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Biomedical subjects

P B McCray

Publications and source records attributed to P B McCray.

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

A novel murine beta -defensin expressed in tongue, esophagus, and trachea.

beta-Defensins are broad spectrum antimicrobial peptides expressed at epithelial surfaces. Two human beta-defensins, HBD-1 and HBD-2, have been identified. In the lung, HBD-2 is an inducible product of airway epithelia and may play a role in innate mucosal defenses. We recently characterized rat homologs (RBD-1, RBD-2) of the human genes and used these sequences to identify novel mouse genes. Mouse beta-defensin-4 (MBD-4) was amplified from lung cDNA using polymerase chain reaction primers designed from conserved sequences of RBD-2 and HBD-2. A full-length cDNA was cloned which encodes a putative peptide with the sequence MRIHYLLFTFLLVLLSPLAAFTQIINNPITCMTNGAICWGPCPTAFRQIGNCGHFKVRCCKIR. The peptide shares approximately 40% identity with HBD-2. MBD-4 mRNA was expressed in the esophagus, tongue, and trachea but not in any of 20 other tissues surveyed. Cloning of the genomic sequence of MBD-4 revealed two nearly (>99%) identical sequences encoding MBD-4 and the presence of numerous additional highly similar genomic sequences. Radiation hybrid mapping localized this gene to a region of chromosome 8 near several other defensins, MBD-2, MBD-3, and alpha-defensins (cryptdins)-3 and -17, consistent with a gene cluster. Our genomic cloning and mapping data suggest that there is a large beta-defensin gene family in mice. Identification of murine beta-defensins provides an opportunity to understand further the role of these peptides in host defense through animal model studies and the generation of beta-defensin-deficient animals by gene targeting.

Amino Acid Sequence↗
PubMed2000

Bactericidal activity of mammalian cathelicidin-derived peptides.

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.

Animals↗
PubMed2000

Human coronavirus 229E infects polarized airway epithelia from the apical surface.

Gene transfer to differentiated airway epithelia with existing viral vectors is very inefficient when they are applied to the apical surface. This largely reflects the polarized distribution of receptors on the basolateral surface. To identify new receptor-ligand interactions that might be used to redirect vectors to the apical surface, we investigated the process of infection of airway epithelial cells by human coronavirus 229E (HCoV-229E), a common cause of respiratory tract infections. Using immunohistochemistry, we found the receptor for HCoV-229E (CD13 or aminopeptidase N) localized mainly to the apical surface of airway epithelia. When HCoV-229E was applied to the apical or basolateral surface of well-differentiated primary cultures of human airway epithelia, infection primarily occurred from the apical side. Similar results were noted when the virus was applied to cultured human tracheal explants. Newly synthesized virions were released mainly to the apical side. Thus, HCoV-229E preferentially infects human airway epithelia from the apical surface. The spike glycoprotein that mediates HCoV-229E binding and fusion to CD13 is a candidate for pseudotyping retroviral envelopes or modifying other viral vectors.

Cell Line↗
PubMed2000

Synergistic and additive killing by antimicrobial factors found in human airway surface liquid.

Airway surface liquid contains multiple factors thought to provide a first line of defense against bacteria deposited in the airways. Although the antimicrobial action of individual factors has been studied, less is known about how they work in combination. We examined the combined action of six antimicrobial peptides found in airway surface liquid. The paired combinations of lysozyme-lactoferrin, lysozyme-secretory leukocyte protease inhibitor (SLPI), and lactoferrin-SLPI were synergistic. The triple combination of lysozyme, lactoferrin, and SLPI showed even greater synergy. Other combinations involving the human beta-defensins, LL-37, and tobramycin (often administered to cystic fibrosis patients by inhalation) were additive. Because the airway surface liquid salt concentration may be elevated in cystic fibrosis patients, we examined the effect of salt on the synergistic combinations. As the ionic strength increased, synergistic interactions were lost. Our data suggest that the antibacterial potency of airway surface liquid may be significantly increased by synergistic and additive interactions between antimicrobial factors. These results also suggest that increased salt concentrations that may exist in cystic fibrosis could inhibit airway defenses by diminishing these synergistic interactions.

Anti-Bacterial Agents↗
PubMed2000

Increasing epithelial junction permeability enhances gene transfer to airway epithelia In vivo.

Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.

Amiloride↗
PubMed2000

Development of retroviral vectors for gene transfer to airway epithelia.

