Inhibition of angiogenic factor production from murine mast cells by an antiallergic agent (epinastine hydrochloride) in vitro.

Angiogenesis is an important event both in the development of allergic inflammatory responses and in the pathophysiology of tissue remodeling in allergic diseases. In the present study, therefore, we examined the influence of antihistamines on angiogenesis through the choice of epinastine hydrochloride (EP) and murine mast cells in vitro. Mast cells (5 x 10(5) cells/mL) presensitized with murine IgE specific for ovalbumin (OVA) were stimulated with 10 ng/mL OVA in the presence of various concentrations of EP for 4 hours. The levels of angiogenesis factors, keratinocyte-derived chemokine (KC), tumor necrosis factor-alpha (TNF), and vascular endothelial growth factor (VEGF) in culture supernatants, were examined by ELISA. We also examined mRNA expression for the angiogenesis factors by RT-PCR. EP significantly inhibited the production of KC, TNF, and VEGF induced by IgE-dependent mechanism at more than 25 ng/mL. Semiquantitative analysis using RT-PCR showed that EP also significantly reduced mRNA expressions for KC, TNF, and VEGF. These results strongly suggest that EP suppresses angiogenesis factor production through the inhibition of mRNA expression in mast cells and results in favorable modification of clinical conditions of allergic diseases.

Relation between hematocrit and optical density in pulse oximetry -In vitro study with Waseda mock circulatory system-.

Optical properties of living tissues have not been well established even today, and bioopticinstrumentations have to be based on empirical formulae. In order to examine optical properties of the tissue having pulsating blood perfusion, we investigated the relation between optical density (defined as O.D.) of whole blood and hematocrit by transmission spectrophotometry. We used Waseda mock circulatory system that simulates blood circulation in the tissue. It was found that with increasing light path length, O.D. per unit light path length due to scattering and absorption effect, tended to become constant in each hematocrit. For wavelengths of 660, 805 and 940 nm, the relations between O.D. of whole blood and hematocrit predicted by Twersky's equation, Loewinger's equation and photon diffusion equation fitted to the data obtained. Meanwhile, for 1300 nm, the relation predicted by Loewinger's equation gave the best fit to the data.

Suppression of matrix metalloproteinase production from nasal fibroblasts by macrolide antibiotics in vitro.

It is well known that low-dose and long-term administration of macrolide antibiotics favourably modify the clinical status of chronic airway inflammatory diseases. However, the therapeutic mode of action of macrolide antibiotics is not well understood. The present study aimed to examine the influence of macrolide antibiotics, roxithromycin (RXM) and josamycin (JM) on matrix metalloproteinase (MMP) production from nasal polyp fibroblasts (NPF) in vitro. NPF, at a concentration of 2.5 x 10(5) cells x mL(-1), were stimulated with tumour necrosis factor (TNF)-alpha in the presence of various concentrations of RXM or JM for 24 h. MMP-2 and -9 levels in culture supernatants were analysed by ELISA, and MMP mRNA expression was examined by RT-PCR. The influence of RXM on nuclear factor (NF)-kappaB and activator protein (AP)-1 activation was also examined. Addition of RXM (but not JM) at 5.0 and 7.5 microg x mL(-1) significantly suppressed the production of MMP-2 and -9 from NPF induced by TNF-alpha stimulation. RXM also suppressed MMP mRNA expression through the inhibition of NF-kappaB and AP-1 activation. The present results suggest that the suppressive activity of roxithromycin on MMP-2 and -9 production is, in part, responsible for the therapeutic action of macrolides on chronic airway inflammatory diseases.

Suppressive activity of fexofenadine hydrochloride on metalloproteinase production from nasal fibroblasts in vitro.

BACKGROUND: Allergic rhinitis (AR) is an inflammatory disease characterized by nasal wall remodelling with intense infiltration of eosinophils and mast cells/basophils. Matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are the major proteolytic enzymes that induce airway remodelling. These enzymes are also important in the migration of inflammatory cells through basement membrane components. OBJECTIVE: We evaluated whether fexofenadine hydrochloride (FEX), the carboxylic acid metabolite of terfenadine with selective H(1)-receptor antagonist activity, could inhibit MMP production from nasal fibroblasts (NFs) in response to TNF-alpha stimulation in vitro. METHODS: NFs were established from nasal polyp-derived fibroblasts (PFs) taken from patients with AR. Nasal mucosal fibroblasts (MFs) were also induced from nasal mucosal tissues from septal deformity patients without allergy. PF and MF (2 x 10(5) cells/mL, each) were stimulated with TNF-alpha in the presence of various concentrations of FEX. After 24 h, culture supernatants were obtained and assayed for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 levels by ELISA. The influence of FEX on mRNA expression of MMPs and TIMPs in 4 h-cultured cells was also evaluated by real-time RT-PCR. Furthermore, nuclear factor-kappa B (NF-kappa B) activation in fibroblasts treated with FEX for 4 h was examined by ELISA. RESULTS: FEX at more than 350 ng/mL inhibited the production of MMP-2 and MMP-9 from both PF and MF in response to TNF-alpha stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected by FEX. FEX also inhibited MMP mRNA expression and NF-kappa B activation in PF and MF after TNF-alpha stimulation. CONCLUSION: The present data suggest that the attenuating effect of FEX on MMP-2 and -9 production from NFs induced by inflammatory stimulation may underlie the therapeutic mode of action of the agent on allergic diseases, including AR.

