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A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of 125I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

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.

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

Liver disease during chemotherapy and after its completion was studied in 103 leukemic children in long-term remission. Seventy developed chronic liver disease during therapy; 22 out of 56 with adequate follow-up showed persisting abnormality or deterioration of liver function after stopping therapy. In 38 studied prospectively, biopsies were obtained at treatment withdrawal. Five showed chronic lobular, 17 chronic persistent, 9 chronic active hepatitis whereas 7 had minimal changes. These children had transiently detectable serum hepatitis-B virus (HBV) markers during (44.4%), at completion of (7.8%) and subsequent to (48.3%) chemotherapy. Serum HBV markers correlated significantly with both severity of histologic changes (P less than 0.05) and persistent biochemical abnormalities for over 6 months after treatment suspension (P less than 0.001). No direct relationship was found between drug administration and liver damage. The data from the study suggest that in leukemic children viral infections contribute to chronic liver damage, which can jeopardize the long-term prognosis of acute leukemia.

Using a commercial source of peroxidase-labelled anti-C3d antibody (Dakopatts), an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify the complement fragment C3d. The technique enables the detection of C3d in plasma, urine and cerebrospinal fluid (CSF). The C3d-ELISA therefore provides a very sensitive technique for the evaluation of complement activation in biological fluids. In both plasma and urine the technique is able to discriminate between samples from normal controls and patients with rheumatoid arthritis in whom complement activation is known to occur. A good correlation was found between results obtained by ELISA and those by laser nephelometry (r = 0.91, P less than 0.0001). Microtitre plates pre-coated with anti-C3d antibody and subsequently stored at -70 degrees C retained the ability to perform in this assay. The sensitivity, short assay time and use of commercial reagents and pre-coated plates give this technique numerous potential applications in the evaluation of complement activation.

To evaluate factors influencing the alteration in subsets of T-lymphocytes, we studied 24 pairs of identical twins discordant for insulin-dependent (type I) diabetes mellitus. Subsets were assessed by monoclonal antibodies and a pure preparation of peripheral blood mononuclear cells obtained by centrifugation of heparinized whole blood with a Ficoll/Triosil gradient. In 12 pairs studied within 5 yr of diagnosis, we observed a reduction in the percentage of cells reacting with OKT8 (recognizing the CD8 antigen present on the suppressor/cytotoxic subset) (P < .05), but a similar level was detected in their nondiabetic cotwins. In 12 pairs studied >5 yr after the diagnosis and in whom the nondiabetic twin is less likely to develop diabetes, the percentage of cells reacting with OKT8 was reduced in both the diabetic (P < .05) and the nondiabetic (P < .01) twins. Reductions were also seen with OKT3 (recognizing the CD3 antigen present on the total T-lymphocyte population) and OKT4 (recognizing the CD4 antigen present on the helper/inducer subset), but only in the diabetic twins from the group with longer discordance. We conclude that a reduced percentage of suppressor/cytotoxic cells is associated with type I diabetes, but the reduction appears to be genetically determined. Total T-lymphocytes are also reduced but mainly in the helper/inducer subset and only in diabetic patients of long duration. Such a reduction cannot therefore be primarily genetically determined.

Circulating immune complexes were found in 15 of 16 patients with fulminant hepatic failure due to viral hepatitis and all of six patients who had had halothane anaesthesia; however, they were found in only five of 32 patients with paracetamol-induced hepatic necrosis. The levels of circulating immune complexes were not related to the severity of the clinical course, development of renal failure, final outcome, or severity of hypocomplementaemia. All the patients had depressed reticuloendothelial function as assessed by the clearance of 125I microaggregated albumin. These findings show that circulating immune complexes in fulminant hepatic failure are not simply a reflection of an immune response to liver antigens released as a result of the hepatic necrosis; nor are they a reflection of the failure of the reticuloendothelial system. This supports the view that circulating immune complexes are associated with immune mediated liver injury and may contribute to the process.

The presence of antibodies to glycosylated albumin was studied by means of a newly developed sandwich enzyme-linked immunosorbent assay in 29 long-standing Type 1 (insulin-dependent) diabetic patients with microvascular complications and in 20 normal subjects. Two types of antibody reactivity were detected. One directed against glucitol-albumin expressing G and M isotypes. The second, predominantly belonging to the IgG class, reacted with an epitope shared by non-glycosylated albumin and the ketoamine adduct of albumin glycosylation. Both types of antibodies, with affinity constant ranging from 10(4) to 10(7) (mol/l)-1 were found in normal and diabetic subjects, but higher titres were significantly more prevalent in the diabetic patients. These antibodies may represent the result of immune tolerance breakdown or, alternatively, be natural antibodies. Although their function remains to be established, their raised prevalence in Type 1 diabetes may be relevant to diabetic microvascular disease.

