Ken-ichi Hisamatsu

Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay

A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4-benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/B(o)%) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N(omega)-nitro-L-arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35.21 +/- 4.87 ng/mL (135.4 +/- 18.7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41.40 +/- 20.96 ng/mL (159.02 +/- 80.6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources.

Interaction between Oral Alpha-Streptococci and Group a Streptococci in Patients with Tonsillitis

The incidence of oral α-streptococci with inhibitory activity against group A streptococci, as a defense mechanism against bacterial infection in the oral cavity, was investigated in 141 patients with streptococcal tonsillitis. The study population included both children (n = 79) and adults (n = 62). Infection by group A streptococci appeared to be more common in children than in adults, as the detection rates of inhibitory α-streptococci in healthy children (29.7%), as well as pediatric patients with tonsillitis (14.9%), were lower than those in adults (63.0%; p < .01). It is possible to consider oral α-streptococci with inhibitory activity to be among the indications for tonsillectomy in patients with streptococcal tonsillitis, since the detection rate of inhibitory α-streptococci in surgical cases (10.9%) was significantly lower than that in nonsurgical cases (31.1 %; p < .01). The high detection rate of these strains during the postoperative state supported the observation that the incidence of group A streptococcal infection was decreased postoperatively. Accordingly, it is useful to investigate bacterial interference between oral α-streptococci and group A streptococci in patients scheduled for tonsillectomy.

Investigation of oral α-streptococcus showing inhibitory activity against pathogens in children with tonsillitis

The incidence of oral alpha-streptococcus with inhibitory activity against group A streptococcus, as a defense mechanism against bacterial infection in the oral cavity, was investigated in pediatric individuals with tonsillitis. Infection by group A streptococcus appeared to be common in children, because the detection rate of inhibitory alpha-streptococcus in healthy children as well as pediatric patients with tonsillitis was lower than in adults and elderly patients. In particular, the detection rate of these strains was predominantly low in patients with beta-streptococcus. Among pediatric patients scheduled for tonsillectomy, the detection rate of inhibitory alpha-streptococcus was low preoperatively. However, the rate was markedly increased after surgery. The high postoperative detection rate of these strains reflected the decreased incidence of group A streptococcal infection. The results of this investigation of bacterial interference between oral alpha-streptococcus and group A streptococcus suggested that surgical treatment is a more effective approach for improving the oral bacterial flora in children with recurrent tonsillitis.

Age‐related Alterations in the Auditory Brainstem Responses and the Compound Action Potentials in Guinea Pigs

The auditory brainstem response (ABR) and the eighth nerve compound action potential (CAP) were measured using click stimuli to investigate the agerelated alteration in the auditory function in 66 guinea pigs consisting of four age groups. With advancing age, a gradual elevation of the thresholds in both the ABR and CAP was clearly seen, together with the prolonged latencies for waves I, II, III, and IV to clicks at 95 dBpeSPL in the ABR. There were some individual differences in either threshold elevation or latency prolongation of both the ABR and CAP in aged guinea pigs. These findings suggest that the effect of individual differences on degenerative aging processes of the auditory system should be considered in selected aged animals, although a significant elevation of the neural auditory threshold is clearly found with advancing age as a whole.

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.

病原菌発育抑制性α-レンサ球菌の口腔内定着状況と喫煙, 含嗽の及ぼす口腔細菌叢への影響について

We investigated the distribution of oral alpha-streptococci with inhibitory activity against pathogens, which compose an oral defense mechanism. Detection rate of alpha-streptococci with inhibitory activity against S. pyogenes and S. aureus derived from the tonsil was higher than in other portions, the tongue, cheek, gingiva, or saliva. It has suggested that tonsillar bacterial flora are mainly a defense mechanism. The oral flora in healthy smokers was compared with healthy non-smokers to investigate the influence of tobacco on oral bacterial flora. The results showed that the detection rate of S. aureus in smokers was higher while that of alpha-streptococci with inhibitory activity against S. aureus was lower. However, the detection rate of alpha-streptococci with inhibitory activity against S. pyogenes in smokers was as high as in non-smokers. It is suggested that it was easy for S. aureus to adhere to the oral mucosa in smokers, and was considered to influence the strain which produces beta-lactamase for medical treatment. We investigated the influence of gargling on oral bacterial flora by comparing the amount of bacteria before and after gargling with popidine-iodine gargle and saline solution. It was shown that alpha-streptococci, a main component of normal oral flora were decreased after gargling in both smokers and non-smokers. Furthermore, it was shown that group A Streptococcus was not decreased after gargling, and it was concluded that use of gargle medicinal mouth wash in bacillus carriers should be studied further.

