Mouse models of liver fibrosis.

Liver fibrosis is the final common pathway in a variety of liver diseases. To model liver fibrosis in mice is important as mechanisms not only of fibrogenesis, but also of fibrolysis, need to be clearly defined. Also, small rodents present a possibility to test potential treatments in vivo. Today, there are several mouse models of liver fibrosis available--induced by administration of hepatotoxins, by bile duct ligation or by immunological mechanisms--and, more and more widespread, transgenic animal models elucidating pathogenesis and common pathways in liver fibrosis. These different mouse models are complementary as they represent different pathways to fibrosis--as also seen in human disease. Recently, several promising treatment methods interfering with cytokine signaling have been published, offering new potential therapeutic interventions. This review seeks to summarize the different methods of fibrosis induction as well as to briefly review some promising new treatment options for fibrosis in the mouse model.

Multiplex neuritis in a patient with autoimmune hepatitis: a case report.

A 37-year old woman presented with a 9-year history of hepatitis of unknown origin and aminotransferases within a 3-fold upper limit of normal. Autoimmune hepatitis (AIH) was diagnosed on the basis of elevated aminotransferases, soluble liver antigen/liver pancreas (SLA/LP) autoantibodies and characteristic histology. Immunosuppressive therapy led to rapid normalization of aminotransferases. Two years later, the patient developed left sided hemisensory deficits under maintenance therapy of prednisolone and azathioprine (AZT). Later she developed right foot drop and paraesthesia in the ulnar innervation territory on both sides. Magnetic resonance imaging (MRI) and cerebral panangiography suggested cerebral vasculitis. Neurological investigation and electromyography disclosed multiplex neuritis (MN) probably due to vasculitis. Consistent with this diagnosis, autoantibodies to extractable nuclear antigens were detectable in serum. Immunosuppression was changed to oral 150 mg cyclophosphamide (CPM0) per day. Prednisolone was increased to 40 mg/d and then gradually tapered to 5 mg. Oral CPM was administered up to a total dose of 40 g and then substituted by 6 times of an intervall infusion therapy of CPM (600 mg/m(2)). Almost complete motoric remission was achieved after 3 mo of CPM. Sensibility remained reduced in the right peroneal innervation territory. Follow-up of cranial MRI provided stable findings without any new or progressive lesions. This is the first report of multiplex neuritis in a patient with autoimmune hepatitis.

Establishment of standardised SLA/LP immunoassays: specificity for autoimmune hepatitis, worldwide occurrence, and clinical characteristics.

BACKGROUND: Antibodies to soluble liver antigen/liver pancreas (SLA/LP) are specific markers of autoimmune hepatitis. Their target antigen has recently been cloned. AIMS: To establish standardised immunoassays using the recombinant antigen, and to assess the frequency and significance of seropositivity in patients from different countries. METHODS: An enzyme linked immunoassay was developed using purified recombinant antigen and validated by testing sera from 200 healthy blood donors and 1026 patients with various liver and non-liver diseases. The assay was then applied to 454 sera from 419 patients with autoimmune hepatitis from different countries. All sera were also tested by inhibition immunoassay and western blot. RESULTS: Antibodies were reliably detected by the recombinant immunoassay and occurred exclusively in patients with autoimmune liver disease. Twenty three of 149 patients from the USA (15%), 23/132 from Brazil (17%), 21/108 from Germany (19%), and 2/30 from Japan (7%) were seropositive. Clinical features at presentation were similar between seropositive and seronegative patients. However, relapse after corticosteroid withdrawal or during maintenance therapy occurred more commonly in seropositive patients. CONCLUSIONS: Antibodies to SLA/LP can be reliably detected by these standardised immunoassays based on recombinant antigen. Antibodies to SLA/LP occur with similar frequencies in different geographical regions, races, and age groups, and are of exquisite diagnostic specificity. Whether SLA/LP positive patients represent a clinically distinct subgroup remains to be determined; relapse during treatment reduction appeared to be more common in the SLA/LP group.

