and used either immediately or frozen in 10% dimethyl-sulfoxide.

Back to Top | Article Outline

HCV and Peptide Antigens

Recombinant HCV core and nonstructural (NS3, NS4, and NS5) (Fitzgerald Industries, Acton MA), HIV (Gp120 peptide, Biosynthesis, TX), PHA (Sigma, St. Louis, MO) antigens were tested endotoxin-free (15). PBMCs were stimulated with antigens (5 μg/mL) in 24-well plates at 37°C in 5% CO2 overnight before use.

Back to Top | Article Outline

ELISpot

ELISpot was performed as described previously (14, 15). Stimulated PBMCs were cultured in triplicate (3×105 cells/200 μL) in immunospot plates in presence of antigens (5 μg/mL) for 72 hr. IFN-γ, IL-10, and IL-17 (BD Bioscience, CA) ELISpot were performed as per manufacturer's instructions and spots analyzed in ImmunoSpot Analyzer (CTL, Cleveland, OH). Cells cultured in medium (CTL) and irrelevant peptide (HIV) were used as negative and PHA as positive control. Spots in the experimental wells +2 standard deviations (SD) of negative control were considered significantly positive and expressed as spm.

Back to Top | Article Outline

Luminex Assay for Serum Cytokines

Serum cytokines and chemokines were measured using human 25-plex immunoassays (Invitrogen, Carlsbad, CA) (15). Plates were read on Luminex xMAP (Fischer, Pittsburgh, PA). Concentrations obtained by the standard curve were expressed in picograms per milliliters.

Back to Top | Article Outline

ELISA for Abs to Col

Abs to various Col were determined by ELISA (16, 42). ELISA plates were coated with recombinant human: Col I (Cell Sciences, Canton, MA), Col II, III, and V (Sigma), and Col IV (Biodesign International, Saco, ME). Serum was tested for binding to Col. Detection done by peroxidase-conjugated goat-anti-human (Jackson Immunoresearch, West grove, PA), developed using tetramethylbenzidine and read at 450 nm. Concentration of Abs was calculated using standard curve of known concentration of anti-Col (Santa Cruz Biotechnology, CA). Positive cutoff was set as +2SD of mean in normal subjects. This was 14 ng/mL for anti-Col I, 2 ng/mL for anti-Col II, 5 ng/mL for anti-Col III, 1 ng/mL for anti-Col IV, and 140 ng/mL for anti-Col V.

Back to Top | Article Outline

Statistical Analysis

Analysis was performed using SPSS version 17 (SPSS Inc., Chicago, IL). Schapiro-Wilks test was used to check for normality and non-normal data log transformed. Kruskal-Wallis and Mann-Whitney U test were used to compare clinical demographics, cellular responses and cytokine and Abs concentrations between groups. Correlation analysis was performed by Spearman rank test. Patient and allograft survival were compared by Kaplan-Meier and log-rank tests. Two-sided level of significance set at P less than 0.05.

Back to Top | Article Outline

ACKNOWLEDGMENTS

The authors thank Dr. Elizabeth M. Brunt in Pathology and Immunology for assistance in discussing histopathology and reviewing the manuscript, and Ms. Billie Glasscock for assistance in manuscript preparation.

