泰山磐石散对复发性流产小鼠母胎界面Th1/Th2细胞因子及妊娠预后的影响

目的:观察泰山磐石散对复发性流产小鼠母胎界面Th1/Th2细胞因子及妊娠预后的影响,为泰山磐石散临床应用提供新的实验依据。方法:采用经典造模方式DBA/2小鼠与CBA/J杂交,获得复发性流产小鼠模型,随机将与DBA/2小鼠合笼的60只CBA/J妊娠小鼠分为模型组、中药低剂量组、中药中剂量组、中药高剂量组和阳性对照组;与BALB/C合笼的10只CBA/J孕鼠作为正常妊娠模型。于妊娠14 d后处死孕鼠,观察小鼠胎盘丢失情况,并提取培养胎界母细胞,24 h后收集细胞上清液,酶联免疫吸附测定(ELISA)法检测上清液中肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)、白细胞介素4(IL-4)和IL-10含量。结果:经泰山磐石散治疗后,与模型组比较,复发性流产小鼠胎盘丢失率明显改善,母胎界面细胞上清液中Th1型细胞因子IFN-γ明显降低,Th2型细胞因子IL-4、IL-10明显升高,Th1/Th2免疫调节失衡明显改善,以中药高剂量组改善最为明显。结论:泰山磐石散能改善复发性流产小鼠胎盘丢失情况,其具体机制可能是通过调节Th1/Th2免疫调节平衡实现。     

Ultrastructural alterations in human decidua in miscarriages compared to normal pregnancy decidua

BACKGROUND: Pregnant endometrial stroma, an immunologically privileged site in the female reproductive system, is enriched by decidual and natural killer (NK) cells. Since the cellular microenvironment in early pregnancy from the decidual tissues of normal and miscarriage cases has gained importance, with special emphasis on cell-to-cell contacts, we aimed to document the plastic structure of the cellular milieu in normal and miscarriage decidua. METHODS: Endometrial biopsies were obtained from women after legal curettage or women who had been treated by curettage after miscarriage. Samples were analysed in a light microscope (LM), a scanning electron microscope (SEM) and a transmission electron microscope (TEM). RESULTS: Decidual cells possess several polyploidic protrusions on cell membranes. NK cells were distributed among decidual cells. Decidual cells were found to develop gap junctions in the interfaces between each other. Their cytoplasms were also found to possess well-developed protein synthesising organelles. Decidual cells obtained from miscarriages showed a moderate degree of degeneration and, in between, a decreased number of junctional complexes. Mononuclear cell infiltration was found to be significantly low. CONCLUSION: We conclude that decidual cells during early pregnancy build a series of miniature cell-cell contacts to assemble a proper endometrial milieu. In contrast, in miscarriage samples, those intercellular communications seem lacking, associated with an increased number of NK cells, a phenomenon which obviously alters proper implantation and leads to the induction of embryonic disgenesis and miscarriage.     

The balance between cytotoxic NK cells and regulatory NK cells in human pregnancy

NK cells kill tumor cells and virus-infected cells as well as secrete a variety of cytokines. These effector functions are regulated by the balance between activating receptor signals and inhibitory receptor signals which are triggered by specific major histocompatibility complex (MHC) or non-MHC ligands. It is thought currently that the balance between immunostimulation and immunoregulation in T cell immunity is achieved by a Th1/Th2/Th3/Tr1 and CD4(+)CD25(+) regulatory T (Treg) cell paradigm. Here, we discuss the cytokine paradigm of NK cells in human pregnancy. During normal, intact pregnancy, peripheral blood NKr1 cells and decidual NK3 cells increase, while these NK cell populations decrease significantly in miscarriage cases, suggesting that an imbalance in NK1/NK2/NK3/NKr1 is correlated with miscarriage. Recent investigations have shown that not only Treg cells, but also regulatory NK (NK reg) cells, play very important roles in the maintenance of pregnancy. We summarize the progress in studying NK reg cells and focus on how NK reg cells and cytotoxic NK cells affect the reproductive immune response.     

