The Study On The Associativity Between Staphylococcal Superantigens With The Development And Recurrence Of Chronic Rhinosinusitis With Nasal Polyps

Background and Objective The etiology study of chronic rhinosinusitis with nasal polyps (CRSwNP) has always been the complicate and popular topic in the field of rhinology. Allergy and chronic inflammation are regarded to play important roles in the development of nasal polyps that is induced by multi-factors. Although proinflammatory cytokines, adhesion molecules on the vascular endothelium and the chemokines in nasal polyps have been well documented, the initial inciting factor that initiates the inflammatory infiltration of nasal lateral wall remains elusive. Recently, with the exploration of the nasal polyps' etiology, scholars in Europe and America have suggested the superantigen hypothesis of CRSwNP which proposes that superantigens, known as exotoxins, produced by colonizing Staphylococcus aureus are capable of playing a key role as the trigger factor in the CRSwNP development. Staphylococcal superantigens, promoting the activation and proliferation of CD4~+ T-cell, CD8~+ T-cell, B-cell, macrophage, antigen presenting cell, eosinophile and epithelium, trigger inflammatory infiltration in the mucus of nasal lateral wall and the production of massive cytokines and inflammatory mediators which are responsible for the inflammation cascade response resulting in the mucosa oedema and nasal polyps happened. At present, the role of superantigens in the CRSwNP develope mechanisms is still unclear. The purposes of the study are listed as follows:1. To explore the relationship between Staphylococcus aureus and the progress of CRSwNP via detection its presence in the secretion obtained from nasal polyp surface of patients with CRSwNP comparing with that in the sinonasal mucus of patients with CRS without nasal polyps (CRSsNP) and normal controls.2. To explore the correlativity and significance between staphylococcal superantigens and the development of CRSwNP through detecting directly the superantigens in the nasal polyps tissue of CRSwNP patients, and in the sinonasal mucosa of CRSsNP and normal controls.Methods1. In the conditions of severe sterile operation, the secretion of nasal polyp surface were obtained from 42 patients with bilateral nasal polyps, and sinonasal mucus surrounding the ostium of maxillary antrum were obtained from 27 patients with CRSsNP as well as from 12 normal controls. Mucus specimens were cultured, separation and identification for bacteria.2. Polyp tissue obtained from patients with CRSwNPs and sinonasal mucosa samples from the middle meatus with CRSsNP were collected at the time of surgery. Nasal mucosal biopsies from the middle turbinate served as the control tissue from the control group. Tissue samples were assayed for the three most common superantigens produced by Staphylococcus aureus (SEA, SEB, TSST-1) using an ELISA technique.Results1. The mucus culture results showed the dominant microbes in sinonasal mucus were normal flora and anaerobes, and the great part of pathogenic bacteria in dispersive samples were staphylococcus including staphylococcus aureus, staphylococcus haemolytieus, staphylococcus schleiferi, staphylococcus epidermidis and staphylococcus xylosus. The percentages of Staphylococcus aureus in sinonasal mucus were 7.14% (3/42) for CRSwNP subjects, 3.70% (1/27) for CRSsNP, and 0.00%(0/12) for normal controls respectively. There were no significant differences between these groups.2. In CRSwNP subjects 23 of 42 samples (54.76%) demonstrated reactivity for at least one staphylococcal superantigen by ELISA technique, including 4 for SEA, 13 for SEB and 8 for TSST-1. Two subjects reacted for two superantigens (SEB and TSST-1, SEA and SEB respectively). One sample was negetive for superantigen in the three CRSwNP patients whose mucus culture results showed Staphylococcus aureus positive. There were no positive results in CRSsNP or control group. There was highly significant difference between the percentage of superantigens positive in CRSwNP and that in CRSsNP or in normal controls.Conclusions1. The presence of Staphylococcus aureus was low in sinonasal mucus of patients with CRSwNP. There was no significant difference comparing with CRSsNP or normals. There should be a further research on this study to provide more accurate documents.2. The presence of staphylococcal superantigens was high in nasal polyps of patients with CRSwNP. There were no suprantigen present in patients with CRSsNP or normals, which suggested there was a correlation between Staphylococcal superantgens and the development of CRSwNP. Background and Objective Recent studies on etiology of chronic rhinosinusitis with nasal polyps (CRSwNP) have suggested a superantigen model that proposes that superantigens produced by colonizing Staphylococcus aureus may serve as the initial inciting factor in the pathogenesis of CRSwNP. Staphylococcal superantigens are