IL-17A promotes the exacerbation of IL-33-induced airway hyperresponsiveness by enhancing neutrophilic inflammation via CXCR2 signaling in mice.

Neutrophilic airway inflammation is a hallmark of patients with severe asthma. Although we have reported that both IL-33 and IL-17A contributed to IgE-mediated neutrophilic inflammation in mice, the relationship remains unclear. In this article, we examined how IL-17A modifies IL-33-induced neutrophilic inflammation and airway hyperresponsiveness (AHR). IL-33 was intratracheally administered to BALB/c mice on days 0-2; furthermore, on day 7, the effect of the combination of IL-33 and IL-17A was evaluated. Compared with IL-33 or IL-17A alone, the combination exacerbated neutrophilic inflammation and AHR, associated with more increased levels of lung glutamic acid-leucine-arginine(+) CXC chemokines, including CXCL1, CXCL2, and CXCL5, and infiltration by alveolar macrophages expressing CXCR2. Treatment with anti-CXCR2 mAb or depletion of alveolar macrophages repressed neutrophilic inflammation and AHR; in addition, depletion of neutrophils suppressed AHR. These findings prompted us to examine the role of CXCR2 in IgE-sensitized mice; a single treatment with anti-CXCR2 mAb in the seventh Ag challenge inhibited late-phase airway obstruction, AHR, and neutrophilic inflammation. In addition to inhibition, multiple treatments during the fourth to seventh challenge attenuated early-phase airway obstruction, eosinophilic inflammation, and goblet cell hyperplasia associated with the reduction of Th2 cytokine production, including IL-4, IL-5, and IL-13. Collectively, IL-33 cooperated with IL-17A to exacerbate AHR by enhancing neutrophilic inflammation via CXCR2 signaling; furthermore, CXCR2 signaling derived Th2 responses. We thus suggest the underlying mechanisms of IL-33 and IL-17A in allergic asthma and CXCR2 as potential therapeutic targets for the disease.

IgA directly inhibits antigen-dependent B cell activation following distinctive distribution of the antigen in mice.

CONTEXT: Serum IgA suppresses immune responses when exposed to antigens recognized by the antibody; however, the underlying mechanism remains unclear. OBJECTIVE: We herein clarified the relationships between changes in antigen distribution and antigen-dependent B cell activation in the presence or absence of IgA against the antigen in mice. MATERIALS AND METHODS: DBA/1J and HR-1 mice were intravenously injected with ovalbumin (OVA) and anti-OVA monoclonal IgA OA-4. The distribution of the antigen and B cell responses were measured. RESULTS: B cell activation by injected OVA, namely, increases in anti-OVA IgG production and the populations of B220(+)GL7(+) and B220(+)CD69(high) splenocytes, was diminished by the co-injection of OA-4. Co-injected OA-4 increased OVA in the serum as well as in the bile and gut. This was coincident with its decrease in the urine due to the inhibition of OVA monomer secretion through the formation of immune complexes. The apparent similarities in the association between fluorescein isothiocyanate (FITC)-OVA and splenic B cells in the presence and absence of OA-4 in vivo appeared to be attributed to compensation between the two effects of OA-4; an increase in serum OVA in vivo and inhibition of the association between OVA and B cells, as suggested by in vitro experiments. DISCUSSION: Based on these results, the stimulation of B cells by OVA may be directly reduced, at least partly, by the neutralization of OVA by OA-4. CONCLUSION: IgA may be an effective drug for the treatment of immune disorders due to its ability to blunt antigen-specific B cell activation.

IgE/antigen-mediated enhancement of IgE production is a mechanism underlying the exacerbation of airway inflammation and remodelling in mice.

IgE is known to enhance some antibody responses to specific antigens, but whether this contributes to allergic asthma remains unclear. We have previously found that repeated antigen challenges in mice sensitized with antigen-specific IgE monoclonal antibody (mAb) exacerbated airway inflammation and remodelling accompanied by increased levels of endogenous antigen-specific IgE and IgG1. Here, we investigated whether IgE/antigen-mediated enhancement of endogenous IgE production contributes to the exacerbation of airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA) -specific IgE mAb were challenged with OVA intratracheally seven times; anti-IgE mAb was intraperitoneally administered 1 day before the fourth challenge. Treatment with anti-IgE mAb inhibited the increased level of endogenous OVA-specific IgE in serum, but not OVA-specific IgG1, and a biphasic increase in airway resistance at the fourth challenge. Furthermore, a biphasic increase in airway resistance, airway hyper-responsiveness to methacholine, OVA-specific IgE and IgG1 production, and infiltrations by neutrophils and eosinophils in the lungs at the seventh challenge were suppressed by treatment; airway remodelling, such as goblet cell hyperplasia and sub-epithelial fibrosis, was also reduced. In addition, the production of interleukin-17A, interleukin-33 and CXCL1 in the lungs related to these IgE-mediated responses was decreased by treatment. Collectively, we found that the mechanism leading to the exacerbation of allergic asthma is closely related to IgE/antigen-mediated enhancement of IgE production, suggesting that this may create a vicious circle leading to the chronic status in asthmatic patients having levels of antigen-specific IgE ready to form complexes with antigen.

