The characteristics of human papillomavirus DNA in head and neck cancers and papillomas.

AIM: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). METHODS: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). RESULTS: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. CONCLUSIONS: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.

High co-prevalence of genogroup 1 TT virus and human papillomavirus is associated with poor clinical outcome of laryngeal carcinoma.

BACKGROUND: The aetiology and factors leading to the progression of laryngeal cancer are still unclear. Although human papillomavirus (HPV) has been suggested to play a role, reports concerning the effect of HPV infection on tumour development are controversial. Recently, transfusion transmitted virus (TTV) was suggested to play a role in certain infections as a causative or coinfecting agent. AIMS: To investigate whether the development and progression of laryngeal squamous cell carcinoma is associated with coinfection with TTV and HPV. METHODS: The prevalence of TTV and HPV was investigated using the polymerase chain reaction in tissue samples from 40 healthy individuals, 10 patients with recurrent papillomatosis, five patients with papillomatosis with malignant transformation, and 25 patients with laryngeal carcinoma. The obtained prevalence data were compared and analysed statistically. RESULTS: In the 11 patients with carcinoma who had metastasis or relapse there was a high rate of coinfection with genogroup 1 TTV and HPV (eight of 11), whereas in the 14 without tumour progression no coinfection was found. Coinfection was associated with significantly lower tumour free survival in patients with carcinoma (p < 0.001). Furthermore, four of five patients who had papillomatosis with malignant transformation were coinfected with genogroup 1 TTV and HPV. CONCLUSIONS: Although the nature of cooperation between HPV and TTV needs to be investigated further, coinfection with genogroup 1 TTV and HPV appears to be associated with poor clinical outcome in laryngeal cancer.

The distribution of bromine content of dibromodulcitol in the central nervous system of patients with malignant gliomas.

The bromine content of human gliomas and white matter was determined by neutron activation analysis (NAA) following p.o. administration of a single dose of 400-500 mg/m2 dibromodulcitol (DBD). In another group of patients with brain gliomas, the bromine content was measured subsequent to application of a single dose of 334 mg/m2 of sodium bromide (equivalent dose regarding the bromine content of DBD). The bromine content of these two groups was compared to the values found in a third control group of untreated patients. The amount of bromine after DBD application was three to four times higher than in the untreated samples and the average accumulation ratio of 1.8 +/- 0.4 proved to be nearly identical both in tumour and white matter. The bromine values after NaBr treatment showed a different pattern of distribution. The accumulation was higher in the tumour tissue than in the normal white matter. These findings demonstrate that the pharmacokinetic properties of DBD- and NaBr-derived bromine are different, suggesting that the increase of bromine after DBD administration could be due to covalently bound bromine in DBD.

Intracellular calcium and outer hair cell electromotility.

The influence of increased intracellular calcium level on outer hair cell (OHC) electromotility was examined by means of transcellular electrical stimulation in a partitioning microchamber. Electromotile activity was measured before and after application of the calcium ionophore ionomycin, which promotes the inflow of extracellular calcium, as well as its release from intracellular calcium stores. The ionomycin solvent, dimethyl sulphoxide (DMSO), by itself elicited a significant decrease in the magnitude of OHC electromotility. The DMSO effect was counteracted by 10 microM ionomycin and was reversed by 50 microM ionomycin. The increase in electromotility is partially mediated by a calmodulin-dependent mechanism, since W7, a calmodulin antagonist, attenuated the 50 microM ionomycin-induced motility increase. Our results suggest that the electromotility magnitude increase in isolated OHCs due to ionomycin is a calcium/calmodulin-dependent phenomenon.

Impaired bronchomotor responses to field stimulation in guinea-pigs with cisplatin-induced neuropathy.

Pre-treatment with cisplatin (3 mg/kg) i.p. once a day over 6 days induced sensory neuropathy as confirmed by femoral nerve conduction velocity test and significantly decreased contractions induced by electrical field stimulation (100 stimuli, 20 V, 0.1 ms, 20 Hz) in isolated main bronchial rings from guinea-pigs. The field stimulation-induced non-adrenergic, non-cholinergic (NANC) relaxations, however, were amplified in rings from animals with cisplatin neuropathy. The NANC relaxation response was completely blocked by 30 microM N(G)-nitro-L-arginine methyl ester in preparations from both control and cisplatin-treated animals. Superoxide dismutase (40 units/ml) was without effect on NANC relaxation in control rings, however, it substantially decreased NANC relaxation in preparations from animals with cisplatin neuropathy. These results show that cisplatin-induced sensory neuropathy is accompanied by attenuation of neural bronchoconstriction and an enhanced NANC relaxation. The latter is in part attained by an increased peripheral superoxide production.

