Clinical transplantation of a tissue-engineered airway.

BACKGROUND: The loss of a normal airway is devastating. Attempts to replace large airways have met with serious problems. Prerequisites for a tissue-engineered replacement are a suitable matrix, cells, ideal mechanical properties, and the absence of antigenicity. We aimed to bioengineer tubular tracheal matrices, using a tissue-engineering protocol, and to assess the application of this technology in a patient with end-stage airway disease. METHODS: We removed cells and MHC antigens from a human donor trachea, which was then readily colonised by epithelial cells and mesenchymal stem-cell-derived chondrocytes that had been cultured from cells taken from the recipient (a 30-year old woman with end-stage bronchomalacia). This graft was then used to replace the recipient's left main bronchus. FINDINGS: The graft immediately provided the recipient with a functional airway, improved her quality of life, and had a normal appearance and mechanical properties at 4 months. The patient had no anti-donor antibodies and was not on immunosuppressive drugs. INTERPRETATION: The results show that we can produce a cellular, tissue-engineered airway with mechanical properties that allow normal functioning, and which is free from the risks of rejection. The findings suggest that autologous cells combined with appropriate biomaterials might provide successful treatment for patients with serious clinical disorders.

The mucosal immune response to laryngopharyngeal reflux.

RATIONALE: Laryngopharyngeal reflux (LPR) affects up to 20% of Western populations. Although individual morbidity is usually moderate, treatment costs are high and there are associations with other diseases, including laryngeal cancer. To date, there have been no studies of the mucosal immune response to this common inflammatory disease. OBJECTIVES: To determine the mucosal immune response to LPR. METHODS: We performed a prospective immunologic study of laryngeal biopsies from patients with LPR and control subjects (n = 12 and 11, respectively), and of primary laryngeal epithelial cells in vitro. MEASUREMENTS AND MAIN RESULTS: Quantitative multiple-color immunofluorescence, using antibodies for lymphocytes (CD4, CD8, CD3, CD79, CD161), granulocytes (CD68, EMBP), monocytic cells (CD68, major histocompatibility complex [MHC] class II), and classical and nonclassical MHC (I, II, beta(2)-microglobulin, CD1d). Univariate and multivariate analysis and colocalization measurements were applied. There was an increase in percentage area of mucosal CD8(+) cells in the epithelium (P < 0.005), whereas other leukocyte and granulocyte antigens were unchanged. Although epithelial MHC class I and II expression was unchanged by reflux, expression of the nonclassical MHC molecule CD1d increased (P < 0.05, luminal layers). In vitro, laryngeal epithelial cells constitutively expressed CD1d. CD1d and MHC I expression were inversely related in all subjects, in a pattern which appears to be unique to the upper airway. Colocalization of natural killer T (NKT) cells with CD1d increased in patients (P < 0.01). CONCLUSIONS: These data indicate a role for the CD1d-NKT cell axis in response to LPR in humans. This represents a useful target for novel diagnostics and treatments in this common condition.

Campylobacter and IFNgamma interact to cause a rapid loss of epithelial barrier integrity.

BACKGROUND: The intestinal epithelium is a single layer of polarized cells and is the primary barrier separating foreign antigen and underlying lymphoid tissue. IFNgamma alters epithelial barrier function during inflammation by disrupting tight cell junctions and facilitating the paracellular transport of luminal antigens. The aim of this work was to determine whether Campylobacter infection of cells exposed to IFNgamma would lead to greater disruption of cell monolayers and hence increased bacterial translocation. METHODS: Monolayers were polarized on Transwell polycarbonate membranes for 14 days and then cultured in the presence or absence of 100 U/mL IFNgamma. Campylobacter was added to the apical side of the monolayer at an MOI of 30. Transepithelial electrical resistance (TEER) was recorded and bacteria in the basal well counted every 2 hours. Cells were stained for occludin, actin, and nuclear DNA, and cell viability determined by measurement of apoptosis. RESULTS: In the presence of IFNgamma, TEER dropped significantly after 18 hours, indicating a reduction in barrier function. A further significant decrease was seen in the presence of both IFNgamma and Campylobacter, indicating a synergistic effect, and cellular morphology and viability were affected. Bacterial translocation across the monolayer was also significantly greater in the presence of IFNgamma. CONCLUSIONS: These combined effects indicate that Campylobacter infection concomitant with intestinal inflammation would result in a rapid and dramatic loss of epithelial barrier integrity, which may be a key event in the pathogenesis of Campylobacter-mediated colitis and the development of bloody diarrhea.

