ABCMdb

PMID: 15246977

van Heeckeren AM, Schluchter MD, Drumm ML, Davis PB
Role of Cftr genotype in the response to chronic Pseudomonas aeruginosa lung infection in mice.
Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L944-52. Epub 2004 Jul 9., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and ⌬F508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Login to comment 15 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and ⌬F508, all backcrossed to the C57BL/6J background) were compared with respect to growth and in their ability to respond to lung infection elicited with Pseudomonas aeruginosa-laden agarose beads. Login to comment 17 ABCC7 p.Tyr122* ABCC7 p.Ser489* The inflammatory responses to P. aeruginosa-laden agarose beads were comparable in mice of all four Cftr mutant genotypes with respect to absolute and relative cell counts in bronchoalveolar lavage fluid, and cytokine levels (TNF-␣, IL-1beta, IL-6, macrophage inflammatory protein-2, and keratinocyte chemoattractant) and eicosanoid levels (PGE2 and LTB4) in epithelial lining fluid: the few small differences observed occurred only between cystic fibrosis mice bearing the S489X mutation and those bearing the knockout mutation Y122X. Login to comment 33 ABCC7 p.Ser489* This latter model has shown that the inflammatory response to these P. aeruginosa-laden agarose beads is excessive in cystic fibrosis mice bearing the S489X knockout mutation compared with wild-type littermates (9, 26, 27). Login to comment 35 ABCC7 p.Ser489* This latter model has shown that the inflammatory response to these P. aeruginosa-laden agarose beads is excessive in cystic fibrosis mice bearing the S489X knockout mutation compared with wild-type littermates (9, 26, 27). Login to comment 37 ABCC7 p.Arg117His On the other hand, if it is the lack of CFTR in the apical membrane that is the primary driver of the inflammatory response, cystic fibrosis mice bearing the R117H allele should have signifi- Address for reprint requests and other correspondence: A. Login to comment 39 ABCC7 p.Arg117His On the other hand, if it is the lack of CFTR in the apical membrane that is the primary driver of the inflammatory response, cystic fibrosis mice bearing the R117H allele should have signifi- Address for reprint requests and other correspondence: A. Login to comment 46 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* In these experiments we tested cystic fibrosis mice bearing the ⌬F508, R117H, Y122X, or S489X genotypes, all backcrossed to the common C57BL/6J genetic background, using the mucoid P. aeruginosa agarose bead model to compare their inflammatory responses. Login to comment 48 ABCC7 p.Ser489* MATERIALS AND METHODS Mice Breeding pairs of heterozygote congenic mice (ՆN10) bearing the S489X mutation (B6.129P2-Cftrtm1Unc , stock no. Login to comment 49 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* In these experiments we tested cystic fibrosis mice bearing the ⌬F508, R117H, Y122X, or S489X genotypes, all backcrossed to the common C57BL/6J genetic background, using the mucoid P. aeruginosa agarose bead model to compare their inflammatory responses. Login to comment 51 ABCC7 p.Ser489* MATERIALS AND METHODS Mice Breeding pairs of heterozygote congenic mice (ՆN10) bearing the S489X mutation (B6.129P2-Cftrtm1Unc , stock no. Login to comment 53 ABCC7 p.Arg117His ABCC7 p.Tyr122* Nomenclature rules available on the Jackson Laboratory website were followed; B6.129S6-Cftrtm2Uth mice bear the R117H mutation, and B6.129S6-Cftrtm3Uth mice bear the Y122X mutation. Login to comment 54 ABCC7 p.Ser489* Cystic fibrosis mice for these strains are indicated by their Cftr mutation (e.g., B6.129P2-Cftrtm1Unc are referred to as cystic fibrosis mice bearing the S489X mutation). Login to comment 56 ABCC7 p.Arg117His ABCC7 p.Tyr122* Nomenclature rules available on the Jackson Laboratory website were followed; B6.129S6-Cftrtm2Uth mice bear the R117H mutation, and B6.129S6-Cftrtm3Uth mice bear the Y122X mutation. Login to comment 57 ABCC7 p.Ser489* Cystic fibrosis mice for these strains are indicated by their Cftr mutation (e.g., B6.129P2-Cftrtm1Unc are referred to as cystic fibrosis mice bearing the S489X mutation). Login to comment 58 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* Cystic fibrosis mice bearing the severe mutations S489X, Y122X, and ⌬F508 were fed the liquid elemental diet Peptamen (Nestle Clinical Nutrition, Deerfield, IL) after weaning, whereas cystic fibrosis mice bearing the mild mutation R117H were fed a standard rodent chow until 1 wk before the start of the experiment at which point they were fed Peptamen until the termination of the experiment. Login to comment 61 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* Cystic fibrosis mice bearing the severe mutations S489X, Y122X, and ⌬F508 were fed the liquid elemental diet Peptamen (Nestle Clinical Nutrition, Deerfield, IL) after weaning, whereas cystic fibrosis mice bearing the mild mutation R117H were fed a standard rodent chow until 1 wk before the start of the experiment at which point they were fed Peptamen until the termination of the experiment. Login to comment 75 ABCC7 p.Tyr122* ABCC7 p.Ser489* At 3 wk of age mice were weighed and then weaned, at which point cystic fibrosis mice bearing the severe Cftr mutations S489X, Y122X, and ⌬F508 were fed a liquid diet. Login to comment 78 ABCC7 p.Tyr122* ABCC7 p.Ser489* At 3 wk of age mice were weighed and then weaned, at which point cystic fibrosis mice bearing the severe Cftr mutations S489X, Y122X, and ⌬F508 were fed a liquid diet. Login to comment 121 ABCC7 p.Tyr122* ABCC7 p.Tyr122* ABCC7 p.Ser489* Cystic fibrosis mice bearing the severe Cftr mutations Y122X, S489X, and ⌬F508 weighed significantly less (P Ͻ 0.05) than homozygote wild-type controls at 7, 14, and 21 days of life with one exception; cystic fibrosis mice with the Y122X mutation did not differ significantly from wild-type mice at 7 days of age (P Ͼ 0.05), but sample sizes were small. Login to comment 122 ABCC7 p.Arg117His The weight of cystic fibrosis mice bearing the mild R117H Cftr mutation did not differ significantly from wild-type controls at 7 days of age (P Ͼ 0.05) and weighed significantly more than mice bearing the severe mutations at 7 and 14 days of age (P Ͻ 0.05); however, they weighed significantly less than wild-type controls thereafter (P Ͻ 0.05) and were not significantly different from cystic fibrosis mice bearing the severe Cftr mutations at 21 days of life (P Ͼ 0.05). Login to comment 124 ABCC7 p.Tyr122* ABCC7 p.Tyr122* ABCC7 p.Ser489* Cystic fibrosis mice bearing the severe Cftr mutations Y122X, S489X, and ⌬F508 weighed significantly less (P Ͻ 0.05) than homozygote wild-type controls at 7, 14, and 21 days of life with one exception; cystic fibrosis mice with the Y122X mutation did not differ significantly from wild-type mice at 7 days of age (P Ͼ 0.05), but sample sizes were small. Login to comment 125 ABCC7 p.Arg117His The weight of cystic fibrosis mice bearing the mild R117H Cftr mutation did not differ significantly from wild-type controls at 7 days of age (P Ͼ 0.05) and weighed significantly more than mice bearing the severe mutations at 7 and 14 days of age (P Ͻ 0.05); however, they weighed significantly less than wild-type controls thereafter (P Ͻ 0.05) and were not significantly different from cystic fibrosis mice bearing the severe Cftr mutations at 21 days of life (P Ͼ 0.05). Login to comment 128 ABCC7 p.Ser489* The amiloride response was significantly different between wild-type mice and S489X mice (P ϭ 0.005). Login to comment 130 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* Absolute body weight of cystic fibrosis mice before weaning Cftr Genotype Sample Size Age, days 7 14 21 S489X/S489X 14 3.0Ϯ0.6 5.8Ϯ0.9 7.0Ϯ1.1 Y122X/Y122X 4 3.6Ϯ1.7 6.1Ϯ2.3 7.2Ϯ2.5 ⌬F508/⌬F508 10 3.1Ϯ0.9 5.6Ϯ1.2 6.7Ϯ0.8 R117H/R117H 15 4.7Ϯ0.7* 7.1Ϯ0.9* 7.9Ϯ1.8 Data are represented as means Ϯ SD. Login to comment 131 ABCC7 p.Ser489* The amiloride response was