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10. A simple technique for assaying alpha-1 antitrypsin activity
VOLUME 47 NUMBER 2
Abstracts of papers
89
1970). In the present study, these findings were confirmed with sera from individuals showing immediate hypersensitivity reactions to a variety of pollens, drugs, foods, organic dusts, and parasites. Characteristic morphological changes in rat peritoneal mast cells were observed in the presence of both allergic sera and antigen. The cytologic factor in these sera had physiochemica,! properties similar to those of IgE-type reagins. In addition, cytologic changes took place in the presence of either allergic sera or two IgE myeloma proteins and monospecific anti-IgE sera, whereas mast cells incubated with allergic sera failed to give a response on challenge with antisera to human IgG, IgA, IgM, or IgD immunoglobulins. To determine whether IgE-type reagins bind specifically to rat mast cells, an immunofluorescent study was undertaken. Mast cells sensitized with ragweedallergic sera showed specific immunofluorescence with antihuman IgE or antihuman lightchain (K) globulin preparations tagged with fluorescein-isothiocyanate, whereas similarly tagged antihuman IgG, IgA, IgM, or IgD globulin preparations were inactive in this respect. Whether the specific cytologic changes in the rat mast cells are due primarily to (1) cell-bound reagin interacting with allergen or (2) preformed reagin-allergen complexes binding to these cells is presently under investigation.
9. Demonstration of passive sensitization of monkey mast cells by IgE. Kimishige Ishizaka, M.D., Teruko Ishizaka, M.D., and Hisao Tomioka, M.D., Baltimore, Md. Localization of IgE in monkey tissues was studied with the use of E myeloma protein. When isil-labeled E myeloma protein was injected into monkey skin, 90 to 95 per cent of the protein disappeared from the skin site within 2 to 3 days. However, disappearance rate of the protein after this period became very slow. The time for a half loss of E myeloma protein from the site was 8 to 10 days, as compared with about 2 days for IgG. When the skin sites sensitized with E myeloma protein were challenged with is^l-labeled anti-IgE and the tissues were examined by autoradiography, the antibody was detected on mast cells. Binding of IgE with mast cells was demonstrated in other tissues. When monkeys were sensitized with an intravenous injection of E myeloma protein and tissues from the sensitized animal were treated with i25i-anti-IgE in the presence of ethylenediaminetetraacetic acid (EDTA), mast cells in omentum and in the lamina propria of intestine were labeled. Cell suspensions were obtained from lung tissues of the sensitized monkeys and treated with i25l-anti-IgE or -anti-IgG. Mast cells in the suspension were labeled with anti-IgE, whereas macrophages and neutrophils but not mast cells were labeled with anti-IgG. Incubation of such cell suspensions with the antibody specific for Fd portion of the E myeloma protein resulted in the release of both histamine and slow-reacting substance of anaphylaxis (SES-A). The results suggest that the release of both chemical mediators is caused by the combination of the antibody with mast cell-bound E myeloma protein.
10. A simple technique for assaying alpha-1 antitrypsin activity. Guillermo V. Villacorte, M.D., M. Eleanor Mischel, O.S.B., and Robert G. Townley, M.D., Omaha, Neb. Detection of qualitative and quantitative deficiency of serum alpha-1 antitrypsin is important in hereditary emphysema. The present metholology is cumbersome. A simple, rapid, semiquantitative technique based on the ability of alpha-1 antitrypsin to inhibit trypsin digestion of denatured fibrinogen in an agar plate (a modification of Cawley's method) has therefore been developed. Wells in the fibrinogen-agar plates were filled with 2 fd aliquots of previously incubated 1:1 serum-trypsin mixtures. After 4 hours of incuba-
90
American Academy of Allergy
J. ALLERG. FEBRUARY 1971
tion in a moist chamber, the diameters of fibrinolysis were measured, and percentages of inhibition were calculated relative to the diameter of the 1:1 saline-trypsin standard. In general, results parallel both the serum concentrations as determined by single radial immunodiffusion and trypsin inhibitory capacity, as assayed by the method of Erickson, of alpha-1 antitrypsin in a representative group of patients and normal control subjects. The degree of inhibition of trypsin activity ranged from 0 to 1.5 per cent in homozygous subjects; 3 to 9 per cent in heterozygous subjects; and 15 to 24 per cent in normal subjects. Thus, it would appear that this technique can easily distinguish normal individuals from both the homozygous and heterozygous persons, although it can not as readily separate the latter two from one another.
