Phenotypic Analysis of Adhesion Molecules in First-Trimester Decidual Tissue From Chorion Villus Samples

PROBLEM: First-trimester decidua contain CD56^bright /CD16^neg lymphocytes that phenotypically resemble a subset of peripheral blood natural killer (NK) cells. Factors controlling their localization to decidua are unknown, but they may relate to trophoblast invasion, endometrial decidualization, and adhesion molecule expression. METHOD OF STUDY: Ninety-one chorion villous samples (CVS) were screened for the presence of decidua, and selected specimens were analyzed for the expression of adhesion molecule pairs using a panel of monoclonal antibodies. RESULTS: Lymphoid cells in CVS-derived decidua expressed CD56, PECAM, intracellular adhesion molecule-1 (ICAM-1), and the integrins LFA-1, αEβ7, and α4β1, and co-receptors for these molecules were expressed by decidual stromal cells, invasive trophoblasts, and venule endothelium. Some endothelium expressed a cuboidal phenotype. CONCLUSIONS: The expression of complimentary pairs of adhesion molecules by maternal lymphoid cells and by either extravillous cytotrophoblast or decidual stromal cells supports the hypothesis that these cells interact within decidua. Also, both LFA-1 and α4β1 may contribute to decidual localization of CD56^bright NK cells.     

655例孕早期直接法绒毛染色体检查结果及分析

报道６５５例孕早期（７－１０周）直接法绒毛染色体检查结果。实验失败１４例，成功率为９７．８６％（６４１／６５５）。核型异常者３５例，占５．４６％（３５／６５５－１４）；其中不平衡者１９例。夫妇一方染色体异常为指征的绒毛核型异常率最高，为５３．３３％（１６／３０）；其次为生过染色体异常儿者，绒毛核型异常率为９．０９％（６／６６）。提示这些类型的孕妇进行产前诊断的必要性。     

ORIGINAL ARTICLE: Peripheral Blood NK Cells Reflect Changes in Decidual NK Cells in Women With Recurrent Miscarriages

Citation Park DW, Lee HJ, Park CW, Hong SR, Kwak‐Kim J, Yang KM. Peripheral blood NK cells reflect changes in decidual NK cells in women with recurrent miscarriages. Am J Reprod Immunol 2010; 63: 173–180 Problem We aimed to investigate if peripheral blood natural killer (pNK) cell levels are correlated with decidual NK (dNK) cell levels, and if chemokine expression has any role in dNK cell regulation. Method of study Decidual tissues of women having two or more miscarriages with normal karyotype were collected after miscarriage and an immuno‐histochemisty study was made. pNK cells were evaluated using flow cytometric analysis. Results The %CD3^?/56⁺ and %CD3^?/56⁺/16⁺ pNK cells showed a significant correlation with mean number of CD56⁺ dNK cells. The number of decidual CD16⁺ cells was significantly higher in women with elevated pNK (≥15%) than that of normal pNK (<15%). The %CD3^?/56⁺ and %CD3^?/56⁺/16⁺ pNK cells showed an inverse correlation with duration of gestation. The CCL3⁺ and CXCL12⁺ cells were present in the decidua; however, staining intensity was not correlated with number of dNK cells. Conclusion The pNK cell levels reflect changes in dNK cell levels. This implicates that pNK cell level is a clinically useful marker to predict pregnancy outcome. Further study is needed to examine if elevated pNK cells enhance recruitment of dNK cells in the decidua.     

Phenotypic Characterization of CD7 +, CD3 +, and CD8+ Lymphocytes From First Trimester Human Decidua Using Two-Color Flow Cytometry

PROBLEM: There is increasing evidence that decidual lymphocytes play a major role in local interactions at the fetomaternal interface. METHOD: In this paper we use two-color flow cytometry to delineate the phenotype of lymphocytes obtained from human early decidua by mechanical dispersal technique. RESULTS: The most abundant decidual lymphocytes expressed CD7, CD38, CD56, and CD2 markers, relatively small proportions of CD3 ⁺, CD8 ⁺, CD4 ⁺, CD16 ⁺, CD45RA⁺, CD11b⁺, and Leu8⁺ cells were also present. The vast majority of decidual CD7⁺ lymphocytes expressed CD38, CD2, and CD56 markers and were CD3 ^?, CD8 ^?, CD 16 ^?, and CD57 ^?. Decidual CD3⁺ lymphocytes were weakly staining for TCR α/β, lacked T-cell receptor (TCR) γ/δ molecules, and ～40% of them expressed HLA-DR. All decidual CD8⁺ lymphocytes were CD2⁺ and the majority of them expressed CD38, CD56, and CD7 markers and were CD3^?at the same time CD8 ⁺ CD7^? lymphocytes were found in decidua. According to the expression of the CD45RA marker, decidual CD8 ⁺ lymphocytes could be divided into two subsets: CD8 ⁺ CD45RA ⁺ CD56 ⁺ and CD8 ⁺ CD45RA ^?. CONCLUSIONS: These data clearly demonstrate that decidual lymphocytes display phenotypical features different from those of their counterparts in peripheral blood.     

