可生物降解高分子乙型肝炎疫苗微球的免疫原性研究

目的　研究聚乳酸 聚乙二醇共聚物 [poly dl lactide poly(ethyleneglycol) ,PELA]包被汉逊酵母表达的HBsAg制备所得乙型肝炎PELA疫苗微球 (PELA·HBsAg)的免疫原性。方法　应用PELA·HBsAg皮下免疫BALB c小鼠 ,在体液免疫方面 ,采用固相放免法测定抗 HBs的水平 ,对抗 HBs中IgG的亚类进行了ELISA测定。在细胞免疫方面 ,于免疫后用3H TdR法测定单个核细胞 (MNC)对ConA、LPS和HBsAg的增殖反应性 ;小鼠迟发型超敏反应 (DTH)的发生程度 ;淋巴细胞亚群分析 ;检测HBsAg特异性CTL活性等评价细胞免疫。结果　①免疫 4周后 ,PELA·HBsAg与HBsAg混合 ,产生抗 HBs的滴度高于铝佐剂的HBsAg[Al(OH) 3 HBsAg]的 2倍多 ;②PELA·HBsAg与HBsAg混合以及PELA·HBsAg产生的IgG2a与IgG的比例显著高于与铝佐剂的HBsAg ;③PELA·HBsAg免疫 2 5d小鼠脾细胞的MNC体外经ConA、LPS刺激后SI最高 ,达到 133.5 9、4 7.4 8,显著高于Al(OH) 3 HBsAg组 (2 8.98、2 1.81)和HBsAg组 (34.95、14 .75 ) ,P <0 .0 5 ;④PELA·HBsAg免疫小鼠后对SRBC所诱导的DTH反应程度有显著的增强作用 (P <0 .0 5 )。⑤PELA·HBsAg与HBsAg混合后免疫小鼠的脾细胞对P815 HBs细胞的特异杀伤率显著高于其它组别 (P <0 .0 5 )。结论　以PELA为佐剂的HBsAg所     

不同种类乙型肝炎表面抗原诱导小鼠早期细胞免疫应答的比较

目的 比较不同表达系统来源的乙型肝炎(乙肝)表面抗原(HBsAg)免疫小鼠诱导早期脾淋巴细胞抗原特异性细胞免疫应答的特点,探讨影响乙肝疫苗保护效果的因素.方法 3种HBsAg(汉逊酵母、CHO细胞和血源)分别皮下接种不同组小鼠(BALB/c,H-2d),每只3μg,于免疫后4d分离脾单个核细胞(MNC),经细胞分选仪(MACs)分选后,获得纯度高于90％的CD4+和CD8+T细胞,应用ELISPOT测定MNC、CD4+和CD8+T细胞体外刺激后所产生的细胞因子IFN-γ、IL-2斑点数( SFC).结果 细胞分选后,汉逊抗原诱导CD8+T细胞分泌IFN-γ的水平和CD4+T细胞分泌IL-2水平均显著高于CHO抗原组(8/10、2/10,P=0.035;6/10、0/10,P=0.005).汉逊抗原组与血源抗原组CD8+T细胞经体外刺激诱导IFN-y全部阳转(10/10),但分泌水平上汉逊抗原组显著高于血源抗原组(t=2.479,P=0.035);汉逊抗原组诱导CD4+T细胞IFN-γ阳转率及分泌水平均显著高于血源抗原组(10/10、4/10,P=0.005;t=3.967,P=0.003).结论 乙肝抗原免疫小鼠4d即可诱导细胞免疫应答,且汉逊酵母抗原早期诱导抗原特异性IFN-γ、IL-2的能力显著高于CHO和血源抗原,与其临床考核母婴传播阻断HBV保护率显著优于CHO和血源乙肝疫苗相一致,为及时接种乙肝疫苗的必要性和高危新生儿选择接种乙肝疫苗的类型提供依据.     

