Circulating intracellular adhesion molecule-1 concentrations following bronchial provocation in atopic asthma.

A house dust bronchial provocation test (BPT) was used to investigate the effect of allergen-induced airway inflammation and airway hyperresponsiveness (AHR) on the level of circulating intracellular adhesion molecule-1 (c-ICAM-1). The concentration of c-ICAM-1 was measured by the sandwich ELISA while the level of eosinophil cationic protein (ECP) in the sputum was determined by RIA. The parameter used for quantification of AHR was the minimum dose of methacholine (Mch) required to produce a fall in respiratory resistance and was expressed as log Dmin. Fourteen subjects with mild atopic asthma participated in this study. Ten patients (dual asthmatic response group; DAR group) developed a late asthmatic response (LAR) following an immediate asthmatic response (IAR). Four subjects (IAR alone group) exhibited only IAR following BPT. In both groups, the mean baseline concentration of c-ICAM-1 did not change 6 h after BPT (from 195.3+/-20.3 to 220.9+/-27.6 and from 215.5+/-23.5 to 231.3+/-30.5 ng/ml, respectively). However, BPT produced a significant increase in the mean concentration of c-ICAM-1 24 h later in the DAR group (257.3+/-41.14 ng/ml, p < 0.05), but not in the IAR alone group (225.5+/-18.1 ng/ml). BPT also increased ECP levels in the sputum from a baseline value 24 h after BPT in the DAR group (from 30.2+/-10.1 to 68.8+/-19.8 ng/ml; p<0.05), but not in the IAR alone group (from 28.1+/-8.3 to 43.3+/-23.7 ng/ml). There was a significant (p<0.05) correlation between c-ICAM-1-concentrations and sputum ECP levels 24 h after BPT in each group. Furthermore an inverse and significant (p<0.05) correlation was found between c-ICAM-1 concentrations and percent changes in log Dmin 24 h after BPT in each group. Our results suggest that increased concentrations of c-ICAM-1 after BPT may reflect the upregulated expression of airway ICAM-1 during allergen-induced airway inflammation. We propose that c-ICAM-1 is a useful marker for allergic inflammation, particularly that of eosinophilic infiltration into the airway, an essential feature of asthma.

[Endothelin-1 levels in sputum and plasma of asthmatic patient after allergen provocation].

Recently, endothelin-1 (ET-1) has a potent of contractive effect to bronchial smooth muscle, and it suggests that ET-1 contributes to pathophysiology of bronchial asthma. To study the role of endothelin-1 (ET-1) in allergic asthma, we measured ET concentration in plasma and in sputum of nine allergic subjects following allergen provocation by using sandwich-enzyme immunoassay (EIA). ET-1 concentration in plasma were higher during IAR (2.31 +/- 0.24 pg/ml) (0.05 < p < 0.1) and LAR (2.55 +/- 0.27 pg/ml) (0.05 < p < 0.1) than prechallenge value (1.99 +/- 0.23 pg/ml) but there was no significant difference. On the other hand ET-1 concentration in sputum were significantly higher during IAR (14.75 +/- 2.77 pg/ml) (p < 0.05) and LAR (18.51 +/- 4.57 pg/ml) (p < 0.05) than prechallenge values (4.29 +/- 2.55 pg/ml). Thus these results suggest that ET-1 play a role of allergic bronchoconstraction (IAR and LAR) after allergen challenge.

Cytokine concentrations in sputum of asthmatic patients.

To examine whether levels of inflammatory cytokines and eosinophil cationic protein (ECP) in the sputum reflect the severity of bronchial asthma, we measured their levels in the sputum of symptomatic and asymptomatic asthmatics. Interleukin (IL)-1 beta, IL-5, IL-6, IL-8, RANTES, tumor necrosis factor-alpha and ECP concentrations in the sputum of symptomatic patients were significantly higher than in asymptomatic subjects. These findings suggest that these inflammatory cytokines are involved in the exacerbation of asthma.

Effect of azelastine on endotoxin-induced airway hyperresponsiveness in mice.

