Annexin A1 as a target for managing murine pristane-induced systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a polygenic pathological disorder which involves multiple organs. Self-specific B cells play a main role in the lupus pathogenesis by generating autoantibodies as well as by serving as important autoantigen-presenting cells. Autoreactive T lymphocytes, on the other hand, are responsible for B cell activation and proliferation, and cytokine production. Therefore, both factors promote the idea that a down-modulation of activated self-reactive T and B cells involved in the pathogenic immune response is a reasonable approach for SLE therapy. Annexin A1 (ANX A1) is expressed by many cell types and binds to phospholipids in a Ca²⁺ dependent manner. Abnormal expression of ANX A1 was found on activated B and T cells in both murine and human autoimmunity, suggesting its potential role as a therapeutic target. While its role on T lymphocytes is through formyl peptide receptor-like molecules (FPRL), and the formed ANX A1/FPRL pathway modulates T cell receptor signalling, there is still no fool-proof data available for the role of ANX A1 in B cells. We employed a lupus model of Balb/c mice with pristane-induced SLE which very closely resembles human lupus. In the present study, we investigated the possibility to modulate the autoimmune response in a pristane-induced mouse model of SLE using an anti- ANX A1 antibody. Administration of this monoclonal antibody resulted in the inhibition of T-cell activation and proliferation, suppression of IgG anti-dsDNA antibody-secreting plasma cells and of proteinuria, decreased disease activity and prolonged survival compared to control group.     

锯缘青蟹原肌球蛋白的过敏动物模型研究

目的建立锯缘青蟹(Scylla serrata)原肌球蛋白(tropomyosin,TM)的食物过敏性小鼠模型,并对其致敏性进行评价。方法锯缘青蟹TM以灌胃方式刺激小鼠使其致敏,并对小鼠粪便组胺、血清IgE以及脾脏细胞IL-4、IL-13、IFN-λ等淋巴因子的水平进行检测。结果 TM组小鼠均出现了不同程度的过敏症状,其血清IgE的含量是阴性对照组(生理盐水)小鼠的2倍;TM组实验小鼠IL-4、IL-13、IFN-λ的水平分别为(17.508±2.189)pg/ml、(3.150±0.434)pg/ml、(15.056±1.974)pg/ml,粪便中组胺含量为6 244.651 nmol/l,与阴性对照组小鼠相比均有显著差异(P<0.05)。结论锯缘青蟹TM以灌胃方式刺激小鼠,TM组小鼠出现了不同程度的过敏症状,其粪便组胺、血清IgE以及脾脏细胞IL-4、IL-13、IFN-λ等淋巴因子的水平与阴性对照组小鼠相比均有显著差异(P<0.05),建立的TM过敏动物模型可望为蟹类过敏的诊断和免疫治疗提供技术依据。     

The improvement of in vivo model (Balb/c mice) for cervical carcinogenesis using diethylstilbestrol (DES)

Cervical cancer is the most common gynecological cancer and one of the major causes of female cancer-related death worldwide particularly in developing countries. Thus far, there are a few in vivo models have been developed in investigating this type of cancer. In this study, we induced cervical cancer in Balb/c mice by exploiting the carcinogenic property of diestylstilbestrol (DES). The Balb/c pregnant mice were given subcutaneous (SC) injection of 67 μg/kg body weight of DES on GD 13, and the mice gave birth approximately at gestation day 19–22. Female offspring were reared and the body weight was recorded once weekly. The female offspring were sacrificed at age of 5 months. Upon termination, blood was collected in a plain tube via cardiac puncture and the reproductive tracts were collected and weighed. The reproductive tract sections were stained using H&E for observation of pathological changes. The progression of disease state was monitored by measuring the level of serum interleukin (IL-6) using the Mouse IL-6 ELISA Assay Kit (BD OptEIA?, USA). All parameters were compared with Not-induced group. The outcome of this study demonstrated a significant difference in body weight gain, reproductive organ weight, diameter of cervix and the level of serum IL-6 in the Induced group as compared to the Not-induced group (P < 0.05). Histopathological findings revealed the presence of adenosis only in the Induced group. It shows that DES could be employed as an agent to induce cervical carcinogenesis in animal model. In addition to that, new potential anti-cancer agents from various sources could be further evaluated using this technique.     