Human Lymphocytes Obtained from Decidual Tissue Express Killer Activatory Receptors as Well as Killer Inhibitory Receptors: Analysis Using a Single Strand Conformation Polymorphism Method

PROBLEM: The objective was to clarify whether lymphocytes in decidual tissue express only killer inhibitory receptors (KIRs) or both KIRs and killer activatory receptors (KARs). METHOD OF STUDY: A polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis method, using cDNA as a template, was applied to detect as many natural killer receptors (NKRs) expressed on natural killer (NK) cells as possible and to distinguish between KIRs and KARs. Using this method, we analyzed NKRs on lymphocytes in decidual tissue and maternal peripheral blood mononuclear cells (PBMCs) (n = 10) from the same individuals. RESULTS: More than 20 SSCP bands were detected for NKRs on both the lymphocytes in decidual tissue and the maternal PBMCs. The SSCP band patterns were different for each individual. KARs mRNA was detected in lymphocytes in decidual tissue according to the SSCP analysis results. CONCLUSION: Lymphocytes in decidual tissue express not only KIRs but also KARs; this finding suggests their roles in placentation and the maintenance of pregnancy.     

42例孕妇孕早期绒毛组织细胞遗传学分析

目的评价早孕期B超引导下经腹绒毛取材组织样本的细胞遗传学分析在产前诊断中的应用价值。方法回顾分析2010年1月到2011年6月42例早孕期产前细胞遗传学诊断的病例资料。结果 42例取材时间9～13周,穿刺成功率100%,培养成功率为97.6%,平均培养时间为10天,发现异常核型4例。结论孕9周后行B超引导经腹绒毛取材是一项安全可行的早孕期介入性产前诊断方法。绒毛细胞长期培养法和染色体制备方法简单、技术稳定、结果可靠,可用于胎儿染色体疾病的产前诊断。     

南宁地区313例绒毛细胞染色体核型分析

目的探讨通过绒毛细胞培养及染色体核型分析,为产前遗传咨询提供可靠的实验室依据。方法对2006年至2011年间在我院遗传门诊就诊的313例有产前诊断指征的妊娠妇女,在超声的引导下进行腹穿绒毛活检,获取绒毛组织进行培养并进行染色体制备分析。结果 313例标本中共300例培养成功,13例培养失败,成功率为95.8%;其中核型正常288例,异常36例。结论绒毛细胞培养技术稳定可靠,能早期明确染色体异常,为产前遗传咨询提供依据,降低出生缺陷的发生。     

绒毛细胞长期培养法在产前诊断及早孕和早中孕期自然流产病因检测中的应用

目的探讨绒毛细胞长期培养法在产前诊断及早孕和早中孕期自然流产病因检测中的应用价值及两者有无差异。方法采用绒毛细胞长期培养法分别对147例B超引导下经腹绒毛取材的产前诊断病例及384例自然流产患者的流产绒毛进行染色体培养制作、核型分析以及结果对照比较。结果产前诊断绒毛培养成功率为96.60%,明显高于自然流产绒毛的培养成功率（87.24%）;两者的平均培养天数无明显差异（11.5天vs12.3天）;流产绒毛染色体异常率（63.88%）明显高于产前诊断绒毛（9.15%）。结论绒毛细胞长期培养法是一种有效的检测绒毛染色体的方法 ,同时适用于产前诊断绒毛和流产绒毛;绒毛标本的无菌性、新鲜程度对绒毛细胞培养成功率有着重要影响。     

Mechanisms underlying recruitment and accumulation of decidual NK cells in uterus during pregnancy

