PubMed Health⌕ Search

Biomedical subjects

P B McCray

Publications and source records attributed to P B McCray.

At least 55 records · Page 3Linked to original sources
PubMed1998

Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia.

To better understand mechanisms that limit rAAV transduction in the lung, we have evaluated several unique features of rAAV infection in polarized primary airway epithelial cultures. rAAV was found to transduce the basolateral surface of airway epithelia 200-fold more efficiently than the apical membrane. These differences in membrane infection correlated with the abundance of apical heparan sulfate proteoglycan (AAV-2 receptor) and virus binding. UV irradiation augmented rAAV transduction greater than 20-fold, only when virus was applied to the apical membrane. Ultrastructural analysis of UV-irradiated primary cultures demonstrated significant changes in microvilli architecture following exposure to 25 J/m2 UV. Although virus binding and the abundance of heparan sulfate proteoglycan were not increased at the apical membrane following UV irradiation, increased receptor-independent endocytosis of fluorescent beads was seen at the apical membrane following UV irradiation. We hypothesize that endocytotic processes associated with apical membrane-specific pathways of viral entry, and/or processing of virus to the nucleus, may be altered following UV irradiation. Interestingly, UV irradiation had an inhibitory effect on rAAV transduction from the basolateral membrane, which correlated with a decrease in the abundance of heparan sulfate proteoglycan at the basal membrane. In summary, these findings suggest that independent pathways of viral transduction may occur in the apical and basolateral compartments of polarized airway epithelia.

Bronchi↗
PubMed1998

Production of beta-defensins by human airway epithelia.

Human beta-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1beta stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.

Cells, Cultured↗
PubMed1998

Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation.

We cloned a second human beta-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40-100kb of HNP-1, while HBD-2 was about 500-600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.

Base Sequence↗
PubMed1998

Effects of keratinocyte and hepatocyte growth factor in vivo: implications for retrovirus-mediated gene transfer to liver.

We have previously shown that intravenous administration of keratinocyte growth factor (KGF) induces hepatocyte proliferation, allowing for efficient and noninvasive in vivo gene transfer with high-titer retroviral vectors in mice. The distinctive periportal distribution of transduced cells led us to investigate the ability of virus-sized particles to perfuse the liver adequately after growth factor treatment. We found that perfusion was adequate, and that transduction was limited to the periportal region because only those cells were stimulated to divide. Cells in this region also showed increased expression of Ram-1, the receptor for the murine Moloney leukemia virus (MoMLV) amphotropic envelope, after KGF treatment. In further studies we found that recombinant hepatocyte growth factor (HGF) induces a different population of hepatocytes to divide and upregulate Ram-1. The differential pattern of induction suggested that combining KGF and HGF would improve gene transfer efficiency further. Indeed, simultaneous delivery of both growth factors leads to an overall increase in the number of proliferating cells. Importantly, when coupled with MoMLV delivery, efficiency of gene transfer increased. These results confirm the utility of growth factors for noninvasive hepatic gene transfer in mice, and demonstrate how experiments to define the mechanism of transduction can be taken advantage of to develop improved gene transfer protocols.

Animals↗
PubMed1998

Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.

Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.

Adult↗
PubMed1998

Urinary bombesin-like peptide levels in infants and children with bronchopulmonary dysplasia and cystic fibrosis.