Frequency of the recessive 30delG mutation in the GJB2 gene in Northeast-Hungarian individuals and patients with hearing impairment.

Mutations in the GJB2 gene, which encodes a gap junction protein (connexin 26) account for up to 50% of cases of congenital autosomal recessive non-syndromic hearing impairment. A single mutation, 30delG, is responsible for 70% of this autosomal recessive hearing loss in Europe. This study describes the 30delG mutation analysis of 23 Hungarian families (64 individuals) with at least two subjects with congenital non-syndromic hearing defect and of 52 unrelated individuals from the Northeastern population of Hungary. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. DNA was tested by PCR based restriction enzyme assay (BSiYI). Sixty-four percent of the patients displayed this one base deletion in GJB2. Out of these, 65.9% were homozygous for this mutation and 34.1% were heterozygotes. The latter showed compound heterozygosity since in these 14 patients, eight previously reported different nucleotide changes were observed on the second allele. The carrier frequency of the 30delG mutation among control group was one in 10.4 (9.6%). This high frequency of 30delG corresponds more to frequencies reported in Southern than in North Europeans.

Human otosclerotic bone-derived peptide decreases the gain of the electromotility in isolated outer hair cells.

An otosclerotic peptide (OP) (Sziklai et al., 1985a.b) was purified from perilymph and stapes footplate of otosclerotic patients by Sephadex G-25 gel column chromatography and subsequent isotachophoretic (ITP) separation. The transfer function of the electromotility was measured by inserting the isolated outer hair cells (OHC) into a partitioning microchamber (Evans et al., 1991) and applying a series of pairs of brief square-pulse stimuli with opposite polarity and with increasing magnitude. Somatic length changes of the inserted part of the OHCs were measured by an optoelectronic system. The isotachophoretically homogeneous peptide exerted a gain and magnitude decreasing effect on the transfer function of electromotility of isolated OHCs of the guinea pig, in vitro. The operating point of the electromotility did not change due to the effect of the peptide. The peptide decreased the electromotile performance within a minute and bath exchange to normal saline did not completely restore the transfer curve to baseline. Application of caffeine to the cells already under the effect of the otosclerotic peptide produced an opposite effect: gain and magnitude increase. Simultaneous application of acetylcholine (ACh) did not antagonize the effect of OP. The underlying mechanism of the action of OP on the transfer function of electromotility of OHCs is postulated to involve the modulation of intracellular Ca2- concentration.

Effect of acetylcholine and GABA on the transfer function of electromotility in isolated outer hair cells.

Outer hair cells (OHC) from high- and low-frequency regions were separately isolated from guinea pig cochleas. The cells were inserted with their ciliary pole first into a partitioning microchamber so that only 20-50% of the cell length was excluded. Somatic length changes due to transcellular electrical stimulation were measured at the cuticular plate in the inserted portion of the cells. Transfer curves of electromotility of the OHCs were obtained by both a series of brief (2.5 ms) and longer (30 ms) square pulses with opposite polarity and linearly increasing size from 40 to 280 mV in both negative and positive directions. Alterations in the transient and steady-state electromotility transfer curves were examined by application of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) to the synaptic pole. ACh, in the concentration range of 10-30 microM, evoked a significant magnitude and gain increase of electromotility in both transient and steady-state responses without a measurable shift in the operating point of the displacement-voltage transfer curve. A tonotopic response magnitude difference is found for ACh challenge. Basal turn OHCs responded with greater magnitude increase (+90% increase from control) than apical turn OHCs (+40%). GABA exerted an opposite effect, again in a location-dependent manner. Magnitude response decreased about 30% for long cells and 14% for short ones. Atropin, a muscarinic receptor antagonist, completely blocked the increase in electromotility response due to ACh. However, D-tubocurarine, a nicorinic receptor antagonist, while not blocking the ACh effect, altered the cell's apparent operating point. Bicuculline methiodide, a GABAA-receptor antagonist, completely arrested GABA influences on the electromotility response. These results suggest that both ACh and GABA can change the electromotile activity of OHCs, in a tonotopically biased manner. ACh challenge evokes greater magnitude responses in basal turn OHCs, whereas GABA induces greater motility response decrease in apical turn OHCs. The control of the gain and magnitude of electromotility by the transmitter substances appear to involve at least two mechanisms. One is probably related to conformational changes of the voltage-to-movement converter molecules and a change in their number in an effective operational pool, the other operates via changing the electrical resistance of the basolateral cell membrane.

