Topical ocular treatment with monoclonal antibody Fab fragments targeting Japanese cedar pollen Cry j 1 inhibits Japanese cedar pollen-induced allergic conjunctivitis in mice.

Fab fragments (Fabs) of antibodies having the ability only to bind to specific allergens lack effector functions due to the absence of the Fc portion. In the present study, we examined whether IgG1 monoclonal antibody (mAb) Fabs targeting Japanese cedar pollen (JCP) Cry j 1 were able to regulate JCP-induced allergic conjunctivitis in mice. BALB/c mice actively sensitized with JCP were repeatedly challenged by topical administration of JCP eye drops. Fabs prepared by the digestion of anti-JCP IgG1 mAbs (P1-3 and P1-8) with papain were applied to the eye 15min before the JCP challenges followed by measurement of the clinical conjunctivitis score. In the in vitro experiments, P1-3 and P1-8 showed specific binding to JCP Cry j 1. Furthermore, intact P1-3 binding to Cry j 1 was inhibited by P1-3 Fabs, but not P1-8 Fabs; additionally, P1-8 Fabs, but not P1-3 Fabs, suppressed the intact P1-8 binding, suggesting that the epitopes of Cry j 1 recognized by P1-3 and P1-8 were different. Topical ocular treatment with P1-3 Fabs or P1-8 Fabs was followed by marked suppression of JCP-induced conjunctivitis (P<0.01). In histological evaluation, P1-8 Fabs showed a reduction in eosinophil infiltration in the conjunctiva (P<0.01). These results demonstrated that topical ocular treatment with IgG1 mAb Fabs to Cry j 1 was effective in suppressing JCP-induced allergic conjunctivitis in mice. Furthermore, it suggests the possibility that some epitopes recognized by Fabs could be used as a tool to regulate allergic conjunctivitis.

Effect of Ganoderma lucidum on pollen-induced biphasic nasal blockage in a guinea pig model of allergic rhinitis.

Ganoderma lucidum (GL), an oriental medical mushroom, has been used in Asia for the prevention and treatment of a variety of diseases. However, the effect of GL on allergic rhinitis has not been well defined. The current study describes the inhibitory effect of GL on the biphasic nasal blockage and nasal hyperresponsiveness induced by repeated antigen challenge in a guinea pig model of allergic rhinitis. Intranasally sensitized guinea pigs were repeatedly challenged by inhalation of Japanese cedar pollen once every week. Ganoderma lucidum was orally administered once daily for 8 weeks from the time before the first challenge. The treatment with GL dose-dependently inhibited the early and late phase nasal blockage at the fifth to ninth antigen challenges. Furthermore, nasal hyperresponsiveness to intranasally applied leukotriene D4 on 2 days after the eighth antigen challenge was also inhibited by the treatment with GL. However, Cry j 1-specific IgE antibody production was not affected by the treatment. In conclusion, we demonstrated that the pollen-induced biphasic nasal blockage and nasal hyperresponsiveness were suppressed by the daily treatment with GL in the guinea pig model of allergic rhinitis. These results suggest that GL may be a useful therapeutic drug for treating patients with allergic rhinitis.

Inhibition of hematopoietic prostaglandin D synthase improves allergic nasal blockage in guinea pigs.

Although it has been suggested that prostaglandin (PG) D(2) is involved in the pathogenesis of allergic rhinitis, whether the inhibition of hematopoietic PGD(2) synthase (H-PGDS) shows beneficial effects on allergic rhinitis has been unclear. We evaluated the effects of a selective H-PGDS inhibitor, TFC-007, on nasal symptoms on Japanese cedar pollen-induced allergic rhinitis of guinea pigs. Sensitized animals were challenged with the pollen once a week. TFC-007 (30mg/kg, p.o.) given once before a challenge almost completely suppressed PGD(2) production in the nasal tissue early and late after the challenge. Although pre-treatment did not affect the incidences of sneezing and early phase nasal blockage, late phase nasal blockage was partially but significantly attenuated; however, nasal eosinophilia was not suppressed. In contrast, when TFC-007 was given once 1.5h after the challenge, the late phase response was not affected. Collectively, PGD(2) produced by H-PGDS early after an antigen challenge can participate in the induction of late phase nasal blockage, although the mechanism may be independent of eosinophil infilatration. The strategy for H-PGDS inhibition may be beneficial for allergic rhinitis therapy.

