Antifibrotic therapies in the liver.

Significant progress has been made in understanding the principles underlying the development of liver fibrosis. This includes appreciating its dynamic nature, the importance of active fibrolysis in fibrosis regression, and the plasticity of cell populations endowing them with fibrogenic or fibrolytic properties. This is complemented by an increasing array of therapeutic targets with known roles in the progression or regression of fibrosis. With a key role for fibrosis in determining clinical outcomes and encouraging data from recently Food and Drug Administration-approved antifibrotics for pulmonary fibrosis, the development and validation of antifibrotic therapies has taken center stage in translational hepatology. In addition to summarizing the recent progress in antifibrotic therapies, the authors discuss some of the challenges ahead, such as achieving a better understanding of the interindividual heterogeneity of the fibrotic response, how to match interventions with the ideal patient population, and the development of better noninvasive methods to assess the dynamics of fibrogenesis and fibrolysis. Together, these advances will permit a better targeting and dose titration of individualized therapies. Finally, the authors discuss combination therapy with different antifibrotics as possibly the most potent approach for treating fibrosis in the liver.

Antigen presentation by liver cells controls intrahepatic T cell trapping, whereas bone marrow-derived cells preferentially promote intrahepatic T cell apoptosis.

Systemic activation and proliferation of CD8(+) T cells result in T cell accumulation in the liver, associated with T cell apoptosis and liver injury. However, the role of Ag and APC in such accumulation is not clear. Bone marrow chimeras were constructed to allow Ag presentation in all tissues or alternatively to restrict presentation to either bone marrow-derived or non-bone marrow-derived cells. OVA-specific CD8(+) T cells were introduced by adoptive transfer and then activated using peptide, which resulted in clonal expansion followed by deletion. Ag presentation by liver non-bone marrow-derived cells was responsible for most of the accumulation of activated CD8(+) T cells. In contrast, Ag presentation by bone marrow-derived cells resulted in less accumulation of T cells in the liver, but a higher frequency of apoptotic cells within the intrahepatic T cell population. In unmodified TCR-transgenic mice, Ag-induced T cell deletion and intrahepatic accumulation of CD8(+) T cells result in hepatocyte damage, with the release of aminotransaminases. Our experiments show that such liver injury may occur in the absence of Ag presentation by the hepatocytes themselves, arguing for an indirect mechanism of liver damage.

Involvement of CD1 in peripheral deletion of T lymphocytes is independent of NK T cells.

During peripheral T cell deletion, lymphocytes accumulate in nonlymphoid organs including the liver, a tissue that expresses the nonclassical, MHC-like molecule, CD1. Injection of anti-CD3 Ab results in T cell activation, which in normal mice is followed by peripheral T cell deletion. However, in CD1-deficient mice, the deletion of the activated T cells from the lymph nodes was impaired. This defect in peripheral T cell deletion was accompanied by attenuated accumulation of CD8(+) T cells in the liver. In tetra-parental bone marrow chimeras, expression of CD1 on the T cells themselves was not required for T cell deletion, suggesting a role for CD1 on other cells with which the T cells interact. We tested whether this role was dependent on the Ag receptor-invariant, CD1-reactive subset of NK T cells using two other mutant mouse lines that lack most NK T cells, due to deletion of the genes encoding either beta(2)-microglobulin or the TCR element J alpha 281. However, these mice had no abnormality of peripheral T cell deletion. These findings indicate a novel role for CD1 in T cell deletion, and show that CD1 functions in this process through mechanisms that does not involve the major, TCR-invariant set of NK T cells.

The liver as a site of T-cell apoptosis: graveyard, or killing field?

The liver is a site at which apoptotic CD8+ cells accumulate during the clearance phase of peripheral immune responses. Normal mouse liver contains an unusual mixture of lymphocytes in which natural killer (NK) and NK-T cells are abundant and apoptotic T cells are present, and we interpret these cell populations as, respectively, agents and targets of an intrahepatic T-cell trapping and killing mechanism. In support of this idea, direct perfusion of activated lymphocyte populations through the normal liver results in the selective retention of activated CD8+ T cells. T cells trapped in this manner undergo apoptosis in the liver. This mechanism could explain the importance of the liver in oral tolerance, the phenomenon of tolerance induced by portal vein infusion of antigenic cells, the tolerance to allogeneic liver allografts, and the persistence of some liver pathogens including hepatitis C.

