Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse.

PURPOSE: Radiation therapy cures malignant tumors of the head and neck region more effectively when it is combined with application of the anti-EGFR monoclonal antibody cetuximab. Despite the successes achieved, we still do not know how to select patients who will respond to this combination of anti-EGFR monoclonal antibody and radiation. This study was conducted to elucidate possible mechanisms which cause the combined treatment with cetuximab and irradiation to fail in some cases of squamous cell carcinomas. METHODS AND MATERIALS: Mice bearing FaDu and A431 squamous cell carcinoma xenograft tumors were treated with cetuximab (total dose 3 mg, intraperitoneally), irradiation (10 Gy) or their combination at the same doses. Treatment was applied when tumors reached 8mm in size. To collect samples for further protein analysis (two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry MALDI-TOF/TOF, Western blot analysis, and ELISA), mice from each group were sacrificed on the 8th day after the first injection of cetuximab. Other mice were subjected to tumor growth delay assay. RESULTS: In FaDu xenografts, treatment with cetuximab alone was nearly as effective as cetuximab combined with ionizing radiation, whereas A431 tumors responded to the combined treatment with significantly enhanced delay in tumor growth. Tumors extracted from the untreated FaDu and A431 xenografts were analysed for protein expression, and 34 proteins that were differently expressed in the two tumor types were identified. The majority of these proteins are closely related to intratumoral angiogenesis, cell adhesion, motility, differentiation, epithelial-to-mesenchymal transition (EMT), c-myc signaling and DNA repair. CONCLUSIONS: The failure of cetuximab to enhance radiation response in FaDu xenografts was associated with the initiation of the program of EMT and with c-myc up-regulation in the carcinoma cells. For this reason, c-myc and EMT-related proteins (E-cadherin, vimentin) may be considered as potential biomarkers to predict squamous cell carcinoma response after treatment with cetuximab in combination with radiation.

Balance ability and muscle response of the preferred and nonpreferred leg in soccer players.

The aim of this study was to evaluate balance abilities and electromyographic (EMG) latency times of the preferred and nonpreferred leg in soccer players. Whereas side differences between the two legs in force, kicking speed, and joint laxity have been demonstrated in athletes in previous studies, no data are so far available on balance differences. Low balance ability is generally associated with an increased risk of ligament injuries, and the detection of a possible asymmetry in balance is important because a bilateral difference may be a contributing factor to injury. Twenty-one amateur soccer players were tested. Two different balance test instruments were used: the Biodex Stability System and the Tetrax System. For the evaluation of muscle latency times, EMGs were recorded by means of the EquiTest system. None of the tests performed in this study revealed statistically significant differences in balance ability between the preferred and the nonpreferred leg. The investigations of balance function and muscle response in amateur soccer players did not reveal significant differences between the preferred and nonpreferred leg in the current study. However, a certain tendency to better balance in the nonpreferred leg was observed.

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells.

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

Antimicrobial activity of dexamethasone and its combination with N-chlorotaurine.

OBJECTIVE: To investigate the antimicrobial effect of dexamethasone phosphate, the endogenous antiseptic N-chlorotaurine (NCT), and their combination on ear, nose, and throat microorganisms. DESIGN: In vitro study. SUBJECTS: Strains of Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus milleri, Aspergillus flavus, and Aspergillus fumigatus. INTERVENTIONS: Bacterial and fungal strains were cultured with 0.1% dexamethasone with and without a low (0.1%) or high (1%) concentration of NCT. The killing effects of dexamethasone, NCT, and the combination were monitored. RESULTS: Dexamethasone killed S. milleri and A. flavus after incubation times of 24 to 48 hours. The low concentration of NCT caused a 90% reduction of S. aureus and P. aeruginosa within 30 minutes and 99.9% reduction within 50 minutes. The high concentration of NCT reduced viable counts of S. aureus and P. aeruginosa to the detection limit within 10 minutes. The low-concentration combination (0.1% dexamethasone and 0.1% NCT) showed significant (P < .01) synergistic killing of S. aureus with 2- to 3-fold shorter killing times. The high-concentration combination (0.1% dexamethasone and 1% NCT) demonstrated more rapid killing than NCT alone in both S. aureus and P. aeruginosa. CONCLUSIONS: With short and intermediate exposure times, the combination of dexamethasone and NCT showed significantly stronger antimicrobial effects than treatment with NCT alone. Significant killing of S. milleri, A. flavus, and A. fumigatus was observed after extended exposure to dexamethasone. The combined application of dexamethasone and NCT might be a promising therapeutic option, producing high efficacy with low side effects.

