Sources of stress and worry in the development of stress-related mental health problems: A longitudinal investigation from early- to mid-adolescence.

BACKGROUND AND OBJECTIVES: Stress and stress-related mental health complaints are common and increasing among adolescents, especially girls. Identifying typical sources of stress as well as central intervention targets is an important effort in the development of effective prevention and treatment protocols. This study investigated worry as potential mediator in the development of mental health problems in response to common stressors in adolescence. We also examined to what sources adolescents ascribe their stress over the years from the 7th through the 9th grade. DESIGN: Prospective cohort study. METHODS: Self-reported subjective stressor load, worry, anxiety and depressive symptoms were assessed in a sample of Swedish 7th graders (N = 1137; 46% girls, mean age 13.2) with follow-up assessments one and two years later. RESULTS: School was the most common source of stress across all time-points, with girls reporting considerable more stress than boys. Worry mediated the relationship between overall stressor load and depressive symptoms and anxiety over time and was not moderated by gender. CONCLUSIONS: Worry may be an important target in stress prevention and efforts to prevent stress-related problems would benefit from focusing on early adolescence as especially school stress is already relatively common in grade 7.

In vitro evaluation of the astatinated chimeric monoclonal antibody U36, a potential candidate for treatment of head and neck squamous cell carcinoma.

PURPOSE: The purpose of this study was to analyse the properties of the astatinated chimeric MAb (cMAb) U36 as a conjugate to selectively target and eradicate head and neck squamous cell carcinoma (HNSCC). METHODS: cMAb U36 was labelled with 211At via the linker N-succinimidyl 4-(trimethylstannyl)benzoate (SPMB). The quality of the conjugate was extensively evaluated for binding and internalisation capacity, and compared with 125I-SPMB-cMAb U36. The cellular toxicity of the astatinated conjugate was assessed in two types of in vitro growth assay and compared with 131I-labelled cMAb U36 (directly labelled). RESULTS: Comparisons between 211At-cMAb U36 and 125I-cMAb U36 demonstrated an optimal functional capacity of the labelled products. Immunoreactivity and affinity assays showed high immunoreactive fractions (>93%), and an affinity in good agreement between the astatinated and iodinated antibodies. For both conjugates, specific binding to HNSCC cells could be demonstrated, as well as some internalisation. Retention of the astatinated conjugate was just slightly lower than for the iodinated conjugate and still reasonable for therapeutic use (31+/-2% vs 42.6+/-1.0% at 22 h), demonstrating no adverse effects from astatination of the antibody. Studies on cellular toxicity demonstrated a dose-dependent and antigen-specific cellular toxicity for 211At-cMAb U36, with about 10% cell survival at 50 decays per cell. The 131I-labelled conjugate was not as efficient, with a surviving cell fraction of about 50% at 55 decays per cell. CONCLUSION: These results indicate that 211At-cMAb U36 might be a promising future candidate for eradicating HNSCC micrometastases in vivo.

Expression of EGFR, HER2, HER3, and HER4 in metastatic squamous cell carcinomas of the oral cavity and base of tongue.

The expressions of all four receptors in the epidermal growth factor receptor family, EGFR. HER2, HER3, and HER4 were evaluated by immunohistochemistry in 19 cases of metastatic squamous cell carcinoma of the oral cavity and base of tongue. EGFR had a similar and high expression in both primary tumours and the corresponding metastases, while the expression in normal epithelium was lower in most cases. HER2 was not expressed to the same extent as EGFR. However, when HER2 was well expressed, it was in most cases expressed to the same extent and intensity in the primary tumours, metastases, and normal epithelium. The expression of HER3 and HER4 varied and was mainly cytoplasmic in all cases studied. No overexpression of HER3 and HER4 in tumours was seen as compared to normal epithelium. In order to further investigate the distribution of HER3, two HER3 expressing cell lines originating from tongue cancer were analysed in vitro, using radiolabelled anti-HER3 antibodies directed to the extracellular domains of the receptor. The results indicated that HER3 was not present in measurable amounts in the cellular membrane. There is a need for improved diagnostics and therapy for the studied type of tumours, e.g. using radiolabelled antibodies or ligands, and EGFR seemed suitable as target since the expression was high, membrane associated and similar in the primary tumours and the corresponding metastases.

Hearing loss in relation to round window membrane morphology in experimental chronic otitis media.

