Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice

This article is available open access through the publisher’s website at the link below. Copyright © 2008 The American Society of Gene Therapy.Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero –injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (CftrtmlCam) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr–/– mice treated with the CFTR-adenovirus and those treated with the control vector.Sport Aiding Medical Research for Kids, the Cystic Fibrosis Trust, and the Katharine Dormandy Trust

The state of integrated disease surveillance in seven countries: a synthesis report

publishedVersio

The K+ Channel Opener 1-EBIO Potentiates Residual Function of Mutant CFTR in Rectal Biopsies from Cystic Fibrosis Patients

BACKGROUND: The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K⁺ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl⁻ secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown. METHODS: We studied the effects of 1-EBIO on CFTR-mediated Cl⁻ secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl⁻ secretion. RESULTS: Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl⁻ secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl⁻ secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca²⁺-activated and clotrimazole-sensitive KCNN4 K⁺ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl⁻ secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl⁻ conductance. CONCLUSIONS: We conclude that 1-EBIO potentiates Cl⁻secretion in native CF tissues expressing CFTR mutants with residual Cl⁻ channel function by activation of basolateral KCNN4 K⁺ channels that increase the driving force for luminal Cl⁻ exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF

Lubiprostone ameliorates the cystic fibrosis mouse intestinal phenotype

<p>Abstract</p> <p>Background</p> <p>Cystic fibrosis (CF) is caused by mutations in the <it>CFTR </it>gene that impair the function of CFTR, a cAMP-regulated anion channel. In the small intestine loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have small intestinal bacterial overgrowth, an altered innate immune response, and impaired intestinal transit. We investigated whether lubiprostone, which can activate the CLC2 Cl^-channel, would improve the intestinal phenotype in CF mice.</p> <p>Methods</p> <p><it>Cftr^tm1UNC</it>(CF) and wildtype (WT) littermate mice on the C57BL/6J background were used. Lubiprostone (10 μg/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in periodic acid-Schiff base, Alcian blue stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16<it>S </it>gene. Gastric emptying and small intestinal transit in fasted mice were assessed using gavaged rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray and qRT-PCR.</p> <p>Results</p> <p>Crypt width in control CF mice was 700% that of WT mice (<it>P </it>< 0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (<it>P </it>= 0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (<it>P </it>= 0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (<it>P </it>= 0.005). Lubiprostone increased gastric emptying at 20 min postgavage in both WT (<it>P </it>< 0.001) and CF mice (<it>P </it>< 0.001). Lubiprostone enhanced small intestinal transit in WT mice (<it>P </it>= 0.024) but not in CF mice (<it>P </it>= 0.377). Among other innate immune markers, expression of mast cell genes was elevated 4-to 40-fold in the CF intestine as compared to WT, and lubiprostone treatment of CF mice decreased expression to WT control levels.</p> <p>Conclusions</p> <p>These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion.</p

The effect of fluoride on the structure, function, and proteome of intestinal epithelia.

Fluoride exposure is widespread, with drinking water commonly containing natural and artificially added sources of the ion. Ingested fluoride undergoes absorption across the gastric and intestinal epithelia. Previous studies have reported adverse gastrointestinal effects with high levels of fluoride exposure. Here, we examined the effects of fluoride on the transepithelial ion transport and resistance of three intestinal epithelia. We used the Caco-2 cell line as a model of human intestinal epithelium, and rat and mouse colonic epithelia for purposes of comparison. Fluoride caused a concentration-dependent decline in forskolin-induced Cl- secretion and transepithelial resistance of Caco-2 cell monolayers, with an IC50 for fluoride of about 3 mM for both parameters. In the presence of 5 mM fluoride, transepithelial resistance fell exponentially with time, with a t1/2 of about 7 hours. Subsequent imaging by immunofluorescence and scanning electron microscopy showed structural abnormalities in Caco-2 cell monolayers exposed to fluoride. The Young's modulus of the epithelium was not affected by fluoride, although proteomic analysis revealed changes in expression of a number of proteins, particularly those involved in cell-cell adhesion. In line with its effects on Caco-2 cell monolayers, fluoride, at 5 mM, also had profound effects on Cl- secretion and transepithelial resistance of both rat and mouse colonic epithelia. Our results show that treatment with fluoride has major effects on the structure, function, and proteome of intestinal epithelia, but only at concentrations considerably higher than those likely to be encountered in vivo, when much lower fluoride doses are normally ingested on a chronic basis