significantly different between wild-type mice and S489X mice (P ϭ 0.005). Login to comment 132 ABCC7 p.Ser489* *Significantly different from cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation of the same age as assessed by one-way ANOVA; pairwise comparisons made by Tukey`s test. Login to comment 133 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* Absolute body weight of cystic fibrosis mice before weaning Cftr Genotype Sample Size Age, days 7 14 21 S489X/S489X 14 3.0Ϯ0.6 5.8Ϯ0.9 7.0Ϯ1.1 Y122X/Y122X 4 3.6Ϯ1.7 6.1Ϯ2.3 7.2Ϯ2.5 ⌬F508/⌬F508 10 3.1Ϯ0.9 5.6Ϯ1.2 6.7Ϯ0.8 R117H/R117H 15 4.7Ϯ0.7* 7.1Ϯ0.9* 7.9Ϯ1.8 Data are represented as means Ϯ SD. Login to comment 135 ABCC7 p.Ser489* ABCC7 p.Ser489* *Significantly different from cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation of the same age as assessed by one-way ANOVA; pairwise comparisons made by Tukey`s test. Login to comment 138 ABCC7 p.Ser489* ABCC7 p.Ser489* Inflammatory Responses of Cystic Fibrosis Mice Experiments were conducted with each genotype compared in the same experiments to mice bearing the S489X mutation. Login to comment 141 ABCC7 p.Ser489* Nevertheless, each genotype can be compared directly with S489X under identical conditions. Login to comment 142 ABCC7 p.Tyr122* ABCC7 p.Ser489* That is, when comparisons were made between mice bearing the S489X mutation and those bearing the Y122X mutation, we combined data from experiments 68, 72, 80, and 94, taking into account any differences between the experiments. Login to comment 143 ABCC7 p.Arg117His ABCC7 p.Ser489* ABCC7 p.Ser489* Similarly, comparisons between mice bearing the S489X mutation and those bearing the ⌬F508 mutation were made by combining data from experiments 68, 75, 90, and 94, and by combining data from experiments 64 and 94, comparisons were made between mice bearing the S489X mutation and those bearing the R117H mutation. Login to comment 145 ABCC7 p.Tyr122* ABCC7 p.Ser489* That is, when comparisons were made between mice bearing the S489X mutation and those bearing the Y122X mutation, we combined data from experiments 68, 72, 80, and 94, taking into account any differences between the experiments. Login to comment 146 ABCC7 p.Arg117His ABCC7 p.Ser489* ABCC7 p.Ser489* ABCC7 p.Ser489* Similarly, comparisons between mice bearing the S489X mutation and those bearing the ⌬F508 mutation were made by combining data from experiments 68, 75, 90, and 94, and by combining data from experiments 64 and 94, comparisons were made between mice bearing the S489X mutation and those bearing the R117H mutation. Login to comment 149 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* There were no significant differences in starting weight between cystic fibrosis mice bearing the S489X mutation and those bearing any other Cftr mutation (Fig. 3A). Login to comment 150 ABCC7 p.Ser489* There were no significant differences in weight loss between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation. Login to comment 152 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* After differences between the experiments are taken into consideration (Fig. 3B), weight loss in cystic fibrosis mice bearing the S489X mutation is significantly greater than those bearing the Y122X mutation on days 1, 2, and 3 (P Ͻ 0.05) and significantly less than those bearing the R117H mutation on days 1 and 2 (P Ͻ 0.05). Login to comment 153 ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* There were no significant differences in weight loss between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 mutation. Login to comment 156 ABCC7 p.Tyr122* ABCC7 p.Ser489* When stratifying for experiment, we found significant differences (P Ͻ 0.05) between cystic fibrosis mice bearing the Y122X mutation and those bearing the S489X mutation with Fig. 1. Login to comment 157 ABCC7 p.Tyr122* ABCC7 p.Ser489* Cystic fibrosis mice bearing the S489X mutation were removed from this study to be used in other studies starting at 6 wk of age, and data are censored