Cindy Claeys

Impact of RNA quality on reference gene expression stability.

Gene expression quantification methods are important tools in the understanding of the molecular events underlying human diseases and in the identification of diagnostic and therapeutic targets. Generally, the messenger RNA (mRNA) used for these analyses is derived from human biopsies obtained after surgery. As a consequence, several steps during tissue handling have to be carefully controlled in order to preserve the quality and integrity of the RNA material. It is well known that RNA is sensitive to degradation by postmortem processes and inadequate sample handling or storage (1). However, RNA integrity control is often not systematically performed prior to (PCR-based) downstream analyses. While in the past, RNA quality could often not be assessed due to the limited availability of the precious sample (e.g., from microdisected cells or small biopsies), the advent of capillary gel electrophoresis and (sample retention) spectrophotometry technologies (e.g., NanoDrop® ND-1000; NanoDrop Technologies, Wilmington, DE, USA ) has addressed this issue, allowing quality estimations using only nanograms (or even picograms) of total RNA (2). In addition, amplification of RNA is now an alternative method to obtain sufficient amounts to conduct gene expression studies when postmortem tissues are scarce; however, assessment of RNA quality based on the 18S and 28S ribosomal RNA bands is often not possible anymore after amplification. Furthermore, it remains to be determined whether the amplified mRNA can faithfully be used to assess RNA quality of the starting material. Apart from RNA quality, the choice of a proper set of reference genes for accurate normalization is another crucial factor with a profound impact on the reliability of the obtained gene expression levels (3). Reference genes are expressed constitutively in every cell; however, their expression can be regulated with diseases state, during cellular proliferation, due to cellular composition and by mitogenic stimuli (e.g., growth factors) (4,5). Furthermore, it is now known that life styles and genetic make-up of individuals can influence mRNA expression (6). That is why the validation of the expression stability of reference genes remains an important step to ensure the accuracy and reliability of gene expression studies. The objective of this study was to analyze the influence of RNA degradation on the stability and expression pattern of different internal control genes. To this purpose, 10 commonly used reference genes were quantified in both intact and degraded RNA from clinical specimens obtained from ethmoidal and maxillary sinuses collected from patients with nasal polyposis (NP) and chronic rhinosinusitis (CRS). Sixteen clinical tissue samples (30 mg) were homogenized in Tri-reagent buffer (Sigma, St. Louis, MO, USA) (1 mL/50–100 mg of tissue) in a chilled pestle mortar. Total RNA isolation and cDNA synthesis were performed as described previously (7). RNA Impact of RNA quality on reference gene expression stability

Pattern of inflammation and impact of Staphylococcus aureus enterotoxins in nasal polyps from southern China

Background This study analyzes the impact of Staphylococcus aureus enterotoxins (SAEs) and the inflammatory pattern in polyps from China. Methods Nasal tissue was obtained from 27 consecutive bilateral nasal polyps and 15 control patients and assayed for eotaxin, interleukin-5, soluble interleukin-2 receptor, transforming growth factor (TGF) β1, myeloperoxidase, eosinophil cationic protein, total IgE, and specific IgE to SAEs. Activated eosinophils were stained using EG2 antibodies in polyps from Chinese and comparative white patients. Results The number of EG2+ eosinophils was significantly lower in polyps from Chinese patients versus white patients. Chinese polyps showed significantly increased IgE and soluble interleukin-2 receptor versus control samples, whereas TGF-β1 was significantly decreased. Ten of 27 samples in the polyp group versus 0 of 15 controls contained SAE-IgE (p < 0.01). TGF-β1 was significantly down-regulated in SAE+ samples versus SAE- samples (p = 0.04). Conclusion Nasal polyps from China are characterized by B- and T-cell activation, a minor eosinophilic inflammation compared with polyps from white subjects, and a decrease in TGF-β1 in comparison with control inferior turbinate tissue. One-third of patients with polyps showed an IgE response to SAEs.

