Claudina Perez-Novo

Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease

BACKGROUND Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by the accumulation of inflammatory cells; however, an eosinophil predominance is seen in white (Belgian), but not Asian (south Chinese), patients with polyps. OBJECTIVE We sought to investigate the association of inflammatory cell predominance with regulatory T-cell and T-effector cell patterns. METHODS Nasal mucosal tissue was obtained from 26 consecutive Belgian patients with CRSwNP and 21 Belgian control subjects and 29 south Chinese patients with CRSwNP and 29 south Chinese control subjects, who all underwent phenotyping, including nasal endoscopy and computed tomographic scanning. Tissues were investigated for granulocytes and their products and T-effector/regulatory T cells and related cytokines. RESULTS Both CRSwNP groups were comparable in terms of symptoms, computed tomographic scan results, and nasal endoscopy results, but asthma comorbidity was significantly higher in white patients. Tissue from white patients with CRSwNP was characterized by eosinophilic inflammation (eosinophil cationic protein/myeloperoxidase ratio > 2), whereas samples from Asian patients were biased toward neutrophilic inflammation (eosinophil cationic protein/myeloperoxidase ratio = 0.25). Both CRSwNP groups demonstrated significant upregulation of the T-cell activation marker soluble IL-2 receptor alpha and significant downregulation of Foxp3 expression and TGF-beta1 protein content versus their respective control groups. However, whereas white patients displayed a significant increase in T(H)2 cytokine and related marker levels versus control subjects and versus Asian patients, the latter showed a T(H)1/T(H)17 cell pattern versus control tissue. CONCLUSION Nasal polyps (CRSwNP) from white and Asian patients are both characterized by T-cell activation and impaired regulatory T-cell function; however, T-effector cells in the samples from white patients were T(H)2-biased, whereas samples from their Asian counterparts demonstrated a T(H)1/T(H)17 polarization.

T-cell regulation in chronic paranasal sinus disease.

BACKGROUND Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by a T(H)2-skewed eosinophilic inflammation, whereas chronic rhinosinusitis without nasal polyps (CRSsNP) represents a predominant T(H)1 milieu. OBJECTIVE We aimed to study the direct tissue expression of transcription factors for T-cell subpopulations, including T regulatory cells, in relation to the cytokine expression patterns in the different disease subgroups. METHODS The expression of forkhead box P3 (FOXP3), T-box transcription factor (T-bet), GATA-3, retinoid acid-related orphan receptor C (RORc), the suppressive cytokines TGF-beta1 and IL-10, and T(H)1/ T(H)2/ T(H)17 cytokines (IFN-gamma, IL-4, IL-5, IL-13, IL-17) were analyzed by means of RT-PCR in 13 CRSsNP, 16 CRSwNP, and 10 control samples. Additional protein measurements were performed for TGF-beta1 and IFN-gamma. RESULTS In CRSwNP, we observed a significantly lower FOXP3 mRNA and TGF-beta1 protein expression, but a significantly higher T-bet, GATA-3, IL-5, and IL-13 mRNA expression compared with controls, whereas RORc was not significantly different compared with controls. In CRSsNP, FOXP3, T-bet, GATA-3, and RORc expression was not significantly different from controls, whereas TGF-beta1 mRNA, IFN-gamma mRNA, and protein were significantly higher in CRSsNP compared with controls. For IL-17, no significant differences were noted among all groups. CONCLUSION We demonstrate for the first time a decreased FOXP3 expression accompanied by an upregulation of T-bet and GATA-3 and a downregulation of TGF-beta1 in CRSwNP versus controls and CRSsNP.