Carbenoxolone, a new inducer of heat shock protein 70.

Heat shock protein 70 (Hsp70) is capable of protecting cells, tissues, organs, and animals from a subsequent, normally lethal heating, as well as from numerous disease states. Therefore, it would be of great therapeutic benefit to discover compounds that are clinically safe yet able to induce Hsp70 in patients. Carbenoxolone, an antiulcer drug, protects gastric mucosal cells against irritants in vivo and in vitro. We assessed here whether carbenoxolone induces Hsp70 expression in human cell lines. We found that carbenoxolone increased the expression of Hsp70 protein and mRNA, and Hsp70 promoter activity.

Modulation of Th2 type cytokine production from human peripheral blood leukocytes by a macrolide antibiotic, roxithromycin, in vitro.

The influence of a macrolide antibiotic, roxithromycin (RXM), on Th1 and Th2 cytokine productions from human peripheral blood T cells was examined under stimulation with co-stimulatory molecules. Peripheral blood T cells prepared from both healthy and allergic rhinitis donors were cultured in the presence of RXM on anti-CD3 mAb and anti-CD26 mAb-coated wells, anti-CD3 mAb and anti-CD28 mAb-coated wells, and anti-CD3 and PMA. T-cell proliferation, along with the concentration of interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-5 were measured. RXM did not affect T-cell proliferation induced by several ways of co-stimulatory activation as assessed by 3H-thymidine incorporation into DNA. RXM also had no effect on IL-2 and IFN-gamma secretion by T cells prepared from both healthy and allergic rhinitis donors. On the other hand, RXM markedly inhibited both IL-4 and IL-5 secretions under each of the co-stimulatory conditions in a dose-dependent manner. These results indicate that RXM inhibits specifically Th2 cytokine secretion from T cells induced by co-stimulatory molecule stimulations. This inhibitory action of RXM may be partially responsible for attenuating effect of the agent on the inflammatory diseases.

Enhancement of endogenous corticosterone levels by a macrolide antibiotic, roxithromycin in mice.

The influence of roxithromycin (RXM), a macrolide antibiotic, on endogenous corticosterone (CS) levels was examined in BALB/c mice. Mice were sensitized intraperitoneally with two doses of Keyhole Limpet Hemocyanin at 1 week intervals. Mice were given orally 2.5 mg/kg RXM once a day for 14 days starting 7 days after the first sensitization. RXM administration caused markedly increase in endogenous plasma CS levels which was peaked at 60 min after the administration. However, josamycin did not influence on endogenous CS levels in plasma. Injection of dexamethasone inhibits the plasma CS hyperproduction induced by RXM treatment.

Suppression of co-stimulatory molecule expressions on splenic B lymphocytes by a macrolide antibiotic, roxithromycin in vitro.

The influence of a macrolide antibiotic, roxithromycin (RXM), on co-stimulatory molecule expression was examined using in vitro cell culture technique. Spleen cells obtained from BALB/c mice 10 days after immunization with 8.0 micrograms of haemocyanin absorbed to 4.0 mg aluminum hydroxide were cultured in the presence of 100.0 micrograms/ml haemocyanin and various concentrations of RXM for 72 h. Low concentrations (1.0 and 2.5 micrograms/ml) of RXM did not influence cell activation induced by antigenic stimulation, whereas RXM showed a suppressive effect on blastic activity of the cells when the agent was added to the cultures at more than 5.0 micrograms/ml. RXM did not affect blastic activity of splenic T cells by anti-CD3 monoclonal antibody stimulation even when the cells were cultured in the presence of 10.0 micrograms/ml RXM. Addition of anti-CD80 and anti-CD86 monoclonal antibody to cell cultures caused significant suppression of cell activation by antigenic stimulation. We next examined the influence of RXM on co-stimulatory molecule expressions on splenic B cells in response to antigenic stimulation. Addition of RXM at a concentration of 5.0 micrograms/ml into cell cultures remarkably suppressed co-stimulatory molecule, CD40, CD80 and CD86, expressions, which enhanced by antigenic stimulation in vitro.

