大鼠抗原诱导性葡萄膜视网膜炎实验研究

目的：观察Ｗistar大鼠抗原诱导性葡萄膜视网膜炎（antigen-induced uveoretinitis，ＡＩＵ）的临床、组织学和兔疫学变化。方法：使用牛Ｓ抗原和福氏完全佐剂的混合乳剂免疫大鼠１４d后，将Ｓ抗原接种于玻璃体腔，复制ＡＩＵ动物模型。观察其眼部临床表现、组织学改变、血清抗体效价、淋巴细胞增殖反应以及抗原特异性迟发型超敏反应（delayed - type hypersensitivity,ＤＴＨ）。结果：ＡＩＵ表现以眼后节段为主的全葡萄膜炎，平均     

淋球菌重组与减毒鼠伤寒沙门氏菌活疫苗口服免疫的初步研究

在克隆淋球菌膜抗原CppB基因并在大肠杆菌和减毒鼠伤寒沙门氏菌中获得高效表达的基础上,用表达CppB-LacZ融合蛋白的重组减毒鼠伤寒沙门氏菌口服免疫Balb/c小鼠。ELISA检测表明被免疫小鼠血清中特异性抗体滴度可达1:160,并诱发了DTH反应,说明产生了特异的免疫反应。重组菌可在肠道中较持久地寄生,对小鼠未见有明显的毒副反应。     

新疆荒漠肉苁蓉粗多糖配伍流感病毒疫苗的抗原节约作用

目的探究新疆荒漠肉苁蓉粗多糖(crude polysaccharides fromCistanche deserticola Y.C.Ma, CPCD)配伍流感病毒疫苗(influenza virus vaccine, IVV)的抗原节约作用。方法一定剂量的CPCD配伍低剂量(0.01 μg)或高剂量(0.1 μg)的IVV皮下途径免疫小鼠, 血凝抑制(hemagglutinin inhibition, HI)试验检测血清中HI抗体滴度;间接ELISA法检测血清中特异性抗体及亚型的水平;MTT法检测脾脏淋巴细胞增殖水平;流式细胞术检测脾脏细胞中T淋巴细胞亚群及胞内细胞因子IFN-γ的表达水平。结果 CPCD可以显著增强血清中HI抗体滴度(234.67±47.70vs 149.33±47.70,P<0.05), 促进特异性IgG(A450值:1.16±0.63vs 0.30±0.21,P<0.05)和IgG1(A450值:1.09±0.60vs 0.26±0.21,P<0.05)抗体水平的提高, 并促进脾脏淋巴细胞增殖(P<0.05)。CPCD还可显著提高CD4+...     

表达HPV6b L1-E7嵌合蛋白的减毒沙门菌诱导小鼠免疫应答

目的　探讨表达人乳头状瘤病毒 6b型 (HPV6b)L1 E7嵌合蛋白的减毒沙门菌诱导小鼠粘膜及系统免疫反应的可行性。方法　对重组减毒沙门菌S .BRD5 0 9 pTETnir15 6bL1E7体外厌氧诱导其表达 ,Westernblot方法鉴定表达的L1 E7蛋白。此重组沙门菌经滴鼻和口服联合粘膜免疫BALB c小鼠 ,采用ELISA方法及足垫肿胀试验分别检测免疫小鼠血清及阴道冲洗液中特异性抗体和DTH反应。结果　Westernblot分析显示 ,重组沙门菌在预计相对分子质量 (Mr) 5 6× 10 3 处有特异染色带 ,提示此重组沙门菌能表达特异的HPV6bL1 E7蛋白。ELISA检测结果表明实验组小鼠阴道冲洗液中HPV6bL1特异性sIgA显著升高 ,和对照组比较两者差异有显著性 (P <0 .0 1) ,但小鼠血清中HPV6bL1IgG抗体无明显变化 ,实验组和对照组两者差异无显著性。DTH结果显示 :与对照组比较 ,实验组小鼠接受HPV6bL1 E7抗原刺激后产生明显阳性反应 ,提示重组沙门菌免疫小鼠产生了针对HPV6bL1 E7抗原的特异性细胞免疫反应。结论　构建的重组减毒沙门菌S .BRD5 0 9 pTETnir15 6bL1E7可激发有效的粘膜免疫及细胞免疫反应 ,为进一步研制新型HPV粘膜疫苗奠定了实验基础。     

