Tumor risk assessment by means of immunocytochemical detection of early pre-malignant changes in buccal smears.

Increasing incidence of carcinomas in the upper aero-digestive tract, both in Germany and in other European countries requires development of new preventive strategies. The cure rate at advanced tumor stages remains poor in spite of a variety of available therapeutic methods. In the present study the quantitative assessment of a pre-malignant mucosa lesion within a field cancerization was performed by means of immunocytochemical methods. This may allow individuals with an increased risk of developing malignant disease to be identified. Cytosmears taken from healthy buccal mucosa of tumor patients (n=50) and from healthy probands (n=100) with different tobacco and alcohol consumption levels were examined with regard to identifying increased expression of the proliferation markers (PCNA, MIB1), of the tumor suppressor gene product p53 as well as the oncogene product cyclin D1. There was a significant difference in expression of investigated proliferation markers between tumor patients and healthy probands (p<0.0001). When comparing the rate of positively marked cell nuclei to cigarette pack years the marker cyclin D1 and MIB1 show an increased rate in the groups with high tobacco consumption as compared to the group with a low exposure (p>0.05). It could be possible to use the marker MIB1 and cyclin D1 to screen risk groups, since the relative morbidity risk (odds ratio) increases (by 45-62 times) if the threshold value of 4 positively marked cell nuclei is exceeded.

Influence of interleukin-13 on beta-catenin levels in eosinophilic chronic rhinosinusitis cell culture.

Chronic rhinosinusitis (CRS) is one of the most common chronic diseases. The etiology and classification of CRS, with and without nasal polyps, remain unclear. Eosinophils and their products are important in the pathophysiology of allergic diseases and in host immunity to certain organisms. Interleukin 13 (IL-13) plays a pivotal role in eosinophilic inflammation. The migration of epithelial cells requires permanent re-establishment of the intercellular connection. Intercellular connections are maintained by the modulation of adherens junctions consisting of an E-cadherin/beta-catenin complex. In our study we examined the eosinophilic and non-eosinophilic paranasal mucosa obtained from two patients undergoing functional endoscopic sinus surgery. Cell cultures were incubated with human recombinant IL-13 for up to 72 h and beta-catenin concentration was determined with ELISA techniques. Furthermore, immunostaining for beta-catenin was used for the semi-quantitative description of specimens. We were able to ascertain a significant increase in beta-catenin expression in the eosinophilic paranasal cell culture after IL-13 administration compared to the non-eosinophilic culture. Immunostaining for beta-catenin was restricted to the membrane of the cells. Concerning the increased mural expression of beta-catenin, we presume that a fibrotic reaction similar to asthma and chronic obstructive pulmonary disease occurs in patients suffering from CRS. Furthermore, beta-catenin overexpression might be responsible for mucosal thickening and IL-13 seems to be an important marker in eosinophilic CRS.

Effect of vascular cell adhesion molecule 1 on fibroblasts in human eosinophilic chronic rhinosinusitis.

The pathogenesis of eosinophilic chronic rhinosinusitis (ECRS) is still unclear. Paranasal mucosa inflammation is thought to be related to eosinophilic infiltration. This infiltration seems to induce changes in the expression of cell adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1). The E-cadherin-beta-catenin complex maintains the integrity of the epithelium. Downregulation of beta-catenin and E-cadherin is a pivotal factor for progressive cell growth. This study aimed to assess which cytokines regulate the expression of the adhesion molecule E-cadherin and the multi-functional protein beta-catenin, which plays a key role in cadherin-mediated anchoring in ECRS. Cultured ECRS specimens were incubated with human VCAM-1. After a period of up to 72 h, expression of E-cadherin and beta-catenin was determined using cytokine immunoassay and immunohistochemistry. In ECRS, significant increases in E-cadherin expression were found in fibroblast cell cultures. Stimulation with VCAM-1 did not produce a significant alteration in the expression of the adherens junction protein beta-catenin. In addition, VCAM-1 did not decrease the levels of membrane staining for adherens junction proteins. The selective increase in E-cadherin expression in eosinophilic fibroblast cultures might be explained by a higher concentration of the Th2-type cytokines in these cultures. The tissue remodelling observed during chronic eosinophilic inflammation offers new insight into the pathogenesis of ECRS.