分娩发动前后胎盘中白细胞抗原G1 mRNA和NK细胞的变化

目的:检测胎盘及蜕膜组织中白细胞抗原G1(HLA-G1)mRNA和NK细胞在足月妊娠分娩发动前后的变化,探讨其在分娩发动中的作用。方法:通过RT-PCR法检测足月妊娠晚期未临产组(剖宫产组)和临产组胎盘组织中HLA-G1 mRNA的表达,并用免疫组织化学方法测定蜕膜中NK细胞的数量。结果:与未临产组相比临产组胎盘组织中HLA-G1 mRNA表达明显下降,差异有统计学意义(P<0.05);临产组蜕膜中NK细胞数量明显多于未临产组(P<0.05)。结论:分娩发动时胎盘组织表达HLA-G1mRNA下降,蜕膜组织中NK细胞数量明显增多,推测HLA-G1表达下降激活NK细胞可能参与了分娩发动。     

Murine CD45+CD86+ cells isolated from para-aortic lymph nodes in an abortion-prone model

The present study aims to address whether the analysis of CD45+CD86+ cells isolated from para-aortic lymph nodes (pLNs) is valuable in assessment of the status of local immunity at the murine feto-maternal interface. CBA/J x DBA/2 mice, virgin CBA/J mice, and CBA/J x BALB/c mice were used as an abortion-prone model (group A), nonpregnant controls (group N), and fertile controls (group F), respectively. The percentage of CD45+CD86+ cells in the CD45+ cell group (CD45+CD86+ percentage for short) and the absolute number of these cells were determined by means of flow cytometry (FCM), using mononuclear cells isolated from pLNs collected 5.5, 9.5, and 13.5 days post-coitum (dpc), respectively, and mononuclear cells isolated from placentas 13.5 dpc. To clarify the identity of these CD86+ cells, FCM was also performed with CD3, CD19, and DX5 as specific markers for murine T-cells, B-cells, and NK cells, respectively. Both resorption rate and absolute number of resorptions were significantly higher in group A (29.3%, 1.8+/-1.0) than in group F (4.8%, 0.3+/-0.5, P<0.001, respectively). Similarly, both cell percentage and absolute number of CD45+CD86+ cells in pLNs collected 13.5 dpc were significantly higher in group A than in group F (27.5+/-14.0% versus 12.3+/-7.1%, and 1362+/-687 versus 615+/-353, P=0.001, respectively). The CD45+CD86+ percentage was around 7.5% in nonpregnant CBA/J mice, similar to the 10.6% in CBA/JxDBA/2 mice 5.5 dpc, but had increased dramatically, to 23.9%, by 9.5 dpc (P<0.001 versus nonpregnant mice and P=0.002 versus CBA/JxDBA/2 mice 5.5 dpc), and remained at a higher level (27.5%) until 13.5 dpc. However, this trend was not observed in group F during pregnancy. The increased CD45+CD86+ percentage at day 9.5 of gestation, when resorption begins, may support the assumption that CD45+CD86+ cells play a role in the course of embryo resorption. Lymphocyte phenotypic analysis in the lymph nodes that drain the pregnant uterus may be helpful to assess the status of local immunity at the feto-maternal interface.     

口服卵清蛋白和滋养细胞膜抗原诱导小鼠妊娠免疫耐受效果的观察

目的　观察口服卵清蛋白 (OVA)和滋养细胞膜抗原 (TMA)诱导小鼠妊娠免疫耐受效果 ,探讨原因不明复发性自然流产的口服免疫疗法。方法　建立自然流产小鼠模型CBA/J×DBA/ 2 ,分不同剂量观察经口服途径给妊娠CBA/J小鼠OVA和TMA1、TMA2 3种抗原后胚胎丢失率的变化。实验分为 4组 :(1 )口服抗原 (即OVA、TMA1和TMA2 )免疫组 :分为低剂量单次 (即OVA 2 0 μg× 1次 ,TMA1和TMA2均为 50 0 μg× 1次 )、低剂量 5次 (即OVA 2 0 μg× 5次、2 0 0 μg× 5次、2mg× 5次 ,TMA1和TMA2均为 50 0 μg× 5次、1mg× 5次、2mg× 5次 )和高剂量单次 (即OVA 2 0mg× 1次 ,TMA1和TMA2均为 8mg× 1次 ) ;(2 )另设 3个对照组 ,即未免疫组 (未予任何处理 )、主动免疫组 (腹腔注射雄性BALB/c小鼠脾细胞 )和空白对照组 (口服生理盐水 )。结果　 4组小鼠的胚胎丢失率分别为 :未免疫组 2 9% ,主动免疫组 8% ,空白对照组 2 8% ;口服抗原免疫组中 ,口服OVA 2 0 μg× 1次、2 0 μg× 5次、2 0 0 μg× 5次、2mg× 5次、2 0mg× 1次者分别为 2 6 %、6 %、1 0 %、4 %和 1 0 % ,口服TMA1及TMA2 50 0 μg× 1次、50 0 μg× 5次、1mg× 5次、2mg× 5次、8mg× 1次者分别为 2 2 %及 2 6 %、2 3 %及 4 %、2 7%及1 1 %、2 7%及 3 %、2 4 %     

