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Title: The role of humoral mediators in immunological reactions to streptokinase
Author: Afshari, Afshin
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1995
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Streptokinase is now a commonly administered thrombolytic agent in AMI patients. One of the main disadvantages of its administration is that as a foreign antigen it may provoke hypersensitivity reactions due to previous streptococcal infections, and' pre-existing antibodies may reduce efficacy of treatment. The aim of this study was to look at role of humoral immunity to streptokinase. To this end three areas were studied; 1) Specific anti-streptokinase antibodies .2) Role of complement and 3) Role of the immune complex removal. ELISA's were developed to measure the levels of IgG, IgA, IgM, IgE and neutralising anti-streptokinase antibodies in a noraial population. Only a small proportion of the normal individuals had elevated levels of anti-streptokinase antibodies (IgG 1.68%, IgA 2.7%, IgM 9.9% and neutralising anti-streptokinase antibodies 0.4%). The levels of anti-streptokinase antibodies in 20 patients with AMI (10 patients treated with streptokinase and 10 with r-tPA) were compared to the normals. Administration of streptokinase in 10 patients studied, resulted in an immediate fall in level of anti-streptokinase antibodies and developed a specific response. The levels of anti-streptokinase antibodies in patients treated with streptokinase and reperfused early were within the normal ranges, and levels rose in the late reperfusion or non reperfusion groups. Of the 10 patients given streptokinase, one patient with elevated levels of IgG and IgA developed serum sickness. The levels of specific IgM anti-streptokinase antibodies in patients with rheumatoid arthritis were significantly elevated. This did not correlate with IgG anti-mycobacterium heat shock protein 65 (mHSP65). In patients with Henoch Schonlein Purpura there was a correlation between IgG anti-mHSP65 levels and IgG, IgA and neutralising anti-streptokinase antibodies levels. The interactions of complement system and immune complexes was studied by measurement of complement activation products Cls:Cl-INH, C3b-P and C5b-9 using modified techniques introduced by Auda et al 1990. An indirect ELISA for measuring serum C3d levels and a modified ELISA for detecting erythrocyte (E) with bound C3d were developed to evaluate the relationship of bound C3d and the levels of free complement components. Patients treated with r-tPA did not generate complement activation products. All the ten patients administered streptokinase generated Cls:Cl-INH and only one patient had increased levels of C3b-P and C5b-9. The extent of complement activation correlated with the pre-treatment levels of anti-streptokinase antibodies. In vitro effects of the ICs on complement activation showed that the presence of erythrocytes did not modulate significantly C3d or Cls:Cl-INH levels generated by the ICs, however, they exerted an influence on C3b-P through the actions of CRL The kinetics and dose response of streptokinase ICs, C3c and C3d binding to erythrocytes (E's) were studied by flow cytometry and its relationship with free complement activation was determined by measuring the free complement activation products in the accompanying supernatants. This study showed that the binding of ICs to E's depended significantly on the extent of complement activation (p < 0.001) and the dose of ICs (p < 0.001). The clearance of ICs during streptokinase treatment was studied during a period of 30 minutes after streptokinase administration in 4 patients with AMI by detecting E bound ICs using FACscan analysis. Streptokinase ICs were detectable on E's in one of patients. This patient generated high levels of complement activation products which correlated with E's bound ICs (p < 0.001), thus the clearance of streptokinase ICs depends on the extent of complement activation and the observed adverse reaction in patient CD (With elevated levels of IgA) is due to poor complement activation by the formed ICs. Study of anti-streptokinase ICs and IgG interactions with E's revealed that they also bound to E's in absence of NHS. A haemagglutinin test among normal individuals and patient groups revealed that the of binding of rabbit IgG to human erythrocytes is not a non-specific adsorption. In addition, F(ab)2 fragment did not bind to the E's as determined by FACScan analysis. Using flow cytometry human IgG (Fc fragment) inhibited the binding of rabbit IgG to human erythrocytes indicated that both rabbit IgG and Fc fragment of human IgG bind to the same sites on erythrocytes. Using a set of monoclonal anti-human FcyR antibodies, FcyRI (CD64) was detectable on erythrocytes. The pattern of mouse IgG subclasses binding to human E's (IgG3 > IgG2b >IgG2a) was consistent with the pattern of CD 16 (isoforms A and B) and CD64 (isoform A). These findings indicate the presence of FcyR on the erythrocytes, which may play an important role in handling of immune complexes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral