Development and verification of double antibody sandwich ELISA for determination of ovalbumin / 中国生物制品学杂志

@#Objective To develop and verify a double antibody sandwich ELISA detection method for the determination of ovalbumin(OVA),in order to determine the OVA content in influenza vaccines.Methods The rabbit anti-OVA polyclonal antibody was used as coating antibody and HRP labeled rabbit anti-OVA polyclonal antibody as detection antibody to develop a double antibody sandwich ELISA for OVA.The antibody concentration(2,1,0.5 and 0.25 pg/mL) for coating,enzyme-labeled antibody concentration(0.5,0.25 and 0.125 μg/mL),and kinds of blocking reagent(blocking with1% BSA,blocking with 2% BSA,blocking with 1% BSA and 1% sucrose,and blocking with 2% BSA and 2% sucrose,using nonblocking as control) were optimized,and the Cut-off value was determined as the judgment standard.The developed method was verified for the linear range and detection limit,specificity,repeatability and accuracy.Results The optimum detection conditions were as follows:the concentration of coating antibody was 1 μg/mL,the concentration of enzyme-labeled antibody was 0.25 μg/mL;The blocking reagent was 2% BSA and 2% sucrose;The Cut-off value was 0.051 66.The linear range of the method was 5～0.313 ng/mL,and the detection limit was 0.078 ng/mL;It did not react with influenza virus,bovine serum albumin(BSA) and Vero cell supernatant;The intraplate and interplate coefficient of variation(CV) were between 2.562%～13.887% and 4.000%～16.497% respectively;The coincidence rate between the results of this method and the Germany SERAMUN OVA quantitative test kit ranged from 93.79% to 107.05%.Conclusion The developed OVA double antibody sandwich ELISA has good specificity,repeatability,linear range and accuracy with convenience and lower cost,which might be used for the quantitative detection of OVA.

Comparative study on establishing an immune tolerance model for allergic conjunctivitis in mice with different methods / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To establish an immune tolerance model for allergic conjunctivitis in newborn mice with different methods and observe the impact of environmental factors on allergic conjunctivitis in early life.METHOD: A total of 50 Balb/c newborn mice were randomly divided into blank control group, ovalbumin(OVA)+subcutaneous injection group, OVA+nebulized inhalation group, OVA+gastric group, ragweed pollen(RW)+subcutaneous injection group, RW+nebulized inhalation group, RW+gastric group, house dust mite(HDM)+subcutaneous injection group, HDM+nebulized inhalation group, HDM+intragastric group(n=5 animals/group). Except for the blank control group, mice in each group were individually exposed to the corresponding antigens to induce immune tolerance early in life and stimulated with the corresponding antigens in adulthood. The ocular surface was visualized by anterior segment photography. The relative expression level of conjunctival RANTES and IL-17 mRNA was measured by RT-qPCR and serum IL-17 concentration was measured by ELISA.RESULTS: Compared with the blank control group, the relative expression level of conjunctiva IL-17 mRNA in RW+gastric group was the highest, and it was the lowest in RW+subcutaneous group(all P<0.05). The relative expression level of conjunctiva RANTES mRNA was the highest in RW+gastric group(P<0.001). Compared with the blank control group, the serum concentration of IL-17 was increased in all treatment groups except OVA+nebulizer group and RW+subcutaneous group(P<0.05).CONCLUSION: The immune tolerance of allergic conjunctivitis induced by subcutaneous injection of antigen was the most suitable method in the early life of mice.

Mechanism of mixed probiotics relieves food allergy in infant mice through the programmed cell death 1/programmed cell death ligand 1 pathway / 中华实用儿科临床杂志

Objective:To investigate the effects of mixed probiotics on food allergy and the underlying mechanism.Methods:BALB/c mice on the 15 th day of pregnancy were randomly (random number table method) classified into the control group, food allergy model group and mixed probiotics group.Mice in the food allergy model and mixed pro-biotics group were subjected to ovalbumin (OVA) sensitization after birth, and those in the mixed probiotics group were then given probiotic solution by gavage from day 21 to day 35.Mice in control group were similarly given 9 g/L saline.Twenty-four hours after the last OVA sensitization, intestinal histopathological sections were prepared to observe intestinal pathological changes.Blood smears were prepared to detect eosinophil count.In addition, serum samples were collected to measure cytokine levels and OVA specific antibodies.The number of dendritic cells (DCs) and regulatory T cells (Tregs) in mouse mesenteric lymph nodes was calculated.Differences among 3 groups were compared by the One- Way ANOVA or Kruskal- Wallis H test. Results:Compared with those of food allergy model group, diarrhea score, the ratio of eosinophils and serum levels of interleukin(IL)-4, IL-5, IL-13, mast cell protease 1 (MCPT-1), and OVA specific antibodies IgE and IgG were significantly lower in mixed probiotics group[(2.00±0.71) points vs.(3.22±0.97) points, (2.28±1.61)% vs.(10.99±2.26)%, (413.68±22.81) ng/L vs.(708.78±27.66) ng/L, (36.64±3.74) ng/L vs.(46.05±4.95) ng/L, (201.37±65.61) ng/L vs.(495.22±96.66) ng/L, (31 924.15±1 177.77) ng/L vs.(36 175.77±618.29) ng/L, (9.10±8.08) ng/L vs.(19.69±0.84) ng/L, (30.50±8.81) ng/L vs.(190.32±6.40) ng/L], while IL-10 level was significantly higher[(164.12±3.88) ng/L vs.(123.90±7.31) ng/L] ( t=3.37, 8.72, 16.07, 3.90, 7.40, 7.95, 3.91, 44.00 and 7.76, respectively, all P<0.01). Compared with those of food allergy model group, programmed cell death ligand 1 (PD-L1) level on the surface of CD 103+ DCs and CD 103+ CD 80-CD 40-DCs, the proportion of Tregs in CD4 + T cells, and the level of programmed cell death 1 (PD-1) on the surface of Tregs were significantly higher in mixed probiotics group[(75.59±0.45)% vs.(45.60±4.73)%, (67.56±1.87)% vs.(37.12±6.07)%, (8.24±0.69)% vs.(6.20±0.66)%, (11.25±3.12)% vs.(4.08±2.33)%]( t=7.88, 4.48, 3.63 and 3.71, all P<0.01). Conclusions:Mixed probiotics can alleviate the symptoms of food allergy and inflammatory response of young rats through mediating Tregs via the PD-1/PD-L1 pathway.

