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44. 3H-thymidine uptake response of lymphocytes from patients with intrinsic asthma upon in vitro challenge with bacterial antigens
106
American Academy of Allergy
J. ALLERG. FEBRUARY 1971
blocked by methyl-a-D-mannoside (an inhibitor of Con A). It is concluded that Con A-stimulated lymphocytes are cytotoxic, an effect possibly due to lymphocytotoxin release.
43. The lack of detectable circulating antigen-reactive cells during local immunization by food antigen. Salmon S. Goldberg, M.D., Richard M. Rothberg, M.D., Sumner C. Kraft, M.D., and Raymond D. A. Peterson, M.D., Chicago, 111. roUowing the ingestion of low concentrations of bovine serum albumin (BSA), circulating anti-BSA originating in gut-associated lymphoid tissue is indistinguishable from circulating anti-BSA produced following parenteral immunization (J. Immun. 98: 386, 1967). To test for circulating antigen-reactive cells (AEC) during oral immunization, circulating lymphocytes from rabbits ingesting either 0.1 or 2.0 per cent BSA were cultured in the presence or absence of BSA. Every week, lymphocyte responsiveness to BSA was estimated by sH-thymidine uptake, and circulating anti-BSA concentrations were measured by the ammonium sulfate method. The results were compared with data obtained from rabbits immunized to BSA administered intravenously or suboutaneously-inoil. During the first 10 weeks: (1) The animals orally immunized with either 0.1 or 2.0 per cent BSA and the animals immunized subcutaneously-in-oil had comparable amounts of circulating anti-BSA. (2) After 14 to 21 days, circulating AEC consistently were detected in the subcutaneously immunized group. (3) The intravenously immunized animals had low concentrations of circulating anti-BSA but AEC were detectable. (4) Circulating AEC very rarely were detected in orally immunized animals. Discontinuing antigen ingestion led to diminshed antibody levels and the continued absence of detectable AEC. Following subcutaneous challenge with a dose of BSA which was adequate to produce circulating AEC in nonimmunized rabbits, orally immunized animals developed an increased rate of antibody synthesis without detectable AEC. One week following a subsequent intravenous challenge with BSA, however, 64 per cent of orally immunized rabbits had detectable AEC. These results and studies by others suggest that during oral immunization AEC may be localized primarily to gut-associated lymphoid tissue and that cellmediated immunity may comprise a part of the secretory immunologic system.
44. ^H-thymidine uptake response of lymphocytes from patients with intrinsic asthma upon in vitro challenge with bacterial antigens. Clarence M. Virtue, M.D., Heinz J. Wittig, M.D., and Terrence J. Cook, M.D., Gainesville, Fla. Chronic infection of the respiratory tract is often thought to play an important role in the course of intrinsic asthma. To study this further, the response of lymphocytes from patients with intrinsic asthma was investigated by in vitro challenge with various bacterial antigens and assayed by liquid scintillation counting of sH-thymidine uptake. Lymphocytes from normal individuals were also run in an identical manner for comparison. All lymphocytes were cultured at 37° C. in TC 199 Hepes medium containing 10 per cent fetal calf serum. Cells were challenged with a commercial mixed staphylococcal-streptococcal-pneumococcal bacterial antigen and, separately, with lyophilized M. influenza bacterial antigen. sH-thymidine label was added to all tubes on the third day. Cultures were terminated on the fourth day and lymphocytes harvested by trichloroacetic acid (TCA) precipitation. After drying, the precipitate was solubilized and placed in scintillation fluid for counting in a Packard liquid scintillation spectrometer. Lymphocytes from 16 intrinsic asthmatic patients were compared with those from normal control subjects. The mean of the unstimulated background counts from all patients with intrinsic asthma was 250 per cent above the mean of the control values. Upon challenge with mixed bacterial antigen, the majority of both patients and control subjects had 300 per cent or greater increase in counts. E. influenza antigen produced a drop in counts between 20 to 70 per cent below unstimulated background in all patients and control subjects.
VOLUME A7
Abstracts of papers
107
NUMBER 2
Lymphocytes from intrinsic asthmatic patients respond to challenge with mixed bacterial and also K. influensa antigen in the same manner and degree as do control subjects. However, unstimulated lymphocytes of some p a t i e n t s with intrinsic asthma appear to have a significantly higher turnover t h a n do those of normal control subjects. The reasons for this are unexplained, and further investigation is indicated.
