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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.

Background: The intestinal epithelium is a single layer of polarized cells and is the primary barrier separating foreign antigen and underlying lymphoid tissue. IFNγ 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 IFNγ 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 IFNγ. 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 IFNγ, TEER dropped significantly after 18 hours, indicating a reduction in barrier function. A further significant decrease was seen in the presence of both IFNγ 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 IFNγ. 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. (Inflamm Bowel Dis 2007)

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.

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.

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.

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 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 perhaps surprising that our understanding of the immunology of this organ remains relatively limited. However, 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 other inflammatory diseases of the respiratory and alimentary tracts such as asthma and inflammatory bowel disease.

Summary 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. Funding Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Fondo de Investigacion Sanitaria, Spain; Charles Courtenay-Cowlin Fund, University of Bristol; UK Arthritis Research Campaign; and the James Tudor Foundation.

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.To determine the mucosal immune response to LPR.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.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 (P0.005), 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 (P0.05, 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 (P0.01).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.

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.

Background. Mucosal immunity is incomplete at birth1,2 and may depend early exposure to bacteria. Objectives. Determine how early neonatal exposure to bacteria alters leukocytes in mammalian upper airways. Method. Five sows were delivered by Caesarean section into a sterile tent. Group A (n = 9) was maintained germ-free. Others were colonised with Schaedler flora plus Staphylococcus (Group B, n = 6) or Bacillus (Group C, n = 5). In group A, two animals were killed at birth and two at 14 days. Remaining animals were killed at a median 24 days. CD4 (helper T-cells), MHC II (monocytic cells) and CD16 (monocyte activation) expression was measured in upper airway mucosa by quantitative immunofluorescence histology. Results. CD4+ cells were rare in Group A, but increased significantly in colonised animals (GpB P = 0.004, GpC P = 0.018). In Group A, MHC II and CD16 expression was low and unaltered by age. Group B exhibited a significant increase in both antigens compared to Group A (MHC II P = 0.002; CD16 P = 0.017). Although higher, levels in Group C were statistically similar to Group A (MHC II P = 0.76, cd16 P = 0.883). There was significantly less co-localisation of CD4+ cells with myeloid cells in Group A (P

Objectives. To determine the symptomatic and mucosal immune response to environmental pollution in UK population. Methods. We performed a prospective, matched questionnaire and laryngeal biopsy study in normal persons (n = 63). Subjects completed a validated health questionnaire. Levels of exposure to pollutants for the year prior to the study were determined via area-based measurements of environmental particulates and composite air quality indices (CAQI). Quantitative multiple-colour immunofluorescence was used to measure in vivo expression of antigens in laryngeal mucosal pinch biopsies: CD45 (RO, RC: leucocytes), CD4, CD8, CD79, (lymphocytes), MHC I, II (antigen presenting molecules). Multiple linear regression, correcting for age, was used to explore the relationship between dependent variables and pollution measures. Results. Complete data were available for 49 (questionnaire) and 54 (immunohistology) subjects. High levels of particulates and CAQI were associated with self-reported frequent sore throats (P = 0.04 and 0.01, respectively), and with a trend towards increased asthma (P = 0.07 and 0.08). Dual HLA-DQ (MHC II) and CD45 (leucocyte) expression by laryngeal epithelium was significantly correlated with levels of particulates (P = 0.03) and CAQI (P = 0.04). CAQI alone was significantly associated with CD4+ expression (P = 0.002). Conclusions. Pollution is known be associated with an increased prevalence of asthma, but this is the first report of an association with throat symptoms. We hypothesise that environmental pollution may contribute to pharyngeal inflammation by selective up-regulation of the MHC Class II molecule, HLA-DQ in epithelium and subsequent activation of leucocytes.

