Animals ; Asthma ; immunology ; Diabetes Mellitus, Experimental ; immunology ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Male ; Ovalbumin ; immunology ; Rats ; Rats, Sprague-Dawley ; Streptozocin ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo establish a food allergy model in Brown Norway (BN) rats by gavage of ovalbumin (OVA) without any adjuvant, and to evaluate this model.

METHODSA total of 20 male BN rats aged 3 weeks were randomly divided into allergy group and control group (n=10 each). BN rats in the allergy group were given OVA 1 mg per day by gavage, and all the rats were treated for 41 days continuously. On day 42, the rats in the allergy group were given OVA 100 mg by gavage for challenge. The rats in the control group were given normal saline of the same volume by gavage. Differences in body length, body weight, and food intake were compared between the two groups on days 7, 14, 21, 28, 35, and 42. ELISA was used to measure the serum OVA-IgE level and plasma histamine level after challenge on day 42, and the changes in rats' appearance and fecal properties were observed. The model of food allergy was considered successful when the serum OVA-IgE level in the allergy group was no less than the mean serum OVA-IgE level + 3 standard deviation in the control group.

RESULTSThere were no significant differences in body length, body weight or food intake between the allergy and control groups at all time points (P>0.05). On day 21, the control group had a significantly higher food intake than the allergy group (P<0.05). On day 42 after challenge, the allergy group showed significantly higher serum OVA-IgE and plasma histamine levels than the control group (P<0.05). The sensitization rate (rate of successful modeling) was 90%. The fecal properties showed no significant differences between the two groups.

CONCLUSIONSOVA by gavage without any adjuvant can successfully establish the model of food allergy in BN rats and has a high success rate. Food allergy induced by OVA may reduce food intake within a short period of time, but no influence on rats' body length or body weight has been observed.

Animals ; Disease Models, Animal ; Food Hypersensitivity ; etiology ; immunology ; Histamine ; blood ; Immunoglobulin E ; blood ; Male ; Ovalbumin ; immunology ; Rats ; Rats, Inbred BN

BACKGROUNDThe hygiene hypothesis has been proposed to explain the pathogenesis of asthma. Allergen exposure was shown to inhibit asthma in an animal model. But the optimal timing of allergen exposure remains unclear. This study aims to explore the time effcct of allergen exposure and the possible mechanisms.

METHODSNeonate Wistar rats were randomly divided into asthma group, control group and day 1, day 3, day 7, and day 14 groups. The day 1, day 3, day 7 and day 14 groups were injected with ovalbumin (OVA) subcutaneously on days 1, 3, 7 and 14 after birth, respectively. Six weeks later, all groups, except the control group, were sensitized and stimulated with OVA to make the asthma model. We observed the pulmonary pathologic changes, detected the regulatory T cells, and CD28 expression level in thymus and spleen by flow cytometry.

RESULTSThe asthmatic inflammation in the day 1, day 3 and day 7 groups, but not the day 14 group, was alleviated. The asthma group and day 14 group had lower proportions of regulatory T cells in the thymus compared with the control group, day 1, day 3, and day 7 groups. There was no significant difference in the CD28 expression levels on the regulatory and conventional T cells among groups. But the control group and the day 1, day 3, and day 7 groups had relatively higher proportions of CD28 positive regulatory T cells in the thymus than the day 14 group and the asthma group.

CONCLUSIONSThere is a "time-window" for early allergen exposure. The impairment of regulatory T cells may promote the development of asthma. Allergen exposure in the "time-window" can make the thymus produce normal quantity of regulatory cells. The CD28 signal on regulatory T cells may participate in the production of regulatory T cells.