Vasopressin entry into the inner ear fluids of the rat.

The entry of arginine-vasopressin (AVP) and sucrose into cochlear endolymph, perilymph of scala vestibuli (PLV), perilymph of scala tympani (PLT), and cisternal cerebrospinal fluid (CSF) was studied, in anesthetized rats, after the administration into the cerebral lateral ventricle of a 10 microliter solution containing the radioactive tracers. Both tracers were detected in PLV, PLT, and CSF but not in endolymph. Monoexponential decay curves were calculated for PLV, PLT, and CSF, and for each tracer no difference was found between the regression lines calculated for the different fluids. These results indicate that (i) injection into the cerebral lateral ventricle is a useful tool to study the permeability of the cochlear epithelium to different solutes and (ii) no specific transport system exists for AVP across the cochlear epithelium, suggesting that AVP may exert its effect via the perilymphatic side of the stria vascularis.

Cyclic GMP and outer hair cell electromotility.

The aim of this study is to examine the effect of phosphorylation pathways on the electrically evoked fast motile response of isolated outer hair cells (OHCs). Transcellular electrical stimulation was applied in the microchamber to guinea pig OHCs and motility was measured before and after drug application. Forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (PMA, protein kinase C activator) and dibutyryl 3',5'-cyclic guanosine monophosphate (cGMP agonist) were studied. As controls, L15 medium and dimethyl-sulfoxide (DMSO) were used. In each group, 12 cells were measured. Forskolin and PMA were dissolved in 0.1% DMSO to render them membrane permeable. DMSO by itself caused a statistically significant electromotility magnitude decrease. Forskolin and PMA could not reverse the motility decrease due to DMSO, the effects seen in their presence were the same as observed with DMSO alone. Thus, neither 3',5'-cyclic AMP-dependent protein kinase nor calcium/phospholipid-dependent protein kinase appear to have modulatory effects on electromotility. Dibutyryl cGMP (DBcGMP), in concentrations of 200 microM, elicited a significant electromotility magnitude increase. The DBcGMP effect could be inhibited by co-application of 200 microM DBcGMP and 100 microM 8-Rp-pCPT-cGMPS (8-4-chlorophenylthio-guanosine 3',5'-cyclic monophosphothioate, Rp isomer, a cGMP antagonist). Our results suggest that OHC electromotility is modulated by a cGMP-dependent pathway.

Time-related alteration of endolymph composition in an experimental model of endolymphatic hydrops.

The electrochemical changes of the inner ear fluids were studied in the guinea pig during the development of endolymphatic hydrops in an experimental model of Meniere's disease obtained by the blockage of the vestibular aqueduct. The endocochlear potential (first and third turns) was recorded, and the sodium, potassium, and chloride concentrations, and osmolality of the endolymph (first and third turns) and perilymph were determined at different intervals from 2 to 24 weeks after the induction of the hydrops. The development of hydrops was monitored by the compound action potential once a week during the observation period. In normal, nonoperated guinea pigs, longitudinal endolymphatic gradients of endocochlear potential, potassium and chloride concentrations, and osmolality, increasing from the apex to the base of the cochlea, were observed. After 2 weeks of hydrops, no alteration of this pattern was detected. After 6 and 9 weeks of hydrops, a progressive decrease of endocochlear potential, potassium and chloride concentrations, and osmolality was noticed at the first turn (6 and 9 weeks) and then at the third turn (9 weeks) which resulted in the disappearance of longitudinal gradients. At 24 weeks, the endocochlear potential was still diminished by 60%, whereas potassium and chloride concentrations and osmolality increased as compared to 9-week values but remained lower than in controls. The changes in composition of endolymph induced by the development of the hydrops could be related to the progressive alteration of the ionic permeability of the cochlear epithelium, which should be localized at the distended Reissner's membrane.

Changes in brain activation caused by caloric stimulation in the case of cochleovestibular denervation--PET study.

