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To determine the clinical, biochemical, and histological features, and outcome of childhood autoimmune hepatitis (AIH), we reviewed the medical records of 52 children with AIH, 32 (median age: 10 [2-15] years) anti-nuclear and/or smooth muscle antibody (ANA/SMA) positive, 20 (7 [0.8-14] years) liver/kidney microsomal antibody (LKM-1) positive, with median follow-up of 5 years (range 0.3-19). At presentation: 56% had symptoms of prolonged acute hepatitis; LKM-1 positive were younger (P = .011), with higher bilirubin (P = .007), and AST (P = .047); ANA/SMA positive had lower albumin (P = .023); 69% ANA/SMA positive, and 38% LKM-1 positive were cirrhotic (P = .080). ANA/SMA positive had increased frequency of HLA haplotype A1/B8/DR3/DR52a compared with controls (53% vs. 14%, P < .001). Of six (5 LKM-1 positive) with fulminant hepatitis, four were transplanted, one died, and one ANA/SMA positive improved with immunosuppression. Of 47 treated with immunosuppression, 2 (1 LKM-1 positive) died with no remission and 4 (2 LKM-1 positive) were transplanted 8 to 14 years after diagnosis. Immunosuppression was stopped successfully in 19% of ANA/SMA positive after a median of 3 years of treatment, but in none of LKM-1 positive. Baseline bilirubin and international normalized prothrombin ratio (INR) were independent variables predictive of outcome. In conclusion, ANA/SMA positive and LKM-1 positive AIH in childhood have clinical, biochemical, and histological differences, but similar severity and long-term outcome.

Susceptibility to autoimmune hepatitis in white patients is associated with the human leukocyte antigen class II antigens DR3 and DR4. To analyze the molecular basis of these associations, we used oligonucleotide probes to determine the DRB, DQA and DQB hypervariable nucleotide sequences in 119 patients with autoimmune hepatitis and 177 matched controls. DRB3*0101, which encodes DR52a, predisposed patients most strongly to the disease. It was present in 58% of patients and 25% of controls (corrected P < 0.000005), whereas DQA1*0101 and 0102 conferred protection in males only. The DR4 subtype, DRB1*0401, was raised in the DRB3*0101-negative patients; 81% possessed either DRB3*0101 or DRB1*0401, compared with 42% of controls (corrected P < 0.0000001). These alleles encode the amino acid sequence Leu-Leu-Glu-Gln-Lys-Arg at positions 67 to 72 of the DR beta polypeptide, which was present in 94% of patients and 64% of controls (corrected P < 0.000001) and in all patients who tested positive for autoantibodies to the hepatic asialoglycoprotein receptor. The patients with DRB1*0401 had less severe disease, relapsed less frequently and were first seen significantly later in life than those patients with DRB3*0101; and whereas a single copy of DRB1*0401 predisposed to autoimmune hepatitis, DRB3*0101-associated susceptibility had a dose-related effect. These data provide evidence that specific residues in the DR beta polypeptides predispose to autoimmune hepatitis in white patients and genes linked to DRB3*0101 and DRB1*0401 may determine two clinically distinct disease patterns.

The portal tract mononuclear cell infiltrate has been characterised in 28 liver biopsy samples showing features of chronic aggressive hepatitis from 12 patients with autoimmune chronic active hepatitis, 12 with primary sclerosing cholangitis, and four with other chronic liver diseases (two with alpha 1-antitrypsin deficiency, one with Wilson's disease, and one with chronic hepatitis B infection). In all patients liver disease had started in childhood. The mononuclear cell infiltrate was investigated by a two step immunoperoxidase technique using monoclonal antibodies to: total, alpha/beta T cell receptor positive, helper/inducer, suppressor/cytotoxic T lymphocytes; B lymphocytes; killer/natural killer cells; monocyte/macrophages; and to the activation markers HLA-DR antigens, interleukin 2 receptor (IL-2R), transferrin receptor, and 4F2Ag. In all samples the infiltrate consisted of mainly alpha/beta T cell receptor T lymphocytes. Although T helper/inducer cells predominated in patients with autoimmune chronic active hepatitis, T suppressor/cytotoxic lymphocytes were preponderant in patients with primary sclerosing cholangitis and the other chronic liver diseases. Killer/natural killer cells accounted for up to 25% of the mononuclear cell infiltrate in patients with autoimmune chronic active hepatitis, being rare or absent in the other diseases. Monocytes/macrophages were always found, but they were more numerous in primary sclerosing cholangitis than in the other chronic liver diseases. B lymphocytes were rare or absent in all subjects. Activated mononuclear cells were present in all subjects, but although in patients with autoimmune chronic active hepatitis and primary sclerosing cholangitis most cells of the infiltrate expressed HLA-DR antigens and up to 75% IL-2R, in other forms of chronic liver diseases HLA-DR positive cells were less common and IL-2R positive cells ere rare or absent. These results show that the cells responsible for the histological characteristics of chronic aggressive hepatitis vary in their functional phenotype and state of activation according to the type of underlying liver disorder, confirming the involvement of different pathogenetic mechanisms.