We examined the role of lipopolysaccharide (LPS) on the pulmonary inflammatory process in mice, including the release of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) by macrophages, and investigated the mechanism of action of azelastine (AZ) on the release of these two cytokines. Intratracheal instillation of 1.0 microgram/ml LPS into BALB/c mice caused a significant increase in both IL-1 beta and TNF-alpha in aqueous lung extracts. These changes were modified, under control conditions, by a single dose of 0.05 mg/kg of AZ administered 1 and 11 h after LPS infusion. Intratracheal instillation of LPS also caused a significant increase in bronchial hyperresponsiveness to methacholine (Mch). AZ significantly inhibited Mch responsiveness in LPS-infused mice compared with nontreated control mice. Our results suggested that intratracheal instillation of LPS induces the secretion of macrophage cytokines in the airways of mice, accounting, at least partly, for LPS-induced airway hyperresponsiveness. Our results also indicate that the attenuating effect of AZ on LPS-induced airway hyperresponsiveness could be explained by its inhibitory effect on macrophage cytokine production.

Effect of IPD-1151T (Suplatast Tosilate) on airway hyperresponsiveness in mice.

We investigated the effect of IPD-1151T (Suplatast Tosilate, (+/-)-[2-[4-(3-ethoxy-2-hydroxypropoxy) phenyl-carbamoyl]-ethyl] dimethylsulfonium p-toluenesulfonate) against allergic bronchoconstriction induced by allergen and methacholine (MCh).BALB/c mice were sensitized by intraperitoneal injection of 0.5 microgram of keyhold limpet hemocyanin mixed with Al(OH)3. IPD-1151T was administered orally once a day for 5 or 14 days in doses of 10, 30 or 100 mg/kg body weight. Bronchoconstriction was measured 24 hrs after the final drug administration. IPD-1151T inhibited both antigen-mediated allergic bronchoconstriction and induction of bronchial hyperresponsiveness after sensitization or allergen challenge in actively sensitized mice. The degree of inhibition was proportional to the dose and frequency of oral administration of the agent.

Effect of methotrexate on asthmatic reaction in sensitized guinea pigs.

To understand the mechanism of antiasthmatic property of the antimetabolite agent, methotrexate (MTX), we examined its effect on time-related changes in specific airway resistance, bronchial responsiveness, and accumulation of lymphocytes and eosinophils in lung tissue and the bronchial lumen, before and after antigen challenge in ovalbumin (OA)-sensitized guinea pigs. Intraperitoneal administration of MTX significantly inhibited the antigen-induced late asthmatic responses (LAR) in actively sensitized animals in dose-dependent manner. Examination of bronchoalveolar lavage fluid (BALF) revealed that 0.25, 0.5 and 1.0 mg/kg body weight of MTX significantly inhibited the recruitment of eosinophils, lymphocytes (6 and 24 h after antigen challenge) and neutrophils (0.5, 6 and 24 h after antigen challenge) in the airways in a dose-dependent manner. Histological examination of lung tissue revealed that MTX significantly inhibited eosinophil infiltration into the airway (6 and 24 h after antigen challenge). Furthermore, MTX significantly inhibited the infiltrations of PKH-2-labeled peripheral blood mononuclear cells (mostly lymphocytes) into the airways (24 h after antigen challenge). MTX also inhibited airway hyperresponsiveness to methacholine following OA challenge in a dose-dependent manner. We conclude that the antiasthmatic effect of methotrexate is mainly due to inhibition lymphocytes and eosinophil infiltration into the airway.

Effect of roxithromycin on respiratory bacterial infection in mice.

The effects of roxithromycin (RXM), an antibiotic of the macrolide family, on respiratory bacterial infection in mice were examined. BALB/c mice were administered with RXM orally at a dose of 5.0 or 2.5 mg/kg once per day for 14 days. On day 2 after the final drug administration, the mice were nasally infected with Haemophilus influenzae. RXM dose dependently inhibited the pathological changes in lung tissues induced by H. influenzae infection. RXM also enhanced 2',5'-oligoadenilate synthetase production in response to infection.