淋巴细胞中NF-κB p65活性水平在Fx1A诱导的口服耐受大鼠中的变化

目的：探讨核因子-κB(NF-κB)p65在口服耐受发生中的活性改变及其意义。方法：雌性Wistar大鼠30只随机分成两组：①口服耐受组，用小剂量(每次8mg／2mL PBS)的Fx1A抗原灌胃。②对照组，用PBS2mL／次灌胃。观察大鼠迟发型超敏反应(DTH)，进行脾淋巴细胞增殖试验，了解大鼠免疫功能的改变。以免疫组化和ELISA法，检测大鼠淋巴组织中NF-κB p65的活性及TGF-β1的表达。结果：与对照组相比较，口服耐受组大鼠DTH和抗原特异性淋巴细胞增殖反应明显受抑制；肠黏膜Peyer’s淋巴结(PP结)中NF-κB p6S的活性及TGF-β1的表达明显增加。脾淋巴组织中NF-κB p65的活性降低，但TGF-B1的表达明显增加。结论：低剂量Fx1A抗原诱导的口服耐受的发生，可能与不同部位免疫细胞内NF-κB p65的活性改变有关。     

抗SARS-CoV-2相关线性表位抗体诱发小鼠神经炎性反应

目的 评价已知的SARS-CoV-2刺突蛋白线性抗原表位相关非病毒中和抗体的潜在生物学功能。方法 根据报道优选出COVID-19患者中响应频率最高、抗体存留时间最长的线性抗原表位S2-78为研究对象。用血蓝蛋白偶联的S2-78多肽(KLH-S2-78)免疫小鼠后，对其血清中的anti-S2-78 IgG抗体水平、小鼠行为学表型和大脑中小胶质细胞数量密度进行检测。结果 KLH-S2-78免疫小鼠外周血中出现高滴度的anti-S2-78 IgG抗体(最高滴度为25 600,A₄₅₀=0.305 5),呈现为感觉运动门控缺陷、嗅觉功能受损和自发活动能力受损的精神病样行为学表型，大脑前额叶皮质和海马体中小胶质细胞数量密度显著增加(P<0.05),处于中枢炎性反应应激状态。结论 Anti-S2-78 IgG抗体有可能通过激发机体的炎性反应导致大脑神经炎性损伤。     

IL-18基因佐剂协同HSV-1 gB DNA疫苗免疫诱导的特异性免疫应答

目的研究IL-18 cDNA协同单纯疱疹病毒Ⅰ型（HSV-1）糖蛋白B（gB）核酸疫苗免疫对机体体液免疫和细胞免疫应答的影响。方法利用pcDNA3载体分别构建HSV-1gB和IL-18的真核表达质粒pgB和pIL-18,分pcDNA3、pgB和pgB＋pIL-18三组,肌肉注射免疫接种BALB/c小鼠3次,每次间隔2周,每次接种质粒100μg,第3次免疫后2周,用ELISA检测特异性抗体滴度;利用^3H-TdR掺入法进行T细胞特异性抗原刺激实验;耳廓肿胀实验检测迟发型超敏（DTH）反应。结果与pgB单独免疫组相比,pIL-18的协同免疫可以显著增强ELISA特异性抗体滴度、T细胞增殖反应和DTH反应。结论IL-18cD-NA的协同免疫可以显著提高pgBDNA疫苗诱导的体液免疫和细胞免疫水平,增强核酸疫苗对机体的免疫保护作用。     

