Is PBC a viral infectious disease?

The human betaretrovirus and the closely related mouse mammary tumor virus have been linked with the development of cholangitis and mitochondrial antibody production in patients with primary biliary cholangitis (PBC) and mouse models of autoimmune biliary disease, respectively. In vitro, betaretroviruses have been found to stimulate the expression of mitochondrial autoantigens on the cell surface of biliary epithelial cells. In vivo, both mitochondrial autoantigens and viral proteins have been shown to be co-expressed in biliary epithelium and lymphoid tissue. Notably, both mice and humans make poor antibody responses to betaretrovirus infection, whereas proinflammatory responses to viral proteins have been observed in T lymphocyte studies. Furthermore, proviral integration studies have confirmed the presence of human betaretrovirus in biliary epithelium of patients with PBC. Preliminary proof of principal studies using combination antiretroviral therapy have shown that suppression of viral expression is associated with sustained biochemical response. As the previous regimen used was poorly tolerated, further randomized controlled trials are planned to determine whether betaretrovirus infection plays an important role in the development of PBC.

Combination antiretroviral studies for patients with primary biliary cirrhosis.

Following the characterization of a human betaretrovirus in patients with primary biliary cirrhosis (PBC), pilot studies using antiretroviral therapy have been conducted as proof of principal to establish a link of virus with disease and with the eventual aim to find better adjunct therapies for patients unresponsive to ursodeoxycholic acid. In the first open label pilot study, the reverse transcriptase inhibitor lamivudine had little demonstrable biochemical or histological effect after 1 year. Whereas, lamivudine in combination with zidovudine was associated with a significant reduction in alkaline phosphatase as well as improvement in necroinflammatory score, cholangitis and ductopenia over a 12 mo period. A double blind, multi-center randomized controlled trial using lamivudine with zidovudine for 6 mo confirmed a significant reduction in alkaline phosphatase, ALT and AST in patients on antiviral therapy. However, none of the patients achieved the stringent endpoint criteria for normalization of alkaline phosphatase. Furthermore, some patients developed biochemical rebound consistent with drug resistance. A major fault of these studies has been the inability to measure the viral load in peripheral blood and therefore, provide a direct correlation between improvement of hepatic biochemistry and reduction in viral load. Nevertheless, viral mutants to lamivudine with zidovudine were later characterized in the NOD.c3c4 mouse model of PBC that has been used to test other antiretroviral regimens to betaretrovirus. The combination of tenofovir and emtricitabine reverse transcriptase inhibitors and the HIV protease inhibitor, lopinavir were found to abrogate cholangitis in the NOD.c3c4 mouse model and the same regimen normalized the liver tests in a PBC patient with HIV and human betaretrovirus infection. This combination antiretroviral therapy has now been used in a double blind randomized controlled crossover study for patients with PBC followed by an open label extension study. Only a third of the PBC patients were able to tolerate the lopinavir but those maintained on tenofovir, emtricitabine and lopinavir experienced sustained and clinically meaningful reduction in hepatic biochemistry. While we await the histological and virological evaluation, it is clear that better tolerated regimens of antiretroviral treatment will be required in future clinical trials.

Impact of combination antiretroviral therapy in the NOD.c3c4 mouse model of autoimmune biliary disease.

BACKGROUND & AIMS: The NOD.c3c4 mouse model develops autoimmune biliary disease characterized by spontaneous granulomatous cholangitis, antimitochondrial antibodies and liver failure. This model for primary biliary cirrhosis (PBC) has evidence of biliary infection with mouse mammary tumour virus (MMTV), suggesting that the virus may have a role in cholangitis development and progression of liver disease in this mouse model. We tested the hypothesis that MMTV infection is associated with cholangitis in the NOD.c3c4 mouse model by investigating whether antiretroviral therapy impacts on viral levels and liver disease. METHODS: NOD.c3c4 mice were treated with combination antiretroviral therapy. Response to treatment was studied by measuring MMTV RNA in the liver, liver enzyme levels in serum and liver histology using a modified Ishak score. RESULTS: Combination therapy with the reverse transcriptase inhibitors, tenofovir and emtricitabine, resulted in a significant reduction in serum liver enzyme levels, attenuation of cholangitis and decreased MMTV levels in the livers of NOD.c3c4 mice. Furthermore, treatment with the retroviral protease inhibitors, lopinavir and ritonavir, in addition to the reverse transcriptase inhibitors, resulted in further decrease in MMTV levels and attenuation of liver disease in this model. CONCLUSIONS: The attenuation of cholangitis with regimens containing the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the protease inhibitors, lopinavir and ritonavir, suggests that retroviral infection may play a role in the development of cholangitis in this model.

