Immunogenic comparison for two different recombinant chimeric peptides (CP12 and CP22) containing one or two copies of three linear B cell epitopes from β-hCG subunit

To develop a superior chimeric peptide (CP) vaccine of human chorionic gonadotropin (hCG), two CP antigens (named CP12 and CP22) encoding one or two copies of three linear B cell epitopes from the β-hCG subunit and six foreign T cell epitopes, including two promiscuous TCEs from hepatitis B surface antigen and tetanus toxoid, were constructed and biosynthesized. The hCG CP12 and CP22 of 21 or 23 kDa, respectively, were expressed in Escherichia coli at the level of ∼1% of total cell proteins when inserted into thermo-inducible pBV221 expression vector. The purified CP12 and CP22 proteins with >95% relative homogeneity are immunogenic, and elicited antibodies against the β5, β9 and β8 BCEs of β-hCG in both rabbits and three different inbred strains of mice. A mouse uterine weight study in Balb/c mice demonstrated that the CP12 and CP22 antigens with an additional β5 neutralizing epitope enhanced the in vivo bio-neutralization capacity of the induced antibodies compared to the C-terminal immunogen of β-hCG. We propose that the biosynthesized CP22, possessing with two copies of three BCEs, represents a novel candidate antigen for an hCG contraceptive or tumor therapeutic vaccine.     

Population coverage analysis of T-Cell epitopes of Neisseria meningitidis serogroup B from Iron acquisition proteins for vaccine design

Although the concept of Reverse Vaccinology was first pioneered for sepsis and meningococcal meningitidis causing bacterium, Neisseria meningitides, no broadly effective vaccine against serogroup B meningococcal disease is yet available. In the present investigation, HLA distribution analysis was undertaken to select three most promiscuous T-cell epitopes out of ten computationally validated epitopes of Iron acquisition proteins from Neisseria MC58 by using the population coverage tool of Immune Epitope Database (IEDB). These epitopes have been determined on the basis of their binding ability with maximum number of HLA alleles along with highest population coverage rate values for all the geographical areas studied. The comparative population coverage analysis of moderately immunogenic and high immunogenic peptides suggests that the former may activate T-cell response in a fairly large proportion of people in most geographical areas, thus indicating their potential for development of epitope-based vaccine.     

甲型H1N1流感病毒感染不同免疫缺陷小鼠的比较

目的宿主免疫系统的功能状态在病毒的感染中起着至关重要的作用,本实验观察了不同免疫缺陷小鼠感染甲型H1N1流感病毒的差异。方法使用六个品系的近交系小鼠,经乙醚麻醉后进行滴鼻攻毒,分析其在病毒感染后存活率、体重变化和肺组织病理改变的异同。结果感染H1N1病毒的6种小鼠在观察的14d内,野生型的C57BL／6小鼠感染开始体重缓慢下降,感染后期有所回升,有半数存活;BALB／c小鼠和四种免疫缺陷品系小鼠感染病毒后体重随病情发展快速下降,死亡率均为100％。野生型C57BL／6小鼠感染初期为较弥漫的间质性肺炎,后期病变逐渐局限;BALB／c小鼠和四种免疫缺陷品系小鼠感染病毒后出现弥漫的中重度间质性肺炎,细支气管上皮有变性坏死,但炎症细胞明显少于C57BL／6小鼠。结论在甲型H1N1流感病毒的初次感染中固有免疫和特异性免疫分别在感染的初期和后期起主要作用,宿主免疫系统的功能状态影响着甲型H1N1病毒感染和预后。     

Caspase Work Model During Pathogen Infection

Caspases are an evolutionarily conserved family of aspartate-specific cystein-dependent proteases with essential functions in apoptosis and normally exist in cells as inactive proenzymes. In addition to the inflammatory caspases, the initiator and effector caspases have been shown to have an important role in regulating the immune response, but are involved in different ways. We give a brief introduction on the benefit of apoptosis on the clearance of invasive pathogens, and the caspase functions involved i...     

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.     

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.     

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c⁺), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45^hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.     

