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27B. Nonimmunologic inhibition of B. subtilis alkaline proteinases by human serum components
98
American Academy of Allergy
J. ALLER6. FEBRUARY 1971
and immunologic features of this disease, 249 workers in a detergent factory were evaluated. Sixty-eight employees had clear-cut respiratory symptoms of either rhinitis or asthma on exposure to B. suMilis enzyme. Of these 68 workers, 62 were atopic and 58 had positive immediate skin reactivity to prick tests with 0.05 or 0.50 mg. per milliliter of B. subtilis enzyme. Of the 181 asymptomatic workers, 60 had positive skin reactivity to the enzyme. Only one of 50 atopic patients in a general allergy clinic manifested a positive skin reaction to 5.0 mg. per milliliter of enzyme solution. The skin-sensitizing antibody to B. suhtilis enzyme was heat labile and was successfully transferred to the skin of man and transferred systemically to monkey. Precipitating antibody to the enzyme was found in serum of several workers but in no higher proportion than normal control subjects. Pulmonary functions tests in several acutely ill workers showed marked airway obstruction. Follow-up tests performed 2 weeks to 6 months after cessation of symptoms showed only mild hyperinflation. An "epidemic" of bronchospasm involving a large number of workers was traced to the freezing over of an air duet on the plant roof. Environmental precautions including twice-a-shift air monitoring, enclosure, and automation of several operations, and use of space suits, disposable gloves, and masks has resulted in a marked decline in respiratory symptoms.
27A. Antigenic composition of B. subfilis enzyme preparations. Jerry Dolovich, M.D., Maria Teresa Debanne, Ph.D., and Victoria Monte-Wicher, Ph.D., Hamilton, Ontario, Canada. The numbers of antigenic components, some quantitative relationships, and the presence of proteinase and amylase activities in B. subtilis enzyme preparations from diverse sources have been examined. Antisera to water-soluble portions of two nonpurified preparations used commercially and herein designated NPPA and NPPB and antisera to purified preparations were produced in rabbits. Antigenic composition was examined qualitatively by double diffusion in agar and Immunoelectrophoresis and quantitatively with monospecific antisera and purified reference antigens in the single radial diffusion method. Proteinase and amylase activities were detected with alpha casein and hydrolysed starch substrates, respectively. NPPA contained greater than 4 antigenic components including subtilopeptidase A (SPA) as the predominant alkaline proteinase (AP) and a trace quantity of amylase. There were greater than 5 antigenic components including subtilisin B (SLB), the predominant AP, and amylase in NPPB. There is no cross-reactivity between SPA and SLB. Two crystalline preparations of SPA and one of SLB each contained one readily detectable antigen shown to be proteinase by the demonstration of caseinolytic activity in precipitin lines; there was no amylase. Two SLB preparations described as crystalline and a third as purified all contained 3 or more antigens; amylase was present in 2. In addition to these components, all AP preparations, with one exception, contained trace quantities of the alternate AP. The heterogeneity of these preparations must be considered in any interpretations of allergic reactions to B. suhtilis preparations.
27B. Nonimmunologic inhibition of B. subtilis alkaline proteinases by human serum components. Victoria Wicher, Ph.D., and Jerry Dolovich, M.D., Hamilton, Ontario, Canada. Observed interactions of the bacillopeptidases, subtilopeptidase A (SPA) and subtilisin B (SLB), with human serum aj-macroglobulin (a^-M.) and Oj-a-ntitrypsin (dj-AT) are considered to be potential determinants of the allergenicity of these enzymes and the reac-
VOLUME i,7 NUMBER 2
Abstracts of papers
99
tions they produce. The binding of SPA and SLB to a2-M and cti-AT and the resulting patterns of inhibition are described. i25l-labeled SPA and SLB were utilized in an examination of binding. Normal human serum (NHS) fractions were prepared by gel filtration on Sephadex G-200. Proteolytic activity was detected with an alpha casein substrate. 1251-iabeled SPA and SLB were found to bind to the cij-M and ai-AT immunoelectrophoretio arcs of NHS. The antigenic reactivity of SPA is retained in the aj-M-SPA complex. dj-M- and ai-AT-containing NHS fractions inhibit the caseinolytic activities of SPA and SLB, and the inhibitory components have a, and aj mobilities, respectively. arATdeficient sera have diminished inhibitory capacities. Electrophoresis preparations of proteinase-serum fraction mixtures developed for proteinase activtiy revealed that the aj-MSPA complex has residual proteinase activity which is resistant to inhibition by ai-AT. B. subtilis alkaline proteinases are bound and inactivated by aj-M and aj-AT. The SPA of the a2-M-complex retains antigenic reactivity and has residual proteinase activity which resists inhibition by aj-AT. These interactions may have profound effects upon the allergenicity and reactivity of these enzymes.