Effect of Echinococcus granulosus hydatid cyst fluid protein on allergic rhinitis induced by ovalbumin in mice / 中国血吸虫病防治杂志

Objective To investigate the protective effect of Echinococcus granulosus hydatid cyst fluid protein (HCFP) on ovalbumin (OVA)-induced allergic rhinitis (AR) in mice. Methods Twenty-four BALB/c mice at ages of 8 to 10 weeks, each weighing approximately 20 g, were randomly divided into four groups, including groups A (blank control group), B (blank intervention group), C (AR model group) and D (AR+HCFP intervention group), with 6 mice in each group. On days 0, 2, 4, 6, 8, 10 and 12, mice in groups A, B, C and D were injected with 200 μL sterile phosphate buffered saline (PBS), 200 μL sterile PBS containing 20 μg HCFP, 200 μL sterile PBS containing 50 μg OVA and 5 mg Al(OH)3 gel, and 200 μL sterile PBS containing 50 μg OVA, 5 mg Al(OH)3 gel and 20 μg HCFP, respectively. On days 14 to 20, mice in groups A, B, C and D were administered with 40 μL sterile PBS, 40 μL sterile PBS containing 20 μg HCFP, 40 μL sterile PBS containing 2 mg OVA and 40 μL sterile PBS containing 2 mg OVA and 20 μL HCFP by nasal drop, respectively. Mouse behavioral changes were observed and behavioral scores were estimated. The serum levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-5, IL-10, transforming growth factor-β (TGF-β) and OVA-specific IgE antibody (OVA-sIgE) were measured using enzyme-linked immunosorbent assay (ELISA), and the pathological changes of mouse nasal mucosa were observed by hematoxylin and eosin (HE) staining. Results The mean behavioral score was significantly greater in Group C (6.83 ± 0.50) than in groups A (1.17 ± 0.52) and B (1.33 ± 0.52) (P < 0.05), while a lower mean behavioral score was estimated in Group D (3.50 ± 0.50) than in Group C (P < 0.05). There were significant differences among the groups in terms of serum IFN-γ (F = 4.08, P < 0.05), IL-4 (F = 275.90, P < 0.05), IL-5 (F = 96.82, P < 0.05), IL-10 (F = 77.67, P < 0.05), TGF-β (F = 9.98, P < 0.05) and OVA-sIgE levels (F = 44.69, P < 0.05). The serum IFN-γ level was significantly lower in Group C than in groups A, B and C (P < 0.05), and the serum levels of IL-4, IL-5 and OVA-sIgE were significantly higher in Group C than in groups A, B and C (P < 0.05), while the serum IL-10 and TGF-β levels were significantly greater in Group D than in Group C (P < 0.05). Microscopy showed apparent loss of nasal mucosa cilia, increased number and enlargement of goblet cells, interstitial edema and submucous vascular dilation in Group C, while the pathological changes of nasal mucosa were alleviated in Group D relative to Group C. Conclusions E. granulosus HCFP has a protective activity against OVA-induced allergic rhinitis in mice.

Effects of early allergen exposure on airway inflammation responses and airway hyper-reactivity in an asthma mouse model / 中华微生物学和免疫学杂志

