Tracheal Xenografts Research Articles

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation invitro and in tracheal xenografts invivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Orthotopic tracheal transplantation using human bronchus: an original xenotransplant model of obliterative airway disorder.

Bronchiolitis obliterans syndrome (BOS) is a main cause of allograft dysfunction and mortality after lung transplantation (LTx). A better understanding of BOS pathogenesis is needed to overcome this treatment-refractory complication. Orthotopic tracheal transplantation using human bronchus was performed in Brown Norway (BN) and nude (RNU) rats. Allografts were recovered in both strains at Day 7 (BN7 , n = 6; RNU7 , n = 7) or Day 28 (BN28 , n = 6; RNU28 , n = 6). Immune response of the host against the bronchial graft was assessed. Human samples from BOS patients were used to compare with the histological features of the animal model. Obstruction of the allograft lumen associated with significant infiltration of CD3+ and CD68+ cells was observed in the BN group on Day 28. Immune response from type 1 T-helper cells against the tracheal xenograft was higher in BN animals compared to nude animals on Days 7 and 28 (P < 0.001 and P = 0.035). Xenoreactive antibodies were significantly higher at Day 7 (IgM) and Day 28 (IgG) in the BN group compared to RNU (respectively, 37.6 ± 6.5 vs. 5.8 ± 0.7 mean fluorescence, P = 0.039; and 22.4 ± 3.8 vs. 6.9 ± 1.6 mean fluorescence, P = 0.011). Immunocompetent animals showed a higher infiltration of S100A4+ cells inside the bronchial wall after 28 days, associated with cartilage damage ranging from invasion to complete destruction. In vitro expression of S100A4 by human fibroblasts was higher when stimulated by mononuclear cells (MNCs) from BN rats than from RNU (2.9 ± 0.1 vs. 2.4 ± 0.1 mean fluorescence intensity, P = 0.005). Similarly, S100A4 was highly expressed in response to human MNCs compared to stimulation by T-cell-depleted human MNCs (4.3 ± 0.2 vs. 2.7 ± 0.1 mean fluorescence intensity, P < 0.001). Obliterative bronchiolitis has been induced in a new xenotransplant model in which chronic airway obstruction was associated with immune activation against the xenograft. Cartilage infiltration by S100A4+ cells might be stimulated by T cells.

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

Co-culture-expanded human basal epithelial stem cells for application in tracheal tissue engineering

BackgroundStem-cell-based tracheal replacement has been used to treat patients with end-stage airway disease in compassionate cases. Clinical experience suggests that restoration of an epithelial barrier is a priority to improve outcomes, but recent data suggest that a substantial number of autologous epithelial cells are required for effective engraftment. Existing cell culture methods fail to expand the airway epithelial progenitor pool, presenting a considerable hurdle for progression to clinical trials. We aimed to assess feeder layers and a Rho-associated kinase inhibitor as a method to expand human respiratory epithlieum for airway bioengineering. MethodsHuman epithelial cells from endobronchial biopsy samples were cultured on mouse 3T3-J2 fibroblast feeder layers in medium containing Y-27632, a Rho-associated kinase inhibitor (3T3+Y). Air-liquid interface and tracheosphere assays were done to determine differentiation capacity, and high-speed video microscopy to observe ciliary behaviour. Telomerase expression was measured with immunofluorescence and western blotting and telomere length with real-time PCR. Cells were karyotyped and engraftment potential tested in ex-vivo and in-vivo xenograft models. Findings3T3+Y allowed for rapid and sustained expansion of airway epithelial basal cells. Population doublings demonstrated that cultures derived from donor biopsy samples could provide sufficient cell numbers to recellularise human tracheal scaffolds. At clinically applicable passages, cells were capable of airway differentiation in vitro, forming both ciliated and goblet lineages. Ciliary beat frequency and beat pattern were within normal range. Cells were karyotypically normal despite extensive expansion. Protein expression of telomerase was increased in 3T3+Y compared with that in conventional growth medium, and telomere length appeared to stabilise. Cultured cells repopulated a decellularised tracheal scaffold ex vivo and restored a differentiated epithelium in an in-vivo tracheal transplantation xenograft model. InterpretationThe findings show that 3T3+Y generates large numbers of airway basal epithelial stem cells that retain qualities desirable for clinical transplantation. These data have important implications for personalised autologous airway epithelial cell therapy because cells could be obtained rapidly and in an appropriate number. Preclinical and clinical validation is required. FundingWellcome Trust.