Natural killer (NK) cells represent the most prominent immune cell type found in the uterus in the first trimester of human pregnancy and in the secretory phase of menstrual cycle. The role of NK cells in pregnancy has been largely discussed over the past years and it is now becoming increasingly clear that they may influence pregnancy outcome at several levels. In normal pregnancy, it appears that the major function of NK cells is to provide benefit by secreting a number of cytokines, chemokines and angiogenic factors rather than to exert a cytotoxic activity. However, the origin of decidual NK cells is still debated and it remains unclear whether they can derive from NK cell populations recruited from peripheral blood and/or other tissues or from self renewal of NK cell progenitors present in the uterus prior to pregnancy or recruited from other tissues. Here, we review the molecular mechanisms underlying peripheral blood NK cell recruitment and its role in the accumulation of NK cells in the decidua during early pregnancy.     

Comparative Analysis of the Immunophenotypes of Decidual and Peripheral Blood Large Granular Lymphocytes and T Cells During Early Human Pregnancy

PROBLEM : The functional role of the leukocytes in the decidua is not clear. They may regulate the maternal immune response to the fetal allograft. However, the factors controlling maternal and fetal communication have not yet been identified. METHOD : A comparative analysis of the phenotypes of decidual and peripheral blood large granular lymphocytes (LGLs) and T lymphocytes in early human pregnancy was performed on decidual tissue and blood samples obtained from ten patients at therapeutic abortion. RESULTS : Whereas most of the decidual LGLs were found to have a CD56^bright++ phenotype, most of the peripheral blood NK cells (90%) showed the classical CD56^dim+ phenotype, and only a small proportion were CD56^bright+ cells. Another striking difference was found in the expression of very late antigen 1 (VLA-1, CD49a): Almost all the decidual CD56^bright++ LGLs, but virtually none of the peripheral blood CD56⁺ NK cells expressed this antigen. Further differences were found in the expression of CD16, CD44, CD45RA, CD54, and CD57. There were also differences in phenotype between T cells derived from decidual tissue and those derived from peripheral blood. Approximately 31% of the CD3⁺ decidual T cells expressed VLA-1, but this antigen was virtually absent on peripheral blood T cells. A further difference was seen in the expression of HLA-DR. This activation antigen was found on 32 ± 13% of the decidual T cells but only 8 ± 5% of the peripheral blood T cells. Additionally, the proportion of cells expressing CD38 was higher among decidual than peripheral blood T cells. CONCLUSION : The findings suggest that both decidual LGLs and a subset of decidual T cells are activated and possibly play a role in the control of trophoblast growth and placental development.     

An evaluation of maternal cell contamination in cultures of chorionic villi for the prenatal diagnosis of chromosome abnormalities

Culture of chorionic villus cells provides a method of obtaining chromosomes of excellent quality for first trimester prenatal diagnosis. Concern exists that maternal cells present in the biopsy may contaminate the culture and lead to misdiagnosis. This study has confirmed that karyotypes obtained from female villus cultures were non-maternal by establishing the presence of paternal markers using Q- and C-banding. Male cultures were harvested serially to investigate the possibility of maternal cell overgrowth. Of 82 successful cultures investigated, 37 were male and 45 female and of the males 4 contained a mixture of male and female cells. Thorough dissection of the material is essential if maternal cell contamination is to be mininimised. The use of heteromorphic chromosome markers to establish that fetal cells have grown provides valuable reassurance.     

Characterization of NKT Cells in Human Peripheral Blood and Decidual Lymphocytes

PROBLEM: To examine whether natural killer (NKT) cells are present in human pregnancy decidua. METHOD OF STUDY: We calculated the percentage of CD3+CD161+Valpha 24+-NKT cells in peripheral blood and early pregnancy decidua, and analyzed intracellular cytokines, interleukin (IL)-4 and interferon (IFN)gamma in NKT cells using flow cytometry. RESULTS: A distinct subset of CD3+ CD161+ lymphocytes expressing an invariant antigen receptor encoded by the Valpha24 and Vbeta11 segment was accumulated in the decidua. In pregnant subjects the percentages of NKT cells were significantly increased in the decidua compared with peripheral blood. Both NKT cells in the decidua and the peripheral blood had an ability to rapidly produce cytokine associated with Th1 (IFNgamma) and Th2 (IL-4). Interestingly, the percentages of IL-4 and IFNgamma producing NKT cells were significantly higher in the decidua compared with the peripheral blood. CONCLUSIONS: These findings suggest that NKT cells might control the Th1/Th2 balance by producing IL-4 and IFNgamma at the feto-maternal interface.     

