Juergen Brieger

Fungal DNA is present in tissue specimens of patients with chronic rhinosinusitis

Background It has been postulated that fungal organisms might represent the immunologic target initiating and maintaining the disease process in patients with chronic rhinosinusitis (CRS). The presence of fungi in nasal mucus has been established by different groups, but so far it has not been shown how the immune system could even recognize such extramucosal—extracorporal—fungal targets. The aim of this study was to determine whether fungal DNA is present in tissue specimens taken from patients with polypoid CRS. Methods Twenty-seven surgical specimens were collected from patients suffering from CRS. Fifteen surgical specimens from healthy ethmoidal mucosa served as controls. A second set of controls consisted of five surgical specimens of acoustic neuroma, which were included to rule out contamination within the protocol. All paranasal tissue samples were treated and rinsed carefully with a solution of Dithiothreitol to digest any nasal mucus and ensure that only tissue was examined. A highly sensitive two-step polymerase chain reaction (PCR) was applied to detect fungal DNA, using one universal primer for unspecific detection of fungal DNA and a second primer pair specific for Alternaria. Results Fungal DNA was detected in all 27 CRS specimens equally with both PCR primers. Controls from healthy paranasal mucosa were positive using the panfungal primers in 10 of 15 cases but were all negative for Alternaria DNA. PCR was negative for fungal DNA in all five neuroma specimens. Conclusions Fungal DNA can be detected within sinonasal tissue specimens of patients suffering from CRS. These findings need to be discussed with respect to the proposed hypothesis of the immune system recognizing extramucosal organisms and initiating an immune response in sensitized patients.

Expression, localization, and significance of vascular permeability/vascular endothelial growth factor in nasal polyps.

Background The exact etiologic mechanisms leading to the formation of nasal polyps have remained largely obscure. A key phenomenon of this specific type of chronic inflammatory disease in nasal respiratory mucosa is remarkable edema. Vascular permeability/vascular endothelial growth factor (VPF/VEGF) plays an important role in inducing angiogenesis and modulating capillary permeability. Objective To study the expression and localization of VPF/VEGF as a putative key factor in nasal polyp development. Methods Specimens of nasal polyps (n = 12) were harvested during endonasal sinus surgery in patients with polypous chronic rhinosinusitis. Specimens of healthy nasal respiratory mucosa (n = 12) served as controls and were obtained from inferior turbinates of patients undergoing surgery for nasal obstruction without signs and symptoms of inflammatory disease. Frozen sections were immunohistochemically stained for VPF/VEGF and quantitatively analyzed, using computer-based image analysis. Results The expression of VPF/VEGF in specimens of nasal polyps was significantly stronger than in specimens of healthy nasal mucosa of controls. VPF/VEGF in polypous tissue was mainly localized in vascular endothelial cells, in basal membranes and perivascular spaces, and in epithelial cells. Conclusion The markedly increased expression in nasal polyps as opposed to healthy nasal mucosa suggests that VPF/VEGF may play a significant role in both the formation of nasal polyps and in the induction of heavy tissue edema. This finding is discussed with respect to the differential expression of cyclooxygenase (COX) isoenzymes-1 and -2 (COX-1 and COX-2) in nasal polyps.

Genotoxic effects of zinc oxide nanoparticles

The potential toxicity of nanoparticles has currently provoked public and scientific discussions, and attempts to develop generally accepted handling procedures for nanoparticles are under way. The investigation of the impact of nanoparticles on human health is overdue and reliable test systems accounting for the special properties of nanomaterials must be developed. Nanoparticular zinc oxide (ZnO) may be internalised through ambient air or the topical application of cosmetics, only to name a few, with unpredictable health effects. Therefore, we analysed the determinants of ZnO nanoparticle (NP) genotoxicity. ZnO NPs (15-18 nm in diameter) were investigated at concentrations of 0.1, 10 and 100 μg mL(-1) using the cell line A549. Internalised NPs were only infrequently detectable by TEM, but strongly increased Zn(2+) levels in the cytoplasm and even more in the nuclear fraction, as measured by atom absorption spectroscopy, indicative of an internalised zinc and nuclear accumulation. We observed a time and dosage dependent reduction of cellular viability after ZnO NP exposure. ZnCl2 exposure to cells induced similar impairments of cellular viability. Complexation of Zn(2+) with diethylene triamine pentaacetic acid (DTPA) resulted in the loss of toxicity of NPs, indicating the relevant role of Zn(2+) for ZnO NP toxicity. Foci analyses showed the induction of DNA double strand breaks (DSBs) by ZnO NPs and increased intracellular reactive oxygen species (ROS) levels. Treatment of the cells with the ROS scavenger N-acetyl-l-cysteine (NAC) resulted in strongly decreased intracellular ROS levels and reduced DNA damage. However, a slow increase of ROS after ZnO NP exposure and reduced but not quashed DSBs after NAC-treatment suggest that Zn(2+) may exert genotoxic activities without the necessity of preceding ROS-induction. Our data indicate that ZnO NP toxicity is a result of cellular Zn(2+) intake. Subsequently increased ROS-levels cause DNA damage. However, we found evidence for the assumption that DNA-DSBs could be caused by Zn(2+) without the involvement of ROS.