Back to Top | Article Outline

REFERENCES

1. Kim WR. The burden of hepatitis C in the United States. Hepatology 2002; 36(5 suppl 1): S30.
2. Buti M, San Miguel R, Brosa M, et al. Estimating the impact of hepatitis C virus therapy on future liver-related morbidity, mortality and costs related to chronic hepatitis C. J Hepatol 2005; 42: 639.
3. Organ Procurement and Transplant Network. Online Data, 2011.
4. Nocito A, El-Badry AM, Clavien PA. When is steatosis too much for transplantation? J Hepatol 2006; 45: 494.
5. Doyle MB, Vachharajani N, Wellen JR, et al. Short- and long-term outcomes after steatotic liver transplantation. Arch Surg 2010; 145: 653.
6. Verran D, Kusyk T, Painter D, et al. Clinical experience gained from the use of 120 steatotic donor livers for orthotopic liver transplantation. Liver Transpl 2003; 9: 500.
7. Selzner M, Clavien PA. Fatty liver in liver transplantation and surgery. Semin Liver Dis 2001; 21: 105.
8. Angulo P. Nonalcoholic fatty liver disease and liver transplantation. Liver Transpl 2006; 12: 523.
9. Gane EJ. The natural history of recurrent hepatitis C and what influences this. Liver Transpl 2008; 14(suppl 2): S36.
10. Feray C, Samuel D, Thiers V, et al. Reinfection of liver graft by hepatitis C virus after liver transplantation. J Clin Invest 1992; 89: 1361.
11. Berenguer M, Lopez-Labrador FX, Wright TL. Hepatitis C and liver transplantation. J Hepatol 2001; 35: 666.
12. Hassoba H, Leheta O, Sayed A, et al. IL-10 and IL-12p40 in Egyptian patients with HCV-related chronic liver disease. Egypt J Immunol 2003; 10: 1.
13. Sobue S, Nomura T, Ishikawa T, et al. Th1/Th2 cytokine profiles and their relationship to clinical features in patients with chronic hepatitis C virus infection. J Gastroenterol 2001; 36: 544.
14. Bharat A, Barros F, Narayanan K, et al. Characterization of virus- specific T-cell immunity in liver allograft recipients with HCV-induced cirrhosis. Am J Transplant 2008; 8: 1214.
15. Basha HI, Subramanian V, Seetharam A, et al. Characterization of HCV-specific CD4+Th17 immunity in recurrent hepatitis C-induced liver allograft fibrosis. Am J Transplant 2011; 11: 775.
16. Borg BB, Seetharam A, Subramanian V, et al. Immune response to extracellular matrix collagen in chronic hepatitis C-induced liver fibrosis. LiverTranspl 2011; 17: 814.
17. Wiesner RH, Sorrell M, Villamil F. Report of the first International Liver Transplantation Society expert panel consensus conference on liver transplantation and hepatitis C. Liver Transpl 2003; 9: S1.
18. Briceño J, Ciria R, Pleguezuelo M, et al. Impact of donor graft steatosis on overall outcome and viral recurrence after liver transplantation for hepatitis C virus cirrhosis. Liver Transpl 2009; 15: 37.
19. Burra P, Loreno M, Russo FP, et al. Donor livers with steatosis are safe to use in hepatitis C virus-positive recipients. Liver Transpl 2009; 15: 619.
20. Anderson CD, Upadhya G, Conzen KD, et al. Endoplasmic reticulum stress is a mediator of posttransplant injury in severely steatotic liver allografts. Liver Transpl 2011; 17: 189.
21. Selzner M, Rudiger HA, Sindram D, et al. Mechanisms of ischemic injury are different in the steatotic and normal rat liver. Hepatology 2000; 32: 1280.
22. McCormack L, Petrowsky H, Jochum W, et al. Use of severely steatotic grafts in liver transplantation: A matched case-control study. Ann Surg 2007; 246: 940.
23. Baron PW, Sindram D, Higdon D, et al. Prolonged rewarming time during allograft implantation predisposes to recurrent hepatitis C infection after liver transplantation. Liver Transpl 2000; 6: 407.
24. Watt KD, Lyden ER, Gulizia JM, et al. Recurrent hepatitis C posttransplant: Early preservation injury may predict poor outcome. Liver Transpl 2006; 12: 134.
25. Banff schema for grading liver allograft rejection: An international consensus document. Hepatology 1997; 25: 658.
26. Batts KP, Ludwig J. Chronic hepatitis. An update on terminology and reporting. Am J Surg Pathol 1995; 19: 1409.
27. Gruener NH, Jung MC, Schirren CA. Recurrent hepatitis C virus infection after liver transplantation: Natural course, therapeutic approach and possible mechanisms of viral control. J Antimicrob Chemother 2004; 54: 17.
28. Ploeg RJ, D'Alessandro AM, Knechtle SJ, et al. Risk factors for primary dysfunction after liver transplantation—A multivariate analysis. Transplantation 1993; 55: 807.
29. Strasberg SM, Howard TK, Molmenti EP, et al. Selecting the donor liver: Risk factors for poor function after orthotopic liver transplantation. Hepatology 1994; 20 (4 Pt 1): 829.
30. Charlton M, Seaberg E. Impact of immunosuppression and acute rejection on recurrence of hepatitis C: Results of the National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database. Liver Transpl Surg 1999; 5 (4 suppl 1): S107.
31. Papatheodoridis GV, Davies S, Dhillon AP, et al. The role of different immunosuppression in the long-term histological outcome of HCV reinfection after liver transplantation for HCV cirrhosis. Transplantation 2001; 72: 412.
32. Ghobrial RM, Steadman R, Gornbein J, et al. A 10-year experience of liver transplantation for hepatitis C: Analysis of factors determining outcome in over 500 patients. Ann Surg 2001; 234: 384.
33. Teramoto K, Bowers JL, Khettry U, et al. A rat fatty liver transplant model. Transplantation 1993; 55: 737.
34. Chui AK, Shi LW, Rao AR, et al. Donor fatty (steatotic) liver allografts in orthotopic liver transplantation: A revisit. Transplant Proc 2000; 32: 2101.
35. Garcia-Retortillo M, Forns X, Feliu A, et al. Hepatitis C virus kinetics during and immediately after liver transplantation. Hepatology 2002; 35: 680.
36. Gane EJ, Naoumov NV, Qian KP, et al. A longitudinal analysis of hepatitis C virus replication following liver transplantation. Gastroenterology 1996; 110: 167.
37. Rosen HR. Hepatitis C virus in the human liver transplantation model. Clin Liver Dis 2003; 7: 107.
38. Oyanagi Y, Takahashi T, Matsui S, et al. Enhanced expression of interleukin-6 in chronic hepatitis C. Liver 1999; 19: 464.
39. Bortolami M, Kotsafti A, Cardin R, et al. Fas/FasL system, IL-1beta expression and apoptosis in chronic HBV and HCV liver disease. J Viral Hepat 2008; 15: 515.
40. Lemmers A, Moreno C, Gustot T, et al. The interleukin-17 pathway is involved in human alcoholic liver disease. Hepatology 2009; 49: 646.
41. Zhang JY, Zhang Z, Lin F, et al. Interleukin-17-producing CD4(+) T cells increase with severity of liver damage in patients with chronic hepatitis B. Hepatology 2010; 51: 81.
42. Bharat A, Saini D, Steward N, et al. Antibodies to self-antigens predispose to primary lung allograft dysfunction and chronic rejection. Ann Thorac Surg 2010; 90: 1094.
43. Fedoseyeva EV, Zhang F, Orr PL, et al. De novo autoimmunity to cardiac myosin after heart transplantation and its contribution to the rejection process. J Immunol 1999; 162: 6836.
44. Bhogal RK, Bona CA. B cells: No longer bystanders in liver fibrosis. J Clin Invest 2005; 115: 2962.
45. Abou-Shady M, Friess H, Zimmermann A, et al. Connective tissue growth factor in human liver cirrhosis. Liver 2000; 20: 296.
Keywords:

Allograft Steatosis; Hepatitis C; Recurrence; Fibrosis; Liver transplantation

© 2011 Lippincott Williams & Wilkins, Inc.