In Vitro Genotoxic and Cytotoxic Effects of Protein Somatic Products from Helminths on Donor Blood Lymphocytes

Protein somatic products of adult helminths Hymenolepis nana and Toxocara canis and secretory-excretory somatic product of Trichinella spiralis larvae in vitro produced genotoxic and cytotoxic effects on donor blood lymphocytes, which manifested in accumulation of single-chain breaks, alkaline-labile sites in nuclear DNA, and apoptotic cells. This effect depended on the concentration of parasitic protein products during coculturing.     

The In Vitro Effect of a Calf Thymus Extract (Thymostimulin) on T Cell Phenotypes in Cord Blood Lymphocytes

ABSTRACT: An investigation was carried out on the in vitro effect of a calf thymus extract, thymostimulin, on the distribution of T cell phenotypes as defined by OKT3, OKT4, and OKT8 murine monoclonal antibodies and on E-rosetting cells in human cord blood lymphocytes from healthy newborns. The percentages of E-rosette-forming lymphocytes and OKT3⁺ total T population were lower in newborns than in adults (E-rosettes: 43.8% ± 13% vs 57.9% ± 7.9%, p < 0.01; OKT3⁺ cells: 53.3% ± 15.5% vs 79.9% ± 4.7%, p < 0.01), while the OKT4⁺/OKT8⁺ (helper/suppressor) cell ratio was normal in both (newborns: 3.40; adults: 2.44—NS). Thymostimulin increased the number of E-rosette-forming cells from 43.8% ± 13% to 49.9% ± 12.7% (p < 0.01), as well as the percentage of phenotypic T lymphocytes. The highest increases were observed in the OKT4⁺ cells (37.7% ± 14% to 49.1% ± 13.8%, p < 0.001), while smaller changes were observed in the OKT3⁺ cells (53.3% ± 15.5% to 58.1% ± 13.3%, p < 0.02) and OKT8⁺ cells (12.8% ± 6.4% to 16.6% ± 6.5%, p < 0.02). The results of the present study suggest that thymostimulin mainly provokes an increase in the helper T cell phenotype in cord blood lymphocytes.     

IFN-γ/IL-4对早孕期蜕膜细胞和绒毛滋养层细胞的体外影响

探讨Th1/Th2型细胞因子对人孕早期绒毛滋养层细胞和蜕膜细胞活性及其内分泌功能的影响。绒毛滋养层细胞和蜕膜细胞活性采用MTT法进行检测 ,绒毛组织分泌的人绒毛膜促性腺激素 (hCG )和蜕膜组织分泌的催乳素 (PRL )均采用放免法 (RIA )进行分析测定。结果 :(1)一定浓度范围的Th1型细胞因子IFN γ (10～ 10 0 0ng/ml)和Th2型细胞因子IL 4 (1～ 10ng/ml)对绒毛滋养层细胞活性及其hCG分泌分别有不同程度的抑制作用 (P <0 0 1)和促进作用 (P <0 0 1) ;(2 )Th1型细胞因子IFN γ低浓度时 (1～ 10ng/ml)对蜕膜细胞活性及PRL分泌有刺激作用 (P <0 0 5 ) ,高浓度时 (10 0～ 10 0 0ng/ml)则有抑制作用 (P <0 0 1) ,Th2型细胞因子IL 4对蜕膜上述功能的调节作用与对绒毛滋养层的调节作用相似。Th1/Th2型细胞因子可能是通过影响绒毛滋养层细胞和蜕膜细胞活性及其内分泌功能而在早期妊娠中起重要的免疫调节作用     

不同临床期多发性骨髓瘤患者细胞免疫功能流式细胞术分析

本文应用流式细胞仪（ＦＣＭ）分析了２３例不同临床期多发性骨髓瘤（ＭＭ）患者Ｔ细胞亚群、ＮＫ细胞及ＣＤ１９＋Ｂ淋巴细胞变化。结果显示：初诊未化疗ＭＭ患者ＣＤ４、ＣＤ２５、ＣＤ１９细胞百分率ＣＤ４／ＣＤ８比值均下降，ＣＤ８细胞百分率升高；稳定期ＭＭ患者除ＣＤ２５、ＣＤ１９细胞百分率下降外，其他指标均正常；进展期ＭＭ患者ＣＤ３、ＣＤ４细胞百分率和ＣＤ４／ＣＤ８比值下降，ＣＤ８、ＣＤ１９细胞百分率升高；不同临床期ＭＭ患者ＮＫ细胞与正常人比较无显著差异（Ｐ＞０．０５）。研究结果表明：不同临床期ＭＭ患者细胞免疫功能不同，细胞免疫调节异常在ＭＭ发病机制及稳定期脱逸过程中可能起一定作用。     