Influenza virus A stimulates expression of eotaxin by nasal epithelial cells.

BACKGROUND: Respiratory virus is one of the most common causes of airway inflammation, but its pathogenic mechanisms are not well understood. Eotaxin is a potent eosinophil chemoattractant and is a selective agonist for C-C chemokine receptor 3 (CCR3). Although it has recently been demonstrated that epithelial cells express eotaxin, both in vivo and in vitro, there are few data concerning the expression in viral infection. OBJECTS: We hypothesized that eotaxin may play an important role in attracting inflammatory cells into the airway after viral infection and analysed whether viral infection induces eotaxin in nasal epithelial cells in vitro. METHODS: Nasal epithelial cells obtained from polypectomy for nasal polyp were infected with influenza virus A (subtype H3N2). The cells and supernatants were collected 8, 24 and 48 h after infection. Eotaxin mRNA was analysed by RT-PCR. Eotaxin concentration in the supernatants was analysed by enzyme-linked immunosorbent assay. We also examined a blocking assay to analyse the intervention of pro-inflammatory cytokines, TNF-alpha and IL-1beta in eotaxin production induced by influenza virus. RESULTS: The results showed that eotaxin was expressed constitutively in uninfected cells, but was up-regulated for both mRNA and protein levels in infected cells. Blocking experiments using anti-TNF-alpha and anti-IL-1beta antibodies showed no effects of these agents on the level of eotaxin. In addition, UV-inactivated virus did not enhance the expression of eotaxin. CONCLUSIONS: These results suggest that influenza virus A infection in nasal epithelial cells stimulates the expression of eotaxin, and may play an important role in the pathogenesis of airway inflammation by inducing eotaxin.

[The development of language and problems in 30 mild, moderate, and moderately severe hearing-impaired children].

Some mild, moderate, and moderately severe-hearing impaired children have poor language and educational problems despite comparatively good hearing. We studied 30 mild, moderate, and moderately severe hearing-impaired children cared for at Showa University and Jiseikai Hospitals. Their ages ranged from 3 to 14 years and average hearing from 35.0 dB HL to 68.8 dB HL. Our findings were as follows: (1) The average age of suspected hearing problem onset was 2 years 10 months. On the average, delayed diagnosis was made at 4 years 2 months and children were fitted with hearing aids at 5 years 3 months. (2) Over 25% them wore hearing aids infrequently. (3) Language delay was observed in 14 of 24 children examined using the WISC-III test. Many wore hearing aids infrequently and exhibited inadequate oral communication in Japanese due, for example, to deaf parents or children educated overseas. (4) According to a questionnaire, many mothers usually talked to the children aware of their hearing condition. But almost mothers of children with delayed development could not teach children if they couldn't hear, and only repeated same words for children's clarification, e.g., "Pardon?". It is important to detect hearing impairment in children as early as possible. Guidance by specialists and communication training are very important, especially for children who are mild, moderate, and moderately severe hearing-impaired.

Influence of a macrolide antibiotic, roxithromycin, on mast cell growth and activation in vitro.

BACKGROUND: Long-term administration of macrolide antibiotics is recognized to be able to favorably modify the clinical condition of inflammatory diseases, such as diffuse panbronchiolitis and cystic fibrosis. However, the precise mechanisms by which macrolide antibiotics could improve clinical conditions of the patients are not well understood. AIM: The present study was designed to examine the influence of macrolide antibiotics on effector cell functions responsible for inflammation through the choice of roxithromycin (RXM) and mast cell. METHODS: Mast cells were induced by long-term culture of splenocytes from BALB/c mice. RXM was added to the cultures at seeding and then every 4-5 days, when the culture medium was replaced with a fresh one. The influence of RXM on mast cell growth was evaluated by counting the number of cells grown on the 16th day. We also examined the influence of RXM on mast cell activation by examining histamine release and inflammatory cytokine secretion. RESULTS AND CONCLUSION: RXM could not inhibit mast cell growth, even when splenocytes were exposed to 100 microg/ml of RXM throughout the entire culture periods. RXM also could not suppress histamine release from cultured mast cells in response to non-immunological and immunological stimulations. However, RXM could suppress inflammatory cytokine, interleukin-1beta, interleukin-6, granulocyte macrophage-colony stimulating factor and tumor necrosis factor-alpha, secretions induced by concanavalin A stimulation at a concentration of as little as 0.5 microg/ml. These results may suggest that RXM modulated the ability of mast cells to secrete inflammatory cytokines and results in improvement of clinical condition of chronic inflammatory diseases.

The inhibitory effect of anti-allergic agent suplatast tosilate (IPD-1151T) on methacholine- and allergen-induced bronchoconstriction in sensitized mice. asakazu@med.showa-u.dc.jp.