Are CD57+ Natural Killer cells really important in early pregnancy failure?

Objective  The aim of this study was to compare the CD57+ Natural Killer (NK) cell counts in normal pregnancies and in cases grouped according to different types of early pregnancy failure. Materials and methods  A prospective case control study which was set in Baskent University Faculty of Medicine, Obstetrics and Gynecology Department. A total of 119 women whose pregnancies ended in the first trimester were divided into elective pregnancy termination, incomplete miscarriage, intrauterine demise, ectopic pregnancy and recurrent pregnancy loss groups. CD57+ NK cells were stained and counted in the histologic preparations of the decidua in all of these groups. Results  CD57+ NK cell counts were 2.14 ± 1.42 in control, 2.24 ± 1.92 in incomplete miscarriage, 1.82 ± 1.34 in intrauterine demise, 2.54 ± 1.80 in ectopic pregnancy and 3.42 ± 2.15 in recurrent pregnancy failure group. There were no statistically significant differences between the control group and the other four groups with respect to the CD57+ NK cell counts. Conclusion  This study suggests that CD57+ NK cell count is not associated with early pregnancy failure. This study was supported by Baskent University Grant KA06/238 after Institutional Review Board Approval.     

Interleukin-17 expression in the human placenta

Interleukin (IL)-17 is a proinflammatory cytokine with pleiotropic activities including inducing neovascularization and production of proangiogenic molecules. As pregnancy outcome depends on the balance of Th1-like/Th2-like cytokines and an increased blood supply to the fetoplacental unit, the expression of IL-17 mRNA and protein in human placental tissues was investigated. IL-17 mRNA was expressed by purified cytokeratin-positive term placental trophoblast cells, HLA-G+ extravillous trophoblast cells and placental macrophages (Hofbauer cells). IL-17 localized in both cyto- and syncytiotrophoblasts of normal term pregnancy, spontaneous miscarriage and in molar pregnancy. In spontaneous miscarriage and molar pregnancy extravillous trophoblast cells were consistently immunoreactive for IL-17. IL-17 expression in human placenta may play a key role in angiogenesis and/or immunoregulation in the establishment of pregnancy.     

Morphometric analysis of the histology of spontaneous fetal resorption in a murine pregnancy

Immunopathology of the spontaneous resorption phenomenon in the CBA x DBA/J murine model was explored using morphometric analysis. Accompanying the previously reported presence of natural killer (NK) cells in resorptive feto-placental units we find major changes in tissue morphology indicating that early infiltration of the feto-placental unit by maternal leukocytes plays a direct role with NK cells in fetal demise. Total number of cell nuclei per field and total nuclear area per field were significantly elevated in feto-placental units containing abnormally increased NK cell presence before detectable resorption as early as day 7 of gestation. This difference persisted throughout all stages of early gestation up to and including the final resorption event at day 10 to 12. Increases in cell density were also detected in areas of the embryonic unit not associated with NK infiltration. These results demonstrate that the spontaneous resorption phenomenon in this model involves: (i) Early (day 7-8) cellular infiltration of the decidual-ectoplacental cone junction associated with the presence in this area of NK cells. (ii) Late (day 8-9) cellular infiltration of the ectoplacental cone.     