Objective:To investigate the effects of early allergen exposure on airway inflammation responses and airway hyper-reactivity in a mouse model of asthma and the possible mechanism.Methods:Neonate BALB/c mice were randomly divided into three groups of normal control group, asthma group and early exposure group with eight in each group. The mice in the early exposure group were subcutaneously injected with 0.1 ml of ovalbumin (OVA) once a day for 10 consecutive days on the third day after birth and the other two groups were treated with 0.1 ml of PBS. Six weeks after birth, the mice in the asthma group and the early exposure group were sensitized with 0.2 ml of OVA allergen once a day for five consecutive days and then challenged with OVA 7 d after sensitization. The normal group was sensitized and challenged with PBS. Pathological changes in 1ung tissues were detected by HE or PAS staining. Flow cytometry was performed to quantify T cell subsets in bronchoalveolar lavage fluid (BALF). Th2-related cytokines (IL-4, IL-5 and IL-13) in BALF and the levels of OVA-specific IgE and IgG in serum were measured by ELISA. The percentages of CD4 + IFN-γ + T(Th1), CD4 + IL-4 + T(Th2) and CD4 + CD25 + Foxp3 + Treg cells in lung tissues were detected by flow cytometry. The airway resistance was detected using a small animal lung function testing system. Results:The pathological changes in lung tissues were less severe in the early exposure group than in the asthma group. However, PAS staining results showed no significant difference in cell metaplasia was found between the two groups. Compared with the asthma group, early exposure to OVA allergen significantly reduced the percentage of Th2 cells [(16.2±2.4)% vs (19.4±3.4)%, P<0.05] and increased the proportion of Treg cells [(3.7±0.4)% vs (2.1±1.4)%, P<0.05]. Moreover, the levels of IL-4 [(65.3±17.1) pg/ml vs (144.4±23.1) pg/ml] and IL-5 [(103.3±21.1) pg/ml vs (198.3±23.1) pg/ml] in BALF were significantly down-regulated in the early exposure group. A reduced production of OVA-specific IgE but not IgG in serum and an inhibited airway hyper-reactivity following OVA challenge were observed in the early exposure group. Conclusions:Early exposure to OVA allergen could offer a protective effect against OVA re-exposure-induced pathological changes, inflammatory responses and airway hyper-reactivity in grown-up mice.

Immunomodulatory effect of Quercetin on dysregulated Th1/Th2 cytokine balance in mice with both type 1 diabetes and allergic asthma

T-helper subtype imbalance is intricate in type 1 diabetes (T1D) and asthma initiation. The role of quercetin in immunedysregulation in comorbid conditions of T1D and asthma is not available. In this study, it was thought worthy toevaluate the role of quercetin on modulating Th1/Th2 cytokine dysregulation in comorbid diabetic asthma. Male Balb/cmice were injected intravenously with alloxan (80 mg/kg) to persuade T1D. Succeeding diabetes introduction, twointraperitoneal sensitizing doses of ovalbumin emulsion (50 µg ovalbumin blended with 2.5 mg alum/sensitization) weregiven on days 3 and 8. Mice were given intranasal challenges of ovalbumin (100 µg ovalbumin/25 µl of sterile saline) ondays 13–15. Oral quercetin treatment (10–30 mg/kg) was given daily on days 3–15. Nasal hyperresponsiveness (NHR)was recorded immediately after Ova challenge on day 16. Bronchoalveolar lavage fluid (BALF), blood, and lungs werecollected 1-hour post NHR for further analysis. Quercetin treatment significantly decreased eosinophils, interleukin-4while increasing interferon-gamma in blood, and BALF and reduced the allergic airway inflammation by inhibitinginflammatory cell infiltration and mucous cell metaplasia. Furthermore, quercetin with a dose of 30 mg/kg demonstrateda significant glucose reduction. Thus, quercetin exerted dose-dependent anti-asthma activity by modulating Th1/Th2balance with glucose-lowering potential in comorbid mice.

Effect of Lavandula dentata extract on Ovalbumin-induced Asthma in Male Guinea Pigs / Efeito do extrato de Lavandula dentata na asma induzida por ovalbumina em cobaias machos

Abstract Asthma is an inflammatory disease of the lungs, and it causes oxidative stress. Lavandula dentata is an aromatic herb with anti-oxidative and anti-inflammatory activities. This study examined the activity of L. dentata extract on a guinea pig model of asthma. Adult males were divided into five groups: First group was control, second was asthma model induced by OVA, third was treated with L. dentata extract orally (300 mg/kg) for 21 days; the fourth was an asthma model with L. dentata extract (300 mg/kg) and fifth was treated with Tween 80 for 21 days. OVA treatment increased IgE, triglycerides, total cholesterol, glucose levels in serum, WBC count in blood and MDA in lungs. Also, OVA reduced SOD activity, GSH content in lungs, and GGT activity in serum (p<0.05). L. dentata extract treatment in asthma model reduced elevated IgE, triglycerides, total cholesterol, glucose levels in serum, and MDA in lungs (p<0.05), while it increased GSH content in lungs (p<0.05). These results suggest the possibility that L . dentata extract can exert suppressive effects on asthma, and may provide evidence that it is a useful agent for the treatment of allergic airway disease, it also limits oxidative stress induced by OVA. L. dentata extract appears to have hypolipidemic and hypoglycemic activities.

Resumo A asma é uma doença inflamatória dos pulmões e causa estresse oxidativo. Lavandula dentata é uma erva aromática com atividades anti-oxidantes e antiinflamatórias. Este estudo examinou a atividade do extrato de L. dentata em um modelo de asma de cobaia. Os machos adultos foram divididos em cinco grupos: o primeiro grupo foi controle, o segundo modelo foi o da asma induzido pela OVA, o terceiro foi tratado com extrato de L. dentata por via oral (300 mg / kg) por 21 dias; o quarto foi um modelo de asma com extrato de L. dentata (300 mg / kg) e o quinto foi tratado com Tween 80 por 21 dias. O tratamento com OVA aumentou a IgE, os triglicerídeos, o colesterol total, os níveis de glicose no soro, a contagem de leucócitos no sangue e o MDA nos pulmões. Além disso, o OVA reduziu a atividade da SOD, o conteúdo de GSH nos pulmões e a atividade da GGT no soro (p <0,05). O tratamento com extrato de L. dentata no modelo de asma reduziu a IgE elevada, triglicérides, colesterol total, níveis séricos de glicose e MDA nos pulmões (p <0,05), enquanto aumentou o conteúdo de GSH nos pulmões (p <0,05). Estes resultados sugerem a possibilidade do extrato de L. dentata poder exercer efeitos supressores sobre a asma, e pode fornecer evidências de que é um agente útil para o tratamento de doenças alérgicas das vias aéreas, além de limitar o estresse oxidativo induzido pela OVA. O extrato de L. dentata parece ter atividades hipolipemiantes e hipoglicêmicas.

Reversal of Olfactory Disturbance in Allergic Rhinitis Related to OMP Suppression by Intranasal Budesonide Treatment

PURPOSE: We evaluated the severity of olfactory disturbance (OD) in the murine model of allergic rhinitis (AR) and local allergic rhinitis (LAR) in mice. We also investigated the therapeutic effect of an intranasal steroid on OD. METHODS: Forty BALB/c mice were divided into 5 groups (n = 8 for each). The control group was sensitized intraperitoneally (i.p.) and challenged intranasally (i.n.) with saline. Mice in the AR group got i.p. and i.n. ovalbumin (OVA) administration for AR induction. The LAR group was challenged i.n. with 1% OVA for inducing local nasal allergic inflammation, without inducing the systemic allergy. The OD group got an i.p. methimazole administration (75 mg/kg) to induce total destruction of olfactory mucosa. Mice in the intranasal budesonide group received i.n. budesonide (12.8 μg per time, 30 minutes after the i.n. OVA challenge) while using OVA to cause systemic allergies. We conducted a buried-food pellet test to functionally assess the degree of OD in each group by measuring the time taken until finding hidden food. We evaluated the damage to olfactory epithelium using histopathologic evaluation and compared the degree of olfactory marker protein (OMP) expression in olfactory epithelium using immunofluorescent staining. RESULTS: Mice of the AR (81.3 ± 19.8 seconds) and LAR groups (66.2 ± 12.7 seconds) spent significantly more time to detect the pellets than the control group (35.6 ± 12.2 seconds, P < 0.01). After treatment, the intranasal budesonide group exhibited significantly better results (35.8 ± 11.9 seconds) compared with the AR and LAR groups (P < 0.01). The AR and LAR groups showed considerable olfactory epithelial damage and suppression of OMP expression compared with the control group. In the intranasal budesonide group, the olfactory lesions and OMP expression had improved substantially. CONCLUSIONS: OD may be caused by olfactory epithelial damage and suppression of OMP expression in nasal allergic inflammation and could be reversed using an intranasal steroid.

Development of Protocol for Production of Primary Antibody against Ovalbumin Protein in Chicken for Detection of the Protein through Western Blotting

Ovalbumin, a major protein of egg white plays many roles including providing nutrition to the developing embryo, acting as coagulating agent, folliculogenesis and angiogenesis in chicken and other animals. This protein is expressed mainly in magnum and then deposited over the yolk of the oocyte/zygote. Hence, it is important in formation of egg and is an essential target to measure. We cloned chicken ovalbumin CDS in pAcGFP-C1 vector and has been initially expressed in chicken primary magnum cell culture. The ovalbumin protein tagged with 6x Histidine was purified from cell culture and used for production of primary antibody in rat. The ovalbumin protein along with freund’s adjuvant was injected to the rat, booster was given, and finally, hyper-immune sera was collected from rat. The antisera was purified for isolation of IgG. The IgG was used as primary antibody for Western blotting. Through Western blotting, ovalbumin protein isolated from chicken magnum was detected and the protocol was established to detect chicken ovalbumin protein.

Ocular Tolerability And Efficacy Of Ebastine Colloidal Formulation In Allergic Conjunctivitis

Allergic conjunctivitis (AC), defined by ocular itching, hyperemia, lacrimation and edema, impairs the quality of life across the globe. Ebastine is available as an oral antihistamine formula, such as tablets and syrup, for allergic disorders. Topical antihistamines are preferred over oral agents since their direct application at the site of action results in rapid onset and superior efficacy with less systemic side effects. The objective of the present work was to evaluate the antiallergic potential of optimized ebastine (1% w/v) colloidal ocular formulation by performing in vitro study like hen's egg chorioallantoic membrane test (HET-CAM) for tolerability and in vivo efficacy study in ovalbumin (OA)-induced allergic conjunctivitis (AC) with acute ocular irritation study. Eye scratching behavior and edema were evaluated after topical antigen challenge. Edema was scored at periodic interval after the instillation of ovalbumin followed by histopathology. The results showed that ebastine (1% w/v) colloidal ocular formulation was effective in inhibiting symptoms of eye inflammation induced by ovalbumin. Further, the study indicated that said formulation has a quick onset and the duration of effect sufficient to provide relief from symptoms for 24 hr. Ocular irritation by HET-CAM assay showed that the developed formulation does not cause any irritation to the blood vessels. Acute ocular irritation test was performed using rabbits and results showed that developed formulation was non-irritant to the eye. The present study revealed that the ocular ebastine formulation could offer a novel therapeutic opportunity against IgE-mediated allergic conjunctivitis.

Protective effect of excretory-secretory protein from adult Trichinella spiralis on ovalbumin-induced allergic rhinitis in mice / 中国血吸虫病防治杂志

Objective To investigate the protective effect of excretory-secretory protein (AES) from adult Trichinella spiralis on ovalbumin (OVA)-induced allergic rhinitis in mice. Methods Eighteen female BALB/c mice were randomly divided into three groups, including the blank control group (Group A), OVA-induced rhinitis group (Group B) and AES treatment group (Group C). Mice in Group A were given PBS. Mice in Group B were intraperitoneally injected with antigen adjuvant suspension for systemic sensitization, once every other day for seven times; then, local excitation was intranasally induced with 5% OVA solution once a day for seven times to establish a mouse model of allergic rhinitis. In addition to induction of allergic rhinitis, mice in Group C were given 25 μg AES at baseline sensitization and local excitation. Following the final challenge, mice were observed for 30 min in each group, and the behavioral score was evaluated. The serum levels of IFN-γ, IL-4, IL-5, IL-10 and TGF-β were determined using an enzyme-linked immunosorbent assay in mice, and the pathological changes of mouse nasal mucosa were observed under a microscope. Results There was a significant difference in the mouse behavioral scores among the three groups (F = 110.12, P < 0.01). The mouse behavioral score was significantly higher in Group B than in Group A (7.17 ± 0.75 vs. 1.33 ± 0.52, P < 0.01), and more remarkable pathological damages of mouse nasal mucosa were seen in Group B than in Group A, while the mouse behavioral score was significantly decreased in Group C than in Group B (P < 0.01), and the pathological damages of mouse nasal mucosa remarkably alleviated in Group C relative to Group B. There was a significant difference in serum IFN-γ level among the three groups (F = 7.50, P < 0.01) and the serum IFN-γ level in Group B was significantly lower than in group A and C (both P < 0.05). There were significant differences in serum IL-4 (F = 470.81, P < 0.01) and IL-5 levels (F =68.20, P < 0.01) among the three groups, and significantly greater serum IL-4 and IL-5 levels were detected in Group B than in Group A (P < 0.01), while significantly lower serum IL-4 and IL-5 levels were detected in Group C than in Group B (P < 0.01). There were significant differences in serum IL-10 (F = 174.91, P < 0.01) and TGF-β levels (F = 9.39, P < 0.01) among the three groups, and significantly greater serum IL-10 and TGF-β levels were seen in Group C than in Group B (both P < 0.05). Conclusion T. spiralis AES has a remarkable protective activity against OVA-induced allergic rhinitis in mice.

Protective effect of excretory-secretory protein from adult Trichinella spiralis on ovalbumin-induced allergic rhinitis in mice / 中国血吸虫病防治杂志

Objective To investigate the protective effect of excretory-secretory protein (AES) from adult Trichinella spiralis on ovalbumin (OVA)-induced allergic rhinitis in mice. Methods Eighteen female BALB/c mice were randomly divided into three groups, including the blank control group (Group A), OVA-induced rhinitis group (Group B) and AES treatment group (Group C). Mice in Group A were given PBS. Mice in Group B were intraperitoneally injected with antigen adjuvant suspension for systemic sensitization, once every other day for seven times; then, local excitation was intranasally induced with 5% OVA solution once a day for seven times to establish a mouse model of allergic rhinitis. In addition to induction of allergic rhinitis, mice in Group C were given 25 μg AES at baseline sensitization and local excitation. Following the final challenge, mice were observed for 30 min in each group, and the behavioral score was evaluated. The serum levels of IFN-γ, IL-4, IL-5, IL-10 and TGF-β were determined using an enzyme-linked immunosorbent assay in mice, and the pathological changes of mouse nasal mucosa were observed under a microscope. Results There was a significant difference in the mouse behavioral scores among the three groups (F = 110.12, P < 0.01). The mouse behavioral score was significantly higher in Group B than in Group A (7.17 ± 0.75 vs. 1.33 ± 0.52, P < 0.01), and more remarkable pathological damages of mouse nasal mucosa were seen in Group B than in Group A, while the mouse behavioral score was significantly decreased in Group C than in Group B (P < 0.01), and the pathological damages of mouse nasal mucosa remarkably alleviated in Group C relative to Group B. There was a significant difference in serum IFN-γ level among the three groups (F = 7.50, P < 0.01) and the serum IFN-γ level in Group B was significantly lower than in group A and C (both P < 0.05). There were significant differences in serum IL-4 (F = 470.81, P < 0.01) and IL-5 levels (F =68.20, P < 0.01) among the three groups, and significantly greater serum IL-4 and IL-5 levels were detected in Group B than in Group A (P < 0.01), while significantly lower serum IL-4 and IL-5 levels were detected in Group C than in Group B (P < 0.01). There were significant differences in serum IL-10 (F = 174.91, P < 0.01) and TGF-β levels (F = 9.39, P < 0.01) among the three groups, and significantly greater serum IL-10 and TGF-β levels were seen in Group C than in Group B (both P < 0.05). Conclusion T. spiralis AES has a remarkable protective activity against OVA-induced allergic rhinitis in mice.

