Standardised assessment of membrane proteinase 3 expression. Analysis in ANCA-associated vasculitis and controls.

OBJECTIVES: Increased numbers of neutrophils expressing proteinase 3 on their membrane (mPR3) have been reported in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and are suggested to be involved in AAV immunopathogenesis. In most studies, neutrophils were analysed for mPR3 expression without priming with TNFalpha, suggesting that mPR3 expression on neutrophils is dependent on other priming events, such as isolation procedures . These priming events can be variable. Therefore, we analysed mPR3 expression on neutrophils before and after priming with TNFalpha to assess whether standardised assessment of mPR3 expression requires priming. Using neutrophils before and after priming with TNFalpha, we assessed percentages of mPR3(+) neutrophils in patients with AAV and in disease and healthy controls. METHODS: Neutrophils from patients with PR3-AAV and MPO-AAV, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), and from healthy controls were analysed before and after priming with TNFalpha for mPR3 expression. RESULTS: 42% of all individuals analysed showed minimal expression for mPR3 on all neutrophils before priming with TNFalpha, whereas after priming a clear mPR3(+) subset was observed next to mPR3(-) neutrophils, corresponding to bimodal mPR3 expression. In patients with PR3-AAV or MPO-AAV, the percentage of mPR3(+) neutrophils after priming with TNFalpha was significantly increased (p<0.01 and p<0.05, respectively) compared with healthy controls. Percentages of mPR3(+) PMN were also increased in patients with SLE (p<0.01) but not in RA. CONCLUSION: Standardised assessment of proteinase 3 on the membrane of neutrophils requires priming with TNFalpha. Percentages of mPR3(+) PMN are increased in AAV and SLE, but not in RA.

Practical pharmacogenetics: the cost effectiveness of screening for thiopurine s-methyltransferase polymorphisms in patients with rheumatological conditions treated with azathioprine.

OBJECTIVE: Thiopurine S-methyltransferase (TPMT), which catalyzes the inactivation of azathioprine (AZA), exhibits genetic polymorphism that results in dose related, serious toxicities (mainly hematological cytopenias) in 10-15% of individuals treated with AZA. Polymerase chain reaction (PCR) tests provide a sensitive, specific means of prospectively identifying these patients before AZA therapy and minimizing toxicity through dosage reduction. Our objective was to model the cost effectiveness of the 2 alternative AZA treatment strategies in rheumatologic conditions: (1) utilizing PCR to determine polymorphisms leading to TPMT deficiencies prior to AZA therapy with a reduction in dose; and (2) no testing. The analysis was conducted from a third party payer perspective over one year. METHODS: A decision analytic model was applied to map the costs and outcomes of patients under both strategies. Data applied to the model included the positive and negative predictive values of the PCR, the probabilities of adverse events due to AZA, and the costs associated with their management. Sources of data included published clinical trials, diagnostic test evaluations, surveillance trials, and economic evaluations. RESULTS: Dose related toxicities resulted in AZA discontinuation rates of 10-20%. The usual dosing strategy cost $677 Cdn per patient, whereas the genotype directed dosing strategy cost $663 Cdn per patient. In the genotype dosing strategy, the number needed to treat to avoid one adverse event over 6 months was 20. Thus, the genotype based dosing strategy dominated the usual dosing strategy. One-way sensitivity analyses revealed that the estimates were robust to ranges of +/- 30% for the costs, the properties of the PCR test, and the probability of adverse events. CONCLUSION: The introduction of PCR testing to identify TPMT polymorphisms prior to AZA treatment may represent good value in certain health care settings.

Assessment of in vivo frequency of mutated T cells in patients with systemic lupus erythematosus.

The frequency of mutant T cells (FMC) in blood lymphocytes from patients with systemic lupus erythematosus (SLE) was measured by growing cells in the presence and in the absence of 6-thioguanine. Patients with SLE had a spectrum of FMC ranging from normal to about 100 times normal. This high FMC among cells from SLE patients appears to reflect excessive in vivo activation and proliferation during the course of the disease. This represents the first demonstration of such a T cell abnormality in SLE; it supports the hypothesis that SLE T cells demonstrate increased in vivo division and/or survival.

Assessment of kininases in rheumatic diseases and the effect of therapeutic agents.

Bradykinin is degraded in human plasma by a carboxypeptidase to yield desArg9-bradykinin (DBK) which is then digested by angiotensin-converting enzyme (ACE) to the pentapeptide Arg-Pro-Pro-Gly-Phe and the tripeptide Ser-Pro-Phe. We have studied the rate of kinin degradation by each of these enzymes in patients with rheumatoid arthritis (RA) and with systemic lupus erythematosus (SLE), compared with the degradation rate in degenerative joint disease and normal subjects. Carboxypeptidase activity was the same in all individuals, but ACE activity was increased in the RA and SLE patients. We examined the effects of aspirin, sodium salicylate, auranofin, penicillamine, and corticosteroids on kinin metabolism, and all of these were marked inhibitors of ACE; however, only penicillamine had any demonstrable inhibition of carboxypeptidase. These observations suggest rapid degradation of DBK in patients with untreated RA and SLE, whereas drugs utilized in therapy have the opposite effects. Studies to examine the role of DBK in disease manifestations are in progress.