Natural autoantibodies in nude and normal outbred (Swiss) and inbred (BALB/c) mice

Spleen cells from adult unprimed outbred (Swiss) and inbred (BALB/c) mice, either normal (no) or athymic-nude (nu) as well as spleen cells from Swiss nude mice bearing two different human tumors (BUR and PINQ), were fused with the mouse non-secreting myeloma cell line P3X63 Ag8-653. The supernatants of immunoglobulin secreting hybrids, all containing IgM, were screened for antibody activity against macromolecular antigens (autologous: actin, tubulin, myosin, dsDNA) and haptens (TNP, NP, NIP and NBrP). Furthermore, their idiotypic determinants were analyzed using a rabbit anti-idiotype which recognizes a major cross-reactive idiotype (IdD23) of BALB/c natural polyreactive autoantibodies. In all the mice studied, we identified: (1) hybrids reacting strongly with one or more haptens (10.7 to 37.8%) and (2) hybrids secreting natural monoclonal autoantibodies (NMoAb) with broad reactivities (polyreactive and/or oligoreactive) against autoantigens and/or haptens (11.4 to 26.8%). The results indicate that: (1) cells secreting natural autoantibodies with broad reactivities exist in both normal and nude mice, independently of the genetic background (inbred/outbred) of the mouse. However, in nude mice, the natural autoantibodies exhibit a more restricted pattern of reactivity (oligoreactive) compared to those of normal mice, and do not express the common idiotype IdD23 of natural polyreactive autoantibodies. (2) Tumors grafted into nude mice seem to induce the expression of polyreactive autoantibodies bearing the IdD23.     

TSHR大分子及其活性多肽在BALB/c小鼠体内的免疫原特性研究

目的：观察促甲状腺激素受体(TSHR)及其活性片段aa352-366在BALB/c小鼠体内的免疫原特性研究，探讨TSHR分子结构与功能的关系。 方法: 将血蓝蛋白(KLH)偶联的多肽TSHRaa352-366-KLH和多肽加受体混合组-TSHRaa352-366-KLH 加豚鼠TSHR(gTSHR),间隔15 d分两组分别注入BALB/c小鼠腹腔内，对照组注射KLH， 测定各时相血清中甲状腺激素及促甲状腺激素受体抗体水平；90 d后处死动物，检测小鼠甲状腺组织TSHR mRNA水平及其病理变化。 结果: 与各组自身0 d的数据比较，多肽组小鼠血清TT3、TT4水平降低(P<0.01)，TRAb升高(P<0.01)； 多肽加受体混合组TT3、TT4水平降低(P<0.01)，TRAb(P<0.01)、TBAb(P<0.05)和TSAb(P<0.05)均升高。 此外，混合组甲状腺组织TSHR mRNA水平明显高于正常组(P<0.05)；多肽组小鼠甲状腺组织显示滤泡上皮细胞扁平、核缺失、皱缩及胶质浓缩等甲减的病理改变, 而混合组则呈现不均一的病理变化。 结论: TSHR以及活性片段hTSHRaa 352-366都具有免疫原性， 刺激小鼠血清TRAb、TSAb和TBAb的产生，引起甲状腺组织的病理改变。     

Coexistence of antigen-specific TH1 and TH2 cells in genetically susceptible BALB/c mice infected with Leishmania major

CD4-positive T cell clones with specificity for the protozoan parasite Leishmania major (L. major) of both the protective TH1 and the disease-exacerbating TH2 subtype were isolated from a diseased L. major-infected mouse of the susceptible BALB/c strain. In addition, TH2 cells were isolated from the lesion-draining lymph nodes of an animal clinically healed nine months after sublethal irradiation and subsequent infection. Our data support the notion that the differential outcome of the disease in non-irradiated versus irradiated BALB/c mice reflects the regulation of the two CD4+ T cell subsets. These data also argue against the possibilities that: 1) TH2 cells and their precursors are totally eliminated by irradiation and that 2) TH2 cells are capable of completely hindering the expansion of TH1 cells in diseased animals.     