Effects of lactoferrin on infectious diseases in Japanese summer: A randomized,double-blinded,placebo-controlled trial

PurposeTo investigate the effects of lactoferrin (LF) on infectious diseases in Japanese summer.MethodsAn intake of placebo, 200 mg, or 600 mg of LF were administered to healthy adults in Kyushu University of Health and Welfare for 12 weeks in a randomized, double-blinded, placebo-controlled parallel-group comparative trial. The primary endpoints were the prevalence and duration of infectious diseases and changes in immune parameters.ResultsThree hundred and ten subjects were randomized (placebo, n = 104; 200 mg, n = 103; 600 mg, n = 103). Twenty subjects were lost to the follow-up, leaving 290 for a full analysis set (n = 99; n = 95; n = 96). The duration (day) of total infectious diseases was shorter in the 200 mg group (2.0, p = 0.045) and 600 mg group (2.0, p = 0.010) than in the placebo group (3.0). The duration of summer colds was shorter in the 600 mg group (2.0, p = 0.036) than in the placebo group (3.0). No significant differences were observed in the prevalence of infectious diseases or changes in immune parameters. In exploratory investigations, changes in the neutrophil phagocytic capacity, cortisol concentrations, and T score of “Vigor/Activity” in the Profile of Mood States 2 were greater in the 600 mg group than in the placebo group, when analysis was done on the lower half groups at the baseline. Adverse events were similar in each group and none had a causal relationship with the intake of the test foods.ConclusionsIn summer, the intake of LF attenuates infectious diseases, including summer colds.     

Engineered recombinant ovomucoid third domain can desensitize Balb/c mice of egg allergy

The purpose of this study was to determine the in vivo desensitization efficacy of a hypoallergenic variant of egg white ovomucoid third domain (DIII) in Balb/c mice model. We mapped the immunodominant B-cell epitopes of ovomucoid in Balb/c mice. A hypoallergenic ovomucoid third domain (GMFA) mutant isoform having ablated allergenicity against egg allergic patient's sera was used to desensitize DIII-sensitized Balb/c mice by intraperitoneal injections. Ovomucoid DIII generated high levels of plasma histamine and specific immunoglobulin (Ig)E levels, and increased Th2 type cytokine (IL-4). On the other hand, the allergic response of mice desensitized with the GMFA was found to be significantly inhibited and abrogated by prevention of anaphylaxis reactions, low histamine levels and increased Th1-type cytokine (INF-gamma). It was found that significantly higher levels of IL-10 and IL-12 were secreted in the desensitized group. Desensitization with the GMFA antigen also suppressed synthesis of DIII specific-IgE levels and enhanced specific IgG2a and IgG levels compared with the group treated with the DIII antigen. The present results indicated that hyposensitization with the GMFA can desensitize or down-regulate the allergic response in Balb/c mice and this hypoallergenic variant of ovomucoid DIII can shift an ongoing allergen-specific Th2 response towards a Th1 skewed response.     

Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(-), E2b(-)] adenoviral vectors

The systemic delivery of [E1(-)] adenoviral (Ad) vectors encoding a transgene results in efficient viral uptake and abundant transgene expression in the liver. However, [E1(-)]Ad vector persistence is transient due to cytotoxic T lymphocyte (CTL)-mediated loss of the Ad-infected cells. Our laboratory has previously demonstrated that additional modifications to the [E1(-)]Ad vector genome, by deletion of the Ad E2b genes, significantly decreased virus-genome-derived gene expression and simultaneously improved the long-term performance of the resultant [E1(-), E2b(-)]Ad vector. In this study, we confirmed that [E1(-), E2b(-)]Ad vector genomes could persist equally well in C57Bl/6 or Balb/c mouse hepatocytes. Despite vector genome persistence, we observed a strain-dependent variability in the duration of CMV enhancer/promoter-driven transgene expression in the liver. While Balb/c mice rapidly shut down [E1(-), E2b(-)]Ad-derived transgene expression, C57Bl/6 mice allowed for prolonged transgene expression. This occurred even when both strains were crossed into a severe combined immune-deficient background, demonstrating that host adaptive immune responses are not responsible for the phenomenon. Furthermore, differential methylation of the CMV enhancer/promoter was also not demonstrated in either strain of mouse, eliminating this mechanism as causative. Thus, alternative mechanisms for this phenomenon are discussed.     

Pharmacogenetic study of anxiolytic effects of new cholecystokinin receptor antagonists in animals with different levels of emotionality

Effects of two new peptide antagonists of central cholecystokinin receptors, GB-101 (0.05-0.40 mg/kg) and GB-115 (0.006-0.100 mg/kg), on the behavior of inbred animals differing by the reactions to emotional stress were studied in standard tests for evaluation of tranquilizers. The test drugs dose-dependently increased the total locomotor activity in the open field test in Balb/c mice and had no effect on the behavior of C57Bl/6 mice. GB-101 (0.4 mg/kg) and GB-115 (0.025 and 0.05 mg/kg) produced an anxiolytic effect on MR rats (but not on MNRA rats) in the elevated plus-maze and conflict tests. These data confirm anxioselective effects of the test compounds.     

Atropine, a muscarinic cholinergic receptor antagonist increases serotonin, but not dopamine levels in discrete brain regions of mice

We investigated the effects of atropine, a muscarinic acetylcholine (ACh) receptor antagonist, on the level of serotonin in discrete brain regions, the nucleus raphe dorsalis (NRD), nucleus caudatus putamen (NCP), cerebral cortex and the cerebellum. Biogenic amines were assayed employing HPLC electrochemistry in these regions 30 min following different doses of atropine (5, 10, 25mg/kg; i.p.), and at various time points (15, 30, 60, 120 min) after 25mg/kg of the drug. The cholinergic receptor antagonist caused a dose-dependent alteration in the level of serotonin in NRD, but the increase was not dose-dependent for other regions studied. The metabolite of serotonin, 5-hydroxyindoleacetic acid was unaffected. Atropine did not affect the levels of dopamine or its metabolites dihydroxyphenyl acetic acid and homovanillic acid. The present study suggests significant effect of this antimuscarinic agent on the synthesis of serotonin in the central serotoninergic pathways, which may have clinical relevance.     

Toll-like receptor 7 agonist induces hypoplasia of the biliary system in a neonatal mouse model

Background/Purpose

Viral infections and innate immunity signaling, especially Toll-like receptor 7 (TLR7) have been implicated in the pathogenesis of biliary atresia (BA). Administration of rhesus rotavirus-type A to newborn Balb/c mice produces inflammatory obstruction of bile ducts, which resembles human BA. However, whether activation of TLR7 signaling plays a role in neonatal hepatobiliary injury remains to be investigated.

Differential responses to natural and recombinant allergens in a murine model of fish allergy

Aerosolized fish proteins are an important cause of allergic airway reactions in both the domestic and the occupational environment. The aim of this study was to investigate inhalant fish-induced allergy in a mouse model and compare immune responses generated by raw and heat-treated fish extracts as well as natural and recombinant forms of the major fish allergen parvalbumin. Mice were sensitized with raw or cooked pilchard extract and challenged intranasally with cooked pilchard extract, purified natural pilchard parvalbumin or recombinant carp parvalbumin (rCyp c1.01). Cooked pilchard extract predominantly sensitized mice to parvalbumin and induced specific IgG1 and IgE antibodies against both pilchard parvalbumin and rCyp c1.01, whereas additional allergens were recognized by mice sensitized with raw extract, including a 36 kDa allergen that was also recognized by fish processing workers and was identified as glyceraldehyde-3-phosphate dehydrogenase. Mice challenged with cooked extract and purified pilchard parvalbumin had increased Th2 cytokine production in mediastinal lymph node cells and splenocytes, whereas mice challenged with rCyp c1.01 did not. This study identifies a new IgE-binding protein that may be important in occupational allergy to fish and demonstrates the feasibility of testing recombinant allergens for immunotherapeutic potential in vivo.     

原花青素A-1调节ConA刺激小鼠脾细胞分泌Th1/Th2细胞因子的影响

目的研究从碎米花杜鹃叶中分离得到的化合物原花青素A-1(proanthocyanidin A-1,简称PAA-1)对Con A刺激的小鼠脾细胞细胞因子分泌水平的调节作用,确定其免疫增强的作用机制。方法采用双抗夹心ELISA法,通过不同浓度PAA-1协同Con A体外刺激,检测其对小鼠脾细胞分泌的各种细胞因子分泌量的影响。结果 1)PAA-1各浓度在体外能增加Con A刺激的脾细胞分泌的辅助性T细胞亚群(Th1)细胞因子(IL-2、IL-12、IFN-γ、TNF-α)的分泌量;2)PAA-1对Con A刺激的脾细胞分泌的抑制Th2细胞因子(IL-4、IL-10)的分泌量起抑制作用。结论 PAA-1是通过增加Con A刺激的小鼠脾细胞Th1细胞因子的分泌量,来发挥免疫增强作用,并使Th1/Th2的平衡向Th1方向移动,这对许多的Th2占优势的免疫紊乱性疾病的治疗具有重要的意义,为其开发为新型免疫增强剂提供试验依据。     

微小隐孢子虫子孢子表面蛋白质粒DNA接种诱导Balb/c小鼠的免疫应答

目的　探讨微小隐孢子虫子孢子表面蛋白CP2 3重组质粒pCR3.1～ 2 3DNA疫苗诱导机体产生体液和细胞免疫应答的效果。方法　用重组的DNA疫苗于Balb/c小鼠后腿胫骨前肌注射免疫 ,于 0、3、6周共免疫 3次 ,10 0 μg/次。免疫后不同时间检测体液和细胞免疫应答指标。并用 1× 10 6卵囊进行攻虫试验。结果　pCR3.1～ 2 3可诱导机体产生相应的特异性抗体 ,对C .parvum卵囊攻击具有保护作用。结论　微小隐孢子虫子孢子表面蛋白CP2 3重组质粒pCR3.1～ 2 3有可能作为侯选的隐孢子虫DNA疫苗 ,值得进一步深入研究。     

Comparative study of the adjuvanticity of Bacillus thuringiensis Cry1Ab protein and cholera toxin on allergic sensitisation and elicitation to peanut

We aimed to investigate the adjuvanticity of Cry1Ab on allergic sensitisation and elicitation to peanut in mice, in comparison with cholera toxin (CT), a known mucosal Th2 adjuvant. Balb/c mice were gavaged with phosphate buffer saline (PBS) (control), peanut protein extract (PE) alone or PE co-administered either with CT or Cry1Ab. Peanut-specific IgE, IgG1 and IgG2a and Th1/Th2/Th17 cytokine secretion by splenocytes were assayed. Gavaged mice were further challenged with PE. Markers of the immediate and late phases of the allergic reaction were assayed in bronchoalveolar lavages (BAL) fluids collected 10 minutes or 36 hours after airway challenge, respectively. Sensitisation to peanut was only observed in mice receiving PE plus CT, as demonstrated by specific IgE and IgG1 production in sera and secretion of Th2 (and Th17) cytokines by splenocytes. Following challenge, mice sensitised with PE plus CT produced leukotrienes (LT) E4 and C4, then Th2/Th17-cytokines and showed eosinophil/neutrophil influx in BAL. Interestingly, LT production, Th2/Th17 cytokine release and eosinophil influx were also significant in mice gavaged with PE plus Cry-1Ab, but not with PBS or PE alone. Cry1Ab did not demonstrate adjuvant effects on oral sensitisation to peanut. However, this study shows possible adjuvant properties of Cry1Ab on the elicitation of the allergic reaction, at least in the Balb/c mouse model and in the experimental conditions used.     

Treatment with chitin microparticles is protective against lung histopathology in a murine asthma model

BACKGROUND: Chitin, a natural polysaccharide extracted from shrimp, is a potent T and B cell adjuvant when delivered in the form of chitin microparticles and can shift a polarized T-helper type 2 (Th2) immune response towards a Th1 response. OBJECTIVE: We investigated the beneficial effects of the intranasal application of chitin microparticles in newborn mice before and after the establishment of a model of allergic asthma. METHODS: Mice were grouped as asthma (A), primary prevention (PP), treatment (T), primary prevention+treatment (PPT) and control (C) groups. All mice except controls were sensitized with ovalbumin intraperitoneally and challenged intratracheally to establish the asthma model. Mice in the PP and PPT groups received chitin microparticles intranasally during the newborn period before sensitization. Mice in the PPT and T groups received intranasal chitin microparticles after challenge. Airway histopathology was evaluated in all groups. RESULTS: All of the airway histopathologic parameters of small and medium-sized airways of the T and PPT groups were significantly ameliorated when compared with the asthma model group. In the large airways, thicknesses of basement membrane, epithelium and subepithelial smooth muscle layers of the PPT group and basement membrane thicknesses of the T group were also significantly lower compared with the asthma model group. Comparison of the PP group with the asthma model group revealed significantly reduced goblet cell numbers and significantly reduced epithelial and basement membrane thicknesses in small and medium airways, in addition to significantly reduced basement membrane thicknesses in the medium-sized airways. CONCLUSION: Intranasal application of microgram quantities of chitin microparticles had a beneficial effect in preventing and treating histopathologic changes in the airways of asthmatic mice.     