Effect of the C3a-receptor antagonist SB 290157 on anti-OVA polyclonal antibody-induced arthritis.

It was investigated whether the C3a-receptor antagonist (C3aRA) SB 290157 was involved in the suppression of anti-OVA pAb-induced arthritis because it is well known that anaphylatoxin C3a plays a crucial role in the development of an effective inflammatory response during complement activation. Anti-OVA pAb-induced arthritis was induced in DBA/1J mice by administration of anti-OVA pAb 0.5 h prior to intra-articular (i.a.) injection of OVA (0 h). Two peaks of joint swelling were observed at 0.5 and 3 h. The role of C3aRA in arthritis was investigated by injection of SB 290157 at concentrations of 10 and 30 mg/kg at 0 and 2 h. The antagonist was able to reduce joint swelling only at 3 h, and about 50% inhibition of joint swelling was observed with the concentration of 30 mg/kg. The C3 level was significantly decreased at 3 h compared with naïve mice showing complement consumption. Furthermore, the C3 activation was observed and increased corresponding to the graded concentration of anti-OVA pAb. The results also revealed that the C3aRA was able to reduce the expression of IL-1beta in synovial tissue. Taken together, the results suggested that C3aRA may be effective in the inhibition of arthritis.

Absence of nasal blockage in a Japanese cedar pollen-induced allergic rhinitis model mouse.

BACKGROUND: Japanese cedar pollen-induced allergic rhinitis in a guinea pig model clearly induced not only sneezing but also biphasic nasal blockage. To date, there have only been a few reports on models of murine allergic rhinitis which clearly show nasal blockage. Therefore, in order to try and develop such a model, we administered multiple dosages of intranasal pollen or purified antigen protein Cry j 1. METHODS: B10.S mice were sensitized by intranasal instillations of either pollen extract or Cry j 1 twice a day for 7 days, which was adsorbed on Al(OH)(3). Subsequently, once a week, the mice were given multiple intranasal instillation challenges of either the pollen suspension or Cry j 1 and the frequency of sneezing was observed after respective challenges were made. Specific airway resistance (sRaw) was measured as an indicator for nasal blockage. Cry j 1-specific IgE levels were measured using an enzyme-linked immunosorbent assay. RESULTS: The serum Cry j 1-specific IgE level showed clear elevation only in the group sensitized by Cry j 1 + Al(OH)(3) and then challenged by Cry j 1. No elevations were seen in the groups sensitized by pollen extract + Al(OH)(3) followed by a pollen suspension challenge. There was an immediate increase in sneezing after challenges in all of the sensitized-challenged groups. Nevertheless, no increases in sRaw in any of the groups were detected at any of the time points during the 8 hours following the challenges. CONCLUSIONS: Cry j 1 may be more effective than crude antigens for efficient sensitization/challenge in mice. No increase in sRaw occurred, even in mice that possessed high amounts of Cry j 1-specific IgE and that exhibited sneezing.

Effect of TA-270, a novel quinolinone derivative, on antigen-induced nasal blockage in a guinea pig model of allergic rhinitis.

TA-270 (4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone) is a novel quinolinone derivative that has been demonstrated to possess an anti-oxidative activity against peroxynitrite, a potent oxidant, that is generated by the reaction of nitric oxide with superoxide anions. The current study describes the inhibitory effect of TA-270 on the biphasic nasal blockage induced by repeated antigen challenge in an allergic rhinitis guinea pig model. In the present in vitro study, TA-270 potently inhibited the oxidative reaction induced by peroxynitrite (IC(50)=79 nM). In addition, TA-270 (0.3-30 mg/kg, p.o.) dose-dependently inhibited peroxynitrite (3 mM, 10 mul/nostril)-induced nasal blockage in guinea pigs. In the antigen-induced allergic rhinitis model, TA-270 (0.3, 3, and 30 mg/kg, p.o.) given 1 h before the antigen challenge suppressed early phase nasal blockage by 36%, 42%, and 63%, respectively. Furthermore, TA-270 (0.3, 3, and 30 mg/kg, p.o.) showed a relatively strong suppression of late phase nasal blockage (39%, 62%, and 72%, respectively). The late phase nasal blockage was significantly inhibited (61%) even when TA-270 (30 mg/kg, p.o.) was administered 18 h before the antigen challenge. In conclusion, TA-270 improved antigen-induced nasal blockage, probably through its peroxynitrite scavenging action, and the effect was sustained for at least 18 h. Thus, TA-270 would be expected to relieve nasal blockage in allergic rhinitis patients.

Effect of local nasal immunotherapy on nasal blockage in pollen-induced allergic rhinitis of Guinea pigs.

BACKGROUND: As a non-injection route for immunotherapy, local nasal immunotherapy has been examined in allergic rhinitis patients. However, it is unclear how the immunotherapy affects sneezing, biphasic nasal blockage and nasal hyperresponsiveness. Thus, we evaluated the therapeutic effects of nasal immunotherapy on the symptoms of guinea pig allergic rhinitis. Additionally, we also evaluated whether the immunotherapy relieved pollen-induced allergic conjunctivitis. METHODS: Sensitized animals were repeatedly challenged by pollen inhalation once every week. After the 7th challenge, the pollen extract was intranasally administered 6 times a week until the 30th challenge. Sneezing frequency was counted after each of the challenges. As an indicator of nasal blockage, changes in specific airway resistance were measured. Nasal hyperresponsiveness was assessed by measuring leukotriene D(4)-induced nasal blockage. Additionally, during the immunotherapy, we applied pollen onto the ocular surface to induce the allergic conjunctivitis symptoms. RESULTS: At the 11th-30th challenges, the nasal immunotherapy showed inhibition or a tendency to inhibit the biphasic nasal blockage although the inhibitions were variable at respective challenges. The development of nasal hyperresponsiveness was markedly suppressed by the immunotherapy. Nevertheless, neither sneezing nor antigen-specific IgE antibody production was substantially influenced by the immunotherapy. On the other hand, the nasal immunotherapy did not affect the induction of allergic conjunctivitis symptoms. CONCLUSIONS: Local nasal immunotherapy may be clinically useful for allergic nasal blockage associated with nasal hyperresponsiveness. The mechanisms responsible for this effectiveness might not be related to IgE production. Additionally, the effectiveness for nasal tissue was dissociated from that seen for the ocular tissue.

Effects of multiple dexamethasone treatments on aggravation of allergic conjunctivitis associated with mast cell hyperplasia.

In a Japanese cedar pollen-induced allergic conjunctivitis model in guinea pigs, symptoms were aggravated by repeated pollen challenges. In addition, the number of mast cells in the conjunctiva was increased by multiple challenges. The amount of a mast cell mediator, histamine in ophthalmic lavage fluid was also increased by multiple challenges. In the present study, we evaluated the effects of multiple dexamethasone treatments to assess the relationship between the aggravation of symptoms and mast cell hyperplasia. Sensitized guinea pigs were challenged by dropping a pollen suspension onto their eye surface once a week until the 15th challenge. Dexamethasone (10 mg/kg, p.o.) was administered once 3 h before the 15th challenge or 3 h before every 1st--15th challenge. Mast cells in the conjunctival tissue were detected by toluidine blue staining. Histamine was fluorometrically assayed by high-performance liquid chromatography. Serum Cry j 1-specific IgE titer was measured by an enzyme-linked immunosorbent assay. The results indicated that a single treatment with dexamethasone did not affect the 15th challenge-induced symptoms; however, multiple treatments with the corticosteroid suppressed not only conjunctivitis symptoms after every challenge but also the mast cell hyperplasia and the increase in histamine in the lavage fluid. Conversely, the increase in the IgE titer in the serum was not affected by multiple treatments with dexamethasone. In conclusion, increased numbers of mast cells in the conjunctival tissue may be associated with the aggravation of allergic conjunctivitis symptoms.

Involvement of peroxynitrite in pollen-induced nasal blockage in guinea pigs.

Nitric oxide (NO) has been implicated in early and late phase nasal blockage in a Japanese cedar pollen-induced experimental allergic rhinitis guinea pig model. In this study, we investigated the role of peroxynitrite, which is formed by a rapid reaction of NO with superoxide anion, in the antigen-induced biphasic nasal blockage. Sensitized guinea pigs were repeatedly challenged by pollen inhalation once every week. The peroxynitrite scavenger, ebselen (30 mg/kg), or the xanthine oxidase inhibitor, allopurinol (50 mg/kg), was intraperitoneally administered 30 min before the antigen challenge. The late phase nasal blockage induced 4 h after the challenge was largely suppressed by ebselen (57% inhibition; P<0.05) and allopurinol (47% inhibition; P<0.05), but neither ebselen nor allopurinol influenced the early phase response. On the other hand, the intranasal instillation of peroxynitrite (10(-3) and 10(-2) M, 10 microl/nostril) caused a remarkable dose-dependent nasal blockage in the sensitized guinea pig. These results suggest that peroxynitrite plays a major role in the late phase nasal blockage induced by the antigen challenge in sensitized guinea pigs.

Polycyclic aromatic hydrocarbons aggravate antigen-induced nasal blockage in experimental allergic rhinitis.

It has been hypothesized that air pollution has played a role in the increase in allergy prevalence. However, it remains unclear what exact roles are played by polycyclic aromatic hydrocarbons (PAHs), which are encountered in the environment in the form of air pollution, in allergic rhinitis. Thus, we examined whether benzo(a)pyrene (BaP) and 1-nitropyrene (1-NP), representative PAHs, aggravate allergic rhinitis symptoms, using a guinea-pig model. Sensitized animals were repeatedly challenged by inhalation of Japanese cedar pollen once a week. BaP or 1-NP was daily and intranasally administered for 2 weeks (short-term treatment) or for 22 weeks from the time before the sensitization period (long-term treatment). The short-term treatment affected neither nasal blockage nor sneezing induced by antigen. In contrast, the long-term treatment aggravated the antigen-induced nasal blockage that was induced 7 weeks after the start of the treatment with BaP or 1-NP. This aggravation continued during the intranasal treatment with PAH. However, neither sneezing nor Cry j 1-specific IgE antibody production was affected even by the long-term treatment. In conclusion, the long-term treatment with BaP and 1-NP can aggravate allergic rhinitis. The mechanisms underlying this aggravation are not associated with production of Cry j 1-specific IgE.

Different mechanisms between thromboxane A2- and leukotriene D4-induced nasal blockage in guinea pigs.

Although thromboxane (TX)A2 is involved in allergic rhinitis, the mechanisms inducing nasal blockage have not been elucidated. We evaluated the roles of nasal mucosal vascular changes following intranasal instillation of the TXA2 analog U-46619 or leukotriene (LT)D4 to induce nasal blockage in a guinea pig model of allergic rhinitis. Both U-46619- and LTD4-induced nasal blockages in sensitized animals were swiftly and completely suppressed by a vasoconstrictor, naphazoline. The nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester relieved LTD4-induced nasal blockage, but not U-46619-induced nasal blockage. Although both agonists produced vasodilatation of nasal mucosa in vivo, LTD4 caused vasodilatation while U-46619 caused vasoconstriction in vitro. Both LTD4- and U-46619-induced nasal blockages in vivo should depend on vasodilatation of nasal mucosa. LTD4-induced nasal blockage is induced by direct vasodilatation via nitric oxide. In contrast, U-46619-induced nasal blockage may be associated with contraction of a certain vein that should exist at the exit of capacitance vessels, leading to congestion of the nasal mucosa.