by death in cystic fibrosis mice bearing the Y122X mutation starting after 6 wk of age. Login to comment 159 ABCC7 p.Arg117His ABCC7 p.Ser489* L947ROLE regard to TNF-␣ and IL-1beta concentrations in the epithelial lining fluid, although there were no significant differences in cytokine levels between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 or R117H mutations. Login to comment 160 ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* Cystic fibrosis mice bearing the S489X mutation were removed from this study to be used in other studies starting at 6 wk of age, and data are censored by death in cystic fibrosis mice bearing the Y122X mutation starting after 6 wk of age. Login to comment 162 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Ser489* ABCC7 p.Ser489* L947ROLE regard to TNF-␣ and IL-1beta concentrations in the epithelial lining fluid, although there were no significant differences in cytokine levels between cystic fibrosis mice bearing the S489X mutation and those bearing the ⌬F508 or R117H mutations. Login to comment 163 ABCC7 p.Ser489* In direct comparisons when stratifying for experiment, we found no significant differences in epithelial lining fluid concentrations of IL-6, MIP-2, KC, LTB4, or PGE2 (P Ͼ 0.05) between mice bearing the S489X mutation and those bearing any of the other Cftr mutations. Login to comment 164 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* In a comparison of S489X vs. Y122X, ⌬F508, and R117H genotypes, the available sample sizes provide 80% power to detect Fig. 2. Login to comment 165 ABCC7 p.Arg117His ABCC7 p.Ser489* Cystic fibrosis mice bearing the R117H mutation had significantly lower relative neutrophil numbers and greater absolute alveolar macrophage numbers (Table 5) compared with those bearing the S489X mutation in direct comparisons when stratifying for experiment. Login to comment 167 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* In a comparison of S489X vs. Y122X, ⌬F508, and R117H genotypes, the available sample sizes provide 80% power to detect Fig. 2. Login to comment 168 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* The difference in nasal PD from the start of the response to 1 min later was determined, and representative tracings are shown to the end of the tracing period from wild type (A), S489X (B), Y122X (C), ⌬F508 (D), and R117H (E) mice. Login to comment 170 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* Nasal potential differences in wild-type mice and mice bearing mutations in Cftr Cftr Mutation Sample Size Amiloride, mV Chloride-free, Amiloride, Forskolin, mV None 3 -2.1Ϯ1.5 14.4Ϯ1.2 S489X 7 -13.2Ϯ5.1* -2.6Ϯ2.2* Y122X 3 -10.9Ϯ3.7 -0.8Ϯ1.3* ⌬F508 6 -7.6Ϯ3.5 -1.8Ϯ0.8* R117H 3 -8.5Ϯ0.1 0.1Ϯ0.8* Data are means Ϯ SD. Login to comment 171 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* The difference in nasal PD from the start of the response to 1 min later was determined, and representative tracings are shown to the end of the tracing period from wild type (A), S489X (B), Y122X (C), ⌬F508 (D), and R117H (E) mice. Login to comment 173 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* Nasal potential differences in wild-type mice and mice bearing mutations in Cftr Cftr Mutation Sample Size Amiloride, mV Chloride-free, Amiloride, Forskolin, mV None 3 -2.1Ϯ1.5 14.4Ϯ1.2 S489X 7 -13.2Ϯ5.1* -2.6Ϯ2.2* Y122X 3 -10.9Ϯ3.7 -0.8Ϯ1.3* ⌬F508 6 -7.6Ϯ3.5 -1.8Ϯ0.8* R117H 3 -8.5Ϯ0.1 0.1Ϯ0.8* Data are means Ϯ SD. Login to comment 176 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* Starting sample sizes in each experiment Experiment Mouse Strain by Cftr Mutation S489X Y122X ⌬F508 R117H 64 9 (1) 0 0 9 (1) 68 8 4 5 0 72 8 (1)* 8 0 0 75 7 (1) 0 11 (1) [2]* 0 80 9* 10 0 0 90† 10 0 10 0 94† 9 7* 9 (1) 9 (1)* Total 60 (3) 29 35 (2) [2] 18 (2) The number of mice that died due to surgical complications or pulmonary obstruction is noted in parentheses and brackets, respectively. Login to comment 178 ABCC7 p.Tyr122* ABCC7 p.Ser489* When comparing the