Objectives. To determine whether laryngeal reflux (LPR) affects the population size and distribution of laryngeal mucosal flora in humans; and whether LPR alters the integrity of the laryngeal mucosa. Further, to specifically assess whether Helicobacter pylori (H. pylori) is present in the laryngeal mucosa and if LPR is associated with this. Methods. Laryngeal mucosal biopsies were taken from patients diagnosed LPR-positive or LPR-negative, none of whom had laryngeal disease. Presence of bacteria and epithelial integrity were determined by: fluorescent in situ hybridisation using oligonucleotide probes specific to all Eubacteria and H. pylori; real-time PCR specific for H. Pylori and all bacteria; and immunofluorecence using an antibody to occludin. Results. Helicobacter pylori was discovered in all the biopsies tested – both in those taken from patients who did not have LPR and those that did – with no significant difference in its distribution between the two groups. Helicobacter pylori was found in both the epithelium and lamina propria of the laryngeal mucosa. Further, an increase in the number of total Eubacteria present in the laryngeal mucosa was found to be significantly associated with LPR (LPR mean 58.2 se 19.0 bacteria/field of view, non-LPR mean 17.3 se 4.7; P = 0.02). Conclusions. The association between LPR and an increase in total Eubacteria in the laryngeal mucosa presents a possible aetiology for pathological symptoms associated with LPR. The fact that H. pylori was present in all of the laryngeal mucosal biopsies has significant implications for upper airway research.

Objectives: Damage to epithelial integrity has been implicated in inflammatory disease and neoplasia. Bacterial species such as Pseudomonas and Helicobacter have been shown to cause disease by tight cell junction breakdown and loss of epithelial integrity in the gut (1) There has been one description of the bacterial flora of the human larynx and its variation between individuals, using traditional culture techniques. (2) Sub-site variation has not been characterised. We have compared methods of bacterial species identification and laryngeal sub-site variation. Methods: Bacteria were identified by sequencing of variable regions of the 16S RNA gene, obtained by PCR amplification of DNA from supra- and sub-glottic laryngeal biopsies from pigs. Sequences were aligned with those of known bacteria using the blast nucleotide search engine. Concurrently, bacterial swabs from each larynx subsite were plated on to blood agar. Bacterial colonies isolated by this method were identified by 16S sequencing. Results: A total of 6 Bacterial species were identified in the supra-glottis compared to eight identified in the sub-glottis by culture techniques. 10 bacterial species were identified in the supra-glottis by cloning techniques, including fastidious species. Conclusion: Bacterial species vary between supra and sub-glottis.Cloning techniques are superior at identifying bacterial species when compared with traditional culture techniques. References 1 Sawada N et al. (2003) Med. Electron Microsc.36,147–156 2 Tanaka I et al. (1996)Acta Otoloaryngol.525 (Suppl.), S44–50

Objectives. We recently demonstrated an increase in the MHC-associated molecule, β-2-microglobulin in laryngeal epithelium from patients with laryngopharyngeal reflux (LPR). This could not be accounted for by an increase in MHC Class I, particularly in the superficial layers where Class I is weakly expressed. We hypothesised that this reflected an increase in CD1d, a non-classical MHC molecule. Methods. Posterior laryngeal biopsies were obtained from 10 controls and 11 LPR patients. Multicolour immunofluorescence microscopy was used to measure expression of CD1d, CD161 (NK/NKT marker), CD4/8 T-cells, macrophage, neutrophil and eosinophil antigens. Two-way analysis of variance and unpaired T-tests were performed on transformed data. Results. Expression of CD1d increased from basal to superficial layers, and was marginally raised in LPR (P = 0.067), whilst CD161 increased significantly (P = 0.02). While CD161(+) cells were distributed throughout the epithelium, CD8(+) T-cells were localised to the deep, MHC class I-expressing layers. EMBP was significantly reduced in LPR (P = 0.01). Conclusions. CD1d and MHC class I exhibit reciprocal expression patterns, which are reflected by the distribution of cells expressing their respective ligands (NKT cells and CD8(+) T-cells). An increase in CD1d in LPR may be responsible for the increase in β-2-microglobulin observed, and is associated with a significant increase in NKT-cells. The CD1d-NKT cell axis may be a crucial part of maintaining tolerance in the laryngeal mucosa and changes in this system may be responsible for the persistence of inflammation in LPR.