Tetsuya Ganbo

Cytotoxicity of human eosinophil granule major basic protein to human nasal sinus mucosa in vitro

The toxic effects of the human eosinophil granule major basic protein (MBP), reduced and alkylated, were studied on human nasal mucosa in vitro. With a microscope coupled with a television monitor (magnification x 2500) and videotape recorder, we investigated the effects of MBP on the mucosa and the ciliary activity of single cells. In nasal mucosal specimens from normal individuals, MBP, 5 mumol/L and 10 mumol/L, significantly inhibited (p less than 0.01) ciliary activity by 4 and 1 hours of exposure, respectively. At these same MBP concentrations, the mucosal surface profiles were altered by 4 hours of exposure, and ciliostasis was 75% to 100% complete by 9 and 6 hours, respectively. In a mucosal specimen from a patient with nasal allergy, 1 mumol/L of MBP significantly inhibited (p less than 0.01) ciliary activity by 1 hour; alteration of the mucosal surface profile appeared by 3 hours of exposure, and ciliostasis was 75% to 100% by 13 hours. Similar alterations of the mucosal surface profile were observed with specimens from a second patient with allergies; in contrast, 1 mumol/L of MPB had no effect on specimens from a nonallergic patient. These results indicate that MBP damages human upper respiratory epithelium, causing ciliostasis and alteration of the epithelial surface at concentrations likely achieved in vivo. Furthermore, the mucosal specimens from two allergic patients were damaged by concentrations of MBP that had no effect on mucosal specimens from a normal individual.

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.

Mucosal Dysfunction and Damage Induced by Platelet Activating Factor (PAF)

The effect of PAF on human nasal mucosa was investigated in vitro. Normal paranasal sinus mucosa was obtained from the ethmoid sinuses by surgical procedure and incubated in the form of tissue culture. Ciliary movement was viewed under an inverted microscope and recorded on video tapes, and its activity was measured photoelectrically. Morphological alterations were examined by light and electron microscopy. PAF inhibited ciliary activity of human nasal mucosa, in a time and a dose dependent manner, at concentrations from 10(-6)M to 10(-10)M, while no significant change was observed at 10(-11) M. Lyso-PAF exhibited minimal effect on the mucosa at a concentration of 10(-6) M. Morphological alterations of the epithelial layer of the mucosa such as edema, cell exfoliation and desquamation were found to increase across time. Ultrastructural alterations were observed prior to inhibition of ciliary activity. These data indicate the cytotoxic effect of PAF on human paranasal sinus mucosa.

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.

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 eusiachian tube.

Platelet activating factor induced respiratory mucosal damage.

Human sinus mucosal specimens from eight normal individuals were exposed to platelet activating factor (PAF) at concentrations ranging from 10−6 M to 10−11 M in a humidified CO2 chamber at 37°C. The mucosal surface of the specimens was recorded on video tapes and magnified 2,500 times on a 19-inch television (TV) monitor. Ciliary activity of each ciliated cell was photoelectrically measured on the TV monitor in real time. PAF induced mucosal damage which resulted in a coarse profile including ciliostasis and exfoliation of epithelial cells. The length of the incubation period in which the initial coarse profile occurred on the mucosal surface inversely correlated with the concentration of exposed PAF ranging from 10−6 M to 10−10 M with r=−0.712 (p<2×10−4). Both the control medium and 10−8 M lysoPAF showed no effect on ciliary activity or mucosal surface alteration even after 24 hr of exposure. Significant ciliary inhibition was noted after 6 hr of exposure to PAF at concentrations of 10−8 M and 10−10 M (p<0.05). After 10 hr of exposure, significant ciliary inhibition (p<0.01) was noted at all concentrations. Inhibition occurred in a time- and dose-dependent manner. The length of the incubation period in which initial ciliostatis occurred and the level of PAF concentration showed an inverse correlation with r=−0.918 (p<10−6). These results indicate that PAF is cytotoxic to human respiratory mucosa.

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.

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.

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.

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/L PAF, 10−5 mol/L prostaglandin E2 (PGE2), 10−5 mol/L PAF 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.