Human Ciliated Airway Epithelium Research Articles

P256 P2X4 and nNOS Expression in Human Ciliated Airway Epithelium

BackgroundCalcium (Ca2+) and nitric oxide (NO) modulate ciliary beat frequency (CBF). NO synthase (NOS) isoenzymes, responsible for NO production, localise in human airway epithelium and constitutive NOS activity is dependant...

Comparison of differing cytopathic effects in human airway epithelium of parainfluenza virus 5 (W3A), parainfluenza virus type 3, and respiratory syncytial virus

Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.

Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is critical to develop suitable in vitro platforms to isolate and characterize the replication kinetics and pathogenesis of recently identified human pathogens. HCoV-HKU1, a human coronavirus identified in a clinical sample from a patient with severe pneumonia, has been a major challenge for successful propagation on all immortalized cells tested to date. To determine if HCoV-HKU1 could replicate in in vitro models of human ciliated airway epithelial cell cultures (HAE) that recapitulate the morphology, biochemistry, and physiology of the human airway epithelium, the apical surfaces of HAE were inoculated with a clinical sample of HCoV-HKU1 (Cean1 strain). High virus yields were found for several days postinoculation and electron micrograph, Northern blot, and immunofluorescence data confirmed that HCoV-HKU1 replicated efficiently within ciliated cells, demonstrating that this cell type is infected by all human coronaviruses identified to date. Antiserum directed against human leukocyte antigen C (HLA-C) failed to attenuate HCoV-HKU1 infection and replication in HAE, suggesting that HLA-C is not required for HCoV-HKU1 infection of the human ciliated airway epithelium. We propose that the HAE model provides a ready platform for molecular studies and characterization of HCoV-HKU1 and in general serves as a robust technology for the recovery, amplification, adaptation, and characterization of novel coronaviruses and other respiratory viruses from clinical material.

Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys

Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive ( ts) and non- ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32 °C and exhibited little productive replication at 37 °C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates.

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Physicochemical properties of gene transfer vectors play an important role in both transduction efficiency and biodistribution following airway delivery. Adeno-associated virus (AAV) vectors are currently used in many gene transfer applications; however, the respiratory epithelium remains a challenging target. We synthesized two cationic sterol-based lipids, dexamethasone-spermine (DS) and disubstituted spermine (D(2)S) for pulmonary gene targeting. Scanning and transmission electron micrographs (TEM) confirmed that AAV/lipid formulations produced submicron-sized clusters. When AAV2/9 or AAV2/6.2 were formulated with these cationic lipids, the complexes had positive zeta potential (zeta) and the transduction efficiency in cultured A549 cells increased by sevenfold and sixfold, respectively. Transduction of cultured human airway epithelium with AAV2/6.2-lipid formulations also showed approximately twofold increase in green fluorescence protein (GFP) positive cells as quantified by flow cytometry. Intranasal administration of 10(11) genome copies (GC) of AAV2/9 and AAV2/6.2 coformulated with lipid formulations resulted in an average fourfold increase in transgene expression for both vectors. Formulation of AAV2/9 with DS changed the tropism of this vector for the alveolar epithelium, resulting in successful transduction of conducting airway epithelium. Our results suggest that formulating AAV2/9 and AAV2/6.2 with DS and D(2)S can lead to improved physicochemical characteristics for in vivo gene delivery to lung.

Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission.

Transduction Efficiencies of Novel AAV Vectors in Mouse Airway Epithelium In Vivo and Human Ciliated Airway Epithelium In Vitro

We have characterized the ability of adeno-associated virus (AAV) serotypes 1-9 in addition to nineteen novel vectors isolated from various tissues, to transduce mouse and human ciliated airway epithelium (HAE). Vectors expressing alpha-1-antitrypsin (AAT) and beta-galactosidase were co-instilled into the mouse lung. Of all the vectors tested rh.64R1, AAV5 and AAV6 were the most efficient. The high transduction observed in mouse was reproduced in HAE cell cultures for both rh.64R1 and AAV6 but not for AAV5. Since AAV6 was the most efficient vector in mouse and HAE we also tested the transduction efficiencies of the AAV6 singleton vectors (i.e., AAV6 variants with targeted mutations) in these models. Of these, AAV6.2 transduced mouse airway epithelium and HAE with greater efficiency than all other AAV vectors tested. We demonstrated that AAV6.2 exhibits improved transduction efficiency compared to previously reported AAVs in mouse airways and in culture models of human airway epithelium and that this vector requires further development for preclinical and clinical testing.

Transduction Efficiencies of Novel AAV Vectors in Mouse Airway Epithelium In Vivo and Human Ciliated Airway Epithelium In Vitro

Transduction Efficiencies of Novel AAV Vectors in Mouse Airway Epithelium In Vivo and Human Ciliated Airway Epithelium In Vitro

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV.

Role of interferon in the replication of human parainfluenza virus type 1 wild type and mutant viruses in human ciliated airway epithelium.