Frequent proviral integration of the human betaretrovirus in biliary epithelium of patients with autoimmune and idiopathic liver disease.

BACKGROUND: A human betaretrovirus (HBRV) has been linked with primary biliary cirrhosis (PBC) following the detection of viral particles in biliary epithelium by electron microscopy and cloning of the betaretrovirus genome from biliary epithelium and peri-hepatic lymph nodes. Evidence for viral infection was found in the majority of PBC patients' peri-hepatic lymph node samples. However, less than a third of the liver samples had detectable HBRV, whereas others were unable to detect betaretrovirus infection or noted the presence of virus in the liver of patients with other diagnoses. AIMS: To address the hypothesis that the betaretrovirus may be below the limits of detection in the liver, biliary epithelial cells (BEC) were investigated for the evidence of infection. METHODS: Ligation-mediated PCR and next generation sequencing were used to detect proviral integrations in liver, lymph nodes and BEC isolated from liver transplant recipients. Hybridisation-based assays were used to detect betaretroviral RNA in BEC. RESULTS: Unique HBRV integrations and betaretrovirus RNA were detected in the majority of biliary epithelia derived from patients with PBC, autoimmune hepatitis and cryptogenic liver disease but rarely in other liver transplant recipients with primary sclerosing cholangitis and other hepatic disorders. HBRV integrations were commonly found in PBC patients' lymph nodes but rarely in whole liver samples. CONCLUSIONS: Human betaretrovirus infection is frequently observed at the site of disease in patients with primary biliary cirrhosis and also in biliary epithelium of patients with autoimmune hepatitis and cryptogenic liver disease.

Liver transplantation for primary biliary cirrhosis in a hepatitis endemic region: a single-center Asian experience.

From March 1984 to November 2008, we performed 539 primary liver transplantations (LTs). Nineteen (19, 3.5%) were transplanted for end-stage liver disease secondary to primary biliary cirrhosis (PBC). There were 17 (89%) female and 2 (11%) male recipients. The overall mean age was 50.3 ± 6.3 yr. The mean model for end-stage liver disease, and Child-Turcotte-Pugh scores were 20.7 ± 2.1, and 11.0 ± 0.5, respectively. There were 2 (11%) United Network for Organ Sharing status 3, 16 (84%) 2B, and 1 (5%) 2A patients. Fourteen patients (14, 73.7%) underwent living donor LT, and five patients (26.3%) received deceased donor LT. The primary immunosuppression consisted of cyclosporine (n = 5) and tacrolimus (n = 14). Liver function returned to normal one month after transplantation. The overall mean follow-up was 5.8 ± 0.8 yr (range, four months to 15.7 yr). The overall one-, three-, and five-yr survival rates were 94.7%, 89.2%, and 89.2%, respectively. Without hepatitis B virus (HBV) prophylaxis, one patient acquired de novo HBV infection after receiving a graft from an anti-HBc(+) donor. Another patient developed recurrent hepatitis C infection and expired 25 months after transplantation. Our results showed that HBV prophylaxis was effective not only against de novo infection, but it also worked on pre-transplant HBV carrier with PBC and helped in virus clearance.

Linking human beta retrovirus infection with primary biliary cirrhosis.

