Significance of spiral ligament fibrocytes with cochlear inflammation.

It has been recently suggested that the spiral ligament fibrocytes, which interconnect with the basal cells of the stria vascularis via gap junctions, may be critical in maintaining cochlear homeostasis. In animal models of pathological conditions such as labyrinthitis and otitis media, reduced immunostaining for gap junction protein connexin 26 is observed in the spiral ligament. This suggests that disruption of the spiral ligament fibrocytes could be among the causes of cochlear dysfunction due to cochlear inflammation. Cultured spiral ligament fibrocytes have been shown to secrete chemokines and other mediators after stimulation of proinflammatory cytokine TNF-alpha or IL-1beta. Each of these mediators might induce inflammatory cell movement, which would prolong the inflammatory response. It is reasonable that such enhanced biological defense ability could be the cause of spiral ligament fibrocyte damage.

Classification and culture of spiral ligament fibrocytes from mice.

In this study, we established an immunocytochemical strategy to classify the fibrocytes of the murine spiral ligament (SL), and SL cultures were characterized. Similar to those in other mammals, three different types of fibrocytes were identified. Type I fibrocytes, which are found lateral to the stria vascularis, showed positive immunoreactivity for caldesmon and S-100 protein and were not stained for sodium-potassium-adenosinetriphosphatase (Na-K-ATPase). Type II fibrocytes are located lateral to the spiral prominence epithelium and suprastrial region, and they were distinguishable by their positive staining for Na-K-ATPase. Type III fibrocytes, which are found adjacent to bone in the inferior region of the SL, contained caldesmon but not S-100 or Na-K-ATPase. Secondary cultures from the SL were positive for caldesmon and S-100 and negative for Na-K-ATPase, suggesting that these cells were type I fibrocytes. The present immunocytochemical approach was useful for the classification of murine fibrocyte cultures, and these cultures may benefit future immunological studies of the inner ear because mice have been well characterized immunologically.

Age-related changes in the murine cochlear lateral wall.

Cochleas from C57BL/6 mice were investigated electrophysiologically and histochemically to evaluate the pathology of presbycusis. The average auditory brainstem response thresholds from 6-week-old mice were significantly lower than those of 6-month-old mice and those of 1-year-old mice. Histologic observation revealed changes in the cochlea after age 6 months. Conventional hematoxylin and eosin (H&E) staining showed disorganization of the organ of Corti, a decrease in the number of spiral ganglion cells, and atrophy of the stria vascularis. Although H&E staining and type II collagen immunolabeling did not show obvious changes in the spiral ligament (SL), the density of connexin 26 staining was reduced in this region. Sodium-potassium-adenosinetriphosphatase immunolabeling was increased in the SL, whereas its average density was not significantly altered in the stria vascularis. These results suggest that the SL could be among the regions responsible for cochlear malfunction with aging.

[Clinical study of bilateral parotid tumors].

We studied retrospectively 212 patients with parotid tumors who were treated in our hospital between October 1981 and March 1998. One hundred seventy-two of the tumors were benign, and 40 of them were malignant. The tumors were bilateral in 13 patients. Since 1992, we have treated at least 1 bilateral parotid tumor patient per year, and the number of patients with bilateral parotid tumors has tended to increase. Histologically, adenolymphomas occurred in 11 patients, and there was one occurrence of pleomorphic adenoma and one occurrence of basal cell adenoma. Eighty-five percent of all bilateral parotid tumors were adenolymphomas, and the bilateral parotid tumors comprised twenty percent (11 of 53 patients) of all adenolymphomas that we encountered. Among the 13 patients with bilateral parotid tumors, 1 patient experienced them heterochronously. In 7 of the 13 patients the tumor on the opposite side was found by diagnostic imaging. One patient showed recurrence in both parotid glands 4 years after initial surgery. Comparing bilareral adenolymphomas with unilateral adenolymphomas, there was no significant difference in the age or sex of the patients. Regarding bilateral adenolymphoma, 4 patients showed a solitary tumor on either side, 4 patients showed a solitary tumor on one side and multiple tumors on the other side, and 4 patients showed multiple bilateral tumors. Regarding unilateral adenolymphoma, 38 patients showed solitary tumors and 4 patients showed multiple tumors. Bilateral adenolymphomas were more multicentric than unilateral adenolymphomas. Epstein-Barr virus-encoded RNA (EBER) was detected in 11 of the 12 bilateral adenolymphomas and in 18 of 35 patients with unilateral adenolymphoma, by in situ hybridization. EBER was detected more frequently in the multiple unilateral adenolymphomas than in the solitary unilateral adenolymphomas. Based on our experience, the bilateral parotid tumor is not rare. Care should be taken to observe the other side of the parotid gland with parotid tumors that are suspected adenolymphomas. Imaging may be helpful for the detection of bilateral tumors. A relationship may exist between Epstein-Barr virus and adenolymphoma multicentricity.