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.

Expression of HLA DR antigens on T lymphocytes indicates that these cells are actively involved in an immune response. Raised levels of activated T lymphocytes were found in 14 of 15 recently diagnosed but in only 7 of 28 long-standing insulin-dependent diabetics. 9 of the recently diagnosed patients retested 6 months later still had high levels of activated T lymphocytes. Even long-standing insulin-dependent diabetics had significantly higher levels of activated T lymphocytes than non-insulin-dependent diabetics and healthy controls. 5 of 7 unaffected co-twins of recently diagnosed insulin-dependent diabetics had high levels of activated T cells; this increase persisted in 2 retested 6 months later, when mildly impaired glucose tolerance had developed. These results confirm that there is an active cellular immune reaction in newly diagnosed insulin-dependent diabetics which may precede the disease and persist for at least 6 months after its appearance.

A new nephelometric technique to measure C3d as an indicator of complement activation, is described. C3d is isolated at high concentration of polyethyleneglycol (PEG), incubated with commercially available anti-C3d antiserum at a final concentration of 2.5% PEG and then measured in a Behring Laser Nephelometer. In contrast to previously available techniques our assay detects the low concentrations of C3d present in all normal subjects, which result from the continuous C3 catabolism occurring in vivo. We have also measured C3d blood concentrations in a large number of patients with diseases associated with complement activation. Raised C3d concentrations were found in 68% of rheumatoid arthritis, 57% of primary biliary cirrhosis, 38% of chronic active hepatitis, 100% of Gram-negative bacteraemia and 100% of malaria. The nephelometric technique has proved to be sensitive, economical and fast, and could be adapted for routine determination of C3d blood concentrations to monitor disease activity and response to treatment.

Opsonisation of heat-killed bakers yeast and C3 and C4 concentrations were determined in sera from 69 children with liver disease and 39 controls as simple screening procedures for abnormalities in the complement system. Where significant defects in these moieties were encountered, the activity of the following was measured: total haemolytic complement, C4, C5, total alternative pathway, factor B and D activities were measured. Complement activation was detected by the presence of C3d in EDTA plasma samples. Yeast opsonisation, C3 and C4 concentrations and activities of all complement components measured were significantly (P less than 0.001) reduced in all of 10 children with fulminant hepatic failure (FHF) and six with chronic active hepatitis (CAH). There was no consistent relationship between complement deficiency and standard tests of liver function. C3 breakdown products were identified in plasma from five patients with CAH and complement deficiency but not in three patients with complement deficiencies associated with FHF. Complement concentration and activity improved in children recovering from FHF and in CAH treated by immunosuppressants. Yeast opsonisation index was raised significantly without parallel increase in C3 or C4 concentration in eight of nine children with biliary atresia before surgery and 11 of 13 with alpha-1-antitrypsin deficiency regardless of age or the presence of active liver disease. Yeast opsonisation indices became normal after successful surgery in patients with biliary atresia.

Circulating antibodies reacting specifically with the cell membrane of hepatocytes isolated from halothane-anesthetized rabbits were detected in nine of 11 patients with fulminant hepatic failure after helothane-induced anesthesia. The immunoglobulin deposition, as revealed by immunofluorescence, showed a granular pattern on the hepatocyte surface membrane. Preincubation of halothane-pretreated, but not of control, hepatocytes with serum containing this antibody rendered them susceptible to cytotoxic effects of normal lymphocytes in vitro. Control studies using serum from subjects repeatedly exposed to halothane without the development of liver damage, and from patients with viral and toxic liver injury have confirmed the specificity of these findings to serve halothane-associated liver injury. These results provide further evidence of an immunologic component in this condition.

Lettre a l'editeur: confirmation de concentrations basses de C3 et C4 (doses par nephelometrie) dans le diabete de type I au debut, dues a une synthese defectueuse et pas a une augmentation de la consommation

Recent evidence, including the presence of circulating immune complexes, suggests that abnormalities of humoral immunity may be important in the pathogenesis of primary sclerosing cholangitis. The aim of the present study was to determine whether activation of the complement system is present in patients with this disease, as this would be supportive evidence of a role for circulating immune complexes in primary sclerosing cholangitis. Plasma complement fragments C3d and C4d, and serum C3 and C4, their respective parent molecules, have been assayed. Both C3d and C4d were elevated in patients with primary sclerosing cholangitis compared with patients with extrahepatic obstructive cholestasis and normal controls (p less than 0.01 in all instances). C3 was elevated in both patient groups, in whom it was similar, compared with normal controls (p less than 0.001 in both cases), whereas C4 was similar in all groups. Elevated levels of circulating immune complexes were identified in 21 of 24 (88%) patients with primary sclerosing cholangitis, but in none of the normal controls. These findings support the hypothesis that in primary sclerosing cholangitis circulating immune complexes are associated with activation of complement via the classical pathway.

To investigate the possibility that low complement concentrations in the plasma and ascites of patients with severe liver disease could be secondary to complement consumption, complement activation was studied in 32 patients with severe liver disease, 11 of whom had spontaneous bacterial peritonitis (SBP). In patients with SBP, plasma C3 and C4 were significantly lower than in uninfected patients (mean values 0.74 v 1.13 g/l, p less than 0.01 and 0.20 v 0.28 g/l, p less than 0.05 respectively). Plasma complement activation via the classical pathway, as shown by C4d/C4, was significantly increased in patients with SBP compared with uninfected patients (37.3 v 22.2, p less than 0.01) as was C3d/C3 (14.0 v 8.11, p less than 0.01), but there was no significant difference in Ba/B between SBP and uninfected patients. Ascitic C3 concentrations were higher in patients without SBP than in infected patients (0.37 v 0.08 g/l, p less than 0.05), as were factor B values (0.11 v 0.03 g/l, p less than 0.05). There was no significant difference in ascitic C4 concentrations in patients with SBP compared with uninfected patients (0.03 v 0.07 g/l). Although consumption of C3, as shown by C3d/C3 in ascites, was increased in infected patients compared with uninfected patients (79.1 v 36.1, p less than 0.05), there was no difference in ascitic complement activation between the groups for either the classical or alternative pathways. In SBP, decreased plasma C3 and C4 are primarily caused by increased activation of the classical pathway and not impaired hepatic synthesis. Activation and consumption of C3 is one factor causing the low ascitic C3 concentrations observed in SBP.

After hepatitis B virus (HBV) infection, liver injury and viral control have been thought to result from lysis of infected hepatocytes by virus-specific cytotoxic T cells. Patients are usually studied only after developing significant liver injury, and so the viral and immune events during the incubation phase of disease have not been defined. During a single-source outbreak of HBV infection, we identified patients before the onset of symptomatic hepatitis. The dynamics of HBV replication, liver injury, and HBV-specific CD8+ and CD4+ cell responses were investigated from incubation to recovery. Although a rise in alanine transaminase (ALT) levels was present at the time of the initial fall in HBV-DNA levels, maximal reduction in virus level occurred before significant liver injury. Direct ex vivo quantification of HBV-specific CD4+ and CD8+ cells, by using human leukocyte antigen (HLA) class I tetramers and intracellular cytokine staining, showed that adaptive immune mechanisms are present during the incubation phase, at least 4 weeks before symptoms. The results suggest that the pattern of reduction in HBV replication is not directly proportional to tissue injury during acute hepatitis B in humans. Furthermore, because virus-specific immune responses and significant reductions in viral replication are seen during the incubation phase, it is likely that the immune events central to viral control occur before symptomatic disease.

Immune reactions to hepatocyte membrane antigen has been suggested to play an important role in liver damage in several conditions. Antigens on the surface of cells are recognised in association with glycoproteins coded for MHC. In this study we have investigated expression of MHC class I & II antigens on isolated hepatocytes from 24 children with liver disease: 2 autoimmune chronic active hepatitis, 4 primary sclerosing cholangitis, 5 alpha-1 antitrypsin deficiency, 3 post-liver transplant, 3 hepatitis B related chronic liver disease, 7 minor histological changes. Hepatocytes were isolated from liver biopsies by mechanical disruption. Class I(HLA-A, B & C) antigens were detected by W6/32 monoclonal antibody(Mab) and Class II(HLA-DR) antigens by L243 Mab. Class I antigens were present on isolated hepatocytes in all cases while Class II were never detected. Positive stain for Class II was seen on Kuppfer cells. Expression of HLA-DR antigens on hepatocytes could be induced by a 72hr.incubation with 200 U of recombinant gamma interferon. These data suggest that immune recognition of hepatocyte membrane antigens may not be class II restricted. Alternatively, the HLA-DR positive Kuppfer cells might act as liver antigens presenting cells.

