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89 Bulk culture of human blood basophils from patients with chronic myelogenous leukemia (CML)
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BULK CULTURE OF HUMAN BLOOD BASOPHILS FROM ~TIENTS wriw CHRONIC MYELOGENOUS LEUKEMIA ICML). M F Mullarkey, M.D., L. Sorenson, B.A., M.A: Bean, M.D., E.Y. Chi, Ph.D., and W.R. Henderson, M.D., Seattle, Washington. Peripheral blood from three oatients with CML was. cultured in 50 ml volumes in the presence of interleuken-Z-depleted media obtained from a clone of human T-cell leukemia. Replicate CML-1 cultures begun with 105 basophils (B's)/ml produced 4xl05/ml B's in 16 days and were viable at 40 days. Replicate CML-2 begun with 3x105/ml B's increased to 6xl05/ml in 20 days and were viable at 30 days. CML-3 cells were initiated with 2.5x105/ml B's, produced 6.8x105/ml B's at 18 days and were still viable at 27 days. Peripheral blood B's from normal donors showed increased longevity in the presence of growth factors. Alcian blue stains and electron microscopy demonstrated basophil morphology. B's released histamine in a dose-dependent fashion to Ca ionophore but not to 48/80. B's were passively sensitized to IgE specific to timothy pollen and released histamine on challenge with pollen. Histamine content per B ranged from 0.4 to 0.7 pg/cell. We conclude that with this technique, human B's may be easily produced, in large quantities, from the blood of patients with CML.
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92 THE ROLE OF ESTERASES IN THE HECHRNISn OF HIs’TAHINE RELEASE FWUN HUHAN BASOPHILS. H.L. 4e1er. L.,I. 5:3n3*. C.L. Crosz. B. Papirneister. 3ritj j.E. Cl:duff. USAMICD. AYG. tib 21010 D~isooropvlfiuerophosohate(DFPi. srl esterase inhibitor, has lIeera (used to study the mediate hyoersensitivity reaoaion. The m~n~b~t:onof I$-Jewndent nistaminerelease by DFP(>I0 -iI) led to ?rteconclusion that release rechahisr required activation of 3rl esterase inhibitable by DFP. Nouever.at concentrationsof esterase :rihlbitor whichinhibited histwine release ue also cgservedeffects m tne cellular oetabolisa of leukocytes, $ne NAB levels in the leukocytesfell at concentratiMs of DFP?lS ti DFPandthe decrease in tJADparalleied the DFPmhibition of histaminerelease. Our data raise questions concernIn? the requirewrit for the activation of an &era% Ia the release of histarine from basoohils. However.wehavefowl anotherrole for esterasesin the release of histame. BFP and soother small wlecular esterase inhibitor pinacolyl wthglphosphooofluoridate(sonan). a phosphonate.directi\ DFP a’ f;$ aixed -$umn leukocytes. released his&me concentrationsof 5x10 h to 3x10 tl released histaoioe while es4y ase intjbitor , released histame at 501an. a strer concentrationsfret 5x10 and5x10 W. Ihe release appearedto be bv a honlytic aechanisrsince it uas 9th calciw and temperature dependmt and neither LDH nor NRD #as released into the supernatant. Ihis directly induced release of histaaine by the e&erase inhibitors indicates that an e&erase is involved in the regulation of histaaine release. It nowappearsthat this esterase acts to prevent OF reduce histamine release. Ye also used the reversible esterase inhibitors pyridostigmioe andphysostiWne. Cells we pteincubatedwith pyridost@oineor physostipminebefore exposureto sown. Preincubatlon uith physostiaine resulted in a .dosedepend& inhibitioh of swan initiated histmioe release. but preinwbatiM uith pyridostipaine did not result in inhibition of the sooandependent histame release. It appearsthat the effect of 5om is on an intracellular esterase since the neutral carbamte, phpstlgrirbe, uw able to coBpete for the active site and block srman &pm&t histaame release while the charqed earbarate. pyridostigrin, had ho effect m hirtaeine release. 127
HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC) VS. AMES SERALYZER (AS) FOR SERUM THEOPHYLLINF DETERMINATIONS. L. AmdalI G.G. Shgiro, C.W. Bierman C T Fu'rTk-~~~s~~~'Tse~;a‘~e _-L-Y-T------L_I_.-. .-.- ._ Washlngton. Comparison of HPLC and AS was decigned to test whether AS is adequately re?iable for therapeutic monitoring of theophyll lne concentrations in a clinic setting* Seventeen patients had blood drawn by micro method. Mean level by AS was 6.59 my/ml IC 3.79 (SD) and by HPLC 9.47pg/ml 2 6.96mg, r = O.?O. In several instances AS underestimated levels appreciably, elg. AS 15.5 US/ml, HPLC For twenty patients with blood 31.8uu/ml. drawn by venipuncture, means for the two methods-were almost identical r = -38. Determinations of levels in serum spiked with theophylline were obtained with HPLC and AS using two different supply sets (1 and 2). The following examples of variability were documented: HPLC Set 1 set 2 Sample 1 16.3 19.4 19.9 Samole 2 27.0 34.6 30.0 44.0 57.1 68.6 Sample 3 Studies of spiked serum showed significant variahility both for instances when hemolyzed serum was used and when it was not. Ames Seralyzer and HPLC determinations were most dissimilar in the higher concentration range. Our experience that the Seralyzer underestimates toxic levels and that there is great variability between supply kits should be noted by practitioners considering this svstem.
