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.     

Simultaneous enantioselective separation of azelastine and three of its metabolites for the investigation of the enantiomeric metabolism in rats. I. Liquid chromatography-ionspray tandem mass spectrometry and electrokinetic capillary chromatography

Enantioselective separation methods and the enantioselective determination of the anti-allergic drug azelastine and of three of its main phase I metabolites in a biological matrix underwent chromatographic and electrophoretic investigations. An enantioselective assay of a coupling of HPLC using a beta-cyclodextrin chiral stationary phase to ionspray tandem mass spectrometry is presented. Additionally, this assay is compared to another enantioselective assay using electrokinetic capillary chromatography with beta-cyclodextrin and carboxymethyl-beta-cyclodextrin in polyacrylamide-coated capillaries. For capillary electrophoresis (CE) the importance of polyacrylamide coating for the validation of this separation method is highlighted. Extracted rat plasma samples of enantioselective metabolism studies were measured by both validated assays. Differences in the pharmacokinetics and pharmacodynamics were evaluated for the main substance azelastine and its main metabolite demethylazelastine. So, a first hint about the enantioselectivity of biotransformation of azelastine in rats was seen after oral application of either enantiomer or the racemate to rats.     

Anti-allergic rhinitis effect of caffeoylxanthiazonoside isolated from fruits of Xanthium strumarium L. in rodent animals

The fruits of Xanthium strumarium L. (Asteraceae) have been used extensively in China for treatment of various diseases such as allergic rhinitis (AR), tympanitis, urticaria and arthritis or ozena. This study was designed to systemically investigate the effects of the caffeoylxanthiazonoside (CXT) isolated from fruits of X. strumarium on AR in rodent animals. Animals were orally administered with CXT. Anti-allergic activity of CXT was evaluated by passive cutaneous anaphylaxis test (PCA); acetic acid-induced writhing tests were used to evaluate the analgesic effects of CXT; acetic acid-induced vascular permeability tests were performed to evaluate anti-inflammatory effect of CXT. Then, the model AR in rats was established to evaluate the effects of CXT on AR with the following tests: the sneezing and nasal scratching frequencies, IgE level in serum, and histopathological examinations. Our results demonstrated that CXT had favorable anti-allergic, anti-inflammatory and analgesic effects. Additionally, we found that CXT was helpful to ameliorate the nasal symptoms and to down-regulate IgE levels in AR rats. Thus, we suggested that CXT can be treated as a candidate for treating AR.     

An oral sensitization model in Brown Norway rats to screen for potential allergenicity of food proteins.

We developed an oral sensitization protocol for food proteins for the rat. Young Brown Norway (BN) rats were exposed to 1 mg ovalbumin (OVA) by daily gavage dosing for 42 days without the use of an adjuvant. OVA-specific IgE and IgG responses were determined by ELISA. On an oral challenge with OVA some clinical symptoms of food allergy-like effects on the respiratory system, blood pressure, and permeability of the gastrointestinal barrier were studied. In addition, BN rats were orally exposed to a total hen egg white protein (HEW) extract and cow's milk (CM) and the specificities of induced antibody responses were compared with the specificities of antibodies in sera from egg- and milk-allergic patients using immunoblotting. Animals orally exposed to the allergens developed specific IgE and IgG antibodies which recognized the same proteins compared with antibodies from egg- or CM-allergic patients. Among the various clinical symptoms of food allergy, gut permeability was increased after an oral challenge. In addition, some animals demonstrated a temporary decrease in breathing frequency or systolic blood pressure. The results obtained show that the Brown Norway rat is a suitable animal model for inducing specific IgG and IgE responses on daily intragastric dosing of OVA without the use of an adjuvant. Moreover, local immune-mediated effects on oral challenge are observed. The observation that enterally exposed BN rats and food-allergic patients demonstrate antibody responses to a comparable selection of proteins on exposure to different protein mixtures (HEW and CM) further supports the suitability of the BN rat as an animal model for food allergy research and for the study of the allergenicity of (novel) food proteins.     

Oral Administration of Alginic Acid Oligosaccharide Suppresses IgE Production and Inhibits the Induction of Oral Tolerance

We have found that alginic acid oligosaccharide (ALGO) enhanced Th1 by promoting IL-12 production, suggesting that ALGO can be applied as an anti-allergic food. In this study we examined both positive and negative functions of ALGO. First we investigated the anti-allergic activity of ALGO, as a positive function, when orally administered. IgE production was significantly inhibited in mice fed ALGO as compared to control mice. This result indicates that ALGO had anti-allergic activity even when orally administered. On the other hand, we also found a negative function of ALGO. Oral co-administration of a protein antigen and ALGO inhibited the induction of oral tolerance to the protein. These data indicate the potential of ALGO as an anti-allergic food material and the necessity of further examination to determine a safe method application.     

Oral administration of alginic acid oligosaccharide suppresses IgE production and inhibits the induction of oral tolerance

We have found that alginic acid oligosaccharide (ALGO) enhanced Th1 by promoting IL-12 production, suggesting that ALGO can be applied as an anti-allergic food. In this study we examined both positive and negative functions of ALGO. First we investigated the anti-allergic activity of ALGO, as a positive function, when orally administered. IgE production was significantly inhibited in mice fed ALGO as compared to control mice. This result indicates that ALGO had anti-allergic activity even when orally administered. On the other hand, we also found a negative function of ALGO. Oral co-administration of a protein antigen and ALGO inhibited the induction of oral tolerance to the protein. These data indicate the potential of ALGO as an anti-allergic food material and the necessity of further examination to determine a safe method application.     

Effects of a newly synthesized leukotriene antagonist, NZ-107, on immediate-type hypersensitivity reaction in rats and guinea-pigs.

The effects of 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone (NZ-107) on immediate type hypersensitivity reactions in rats and guinea-pigs were studied. 1. When NZ-107, at a dose of 50 mg/kg (i.p.) or 100 mg/kg (orally), was administered to rats, 48-h homologous passive cutaneous anaphylaxis (PCA) reaction and histamine-, leukotriene C4 (LTC4)- and leukotriene D4 (LTD4)-induced skin reactions were suppressed by the agent. 2. NZ-107 (10(-6) g/ml) inhibited both LTC4- and LTD4-induced contractions of isolated rat stomach smooth muscle. 3. NZ-107 inhibited antigen-induced histamine release from rat peritoneal mast cells by 26% at a concentration of 10(-4) g/ml. 4. NZ-107, at doses of 25 and 50 mg/kg (orally), significantly inhibited guinea-pig 3-h heterologous PCA reaction. 5. NZ-107 inhibited antigen-induced histamine release from guinea-pig lung tissue by 17% and 48% at concentrations of 5 x 10(-5) and 10(-4) g/ml, respectively. 6. NZ-107, at doses of 25 and 50 mg/kg (i.p.), inhibited antigen-induced bronchoconstriction and eosinophil accumulation in the bronchoalveolar lavage fluid (BALF) of guinea-pigs. These results suggest that NZ-107 has anti-allergic action including inhibition of eosinophil accumulation in an antigen-challenged airway lesion in rats and guinea-pigs. The anti-allergic action of this agent is thought to be due to its action as a histamine and LT antagonist and its consequent inhibition of antigen-induced histamine release.     

Efficacy of wogonin in the production of immunoglobulins and cytokines by mesenteric lymph node lymphocytes in mouse colitis induced with dextran sulfate sodium

We previously examined wogonin, isolated from Scutellaria baicalensis, chemical mediators, and IgE by mesenteric lymph node (MLN) lymphocytes in rats. The present study explores the effect of wogonin on the MLN lymphocyte function of mice given orally at 20 mg/kg for 2 weeks with dextran sulfate sodium (DS)-induced colitis. The results indicate that IgA levels in MLN lymphocytes were high, while IgE was low, in mice given wogonin compared to those given water. Also, fecal IgA concentration of DS in the wogonin group mice was significantly higher than in the DS group. Concentrations of interferon-gamma and interleukin (IL)-2 of T cells by concanavalin A treatment was significantly higher in the wogonin fed group than in the normal group. Activation-induced IL-4, IL-5 and IL-10 secretion was lower in wogonin fed mice compared control mice after DS-induced colitis. For these reasons, we conclude that wogonin can alleviate the inflammation in DS-induced colitis brought about by an abnormal Th(2) response.     

Serum IgE levels in rats infected with Paragonimus westermani]

Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20-25 metacercariae of P. westermani from Cambaroides similis. Before and after infection (1, 2, 3, 4, 6, 8 weeks) of P. westermani, the blood was collected from the retro-orbital venous plexus of rats and kept serum at -70 degrees C. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0.18 +/- 0.042 at 2 weeks, 0.28 +/- 0.151 at 4 weeks and 0.43 +/- 0.055 at 8 weeks after infection. The absorbances of non-infected rats ranged 0.07 +/- 0.021-0.12 +/- 0.025. 2. Specific IgG values were slightly increased at 3 weeks (0.20 +/- 0.032) and gradually increased up to 8 weeks (0.31 +/- 0.067) after infection. The absorbances of non-infected rats ranged 0.11 +/- 0.035-0.18 +/- 0.019. The present results suggested that P. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.     