Autoimmunity to the p53 protein is a feature of systemic lupus erythematosus (SLE) related to anti-DNA antibodies.

The induction of anti-DNA autoantibodies in systemic lupus erythematosus (SLE) patients is problematic because mammalian DNA is poorly immunogenic at best. Here we demonstrate a chain of connected antibodies in SLE patient sera that could account for the induction of anti-DNA antibody, and possibly for some of the pathogenic features of SLE. We now report that SLE patients, in addition to anti-DNA, produce antibodies to the carboxy-terminal domain of the tumour suppressor molecule p53; this p53 domain recognizes damaged DNA. Hence, these anti-p53 antibodies could mimic damaged DNA immunologically. Indeed, SLE sera do contain anti-idiotypic antibodies to a prototypic anti-p53 antibody. Moreover, SLE anti-DNA antibodies also recognize this type of anti-p53 antibody. Indeed, binding of affinity-purified anti-DNA both to DNA and to the anti-p53 antibody could be blocked by a p53 peptide derived from the DNA-binding domain. This mimicry of the p53 DNA-binding domain by the SLE anti-DNA antibodies is functional: activation of the p53 molecule could be inhibited by such anti-DNA antibodies. Thus, anti-DNA antibodies may arise in SLE patients by a chain of idiotypic autoimmunity centered around p53 autoimmunity. The SLE anti-DNA and anti-p53 antibodies can functionally block p53 activation, and so could affect apoptosis.

[Presence of SLA/LP autoantibodies in patients with primary biliary cirrhosis as a marker for secondary autoimmune hepatitis (overlap syndrome)]. / Nachweis von SLA/LP-Autoantikörpern bei Patienten mit primär biliärer Zirrhose als Marker für eine sekundäre autoimmune Hepatitis (Overlapsyndrom).

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate whether the presence of SLA/LP-autoantibodies in PBC-patients gives evidence for a secondary AIH, also called AIH/PBC-overlap-syndrome. PATIENTS AND METHODS: Out of 233 consecutive patients with PBC who had been followed between October 1980 and April 2000, we evaluated the data of anti-SLA/LP-positive patients and compared them to patients with an anti-SLA/LP-negative AIH/PBC overlap syndrome as well as to patients with a classical course of AIH and PBC. RESULTS: In total we could identify nine PBC patients with anti-SLA/LP antibodies (six women/three men) or 3.9% of the study population, Anti-SLA/LP-positive PBC patients were slightly younger at diagnosis in comparison to anti-SLA/LP-negative PBC-patients (49.9 vs. 53.2 years). Transaminases and gamma-globulins were significantly higher in anti-SLA/LP-positive PBC-patients in comparison to anti-SLA/LP-negative PBC-patients (mean: 235 vs. 55 IU/l and 27.6 vs. 19.5 g/l). Anti-SLA/LP-positive patients significantly more frequently had an HLA-type that is characteristic for AIH (B8; DR3; DR4). Immunosuppressive therapy reduced inflammatory activity and cholestasis significantly. Relapses were frequent after reduction or discontinuation of immunosuppressive therapy. CONCLUSION: The presence of SLA/LP autoantibodies in PBC patients has a high specificity for a secondary AIH (AIH/PBC overlap syndrome). These patients have a good response to immunosuppressive therapy. The autoantibody profile and immunogenetics may help in future to identify PBC patients that benefit most from immunosuppressive therapy.

Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis.

