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W H Colledge

Publications and source records attributed to W H Colledge.

At least 37 records · Page 2Linked to original sources
PubMed2000

A murine tracheal culture system to investigate parameters affecting gene therapy for cystic fibrosis.

Cystic fibrosis (CF) is a life-threatening condition caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Delivery of the CFTR gene to the airways offers a potential treatment for CF but requires improvement in efficiency to obtain clinical benefit. We have developed a murine tracheal culture system that maintains tissue integrity as judged by normal histological appearance, high transepithelial resistance and electrophysiological responses similar to fresh tissue. This ex vivo system allows precise control of gene delivery parameters to a structure that retains the in vivo cellular architecture. We have demonstrated correction of CFTR-dependent Cl- secretion following ex vivo delivery of the CFTR gene to tracheas from CF null mice. We have used this system to examine parameters affecting liposome-mediated gene delivery to the upper airway such as plasmid dose. We have also found that a contact time of 1 min for the transfection mixture is sufficient to achieve significant DNA binding and maximal reporter gene expression.

Animals↗
PubMed2000

Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis.

The major cause of mortality in patients with cystic fibrosis (CF) is lung disease. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in the airways is a potential treatment. Clinical studies in which the CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in modest, transient gene expression. It seems likely that repeated administration of the gene transfer vector will be required for long-term gene expression. We have undertaken a double-blinded study in which multiple doses of a DNA/liposome formulation were delivered to the nasal epithelium of CF patients. Ten subjects received plasmid DNA expressing the CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes, whilst two subjects received placebo. Each subject received three doses, administered 4 weeks apart. There was no evidence of inflammation, toxicity or an immune response towards the DNA/liposomes or the expressed CFTR. Nasal epithelial cells were collected 4 days after each dose for a series of efficacy assays including quantitation of vector-specific DNA and mRNA, immunohistochemistry of CFTR protein, bacterial adherence, and detection of halide efflux ex vivo. Airway ion transport was also assessed in vivo by repeated nasal potential difference (PD) measurements. On average, six of the treated subjects were positive for CFTR gene transfer after each dose. All subjects positive for CFTR function were also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein. The efficacy measures suggest that unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy.

Adolescent↗
PubMed2000

Genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and colon.

The action of the isoflavone genistein on the cystic fibrosis transmembrane conductance regulator (CFTR) has been studied in many cell systems but not in intact murine tissues. We have investigated the action of genistein on murine tissues from normal and cystic fibrosis (CF) mice. Genistein increased the short-circuit current (I(sc)) in tracheal (16.4 +/- 2.8 microA/cm(2)) and colonic (40.0 +/- 4.4 microA/cm(2)) epithelia of wild-type mice. This increase was inhibited by furosemide, diphenylamine-2-carboxylate, and glibenclamide, but not by DIDS. In contrast, genistein produced no significant change in the I(sc) of the tracheal epithelium (0.9 +/- 1.1 microA/cm(2)) and decreased the I(sc) of colons from CF null (-13.1 +/- 2.3 microA/cm(2)) and DeltaF508 mice (-10.3 +/- 1.3 microA/cm(2)). Delivery of a human CFTR cDNA-liposome complex to the airways of CF null mice restored the genistein response in the tracheas to wild-type levels. Tracheas from DeltaF508 mice were also studied: 46% of trachea showed no response to genistein, whereas 54% gave an increase in I(sc) similar to that in wild type. We conclude that genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and distal colon.

Animals↗
PubMed2000

Murine CFTR channel and its role in regulatory volume decrease of small intestine crypts.

Cystic fibrosis (CF) is caused by mutations in the secretory Cl(-) channel CFTR (cystic fibrosis transmembrane conductance regulator). Variation in the severity of disease has been attributed to mutations in the CFTR gene that cause different degrees of dysfunction of the CFTR Cl(-) channel. However, studies of mouse models of CF indicate that the severity of intestinal pathology is not correlated with activity of the CFTR chloride channel. This observation suggests that other 'environmental' factors might be important in determining the severity of disease. In this respect, we have identified and characterised an additional cellular defect in intestinal epithelial cells of CF mice, the inability of these cells to regulate their volume after hypotonic challenge. Here, we review the function of murine CFTR as both a Cl(-) channel and as a regulator of volume-dependent homeostatic cell mechanisms.

Animals↗
PubMed2000

Sodium channel blockers and uridine triphosphate: effects on nasal potential difference in cystic fibrosis mice.