Compared to normal infants and children, there are increased numbers of neuroendocrine cells with bombesin-like peptide (BLP) immunostaining in the lungs of infants and children with bronchopulmonary dysplasia (BPD) and cystic fibrosis (CF). However, there are no data documenting levels of urinary BLP in normal infants and children, or in children with lung disease. We therefore determined the normal developmental pattern for urinary BLP excretion in healthy infants and children, and in infants and children with BPD and CF, and correlated these findings with the subjects' clinical course. We measured urinary BLP levels in 110 subjects: 54 controls, 33 with BPD, and 23 with CF. An age-dependent decline in urinary bombesin levels was evident in the control and BPD subjects, but not in those with CF. There were no statistically significant differences in BLP levels between normal infants and those with BPD. Mean BLP levels were higher in the more immature preterm infants with BPD who required increased ventilatory support. The highest mean BLP levels were documented in BPD infants under age 3 months (882 fmol/mg creatinine), in controls between 3 and 12 months of age (625 fmol/mg creatinine), and in 12-60-month-old CF subjects (486 fmol/mg creatinine). Thus there is an age-dependent decline in BLP levels in controls and BPD, but not in CF. We speculate that the elevated urinary BLP levels in infants and children with BPD and CF may reflect increased pulmonary neuroendocrine cell activity in these conditions, due to the epithelial regenerative response to airway damage and repair.

Aging↗
PubMed1998

Transient surfactant protein B deficiency in a term infant with severe respiratory failure.

A 38-day-old male infant with persistent pulmonary hypertension and respiratory failure since birth was found to have a complete absence of surfactant protein B (SP-B) along with an aberrant form of SP-C in his tracheal aspirate fluid, findings consistent with the diagnosis of hereditary SP-B deficiency. Surprisingly, SP-B and SP-B messenger ribonucleic acid were present in lung biopsy tissue. However, DNA sequence analysis demonstrated a point mutation in exon 5 of one of the SP-B gene alleles. The infant's mother was found to be a carrier of this mutation. The infant's other SP-B allele did not differ from the published DNA sequence for the SP-B gene. We conclude that this patient had a transient deficiency of SP-B, in contrast to that of previously described infants with irreversible respiratory failure caused by hereditary SP-B deficiency. We recommend that infants with suspected SP-B deficiency have serial analysis of tracheal fluid samples for both SP-B and SP-C before lung biopsy, along with genetic analysis for the known SP-B mutations. We speculate that the new mutation found in one of this patient's SP-B genes was in part responsible for the transient deficiency of SP-B.

Exudates and Transudates↗
PubMed1998

Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia.

Gene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl- transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.

Bronchi↗
PubMed1998

Developmental regulation of epithelial sodium channel subunit mRNA expression in rat colon and lung.

Na+ absorption via amiloride-sensitive Na+ channels is of critical importance in the transition between fetal and neonatal life in several tissues, including the colon, lung, and kidney. To characterize and contrast the mRNA expression of each of the three epithelial Na+ channel complex (ENaC) subunits, we conducted RNase protection assays (RPA) and in situ hybridization in colon and lung in fetal (17, 19, 20, and 21 days) and postnatal (1, 3, 9, 15, and 30 days) rats (r). In the colon the alpha-, beta-, and gamma-rENaC subunits showed quantitatively different but qualitatively similar expression. All three subunits gradually increased in abundance from fetal day 19 through day 30 of life. The amount of each subunit on day 30 was approximately three times the amount at day 1. In situ hybridization showed that each subunit was localized to the surface epithelial cells with minimal expression in the crypts. The lung showed a completely different pattern. In contrast to the colon, the total amount of alpha-rENaC mRNA (by RPA) in the lung increased dramatically from fetal day 19 to 21, whereas beta- and gamma-rENaC showed modest prenatal increases. The amounts of all three mRNAs fell after birth through day 9 (to about 75% of the day 1 value). On days 15 and 30 the amount of mRNA rose to approach the values on day 1. alpha-rENaC mRNA abundance always exceeded beta- and gamma-rENaC, and the quantitative expression was different for alpha- than for beta- and gamma-rENaC. In situ hybridization studies showed that all three subunits were expressed in epithelial cells of the bronchi, bronchioles, and alveoli and not in blood vessels. These studies show striking developmental heterogeneity in rENaC mRNA expression between lung and colon, probably reflecting different developmental regulatory mechanisms in these organs.

Animals↗
PubMed1997

Alveolar macrophages inhibit retrovirus-mediated gene transfer to airway epithelia.