Sm抗原纯化及ELISA检测Sm抗体的初步研究

从小牛胸腺中盐析出来的Sm/RNP提取物经DEAE Bio-Ge1 A层析即为Sm抗原。在PAGE检测时Sm抗原在BSA之前有几条染色带。 血清学研究显示,Sm抗原仅与Sm阳性和Sm/RNP阳性血清发生反应,不与RNP阳性血清发生反应。经Sm抗原吸收后,Sm阳性和Sm/RNP阳性血清与Sm抗原的反应降低至接近正常人水平,而上述血清与RNP抗原的反应设有明显降低。结果表明,Sm与RNP已完全分开。ELISA方法检测Sm抗体有10/18的SLE和2/4MCTD是阳性,在14例RA、硬皮病和其它疾病中为阴性。     

人特异性T细胞系间接识别猪抗原

SLA (swineleukocyteantigen )特异性CD4+T细胞系和自身APC以及猪PBMC共同孵育时 ,T细胞增殖反应很强 ,而同仅有自身APC或猪PBMC刺激时的T细胞增殖有显著差异。提示抗原特异性CD4+T细胞系以间接途径识别SLA抗原。该识别能被抗人CD4和HLAII类抗体阻断 ,而抗SLAII类抗体阻断作用很弱 ,表明HLAII类分子在间接识别中起重要作用 ,这一结果不同于直接从外周血中分离的未经抗原致敏的T细胞。直接识别猪APC递呈的抗原。有可能异种移植初期以直接识别为主导 ,随着移植物存活时间的延长逐渐转为间接识别     

幽门螺杆菌全菌抗原口服免疫BLAB／c小鼠的免疫应答机理研究

目的 分析幽门螺杆菌全菌抗原的口服免疫应答反应。方法 ELISA分析免疫小鼠血清、唾液、粪便提取物特异抗体水平,ELISPOT分析胃粘膜、派伊尔小结（PP）抗原行异性抗体分泌细胞（ASC）,RT－PCR分析PP T细胞因子mRNA表达水平。结果 ①口服免疫可诱导强烈的血清IgG反应和唾液、粪便提取物特sIgA反应;②胃粘膜、PP结产生大量抗原特异性抗体分泌细胞（ASC）,尤以sIgA-ASC型居多,PP结抗原特异性形成细胞（ASC）数量与特异抗体水平密切相关;③加佐剂免疫组小鼠PP T细胞,体外抗原刺激下,早期高表达IFN－γ晚期高表达IL－4。结论 全菌抗原和粘膜佐剂免疫可诱导H.pylori特异的系统、粘膜免疫应答,局部sIgA可能在抗H.pylori感染中具有重要作用,肠粘膜免疫主要诱导部位PP早期表现为TH1型优势应答,晚期则转为TH2型优势应答。     

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.     

Oral administration of bovine whey proteins to mice elicits opposing immunoregulatory responses and is adjuvant dependent

Most studies investigating the induction of oral tolerance (OT) use purified proteins such as ovalbumin (OVA), bovine serum albumin (BSA) and beta-lactoglobulin (beta-LG). Little information is available regarding the induction of OT to a protein mixture, e.g. cow's milk. In this study we compared the regulatory mechanisms induced after the oral administration of a whey protein concentrate (WP) derived from cow's milk following immunization with two different adjuvants, complete Freund's adjuvant (CFA) and alum. OVA was used as a control antigen. Animals were given a single feed of these proteins at an equivalent dose of 1 mg/g body weight before they were immunized seven days later with the antigen in Freund's adjuvant or alum. Delayed type hypersensitivity (DTH) responses were suppressed by both a feed of WP and OVA after immunization with CFA. However, only OVA feeding suppressed antigen specific IgG responses. In an attempt to investigate whether WP would tolerize the more susceptible IgE responses, alum immunization replaced CFA as the adjuvant used for systemic immunizations. WP, after a single feed, significantly primed for DTH and IgE responses indicating oral sensitization to WP. In contrast, OVA suppressed DTH, IgE and IgG responses. Antigen specific proliferation of mononuclear cells was suppressed in mice fed OVA, but primed in those fed with WP. In addition cells taken from sensitized mice fed WP up-regulated levels of specific interleukin (IL) -4, -10 and -12 in vitro whereas these cytokines were suppressed in cultures from tolerant WP fed mice. Global suppression was obtained in cultures from tolerant OVA fed mice. TGF-beta was not detected in draining PLN cell cultures of either tolerant or sensitized mice. These data suggest that a whey protein mixture induces divergent responses following immunization with either CFA or alum despite being fed at an identical dose. We suggest that that the choice of the adjuvant may determine the immunoregulatory outcome and this is also reflected by the systemic cytokine profile.     