Leukotriene D4-induced mucosal damage during long observation periods in vitro

The effect of leukotriene D4 (LTD4) on human paranasal sinus mucosa was investigated for over 1 hour up to 24 hours using a VTR system at 2,500 X in vitro. Ethmoidal sinus mucosal specimens were incubated in tissue culture, and after exposure to LTD4 the mucosal surface profile was viewed under an inverted phase-contrast microscope equipped with a VTR system on a TVscreen. LTD4-induced ultrastructural alterations, ciliostasis, alterations consisted of a coarse profile and epithelial cell exfoliation. Ciliary activity was photoelectorically measured on the screen hourly. The mucosal specimens were morphologically examined before and after exposure to LTD4 by transmission electron microscopy. LTD4 inhibited ciliary activity in a time- and dose-dependent manner at concentrations ranging from 10(-6)M to 10(-10)M, whereas LTE4 had a minimal effect on the mucosa even at the concentration of 10(-6) M. Irrigation of the mucosa with culture medium after 5-min of exposure to 10(-8) M LTD4 delayed appearance of ciliary inhibition and alteration of the mucosal surface profile, but had no effect after 15-min exposure. These effects of LTD4 on the mucosa were blocked by preincubation with the LT antagonists, FPL-55712 and Ly-171883. These results demonstrate the cytotoxic effect of LTD4 on human airway epithelium in vitro.

The effect of compound nasal surgery on obstructive sleep apnea syndrome.

Background Nasal surgery often fails to ameliorate the symptoms of obstructive sleep apnea syndrome (OSAS). We developed a compound nasal surgery (CNS) method that consists of septoplasty combined with submucosal inferior turbinectomy and posterior nasal neurectomy to ensure low nasal resistance during sleep. Objective To clarify the effect of CNS on OSAS, pre- and postoperative changes in sleep-related events were studied by using polysomnography, the Epworth sleepiness scale (ESS), the visual analog scale for snoring, and health-related quality of life (QOL). Methods Forty-five consecutive patients with OSAS and with nasal problems underwent CNS. Three months later, the postoperative effect on OSAS was assessed by using polysomnography findings, daytime sleepiness by the ESS, nasal allergy symptoms, and health-related QOL. Snoring was assessed by the family by using a visual analog scale. Results The indices of apnea, apnea-hypopnea, oxygen desaturation, and arousal; the ESS; allergic symptom score; health-related QOL; and snoring on a visual analog scale were all significantly improved. Conclusions CNS improves OSAS events without any pharyngeal surgical procedure in selected patients. If high nasal resistance associated with OSAS is present, then CNS should thus be considered.BACKGROUND Nasal surgery often fails to ameliorate the symptoms of obstructive sleep apnea syndrome (OSAS). We developed a compound nasal surgery (CNS) method that consists of septoplasty combined with submucosal inferior turbinectomy and posterior nasal neurectomy to ensure low nasal resistance during sleep. OBJECTIVE To clarify the effect of CNS on OSAS, pre- and postoperative changes in sleep-related events were studied by using polysomnography, the Epworth sleepiness scale (ESS), the visual analog scale for snoring, and health-related quality of life (QOL). METHODS Forty-five consecutive patients with OSAS and with nasal problems underwent CNS. Three months later, the postoperative effect on OSAS was assessed by using polysomnography findings, daytime sleepiness by the ESS, nasal allergy symptoms, and health-related QOL. Snoring was assessed by the family by using a visual analog scale. RESULTS The indices of apnea, apnea-hypopnea, oxygen desaturation, and arousal; the ESS; allergic symptom score; health-related QOL; and snoring on a visual analog scale were all significantly improved. CONCLUSIONS CNS improves OSAS events without any pharyngeal surgical procedure in selected patients. If high nasal resistance associated with OSAS is present, then CNS should thus be considered.

Investigation of the nasopharyngeal bacterial flora in children with otitis media with effusion.

A study was undertaken to evaluate the incidence of alpha-streptococci with inhibitory activity against pathogens, a defense mechanism of the normal flora against bacterial infection, in the nasopharynx and tonsils of 37 children with otitis media with effusion (OME). In the patients with OME, the detection rates of alpha-streptococci with inhibitory activity against Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus and group A streptococci in the nasopharynx were significantly lower than those of alpha-streptococci isolated from the tonsils. Furthermore, the detection rates of nasopharyngeal alpha-streptococci with inhibitory activity against H. influenzae and S. pneumoniae in OME patients were lower than those in patients with tonsillitis. In conclusion, low nasopharyngeal levels of alpha-streptococci with inhibitory activity against pathogens appear to render children susceptible to attacks of OME. We suggest that it is important to investigate the relationships between the prevalence of pathogens and the inhibitory activity of alpha-streptococci against them in the nasopharynx.

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.