S489X to Y122X or ⌬F508 strains, we could detect even smaller differences with high power. Login to comment 180 ABCC7 p.Ser489* Kruskal-Wallis tests were performed only on data from the S489X mice, comparing responses across experiments for the weight, cytokine, and cell count data. Login to comment 181 ABCC7 p.Tyr122* ABCC7 p.Ser489* When comparing the S489X to Y122X or ⌬F508 strains, we could detect even smaller differences with high power. Login to comment 183 ABCC7 p.Ser489* Kruskal-Wallis tests were performed only on data from the S489X mice, comparing responses across experiments for the weight, cytokine, and cell count data. Login to comment 191 ABCC7 p.Ser489* *Significantly different from cystic fibrosis mice bearing the S489X mutation at the same time point, after differences between experiments are taken into consideration. Login to comment 193 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* ABCC7 p.Ser489* Inflammatory mediators in epithelial lining fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X TNF-␣ 7.8* (5.1/13.1) 10.3 (6.0/14.3) 8.0 (6.7/12.2) 9.5 (7.2/12.4) 14.7 (5.9/23.5) 13.6 (9.1/22.3) IL-1beta 14.5* (5.6/19.3) 16.0 (7.5/25.7) 14.4 (10.0/20.2) 16.0 (8.2/20.2) 12.2 (3.0/32.6) 18.2 (8.1/24.3) IL-6 12.3 (5.9/28.4) 14.6 (7.9/33.1) 11.9 (7.8 (22.8) 13.4 (8.0/19.2) 16.6 (7.3/24.7) 20.8 (9.0/62.4) MIP-2 55.8 (12.1/86.3) 66.9 (34.8/107.3) 90.8 (58.8/129.7) 102.2 (48.0/140.8) 55.6 (21.2/202.5) 96.0 (37.6/131.5) KC 4.1 (3.3/5.5) 5.9 (3.5/7.8) 5.4 (4.1/9.4) 6.3 (4.8/8.6) 12.8 (5.0/15.8) 7.4 (4.6/10.7) LTB4 1.2 (0.2/3.3) 2.3 (1.3/7.8) 5.9 (3.6/10.5) 7.1 (2.3/10.5) 2.1 (1.2/16.7) 2.3 (1.3/7.8) PGE2 4.4 (2.3/6.0) 8.0 (4.4/12.2) 11.4 (7.3/16.6) 12.9 (7.6/22.0) 9.2 (5.9/13.5) 8.0 (4.4/12.2) Data are pooled from available data, are represented as the median (25th/75th percentiles), and are expressed as ng/ml epithelial lining fluid (ELF). Login to comment 194 ABCC7 p.Ser489* *Significantly different from cystic fibrosis mice bearing the S489X mutation at the same time point, after differences between experiments are taken into consideration. Login to comment 195 ABCC7 p.Ser489* *Significantly different from mice bearing the S489X mutation in the same comparison group adjusting for differences between experiments (P Ͻ 0.05). Login to comment 196 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* ABCC7 p.Ser489* Inflammatory mediators in epithelial lining fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X TNF-␣ 7.8* (5.1/13.1) 10.3 (6.0/14.3) 8.0 (6.7/12.2) 9.5 (7.2/12.4) 14.7 (5.9/23.5) 13.6 (9.1/22.3) IL-1beta 14.5* (5.6/19.3) 16.0 (7.5/25.7) 14.4 (10.0/20.2) 16.0 (8.2/20.2) 12.2 (3.0/32.6) 18.2 (8.1/24.3) IL-6 12.3 (5.9/28.4) 14.6 (7.9/33.1) 11.9 (7.8 (22.8) 13.4 (8.0/19.2) 16.6 (7.3/24.7) 20.8 (9.0/62.4) MIP-2 55.8 (12.1/86.3) 66.9 (34.8/107.3) 90.8 (58.8/129.7) 102.2 (48.0/140.8) 55.6 (21.2/202.5) 96.0 (37.6/131.5) KC 4.1 (3.3/5.5) 5.9 (3.5/7.8) 5.4 (4.1/9.4) 6.3 (4.8/8.6) 12.8 (5.0/15.8) 7.4 (4.6/10.7) LTB4 1.2 (0.2/3.3) 2.3 (1.3/7.8) 5.9 (3.6/10.5) 7.1 (2.3/10.5) 2.1 (1.2/16.7) 2.3 (1.3/7.8) PGE2 4.4 (2.3/6.0) 8.0 (4.4/12.2) 11.4 (7.3/16.6) 12.9 (7.6/22.0) 9.2 (5.9/13.5) 8.0 (4.4/12.2) Data are pooled from available data, are represented as the median (25th/75th percentiles), and are expressed as ng/ml epithelial lining fluid (ELF). Login to comment 198 ABCC7 p.Ser489* *Significantly different from mice bearing the S489X mutation in the same comparison group adjusting for differences between experiments (P Ͻ 0.05). Login to comment 200 ABCC7 p.Gly551Asp ABCC7 p.Ser489* This has been demonstrated for cystic fibrosis mice bearing the S489X mutation both backcrossed to the C57BL/6 background (9) and on a mixed genetic background of 129P2 and C57BL/6 (10) and for cystic fibrosis mice bearing the G551D mice on a mixed genetic background of CD-1 and 129/Sv (17), in three different laboratories, each with one of two mucoid clinical strains of P. aeruginosa. Login to comment 203 ABCC7 p.Gly551Asp ABCC7 p.Ser489* This has been