11. The effect of intermittent positive-pressure breathing on airway resistance in normal and asthmatic children. Ralph E. Moore, M.D., Margo A . Pinney, B.S.N., and Ernest K. Cotton, M.D., Denver, Colo. Mechanical irritation of the upper airway is known to increase total lung resistance. This study examines the effect of mechanical stimulation as initiated by intermittent positive-pressure breathing (IPPB) on the airways of normal and asthmatic children. The effect of IPPB on airway resistance was studied in 8 normal and 20 asthmatic children. IPPB with water was administered with a Handi Vent at a pressure of 15 em. of HjO for 30 breaths, and on another study day mist was administered by a compressor-driven DeVilbiss No. 40 nebulizer for 30 breaths. Airway resistance (EAW) and thoracic gas volume (TGV) were measured in a body plethysmograph. The product of airway resistance and thoracic gas volume. RAW X TGV, was used as a manifestation of variation in airway resistance. Airway resistance was measured before, immediately, and 15 minutes after treatment. Two of the 8 normal patients had a significant increase in resistance following IPPB. There was no significant change in resistance following the DeVilbiss nebulizer treatment in normal patients. In the asthmatic group, 9 of 16 patients who received IPPB had a significant increase in airway resistance. Only 2 of 13 patients who received mist by the DeVilbiss nebulizer had a significant increase in airway resistance. The results indicate that IPPB treatment may cause an increase in airway resistance in both normal and asthmatic children.
12. Mouse "asthma": Induction after pertussis vaccination. John T. Connell, M.D., New York, N. Y . Mice are resistant to histamine but become anaphylactically sensitive to it after pertussis vaccination. Will they also exhibit bronchospasm to sublethal doses of histamine? I determined pulmonary resistance (R) in mice by measuring alveolar pressure ( P A ) at known air flow rates (V): R =A^-
B was determined in 6 Webster mice during a control
period and every 10 minutes for 2 hours after intramuscular injection of 0.4 mg. of histamine base. E was unchanged after histamine, i.e., bronchospasm did not occur. The animals were then given 0.5 c.c. of pertussis vaccine intramuscularly. Three days later 0.4 mg. of histamine intramuscularly was repeated and B determined. R did not change in 5 mice but increased slightly in one. Repetition of histamine on the eighth day after pertussis caused a 100 per cent increase in R in 5 mice and a 25 per cent increase in the sixth. Bronchial obstruction was greatest 30 to 80 minutes after histamine and was reversible. The results suggest that bronchial sensitivity of mice to histamine increases after pertussis vaccination. Five of the 6 mice were challenged 8 days after pertussis vaccination with aerosols of histamine. My present aerosol technique is crude and amounts actually inhaled are
Abstracts of papers
89
1970). In the present study, these findings were confirmed with sera from individuals showing immediate hypersensitivity reactions to a variety of pollens, drugs, foods, organic dusts, and parasites. Characteristic morphological changes in rat peritoneal mast cells were observed in the presence of both allergic sera and antigen. The cytologic factor in these sera had physiochemica,! properties similar to those of IgE-type reagins. In addition, cytologic changes took place in the presence of either allergic sera or two IgE myeloma proteins and monospecific anti-IgE sera, whereas mast cells incubated with allergic sera failed to give a response on challenge with antisera to human IgG, IgA, IgM, or IgD immunoglobulins. To determine whether IgE-type reagins bind specifically to rat mast cells, an immunofluorescent study was undertaken. Mast cells sensitized with ragweedallergic sera showed specific immunofluorescence with antihuman IgE or antihuman lightchain (K) globulin preparations tagged with fluorescein-isothiocyanate, whereas similarly tagged antihuman IgG, IgA, IgM, or IgD globulin preparations were inactive in this respect. Whether the specific cytologic changes in the rat mast cells are due primarily to (1) cell-bound reagin interacting with allergen or (2) preformed reagin-allergen complexes binding to these cells is presently under investigation.
9. Demonstration of passive sensitization of monkey mast cells by IgE. Kimishige Ishizaka, M.D., Teruko Ishizaka, M.D., and Hisao Tomioka, M.D., Baltimore, Md. Localization of IgE in monkey tissues was studied with the use of E myeloma protein. When isil-labeled E myeloma protein was injected into monkey skin, 90 to 95 per cent of the protein disappeared from the skin site within 2 to 3 days. However, disappearance rate of the protein after this period became very slow. The time for a half loss of E myeloma protein from the site was 8 to 10 days, as compared with about 2 days for IgG. When the skin sites sensitized with E myeloma protein were challenged with is^l-labeled anti-IgE and the tissues were examined by autoradiography, the antibody was detected on mast cells. Binding of IgE with mast cells was demonstrated in other tissues. When monkeys were sensitized with an intravenous injection of E myeloma protein and tissues from the sensitized animal were treated with i25i-anti-IgE in the presence of ethylenediaminetetraacetic acid (EDTA), mast cells in omentum and in the lamina propria of intestine were labeled. Cell suspensions were obtained from lung tissues of the sensitized monkeys and treated with i25l-anti-IgE or -anti-IgG. Mast cells in the suspension were labeled with anti-IgE, whereas macrophages and neutrophils but not mast cells were labeled with anti-IgG. Incubation of such cell suspensions with the antibody specific for Fd portion of the E myeloma protein resulted in the release of both histamine and slow-reacting substance of anaphylaxis (SES-A). The results suggest that the release of both chemical mediators is caused by the combination of the antibody with mast cell-bound E myeloma protein.
