Comparative analysis of the impact of e-cigarette vapor and cigarette smoke on human gingival fibroblasts.

Human gingival fibroblasts (HGF) play a vital role in wound healing, oral cancer, and are among the first cells being exposed to e-cigarette vapor (eCV) or cigarette smoke (CS) during inhalation. Although the cell-damaging effect of CS has been well studied, the effects of eCV on gingival cells are still unclear. The aim of this in vitro study was to compare the effects of eCV and CS on HGF in terms of proliferation, metabolic activity, cell death, and formation of reactive oxygen species (ROS). After 24 h cell numbers in CS-exposed cells in contrast to eCV-exposed cells were significantly decreased compared to the control. At later points in time, such differences could no longer be observed. Compared to the control, HGF stimulated with eCV showed a significantly higher metabolic activity 1 h, 24 h, and 48 h after exposure. 24 h after exposure, the metabolic activity was increased in both test groups. No caspase 3/7 activation nor significant differences in the amount of apoptosis/necrosis among the groups were seen. Only in CS-exposed cells ROS formation was increased at 1 h, 3 h, and 6 h after exposition. In conclusion, when compared to conventional CS, a less harmful effect of eCV on HGF can be assumed.

Osseous response on linear and cyclic RGD-peptides immobilized on titanium surfaces in vitro and in vivo.

Biomimetic surface modifications of titanium (Ti) implants using the Arg-Gly-Asp-sequence (RGD) are promising to accelerate bone healing in cases of medical implants. Therefore, we compared the impact of linear and cyclic RGD (l- and c-RGD) covalently coupled onto Ti surfaces on the osseous response in vitro and in vivo. In vitro, osteoblasts' behavior on different surfaces (unmodified, amino-silanized [APTES], l- and c-RGD) was analysed regarding adhesion (fluorescence microscopy), proliferation (resazurin stain) and differentiation (reverse transcription polymerase chain reaction on alkaline phosphatase and osteocalcin). In vivo, osteosynthesis screws (unmodified n = 8, l-RGD n = 8, c-RGD n = 8) were inserted into the proximal tibiae of 12 rabbits and evaluated for bone growth parameters (bone implant contact [%] and vertical bone apposition [VBA;%]) at 3 and 6 weeks. In vitro, c- as well as l-RGD surfaces stimulated osteoblasts' adherence, proliferation and differentiation in a similar manner, with only subtle evidence of superiority of the c-RGD modifications. In vivo, c-RGD-modifications led to a significantly increased VBA after 3 and 6 weeks. Thus, coating with c-RGD appears to play an important role influencing osteoblasts' behaviour in vitro but especially in vivo. These findings can be applied prospectively to implantable biomaterials with hypothetically improved survival and success rates. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 106A: 419-427, 2018.

Tissue engineered pre-vascularized buccal mucosa equivalents utilizing a primary triculture of epithelial cells, endothelial cells and fibroblasts.

Artificial generated buccal mucosa equivalents are a promising approach for the reconstruction of urethral defects. Limiting in this approach is a poor blood vessel supply after transplantation, resulting in increased morbidity and necrosis. We generated a pre-vascularized buccal mucosa equivalent in a tri-culture of primary buccal epithelial cells, fibroblasts and microvascular endothelial cells, using a native collagen membrane as a scaffold. A successful pre-vascularization and dense formation of capillary-like structures at superficial areas was demonstrated. The lumen size of pre-formed blood vessels corresponded to the capillary size in vivo (10-30 µm). Comparing native with a highly cross-linked collagen membrane we found a distinct higher formation of capillary-like structures on the native membrane, apparently caused by higher secretion of angiogenic factors such as PDGF, IL-8 and angiopoietin by the cells. These capillary-like structures became functional blood vessels through anastomosis with the host vasculature after implantation in nude mice. This in vitro method should result in an accelerated blood supply to the biomaterial with cells after transplantation and increase the succes rates of the implant material.

No acute effects of an exposure to 50 ppm methyl methacrylate on the upper airways.