To investigate the function of activated T lymphocytes in autoimmune chronic active hepatitis, 7 of 15 T-cell clones from the peripheral blood of 8 patients were studied. These clones showed specificity for liver-membrane antigen with proliferation when stimulated by rabbit liver cell membranes. 6 of these clones reacted with liver-specific lipoprotein complex, and 1 clone (and 3 subclones) responded to the asialoglycoprotein receptor (ASGPR), both known targets of immune attack in autoimmune chronic active hepatitis. 2 of these clones stimulated autologous B lymphocytes to produce liver-membrane-specific autoantibodies and antibody to the ASGPR. These results suggest that liver-membrane-specific activated T lymphocytes in peripheral blood may be important in the autoimmune attack of chronic active hepatitis.

Children with uncontrolled autoimmune chronic active hepatitis have increased numbers of activated T lymphocytes expressing interleukin 2 receptors (IL2R). A soluble form of IL2R has recently been described whose proposed role is to downregulate T cell activation by competing for interleukin 2. We investigated whether a deficiency of soluble IL2R could account for the high concentrations of IL2R positive T lymphocytes in autoimmune chronic active hepatitis. Soluble IL2R was measured by enzyme-linked immunosorbent assay in the serum of 16 children with autoimmune chronic active hepatitis, eight with chronic liver disease due to hepatitis B virus infection, seven with Wilson's disease, nine with alpha 1 antitrypsin deficiency, and 15 healthy age matched controls. Soluble IL2R concentration was significantly higher in patients with autoimmune chronic active hepatitis than in healthy controls (mean (SEM) 475 (75) U/ml, 145 (8) U/ml respectively, p less than 0.01). Eleven patients who had active disease had significantly higher soluble IL2R concentrations (590 (89) U/ml) than the five cases with inactive disease (220 (36) U/ml, p less than 0.01). No difference was found between the controls and the patients with chronic liver disease due to hepatitis B infection, Wilson's disease, and alpha 1 antitrypsin deficiency. Percentages and absolute numbers of surface IL2R positive T cells as detected by immunofluorescence were significantly higher in the patients with autoimmune chronic active hepatitis (11.8% (1); 274/microliters (31)) than in controls (0.2% (0.1); 5/microliters (2), p less than 0.001), the highest values being found in those with uncontrolled disease. A significantly positive correlation was observed between concentrations of soluble IL2R and the percentage of T cells expressing IL2 receptors (r=0.67, p<0.001). These results indicate that the high levels of IL2R positive T lymphocytes characteristic of autoimmune chronic active hepatitis are not due to a deficiency of soluble IL2 receptors.

To investigate mechanisms of hepatocyte injury, lymphocytes from 41 children with chronic liver disease were incubated with autologous liver cells in a microcytotoxicity assay. Cytotoxicity was significantly increased in 18 of 25 patients with chronic hepatitis B virus (HBV) infection, in five of nine with "autoimmune" chronic active hepatitis (CAH), and in only one of seven with histologically inactive liver disorders. There was a good correlation between cytotoxicity and biochemical and histologic markers of disease activity in children with autoimmune CAH, whereas in HBsAg-positive disease a positive correlation was found only with serum alanine aminotransferase (SGPT). Children with autoimmune CAH receiving steroid treatment had normal cytotoxicity, whereas increased values were found in two of three HBsAg-positive patients receiving prednisolone. Fractionation studies revealed that non-T cells were cytotoxic in both autoimmune and HBcAg-positive chronic liver disease. T cell cytotoxicity was exclusively found in children with chronic HBV infection, particularly with HBc antigenemia. Blocking experiments showed that T-lymphocytes from HBsAg-positive children reacted with HBV core antigen on the hepatocyte surface. Non-T cells were directed against hepatocyte membrane antigens in both HBsAg-positive and HbsAg-negative children. These results suggest that different immune mechanisms of liver damage are involved in autoimmune and HBsAg-positive chronic liver disease.

