Staphylococcus aureus enterotoxin B regulates prostaglandin E-2 synthesis, growth, and migration in nasal tissue fibroblasts

Background. Superantigens and eicosanoids are important amplifiers and regulators of inflammation in airway diseases. We therefore studied the possible influence of Staphylococcus aureus enterotoxin B ( SEB) on the cyclooxygenase ( COX) pathway and basic functions of airway structural cells. Methods. Fibroblasts were isolated from nasal inferior turbinate tissue and cultured in the presence of different concentrations of SEB. Preincubation with interferon ( IFN)-gamma was performed to induce expression of major histocompatibility complex ( MHC) class II receptors. Prostaglandin E2 ( PGE(2)) production was assayed by enzyme-linked immunosorbent assay, and levels of COX-2 and prostanoid E receptors 1-4 ( EP1-4) were assayed by real-time polymerase chain reaction. Migration and growth tests were performed, and SEB was localized within the cells by confocal microscopy. Results. Stimulation with IFN-gamma and SEB significantly down-regulated PGE2, COX-2, and EP2 expression but not EP1, EP3, or EP4 expression. The enterotoxin blocked cell growth but increased the fibroblast migration rate. SEB was localized within the cell in the presence and absence of MHC-II, suggesting that mechanisms other than conventional binding may allow the enterotoxin to enter the cell. Conclusions. These findings may have major implications for our understanding of the role played by bacterial superantigens in regulating the inflammatory and remodeling mechanisms of upper airway diseases and hence may help elucidate the pathophysiology of these diseases

Impact of the patient’s body position on the intraabdominal workspace during laparoscopic surgery

BACKGROUND: The effects of the patient's body position on the intraabdominal workspace in laparoscopic surgery were analyzed. METHODS: The inflated volume of carbon dioxide was measured after insufflation to a preset pressure of 15 mmHg for 20 patients with a body mass index (BMI) greater than 35 kg/m(2). The patients were anesthetized with full muscle relaxation. The five positions were (1) table horizontal with the legs flat (supine position), (2) table in 20 degrees reverse Trendelenburg with the legs flat, (3) table in 20 degrees reverse Trendelenburg with the legs flexed 45 degrees upward at the hips (beach chair position), (4) table horizontal with the legs flexed 45 degrees upward at the hips, and (5) table in 20 degrees Trendelenburg with the legs flat. The positions were performed in a random order, and the first position was repeated after the last measurement. Repeated measure analysis of variance was used to compare inflated volumes among the five positions. RESULTS: A significant difference in inflated volume was found between the five body positions (P = 0.042). Compared with the mean inflated volume for the supine position (3.22 +/- 0.78 l), the mean inflated volume increased by 900 ml for the Trendelenburg position or when the legs were flexed at the hips, and decreased by 230 ml for the reverse Trendelenburg position. CONCLUSIONS: The Trendelenburg position for lower abdominal surgery and reverse Trendelenburg with flexing of the legs at the hips for upper abdominal surgery effectively improved the workspace in obese patients, even with full muscle relaxation.status: publishe

Classification and management of allergic rhinitis patients in general practice during pollen season

Background: Allergic rhinitis (AR) represents a major challenge in primary care. The Allergic Rhinitis and its Impact on Asthma (ARIA) group proposed a new classification for AR and developed evidence-based guidelines for the management of this disease. We conducted this study to further characterize the classes of AR described by ARIA, and to evaluate whether the management of AR in general practice is in accordance with the ARIA guidelines. Methods: During the pollen season of 2003, 95 Belgian general practitioners (GPs) enrolled 804 patients who presented with symptoms of AR. For each patient, a questionnaire comprising the clinical presentation and management was completed. Results: In 64% of the patients, AR was classified as intermittent and in 36% as persistent. Persistent rhinitis caused more discomfort than intermittent rhinitis. Only 50% of the patients had ever undergone allergy testing. Among them, 51% were allergic to both seasonal and perennial allergens. Eighty-two per cent of the persistent rhinitics were allergic to at least one seasonal allergen and 72% of the intermittent rhinitics to at least one perennial allergen. When compared strictly with the ARIA recommendations, 49% of the patients with mild and/or intermittent AR were overtreated, whereas about 30% of those with moderate/severe persistent rhinitis were undertreated. Conclusion: This study confirms that the previous classification of AR into 'seasonal' and 'perennial' is not satisfactory and that intermittent and persistent AR are not equivalent to seasonal and perennial AR respectively. Furthermore, persistent rhinitis has been shown to be a distinct disease entity. Further efforts are required to disseminate and implement evidence-based diagnostic and treatment guidelines for AR in primary care practice

