乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

表皮生长因子和降钙素基因相关肽在高频电磁场 治疗急性胃损伤中作用研究

目的：探讨高频电磁场（high-frequency electromagnetic fields,HEMFs）曝露治疗急性胃损伤的作用机制。方法：选用健康SD大鼠,以Indomethacin灌胃法复制胃粘膜急性损伤模型,观察40．68MHz HEMFs（波长为7．3m）曝露（微热量,30—50mA,15min,1／d）治疗1或6次后,大鼠胃粘膜损伤程度（胃损伤指数和病理损伤积分）、胃粘膜血流量、血浆表皮生长因子（epidermal growth factor,EGF）和降钙素基因相关肽（calcitonin gene—related peptide,CGRP）水平。结果：HEMFs曝露1次后,胃粘膜血流量较对照组明显升高（P〈0．05）,大鼠胃粘膜损伤程度、血浆EGF和CGRP水平较对照组均无显著性差异（P均〉0．05）;HEMFs曝露6次后,胃粘膜损伤程度较对照组明显改善（P均〈0．05）,胃粘膜血流量显著升高（P〈0．05）,血浆EGF和CGRP水平较对照组无显著性差异（P均〉0.05）。结论：HEMFs曝露治疗大鼠胃粘膜急性损伤可能与其增加胃粘膜血流量有关,与血EGF和CGRP无关。     

Beta-glucan derived from zymosan acts as an adjuvant for collagen-induced arthritis

Collagen-induced arthritis (CIA) is an experimental model of rheumatoid arthritis (RA) and has helped researchers to analyze the pathogenesis of inflammatory joint disease. In classical CIA, Freund's complete adjuvant (FCA), which contains heat-killed Mycobacterium tuberculosis, is used as an adjuvant. In our previous study, we reported that particles of beta-glucan, OX-CA, derived from Candida albicans, acted as a proper adjuvant in the CIA model. In this study, to establish pure beta-glucan as an adjuvant for CIA, we tested a commercially available preparation of Zymosan A (ZYM) and modified its products. beta-Glucan fractions of ZYM were prepared by oxidation with various concentrations of NaClO. The oxidized ZYM (OX-ZYM) was mainly composed of beta-glucan. In this study, we examined its effect as an adjuvant for CIA. DBA/1 mice injected with CII and OX-CA developed arthritis 7-10 days after receiving booster injections; the OX-ZYM fractions induced arthritis with the same time course. 0.01% OX-ZYM (oxidized with a 0.01% NaClO solution) caused arthritis faster than 0.1% OX-ZYM or 0.5% OX-ZYM. In conclusion, beta-glucan derived from ZYM by brief oxidation with NaClO is a suitable adjuvant for a CIA model with anti-CII antibody production.     

Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.     

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosal immunization in a mouse model

BACKGROUND: A critical component of the host defense against enteric infections is the immunological response of the mucosal membrane, a major starting point of infectious disease, such as typhoid fever. The mucosal immune system consists of an integrated network of lymphoid tissues, mucous membrane-associated cells, and effector molecules. In the present study, we developed a recombinant Bifidobacterium animalis (B. animalis) genetically modified with the Salmonella flagellin gene for mucosal immunization as an oral typhoid vaccine. METHODS: We constructed an oral vaccine against Salmonella typhimurium, consisting of recombinant B. animalis containing the flagellin gene of Salmonella. The recombinant B. animalis was administered orally to mice every other day for 6 weeks. Anti-flagellin antibodies in the serum and stools were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We detected significantly higher levels of flagellin-specific IgA in the serum and stools of the mice treated with the recombinant B. animalis containing the flagellin gene than was seen in those treated with parental B. animalis. CONCLUSIONS: Our findings suggest that an oral vaccination using recombinant B. animalis genetically modified with the flagellin gene of Salmonella may be effective against Salmonella infections.     