PURPOSE: The German MAK value of methyl methacrylate has been fixed at 50 ppm. The aim of this study was to evaluate possible acute effects of an exposure to 50 ppm methyl methacrylate on the upper airways of human subjects. METHODS: Twenty healthy subjects were exposed to 50 ppm methyl methacrylate and to air (sham) in an exposure chamber for 4 h according to a crossover design. Symptoms were assessed by the SPES questionnaire. Olfactory thresholds for n-butanol and mucociliary transport time were measured before and after exposure. Concentrations of interleukin 1ß and interleukin 8 were determined in nasal secretions taken after exposure. mRNA levels of interleukins 1ß, 6 and 8, tumor necrosis factor α, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1, and cyclooxygenases 1 and 2 were measured in nasal epithelial cells, obtained after exposure. Possible effects were investigated by semiparametric and parametric crossover analyses. RESULTS: The score of the item "irritation to the nose" was slightly elevated following exposure to methyl methacrylate (p ≤ 0.01). Olfactory functioning was not impaired. Mucociliary transport time did not change. Neither concentrations of interleukins in nasal secretions nor mRNA levels were elevated. CONCLUSION: Only minor irritating effects on the nose were observed. The acute exposure to 50 ppm methyl methacrylate did not cause any adverse effects. However, the results cannot be extrapolated to chronic exposure.

17ß-Estradiol reduces nitric oxide production in the Guinea pig cochlea.

Intense noise exposure and the application of ototoxic substances result in increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO). In order to reduce the free NO concentration in the inner ear under pathological conditions, the use of natural cytoprotective substances such as 17ß-estradiol is a promising therapeutic concept. In male guinea pigs the organ of Corti and the lateral wall were isolated from the cochlea and afterwards incubated for 6 h in cell-culture medium. 17ß-Estradiol was adjusted in 2 concentrations to organ cultures of the right ears (12 animals per concentration). The left ears were used as controls. The NO production was quantified in the supernatant by chemiluminescence after incubation. Depending on the concentration, 17ß-estradiol reduced NO in the organ of Corti by 43% (p=0.015) and 46% (p=0.026), respectively. In the lateral wall, the NO concentration was reduced by 24%, but without statistical significance (p=0.86). However, when analyzing the association between the 2 cochlear regions for each animal separately, the NO concentrations were lower in nearly all 17ß-estradiol-treated ears compared to controls. In order to demonstrate the flexibility of the organ culture system, the NO donor DETA NONOate and the nitric oxide synthase inhibitors L-NAME and L-NMMA were applied. The electron microscopic analysis revealed a well-preserved cochlear cell morphology after incubation. The ability of 17ß-estradiol to influence the NO production preferentially in the organ of Corti might offer new therapeutic perspectives for inner ear protection.

Acute effects of an exposure to 100 ppm 1-methoxypropanol-2 on the upper airways of human subjects.

The German MAK value of 1-methoxypropanol-2 has been fixed at 100 ppm. The aim of this study was to evaluate possible acute effects of an exposure to 100 ppm 1-methoxypropanol-2 on the upper airways of human subjects. Twenty subjects were exposed in a crossover design to 100 ppm 1-methoxypropanol-2 and to air in an exposure chamber for 4h. Subjective symptoms were assessed by questionnaire. Olfactory thresholds for n-butanol and mucociliary transport time were measured before and after exposure. Concentrations of interleukin 1ß and interleukin 8 were determined in nasal secretions taken after exposure. mRNA levels of interleukins 1ß, 6 and 8, tumor necrosis factor α, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1, and cyclooxygenases 1 and 2 were measured in nasal epithelial cells, obtained after exposure. Possible effects were investigated by semiparametric and parametric cross-over analyses. Subjects did not have any subjective irritating symptoms. The olfactory threshold was slightly elevated following exposure to 1-methoxypropanol-2. Mucociliary transport time did not change. Neither concentrations of interleukins in nasal secretions nor mRNA levels except for interleukin 1ß were higher after exposure to 1-methoxypropanol-2. In conclusion, the acute exposure to 100 ppm 1-methoxypropanol-2 did not cause clear-cut adverse effects in test subjects.

Prospective, blinded comparison of cytology and DNA-image cytometry of brush biopsies for early detection of oral malignancy.