demonstrated for cystic fibrosis mice bearing the S489X mutation both backcrossed to the C57BL/6 background (9) and on a mixed genetic background of 129P2 and C57BL/6 (10) and for cystic fibrosis mice bearing the G551D mice on a mixed genetic background of CD-1 and 129/Sv (17), in three different laboratories, each with one of two mucoid clinical strains of P. aeruginosa. Login to comment 204 ABCC7 p.Ser489* Correcting the Cftr defect in the gut of cystic fibrosis mice bearing the S489X mutation, by transgenic provision of human CFTR driven by the fatty acid binding protein promoter, results in a much more robust cystic fibrosis mouse that grows normally and does not have intestinal obstruction on a diet of normal mouse chow. Login to comment 207 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* Correcting the Cftr defect in the gut of cystic fibrosis mice bearing the S489X mutation, by transgenic provision of human CFTR driven by the fatty acid binding protein promoter, results in a much more robust cystic fibrosis mouse that grows normally and does not have intestinal obstruction on a diet of normal mouse chow. Login to comment 208 ABCC7 p.Arg117His None of the cystic fibrosis mice studied here grows as well as their wild-type littermates, although the cystic fibrosis mice bearing the R117H mutation maintain weight better at week 1 of life. Login to comment 210 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* In this model of lung infection and inflammation, four different genotypes of cystic fibrosis mice were tested: two knockout mice, Y122X and S489X; mice homozygous for the major processing mutation in cystic fibrosis, ⌬F508; and mice homozygous for a channel mutant, R117H, which reaches the plasma membrane but does not function normally. Login to comment 211 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* None of the cystic fibrosis mice studied here grows as well as their wild-type littermates, although the cystic fibrosis mice bearing the R117H mutation maintain weight better at week 1 of life. Login to comment 213 ABCC7 p.Ser489* Previous results indicate that cystic fibrosis mice bearing the ⌬F508 mutation display the cystic fibrosis phenotype established for the S489X knockout mouse (5) with regard to the nasal potential difference (3, 12). Login to comment 214 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* Here we show that cystic fibrosis mice bearing the Cftr mutations S489X, ⌬F508, Y122X, and R117H on the congenic C57BL/6J background also display the cystic fibrosis electrophysiological phenotype. Login to comment 217 ABCC7 p.Trp1282* However, this will not explain the results in IB-3 cells, which are compound heterozygous cystic fibrosis cells containing W1282X and ⌬F508 (6). Login to comment 220 ABCC7 p.Trp1282* However, this will not explain the results in IB-3 cells, which are compound heterozygous cystic fibrosis cells containing W1282X and ⌬F508 (6). Login to comment 222 ABCC7 p.Arg117His The R117H allele, unlike those of the other genotypes studied here, is expected to reach the plasma membrane. Login to comment 223 ABCC7 p.Arg117His Although the R117H animals have weight comparable to wild-type littermates at 1 wk of age, as early as 2 wk of age Table 5. Login to comment 224 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* ABCC7 p.Ser489* Cell numbers in BAL fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X %AM 5.3 (2.8/41.1) 6.0 (4.3/11.9) 5.8 (3.3/8.7) 4.3 (2.4/5.9) 12.7 (5.8/25.0) 4.7 (4.3/7.4) %PMN 94.3 (58.9/96.9) 94.0 (87.3/95.6) 94.2 (91.3/96.7) 95.3 (94.1/97.6) 87.3* (75.0/94.2) 95.0 (92.6/95.3) %Lymph 0.0 (0.0/0.7) 0.0 (0.0/1.3) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) AM/ml 36 (12/66) 34 (15/72) 47 (23/88) 60 (12/126) 104* (32/168) 78 (56/102) PMN/ml 787 (14/1,449) 801 (422/1,528) 850 (396/1,310) 1,378 (423/1,714) 857 (415/1,549) 1,520 (1,125/1,716) Data are pooled from available data and are represented as the median (25th/75th percentiles). Login to comment 225 ABCC7 