Upregulation of lymphotoxin beta expression in liver progenitor (oval) cells in chronic hepatitis C.

BACKGROUND: Bipotent liver progenitor (oval) cells with the ability to differentiate into hepatocytes and biliary epithelium have recently been identified in human subjects with hepatitis C. Animal studies suggest that members of the tumour necrosis factor family, including lymphotoxin beta (LT-beta), regulate oval cell proliferation in liver disease, but its role in human liver disease is unclear. AIMS: This study seeks to establish a role for LT-beta in hepatitis C related liver injury and to provide evidence that its increased expression is related to the presence of oval cells. METHODS: Liver biopsy specimens were obtained from patients with chronic hepatitis C virus (HCV) infection (n=20). Control liver samples (n=5) were obtained from liver resection or transplant surgery. LT-beta expression in liver biopsy specimens was studied using quantitative real time polymerase chain reaction and immunohistochemistry. RESULTS: LT-beta mRNA levels were similar in control and HCV liver in the absence of fibrosis. In subjects with portal fibrosis, LT-beta mRNA levels were elevated 2.2-fold over control liver levels (p=0.04). In subjects with bridging fibrosis, LT-beta mRNA levels increased 4.4-fold over control liver levels (p=0.02). LT-beta mRNA levels in subjects with established cirrhosis were increased 3.3-fold compared with controls and 2.6-fold compared with mild liver damage (p=0.02). Immunohistochemical analysis established that LT-beta was expressed by oval cells, inflammatory cells, and small portal hepatocytes. CONCLUSIONS: In chronic HCV infection, LT-beta expression is observed in multiple hepatic cell types, including oval cells. LT-beta expression is significantly increased when fibrosis or cirrhosis is present, suggesting a role for LT-beta in the pathogenesis of chronic hepatitis C and a possible role in oval cell mediated liver regeneration.

Characterization of the murine CD30 ligand (CD153) gene: gene structure and expression.

CD153 (CD30 ligand) has been described as a 40-kDa type II transmembrane glycoprotein belonging to the TNF superfamily and is expressed primarily by activated T cells, B cells and monocytes. In this study, we have determined that the murine CD153 gene consists of four exons, with three intervening introns, spaced over approximately 26 kb of genomic sequence. Sequence analysis of the murine CD153 promoter and 5' flanking region revealed the presence of a TATA box element immediately upstream of two tsp sites, together with putative binding motifs for a variety of lymphoid-specific transcription factors. 5'RACE analysis of LPS-stimulated RAW264.7 macrophage cDNA identified at least four transcriptional start sites for murine CD153, with two sites occurring downstream of the previously predicted translation initiation codon. Additionally, 5' RACE analysis identified multiple murine CD153 polyadenylation sites. Our results indicate that primary murine CD153 transcripts may vary from 26 kb to approximately 28 kb in length.

A modified choline-deficient, ethionine-supplemented diet protocol effectively induces oval cells in mouse liver.

Several reliable and reproducible methods are available to induce oval cells in rat liver. Effective methods often involve inhibiting proliferation in hepatocytes using an alkylating agent, then subjecting the rat to partial hepatectomy (PH). The surgery is difficult to perform reproducibly in mice. Approaches that do not include partial hepatectomy, such as administration of D-galactosamine, are ineffective in mice. We found that a choline-deficient, ethionine-supplemented (CDE) diet, which is very effective in rats, leads to high morbidity and mortality when administered to mice. This article outlines an alternative protocol by which a CDE diet can be administered to mice. This diet is shown to be highly effective for oval cell induction, without causing high mortality. It takes less time and is at least as effective as other commonly used protocols for inducing oval cells in mice.

Involvement of Sp1 and microsatellite repressor sequences in the transcriptional control of the human CD30 gene.

CD30, as a member of the tumor necrosis factor (TNF) receptor family, is expressed on the surface of activated lymphoid cells. CD30 overexpression is a characteristic of lymphoproliferative diseases such as Hodgkin's/non-Hodgkin's lymphomas, embryonal carcinoma, and a number of Th2-associated diseases. The CD30 gene has been mapped to a region of the murine genome that is involved in susceptibility to systemic lupus erythematosus. Functionally, CD30 may play a role in the deletion of autoreactive T cells. We were interested in determining the molecular nature of CD30 overexpression. Sequence comparison has revealed significant identity between the TATA-less human and murine CD30 promoters; they share a number of common consensus binding motifs. Transfection assays identified three regions of transcriptional importance; the region between position -1.2 kb and -336 bp, containing a CCAT microsatellite sequence, a conserved Sp1 site at positions -43 to -38, and a downstream promoter element (DPE) at positions +24 to +29. EMSA and DNase I footprinting showed specific DNA-protein interactions of the CD30 promoter with the Sp1 site and the CCAT repeat region. The DPE element was shown to be essential for start site selection. We conclude that the conserved Sp1 site at -43 to -38 is associated with maximum reporter gene activity, the DPE element is required for start site selection, and the CCAT tetranucleotide repeats act to repress transcription. We also have shown that the microsatellite is multiallelic, when we screened a random healthy population. Further studies are required to determine whether microsatellite instability in the repressor predisposes susceptible individuals to CD30 overexpression.

Mapping of a candidate region for susceptibility to inclusion body myositis in the human major histocompatibility complex.

Inclusion body myositis (IBM) is a form of idiopathic inflammatory myopathy of unknown aetiology. A strong association with HLA class II (HLA-DR3) suggested a role for genes in the human major histocompatibility complex (MHC) in the predisposition to this disease. In this study, we have taken advantage of the ancestral haplotype (AH) concept and historical recombinations to map for a possible susceptibility gene(s) in the MHC. We performed detailed typing of three MHC-related HSP70 genes and defined allelic combinations in the context of MHC AH. We also modified existing methods to give a simple and accurate method for typing two TNF microsatellites. Using the HSP70 and TNF markers and HLA-DR, -B, and C4 typing of our patients with IBM, we defined a potential site for the MHC-associated susceptibility gene(s) in the region between HLA-DR and C4.

Analysis of the human and mouse promoter region of the non-Hodgkin's lymphoma-associated CD30 gene.

To investigate the regulation of CD30 at the level of transcription, we have isolated and compared the promoter sequence of human and murine CD30. Analysis of the human and mouse promoter identified a number of potential transcription factor binding sites, including ETS, MZF, AP-1, IK2, CREB, Stat, USF, and Spl. The absence of TATA or CAAT boxes and the identification of one major and three minor transcription initiation sites for CD30 suggest that it is a member of the class of TATA-less promoters that use initiator elements to correctly position the RNA polymerase. Comparison of the murine and human CD30 promoters identified a number of highly conserved regions, including an Spl site 40 bp upstream from the major start site and a downstream promoter element (DPE) that may be involved in directing transcriptional initiation of the CD30 gene. Functional analysis of the human CD30 promoter in transfected Jurkat T cells provided further evidence that these conserved regions are important regulatory elements in the CD30 promoter.

Characterisation of the human CD30 ligand gene structure.

The gene for the human CD30 ligand was molecularly cloned, sequenced and characterised. The gene structure consisted of four exons and three intervening introns spaced over 17.1 kb of genomic DNA. The 5' flanking region of the CD30L gene was sequenced and analysis of the region revealed the presence of several regulatory regions including a poly-dT element directly upstream from the transcription start site and consensus sequences for AP4, IK2, MZF1, E47 and ELK/cETS1. The absence of a canionical TATA motif suggests CD30L gene expression is regulated by a TATA-less promoter.