Notes: Twenty-two neuroendocrine tumours of the larynx were investigated using a panel of immunocytochemical markers. Three were small cell carcinomas, eight were large cell neuroendocrine carcinomas and 11 were paragangliomas. Twenty were positive for protein gene product 9.5, 19 for neuron-specific enolase, 15 for chromogranin A, nine for bombesin, eight for substance P, eight for neuropeptide Y, eight for metenkephalin, seven for somatostatin, five for calcitonin, eight for calcitonin gene-related peptide and one for vasoactive intestinal polypeptide. Bombesin immunoreactivity was largely restricted to the small cell carcinomas and large cell neuroendocrine carcinomas and neuropeptide Y, metenkephalin and substance P to the parangangliomas. This comprehensive immunocytochemical analysis of neuroendocrine tumours of the larynx demonstrates that these tumours represent special entities but have similar patterns of immunostaining to those of neuroendocrine tumours in other sites.

Notes: We have examined the effect of prolonged treatment with topical corticosteroid on allergen-induced early and late nasal responses and the associated inflammatory cell infiltrate in grass pollen sensitive allergic rhinitics. Following a randomized double-blind 6 week treatment period with fluticasone propionate 200 μg aqueous nasal spray twice daily or matched placebo spray, nasal provocation was performed using Timothy grass pollen extract. Nasal symptoms were recorded at intervals from 0 to 24 h. Nasal biopsies were performed before treatment and at 24 h after allergen and processed for immunohistology. When corticosteroid-treated patients were compared with the placebo group there was an approximately 50% decrease in the size of the early (0-60 min) response and almost complete inhibition of late (1–24 h) nasal symptoms after allergen challenge. After allergen challenge markedly fewer T lymphocytes and CD25+ (interleukin-2 receptor bearing) cells were observed in both the epithelium and submucosa in fluticasone treated patients compared with the placebo group. Significantly less total and activated eosinophils were observed, particularly within the nasal epithelium. Submucosal mast cell counts were decreased, whereas increased numbers of submucosal neutrophils were observed. These results confirm that topical corticosteroid treatment inhibits allergen-induced early and late nasal responses. This may possibly occur following a decrease in T lymphocytes and/or mast cells and their products and a consequent reduction in tissue eosinophilia.

Notes: Background In bronchial mucosa, T cells are in close association with fibroblasts. This cell contact raises the possibility of cross-talk between the two cell types through cytokines, such as interleukin-4 (IL-4).Objective We postulated that IL-4 may modulate collagen synthesis and degradation in the fibroblasts of asthmatics.Methods Bronchial fibroblasts from asthmatics (BAF) and normal controls (BNF) were stimulated with IL-4. Procollagen I gene expression and protein production were measured by real-time PCR, RT-PCR, and radioimmunoassay. The effect of IL-4 on the regulation of procollagen I (α1) promoter was studied through transient cell transfections. The implication of Sp1 and AP-1 in regulating IL-4-induced procollagen I (α1) production was determined. The effect of IL-4 on metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) production and gene expression was evaluated.Results Following IL-4 stimulation, there was a significant increase in the expression of mRNA of procollagen I (α1) by human bronchial fibroblasts of asthmatics and controls. IL-4 has a dose–response effect on mRNA, with a maximal effect at 5 ng/mL, as determined by real-time PCR. The maximal increase in procollagen I (α1) was observed at 6 h after IL-4 stimulation in both BNF and BAF. BAFs have a greater increase in the procollagen I (α1)/β2 microglobulin ratio after 6 h of IL-4 stimulation (4.1×10−2±0.03 to 20.8×10−2±0.1) compared with BNF (2.9×10−2±0.006 to 9.2×10−2±0.08) (P=0.001). In transient transfection experiments, IL-4 increased promoter activity by threefold in BAF and BNF. Sp1 was up-regulated after IL-4 stimulation and AP-1 was down-regulated as shown by electrophoretic mobility shift assay. IL-4 decreased MMP-2 protein and mRNA levels, and did not alter TIMP-2 production.Conclusions IL-4 positively regulates procollagen I (α1) transcription by direct promoter activation and increases the TIMP-2/MMP-2 ratio, thereby supporting the profibrotic effect of this cytokine. Thus, this study emphasizes that IL-4 may be considered as a link between inflammation and collagen deposition observed in asthmatic airways.

