Dinitrophenyl-human Serum Albumin Research Articles

Effect of stearoyl-coenzyme a desaturase 1 (SCD1) on the function of mast cells

Background: The prevalence of childhood asthma and obesity is increasing, while obesity increases the risk and severity of asthma. Lipid metabolism has been considered as an important factor in the pathogenesis of obesity-associated asthma. Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that catalyzes the production of monounsaturated fatty acids (MUFA).Methods: In the present study, the microarray data retrieved from the Gene Expression Comprehensive Database (GEO) was analyzed to further clarify the impact of SCD1 on Mast cell activation related lipid mediators and the correlation between SCD1 and obesity asthma in the population.Results: SCD1 was highly expressed in IgE-activated bone marrow-derived mast cells (BMMCs). Meanwhile, SCD1 was also verified expressed highly in dinitrophenyl human serum albumin (DNP-HAS) stimulated RBL-2H3 cells. The expression of SCD1 was up-regulated in peripheral blood leukocytes of asthmatic children, and was positively correlated with skinfold thickness of upper arm, abdominal skinfold and body mass index (BMI). Inhibition of SCD1 expression significantly suppressed the degranulation, lipid mediator production, as well as the migration ability in DNP-HAS-stimulated RBL-2H3 cells.Conclusion: SCD1 is involved in obese-related asthma through regulating mast cells.

Three phlorotannins from Sargassum carpophyllum are effective against the secretion of allergic mediators from antigen-stimulated rat basophilic leukemia cells

Sargassum carpophyllum (Sargassaceae) is a brown seaweed that contains phlorotannins, which are phloroglucinol polymers with reported anti-inflammatory activities. The phlorotannins 2-[2-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy]-1,3,5-benzenetriol (1), 2,2′-[[2-(3,5-dihydroxyphenoxy)-5-hydroxy-1,3-phenylene]bis(oxy)]bis(1,3,5-benzenetriol) (2), and 2-[2-[4-[2-(3,5-dihydroxyphenoxy)-3,5-dihydroxyphenoxy]-3,5-dihydroxyphenoxy]-3,5-dihydroxyphenoxy]-1,3,5-benzenetriol (3) were isolated from S. carpophyllum. Here, we evaluated the anti-allergic activities of these compounds and comprehensively explored their effects on intracellular protein levels. Immunoglobulin E-sensitized rat basophilic leukemia cells pretreated with any of these three compounds exhibited reduced β-hexosaminidase, prostaglandin D2, and tumor necrosis factor-α secretion compared with dinitrophenyl-human serum albumin (DNP-HSA)-stimulated cells. Reduction of β-hexosaminidase release was dose-dependent but the half-maximal inhibitory concentrations of the compounds were similar (36–51 μM). Proteomics analysis revealed that the three compounds up-regulated 25 proteins and down-regulated 33 proteins compared with DNP-HSA stimulation alone, and slightly suppressed proteasome 5 expression linked to the regulation of IκB. These results demonstrate that these phlorotannins are potentially useful for preventing immediate hypersensitivity. S. carpophyllum may be a functional food.

Cornus officinalis Ethanolic Extract with Potential Anti-Allergic, Anti-Inflammatory, and Antioxidant Activities.

Atopic dermatitis (AD) is an allergic and chronic inflammatory skin disease. The present study investigates the anti-allergic, antioxidant, and anti-inflammatory activities of the ethanolic extract of Cornus officinalis (COFE) for possible applications in the treatment of AD. COFE inhibits the release of β-hexosaminidase from RBL-2H3 cells sensitized with the dinitrophenyl-immunoglobulin E (IgE-DNP) antibody after stimulation with dinitrophenyl-human serum albumin (DNP-HSA) in a concentration-dependent manner (IC50 = 0.178 mg/mL). Antioxidant activity determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power assay, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity, result in EC50 values of 1.82, 10.76, and 0.6 mg/mL, respectively. Moreover, the extract significantly inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production and the mRNA expression of iNOS and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) through attenuation of NF-κB activation in RAW 264.7 cells. COFE significantly inhibits TNF-α-induced apoptosis in HaCaT cells without cytotoxic effects (p < 0.05). Furthermore, 2-furancarboxaldehyde and loganin are identified by gas chromatography/mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, respectively, as the major compounds. Molecular docking analysis shows that loganin, cornuside, and naringenin 7-O-β-D-glucoside could potentially disrupt the binding of IgE to human high-affinity IgE receptors (FceRI). Our results suggest that COFE might possess potential inhibitory effects on allergic responses, oxidative stress, and inflammatory responses.

