GM－CSF对人单核细胞HLA－DR抗原表达的影响

本文采用ＥＬＩＳＡ方法，研究了重组的人粒细胞一巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）对体外培养的正常人外周血单核细胞ＨＬＡ－ＤＲ抗原表达的影响。结果表明ＧＭ－ＣＳＦ能提高单核细胞ＨＬＡ－ＤＲ抗原表达，并且呈剂量依赖关系，最适剂量（５００ｕ／ｍｌ）时能使ＤＲ抗原表达量提高到对照组的２．５倍。ＧＭ－ＣＳＦ对ＨＬＡ－ＤＲ抗原表达的诱导作用并不是通过诱生的ＩＦＮ－γ的作用而实现的，因为有中和活性的ＩＦＮ－γ单抗并不能阻断这种效应。ＧＭ－ＣＳＦ能够增强低剂量ＩＦＮ－γ（５ｕ／ｍｌ）对单核细胞ＨＬＡ－ＤＲ抗原的诱导作用，但未观察到对高剂量ＩＦＮ－γ（１００ｕ／ｍｌ）的ＨＬＡ－ＤＲ抗原的诱导增强作用。     

GM－CSF对人单核细胞HLA－DR抗原表达的影响

本文采用ＥＬＩＳＡ方法，研究了重组的人粒细胞一巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）对体外培养的正常人外周血单核细胞ＨＬＡ－ＤＲ抗原表达的影响。结果表明ＧＭ－ＣＳＦ能提高单核细胞ＨＬＡ－ＤＲ抗原表达，并且呈剂量依赖关系，最适剂量（５００ｕ／ｍｌ）时能使ＤＲ抗原表达量提高到对照组的２．５倍。ＧＭ－ＣＳＦ对ＨＬＡ－ＤＲ抗原表达的诱导作用并不是通过诱生的ＩＦＮ－γ的作用而实现的，因为有中和活性的ＩＦＮ－γ单抗并不能阻断这种效应。ＧＭ－ＣＳＦ能够增强低剂量ＩＦＮ－γ（５ｕ／ｍｌ）对单核细胞ＨＬＡ－ＤＲ抗原的诱导作用，但未观察到对高剂量ＩＦＮ－γ（１００ｕ／ｍｌ）的ＨＬＡ－ＤＲ抗原的诱导增强作用。     

抗肾综合征出血热病毒人单克隆抗体杂交瘤细胞株的建立及初步鉴定

以亲和层析技术纯化的肾综合征出血热病毒（ＨＦＲＳＶ）结构蛋白（５５０００，６７０００）为特异性抗原，体外免疫正常人外周血淋巴细胞（ＰＢＬ），然后将体外免疫的淋巴细胞与人－鼠种间杂交瘤细胞（Ｋ６Ｈ６／Ｂ５）融合，经ＥＬＩＳＡ间接法筛选出２株（２Ｄ５，１Ｂ７）分泌抗－ＨＦＲＳＶ人单克隆抗体（Ｈ－ＭｃＡｂ）杂交瘤细胞株，４次克隆化后，１００％阳性。对２Ｄ５株初步鉴定表明，其抗体类型为ＩｇＧ１；培养上清中抗体浓度为２０～３０μｇ／ｍｌ；特异性免疫荧光反应证明２Ｄ５株Ｈ－ＭｃＡｂ是ＨＦＲＳＶ特异性；中和效价为１∶１６０；体外连续传代６个月仍稳定分泌抗体。     

α—干扰素对正常与甲亢甲状腺细胞生成c—AMP的调节作用

采用甲状腺细胞单层培养及标记免疫分析法，对比研究α－干扰素（ＩＦＮ－α）对正常与Ｇｒａｖｅｓ病（ＧＤ）甲状腺细胞生成ｃＡＭＰ的影响，结果显示：（１）基础状态下，１０＾－３、１０＾－１和１０ｕ／ｍｌ的ＩＦＮ－α可刺激正常甲状腺细胞产生ＣＡＭＰ，刺激强度随ＩＦＮ－α浓度的增大而减弱，当试验剂量达１０＾３ｕ／ｍｌ时，ＣＡＭＰ的生成量与无刺激的对照组比较无统计学差异，此浓度区间的ＩＦＮ－α对ＧＤ甲状腺细胞     

