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Biomedical subjects

R C Boucher

Publications and source records attributed to R C Boucher.

At least 109 records · Page 6Linked to original sources
PubMed1996

Cytokine production by cultured human bronchial epithelial cells infected with a replication-deficient adenoviral gene transfer vector or wild-type adenovirus type 5.

Exposure of animals to adenoviral gene transfer vectors has been associated with respiratory tract inflammation. The pathogenesis of this inflammation is unclear. One hypothesis is that viral vectors directly induce production of inflammatory cytokines by host cells in the airways. We exposed cultured human lung cells to an adenovirus-5--based vector containing the cytomegalovirus promoter and lacZ reporter gene (Ad.CMV.lacZ) and to wild-type adenovirus 5 (wtAd5) and measured subsequent release of cytokines into cell culture supernatants. Inoculation of human bronchial epithelial (HBE) cells with Ad.CMV. lacZ at 10(1) to 10(4) plaque-forming units (pfu)/cell resulted in dose-related expression of lacZ by both X-gal staining and immunohistochemistry but did not increase release of interleukin (IL)-8 or IL-6 at 24, 48, or 96 h after inoculation. In the same cultures, tumor necrosis factor-alpha induced marked increases in release of both IL-8 and IL-6 at 24 and 48 h after stimulation. Similar data were observed in the BEAS-2B HBE cell line. HBE cells incubated with wtAd5 at doses of 10(1) to 10(3) pfu/cell did not release increased amounts of IL-6 or IL-8 up to 48 h after inoculation, though wild-type respiratory syncytial virus (3 pfu/HBE cell) infection resulted in increases in both cytokines. Human alveolar macrophages obtained by bronchoalveolar lavage also showed no increases in cytokine release after incubation with Ad.CMV.lacZ, though relatively little gene transfer occurred in macrophages. These data do not support a role for direct induction of airway epithelial or alveolar macrophage inflammatory cytokines in the pathogenesis of inflammation associated with exposure of airways to adenovirus or to adenoviral gene transfer vectors.

Adenoviruses, Human↗
PubMed1995

Membrane-restricted regulation of Ca2+ release and influx in polarized epithelia.

Epithelial cells exist in a complex setting in which responses to mucosal or serosal environments are mediated by receptors expressed on specialized cellular domains, such as apical versus basolateral cell membranes. We investigated whether airway epithelia can react selectively through G-protein-coupled receptors to stimuli in the mucosal or serosal environments by measuring inositol phosphate and intracellular Ca2+ responses in polarized human nasal epithelial monolayers. We report here that unilateral ATP (10(-4) M) administration stimulated P2 purinoceptors and tapped pools of intracellular Ca2+ associated with the plasma membrane ipsilateral but not contralateral to stimulated receptors. Similarly, activation of plasma membrane Ca2+ influx by ATP was confined to the membrane ipsilateral to receptor stimulation. These findings demonstrate that polarized epithelia restrict P2 receptor-mediated responses to a single domain of the cell, reflecting membrane-specific generation and catabolism of inositol phosphates and confinement of calcium influx regulation to the membrane ipsilateral to the stimulated receptors.

Adenosine Triphosphate↗
PubMed1995

CFTR as a cAMP-dependent regulator of sodium channels.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.

3T3 Cells↗
PubMed1995

In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis.

Cystic fibrosis (CF) is a monogenetic disease that is associated with chronic airways disease and early death. The pulmonary disease reflects mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and associated abnormal epithelial ion transport, including defective cAMP-mediated (CFTR) Cl- secretion and an accelerated rate of basal Na+ transport. With the development of vectors for gene therapy, the airway epithelium of CF patients has been targeted for studies of gene transfer. The biological efficacy of gene transfer of the normal CFTR cDNA into CF respiratory epithelia can be assessed by in vivo measurements of the transepithelial potential difference (PD), a parameter of ion transport that reflects the expression and function of CFTR. This paper describes techniques that can be used to discriminate in vivo between the ion transport phenotype of normal subjects and patients with cystic fibrosis. Protocols are outlined to allow assessment of individual components of the electrolyte transport phenotype, i.e., the magnitude of the basal and cAMP-mediated (CFTR) Cl- secretion, and the rate of Na+ transport. The physiologic basis of the protocols and important technical features of these measurements are defined. If performed properly, the in vivo nasal PD technique clearly discriminates between normal subjects and cystic fibrosis patients, and can yield estimates of the biological efficacy of gene transfer to achieve correction of the electrolyte transport defects in CF patients.