Enhanced release of IgE-dependent early phase mediators from nasal polyp tissue

Background: The mast cell is a crucial effector cell in allergic rhinitis and other inflammatory diseases. During the acute allergic reaction preformed mediators such as histamine, but also de novo produced mediators such as leukotrienes (LTC4/D-4/E-4) and prostaglandins (PGD(2)) are released. Mast cells represent targets for therapeutic intervention, and thus a human ex-vivo model to stimulate mast cells taken from mucosal sites would be instrumental for drug intervention studies. We have aimed to activate mast cells within ex-vivo human nasal tissue by IgE/anti-IgE specific (epsilon chain specific) stimulations and in this respect to test the usability of nasal polyps versus inferior turbinates Methods: Biopsy samples were collected from patients with nasal polyps and inferior turbinates from patients who underwent sinus or septal surgery. Tissue fragments were primed with IgE 1 mu g/ml for 60 minutes and then stimulated for 30 minutes with tissue culture medium (negative control), anti-IgE 10 mu g/ml, anti-IgE 30 mu g/ml and ionomycin 10 mu M (positive control). Histamine, leukotrienes and PGD2 were measured in supernatants. To help provide an understanding of the extent of the response, the number of tryptase and Fc epsilon RI alpha positive cells was evaluated by means of immunohistochemistry and the Fc epsilon RI alpha-chain was measured by means of quantitative PCR in the nasal polyp and inferior turbinate tissues. Finally, the correlation between IgE concentrations in the nasal tissue and the release of mediators was analysed. Results: Stimulations with anti-IgE on IgE-primed nasal tissue fragments lead to a concentration-dependent release of histamine, leukotrienes and PGD(2). The release of these early phase mediators was significantly higher in nasal polyps compared to inferior turbinates, although tryptase, Fc epsilon RI alpha positive cells and Fc epsilon RI alpha-chain transcripts were equally present in both groups. No correlation was found between baseline concentrations of IgE, and the release of histamine, LTC4/LTD4/LTE4 and PGD2 after stimulation. Conclusion: This human nasal challenge model mimics the allergic early phase reaction. The release of histamine, cys-leukotrienes and PGD(2) was significantly higher in nasal polyps versus inferior turbinates, however, this observation could not be explained by differences in mast cell or Fc epsilon RI+ cell numbers

Differential expression of the interleukin 5 receptor alpha isoforms in blood and tissue eosinophils of nasal polyp patients

Given the key role of interleukin-5 (IL-5) in eosinophil function, we investigated the regulated expression of the membrane-anchored (TM-IL-5R alpha) isoform, or a secreted (SOL IL-5R alpha) isoform, on both protein and transcript level in vitro and in vivo. A real-time PCR, FACS and ELISA were established to determine IL-5R alpha isoform expression in peripheral blood and nasal tissue from control subjects and nasal polyp (NP) patients with or without asthma. Human peripheral blood eosinophils were incubated with IL-5 and were analyzed for SOL-IL-5R alpha and TM-IL-5R alpha mRNA and protein levels in comparison with CD-69 expression. SOL-IL-5R alpha and TM-IL-5R alpha mRNA and protein expression was significantly increased in NP vs controls. In polyp tissue, SOL-IL-5R alpha expression correlated to disease severity and eosinophils counts, whereas TM-IL-5R alpha levels were inversely correlated to eosinophils counts and SOL-IL-5R alpha expression. FACS analysis revealed increased CD-69 and decreased TM-IL-5R alpha expression in NP tissue eosinophils vs blood eosinophils. Incubation of blood eosinophils with IL-5 caused up-regulation of CD-69 and down-regulation of TM-IL-5R alpha after 2 and 24 h. The expression of SOL-IL-5R alpha and TM-IL-5R alpha differs according to the eosinophil activation state and localization in the body (blood vs tissue) and may therefore be involved in the fine-tuning of the eosinophil homeostasis. Exposure of eosinophils to IL-5 reduces their responsiveness to IL-5 by regulated expression of the IL-5R alpha isoforms. Since, TM-IL-5R alpha is down-regulated and SOL-IL-5R alpha (antagonistic) is upregulated in NP tissue, our findings are important to understand the clinical trials with anti-IL-5 in humans

Quantitative Real Time Polymerase Chain Reaction for measurement of human Interleukin – 5 receptor alpha spliced isoforms mRNA

BACKGROUND: Expression of human Interleukin-5 receptor alpha (hIL-5Rα) is controlled by alternative splicing, which generates two different transcripts encoding a membrane-anchored and a soluble form of the receptor, respectively. Although the study of the expression and regulation of hIL-5Rα is of crucial importance in the field of immunological processing, methods and techniques until now described lack sufficient sensitivity for detection of small differences in the expression of these isoforms. The aim of this study was to develop a reliable and sensitive real-time quantitative PCR assay to analyse the expression level of each isoform. METHODS: For the quantitative real-time PCR assay, two standard curves specific for each splice variant were constructed. PCR amplifications were performed on CDNA from peripheral blood, eosinophilic chronic rhinosinusitis and normal nasal tissue using a common forward and two specific reverse primers, in combination with SYBR Green I as the detection format. RESULTS AND CONCLUSION: We have developed an accurate and reliable assay for quantification of interleukin-5 receptor alpha mRNA isoforms over a broad dynamic range of input molecules. Importantly, excess of one isoform did not influence accurate quantification of the other isoform. Quantification of hIL-5Rα variants in human samples demonstrated an overexpression of both membrane-anchored and soluble encoding variants in eosinophilic chronic rhinosinusitis tissue and peripheral blood in patients with eosinophilic chronic rhinosinusitis compared to healthy subjects. The implementation of this assay will allow a better understanding of the regulatory mechanisms of the hIL-5Rα gene and hence its role in the pathogenesis of chronic inflammatory diseases

Staphylococcal enterotoxin B influences the DNA methylation pattern in nasal polyp tissue : a preliminary study

Staphylococcal enterotoxins may influence the pro-inflammatory pattern of chronic sinus diseases via epigenetic events. This work intended to investigate the potential of staphylococcal enterotoxin B (SEB) to induce changes in the DNA methylation pattern. Nasal polyp tissue explants were cultured in the presence and absence of SEB; genomic DNA was then isolated and used for whole genome methylation analysis. Results showed that SEB stimulation altered the methylation pattern of gene regions when compared with non stimulated tissue. Data enrichment analysis highlighted two genes: the IKBKB and STAT-5B, both playing a crucial role in T- cell maturation/activation and immune response