^125碘—标记弓形虫感染孕鼠后胎鼠脑放射示踪研究

应用１２５碘－标记弓形虫经尾静脉感染受孕１４ｄ的昆明种小鼠，动态观察分布和受侵细胞变化情况，结果显示：孕鼠脑感染率为３０．６０％，胎鼠头感染率为７１．２０％，胎鼠头／躯干的放射性比值为１．３２～５．９５，随受染时间的延长而增高。放射自显影的光电镜观察提示，弓形虫感染后４～８ｈ虫体即在胎鼠脑胶质细胞内被发现。４８ｈ细胞核内可见，细胞退变崩解。７２ｈ脑神经细胞显著减少。实验结果为妊娠期感染弓形虫易导致胎儿神经管畸形提供实验病理学基础。     

用足孕期畸形学指标评价毫米波辐射对小鼠植入后胚胎生长发育…

本实验用频率３６．１１ＧＨＺ，功率密度为１０．０ｍＷ／ｃｍ＾２的毫米波，在小鼠怀孕６－１５ｄ时进行２ｈ／ｄ的照射，在孕期终了用足孕期畸形学指标进行分析。结果显示，毫米波照射可导致足孕期孕期体重，体重增加数和胎鼠体重的明显降低，胎盘重明显减轻，足孕时胎仔身长及尾长均减短，照射未导致孕鼠脑，肝，肾，卵巢等及肝器重量及脏器／体重比值，活胎数，死胎数，吸收数等指标发生明显变化，亦未导致胎鼠外表发生畸形，内     

毫米波辐照对小鼠植入后胚胎发育的影响：：Ⅰ.足孕期畸形学评价

本文采用频率36.11GHz,功率密度为7.2mW/cm~2的毫米波,在小鼠怀孕6—15天时进行2小时/天的照射,在孕期终了(18天)用足孕期畸形学指标进行分析。结果发现,足孕期孕鼠体重、孕鼠体重增加数、平均孕鼠体重变化百分率,孕鼠脑、肝、肾,卵巢等脏器重量及脏器/体重比值,活胎数、吸收数、死胎数、总植入数,平均胎鼠体重、胎盘重、胎鼠身长、尾长、性别比等指标无明显变化;亦未导致胎鼠外表、内脏发生畸形,或骨骼畸形增多。孕鼠主要脏器组织形态学检查无明显改变。     

小剂量^6^0Coγ射线辐照对小鼠植入后期胚胎发育的影响

本文采用＾６＾０Ｃｏγ射线，在小鼠怀孕６－１５天时进行０．２Ｇｙ／ｄ的照射，在孕期终了（１８天）用足孕期畸形学指标及肠道的免疫组织化学进行分析。结果发现，照射组孕鼠足孕时体重，体重增加数，平均体重变化百分率明显低于对照组，照射组胎仔足孕时平均胎重，身长，尾长均明显低于或短于对照组胎仔，照射组胎仔肠道每平方毫米的５－ＨＴ阳性细胞数明显少于对照组胎仔。孕鼠脑，肝，肾，卵巢等脏器重量及脏器／体重比值，活     

用足孕期畸形学指标评价毫米波辐射对小鼠植入后胚胎生长发育的影响

本实验用频率36.11GHz、功率密度为10.0mW/cm~2的毫米波,在小鼠怀孕6～15d时进行2h/d的照射,在孕期终了用足孕期畸形学指标进行分析。结果显示,毫米波照射可导致足孕期孕鼠体重、体重增加数和胎鼠体重的明显降低,胎盘重明显减轻,足孕时胎仔身长及尾长均减短,照射未导致孕鼠脑、肝、肾、卵巢等脏器重量(右肾除外)及脏器/体重比值、活胎数、死胎数、吸收数等指标发生明显变化,亦未导致胎鼠外表发生畸形、内脏发生畸形,或骨骼畸形增多。     

重组人白介素2对小鼠弓形虫垂直传播的影响

目的　研究rhuIL - 2对弓形虫垂直传播的影响。方法　用不同剂量rhuIL - 2处理感染弓形虫孕鼠 ,于妊娠第12天取孕鼠子宫 ,观察记录平均活胎率的变化 ,并将胚胎及胎盘固定 ,做免疫组化染色 ,观察胎盘胎鼠感染情况 ,计算孕鼠的垂直传播率。结果　高剂量组与感染组相比活胎率明显升高 (P <0 0 5 ) ;两处理组的垂直传播率均较感染组明显下降 (P<0 0 5 ) ;免疫组化结果显示 ,胎盘滋养层细胞、绒毛间隙、蜕膜细胞均有大量弓形虫抗原表达 ;部分孕鼠的胎盘有弓形虫抗原表达 ,但其胚胎组织弓形虫抗原的表达却呈阴性。结论　rhuIL - 2一定程度上可以降低弓形虫的垂直传播率 ,对感染孕鼠及胚胎起到一定保护作用。胎盘作为局部免疫屏障具有重要的研究价值。     