Human parainfluenza virus type 1 (HPIV1) is a significant cause of pediatric respiratory disease in the upper and lower airways. An in vitro model of human ciliated airway epithelium (HAE), a useful tool for studying respiratory virus-host interactions, was used in this study to show that HPIV1 selectively infects ciliated cells within the HAE and that progeny virus is released from the apical surface with little apparent gross cytopathology. In HAE, type I interferon (IFN) is induced following infection with an HPIV1 mutant expressing defective C proteins with an F170S amino acid substitution, rHPIV1-C(F170S), but not following infection with wild-type HPIV1. IFN induction coincided with a 100- to 1,000-fold reduction in virus titer, supporting the hypothesis that the HPIV1 C proteins are critical for the inhibition of the innate immune response. Two recently characterized live attenuated HPIV1 vaccine candidates expressing mutant C proteins were also evaluated in HAE. The vaccine candidates, rHPIV1-C(R84G/Delta170)HN(T553A)L(Y942A) and rHPIV1-C(R84G/Delta170)HN(T553A)L(Delta1710-11), which contain temperature-sensitive (ts) attenuating (att) and non-ts att mutations, were highly restricted in growth in HAE at permissive (32 degrees C) and restrictive (37 degrees C) temperatures. The viruses grew slightly better at 37 degrees C than at 32 degrees C, and rHPIV1-C(R84G/Delta170)HN(T553A)L(Y942A) was less attenuated than rHPIV1-C(R84G/Delta170)HN(T553A)L(Delta1710-11). The level of replication in HAE correlated with that previously observed for African green monkeys, suggesting that the HAE model has potential as a tool for the preclinical evaluation of HPIV1 vaccines, although how these in vitro data will correlate with vaccine virus replication in seronegative human subjects remains to be seen.

Role of Interferon in the Replication of Human Parainfluenza Virus Type 1 Wild Type and Mutant Viruses in Human Ciliated Airway Epithelium

Role of Interferon in the Replication of Human Parainfluenza Virus Type 1 Wild Type and Mutant Viruses in Human Ciliated Airway Epithelium

Glycocalyx Restricts Adenoviral Vector Access to Apical Receptors Expressed on Respiratory Epithelium In Vitro and In Vivo: Role for Tethered Mucins as Barriers to Lumenal Infection

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.

Identification of Na(+)-H+ exchange in human normal and cystic fibrotic ciliated airway epithelium.

The pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein was used to measure intracellular pH (pHi) and test for Na(+)-H+ exchange in single ciliated human nasal epithelial (HNE) cells from normal and cystic fibrosis (CF) subjects. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered NaCl Ringer solution, average pHi for normal and CF ciliated HNE cells was 7.15 +/- 0.02 and 7.18 +/- 0.03, respectively. Utilizing the NH+4-loading technique to acid load cells, we found that pHi decreased from approximately 7.2 to approximately 6.6; subsequent alkalinization of normal and CF ciliated cells required Na+ and was independent of Cl- but was blocked by amiloride (500 microM). In the presence of extracellular Na+, initial rates (delta pHi/min) of recovery from an acid load in normal and CF cells were 0.09 +/- 0.03 and 0.12 +/- 0.05, respectively. Pretreatment of cells with phorbol 12-myristate 13-acetate (PMA, 100 nM) caused initial rates of recovery to increase over control values by approximately twofold in both cell preparations. Other studies showed that the native H+ conductance of the plasma membrane was negligible in both normal and CF cell preparations. The results provide evidence in support of an amiloride-sensitive PMA-activated Na(+)-H+ exchanger in ciliated HNE cells from normal and CF donors.

Effects of bradykinin on intracellular calcium regulation in human ciliated airway epithelium.

The Ca(2+)-mobilizing action of bradykinin (BK) was investigated in ciliated human nasal epithelial (HNE) cells utilizing fura-2 fluorescence and microspectrofluorimetry. In ciliated cells, basal intracellular Ca2+ concentration ([Ca2+]i) was 123 +/- 3 nM (n = 142). BK caused [Ca2+]i to increase (spike) rapidly (within 6 s) to greater than 550 nM by releasing Ca2+ from intracellular pools. The mean effective dose for the process was 1.5 x 10(-7) M BK. The spike was due to the activation of a beta 2-receptor. The spike was unaffected by inhibitors of either cyclooxygenase or voltagegated Ca2+ channels. After the spike, [Ca2+]i decreased to a plateau level (120-250 nM). This plateau persisted (up to 10 min) until the addition of La3+ (0.3 x 10(-3) M) or until the removal of either extracellular Ca2+ or the agonist. No changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels were detected after BK exposure. Additional studies revealed that indomethacin (10(-6) M), isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (1 mM) had no effect on [Ca2+]i in ciliated HNE cells. In summary, these data suggest that 1) BK mediates both Ca2+ release from internal pools and Ca2+ entry into the cytoplasm from the extracellular space, and 2) unlike the response to cultured dog airway epithelia, the release of [Ca2+]i in response to BK does not appear to be mediated by either cyclooxygenase pathway or adenylate cyclase-cAMP systems.