45. In vitro functions of lymphocytes in human atopy. Malcolm N. Blumenthal, M.D., Helen Hallgren, B.S., and Ed Yunis, M.D., Minneapolis, Minn. The functions of sensitized lymphocytes in human atopy have not yet been determined. This study investigates lymphocytes obtained from patients with ragweed atopy regarding their ability to undergo blast transformation and to cause destruction of fibroblasts when exposed to ragweed antigen. The role played by I g E , IgM, and IgG in these reactions is also explored. Thirty-three patients were divided into three groups: A, ragweed sensitive, nontreated; B, ragweed sensitive, t r e a t e d ; and C, nonatopic patients. The cytotoxic effects on human fibroblasts mediated by lymphocytes and ragweed antigen or of antisera to human I g E (E-chain specific) was studied. Optimal concentrations of ragweed antigen were 500 and 1,000 fig and of antisera to I g E were a 1:5 dilution. I n ragweed-sensitive patients positive cytotoxic effects were noted in 3 of 6 patients using ragweed and 2 of 4 patients using anti-IgE sera. All appropriate controls were negative. The lymphocyte transformation produced by ragweed was studied using ragweed antigen. The cultures were assayed for blast transformation by measuring the u p t a k e of radioactive thymidine. Positive transformation was obtained in 10 of 14 patients from Group A and 2 of 6 patients from Group B, with the optimal dose of ragweed antigen being 500 and 1,000 fig. No significant stimulation was noted in Group C. The lymphocyte transformation following the addition of 1:5 dilutions of specific antisera to I g E , IgG, and I g M was also studied. Postive transformation was noted in the majority of all three groups using anti-IgE sera. No significant transformation was noted in all groups using antisera to IgM. Greater transformation was noted following the addition of antisera to IgG in Groups B and C as compared to Group A. Our findings suggest t h a t the in vitro responses of circulating cells can be associated with clinical ragweed atopy. The type and degree of response may be related to the degree of clinical sensitivity.
46. Fate of inhaled whole pollen in asthmatics. Archie F. Wilson, M.D., Ph.D., Harold S. Novey, M.D., Edgar L. Surprenant, M.D., and Leslie R. Bennett, M.D., Irvine and Los Angeles, Calif. Pollen grains are considered important allergens in producing asthma despite sizes which should preclude passage past the largest airways. A study was designed to follow the p a t h of inhaled pollen and to determine whether its deposition correlated with the focal areas of decreased ventilation and perfusion found in the lungs of patients having asthma. A method was developed to t a g t h e outer coat of various pollen with ssmTc. No change in antigenic or morphologic characteristics was detected. I n t a c t Poa pratensis pollen labeled with technetium was inhaled by 5 sensitive subjects who were clinically well. The distribution of pollen, spirometry, arterial blood gases, and regional ventilation and perfusion (using radioxenon and a scintillation camera) were monitored over several hours. Initially, each subject noted mild upper a i r w a y irritation associated w i t h a minor and transient fall in arterial 0 , and spirometric measurements but little or no change in t h e distribution of ventilation and perfusion. However, 4 developed marked asthmatic symptoms 6 to 10 hours later. The one who failed to develop asthma inhaled the smallest amount of pollen. Radiopollen was never demonstrated in the lungs. I t always deposited in the oropharynx and was rapidly swallowed. 99mTo was detected almost immediately in the esophagus, stomach, and later in the blood and urine.
American Academy of Allergy
J. ALLERG. FEBRUARY 1971
blocked by methyl-a-D-mannoside (an inhibitor of Con A). It is concluded that Con A-stimulated lymphocytes are cytotoxic, an effect possibly due to lymphocytotoxin release.
43. The lack of detectable circulating antigen-reactive cells during local immunization by food antigen. Salmon S. Goldberg, M.D., Richard M. Rothberg, M.D., Sumner C. Kraft, M.D., and Raymond D. A. Peterson, M.D., Chicago, 111. roUowing the ingestion of low concentrations of bovine serum albumin (BSA), circulating anti-BSA originating in gut-associated lymphoid tissue is indistinguishable from circulating anti-BSA produced following parenteral immunization (J. Immun. 98: 386, 1967). To test for circulating antigen-reactive cells (AEC) during oral immunization, circulating lymphocytes from rabbits ingesting either 0.1 or 2.0 per cent BSA were cultured in the presence or absence of BSA. Every week, lymphocyte responsiveness to BSA was estimated by sH-thymidine uptake, and circulating anti-BSA concentrations were measured by the ammonium sulfate method. The results were compared with data obtained from rabbits immunized to BSA administered intravenously or suboutaneously-inoil. During the first 10 weeks: (1) The animals orally immunized with either 0.1 or 2.0 per cent BSA and the animals immunized subcutaneously-in-oil had comparable amounts of circulating anti-BSA. (2) After 14 to 21 days, circulating AEC consistently were detected in the subcutaneously immunized group. (3) The intravenously immunized animals had low concentrations of circulating anti-BSA but AEC were detectable. (4) Circulating AEC very rarely were detected in orally immunized animals. Discontinuing antigen ingestion led to diminshed antibody levels and the continued absence of detectable AEC. Following subcutaneous challenge with a dose of BSA which was adequate to produce circulating AEC in nonimmunized rabbits, orally immunized animals developed an increased rate of antibody synthesis without detectable AEC. One week following a subsequent intravenous challenge with BSA, however, 64 per cent of orally immunized rabbits had detectable AEC. These results and studies by others suggest that during oral immunization AEC may be localized primarily to gut-associated lymphoid tissue and that cellmediated immunity may comprise a part of the secretory immunologic system.
