Roberto J. Firpi

An immunomodulatory role for CD4+CD25+ regulatory T lymphocytes in hepatitis C virus infection

The CD4+CD25+ regulatory T lymphocytes have been implicated in suppressing T cell immune responses. Our aim was to characterize the frequency, phenotype, function, and specificity of CD4+CD25+ T cells in hepatitis C virus (HCV) infection. Peripheral CD4+CD25+ cells from recovered (n = 15), chronic infected (n = 30), and normal control (n = 15) subjects were analyzed ex vivo for quantitation, phenotype, and effect on HCV‐specific interferon gamma production and proliferation. CD4+CD25+ specificity was determined by intracellular cytokine staining for interleukin 10 (IL‐10). A higher proportion of CD4+CD25+ were found in chronic infection (mean, 3.02%) when compared with recovered (1.64%, P = .001) and normal controls (2.27%, P = .02). CD4+CD25+ cells display CD45ROhigh, CD45RAlow, CD28high, CD62Lhigh, and CD95high phenotype. HCV‐specific interferon gamma activity was enhanced in peripheral blood mononuclear cells depleted of CD4+CD25+ and suppressed in peripheral blood mononuclear cells enriched with CD4+CD25+. Depletion of CD4+CD25+ cells also enhanced HCV‐specific CD4+ and CD8+ T cell proliferation. Cytokine analysis suggested CD4+CD25+ cells secrete transforming growth factor beta (TGF‐β1) and IL‐10. The inhibitory role for TGF‐β1 was confirmed by anti–TGF‐β1. Transwell studies showed CD4+CD25+ mediated suppression to be dose dependent and requiring cell contact. CD4+CD25+ cells showed HCV‐specificity through IL‐10 production, with a frequency ranging from 1.9% to 5.3%. A positive correlation was detected between CD4+CD25+ T cell frequency and HCV RNA titer, whereas an inverse relation was found with liver inflammatory activity. In conclusion, CD4+CD25+ T lymphocytes constitute a highly differentiated population and appear to play a role in viral persistence by suppressing HCV‐specific T cell responses in a cell–cell contact manner. (HEPATOLOGY 2004;40:1062–1071.)

Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation.

Cyclosporine is an immunosuppressive agent widely used in the management of liver transplant recipients. Cyclosporine has been shown to have antiviral activities against HIV, herpes simplex, and vaccinia viruses. The aim of this study was to determine the effect of Cyclosporine in viral clearance in the liver transplant recipients during therapy with combination of interferon and ribavirin, and to determine the anti‐viral potential of Cyclosporine in vitro. Immunosuppression consisted of either Cyclosporine or Tacrolimus‐based therapy. Both groups received therapy with interferon and ribavirin for 48 weeks when evidence of progressive histologic disease was determined. We found that subjects on Cyclosporine‐based immunosuppression (n = 56) had a higher sustained virological response of 46% compared to 27% in the patients on Tacrolimus‐based therapy (n=59, P = 0.03). In vitro studies were performed to evaluate the antiviral effect of Cyclosporine in the replicon system. These studies showed that Cyclosporine inhibits hepatitis C viral replication in a dose‐dependent manner. Combination of Cyclosporine with interferon showed additive effect, and its function is independent of interferon signaling pathways. In conclusion, Cyclosporine may offer an advantage to Tacrolimus in those patients undergoing interferon‐based therapy and should be studied in a prospective randomized trial. Liver Transpl 12:51–57, 2006.

Sustained viral response to interferon and ribavirin in liver transplant recipients with recurrent hepatitis C