Thymic stromal lymphopoietin-induced interleukin-17A is involved in the development of IgE-mediated atopic dermatitis-like skin lesions in mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with elevated levels of allergen-specific IgE. Although thymic stromal lymphopoietin (TSLP) and interleukin-17A (IL-17A) have been considered as important factors in allergic diseases, their relationships in AD have not been fully defined. Here, we show the contribution of TSLP-induced IL-17A responses to IgE-mediated AD-like skin lesions. BALB/c mice passively sensitized by intraperitoneal injections of ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were challenged with OVA applied to the skin six times. Treatment with anti-TSLP mAb during the second to sixth challenges inhibited IgE-mediated AD-like skin lesions and IL-17A production in lymph nodes. Furthermore, the increased number of IL-17A-producing CD4(+) and γδ T cells in lymph nodes and neutrophilic inflammation in the skin were reduced by anti-TSLP mAb. These findings prompted us to examine the roles of IL-17A. Treatment with anti-IL-17A mAb suppressed the AD-like skin lesions and neutrophilic inflammation; anti-Gr-1 mAb also inhibited them. Furthermore, treatment with CXCR2 antagonist reduced the AD-like skin lesions and neutrophilic inflammation accompanied by the reduction of IL-17A production; the increased CXCR2 expression in the epidermal cells was suppressed by anti-TSLP mAb. Meanwhile, these treatments, except for anti-Gr-1 mAb, inhibited the increased mast cell accumulation in the skin. Collectively, the mechanism of IgE mediating IL-17A-producing CD4(+) and γδ T cells through TSLP by repeated antigen challenges is involved in AD-like skin lesions associated with skin inflammation, such as neutrophil and mast cell accumulation; TSLP may regulate CXCR2 signalling-induced IL-17A production.

Intratracheal exposure to Fab fragments of an allergen-specific monoclonal antibody regulates asthmatic responses in mice.

Fab fragments (Fabs) maintain the ability to bind to specific antigens but lack effector functions due to the absence of the Fc portion. In the present study, we tested whether Fabs of an allergen-specific monoclonal antibody (mAb) were able to regulate asthmatic responses in mice. Asthmatic responses were induced in BALB/c mice by passive sensitization with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) (day 0) and by active sensitization with OVA (days 0 and 14), followed by intratracheal (i.t.) challenge with OVA on day 1 and days 28, 29, 30 and 35. Fabs prepared by the digestion of an anti-OVA IgG1 (O1-10) mAb with papain were i.t. administered only once 30 min before antigenic challenge on day 1 or day 35. The results showed that i.t. administration of O1-10 Fabs with OVA markedly suppressed the early and/or late phases of asthmatic responses caused by passive and active sensitization. Similar results were obtained when Fabs of anti-OVA IgG2b mAb (O2B-3) were i.t. administered. In contrast, neither i.t. injection of intact 01-10/O2B-3 nor systemic injection of O1-10 Fabs suppressed the asthmatic responses. In vitro studies revealed that the capture of OVA by O1-10 Fabs prevented the subsequent binding of intact anti-OVA pAbs to the captured OVA. These results suggest that asthmatic responses may be down-regulated by the i.t. exposure to Fabs of an allergen-specific mAb via a mechanism involving the capture of allergen by Fabs in the respiratory tract before the interaction of intact antibody and allergen essential for the induction of asthmatic responses.

Complement C3a-induced IL-17 plays a critical role in an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness via neutrophilic inflammation in mice.