Analysis of IgE turnover in non-sensitized and sensitized rats

BACKGROUND: Although the levels of immunoglobulin E (IgE) in the circulating blood are often elevated in patients with allergic diseases, such levels cannot always be considered as pathognomonic signs of allergy. The induction of allergic reactions in the tissue was inferred to be related to the amount of IgE passing through the vascular wall. AIMS: We attempted to clarify which compartment, the intravascular or extravascular, plays an important role in the regulation of the turnover of rat IgE. METHODS: The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. RESULTS: The transfer rate constants from the central to tissue compartment (Kct) were larger than those from the tissue to central compartment (Ktc) irrespective of the sensitized state. The value of the distribution volume of the tissue compartment (Vt) was larger than that of the distribution volume of the central compartment (Vc) irrespective of the sensitized state. CONCLUSIONS: These Findings suggest that the short half-life of rat IgE in the circulation could be attributable to the distribution of IgE from the intravascular to the extravascular compartment.     

Urinary excretion of furosemide in rats with HgCl2-induced acute renal damage.

To examine the influence of mercuric chloride (HgCl2)-induced acute renal damage on urinary excretion of furosemide, HgCl2 (1 mg/kg) or its vehicle alone was given intraperitoneally to Wistar rats. The following two experiments were done. Study I: Three percent body weight (b.w.) of 1% NaCl solution or furosemide (30 mg/kg) in 3% b.w. of 1% NaCl solution was given orally before and after HgCl2 treatment, and an 8-hour urine was collected. Study II: Furosemide (30 mg/kg) was given orally, and blood samples were obtained at 1, 2, 3, 4, 6 and 8 hours after administration. Urinary excretion of N-acetyl-beta-D-glucosaminidase increased, and urine volume and urinary excretions of furosemide and sodium decreased in the HgCl2-treated rats. There were significant correlations between the urinary furosemide and its diuretic effects. Regression lines after HgCl2 were significantly different from those before treatment. The values of absorption as well as elimination rate constant were smaller, while the time to maximum concentration and the elimination half-life were longer in the HgCl2-treated rats compared to vehicle-treated animals. These results suggest that the urinary excretion of furosemide and the responsiveness of renal tubular cells to this agent are impaired in rats with HgCl2-induced acute renal damage.     

Hydrogen-Related Enhancement of In Vivo Antioxidant Ability in the Brain of Rats Fed Coral Calcium Hydride

This study explored the effect of coral calcium hydride (CCH) on rat intrahippocampal antioxidant ability by measuring the PCAM nitroxide radical decay ratio when CCH was (a) co-perfused into the hippocampus and (b) fed orally to the rats for 4 weeks under a freely moving state. Estimation of the in vivo antioxidant effect was obtained by administration of the blood–brain barrier-permeable PCAM nitroxide radical and the measured PCAM radical decay ratio then correlated to the amount of antioxidant in the brain using electron spin resonance (ESR) spectroscopy combined with microdialysis. The half-life periods of PCAM in rats treated with CCH in both the co-perfusion and orally fed groups were significantly shorter compared to the control group. These results clarify the mechanism that CCH may exert antioxidant activity by significantly enhancing the basal endogenous antioxidant ability in the hippocampus through a synergistic effect with α-tocopherol and ascorbic acid.     

Motor activity as an unbiased variable to assess anaphylaxis in allergic rats

The release of mediators by mast cells triggers allergic symptoms involving various physiological systems and, in the most severe cases, the development of anaphylactic shock compromising mainly the nervous and cardiovascular systems. We aimed to establish variables to objectively study the anaphylactic response (AR) after an oral challenge in an allergy model. Brown Norway rats were immunized by intraperitoneal injection of ovalbumin with alum and toxin from Bordetella pertussis. Specific immunoglobulin (Ig) E antibodies were developed in immunized animals. Forty days after immunization, the rats were orally challenged with the allergen, and motor activity, body temperature and serum mast cell protease concentration were determined. The anaphylaxis induced a reduction in body temperature and a decrease in the number of animal movements, which was inversely correlated with serum mast cell protease release. In summary, motor activity is a reliable tool for assessing AR and also an unbiased method for screening new anti-allergic drugs.     

Assessment of the Sensitizing Potential of Processed Peanut Proteins in Brown Norway Rats: Roasting Does Not Enhance Allergenicity

Background

IgE-binding of process-modified foods or proteins is the most common method for examination of how food processing affects allergenicity of food allergens. How processing affects sensitization capacity is generally studied by administration of purified food proteins or food extracts and not allergens present in their natural food matrix.