Gene Delivery to the Airway

This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts, as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols.

TFF (Trefoil Factor Family) Peptides and their Potential Roles for Differentiation Processes During Airway Remodeling

Several lines of defense maintain the surface integrity of the delicate airway epithelium which is regularly subjected to severe trauma. These defense mechanisms include protection by the mucus layer, rapid repair by restitution (cell migration) and regeneration via proliferation and differentiation. Luminal surveillance peptides such as epidermal growth factor (EGF) and trefoil factor family (TFF) peptides support synergistically these processes. TFFs are well known particularly for their key role in mucosal restitution and there is an increasing body of evidence that TFFs also support mucosal differentiation processes. Mucus overproduction during inflammatory and obstructive airway diseases is a partial consequence of an increase in the number of goblet cells due to cell division (goblet cell hyperplasia) or differentiation (goblet cell metaplasia). Particularly the latter process reflects the plasticity of the airway epithelium and causes intense airway remodeling. Goblet cells are derived, at least in part, from Clara cells, which trans-differentiate from a serous into a mucous phenotype. This process is critically dependent upon IL-13. In a recent report (Kouznetsova et al. AJRCMB 36:286-297, 2007) using a murine asthma model it was shown that trans-differentiating Clara cells specifically express Tff1 which is stored in a specific subset of secretory granules. This points to a role for Tff1 as an autocrine factor for the trans-differentiation of Clara cells toward goblet cells. Such a role of TFFs for differentiation processes of the airways is supported by another recent study (LeSimple et al. AJRCMB 36:296-303, 2007) where induction of TFF3 synthesis was shown with differentiation in in vivo humanized tracheal xenograft and in vitro air-liquid interface culture models. Furthermore, exogenous TFF3 promoted differentiation of ciliated cells in an EGF receptor-dependent manner. Taken together, both studies imply that TFFs may play key roles for various differentiation processes of the airways and they could be promising novel targets in order to treat severe chronic and acute airway diseases.

In vivomodels of human airway epithelium repair and regeneration

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. Thein vivostudy 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 reconstitute a functional respiratory epithelium after several weeks.Humanised tracheal xenograft models have been developed in immunodeficient nude and severe combined immunodeficient (SCID) mice in order to mimic the natural regeneration process of the human airway epithelium and to analyse the cellular and molecular events involved during the different steps of airway epithelial reconstitution. These models represent very powerful tools for analysing the modulation of the biological functions of the epithelium during its regeneration. They are also very useful for identifying stem/progenitor cells of the human airway epithelium.A better knowledge of the mechanisms involved in airway epithelium regeneration, as well as the characterisation of the epithelial stem and progenitor cells, 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.

Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo

Recent work has indicated that adult bone marrow-derived cells have the ability to contribute to both the haematopoietic system and other organs. Haematopoietic reconstitution by whole bone marrow and selected but not fully characterised cell populations have resulted in reports indicating high-level repopulation of lung epithelia. The well-characterised cells from the side population have a robust ability for haematopoietic reconstitution. We have used freshly isolated side population cells derived from ROSA26 adult bone marrow and demonstrate that despite being unable to contribute to embryos following blastocyst injection, or air liquid interface cultures or denuded tracheal xenografts, they could contribute to the tracheal epithelium in vivo. Epithelial damage is reported to be important in encouraging the recruitment of marrow-derived stem cells into non-haematopoietic organs. Here we demonstrate that mice engrafted with side population cells have donor-derived cells present in the epithelial lining of the trachea following damage and repair. Donor-derived cells were found at a frequency of 0.83%. Widefield and confocal microscopy revealed donor cells that expressed cytokeratins, indicative of cells of an epithelial nature. These results imply that SP haematopoietic stem cells from the bone marrow do not have the ability to contribute to airway epithelia themselves but require factors present in vivo to allow them to acquire characteristics of this tissue.

Lysozyme Secretion by Submucosal Glands Protects the Airway from Bacterial Infection

Submucosal glands are abundant (approximately 1 gland/mm2) secretory structures in the tracheobronchial airways of the human lung. Because submucosal glands express antibacterial proteins, it has been proposed that they contribute to lung defense. However, this concept is challenged by the fact that mice do not have submucosal glands in their bronchial airways, yet are quite resistant to bacterial lung infection. The contribution of airway submucosal glands to host defense is also debated as a pathophysiologic component of cystic fibrosis lung disease. Here, we asked whether submucosal glands protect airways against bacterial infection. By comparing tracheal xenograft airways with and without glands, we found that the presence of glands enhanced bacterial killing in vivo and by airway secretions in vitro. Moreover, immunodepletion studies suggested that lysozyme is a major antibacterial component secreted by submucosal glands. These studies provide evidence that submucosal glands are a major source of antibacterials critical for maintaining sterile airways.