BACKGROUND: Autoantibodies are a hallmark of autoimmune hepatitis, but most are not disease specific. Autoantibodies to soluble liver antigen (SLA) and to liver and pancreas antigen (LP) have been described as disease specific, occurring in about 30% of all patients with autoimmune hepatitis, but no standardised assays are available. Methods We tested 2000 serum samples from patients with various liver diseases and controls for SLA autoantibodies by inhibition ELISA. Serum samples positive for SLA antibodies were used for immunoscreening of cDNA expression libraries. Identified clones were tested against a panel of serum samples positive for SLA and LP autoantibodies and control serum samples, and the epitope mapped by deletion mutants and exonuclease digestion. FINDINGS: SLA and LP autoantibodies were identical. Of 2000 serum samples screened, 35 were positive for SLA autoantibodies. These positive samples came from patients with autoimmune hepatitis; three from patients with an overlap syndrome (primary biliary cirrhosis and secondary autoimmune hepatitis). Expression cloning and absorption experiments identified a 422 aminoacid protein present in two splice variants as the sole target antigen. Aminoacids 371-409 were critical for immune recognition. INTERPRETATION: The identified cDNA encodes the primary target antigen of SLA/LP autoantibodies. The SLA/LP antigen has a previously unknown aminoacid sequence, and presumably codes for an unindentified enzyme, suggested to be UGA-suppressor tRNA-associated protein. SLA/LP autoantibodies are disease specific and recognise a dominant epitope, suggesting a specific antigen-driven immune response. Identification of the SLA/LP target antigen will allow establishment of a reliable, widely available diagnostic assay. Furthermore, its role in the pathogenesis of autoimmune hepatitis can now be studied.

Systemic lupus erythematosus in mice, spontaneous and induced, is associated with autoimmunity to the C-terminal domain of p53 that recognizes damaged DNA.

The tumor suppressor molecule p53 features a regulatory domain at the C terminus that recognizes damaged DNA. Since damaged DNA might be involved in activating anti-DNA autoantibodies, we tested whether autoimmunity to the C terminus of p53 might mark murine systemic lupus erythematosus (SLE). We now report that MRL / MpJ-Fas(lpr) mice, which spontaneously develop SLE, produce antibodies both to the C terminus of p53 and to a monoclonal antibody (PAb-421) that binds the p53 C terminus. Anti-idiotypic antibodies to PAb-421 (sampled as monoclonal antibodies) could also bind DNA. Thus, the PAb-421 antibody mimics DNA, and the anti-idiotypic antibody to PAb-421 mimics the p53 DNA-binding site. This mimicry was functional; immunization of BALB / c mice to PAb-421 induced anti-DNA antibodies and antibodies to the C terminus of p53, and most of the mice developed an SLE-like disease. Immunization of C57BL / 6 mice to PAb-421 induced antibodies to p53, but not to its C-terminal domain. The C57BL / 6 mice also did not develop anti-DNA antibodies or the SLE-like disease. Thus, network autoimmunity to the domain of p53 that recognizes damaged DNA can be a pathogenic feature in SLE in genetically susceptible strains of mice.

Immunity to p53 induced by an idiotypic network of anti-p53 antibodies: generation of sequence-specific anti-DNA antibodies and protection from tumor metastasis.

The general overexpression of p53 by different types of tumor cells suggests that p53 immunity might be generally useful for tumor immunotherapy. We describe here the induction of immunity to p53 and resistance to tumor metastasis using an idiotypic network. Mice were immunized with domain-specific anti-p53 monoclonal antibodies (Ab1): PAb-248 directed to the N-terminus; PAb-246 directed to the specific DNA-binding region; or PAb-240 directed to a mutant p53 that does not bind specific DNA. Immunized mice responded by making anti-idiotypic antibodies (Ab2) specific for the Ab1 inducer. Ab1 PAb-246 induced Ab2 that, like p53 itself, could bind the specific DNA oligonucleotide sequence of the p53 responsive element. Mice immunized with Ab1 PAb-240 or PAb-246 spontaneously made Ab3 anti-p53 antibodies that reflected the specificity of their Ab1 inducers: Ab1 PAb-246 induced Ab3 specific for wild-type p53; PAb-240 induced Ab3 specific for mutant p53. Ab1 PAb-248 induced only Ab2. The spontaneously arising Ab3 were of T cell-dependent IgG isotypes. Peptides from the complementarity determining regions of the Ab1 antibodies PAb-240 and PAb-246 could also induce Ab3 anti-p53. Finally, mice that produced Ab3 anti-p53 acquired resistance to tumor metastases. Therefore, an anti-idiotypic network built around certain domains of p53 seems to be programmed within the immune system, specific Ab2 antibodies can mimic the DNA binding domain of p53, and Ab3 network immunity to p53 can be associated with resistance to tumor cells.