In vitro and in vivo Antiallergic Effects of Taurine on Allergic Rhinitis

Background: The current treatment for allergic rhinitis (AR) is inadequate. Objective: The present study aimed to investigate the therapeutic effect of taurine on AR and to identify the underlying molecular mechanisms. Methods: The serum level of the antioxidant enzyme extracellular superoxide dismutase (SOD3) was determined in AR patients and in healthy controls. The antiallergic inflammatory effects of taurine were evaluated in a dinitrophenyl-human serum albumin (DNP-HSA)-stimulated human mast cell line (HMC-1) and in an ovalbumin (OVA)-induced AR mouse model. Results: Clinically, a reduction in serum level of SOD3 was observed in AR patients. Taurine treatment led to dose-dependent increases in SOD3 at both protein and mRNA levels in HMC-1 cells. SOD3 production was regulated by peroxisome proliferator-activated receptor-γ (PPAR-γ) in response to taurine. SOD3 overexpression inhibited the release of proinflammatory cytokines including tumor necrosis factor-α (, interleukin (IL)-4, and IL-6. Its overexpression also ameliorated the loss of interferon-γ. SOD3 and PPAR-γ influenced inflammatory cytokine production via regulation of the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). An OVA-induced AR animal model study showed that taurine was efficacious in alleviating allergic inflammatory reactions by relieving behavior symptoms of AR mice and reducing eosinophilic and mast cell infiltration into the nasal cavity. In addition, taurine treatment increased the production of SOD3 and PPAR-γ, which, in turn, suppressed expression of proinflammatory cytokines through phosphorylation of ERK1/2. Conclusion: Taurine could potentially serve as a therapeutic treatment for allergic disorders.

Seleno-L-Methionine Suppresses Immunoglobulin E-Mediated Allergic Response in RBL-2H3 Cells.

The effect of seleno-L-methionine (SeMet) on immunoglobulin (Ig) E-mediated allergic responses were investigated using rat basophilic leukemia RBL-2H3 cells. Cells were first treated with or without SeMet, sensitized with anti-dinitrophenyl IgE and stimulated with the antigen dinitrophenyl-human serum albumin, before the measurement of degranulation, calcium mobilization, mRNA expression and protein secretion of interleukin (IL)-4 and tumor necrosis factor (TNF)-α, and phosphorylation of spleen tyrosine kinase (Syk), Akt, and mitogen-activated protein kinases (MAPKs). The antigen-induced β-hexosaminidase release, a degranulation marker, was significantly inhibited by SeMet treatment. SeMet also significantly suppressed antigen-induced calcium mobilization. Antigen-induced increases in the mRNA expression and protein secretion of IL-4 and TNF-α were both significantly attenuated by SeMet treatment. In addition, SeMet significantly suppressed antigen-induced phosphorylation of Syk, Akt, and MAPKs. These results demonstrate that SeMet suppresses antigen-induced degranulation, and mRNA expression and protein secretion of IL-4 and TNF-α, and inhibits antigen-induced mobilization of calcium and activation of Syk, Akt, and MAPKs. Our study provides valuable information that may be useful in the prevention and treatment of allergic diseases.

Licarin A is a candidate compound for the treatment of immediate hypersensitivity via inhibition of rat mast cell line RBL-2H3 cells.

