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31. Isolation and characterization of the dialysate from Bermuda grass pollen
100
American Academy of Allergy
J. AUERG. FEBRUARY 1971
there was a significant increase in serum free T, in the group immunized with rabbit tissue. In vitro incorporation of i^C-amino acids into gamma globulin by thyroid glands from the HTg-immunized animals exceeded incorporation by control thyroids, but the difference fell short of statistical significance. Thyroiditis and the production of materials that affect the McKenzie bioassay appear to be completely separable phenomena. This study provides further evidence that thyroglobulin is not the important antigen leading to the production of thyroid-stimulating globulin.
30. Effects of 6-mercaptopurine on guinea pig cellular and humoral immune mechanisms. S. Michael Phillips, M.D., Boston, Mass., and Burton Zweiman, M.D., Philadelphia, Pa. We have previously reported that lymphocytes of 6-mercaptopurine (6-MP)-treated guinea pigs with depressed tuberculin hypersensitivity showed normal in vitro proliferative responses to phytohemagglutinin (PHA) or tuberculin. However, there were some defects in monocyte-macrophage function. This report describes continuing studies of 6-MP immunosuppressive mechanisms. After base-line studies and injection of complete Freund's adjuvant on Day 0, Group A received 6-MP, 40 mg. per kilogram on Days 4 to 14; Group B received placebo. Results: Similar responses for Groups A and B in: (1) weekly patterns of in vitro lymphocyte responses to PHA and tuberculin; (2) migration inhibitory factor (MIF) production by lymphocytes incubated with tuberculin; (3) circulating antibody production and in vitro lymphocyte proliferative responses to soluble antigen (human gamma globulin, HGG) given on Day 0, though delayed skin test reaction to this antigen suppressed in Group A; (4) wheal and flare responses to histamine and compound 48-80. Different responses between Groups A and B in: (A) depressed in vivo monocyte-macrophage kinetics in Group A measured by isotope uptake; (B) depletion of certain areas in regional nodes of Group A; (C) "rebound" in immune responsiveness in group A after 6-MP stopped. In 13 of 14 of Group A, tuberculin skin test reactivity was reconstituted shortly after receipt of marrow (but not node) cells from normal unsensitized donors. Isotope studies show many inflammatory cells of donor origin in these skin test reactions. Results suggest a greater suppressive effect of 6-MP on cellular rather than humoral immunity, with a major action on effector inflammatory cells of marrow origin. 3 1 . Isolation and characterization of the dialysate from Bermuda grass pollen.
Roberta L. Meyers, Ph.D., Los Angeles, Calif., Aline "W. Bems-Mason, B.A., Kent H. Thayer, M.D., Bruce H. Feldman, M.D., and Harold N. Eosengren, M.D., Long Beach, Calif. Previous studies on the isolation of haptenic material from the dialysates of timothy grass pollen (Malley, J. Immun. 93: 420, 1964) and ragweed pollen (Meyers, Fed. Proc. 25: 729, 1966) led us to investigate whether haptenic material occurs in Bermuda grass pollen, which is the major allergen in the southwestern United States. Bermuda grass pollen (Cynodon daotylon) was extracted with water at neutral pH and the dialysate recovered. Sephadex G-25 or Sephadex G-50 chromatography of the dialysate gave two peaks, I and II. Peak I reacted with rabbit anti-water soluble cynodon (WSC) and rabbit anti-whole cynodon dialysate (WCD) in Ouchterlony gel diffusion, whereas peak I I did not react with the antisera. Peak I I inhibited precipitation of WSC and rabbit antiWSC in Ouchterlony gel diffusion and inhibited the quantitative precipitation reaction between WSC and anti-WSC. Patients sensitive to cynodon reacted to peak I just as strongly as to a standard cynodon extract. However, they had a markedly reduced skin test reactivity to peak II. The
VOLUME 47 NUMBER 2
Abstracts of papers
101
results indicate the haptenic nature of the Sephadex G-50 peak II from the dialysate of eynodon extract is similar to that found in timothy and ragweed pollens. 3 2 . Eosinophilic leukocytes and antigen-antibody complexes. T a k e r u Ishikawa, M.D., K o n r a d Wicher, Ph.D., a n d C a r l E . Arbesman, M.D., Buffalo, N . Y. The attraction of eosinophils to antigen-antibody (Ag-Ab) complexes composed of parasitic helminths extracts and their corresponding antibodies has been reported. It was of interest to study this phenomenon employing allergen-antibody or immunoglobulin-antibody complexes. Soluble Ag-Ab complexes were prepared with antigens of ragweed, bovine gamma globulin, and crystallized egg albumin and their homologous rabbit antisera. In addition, ragweed extract was combined with sera from ragweed-sensitive patients. Blood from six different individuals (two were atopic) with high eosinophilia was used as the source for these cells. The eosinophils were washed and mixed with the Ag-Ab complexes, the antigen alone, and the antisera alone. The attraction of the eosinophils to the above reagents was examined by immunofluorescent technique using conjugated guinea pig antiserum to rabbit IgQ- and (conjugated) rabbit antisera to human immunoglobulins (Gr, A, M, D, E). To avoid nonspecific staining, the eosinophils were treated with bovine serum albumin conjugated with Bhodamine B prior to application of various conjugates. Only the rabbit and human Ag-Ab complexes were detected on the eosinophils. Neither the antigens nor antibodies alone could be detected. Only anti-IgG and -IgM conjugates demonstrated fluorescence in the human system. Similar results were obtained using radioautography.
