Cytokines are a series of small polypeptides which play important roles in bodie's defense system and homeostasis. Many kinds of cytokines are produced from a variety of cell types and they function through autocrine, paracrine and endocrine mechanisms. Cytokines regulate the growth, differentiation and migration of cells and they are involved in the immune response, inflammation, wound healing and erythropoiesis of the body. Moreover, cytokines affect the tumor growth and metastasis. Cytokines exhibit their function in either negative or positive manner. Cytokines bind specific membrane receptors and elicit physiological functions through the receptor-mediated signaling pathway. To date, nearly 100 cytokines have been reported and their list is still increasing. Thus, it is difficult to review all the cytokines but we classified major cytokines (about 50) into six groups based on their producing cell types and common functions. Wound healing was described with respect to cytokine's interplay in physiology and the EGF receptor system was discussed as a typical example of the molecular mechanisms of signaling pathway.
To elucidate the role of cytokines in the pathogenesis of otitis media with effusion (OME), levels of IL-1β, TNF-α, IL-6, and IL-8 in the middle ear effusions (MEEs) collected from 62 ears of patients with OME were determined by enzyme-linked immunosorbent assay (ELISA). MEEs were estimated by the naked eye to be either a mucoid or a serous variety and were classified into neutrophil-rich, lymphocyte-rich, and few cell types by cytological analysis. Following the centrifugation of the diluted samples of MEEs, the supernatants were stored for cytokine-specific ELISA and ribonucleic acids were extracted from the pellets for RT-PCR assay. Endotoxin levels and the activities of lysozyme and collagenase were also measured in the supernatants of MEEs and compared with the concentrations of cytokines. Levels of IL-1β, IL-8, endotoxin, and the activities of lysozyme and collagenase were higher in mucoid type MEEs than in serous type MEEs, and were significantly increased in neutrophil-rich group. Significant correlation was observed between the levels of endotoxin and that of IL-1β and IL-8. The incidence of IL-6 and IL-8 detected by ELISA was higher than that detected by RT-PCR, suggesting that these cytokines might be produced by middle ear mucosa as well as inflammatory cells contained in MEEs. Erythromycin therapy is known to be effective for OME and decreased the levels of IL-1β, IL-6, IL-8, endotoxin, and the activities of lysozyme and collagenase in MEEs. The findings suggest that the production of cytokines in MEEs might be influenced by the presence of endotoxin and that these factors are associated with the persistence of MEEs.
Effects of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on healing proces of the tympanic membrane perforation were investigated in guinea pigs. The tympanic membrane perforations were examined histologically and immunohistochemically with bromodeoxyuridine and labeling index of bromodeoxyuridine at the margin of perforation was calculated. The tympanic membrane perforations were closed more rapidly in the PDGF-treated than in the EGF-treated ears. Significant proliferation of the epithelial layer was obtained with both treatments. However, proliferation of the middle layer of the tympanic membrane was greater in the PDGFtreated than the EGF-treated ears. These results suggested that proliferation of the middle layer stimulates the epithelial layer and that proliferation of the epithelial layer directly fastens the closure of perforation of the tympanic membrane.
In order to elucidate the mechanism of proliferation of cholesteatoma epidermis, the relationship with various kinds of growth factors, receptors and cytokines was studied. At the same time, programmed cell death was also studied. When expression of PCNA protein was investigated, the results suggested hyperproliferative nature of cholesteatoma epidermis. Expression of IL-1 alpha protein was observed in subepidermal cholesteatoma epidermis showing strong inflammation, indicating a correlation with the state of subepidermal inflammation. TGF alpha protein was expressed at a high frequency in cholesteatoma tissue and normal skin tissue, indicating a correlation with the degree of infiltration by subepidermal inflammatory cells. Although expression of EGF mRNA was also increased in cholesteatoma, cholesteatoma was characterized by overexpression of EGF receptor mRNA (EGF-R mRNA) in all layers of the epidermis. Increased expression of KGF mRNA was noted as a factor which affects the effects of subepidermal inflammation on epidermal proliferation. In the cholesteatoma epidermis, overexpression of KGF-R, as well as EGF-R, was observed, which suggested paracrine regulation of KGF and KGF-R. When the relationship between proliferation of cholesteatoma epidermis and programmed cell death was investigated based on tissue sections, cells undergoing apoptosis were observed in the prickled cell layer and granular layer excluding the basal cell layer, same as normal skin tissue of the external ear canal. From these findings, it is speculated that apoptosis, as well as proliferation ability of cells, is also increased in cholesteatoma epidermis to keep the balance of proliferation. In other words, proliferation of cholesteatoma epidermis is stimulated by the action of cytokines, growth factors and subepidermal inflammatory cells, while it is compensated by apoptosis, or programmed cell death.