We previously demonstrated that some phenylpropanoids are capable of inhibiting activated mast cells. This study evaluated the anti-allergic effects of licarin A, a neolignan isolated from various plants, on antigen-stimulated rat mast cell line. The inhibitory effects of licarin A on histamine release, tumour necrosis factor-α (TNF-α) and prostaglandin D2 (PGD2) production, and cyclooxygenase-2 (COX-2) expression in dinitrophenyl-human serum albumin (DNP-HSA) rat basophilic leukemia cells (DNP-HSA-stimulated RBL-2H3 cells), were investigated by spectrofluorometry, ELISA and immunoblotting. Licarin A significantly and dose-dependently reduced TNF-α production (IC50 12.6 ± 0.3 μm) in DNP-HSA-stimulated RBL-2H3 cells. Furthermore, the levels of PGD2 secretion in DNP-HSA-stimulated cells pretreated with licarin A were lower than those stimulated with DNP-HSA alone (positive control). Treatment with licarin A at 20 μm produced slight suppression of DNP-HSA-induced increases in COX-2 mRNA and protein levels. We identified several signalling pathways that mediated these pharmacological effects. Licarin A treatment tended to reduce phosphorylated protein kinase C alpha/beta II (PKCα/βII) and p38 mitogen-activated protein kinase (MAPK) protein levels. Our results demonstrate that licarin A reduces TNF-α and PGD2 secretion via the inhibition of PKCα/βII and p38 MAPK pathways; this compound may be useful for attenuating immediate hypersensitivity.

Elemol from Chamaecyparis obtusa ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis.

Chamaecyparis obtusa has been traditionally used as an antibiotic agent and in cosmetics for the prevention of microorganism infection and skin troubles. Atopic dermatitis (AD) is a chronic inflammatory skin disease that encompasses immunologic responses, susceptibility factors and compromised skin-barrier function. Use of plant medicines in therapeutic treatment of AD has recently been suggested as an alternative therapeutic option. The present study examined the effect of elemol, an active component of Chamaecyparis obtusa, on AD using in vivo and in vitro models. RBL-2H3 cells were stimulated with concanavalin A and dinitrophenyl human serum albumin, and atopic dermatitis was induced in BALB/c mice by topical application of 2,4-dinitrochlorobenzene (DNCB) prior to elemol treatment. The mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction, and the levels of β-hexosaminidase and serum immunoglobulin E (IgE) were examined by ELISA. Histological changes were also performed by microscopy. Elemol attenuated the onset of AD-like skin lesions, reduced serum IgE levels and decreased mast cell infiltration into the dermis and hypodermis. In addition, elemol downregulated the transcriptional expression of several pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IκBα, in the skin of the DNCB-induced animal models of AD. In the RBL-2H3 mast cell line, elemol significantly inhibited the mRNA expression of IL-4 and IL-13, and further attenuated the release of β-hexosaminidase from mast cells. Histological examination revealed that elemol significantly ameliorated the DNCB-induced dermal destruction in mice. The results of the present study suggested that elemol may have therapeutic potential in the treatment of AD due to its immunosuppressive effects.

Resveratrol Suppresses Cytokine Production Linked to FcεRI-MAPK Activation in IgE-Antigen Complex-Exposed Basophilic Mast Cells and Mice.

A complicated interplay between resident mast cells and other recruited inflammatory cells contributes to the development and progression of allergic inflammation entailing the promotion of T helper 2 (Th2) cytokine responses. The current study examined whether resveratrol suppressed the production of inflammatory Th2 cytokines in cultured rat basophilic leukemia RBL-2H3 cells. Cells pre-treated with resveratrol nontoxic at 1–25 μM were sensitized with anti-dinitrophenyl (anti-DNP), and subsequently stimulated by dinitrophenyl-human serum albumin (DNP–HSA) antigen. Resveratrol dose-dependently diminished the secretion of interleukin (IL)-3, IL-4, IL-13 as well as tumor necrosis factor (TNF)-α by the antigen stimulation from sensitized cells. It was found that resveratrol mitigated the phosphorylation of p38 MAPK, ERK, and JNK elevated in mast cells exposed to Fc epsilon receptor I (FcεRI)-mediated immunoglobulin E (IgE)-antigen complex. The FcεRI aggregation was highly enhanced on the surface of mast cells following the HSA stimulation, which was retarded by treatment with 1–25 μM resveratrol. The IgE-receptor engagement rapidly induced tyrosine phosphorylation of c-Src-related focal adhesion protein paxillin involved in the cytoskeleton rearrangement. The FcεRI-mediated rapid activation of c-Src and paxillin was attenuated in a dose-dependent manner. In addition, the paxillin activation entailed p38 MAPK and ERK-responsive signaling, but the JNK activation was less involved. Consistently, oral administration of resveratrol reduced the tissue level of phosphorylated paxillin in the dorsal skin of DNP–HSA-challenged mice. The other tyrosine kinase Tyk2-STAT1 signaling was activated in the dorsal epidermis of antigen-exposed mice, which was associated with allergic inflammation. These results showed that resveratrol inhibited Th2 cytokines- and paxillin-linked allergic responses dependent upon MAPK signaling. Therefore, resveratrol may possess the therapeutic potential of targeting mast cells in preventing the development of allergic inflammation.