Progression of chronic pulmonary tuberculosis in mice intravenously infected with ethambutol resistant Mycobacterium tuberculosis

Purpose: Ethambutol (EMB) is an important first line drug, however little information on its molecular mechanism of resistance and pathogenicity of resistant isolates is available. Present work was designed to study virulence of the EMB resistant M. tuberculosis strains and the host responses in-vivo on infection of EMB resistant M. tuberculosis using Balb/c mouse model of infection. Methods: Three groups of Balb/c mice (female, age 4-6 wk; 21 mice in each group) were infected intravenously with 106 CFU of M. tuberculosis H37Rv and two EMB resistant clinical isolates. Age and sex matched control animals were mock inoculated with Middlebrook 7H9 broth alone. At 10, 20, 30, 40, 50, 60, and 70 days post-infection three animals from each group were sacrificed by cervical dislocation and lung tissue was collected for further analysis. Results: Infection with EMB resistant M. tuberculosis led to progressive and chronic disease with significantly high bacillary load (p=0.02). Massive infiltration and exacerbated lung pathology with increased expression of IFN-γand TNF-αwas observed in lungs of mice infected with EMB resistant strains. The present study suggests that infection with EMB resistant M. tuberculosis leads to chronic infection with subsequent loss of lung function, bacterial persistence with elevated expression of TNF-αresulting in increased lung pathology. Conclusion: These findings highlight that EMB resistant M. tuberculosis regulates host immune response differentially and its pathogenicity is different from drug sensitive strains of M. tuberculosis.     

The Effect of Different Doses of Cigarette Smoke in a Mouse Lung Tumor Model

Few studies have used Balb/c mice as an animal model for lung carcinogenesis. In this study, we investigated the effect of different doses of cigarette smoking in the urethane-induced Balb/c mouse lung cancer model. After injection of 3mg/kg urethane intraperitoneally, the mice were then exposed to tobacco smoke once or twice a day, five times a week, in a closed chamber. The animals were randomly divided into four groups. The control group (G0) received urethane only. The experimental groups (G1, G2 and G3) received urethane and exposure to the smoke of 3 cigarettes for 10 minutes once a day, 3 cigarettes for 10 minutes twice a day, and 6 cigarettes for 10 minutes twice a day, respectively. The mice were sacrificed after 16 weeks of exposure, and the number of nodules and hyperplasia in the lungs was counted. The results showed no statistically significant difference in the mean number of nodules and hyperplasia among the different groups, suggesting that the Balb/c mice are not suitable to study the pathogenesis of tobacco smoking-induced tumor progression in the lungs.     

Th2 cells promote eosinophil-independent pathology in a murine model of allergic bronchopulmonary aspergillosis

Repeated inhalation of airborne conidia derived from the fungus Aspergillus fumigatus (Af) can lead to a severe eosinophil-dominated inflammatory condition of the lung termed allergic bronchopulmonary aspergillosis (ABPA). ABPA affects about 5 million individuals worldwide and the mechanisms regulating lung pathology in ABPA are poorly understood. Here, we used a mouse model of ABPA to investigate the role of eosinophils and T cell-derived IL-4/IL-13 for induction of allergic lung inflammation. Selective deletion of IL-4/IL-13 in T cells blunted the Af-induced lung eosinophilia and further resulted in lower expression of STAT6-regulated chemokines and effector proteins such as Arginase 1, Relm-α, Relm-β, and Muc5a/c. Eosinophil-deficient ΔdblGata mice showed lower IL-4 expression in the lung and the number of Th2 cells in the lung parenchyma was reduced. However, expression of the goblet cell markers Clca1 and Muc5a/c, abundance of mucin-positive cells, as well as weight gain of lungs were comparable between Af-challenged ΔdblGata and WT mice. Based on these results, we conclude that T cell-derived IL-4/IL-13 is essential for Af-induced lung eosinophilia and inflammation while eosinophils may play a more subtle immunomodulatory role and should not simply be regarded as pro-inflammatory effector cells in ABPA.