Selective retention of activated CD8+ T cells by the normal liver.

Activation-induced cell death resulting in peripheral deletion of CD8+ T cells is associated with the accumulation of large numbers of apoptotic T cells in the liver. The hypothesis that this accumulation results from the intrahepatic trapping of T cells from the circulating pool predicts that the liver should retain T cells, which subsequently undergo apoptosis. Here we test this prediction. Perfusion of the liver with lymphocyte mixtures showed retention of activated, but neither resting nor apoptosing, T cells. This trapping was selective for CD8+ cells and was mediated primarily by ICAM-1 constitutively expressed on sinusoidal endothelial cells and Kupffer cells. T cells trapped in the liver became apoptotic. The normal liver is therefore a "sink" for activated T cells.

IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells.

The liver contains abundant cytotoxic cells, including NK-T cells, NK cells, and CTLs. However, the regulation of this cytotoxicity is not fully understood. In this study, we investigated the effect of a recently described cytokine, IL-18, which is present in large quantities in the liver, on the cytotoxicity of intrahepatic lymphocyte subpopulations. This effect of IL-18 was assessed by assaying the in vitro cytotoxicity of purified NK-T, NK, and T cells against a CD95- and perforin-sensitive T cell line, Jurkat. The results show that IL-18 enhances the killing activity of liver NK-T cells by a CD95-independent, perforin-dependent pathway. IL-18 also augments liver NK cell activity, but the exact mechanisms of this killing remain to be elucidated. Finally, the augmentation of the killing activities of liver NK-T and NK cells by IL-18 is not due to soluble TNF-alpha, because none of these cell populations had detectable TNF-alpha production.

TCR ligation on CD8+ T cells creates double-negative cells in vivo.

The lack of CD95 in mice is associated with an accumulation of TCR alphabeta+ CD4- CD8- (double-negative (DN)) cells in the lymph nodes (LNs) and other organs. To test the hypothesis that these DN cells arise from TCR alphabeta+ CD8+ cells after activation via the TCR, we have crossed an MHC class I-restricted TCR transgene (tg) onto the lpr genotype to generate two TCR-transgenic experimental groups, TCRtg+ lpr/+ (CD95-intact) and TCRtg+ lpr/lpr (CD95-deficient). Specific peptide administration resulted in peripheral deletion of TCR alphabeta cells from the LNs of CD95-intact and CD95-deficient mice. On day 3 after peptide administration in the CD95-deficient but not the CD95-intact mice, there was a ninefold increase in the percentage of DN cells in the LN; this increase returned to control levels by day 10. Peripheral deletion was associated with an accumulation of TCR alphabeta+ CD8high cells in the livers of mice of both genotypes by day 3, which returned to control levels by day 10 without an increase in the percentage or total number of DN cells. Our data show that the in vivo stimulation of TCR alphabeta+ CD8+ cells in the absence of CD95 results in an initial accumulation and an eventual loss of DN cells. This identifies a role for CD95 after TCR alphabeta stimulation in the efficient removal of TCR alphabeta+ CD8+ cells after the down-regulation of CD8. CD95 is not essential for this process, because other mechanisms can compensate, but such mechanisms are less efficient in the LN.

Antibiotic prophylaxis for ERCP: a randomized clinical trial comparing ciprofloxacin and cefuroxime in 200 patients at high risk of cholangitis.

OBJECTIVE: To compare the efficacy and safety of oral ciprofloxacin and intravenous cefuroxime in patients at high risk of cholangitis after endoscopic retrograde cholangiopancreatography (ERCP). DESIGN: Prospective, randomized study. SETTING: A primary and tertiary referral centre. PATIENTS: A series of 232 consecutive patients who either had radiological evidence of biliary obstruction or were aged over 70 years were randomly assigned to receive either oral ciprofloxacin or intravenous cefuroxime before and after ERCP. Two-hundred and nine patients finished the study, with 23 being excluded because of withdrawal of consent or incomplete ERCP. INTERVENTIONS: Patients underwent ERCP: blood samples were taken for culture, full blood count and biochemistry before and after the procedure. Clinical follow-up was carried out 7 and 42 days after ERCP. MAIN OUTCOME MEASURES: Clinical, bacteriological or biochemical evidence of cholangitis, septicaemia or adverse drug reactions, and the cost of both protocols. RESULTS: Follow-up was recorded in all 209 patients who completed the study. By 42 days after ERCP, three patients had died (cholangiocancer, pancreatic cancer and renal failure). Cholangitis was diagnosed in one patient from each of the two trial groups. Blood cultures from the patient on ciprofloxacin gave negative results, but a post-ERCP blood sample from the patient on cefuroxime grew Pseudomonas aeruginosa, which was sensitive to ciprofloxacin. There were no serious side-effects in either study group, but two patients assigned to ciprofloxacin became too nauseous to take the medication. The cost of the cefuroxime protocol was l7.56 pounds per patient, compared with 4.76 pounds per patient for the ciprofloxacin protocol. CONCLUSION: A pre- and post-ERCP oral ciprofloxacin regime is safe and provides effective prophylaxis against ERCP-induced cholangitis and septicaemia in high-risk patients. It is also more economical than a regime of intravenous cefuroxime and does not require nursing staff with training in intravenous techniques.

Role of human papillomavirus in determining the HLA associated risk of cervical carcinogenesis.

AIMS: To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC). METHODS: Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval. RESULTS: Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05). CONCLUSIONS: There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC.

Defining the immunogenetic susceptibility to primary biliary cirrhosis.

Primary biliary cirrhosis is a chronic cholestatic disease, thought to be immune-mediated with genetic susceptibility encoded in the major histocompatibility complex. In northern Europeans, the best established associations are with HLA-DR8 and the complement allele, C4B2. These associations could be due to a single susceptibility locus on an extended haplotype linking HLA-DR8 and C4B2 or to both HLA-DR8 and C4B2 independently conferring disease susceptibility. C4B2 genotyping was performed on 64 patients with primary biliary cirrhosis and 61 controls matched for ethnic background and frequency of HLA-DR8. C4B2 was associated with HLA-DR8 (p < 0.05) in PBC. No difference in the frequency of C4B2 was detected between control and disease populations, suggesting that HLA-DR8 and C4B2 are in linkage disequilibrium and that C4B2 is not a susceptibility locus for PBC. Taq I polymorphisms were screened in the disease and control populations with the cosmid probe G91, located midway between the HLA-DR and complement loci. One G91 restriction fragment (G91A) was found to be associated with both HLA-DR8 and C4B2, at equal frequency in health and disease, providing evidence of an HLA-DR8-G91A-C4B2 extended haplotype. The frequency of G91A was the same in the disease and control populations, suggesting that G91A does not confer disease susceptibility. These findings establish G91 as the telomeric boundary for disease susceptibility associated with HLA-DR8, encoded on chromosome six. These studies help define the immunogenetic susceptibility locus for primary biliary cirrhosis.

HLA DR4 is a marker for rapid disease progression in primary sclerosing cholangitis.

BACKGROUND/AIMS: Primary sclerosing cholangitis (PSC) is an inflammatory disease of the biliary tree associated with an increase in the HLA alleles DR3, DR52a, DR2, Dw2, and a decrease in DR4. However, it is not certain which of these alleles provides the primary associations. Our aim was to establish the primary HLA associations with PSC and to assess the ability of HLA alleles to mark for disease progression. METHODS: By applying molecular techniques to archival tissue, we have genotyped 83 PSC patients from two populations and 131 controls for the alleles HLA DR2, DR3, DR4, DRw12, DR52a, and Dw2. RESULTS: HLA DR3, DR52a, DR2, and Dw2 were all significantly increased in PSC, with the relative risk for DR52a and Dw2 being greater than for DR3 and DR2, respectively. HLA DR4 was significantly decreased, but this may be artifactual to the DR3, DR2 increase. HLA DR4 and not DR52a marks for rapid disease progression in both our PSC populations. CONCLUSIONS: HLA DR52a and Dw2 are the best candidate alleles for providing the known HLA association with PSC. HLA DR4 and not DR52a marks for rapid disease progression in our two PSC populations.