Immunologic response of the laryngeal mucosa to extraesophageal reflux.

OBJECTIVES: Extraesophageal reflux is common. The treatment costs are high, and there are associations with other diseases, including laryngeal cancer. Our studies of the mucosal immune response to this common inflammatory disease suggest an important role for the nonclassic antigen-presenting molecule CD1d in the response to inflammation. This study was performed to further explore the relationship between the CD1d-NKT cell-iGb3 axis and reflux. METHODS: We carried out a prospective study of laryngeal biopsies from 12 patients with laryngopharyngeal reflux and 11 controls. Quantitative multiple-color immunofluorescence using antibodies for lymphocytes (CD3, CD161) and classic and nonclassic major histocompatibility complex (I, II, beta2m, CD1d) was performed, and univariate and multivariate analysis and co-localization measurements were applied. RESULTS: Epithelial major histocompatibility complex class I and II expression was unchanged by reflux, but expression of CD1d increased (p < 0.05; luminal layers) and confidence intervals diminished in the reflux group. Co-localization of NKT cells with CD1d increased in patients (p < 0.01); iGb3 exhibited strong expression throughout all layers of the laryngeal epithelium. CONCLUSIONS: These data indicate a role for the CD1d-NKT cell-iGb3 axis in response to extraesophageal reflux in humans. This represents a useful target for novel diagnostics and treatments for this common condition.

The isolation and characterisation of primary human laryngeal epithelial cells.

We have shown that the larynx has a prominent immunological component that varies between individuals, and which is influenced by lifestyle factors implicated in the pathogenesis of the inflammatory and neoplastic diseases of the larynx. In order to explore the mechanisms of such links between laryngeal mucosal immunity and the development of lifestyle-related disease, reliable in vitro models are essential. In this study, we isolated and characterised primary laryngeal epithelial cells from normal individuals and show they can be cultured and manipulated to express MHC class II molecules in vitro.

A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: from concept to clinical trial.

Cell and tissue engineering are now being translated into clinical organ replacement, offering alternatives to fight morbidity, organ shortages and ethico-social problems associated with allotransplantation. Central to the recent first successful use of stem cells to create an organ replacement in man was our development of a bioreactor environment. Critical design features were the abilities to drive the growth of two different cell types, to support 3D maturation, to maintain biomechanical and biological properties and to provide appropriate hydrodynamic stimuli and adequate mass transport. An analytical model was developed and applied to predict oxygen profiles in the bioreactor-cultured organ construct and in the culture media, comparing representative culture configurations and operating conditions. Autologous respiratory epithelial cells and mesenchymal stem cells (BMSCs, then differentiated into chondrocytes) were isolated, characterized and expanded. Both cell types were seeded and cultured onto a decellularized human donor tracheal matrix within the bioreactor. One year post-operatively, graft and patient are healthy, and biopsies confirm angiogenesis, viable epithelial cells and chondrocytes. Our rotating double-chamber bioreactor permits the efficient repopulation of a decellularized human matrix, a concept that can be applied clinically, as demonstrated by the successful tracheal transplantation.

Smoking influences the immunological architecture of the human larynx.

Little is known about the effects of demographic and lifestyle factors on laryngeal mucosal immunology. Pinch biopsies of laryngeal mucosa were studied from 63 patients without laryngeal disease. Areas of positive staining for HLA-DR, HLA-DQ, HLA-DP, CD45, CD45RA, CD45RO, CD4, CD8, and CD79 were calculated. Patients were stratified according to gender and smoking status. Analysis of covariance showed current cigarette smokers had increased numbers of CD4+ T cells and there was an association between older age and greater CD4+ T cell numbers in both epithelium and lamina propria. Older age and female gender were associated with decreased lamina propria CD4+ CD45RO+ T cells and an increase in CD4+ CD45RO- T cells. T cell populations in the larynx may therefore be influenced by smoking, age and gender. We hypothesize that smoking induces changes in normal immunological function of the larynx, which may contribute to the etiology of inflammatory disease and cancer.