Assessment of three simple techniques for the screening of circulating immune complexes: Correlation with a parameter of complement consumption

The sera of 36 normal controls, 45 patients with various diseases and 11 pregnant women were screened for circulating immune complexes using three relatively simple and inexpensive techniques. These included inhibition of agglutination of IgG coated latex particles with a serum having rheumatoid factor activity, polyethylene glycol precipitation and anti-complementary activity test. The circulating immune complexes were detected in a significantly higher proportion of patients as compared to normal controls. In the patients, the presence of circulating immune complexes did not always correlate with clinically detectable immunoinflammatory tissue damage indicating that pathogenic as well as nonpathogenic immune complexes were being detected by the above mentioned techniques. The alpha-1-antitrypsin/C3 ratio, however, correlated well with clinically apparent immuno-inflammation.     

Functional HY-specific CD8+ T cells are found in a high proportion of women following pregnancy with a male fetus

Recent studies have demonstrated that fetal cells can be detected in the maternal circulation during virtually all human pregnancies. These fetal cells can engraft and may be isolated for many decades after pregnancy, leading to a state that may be maintained by the passage of pregnancy-associated progenitor cells. The clinical consequences of fetal cell microchimerism are unclear but may be potentially detrimental or valuable to the mother. One possibility is the generation of an alloreactive immune response by the mother to antigens expressed by the fetus; for example, the HY protein encoded by the Y chromosome. To test this we have screened a cohort of women with a range of parity histories within 8 yr of their last pregnancy for the presence of an HY-specific CD8+ T-cell response. Fluorescent HLA-peptide (HY) tetramers were used to stain short-term T-cell cultures from these women for analysis by flow cytometry. Responses were detected in 37% of women with a history of pregnancies that produced males, and this value rose to 50% in women with two or more pregnancies that produced males. HY-specific CD8+ T cells also could be detected directly in the peripheral blood of women with a history of at least two pregnancies that produced males. These HY-specific CD8+ T cells produced interferon gamma (IFNG) following peptide stimulation, demonstrating their functional capacity. In conclusion, our data indicate that alloreactive CD8+ T cells are generated frequently following normal pregnancy and retain functional capability for years following pregnancy.     

Circulating immune complexes in cervical cancer patients as detected by C1q binding

In a retrospective study in women with cervical cancer, circulating immune complex levels were measured by radioimmunoprecipitation with 125I-C1q. Sera from 46 patients with cervical cancer and 35 normal controls were examined. Significantly higher levels of immune complexes were detected in cancer patients compared with controls. Mean value of binding capacity in patients was 49.8%, and by contrast, in the controls was 27.4% (two-tail test = 0). Increases in tumor mass were associated with high levels of circulating immune complexes. The presence of immune complexes in circulation statistically correlated with disease activity, however, the assay used still had limited value for diagnosis or aiding in therapeutic decisions. Nevertheless, the future holds promise for such uses.     

Circulating immune complexes in patients with breast cancer.

In a retrospective study in women with breast cancer circulating immune complex levels were measured by radioimmunoprecipitation with 125I-Clq. Before operation all the patients showed plasma immune complex levels significantly higher than those in controls. Twelve months after mastectomy patients identified clinicopathologically as having a good prognosis had almost normal levels of immune complexes. By contrast, patients with detectable dissemination on diagnosis or those who died within 22 months after mastectomy had significantly raised plasma levels. The tumour-specific nature of the immune complexes detected remains to be shown and suggestions about the applicability of this test not only for prognosis but also for monitoring the course of malignant diseases need to be confirmed by further investigations.     