Retroviral vectors offer several potential advantages for attaining persistent expression of a therapeutic gene in airway epithelia for diseases such as cystic fibrosis. However, several problems have limited their application. Developments in vector production and the advent of lentiviral vectors have increased the investigation of recombinant retrovirus for gene transfer to airway epithelia. In addition, an improved understanding of some of the barriers limiting gene transfer has led to increased transduction efficiencies. The development of novel vector formulations and the use of new envelope pseudotypes are examples of recent findings that are leading to advances in this field.

Animals↗
PubMed1999

Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype.

The epithelial Na+ channel (ENaC) is composed of three homologous subunits: alpha, beta and gamma. We used gene targeting to disrupt the beta subunit gene of ENaC in mice. The betaENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the -/- mice, we found an increased urine Na+ concentration despite hyponatremia and a decreased urine K+ concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to alphaENaC-deficient mice, which die because of defective lung liquid clearance, neonatal betaENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo, the beta subunit is required for ENaC function in the renal collecting duct, but, in contrast to the alpha subunit, the beta subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the betaENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.

Aldosterone↗
PubMed1999

Production of beta-defensin antimicrobial peptides by the oral mucosa and salivary glands.

beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human beta-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected beta-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether beta-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1beta (IL-1beta) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased approximately 16-fold with IL-1beta treatment and approximately 5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human beta-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1beta and LPS. Human beta-defensins may play an important role in the innate defenses against oral microorganisms.

3T3 Cells↗
PubMed1999

Differences in the concentrations of small, anionic, antimicrobial peptides in bronchoalveolar lavage fluid and in respiratory epithelia of patients with and without cystic fibrosis.

Affinity-purified rabbit polyclonal (PAB96-1) and mouse monoclonal (1G9-1C2) antibodies to synthetic H-DDDDDDD-OH, an antimicrobial anionic peptide (AP) originally isolated from ovine pulmonary surfactant, were prepared and used to assess the concentrations of AP-like molecules in human respiratory tract samples. In bronchoalveolar lavage fluids, concentrations of AP-like molecules measured by enzyme-linked immunosorbent assay were significantly lower in 13 patients with cystic fibrosis (CF) (mean +/- standard deviation [SD], 0.78 +/- 0.46 mM) than in 34 patients without CF (1. 30 +/- 0.66 mM) (P = 0.01). In pulmonary tissues of three patients without CF, very little antigen was stained in the apical cytoplasm of the bronchial and bronchiolar epithelium yet robust staining was seen in the alveolar epithelium. In pulmonary tissues of three patients with CF, robust staining of antigen was seen in the apical cytoplasm of the bronchial and bronchiolar epithelium yet no staining was seen in the alveolar epithelium. These results show that AP-like molecules are present in healthy human respiratory tract samples and differ in concentration and location of expression in patients with and without CF.

Animals↗
PubMed1999

Molecular cloning and characterization of rat genes encoding homologues of human beta-defensins.

beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that may play a role in mucosal defenses of several organs. They have been isolated in several species, and in humans, two beta-defensins have been identified. Here, we report the identification of two genes encoding beta-defensin homologues in the rat. Partial cDNAs were found by searching the expressed-sequence-tag database, and primers were designed to generate full-length mRNA coding sequences. One gene was highly similar to the human beta-defensin-1 (HBD-1) gene and mouse beta-defensin-1 gene at both the nucleic acid and amino acid levels and was termed rat beta-defensin-1 (RBD-1). The other gene, named RBD-2, was homologous to the HBD-2 and bovine tracheal antimicrobial peptide (TAP) genes. The predicted prepropeptides were strongly cationic, were 69 and 63 residues in length for RBD-1 and RBD-2, respectively, and contained the six-cysteine motif characteristic of beta-defensins. The beta-defensin genes mapped closely on rat chromosome 16 and were closely linked to the alpha-defensins genes, suggesting that they are part of a gene cluster, similar to the organization reported for humans. Northern blot analysis showed that both RBD-1 and RBD-2 mRNA transcripts were approximately 0.5 kb in length; RBD-1 mRNA was abundantly transcribed in the rat kidney, while RBD-2 was prevalent in the lung. Reverse transcription-PCR indicated that RBD-1 and RBD-2 mRNAs were distributed in a variety of other tissues. In the lung, RBD-1 mRNA expression localized to the tracheal epithelium while RBD-2 was expressed in alveolar type II cells. In conclusion, we characterized two novel beta-defensin homologues in the rat. The rat may be a useful model to investigate the function and contribution of beta-defensins to host defense in the lung, kidney, and other tissues.