OBJECTIVES: Adjunctive techniques like DNA image cytometry (DNA-ICM) have been attributed to enhance the diagnostic performance of oral brush biopsies. The aim of the study was an evaluation of brush biopsies, analysed according to morphological criteria and by DNA-ICM vs. histological findings in a blinded prospective trial. MATERIALS AND METHODS: Eighty eight brush biopsies of 70 patients were sampled. Only clinical suspicious but not evident malignant oral lesions were included. Clinical diagnosis was leukoplakia (n = 36), lichen planus (n = 18), verruciform erythroplakia (n = 12), erythroleukoplakia (n = 9), erosion (n = 7) and induration (n = 6). Evaluation was conducted via histology, cytology and DNA-ICM. RESULTS: Histological diagnosis revealed eight cases of squamous intraepithelial dysplasia (SIN 1 n = 6, SIN 2 n = 2), four cases of carcinoma-in situ and 25 cases of oral T1-cancer. Remaining cases were leukoplakia (n = 28), lichen planus (n = 15) and local inflammation (n = 8). Brush biopsy detected malignant lesions including SIN>1 with a sensitivity of 55% and a specificity of 100%. DNA-ICM had a sensitivity of 70% and a specificity of 100%. The combination of both methods showed a sensitivity of 76% and a specificity of 100%. The predominant reason for false negative results were sampling errors with insufficient cells (86% in brush biopsy and 100% in DNA-ICM). CONCLUSION: DNA-ICM has the potential to substantially improve the sensitivity of a pure morphological interpretation of oral brush biopsies. Method inherent sampling errors may be accountable for a lower sensitivity compared to conventional histological diagnosis. Therefore, DNA-ICM should not be used to rule out malignancy, when lesions are already clinically suspicious for oral cancer.

Associations between single-nucleotide polymorphisms of the VEGF gene and long-term prognosis of oral squamous cell carcinoma.

INTRODUCTION: Functional polymorphisms (SNPs) of the vascular endothelial growth factor (VEGF) are associated with the incidence of oral squamous cell carcinoma (OSCC). An impact of VEGF-SNPs on prognosis of OSCC patients seems possible. Therefore, correlations between prognostic parameters of OSCC patients and five VEGF-SNPs were determined. MATERIALS AND METHODS: In a retrospective long-term study, in 113 OSCC patients that underwent curative resections, five VEGF-SNPs (-1154 G/A, +405 G/C, +936 C/T, -2578 C/A, and -460 C/T) were analyzed. Associations between SNPs and prognosis (incidence of local recurrent disease, second cancer, metastases, death, total disease-free survival) were examined. RESULTS: After a mean follow-up time of 57.6 months, 32 patients had local recurrences; 15 patients had second cancer, 15 patients metastases, and 23 patients died. The mean disease-free survival was 43.1 months. A significant increased incidence of OSCC in smokers with the VEGF -2578 A/C and -460 C/T SNP was seen (each P < 0.0001). In univariate analysis, patients with advanced OSCCs (T > 2 or N > 0) together with the -1154 A/A allele had a significant worse survival and a worse disease-free survival (both P < 0.04). The same was seen for the +405 G/G SNP (both P = 0.002). In multivariate analysis, only the negative influence of the +405 G/G SNP on survival in advanced OSCCs (T > 2) could be confirmed (P = 0.002). DISCUSSION: Possible reciprocal interactions between smoking and VEGF-SNP function were observed. Multivariate analysis confirmed the VEGF +405 G/G genotype to be associated with poor survival in advanced OSCCs; a further use of this haplotype as biomarker has to be discussed.

Lack of neurofibromatosis type 2 gene promoter methylation in sporadic vestibular schwannomas.

BACKGROUND: Vestibular schwannomas (VS) are benign tumors of the nervous system that are usually sporadic but also occur in the inherited disorder neurofibromatosis type 2 (NF2). In VS, losses of chromosomal material and mutations of the NF2 gene have been established to be causative. For a subset of VS without detectable gene alterations, promoter inactivation by hypermethylation has been suggested. However, published data are very limited and contradictory. METHODS: We analyzed NF2 gene promoter methylation in 35 sporadic VS by methylation-specific PCR. RESULTS: Twenty-three of the tumors were informative, showing no promoter methylation. In the remaining 12 tumors, promoter methylation could neither be verified nor excluded. CONCLUSIONS: Our study suggests that NF2 gene inactivation by promoter hypermethylation is a rare or very uncommon mechanism of NF2 gene inactivation in sporadic VS. Other mechanisms destabilizing the NF2 gene product, yet to be identified, might play a role in the genesis of VS apart from the loss or mutation of the NF2 gene.