大鼠经鼻腔乙酰胆碱受体耐受诱导的试验研究

用乙酰胆碱受体（ＡＣｈＲ）加完全弗氏佐剂（ＣＦＡ）免疫大鼠前，鼻腔给予ＡＣｈＲ可有效地预防实验性自身免疫性重症肌无力（ＥＡＭＣ）。本文结果表明，与非耐受对照组比，鼻腔ＡＣｈＲ耐受组大鼠免疫后，国窝、腹股沟淋巴结（ＰＩＬＮ）的单个核细胞（ＭＮＣ）ＡＣｈＲ特异的淋巴细胞增殖反应受到明显抑　制，鼻腔耐受组ＰＩＬＮ中ＡＣｈＲ特异的ＣＤ４~＋／ＣＤ８~－克隆增生也明显受抑制，差异均有显著性。此外，鼻腔耐受组ＡＣｈＲ特异性皮肤迟发型过敏反应（ＤＴＨ）也显著降低。提示鼻腔ＡＣｈＲ耐受后引起的特异性Ｔ淋巴细胞反应的低调可能是临床肌无力抑制的基础。     

Suppression of hen egg lysozyme-induced arthritis by intravenous antigen administration: no role in this for antigen-driven bystander suppression.

The induction of tolerance, particularly by intervention before established immunity, is widely accepted. We studied the effects of intravenous (i.v.) administration of hen egg lysozyme (HEL), before as well as after immunization, on a HEL-induced arthritis. Arthritis and also cartilage destruction were almost completely suppressed when 100 micrograms HEL was injected before immunization. Antigen-specific proliferative T cell responses and IL-2 production in vitro were inhibited. Antigen-specific immunoglobulin and IgG1 titres were equal in control and tolerized mice, in contrast to lowered IgG2a titres in tolerized animals. Detailed histological studies showed that the immune complex-dependent polymorphonuclear cell phase (< 24 h after arthritis induction) was equal for control and HEL-injected mice. Only in the T cell-dependent phase of the arthritis (> 24 h), did suppression become pronounced in tolerized mice. I.v. administration of 100 micrograms HEL after immunization could only marginally reduce infiltrate and exudate, and no reduction of cartilage destruction was seen. An elegant way to interfere in an established immunity can be offered by creation of bystander suppression. We show that i.v. administration of HEL followed by triggering with HEL, at the moment either of immunization or of arthritis induction, does not reduce a methylated bovine serum albumin (BSA)-arthritis. We conclude that arthritis can be suppressed almost totally when HEL is injected intravenously before immunization. Treatment after immunization is less effective. The i.v. induced suppression is T cell-mediated and and antigen-specific: no bystander suppression circuit can be generated.     

