Cellular distribution of mucosa-associated lymphoid tissue with otitis media in children.

This study examined mucosa-associated lymphoid tissue (MALT) in the eustachian tube (ET), middle ear (ME), and mastoid antrum (MA) in 163 celloidin-embedded temporal bones from children with or without otitis media. Otitis media was defined by the presence of histopathologically identified inflammatory cell infiltration in the mucosa or cavity of the ME. We found MALT in the ET in 30 cases (46.2%), in the ME in 19 cases (29.2%), and in the MA in 4 cases (6.2%) out of 65 cases of otitis media, and in the ET in 7 (7.1%), in the ME in 0, and in the MA in 0 out of 98 specimens without otitis media. No MALT appeared in any children under the age of 1 month. Immunohistochemical methods were used to investigate MALT in 12 horizontally cut temporal bones with OM. The follicular area contained OPD4-positive (helper-inducer T) cells and a few CD8-positive (cytotoxic and suppressor T) cells, whereas the parafollicular area contained OPD4-positive and CD8-positive T cells. CD57-positive (natural killer) cells were confined to the germinal center. CD30-positive (activated T and B) cells were observed throughout the follicles. A few CD15-positive (granulocyte, monocyte) cells were found in the follicles. Histopathologic and immunohistochemical findings were indistinguishable for MALT in the ET, ME, and MA. Our results suggest that MALT may be a mechanism for producing a rapid and massive local immune reaction to repeated bacterial infections via the ET.

Inflammatory response to chronic otitis media in DiGeorge syndrome: a case study using immunohistochemistry on archival temporal bone sections.

Immunohistochemical analyses were conducted on archival celloidin-embedded human temporal bone sections from an 8-month-old boy with chronic otitis media and DiGeorge syndrome. We employed antigen retrieval methods with saturated sodium hydroxide-methanol solution, microwave incubation, and proteolytic treatment to demonstrate the distribution of T-lymphocytes, B-lymphocytes, macrophages, and intercellular adhesion molecule 1 (ICAM-1) expression in the middle ear. B-lymphocytes and macrophages were observed predominantly within the middle ear mucosa. T-lymphocytes were rare. Further, ICAM-1 was expressed in the vascular endothelium of the lamina propria, as well as infiltrating mononuclear cells. This suggests that the expression of ICAM-1 can be induced in the middle ear with otitis media, even if T-lymphocytes are depressed in a cell-mediated immunodeficiency disorder such as DiGeorge syndrome.

Postnatal development of Eustachian tube: a computer-aided 3-D reconstruction and measurement study.

The postnatal development of the Eustachian tube (ET) and its surrounding structures was investigated by means of computer-aided three-dimensional (3-D) reconstruction methods in 13 normal human temporal bones, obtained from individuals 3 months to 71 years old. The cross-sectional area, width and height of the lumen in most of the cartilaginous portion of the ET were significantly smaller in children than in adults. In particular, there was a marked, age-associated difference in the shape of the lumen in the cartilaginous portion of the ET. In adults, the cross-sectional area of the lumen declined monotonically between a large opening at the pharyngeal orifice and the narrowest portion of the ET (near the border of the cartilaginous and junctional regions). In children, by contrast, the ET lumen was uniformly smaller over the first 80% of its length from the pharyngeal orifice. It is suggested that this immature morphology of the ET lumen may confer increased risk of developing otitis media during childhood.

Immunohistochemistry of lymphocytes and macrophages in human celloidin-embedded temporal bone sections with acute otitis media.

Immunohistochemical analyses were used to investigate the distribution of lymphocytes and macrophages in routine human temporal bone sections obtained from a subject with acute suppurative otitis media. Primary antibodies specific for human CD3 and CD43 (T-lymphocytes), CD20 (B-lymphocytes), CD45 (leukocyte common antigen), and CD68 (macrophages) were used. As a pretreatment, the sections were soaked in antigen retrieval solution (saturated sodium hydroxide-methanol solution in methanol at a ratio of 1:3). A second antigen retrieval procedure (microwave treatment in 1% zinc sulfate) was also employed for identifying CD3-positive cells. Then the avidin-biotin-peroxidase complex technique was performed. Positive reactions to all antibodies but anti-CD68 were observed in the mucosa of the eustachian tube, tympanic cavity, and mastoid air cells. Particularly, cells positive to anti-CD3 or anti-CD43 were making a diffuse invasion upon the lamina propria. CD68-positive cells were scattered only in the effusion of mastoid air cells. These results suggest that the retrospective immunohistochemical study of archival temporal bone sections is a promising approach to investigate the pathogenesis of otitis media.