The VEGF/VEGF-R axis in sporadic vestibular schwannomas correlates with irradiation and disease recurrence.

BACKGROUND/AIMS: The molecular mechanisms downstream of mutated neurofibromatosis type 2 (NF2) gene resulting in the growth and development of vestibular schwannoma (VS) are controversial. Several lines of evidence suggest the involvement of the vascular endothelial growth factor (VEGF) pathway in VS development. Given that recent studies of VEGF blockade in patients with NF2-associated VS showed positive effects on VS growth control, we initiated this comprehensive study of the VEGF pathway in sporadic VS. METHODS: A tissue microarray analysis of 182 sporadic VS was conducted. The expression of VEGF and its receptors as well as the proliferative activity of the tumors were quantified. The expression data were correlated to tumor volumes and diameters as well as to tumor recurrence and previous irradiation. RESULTS: All studied tumors expressed VEGF and its receptors. Proliferative activity was related to the growth characteristics of the tumors. Moreover, we found significantly higher VEGF levels in recurrent tumors (p = 0.0387) and in preoperatively irradiated tumors (p = 0.0213). CONCLUSION: Our data suggest a relevant role of the VEGF pathway in VS growth and therapy outcome. Therefore, targeting this pathway using antiangiogenic compounds might be beneficial for patients with sporadic VS, especially those with recurrent or irradiated tumors.

Trifunctional antibodies induce efficient antitumour activity with immune cells from head and neck squamous cell carcinoma patients after radio-chemotherapy treatment.

BACKGROUND Trifunctional antibodies, such as catumaxomab (anti-EpCAM×anti-CD3) and ertumaxomab (anti- HER-2/neu×anti-CD3), transiently link immune effector cells to tumour cells, which results in cellular cytotoxicity towards the tumour cells. A functional immune system is therefore essential for effective anti-tumour activity. However, the commonly observed haematotoxicity of chemotherapeutics and radiation therapy may be associated with some degree of immunosuppression. Combining chemotherapy and trifunctional antibodies in cancer treatment requires understanding of the impact of chemotherapeutics on immune cell function and, thus, on the activity of trifunctional antibodies. METHODS The effect of chemotherapeutic treatment on trifunctional antibody-mediated anti-tumour activity was assessed in vitro. Blood samples were collected from 12 head and neck squamous cell carcinoma patients after chemotherapy (5-fluorouracil, cisplatin) and radiotherapy, and from one healthy control donor. The immune cell status was analysed and mononuclear cells (MNC) were isolated. The potency of catumaxomab and ertumaxomab was assessed in a cytotoxicity assay using MNC isolated from each patient sample in co-culture with a tumour target cell line. The release of infl ammatory cytokines was also monitored in the cell culture supernatant. RESULTS Most patients included in this study had decreased immune cell counts during the course of chemotherapy. Nonetheless, an effective and concentration-dependent anti- tumour activity mediated by trifunctional antibodies was demonstrated using these patient immune effector cells. The immune response activity of the patient immune cells was not impaired one week after cisplatin administration or even three days after the last 5-fluorouracil treatment. CONCLUSION This study shows for the first time that immune effector cells from cancer patients undergoing standard chemotherapy and radiotherapy can be activated by trifunctional antibodies for efficient killing of tumour cells.

[Possible molecular mechanisms of spontaneous remission in sudden idiopathic hearing loss]. / Mögliche molekulare Mechanismen einer Spontanremission nach Hörsturz.

According to current knowledge, it must be assumed that temporary idiopathic hearing loss and its spontaneous remission are based on mechanical and/or pathological alterations in the inner ear. The causal mechanisms might be based on inter-individual variations. Induced by dose-dependent activators, temporary as well as permanent damage might occur. Sudden hearing loss may be initiated by an increase in the local nitric oxide (NO) concentration. Spontaneous remission, i.e. functional restoration, can be explained by a local decrease in the NO concentration. In this context, regulatory systems such as the gap-junction system, blood vessels or synapses might be affected. In addition, alterations in the hormone level of estrogen and mineralocorticoids, as well as cellular glutathione and vitamin levels, might lead to temporary alterations in the inner ear. Recent experimental findings indicate a role for the shuttle protein Survivin in the spontaneous remission of sudden hearing loss.

