Ratio of myeloid and plasmacytoid dendritic cells and TH2 skew in CRS with nasal polyps.

BACKGROUND: The role of myeloid and plasmacytoid dendritic cells and its consequences for the T(H)2 skew in chronic rhinosinusitis (CRS) with nasal polyps (CRSNP(+)) should be detailed. METHODS: In 18 CRS patients without nasal polyps (CRSNP(-)), 35 CRSNP(+) patients and 22 patients with nasal structural abnormalities without rhinosinusitis (controls), dendritic cells (DC) were differentiated into myeloid (mDC) and plasmacytoid (pDC) subtypes using an antibody cocktail including CD1c (BDCA-1) and CD303 (BDCA-2) in peripheral blood mononuclear cells (PBMC) and single cell preparations of sinonasal mucosa by flow cytometry. Moreover, cells were analysed for expression of CD45, CD3, CD4, CXCR3 (T(H)1) and CCR4 (T(H)2) and IFN-gamma, IL-5, TGF-beta1, TGF-beta2, ECP and total IgE in nasal secretions were determined. As a possible confounder, Staphylococcus aureus in nasal lavages was detected. RESULTS: The tissue mDC/pDC-ratio was 1.7 (1.0-2.4) in controls, 3.0 (1.8-4.0) in CRSNP(-) and 0.8 (0.6-1.0) in CRSNP(+) (P < 0.01). In tissue samples, the T(H)1/T(H)2 ratio was 12.6 (6.4-16.0) in controls, 12.5 (6.9-21.2) in CRSNP(-) and 1.8 (1.3-3.6) in CRSNP(+) (median and interquartile range, P < 0.001). Less pronounced differences were found in PBMC. S. aureus detection rates or TGF-beta levels did not differ between patient groups and S. aureus detection had no influence on the parameters investigated. CONCLUSION: A significant T(H)2 skew in CRSNP(+) could be confirmed on the cellular level. It was driven by low myeloid dendritic cell numbers. The T(H)2 skew did not correlate with S. aureus detection. The data support the concept that CRSNP(+) and CRSNP(-) are pathophysiologically distinct.

Absence of inflammatory response from upper airway epithelial cells after X irradiation.

Radiotherapy of head and neck tumors causes adverse reactions in normal tissue, especially mucositis. The dose- and time-dependent response of upper airway cells to X radiation should be analyzed in terms of the pro-inflammatory potential. Immortalized BEAS-2B lung epithelial cells were treated with 2, 5 and 8 Gy. Out of 1232 genes, those that were transcribed differentially after 2, 6 and 24 h were assigned to biological themes according to the Gene Ontology Consortium. Enrichment of differentially regulated gene clusters was determined with GOTree ( http://bioinfo.vanderbilt.edu/gotm ). Eleven cytokines were measured in culture supernatants. The cell cycle response up to 24 h and induction of apoptosis up to 4 days after exposure were determined by flow cytometry. A significant dose- and time-dependent gene activation was observed for the categories response to DNA damage, oxidative stress, cell cycle arrest and cell death/apoptosis but not for immune/inflammatory response. This correlated with functional G(2) arrest and apoptosis. Pro-inflammatory cytokines accumulated in supernatants of control cells but not of X-irradiated cells. The complex gene expression pattern of X-irradiated airway epithelial cells is accompanied by cell cycle arrest and induction of apoptosis. In vivo, this may impair the epithelial barrier. mRNA and protein expression suggest at most an indirect contribution of epithelial cells to early radiogenic mucositis.

Staphylococcus aureus in nasal lavage and biopsy of patients with chronic rhinosinusitis.