HBV截短core基因融合preS1基因在BCG中的表达及其免疫应答

目的:探讨HBV相关抗原在卡介苗(BCG)中的有效表达及其刺激产生的体液和细胞免疫应答效果。方法:将带有HBV截短C基因和preS1基因片段的穿梭载体转化BCG菌株,筛选重组菌,SDS-PAGE和Western blot实验分析其抗原表达特性;分别以生理盐水、BCG、BCG-pDE22、BCG-pDE22-CS1皮下注射BALB/c小鼠,进行连续抗体测定和细胞毒性T细胞杀伤实验。结果:与对照组相比,重组BCG可表达Mr为24000的新生蛋白,与CS1融合蛋白大小相符,Westernblot实验分析显示出良好的抗原结合特性;带有HBVCS1抗原基因的重组BCG免疫组抗体滴度显著升高,特异性杀伤能力更强。结论:BCG可以作为HBV相关抗原的活载体,为研制HBV新型疫苗提供了实验依据。     

T-lymphocytes in experimentalLeishmania amazonensis infection: comparison between immunized and naive BALB/c mice

Highly susceptible naive BALB/c mice or mice that had previously been immunized i.v. with solubilized homologous antigen (partially resistant) were infected withLeishmania amazonensis. Histologically, the main differences between the two groups were lymphocytic infiltration and macrophage activation. Assays of T-cell function at 3 and 10 weeks after infection revealed that purified T-cells did not proliferate following treatment with leishmania antigen. A mitogenic anti-CD3 monoclonal antibody (mAb) failed to activate T-cells after 3 weeks of infection as judged by proliferation and IL-2 secretion assays. After 10 weeks of infection, anti-CD3 mAb fully activated T-cells to proliferation and IL-2 secretion. On the other hand, T-cells released IL-3 in response to leishmania antigen, anti-CD3 mAb and anti-Thy1 mAb at 3 and 10 weeks post-infection. Surprisingly, a mitogenic anti-Thy 1 mAb (G7) fully activated T-cells even at 3 weeks of infection as judged by proliferative and IL-2 secretion assays. No significant differences were found in the proliferative or interleukin secretory responses of T-cells from animals that had been infected in either the presence or the absence of prior immunization. Since the Thy1 triggering pathway has different accessory cell and cytokine requirements than does the CD3: TCR lymphocytes activation pathway, it is possible that immunization was more effective in changing the cellular interactions of the T-lymphocyte than in altering its intrinsic capabilities.     

Early ultrastructural changes in the biliary epithelial cells of BALB/c and DDY mice immunized with swine serum

Electron microscopic observations were carried out on the biliary epithelial cells of BALB/c and DDY mice which had received an intraperitoneal injection of 0.2 ml of swine serum twice a week for 2 or 4 weeks. The most characteristic feature of the biliary epithelial cells of BALB/c mice was a marked increase in the number of vesicles having a close spatial relationship with the well-developed Golgi apparatus or rough endoplasmic reticulum (rER). In contrast, marked dilation of rER filled with moderately electron-dense material was conspicuous in the biliary epithelial cells of DDY mice. A prominent increase in the number of blebs and lateral and basal cytoplasmic protrusions in the dilated intercellular space of the biliary epithelium, and submucosal eosinophil infiltration, collagen fiber proliferation and gland hyperplasia with increased mucin secretion were common to both strains. This experimental model of bile duct disease also seems to be useful for investigating alteration of protein synthesis and secretion in epithelial cells.     

Protective effect of isoprinosine in genetically susceptible BALB/c mice infected with Leishmania major.

The effects of an immunopotentiating drug Inosine Pranobex (isoprinosine) were investigated in an experimental cutaneous leishmaniasis model. The highly susceptible BALB/c mice treated orally with isoprinosine developed significantly delayed onset of disease when infected with Leishmania major compared to untreated mice. The drug itself is not toxic to the parasite up to millimolar levels in vitro. The increase in resistance to L. major infection is accompanied by a marked decrease in the CD4+/CD8+ ratio and the leishmanial antigen-specific proliferative response of the spleen cells of isoprinosine-treated mice compared to untreated mice. There was a significant increase in the production of IFN-gamma but a decrease in the secretion of IL-3 and IL-4 by the spleen cells of isoprinosine-treated mice in response to concanavalin A with or without L. major infection compared to untreated controls. There was, however, no significant difference in the level of IL-2 production by the spleen cells between mice with or without isoprinosine treatment. These data are consistent with the interpretation that isoprinosine potentiates the resistance to leishmanial infection by up-regulating the host-protective Th1 cells and down-regulating the disease-promoting Th2 cells or, alternatively, by increasing CD8+ T-cell function.     

