Detection of modifier loci influencing the lung phenotype of cystic fibrosis knockout mice

The variable severity of lung disease associated with cystic fibrosis (CF) cannot be explained by the genotype of the cystic fibrosis transmembrane conductance regulator (CFTR) locus alone. Lung disease has been reported in a congenic CF mouse model of C57BL/6J genetic background (B6 CF), in the absence of detectable infection, but not in CF mice of mixed genetic background, nor in wild-type animals maintained in identical environments. In this report, studies are presented to show that the same CF mutation in mice of a BALB/c background (BALB CF) results in minimal lung disease. By 12 weeks of age B6 CF mice developed a lung disease consisting of mononuclear cell interstitial infiltrate and fibrosis, and BALB CF or littermate control mice developed minimal histopathology. Therefore, it is possible to identify the chromosomal locations of genes that can contribute to the susceptibility to lung disease in B6 CF mice compared with BALB CF mice by means of a quantitative trait loci (QTL) mapping strategy based on the variable histology of the (B6 × BALB) F2 CF mice. Significant linkage of the fibrotic lung phenotype was detected for a region on Chromosome (Chr) 6, defined by markers D6Mit194 to D6Mit201, and suggestive linkage was found for regions on Chr 1, 2, 10, and 17. Additional loci, suggestive of linkage, were also detected for the interstitial thickening phenotype. Most of these putative loci are specific to the sex of the animals. These results suggest that multiple genes can influence the severity of CF lung disease in mice.     

Intestinal phenotype of variable-weight cystic fibrosis knockout mice

Cystic fibrosis (CF) transmembrane conductance regulator (Cftr) knockout mice present the clinical features of low body weight and intestinal disease permitting an assessment of the interrelatedness of these phenotypes in a controlled environment. To identify intestinal alterations that are affected by body weight in CF mice, the histological phenotypes of crypt-villus axis height, goblet cell hyperplasia, mast cell infiltrate, crypt cell proliferation, and apoptosis were measured in a population of 12-wk-old (C57BL/6 x BALB/cJ) F2 Cftr(tm1UNC) and non-CF mice presenting a range of body weight. In addition, cardiac blood samples were assessed, and gene expression profiling of the ileum was completed. Crypt-villus axis height decreased with increasing body weight in CF but not control mice. Intestinal crypts from CF mice had fewer apoptotic cells, per unit length, than did non-CF mice, and normalized cell proliferation was similar to control levels. Goblet cell hyperplasia and mast cell infiltration were increased in the CF intestine and identified to be independent of body weight. Blood triglyceride levels were found to be significantly lower in CF mice than in control mice but were not dependent on CF mouse weight. By expression profiling, genes of DNA replication and lipid metabolism were among those altered in CF mice relative to non-CF controls, and no differences in gene expression were measured between samples from CF mice in the 25th and 75th percentile for weight. In this CF mouse model, crypt elongation, due to an expanded proliferative zone and decreased apoptosis, was identified to be dependent on body weight.     

Surface-lining layer of airways in cystic fibrosis mice

Lung disease is the major cause of death in individuals suffering from cystic fibrosis (CF), with abnormal lung-lining fluids occurring as early as early infancy. However, the precise etiology of CF lung disease is still poorly understood. We investigated the structural components of the airway surface-lining layer in targeted Cftrtm1HGU/Cftrtm1HGU mutant mice and non-CF controls. Five lungs per animal group were fixed by intravascular triple perfusion. The ultrastructure of the surface-lining layer of large and small intrapulmonary conducting airways was systematically investigated according to a standard protocol in transmission and scanning electron micrographs. In both animal groups, the surface-lining layer consisted of an aqueous phase and an osmiophilic film of variable thickness at the air-fluid interface. The aqueous phase usually did extend <1 microm beyond the uppermost tips of the epithelial cells in both animal groups. The aqueous phase of the small airways was slightly more electron dense in Cftrtm1HGU/Cftrtm1HGU than in non-CF mice. Neither the ultrastructure of the surfactant film at the air-fluid interface nor the forms assumed by the osmiophilic structures associated with surfactant turnover in the aqueous layer differed significantly in Cftrtm1HGU/Cftrtm1HGU and non-CF mice. Hence, there were no signs of any ultrastructural abnormalities in the surface-lining layer of young adult Cftrtm1HGU/Cftrtm1HGU mice before infection with CF-related pathogens.     