No evidence for a correlation of glutathione S-tranferase polymorphisms and chronic rhinosinusitis.

OBJECTIVE: Cellular detoxification mechanisms are mandatory for cellular protection against oxidative stress and reactive oxygen species. One major group of antioxidative active enzymes involved in cellular detoxification are the Glutathione S-Transferases (GST). Multiple subtypes like GSTM1, GSTP1, and GSTT1 and variants of them are known, arising from allelic variations of the GST loci. Moreover, functional variants occur in high percentages and have been associated with diseases like bronchial asthma and bronchial hyperresponsiveness. The interplay of oxidative stress, detoxifying genes like GSTs and the genesis of respiratory tract illness is under contradictory debate. In this study, we analysed the potential association of GST-polymorphisms and chronic rhinosinusitis (CRS). METHODS: In total 170 nasal tissue samples, 49 tissue samples from patients with CRS without nasal polyps, 69 tissue samples from CRS with nasal polyps and 52 healthy tissue controls of the inferior turbinate were analysed for their individual GST-status. Genotypes for GSTM1 (null versus present), GSTT1 (null versus present), and GSTP1 (Ile105Val) were determined by Polymerase Chain Reaction. The respective genotypes were correlated to the incidence of CRS with and without nasal polyps in aspirin-tolerant and intolerant patients and to the individual health status concerning asthma and allergies. RESULTS: No correlation between any GST-polymorphism and CRS with and without nasal polyps or allergies or asthma or aspirin-intolerance was observed. CONCLUSION: Our results do not suggest that there is a relevant genetic predisposition considering the individual GST-status for the susceptibility of nasal respiratory epithelia leading to CRS.

Immobilisation of linear and cyclic RGD-peptides on titanium surfaces and their impact on endothelial cell adhesion and proliferation.

Functional coatings on titanium vascular stents and endosseous dental implants could probably enhance endothelial cell (EC) adhesion and activity with a shortening of the wound healing time and an increase of peri-implant angiogenesis during early bone formation. Therefore, the role of the structure of linear and cyclic cell adhesive peptides Arg-Gly-Asp (l-RGD and c-RGD) on differently pre-treated titanium (Ti) surfaces (untreated, silanised vs. functionalised with l- and c-RGD peptides) on EC cell coverage and proliferation was evaluated. After 24 h and after 3 d, surface coverage of adherent cells was quantified and an alamarBlue® proliferation assay was conducted. After 24 h, l-RGD modified surfaces showed a significantly better coverage of adhered cells than untreated titanium (p=0.01). Differences between l-RGD surfaces and silanised Ti (p=0.066) as well as between l-RGD and c-RGD surfaces (p=0.191) were not significant. After 3 d, c-RGD surfaces showed a significantly higher cell coverage than untreated Ti, silanised and l-RGD titanium surfaces (all p<0.0001). After 24 h, c-RGD modified surfaces showed significant higher cell proliferation compared to untreated Ti (p=0.003). However, there were no differences in proliferation between c-RGD and l-RGD (p=0.126) or c-RGD and silanised titanium (p=0.196). After 3 d, proliferation on c-RGD surfaces outranged significantly untreated titanium (p=0.004), silanised (p=0.001) and l-RGD surfaces (p=0.023), whereas no significant difference could be found between untreated Ti and l-RGD surfaces (p=0.54). According to these results, the biomimetic coating of c-RGD peptides on conventional titanium surfaces showed a positive effect on EC cell coverage and proliferation. We were able to show that modifications of titanium surfaces with c-RGD are a promising approach in promoting endothelial cell growth.

Genomic changes in salivary gland pleomorphic adenomas detected by comparative genomic hybridization.

The aim of this study was to investigate whether so far unknown chromosomal alterations in pleomorphic adenoma (PA) exist. To this end, tissue samples from 18 patients with parotid gland PA were studied by comparative genomic hybridization (CGH) using Phi-29-DNA-polymerase for DNA amplification. The most common aberrations were losses of chromosomal material of 19p (6/18), 9q, 16p, and 19q (in 3 out of 18 patients each). Additional losses were observed on 4p, 5q, and 17q (2 / 18 each). Gains involved chromosomes 2p, 4p, 6p, 17q, and 21q (2 / 18 each). Losses of 19p have been associated with inactivation of tumor-suppressor genes in carcinomas previously. As a result, pleomorphic adenomas show a considerable diversity of chromosomal gains and losses detected by CGH. The 19p arm, and particularly its 19p13 region, need be further studied to elucidate the potential impact of associated lost tumor suppressor genes on PA development.