Sodium channel inhibitors block the enhanced Na+ reabsorption in cystic fibrosis (CF). Extracellular nucleotides facilitate Cl- secretion via Ca2+ gated Cl- channels. A combination of these effects may produce less viscid secretions in CF which are easier to expectorate. This study examined the effects of combining sodium channel blockers with uridine triphosphate (UTP) on nasal membrane potential difference (PD) in CF insertional null mutant mice (cftr(tm1HGU)), deltaF508 homozygous mice (cftr(tm1Cam)) and matched control animals. Median basal PD in the insertional CF mice and deltaF508 CF mice were -28 and -34 mV respectively. These values were significantly different to the control animals (-20 mV). Amiloride and loperamide reduced the PD in cftr(tm1HGU) CF mice (deltaPD 13 mV & 15 mV respectively) suggesting Na+ blockade. The subsequent addition of UTP in a chloride-free vehicle increased the PD (deltaPD -8- -12.5 mV). DeltaF508 mice showed significantly greater responses compared with CF insertional null mutant mice (p<0.05). The action of UTP was brief and not prolonged by the addition alpha-beta-methylene-adenosine 5' diphosphate. Suramin, a competitive antagonist of P2 purinoceptors blocked the action of UTP. In conclusion, this study demonstrated dose dependant nasal membrane potential changes in differences mice with uridine triphosphate in the presence of sodium channel blockers suggestive of chloride secretion. More stable analogues of uridine triphosphate in combination with long acting sodium channel blockers such as loperamide may have therapeutic potential in cystic fibrosis.

Amiloride↗
PubMed1999

A retroviral gene trap insertion into the histone 3.3A gene causes partial neonatal lethality, stunted growth, neuromuscular deficits and male sub-fertility in transgenic mice.

Spermatogenesis is a complex developmental pro-cess involving cell division and differentiation. Approximately half of all sterile males have defects in spermatogenesis or sperm function. An insight into the molecular control points regulating this process might help in treating male infertility. Gene trapping in embryonic stem cells and the generation of transgenic mice represents one route to identify genes expressed during spermatogenesis. The trapped gene is tagged with a lacZ reporter gene so that the expression pattern of the gene can be visualized by staining for beta-galactosidase activity. We have screened transgenic mouse lines for expression of trapped genes in the gonads. One such trap event was shown to be in the replacement histone 3.3A gene ( H3.3A ). This gene was expressed ubiquitously during embryonic development until 13.5 days post-coitum and in the adult heart, kidney, brain, testes and ovaries. This mutation resulted in postnatal death of 50% of homozygous mutants. Surviving mutants displayed reduced growth rates when competing with wild-type siblings for food. Mutant mice also had a neuro-muscular deficit and males displayed reduced copulatory activity. When copulations did occur, these resulted in very few pregnancies, suggesting that mutations in the H3.3A gene may contribute to some cases of impaired fertility in man.

Animals↗
PubMed1998

Salmonella typhi uses CFTR to enter intestinal epithelial cells.

Homozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). In the heterozygous state, increased resistance to infectious diseases may maintain mutant CFTR alleles at high levels in selected populations. Here we investigate whether typhoid fever could be one such disease. The disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. We found that S. typhi, but not the related murine pathogen S. typhimurium, uses CFTR for entry into epithelial cells. Cells expressing wild-type CFTR internalized more S. typhi than isogenic cells expressing the most common CFTR mutation, a phenylalanine deleted at residue 508 (delta508). Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Heterozygous deltaF508 Cftr mice translocated 86% fewer S. typhi into the gastrointestinal submucosa than wild-type Cftr mice; no translocation occurred in deltaF508 Cftr homozygous mice. The Cftr genotype had no effect on the translocation of S. typhimurium. Immunoelectron microscopy revealed that more CFTR bound to S. typhi in the submucosa of Cftr wild-type mice than in deltaF508 heterozygous mice. We conclude that diminished levels of CFTR in heterozygotes may decrease susceptibility to typhoid fever.

Animals↗
PubMed1998

Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens.

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin/BM40) is a secreted Ca2+-binding glycoprotein that interacts with a range of extracellular matrix molecules, including collagen IV. It is widely expressed during embryogenesis, and in vitro studies have suggested roles in the regulation of cell adhesion and proliferation, and in the modulation of cytokine activity. In order to analyse the function of this protein in vivo, the endogenous Sparc locus was disrupted by homologous recombination in murine embryonic stem cells. SPARC-deficient mice (Sparctm1Cam) appear normal and fertile until around 6 months of age, when they develop severe eye pathology characterized by cataract formation and rupture of the lens capsule. The first sign of lens pathology occurs in the equatorial bow region where vacuoles gradually form within differentiating epithelial cells and fibre cells. The lens capsule, however, shows no qualitative changes in the major basal lamina proteins laminin, collagen IV, perlecan or entactin. These mice are an excellent resource for further studies on how SPARC affects cell behaviour in vivo.