不同来源乙肝表面抗原的细胞免疫学对比研究

目的　研究不同来源乙肝表面抗原诱导CD8 抗原特异性的细胞毒T淋巴细胞反应(CTL)的差异及T细胞产生细胞因子的能力 ,从而对各种来源乙型肝炎表面抗原 (HBsAg)的免疫原性有更充分的评价。方法　不同来源HBsAg分别免疫BALB c小鼠 ,于免疫后不同时间制备脾脏单个核细胞 (MNC) ,再以相应的HBsAg体外刺激 ,酶联免疫方法 (ELISA)测定细胞因子分泌水平 ;以Na2 5 1CrO4标记靶细胞 ,测定CTL反应活性 ;应用流式细胞仪分析T细胞亚群。结果　HM HBsAg免疫小鼠 10d后的脾细胞产生IFN γ水平最高 ,而在免疫后 2 0d和 2 5d ,IL 2的水平显著高于其它各组 ,CD3 分子脾淋巴细胞显著高于其它各实验组 (P <0 .0 5) ;plasma HBsAg免疫小鼠的脾淋巴细胞产生IFN γ和IL 2水平较低 ,但在免疫 2 5d对P815 HBs的杀伤性最强 ;HM HBsAg和SSC HBsAg免疫后CD4 CD8- 脾淋巴细胞比例显著增高 (P <0 .0 5) ;CD4- CD8 细胞所占百分比各组间及与空白对照组间差异无显著性 (P >0 .0 5)。结论　不同来源HBsAg诱导的细胞免疫反应类型和程度不同     

IL-3、IL-6联合转基因的基质细胞促进异基因小鼠骨髓移植后的免疫重建

探讨转染IL 3和IL 6基因的骨髓基质细胞系QXMSC1对异基因骨髓移植 (BMT )小鼠免疫功能重建的促进作用。用逆转录病毒载体将小鼠IL 3和IL 6基因转染到骨髓基质细胞系QXMSC1(H 2 d) ,分别建立基质细胞系QXMSC1IL 3、QXMSC1IL 6及QXMSC1IL 3/IL 6 ,供体BALB/c(H 2 d)小鼠骨髓去除T细胞 ,致死量照射 (9 0Gy)受体小鼠C5 7BL/ 6 (H 2 b)后 ,输入供体骨髓细胞 (1× 10 5/只 )的同时输入基质细胞QXMSC1IL 3/IL 6 (5× 10 5/只 )。在骨髓移植后 30d、 6 0d ,检测BMT小鼠脾细胞对LPS、ConA的反应强度、脾细胞中辅助性T淋巴细胞前体频数 (HTLp )、杀伤性T淋巴细胞前体频数 (CTLp )、抗体生成细胞 (PFC )的能力及对迟发型超敏反应 (DTH )的能力来评价BMT后免疫功能的恢复情况。异基因BMT并输入基质细胞系QXMSC1IL 3/IL 6可明显增强BMT后淋巴细胞对LPS、ConA反应强度 ,小鼠对异基因小鼠脾细胞DTH反应增强 ,脾淋巴细胞HTLp、CTLp及PFC数明显增加。基质细胞QXMSC1可作为有效的基因载体明显促进异基因骨髓移植小鼠免疫功能重建。联合转入IL 3和IL 6对BMT后免疫功能的恢复有协同作用     

西洋参茎叶总皂甙在小鼠体内外的免疫效应

本文研究了西洋参茎叶总皂甙(PNS)对小鼠免疫功能的影响,发现 PNS 能在体内外协同ConA 促进小鼠脾细胞增殖;协同 ConA 增强小鼠脾 T 细胞产生淋巴因子的能力;明显增强小鼠脾 NKC 活性;PNS 在体内还能增强 LPS 刺激小鼠脾细胞的增殖反应;增强小鼠对胸腺依赖性抗原(SRBC)的初次抗体应答能力.     

重组白细胞介素2促进小鼠胸腺细胞发育及辐射损伤小鼠免疫功能的恢复

经亚致死量照射的BALB/c小鼠胸腺内残留的胸腺干细胞经历与胚胎胸腺发育十分相似的过程,体内给予IL-2(6×10~5u/kg)可明显促进胸腺细胞由CD4~-CD8~-向CD4~+CD8~+再向CD4~+CD8~-/CD4~-CD8~-分化并明显促进胸腺细胞增殖和亚致死量照射小鼠脾细胞对ConA、LPS 的增殖能力,促进同种异型细胞的混合淋巴细胞反应(MLR)和绵羊红细胞(SRBC)的抗体形成细胞数(PFC)等多项免疫功能的恢复。     