The present study was performed to test the effect of single and repeated Pseudomonas aeruginosa exotoxin A (PaExoA) instillations in the middle ear of the rat. The hearing level was examined by the ABR technique, round window membrane (RWM) thickness was measured and morphology was studied by light microscopy. The results showed both reversible and permanent hearing loss (HL). In animals that received a single dose of PaExoA, the RWM thickness doubled initially and remained thickened during the observation period. When PaExoA was instilled on several occasions, RWM thickness doubled, before decreasing to near-control levels. This study confirms the toxicity of PaExoA and the partially reversible HL occurring after a single application of the toxin. The diminished effect of repeated toxin instillations--despite the decreasing thickness of the RWM--is discussed.

How can the hooded seal dive to a depth of 1000 m without rupturing its tympanic membrane? A morphological and functional study.

Recent studies using a satellite-linked dive recorder have shown that the hooded seal (Cystophora cristata), a common Arctic pinniped, can dive to a depth of > 1000 m and stay submerged for close to 1 h. At these depths the water pressure reaches 100 atm, entailing obvious risk of serious damage to the hearing apparatus, mainly the tympanic membrane (TM) and middle ear (ME). We dissected and photodocumented the temporal bones of five newborn and three adult hooded seals in order to study the temporal bone structure and reveal its protective mechanisms for extreme pressure changes. Specimens were sectioned and stained for light microscopy. The thicknesses of the pars tensa and pars flaccida were found to average 60 and 180 microm, respectively. The ME cavity hosts a cavernous tissue of thin-walled vessels beneath the modified respiratory epithelium. The ME and external ear canal (EAC) volumes can be altered appreciably by filling/emptying the cavernous tissue with blood. The ossicles were fixed by contracting the tensor tympani and stapedius muscles simultaneously with complete occlusion of the EAC. According to Boyle's law, the volume of the gas-filled ME cavity at a depth of 1000 m is only 1% of its volume at the surface of the sea. Ascent from such a depth allows the gas in the ME cavity to expand, causing the TM to bulge laterally. This movement is counteracted by a reduction in the blood volume inside the cavernous sinuses, action in the tensor tympani and stapedius muscles and discharge of gas through the Eustachian tube. The presence of a firm, broad-based exostosis in the floor of the EAC lateral to the TM helps to obstruct the EAC.

Localization of nitric oxide synthase isoforms in the human cochlea.

The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.

Detection of nitric oxide in the guinea pig inner ear, using a combination of aldehyde fixative and 4,5-diaminofluorescein diacetate.

Localization of nitric oxide (NO) production sites in the inner ear of the guinea pig was investigated using a combination of glutaraldehyde fixative and a new fluorescence NO indicator. 4,5-diaminofluorescein diacetate (DAF-2DA). The cochlea and vestibular end organs were examined to locate NO production sites. The fluorescence persisted after glutaraldehyde fixation and embedding with water-soluble resin. NO production in the cochlea was observed in the outer and inner hair cells, nerve endings, nerve fibers and supporting cells of the organ of Corti, stria vascularis, spiral ligament, ganglion cells, etc. In the vestibular end organs, both type I and type II sensory cells, nerve fibers, blood vessels and dark cells displayed fluorescence. This localization was exactly identical to that of NO synthase. Thus, detection of intracellular NO production by using a combination of glutaraldehyde fixation and DAF-2DA is useful for examining the function of NO in cells, both in situ and in vivo.

Direct evidence of nitric oxide production in the guinea pig organ of Corti.

Production of nitric oxide (NO) in the organ of Corti of the guinea pig was investigated using the new fluorescence indicator 4,5-diaminofluorescein diacetate for direct detection of NO. The organ of Corti, lateral wall of the cochlea and isolated outer and inner hair cells were examined to locate NO production sites. The fluorescence intensities were augmented by stimulation with L-arginine or glutamate, and significantly increased after inoculation with lipopolysaccharide. This is the first direct evidence of NO production in the cochlea. NO may play an important role in the physiology of the organ of Corti and may also be involved in hearing disorders.

Nitric oxide in guinea pig vestibular sensory cells following gentamicin exposure in vitro.

Gentamicin-induced production of nitric oxide (NO) in the vestibular end organs of the guinea pig was investigated using the new fluorescence indicator 4,5-diaminofluorescein diacetate for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with gentamicin. This increase in fluorescence was inhibited by the presence of the non-specific inhibitor for nitric oxide synthase, L-N(G)-nitroarginine methylester, and by the non-specific N-methyl-D-aspartic acid antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate. These findings indicate that NO may play an important role in the ototoxicity of aminoglycoside.

Pharmacological models for inner ear therapy with emphasis on nitric oxide.