28. The autoantigenic determinant groups in thyroid autoallergy. Sidney Shtilman, Ph.D., and Mohammad Ghayasuddin, Ph.D., New York, N. Y. The antigenic composition of the thyroglobulin (Tg) molecule has been explored in some detail, especially with regard to autoallergy to the thyroid gland. It had been shown that there is a greater reactivity of Tg with heteroantibodies as compared to autoantibodies, suggesting the presence of at least two kinds of antigenic determinant groups on this molecule. Enzymatic hydrolysis has now been used to obtain fragments that might differ in bearing the heteroantigenic and the autoantigenic determinants of the parent molecule. Babbit Tg was treated with trypsin at 37° C. in a ratio of 1:100, After 10 hours the digestion was stopped by adding soybean inhibitor. This digest, in the ultracentrifuge, revealed three components; the major boundary, nearly 90 per cent of the total protein, had a sedimentation coefficient of 1.2S. Sephadex gel filtration, using Gr-200 and then G-lOO, was used to purify these fragments. Several fractions have been characterized by various immunochemical methods, as well as by ultracentrifugation and zone electrophoresis. When tested against the two types of antisera, one fraction reacted against both kinds of serum, giving four lines of precipitation against the heteroantibodies, but only one line against the autoantibodies. These and other observations have confirmed our hypothesis that there is on the thyroglobulin molecule a smaller number of antigenic determinants that can precipitate with autoantibodies as compared to heteroantibodies.
29. Comparative effects of immunization of rabbits with human thyroglobulin and human and rabbit thyroid microsomal fractions. G. N. Beall, M.D., I. J. Chopra, M.D., and D. H. Solomon, M.D., Torrance, Calif. We have previously immunized rabbits with materials derived from the human thyroid in an attempt to produce an experimental model of Graves' disease. Such rabbits developed a thyroid-stimulating globulin in their serum, but similarly immunized baboons did not; instead they developed a severe thyroiditis. In an effort to delineate these phenomena further, we immunized rabbits with human thyroglobulin (HTg) and human and rabbit thyroid microsomal fractions. The sera were assayed for thyroid-stimulating substances with the McKenzie bioassay. Five of 10 animals immunized with human thyroglobulin developed histological evidence of thyroiditis, but none had thyroid-stimulating activity in its serum. Only one of 26 animals immunized with rabbit or human thyroid microsomal fractions had any histological abnormality in the thyroid, but the sera of 8 gave positive McKenzie bioassay responses. Although the frequency of the latter response was not greater after immunization with rabbit as compared to human thyroid microsomal fraction.
American Academy of Allergy
J. ALLER6. FEBRUARY 1971
and immunologic features of this disease, 249 workers in a detergent factory were evaluated. Sixty-eight employees had clear-cut respiratory symptoms of either rhinitis or asthma on exposure to B. suMilis enzyme. Of these 68 workers, 62 were atopic and 58 had positive immediate skin reactivity to prick tests with 0.05 or 0.50 mg. per milliliter of B. subtilis enzyme. Of the 181 asymptomatic workers, 60 had positive skin reactivity to the enzyme. Only one of 50 atopic patients in a general allergy clinic manifested a positive skin reaction to 5.0 mg. per milliliter of enzyme solution. The skin-sensitizing antibody to B. suhtilis enzyme was heat labile and was successfully transferred to the skin of man and transferred systemically to monkey. Precipitating antibody to the enzyme was found in serum of several workers but in no higher proportion than normal control subjects. Pulmonary functions tests in several acutely ill workers showed marked airway obstruction. Follow-up tests performed 2 weeks to 6 months after cessation of symptoms showed only mild hyperinflation. An "epidemic" of bronchospasm involving a large number of workers was traced to the freezing over of an air duet on the plant roof. Environmental precautions including twice-a-shift air monitoring, enclosure, and automation of several operations, and use of space suits, disposable gloves, and masks has resulted in a marked decline in respiratory symptoms.
27A. Antigenic composition of B. subfilis enzyme preparations. Jerry Dolovich, M.D., Maria Teresa Debanne, Ph.D., and Victoria Monte-Wicher, Ph.D., Hamilton, Ontario, Canada. The numbers of antigenic components, some quantitative relationships, and the presence of proteinase and amylase activities in B. subtilis enzyme preparations from diverse sources have been examined. Antisera to water-soluble portions of two nonpurified preparations used commercially and herein designated NPPA and NPPB and antisera to purified preparations were produced in rabbits. Antigenic composition was examined qualitatively by double diffusion in agar and Immunoelectrophoresis and quantitatively with monospecific antisera and purified reference antigens in the single radial diffusion method. Proteinase and amylase activities were detected with alpha casein and hydrolysed starch substrates, respectively. NPPA contained greater than 4 antigenic components including subtilopeptidase A (SPA) as the predominant alkaline proteinase (AP) and a trace quantity of amylase. There were greater than 5 antigenic components including subtilisin B (SLB), the predominant AP, and amylase in NPPB. There is no cross-reactivity between SPA and SLB. Two crystalline preparations of SPA and one of SLB each contained one readily detectable antigen shown to be proteinase by the demonstration of caseinolytic activity in precipitin lines; there was no amylase. Two SLB preparations described as crystalline and a third as purified all contained 3 or more antigens; amylase was present in 2. In addition to these components, all AP preparations, with one exception, contained trace quantities of the alternate AP. The heterogeneity of these preparations must be considered in any interpretations of allergic reactions to B. suhtilis preparations.