Objectives

The aim was to investigate if thermal processing increases sensitization potential of whole peanuts via the oral route. In parallel, the effect of heating on sensitization potential of the major peanut allergen Ara h 1 was assessed via the intraperitoneal route.

Methods

Sensitization potential of processed peanut products and Ara h 1 was examined in Brown Norway (BN) rats by oral administration of blanched or oil-roasted peanuts or peanut butter or by intraperitoneal immunization of purified native (N-), heated (H-) or heat glycated (G-)Ara h 1. Levels of specific IgG and IgE were determined by ELISA and IgE functionality was examined by rat basophilic leukemia (RBL) cell assay.

Results

In rats dosed orally, roasted peanuts induced significant higher levels of specific IgE to NAra h 1 and 2 than blanched peanuts or peanut butter but with the lowest level of RBL degranulation. However, extract from roasted peanuts was found to be a superior elicitor of RBL degranulation. Process-modified Ara h 1 had similar sensitizing capacity as NAra h 1 but specific IgE reacted more readily with process-modified Ara h 1 than with native.

Conclusions

Peanut products induce functional specific IgE when dosed orally to BN rats. Roasted peanuts do not have a higher sensitizing capacity than blanched peanuts. In spite of this, extract from roasted peanuts is a superior elicitor of RBL cell degranulation irrespectively of the peanut product used for sensitization. The results also suggest that new epitopes are formed or disclosed by heating Ara h 1 without glucose.     

Lymphocytes bearing Fc receptors for IgE. II. Induction of Fcepsilon-receptor bearing rat lymphocytes by IgE.

The proportion of lymphocytes bearing receptors for IgE (FcepsilonR) markedly increased after infection of rats with Nippostrongylus brasiliensis (Nb). The FcepsilonR-bearing lymphocytes from the infected animals bound more IgE-coated erythrocytes in rosette assay than FcepsilonR-bearing cells from normal rats, suggesting that the number of FcepsilonR per cell may also increase following the infection. In contrast, the number of IgE-receptors on peritoneal mast cells did not change after Nb infection. The increase in the proportion of FcepsilonR-bearing lymphocytes in Nb-infected rats is probably due to an increased concentration of IgE in the environment. The proportion of FcepsilonR-bearing cells in normal rat lymphocyte suspensions increased by culture of the cells with rat IgE of 1 microgram/ml or higher concentration. Other immunoglobulins such as rat IgG, human IgE, or rabbit IgG failed to induce either FcepsilonR-bearing cells or FcgammaR-bearing cells. It was also found that induction of Fc receptors by rat IgE is confined to FcepsilonR. Kinetic studies on the induction of FcepsilonR-bearing lymphocytes in vitro showed that the proportion of these cells in lymphocyte suspensions increased within 8 hr incubation with rat IgE but not within 4 hr. Evidence was obtained that both RNA synthesis and protein synthesis, but no DNA synthesis, are required for the induction of FcepsilonR-bearing cells or the expression of the receptors on the cell surface.     

Effect of myeloma IgE injections on passive and active cutaneous anaphylaxis in rats

The ability of injected rat IgE myeloma protein IR162 to inhibit passive and active cutaneous anaphylaxis in Lewis rats was investigated. IgE injected i.p. 24 hr before the sensitization with IgE anti-ovalbumin (OVA) completely inhibited both IgE- and IgG2a-induced passive cutaneous anaphylactic (PCA) reactions at a dose (2.5 mg/100 g body weight) that resulted in peak serum concentrations of 150 micrograms IgE IR162/ml. Peak IgE IR162 serum concentrations of 20 to 60 micrograms/ml inhibited the PCA reaction in approximately 50% of the rats. Intracutaneous injection of a mixture of myeloma IgE and anti-OVA IgE in a ratio of 100:1 or more also inhibited the PCA reaction. In contrast, the PCA reaction was not inhibited by seven daily doses of IgE beginning 24 hr after passive sensitization. Likewise, the cutaneous anaphylactic reaction elicited in rats 14 days after immunization with OVA and Bordetella pertussis was not prevented by daily injections of myeloma IgE despite a 1000- to 3000-fold excess of the myeloma IgE to anti-OVA IgE serum concentration. The data demonstrate that parenteral administration of myeloma IgE inhibits the PCA reaction only when given before passive sensitization and does not prevent cutaneous anaphylaxis in actively immunized rats. Because myeloma IgE failed to inhibit anaphylactic reactions in actively immunized rats, it is questionable whether administering human IgE-derived synthetic peptides or recombinant DNA-produced IgE fragments will be able to prevent allergic diseases by blocking the IgE Fc receptors on mast cells.     