Allergen-specific IgE plays an essential role in the pathogenesis of allergic asthma. Although there has been increasing evidence suggesting the involvement of IL-17 in the disease, the relationship between IL-17 and IgE-mediated asthmatic responses has not yet been defined. In this study, we attempted to elucidate the contribution of IL-17 to an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness (AHR). BALB/c mice passively sensitized with an OVA-specific IgE mAb were challenged with OVA intratracheally four times. The fourth challenge caused a late-phase increase in airway resistance associated with elevated levels of IL-17(+)CD4(+) cells in the lungs. Multiple treatments with a C3a receptor antagonist or anti-C3a mAb during the challenges inhibited the increase in IL-17(+)CD4(+) cells. Meanwhile, a single treatment with the antagonist or the mAb at the fourth challenge suppressed the late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid. Because IL-17 production in the lungs was significantly repressed by both treatments, the effect of an anti-IL-17 mAb was examined. The late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid was inhibited. Furthermore, an anti-Gr-1 mAb had a similar effect. Collectively, we found that IgE mediated the increase of IL-17(+)CD4(+) cells in the lungs caused by repeated Ag challenges via C3a. The mechanisms leading to the IgE-mediated late-phase asthmatic response and AHR are closely associated with neutrophilic inflammation through the production of IL-17 induced by C3a.

Intravenous IgA complexed with antigen reduces primary antibody response to the antigen and anaphylaxis upon antigen re-exposure by inhibiting Th1 and Th2 activation in mice.

CONTEXT: Serum IgG, IgE and IgM have been shown to enhance the primary antibody responses upon exposure to the soluble antigens recognized by those antibodies. However, how IgA affects these responses remains unknown. OBJECTIVE: We investigated the effects of intravenously administered monoclonal IgA on the immune responses in mice. MATERIALS AND METHODS: DBA/1J mice were immunized with ovalbumin in the presence or absence of anti-ovalbumin monoclonal IgA. The Th1 and Th2 immune responses to ovalbumin and the anaphylaxis induced by re-exposure to ovalbumin were measured. RESULTS: IgA complexed with antigen attenuated the primary antibody responses to the antigen in mice, in contrast to IgG2b and IgE. The primary antibody responses, i.e. the de novo synthesis of anti-ovalbumin IgG2a, IgG1 and IgE in the serum, and the subsequent anaphylaxis induced with re-exposure to ovalbumin were reduced by the co-injection of anti-ovalbumin monoclonal IgA at ovalbumin immunization. The Th1, Th2 and Tr1 cytokines interferon-γ, interleukin-4 and interleukin-10, respectively, released from ovalbumin-restimulated cultured splenocytes collected from allergic mice were also reduced by the treatment. The induction of interferon-γ and interleukin-4 secretion by splenocytes from ovalbumin-immunized mice stimulated in vitro with ovalbumin was also significantly reduced by the antigen complexed with anti-ovalbumin IgA. CONCLUSION: These data suggest that the direct inhibition of Th1 and Th2 activation by anti-ovalbumin monoclonal IgA participates in the inhibition of the primary antibody responses. IgA plays important immunosuppressive roles under physiological and pathological conditions and is a promising candidate drug for the treatment of immune disorders.

Interleukin-33 and alveolar macrophages contribute to the mechanisms underlying the exacerbation of IgE-mediated airway inflammation and remodelling in mice.

Allergen-specific IgE has long been regarded as a major molecular component of allergic asthma. Additionally, there is increasing evidence of the important roles of interleukin-33 (IL-33) in the disease. Here, we show that IL-33 and alveolar macrophages play essential roles in the exacerbation of IgE-mediated airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were challenged with OVA seven times intratracheally. The seventh challenge exacerbated airway inflammation and remodelling compared with the fourth challenge; furthermore, markedly increased expression of IL-33 in the lungs was observed at the fourth and seventh challenges. When anti-IL-33 or anti-ST2 antibody was administered during the fourth to seventh challenge, airway inflammation and remodelling were significantly inhibited at the seventh challenge. Because increases of IL-33(+) and ST2(+) alveolar macrophages and ST2(+)  CD4(+) T cells in the lungs were observed at the fourth challenge, the roles of macrophages and CD4(+) cells were investigated. Depletion of macrophages by 2-chloroadenosine during the fourth to seventh challenge suppressed airway inflammation and remodelling, and IL-33 production in the lung at the seventh challenge; additionally, anti-CD4 mAb inhibited airway inflammation, but not airway remodelling and IL-33 production. Meanwhile, treatment with 2-chloroadenosine or anti-CD4 mAb decreased IL-33-induced airway inflammation in normal mice; airway remodelling was repressed only by 2-chloroadenosine. These results illustrate that macrophage-derived IL-33 contributes to the exacerbation of IgE-mediated airway inflammation by mechanisms associated with macrophages and CD4(+) cells, and airway remodelling through the activation of macrophages.