Peripheral Dendritic Cells and CD4+CD25+Foxp3+ Regulatory T Cells in the First Trimester of Normal Pregnancy and in Women with Recurrent Miscarriage

The development of pregnancy is possible due to initiation of immune response in the body of the mother resulting in immune tolerance. Miscarriage may be caused by the impaired maternal immune response to paternal alloantigens located on the surface of trophoblast and fetal cells. The aim of the study was to compare the population of circulating dendritic cells (DCs) and CD4+CD25+Foxp3+ regulatory T cells (TREGs) in the first trimester of a normal pregnancy and in women with recurrent miscarriage and an attempt to determine the relationship between these cells and the role they may play in human reproductive failures. The study was conducted in a group of 33 first trimester pregnant women with recurrent miscarriage and in a group of 20 healthy pregnant women in the first trimester of normal pregnancy. Among mononuclear cells isolated from peripheral blood, the populations of DCs and TREGs were assessed by flow cytometry. The percentage of myeloid DCs and lymphoid DCs showed no significant difference between study and control group. Older maternal age and obesity significantly reduced the pool of circulating myeloid and lymphoid DCs (R=-0.39, p=0.02). In miscarriages the percentage of circulating TREGs was significantly lower compared to normal pregnancies (p=0.003). Among the analysed factors the percentage of TREGs was the most sensitive and the most specific parameter which correlated with the pregnancy loss. The reduction in the population of circulating TREGs suggests immunoregulatory mechanisms disorder in a pregnancy complicated by miscarriage.     

Fetal-specific CD8+ cytotoxic T cell responses develop during normal human pregnancy and exhibit broad functional capacity

Tolerance of the semiallogeneic fetus presents a significant challenge to the maternal immune system during human pregnancy. T cells with specificity for fetal epitopes have been detected in women with a history of previous pregnancy, but it has been thought that such fetal-specific cells were generally deleted during pregnancy as a mechanism to maintain maternal tolerance of the fetus. We used MHC-peptide dextramer multimers containing an immunodominant peptide derived from HY to identify fetal-specific T cells in women who were pregnant with a male fetus. Fetal-specific CD8(+) T lymphocytes were observed in half of all pregnancies and often became detectable from the first trimester. The fetal-specific immune response increased during pregnancy and persisted in the postnatal period. Fetal-specific cells demonstrated an effector memory phenotype and were broadly functional. They retained their ability to proliferate, secrete IFN-γ, and lyse target cells following recognition of naturally processed peptide on male cells. These data show that the development of a fetal-specific adaptive cellular immune response is a normal consequence of human pregnancy and that unlike reports from some murine models, fetal-specific T cells are not deleted during human pregnancy. This has broad implications for study of the natural physiology of pregnancy and for the understanding of pregnancy-related complications.     

The urinary excretion of pregnanediol during pregnancy determined by gas-liquid chromatography. I. Its evolution throughout the normal and pathological pregnancy (author's transl)]

Employing the technique described by Van Kampen and Anker, modified by Macarulla et al., 180 pregnant women have been studied (66 normals and 114 with different pathology: infertility, toxemia, diabetes, Rh isoinmunization, gemelar pregnancy and abortions), taking 319 determinations of pregnanediol in 24 hours urine samples. The analysis of the results show in normal pregnancy a progressive increase of the urinary pregnanediol from the beginning of gestation, this increase being more intense from the 20th week, reaching the maximum value in the 37th week and from this point descending slowly. In patients with toxemia, the values of pregnanediol (in the majority of the cases) are decreased, while in pregnant women with antecedents of infertility are increased from the 36th week of pregnancy, although they had protective treatment from first months of pregnancy. No manifest deviations of urinary pregnanediol from the normal values exist in diabetic pregnant women, Rb isoinmunization or gemelar pregnancies. In aborted pregnancies the pregnanediol values are markedly decreased without a tendency to increase, contrary to the threats of abortion in full-term pregnancies.     

Placentation in cloned cattle: structure and microvascular architecture

To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies established by somatic nucleus transfer (NT), the umbilical cord and fetal membranes were edematous. Placentomal fusion was common, resulting in increased size and a decreased number of placentomes. Extensive areas of the chorioallantoic membrane were devoid of placentomes. An increased number of functional or accessory microcotyledons (<1 cm) were present at the maternally oriented surface of fetal membranes. Extensive areas of extravasated maternal blood were present within the placentomes and in the interplacentomal region. The crypts on the caruncular surface were dilated and accommodated complexes of more than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops and sinusoidal dilations as in normal pregnancies. At the materno-fetal interface, however, the trophoblast and uterine epithelium had normal histology. In conclusion, the NT placentas had a range of pathomorphological changes; this was likely associated with the poor clinical outcome of NT pregnancies.     