Reappraisal of peripheral NK cells in women with recurrent miscarriage

The aim of this study was to examine the relationship between peripheral natural killer (NK) cells and recurrent miscarriage by improved methods. Peripheral NK cell measurement was carried out using flow cytometry of morning blood samples obtained in the early follicular phase, analysed within 8 h of collection. Eighty-five Chinese women with recurrent miscarriage who previously tested negative for autoantibodies, and 27 control subjects who were not using any hormonal methods for contraception, were recruited. No significant difference was found in the number of peripheral NK cells and their subsets between women with recurrent miscarriages and control subjects. Only 5% of women with recurrent miscarriage had high peripheral NK cells. The number of previous miscarriages did not appear to have an impact on the number of NK cells. In conclusion, there appears to be limited value in the routine measurement of peripheral NK cells in women with recurrent miscarriage.     

Evaluation of KIR genes in recurrent miscarriage

Purpose

Natural killer (NK) cells express killer immunoglobulin-like receptors (KIRs) which recognize HLA class I molecules on trophoblasts. KIRs could either activate NK cells or inhibit them to produce soluble factors necessary for the maintenance of pregnancy, thus they are suspected of being involved in the causes of recurrent miscarriage. The aim of this study was to evaluate whether there is any possible association between KIR genes, genotypes and recurrent miscarriage.

Methods

The present study was carried out on 40 women who had unexplained recurrent miscarriage and 90 controls. Sequence-specific oligonucleotide probes analysis were used to investigate 16 KIR genes. All data were statistically analyzed by Fisher Exact Test.

Results

The rate of Bx genotypes that consists elevated number of activating KIR genes was significantly higher (p = 0.014) in women with recurrent miscarriage when compared with the control group. Additionally, the frequency of AA genotype (AA1) of the subjects in the study group was significantly lower than the frequency of the subjects in the control group (p = 0,014). Furthermore, there were no statistically significant differences in the frequencies of the individual KIR genes between women with recurrent miscarriage and the control group.

Conclusions

Inclined balance of KIRs toward an activating state in NK cells may contribute to recurrent miscarriage.     

淋巴细胞免疫治疗对小鼠自然流产模型母胎界面CD80~+表达及妊娠结局的影响

目的:评价CBA/J×DBA/2小鼠配对组合作为反复自然流产模型的生殖力特点,及其与母胎交界CD80表达间的关系,并研究淋巴细胞免疫治疗(lymphocyte immunotherapy,LIT)对CD80表达水平的影响。方法:对CBA/J×DBA/2小鼠的生殖力特点进行为期120d的观察,并与生殖力正常的4种对照组进行比较。另计算15对CBA/J×DBA/2小鼠孕13d的胚胎吸收率,并用CD80-FITC和CD45-PE双色流式细胞术检测CD80细胞在母胎交界面的构成比。为了明确CD80~+细胞的身份,检测了CD3、DX5(NK细胞)和MHC-Ⅱ在CD80细胞群中的表达水平。此外,检测LIT组与未治疗组CBA/J×DBA/2小鼠胚胎吸收率和CD80细胞的阳性率。结果:CBA/J×DBA/2小鼠的流产特点是为孕10d左右的反复流产。CBA/J×DBA/2小鼠孕13d的胚胎吸收率显著高于BALB/c×DBA/2小鼠(30.8％±16.6％vs.7.7％±6.7％,P相似文献   

Interleukin-6 in pregnancy and gestational disorders

IL6 is a multifunctional cytokine with pivotal roles in the inflammatory response and in directing T cell differentiation in adaptive immunity. IL6 is widely expressed in the female reproductive tract and gestational tissues, and exerts regulatory functions in embryo implantation and placental development, as well as the immune adaptations required to tolerate pregnancy. Here, we summarise the current understanding of how membrane-bound and soluble receptors mediate IL6 signalling to regulate leukocytes and non-haemopoietic cells. We review the published literature regarding the expression and actions of IL6 in the uterus, decidua and placenta, and studies implicating this cytokine in pregnancy disorders. Elevated IL6 is frequently evident in the altered cytokine profiles characteristic of unexplained infertility, recurrent miscarriage, preeclampsia and preterm delivery. Notably, there is compelling evidence indicating altered systemic IL6 trans-signalling in women prone to recurrent miscarriage, with excessive IL6 bioavailability potentially inhibiting generation of CD4+ T regulatory cells required for pregnancy tolerance. Insufficient local IL6 may also contribute to fetal loss, since IL6 expression is reduced in the endometrium of women with recurrent miscarriage, and in the fetal-placental tissue of CBA×DBA/2 mice. Consistent with the role of IL6 in key reproductive events, Il6 null mutant mice exhibit elevated fetal resorption and delayed parturition. Investigation of the association between IL6 signalling components and T cell responses in pregnant women, as well as detailed analysis of the maternal immune response in IL6-deficient mice, is now required to define the mechanisms by which this cytokine exerts influence on reproductive success.     

