I. M. Roitt

GALACTOSYLATION OF IgG ASSOCIATED OLIGOSACCHARIDES: REDUCTION IN PATIENTS WITH ADULT AND JUVENILE ONSET RHEUMATOID ARTHRITIS AND RELATION TO DISEASE ACTIVITY

The prevalence of agalactosyl N-linked oligosaccharides on serum IgG was determined for patients with juvenile onset and with adult rheumatoid arthritis. A significant difference in the prevalence of these structures from age matched controls was found in both types of arthritis. In patients with adult onset rheumatoid arthritis, the results showed a strong correlation between the prevalence of IgG-associated agalactosyl oligosaccharides and disease activity. A correlation between disease activity and agalactosyl structures was also seen in a retrospective analysis of serial IgG samples from patients with juvenile onset disease. The finding that childhood onset arthritis and adult rheumatoid arthritis share a defect of glycosylation of serum IgG suggests that there may be a greater similarity between these two varieties of rheumatoid arthritis than has been hitherto considered. The observation that the incidence of agalactosyl oligosaccharides on IgG fluctuates with disease activity provides indirect evidence for a seminal role for this change of glycosylation in the inflammatory process which, in rheumatoid arthritis, is focused on the synovial tissues and results in bone erosions and joint destruction.

POSSIBLE ROLE OF MYCOPLASMA FERMENTANS IN PATHOGENESIS OF RHEUMATOID ARTHRITIS

Abstract Mycoplasma fermentans membranes inhibited leucocyte migration in twenty-nine (67%) out of forty-three patients with rheumatoid arthritis. No inhibition was observed with leucocytes from osteoarthritic patients or healthy controls. M. fermentans is often present in affected joints in rheumatoid arthritis, and it is postulated that the chronic course of the inflammatory process is due to a hypersensitivity response to the organism. Immunoglobulin present in the surrounding fluid is bound firmly to the mycoplasma membrane and could be the stimulus for production of rheumatoid factors. If rheumatoid arthritis is due to an infectious process new chemotherapeutic approaches should be possible.

Changes in normal glycosylation mechanisms in autoimmune rheumatic disease.

To investigate potential mechanisms controlling protein glycosylation we have studied the interrelationship between lymphocytic galactosyltransferase (GTase) activity and serum agalactosylated immunoglobulin G levels (G(0)) in healthy individuals and patients with rheumatoid arthritis and non-autoimmune arthritis. In RA there was reduced GTase activity and increased G(0). A positive linear correlation between B and T cell GTase was found in all individuals. The relationship between GTase and G(0) was found to be positive and linear in the control population and negative and linear in the RA population. Sulphasalazine therapy maintained normal levels of GTase and caused a reduction in G(0) in the RA population. IgG anti-GTase antibodies (abs) were significantly increased in the RA population, whereas IgM anti-GTase abs were significantly decreased in both the RA and the non-autoimmune arthritis groups. These data describe a defect in RA lymphocytic GTase, with associated abnormal G(0) changes, which is corrected by sulphasalazine. A possible regulatory mechanism controlling galactosylation in normal cells is suggested, in which there is parallel control of B and T cell GTase. IgM anti-GTase abs may be integrated into this normal regulatory process. This is disrupted in RA, where the positive feedback between GTase and G(0) is lost and there is an associated increase in IgG anti-GTase abs, which may result from isotype switching as IgM anti-GTase abs are reduced. We suggest that these mechanisms are of relevance to the pathogenesis of RA, and that their manipulation may form part of a novel therapeutic approach.

REDUCED B-CELL GALACTOSYLTRANSFERASE ACTIVITY IN RHEUMATOID ARTHRITIS

Autosensitisation to IgG may be important in the pathogenesis of rheumatoid arthritis and could be related to reduced glycosylation of the oligosaccharides in the C gamma 2 region of serum IgG. The activity of galactosyltransferase, the enzyme that catalyses the addition of galactose to the oligosaccharide chains, was measured in the circulating B cells of seventeen patients with classic rheumatoid arthritis. It was significantly lower than that of a group of eleven controls (p less than 0.001) or of nine age-matched controls (p less than 0.001). In contrast, the enzyme activity of the T cells was within the range of that in nine age-matched controls, and enzyme activity in monocyte-rich mononuclear-cell populations was higher than in controls, possibly reflecting stimulation of the monocytes in rheumatoid arthritis. These findings suggest that galactosyltransferase may regulate the degree of glycosylation during IgG synthesis and could therefore be implicated in the rheumatoid inflammatory process.

