William R. Roche

Immunohistochemistry on Resin Sections: A Comparison of Resin Embedding Techniques for Small Mucosal Biopsies

We have modified resin embedding methods to provide optimal information from endoscopic biopsies. Mucosal biopsies were fixed either in buffered formalin and processed for embedding in Araldite or in acetone containing protease inhibitors and embedded in glycol methacrylate (GMA). GMA embedding generated an immunophenotypic profile similar to that obtained in frozen sections while yielding far superior morphology and greater numbers of sections from small biopsies. The phenotypic markers included those for T cells, macrophages, mast cells, eosinophils and neutrophils. We have also demonstrated collagens, cell adhesion molecules and integrin molecules. Sections of similar quality were obtained with Araldite but the repertoire of antibodies was restricted to those which can be applied to formalin fixed, paraffin embedded tissues. We suggest that for optimal results, small biopsies to be subjected to immunochemistry are fixed in acetone at -20 C with the inclusion of protease inhibitors and embedded in GUIA with careful temperature control.

The bronchial epithelium as a key regulator of airway inflammation and remodelling in asthma

While asthma is an inflammatory disorder of the airways involving mediator release from mast cells and eosinophils and orchestrated by T cells, inflammation alone is insufficient to explain the chronic nature of the disease and its progression. Evidence is presented that the epithelium is fundamentally disordered in chronic asthma manifest by increased fragility, and an altered phenotype to one that secretes mucus, mediators, cytokines, chemokines and growth factors. Epithelial injury is mediated by exogenous factors such as air pollutants, viruses and allergens as well as by endogenous factors including the release of proteolytic enzymes from mast cells (tryptase, chymase) and eosinophils (MMP‐9). Following injury, the normal epithelium should respond with increased proliferation driven by ligands acting on epidermal growth factor (EGF) receptors or through transactivation of the receptor. The epithelial response to these stimuli in asthma appears to be impaired despite upregulation of CD44 capable of enhancing presentation of EGF ligands to epidermal growth factor receptors (EGFR). Because the epithelium is ‘held’ in this repair phenotype, it becomes a continuous source of proinflammatory products as well as growth factors that drive airway wall remodelling.

Markers of eosinophilic inflammation and tissue re-modelling in children before clinically diagnosed bronchial asthma.

Chronic inflammatory changes in the bronchial mucosa have been well documented in patients with established asthma. Much less is known of the changes, which occur in the airways of children early in the evolution of their disease with most of the information based on indirect markers of inflammation only. We evaluated markers of inflammation and tissue re‐modelling in bronchial biopsies from children with early respiratory symptoms before a clear clinical diagnosis of bronchial asthma could be made. We examined bronchial biopsies performed in 27 children between the ages of 1.2 and 11.7 yr who were bronchoscoped for a clinical indication because of recurrent or chronic respiratory symptoms. The patients were re‐evaluated 22–80 months after the original bronchoscopy to determine whether or not they had subsequently developed bronchial asthma. There were more eosinophils in the bronchial mucosa (129.4 vs. 19.1 cells/mm2 of lamina propria, p < 0.001) and the thickness of the subepithelial lamina reticularis was greater (4.65 vs. 3.72 μm, p = 0.044) in children with bronchial asthma diagnosed at follow‐up, compared with the children who did not progress to asthma. Eosinophilic inflammation and airway re‐modelling occur early in the natural history of bronchial asthma and are present even before asthma would be diagnosed based on clinical symptoms. Recognition of these changes and their significance for clinical disease should emphasize the need for timely detection and diagnosis of asthma in children to facilitate the early introduction of anti‐asthma therapy.

Weekend hospitalization and additional risk of death: An analysis of inpatient data

Objective To assess whether weekend admissions to hospital and/or already being an inpatient on weekend days were associated with any additional mortality risk. Design Retrospective observational survivorship study. We analysed all admissions to the English National Health Service (NHS) during the financial year 2009/10, following up all patients for 30 days after admission and accounting for risk of death associated with diagnosis, co-morbidities, admission history, age, sex, ethnicity, deprivation, seasonality, day of admission and hospital trust, including day of death as a time dependent covariate. The principal analysis was based on time to in-hospital death. Participants National Health Service Hospitals in England. Main Outcome Measures 30 day mortality (in or out of hospital). Results There were 14,217,640 admissions included in the principal analysis, with 187,337 in-hospital deaths reported within 30 days of admission. Admission on weekend days was associated with a considerable increase in risk of subsequent death compared with admission on weekdays, hazard ratio for Sunday versus Wednesday 1.16 (95% CI 1.14 to 1.18; P < .0001), and for Saturday versus Wednesday 1.11 (95% CI 1.09 to 1.13; P < .0001). Hospital stays on weekend days were associated with a lower risk of death than midweek days, hazard ratio for being in hospital on Sunday versus Wednesday 0.92 (95% CI 0.91 to 0.94; P < .0001), and for Saturday versus Wednesday 0.95 (95% CI 0.93 to 0.96; P < .0001). Similar findings were observed on a smaller US data set. Conclusions Admission at the weekend is associated with increased risk of subsequent death within 30 days of admission. The likelihood of death actually occurring is less on a weekend day than on a mid-week day.

The site of disruption of the bronchial epithelium in asthmatic and non-asthmatic subjects.