A morphological and immunological study of human placental bed biopsies in miscarriage

Placental bed biopsies were examined in three groups of pregnant women for maternal vascular response to placentation as well as for the presence of cells associated with local immunosuppression activity. In the group of women undergoing legal abortion, the histological appearance of trophoblastic invasion was normal in all but one, and the proportion of immunosuppressor cells was also normal. In the missed miscarriage group the histological appearances were abnormal except in one patient. In women with a history of recurrent miscarriage who had miscarried after immunization, placentation was normal in some and defective in others. Immunosuppressor cells appeared to be diminished in number, although there was no correlation between the cytotoxic status of their sera and their pregnancy outcome.     

A morphological and immunological study of human placental bed biopsies in miscarriage

Summary. Placental bed biopsies were examined in thrce groups of pregnant women for maternal vascular response to placentation as well as for the presence of cells associated with local immunosuppression activity. In the group of women undergoing legal abortion, the histological appearance of trophoblastic invasion was normal in all but one, and the proportion of immunosuppressor cells was also normal. In the missed miscarriage group the histological appearances were abnormal except in one patient. In women with a history of recurrent miscarriage who had miscarried after immunization, placentation was normal in some and defective in others. Immunosuppressor cells appeared to be diminished in number, although there was no correlation between the cytotoxic status of their sera and their pregnancy outcome.     

分泌期及早期子宫内膜巨噬细胞及自然杀伤细胞的测定

目的 探讨子宫内膜局部免疫微环境对妊娠的影响。方法 对１９例孕６￣９周的正常妊娠和１１例正常分泌期妇女,采用流式细胞技术检测蜕膜及内膜的巨噬细胞及自然杀伤（ＮＫ）细胞。结果 蜕膜组织中巨噬细胞（白细胞分化抗原分化簇（ＣＤ）１４阳性即ＣＤ１４」及ＮＫ细胞（ＣＤ５６阳性即ＣＤ５６）含量较分泌期子宫内膜组织增加,差异有极显著性。ＮＫ细胞的ＣＤ５６ＣＤ１６亚群含量增加,差异有极显著性,而ＣＤ５６ＣＤ１６亚     

Allogeneic reactivity of maternal lymphoid cells during the course of gestation. Modifications and sex differences in a local GVH assay

The immune reactivity of lymphoid cells from pregnant mice was studied during the course of pregnancy in primiparous and multiparous animals either " isopregnant " (male and female of same strain) or " allopregnant " (male and female differing at H-2), using a local GVH assay (CBA lymphoid cells injected into (CBA X A/J)F1 recipients). The findings were as follows: The lymphoid cell number in the para-aortic lymph nodes ( PALN ) was increased at all stages of gestation. The peak occurred in the 2nd week in primiparity and as early as 60 h after fertilization in multiparity. PALN cell alloreactivity was weak at the beginning and higher than normal in the third week of pregnancy. Spleen cell alloreactivity was increased in the second week and decreased in the third week in primiparous compared with multiparous animals. Anti-paternal alloreactivity exhibited by spleen cells of allogestation was decreased (as compared to cells of isogestation ) especially in primiparous mice, particularly in the third week. At this time, the anti-paternal alloreactivity of PALN cells was increased. The influence of the recipient's sex on GVHR intensity was reversed when the cells were obtained from a pregnant donor, becoming stronger in male compared with female hybrids.     