An indirect immunofluorescence technique was used to investigate the immunoglobulin class, IgG subclass, light chain type and complement fixing ability of anti-liver kidney microsomal antibody (anti-LKM) in the sera of six children and five adults with autoimmune chronic active hepatitis (aCAH). Anti-LKM titres ranged from 1/80 to 1/20,480. In the children, the antibody belonged to the IgG1 subclass alone (titre 1/80-1/20,480) and was able to fix complement (titre 1/40-1/5120). In the adult group, antibody belonged to the IgG1 subclass in three cases (titre 1/40-1/640) whilst two belonged to both IgG1 (titre 1/640) and IgG4 (titre 1/40, 1/640). Such subclass restriction is similar to that found in other autoimmune disorders and may be genetically determined. Investigation of the light chain constituent of anti-LKM revealed that the kappa to lambda ratio was consistent with a polyclonal antibody response. To investigate the nature of the antigen to which anti-LKM is directed, the ability of these sera to bind to the surface membrane of isolated human hepatoma cells (Alexander cells) was investigated. Four of the eleven sera showed significant binding activity. Prior incubation of these four sera with Alexander cells abolished their membrane binding activity, but did not alter the anti-LKM titre. These results suggest that anti-LKM binds to cytoplasmic constituents alone and does not cross-react with surface antigens.

Anti-lymphocytic antibodies (ALA) have been described in a variety of autoimmune disorders. We have investigated the presence of ALA in autoimmune chronic active hepatitis (aCAH) starting in childhood. Using a modified Terasaki technique ALA were found in 17 of 18 patients with aCAH but in only one of 15 patients with alpha-1-anti-trypsin deficiency or Wilson's disease and three of 27 age-matched healthy controls (P less than 0.0005 for both). Sera from 12 patients with uncontrolled aCAH had significantly higher cytotoxicity values than sera from six children with inactive diseases (P less than 0.01). ALA were directed to T but not B lymphocytes and were not reactive with specific HLA antigens. No preferential killing was observed against CD4 or CD8 positive T lymphocytes. Characterization of ALA revealed them to be cold-reactive IgM. The possible role of ALA in aCAH is discussed.

Children with primary sclerosing cholangitis or autoimmune chronic active hepatitis have similar high levels of immunoglobulin G and non-organ-specific autoantibodies and may have similar histological features. To investigate a possible immunopathogenesis of primary sclerosing cholangitis, we have studied a series of regulatory and/or effector immune mechanisms in eight children with primary sclerosing cholangitis, comparing them to 14 children with autoimmune chronic active hepatitis and 24 healthy children as controls. Antibodies to a liver membrane protein preparation were found in all children with autoimmune chronic active hepatitis tested and in seven of eight with primary sclerosing cholangitis, whereas antibodies against the hepatic asialoglycoprotein receptor were present in three of six patients with autoimmune chronic active hepatitis and in two of the eight with primary sclerosing cholangitis. Lymphocyte cytotoxicity values to autologous hepatocytes were similarly elevated in primary sclerosing cholangitis (median: 50%; range: 38 to 83%) and in autoimmune chronic active hepatitis (median: 52%; range 37 to 87%) compared to controls (median: 8%; range: 0 to 27%) (p less than 0.01 for both). In contrast, T suppressor cell number and function were normal in patients with primary sclerosing cholangitis (median: 23%; range: 19 to 28%; and median: 54%; range: 44 to 61%), but significantly decreased in patients with autoimmune chronic active hepatitis (median: 15%; range: 9 to 21%; and median: 9%; range: -40 to +21%) when compared to controls (median: 24%; range: 16 to 29%; and median: 53%; range: 8 to 77%) (p less than 0.01 for both).(ABSTRACT TRUNCATED AT 250 WORDS)

Of 26 patients with autoimmune chronic active hepatitis (CAH) starting in childhood 18 (69%) had low C4 and 5 (19%) had low C3 serum levels. Impaired hepatic synthesis and immune-consumption were unlikely since transferrin levels were normal in all patients, albumin levels were persistently low in only 3, and only 3 had raised levels of activation fragment C3d. C4d was normal in all patients studied. In the families of 12 probands with low C4, 7 parents had low C4 and 2 had levels which were at the lower limit of normal. 5 of 10 siblings from 5 families had low C4. These results suggest that low C4 levels in CAH are genetically determined. C4 phenotyping in 20 patients and in 26 parents showed that 90% and 81%, respectively, had null allotypes at either the C4A or C4B locus compared with 59% in controls, indicating that defective expression of structural genes may contribute to the observed C4 deficiency.