Regulation of matrix metalloproteinases (MMP)-2/-9 expression in eosinophilic chronic rhinosinusitis--cell culture by interleukin-5 and -13?

BACKGROUND: Eosinophils are a prominent immunological feature of chronic rhinosinusitis (CRS). Cytokines in the respiratory mucosa may be the key to upper airway pathophysiology. Matrix metalloproteinases (MMP) represent an entire group of Zn2+ dependent endopeptidases with the potential to alter the extracellular matrix (ECM). In this study epithelial cultures of CRS were treated with interleukin (IL)-5 or IL-13 and subsequent levels of metalloproteinases were determined. MATERIALS AND METHODS: The cells for CRS culture were obtained from patients undergoing functional endoscopic sinus surgery. After 8-72 hours incubation with 0.2-0.4 ng/ml IL-5 or 3-6 ng/ml IL-13, the expression of the MMP-2 and -9 in the CRS cultures was analysed. RESULTS: After 72 hours incubation with IL-5, the relative levels of MMP-2 showed no significant alteration in protein expression in comparison with the control groups. Incubation with IL-13 revealed a statistically insignificant decrease of the relative MMP-9 expression in ECRS compared to the control group (p>0.1). CONCLUSION: Alterations of MMP-2 and -9 expression may play a role in ECRS, but the association with IL-5 and IL-13 remains unclear.

In vitro study of interleukin-5 (IL-5) in human eosinophilic chronic rhinosinusitis cell culture.

BACKGROUND: Eosinophilic chronic rhinosinusitis (ECRS) is an inflammatory disease in which the epithelial mesenchymal unit appears to be important in regulating the pathological mechanisms. Changes in adhesion molecule (AM) expression by inflammatory cells have been reported. The damage of respiratory epithelium in allergic diseases has a close correlation with the extent of eosinophil infiltration. In our study, we investigated the effect of IL-5 on beta-catenin and E-cadherin levels in ECRS. MATERIALS AND METHODS: ECRS cell cultures were incubated with IL-5 and beta-catenin/E-cadherin levels were analysed after 8-72 hours using cytokine immunoassay and immunohistochemistry. RESULTS: Eight hours of incubation with IL-5 resulted in 0.19 ng/ml E-cadherin (15.27 ng/ml beta-catenin), whereas in the control 0.14 ng/ml (15.45 ng/ml beta-catenin) was detectable. After 24 and 48 hours, 0.18 ng/ml (16.47 ng/ml beta-catenin) and 0.33 ng/ml (17.88 ng/ml beta-catenin) were measured in the incubated cell cultures, respectively; 72 hours of incubation with IL-5 resulted in 0.14 ng/ml (19.36 ng/ml beta-catenin), whereas 0.17 ng/ml (20.09 ng/ml beta-catenin) was determined in the controls. This study demonstrated a significant decrease in E-cadherin expression in cell cultures after stimulation with IL-5, especially in incubation-time adjusted analysis. However, the immunostaining was restricted to the membrane of the cells. CONCLUSION: In regard to the increased mural expression of AM, we believe that a fibrotic reaction similar to that in chronic obstructive pulmonary disease takes place in patients suffering from ECRS.

VCAM-1 increases levels of HGF in eosinophilic chronic rhinosinusitis cell culture.