Influence of class M1 glutathione S-transferase (GST Mu) polymorphism on GST M1 gene expression level and tumor size in oral squamous cell carcinoma.

Glutathione S-transferases (GST) are antioxidant enzymes and oxidative stress markers in oral carcinogenesis. They present a system of polymorphic proteins. Some variants are associated with increased sensitivity to toxic compounds, as it is known for the GSTM1-null variant allele. However, the influence of the GSTM1 allele variant genotype on GSTM1-mRNA quantity in oral squamous cell carcinoma (OSCC) and normal mucosa as well as the impact on prognosis remains unclear. The genotype for GSTM1 (mutation vs. wild type) was determined by polymerase chain reaction (PCR) using genomic DNA extracted from peripheral blood from 28 OSCC patients. From the same patients, 28 pairs of OSCC cells and normal oral mucosal cells were obtained by brush biopsy. mRNA was extracted from these paired samples and the expression levels of GSTM1 were examined by real-time reverse transcriptase qPCR (RT-qPCR). The mRNA expression of the OSCC samples was normalized against an external standard, as well as to the corresponding normal mucosa. The coincidence of GSTM1 genotype and GSTM1-mRNA-expression level was examined. In 15 patients (54%), the null genotype GSTM1 was present. In the GSTM1-null allele group, the GSTM1 gene expression level was determined at 1.63 (mean: 3.08; SD 3.4) folds vs. 3.6 (mean: 10.5; SD 14.2) folds in the group with the positive genotype (p=0.06), if calibrated vs. individual normal mucosa. More T3 and T4 OSCCs (+38%), higher UICC stadia (+38%) and more lymphatic metastasis (+28%) were seen in the group with the negative allele. Furthermore, positive GSTM1 genotype and enhanced GSTM1 gene expression was accompanied with increased tumor size, lymphatic metastasis status and UICC stadium. A coincidence of null type GSTM1 and lowered GSTM1 gene expression was observed. The larger tumors and more frequent lymph node metastases in this group could be explained by the insufficient cell protection by GST.

[Protection of the cochlea by ascorbic acid in noise trauma]. / Protektion der Kochlea durch Vitamin C bei Lärmexposition.

BACKGROUND: The protective effect of ascorbic acid against noise-induced hearing loss and increased nitric oxide (NO) formation after noise exposure have already been demonstrated in animal models. However, the influence of ascorbic acid on noise-induced NO production within the cochlea is still unclear. METHODS: Guinea pigs (n=48) were fed for 7 days with low [25 mg/kg bodyweight (bw)/day] and high (525 mg/kg bw/day) doses of ascorbic acid. Then half of the animals were exposed to noise (90 dB for 1 h). The hearing levels were recorded beforehand, on the 3rd and 7th days after feeding, and directly after noise exposure. Finally, the organ of Corti and the lateral wall were removed from the inner ear and incubated separately for 6 h in culture medium, and the nitrite content was determined in the supernatant. RESULTS: Compared with low-dose feeding, feeding of high doses of ascorbic acid resulted in a reduction of hearing impairment of about 8 dB after noise exposure. A correlation between hearing improvement and decreased NO production was detectable for both cochlea regions but was more pronounced in the lateral wall. CONCLUSION: A high dose of ascorbic acid lowers NO production in the inner ear, reduces hearing loss, and protects the cochlea from nitroactive stress.

No acute effects of an exposure to 50 ppm acetaldehyde on the upper airways.

