Spontaneous proliferative activity of lymphocytes in normal and EPH gestosis complicated pregnancy]

The spontaneous proliferative activity of lymphocytes and CD4+/CD8+ ratio has been estimated in normal and EPH gestosis-affected pregnancy; also the effects of autologous and allogenic sera on this lymphocyte activity was evaluated. Although in normal pregnancy there was a very low CD4+/CD8+ ratio, the spontaneous proliferative activity of lymphocytes and the effects of autologous sera exerted thereupon were the same in either healthy pregnant women or in non-pregnant controls, what indicates a normal cellular immunity in physiological pregnancy. In gestosis an increased proliferative activity of lymphocytes and very high CD4+/CD8+ ratio was observed, when referred to non-pregnant controls. The serum of normal pregnant women did not reveal any immunosuppressive features against the activity of both autologous lymphocytes and gestosis--affected pregnancy, whereas it clearly inhibited the lymphocyte proliferation in non-pregnant controls. A different model of the autologous sera effects on the lymphocyte activity in gestosis women--when compared with the other examined women--is an evidence for altered immuno-modulating parameters of the serum in question.     

Effects of sera of gynecological cancer and pregnancy on cellular immunity

The effects of sera from untreated gynecological cancer patients in 1st, 2nd, 3rd trimesters of normal pregnancy on PHA and MLR induced lymphocytes blastformation, natural killer cell activity and killer T cell induction were analysed. 1. The 3H-thymidine uptake of lymphocytes by PHA and MLR significantly decreased in cancerous and 3rd trimester pregnant sera. It is thought that the existence of non specific immunosuppressive factors in these conditions, and this phenomenon might be caused by coating and/or masking of the surface of the effector cell. The factors might have an effect on the proliferation phase of lymphocyte blastoformation. 2. Normal human lymphocytes pretreated with cancerous sera inhibited natural killer cell activity. This phenomenon could not be demonstrated with pregnant sera. 3. Cancerous and 3rd trimester pregnant sera suppressed the process of killer T cell induction caused by alloantigen.     

Cell-mediated immunity in normal pregnancy and pre-eclampsia

The in vitro responses of maternal lymphocytes to phytohaemagglutinin (PHA), concanavalin A (Con A) and purified protein derivative of tuberculin (PPD) were determined in uncomplicated and pre-eclamptic pregnancies and nongravid female controls. PHA, Con A and PPD responses were significantly lower in both pregnant groups compared to nongravid controls. No difference was observed in lymphocyte reactivity between women with pre-eclampsia or uncomplicated pregnancy. Both pregnant groups exhibited normal numbers of circulating T and B lymphocytes. The number of active E rosette-forming cells was significantly higher in peripheral blood of pre-eclamptic patients.     

Origin of raised maternal serum alpha-fetoprotein levels in second-trimester oligohydramnios.

Concanavalin A (Con A) subtyping of alpha-fetoprotein (AFP) revealed higher concentrations of AFP non-reactive with Con A in sera of 12 pregnant women with second-trimester oligohydramnios and raised total serum AFP levels than in sera of 42 pregnant women with raised total serum AFP levels and a normal amniotic fluid volume. This suggests that in oligohydramnios the origin of excess AFP in the maternal compartment is amniotic fluid. It is proposed that oligohydramnios and the associated raised maternal serum AFP levels are caused by damage of the fetal membranes prior to 16 weeks of gestation resulting in leakage of amniotic fluid to the decidual tissue and resorption in the maternal circulation.     

