Comparison of histopathology in acute allograft rejection and recurrent hepatitis C infection after liver transplantation

Recurrent hepatitis C infection after orthotopic liver transplantation (OLT) is frequent and may occur as early as a few weeks postoperatively. Early histopathological features of recurrent hepatitis C virus (HCV) infection may be modified by immunosuppressive therapy and can be difficult to differentiate from acute allograft rejection (AAR). Thus, we retrospectively compared histopathological features of liver biopsy specimens from two carefully selected patient groups: one with unequivocal recurrent hepatitis C, the other with unequivocal AAR. Index biopsy specimens obtained at the time of the appearance of liver test abnormalities after OLT and all serial liver biopsy specimens (2 to 13 per patient) were assessed under code and scored semiquantitatively for 44 histopathological variables. The index biopsy specimens from patients with recurrent HCV infection and AAR index biopsies (AAR-Ib) differed significantly (P < .05) for 11 features (10 features were statistically associated with AAR and 1 with early recurrence of HCV infection). Statistically significant features associated with AAR included bile duct injury with overlapping nuclei, lymphocytic infiltrates and necrosis, endothelialitis, portal inflammatory infiltrates containing eosinophils and polymorphonuclear leukocytes, hepatocyte mitoses, and zone 3 canalicular cholestasis. In contrast, the only statistically significant feature associated with early recurrent HCV was sinusoidal dilatation. Stepwise discriminant analysis showed that the presence of eosinophils in the portal inflammatory infiltrate, bile duct necrosis, and bile duct lymphocytic infiltrates were independently associated with AAR. However, serial biopsy specimens from patients with recurrent HCV infection showed statistically significant progression in scores for portal inflammation, portal lymphoid aggregates, and lobular inflammation. We conclude that (1) multiple histopathological features are associated with AAR; (2) early recurrent HCV infection is characterized by elevated alanine aminotransferase levels, positive HCV RNA by polymerase chain reaction (PCR), and absence of diagnostic histopathological features; and (3) serial biopsies are needed to demonstrate progression of histopathological features of recurrent hepatitis C. (Liver Transpl Surg 1997 Jul;3(4):398-406)     

Hepatocellular MxA protein expression supports the differentiation of recurrent hepatitis C disease from acute cellular rejection after liver transplantation

MacQuillan GC, de Boer WB, Allan JE, Platten MA, Reed WD, Jeffrey GP. Hepatocellular MxA protein expression supports the differentiation of recurrent hepatitis C disease from acute cellular rejection after liver transplantation.
Clin Transplant 2009 DOI: 10.1111/j.1399‐0012.2009.01068.x
© 2009 John Wiley & Sons A/S. Abstract: Differentiation of recurrent hepatitis C virus (HCV) disease from acute cellular rejection (ACR) following liver transplantation can be difficult. Previously, we have found that MxA protein, a specific and sensitive marker for type 1 interferon production, is strongly expressed in chronic HCV disease. Here, we investigate MxA expression as a marker for recurrent HCV disease in the livers of 14 adult HCV patients who underwent liver transplantation. Serial liver biopsies available for 12 of these patients were stained for MxA protein and scored using a semi‐quantitative approach. Hepatocellular MxA protein levels were significantly up‐regulated (p = 0.025) in recurrent HCV disease in comparison to ACR. In biopsies that showed histological changes consistent with recurrent HCV disease, strong hepatocellular MxA staining was present in 14/18 (78%). In the liver biopsies with histological evidence of ACR, strong MxA hepatocellular staining was present in only three of 10 (30%). Thus, assessment of hepatocellular MxA protein expression can contribute to the differential diagnosis of ACR and recurrent HCV disease following liver transplantation. In conclusion, analysis of intrahepatic MxA levels has the potential to reduce the inappropriate use with high‐dose pulsing of steroids post‐operatively.     

Hepatic stellate cells in biopsies from liver allografts with acute rejection

BACKGROUND: [corrected] Hepatic stellate cells (HSCs) are nonparenchymal elements that play a major role in fibrogenesis due to various pathologies. HSCs are easily activated by certain injuries, which produce contraction and relaxation of HSCs, resulting in hepatic microcirculatory disturbances. The present study sought to analyze the expression of alpha-smooth muscle actin (alpha-SMA) positive HSCs in liver allografts during acute rejection episodes (ARE), determining whether it was related to the pathogenesis of this immune response. MATERIALS AND METHODS: Using immunohistochemistry and a semiquantitative scoring system, the expression of alpha-SMA in HSCs was analyzed in liver allografts with ARE (group 1, n = 64) or without ARE (group 2, n = 20). Normal liver tissue from transplant donors (group 3, n = 53) served as the control materials. RESULTS: Significantly more alpha-SMA positive HSCs were found in group 2 than in the other two groups (P < .05). The minimal difference observed between groups 1 and 3 was not statistically significant. As well, no statistical association was found between expression of alpha-SMA and the clinical parameters of age, gender, etiology of liver failure, donor type (partial or whole), posttransplantation period, and liver function tests. CONCLUSIONS: While these results represent preliminary findings, it may be possible that HSC expression is a protective mechanism during ARE in hepatic allograft patients. If this is true, enhanced expression of this protein may mitigate ARE in liver allograft patients.     