Efficient induction of arthritis in mice by an arthritogenic 4-clone cocktail of anti-type II collagen monoclonal antibodies recognizing different epitopes of the antigen.

We previously reported that a combination of 4 monoclonal antibodies (mAbs) (cocktail A) to type II collagen (CII), including immunoglobulin G (IgG)2b (C2B-9 and C2B-14) and IgG2a (C2A-7 and C2A-12), induced arthritis in DBA/1J mice. In this study, we found that C2B-9 and C2A-7 as well as C2B-14 and C2A-12 recognized the same or similar epitopes on CII. Based on these data, we hypothesized that the combination of more than 3 mAbs recognizing different epitopes on CII may more efficiently induce arthritis. Therefore, in addition to C2B-9 and C2B-14, which show high binding activity to CII compared with C2A-7 and C2A-12, we developed two more mAbs including IgG2b (C2B-17) and IgG2a (C2A-16), to make a new cocktail (cocktail B) consisting of these 4 mAbs. To compare the ability of cocktail B to induce arthritis with cocktail A, DBA/1J mice were injected with these cocktails. The results showed that cocktail B was able to induce more severe arthritis than cocktail A, especially more markedly affecting rear paws. Histologically, there was more marked proliferation of synovial tissues, massive infiltration by inflammatory cells, and severe destruction of cartilage and bone in mice treated with cocktail B than with cocktail A. Collectively, the new combination of 4 mAbs recognizing different respective epitopes appears to effectively induce arthritis in mice. Thus, the results may provide insights into the selection of mAbs associated with the development of arthritis.

Exposure to multiwalled carbon nanotubes and allergen promotes early- and late-phase increases in airway resistance in mice.

The facilitating effects of multiwalled carbon nanotubes (MWCNT) on allergic asthma have not been sufficiently examined, although MWCNT appear to significantly increase the risk of health problems from occupational or environmental exposure. In this study, we examined whether sensitization by the combination of MWCNT with ovalbumin (OVA) promotes allergic asthmatic responses. BALB/c mice administered vehicle, MWCNT, OVA, or MWCNT+OVA through an intranasal route were challenged with OVA intratracheally four times. In the MWCNT+OVA group, the fourth challenge caused not only early- but also late-phase increases in airway resistance, although these responses were not observed in the vehicle, MWCNT, or OVA group; furthermore, the extents of the early and late responses were comparable to those in mice systemically sensitized with OVA+alum. Sensitization with MWCNT and OVA promoted airway inflammation and goblet cell hyperplasia in the lung compared with the vehicle, MWCNT or OVA group. In addition, adjuvant activity for OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a production in serum and increased levels of interleukin-4 (IL-4), IL-5, IL-13, and IL-17 in the lung tissue were observed. In conclusion, these results suggest that exposure to MWCNT and antigen can induce a biphasic increase in airway resistance, airway inflammation, goblet cell hyperplasia, and the production of antigen-specific antibodies. This study highlights the risk of exposure to a combination of MWCNT with antigen.

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.

Complete dependence on CD4+ cells in late asthmatic response, but limited contribution of the cells to airway remodeling in sensitized mice.

It is known that the late asthmatic response (LAR), a characteristic feature of asthma, is closely associated with CD4+ Th2 cell-mediated allergic inflammation. Airway remodeling is also a pathogenesis of asthma, but literature reporting roles of CD4+ cells in the remodeling is controversial. There has been no study that simultaneously assessed the roles of CD4+ cells in both LAR and airway remodeling. Sensitized mice were intratracheally challenged with ovalbumin 4 times. Treatment with an anti-CD4 monoclonal antibody (mAb) before the 1st challenge almost completely abolished increase in CD4+ cells in the tissues after the 4th challenge. The late phase increase in airway resistance after the 4th challenge was also completely inhibited by anti-CD4 mAb. Parameters of airway remodeling, subepithelial fibrosis and epithelial thickening were attenuated by treatment, whereas the inhibition was only 30% - 40%. Bronchial smooth muscle thickening was not affected. Because interleukin (IL)-5 production as well as eosinophilia was effectively suppressed by anti-CD4 mAb, the effect of anti-IL-5 mAb was also examined, resulting in no inhibition of airway remodeling. Collectively, although the LAR was completely dependent on CD4+ cell activation, airway remodeling was only partially dependent on the cell.

Complement C3a regulates late asthmatic response and airway hyperresponsiveness in mice.