Cholesteatoma otitis media is characterized by invasion of keratinized epithelium into the middle ear and marked bone destruction on the surface of bone adjacent to the subepithelial granulation tissue. To clarify the roles of cytokines in the mechanisms of cholesteatoma-induced bone destruction, surgical specimens of cholesteatoma tissues were maintained in organ culture under a variety of conditions, and the culture media were assayed for the cytokine concentration and bone resorbing activity. As a result, cholesteatoma tissue produced the intense bone resorbing activity attributable to interleukin (IL)-1α. IL-1 α and tumor necrosis factor (TNF)α proved to form a vicious circle by stimulating their own production. IL-6 was an indirect feedback inhibitor of IL-1α through suppression of TNF α. Some of anti-inflammatory cytokines such as IL-1 receptor antagonist (IL-lra) and IL-10 were detected in cholesteatoma culture media, however, their amounts were not enough to suppress the pro-inflammatory cytokines. Anti-inflammatory or immunosuppressive agents such as dexamethasone or cyclosporin A did not block the cholesteatoma-derived bone resorbing activity but reduced the generation of IL-1 α or TNF α. These results indicate that IL-1 α is a key mediator inducing bone destruction and the proinflammatory cytokines formed a complicated cytokine network in cholesteatoma otitis media. Moreover, the insufficiency of anti-inflammatory cytokines in cholesteatoma and the difficulty of artificial regulation of bone resorption were thought to relate to intractability or perpetuation of this disease.
During the past decade, techniques related to molecular biology has made remarkable progress and their application to clinical otorhinolaryngology has advanced. I herein introduce general basis related to molecular biology and their application in cloning genes related to apoptosis. Investigation of the relationship between cell cycle related gene products and the clinical outcome of head and neck cancer is also mentioned.
Rapid progress in molecular biology leads to the application of new techniques to the inner ear research. Now we have been able to identify inner ear-specific molecules, novel molecules and registered molecules in the gene bank from inner ear tissues. Analysis of glutamate receptor gene family expressed in the rat inner ear has been performed in our laboratory. As for AMPA type glutamate receptor, a dominant expression of GIuR-2 subunit in the rat cochlea is confirmed. A cochlea-specific short GluR-3 subunit (sGluR) is also identified in the rat cochlea and is shown to serve as an inhibitory subunit, whose inhibitory effect is possibly caused by a conformational change of glutamate receptor complex. Expression of water channels (aquaporins; AQPs) in the rat inner ear tissues has been also analyzed in our laboratory. AQP-2, an ADH (antidiuretic hormone)-sensitive water channel, is identified in the rat cochlea as well as the endolymphatic sac. The expression of inner ear AQP-2 could be regulated by ADH via adenylate cyclase-cyclic AMP cascade, as in the kidney collecting duct cells. It has been suggested that AQP-2 and other members of AQP family could serve as key molecules in regulation of the homeostasis of inner ear fluids. Mutations in molecules of this gene family, therefore, might be quite possible in pathogenesis of several inner ear diseases such as Meniere's disease.
The localization of nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase was investigated in the vestibular organ of the pigmented guinea pig by a histochemical method. Intense reactivity to NADPH-diaphorase was found in the cytoplasm of both type I and type II vestibular sensory cells, whereas there was no reactivity in their nuclei and sensory hairs. The afferent nerve chalices were usually not stained. Supporting cells displayed no staining. The cytoplasm of other fluid transporting cells, as well as of vestibular ganglion cells, revealed weak to moderate NADPH-diaphorase activity. These findings support the hypothesis that nitric oxide is a mediator of neurotransmission in the vestibular system in sensory cells and ganglia. Nitric oxide in the fluid transporting cells may play an important role in maintaining endolymph and ion homeostasis.
The localization of nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase activity was investigated in the endolymphatic sac of the pigmented guinea pig by histochemical method. Intense reactivity to NADPH-diaphorase was found in the cytoplasm of epithelial cells, whereas there was no reactivity in their nuclei. NADPH-diaphorase activity was also noted around arteries and veins. The present findings support the hypothesis that nitric oxide in the epithelial cells may play an important role in the active intracellular transport of endolymph and ions. Nitric oxide may be crucially involved in regulating the blood flow of the endolymphatic sac. We conclude that nitric oxide may be an important factor in a local feedback system in the endolymphatic sac, regulating homeostasis of endolymph volume, of pressure, and of electrolyte balance.
Intracellular Ca2+ concentration was raised by photolysis of the caged calcium compound, Nitr-5, and by the intra-pipette perfusion technique. Increase of intracellular Ca2+ induced an outward current at the membrane potential of -40mV when recorded with an intracellular medium of 160mM KCl. This outward current was carried through the Ca2+-activated K channel. While increase of intracellular Ca2+ induced an inward current at -50mV with an 160mM CsCl-based intracellular medium. This inward current was carried through the Ca2+-activated non-selective cation channel. The amplitude of the mechano-electrical transduction current was reduced by the increase of intracellular Ca2+ both by the photolysis and by the intra-pipette perfusion methods.