Characterization of CD4, CD8, CD56 positive lymphocytes and C4d deposits to distinguish acute cellular rejection from recurrent hepatitis C in post-liver transplant biopsies

INTRODUCTION: Hepatitis C viral (HCV) infection is the most common cause for liver transplantation (LTx) in USA. Hepatitis C viral recurrence in liver allograft is almost universal, which is often difficult to distinguish from acute cellular rejection (ACR). AIM: Aim of the present study is to examine the differences between distribution of CD4, CD8, CD56 positive lymphocytes, and C4d deposits in patients with ACR and recurrent HCV. PATIENTS AND METHODS: As a pilot project, a group of five post-LTx HCV RNA negative patients, strongly suspicious for ACR based on clinical findings and history of medication non-compliance and another group of five post-LTx HCV positive, medication compliant patients with abnormal liver function were retrospectively selected. Liver biopsies of these patients were stained with monoclonal CD4, CD8, CD56, and polyclonal C4d antibodies and compared. RESULTS: Mean CD4, CD8, and CD56 counts in ACR group were 156.7 +/- 17.6, 35.4 +/- 8.8, and 1.0 +/- 1.8/HPF, respectively and were 89.7 +/- 41.3, 20.3 +/- 23.2, and 0.6 +/- 0.9/HPF, respectively in HCV recurrence group. Biopsies of four of five patients with ACR demonstrated moderate to strong C4d staining, whereas all patients with recurrent HCV had none to mild C4d staining. CONCLUSION: Mean CD4, CD8, and CD56 were similar for acute rejection and recurrent HCV infection. However, 80% of patients with ACR showed moderate to strong staining for C4d and all recurrent HCV patients showed none to mild C4d staining.     

Granzyme expression in fine-needle aspirates from liver allografts is increased during acute rejection

We investigated whether determination in fine-needle aspiration biopsy (FNAB) specimens of cells expressing granzymes (Grs) and Fas ligand would provide a reliable, easy, and quantitative measure of rejection activity in the transplanted liver. Retrospectively, 13 FNAB specimens obtained during clinical acute rejection, 10 FNAB specimens obtained during subclinical rejection, 12 FNAB specimens obtained during cytomegalovirus (CMV) infection, and 26 FNAB specimens obtained in the absence of rejection or infection were included on the study. Cytospin preparations of FNAB and peripheral-blood specimens were immunocytochemically stained for Fas-ligand and Gr, and increments in the liver were calculated by subtracting frequencies of positive cells in blood from those in FNAB specimens. Only sporadically Fas ligand–expressing, but many Gr-expressing, cells were detected in FNAB specimens. Increments in Gr-positive (Gr⁺) cells were significantly greater in FNAB specimens obtained during clinical rejection (median, 70 Gr⁺ cells; range, 0 to 312 Gr⁺ cells; P = .006) and tended to be greater in FNAB specimens obtained during subclinical rejection (median, 62 Gr⁺ cells; range, 5 to 113 Gr⁺ cells; P = .09) compared with those obtained in the absence of rejection (median, 16 Gr⁺ cells; range, 0 to 103 Gr⁺ cells). Increments obtained during clinical or subclinical rejection did not differ from those obtained during CMV infection (median, 27 Gr⁺ cells; range, 6 to 212 Gr⁺ cells). With the exclusion of specimens obtained during CMV infection, the sensitivity of Gr determination in FNAB specimens for the diagnosis of acute rejection (either clinical or subclinical) was 70%, and specificity, 69%. In FNAB specimens obtained during clinical and subclinical acute rejection episodes after liver transplantation, increased numbers of Gr-expressing cells were present; in the absence of CMV infection, their quantification provides a measure for rejection activity with moderate accuracy. (Liver Transpl 2002;8:952-956.)     