33. Measurement of binding of tubercle bacilli components by antibodies in sera from tuberculous and nontuberculous individuals. Emil J. Bardana, Jr., M.D., Percy Minden, M.D., J. Kenneth McClatchy, Ph.D., and Richard S. Farr, M.D., Denver, Colo. Previous studies from this laboratory employing radioimmunodiffusion indicated that antibodies to partially purified isolates from tubercle bacilli were present in sera from tuberculous patients and from some normal control subjects. Since similar findings have been reported with other tests, studies were undertaken using a primary binding test to quantitate this binding. Heat-killed suspensions of Mycohacterium tuberculosis strain II37Kv were sonicated and centrifuged. The supernate was treated with manganese chloride and cetyl pyridinium chloride (CPC) to precipitate nucleic acids and acidic polysaccharides, respectively. The new supernate (CPC-S) was labeled with i3il (isiI-CPC-S) and was the test antigen. Antigen-antibody complexes in a 1:10 dilution of serum were precipitated by heterologous antihuman IgG. Eesults were expressed as the percentage of isiI-CPC-S precipitated. The per cent of antigen bound by all the sera tested ranged from only 8.0 to 19.6 per cent. However, sera from 24 of 27 patients with active tuberculosis bound more than 15 per cent, whereas sera from 16 of 20 healthy individuals and sera from 14 of 17 patients with chronic nontuberculous diseases bound less than 15 per cent. It would appear, therefore, that 131I-CPC-S consists of a number of components of which only a small portion are critical to the measurement of specific antibodies from tuberculous patients. It is likely that some of the antigen-binding capacities of some of the control sera may have resulted from subclinical tuberculous infections, exposure to saprophytic mycobacteria, and/or the exposure to antigenic groups shared by mycobacteria and non-acid-fast bacteria. Studies using more highly purified antigens are in progress to investigate these possibilities.
34. Ciliocytophthoria and differential diagnosis of asthma. Harold R. Styler, M.D., Santa Barbara, Calif.
American Academy of Allergy
J. AUERG. FEBRUARY 1971
there was a significant increase in serum free T, in the group immunized with rabbit tissue. In vitro incorporation of i^C-amino acids into gamma globulin by thyroid glands from the HTg-immunized animals exceeded incorporation by control thyroids, but the difference fell short of statistical significance. Thyroiditis and the production of materials that affect the McKenzie bioassay appear to be completely separable phenomena. This study provides further evidence that thyroglobulin is not the important antigen leading to the production of thyroid-stimulating globulin.
30. Effects of 6-mercaptopurine on guinea pig cellular and humoral immune mechanisms. S. Michael Phillips, M.D., Boston, Mass., and Burton Zweiman, M.D., Philadelphia, Pa. We have previously reported that lymphocytes of 6-mercaptopurine (6-MP)-treated guinea pigs with depressed tuberculin hypersensitivity showed normal in vitro proliferative responses to phytohemagglutinin (PHA) or tuberculin. However, there were some defects in monocyte-macrophage function. This report describes continuing studies of 6-MP immunosuppressive mechanisms. After base-line studies and injection of complete Freund's adjuvant on Day 0, Group A received 6-MP, 40 mg. per kilogram on Days 4 to 14; Group B received placebo. Results: Similar responses for Groups A and B in: (1) weekly patterns of in vitro lymphocyte responses to PHA and tuberculin; (2) migration inhibitory factor (MIF) production by lymphocytes incubated with tuberculin; (3) circulating antibody production and in vitro lymphocyte proliferative responses to soluble antigen (human gamma globulin, HGG) given on Day 0, though delayed skin test reaction to this antigen suppressed in Group A; (4) wheal and flare responses to histamine and compound 48-80. Different responses between Groups A and B in: (A) depressed in vivo monocyte-macrophage kinetics in Group A measured by isotope uptake; (B) depletion of certain areas in regional nodes of Group A; (C) "rebound" in immune responsiveness in group A after 6-MP stopped. In 13 of 14 of Group A, tuberculin skin test reactivity was reconstituted shortly after receipt of marrow (but not node) cells from normal unsensitized donors. Isotope studies show many inflammatory cells of donor origin in these skin test reactions. Results suggest a greater suppressive effect of 6-MP on cellular rather than humoral immunity, with a major action on effector inflammatory cells of marrow origin. 3 1 . Isolation and characterization of the dialysate from Bermuda grass pollen.