Age of Onset↗
PubMed1998

Thymic lymphomas in mice with a truncating mutation in Brca2.

Inherited mutations in the BRCA2 gene predispose women to breast and ovarian cancer. We created a mutation in the mouse Brca2 gene that terminates translation in exon 11 at 45% of the normal transcript length. Ninety % of Brca2(tm1Cam) homozygous mutant mice die prenatally or perinatally. The location of the Brca2(tm1Cam) mutation differs from those reported previously, and this phenotype suggests a correlation with genotype analogous to that previously reported in humans. Although heterozygote mice have remained free of tumors for 10 months, Brca2(tm1Cam) homozygous mutants that survived to adulthood died with thymic lymphomas between 12 and 14 weeks of age.

Alleles↗
PubMed1998

Crouzon-like craniofacial dysmorphology in the mouse is caused by an insertional mutation at the Fgf3/Fgf4 locus.

Retroviral insertional mutagenesis by means of ES cells has resulted in a new autosomal dominant mutation causing craniofacial dysmorphology in the mouse (Bulgy-eye, Bey). Heterozygous Bey mice are viable and fertile but show facial shortening with increased interorbital distance and precocious closure of several cranial sutures (craniosynostosis). These features provide a murine phenocopy for a large class of human craniofacial dysmorphology syndromes associated with craniosynostosis, particularly Crouzon syndrome. The retroviral vector integration responsible for the Bey mutation is inserted in the intragenic region between Fgf3 and Fgf4. Transcript analysis demonstrates that expression of both Fgf3 and Fgf4 is up-regulated in the cranial sutures of Bey mice. Many of these human craniosynostosis syndromes are caused by mutations in the extracellular domain of receptors for fibroblast growth factors that result in constitutive receptor activation. Our data confirm that fibroblast growth factor signalling pathways are involved in craniofacial development and suggest that some human malformation pedigrees or sporadic craniosynostosis may be caused by mutations that deregulate expression of the Fgf ligands.

Animals↗
PubMed1998

Disruption of murine alpha-enolase by a retroviral gene trap results in early embryonic lethality.

Gene trapping with the retroviral ROSA beta geo vector was used to generate lines of mice carrying disrupted genes. Both cDNA and genomic flanks have been cloned from a number of these lines. One mutation has been shown to disrupt the alpha-enolase gene by insertion of the splice-trap vector into the first intron. In adult mice, lacZ expression was detected only in testes. Embryonic expression was detected from 10.5-day postcoitum embryos and was seen as a diffuse staining pattern over much of the embryo, consistent with the housekeeping gene function of alpha-enolase. This mutation results in an early recessive embryonic lethality. Mice heterozygous for the mutation have no obvious phenotype. Mutations of this gene in humans are reported to be associated with rare autosomal-dominant, non-spherocytic haemolytic anaemia. This phenotype is not reproduced in mice heterozygous for this mutation.

Animals↗
PubMed1998

Involvement of Brca2 in DNA repair.

Abnormalities precipitated by a targeted truncation in the murine gene Brca2 define its involvement in DNA repair. In culture, cells harboring truncated Brca2 exhibit a proliferative impediment that worsens with successive passages. Arrest in the G1 and G2/M phases is accompanied by elevated p53 and p21 expression. Increased sensitivity to genotoxic agents, particularly ultraviolet light and methylmethanesulfonate, shows that Brca2 function is essential for the ability to survive DNA damage. But checkpoint activation and apoptotic mechanisms are largely unaffected, thereby implicating Brca2 in repair. This is substantiated by the spontaneous accumulation of chromosomal abnormalities, including breaks and aberrant chromatid exchanges. These findings define a function of Brca2 in DNA repair, whose loss precipitates replicative failure, mutagen sensitivity, and genetic instability reminiscent of Bloom syndrome and Fanconi anemia.

Animals↗
PubMed1998

Mutation analysis of the c-mos proto-oncogene in human ovarian teratomas.

Female transgenic mice lacking a functional c-mos proto-oncogene develop ovarian teratomas, indicating that c-mos may behave as a tumour-suppressor gene for this type of tumour. We have analysed the entire coding region of the c-MOS gene in a series of human ovarian teratomas to determine whether there are any cancer-causing alterations. DNA from twenty teratomas was analysed by single-strand conformational analysis (SSCA) and heteroduplex analysis (HA) to screen for somatic and germline mutations. In nine of these tumours the entire gene was also sequenced. A previously reported polymorphism and a single new sequence variant were identified, neither of which we would predict to be disease-causing alterations. These results suggest that mutations in the coding region of the c-MOS gene do not play a significant role in the genesis of human ovarian teratomas.

