Witold Szyfter

Triggering of Toll-like Receptor 4 Expressed on Human Head and Neck Squamous Cell Carcinoma Promotes Tumor Development and Protects the Tumor from Immune Attack

Toll-like receptors (TLR) expressed on inflammatory cells play a key role in host defense against pathogens, benefiting the host. TLR are also expressed on tumor cells. To evaluate the role of TLR in tumor cells, we investigated TLR4 signaling effects on human head and neck squamous cell carcinoma (HNSCC). Tumor tissues were obtained from 27 patients with laryngeal and 12 with oral cavity cancers. Normal mucosa was obtained from 10 patients with nonneoplastic disorders. Smears for bacteria were taken from all patients during surgery. TLR4 expression in tumors and HNSCC cell lines (PCI-1, PCI-13, and PCI-30) was detected by reverse transcription-PCR and immunohistochemistry. Cell growth, apoptosis, nuclear factor-kappaB (NF-kappaB) translocation, and MyD88 and IRAK-4 expression, as well as Akt phosphorylation were measured following tumor cell exposure to the TLR4 ligand lipopolysaccharide (LPS). Tumor cell sensitivity to NK-92-mediated lysis was evaluated in 4-hour (51)Cr-release assays. Cytokine levels in HNSCC supernatants were measured in Luminex-based assays. TLR4 was expressed in all tumors, HNSCC cell lines, and normal mucosa. The TLR4 expression intensity correlated with tumor grade. LPS binding to TLR4 on tumor cells enhanced proliferation, activated phosphatidylinositol 3-kinase/Akt pathway, up-regulated IRAK-4 expression, induced nuclear NF-kappaB translocation, and increased production (P<0.05) of interleukin (IL)-6, IL-8, vascular endothelial growth factor, and granulocyte macrophage colony-stimulating factor. TLR4 triggering protected tumor cells from lysis mediated by NK-92 cells. TLR4 ligation on tumor cells supports HNSCC progression.

An evaluation of the preservation of residual hearing with the Nucleus® Contour Advance™ electrode

Conclusion. Our study results confirm that it is possible to preserve preoperative hearing levels in the majority of subjects when using the Nucleus 24 Contour Advance provided that there is adherence to the major principles of ‘soft surgery’. Our study group demonstrated that 71–86% of subjects showed preservation of preoperative hearing thresholds at 6 months to varying degree. Objectives. The aim of the study was to assess the degree of residual hearing preserved postoperatively in a group of standard cochlear implant (CI) candidates following implantation via soft surgery with a Nucleus® 24 Contour Advance™ CI. Surgical technique variations from the soft surgery guidelines provided were assessed and their potential impact upon the conservation of residual hearing was examined. Subjects and methods. A prospective multicentre study involving a within-subject repeated measures design with each subject acting as their own control was performed. Pure-tone audiometric thresholds were assessed and compared in both implanted and contralateral ears for each subject preoperatively as baseline measures and at 6 months postoperatively. Surgeons were asked to complete a questionnaire to capture various aspects of the surgical technique used for each subject. Variations in the surgical technique performed were examined for potential correlation with conservation of residual hearing. Twenty-eight adult subjects, with a severe to profound hearing impairment, were enrolled in the study across eight implant clinics in four countries. Results. In all, 36% of subjects demonstrated preservation of thresholds to within 10 dB of preoperative thresholds across the frequency range (0.25, 0.5, 1.0, 2.0 and 4.0 KHz) and for the low frequency range (0.25–1.0 KHz). Approximately two-thirds of subjects demonstrated preservation of preoperative thresholds to within 20 dB. Preservation of low frequency thresholds post-implant was shown to correlate moderately with cochleostomy site, being more likely for subjects with a site anterior-inferior to the round window but also possible with inferior locations; weakly with cochleostomy size, being more likely when smaller than 1.2 mm; and also with the use of Healon® as a sealant and lubricant. Preservation of hearing thresholds across up to 4000 Hz was shown to correlate weakly with the use of suction following opening of the endostium and with bone dust contamination, both having a negative effect upon preservation, while no correlation was observed with the preservation of thresholds for low frequencies alone.

Immunohistochemical analysis of growth mechanisms in juvenile nasopharyngeal angiofibroma

Angiogenic factors are discussed to participate in growth and promotion of juvenile nasopharyngeal angiofibroma (JNA). However, only few data are available and mechanisms remain unclear. In the presented study we analysed the expression and subcellular distribution of several angiogenic growth factors and receptors potentially involved in JNA-growth and -vascularisation. In a retrospective, descriptive, multicenter-study, we analysed 13 formalin-fixed, paraffin-embedded or cryopreserved JNA-tumors (eleven primary tumors and two recurrent ones) after immunohistochemical staining. We used monoclonal antibodies specific for transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF), the VEGF-receptors 1 and -2 (FLT-1 and FLK-1), and the hypoxia inducible factor (Hif-1α). Data were compared to the vessel density. Quantitative analysis of staining intensities was performed by a computer assisted quantification technique. Endothelial and stromal compartments of the samples were analysed separately. Data were compared to vessel densities and patients data. The VEGF-Receptor-2 (FLK) was frequently unregulated in the stroma and endothelium of those samples with high vessel densities. Similarly, we observed high bFGF- and TGF-β1 levels in the stroma of strong vascularised samples. No correlations of expression levels to patients’ data were found. The reported data support the concept of JNA-growth and -vascularisation driven by factors released from stromal fibroblasts. Therefore, inhibition of these factors might be beneficial for the therapy of inoperable JNA.