CELL-MEDIATED (DELAYED) HYPERSENSITIVITY IN PATIENTS WITH SUMMER HAY-FEVER

Abstract Pollen-induced lymphocyte transformation in hay-fever subjects with clinical immediate hypersensitivity reflects the presence of a sensitised thymus-dependent cell population, which also gives rise to positive macrophage and leucocyte migration-inhibition tests in vitro and delayed-type skin tests in vivo. It is postulated that the thymus-dependent (T-) lymphocytes cooperate in the production of IgE antibody by B-lymphocytes of the plasma-cell series. Lymphocyte transformation per se should still be regarded as a manifestation of cell-mediated (delayed) hypersensitivity in vitro, whatever the apparent clinical correlations may be.

THE THYROID CYTOTOXIC AUTOANTIBODY

The presence of circulating autoantibodies in patients with Hashimotos disease suggests that autoimmunity is implicated in the disease process, but the ultimate proof of an autoimmune patho-genesis of thyroiditis and other human diseases must be obtained by demonstrating the autoag-gressive action of antibodies or immunologically competent cells on the living tissue in its normal environment. The demonstration by Pulvertaft, Doniach, Roitt and Hudson (1, 2) of a serum factor capable of destroying human thyroid cells in tissue culture is an advance in this direction. This finding has been confirmed and amplified by Irvine (3, 4) and by Goudie and McCallum (5). Further investigations of this phenomenon are reported in the present paper. The cytotoxic factor is shown to be an antibody reacting with the thyroid micro-somal antigen; its mode of action has been further studied and its incidence determined in a variety of thyroid conditions and in persons without overt thyroid disease. METHODS AND MATERIALS Tissue culture. Primary monolayer cultures of trypsin-dispersed thyroid epithelium were set up in chambers prepared on microscope slides by the method of Pulver-taft and co-workers (1). The chambers consisted of a plastic ring attached to a histology slide with silicone grease, and sealed with a coverslip. Fresh thyroid tissue was dispersed for 1 hour in 0.25 per cent trypsin (Difco, 1: 250), and after centrifugation a suitable inoculum was prepared by adding a concentrated suspension of cells to Parker 199 medium until a density of 5 epithelial clumps per field was seen when a drop was viewed through a 1oX objective. The ideal cell concentration resulted in the growth of 30 to 50 cell clumps in a control chamber. Each chamber contained 0.1 ml of test serum, 0.1 ml fresh normal serum, and 0.6 ml of cell inoculum. The fresh normal serum used routinely had a complement titer of between 160 and 320 minimal hemolytic doses (MHD) per ml, as determined by the micromethod of Donnelley (6), and was known to support good cell growth. Fresh normal serum was always used in tests with serum fractions or absorbed sera. Guinea pig serum could be used as a source of complement but was occasionally cytotoxic. The cultures were incubated for 18 to 24 hours at 370C. The sensitivity of each gland was established by including as controls known weakly cytotoxic sera and potent Hashimoto sera, sometimes set up in dilutions. With sensitive glands, all the cells …

Leucocyte Migration Inhibition in Thyroid Disease

Leucocyte migration inhibition tests (LMT) were carried out in 49 patients with thyroid disease and 47 healthy controls using purified human thyroid microsomes and thyroglobulin as the antigens. Patie

Lymphocytes from patients with rheumatoid arthritis produce agalactosylated IgG in vitro

The percentage of oligosaccharide chains lacking galactose was measured in IgG obtained from pokeweed mitogen‐activated cultures of blood lymphocytes from patients with rheumatoid arthritis and controls. Secreted IgG from rheumatoid arthritis lymphocytes was deficient in galactose compared with IgG from the lymphocytes of controls. This confirms that agalactosylation is a significant feature of the disease and demonstrates that it can occur at the B cell level and is not merely a post‐secretory event.

IgE IN LYMPHOID CELLS FROM POLLEN-STIMULATED CULTURES

Abstract Lymphocytes from atopic patients allergic to grass pollen transform in culture in response to the specific allergen. A small proportion of the transformed cells were shown to contain IgE. A comparable number of cells were stained by fluorescein-conjugated pollen extract. The relationship of the lymphocyte transformation to delayed-type hypersensitivity in atopic individuals is discussed.

Immunofluorescent technique for the detection of monoclonal internal image anti-idiotypic antibodies of hepatitis B surface antigen

Monoclonal anti-idiotypic antibodies to HBsAg were screened by immunofluorescence for the presence of a subset behaving as the internal image of the original antigen. We describe the technique and the criteria fulfilled to establish that 2/6 monoclonals studied act as the internal images of the a determinant of hepatitis B surface antigen.