Analysis of differentially expressed proteins in oral squamous cell carcinoma by MALDI-TOF MS.

PURPOSE To explore the presence of differentially expressed proteins in OSCC for discrimination of tumour and normal mucosa to establish potential biomarkers and therapeutic targets. EXPERIMENTAL DESIGN Paired protein samples of 12 individuals (tongue cancer and non-cancerous mucosa) were separated by two-dimensional polyacrylamid gel electrophoresis. The protein patterns were compared pairwise and protein spots were quantified. We identified about 70 regulated proteins which we subsequently identified by MALDI-TOF mass spectrometry. RESULTS Cancerous and non-cancerous tissues could be most precisely distinguished by a panel of proteins. They include the heat shock proteins (hsp)70 and 90, keratins (ck) 5, 6, 13, 14, 16, 17 and 19, beta globin, alpha-2-actin, stratifin, tropomyosin, calreticulin precursor, beta-2-tubulin, galectin7, thioredoxin, involucrin, adenylyl-cyclase-associated protein, disulfide isomerase-associated protein, thyrosine 3-monooxygenase, MYL2 and the s100 calcium binding protein. MYL3, cardiac muscle alpha actin 1 proprotein and transferrin were under-represented in OSCC. Six biomarkers, ck6 und ck13, beta globin, alpha-2-actin, hsp70 and hsp90 discriminated best between cancerous and non-cancerous oral tissues. All over-expressed proteins were analysed by STRING-analysis to highlight experimentally determined and computationally predicted interactions between the proteins. Especially involucrin, hsp70, calreticulin precursor, stratifin, (ck) 5, 6, 14, 19, tyrosine 3-monooxygenase, beta-2-tubulin and disulfide isomerase associated protein showed multiple relations. CONCLUSION We identified six proteins which are differentially expressed in most OSCC compared to healthy tissues. Of those, by string analysis, multiple interaction partners are assumed for hsp70. This protein is supposed to be the most promising candidate as marker molecule and target for OSCC therapy.

Expression of Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor in Sporadic Vestibular Schwannomas Correlates to Growth Characteristics

Hypothesis: Expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) may have an impact on the growth characteristics of sporadic vestibular schwannomas (VSs). Background: Vestibular schwannoma is a benign, slow-growing neoplasm that accounts for 6% of all intracranial tumors. The biological backgrounds for neoplastic growth and especially for the various growth patterns of VS remain largely unknown. Because several angiogenic and cytotrophic factors have been described to be involved in the growth of malignant tumors, we initiated this study to examine 2 major representatives of such growth factors in VS and their possible correlation to the growth characteristics of sporadic VSs. Methods: Surgical specimens from 17 patients with sporadic VS were examined, and the expression of 2 major angiogenic and neurotrophic factors, bFGF and VEGF, was quantitatively analyzed at the mRNA and protein levels. The microvessel density (MVD) was defined by CD31 staining. Results: All tumors showed expression of bFGF and VEGF at both the mRNA and protein levels. The mRNA expression and the protein expression of both growth factors correlated positive to tumor volume, to tumor growth index, and to MVD. Conclusion: The bFGF and VEGF mRNA expression and the bFGF and VEGF protein expression in sporadic VS correlates to the tumour volume, to the tumor growth index, and to the MVD. This might indicate an angiogenic and neurotrophic influence of these factors and a possible involvement in the growth of sporadic VS.

New immunohistologic findings on the differential role of cyclooxygenase 1 and cyclooxygenase 2 in nasal polyposis.

Background Cyclooxygenase 1 (Cox-1) plays a key role in arachidonic acid metabolism and in the pathophysiology and immunology of nasal polyposis in patients suffering from aspirin intolerance. We hypothesize that Cox-2 also might be relevant in the etiology of nasal polyps of aspirin-tolerant patients by their effects on inflammatory mediators as well as on microvascular permeability. Methods Fifty-two surgical specimens were immunohistochemically labeled for Cox-1 and Cox-2. Specimens were taken from chronically inflamed mucosa (n = 19) and from nasal polyps (n = 19) during endonasal sinus surgery. Controls were obtained from healthy nasal respiratory mucosa (n = 14), harvested during turbinate surgery in patients with nasal obstruction without inflammatory disease. Staining intensities were semiquantitatively assessed and statistically analyzed. Results In chronically inflamed tissue the expression of Cox-1 and Cox-2 was strongly labeled. However, in nasal polyps the staining pattern of Cox-1 was similar, but Cox-2 expression in epithelial cells was significantly less than in inflamed, nonpolypous specimens. Conclusion These data suggest that while Cox-1 is strongly up-regulated, Cox-2 expression is significantly lower in epithelial cells of nasal polyps than in those of chronic sinusitis without polyps. The relevance of this finding has to be discussed with respect to the regulatory function of Cox on the inflammatory reaction in nasal respiratory mucosa and its hypothetical role in alterations of capillary permeability via vascular permeability factor/vascular endothelial growth factor.

