中国流行株HIV-1Gag与IFN-α2b重组质粒的构建与实验免疫研究

目的探讨中国流行株 ＨＩＶ－ １Ｇａｇ与 ＩＦＮ－ α２ｂ共表达重组核酸疫苗质粒的免疫效果。方法以 核酸疫苗质粒ｐＩＲＥＳ１ｎｅｏ为表达载体,构建重组核酸疫苗质粒ｐＩＲＥＳ１－Ｇａｇ、ｐＩＲＥＳ１－Ｇａｇ－ＩＦＮ,通过间接免疫荧光 试验、ＤｏｔＥＬＩＳＡ检测Ｃａｇ／ｈＩＬ－２／ｈＩＬ－６基因的表达产物。另将此重组核酸疫苗质粒免疫ＢＡＬＢ／ｃ小鼠,进行淋巴细 胞转化试验、ＣＤ４＋、ＣＤ８＋Ｔ淋巴细胞数量测定、细胞毒性Ｔ淋巴细胞（ＣＴＬ）特异性杀伤作用检测及血清抗体检测。 结果构建的重组质粒转染ＢＨＫ细胞后可表达目的基因,免疫小鼠后可有效地刺激淋巴细胞增殖,诱导特异性 ＣＴＬ反应,当和 ＩＦＮ－α２ｂ共表达时免疫效果更加显著。结论与 Ｇａｇ蛋白共表达的 ＩＦＮ－ａ２ｂ能够进一步提高细胞 免疫与体液免疫水平,构建的重组质粒为ＨＩＶ－１ＤＮＡ疫苗的研究奠定了一定实验基础。     

《中国生物制品学杂志》2001年第14卷总目录

论 著 噬菌体展示短肽模拟腺病毒表位的研究（１） 腺病毒载体介导的ＨＳＶ－ｔｋ基因杀伤肿瘤细胞的研究（４） 荷瘤小鼠骨髓来源的树突状细胞诱导产生的抗肿瘤 免疫反应（７） 中国流行株ＨＩＶ－１　Ｇａｇ与ＩＦＮ－α２ｂ重组质粒的构 建与实验免疫研究（１０） 利用细菌／杆状病毒系统在昆虫细胞中表达ＨＩＶ－１ Ｇａｇ蛋白（１４） ＨＣＶ Ｃ区基因片段的克隆、表达及纯化（１７） 人角质细胞生长因子的克隆及表达（２１） 抗轮状病毒ＩｇＹ的研制（２４） 口服双歧杆菌、酷酸梭菌活菌制剂（肠康平）的药效作 用（２７） 应用ＰＣＲ…     

EHV-1_(CN)与IL-2基因共表达核酸疫苗诱导产生CTL的观察

目的 观察ＨＩＶ－１ＣＮ　ｇｐ１２０基因与ＩＬ－２基因共表达核酸疫苗质粒ｐＧＰＩＬ－２诱导产生ＣＴＬ。方法 脂质体介导共表达中国流行株ＨＩＶ－１Ｇａｇ－ｇｐ１２０基因与ＩＬ－２基因的核酸疫苗质粒ｐＧＩＬ－２转染ＢＨＫ－２１细 胞,以间接免疫光法鉴定其表达。取ｐＧＰＩＬ－２免疫鼠脾细胞,检测ｐＧＰＩＬ－２诱导的细胞毒性Ｔ细胞杀伤活性。 结果ｐＧＰＩＬ－２可有效地诱导ＣＩＬ的产生,并可杀伤ＨＩＶ－１ＣＮ嵌合基因转染的靶细胞。结论 为进一步设计中国流行株ＨＩＶ－１核酸疫苗提供了重要实验依据。     