Adolescent↗
PubMed1995

Effect of adenoviral vector infection on cell proliferation in cultured primary human airway epithelial cells.

Although recombinant adenoviruses are used as vectors for delivering therapeutic genes to the airways of cystic fibrosis (CF) patients, the effects of these vectors on the kinetics of airway epithelial cell growth have not been investigated. We tested whether E1, E3-deleted Ad vectors (Ad5-CMV-lacZ) affect the kinetics of cell proliferation of human airway epithelial cells in primary culture. There was a dose-dependent relationship between the vector multiplicity of infection (moi) and the efficiency of Ad-mediated lacZ gene transfer. Growth curves of cells exposed to vector were shifted to the right as compared to vehicle in a dose-dependent manner. The vector-induced slowing of cell proliferation resulted from both (i) increased apoptotic cell death and (ii) lower recruitment into S phase. UV inactivation of the vector genes abolished the effects on cell proliferation. These data demonstrate that as the moi of vectors is increased to achieve effective gene transfer, apoptosis and slowing of the cell cycle of infected cells increases concomitantly. The identification and inactivation of these vector effects on human airway cells may be important for reducing the toxicity of adenovirus vectors for gene therapy of CF airways.

Adenoviruses, Human↗
PubMed1995

Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by diadenosine tetraphosphate.

1. The human P2U-purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis. 2. High basal levels of inositol phosphates occurred in P2U-purinoceptor-expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3. UTP, ATP and ATP gamma S were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 microM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y-, P2X-, and P2T-purinoceptor agonists, 2MeSATP, alpha,beta-MeATP, and 2MeSADP, respectively. 4. Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U-purinoceptor with an EC50 (720 nM) in the range of the most potent P2U-purinoceptor agonists. Ap4A may be a physiologically important activator of P2U-purinoceptors.

Adenine↗
PubMed1995

Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP.

Luminal extracellular ATP evoked a bumetanide-sensitive short-circuit current in cultured T84 cell epithelia (90.2 +/- 18.2 microA/cm2 at 100 microM ATP, apparent 50% effective concentration, 11.5 microM). ATP appeared to increase the Cl- conductance of the apical membrane but not the driving force for Cl- secretion determined by basolateral membrane K+ conductance. Specifically, the magnitude of Cl- secretion stimulated by ATP was independent of basal current, and forskolin pretreatment abolished subsequent stimulation of Cl- secretion by ATP. Whereas ATP stimulated modest production of adenosine 3',5'-cyclic monophosphate (cAMP) by T84 cells, ATP caused smaller increases in intracellular Ca2+ and inositol phosphate activities than the Ca(2+)-signaling Cl- secretagogue carbachol. An inhibitor of 5'-nucleotidase, alpha,beta-methyleneadenosine 5'-diphosphate, blocked most of the response to luminal ATP. The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline blocked both the luminal ATP-dependent generation of cAMP and Cl- secretion when administered to the luminal but not submucosal bath. These results demonstrate that the Cl- secretion stimulated by luminal ATP is mediated by a A2-adenosine receptor located on the apical cell membrane. Thus metabolism of extracellular ATP to adenosine regulates the activity of cystic fibrosis transmembrane conductor regulator (CFTR) in the apical membrane of polarized T84 cells.

Adenosine Triphosphate↗
PubMed1995

cAMP- but not Ca(2+)-regulated Cl- conductance in the oviduct is defective in mouse model of cystic fibrosis.

Defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- transport in cystic fibrosis (CF) reflects defects in the cystic fibrosis transmembrane conductance regulator (CFTR). A moderate level of CFTR mRNA expression has been found in rodent and human oviductal epithelium, but unlike other CFTR-expressing tissues, the oviduct in CF patients is apparently normal. The present study was carried out to investigate the relative magnitude of the cAMP- and intracellular Ca2+ (Cai2+)-regulated Cl- secretion in primary cultures of the oviduct from normal and CF mice generated by targeted disruption of the murine CF gene. Normal oviductal epithelium exhibited a basal equivalent short-circuit current (Ieq) of 20.3 +/- 1.7 muA/cm2. CF oviduct exhibited a lower basal Ieq of 4.5 +/- 1.9 muA/cm2. In normal mice, forskolin (10(-5) M, apical) elicited a slowly developing sustained rise in Ieq, whereas ionomycin (5 x 10(-6) M, apical) and ATP (10(-4) M, apical) induced larger increases in Ieq consisting of a prompt, transient response followed by a slowly decreasing component. The Ieq response to forskolin was totally abolished in CF mouse oviducts, but the magnitudes of the peak Ieq responses to ionomycin and ATP were not different from normal. The time courses of the ionomycin- and ATP-evoked responses, however, were significantly more transient in CF than in normal oviducts. These results demonstrate that CF mouse oviduct exhibits defective cAMP- but not Cai(2+)-mediated Cl- secretion. The relatively high level of functional expression of the alternative Cai(2+)-activated Cl- secretory pathway in the mouse oviduct may contribute to the absence of major pathology in the CF oviduct.

Adenosine Triphosphate↗
PubMed1995

Relative expression of the human epithelial Na+ channel subunits in normal and cystic fibrosis airways.

The availability of the newly cloned subunits (alpha, beta, gamma) of the epithelial Na+ channel (ENaC) permits molecular studies of the pathogenesis of the abnormal Na+ transport rates of cystic fibrosis (CF) airway epithelia. Northern analyses of airway epithelia showed that both normal and CF airway epithelia express ENaC subunit mRNAs in a ratio of alpha > beta > gamma. In situ hybridization studies revealed expression of all three ENaC subunits in the superficial epithelium and the alpha- and beta-subunits in the gland ductular and acinar epithelium of both normal and CF airways. Ribonuclease protection assays revealed that the steady-state levels of alpha-, beta-, and gamma-ENaC mRNAs were similar in CF and normal airway superficial epithelia. These findings indicate that 1) Na+ transport defects in CF airways disease may be expressed in glandular acinar and ductal epithelium as well as superficial epithelium, and 2) the molecular pathogenesis of Na+ hyperabsorption in CF airways does not reflect increased levels of Na+ channel mRNAs, and probably number, but reflects an absence of the normal inhibitory regulation of Na+ channels by CF transmembrane conductance regulator proteins.

Base Sequence↗
PubMed1995

Bioelectric properties of cultured monolayers from epithelium of distal human fetal lung.

Throughout intrauterine life, Cl(-)-rich liquid is secreted by the pulmonary epithelium. To evaluate the role of the most distal epithelium in liquid secretion, we measured bioelectric properties of monolayers composed of epithelial cells from acinar structures of postmortem human fetal lung (mean gestation, 22.3 wk; range, 18-24 wk). These monolayers formed high-resistance (R) barriers (mean R = 363 Ohm/cm2) when cultured in hormone-supplemented, serum-free medium. The transepithelial electrical potential difference (4.0 mV, lumen negative), was similar to that of whole fetal sheep lung in vivo. Equivalent short-circuit current (Ieq) was inhibited by apical amiloride (-20%), 5-(N-ethyl-N-isopropyl)-amiloride (-33 to -49%), or diphenylamine-2-carboxylate (DPC; -26%), and by basolateral ouabain (-77%), whereas apical 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) had no effect. Bumetanide added to the basolateral bath did not affect resting Ieq, but inhibited Ieq (-19%) in monolayers pretreated with apical amiloride, basolateral terbutaline, and apical ATP, and also inhibited Ieq (-22%) of monolayers pretreated with basolateral amiloride and DIDS. Ieq was stimulated by terbutaline (90-128%), ATP (70-186%), and ionomycin (141%). Stimulation of Ieq by these agents is compatible with induction of Cl- secretion through two pathways: channels that are opened by a rise in adenosine 3',5'-cyclic monophosphate, and channels that are opened by a rise in intracellular Ca2+. Inhibition of Ieq by apical DPC implies that Cl- secretion may contribute to basal Ieq.