Suppression of adjuvant arthritis in rats by induction of oral tolerance to mycobacterial 65-kDa heat shock protein

Oral administration of mycobacterial 65-kDa heat shock protein (HSP) given daily for 5 days prior to immunization with Mycobacterium tuberculosis (Mt) suppressed the development of adjuvant arthritis (AA) in rats. AA was significantly suppressed by 30 and 300 μg HSP, and variably by 0.3, 3 μg or 1 mg. Histological analysis of joint samples obtained from control and test rats confirmed the suppression of AA in the fed group. Feeding Mt or hen egg lysozyme (HEL) failed to affect AA, indicating that the suppression was HSP specific. The oral administration of 30 μg HSP decreased both delayed-type hypersensitivity (DTH) reactions and proliferative responses to HSP and Mt. In addition, the proliferation of lymph node cells (LNC) from Mt-sensitized rats was inhibited by the addition of spleen cells (SPC) from HSP-fed animals, possibly by the secretion of transforming growth factor (TGF)-β. Spleen cells obtained from tolerized donors were capable of transferring the tolerance to naive recipients. These results demonstrate that feeding HSP is an effective way to suppress AA and that the suppression of AA may be mediated by regulatory T cells generated following oral administration of mycobacterial 65-kDa HSP.     

口服髓鞘碱性蛋白诱导免疫耐受治疗实验性自身免疫性脑脊髓炎的研究

目的建立实验性自身免疫性脑脊髓炎（ＥＡＥ）动物模型,探讨口服自身抗原诱导免疫耐受对大鼠ＥＡＥ的防治作用。方法在普通Ｗｉｓｔａｒ大鼠经１次足跖真皮内注射完全弗氏佐剂－豚鼠全脊髓匀浆或完全弗氏佐剂－髓鞘碱性蛋白乳剂加百日咳疫苗诱发ＥＡＥ疾病;另在大鼠致炎前及发病后口服髓鞘碱性蛋白（ＭＢＰ）,观察其对ＥＡＥ的防治作用。结果在Ｗｉｓｔａｒ大鼠成功地诱发了ＥＡＥ,发病率将近９０％。大鼠在致炎前口服ＭＢＰ,可明显推迟ＥＡＥ发病时间,降低ＥＡＥ发病率,使神经组织病理改变明显改善。大鼠发生ＥＡＥ后给予ＭＢＰ,可明显控制发病动物的病情,使病程缩短及减轻患鼠神经组织的炎症程度。另外,在耐受鼠由ＭＢＰ引起的迟发型超敏反应（ＤＴＨ）以及体外针对ＭＢＰ的淋巴细胞增殖反应也明显受到抑制。结论口服ＭＢＰ可引起特异性的免疫耐受,从而产生对实验性自身免疫性脑脊髓炎的防治作用     

Suppression of an established DTH response to ovalbumin in mice by feeding antigen after immunization

Experiments were designed to examine whether systemic delayed-type hypersensitivity responses (DTH) to ovalbumin (OVA) can be suppressed when antigen is fed after immunization, and to investigate the immunological mechanisms involved. A single 25 mg feed of OVA given 7 or 14 days after immunization with OVA in complete Freund's adjuvant (CFA) suppressed the DTH response of BDF1 mice, but had no significant effect on the serum IgG antibody response. DTH suppression was greatest when antigen was fed soon after immunization, and became less pronounced as the time interval between feeding and immunization increased. The phenomenon was also demonstrated in mice of the BALB/c strain. Cell transfer experiments suggested that the post-immunization suppression was not due to a population of suppressor cells that have been described previously in association with classical oral tolerance for DTH. We conclude that there are separate and distinct mechanisms for the prevention of induction of DTH by antigen feeding in naive mice and the suppression of expression of DTH in sensitized animals.     