小鼠Cpne5基因mRNA水平的表达分布及其在胚胎的表达定位

目的明确小鼠Cpne5基因在mRNA水平的组织表达分布及其在胚胎的表达定位。方法提取新生、成年小鼠主要脏器的总RNA,利用RT-PCR法检测Cpne5 mRNA的表达量;合成针对Cpne5 mRNA的杂交探针,取不同胎龄胎鼠进行整体原位杂交。结果 Cpne5 mRNA在成年小鼠10种脏器组织均有不同程度的表达,以大脑,小脑,睾丸和肺脏表达量较高;而肝脏和肌肉未表达。新生小鼠9种脏器中,Cpne5表达量最高的是脑组织,眼和肾次之,肺和肝脏表达量很少。制备并评估Cpne5原杂探针的产量,SP6转录的反义探针浓度为100ng/μL,T7转录的正义探针浓度为10ng/μL,原位杂交结果显示Cpne5 mRNA主要表达于胎鼠的端脑、间脑、中脑及菱脑原节。结论在新生和成年小鼠的脑组织中Cpne5基因mRNA高水平表达,并在胎鼠发育过程中定位于胎脑。     

STAg联合ESA鼻内免疫小鼠对弓形虫垂直传播的阻断作用

观察弓形虫可溶性速殖子抗原（Soluble tachyzoite antigen,STAg）与排泄-分泌抗原（Excreted/secreted antigens,ESA）单独或联合鼻内免疫小鼠对弓形虫垂直传播的阻断作用。分别用PBS 20μL和STAg 20μg、ESA 20μg及联合抗原（STAg 10μg＋ESA 10μg）鼻内免疫BALB/c处女鼠2次,间隔2周。末次免疫后第13天处女鼠与雄鼠以2∶1同居。妊娠第8天用弓形虫速殖子（8000个/只）灌胃攻击所有孕鼠。妊娠第18天处死,计算活胎率,并测定孕鼠肝、胎盘和胎鼠脑组织弓形虫抗原和孕鼠脾组织内虫荷,同时测定孕鼠小肠冲洗液SIgA、血清IgG水平。结果表明,ESA组和联合抗原组活胎率显著高于PBS组,STAg组、ESA组和联合抗原组胎盘及胚胎感染率均低于PBS组,联合抗原组最低。孕鼠在弓形虫速殖子攻击后,PBS组明显出现竖毛、倦怠、腹部塌陷等异常表现,感染率为100%,死亡率为22.22%,而STAg组、ESA组和联合抗原组有轻微症状,感染率分别为85.71%、57.14%、57.14%,死亡率分别为16.67%、0%、0%。STAg组、ESA组和联合抗原组脾组织内虫荷均显著低于PBS组,减虫率分别为72.19%、72.29%、78.45%,ESA组和联合抗原组小肠冲洗液特异性SIgA均显著高于PBS组,联合抗原组最高,血清特异性IgG抗体水平差异不显著。由此可见,ESA单独或与STAg联合免疫可诱导孕鼠小肠液高水平SIgA,显著降低脾组织虫荷,显著提高胚胎存活率,表现出明显的垂直传播阻断的作用。     

DIG—DNA探针检测弓形虫核酸

应用ＰＣＲ技术，对弓形虫Ｂ１基因的部分序列进行体外扩增，获得２０７ｂｐ的特异性核酸片段，并通过地高辛配基（Ｄｉｇ－１１－ｄＵＴＰ）标记作为探针，通过斑点杂交试验检测弓形虫核酸。实验表明，该探针能与ＲＨ、ＳＨ１、ＺＳ１、ＺＳ２及ＧＬ１株弓形虫核酸特异性杂交，最低检测量为２５ｐｇ纯化弓形虫核酸。在急性感染弓形虫的小鼠，该探针可于感染后第２天（２４ｈ后），从组织（肝、脾、及肾）中检测到弓形虫核酸，第３天（４８ｈ后）可从部分小鼠（１／５）外周血中检测到弓形虫核酸。表明该探针具有特异、敏感、快速、实用的特点，可望用于弓形虫病的早期诊断，适用于基层及流行病学调查。     