Activation and Function of the Rap1 Gtpase in B Lymphocytes

Rap1 is a monomeric GTPase that is closely related to Ras. In this review, we summarize our recent work showing that the B cell antigen receptor (BCR), as well as chemokine receptors, activate Rapl via a pathway that involves phospholipase C-dependent production of diacylglycerol (DAG). The possible identities of the DAG-regulated guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that regulate the activation of Rap1 by the BCR and chemokine receptors will be discussed. Although initially thought to be an antagonist of Ras-mediated signaling, Rap1 does not appear to modulate the ability of the BCR to activate downstream targets of Ras. Instead, activation of Rap1 promotes B cell adhesion as well as B cell migration toward chemokines. Thus, Rap1 may play a key role in a number of processes that are essential for B cell development and activation.     

Down-Regulated Expression of Perforin-Positive/CD16+ Cells in the Peripheral Blood Lymphocytes in the First Trimester of Pregnancy and Up-Regulation at the End of Pregnancy

PROBLEM: Immunophenotypic profiles of perforin (P)-positive peripheral blood lymphocytes in the first trimester and at the term of human pregnancy were analyzed. METHOD OF STUDY: Perforin expression in peripheral blood lymphocyte subsets was measured by simultaneous detection of P (intracellular antigen) and cell-surface antigens (CD3, CD4, CD8, CD16, and CD56) by flow cytometry in nonpregnant (NP) and pregnant women in the first trimester (FTP) and at the time of parturition (TP). RESULTS: The percentage of total P⁺ cells in peripheral blood compared to nonpregnant women was slightly lower in the FTP but significantly higher at TP. Perforin-positive cells were significantly elevated in T lymphocyte subsets (CD3⁺P⁺, CD4⁺P\ CD8⁺P⁺) in both the FTP and TP groups, as was the percentage of CD56⁺P⁺ cells. Profound changes in the CD16⁺ subpopulation were found in the FTP group compared to both the NP and TP groups (a drastic decrease of CD16⁺P⁺ cells; CD16⁺ cells among P⁺ cells; P⁺ cells among CD16⁺ cells). A considerable part of CD3⁺ cells in both the FTP and TP groups are CD3⁺CD56⁺P⁺. The average fluorescence intensity (AFI) for P (a measure of P content per cell) was significantly decreased in FTP and increased in TP groups. CONCLUSIONS: The CD16 molecule is FcγRIIIA which is the only Fc receptor responsible for antibody dependent cell-cytotoxicity (ADCC) of NK and T-cells. In the first-trimester human pregnancy this mechanism is severely down-regulated compared to both the NP and TP groups.     

小鼠骨髓来源树突状细胞的分离与扩增培养

目的 :探讨树突状细胞 (DC)分离纯化及其体外扩增的方法。方法 :无菌制备C57BL/6小鼠骨髓 ;依次用红细胞裂解液去除红细胞 ,通过半粘附法去除T、B细胞 ,又在粒细胞巨噬细胞集落刺激因子 (GM CSF)和白细胞介素 4 (IL 4 )协同诱导下培育 ,DC前体分化发育成DC并扩增。在第 7天用脂多糖 (LPS)和肿瘤坏死因子 a (TNF a)刺激 4 8h ,检测细胞因子白介素 12 (IL 12 )浓度及细胞表面标志CD11c、CD80、CD86和MHCⅡ。结果 :DC细胞数增加 ,其形态在光镜下多为特征性星形 ,也有梭形和多角形 ;至培养第 9天DC细胞表面标志CD11c、CD80、CD86、MHCⅡ阳性率分别为 86 .32± 12 .14 %、76 .4 2± 8.4 5%、77.12± 9.0 5%、6 8.4 5± 6 .84 % ,IL 12浓度较未用LPS和TNF a组明显增加 (P <0 .0 1)。结论 :①结果所得细胞的形态和功能符合DC ;②用LPS和TNF a刺激可以获得成熟DC。