Detection of reactivation and size variation in the regulatory region of JC virus in brain tissue.

AIMS: To develop a sensitive and specific polymerase chain reaction (PCR) based system for detecting genomic variation in JC virus. To apply this system to formalin fixed, paraffin wax embedded brain tissue from patients with and without progressive multifocal leucoencephalopathy (PML). METHODS: A pair of primers (JC1 and JC2) were designed to be complementary to the early and late regions of JC and BK polyomaviruses, respectively. A third primer (JC3), internal to JC1 and JC2, was designed to be specific for JC virus. The specificity of JC3 was investigated by amplifying plasmids with BK or JC virus genomes. Sensitivity was estimated by titration of a plasmid containing JC virus genome. Seven brains from patients with PML (PMLB) and 30 from patients without PML (non-PMLB) were amplified using JC1 and JC2, followed by JC1 and JC3. Amplification of the beta globin gene was used as an amplification control. RESULTS: Amplification with JC1 and JC2 was common for JC and BK viruses, but with JC1 and JC3 it was specific for JC virus. The sensitivity of the system was 25 copies of JC plasmid per 10 microliters of digested tissue. Five out of seven PMLB and 28 of the 30 non-PMLB amplified for beta globin, but only the PMLB gave a signal with polyoma primers. Hypervariation of the length of the regulatory region of the JC isolates in the PML tissues was consistent with the presence of multiple strains of JC. CONCLUSIONS: Variation in the regulatory region of JC virus can be specifically and sensitively detected from routinely processed, paraffin wax embedded brain tissue. Variation in the regulatory region is common in PML derived JC strains, but JC virus was not detectable in non-PMLB tissue.

Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV.

A survey of cytomegalovirus (CMV) DNA in primary sclerosing cholangitis (PSC) liver tissues using a sensitive polymerase chain reaction (PCR) based assay.

Reactivation of cytomegalovirus (CMV) has been implicated as a possible etiological agent in primary sclerosing cholangitis (PSC) partly because of the ability of CMV infection to cause hepatobiliary damage, and further because of the recent recognition of a PSC-like syndrome in AIDS patients, many of whom have hepatobiliary infection with CMV. Direct evidence of CMV infection in PSC has come from a study detecting CMV DNA in 7/7 PSC livers, but only 5/20 controls. We have developed an assay for CMV-DNA by amplification of the immediate early region of CMV using the polymerase chain reaction, followed by Southern blotting and 32P oligoprobing of the amplification product. This system has an average sensitivity of at least 25 copies of CMV-DNA per 5000 formalin-fixed paraffin-embedded cells. 37 PSC and 19 control samples of formalin-fixed paraffin-embedded hepatobiliary tissues were studied. Amplification for the beta-globin in each sample was used as an amplification control, and fetal lung with known CMV infection as the CMV-positive control. 37/37 PSC tissues amplified for beta-globin, and one of these was positive for CMV-DNA. All 19 controls amplified for beta-globin, with none being positive for CMV. The lack of CMV-DNA in 35/36 PSC samples at a level of 25 copies per 5000 cells, we believe, rules out any significant CMV reactivation in these tissues, and suggests that CMV replication and re-activation is not responsible for the progression of PSC.

Analysis of complex genetic systems by ARMS-SSCP: application to HLA genotyping.

We have developed a new method for analysis of complex genetic systems by a combination of the Amplification Refractory Mutation System (ARMS) and Single-Strand Conformation Polymorphism (SSCP) analysis: ARMS-SSCP. Thus, a complex allelic series is subdivided into a number of groups by ARMS followed by the identification of specific alleles using SSCP analysis. We have shown that the HLA alleles at the DRB3 and DQB1 loci were distinguishable from each other using ARMS-SSCP. In 36 individuals typed for the DRB3 and 48 individuals typed for the DQB1 loci, ARMS-SSCP results were in complete agreement with those obtained using the established method of sequence-specific oligonucleotide (SSO) hybridisation. With silver staining, ARMS-SSCP is a rapid, non-radioactive and reliable method which also offers the possibility for detecting new HLA alleles. We have demonstrated that ARMS-SSCP can be performed using fluorescent PCR primers, a feature which gives the method potential for automation.