Relevant histopathologic findings that distinguish acute cellular rejection from cholangitis in hepatic allograft biopsy specimens

BACKGROUND AND AIM: Histopathologic differential diagnosis of acute cellular rejection (ACR) and cholangitis continue to pose important problems following liver transplantation. The purpose of the present study was to evaluate the histopathologic features of ACR versus cholangitis. METHODS: The following variables were evaluated among 36 hepatic allograft biopsy specimens, consisting of 21 with ACR (group 1) and 15 with cholangitis (group 2) for ductal neutrophilic infiltration, presence/density of portal eosinophilia, centrilobular necrosis, central/portal vein endothelialitis, pericentral inflammation, hepatocyte ballooning, hepatocanalicular/ductular cholestasis, hepatocyte apoptosis, lobular inflammation, ductular proliferation, periductal fibrosis/edema, ductular epithelial damage, and portal inflammation. Only the first biopsy samples of the ACR group were included in this study. RESULTS: The incidences of ductal neutrophilic infiltration (93.3% vs 19%), hepatocanalicular cholestasis (86.7% vs 47.6%), ductular cholestasis (60% vs 0%), ductular proliferation (93.3% vs 4.8%), and periductal fibrosis/edema (93.3% vs 19%) were significantly greater in group 2 than group 1 (P < .05). In contrasts the incidences of portal eosinophilia (mean +/- SD, 3.37 +/- 3.9 vs 0.73 +/- 0.8), dense portal eosinophilia (mean +/- SD, 0.33 +/- 0.31 vs 0.11 +/- 0.16), central vein endothelialitis (0% vs 57.1%), portal vein endothelialitis (20% vs 95.2%), apoptosis (40% vs 71.4%), and necroinflammation (0% vs 90.5%) were significantly higher in group 1 (P < .05). The other parenchymal histopathologic changes and features of portal inflammation were similar in the 2 groups. CONCLUSION: In the differential diagnosis, ductal changes (cholestasis, neutrophilic infiltration, proliferation, and periductal fibrosis/edema) favor cholangitis, whereas the presence and density of portal eosinophilia favor ACR. Portal inflammation is not a distinctive morphological finding.     

Fine-needle aspiration biopsy in the monitoring of liver allografts. Different cellular findings during rejection and cytomegalovirus infection

Fine-needle aspiration biopsies were used for clinical monitoring of liver allografts; 21 patients with an inflammatory episode of acute rejection (12.0 +/- 3.3 CIU at the peak) were studied; all episodes were reversible. The inflammatory infiltrate consisted mainly of lymphoid cells, including lymphoid blasts, with increased numbers of class II and IL-2-receptor expressing lymphocytes. No lymphoid activation was seen in corresponding blood specimens. A rapid response to antirejection therapy with high-dose steroids was recorded by FNAB. Another group of 7 recipients without rejection developed a severe CMV disease. The CMV disease was associated with mild inflammation in the FNAB (3.2 +/- 0.9 CIU at the peak), but fewer blast cells and activated cell types were recorded in the graft, and blast cells and lymphocyte activation were seen in the blood specimens, as well. The inflammation disappeared from the FNABs during successful antiviral treatment. The cellular findings of rejection and CMV infection were significantly different, and the presence or absence of rejection could be firmly established by FNAB.     

Platelet-derived growth factor gene polymorphism in recurrent hepatitis C infection after liver transplantation

BACKGROUND: Recurrent hepatitis C virus (HCV) infection is particularly aggressive in the post liver transplantation setting, with rapid progression of liver fibrosis. Platelet-derived growth factor (PDGF) is reportedly involved in the pathogenesis of liver fibrosis. The aim of this study was to evaluate the possible contribution of molecular variants of the PDGF-B gene to recurrent HCV infection after liver transplantation. METHODS: DNA was extracted from peripheral blood mononuclear cells of 40 patients who underwent liver transplantation for chronic HCV infection and genotyped for polymorphisms in PDGF-B at positions +1135 (A to C) and +286 (A to G). Intrahepatic PDGF-B expression was detected by immunohistochemistry and assessed semiquantitatively. Forty-seven healthy individuals served as controls. RESULTS: Recurrent HCV infection occurred in 34 patients (85%) after a median interval of 10.5 months (range 1.5-60.0). A statistically significant difference was observed in the distribution of the PDGF-B gene polymorphism at position +1135, but not +286 between patients and controls (P=0.05). The A/A genotype occurred at a highly significantly increased rate in patients with recurrent HCV infection than in those without (64.7% vs. 16.67%, P=0.0001), and in patients with severe than in those with nonsevere recurrence (100% vs. 53.85%, P=0.05). The expression level of intrahepatic PDGF-B was found to be highly correlated with the fibrosis stage (P<0.0001). Further analysis yielded a highly statistically significant relationship between the PDGF-B gene polymorphism at position +1135 and clinical parameters of disease severity. CONCLUSIONS: PDGF-B gene polymorphism appears to be associated with severe recurrent HCV infection after liver transplantation. PDGF-B may play an essential role in the development and progression of hepatic fibrosis. These findings, if confirmed, may have important therapeutic implications.     