Inhibitory effects of Acorus calamus extracts on mast cell-dependent anaphylactic reactions using mast cell and mouse model

Ethnopharmacological relevanceAcorus calamus Linn. (Araceae) is a traditional herbal plant used for centuries to treat various allergic symptoms including asthma and bronchitis. Aim of the studyThe present study was focused to provide a pharmacological basis for the traditional use of Acorus calamus in allergic symptoms using the mast cell-dependent anaphylactic reactions in in vitro and in vivo models. Materials and methodsCell viabilities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Dinitrophenyl-human serum albumin (DNP-HSA) induced β-hexosaminidase and interleukin (IL)-4 productions in IgE-sensitized rat basophilic leukaemia (RBL-2H3) cells were measured by enzymatic assay and enzyme-linked immunosorbent assay (ELISA). Passive cutaneous anaphylaxis (PCA) reaction mouse model was implemented for in vivo studies. ResultsHot water (HW), butylene glycol (BG), hexane (HE) and steam distilled (SD) extracts of Acorus calamus showed different cytoxicity levels evaluated in RBL-2H3 cells. Sub-toxic doses of HW extract suppressed the β-hexosaminidase secretion and IL-4 production significantly and dose dependently in DNP-HSA induced IgE-sensitized RBL-2H3 cells compared to other extracts of Acorus calamus. Further, in vivo studies also revealed that the HW extract significantly inhibited the PCA reaction in mouse compared to the normal control group. ConclusionHW extract of Acorus calamus most effectively inhibited degranulation and IL-4 secretion in DNP-HSA-stimulated RBL-2H3 cells and also reduced the mast cell-mediated PCA reaction in mouse, providing a therapeutic evidence for its traditional use in ameliorating allergic reactions.

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.

Inhibition of the Antigen-Induced Activation of RBL-2H3 Cells by Gab2 siRNA

Gab2 plays an important role in FcepsilonRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 (scaffolding adapter Grb2-associated binder 2) siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2-mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl (DNP)-specific IgE overnight and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of beta-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine, reduced the production of IL-4 and TNF-alpha and inhibited the phosphorylation of Akt, PKCdelta and p38 mitogen-activated protein kinase (MAPK). Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the FcepsilonRI-mediated Ca(2+)-independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated.

Inhibition of the antigen-induced activation of RBL-2H3 cells by sinomenine

The present study was designed to investigate the effect of sinomenine (SIN), an alkaloid extracted from sinomenium acutum, on the antigen-induced activation of RBL-2H3. For this investigation, the RBL-2H3 cells were sensitized with dinitrophenyl (DNP)-specific IgE overnight in 1.0 ml of Eagle's MEM (EMEM), and varying doses of SIN were added to the culture medium for 30 min and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. The effects of SIN on antigen-induced release of β-hexosaminidase were measured by enzymatic assay, calcium influx by FACS, cytokines by ELISA, and signaling events by immunoblotting. The results showed that treatment with SIN was followed by a decrease in FcεRI-mediated mast cell release of β-hexosaminidase, production of IL-4 and TNF-α, phosphorylation of Gab2 (Scaffolding adapter Grb2-associated binder 2), Akt and p38 mitogen-activated protein kinase (MAPK). In addition, SIN had no effect on the phosphorylation of LAT and no significant difference on calcium mobilization was observed between control and SIN treated group. These results suggested that SIN might suppress the antigen-induced activation of RBL-2H3 cells via a Ca 2+ independent pathway.

