Shawn T. Wasilenko

Clinical trial: randomized controlled study of zidovudine and lamivudine for patients with primary biliary cirrhosis stabilized on ursodiol

Background  A human betaretrovirus has been characterized from patients with primary biliary cirrhosis (PBC). Uncontrolled studies using combination anti‐retroviral therapy have reported significant biochemical and histological improvement.

Metagenomic Analysis of Microbiome in Colon Tissue from Subjects with Inflammatory Bowel Diseases Reveals Interplay of Viruses and Bacteria

Abstract:Inflammatory bowel diseases (IBD), Crohns disease and ulcerative colitis, are poorly understood disorders affecting the intestinal tract. The current model for disease suggests that genetically susceptible patients develop intolerance to gut microflora, and chronic inflammation develops as a result of environmental insults. Although interest has mainly focused on studying genetic variants and gut bacterial flora, little is known about the potential of viral infection to contribute to disease. Accordingly, we conducted a metagenomic analysis to document the baseline virome in colonic biopsy samples from patients with IBD in order to assess the contribution of viral infection to IBD. Libraries were generated from colon RNA to create approximately 2 GB sequence data per library. Using a bioinformatic pipeline designed to detect viral sequences, more than 1000 viral reads were derived directly from tissue without any coculture or isolation procedure. Herein, we describe the complexity and abundance of viruses, bacteria/bacteriophage, and human endogenous retroviral sequences from 10 patients with IBD and 5 healthy subjects undergoing surveillance colonoscopy. Differences in gut microflora and the abundance of mammalian viruses and human endogenous retroviruses were readily detected in the metagenomic analyses. Specifically, patients with herpesviridae sequences in their colon demonstrated increased expression of human endogenous viral sequences and differences in the diversity of their microbiome. This study provides a promising metagenomic approach to describe the colonic microbiome that can be used to better understand virus–host and phage–bacteria interactions in IBD.

Identification of Hepatotropic Viruses from Plasma Using Deep Sequencing: A Next Generation Diagnostic Tool

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.

Mouse mammary tumor virus in anti-mitochondrial antibody producing mouse models

BACKGROUND & AIMS A human betaretrovirus resembling the mouse mammary tumor virus (MMTV) has been characterized in primary biliary cirrhosis (PBC) and associated with aberrant pyruvate dehydrogenase complex (PDC)-E2-like expression. As MMTV is prevalent in mice as either an exogenous or endogenous infection, we tested the hypothesis that MMTV is linked with anti-mitochondrial antibody (AMA) production in models with severe immune dysfunction. METHODS Evidence for MMTV was assessed in the liver and spleen of mice by PCR and immunochemistry and PDC-E2-like protein by immunochemistry. ELISA and Western blot were used to investigate AMA and anti-MMTV antibody production. RESULTS Increased MMTV gag or env expression was detected in the livers of AMA producing mice including NOD.c3c4, CD4 directed dominant negative TGF-β receptor II and IL-2 receptor α knockout mice as well as the NOD parental strain when compared to healthy strains and biliary disease control mice. The NOD.c3c4 mice expressed MMTV surface and capsid proteins and aberrant PDC-E2-like protein in the bile ducts, whereas IL-2 receptor α knockout mice, NOD.c3c4 and the NOD mice expressed MMTV proteins and aberrant PDC-E2-like protein in the spleen. A significant correlation between anti-MMTV antibody production and AMA production was observed in the sera of NOD and NOD.c3c4 mice (p<0.0001). CONCLUSIONS The association of betaretroviral protein production and aberrant PDC-E2-like protein expression in the NOD.c3c4, NOD, and the IL-2 receptor α knockout mice is comparable to observations in patients with PBC. The correlation of AMA and anti-MMTV suggests the hypothesis that MMTV infection may trigger the production of AMA.

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.

Primary biliary cirrhosis, bacteria and molecular mimicry: what's the molecule and where's the mimic?