Antibacterial activity of mometasone furoate.

OBJECTIVE: To test the antibacterial properties of the topical corticoid mometasone furoate, which is used as a nasal spray. DESIGN: The activity of mometasone (0.01%, 0.1%, and 0.5%) in buffer solution against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pyogenes, and Streptococcus milleri was tested by quantitative killing assays. SETTING: In vitro study. MAIN OUTCOME MEASURE: Reduction of viable bacteria and fungi in quantitative killing assays. RESULTS: Mometasone (0.5%) reduced viable counts of S pyogenes and S milleri by 99.99% and 99.00%, respectively, after 24 hours of incubation, whereas colony-forming units (CFUs) of S aureus, P aeruginosa, and E coli were not affected by the corticoid. Mometasone (0.1%) caused a decrease in CFUs of S pyogenes of 99.90% to 99.99%, while it led to a 99.00% reduction in CFUs of S milleri, but only if low bacterial counts of 1 x 10(4) CFUs/mL were incubated. By contrast, the use of mometasone at a low concentration (0.01%) demonstrated an increase in CFUs of S milleri if the baseline bacterial count was low (1 x 10(4) CFUs/mL). CONCLUSION: Mometasone demonstrates antimicrobial activity against streptococci.

Influence of essential and fatty oils on ciliary beat frequency of human nasal epithelial cells.

BACKGROUND: In alternative and complementary medicine, the use of essential and fatty oils has become more and more popular. In addition to conventional medical therapies, self-medication is showing increasing popularity, using agents with unclear compounds and poorly controlled dosages. Among other disorders, these alternative treatments are used in bronchitis and rhinitis, including some topical applications. Thus, the influence on ciliated epithelia should be evaluated, because a disturbance of the ciliary function can lead to recurrent sinusitis and chronic rhinosinusitis. The aim of this study was to test the influence of fatty and essential oils on the ciliary beat frequency (CBF) of nasal mucosa in vivo. METHODS: The influence of sesame oil, soy oil, peanut oil, Miglyol 840, thyme oil, lavender oil, eucalyptus oil, and menthol on the ciliary activity of nasal brushings was evaluated by digital high-speed imaging. RESULTS: The presence of most fatty oils resulted in an increase in CBF, the effect being highest for peanut oil. Miglyol 840 had no significant influence on CBF. The essential oils were tested at a concentration of 0.2 and 2%. Thyme oil did not affect CBF, whereas the presence of all other essentials oils resulted in an increase in CBF; the effect was higher at 0.2% than at 2%. CONCLUSION: Except thyme oil and Miglyol 840, all tested oils caused an increase in CBF. Interestingly, the 0.2% concentrations of essential oils resulted in stronger effects when compared with the 2% concentrations.

Antitumor activity of CTFB, a novel anticancer agent, is associated with the down-regulation of nuclear factor-kappaB expression and proteasome activation in head and neck squamous carcinoma cell lines.

This study aimed to characterize the antitumor activity of 5-Chloro-N-[2-[2-(4-chloro-phenyl)-3-methyl-butoxy]-5-trifluoromethyl-phenyl]-2-hydroxy-benzamide (CTFB), a novel anticancer agent, in head and neck cancer cell lines, FaDu, SCC-25 and cisplatin-resistant CAL-27. CTFB was generated as a result of an extensive medicinal chemistry effort on a lead compound series discovered in a high-throughput screen for inducers of apoptosis. All cell lines showed significant growth delay in response to CTFB treatment at a concentration of 1 micromol/L with 17.16 +/- 2.08%, 10.92 +/- 1.22%, and 27.03 +/- 1.86% of cells surviving at 120 h in FaDu, CAL-27, and SCC-25, respectively. To define proteins involved in the mechanism of action of CTFB, we determined differences in the proteome profile of cell lines before and after treatment with CTFB using two-dimensional difference gel electrophoresis followed by computational image analysis and mass spectrometry. Eight proteins were found to be regulated by CTFB in all cell lines. All these proteins are involved in cytoskeleton formation and function and/or in cell cycle regulation. We showed that CTFB-induced cell growth delay was accompanied by cell cycle arrest at the G(0)-G(1) phase that was associated with the up-regulation of p21/WAF1 and p27/Kip1 expression and the down-regulation of cyclin D1. Furthermore, we showed that activity of CTFB depended on the down-regulation of nuclear factor-kappaB (NF-kappaB) and NF-kappaB p65 phosphorylated at Ser(536). The level of proteasome activity correlated with the response to CTFB treatment, and the down-regulation of NF-kappaB is accompanied by enhanced proteasome activity in all investigated head and neck cancer cell lines. In this report, we show that CTFB reveals multiple effects that lead to delayed cell growth. Our data suggest that this compound should be studied further in the treatment of head and neck cancer.