Several environmental agents have been linked with primary biliary cirrhosis (PBC) that include bacteria, xenobiotics and viruses. A human beta retrovirus (HBRV) related to mouse mammary tumor virus has been cloned and characterized from patients with PBC. This agent can be detected in the majority of patients' perihepatic lymph nodes by immunochemistry and RT-PCR. The HBRV has recently been isolated in culture and integration sites have been identified in the genome of patients to provide convincing evidence of beta retrovirus infection in patients. Three lines of evidence support a role for the virus in PBC. First, the beta retrovirus is linked with aberrant expression of mitochondrial protein(s) on the biliary epithelium cell (BEC) surface, a disease specific phenotype. Second, the related agent, mouse mammary tumor virus has been linked with autoimmune biliary disease in the NOD.c3c4 mouse model for PBC. In this mouse model, the virus is localized to diseased biliary epithelium that also display aberrant expression of the mitochondrial autoantigens. In translational studies, both patients with PBC and NOD.c3c4 mice demonstrate significant improvement in biliary disease with combination antiviral therapy. An overview of the biological relevance of the beta retrovirus infection in PBC will be discussed in this review.

Cyclosporine A inhibits in vitro replication of betaretrovirus associated with primary biliary cirrhosis.

BACKGROUND/AIM: Up to one-third of patients with primary biliary cirrhosis (PBC) experience recurrent disease following liver transplantation, which is associated with earlier and more severe recurrence in patients treated with tacrolimus as compared with cyclosporine A (CsA). As the latter has known antiviral activity, we hypothesized that CsA has the ability to inhibit the betaretrovirus characterized from patients with PBC. METHODS: We investigated whether CsA, the cyclosporine analogue NIM811, tacrolimus and other compounds can modulate the mouse mammary tumour virus production from Mm5MT cells. Viral load was evaluated in the cell supernatants by quantifying reverse transcriptase (RT) levels and betaretrovirus RNA. RESULTS: A significant correlation was observed with increasing concentrations of CsA and NIM811, and decreasing of RT levels (rho-0.59, P=0.04 and rho-0.74, P=0.006 respectively), whereas tacrolimus had no significant effect (rho-0.27, P=0.4). At a dose of 3 microg/ml, CsA, NIM811 and the human immunodeficiency virus aspartyl protease inhibitor, lopinavir, were all associated with greater than three-fold reduction in the betaretrovirus RNA production from Mm5MT cells as compared with tacrolimus (P<0.005). CONCLUSIONS: These studies demonstrate that the cyclophilin inhibitors CsA and NIM811 have antiviral activity against betaretrovirus production in vitro.

Overlap syndrome triggered by acute viral hepatitis A.

We report the case of a 24-year-old woman with an autoimmune hepatitis/primary biliary cirrhosis overlap syndrome triggered by an acute hepatitis A. A number of viruses have been proposed as potential triggers of autoimmune hepatitis in patients with genetic predisposition. To date, approximately 10 cases of type 1 autoimmune hepatitis following hepatitis A virus infection have been published in the medical literature. To our knowledge, this is the first case of overlap syndrome triggered by an acute hepatitis A.

Occult hepatitis B virus infection in patients with autoimmune liver diseases.

BACKGROUND: Occult hepatitis B virus (HBV) infection is characterized by undetectable serum HBV surface antigen (HBsAg) but detectable HBV-DNA in serum or liver. AIMS: To determine the prevalence and clinical impact of occult HBV in autoimmune liver diseases as similar data are missing. METHODS: One hundred and ninety-six sera samples from HBsAg-negative patients, including 66 autoimmune hepatitis (AIH), 93 primary biliary cirrhosis (PBC) and 37 primary sclerosing cholangitis (PSC), were investigated for HBV-DNA using the polymerase chain reaction (PCR) before treatment initiation. One hundred and three serial samples from 38 AIH patients under immunosuppression and 282 selected blood donors (HBsAg negative; antibodies to HBV-core antigen positive) were also investigated. Fourteen available paraffin-embedded AIH liver samples were also investigated for HBV-DNA by nested-PCR. RESULTS: Hepatitis B virus DNA was detected in the serum of 24/196 patients (12.2%) and 0/282 donors (P=0.0000). Nine patients had AIH (13.6%), eight had PBC (8.6%) and seven had PSC (18.9%) (P=0.0000 vs healthy). HBV-DNA detection in AIH livers was higher than in serum. HBV-DNA was associated neither with HBV markers nor with epidemiological, laboratory and clinical data. Serial testing of AIH patients revealed two HBV-DNA-negative patients before treatment becoming positive during treatment, while all HBV-DNA-positive patients before immunosuppression became negative. CONCLUSION: Based mainly on serum HBV-DNA, we found a significant proportion of autoimmune liver disease patients with occult HBV compared with donors. However, taking into account our results in a small number of liver tissues, it should be emphasized that occult HBV could be even higher when both serum and liver specimens are investigated. Occult HBV does not seem to affect the clinical and laboratory features of the diseases, while AIH patients with occult HBV under immunosuppression do not deteriorate during follow-up.

