Gene-expression profiles in human nasal polyp tissues and identification of genetic susceptibility in aspirin-intolerant asthma.

BACKGROUND: Aspirin-intolerant asthma (AIA) is a subtype of asthma induced by non-steroidal anti-inflammatory drugs and characterized by an aggressive mucosal inflammation of the lower airway (asthma) and the upper airways (rhinitis and nasal polyp). The lower airway lesion and the nasal polyp in AIA are postulated to have common pathogenic features involving aspirin sensitivity that would be reflected in the gene expression profile of AIA polyps. OBJECTIVE: This study was conducted to clarify the pathogenesis of AIA using gene expression analysis in nasal polyps, and identify genetic susceptibilities underlying AIA in a case-control association study. METHODS: Global gene expression of nasal polyps from nine AIA patients was examined using microarray technology in comparison with nasal polyps from five eosinophilic sinusitis (ES) patients, a related disease lacking aspirin sensitivity. Based on the AIA-specific gene expression profile of nasal polyp, candidate genes for AIA susceptibility were selected and screened by a case-control design of 219 AIA patients, 374 non-asthmatic control (CTR), and 282 aspirin-tolerant asthmatic (ATA) subjects. RESULTS: One hundred and forty-three elevated and three decreased genes were identified as AIA-specific genes that were enriched in immune response according to Gene Ontology analysis. In addition, a k-means-based algorithm was applied to cluster the genes, and a subclass characteristic of AIA comprising 18 genes that were also enriched in immune response was identified. By examining the allelic associations of single nucleotide polymorphisms (SNPs) of AIA candidate genes relevant to an immune response with AIA, two SNPs, one each of INDO and IL1R2, showed significant associations with AIA (P=0.011 and 0.026 after Bonferroni's correction, respectively, in AIA vs. CTR). In AIA-ATA association analysis, modest associations of the two SNPs with AIA were observed. CONCLUSION: These results indicate that INDO and IL1R2, which were identified from gene expression analyses of nasal polyps in AIA, represent susceptibility genes for AIA.

Esterase-like activity of human serum albumin II: reaction with N-trans-cinnamoylimidazoles.

To elucidate the details of the esterase activity of human serum albumin, the reaction of N-trans-cinnamoylimidazoles with albumin was investigated kinetically at various pHs at 25 degrees. The reaction consisted of the acylation of albumin (probably the tyrosine-411 residue) by the substrate and the deacylation of cinnamoyl-albumin. The acylation was approximately 10--100-fold faster than the spontaneous hydrolysis of the substrate over the pH range examined. The pH profile for the deacylation rate constant indicated the participation of a group having a pKa of approximately 9.4. The deacylation was subjected to the effect of deuterium oxide. The electron-withdrawing substituent facilitated the deacylation; the Hammett rho value was 1.63. These results suggest that the deacylation proceeded via general base catalysis by this group.

Kinetics of the rapid modification of human serum albumin with trinitrobenzenesulfonate and localization of its site.

The rapid reaction of human serum albumin with trinitrobenzenesulfonate (I) and the location of the reactive site were investigated to characterize the chemical modification of albumin by I. The modification proceeds through trinitrophenylation of a lysine residue of albumin and monoaddition of the byproduct, sulfite ion, to the trinitrophenylalbumin, as reported previously. The individual kinetic parameters for both reactions were determined at various pH values and 25 degrees. The epsilon-amino group of the lysine residue which has a pKa value of approximately 8.9 was the reactive group involved in the trinitrophenylation. The dissociation constant of the sulfite monoadduct was about 10-fold smaller than that of the monoadduct of the model compound trinitrophenyl alpha-acetyllysine. The modification of albumin by I reduced the fluorescence intensity of the tryptophan-214 residue in the albumin amino acid sequence. Acetylation of the lysine-199 residue with aspirin and 5-nitroaspirin decreased the trinitrophenylation rate of albumin with I. These results on the fluorescence spectroscopy and the effect of the acetylation suggest that the reactive group for I is the lysine-199 residue located near the tryptophan-214 residue.

Characterization of benzodiazepinooxazoles: crystal and molecular structure of trans and cis isomers of oxazolam.

