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New insight into the clinical characteristics of adults vs children with severe asthma
J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER I
greater rate than those with mild/moderate asthma? Do severe asthmatics lose responsiveness to GC over time? Do any therapies alter disease progression over time? remain to be studied. We evaluated 260 patients with severe asthma over the past 6 years, entering them into a comprehensive database: 27.1 +_.1.1 yr; range 2.3 to 74 years; 56.5% female; 47% children; 78.9% Caucasian, 18.9% African-American. The majority (202 or 78%) of asthmatics had onset of their disease in childhood (CO). Correlations of asthma duration with multiple parameters of disease severity were evaluated using simple regression analyses. Significant relationships were seen between asthma duration and worsening lung function (FEV 1, FVC, FEV1/FVC ratio, Raw, SGaw), oral GC dose, duration of oral GC therapy, and GC-induced adverse effects. Of interest, asthma duration was inversely related to in vitro GC responsiveness. Despite escalating oral and high dose inhaled GC use, the FEV 1 declined at a rate of 0.65% predicted per yr- a value not greater than previously reported in less severe asthma; notably, the Y-intercept of our cohort was only 79% of predicted vs. - 100% predicted in other studies. This suggests a significant loss of lung function soon after asthma onset. Similar relationships were not noted between duration and disease severity in patients with adult onset ( A t ) asthma. They had a significantly later onset (35.6 vs. 3.5 yr; p<0.0001) and shorter duration (10.3 vs. 18.1 yr; p<0.001) of asthma compared to CO asthmatics. Despite significant differences in duration, A t asthmatics had significantly lower lung function parameters (FEV 1 FVC, FEV1/FVC, Raw, Sgaw) than CO asthmatics. Of note, there were no differences in oral GC requirement or duration of GC therapy. A t asthma appears to be a distinct form of asthmadespite shorter duration of the disease; A t asthmatics had similar clinical parameters with greater airflow obstruction. The results of our cross-sectional study of severe asthma suggest that in patients with CO asthma, duration of the disease relates significantly to multiple parameters of disease severity. Second, CO asthmatics lose responsiveness to the anti-inflammatory effects of GCs over time. Whether this is due to effects of increasing age or tolerance must be determined. Third, in patients with CO asthma, there is progressive loss of lung function despite aggressive inhaled and systemic GC therapy, suggesting there are GC-insensitive _+GC-independent aspects of the disease.
1t
~1~'~ New Insight Into the Clinical Characteristics el Adults vs Children With Severe Asthma
II IJllJI
Henry Avner Jenkins, Stanley J Szefler, Ronina A Covar, Monica Jones, Eleanor E Brown, Erwin W Gelfand, Joseph D Spahn National Jewish Medical and Research Center, Denver, CO Large gaps in our knowledge pertaining to the clinical characteristics of severe asthma exist, as illustrated by the recent NHLBI workshop, "Pathophysiology of Severe Asthma" (1). The clinical pharmacology service at NJMRC has evaluated 260 severe asthmatics (mean age 27.1 +_1.1 yr; range 2.3 to 74) over the last 6 years. An extensive database has been collected that includes demographic, pharmacokinetic and lung function information. The cohort consisted of 56.5% females; 78.9% were Caucasian, and 18.9% African American. 76% required chronic oral GC therapy (27.5 +_3.1 mg/d), all were on high dose inhaled glucocorticoid (GC) therapy (1151 ___ 47 mcg/d), and 25% had at least 1 previous intubation. Adults (A) accounted for 53.1% (n=138) of the entire cohort and differed significantly from Children (C) (n=122) in many parameters including: female predominance (72%) (P<0.0001), lower total lgE (233-+55.9 vs. 512-+86.5 ku/1, p=0.007) higher admission oral GC dose (35 vs. 20 mg/d, p<0.01), longer duration of chronic oral GC use (5.3 vs. 2.3 yr, p<0.0001), anda greater number of GC associated adverse effects, (p<0.0001). In addition, 82% o f A vs 63% of C were receiving oral GCs on admission. GC pharmacokinetic assays found A to have significantly slower prednisolone clearance (188+_7 vs 220-+6.5 ml/min/l.73m2, p=0.0008), while in vitro analysis of GC responsiveness using a lymphocyte stimulation assay found A to be less responsive to GCs [hydrocortisone log IC50 2.41 vs 1.96 nM, p<0.0001, dexamethasone log IC50 1.21 vs 0.77 nM, p<0.002), fluticasone propionate log IC50 0.14 vs 0.67 nM, p<0.0005)]. Lung function studies revealed C to have greater airtrapping (RV 255 +_9.9 vs. 195_+8.1%, p<0.0001) but less airflow obstruction (FEV 174.5_+2.1 vs. 57.7+_1.9%, p<0.0001) than A. Of note, 41% of C