Detection of new antigenic proteins in Japanese cedar pollen.

In Japan, an increasing number of people suffer from pollenosis, a typical atopic disease. Japanese cedar (Cryptomeria japonica) pollen is the most common allergen that causes pollenosis. Although Cry j 1 and Cry j 2 are the common allergenic proteins contained in the pollen, there is a small population of patients who exhibit positive skin reactions to the pollen extract but are negative for both Cry j 1 and Cry j 2. This suggests that the pollen may contain other antigenic proteins besides these two molecules. In this study, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to examine partially purified pollen extract (PPPE) and determine if there were other proteins that reacted to anti-PPPE sera collected from sensitized guinea pigs, mice and rabbits. Subsequently, we detected four bands of proteins with molecular weights around 7, 15, 20 and 31 kDa, which were different from those normally seen for Cry j 1 and Cry j 2. Among these, the 7, 15 and 20 kDa proteins could not be detected when anti-Cry j 1 monoclonal anti-body (mAb) and anti-Cry j 2 mAb were used as antibodies for Western blotting. Therefore, these three proteins may differ from Cry j 1 and Cry j 2 not only in molecular weight but also in antigenicity. In conclusion, three new antigenic proteins that are not identical to Cry j 1 and Cry j 2 have been shown to exist in Japanese cedar pollen. Structural analyses of these proteins may be useful in the development of new therapeutic methods, including specific immunotherapy for pollenosis.

Multiple cedar pollen challenge diminishes involvement of histamine in allergic conjunctivitis of Guinea pigs.

It has been reported that antihistamines do not fully modify symptoms of allergic conjunctivitis in clinical settings, suggesting that histamine is not the only contributor to symptom generation in the disease. However, in the majority of experimental allergic conjunctivitis models, antihistamines are very effective in the reduction of symptoms. In the present study, we used our recently developed guinea pig model of allergic conjunctivitis and evaluated whether involvement of histamine in the induction of symptoms of allergic conjunctivitis is altered by multiple antigen challenges. Guinea pigs were sensitized by intraperitoneal injection of Japanese cedar pollen extracts adsorbed on aluminum hydroxide gel, and then challenged by dropping a pollen suspension without the adjuvant on each eye once a week until the 15th challenge. The magnitude of the conjunctivitis intensity score (CIS), itch-associated scratching response and albumin leakage were found to increase with repeated challenges. At the 1st-3rd challenges, histamine H(1) receptor antagonist, mepyramine (10 mg/kg, p.o.), strongly reduced all these symptoms. However, symptoms at the 5th-15th challenges were not inhibited by mepyramine. On the other hand, a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (10 mg/kg, i.v.), potently inhibited the increase of CIS and albumin leakage at the 15th challenge. In conclusion, histamine involvement in the induction of conjunctivitis symptoms in our model was diminished by multiple antigen challenges. The allergic conjunctivitis at the chronic stage is partly mediated by nitric oxide (NO) derived from NOSs that may be activated by mediators other than histamine. The histamine-independent allergic conjunctivitis may be useful for analyzing mechanisms underlying chronic conjunctivitis.

Kinins are involved in the development of allergic nasal hyperresponsiveness in guinea pigs.