BACKGROUND: Staphylococcus aureus may play a relevant etiologic role in chronic rhinosinusitis (CRS) and may explain the T(H2) shift observed in CRS with nasal polyps (CRSNP(+)). Naturally occurring S. aureus small colony variants (SASCV) escape immune surveillance, antibiotic treatment and microbiologic routine diagnostic techniques. The frequency of S. aureus and SASCV in CRS patients and S. aureus-related effects on the local immune response should be prospectively investigated. METHODS: Nasal lavages and mucosal biopsies of CRS patients were examined with bacterial culture suitable for detecting SASCV, real time PCR and fluorescence in situ hybridization. To assess the effects of S. aureus positivity, interleukin-5 (IL-5), interferon-gamma, total immunoglobulin E (IgE), eotaxin, granulocyte-colony stimulating factor, and eosinophil cationic protein in nasal lavages were determined and gene transcription analysis of nasal biopsies from S. aureus positive and negative CRSNP(+) patients was performed. RESULTS: Thirty-one CRSNP(+) patients, 13 CRS patients without polyps, and 21 control patients were evaluated. Staphylococcus aureus was detected by any method in 25 patients (39%). Staphylococcus aureus detection rates did not differ between the three disease groups (P = 0.3). Staphylococcus aureus small colony variants were not found. In nasal lavages, IL-5 and total IgE levels were higher in CRSNP(+) patients than in CRSNP(-) patients or controls (P < 0.05). Staphylococcus aureus positivity did not influence biomarker concentrations in nasal lavages. Genes for T(H2) cytokines were not differentially transcribed. CONCLUSIONS: We could not observe a higher prevalence of S. aureus in CRS patients with or without nasal polyps than in controls. We could not substantiate that S. aureus intensifies the T(H2) shift in CRSNP(+) patients. Staphylococcus aureus small colony variants were not detected in any sample.

Long-term treatment with aspirin desensitization: a prospective clinical trial comparing 100 and 300 mg aspirin daily.

BACKGROUND: The daily dose of aspirin in desensitization in aspirin-sensitive asthmatics with nasal polyps is still a matter of debate. AIMS OF THE STUDY: To compare two doses of aspirin during the first year of desensitization and to evaluate long-term effects on nasal/pulmonary symptoms. METHODS: Patients with positive aspirin provocation test were treated with either 100 or 300 mg aspirin daily. RESULTS: In all patients taking 100 mg aspirin (n = 7) recurrent nasal polyps were observed. No patient experienced reduction of asthma medication or improvement of pulmonary function. In the 300 mg group no recurrent nasal polyps were seen. Asthma medication could be reduced in three patients, pulmonary function was improved in five patients. Thirty-nine consecutively desensitized patients, taking 300 mg aspirin, showed significant improvement of olfaction and polyp-free nasal passages during the first year of therapy. After a median follow-up of 27 months no sinus revision surgery was necessary. CONCLUSIONS: Aspirin desensitization followed by 300 mg aspirin daily is efficacious and results in polyp-free nasal airways, improvement of sense of smell, and reduction of the need for sinus revision surgery for recurrent nasal polyps. Aspirin in a dose of 100 mg daily is not sufficient to effectively reduce nasal and bronchial or pulmonary symptoms and to prevent recurrent nasal polyps by at least the first 12 months of treatment.

Growth factors and scaffold composition influence properties of tissue engineered human septal cartilage implants in a murine model.

Several surgical disciplines apply cartilage grafts for reconstructive purposes and have to overcome the scarcity of donor sites for this unique tissue. Employing the techniques of tissue engineering, cartilage might be generated in reasonable amounts for clinical purposes. Application of growth factors together with biochemical and biomechanical scaffold properties influence the process of ex vivo transplant production. The aims of this study are: 1) to investigate the influence of IGF-1 and TGFbeta-2 on tissue engineered human septal cartilage in vitro and in vivo after transplantation in nude mice; 2) to analyse the effect of the polydioxanone (PDS) content of the biodegradable Ethisorb E210 scaffold on the properties of the implanted constructs. Cells were three-dimensionally cultured on biodegradable Ethisorb E210 (PGA-PLA-copolymer fleeces with polydioxanone (PDS) adhesions), or on E210 scaffolds with a reduced polydioxanone content. Wet weight (ww), GAG-, and hydroxyprolin-content, as well as the cellularity of the neocartilage constructs were quantitatively evaluated. Additionally, the in vivo resorption of the two types of cell carriers was monitored. Addition of growth factors clearly increased the wet weight of the in vitro cultured constructs before transplantation. After transplantation, high PDS content improved the in vivo stability and macroscopic morphometric appearance of the tissue engineered specimens and led to enhanced deposition of glycosaminoglycans in transplanted constructs. Hydroxyproline content of the implants was not affected by either growth factors or PDS content. These data suggest a role for IGF-1 and TGFbeta-2 in preparative in vitro culture of chondrocytes before implantation, while PDS content of the scaffold is important for in vivo properties of the implanted material.