腺病毒载体介导的CTLA4-Ig基因转移促进异基因小鼠皮肤移植耐受

目的　探讨CTLA4 Ig重组腺病毒 (AdCTLA4 Ig)促进“脾细胞 (spleencells ,SP) 环磷酰胺 (cyclophosphamide ,CP)”系统诱导移植耐受的作用及其机制。方法　BALB c(H 2 d)小鼠经尾静脉注射C57BL 6(H 2 b,B6)小鼠脾细胞和AdCTLA4 Ig颗粒 ,48h后腹腔注射环磷酰胺 ,并同天进行皮肤移植 ,于耐受 2 5d时对受体小鼠进行混合淋巴细胞反应 (mixedlymphocytereaction ,MLR)、迟发型超敏反应 (delayedtypehypersensitivity ,DTH)等耐受状态的检查。结果　B6小鼠的皮肤移植物在耐受的BALB c小鼠中存活期特异延长。MLR和DTH检验证明BALB c小鼠对B6小鼠的脾细胞产生特异性耐受 ,对无关第三者ICR小鼠的脾细胞仍表现出强烈免疫应答。结论　AdCTLA4 Ig颗粒可以明显促进“细胞 环磷酰胺”诱导的异基因皮肤移植耐受 ;克隆排除和克隆无能是耐受诱导的主要因素     

环磷酰胺免疫删减法制备细胞表面抗原单克隆抗体小鼠模型的建立

目的:观察不同剂量环磷酰胺(CP)诱导免疫耐受的作用, 建立一个免疫删减法制备细胞表面抗原单克隆抗体(mAb)的动物免疫模型.方法:雌性 BALB/c小鼠经CHO细胞免疫后分别给予不同剂量的CP诱导免疫耐受, 选择血清抗体效价最低的一组小鼠按常规方式腹腔注射CHO-TM5细胞进行免疫.ELISA测定各小鼠血清中CHO抗体及CHO-TM5抗体效价, 检测血清抗体对CHO细胞与CHO-TM5细胞结合反应的差异性.结果:BALB/c小鼠腹腔注射不同剂量的CP后, 各剂量组均出现不同程度的免疫耐受作用.CP 100 mg/kg组与CP 125 mg/kg组小鼠血清抗体效价较低, 接近阴性对照组.ELISA检测显示CP 100 mg/kg组血清抗体与CHO-TM5细胞呈阳性结合反应, 不与CHO细胞结合;而CP 0 mg/kg组血清抗体与CHO细胞与CHO-TM5细胞的结合反应均呈阳性.结论:CP 100 mg/kg具有良好诱导小鼠对CHO和CHO-TM5细胞的共同抗原表位产生免疫耐受, 相对增强对CHO-TM5细胞目的抗原hTM的免疫应答的作用, 为免疫删减法制备细胞表面抗原mAb提供了一个理想的动物免疫模型.     

抗MHC-Ⅰ类分子单抗促进异基因小鼠皮肤移植耐受

目的通过静脉注射异基因脾细胞和抗H-2b单抗加腹腔注射环磷酰胺诱导成年B6小鼠对异基因皮肤移植物产生免疫耐受.方法经C57BL/6(H-2b,B6)小鼠尾静脉注射BALB/c小鼠(H-2d)脾细胞,2 d后腹腔注射环磷酰胺(CP),随后2次尾静脉注射抗小鼠的H-2b单克隆抗体,然后进行皮肤移植,并于耐受30d对受体B6小鼠作混合淋巴细胞反应(mixed lymphocyte reaction,MLR)、迟发型超敏反应(delayed type hypersensitivity,DTH)等耐受状态检测.结果 BALB/c小鼠的皮肤移植物在耐受B6小鼠中存活期特异延长.MLR和DTH检验证明:B6小鼠对BALB/c小鼠的脾细胞产生特异性耐受,对无关第3者KM小鼠的脾细胞仍表现出强烈的免疫反应.结论抗MHC-Ⅰ类分子单克隆抗体可明显促进"细胞+CP"诱导的异基因皮肤移植耐受;克隆排除是诱导耐受的主要因素.     