Effects of influenza A virus on lectin-binding patterns in murine nasopharyngeal mucosa and on bacterial colonization.

To clarify the role of viral infection in otitis media, we intranasally inoculated mice with influenza A virus and examined histologic changes in the nasopharyngeal mucosa using a battery of lectins. Additionally, live Haemophilus influenzae or Streptococcus pneumoniae was injected into the nasopharynx after virus inoculation, and the clearance of bacteria from the nasopharynx was examined. Staining of the mucous blanket and epithelial cell surfaces in the nasopharynx with peanut agglutinin, succinyl wheat-germ agglutinin, and Bandeiraea simplicifolia agglutinin was significantly enhanced with intranasal virus inoculation when compared with that in control animals. The nasopharynx was moderately stained with Maachia amurensis agglutinin and wheat-germ agglutinin in control animals, and the staining was enhanced after virus inoculation. These findings were most remarkable 5 and 9 days after virus inoculation. The numbers of bacteria cultured from the nasopharynx were significantly increased when bacteria were injected 5 days after virus inoculation. These results suggest that an alteration in the glycoconjugate structure lining the nasopharyngeal mucosa caused by the influenza virus might be associated with the reduction in bacterial clearance.

Effect of proinflammatory cytokines on cultured spiral ligament fibrocytes.

To clarify the effect of proinflammatory cytokines on spiral ligament (SL) fibrocytes, in vitro studies were performed using secondary cell cultures. Cultures from murine SL fibrocytes were stimulated by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha, and secretion of various mediators was measured by enzyme-linked immunosorbent assay. After stimulation with the proinflammatory cytokines, IL-6, TNF-alpha, monocyte chemoattractant protein-1, KC, macrophage inflammatory protein-2, soluble intercellular adhesion molecule-1, and vascular endothelial growth factor levels were elevated. Secretion of these chemokines and other mediators could induce inflammatory cell movement, which would prolong the inflammatory response, leading to fibrocyte damage. Given that SL fibrocytes may play a role in cochlear fluid and ion homeostasis, such fibrocyte disruption could cause cochlear malfunction.

The influence of pneumococcal otitis media on the cochlear lateral wall.

The cochlear influence of otitis media was investigated in order to identify damaged regions causing cochlear malfunction. BALB/c mice were challenged with viable Streptococcus pneumoniae into the middle ear cavity and were killed 1 day to 1 month later for immunohistochemical analysis. Otitis media was induced in all of the animals, and some showed inflammatory cells in the cochlea. Although other changes were not obvious by hematoxylin and eosin staining, immunohistochemistry showed the presence of fibrinogen in the cochlea, mainly in the lower portion of the spiral ligament and in the spiral limbus. Immunostaining for connexin 26 was decreased in the spiral ligament, accompanied by marked fibrinogen staining. Immunostaining for sodium-potassium-adenosine triphosphatase in the stria vascularis and in the type II fibrocytes of the spiral ligament was not affected obviously. The presence of fibrinogen in the cochlea suggests disruption of the blood-labyrinth barrier caused by the middle ear inflammation. Changes in connexin 26 staining suggest the possibility that the spiral ligament could be among the regions responsible for the cochlear malfunction.

Changes in immunostaining of inner ears after antigen challenge into the scala tympani.

