ABCMdb

ABCC7 p.Ser489*

ClinVar:	c.1466C>A , p.Ser489* D , Pathogenic
CF databases:	c.1466C>A , p.Ser489* D , CF-causing

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[hide] A novel mutation in the CFTR gene correlates with ... J Med Genet. 2000 Mar;37(3):215-8. Wang J, Bowman MC, Hsu E, Wertz K, Wong LJ
A novel mutation in the CFTR gene correlates with severe clinical phenotype in seven Hispanic patients.
J Med Genet. 2000 Mar;37(3):215-8., [PMID:10777364]

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570 SYLVAIN R RIVARD* CHRISTIAN ALLARD† JEAN-PIERRE LEBLANC† MARCEL MILOT† GERVAIS AUBIN† FERNAND SIMARD† CLAUDE FÉREC‡ MARC DE BRAEKELEER†§¶ *Département des Sciences Fondamentales, Université du Québec à Chicoutimi, Canada Table 1 Distribution of cystic fibrosis patients diagnosed before the age of 5 by age groups in Saguenay-Lac-Saint-Jean, (A) by genotype, (B) by mutation 0-10 years 10.1-20 years Over 20 years All ages No % No % No % No % (A) Genotype F508/ F508 15 (1) 40.5 21 (2) 36.2 18 (3) 42.9 54 (6) 39.4 F508/621+1G→T 12 (1) 32.4 16 (1) 27.6 10 (1*) 23.8 38 (3*) 27.7 F508/A455E 1 2.7 6 10.3 5 11.9 12 8.8 F508/I148T 1 2.7 1 1.7 2 1.5 F508/Y1092X 3 (1) 5.2 1 2.4 4 (1) 2.9 F508/Q890X 1 2.4 1 0.7 F508/R1158X 1 2.4 1 0.7 621+1G→T/621+1G→T 2 (1) 5.4 4 6.9 1 2.4 7 (1) 5.1 621+1G→T/A455E 1 2.7 4 6.9 3 7.1 8 5.8 621+1G→T/711+1G→T 2 (1) 5.4 2 (1) 3.4 4 (2) 2.9 621+1G→T/Y1092X 1 2.7 1 0.7 621+1G→T/S489X 1 2.7 1 0.7 621+1G→T/G85E 1 (1) 1.7 1 (1) 2.4 2 (2) 1.5 A455E/R117C 1 2.7 1 0.7 N1303K/I148T 1 2.4 1 0.7 Total 37 58 42 137 Death (4) 10.8 (6) 10.3 (5*) 11.9 (15*) 10.9 (B) Mutation F508 16 (1) 43.2 25 (3) 43.1 21 (3) 51.2 62 (7) 45.6 621+1G→T 18 (3) 48.6 23 (3) 39.7 12 (2*) 29.3 53 (8*) 39.0 A455E 3 8.1 10 17.2 8 19.5 21 15.4 Total 37 58 41 136 Death (4) 10.8 (6) 10.3 (5*) (12.2) (15*) (11.0) ( ): Number of deaths. Login to comment

[hide] CFTR modulates lung secretory cell proliferation a... Am J Physiol Lung Cell Mol Physiol. 2000 Aug;279(2):L333-41. Larson JE, Delcarpio JB, Farberman MM, Morrow SL, Cohen JC
CFTR modulates lung secretory cell proliferation and differentiation.
Am J Physiol Lung Cell Mol Physiol. 2000 Aug;279(2):L333-41., [PMID:10926557]

Abstract [show]
We have permanently reversed the lethal phenotype in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-deficient (knockout) mouse after in utero gene therapy with an adenovirus containing the cftr gene. The gene transfer targeted somatic stem cells in the developing lung and intestine, and these epithelial surfaces demonstrated permanent developmental changes after treatment. The survival statistics from the progeny of heterozygote-heterozygote matings after in utero cftr gene treatment demonstrated an increased mortality in the homozygous normal pups, indicating that overexpression during development was detrimental. The lungs of these pups revealed accelerated secretory cell proliferation and differentiation. The extent of proliferation and differentiation in the secretory cells of the lung parenchyma after in utero transfer of the cftr gene was evaluated with morphometric and biochemical analyses. These studies provide further support of the regulatory role of the cftr gene in the development of the secretory epithelium.

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54 METHODS Colony Maintenance The S489X cftr-mutant UNC mouse strain was obtained from Jackson Laboratories as a fifth-generation backcross. Login to comment
57 Under these conditions, the S489X cftr-mutant mice develop intestinal obstruction, and Ͻ5% survive into adulthood. Login to comment
72 Wild-type CFTR and the knockout S489X allele were amplified in parallel reactions with conditions provided by Jackson Laboratories. Login to comment

[hide] In vivo alterations of IFN regulatory factor-1 and... J Clin Invest. 2000 Aug;106(3):403-10. Kelley TJ, Elmer HL
In vivo alterations of IFN regulatory factor-1 and PIAS1 protein levels in cystic fibrosis epithelium.
J Clin Invest. 2000 Aug;106(3):403-10., [PMID:10930443]

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Inducible nitric oxide synthase-2 (NOS2) expression has been shown to be reduced in cystic fibrosis (CF) epithelial cells. Reduced NOS2 expression is unexpected, given the inflammatory nature of CF airway disease, and is an indication that cell-signaling mechanisms necessary for proper NOS2 regulation are probably altered in CF epithelium. Therefore, we examined the expression levels of regulatory factors necessary for NOS2 expression in CF epithelium and showed that IFN regulatory factor-1 (IRF-1) is necessary for full NOS2 expression. Mice lacking IRF-1 expression have diminished epithelial NOS2 expression, as well as reduced NO-dependent chloride transport across the nasal epithelia. Furthermore, IRF-1 protein expression is reduced in nasal and intestinal epithelial cells from CF mice, suggesting a possible mechanism for the CF-related reduction of epithelial NOS2 expression. Active signal transducer and activator of transcription-1 (Stat1) is necessary for both NOS2 and IRF-1 expression. We found that protein levels of Stat1 were increased in CF cells, but that the active phosphorylated form of Stat1 was bound to the protein inhibitor of activated Stat1 (PIAS1). We propose that increased levels of PIAS1 diminish certain cell-signaling pathways, resulting in reduced IRF-1 and NOS2 expression in CF epithelial cells.

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324 Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

[hide] Pseudomonas aeruginosa induction of apoptosis in r... Am J Respir Cell Mol Biol. 2000 Sep;23(3):304-12. Rajan S, Cacalano G, Bryan R, Ratner AJ, Sontich CU, van Heerckeren A, Davis P, Prince A
Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors.
Am J Respir Cell Mol Biol. 2000 Sep;23(3):304-12., [PMID:10970820]

Abstract [show]
Airway epithelial cells can respond to infection by activating several signaling pathways. We examined the induction of apoptosis in response to Pseudomonas aeruginosa PAO1 in normal cells and several cystic fibrosis (CF) and corrected cell lines. Epithelial cells in monolayers with tight junctions, confirmed by apical ZO-1 staining demonstrated by confocal microscopy, were entirely resistant to PAO1-induced apoptosis. In contrast, cell lines such as 9HTEo(-) cells that do not form tight junctions were susceptible, with 50% of the population apoptotic after 6 h of exposure to PAO1. CF transmembrane conductance regulator (CFTR) dysfunction caused by different mechanisms (trafficking mutations, overexpression of the regulatory domain or antisense constructs) did not alter rates of apoptosis, nor were differences apparent in terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling detection of apoptotic airway cells from PAO1 infected cftr -/- or control mice. Bacterial expression of specific adhesins, complete lipopolysaccharide, and a functional type III secretion system were all necessary to evoke apoptosis even in susceptible epithelial cells. Unlike other mucosal surfaces, the airway epithelium is highly resistant to apoptosis, and this response is activated only when the appropriate epithelial conditions are present as well as fully virulent P. aeruginosa capable of coordinately expressing both adhesins and cytotoxins.

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84 Immunohistochemistry Mice homozygous for the CFTR S489X (null) mutation or wild-type littermate controls (cftr -/- and ϩ/ϩ) were inoculated with P. aeruginosa impregnated in agar beads as previously described (10). Login to comment

[hide] Expression of nucleotide-regulated Cl(-) currents ... Am J Physiol Cell Physiol. 2000 Nov;279(5):C1578-86. Thomas EJ, Gabriel SE, Makhlina M, Hardy SP, Lethem MI
Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines.
Am J Physiol Cell Physiol. 2000 Nov;279(5):C1578-86., [PMID:11029305]

Abstract [show]
The dominant route for Cl(-) secretion in mouse tracheal epithelium is via Cl(-) channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(-/-)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/-)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10(-4) M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current (I(sc)) and resistance of both cell lines, with larger increases in I(sc) being elicited by ionomycin (10(-6) M). Both cell lines expressed P(2)Y(2) receptors and responded to the purinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10(-4) M) with an increase in I(sc). This response could be inhibited by DIDS and was abolished in the presence of Cl(-)-free Ringer solution. Reducing the mucosal Cl(-) concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(-/-) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I(sc) and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl(-) currents and may prove valuable tools in studying the properties of this pathway.

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42 By use of this protocol, the CFTR knockout (S489X) gave a 210-bp product and the wild-type CFTR yielded a 190-bp product. PCR products were identified by electrophoresis on 2% (wt/vol) and 3% (wt/vol) agarose gels for TAg and CFTR products, respectively. Login to comment
90 The CFTR and TAg genotype of each cell line was confirmed by PCR, which indicated that MTE18- (-/-) cells were homozygous for the S489X CFTR knockout, MTE7b-(ϩ/-) cells were heterozygous for this mutation (Fig. 1E), and both cell lines were positive for TAg (Fig. 1F). Login to comment
155 In the present study, two such cell lines have been generated: one from a mouse homozygous for the S489X CFTR knockout and one from a mouse heterozygous for this mutation. Login to comment

[hide] Generation and phenotype of cell lines derived fro... Am J Physiol Cell Physiol. 2001 Jan;280(1):C228-36. Takacs-Jarrett M, Sweeney WE, Avner ED, Cotton CU
Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2K(b)-tsA58 transgene.
Am J Physiol Cell Physiol. 2001 Jan;280(1):C228-36., [PMID:11121394]

Abstract [show]
Tracheal, renal, salivary, and pancreatic epithelial cells from cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) -/-] and non-CF mice that carry a temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were isolated and maintained in culture under permissive conditions (33 degrees C with interferon-gamma). The resultant cell lines have been in culture for >1 year and 50 passages. Each of the eight cell lines form polarized epithelial barriers and exhibit regulated, electrogenic ion transport. The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl(-) permeability and cAMP-stimulated Cl(-) secretion. In contrast, the four CFTR -/- cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated Cl(-) secretory responses. Ca(2+)-activated Cl(-) secretion is retained in both CF and non-CF cell lines. Thus we have generated genetically well-matched epithelial cell lines from several tissues relevant to cystic fibrosis that either completely lack CFTR or express endogenous levels of CFTR. These cell lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.

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41 The goal of this work was to cross the ImmortoMouse (26) with the University of North Carolina (UNC) CF knockout mouse (CFTR S489X) (40) and develop genetically well-matched, conditionally immortalized CF and non-CF epithelial cell lines. Login to comment
42 METHODS Animals Male mice, homozygous for a temperature-sensitive SV40 large T antigen transgene (ImmortoMouse; CBA/ca X C57B1/10 strain; Charles River Laboratories) (26), were bred with female mice that were heterozygous for the S489X CFTR mutation (UNC; CFTR ϩ/-; C57BL/6J X F129 strain) (40). Login to comment
50 Primers 5 and 6 were used to amplify the S489X neodisrupted allele of CFTR (NEO). Login to comment
145 The CF cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) were negative for the wild-type CFTR allele (faint 200-bp bands are non-CFTR PCR products) and positive for the S489X neodisrupted allele of CFTR. Login to comment
151 Magnification, ϫ270. both the wild-type CFTR allele and the S489X neodisrupted allele, whereas one of the non-CF cell lines (mSEC1) was positive for the wild-type CFTR allele and negative for the S489X neodisrupted allele of CFTR. Login to comment
179 Wild-type cystic fibrosis transmembrane conductance regulator (CFTR), neomycin disrupted S489X CFTR (NEO), and Immorto (IM) alleles were identified from PCR products separated by gel electrophoresis. Login to comment

[hide] Role of CFTR in autosomal recessive polycystic kid... J Am Soc Nephrol. 2001 Apr;12(4):719-25. Nakanishi K, Sweeney WE Jr, Macrae Dell K, Cotton CU, Avner ED
Role of CFTR in autosomal recessive polycystic kidney disease.
J Am Soc Nephrol. 2001 Apr;12(4):719-25., [PMID:11274233]

Abstract [show]
An extensive body of in vitro data implicates epithelial chloride secretion, mediated through cystic fibrosis transmembrane conductance regulator (CFTR) protein, in generating or maintaining fluid filled cysts in MDCK cells and in human autosomal dominant polycystic kidney disease (ADPKD). In contrast, few studies have addressed the pathophysiology of fluid secretion in cyst formation and enlargement in autosomal recessive polycystic kidney disease (ARPKD). Murine models of targeted disruptions or deletions of specific genes have created opportunities to examine the role of individual gene products in normal development and/or disease pathophysiology. The creation of a murine model of CF, which lacks functional CFTR protein, provides the opportunity to determine whether CFTR activity is required for renal cyst formation in vivo. Therefore, this study sought to determine whether renal cyst formation could be prevented by genetic complementation of the BPK murine model of ARPKD with the CFTR knockout mouse. The results of this study reveal that in animals that are homozygous for the cystic gene (bpk), the lack of functional CFTR protein on the apical surface of cystic epithelium does not provide protection against cyst growth and subsequent decline in renal function. Double mutant mice (bpk -/-; cftr -/-) developed massively enlarged kidneys and died, on average, 7 d earlier than cystic, non-CF mice (bpk -/-; cftr +/+/-). This suggests fundamental differences in the mechanisms of transtubular fluid secretion in animal models of ARPKD compared with ADPKD.

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38 The absence of functional CFTR in the animal model for CF (CFTR S489X or UNC) used in this study has been demonstrated extensively by Western analysis, ribonuclease protection assay, electrophysiologic analysis, and PCR analysis. Login to comment

[hide] Nitric oxide deficiency contributes to impairment ... Am J Respir Cell Mol Biol. 2001 May;24(5):621-6. Mhanna MJ, Ferkol T, Martin RJ, Dreshaj IA, van Heeckeren AM, Kelley TJ, Haxhiu MA
Nitric oxide deficiency contributes to impairment of airway relaxation in cystic fibrosis mice.
Am J Respir Cell Mol Biol. 2001 May;24(5):621-6., [PMID:11350833]

Abstract [show]
The pulmonary disease of cystic fibrosis (CF) is characterized by persistent airway obstruction, which has been attributed to chronic endobronchial infection and inflammation. The levels of exhaled nitric oxide (NO) are reduced in CF patients, which could contribute to bronchial obstruction through dysregulated constriction of airway smooth muscle. Because airway epithelium from CF mice has been shown to have reduced expression of inducible NO synthase, we examined airway responsiveness and relaxation in isolated tracheas of CF mice. Airway relaxation as measured by percent relaxation of precontracted tracheal segments to electrical field stimulation (EFS) and substance P, a nonadrenergic, noncholinergic substance, was significantly impaired in CF mice. The airway relaxation in response to prostaglandin E2 was similar in CF and non-CF animals. Treatment with the NO synthase inhibitor NG-nitro-L-arginine methylester reduced tracheal relaxation induced by EFS in wild-type animals but had virtually no effect in the CF mice. Conversely, exogenous NO and L-arginine, a NO substrate, reversed the relaxation defect in CF airway. We conclude that the relative absence of NO compromises airways relaxation in CF, and may contribute to the bronchial obstruction seen in the disease.

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23 To test this hypothesis, we compared the airway relaxant response of CF mice homozygous for the null S489X mutation with that of their normal littermates. Login to comment
28 Materials and Methods Animals Male and female mice homozygous for the S489X (CFTR-/- ) mutation of the CFTR and their normal littermates (CFTRϩ/ϩ ) were studied (18, 19). Login to comment
35 of Pediatrics, Washington University School of Medicine, St. Louis, MO. Abbreviations: analysis of variance, ANOVA; cystic fibrosis, CF; CF transmembrane conductance regulator, CFTR; mice homozygous for the S489X mutation of the CFTR, CFTR-/- mice; normal littermates of CFTR-/- mice, CFTRϩ/ϩ mice; electrical field stimulation, EFS; inducible NOS, iNOS; NG-nitro-L-arginine methylester, L-NAME; neuronal NOS, nNOS; nitric oxide, NO; NO synthase, NOS; prostaglandin, PG; standard error of the mean, SEM; substance P, SP; percentage of maximal tracheal tension, %T max. Login to comment

[hide] Transgenic cystic fibrosis mice exhibit reduced ea... J Immunol. 2001 Jun 15;166(12):7410-8. Schroeder TH, Reiniger N, Meluleni G, Grout M, Coleman FT, Pier GB
Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract.
J Immunol. 2001 Jun 15;166(12):7410-8., 2001-06-15 [PMID:11390493]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.

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5 We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the ⌬F508 Cftr allele or an allele with a Cftr stop codon (S489X). Login to comment
6 Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 ⌬F508 Cftr mice, whereas ⌬F508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. Login to comment
47 Transgenic CF mice included the following strains: C57BL/6 mice homozygous for the ⌬F508 allele of mouse Cftr (B6.129S6-Cftrttm1Kth ) (10), noninbred homozygous ⌬F508 Cftr mice (11), and doubly transgenic S489X Cftr-fatty acid binding protein (FABP)huCftr mice (no murine Cftr protein but expressing wild-type human (hu) CFTR protein in the gastrointestinal tract from the huCFTR gene under the control of the mouse FABP promoter) (12). Login to comment
48 The doubly transgenic S489X Cftr-FABPhuCftr mice were bred as homozygotes. Login to comment
113 Plating serial dilutions of the gastrointestinal organs and the nasopharyngeal anatomic area of intranasally infected S489X-FABPhuCftr mice revealed that the applied bacteria were not swallowed but remained in the nasopharynx of these transgenic CF mice (data not shown). Login to comment
122 We first compared the total and internalized amount of bacteria in the lungs 4.5 h after tracheal infection of anesthetized wild-type C57BL/6 mice with that in two strains of transgenic CF mice: noninbred S489X FABPhuCftr mice and inbred B6.129S6-Cftrttm1Kth . Login to comment
123 We found that lung cells of wild-type C57BL/6 mice internalized 7.6 times more P. aeruginosa PAO1 bacteria than did the C57BL/6 mice homozygous for the ⌬F508 Cftr allele (B6.129S6-Cftrttm1Kth mice) and 3.9 times more bacteria than did the S489X-FABPhuCftr mice ( p Ͻ 0.001, ANOVA and Fisher PLSD; Fig. 1A). Login to comment
125 Coincident with the impaired internalization of P. aeruginosa in the respiratory tract of the two transgenic CF mouse strains, the total level of the infecting bacteria measured in the lungs was increased 22-fold in homozygous ⌬F508 Cftr C57BL/6 mice and 30-fold in S489X-FABPhuCftr mice, whereas in wild-type mice the increase in the bacterial burden over the inoculating dose was only 10-fold (Fig. 1B; both CF strains p Ͻ 0.01, ANOVA and Fisher PLSD compared with wild-type mice). Login to comment
134 Chroneos et al. (7) recently reported that there was no difference in clearance of P. aeruginosa from the lungs of S489X-FABPhuCftr mice compared with wild-type mice. Login to comment
198 Chroneos et al. (7) recently reported no difference in clearance of P. aeruginosa strain FRD-1 from the lungs of S489X-FABPhuCftr mice in comparison to wild-type mice, 24 h after a dose of 1.5 ϫ 107 CFU delivered intratracheally. Login to comment
204 Chroneos et al. (7) also studied strain PAO1, as we did, but they only compared clearance between wild-type mice and double-transgenic S489X-FABPhuCftr mice that had been further engineered to overexpress huCFTR in the lungs under the control of the surfactant protein C (SP-C) promoter (SP-ChuCFTR mice). Login to comment
208 For strain PAO1, Chroneos et al. (7) did not compare clearance in the transgenic S489X-FABPhuCFTR mice lacking CFTR in the lung with wild-type mice. Login to comment
263 This occurred in both inbred and outbred transgenic mouse lines homozygous for the ⌬F508 allele of Cftr and in S489X Cftr-FABPhuCftr mice, which have no intact CFTR protein. Login to comment

[hide] Antioxidant imbalance in the lungs of cystic fibro... Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L31-8. Velsor LW, van Heeckeren A, Day BJ
Antioxidant imbalance in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice.
Am J Physiol Lung Cell Mol Physiol. 2001 Jul;281(1):L31-8., [PMID:11404242]

Abstract [show]
Recent studies suggest that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein modulates epithelial reduced glutathione (GSH) transport and when defective creates an antioxidant imbalance. To test whether the CFTR protein modulates lung antioxidant defenses in vivo, epithelial lining fluid (ELF) and lung tissue from CFTR knockout (CFTR-KO) and wild-type (WT) mice were compared for GSH content and the activities of glutathione reductase, glutathione peroxidase, and gamma-glutamyltransferase. In the CFTR-KO mice, the ELF concentration of GSH was decreased (51%) compared with that in WT mice. The concentration of GSH in the lung tissue of CFTR-KO mice, however, was not significantly different from that in WT mice. The activities of glutathione reductase and glutathione peroxidase in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice (48 and 28%, respectively). Tissue lipid and DNA oxidation were evaluated by measurement of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine, respectively. The levels of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice. These data support our hypothesis that a mutation in the CFTR gene can affect the antioxidant defenses in the lung and may contribute to the exaggerated inflammatory response observed in CF.

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42 The CFTR-KO mice used in this study were congenic B6.129P2-Cftrtm1Unc , which possess the S489X mutation in CFTR that renders the CFTR protein nonfunctional (9, 40). Login to comment
44 In this study, the CFTR-KO mice, homozygous for the S489X mutation, were compared with their WT littermates. Login to comment

[hide] Enhanced susceptibility to pulmonary infection wit... Infect Immun. 2001 Aug;69(8):5138-50. Sajjan U, Thanassoulis G, Cherapanov V, Lu A, Sjolin C, Steer B, Wu YJ, Rotstein OD, Kent G, McKerlie C, Forstner J, Downey GP
Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice.
Infect Immun. 2001 Aug;69(8):5138-50., [PMID:11447196]

Abstract [show]
Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate of Burkholderia cepacia (genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs of Cftr (m1unc-/-) (Cftr(-/-)) and congenic Cftr(+/+) controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptible Cftr(-/-) mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from the Cftr(-/-) mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages from Cftr(-/-) mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.

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310 In contrast, when another strain of CFTR-deficient mice (Cftrm1UNC ; S489X null mutant) was challenged with S. aureus, no differences in bacterial clearance between CFTR-deficient mice and the controls were demonstrated (55). Login to comment

[hide] Mineral content of calcified tissues in cystic fib... Biol Trace Elem Res. 2001 Oct;83(1):69-81. Gawenis LR, Spencer P, Hillman LS, Harline MC, Morris JS, Clarke LL
Mineral content of calcified tissues in cystic fibrosis mice.
Biol Trace Elem Res. 2001 Oct;83(1):69-81., [PMID:11694004]

Abstract [show]
Although abnormal hard tissue mineralization is a recognized complication of cystic fibrosis (CF), the pathogenesis leading from the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is poorly understood. We hypothesized that CFTR plays a direct role in the mineralization of bone and teeth and tested the hypothesis using CF mouse models [CFTR(-) mice]. In vivo measurements by dual-emission X-ray absorpitometry (DEXA) indicated that bone mineral density (BMD) was reduced in CF mice as compared to gender-matched littermates. However, no change was evident after correction of BMD for the covariant of body weight. The latter finding was confirmed in isolated femurs and nasal bones by standard dry-ashing and instrumental neutron activation analysis (INAA). INAA of the continuously growing hypsodont incisor teeth from CFTR(-) mice revealed reduced Ca and normal P in the enamel layer--a finding consistent with changes in the deciduous teeth of CF children. Interestingly, enamel fluoride was increased in the CFTR(-) incisors and may associate with abnormal enamel crystallite formation. The iron content of the incisor enamel was reduced, explaining the loss of yellow pigmentation in CFTR(-) incisors. In contrast to the incisors, the mineral content of the slow-growing brachydont molar teeth was not different between CFTR(-) and CFTR(+) mice. It was concluded that CFTR does not play a direct role in the mineralization of bones or brachydont teeth in mice. Functional CFTR is apparently required for normal mineralization of the hypsodont incisors. However, multiple changes in the mineral composition of the CF incisors suggest an indirect role for CFTR, perhaps by maintaining a normal salivary environment for continuous tooth eruption.

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36 MATERIALS AND METHODS Animals This study was performed on CF mice with the S489X mutation (cftrtm1Unc) (14) and ∆F508 mutation (cftrtm1kth) (15). Login to comment

[hide] A(2) adenosine receptors regulate CFTR through PKA... Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L12-25. Cobb BR, Ruiz F, King CM, Fortenberry J, Greer H, Kovacs T, Sorscher EJ, Clancy JP
A(2) adenosine receptors regulate CFTR through PKA and PLA(2).
Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L12-25., [PMID:11741811]

Abstract [show]
We investigated adenosine (Ado) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo. A(2B) Ado receptors were identified in Calu-3, IB-3-1, COS-7, and primary human airway cells. Ado elevated cAMP in Calu-3, IB-3-1, and COS-7 cells and activated protein kinase A-dependent halide efflux in Calu-3 cells. Ado promoted arachidonic acid release from Calu-3 cells, and phospholipase A(2) (PLA(2)) inhibition blocked Ado-activated halide efflux in Calu-3 and COS-7 cells expressing CFTR. Forskolin- and beta(2)-adrenergic receptor-stimulated efflux were not affected by the same treatment. Cytoplasmic PLA(2) (cPLA(2)) was identified in Calu-3, IB-3-1, and COS-7 cells, but cPLA(2) inhibition did not affect Ado-stimulated cAMP concentrations. In cftr(+) and cftr(-/-) mice, Ado stimulated nasal Cl(-) secretion that was CFTR dependent and sensitive to A(2) receptor and PLA(2) blockade. In COS-7 cells transiently expressing DeltaF508 CFTR, Ado activated halide efflux. Ado also activated G551D CFTR-dependent halide efflux when combined with arachidonic acid and phosphodiesterase inhibition. In conclusion, PLA(2) and protein kinase A both contribute to A(2) receptor activation of CFTR, and components of this signaling pathway can augment wild-type and mutant CFTR activity.

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108 The cftr(-/-) mice carried two copies of the human cftr cDNA, which contains a stop codon at position 489 (S489X). Login to comment

[hide] Functional IL-10 deficiency in the lung of cystic ... J Immunol. 2002 Feb 15;168(4):1903-10. Soltys J, Bonfield T, Chmiel J, Berger M
Functional IL-10 deficiency in the lung of cystic fibrosis (cftr(-/-)) and IL-10 knockout mice causes increased expression and function of B7 costimulatory molecules on alveolar macrophages.
J Immunol. 2002 Feb 15;168(4):1903-10., 2002-02-15 [PMID:11823525]

Abstract [show]
Alveolar macrophages are poor APCs that only minimally express B7 costimulatory molecules. Because our previous data suggest that bronchial epithelial cells constitutively secrete IL-10, and IL-10 inhibits B7 expression in vitro, we hypothesized that this IL-10 is responsible for suppressing B7 expression on macrophages that enter the airways. Furthermore, because we have shown that cystic fibrosis (CF) lungs are deficient in IL-10, we hypothesized that bronchoalveolar macrophages (BALMs) from cystic fibrosis transmembrane conductance regulator (CFTR)(-/-) as well as IL-10(-/-) mice might express increased B7. Immunofluorescence for B7 was positive on BALMs from CF patients and CFTR(-/-) and IL-10(-/-) mice, but was negative on controls. FACS showed that 63.9% of BALMs from IL-10(-/-) mice were B7-1 positive, as were 67.4% of BALMs from CFTR(-/-) mice, whereas <7% of BALMs from wild-type controls were positive. Using BALMs to costimulate splenic T cells with anti-CD3 as a mitogen showed 9202 +/- 2107 cpm [(3)H]thymidine incorporation for BALMs from IL-10(-/-) mice and 4082 +/- 1036 cpm for BALMs from CFTR(-/-) mice, but <200 cpm with BALMs from either type of +/+ mouse. Treatment of CFTR(-/-) mice with recombinant mouse IL-10 reduced the B7 expression and costimulatory activity of the BALMs. These data suggest that the IL-10 secreted in the healthy lung may be responsible for the absence of B7 and poor costimulatory activity of BALMs and that reductions of pulmonary IL-10 in CF may enhance B7 expression and local immune responses.

