Different role of serum components and cytokines on alveolar macrophage activation by soluble fungal (1-->3)-beta-D-glucan.

In this study, we investigated the mechanism of alveolar macrophage activation by systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395. Multiple i.v. administration (10 mg/kg; once daily for 10 consecutive days) of SSG enhanced some functions of alveolar macrophages, such as lysosomal enzyme activity and nitric oxide secretion, on day 1 after the last administration, and it also elevated the concentrations of serum protein, interferon gamma and SSG in bronchoalveolar lavage fluid on the same day. On the in vitro assay system, stimulation by SSG alone (500 microg/ml) slightly augmented the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide production of cells. Stimulation by serum (1 or 10% mouse serum) or serum components, such as fibronectin (25 microg/ml) and albumin (500 microg/ml), alone strongly augmented only the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide secretion from cells, and no synergism or additive-like effect was observed between serum components and SSG. In contrast, stimulation by crude lymphokine (5%) or recombinant murine interferon gamma (100 U/ml) alone did not induce augmentation of lysosomal enzyme activity and nitric oxide production of alveolar macrophages in vitro, but when cells were incubated together with crude lymphokine or recombinant murine interferon gamma and SSG (500 microg/ml), a significant combined effect was observed on both functions of alveolar macrophages. In addition, pretreatment of crude lymphokine or recombinant murine interferon gamma enhanced the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface in vitro though pretreatment by serum components had no effect. Based on these findings, the enhancement of alveolar macrophage functions by systemic administration of SSG appears to be mediated, at least in part, by both the simple effect of serum components including fibronectin and albumin leaked from pulmonary peripheral blood into the alveoli and the synergistic effect between lymphokines released from activated pulmonary T cells and SSG itself entering the alveoli after SSG injection via the priming effect of lymphokines which enhances the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface.

Roles of mast cells and sensory nerves in cutaneous vascular hyperpermeability and scratching behavior induced by poly-L-arginine in rats.

We investigated whether the polycation poly-L-arginine elicited cutaneous vascular hyperpermeability and scratching behavior and, if so, whether these responses involved mast cells and sensory nerves in rats. Intradermal injections of poly-L-arginine induced vascular hyperpermeability and scratching behavior. Combined treatment with chlorpheniramine and methysergide almost completely suppressed the poly-L-arginine (50 microg/site)-induced plasma leakage. Capsaicin desensitization and the tachykinin NK(1) receptor antagonist LY303870, (R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane, partially inhibited the leakage. In mast cell-deficient rats, poly-L-arginine only minimally induced plasma leakage. On the other hand, capsaicin desensitization and LY303870, but not chlorpheniramine or methysergide, suppressed the poly-L-arginine (200 microg/site)-induced scratching. Moreover, poly-L-arginine elicited the scratching even in mast cell-deficient rats. These results suggest that substance P is at least partly involved in both the cutaneous plasma leakage and the scratching behavior induced by poly-L-arginine. Moreover, mast cell-derived amines are suggested to be involved in the plasma extravasation but scarcely, if any, in the scratching behavior.

Erdosteine enhances mucociliary clearance in rats with and without airway inflammation.

Erdosteine is a new homocysteine-derived expectorant and has been reported to have many mucolytic effects. In this report, we studied the activities of erdosteine on mucociliary clearance in normal and airway-inflammation-induced rats. In normal rats, erdosteine at doses of 100-600 mg/kg significantly promoted mucociliary clearance. However, erdosteine did not change the concentrations of mucopolysaccharides in bronchoalveolar lavage fluid (BALF). In the LPS-instillated rats, the mucociliary clearance was inhibited and the number of inflammatory cells, albumin concentration, and mucopolysaccharides concentration in BALF were increased. Erdosteine at doses of 100-600 mg/kg significantly attenuated the inhibition of mucociliary clearance and the increase of inflammatory cells, however, it did not prevent the increase of albumin and mucopolysaccharides. Other mucolytic drugs which are ambroxol and S-carboxymethylcysteine, had no effect. These results indicate that erdosteine promotes the mucociliary clearance in normal and airway-inflammation-induced rats.

Study of in vitro cytotoxicity of a water soluble organic arsenic compound, arsenosugar, in seaweed.