Establishment of a mouse model of atopic dermatitis induced by topical application of ovalbumin and calcipotriol / 中华皮肤科杂志

Objective To explore a method for rapidly establishing a mouse model of atopic dermatitis (AD).Methods C57BL/6 mice served as model animals,and were randomly divided into 3 groups:calcipotriol + ovalbumin (OVA) group (n =6) topically treated with calcipotriol and OVA on the mouse ears,calcipotriol group (n =6) topically treated with calcipotriol on the ears,and control group (n =3) topically treated with 75％ alcohol on the ears.The treatment lasted 12 days.Before the model establishment and on day 14,the photos of the mouse ears were taken,and ear thickness was measured;moreover,blood samples were obtained from the mouse caudal vein,and serum levels of total IgE and OVAspecific IgE were detected.On day 14,the skin tissues of mouse auricles were resected and subjected to histopathological examination.Results On day 14,erythematous swelling,dryness and desquamation occurred on the mouse ear skin in the calcipotriol + OVA group and calcipotriol group,and both the two groups showed significantly increased ear thickness compared with those before the model establishment (both P ＜ 0.001).However,there was no significant difference in the ear thickness between the calcipotriol + OVA group (0.355 ± 0.03 mm) and calcipotriol group (0.370 ± 0.05 mm,q =0.674,P =0.231).Histopathological examination of the ear skin showed more obvious epidermal hyperplasia and infiltration of dermal inflammatory cells including eosinophils and mastocytes in the calcipotriol + OVA group compared with the calcipotriol group and control group.Immunohistochemical study revealed that there was no significant difference in the expression of thymic stromal lymphopoietin (TSLP) and interferon (IFN)-γ among the 3 groups (both P ＞ 0.05),while the expression of interleukin (IL)-13 significantly differed among the 3 groups (F =5.159,P =0.032),and was significantly higher in the calcipotriol + OVA group (77.12 ± 5.46) than in the control group (55.49 ± 9.92,q =3.170,P =0.021).On day 14,the calcipotriol + OVA group and calcipotriol group both showed markedly increased total serum IgE levels compared with those before the treatment,and the calcipotriol + OVA group showed a more significant increase (8 278.56 ± 3 297.68 vs.892.64 ± 82.83 μ g/L,t =4.132,P =0.026).Meanwhile,the serum level of OVA-specific IgE was significandy higher in the calcipotriol + OVA group (192.846 ± 15.391 μg/L) than in the calcipotriol group (8.492 ±:3.879 μg/L,q =22.476,P ＜ 0.001) on day 14.Conclusion The mouse model of allergeninduced AD can be rapidly established by topical application of calcipotriol and OVA for 12 consecutive days,which lays a foundation for further study on allergen-related pathogenesis of AD.

Suppressive Effect of 4-Hydroxy-2-(4-Hydroxyphenethyl) Isoindoline-1,3-Dione on Ovalbumin-Induced Allergic Asthma

4-Hydroxy-2-(4-hydroxyphenethyl)isoindoline-1,3-dione (PD1) is a synthetic phthalimide derivative of a marine compound. PD1 has peroxisome proliferator-activated receptor (PPAR) γ agonistic and anti-inflammatory effects. This study aimed to investigate the effect of PD1 on allergic asthma using rat basophilic leukemia (RBL)-2H3 mast cells and an ovalbumin (OVA)-induced asthma mouse model. In vitro, PD1 suppressed β-hexosaminidase activity in RBL-2H3 cells. In the OVA-induced allergic asthma mouse model, increased inflammatory cells and elevated Th2 and Th1 cytokine levels were observed in bronchoalveolar lavage fluid (BALF) and lung tissue. PD1 administration decreased the numbers of inflammatory cells, especially eosinophils, and reduced the mRNA and protein levels of the Th2 cytokines including interleukin (IL)-4 and IL-13, in BALF and lung tissue. The severity of inflammation and mucin secretion in the lungs of PD1-treated mice was also less. These findings indicate that PD1 could be a potential compound for anti-allergic therapy.

Preparation of bacterial outer membrane vesicle coated nanoparticle loaded with drug and evaluation of its nasal immune effect in mice / 上海交通大学学报（医学版）