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43 Mice homozygous for the S489X (B6.129P2-Cftrtm1Unc ) mutation of the CFTR gene, congenic (n ϭ 10 generations) onto C57BL/6J background, (18) and their normal littermates, designated as CFTR-/- and CFTRϩ/ϩ , respectively, were bred in our Animal Core facility. Login to comment

[hide] Functional evidence of CFTR gene transfer in nasal... Mol Ther. 2002 Apr;5(4):413-9. Ziady AG, Kelley TJ, Milliken E, Ferkol T, Davis PB
Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA complexes targeted to the serpin-enzyme complex receptor.
Mol Ther. 2002 Apr;5(4):413-9., [PMID:11945068]

Abstract [show]
Molecular conjugates that target the serpin-enzyme complex receptor transfer the cDNA encoding human cystic fibrosis transmembrane conductance regulator (CFTR) to the nasal epithelium of cystic fibrosis mutant mice. These complexes effect partial correction of the chloride transport defect as assessed by in vivo nasal potential difference measurements, produce immunohistochemical staining for CFTR, and restore expression of nitric oxide synthase-2 (NOS-2), which is downregulated in the epithelium of mice and humans with cystic fibrosis. Complexes that lack the receptor ligands were ineffective, so receptor access was essential. Mice treated with receptor-targeted lacZ showed beta-galactosidase expression in epithelial cells and submucosal glands, but no electrophysiologic correction or NOS-2 expression, so simply accessing the serpin-enzyme complex receptor was not sufficient to produce the observed electrophysiologic or immunohistochemical changes. Correction of the cAMP-stimulated chloride transport was dose related at days 7 and 12 after complex administration, but, for most animals, nasal potential difference had returned to baseline by day 18. Molecular conjugates targeting the serpin-enzyme complex receptor, used to compact plasmid DNA, hold promise for gene therapy of cystic fibrosis.

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172 We studied 8to 10-week-old B6.129P2-cftrtm1Unc mice (CF knockout, S489X mice) [25] fed a liquid diet [26], or STOCK Cftrtm1Unc - TgN(FABPCFTR)#Jaw mice [27], which are CF mutant mice with the defect corrected only in intestinal epithelium by transgenic human CFTR. Login to comment

[hide] Characterization of LPS-induced lung inflammation ... J Appl Physiol. 2002 May;92(5):2169-76. Freedman SD, Weinstein D, Blanco PG, Martinez-Clark P, Urman S, Zaman M, Morrow JD, Alvarez JG
Characterization of LPS-induced lung inflammation in cftr-/- mice and the effect of docosahexaenoic acid.
J Appl Physiol. 2002 May;92(5):2169-76., [PMID:11960971]

Abstract [show]
The mechanism by which Pseudomonas causes excessive inflammation in the cystic fibrosis lung is unclear. We have reported that arachidonic acid is increased and docosahexaenoic acid (DHA) decreased in lung, pancreas, and ileum from cftr-/- mice. Oral DHA corrected this defect and reversed the pathology. To determine which mediators regulate inflammation in lungs from cftr-/- mice and whether inhibition occurs with DHA, cftr-/- and wild-type (WT) mice were exposed to aerosolized Pseudomonas lipopolysaccharide (LPS). After 2 days of LPS, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2, and KC levels in bronchoalveolar lavage fluid were increased in cftr-/- compared with WT mice and not suppressed by pretreatment with oral DHA. Neutrophil levels were not different between cftr-/- and WT mice. After 3 days of aerosolized LPS, neutrophil concentration, TNF-alpha, and the eicosanoids 6-keto-PGF1alpha, PGF2alpha, PGE2, and thromboxane B2 were all increased in bronchoalveolar lavage fluid from cftr-/- mice compared with WT controls. Oral DHA had no significant effect on TNF-alpha levels in cftr-/- mice. In contrast, neutrophils and eicosanoids were decreased in cftr-/- but not in WT mice treated with DHA, indicating that the effects of DHA on these inflammatory parameters may be related to correction of the membrane lipid defect.

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28 To circumvent this problem, a model has been established whereby instillation of agarose beads coated with Pseudomonas into the lungs of S489X cftr-/- mice has been shown to result in increased inflammation and mortality compared with that observed in wild-type (WT) mice (13, 14). Login to comment
29 To circumvent this problem, a model has been established whereby instillation of agarose beads coated with Pseudomonas into the lungs of S489X cftr-/- mice has been shown to result in increased inflammation and mortality compared with that observed in wild-type (WT) mice (13, 14). Login to comment

[hide] Gene complementation of airway epithelium in the c... Hum Mol Genet. 2002 May 1;11(9):1059-67. Oceandy D, McMorran BJ, Smith SN, Schreiber R, Kunzelmann K, Alton EW, Hume DA, Wainwright BJ
Gene complementation of airway epithelium in the cystic fibrosis mouse is necessary and sufficient to correct the pathogen clearance and inflammatory abnormalities.
Hum Mol Genet. 2002 May 1;11(9):1059-67., 2002-05-01 [PMID:11978765]

Abstract [show]
Increasingly, cystic fibrosis (CF) is regarded as an inflammatory disorder where the response of the lung to Pseudomonas aeruginosa is exaggerated as a consequence of processes mediated by the product of the CF gene, CFTR. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type(s) within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance. We generated G551D CF mice transgenically expressing the human CFTR gene in two tissue compartments previously demonstrated to mediate a CFTR-dependent inflammatory response: lung epithelium and alveolar macrophages. Following chronic pulmonary infection with P. aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals. Additionally, these data indicate the general role for epithelial cell-mediated events in the response of the lung to bacterial pathogens and the importance of CFTR in mediating these processes.

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24 CF mice with the null S489X mutation (Cftrm1UNC ) when infected with P. aeruginosa embedded in agar beads produce more proinflammatory cytokines such as tumor necrosis factor a (TNF-a), macrophage inflammatory protein 2 (mip-2) and KC compared with wild-type animals (23). Login to comment

[hide] CFTR is a pattern recognition molecule that extrac... Proc Natl Acad Sci U S A. 2002 May 14;99(10):6907-12. Epub 2002 May 7. Schroeder TH, Lee MM, Yacono PW, Cannon CL, Gerceker AA, Golan DE, Pier GB
CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-kappa B translocation.
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6907-12. Epub 2002 May 7., 2002-05-14 [PMID:11997458]

Abstract [show]
Immune cells are activated during cellular responses to antigen by two described mechanisms: (i) direct uptake of antigen and (ii) extraction and internalization of membrane components from antigen-presenting cells. Although endocytosis of microbial antigens by pattern recognition molecules (PRM) also activates innate immunity, it is not known whether this involves extraction and internalization of microbial surface components. Epithelial cells on mucosal surfaces use a variety of receptors that are distinct from the classical endocytic PRM to bind and internalize intact microorganisms. Nonclassical receptor molecules theoretically could act as a type of endocytic PRM if these molecules could recognize, bind, extract, and internalize a pathogen-associated molecule and initiate cell signaling. We report here that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the outer core oligosaccharide of the lipopolysaccharide (LPS) in the outer membrane of Pseudomonas aeruginosa satisfies all of these conditions. P. aeruginosa LPS was specifically recognized and bound by CFTR, extracted from the organism's surface, and endocytosed by epithelial cells, leading to a rapid (5- to 15-min) and dynamic translocation of nuclear transcription factor NF-kappa B. Inhibition of epithelial cell internalization of P. aeruginosa LPS prevented NF-kappa B activation. Cellular activation depended on expression of wild-type CFTR, because both cultured Delta F508 CFTR human airway epithelial cells and lung epithelial cells of transgenic-CF mice failed to endocytose LPS and translocate NF-kappa B. CFTR serves as a critical endocytic PRM in the lung epithelium, coordinating the effective innate immune response to P. aeruginosa infection.

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20 Noninbred, homozygous ⌬F508 cftr mice (5) and doubly transgenic S489X cftrFABPhuCFTR mice expressing no murine CFTR protein, but expressing wt human CFTR protein in the gastrointestinal tract from the CFTR gene under the control of the mouse fatty acid binding protein promoter (6), were bred in our facility (7). Login to comment
91 There was no translocation of NF-␬B after intratracheal injection of P. aeruginosa into doubly transgenic S489X cftrFABPhuCFTR mice, which lacked expression of murine CFTR in any tissue but expressed human CFTR in the gastrointestinal tract (ref. Login to comment

[hide] Cystic fibrosis: a worldwide analysis of CFTR muta... Hum Mutat. 2002 Jun;19(6):575-606. Bobadilla JL, Macek M Jr, Fine JP, Farrell PM
Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening.
Hum Mutat. 2002 Jun;19(6):575-606., [PMID:12007216]

Abstract [show]
Although there have been numerous reports from around the world of mutations in the gene of chromosome 7 known as CFTR (cystic fibrosis transmembrane conductance regulator), little attention has been given to integrating these mutant alleles into a global understanding of the population molecular genetics associated with cystic fibrosis (CF). We determined the distribution of CFTR mutations in as many regions throughout the world as possible in an effort designed to: 1) increase our understanding of ancestry-genotype relationships, 2) compare mutational arrays with disease incidence, and 3) gain insight for decisions regarding screening program enhancement through CFTR multi-mutational analyses. Information on all mutations that have been published since the identification and cloning of the CFTR gene's most common allele, DeltaF508 (or F508del), was reviewed and integrated into a centralized database. The data were then sorted and regional CFTR arrays were determined using mutations that appeared in a given region with a frequency of 0.5% or greater. Final analyses were based on 72,431 CF chromosomes, using data compiled from over 100 original papers, and over 80 regions from around the world, including all nations where CF has been studied using analytical molecular genetics. Initial results confirmed wide mutational heterogeneity throughout the world; however, characterization of the most common mutations across most populations was possible. We also examined CF incidence, DeltaF508 frequency, and regional mutational heterogeneity in a subset of populations. Data for these analyses were filtered for reliability and methodological strength before being incorporated into the final analysis. Statistical assessment of these variables revealed that there is a significant positive correlation between DeltaF508 frequency and the CF incidence levels of regional populations. Regional analyses were also performed to search for trends in the distribution of CFTR mutations across migrant and related populations; this led to clarification of ancestry-genotype patterns that can be used to design CFTR multi-mutation panels for CF screening programs. From comprehensive assessment of these data, we offer recommendations that multiple CFTR alleles should eventually be included to increase the sensitivity of newborn screening programs employing two-tier testing with trypsinogen and DNA analysis.

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112 Jewish 1) 405+1G®A (48.0%) 3) W1282X (17.0%) - - 4 23 Kerem et al. [1995] (Tunisia) 2) DF508 (31.0%) 4) 3849+10KbC®T (4.0%) Jewish 1) G85E 4) G542X - - 6 10 Kerem et al. [1995] (Turkey) 2) DF508 5) 3849+10KbC®T 3) W1282X 6) W1089X Jewish (Yemen) None - - 0 5 Kerem et al. [1995] Lebanon 1) DF508 (35.0%) 6) 4096-28G®A (2.5%) - - 9 40 Desgeorges et al. [1997] 2) W1282X (20.0%) 7) 2789+5G®A (2.5%) 3) 4010del4 (10.0%) 8) M952I (2.5%) 4) N1303K (10.0%) 9) E672del (2.5%) 5) S4X (5.0%) Reunion ∆F508 (52.0%) 1717-1G→A (0.7%) 90.4 81.7 9 138 Cartault et al. [1996] Island Y122X (24.0%) G542X (0.7%) 3120+1G→A (8.0%) A309G (0.7%) A455E (2.2%) 2789+5G→A (0.7%) G551D (1.4%) Saudi North: 3) H139L - - North 1 49 families El-Harith et al. [1997]; Arabia 1) 1548delG 4) L1177X Central 3 Kambouris et al. [1997]; Central: 5) DF508 South 4 Banjar et al. [1999] 1)I1234V 6) 3120+1G®A West 9 2)1548delG 7) 425del42 East 6 3)DF508 8) R553X South: 9) N1303K 1) I1234V East: 2) 1548delG 1) 3120+1G®A 3) 711+1G®T 2) H139L 4) 3120+1G®A 3) 1548delG West: 4) DF508 1) I1234V 5) S549R 2) G115X 6) N1303K Tunisia ∆F508 (17.6%) G85E (2.6%) 58.7 34.5 11 78 Messaoud et al. [1996] G542X (8.9%) W1282X (2.6%) 711+1G→T (7.7%) Y122X (1.3%) N1303K (6.4%) T665S (1.3%) 2766del8NT (6.4%) R47W+D1270N (1.3%) R1066C (2.6%) Turkeye ∆F508 (24.5%) 1066L (1.3%) 80.6 65.0 36 1067/670 Yilmaz et al. [1995]; Estivill et al. 1677delTA (4.1%) E822X (1.3%) [1997]; Onay et al. [1998]; 2789+5G→A (3.9%) 2183+5G→A+2184insA (1.3%) Macek et al. [2002] 2181delA (3.8%) D110H (0.8%) R347H (3.6%) P1013L (0.8%) N1303K (2.9%) 3172delAC (0.8%) 621+1G→T (2.6%) 1259insA (0.8%) G542X (2.6%) M1028I (0.8%) TABLE 1. Continued. Estimated Projected detection of Number of Number of Country/ allele two CFTR mutations chromosomes Region Mutation array detectiona mutationsb includedc (max/min)d Reference WORLDWIDEANALYSISOFCFTRMUTATIONS587 E92K (2.6%) 4005+1G→A (0.7%) A96E (2.6%) W1282X (0.7%) M152V (2.6%) I148T (0.6%) 2183AA→G (2.5%) R1162X (0.6%) 296+9A→T (1.6%) D1152H (0.6%) 2043delG (1.4%) W1098X (0.6%) E92X (1.4%) E831X (0.6%) K68N (1.4%) W496X (0.6%) G85E (1.3%) F1052V (0.5%) R1158X (1.3%) L571S (0.5%) United Arab S549R (61.5%) ∆F508 (26.9%) 88.4 78.1 2 86/52 Frossard et al. [1988]; Emirates Frossard et al. [1999] North/Central/South Americas Argentina ∆F508 (58.6%) N1303K (1.8%) 69.1 47.7 5 326/228 CFGAC [1994]; Chertkoff et al. W1282X (3.9%) 1717-1G→A (0.9%) [1997] G542X (3.9%) Brazilf ∆F508 (47.7%) W1282X (1.3%) 66.8 44.6 10 820/500 CFGAC [1994]; Cabello et al. (total) G542X (7.2%) G85E (1.3%) [1999]; Raskin et al. [1999]; R1162X (2.5%) R553X (0.7%) Bernardino et al. [2000] R334W (2.5%) L206W (0.6%) N1303K (2.4%) 2347delG (0.6%) South East: >∆F508, G542X South: >N1303K Brazil ∆F508 (31.7%) N1303K (2.5%) 42.5 18.1 3 120 Parizotto and Bertuzzo [1997] (Sao Paulo) G542X (8.3%) Canada ∆F508 (59.0%) G542X (0.5%) 98.5 97.0 13 381/200 Rozen et al. [1992]; (Lac St. Jean) 621+1G→T (24.3%) N1303K (0.5%) De Braekeleer et al. [1998] A445E (8.2%) Q890X (0.5%) Y1092X (1.2%) S489X (0.5) 711+1G→T (1.0%) R117C (0.5%) I148T (1.0%) R1158 (0.5%) G85E (0.8%) Canada ∆F508 (71.4%) ∆I507 (1.3%) 90.9 82.6 7 77 Rozen et al. [1992] (Quebec City) 711+1G→T (9.1%) Y1092X (1.3%) 621+1G→T (5.2%) N1303K (1.3%) A455E (1.3%) Canada ∆F508 (70.9%) W1282X (0.9%) 82.0 67.2 10 632 Kristidis et al. [1992] (Toronto) G551D (3.1%) R117H (0.9%) G542X (2.2%) 1717-1G→A (0.6%) 621+1G→T (1.3%) R560T (0.6%) N1303K (0.9%) ∆I507 (0.6%) Chile ∆F508 (29.2%) R553X (4.2%) 33.4 11.2 2 72 Rios et al. [1994] Columbia 1) DF508 (35.4%) 3) N1303K (2.1%) - - 4 48 Restrepo et al. [2000] 2) G542X (6.3%) 4) W1282X (2.1%) Ecuador 1) DF508 (25%) - - 1 20 Paz-y-Mino et al. [1999] (Continued) BOBADILLAETAL. Login to comment

[hide] Hypersusceptibility of cystic fibrosis mice to chr... Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1949-54. Epub 2003 Feb 10. Coleman FT, Mueschenborn S, Meluleni G, Ray C, Carey VJ, Vargas SO, Cannon CL, Ausubel FM, Pier GB
Hypersusceptibility of cystic fibrosis mice to chronic Pseudomonas aeruginosa oropharyngeal colonization and lung infection.
Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1949-54. Epub 2003 Feb 10., 2003-02-18 [PMID:12578988]

Abstract [show]
No transgenic cystic fibrosis (CF) mouse model developed to date mimics the major clinical phenotype found in humans with CF, chronic Pseudomonas aeruginosa lung infection. In a transgenic CF transmembrane conductance regulator (cftr) mouse colony, we found WT, heterozygous, and homozygous CF mice housed in the same cage became chronically colonized in the oropharynx with environmental P. aeruginosa when the bacterium was present in drinking water. Elimination of P. aeruginosa from drinking water resulted in clearance in most WT and CF heterozygous, but not homozygous mice. For experimental evaluation, a combination of specific animal husbandry techniques and an oral infection route showed cftr(-/-) mice but not WT mice can be chronically colonized by P. aeruginosa with subsequent lung translocation, yielding a pathologic picture indicative of chronic lung infection. In some instances, mucoid isolates of P. aeruginosa were recovered from lungs, indicating conditions were present for conversion to mucoidy. Overexpression of human CFTR in the lungs of WT mice markedly accelerated the clearance rate of P. aeruginosa, demonstrating that lung levels of CFTR play an important role in defense against infection. P. aeruginosa mutants unable to express the surface polysaccharide alginate or the global regulator GacA were deficient in their ability to colonize the mice. CF mice made potent immune responses to P. aeruginosa outer membrane antigens. Overall, we found that under the proper conditions, transgenic CF mice are hypersusceptible to P. aeruginosa colonization and infection and can be used for evaluations of lung pathophysiology, bacterial virulence, and development of therapies aimed at treating CF lung disease.

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19 Cftrtm1Unc -TgN(FABPCFTR) [fatty acid-binding protein (FABP)- CFTR] mice have a stop codon in the murine cftr gene (S489X) but also express human CFTR in the gut epithelium due to transgenic introduction of Cftr under the control of the FABP promoter (10). Login to comment

[hide] Delivery of CFTR by adenoviral vector to cystic fi... Am J Physiol Lung Cell Mol Physiol. 2004 Apr;286(4):L717-26. Epub 2003 Sep 26. Van Heeckeren AM, Scaria A, Schluchter MD, Ferkol TW, Wadsworth S, Davis PB
Delivery of CFTR by adenoviral vector to cystic fibrosis mouse lung in a model of chronic Pseudomonas aeruginosa lung infection.
Am J Physiol Lung Cell Mol Physiol. 2004 Apr;286(4):L717-26. Epub 2003 Sep 26., [PMID:14514520]

Abstract [show]
In cystic fibrosis (CF) there is an excessive inflammatory response to lung infections with Pseudomonas aeruginosa, which causes significant morbidity and mortality. Mice deficient in the cystic fibrosis conductance transmembrane regulator homolog (Cftr) have exaggerated production of proinflammatory cytokines in epithelial lining fluid and increased mortality in response to chronic bronchopulmonary infection with mucoid P. aeruginosa, compared with infected wild-type littermates. Whether delivery of CFTR to CF airways by an adenoviral vector (Ad2/CFTR-16) decreases cytokine production and mortality in response to chronic bronchopulmonary infection with mucoid P. aeruginosa was tested. CF mice [stock Cftrtm1Unc-TgN(FABPCFTR)#Jaw] were anesthetized with isoflurane and inoculated intranasally with either Ad2/CFTR-16, diluent (sucrose), or empty vector (Ad2/EV). Two weeks later, mice were anesthetized with 2.5% Avertin and inoculated transtracheally with P. aeruginosa-laden agarose beads (PA M57-15). The cumulative 10-day survival of mice pretreated with Ad2/CFTR-16 was significantly higher compared with mice pretreated with sucrose but not significantly higher than mice pretreated with Ad2/EV. After adjusting for differences in experiment, we found weight loss at 3 days for mice treated with Ad2/CFTR-16 to be significantly less than for the sucrose- or Ad2/EV-treated groups. However, cytokine responses were similar in all groups 3 days after infection. In conclusion, the observed survival advantage of adenoviral delivery of CFTR to the CF lung may be due either to CFTR expression or possibly to proinflammatory effects of the adenoviral vector, or both.

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222 L722 Stock Cftrtm1Unc -TgN(FABPCFTR)#Jaw mice bear the S489X mutation in Cftr and the human CFTR transgene driven by the fatty acid binding promoter (FABP), which drives CFTR expression primarily in the intestinal tract. Login to comment

[hide] Abnormal Paneth cell granule dissolution and compr... Am J Physiol Gastrointest Liver Physiol. 2004 Jun;286(6):G1050-8. Epub 2004 Jan 8. Clarke LL, Gawenis LR, Bradford EM, Judd LM, Boyle KT, Simpson JE, Shull GE, Tanabe H, Ouellette AJ, Franklin CL, Walker NM
Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice.
Am J Physiol Gastrointest Liver Physiol. 2004 Jun;286(6):G1050-8. Epub 2004 Jan 8., [PMID:14715526]

Abstract [show]
Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.

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25 The obstructive syndrome and histopathological appearance are accurately reproduced in CF mouse models, in particular, CFTR knockout mice, such as the S489X mutant mouse (42). Login to comment
54 The experiments in this study used gene-targeted weanling mice (4-6 wk old) that were homozygous for the S489X mutation (cftrtm1Unc ) (42) and maintained on a C57BL/6J background (Ͼ6 generations). Login to comment

[hide] Ventilatory responses to hypercapnia and hypoxia i... Pediatr Res. 2004 May;55(5):738-46. Epub 2004 Feb 5. Bonora M, Bernaudin JF, Guernier C, Brahimi-Horn MC
Ventilatory responses to hypercapnia and hypoxia in conscious cystic fibrosis knockout mice Cftr-/-.
Pediatr Res. 2004 May;55(5):738-46. Epub 2004 Feb 5., [PMID:14764916]

Abstract [show]
This study was designed to examine the ventilatory performance and the lung histopathology of cystic fibrosis knockout mice (Cftr-/-) compared with heterozygous (Cftr+/-) or wild-type (Cftr+/+) littermates. Ventilation was recorded in conscious animals using whole-body plethysmography. Tidal volume (VT), respiratory frequency (f), and minute ventilation (VE) were measured during air breathing and in response to various levels of hypercapnia (2, 4, 6, or 8% CO2) or hypoxia (14, 12, 10, or 8% O2). The results for Cftr+/- and Cftr+/+ were pooled into one control group because they did not differ. In air and in response to hypercapnia, VE, VT, and f were similar in Cftr-/- mice and in controls. During graded hypoxia, VE was decreased in Cftr-/- mice at 10 and 8% O2 because of a lower f. Histology showed neither inflammation nor obstruction of airways in Cftr-/- mice. Morphometric analysis showed alveolar dilation as a result of either distension or impaired development. In conclusion, cystic fibrosis knockout mice have normal baseline breathing and ventilatory response to hypercapnia but a decreased ventilatory response to severe hypoxia. This latter result associated with the morphometric analysis suggests that Cftr-/- mice may exhibit immaturity of the respiratory system.

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27 Female and male 129/BC mice heterozygous for the S489X mutation were crossed to obtain homozygous S489X mice (7). Login to comment
33 Mice homozygous for the S489X mutation were classified as Cftr-/- , mice heterozygous for the S489X mutation as Cftrϩ/- , and wild-type mice as Cftrϩ/ϩ . Login to comment
149 However, minor changes in the respiratory tract have been described in the models with the S489X Cftr mutation. Login to comment

[hide] Additional disruption of the ClC-2 Cl(-) channel d... J Biol Chem. 2004 May 21;279(21):22276-83. Epub 2004 Mar 7. Zdebik AA, Cuffe JE, Bertog M, Korbmacher C, Jentsch TJ
Additional disruption of the ClC-2 Cl(-) channel does not exacerbate the cystic fibrosis phenotype of cystic fibrosis transmembrane conductance regulator mouse models.
J Biol Chem. 2004 May 21;279(21):22276-83. Epub 2004 Mar 7., 2004-05-21 [PMID:15007059]

Abstract [show]
Cystic fibrosis is a fatal inherited disease that is caused by mutations in the gene encoding a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). It has been suggested that the cystic fibrosis phenotype might be modulated by the presence of other Cl(-) channels that are coexpressed with CFTR in some epithelial cells. Because the broadly expressed plasma membrane Cl(-) channel, ClC-2, is present in the tissues whose function is compromised in cystic fibrosis, we generated mice with a disruption of both Cl(-) channel genes. No morphological changes in their intestine, lung, or pancreas, tissues affected by cystic fibrosis, were observed in these mice. The mortality was not increased over that observed with a complete lack of functional CFTR. Surprisingly, mice expressing mutant CFTR (deletion of phenylalanine 508), survived longer when ClC-2 was disrupted additionally. Currents across colonic epithelia were investigated in Ussing chamber experiments. The disruption of ClC-2, in addition to CFTR, did not decrease Cl(-) secretion. Colon expressing wild-type CFTR even secreted more Cl(-) when ClC-2 was disrupted, although CFTR transcript levels were unchanged. It is concluded that ClC-2 is unlikely to be a candidate rescue channel in cystic fibrosis. Our data are consistent with a model in which ClC-2 is located in the basolateral membrane.

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57 EXPERIMENTAL PROCEDURES Mice-ClC-2 knock-out mice, described previously (24) and backcrossed for seven generations into C57Bl6, were mated with heterozygous CFTR ⌬F508 mice (6) (CFTRkth , called Cftr⌬F/⌬F ) and CFTR S489X mice (5)(CFTRunc , called Cftr-/- ) (26) (both in C57Bl6 background) obtained from Jackson Laboratories. Login to comment

[hide] Role of Cftr genotype in the response to chronic P... Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L944-52. Epub 2004 Jul 9. 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., [PMID:15246977]

Abstract [show]
Patients with cystic fibrosis have a lesion in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which is associated with abnormal regulation of other ion channels, abnormal glycosylation of secreted and cell surface molecules, and vulnerability to bacterial infection and inflammation in the lung usually leading to the death of these patients. The exact mechanism(s) by which mutation in CFTR leads to lung infection and inflammation is not clear. Mice bearing different mutations in the murine homolog to CFTR (Cftr) (R117H, S489X, Y122X, and DeltaF508, 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. Body weights of mice bearing mutations in Cftr were significantly smaller than wild-type mice at most ages. 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-alpha, 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. Thus we cannot implicate either misprocessing of CFTR or failure of CFTR to reach the plasma membrane in the genesis of the excess inflammatory response of CF mice. Therefore, it appears that any functional defect in CFTR produces comparable inflammatory responses to lung infections with P. aeruginosa.