In the present study, we demonstrated the cytotoxic effect of a dimethylarsenic compound in seaweed, (R)-(2',3'-dihydroxypropyl) 5- deoxy-5-dimethylarsinoyl-beta-D-riboside, namely arsenosugar (AsSug), on mammalian cells, murine macrophages, in comparison with that of an inorganic arsenical, arsenite, in vitro. More than 99.5% pure AsSug was synthesized. Arsenite was strongly and equally toxic to both peritoneal macrophages (PMs) and alveolar macrophages (AMs), and the concentration of arsenite that inhibited the viability of cells by 50% compared to the viability of control cells (50% inhibitory concentration; IC50) was 5 microM. In contrast, AsSug showed no cytotoxicity to both PMs and AMs at the microM concentration level; however, it induced different and interesting cellular responses in both macrophages at high concentrations, 1-10 mM. AsSug enhanced the viability of PMs at an optimal dose of 5 mM; conversely, it showed weak but significant cytotoxicity to AMs (IC50 = 8 mM).

Induction of chromosomal aberrations in cultured human fibroblasts by inorganic and organic arsenic compounds and the different roles of glutathione in such induction.

Clastogenic effects of a variety of arsenic compounds were examined on cultured human fibroblasts. The following compounds were tested: inorganic arsenicals (arsenite and arsenate), the major metabolites of inorganic arsenicals in human and experimental animals [methylarsonic acid (MAA), dimethylarsinic acid (DMAA) and trimethylarsine oxide (TMAO)], and water-soluble organoarsenic derivatives [2', 3'-dihydroxypropyl-5-deoxy-5-dimethylarsinoyl-beta-D-riboside (arsenosugar), arsenocholine, arsenobetaine and tetramethylarsonium iodide] found in marine organisms. Arsenic compounds induced mainly chromatid gaps and chromatid breaks. The rank order of compounds in terms of clastogenic potency was arsenite > arsenate > DMAA > MAA > TMAO. DMAA was very potent and caused chromosome pulverizations in most metaphases when present at doses higher than 7 x 10(-3) M. Arsenosugar, arsenocholine, arsenobetaine and tetramethylarsonium iodide were less effective. Depletion of cellular glutathione (GSH) with L-buthionine-SR-sulfoximine (BSO), increased the incidence of chromosomal aberrations induced by arsenite, arsenate and MAA, and markedly suppressed the clastogenic effects of DMAA. DMAA was highly clastogenic even in GSH-depleted cells when the cells were incubated with DMAA in the presence of GSH (5 and 10 mM). These results suggest that GSH might play a role in protecting cells against the clastogenic effects of arsenite, arsenate and MAA. GSH might be involved in the expression of clastogenic actions of DMAA.

Polymer-mediated extraction of 8-quinolinolato metal chelates for the determination of metal ions in water with graphite furnace atomic absorption spectrometry.

Water-insoluble 8-quinolinolato metal chelates were formed and were stably solubilized in the aqueous solution of a water-soluble polymer, poly (N-isopropylacrylamide)(PNIPAAm), at room temperature. When the solution was heated at 50 degrees C, PNIPAAm precipitated and then formed a gum-like aggregate (polymer phase) having a very small volume. Accompanying the polymer precipitation, hydrophobic 8-quinolinolato chelates with cobalt(II), iron(III), nickel(II), and copper(II) ions were efficiently incorporated into the polymer phase. At 0.5% (w/v) of PNIPAAm and 8.0 mM of 8-quinolinol, the recoveries in the incorporation of four metal chelates were quantitative. The fluorescence spectra of a probe suggests that the hydrated polymer in the aqueous solution provides hydrophobic portions which can incorporate hydrophobic metal chelates. The polymer phase was easily taken out from the solution and was dissolved with a small amount of acetonitrile. The resulting solution could be directly introduced into a graphite furnace of atomic absorption spectrometry. The signal intensities for the absorbance of cobalt after concentrating the chelate were 100-fold greater than those before the concentration.

Protective effect of erdosteine against hypochlorous acid-induced acute lung injury and lipopolysaccharide-induced neutrophilic lung inflammation in mice.

The effect of erdosteine, a mucoactive drug, on hypochlorous acid (HOCl)-induced lung injury, and the lipopolysaccharide (LPS)-induced increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the airway, was investigated. Male BALB/c mice were orally administered erdosteine (3-100 mgkg(-1)), ambroxol hydrochloride (ambroxol) (3-30 mgkg(-1)), S-carboxymethyl-L-cysteine (S-CMC) (100-600 mgkg(-1)) or prednisolone (10 mgkg(-1)), 1 h before intratracheal injection of HOCl or LPS. In the HOCl-injected mice, erdosteine markedly suppressed increases in the ratios of lung wet weight to bodyweight and lung dry weight to bodyweight, whereas the other mucoactive drugs ambroxol and S-CMC had little effect. Erdosteine also inhibited the LPS-induced neutrophil influx, although it did not affect the increased level of TNF-alpha in the bronchoalveolar lavage fluid. The results suggest that attenuation of reactive oxygen species and neutrophil recruitment is involved in the clinical efficacy of erdosteine in the treatment of chronic bronchitis.