Human fetal trachea-scid mouse xenografts: efficacy of vesicular stomatitis virus-G pseudotyped lentiviral-mediated gene transfer

Background/Purpose Postnatal gene transfer in respiratory epithelium has been inefficient, particularly in submucosal gland cells, the target cells for cystic fibrosis transmembrane regulator (CFTR) gene transfer in cystic fibrosis. The authors hypothesized that fetal tracheobronchial epithelium may be more receptive to gene transfer in that precursor cells of the respiratory epithelium may be more accessible. Methods Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral vector was first tested in human fetal tracheal organ culture then validated in a human fetal tracheal xenograft model in severe combined immunodeficiency (SCID) mice. Between 7 × 10 7 and 1.6 × 10 8 transducing units of lentiviral vector encoding the transgene LacZ under the control of the human CMV promoter were administered onto the lumenal surface of the xenografts (n = 6). Biopsy specimens were taken from the xenografts at one month (n = 1), 2 months (n = 2), 3 months (n = 1), 6 months (n = 4), and 9 months (n = 1) after vector administration. Analysis of transgene expression was performed on X-gal stained sections. Results Transgene expression was observed in 20.2% to 99% of the surface epithelial cells (mean, 70.8 ± 32.3% SD) and in 28.4% to 99% of the submucosal gland cells (mean, 68.5 ± 26.2% SD) out to 9 months after vector administration in the tracheal xenografts, equivalent to 63 weeks postconceptual age. No staining was seen in the controls. Conclusions Excellent gene transfer in human fetal tracheal xenografts after VSV-G pseudotyped lentiviral vector administration, which may result from more accessible target precursor cells during development, suggests the feasibility of fetal gene therapy for the treatment of congenital airway disease such as cystic fibrosis.

Targeted delivery of antiprotease to the epithelial surface of human tracheal xenografts.

The cystic fibrosis (CF) lung is uniquely susceptible to Pseudomonas aeruginosa, and infection with this organism incites an intense, compartmentalized inflammatory response that leads to chronic airway obstruction and bronchiectasis. Neutrophils migrate into the airway, and released neutrophil elastase contributes to the progression of the lung disease characteristic of CF. We have developed a strategy that permits the delivery of antiproteases to the inaccessible CF airways by targeting the respiratory epithelium via the human polymeric immunoglobulin receptor (hpIgR). A fusion protein consisting of a single-chain Fv directed against secretory component, the extracellular portion of the pIgR, linked to human alpha1-antitrypsin is effectively ferried across human tracheal xenografts and delivers the antiprotease to the apical surface to a much greater extent than occurs by passive diffusion of human alpha1-antitrypsin alone. Targeted antiprotease delivery paralleled hpIgR expression in the respiratory epithelium in vivo and was not increased by escalating dose, so airway penetration was receptor-dependent, not dose-dependent. Thus, this approach provides us with the ability to deliver therapeutics, like antiproteases, specifically to the lumenal surface of the respiratory epithelium, within the airway surface fluid, where it will be in highest concentration at this site.

Fluticasone propionate inhibits lipopolysaccharide-induced proinflammatory response in human cystic fibrosis airway grafts.

Airway inflammation, one of the major factors leading to lung damage in cystic fibrosis (CF) patients, is associated with an abnormal increase in proinflammatory cytokines. In this work, we demonstrate the increased release of the proinflammatory cytokines after lipopolysaccharide (LPS) stimulation: human interleukin (hIL)-8 in CF and non-CF airway xenografts, and hIL-6 and human growth-related oncogene-alpha (hGRO-alpha), which could be only analyzed in non-CF xenografts. Under basal conditions, we observed that hIL-8 was higher in CF xenografts compared with non-CF. We also report the anti-inflammatory effect of a glucocorticoid, fluticasone propionate (FP), on CF airway epithelium using a humanized model of airway inflammation developed in nude mice. In CF and non-CF tracheal xenografts, airway inflammation was induced by inoculating Pseudomonas aeruginosa LPS (4 h; 1 microg/ml) in the lumen of the xenografts. FP pretreatment (2 h; 10(-8) M) followed by P. aeruginosa LPS stimulation induced a significant reduction of LPS-induced hIL-8 release in airway liquid collected from CF and non-CF tracheal xenografts (85 and 80%, respectively). In non-CF tracheal xenografts, FP treatment before LPS stimulation induced a significant decrease in hIL-6 and hGRO-alpha. From these data, we suggest that FP exerts anti-inflammatory properties that may be appropriate to CF therapy, at an early stage of the disease. In addition, these results demonstrate that the humanized airway model of inflammation provides a relevant tool for analyzing the effects of anti-inflammatory drugs in different diseases in which airway inflammation is implicated.