OBJECTIVE: German MAK value of acetaldehyde has been fixed at 50 ppm to prevent from irritating effects. The threshold value is mainly based on animal experiments. The aim of this study was to evaluate acute effects of an exposure to 50 ppm acetaldehyde on the upper airways of human subjects. METHODS: Twenty subjects were exposed to 50 ppm acetaldehyde and to air in an exposure chamber for 4 h according to a crossover design. Subjective symptoms were assessed by questionnaire. Olfactory threshold for n-butanol and mucociliary transport time were measured before and after exposure. Concentrations of interleukin 1beta and interleukin 8 were determined in nasal secretions taken after exposure. mRNA levels of interleukins 1beta, 6 and 8, tumour necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1, and cyclooxygenases 1 and 2 were measured in nasal epithelial cells, gained after exposure. Possible effects were investigated by semiparametric and parametric crossover analyses. RESULTS: Exposure to acetaldehyde did not cause any subjective irritating symptoms. Olfactory threshold did not change. Mucociliary transport time increased insignificantly after exposure to acetaldehyde. Neither concentrations of interleukins in nasal secretions nor mRNA levels of inflammatory factors were higher after exposure to acetaldehyde. CONCLUSION: An acute exposure to 50 ppm acetaldehyde did not cause any adverse effects in test subjects.

Proangiogenic effects of ionizing irradiation on squamous cell carcinoma of the hypopharynx.

OBJECTIVE: There is experimental evidence that ionizing irradiation affects a proangiogenic response. However, the relevance of this effect on tumour growth in vivo is not in detail investigated yet. The present objectives were to examine the influence of ionizing radiation on the expression of the vascular endothelial growth factor (VEGF) and its receptors (flt-1 and flk-1), the microvessel density and the tumour proliferation, in head and neck squamous cell carcinoma (HNSCC). METHODS: We used a HNSCC-cell line, derived from a hypopharyngeal tumour, for subcutaneous injection in 16 athymic nude mice. After reaching an average diameter of 12-14 mm the xenografts were randomised and 8 out of the 16 animals (therapy group) were irradiated with a single fraction of 6 Gy while the control group remained without any intervention. The irradiated and the respective control tumours were prepared after 7 (T7) and 70 days (T70) for immunohistochemical analysis. The expression of VEGF, its receptors flk-1 and flt-1, the vessel density (CD31) and the proliferation rate (Ki67) were quantified. RESULTS: At the point of time T7 we observed a reduction of the tumour growth rate, of the proliferative activity and of the VEGF- as well as of the VEGF-R-expression. At the point of time T70 we found increased values for proliferation, microvessel density, VEGF- and flk-1 expression in the therapy group compared to the therapy group at T7 as well as to the control group at T70. CONCLUSION: These changes might suggest a long-term proangiogenic effect of irradiation, which might result in growth promotion of the remaining tumour after the end of therapy.

Image cytometric DNA analysis of mucosal biopsies in patients with primary achalasia.

AIM: To determine DNA aneuploidy in mucosal biopsies of achalasia patients for subsequent rapid diagnosis. METHODS: Biopsies from the middle third of the esophagus were obtained in 15 patients with achalasia. Immunohistochemical staining was carried out with monoclonal antibodies MIB-1 for Ki67 and PAb 1801 for p53, in addition to the conventional histologic examination for dysplasia. Nuclei of fresh biopsy material were enzymatically and mechanically isolated, and the DNA content was determined with image cytometry after Feulgen staining. DNA grading of malignancy was assessed according to Boecking to determine the variability of DNA values noted around the normal diploid peak. Further indices measured included the aneuploid rate, and the 5c-, 7c- and 9c-exceeding rate. RESULTS: The histological examination did not demonstrate dysplasia; while MIB-1 (basal) showed a positive reaction in 8/15 achalasia specimens, p53 was negative in all specimens. Image cytometric DNA analysis detected aneuploidy in 4/15 (26.7%) specimens. Samples from 15 patients with squamous cell carcinoma as well as specimens obtained exclusively 2 cm proximal to the tumor served as reference tests. All carcinomas (15/15) as well as 9 of the peritumoral samples (9/15) were aneuploid. The comparison of biopsies from achalasia patients with peritumoral and carcinoma specimens revealed statistically significant differences regarding the aneuploid rate (diploid: P < 0.0001; tetraploid: P = 0.001), grading of malignancy according to Boecking (P < 0.0001) and the 5c- (P < 0.0001), 7c- (P < 0.0001), and 9c- (P = 0.0001) exceeding rate with progredient DNA alterations in the respective order. CONCLUSION: The finding that DNA aneuploidy was identified by image cytometry in esophageal specimens of patients with achalasia, which may be due to specific chromosomal alterations presenting as precancerous lesions in 27% of patients, leads us to conclude that image cytometry represents a valuable screening tool.