Mucolytic and antitussive effects of erdosteine.

To investigate the influence of erdosteine, a new homocysteine-derived expectorant, on airway clearance we studied the effects of the drug on the viscosity of mucin, on the mucociliary transport rate in quails, on airway secretion in rats and on the cough reflex in guinea-pigs. The active metabolite of erdosteine, M1 (10 microM to 1 mM), significantly reduced the viscosity of porcine stomach mucin. Erdosteine by itself did not reduce viscosity. Erdosteine significantly promoted mucociliary transport in quails and increased airway secretion in rats. The effect was still apparent 24h after administration. Erdosteine significantly suppressed citric acid-induced cough reflexes in guinea-pigs but did not suppress mechanical stimuli-induced cough reflexes. Erdosteine suppressed the reduction of the recovery volume of bronchoalveolar lavage fluid and albumin leakage into the fluid in citric acid-exposed guinea-pigs. These results indicate that erdosteine removes sputum by reducing its viscosity, and by promoting mucociliary transport and sustained enhancement of airway secretion. It also suppressed the chemical stimulation-induced cough reflex and plasma leakage into the airway. These results suggest that erdosteine is an excellent expectorant with several modes of action.

Occurrence of organo-arsenicals in jellyfishes and their mucus.

Water-soluble arsenic compound fractions were extracted from seven species of jellyfishes and subjected to analysis by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for arsenicals. A low content of arsenic was found to be the characteristic of jellyfish. Arsenobetaine (AB) was the major arsenic compound without exception in the tissues of the jellyfish species and mucus-blobs collected from some of them. Although the arsenic content in Beroe cucumis, which preys on Bolinopsis mikado, was more than 13 times that in B. mikado, the chromatograms of these two species were similar in the distribution pattern of arsenicals. The nine species of jellyfishes including two species treated in the previous paper can be classified into arsenocholine (AC)-rich and AC-poor species. Jellyfishes belonging to Semaostamae were classified as AC-rich species.

Ability of histamine to increase nasal mucosal permeability to macromolecules in guinea pigs.

The effect of histamine on nasal mucosal permeability against an antigen was investigated by using modified passive cutaneous anaphylaxis (PCA) reactions in normal and actively sensitized guinea pigs. The administration of a dinitrophenyl-coupled Ascaris (DNP-Ascaris) solution as an antigen into the nasal cavity caused PCA reactions in the dorsal skin of normal guinea pigs. The administration of histamine into the nasal cavity before the antigen treatment significantly enhanced the anaphylactic responses. The PCA reactions did not occur when ovalbumin (OA) was administered intranasally in normal guinea pigs. In guinea pigs sensitized against DNP-Ascaris, however, PCA reactions to anti-OA antiserum were elicited by the intranasal administration of OA. The intranasal administration of histamine before the antigen treatment also enhanced anaphylactic responses in sensitized guinea pigs. These results indicate that histamine increases nasal mucosal permeability and that this may be one of the causes of nasal hypersensitivity in nasal allergy.

[The effect of KW-4679, an antiallergic drug, on experimental allergic rhinitis].

Effect of KW-4679, an antiallergic agent, on the experimental allergic rhinitis was studied in guinea pigs. 1) KW-4679 inhibited the sneeze response and the nasal rubbing induced after the intranasal administration of antigen in sensitized guinea pigs. The inhibitions were dose-related and significant at 0.03 mg/kg p.o. for sneeze response and 1 mg/kg p.o. for nasal rubbing, respectively. 2) The nasal vascular permeability was increased after the intranasal administration of antigen in sensitized guinea pigs. KW-4679 significantly reduced the increase of the nasal vascular permeability at 1 mg/kg p.o. or higher. From these results, KW-4679 may have inhibitory effects on sneeze, nasal irritation and rhinorrhea in clinical use.

Effect of oxatomide on experimental allergic rhinitis in guinea pigs.

