Showing papers by "Joseph M. Pilewski published in 1995"

Retrovirus-mediated gene transfer in lungs of living fetal sheep

Abstract: In utero somatic gene transfer may be a useful therapeutic strategy for a variety of inherited disorders. In the present study, we demonstrate transgene expression in the airways of fetal lamb lungs, 2-3 weeks after injection of Moloney murine leukemia retrovirus based vectors containing cDNA for beta-galactosidase (lacZ) or human interleukin receptor antagonist protein (IRAP), into the fluid filled future airspace of fully catheterized twin fetal lambs (104-117 days gestational age; term 147 days). Expression of lacZ or IRAP was limited to the twin that received the respective vector and was apparent, at light microscopic level, in the epithelium and submucosal space of proximal airways, and to a lesser extent, in the respiratory epithelium of the distal airways. These data demonstrate for the first time that transfer of foreign DNA to fetal lung can be accomplished. These findings support the use of retroviral vectors for somatic lung DNA transfer and suggest that inherited disorders such as cystic fibrosis may be approached therapeutically via gene transfer, in utero.

Localization of Multiple Functional Domains on Human PECAM-1 (CD31) by Monoclonal Antibody Epitope Mapping

Abstract: PECAM-1, a cell adhesion molecule of the immunoglobulin gene (Ig) superfamily, has been implicated in white cell transmigration, integrin activation on lymphocytes, and cell-cell adhesion. The purpose of this investigation was to identify specific regions of the PECAM-1 extracellular domain mediating these functions by identifying the location of epitopes of bioactive anti-PECAM-1 monoclonal antibodies. The binding regions of mAbs important in PECAM-1-mediated leukocyte transmigration (Hec 7.2 and 3D2) were mapped to N-terminal Ig-like domains. The epitopes of monoclonal antibodies that activated integrin function on lymphocytes were dispersed over the entire extracellular region, but those that had the strongest activating effect were preferentially localized to the N-terminus of the molecule. The binding regions of mAbs that blocked PECAM-1-mediated heterophilic L-cell aggregation were located either in Ig-like domain 2 (NIH31.4) or Ig-like domain 6 (4G6 and 1.2). Site-directed mutagenesis further pinpo...

ICAM-1 expression on bronchial epithelium after recombinant adenovirus infection.

Abstract: Early experience with recombinant adenoviruses for gene transfer to airway epithelium suggests that these vectors are associated with the development of inflammation. The mechanisms for this are unclear, but previous work has shown that respiratory viruses can cause increased expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells. We therefore hypothesized that recombinant adenoviruses may induce ICAM-1 expression and thereby facilitate the development of airway inflammation. To address this, primary cultures of human bronchial epithelial cells were examined for ICAM-1 expression by flow cytometry after infection with a serotype 5, E1/E3-deleted recombinant adenovirus containing the Escherichia coli LacZ reporter gene driven by the cytomegalovirus promoter (Ad.CMVlacZ). Compared with control cells, ICAM-1 expression was unchanged after infection with Ad.CMVlacZ, but increased after infection with wild-type adenovirus. Treatment of Ad.CMVlacZ-infected cells with interferon-gamm...

Adenovirus-mediated gene transfer to human bronchial submucosal glands using xenografts.

Abstract: The cystic fibrosis (CF) transmembrane conductance regulator has been localized to both submucosal glands and surface epithelium, suggesting that both glandular and surface epithelium may be important targets for gene therapy. To determine the distribution and efficiency of recombinant adenovirus-mediated gene transfer to human airway submucosal glands, an in vivo model was developed by heterotopically transplanting human bronchial segments from both normal and CF lung tissue into severe combined immunodeficient mice. A serotype 5 E1-deleted recombinant adenovirus containing a lacZ reporter gene driven by the cytomegalovirus promoter (H5.010CMVlacZ) was used to infect the xenografts. Transgene expression was correlated with three factors: 1) viral titer, 2) penetration of microspheres, and 3) dwell time of the viral instillate. At viral titers ranging from 10(8) to 10(11) plaque forming units/ml, expression of the lacZ gene was observed in a subpopulation of epithelial cells within approximately 40% of submucosal glands, with more efficient gene transfer to the ducts than the secretory tubules. Within individual glands, gene transfer varied from < 1% to approximately 20% of submucosal cells, including duct, mucous, and serous cells. Adenovirus-sized fluorescent microspheres were found to penetrate only some of the submucosal glands, suggesting that the gene transfer efficiency to submucosal tubules is due to limited viral particle penetration rather than tropism. Gene transfer to surface epithelial cells was inefficient. However, the percentage of transduced surface epithelial cells increased from < 1% to 5-10% as the dwell time of viral solution was increased from 5 min to 1 h, indicating that the time allowed for virus binding and entry is important for gene transfer efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus-mediated gene transfer to human brochial submucosal glands using xenografts