Allergic asthma is a chronic inflammatory disorder of the airways characterized by biphasic airway obstruction and airway hyperresponsiveness. In this study, we attempted to elucidate the contribution of the complement C3a to these asthmatic symptoms. BALB/c mice sensitized by i.p. injections of OVA plus alum were challenged with OVA intratracheally four times. The fourth challenge caused a biphasic asthmatic response peaking at 10 min and 3-4 h, as well as airway hyperresponsiveness to methacholine. Histological examination revealed increased expression of C3a receptors in the lung on the fourth challenge. Additionally, the C3 level in serum 4 h after the fourth challenge was significantly reduced compared with that before the challenge. When a C3a receptor antagonist, SB290157, was administered i.p. 30 min before the fourth challenge, the late-phase asthmatic response and airway hyperresponsivness induced by the fourth challenge were significantly inhibited, although the early-phase response was not influenced. In bronchoalveolar lavage fluid, neutrophil infiltration 24 h after the fourth challenge was reduced by the treatment. On the other hand, SB290157 suppressed the increased expression of IL-1beta in the lung in this model, and the intratracheal administration of IL-1beta induced airway obstruction, airway hyperresponsiveness, and neutrophil infiltration in normal mice. These results illustrate that C3a is involved in the development of the late asthmatic response and airway hyperresponsiveness. The mechanism leading to the development of these symptoms may correlate with the recruitment of neutrophils and/or the production of IL-1beta induced by C3a.

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.

Intratracheal sensitization/challenge-induced biphasic asthmatic response and airway hyperresponsiveness in guinea pigs.

In most experimental model of asthma using guinea pigs, the animals are made to inhale an aerosolized antigen which passes through the nasal cavity. In the present study, we attempted to create an animal model of asthma showing a biphasic asthmatic response and airway hyperresponsiveness, in which the allergic responses are restricted to the lung. Guinea pigs were sensitized by the intratracheal instillation of ovalbumin (OVA)+Al(OH)3 once a day for 7 d, and then intratracheally challenged with OVA 12 d after the last sensitization. The change in specific airway resistance (sRaw) and airway responsiveness to histamine were measured. Pranlukast (100 mg/kg), theophylline (50 mg/kg), and dexamethasone (10 mg/kg) were orally administered 18 and 2 h before the antigen challenge. The challenge caused a marked biphasic elevation of sRaw with peaks at 5 min and 4 h. At 24 h, airway hyperresponsiveness to histamine was observed. Pranlukast, theophylline, and dexamethasone suppressed the late asthmatic response and airway hyperresponsiveness. The early asthmatic response was inhibited by theophylline and dexamethasone. In conclusion, the intratracheal sensitization and challenge caused a biphasic asthmatic response and airway hyperresponsiveness in guinea pigs. This model may be useful for the evaluation of anti-asthma drugs.

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice.

BACKGROUND AND AIM: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice. MATERIAL AND METHODS: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-gamma as well as Th2 cytokines such as IL-4 and IL-5 were measured. RESULTS: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-gamma. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells. CONCLUSION: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.

Effects of focused ultrasound sonodynamic treatment on the rat blood-brain barrier.

BACKGROUND: Ultrasound has recently been applied to the treatment as well as the diagnosis of various pathologies, and its antitumor effects in the treatment of human cancer and experimental models of cancer have been demonstrated. In addition, it is possible that certain photosensitizers will enhance the antitumor effects of ultrasound. However, very few studies have been reported on how the blood-brain barrier is affected by sonodynamic therapy. The purpose of this study was to evaluate disruption of the blood-brain barrier with focused ultrasound with a photosensitizer, for clinical application of sonodynamic therapy to brain tumors. MATERIALS AND METHODS: Rat brains were subjected to focused ultrasound irradiation via a transducer with or without prior intravenous injection of photosensitizer, and lesions were examined histologically by electron microscopy. RESULTS: Electron microscopically, swelling of astroglial processes, denatured cells, protoplasm of endothelial cells, and mitochondria were observed in the center and border of regions of ultrasonic irradiation. There were numerous pinocytotic vesicles in the cytoplasm of the endothelial cells. In addition, disruption of the cytoplasmic membrane of endothelial cells and astroglia was found in these regions. CONCLUSION: These findings suggest that sonodynamic therapy with a photosensitizer affects the blood-brain barrier, and that blood vessel permeability increases not only as a result of destruction of the blood-brain barrier but also by disruption of the cytoplasmic membrane of endothelial cells.