Untreated Chronic Rhinosinusitis: A Comparison of Symptoms and Mediator Profiles

Objectives/Hypothesis Since the early 1990s, the knowledge of inflammatory mediators involved in chronic rhinosinusitis has increased extensively. However, until the present no data on trends in mediator levels in the natural course of the exacerbation‐free interval of chronic rhinosinusitis have been published. The purpose of the study was to examine how levels of inflammatory mediators and clinical findings in chronic rhinosinusitis relate over time in the absence of acute exacerbation.

Reactive oxygen species activation of MAPK pathway results in VEGF upregulation as an undesired irradiation response

BACKGROUND Radioresistance limits the effectiveness of radiotherapy in head and neck squamous cell carcinoma. We previously demonstrated post-radiogenic mitogen-activated protein kinase (MAPK) pathway activation and vascular endothelial growth factor (VEGF) release resulting in reduced tumor cell response. Here, we examined the association of this mechanism with the induction of reactive oxygen species (ROS) under irradiation (IR). METHODS Intracellular ROS after IR were measured. We modeled radiation-induced ROS by exposure of two SCC lines to H2 O2 and evaluated the impact of irradiation and ROS on ERK phosphorylation by Western blot, immunohistochemistry, and ELISA. RESULTS We found elevated pERK levels after treatment with IR and H2 O2 , which could be distinctly suppressed by U0126. Immunohistochemistry and ELISA revealed increased intracellular VEGF levels after H2 O2 application. CONCLUSIONS Our data show that irradiation-induced ROS activate the MAPK pathway and release of VEGF. As VEGF is known to be released after cellular distress resulting in cytoprotection, the described mechanism is potentially of importance for therapy success.

Demethylation treatment restores hic1 expression and impairs aggressiveness of head and neck squamous cell carcinoma

Promoter hypermethylation of tumor suppressor genes is a common feature of primary cancer cells. However, at date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis are not yet been well defined. In the present study we analysed the methylation status of the gene hypermethylated in cancer-1 (hic1), a gene located on chromosome 17p13.3, a region frequently lost in HNSCC. We analysed 22 HNSCC samples and three cell lines using methylation specific PCR (MSP). We found hic1 methylated in 21 out of 22 samples and in all three cell lines. Treatment of the cell lines with the demethylating agent 5-Azacytidin (5-Aza) resulted in the demethylation of the hic1 promoter and reactivation of hic1 expression as determined by MSP, qPCR and Western blot. Functional analyses revealed decreased proliferative activity and colony forming ability of treated cells. In summary, we found in HNSCC hic1 regulated by promoter methylation. 5-Aza application resulted in the reexpression of hic1 and was followed by decreased aggressiveness of the cancer cells. Our data indicate that hic1 might be a player in HNSCC development and suggest further evaluation of 5-Aza for HNSCC treatment.

Pharmacological genome demethylation increases radiosensitivity of head and neck squamous carcinoma cells

Aberrant inactivation of tumor suppressor genes by promoter hypermethylation has been recognized as a crucial step of tumor development and is related to aggressiveness and therapy resistance. To identify potential novel treatment strategies, we evaluated pharmacological genome demethylation for the increase of irradiation treatment effectiveness in head and neck squamous cell carcinoma (HNSCC) in this in vitro study. HNSCC cells were cultured with 2 different concentrations of 5-azacytidine (5-Aza) for 72 h, followed by a single fraction irradiation with 4 or 50 Gy, respectively. To show successful genome demethylation, the methylation status of the tumor suppressor gene hic1 (hypermethylated in cancer) promoter was analyzed by methylation specific PCR (MSP) as well as hic1 transcription by quantitative RT-PCR. Survival, apoptosis, viability, and migration of the tumor cells were analyzed as functional parameters of combined treatment response. After 5-Aza treatment the hic1 promoter was demethylated and gene transcription restored demonstrating genome demethylation. 5-Aza treated cells tended to be less viable and showed decreased survival indicated by lower colony numbers. Apoptosis and migration were not affected. The combined application of irradiation and 5-Aza significantly reduced survival compared to the single treatments. Accordingly, apoptosis was strongly increased after combined 4 Gy/5-Aza treatment. Viability was not additionally affected by combined treatment. Migration was affected weakly by combined high dosage irradiation/5‑Aza treatment. Our data show that the combined application of 5-Aza and irradiation is effective in vitro. A demethylating concept prior to irradiation should be further evaluated for its potential to reduce irradiation resistance.