The stereochemistry of the trans and cis isomers of 2(S)- and 2(R)-oxazolam is described. A total of four molecular structures of the 11b(R) and 11b(S) diastereomers of 2(S)- and 2(R)-oxazolam have been revealed by X-ray diffraction techniques. 2(S)-Oxazolam crystallizes in space group P2(1)/c, with cell dimensions a = 16.180(2) A, b = 8.791(2) A, c = 13.574(2) A, and beta = 102.07(1) degrees. The molecule in an asymmetric unit exhibited disorder at the 2-carbon (C2) atom, and the structures for trans and cis isomers have been resolved crystallographically. 2(S)-Oxazolam forms polymeric chains with intermediation of ethanol through hydrogen bonding. 2(R)-Oxazolam crystallizes in space group P1, with cell dimensions a = 7.754(1) A, b = 8.654(1) A, c = 15.795(2) A, alpha = 104.09(1) degrees, beta = 91.19(1) degrees, and gamma = 114.00(1) degrees. A similar disorder at the C2 position has been elucidated by a mixture of trans- and cis-2(R)-oxazolams. The two 2(R)-oxazolam molecules form a dimer with a hydrogen bond net. The structural details, focusing on the conformation and molecular complex formation of both oxazolams, have been discussed in connection with the oxazolidine ring stability and the crystal polymorph.

Immune responses against Streptococcus pyogenes in human palatine tonsils.

We investigated cellular immunity against Streptococcus pyogenes in human tonsils by measuring antigen-specific immunoglobulin-secreting cells and the production of cytokines from CD4+ T cells in response to M proteins. The incidence of S pyogenes in tonsils was significantly higher in patients with recurrent tonsillitis (RT) than in patients with tonsillar hypertrophy (TH). M protein-specific immunoglobulin A (IgA) and immunoglobulin G spot-forming cells were increased in patients with RT compared with patients with TH. In RT the number of M protein-specific IgA spot-forming cells was significantly greater in the S pyogenes-negative subjects than in the S pyogenes-positive subjects. Proliferation of CD4+ T cells and production of interferon-gamma (IFN-gamma) and interleukins -2, -4, -5, and -6 (IL-2, IL-4, IL-5, and IL-6) from those T cells were observed in response to M protein. The concentrations of IFN-gamma and IL-4 were higher in RT than in TH. These findings suggest that S pyogenes is associated with the pathogenesis of RT and that immune responses against M protein may play an important role in preventing the colonization of this bacteria in tonsils.

Latissimus dorsi myocutaneous-iliac bone flap for reconstruction of massive defects of mandible and oral basis.

Recent advances in reconstructive surgery for head and neck cancer have improved the cure rate of advanced carcinoma, and the function of the organ. However, it still remains difficult to repair the mandible and oral floor. We devised a combined flap of myocutaneous latissimus dorsi and iliac bone, and applied it to two patients with advanced carcinoma of the oral cavity that invaded to the mandible (T4N3M0). Each patient received preoperative irradiation, totalling 30 Gy and 40 Gy. Two weeks before the extensive resection, a sufficient bony mass for the presumed mandibular defect was taken from the iliac crest and transplanted beneath the latissimus dorsi muscle. Defects of the mandible and oral floor were reconstructed using this combined flap immediately after the resection. The patients began to eat 2 weeks after surgery.

Effects of oral and systemic immunization on nasopharyngeal clearance of nontypeable Haemophilus influenzae in BALB/c mice.

BALB/c mice were immunized orally or subcutaneously with formalin-killed nontypeable Haemophilus influenzae (NTHi). Salivary immunoglobulin A (IgA) antibody titers against NTHi were significantly increased by oral immunization, but not by subcutaneous immunization. Both immunization procedures remarkably increased the levels of serum antibody activities of both IgA and immunoglobulin G. Live NTHi were then inoculated into the naso-pharynx, and the clearance of the pathogen from the nasopharynx was observed. Significantly fewer bacteria were present in the nasopharynx of the orally immunized mice than in the control mice. However, there was no significant difference between the subcutaneously immunized mice and the control mice. The results indicate that oral immunization can enhance the ability of mice to clear NTHi from the nasopharynx.

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.

Immune responses of adenoidal lymphocytes specific to Haemophilus influenzae in the nasopharynx.

OBJECTIVES: Investigate immune responses of adenoidal lymphocytes against outer-membrane protein P6 purified from nontypeable Haemophilus influenzae (HI). Clarify the role of adenoids in regulating the colonization of HI in the nasopharynx. STUDY DESIGN: Microbiological and immunological examinations of adenoids obtained from 21 children, 15 boys and five girls, from 1 to 13 years of age (median, 5 y), suffering from adenoidal hypertrophy complicated by otitis media with effusion (OME). METHODS: The incidence of HI in adenoids was compared with the number of P6-specific immunoglobulin (Ig) A-secreting cells in adenoids, determined by enzyme-linked immunoassay. RESULTS: Quantitative culture assay showed significant correlation between the numbers of HI in adenoids and those in nasopharyngeal secretions (NS). In children aged 5 years and younger, the numbers of P6-specific IgA-secreting cells in adenoids were significantly correlated with IgA antibody titers in NS (r = 0.68, P < .05). The numbers of P6-specific IgG- and IgA-secreting cells were lower in children aged 6 years and older than in children aged 5 years and younger. Furthermore, the number of P6-specific IgA-secreting cells was significantly increased in HI-negative subjects when compared with HI-positive subjects (P < .05). CONCLUSIONS: Adenoids play an important role as an effector site of the mucosal immune system in the upper respiratory tract. IgA immune responses in adenoids are responsible for the clearance of HI from the nasopharynx.