We have investigated the effect of oxatomide, an antiallergic agent, on experimental allergic rhinitis in sensitized guinea pigs. Oxatomide (1 and 10 mg/kg, p.o.) significantly inhibited the sneeze response and nasal rubbing after antigen challenge. Oxatomide (10 and 30 mg/kg) reduced the increase in nasal vascular permeability induced by the antigen-antibody reaction. The decreases in nasal cavity volume caused by nasal mucosal swelling 10 min, 30 min and 6 hr after antigen challenge were significantly inhibited by oxatomide (30 mg/kg). These results indicate that oxatomide inhibits the experimental allergic rhinitis in guinea pigs.

Inorganic and methylated arsenic compounds induce cell death in murine macrophages via different mechanisms.

We demonstrate in this study the cytotoxic effects of inorganic arsenicals, arsenite and arsenate, and organic arsenic compounds, monomethylarsonic acid (MAA), dimethylarsinic acid (DMAA), and trimethylarsine oxide (TMAO), which are metabolites of inorganic arsenicals in human bodies, using murine macrophages in vitro. Inorganic arsenicals, both arsenite and arsenate, are strongly toxic to macrophages, and the concentration that decreased the number of surviving cells to 50% of that in untreated controls (IC50) was 5 or 500 microM, respectively. These inorganic arsenicals mainly caused necrotic cell death with partially apoptotic cell death; about 80% of dead cells were necrotic, and 20% were apoptotic. The inorganic arsenicals also induced marked release of an inflammatory cytokine, tumor necrosis factor alpha (TNF alpha), at cytotoxic doses. This strong cytotoxicity of an inorganic arsenical, arsenite, might be mediated via active oxygen and protease activation because it was inhibited by the addition of some antioxidant reagents, such as superoxide dismutase (SOD), catalase, and GSH, or by a peptide inhibitor of interleukin-1 beta-converting enzyme (ICE). It is likely that these immunotoxic effects of inorganic arsenicals may evoke both immunosuppression and inflammation, and they may be central factors causing carcinogenesis and severe inflammatory responses, such as hepatomegaly and splenomegaly, in chronic arsenicosis patients who daily ingested arsenic-contaminated well water. In contrast, the cytotoxic effects of methylated arsenic compounds were lower than those of inorganic arsenicals. The IC50 value of DMAA was about 5 mM, and MAA and TMAO had no toxicity even at concentrations over 10 mM. Additionally, these methylated chemicals suppressed the TNFalpha release from macrophages. DMAA induced mainly apoptotic cell death in macrophages as indicated by cellular morphological changes, condensed nuclei, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), and DNA fragmentation. However, the cytotoxicity of DMAA might be induced via a different mechanism from that of inorganic arsenicals because it was not abolished by the additions of SOD, catalase, or ICE inhibitor. Conversely, GSH enhanced the toxicity of DMAA. These data suggest that methylation of inorganic arsenicals in mammals plays an important role in suppression of both severe immunosuppression and inflammatory responses caused by inorganic arsenicals.

Toxicity and metabolism of trimethylarsine in mice and hamsters.

Trimethylarsine (TM-As) proved to be an arsenic compound of low toxicity, with a po LD50 of 7870 mg/kg in mice. A single po dose of 10 mg/kg of TM-As caused no hemolysis, but a single po dose of 750 mg/kg induced mild, transient hemolysis in hamsters. TM-As was very rapidly eliminated into the urine, with a biological half-life of 3.7 hr. TM-As was oxidized in vivo to form trimethylarsine oxide (TMAO) and excreted as such into the urine. TM-As was never demethylated in vivo. A mechanism was demonstrated by which a part of TM-As was eliminated directly into the expired air. We drew a conclusion that TM-As is far less an toxic than arsine, most probably due to its in vivo conversion to TMAO.

Better grading systems for evaluating the degree of lymph node invasion in cancer of the thoracic esophagus.

To evaluate the quality of various grading systems for lymph node invasion in cancer of the thoracic esophagus, the surgical results of 142 patients who underwent systematic lymph node dissection with curative intent were analyzed. The survival probability of patients in the same grade was modeled using a Weibull distribution and the parameters were estimated by the maximum likelihood principle. The quality of each grading system was measured by the Akaike Information Criterion (AIC) of the estimated statistical model, by which the smaller the AIC of a grading system, the smaller the loss of information for predicting outcomes. The AIC of the TNM grading of the International Union Against Cancer, the grading of the Japanese Society for Esophageal Diseases, and the grading designed according to the total number of positive lymph nodes became substantially smaller in that order. The AIC of grading systems variously designed on rather simple criteria were examined with the aim of creating a better grading system. It was concluded that a grading system based on the total number of positive nodes and the state of the paratracheal and/or middle mediastinal node groups was better than the other systems examined.

Glutathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALB/c 3T3 cells.

The cytotoxicity of arsenic compounds towards BALB/c 3T3 cells in culture was investigated, together with the role of glutathione (GSH) in the induction of the cytotoxic effects. The rank order of cytotoxicity was as follows: arsenite (As3+) > arsenate (As5+) > dimethylarsinic acid (DMAA) > methylarsonic acid (MAA) > trimethylarsine oxide (TMAO). Arsenobetaine, arsenocholine and the tetramethylarsonium ion were less toxic. Depletion of GSH enhanced the cytotoxic effects of As3+, As5+, MAA and TMAO, while the cytotoxicity of DMAA was markedly reduced by depletion of GSH. These results suggest that GSH plays a role in protecting the cells against the toxic effects of As3+, As5+, MAA and TMAO while it is involved in the induction of the cytotoxic effects of DMAA.

Effect of oxatomide nasal spray on experimental allergic rhinitis in guinea pigs and rats.

We investigated the effect of topically applied oxatomide, an antiallergic agent, on the assault of allergic rhinitis in actively sensitized guinea pigs. Topical application of oxatomide nasal spray (0.025%) reduced the severity of allergic rhinitis which was assessed by determining dye leakage and histamine released to the nasal cavity of guinea pigs. Furthermore, oxatomide nasal spray treatment significantly prevented the increase in dye leakage induced by histamine administration in guinea pigs and rats. These results indicate that the topical application of oxatomide inhibits both the release and the action of histamine. Therefore, oxatomide nasal spray may be beneficial for treatment of allergic rhinitis.

The effect of KW-4679, an antiallergic drug, on experimental allergic rhinitis in guinea pigs: effects on nasal blockage.

We investigated the effect of KW-4679 (Z-11-(dimethylaminopropyliden)-6,11-dihydrodibenzoxepin-2-a cetic acid hydrochloride), an antiallergic agent, on the nasal blockage induced by antigen challenge into the nostrils of actively sensitized guinea pigs. The change of the nasal cavity volume caused by nasal mucosal swelling after antigen challenge was measured by acoustic rhinometry. Oral administration of KW-4679 (0.01-10 mg/kg) significantly inhibited the decrease in the nasal cavity volume at 10 min, 30 min and 6 hr after antigen challenge. Ketotifen (1-10 mg/kg, p.o.) also inhibited the decrease in the nasal cavity volume after antigen challenge. These results indicate that KW-4679 may be useful for the treatment of allergic rhinitis.

Accuracy of measurement of acoustic rhinometry applied to small experimental animals.

Nasal obstruction is one of the major symptoms of allergic rhinitis. In the study of the mechanism of nasal obstruction, experiments on animal are useful. In adult humans, acoustic rhinometry has been used to evaluate nasal obstruction by determining nasal cavity dimensions in terms of cross-sectional areas as a function of the distance from the nostril. We modified the equipment used on humans to assess dimensions of nasal airway geometry of small experimental animals. The purpose of this study was to investigate the accuracy of measurement of the modified acoustic rhinometry applied to small experimental animals using nasal cavity models and guinea pigs. Measurement of the nasal cavity models (made of cylindrical silicone tubes) showed that the acoustic rhinometry estimated 85.5% of actual area and 79.0% of actual volume. In guinea pigs, nasal cavity volume determined by the acoustic rhinometry was 73.7 +/- 20.0% of actual volume. The actual volume was estimated by impression material instilled into the nasal cavity of the animals (IM volume), and volume determined by acoustic rhinometry significantly correlated with IM volume. Furthermore, there was a significant negative correlation between the volume and nasal airway resistance in guinea pigs. Measurement of the nasal airway resistance is the method frequently used in the evaluation of the nasal obstruction in guinea pigs. These results suggest that acoustic rhinometry is useful in evaluating nasal obstruction in small experimental animals.

Identification of arsenobetaine as a major arsenic compound in muscle of two demersal sharks, shortnose dogfish Squalus brevirostris and starspotted shark Mustelus manazo.

The major water-soluble arsenic compound was isolated from the muscle of shortnose dogfish Squalus brevirostris and of starspotted shark Mustelus manazo, both of which are demersal sharks. The isolated compound was identified to be arsenobetaine by its chromatographic and spectrometric analyses.