We evaluated roles of kinins in allergen-induced nasal blockage and sneezing, and development of nasal hyperresponsiveness to leukotriene D4 in a Japanese cedar pollen-induced allergic rhinitis model of guinea pigs. Sensitised guinea pigs were repeatedly challenged by pollen inhalation once every week. Neither a bradykinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin nor a bradykinin B2 receptor antagonist, icatibant suppressed allergen-induced sneezing and nasal blockage. However, development of nasal hyperresponsiveness to leukotriene D4 was significantly suppressed by them. The amount of bradykinin in nasal cavity lavage fluid was immediately increased after the challenge. In non-sensitised animals, hyperresponsiveness to leukotriene D4 was developed by a bradykinin B2 receptor agonist, bradykinin, but not by a bradykinin B1 receptor agonist, des-Arg10-kallidin, while in the sensitised-challenged animal, both agonists developed hyperresponsiveness. In conclusion, the nasal hyperresponsiveness appeared to be induced by kinins produced in response to the antigen challenge through activation of not only bradykinin B2 but also B1 receptors.

[Studies on the experimental allergic rhinitis induced by Japanese cedar pollen--role of cysteinyl leukotrienes in nasal allergic symptoms].

Cysteinyl leukotrienes (CysLTs: LTC4, LTD4, and LTE4) are a family of potent inflammatory mediators that appear to contribute to the pathophysiologic features of allergic rhinitis. Because treatment with a CysLT1 receptor antagonist and a 5-lipoxygenase inhibitor modified allergen-induced nasal blockage in patients with allergic rhinitis, and CysLTs were detected in nasal cavity lavage fluid, it has been suggested that CysLTs act as significant inflammatory mediators in allergic rhinitis. The role of CysLTs was evaluated in our experimental allergic rhinitis model in sensitized guinea pigs which shows biphasic nasal blockage, sneezing and nasal hyperresponsiveness to LTD4 induced by repetitive inhalation challenge with Japanese cedar pollen. In this model, the CysLT1 receptor antagonist pranlukast suppressed the late-phase nasal blockage but not early blockage and sneezing. Nasal hyperresponsiveness (nasal blockage) to LTD4 was largely blocked by pranlukast, naphazoline, and N omega-nitro-L-arginine-methyl ester. The results demonstrate that nasal blockage induced by CysLTs is mainly due to dilatation of nasal blood vessels, which can be induced by the nitric oxide produced through CysLT1 receptor activation. On the other hand, when pollen inhalation challenge was performed in the presence of nasal hyperresponsiveness, antigen-induced biphasic nasal blockage and sneezing were considerably enhanced and CysLTs contributed to both symptoms, suggesting that nasal hyperresponsiveness induces aggravation of antigen-induced nasal symptoms. The results presented in this study further suggest that our model is a good representative of human allergic rhinitis and offer evidence that CysLTs are chemical mediators mainly responsible for allergic nasal symptoms.

Acquired nasal hyperresponsiveness aggravates antigen-induced rhinitis in the guinea pig.

Whether a state of nasal hyperresponsiveness influences antigen-induced biphasic nasal blockage and sneezing were examined using a guinea pig model of allergic rhinitis. Sensitized animals were challenged with an antigen, Japanese cedar pollen, once every week. Before the 13th challenge, the animals were randomly divided into 2 groups, and then the 13th challenge was performed (Groups A-0 and B-0). The 14th challenge was done on day 2 (Group A-2) and on day 7 (Group B-7) after the 13th challenge, on which nasal hyperresponsiveness was present and absent, respectively. Biphasic nasal blockage and sneezing after the challenge in Group A-2 were more severe than those in Group A-0, while those of Group B-7 were almost the same as those of Group B-0. An anti-histaminic, mepyramine, inhibited sneezing but not the biphasic nasal blockage in Group B-7. A cysteinyl leukotriene (CysLT) antagonist, pranlukast, suppressed the late nasal blockage but not the early blockage and sneezing in Group B-7. In contrast, in Group A-2, mepyramine significantly attenuated not only sneezing but also the early nasal blockage. Pranlukast significantly inhibited both nasal blockage and sneezing in Group A-2. In conclusion, nasal hyperresponsiveness aggravated the antigen-induced nasal responses, to which histamine and CysLTs considerably contributed.