TNF-alpha from monocyte of patients with pre-eclampsia-induced apoptosis in human trophoblast cell line

OBJECTIVE: In pre-eclampsia, fetal growth restriction is frequently observed, and the possible involvement of inhibitory substances on trophoblast cell proliferation and differentiation has been suggested. The objective of this study was to investigate the effects humoral factors, such as cytokines, produced in immune cells on proliferation of an immortalized trophoblastic cell line (TCL) that we established. METHODS: Serum and lymphocyte layers were isolated from the blood of normal pregnant and preeclamptic women. The lymphocyte layer was further fractionated into different immune cell populations by the Stem Sep method. Immortalized trophoblastic cells were cultured with the sera diluted. The cytokine concentrations in the supernatants of lymphocyte cultures were compared between normal pregnancy and pre-eclampsia. The number, DNA content and induced apoptosis were examined on the immortalized trophoblastic cells at the end of culture. RESULTS: The sera from preeclamptic women significantly inhibited the immortalized trophoblastic cell proliferation in comparison with those from normal pregnancy. Among the lymphocyte fractions, monocytes significantly inhibited the immortalized trophoblastic cell proliferation. The monocytes from preeclamptic women were found to produce higher levels of tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant than those from normal pregnant women. The coculture with the monocytes from preeclamptic women increased the frequency of TUNEL-positive TCL cells. TNF-alpha inhibited immortalized trophoblastic cell proliferation in a dose-dependent manner and induced apoptosis. CONCLUSION: The present results suggest that monocytes are activated and that cytokines, such as TNF-alpha, which is produced by monocytes, induce apoptosis and inhibit proliferation of trophoblast cells in pre-eclampsia.     

早孕妇女血清及蜕膜组织液对自然杀伤细胞活性及淋巴细胞增殖的影响

目的　探讨早孕妇女血清及蜕膜组织液对自然杀伤 (NK)细胞及淋巴细胞功能的影响。方法　采用同位素掺入法 ,检测 3 2例正常早孕妇女血清 (早孕血清组 )及蜕膜组织液 (蜕膜组织液组 )对正常妇女外周血中NK细胞活性 (% )及淋巴细胞增殖功能的影响 ,并采用 2 5例正常未孕妇女的血清作对照 (对照组 )。结果　早孕血清组、蜕膜组织液组及对照组的NK细胞活性分别为 (3 5 .8±3 .0 ) %、(19.1± 2 .9) %及 (46.1± 3 .2 ) % ,淋巴细胞增殖功能 [每分钟同位素放射次数 (CPM) ]分别为(3 689± 4 5 6)CPM、(2 0 5 9± 2 88)CPM及 (4883± 64 3 )CPM ,3者间差异均有极显著性 (P <0 .0 1) ,且蜕膜组织液组均显著低于早孕血清组 (P <0 .0 0 5 )。结论　正常早孕妇女血清及蜕膜组织液具有免疫抑制作用 ,而蜕膜组织液可能是调节早期妊娠子宫局部免疫功能的重要因素之一。     

Lymphocyte transformation during pregnancy: an analysis using whole-blood culture

In an effort to provide information on maternal immunocompetence, a study was undertaken to measure lectin-induced transformation of maternal lymphocytes. Whole-blood culture was used. The phytohemagglutinin (PHA)-induced lymphocyte transformation of 137 pregnant women was significantly (P less than 0.05 to 0.001) diminished until 21 weeks' gestation, and concanavalin A (Con A)-induced lymphocyte transformation of 116 pregnant women was also significantly (P less than 0.01 to 0.001) diminished at all stages of gestation, as compared with those of 39 nonpregnant women. In addition, the Con A-induced lymphocyte transformation of ovarian venous blood and uterine venous blood from a pregnant woman was prominently diminished, as compared with that of basilic venous blood. It is suggested that there are maternal T-cell subpopulations that have different reactivities to PHA and Con A.     

ELISA test for the detection of an immunological blocking factor in human pregnancy serum

We have previously shown that progesterone-treated lymphocytes of healthy pregnant women can produce a 34 kDa protein, progesterone-induced blocking factor (PIBF) capable of blocking lymphocyte function in vitro. Lymphocytes of women with idiopathic threatened pre-term delivery failed to produce this factor. On the assumption that the PIBF appears in the serum of healthy pregnant women but not in that of women at risk for immunologically based abortion of pre-term delivery, an ELISA has been designed for detecting the blocking factor in serum samples. Sera obtained at the time of delivery, as well as those from women with pre-term deliveries or miscarriages, contained significantly less PIBF than sera of 209 healthy pregnant women. Data obtained by testing sera of women at 16 weeks gestation whose pregnancies ended in spontaneous abortion showed that the predictive value of the test depended on the time interval between blood sampling and the onset of abortion. In 11 of 13 women with uterine contractions the outcome of pregnancy was predictable by normal or lower than normal levels of PIBF in the sera taken at the time the symptoms were presented. The data suggest that determination of PIBF in pregnancy serum might contribute to the diagnosis of immunologically bases pre-term disruption of pregnancy.     

Immunosuppressive activity of human amniotic fluid of normal and abnormal pregnancies

Twenty specimens of amniotic fluid (AF) obtained between week 16 and 18 of gestation from normal pregnant women and six specimens from pregnant women in which trisomia of chromosome 21 was found were tested for immunosuppressive activity. Incubation of normal human donor lymphocytes with 0.2-1 mL of AF from normal pregnant women for one hour at 37 degrees C was sufficient for induction of significant inhibition of the ability of these cells to induce a local xenogeneic graft-versus-host reaction (GVHR) as well as inhibition of E and E-active rosette formation, the GVHR being the most sensitive test. On the other hand, amniotic fluid obtained from the six pregnant women in which trisomia of chromosome 21 was found showed no inhibitory activity in either the E or E-active rosette formation, nor in the local xenogeneic graft-versus-host reaction. AF from all the women tested was found to have no effect on phenotype expression of the lymphocytes, as tested by the monoclonal antibodies OKT4+ and OKT8+, nor on B-lymphocytes, as tested by surface immunoglobulins. No correlation was found between the alpha-fetoprotein levels in the sera of those women and the immunosuppressive activity. These findings indicate that genetic defects of the conceptus are not limited to the embryo but may affect the composition of immunosuppressive components present in normal amniotic fluid.     

正常孕妇,新生儿及异常胎儿血清中人微小病毒B19感染的研究

目的：了解人微小病毒Ｂ１９在母婴中的感染状况以及该病毒对胎儿的危害。方法：应用聚合酶链反应技术（ＰＣＲ）检测３５０例正常孕妇及新生儿血清、９例异常胎儿组织血清人微小病毒Ｂ１９ＤＮＡ。结果：正常孕妇血清Ｂ１９ＤＮＡ阳性率为１．１４％，新生儿脐血阳性率为０．２８％；９例异常胎儿中有６例孕妇血清及脐血Ｂ１９ＤＮＡ阳性（６／９）。应用原位杂交的方法在ＰＣＲ阳性的异常胎儿脑和脾组织中也检出了Ｂ１９ＤＮＡ阳性颗粒。结论：应用ＰＣＲ法检查孕妇及新生儿Ｂ１９病毒具有高度敏感、简便、特异的特点，而原位杂交方法可对病毒感染进行定位，以了解感染部位的病理变化。     

Immunosuppressive activity and alpha interferon concentrations in human embryo culture media as an index of potential for successful implantation.

OBJECTIVE: To evaluate potential correlations between establishment of pregnancy and immunosuppressive activity secreted by the preimplantation embryo. DESIGN: To evaluate immunosuppressive activity, supernatants from preimplantation embryos were assessed for their ability to inhibit lymphocyte proliferation. Additionally, alpha interferon concentrations were also measured in these supernatants. We compared these parameters from embryo culture supernatants of women who did and did not achieve pregnancy after in vitro fertilization (IVF). Immunosuppression was assessed using a lymphocyte proliferation assay with concanavalin A (Con A) and phytohemagglutinin (PHA) as mitogens. SETTING: In vitro fertilization program at the University of Utah Medical Center. PARTICIPANTS: Couples less than 40 years of age, with normal semen quality and bilateral tubal obstruction. RESULTS: Immunosuppression calculated using the stimulation index (mean +/- SEM) in pregnant and nonpregnant women, respectively, were: Con A: 43.9 +/- 3.9 versus 19.1 +/- 10.1, P less than 0.04. PHA: 23.6 +/- 5.6 versus 12.5 +/- 12.8, P less than 0.02. Alpha interferon levels (mean +/- SD) in pregnant and nonpregnant women were not significantly different: 23.98 +/- 9.6 U/mL versus 24.79 +/- 2.5 U/mL. CONCLUSIONS: We conclude that pre-embryos with the capacity for successful implantation secrete greater amounts of immunosuppressive factors than those destined not to implant, as measured by Con A and PHA lymphocyte proliferation assays. Refinement of assay techniques and identification of the substances involved could have significant impact on IVF programs.     