We found a significant increase of activated circulating T lymphocytes expressing interleukin 2 receptor (IL-2r) (mean +/- s.e.m. 11.0 +/- 1.1%) or DR antigen (5.0 +/- 0.49%) in patients with autoimmune chronic active hepatitis (CAH) starting in childhood when compared to healthy controls (0.14 +/- 0.09%, P less than 0.001 and 2.8 +/- 0.06%, P less than 0.01). Patients with liver disorders due to Wilson's disease (IL-2r 0.64 +/- 0.25%, DR 3.5 +/- 0.22%) or alpha-1-antitrypsin deficiency (IL-2r 0.1 +/- 0.06%, DR 2.8 +/- 0.35%) had levels similar to controls. Levels of both IL-2r and DR positive T lymphocytes were higher in patients with uncontrolled CAH (IL-2r 18.0 +/- 1.01%; DR 6.3 +/- 0.78%) than in patients with inactive disease (IL-2r 3.2 +/- 1.4%, P less than 0.001; DR 3.0 +/- 0.13%, P less than 0.01). In patients with active disease levels of IL-2r positive cells were higher than DR positive cells (P less than 0.001). Only 21% of activated T cells coexpressed the two markers of activation. Sixty-seven percent of IL-2r positive T lymphocytes were helper/inducer and 25% suppressor/cytotoxic, while 66% of the DR positive T cells were suppressor/cytotoxic and 31% helper/inducer. The finding that the highest levels of activated T lymphocytes are present in patients with uncontrolled CAH suggests that these cells are involved in its pathogenesis. The preferential increase of activated helper/inducer cells might explain the enhanced immune reactivity characteristic of autoimmune CAH.

Autoimmune chronic active hepatitis (aCAH) and primary sclerosing cholangitis (PSC) are liver disorders occurring in childhood in which non-organ specific autoantibodies, such as anti-nuclear antibody (ANA) are frequently found. Antibodies to double stranded DNA (dsDNA), which are typically present in systemic lupus erythematosus (SLE), have been detected in both acute and chronic liver diseases in adults. In this study, using a radioimmunoassay technique widely employed to measure antibodies to dsDNA, we have demonstrated significantly increased levels (median and range; 11.9, 1.0-36.5 U/ml) in 21 children with aCAH compared with normal children (1.0, 0.7-2.1 U/ml; p less than 0.01). Five children with aCAH had levels in the range considered diagnostic for SLE (greater than 25 U/ml) and of these, three had ANA and two had anti-liver kidney microsomal antibody. In addition, one child had antibodies to dsDNA as detected by the Crithidia luciliae test. DNA binding in aCAH was correlated with serum aspartate-amino transferase levels (r = 0.68; p less than 0.001), suggesting a direct relationship with disease activity. In PSC, levels of antibodies to dsDNA were significantly increased compared to normal controls (median and range; 7.0, 5.6-10.2 U/ml; p less than 0.01) but were not as high as those found in aCAH.

In autoimmune chronic active hepatitis (aCAH), autoaggression is believed to derive from a defect in immunoregulation. Antigen non-specific Concanavalin A (Con A) induced suppressor cell function has been reported to be impaired. In 11 children with aCAH we have investigated inhibition of production of a specific antibody (anti-tetanus toxoid, anti-TT) by suppressor cells induced either by a non-specific stimulus (Con A) or by the specific antigen (tetanus toxoid, TT). Con A induced suppression of anti-TT was significantly lower in patients (15.7 +/- 2.5%) than in controls (46.7 +/- 4.4%; P less than 0.01). In contrast, high dose tetanus toxoid induced suppression was similar in patients and controls (69.8 +/- 4.2, 72.0 +/- 3.6%, respectively). Both groups had similar serum anti-TT levels and in vitro production of anti-TT in response to optimal dose of TT. Our data indicate that antibody production to a T cell-dependent antigen is under the control of at least two regulatory mechanisms, one antigen specific and one antigen non-specific, only the latter being defective in aCAH.

Con A stimulated suppressor cell function and the proportion of suppressor T cells were reduced in children with untreated chronic active hepatitis (CAH) but were normal in corticosteroid treated CAH patients, patients with severe acute hepatitis and inactive chronic liver disease. Adults with CAH also have defective suppressor function hut a normal proportion of T suppressor cells. This difference may account for the observation that relapse after treatment withdrawal is less frequent in children than in adults. [ABSTRACT FROM AUTHOR]

In a micrototoxicity assay, lymphocytes from ten out of sixteen patients with HBsAg-negative chronic active hepatitis have been shown to be cytotoxic to autologous hepatocytes isolated from percutaneous liver biopsies. This cytotoxicity was demonstrable in all six untreated patients but in only four out of ten receiving immunosuppressive treatment, the presence of cytotoxicity showing a significant association with the activity of the disease assessed histologically. The addition of excess purified lipoprotein (LSP), derived from the hepatocyte plasma membrane, blocked the reaction in all cytotoxic cases, indicating that LSP was the major target antigen. Enriched fractions of T cells were cytotoxic in only one case, whereas non-T cell fractions were cytotoxic in the other ten cases investigated in this way. For optimum T cell cytotoxicity, effector and target cells must share histocompatibility determinants and the results of this study using an autologous system show conclusively that the lymphocyte cytotoxicity found in HBsAg-negative chronic active hepatitis is mediated by a non-T cell population. [ABSTRACT FROM AUTHOR]