Gene transfer with integrating vectors such as recombinant retrovirus has the potential to correct inherited lung diseases permanently. As a gene therapy target, the pulmonary epithelium presents several challenges to vector delivery in vivo. Many of the host defenses that have evolved to prevent infection from inhaled bacteria or viruses represent potential barriers to gene transfer to the lung. We performed in vitro studies to determine whether two components of the innate immune system of the lung, airway surface fluid and alveolar macrophages, inhibit retroviral gene transfer to airway epithelia. Human alveolar macrophages obtained by bronchoalveolar lavage from normal subjects were left untreated or activated with lipopolysaccharide (LPS) for 3 hr in the presence of subconfluent human bronchial epithelial cells (HBE); than 4 x 10(5) cfu DA-luciferase retrovirus was added. Three days after infection, luciferase activity was measured in cell lysates. When the epithelial cells were co-cultured with LPS-activated macrophages, retroviral gene transfer to HBE cells was reduced by approximately 60%. Nonactivated macrophages decreased the transfection to approximately 55% of control values. In control experiments with either activated or inactivated macrophages but without epithelia, no luciferase activity was detected, suggesting that terminally differentiated alveolar macrophages are not infected by the recombinant retrovirus. Pretreatment of alveolar macrophages with dexamethasone restored gene transfer to approximately 60% of control values. In contrast, incubation of retrovirus with airway surface fluid had no inhibitory effect on gene transfer. These experiments document that AM inhibit retrovirus-mediated gene transfer to airway epithelia in vitro, and may represent a barrier to retroviral gene transfer in vivo. These barriers may be overcome, at least partially, with pharmacological agents.

Antineoplastic Agents, Hormonal↗
PubMed1997

A classification scheme for paradoxical vocal cord motion.

Paradoxical vocal cord motion (PVCM) is characterized by the inappropriate adduction of the true vocal cords during inspiration. Multiple causes have been proposed for this group of disorders, which share the common finding of mobile vocal cords that adduct inappropriately during inspiration and cause stridor by approximation. Management of this group of disorders has been complicated by the lack of a classification scheme to include all types of PVCM. We propose that PVCM be classified according to its underlying etiology and recognize the following causes of the disorder: 1. brainstem compression; 2. cortical or upper motor neuron injury; 3. nuclear or lower motor neuron injury; 4. movement disorder; 5. gastroesophageal reflux; 6. factitious or malingering disorder; 7. somatization/conversion disorder. Case reports are presented to illustrate the characteristic features and diagnostic evaluation used in assessing patients with PVCM. Management varies depending on the cause of PVCM and entails speech therapy, pharmacologic therapy, behavioral modification, and/or surgical intervention. Recognition of the multiple causes of PVCM allows otolaryngologists to formulate well-directed diagnostic evaluation and treatment.

Adolescent↗
PubMed1997

Human airway epithelia express a beta-defensin.

Recent evidence suggests that the surface fluid secreted by human airway epithelial cells contains antimicrobial activity. The identification of the factor(s) responsible for the antibacterial activity in airway surface fluid may bring new insights into lung defense mechanisms and the pathogenesis of disease. Defensins are small cationic peptides with broad-spectrum antimicrobial activity, and are therefore candidates for the bacterial killing factor in airway surface fluid. They are produced in mucosal epithelia and neutrophils of several species. To date no defensins have been isolated from human airway epithelia; however, such peptides are produced by bovine airway epithelia. A cDNA fragment for a human beta-defensin-1 (hBD-1) was recently cloned from the kidney. We cloned the full-length coding sequence for hBD-1 from human airway epithelia. The amino acid sequence for hBD-1 shared homology with the signal/propiece and mature peptides of other beta-defensins, including bovine tracheal antimicrobial peptide and lingual antimicrobial peptide. By Northern analysis, a single approximately 400-nt hBD-1 transcript was expressed in airway epithelia. Ribonuclease protection analysis demonstrated that hBD-1 transcripts were more abundant in the conducting airways than the gas exchange regions of human lung and that the expression was developmentally regulated. Human beta-defensin-1 may contribute to the antimicrobial activity of airway surface fluid and play a role in the mucosal defenses of the lung.