TGF-β signaling and collagen deposition in chronic rhinosinusitis

BACKGROUND Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. OBJECTIVE The objective was to analyze the presence of TGF-beta isoforms, receptors, intracellular signaling, and collagen deposition in chronic rhinosinusitis. METHODS Sinonasal mucosal samples obtained from chronic rhinosinusitis with nasal polyps (CRSwNP; n = 13), chronic rhinosinusitis without nasal polyps (CRSsNP; n = 13), and controls (n = 10) were analyzed for TGF-beta isoforms 1 and 2 by means of ELISA and IHC, and for TGF-beta R1, 2, and 3 by RT-PCR and IHC. As downstream proteins, phospho-Smad 2 (pSmad 2) and collagen were analyzed by performing immunostaining and picrosirius red staining, respectively. RESULTS TGF-beta 1 and 2 protein concentrations, TGF-beta receptor (R) I and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly higher in CRSsNP versus controls. In CRSwNP, TGF-beta 1 protein concentration, TGF-beta RII and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly lower versus controls. Only TGF-beta 2 protein was found higher in CRSwNP versus controls. CONCLUSION A high TGF-beta 1 protein expression, increased TGF-beta RI expression, and a high number of pSmad 2-positive cells all indicate an enhanced TGF-beta signaling in CRSsNP, whereas a low TGF-beta 1 protein concentration, a decreased expression of TGF-beta RII, and a low number of pSmad 2-positive cells in CRSwNP indicate a low level of TGF-beta signaling in CRSwNP. These findings are compatible with the remodeling patterns observed, reflected by a lack of collagen in CRSwNP, and excessive collagen production with thickening of the collagen fibers in the extracellular matrix in CRSsNP.

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

Clinical and immunological determinants of severe/refractory asthma (SRA): association with Staphylococcal superantigen-specific IgE antibodies

To cite this article: Kowalski ML, Cieślak M, Pérez‐Novo CA, Makowska JS, Bachert C. Clinical and immunological determinants of severe/refractory asthma (SRA): association with Staphylococcal superantigen‐specific IgE antibodies. Allergy 2011; 66: 32–38.

Aspirin Sensitivity and IgE Antibodies to Staphylococcus aureus Enterotoxins in Nasal Polyposis: Studies on the Relationship

Background: Nasal polyposis is a multifactorial disease characterized by a chronic eosinophilic inflammation of the sinus mucosa, often associated with asthma and aspirin sensitivity. We have recently shown that the presence of IgE antibodies to Staphylococcus aureus enterotoxins (SAEs) was related to the severity of eosinophilic inflammation in nasal polyp tissue. In this study, we therefore aimed to determine, whether aspirin sensitivity was related to an immune response to SAEs, and how both criteria would be related to eosinophilic inflammation. Methods: 40 subjects with nasal polyposis (NP) were classified as aspirin-sensitive (n = 13, ASNP) or aspirin-tolerant (n = 27, ATNP) based on a bronchial aspirin challenge test. Homogenates prepared from nasal polyp tissue and inferior nasal turbinates from healthy subjects (n = 12) were analyzed for concentrations of IL-5 by enzyme immunoassay and for ECP, total and IgE to a mix of SAEs (A, C, TSST-1) using the ImmunoCAP system. Results: Concentrations of IL-5, ECP, total IgE, and IgE to an SAE mix were significantly increased in ASNP compared with ATNP patients and controls. In addition, a subgroup analysis showed an increase in eosinophilic markers in ATNP-SAE(+) compared to ATNP-SAE(–). This relationship, however, was not found in ATNP-SAE(+) and ATNP-SAE(–) subjects, indicating that SAE immune response is overlapped or not relevant in this condition. Conclusions: Aspirin sensitivity was associated with increased concentrations of eosinophil-related mediators, as well as IgE antibodies to SAEs in nasal polyp tissue. However, a direct impact of S. aureus could not be established. It seems that aspirin sensitivity and immune reactions to SAEs are independently related to eosinophilic inflammation.

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.

Pathophysiology of Chronic Rhinosinusitis

Chronic rhinosinusitis (CRS), a disease presenting with chronic symptoms such as nasal obstruction, rhinorrhea, hyposmia and facial pain, is highly prevalent and has a considerable impact on quality of life and health care expenditures. The disease is characterized by chronic inflammation of the sinonasal mucosa and can present with nasal polyps. Current consensus classifies CRS into CRS with nasal polyps and CRS without nasal polyps. This review illustrates the diversity of pathophysiological observations in CRS and highlights selected etiological hypotheses. A wide spectrum of alterations is described regarding histopathology, pattern of T cells and inflammatory effector cells, remodeling, immunoglobulin production, chemokine and eicosanoid production, and the role of microorganisms. The pathophysiological diversity observed in CRS seems to stand in contrast to its nonspecific clinical presentation, but is of the utmost importance in the development and application of highly individualized treatments. Identification of specific disease subgroups and their etiologies is an important and challenging task for future research.

Mixed T Helper Cell Signatures In Chronic Rhinosinusitis with and without Polyps

In chronic rhinosinusitis (CRS) different phenotypes have been reported based on cytokine profile and inflammatory cell patterns. The aim of this study was to characterize the intracytoplasmatic cytokines ofTcells infiltrating theinflamed sinonasal mucosa. Methods Infiltrated T cells and tissue homogenates from sinonasal mucosal samples of 7 healthy subjects, 9 patients with CRS without nasal polyp (CRSsNP), 15 with CRS with nasal polyps (CRSwNP) and 5cystic fibrosis patients (CF-NP) were analyzed for cytokine expression using flow cytometry and multiplex analysis respectively. Intracytoplasmic cytokinesin T cells were analyzed after stimulation of nasal polyps with Staphylococcus aureus enterotoxin B for 24 hours. Results The number of T cellsper total living cells was significantly higher in patients with CRSwNP vs. CRSsNP and controls. 85% of the CD4+ Tcells showed to be memory T cells. The effector T cells present in all tissues have apredominantTh1 phenotype. Only in CRSwNP, a significantfraction of T cellsproduced the Th2 cytokinesIL-4 and IL-5, while nasal polyps from CF patients were characterized by a higher CD4/CD8 T cell ratio and an increased number of Th17 cells. 24 h stimulation with SEB resulted in a significant induction of CD4+ T cells producing IL-10 (Tr1 cells). Conclusion T cell cytokine patternsin healthy and inflamed sinonasal mucosa revealed that Th2 cells (IL-4 and IL-5 producing cells) are significantly increased in CRSwNP mucosal inflammation. Exposure to SEB stimulates Tr1 cellsthat may contribute to the Th2 bias in CRSwNP.

Prevalence of chronic rhinosinusitis in Sao Paulo

BACKGROUND Glucocorticoids (GCs) are considered drugs of choice for treating nasal polyps (NPs). However, a subset of patients shows a limited clinical response even to high doses of GCs. Altered expression of glucocorticoid receptors (GRs), namely GR-alpha; and GR-beta;, is a potential mechanism underlying GC insensitivity. GCs modulate the expression of several cytokines, including transforming growth factor-beta (TGF-beta), which may contribute to cellular proliferation in NPs. The study investigates some biomolecular features of GC-resistant NPs, and examines possible differences from normal mucosa (NM). METHODOLOGY Radioligand binding assay (binding) was used to determine GR-alpha; binding capacity; Western blotting was used to evaluate GR-alpha;, GR-beta;, and TGF-beta; expression and GR-alpha; subcellular distribution. NPs were sampled in 32 patients during ethmoidectomy; NM was taken from 15 healthy patients during rhinoplasty. RESULTS GR-alpha; was present in NPs and NM, with lower affinity for the ligand in NPs. GR-alpha; was prevalent in the cytosol of NPs that were GR-alpha-negative to the binding assay. GR-beta was expressed in NPs and absent in the majority of NM. TGF-beta1 expression was higher in NPs than in NM. CONCLUSIONS GR-beta and TGF-beta1 might be involved in NP pathogenesis, but their role in modulating GC sensitivity is still unclear.INTRODUCTION Studies designed to investigate chronic rhinosinusitis (CRS) epidemiology play an important role to assess population`s distribution and risk factors to result in the development and promotion of public health policies. METHOD This study design is a survey carried out with a complex two-stage cluster sampling plan. Personal interviews were carried out with 2,003 individuals. The questionnaire included the epidemiological criteria for CRS. Demographic data, history of physician-diagnosed respiratory diseases (asthma, sinusitis, rhinitis), smoking, family income, educational attainment, and household characteristics were also evaluated. RESULTS The overall response rate was 93.9% of the households. Mean age was 39.8 +- 21 years; 45.33% were male. The overall prevalence of CRS in the city of Sao Paulo was 5.51%. We found a significant association between diagnosis of CRS and diagnosis of asthma and CRS and diagnosis of rhinitis and a significant association between presence of CRS and belonging to the low-income subgroup. CONCLUSION The municipality of Sao Paulo has an urban population of 11 million. According to the present study, the prevalence of CRS is 5.51%, which represents more than 500,000 individuals affected by this condition in the city.