The influence of an anti-allergic agent, suplatast tosilate (IPD-1151T; (+/-)-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenyl-carbamoyl]-ethyl] dimethylsulfonium p-toluenesulfonate) on allergic bronchoconstriction induced by allergen and methacholine (MCh) were examined in mice. BALB/c mice were sensitized by intraperitoneal injection of dinitrophenylated-keyhole limpet hemocyanin (DNP-KLH) mixed with A1(OH)3 (DNP-KLH). IPD-1151T was administered orally once a day for either 5 or 14 days in doses of 10, 30 or 100 mg/kg. Bronchoconstriction was measured 24h after the final drug administration. IPD-1151T inhibited both antigen- and MCh-mediated bronchoconstriction in actively sensitized mice. The inhibition induced was closely related to the dose and frequency of oral administration of the agent. We also examined the effect of IPD-1151T on IgE production in response to DNP-KLH immunization. IPD-1151T inhibited dose-dependently both total and specific IgE concentrations in serum prepared from mice 15 days after immunization. These results strongly indicate that IPD-1151T inhibits IgE production in vivo and results in attenuating effect on bronchoconstriction.

Suppressive effects of co-stimulatory molecule expressions on mouse splenocytes by anti-allergic agents in vitro.

The influence of anti-allergic drugs, epinastine hydrochloride (EP) and disodium cromoglycate (DSCG), on the co-stimulatory molecule expression was examined using in vitro cell culture technique. Spleen cells obtained from BALB/c mice 10 days after immunization with haemocyanin absorbed to aluminium hydroxide were cultured in the presence of 100.0 microg/ml haemocyanin and various concentrations of the agents. Low concentrations (<1.5 x 10(-4)M) of EP and DSCG did not influence spleen cell blastic activity induced by antigenic stimulation, whereas these agents caused significant inhibition of spleen cell activation when 2 x 10(-4) M of the agents were added to cell cultures. EP and DSCG also did not affect blastic activity of sensitized splenic T cells by anti-CD3 monoclonal antibody stimulation even when these cells were cultured in the presence of 2 x 10(-4) M of the agents. We next examined the influence of EP and DSCG on the expression of co-stimulatory molecules on spleen cells in response to antigenic stimulation. Sensitized spleen cells were cultured in the presence of 2 x 10(-4)M of the agents and the expression of molecules were examined by flow cytometer 24h later. EP and DSCG suppressed the expression of costimulatory molecules, CD40 and CD80, but not CD86, on splenic B cells which were enhanced by antigenic stimulation in vitro.

Localization of mRNA for SHIP2, SH2 domain-containing inositol polyphosphate 5-phosphatase, in the brain of developing and mature rats.

The localization of mRNA for SHIP2, SH2 domain containing inositol 5-phosphatase SHIP isozyme, was examined by in situ hybridization histochemistry in the brain of developing and mature rats. SHIP2 mRNA was first detected in the ventricular germinal zone at embryonic stages. As the postnatal development proceeded, the expression signal was evident in cell of the white matters, presumptive oligodendrocytes, and no significant expression was seen in neurons throughout the development.

[Intracellular calcium kinetics after odorant stimulus in olfactory receptor cells isolated from mice].

cAMP and IP3 act as secondary messengers in olfactory signal transduction and when activated, stimulate calcium levels in olfactory receptor cells. Little is known however, about the causal mechanism. We studied calcium kinetics in mouse olfactory receptor cells after odorant stimuli. Olfactory receptor cells were isolated from female BALB/c mice, treted with trypsin, and stained with Fura-2/AM. Changes in intracellular Ca2+ concentrations in stained cells were measured with a fluorescent microscopic image-processing device (ARGUS-50; Hamamatsu Photonix, Japan). We found that intracellular Ca2+ concentrations rose after exposure to a set of odorants, including 3-ethoxy-4-hydroxy-benzaldehyde, caprylic acid, heptanoic acid, nonanoic acid, eugenol, phenethyl alcohol, and n-amyl acetate. Adding 2', 5'-dideoxyadenosine, a cAMP inhibitor, beforehand suppressed olfactory receptor cell response to odorants. Intracellular Ca2+ concentrations increased substantially in response to stimulation by odorants in calcium-free Ringer's solution, but only a slight increase was seen in intracellular calcium concentration in response stimulation by a high concentration of K+ (145.6 mM) in calcium-free Ringer's solution. The increase in intracellular Ca2+ concentration after odorant stimuli was suppressed when olfactory receptor cells were pretreated with ryanodine, which releases Ca2+ from intracellular stores. These findings suggest that elevated Ca2+ concentrations may be involved in releasing Ca2+ from intracellular calcium stores in mouse olfactory receptor cells, in which cAMP functions as a secondary messenger in olfactory signal transduction.