27B. Nonimmunologic inhibition of B. subtilis alkaline proteinases by human serum components. Victoria Wicher, Ph.D., and Jerry Dolovich, M.D., Hamilton, Ontario, Canada. Observed interactions of the bacillopeptidases, subtilopeptidase A (SPA) and subtilisin B (SLB), with human serum aj-macroglobulin (a^-M.) and Oj-a-ntitrypsin (dj-AT) are considered to be potential determinants of the allergenicity of these enzymes and the reac-
VOLUME i,7 NUMBER 2
Abstracts of papers
99
tions they produce. The binding of SPA and SLB to a2-M and cti-AT and the resulting patterns of inhibition are described. i25l-labeled SPA and SLB were utilized in an examination of binding. Normal human serum (NHS) fractions were prepared by gel filtration on Sephadex G-200. Proteolytic activity was detected with an alpha casein substrate. 1251-iabeled SPA and SLB were found to bind to the cij-M and ai-AT immunoelectrophoretio arcs of NHS. The antigenic reactivity of SPA is retained in the aj-M-SPA complex. dj-M- and ai-AT-containing NHS fractions inhibit the caseinolytic activities of SPA and SLB, and the inhibitory components have a, and aj mobilities, respectively. arATdeficient sera have diminished inhibitory capacities. Electrophoresis preparations of proteinase-serum fraction mixtures developed for proteinase activtiy revealed that the aj-MSPA complex has residual proteinase activity which is resistant to inhibition by ai-AT. B. subtilis alkaline proteinases are bound and inactivated by aj-M and aj-AT. The SPA of the a2-M-complex retains antigenic reactivity and has residual proteinase activity which resists inhibition by aj-AT. These interactions may have profound effects upon the allergenicity and reactivity of these enzymes.
28. The autoantigenic determinant groups in thyroid autoallergy. Sidney Shtilman, Ph.D., and Mohammad Ghayasuddin, Ph.D., New York, N. Y. The antigenic composition of the thyroglobulin (Tg) molecule has been explored in some detail, especially with regard to autoallergy to the thyroid gland. It had been shown that there is a greater reactivity of Tg with heteroantibodies as compared to autoantibodies, suggesting the presence of at least two kinds of antigenic determinant groups on this molecule. Enzymatic hydrolysis has now been used to obtain fragments that might differ in bearing the heteroantigenic and the autoantigenic determinants of the parent molecule. Babbit Tg was treated with trypsin at 37° C. in a ratio of 1:100, After 10 hours the digestion was stopped by adding soybean inhibitor. This digest, in the ultracentrifuge, revealed three components; the major boundary, nearly 90 per cent of the total protein, had a sedimentation coefficient of 1.2S. Sephadex gel filtration, using Gr-200 and then G-lOO, was used to purify these fragments. Several fractions have been characterized by various immunochemical methods, as well as by ultracentrifugation and zone electrophoresis. When tested against the two types of antisera, one fraction reacted against both kinds of serum, giving four lines of precipitation against the heteroantibodies, but only one line against the autoantibodies. These and other observations have confirmed our hypothesis that there is on the thyroglobulin molecule a smaller number of antigenic determinants that can precipitate with autoantibodies as compared to heteroantibodies.
29. Comparative effects of immunization of rabbits with human thyroglobulin and human and rabbit thyroid microsomal fractions. G. N. Beall, M.D., I. J. Chopra, M.D., and D. H. Solomon, M.D., Torrance, Calif. We have previously immunized rabbits with materials derived from the human thyroid in an attempt to produce an experimental model of Graves' disease. Such rabbits developed a thyroid-stimulating globulin in their serum, but similarly immunized baboons did not; instead they developed a severe thyroiditis. In an effort to delineate these phenomena further, we immunized rabbits with human thyroglobulin (HTg) and human and rabbit thyroid microsomal fractions. The sera were assayed for thyroid-stimulating substances with the McKenzie bioassay. Five of 10 animals immunized with human thyroglobulin developed histological evidence of thyroiditis, but none had thyroid-stimulating activity in its serum. Only one of 26 animals immunized with rabbit or human thyroid microsomal fractions had any histological abnormality in the thyroid, but the sera of 8 gave positive McKenzie bioassay responses. Although the frequency of the latter response was not greater after immunization with rabbit as compared to human thyroid microsomal fraction.