Immunosuppressive effects of rabbit blastocoelic fluid and embryo culture medium

In order to understand the mechanism of the feto-maternal immune relationship, we assayed the immunosuppression activities of fresh blastocoelic fluid and decomplemented peripheral serum collected from day-9 pregnant white New Zealand rabbits and of rabbit embryo culture medium (ECM). Because the viability of the human lymphocytes was not affected by either of these biological fluids and since they were easy to obtain in sufficient quantities, they were used uniformly in all the experiments. Immunosuppressive effect was calculated by the relative inhibition of proliferation of Con A-stimulated lymphocytes. The immunosuppressive effect of blastocoelic fluid of the 9-day pregnant rabbits was significantly higher than that of autologous decomplemented serum (P less than 0.001). The inhibition by the serum was non-specific because sera from non-pregnant animals as well as sera from different stages of pregnancy and pseudo-pregnancy showed the same level of inhibition. The ECM of 6.5-7-day-old embryo showed a pronounced immunosuppressive effect. When embryos of 1,3 and 5 days were cultured and their culture media were assayed only with 5-day-old embryo the effect had begun to appear, but it was far less than that of 7-day-old embryo (P less than 0.02). The suppressive activity of both the blastocoelic fluid and ECM was not due to cytotoxic effect, since this fluid supported the in vitro growth of single-cell rabbit embryos up to the stage of blastocyst. These results suggest that the immunologic tolerance of the embryo might be due to the immunosuppressors secreted by the embryo and that there might be a localized effect at the implantation site rather than a maternal systemic immunosuppressive effect.     

Status of autoantibodies to zona pellucida in human reproduction

The sera of 80 patients were tested by the indirect immunofluorescence technique to evaluate the presence of autoantibodies to zona pellucida in human reproduction. The incidence of positive anti-zona activity was 71.4% (25/35) in the infertile women, 40.0% (6/15) in the normal, nonpregnant fertile women, 20.0% (3/15) in the normal pregnant women and 26.7% (4/15) in the normal fertile men, when unabsorbed sera were tested. To overcome false positive reactions due to nonspecific serum components, all the positive sera were absorbed with formalinized porcine red blood cells and retested. However, after absorption, anti-zona activity was lost in all the positive sera from non-pregnant fertile women, pregnant women and fertile men; but it was retained in 51.4% (18/35) of the infertile women. These positive sera (18) were further absorbed with zona-coated eggs and retested. Fluorescence was lost in all the positive sera, thus demonstrating the presence of antibodies specific to zona antigen in the infertile women. The study also revealed that autoantibodies to zona were seen more often in patients of greater age (26 to 40 years) and in those who had been infertile for a greater period of time (greater than 6 years).     

Interferon-like substance in pregnancy sera

Interferon(IFN) can be produced from lymphocytes in vitro as well as in vivo using various immune stimuli. This paper shows that the amount of IFN or IFN-like substance increases in pregnancy sera as pregnancy proceeds. 1) IFN activity was detected in 45% of 97 pregnant women overall and more frequently as pregnancy went on. 2) The IFN was trypsin sensitive, beat labile, acid sensitive and species specific. 3) Maternal lymphocytes produced approximately twice as much interferon as cord blood lymphocytes did by phytohemagglutinin stimulation. 4) IFN was detected in half of the mixed lymphocyte cultures(MLR) containing cord blood and maternal blood. Whether or not they can produce IFN may be an indication of the immunological competence of pregnant women and the interferon may contribute to the immunoregulation of fetal acceptance.     