Abstracts
S353
vs 18% of A had FEVI'S of >80%, and only 28% C vs 67% of A had FEV 1 values of <60% predicted (p<0.0001). Similarities between A and C with severe asthma include: requirement for chronic oral GC therapy, a history of intubation (27 vs 23.5%), and total eosinophil counts (243 vs 252 cumm; p=0.8), respectively. However, major differences exist between the two groups (greater admission dose and duration of oral GC use, diminished GC responsiveness in vitro, greater number of GC adverse effects, and greater degree of airflow obstruction noted among A) suggesting that they are pathophysiologically distinct diseases. Furthermore, pulmonary function criteria of the guidelines on severity classification do not appear to be applicable to children on anti-inflammatory therapy. Only 28% of our asthmatic children would be classified as severe, while over 40% would be considered mild persistent based on NHLBI FEV 1 criteria. It may be time for a reappraisal of lung function values in childhood asthma, lj Allergy Clin Immuno12000; 106:1033-1042.
1101 Identification and Characterization °f K+ Channels in Human Eosinophils A Schwingshackl, M Duszyk, Redwan Moqbel University of Alberta, Edmonton, AB, Canada BACKGROUND" Eosinophils are important effector cells in allergic disease including bronchial asthma and are thought to contribute to the inflammation underlying the pathenogenesis of this disorder. K ÷ channels play an important role in vital cellular functions such as stimulus-receptor coupling, volume regulation and membrane potential setting in many cell types. However, little is known about these channels in eosinophils. It has been suggested K ÷ currents may play a role in eosinophil superoxide production and promote cell shrinkage during eosinophil apoptosis. Eosinophils express subunits of sulfonylurea receptors that are part of ATPdependent (KAT e ) K + channels. Previous studies have shown that eosinophils also express inwardly-rectifying K + channels of the Kir 2.1 type but their functional significance remains unknown. A new group of K ÷ channels, two pore-domain channels (e.g. TWlK and TASK), have recently been found in peripheral blood leukocytes. However, their expression in eosinophils is unknown. OBJECTIVE: To identify and to characterize the different types of K ÷ channels in human eosinophils. M E T H O D S : Human peripheral blood eosinophils were purified (<98%) from atopic asthmatic volunteers by anti-CD16 negative immunoselection. K + channel gene expression was studied using RT-PCR. Genes were cloned using a TA Cloning Kit and sequenced using a Beckman Instruments ABI373A sequenator. Whole cell currents were measured using the amphotericin B-perforated patch clamp technique. Data obtained at +80 mV were used for statistical analysis. RESULTS: We have studied mRNA expression of intermediate (IK) and large conductance (BK) calcium-activated K ÷ channels, voltage-gated K + channels (Kv), as well as TWIK-1 and TASK-2. Human peripheral blood eosinophils expressed mRNA for IK, Kv and TWIK-1, but not for BK channels. The identity of these mRNAs was confirmed by sequencing the PCR products. In patch clamp experiments, glibenclamide ( 100 ~tM), a blocker of KATe, and clotrimazole (30 ~tM), a blocker oflK, had no effect on baseline whole cell currents. These results imply that KAT e and IK are usually closed under resting conditions and do not contribute to baseline currents. However, these channels could be activated by specific openers, diazoxide (100 ~M) and 1-EBIO (500 I-tM), respectively. Both openers increased the whole cell current by 64_+20% (p<0.05) and 73_+19% (p<0.05), respectively. Furthermore, 4-aminopyridine (2 mM), a blocker of Kv, decreased whole cell currents by 47+_7% (p<0.01). Baseline and stimulated whole cell currents in eosinophils showed neither time- nor voltage dependency. CONCLUSION: Our results show that human eosinophils express both message and protein for several members of K ÷ channel families. The fact that Kv channels are activated under resting conditions implies that these channels may be major contributors in setting the resting membrane potential in our cells. In addition, the presence of KATe and IK channels suggests that, with appropriate stimulation, they might be involved in eosinophil mediator release similarly to the secretory mechanism described in the pancreas.