Brain phospholipid arachidonic acid half-lives are not altered following 15 weeks of N-3 polyunsaturated fatty acid adequate or deprived diet

Previous studies have infused radiolabeled arachidonic acid (AA) into rat brains and followed AA esterification into phospholipids for up to 24 h; however, the half-life of AA in rat brain phospholipids is unknown. Eighteen day old rats were fed either an n-3 PUFA adequate or deprived diet for 15 weeks. Following the 15 weeks, 40 µCi of [³H] AA was injected intracerebroventricularly into the right lateral ventricle using stereotaxic surgery and returned to their dietary treatment. From 4–120 days after [³H] AA administration, brains were collected for chemical analyses. The half-life of AA in rat brain phospholipids was 44 ± 4 days for the n-3 PUFA adequate group and 46 ± 4 days for the n-3 PUFA deprived group, which closely approximates the predicted half-life previously reported, based on the rate of entry from the plasma unesterified pool, suggesting the plasma unesterified pool is a major contributor to brain uptake of AA. Furthermore, unlike a previous report in which the half-life of brain phospholipid docosahexaenoic acid (DHA) was increased in n-3 PUFA deprived rats, n-3 PUFA deprivation did not significantly alter the AA half-life, suggesting different mechanisms exist to maintain brain concentrations of AA and DHA.     

Reversible drug effects on the metabolic activation of polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) were effectively inhibited by chlorpromazine (10 microM) and azelastine (20 microM) in terms of superoxide generation, and restored by the addition of dodecylbenzenesulfonic acid (DBS) in a range of concentrations from 20 to 40 microM. The stimulation of superoxide generation by DBS was also inactivated by dodecylamine (DA) but was restored by the subsequent addition of DBS. A dose dependent competitive inhibition and activation of leukocytes was observed between azelastine (10 microM) and DBS (20 microM). The release of arachidonic acid from leukocytes activated by the chemotactic peptide was decreased by DA or chlorpromazine, but could be restored by DBS. The changes in membrane potential of leukocytes as monitored by cyanine dye were also decreased by DA, chlorpromazine or azelastine. These observations indicate that some cationic drugs reversibly inhibit membrane bound enzymes or receptors. The physiological responses of these inhibited PMNs can then be restored by appropriate anionic amphiphiles.     

Cumulation of active metabolites of levo-alpha-acetylmethadol in the rat fetus and neonate

Levo-alpha-acetylmethadol (LAAM, 0.2 or 2.0 mg/kg/day) was orally administered to female Sprague-Dawley rats for one month prior to and throughout pregnancy. The rats were killed on the 18th day of gestation along with a group of 18-day pregnant females given a single oral 2.0 mg/kg dose of LAAM 24 hours earlier. Although cumulation of LAAM or its active metabolites was not seen in plasma or brain of pregnant rats given drug chronically, significant cumulation was observed in whole fetus and in fetal brain. In addition, a 2–3 fold elevation in the concentrations, and an even greater elevation of total content, was noted in the newborn pup. These data suggest that opiate intoxication soon after birth may be a factor responsible for the increased morbidity and mortality of rat pups prenatally exposed to LAAM.     

Inhibitory effect of polyphenol-enriched apple extracts on mast cell degranulation in vitro targeting the binding between IgE and FcepsilonRI

Extracts from immature fruit of the apple (Rosaceae, Malus sp.), which contain procyanidins (polymers of catechins) as the major ingredients, are known to inhibit histamine release from mast cells. We analyzed in this study the mechanism for the anti-allergic activity of two polyphenol-enriched apple extracts. These extracts, termed "crude apple polyphenol (CAP)" and "apple condensed tannin (ACT)", reduced the degranulation of mast cells caused by cross-linking of the high-affinity receptor for IgE (FcepsilonRI) with IgE and the antigen in a dose-dependent manner. Furthermore, western blotting revealed that phosphorylation of the intracellular signal-transduction molecules caused by cross-linking of FcepsilonRI was markedly decreased by the addition of CAP or ACT. We then analyzed the effects of CAP and ACT on the binding of the IgE antibody to FcepsilonRI on mast cells, which is the first key step in the allergic reaction mediated by mast cells, and found that this binding was markedly inhibited by both CAP and ACT. These results indicate that the inhibition of binding between FcepsilonRI and IgE by either CAP or ACT was the probable cause of the suppression of mast cell activation. This is the first report demonstrating the molecular mechanism for the anti-allergic effect of procyanidin-enriched extracts from apples.