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15 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 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
35 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
49 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 MATERIALS AND METHODS Mice Breeding pairs of heterozygote congenic mice (ՆN10) bearing the S489X mutation (B6.129P2-Cftrtm1Unc , stock no. Login to comment
57 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
61 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
78 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
124 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
131 The amiloride response was significantly different between wild-type mice and S489X mice (P ϭ 0.005). Login to comment
133 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 *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 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 Nevertheless, each genotype can be compared directly with S489X under identical conditions. Login to comment
145 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 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 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
152 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 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 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
160 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 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 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
165 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 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
171 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 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 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
181 When comparing the S489X to Y122X or ⌬F508 strains, we could detect even smaller differences with high power. Login to comment
183 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
194 *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
196 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 *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
203 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
207 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
210 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 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 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 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
227 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 *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
242 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 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
13 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
33 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
46 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 MATERIALS AND METHODS Mice Breeding pairs of heterozygote congenic mice (ՆN10) bearing the S489X mutation (B6.129P2-Cftrtm1Unc , stock no. Login to comment
54 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 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 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 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
128 The amiloride response was significantly different between wild-type mice and S489X mice (P ϭ 0.005). Login to comment
130 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
132 *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
142 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 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
150 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
157 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 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
164 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 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 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
178 When comparing the S489X to Y122X or ⌬F508 strains, we could detect even smaller differences with high power. Login to comment
180 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 *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 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
195 *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 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 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
224 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
239 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 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

[hide] The "Goldilocks effect" in cystic fibrosis: identi... BMC Genet. 2004 Jul 27;5:21. Cohen JC, Lundblad LK, Bates JH, Levitzky M, Larson JE
The "Goldilocks effect" in cystic fibrosis: identification of a lung phenotype in the cftr knockout and heterozygous mouse.
BMC Genet. 2004 Jul 27;5:21., 2004-07-27 [PMID:15279681]

Abstract [show]
BACKGROUND: Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype. RESULTS: Using measurements of pulmonary mechanics, a definitive lung phenotype was demonstrated in the cftr-/- mouse. Lungs showed decreased compliance and increased airway resistance in young animals as compared to cftr+/+ littermates. These changes were noted in animals less than 60 days old, prior to any long term inflammatory effects that might occur, and are consistent with structural differences in the cftr-/- lungs. Surprisingly, the cftr+/- animals exhibited a lung phenotype distinct from either the homozygous normal or knockout genotypes. The heterozygous mice showed increased lung compliance and decreased airway resistance when compared to either homozygous phenotype, suggesting a heterozygous advantage that might explain the high frequency of this mutation in certain populations. CONCLUSIONS: In the mouse the gene dosage of cftr results in distinct differences in pulmonary mechanics of the adult. Distinct phenotypes were demonstrated in each genotype, cftr-/-, cftr +/-, and cftr+/+. These results are consistent with a developmental role for CFTR in the lung.

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22 PV curves were measured in triplicate, starting from positive end-expiratory pressure (PEEP) values of 0, 3, and 6 cmH2O in S489X mice at 30-60 days of age following genotyping for the normal and mutant cftr alleles. Login to comment
79 Methods Mouse strain The S489X mouse 5th generation backcross to C57Bl/6 has been maintained by random mating for the past 8 years. Login to comment
81 Mice 30-60 days of age from our S489X mouse colony were genotyped for the normal and mutant cftr alleles. Login to comment

[hide] A large-scale study of the random variability of a... Eur J Hum Genet. 2005 Feb;13(2):184-92. Modiano G, Bombieri C, Ciminelli BM, Belpinati F, Giorgi S, Georges M, Scotet V, Pompei F, Ciccacci C, Guittard C, Audrezet MP, Begnini A, Toepfer M, Macek M, Ferec C, Claustres M, Pignatti PF
A large-scale study of the random variability of a coding sequence: a study on the CFTR gene.
Eur J Hum Genet. 2005 Feb;13(2):184-92., [PMID:15536480]

Abstract [show]
Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (n(g) approximately 100-150 genes). In the present investigation, a large random European population sample (average n(g) approximately 1500) was studied for a single gene, the CFTR (Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q > 0.005), much lower than that of the synonymous (S) substitutions, but they showed a similar rate of subpolymorphic (q < 0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic.

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33 In the Tajima`s test,19 the null hypothesis of neutrality is rejected if a statistically significant difference between p Common and rare nonsynonymous and synonymous cSNSs G Modiano et al European Journal of Human Genetics Table 1 List of the 61 cSNSsa encountered in the present survey The random samples of genes (and the technique utilized) cSNS variants found NE Italy (DGGE) Central Italy (DGGE) Southern France (DGGE) Northern France (DHPLC) Spain (SSCA) Czechia (DGGE) Hb Â 104 Exon Exon Length (bp) Ref. no. SNS SASc 1st 100d 2nd 500 1st 100d 2nde 1st 100d 2nd 500 1st 100 2nde 82d 72 Abs. Freq. Total sample size q Â 104 se Â 104 NSf Sf 1g 53 0 0 0 0 0/452 0 924 2 111 1 223C4T R31C 1 1 1/500 1 1 0 0/450 0 5 (11) 1 932 (2 432) 45.23 13.61 90 2 224G4T R31L 0 0 0/500 0 0 0 1/450 0 1 1 932 5.17 5.17 10 3 257C4T S42F 0 0 1/500 0 0 0 0/450 0 1 1 932 5.17 5.17 10 3 109 4 334A4G K68E 1 0 0 0/498 0 0 0 0/452 0 0 1 2 504 3.99 3.99 8 5 352C4T R74W 0 0 0 0/498 0 0 0 1/452 0 0 1 2 504 3.99 3.99 8 6 356G4A R75Q 1 7 1 7/498 2 9 2 9/452 0 2 40 (40) 2 504 (2 544) 157.23 24.66 310 7 386G4A G85E 0 0 1 1/498 0 0 0 0/452 0 0 2 2 504 7.99 5.65 16 4 216 8 482G4A R117H 0 0 0 0/292 0 2 0 1/456 0 0 3 2 302 13.03 7.52 26 9 528T4G I132M 0 0 0 0/292 0 0 0 1/456 0 0 1 2 302 4.34 4.34 8 10 575T4C I148T 1 2 0 1/292 0 0 0 1/456 0 1 6 2 302 26.06 10.63 52 5 90 11 640C4T R170C 0 0 0 0/6 0 0 1/448 0 1 1 436 6.96 6.96 14 12 641G4A R170H 1 1 0 0/6 0 0 2/448 0 4 (4) 1 436 (1 930) 20.73 10.35 41 6a 164 0 0 0/6 0 0 0/432 0 0 992 6b 126 0 0 0/6 0 0 0/454 0 942 7 247 0 0 0/6 0 0 0/796 0 1 284 8 93 13 1281G4A L383 0 0 0 0/6 0 0 1/456 0 0 1 1 516 6.60 6.60 13 9 183 14 1402G4A G424S 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 15 1459G4T D443Y 0 0 0/6 0 0 1/454 0 1 940 10.64 10.64 21 10 192 16 1540A4G M470Vh 42 197 30 37/96 39 199 (i) (i) 27 571(736) 1 484 (1 912) 3849.37 111.28 4 735 17 1598C4A S489X 0 0 0 0/96 0 0 0 1/796 0 1 2 374 4.21 4.21 8 18 1648A4G I506V 1 0 0 0/96 0 0 0 0/796 0 1 2 374 4.21 4.21 8 19 1655T4G F508C 0 1 0 0/96 0 0 0 1/796 0 2 2 038 8.42 5.96 17 20 1716G4A Q528 2 16 1 0/96 0 19 i I 5 43 (58) 1 478 (2 024) 286.56 37.08 557 11 95 21 1756G4T G542X 0 2 0 0/134 0 0 0/796 0 0 2 1 984 10.08 7.12 20 22 1764T4G G544 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 23 1784G4A G551D 0 0 0 0/134 0 0 1/796 0 0 1 1 984 5.04 5.04 10 12 87 24 1816G4A V562I 0 0 0 0 1 0 0/450 0 0 1 (1) 2 004 (2 504) 3.99 3.99 8 25 1816G4C V562L 0 0 0 1 0 0 1/450 0 0 2 (3) 2 004 (2 504) 11.98 6.91 24 26 1859G4C G576A 1 2 0 1 11 0 8/450 0 0 23 (27) 2 004 (2 538) 106.38 20.36 213 13 724j 449 27 1997G4A G622D 0 0 0/80 0/96 1 0 0 0/444 0 1 2 002 5.00 5.00 10 28 2082C4T F650 1 0 0/80 0/20 0 0 0 0/444 0 1 (1) 1 926 (2 412) 4.15 4.15 8 29 2134C4T R668C 1 2 0/80 0/96 1 11 0 12/444 0 27(32) 2 002 (2 558) 125.10 21.98 247 275 30 2377C4T L748 0 0 0/6 0 1 1 388 25.77 25.77 52 14a 129 31 2670G4A W846X 0 0 0/6 0 1 0/452 0/80 0 1 1 010 9.90 9.90 20 32 2694T4G T854 33 23 0/6 33 38 149/452 14/80 11 301 1 010 2980.20 143.92 4 184 33 2695G4A V855I 0 0 0/6 0 0 1/452 0/80 0 1 1 010 9.90 9.90 20 14b 38 0 0 0 0/520 0 0 0 0/446 0 2 448 15 251 34 2816G4C S895T 0 0 0/6 0 0 2/436 0 0 2 996 20.08 14.18 40 35 2831A4C N900T 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 36 2988G4C M952I 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 37 3030G4A T966 (2)k (1)k 0 6/436 0 6 (25)k 618 (1814)k 137.82 27.37 272 38 3032T4C L967S 0 0 0/6 0 0 1/436 0 0 1 996 10.04 10.04 20 16 80 0 0 0/498 0 0 0/450 0 0 1 502 17a 151 39 3123G4C L997F 0 2 2 1/494 0 7 1 4/454 0 0 17 2 502 67.95 16.42 135 40 3157G4A A1009T 0 2 0 0/494 0 0 0 0/454 0 0 2 2 502 7.99 5.65 16 41 3212T4C I1027T 1 0 0 0/494 0 0 0 0/454 0 0 1 2 502 4.00 4.00 8 17b 228 42 3286T4G F1052V 1 1 0 1/194 0 0 0 0/452 0 0 3 (3) 2 200 (2 240) 13.39 7.73 27 43 3337G4A G1069R 0 1 0 0/194 0 0 0 0/452 0 0 1 2 200 4.55 4.55 9 CommonandrarenonsynonymousandsynonymouscSNSs GModianoetal 186 EuropeanJournalofHumanGenetics 44 3345G4T Q1071H 0 0 0 0/194 0 1 0 0/452 0 0 1 2 200 4.55 4.55 9 45 3417A4T T1995 1 3 0 0/194 1 1 0 0/452 0 0 6 (8) 2 200 (2 506) 31.92 11.27 64 46 3419T4G L1096R 0 0 0 0/194 1 0 0 0/452 0 0 1 2 200 4.55 4.55 9 47 3477C4A T1115 0 0 0 0/194 0 0 0 1/452 0 0 1 2 200 4.55 4.55 9 18 101 48 3523A4G I1131V 0 0 1 0/10 0 0 0/448 0 0 1 (2) 1 512 (1 908) 10.48 7.07 21 49 3586G4C D1152H 0 0 0 0/10 0 0 1/448 0 0 1 1 512 6.61 6.61 13 19 249 50 3617G4T R1162L 0 0 1 1/494 0 0/260 0 0/454 0 0 2 2 262 8.84 6.25 18 51 3690A4G Q1186 0 0 0 0/494 0 0/260 0 0/454 1 0 1 2 262 4.42 4.42 9 52 3813A4G L1227 0 1 0 0/494 0 0/260 0 0/454 0 0 1 2 262 4.42 4.42 9 53 3837T4G S1235R 1 1 0 1/494 0 4/260 0 7/454 0 1 15 (15) 2 262 (2 310) 69.94 16.71 140 20 156 54 4002A4G P1290 2 3 0/6 3 5 18/454 3/80 2 36 1 012 357.73 58.22 690 21 90 55 4009G4A V1293I 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 56 4029A4G T1299 1 0 0/6 0 1/300 0 1/456 0 0 3 (8) 1 316 (2 330) 34.33 12.12 69 57 4041C4G N1303K 1 0 0/6 0 0/300 0 0/456 0 0 1 1 316 7.60 7.60 15 58 4085T4C V1318A 0 0 0/6 0 0/300 0 1/456 0 0 1 1 316 7.60 7.60 15 22 173 0 0 0/18 0 0 0/450 0 0 1 022 23 106 0 0 0 0/6 0 0 0/448 0 1 436 24l 198+3 59 4404C4T Y1424 1 0 0/6 1 2 5/420 0 2 11 (32) 980 (2 516) 127.19 22.34 251 60m 4521G4A Q1463 (21) (16) (3/32) (14/80) (30) (94/420) 15/76 (17) 15 (227) 76 (1052) 2142.86 131.07 3 367 61 4563T4C D1477 0 0 0/6 0 1 0/420 0 0 1 980 10.20 10.20 20 Totals 6 525 9 584 16 109 The bracketed figures include also the RFLP analysis data (see Materials and methods); the NE Italy, Central Italy, Southern and Northern France are each subdivided into two samples where the 1st is made up of 100 genes. 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[hide] Chloride conductance of CFTR facilitates basal Cl-... Am J Physiol Gastrointest Liver Physiol. 2005 Jun;288(6):G1241-51. Epub 2005 Jan 13. Simpson JE, Gawenis LR, Walker NM, Boyle KT, Clarke LL
Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum.
Am J Physiol Gastrointest Liver Physiol. 2005 Jun;288(6):G1241-51. Epub 2005 Jan 13., [PMID:15650130]

Abstract [show]
Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl(-)/HCO(3)(-) exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl(-)/HCO(3)(-) exchange rate was reduced by approximately 35% in CF compared with WT villous epithelium. Cl(-)/HCO(3)(-) exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl(-)(out)/HCO(3)(-)(in) exchange in both epithelia. In anion substitution studies, anion(in)/HCO(3)(-)(out) exchange rates were greater in WT epithelium using Cl(-) or NO(3)(-), but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO(4)(2-). Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl(-)/HCO(3)(-) exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl(-)/HCO(3)(-) exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl(-) channel activity of CFTR facilitates apical membrane Cl(-)(in)/HCO(3)(-)(out) exchange by providing a Cl(-) "leak" under basal conditions.

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50 The experiments in this study used mice 8-16 wks old that were either homozygous for the S489X mutation of the murine homolog of CFTR (cftrtm1UNC ) or homozygous for the mCFTR ⌬F508 mutation (cftrtm1Kth ) and maintained on a C57BL/6J background (54, 64). Login to comment
51 The experiments in this study used mice 8-16 wks old that were either homozygous for the S489X mutation of the murine homolog of CFTR (cftrtm1UNC ) or homozygous for the mCFTR ⌬F508 mutation (cftrtm1Kth ) and maintained on a C57BL/6J background (54, 64). Login to comment

[hide] Susceptibility to typhoid fever is associated with... Hum Genet. 2005 Oct;118(1):138-40. Epub 2005 Oct 28. van de Vosse E, Ali S, de Visser AW, Surjadi C, Widjaja S, Vollaard AM, van Dissel JT
Susceptibility to typhoid fever is associated with a polymorphism in the cystic fibrosis transmembrane conductance regulator (CFTR).
Hum Genet. 2005 Oct;118(1):138-40. Epub 2005 Oct 28., [PMID:16078047]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is the affected protein in cystic fibrosis (CF). The high rate of CF carriers has led to speculation that there must be, similar to the sickle cell haemoglobin advantage in malaria, a selective advantage for heterozygotes. Such a selective advantage may be conferred through reduced attachment of Salmonella typhi to intestinal mucosa, thus providing resistance to typhoid fever. We tested this hypothesis by genotyping patients and controls in a typhoid endemic area in Indonesia for two highly polymorphic markers in CFTR and the most common CF mutation. We found an association between genotypes in CFTR and susceptibility to typhoid fever (OR=2.6). These analyses suggest that the role CFTR plays in vitro in S. typhi infection is also important for infection in the human population.

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12 A CFTR null allele (S489X) in mice was found to confer resistance to cholera toxin (Gabriel et al. 1994), however, this particular mutation is very rare in humans (found in one patient) and may not be comparable to F508del, W1282X and 1677del TA in survival in a population. Login to comment

[hide] Pulmonary neuroendocrine cells, airway innervation... Am J Respir Cell Mol Biol. 2006 Sep;35(3):320-6. Epub 2006 Apr 13. Pan J, Luk C, Kent G, Cutz E, Yeger H
Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice.
Am J Respir Cell Mol Biol. 2006 Sep;35(3):320-6. Epub 2006 Apr 13., [PMID:16614351]

Abstract [show]
The amine- and peptide-producing pulmonary neuroendocrine cells (PNEC) are widely distributed within the airway mucosa of mammalian lung as solitary cells and innervated clusters, neuroepithelial bodies (NEB), which function as airway O2 sensors. These cells express Cftr and hence could play a role in the pathophysiology of cystic fibrosis (CF) lung disease. We performed confocal microscopy and morphometric analysis on lung sections from Cftr-/- (null), Cftr+/+, and Cftr+/- (control) mice at developmental stages E20, P5, P9, and P30 to determine the distribution, frequency, and innervation of PNEC/NEB, innervation and cell mass of airway smooth muscle, and neuromuscular junctions using synaptic vesicle protein 2, smooth muscle actin, and synaptophysin markers, respectively. The mean number of PNEC/NEB in Cftr-/- mice was significantly reduced compared with control mice at E20, whereas comparable or increased numbers were observed postnatally. NEB cells in Cftr null mice showed a significant reduction in intracorpuscular nerve endings compared with control mice, which is consistent with an intrinsic abnormality of the PNEC system. The airways of Cftr-/- mice showed reduced density (approximately 20-30%) of smooth muscle innervation, decreased mean airway smooth muscle mass (approximately 35%), and reduced density (approximately 20%) of nerve endings compared with control mice. We conclude that the airways of Cftr-/- mice exhibit heretofore unappreciated structural alterations affecting cellular and neural components of the PNEC system and airway smooth muscle and its innervation resulting in blunted O2 sensing and reduced airway tonus. Cftr could play a role in the development of the PNEC system, lung innervation, and airway smooth muscle.

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131 Because NEB represent airway O2 sensors (analogous to carotid body chemoreceptors) involved in the control of breathing (2, 4), the previously mentioned abnormalities could partly account for recent observation of decreased ventilatory responses to hypoxia observed in Cftr-/- mice using whole-body plethysmography (20) and decreased compliance and increased airway resistance in the S489X Cftr-/- mouse (21). Login to comment

[hide] Acute Pseudomonas challenge in cystic fibrosis mic... J Allergy Clin Immunol. 2006 May;117(5):1163-9. Saadane A, Soltys J, Berger M
Acute Pseudomonas challenge in cystic fibrosis mice causes prolonged nuclear factor-kappa B activation, cytokine secretion, and persistent lung inflammation.
J Allergy Clin Immunol. 2006 May;117(5):1163-9., [PMID:16675347]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is characterized by an excessive and prolonged inflammatory response to Pseudomonas aeruginosa in the lung. There are high levels of cytokines and chemokines and an exaggerated PMN influx causing significant morbidity and mortality. OBJECTIVE: To compare the kinetics of the inflammatory response with the kinetics of clearance of acute bacterial challenge in the lungs of CF and wild-type (WT) mice. METHODS: We challenged CF knockout (KO) and WT mice intratracheally with P aeruginosa in suspension and evaluated bacteria counts, nuclear factor-kappaB (NF-kappaB), and inhibitor of NF-kappaB alpha protein (I-kappaBalpha) in lung tissue, cytokines, and PMN in bronchoalveolar lavage (BAL). RESULTS: Both groups of mice cleared the infection with the same kinetics. CF-KO mice had more PMN in BAL than WT mice. CF-KO mice had high concentrations of proinflammatory cytokines in BAL on days 2 and 4, whereas cytokines in BAL from WT mice were only slightly elevated. CF-KO mice failed to regenerate I-kappaBalpha once it was degraded, and consequently had prolonged and excessive activation of NF-kappaB for the entire 6-day duration of the study. In contrast, WT mice showed only slight NF-kappaB activation, which plateaued at day 4. CONCLUSION: These data suggest that NF-kappaB is dysregulated in CF lung infection and could be a good target for therapy. Prolonged responses to initial acute infections may contribute to the eventual establishment of chronic persistent inflammation. CLINICAL IMPLICATIONS: Dysregulation of the I-kappaB/NF-kappaB pathway in cystic fibrosis leads to prolonged cytokine secretion and persistent inflammation in response to acute challenges and may be important in the development of chronic lung inflammation and infection.

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28 METHODS Mice Male CF-KO mice 7 to 10 weeks old homozygous for the S489X (B6.129P2-CftrtmtUnc ) (null) mutation17 and normal wt/wt (WT) littermates were used after genotyping by polymerase chain reaction on tail snip DNA. Pups were weaned at 10 days of life and fed Peptamen (Clintech Nutrition Co, Deerfield, Ill) to avoid intestinal obstruction.18 All mice were maintained in sterile microisolator cages with sterile water. Login to comment

[hide] The cystic fibrosis transmembrane conductance regu... J Med Genet. 2006 Jun;43(6):e29. Jin R, Hodges CA, Drumm ML, Palmert MR
The cystic fibrosis transmembrane conductance regulator (Cftr) modulates the timing of puberty in mice.
J Med Genet. 2006 Jun;43(6):e29., [PMID:16740913]

Abstract [show]
BACKGROUND: Delayed puberty is common among individuals with cystic fibrosis (CF) and is usually attributed to chronic disease and/or poor nutrition. However, it has recently been recognised that pubertal delay can occur even in the setting of good nutritional and clinical status. This finding, along with evidence that Cftr is expressed in rat brain, human hypothalamus, and a gonadotropin releasing hormone secreting cell line, raises the possibility that some of the pubertal delay in CF could stem directly from alterations in Cftr function that affect the hypothalamic-pituitary-gonadal axis. METHODS: To examine this hypothesis, we investigated pubertal timing (as assessed by vaginal opening (VO)) in a mouse model of CF (Cftr(tm1Unc)) engineered to produce a truncated Cftr mRNA and referred to as S489X. Homozygous knockout, heterozygote, and wild type (WT) female mice were examined. RESULTS: As expected, the S489X-/S489X- knockout mice, which have chronic inflammation and gastrointestinal disease, grew more slowly and had later onset of puberty than WT animals. We anticipated that the S489X-/S489X+ heterozygotes, which have no clinical CF phenotype, might display an intermediate timing of puberty. Surprisingly, however, these mice had earlier VO than WT. These findings were confirmed in a second, independent model of CF engineered to generate the deltaF508 mutation in mice. Again, the homozygotes displayed later pubertal timing, while the heterozygotes displayed earlier VO than the WT animals. CONCLUSIONS: These data provide further evidence that Cftr can directly modulate the reproductive endocrine axis and raise the possibility that heterozygote mutation carriers may have a reproductive advantage.

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131 Methods: To examine this hypothesis, we investigated pubertal timing (as assessed by vaginal opening (VO)) in a mouse model of CF (Cftrtm1Unc ) engineered to produce a truncated Cftr mRNA and referred to as S489X. Login to comment
146 First, B6 mice heterozygous for the S489X mutation that generates a stop codon in the coding sequence of exon 10 of Cftr (B6.129P2-Cftrtm1Unc /J),20 were purchased from Jackson Laboratory (Bar Harbor, ME; stock no. Login to comment
148 This mutation leads to a truncated protein product similar to many human CF mutations.20 Heterozygous S489X mice (referred to as S489X2 /S489X+ ) were bred and the progeny homozygote, heterozygote, or negative (wild type, WT) for the S489X mutation were studied. Login to comment
153 In the colony described here, the DF508 and S489X lines are backcrossed to B6 mice every five generations to ensure genetic fidelity and prevent genetic drift. Login to comment
154 Live animals were genotyped at 7 days after date of birth by PCR analysis of DNA extracted from a toe removed for mouse identification.24 WT mice from the S489X or DF508 matings are both predicted to be indistinguishable from B6 mice due to the backcrossing of these alleles. Login to comment
155 Because there were no observable phenotypic differences between the WT mice generated from the two mating strategies (mean timing of vaginal opening (VO) 31.3¡1.4 days for S489X WT (n = 14) and 31.3¡1.5 days for DF508 WT (n = 12)), WT animals were assessed as a single group. Login to comment
157 For the S489X mice, the p values for comparison of homozygote and heterozygote mice to WT mice were ,0.00001 and 0.00004, respectively; for the DF508 mice, the p values for comparison of homozygote and heterozygote mice to WT mice were 0.00007 and 0.0002, respectively. Login to comment
172 S489X- /S489X+ 100 90 70 80 60 40 50 30 20 0 10 80 Age (days) S489X- /S489X - MicewithVO(%) 20 7570656055504540353025 WT Figure 1 Comparison of the timing of vaginal opening (VO) for S489X mice. Login to comment
180 RESULTS VO occurred significantly later in S489X homozygous knockout (S489X2 /S489X2 ) mice than in WT B6 mice (table 1, fig 1). Login to comment
181 The S489X CF mice also grew more slowly than WT mice and had not even reached a mean weight of 15 g by 50 days of age; thus, despite the delay in VO, the S489X2 / S489X2 mice experienced VO at significantly lower mean body weights than the WT animals. Login to comment
182 To investigate the direct effects of Cftr on pubertal timing without the confounding effects of chronic disease, mice heterozygous for the S489X mutation were examined. Login to comment

[hide] Mitochondrial oxidative stress in the lungs of cys... Am J Respir Cell Mol Biol. 2006 Nov;35(5):579-86. Epub 2006 Jun 8. Velsor LW, Kariya C, Kachadourian R, Day BJ
Mitochondrial oxidative stress in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice.
Am J Respir Cell Mol Biol. 2006 Nov;35(5):579-86. Epub 2006 Jun 8., [PMID:16763223]

Abstract [show]
Cystic fibrosis is a fatal genetic disorder involving dysfunction of the cystic fibrosis transmembrane regulator protein (CFTR) resulting in progressive respiratory failure. Previous studies indicate that CFTR regulates cellular glutathione (GSH) transport and that dysfunctional CFTR is associated with chronic pulmonary oxidative stress. The cause and the source of this oxidative stress remain unknown. The current study examines the role of the mitochondria in CFTR-mediated pulmonary oxidative stress. Mitochondrial GSH levels and markers of DNA and protein oxidation were assessed in the lung mitochondria from CFTR-knockout mice. In addition, in vitro models using human CFTR-sufficient and -deficient lung epithelial cells were also employed. Mitochondrial GSH levels were found to be decreased up to 85% in CFTR-knockout mice, and 43% in human lung epithelial cells deficient in CFTR. A concomitant 29% increase in the oxidation of mitochondrial DNA, and a 30% loss of aconitase activity confirmed the existence of a mitochondrial oxidative stress. Flow cytometry revealed significantly elevated levels of cellular reactive oxygen species (ROS) in CFTR-deficient human lung cells. These studies suggest that dysfunctional CFTR leads to an increase in the level of ROS and mitochondrial oxidative stress. This oxidative stress, however, appears to be a consequence of lower mitochondrial GSH levels and not increased oxidation of GSH. Further studies are needed to determine how CFTR deficiency contributes to mitochondrial oxidative stress and the role this plays in CFTR-mediated lung pathophysiology.

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40 Animal Care and Use This study compared the mitochondrial consequences of the CFTR mutation from two congenic CFTR KO strains (S489X and FABP breeding stock were kind gifts from Dr. Anna van Heeckeren, Case Western Reserve University, Cleveland, OH) with C57B6 control mice. Login to comment
41 In the S489X congenic C57B6 strain, a mutation creates a stop codon and produces a truncated CFTR protein (19). Login to comment
43 To avoid this problem, S489X mice are maintained on a liquid diet (Peptamen; Nestle, Glendale, CA). Login to comment
45 With the exception of the liquid diet for the S489X mice, all mice were provided solid mouse chow and autoclaved tap water ad libitum. Login to comment
138 In the S489X CFTR KO mice on a liquid diet, mitochondrial GSH concentrations were decreased 43% compared with controls. Login to comment
148 Mitochondria isolated from the lungs of S489X and FABP CFTR KO mice displayed significantly lower levels of GSH than mitochondria from control mice. Login to comment
206 Both S489X and FABP CFTR KO mice had significantly lower mitochondrial GSH concentrations than control mice. Login to comment
208 Between the two CFTR KO mouse strains, however, the FABP GSH levels were considerably lower than the levels in the S489X mice. Login to comment
209 The S489X mutation generates a stop codon and produces a truncated protein (19). Login to comment
210 The S489X CFTR KO mouse must be maintained on a liquid diet to prevent fatal bowel obstructions. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Diabetes. 2006 Jul;55(7):1939-45. Stalvey MS, Muller C, Schatz DA, Wasserfall CH, Campbell-Thompson ML, Theriaque DW, Flotte TR, Atkinson MA
Cystic fibrosis transmembrane conductance regulator deficiency exacerbates islet cell dysfunction after beta-cell injury.
Diabetes. 2006 Jul;55(7):1939-45., [PMID:16804061]

Abstract [show]
The cause of cystic fibrosis-related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR(-/-)), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced beta-cell injury. Fasting blood glucose levels were similar among age-matched non-STZ-administered animals, but they were significantly higher in CFTR(-/-) mice 4 weeks after STZ administration (288.4 +/- 97.4, 168.4 +/- 35.9, and 188.0 +/- 42.3 mg/dl for CFTR(-/-), C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTR(-/-) mice. STZ reduced islets among all strains; however, only CFTR(-/-) mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatus-sensitized CFTR(-/-) mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTR(-/-) state.

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38 RESEARCH DESIGN AND METHODS We used mice with the CFTR S489X-/- neo insertion, developed initially at the University of North Carolina (20). Login to comment
42 CFTR S489X-/- FABP-hCFTRϩ/ϩ (hereafter designated CFTR-/- ), C57BL/6J, and FVB/NJ mice were housed under specific pathogen-free conditions at the University of Florida Animal Care Services according to National Institutes of Health guidelines and allowed food and water ad libitum. Login to comment
56 CFTR S489X-/- animals (4-6 weeks old) were sensitized to Aspergillus fumigatus crude protein extract (Greer Laboratories, Lenoir, NC). Login to comment
66 Lung, intestine, and pancreatic mRNA from CFTR S489X-/- FABP-hCFTR؉/؉ mice was used to analyze the expression pattern of the hCFTR driven by the FABP promoter by RT-PCR, using gene-specific primers for hCFTR and B-actin control primers. Login to comment
180 Interestingly, the CFTR S489X-/- FABP-hCFTRϩ/ϩ model used in this experiment has not been previously reported to have pancreatic disease. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Dev Dyn. 2006 Oct;235(10):2736-48. Cohen JC, Larson JE
Cystic fibrosis transmembrane conductance regulator (CFTR) dependent cytoskeletal tension during lung organogenesis.
Dev Dyn. 2006 Oct;235(10):2736-48., [PMID:16906518]

Abstract [show]
There is growing evidence for the role of CFTR (cystic fibrosis transmembrane conductance regulator) in lung development and differentiation. The mechanism by which the chloride channel could affect lung organogenesis, however, is unknown. In utero CFTR gene transfer in the fetal lungs of mice, rats, and non-human primates was shown previously to alter lung structure and function. A study of the genes altered in the fetal rat lung following CFTR overexpression was initiated in an effort to determine the molecular mechanism for CFTR-dependent differentiation. In utero gene transfer with recombinant adenoviruses carrying either a reporter gene or CFTR resulted in the increased expression of a number of genes upon microarray analysis. The majority of the genes overexpressed in the CFTR-treated lungs were primarily associated with muscle structure and function. Histological and biochemical characterization of these proteins including myosin heavy chain, heat shock protein 27, and isoforms of myosin light chain showed that CFTR overexpression had a profound effect on smooth muscle contraction-related proteins. The CFTR-dependent regulation of smooth muscle contraction related proteins was shown to be related to chloride and extracellular ATP and was dependent upon the PI3 Kinase and Phospholipase C pathways. The changes in smooth muscle proteins were consistent with CFTR-dependent contractions of the embryonic airway smooth muscle. An assay was developed using fluorescent polystyrene beads to show that CFTR did indeed increase amniotic fluid flow into the fetal lung. Increased amniotic fluid pressure was shown previously to be associated with stretch-induced differentiation of the lung. Evaluation of neonatal respiratory function showed that CFTR-dependent muscle contractions and increased amniotic fluid pressure resulted in accelerated maturation of the neonatal rat lung. In addition, these CFTR-dependent changes were shown to be sufficient to reverse the lung phenotype of the CFTR knockout mouse. Mechanical forces influence lung development through pulmonary distension. CFTR overexpression in the fetal lung altered differentiation and development in the lung. These results are consistent with CFTR influencing lung development by regulating the muscle contractions associated with cytoskeletal tension and stretch induced differentiation. Deficiency of CFTR altering lung development would contribute significantly to the Cystic Fibrosis disease phenotype.

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263 CFTR knockouts, S489X mice 5th generation backcross, with C57Bl/6 were used. Login to comment

[hide] Early embryonic renal tubules of wild-type and pol... J Am Soc Nephrol. 2006 Dec;17(12):3424-37. Epub 2006 Nov 15. Magenheimer BS, St John PL, Isom KS, Abrahamson DR, De Lisle RC, Wallace DP, Maser RL, Grantham JJ, Calvet JP
Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation.
J Am Soc Nephrol. 2006 Dec;17(12):3424-37. Epub 2006 Nov 15., [PMID:17108316]

Abstract [show]
Metanephric organ culture has been used to determine whether embryonic kidney tubules can be stimulated by cAMP to form cysts. Under basal culture conditions, wild-type kidneys from embryonic day 13.5 to 15.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-d period. Treatment of these kidneys with 8-Br-cAMP or the cAMP agonist forskolin induced the formation of dilated tubules within 1 h, which enlarged over several days and resulted in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Tubule dilation was reversible upon withdrawal of 8-Br-cAMP and was inhibited by the cAMP-dependent protein kinase inhibitor H89 and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)172. For further testing of the role of CFTR, metanephric cultures were prepared from mice with a targeted mutation of the Cftr gene. In contrast to kidneys from wild-type mice, those from Cftr -/- mice showed no evidence of tubular dilation in response to 8-Br-cAMP, indicating that CFTR Cl(-) channels are functional in embryonic kidneys and are required for cAMP-driven tubule expansion. A requirement for transepithelial Cl(-) transport was demonstrated by inhibiting the basolateral Na(+),K(+),2Cl(-) co-transporter with bumetanide, which effectively blocked all cAMP-stimulated tubular dilation. For determination of whether cystic dilation occurs to a greater extent in PKD kidneys in response to cAMP, Pkd1(m1Bei) -/- embryonic kidneys were treated with 8-Br-cAMP and were found to form rapidly CFTR- and Na(+),K(+),2Cl(-) co-transporter-dependent cysts that were three- to six-fold larger than those of wild-type kidneys. These results suggest that cAMP can stimulate fluid secretion early in renal tubule development during the time when renal cysts first appear in PKD kidneys and that PKD-deficient renal tubules are predisposed to abnormally increased cyst expansion in response to elevated levels of cAMP.

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35 Cftrm1UNC (S489X) mice produce no stable CFTR mRNA or protein and therefore are considered to have a null phenotype (32,33). Login to comment

[hide] Parthenolide inhibits IkappaB kinase, NF-kappaB ac... Am J Respir Cell Mol Biol. 2007 Jun;36(6):728-36. Epub 2007 Feb 1. Saadane A, Masters S, DiDonato J, Li J, Berger M
Parthenolide inhibits IkappaB kinase, NF-kappaB activation, and inflammatory response in cystic fibrosis cells and mice.
Am J Respir Cell Mol Biol. 2007 Jun;36(6):728-36. Epub 2007 Feb 1., [PMID:17272824]

Abstract [show]
Cystic fibrosis (CF) is characterized by prolonged and excessive inflammatory responses in the lung and increased activation of NF-kappaB. Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for anti-inflammatory activity. Several studies suggest that this compound inhibits the NF-kappaB pathway, but the exact site is controversial. We hypothesized that parthenolide might ameliorate the excessive inflammatory response in CF models by inhibiting activation of NF-kappaB. This was tested in vitro, using two pairs of cell lines with defective versus normal CF transmembrane conductance regulator (CFTR) (antisense/sense transfected 16 HBE and IB-3/S9), and in vivo, using CFTR-knockout (KO) mice. All cell lines were pretreated with parthenolide and then stimulated with IL-1beta and/or TNF. Parthenolide significantly inhibited IL-8 secretion induced by these cytokines and prevented NF-kappaB activation, IkappaBalpha degradation, and IkappaB Kinase complex activity. CFTR-KO and wild-type mice were pretreated with parthenolide or vehicle alone then challenged intratracheally with LPS. Bronchoalveolar lavage was performed 3, 6, and 8 h later. Parthenolide pretreatment inhibited PMN influx as well as cytokine and chemokine production. This was also associated with inhibition of IkappaBalpha degradation and NF-kappaB activation. We thus conclude that parthenolide inhibits IkappaB kinase, resulting in stabilization of cytoplasmic IkappaBalpha, which in turn leads to inhibition of NF-kappaB translocation and attenuation of subsequent inflammatory responses. IkappaB kinase may be a good target, and parthenolide and/or feverfew might be promising treatments for the excessive inflammation in CF.

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61 These are stock CftrtmtUnc -TgN(FABPCFTR)#Jaw mice bearing the S489X mutation in cftr and also a transgene for full-length human CFTR driven by the fatty acid-binding promoter (FABP), which restores CFTR function in the intestinal tract. Login to comment

[hide] A role for CFTR in the elevation of glutathione le... Am J Physiol Lung Cell Mol Physiol. 2007 Jun;292(6):L1590-7. Epub 2007 Mar 16. Kariya C, Leitner H, Min E, van Heeckeren C, van Heeckeren A, Day BJ
A role for CFTR in the elevation of glutathione levels in the lung by oral glutathione administration.
Am J Physiol Lung Cell Mol Physiol. 2007 Jun;292(6):L1590-7. Epub 2007 Mar 16., [PMID:17369290]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is the only known apical glutathione (GSH) transporter in the lung. The purpose of these studies was to determine whether oral GSH or glutathione disulfide (GSSG) treatment could increase lung epithelial lining fluid (ELF) GSH levels and whether CFTR plays a role in this process. The pharmacokinetic profile of an oral bolus dose of GSH (300 mg/kg) was determined in mice. Plasma, ELF, bronchoalveolar lavage (BAL) cells, and lung tissue were analyzed for GSH content. There was a rapid elevation in the GSH levels that peaked at 30 min in the plasma and 60 min in the lung, ELF, and BAL cells after oral GSH dosing. Oral GSH treatment produced a selective increase in the reduced and active form of GSH in all lung compartments examined. Oral GSSG treatment (300 mg/kg) resulted in a smaller increase of GSH levels. To evaluate the role of CFTR in this process, Cftr knockout (KO) mice and gut-corrected Cftr KO-transgenic (Tg) mice were given an oral bolus dose of GSH (300 mg/kg) and compared with wild-type mice for changes in GSH levels in plasma, lung, ELF, and BAL cells. There was a twofold increase in plasma, a twofold increase in lung, a fivefold increase in ELF, and a threefold increase in BAL cell GSH levels at 60 min in wild-type mice; however, GSH levels only increased by 40% in the plasma, 60% in the lung, 50% in the ELF, and twofold in the BAL cells within the gut-corrected Cftr KO-Tg mice. No change in GSH levels was observed in the uncorrected Cftr KO mice. These studies suggest that CFTR plays an important role in GSH uptake from the diet and transport processes in the lung.

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43 C57BL/6J congenic Cftr KO (S489X) mice (22.3 Ϯ 0.2 g) that possessed the S489X mutations in the murine equivalent to CFTR (47) and C57BL/6J congenic gut-corrected Cftr KO-Tg mice (23.7 Ϯ 0.3 g) that had intestinal specific expression of normal human Cftr driven by the fatty acid binding promoter (57) were employed in these studies. Login to comment

[hide] Impact of nutrition on phenotype in CFTR-deficient... Pediatr Res. 2007 Nov;62(5):528-32. Cottart CH, Bonvin E, Rey C, Wendum D, Bernaudin JF, Dumont S, Lasnier E, Debray D, Clement A, Housset C, Bonora M
Impact of nutrition on phenotype in CFTR-deficient mice.
Pediatr Res. 2007 Nov;62(5):528-32., [PMID:17805210]

Abstract [show]
To elucidate the impact of nutrition in cystic fibrosis (CF), we compared the phenotypic traits of Cftr -/- mice fed either a lipid-enriched liquid diet (Peptamen) or a standard chow combined with polyethylenglycol osmotic laxative (PEG), two strategies commonly used to prevent intestinal obstruction in CF mice. Survival, growth, liver, and ventilatory status were determined in Cftr -/- and Cftr +/+ mice, followed-up until 120 d. Ventilation was recorded in conscious animals using whole-body plethysmography. We found that the survival rate was similar in Peptamen and PEG Cftr -/- mice. Cftr -/- mice had lower minute ventilation than Cftr +/+ mice, whatever the diet. Both Cftr -/- and Cftr +/+ mice fed Peptamen displayed preadult growth delay compared with PEG-treated animals. Despite subsequent growth catch-up, Cftr -/- mice remained smaller than Cftr +/+ mice, whatever the diet. All Peptamen fed Cftr -/- mice showed hepatomegaly and liver steatosis, which also occurred but to a lesser extent in Peptamen fed Cftr +/+ animals. Therefore, while both treatment strategies are similarly efficient to avoid high mortality at weaning, Peptamen induces preadult growth delay and liver steatosis. These effects of diet are important to consider in future animal studies and also prompt to evaluate high-energy diets in CF patients.

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21 Animals used in this study were male CF mice homozygous for the S489X mutation (Cftrtm1Unc ) (6) back-crossed into C57BL/6 (three generations) (Cftr-/- ) and their normal littermates (Cftrϩ/ϩ ). Login to comment

[hide] Partial correction of the CFTR-dependent ABPA mous... J Gene Med. 2008 Jan;10(1):51-60. Mueller C, Torrez D, Braag S, Martino A, Clarke T, Campbell-Thompson M, Flotte TR
Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene.
J Gene Med. 2008 Jan;10(1):51-60., [PMID:18023072]

Abstract [show]
Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes.

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35 Mouse strains The primary CFTR knockout strain used for these studies was the CFTR S489X -/- neo insertion in C57BL/6 mice developed initially at the University of North Carolina [41] and then modified with the transgenic overexpression of gut-specific expression of human CFTR from the fatty acid binding protein (FABP) promoter in order to prevent intestinal obstruction and improve viability [42]. Login to comment
46 Aspergillus sensitization and challenge Five- to six-week old CFTR S489X -/-; FABP-hCFTR (+/+), and C57BL/6J mice were house in the SPF mouse colony of the University of Florida according to NIH guidelines and were allowed food and water ad libitum. Login to comment

[hide] Infertility in females with cystic fibrosis is mul... Endocrinology. 2008 Jun;149(6):2790-7. Epub 2008 Mar 6. Hodges CA, Palmert MR, Drumm ML
Infertility in females with cystic fibrosis is multifactorial: evidence from mouse models.
Endocrinology. 2008 Jun;149(6):2790-7. Epub 2008 Mar 6., [PMID:18325992]

Abstract [show]
Infertility is commonly associated with cystic fibrosis (CF). Although infertility in men with CF has been thoroughly investigated, the infertility observed in women with CF has not been well studied. To investigate female infertility associated with CF, we used two independently derived mouse models of CF. Both of these models displayed decreased fertility characterized by a reduction in litter number and litter size. Our findings suggest that much of the reduced fertility in these mice originates from decreased fertilization due to inadequate sperm transport within the female reproductive tract. However, our data indicate that additional reproductive phenotypes in the CF female mice also contribute to the reduced fertility including small ovarian and uterine size, aberrant estrous cycles, and decreased oocyte ovulation rates. These data, along with previous work demonstrating that the gene mutated in CF, the cystic fibrosis transmembrane conductance regulator (CFTR), is normally expressed in tissues vital to reproduction, raises the possibility that CFTR may have a direct effect on fertility. If so, CFTR may also play an important role in normal female fertility within the general population.

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36 Materials and Methods Mouse strains The CF mouse models (Cftr-/- ) used in these studies contained either the Cftrtm1Unc mutation (referred to as the S489X mutation in this manuscript), which generates a stop codon in the coding region of exon 10 of Cftr (32) (Jackson Laboratory, Bar Harbor, ME; stock no. Login to comment
76 Results Fertility in Cftr mutant females To determine the effect of the absence of Cftr on female fertility, two independently derived mouse models of CF (S489X and ⌬F508) and wild-type females were mated to wild-type males for 5 months and examined daily for subsequent birth of litters and number of pups. Login to comment
95 Fertility of wild-type and Cftr mutant females Mouse type Mice, n Females with no litters, n (%) Average litter per month Average no. of pups/litter Wild type 18 0 (0) 0.92 Ϯ 0.13 6.56 Ϯ 2.36 ⌬F508 14 5 (35.7) 0.32 Ϯ 0.26a 3.81 Ϯ 1.43a S489X 10 2 (20) 0.36 Ϯ 0.36a 3.55 Ϯ 1.92a Both mutations 24 7 (29.2) 0.34 Ϯ 0.30a 3.70 Ϯ 1.61a a P Ͻ 0.001 vs. wild type. TABLE 2. Login to comment
96 Uterine and ovarian size in wild-type and Cftr mutant females Mouse type Mice, n Average ovarian weight (mg) Average uterine weight (mg) Average mouse weight (g) 6-8 wk Wild type 7 3.1 Ϯ 0.6 61.4 Ϯ 9.8 19.0 Ϯ 1.45 ⌬F508 6 1.4 Ϯ 0.2a 27.4 Ϯ 6.4a 16.4 Ϯ 1.59b S489X 6 1.8 Ϯ 0.6a 26.6 Ϯ 14.3a 15.9 Ϯ 1.46a Both mutations 12 1.6 Ϯ 0.5a 27.0 Ϯ 10.6a 16.1 Ϯ 1.48a 14-16 wk Wild type 11 4.2 Ϯ 1.1 74.8 Ϯ 11.8 21.6 Ϯ 1.92 ⌬F508 6 2.6 Ϯ 0.6a 45.8 Ϯ 20.5b 18.6 Ϯ 0.88a S489X 8 2.8 Ϯ 0.6a 50.6 Ϯ 17.1b 19.4 Ϯ 1.33a Both mutations 14 2.7 Ϯ 0.6a 48.2 Ϯ 18.2b 19.0 Ϯ 1.11a a P Ͻ 0.005 vs. wild type. Login to comment
123 Wild-type and Cftr mutant females` response to exogenous hormones Mouse type Mice, na Average ovarian weight (mg) Average uterine weight (mg) Average mouse weight (g) Oocytes ovulated, n Wild type 10 7.3 Ϯ 2.3 110 Ϯ 10 21.5 Ϯ 3.08 24.3 Ϯ 9.62 ⌬F508 9 7.5 Ϯ 1.9 110 Ϯ 30 19.8 Ϯ 2.10b 24.2 Ϯ 7.87 S489X 6 6.0 Ϯ 1.7 92 Ϯ 14 19.4 Ϯ 1.85b 18.6 Ϯ 5.64 Both mutations 15 6.9 Ϯ 1.8 100 Ϯ 25 19.6 Ϯ 1.98b 22.2 Ϯ 7.47 a Ovulated oocytes were present in all injected females. Login to comment
125 Characterization of estrous cycle in wild-type and Cftr mutant females Mouse type Mice, n Mice with no estrus, n (%) Total no. of estrus Avg no. of cycles/mousea Cycle lengtha Wild type 14 0 (0) 58 4.14 Ϯ 0.86 5.11 Ϯ 1.58 ⌬F508 18 4 (22.2) 29 2.07 Ϯ 1.20b 11.79 Ϯ 5.64b S489X 12 5 (41.7) 16 2.29 Ϯ 1.44b 11.19 Ϯ 6.29c Both mutations 30 9 (30.0) 45 2.14 Ϯ 1.28b 11.59 Ϯ 5.71b a Calculated using only females with at least one estrous cycle. Login to comment
156 Fertilization of oocytes 48 h after hCG in wild-type and mutant females Mouse type (no. of mice) Total cells, n Cells at stage, % Oocyte One cell Two cell Three cell Four cell Wild type (4) 71 8.5 1.4 76.0 5.6 8.5 ⌬F508 (4) 63 98.4 0 1.6 0 0 S489X (4) 65 100 0 0 0 0 Both mutations 128 99.2 0 0.8 0 0 TABLE 6. Login to comment
157 In vitro fertilization of oocytes from wild-type and Cftr mutant females Mouse type (no. of mice) Fertility rate (%)a Wild type (4) 49 of 72 (68.1) ⌬F508 (4) 52 of 74 (70.3) S489X (4) 46 of 68 (67.6) Both mutations (8) 98 of 142 (69.0) a Fertility rate is expressed as the number two-cell embryos over the total number of oocytes (percent). Login to comment
190 Number of oviductal sperm and capacitation in wild-type and Cftr mutant females Mouse type (no. of mice) Total no. of sperm (average per oviduct) Capacitation (n ϭ 200 per mouse) (%) Wild type (3) 1536 Ϯ 291 100 ⌬F508 (3) 142 Ϯ 105a 100 S489X (3) 132 Ϯ 133a 100 Both mutations (6) 137 Ϯ 114a 100 a P Ͻ 0.001 vs. wild type. Login to comment

[hide] Distribution of CFTR mutations in Saguenay- Lac-Sa... Genet Med. 2008 Mar;10(3):201-6. Madore AM, Prevost C, Dorfman R, Taylor C, Durie P, Zielenski J, Laprise C
Distribution of CFTR mutations in Saguenay- Lac-Saint-Jean: proposal of a panel of mutations for population screening.
Genet Med. 2008 Mar;10(3):201-6., [PMID:18344710]

Abstract [show]
PURPOSE: Saguenay-Lac-Saint-Jean is a region located in the northeastern part of the Province of Quebec, Canada, and is characterized by a founder effect. In this region, it has been documented that the incidence of cystic fibrosis reached 1/902 live births between 1975 and 1988, three times higher than the average incidence of 1/2500 live births reported in other Caucasian populations. This corresponds to a carrier rate of 1/15. METHODS: Using genotyping data from the Canadian Consortium for Cystic Fibrosis Genetic Studies, this article describes the cystic fibrosis transmembrane conductance regulator profile of the cystic fibrosis population living in the Saguenay-Lac-Saint-Jean region and compares it with cystic fibrosis populations living in three other regions of the Province of Quebec. RESULTS: Significant differences in allelic frequencies of common mutations (as DeltaF508, 621 + 1G>T and A455E), and in percentage of covered allele with three or six mutations, were found in Saguenay-Lac-Saint-Jean compared to other regions. Based on this result, two mutation panels exceeding 90% sensitivity threshold are now proposed for cystic fibrosis carrier screening in this region. CONCLUSION: The implementation of the proposed carrier screening program could diminish the incidence of this disease in this region and allow future parents to make informed decisions about family planning.

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48 Altogether, the six mutations represent 95.89% of the CFTR allele of CF patients in the SLSJ population, whereas the proportions are 86.85, 85.27, and Table 2 Cystic fibrosis mutations present in the four populations studied Mutationa Allelic frequency (number of alleles [%]) Populationb 1 2 3 4 „F508 106 (62.35) 55 (72.37) 398 (72.36) 67 (57.78) 621 ؉ 1G>T 42 (24.71) 6 (7.89) 30 (5.45) 1 (0.85) A455E 12 (7.06) 2 (2.63) 14 (2.55) 1 (0.85) 3199del6 1 (0.59) 1 (1.32) 7 (1.27) 1 (0.85) 711 ؉ 1G>T 1 (0.59) 1 (1.32) 15 (2.73) 1 (0.85) Y1092X 1 (0.59) 1 (1.32) 5 (0.91) 0 R117C 2 (1.18) 0 0 0 ‚I507 1 (0.59) 2 (2.63) 10 (1.82) 0 L206W 1 (0.59) 1 (1.32) 9 (1.64) 0 R1158X 1 (0.59) 0 0 0 S489X 1 (0.59) 0 1 (0.18) 0 R553X 0 2 (2.63) 2 (0.36) 0 R334W 0 1 (1.32) 2 (0.36) 0 G542X 0 0 10 (1.82) 0 G85E 0 0 6 (1.09) 5 (4.24) N1303K 0 0 5 (0.91) 1 (0.85) IVS8-5T 0 0 4 (0.73) 0 W1282X 0 0 3 (0.55) 7 (5.93) R347P 0 0 1 (0.18) 2 (1.69) V520F 0 0 1 (0.18) 0 I1027T 0 0 1 (0.18) 0 R1066C/IVS 0 0 1 (0.18) 0 Q1313X 0 0 1 (0.18) 0 1898ϩ3GϾA 0 0 1 (0.18) 0 2183AAϾG 0 0 1 (0.18) 0 2951insA 0 0 1 (0.18) 0 G551D 0 0 0 2 (1.69) 1525-iG-A 0 0 0 2 (1.69) Y109C 0 0 0 1 (0.85) S549N 0 0 0 1 (0.85) 3154del1G 0 0 0 1 (0.85) UNKNOWN 1 (0.59) 4 (5.26) 20 (3.82) 25 (21.19) Number of alleles genotypedc 170 (100) 76 (100) 550 (100) 118 (100) a The six mutations included in the panels proposed are in bold. Login to comment

[hide] Adenovirus-mediated in utero expression of CFTR do... Mol Ther. 2008 May;16(5):812-8. Epub 2008 Mar 11. Davies LA, Varathalingam A, Painter H, Lawton AE, Sumner-Jones SG, Nunez-Alonso GA, Chan M, Munkonge F, Alton EW, Hyde SC, Gill DR
Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.
Mol Ther. 2008 May;16(5):812-8. Epub 2008 Mar 11., [PMID:18388934]

Abstract [show]
Gene therapy is being investigated in the treatment of lung-related aspects of the genetic disease, Cystic fibrosis (CF). Clinical studies have demonstrated CF transmembrane conductance regulator (CFTR) expression in the airways of adults with CF using a variety of gene transfer agents. In utero gene therapy is an alternative approach that facilitates vector transduction of rapidly expanding populations of target cells while avoiding immune recognition of the vector. In CF, in utero gene transfer could potentially delay the onset of disease symptoms in childhood and compensate for the role, if any, that CFTR plays in the developing organs. Previously published studies have suggested that transient expression of CFTR in utero was sufficient to rescue the fatal intestinal defect in S489X Cftr(tm1Unc)/Cftr(tm1Unc) knockout mice. We replicated these studies using an identical CFTR-expressing adenoviral vector and CF mouse strain in sufficiently large numbers to provide robust Kaplan-Meier survival data. Although each step of the procedure was carefully controlled and vector-specific CFTR expression was confirmed in the fetal organs after treatment, there was statistically no significant improvement in the survival of mice treated in utero with AdCFTR, compared with contemporaneous control animals.