Serum adenosine deaminase activity and its isoenzyme pattern in women with normal pregnancies

Adenosine deaminase (ADA) is a purine enzyme which is essential for the proliferation, maturation and function of lymphoid cells, and congenital deficiency of this enzyme is associated with severe combined immunodeficiency disease. The activity of ADA has changed in diseases characterized by the alteration of cell-mediated immunity such as rheumatoid arthritis, systemic lupus erythematosus and tuberculosis, so ADA has been considered as a nonspecific marker of cell-mediated immunity. In this study we examined changes in serum total ADA activity and the patterns of two ADA isoenzymes, ADA1 and ADA2 in normal pregnant women, and evaluated the possible role of the alteration of cell-mediated immunity during normal pregnancy as causes of changes in ADA activity. We measured serum activities of total ADA, ADA1 and ADA2 in normal pregnant women in the third trimester (n=24) and age-matched healthy nonpregnant women (n=24). Peripheral blood lymphocytes and monocytes were also measured. In normal pregnant women, serum total ADA activity averaged 10.5 +/- 0.5 U/L, which was significantly lower than in nonpregnant women (14.0 +/- 0.5 U/L ) (p<0.05), and mean serum ADA2 activity also significantly reduced that of nonpregnant women (p<0.05). There was no significant difference in ADA1 activity in normal pregnant and nonpregnant women. The decrease in total ADA activity was accompanied by the decrease in lymphocyte count. These results suggest that reduced serum total ADA activity reflects decrease in ADA2 activity, and which may be in part associated with depressed cell-mediated immunity during normal pregnancy.     

妊娠肝内胆汁淤积症患者的胎儿淋巴细胞研究

目的探讨胎儿淋巴细胞在妊娠肝内胆汁淤积症(ICP)发病中的作用。方法采用单向混合淋巴细胞反应法,检测20例ICP患者(ICP组)及20例正常孕妇(对照组)的脐血胎儿淋巴细胞与母体外周血已灭活的淋巴细胞、皮肤组织可溶性抗原、蜕膜组织可溶性抗原的增殖反应情况。结果(1)ICP组脐血胎儿淋巴细胞与母体已灭活的淋巴细胞混合反应中,胎儿淋巴细胞的增殖反应程度为2．75±0．36,显著高于对照组的1．45±0．19,两组比较,差异有统计学意义(P＜0．05);(2) ICP组脐血胎儿淋巴细胞与母体蜕膜组织可溶性抗原混合反应中,胎儿淋巴细胞的增殖反应程度为1．45±0．19,显著高于对照组的0．67±0．24,两组比较,差异有统计学意义(P＜0．05);(3)ICP组脐血胎儿淋巴细胞与母体皮肤组织可溶性抗原反应中,胎儿淋巴细胞的增殖反应程度为1．22±0．44,显著高于对照组的0．66±0．27,两组比较,差异有统计学意义(P＜0．05)。结论胎儿淋巴细胞可能是ICP发病过程中的主要效应细胞之一;母-胎间免疫失衡是ICP发病的重要机制之一。     

Mitogenic responses of peripheral blood lymphocytes from pregnant and ovarietomized heifers and their modulation by serum