Adult↗
PubMed1997

Ursodeoxycholic acid improves cholestasis in infants with cystic fibrosis.

OBJECTIVE: To describe two infants with cholestatic jaundice treated with ursodeoxycholic acid (UDCA). CASE SUMMARY: Two infants with cystic fibrosis (CF)-associated hepatobiliary disease, manifesting as cholestatic jaundice and elevated liver enzymes within the first 6 weeks of life, had improved biochemical indices of liver function following treatment with UDCA 20-40 mg/kg/d. DISCUSSION: To our knowledge, this is the first report of UDCA treatment in infants with CF-associated cholestatic jaundice. Infants and children require treatment with increased doses of UDCA to compensate for reduced intestinal absorption of bile acid and immaturity of the enterohepatic circulation. CONCLUSIONS: UDCA appears to be a cost-effective treatment for CF-associated hepatobiliary disease in infants and children.

Cholagogues and Choleretics↗
PubMed1996

Proliferation induced by keratinocyte growth factor enhances in vivo retroviral-mediated gene transfer to mouse hepatocytes.

Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.

Animals↗
PubMed1996

Localization of epithelial sodium channel subunit mRNAs in adult rat lung by in situ hybridization.

The transport of Na+ through amiloride-sensitive sodium channels (ENaC) plays a major role in the absorption of fluid across the pulmonary epithelium. The proteins forming the ENaC channel are encoded by three genes in the rat (alpha-, beta-, and gamma-rENaC). According to Northern blot, all three subunit mRNAs were expressed in adult rat lung. Each subunit was expressed as a single transcript of approximately 3.7, 2.2, and 3.2 kb for alpha-, beta-, and gamma-rENaC, respectively. To localize the alpha-, beta-, and gamma-rENaC subunit mRNAs, we used in situ hybridization. Frozen and paraffin-embedded tissues were hybridized with sense and antisense 35S-labeled riboprobes. The alpha-rENaC mRNA was most abundant and was expressed diffusely in epithelia of the trachea, bronchi, bronchioles, and alveoli. At the alveolar level, alpha-rENaC was expressed in type II cells. The beta- and gamma-rENaC mRNAs were most abundant in the bronchial and bronchiolar epithelia. All three subunits were expressed in the renal cortical collecting duct in a pattern similar to that previously reported by other investigators. Thus the rENaC subunit mRNAs are expressed in regions of the lung where functional Na+ absorption is found. These results are consistent with an important role for ENaC in the absorption of Na+ and fluid across the pulmonary epithelium in all regions of the lung.

Animals↗
PubMed1996

Keratinocyte growth factor stimulates CFTR-independent fluid secretion in the fetal lung in vitro.

Keratinocyte growth factor (KGF) caused cystic dilation of mouse fetal lung explants in vitro, markedly increasing the luminal volume of lung buds and disrupting branching morphogenesis. Effects of KGF were dose dependent, were detected within 4 h of treatment, and were blocked by cycloheximide but not by actinomycin D, indicating that de novo protein synthesis mediated the response. Effects of KGF were inhibited by bumetanide, an inhibitor of the Na(+)-K(+)-Cl- cotransporter, and ouabain, an inhibitor of the Na(+)-K(+)-ATPase. KGF stimulated fluid secretion equally in lung buds from cystic fibrosis transmembrane conductance regulators (CFTR) -/- and wild-type embryos, indicating that the effects were mediated by CFTR-independent Cl- transport. Microelectrode studies demonstrated that, whereas KGF did not acutely alter the transepithelial potential difference (PD) across the respiratory epithelium, the PD decreased while luminal volume increased during chronic exposure. KGF inhibited expression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) mRNA, suggesting that KGF may inhibit Na+ absorption, which may contribute to KGF-induced fluid accumulation. KGF-induced fluid accumulation is driven by CFTR-independent Cl- transport and associated with decreased expression of alpha-ENaC.

Animals↗