Professor James Neuberger
Based: University Hospital Birmingham
Donor: British Liver Trust and PBC Foundation
Amount: £71,910 (payable over three years)
Start date: September 1997
Title of study: An investigation into the pathogenesis of PBC
Download Final report - Professor Neuberger.pdf
In 2000 Professor Neuberger wrote:
"Three years ago the PBC Foundation and the British Liver Trust gave me a three year grant to fund a research fellow to look further at the aetiology of primary biliary cirrhosis (PBC). This funding ended at the end of September 2000 and this report highlights the work done during the tenure of the funding.
Background
PBC is a disease of unknown aetiology. At the present time there is no specific treatment. Ursodeoxycholic acid (UDCA) may slow progression but liver transplantation remains the only definitive therapeutic option. PBC is characterised by the presence of antimitochondrial antibodies. These are specific antibodies which are found in nearly all people with PBC and not in any other group of people with liver disease. Thus, understanding more about the AMA may allow elucidation of the mechanism of disease and may lead to possible therapeutic interventions.
Yeaman and colleagues in Newcastle and Gershwin and colleagues in North America showed that the antigen recognised by AMA was a protein known as E2. This is an enzyme which is normally located in the mitochondrion within the cell. Mitochondria are found in almost all cells except red blood cells. We and subsequently others have shown that in the liver of people with PBC, the E2 antigen is present on the cell membrane of the biliary epithelial cells. This may account for the association between AMA and PBC; further understanding of the mechanisms leading to membrane expression of E2 on the target cells may help elucidate the pathogenesis of PBC.
Work Completed to Date
1. Characterisation of membrane E2
Preliminary results (published in Lancet) showed that normal human biliary epithelial cells do not have E2 on the membrane. However, when normal biliary epithelial cells are incubated for a short while with homogenates of lymph nodes from people with PBC, then the characteristic features of PBC can be induced on these biliary epithelial cells.
The initial work undertaken and supported by the BLT and PBC Foundation was to characterise this observation further. Firstly, we used a larger number of lymph nodes from both patients with PBC and normal controls. This confirmed our initial observations. We then studied other liver diseases such as primary sclerosing cholangitis (PSC), alcoholic liver disease and others to show that the findings were specific for PBC.
We then went on to characterise the membrane expression of E2 and were able to show that not only was there increased E2 within the cells but there was membrane-bound E2. This we showed with confocal laser microscopy and with electron microscopy.
Subsequent studies were undertaken to look for factors that might be responsible for this transmission. In the first set of experiments we showed the supernatants from the culture median could have the same effect as PBC lymph node homogenates. It proved easier to work with the supernatants since this is a cleaner preparation. Subsequently work was undertaken to characterise the transmissible effect further. In brief, we showed that this was not due to serum IgA released in the medium and taken up by the biliary epithelial cells.
We were able to show that high speed centrifugation resulted in abolition of the effect. This raised the possibility that a viral or infectious agent could be responsible. Irradiating the supernatant, at a dose that did not alter biological activity of measured cytokines, abolished inducing effect. These studies have subsequently been written up for publication.
Our current approach is to look in vitro to determine whether co-incubation with anti-viral drugs has any effect on aberrant E2 expression. Preliminary data are encouraging.
2. Viral studies
The experiments above indicated the possibility that the transmissible effect was induced by a virus. We, therefore, undertook collaborative studies with Dr William Carman at the Institute of Virology, University of Glasgow, and Dr Andrew Mason at the Ochsner Foundation in New Orleans. We have worked extensively with these groups providing information and resources. Electron microscopy studies done both in Birmingham and in Glasgow have raised the possibility of viral particles in the supernatant and in biliary epithelial cells from people with PBC.
We prepared sufficient DNA from biliary epithelial cells in both normal and PBC people to enable a CDNA library to be cloned. Samples were sent both to Glasgow and New Orleans. A number of sequences were identified which may be related to a viral agent which is a retrovirus. Studies using Southern Blotting technique and PCR revealed that the virus is not endogenously coded.
Further studies were undertaken using RT-PCR. The viral sequences was found in 90% of biliary epithelial cells from PBC people and less than 10% of controls. In the supernatants again we were able to show sequences in 55% of patients and none of controls. Similar proportions were seen in serum and liver of people with PBC and controls.
More studies are currently underway to look for evidence of viral reactivity and confirm the specificity looking at other sequences from the same virus.
Two papers looking at the evidence for a specific viral sequence and for its relationship with the antigen are currently being prepared jointly with the two collaborative teams.
Overall, thanks to the support of the British Liver Trust and PBC Foundation, we feel that major advances have been made. We have shown that the PBC phenotype in biliary epithelial cells can be induced by a factor present in lymph nodes of people with PBC. The nature of this agent is not clear. We believe there is suggestive evidence there may be a virus involved and are in the process of showing whether this virus is strongly associated with PBC.
During the next year our efforts will be focussed on confirming this hypothesis. It is important that these findings are taken cautiously. Prior to publishing we need to be absolutely sure of our data. Although some centres, on the basis of our published abstracts, have started treating patients with anti-viral drugs (and anecdotally with some success), we feel that this is premature. We plan 3 major publications arising from this work and once they are in press, we will be in a strong position to go for national funding and consider a therapeutic trial of therapy in patients with PBC."