The genetic factors influencing the development of trichotillomania

Trichotillomania (TTM), an obsessive-compulsive spectrum disorder (OCSD), is a psychiatric condition characterized by repetitive hair pulling. Evidence from family and twin studies suggest a heritable link of TTM. Functional polymorphisms in genes involved in neuronal pathways might influence the susceptibility to TTM. This review is an attempt to compile the genetic factors reported to modify the development of TTM.     

Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis.     

Mineral content of calcified tissues in cystic fibrosis mice

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. Preliminary reports published in Pediatric Pulmonology, 14, 253A (1997) and 15, 253A (1998).     

Disrupted tight junctions in the small intestine of cystic fibrosis mice

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 TNFα, 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 TNFα may be a causative factor.     

Linkage disequilibrium, cystic fibrosis, and genetic counseling.

Strong linkage disequilibrium occurs between the cystic fibrosis (CF) locus and polymorphisms detected with the DNA probes XV-2c and KM-19. In a North American population, 86% of CF chromosomes occur with a haplotype which occurs on only 14% of normal chromosomes. An individual homozygous for the highest-risk haplotype has an 81-fold greater probability of carrying a CF allele than does an individual homozygous for the lowest-risk haplotype. The linkage-disequilibrium data can be used for prenatal diagnosis and genetic counseling in CF families. The data are useful in 1-in-4-risk pregnancies when DNA is not available from the propositus and in counseling close relatives of CF families. Serious problems arise with some pregnancies which remain at intermediate risks after analysis, and families are left with difficult decisions. It is not clear that genetic testing for couples at less than 1-in-4 risk is cost-effective or standard care, but use of linkage-disequilibrium data will provide more accurate risk probabilities in a substantial proportion of cases if such testing is carried out. Our results emphasize the need for a specific biological or molecular carrier test. This experience in using linkage-disequilibrium and linkage data in combination for genetic counseling provides a model system for the diagnosis of other disorders.     

Physiological and genetic factors influencing fruit cracking

Discovering the complexity of seed structure and function along with a number of vital processes such as seed growth and development, germination are important factors in unlocking the secrets of consistent crop yield. Fenugreek (Trigonella foenum-graecum L.), a multi-purpose annual, dryland-adapted, forage, legume crop is cultivated in different parts of the world with great potential for introduction under suitable agro-climatic zones in sub-Saharan Africa and Latin America. Fenugreek seed is used extensively for its medicinal, pharmaceutical and nutraceutical properties. It is effective in the treatment of diabetes, hyperglycaemia (thyroxine-induced type) and hypercholesterolemia. This review discusses seed physiological processes and several important biochemical seed constituent, e.g., steroidal sapogenins (diosgenin), polysaccharide fiber (galactomannan), amino acid (4-hydroxyisoleucine), etc, with important medicinal and pharmacological characteristics impacting human and animal health. However, there are noticeable differences in the quality of several phytochemicals found in fenugreek seed possibly due to variations in plant genotypes and agro-climatic conditions under which the crop is grown. Hence, it is important to note that for consistent seed yield and quality of fenugreek cultivars there is an urgent need for continuing efforts in genetic improvements and in developing high yielding, disease and drought-resistant varieties suitable for different agro-climatic conditions. Therefore, in addition to the physico-biochemistry of fenugreek seed different approaches for genetic improvement have also been discussed.     

What genetic risk should suggest prenatal diagnosis for cystic fibrosis?

Prenatal diagnosis of cystic fibrosis is today possible by chemical study of amniotic fluid during the 18th week of pregnancy. We have, among 90 families seen for genetic counseling between 1972 and 1985, estimated the risk of recurrence; it was of 0.25 in 55 cases; between 0.05 and 0.16 in 30 cases, greater than 0.01 in 16 cases. Before suggesting a prenatal diagnosis, it is necessary to take as basis the risk of abortion and the reliability of the method. The first risk is well known, less 0.005, in these young women; reliability can be estimated: almost 0.01 of false wrong negative, and this number is probably overvalued if all technical conditions are perfect. In front of these risks, it seems possible to propose a prenatal diagnosis from a risk of 0.01, the families being informed of all risks. We think so to hearten some families and to make possible for them to live quietly these pregnancies, probably non undertaken without our help.     

Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal tracheal xenografts in SCID mice

We measured thebioelectric properties of 14 cystic fibrosis (CF) and 33 non-CF humanfetal tracheal xenografts in severe combined immunodeficiency (SCID)mice. All xenografts exhibited a mature airway-type epitheliumirrespective of their gestational age, duration of engraftment, andgenotype. The in vivo potential difference and the in vitro baselineshort-circuit current(I_sc) weresignificantly higher in non-CF than in CF xenografts. In non-CFxenografts, sequential addition of amiloride, forskolin, and ATPresulted in a 39.4% decrease, a 24.1% increase, and a 43.6% increasein I_sc,respectively. In CF xenografts, forskolin had no significant effect onI_sc, whereasamiloride- and ATP-induced changes inI_sc wereproportionally higher than in non-CF xenografts (60.0 and+68.8%, respectively). These results indicate that the bioelectricproperties of non-CF xenografts are similar to those of postnatalairways and that CF xenografts exhibit lower baseline electrogenicactivity than non-CF xenografts but similar regulation of ion transportprocesses to postnatal CF airways. This model of mature human fetaltracheal mucosa may help gain insight into early CF airwaypathogenesis.

     

Physical and genetic analysis of cosmids from the vicinity of the cystic fibrosis locus.

Cosmid libraries have been constructed from DNA of somatic cell hybrid cell lines, each containing a fragment of human chromosome seven and including sequences closely linked to cystic fibrosis (CF). Cosmids containing human DNA as insert were isolated from the library. Three cosmids, when used as probes to total genomic DNA, detected polymorphic loci, each of which was shown to be in strong linkage disequilibrium with CF. Restriction endonuclease digestion of cosmid clones and use of a new, rapid method of chromosome walking based on competitive hybridisation of cosmid inserts has allowed identification of several groups of overlapping cosmids ("contigs") from the vicinity of CF.     

Analysis of 112 haplotypes carrying the cystic fibrosis gene. Applications in genetic counseling

Cystic fibrosis (CF) is always a common lethal genetic disease. The locus is localized to human chromosome 7q22-7q31. Genetic linkage between the CF locus and polymorphic DNA marker is used to realize family studies. We have genotyped 56 families (352 patients) with a CF child. The informativeness with the six markers (Met D/Taq I, Met H/Taq I, Met H/Msp 1, XV2c/Taq 1, km19/pst pJ 3.11/Msp 1) is important (96%). The linkage desequilibrium between alleles detected by XV2 c and Km 19 described by Estivill and al, is also showed in our population. The haplotype B (Km 19 = 6.6 kb, XV2 c = 2.1 kb) is present on 84% of our 112 CF chromosomes. We have established the frequencies of the 10 possible genotypes in the pool of the 112 CF chromosomes and in the pool of the normal chromosome and according to Bayes obtained the predictive positive value to be heterozygote. It is possible to precise the genetic counselling in these families.     

Characteristics of behavior of knockout mice with genetic monoamine oxidase A deficiency

The effect of deletion of monoamine oxidase A (MAO A) in the gene encoding on behavior of transgenic Tg8 mice was studied. A decrease in the amplitude of acoustic startle reflex rather than the prepulse inhibition was found in lacking MAO A Tg8 mice, as compared with the control C3H strain. The exploratory activity in the hole-board test in Tg8 was decreased as well as the number of crossed lines in the light-dark test. Tg8 mice showed decreased latency and increased intensity of intermale aggression. At the same time, no difference was found between Tg8 and C3H mice in locomotor activity, in the expression of sexual motivation, and in the behavior in the elevated plus-maze test. No predisposition to catalepsy was shown.     