Molecular and cellular alterations in tobacco smoke-associated larynx cancer

Tumours of head and neck belong to the most frequent types of cancer world-wide. In Poland, mortality from larynx cancer among males has been continuously increasing during the last decades up to 8.4 deaths per 100,000 men in 1993, which exceeds epidemiological records from other countries. The aetiology of laryngeal cancer is strongly associated with exposure to carcinogens present in tobacco smoke. The review describes a sequence of molecular and cellular events from carcinogenic exposure, DNA adduct formation, detection of mutations in the p53 gene, loss of heterozygosity (LOH) in chromosomal loci encoding the p53 and p16 genes, and loss of control of the cell cycle. The section concerning DNA adducts includes a discussion of the role of such confounders as exogenous exposure, the age and sex of the subject, and disease progression. The significance of genetic factors as individual risk determinants is discussed in relation to bleomycin-induced chromosome instability and in connection with the occurrence of defects in genes encoding detoxifying enzymes. The question concerning the substantial difference between men and women in larynx cancer morbidity and mortality remains open, even when the significantly higher adduct formation in male DNA compared with female material was taken into account. Preliminary experiments suggest a role of the frequently observed loss of the Y-chromosome.

Oxidative DNA base modifications and polycyclic aromatic hydrocarbon DNA adducts in squamous cell carcinoma of larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.

Screening of the General Polish Population for Deafness-Associated Mutations in Mitochondrial 12S rRNA and tRNASer(UCN) Genes

Mutations in mitochondrial DNA are associated potentially with nonsyndromic and aminoglycoside-induced hearing loss. Several nucleotide changes associated with hearing impairment were described; however, a variable frequency of deafness-associated mutations in different populations has been observed. The aim of the present study was to determine the frequency of pathological mutations in mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes in a group of 500 individuals representative of the general population of Poland. Mutational screening of 12S rRNA revealed the presence of three deafness-associated mutations, A827G, T961C, and A1555G, and one potentially pathogenic substitution, T669C. The carrier frequency of pathological mutations was estimated to be 1.2% (6/500) in the general Polish population. A deafness-associated G7444A mutation in the precursor of tRNA(Ser(UCN)) gene was identified in 8/500 (1.6%) unrelated blood donors. Seven nucleotide changes identified in 12S rRNA (G709A, G750A, G930A, T1243C, T1420C, and G1438A) and tRNA(Ser(UCN)) (C7476T), based on a frequency exceeding 1.0%, were considered as polymorphisms of 12S rRNA and tRNA(Ser(UCN)) in the studied population. Mitochondrial 12S rRNA gene seems to be the hot spot for deafness-associated mutations in the Polish population. The relatively high carrier frequency of tRNA(Ser(UCN)) G7444A (1/62) suggests that this substitution might be a nonpathogenic polymorphism in the Polish population.

Polish Universal Neonatal Hearing Screening Program-—4-year experience (2003—2006)

OBJECTIVE The aim of this paper is to share our experience and observations in running the Universal Neonatal Hearing Screening Program on a national level, present results and indicate some problems that have arisen during these 4 years. METHODS Polish Universal Neonatal Hearing Screening Program started back in 2002 in all neonatal units in Poland. Implemented testing methods consisted of test of transient evoked otoacoustic emission (TEOAE) performed in all new born children in their first 2-3 days of life and auditory brainstem response testing (ABR) conducted on children, who did not meet the TEOAE pass criteria. Additional questionnaire registered information on ototoxic drugs and family history of hearing impairment in every newborn. Diagnosed children were further referred for treatment and rehabilitation. RESULTS After 4 years of running the program (between 2003 and 2006) a total number of 1,392,427 children were screened for hearing impairment, what stands for 96.3% of all delivered babies, registered in Poland. The screening program enabled to identify and refer for further treatment 2485 children with various types of hearing loss, 312 with profound (0.02% of population) and 145 with severe sensorineural hearing loss (0.11% of population). CONCLUSIONS Our results indicate the accuracy of newborn hearing screening which remain an issue. Although improvement is needed in both intervention systems and diagnostic follow-up of hospitals, the Polish Universal Neonatal Hearing Program fully has achieved the main goal, the identification and treatment of hearing impaired children.