Feasibility of cryopreserved tracheal xenotransplants with the use of short-course immunosuppression

Objective: We evaluated the feasibility of discordant xenotransplantation of the cryopreserved trachea with intermittent immunosuppression to help solve the shortage of donor tracheas. Methods: Two experiments were performed with heterotopic transplantation models in 14 guinea pigs and 85 rats. So that the minimal dose of FK506 for viable fresh xenografts could be determined, FK506 was given in escalating doses (0, 1.5, 2.5, and 3.5 mg/kg) for recipient animals after xenogeneic transplantation. With the goal of obtaining a long-term survival of the xenografts, the effect of cryopreservation on xenografts was assessed and thereafter different cycles of immunosuppression every third week were evaluated in fresh or cryopreserved xenografts in the second experiment. Results: An FK506 dosage of more than 2.5 mg/kg per day was much more effective than smaller dosages, as demonstrated by morphologic assessment. A higher dosage of FK506 potentially delayed the rejection of xenografts and can thus maintain tracheal xenograft viability for less than 4 weeks in rat recipients. In experiment 2, the cryopreserved xenografts showed less histologic viability than fresh xenografts but greater patency of the lumen. The patency of cryopreserved xenografts was favorably maintained for a longer period than that of fresh xenografts with either the same number or more cycles of immunosuppression. Conclusions: We conclude that the synergistic effect of cryopreservation and adequate intermittent immunosuppression may enable tracheal xenografts to remain viable over longer periods. (J Thorac Cardiovasc Surg 2001;121:241-8)

New models of the tracheal airway define the glandular contribution to airway surface fluid and electrolyte composition.

Antibacterial defenses in the airway are dependent on multifactorial influences that determine the composition of both fluid and/or electrolytes at the surface of the airway and the secretory products that aid in bacterial killing and clearance. In cystic fibrosis (CF), these mechanisms of airway protection may be defective, leading to increased colonization with Pseudomonas aeruginosa. Submucosal glands, a predominant site of cystic fibrosis transmembrane conductance regulator (CFTR) protein expression in the airway, have been hypothesized to play an important role in protection of the airway. Furthermore, recent studies have suggested that the salt concentration at the airway surface may be a key factor in regulating the activity of antibacterial substances in the airway. To explore these issues, we have used a new model of the ferret tracheal airway to evaluate the contribution of submucosal glands in regulating airway surface fluid and electrolyte composition. Using tracheal xenograft models with and without submucosal glands, we have characterized several aspects of airway physiology that may be important in defining antibacterial properties. These endpoints included the contribution of submucosal glands in defining bioelectric properties of the surface airway epithelium, airway surface fluid (ASF) chloride composition, ASF volumes, and secretion of the antibacterial factor lysozyme. Findings from these studies demonstrate a significantly elevated secreted fluid volume (Vs) and chloride concentration ([Cl](s)) in ASF from airways with submucosal glands (Vs = 47 +/- 4 microl; [Cl](s) = 128 +/- 5 mM), as compared with xenograft airways without glands (Vs = 36 +/- 2 microl; [Cl](s) = 103 +/- 6 mM). Furthermore, a temperature labile factor secreted by submucosal glands appears to alter the baseline activation of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and/or diphenylamine-2-carboxylic acid-sensitive chloride channels in the surface airway epithelium. Lastly, the lysozyme content of tracheal airways with submucosal glands was 8.5-fold higher than were airways without glands. These studies demonstrate that submucosal glands affect both the ionic composition and bioelectric properties of the airway and suggest that models evaluating antibacterial properties of the airway in CF should take into account the contribution of glands in airway physiology.