Characteristics of the protective response of BALB/c mice immunized with a purified Plasmodium yoelii schizont antigen

The response of BALB/c mice to immunization with a 230,000 mol. wt protective antigen purified from the blood stages of Plasmodium yoelii was analysed. The protective response to immunization was adjuvant-dependent, and saponin was found to be the most effective adjuvant tested. The intraperitoneal route was found to be superior to the subcutaneous route for protective immunization. Maximal protection was achieved using two immunizations with greater than 12.5 micrograms of antigen plus saponin, but even under these conditions challenge infection developed normally for 5 days, followed by the appearance of crisis forms and rapid clearance of parasites. Effective immunization induced high antibody titres, but serum from immunized mice was not protective on passive transfer. On the other hand, hyperimmune serum from mice recovered and rechallenged with live P. yoelii was protective on passive transfer, even though the titre of antibody specific for the 230,000 mol. wt antigen in this serum was the same as that in the serum from mice immunized with the purified antigen. It was concluded that the 230,000 mol. wt antigen may provide protection against P. yoelii via the cell-mediated immune effector pathway described by Playfair et al. (1979), and that hyperimmune serum contains protective antibodies specific for P. yoelii antigens other than the 230,000 mol. wt antigen.     

HPV6b L1/Ct MOMP多表位嵌合DNA诱导BALB/c小鼠产生特异性细胞免疫的研究

目的 探讨人乳头瘤病毒(HPV)6b结构蛋白L1和沙眼衣原体(Ct)主要外膜蛋白(MOMP)多表位嵌合DNA(HPV6b L1/Ct MOMP)诱导BALB/c小鼠产生特异性细胞免疫效应,及HPV6b L1对Ct MOMP多表位基因诱导细胞免疫的增强作用.方法 将Ct MOMP多表位基因连接在经真核密码子优化的HPV6b L1羧基端,构建嵌合重组质粒pcDNA3.1(+)/HPV6b L1/Ct MOMP多表位,经酶切和测序证实后,转染COS-7细胞,间接免疫荧光鉴定其在真核细胞中的表达;将纯化后的嵌合重组质粒肌肉注射免疫BALB/c小鼠,并设置pcDNA3.1(+)/Ct MOMP多表位质粒、pcD-NA3.1(+)和PBS三组对照.采用0、2、4周免疫程序,DNA剂量为每只150μg/次.于末次免疫后2周,分别用乳酸脱氢酶(LDH)释放法和细胞内细胞因子染色-流式细胞术(ICS-FAGS)检测小鼠脾细胞对Ct MOMP多表位蛋白和HPV6b L1蛋白的特异性CTL活性,胞内细胞因子IFN-γ、IL-4和IL-10产生水平等细胞免疫应答指标.结果免疫后,在效靶比(E∶T)分别为40∶1、20∶1时,pcDNA3.1(+)/HPV6b L1/Ct MOMP多表位嵌合重组质粒免疫组小鼠产生了针对Ct MOMP多表位蛋白特异性CTL杀伤活性(44.56%±4.02%,35.35%±2.89%)和HPV6b L1蛋白特异性CTL杀伤活性(27.08%±2.04%,21.68%±4.06%),与各对照组比较差异有统计学意义(F=72.87,F=114.55,P＜0.05;F=30.04,F=10.47,P＜0.05),且显著高于pcDNA3.1(+)/Ct MOMP多表位质粒组(35.50%±2.68%,30.24%±1.75%;12.27%±3.36%,9.32%±3.07%;P＜0.05);而pcDNA3.1(+)/Ct MOMP多表位质粒组小鼠仅显示了Ct MOMP多表位特异性的CTL杀伤活性,与对照组比较差异也有统计学意义(F=58.85,F=120.21;P＜0.05).胞内细胞因子IFN-γ的产生水平,pcDNA3.1(+)/HPV6b L1/Ct MOMP多表位嵌合质粒组(4.34%±0.06%)高于pcDNA3.1(+)/Ct MOMP多表位质粒组(3.14%±0.18%),与对照组比较差异有统计学意义(F=473.83,P＜0.05),而重组质粒组较空载体组和PBS对照组差异也有统计学意义(F=211.72,P＜0.05);但IL-4和IL-10水平则各组间差异无统计学意义(F=0.97,P＞0.05;F=2.25,P＞0.05).结论 pcDNA3.1(+)/HPV6b L1/Ct MOMP多表位嵌合DNA质粒可诱导BALB/c小鼠同时产生针对HPV6b L1蛋白和Ct MOMP多表位蛋白特异性的细胞免疫效应;真核密码子优化的HPV6b L1能显著增强Ct MOMP 多表位基因诱导的小鼠特异性细胞免疫效应.     