BACKGROUND: Eosinophilic chronic rhinosinusitis (ECRS) is one of the most common diseases worldwide. To date the underlying cause remains unclear and no drug has been accredited for first-line therapy. VCAM-1 has been reported to play a pivotal role in establishing ECRS. Other authors have reported that inflammatory cytokines may mediate changes in the underlying epithelium in the sinuses through hepatocyte growth factor HGF. In our study, the effect of VCAM-1 on HGF levels was investigated. MATERIALS AND METHODS: ECRS cell cultures were incubated with VCAM-1 and HGF levels were determined after 16, 24, 48 and 72 hours. RT-PCR was enrolled to depict the HGF-RNA levels. RESULTS: Sixteen hours of incubation showed 28.5 pg/ml HGF, whereas in the control 16.3 pg/ml was detectable. After 24 and 36 hours, 37 pg/ml and 43.5 pg/ml HGF were measured in the incubated cell cultures, respectively; 72 hours of incubation with VCAM-1 resulted in 50.6 pg/ml HGF, whereas 23.5 pg/ml was determined in the controls. The RT-PCR for HGF also showed increased concentration in the incubated cells after 72 hours. CONCLUSION: VCAM-1 induced an increase in levels of HGF in the ECRS cell cultures. The rising transcriptional activity was demonstrated by means of RT-PCR. The levels of HGF were within physiological ranges, suggesting that a misbalance between HGF and VCAM-1 resulted in the establishment of ECRS. Further experiments are necessary to reveal the role of HGF in the development of ECRS. This is the first report about the effect of VCAM-1 on growth factors in ECRS cell culture.

Current advances in the basic research and clinical management of sinonasal inverted papilloma (review).

Inverted papilloma (IP) is a benign sinonasal lesion that has a known propensity for recurrence, local aggressiveness and an association with transformation to squamous cell carcinoma. Due to the high rate of recurrence, association with malignancy and a tendency of multicentricity, the surgical approaches to treatment are controversial. Over the years there has been a slow evolution from aggressive (en bloc) resection by lateral rhinotomy to endoscopic techniques. This progress corresponds to the advances that have been made in endoscopic sinus surgery over the past 15 years. Technological advances have allowed the detection of sinonasal IP before its extension beyond the sinonasal region, thus enabling minimally invasive techniques to be used in the treatment of selected cases of IP. Differences in recurrence rates were not observed for endoscopic management as compared with lateral rhinotomy or sublabial degloving approaches. In terms of aetiology there is certain evidence that the presence of HPV in IP could be predictive of malignant transformation. Although IPs are monoclonal proliferations, they do not fit the profile of a prototypic precursor lesion. In contrast, an increased EGFR and TGF-alpha expression is associated with early events in IP carcinogenesis. Parameters such as hyperkeratosis, squamous epithelial hyperplasia and a high mitotic index are negative prognostic indicators, which could be useful in the future follow-up of patients with IP. Present literature should encourage us to recommend the use of a uniformly accepted staging system. The propensity for delayed recurrences and the maximal 13% incidence of malignant transformation mandates careful, long-term follow-up.

Saliva as a biomarker for head and neck squamous cell carcinoma: in vitro detection of cytotoxic effects by using the plating efficiency index.

Worldwide, the incidence of head and neck squamous cell carcinoma (HNSCC) is increasing. The development of an effective prevention program would provide a promising opportunity to control this disease. We applied the 'plating efficiency index' of Chinese hamster lung fibroblasts to demonstrate the presence of cytotoxic effects in saliva samples from cancer patients as well as from healthy probands. Correlations between individual risk factors and the cytotoxic effects of saliva specimens were analysed and evaluated. Saliva samples were obtained from male patients (n=43) with carcinomas of the upper aerodigestive tract, and from a healthy age-matched male control group (n=131) with different tobacco and alcohol consumption habits. The extraction of non-stimulated sober saliva was chosen for this investigation. In vitro cytotoxicity of the saliva was detected using the permanent mammalian cell line V79 (lung fibroblasts) of Chinese hamsters. The determination of the plating efficiency index was made in the logarithmic growth phase of the initial cell culture. A reduction in cell colonies (plating efficiency) of at least 50% was defined as strongly cytotoxic. A significant direct correlation was found between daily tobacco consumption and the reduction of plating efficiency (p<0.0001). We found only a moderate increase in the cytotoxicity of tested saliva samples in correlation with daily alcohol uptake. The difference between tumor patients and healthy probands was highly significant (chi(2) test; p=0.001). Using the method of logistic regression analysis, we found a 3.6-fold increased cancer risk in probands with cytotoxic saliva (p<0.001). According to our results, the plating efficiency index seems to be a suitable method for the detection of increased cancer risk. In combination with several effective biomarkers on cytogenetic end-points, it may help to establish biomonitoring programs for secondary cancer prevention.

Sulindac sulfone modulates beta-catenin in human cholesteatoma cell culture.

BACKGROUND: External auditory canal cholesteatoma (EACC) is a chronic inflammation of the bony ear meatus. Its etiology is not clearly understood. Other than surgical intervention, conservative methods are investigated for different cholesteatomas. Inducing apoptosis seems to be an appropriate strategy. Sulindac sulfone is a new class of targeted and pro-apoptotic drugs. It provokes apoptosis by inducing phosphorylation of beta-catenin, which is a multifunctional protein in the cell-cell adhesion complex. METHODS: EACC-cell cultures were incubated with different concentrations of sulindac sulfone (400 and 800 micromol). After 16, 24, and 48 h, beta-catenin concentrations were determined by ELISA, Western blot, and immunohistochemical analysis. RESULTS: After 48 h incubation with 400 micromol sulindac sulfone, the average level of beta-catenin showed a decrease of 46% (0.004337 microg/mL) from those determined at 16 h with the same concentration of sulindac sulfone. At 800 micromol sulindac sulfone, the treated cell culture showed a reduction of 66.2% (0.003443 microg/mL). Comparing total protein content and the fraction of beta-catenin at different points in time, the concentration of beta-catenin decreased in both EACC cell cultures, 400 micromol (minus 63%) and 800 micromol (minus 81%). CONCLUSIONS: The results presented in this paper are the first to demonstrate the chemopreventive effects of the agent sulindac sulfone on cholesteatomas. The greatest decrease of beta-catenin was observed between 16 and 24 h incubation. The inhibitory effect of sulindac sulfone as a local treatment seems to be a useful additional tool for nonsurgical approach to the therapy of EACCs.

A three-step purification method of large quantities of human recombinant alpha endothelial cellular growth factor for clinical use.

The endothelial cellular growth factor alpha-ECGF is a candidate drug for the induction of therapeutic neoangiogenesis. Its use in extensive experimental and clinical trials is hampered by the fact that currently published purification procedures allow only small yields, and the absence of pyrogenic impurities is not demonstrated. The rh alpha-ECGF was expressed in E. coli. Isolation of rh alpha-ECGF from E. coli lysates to apparent homogenicity was achieved by a three step purification procedure involving ionic exchange, heparin-sepharose and polymyxin B chromatography. By this method, 200 mg of rh alpha-ECGF was purified from 15 g wet weight E. coli bacteria. The isolated protein of 18 kDa appeared as a single band after SDS gel electrophoresis and subsequent silver-staining. The biological activity was expressed in the chorion-allantois-membrane assay and in the 3H-thymidine proliferation in baby hamster kidney cells. Drug trials with rabbits revealed no increase in body temperature after intravenous injections with 1 mg rh-ECGF.

Chondrosarcoma of the larynx and review of the literature.