Myringoplasty by Adhesive Method (Yuasa, et al. 1989) has been performed using a microscope and small-diameter rigid endoscopes. Two endoscopes were used, one with 0° straight forward view and one with 30° forward-oblique view. Both endoscopes had an outer diameter of 1.7mm and a visibility angle of 60° A CCD camera was attached to the endoscope to show the operating field on a TV monitor. 125 ears were surgically treated between September 1991 and October 1996. A microscope alone was used in 81 ears (group A) and both microscope and endoscope were used in 44 ears (group B). There were no significant differences between the two groups in background factors including patient age, etiology and size of tympanic membrane perforations. Surgical damage to the external auditory canal was smaller in group B than in group A, although the success rate of perforation closure and postoperative hearing were similar in both groups. The endoscope has advantages to the microscope in term of observation and recording of the operative field. However, for practical application, the surgical procedures under endoscope were deemed suitable only when those were difficult to perform under a microscope.
We performed tympanoplasty in middle ear cholesteatoma in 3 patients with Down's syndrome. The most characteristic findings were a narrow osseous external meatus, a poorly developed mastoid cavity, and the low set tegmen of middle cranial fossa. Tympanoplasty modified type III with enlargement of the external meatus by canal-wall-down technique and meatoplasty were performed in addition to complete remoral of cholesteatoma. Only the peripheral mastoid cavity was locally obliterated by bone pate, in order to make observation and treatment easy in the ear without a dead angle. This also allowed us to carry out early epithelization in the external meatus. A tympanostomy tube was inserted at the same time as tympanoplasty was performed in a single case with pars tensa cholesteatoma. The confidence from patient and co-operation of a local clinic before operation are necessary to allow the sufficient post-operative examination and treatment.
A two-year-old girl had severe bilateral congenital sensorineural hearing loss. When myringotomy was performed for her left ear for treatment of serous otitis media (SOM), cerebrospinal fluid (CSF) otorrhea was unexpectedly found and Mondini dysplasia was diagnosed by CT. A total amount of otorrhea was estimated up to 421ml per day at the third day after myringotomy. Two years after that episode she developed meningitis twice. At exploratory tympanotomy CSF leakage around the stapedial footplate was found. Then the inner ear was obliterated tightly with 8 small pieces of fascia of temporal muscle, and the oval window was covered with fascia. After that two pieces of auricural cartilaginous chip were placed. A spinal drainage was performed because the CSF leakage of the left ear was excessive. Three months after the operation she developed acute otitis media of the right ear and right myringotomy was performed. Soon after she developed meningitis and CSF otorrhea. At tympanotomy of her right ear it was revealed that CSF leaked around the stapedial footplate. We obliterated it as in the left ear. Both stapes were anomalous. After those operations she has remained free from meningitis and good aeration of both middle ears and mastoid cells was observed.
The Tullio phenomenon, a well-known vestibular symptoms induced by loud sounds, is usually associated with perilymph fistula and Meniere's disease. It is rarely seen in an otological practice. We report a case of Tullio phenomenon in a 73-year-old male patient with perilymph fistula. The patient presented to the Department of Otolaryngology, Keio University Hospital with complaints of vertigo and postural imbalance caused by loud sounds. The symptoms were developed 3 weeks earlier and had been especially exacerbated by car horns. The patient had a 2-year history of fluctuations of hearing in the left ear and occasional attacks of vertigo, which typically occurred when belching or inserting a finger in the left external auditory meatus. Pure tone audiometry revealed a moderate degree of sensorineural hearing loss in the left ear. While examination with Frenzel's glasses showed no spontaneous or positional nystagmus, the application of negative pressure to the left external auditory meatus elicited a horizontal nystagmus beating toward the left. Further vestibular tests were performed and vertigo and nystagmus were occurred when pure tones (100 dBHL) were applied to the left ear. Exposure to 500Hz tones caused the nystagmus toward the right, and it reversed when 1kHz, 2kHz, and 4kHz tones were applied. When standing, the patient consistently tilted to the left with tones at 1kHz. The fistula sign was again positive, but this time the induced nystagmus toward the right was detected regardless of whether positive or negative pressure was applied on the external meatus. A positive glycerol test indicated the presence of endolymphatic hydrops. Surgical exploration of the left middle ear revealed intermittent leakage of perilymph from both oval and round windows. The fistulas were closed using fascia and fibrin glue. The patient had no attack of vertigo for 7 months after surgery, and his hearing was also improved. As a conclusion, we assumed that the inconsistent findings of preoperative nystagmus induced by sound and pressure were due to the intermittent outflow of perilymph from the two fistulas.