Inactivation of the Skin-Sensitizing Antibodies of Human Allergy by Thiols

Abstract

The skin-sensitizing antibody or reagin is considered to be an important pathogenetic factor in human atopic allergy. Although many of the unique characteristics of this antibody are well known, a clear understanding of its physicochemical properties has been impeded by discordant findings ( 1 ) . One method which has contributed valuable insights into antibody structure and classification is in vitro exposure to sulfhydryl (SH) compounds (2). This treatment inactivates high molecular weight ( 19s) antibodies (3), dissociating them into 7s subunits, presumably through reductive cleavage of disulfide bridges ( 2 ) . Ordinary 7s ( 72-globulin) antibodies are not affected(3) unless a denaturing agent is used in conjunction with SH treatment (4). Since the effect of ,SH compounds on the activity of human reagins was unknown, this investigation was undertaken. Data will be presented to show that in vitro SH treatment of the reagin-containing sera of allergic patients destroys the skin-sensitizing activity of these sera.Materials and methods. After screening donors for a history of hepatitis, Wassermann negative sera from 16 allergic children and adults were selected for their ability to passively sensitize non-atopic human skin to subsequent challenge with the appropriate allergen (s) (the Prausnitz-Kustner or P-K reaction). The sera were sterilized by Seitz or Millipore filtration and stored at 4°C. The reagins represented included those to pollens, animal danders, molds and foods. The thiols employed in the majority of experiments were /I-mercaptoethanol (ME) and DL-penicillamine ; L-cysteine and /I-mercaptoethylamine (MEA) were also used in certain later studies.In a typical experiment, to each allergic serum diluted 1 :3 with triethanolamine buffered saline (TBS) (pH 7.35, p = 0.15) was added an equal volume of 0.2M thiol which had been freshly prepared in TBS and adjusted, when necessary, to pH 7.2-7.3. Thus, the final thiol concentration was 0.1M and the find serum dilution was 1:6, giving a protein concentration of approximately 1%. Untreated controls consisted of aliquots of the same sera diluted equally with TBS alone and thereafter handled identically to the thioltreated aliquots. Incubation in sterile, closed tubes was usually at room temperature for 24-48 hours in experiments with ME or MEA, and at 37°C for 24 hours with penicillamine or cysteine. After incubation, treated and control sera were dialyzed (separately) for 36-48 hours at 4°C against TBS with sufficient changes to reduce the calculated thiol concentration to 1WM or less. The dialyzed sera were then sterilized by Millipore or Seitz filtration. After 48 hours were allowed for bacterial cultures, bioassays were begun.Antibody activity in the control and treated sera was assayed, usually without further dilution of the sera, by the P-K reaction in normal, non-allergic human volunteers unreactive in direct intradermal skin tests with the allergens concerned. Each treated serum and its untreated control were uniformly by guest on July 29, 2015 ebm.s...

Slowly Sedimenting Mercaptoethanol-resistant Antinuclear Factors Related Antigenically to M Immunoglobulins (γ1M-Globulin) in Patients with Systemic Lupus Erythematosus *

“…However, double diffusion studies were performed with the Sephadex fractions from seven of the sera; the results are listed in Table II were nine males, four of whom had the unusual ANF. All eight patients with the unusual ANF had positive LE cell preparations on the same sample of blood, whereas only 16 of the 45 (4,5,7,8,26). In previous reports on ANF's, relatively small numbers of sera have been studied, and only limited attempts have been made to characterize them further.…”

“…fraction, although some have sedimentation coefficients ranging from 10 to 15-S, and their antibody activity is frequently destroyed by treatment with thiol compounds (3)(4)(5). Antibody activity has recently also been reported to be associated with certain low molecular weight urine y-globulins (6) and with proteins with intermediate sedimentation coefficients whose precise nature remains to be determined (7,8).…”

The Properties of Fragments of Skin-Sensitizing and Blocking Antibodies as Revealed by the Prausnitz-Küstner, Passive Cutaneous Anaphylaxis and Hemagglutination Reactions

“…Congr. of Allergology, New York, 19C1 (7) and were quoted in a recent publication by I.eddy et al (8). ** These sera will be referred to hereafter as human immune sera.…”

In vitro and in vivo inactivation of reagins with mercaptans

“…Skin-sensitizing antibodies, unlike immune antibodies (IgG), are sensitive to reducing agents at low concentrations and are inactivated by treatment with 0-1 M mercaptoethanol (Leddy et al, 1962;Gyenes et ai, 1964). If reagins could be inactivated in vivo by the administration of mercaptans to man, without concomitant inactivation of blocking antibody or without changing other vital functions, this procedure might have therapeutic potential for the treatment of allergic conditions mediated by skin-sensitizing antibodies.…”

“…If reagins could be inactivated in vivo by the administration of mercaptans to man, without concomitant inactivation of blocking antibody or without changing other vital functions, this procedure might have therapeutic potential for the treatment of allergic conditions mediated by skin-sensitizing antibodies. The possibility that reagins might be destroyed or inactivated in vivo by the administration of mercaptans, a procedure which had been previously shown to be effective in vitro with mercaptoethanol (Leddy et al, 1962;Gyenes et al, 1964), has been explored in this study.…”