小檗碱对DNFB诱导的小鼠迟发型超敏反应的影响

目的:研究小檗碱(Ber)对二硝基氟苯(DNFB)诱导的小鼠迟发型超敏反应(DTH)的影响,探讨其免疫抑制作用的机制。方法:BALB/c小鼠分为对照组、DTH组与Ber处理的DTH组。对照组以不含DNFB的溶剂处理,DTH组以5g/LDNFB腹部致敏、耳廓激发的方法建立模型,Ber处理的DTH组每天腹腔注射(i.p)Ber,连续7d,总剂量为30mg/kg体质量。激发后48h,取小鼠双耳称重了解Ber对炎症抑制率的影响。镜下观察耳廓局部组织的变化;以结晶紫法检查Ber对脾脏T淋巴细胞与细胞外基质(ECM)黏附作用的影响;以膜联蛋白V(AnnexinV)染色检查淋巴结细胞的凋亡率。结果:双耳称重的结果显示,Ber处理的DTH组与DTH组具有显著的差异(P<0.05),能明显抑制DNFB引起的炎症反应;耳廓局部组织学检测也表明,Ber能够抑制DNFB诱导的DTH,明显减少淋巴细胞的浸润。Ber处理的DTH组的T细胞与ECM的黏附能力明显降低(P<0.05)。Ber处理的DTH组与DTH组和对照组的细胞凋亡率均无明显差异(P>0.05)。结论:Ber对DNFB诱导的小鼠DTH具有明显的抑制作用,降低小鼠T细胞与ECM的黏附能力可能是其抑制DTH的机制之一。     

Host defense in cryptococcosis. I. An in vivo model for evaluating immune response.

An inbred mouse model was used to evaluate in vivo host immune response to Cryptococcus neoformans. Within 1 week of immunization, mice developed delayed type hypersensitivity reactions (DTH) to cryptococcal extracts derived from either culture filtrates or disrupted cells. There was no significant cross reactivity with extracts of other fungi. Previous immunization provided considerable protection against subsequent challenge with multiple strains of cryptococci. DTH also developed after nonimmunized mice were challenged with C. neoformans; however, in this situation DTH was not associated with prolonged survival. These studies indicate that mice can be immunized and protected against cryptococcosis and that this protection is associated with acquisition of DTH to cryptococcal antigens.     

Secondary delayed-type hypersensitivity to sheep red blood cells and minor histocompatibility antigens in mice: transfer of memory by recirculating thoracic duct lymphocytes

Secondary delayed-type hypersensitivity (DTH) in mice to sheep red blood cells (SRBC) and minor histocompatibility (H) antigens is dependent on long-lived memory T cells. In this paper we investigated whether these memory T cells recirculate. It was shown that late phase "immune' thoracic duct lymphocytes (TDL) from mice which were immunized with SRBC or non-H-2-incompatible spleen cells several weeks previously could adoptively transfer secondary DTH to these antigens. Passing the immune TDL through intermediate recipients demonstrated that these SRBC- or minor H-antigen-reactive memory T cells recirculate from blood to lymph. In contrast to mice immunized with minor H antigens, no secondary type DTH reactivity could be demonstrated in mice immunized with H-2-incompatible spleen cells. Also, after adoptive transfer of TDL from mice immunized with H-2-alloantigens, it was impossible to demonstrate an accelerated DTH reactivity.     

Essential pathogenic role for endogenous interferon-gamma (IFN-γ) during disease onset phase of murine experimental autoimmune orchitis. I. In vivo studies

We previously found that immunization of CH3/He male mice with syngeneic testicular germ cells (TGC) without the aid of any adjuvants was sufficient to induce DTH to TGC and experimental autoimmune orchitis (EAO). To evaluate the role of endogenous IFN-γ in this model, C3H/He mice immunized subcutaneously with TGC on days 0 and 14 received a single injection of anti-murine IFN-γ MoAb on day 15, 20 or 25. On day 45, DTH to TGC was tested, testis specimens were collected for histological examination, and blood samples collected for IFN-γ measurement. The results showed that whilst MoAb treatment on day 15 or 25 did not influence DTH responses, EAO development, and appearance of IFN-γ in the circulation, treatment on day 20 significantly suppressed all of them. Thus, a single injection with anti-IFN-γ MoAb may successfully down-regulate testicular autoimmunity, provided that the treatment is given at an optimal time point during disease development.     