百令胶囊联合罗氟司特治疗老年支气管哮喘的效果及对免疫功能的影响

目的:研究百令胶囊联合罗氟司特治疗老年支气管哮喘的效果及对免疫功能的影响。方法:选择2018年3月~2019年3月我院第六派驻门诊部收治的110例老年支气管哮喘患者,随机分为两组,每组各55例。对照组患者口服罗氟司特片,每次500 mg,每天1次。观察组患者联合服用百令胶囊,每次5粒,每天3次。比较两组的临床控制率,治疗前后两组诱导痰中炎症因子、中性粒细胞、呼出气一氧化氮(fractional exhaled nitric oxide,FeNO)水平和免疫功能的变化。结果:治疗后,观察组的临床控制率为70.91%(39/55),明显高于对照组[40.00%(22/55)](P<0.05);两组的CD4+、CD4+/CD8+和CD3+均较治疗前明显升高,且观察组的CD4+、CD4+/CD8+和CD3+明显高于对照组(P<0.05);两组患者诱导痰中中性粒细胞绝对值、中性粒细胞百分比、白细胞介素-4(interleukin-4,IL-4)、FeNO、白细胞介素-17(interleukin-17,IL-17)均较治疗前明显降低(P<0.05),诱导痰中的干扰素-γ(Interferon gamma,IFN-γ)均较治疗前明显升高(P<0.05),且观察组诱导痰中的组中性粒细胞绝对值、中性粒细胞百分比、IL-4、FeNO、IL-17明显低于对照组(P<0.05),诱导痰中的IFN-γ均明显高于对照组(P<0.05)。结论:百令胶囊联合罗氟司特对老年支气管哮喘患者具有显著的疗效,其机制可能与抑制中性粒细胞的激活、调节T淋巴细胞亚群的平衡和抑制相关炎症因子的释放相关。     

小青龙汤加味联合盐酸左西替利嗪胶囊治疗鼻鼽的疗效及免疫功能探究

摘要 目的：探究小青龙汤加味联合盐酸左西替利嗪胶囊治疗鼻鼽的疗效及免疫功能。方法：选择2018年12月至2020年4月陕西省中医医院诊治的鼻鼽患者102例,按照随机的数字表法分为两组,研究组60例,给予小青龙汤加味联合盐酸左西替利嗪胶囊治疗;对照组62例,给予盐酸左西替利嗪胶囊治疗,两组均持续治疗28 d,对比两组治疗的有效率、治疗前后症候积分、免疫指标免疫球蛋白E（immunoglobulin E,IgE）、白介素（interleukin,IL）IL-4、IL-12、IL-17A、IL-35 及不良反应的情况。结果：治疗前,两组的症候积分比较无差异（P>0.05）,治疗28 d后,两组的症候积分均显著降低,且观察组显著低于对照组（P<0.05）。治疗后观察组的总有效率为91.67 %,显著高于对照组（75.81 %,P<0.05）。治疗前,两组的IgE、IL-4、IL-12、IL-17A、IL-35 的水平对比无差异（P>0.05）,治疗后28 d,两组IgE、IL-4、IL-17A水平均降低,IL-12、IL-35 的水平升高,且研究组均优于对照组（P<0.05）。治疗期间观察组不良反应的发生率为8.33 %,与对照组对比无差异（11.29 %,P>0.05）。结论：小青龙汤加味联合盐酸左西替利嗪胶囊治疗鼻鼽,能够显著改善患者的症候,提高疗效,改善患者的免疫功能,同时有助于矫正体内免疫功能失衡,系可能是治疗鼻鼽的机制,同时不会增加不良反应。     

奥沙利铂和氟尿嘧啶联合贝伐珠单抗对晚期胃癌患者免疫功能、肿瘤标志物和预后的影响

摘要 目的：探讨奥沙利铂和氟尿嘧啶联合贝伐珠单抗对晚期胃癌患者免疫功能、肿瘤标志物和预后的影响。方法：选取2014年3月到2017年5月期间我院收治的晚期胃癌患者62例,根据住院号单双数分为对照组和观察组,各31例。对照组给予奥沙利铂和氟尿嘧啶治疗,观察组在对照组的基础上联合贝伐珠单抗治疗,对比两组临床有效率、免疫功能、肿瘤标志物、不良反应和预后。结果：观察组治疗后的临床有效率较对照组高（P<0.05）。两组治疗后免疫球蛋白（Ig）A、IgG、CD4⁺/CD8⁺、CD4⁺、IgM均升高,且观察组高于对照组（P<0.05）,两组治疗后CD8⁺均降低,且观察组低于对照组（P<0.05）。两组治疗后癌胚抗原( CEA) 、糖类抗原199（CA199）、糖类抗原72-4（CA72-4）均降低,且观察组低于对照组（P<0.05）。观察组的不良反应总发生率较对照组低（P<0.05）。观察组复发率、癌症转移率低于对照组,且3年生存率高于对照组（P<0.05）。结论：奥沙利铂和氟尿嘧啶联合贝伐珠单抗治疗晚期胃癌患者,可有效缓解病情,提高免疫功能,同时还降低不良反应发生率,获得良好的预后。