N-chlorotaurine--a new safe substance for postoperative ear care.

OBJECTIVES: The new endogenous substance N-chlorotaurine (NCT), which for the first time was synthesized and introduced into clinical practice by our research group, was now used for postoperative ear care following tympanoplasty. The antiseptic and drying effect of NCT was evaluated and compared to the standard postoperative procedure in our department. METHODS: Prospective randomized trial on 10 patients. Local irrigations of the external auditory canal with 1% NCT solution were performed once daily until the canal was dry. RESULTS: NCT was well tolerated. The external auditory canal showed no signs of infections and dried significantly more rapidly than the control group. In contrast to previous suppositions, epithelialization proceeded without complications and was identical in both groups. CONCLUSIONS: As NCT in our first postoperative patient study was effective in preventing infections and drying of the outer ear canal was faster without impairing epithelialization, this agent seems useful for postoperative ear care.

A novel N-chlorotaurine-corticosteroid combination as a preservative-free local disinfectant: influence on the ciliary beat frequency in vitro.

CONCLUSIONS: The combination of N-chlorotaurine (NCT) and a corticosteroid seems to be a very promising substance for the local therapy of ENT infections. As it can be used without any preservatives, the effect on the ciliary beat frequency (CBF) is much less than that of products containing benzalkonium chloride (BAC). The in vitro results obtained in this study encourage us to perform clinical trials on this novel combination for intranasal application. OBJECTIVE: To investigate the influence of a novel mixture of NCT and a corticosteroid [fluticasone propionate (FP)] on the CBF of human ciliated cells in vitro. MATERIAL AND METHODS: The study was designed as an in vitro study. CBF was measured by means of a photometric technique involving the combination of a light microscope, a photometer, a photographic multiplier and a computerized analyzing unit. RESULTS: The combination of 1% NCT + 0.5 mg/ml FP decreased the CBF to 42.17% of its original value after 20 min. Treatment with BAC lowered the CBF depending on the concentration to 96.61% of its original value with 0.04 mg/ml, to 91.90% with 0.1 mg/ml, to 63.46% with 0.2 mg/ml and to 0% with 0.5 mg/ml. After rinsing in saline, the CBF of samples treated with 1% NCT + 0.5 mg/ml FP recovered to 68.93% of its original value.

Protein sites of attack of N-chlorotaurine in Escherichia coli.

N-Chlorotaurine sodium (NCT) is a promising microbicidal agent for topical treatment of infections. Its targets of attack in Escherichia coli have been investigated by proteomics. Incubation in 1% NCT for 10 and 30 min revealed a change of the charge and a separation of numerous proteins into a series of spots with a different pI. Charge differences could be related to oxidation of cysteine residues to their corresponding sulfonic acids. Heat shock protein 60 appeared, while ribosome-releasing factor, d-ribose periplasmic binding protein, and malonyl-CoA transacylase spots decreased. These results indicate penetration of oxidation capacity into the bacteria and destruction of essential proteins by NCT.

H. pylori infection increases levels of exhaled nitrate.

BACKGROUND: Helicobacter pylori infection is one of the most common chronic bacterial infections worldwide. Despite the existence of a breath test for the diagnosis of H. pylori infection, no study has described the composition of volatile compounds, especially the levels of nitrate, in the exhaled air of patients with H. pylori infection. MATERIALS AND METHODS: The volatile compounds in the exhaled air of 14 patients suffering from H. pylori gastritis and 11 controls were analyzed using proton transfer reaction-mass spectrometry. Gastric biopsy was used to establish diagnosis of current H. pylori infection. RESULTS: Comparing mass spectra between groups, Mass 28 (hydrogen cyanide, HCN) and Mass 64 (hydrogen nitrate, H2NO3) were found to be significantly elevated in patients with H. pylori infection. CONCLUSIONS: The main result of the present study is that in H. pylori-infected patients, levels of exhaled hydrogen nitrate and hydrogen cyanide are found to be significantly elevated. However, further studies are necessary to find out whether the differences in the detected mass spectrum are specific enough to differentiate patients with H. pylori gastritis from healthy controls.