Ⅰ型人类免疫缺陷病毒包膜gp120与乙型肝炎病毒表面抗原免疫原性比较

目的 比较Ⅰ型人类免疫缺陷病毒(HIV-1)及乙型肝炎病毒(HBV)包膜蛋白初次免疫及加强免疫后诱导产生抗体的规律,为提高HIV-1包膜蛋白诱导保护性抗体产生能力提供创新思路.方法 以10周龄雌性C57BL/6小鼠为动物模型,分别用HIV-1 06044毒株gp120三聚体(gp120T)、HBV表面抗原(HBsAg)蛋白与AddaVax佐剂免疫小鼠,背部皮下注射,共免疫3次,每次免疫间隔3周,第一、第二次免疫后7d和第三次免疫后3d、7d取血;第一次免疫后7d、第三次免疫后3d、7d取脾组织.用酶联免疫吸附实验(ELISA)及酶联免疫斑点实验(ELISpot)方法检测免疫小鼠血浆特异性结合抗体滴度及抗体分泌细胞(ASC)数量.结果 gp120T和HBsAg两种蛋白初次免疫后,动物均未产生明显的特异性抗体.两种蛋白加强免疫后特异性抗体水平明显升高,gp120T一次加强免疫及两次加强特异性抗体滴度逐渐升高,而HBsAg一次加强抗体滴度已经接近两次加强的水平.二次加强免疫后,gp120T和HBsAg免疫鼠脾脏特异性ASC数量差异不显著.结论 HIV-1包膜gp120T加强免疫诱导抗体水平达到高峰慢于HBsAg加强免疫,即加强免疫后gp120T诱导的回忆反应慢于HBsAg.     

The effects of Con A-induced lymphokines from the T-lymphocyte subpopulations on human monocyte leishmanicidal capacity and H2O2 production.

Immunization of BALB/c mice with measles virus inactivated with beta-propiolactone and mixed with 100 micrograms of the cationic surface-active lipid dimethyl dioctadecyl ammonium bromide (DDA) primes for a strong virus-specific delayed-type hypersensitivity (DTH) response that peaks 1 week later. Optimal immunization and challenge doses were found to be 8 and 4 micrograms/mouse, respectively, and pretreatment with 200 mg of cyclophosphamide/kg 2 days prior to immunization significantly enhanced the DTH response. When compared to Freund's complete and incomplete adjuvants, DDA was superior for induction of DTH to inactivated purified measles virus. As DDA could be administered to animals at a site different from the measles virus antigens, or 1 day previously, and still significantly enhance the DTH response, DDA is probably acting more as an immune modulator than as a simple adjuvant. The conditions for an optimal DTH response to measles virus were also shown to be applicable to other enveloped viruses, for example, a strong DTH response was similarly generated to inactivated purified influenza PR8 virus and to herpes simplex virus type I antigens present in plasma membranes isolated from infected Vero cells.     

Development of specific and cross-reactive lymphocyte proliferative responses during chronic immunizing infections with Rickettsia tsutsugamushi

The development of antigen-responsive lymphocytes was followed in mice immunized with the Gilliam, Karp, or Kato strains of Rickettsia tsutsugamushi by utilizing an in vitro lymphocyte proliferation assay. Subcutaneous immunization with viable rickettsiae of all three strains resulted in the appearance of lymphocytes in the spleen responding to irradiated tissue culture-grown rickettsiae used as stimulating antigens. Although all animals demonstrated antigen-induced proliferation elicited by homologous antigen by 14 days after immunization, the time of peak responsiveness varied, depending on the strain of rickettsiae used for immunization. In all cases, peak proliferative responses occurred at a time after immunization that was after the previously reported time after immunization at which resistance to rechallenge was observed. Reactivity to heterologous strains of R tsutsugamushi developed roughly in parallel with homologous reactivity in Karp- and Gilliam-immunized mice, with a marked degree of heterologous reactivity evident. Kato-immunized mice demonstrated greater reactivity to heterologous antigens early in the development of antigen reactivity and demonstrated a somewhat greater degree of cross-reactivity, relative to homologous responses, than the other groups. It was found that nylon wool-nonadherent immune cells, if cultured with antigen and adherent cells obtained from normal spleens or peritoneal exudates, responded in culture. The thymus-derived lymphocyte nature of the responding cell was further suggested when treatment of immune spleen cells with anti-Thy 1.2 serum and complement eliminated antigen response.     