表达TSHR-A亚单位的腺病毒免疫BALB/c雌性小鼠建立甲亢模型

应用表达TSHR-A亚单位的重组腺病毒免疫BALB/c小鼠建立甲亢模型。分别用1010和108病毒颗粒/50μl PBS的腺病毒免疫6~8周雌性BALB/c小鼠,在完成3次免疫后通过检测血清T4、TRAb水平,甲状腺体积及甲状腺病理来鉴定动物模型。在所有接受免疫的小鼠中,甲亢的发生率为75%,表现为血清T4、TRAb增高,甲状腺体积增大,甲状腺病理表现符合甲亢。低免疫剂量组和高免疫剂量组小鼠血清T4较对照组均显著增高,两组间无显著性差异;低免疫剂量组和高免疫剂量组小鼠血清TRAb水平均显著高于对照组,前者血清TRAb水平低于后者。该模型具有成模率高,稳定性好的特点。低免疫剂量建立的动物模型更符合GD特点。     

BALB/c mice infected with Angiostrongylus cantonensis: A new model for demyelination in the brain

In this study, we present a new model for demyelination of the central nervous system (CNS). BALB/c mice were infected with Angiostrongylus cantonensis and analyzed 7, 14, and 21 days postinfection. Neurological scale evaluation, magnetic resonance imaging (MRI), histology, real-time quantitative polymerase chain reaction, and western blotting were all performed on days 7, 14, and 21. The results showed that the neurological functions and weight of A. cantonensis-infected mice decreased markedly after 21 days of infection. MRI showed subdural effusion and white high signals in the corpus callosum in both T1WI and T2WI, while hematoxylin and eosin and luxol fast blue staining showed hemorrhage and demyelination in the corpus callosum. Transmission electron microscopy revealed that the ultrastructure of the myelin sheath in the corpus callosum was dispersed or disintegrated. The percentage of myelinated axons was significantly decreased, and the g-ratio was lower than that in the normal group. Both protein and mRNA levels of myelin basic protein decreased markedly at 21 days postinfection. Immunofluorescence revealed that the number of CC1 positive cells in the corpus callosum also decreased, which confirmed the damage of A. cantonensis to oligodendrocytes. Our experiments confirmed that A. cantonensis infection caused demyelination in the CNS of BALB/c mice after 21 days, and its clinical manifestations and pathological changes were similar to those of multiple sclerosis and other CNS demyelination models. Thus, mice infected with A. cantonensis could be used as a new model to study acute demyelination of the CNS.     

Specific inflammatory cell infiltration of hepatic schizonts in BALB/c mice immunized with attenuated Plasmodium yoelii sporozoites.

We compared immunization of BALB/c mice with radiation-attenuated versus killed sporozoites of the rodent malaria parasite, Plasmodium yoelii. We employed a suboptimal schedule of only two immunizations, in expectation that some parasites might break through the resultant low level immunity and that it might thus be possible to study the response of the host against these 'breakthrough' schizonts. As a measure of protective immunity, we used histological means to determine the percentages of challenge sporozoites prevented from completing development into hepatic schizonts within the liver. Immunization with attenuated sporozoites led to almost complete protection, whereas immunization with similar dosages of killed sporozoites led to approximately a 75% protection. Fluorescent antibody titers against sporozoites were similar in both sets of immunized animals. However, serum from mice immunized with attenuated sporozoites had a protective effect upon passive transfer into immunologically naive mice subsequently challenged with normal sporozoites; serum from mice immunized with killed sporozoites had no such effect. When mice suboptimally immunized with attenuated sporozoites were challenged, we observed breakthrough schizonts being infiltrated with inflammatory cells, primarily mononuclear cells, and neutrophils; partial depletion of CD4+ or CD8+ cells within these mice prior to challenge prevented the infiltration of breakthrough schizonts. Thus, cellular infiltration of schizonts was apparently secondary to earlier action by lymphocytes. This infiltration was also not observed in mice immunized with killed sporozoites. The more effective protective immunity induced by attenuated sporozoites could be due to their ability to release antigen into the cytoplasm of hepatocytes that they invade or their ability to continue differentiating, thereby presenting new antigens that are not seen after immunization with killed sporozoites.     