capable of activating and proliferating clonally the T-cell by binding the T-cell receptor beta variable region (TCRVβ). The generalized activation of T-cell population results in producing massive cytokines, and triggering the initial damage of the lateral wall mucosa in nasal cavity. To verification that superantigens are present in the local mucosa of patients with CRSwNP and bind with TCRVβresulting in remarkable activation of T-cell, is necessary for demonstrating the superantigen hypothesis. Recent reports on this hypothesis have identified Staphylococcal superantigens in sinonasal mucus of patients with CRSwNP. Although some reports have focused on detecting the expression of TCRVβin nasal polyps, there were few studies on the expression of TCRVβ~+ T-cell in the peripheral blood of CRSwNP patients, and on the contributions of different superantigens to the selective expression of TCRVβfamily numbers. The purpose of the study was listed as follows:1. To investigate the effect of staphylococcal superantigens on T-cell via determiningthe expression of TCRVβin peripheral blood and local nasal tissue of patientswith CRSwNP or CRSsNP and normal controls. 2. To analyze the effect of superantigens on the dominant expression of TCRVβgene spectra and investigate the possible mechanism of superantigens as etiological angents on CRSwNP by determining the expression of twenty-four subtypes of TCRVβfamily members mRNA in nasal polyp of patients with CRSwNP in comparison with that of patients with CRSsNP and normal controls.Methods1. Sinonasal polyp or mucosa tissue and blood samples obtained from 42 CRS patients with bilateral nasal polyps, 27 with CRSsNP and twelve normal subjects were analyzed by flow cytometry to determine the expression of TCRVβ~+ T-cell.2. The tissue samples were detected for the expression of TCRVβgene family mRNA of total 24 subtypes using the technique of reverse transcriptase-polymerase chain reaction (RT-PCR) to explore the selective action of superantigens on the gene expression of TCRVβfamily.Results1. The mean percentages of TCRVβin peripheral blood were 51.64% for CRSwNP group, 37.68% for CRSsNP, 12.50% for normal controls, 53.19% for CRSwNP with superantigen positive, 49.31% for CRSwNP with superantigen negative respectively. Analysis of the variants demonstrated that the positive rate of TCRV 3 in CRSwNP and the two subgroups based on superantigen positive or negative was highly significant versus that in normal group(P<0.01), while no statistically significant difference versus CRSsNP. There was significant difference between CRSsNP and normal groups (P<0.05). There was clearly an increasing tendency paralleling the percentage of TCRVβin the superantigen-positive subgroup with that in the superantigen-negative subgroup, although no statistical significance.2. The mean percentages of TCRVβin local nasal tissue were 54.33% for CRSwNP group, 41.95% for CRSsNP, 27.13% for normal controls, 57.17% for CRSwNP with superantigen positive, 50.92% for CRSwNP with superantigen negative respectively. Comparisons on the variable expression of TCRVβshowed no significant difference between the CRSwNP versus CRSsNP, while there was statistical difference between superantigen-positive subgroup and CRSsNP (P<0.05). The difference between CRSsNP and normal group didn't reach significance. The similar increasing tendency was present in the polyp tissue of superantigen-positive subgroup in comparison with superantigen-negative subgroup.3. There was an difference on the mRNA expression of TCRVβfamily members. The range of Vβexpressed gene fragments was from three to sixteen in CRSwNP individuals. 18 or so Vβsubtypes expressed in CRSsNP patients, and all 24 expressed in normal controls.4. There was an close correlation between expression intensity of TCRVβfamily members and staphylococcal superantigens. Vβ3, 14, 15, 17 and 20 were highly expressed in patients with SEB positive by ELISA, as well as Vβ2 and 3 6.1-6.3 in patients with TSST-1 positive. There was no dominant expression of Vβfragment in subjects with superantigens negative.Conclusions1. The amount of TCRVβ~+ T-cell in sinonasal mucosa and blood samples of CRSwNP patients increased, especially in superantigen-positive nasal polyps with significance.2. The selective expression of TCRVβgene-spectra is corresponding with the target of different superantigens in the nasal polyp tissue of patients with superantigen-positve CRSwNP.3. Staphylococcal superantigens are capable of activating Vβ~+ T-cell by binding with specific receptor of TCRVβsubtypes, which suggests the possibility of superantigens as etiological agents of CRSwNP. Background and Objective Glucocorticoid (GC) is the first choice in treatment of chronic rhinosinusitis. However, some patients were insensitive or resistant to glucocorticoid, which relapsed, even received regular glucocorticoid treatment. This may be one of reasons of high relapse rate of chronic rhinosinusitis with nasal polyps