Roberta L. Meyers, Ph.D., Los Angeles, Calif., Aline "W. Bems-Mason, B.A., Kent H. Thayer, M.D., Bruce H. Feldman, M.D., and Harold N. Eosengren, M.D., Long Beach, Calif. Previous studies on the isolation of haptenic material from the dialysates of timothy grass pollen (Malley, J. Immun. 93: 420, 1964) and ragweed pollen (Meyers, Fed. Proc. 25: 729, 1966) led us to investigate whether haptenic material occurs in Bermuda grass pollen, which is the major allergen in the southwestern United States. Bermuda grass pollen (Cynodon daotylon) was extracted with water at neutral pH and the dialysate recovered. Sephadex G-25 or Sephadex G-50 chromatography of the dialysate gave two peaks, I and II. Peak I reacted with rabbit anti-water soluble cynodon (WSC) and rabbit anti-whole cynodon dialysate (WCD) in Ouchterlony gel diffusion, whereas peak I I did not react with the antisera. Peak I I inhibited precipitation of WSC and rabbit antiWSC in Ouchterlony gel diffusion and inhibited the quantitative precipitation reaction between WSC and anti-WSC. Patients sensitive to cynodon reacted to peak I just as strongly as to a standard cynodon extract. However, they had a markedly reduced skin test reactivity to peak II. The
VOLUME 47 NUMBER 2
Abstracts of papers
101
results indicate the haptenic nature of the Sephadex G-50 peak II from the dialysate of eynodon extract is similar to that found in timothy and ragweed pollens. 3 2 . Eosinophilic leukocytes and antigen-antibody complexes. T a k e r u Ishikawa, M.D., K o n r a d Wicher, Ph.D., a n d C a r l E . Arbesman, M.D., Buffalo, N . Y. The attraction of eosinophils to antigen-antibody (Ag-Ab) complexes composed of parasitic helminths extracts and their corresponding antibodies has been reported. It was of interest to study this phenomenon employing allergen-antibody or immunoglobulin-antibody complexes. Soluble Ag-Ab complexes were prepared with antigens of ragweed, bovine gamma globulin, and crystallized egg albumin and their homologous rabbit antisera. In addition, ragweed extract was combined with sera from ragweed-sensitive patients. Blood from six different individuals (two were atopic) with high eosinophilia was used as the source for these cells. The eosinophils were washed and mixed with the Ag-Ab complexes, the antigen alone, and the antisera alone. The attraction of the eosinophils to the above reagents was examined by immunofluorescent technique using conjugated guinea pig antiserum to rabbit IgQ- and (conjugated) rabbit antisera to human immunoglobulins (Gr, A, M, D, E). To avoid nonspecific staining, the eosinophils were treated with bovine serum albumin conjugated with Bhodamine B prior to application of various conjugates. Only the rabbit and human Ag-Ab complexes were detected on the eosinophils. Neither the antigens nor antibodies alone could be detected. Only anti-IgG and -IgM conjugates demonstrated fluorescence in the human system. Similar results were obtained using radioautography.
33. Measurement of binding of tubercle bacilli components by antibodies in sera from tuberculous and nontuberculous individuals. Emil J. Bardana, Jr., M.D., Percy Minden, M.D., J. Kenneth McClatchy, Ph.D., and Richard S. Farr, M.D., Denver, Colo. Previous studies from this laboratory employing radioimmunodiffusion indicated that antibodies to partially purified isolates from tubercle bacilli were present in sera from tuberculous patients and from some normal control subjects. Since similar findings have been reported with other tests, studies were undertaken using a primary binding test to quantitate this binding. Heat-killed suspensions of Mycohacterium tuberculosis strain II37Kv were sonicated and centrifuged. The supernate was treated with manganese chloride and cetyl pyridinium chloride (CPC) to precipitate nucleic acids and acidic polysaccharides, respectively. The new supernate (CPC-S) was labeled with i3il (isiI-CPC-S) and was the test antigen. Antigen-antibody complexes in a 1:10 dilution of serum were precipitated by heterologous antihuman IgG. Eesults were expressed as the percentage of isiI-CPC-S precipitated. The per cent of antigen bound by all the sera tested ranged from only 8.0 to 19.6 per cent. However, sera from 24 of 27 patients with active tuberculosis bound more than 15 per cent, whereas sera from 16 of 20 healthy individuals and sera from 14 of 17 patients with chronic nontuberculous diseases bound less than 15 per cent. It would appear, therefore, that 131I-CPC-S consists of a number of components of which only a small portion are critical to the measurement of specific antibodies from tuberculous patients. It is likely that some of the antigen-binding capacities of some of the control sera may have resulted from subclinical tuberculous infections, exposure to saprophytic mycobacteria, and/or the exposure to antigenic groups shared by mycobacteria and non-acid-fast bacteria. Studies using more highly purified antigens are in progress to investigate these possibilities.
34. Ciliocytophthoria and differential diagnosis of asthma. Harold R. Styler, M.D., Santa Barbara, Calif.