HUMORAL IMMUNE-MEDIATED ACUTE, ANTIGEN-INDUCED ARTHRITIS IN RATS IS SUPPRESSED BY THE INDUCING ANTIGEN ADMINISTERED ORALLY BEFORE, BUT NOT AFTER, IMMUNIZATION

Acute ovalbumin-induced arthritis (OIA), which is mediated by Arthus reaction to ovalbumin (OVA) in the joint space, can be induced by immunization of rats with OVA followed by the intraarticular injection of OVA. Because oral administration of antigen induces immunological unresponsiveness, we studied for the first time the effects of oral administration of OVA on acute OIA. The oral administration of OVA before immunization significantly suppressed the development of acute OIA, in accordance with decreases in both the anti-OVA IgG antibody production and in vitro lymphocyte proliferative responses to OVA. However, the oral administration of OVA after immunization did not show any decrease in antibody production or in vitro lymphocyte proliferation to OVA, or in the severity of acute OIA. These results indicate that the induction of oral tolerance to OVA in OIA is possible by oral administration of OVA before, but not after, immunization with the antigen. This supports the concept of using antigen feeding as a treatment for certain humoral immune-mediated diseases.     

Oral recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing HIV-1 antigens as a freeze-dried vaccine induces long-term,HIV-specific mucosal and systemic immunity

Induction of HIV-1-specific immune responses was evaluated using a recombinant BCG (rBCG) vector-based vaccine expressing HIV-1 Env V3 peptide (rBCG-pSOV3J1). rBCG-pSOV3J1 was manufactured as a freeze-dried preparation based on good laboratory practice guidelines. Guinea pigs were immunized with the freeze-dried rBCG vaccine by oral administration to test the effectiveness of what is generally considered the most convenient and practical route for vaccination. While delayed-type hypersensitivity (DTH) skin reactions to purified protein derivative were not detected in any of the animals receiving oral rBCG-pSOV3J1, HIV-1 V3J1 antigen-specific DTH responses were detected in all of the immunized guinea pigs 1.5 years after immunization. In addition, significant proliferative responses against HIV-1 V3J1 antigen were measured in peripheral blood mononuclear cells and splenocytes from all animals receiving oral rBCG. Interestingly, intestinal intraepithelial lymphocytes from the animals also exhibited high levels of proliferative activity against HIV-1 V3J1 antigen. These results suggest that oral vaccination of guinea pigs with freeze-dried rBCG-pSOV3J1 induces high levels of functional T cells specific for HIV-1 antigens in both mucosal and systemic compartments and suggest that this approach has potential for use as a vaccine against HIV-1.     

Oral Tolerization Leads to Active Suppression and Bystander Tolerance in Adult Rats while Anergy Dominates in Young Rats

Oral tolerance was induced in 4-week-old (young) and 12-week-old (adult) rats by feeding ovalbumin (OvA)-containing pellets during 4 weeks. Seven weeks after removal of the OvA-pellets the rats were immunized with a mixture of OvA and human serumalbumin (HSA) in Freund's complete adjuvant (FCA), and the following immune response was monitored. Both the young and adult groups of OvA-fed rats had significantly suppressed OvA-specific delayed-type hypersensitivity (DTH) responses and T-cellproliferation, reflecting a long-lasting T-cell tolerance to OvA both in vivo and in vitro. Furthermore, spleen cells from rats tolerized as adults were able to suppress the proliferation of primed T-cells from normal immunized rats, demonstrating thepresence of antigen-specific suppressive cells. Accordingly, the adult rats showed bystander suppression of the response to HSA with respect to DTH-reaction, specific proliferation, and reduced enlargement of the draining lymph nodes after immunization.There was no evidence of active suppression in vitro or bystander tolerance in the orally tolerized young group, indicating that anergy rather than active suppression was prevalent in these rats. Furthermore, in the young group there was no suppression of the antibody response since the IgG and IgE anti-OvA antibody levels were indistinguishable from those of the controls. Contrary to the young rats, the adult fed group showed transiently elevated levels of IgG anti-OvA antibodies at 1 weekpost-immunization, followed by a subsequent significantly suppressed IgG antibody response. In conclusion, the results demonstrate that the induction of anergy or active suppression after antigen feeding can be determined by the age at which the antigenis introduced to the mucosal immune system.