There are a number of well-known stimulation methods for the investigation of the central projection of the vestibular system. In addition to optokinetic, galvanic and neck vibration tests, the most widespread method is caloric stimulation. These listed methods cause not only vestibular, but also other effects on the central nervous system (CNS) (acoustic, tactile and nociceptive). In this paper, positron emission tomography (PET) was used to investigate whether caloric stimulation contains a non-vestibular (extravestibular) component, which would cause a distortion in the cortical activity and therefore in the vestibular effect on the CNS. Caloric stimulation was carried out in six patients who had been operated on due to cerebello-pontine angle tumour. These patients suffered post-operatively from a complete lesion of the vestibular system and anacusis on the operated side. Ipsilaterally activated areas were the inferior pole of the post-central gyrus and temporoparietal junction, caudal part of the post-central gyrus (SI, SII), inferior parietal lobule and medial frontal gyrus. Contralaterally activated areas were the anterior cingulate gyrus, medial frontal gyrus, posterior part of the insula, post-central gyrus and temporoparietal junction (SII). Ipsilaterally deactivated areas were the caudal and cranial part of the medial occipital gyrus (V2, V3, V4, V5). Contralaterally deactivated areas were the lingual gyrus, inferior occipital gyrus (V2, V3) and fusiform gyrus. On the basis of these data, it was postulated that, during caloric stimulation, extravestibular reaction also occurs, which corresponds to the subjective feeling of heat and pain. The deactivation of the occipital cortex due to an extravestibular effect was demonstrated. This is the first observation to suggest the possibility of nociceptivevisual interaction.

Distribution of toxic and radiation components in air particulates.

The concentrations of several toxic heavy metals and volatile organic compounds (VOCs) in various types of Hungarian fly-ash fine particulates were investigated by means of instrumental neutron activation analysis, X-ray fluorescence analysis and gas chromatography, coupled with mass spectrometry. Within a power station, particulate samples were taken from the boiler zone (BO), from the electrostatic dust filter chamber (FI) and from the flue-gas at the top of the stack (ST). Enrichment rates of the toxic metals both in FI and ST particulate fractions related to the BO concentrations were calculated to enable the temperature dependence on the adsorption of the toxic components to be studied. In addition, both the total amounts of the VOCs and their partial distributions in accordance with the number of carbon atoms were also studied in fly-ash particulates. From them, 31 organic species were identified and determined. Since Hungarian brown coals have high uranium and thorium contents, the specific radioactivities of the daughter isotopes of both the 232Th and 238U decay series were also measured and are discussed.

Acetylcholine controls the gain of the voltage-to-movement converter in isolated outer hair cells.

Sensitivity of the electromotility of isolated guinea pig outer hair cells (OHCs) to acetylcholine (ACh) was examined. OHCs were held in a partitioning microchamber in a position so that their ciliary poles were inserted and their somatic length changes measured. Transcellular square-wave stimuli were delivered to record voltage-to-movement conversion of the cells. ACh was applied to the synaptic poles. The transfer function of electromotility intimated shifts of the membrane potential with concomitant gain changes: gain decreases when the membrane potential is shifted in hyperpolarization direction and gain increases when the membrane potential is shifted in the opposite direction due to application of ACh in 100-500 microM concentration. Clear gain increase of the electromotility in basal turn OHCs to ACh was observed, whereas inconsistent results for apical turn OHCs were found. The latter is probably due to the pharmacological dose of ACh used. This possibility is further supported by results in which ACh abolished the sensitivity of the magnitude response of electromotility to DC depolarizing bias.

Cochlear implantation improves hearing in the contralateral ear.

The electrical stimulation of deaf ears by cochlear implants (CI) improved contralateral hearing in seven patients: 5 intracochlear (IC) (analogue processor) and 2 extracochlear (EC) (digital processor) implants. Our patients obtained best speech discrimination scores (98% + 2%, n = 11) when lip reading and CI were combined with a high-power hearing aid fitted into the contralateral ear. Contralateral hearing improvement was 10-15 dB sound pressure level (SPL) at frequencies up to 4000 Hz; hearing in a previously totally deaf ear recovered 4-6 months after CI. The phenomenon was independent of the type of speech-encoding (analogue or digital). Five out of 6 reimplanted patients who first received an 8-channel digital-pulsatile EC reported better speech discrimination after the second CI, when IC devices were implanted with analogue speech processors. These results suggest that EC implantation should be considered in childhood as a first choice, since i) It does not exclude the possibility of a second IC implantation with the same success rate as for the first; ii) The inner ear sensory epithelium remains intact after EC implantation, and consequently, if any regenerating process takes place in the implanted ear due to electrical stimulation it may provide more physiological hearing later on.