Lymphocyte-enriched peripheral blood mononuclear leukocytes (PBL) were obtained concurrently from a group of gestating heifers at intervals throughout pregnancy and from a group of ovariectomized heifers of the same age. The cells were suspended in media containing either ovariectomized heifer sera or sera from heifers at the homologous stage of pregnancy, and incubated with various concentrations of Concanavalin A (Con A), Pokeweed Mitogen (PWM) or phytohemagglutinin (PHA). Their mitotic response to the lectins was then assessed by determining [³H]thymidine incorporation into DNA. The response of PBL from pregnant heifers gradually increased as pregnancy progressed when compared with the response of PBL from ovariectomized heifers. The increased response continued until parturition but was not detectable at two weeks postpartum. This pattern was demonstrated for all three mitogens, although the effect was more pronounced with Con A and PWM than PHA. Sera from mid-trimester pregnant animals suppressed the mitotic response of PBL from both pregnant and ovariectomized heifers. This suppressive effect was not detectable in sera from pregnant animals in early or late stages of pregnancy.     

Serum factors influencing spontaneous rosette formation by lymphocytes of pregnant women.

The effect of sera from pregnant women on the percentage of spontaneous rosette-forming peripheral lymphocytes was investigated. Pregnancy lymphocytes displayed a significantly lower capacity to bind SRBC than control male lymphocytes. However, after an exhaustive washing, it was possible to demonstrate a significant increase of spontaneous rosettes formed by pregnancy lymphocytes. It was found that the incubation of pregnancy-washed lymphocytes with pregnancy but not homologous male serum restored to depressed levels the values of rosette-forming peripheral lymphocytes. This blocking activity was significantly higher with autologous serum than homologous pregnancy serum. Control lymphocytes were unaffected both by washing and by incubation with pregnancy sera. The blocking activity was found in the same ion-exchange chromatography fraction of pregnancy serum where paternal HLA antigens could be demonstrated, and was reproduced by a soluble HLA preparation from the husband's lymphocytes.     

Activity of natural killer cells in normal pregnancy and edema-proteinuria-hypertension gestosis

Natural killer cytotoxicity of peripheral blood lymphocytes in normal pregnancy and edema-proteinuria-hypertension (EPH) gestosis was investigated and compared to natural killer cytotoxicity in lymphocytes of normal nonpregnant control donors. These lymphocytes were also compared for their ability to respond to interferon treatment in vitro. Natural killer activity was found to be only slightly decreased in normal pregnant women but was found to be increased in patients with EPH gestosis. Interferon treatment of peripheral lymphocytes caused strong enhancement of natural killer activity in lymphocytes of normal pregnant women but resulted in only weak activities in lymphocytes from patients with EPH gestosis. We consequently concluded that pre-natural killer lymphocyte subpopulations from patients with EPH gestosis have already been activated by a presently still unknown stimulator.     

Effect of physiologic levels of glutamine on ICAM-1 expression in endothelial cells activated by preeclamptic plasma

OBJECTIVE: To investigate whether the plasma glutamine (GLN) concentration would be depleted in pregnant women with preeclampsia and whether administering GLN comparable to physiologic levels would decrease cellular adhesion molecule expression in human umbilical vein endothelial cells (HUVECs) induced by plasma in preeclamptic women. STUDY DESIGN: We assessed plasma GLN levels from blood samples collected from 20 women with preeclampsia and 10 normal pregnant women. HUVECs were cultured in medium-199, containing fetal calf serum, antibiotics and growth factor, at different concentrations (0, 300, 500 microM) of GLN for 24 hours. We stimulated those cells for 1.5-6.0 hours with sera from patients with preeclampsia and then determined the expression of intercellular cell adhesion molecules (ICAM)-1 and vascular cell adhesion molecules (VCAM)-1 on endothelial cells by flow cytometry. RESULTS: Women with preeclampsia had significantly lower plasma GLN concentrations as compared with normal pregnant women. There were no differences in VCAM-1 expression in HUVECs among various GLN concentrations at each time point. However, ICAM-1 expression in HUVECs was significantly lower in the 500-microM GLN group than in the 0- and 300-microM groups at 3, 4.5 and 6 hours. CONCLUSION: This study showed that plasma from women with preeclampsia had significantly lower GLN levels than that from normal pregnant women and that administering GLN at physiologic levels reduces HUVEC ICAM-1 expression induced by preeclamptic plasma.