Primary biliary cirrhosis (PBC) is an enigmatic disease. While it is thought that an environmental agent precipitates disease in a genetically susceptible individual, we are still none the wiser about the specific genes and microbes that intersect to cause PBC. For quite some time, bacterial proteins have been touted as the molecular mimic for breaking tolerance to mitochondrial proteins in PBC. This notion has gained considerable momentum, as the majority of patients with PBC make antimitochondrial antibodies (AMA) and T-lymphocyte responses to the inner lipoyl domain of the pyruvate dehydrogenase complex (PDC)-E2 and other lipoylated mitochondrial enzyme complexes (1–3). PDC-E2 and related proteins are found in all organisms that use oxidative phosphorylation and it has been hypothesized that infection with a spectrum of several bacteria including Escherichia, Mycobacteria, Chlamydia, Helicobacter and Novosphingobium can trigger PBC by breaking tolerance to human PDC-E2 (1). In this issue of Liver International, Berg and colleagues add Mycobacteria pneumoniae to the long list of pathogens with a potential role in propagating PBC by showing that sera from PBC patients has reactivity against recombinant PDC-E2 encoded by M. pneumoniae (4). The authors also showed that bacterial reactivity was not blocked by excess porcine PDC-E2 in a proportion of patients and some PBC patients recognized M. pneumoniae PDC-E2 but not porcine PDC-E2. Based on these findings the authors hypothesize that infection with M. pneumoniae results in an immune response directed against PDC-E2 present on the bacterial cell membrane to initiate PBC. Many investigators have posited a bacterial etiology of PBC based on PDC-E2 cross-reactivity (1) but is there additional evidence to support the bacterial molecular mimicry theory? Originally, molecular mimicry was adopted as an attractive model for many autoimmune disorders because it provided a link with infection and loss of tolerance to host proteins (5). The hypothesis stemmed from the idea that microbes share immunodominant epitopes with the host. These molecular mimics either allowed the microbe to escape immune recognition or, alternatively, provoked loss of tolerance to the host protein once the microbe was recognized. Although this idea has been circulating for over 40 years, there is no convincing evidence that human autoimmune disorders are actually triggered by molecular mimicry (5). Indeed, only one animal model of herpes virus induced stromal keratitis in mice has linked molecular mimicry with disease and even then, viral replication is still required to perpetuate disease (6). So why has it been so difficult to prove that molecular mimicry triggers human autoimmune diseases? Certainly, it is problematic to demonstrate causality for any microbial infection in chronic, multi-factorial complex disorders. But shouldn’t we be looking for alternative models for breaking tolerance now that molecular mimicry hypothesis has been ongoing for four decades without concrete evidence that it triggers human disease? There are additional layers of complexity in establishing the autoimmune pathogenesis of PBC. For example, it is fair to ask how an autoimmune response to a protein located on the inner mitochondrial membrane of nearly all nucleated cells causes an organ-specific disease targeted to biliary epithelium. Some light has been thrown on this mystery by studies showing aberrant expression of large amounts of PDC-E2 on the cell surface of biliary epithelium and lymph node macrophages from patients with PBC (2). It was originally thought that the protein found on biliary epithelium was a molecular mimic of PDC-E2 as not all preparations of AMA reacted with the antigen (7). However, subsequent studies performed on biliary epithelium isolated from PBC patients’ livers revealed that these proteins had the same molecular weight as human PDC-E2 and the related PDC-E3 binding protein that is also reactive with AMA (8). The search for bacterial PDC-E2 in the liver has been unrewarding and the consensus is that specific bacteria are not found in the biliary epithelium or the liver of patients with PBC, apart from a couple of reports to the contrary (1). So it appears as though the trigger to break tolerance is actually human PDC-E2 or a modified form of the protein located on the biliary epithelium cell surface rather than a bacterial molecular mimic (2, 3). As bacteria have not been reproducibly detected in the liver of PBC patients, the current molecular mimicry model proposes that patients with PBC suffer recurrent urinary tract or other bacterial infections. In this regard, it is noteworthy that in a large survey of North American patients, a history of urinary tract infections were recorded in 59% of PBC patients and 52% of control subjects (9). The caveat here is that these events only trigger PBC in susceptible patients. The molecular mimicry model predicts that bacterial infection provokes an

Other potential medical therapies: the use of antiviral agents to investigate and treat primary ciliary cirrhosis.

A human betaretrovirus has been characterized in patients with primary biliary cirrhosis (PBC) and the related mouse mammary tumor virus linked with autoimmune biliary disease in the NOD.c3c4 mouse model. Translational studies have been performed in patients who have PBC to investigate the role of viral infection in disease. Patients treated with Combivir experienced significant improvements in hepatic biochemistry, clinical symptoms, and histology with evidence of reversal of ductopenia. Preliminary studies suggest that the NOD.c3c4 mouse model of PBC provides a good model to test safer and more potent drug regimens for future use in trials for patients who have PBC.

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

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.

Duplication of MER115 on chromosome 4 in patients with primary biliary cirrhosis

Background: Primary biliary cirrhosis (PBC) is a complex disease with genetic and environmental influences. The disease is more prevalent in families with PBC and candidate gene case–control studies have linked PBC with DRB1*08 human leucocyte antigen class II alleles.

Characterization of a human betaretrovirus in patients with primary biliary cirrhosis

Our laboratory has characterized and cloned a human betaretrovirus resembling the mouse mammary tumor virus (MMTV) from biliary epithelium and lymph nodes of patients with the autoimmune biliary disease, primary biliary cirrhosis (PBC). Evidence for betaretrovirus infection has been detected mainly in patients’ lymphoid tissue by RT-PCR and immunohistochemistry in approximately 75% of PBC patients. However, the viral burden was observed to be far less in the liver and we could only identify viral sequence in a third of patients using RT-PCR. Other labs have been unable to confirm our data using PCR of hepatic DNA. Not surprisingly, the betaretroviral association with PBC has become a controversial issue. In order to provide more robust evidence of infection in patients with liver disease, we have isolated an infectious betaretrovirus from lymph nodes. We cloned and sequenced the proviral isolates and confirmed viral particles by electron microscopy. We have also conducted ligation-mediated PCR studies with next generation sequencing and found proviral integration sites both in vivo and in vitro. To date, we have identified more than 3,000 integration sites in perihepatic lymph nodes, liver and isolated biliary epithelium in the majority of patients with PBC and seldom in patients with other liver diseases. These data confirm the presence of an MMTV-like virus in patients with autoimmune liver disease. Ongoing studies using biliary disease mouse models with MMTV infection and randomized controlled anti-retroviral trials for patients with PBC will be required to further characterize the association of human betaretrovirus infection with PBC.