病毒性肝炎患者α干扰素中和抗体的发生率及其意义

目的：探讨α干扰素（ＩＦＮα）中和抗体产生的规律及其意义。方法：采用抗病毒中和生物测定法检测３２例健康人和１１６例ＩＦＮα治疗的慢性肝炎患者血清中的ＩＦＮα的中和抗体（ＮＡ）。结果：健康人及ＩＦＮα治疗前的患者均未检出ＮＡ。治疗后共２０例（１７２％）阳性，其中４例是在治疗后第２个月检出，２０例第６个月全部阳性，ＩＦＮ－α２ａ、ＩＦＮ－α２ｂ、ＩＦＮ－α１ｂ治疗组的ＮＡ阳性率分别为３４６％、１３２％、１１５％。ＮＡ阳性组、ＮＡ高滴度组的病毒清除率显著低于ＮＡ阴性组、ＮＡ低滴度组（Ｐ＜００１），相反病毒复发率在ＮＡ阳性组显著高于ＮＡ阴性组（Ｐ＜００５）。治疗前ＡＬＴ高水平组的ＮＡ阳性率显著低于ＡＬＴ低水平组（Ｐ＜００５），而前者的病毒阴转率显著高于后者（Ｐ＜００５）。结论：提示ＩＦＮα治疗后ＮＡ发生率较低，其产生有一定的时间规律。ＮＡ可影响治疗效果，对治疗前ＡＬＴ水平的了解有助于病例的选择     

HCMV感染对单核细胞HLA－DR表达的影响

本文采用细胞ＥＬＩＳＡ法，研究发现人巨细胞病毒（ＨＣＭＶ）感染对单核细胞ＨＬＡ－ＤＲ的影响。结果表明ＨＣＭＶ感染后１ｄ，单核细胞ＨＬＡ－ＤＲ表达显著增高（Ｐ＜０．０１），以后逐渐降低，ｄ５降至对照水平；ＩＦＮγ（５００Ｕ／ｍｌ）．ＴＮＦ（２５０Ｕ／ｍｌ）、ＩＬ－６（５００／ｍｌ）、ＩＬ－１（５００／ｍｌ）均能不同程度地刺激单核细胞ＨＬＡ－ＤＲ表达；ＨＣＭＶ感染后，细胞因子刺激ＨＬＡ－ＤＲ表达的水平在感染后ｄ５，较对照组均显著降低（Ｐ＜０．０１）；ＩＬ－１＋ＩＦＮ－γ及ＴＮＦ＋ＩＦＮ－γ在刺激单核细胞ＨＬＡ－ＤＲ表达时有协同作用；ＨＣＭＶ感染后，ＩＦＮ－γ＋ＩＬ－１及ＴＮＦ＋ＩＦＮ协同刺激单核细胞ＨＬＡ－ＤＲ表达水平较对照组显著降低（Ｐ＜０．０１）。结果提示：在ＨＣＭＶ感染引起免疫抑制过程中，其引起单核细胞ＨＬＡ－ＤＲ表达降低是一重要机制。     

HCMV感染对单核细胞HLA－DR表达的影响

本文采用细胞ＥＬＩＳＡ法，研究发现人巨细胞病毒（ＨＣＭＶ）感染对单核细胞ＨＬＡ－ＤＲ的影响。结果表明ＨＣＭＶ感染后１ｄ，单核细胞ＨＬＡ－ＤＲ表达显著增高（Ｐ＜０．０１），以后逐渐降低，ｄ５降至对照水平；ＩＦＮγ（５００Ｕ／ｍｌ）．ＴＮＦ（２５０Ｕ／ｍｌ）、ＩＬ－６（５００／ｍｌ）、ＩＬ－１（５００／ｍｌ）均能不同程度地刺激单核细胞ＨＬＡ－ＤＲ表达；ＨＣＭＶ感染后，细胞因子刺激ＨＬＡ－ＤＲ表达的水平在感染后ｄ５，较对照组均显著降低（Ｐ＜０．０１）；ＩＬ－１＋ＩＦＮ－γ及ＴＮＦ＋ＩＦＮ－γ在刺激单核细胞ＨＬＡ－ＤＲ表达时有协同作用；ＨＣＭＶ感染后，ＩＦＮ－γ＋ＩＬ－１及ＴＮＦ＋ＩＦＮ协同刺激单核细胞ＨＬＡ－ＤＲ表达水平较对照组显著降低（Ｐ＜０．０１）。结果提示：在ＨＣＭＶ感染引起免疫抑制过程中，其引起单核细胞ＨＬＡ－ＤＲ表达降低是一重要机制。     

干扰素对人胃癌细胞IAP—2表达的调节

应用基因重组干扰素－α和－γ体外诱导三侏人胃癌细胞系ＳＧＣ７９０１，ＭＧＣ８０３和ＭＫＮ４５，ＡＢＣ－ＣＥＬＩＳＡ法测定诱导组和对照细胞表面免疫抑制酸性蛋白Ⅱ型的表达量。在基础培养条件下，三株细胞表达ＩＡＰ－２均呈低水平。低浓度（＜１０００ｕ／ｍｌ）ＩＦＮ－α或－γ反使其ＩＡＰ－２表达量显著减少。这些结果提示：（１）ＩＦＮ－α和－γ可能对胃癌细胞ＩＡＰ－２表达呈双向调节作用；（２）癌细胞ＩＡＰ－２     

携带hIL-2cDNA的逆转录病毒载体的构建及其在人肝癌细胞中的特异表达研究

将人白细胞介素２（ｈＩＬ-２）ｃＤＮＡ克隆到逆转录病毒载体ＭＮＳＭ的ＰＬ位点　，分别构建了转录受ＳＶ４０早期启动子和人甲胎蛋白增强子调控的重组逆转录病毒载体ＭＮＳＩ和ＭＮＳＩＡ。用脂质体转染法将ＭＮＳＩ和ＭＮＳＩＡ分别转导ＰＡ３１７包装细胞，测质粒转染率为（５～２０）×１０~(－３)克隆／μｇＤＮＡ·１０~６细胞，病毒感染率为（５．４～４５０）×１０~４ＣＦＵ／ｍｌ。重组病毒在４μｇ／ｍｌ ｐｏｌｙｂｒｅｎｅ存在条件下感染人肝癌细胞、肾癌细胞和黑色素瘤细胞，Ｎｅｏ~Ｒ克隆经Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证明ｈＩＬ－２ｃＤＮＡ转入人肿瘤细胞并整合，　Ｒ　ＮＡ斑点杂交及ＩＬ－２活性表达分析证明，人甲胎蛋白增强子可促进异源启动子启动ｈＩＬ－２ｃＤ－ＮＡ在合成甲胎蛋白的人肝癌细胞中高效特异转录和表达。该研究对肝癌特异性免疫增强基因治疗有重要意义。     

细胞因子调节人肝癌细胞ICAM—1分子的表达及其对CD3AK杀伤功能…

应用基因重组人干扰素α（ＩＦＮ－α）、干扰素γ（ＩＦＮ－γ）和肿瘤坏死因子（ＴＮＦ）体外处理人肝癌细胞系Ｈ－７４０２，ＡＢＣ－ＣＥＬＩＳＡ法检测各处理组和对照组细胞间粘附分子－１（ＩＣＡＭ－１）的表达水平，用ＬＤＨ释放法观察ＣＤ３单克隆缺本活化的杀伤细胞对各处理条件下Ｈ－７４０２细胞的杀伤活性。     

Phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (PEDV) neutralizing epitopes

Seven-mer phage random peptide libraries were panned against 2C10, a monoclonal antibody that showed neutralizing activities against PEDV. Recombinant M13 phages displaying the peptides SHRLP(Y/Q)(P/V) or GPRPVTH on the g3p minor coat protein showed strong binding affinity with 2C10 (70% and 30% of recovered phages, respectively) after multiple panning. Sequence analysis suggested that these peptides are similar with (1368)GPRLQPY(1374) found at the carboxy-terminal of the S protein. In neutralization inhibition assays, the two peptide motifs and a 24-mer synthetic peptide corresponding to the C-terminal endodomain of PEDV S protein were observed to compete for the antigen binding site of 2C10, as demonstrated by the loss or reduction of neutralizing activity of the monoclonal antibody. This new finding suggests that the newly discovered peptide motifs mimic a neutralizing epitope PEDV.     

