Louisa Rees

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 recipients 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.

Norepinephrine increases the pathogenic potential of Campylobacter jejuni

Background:Campylobacter jejuni can cause a spectrum of diseases in humans, ranging from enteritis and diarrhoea to severe inflammation, profuse bloody diarrhoea and chronic relapsing infection. Norepinephrine (NE) levels in the intestine increase under conditions of stress and trauma, and are thought to result in spill over of NE into the intestinal lumen. NE is known to stimulate the growth of a range of bacterial species, and to increase the pathogenicity of Escherichia coli. Aim: To determine the effects of NE on the pathogenic potential of C jejuni in a model system. Methods:C jejuni was grown in iron-replete and iron-limited media in the presence and absence of 100 μM NE. Several virulence-associated characteristics, including motility and cell invasion, were measured. Results: When C jejuni was grown in iron-limited media in the presence of NE, growth rate, motility and invasion of cultured epithelial cells were increased compared with cultures grown in the absence of NE. Bacteria exposed to NE during growth also caused greater subsequent disruption of cultured epithelial cell monolayers, inducing widespread breakdown of tight junctions. Conclusion: Exposure to NE causes an increase in the virulence-associated properties of Campylobacter. Stress and concomitant infection could therefore be contributory factors to the variable presentation of this disease.

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.

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.

At the crossroads: mucosal immunology of the larynx.

The larynx sits at the crossroads between gastrointestinal and respiratory tracts. Besides its intrinsic importance in breathing, swallowing and voice production, the larynx is also exposed to unique immunological challenges. Given the propensity of chronic inflammatory conditions such as chronic laryngitis, which affects up to 20% of Western populations, it is surprising that our understanding of the immunology of this organ remains relatively limited. Recent work on the immunological architecture of the laryngeal mucosa, and its changes that result from external challenges and inflammatory conditions, provided valuable insight into the fascinating immunology of this organ. The lessons learnt from these investigations may go beyond devising improved therapy for chronic laryngeal inflammation. Establishing whether and how the laryngeal mucosa may be involved in the modulation of wider mucosal responses may provide novel routes to the treatment of inflammatory diseases of the respiratory and alimentary tracts such as asthma and inflammatory bowel disease.

Campylobacter and IFNγ 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. IFN&ggr; 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 IFN&ggr; 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 IFN&ggr;. 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 IFN&ggr;, TEER dropped significantly after 18 hours, indicating a reduction in barrier function. A further significant decrease was seen in the presence of both IFN&ggr; 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 IFN&ggr;. 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, β2m, 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.

Laryngopharyngeal reflux and the flora of the larynx

OBJECTIVES: To describe our experience with Gore-Tex medialization laryngoplasty and the treatment of dysphagia as well as examine the anatomy of Gore-Tex and silastic medialization laryngoplasty in two fresh cadaver larynges. METHODS: Retrospective case review/anatomic cadaver dissection. Tertiary referral center. Between April 2000 and September 2008, 189 Gore-Tex medialization laryngoplasties were performed on 180 patients by the senior author. Complete records and analyses were available and performed on 121 procedures for 113 patients. In addition, two fresh cadaver larynges were dissected for anatomic analysis. Main outcome measures were discontinuation of g-tube use or avoidance of g-tube, as well as clinical subjective improvement in swallowing function. Cadaver dissection measurements were made to assess true vocal fold lengthening following silastic and GoreTex medialization laryngoplasty, as well as photodocumentation of implant location. RESULTS: 57/113 (50%) of patients had complaints of dysphagia at presentation with 47/57 (82%) having an objective swallowing evaluation. 32/47 (68%) documented penetration and/or aspiration. 20/57 (35%) patients with dysphagia required g-tubes for alimentation. 11/20 (55%) patients were able to discontinue g-tube use following Gore-Tex medialization laryngoplasty and an additional 5 patients with aspiration were able to avoid g-tubes with Gore-Tex medialization laryngoplasty and swallowing therapy. Cadaver dissection demonstrated increased true vocal cord lengthening with Gore-Tex and a more posterior position than can be achieved with silastic medialization. CONCLUSIONS: Gore-Tex medialization laryngoplasty is a well tolerated and described treatment for the management of glottal incompetence. The procedure is an appropriate adjunct in dysphagia management for the appropriate patient population.

Helicobacter pylori Is Present in Head and Neck Cancer Lesions

Objective: To investigate the presence of Helicobacter pylori (H pylori), a key pathogen in chronic gastritis and recognized as a major cause of gastric cancer, in cancer samples and adjacent normal tissues of the head and neck region. Method: Twenty-eight fresh-frozen HNSCC samples and 11 biopsies from normal tissue were obtained from fully informed and consented patients at GSHU. From 8 of these cancer samples adjacent normal tissue was available. FISH using a probe for H pylori was used to examine sections of these samples and enumerate the bacterium. Results: The average number of H pylori per biopsy section was significantly higher in the SCC group (P < .03) than controls for all sites examined. Most interestingly, in paired SCC and adjacent normal biopsies from the same patient the average number of H pylori per biopsy was significantly higher in the SCC tissue compared with the adjacent normal tissue (P < .006). These data indicate that H pylori shows a numerical and spatial association with squamous cell cancer lesions of the head and neck. Conclusion: The fact that H pylori is present in higher numbers in the tumor tissue compared with adjacent tissue and tissue obtained from healthy individuals suggests either a predisposition for the colonization of SCC with H pylori, or a causative role for the bacterium in cancer at these sites.