Tolerability of N-chlorotaurine in chronic rhinosinusitis applied via yamik catheter.

OBJECTIVES: A rational approach in the treatment of chronic rhinosinusitis (CRS) is the intranasal application of antiseptic agents, due to the pathogenetic role of bacteria and fungi. N-Chlorotaurine (NCT), a mild endogenous oxidant with broad-spectrum antimicrobial activity, has been tested for the first time in CRS. METHODS: This one-arm phase IIa clinical study is the first step in the clinical development of this promising substance for local therapy of CRS. The nasal and paranasal cavities of 12 patients were rinsed with 10-20 ml of 1% aqueous NCT solution, applied via a novel catheter system (YAMIK). Treatment consisted of three lavages per week for 4 weeks. RESULTS: NCT caused neither alterations of the mucosa nor burning pain during application. Nevertheless, the insertion of the catheter, the insufflation of the posterior cuff and the overpressure inside the sinuses after infiltration led to moderate pain in some patients. Mucosal swelling decreased in all subjects, nasal breathing could be improved in nine patients and impaired olfaction in seven. Polyps did not disappear within the 1-month period of the study. CONCLUSIONS: The good tolerability and possible beneficial effects of NCT encourage its further investigation in CRS. Despite some limitations the YAMIK catheter proved to be a convenient and safe device for rinsing the nasal and paranasal sinuses.

Solitary subglottic neurofibroma: a report of an unusual manifestation.

Neurofibromas of the larynx are extremely rare, especially in the subglottic part. Most of the patients with neurofibromas suffer from neurofibromatosis type 1 (NF1, von Recklinghausen's disease), which is characterized by cafe-au-lait spots and neurofibromas of any type. We present a case of a solitary neurofibroma in the subglottic region that relapsed 4 years after the primary diagnosis. The clinical findings, histological results and therapy are discussed.

Influence of amphotericin B on the ciliary beat frequency of nasal mucosa.

OBJECTIVE: A new therapeutic approach in eosinophilic fungal rhinosinusitis is topical application of antifungals such as amphotericin B. The tolerability of a recently applied concentration of this drug by human nasal mucosa was tested in vitro. METHODS: Ciliary beat frequency (CBF) was measured by a photometric technique, combining a light microscope, a photometer, a photograph-multiplier, and a computed analyzing unit. RESULTS: Treatment for 20 minutes with amphotericin B finally diluted in saline to 0.1 mg/mL caused no decrease in CBF. By contrast, both amphotericin B diluted in distilled water and distilled water without additives lowered CBF about 50%. This decrease was not reversible after reincubation for 20 minute in saline. CONCLUSIONS: The results confirm the good tolerability of topical amphotericin B in the nasal and paranasal cavity. Because of the negative effect of distilled water, final dilution in physiologic solvents should be considered.

High-resolution image cytometry on smears of normal oral mucosa: a possible approach for the early detection of laryngopharyngeal cancers.

BACKGROUND: The purpose of this study was to investigate the possibility of identifying laryngopharyngeal cancers by nuclear chromatin texture feature analysis of cell nuclei from mucosal scrapings obtained from clinically and cytologically noncancerous areas of the soft palate in patients with cancer. METHODS: The collective consisted of 68 controls and 77 cases of laryngopharyngeal carcinomas. After Feulgen staining, 3000 cell nuclei were automatically measured using a high-resolution image analyser (CytoSavant Oncometrics, Vancouver, BC, Canada). Texture features were extracted for calculation of a discriminant function, which allows the two groups to be distinguished. RESULTS: Two parameters allowed the two populations to be distinguished. The classifier reached an overall performance of 72.7% sensitivity, 82.4% specificity, a positive predictive value of 80.5%, a negative predictive value of 75.1%, and an area under the receiver operating characteristics (ROC) curve of 0.7754. CONCLUSION: Our work shows that subtle changes in the chromatin distribution in cell nuclei from ostensibly normal cells in the vicinity of carcinomas are demonstrable in the oral cavity of patients suffering from laryngopharyngeal cancers. It may be possible to develop this method into a valuable clinical tool to reduce the high rate of delayed diagnosis of oral and laryngopharyngeal cancers.

Acute otitis externa: efficacy and tolerability of N-chlorotaurine, a novel endogenous antiseptic agent.