Leukotriene C4 inhibits ciliary activity in the presence of gamma-glutamyl transpeptidase.

We incubated human paranasal sinus mucosa in tissue culture with each of leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4). Ciliary beat frequency (CBF) was measured photoelectrically, and the concentrations of leukotrienes in the incubation medium were determined over time. LTC4 significantly decreased CBF with 2-h, 4-h, and 8-h exposures to 10(-6) M, 10(-8) M, and 10(-10) M, respectively. Moreover, LTC4 dose dependently reduced CBF to 81.4% of the initial value after 6-h exposure to 10(-6) M, to 82.5% after 8-h exposure to 10(-8) M, and to 89.7% after 12-h exposure to 10(-10) M. LTD4 also exhibited progressive ciliary inhibition, while LTE4 had a minimal effect on CBF. In the medium, LTC4 was changed to LTD4 and further to LTE4. gamma-Glutamyl transpeptidase (gamma-GTP), an enzyme that converts LTC4 to LTD4, was detected in the mucosa. Serine-borate complex, an inhibitor of gamma-GTP, blocked the inhibitory effect of LTC4 on CBF. These findings suggest that LTC4 may induce ciliary inhibition indirectly by conversion to LTD4.

Effects of platelet activating factor on mucociliary clearance of the eustachian tube.

The effects of platelet activating factor (PAF) on mucociliary clearance of the eustachian tube were investigated both in vitro and in vivo. Normal ciliated epithelium was obtained from the eustachian tube of guinea pigs and incubated with PAF at concentrations ranging from 10(-10) to 10(-6) mol/L. Ciliary activity was observed under an inverted microscope and quantified photoelectrically. The PAF dose-dependently inhibited ciliary activity. One milliliter each of 10(-5) mol/LPAF, 10(-5) mol/L prostaglandin E2 (PGE2), 10(-5) mol/LPAF and PGE2, or the control solution (0.1 v/v% methanol-phosphate-buffered saline) was directly injected into the tympanic bullae of anesthetized chinchillas. The middle ear was examined by otomicroscopy, tympanometry, and auditory brain stem response in relation to time. The PAF delayed middle ear clearance, and the PGE2 augmented its delay. These findings suggest that PAF inhibits mucociliary clearance of the eustachian tube from the middle ear, and that PGE2 plays an important role in the augmentation of inflammatory disorders.

Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa.

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10(-6)M to 10(-9)M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10(-8)M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a gamma-glutamyl transpeptidase (gamma-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by gamma-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.

The effects of leukotrienes C4 and D4 on ciliary activity of human paranasal sinus mucosa in vitro.

The effects of leukotrienes C4 and D4 on ciliary activity of human paranasal sinus mucosa were investigated in vitro. Normal mucosa was surgically obtained from human paranasal sinuses and incubated in the form of tissue culture. Ciliated cells were magnified under an inverted microscope, and ciliary activity was photoelectrically measured. LTD4 progressively inhibited ciliary activity, and showed a more potent effect on ciliary activity compared to LTC4. The concentrations of LTC4 and LTD4 in the incubation medium were determined by radioimmunoassay when the mucosa was incubated with 10(-8) M LTC4. The concentration of LTD4 gradually increased and after 90 min reached the maximum of 0.71 x 10(-8) M, while that of LTC4 was reduced to about 10% of its initial concentration within 60 min. These results suggested the possible conversion of LTC4 to LTD4 on the mucosa, and that LTC4 can inhibit ciliary activity by means of LTD4.

Effect of ibudilast on ciliary activity of human paranasal sinus mucosa in vitro.

The effect of ibudilast (CAS 50847-11-5, 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine, KC-404), an anti-asthmatic drug, on ciliary beat frequency (CBF) of human paranasal sinus mucosa was examined in vitro. Ciliary activation was observed after a 10-min exposure to 4.6 x 10(-6) mol/l ibudilast. Ibudilast dose-dependently increase CBF at the concentrations ranging from 4.6 x 10(-7) mol/l to 4.6 x 10(-5) mol/l. Propranolol inhibited ciliary activity induced by ibudilast; however, neither indometacin nor verapamil affected the activation of ibudilast on CBF. Platelet activating factor (PAF) and Leukotriene D4 (LTD4) are chemical mediators inducing mucosal dysfunction and damage. Ibudilast prevented ciliary inhibition induced by PAF and LTD4. These findings indicated that ibudilast activates CBF and inhibits the effect of PAF and LTD4 on ciliated cells, and consequently improves the pathogenesis of allergic disorders such as the inhibited mucociliary transport system and airway hyperresponsiveness.