greater rate than those with mild/moderate asthma? Do severe asthmatics lose responsiveness to GC over time? Do any therapies alter disease progression over time? remain to be studied. We evaluated 260 patients with severe asthma over the past 6 years, entering them into a comprehensive database: 27.1 +_.1.1 yr; range 2.3 to 74 years; 56.5% female; 47% children; 78.9% Caucasian, 18.9% African-American. The majority (202 or 78%) of asthmatics had onset of their disease in childhood (CO). Correlations of asthma duration with multiple parameters of disease severity were evaluated using simple regression analyses. Significant relationships were seen between asthma duration and worsening lung function (FEV 1, FVC, FEV1/FVC ratio, Raw, SGaw), oral GC dose, duration of oral GC therapy, and GC-induced adverse effects. Of interest, asthma duration was inversely related to in vitro GC responsiveness. Despite escalating oral and high dose inhaled GC use, the FEV 1 declined at a rate of 0.65% predicted per yr- a value not greater than previously reported in less severe asthma; notably, the Y-intercept of our cohort was only 79% of predicted vs. - 100% predicted in other studies. This suggests a significant loss of lung function soon after asthma onset. Similar relationships were not noted between duration and disease severity in patients with adult onset ( A t ) asthma. They had a significantly later onset (35.6 vs. 3.5 yr; p<0.0001) and shorter duration (10.3 vs. 18.1 yr; p<0.001) of asthma compared to CO asthmatics. Despite significant differences in duration, A t asthmatics had significantly lower lung function parameters (FEV 1 FVC, FEV1/FVC, Raw, Sgaw) than CO asthmatics. Of note, there were no differences in oral GC requirement or duration of GC therapy. A t asthma appears to be a distinct form of asthmadespite shorter duration of the disease; A t asthmatics had similar clinical parameters with greater airflow obstruction. The results of our cross-sectional study of severe asthma suggest that in patients with CO asthma, duration of the disease relates significantly to multiple parameters of disease severity. Second, CO asthmatics lose responsiveness to the anti-inflammatory effects of GCs over time. Whether this is due to effects of increasing age or tolerance must be determined. Third, in patients with CO asthma, there is progressive loss of lung function despite aggressive inhaled and systemic GC therapy, suggesting there are GC-insensitive _+GC-independent aspects of the disease.
1t
~1~'~ New Insight Into the Clinical Characteristics el Adults vs Children With Severe Asthma
II IJllJI
Henry Avner Jenkins, Stanley J Szefler, Ronina A Covar, Monica Jones, Eleanor E Brown, Erwin W Gelfand, Joseph D Spahn National Jewish Medical and Research Center, Denver, CO Large gaps in our knowledge pertaining to the clinical characteristics of severe asthma exist, as illustrated by the recent NHLBI workshop, "Pathophysiology of Severe Asthma" (1). The clinical pharmacology service at NJMRC has evaluated 260 severe asthmatics (mean age 27.1 +_1.1 yr; range 2.3 to 74) over the last 6 years. An extensive database has been collected that includes demographic, pharmacokinetic and lung function information. The cohort consisted of 56.5% females; 78.9% were Caucasian, and 18.9% African American. 76% required chronic oral GC therapy (27.5 +_3.1 mg/d), all were on high dose inhaled glucocorticoid (GC) therapy (1151 ___ 47 mcg/d), and 25% had at least 1 previous intubation. Adults (A) accounted for 53.1% (n=138) of the entire cohort and differed significantly from Children (C) (n=122) in many parameters including: female predominance (72%) (P<0.0001), lower total lgE (233-+55.9 vs. 512-+86.5 ku/1, p=0.007) higher admission oral GC dose (35 vs. 20 mg/d, p<0.01), longer duration of chronic oral GC use (5.3 vs. 2.3 yr, p<0.0001), anda greater number of GC associated adverse effects, (p<0.0001). In addition, 82% o f A vs 63% of C were receiving oral GCs on admission. GC pharmacokinetic assays found A to have significantly slower prednisolone clearance (188+_7 vs 220-+6.5 ml/min/l.73m2, p=0.0008), while in vitro analysis of GC responsiveness using a lymphocyte stimulation assay found A to be less responsive to GCs [hydrocortisone log IC50 2.41 vs 1.96 nM, p<0.0001, dexamethasone log IC50 1.21 vs 0.77 nM, p<0.002), fluticasone propionate log IC50 0.14 vs 0.67 nM, p<0.0005)]. Lung function studies revealed C to have greater airtrapping (RV 255 +_9.9 vs. 195_+8.1%, p<0.0001) but less airflow obstruction (FEV 174.5_+2.1 vs. 57.7+_1.9%, p<0.0001) than A. Of note, 41% of C
