Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.     

树突状细胞对海藻酸钙纳米胶囊的吞噬作用与功能诱导

本研究采用量子点标记方法,证实了人外周血来源树突状细胞对海藻酸钙纳米胶囊的吞噬作用,并对其体外成熟诱导和接受偶联有牛血清白蛋白的纳米胶囊刺激之后的自体T淋巴细胞免疫作用进行了探讨。实验结果表明,树突状细胞在吞噬海藻酸钙纳米胶囊后被诱导成熟,而且偶联有牛血清白蛋白的纳米胶囊可诱导自体T淋巴细胞增殖,显示该海藻酸钙纳米胶囊可能成为具有载体功能的癌症细胞免疫治疗佐剂。     

Abdominal paracentesis drainage attenuates intestinal mucosal barrier damage through macrophage polarization in severe acute pancreatitis

Abdominal paracentesis drainage (APD), as an effective treatment of severe acute pancreatitis (SAP) in clinical settings, can ameliorate intestinal barrier damage and the overall severity of SAP. However, the mechanism underlying therapeutic effects of APD on damaged intestinal mucosal barrier during SAP is still unclear. Here, SAP was induced by injecting 5% Na-taurocholate retrograde into the biliopancreatic duct of rats to confirm the benefits of APD on enteral injury of SAP and further explore the possible mechanism. Abdominal catheter was placed after SAP was induced in APD group. As control group, the sham group received no operation except abdominal opening and closure. By comparing changes among control group, sham group, and APD group, APD treatment obviously lowered the intestinal damage and reduced the permeation of intestinal mucosal barrier, which was evidenced by intestinal H&E staining, enteral expression of tight junction proteins, intestinal apoptosis measurement and detection of serum diamine oxidase, intestinal fatty acid binding protein and D-lactic acid. Furthermore, we found that APD polarized intestinal macrophages toward M2 phenotype by the determination of immunofluorescence and western blotting, and this accounts for the benefits of APD for intestinal injury in SAP. Importantly, the protective effect against intestinal injury by APD treatment was mediated through the inhibited ASK1/JNK pathway. In summary, APD improved the intestinal mucosal barrier damage in rats with SAP through an increasing portion of M2 phenotype macrophages in intestine via inhibiting ASK1/JNK pathway.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

表面展示新城疫病毒部分F蛋白的重组干酪乳杆菌的构建及其免疫原性

本研究旨在构建重组干酪乳杆菌pLA-Newcastlediseasevirus (NDV)-F/Lactobacillus casei,获得表达产物,并探讨其免疫效果。利用PCR扩增携带部分主要抗原表位的NDV F基因,与穿梭质粒pLA连接转化至大肠杆菌BL21 (DE3)中,筛选阳性重组质粒,将其电转化至干酪乳杆菌中,构建重组干酪乳杆菌pLA-NDV-F/L. casei,应用PCR鉴定阳性菌株,Western blotting鉴定重组菌反应原性,间接免疫荧光、流式细胞术和激光共聚焦检测蛋白表达情况。试验选用14日龄雏鸡,各组免疫方式为口服+滴鼻。设立pLA-NDV-F/L. casei两次免疫组和三次免疫组、弱毒疫苗组、 pLA/L.casei、未攻毒PBS组和攻毒PBS组。间接ELISA方法检测雏鸡血清IgG、肠道、鼻腔、肺脏中sIgA抗体效价,评价试验组雏鸡攻毒保护率。结果表明,有94.10%的重组菌表达了F蛋白,且高效表达在干酪乳杆菌细胞表面,蛋白大小为62kDa,并能与抗NDV阳性血清特异性结合。各免疫组anti-F IgG和s Ig A抗体水平显著高于对照组,p LA-NDV-F/L. casei三次免疫组抗体持续时间比两次免疫组延长28 d,抗体峰值没有显著差异。免疫pLA-NDV-F/L. casei三次、两次、弱毒疫苗、pLA/L. casei和PBS的攻击保护率分别为80%、80%、90%、0%和0%。因此,利用干酪乳杆菌表达体系成功表达了携带部分抗原表位的NDVF基因,具备良好的反应原性和免疫原性,可诱导机体产生保护性免疫应答。     

DNA疫苗免疫佐剂的研究进展

DNA疫苗是最近几年从基因治疗研究领域发展起来的一种新型疫苗,它能诱导机体产生持久的体液免疫和细胞免疫应答,能够抗病毒,细菌和寄生虫的感染,对自身免疫性疾病和过敏性疾病有一定的疗效作用。但与传统的灭活疫苗相比,其免疫效价还比较低,最近的研究表明：联合使用DNA疫苗和疫苗佐剂如细胞因子,协同刺激分子等有助于提高DNA疫苗的免疫效价,这一发现有利于研制更有效的DNA疫苗,本文就通过使用免疫佐剂提高DNA免疫效价的最新进展做一综述。     

N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine

DNA vaccines consisted of tumor-associated antigen (TAA) are well suited for immunotherapy against tumor. The construct can contain TAA fused to an appropriate molecule (biologic adjuvant) to improve the efficacy of anti-tumor immune response. Heat shock protein 70 (HSP70) has been shown to be an excellent candidate, capable of cross-priming TAA by antigen presenting cells leading to a robust T-cell response. However, the relationship between strong T-cell responses and tumor rejection is not always mutually exclusive, for which TAA loss or activation of suppressive mechanisms may occur. HSP70 fused to downstream of Her2/neu as DNA vaccine has been shown to be efficient against Her2-expressing tumors. In this study, we examined if N-terminally fusion of Her2/neu to HSP70 could also improve efficiency of Her2/neu DNA vaccine. Therefore, mice with an established Her2/neu expressing tumor were immunized with DNA vaccine consisting of extracellular and trans-membrane domain (EC+TM) of rat Her2/neu alone or N-terminally fused to HSP70 and immune response was evaluated. Administration of rat Her2/neu led to partial control of tumor progression. Surprisingly, fusion of HSP70 to N-terminal of rat Her2/neu led to tumor progression. Our result proposes that fusion direction of biologic adjuvant is an important consideration when Her2/neu is used.     

CpG对乙型肝炎基因重组(CHO细胞)疫苗免疫效果的影响

为了研究CpG-寡脱氧核苷酸(CpG-OPN)作为佐剂对乙型肝炎基因重组(CHO细胞)疫苗(简称乙肝疫苗)免疫效果的影响,以乙肝疫苗加Al(OH)3、疫苗加CpG和疫苗加Al(OH)3与CpG3三种配伍方式,通过腹腔、皮下或肌内3种不同途径免疫Balb/c小鼠,观察不同免疫途径和不同配伍的免疫效果.同时又将疫苗与CpG混合后在4℃存放6个月再免疫小鼠,观察CpG的稳定性.结果表明:①3种免疫途径中以肌内注射效果最好,这在使用CpG的实验组尤为明显,在该组肌内免疫的ED50比腹腔的低了10倍,而诱发的抗体滴度提高了3倍;②疫苗与CpG、Al(OH)3联合使用的免疫效果最好,在肌内免疫时联合使用的免疫效果比疫苗+Al(OH)3提高4倍,比疫苗+CpG提高7倍;③疫苗+Al(OH)3免疫时,表现为IgG1抗体亚型占优势,而再加入CpG后则IgG1和IgG2a均升高,以IgG2a最显著;④疫苗与CpG混合后4℃保存半年,不影响其活性.     

肿瘤DNA疫苗佐剂研究进展

对近年来在增加肿瘤DNA疫苗免疫原性、提高肿瘤DNA疫苗效力方面所取得的进展予以综述。简要阐述了以下几种较有效的增强肿瘤DNA疫苗效力策略的机制和进展：（1）以细胞因子表达质粒为佐剂;（2）以质粒编码的趋化因子、协同刺激分子、共刺激分子、补体为佐剂;（3）以CPGODN为佐剂;（4）其他一些佐剂。     

Aberrant,ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy.