David B. G. Oliveira

Gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) may not be sensitive indicators of hepatocellular damage in patients taking anticonvulsant drugs as raised levels may only reflect enzyme induction. Aspartate aminotransferase (AST) is a specific, but relatively insensitive marker of liver damage and has a poor correlation with liver histology. Serum F protein is found in high concentration in the liver and levels are not influenced by enzyme induction. We measured serum F protein levels in patients taking carbamazepine (CBZ) and phenytoin (PHT) as monotherapy and in patients receiving multiple drugs. We compared the results with patients taking sodium valproate (VPA). Serum F protein levels were elevated in 6%, 22% and 13% of patients receiving CBZ, PHT and VPA, respectively. Raised GGT levels were reported for both the CBZ (26%) and PHT (78%) groups. Raised ALP levels were observed in 16%, 25% and 4% of the CBZ, PHT and VPA groups, respectively. Raised levels of serum F protein in the VPA group and the absence of any associated increases in either GGT or AST may further support the suggestion that serum F protein is an indicator of hepatocellular dysfunction associated with anticonvulsant therapy. However, further correlation with liver histology is required.

Anti-glomerular basement membrane (GBM) disease is characterized by the production of an autoantibody with very restricted specificity, with no evidence of polyclonal B cell activation. It was therefore surprising to find that in a solid-phase ELISA a proportion of anti-GBM sera showed significant binding to pyruvate dehydrogenase (PDH), a reactivity usually associated with the antimitochondrial autoantibodies (AMA) found in primary biliary cirrhosis (PBC). The specificity of this reactivity was confirmed by inhibition and competition experiments. The AMA found in anti-GBM sera were of much lower affinity than those found in PBC sera, and recognized a more restricted set of species (mainly the 55-kD and occasionally the 74-kD component of PDH). However, it was possible to block the binding in a Western blot of an anti-GBM serum to both the 55-kD and 74-kD species with F(ab')2 fragments prepared from a PBC serum. Although AMA have been found in diseases other than PBC, such diseases have usually been characterized by polyclonal B cell activation. The stimulus to the production of AMA in anti-GBM disease, and their significance in pathogenesis (if any), are unknown.

We have studied the total IgG subclass and anti-mitochondrial antibody (AMA) specific IgG subclass distribution in primary biliary cirrhosis (PBC) sera. In order to solve the problems caused by the differing affinities of subclass specific monoclonals and the competitive inhibition of antibodies in a whole serum assay, six sera were separated into subclass-specific fractions by affinity-depletion chromatography. AMA subclass distribution of 20 further sera from patients with PBC was assessed using conventional methods and the results were calibrated against one of the fractionated sera. Light chain distribution and AMA functional affinity were also assessed for the fractionated subclasses. Total amounts of IgG3 were significantly increased compared with normal controls. AMA were found in all IgG subclasses and not restricted predominantly to IgG3 as previously described. The functional affinity of IgG3 AMA is generally lower as compared with that of other subclasses. No light chain restriction was found.

To determine the role of prophylactic N-acetylcysteine in the prevention of contrast-induced nephrotoxicity. One hundred and sixteen patients undergoing noncoronary angiography, with or without pre-existing renal impairment, were randomly assigned to receive prophylactic oral N-acetylcysteine or no treatment. Serum creatinine (sCr) was measured prior to angiography and 48 hr after the procedure. Urine samples were collected before and after the examination for measurement of malondialdehyde (MDA) concentration. Contrast-induced nephrotoxicity (CIN) was defined as a rise in serum creatinine of 0.5 mg/dl (44 mmol/l) at 48 hr. Complete data were available on 106 patients, 53 of whom had received N-acetylcysteine. There were no significant differences between the two groups in baseline characteristics, type of angiogram, or volume and concentration of contrast used. Three patients (2.8%), all of whom had received N-acetylcysteine, developed CIN. In the N-acetylcysteine group, the mean serum creatinine in patients with renal impairment was 151.0 ± 44.2 μmol/l prior to the procedure and 155.6 ± 48.6 μmol/l (p = 0.49) after the procedure. Respective values for those without renal impairment were 79.6 ± 15.1 μmol/l and 81.2 ± 20.0 μmol/l (p = 0.65). In the group that had not received N-acetylcysteine, the mean serum creatinine levels before and after the procedure were 150.0 ± 58.1 and 141.4 ± 48.0 μmol/l (p = 0.17) in patients with renal impairment and 79.7 ± 14.2 and 81.4 ± 15.4 μmol/l (p = 0.34) in those without renal impairment. In both groups, no significant change in urinary MDA concentration was observed. There is no benefit to the prophylactic administration of N-acetylcysteine in patients undergoing peripheral angiography using current contrast media.

