Biomedical subjects
Publications and source records attributed to F Kokubu.
Viral infection induces airway inflammation. It is possible that bronchial epithelium derived chemokine contributes to the migration and accumulation of inflammatory cells in the airway viral infection. We infected bronchial epithelial cells with influenza virus and analysed mRNA expression and production of RANTES. The expression of mRNA and the production of RANTES were detected in infected cells using RT-PCR method and ELISA.
Bronchial asthma is characterized as a chronic inflammation of the airway that causes an infiltration of lymphocytes and eosinophils. Cell to cell interaction or cell to tissue interaction is essential for infiltration of eosinophils to underlying tissues. These phenomena are closely related to the expression of intercellular adhesion molecule-1 (ICAM-1). Inhalation of steroids, such as beclomethasone dipropionate, is commonly used to cure airway inflammation. In this study, we investigated the effect of cytokines on ICAM-1 expression on human bronchial epithelial cell lines, NCI-H292. Moreover, the effect of dexamethasone on ICAM-1 expression stimulated by IL-1 beta, TNF-alpha and IFN-gamma was observed. Treatment with IL-1 beta, TNF-alpha and IFN-gamma dose-dependently increased ICAM-1 expression on NCI-H292 cells. Inhibitory effects were exerted by dexamethasone on ICAM-1 expression in cells stimulated by IL-1 beta and IFN-gamma in a dose-dependent manner, but not in cells stimulated by TNF-alpha. These results suggest that the inhibition of ICAM-1 expression could be related to the pharmacological action of steroid drugs.
The clinical study and in vitro study used with leukocytes were made of a case of food-dependent exercise-induced anaphylaxis induced by shrimp. A 26-year-old man experienced anaphylactic reaction of nasal obstruction, face edema and dyspnea while running 90 minutes after eating shrimp. He experienced similar episodes two years ago in his past history. IgE-RAST was positive for shrimp. Anaphylactic reaction and elevation of plasma histamine levels were verified by exercise challenge test after eating 100 g shrimp. At the same time, we verified the dedine of plasma cAMP levels after eating shrimp. In leukocyte stimulating test used with shrimp antigen, histamine level elevations, which were lower compared with calcium ionophore A23187 (Ca I 10(-6) M) stimulation, were recognized in dose dependent manner in this patient. But in normal subject, histamine level elevations were not recognized. We diagnosed him food-dependent exercise-induced anaphylaxis. It was suggested that there was relation between histamine release and decline of cAMP levels of plasma after eating shrimp in this case.
Inflammation of mucosa and epithelial damage the of bronchi are characteristic pathological features of asthma. Infiltration of T lymphocytes and eosinophils followed by their activation and release of cytokines seems to be a contributing process. We studied the interaction of interleukin 2 (IL-2) and interleukin 5 (IL-5), cytokines which act on eosinophils, using human peripheral lymphocytes. We found that cyclosporin A (CyA) inhibited the IL-5 production from human peripheral lymphocytes induced by IL-2 stimulation. Human peripheral lymphocytes were isolated from healthy volunteers and bronchial asthma patients with mild eosinophilia. Lymphocyte cultures stimulated with IL-2 were cultured at 37 degrees C in a humidified atmosphere of 5% CO2 in air. After 72 hours' incubation, the proliferative response of the lymphocytes was examined by [3H]thymidine uptake, and at the same time IL-5 in the supernatant of the culture medium was assayed by ELISA. The lymphocytes proliferated on IL-2 stimulation in all cases; IL-5 was detected in 3 out of 9 healthy volunteer cases and 7 out of 11 asthma patient cases. CyA added at the beginning of incubation inhibited both of these responses in a dose-dependent manner.