Reverse transcriptase activity in patients with primary biliary cirrhosis and other autoimmune liver disorders.

BACKGROUND: Patients with biliary disease make retroviral antibodies and the Human Betaretrovirus has been characterized in patients with primary biliary cirrhosis. AIM: To screen patients with autoimmune liver disease for evidence of retroviral infection. METHODS: Real-time reverse transcriptase polymerase chain reaction was used to detect Human Betaretrovirus, and a reverse transcriptase assay to measure reverse transcriptase activity in plasma. RESULTS: Using reverse transcriptase polymerase chain reaction, 24% of primary biliary cirrhosis samples were positive for Human Betaretrovirus when compared to 13% with autoimmune hepatitis, 5% of other liver diseases and 3% of the non-liver disease control subjects. Reverse transcriptase activity was found in 73% of patients with autoimmune hepatitis, 42% with primary biliary cirrhosis, 22% of liver patients without viral or autoimmune disease and 7% of subjects without liver disease. In patients with autoimmune liver disease, detection of reverse transcriptase activity was related to higher ALT levels, whereas others stabilized on immunosuppressive therapy either preliver or postliver transplantation were less likely to be reverse transcriptase-positive. CONCLUSIONS: Most patients with autoimmune hepatitis have detectable reverse transcriptase activity. Investigations will be required to assess whether this represents the expression of endogenous retroviruses and retrotransposable elements in inflamed tissue, or signifies the presence of exogenous retroviral infection.

Pilot studies of single and combination antiretroviral therapy in patients with primary biliary cirrhosis.

OBJECTIVE: Preliminary reports suggest that patients with primary biliary cirrhosis (PBC) have evidence of human betaretrovirus infection. The aim of this study was to determine whether antiviral therapy impacts on the disease process. METHODS: We conducted two consecutive open-labeled, nonrandomized, 1-yr pilot studies; the first with lamivudine 150 mg/day and the second with Combivir combination therapy using lamivudine 150 mg and zidovudine 300 mg twice a day. Eleven PBC patients enrolled in each study, seven patients were entered into both studies, and one patient was withdrawn from each study due to side effects. RESULTS: Evaluation of liver biopsies before and after lamivudine therapy showed a 4-5 increase in necroinflammatory score, a 1-1.5 elevation in bile duct injury, with little change in the percentage of portal tracts with bile ducts (50-52%). None of the patients in the lamivudine study normalized alkaline phosphatase. Histological assessment following Combivir therapy revealed a 6 to 4 improvement in necroinflammatory score (p < 0.03, 95% CI: 0.53-2.33), a 3 to 1 reduction in bile duct injury (p < 0.02, 95% CI: 1.08-2.07), and a 45-75% increase in portal tracts with bile ducts (p < 0.05, 95% CI: 0.02-0.29). In the Combivir cohort, five patients normalized alkaline phosphatase and four developed normal AST, ALT, and alkaline phosphatase. CONCLUSIONS: Histological and biochemical endpoints were achieved in the Combivir pilot study suggesting a larger placebo-controlled trial is required as a proof of principle to assess whether antiviral therapy impacts the PBC disease process.

Apocalypsal versus apocryphal: the role of retroviruses in primary biliary cirrhosis.

The etiology of primary biliary cirrhosis (PBC), much like that of other autoimmune diseases, remains enigmatic. In the case of PBC, several candidates have been proposed, including bacteria, chemicals, and retroviruses. The support for a retroviral etiology has led to a pilot therapeutical interventional program. However, there is limited objective data which can be used to support such a thesis. Until reproducible and mechanistically sound data can be offered, we suggest that pharmaceutical trials are unrealistic and should be discouraged.