To study the mechanisms of immune responses and immune injuries in inner ears, labyrinthitis was induced by inoculation of keyhole limpet hemocyanin (KLH) into the scala tympani of systemically sensitized guinea pigs. Inner ears were then immunostained for KLH, immunoglobulin G (IgG), albumin, connexin26 (Cx26), and sodium-potassium adenosine triphosphate (Na,K-ATPase). Inflammatory cells containing KLH were observed in the scala tympani and in the collecting venule of the spiral modiolar vein (SMV). Spiral ligament, spiral limbus, and blood vessels including the SMV were diffusely positive for IgG and albumin. Immunoreactivity for Cx26 and Na,K-ATPase was decreased compared with the normal ears in the fibrocytes of the spiral ligament. These results suggest that inflammatory cells and blood constituents could extravasate into the cochlea from blood vessels and that fibrocyte damage in the spiral ligament could cause cochlear dysfunction.

Induction of antigen-specific antibody-secreting cells in the nasal mucosa by intranasal immunization of BALB/c mice.

Recent studies have demonstrated that antigen-specific IgA immune responses in nasal cavity are induced by intranasal immunization with several antigens. However, the precise mechanisms regulating such immune responses are unclear. In the present study, mice were immunized intranasally with horseradish peroxidase (HRP) plus cholera toxin into the right nostril, and HRP-specific IgA immune responses were investigated. After the immunization, an enzyme-linked immunospot assay showed an increased number of anti-HRP IgA cells in the nasal mucosa. Histochemical staining demonstrated the presence of anti-HRP antibody-secreting cells, mainly in the nasal subepithelial layer. Interestingly, these cells were induced predominantly on the right nostril, where intranasal immunization was performed. These findings suggest that antigen-specific IgA antibodies in the nasal secretions were produced locally from antibody-secreting cells in the nasal mucosa and that IgA immune responses in the nose were compartmentalized. Furthermore, lectin histochemistry showed an accumulation of peanut agglutinin-positive cells in the nasal-associated lymphoreticular tissue, suggesting the development of germinal centers in the immunized animals.

Histological features and prognosis of patients with mucoepidermoid carcinoma of the parotid gland.

Histological features and prognosis of patients with mucoepidermoid carcinoma of the parotid gland were analysed. Tumours from 13 patients were classified according to histological grades and immunoreactivity for HER-2/neu. Surgical resection of the tumour was performed for all patients, and the overall five-year survival rate was 69 per cent. The patients whose histological grades were 1 or 2 showed a 100 per cent five-year survival rate, but no patient with grade 3 survived five years. Also, patients who had tumours that overexpressed HER-2/neu had a lower survival rate (25 per cent) than patients with tumours that had weaker immunostaining (89 per cent). We considered tumours classified as grade 3 plus strong HER-2/neu expression to be 'high malignancy', and compared them with 'low malignancy' tumours that were grade 1 or 2 and had weaker HER-2/neu staining. Patients with high malignancy tumours had shorter recurrence-free intervals and shorter overall survival than patients with low malignancy tumours. The overall survival period of the low malignancy cases was much longer than the recurrence-free interval; unlike that in the high malignancy tumour patients. These results suggest that the combination of histological grades and expression of HER-2/neu may be a useful predictor of the prognosis for mucoepidermoid carcinomata.

Immunological potential of the tympanic membrane. Observation under normal and inflammatory conditions.

PURPOSE: The immunological potential of the murine tympanic membrane was studied under normal and inflammatory conditions. MATERIALS AND METHODS: Experimental otitis media was induced by injecting keyhole limpet hemocyanin into the tympanic cavity of systemically sensitized mice. The animals were killed from 1 day to 2 weeks after the injection, and the distribution of the immunocompetent cells in the tympanic membrane was compared with those in normal animals, by immunohistochemical and toluidine blue staining. RESULTS: Tympanic membranes under normal conditions showed few immunocompetent cells, except mast cells and la-positive dendritic cells (presumably Langerhans' cells) in the pars flaccida and in the annular and manubrial regions of the pars tensa. After the induction of otitis media, lymphocytes migrated into these regions, although the other regions of the pars tensa showed few of these cells. la-positive cells migrated into both pars tensa and pars flaccida. Mast cells did not show obvious changes between the normal and inflammatory conditions. CONCLUSION: The pars flaccida and the annular and manubrial regions of the pars tensa are considered to be the immunologically potential sites, responsible for the immune responses. The remaining greater part of the tympanic membrane can recognize antigens by the migrated Langerhans' cells.

Immunology of the tympanic membrane.