Effect of ischemia-reperfusion on the incidence of acute cellular rejection and timing of histologic hepatitis C virus recurrence after liver transplantation

BACKGROUND: Because of a critical shortage of deceased donor (DD) livers, more extended criteria allografts are being utilized; these allografts are at increased risk for ischemia-reperfusion injury (IRI). We assessed whether, in a large cohort of patients transplanted for hepatitis C virus (HCV) either via a DD or live donor (LD), there was a relationship between the degree of IRI and the frequency and timing of acute cellular rejection (ACR) and histologic HCV recurrence. METHODS: During an 8-year study, patients were separated into four groups based on peak alanine aminotransferase (ALT) levels and three groups based on severity of IRI on postreperfusion liver biopsy. RESULTS: The mean follow-up time of 433 DD and 44 LD recipients was 1212 days. We noted a strong correlation in DD between peak ALT and the histologic degree of IRI (P = .01). There was no difference in the incidence or grade of ACR among the four groups. There was no correlation between the severity of IRI and the incidence or time to histologic recurrence of HCV. CONCLUSIONS: The magnitude of peak ALT correlated with the severity of IRI on postreperfusion liver biopsy. Among this large HCV cohort, there was no correlation between the severity of IRI and the incidence or timing of histologic HCV recurrence or incidence of ACR.     

Cytokine profile of liver- and blood-derived nonspecific T cells after liver transplantation: T helper cells type 1/0 lymphokines dominate in recurrent hepatitis C virus infection and rejection

Orthotopic liver transplantation (OLT) is a successfultreatment in patients with hepatitis C virus (HCV)-associated end-stage liver disease worldwide. T lymphocytes and their cytokines are believed to have a pivotal role in the defense against HCV and in allograft rejection. An immunosuppressive drug regimen may cause a cytokine imbalance toward a T helper (TH) cell type 2 profile that is associated with the persistence of infection and acceptance of the graft. The aim of this study is to assess whether cytokine imbalances toward a TH 1- or TH2-type response may have a role in recurrent HCV infection and rejection after OLT Twenty-one intrahepatic Tcell lines of 15 patients with recurrent HCV infection after OLT (group A) and 15 lines of 11 patients with rejection (group B) were studied. Both patient groups had received liver allografts because of HCV-associated end-stage liver disease. Patients with HCV-induced liver disease who did not undergo OLT served as controls: 17 patients with chronic hepatitis C (CH-C) and 8 patients with cirrhosis. At the time of liver biopsy, 14 blood-derived Tcell lines of 12 patients with recurrent HCV infection, 7 of 10 patients with rejection, and 18 of 18 control patients were also investigated. Regardless of the underlying disease (recurrent HCV infection, rejection, HCVinduced hepatitis, and cirrhosis), all liver tissue-derived Tcell lines produced interferon-'Y; some additionally produced interleukin-4 (IL-4), but none produced IL-10, indicating that the TH0/1 cytokine profile dominates. Tcell lines showing a TH1 cytokine profile derived from intrahepatic T cells could be established significantly more often in recurrent HCV infection and rejection than in controls with CH-C (Fisher's exact test, P < .05). The cytokine profile of intrahepatic T cells did not differ from that obtained in peripheral blood. TH0/1 cytokine profile dominates the intrahepatic and blood-derived immune response in recurrent HCV infection and rejection after OLT regardless of the mode of immunosuppressvn. The lymphokine profile of immunocompromised patients with recurrent HCV infection or rejection does not differ principally from that of patients with HCV-induced hepatitis and cirrhosis, but seems to show a TH1 profile significantly more often.     

Alpha-interferon therapy for chronic hepatitis C may induce acute allograft rejection in kidney transplant patients with failed allografts.