Dendritic cells infected with adenovirus expressing the thyrotrophin receptor induce Graves' hyperthyroidism in BALB/c mice

Dendritic cells (DCs) are the most potent antigen-presenting cells and a prerequisite for the initiation of primary immune response. This study was performed to investigate the contribution of DCs to the initiation of Graves' hyperthyroidism, an organ-specific autoimmune disease in which the thyrotrophin receptor (TSHR) is the major autoantigen. DCs were prepared from bone marrow precursor cells of BALB/c mice by culturing with granulocyte macrophage-colony stimulating factor and interleukin-4. Subcutaneous injections of DCs infected with recombinant adenovirus expressing the TSHR (but not beta-galactosidase) in syngeneic female mice induced Graves'-like hyperthyroidism (8 and 35% of mice after two and three injections, respectively) characterized by stimulating TSHR antibodies, elevated serum thyroxine levels and diffuse hyperplasitc goiter. TSHR antibodies determined by ELISA were of both IgG1 (Th2-type) and IgG2a (Th1-type) subclasses, and splenocytes from immunized mice secreted interferon-gamma (a Th1 cytokine), not interleukin-4 (a Th2 cytokine), in response to TSHR antigen. Surprisingly, IFN-gamma secretion, and induction of antibodies and disease were almost completely suppressed by co-administration of alum/pertussis toxin, a Th2-dominant adjuvant, whereas polyriboinosinic polyribocytidylic acid, a Th1-inducer, enhanced splenocyte secretion of IFN-gamma without changing disease incidence. These observations demonstrate that DCs efficiently present the TSHR to naive T cells to induce TSHR antibodies and Graves'-like hyperthyroidism in mice. In addition, our results challenge the previous concept of Th2 dominance in Graves' hyperthyroidism and provide support for the role of Th1 immune response in disease pathogenesis.     

Evidence for pleomorphism of estrogen receptor capacity and affinity in liver and thymus of immature BALB/c and (NZBxNZW) F1 mice, a model of systemic lupus erythematosus

Binding properties of estrogen receptors in liver, thymus and uterus of BALB/c and (NZBxNZW) F1 mice were compared. (NZBxNZW) F1 mice spontaneously develop an autoimmune disease resembling human systemic lupus erythematosus (SLE). It is hypothesized that estradiol, through its receptors, mediates the progression of murine SLE. High-speed cytosols were prepared from liver, thymus and uterus and incubated with the synthetic estrogen 3H-moxestrol (NEN). Scatchard plots were derived from binding isotherms obtained. Rectilinear Scatchard plots were obtained from all tissues, which is consistent with the presence of only one class of binding sites, or of more than one class but with the same affinity. There were, however, strain differences in that the affinity of the binding reaction was significantly higher in cytosols from BALB/c liver in males and females. In thymus, the situation was reversed in that the affinity was significantly higher in cytosols from (NZBxNZW) F1 mice. Thymic cytosols from BALB/c mice contained significantly more estrogen receptors per mg cytosol protein than did those from (NZBxNZW) F1 mice. The exacerbation of murine SLE may be due, at least in part, to these properties of estrogen receptors in liver and thymus.     