Under normal conditions, tympanic membranes lack immunocompetent cells, except mast cells and Langerhans cells in the pars flaccida, and the annular and manubrial regions of the pars tensa. Lymphocytes migrate to these regions after the induction of immune-mediated otitis media, although the other regions of the pars tensa do not show the presence of these cells. Langerhans cells appear throughout the entire tympanic membrane, in case of otitis media. The unique features of the immunological aspect of the tympanic membrane are discussed.

Changes in immunostaining of cochleas with experimentally induced endolymphatic hydrops.

Cochleas with experimentally induced endolymphatic hydrops were immunostained for Na+,K(+)-ATPase, intracellular Ca(++)-ATPase, carbonic anhydrase, aldehyde dehydrogenase, calcium-binding proteins, vimentin, and the gap junction protein, connexin 26. No changes in immunostaining of hydropic ears were observed 1 week after blockage of the endolymphatic duct. Two weeks to 1 month after the operation, immunostaining of type I fibrocytes in the spiral ligament, which are positive for all but Na+,K(+)-ATPase, was slightly decreased on the operated side. These changes became more pronounced 3 months after the operation. However, staining for Na+,K(+)-ATPase of the stria vascularis and of type II fibrocytes of the spiral ligament was not reduced until 6 months postoperative. The reduction of enzymes and other cell constituents that may be involved in ion balance of cochlear fluids indicates that cells in the spiral ligament play an important role in cochlear homeostasis and that they merit further study in animal and human otopathology.

Immunolocalization of Na+, K(+)-ATPase, Ca(++)-ATPase, calcium-binding proteins, and carbonic anhydrase in the guinea pig inner ear.

The distribution of Na+, K(+)-ATPase, Ca(++)-ATPase, carbonic anhydrase, and calcium-binding proteins were investigated immunohistochemically in paraffin sections of guinea pig inner ears. Marginal cells of the stria vascularis, type II fibrocytes of the spiral ligament, and cells in supralimbal and suprastrial regions, were positive for Na+, K(+)-ATPase. Type I fibrocytes of the spiral ligament were positive for Ca(++)-ATPase, carbonic anhydrase, calmodulin and osteopontin. In the vestibular system, dark cells were positive for Na+, K(+)-ATPase. However, these cells and subepithelial fibrocytes were negative for Ca(++)-ATPase, carbonic anhydrase, and the calcium-binding proteins. In the endolymphatic sac, epithelial cells in intermediate and distal portions were positive for Na+, K(+)-ATPase, but the reaction was less than that in the stria. The same endolymphatic sac cells that were positive for Na+, K(+)-ATPase were also positive for Ca(++)-ATPase and calcium-binding proteins, but negative for carbonic anhydrase. The presence of Ca(++)-ATPase and calcium-binding proteins in the type I fibrocytes of the spiral ligament suggests that these cells are involved in mediating Ca++ regulation. Lower levels of Na+, K(+)-ATPase and the co-existence of Ca(++)-ATPase and calcium-binding proteins in the epithelial cells of the endolymphatic sac indicate that these cells have a distinctive role in ion transport that is different from that of the cells of the stria vascularis and vestibular dark cells.

Distribution of immunocompetent cells in the endolymphatic sac.

To better understand the role of immunocompetent cells in the defense mechanism of the inner ear, the distribution patterns of those cells were investigated in the endolymphatic sac (ES) of mice maintained in three different conditions: germ-free (GF), specific pathogen-free (SPF), and conventional (CV). In another experiment, the recruitment of lymphocyte subsets was examined in the ES of SPF rats undergoing a perilymphatic antigen challenge after systemic presensitization. In the ES of GF mice, no immunocompetent cells were found. In the ES of SPF and CV mice, cells positive for IgG, IgA, IgM, and Lyt-1 were present in much smaller numbers than in the nasal mucosa. Cells positive for Lyt-2 were not seen in the ES of any mice. In the ES of rats that underwent a perilymphatic antigenic stimulation after a systemic presensitization, B lymphocyte subsets (positive for IgG, IgA, IgM) were mobilized in increased numbers, and T cell subsets (helper/inducer and suppressor) were also found 1 week after perilymphatic antigen challenge. These results taken together suggest that the ES is not originally equipped to possess immunocompetent cells and mount an immune response, but that once it has been activated with the inner ear antigenic stimuli, the ES can be the active site of a local immune response of the inner ear.

Distribution of immunocompetent cells in normal nasal mucosa: comparisons among germ-free, specific pathogen-free, and conventional mice.