The High‐Affinity Immunoglobulin‐E Receptor (FceRI) is Endocytosed by an AP‐2/Clathrin‐Independent, Dynamin‐Dependent Mechanism

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI), expressed on mast cells and basophils, initiates the immediate hypersensitivity reaction. Aggregated FcepsilonRI has been reported to rapidly migrate to lipid rafts in RBL-2H3 cells. We confirmed that aggregated FcepsilonRI is found in the lipid raft fractions of cellular lysates. Furthermore, we show that the cross-linked FcepsilonRI remains associated with detergent-resistant structures upon internalization. Previous morphological studies have reported that aggregated FepsiloncRI is endocytosed via clathrin-coated pits, which in general are not lipid raft associated. To address this apparent discrepancy, we employed siRNA to suppress expression of components of the clathrin-mediated internalization machinery, namely, clathrin heavy chain, and the AP-2 (alpha-adaptin or mu2-subunit). Transferrin receptor (TfR) is endocytosed by a clathrin-mediated process and, as expected, each transfected siRNA caused a two to threefold elevation of TfR surface expression and almost completely inhibited its endocytosis. In contrast, there was no effect on surface expression levels of FcepsilonRI nor on the endocytosis of the dinitrophenyl-human serum albumin (DNP-HSA)/IgE/FcepsilonRI complex. On the contrary, internalization of DNP-HSA/IgE/FcepsilonRI was inhibited by overexpression of a dominant-negative dynamin mutant. We conclude that internalization of cross-linked FcRI does not require the AP-2/clathrin complex but is dynamin-dependent and may be lipid raft mediated.

IgE-dependent Activation of Sphingosine Kinases 1 and 2 and Secretion of Sphingosine 1-Phosphate Requires Fyn Kinase and Contributes to Mast Cell Responses

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells results in the production and secretion of sphingosine 1-phosphate (S1P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of sphingosine kinase (SphK1 and SphK2) are expressed and activated upon FcepsilonRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcepsilonRI coupling to SphK1 and -2 and for subsequent S1P production. Normal activation of SphK1 and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK1 and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK1 activation differed from that of SphK2. Whereas SphK1 was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to FcepsilonRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.

Development of the Mouse Passive Abdominal Wall Anaphylaxis (PAA) Method and Application of the Method to Search for Anti-allergic Effect of Foods

Food allergy, which generally belongs to the category of Type I hypersensitivity reaction, is a serious health problem. Previously, we reported two types of mouse anaphylactic models, one using the abdominal wall as a site for both induction and estimation (AW method), and the other estimating the hypotension in mice sensitized passively with mouse monoclonal anti-DNP IgE.In this paper, we propose a passive abdominal wall anaphylaxis (PAA) method for use in screening for anti-allergic substances. Normal mice were intraperitoneally sensitized with mouse monoclonal anti-DNP IgE (5μg/mouse) and 24 hours later, challenged with dinitrophenyl human serum albumin (10μg/50μL/site) on the abdominal wall. The PAA was estimated in terms of the increase of vascular permeability at the challenged abdominal wall site.The PAA was inhibited by the oral preadministration of diphenhydramine in a dosedependent manner (PAA inhibition, PAAI). The anti-allergic activities of such well-known anti-allergic foods as oolong tea, garlic, and leek were confirmed by the PAAI test. Therefore, the PAA method appears to be applicable for the screening of anti-allergic substances in foods, simply, rapidly, and with high sensitivity.

Association Between Immunisation, Reduced Weight Gain and Plasma Cortisol Concentrations in Juvenile Baltic Salmon (Salmo salar)

The changes in plasma cortisol levels, immune response parameters and growth of juvenile Atlantic salmon (Salmo salar) were monitored during a 50 days period following a DNP-HSA (di-nitrophenyl human serum albumin) immunisation program. Antibody titers rose significantly after a single immunisation. An increased plasma cortisol concentration was observed in association with injection of both antigen and saline. A single injection had a significant negative effect on growth of fish and fish subjected to 2 injections with a 25 days interval had an even larger growth reduction. The plasma cortisol concentration and the specific antibody response were compared at an individual level but no correlation was found. Total serum protein increased during the experimental period independently of immunisation. In contrast the total serum immunoglobulin 50 days after the first immunisation was clearly connected to antigen exposure. The observations are discussed in relation to immunophysiological changes during immunisation and stress induction.