Transforming growth factor β1 in nasal remodeling: differences between chronic rhinosinusitis and nasal polyposis

Background Chronic rhinosinusitis (CRS) and nasal polyposis (NP) are histopathologically characterized by different gross morphological aspects. Transforming growth factor (TGF) β1 plays an important role in tissue remodeling, which is poorly understood in chronic diseases of the sinuses. Methods The expression of TGF-β1 was analyzed by enzyme-linked immunosorbent assay, quantitative reverse-transcription polymerase chain reaction, and immunohistochemistry in nasal tissue from controls (n = 6), CRS (n = 19), or NP (n = 19). Results CRS presented significantly higher concentrations of TGF-β1 at protein (p = 0.0008) and mRNA levels (p = 0.025) when compared with NP samples. In CRS, TGF-β1+ staining of the extracellular matrix was found abundantly and related to fibrosis. In contrast, no TGF-β1 staining was found in the pseudocyst areas in NP. Conclusions CRS was histologically characterized by fibrosis, which was reflected by a significantly higher expression of TGF-β1 at RNA and protein levels when compared with NP. We show that TGF-β1 expression is related to fibrosis, differentiating CRS without polyps from NP.

Expression of eicosanoid receptors subtypes and eosinophilic inflammation: implication on chronic rhinosinusitis

BackgroundEicosanoid receptors are G-protein-coupled receptors playing an important immunomodulatory role in airway diseases. However, there is little information on the expression of these receptors and their link with eosinophilic inflammation in paranasal sinus diseases. We aimed with this study to investigate the tissue expression of leukotrienes and prostaglandin E2 receptors in chronic rhinosinusitis patients and the link of this regulation with eosinophilic inflammation.MethodsSamples were prepared from nasal tissue of patients with chronic rhinosinusitis without nasal polyps (CRS, n = 11), with nasal polyps (CRS-NP, n = 13) and healthy subjects (Controls, n = 6). mRNA expression of CysLT1, CysLT2, BLT1, BLT2, E-prostanoid receptors (EP1, EP2, EP3, EP4) and sol-IL-5Rα was determined by real-time PCR. Concentrations of PGE2, LTC4/D4/E4, LTB4 and sol-IL-5Rα were determined by ELISA and of ECP by ImmunoCap. Protein expression and tissue localization of eicosanoid receptors and activated eosinophils were evaluated by immunohistochemistry.ResultsCysLT1 mRNA expression was significantly increased in CRS-NP compared to CRS and controls, and CRS compared to controls, whereas CysLT2 mRNA was enhanced in both CRS groups without differences between them. Levels of both receptors correlated to the number of activated eosinophils, sol-IL-5Rα, ECP and LTC4/D4/E4 concentrations in the disease groups. PGE2 protein concentrations and prostanoid receptors EP1 and EP3 were down-regulated in the CRS-NP tissue vs. CRS and controls, whereas EP2 and EP4 expression was enhanced in CRS and CRS-NP patients vs. controls. No differences in BLT receptors were observed between patients and controls.ConclusionCyLTs receptors are up-regulated in nasal polyp tissue and their expression correlate with eosinophilic inflammation supporting previous results. Eicosanoid receptors mRNA pattern observed suggests that down-regulation of EP1 and EP3 in CRS-NP and up-regulation EP2 and EP4 in CRS and CRS-NP groups may have some role in the development of the diseases and their regulation may not be directly linked to eosinophil activation but involve post-transcriptional events mainly related to other inflammatory cell sources.