Recurrent hepatitis C infection is an important cause of progressive fibrosis, cirrhosis, and graft loss following orthotopic liver transplantation. Treatment for posttransplant recurrence of hepatitis C with interferon‐based therapy is difficult but results in loss of detectable virus in up to 30% of patients. However, the durability of viral clearance and the associated histologic response in this setting is unknown. The aim of this study was to determine whether viral loss in response to antiviral therapy is durable and associated with improvement in liver histology. All liver transplant recipients who received interferon‐based treatment for recurrent hepatitis C virus (HCV) at the University of Florida from 1991 to 2002 were included in this study. Patients who lost detectable HCV after treatment with interferon alone or in combination with ribavirin were followed to assess the durability of viral response and its impact on liver histology. One hundred nineteen transplant recipients were treated with interferon or combination therapy. Twenty‐nine (20 men, 9 women; mean age, 54 yrs [range, 42–74 yrs]) lost detectable HCV RNA and remained virus negative for at least 6 months after discontinuing therapy (sustained viral response[SVR]). The mean follow‐up after discontinuing therapy was 24.7 months (range, 6–70 mos). Our study cohort included one patient with SVR following interferon monotherapy and 28 patients with SVR following combination therapy with interferon plus ribavirin. All patients remained HCV RNA negative (assessed by polymerase chain reaction or branched‐DNA assay) during follow‐up of up to 5 years. Liver histology assessed 2 years after treatment showed less inflammation compared with before treatment in 50% and showed no change in 38%. By 3 to 5 years post‐treatment (n = 15 recipients), inflammation was reduced in 60% and remained unchanged in 33%. Fibrosis stage at 2 years improved by ≥ 1 stage in 27 %, remained unchanged in 38 %, and worsened in 35% despite viral clearance. At 3 to 5 years, the fibrosis stage had improved in 67%, remained unchanged in 13%, and worsened in 20%. Both grade of inflammation and fibrosis stage improved by 3 to 5 years posttreatment compared with baseline histology (p < 0.05). In conclusion, loss of HCV after treatment of recurrent chronic hepatitis C with interferon and ribavirin is durable, and the durability of the SVR is associated with improvement in hepatic inflammation and regression of fibrosis. (Liver Transpl 2004;10:199–207.)

One-year protocol liver biopsy can stratify fibrosis progression in liver transplant recipients with recurrent hepatitis C infection:

Determinants of progression to cirrhosis in hepatitis C virus (HCV) infection have been well described in the immunocompetent population but remain poorly defined in liver transplant (LT) recipients. This cohort study determines the factors contributing to the development of fibrosis and its rate of progression in the allograft. Predictive factors analyzed include: demographics, host and donor factors, surgery‐related variables (cold and warm ischemia time), rejection episodes, cytomegalovirus infection (CMV), and immunosuppression. Over 12 years, 842 adult LTs were performed at our institution; 358 for the indication of HCV. A total of 264 patients underwent protocol liver biopsies at month 4 and yearly after LT. Using the modified Knodell system of Ishak for staging fibrosis, the median fibrosis progression rate was .8 units/year (P < .001). Rapid fibrosis progression (>.8 units/year) was best identified by liver histology performed at 1 year. Donor age > 55 years was associated with rapid fibrosis progression and development of cirrhosis (P < .001). In contrast, donor age < 35 years was associated with slower progression of fibrosis (P = .003). Risk factors for graft loss due to recurrent HCV included recipient age > 35 years (P = .01), donor age > 55 years (P = .005), and use of female donor allografts (P = .03). In conclusion, fibrosis progression in HCV‐infected LT recipients occurs at a rate of .8 units/year. Increased donor age has a major impact on disease progression, graft failure, and patient survival. A liver biopsy performed at 1 year posttransplant can help identify those patients more likely to develop progressive disease and may allow better targeting of antiviral therapy. (Liver Transpl 2004;10:1240–1247.)

Pilot study: fenofibrate for patients with primary biliary cirrhosis and an incomplete response to ursodeoxycholic acid

Aliment Pharmacol Ther 2011; 33: 235–242

The natural history of hepatitis C cirrhosis after liver transplantation

Hepatitis C after liver transplantation leads to graft cirrhosis in up to 30% of patients within 5 years, but limited data exist regarding the clinical course of cirrhosis after transplantation. The aims of this study were to report the natural history of hepatitis C cirrhosis after liver transplantation and to identify risk factors for decompensation and survival. Hepatitis C patients underwent protocol liver biopsies yearly after liver transplantation. After cirrhosis was identified by biopsy, the outcomes of interest were the development of decompensation, death, or retransplantation for hepatitis C. Kaplan‐Meier and Cox regression analysis was used to determine survival and risk factors for decompensation and mortality. Out of 502 liver transplants performed for hepatitis C, 88 patients (18%) had cirrhosis within 3.7 years. Seventy‐one patients were compensated at diagnosis. The cumulative probability of decompensation 1 year after cirrhosis was 30%. A Model for End‐Stage Liver disease score ≥ 16 was predictive of decompensation and poor survival, whereas successful interferon treatment was found to reduce this risk (relative risk = 0.05). Once decompensation occurred, 1‐year survival was 46%. In conclusion, the results confirm an accelerated natural history of hepatitis C cirrhosis after liver transplantation and demonstrate poor survival after decompensation. The Model for End‐Stage Liver Disease can stratify risk for decompensation and survival, whereas successful antiviral therapy may be protective. Liver Transpl 15:1063–1071, 2009.