44. ^H-thymidine uptake response of lymphocytes from patients with intrinsic asthma upon in vitro challenge with bacterial antigens. Clarence M. Virtue, M.D., Heinz J. Wittig, M.D., and Terrence J. Cook, M.D., Gainesville, Fla. Chronic infection of the respiratory tract is often thought to play an important role in the course of intrinsic asthma. To study this further, the response of lymphocytes from patients with intrinsic asthma was investigated by in vitro challenge with various bacterial antigens and assayed by liquid scintillation counting of sH-thymidine uptake. Lymphocytes from normal individuals were also run in an identical manner for comparison. All lymphocytes were cultured at 37° C. in TC 199 Hepes medium containing 10 per cent fetal calf serum. Cells were challenged with a commercial mixed staphylococcal-streptococcal-pneumococcal bacterial antigen and, separately, with lyophilized M. influenza bacterial antigen. sH-thymidine label was added to all tubes on the third day. Cultures were terminated on the fourth day and lymphocytes harvested by trichloroacetic acid (TCA) precipitation. After drying, the precipitate was solubilized and placed in scintillation fluid for counting in a Packard liquid scintillation spectrometer. Lymphocytes from 16 intrinsic asthmatic patients were compared with those from normal control subjects. The mean of the unstimulated background counts from all patients with intrinsic asthma was 250 per cent above the mean of the control values. Upon challenge with mixed bacterial antigen, the majority of both patients and control subjects had 300 per cent or greater increase in counts. E. influenza antigen produced a drop in counts between 20 to 70 per cent below unstimulated background in all patients and control subjects.
VOLUME A7
Abstracts of papers
107
NUMBER 2
Lymphocytes from intrinsic asthmatic patients respond to challenge with mixed bacterial and also K. influensa antigen in the same manner and degree as do control subjects. However, unstimulated lymphocytes of some p a t i e n t s with intrinsic asthma appear to have a significantly higher turnover t h a n do those of normal control subjects. The reasons for this are unexplained, and further investigation is indicated.
45. In vitro functions of lymphocytes in human atopy. Malcolm N. Blumenthal, M.D., Helen Hallgren, B.S., and Ed Yunis, M.D., Minneapolis, Minn. The functions of sensitized lymphocytes in human atopy have not yet been determined. This study investigates lymphocytes obtained from patients with ragweed atopy regarding their ability to undergo blast transformation and to cause destruction of fibroblasts when exposed to ragweed antigen. The role played by I g E , IgM, and IgG in these reactions is also explored. Thirty-three patients were divided into three groups: A, ragweed sensitive, nontreated; B, ragweed sensitive, t r e a t e d ; and C, nonatopic patients. The cytotoxic effects on human fibroblasts mediated by lymphocytes and ragweed antigen or of antisera to human I g E (E-chain specific) was studied. Optimal concentrations of ragweed antigen were 500 and 1,000 fig and of antisera to I g E were a 1:5 dilution. I n ragweed-sensitive patients positive cytotoxic effects were noted in 3 of 6 patients using ragweed and 2 of 4 patients using anti-IgE sera. All appropriate controls were negative. The lymphocyte transformation produced by ragweed was studied using ragweed antigen. The cultures were assayed for blast transformation by measuring the u p t a k e of radioactive thymidine. Positive transformation was obtained in 10 of 14 patients from Group A and 2 of 6 patients from Group B, with the optimal dose of ragweed antigen being 500 and 1,000 fig. No significant stimulation was noted in Group C. The lymphocyte transformation following the addition of 1:5 dilutions of specific antisera to I g E , IgG, and I g M was also studied. Postive transformation was noted in the majority of all three groups using anti-IgE sera. No significant transformation was noted in all groups using antisera to IgM. Greater transformation was noted following the addition of antisera to IgG in Groups B and C as compared to Group A. Our findings suggest t h a t the in vitro responses of circulating cells can be associated with clinical ragweed atopy. The type and degree of response may be related to the degree of clinical sensitivity.
46. Fate of inhaled whole pollen in asthmatics. Archie F. Wilson, M.D., Ph.D., Harold S. Novey, M.D., Edgar L. Surprenant, M.D., and Leslie R. Bennett, M.D., Irvine and Los Angeles, Calif. Pollen grains are considered important allergens in producing asthma despite sizes which should preclude passage past the largest airways. A study was designed to follow the p a t h of inhaled pollen and to determine whether its deposition correlated with the focal areas of decreased ventilation and perfusion found in the lungs of patients having asthma. A method was developed to t a g t h e outer coat of various pollen with ssmTc. No change in antigenic or morphologic characteristics was detected. I n t a c t Poa pratensis pollen labeled with technetium was inhaled by 5 sensitive subjects who were clinically well. The distribution of pollen, spirometry, arterial blood gases, and regional ventilation and perfusion (using radioxenon and a scintillation camera) were monitored over several hours. Initially, each subject noted mild upper a i r w a y irritation associated w i t h a minor and transient fall in arterial 0 , and spirometric measurements but little or no change in t h e distribution of ventilation and perfusion. However, 4 developed marked asthmatic symptoms 6 to 10 hours later. The one who failed to develop asthma inhaled the smallest amount of pollen. Radiopollen was never demonstrated in the lungs. I t always deposited in the oropharynx and was rapidly swallowed. 99mTo was detected almost immediately in the esophagus, stomach, and later in the blood and urine.