Comparison of inhibitory activities of zinc oxide ultrafine and fine particulates on IgE-induced mast cell activation.

The effects of ultrafine and fine particles of zinc oxide (ZnO) on IgE-dependent mast cell activation were investigated. The rat mast cell line RBL2H3 sensitized with monoclonal anti-ovalbumin (OVA) IgE was challenged with OVA in the presence or absence of ZnO particles and zinc sulfate (ZnSO4). Degranulation of RBL2H3 was examined by the release of ß-hexosaminidase. To understand the mechanisms responsible for regulating mast cell functions, the effects of ZnO particles on the levels of intracellular Zn2+, Ca2+, phosphorylated-Akt, and global tyrosine phosphorylation were also measured. IgE-induced release of b-hexosaminidase was obviously attenuated by ultrafine ZnO particles and ZnSO4, whereas it was very weakly inhibited by fine ZnO particles. The intracellular Zn2+ concentration was higher in the cells incubated with ultrafine ZnO particles than in those with fine ZnO particles. Consistent with inhibitory effect on release of b-hexosaminidase, ultrafine ZnO particles and ZnSO4, but not fine ZnO particle, strongly attenuated the IgE-mediated increase of phosphorylated-Akt and tyrosine phosphorylations of 100 and 70 kDa proteins in RBL2H3 cells. These findings indicate that ultrafine ZnO particles, with a small diameter and a large total surface area/mass, could release Zn2+ easily and increase intracellular Zn2+ concentration efficiently, thus decreasing FceRI-mediated mast cell degranulation through inhibitions of PI3K and protein tyrosine kinase activation. Exposure to ZnO particles might affect immune responses, especially in allergic diseases.

Pulmonary emphysema induced by cigarette smoke solution and lipopolysaccharide in guinea pigs.

Exposure of animals to cigarette smoke for longer than 3 months leads to the development of chronic obstructive pulmonary disease (COPD) showing pulmonary emphysema. We attempted to create a COPD model with emphysema that could be established in a shorter period of time. Guinea pigs were intratracheally treated once a day on days 0-3, 5-8, 10-13 and 15-18 with a cigarette smoke solution (CSS), which was prepared by bubbling a stream of smoke into saline. Additionally, lipopolysaccharide (LPS) was administered intratracheally as an exacerbation factor on days 4, 9 and 14. By day 19, there was a gradual elevation of specific airway resistance (sRaw). In addition, both residual volume and functional residual capacity were found to be significantly higher on day 19. In the lungs, there was a marked increase in leukocytes, especially neutrophils. Histologically, we observed epithelial hyperplasia and emphysema. On the other hand, daily oral administration of theophylline during the administration of CSS and LPS suppressed the sRaw increase and the epithelial hyperplasia, but not other functional structural changes. In conclusion, we established an experimental COPD model in guinea pigs by using intratracheal instillations of CSS and LPS over a considerably shorter term than has been reported for other models.

Inhibition of IgE-induced mast cell activation by ethyl tertiary-butyl ether, a bioethanol-derived fuel oxygenate.

OBJECTIVES: The effect of ethyl tertiary-butyl ether (ETBE), which is widely used as a fuel oxygenate commonly produced from bioethanol, on immunoglobulin (Ig)E-dependent mast cell activation was investigated. METHODS: The rat mast cell line RBL2H3 sensitised with monoclonal anti-ovalbumin IgE was challenged with ovalbumin in the presence or absence of ETBE, tert-butanol (TBA), which is the main metabolite of ETBE in humans, and ethanol. Degranulation of RBL2H3 was examined by the release of beta-hexosaminidase. To understand the mechanisms responsible for regulating mast cell function, the effects of ETBE, TBA and ethanol on the levels of intracellular calcium, phosphorylation of Akt (as a marker of phosphatidylinositol 3-kinase) and global tyrosine phosphorylation were also measured as indicators of mast cell activation. KEY FINDINGS: In the presence of ETBE, TBA or ethanol, IgE-induced release of beta-hexosaminidase was decreased. These compounds also attenuated the IgE-mediated increase in the levels of intracellular Ca(2+), phosphorylation of Akt and global tyrosine phosphorylation in RBL2H3 cells. CONCLUSIONS: ETBE, TBA and ethanol inhibited mast cell degranulation by inhibiting the increase in intracellular calcium ion concentration and activation of phosphatidylinositol 3-kinase and protein tyrosine kinase activation, suggesting that exposure to ETBE might affect immune responses, particularly in allergic diseases.