Summary: Mercuric chloride (HgCl₂)‐induced autoimmunity in Brown Norway rats is a spontaneously resolving autoimmune response driven by the activation of T helper type 2 lymphocytes (Th2 cells). Treatment with antibody to OX40‐ligand (OX40‐L) from the time of the first HgCl₂ injection for 12 days had little effect. Delayed treatment commenced 8 days after the first HgCl₂ injection significantly suppressed immunoglobulin E production, splenomegaly, weight loss and mortality. This makes OX40/OX40‐L signalling an attractive therapeutic target for Th2‐driven autoimmune diseases. Intravenous administration of the murine antibody to OX‐40‐L elicited a vigorous anti‐mouse immunoglobulin antibody response that was significantly enhanced compared to the response to control immunoglobulin. It is likely that this response significantly reduced the plasma half‐life of the anti‐OX40‐L antibody and this observation has clear implications for the interpretation of data from experiments where anti‐OX40‐L is used in vivo.

Summary: Administration of mercuric chloride (HgCl2) to Brown Norway rats causes Th2 dominated autoimmunity including a caecal vasculitis. Disease peaks 14 days after starting HgCl2 after which animals immunoregulate spontaneously. In a third phase, if animals are rechallenged with HgCl2 6 weeks later they appear resistant, developing only attenuated disease. Previous studies suggested a role for CD8+ cells as partial mediators of resistance but no groups had studied the role of αβ T cells, γδ T cells or natural killer (NK) cells in resistance. We used adoptive transfer and in vitro cell depletion to show that αβ T cells are also partially responsible for resistance. Donor animals were treated with HgCl2 or saline and killed 21 days later. Cells from donor spleens were transferred into recipient animals which were challenged with HgCl2 and killed 14 days later. Test recipients received spleen cells from HgCl2-treated donors after in vitro depletion of one subset of cells. Recipients receiving spleen cells from saline-treated donors remained susceptible to HgCl2-induced vasculitis; those receiving spleen cells from HgCl2-treated donors were resistant. Animals receiving αβ T-cell-depleted spleen cells from HgCl2-treated donors showed partial reversal of resistance. Our results suggest a role for αβ T cells in the resistant phase of the Brown Norway rat model of vasculitis.

Administration of mercuric chloride to Brown Norway rats results in T helper type 2 (Th2)- dominated autoimmunity characterized by high immunoglobulin E (IgE) concentrations, the production of multiple IgG autoantibodies, including those to glomerular basement membrane (GBM), arthritis and caecal vasculitis. After 14 days animals immunoregulate and auto-immunity resolves even if mercuric chloride injections are continued. In a third phase, if animals are re-challenged with mercuric chloride 6 weeks later, they show only attenuated autoimmunity with lower anti-GBM antibody concentrations and arthritis scores. Resistance to the induction of anti-GBM antibodies can also be achieved following an initial challenge with low-dose (one-tenth standard dose) mercuric chloride. We have now studied this resistant phase in more detail. We have shown, first, that following an initial full-dose mercuric chloride challenge, resistance also affects susceptibility to caecal vasculitis. Second, following an initial full-dose mercuric chloride challenge, the IgE response upon re-challenge is initially accelerated but subsequently enters a resistant phase and third, following an initial challenge with low-dose mercuric chloride, resistance is also seen to the induction of caecal vasculitis but is not seen in IgE serology (where results suggest competing effector and regulatory cell populations). Studying such regulatory phases in animal models of autoimmunity may be of benefit in the future in designing new therapies for human vasculitis.

Administration of mercuric chloride (HgCl(2)) to Brown Norway rats causes Th2-dominated autoimmunity with raised immunoglobulin E concentrations and gut vasculitis, both of which are T-cell dependent, peak at 14 days after starting HgCl(2) and then spontaneously resolve. If animals are re-challenged with HgCl(2) 6 weeks after initial exposure, they are resistant to autoimmunity, developing only attenuated disease. Recently, a separate phase of early caecal vasculitis was described beginning 24 h after initiating HgCl(2) and prior to caecal entry of T cells. Previous work suggested this early vasculitis was alpha beta T-cell independent and implied a role for mast cells. We further tested this hypothesis by performing a histological study during the first 93 h following HgCl(2) challenge defining the precise relationship between gut mast cell degranulation and appearing caecal vasculitis. We also studied whether early caecal vasculitis enters a resistant phase upon re-challenge with HgCl(2). We show a direct correlation between mast cell degranulation and early caecal vasculitis following initial HgCl(2) challenge. We demonstrate resistance to re-challenge in this phase of injury, with results at re-challenge also showing a correlation between mast cell degranulation and early caecal injury.