Inhibition of mucociliary clearance of the eustachian tube by leukotrienes C4 and D4.

The effect of leukotrienes C4 (LTC4) and D4 (LTD4) and prostaglandin E2 (PGE2) on mucociliary clearance of the eustachian tube was investigated in vitro and in vivo. Normal ciliated epithelium was obtained from the eustachian tube of guinea pigs and incubated separately with LTC4, LTD4, and PGE2 at concentrations of 10(-8) mol/L and 10(-6) mol/L. Ciliary activity was measured photoelectrically. Leukotriene D4 progressively inhibited ciliary activity, while PGE2 promoted it. Leukotriene C4 also induced ciliary inhibition. One milliliter each of 10(-5) mol/L LTC4, LTD4, and PGE2 was directly injected into the tympanic bullae of chinchillas under anesthesia. The middle ears were examined by otomicroscopy, tympanometry, and auditory brain stem response over time. Clearance of middle ear effusion was delayed by LTC4 and LTD4, as compared with PGE2 and the control. These findings indicate that LTC4 and LTD4 inhibit mucociliary clearance of the eustachian tube.

Detection of specific IgE antibodies to Japanese cypress pollen in patients with nasal allergy: a comparative study with Japanese cedar.

Japanese cypress pollinosis has recently attracted attention and its clinical relationship with Japanese cedar pollinosis has been pointed out. To compare the two kinds of pollinosis, we retrospectively examined specific IgE antibodies to both pollen of Japanese cypress and cedar in the sera of 150 patients with nasal allergy using AlaSTAT assay. During the season in which the pollens of these two species are dispersed, the positive rates for Japanese cypress and cedar increased to 51.4 and 75.0%, respectively. The percentage of patients positive for both of cypress and cedar was elevated to 51.4%, corresponding to 68.5% of the total patient group positive for cedar. Almost all the cases positive for cypress had IgE antibodies to cedar, the value of which was considerably higher than that of cases positive only for cedar. Furthermore, increases in titers of specific IgE antibodies to cypress was observed in four of six cases, compared between specific IgE antibodies to cypress in pre- and post-dispersion of cypress pollen. These findings suggest the following possibility: (i) there is cross-antigenicity between the two pollen species, and (ii) patients are immunologically affected by cypress pollen to express higher levels of specific IgE antibodies after pollen dispersion.

Eosinophil cationic protein in perennial allergic rhinitis.

For a quantitative investigation of eosinophil activation in perennial allergic rhinitis, eosinophil cationic protein (ECP) concentrations were measured by a radioimmunoassay in serum, nasal secretions (ECPWN) and in the supernatant of these nasal secretions (ECPsup) obtained from normal subjects and allergic patients. Levels of ECPWN and ECPsup were higher than that of ECPserum. ECPsup showed a positive correlation with clinical severity, despite the lack of a significant correlation with eosinophilia in nasal smears. ECPWN and ECPserum showed no significant correlation with any of these clinical parameters. There was a weak tendency toward an increase in histamine sensitivity of the nasal mucosa of allergic patients with higher ECPsup although this was not statistically significant. These results suggest accumulation and activation of eosinophils in the allergic nasal mucosa, and also indicate that ECPsup may be a clinical parameter of perennial allergic rhinitis.

Elevation of soluble interleukin-2 receptor levels in nasal allergy.

To investigate soluble IL-2 receptor (sIL-2R) levels in nasal allergy, the sera and nasal secretions from patients with nasal allergy and from healthy subjects were subjected to a double-epitope enzyme-linked immunosorbent assay. Significant elevation of sIL-2R concentrations in the sera and nasal secretions was observed in the allergy patients (n = 26) compared with those of healthy subjects (n = 9). IL-2R-positive (CD25(+)) cells were observed in the crust formed in an allergic nasal mucosa. The concentration of sIL-2R in the sera correlated neither with the eosinophil count of the peripheral blood count nor with clinical severity. The concentration of sIL-2R in the nasal secretions was significantly higher compared with that in the sera from allergic patients (p < 0.01), whereas no significant difference was observed between sIL-2R levels in the sera and nasal sections from normal subjects. These findings indicate that sIL-2R plays an essential role in allergic processes by regulating IL-2R-positive cells recruited into the nasal mucosa.

Detection of soluble interleukin-2 receptor and soluble intercellular adhesion molecule-1 in the effusion of otitis media with effusion.