Unraveling the complex genetic model for cystic fibrosis: pleiotropic effects of modifier genes on early cystic fibrosis-related morbidities

The existence of pleiotropy in disorders with multi-organ involvement can suggest therapeutic targets that could ameliorate overall disease severity. Here we assessed pleiotropy of modifier genes in cystic fibrosis (CF). CF, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, affects the lungs, liver, pancreas and intestines. However, modifier genes contribute to variable disease severity across affected organs, even in individuals with the same CFTR genotype. We sought to determine whether SLC26A9, SLC9A3 and SLC6A14, that contribute to meconium ileus in CF, are pleiotropic for other early-affecting CF co-morbidities. In the Canadian CF population, we assessed evidence for pleiotropic effects on (1) pediatric lung disease severity (n = 815), (2) age at first acquisition of Pseudomonas aeruginosa (P. aeruginosa) (n = 730), and (3) prenatal pancreatic damage measured by immunoreactive trypsinogen (n = 126). A multiple-phenotype analytic strategy assessed evidence for pleiotropy in the presence of phenotypic correlation. We required the same alleles to be associated with detrimental effects. SLC26A9 was pleiotropic for meconium ileus and pancreatic damage (p = 0.002 at rs7512462), SLC9A3 for meconium ileus and lung disease (p = 1.5 × 10^?6 at rs17563161), and SLC6A14 for meconium ileus and both lung disease and age at first P. aeruginosa infection (p = 0.0002 and p = 0.006 at rs3788766, respectively). The meconium ileus risk alleles in SLC26A9, SLC9A3 and SLC6A14 are pleiotropic, increasing risk for other early CF co-morbidities. Furthermore, co-morbidities affecting the same organ tended to associate with the same genes. The existence of pleiotropy within this single disorder suggests that complementary therapeutic strategies to augment solute transport will benefit multiple CF-associated tissues.     

Studies of factors influencing the mutagenicity of EMS in mice

Three different routes of administration of ethyl methanesulphonate (EMS) (i.p., oral, i.t.) were compared for their relative efficiencies in the induction of dominant lethal effects. Included in the comparisons between oral and i.p. injections, was a preliminary study into the existence of strain differences in sensitivity to EMS between C3D2 F1 hybrid mice and strain DBA/2J mice. No route of administration dependent effects were found between oral and i.p. injections regardless of the test animal used. I.t. injections of EMS did not induce dominant lethal effects. One treatment related strain difference was observed.     

Acute intratracheal Pseudomonas aeruginosa infection in cystic fibrosis mice is age-independent

Background

Since the discovery of the human CFTR gene in 1989 various mouse models for cystic fibrosis (CF) have been generated and used as a very suitable and popular tool to approach research on this life-threatening disease. Age related changes regarding the course of disease and susceptibility towards pulmonary infections have been discussed in numerous studies.

Methods

Here, we investigated Cftr^{TgH(neoim)Hgu} and Cftr^tm1Unc-Tg(FABPCFTR)1Jaw/J CF mice and their non-CF littermates during an acute lung infection with Pseudomonas aeruginosa for age dependent effects of their lung function and immune response.Mice younger than three or older than six months were intratracheally infected with P. aeruginosa TBCF10839. The infection was monitored by lung function of the animals using non-invasive head-out spirometry and the time course of physiological parameters over 192 hours. Quantitative bacteriology and lung histopathology of a subgroup of animals were used as endpoint parameters.

Results

Age-dependent changes in lung function and characteristic features for CF like a shallower, faster breathing pattern were observed in both CF mouse models in uninfected state. In contrast infected CF mice did not significantly differ from their non-CF littermates in susceptibility and severity of lung infection in both mouse models and age groups. The transgenic Cftr^tm1Unc-Tg(FABPCFTR)1Jaw/J and their non-CF littermates showed a milder course of infection than the Cftr^{TgH(neoim)Hgu} CF and their congenic C57Bl/6J non-CF mice suggesting that the genetic background was more important for outcome than Cftr dysfunction.

Conclusions

Previous investigations of the same mouse lines have shown a higher airway susceptibility of older CF mice to intranasally applied P. aeruginosa. The different outcome of intranasal and intratracheal instillation of bacteria implies that infected CF epithelium is impaired during the initial colonization of upper airways, but not in the subsequent response of host defense.     

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.