Allergens ; immunology ; Animals ; Asthma ; etiology ; CD28 Antigens ; analysis ; physiology ; Disease Models, Animal ; Female ; Ovalbumin ; immunology ; Rats ; Rats, Wistar ; Signal Transduction

The role of alveolar macrophages (AMs) in the pathogenesis of asthma is still unknown. The aim of the present study was to investigate the effects of AM in the murine model of asthma. AMs were selectively depleted by liposomes containing clodronate just before allergen challenges, and changes in inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid were measured. AMs were then adoptively transferred to AM-depleted sensitized mice and changes were measured. Phenotypic changes in AMs were evaluated after in vitro allergen stimulation. AM-depletion after sensitization significantly increased the number of eosinophils and lymphocytes and the concentrations of IL-4, IL-5 and GM-CSF in BAL fluid. These changes were significantly ameliorated only by adoptive transfer of unsensitized AMs, not by sensitized AMs. In addition, in vitro allergen stimulation of AMs resulted in their gaining the ability to produce inflammatory cytokines, such as IL-1beta, IL-6 and TNF-alpha, and losing the ability to suppress GM-CSF concentrations in BAL fluid. These findings suggested that AMs worked probably through GM-CSF-dependent mechanisms, although further confirmatory experiments are needed. Our results indicate that the role of AMs in the context of airway inflammation should be re-examined.
Animals ; Asthma/*immunology ; Bronchoalveolar Lavage Fluid/chemistry/cytology/immunology ; Cytokines/biosynthesis/immunology ; Disease Models, Animal ; Female ; Immunization ; Immunomodulation/*immunology ; Inflammation/*immunology ; Leukocytes/immunology ; Macrophages, Alveolar/*immunology ; Mice ; Mice, Inbred C57BL ; Ovalbumin/immunology

EPSAH is an exopolysaccharide from Aphanothece halophytica GR02. The present study was designed to evaluate its toxicity and adjuvant potential in the specific cellular and humoral immune responses in ovalbumin (OVA) in mice. EPSAH did not cause any mortality and side effects when the mice were administered subcutaneously twice at the dose of 50 mg·kg(-1). Hemolytic activity in vitro indicated that EPSAH was non-hemolytic. Splenocyte proliferation in vitro was assayed with different concentrations of EPSAH. The mice were immunized subcutaneously with OVA 0.1 mg alone or with OVA 0.1 mg dissolved in saline containing Alum (0.2 mg) or EPSAH (0.2, 0.4, or 0.8 mg) on Day 1 and 15. Two weeks later, splenocyte proliferation, natural killer (NK) cell activity, production of cytokines IL-2 from splenocytes, and serum OVA-specific antibody titers were measured. Phagocytic activity, production of pro-inflammatory cytokines IL-1 and IL-12 in mice peritoneal macrophages were also determined. EPSAH showed a dose-dependent stimulating effect on mitogen-induced proliferation. The Con A-, LPS-, and OVA-induced splenocyte proliferation and the serum OVA-specific IgG, IgG1, and IgG2a antibody titers in the immunized mice were significantly enhanced. EPSAH also significantly promoted the production of Th1 cytokine IL-2. Besides, EPSAH remarkably increased the killing activities of NK cells from splenocytes in the immunized mice. In addition, EPSAH enhanced phagocytic activity and the generation of pro-inflammatory cytokines IL-1 and IL-12 in macrophages. These results indicated that EPSAH had a strong potential to increase both cellular and humoral immune responses, particularly promoting the development of Th1 polarization.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Cyanobacteria ; chemistry ; Female ; Immunity, Cellular ; Immunity, Humoral ; Immunization ; Interleukin-12 ; immunology ; Interleukin-2 ; immunology ; Killer Cells, Natural ; immunology ; Mice ; Mice, Inbred ICR ; Ovalbumin ; immunology ; Polysaccharides ; administration & dosage ; immunology ; Rabbits ; Th1 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVE: To evaluate the function of Th17 cells in allergic rhinitis,through comparing the symptoms, pathology and and the quantity of Th1, Th2 and Th17 cytokine in normal mice, allergic rhinitis mice and allergic rhinitis mice with IL-17 antibody application. METHOD: Thirty BALB/c mice were randomly divided into three groups, control group, allergic rhinitis group, and therapy group. The allergic rhinitis model was induced by classical method with ovalbumin. The therapy group was treated with IL-17 antibody. The concentration of IL-17, IL-4 and IFN-gamma in serum was measured by enzyme-linked immunosorbent assay (ELISA). Nasal mucosal inflammation was evaluated by HE staining. The expression of RORgammat mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression level of IL-17, IL-4 and RORgammat mRNA in allergic rhinitis group were significantly higher than those of control group and IL-17 antibody treated group (P < 0.05). While the expression level of IFN-gamma in allergic rhinitis group were significantly was lower than those of control group and IL-17 antibody treated group (P < 0.05). The inflammation reaction in therapy group abated with nasal mucosal HE staining. CONCLUSION The large quantity of Th2, Th17 cells were found in allergic rhinitis. It might be associated with the pathogenesis of allergic rhinitis. The control of Th17 cells expression may be an effective way to treat allergic rhinitis.
Animals ; Disease Models, Animal ; Female ; Interferon-gamma ; immunology ; Interleukin-17 ; immunology ; Interleukin-4 ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; immunology ; Rhinitis ; immunology ; metabolism ; Th17 Cells ; immunology ; Th2 Cells ; immunology

