Biomedical subjects

W H Colledge

Publications and source records attributed to W H Colledge.

At least 19 recordsLinked to original sources

PubMed2001

Ultrastructural changes in exocrine tissues of a DeltaF-508 CFTR mouse model.

Cystic fibrosis (CF) is characterized by abnormal secretion from epithelial cells. We wanted to detect changes in the ultrastructural characteristics of cells within a number of exocrine tissues, including the colon, submandibular and parotid salivary glands of DeltaF-508 CFTR animals. Therefore, in the present study a DeltaF-508 CFTR mouse model was compared to control, by applying conventional and complex carbohydrates staining techniques to tissue sections at the electron microscope level. The colon of DeltaF-508 CFTR mice contained thick mucous secretions that harbored many bacteria, along with cytoplasmic fragments and leukocytes. Leukocytes were also seen to infiltrate the cytoplasm of goblet cells. Tissues were taken before, 10 min after isoprenaline, and 30 min after a further injection of methacholine. In the submandibular gland, there is limited secretory activity after isoprenaline treatment, and this increases further with methacholine treatment. Depletion of the secretory granules of acinar cells is observed, following the combined isoprenaline and methacholine treatment, but no significant changes in granule numbers occurred in granular tubule cells. Glycogen, abundant before treatment, is reduced within 10 min of isoprenaline treatment and is completely exhausted by 30 min, especially in the convoluted granular tubule cells. A few secretory granules in acinar and in granular tubule cells of the DeltaF-508 CFTR submandibular glands displayed two electron densities. The secretory responses of the parotid gland cells were similar to those in submandibular gland cells, except that in these DeltaF-508 CFTR cells, secretory granules appeared more polymorphic in structure than those found in control animals.

Animals↗

PubMed2001

Effect of acute saline volume expansion in the anaesthetised DeltaF508 cystic fibrosis mouse.

It has been suggested that CFTR Cl(-) channels in the renal inner medullary collecting duct may be involved in mediating increased renal salt excretion during extracellular fluid volume expansion. To investigate this hypothesis, in-vivo clearance experiments were performed comparing wild-type (WT) and DeltaF508-CFTR transgenic mice (cftr (tm2Cam)). Control animals were given a 0.1-ml bolus of 0.9% saline, followed by I.V. infusion at 0.3 ml x h(-1). Volume expansion was applied by infusing a 1-ml bolus of 0.9% saline followed by infusion at 0.6 ml x h(-1). No significant differences in renal NaCl handling between WT mice ( C(Na)=1.2 +/- 0.3 microl x min(-1), C(Cl)=4.0 +/- 0.5 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=1.7 +/- 0.5 microl x min(-1), C(Cl)=4.1 +/- 0.8 microl x min(-1)) were observed under control conditions. Volume expansion resulted in large significant increases in NaCl clearance in both WT mice ( C(Na)=7.0 +/- 0.9 microl x min(-1), C(Cl)=12.0 +/- 0.6 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=7.2 +/- 1.6 microl x min(-1), C(Cl)=11.0 +/- 2.2 microl x min(-1)). However, there was no significant difference between WT and DeltaF508-CFTR mice. In conclusion, renal NaCl excretion is not significantly different under basal conditions and during saline volume expansion in DeltaF508-CFTR mice. The data suggest that CFTR is not a physiologically important mediator of volume natriuresis.

Anesthesia↗

PubMed2001

Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium.

Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.

Acetylcysteine↗

PubMed2001

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter.

For effective gene therapy of chronic disease, persistent transgene expression at therapeutic levels is required. Clinical studies of airway gene transfer in patients with cystic fibrosis (CF) have resulted in short-lived transgene expression. We used intra-nasal dosing of naked plasmid DNA to the murine lung as a model for investigating the duration of airway gene transfer from a series of reporter expression plasmids. Transgene expression was transient when mediated by the viral promoters CMV, RSV and SV40, falling to less than 10% of peak expression after 2 weeks, although the presence of the adenoviral E4ORF3 gene in cis, resulted in extended duration of reporter activity from the CMV promoter. Transient expression from these promoters was not due to loss of the vector as determined by quantitative TaqMan PCR analysis. However, use of the promoters from the human polybiquitin C (UbC) and the elongation factor 1alpha (EF1alpha) genes resulted in persistent gene expression in the mouse lung. The UbC promoter directed high-level reporter activity which was maintained for up to 8 weeks and was still detectable 6 months after a single administration. Such persistent airway transgene expression from a nonviral vector without the concomitant expression of a potential antigen has not been reported previously. Thus, despite the persistence of vector DNA in vivo, attenuation of promoter function may lead to silencing of transgene expression and careful selection of promoter sequences is recommended for in vivo gene transfer.

Administration, Inhalation↗

PubMed2001

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo.

Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.

Animals↗

PubMed2001

Inflammation in cystic fibrosis airways: relationship to increased bacterial adherence.