Idiotypic immunization induces immunity to mutated p53 and tumor rejection.

The p53 molecule might serve as a common tumor-associated antigen, as the tumor suppressor gene p53 is mutated and the p53 protein is often over-expressed in tumor cells. We report that effective immunity to p53 can be induced through an idiotypic network by immunization of mice with a monoclonal antibody (PAb-240) specific for mutated p53, or with a peptide derived from the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) of this antibody. The immunized mice produced IgG antibodies to p53 and mounted a cytotoxic reaction to a tumor line bearing mutated p53. The idiotypically immunized mice were resistant to challenge with the tumor cells. Thus antibodies to p53 might serve as immunogens for activating resistance to some tumors. At the basic level, these findings indicate that a network of p53 immunity may be organized naturally within the immune system.

Humoral mechanisms in T cell vaccination: induction and functional characterization of anti-lymphocytic autoantibodies.

T cell vaccination, the application of syngeneic attenuated T cells, has been shown to prevent effectively and treat experimental autoimmune diseases, but its mechanisms of action are poorly understood. Here we present data on the induction of a humoral anti-T cell response by T cell vaccination, capable of strongly inhibiting T cell proliferation and of ameliorating experimental autoimmune disease. T cell vaccination in the Lewis rat induced autoantibodies reactive with several syngeneic T cell proteins. These autoantibodies were not detectable in normal Lewis sera as assessed by immunoblotting and flow cytometry with intact syngeneic T cells. The autoantibody reactivity was not restricted to one idiotype, was detected as early as 1 week after vaccination and was dominated by IgG, suggesting the boosting of a naturally preformed humoral network by T cell vaccination. Recovery from passively or actively induced experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, too, could be shown to be associated with the development of anti-T cell autoantibodies. In vitro, both the post-EAE and the post-vaccination sera had a strong suppressive effect on the proliferation of syngeneic T cell clones. This inhibition was shown to be mediated by antibodies and to be partly complement-dependent. In vivo, both kinds of sera were able to ameliorate EAE. This protective effect of the post-vaccination sera was not idiotype-specific, since sera obtained after T cell vaccination with an unrelated T cell clone were similarly effective in suppressing EAE. These results suggest that anti-lymphocytic antibodies might play an immunoregulatory role that can be positively manipulated by T cell vaccination.

Estimation of the frequency of self-reactive T cells in health and inflammatory diseases by limiting dilution analysis and single cell cloning.

Autoreactive T cells have recently been detected not only in autoimmune diseases but also in healthy individuals, but their frequency is thought to be low. The aim of our study was to estimate the frequency of self-reactive T cells by using limiting dilution analyses of peripheral blood lymphocytes. Assessment of self-reactivity in this study was defined as T-cell proliferation to autologous non-T cells in the absence of foreign antigens. When culture conditions were optimized by adding interleukin 2, healthy individuals showed a frequency of self-reactive T cells ranging from 1/60 to 1/600. These results were confirmed by using unseparated peripheral blood leukocytes or Epstein-Barr virus transformed B-cell blasts as stimulators. All cultures were performed exclusively in autologous serum. Single cell cloning from a healthy donor yielded 568 T-cell clones, 12 of which showed self-reactivity giving a frequency of more than 1 in 50 T cells. Eight of these 12 T-cell clones were inhibited by MHC-class II antibodies. Frequency analyses of self-reactive T cells in patients with autoimmune diseases (rheumatoid arthritis, autoimmune hepatitis or primary biliary cirrhosis), with viral hepatitis or with inflammatory bowel diseases showed similar frequencies in all patient groups and no significant differences from normal individuals. In conclusion, we have found a high frequency of self-reactive T cells in both health and disease. We postulate that self-reactive T cells constitute an important part of the physiological T-cell repertoire.