Chondrosarcoma (CS) of the larynx was first described in 1935. Cartilaginous tumours of the larynx are largely rare and there is little literature concerning them. Laryngeal CS manifest with a different pathological behaviour to other malignancies of the larynx and as such the treatment of these neoplasias are different. The purpose of this review is to present a detailed report of the laryngeal CS in recent literature. We present a case of laryngeal CS of the cricoid cartilage and a case of a sarcomatous neoplasm of the vocal cord as a potential differential diagnosis. Although representing a rare malignancy, the last decade has brought new insights in surgical treatment of laryngeal CS and subsequent reduction in recurrence rates, whereas progress in tumour biology and etiological agents is still scarce. We concentrate on new insights in classification, radiologic and pathologic features, and treatment modalities in the last two decades. Based on the literary evidence the authors recommend a conservative laryngeal function-preserving surgery. Total laryngectomy should be reserved to recurrent CS and rare cases of voluminous high-grade CS of the larynx.

The role of S100A1 in external auditory canal cholesteatoma.

S100 proteins were reported to be involved in different biological activities such as transduction of intracellular calcium signalling. It has been reported that each member of the S100 protein family exhibits a distinct tissue-specific pattern of expression. Furthermore, altered S100 protein expression and function correlate with many diseases. The expression of S100A1 was reported to be increased in different tissue with hyperplasia. The external auditory canal cholesteatoma (EACC) is a benign hyperplasia of the auditory meatal skin. However, without treatment, EACC destroys adjacent tissue. Seventeen EACC specimens were collected and investigated immunohistochemically against S100A1. Normal auditory meatal skin served as control. In the EACC, S100A1 showed a more homogeneous staining pattern, positively expressed throughout the epithelial layers. The keratin debris showed no detectable expression of S100A1. In the auditory meatal skin (control), S100A1 was only expressed in the basal layer of the epithelium. We showed that there are different staining patterns in normal auditory meatal skin, middle ear cholesteatoma and external auditory canal cholesteatomas. The immunoreactivity increased with the stage of the disease. Contrary to that reported previously in the middle ear cholesteatomas, the reactivity was strongest in the upper epithelial layers. In our collective, the epithelial matrix of the EACC showed strong reactivity throughout all the layers. Surprisingly, there is no study regarding the connection between growth factors and S100A1. In previous experiments we showed significant increase of growth factors in EACC. This correlates with our new data concerning S100A1. This is the first study to show the different reactivity pattern of S100A1 in the external auditory canal cholesteatoma.

Chemopreventive alteration of the cell-cell adhesion in head and neck squamous cell cancer.

Approximately 310,000 new cases of oral and pharynx cancer account for a major cause of neoplasm related morbidity and mortality world-wide. Unfortunately, the survival rate has not improved significantly in the last decade. The vast majority of head and neck cancer is squamous cell carcinoma. The major adhesion-proteins involved in the development and maintenance of all solid tissue are the Cadherins. Cadherins are the transmembrane components of the adherent junction with interaction with plakoglobin and beta-catenin. Downregulation of Cadherins and catenins is frequently observed in many types of human cancer. Sulindac sulfone is one of the new therapeutic apoptotic agents that show promise in the treatment of cancer. In this study, we incubated sulindac sulfone with a head and neck cancer cell line and investigated the outcome of E-Cadherin. Immunohistochemical and Western blot analyses were then performed, with different concentrations of sulindac sulfone (100, 200, 400, 600, and 800 microMol) for 48 h. At 400 microMol of sulindac sulfone a decrease of 21% was observed; at 600 microMol, 44% decrease of beta-catenin concentration was seen, and incubation with 800 microMol resulted in 73% reduction of secreted beta-catenin. Incubation with sulindac sulfone seemed to stop proliferation; however, with respect to the controls, there was no increased reduction of the total protein. Sulindac sulfone resulted in an increase of E-Cadherin content in the head and neck squamous cell cancer cell line after 48 h of incubation; however, the reactivity was restricted to the adherent junctions. At increasing concentrations of sulindac sulfone, intercellular E-Cadherin immunostaining intensifyied. ELISA also depicted significant rising levels of E-Cadherin. Sulindac sulfone contributes to the inactivation of cGMP phospho-diesterase. Thus, the accumulation of cellular cGMP and protein kinase G is induced. The following degradation of the phosphorylated beta-catenin and the dissociation from the Cadherin-catenin complex releases E-Cadherin. This may also contribute to growth inhibition and co-ordinate with apoptosis induction. It is not really clear as to, which pathway results in the elevation of the E-Cadherin proteins. However, in epithelial cancer cells, the Cadherin-catenin complex serves as a target for the chemopreventive agent, sulindac sulfone.

Hereditary hemorrhagic telangiectasia: an update on clinical manifestations and diagnostic measures.

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease, is an autosomal dominant disorder of the fibrovascular tissue. It is characterized by the classic triad of (muco-)cutaneous telangiectases, arteriovenous malformations with recurrent epistaxis and hemorrhages, and inheritance. A wide variety of clinical manifestations in HHT have been described. In more than 90% of the patients, nosebleeds are the first predominant symptom, therefore ENT physicians often play a key role as far as diagnosis and management of the disease are concerned. In spite of recent diagnostic and therapeutic progress, a cure for this often burdening and handicapping disease is still not available. Apart from affecting the nose, arteriovenous malformations (AVMs) may also affect the skin, lungs, brain, liver and gastrointestinal tract. The two known genes that are implicated in HHT are endoglin (ENG) located on chromosome 9q33-q34 and activin-receptor-like kinase (ALK1) located on chromosome 12q13. Mutations of ENG are observed in HHT type 1 with an incidence up to 40% for pulmonary AVMs, whereas mutations of ALK1 are observed in HHT type 2 with an incidence of only 14% for pulmonary AVMs, which clinically distinguishes these two types of mutation. The emphasis of this paper is mainly on the clinical manifestation, molecular genetics and diagnosis of HHT, taking account of current literature on HHT in order to better understand the complexity of the disease. Recent therapeutic options in the treatment of HHT have been omitted from this paper as they are subject of a following paper. HHT is more common than previously thought and shows a broad range of different clinical organ manifestations that can be sources of substantial morbidity and mortality, making HHT a continuing challenge for many sub-specialties where interdisciplinary diagnostic screening is mandatory in the management of the disease.

Ossifying fibromyxoid tumor of the nasal septum.

The fibromyxoid tumor is quite a rare soft tissue tumor and typically presents as an ossifying fibromyxoid tumor (OFMT) in the subcutis of the extremities of adults. Most authors favour schwannian or chondroid origin of this lesion with somehow uncertain biologic dignity. Local recurrence is seen in 27% of patients after primary excision. We present a case of a fibromyxoid tumor of the nasal septum in a 49-year-old female who complained of nasal airway obstruction and enlargement of the right contour of the nose. Endonasal, endoscopic tumor excision was performed. The tumor contained spindle-shaped and polygonal cells, mucoid pseudocysts and a fibromyxoid stroma with local calcifications. The clinical behaviour of OFMT in general is benign but some reports have documented atypical tumors with histologic signs of malignancy. Complete local resection is the treatment of choice. Because of the high rate of local recurrence, clinical follow-up examinations are necessary.

Patients with hereditary hemorrhagic telangiectasia have increased plasma levels of vascular endothelial growth factor and transforming growth factor-beta1 as well as high ALK1 tissue expression.