The combination of sorafenib with transarterial chemoembolisation for hepatocellular carcinoma

Aliment Pharmacol Ther 2011; 34: 205–213

Early hepatic stellate cell activation is associated with advanced fibrosis after liver transplantation in recipients with hepatitis C

Recurrent hepatitis C after liver transplantation is a serious problem faced by liver transplant recipients. Activation of hepatic stellate cells is an early step in hepatic fibrogenesis. The aim of this study was to evaluate hepatic stellate cell activation, early after liver transplantation, as a predictor for the subsequent development of advanced fibrosis. Forty‐six patients who underwent liver transplantation for hepatitis C and protocol liver biopsies were divided into rapid fibrosers (n = 21), defined as recipients who developed bridging fibrosis or cirrhosis within 2 years of liver transplantation, and slow fibrosers (n = 25). The protocol liver biopsy obtained 4 months after transplantation was stained and quantitated for hepatic stellate cell activation with antibody to alpha smooth muscle actin. Hepatic stellate cell activity was independently associated with rapid fibrosis (odds ratio: 1.6 [95% CI: 1.1,2.2], P = 0.013). The c‐statistics for the receiver operating characteristic curve for stellate cell activity and fibrosis were 0.78 and 0.67, respectively, P = 0.36. The receiver operating characteristic curve for a model including stellate cell activity, histology activity index, and alanine aminotransferase. obtained at month 4 had the best c‐statistic (0.88). In recipients with stage 0 or 1 fibrosis on the month 4 liver biopsy who subsequently developed advanced fibrosis, the c‐statistic for the receiver operating characteristic curves was significantly better for stellate cell activity than for stage of fibrosis (0.77 and 0.51, respectively; P = 0.004). In conclusion, hepatic stellate cell activation early after liver transplantation complements traditional testing for identifying liver transplant recipients with hepatitis C at greatest risk for developing advanced fibrosis. (Liver Transpl 2005;11:1235–1241.)

Management of viral hepatitis in hematologic malignancies

Viral hepatitis is the third major cause of liver dysfunction in allogeneic transplant recipients and has become a significant concern in patients with hematological malignancies receiving chemotherapy. Thus, identification of patients at risk for viral hepatitis is very important when evaluating and treating hematological malignancies. Serologic screening for all patients should include anti-HCV, hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), and hepatitis B core antibody (anti-HBc) testing. Current therapies for hepatitis B (HBV) virus infection are aimed at viral suppression, while treatment for hepatitis C (HCV) virus can eradicate infection in many treated patients. To prevent HBV viral reactivation, prophylaxis with nucleoside analogues should be initiated for all HBsAg-positive patients. HCV infection appears to have little impact on short-term survival after bone marrow transplantation (BMT), but eventually can impact long-term survival due to progression of liver disease. In this review we will highlight the mechanisms of virus reactivation, clinical manifestations, and management strategies to minimize acute and chronic morbidity in this population.

Hepatocellular carcinoma cell supernatants increase expansion and function of CD4(+)CD25(+) regulatory T cells.

Dysfunction of the host immune system in cancer patients can be due to a number of factors, including suppression of tumor-associated antigen reactive lymphocytes by CD4+CD25+ regulatory T (Treg) cells. Several studies suggest that Tregs are elevated in cancer patients and that depletion of Tregs may enhance the antitumor immunity of host, but the pathogenic and mechanistic relationship between cancer and Tregs is still unclear. In this report, we show that Tregs are increased in peripheral blood mononuclear cells (PBMCs) from hepatocellular carcinoma (HCC) patients and positively correlate with tumor burden. When PBMCs are co-cultured with human hepatoma cell lines Huh7, HepG2, and Hclone5, CD4+CD25+-T cell populations increase in frequency and undergo phenotypic and functional changes. CD45RA, CD45RO, CD69, CD62L, GITR, CTLA-4, Ki67, granzyme A, granzyme B, and FOXP3 expression were upregulated in CD4+CD25+ cells after in vitro exposure to HCC cell lines. CD4+CD25+ T cells from PBMCs that were co-cultured with Huh7 cells also have higher suppressor ability compared to that of the CD4+CD25+ T cells from control PBMC. Huh7 culture supernatants appear to promote CD4+CD25+ T-cell proliferation and inhibit CD4+CD25− T-cell proliferation. In conclusion, these results strongly suggest that tumor-related factors not only induce and expand CD4+CD25+ cells, but also enhance their suppressor ability.