Predictive and monitoring value of matrix metalloproteinase‐9 for healing quality after sinus surgery

Functional endoscopic sinus surgery is considered the standard therapeutic procedure for chronic rhinosinusitis and nasal polyposis after failure of medical treatment. We tested the hypothesis that the healing outcome after surgery was correlated to the secretion profile of gelatinase‐B (matrix metalloproteinase‐9 [MMP‐9]) and transforming growth factor‐β1 (TGF‐β1) in nasal fluid. We performed a prospective study in 36 patients bilaterally operated for chronic rhinosinusitits or nasal polyposis and the healing quality was evaluated until 6 months after surgery by standardized nasal endoscopy, using a visual analog scale. Before functional endoscopic sinus surgery and during the postoperative period, TGF‐β1 and MMP‐9 in nasal secretions were measured by enzyme‐linked immunosorbent assay. Both MMP‐9 and TGF‐β1 showed a significant increase initially after surgery. The healing quality after 6 months was significantly and independently correlated to preoperative MMP‐9 concentrations in nasal secretions (p = 0.03), initial disease (p = 0.03), and previous sinus surgery (p = 0.004). Furthermore, concentrations of MMP‐9 were significantly lower in patients with good healing (visual analog scale < 3) from week 3 to month 6 compared to patients with poor healing. MMP‐9 is the first objective factor suitable to predict and monitor the healing quality after sinus surgery, indicating MMP‐9 as a possible therapeutic target.

Eicosanoid Metabolism and Eosinophilic Inflammation in Nasal Polyp Patients with Immune Response to Staphylococcus Aureus Enterotoxins

Background Staphylococcus aureus–derived enterotoxins (SEs) have been implicated in the pathogenesis of airway inflammatory diseases, especially nasal polyposis. However, the exact role of these molecules in the regulation of eicosanoid synthesis in this pathology remains unexplored. We studied the possible impact of SE-induced immune responses on the eicosanoid production in nasal polyp (NP) patients. Methods Tissue sample homogenates from NP patients, with (NP-SEs[+]) and without detectable IgE-antibodies to SEs (NP-SEs[–-]; ImmunoCap system), were assayed for IL-5, myeloperoxidase, leukotriene C4/D4/E4 (LTC4/D4/E4), LTB4, lipoxin A4, total IgE, and eosinophil cationic protein. Results Inflammatory makers, eicosanoids, and total IgE were significantly increased in NP-SEs(+) compared with NP-SEs(–) tissues, with the exception of myeloperoxidase, which was similar in both groups. Eicosanoid concentrations correlated to IL-5 and eosinophil cationic protein; however, only cys-leukotriene levels correlated with IgE-antibodies to SEs, independently of allergy and asthma. Conclusion Eicosanoid synthesis is up-regulated in polyp tissue of patients with immune response to SEs and seems to be related to the inflammatory reaction induced by these enterotoxins.

Staphylococcus aureus Enterotoxin B Regulates Prostaglandin E2 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 (EP(1-4)) 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 PGE(2), COX-2, and EP(2) expression but not EP(1), EP(3), or EP(4) 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.

Matrix metalloproteinases in chronic sinus diseases

Abstract Extracellular matrix (ECM) in nasal polyps is characterized by a subepithelial infiltration of eosinophils and pseudocyst formations. The processes leading to the vacuolisation of the ECM remain unclear but the role of matrix metalloproteinases, because of their proteolytic activities against the different components of the ECM, has been evoked. Immunohistochemistry for MMP-7, MMP-9 and TIMP-1 was performed on frozen sections from 10 controls, 10 patients with nasal polyposis (NP) and 10 with chronic sinusitis (CS). ELISA measurements were performed on tissue homogenates. In NP, when compared to CS or control samples, a staining for MMP-9 and MMP-7 appeared in blood vessels. In NP, MMP-9 was also upregulated in epithelium in glands and MMP-9-positive mononuclear inflammatory cells were increased, especially in the centre of pseudocyst formations. Concentrations of MMP-9 protein were found significantly increased in both CS and NP compared to controls, while MMP-7 was significantly increased in NP compared to controls and CS. TIMP-1 protein was significantly increased in CS, but not in polyps, when compared to normal mucosa. CS and NP show different patterns of MMP-7/-9 and TIMP-1 expression. This difference in regulation of enzymatic proteins could form the basis for the different tissue remodelling processes of these sinus diseases.