OBJECTIVE: The study's objective was to test the tolerability and efficacy of the endogenous antiseptic N-chlorotaurine (NCT) in comparison with a standard clinical treatment according to a phase IIb clinical trial protocol. STUDY DESIGN: The antimicrobial agent NCT was compared with the antibiotic component drops Otosporin (containing neomycin, polymyxin B, and hydrocortisone) for topical treatment of acute otitis externa in a randomized and rater-blinded clinical study. METHODS: Fifty patients suffering from acute otitis externa were divided into two groups according to a randomized list. The test group was treated with 1 mL of 1% aqueous NCT solution, the reference group with 1 mL of Otosporin. The substances were applied to the external ear canal at one daily session until the signs of infection disappeared. Efficacy and tolerability were evaluated daily by visual analogue scale and a six-step infection score. In addition, smears were analyzed to identify the causative pathogens. RESULTS: Both medications were equally well tolerated by the patients. The treatment was successful for all patients of the NCT group, whereas in one patient from the reference group, the infection did not disappear. The inflammation score improved more rapidly in the NCT group, which resulted in an earlier termination of the therapy. This difference became highly significant on days 4 to 7 (P <.01 each). Time needed for disappearance of inflammation (score 0) was 5.6 +/- 1.6 (mean +/- SD, range 3-9) days in the NCT group and 7.4 +/- 1.6 (range 4-10) days in the Otosporin group (P <.001). As expected, microbiologic cultures from ear swabs revealed Pseudomonas aeruginosa (58%) followed by Staphylococcus aureus (18%) as the main causative pathogens. CONCLUSIONS: NCT appears to be well tolerated and more effective than the therapy using antibiotic component drops. Because of its endogenous nature and its higher efficacy, NCT appears to be a good choice for topical treatment of acute otitis externa.

Tolerability of N-chlorotaurine in the guinea pig middle ear: a pilot study using an improved application system.

The tissue tolerance of N-chlorotaurine (NCT), a mild endogenous antimicrobial oxidant, has been investigated by application to the guinea pig middle ear. The animals were implanted with a novel cannula system that allows chronic external drug delivery to the round window niche. In the first part of the study, 3 animals each received 100 microL of 0.1% NCT (5.5 mmol/L) and 1% NCT, respectively, in aqueous solution twice daily for 8 days. In the second part, NCT was dissolved in phosphate-buffered saline solution to 300 milliosmolar (isotonic), and 27 microL was injected in 3 additional animals twice daily for 7 days. The guinea pigs injected with 100 microL of NCT developed immediate dizziness and nystagmus and did not thrive. Other reactions included mucosal thickening in the middle ear, rupture of the tympanic membrane, and blood and gelatinous material in the cochlea accompanied by hair cell loss and a 10- to 90-dB elevation of the hearing threshold as determined by auditory brain stem responses. The effects seemed to be dose-dependent, but the rate of variability was high across animals. In contrast, the guinea pigs treated with 27 microL of isotonic NCT showed no signs of discomfort, no or only moderate thickening of the middle ear mucosa, no shift of the hearing threshold, and no hair cell loss. Positive control animals injected with 10% neomycin sulfate developed extensive hair cell loss. Provided that the membranes of the inner ear are intact and that low single-dose volumes are used to avoid increased middle ear pressure, isotonic NCT seems to be well tolerated in the tympanic cavity. The new drug delivery system proved to be advantageous for ototoxicity studies.

Phenotypic characterization and distribution of dendritic cells in parotid gland tumors.

Evidence on the relationship between the infiltration of dendritic cells (DCs) and prognosis in head and neck tumors exists. Interestingly, only limited information is available regarding the maturation state and distribution of DCs in parotid gland tumors. The purpose of our study was therefore to extend these observations and to investigate in more detail the density and distribution of mature DCs and Langerhans cells (LCs) in parotid gland tumors. We present immunohistochemical evidence of characterization and distribution of DCs and LCs in parotid gland tumors, enclosed pleomorphic adenomas and malignant parotid tumors. Two populations of mature DCs could be identified, P55(+)-DCs and DC-LAMP(+)-DCs, whereas LCs could be identified as Langerin(+)-LCs. The overall impression was that parotid gland tumors contained only few mature DCs and LCs. Considering the sparsity of mature DCs in the malignant tissues, anti-tumor response can be only limited. On the basis of our data, we imply that the application of DC vaccination in combination with other modalities for treatment of parotid gland carcinoma should be taken into account. In this regard, the utilization of DC immunotherapy for management of minimal residual disease after resection of primary tumor can be promising. Putative targets expressed in this type of tumors have to be defined.