Different factors like parenteral nutrition, infusion of fat emulsions, short gestational age, low apgar scores, late enteral feeding, and infections are discussed in the genesis of “idiopathic” neonatal cholestasis. To test the hypothesis that bacterial infections are the main cause of cholestasis in intravenously fed infants two groups were studied retrospectively. Group I: 152 newborn infants who were born from 1973 till 1981 were fed intravenously for at least 7 days and developed severe bacterial infections. Group II: 92 newborn infants who were matched to the group I cases with respect to the year of birth, birth weight and gestational age, apgar scores and duration of parenteral nutrition but did not develop infections. Bacterial infections were diagnosed on the basis of impaired microcirculation with prolonged capillary filling time, shift to the left in the white differential and positive blood culture. Diagnosis of cholestasis was made at total bilirubin of ⋝4 mg/dl with a portion of ⋝40 % conjugated bilirubin. Results: All tO cases of cholestasis were found in group I. All other above mentioned factors were distributed equally to both groups and could not be attributed as a major factor in the etiology of idiopathic neonatal cholestasis. Conclusion: Bacterial infections were the main cause of neonatal cholestasis.

CHARACTERISATION OF ANTI-LIVER KIDNEY MICROSOMAL (LKM) ANTIBODY IN CHILDRE WITH AUTOIMMUNE CHRONIC ACTIVE HEPATITIS (aCAH)

Children with aCAH and those with PSC have similarly high levels of IgG, non organ and liver specific autoantibody & increased lymphocyte cytotoxicity to autologous hepatocytes. However, they differ in several immunological parameters. Only in aCAH there is decreased T suppressor cell number & function and increased circulating activated T helper cells, suggesting different mechanisms for the emergence of autoimmunity. Antinuclear antibody(ANA) is characteristic of both PSC and a sub-group of aCAH. The aim of this study was to characterise ANA in these 2 conditions. Using an indirect immunofluorescence technique and monoclonal antibodies to IgG subclasses, we have determined the immunoglobulin class and IgG subclass of ANA in 5 patients with aCAH[3 female, median age 18yrs, range 15-19yrs] & in 8 with PSC[4 female, median age 8.5yrs, range 3-14yrs]. Patients with aCAH had titre of ANA between 1:10 & 1:640 & in all of them ANA belonged to the IgG class and IgGl subclass. In contrast, in children with PSC ANA(titres 1:10-1:2560) were only IgG in 6 and IgG and IgM in 2. Of 6 patients with ANA titres of more than 1:10, 3 expressed ANA in 2 subclasses[IgGl & IgG3 in 2, IgGl & IgG4 in 1], and 1 patient expressed ANA in all 4 subclasses. These differences in ANA class & subclass between aCAH & PSC could derive from differences in the nature of the nuclear antigen or could indicate that ANA production results from the breakdown of different immunoregulatory mechanisms.

PSC histological features may resemble those of aCAH and increased autoantibody and IgG levels are found in both conditions. PSC, however, is less responsive to immunesuppressants. To investigate whether this derives from differences in regulatory and/or effector immune mechanisms we have studied 7 children with PSC, 14 with aCAH and 26 healthy children as controls. Lymphocyte cytotoxicity values to autologous hepatocytes were similarly elevated in 5 children with PSC (mean±SD, 50±9%) and in 7 with aCAH (53±16%) studied. In contrast, T suppressor number and Con-A induced function were normal in patients with PSC (23.5±3.1% and 50.8±5.3%), but significantly decreased in children with aCAH (15.8±4.7% and 7.6±6.5%), when compared to controls (24.2±5.4%, p

Non organ-specific autoantibodies, in particular antinuclear antibody(ANA) are present in both aCAH and PSC of childhood, suggesting an imbalance in immune-regulation akin to that observed in systemic lupus erythematosus(SLE). The aim of this study was to investigate whether an increase in antibody to double-stranded DNA (dsDNA), typical of SLE, is present in these two conditions. Binding to dsDNA was determined by RIA in 8 ANA positive children with PSC [4 male, median age 7yrs, range 3-14yrs] and in 20 patients with aCAH[5 male, median age 12.5yrs, range 8-21yrs], 6 of whom are ANA positive, the remainder being positive for antibodies to smooth muscle (SMA), liver/kidney microsomes(LKM) and/or gastric parietal cells(GPC). Anti-dsDNA antibodies in the range diagnostic for SLE (>25 U/ml) were found in 5 children with aCAH[2 ANA positive, 2 LKM positive, 1 SMA positive] but in none of the PSC group. The mean dsDNA binding in children with aCAH was 15.0 IU/ml ± SD 10.63 compared to 7.78 U/ml ± SD 1.7 in those with PSC (p < 0.05). Anti-dsDNA antibodies of more than 25 U/ml in aCAH occurred in the absence of ANA. In the PSC group where ANA is commoner & generally of higher titre, anti-dsDNA antibodies did not correlate with ANA titre. This is a further demonstration of different immune dysfunction in these two chronic liver disorders.