Cell Membrane↗
PubMed1995

Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency.

Ciliated airway epithelial cells contribute to mucociliary transport systems via ciliary beating and electrolyte transport mechanisms. Both of these activities are regulated by agonists acting through intracellular calcium- and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent processes (5, 15, 18, 27). This study examines the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the regulation of both ciliary beat frequency (CBF) and electrolyte transport in human airway epithelia (HAE). In a previous report, cGMP production in HAE was observed after stimulation with either C-type natriuretic peptide (CNP) or sodium nitroprusside (SNP) (6). In this study, CNP was found to increase CBF by 30 +/- 6.9%, and this effect was mimicked by the cGMP analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), but not by sodium nitroprusside. CNP-induced increases in CBF do not appear to be mediated by changes in either intracellular calcium or cAMP levels. Using modified Ussing chambers, we also investigated CNP's potential modulation of sodium and chloride transport rates. Neither CNP, nor SNP, nor 8-BrcGMP altered active ion transport rates. We conclude that CNP regulates ciliary beat via cGMP-dependent mechanisms, whereas no effect of CNP or cGMP on ion transport was detected.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Screening Young syndrome patients for CFTR mutations.

Young syndrome is characterized by obstructive azoospermia associated with chronic sinobronchial disease of an infectious nature, but normal sweat-gland and pancreatic function as well as normal nasal potential differences. Congenital bilateral absence of the vas deferens (CBAVD) in some patients arises from mutations within the cystic fibrosis (CF) transmembrane regulator (CFTR) gene. Because of some similarities between Young syndrome, CF, and CBAVD, we evaluated 13 patients with Young syndrome, including screening for more than 30 different mutations within the CFTR gene. The mean age of the patients was 43 yr (range, 32 to 50 yr), and all were of northern European extraction. The sweat chloride concentration was normal in all patients (mean = 29 mEq/L; range, 8 to 43 mEq/L). Most had intermittent bronchial and sinus infections, but none was chronically colonized with Staphylococcus aureus or Pseudomonas aeruginosa. The FEV1 was normal or only mildly reduced in most patients (mean = 74%; range, 48 to 100% predicted). Of 26 Young syndrome chromosomes, we identified one with the recognized CF mutation delta F508. The incidence of CFTR mutations (1 in 26) did not differ significantly from the expected carrier frequency in this population. In summary, it is unlikely that the typical Young syndrome patient has a clinical disease associated with CFTR mutation on both alleles.

Adult↗
PubMed1995

Pharmacologic modulation of salt and water in the airway epithelium in cystic fibrosis.

Cystic fibrosis (CF) is a recessive genetic disease with thickened airway secretions that result from abnormal airway epithelial ion transport, including defective cyclic AMP-mediated Cl- (liquid) secretion and excessive Na+ (liquid) absorption. These abnormalities reflect mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which normally functions as a cyclic AMP-regulated Cl- channel. Aerosolized pharmacologic agents are being tested as novel treatment for these genetic ion transport defects. Amiloride aerosol inhibits excessive Na+ absorption, and pilot studies in adult patients with CF show improved biorheology and mucociliary clearance of airway secretions, as well as slowing of the decline in lung function. Phase III studies of amiloride in adults and adolescents are underway, and short-term safety studies in children are under way. Aerosolized uridine triphosphate (UTP) induces Cl- secretion in CF airway epithelia via non-CFTR Cl- channels. Initial safety studies suggest that acute aerosolized UTP is well tolerated, and acute studies of the effect on mucociliary clearance are underway. Pharmacotherapy that targets abnormal ion transport holds promise for the treatment of CF airway disease.

Adolescent↗
PubMed1995

Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients.