Effects of clarithromycin on cultured human nasal epithelial cells and fibroblasts.

OBJECTIVE/METHODS: Long-term administration of clarithromycin has been reported to be effective in the treatment of chronic sinusitis. To investigate the mechanism underlying the anti-inflammatory activity of clarithromycin, the authors evaluated the effect of clarithromycin on the gene expression of proinflammatory cytokine and the DNA-binding activity of nuclear factor (NF)-kappa B in cultured human nasal epithelial cells and fibroblasts. Cells were incubated with endotoxin purified from nontypable Haemophilus influenzae or interleukin (IL)-1 beta in the presence of clarithromycin. RESULTS: Northern blot analysis revealed that clarithromycin suppressed IL-1 beta gene expression in human nasal epithelial cells stimulated by H. influenzae endotoxin (HIE). Intercellular adhesion molecule-1 gene expression in nasal fibroblasts stimulated by IL-1 beta was also suppressed by clarithromycin. Furthermore, electrophoretic mobility shift assay demonstrated that clarithromycin reduced DNA-binding activity of NF-kappa B in both human nasal epithelial cells and fibroblasts stimulated by HIE or IL-1 beta, respectively. CONCLUSION: The present results suggest that clarithromycin may reduce gene expression of proinflammatory cytokines and adhesion molecules from nasal mucosa at the transcriptional factor level and exert an anti-inflammatory effect on nasal mucosa in chronic sinusitis.

Reflectance spectrophotometric studies on mucosal pathology of the upper airway.

Reflectance spectrophotometry analysis has been applied to the human nasal mucosa in vivo to determine the objective parameters of the colors of the mucous membrane, as well as to study the hemodynamics and enzymatic activities related to respiratory chain metabolism in the nasal mucosa. In 24 normal subjects, there were three main spectral peaks: the peak at around 500 nm (PI), the smallest at 560 nm (P2) and the highest one at 660 nm (P3). In 28 patients with nasal allergy, spectral P3 shifted remarkably toward the shorter wavelength at around 640 nm, compared to that of normal subjects. Additionally, the increment of the absorption band at P2 was also observed. These phenomena could be seen in the nasal mucosa of the subjects after use of decongestant spray. Reflectance spectrophotometric analysis will serve as a non-invasive method of giving us useful information for a further understanding of the mucosal pathophysiology of the upper airway.

Staphylococcus epidermidis and Staphylococcus aureus in otitis media with effusion.

Bacteriologic investigation of middle ear effusion (MEE), external ear canal, and the nasopharynx was carried out on 458 patients with otitis media with effusion. Staphylococcus epidermidis was the most common bacteria in MEE, even after excluding the contaminants from the external ear canal, which had the same value of minimal inhibitory concentration as the paired MEE. The bacterial agreement of S epidermidis between MEE and the nasopharynx was extremely rare in contrast with Haemophilus influenzae, Streptococcus pneumoniae, and Branhamella catarrhalis, although the organism was also frequently isolated from the nasopharynx. Staphylococcus aureus, having the same minimal inhibitory concentration as that in the nasopharynx, was more frequently found in MEE than S epidermidis. The results suggest that S epidermidis found in MEE is not a pathogen, but rather a contaminant in many instances. Staphylococcus aureus seems to be a causative agent in otitis media with effusion.

The role of bacterial adherence in otitis media with effusion.

Adherence of nontypable Haemophilus influenzae and Streptococcus pneumoniae to nasopharyngeal epithelial cells was investigated in vitro. Both strains had higher affinity to the epithelial cells of children than to those of adults. In children, the adherence was significantly greater in patients with otitis media with effusion than in normal subjects. Secretory IgA in nasopharyngeal secretions was found to have antibody activity against the bacteria. Adherence of both bacteria was significantly smaller in the group having secretory IgA antibody activity than in the group having no activity. These results suggest that bacterial adherence to the nasopharynx may play an important role in the pathogenesis of otitis media with effusion in children, and that secretory IgA in nasopharyngeal secretions may be related to the decrease of adherence.

Effect of cyclodextrins on the acid hydrolysis of digoxin.