It is unclear whether inflammation in the cystic fibrosis (CF) lung relates predominantly to bacterial infection, or occurs as a direct consequence of mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. Interleukin (IL)-8 secretion from CF and non-CF cell lines, and from CF and non-CF human primary nasal epithelial cells incubated with or without Pseudomonas aeruginosa, was measured. Activation of nuclear factor-kappaB (NF-kappaB) in unstimulated CF and non-CF nasal epithelial cells, cell lines and murine tissues was measured by gel-shift assays. No significant difference in basal IL-8 production or NF-kappaB activation was observed between CF and non-CF primary nasal cells. However, CF cells exhibited a significantly (p<0.01) increased IL-8 secretion following P. aeruginosa stimulation. Equalization of the increased P. aeruginosa adherence observed in CF cells, to non-CF levels, resulted in comparable IL-8 secretion. Further, IL-8 production did not differ with mutations which result in either correctly localized CFTR, or in partial/total mislocalization of this protein. Similar levels of NF-kappaB activation were observed in a number of organs of wildtype and CF mice. Finally, IL-8 secretion and NF-kappaB activity were not consistently increased in CF cell lines. Cos-7 cell transfection with plasmids expressing deltaF508 or G551D mutant CFTR protein resulted in increased activation of a p50-containing NF-kappaB complex, but IL-8 secretion was similar to wild-type cells. The authors conclude that the stimulus produced by Pseudomonas aeruginosa is the predominant inflammatory trigger in their models.

Adolescent↗

PubMed2001

Acetylcholine induces cytosolic Ca2+ mobilization in isolated distal colonic crypts from normal and cystic fibrosis mice.

In intestinal biopsies from cystic fibrosis (CF) patients acetylcholine fails to elicit a chloride secretion response, and this observation can be explained by a defect in the Ca2+ signalling pathway in CF secretory cells. We tested the hypothesis that in CF intestine, the generation of an intracellular Ca2+ signal upon cholinergic stimulation is absent. A transgenic CF mouse model was used. Electrical measurements on intact jejunum and unstripped colon were performed in Ussing chambers. Intact distal colonic crypts were isolated, and the intracellular Ca2+ concentration was monitored using the Ca2+-sensitive dye fura-2. Acetylcholine increased the short-circuit current generated by wild-type jejunum and colon, but failed to induce a response in CF tissues. Acetylcholine caused a transient elevation in the intracellular Ca2+ concentration in colonic crypts from both wild-type and CF mice; the amplitude and timing of the response in CF crypts was indistinguishable from that in wild-type crypts. The response to acetylcholine was also observed in the absence of extracellular calcium, indicating intracellular stores as the source from which the cytosolic Ca2+ concentration increased. We conclude that the absence of a cholinergically-induced secretory response in CF intestine is not due to a defect in the generation of a Ca2+ signal in intestinal cells upon cholinergic stimulation.

Acetylcholine↗

PubMed2000

Evidence for cystic fibrosis transmembrane conductance regulator-dependent sodium reabsorption in kidney, using Cftr(tm2cam) mice.

The aims of this study were to investigate (a) if renal Na(+) handling was normal in Cftr(tm2cam) delta F508 cystic fibrosis mice, (b) whether adaptation to dietary salt depletion was preserved and (c) whether Cftr(tm2cam) delta F508 mice exhibited enhanced amiloride-sensitive Na(+) absorption. In Na(+)-replete animals (maintained on a 0.32 % NaCl diet) given a 150 mM NaCl i.v. maintenance infusion, there was no difference in fractional Na(+) excretion (FE(Na)) between wild-type (0. 42 +/- 0.06 %, n = 12) and Cftr(tm2cam) delta F508 mice (0.47 +/- 0.13 %, n = 7). Amiloride infusion significantly increased FE(Na) in both wild-type (3.14 +/- 0.83 %, n = 6) and Cftr(tm2cam) delta F508 mice (3. 47 +/- 0.63 %, n = 9), though with no significant difference between genotypes. A 14 day dietary salt restriction (animals maintained on a 0.03 % NaCl diet) and maintenance infusion with a 15 mM NaCl vehicle caused a reduction in FE(Na) to 0.14 +/- 0.05 %, n = 8 in wild-type mice and 0.14 +/- 0.04 %, n = 8 in Cftr(tm2cam) delta F508 mice. No significant difference in the ability to adapt to low salt conditions was apparent comparing the two genotypes. Treatment of salt-restricted mice with amiloride resulted in a blunted natriuresis in both wild-type mice (FE(Na) = 1.10 +/- 0.16 %, n = 7) and Cftr(tm2cam) delta F508 mice (FE(Na) = 1.97 +/- 0.29 %, n = 9). The natriuresis induced by amiloride was significantly greater in Cftr(tm2cam) delta F508 mice than in wild-type controls. In conclusion, Cftr(tm2cam) delta F508 mice exhibit normal renal salt excretion when either salt replete or salt restricted. Enhanced amiloride-sensitive FE(Na) is consistent with increased Na(+) absorption via the amiloride-sensitive sodium channel ENaC, in cystic fibrosis kidney, but this was only observed during salt restriction.

Amiloride↗

PubMed2000

Eomesodermin is required for mouse trophoblast development and mesoderm formation.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Amino Acid Sequence↗

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↗