Differentiated and functional human airway epithelium regeneration in tracheal xenografts

To investigate the regeneration process of a well-differentiated and functional human airway epithelium, we adapted an in vivo xenograft model in which adult human nasal epithelial cells adhere and progressively repopulate denuded rat tracheae grafted in nude mice. The proliferating activity, the degree of differentiation, and the barrier integrity of the repopulated epithelium were studied during the regeneration process at optical and ultrastructural levels with immunocytochemistry and a permeability tracer. Three days after implantation in nude mice, tracheal xenografts were partially repopulated with a flattened nonciliated and poorly differentiated leaky epithelium. By the end of the first week after the graft, cell proliferation produced on the entire surface of the rat trachea an epithelium that was stratified into multiple layers and tightly sealed. During successive weeks, cell proliferation dramatically decreased. Moreover, the epithelium became progressively columnar, secretory, ciliated, and transiently leaky. At 4-5 wk, a fully differentiated pseudostratified functional epithelial barrier impermeable to a low-molecular-weight tracer was reconstituted. The regeneration of a well-differentiated and functional human airway epithelium in rat tracheae grafted in nude mice includes several steps that mimic the regeneration dynamics of airway epithelium after injury.     

Human airway xenograft models of epithelial cell regeneration

Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.     

Repair and regeneration of the airway epithelium

Despite an efficient defence system, the airway surface epithelium, in permanent contact with the external milieu, is frequently injured by inhaled pollutants, microorganisms and viruses. The response of the airway surface epithelium to an acute injury includes a succession of cellular events varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even to complete denudation of the basement membrane. The epithelium has then to repair and regenerate to restore its functions, through several mechanisms including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. The cellular and molecular factors involved in wound repair and epithelial regeneration are closely interacting and imply extracellular matrix proteins, matrix metalloproteinases (MMPs) and their inhibitors as well as cytokines and growth factors secreted by airway epithelial and mesenchymal cells. The development of in vitro and in vivo models of airway epithelium wound repair allowed the study of the spatio-temporal modulation of these factors during the different steps of epithelial repair and regeneration. In this context, several studies have demonstrated that the matrix and secretory environment are markedly involved in these mechanisms and that their dysregulation may induce remodelling of the airway mucosa. A better knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to regenerative therapeutics allowing the reconstitution of a functional airway epithelium in numerous respiratory diseases such as asthma, chronic obstructive pulmonary diseases, cystic fibrosis and bronchiolitis.     

In vivo restitution of airway epithelium

Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-m wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [³H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2–3 m/min) over the denuded basement membrane. Within 8–15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.     

Gene delivery to airway epithelial cells in vivo: a direct comparison of apical and basolateral transduction strategies using pseudotyped lentivirus vectors

Lentivirus vectors are being investigated as gene delivery vehicles for cystic fibrosis airway gene therapy. Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped vectors transduce airway epithelia via receptors that are located predominantly on the basolateral surface of the airway epithelium. Effective transduction with VSV-G-pseudotyped vectors requires the use of a pre-treatment that disrupts epithelial tight junctions, allowing access to these basolateral receptors. In contrast, it has been reported that apically targeted lentiviral vectors allow efficient gene transfer in the absence of any pre-treatment. In a direct comparison of transduction by a VSV-G-pseudotyped vector, in combination with a pre-treatment with lysophosphatidylcholine (LPC), and the same vector pseudotyped with the apically targeted baculovirus GP64 envelope (without any pre-treatment), the GP64 vector was found to be significantly less efficient. However, when a pre-treatment with LPC was used the level of transduction with the GP64-pseudotyped lentiviral vector was not significantly different to that resulting from the VSV-G-pseudotyped vector. The cell types transduced with each vector were essentially the same, with the majority of cells transduced being respiratory (ciliated cells). However, unlike the VSV-G-pseudotyped vector, which results in persisting gene expression, transduction with the GP64-pseudotyped vector resulted in gene expression that declined to undetectable levels over six months, whether or not an LPC pre-treatment was used.     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