The effects of three cyclodextrins (alpha-, beta-, gamma-CyD) on the acid hydrolysis of digoxin were examined. From the high performance liquid chromatographic tracing of each of the four components (digoxin, bisdigitoxoside, monodigitoxoside, digoxigenin) in reaction mixtures, the individual rate constants (K1-K6) were determined by analogue computer simulation. The hydrolysis was suppressed by CyDs in the order of beta-great than gamma-greater than alpha-greater than-CyD, where beta-CyD inhibited the appearance rates of digoxigenin (k3, K5, and K6) significantly. In the dissolution study of digoxin tablets, the increase in dissolution rate and decrease in acid hydrolysis were attained by inclusion complexation. The data are presented suggesting that CyDs are useful for improving the oral bioavailability of digoxin.

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.

Secretory IgA and serum type IgA in nasal secretion and antibody activity against the M protein.

We studied IgA immunoglobulins in nasal secretions in order to clarify mucosal immunity of the nasal cavity and paranasal sinuses during chronic nasal infection. Secretory IgA and serum type IgA of 165 samples of nasal secretions were analyzed quantitatively by use of electroimmunodiffusion techniques, and the specific antibody activity of secretory IgA against the M protein of Streptococcus pyogenes was investigated by use of enzyme-linked immunosorbent assay. Results show that although the secretory IgA content in nasal secretions was elevated in chronic sinusitis, its specific antibody activity against the M protein was lower than that in normal subjects. This evidence suggests that nonspecific secretory IgA antibodies are predominantly produced in chronic sinusitis, and that mucosal immunity preventing the adherence of bacteria is impaired in the diseased mucosa.

Secretory IgA and bacterial adherence to nasal mucosal cells.

The ability of Streptococcus pyogenes to adhere to nasal mucosal cells was investigated by an in vitro assay system in order to clarify the actual role of secretory IgA in mucosal immunity in the nose. The number of bacteria adhering to isolated mucosal cells was significantly larger in patients with chronic sinusitis than in normal control subjects. The ability of bacteria to adhere, however, was significantly lower in the group of subjects having specific secretory IgA antibody activity to the M protein than in those having no secretory IgA activity. Findings demonstrated that mucosal immunity preventing or blocking the adherence of bacteria is impaired in patients with chronic sinusitis.

Inhibition of bacterial adherence by nasopharyngeal secretions.

The role of secretory immunoglobulin (Ig) A in nasopharyngeal secretions in the adherence of Streptococcus pneumoniae and Hemophilus influenzae to nasopharyngeal epithelial cells was investigated in vitro. The adherence was remarkably reduced by treating bacteria with nasopharyngeal secretions, and the antiadhesive activity was significantly greater in nasopharyngeal secretions having secretory IgA antibody activity against bacteria than in those having no activity. Noticeable changes were not observed in the antiadhesive activity caused by absorption of IgG from nasopharyngeal secretions. Results suggest that secretory IgA in nasopharyngeal secretions is related to bacterial adherence and adds to the prevention of nasopharyngeal infections.

Role of superoxide dismutase in otitis media with effusion.

The concentration of superoxide dismutase (SOD) in middle ear effusion (MEE) was measured by the electron spin resonance trapping method in order to clarify the role of SOD in otitis media with effusion (OME) in children. The SOD levels in mucoid MEEs were significantly higher than those in serous and purulent MEEs. There was no significant difference in the levels of SOD between neutrophil-dominant MEEs and mononuclear cell-dominant MEEs, and the levels were negatively correlated with the number of neutrophils in the MEEs. Moreover, the levels were significantly increased in patients having recurrence of MEE within 3 months compared with patients without recurrence of MEE. Although it is known that SOD plays an important role in protecting the host from oxygen radicals, the findings in this study suggest that SOD might be related to the chronicity of OME.

Role of interleukin-1beta in a murine model of otitis media with effusion.

To clarify the role of interleukin-1beta (IL-1beta) in the pathogenesis of otitis media with effusion (OME), we developed and investigated a murine model of this disease. Specific pathogen-free male BALB/c mice received intratympanic injections of 20 microg of endotoxin derived from nontypeable Haemophilus influenzae. Three days after injection, middle ear effusions were observed through the eardrum. Similar pathological changes were observed after inoculation with 100 ng of recombinant IL-1beta. Anti-IL-1 receptor antibodies inhibited the pathological changes induced by the endotoxin. In situ hybridization showed expression of IL-1beta messenger RNA in the epithelium of the middle ear mucosa. These results suggest that IL-1beta might be associated with endotoxin-induced inflammation in the middle ear and might play an important role in the induction of otitis media with effusion.