基因疫苗微粒系统黏膜免疫的研究进展

微粒系统作为一种基因疫苗的黏膜免疫递送系统,能够增强免疫效果,具有同时诱导系统免疫应答和黏膜免疫应答、产生共同黏膜免疫应答、增加病人的顺应性、降低疫苗推广成本等优点.文章综述微粒系统增强基因疫苗黏膜免疫效果的机制、常见的微粒系统、载体材料以及优化微粒系统的研究进展.     

疫苗微粒给药系统的研究进展

微粒作为疫苗载体,在疫苗投递系统中的优势已越来越明显.从疫苗微粒在疫苗的口服给药、皮内注射给药、呼吸道黏膜直接接种和其他接种途径等四个方面对微粒给药系统在疫苗投递中的应用现状进行了总结与评价:同时对疫苗微粒给药系统常用高分子化合物进行综述;从单剂量疫苗、新型疫苗、老疫苗新用等方面对微粒给药系统在疫苗投递中的应用前景进行了综述与总结.     

口服霍乱减毒活疫苗研究进展

理论上口服霍乱减毒活疫苗优于死疫苗,减毒的霍乱弧菌经单剂口服接种即可在肠道定居并迅速产生免疫应答,无需再次接种.在霍乱流行区的部分人群中,由于先存抗体可抑制活菌在肠道定居,从而使疫苗免疫效果下降.目前,已开发了多种针对不同血清群的口服霍乱减毒活疫苗,在志愿者中已证明是安全有效的,疫苗进一步的研究及临床评估正在进行中.     

肠黏膜屏障功能与消化系统疾病关系的研究进展

肠道作为机体与外界相通的器官,其屏障功能对人类健康至关重要。肠黏膜屏障由物理屏障、生物屏障、化学屏障以及免疫屏障四部分组成,防止腔内有害物质侵入黏膜下组织,同时能选择性滤过营养物质和电解质。肠道的这种特性被称为肠黏膜屏障功能。肠黏膜屏障功能障碍会导致黏膜通透性增加,且与多种消化系统疾病（如炎症性肠病、乳糜泻、非酒精性脂肪性肝病等）的发生发展密切相关。本文主要对肠黏膜屏障构成及肠黏膜屏障功能与消化系统疾病发生关系的研究进展进行综述。     

肠道屏障功能评估方法的现状及研究进展

肠道屏障是指肠道能防止肠腔内的有害物质如细菌和毒素等穿过肠黏膜进入体内其他组织器官和血液循环的结构和功能的总和。它可分为肠黏膜外防止细菌易位的支持系统和肠黏膜本身两部分,包括：肠黏膜上皮、肠粘液、肠道菌群、分泌性免疫球蛋白、肠道相关淋巴组织、胆盐、激素和胃酸等。肠道屏障通常可以归纳为机械屏障、化学屏障、生物屏障、免疫屏障四大功能。临床上对肠屏障损伤治疗的需要,以及如何正确测定和评估肠道屏障功能障碍的程度,一直是医学界不懈追求的方向。本文就近年来国内外这方面的研究进展作一综述。     

中药对肠黏膜屏障的作用机制研究进展

肠黏膜屏障能够有效阻止肠内的有害物质穿过肠黏膜进入血液及其他组织,在维持机体与外环境的稳态上发挥着非常重要的作用。但机体在遭受严重应激后,易导致肠黏膜屏障功能下降。近年来,中医药的介入为肠黏膜屏障的保护开辟了一条新道路。中药可通过调控肠上皮细胞间紧密连接、调节肠道微生态平衡及激发肠道免疫应答等作用修复受损的肠黏膜屏障。本文对中药在受损肠黏膜保护及修复中的作用机制进行综述,旨在阐明中药在肠黏膜屏障保护中的重要作用。     

中药\

肠道不仅负责食物的消化和吸收, 还能通过微生态、神经内分泌及黏膜免疫与肠道外组织器官相联系, 参与机体稳态的维持、疾病的发生发展、药物的转运和作用等过程。肠道是多种中药活性成分口服给药的初始作用部位, 现已发现小檗碱、姜黄素和白藜芦醇等物质可通过肠道途径发挥药理效应。从肠道菌群、肠道神经内分泌和肠道黏膜免疫角度综述了中药药理作用的肠道途径研究进展。

     

谷氨酰胺在肠道中的作用研究进展

谷氨酰胺(GLN)是人体内参与氨运输和代谢的重要载体,也是偶联体内代谢过程及维持某些组织器官结构和功能的重要物质。目前认为谷氨酰胺对维护修复肠黏膜屏障功能具有重要的作用。随着对GLN研究的增多,本文就GLN在肠道吸收和肠道免疫方面的相关作用进行阐述。     

新型冠状病毒肺炎对肠道微生物群及肠黏膜屏障缺陷影响机制的研究进展

肠道微生物群与肠黏膜屏障之间的交互作用对维持肠道内环境的稳定起到了至关重要的作用。新型冠状病毒感染可以通过不同方式直接或间接破坏肠道微生物群的多样性,影响肠黏膜屏障的结构与代谢。肠道内稳态与微环境在新型冠状病毒肺炎（COVID-19）的发病机制及全身炎症反应的增强中至关重要,提示肠道微生物群及黏膜屏障可作为逆转该类患者病理进程重要的治疗靶点。就COVID-19所致肠道微生物群及相关肠黏膜屏障缺陷机制的研究进展进行综述。     

肠道菌群在炎症性肠病发病机制与治疗中的作用研究进展

炎症性肠病是一种以肠道炎症和黏膜损伤为特征的慢性疾病,主要包括克罗恩病和溃疡性结肠炎,其发病原因尚不明确。现有研究表明炎症性肠病的发病机制与宿主的遗传易感性、肠道菌群紊乱、肠黏膜屏障破坏和肠黏膜免疫异常等密切相关。总结目前炎症性肠病发病机制、患者肠道菌群结构及治疗方法等相关的研究进展,旨在对今后炎症性肠病的治疗和药物研发有所裨益。     

Chitosan for mucosal vaccination.

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyer's patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.     

Evaluation of nano- and microparticle uptake by the gastrointestinal tract

Numerous papers over the last two decades have demonstrated that particle uptake by the gastrointestinal tract is a reality. In addition, polymeric nano- and microparticles have proved to be useful delivery systems to enhance oral bioavailability of poorly absorbed drugs or to induce mucosal immune response. However, despite the amount of data available, no set criteria are available for the design of a good particulate carrier for oral delivery of peptides or antigens. This is partly due to the publication of conflicting and confusing data. The source of discrepancy is actually multiparametric (e.g., methodology, mode of evaluation, animal species) and is still not fully understood. The purpose of this review is to discuss the advantages and the limitations of the methodologies and the models used to evaluate gastrointestinal uptake of nano- and microparticles.     

Transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal M-cell model

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres (size 0.2 microm) and chitosan microparticles (size 1.7 microm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.     

Transport of Chitosan Microparticles for Mucosal Vaccine Delivery in a Human Intestinal M-cell Model

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14 C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres ®) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres ® (size 0.2 μ m) and chitosan microparticles (size 1.7 μ m) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.     

Biodegradable nanoparticles for oral delivery of peptides: is there a role for polymers to affect mucosal uptake?

Numerous authors have demonstrated uptake of micro- and nanospheres, consisting of natural or synthetic polymeric materials from the gastrointestinal tract over the past two decades. The exploitation of particulate carrier systems for the delivery of peptides and other hydrophilic macromolecules via the oral route remains a challenging task due to morphological and physiological absorption barriers in the gastrointestinal tract. This review examines recent progress in the field of nanoparticle uptake from this site of administration. Since most studies have been performed with poly(styrene) particles of different sizes relatively little is known about both the effect of physicochemical particle properties critical for absorption after peroral application, and the mechanisms of gastrointestinal particle uptake. Apart from particle size, type and composition of the polymers used for micro- or nanoencapsulation are crucial for an uptake and transport across mucosal barriers. Factors such as particle surface charge and hydrophilic/hydrophobic balance of these polymeric materials have not been investigated systematically since adjustment of these particle properties is almost impossible without synthetic modification of the polymers. The current findings will be reviewed and compared to those obtained with nanoparticles consisting of a novel class of charged comb polyesters, poly(2-sulfobutyl-vinyl alcohol)-graft-poly(D,L-lactic-co-glycolic acid), SB-PVAL-g-PLGA, allowing adjustment of physicochemical nanoparticle properties with a single class of polymers.     

Harnessing the antibacterial and immunological properties of mucosal-associated invariant T cells in the development of novel oral vaccines against enteric infections

Enteric infections are a major cause of mortality and morbidity with significant social and economic implications worldwide and particularly in developing countries. An attractive approach to minimizing the impact of these diseases is via the development of oral vaccination strategies. However, oral vaccination is challenging due to the tolerogenic and hyporesponsive nature of antigen presenting cells resident in the gastrointestinal tract. The inclusion of adjuvants in oral vaccine formulations has the potential to overcome this challenge. To date no oral adjuvants have been licenced for human use and thus oral adjuvant discovery remains a key goal in improving the potential for oral vaccine development. Mucosal-associated invariant T (MAIT) cells are a recently discovered population of unconventional T cells characterized by an evolutionarily conserved αβ T cell receptor (TCR) that recognizes antigens presented by major histocompatibility complex (MHC) class I-related (MR1) molecule. MAIT cells are selected intra-thymically by MR1 expressing double positive thymocytes and enter the circulation with a naïve phenotype. In the circulation they develop a memory phenotype and are programmed to home to mucosal tissues and the liver. Once resident in these tissues, MAIT cells respond to bacterial and yeast infections through the production of chemokines and cytokines that aid in the induction of an adaptive immune response. Their abundance in the gastrointestinal tract and ability to promote adaptive immunity suggests that MAIT cell activators may represent attractive novel adjuvants for use in oral vaccination.     