after surgery and other therapy. Glucocorticoid takes effect after its conjunction with glucocorticoid receptor (GR). According to their biological functions, glucocorticoid receptor can be divided into two kinds: GRαand GRβ. After combination with GRα, glucocorticoid makes its function of depressing inflammation and immune, while GRβacts as the endogenous inhibitory receptor of GRαby antagonizing its effect. To date, the pathologic and molecular foundation of glucocorticoid resistance in nasal polyps hasn't been explored. Recent studies mostly focus on evaluating the expression of GRβ. Few researches are done on factors that induced the over-expression GRβand their mechanisms. The purpose of the study was listed as follows:1. To investigate the role of GR in the process of CRSwNP developing and recurring, and to analyze the relationship between GR with GC resistance, via determining the expression of GRαand Grβalong with the infiltrating degree of EOS in the sinonasal mucosa of CRSwNP patients with recurrence or no recurrence and CRSsNP patients, who were all treated by regular glucocorticoid.2. To investigate the functional mechanisms of superantigens on the glucocorticoid insensitivity in CRSwNP by analyzing the contribution of superantigens to the excessive expression of glucocorticoid receptor.Methods1. Sinonasal polyp and mucosa tissue, obtained from CRSwNP patients with 23 superantigens positive, 27 with superantigens negative, 17 with recurrence, 18 without recurrence and from 12 CRSsNP followed by 1.5 - 2.0 years, were determined for the expression of GRαand GRβusing immunohistochemistry technique.2. The paraffin sections in the different courses of disease above mentioned were stained by HE to count the numbers of EOS, and analyze the relationship with staphylococcal superantigens and GR over-expression.Results1. GRαand GRβwere observed in cytoplasm and nucleus of epithelium, glandular and inflammatory cells, especially in the cytoplasm of epithelium and glandular in all subjects.2. The expression of GRαin superantigen-positive nasal polyps was a little higher than that in superantigen-negatives. The expression of GRαin recurred nasal polyps was higher than that in no recurred patients also with subtle difference. There was no clear difference between CRSsNP with controls. The expression of GRαin four CRSwNP groups was increased comparing with CRSsNP or normals, but didn't reach the point of significant difference (P>0.05).3. Concerning the expression of GRβ, there was a clearly increasing tendency in recurred CRSwNP in comparison with CRSwNP without recurrence, as well as in superantigen-positive nasal polyps versus superantigen-negatives, although there were both no significances (P>0.05). The expression of GRβin CRSsNP was higher than that in normal, but no significance was found (P>0.05). There were significant differences comparing the four CRSwNP with CRSsNP or normals.4. The ratio of GRβ/GRαin CRSwNP with positive or negative superantigens was significantly higher than that in normal subjects, as well as CRSwNP with or without recurrence higher than that of normals. These CRSwNP groups compared respectively with CRSsNP, no significant differences were found.5. The amount of EOS in CRSwNP with positive superantigens was higher than that in CRSwNP with negative superantigens, but difference no significance. EOS has an increasing tendency in recurred CRSwNP comparison with CRSwNP without recurrence, but difference wasn't significant. The numbers of EOS in four groups of CRSwNP were significantly higher than that in CRSsNP or in normal controls.Conclusions1. GRαand GRβcan be detected in epithelium, endocrine and inflammatory cells of each group. It located in both nucleus and cytoblasm, mainly in epithelium and the cytoblasm of endocrine.2. Down-regulation of GRa was not found in every step of chronic rhinosinusitis with nasal polyps3. We found high levels of GRβin nasal polyps. The expression of GRβhas an increasing tendency in CRSwNP patients with positive superantigen compared with that negative superantigen, and similar increasing tendency was found in recurrenced CRSwNP in comparison with no recurrenced CRSwNP. The difference of GRβexpression between two superantigen groups was more clearly than that between groups with and without relapse, suggesting superantigens could induce the expression of GRβ.4. The change of GRβ/GRαratio in different courses of CRS was in accordance with over-expression of GRβ. GRαwas the dominantly present GR subtype in normal nasal mucosa, while GRβwas the more than GRαin chronic inflammation.5. The degree of EOS infiltration in different courses of CRS was in accordance with over-expression of GRβ, which suggested the role of GRβin inhibiting hormonal effect.6. There was a relationship between over-expression of GRβand glucocorticoid insensitivity. Staphylococcal superantigens could possibly induce the glucocorticoid resistance by aggravating the expression of GRβ.