Flavonoids alter bone-remodelling in auditory ossicle organ cultures.

The beneficial role of bioflavonoids in an otosclerosis-like bone-remodelling process can be implicated from its interference with bone resorption induced by prostaglandin E2 (PGE2) in cultured guinea pig ossicles. Ipriflavone (7-isopropoxy-isoflavon) and quercetin reduced PGE2-elevated collagenase-like peptidase (Cl-peptidase) activity and potentiated a PGE2-induced decrease in collagen synthesis. The fact that PGE2 effects are mediated through cyclic AMP in bone turnover and flavonoids act synergistically with PGE2 in collagen synthesis confirm a cyclic AMP phosphodiesterase inhibitory role of flavonoids. It has already been attempted to use Ipriflavone medical treatment of otosclerosis. Quercetin, which has a better than Ipriflavone water-solubility seems as promising as Ipriflavone in the control of the otosclerotic bone-remodelling disturbance.

Hyposmotic swelling induces magnitude and gain change in the electromotile performance of isolated outer hair cells.

OHCs were inserted into a partitioning microchamber. Reversible swelling was induced by hyposmotic incubation medium (270 mos.kg H2O-1). The transfer curve of electromotility was measured by stimulating the OHCs with a series of brief (2.5 ms) square-pulses with both polarities and graded magnitudes. Response magnitude and gain of electromotility were increased in swollen OHCs. On the basis of their electromotile performance-characteristics, swelling appeared to depolarize the OHCs. Transition from contraction dominated asymmetry of motility was observed towards a symmetric response. Depolarization, by itself, cannot explain the gain and magnitude increase. Acetylcholine (ACh) responsiveness of swollen OHCs preserves the pre-challenged pattern: ACh further increases gain and magnitude.

Effect of flavonoid on PGE2-induced alterations in percentage collagen synthesis in ossicle organ cultures.

In adult guinea-pig stapes organ cultures, 3H (2,3)-proline incorporation into the collagenase-digestible protein (CDP) and non-collagen protein (NCP) fractions of the ossicles was measured and the percentage of collagen synthesis (PCS) was calculated. Prostaglandin E2 (PGE2), a potent stimulator of bone resorption, inhibited the PCS in low concentrations (5 and 25 microM), whereas it stimulated it in pharmacological concentrations (50 and 100 microM). Ipriflavon, an isoflavone derivative of therapeutical potential in otosclerosis, also reduced the PCS in 1, 10 and 50 microM concentrations. 50 microM Ipriflavon stimulated the PCS inhibited by 5 microM PGE2, but decreased the PGE2-induced PCS enhancement in vitro.

Cathepsin D activity in otosclerotic bone and perilymph.

Otosclerotic bone and perilymph samples obtained during stapedectomy operations from patients suffering from pure conductive hearing loss of at least 40 dBs air-bone gap at 500 Hz. Control perilymph samples derived from patients suffering from round window membrane rupture or Meniere's disease. Enzyme activities were determined using a synthetic fluorogenic substrate by fluorescence spectrophotometry. No significant differences have been found between the protein content of otosclerotic and normal or non-otosclerotic perilymph samples. Cathepsin D activity was significantly higher (p less than 0.001) in the otosclerotic perilymph samples. Otosclerotic stapes footplate contained more activity than normal meatal cortical bone as well, though the difference was not significant. Biological significance of the elevated Cathepsin D activity must be considered.

Examination of noncollagen protein composition of the footplate and superstructure of the otosclerotic stapes.

We have differentially precipitated 2 fractions of noncollagen proteins extracted from separately pooled superstructure and footplate of surgically removed stapes of patients from both sexes, who have suffered from otosclerosis. The two fractions were: EDTA-soluble, CaCl2 precipitable (Fraction-1) and EDTA-soluble, CaCl2 non-precipitable proteins. The protein pattern of these two fractions was compared by isoelectric focusing and two-dimensional gel electrophoresis. The phosphoprotein fraction (Fraction-1) of the otosclerotic stapes footplate contained acidic (pI 3-6), low molecular weight (20-40 kD) proteins, which were not detected in the superstructure and temporal cortical bone. Two-dimensional mapping of the Fraction-2 showed predominantly acidic proteins in the footplate and some basic minor components in the superstructure. The lack of the low molecular weight proteins in the superstructure proves the localisation of the otosclerotic process only in the footplate.