BACKGROUND AND OBJECTIVES: Hereditary hemorrhagic telangiectasia (HHT), an inherited vascular dysplasia, is caused by mutations in endoglin or activin receptor-like kinase (ALK)-1. Haploinsufficiency for these genes is thought to result in an imbalanced angiogenic activity. The aim of this study was to evaluate the plasma levels and the expression profiles of angiogenic and angiogenesis-related factors in the context of HHT. DESIGN AND METHODS: Vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta1 plasma concentrations were determined in 31 HHT patients and 40 healthy controls by ELISA. VEGF and TGF-beta1 plasma concentrations were correlated with the patients' clinicopathological features. Tissue expression of angiogenic and angiogenesis related proteins was determined by immunostaining on nasal cryostat sections from 13 HHT patients and 5 healthy controls. RESULTS: Of the 31 patients, 29 had statistically significantly raised plasma concentrations of VEGF and TGF-beta1 but there was no correlation with specific clinicopathological features. Increased VEGF, TGF-beta1 and ALK1 immunostaining was seen in all 13 investigated patients. beta-smooth muscle actinin immunostaining was increased in 12 patients. Increased endoglin immunostaining was seen in only 9 patients. INTERPRETATION AND CONCLUSIONS: This study provides evidence of the role of VEGF and TGF-beta1 in the pathogenesis of HHT. Plasma concentrations of these two factors may serve as further diagnostic criteria for HHT. For the first time, we report increased TGF-beta1 plasma concentrations and increased TGF-beta1 and ALK1 tissue expression in HHT, which appear not to be specifically associated with either endoglin or ALK1 mutations. The data suggest that HHT is an angiogenic disorder characterized by an over-expression of VEGF, TGF-beta1 and ALK1.

Targeting TGF-beta1 increases hepatocyte growth factor (HGF/SF) levels in external auditory canal cholesteatoma (EACC) epithelial cell culture.

OBJECTIVE: The transforming growth factor (TGF)-beta 1 is known to have pro- and anti-angiogenic actions. Hepatocyte growth factor/scatter factor (HGF/SF) antagonizes TGF-beta1 by stabilizing SMAD transcriptional co-repressor TGIF. HGF/SF is a multifunctional polypeptide with morphogenic, motogenic, angiogenic, and proliferative capabilities. We assume HGF to be a pivotal factor during the pathogenesis of the external auditory canal cholesteatoma (EACC). In this study, we investigate the effect of antisense targeting TGF-beta1 on HGF/SF levels in the epithelial EACC-culture. MATERIALS: For 48 h, epithelial EACC cell culture was incubated with 3 and 6 mumol antisense targeting TGF-beta1, respectively. Levels of HGF/SF were determined and normalized to cellular protein. Untreated EACC cell culture and scrambled TGF-beta1-antisense served as control. In the second experiment, EACC cells were incubated with rh TGF-beta1 (2 and 4 ng/ml) for 48 h and HGF/SF was determined. RESULTS: After incubation with 3 mumol TGF-beta1-antisense, the average level of HGF/SF was measured at 43.68 pg/ml. Incubation with 6 micromol TGF-beta1-antisense showed 64.95 pg/ml. In untreated EACC (control), the average level of HGF/SF after 48 was 34.55 pg/ml. Incubation with scrambled TGF-beta1 oligonucleotide showed an average HGF/SF level of 34.41 (3 micromol) and 35.66 (6 micromol), respectively. The difference between the scrambled antisense and the targeting antisense TGF-beta1 was significant (p<0.05). After incubation with 2 ng/ml TGF-beta1, the HGF/SF levels were at 22.16 pg/ml. TGF-beta1, 4 ng/ml, resulted in 15.33 pg/ml of HGF/SF. The difference of the levels of HGF/SF after incubation with exogenous TGF-beta1 was significant (p<0.05). CONCLUSION: In this study, levels of HGF/SF increased in the epithelial EACC cell culture after incubation with 3 and 6 mumol antisense TGF-beta1 oligonucleotides depending on the concentration of the antisense. In reverse, TGF-beta1 acted as inhibiting cytokine on HGF/SF levels. In conclusion, TGF-beta1 may be a useful therapeutic agent for managing EACC.

Estriol induced squamous metaplasia on the nasal mucosa in patients with hereditary hemorrhagic telangiectasia.

