C. L. Burek

Mercury exposure, malaria, and serum antinuclear/antinucleolar antibodies in amazon populations in Brazil: A cross-sectional study

BackgroundMercury is an immunotoxic metal that induces autoimmune disease in rodents. Highly susceptible mouse strains such as SJL/N, A.SW, B10.S (H-2s) develop multiple autoimmune manifestations after exposure to inorganic mercury, including lymphoproliferation, elevated levels of autoantibodies, overproduction of IgG and IgE, and circulating immune complexes in kidney and vasculature. A few studies have examined relationships between mercury exposures and adverse immunological reactions in humans, but there is little evidence of mercury-associated autoimmunity in humans.MethodsTo test the immunotoxic effects of mercury in humans, we studied communities in Amazonian Brazil with well-characterized exposures to mercury. Information was collected on diet, mercury exposures, demographic data, and medical history. Antinuclear and antinucleolar autoantibodies (ANA and ANoA) were measured by indirect immunofluorescence. Anti-fibrillarin autoantibodies (AFA) were measured by immunoblotting.ResultsIn a gold mining site, there was a high prevalence of ANA and ANoA: 40.8% with detectable ANoA at ≥1:10 serum dilution, and 54.1% with detectable ANA (of which 15% had also detectable ANoA). In a riverine town, where the population is exposed to methylmercury by fish consumption, both prevalence and levels of autoantibodies were lower: 18% with detectable ANoA and 10.7% with detectable ANA. In a reference site with lower mercury exposures, both prevalence and levels of autoantibodies were much lower: only 2.0% detectable ANoA, and only 7.1% with detectable ANA. In the gold mining population, we also examined serum for AFA in those subjects with detectable ANoA (≥1:10). There was no evidence for mercury induction of this autoantibody.ConclusionsThis is the first study to report immunologic changes, indicative of autoimmune dysfunction in persons exposed to mercury, which may also reflect interactions with infectious disease and other factors.

Antibodies to selected minor target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA).

In patients with anti‐neutrophil cytoplasmic antibodies (ANCA)‐associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C‐ANCA) and perinuclear (P‐ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegeners granulomatosis, microscopic polyangiitis and Churg–Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C‐ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P‐ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti‐PR3 and anti‐MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well‐defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF‐positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF‐negative (–) sera (P < 0·01). Patients with IF+ PR3‐MPO‐ sera showed the most varied reactivity to the minor antigens. Among the IF+ groups, the IF+ PR3+/MPO‐ sera showed the lowest reactivity to the minor antigens. Patients with well‐defined ANCA specificities, e.g. the PR3‐ANCA response associated with Wegeners granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes.

IgG subclass distribution of thyroglobulin antibodies in patients with thyroid disease

The IgG subclass distribution of thyroglobulin antibodies (TgAb) has been studied in Hashimoto and Graves’ patients by several investigators with conflicting results, in part explainable by methodological problems. We have recently developed a quantitative ELISA to measure in absolute terms the serum concentration of TgAb subclasses. The aim of the present study was to apply this method in a large series of patients with autoimmune as well as, for the first time, non‐autoimmune thyroid diseases. We examined 28 patients with Hashimotos thyroiditis, 30 with Graves’ disease, 21 with thyroid carcinoma and 18 with non‐toxic goitre, all selected for the presence of TgAbs. The results indicated that TgAbs in thyroid diseases were not restricted to any particular isotype, but comprised all four IgG subclasses. IgG1 was represented similarly in the four groups. The same was true for IgG3, even though its contribution to the total antibody content was very small. IgG4 was the dominant subclass in patients with Graves’ disease, thyroid carcinoma and non‐toxic goitre, probably reflecting a prolonged antigenic challenge. In Hashimotos thyroiditis IgG2 was dominant, possibly because T helper lymphocytes infiltrating the thyroid are typically Th1 type.

Iodination of murine thyroglobulin enhances autoimmune reactivity in the NOD.H2h4 mouse