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We have previously demonstrated that airway responsiveness was enhanced following a late bronchial response (LBR) after an allergen challenge in ovalbumin (OA)-sensitized guinea pigs. The purpose of the present studies was to evaluate whether airway responsiveness to methacholine increased after an immediate bronchial response (IBR) and the possible involvement of the beta-adrenoceptor dysfunction in OA-sensitized guinea pigs. Guinea pigs were actively sensitized by aerosolized OA. Following OA exposure, IBR appeared. After IBR when specific airway resistance returned to the base line value, airway responsiveness to methacholine increased significantly. Before OA exposure, propranolol induced bronchoconstriction (PIB) was not provoked, however, after IBR, PIB was provoked and the guinea pigs died because of severe bronchoconstriction. These results suggest that airway responsiveness to methacholine increases significantly after IBR. Furthermore, the dysfunction of the beta-adrenoceptor may be a mechanism of this hyperresponsiveness in OA-sensitized guinea pigs.
A sulpyrin inhalation test was given to 13 patients with aspirin-induced asthma (AIA) and 8 patients with non-aspirin-induced asthma (non-AIA) to observe the changes in plasma histamine levels before and after challenges. The respiratory function (FEV1.0) was measured before and after sulpyrin inhalation. A decrease of more than 20% the initial value (basal value) was defined as a positive response. Plasma histamine was determined by high-performance liquid chromatography (HPLC). In 11 patients with AIA, a positive response was observed (SIT positive), with a fall of FEV1.0 to 63.70 +/- 4.87% of the basal value. In 2 patients with AIA and 8 patients with non-AIA, no positive response was observed (SIT negative). In patients with SIT positive, plasma histamine levels increased significantly from 0.61 +/- 0.06 ng/ml before challenges to 1.34 +/- 0.22 ng/ml after challenges (p less than 0.01). No significant changes of plasma histamine occurred in the SIT negative patients. These results suggest that mast cells play some role in the mechanism of the development of aspirin-induced asthma.
An (125I) iodohydroxybenzyl pindolol (125IHYP) binding inhibition assay was performed. Various dilutions (1:5-1:500) of sera from asthmatics and controls were incubated with canine lung membranes for 60 minutes at 30 degrees C. 125IHYP was added to the membranes for 30 minutes at room temperature in the presence and absence of 10 microM 1-propranolol, and the samples were washed through a Gelman (Type A-E) glass fiber filter using a washing buffer. Radio activity was measured with Aloka gamma counter. In the presence of various serum dilutions from asthmatics, 125IHYP specific bindings of 0.16 fmol to 4.38 fmol were measured. 125IHYP binding was inhibited in a dose-related and nonspecific manner. Serum, albumin, L-histidine and L-cysteine also inhibited 125IHYP specific binding to beta-receptors. Percentages of inhibition of serum from asthmatics on 125IHYP specific finding to beta-receptors were -17.6% to +9.3%, which were compared with identical dilutions of control serum. There was no significant difference in 125IHYP binding inhibition assay between asthmatics and controls. From these results, development of autoantibody to beta-adrenergic receptors could not be detected in this study.
Immunoglobulin (Ig) gene organization in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, is unique. Homologous Ig heavy chain variable (VH) and constant region (CH) specific probes were used to screen a spleen cDNA library constructed in lambda gt11. Both secretory (SEC) and transmembrane (TM) cDNA clones were recovered; the latter were identified by a negative selection strategy. The complete sequence of the CH portion of a Heterodontus genomic DNA-lambda clone also was determined. The sequences of the individual CH genes differ from each other in all exons. When compared to mammalian prototypes, similarities in exon and intron organization as well as conservation of sequences involved with differential splicing of SEC and TM mRNA indicate that Heterodontus heavy chain genes are of the mu type, although intron lengths are uniformly longer in Heterodontus. Heterodontus genes are not associated, however, with the family of DNA sequences that have been implicated in heavy chain class switching in mammals. Spleen cDNA library screening and RNA blot analyses indicate that mRNAs encoding TM Ig are exceedingly rare. The relationship between this quantitative difference and the distribution of polyadenylation signal sequences suggests that regulation of Ig gene expression in Heterodontus may be highly dependent on position effects.
The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH-D1-D2-JH-CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH-JH-CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH-CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH-D1-D2-JH-CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N-type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T-cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene.