Integrative gene therapy typically requires dividing cells. This requirement has been perceived as an impediment for gene transfer to mature, uninjured airways where proliferation rates are very low. In diseases such as cystic fibrosis (CF) that may be candidates for integrative gene therapy, airway cell turnover is not known but may be increased as a result of chronic inflammation. To determine if cells in airway surface epithelium and submucosal glands of CF patients proliferate at an increased rate, paraffin sections of bronchial segments removed from CF patients (n = 6) at the time of lung transplantation or rapid autopsy and from non-CF patients (n = 4) undergoing lung resection or transplantation were immunostained with PC10, a monoclonal antibody to proliferating cell nuclear antigen (PCNA), a marker of proliferating cells. The PCNA index (percentage of nuclei immunostaining for PCNA) in CF bronchial surface epithelium was 17.0 +/- 4.6% (mean +/- SEM), substantially greater than in non-CF airways (less than 0.2%). Within submucosal glands, PCNA-positive cells were more prevalent in the collecting ducts of CF patients than in those of normal subjects, but only rare mucous or serous cells were PCNA positive. These studies show that airway epithelial cell proliferation rates can be very high in inflamed CF airways. This prevalence of proliferating cells suggests that CF airway epithelium and submucosal gland ducts may be amenable to gene transfer using vectors, such as retroviruses, that require cell replication for stable integrative expression. Further studies are needed to evaluate cell proliferation in CF airways with less extensive airway injury.

Adolescent↗
PubMed1995

Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse.

The absence of pathologic changes in newborn cystic fibrosis (CF) lung suggests that the fetal CF lung is inflated with a normal volume of liquid and that Cl- is secreted through paths other than the cystic fibrosis transmembrane conductance regulator (CFTR)-associated Cl- channel. We studied liquid content of distal lung and transepithelial electrical potential difference (PD) of cultured cystic tracheal explants from 16 to 19 day gestation fetal mice of CFTR (+/-)(heterozygous) females that were mated with CFTR (-/-) "knockout" males. Distal lung water content was not affected by fetal genotype. Basal PDs were not different (CFTR (+/-), 8.6 mV, and CFTR (-/-), 9.1 mV), and PDs of both groups were inhibited by intraluminal injection of amiloride (10(-4) M) (-25%) and after addition of bumetanide (10(-4) M) to the bath (-40%). Terbutaline (3 x 10(-5) M) induced a similar increase in PD (about 65%) in both groups. Intraluminal injection of ionomycin (2 x 10(-5) and 5 x 10(-6) M) raised PD in both groups (CFTR (+/-) by 32 and 27% and CFTR (-/-) by 41 and 11%). All of the increase in PD induced by terbutaline and ionomycin was inhibited by bumetanide. The PD response to terbutaline was not attenuated by pretreatment with ionomycin or the Ca2+ chelator BAPTA (10(-4) M). Ionomycin or ATP, but not terbutaline, increased intracellular Ca2+ concentration of isolated cultured tracheal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.

Adenoviridae↗
PubMed1995

Alteration of sulfation of glycoconjugates, but not sulfate transport and intracellular inorganic sulfate content in cystic fibrosis airway epithelial cells.

The secreted and cell surface high molecular weight glyco-conjugates (HMG) generated by primary cultures of airway epithelial cells from cystic fibrosis (CF) patients are oversulfated. To determine whether this abnormality is maintained in transformed CF airway epithelial cells and whether differences in transport or intracellular accumulation of sulfate can explain this alteration, we assessed sulfate metabolism in paired CF and normal cell lines as well as primary cultures of CF and normal cells. Both 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-inhibitable and -resistant [35S]sulfate efflux and influx were identical for each pair of CF and normal cell lines. Furthermore, cell content of inorganic sulfate was not significantly different in CF and normal cells. However, compared with primary CF cells that oversulfate HMG transformed CF cells oversulfated cell surface HMG but not HMG released into culture medium. Our results suggest that plasma membrane sulfate transport is not altered in CF airway epithelial cells and the abnormal sulfation of HMG may be due to perturbation in intracellular sulfate activation or transfer of activated sulfate to HMG. The relationship of this abnormality to CF transmembrane conductance regulator mutations remains to be determined.

Biological Transport↗