乳糖化人生长激素放射免疫分析方法的研究

目的：探讨鼠肝、肾匀浆中乳糖化生长激素（ｈＧＨ－Ｌ）和人生长激素（ｈＧＨ）的放射免疫分析（ＲＩＡ）方法。方法：取正常小鼠肝或肾匀浆，再在匀浆液中准确加入不同量的ｈＧＨ－Ｌ和ｈＧＨ，制得不同浓度的工作液，参照常规ＲＩＡ方法制作标准曲线。再取静脉注射ｈＧＨ－Ｌ或ｈＧＨ后不同时刻的小鼠肝和肾，经匀浆处理后，应用制作的标准曲线测定肝和肾的ｈＧＨ－Ｌ或ｈＧＨ浓度，以评价ｈＧＨ－Ｌ的体内动力学行为，并为该分析     

Detection of Toxoplasma gondii tachyzoites and bradyzoites in blood, urine, and brains of infected mice.

Different techniques for identifying Toxoplasma gondii were compared. PCR was used to amplify part of the major surface antigen P30 gene of T. gondii. Amplified-DNA detection with the DNA enzyme immunoassay (PCR-DEIA) was more sensitive than ethidium bromide staining after agarose gel electrophoresis and as sensitive as nested PCR. PCR-DEIA, using common enzyme-linked immunosorbent assay (ELISA) methods, avoids agarose gel electrophoresis for the identification of amplified products. T. gondii can also be detected with equal sensitivity in infected fibroblasts, but only after at least 8 days of cell culture. PCR-DEIA is thus recommended because of its sensitivity and convenience for detecting early parasitemia in the surveillance of toxoplasmosis among pregnant women and immunocompromised hosts. The courses of infection in mice infected with two strains of T. gondii were compared. Tachyzoites of the virulent strain T. gondii RH, killing the host in 4 days, were identified in urine specimens and blood samples of mice 24 to 94 h after inoculation but not in brains, but no antibodies were detected. After intraperitoneal inoculation with cysts of the low-level virulence Beverley strain of T. gondii, parasites were identified in blood samples 4 days later and up to 17 days (but not in urine specimens) and in the brain from day 6 through day 525. By ELISA, high antibody titers were found from day 11 to day 525, with parasitemia preceding the appearance of antibodies. The usefulness of PCR-DEIA tests in conjunction with the search for circulating antibodies for the early diagnosis of toxoplasmosis in humans is discussed.     

Effect of pregnancy on resistance to Listeria monocytogenes and Toxoplasma gondii infections in mice.

Studies of the effect of pregnancy on the capacity of mice to resist Listeria monocytogenes and Toxoplasma gondii infection revealed significantly diminished resistance of pregnant mice to infection by both agents as measured by mortality. The development of immunity to listeria was assessed by studying the kinetics of listeria growth in the livers and spleens of virgin and pregnant mice infected intravenously with a sublethal dose of listeria. In both virgin and pregnant mice there was a rise in the number of listeria colony-forming units per organ during the first 3 days after infection. Thereafter, there was a decline in colony-forming units in these organs in virgin mice but a persistence of listeria in spleens and livers of pregnant mice. Paradoxically, during the first 3 days after infection, listeria counts in spleens of virgin mice were significantly higher than those in pregnant mice. Nonspecific resistance to listeria conferred by chronic infection with T. gondii was significantly diminished in pregnant mice when measured by mortality and quantitative cultures of listeria in livers and spleens. These studies demonstrate a remarkably decreased resistance of pregnant mice to two intracellular organisms and a diminished capacity of pregnant mice to develop immunity to listeria. This decrease in resistance may play an important role in congenital transmission of these organisms.     

Interactions between murine AIDS (MAIDS) and toxoplasmosis in co-infected mice.

We coinfected C57B1/6 mice with LP-BM5 murine leukaemia viruses, responsible for murine AIDS (MAIDS), and an avirulent strain of Toxoplasma gondii. Virus-infected mice were infected perorally on day 30 with 10 cysts of T. gondii, and T. gondii-infected mice were challenged with LP-BM5 on day 20, 30 or 60 after parasite inoculation. Uninfected and singly infected mice were used as controls. The kinetics of parasite burden in blood, lungs and brain, together with blood lymphocyte subsets, and spleen and lymph node weights, were serially determined in each group of mice. The kinetics of parasite counts in mice infected by LP-BM5 then by T. gondii were similar to those in mice infected by T. gondii only, except for lung counts, which reached higher values than in animals infected with T. gondii alone, then fell and re-increased until the end of the experiment. The only significant change in parasite burdens when mice were first infected by T. gondii and then by LP-BM5, compared with T. gondii controls, was an increase in lung counts in mice challenged with LP-BM5 20 days after T. gondii inoculation. Whatever the schedule of co-infection, the kinetics of lymphocyte subsets in co-infected mice differed from those in T. gondii- or LP-BM5-infected mice; in dually infected mice CD4+ and CD8+ cell counts were intermediate between values in mice singly infected by the parasite or the virus. Enlargement of spleen and lymph nodes, which is a major criterion of MAIDS progression, was significantly less marked in co-infected mice than in mice infected with LP-BM5 alone. These data point to cross-regulation of T. gondii and LP-BM5 infections, which results in increased susceptibility to T. gondii, and may alter the progression of MAIDS.     

