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Lymphocytic galactosyltransferase in rheumatoid arthritis and other diseases

Axford, John Stewart; (1990) Lymphocytic galactosyltransferase in rheumatoid arthritis and other diseases. Doctoral thesis (M.D), UCL (University College London). Green open access

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Abstract

IgG-Fc galactose (IgG-gal) levels varies with age and reduced serum IgG-gal values have been reported in a restricted number of diseases: rheumatoid arthritis (RA), tuberculosis (TB), Crohn's disease and SLE associated with Sjogren's syndrome. The experimental objective of this study was to determine whether lymphocytic galactosyltransferase (GTase), the enzyme responsible for the transfer of galactose to IgG-Fc within the lymphocytic golgi, is defective in RA, and other diseases, and if it is causally related to IgG carbohydrate abnormalities. A reliable and reproducible assay of lymphocytic GTase, detecting the transfer of radiolabelled galactose to an acceptor glycoprotein, was devised, with a coefficient of variability of 4.4% and the ability to detect GTase from about 5x105 cells. Preliminary experiments with human GTase showed that activity could be detected in RA and control lymphocytes, serum and plasma, although no correlation existed between serum and lymphocytic levels in RA. A control age-matched population (n=32), without autoimmune disease, was used throughout for comparison. In all, except three, IgG-gal values were within 2 standard deviations of the normal mean for the age. No age relationship between IgG-gal and GTase activity was found in the control population. A wide range of GTase activities was noted in the control population and IgG-Fc galactosylation was positively correlated with GTase activity. A similar level of GTase activity was observed in B cell enriched preparations from human tonsils. The GTase activity in whole mononuclear cell preparations, from patients with RA, was significantly elevated. However, removal of monocytes, resulted in a fall in GTase activity, and B and T GTase activities were both reduced when measured individually when compared to healthy controls. This may indicate that T cell glycoproteins could be affected in a similar manner as IgG. A positive correlation between B and T cell GTase activity and a negative correlation between IgG-gal and B and T cell GTase was found. On splitting the rheumatoid arthritis population into groups, dependent upon drug treatment, it was noted that those patients taking sulphasalazine (SASP) had higher mean levels of GTase than the other patients in the study. This effect was shown not to be due to disease activity and was therefore presumed to be related to the actions of SASP itself. SASP is known to have pharmacological potential to cause these effects. In an initial longitudinal analysis of glycosylation parameters in patients with rheumatoid arthritis, a temporal relationship with clinical activity was found. In addition, SASP was shown to maintain GTase levels in other patients. Abnormal GTase activity was observed in clinically inactive patients, which may indicate continuing subclinical cellular dysfunction. Although GTase activity was found to be normal in patients with osteoarthritis when compared to healthy controls, it was decreased in patients with SLE, Crohn's disease and active pulmonary TB. In only the TB patients did GTase return to normal with treatment. CD5 positive B cell GTase activity from patients with chronic lymphocytic leukaemia was determined and a significant decrease in GTase activity was noted when compared to a control population with the same age range. In an attempt to investigate the mechanism of fluctuations in GTase activity the presence of an intracellular inhibitor and specific polymorphisms within the gene encoding the GTase activator (GTA) gene, were looked for. Using normal GTase from human lymphocytes, bovine milk and human milk, no inhibition of activity was detected when mixed with RA lymphocyte extract. In one experiment, synergism was noted in both RA and normal preparations, which may indicate the presence of a co-factor in human lymphocytes. On restriction enzyme digestion of RA and normal lymphocytic DNA, no gross structural changes in the GTA gene was seen and hence gene abnormalities are also unlikely to be responsible for reduced GTase activity. RFLPs were, however, observed using the enzyme Bgl II in both normal and RA DNA. To test whether fluctuations in isoenzymes of GTase may be implicated in the abnormal glycosylation of IgG, and potentially other glycoproteins, GTase activity towards 5 different oligosaccharide acceptors was determined. A decrease in enzyme activity, comparable to ovalbumin, was noted only with asialo-mucin. In order that cells, other than those from the periphery, could be studied in disease, the MRL/lpr mouse, a model for rheumatoid arthritis, was used. GTase from pooled peripheral and splenic B cells was assayed from MRL/lpr, MRL+/+ and CBA/J (control) strains of mice. Splenic B cell GTase activity was found to be similar in both the MRL strains and the CBA/J control strain, whereas enzyme activity was reduced in the peripheral B cells only in the MRL strains. This may suggest that reduced GTase in the MRL mouse is one of a number of factors leading to disease in the lpr strain, which are not present in the +/+ strain, and is not a direct result of the disease itself. In conclusion, GTase activity has been studied in normal individuals and those with RA, SLE, TB, CD and CLL. Reduced levels of GTase are apparent in the presence of disease, irrespective of clinical activity, but in individuals with TB the GTase always returns to normal following antibiotic treatment and often does in patients with RA treated with SASP. There is no evidence to suggest that reduced GTase activity may be due to an intracellular inhibitor of GTase in RA, nor to gross structural changes in the GTA gene. Evidence is provided which suggests that GTase isoenzyme changes may occur in RA. A hypothesis is presented linking isoenzyme changes with glycoprotein abnormalities and the clinical presentation of disease. Similar GTase activity findings are reported in the peripheral, but not the splenic, B cells of the MRL (arthritic) mouse, suggesting that cells being sampled in the periphery are trafficing from areas of inflammation.

Type: Thesis (Doctoral)
Qualification: M.D
Title: Lymphocytic galactosyltransferase in rheumatoid arthritis and other diseases
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10114363
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