To better understand the role of immunocompetent cells in the defense mechanism of the upper respiratory tract against microbial invasions, the distribution patterns of those cells were investigated in nasal mucosa of mice maintained in three different conditions: germ-free (GF), specific pathogen-free (SPF), and conventional (CV) conditions. Immunostaining by the indirect peroxidase method and toluidine blue staining were employed for the detection of immunocompetent cells and mast cells. For immunostaining, anti-IgG, -IgA, and -IgM polyclonal antibodies and anti-Lyt-1, -Lyt-2, and -Mac-1 monoclonal antibodies were used as primary antibodies. In nasal mucosa of CV mice, Mac-1+ cells, mast cells, and all cell types of lymphocyte subsets were present. In nasal mucosa of SPF mice, all cell types were also positive, but fewer in number than those of CV mice. In nasal mucosa of GF mice, IgG+, IgA+, and Lyt-2+ cells were rare, although IgM+ and Lyt-1+ cells were present in small numbers. An electron microscopic study revealed that follicle-like lymphocyte aggregates with high endothelial venules were present in nasal mucosa close to the mucosal epithelia. These findings suggest that lymphocytes are mobilized to nasal mucosa, responding to continuous antigenic stimuli, and play an important role in the local defense mechanism of the upper respiratory tract.

Analysis of immunocompetent cells in the middle ear mucosa.

A quantitative analysis of immunocompetent cells in the middle ear mucosa of mice was carried out by an indirect immunostaining method using various monoclonal antibodies. Mice bred in germ-free, specific pathogen-free, and conventional conditions were used to examine nonimmunized middle ear mucosa. Middle ear mucosae of otitis media-induced mice were also examined. In normal middle ear mucosa, mast cells were substantial, followed by Mac-1-positive cells and lymphocytes. Even though IgA-, IgM-, and Lyt-1-positive cells were seen in the mucosa of conventional mice, IgM-positive cells were seen only in mucosae of specific pathogen-free and germ-free mice. In otitis media-induced mice by inoculation with nontypable Haemophilus influenzae or lipopolysaccharide, Mac-1-positive cells were dominant. Although the numbers of IgM- and Lyt-1-positive cells increased markedly, the numbers of other lymphocyte subsets did not increase until 14 days after inoculation. These findings suggest that the middle ear is immunologically a potential organ as long as it is not exposed to antigenic stimulation. It is considered to be an immunoreactive site only after it has been activated with pathogens.

Functional architecture of the nasopharyngeal tonsil.

The exact architecture of the normal nasopharyngeal tonsil remains obscure because most histopathologic investigations have been based on surgically removed adenoids. We compared enlarged adenoids and normal nasopharyngeal tonsils under both light and electron microscopes. The marked features of clinically enlarged adenoids were a large extension of the reticular epithelium and increased germinal centers. A tendency toward increased stratified squamous epithelium and decreased ciliated epithelium was apparent in enlarged adenoids, possibly due to inflammatory conditions. One type of nonciliated cell seemed to transport foreign material into underlying lymphocytes, as do the M cells of gut-associated lymphoid tissue. This type of nonciliated cell was rarely found in the extended reticular epithelium of enlarged adenoids. These findings suggest a disturbance of the antigen-trapping system and surface protections in adenoidal enlargement.

Experimental otitis media in mice. Immunohistochemical observations.

In order to identify T cells and B cells in the middle ear (ME) mucosa, immunohistochemical observations were performed on mice which were induced with immune-mediated otitis media with effusion (OME). C3H/HeN mice were challenged with dinitrophenylated ovalbumin in the ME, 2 weeks after the systemic immunization. At 1, 3 and 7 days post challenge, the mice were sacrificed, and IgG-, IgA- or IgM-positive cells, as well as Lyt-1- or Lyt-2-positive cells, were observed in the subepithelial layer of ME. IgG- or IgA-positive lymphocytes were detected at 7 days post challenge, whereas IgM-positive cells were rarely seen. At 3 days post challenge, the number of Lyt-1-positive lymphocytes exceeded that of Lyt-2-positive lymphocytes; however, at 7 days post challenge they were similar. Previous studies have suggested that immune complexes are a key factor in the generation of immune-mediated OME. However, it seems possible that delayed type hypersensitivity may contribute to the pathogenic mechanism of OME.