Tumour-induced suppression of delayed-type hypersensitivity in the mouse: independence of cyclophosphamide-sensitive suppressor cells

Delayed-type hypersensitivity (DTH) responses to sheep red blood cells and ovalbumin were markedly reduced in mice receiving viable Landschütz ascites tumour cells at the time of immunization. Similar inhibitory effects were observed in control immunized animals when cell-free ascitic fluid or tumour bearer serum was administered together with antigen at the challenge site. Normal mouse serum exhibited much less pronounced inhibitory activity over the range of concentrations tested. High-dose cyclophosphamide (Cy) prior to immunization enhanced DTH responses to both antigens in normal mice. However, in animals also given tumour or soluble tumour-associated material, the immunosuppressive effect which had been observed in non-Cy-treated controls was preserved. It is concluded that the observed suppression of DTH is not dependent on Cy-sensitive T suppressor cells or on the presence of immunosuppressive factors during induction of the immune response.     

Antigen-Induced Protective and Nonprotective Cell-Mediated Immune Components against Cryptococcus neoformans

Mice immunized with two different cryptococcal antigen preparations, one a soluble culture filtrate antigen (CneF) in complete Freund’s adjuvant (CFA) and the other heat-killed Cryptococcus neoformans cells (HKC), develop two different profiles of activated T cells. CneF-CFA induces CD4⁺ T cells responsible for delayed-type hypersensitivity (DTH) reactivity and for amplification of the anticryptococcal DTH response, whereas HKC induce CD4⁺ and CD8⁺ T cells involved in anticryptococcal DTH reactivity and activated T cells which directly kill C. neoformans cells. The main purpose of this study was to assess the level of protection afforded by each of the two different T-cell profiles against challenge with viable C. neoformans cells, thereby identifying which activated T-cell profile provides better protection. CBA/J mice immunized with CneF-CFA had significantly better protective responses, based on better clearance of C. neoformans from tissues, on longer survival times, and on fewer and smaller lesions in the brain, than HKC-immunized mice or control mice similarly infected with C. neoformans. Both immunization protocols induced an anticryptococcal DTH response, but neither induced serum antibodies to glucuronoxylmannan, so the protection observed in the CneF-CFA immunized mice was due to the activated T-cell profile induced by that protocol. HKC-immunized mice, which displayed no greater protection than controls, did not have the amplifier cells. Based on our findings, we propose that the protective anticryptococcal T cells are the CD4⁺ T cells which have been shown to be responsible for DTH reactivity and/or the CD4⁺ T cells which amplify the DTH response and which have been previously shown to produce high levels of gamma interferon and interleukin 2. Our results imply that there are protective and nonprotective cell-mediated immune responses and highlight the complexity of the immune response to C. neoformans antigens.     

Delayed-Type Hypersensitivity after Immunization with Ultrasonicated Mycobacterium lepraemurium in C3H and C57BL Mice

Subcutaneous immunization with ultrasonicated Mycobacterium lepraemurium (MLMSon) in incomplete Freund's adjuvant (IFA) induced long-lasting skin reactivity with the kinetics of a tuberculin-type delayed hypersensitivity (DTH) reaction in both C3H and C57BL mice. The reactivity generally was stronger in C57BL than in C3H mice, and with increasing doses of MLMSon test antigen the local reaction increased more in C57BL than in C3H mice. Pretreatment of C3H mice with cyclophosphamide before immunization caused a shift in the dose-response curve so that the local reaction increased more with increasing doses of test antigen. Histological examination of the reaction elicited by MLMSon in immunized mice revealed a predominantly mononuclear cell infiltrate, and local reactivity could be transferred by immune cells but not by immune serum. The local reaction elicited by MLMSon exerted an adjuvant effect on the induction of DTH to sheep erythrocytes. Thus, MLMSon in IFA given subcutaneously induced stable DTH that conformed to the criteria for tuberculin-type DTH.