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11 After vector delivery at 16 days of gestation (equivalent to a 15-to 20-week gestation period in humans), transgene expression is observed in the lung, stomach and gut and is highly correlated with increased fetal breathing movements.4 Several CF transgenic mouse strains have been generated in an attempt to model and evaluate potential treatment strategies, but although these mouse models display many of the -CF-related ion-transport bioelectric defects found in human CF tissues, lung pathology is not recapitulated.5 However, some abnormalities of the CF gastrointestinal tract such as intestinal obstruction are observed in mouse models, where they can be very severe, leading to poor survival.6 The CF knockout mouse strain S489X carrying the Cftrtm1Unc mutation develops intestinal obstruction leading to intestinal perforation, peritonitis and death, resulting in only 5% survival to adulthood,7 although these mice can survive given the appropriate dietary intervention.8 Survival of these mice was also reported to increase dramatically when recombinant adenovirus expressing human CF transmembrane conductance regulator (hCFTR) was delivered via intra-amniotic injection to fetal mice in utero.9 The improved survival was observed even though the adenovirus-mediated CFTR expression was transient, implying that continuous expression of CFTR was not required, therefore challenging the widely held view that it is the absence of the CFTR chloride channel that leads to CF disease in the adult. Login to comment
52 Effect of hCFTR expression on survival of cftr -/- mice Any conclusion regarding the effectiveness of in utero hCFTR expression in correcting the lethal intestinal defect in S489X Cftrtm1Unc /Cftrtm1Unc mice will be wholly dependent on the number of Cftr -/- mice surviving after injection of AdCFTR compared with injection of control AdLacZ vector. Login to comment
57 Finally, to assess the impact of in utero delivery of AdCFTR on the survival of Cftr -/- mice, S489X litters were injected at E16 a pCIKCFTR 195 kd 112 kd Band C Band B AdGFP AdCFTR b 0 -0.1 0.0 Rateof 125 |efflux/min 0.1 0.2 0.3 0.4 0.5 1 2 Time (min) 3 4 Untreated AdLacZ AdCFTR T84 cells c 0 0.01 Untreated AdCFTR 0.1 1 10 100 1,000 hCFTRmRNAcopies/ngRNA (3) (6) (7) (4) (13) (4) (+/+) (+/-) (-/-) d Untreated AdCFTR 0 0.01 0.1 1 10 100 hCFTRmRNAcopies/ngRNA (3) (6) (7) (5) (15) (5) (+/+) (+/-) (-/-) Figure 3 Human cystic fibrosis transmembrane conductance regulator (hCFTR) can be detected in lungs and intestines of S489X Cftrtm1Unc mice following intra-amniotic injection of AdCFTR. Login to comment
63 Expression of hCFTR mRNA was confirmed in vivo following intra-amniotic injection of 108 plaque forming units (pfu)/fetus of AdCFTR at E16 to litters resulting from heterozygote matings of S489X Cftrtm1Unc mice. Login to comment
68 Table 1 In utero injection and survival of S489X Cftrtm1Unc /Cftrtm1Unc mice Untreated AdLacZ 107 pfu AdCFTR 107 pfu AdCFTR 108 pfu Litters 21 17 15 13 Injected 0 142 114 102 Lost NA 11 14 6 Born 156 131 100 96 Day 0 -/- 25 20 20 22 +/- 87 54 45 48 +/+ 42 48 30 25 Unknown 2 9 5 1 Day 100 -/- 4 2 2 3 +/- 70 28 39 29 +/+ 33 28 26 13 Unknown 0 0 0 0 Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; pfu, plaque forming units; NA, not applicable. Login to comment
69 Litters from heterozygote S489X matings were injected with AdLacZ or AdCFTR at 16 days of gestation. Login to comment
97 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival (+/+) (+/-) (-/-) a 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival c 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival Untreated AdLacZ 107 AdCFTR 107 AdCFTR 108 b 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival d Figure 4 Survival of S489X Cftrtm1Unc mice following expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) from AdCFTR. Login to comment
98 Kaplan-Meier plots of S489X Cftrtm1Unc survival following intra-amniotic injections. Login to comment
105 It was unfortunate that the S489X Cftrtm1Unc mouse strain dem- onstratedsuchhighlevelsofmaternalcannibalismandneglectlead- ing to a high mortality of newborn pups of all genotypes, especially following surgical interventions. Login to comment
112 An important difference is the ~20% survival of the Cftr -/- mice observed in our facility compared with a previously reported 5%.14 The intestinal pathology and mortality in CF transgenic mouse models can vary dependent on the precise Cftr knockout mutation, environmental influences and the independent segregation of modifier genes.6,17 The survival chances of one CF mouse model Cftrtm1G551D /Cftrtm1G551D were shown to vary between 27 and 67% depending on undetermined environmental factors in the animal breeding facility.18 Interestingly, when we transferred a subset of our S489X Cftr -/- mice to an alternative animal facility they died of intestinal obstruction within a few days (data not shown). Login to comment
115 Unavoidably, our studies used S489X Cftrtm1Unc CF knockout mice as a 10th generation backcross onto to C57BL/6 background, as opposed to a 5th generation backcross.10 In the accompanying paper, Buckley et al.16 attempted independent verification of the original study, using the same adenoviral vector, but using an alternative knockout CF mouse allele (Cftrtm1Cam /Cftrtm1Cam ) on an inbred genetic background (129 S6Sv/Ev) to minimize genetic heterogeneity and the effects of potential modifier genes. Login to comment
124 The Cftr-mutant UNC mouse strain, S489X (Cftrtm1UNC / Cftrtm1UNC ) was obtained as a 10th generation backcross on a C57BL/6 background (Dr Julia Dorin, MRC Human Genetics Unit, Edinburgh, UK) and was maintained via heterozygote matings in open cages with hardwood sawdust and R&M3 breeders` diet (Special Diet Services, Witham, UK) with weaning at 21 days. Login to comment
142 To generate time-mated pregnancies, heterozygote S489X Cftrtm1Unc females were introduced to cages containing heterozygote males overnight with the following day regarded as day 1 of gestation (E1) in resulting pregnancies. Login to comment

[hide] Congenital tracheal malformation in cystic fibrosi... J Physiol. 2008 Jul 1;586(13):3231-43. Epub 2008 May 1. Bonvin E, Le Rouzic P, Bernaudin JF, Cottart CH, Vandebrouck C, Crie A, Leal T, Clement A, Bonora M
Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice.
J Physiol. 2008 Jul 1;586(13):3231-43. Epub 2008 May 1., 2008-07-01 [PMID:18450781]

Abstract [show]
In cystic fibrosis (CF) patients, the major alteration in pulmonary function is due to peripheral airway obstruction. In the present study, we investigated the possibility that alterations in the extrathoracic airways, particularly in the trachea that expresses high levels of CFTR (CF transmembrane conductance regulator), may contribute to respiratory dysfunction. We performed morphological analyses of the trachea and airway functional studies in adult Cftr knockout (Cftr(-/-)) and F508del-CFTR mice and their controls. Macroscopic and histological examination of the trachea showed the presence of one to seven disrupted or incomplete cartilage rings in Cftr(-/-) mice (23/25) while only a few Cftr(+/+) mice (6/25) had one abnormal ring. Tracheal defects were mainly localized in the proximal trachea. In 14 Cftr(-/-) mice, frontal disruption of the first three to six rings below the cricoid cartilage were associated with upper tracheal constriction. Similar tracheal abnormalities were detected in adult F508del-CFTR and in newborn Cftr(-/-) and F508del-CFTR mice. Tracheal and ventilatory function analyses showed in Cftr(-/-) mice a decreased contractile response of the proximal trachea and a reduced breathing rate due to an increase in the inspiratory and expiratory times. In F508del-CFTR mice, the expiratory time was longer than in controls. Therefore, these structural and functional abnormalities detected in adult and newborn CF mouse models may represent congenital malformations related to CFTR dysfunction. These results raise important questions concerning the mechanisms governing tracheal development within the context of CFTR protein dysfunction and the implication of such abnormalities in the pathogenesis of airway disease in CF.

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16 Although CFTR-deficient mice do not develop classical lung disease, some alterations in pulmonary function have been reported in Cftrtm1Unc knockout mice homozygous for the S489X mutation (Cftr-/- ) (Kent et al. 1996; Cohen et al. 2004). Login to comment
32 Animals The study was carried out on two different CF mouse models: (1) Cftrtm1Unc knockout mice homozygous for the S489X-CFTR mutation (Snouwaert et al. 1992), back-crossed into C57BL/6 (three generations) (Cftr-/- ) and their normal littermates (Cftr+/+ ). Login to comment
42 We also performed experiments on newborn homozygous mice for both mutations (S489X and F508del) and their control littermates at 1 day postnatal. Login to comment
144 The high rate of tracheal abnormalities in adult Cftr-/- mice homozygous for the S489X mutation of the CFTR protein, in contrast to that observed in control Cftr+/+ mice, suggests that these abnormalities could be directly Table 2. Login to comment

[hide] Animal models of chronic lung infection with Pseud... Lab Anim. 2008 Oct;42(4):389-412. Epub 2008 Sep 9. Kukavica-Ibrulj I, Levesque RC
Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies.
Lab Anim. 2008 Oct;42(4):389-412. Epub 2008 Sep 9., [PMID:18782827]

Abstract [show]
Cystic fibrosis (CF) is caused by a defect in the transmembrane conductance regulator (CFTR) protein that functions as a chloride channel. Dysfunction of the CFTR protein results in salty sweat, pancreatic insufficiency, intestinal obstruction, male infertility and severe pulmonary disease. In most patients with CF life expectancy is limited due to a progressive loss of functional lung tissue. Early in life a persistent neutrophylic inflammation can be demonstrated in the airways. The cause of this inflammation, the role of CFTR and the cause of lung morbidity by different CF-specific bacteria, mostly Pseudomonas aeruginosa, are not well understood. The lack of an appropriate animal model with multi-organ pathology having the characteristics of the human form of CF has hampered our understanding of the pathobiology and chronic lung infections of the disease for many years. This review summarizes the main characteristics of CF and focuses on several available animal models that have been frequently used in CF research. A better understanding of the chronic lung infection caused particularly by P. aeruginosa, the pathophysiology of lung inflammation and the pathogenesis of lung disease necessitates animal models to understand CF, and to develop and improve treatment.

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170 Table 2 Cystic fibrosis (CF) mouse models CF mice Mutation/molecular strategy Phenotype/limitation CFTR KO CFTRtm1Unc (Snouwaert et al. 1992) Exon 10 replacement, null mutation, inframe stop Severe intestinal phenotype and high mortality; no lung disease CFTRtm1Hgu (Dorin et al. 1992) Exon 10 insertional mutagenesis Intestinal blockage; minor pathology in lungs of one mouse CFTRtm1Cam (Ratcliff et al. 1993) Exon 10 replacement, null mutation Severe intestinal phenotype and high mortality; pathology in lacrimal gland and pancreas of some mice; no lung disease CFTRtm1Bay (O`Neal et al. 1993) Exon 3 insertional duplication, null mutation Severe intestinal phenotype and high mortality; no lung disease CFTRtm1Hsc (Rozmahel et al. 1996) Exon 1 replacement, null mutation Severe intestinal phenotype and high mortality; no lung disease Other mutations CFTRtm1Kth (Zeiher et al. 1995) DF508 by exon 10 replacement High mortality and reduction in size, variable pathology of the gastrointestinal tract, normal lung, pancreas, gallbladder, male reproductive tract, lacrimal gland and submandibular glands CFTRtm1Eur (van Doorninck et al. 1995, French et al. 1996) DF508 by exon 10 'hit and run` Normal survival, growth retarded but no abnormalities or stasis of inspissated mucus in lungs, pancreas, liver bile ducts, vas deferens and salivary glands CFTRtm1G551D (Delaney et al. 1996) G551D by exon 11 replacement Moderate phenotype with reduced incidence of intestinal blockage and 67% survival; no lung disease CFTRtm2Hgu (Dickinson et al. 2000) G480C by exon 10 'hit and run` Normal survival, no reduction in body weight, preserved cAMP-mediated Cl2 response, decreased Ca2þ -related Cl2 response CFTR2/2hCFTR-G542X (Du et al. 2002) CFTR2/2 null mutation that also express a human CFTR-G542X stop mutation under control of the intestinal FABP promoter Suppression of the hCFTR-G542X mutation in vivo by aminoglycosides CFTRtm1Unc -TgN(FABPCFTR) (Zhou et al. 1994) Stop codon in the murine CFTR gene (S489X) but also express human CFTR in the gut epithelium (transgenic introduction of CFTR under FABP promoter) Functional correction of ileal goblet cell and crypt cell hyperplasia and cyclic adenosine monophosphate-stimulated chloride secretion, improved survival Congenic C57BL/6J CFTR2/2 (Durie et al. 2004) Long-lived congenic C57BL/6J CFTR2/2 All organs pathologically affected by the human form of CF Scnn1a-, Scnn1b- and Scnn1c-transgenic mice (Mall et al. 2004) Transgenic mice overexpressing airway-specific ENaC to increase Naþ absorption CF-like lung disease FABP: fatty acid-binding protein, ENaC: epithelial Naþ channels. Login to comment

[hide] cAMP-mediated regulation of cholesterol accumulati... Am J Physiol Lung Cell Mol Physiol. 2008 Nov;295(5):L809-19. Epub 2008 Sep 12. Manson ME, Corey DA, White NM, Kelley TJ
cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells.
Am J Physiol Lung Cell Mol Physiol. 2008 Nov;295(5):L809-19. Epub 2008 Sep 12., [PMID:18790990]

Abstract [show]
The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPC1, is also directly regulated by cAMP; therefore, it is postulated that NPC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of beta-arrestin-2 (betaarr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, Cftr(-/-) MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls.

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124 To verify results in an in vivo model, we excised nasal epithelium from S489X Cftr-/- mice and assayed for pCREB content compared with nasal epithelium from age-matched wild-type mice. Login to comment
121 To verify results in an in vivo model, we excised nasal epithelium from S489X Cftr-/- mice and assayed for pCREB content compared with nasal epithelium from age-matched wild-type mice. Login to comment

[hide] Defective acid sphingomyelinase pathway with Pseud... Am J Respir Cell Mol Biol. 2009 Sep;41(3):367-75. Epub 2009 Jan 23. Yu H, Zeidan YH, Wu BX, Jenkins RW, Flotte TR, Hannun YA, Virella-Lowell I
Defective acid sphingomyelinase pathway with Pseudomonas aeruginosa infection in cystic fibrosis.
Am J Respir Cell Mol Biol. 2009 Sep;41(3):367-75. Epub 2009 Jan 23., [PMID:19168701]

Abstract [show]
Acid sphingomyelinase (ASMase) is a key enzyme in sphingolipid metabolism, which can be activated by various cellular stress mechanisms including bacterial pathogens. Activation of ASMase generates ceramide, which is important for innate immune response to eliminate infected pathogens. The current study reveals a defective ASMase pathway after Pseudomonas aeruginosa infection in both a cystic fibrosis (CF) bronchial epithelial cell line (IB3-1 cell) and in the lungs of CF transmembrane conductance regulator (CFTR) knockout (KO) mice as compared with S9 cells and wild-type C57BL/6 mice. ASMase activity and total ceramide levels significantly increased in S9 cells and C57BL/6 mice with P. aeruginosa infection, but not in IB3-1 cells and CFTR KO mice. The silencing of CFTR by CFTR RNAi in S9 cells significantly decreased ASMase activity after bacterial infection as compared with controls. This study also demonstrates that induction of ASMase is responsible for modulating the immune response to bacterial infection. Blocking ASMase activity with specific ASMase RNAi, an ASMase inhibitor, or an ASMase antibody in S9 cells significantly increased IL-8 levels with P. aeruginosa infection compared with controls. Reciprocally, adding exogenous bacterial sphingomyelinase to IB3-1 cells significantly decreased IL-8 levels compared with untreated cells. In addition, silencing of ASMase in S9 cells also significantly decreased bacterial internalization. Adding exogenous bacterial sphingomyelinase to IB3-1 cells reconstituted the cell death response to P. aeruginosa infection. This study demonstrates that the defective ASMase pathway in CF is a key contributor to the unabated IL-8 response with P. aeruginosa infection and to the compromised host response failing to eradicate bacteria.

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79 Animal Experiments The CFTR KO mice are Cftrtm1Unc-TgN(FABPCFTR) (fatty acid-binding protein [FABP]-CFTR) mice that have a stop codon in the murine CFTR gene (S489X) but also express human CFTR in the gut epithelium due to transgenic introduction of CFTR under the control of the FABP promoter (30). Login to comment

[hide] Side chain structure determines unique physiologic... Hepatology. 2009 Jun;49(6):1972-81. Halilbasic E, Fiorotto R, Fickert P, Marschall HU, Moustafa T, Spirli C, Fuchsbichler A, Gumhold J, Silbert D, Zatloukal K, Langner C, Maitra U, Denk H, Hofmann AF, Strazzabosco M, Trauner M
Side chain structure determines unique physiologic and therapeutic properties of norursodeoxycholic acid in Mdr2-/- mice.
Hepatology. 2009 Jun;49(6):1972-81., [PMID:19475687]

Abstract [show]
24-norursodeoxycholic acid (norUDCA), a side chain-modified ursodeoxycholic acid derivative, has dramatic therapeutic effects in experimental cholestasis and may be a promising agent for the treatment of cholestatic liver diseases. We aimed to better understand the physiologic and therapeutic properties of norUDCA and to test if they are related to its side chain length and/or relative resistance to amidation. For this purpose, Mdr2(-/-) mice, a model for sclerosing cholangitis, received either a standard diet or a norUDCA-, tauro norursodeoxycholic acid (tauro- norUDCA)-, or di norursodeoxycholic acid (di norUDCA)-enriched diet. Bile composition, serum biochemistry, liver histology, fibrosis, and expression of key detoxification and transport systems were investigated. Direct choleretic effects were addressed in isolated bile duct units. The role of Cftr for norUDCA-induced choleresis was explored in Cftr(-/-) mice. norUDCA had pharmacologic features that were not shared by its derivatives, including the increase in hepatic and serum bile acid levels and a strong stimulation of biliary HCO(3)(-)-output. norUDCA directly stimulated fluid secretion in isolated bile duct units in a HCO(3)(-)-dependent fashion to a higher extent than the other bile acids. Notably, the norUDCA significantly stimulated HCO(3)(-)-output also in Cftr(-/-) mice. In Mdr2(-/-) mice, cholangitis and fibrosis strongly improved with norUDCA, remained unchanged with tauro- norUDCA, and worsened with di norUDCA. Expression of Mrp4, Cyp2b10, and Sult2a1 was increased by norUDCA and di norUDCA, but was unaffected by tauro- norUDCA. CONCLUSION:The relative resistance of norUDCA to amidation may explain its unique physiologic and pharmacologic properties. These include the ability to undergo cholehepatic shunting and to directly stimulate cholangiocyte secretion, both resulting in a HCO(3)(-)-rich hypercholeresis that protects the liver from cholestatic injury.

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27 Congenic B6.129P2-Cftrtm1Unc mice, which possess the S489X mutation that blocks transcription of Cftr,13 were fed with Peptamen (Nestle Clinical Nutrition, Deerfield, IL) or with Peptamen containing norUDCA (in a dosage equivalent to that used in Mdr2-/- mice) for 1 week. Login to comment

[hide] Peroxiredoxin 6 fails to limit phospholipid peroxi... PLoS One. 2009 Jun 29;4(6):e6075. Trudel S, Kelly M, Fritsch J, Nguyen-Khoa T, Therond P, Couturier M, Dadlez M, Debski J, Touqui L, Vallee B, Ollero M, Edelman A, Brouillard F
Peroxiredoxin 6 fails to limit phospholipid peroxidation in lung from Cftr-knockout mice subjected to oxidative challenge.
PLoS One. 2009 Jun 29;4(6):e6075., [PMID:19562038]

Abstract [show]
Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult.The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr(-/-) lung when compared to wild-type (Cftr(+/+)) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr(-/-) lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr(-/-) mice compared to Cftr(+/+). Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr(+/+) lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr(-/-) lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr(-/-) lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.

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46 Animals used in this study were Cftr-knockout (Cftr 2/2) mice homozygous for the S489X mutation (B6;129-Cftrtm1Unc) [16] backcrossed into C57BL/6 (three generations) and their wild-type (Cftr+/+) littermates (CDTA, Orle´ans, France). Login to comment

[hide] The triterpenoid CDDO limits inflammation in precl... Am J Physiol Lung Cell Mol Physiol. 2009 Nov;297(5):L828-36. Epub 2009 Aug 21. Nichols DP, Ziady AG, Shank SL, Eastman JF, Davis PB
The triterpenoid CDDO limits inflammation in preclinical models of cystic fibrosis lung disease.
Am J Physiol Lung Cell Mol Physiol. 2009 Nov;297(5):L828-36. Epub 2009 Aug 21., [PMID:19700644]

Abstract [show]
Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-kappaB activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.

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122 Electrophysiological phenotype (i.e., nasal potential difference) and inflammatory responses in the lung to P. aeruginosa are very similar in R117H mice and multiple other CF transgenic mice, including mice bearing the ⌬F508 or S489X mutation (40). Login to comment

[hide] Modulation of exaggerated-IgE allergic responses b... Mol Ther. 2010 Mar;18(3):511-8. Epub 2009 Nov 24. Mueller C, Keeler A, Braag S, Menz T, Tang Q, Flotte TR
Modulation of exaggerated-IgE allergic responses by gene transfer-mediated antagonism of IL-13 and IL-17e.
Mol Ther. 2010 Mar;18(3):511-8. Epub 2009 Nov 24., [PMID:19935781]

Abstract [show]
Asthma and allergic rhinitis are almost invariable accompanied by elevated levels of immunoglobin E (IgE), and more importantly a genetic link between IgE levels and airway hyper-responsiveness has been established. We hypothesized that expression of soluble receptors directed against interleukin (IL)-13 and IL-17e would prevent the cytokines from engaging the cell-bound receptors and therefore help to attenuate allergic responses in a Cftr(-/-)-dependent mouse model of exaggerated-IgE responses. Cftr(-/-) mice were injected with recombinant adeno-associated virus 1 (rAAV1) intramuscularly expressing soluble receptors to IL-17e (IL-17Rh1fc) or IL-13 (IL-13Ralpha2Fc). Total IgE levels, in mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapy, were lower than in the control group. Interestingly Aspergillus fumigatus (Af)-specific IgE levels were undetectable in both the mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapies. Further flow cytometry analysis of intracellular gene expression suggests that blocking IL-17e may be interfering with signaling upstream of CD4+ and CD11b+ cells and reducing IgE levels by affecting signaling on these cell populations. In contrast it appears that IL-13 blockade acts downstream to reduce IgE levels probably by directly affecting B-cell maturation. These studies demonstrate the feasibility of targeting T helper 2 (Th2) cytokines with rAAV-delivered fusion proteins as a means to treat aberrant immune responses.

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189 The primary Cftr knockout strain used for these studies was the CFTR S489X-/- neo insertion in C57BL/6 mice developed initially at the University of North Carolina24 and then modified with the transgenic overexpression of gut-specific expression of human CFTR from the fatty acid-binding protein promoter in order to prevent intestinal obstruction and improve viability.25 These mice have then been backcrossed 10 generations onto a C57BL/6 mouse. Login to comment
194 Six to eight-week-old Cftr S489X-/-; fatty acid-binding protein human CFTR+/+, and wild-type lit- termatemicewerehousedinthespecificpathogen-freemousecolonyofthe University of Massachusetts Medical School (Worcester, MA) according to National Institutes of Health guidelines and were allowed food and water ad libitum. Login to comment

[hide] Pharmacological correction of a defect in PPAR-gam... Nat Med. 2010 Mar;16(3):313-8. Epub 2010 Feb 14. Harmon GS, Dumlao DS, Ng DT, Barrett KE, Dennis EA, Dong H, Glass CK
Pharmacological correction of a defect in PPAR-gamma signaling ameliorates disease severity in Cftr-deficient mice.
Nat Med. 2010 Mar;16(3):313-8. Epub 2010 Feb 14., [PMID:20154695]

Abstract [show]
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (encoded by Cftr) that impair its role as an apical chloride channel that supports bicarbonate transport. Individuals with cystic fibrosis show retained, thickened mucus that plugs airways and obstructs luminal organs as well as numerous other abnormalities that include inflammation of affected organs, alterations in lipid metabolism and insulin resistance. Here we show that colonic epithelial cells and whole lung tissue from Cftr-deficient mice show a defect in peroxisome proliferator-activated receptor-gamma (PPAR-gamma, encoded by Pparg) function that contributes to a pathological program of gene expression. Lipidomic analysis of colonic epithelial cells suggests that this defect results in part from reduced amounts of the endogenous PPAR-gamma ligand 15-keto-prostaglandin E(2) (15-keto-PGE(2)). Treatment of Cftr-deficient mice with the synthetic PPAR-gamma ligand rosiglitazone partially normalizes the altered gene expression pattern associated with Cftr deficiency and reduces disease severity. Rosiglitazone has no effect on chloride secretion in the colon, but it increases expression of the genes encoding carbonic anhydrases 4 and 2 (Car4 and Car2), increases bicarbonate secretion and reduces mucus retention. These studies reveal a reversible defect in PPAR-gamma signaling in Cftr-deficient cells that can be pharmacologically corrected to ameliorate the severity of the cystic fibrosis phenotype in mice.

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154 Eckman, E.A., Cotton, C.U., Kube, D.M. & Davis, P.B. Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment
293 We inbred mice heterozygous for the S489X (B6.129P2-CFTRtm1Unc, or Cftr+/-) mutation for more than ten generations. Login to comment

[hide] The characterization of the first anti-mouse Muc6 ... Histochem Cell Biol. 2010 May;133(5):517-25. Epub 2010 Mar 23. Gouyer V, Leir SH, Tetaert D, Liu Y, Gottrand F, Harris A, Desseyn JL
The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice.
Histochem Cell Biol. 2010 May;133(5):517-25. Epub 2010 Mar 23., [PMID:20309575]

Abstract [show]
Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr-/- mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.

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37 Cftr-knockout (Cftr¡/¡) mice used are homozygous for the S489X-CFTR mutation (B6;129-Cftrtm1Unc ) (Snouwaert et al. 1992). Login to comment
38 The S489X mouse third generation backcross to C57BL/6 are maintained at the French Centre of Transgenesis, Archiving and Animal Models in Orleans (MF Bertrand, CDTA, Orléans, France) by random mating of heterozygous mice and are provided with their wild-type littermates (Cftr+/+) at 21-25 days of age. Login to comment

[hide] Hypertonic saline increases lung epithelial lining... Respir Res. 2010 Aug 27;11:119. Gould NS, Gauthier S, Kariya CT, Min E, Huang J, Brian DJ
Hypertonic saline increases lung epithelial lining fluid glutathione and thiocyanate: two protective CFTR-dependent thiols against oxidative injury.
Respir Res. 2010 Aug 27;11:119., [PMID:20799947]

Abstract [show]
BACKGROUND: Cystic fibrosis is a debilitating lung disease due to mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) and is associated with chronic infections resulting in elevated myeloperoxidase activity and generation of hypochlorous acid (HOCl). CFTR mutations lead to decreased levels of glutathione (GSH) and thiocyanate (SCN) in the epithelial lining fluid (ELF). Hypertonic saline is used to improve lung function however the mechanism is uncertain. METHODS: In the present study, the effect of GSH and SCN on HOCl-mediated cell injury and their changes in the ELF after hypertonic saline nebulization in wild type (WT) and CFTR KO mice was examined. CFTR sufficient and deficient lung cells were assessed for GSH, SCN and corresponding sensitivity towards HOCl-mediated injury, in vitro. RESULTS: CFTR (-) cells had lower extracellular levels of both GSH and SCN and were more sensitive to HOCl-mediated injury. In vivo, hypertonic saline increased ELF GSH in the WT and to a lesser extent in the CFTR KO mice but only SCN in the WT ELF. Finally, potential protective effects of GSH and SCN at concentrations found in the ELF against HOCl toxicity were examined in vitro. CONCLUSIONS: While the concentrations of GSH and SCN associated with the WT ELF protect against HOCl toxicity, those found in the CFTR KO mice were less sufficient to inhibit cell injury. These data suggests that CFTR has important roles in exporting GSH and SCN which are protective against oxidants and that hypertonic saline treatment may have beneficial effects by increasing their levels in the lung.

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48 C57BL/6J congenic gut-corrected Cftr KO-Tg mice that possess a S489X truncated mutation in the murine equivalent to CFTR and have intestinal specific expression of normal human Cftr driven by the fatty acid binding promoter were originally obtained from Case Western Reserve University`s CF Animal Core, as previously described [18]. Login to comment
187 The CFTR KO mice utilized in this study possess the S489X truncation mutation resulting in a non functional CFTR. Login to comment

[hide] Listeria monocytogenes exploits cystic fibrosis tr... Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1633-8. Epub 2011 Jan 10. Radtke AL, Anderson KL, Davis MJ, DiMagno MJ, Swanson JA, O'Riordan MX
Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome.
Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1633-8. Epub 2011 Jan 10., 2011-01-25 [PMID:21220348]

Abstract [show]
Virulence of the intracellular pathogen Listeria monocytogenes (Listeria) requires escape from the phagosome into the host cytosol, where the bacteria replicate. Phagosomal escape is a multistep process characterized by perforation, which is dependent on the pore-forming toxin listeriolysin O (LLO), followed by rupture. The contribution of host factors to Listeria phagosomal escape is incompletely defined. Here we show that the cystic fibrosis transmembrane conductance regulator (CFTR) facilitates Listeria cytosolic entry. CFTR inhibition or mutation suppressed Listeria vacuolar escape in culture, and inhibition of CFTR in wild-type mice before oral inoculation of Listeria markedly decreased systemic infection. We provide evidence that high chloride concentrations may facilitate Listeria vacuolar escape by enhancing LLO oligomerization and lytic activity. We propose that CFTR transiently increases phagosomal chloride concentration after infection, potentiating LLO pore formation and vacuole lysis. Our studies suggest that Listeria exploits mechanisms of cellular ion homeostasis to escape the phagosome and emphasize host ion-channel function as a key parameter of bacterial virulence.

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310 Eckman EA, Cotton CU, Kube DM, Davis PB (1995) Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

[hide] Methods for evaluating inflammation in cystic fibr... Methods Mol Biol. 2011;742:51-76. Ziady AG, Davis PB
Methods for evaluating inflammation in cystic fibrosis.
Methods Mol Biol. 2011;742:51-76., [PMID:21547726]

Abstract [show]
Cystic fibrosis is characterized by excessive pulmonary inflammation, which presents early in life and becomes self-sustaining, eventually leading to the destruction of the lung. Treating inflammation is one of the most pressing needs in CF therapy and has been shown to slow lung function deterioration. However, it remains unclear whether excessive inflammation is a direct result of CFTR dysfunction, and thus innate, or develops in response to early stimulation of inflammatory pathways. Here, we will discuss clinically relevant studies and the methods employed by them. We will focus on investigations in cell and animal models as well as patients. Our discussion will describe the character of pulmonary inflammation in CF and present potential therapeutic approaches that can ameliorate excessive responses and improve disease prognosis.

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598 For example, when bred into the C57BL/6 background R117H mutant mice exhibit CF defects similar in magnitude to those observed in mice bearing the F508del or S489X mutation. Login to comment

[hide] Defective CFTR-dependent CREB activation results i... PLoS One. 2011;6(5):e19120. Epub 2011 May 9. Xu WM, Chen J, Chen H, Diao RY, Fok KL, Dong JD, Sun TT, Chen WY, Yu MK, Zhang XH, Tsang LL, Lau A, Shi QX, Shi QH, Huang PB, Chan HC
Defective CFTR-dependent CREB activation results in impaired spermatogenesis and azoospermia.
PLoS One. 2011;6(5):e19120. Epub 2011 May 9., [PMID:21625623]

Abstract [show]
Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3) (-) transport and the expression of the HCO(3) (-) sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3) (-) could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.