Abstract: The cystic fibrosis (CF) transmembrane conductance regulator has been localized to both submucosal glands and surface epithelium, suggesting that both glandular and surface epithelium may be important targets for gene therapy. To determine the distribution and efficiency of recombinant adenovirus-mediated gene transfer to human airway submucosal glands, an in vivo model was developed by heterotopically transplanting human bronchial segments from both normal and CF lung tissue into severe combined immunodeficient mice. A serotype 5 E1-deleted recombinant adenovirus containing a lacZ reporter gene driven by the cytomegalovirus promoter (H5.010CMVlacZ) was used to infect the xenografts. Transgene expression was correlated with three factors: 1) viral titer, 2) penetration of microspheres, and 3) dwell time of the viral instillate. At viral titers ranging from 10 8 to 10 11 plaque forming units/ml, expression of the lacZ gene was observed in a subpopulation of epithelial cells within ∼ 40% of submucosal glands, with more efficient gene transfer to the ducts than the secretory tubules. Within individual glands, gene transfer varied from < 1% to ∼ 20% of submucosal cells, including duct, mucous, and serous cells. Adenovirus-sized fluorescent microspheres were found to penetrate only some of the submucosal glands, suggesting that the gene transfer efficiency to submucosal tubules is due to limited viral particle penetration rather than tropism. Gene transfer to surface epithelial cells was inefficient. However, the percentage of transduced surface epithelial cells increased from < 1% to 5-10% as the dwell time of viral solution was increased from 5 min to 1 h, indicating that the time allowed for virus binding and entry is important for gene transfer efficiency. This report presents the first definitive demonstration of adenovirus-mediated transgene delivery into human airway submucosal glands

How do cystic fibrosis transmembrane conductance regulator mutations produce lung disease

Abstract: In recent years, several functions of the cystic fibrosis transmembrane conductance regulator have been discovered, yet the pathophysiology of the pulmonary disease in cystic fibrosis remains unclear. At the cellular level, functions of this protein include regulation of chloride and sodium transport at the cell membrane and in intracellular organelles, regulation of protein trafficking, and posttranslational processing of glycoconjugates. Elucidation of these functions has led to several hypotheses to account for how defects in the cystic fibrosis transmembrane conductance regulator produce pulmonary disease, but a clear understanding of the pathophysiologic links between the cellular functions of the cystic fibrosis transmembrane conductance regulator and organ dysfunction has been hampered by the lack of ideal model systems. Current evidence suggests that defects in the cystic fibrosis transmembrane conductance regulator lead to alterations in periciliary fluid homeostasis, mucus hydration, mucin secretion, and apical membrane protein structure. In turn, these alterations impair mucociliary clearance and promote bacterial infection, which then leads to chronic airway inflammation and the development of bronchiectasis.

Modulation of adhesion molecules by cytokines in vivo using human/severe combined immunodeficient (SCID) mouse chimeras

Abstract: Endothelial cell-leukocyte interactions involve multiple cell adhesion molecules acting in a programmed and sequential manner to create a leukocyte-endothelial cell adhesion cascade. To understand this process fully,in vivo models are needed. To accomplish this, we have transplanted pieces of normal human tissues onto immunodeficient mice to create chimeric animals. In one model, human skin is grafted and closely resembles normal skin histologically. The grafts retain their human vasculature and show low baseline expression of E-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1. After intradermal injection of human cytokines, these cell adhesion molecules are markedly upregulated and an active inflammatory reaction ensues, with migration of murine leukocytes. Intravenous injection of an antihuman E-selectin antibody completely inhibits leukocyte accumulation induced by tumor necrosis factor-α but only partially inhibits leukotriene B4-induced inflammation. In a second model, human bronchus was successfully transplanted heterotopically into severe combined immunodeficient mice. Injection of tumor necrosis factor induced upregulation of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 in the submucosal microvessels, with slightly different kinetics than in the skin. In conclusion, human-severe combined immunodeficient chimeric mice represent a useful model system to study the regulation and function of human cell adhesion molecules in anin vivo setting.