肿节风注射液对荷瘤小鼠T淋巴细胞增殖活力及NK细胞活性的影响

目的研究肿节风注射液(ZJF)对荷瘤小鼠T淋巴细胞增殖活力及NK细胞活性的影响,探讨其抗肿瘤作用的机制。方法无菌取C57BL/6纯系小鼠脾脏,制成5×106个/ml的单个脾细胞悬液,MTT法检测不同浓度(50、25、12.5、6.25和3.13 mg/ml,以生药量计)的ZJF对正常小鼠T淋巴细胞增殖的影响。将近交系615小鼠经左腋皮下注射小鼠前胃癌Fc细胞(1×106个/ml,0.2 ml/只),复制荷瘤小鼠模型,并随机分成5组:ZJF低、中、高剂量组(给药剂量分别为2、10、20 mg/kg,以生药量计)、CTX组[0.3 g/(kg·3 d)]及阴性对照组(生理盐水0.1 ml/10 g),MTT法检测ZJF对荷瘤小鼠T淋巴细胞增殖及NK细胞活性的影响。结果 ZJF可显著促进正常小鼠T淋巴细胞的增殖(P<0.05),且呈剂量依赖性;ZJF低、中、高剂量组荷瘤小鼠T淋巴细胞的增殖活力明显高于阴性对照组(P<0.05),且呈剂量依赖性,ZJF中、高剂量组荷瘤小鼠NK细胞的活性明显高于阴性对照组(P<0.05)。结论 ZJF可促进正常小鼠与荷瘤小鼠T淋巴细胞的增殖活力,增强NK细胞活性,为其抗肿瘤作用机制的研究提供了实验依据。     

橡胶防老剂DFC－34的毒性研究

对防老剂ＤＦＣ－３４进行了急性毒性、小鼠骨髓嗜多染红细胞微核、精子畸变及Ａｍｅｓ４项试验。结果表明，防老剂ＤＦＣ－３４同低毒物质，小鼠骨髓嗜多染红细胞微核率和精子畸变率随着剂量的增加而升高，Ａｍｅｓ试验未发现有致突变作用。     

BCG-CpG-DNA免疫活性作用的初步研究

目的研究BCG-CpG-DNA的免疫学活性。方法通过淋巴细胞增殖、T细胞亚群分析、细胞因子产生、对NK细胞杀伤功能影响等试验,检测BCG-CpG-DNA的免疫活性。结果BCG-CpG-DNA能促进小鼠T、B淋巴细胞增殖,活化巨噬细胞分泌IL-12,并能增强ConA诱导IFN-γ产生,提高小鼠脾细胞中CD3+、CD4+、CD8+T细胞亚群的含量,同时增强小鼠NK细胞的杀伤活性。实验组与对照组所有结果差异均有显著意义(P<0.05)。结论BCG-CpG-DNA能活化抗原呈递细胞(APC),并具有较好的免疫活性。     

气化炉急冷室结渣原因探讨

本文探讨了输血引起的免疫抑制与肿瘤生长和转移之间的关系。实验结果表明：接受同种异品系血输注的小鼠，迟发型超敏反应和ＣｏｎＡ诱导的Ｔ淋巴细胞增殖反均应均明显下降，肿瘤的体积、重量和肺表面结节则明显增加。而同种同品系间的输血对受者Ｔ淋巴细胞功能及肿瘤的生长和转移无明显影响。提示，Ｈ－２不相容性输血对肿瘤局部生长和肺转移的促进作用，与Ｔ淋巴细胞活性有关。     

CD40与CD40L研究

ＣＤ4 0是肿瘤坏死因子 (ＴＮＦ)家族成员 ,可在多种细胞表达 ,包括各种抗原递呈细胞 (ＡＰＣ)、Ｂ细胞、成纤维细胞 ,上皮细胞、内皮细胞等。ＣＤ4 0配体(ＣＤ4 0Ｌ)是一相对分子质量 330 0 0ＩＩ型膜糖蛋白 ,也属ＴＮＦ家族 ,ＴＣＲ刺激下可在ＣＤ4 +Ｔ细胞上表达[1] 。最初研究显示ＣＤ4 0 ＣＤ4 0Ｌ的相互作用在胸腺依赖体液免疫反应中起重要作用 ,调节ＣＤ4 +Ｔ细胞和Ｂ细胞的同源化相互作用 ,对Ｂ细胞激活、分化及记忆的产生至关重要 ,同时对激活抗原递呈细胞也非常重要 ,是Ｔ细胞激活的关键[1] 。Ｌｅｆｒａｎｃｏｉｓ等的研究显示 …     

转基因树突状细胞激活细胞毒性T细胞特异性杀伤淋巴瘤的体外实验

目的 探讨转基因树突状细胞激活细胞毒性T细胞产生抗淋巴瘤的特异性细胞免疫反应。方法 采用人骨髓来源的髓系前体细胞,在人细胞因子IL-4、GM-CSF和INF-α诱导下,在体外生成大量树突状细胞。将制备好的含有IgVH1核酸质粒,用脂质体法转染树突状细胞。转染成功的树突状细胞与外周血T淋巴细胞共培养,激活特异性CTL细胞,与阳性表达IgVH1的人淋巴瘤Namalwa细胞反应,用3H-TdR掺入法观察CLLs对瘤细胞的特异性杀伤效应。结果 树突状细胞能够用脂质体方法转染IgVH1核酸质粒,并且有效递呈给外周血T细胞,对表达IgVH1的人淋巴瘤Namalwa细胞产生特异性免疫杀伤活性,与对照组相比差异有显著意义（P<0.01）。结论 体外诱导扩增的 DC能够转染 IgVH1核酸质粒,体外激活T淋巴细胞,产生特异性细胞毒效应。     

10，10’－二烃基－9，9’－联二吖啶烯的电荷转移光谱和氧化反应研究

研究了１０，１０’－二烃基－９，９’－联二吖啶烯的ＤＤＱ的电荷转移光谱，用两种方法计算出了它们的电离热（Ｉｐ）值，并研究了该系列化合物与ＤＤＱ、ＴＣＮＥ和ＣＡ的氧化反应，其氧化结果和用ＨＮＯ＿３氧化所得到的１０，１０’－烃基－９，９’－联二吖啶硝酸盐结果一致．在ＤＤＱ、ＴＣＮＥ和ＣＡ中，只有ＤＤＱ可以和该系列化合物形成ＣＴＣ，其原因是ＤＤＱ有弱的氧化能力而有强的络合能力．     

慢性粒细胞白血病树突状细胞对T细胞增殖作用的研究

目的　体外诱导慢性粒细胞白血病 (CML)患者外周血单个核细胞 (PBMCs)为树突状细胞 (DCs) ,并对其形态、表型及对T细胞刺激增殖作用进行研究。方法　利用GM CSF、IL 4、TNF α体外定向诱导生成树突状细胞 ,对所诱生的细胞进行细胞表型及形态检测 ,用D FISH方法检测所诱生DCs的白血病源性 ,应用MTT法检测所诱生的DCs刺激T细胞增殖的能力。结果　CML患者PBMCs在体外可诱导生成bcr/abl融合基因阳性的DCs(CMLDCs)。CMLDCs对自体T细胞有明显刺激增殖作用 ,而CML细胞无此作用。CMLDCs刺激同一异体T细胞增殖能力弱于正常DCs,但当培养体系中加入 30 0U/ml干扰素 α(IFN α)时可使其刺激能力接近正常DCs。结论　CMLDCs具有刺激自体及异体T细胞增殖的能力 ,但对异体T细胞的刺激作用弱于正常DCs,IFN α可提高CMLDCs刺激T细胞增殖能力     

Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen

Macrophages (MΦ) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and MΦs in immunity against Mycobacterium bovis Bacillus Calmette-Guérin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic MΦs compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in MΦs were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic MΦs were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic MΦs participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation.     

Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant,Petasites japonicus,via the Induction of Murine Dendritic Cell Maturation

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8⁺ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.     

T Lymphocyte Antigen 4-Modified Dendritic Cell Therapy for Asthmatic Mice Guided by the CCR7 Chemokine Receptor

The CD80/CD86-CD28 axis is a critical pathway for immuno-corrective therapy, and the cytotoxic T lymphocyte antigen 4 (CTLA4) is a promising immunosuppressor targeting the CD80/CD86-CD28 axis; however, its use for asthma therapy needs further optimization. A human CTLA4 fused with the IgCγ Fc (CTLA4Ig) and mouse CC chemokine receptor type7 (CCR7) coding sequences were inserted into a recombinant adenovirus (rAdV) vector to generate rAdV-CTLA4Ig and rAdV-CCR7. The naive dendritic cells (DCs) were infected with these rAdVs to ensure CCR7 and CTLA4Ig expression. The therapeutic effects of modified DCs were evaluated. rAdV-CTLA4Ig and rAdV-CCR7 infected DCs improved all asthma symptoms. Inflammatory cell infiltration and cytokine analysis showed that rAdV-CTLA4Ig and rAdV-CCR7-modified DC therapy reduced the number of eosinophils and lymphocyte and neutrophil infiltration in the lung. Interestingly, assessment of the humoral immunity showed that the IL-4 and IFNγ levels of the rAdV-CTLA4Ig and rAdV-CCR7-modified DC-treated mice decreased significantly and did not reverse the Th1/Th2 balance. DCs expressing CCR7 displayed guidance ability for DC migration, primarily for DCs in the inflammatory lung. Additionally, the rAdVs caused an inflammatory response by inducing DC differentiation, inflammatory cell infiltration and changes in cytokines; however, mice transplanted with rAdV-green fluorescent protein (GFP)-infected DCs displayed no asthma manifestations. In conclusion, CTLA4Ig-modified DCs exhibited a therapeutic effect on asthma, and CCR7 may guide DC homing. The combination of these two molecules may be a model for precision-guided immunotherapy.     

In vitro enhancement of dendritic cell-mediated anti-glioma immune response by graphene oxide

Malignant glioma has extremely poor prognosis despite combination treatments with surgery, radiation, and chemotherapy. Dendritic cell (DC)-based immunotherapy may potentially serve as an adjuvant treatment of glioma, but its efficacy generally needs further improvement. Here we explored whether graphene oxide (GO) nanosheets could modulate the DC-mediated anti-glioma immune response in vitro, using the T98G human glioma cell line as the study model. Pulsing DCs with a glioma peptide antigen (Ag) generated a limited anti-glioma response compared to un-pulsed DCs. Pulsing DCs with GO alone failed to produce obvious immune modulation effects. However, stimulating DCs with a mixture of GO and Ag (GO-Ag) significantly enhanced the anti-glioma immune reaction (p < 0.05). The secretion of interferon gamma (IFN-γ) by the lymphocytes was also markedly boosted by GO-Ag. Additionally, the anti-glioma immune response induced by GO-Ag appeared to be target-specific. Furthermore, at the concentration used in this study, GO exhibited a negligible effect on the viability of the DCs. These results suggested that GO might have potential utility for boosting a DC-mediated anti-glioma immune response.     

Review of Dendritic Cells,Their Role in Clinical Immunology,and Distribution in Various Animal Species

Dendritic cells (DCs) are cells derived from the hematopoietic stem cells (HSCs) of the bone marrow and form a widely distributed cellular system throughout the body. They are the most efficient, potent, and professional antigen-presenting cells (APCs) of the immune system, inducing and dispersing a primary immune response by the activation of naïve T-cells, and playing an important role in the induction and maintenance of immune tolerance under homeostatic conditions. Thus, this review has elucidated the general aspects of DCs as well as the current dynamic perspectives and distribution of DCs in humans and in various species of animals that includes mouse, rat, birds, dog, cat, horse, cattle, sheep, pig, and non-human primates. Besides the role that DCs play in immune response, they also play a pathogenic role in many diseases, thus becoming a target in disease prevention and treatment. In addition, its roles in clinical immunology have also been addressed, which include its involvement in transplantation, autoimmune disease, viral infections, cancer, and as a vaccine target. Therefore, based on the current knowledge and understanding of the important roles they play, DCs can be used in the future as a powerful tool for manipulating the immune system.     

Investigating T Cell Immunity in Cancer: Achievements and Prospects

T cells play a key role in tumour surveillance, both identifying and eliminating transformed cells. However, as tumours become established they form their own suppressive microenvironments capable of shutting down T cell function, and allowing tumours to persist and grow. To further understand the tumour microenvironment, including the interplay between different immune cells and their role in anti-tumour immune responses, a number of studies from mouse models to clinical trials have been performed. In this review, we examine mechanisms utilized by tumour cells to reduce their visibility to CD8⁺ Cytotoxic T lymphocytes (CTL), as well as therapeutic strategies trialled to overcome these tumour-evasion mechanisms. Next, we summarize recent advances in approaches to enhance CAR T cell activity and persistence over the past 10 years, including bispecific CAR T cell design and early evidence of efficacy. Lastly, we examine mechanisms of T cell infiltration and tumour regression, and discuss the strengths and weaknesses of different strategies to investigate T cell function in murine tumour models.     