Cyclic AMP-Dependent Regulation of Kv7 Voltage-Gated Potassium Channels

Voltage-gated Kv7 potassium channels, encoded by KCNQ genes, have major physiological impacts cardiac myocytes, neurons, epithelial cells, and smooth muscle cells. Cyclic adenosine monophosphate (cAMP), a well-known intracellular secondary messenger, can activate numerous downstream effector proteins, generating downstream signaling pathways that regulate many functions in cells. A role for cAMP in ion channel regulation has been established, and recent findings show that cAMP signaling plays a role in Kv7 channel regulation. Although cAMP signaling is recognized to regulate Kv7 channels, the precise molecular mechanism behind the cAMP-dependent regulation of Kv7 channels is complex. This review will summarize recent research findings that support the mechanisms of cAMP-dependent regulation of Kv7 channels

A note on the concentrations of steroidal residues in tissues of mature female sheep implanted with trenbolone acetate

Thirty-two Blackface female mature sheep weighing 45 kg on average were blocked by weight and randomly allocated 60 days before slaughter to be untreated controls (C) or subcutaneously implanted with 20 (TA1), 40 (TA2) or 60 (TA3) mg trenbolone acetate (TBA). Samples of blood were collected throughout the study and post-mortem samples of selected body tissues were analysed residues.Concentrations in blood of 17β-hydroxy-trenbolone (TBOH) peaked within 1 to 2 weeks of implantation and declined thereafter. Concentrations in blood of oestradiol-17β (OE) were elevated and generally increased with increasing dose levels of TBA. The highest concentrations of solvent extractable residues (TBOH for group TA3) ranged from 613 ng/kg in kidney to 130 ng/kg for rib muscle. Trenbolone-17β- glucuronide was detected in liver and kidney at maximum concentrations of 157 ng/kg and 94 ng/kg respectively. Implantation with TBA had no effect on concentrations of OE in any of the post-mortem tissues examined.</jats:p

The effect of implantation of trenbolone acetate and oestradiol-17β in wether lambs at two initial live weights on concentrations of steroidal residues and blood glucose, urea and thyroid hormones

AbstractThirty-two Border Leicester ♂ × Scottish Blackface ♀ wether lambs aged about 5 months were divided into two groups on the basis of live weight such that group Gl contained the 16 lightest lambs and group G2 the 16 heaviest. Lambs in group Gl were subdivided equally at random either to be sham-implanted controls (group C1) or to be implanted with 35 mg trenbolone acetate (TBA) + 5 mg oestradiol-17β (group T1) at 24 kg initial live weight. The lambs in group G2 were also subdivided into two groups (groups C2 and T2) and similarly treated approximately 1 month later at 37 kg initial live weight. The lambs were offered ad libitum a good-quality diet. They were slaughtered 60 days after implantation. Comparisons were made for the main effects of hormonal treatment and initial live weight.Concentrations in blood of 17-β-hydroxy-trenbolone (TBOH) and oestradiol-17β (OE) measured by radioimmunoassay peaked within 1 to 3 weeks after implantation and declined thereafter. Maximum concentrations and concentrations at slaughter respectively were 1·46 and 0·32 μg/l (group T1) and 0·78 and 0·28 μg/l (T2) for TBOH and 85 and 33 μg/1 (T1) and 59 and 37 ng/l (T2) for OE. Values up to week 7 were consistently greater in implanted animals in group T1 than in group T2. Hormonal implantation decreased the concentrations of total plasma triiodothyronine and thyroxine and urea and increased values for glucose up to week 5 or 6 after implantation. The animals in group G1 compared with G2 had, on average, variably lower concentrations in plasma of triiodothyronine, thyroxine, glucose and urea.The highest concentrations of solvent-extractable residues were obtained in samples of kidney and liver (up to about 500 ng/kg for TBOH and 180 ng/kg for OE) with intermediate levels for fat and lowest levels for muscle. Conjugated trenbolone ranging from 45 to 186 ng/kg was present in samples of kidney, liver and perinephric fat. Trenbolone acetate was detected only in samples of fat. Variable effects of live weight at implantation on residue levels were recorded.</jats:p