A homologous probe for the constant region of the Heterodontus francisci (horned shark) immunoglobulin heavy chain was used to screen a genomic DNA library constructed in bacteriophage lambda, and a large number of independent clones were recovered. Their hybridization patterns with segment-specific probes are consistent with the close linkage of heavy-chain constant (CH), joining (JH), and variable (VH) gene segments. Differences in the nucleotide sequences of the first CH exon of five genes primarily are localized to 5' positions; extended regions of sequence identity are noted at 3' positions. The predicted amino acid sequences of each gene are different and are related distantly to the corresponding regions of higher vertebrate immunoglobulins. Gene-specific oligodeoxynucleotide probes were used to establish that at least three of the five genes are transcriptionally active. Quantitative gene titration data are consistent with the large numbers of genes suggested by the library screening analyses. In this representative early vertebrate, it appears that (VH-diversity-JH) segments are associated with individual constant region genes that can differ at the predicted protein level.
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Ten patients with allergic asthma were studied to compare the inhibitory effects of ketotifen and disodium cromoglycate on bronchial responses, especially the late asthmatic response, induced by house dust allergen in bronchial provocation tests. In the first study, types of bronchial response to house dust challenge were classified from peak expiratory flow rate measurements made at intervals after the test. Nine patients showed dual asthmatic response (immediate with isolated late response) and 1 patient showed isolated late asthmatic response. The tests were treated at 1-week intervals, patients having been premedicated from the night before the test with either ketotifen (3 X 2 mg oral doses) or disodium cromoglycate (3 X 40 mg by inhalation). The results showed that both drugs produced significant protection against the immediate bronchial response and that ketotifen was at least as effective as disodium cromoglycate in inhibiting the late asthmatic response.
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BACKGROUND: Interleukin (IL)-10 induces a long-term antigen-specific anergy in human CD4+ T cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from house dust mite (Dermatophagoides farinae, Der f)-sensitized asthmatic patients. PBMCs were stimulated with Der f antigen for 72 h immediately after purification or after 48 h of resting culture with medium, and IL-10 and IL-5 in the culture supernatant were measured. PBMCs were also stimulated with Der f antigen for 7 days either immediately after purification or after 48 h of resting culture, after which cells were collected. Secondary proliferative responses of these cells to stimulation for 3 days with Der f antigen and mitomycin C-treated PBMCs as antigen-presenting cells or with phorbol myristate acetate (PMA) plus calcium ionophore were investigated. RESULTS: Stimulation of PBMCs with Der f antigen immediately after purification significantly increased the proliferative response and IL-5 production. Stimulation of PBMCs with Der f antigen after resting culture with medium alone for 48 h significantly decreased IL-5 production and markedly increased IL-10 production. Although activation of cells with Der f antigen immediately after purification significantly increased secondary proliferative responses, stimulation after 48 h of resting culture failed to increase secondary proliferative responses. However, proliferation recovered when cells were activated with PMA plus calcium ionophore. CONCLUSION: These results suggest that antigen-specific Th2 cells are anergized by IL-10 and that Th2 cell tolerance may suppress eosinophilic inflammation in allergic asthma.
Bronchial epithelial cells play an important role in the pathogenesis of some inflammatory diseases of bronchial mucosa. Epithelial-cell-derived cytokines are important in the elucidation of the mechanism by which airway inflammation occurs, especially in respiratory virus infection, because these cells are the primary sites of viral infection. We infected bronchial epithelial cells, NCI-H292, with influenza virus A (H3N2) and examined the concentrations of cytokines, interleukin-6 (IL-6), IL-8 and regulated on activation, normal T cells, expressed and secreted (RANTES), in the culture media of infected cells using the enzyme-linked immunosorbent assay system and gene expression of RANTES on epithelial cells by the reverse-transcriptase-polymerase chain reaction method. We found that significant amounts of IL-6, IL-8 and RANTES were released. RANTES mRNA was also detected in infected bronchial epithelial cells. It is suggested that cytokine production in human bronchial epithelial cells may contribute to the pathogenesis of airway inflammatory disorders.