Objective: To prepare a bacterial outer membrane vesicle (OMV) coated poly (lactic-co-glycolic acid) copolymer (PLGA) nanoparticle loaded with ovalbumin (OVA) and evaluate its intranasal immune effect in mice. Methods: OMV was prepared by ultrafiltration concentration method. OVA loaded PLGA nanoparticle (NP) was prepared by emulsion-solvent evaporation method. OMV coated PLGA nanoparticle (OMV-PLGA NP) loaded with OVA was prepared by extrusion method and characterized. BALB/c mice were intranasally immunized and specific sIgA levels in nasal wash, jejunum and fecal pellet were determined by ELISA. Results: Size of OVA loaded OMV-PLGA NP was (234.4±22.9) nm. The shell-core structure of OVA loaded OMV-PLGA NP was proved by transmission electron microscope. After 14 d of administration, sIgA antibody levels in nasal wash, jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group were the highest in all treated groups. Compared with the group treated with OMV and OVA, OVA-specific sIgA antibody level in nasal wash, jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group was increased 1.6, 2.1 and 1.7 times, respectively. Compared with the group treated with OMV and OVA, OMV-specific sIgA antibody level in nasal wash, jejunum and fecal pellet of OVA loaded OMV-PLGA NP treated group was all increased 1.5 times. Conclusion: This novel nanoparticle drug delivery system can simultaneously delivery OVA and OMV to antigen presenting cells, resulting in stronger mucosal immune response in mice.

Inhibitory effects and mechanism of Triticum aestivum on OVA-induced anaphylaxis in mice / 中华微生物学和免疫学杂志

Objective@#To investigate the inhibitory effects and mechanism of Triticum aestivum (TA) on anaphylaxis in ovalbumin (OVA)-sensitized mice.@*Methods@#Twenty-five female BALB/c mice were randomly divided into five groups (n=5), including control group, OVA group, 100 mg/kg TA group, 200 mg/kg TA group and DEX (dexamethasone) group. OVA (20 μg) and aluminum hydroxide (1 mg) were dissolved in 100 μl of PBS solution and injected into the abdominal cavity of each experimental mouse, while the mice in the control group were injected with 100 μl of 0.9% normal saline. All mice were given drinking water. The above processes were repeated two weeks later, and different concentrations of experimental drugs were diluted in 1% CMC (carboxymethyl cellulose) solution to feed mice. Allergic symptoms and behaviors of each mouse were scored in the process of feeding. On the 12th day after first sensitization, mice in the control and experimental groups were subcutaneously injected in the auricle with 20 μl of 0.9% saline solution and OVA, respectively. The thickness of each mouse′s auricle was measured 6 and 24 hours later. Histopathologic changes in auricle tissues were observed by hematoxylin-eosin(HE) staining 24 hours later. The mice were euthanized by cervical dislocation in the 6th week after first sensitization. Abdominal aorta serum samples were collected and tested for specific inflammatory cytokines (IgE, IgG1, TNF-α, IL-4) and anti-OVA antibodies (IgE, IgG1) by ELISA. ELISA and RT-PCR were used to detect the expression of IFN-γ, IL-4, IL-5, IL-12 and IL-13 after spleen lymphocytes were stimulated with different concentrations of drugs.@*Results@#Both the score of allergic symptoms and behavior and the thickness of the auricle of the OVA group were the highest among all groups (P<0.05). A TA dose-dependent decrease was found in both of the two parameters, which was confirmed by histopathological analysis. ELISA results showed that the plasma levels of IgE, IgG1, IL-4 and TNF-α in the experimental groups were significantly higher than those in the control group, but all of them were decreased in a TA dose-dependent manner. TA at the high concentration of 200 mg/kg was similar to DEX in inhibiting the expression of IgE and TNF-α. RT-PCR results showed that Th1 cytokines (IFN-y and IL-12) in the experimental groups increased significantly as compared with those in the control group (P<0.05). Expression of IFN-y and IL-12 was not significantly affected by TA at various concentrations, but markedly inhibited by DEX. The levels of Th2 cytokines (IL-4, IL-5 and IL-13) in the experiment groups were significantly higher than those in the control group (P<0.05). Compared with the OVA group, the levels of IL-4, IL-5 and IL-13 were significantly reduced in TA groups, especially in the TA 100 μg/ml group in which the expression of the three cytokines was decreased by about 60%, 18% and 20%, respectively.@*Conclusion@#TA helps to maintain immune balance by selectively inhibiting the expression of Th2 cytokines (IL-4, IL-5, IL-13), IgE and IgG and not influencing Th1 cytokines (IFN-γ and IL-12). It has an anti-allergic effect as it effectively suppresses allergic responses. This research provides both theoretical and experimental basis for further development of novel anti-anaphylactic treatment with TA.

Immunopotentiating effects of polysaccharides extracted from cultivated and wild Cistanche desertico-la in Xinjiang / 中华微生物学和免疫学杂志

Objective To compare the immunopotentiating effects of polysaccharides extracted from wild/cultivate Cistanehe deserticola (WCDPS/CCDPS) in Xinjiang. Methods ICR mice were subcu-taneously injected twice with different doses(low,medium and high) of WCDPS and CCDPS in combination with ovalbumin (OVA). OVA-specific antibody IgG,as well as IgG1 and IgG2a subtypes, was determined by ELISA. OVA-specific lymphocyte proliferation was measured by MTT. Expression of CD4+T and CD8+T cells was analyzed by flow cytometry. Results Both WCDPS and CCDPS could significantly improve the production of OVA-specific IgG,IgG1 and IgG2a,promote the proliferation of OVA-specific lymphocytes and increase the expression of CD4+T and CD8+T cells(all P<0.05) with no significant difference between them at the same dosages (P>0.05). WCDPS and CCDPS had no influence on the body weight of mice after im-munization. Conclusion WCDSP and CCDPS could significantly enhance the OVA-specific humoral and cellular immune responses with no statistical difference and are characterized by high safety.