过继转移FasL基因修饰的树突细胞对小鼠自然流产模型胚胎丢失的影响

目的:观察过继转移FasL基因修饰的树突细胞(DC)对小鼠自然流产模型胚胎丢失的影响,探讨它在诱导妊娠免疫耐受中的作用。方法:构建鼠源FasL(mFasL)真核表达载体pcDNA3.1-mFasL,用电转染法将它转染给DBA/2雄鼠骨髓来源的DC,将转染成功的mFasL-DC于交配前经腹腔注射给CBA/J母鼠。实验动物分为6组:(1)正常妊娠模型组(CBA/J×BALB/c);(2)未添加干预的流产模型组(CBA/J×DBA/2);(3)转输DC培养基(DCCM)的流产模型组;(4)转输单纯DC(DC)的流产模型组;(5)转输转染空质粒DC的流产模型组;(6)转输转染mFasL质粒DC的流产模型组,于妊娠第12~14天观察孕鼠胚胎丢失率。结果:转输mFasL-DC后孕鼠胚胎丢失率明显低于未添加干预或转输DC培养基的流产模型组(P<0.01),与转输单纯DC或带空质粒的DC组相比,其胚胎丢失率也明显下降(P<0.05),它与正常妊娠组相比胚胎丢失率无显著差异(P>0.05);转输单纯DC或空质粒的DC组与未添加干预流产模型组相比胚胎丢失率有所下降,但没有统计学差异;转输DC培养基组与未加干预组之间胚胎丢失率无统计学差异(P>0.05)。结论:过继转移mFasL-DC能诱导妊娠免疫耐受,降低小鼠自然流产模型孕鼠胚胎丢失率。     

Changes in natural killer cell activity in normal pregnant and postpartum women: increases in the first trimester and postpartum period and decrease in late pregnancy

Changes in the activity and number of natural killer (NK) cells in peripheral blood in normal pregnant and postpartum women were examined. NK activity was measured in a 4-h 51Cr-release assay and evaluated by conventional relative lytic units and absolute lytic units which represent the total NK activity within a fixed volume of circulating blood. The number of NK cells was analyzed with FITC-conjugated monoclonal antibodies and by use of an automated flow cytometer. Unexpectedly, the relative NK activity increased in the first trimester and also for 1 month postpartum compared to the activity in normal non-pregnant controls. On the other hand, absolute NK activity decreased in the third trimester compared to the activity in normal non-pregnant controls. The percentage of CD57+ cells decreased in the second trimester, but the percentage of CD16+ cells did not change during pregnancy or the postpartum period. The absolute counts of CD57+ cells and CD16+ cells decreased in the second and third trimesters and increased transiently in the postpartum period. These findings indicate that the increased NK activity in the first trimester and at 1 month postpartum is induced by increased cytotoxic activity of individual NK cells, and that the decreased NK activity in late pregnancy is induced by a decrease in the numbers of NK cells. These physiological changes may play an important role in implantation in early pregnancy, protection of the fetal allograft in late pregnancy and in the natural defense against infection during the puerperal period.     

Pregnancy rate and embryo loss in the NK1.1+ T cell-depleted mouse

OBJECTIVE: To determine the role of natural killer cell (NK) 1.1+ T cells in pregnancy and in embryo loss. STUDY DESIGN: Four groups of C57 mice, consisting of 20 animals each, were studied. Groups A and B included pregnant females treated with anti-NK1.1 monoclonal antibodies until they gave birth or non-NK1.1-depleted antibodies, respectively. In order to evaluate the role of NK1.1+ T cells in pregnancy, female mice in group C were treated with anti-NK1.1 monoclonal antibodies every seven days starting seven days prior to mating until they gave birth. Control mice in group D were not NK1.1-depleted. NK1.1+ T cell depletion was determined by flow cytometric analysis. RESULTS: Depletion of NK1.1+ T cells did not significantly change the pregnancy rate, nor did it significantly alter the number of live births. Numbers of live births tended to decrease in NK1.1-depleted mice, with a mean number of live births of 4.66 as compared with 6.09 in NK1.1-depleted and nondepleted mice, respectively. Similarly, mice treated with anti-NK1.1 monoclonal antibodies every seven days starting seven days prior to mating became pregnant at a rate of 40% as compared with 60% in non-NK1.1-depleted controls. A comparable trend was observed in the number of live births, with a mean number of live births of 5.0 as compared with 6.1 in NK1.1-depleted and non-NK1.1-depleted mice, respectively. CONCLUSION: In the NK1.1-depleted mouse model, NK1.1+ T cells do not have any direct effect on pregnancy rate or on early embryo loss.