[Studies on biphasic animal model of asthma--lymphocytes and eosinophils].

To clarify the role of lymphocytes and eosinophils in the late asthmatic response (LAR) observed several hours after OA challenge, and in airway inflammation observed 24 hrs after OA challenge in OA sensitized guinea pigs, we examined the infiltration and/or accumulation of lymphocytes and eosinophils in lung tissues by PKH2 labeling technique and Luna staining technique, respectively. The guinea pigs showed LAR 6 hrs after exposure to antigen, when moderate infiltration of lymphocytes and marked accumulation of eosinophils were observed in lung tissues. At 24 hrs after OA challenge, intensive infiltrations of both lymphocytes and eosinophils into the lung were observed. Pretreatment with MTX (methotrexate) suppressed LAR, inhibited infiltrations of lymphocytes and eosinophils into lung tissues at 6 hrs (LAR) and at 24 hrs after OA challenge, and prevented bronchial hyperresponsiveness to methacholine 48 hrs after antigen exposure in a dose-dependent manner. These findings suggest that lymphocytes and eosinophils are involved in LAR and also in the chronic airway inflammation which is believed to make the bronchial asthmatic condition more severe and more refractory. Also, our results showed that MTX, which has an inhibitory action on such inflammation, may be a useful bronchial asthma.

Genetic polymorphisms of α- and ß-amylase isozymes in wild emmer wheat, Triticum dicoccoides, in Israel.

α- and ß-amylase isozyme diversity was studied electrophoretically by thin-layer polyacrylamide gel isoelectrofocusing in the tetraploid wild emmer wheat, Triticum dicoccoides, the progenitor of all cultivated wheats. We analyzed 225 plants from 23 populations encompassing the ecological spectrum of T. dicoccoides in Israel. The results were as follows: (a) Band and multilocus genotype polymorphisms abound and vary within and between the four amylase components: malt, green (α-amylases), and dry and germinating seeds (ß-amylases). (b) The number of bands of malt, green, and dry and germinating seeds were 20, 6, 11 and 13, respectively, generating 40, 6, 51, and 51 patterns or multilocus genotypes (MGP), respectively. The MGPs vary drastically within and between populations, from monomorphic in some populations with a single pattern to highly polymorphic ones, (c) Mean H e values for malt, green, and germinating and dry seeds are 0.053, 0.055, 0.088, and 0.077, respectively; mean number of bands per individual was 11.8, 4.4, 7.6, and 4.0, respectively, (d) The percentages of 50 bands and 148 multilocus genotype patterns (MGP) (in parenthesis) were classified into widespread, sporadic, and localized: 84.4 (10.8), 8.9 (12.2), 6.7 (77.0), respectively. Notably, 89.2% of the patterns were not widespread, but sporadic and localized, (e) The mean value of genetic distances among populations (Nei's D) for the four amylase groups is D = 0.136, 0.175, 0.288 and 0.307, respectively, not displaying geographical correlates. (f) Most of the α- and ß-amylase diversity is between populations (G st = 68-75%). (g) Significant environmental correlates occur between either bands or patterns and climatic diversity (water and primarily temperature factors). (h) Significant associations of multilocus amylase bands occur across Israel. Like-wise, significant gametic phase disequilibria, D, occur within populations and are positively correlated with climatic variables, primarily that of temperature, (i) Discriminant analyses correctly classified (95-100%) the 23 wild emmer populations into their ecogeographical region and soil type. (j) Autocorrelation analysis showed that there is no correlation between bands and geographic distance and excluded migration as a major factor of amylase differentiation.These results suggest that diversifying climatic and edaphic natural selection rather than stochastisity or migration is the major evolutionary force driving amylase differentiation at both the single and multilocus levels. Furthermore, wild emmer harbors high levels of α- and ß-amylase diversity both as single bands and as multilocus adaptive genetic patterns. These are exploitable both as genetic markers for quantitative loci (QTLs) and as adaptive genetic resources to improve wheat germination and growth through classical breeding and/or biotechnology.