Effect of Semecarpus anacardium Linn. nut milk extract on rat neutrophil functions in adjuvant arthritis

Neutrophils play an important role in the pathogenesis of rheumatoid arthritis (RA) and various inflammatory conditions, by accumulation and liberation of active proteolytic enzymes. The effect of milk extract of Semecarpus anacardium Linn. nuts (SA) at a dosage of 150 mg kg(-1) body weight day(-1) for 14 days on adjuvant arthritis was studied to gain some insight into this intriguing disease in relation to neutrophil functions. The decreased phagocytic function of neutrophils (phagocytic index and avidity index) found in adjuvant arthritis was significantly increased by the administration of the drug SA. Increased levels of reactive oxygen species (superoxide radical, hydroxyl radical, H2O2 and myeloperoxidase), lysosomal enzymes (acid phosphatase and cathepsin D) and increased accumulation of neutrophils in the joints observed in adjuvant arthritic animals were reverted back to near normal levels by treatment with SA. The results of this study indicate that SA can be considered to be a good therapeutic agent for inflammation and arthritis.     

Phagocytosis of N-acetyl-D-glucosamine particles, a Th1 adjuvant, by RAW 264.7 cells results in MAPK activation and TNF-alpha, but not IL-10, production

A practical and highly effective Th1 adjuvant should induce Th1 cytokines (IL-12, IL-18, and TNF-alpha) but not the Th2 cytokine IL-10, an inhibitor of Th1 responses. In this study, phagocytosis of N-acetyl-d-glucosamine polymer (chitin) particles by RAW 264.7 cells, a murine macrophage-like cell line, resulted in phosphorylation of MAPK (p38, Erk 1/2, and JNK), and production of relatively high levels of TNF-alpha and COX-2 with increased PGE(2) release. Similar results were observed in response to oligonucleotides with CpG motifs, mycobacterial components and endotoxin. However, these bacterial components also induced a large amount of IL-10. Chitin particles, in contrast, failed to induce detectable levels of IL-10, although the production of high levels of PGE(2) and TNF-alpha and the activation of MAPK's are potentially positive signals for IL-10 production. Thus, our results indicate that chitin particles act as a unique Th1 adjuvant for macrophages without inducing increased production of IL-10.     

A need for careful evaluation of endotoxin contents in acellular pertussis-based combination vaccines

Two batches each of diphtheria-tetanus-acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from foreign markets were tested by mouse body weight decreasing (BWD) toxicity test and Limulus amaebocyte lysate (LAL) test. Three out of the four imported vaccine batches showed the levels of BWD toxicity even comparable to that of DT-whole cell pertussis vaccine. BWD toxicity test is based on endotoxin dose-dependent weight loss of mice and has been used for controlling endotoxin in DTaP. Although of the strong BWD toxicity of the imported vaccines, there was no marked difference in LAL test results between the imported vaccines and Japanese DTaP. However, one imported DTaP batch showed very strong interference with LAL activity of spiked lipopolysaccharide (LPS). The batch interfered not only with LAL activity but also with pyrogenicity and prostaglandin E₂ induction activity. However, the pyrogenicity of the spiked LPS could be recovered from the precipitated fraction of the batch by treating with phosphate buffer to suggest the possibility of recovering in vivo toxicity. As an adequate in vitro test method could not be identified for controlling the safety of the interfering batch, an appropriate in vivo test would be required for testing such vaccines.     

Study on the immune enhancement of different immunoadjuvants used in the pentavalent vaccine for turbots

In this study, we investigated the immune enhancing effects of different adjuvants used in a pentavalent vaccine for turbots. The pentavalent vaccine consisted of inactive bacterial cells from five common pathogenic strains (Vibrio anguillarum, Vibrio scophtalmi, Edwardsiella tarda, Vibrio harveyi and Vibrio alginolyticus) and the adjuvants were astragalus polysaccharides (APS), propolis, and the Freund’s complete adjuvant (FCA). Turbots were immunized with the pentavalent vaccine alone or with one of the adjuvants, and the immune efficiency was evaluated by measuring the activities of lysozyme (LSZ) and superoxide dismutase (SOD), and serum antibody titers. Fish were also challenged with the pathogens after immunization and the relative percent survival (RPS) was assessed. Our results showed that APS, propolis, and FCA had significant immune-enhancing effects on turbots as shown by the higher titers of antibodies against the pathogens, increased LSZ and SOD activities, and enhanced RPS after challenge with pathogens. Among the three adjuvants, FCA had the most significant immune synergistic effects with the vaccine, and APS and propolis had lower and similar immune synergies.