Ficus carica Polysaccharides Promote the Maturation and Function of Dendritic Cells

Various polysaccharides purified from plants are considered to be biological response modifiers and have been shown to enhance immune responses. Ficus carica L. is a Chinese traditional plant and has been widely used in Asian countries for its anti-tumor properties. Ficus carica polysaccharides (FCPS), one of the most essential and effective components in Ficus carica L., have been considered to be a beneficial immunomodulator and may be used in immunotherapy. However, the immunologic mechanism of FCPS is still unclear. Dectin-1 is a non-toll-like pattern recognition receptor, predominately expressed on dendritic cells (DCs). Activation of DCs through dectin-1 signaling can lead to the maturation of DC, thus inducing both innate and adaptive immune responses against tumor development and microbial infection. In our study, we found that FCPS could effectively stimulate DCs, partially through the dectin-1/Syk pathway, and promote their maturation, as shown by the up-regulation of CD40, CD80, CD86, and major histocompatibility complex II (MHCII). FCPS also enhanced the production of cytokines by DCs, including IL-12, IFN-γ, IL-6, and IL-23. Moreover, FCPS-treated DCs showed an enhanced capability to stimulate T cells and promote T cell proliferation. Altogether, these results demonstrate that FCPS are able to activate and maturate DCs, thereby up-regulating the immunostimulatory capacity of DCs, which leads to enhanced T cell responses.     

Characterization and Differentiation of the Tumor Microenvironment (TME) of Orthotopic and Subcutaneously Grown Head and Neck Squamous Cell Carcinoma (HNSCC) in Immunocompetent Mice

For the development and evaluation of new head and neck squamous cell carcinoma (HNSCC) therapeutics, suitable, well-characterized animal models are needed. Thus, by analyzing orthotopic versus subcutaneous models of HNSCC in immunocompetent mice, we evaluated the existence of adenosine-related immunosuppressive B- and T lymphocyte populations within the tumor microenvironment (TME). Applying the SCC VII model for the induction of HNSCC in immunocompetent C3H/HeN mice, the cellular TME was characterized after tumor initiation over time by flow cytometry. The TME in orthotopic grown tumors revealed a larger population of tumor-infiltrating lymphocytes (TIL) with more B cells and CD4⁺ T cells than the subcutaneously grown tumors. Immune cell populations in the blood and bone marrow showed a rather distinct reaction toward tumor induction and tumor location compared to the spleen, lymph nodes, or thymus. In addition, large numbers of immunosuppressive B- and T cells were identified within the TME but also in secondary lymphoid organs, independently of the tumor initiation site. The altered immunogenic TME may influence the response to any treatment attempt. Moreover, when analyzing the TME and other lymphoid organs of tumor-bearing mice, we observed conditions reflecting largely those of patients suffering from HNSCC suggesting the C3H/HeN mouse model as a suitable tool for studies aiming to target immunosuppression to improve anti-cancer therapies.     

Polysaccharides from European Black Elderberry Extract Enhance Dendritic Cell Mediated T Cell Immune Responses

European black elderberry (Sambucus nigra L.) is a popular way to treat common colds or influenza infections. Mechanistically, this might be due to a direct antiviral effect or a stimulatory effect on the immune system of the host. Here, we evaluated the modulatory effects of black elderberry derived water extract (EC15) and its polysaccharide enriched fractions (CPS, BOUND, and UNBOUND) in comparison to a conventional alcoholic extract (EE25), regarding the phenotypical and functional properties of dendritic cells (DCs), which are essential cells to induce potent T cell responses. Interestingly, the water extract and its polysaccharide fractions potently induced DC maturation, while the ethanol extract did not. Moreover, the capacity to stimulate T cells by these matured DCs, as assessed using MLR assays, was statistically higher when induced by the water extracted fractions, compared to immature DCs. On the other hand, the ethanol extract EE25 did not induce T cell stimulation. Finally, the cytokine expression profiles of these DC—T cell cocultures were assessed and correlated well with increased T cell stimulation. Also, the expression of inflammatory cytokines, such as IL-6, TNF-α, and IFN-γ was highly increased in the presence of the elderberry water extract EC15, and the polysaccharide enriched CPS, BOUND, and UNBOUND fractions, but not by EE25. Thus, from these data, we conclude that the polysaccharides present in water-derived elderberry fractions induce potent immune-modulatory effects, which represents the basis for a strong immune-mediated response to viruses including influenza.