USE OF SERALYZER SYSTEM FOR THEOPNYLLINE LEVEL DETERMINATION IN AN OFFICE SETTING. Gary 2. Lotner, M.D. and David G. Tinkelman, --I___--^-.----- tjr;95;;Atlanta, Georgia Rapid reliable serum theophylline level (STL) determinations are essential to optimize theophylline use in a clinical setting. The purpose of this study was to compare 2 techniques of determining STL, high pressure liquid chromatography (HPLC) and a refle rice metric type of analysis, Seralyze ~Ari~~~~~. Sixty-three individual serum samples for theophylline measurement'were analyzed in replicate using both methods. Venipuncture samples were obtained in all cases and in 34, simultaneous finger stick collections were obtained for additional SA assays. The average time in minutes to perform the STL was 15 for the SA and 180 for HPLC. Analysis of the SlL determined by both methods indicate excellent correlation between the 2 methods for venipuncture blood sampling (r=0.96) and for the finger stick sampling (r=0.92) for STL between 5 and 15 mcg/ml (mean difference was 1.22 mcg/ml). For the few levels below 5 and above 15 mcg/ml the differences between levels were greater (mean difference was 2.32 tncg/ml). We conclude that the SA method of STL determination is accurate, and faster that HPLC and we!? suited for use in a clinical office setting.
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BULK CULTURE OF HUMAN BLOOD BASOPHILS FROM ~TIENTS wriw CHRONIC MYELOGENOUS LEUKEMIA ICML). M F Mullarkey, M.D., L. Sorenson, B.A., M.A: Bean, M.D., E.Y. Chi, Ph.D., and W.R. Henderson, M.D., Seattle, Washington. Peripheral blood from three oatients with CML was. cultured in 50 ml volumes in the presence of interleuken-Z-depleted media obtained from a clone of human T-cell leukemia. Replicate CML-1 cultures begun with 105 basophils (B's)/ml produced 4xl05/ml B's in 16 days and were viable at 40 days. Replicate CML-2 begun with 3x105/ml B's increased to 6xl05/ml in 20 days and were viable at 30 days. CML-3 cells were initiated with 2.5x105/ml B's, produced 6.8x105/ml B's at 18 days and were still viable at 27 days. Peripheral blood B's from normal donors showed increased longevity in the presence of growth factors. Alcian blue stains and electron microscopy demonstrated basophil morphology. B's released histamine in a dose-dependent fashion to Ca ionophore but not to 48/80. B's were passively sensitized to IgE specific to timothy pollen and released histamine on challenge with pollen. Histamine content per B ranged from 0.4 to 0.7 pg/cell. We conclude that with this technique, human B's may be easily produced, in large quantities, from the blood of patients with CML.