Amino Acid Sequence↗
PubMed1999

Human beta-defensin-1 mRNA is transcribed in tympanic membrane and adjacent auditory canal epithelium.

The external auditory canal is less susceptible to infections than the sensitive middle-ear cavity. Since recent research has provided insight to the production of potent antimicrobial peptides from various surface epithelia, we wanted to investigate whether protection of the external auditory canal in part could be explained by the production of human beta-defensin-1 (HBD-1). This particular peptide is known to be constitutively expressed in various surface epithelia, such as airway, skin, and urogenital tissues. By reverse transcriptase PCR we demonstrate HBD-1 mRNA in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNA was not detected in the tympanal epithelium of the eardrum. The widespread presence of mRNA encoding for this broad-spectrum antimicrobial peptide in the meatal skin and tympanic membrane suggests that HBD-1 participates in the innate antimicrobial defense of the external auditory canal and middle-ear cavity.

Defensins↗
PubMed1999

Efficient killing of inhaled bacteria in DeltaF508 mice: role of airway surface liquid composition.

Cystic fibrosis mice have been generated by gene targeting but show little lung disease without repeated exposure to bacteria. We asked if murine mucosal defenses and airway surface liquid (ASL) Cl(-) were altered by the DeltaF508 cystic fibrosis transmembrane conductance regulator mutation. Naive DeltaF508 -/- and +/- mice showed no pulmonary inflammation and after inhaled Pseudomonas aeruginosa had similar inflammatory responses and bacterial clearance rates. We therefore investigated components of the innate immune system. Bronchoalveolar lavage fluid from mice killed Escherichia coli, and the microbicidal activity was inhibited by NaCl. Because beta-defensins are salt-sensitive epithelial products, we looked for pulmonary beta-defensin expression. A mouse homolog of human beta-defensin-1 (termed "MBD-1") was identified; the mRNA was expressed in the lung. Using a radiotracer technique, ASL volume and Cl(-) concentration ([Cl(-)]) were measured in cultured tracheal epithelia from normal and DeltaF508 -/- mice. The estimated ASL volume was similar for both groups. There were no differences in ASL [Cl(-)] in DeltaF508 -/- and normal mice (13.8 +/- 2.6 vs. 17.8 +/- 5.6 meq/l). Because ASL [Cl(-)] is low in normal and mutant mice, salt-sensitive antimicrobial factors, including MBD-1, may be normally active.

Administration, Inhalation↗
PubMed1999

Developmental expression of the epithelial Na+ channel in kidney and uroepithelia.

The epithelial Na+ channel (ENaC) plays an important role in regulating Na+ balance in neonatal and adult life. Using in situ hybridization, we localized alpha-, beta-, and gamma-rat ENaC (rENaC) mRNA in developing rat kidney and uroepithelia. rENaC mRNA was first detectable on fetal day 16, and by fetal day 17, mRNA was abundant in the terminal collecting duct and uroepithelia. After birth, the intensity of the signals for all three subunits increased in the cortical collecting ducts and by 9 days after birth had diminished in the inner medullary collecting ducts. Expression in uroepithelial cells was different. mRNA for beta- and gamma-rENaC, but not alpha-rENaC, was detected in pelvis, ureters, and bladder at all stages of development beyond fetal day 16. By RNase protection assay (RPA), the greatest increase in subunit abundance in the kidney occurred before birth. Between postnatal days 9 and 30, the abundance of beta- and gamma-rENaC decreased relative to alpha-rENaC in outer and inner medulla. The urinary bladder, in contrast, demonstrated the greatest increase in beta- and gamma-rENaC mRNA abundance after birth. We were generally unable to detect alpha-rENaC by RPA in urinary bladder. Feeding weaned rats a diet of high or low NaCl did not change the abundance of any of the subunit mRNAs in bladder. These results demonstrate additional heterogeneity of developmental expression and regulation of ENaC. The differences between the collecting duct and uroepithelial cell rENaC mRNA regulation raise the possibility of significant differences in function.

Aging↗
PubMed1999

Development of the innervation and airway smooth muscle in human fetal lung.