10. A simple technique for assaying alpha-1 antitrypsin activity. Guillermo V. Villacorte, M.D., M. Eleanor Mischel, O.S.B., and Robert G. Townley, M.D., Omaha, Neb. Detection of qualitative and quantitative deficiency of serum alpha-1 antitrypsin is important in hereditary emphysema. The present metholology is cumbersome. A simple, rapid, semiquantitative technique based on the ability of alpha-1 antitrypsin to inhibit trypsin digestion of denatured fibrinogen in an agar plate (a modification of Cawley's method) has therefore been developed. Wells in the fibrinogen-agar plates were filled with 2 fd aliquots of previously incubated 1:1 serum-trypsin mixtures. After 4 hours of incuba-
90
American Academy of Allergy
J. ALLERG. FEBRUARY 1971
tion in a moist chamber, the diameters of fibrinolysis were measured, and percentages of inhibition were calculated relative to the diameter of the 1:1 saline-trypsin standard. In general, results parallel both the serum concentrations as determined by single radial immunodiffusion and trypsin inhibitory capacity, as assayed by the method of Erickson, of alpha-1 antitrypsin in a representative group of patients and normal control subjects. The degree of inhibition of trypsin activity ranged from 0 to 1.5 per cent in homozygous subjects; 3 to 9 per cent in heterozygous subjects; and 15 to 24 per cent in normal subjects. Thus, it would appear that this technique can easily distinguish normal individuals from both the homozygous and heterozygous persons, although it can not as readily separate the latter two from one another.
11. The effect of intermittent positive-pressure breathing on airway resistance in normal and asthmatic children. Ralph E. Moore, M.D., Margo A . Pinney, B.S.N., and Ernest K. Cotton, M.D., Denver, Colo. Mechanical irritation of the upper airway is known to increase total lung resistance. This study examines the effect of mechanical stimulation as initiated by intermittent positive-pressure breathing (IPPB) on the airways of normal and asthmatic children. The effect of IPPB on airway resistance was studied in 8 normal and 20 asthmatic children. IPPB with water was administered with a Handi Vent at a pressure of 15 em. of HjO for 30 breaths, and on another study day mist was administered by a compressor-driven DeVilbiss No. 40 nebulizer for 30 breaths. Airway resistance (EAW) and thoracic gas volume (TGV) were measured in a body plethysmograph. The product of airway resistance and thoracic gas volume. RAW X TGV, was used as a manifestation of variation in airway resistance. Airway resistance was measured before, immediately, and 15 minutes after treatment. Two of the 8 normal patients had a significant increase in resistance following IPPB. There was no significant change in resistance following the DeVilbiss nebulizer treatment in normal patients. In the asthmatic group, 9 of 16 patients who received IPPB had a significant increase in airway resistance. Only 2 of 13 patients who received mist by the DeVilbiss nebulizer had a significant increase in airway resistance. The results indicate that IPPB treatment may cause an increase in airway resistance in both normal and asthmatic children.
12. Mouse "asthma": Induction after pertussis vaccination. John T. Connell, M.D., New York, N. Y . Mice are resistant to histamine but become anaphylactically sensitive to it after pertussis vaccination. Will they also exhibit bronchospasm to sublethal doses of histamine? I determined pulmonary resistance (R) in mice by measuring alveolar pressure ( P A ) at known air flow rates (V): R =A^-
B was determined in 6 Webster mice during a control
period and every 10 minutes for 2 hours after intramuscular injection of 0.4 mg. of histamine base. E was unchanged after histamine, i.e., bronchospasm did not occur. The animals were then given 0.5 c.c. of pertussis vaccine intramuscularly. Three days later 0.4 mg. of histamine intramuscularly was repeated and B determined. R did not change in 5 mice but increased slightly in one. Repetition of histamine on the eighth day after pertussis caused a 100 per cent increase in R in 5 mice and a 25 per cent increase in the sixth. Bronchial obstruction was greatest 30 to 80 minutes after histamine and was reversible. The results suggest that bronchial sensitivity of mice to histamine increases after pertussis vaccination. Five of the 6 mice were challenged 8 days after pertussis vaccination with aerosols of histamine. My present aerosol technique is crude and amounts actually inhaled are