Children with aCAH have impaired Concanavalin A(Con A) induced 'suppression of Pokeweed stimulated immunoglobulin synthesis. Since this system tests non-specific immunoglobulin production, we investigated suppression of in vitro production of antibody to Tetanus toxoid (TT), a T-dependent antigen, by lymphocytes from 11 children with aCAH and 14 age-matched healthy controls, all previously immunized. Both patients and controls had similar serum anti-TT levels (mean ± SEM 1.8 ± 0.85 lU/ml, 2.0 ± 0.86 IU/ml) and in vitro production of anti-TT in response to optimal dose of TT (0.005 IU/ml)(5.3 ± 0.88 × 10−3 IU/ml, 6.7 ± 0.94 × 10−3 IU/ml) as measured by ELISA. Suppressor function was induced by 24 hour incubation of lymphocytes with high dose TT (5 IU/ml) or with 20μg/ml Con A. Washed cells were then added to stimulated autologous lymphocytes. TT-induced suppression of anti-TT production was similar in patients (69.8 ± 4.2) and controls (72 ± 3.8%). In contrast, Con A-induced suppression of anti-TT was significantly lower in patients (15.7 ± 2.5%) than controls (46.7 ± 4.9% p

An increase in IgG levels is characteristic of aCAH but it is not known whether such increase affects all subclasses. Indeed, we have recently shown that the anti-liver/kidney microsomal antibody in aCAH is restricted to IgG1. In this study we have measured IgG subclasses using an ELISA technique. In 57 serum samples from 15 children with aCAH [11 female,age range 3-18yrs]. In all untreated cases the total IgG levels were increased and all subclasses were also increased when compared to 97 age-matched controls. [Patients: IgG1 mean ± SD = 10.4 ± 2.63, IgG2 = 4.8 ± 1.24, IgG3 = 2.8 ± 0.63, IgG4 1.3 ± 0.4g/l. Controls;IgG1 = 6.56 ± 1.87, IgG2 = 2.47 ± 0.76, IgG3 = 0.86 ± 0.31, IgG4=0.312 ± 0.22g/l, the differences being significant for IgG2, p

Comparaison de 2 groupes de malades, dans une region endemique pour l'hepatite virale et dans une region non endemique. Les complications hepatiques peuvent etre dues a une surcharge en fer, malgre la chelation, mais les dosages de transaminase et de ferritine indiquent aussi un role important de l'infection par virus B. Une immunisation serait peut etre indiquee pour les malades atteints de thalassemie

A simple technique for detection of serum autoantibodies that bind to liver membrane antigens on the surface of liver cells has been developed using Alexander cells[PLC/PRF/5]. Alexander cells were incubated with test serum(1:160), washed then adherent antibodies were detected by incubating with 125I-proCein A. Specific binding to cell surface was calculated as the ratio between the counts with patient's serum divided by 2SD above the mean count obtained with serum from controls. The ratio was significantly higher in those with active autoimmune chronic active hepatitis (aCAH)[median 4.5, range 1.1-23.1] when compared to inactive cases [1.5, 0.3-4.1; p

We have shown that children with aCAH have genetically determined low C4 levels but it is not known whether C4 function is also impaired. In the present study we measured haemolytic function of C4 and its concentration in 20 children with aCAH(15 female, median age 13½yrs, range 7-18yrs) and in 19 sex and age matched normal controls. C4 function was measured by a radial haemolytic assay, using C4 deficient guinea-pig serum, the concentration by laser nephelometry. A significant reduction of C4 levels was observed in patients [median 0.15g/l, range 0.04-0.485 g/1] compared to healthy children [0.263g/l, 0.153-0.520g/l, p < 0.002] confirming our previous data. C4 function in 16 normal children was 50% (25-100%) but in 3 was very low (4%, 4%, 122). In patients C4 function was 12%(0-67%), significantly reduced when compared to all controls. (41.5%, 4-100%, p< 0.001). There was no significant correlation between function and concentration values. Such discrepancy between function and concentration could be explained by the marked genetic polymorphism of C4. These data show that C4 function is impaired in aCAH. C4 has a key role in virus neutralization and impairment of its function might enable a virus to initiate the autoimmune process leading to CAH.