OBJECTIVETo investigate the preventive effect of interleukin-18 (IL-18) gene modified mature dendritic cells (mDC) vaccine on airway inflammation in mouse asthma model.

METHODSThe asthma model was induced by injection of ovalbumin (OVA) in BALB/c mice. IL-18 gene modified mouse mature dendritic cells (mDC) were detected by flow cytometry and its capacity of inducing allogeneic T cell responses was examined by mixed lymphocyte reaction (MLR). The OVA-induced asthmatic mice were randomly divided into 6 groups: PBS group, DXM group, mDC group, Ad-LacZ-mDC group, Ad-IL-18-mDC group and control group. The pathological changes in lung tissues were assayed by HE and AB-PAS staining. The numbers of inflammatory cells and percentage of eosinophils (EOS) in bronchoalveolar lavage fluid (BALF) were counted. The levels of IFN-γ IL-4 and IL-13 in culture supernatant of splenocytes were measured by ELISA method. The percentage of CD4(+)CD25(+)Foxp3(+) Treg was assessed by flow cytometry analysis.

RESULTThe vaccine was effective in decreasing the infiltration of EOS and accumulation of airway goblet cells in lung tissues, the numbers of inflammatory cells and percentage of EOS in BALF, and the levels of IL-4 and IL-13 in culture supernatant of splenocytes, and in increasing the levels of IFN-γ in culture supernatant of splenocytes and the percentage of CD4(+)CD25(+)foxP3(+) reg.

CONCLUSIONIL-18 gene modified mDC vaccine has a preventive effect on airway inflammation in OVA-induced asthmatic mice.

Animals ; Asthma ; immunology ; pathology ; prevention & control ; Dendritic Cells ; immunology ; Disease Models, Animal ; Genetic Therapy ; Interleukin-18 ; genetics ; Lung ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; immunology

AIMTo synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody.

METHODSThe hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits.

RESULTSThe results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA1, 40.3 for Hapten-BSA2, 17.8 for Hapten-OVA1, and 54.2 for Hapten-OVA2. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC50 value of 2.21 microg.mL(-1) with a detection limit of 0.12 microg.mL(-1).

CONCLUSIONAntigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.

Animals ; Antibodies ; analysis ; Antibody Affinity ; Antibody Formation ; Antineoplastic Agents, Phytogenic ; immunology ; Enzyme-Linked Immunosorbent Assay ; Haptens ; chemistry ; immunology ; Immune Sera ; chemistry ; Male ; Ovalbumin ; immunology ; Podophyllotoxin ; immunology ; Proteins ; immunology ; Rabbits ; Serum Albumin, Bovine ; immunology