Microparticles and polymers for the mucosal delivery of vaccines

Because microparticles are taken up across the gastrointestinal tract following oral administration, they may be exploited for the oral delivery of labile compounds. For example, microparticles have been used for the oral delivery of peptide and protein drugs, and have resulted in improvements in bioavailability. In addition, microparticles have also been exploited for the oral delivery of vaccines, inducing potent immune responses and protective immunity. However, the extent of uptake of microparticles across the gut may limit their potential for oral delivery. Therefore, intranasal immunization is an attractive approach for the induction of mucosal immunity. Microparticles have also been used for the delivery of vaccines to the respiratory tract. In addition to the use of small microparticles to target antigens to mucosal lymphoid tissues, polymeric coatings may also be employed simply to protect antigens against degradation during transit in the gut. Hence, oral delivery using polymers is not necessarily dependent on the uptake of the delivery system across the gut. Recent studies have indicated that a number of polymeric delivery systems possess significant potential for the development of mucosally administered vaccines. However, further work is needed in a number of areas, including the stabilization of antigens within the polymeric delivery systems.     

In vivo uptake of chitosan microparticles by murine Peyer's patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine and low uptake by the gut associated lymphoid tissue (GALT) still complicate the development of efficient oral vaccines. However, previous studies in our laboratory demonstrated that chitosan microparticles can have suitable size, charge, loading and release characteristics for oral vaccination using ovalbumin as model vaccine. In this study, two different approaches were used to investigate the in vivo uptake of chitosan microparticles by murine Peyer's patches. Firstly, a confocal laser scanning microscopy (CLSM) study was performed to visualize the uptake of fluorescent-labeled chitosan microparticles in the Peyer's patches after intragastrical feeding. Subsequently, the intestinal epithelial uptake of ovalbumin loaded chitosan microparticles was visualized using immunohistochemical staining of ovalbumin. Because the microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. CLSM visualization demonstrated that chitosan microparticles enhance the uptake of fluorescent-labeled ovalbumin by the epithelium of the Peyer's patches. No ovalbumin uptake by the intestinal epithelium was observed when the protein was administered without microparticles. Moreover, immunohistochemical visualization studies revealed that ovalbumin could only be transported into the Peyer's patches after association to chitosan microparticles. Since uptake by Peyer's patches is an essential step in oral vaccination, these in vivo experiments demonstrate that chitosan microparticles are very promising vaccine delivery systems.     

Delivery systems and adjuvants for oral vaccines

The oral route is the ideal means of delivering prophylactic and therapeutic vaccines, offering significant advantages over systemic delivery. Most notably, oral delivery is associated with simple administration and improved safety. In addition, unlike systemic immunisation, oral delivery can induce mucosal immune responses. However, the oral route of vaccine delivery is the most difficult because of the numerous barriers posed by the gastrointestinal tract. To facilitate effective immunisation with peptide and protein vaccines, antigens must be protected, uptake enhanced and the innate immune response activated. Numerous delivery systems and adjuvants have been evaluated for oral vaccine delivery, including live vectors, inert particles and bacterial toxins. Although developments in oral vaccines have been disappointing so far, in terms of the generation of products, the availability of a range of novel delivery systems offers much greater hope for the future development of improved oral vaccines.     

A Method for Oral DNA Delivery with N-Acetylated Chitosan

PURPOSE: The gastrointestinal tract poses a variety of morphological and physiological barriers to the expression of a target gene. In this work, N-acetylated chitosan is used as a gene delivery carrier for solving this problem. METHODS: Plasmid DNAs carrying the lacZ gene and interluekin-10 (IL-10) gene were mixed with N-acetylated chitosan. The N-acetylated chitosan/plasmid DNA complex was mixed into a food paste to feed mice. The transport and distribution characteristics of the plasmid along the intestinal mucosa were identified by beta-galactosidase assay. In addition, the stomach and intestines were subjected to analysis for the production of IL-10. RESULTS: The efficiency of N-acetylated chitosan-mediated gene delivery to the intestines was observed to be higher than that of chitosan alone. In particular, this result was most significant in the case of the duodenum, where the LacZ gene was expressed most effectively through the use of N-acetylated chitosan. It was also demonstrated that the IL-10 gene was successfully transferred to intestines through this method. CONCLUSIONS: A plasmid DNA was able to be orally delivered to the intestines using N-acetylated chitosan as a carrier. Thus, we have developed a dietary dose system for delivering a DNA vaccine for treating gastrointestinal diseases.