In vitro genotoxicity and cytotoxicity of benzalkonium chloride.

Benzalkonium chloride (BAC) acts as a preservative in numerous nasal preparations. Possible genotoxic and cytotoxic effects of BAC in human respiratory epithelial BEAS-2B cells should be investigated in vitro. Cell cultures were exposed for 2h to BAC in concentrations ranging from 0.002% to 0.05%. Methyl methanesulfonate served as positive control, PBS as negative control. The tail moment of single-cell gel-electrophoresis (SCGE) was used to assess BAC-induced DNA damage. Cell viability was measured by trypan blue dye exclusion staining. Additionally, the critical micellar concentration (CMC) of BAC in PBS was detected. The tail moment increased dose dependently with the maximum value at 0.02%, and declined for higher concentrations. Nearly all cells died at low BAC concentrations up to 0.01%. Above this concentration cell viability increased. The CMC of BAC in PBS was estimated to be 0.02%. BAC caused relevant DNA changes in respiratory epithelial cells in vitro at concentrations commonly employed in commercially available nasal preparations. Some of the exposed cells survived. In further studies it could be considered to look whether these cells would still be able to proliferate and possibly fix the damage that they have possibly accumulated into an actual mutation using for example the induction of micronuclei.

Nasal carriage of Staphylococcus aureus in house dust mite allergic patients and healthy controls.

BACKGROUND: Staphylococcal colonization may influence the course of allergic diseases such as atopic dermatitis or allergic rhinitis. The frequency of Staphylococcus aureus (SA) nasal carriage and its possible influence on persistent allergic rhinitis was investigated. METHODS: In nasal lavages from 22 patients with house dust mite allergy and 18 healthy controls, the number of SA colony forming units per ml were assessed and related to nasal symptom scores, the concentrations of three inflammatory cell activation markers, nasal total IgE and 17 cytokines in nasal secretions. RESULTS: SA was found in 15/22 allergic patients and 4/18 controls (P < 0.01). Comparing allergic SA carriers with allergic noncarriers, nasal symptom scores tended to be higher (P < 0.1), and the cell activation markers ECP (10(2.23+/-0.33)vs 10(1.45+/-0.50) ng/ml; P < 0.05) and elastase (10(2.70+/-0.21)vs 10(2.12+/-0.34) ng/ml; P < 0.01), and nasal total IgE-levels (10(1.66+/-0.38)vs 10(1.2+/-0.28) kU/ml; P < 0.05) were significantly higher in allergic SA carriers. Nasal SA carriers had a higher nasal IL-13/IFN-gamma ratio (P < 0.01), and this was correlated with higher nasal total IgE in allergic patients (r = 0.6, P < 0.05). CONCLUSION: Nasal SA carriage is frequent in patients with persistent allergic rhinitis. The data of this study suggest that they are not only secondary bystanders, but actively modulate the disease by promoting local IgE production.

Nasal biomarker profiles in acute and chronic rhinosinusitis.

BACKGROUND: Clinical manifestations of rhinosinusitis include acute rhinosinusitis, chronic rhinosinusitis (CRS) with nasal polyps and CRS without polyps. OBJECTIVE: Possible mechanisms defining these three forms of rhinosinusitis should be investigated assessing biomarker profiles in nasal secretions. METHODS: Fifteen cytokines, three cellular activation markers and total IgE were determined in nasal secretions of seven patients with acute rhinosinusitis, 12 patients with CRS without polyps, 13 patients with CRS with polyps and six healthy controls. Principal component analysis was used to extract relevant factors. RESULTS: Irrespective of the clinical manifestation, all biomarkers assessed were increased in patients with rhinosinusitis when compared with controls (P<0.001). Principal component analysis allowed the extraction of three factors explaining 83% of data variance. The general inflammatory activation was mainly reflected by the first factor. The second factor differentiated acute from CRS. This factor correlated with IL-12, which is involved in pathogen-related immune activation by antigen-presenting cells. It was also positively correlated with IL-4, IL-10 and IL-13, which play an important role in the resolution of infections. The third factor differentiated CRS with polyps from CRS without polyps (P=0.001). It represented IL-5 and nasal IgE (nIgE), whereas eosinophil cationic protein and tryptase were not specific for CRS with polyps. CONCLUSION: In mucosal infection, numerous inflammatory mediators are activated. Simple correlations of few biomarkers with a specific disease process bear the risk of overestimating a possibly unspecific effect. To assess biomarker profiles, more complex analytic tools may be more appropriate to delineate mechanisms underlying mucosal disease. Using principal component analysis, it was found that high nIgE and IL-5 levels are specific for CRS with nasal polyps.