Autoimmune thyroiditis in humans has been linked to excess iodine intake. A causative relationship between dietary iodine and thyroiditis has been clearly established in animal models of thyroiditis, including the NOD.H2h4 mouse strain, which develops enhanced thyroiditis spontaneously after supplementation of drinking water with sodium iodide. To assess the mechanisms by which iodine may contribute to disease pathogenesis, we have purified hypoiodinated thyroglobulin (Lo‐I Tg) from the thyroids of mice fed methimazole and potassium perchlorate. This preparation contained only a trace of iodine and was poorly reactive to monoclonal antibody 42C3, which has been shown previously to distinguish hypoiodinated from normal Tg. A cloned T cell line 2D11 from a diseased NOD.H2h4 mouse proliferated in response to normal Tg, but not to Lo‐I Tg. Serum antibodies from NOD.H2h4 mice with thyroiditis were poorly reactive to Lo‐I Tg. To determine that these changes were due specifically to iodine content, Lo‐I Tg was reiodinated in vitro. Reiodination of Lo‐I Tg partially re‐established the reactivity of NOD.H2h4 serum antibodies. The data demonstrate that the reactivity of thyroglobulin‐specific antibodies and certain T cells are dependent on the iodine content of thyroglobulin. These findings suggest that iodine contributes to autoimmune thyroiditis in the NOD.H2h4 mouse by directly enhancing the antigenicity of thyroglobulin.

Tryptic peptides of human thyroglobulin: II. Immunoreactivity with sera from patients with thyroid diseases.

Tryptic peptides of human thyroglobulin (Tg) were analysed by Western immunoblot for their reactivity to circulating autoantibodies from patients with Hashimotos thyroiditis (HT), Graves’ disease (GD) and thyroid carcinoma, and from normal human controls. Low molecular weight peptides were released after 4h incubation of Tg with trypsin. The sera of thyroid disease patients reacted with several peptides, but predominantly bound three peptides with apparent molecular weights (MWap) of 25 kD, 20 kD, and 15kD; the sera of normal individuals did not bind these fragments of Tg. The pattern of Tryptic peptides recognized by the majority of sera from GD patients differed from that recognized by sera from most patients with HT. Autoantibodies from both groups of patients recognized a 15‐kD peptide with a high frequency, but the sera from 26/43 (60%) GD patients also recognized a peptide with MWap of 25 kD, whereas the sera from 22/35 (63%) of HT patients recognized a 20‐kD peptide. A few sera from patients with thyroid carcinoma reacted with peptides with MWap of 15 and 20‐kD, and none bound the 25‐kD peptide. The immunoreactivity of autoantibodies in HT sera to the 20‐kD peptide paralleled the competitive inhibition of the MoAb 137CI by these sera. In addition, MoAb 137CI and Hashimotos sera showed the same Western immunoblot‐binding pattern to Tg tryptic peptides, suggesting that a Hashimoto‐associated epitope and the 137Cl‐binding site are found on the same peptide. These findings suggest that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.

Tryptic peptides of human thyroglobulin: I. Immunoreactivity with murine monoclonal antibodies.

Human thyroglobulin (Tg) was treated with trypsin at different concentrations of trypsin/Tg for various incubation times at 37°C using non‐reducing conditions. A ratio of trypsin to Tg of 1:100 (w/w) was optimal to release small peptides that were reactive to murine MoAbs to human Tg. Most peptides were released after only 1 h incubation with trypsin, but these peptides were further degraded at longer incubation times. However, a few small peptides. the largest of which with an apparent molecular weight (MWap) of 40kD, resisted tryptic digestion up to at least 12 h of incubation. These resistant peptides were further degraded by trypsin at 18–24 h of incubation. Tryptic peptides of Tg, released at I h and 4 h of incubation, were analysed for their immunoreactivity to 16 well characterized anti‐Tg MoAbs by Western immunoblot. Patterns of peptide recognition of these MoAbs were generally unique. Eight MoAbs reacted with peptides of MWapof 10–25 kD and above. Four other MoAbs reacted with peptides of MWap of 25 43 kD and above, and the remaining four reacted with peptides of MWap >43kD. Nine of these MoAbs failed to recognize peptides after reduction, suggesting that the MoAbs bind conformation‐dependent epitopes. The above information will promote the development of models relating the structure of Tg to the autoimmune process, and may provide an understanding of those regions of Tg responsible for the induction of autoimmune thyroiditis.

Heritability analysis of IgG4 antibodies in autoimmune thyroid disease

Abstract A study of IgG4 autoantibody levels in juvenile thyroid disease patients showed evidence of heritability using the ROMP screening method. These levels increased with time despite the fact that total IgG antibody decreased with time. Evidence of heritability was demonstrated only in patients with high titers of autoantibodies to both thyroglobulin (Tg) and thyroperoxidase (TPO) unlike family members who may show high titers of one or the other and be asymptomatic at the time of sampling. Since high and low IgG4 levels give different heritability plots, these findings may represent a more severe fibrotic form of thyroiditis with a distinct genetic background. Hence a simple predictive approach is offered by this screening tool for the disease in patients and family members which may be helpful in the future to identify IgG4-related thyroiditis early in the course of disease without the requirement for biopsy.