Differentiation of human airway epithelia is dependent on erbB2

A clinical case documented a reversible change in airway epithelial differentiation that coincided with the initiation and discontinuation of trastuzumab, an anti-erbB2 antibody. This prompted the investigation into whether blocking the erbB2 receptor alters differentiation of the airway epithelium. To test this hypothesis, we treated an in vitro model of well-differentiated human airway epithelia with trastuzumab or heregulin-alpha, an erbB ligand. In addition, coculturing with human lung fibroblasts tested whether in vivo subepithelial fibroblasts function as an endogenous source of ligands able to activate erbB receptors expressed by the overlying epithelial cells. Epithelia were stained with hematoxylin and eosin and used for morphometric analysis. Trastuzumab treatment decreased the ciliated cell number by 49% and increased the metaplastic, flat cell number by 640%. Heregulin-alpha treatment increased epithelial height and decreased the number of metaplastic and nonciliated columnar cells, whereas it increased the goblet cell number. We found that normal human lung fibroblasts express transforming growth factor-alpha, heparin-binding epidermal-like growth factor, epiregulin, heregulin-alpha, and amphiregulin, all of which are erbB ligands. Cocultures of airway epithelia with primary fibroblasts increased epithelial height comparable to that achieved following heregulin-alpha treatment. These data show that erbB2 stimulation is required for maintaining epithelial differentiation. Furthermore, the mesenchyme underlying the airway epithelium secretes a variety of erbB ligands that may direct various pathways of epithelial differentiation.     

Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model

BACKGROUND: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma. METHODS: HIV-based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk-1) in MSC on growth of Raji cells was determined, both in vitro and in vivo. RESULTS: All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 +/- 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter-donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG-pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co-injection of MSC stably expressing tsFlk-1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP-expressing MSC were used. CONCLUSIONS: Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter-donor variation in transduction efficiency. Gene-modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies.     

Orthogonal arrays in normal and injured respiratory airway epithelium

Orthogonal arrays are found on plasma membranes of glial cells, in the central nervous system, on muscle plasma membranes at neuromuscular junctions, and on a variety of epithelial cells. These structures have been correlated with ion flux. With the aid of freeze fracture technique, orthogonal particle arrays were found on plasma membranes on airway epithelial cells of rats and hamsters. They have been found in abundance at the base of secretory cells throughout normal airway epithelium. These structures were found to increase in number during regeneration in response to injury and they were found in great numbers on plasma membranes of all airway cells in response to acute and chronic NO2 exposure. The lateral and basal plasma membranes of the respiratory epithelium are a new source for studying orthogonal arrays. The normal number and distribution of these arrays can be perturbed in response to mechanical and chemical injury.     

Functional expression of connexin30 and connexin31 in the polarized human airway epithelium

Gap junctions are documented in the human airway epithelium but the functional expression and molecular identity of their protein constituents (connexins, Cx) in the polarized epithelium is not known. To address this question, we documented the expression of a family of epithelial Cx (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, and Cx43) in primary human airway epithelial cells (AEC) grown on porous supports. Under submerged conditions, AEC formed a monolayer of airway cells whereas the air-liquid interface induced within 30-60 days AEC differentiation into a polarized epithelium for up to 6-9 months. Maturation of AEC was associated with the down-regulation of Cx26 and Cx43. The well-differentiated airway epithelium exhibited gap junctional communication between ciliated and between ciliated and basal cells. Interestingly, Cx30 was mostly present between ciliated cells whereas Cx31 was found between basal cells. These results are supportive of the establishment of signal-selective gap junctions with maturation of AEC, likely contributing to support airway epithelium function. These results lay the ground for studying the role of Cx-mediated cell-cell communication during repair following AEC injury and exploring Cx-targeted interventions to modulate the healing process.     

Cell wall-associated protein A as a tool for immunolocalization of Staphylococcus aureus in infected human airway epithelium.

Staphylococcus aureus is a common human pathogen involved in non-bronchial diseases and in genetic and acquired bronchial diseases. In this study, we applied an immunolabeling approach for in vivo and in vitro detection of S. aureus, based on the affinity of staphylococcal protein A (SpA) for the Fc region of immunoglobulins, especially IgG. Most strains of S. aureus, including clinical strains, can be detected with this labeling technique. The approach can be used for detection and localization with transmission electron microscopy or light-fluorescence microscopy of S. aureus in infected tissues such as human bronchial tissue from cystic fibrosis (CF) patients. The methodology can also be applied to cell culture models with the aim of characterizing bacterial adherence to epithelial cells in backscattered electron imaging with scanning electron microscopy. Application to the study of S. aureus adherence to airway epithelium showed that the bacteria did not adhere in vivo to intact airway epithelium. In contrast, bacteria adhered to the basolateral plasma membrane of columnar cells, to basal cells, to the basement membrane and were identified beneath the lamina propria when the epithelium was injured and remodeled, or in vitro when the epithelial cells were dedifferentiated.     

Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha

The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FR alpha), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FR alpha on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FR alpha-blocking antibodies, we demonstrated FR alpha-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FR alpha was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.     

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.     

Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.     

Effects of dynamic compression on lentiviral transduction in an in vitro airway wall model

Asthmatic patients are more susceptible to viral infection, and we asked whether dynamic strain on the airway wall (such as that associated with bronchoconstriction) would influence the rate of viral infection of the epithelial and subepithelial cells. To address this, we characterized the barrier function of a three-dimensional culture model of the bronchial airway wall mucosa, modified the culture conditions for optimization of ciliogenesis, and compared epithelial and subepithelial green fluorescent protein (GFP) transduction by a pWpts-GFP lentivirus, pseudotyped with VSV-G, under static vs. dynamic conditions. The model consisted of human lung fibroblasts, bronchial epithelial cells, and a type I collagen matrix, and after 21 days of culture at air liquid interface, it exhibited a pseudostratified epithelium comprised of basal cells, mucus-secreting cells, and ciliated columnar cells with beating cilia. Microparticle tracking revealed partial coordination of mucociliary transport among groups of cells. Slow dynamic compression of the airway wall model (15% strain at 0.1 Hz over 3 days) substantially enhanced GFP transduction of epithelial cells and underlying fibroblasts. Fibroblast-only controls showed a similar degree of transduction enhancement when undergoing dynamic strain, suggesting enhanced transport through the matrix. Tight junction loss in the epithelium after mechanical stress was observed by immunostaining. We conclude that dynamic compressive strain such as that associated with bronchoconstriction may promote transepithelial transport and enhance viral transgene delivery to epithelial and subepithelial cells. This finding has significance for asthma pathophysiology as well as for designing delivery strategies of viral gene therapies to the airways.     

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.     

Remission of Invasive,Cancer Stem-Like Glioblastoma Xenografts Using Lentiviral Vector-Mediated Suicide Gene Therapy

Background

Glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model.

Methodology/Principal Findings

We used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin- and SOX2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p<0.001) compared to control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect.

Conclusions/Significance

In conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. The inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high reproducibility of the invasive glioma animal model for translational research.     

Modeling the airway epithelium in allergic asthma: Interleukin-13-induced effects in differentiated murine tracheal epithelial cells

Summary Mucous cells of the airway epithelium play a crucial role in the pathogenesis of human inflammatory airway diseases. Therefore, it is of importance to complement in vivo studies that use murine models of allergic asthma with in vitro mechanistic studies that use murine airway epithelial cells, including mucus-containing cells. In this study, we report the development and characterization of an in vitro culture system for primary murine tracheal epithelial (MTE) cells comprising ciliated cells and a substantial number of mucous cells. The increase in mucous cell number over that observed in the native murine airway, or in previously described murine cultures, creates a culture intermediate between the in vivo murine airway epithelium and in vitro cultures of human airway epithelial cells. To establish the usefulness of this culture system for the study of epithelial effects during inflammatory airway diseases, the cells were exposed to interleukin (IL)-13, a central inflammatory mediator in allergic asthma. The IL-13 induced two characteristic epithelial effects, proliferation and modulation of MUC5AC gene expression. There was a concentration dependence of these events, wherein high concentrations of IL-13 (10 ng/ml) induced proliferation, whereas lower concentrations (1 ng/ml) increased MUC5AC mRNA (where mRNA is messenger RNA). Interestingly, these effects occurred in an inverse manner, with the high concentration of IL-13 also provoking a significant decrease in MUC5AC gene expression. Thus, MTE cells cultured in this manner may provide an important link between experimental findings from animal models of allergic asthma and their application to human disease.     

Capacity of human beta-defensin expression in gene-transduced and cytokine-induced cells

The purpose of this study was to determine the capacity of cells transduced with human beta-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9-99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabeNeo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFalpha. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.