BACKGROUND: In hepatitis C virus (HCV) positive kidney transplant (KT) patients, the use of alpha-interferon (alphaIFN) is contraindicated due to the risk of acute rejection (AR). Conversely, if these HCV(+) KT patients lose their allograft, re-transplantation might be contemplated provided alphaIFN therapy has been attempted. METHODS: Between 01/01/1989 and 31/12/1994, 261 kidney transplantations were performed; of these 174 were HCV(-) (group I) and 87 were HCV(+) (group II). RESULTS: At last follow-up (2006), in group I, the number of patients with a functioning graft, the number of patients who died with a functioning graft, and the number of patients who lost their graft before or after month (M) 12 were 92 (52.8%), 14 (8%), 20 (11.5%) and 48 (27.7%), respectively. In group II, the corresponding figures were 22 (25.3%; P < 0.0001), 8 (9.1%; ns), 9 (10.3%; ns) and 48 (55.3%; P < 0.0001). In group I, 19 of 48 (39.5%) patients with failed allografts after M12 underwent transplantectomy (TX) compared to 14 of 48 (29%; ns) in group II. In group II, 11 of 48 (23%) patients were offered alphaIFN therapy after their allograft failed: of these, four (36.3%) developed AR during alphaIFN therapy leading to TX. Histology, in addition to chronic allograft lesions, showed acute cellular and vascular lesions. In patients who were not offered alphaIFN therapy, TX was performed less frequently, i.e. in only six cases (16.2%). CONCLUSIONS: We conclude that even alphaIFN-treated KT patients with a failed allograft can experience acute allograft rejection that requires transplantectomy during therapy.     

Expression of adhesion molecules and their ligands in liver allografts during cytomegalovirus (CMV) infection and acute rejection

Abstract  Vascular adhesion molecules and their ligands are important in leukocyte-endothelial cell interactions and in T-cell activation of rejection cascade. Also, cytomegalovirus (CMV) infection is suggested to be involved in the mechanisms of rejection. In this study, the expression of vascular adhesion molecules ICAM-1, VCAM-1 and ELAM-1 in the liver allografts, the number of leukocytes positive for their ligands LFA-1, VLA-4 and SLex, and activation markers (class II, IL2-receptor) were investigated in liver allografts during CMV infection and acute rejection and compared to grafts with normal function and histology. The adhesion molecules, their ligands and activation markers were demonstrated from liver biopsy frozen sections by the immunoperoxidase technique and monoclonal antibodies. A significant induction of ICAM-1 and VCAM-1 was seen in vascular and sinusoidal endothelium associated with both CMV and rejection, and induction of ELAM-1 in vascular endothelium in rejection only. In both cases, the number of leukocytes expressing LFA-1 was significantly increased, but VLA-4-positive cells were more characteristic for CMV and SLex-positive cells more for rejection. IL2-receptor positivity was practically seen in rejection only, but class II-expressing cells were increased during both CMV infection and rejection. In conclusion, adhesion molecules were induced and the infiltrating cells expressed their ligands both in liver rejection and during CMV infection, although the expression pattern was slightly different.     

Effects of interferon treatment on liver histology and allograft rejection in patients with recurrent hepatitis C following liver transplantation.

Recurrent hepatitis C after liver transplantation remains a significant cause of graft loss and retransplantation. Although treatment of recurrent hepatitis C with interferon-based regimens has become widely accepted as safe and can lead to sustained virologic clearance of hepatitis C virus (HCV) RNA, long-term histologic improvement and the risk of precipitating graft rejection remain controversial. The present study is a retrospective evaluation of the clinical and histological consequences of treating recurrent hepatitis C with interferon-based therapy in a selected group of liver transplant recipients. Twenty-three liver transplant recipients with recurrent hepatitis C and histologic evidence of progressive fibrosis completed at least 6 months of interferon, 83% of whom received pegylated-interferon alpha-2b; only 4 tolerated ribavirin. Overall, 11 patients (48%) had undetectable HCV RNA at the end of 6 months of treatment. Of these patients, 3 remained HCV RNA-negative on maintenance interferon monotherapy for 33 months, and the other 8 (35%) completed treatment and remained HCV RNA-undetectable 24 weeks after discontinuation of interferon. Overall necroinflammatory activity in liver biopsies obtained 2 years after HCV RNA became undetectable decreased significantly (7.73 +/- 2.37 vs. 5.64 +/- 2.94 units before and after treatment, respectively; P =.016). However, 5 of these 11 patients had no histologic improvement in follow-up liver histology. Liver biopsies in the 12 nonresponders demonstrated disease progression. Of the 23 patients treated with interferon, 8 (35%) had evidence of acute or chronic rejection on posttreatment liver biopsy, most of whom had no previous history of rejection (P <.01 for comparison of pretreatment and posttreatment prevalence of histologic rejection), and 2 experienced graft loss from chronic rejection, requiring retransplantation. In conclusion, interferon treatment of recurrent hepatitis C does not consistently improve histologic disease after virologic response, and it may increase the risk of allograft rejection.