91
92 THE ROLE OF ESTERASES IN THE HECHRNISn OF HIs’TAHINE RELEASE FWUN HUHAN BASOPHILS. H.L. 4e1er. L.,I. 5:3n3*. C.L. Crosz. B. Papirneister. 3ritj j.E. Cl:duff. USAMICD. AYG. tib 21010 D~isooropvlfiuerophosohate(DFPi. srl esterase inhibitor, has lIeera (used to study the mediate hyoersensitivity reaoaion. The m~n~b~t:onof I$-Jewndent nistaminerelease by DFP(>I0 -iI) led to ?rteconclusion that release rechahisr required activation of 3rl esterase inhibitable by DFP. Nouever.at concentrationsof esterase :rihlbitor whichinhibited histwine release ue also cgservedeffects m tne cellular oetabolisa of leukocytes, $ne NAB levels in the leukocytesfell at concentratiMs of DFP?lS ti DFPandthe decrease in tJADparalleied the DFPmhibition of histaminerelease. Our data raise questions concernIn? the requirewrit for the activation of an &era% Ia the release of histarine from basoohils. However.wehavefowl anotherrole for esterasesin the release of histame. BFP and soother small wlecular esterase inhibitor pinacolyl wthglphosphooofluoridate(sonan). a phosphonate.directi\ DFP a’ f;$ aixed -$umn leukocytes. released his&me concentrationsof 5x10 h to 3x10 tl released histaoioe while es4y ase intjbitor , released histame at 501an. a strer concentrationsfret 5x10 and5x10 W. Ihe release appearedto be bv a honlytic aechanisrsince it uas 9th calciw and temperature dependmt and neither LDH nor NRD #as released into the supernatant. Ihis directly induced release of histaaine by the e&erase inhibitors indicates that an e&erase is involved in the regulation of histaaine release. It nowappearsthat this esterase acts to prevent OF reduce histamine release. Ye also used the reversible esterase inhibitors pyridostigmioe andphysostiWne. Cells we pteincubatedwith pyridost@oineor physostipminebefore exposureto sown. Preincubatlon uith physostiaine resulted in a .dosedepend& inhibitioh of swan initiated histmioe release. but preinwbatiM uith pyridostipaine did not result in inhibition of the sooandependent histame release. It appearsthat the effect of 5om is on an intracellular esterase since the neutral carbamte, phpstlgrirbe, uw able to coBpete for the active site and block srman &pm&t histaame release while the charqed earbarate. pyridostigrin, had ho effect m hirtaeine release. 127
HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC) VS. AMES SERALYZER (AS) FOR SERUM THEOPHYLLINF DETERMINATIONS. L. AmdalI G.G. Shgiro, C.W. Bierman C T Fu'rTk-~~~s~~~'Tse~;a‘~e _-L-Y-T------L_I_.-. .-.- ._ Washlngton. Comparison of HPLC and AS was decigned to test whether AS is adequately re?iable for therapeutic monitoring of theophyll lne concentrations in a clinic setting* Seventeen patients had blood drawn by micro method. Mean level by AS was 6.59 my/ml IC 3.79 (SD) and by HPLC 9.47pg/ml 2 6.96mg, r = O.?O. In several instances AS underestimated levels appreciably, elg. AS 15.5 US/ml, HPLC For twenty patients with blood 31.8uu/ml. drawn by venipuncture, means for the two methods-were almost identical r = -38. Determinations of levels in serum spiked with theophylline were obtained with HPLC and AS using two different supply sets (1 and 2). The following examples of variability were documented: HPLC Set 1 set 2 Sample 1 16.3 19.4 19.9 Samole 2 27.0 34.6 30.0 44.0 57.1 68.6 Sample 3 Studies of spiked serum showed significant variahility both for instances when hemolyzed serum was used and when it was not. Ames Seralyzer and HPLC determinations were most dissimilar in the higher concentration range. Our experience that the Seralyzer underestimates toxic levels and that there is great variability between supply kits should be noted by practitioners considering this svstem.
USE OF SERALYZER SYSTEM FOR THEOPNYLLINE LEVEL DETERMINATION IN AN OFFICE SETTING. Gary 2. Lotner, M.D. and David G. Tinkelman, --I___--^-.----- tjr;95;;Atlanta, Georgia Rapid reliable serum theophylline level (STL) determinations are essential to optimize theophylline use in a clinical setting. The purpose of this study was to compare 2 techniques of determining STL, high pressure liquid chromatography (HPLC) and a refle rice metric type of analysis, Seralyze ~Ari~~~~~. Sixty-three individual serum samples for theophylline measurement'were analyzed in replicate using both methods. Venipuncture samples were obtained in all cases and in 34, simultaneous finger stick collections were obtained for additional SA assays. The average time in minutes to perform the STL was 15 for the SA and 180 for HPLC. Analysis of the SlL determined by both methods indicate excellent correlation between the 2 methods for venipuncture blood sampling (r=0.96) and for the finger stick sampling (r=0.92) for STL between 5 and 15 mcg/ml (mean difference was 1.22 mcg/ml). For the few levels below 5 and above 15 mcg/ml the differences between levels were greater (mean difference was 2.32 tncg/ml). We conclude that the SA method of STL determination is accurate, and faster that HPLC and we!? suited for use in a clinical office setting.