Notes: Background Pathophysiology of corticosteroid (CS)-resistant asthma remains incompletely understood.Objective To determine if failure of asthma to clinically improve with CS is due to a defective response of airway bronchial inflammation to these drugs.Methods Twenty-one asthmatics having a decreased baseline FEV1 that improved ≥ 30% with inhaled β2 agonist got bronchial biopsies before and at the end of an oral CS treatment (methylprednisolone 40 mg daily for 14 days). They were arbitrarily divided into two groups according to baseline FEV1 improvement following this treatment: ≥ 23% designated as CS-sensitive (CSS) (n = 10) and 〈 15% as CS-resistant (CSR) (n = 11).Results Before oral CS, counts of bronchial mucosa inflammatory cells identified by immunohistochemistry (CD3, MBP, tryptase, CD68, neutrophil elastase and CD25 for lymphocytes, eosinophils, mast cells, macrophages, neutrophils and IL-2 receptors, respectively) were similar in CSS and CSR subjects. Oral CS decreased CD3+ cell counts (medians: 60–20 cells/mm2; P = 0.014) and MBP+ cell counts (medians: 19–4 cells/mm2; P = 0.03) in CSS asthmatics, but only tryptase+ cell counts in CSR asthmatics (medians: 30–18 cells/mm2; P = 0.05). Few bronchial neutrophil elastase+ cells were observed and their counts were similar in the two groups of asthmatics before and when on oral CS (all medians: = 2 cells/mm2).Conclusions These data show that, in these subjects with moderate to severe asthma, lymphocytes and eosinophils constitute most of the inflammatory cells infiltrating the bronchial mucosa. They also demonstrated that clinical impaired response to CS is associated with a persistent bronchial mucosa cellular infiltrate despite oral CS treatment. Additional studies are required to determine the role of this CS-resistant bronchial inflammation in the impaired asthma clinical response to these drugs.

Notes: Background Asthmatic airways are characterized by infiltration with a variety of inflammatory cells such as mast cells and eosinophils. Stem cell factor (SCF) is an important activating and chemotactic factor for both mast cells and eosinophils. In addition, it is a critical growth and differentiation factor for mast cells.Objectives To investigate the contribution of SCF to the pathogenesis of asthma, we examined the expression of SCF and its receptor c-kit in bronchial biopsies and bronchoalveolar lavage (BAL) specimens obtained from asthmatic subjects (n=13) and non-asthmatic control subjects (n=10).Methods SCF and c-kit were detected by in situ hybridization (ISH) and immunocytochemistry (ICC). In order to phenotype the cells expressing SCF and c-kit in asthmatic tissue and BAL cells, combined ISH and ICC were also performed.Results There was a significant difference (P〈0.001) in the SCF mRNA expression in asthmatic airway epithelium (70.38±12.33% positive cells) compared with controls (12.7±17.21% positive cells). There was also a significant difference in subepithelial SCF-mRNA expression, being higher in asthmatics (P〈0.001). A significant difference was also found in c-kit receptor mRNA expression in asthmatic biopsies both in epithelium (P〈0.001) and subepithelium (P〈0.05) compared with controls. ICC results were consistent with the ISH for both SCF and c-kit receptor from asthmatics and controls. The SCF and c-kit receptor mRNA and immunoreactivity in cells recovered from bronchial washing were also significantly higher in asthmatics compared with controls (P〈0.05). While SCF expression was localized predominantly in the epithelial layer in bronchial biopsy tissues, alveolar macrophages were found to be the major source of SCF in bronchial washing from asthmatic subjects.Conclusion The results of this study demonstrate the increased expression of SCF and its receptor, c-kit within human asthmatic airways, which suggests an important role of this cytokine in the pathophysiology of asthma.

Notes: Background Remodelling of the asthmatic airway includes increased deposition of proteoglycan (PG) molecules. One of the stimuli driving airway remodelling may be excessive mechanical stimulation.Objective We hypothesized that fibroblasts from asthmatic patients would respond to excessive mechanical strain with up-regulation of message for PGs.Methods We obtained fibroblasts from asthmatic patients (AF) and normal volunteers (NF) using endobronchial biopsy. Cells were maintained in culture until the fifth passage and then grown on a flexible collagen-coated membrane. Using the Flexercell device, cells were then subjected to cyclic stretch at 30% amplitude at 1 Hz for 24 h. Control cells were unstrained. Total RNA was extracted from the cell layer and quantitative RT-PCR performed for decorin, lumican and versican mRNA.Results In unstrained cells, the expression of decorin mRNA was greater in AF than NF. With strain, NF showed increased expression of versican mRNA and AF showed increased expression of versican and decorin mRNA. The relative increase in versican mRNA expression with strain was greater in AF than NF.Conclusions These data support the hypothesis that proteoglycan message is increased in asthmatic fibroblasts subject to mechanical strain. This finding has implications for the mechanisms governing airway wall remodelling in asthma.

Notes: Using the technique of in situ hybridization, we have attempted to identify messenger RNA for tumour necrosis factor-alpha (TNFα) in cells infiltrating allergen-induced late phase reaetion (LPR) of the skin and the nose ofatopic subjects. We have also compared the number of TNFα mRNA positive cells in bronchoalveolar lavage (BAL) from atopic asthmatics and normal controls. Twenty-four hours after local allergen challenge, 12/14 skin biopsies and 9/10 nasal biopsies had positive hybridization signals for TNFα mRNA whereas only 4/14 and 2/10 biopsies were positive in the relevant diluent controls. Compared with diluent sites significantly increased numbers of cells expressing mRNA for TNFα were observed in the LPR of skin (P 〈0.004) and nose(P 〈0.006). All BAL from asthmatics (n= 10) and from normal volunteers (n= 10) had cells showing positive hybridization signals for TNFα mRN A but these were at increased frequency in asthmatics (P〈 0.001). These results suggest that TNFα may be an important cytokine in atopic allergic inflammation.