Biological markers in nasal secretions.

Biological markers in nasal secretions provide valuable information on nasal pathophysiology. However, published data on biomarker concentrations in nasal fluids are remarkably inconsistent, and the bias due to different sampling techniques, has not yet been systematically evaluated. Concentrations of various protein were repeatedly determined in nasal secretions of 16 healthy volunteers. The proteins were detected by using: 1) alpha2-macroglobulin as a marker for plasma contamination; 2) lactoferrin as a marker for glandular secretion; 3) lactate dehydrogenase as a marker for tissue injury; and 4) interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha, and eosinophil cationic protein and tryptase as indicators for tissue inflammation. A total of four different sampling methods, including nasal lavage (NL) and a new polyurethane foam sampler technique (PFST) were employed. Analyte concentrations in NL were approximately 10-times lower than in specimens obtained by PFST. Due to the unpredictable dilution during NL, various analytes were below the detection limit of the high sensitivity assays employed. With PFST, concentrations below the detection limit rarely occurred. The specimens did not significantly differ regarding plasma contamination, glandular secretion or tissue injury. The considerable variability of reported analyte concentrations in nasal secretions mainly results from different sampling techniques. To collect nasal secretions, samplers are considered superior to nasal lavage techniques.

H-2(d) mice born to and reared by HBeAg-transgenic mothers do not develop T cell tolerance toward the hepatitis B virus core gene products.

The function of the secretory core gene product (HBeAg) of the human hepatitis B virus (HBV) is unknown. It has been proposed that this protein may be passed from the mother to her offspring at the perinatal stage where it might induce immune tolerance. In a previous study we have shown that the murine placenta presents an efficient barrier for the HBe protein and that H-2(b) mice born to HBeAg-positive transgenic mothers do not develop tolerance of specific cytotoxic T cells. In the present work we demonstrate that transgenic mice expressing high serum levels of HBeAg secrete only small amounts of this protein into their milk and excrete minute amounts of the viral gene product in their urine. Furthermore, it is shown that nontransgenic H-2(d) mice born to and reared by HBeAg-positive mothers exhibit a reactivity of HBc/eAg-specific CD4(+) Th cells and CD8(+) cytotoxic T cells comparable to that of normal isogenic control mice. In accordance with this observation the humoral immune responses directed against the HBeAg were comparable between these two groups of animals. This finding indicates that H-2(d) mice potentially exposed to small amounts of maternal HBeAg transferred by the transplacental, lactogenic, or renal route do not develop tolerance toward the HBV core gene products. These data challenge the hypothesis that a potential function of the HBeAg may be to operate as a tolerogen at the perinatal developmental stage.

The hepatitis B virus e antigen cannot pass the murine placenta efficiently and does not induce CTL immune tolerance in H-2b mice in utero.

The function of the secretory core gene product (HBeAg) of the human hepatitis B virus is unclear. It has been discussed that this protein may be passed from the mother to the fetus, where it might induce immunologic tolerance. Here we have examined this possibility with transgenic mice expressing high levels of HBeAg. Analysis of serum samples obtained from nontransgenic fetuses which developed in HBeAg-positive mothers showed no evidence that the HBeAg can pass the placenta. Moreover, direct examination of the HBeAg- and HBcAg-specific cytotoxic T-cell immune response of H-2b mice which developed in either transgenic or nontransgenic mothers revealed no indication that mice which could have been exposed to the HBeAg in utero become tolerant to HBV core gene products. From these data we conclude that the placenta represents an efficient barrier for HBeAg transfer and that the HBeAg does not tolerize cytotoxic T cells, at least in mice of the H-2b haplotype.