p.Arg117His The R117H allele, unlike those of the other genotypes studied here, is expected to reach the plasma membrane. Login to comment 226 ABCC7 p.Arg117His Although the R117H animals have weight comparable to wild-type littermates at 1 wk of age, as early as 2 wk of age Table 5. Login to comment 227 ABCC7 p.Arg117His ABCC7 p.Tyr122* ABCC7 p.Ser489* ABCC7 p.Ser489* ABCC7 p.Ser489* ABCC7 p.Ser489* Cell numbers in BAL fluid Parameter Comparison 1 Comparison 2 Comparison 3 Y122X S489X ⌬F508 S489X R117H S489X %AM 5.3 (2.8/41.1) 6.0 (4.3/11.9) 5.8 (3.3/8.7) 4.3 (2.4/5.9) 12.7 (5.8/25.0) 4.7 (4.3/7.4) %PMN 94.3 (58.9/96.9) 94.0 (87.3/95.6) 94.2 (91.3/96.7) 95.3 (94.1/97.6) 87.3* (75.0/94.2) 95.0 (92.6/95.3) %Lymph 0.0 (0.0/0.7) 0.0 (0.0/1.3) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) 0.0 (0.0/0.0) AM/ml 36 (12/66) 34 (15/72) 47 (23/88) 60 (12/126) 104* (32/168) 78 (56/102) PMN/ml 787 (14/1,449) 801 (422/1,528) 850 (396/1,310) 1,378 (423/1,714) 857 (415/1,549) 1,520 (1,125/1,716) Data are pooled from available data and are represented as the median (25th/75th percentiles). Login to comment 230 ABCC7 p.Ser489* *Significantly different from mice bearing the S489X mutation in the same comparison group adjusting for differences between experiments (P Ͻ 0.05). Login to comment 233 ABCC7 p.Gly551Asp These data are concordant with those of McMorran et al. (17), who studied mice bearing the G551D allele, which reaches the plasma membrane but fails to be activated to open the channel. Login to comment 236 ABCC7 p.Gly551Asp These data are concordant with those of McMorran et al. (17), who studied mice bearing the G551D allele, which reaches the plasma membrane but fails to be activated to open the channel. Login to comment 239 ABCC7 p.Ser489* ABCC7 p.Ser489* Each genotype was studied at the same time as cystic fibrosis mice bearing the S489X mutation, so that direct comparisons are possible (same preparation of agarose beads, same circumstances in the animal facility), and there were no substantive differences between cystic fibrosis mice bearing the S489X mutation and any other genotype. Login to comment 240 ABCC7 p.Tyr122* ABCC7 p.Ser489* Only the cystic fibrosis mice bearing the Y122X mutation differed in two cytokines from S489X, and this may represent the effect of multiple comparisons, rather than a real difference between them. Login to comment 242 ABCC7 p.Ser489* ABCC7 p.Ser489* Each genotype was studied at the same time as cystic fibrosis mice bearing the S489X mutation, so that direct comparisons are possible (same preparation of agarose beads, same circumstances in the animal facility), and there were no substantive differences between cystic fibrosis mice bearing the S489X mutation and any other genotype. Login to comment 243 ABCC7 p.Tyr122* ABCC7 p.Ser489* Only the cystic fibrosis mice bearing the Y122X mutation differed in two cytokines from S489X, and this may represent the effect of multiple comparisons, rather than a real difference between them. Login to comment 245 ABCC7 p.Gly551Asp However, together with clinical observations that patients with severe mutations all seem to have similar lung disease, whether the mutations are stop codons, processing mutants, or activation mutants that reach the membrane, such as G551D, our data suggest that lack of function of CFTR, and not the absence of CFTR at the membrane or the presence of misprocessed CFTR in the cells, is the predominant factor that accounts for the excess airway inflammation. Login to comment 248 ABCC7 p.Gly551Asp However, together with clinical observations that patients with severe mutations all seem to have similar lung disease, whether the mutations are stop codons, processing mutants, or activation mutants that reach the membrane, such as G551D, our data suggest that lack of function of CFTR, and not the absence of CFTR at the membrane or the presence of misprocessed CFTR in the cells, is the predominant factor that accounts for the excess airway inflammation. Login to comment