Effect of administration of a recombinant adenovirus expressing the genes for IFN-gamma and interleukin-12 on acute murine toxoplasmosis.

The effect of recombinant murine interferon-gamma (rMuIFN-gamma) produced from an adenovirus construct on Toxoplasma gondii in tissue culture and on the outcome of a T. gondii infection in mice was determined. Supernatants from AdCMVMuIFN-gamma-infected mouse lung epithelial (MuLE) cells were evaluated for the ability to produce biologically active IFN-gamma by measuring the capacity of the supernatants to activate peritoneal macrophages for killing of T. gondii. The bioactivity of IFN-gamma in supernatants increased with increasing multiplicity of infection (moi). Replication was inhibited 43%, 67%, and 70% by supernatants from MuLE cells infected with AdCMVMuIFN-gamma moi 5, 10, and 50, respectively, (p < 0.01 compared with controls). Bioactivity of IFN-gamma also increased as the length of time after infection increased. T. gondii replication was inhibited 28% and 36%, respectively, by AdCMVMuIFN-gamma-infected MuLE cell supernatants recovered at 24 and 48 h (p < 0.01 compared with control). In vivo administration of AdCMVMuIFN-gamma exhibited 33% mortality by day 9 in mice acutely infected with T. gondii compared with 100% mortality in control mice (p = 0.045). Administration of AdCMVIL-12 reduced mortality to 40% compared with control mice. However, this reduction was not significant (p = 0.08). Overall survival was extended 2 days with AdCMVMuINF-gamma administration and 5 days with AdCMVIL-12. AdCMVMuIFN-gamma in vitro inhibits T. gondii, and in vivo AdCMVMuIFN-gamma and AdCMVIL-12 lead to increased survival in mice.     

孕期弓形虫感染对小鼠胎盘补体调节蛋白表达水平的影响

目的 通过检测弓形虫感染对C57BL/6孕鼠胎盘补体调节蛋白的表达水平,探索孕期弓形虫感染致不良妊娠结局的分子免疫机制.方法 将C57BL/6孕鼠随机分为感染组和正常对照组,每组12只,感染组每只孕鼠于孕8 d腹腔注射200个弓形虫强毒株(RH株)速殖子,对照组于相应时间注射等量生理盐水.所有孕鼠均于孕14 d无菌分离胚胎组织,观察胎鼠发育情况.采用实时荧光定量PCR检测胎盘补体调节蛋白Crry、GPI-DAF及CD59a的mRNA的表达水平;采用流式细胞术检测胎盘单细胞悬液中Crry、GPI-DAF阳性细胞率.结果 弓形虫感染导致感染组死胎率显著升高(感染组死胎率为80.95%,对照组为4.41%),两组之间差异有统计学意义(P＜0.01);弓形虫感染组及对照组Crry、GPI-DAF和CD59a的mRNA表达水平分别为0.786±0.199、0.594±0.096、0.880±0.179和0.550±0.077、0.221±0.074、0.591±0.075,3类补体调节蛋白感染组与对照组比较差异均有统计学意义(P＜0.01);感染组和对照组Crry、GPI-DAF的阳性细胞百分率分别为(10.03±2.11)%、(2.95±1.04)%和(3.15±1.32)%、(0.66±0.26)%,两类补体调节蛋白感染组与对照组比较差异均有统计学意义(P＜0.01).结论 弓形虫感染导致孕鼠胎盘3种补体凋节蛋白Crry、GPI-DAF及CD59a表达水平均升高,打破了母胎界面维持正常妊娠所需的免疫微环境,这可能是孕期弓形虫感染致不良妊娠结局的分子免疫机制之一.