Human and porcine fetal airways have been shown to contract spontaneously from the first trimester, the latter also contracting in response to neural stimulation. Our object was to map immunohistochemically the innervation and its relationship to the airway smooth muscle (ASM) in the human fetal lung from early gestation to the postnatal period. Whole mounts of the bronchial tree were stained with antibodies to the pan-neuronal marker protein gene product 9.5, the Schwann cell marker S-100, and the ASM contractile protein alpha-actin, and imaged using confocal microscopy. By the end of the embryonic period (53 d gestation), the branching epithelial tubules in the primordial lung were covered with ASM to the base of the terminal sacs. An extensive plexus of nerve trunks containing nerve bundles, forming ganglia, and Schwann cells ensheathed the ASM. By 16 wk (canalicular stage), maturation of the innervation was advanced with two major nerve trunks running the length of the bronchial tree, giving rise to varicosed fibers lying on the ASM. An extensive nerve plexus in the mucosa was also present. The distal airways of infants who had died of Sudden Infant Death Syndrome were also covered with smooth muscle and were well innervated. Thus, an essentially complete coat of ASM and an abundant neural plexus ensheathing the airways are an integral part of the branching epithelial tubules from early in lung development.

Embryo, Mammalian↗
PubMed1999

Early-onset inflammatory responses in vivo to adenoviral vectors in the presence or absence of lipopolysaccharide-induced inflammation.

Adenoviral vectors (Ad) have potential for use in pulmonary gene transfer for treating cystic fibrosis (CF). However, Ad may induce inflammation even in the absence of gene expression. Endotoxin from gram-negative bacteria in the airways of CF patients may also induce inflammation, and may further inhibit vector delivery and gene transfer. We used a mouse model to study the time course of Ad-induced lung inflammation and to assess additivity with lipopolysaccharide (LPS)-induced inflammatory responses. C3H/HeJ endotoxin-resistant (RES) mice hyporesponsive to inflammatory stimuli and normoresponsive C3HeB/FeJ endotoxin-sensitive (SEN) mice were studied to characterize inflammatory responses that follow intratracheal instillation of inactivated Ad, with or without simultaneous inhalation exposure to LPS. Instillation of 10(10) Ad particles dramatically increased bronchoalveolar lavage fluid (BALF) concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 at 3 to 6 h and induced profound neutrophilia, maximal at 12 to 24 h. SEN mice had tenfold greater responses than did RES mice at 6, 12, and 24 h. Mice exposed to Ad alone, LPS alone, or Ad + LPS had significant inflammation at the 3-h time point as demonstrated by BALF neutrophils, TNF-alpha, and IL-6. With all three treatments, SEN mice had a five- to 300-fold greater response than did RES mice. Importantly, Ad + LPS yielded no greater inflammatory response than LPS without Ad. These data demonstrate that replication-deficient Ad induce early inflammation and LPS-induced inflammation is not augmented by concurrent treatment with Ad.

Adenoviridae↗
PubMed1999

Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect.

Several problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl(-) transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus-based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl(-) transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing beta-galactosidase transduced 1-14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF. This article may have been published online in advance of the print edition.

Animals↗
PubMed1999

KGF and FGF-10 stimulate liquid secretion in human fetal lung.

During fetal life, the pulmonary epithelium secretes liquid that distends the airways and is important for normal lung growth and development. The factors regulating human fetal lung liquid secretion are poorly understood; however, recent studies in murine models show that keratinocyte growth factor (KGF, FGF-7) and fibroblast growth factor 10 (FGF-10) stimulate liquid secretion. We asked whether KGF and FGF-10 stimulate liquid secretion in human fetal lung. First trimester fetal lung explants developed dose-dependent increases in intraluminal volume in response to KGF and FGF-10. Although there were no acute changes in explant transepithelial potential difference in response to KGF (0.1-1000 ng/mL), exposure to 5-50 ng/mL KGF over 60 h depolarized transepithelial potential difference compared with controls. We used ribonuclease protection assays to quantitate the ontogeny and regulation of mRNA expression for KGF and its receptor. Both mRNA were expressed in fetal and postnatal lung. Because the promoter region of the human KGF gene contains cAMP and IL-6 response elements, we asked whether cAMP or IL-6 stimulated expression of KGF or its receptor. We have previously shown that cAMP stimulates liquid secretion in this model. Both cAMP and IL-6 significantly increased expression of KGF but not KGF receptor during a 48-h experiment. Thus, stimulation of liquid secretion in explant models by cAMP may be mediated in part by induction of KGF expression. KGF and FGF-10 may be important paracrine factors regulating liquid secretion in human fetal lung.

Antigen-Antibody Reactions↗