Immunosuppressive drugs have different effect on B lymphocyte subsets and IgM antibody production in immunized BALB/c mice

Infections are frequently associated with immunosuppressive therapy currently used to prevent organ rejection or treat autoimmune diseases. Such drugs suppress antibody production despite having different mechanisms of action. Antibodies are produced by a non-homogenous population of B lymphocyte subsets. B-1 cells produce natural antibodies and protect immediately after infection, while B2 cells produce antigen-specific IgM antibodies in a later response to infection. To understand how the immunosuppressive drugs affect antibody production by B cell populations, we immunized BALB/c mice with different antigens followed by administration of various immunosuppressive drugs. B-1a and B-1b lymphocytes from spleens of sacrificed animals were analyzed by flow cytometry, natural and antigen –specific IgG and IgM antibodies were determined by nephelometry and ELISA assays. Results showed that prednisone (PDN), cyclophosphamide (CYC), methotrexate (MTX), mycophenolate mofetil (MMF) and azathioprine (AZA) decreased more than 60% of B-1a lymphocytes while cyclosporine (CsA) had little effect. Three drugs PDN, AZA and CYC suppressed the B-2 cells on day 30, while MTX affected this subpopulation early on day 5. Antigen-specific IgM antibodies were dramatically suppressed after 15 days of immunization in animals receiving PDN, CYC or AZA, while MMF, CsA and MTX showed little effect. Natural antibodies were equally decreased in all animals regardless of the specific drug used in treatment. These results will help to choose single or combinations of immunosuppressive drugs in the clinical setting.     

Comparison of ascites production for monoclonal antibodies in BALB/c and BALB/c-derived cross-bred mice

BALB/c male mice were mated with either Swiss-Webster or MF1 females to produce first generation cross-bred offspring. Hybridoma cell lines, from the fusion of P3-NS1-Ag4/1 myeloma cells with spleen cells sensitised to the porcine coronavirus causing transmissible gastroenteritis, were injected intraperitoneally into these mice to produce ascitic fluid containing monoclonal antibodies. Mice of 11 weeks of age weighing between 26 and 34 g were used. The volume of ascites produced by mice injected with four of the five hybrid cell lines tested was greater in the cross-bred offspring than in the BALB/c parent. The fifth cell line gave comparable volumes in the MF1 cross-breed and BALB/c parent but a lesser volume in the Swiss-Webster cross-breed. The antibody titres of the ascites as determined by virus neutralisation, radioimmune and indirect immune fluorescence assays, did not differ significantly between mouse types. The ability to use all offspring from a litter of cross-bred mice, irrespective of sex, and the increased volume of ascitic fluid formed in each mouse, permits fewer animals to be used for the production of ascites in these strains, thereby offering considerable economic and ethical advantages over the use of BALB/c mice.     

日本血吸虫重组Bb(pGEX-Sj14-3-3)疫苗免疫BALB/c小鼠诱导免疫应答的动态检测

目的 研究日本血吸虫重组双歧杆菌属两歧双歧杆菌(Bb) (pGEX-Sjl4-3-3)疫苗免疫小鼠后其免疫应答的动态变化.方法 将重组疫苗分别采用口服灌胃和鼻内接种免疫BALB/c小鼠,分别于免疫后2、4、6、8、10、12、14、16、18、20和22周ELISA法测小鼠血清中IgG及其亚类、IgE和IgA及脾细胞培养液中IFN-γ、IL-12、TNF-α和IL-10水平;MTT法测定脾细胞增殖;流式细胞仪检测脾细胞凋亡率及CD4+和CD8+T细胞亚群百分率.结果 口服组血清IgG、IgG1、IgG2a、IgG2b、IgG3、IgE和IgA水平分别在免疫后8、6、6、4、8、10和6周达峰值.脾淋巴细胞增殖和凋亡水平均在免疫后4周达峰值;CD4+T细胞于8周达峰值,CD8+T细胞于14周达峰值.IFN-γ、IL-12、TNF-α和IL-10水平分别于8、8、6和4周达峰值.鼻内接种组血清IgG、IgG1、IgG2a、IgG2b、IgG3、IgE和IgA分别于4、6、4、4、8、10和8周达峰值.脾淋巴细胞增殖及凋亡水平也均在4周达峰值;CD4+T细胞于8周达峰值,CD8+T细胞于4周达峰值.IFN-γ、IL-12、TNF-α和IL-10水平分别于2、2、4和4周达峰值.口服及鼻腔内接种是两种较好的免疫途径,且后者优于前者.结论 该疫苗可诱导小鼠产生有效的免疫应答.     

杂色曲霉素对新生乳鼠致癌作用的实验研究

我们采用单次杂色曲霉素灌喂新生乳鼠,以观察其诱发肺癌的作用。