Abstract：

Objective To investigate the expression of complement regulatory proteins on placentas of pregnant C57BL/6 mice infected with Toxoplasma gondii in order to explore the molecular immunological mechanism for abnormal pregnancy induced by T. gondii infection. Methods Twenty-four pregnant C57BL/6 mice were randomly divided into two groups equally. The infection group was intraperitoneally injected with 200 of living T, gondii RH strain tachyzoites on the 8th day of gestation, and the normal group of mice was injected with physiological saline. All mice were killed on day 14 after gestation and placentas were collected. The expression levels of Crry, GPI-DAF and CD59a mRNA were analyzed by real-time quantitative PCR, and the positive rates of Crry and GPI-DAF were measured with flow cytometry. Results The died fetus rates of infected group and control were 80. 95% and 4. 41% , respectively. The infected group was significantly higher than of that the control group (P＜0.01). The expression levels of Crry, GPT-DAF and CD59a mRNA in the infected and control group were 0.786 ±0. 199, 0.594 ±0.096, 0.880 ±0. 179 and 0.550 ±0.077, 0.221 ±0.074, 0.591 ± 0.075 , respectively, and the difference of three kind of complement regulation proteins between two groups was all significant (P＜0.01). The positive percentages of Crry and GPI-DAF cells of infected and control group were (10. 03 ± 2. 11) % , (2.95 ±1.04)% and (3. 15 ± 1. 32) % , (0. 66 ±0. 26) % , respectively, and the difference of the two kind complement regulation proteins between two groups was also significant ( P ＜ 0. 01). Conclusion The expression level of mouse placental complement regulatory proteins was increased after infection with T. gondii, and then immunological microenvironment at the fetomaternal interface was destroyed. It may be one of important immunological mechanism for abnormal pregnancy induced by T. gondii infection.     

Opportunistic infections and retrovirus-induced immunodeficiency: studies of acute and chronic infections with Toxoplasma gondii in mice infected with LP-BM5 murine leukemia viruses.

Mice infected with LP-BM5 murine leukemia viruses develop a syndrome, termed mouse AIDS (MAIDS), characterized by increasingly severe immunodeficiency and progressive lymphoproliferation. Virus-infected mice were examined for the ability to resist acute infection and to control chronic infection with the protozoan Toxoplasma gondii, a major opportunistic pathogen of individuals infected with human immunodeficiency virus. Mice infected with the retroviruses for 2 or 4 weeks responded normally to challenge with the parasite, but mice inoculated with the protozoan 8 or 12 weeks after viral infection died with acute disease due to T. gondii. Increased sensitivity to acute infection was associated with a reduced ability to produce gamma interferon (IFN-gamma) and with established changes in CD4+ T-cell function. Mice latently infected with T. gondii and then inoculated with the retrovirus mixture were found to reactivate the parasite infection, with 30 to 40% of dually infected animals dying between 5 and 16 weeks after viral infection. Reactivation was associated with reduced proliferation and impaired production of IFN-gamma in response to stimulation with soluble T. gondii antigens or to concanavalin A. Continuing resistance to lethal reactivation in the remaining mice was shown to require CD8+ T cells and expression of IFN-gamma. In addition, it was found that chronic infection with T. gondii altered the course of MAIDS by inhibiting the progression of splenomegaly and immunodeficiency and reducing the expression of both the helper and etiologic defective viruses. These results support previous studies which indicate that infection with T. gondii is controlled by synergistic interactions between CD4+ and CD8+ T cells, the functions of which are progressively impaired during the course of MAIDS.     

Resistance to Toxoplasma gondii in mice infected as neonates or exposed in utero.

Mice were exposed to the protozoan parasite Toxoplasma gondii in utero or were infected as neonates in order to identify and characterize resistance mechanisms that function protectively during the first weeks after birth. About one-half of the mice born of mothers fed T. gondii cysts at 11 days of gestation survived to weaning age or beyond. No effect of major histocompatibility complex (MHC) haplotype on early survival was observed in a group of backcross progeny; however, long-term survival was strongly dependent on MHC haplotype. The ability of mice infected as neonates to survive until weaning was found to depend on gamma interferon and on Thy-1+ cells but not on CD4+ or CD8+ cells. Mice that survived to maturity after infection as neonates were slightly more resistant to challenge with virulent T. gondii parasites than were sham-infected controls but were less resistant than were mice infected as adults. Together the results indicate the following. (i) Mice congenitally infected with T. gondii have a gamma interferon-dependent mechanism of early resistance that involves Thy-1+ cells but not CD4+ or CD8+ cells. (ii) This mechanism is not under MHC-linked genetic control. (iii) Mice that exhibit long-term survival after congenital infection acquire a modest degree of protection against reinfection with virulent organisms. (iv) The extent of long-term survival of congenitally infected neonates, like that in mice infected as adults, is influenced by MHC genes, presumably via MHC-restricted CD4+ and/or CD8+ cells.     

TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection

To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.