BACKGROUND: Attention has recently been focused on the basal cells of the tracheobronchial epithelium as the mechanism of anchorage of the tall columnar cells, which themselves do not appear to form hemidesmosomes with the basement membrane of the epithelium. Residual basal cells have been described as remaining attached to the basement membrane after epithelial denudation. This led this group to formulate the hypothesis that there may be a potential plane of cleavage between the basal cells and the overlying columnar cell layer within the bronchial epithelium, which becomes disrupted in asthma. METHODS: Bronchoalveolar lavage samples were obtained during bronchoscopy from eight patients with atopic asthma and four normal controls. Ultrathin sections of lavage cell pellets were examined by electron microscopy and the number of columnar and basal cells found in each epithelial cell cluster was counted. Cytocentrifuge preparations of the lavage samples from the same subjects were also examined for free epithelial cells and epithelial cell clusters. RESULTS: Electron microscopic examination of the cell pellets showed that basal cells were present in very small numbers in the epithelial clusters in all subjects (mean 0.03 (SE 0.02)/cluster) and the ratio of columnar cells to basal cells was far greater than was encountered in the intact bronchial epithelium (167 nu 4). The cytocentrifuge preparations showed an increased number of epithelial cell clusters and epithelial cells in the asthmatic patients. Although these clusters were similar in size in the two groups of subjects (6.3 nu 5.1 cells/cluster) the ratio of free epithelial cells to cells within the cluster was higher in the non-asthmatic subjects. CONCLUSIONS: It is proposed that shedding of epithelial cells occurs along a suprabasal plane and that there is a potential plane of cleavage between the suprabasal and the basal cell layers, which might be more vulnerable to the various insults.

Human mast cells express stem cell factor.

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT‐PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase‐only (MCT) and tryptase/chymase (MCTC) subsets. SCF protein product was found in conjunctival MCT and MCTC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross‐linkage of IgE receptor (Fcε–RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor‐mediated pathways.

Seasonal allergic conjunctivitis is accompanied by increased mast cell numbers in the absence of leucocyte infiltration

Background Seasonal allergic conjunctivitis (SAC) is the most common allergic disease to affect the eye. occurring alone or in association with allergic rhinitis. Infiltration with mast cells and eosinophils is characteristic of the chronic forms of allergic conjunctivitis such as vernal and atopic keratoconjunctivitis. and these cell types also contribute significantly to allergic inflammation in the skin. Indirect evidence for a similar pattern of cellular events in SAC comes from studies which demonstrate raised eosinophil and neutrophil numbers in conjunctival scrapings and elevated levels of mast cell tryptase in tears following allergen challenge.

Co-localization of immunoreactive transforming growth factor-beta1 and decorin in bronchial biopsies from asthmatic and normal subjects

Airway wall remodelling is an established pathological feature of asthma but its causes are not well understood. One cytokine of potential relevance is transforming growth factor‐beta1 (TGF‐β1). The immunolocalization of TGF‐β1 and of its small binding proteoglycan decorin have been examined in the airways of normal subjects and atopic asthmatics. Bronchial biopsy specimens were obtained by fibreoptic bronchoscopy, processed into glycolmethacrylate resin, and stained immunohistochemically using specific antibodies. Immunoreactive TGF‐β1 was principally localized extracellularly in association with subepithelial connective tissue. Some staining of bronchial epithelial cells was also evident, but otherwise there was little intracellular staining. The overall pattern of immunohistochemical staining was indistinguishable in biopsy specimens from asthmatic and control subjects. Comparison of adjacent sections demonstrated the co‐localization of immunoreactivity for TGF‐β1 and decorin in the mucosa. It is concluded that immunoreactive TGF‐β1 in human airways is principally extracellular and that matrix‐associated TGF‐β1 is likely to be bound at least in part to decorin. This interaction may provide a reservoir of TGF‐β1 that can be released in an active form in response to appropriate stimuli. Copyright

Immunolocalization of cytokines to mast cells in normal and allergic conjunctiva

Background Recently, the potential role of mast cells in allergic reactions has been extended by the discovery that these cells synthesize, store and secrete multifunctional cytokines. Seasonal allergic conjunctivitis is characterized as an immediate hypersensitivity reaction, in which allergen binds to specitic IgE on mast cells, leading to release of preformed and newly synthesized inflammatory mediators.

Invited Lecture: Activation of the Epithelial Mesenchymal Trophic Unit in the Pathogenesis of Asthma

Background: A recent NIH Workshop and an ERS Task Force concluded that more work was needed to understand mechanisms of severe and chronic asthma. This report describes a series of studies that identify aberrant epithelial mesenchymal signalling in the airways as an important event in maintaining inflammation and driving remodelling in response to environmental injury. Methods: Immunohistochemistry, genotyping and functional studies conducted on cultured asthmatic cells and mucosal biopsies were used to identify biochemical pathways involved in epithelial injury and repair in asthma and their relationship to disease severity. Results: Our findings suggest that the asthmatic state results from an interaction between a susceptible epithelium and Th-2-mediated inflammation to alter the communication between the epithelium and the underlying mesenchyme – the epithelial mesenchymal trophic unit – leading to disease persistence, airway remodelling and refractoriness to corticosteroid treatment. Conclusions: Asthma is more than an inflammatory disorder, but requires engagement of important signalling pathways involved in epithelial repair and tissue remodelling. These pathways involving EGFRs and TGF-βRs provide targets against which to develop novel therapies for chronic asthma.