High-dose-rate and pulsed-dose-rate brachytherapy in palliative treatment of head and neck cancers

PURPOSE The main purpose of the study was to assess the results of high-dose-rate brachytherapy (HDRBT) and pulsed-dose-rate brachytherapy (PDRBT) in the palliative treatment of patients with locally or regionally recurrent head and neck cancers. The detailed aims concerned the evaluation of these methods in the context of local control, survival, and complications rates in patients subgrouped by different parameters such as age, gender, primary and recurrent tumor localization, tumor size, treatment method (HDR/PDR), primary treatment method, and radiation dose applied. METHODS AND MATERIALS PDRBT and HDRBT were used in 106 and 50 patients, respectively. In 8 patients, BT procedures were performed in combination with simultaneous chemotherapy. Sixteen patients were additionally treated with interstitial hyperthermia. All patients were regularly followed up within 6 months. Local control, complications, and survival were assessed. Materials included 156 patients with head and neck cancers treated palliatively with HDRBT and PDRBT in the Department of Otolaryngology of Poznań University of Medical Sciences and in the Department of Brachytherapy of Greater Poland Cancer Center from January 2002 to November 2008. RESULTS Complete and partial remissions 6 months after finishing the treatment were achieved in 37.7% of patients, whereas survival rates 12 and 24 months after brachytherapy were estimated for 40% and 17%, respectively. The overall complications rate was 35%. CONCLUSIONS Our results suggest that HDRBT and PDRBT constitute a safe alternative in the palliative treatment of patients with locally or regionally recurrent head and neck cancers with a relapse in a previously irradiated area, which were not qualified or rejected surgery. It gives a good palliative effect with acceptable complication rate.

Otologic symptoms as initial manifestation of wegener granulomatosis: diagnostic dilemma.

Objective: To show 7 cases of Wegener granulomatosis (WG) with early aural symptoms and to discuss the problems of otologic manifestation in WG. Study Design: Retrospective case review. Setting: Tertiary care university hospital. Patients: All patients were administered to the ENT University Department in Poznań in years 2002-2008 because of otitis media with effusion, facial palsy, sensorineural profound hypoacusis, hypoacusis combined with purulent discharge, and facial nerve palsy or progression of mixed type hypoacusis. Interventions: Diagnostics and treatment. Main Outcome Measures: Otologic symptoms as initial manifestation of WG diagnostic dilemma. Results: The authors want to underline the young age of the patients, ranging from 32 to 46 years. The outcome of initially otologic cases, which developed generalized form of WG, was poor (the first patient died after 2 months, the second patient died after 7 days, the third had the pulmonary insufficiency in 2 months of observation, and the fourth had severe renal failure in 1 month), whereas the patients with localized disease have been successfully under control for 1 to 5 years. Conclusion: As WG often presents otologic symptoms, as an initial sign in some cases, it is important to take WG into consideration in atypical inflammatory states of the ear. The otologic onset of WG is very insidious, and prompt diagnosis in early stage of disease is a challenge. Focal and localized disease in the aural region might possibly require less aggressive therapy than acute-onset multi-organ disease and is connected with better prognosis.

Toll-like receptors 2, 3 and 4 (TLR-2, TLR-3 and TLR-4) are expressed in the microenvironment of human acquired cholesteatoma

Human toll-like receptors (TLR 1-10) are crucial in the induction and activation of innate immunity in the course of an infection. They are expressed mainly on the cells of the immune system, and also on some epithelia and endothelia. Their ligands so called pathogen associated molecular patterns are abundant on invading microbes. TLR-ligand binding results in cell signal transduction and subsequent production of various proinflammatory cytokines such as IL-1 and TNF-α. Acquired cholesteatoma is formed during chronic otitis media in the proportion of cases. It has adverse effects on ear structures, resulting in osteolysis and bone resorption. Its formation and pathogenesis are not fully understood. The current study attempted to search the possible role of TLRs in this somewhat awkward pathological condition. Surgical specimens of human acquired cholesteatoma (n=15) and normal external auditory canal skin (n=5, control tissues) were tested by immunohistochemistry for the presence of TLRs. Three TLRs were examined: TLR-2, TLR-3 and TLR-4. All TLRs tested were demonstrated in matrix (layer of keratinizing epithelium) and perimatrix (granulation tissue) of this inflammatory tumour. Expression of particular TLRs within the keratinizing epithelium was distinct and uneven. In the perimatrix, numerous T (CD3+) cells were seen and relatively few macrophages (CD11c+, HLA-DR+). There was a weak expression of all TLRs on normal (non-inflammatory) skin. Expression of TLR-3 both on the epithelium and some cells within the perimatrix and the presence of T cells may suggest that apart from innate immune responses, mechanisms of adaptive immunity also operate in cholesteatoma. Weak expression of these receptors on normal skin may also suggest the important role of TLRs in the etiopathogenesis of cholesteatoma.