Nitric oxide (NO)-mediated neurotoxicity may be an appropriate pathophysiological model with which to explain a variety of inner ear diseases characterized by acute or progressive hearing loss, tinnitus and vertigo. The localization of NO synthase (NOS) isoforms was examined in the inner ear of the pigmented guinea pig after intratympanic injection of 1 mg lipopolysaccharide (LPS) or 5 mg gentamicin (GM) using an immunohistochemical method, revealing the expression of NOS II in the inner ear. Production of NO in the isolated organ of Corti and utricle or in the isolated vestibular and cochlear hair cells after stimulation with L-arginine, glutamate, GM and LPS was investigated using the fluorescence indicator 4,5-diaminofluorescein diacetate. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, glutamate, GM and LPS. A significant increase in NO production was also noted in the LPS-treated animals. These findings imply that NO from constitutive NOS may mediate ototoxicity in the early phase, whereas NO from NOS II may contribute to the late phase of tissue damage in the inner ear. Based on this hypothesis, reduction of glutamatergic excitotoxicity and inhibition of NOS, scavenging superoxide and scavenging peroxynitrite are thought to attenuate NO-mediated otoneurotoxicity.

Glutamate-induced production of nitric oxide in guinea pig vestibular sensory cells.

Glutamate-induced production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with glutamate, NMDA and AMPA. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role in the glutamate-induced ototoxicity and also be involved in disease of the inner ear.

Alpha and beta subunits of acetylcholine receptors in the human inner ear.

The localization and distribution of nicotinic acetylcholine receptors (n-ACh-r) was characterized by studying alpha and beta subunits in the adult human inner ear by FITC fluorescence technique. In the cochlea, distinct fluorescence staining occurred for beta subunits in outer hair cells (OHCs), but no alpha subunits were identified. Beta subunits differ quantitatively between the three rows of OHCs, decreasing along a base-to-apex gradient in the cochlea. Both alpha and beta subunits were identified on spiral ganglion cells, adjacent nerve fibres and in vestibular hair cells (HCs). It would appear that they form an active complex in n-ACh-r at these locations.

Otoprotectant minimizes hearing defects caused by Pseudomonas aeruginosa exotoxin A.

Exotoxin A, produced by Pseudomonas aeruginosa (PaExoA), penetrates from the middle ear in to the cochlea and causes sensorineural hearing loss (SNHL). In this investigation we studied electrophysiological changes in the albino rat following instillation of PaExoA and N(G)-nitro-L-arginine methyl ester (L-NAME), a known inhibitor of nitric oxide synthesis, into the middle ear. Hearing thresholds were measured by auditory brainstem response (ABR) technique. Latency/intensity curves were constructed to distinguish between cochlear and conductive components of hearing loss. PaExoA caused damage to cochleae and SNHL, mainly at high frequencies. This impairment was blocked by (L-NAME). It would appear that nitric oxide may be a significant link in the mechanism of SNHL caused by bacterial toxin. L-NAME acts as an otoprotectant against the deleterious action of PaExoA.

Glutamate is the afferent neurotransmitter in the human cochlea.

Glutamate, the most important afferent neurotransmitter in the auditory system, is thought to be the afferent transmitter between the cochlear inner hair cells and afferent neurons, hitherto visualized only in the cochlea of animal species. It has been identified for the first time in sections from the human inner ear. L-glutamate, NMDAR2B and the enzyme glutamine synthetase were identified by using monoclonal antibodies. The distribution pattern of the transmitter L-glutamate in the human cochlea is similar to that observed in other mammals. L-glutamate was identified adjacent to outer and inner hair cells and in the spiral ganglion. Similar distributions were found for glutamine synthetase and the ionotropic NMDA receptor subunit NMDAR2. The identification of neurotransmitters and their receptors in the human cochlea has implications for the pharmacotherapy of inner ear diseases.

Dexamethasone modifies the effect of Pseudomonas aeruginosa exotoxin A on hearing.

In the present study, the protective effect of dexamethasone was analysed following exposure of the cochlea to Pseudomonas aeruginosa Exotoxin A (PaExoA). Four groups of albino Sprague-Dawley rats were used. 20 microl saline was instilled through the tympanic membrane into the round window niche (group A, n = 4); 1 microg/20 microl dexamethasone sodium 21-phosphate (dexamethasone) solution was instilled (group B, n = 4); 1 microg/20 microl PaExoA solution was initially instilled followed 1 h later by 20 microl saline (group C, n = 6); and 1 microg/20 microl PaExoA solution was initially instilled followed 1 h later by 1 microg/20 microl dexamethasone solution (group D, n = 6). Frequency-specific (4, 8, 10, 12, 16 and 20 kHz) auditory brainstem responses (ABR) were used to ascertain the threshold prior to exposure and 1, 2, 3, and 5 days and 1 and 2 weeks afterwards. No threshold change was observed in groups A and B, whereas the animals in groups C and D showed some threshold elevation, that in D being smaller than that in C. There was a significant difference at the frequencies 12, 16 and 20 kHz, 2 and 5 days after exposure. The intensity-latency (I-L) curve showed that in group D the cochlear component almost disappeared at high frequency one week after exposure. Our results indicate that dexamethasone can modify the effect of PaExoA caused by non-specific inflammation.