Obliterative airway disease and graft stenting in pig-to-dog tracheal xenotransplantation

Obliterative airway disease occurring in concordant tracheal xenografts in rodent models is histologically similar to obliterative bronchiolitis in human lung allografts. We studied whether obliterative airway disease would occur in a large animal-discordant model. Pig and dog tracheas were cryopreserved for 7 to 14 days, and 18 recipient dogs given splenectomy 7 days before transplantation, then seven tracheal rings were removed and a corresponding five-ring donor tracheal segment was transplanted to the excised site. Grafts were wrapped with pedicled omentum and inmmunosuppression was conducted with tacrolimus or deoxyspergualin. Graft status was observed by bronchoscopy. Dogs were classified into three groups. Group 1 consisted of dog-to-dog allotransplantation animals (control group, n = 5), Group 2 of pig-to-dog xenotransplantation animals (n = 8), and Group 3 of pig-dog xenotransplantation animals who also underwent graft stenting immediately after transplantation (n = 5). Grafts healed well in 4 of 5 Group 1 dogs. Tracheal stricture began on day 5 post transplantation and the lumen was obstructed by fibrosis by days 8 to 14 in all Group 2 dogs. All Group 3 dogs remained in good respiratory status until death. Obliterative airway disease developed quickly in pig-to-dog discordant tracheal xenografts. Graft stenting is a feasible treatment for managing of tracheal obstruction.

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.

Substance P and neurokinin-1 receptor expression by intrinsic airway neurons in the rat.

Tachykinins and their receptors are involved in the amplification of inflammation in the airways. We analyzed the expression of preprotachykinin-A (PPT-A) and neurokinin-1 (NK-1) receptor genes by intrinsic airway neurons in the rat. We also tested the hypothesis that PPT-A-encoded peptides released by these neurons fulfill the requisite role of substance P in immune complex injury of the lungs. We found that ganglion neurons in intact and denervated airways or in primary culture coexpress PPT-A and NK-1 receptor mRNAs and their protein products. Denervated ganglia from tracheal xenografts (nu/nu mice) or syngeneic lung grafts had increased PPT-A mRNA contents, suggesting preganglionic regulation. Formation of immune complexes in the airways induced comparable inflammatory injuries in syngeneic lung grafts, which lack peptidergic sensory fibers, and control lungs. The injury was attenuated in both cases by pretreatment with the NK-1 receptor antagonist LY-306740. We conclude that tachykinins released by ganglia act as a paracrine or autocrine signal in the airways and may contribute to NK-1 receptor-mediated amplification of immune injury in the lungs.

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.

The novel Trk receptor tyrosine kinase inhibitor CEP-701 (KT-5555) exhibits antitumor efficacy against human pancreatic carcinoma (Panc1) xenograft growth and in vivo invasiveness.

The survival rate for patients with pancreatic ductal adenocarcinoma (PDAC) is among the poorest for all cancers. The factors that contribute to this poor prognosis are lack of effective early detection, high rate of metastases and a generally refractory response to available treatment modalities. The most commonly used treatment methods--chemotherapy and radiation therapy--are mainly used for symptom palliation, with surgery being the only "curative" treatment option. The use of combinations of treatment modalities is the only therapy available to patients with locally advanced disease or that which is surgically unresectable. These options are still not sufficient to increase patient survival time significantly. The aggressive behavior and poor prognosis of this cancer is associated with an increased expression of many growth factors and their cognate receptors. We have demonstrated previously the aberrant expression of the Trk receptors (Trks A, B, and C) in PDAC specimens and human PDAC-derived cell lines and a biphasic, dose-dependent response of specific neurotrophic agents on the in vitro invasiveness of PDAC cells. Based on these data we have evaluated the therapeutic potential of inhibiting neurotrophin-Trk interactions using a selective Trk tyrosine kinase inhibitor (CEP-701) on subcutaneous (s.c.) and tracheal xenografts derived from the poorly differentiated PDAC cell line, Panc1. We demonstrate that CEP-701 administration at 10 mg/kg s.c. BID for 21 days inhibited tumor growth of the Panc1 s.c. xenografts in a statistically-significant manner (p < 0.01) compared to vehicle controls, in the absence of morbidity and mortality. A T/C value of 25% was observed for CEP-701-treated s.c. xenografts. In addition, CEP-701 administration inhibited tumor cell invasion in the s.c. tracheal xenograft model of in vivo invasiveness. Taken together, these data suggest that further studies are warranted to evaluate CEP-701 as a potential therapeutic agent in the treatment of PDAC.