AIM: To evaluate the effect of Qi'ao Deocoction (QAD) on the inflammation and hyperresponsiveness of asthma mice. METHODS: 120 Balb/C mice were randomly divided into six groups: normal group, model group, dexamethasone group, high dose QAD group, medium dose QAD group and low dose QAD group. The asthma model was reproduced in Balb/C mice sensitized by ovalbumin, challenged by OVA and LPS. The mice of the normal group were sensitized, challenged and intranasally instilled by PBS. On day 28-34, 6.7, 13.4 and 26.8 g · kg(-1) Qi'ao Decoction were administrated; 0.002 4 g · kg(-1) dexamethasone solution was given to the dexamethasone group; normal and model groups were given the same amount of normal saline. Bronchoalveolar lavage fluid, airway hyperresponsiveness, lung histopathology and cytokines were then collected and analyzed. RESULTS: Compared with normal group, total cellular score, the number of macrophages, lymphocytes, eosinophils and neutrophils of model group significantly increased (P < 0.01). Compared with model group, the administration of dexamethasone induced a significant decrease in eosinophils and neutrophils (P < 0.05, P < 0.01). The number of eosinophils, which plays an important role in airway inflammatory reaction of asthma, of the three QAD groups all decreased (P < 0.01). RL before and after Ach (5 mg · mL(-1)) stimulation in the model group both overtook that in the normal group (P < 0.01). Compared with model group, dexamethasone group, high dose QAD group, medium dose QAD group and low dose QAD group groups all had significantly lower RL before and after Ach stimulation (P < 0.01). Normal pulmonary histopathology was found in the normal group. In the model group, mice exhibited marked increases in inflammatory cell infiltration, mostly including neutrophils and macrophages, perivascular inflammation and thickened alveolus wall (P < 0.01). Dexamethasone application mitigated inflammation around the bronchi (P < 0.05). These histopathological changes were ameliorated in the three decoction groups (P < 0.01, P < 0.05). In addition, alveolus and airway wall lesions of medium dose QAD group and high dose QAD group were reduced, the number of inflammatory cells infiltrated around the walls decreased, no clear degeneration of bronchial epithelial cells was found, and exudates in bronchi declined in different degrees. Compared with normal group, IFN-γ and IL-12 of model group significantly decreased, while IL-4 increased, showing statistic difference (P < 0.05). Compared with model group, IFN-γ and IL-12 level of dexamethasone group went up too, but IL-4 declined (P < 0.05). The level of IFN-γ of medium dose QAD group and high dose QAD group both increased; IL-4 and IL-12 of medium dose group were found significant differences (P < 0.05); but none of the cytokines of low dose QAD group showed statistical significance (P > 0.05). CONCLUSION QAD can significantly inhibit airway inflammation and airway hyperresponsiveness of mice with severe asthma induced by ovalumin and lipopolysaccharide, adjust the balance of cytokines, and improve lung histopathological condition. So, it exhibits great effect on severe asthma.
Animals ; Asthma ; chemically induced ; drug therapy ; immunology ; pathology ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Interleukin-12 ; immunology ; Interleukin-4 ; immunology ; Lipopolysaccharides ; adverse effects ; immunology ; Lung ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; adverse effects ; immunology

To establish a new immune assay for Penicillic Acid (PA) from Penicillium cyclopium, we studied the synthesis of conjugated complete antigens for penicillic acid. PA was conjugated to bovine serum album (BSA) and ovalbumin (OVA) by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC). The artificial antigens PA-BSA and PA-OVA were identified by ultraviolet spectrometric scanning, SDS-PAGE and immunization. Results showed that the absorption peak of conjugation were different from that of the carrier protein alone and of the PA. The conjugated ratio of PA and BSA was 23.2:1 and that of PA and OVA was 10.4:1. Balb/c mice were immunized by the artificial antigen of PA-BSA, with PA-OVA as coating antigen. The average titer of antiserums was more than 12 800 by indirect ELISA. The obtained antigens offered a basis for developing immunoassay method.
Animals ; Antibodies ; blood ; Antigens ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Ethyldimethylaminopropyl Carbodiimide ; analogs & derivatives ; chemistry ; Immunization ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; immunology ; Penicillic Acid ; immunology ; metabolism ; Penicillium ; immunology ; metabolism ; Serum Albumin, Bovine ; immunology