BACKGROUND: The aim of this study was to evaluate by light and electron microscopy the effect of topical estriol on the nasal mucosa in patients with hereditary hemorrhagic telangiectasia (HHT). METHODS: Twelve patients were instructed to apply twice daily 0.1% estriol as a nose ointment over a period of 12 months. Written consent was obtained from each patient, allowing biopsy specimens of the nasal mucosa to be taken prior to and 3, 6 and 12 months after estriol application. RESULTS: Metaplastic change of the nasal mucosa was observed 6 months after topical estriol application. The former ciliated columnar epithelium changed into a keratinizing squamous epithelium. The effect was reversible after discontinuation of estriol application. CONCLUSIONS: For the first time, we could outline the effect of topical estriol on the nasal mucosa. These histomorphological findings, and the fact that estriol is a low-potency metabolite of estradiol, make estriol a valuable agent in the treatment of HHT patients.

Effect of vascular endothelial growth factor on fibroblasts from external auditory canal cholesteatoma.

BACKGROUND: EACC is a disease of the external auditory canal resulting in destruction of adjacent tissue. However, the role of the surrounding mesenchymal fibroblasts of the perimatrix still remains unclear. In this study we treat isolated fibroblasts of EACC with VEGF and determine FGF-2 levels. We also treat the fibroblast cultures with FGF-2 and measured VEGF levels. METHODS: All EACC cell cultures were obtained from five patients undergoing surgery and used at passage 3. After 1-4 days incubation with 50 ng/mL FGF-2, and 1-8 days incubation with 50 pg/mL VEGF incubation, the expression of the FGF-2 and VEGF protein in the supernatants of the HGF/SF-treated and -untreated culture cell lines was analyzed, respectively. RESULTS: After 8 days of incubation with 50 ng/mL VEGF, the levels of FGF-2 decreased. However, after 4 days of incubation with FGF-2 the VEGF levels increased significantly in treated tissue culture (p <0.05) in comparison to untreated EACC fibroblasts. The total protein concentration showed no significant difference in both cultures (p >0.05). CONCLUSIONS: In summary, exogenous FGF-2 increased fibroblast expression of VEGF, which is a major autocrine mediator of FGF-2-induced angiogenesis and proliferation. However, incubation with VEGF resulted in decrease of FGF-2 levels. Regarding the slow growth of the fibroblasts, they may not be as likely to exhibit a reactive or invasive phenotype as seen in middle ear cholesteatoma fibroblasts.

Angiogenesis in hereditary hemorrhagic telangiectasia: VEGF165 plasma concentration in correlation to the VEGF expression and microvessel density.

Angiogenesis defines the physiologic process of capillary blood vessel formation. It is a multistep process, which is controlled by a large number of pro- and anti-angiogenic factors. Vascular endothelial growth factors (VEGF) are the best characterized pro-angiogenic factors, which play a key role in angiogenesis. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant, multi-systemic disorder of angiogenesis, clinically characterized by severe and recurrent hemorrhages. We assume in HHT patients with increased VEGF plasma levels a high VEGF expression also correlates with the degree of microvesssel density (MVD). In 41 HHT patients and 47 healthy patients, the VEGF165 plasma concentration was determined by standard ELISA technique. Cryostat sections of 13 HHT patients were immunostained for VEGF and endothelial cells by an anti-vWF monoclonal antibody using a standard streptavidinbiotin complex procedure. The degree of vessel density was quantified by light microscopy (x200) within 'hot spot' areas of the HHT-tissue. All HHT cryostat sections showed a medium to strong staining for VEFG compared to healthy control tissue. The VEGF expression correlated with the VEGF165 plasma concentration and the mean MVD in HHT patients. HHT patients with medium VEGF staining revealed significantly lower VEGF165 plasma concentrations and a lower mean MVD (204+/-12 vessels/per microscopic field) than HHT patients with strong VEGF staining (327+/-76 vessels/per microscopic field). High VEGF expression in patients with HHT in correlation to their VEGF165 plasma levels and the microvessel density may support the theory that VEGF functions as an important regulator and key protein of angiogenesis, even in HHT.