Role of mast cells in Staphylococcus aureus enterotoxin B-induced atopic dermatitis-like skin inflammation in mice / 中华皮肤科杂志

Objective To investigate the role of mast cells in Staphylococcus aureus enterotoxin B (SEB)-induced atopic dermatitis (AD)-like skin inflammation in BALB/c mice.Methods A total of 24 BALB/c mice were randomly and equally divided into 4 groups to be topically treated with ovalbumin (OVA group),SEB (SEB group),OVA + SEB (OVA + SEB group) and sodium chloride physiological solution (control group) respectively,so as to establish mouse models of epicutaneously induced AD-like skin inflammation.The AD-like skin lesions were evaluated by clinical observation and eczema area and severity index (EASI).Biopsy specimens were obtained from lesional skin of mice and then subjected to toluidine blue staining and immunohistochemical staining to count the mast cells,observe the morphology and distribution of mast cells,and calculate the percentage of degranulated mast cells.Results After 7-week treatment,the OVA group,SEB group and OVA + SEB group all showed severer local skin inflammation,higher EASI scores and denser infiltration of inflammatory cells compared with the control group.Moreover,the OVA + SEB group showed significantly severer local skin inflammation,skin lesions and degree of infiltration of inflammatory cells compared with the OVA group and SEB group (all P ＜ 0.05).The number of mast cells in the dermis of AD-like skin lesions per high-power field (× 400) was significantly higher in the OVA group (median [quartile range]:10.625 [3.675]),SEB group (11.000 [4.163]) and OVA + SEB group (13.875 [8.813]) than that in the control group (5.925 [2.088],all P ＜ 0.05).The SEB group (71.083％ ± 14.519％) and OVA + SEB group (58.767％ ±.16.978％) both showed significantly higher percentage of degranulated mast cells compared with the OVA group (24.050％ ± 11.161％,both P ＜ 0.05) and control group (23.617％ ± 8.132％,both P ＜ 0.05).Bivariate correlation analysis showed that the number of mast cells in the skin lesions was positively linearly correlated with the EASI scores (P ＜ 0.05).Conclusions Epicutaneous application of SEB can induce AD-like skin lesions in mice,and can exacerbate the severity of OVA-induced AD-like skin lesions.Mast cell proliferation,activation/degranulation and tryptase release may participate in the inflammation.

Establishment of a Brown-Norway rat model of cough variant asthma / 中国比较医学杂志

Objective To establish a simple animal model of cough variant asthma(CVA)through sensitizing Brown-Norway(BN)rats with ovalbumin(OVA). Methods A total of 36 BN rats were randomly divided into three groups, including the normal control group,the model control group and the montelukast group. BN rats in the model group and the montelukast group were intraperitoneally administered with 2.0 mg of OVA and 100 mg of Al(OH)3,and the same volume of sterile saline was given to the normal group by intraperitoneal injection. Boosting was carried out by intraperitoneal administration with 0.01 mg of OVA and 100 mg of Al(OH)33 weeks later,and the rats in the normal group were injected with the same dose of physiological saline. Three weeks later,the actively sensitized BN rats were challenged with aerosolized OVA for 7 times on alternative days,and the rats in the normal group were treated with sterile saline instead of OVA. At the same time, the montelukast group was given 1.3 mg/kg of montelukast 30 minutes before atomization by intragastric administration once a day for 2 weeks,and the normal group and the model group were given the same volume of water. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Results Compared with the normal group, the times of coughing(P< 0.01)and the lung resistance(RL)(P< 0.05)in the model group were significantly increased,while the lung compliance(Cdyn)was significantly decreased(P< 0.05). There was a significant difference(P < 0.05)in the times of coughing caused by capsaicin between the model group and the montelukast group. Compared with the model group,RLin the montelukast group was decreased significantly(P< 0.05), and Cdynwas increased significantly(P< 0.05). Conclusions This rat model of CVA is similar to a variety of clinical features of CVA and is easy to operate. Thus it can be used as an effective animal model of CVA.

Mechanism of the expression of IL-25 in the lung during asthma / 西安交通大学学报(医学版)

Objective To study the molecular mechanism of interleukin 25 (IL-25)expression in the lung of asthmatic rats.Methods The expressions of IL-25 mRNA and protein in the lungs were detected by Real-time PCR and ELISA,respectively.The levels of IL-25 mRNA and protein were detected by ovalbumin (OVA)in human bronchial epithelial cells.And the transcription factors that regulate IL-2 5 expression were explored through site prediction.Results The expressions of IL-25 mRNA and protein in the lung of OVA-induced asthma rats were significantly increased during animal experiments.Cell experiments showed that OVA could increase the expression of IL-2 5 in human bronchial epithelial cells in a dose-dependent manner,and OVA could upregulate the expression of transcription factor AP1.AP1 was found in the promoter region of IL-25 by site prediction.The AP1 inhibitor (T5224)significantly reduced the expression of IL-25 in OVA-induced human bronchial epithelial cells. Conclusion The molecular mechanism of IL-25 expression induced by OVA in asthma is related to the increase of transcription factor AP1 .