We measured sIL-2R, TNF-alpha and sICAM-1 in the sera and middle ear effusions (MEEs) of patients with otitis media with effusion (OME). Although there was no signmcant difference between the sIL-2R levels of the serous and mucoid MEEs, they were significantly higher than serum sIL-2R levels of OME patients and healthy controls. TNF-alpha levels of the mucoid MEEs were significantly higher than those of the serous type. However, TNF-alpha was rarely detected in the sera of OME patients or healthy controls. We observed significant differences between the serous and mucoid MEEs with respect to their sICAM-1 levels, which were also higher than serum slCAM-1 levels of OME patients and healthy controls. Our findings suggested that IL-2, TNF-alpha and ICAM-1 could be significantly involved in the pathogenesis of OME through the cytokine network.

Tumour necrosis factor-alpha in nasal allergy.

Tumour necrosis factor-alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay and eosinophil cationic protein (ECP) by radio-immunoassay to evaluate TNF-alpha in nasal allergy. There was no significant difference either between the mean concentrations of TNF-alpha in nasal secretions from the patients with perennial nasal allergy and those of normal subjects, or between the TNF-alpha and ECP concentrations. However, reverse transcription polymerase chain reaction showed a specific increase of TNF-alpha mRNA and IFN-gamma mRNA in allergic nasal mucosa after allergen challenge in vitro. These findings suggest a possibility that T cell-derived IFN-gamma up-regulates macrophages to elaborate TNF-alpha, which may play a role in amplifying allergic inflammation in the nose through the cytokine network.

Turn-over of PAF in cultures of human paranasal sinus mucosa.

The change of PAF concentration in the culture medium was investigated by radioimmunoassay when 10(-8) M PAF or 10(-8) M lyso-PAF was incubated with a piece of normal human paranasal sinus mucosa. The PAF concentration in the medium of the former group was halved within 11.3 min and reduced to less than 5% of the initial concentration within 60 min. However, there was no significant difference in the reduction of PAF concentrations in the medium between groups with or without the mucosa. When 10(-8) M lyso-PAF was incubated with a piece of mucosa, PAF gradually increased and reached the maximum of 0.36 x 10(-8) M at 20 min, and thereafter quickly decreased to a non-detectable level.

The Effect of Lyso-PAF on Ciliary Activity of Human Paranasal Sinus Mucosa in vitro.

The effect of lyso-PAF on ciliated cells was investigated in vitro. Normal mucosa was surgically obtained from human paranasal sinuses and incubated in the form of tissue culture. Ciliated epithelium was magnified under an inverted microscope, and ciliary movement was photo-electrically measured. Ciliary activity was significantly inhibited by 10(-8) M lyso-PAF and could be restored. The effect of lyso-PAF was completely blocked by CV-6209, a specific PAF antagonist. The PAF concentration in the incubation medium of lyso-PAF was determined by radioimmunoassay, because PAF is a well known inhibitor of ciliary activity. PAF gradually increased and after 20 min reached its maximal level. These findings indicated the existence of an enzyme in the paranasal sinus mucosa, by which lyso-PAF is converted to PAF, and that lyso-PAF can inhibit ciliary activity by means of PAF.

Clinical efficacy of Tranilast on otitis media with effusion in children.

In this open randomized study, we evaluated the efficacy of Tranilast, one of the anti-inflammatory drugs, on otitis media with effusion in children. Sixty-two patients (103 ears) were divided into two groups: Group A was given Tranilast and local treatment (nasal and tubal); Group B only received local treatment (control for Group A). The overall improvement rating assessed as "moderately improved or above" for Group A was 63.6%, Group B 47.9%. There was a significant improvement in Group A as compared to Group B (p < 0.05). In subjects who suffered from otitis media with effusion for over 2 months. Group A exhibited 50.0% of efficacy while Group B only 15.4% (p < 0.05).

Study on the metabolism of lyso-platelet activating factor (lyso-PAF) in human paranasal sinus mucosa. The cultured ciliated epithelium can convert lyso-PAF to PAF.

The conversion of lyso-platelet activating factor (lyso-PAF) to PAF in cultured paranasal sinus mucosa obtained from normal human subjects was studied. The PAF concentration in the medium was determined after addition of lyso-PAF. PAF became detectable at 10 minutes after the addition of 10(-8)M lyso-PAF, and reached a maximum concentration (3.25 x 10(-9)M) at 20 minutes. The PAF level then gradually declined to become undetectable at 60 minutes after addition of lyso-PAF. Thus PAF is very unstable having a half-life calculated to be 12.8 minutes with an elimination constant of k = 0.05377 minutes-1. In contrast, lyso-PAF is known to be a stable metabolite of PAF as well as a precursor of PAF. The results obtained from this study suggest that the turnover of lyso-PAF to PAF may play a role in evoking prolonged inflammation in target organs or tissues.