Effects of Poncirus trifoliata on type I hypersensitivity reaction.

A study was carried out to examine the effect of an aqueous extract from immature fruit of Poncirus trifoliata L. (Rutaceae) (PTIFE) on the type I hypersensitivity reaction. Forty-eight hour PCA (passive cutaneous anaphylaxis) in rats was significantly inhibited by the oral administration of PTIFE (200 mg/kg). It also inhibited histamine release from rat peritoneal mast cells (RPMC) induced by mouse anti-dinitrophenyl (DNP)-IgE and dinitrophenyl-human serum albumin (DNP-HSA). These results suggest that PTIFE has anti-allergic action against the type I hypersensitivity reaction.

Effect of azelastine on airway hyperresponsiveness mediated by stimulated macrophages

The effect of the anti-allergic drug azelastine, 4-( p-chlorobenzyl)-2-(hexahydro-1-methyl-1 H-azepine-4-yl)-1-(2H)-phthalazione), on airway hyperresponsiveness induced by immunologically stimulated pulmonary alveolar macrophages was investigated in canine bronchial segments under isometric conditions in vitro. Macrophages stimulated with anti-dinitrophenyl immunoglobulin E (IgE) antibody and dinitrophenyl-human serum albumin potentiated the contractile responses to electrical field stimulation at all frequencies, an effect that was abolished by azelastine (3 × 10 −5 M). In contrast, azelastine had no effect on the potentiation of the contractile responses to electrical stimulation by U46619, a thromboxane A 2 mimetic. The IgE-mediated release of thromboxane A 2 from macrophages was inhibited by azelastine in a concentration-dependent fashion, the maximal decrease and the concentration required to produce a half-maximal effect being 84 ± 6% ( P < 0.001) and 16 μM, respectively. These results suggest that azelastine may attenuate macrophage-induced parasympathetic airway hyperresponsiveness through an inhibition of the release of thromboxane A 2.

Preferential transport of IgA and IgA-immune complexes to bile compared with other external secretions

When radiolabeled dinitrophenyl-human serum albumin was injected intravenously in mice together with M315 IgA, labeled antigen was specifically transported into bile, but not into saliva, urine, milk or bronchial and intestinal secretions. Ultracentrifugal analysis showed that the antigen transported into bile was intact and partly complexed with IgA. The radioactivity that was present in other secretions regardless of M315 IgA, represented free and degraded fragments of antigen. M315 IgA alone was readily transported into bile, where it was detected at high titer by hemagglutination, but not into other secretions, apart from milk which contained only very low titers. The liver therefore appears to be singularly capable of transporting both free and complexed IgA into its secretion, the bile.

Role of serum IgA. Hepatobiliary transport of circulating antigen.

The IgA mediated hepatobiliary excretion of antigen from the circulation was studied using a radiolabeled haptenated protein (dinitrophenyl-human serum albumin) injected intravenously in mice together with monoclonal anti-dinitrophenyl antibodies of different immunoglobulin classes. Antibodies were obtained from ascitic fluids of mice bearing the MOPC315 myeloma (IgA), or immune spleen cell hybridomas (IgG and IgM). IgA antibody brought about the transport of large amounts of antigen from the circulation to the bile during 1-3h. Analysis of bile by gel filtration showed that a large part of the transported antigen remained intact and complexed with IgA. Neither IgA of different specificity nor anti-dinitrophenyl IgM medicated biliary transport of antigen. With anti-dinitrophenyl IgG, only small amounts of low molecular weight fragments of labeled antigen were found in he bile. Preformed immune complex of radiolabeled antigen and IgA antibody were rapidly transported from the circulation to the bile, resulting in threefold-higher levels of radioactivity in bile than in serum. It is proposed that an important function of serum IgA is to mediate the hepatobiliary excretion of corresponding circulating antigens.