Abstracts
S353
vs 18% of A had FEVI'S of >80%, and only 28% C vs 67% of A had FEV 1 values of <60% predicted (p<0.0001). Similarities between A and C with severe asthma include: requirement for chronic oral GC therapy, a history of intubation (27 vs 23.5%), and total eosinophil counts (243 vs 252 cumm; p=0.8), respectively. However, major differences exist between the two groups (greater admission dose and duration of oral GC use, diminished GC responsiveness in vitro, greater number of GC adverse effects, and greater degree of airflow obstruction noted among A) suggesting that they are pathophysiologically distinct diseases. Furthermore, pulmonary function criteria of the guidelines on severity classification do not appear to be applicable to children on anti-inflammatory therapy. Only 28% of our asthmatic children would be classified as severe, while over 40% would be considered mild persistent based on NHLBI FEV 1 criteria. It may be time for a reappraisal of lung function values in childhood asthma, lj Allergy Clin Immuno12000; 106:1033-1042.
1101 Identification and Characterization °f K+ Channels in Human Eosinophils A Schwingshackl, M Duszyk, Redwan Moqbel University of Alberta, Edmonton, AB, Canada BACKGROUND" Eosinophils are important effector cells in allergic disease including bronchial asthma and are thought to contribute to the inflammation underlying the pathenogenesis of this disorder. K ÷ channels play an important role in vital cellular functions such as stimulus-receptor coupling, volume regulation and membrane potential setting in many cell types. However, little is known about these channels in eosinophils. It has been suggested K ÷ currents may play a role in eosinophil superoxide production and promote cell shrinkage during eosinophil apoptosis. Eosinophils express subunits of sulfonylurea receptors that are part of ATPdependent (KAT e ) K + channels. Previous studies have shown that eosinophils also express inwardly-rectifying K + channels of the Kir 2.1 type but their functional significance remains unknown. A new group of K ÷ channels, two pore-domain channels (e.g. TWlK and TASK), have recently been found in peripheral blood leukocytes. However, their expression in eosinophils is unknown. OBJECTIVE: To identify and to characterize the different types of K ÷ channels in human eosinophils. M E T H O D S : Human peripheral blood eosinophils were purified (<98%) from atopic asthmatic volunteers by anti-CD16 negative immunoselection. K + channel gene expression was studied using RT-PCR. Genes were cloned using a TA Cloning Kit and sequenced using a Beckman Instruments ABI373A sequenator. Whole cell currents were measured using the amphotericin B-perforated patch clamp technique. Data obtained at +80 mV were used for statistical analysis. RESULTS: We have studied mRNA expression of intermediate (IK) and large conductance (BK) calcium-activated K ÷ channels, voltage-gated K + channels (Kv), as well as TWIK-1 and TASK-2. Human peripheral blood eosinophils expressed mRNA for IK, Kv and TWIK-1, but not for BK channels. The identity of these mRNAs was confirmed by sequencing the PCR products. In patch clamp experiments, glibenclamide ( 100 ~tM), a blocker of KATe, and clotrimazole (30 ~tM), a blocker oflK, had no effect on baseline whole cell currents. These results imply that KAT e and IK are usually closed under resting conditions and do not contribute to baseline currents. However, these channels could be activated by specific openers, diazoxide (100 ~M) and 1-EBIO (500 I-tM), respectively. Both openers increased the whole cell current by 64_+20% (p<0.05) and 73_+19% (p<0.05), respectively. Furthermore, 4-aminopyridine (2 mM), a blocker of Kv, decreased whole cell currents by 47+_7% (p<0.01). Baseline and stimulated whole cell currents in eosinophils showed neither time- nor voltage dependency. CONCLUSION: Our results show that human eosinophils express both message and protein for several members of K ÷ channel families. The fact that Kv channels are activated under resting conditions implies that these channels may be major contributors in setting the resting membrane potential in our cells. In addition, the presence of KATe and IK channels suggests that, with appropriate stimulation, they might be involved in eosinophil mediator release similarly to the secretory mechanism described in the pancreas.