Antibodies with preselected specificities to protein regions evoked by immunization with free synthetic peptides: dose response to myoglobin antigenic sites reveal an optimum dose for each antigenic site

Previously, we have shown by radioimmune antibody binding studies that immunization with each of the five synthetic antigenic sites of sperm-whale myoglobin (Mb), in their free form (i.e. not coupled to a carrier), resulted in the formation of antibodies that bound specifically to Mb and exclusively to the peptide that was used in immunization. In the present series of studies we have immunized separate groups of Balb/cByJ mice with different amounts of each of the free synthetic antigenic sites so as to determine the optimum immunizing dose for eliciting serum antibodies that bind specifically to Mb. The synthetic peptides corresponded to: Site 1 (peptide 15-22), Site 2 (peptide 56-62), Site 3 (peptide 94-100), Site 4 (peptide 113-120, and Site 5 (peptide 145-151). The dose range of each of the antigenic sites used in immunization was from 6 micrograms to 100 micrograms. Radioimmune antibody binding studies indicated that there is an optimal immunizing dose for each of the five antigenic sites which was smaller than anticipated. The significance of these findings is discussed.     

A novel domain antibody rationally designed against TNF-alpha using variable region of human heavy chain antibody as scaffolds to display antagonistic peptides

Neutralizing of TNF-alpha has been proved effective in treatment of some autoimmune diseases, e.g. rheumatoid arthritis and Crohn's disease. Low molecular weight synthetic peptides can mimic the binding sites of TNF-alpha receptors and block the activity of TNF-alpha. In order to stabilize the conformation, increase the affinity and bioactivity, in this study, heavy chain variable region of human antibody was used as a scaffold to simultaneously display three peptides, which were designed on the interaction between TNF-alpha and it's neutralizing monoclonal antibody. On the basis of the structural character and physical-chemical property of the families of seven kinds of heavy chain variable regions (VH) in human antibodies, the fifth type of VH was screened as scaffold to display the antagonist peptide. Based on the computer-guided molecular design method, a novel domain antibody against TNF-alpha (named as ATD5) was designed as TNF-alpha antagonist. The theoretical study showed that ATD5 was more stable than displayed antagonist peptide. The binding activity with TNF-alpha was higher than free peptides. After expression and purification in Escherichia coli, ATD5 could bind directly with TNF-alpha and inhibit the binding of TNF-alpha to its two receptors, TNFR1 and TNFR2. ATD5 could also reduce the TNF-alpha-mediated cytotoxicity and inhibit TNF-alpha-mediated caspase activation on L929 cells in a dose dependent manner. The activity of ATD5 was significantly stronger than three peptides displayed by ATD5. This study provides a novel strategy for the development of new TNF-alpha inhibitors. This study demonstrates that it is possible to screen potential antagonists of TNF-alpha using in vitro analysis systems in combination with the computer-aided modeling method.     

Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot-and-mouth disease virus.

The multiple antigenic peptide (MAP) system for presenting epitopes to the immune system has been studied with an immunogenic foot-and-mouth disease virus (FMDV) peptide comprising amino acids 141-160 of protein VP1. Neutralizing antibody responses known to protect guinea-pigs against challenge infection were obtained with a single inoculation of 0.8-4 micrograms of peptide, presented as an octamer or a tetramer, whereas 20 micrograms of a dimer were required to evoke a similar level of antibody. A monomeric preparation did not elicit measurable levels of neutralizing antibody at doses up to 20 micrograms. The octameric MAP was also immunogenic using an aluminum hydroxide adjuvant. Antibodies elicited by the octameric, tetrameric and dimeric constructs differed qualitatively in their reaction with sequences within the 141-160 peptide. Those against the octamer reacted poorly with peptides within the 141-160 sequence, whereas those elicited by the tetramer and dimer reacted preferentially with the peptides covering the N-terminal region. The levels of neutralizing antibody obtained with the octamer and tetramer compare favourably with those obtained when the FMDV peptide is attached to carrier proteins but are lower than those obtained when it is presented as part of a peptide-hepatitis B virus core particle. Nevertheless, the ability to elicit protective levels of neutralizing antibody without the use of a carrier protein would be a distinct advantage in the development of synthetic peptide vaccines.     

Antibodies with Preselected Specificities to Protein Regions Evoked by Immunization with Free Synthetic Peptides: Dose Response to Myoglobin Antigenic Sites Reveal an Optimum Dose for Each Antigenic Site

Previously, we have shown by radioimmune antibody binding studies that immunization with each of the five synthetic antigenic sites of sperm-whale myoglobin (Mb), in their free form (i.e. not coupled to a carrier), resulted in the formation of antibodies that bound specifically to Mb and exclusively to the peptide that was used in immunization. In the present series of studies we have immunized separate groups of Balb/cByJ mice with different amounts of each of the free synthetic antigenic sites so as to determine the optimum immunizing dose for eliciting serum antibodies that bind specifically to Mb. The synthetic peptides corresponded to: Site 1 (peptide 15-22), Site 2 (peptide 56-62), Site 3 (peptide 94-100), Site 4 (peptide 113-120), and Site 5 (peptide 145-151). The dose range of each of the antigenic sites used in immunization was from 6 μg to 100 μg. Radioimmune antibody binding studies indicated that there is an optimal immunizing dose for each of the five antigenic sites which was smaller than anticipated. The significance of these findings is discussed.     

Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays

We describe a novel approach to detect antibodies to synthetic peptide antigens in solid-phase radioimmunoassays, using a multiple antigen peptide (MAP) system. The MAPs consist of multiple copies of peptides that are synthesized as single units on a branching lysyl matrix using a solid-phase peptide synthesis method. The efficacy of the MAP approach in solid-phase immunoassays was compared with the conventional approach using a monomeric peptide of the immunodominant epitope of the circumsporozoite proteins of two species of malaria. Two monomeric peptides with 12 and 17 residues were found to bind poorly to plastic surfaces at a concentration up to 30 micrograms/ml, and showed no immunoreactivity to specific polyclonal or monoclonal antibodies, while the corresponding MAP-containing peptides showed excellent binding capacity and immunoreactivity at a concentration of 0.11 microgram/ml. The immunoreactivity of MAP-containing peptides was also superior to that of monomeric peptides conjugated to a protein carrier. The effects of various arrangements of lysyl branching of MAP on antigenicity were also studied, and the optimal number for lysyl branching of MAP was found to be octameric. Thus, the MAP, by enhancing the coating capacity and the avidity of synthetic peptides, provides increased sensitivity and reliability for the use of synthetic peptide to study antigen-antibody interactions on solid surfaces.     

Monoclonal antipeptide antibodies recognize epitopes upon VP4 and VP7 of simian rotavirus SA11 in infected MA104 cells

Summary To study morphogenetic events of rotavirus SA11-infected MA104 cells with strictly defined reagents we produced monoclonal antibodies against synthetic peptides from both outer capsid proteins VP4 (aa residues 228–241: QNTRNIVPVSIVSR) and VP7 (aa residues 319–326: SAAFYYRV) of simian rotavirus SA11. Two of the selected monoclonal antibodies proved to be reactive with determinants of SA11-infected MA104 rhesus monkey kidney cells, with purified SA11 as well as with the particular peptides used for immunization. The anti-VP4 antibody had a demonstrable neutralizing titer of 200 (50% focus reduction) whereas the anti-VP7 MuMAb revealed no detectable neutralizing activity. In peptide-inhibition experiments, the corresponding peptide inhibited its MuMAb whereas the noncorresponding peptide had no effect on antibody binding to intracellular viral antigen. Localization of VP7 was preceded by VP4 as shown by immunofluorescence microscopy.     

Functional domains of human interferon gamma probed with antipeptide antibodies

Functional domains of biologically active polypeptide molecules can be sought by raising antibodies to synthetic peptides. Human interferon gamma (HuIFN gamma) was thus studied, using two peptides based on candidate regions representing amino acids 7-16 and 121-130 of the HuIFN gamma molecule. These were conjugated to bovine serum albumin prior to immunization of rabbits. High titres of antipeptide antibodies which recognized the synthetic peptides were elicited in all of the four rabbits injected. The antipeptide antibodies from one of the rabbits immunized with the C-terminal (121-130) peptide detected native HuIFN gamma at a concn as low as 300 IU/ml, but the antipeptide antibodies from the rabbit immunized with the N-terminal (7-16) peptide did not detect HuIFN gamma. The IFN gamma-reactive antipeptide antibodies (anti-121-130) did not neutralize the antiviral activity of HuIFN gamma in a cytoprotection assay. These data and other studies establish that the C-terminus of HuIFN gamma is not essential for full antiviral activity and indicate an application of antipeptide antibodies in the analysis of structure-function relationships of cytokine molecules.     

Synthetic peptide with antiproliferative activity: a short C-terminal fragment of the human interferon alpha-2 molecule.

The biological activity of six synthetic peptides of the 124-144 region of the human interferon alpha-2 (IFN alpha-2) molecule was studied. Peptides were examined for their ability to inhibit mitogen induced proliferation of human blood cells in vitro. Only the peptide corresponding to the amino acid sequence 124-138 (2438) possessed IFN-like antiproliferative activity. Other tested synthetic peptides did not affect cell proliferation in this experimental system. As with the native IFN alpha-2 molecule, the inhibitory effect of the peptide 2438 was dose-dependent. On simultaneous addition of peptide 2438, antiproliferative activity of IFN alpha-2 was enhanced. Direct cytotoxic effects of synthetic peptide 2438 were not revealed. These results suggest that a synthetic peptide corresponding to the 124-138-amino acid sequence of the human IFN alpha-2 molecule serves as a cytostatic agent.     

Multiepitope vaccines intensively increased levels of antibodies recognizing three neutralizing epitopes on human immunodeficiency virus-1 envelope protein

A few neutralizing antibodies against human immunodeficiency virus-1 (HIV-1) envelope proteins have been shown to be highly effective at neutralizing different strains in vitro, and exist at very low levels in the sera of HIV-1-infected individuals. Based on our hypothesis that epitope vaccination may be a novel strategy for inducing high levels of antibodies against HIV-1, we prepared multiepitope vaccines using three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope proteins. The PI [C-G-(ELDKWA-GPGRAFY)2-K] and PII (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) peptides were synthesized and conjugated to a carrier protein, bovine serum albumin (BSA). After vaccination, both the PI-BSA and PII-BSA multiepitope vaccines induced high levels of epitope-specific antibodies to the three neutralizing epitopes (antibody titre: 1 : 12,800-102,400). The recombinant glycoprotein 160 (rgp160) subunit vaccine induced strong antibody responses to rgp160, but only very weak epitope-specific antibody responses to the three epitopes. The epitope-specific antibodies were isolated from rabbit sera by single epitope-peptide-conjugated sepharose columns. A yield of 51 microg of epitope-specific antibodies/ml of serum (mean value) was obtained and identified to recognize these epitopes, while 0.35 microg of protein was isolated from 1 ml of pooled preserum by C-(ELDKWAG)4- or C-(RILAVERYLKD-G)2-K- and C-(GPGRAFY)4-sepharose columns. The levels of these epitope-specific antibodies induced in rabbits were much greater than 1 microg/ml, a level that is considered to confer long-term protection against some viruses. Moreover, these antibodies recognized the neutralizing epitopes on peptides and rgp41. Based on the fact that a very low level of ELDKWA epitope-specific antibodies exist in HIV-1-infected individuals, these results suggesting that synthetic epitope vaccines could induce high levels of multiepitope-specific neutralizing antibodies indicate a new strategy for developing an effective neutralizing antibody-based epitope/peptide vaccine against HIV-1.