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29 Results Impaired spermatogenesis in CF mice models To investigate possible involvement of CFTR in spermatogenesis, we used a CFTR knockout (Cftrtm1Unc , also referred as S489X) mice. Login to comment
30 Since most homozygous S489X CF mice die at a young age or less frequently available, heterozygous mice were used for quantitative measurement. Login to comment
31 Morphological study showed that testis tissue size and weight were significantly lower in heterozygous S489X CF mice than that of wild-type control (Fig. 1A). Login to comment
32 Daily sperm production (DSP), which is often used to evaluate the spemiogenesis in the testis [21], was significantly reduced in heterozygous S489X CF mice compared with wide-type control (Fig. 1B). Login to comment
33 The sperm number recovered from the epididymis of the heterozygous S489X CF mice was also significantly lower than that of wide-type control (Fig. 1C). Login to comment
34 H&E staining showed slight decrease in diameter of seminiferous tubules (Fig. 1D,E) and slight cytoplasmic shrinkage of spermatocytes and round spermatids (Fig. S1) in S489X CF mice. Login to comment
35 Realtime PCR results showed significant reduction of Protamine-2, a specific marker of spermatid and spermatozoa, in heterozygous and homozygous S489X CF mice at mRNA level (Fig. 1F). Login to comment
36 Immunofluorescence results further demonstrated the down-regulation of Protamine-2 in S489X CF mice at protein level (Fig. 1G). Login to comment
37 CREM, a spermatid-specific transcription factor, was also down-regulated in the homozygous S489X CF mice compared to wild-type as indicated by Western blot and immunofluorescent staining respectively (Fig. 1H&J), suggesting defect in spermatogenesis in CF mice. Login to comment
38 Interestingly, immunofluorescence result showed that cAMP-responsive element binding protein (CREB), an important transcription factor in Sertoli cells during spermatogenesis, was decreased in the Sertoli cells of homozygous S489X CF mice, as compared to heterozygotes and wild-type (Fig. 1I). Login to comment
39 Western blot result also showed downregulation of phosphorylated and total CREB in homozygous S489X CF mice compared to wild-type and heterozygotes (Fig.1K). Login to comment
70 (A) Significantly reduced testis size and weight seen in heterozygous S489X mice compared with wild-type mice (n = 3; *:p,0.05). Login to comment
71 (B) Reduced daily sperm production (DSP) values retrieved from heterozygous S489X testes (n = 4; *:p,0.05). Login to comment
72 (C) Reduced sperm numbers recovered from the epididymis of heterozygous S489X mice (+/+ n = 6, +/2 n = 4; *:p,0.05). Login to comment
73 (D) H&E staining of the cross sections of wild2type, heterozygous and homozygous S489X CF mice testes. Login to comment
78 (F) Realtime PCR of protamine-2 in S489X CF mice testes. Login to comment
79 Protamine-2 mRNA level is significantly lowered in heterozygous and homozygous S489X CF mice compared to their wildtype littermates. Login to comment
80 (G) Immunofluorescent staining of Protamine-2 in +/+, +/2 and 2/2 S489X CF mice testes (stage VII-VIII). Login to comment
111 (H) Immunofluorescent staining of CREM in S489X CF mice testes (stage VIII-I). Login to comment
115 (I) Immunofluorescent staining of CREB in S489X CF mice testes shows stronger immunoactivity in +/+ and +/2 mice Sertoli cell compared to that of 2/2. Login to comment
117 (J) Western blot analysis of CREM expression in S489X CF testes. Login to comment
119 (K) Western blot analysis of CREB expression in S489X CF testes. Login to comment
126 Materials and Methods Animal and cell culture cftrtm1Unc (S489X) mice [45] were from Jackson`s laboratory and maintained in LASEC of CUHK. Login to comment

[hide] Mouse models of cystic fibrosis: phenotypic analys... J Cyst Fibros. 2011 Jun;10 Suppl 2:S152-71. Wilke M, Buijs-Offerman RM, Aarbiou J, Colledge WH, Sheppard DN, Touqui L, Bot A, Jorna H, de Jonge HR, Scholte BJ
Mouse models of cystic fibrosis: phenotypic analysis and research applications.
J Cyst Fibros. 2011 Jun;10 Suppl 2:S152-71., [PMID:21658634]

Abstract [show]
Genetically modified mice have been studied for more than fifteen years as models of cystic fibrosis (CF). The large amount of experimental data generated illuminates the complex multi-organ pathology of CF and raises new questions relevant to human disease. CF mice have also been used to test experimental therapies prior to clinical trials. This review recapitulates the major phenotypic traits of CF mice and highlights important new findings including aberrant alveolar macrophages, bone and cartilage abnormalities and abnormal bioactive lipid metabolism. Novel data are presented on the intestinal and nasal physiology of F508del-CFTR CF mice backcrossed onto different genetic backgrounds. Caveats, and sources of variability including age, gender and animal husbandry, are discussed. Interspecies differences limit comparison of lung pathology in CF mice to the human disease. The recent development of genetically modified pigs and ferrets heralds the application of more advanced animal models to CF research and drug development.

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67 Unfortunately, there is no Table 1 CFTR mutant mice Mouse Mutation Cftr mRNA Genetic Survival to Body wt Contact References background maturity Null mutations Cftrtm1Unc S489X Ex10 R Not detectable* C57Bl/6 <5% 10-25% reduction BH Koller/Jax Labs [113,158] Cftrtm1Cam R487X Ex10 R Not detectable 129S6/Sv/Ev <5% 20% reduction WH Colledge [159] Cftrtm1Hsc M1X Ex1 R Not detectable CD1 x 129 25% Delayed LC Tsui [160] Cftrtm3Bay Ex2 R Not detectable C57Bl/6 x 129 40% Reduced AT Beaudet [161] Cftrtm3Uth Y122X Ex4 R Not detectable C57Bl/6 25% 25-50% reduction M Capecchi/PB Davis [113,162] Hypomorphic mutations Cftrtm1Hgu ** Ex10 I 10% of wt MF1 x 129 90% No reduction J Dorin [113] Cftrtm1Bay Ex3 I <2% wt C57Bl/6 x 129 40% 70% reduction AL Beaudet [163] F508del mutations Cftrtm2Cam F508del R 30% of wt 129S6/Sv/Ev <5% 10-20% reduction WH Colledge [164] Cftrtm1Kth F508del R Low in intestine C57Bl/6 x 129 40% 10-50% reduction KR Thomas/Jax labs [165] Cftrtm1Eur F508del (H&R) Normal levels FVB/129; FVB 90% 10-20% reduction BJ Scholte [9] C57Bl/6 Other point mutations Cftrtm2Hgu G551D R 50% of wt CD1/129 65% 30-50% reduction J Dorin [11] Cftrtm3Hgu G480C (H&R) Normal levels C57Bl/6/129 Normal No reduction J Dorin [166] Cftrtm2Uth R117H R 5-20% of wt C57/Bl6 95% 10-25% reduction M Capecchi/PB Davis [113,162] Transgenes Mouse Transgene Promoter Expression Phenotype References Tg(FABPCFTR) CFTR Rat intestinal fatty acid Intestinal villus epithelia Rescue of CF intestinal pathology [167] binding protein Tg(CCSPScnn1b) Scnn1b Clara cell secretory Airway surface epithelia Na+ hyperabsorption [13] protein (CCSP) Reduced airway surface fluid volume Mucus accumulation, CF-like lung disease; 40% survival. Login to comment

[hide] Loss of CFTR Affects Biliary Epithelium Innate Imm... Gastroenterology. 2011 Oct;141(4):1498-1508.e5. Epub 2011 Jun 26. Fiorotto R, Scirpo R, Trauner M, Fabris L, Hoque R, Spirli C, Strazzabosco M
Loss of CFTR Affects Biliary Epithelium Innate Immunity and Causes TLR4-NF-kappaB-Mediated Inflammatory Response in Mice.
Gastroenterology. 2011 Oct;141(4):1498-1508.e5. Epub 2011 Jun 26., [PMID:21712022]

Abstract [show]
BACKGROUND & AIMS: Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins. METHODS: Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftr(tm1Unc) (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor kappaB (NF-kappaB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs. RESULTS: DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-kappaB. LPS-mediated activation of NF-kappaB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-kappaB. CONCLUSIONS: CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-kappaB. These findings might be used to develop therapies for CF-associated cholangiopathy.

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47 Congenic B6.129P2-Cftrtm1Unc mice, (harbouring a mutation, S489X, blocking CFTR transcription), and their wild type littermates were used as an accepted model for the human CF disease13 . Login to comment
25 Animals and Experimental Protocol Experiments were performed according to protocols approved by the Yale University Institutional Animal Care and Use Committee. Congenic B6.129P2-Cftrtm1Unc mice (harboring a mutation, S489X, blocking CFTR transcription) and their WT littermates were used as an accepted model for the human CF disease.13 Genotyping for each mouse and breeding were performed as described14,15 (see Supplementary Materials and Methods). Login to comment

[hide] The relationship of chronic mucin secretion to air... Am J Respir Cell Mol Biol. 1998 Dec;19(6):853-66. Cressman VL, Hicks EM, Funkhouser WK, Backlund DC, Koller BH
The relationship of chronic mucin secretion to airway disease in normal and CFTR-deficient mice.
Am J Respir Cell Mol Biol. 1998 Dec;19(6):853-66., [PMID:9843919]

Abstract [show]
In the cystic fibrosis (CF) patient, lung function decreases throughout life as a result of continuous cycles of infection, particularly with Pseudomonas aeruginosa and Staphylococcus aureus. The mechanism underlying the pathophysiology of the disease in humans has not been established. However, it has been suggested that abnormal, tenacious mucus, resulting perhaps from improper hydration from loss of Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) protein, impairs clearance of bacteria from the CF airway and provides an environment favorable to bacterial growth. If this hypothesis is correct, it could explain the absence of respiratory disease in CFTR-deficient mice, since mice have only a single submucosal gland and display few goblet cells in their lower airways, even when exposed to bacteria. To test this hypothesis further, we induced allergic airway disease in CFTR-deficient mice. We found that induction of allergic airway disease in mice, unlike bacterial infection, results in an inflammatory response characterized by goblet cell hyperplasia, increased mucin gene expression, and increased production of mucus. However, we also found that disease progression and resolution is identical in Cftr-/- mice and control animals. Furthermore, we show that the presence of mucus in the Cftr-/- airway does not lead to chronic airway disease, even upon direct inoculation with S. aureus and P. aeruginosa. Therefore, factors in addition to the absence of high levels of mucus secretion protect the mouse from the airway disease seen in human CF patients.

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18 We used this technique to create a mouse line (Cftrm1Unc ) in which the Cftr gene was inactivated by introduction of an in-frame stop codon (termed allele S489X) that resulted in the production of a truncated gene product similar to that seen in some human CF patients (4). Login to comment
115 Results Induction of Goblet Cell Hyperplasia in BALB/c, C57BL/6, DBA/2, and 129/SvEv Strains The genetic background on which the S489X mutation is carried consists of a mixture of the BALB/c, C57BL/6, DBA/2, and 129/SvEv mouse strains. Login to comment

[hide] CFTR is the primary known apical glutathione trans... Free Radic Biol Med. 2012 Apr 1;52(7):1201-6. Epub 2012 Jan 12. Gould NS, Min E, Martin RJ, Day BJ
CFTR is the primary known apical glutathione transporter involved in cigarette smoke-induced adaptive responses in the lung.
Free Radic Biol Med. 2012 Apr 1;52(7):1201-6. Epub 2012 Jan 12., [PMID:22266045]

Abstract [show]
One of the most abundant antioxidants in the lung is glutathione (GSH), a low-molecular-weight thiol, which functions to attenuate both oxidative stress and inflammation. GSH is concentrated in the epithelial lining fluid (ELF) of the lung and can be elevated in response to the increased oxidant burden from cigarette smoke (CS). However, the transporter(s) responsible for the increase in ELF GSH with cigarette smoke is not known. Three candidate apical GSH transporters in the lung are CFTR, BCRP, and MRP2, but their potential roles in ELF GSH transport in response to CS have not been investigated. In vitro, the inhibition of CFTR, BCRP, or MRP2 resulted in decreased GSH efflux in response to cigarette smoke extract. In vivo, mice deficient in CFTR, BCRP, or MRP2 were exposed to either air or acute CS. CFTR-deficient mice had reduced basal and CS-induced GSH in the ELF, whereas BCRP or MRP2 deficiency had no effect on ELF GSH basal or CS-exposed levels. Furthermore, BCRP or MRP2 deficiency had little effect on lung tissue GSH. These data indicate that CFTR is predominantly involved in maintaining basal ELF GSH and increasing ELF GSH in response to CS.

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39 Male CFTR KO mice that possess the S489X truncation mutation with gut-corrected recombinant human CFTR were obtained from our in-house colony as previously reported [6]. Login to comment

[hide] Lymphocyte CFTR promotes epithelial bicarbonate se... J Cell Physiol. 2012 Dec;227(12):3887-94. doi: 10.1002/jcp.24101. Tang XX, Fok KL, Chen H, Chan KS, Tsang LL, Rowlands DK, Zhang XH, Dong JD, Ruan YC, Jiang X, Yu SS, Chung YW, Chan HC
Lymphocyte CFTR promotes epithelial bicarbonate secretion for bacterial killing.
J Cell Physiol. 2012 Dec;227(12):3887-94. doi: 10.1002/jcp.24101., [PMID:22552906]

Abstract [show]
The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in lymphocytes has been reported for nearly two decades; however, its physiological role remains elusive. Here, we report that co-culture of lymphocytes with lung epithelial cell line, Calu-3, promotes epithelial HCO(3)- production/secretion with up-regulated expression of carbonic anhydrase 2 and 4 (CA-2, CA-4) and enhanced bacterial killing capability. The lymphocyte-enhanced epithelial HCO(3)- secretion and bacterial killing activity was abolished when Calu3 cells were co-cultured with lymphocytes from CFTR knockout mice, or significantly reduced by interfering with E-cadherin, a putative binding partner of CFTR. Bacterial lipopolysaccharide (LPS)-induced E-cadherin and CA-4 expression in the challenged lung was also found to be impaired in CFTR knockout mice compared to that of the wild-type. These results suggest that the interaction between lymphocytes and epithelial cells may induce a previously unsuspected innate host defense mechanism against bacterial infection by stimulating epithelial HCO(3)- production/secretion, which requires CFTR expression in lymphocytes.

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38 Materials and Methods Animals The Cftrtm1Unc mouse contains a mutant allele designated as S489X (Snouwaert et al., 1992). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2. Ooi CY, Durie PR
Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis.
J Cyst Fibros. 2012 Sep;11(5):355-62. doi: 10.1016/j.jcf.2012.05.001. Epub 2012 Jun 2., [PMID:22658665]

Abstract [show]
BACKGROUND: The pancreas is one of the primary organs affected by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. While exocrine pancreatic insufficiency is a well-recognized complication of cystic fibrosis (CF), symptomatic pancreatitis is often under-recognized. RESULTS: The aim of this review is to provide a general overview of CFTR mutation-associated pancreatitis, which affects patients with pancreatic sufficient CF, CFTR-related pancreatitis, and idiopathic pancreatitis. The current hypothesis regarding the role of CFTR dysfunction in the pathogenesis of pancreatitis, and concepts on genotype-phenotype correlations between CFTR and symptomatic pancreatitis will be reviewed. Symptomatic pancreatitis occurs in 20% of pancreatic sufficient CF patients. In order to evaluate genotype-phenotype correlations, the Pancreatic Insufficiency Prevalence (PIP) score was developed and validated to determine severity in a large number of CFTR mutations. Specific CFTR genotypes are significantly associated with pancreatitis. Patients who carry genotypes with mild phenotypic effects have a greater risk of developing pancreatitis than patients carrying genotypes with moderate-severe phenotypic consequences at any given time. CONCLUSIONS: The genotype-phenotype correlation in pancreatitis is unique compared to other organ manifestations but still consistent with the complex monogenic nature of CF. Paradoxically, genotypes associated with otherwise mild phenotypic effects have a greater risk for causing pancreatitis; compared with genotypes associated with moderate to severe disease phenotypes. Greater understanding into the underlying mechanisms of disease is much needed. The emergence of CFTR-assist therapies may potentially play a future role in the treatment of CFTR-mutation associated pancreatitis.

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855 CFTR mutation Total PI Total PI + PS PIP score CFTR mutation Total PI Total PI + PS PIP score 621+1G>T 96 96 1.00 G542X 74 75 0.99 711+1G>T 36 36 1.00 F508del 1276 1324 0.96 I507del 34 34 1.00 1717-1G>A 20 21 0.95 R553X 24 24 1.00 W1282X 19 20 0.95 Q493X 11 11 1.00 N1303K 45 48 0.94 S489X 11 11 1.00 R1162X 12 13 0.92 1154insTC 10 10 1.00 Y1092X 12 13 0.92 3659delC 9 9 1.00 I148T 10 11 0.91 CFTRdele2 7 7 1.00 V520F 9 10 0.90 4016insT 7 7 1.00 G551D 59 67 0.88 E60X 7 7 1.00 L1077P 5 6 0.83 R560T 7 7 1.00 R1066C 5 6 0.83 R1158X 7 7 1.00 2184insA 9 12 0.75 3905insT 6 6 1.00 2143delT 3 4 0.75 I148T;3199del6 5 5 1.00 1161delC 3 4 0.75 2183AA>G 5 5 1.00 3120+1G>A 3 4 0.75 1898+1G>A 5 5 1.00 S549N 3 4 0.75 2347delG 4 4 1.00 G85E 16 22 0.73 Q1313X 3 3 1.00 R117C 2 3 0.67 Q220X 3 3 1.00 M1101K 19 30 0.63 2184delA 3 3 1.00 P574H 3 5 0.60 1078delT 3 3 1.00 474del13BP 1 2 0.50 L1254X 3 3 1.00 R352Q 1 2 0.50 E585X 3 3 1.00 Q1291H 1 2 0.50 3876delA 2 2 1.00 A455E 18 37 0.49 S4X 2 2 1.00 R347P 6 15 0.40 R1070Q 2 2 1.00 2789+5G>A 6 16 0.38 F508C 2 2 1.00 L206W 6 18 0.33 DELI507 2 2 1.00 IVS8-5T 4 16 0.25 Q1411X 2 2 1.00 3272-26A>G 1 4 0.25 365-366insT 2 2 1.00 R334W 1 10 0.10 R709X 2 2 1.00 3849+10kbC>T 2 22 0.09 1138insG 2 2 1.00 P67L 1 14 0.07 CFTRdele2-4 2 2 1.00 R117H 1 25 0.04 3007delG 2 2 1.00 R347H 0 5 0.00 Q814X 2 2 1.00 G178R 0 3 0.00 394delTT 2 2 1.00 E116K 0 2 0.00 406-1G>A 2 2 1.00 875+1G>C 0 2 0.00 R75X 2 2 1.00 V232D 0 2 0.00 CFTRdel2-3 2 2 1.00 D579G 0 2 0.00 E193X 2 2 1.00 L1335P 0 2 0.00 185+1G>T 2 2 1.00 Mild mutations (based on PIP scores) are shaded in gray. Login to comment

[hide] Impaired CFTR-dependent amplification of FSH-stimu... J Clin Endocrinol Metab. 2012 Mar;97(3):923-32. Epub 2011 Dec 14. Chen H, Guo JH, Lu YC, Ding GL, Yu MK, Tsang LL, Fok KL, Liu XM, Zhang XH, Chung YW, Huang P, Huang H, Chan HC
Impaired CFTR-dependent amplification of FSH-stimulated estrogen production in cystic fibrosis and PCOS.
J Clin Endocrinol Metab. 2012 Mar;97(3):923-32. Epub 2011 Dec 14., [PMID:22170719]

Abstract [show]
CONTEXT: Estrogens play important roles in a wide range of physiological and pathological processes, and their biosynthesis is profoundly influenced by FSH that regulates the rate-limiting enzyme aromatase-converting estrogens from androgens. Abnormal estrogen levels are often seen in diseases such as ovarian disorders in polycystic ovarian syndrome (PCOS), an endocrine disorder affecting 5-10% of women of reproductive age, and cystic fibrosis (CF), a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). OBJECTIVES: We undertook the present study to investigate the mechanism underlying these ovarian disorders, which is not well understood. RESULTS: FSH-stimulated cAMP-responsive element binding protein phosphorylation, aromatase expression, and estradiol production are found to be enhanced by HCO3- and a HCO3- sensor, the soluble adenylyl cyclase, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/DeltaF508 mutant mice. CFTR expression is found positively correlated with aromatase expression in human granulosa cells, supporting its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in PCOS rodent models and human patients. CONCLUSIONS: CFTR regulates ovarian estrogen biosynthesis by amplifying the FSH-stimulated signal via the nuclear soluble adenylyl cyclase. The present findings suggest that defective CFTR-dependent regulation of estrogen production may underlie the ovarian disorders seen in CF and PCOS.

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19 In line with the observation in CF patients, CF mouse models with S489X (17) and DF508 mutation (18) in CFTR also display ovarian disorders with delayed puberty, reduced ovarian size and weight, decreased ovulated oocyte number, longer estrous cycle length, or even the absence of estrous cycle (19). Login to comment
23 Materials and Methods Animals Female ICR (Institute for Cancer Research) mice, cftrtm1Unc (S489X) mice (17), cftrtm1Kth (DF508) mice (18), and Sprague-Dawley (SD) rats were maintained in the Laboratory Animal Service Center, the Chinese University of Hong Kong. Login to comment
146 A, Immunofluorescent staining of CREB and phospho-CREB in wild-type (WT) and homozygous CFTR knockout (S489X) mice ovaries. Login to comment
183 The expression levels of CREB and phospho-CREB were reduced in the ovaries of homozygous CFTR knockout (S489X) mice compared with the wild-type control, as shown by immunofluorescent staining (Fig. 4A), suggesting that the cAMP-CREB signaling pathway is impaired in CFTR knockout mice. Login to comment

[hide] Abnormal expression of Muc5b in Cftr-null mice and... Histochem Cell Biol. 2011 Dec;136(6):699-708. Epub 2011 Oct 18. Valque H, Gouyer V, Husson MO, Gottrand F, Desseyn JL
Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice.
Histochem Cell Biol. 2011 Dec;136(6):699-708. Epub 2011 Oct 18., [PMID:22005837]

Abstract [show]
Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The "nonmammary" mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr( -/- ) mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr( -/- ) mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen's kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.

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53 The Cftr-knockout mice used are homozygous for the S489X-CFTR mutation (B6;129-Cftr tm1Unc) (Snouwaert et al. 1992). Login to comment
54 The S489X mouse third-generation backcross to C57BL/6 were maintained at the French Centre of Transgenesis, Archiving and Animal Models in Orléans (MF Bertrand, CDTA, Orléans, France) by random mating of heterozygous mice and were obtained with their wild-type littermates (Freedman et al. 1999) at 21-25 days of age. Cftr¡/¡ mice may die shortly after weaning because of intestinal obstruction, and both Cftr¡/¡ and Cftr+/+ mice received polyethylene glycol 4000 added to the drinking water (44.4 g/l) to increase the survival of the Cftr¡/¡ mice. Login to comment

[hide] Validation of high-resolution DNA melting analysis... J Mol Diagn. 2008 Sep;10(5):424-34. Epub 2008 Aug 7. Audrezet MP, Dabricot A, Le Marechal C, Ferec C
Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
J Mol Diagn. 2008 Sep;10(5):424-34. Epub 2008 Aug 7., [PMID:18687795]

Abstract [show]
High-resolution melting analysis of polymerase chain reaction products for mutation scanning, which began in the early 2000s, is based on monitoring of the fluorescence released during the melting of double-stranded DNA labeled with specifically developed saturation dye, such as LC-Green. We report here the validation of this method to scan 98% of the coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We designed 32 pairs of primers to amplify and analyze the 27 exons of the gene. Thanks to the addition of a small GC-clamp at the 5' ends of the primers, one single melting domain and one identical annealing temperature were obtained to co-amplify all of the fragments. A total of 307 DNA samples, extracted by the salt precipitation method, carrying 221 mutations and 21 polymorphisms, plus 20 control samples free from variations (confirmed by denaturing high-performance liquid chromatography analysis), was used. With the conditions described in this study, 100% of samples that carry heterozygous mutations and 60% of those with homozygous mutations were identified. The study of a cohort of 136 idiopathic chronic pancreatitis patients enabled us to prospectively evaluate this technique. Thus, high-resolution melting analysis is a robust and sensitive single-tube technique for screening mutations in a gene and promises to become the gold standard over denaturing high-performance liquid chromatography, particularly for highly mutated genes such as CFTR, and appears suitable for use in reference diagnostic laboratories.

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97 Differences in melting plots obtained for a heterozygous F508del and two compounds heterozygous (F508del/S492F and F508del/S489X). Login to comment

[hide] Regulation of Ca(2+)-activated chloride channels b... Biochem Biophys Res Commun. 2004 Apr 9;316(3):612-7. Perez-Cornejo P, Arreola J
Regulation of Ca(2+)-activated chloride channels by cAMP and CFTR in parotid acinar cells.
Biochem Biophys Res Commun. 2004 Apr 9;316(3):612-7., [PMID:15033444]

Abstract [show]
The effect of intracellular cAMP and cystic fibrosis conductance regulator (CFTR) protein on the calcium-activated chloride current (ICaCl) present in parotid acinar cells was studied using the patch clamp technique. Application of 1 mM of 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP), a permeable analog of cAMP, inhibited ICaCl only at positive potentials. This inhibition was partially abolished in cells dialyzed with 20 nM PKI 6-22 amide, a potent peptide that specifically inhibits PKA. Because cAMP is an activator of the CFTR Cl- channel, a known regulator of ICaCl, we also investigated if the inhibition of ICaCl was mediated by activation of CFTR. To test this idea, we added 1 mM CPT-cAMP to acinar cells isolated from knockout animals that do not express the CFTR channel. In these cells the cAMP effect was totally abolished. Thus, our data provide evidence that cAMP regulates ICaCl by a dual mechanism involving PKA and CFTR.

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109 To directly test this idea, we used a CFTR knockout model mice that express the S489X mutation, which results in a nonfunctional truncated channel protein [24]. Login to comment
108 To directly test this idea, we used a CFTR knockout model mice that express the S489X mutation, which results in a nonfunctional truncated channel protein [24]. Login to comment

[hide] Spectrum of CFTR mutations in cystic fibrosis and ... Hum Mutat. 2000;16(2):143-56. Claustres M, Guittard C, Bozon D, Chevalier F, Verlingue C, Ferec C, Girodon E, Cazeneuve C, Bienvenu T, Lalau G, Dumur V, Feldmann D, Bieth E, Blayau M, Clavel C, Creveaux I, Malinge MC, Monnier N, Malzac P, Mittre H, Chomel JC, Bonnefont JP, Iron A, Chery M, Georges MD
Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.
Hum Mutat. 2000;16(2):143-56., [PMID:10923036]

Abstract [show]
We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

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105 d G149R, S489X, S492F, S549R, 1898+1G>A, 2622+1G>A, G970R, R1066H, W1204X, 3850-1G>A, Q1313X. Login to comment

[hide] Modification of development by the CFTR gene in ut... Mol Genet Metab. 1998 Nov;65(3):203-12. Morrow SL, Larson JE, Nelson S, Sekhon HS, Ren T, Cohen JC
Modification of development by the CFTR gene in utero.
Mol Genet Metab. 1998 Nov;65(3):203-12., [PMID:9851885]

Abstract [show]
The in utero infection of rats at 16-17 days gestation with a recombinant adenovirus carrying the human cystic fibrosis transmembrane conductance regulator (cftr) gene resulted in altered lung development and morphology. These structural alterations prompted an evaluation of concurrent functional changes in the cftr-treated lung. CFTR protein could be detected in treated lungs for up to 30 days postinfection, although it was not detected in the intestines at this time. Increased levels of secreted glycoconjugates and lipids were found in lungs treated in utero with human cftr and large vacuoles containing glycoconjugates were detected within cells of the intestines. The scope and durability of these changes suggested that in utero cftr treatment influenced the activity of secretory cells in the developing lung. Altered secretory products in the lungs of cystic fibrosis patients are thought to be associated with increased susceptibility to Pseudomonas aeruginosa infection. We challenged 3-month-old rats (treated in utero with the human cftr gene) with a lethal, intratrachial dose of this bacteria. Rats treated with cftr exhibited enhanced resistance to Pseudomonas infection when compared to controls. These animals displayed little or no associated inflammatory response. No evidence of the adenovirus transgene was detectable at the time of P. aeruginosa inoculation, indicating that continuous ectopic expression of hcftr was not required for enhanced protection. These data demonstrate that in utero, cftr expression influenced the development and function of cells involved in the primary host defense against bacterial infection in the lung.

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22 A recombinant adenovirus containing human cftr (Av1CF2 [10]) was used for in utero gene transfer into the S489X cftr knockout mouse (C57Bl/ 1 To whom correspondence should be addressed at Ochsner Medical Foundation, Department of Molecular Genetics, 1516 Jefferson Highway, New Orleans, LA 70121. Login to comment
29 S489X cftr (-/-) mice develop a lethal intestinal obstruction resulting in less than 5% survival to adulthood (Ͼ40 days) (11). Login to comment
28 S489X cftr (2/2) mice develop a lethal intestinal obstruction resulting in less than 5% survival to adulthood (.40 days) (11). Login to comment

[hide] In-frame elimination of exon 10 in Cftrtm1Unc CF m... Gene. 1998 Apr 28;211(1):117-23. Xu Z, Gupta V, Lei D, Holmes A, Carlson E, Gruenert DC
In-frame elimination of exon 10 in Cftrtm1Unc CF mice.
Gene. 1998 Apr 28;211(1):117-23., [PMID:9573345]

Abstract [show]
Cystic fibrosis (CF) is a severe autosomal-linked inherited disease in humans. Transgenic CF animals play a crucial role in the study of molecular mechanisms underlying disease pathology. In the present study, CFTR mRNA expression was examined in different tissues from one CF mouse model that contains a disruption in exon 10 sequence of the CF transmembrane conductance regulator (CFTR) gene. Multiple tissue samples were collected from new-born normal (+/+), homozygous (-/-), and heterozygous (+/-) mice and compared for their CFTR mRNA expression. Total RNA samples were prepared from eight different tissues (nasal mucosa, trachea, lung, colon, intestine, pancreas, liver, gonads, and brain) and then analyzed by reverse transcription polymerase chain reaction (RT-PCR) amplification. A 329-bp fragment comprising exon 9 through exon 11 of the CFTR gene was amplified from all tissues of the normal mouse. In contrast, a 137-bp fragment was observed in tissue samples from homozygous CF mice. Both the 329-bp and 137-bp fragments were detected in samples from heterozygous mice. Direct sequencing analysis of the amplified fragments showed an exon 9-exon 11 splice junction, indicating that the entire exon 10 sequence was eliminated from homozygous CF mice. RT-PCR analysis of the 3' end of CFTR mRNA showed the presence of a 682-bp, exon 20-24 fragment in -/- and +/- mice. These results demonstrate that an alternately spliced CFTR mRNA is produced in this CF 'knock-out' mouse. A semi-quantitative comparison of the wild-type and exon 10 minus CFTR (CFTR-E10) mRNA in heterozygote animals indicated that less (but a detectable amount) mutant CFTR mRNA was present in all organs tested. There was, however, a significant reduction of CFTR-E10 mRNA in the liver and the pancreas. Since the deletion of exon 10 is in-frame, the significance of the CFTR-E10 mRNA in terms of CFTR protein and function requires further analysis.

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45 Tissue harvest pathway in mouse epithelial cells that may compensate for the alteration in CFTR (Clarke et al., 1992a,b; Alton The Cftrtm1Unc mouse contains a mutant allele designated as S489X (Snouwaert et al., 1992). Login to comment
46 Tissue harvest pathway in mouse epithelial cells that may compensate for the alteration in CFTR (Clarke et al., 1992a,b; Alton The Cftrtm1Unc mouse contains a mutant allele designated as S489X (Snouwaert et al., 1992). Login to comment

[hide] Is meconium ileus genetically determined or associ... J Med Genet. 1998 Mar;35(3):262-3. De Braekeleer M, Allard C, Leblanc JP, Aubin G, Simard F
Is meconium ileus genetically determined or associated with a more severe evolution of cystic fibrosis?
J Med Genet. 1998 Mar;35(3):262-3., [PMID:9541118]

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16 Although the A455E mutation is Table 1 Distribution of meconium ileus among CFTR genotypes in Saguenay Lac-Saint-Jean No of CF Proportion No of CFpatients Proportion Proportion ofMI Genotypes patients (%) with meconium ileus (%) among genotypes AF508/AF508 52 39.4 5 26.3 9.6 AF508/621+G-*T 34 25.8 9 47.4 26.5 AF508/A455E 14 10.6 0 0.0 0.0 621+1G-*T/A455E 8 6.1 0 0.0 0.0 621+1G-*T/G85E 2 1.5 1 5.3 50.0 621+1G-*T/Y1092X 1 0.8 0 0.0 0.0 AF508/Y1092X 4 3.0 1 5.3 25.0 A455E/R117C 1 0.8 0 0.0 0.0 AF508/I148T 2 1.5 0 0.0 0.0 621+1G-*T/ 4 3.0 0 0.0 0.0 711 +1G-*T 621+1G-4T/S489X 1 0.8 0 0.0 0.0 AF508/Q890X 1 0.8 1 5.3 100.0 621+1G->T/ 6 4.5 2 10.5 33.3 621+1G-sT AF508/unknown 1 0.8 1 5.3 100.0 Unknown/unknown 1 0.8 0 0.0 0.0 Table 2 Main clinicalfindings in patients with meconium ileus With MI Without MI p value No of patients 18 18 Sex (M/IF) 6/12 6/12 No of patients alive 16 17 Mean age (SD) 16.75 (9.7) 16.70 (7.9) p=0.99 Mean birth weight (SD) 3.24 (0.40) 3.02 (0.47) p=O.18 Mean birth height (SD) 50.0 (2.27) 50.0 (2.58) p=0.86 Currentweightcentile (SD) 26.7 (24.5) 14.1 (18.0) p=0.06 Current height centile (SD) 29.9 (25.1) 20.6 (25.6) p=0.33 Sweat chloride concentration (mEq/l) 105.9 (6.5) 101.1 (9.8) p=O.12 Mean FVC (SD) 89.7 (24.4) 93.0 (17.0) p=0.75 Mean FEV (SD) 73.1 (23.9) 75.4 (18.7) p=0.81 Mean Shwachman score (SD) 82.8 (11.8) 79.2 (12.6) p=0.36 Colonisation with Pseudomonas aeruginosa 13 14 p=0.70 Staphyloccoccus aureus 16 17 p=0.55 Haemophilus influenzae 13 14 p=0.70 Pseudomonas maltophilia 4 6 p=0.46 Pseudomonas cepacia 0 1 Pancreatic insufficiency 18 18 DIOS 7 1 p=0.016 Rectal prolapse 1 2 p=0.55 Recurrent abdominal pain 6 1 p=0.035 Diabetes mellitus 5 0 p=0.016 Liver complications 3* 0 p=0.07 Nasal polyposis 6 6 p=1.00 DIOS=distal intestinal obstruction syndrome. Login to comment

[hide] Studies of the role for NSP4 in the pathogenesis o... J Infect Dis. 1998 Feb;177(2):455-8. Angel J, Tang B, Feng N, Greenberg HB, Bass D
Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea.
J Infect Dis. 1998 Feb;177(2):455-8., [PMID:9466536]

Abstract [show]
A rotavirus (RV) nonstructural protein, NSP4, has recently been proposed to function as an enterotoxin in the pathogenesis of RV diarrhea. The role of NSP4 in the pathogenesis of RV diarrhea was examined by infecting cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice with virulent murine RV and by comparing deduced amino acid sequences of RV gene 10 encoding NSP4 from three distinct sets of virulent and tissue culture-adapted avirulent variant RVs. Homozygous CFTR (CFTR-/-) mice, which do not respond to any known intestinal secretagogues, experienced diarrhea comparable to that in normal CFTR+/+ littermates after RV challenge. Comparison of amino acid sequences of NSP4 from virulent and attenuated pairs of RVs failed to show consistent or significant changes. Together, these data suggest that enterotoxigenic properties of RV NSP4 are not critical in the pathogenesis of murine RV diarrhea and that attenuation of murine RVs is not usually mediated by mutations in the gene encoding NSP4.

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14 They were produced by introducing an in-frame stop to be capable of inducing a calcium-mediated increase in apical codon in the coding sequence of exon 10 at aa 489 (S489X) [7]. Login to comment
15 They were produced by introducing an in-frame stop to be capable of inducing a calcium-mediated increase in apical codon in the coding sequence of exon 10 at aa 489 (S489X) [7]. Login to comment

[hide] Gene therapy for cystic fibrosis. Lancet. 1997 Apr 26;349(9060):1249-50. Alton E, Smith S, Geddes D
Gene therapy for cystic fibrosis.
Lancet. 1997 Apr 26;349(9060):1249-50., [PMID:9130964]

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12 2 Eckman EA, Cotton CU, Kube DM, Davis PB. Dietary changes improve survival of Cftr S489X homozygous mutant mouse. Login to comment
13 Dietary changes improve survival of Cftr S489X homozygous mutant mouse. Login to comment

[hide] SSCP analysis: a blind sensitivity trial. Hum Mutat. 1997;10(1):65-70. Jordanova A, Kalaydjieva L, Savov A, Claustres M, Schwarz M, Estivill X, Angelicheva D, Haworth A, Casals T, Kremensky I
SSCP analysis: a blind sensitivity trial.
Hum Mutat. 1997;10(1):65-70., [PMID:9222762]

Abstract [show]
Studies of the sensitivity of SSCP analysis usually have been performed under conditions contrary to the rules of quality control trials and have produced widely different results. We have performed a blind trial of the sensitivity of SSCP analysis for the detection of mutations in fragments up to 500 bp in length under a fixed single set of electrophoretic conditions. The mutation detection rate was 84%. In addition, we have identified a second mutation in nine samples. All these mutations are polymorphisms, including a novel polymorphism 1248 + 52T/C first reported in the present work.

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22 List of Mutations Included in the Experiment and Original Method of Detection Used by the Referring Laboratory Referring Probe Original method laboratory no.a Mutation Exon of detection Original SSCP conditions Institut de 1 1677delTA 10 Heteroduplexes Recerca 1 1859G/C 12 DDGE Oncologica, 3 W1282X 20 SSCPb 6% 19:1 (AA:bisAA) 4°C 5h 30W Department 4 delF508 10 Heteroduplexes de Genetica 4 Q1313X 20 SSCPb 6% 19:1 (AA:bisAA) 4°C 5h 30W Molecular, 5 1609delCA 10 SSCPb 6% 19:1 (AA:bisAA) RT 28h 10W10% glycerol Barcelona, 7 T582R 12 DGGE Spain 8 1898+3G→A ivs 12 DGGE Molecular 910085 1161delC 7 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Genetics 860176 1138insG 7 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Laboratory, 930215 1154insTC 7 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Royal 930838 delF508 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Manchester 930127 delI507 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Children`s 931205 Q493X 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm Hospital, 900592 V520F 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm UK G12984 S489X 10 SSCP/Heteroduplexes 9% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 910143 G551D 11 ARMS 930274 S549N 11 SSCP/Heteroduplexes 10% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 920132 1811+1G→C ivs 11 SSCP/Heteroduplexes 10% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 930140 1898+1G→A ivs 12 SSCP/Heteroduplexes 930334 W1282X 20 SSCP/Heteroduplexes 7.25% 49:1 (AA:bisAA) 4°C 20 h 10V/cm 140735 3850-1G→A 20 SSCP/Heteroduplexes 7.25% 49:1 (AA:bisAA) 4°C 20 h 10 V/cm Laboratoire 293 G551D 11 SSCPb 5% 19:1 (AA:bisAA) 4°C 5 h 50W and de Biochimie 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol Genetique, 324 S549R 11 ASO Hybridization Centre 649 1898+1G→A ivs 12 DGGE Hospitalier 583 E585X 12 DGGE Universitaire 710 L967S 15 DGGE Montpellier, 325 S945L 15 SSCPb 5% 19:1 (AA:bisAA) 4° 5h 50W and France 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 473 N1303H 21 SSCPb 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 216 300delA 3 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 287 394delTT 3 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 559 R74W 3 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 237 P67L 3 DGGE 1023 R75X 3 DGGE 885 1215delG 7 DGGE 113 Y122X 4 DGGE, SSCP 356 621+1G→T ivs 4 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 709 621+2T→G ivs 4 SSCP 5% 19:1 (AA:bisAA)4°C 5h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol 802 I148T 4 DGGE 1016 Q98R 4 DGGE V75 R117H 4 SSCP 5% 19:1 (AA:bisAA) 4°C 5 h 50W and 5% 19:1 (AA:bisAA) RT 18h 8W 10%glycerol a Identification numbers given by referring laboratories. Login to comment
57 Type of Mutations Detected by SSCP Analysis in This Study Type of mutation Mutation Mutation characteristics Detected by SSCP analysis Deletions 1677delTA deletion of TA from 1677 Yes delF508 deletion of 3 bp from 1655 Yes delI507 deletion of 3 bp from 1648 Yes 1609delCA deletion of CA from 1609 Yes 1161delC deletion of C at 1161 Yes 300delA deletion of A at 300 Yes 394delTT deletion of TT from 394 Yes 1215delG deletion of G at 1215 No Insertions 1138insG insertion of G after 1138 Yes 1154insTC insertion of TC after 1154 Yes Base 1859G/C Yes substitutions W1282X G→A at 3978 Yes Q1313X C→T at 4069 Yes T582R C→G at 1877 Yes 1898+3G→A A→G at 1898+3 Yes Q493X C→T at 1609 Yes V520F G→T at 1690 Yes S489X C→A at 1598 Yes G551D G→A at 1784 No S549N G→A at 1778 Yes 1811+1G→C G→C at 1811+1 Yese 1898+1G→A G→A at 1898 Yes 3850-1G→A G→A at 3850-1 Yes S549R T→G at 1779 Yes E585X G→T at 1885 Yes L967S C→T at 2966 Yes S945L C→T at 2966 No N1303H A→C at 4039 Yes R74W C→T at 352 Yes P67L C→T at 332 Yes R75X C→T at 355 Yes Y122X T→A at 498 No 621+1G→T G→T at 621+1 No 621+2T→G T→G at 621+2 No I148T T→C at 575 Yes Q98R A→G at 425 Yes R117H G→A at 482 Yes FIGURE 1. Login to comment

[hide] Dietary changes improve survival of CFTR S489X hom... Am J Physiol. 1995 Nov;269(5 Pt 1):L625-30. Eckman EA, Cotton CU, Kube DM, Davis PB
Dietary changes improve survival of CFTR S489X homozygous mutant mouse.
Am J Physiol. 1995 Nov;269(5 Pt 1):L625-30., [PMID:7491981]

Abstract [show]
Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Because the period of highest mortality is during the week after weaning, we hypothesized that providing a low-residue liquid diet would improve survival and growth. When 99 CF mice that survived to 10 days of age were fed Peptamen (Clintec Nutrition), an elemental liquid diet, and housed on corn-cob bedding, 88% of them survived to maturity (50 days). The diet causes only minor histologic and ion transport changes in the intestines of normal mice and does not reduce growth rate or size. CF mice raised on Peptamen continue to display severe pathological changes in the intestine and completely lack a adenosine 3',5'-cyclic monophosphate-inducible chloride current in the cecum. This combination of dietary and bedding changes provides a reliable method for keeping CF mice alive well into adulthood and will be useful for the evaluation of the effect and duration of potential therapies for CF.

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0 269:L625-L630, 1995. ;Am J Physiol Lung Cell Mol Physiol E. A. Eckman, C. U. Cotton, D. M. Kube and P. B. Davis homozygous mutant mouse Dietary changes improve survival of CFTR S489X You might find this additional info useful... 25 other HighWire-hosted articles:This article has been cited by http://ajplung.physiology.org/content/269/5/L625#cited-by including high resolution figures, can be found at:Updated information and services http://ajplung.physiology.org/content/269/5/L625.full can be found at:Molecular Physiology American Journal of Physiology - Lung Cellular andaboutAdditional material and information http://www.the-aps.org/publications/ajplung This information is current as of October 25, 2012. Login to comment
6 It is published 12 times a year (monthly) by the American covering the broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of publishes original researchAmerican Journal of Physiology - Lung Cellular and Molecular Physiology Dietary changes improve survival of CFTR S489X homozygous mutant mouse ELIZABETH A. ECKMAN, CALVIN U. COTTON, DIANNE M. KUBE, AND PAMELA B. DAVIS Departments of Pediatrics, Molecular Biology and Microbiology, and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106 E&man, Elizabeth A., Calvin U. Cotton, Dianne M. Kube, and Pamela B. Davis. Login to comment
7 Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment
12 13): L625-L630,1995.- Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Login to comment
24 Snouwaert et al. [lo; University of North Carolina (UNC)] and Ratcliff et al. (7; University of Cambridge) designed targeting constructs to replace a portion of the CFTR gene with two copies of the neomycin resistance gene (S489X mutation) or a hypoxanthine phosphoribosyl transferase (HPRT) mini-gene, respectively. Login to comment
40 When S489X CFTR (-/-) mice are fed Peptamen complete elemental liquid diet and bedded on corn cob pellets instead of the traditional wood shavings, 88% of these animals which are alive at 10 days of age survive to maturity. Login to comment
43 UNC mice heterozygous for the S489X mutation (CFTR + /-> were purchased from the Jackson Laboratories 1040-0605/95 $3.00 Copyright o 1995 the American Physiological Society L625 (Bar Harbor, ME). Login to comment
54 Primers III and IV were used to amplify the S489X neo-disrupted allele. Login to comment
57 Both wild-type CFTR and S489X alleles were amplified in a single reaction. Login to comment
60 Expected product sizes were N 500 base pairs (bp) for the wild-type allele (primers I and II) and 650 bp for the S489X allele (primers III and IV). Login to comment
129 DISCUSSION The UNC S489X homozygous CF mice have a severe phenotype resulting from complete inactivation of the murine CFTR gene. Login to comment
148 Indeed, a few of the S489X homozygous mice placed on this regimen actually succumbed to intestinal obstruction, so it is possible that the survivors suffered intermittent bouts of this complication and consequently were runts. Login to comment
151 S489X CF knockout mice transgenic for human CFTR under control of the rat intestinal fatty acid-binding gene promoter express CFTR predominantly, but not exclusively, in the intestine and are protected against death from intestinal obstruction even without special diet or Forskolin JI + CFTR (+/&) Solid Food -O- CFTR (+/&) Peptamen +I+ CFTR (4) Peptamen -6 4 -2 0 2 4 6 8 10 Time (min) Fig. 4. Login to comment

[hide] Relationship of a non-cystic fibrosis transmembran... Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):479-83. Clarke LL, Grubb BR, Yankaskas JR, Cotton CU, McKenzie A, Boucher RC
Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice.
Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):479-83., [PMID:7507247]

Abstract [show]
Although loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- channel function is common to all epithelia in cystic fibrosis (CF) patients, the severity of disease varies in different organs. We hypothesized that differences in disease severity in CF relate to the expression of an "alternative" plasma membrane Cl- conductance. In CF mice [Cftr(-/-); mice homozygous for Ser-489 to Xaa mutation], which do not express cAMP CFTR-mediated Cl- secretion, we surveyed organs that exhibit a range of disease severity for a Ca(2+)-mediated apical membrane epithelial Cl- conductance. This alternative conductance (Cl-a) was detected in epithelia of organs from CF mice that exhibit a mild disease phenotype (airway, pancreas) but not in epithelia with a severe phenotype (small, large intestine). We conclude that (i) there is an intracellular Ca(2+)-regulated Cl- conductance that is molecularly distinct from CFTR; and (ii) the level of expression of this alternative Cl- conductance in the epithelium is an important determinant of the severity of organ-level disease in CF.

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18 Early in life, Cftr(-/-) mice [mice homozygous for the Ser-489 to Xaa (S489X) mutation] exhibit severe intestinal disease and little pulmonary disease, which resembles the progression in these organs in CF patients, including those with chain-termination mutations similar to Cftr(-/-) mice (13). Login to comment

[hide] Glycoconjugate metabolism in a cystic fibrosis kno... Mol Genet Metab. 2001 Feb;72(2):122-31. Mailleau C, Paul A, Colin M, Xing PX, Guernier C, Bernaudin JF, Capeau J, Brahimi-Horn MC
Glycoconjugate metabolism in a cystic fibrosis knockout mouse model.
Mol Genet Metab. 2001 Feb;72(2):122-31., [PMID:11161838]

Abstract [show]
Cystic fibrosis knockout mice (cftr(-/-)) die prematurely of obstruction of the intestine which may result from accumulation of dehydrated glycoconjugate-containing mucus. We noted an increase in the specific activity of [(14)C]glucosamine-labeled high-molecular weight glycoconjugates, probably mucin, in the lumen of the intestine of cftr(-/-) (homozygous) mice compared to cftr(+/+) (wild-type) and cftr(+/-) (heterozygous) mice and a decrease in the turnover of glycoconjugates of several organs of the cftr(-/-) mice. No difference in the anionic composition of secreted intestinal glycoconjugates was detected and no difference in the amount of mucin 1 (Muc1) was found in the small intestine, colon, pancreas, and lungs of the different genotypes. In addition, the spleen of the cftr(-/-) mice was significantly smaller than that of control mice and the small intestine and colon were, respectively, longer and shorter compared to control mice. These results indicate modified glycoconjugate metabolism in cystic fibrosis knockout mice and morphologic changes to the spleen and intestine where the latter modifications are possibly related to the intestinal malabsorption associated with cystic fibrosis.

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22 The cftr gene was inactivated by introduction of an in-frame stop codon (the allele S489X) resulting in a truncated gene product. Login to comment
34 Female and male 129/BC mice heterozygous for the S489X mutation were crossed to obtain homozygous S489X mice (12). Login to comment
42 For the purposes of this study wild-type mice were classified as cftraf9;/af9; , mice heterozygous for the S489X mutation as cftraf9;/afa; , and mice homozygous for the S489X mutation as cftrafa;/afa; . Login to comment

[hide] Ursodeoxycholic acid stimulates cholangiocyte flui... Gastroenterology. 2007 Nov;133(5):1603-13. Epub 2007 Sep 2. Fiorotto R, Spirli C, Fabris L, Cadamuro M, Okolicsanyi L, Strazzabosco M
Ursodeoxycholic acid stimulates cholangiocyte fluid secretion in mice via CFTR-dependent ATP secretion.
Gastroenterology. 2007 Nov;133(5):1603-13. Epub 2007 Sep 2., [PMID:17983806]

Abstract [show]
BACKGROUND & AIMS: Cholangiopathies are characterized by impaired cholangiocyte secretion. Ursodeoxycholic acid (UDCA) is widely used for cholangiopathy treatment, but its effects on cholangiocyte secretory functions remain unclear and are the subject of this study. METHODS: Polarized mouse cholangiocytes in tubular (isolated bile-duct units [IBDU]) or monolayer configuration were obtained from wild-type (WT) and B6-129-Cftr(tm1Kth) and Cftr(tm1Unc) mice that are defective in CFTR, an adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl(-) channel expressed in cholangiocytes. Fluid secretion was assessed by video-optical planimetry, Cl(-) and Ca(2+) efflux by microfluorimetry (6-methoxy-N-ethylquinolinium chloride, fura-2, and fluo-4), adenosine triphosphate (ATP) secretion by luciferin-luciferase assay, and protein kinase C (PKC) by Western blot. RESULTS: UDCA stimulated fluid secretion and Cl(-) efflux in WT-IBDU but not in CFTR-KO-IBDU or in WT-IBDU exposed to CFTR inhibitors. UDCA did not affect intracellular cAMP levels but increased [Ca(2+)]i in WT and not in CFTR-KO cholangiocytes. UDCA stimulated apical ATP secretion in WT but not in CFTR-KO cholangiocytes. UDCA-stimulated [Ca(2+)]i increase was inhibited by suramin, a purinergic 2Y-receptor inhibitor. UDCA stimulated the translocation of PKC-alpha and PKC-epsilon to the plasma membrane. UDCA-stimulated secretion was inhibited by 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and by phospholipase C and PKC inhibitors. UDCA increased ATP output in isolated perfused livers from WT but not from CFTR-KO mice. CONCLUSIONS: Our data indicate that UDCA stimulates a CFTR-dependent apical ATP release in cholangiocytes. Secreted ATP activates purinergic 2Y receptors, and, through [Ca(2+)]i increase and PKC activation stimulates Cl(-) efflux and fluid secretion. These data support the concept that CFTR plays a role in modulating purinergic signaling in secretory epithelia and suggest a novel mechanism explaining the choleretic effect of UDCA.

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24 The composition (in mmol/L) of perfusion buffers used was essentially as described.3,4,13 Experimental Animals Congenic B6.129P2-Cftrtm1Unc mice, which possess the S489X mutation that blocks transcription of CFTR,14 and èc;F508-CFTR (B6-129-Cftrtm1Kth) mice with targeted mutation corresponding to the èc;F508 mutation in human, were used as described.15 For further information, see the supplementary online material (see Supplementary Material online at www.gastrojournal.org). Login to comment

[hide] CFTR mutations impart elevated immune reactivity i... Cytokine. 2008 Oct;44(1):154-9. doi: 10.1016/j.cyto.2008.07.468. Epub 2008 Sep 7. Stalvey MS, Brusko TM, Mueller C, Wasserfall CH, Schatz DA, Atkinson MA, Flotte TR
CFTR mutations impart elevated immune reactivity in a murine model of cystic fibrosis related diabetes.
Cytokine. 2008 Oct;44(1):154-9. doi: 10.1016/j.cyto.2008.07.468. Epub 2008 Sep 7., [PMID:18778952]

Abstract [show]
Increased life expectancy in cystic fibrosis (CF) is accompanied by an increasing incidence of CF related diabetes (CFRD). Altered immune reactivity occurs in CF, which we hypothesize, is exacerbated by hyperglycemia. Cystic fibrosis transmembrane conductance regulator deficient (CFTR-/-) mice were rendered hyperglycemic by streptozotocin (STZ) to test this hypothesis. CFTR-/-, C57BL/6J, and FVB/NJ mice received either STZ or lactated ringers (LR) (n=5-10). Four weeks later, splenocytes were harvested, mitogen stimulated, and analyzed for cytokine production (IL-2, IL-4, and IL-10) along with stimulation indices (SI). SI of STZ-treated CFTR-/- were elevated compared to LR-treated mice, although both were greater than C57BL/6J and FVB/NJ (p<0.05). Fasting glucose levels of STZ-treated CFTR-/- mice correlated with SI (p<0.003). Stimulated IL-10 concentrations were elevated in STZ-treated CFTR-/- compared to LR-treated animals and controls (p<0.05). IL-2 levels were greater in CFTR-/- mice compared to controls (p<0.05), but unrelated to STZ. Reinforcing generalized cytokine up-regulation in CFTR-/-, IL-4 levels were greater in CFTR-/- mice compared to C57BL/6J, but FVB/NJ mice demonstrated greatest concentrations following STZ. These results suggest that, hyperglycemia may exacerbate the clinical course in CF by impacting immune reactivity. There is clear need to maximize metabolic management in CFRD.

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64 Mouse strains Our experiments utilized a mixed background mouse model, originating from C57BL/6J mice with the CFTR S489X / neo insertion, as developed initially at the University of North Carolina [28]. Login to comment
69 CFTR S489X / FABP-hCFTR+/+ (hereafter, designated CFTR / ), C57BL/6J and FVB/NJ mice were housed under SPF conditions at the University of Florida Animal Care Services according to NIH guidelines, with allowances of food and water ad libitum. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Gut Microbes. 2013 Jan-Feb;4(1):41-7. doi: 10.4161/gmic.22430. Epub 2012 Oct 12. Lynch SV, Goldfarb KC, Wild YK, Kong W, De Lisle RC, Brodie EL
Cystic fibrosis transmembrane conductance regulator knockout mice exhibit aberrant gastrointestinal microbiota.
Gut Microbes. 2013 Jan-Feb;4(1):41-7. doi: 10.4161/gmic.22430. Epub 2012 Oct 12., [PMID:23060053]

Abstract [show]
The composition of the gastrointestinal microbiome is increasingly recognized as a crucial contributor to immune and metabolic homeostasis-deficiencies in which are characteristic of cystic fibrosis (CF) patients. The murine model (CFTR (-/-) , CF), has, in previous studies, demonstrated characteristic CF gastrointestinal (GI) manifestations including slowed transit and significant upregulation of genes associated with inflammation. To determine if characteristics of the microbiome are associated with these phenotypes we used a phylogenetic microarray to compare small intestine bacterial communities of wild type and congenic CF mice. Loss of functional CFTR is associated with significant decreases in GI bacterial community richness, evenness and diversity and reduced relative abundance of putative protective species such as Acinetobacter lwoffii and a multitude of Lactobacilliales members. CF mice exhibited significant enrichment of Mycobacteria species and Bacteroides fragilis, previously associated with GI infection and immunomodulation. Antibiotic administration to WT and CF animals resulted in convergence of their microbiome composition and significant increases in community diversity in CF mice. These communities were characterized by enrichment of members of the Lactobacillaceae and Bifidobacteriaceae and reduced abundance of Enterobacteriaceae and Clostridiaceae. These data suggest that Enterobacteria and Clostridia species, long associated with small intestinal overgrowth and inflammatory bowel disease, may suppress both ileal bacterial diversity and the particular species which maintain motility and immune homeostasis in this niche. Thus, these data provide the first indications that GI bacterial colonization is strongly impacted by the loss of functional CFTR and opens up avenues for alternative therapeutic approaches to improve CF disease management.

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237 Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

[hide] Ventilatory pattern and energy expenditure are alt... J Cyst Fibros. 2013 Jul;12(4):345-51. doi: 10.1016/j.jcf.2012.11.008. Epub 2013 Jan 3. Darrah RJ, Bederman IR, Mitchell AL, Hodges CA, Campanaro CK, Drumm ML, Jacono FJ
Ventilatory pattern and energy expenditure are altered in cystic fibrosis mice.
J Cyst Fibros. 2013 Jul;12(4):345-51. doi: 10.1016/j.jcf.2012.11.008. Epub 2013 Jan 3., [PMID:23290341]

Abstract [show]
BACKGROUND: Altered ventilatory pattern and increased energy expenditure are facets of the complex cystic fibrosis (CF) phenotype. It is not known whether these are inherent attributes of CF, secondary consequences of lung infection or other disease complications. METHODS: Studies were performed in congenic C57BL/6J, F508del (Cftr((tm1kth))) and CF gut-corrected (F508del) mice. Ventilatory patterns were measured using whole-body plethysmography. Indirect calorimetry was used to determine oxygen consumption, carbon dioxide production and resting energy expenditure. RESULTS: CF mice (F508del and F508del gut-corrected) have a significantly faster respiratory rate and increased ventilatory pattern variability as compared to non-CF mice. F508del but not CF gut-corrected mice had significantly increased energy expenditure per gram body weight. CONCLUSIONS: CF mice exhibit a faster, more variable ventilatory pattern. These changes were present in the absence of detectable infection or illness due to gastrointestinal obstruction. Increased resting energy expenditure does not completely account for these differences.

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169 However, these studies were conducted in a different mouse strain (129/BC) with a different Cftr mutation (S489X) than the mice used in our study. Login to comment

[hide] Disrupted tight junctions in the small intestine o... Cell Tissue Res. 2014 Jan;355(1):131-42. doi: 10.1007/s00441-013-1734-3. Epub 2013 Oct 30. De Lisle RC
Disrupted tight junctions in the small intestine of cystic fibrosis mice.
Cell Tissue Res. 2014 Jan;355(1):131-42. doi: 10.1007/s00441-013-1734-3. Epub 2013 Oct 30., [PMID:24169862]

Abstract [show]
The tight junction (TJ) is the major determinant of paracellular permeability, which in the gut protects the body from entry of harmful substances such as microbial components. In cystic fibrosis (CF), there is increased permeability of the small intestine both in humans and in CF mice. To gain insight into the mechanisms of increased intestinal permeability in CF, I analyze the composition of the TJ in a cystic fibrosis transmembrane conductance regulator (Cftr) knockout mouse model. Significant changes in TJ gene expression in the CF intestine were found for Cldn1, Cldn7, Cldn8 and Pmp22, which were expressed at lower levels and Cldn2 that was expressed at a higher level. Protein levels of claudin-2 were increased in the CF intestine as compared to wild-type, while other TJ proteins were not significantly different. In the villus epithelium of the CF intestine, all TJ components analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces. The pore-forming claudin-2 in the CF intestine showed more intense staining but was correctly localized to the TJ, principally in the crypts that are enlarged in CF. The cytokine TNFalpha, known to affect TJ, was elevated to 160% of wild-type in the CF intestine. In summary, there is a dramatic redistribution of claudin proteins from the TJ/lateral membrane to the basal cytoplasm of the villus epithelium in the CF intestine. These changes in TJ protein localization in CF are likely to be involved in the increased permeability of the CF small intestine to macromolecules and TNFalpha may be a causative factor.

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208 Am J Pathol 164:1481-1493 Eckman EA, Cotton CU, Kube DM, Davis PB (1995) Dietary changes improve survival of CFTR S489X homozygous mutant mouse. Login to comment

[hide] Parasympathetic innervation regulates tubulogenesi... Dev Cell. 2014 Aug 25;30(4):449-62. doi: 10.1016/j.devcel.2014.06.012. Nedvetsky PI, Emmerson E, Finley JK, Ettinger A, Cruz-Pacheco N, Prochazka J, Haddox CL, Northrup E, Hodges C, Mostov KE, Hoffman MP, Knox SM
Parasympathetic innervation regulates tubulogenesis in the developing salivary gland.
Dev Cell. 2014 Aug 25;30(4):449-62. doi: 10.1016/j.devcel.2014.06.012., [PMID:25158854]

Abstract [show]
A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(-) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis.

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260 (H) Comparison of wild-type and Cftr-deficient (S489X) salivary glands from 4-month female mice. Login to comment

[hide] Improving newborn screening for cystic fibrosis us... Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209. Baker MW, Atkins AE, Cordovado SK, Hendrix M, Earley MC, Farrell PM
Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study.
Genet Med. 2015 Feb 12. doi: 10.1038/gim.2014.209., [PMID:25674778]

Abstract [show]
Purpose:Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology.Methods:An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation.Results:The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified.Conclusion:The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.Genet Med advance online publication 12 February 2015Genetics in Medicine (2015); doi:10.1038/gim.2014.209.

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15 Correspondence: Mei W. Baker (mwbaker@wisc.edu) Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study Mei W. Baker, MD1,2 , Anne E. Atkins, MPH2 , Suzanne K. Cordovado, PhD3 , Miyono Hendrix, MS3 , Marie C. Earley, PhD3 and Philip M. Farrell, MD, PhD1,4 Table 1ߒ CF-causing or varying consequences mutations in the MiSeqDx IUO Cystic Fibrosis System c.1521_1523delCTT (F508del) c.2875delG (3007delG) c.54-5940_273ߙ+ߙ10250del21kb (CFTRdele2,3) c.3909C>G (N1303K) c.3752G>A (S1251N) Mutations that cause CF when combined with another CF-causing mutation c.1624G>T (G542X) c.2988ߙ+ߙ1G>A (3120ߙ+ߙ1G->A) c.3964-78_4242ߙ+ߙ577del (CFTRdele22,23) c.613C>T (P205S) c.1021T>C (S341P) c.948delT (1078delT) c.2988G>A (3120G->A) c.328G>C (D110H) c.200C>T (P67L) c.1397C>A (S466X(C>A)) c.1022_1023insTC (1154insTC) c.2989-1G>A (3121-1G->A) c.3310G>T (E1104X) c.3937C>T (Q1313X) c.1397C>G (S466X(C>G)) c.1081delT (1213delT) c.3140-26A>G (3272-26A->G) c.1753G>T (E585X) c.658C>T (Q220X) c.1466C>A (S489X) c.1116ߙ+ߙ1G>A (1248ߙ+ߙ1G->A) c.3528delC (3659delC) c.178G>T (E60X) c.115C>T (Q39X) c.1475C>T (S492F) c.1127_1128insA (1259insA) c.3659delC (3791delC) c.2464G>T (E822X) c.1477C>T (Q493X) c.1646G>A (S549N) c.1209ߙ+ߙ1G>A (1341ߙ+ߙ1G->A) c.3717ߙ+ߙ12191C>T (3849ߙ+ߙ10kbC->T) c.2491G>T (E831X) c.1573C>T (Q525X) c.1645A>C (S549R) c.1329_1330insAGAT (1461ins4) c.3744delA (3876delA) c.274G>A (E92K) c.1654C>T (Q552X) c.1647T>G (S549R) c.1393-1G>A (1525-1G->A) c.3773_3774insT (3905insT) c.274G>T (E92X) c.2668C>T (Q890X) c.2834C>T (S945L) c.1418delG (1548delG) c.262_263delTT (394delTT) c.3731G>A (G1244E) c.292C>T (Q98X) c.1013C>T (T338I) c.1545_1546delTA (1677delTA) c.3873ߙ+ߙ1G>A (4005ߙ+ߙ1G->A) c.532G>A (G178R) c.3196C>T (R1066C) c.1558G>T (V520F) c.1585-1G>A (1717-1G->A) c.3884_3885insT (4016insT) c.988G>T (G330X) c.3197G>A (R1066H) c.3266G>A (W1089X) c.1585-8G>A (1717-8G->A) c.273ߙ+ߙ1G>A (405ߙ+ߙ1G->A) c.1652G>A (G551D) c.3472C>T (R1158X) c.3611G>A (W1204X) c.1679ߙ+ߙ1.6kbA>G (1811ߙ+ߙ1.6kbA->G) c.274-1G>A (406-1G->A) c.254G>A (G85E) c.3484C>T (R1162X) c.3612G>A (W1204X) c.1680-1G>A (1812-1G->A) c.4077_4080delTGTTinsAA (4209TGTT->AA) c.2908G>C (G970R) c.349C>T (R117C) c.3846G>A (W1282X) c.1766ߙ+ߙ1G>A (1898ߙ+ߙ1G->A) c.4251delA (4382delA) c.595C>T (H199Y) c.1000C>T (R334W) c.1202G>A (W401X) c.1766ߙ+ߙ3A>G (1898ߙ+ߙ 3A->G) c.325_327delTATinsG (457TAT->G) c.1007T>A (I336K) c.1040G>A (R347H) c.1203G>A (W401X) c.2012delT (2143delT) c.442delA (574delA) c.1519_1521delATC (I507del) c.1040G>C (R347P) c.2537G>A (W846X) c.2051_2052delAAinsG (2183AA->G) c.489ߙ+ߙ1G>T (621ߙ+ߙ 1G->T) c.2128A>T (K710X) c.1055G>A (R352Q) c.3276C>A (Y1092X (C>A)) c.2052delA (2184delA) c.531delT (663delT) c.3194T>C (L1065P) c.1657C>T (R553X) c.3276C>G (Y1092X (C>G)) c.2052_2053insA (2184insA) c.579ߙ+ߙ1G>T (711ߙ+ߙ 1G->T) c.3230T>C (L1077P) c.1679G>A (R560K) c.366T>A (Y122X) c.2175_2176insA (2307insA) c.579ߙ+ߙ3A>G (711ߙ+ߙ 3A->G) c.617T>G (L206W) c.1679G>C (R560T) - c.2215delG (2347delG) c.579ߙ+ߙ5G>A (711ߙ+ߙ 5G->A) c.1400T>C (L467P) c.2125C>T (R709X) - c.2453delT (2585delT) c.580-1G>T (712-1G->T) c.2195T>G (L732X) c.223C>T (R75X) - c.2490ߙ+ߙ1G>A (2622ߙ+ߙ1G->A) c.720_741delAGGGAG AATGATGATGAAGTAC (852del22) c.2780T>C (L927P) c.2290C>T (R764X) - c.2583delT (2711delT) c.1364C>A (A455E) c.3302T>A (M1101K) c.2551C>T (R851X) - c.2657ߙ+ߙ5G>A (2789ߙ+ߙ5G->A) c.1675G>A (A559T) c.1A>G (M1V) c.3587C>G (S1196X) - Mutations/variants that were validated in this study are in bold. CF, cystic fibrosis. Table 1ߒ Continued on next page reduce carrier detection and potentially improve the positive predictive value (PPV), the NBS goals of equity and the highest possible sensitivity become more difficult to achieve. Login to comment

[hide] The improvement of the best practice guidelines fo... Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99. Girardet A, Viart V, Plaza S, Daina G, De Rycke M, Des Georges M, Fiorentino F, Harton G, Ishmukhametova A, Navarro J, Raynal C, Renwick P, Saguet F, Schwarz M, SenGupta S, Tzetis M, Roux AF, Claustres M
The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus.
Eur J Hum Genet. 2015 May 27. doi: 10.1038/ejhg.2015.99., [PMID:26014425]

Abstract [show]
Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.European Journal of Human Genetics advance online publication, 27 May 2015; doi:10.1038/ejhg.2015.99.

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79 (unknown) Q39X c.115C4T p.Gln39* P67L c.200C4T p.Pro67Leu R75X c.223C4T p.Arg75* 405+1G4A c.273+1G4A 406-1G4A c.274-1G4A E92X c.274G4T p.Glu92* E92K c.274G4A p.Glu92Lys Q98X c.292C4T p.Gln98* 457TAT4G c.325_327delTATinsG p.Tyr109Glyfs*4 D110H c.328G4C p.Asp110His R117C c.349C4T p.Arg117Cys Y122X c.366 T4A p.Tyr122* 574delA c.442delA p.Ile148Leufs*5 444delA c.313delA p.Ile105Serfs*2 663delT c.531delT p.Ile177Metfs*12 G178R c.532G4A p.Gly178Arg 711+3 A4G c.579+3 A4G 711+5G4A c.579+5G4A 712-1G4T c.580-1G4T H199Y c.595C4T p.His199Tyr P205S c.613C4T p.Pro205Ser L206W c.617 T4G p.Leu206Trp Q220X c.658C4T p.Gln220* 852del22 c.720_741delAGGGAGAAT GATGATGAAGTAC p.Gly241Glufs*13 1078delT c.948delT p.Phe316Leufs*12 G330X c.988G4T p.Gly330* Table 1 (Continued ) HGVS nomenclature Legacy name cDNA nucleotide name Protein name R334W c.1000C4T p.Arg334Trp I336K c.1007 T4A p.Ile336Lys T338I c.1013C4T p.Thr338Ile 1154insTC c.1021_1022dupTC p.Phe342Hisfs*28 S341P c.1021 T4C p.Ser341Pro R347H c.1040G4A p.Arg347His 1213delT c.1081delT p.Trp361Glyfs*8 1248+1G4A c.1116+1G4A 1259insA c.1130dupA p.Gln378Alafs*4 W401X(TAG) c.1202G4A p.Trp401* W401X(TGA) c.1203G4A p.Trp401* 1341+1G4A c.1209+1G4A 1461ins4 c.1329_1330insAGAT p.Ile444Argfs*3 1525-1G4A c.1393-1G4A S466X c.1397C4A or c.1397C4G p.Ser466* L467P c.1400 T4C p.Leu467Pro S489X c.1466C4A p.Ser489* S492F c.1475C4T p.Ser492Phe 1677delTA c.1545_1546delTA p.Tyr515* V520F c.1558G4T p.Val520Phe 1717-1G4A c.1585-1G4A 1717-8G4A c.1585-8G4A S549R c.1645 A4C p.Ser549Arg S549N c.1646G4A p.Ser549Asn S549R c.1647 T4G p.Ser549Arg Q552X c.1654C4T p.Gln552* A559T c.1675G4A p.Ala559Thr 1811+1.6kbA4G c.1680-886 A4G 1812-1G4A c.1680-1G4A R560K c.1679G4A p.Arg560Lys E585X c.1753G4T p.Glu585* 1898+3 A4G c.1766+3 A4G 2143delT c.2012delT p.Leu671* 2184insA c.2052_2053insA p.Gln685Thrfs*4 2184delA c.2052delA p.Lys684Asnfs*38 R709X c.2125C4T p.Arg709* K710X c.2128 A4T p.Lys710* 2307insA c.2175dupA p.Glu726Argfs*4 L732X c.2195 T4G p.Leu732* 2347delG c.2215delG p.Val739Tyrfs*16 R764X c.2290C4T p.Arg764* 2585delT c.2453delT p.Leu818Trpfs*3 E822X c.2464G4T p.Glu822* 2622+1G4A c.2490+1G4A E831X c.2491G4T p.Glu831* W846X c.2537G4A p.Trp846* W846X (2670TGG4TGA) c.2538G4A p.Trp846* R851X c.2551C4T p.Arg851* 2711delT c.2583delT p.Phe861Leufs*3 S945L c.2834C4T p.Ser945Leu 2789+2insA c.2657+2_2657+3insA Q890X c.2668C4T p.Gln890* L927P c.2780 T4C p.Leu927Pro 3007delG c.2875delG p.Ala959Hisfs*9 G970R c.2908G4C p.Gly970Arg 3120G4A c.2988G4A function variants that cause CF disease when paired together; (ii) variants that retain residual CFTR function and are compatible with milder phenotypes such as CFTR-RD; (iii) variants with no clinical consequences; and (iv) variants of unproven or uncertain clinical relevance. Login to comment

[hide] Prevalence of meconium ileus marks the severity of... Genet Med. 2015 Jun 18. doi: 10.1038/gim.2015.79. Dupuis A, Keenan K, Ooi CY, Dorfman R, Sontag MK, Naehrlich L, Castellani C, Strug LJ, Rommens JM, Gonska T
Prevalence of meconium ileus marks the severity of mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.
Genet Med. 2015 Jun 18. doi: 10.1038/gim.2015.79., [PMID:26087176]

Abstract [show]
RATIONALE: Meconium ileus (MI) is a perinatal complication in cystic fibrosis (CF), which is only minimally influenced by environmental factors. We derived and examined MI prevalence (MIP) scores to assess CFTR phenotype-phenotype correlation for severe mutations. METHOD: MIP scores were established using a Canadian CF population (n = 2,492) as estimates of the proportion of patients with MI among all patients carrying the same CFTR mutation, focusing on patients with p.F508del as the second allele. Comparisons were made to the registries from the US CF Foundation (n = 43,432), Italy (Veneto/Trentino/Alto Adige regions) (n = 1,788), and Germany (n = 3,596). RESULTS: The prevalence of MI varied among the different registries (13-21%). MI was predominantly prevalent in patients with pancreatic insufficiency carrying "severe" CFTR mutations. In this severe spectrum MIP scores further distinguished between mutation types, for example, G542X (0.31) with a high, F508del (0.22) with a moderate, and G551D (0.08) with a low MIP score. Higher MIP scores were associated with more severe clinical phenotypes, such as a lower forced expiratory volume in 1 second (P = 0.01) and body mass index z score (P = 0.04). CONCLUSIONS: MIP scores can be used to rank CFTR mutations according to their clinical severity and provide a means to expand delineation of CF phenotypes.Genet Med advance online publication 18 June 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.79.

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63 Canadian studies for CF modfier genes 2,492 3,153 43,432 3,596 1,788 2,230 23,397 16,023 3 716 3,438 860 15% (19%) 1,902 2,576 PIP and MIP derivation FEV1 and zBMI modeling MIP calculation following correction of MI variable 23,301 2,413 510 21% (25%) 20% (23%) 13% (15%) Total F508del/others MI prevalence uncorrected (estimated) Missing or incomplete genotype Available for analysis Canadian CF patient registry, born after 1980 US CF patient registry German CF patient registry CF patient registry, North Italy Table 1ߒ Meconium ileus prevalence scores for the most common cystic fibrosis-causing variants p. F508del/other variants Class PIP Canada, (n) MIP, (n) Canada United States Germany Italy HGVS Legacy name c.262_263delTT 394delTT I 0.38 (50) c.3472C>T R1158X I 0.37 (35) c.1558G>T V520F 0.35 (43) c.3484C>T R1162X I 0.34 (135) 0.17 (14) 0.22 (45) c.2012delT 2143delT I 0.33 (13) c.3276C>A or G Y1092X I 0.92 (13) 0.09 (12) 0.33 (55) c.3846G>A W1282X I 1.00 (13) 0.29 (13) 0.32 (442) 0.17 (20) c.1477C>T Q493X I 1.00 (11) 0.19 (11) 0.32 (102) c.3528delC 3659delC I 0.31 (139) c.579ߙ+ߙ1G>T 711ߙ+ߙ1G>T 0.97 (39) 0.30 (38) 0.31 (54) c.178G>T E60X I 0.30 (66) c.1657C>T R553X I 1.00 (16) 0.28 (16) 0.30 (415) 0.24 (107) c.1585-1G>A 1717-1G>A I 1.00 (12) 0.23 (12) 0.29 (367) 0.22 (38) 0.16 (22) c.1766ߙ+ߙ1G>A 1898ߙ+ߙ1G>A 0.29 (139) c.1624G>T G542X I 0.99 (73) 0.31 (72) 0.29 (976) 0.21 (79) 0.22 (33) c.1521_1523delCTT F508del II 0.99 (1292) 0.22 (1260) 0.27 (15391) 0.21 (1910) 0.20 (230) c.1679G>C R560T II 0.27 (123) c.3744delA 3876delA 0.27 (22) c.2128A>T K710X I 0.26 (12) c.1519_1521delATC I507del II 1.00 (20) 0.21 (19) 0.25 (162) c.3909C>G N1303K II 0.98 (40) 0.13 (39) 0.25 (534) 0.23 (80) 0.14 (62) c.489ߙ+ߙ1G>T 621ߙ+ߙ1G>T I 1.00 (90) 0.24 (88) 0.25 (369) 0.21 (11) c.3266G>A W1089X I 0.25 (17) c.1675G>A A559T 0.24 (21) c.988G>T G330X 0.24 (10) c.3773_3774insT 3905insT 0.23 (78) c.2988ߙ+ߙ1G>A 3120ߙ+ߙ1G>A 0.22 (121) c.443T>C I148T;3199del6 1.00 (15) 0.22 (15) c.2052delA 2184delA I 0.21 (89) 0.22 (10) c.2051_2052delAAinsG 2183AA>G 0.20 (73) 0.20 (42) c.948delT 1078delT 0.19 (20) c.1652G>A G551D III 0.96 (54) 0.08 (53) 0.15 (979) 0.09 (84) c.254G>A G85E 0.50 (24) 0.06 (24) 0.14 (137) 0.00 (10) c.3196C>T R1066C 0.14 (42) c.1466C>A S489X 1.00 (14) 0.14 (14) c.3808G>A D1270N 0.13 (19) c.1055G>A R352Q 0.12 (18) c.579ߙ+ߙ5G>A 711ߙ+ߙ5G>A 0.12 (30) c.2175_2176insA 2307insA 0.11 (24) c.349C>T R117C 0.10 (37) c.1040G>C R347P IV 0.18 (11) 0.19 (11) 0.10 (130) 0.02 (56) c.350G>A R117H IV 0.05 (21) 0.00 (21) 0.07 (666) 0.02 (19) c.2657ߙ+ߙ5G>A 2789ߙ+ߙ5G>A V 0.25 (20) 0.00 (20) 0.06 (271) 0.01 (21) c.1040G>A R347H 0.06 (55) c.2988G>A 3120G->A 0.06 (36) c.328G>C D1152H IV 0.06 (124) c.3717ߙ+ߙ12191C>T 3849ߙ+ߙ10kbC>T V 0.07 (14) 0.00 (14) 0.05 (299) 0.01 (42) 0.00 (15) c.1364C>A A455E V 0.16 (45) 0.01 (41) 0.05 (109) c.1000C>T R334W IV 0.18 (11) 0.00 (10) 0.05 (92) c.617T>G L206W 0.06 (18) 0.05 (17) 0.04 (52) c.3302T>A M1101K 0.04 (17) c.200C>T P67L V 0.07 (14) 0.00 (14) Meconium ileus prevalence (MIP) and pancreas insufficiency prevalence (PIP) scores are presented. Login to comment

[hide] Ouabain Regulates CFTR-Mediated Anion Secretion an... J Membr Biol. 2015 Dec;248(6):1145-57. doi: 10.1007/s00232-015-9832-7. Epub 2015 Aug 20. Jansson K, Venugopal J, Sanchez G, Magenheimer BS, Reif GA, Wallace DP, Calvet JP, Blanco G
Ouabain Regulates CFTR-Mediated Anion Secretion and Na,K-ATPase Transport in ADPKD Cells.
J Membr Biol. 2015 Dec;248(6):1145-57. doi: 10.1007/s00232-015-9832-7. Epub 2015 Aug 20., [PMID:26289599]

Abstract [show]
Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) requires the transepithelial secretion of fluid into the cyst lumen. We previously showed that physiological amounts of ouabain enhance cAMP-dependent fluid secretion and cyst growth of human ADPKD cyst epithelial cells in culture and formation of cyst-like dilations in metanephric kidneys from Pkd1 mutant mice. Here, we investigated the mechanisms by which ouabain promotes cAMP-dependent fluid secretion and cystogenesis. Ouabain (3 nM) enhanced cAMP-induced cyst-like dilations in embryonic kidneys from Pkd1 (m1Bei) mice, but had no effect on metanephroi from Pkd1 (m1Bei) mice that lack expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, ouabain stimulation of cAMP-induced fluid secretion and in vitro cyst growth of ADPKD cells were abrogated by CFTR inhibition, showing that CFTR is required for ouabain effects on ADPKD fluid secretion. Moreover, ouabain directly enhanced the cAMP-dependent Cl(-) efflux mediated by CFTR in ADPKD monolayers. Ouabain increased the trafficking of CFTR to the plasma membrane and up-regulated the expression of the CFTR activator PDZK1. Finally, ouabain decreased plasma membrane expression and activity of the Na,K-ATPase in ADPKD cells. Altogether, these results show that ouabain enhances net fluid secretion and cyst formation by activating apical anion secretion via CFTR and decreasing basolateral Na(+) transport via Na,K-ATPase. These results provide new information on the mechanisms by which ouabain affects ADPKD cells and further highlight the importance of ouabain as a non-genomic stimulator of cystogenesis in ADPKD.

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35 Cftrm1UNC (S489X) mice, which exhibit a null CFTR phenotype due to unstable CFTR expression at the mRNA and protein level (Takacs-Jarrett et al. 2001), were also used in this study. Login to comment

[hide] A murine model of cystic fibrosis. Am J Respir Crit Care Med. 1995 Mar;151(3 Pt 2):S59-64. Snouwaert JN, Brigman KK, Latour AM, Iraj E, Schwab U, Gilmour MI, Koller BH
A murine model of cystic fibrosis.
Am J Respir Crit Care Med. 1995 Mar;151(3 Pt 2):S59-64., [PMID:7533607]

Abstract [show]
We have generated a mouse line in which the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been mutated by gene targeting. Like human cystic fibrosis (CF) patients, mice lacking a functional CFTR gene, referred to as CFTR(-/-) mice, show increased numbers of goblet cells and obstruction of glands with inspissated eosinophilic secretions. The obstruction of glands often results in the destruction of gland-containing tissues in these animals. However, unlike the case in human CF patients, the most severe pathological changes in these mice were found, on preliminary analysis, to be confined to the intestinal tract and gallbladder. Although respiratory failure is the primary cause of death among humans with CF, we found only minor pathological alterations in the lungs and upper airways of our CFTR(-/-) animals. Possible explanations for the apparent lack of respiratory disease are the young age at which the animals were examined and the pathogen-free environment in which they were housed. In this manuscript, we examine the respiratory and other organ systems of CFTR(-/-) mice that have survived to adulthood. We also report on initial experiments in which CFTR(-/-) mice have been exposed to bacterial pathogens, and we present data on a single animal that displayed severe respiratory disease.

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33 The resulting mutation, which we have designated S489X, should result in a truncated gene product similar to that 560 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 151 1995 seen with several types of human CF mutations. Login to comment
36 Once cell lines carrying the S489X mutation were isolated, they were used to generate mice carrying the same mutation. Login to comment
37 This was accomplished by injecting the cells carrying the S489X mutation into mouse blastocysts and introducing the injected blastocysts into the uterus of a pseudopregnant female. Login to comment
39 These animals were therefore able to pass the ES cell genome, including the S489X mutation, on to their offspring. Login to comment
40 Offspring receiving the ES cell genome from one chimeric parent were initially identified based on coat color, and those heterozygous for he S489X mutation were subsequently identified by PCR analysis of tail DNA. Login to comment
46 RESULTS Survival We reported previously that genetic analysis of the offspring of CFTR(-I-) mice indicated that there was no prenatal mortality associated with the S489X mutation, because the ratio of offspring lacking the S489X mutation to those heterozygous or homozygous for the mutation did not deviate significantly from the expected Mendelian ratio of 1:2:1. Login to comment
82 Another possibility is that the S489X mutation, because it results in a truncated CFTR protein, may result in more severe intestinal complications than the ..1.508 mutation, which is the most common cause of CF in humans. Login to comment
102 Similar to the S489X mutation in our CFTR(-1-) mice, the W1282X mutation is expected to result in a truncated CFTR protein. Login to comment
163 However, the phenotype associated with the S489X mutation also differs in obvious ways from the symptoms commonly associated with human CF. Login to comment
172 With respect to genetic influences, it is possible that since the S489X mutation is still on a heterogeneous genetic background, and since only a small percentage of CFTR( -1-) mice survive, we may have selected for genotypes that compensate to some degree for the effects of the S289X mutation. Login to comment