Localization of soluble guanylate cyclase activity in the guinea pig inner ear.

The aim of this study was to characterize the nitric oxide (NO) receptor soluble guanylate cyclase (sGC), to determine the cells targeted by NO and to elucidate the function of the NO/cGMP pathway in the inner ear. sGC activity in the inner ear was localized by immunohistochemical detection of NO-stimulated cGMP. Soluble guanylate cyclase activity in the cochlea was detected in the nerve endings underneath the outer and inner hair cells, supporting cells, stria vascularis and vessels. In the vestibular organs, sGC activity was detected in the cytoplasm of sensory cells, nerve fibres, dark cells and transitional cells and vessels. These findings suggest that the NO/cGMP pathway may be involved in regulatory processes in neurotransmission, blood flow and inner ear fluid homeostasis.

Direct evidence of nitric oxide production in guinea pig vestibular sensory cells.

Production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. The utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, and significantly increased after inoculation with LPS. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role for the vestibular physiology and also be involved in disease of the inner ear.

Lipopolysaccharide-induced expression of inducible nitric oxide synthase in the guinea pig organ of Corti.

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the cochlea of the guinea pig could elicit formation of inducible nitric oxide synthase (iNOS). Immunohistochemical study revealed that immunoreactivity to iNOS was seen below outer hair cells representing nerve fibers and synaptic nerve endings. iNOS-staining could also be observed in phalangeal dendrites of Deiter's cells pointing to the cuticular membrane, Hensen's cells and on stria vascularis 48 h after inoculation with LPS. Immunohistochemical investigation with a specific anti-nitrotyrosine antibody also revealed intense immunoreactivity identical to that of iNOS, suggesting formation of peroxynitrite in the organ of Corti by the reaction of NO with O(2)(-). On the basis of these findings, it can be concluded that NO together with O(2)(-), which form the more reactive peroxynitrite, are the most important pathogenic agents in LPS-induced damage of cochlea in the guinea pig.

Exostoses and cavernous venous formation in the external auditory canal of the hooded seal as a functional physiological organ.

Exostoses of the external auditory canal (EAC) develop after protracted mechanical, chemical or thermal irritation in particular. This is a common disorder among aquatic sportsmen and has been considered unique to Man. We dissected and photodocumented the EACs of 5 newborn and 3 adult Hooded Seals (Cystophora cristata). Serial sections of the EACs were prepared for light microscopic evaluation after staining with haematoxylin-eosin or toluidine blue. All EACs exhibited a firm, broad-based. mountain peak-shaped exostosis on the floor of the meatus, lateral to the eardrum. In addition, the meatal skin of the bony EAC harboured large venous sinuses. The exostosis and venous sinuses of the seal EAC participate in the protection of the sensitive hearing apparatus, particularly the pars tensa portion of the drum, during divine.

Free radicals in the guinea pig inner ear following gentamicin exposure.

The purpose of this study was to investigate the occurrence of free radicals, nitric oxide (NO), superoxide (O-2) and peroxynitrite, in the inner ear of the guinea pig following intratympanic injection with 5 mg of gentamicin (GM). Forty-eight hours after GM injection, varying degrees of degeneration of the inner ear were observed. Immunohistochemical study revealed immunoreactivity to NO synthase II (which generates NO) and to xanthine oxidase (which generates O-2) in both the vestibular organ and the organ of Corti. Immunohistochemical investigation, using a specific antinitrotyrosine antibody, also showed intense staining, suggesting formation of peroxynitrite in the inner ear through the reaction of NO with O-2. Scanning electron-microscopic study showed that the ototoxic effects could be blocked with N-nitro-L-arginine methylester, a competitive inhibitor of NO synthase, with superoxide dismutase, an O-2 scavenger, and with ebselen, a scavenger of peroxynitrite. On the basis of these findings, it can be concluded that NO together with O-2, which form more reactive peroxynitrite, play an important role in GM ototoxicity in the guinea pig.