Lack of requirement for CD8+ cells in recovery from and resistance to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a model of T-cell mediated autoimmune disease. Active disease is mediated by myelin basic protein specific CD4+ T-cells, whose adoptive transfer can also induce passive disease. In the Lewis rat EAE is a transient disease inducing lasting resistance to rechallenge. The mechanisms of recovery and resistance are poorly understood. CD8+ suppressor T-cells have mostly been thought to be central, especially in resistance to reinduction of the disease. In this study we showed by complete depletion of CD8+ cells that this subset does not influence either recovery or resistance to EAE in the Lewis rat. This was further confirmed by depleting CD8+ cells only after recovery from acute EAE. Such depletion did not diminish the effective resistance to rechallenge. Recovery from and resistance to EAE appear not to require the presence of CD8+ cells.

Expression of the 60 kDa heat shock protein in normal and inflamed liver.

The 60 kDa heat shock proteins (HSP 60) have been well conserved throughout evolution and are highly immunogenic. Cross-reactivity between bacterial and mammalian HSP 60 is considered a likely mechanism in the pathogenesis of autoimmune diseases. T cell and B cell reactivity to HSP 60 is found in patients with rheumatoid or juvenile arthritis, and the expression of HSP 60 in the inflamed joint is found to be increased. In this study the presence of HSP 60 was demonstrated in normal and inflamed lives. HSP 60 was found to be predominantly expressed in hepatocytes and Kupffer cells, and mainly localized in mitochondria. Heat stress in the form of a 1 h incubation at 42 degrees C increased HSP 60 expression. The expression of HSP 60 in chronic active hepatitis was found to be markedly increased, with predominant expression in areas of inflammatory infiltrates. This increased expression in the inflamed liver was found both in viral and autoimmune hepatitis. High expression of HSP 60 in chronic active hepatitis was entirely due to self (i.e. human) HSP 60; no additional bacterial HSP 60 could be detected. Increased expression of HSP 60 in chronic active hepatitis suggests that immune reactions to HSP 60 may play a role in the immunopathogenesis and perpetuation of chronic inflammatory liver disease.

Induction of the anti-ergotypic response.

The injection of syngeneic activated T cells into rodents can induce a T cell response against activation markers of the T cells, ergotopes. The responding anti-ergotypic T cells have been shown to suppress experimental autoimmune encephalomyelitis (EAE). This paper reports the characteristics of the anti-ergotypic response. It was found that irradiated activated T cells were as good as untreated living activated T cells in inducing anti-ergotypic cells in vivo. Glutardialdehyde-fixed (0.3%) cells were poor stimulators in vivo and non-stimulatory in vitro. Dilution of glutardialdehyde to 0.003% before fixation preserved the stimulatory capacity in vitro. Fixation or irradiation of T cells at different times after activation showed that the stimulatory ergotope appears only after more than 12 h of activation. This ergotope is not secreted by activated T cells, but is a structural component of the activated T cell. Injection of solubilized proteins from activated T cells, but not of supernatants from activated T cells, was able to induce an anti-ergotypic response in vivo. In vitro supernatants from activated T cells also were not stimulatory to anti-ergotypic T cells. The anti-ergotypic response could be measured in draining lymph nodes 3 days after injection, reached a maximum after 7-10 days and subsided thereafter. It was earlier and stronger than the anti-idiotypic response. Induction of the response was dose dependent. As few as 100 cells were able to induce a marked anti-ergotypic response. The ease of the induction and the strength of the anti-ergotypic response suggest a physiological role in immunoregulation.