DNA Mutational Analysis↗
PubMed1998

Basal chloride currents in murine airway epithelial cells: modulation by CFTR.

We have isolated ciliated respiratory cells from the nasal epithelium of wild-type and cystic fibrosis (CF) null mice and used the patch-clamp technique to investigate their basal conductances. Current-clamp experiments on unstimulated cells indicated the presence of K+ and Cl- conductances and, under certain conditions, a small Na+ conductance. Voltage-clamp experiments revealed three distinct Cl- conductances. Itv-indep was time and voltage independent with a linear current-voltage (I-V) plot; Iv-act exhibited activation at potentials greater than +/- 50 mV, giving an S-shaped I-V plot; and Ihyp-act was activated by hyperpolarizing potentials and had an inwardly rectified I-V plot. The current density sequence was Ihyp-act = Iv-act >> Itv-indep. These conductances had Cl(-)-to-N-methyl-D-glucamine cation permeability ratios of between 2.8 and 10.3 and were unaffected by tamoxifen, flufenamate, glibenclamide, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by Zn2+ and Gd3+. Itv-indep and Iv-act were present in wild-type and CF cells at equal density and frequency. However, Ihyp-act was detected in only 3% of CF cells compared with 26% of wild-type cells, suggesting that this conductance may be modulated by cystic fibrosis transmembrane conductance regulator (CFTR).

Animals↗
PubMed1998

Restoration by intratracheal gene transfer of bicarbonate secretion in cystic fibrosis mouse gallbladder.

Gallbladders from cystic fibrosis (CF) mice (Cftrtm1Cam and Cftrtm2Cam) were examined with the short-circuit current technique. The tissues failed to show any electrogenic anion transport in response to forskolin (cAMP stimulus) but responded to the Ca2+ ionophore ionomycin. Administration of the plasmid pTrial10-CFTR2 complexed with cationic liposomes (3beta-[N-(dimethylaminoethane)-carbamoyl]cholesterol and L-alpha-phosphatidylethanolamine dioleolyl) to the airways restored the phenotype of CF gallbladders to that of the wild type, but did not do so when given orally. Formation of human CFTR mRNA in gallbladders of transfected CF null mice was demonstrated. Using the reporter genes pCMV-luc and pCMV-LacZ, we showed that 1) the intratracheal route was more effective than the oral,intravenous, intramuscular, subcutaneous, or intraperitoneal routes in expressing luciferase activity in the gallbladder and 2) beta-galactosidase staining after pCMV-LacZ was confined to the columnar epithelium lining the gallbladder without any discernible activity in it smooth muscle. The discovery of an unusual route for gene transfer to the biliary system may give useful insight into counteracting the consequences of biliary fibrosis in human CF patients.

Animals↗
PubMed1997

A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca(2+)-dependent HCO3- secretion.

1. Most segments of the gastrointestinal tract secrete HCO3-, but the molecular nature of the secretory mechanisms has not been identified. We had previously speculated that the regulator for intestinal electrogenic HCO3- secretion is the cystic fibrosis transmembrane regulator (CFTR) channel. To prove this hypothesis, we have now measured HCO3- secretion by pH-stat titration, and recorded the electrical parameters of in vitro duodenum, jejunum and ileum of mice deficient in the gene for the CFTR protein ('CF-mice') and their normal littermates. 2. Basal HCO3- secretory rates were reduced in all small intestinal segments of CF mice. Forskolin, PGE2, 8-bromo-cAMP and VIP (cAMP-dependent agonists), heat-stable enterotoxin of Escherichia coli (STa), guanylin and 8-bromo-cGMP (cGMP-dependent agonists) and carbachol (Ca2+ dependent) stimulated both the short-circuit current (Isc) and the HCO3- secretory rate (JHCO3-) in all intestinal segments in normal mice, whereas none of these agonists had any effect on JHCO3- in the intestine of CF mice. 3. To investigate whether Cl(-)-HCO3- exchangers, which have been implicated in mediating the response to some of these agonists in the intestine, were similarly active in the small intestine of normal and CF mice, we studied Cl- gradient-driven 36Cl- uptake into brush-border membrane (BBM) vesicles isolated from normal and CF mouse small intestine. Both the time course and the peak value for 4,4'-diisothiocyanostilbene-2',2-disulphonic acid (DIDS)-inhibited 36Cl- uptake was similar in normal and CF mice BBM vesicles. 4. In summary, the results demonstrate that the presence of the CFTR channel is necessary for agonist-induced stimulation of electrogenic HCO3- secretion in all segments of the small intestine, and all three intracellular signal transduction pathways stimulate HCO3- secretion exclusively via activation of the CFTR channel.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.

Animals↗