Biological aging and the etiology of aneuploidy

A prevalent hypothesis concerning the cause of the rise in aneuploid conceptions with maternal age is that the changes that accompany normal ovarian aging increase the rate of meiotic errors in the oocyte. Biological aging of the ovary is accompanied by a decline in both the total oocyte pool and the number of antral follicles maturing per cycle, as well as changes in the levels of circulating reproductive hormones. The biological aging hypothesis predicts that aneuploidy rates should be higher in women with a prematurely reduced oocyte pool, and that women with trisomic conceptions should show signs of earlier ovarian aging than women of the same chronological age without trisomic conceptions. Comprehensive studies of aneuploidy in groups of women with known causes of premature ovarian failure remain to be done, though anecdotal evidence does suggest increased rates of pregnancy loss and aneuploidy. Smoking, which is a well-documented cause of earlier ovarian aging, is not associated with an increase in aneuploid conceptions. Evidence from women with unilateral ovariectomies is inconsistent. Support for the biological aging hypothesis was provided by one study showing that menopause occurred about a year earlier in women with a trisomic spontaneous abortion compared to women with chromosomally normal conceptions. Associations between high FSH and pregnancies with Down syndrome and chromosomally abnormal spontaneous abortions have also been reported. However, the most direct test of the hypothesis, which compared antral follicle counts and hormonal levels in women with trisomic pregnancies and those with chromosomally normal pregnancies, failed to find a difference in the expected direction. A prospective study of FSH levels in women with subfertility also failed to find an association with the rate of pregnancy loss. The bulk of evidence thus suggests that, if the processes of biological aging are indeed related to aneuploidy, they probably involve factors other than those measured by oocyte or antral follicle pool size and reproductive hormone levels.     

Complement-binding activity of myeloma and normal immune complexes

The comparison of complement-fixing capacity of simulated immune complexes formed by normal IgG and IgG, isolated from serum of patients with multiple myeloma, has been performed. In both cases a non-linear dependence of complement-fixing capacity on the complex molecular mass was demonstrated, it being higher for myeloma proteins. Complement supplementation to high molecular complexes leads to their collapse, with normal immune complexes destroyed at lower molecular masses. Heat-aggregation of myeloma immunoglobulins leads to the formation of simulated immune complexes of lower molecular mass compared to normal proteins.     

Stimulation of lymphocytes by platelet-antibody-complexes and their role in the pathogenesis of idiopathic thrombocytopenia

The effect of washed human platelets, platelet lysates, and platelet antibody complexes on 14C-thymidine incorporation by human lymphocytes was studied. For sensitization of platelets, HLA-specific alloantibodies as well as platelet autoantibodies were used. Lymphocytes for in vitro cultures were collected from unsensitized individuals, healthy women with proven fetomaternal immunization against HLA antigens and patients with idiopathic thrombocytopenic purpura (ITP). Unsensitized platelets have a dose-dependent inhibitory effect on the in vitro proliferation of normal lymphocytes induced by mitogens (PHA, ConA, PWM). Platelet antibody complexes (allo- and autoantibodies; allogenic and autologous lymphocyte-platelet combinations) did not stimulate 14C-thymidine incorporation. Lymphocytes from ITP patients showed a significantly reduced stimulatory response toward PHA compared to normal persons. These findings are discussed in the light of our present knowledge regarding the role of cellular immune reactions in the pathogenesis of ITP.     

Pregnancy-associated exosomes and their modulation of T cell signaling

Exosome release by viable cells is a feature of activated cell types, including tumors, fetal cells, and cells of the immune system. Exosomes critically regulate immune activation, by mediating activation-induced cell death. Fetal cells may mimic these events to selectively delete reactive lymphocytes. In this study the presence and composition of placenta-derived exosomes are demonstrated in the maternal circulation along with their consequences on T cell activation markers. For all pregnant patients, exosomes were isolated from sera obtained between 28 and 30 wk gestation. For pregnant women, subsequently delivering at term, circulating levels of placental exosomes were 1.8 times greater than those delivering preterm (p < 0.0001). Exosomes isolated from pregnancies subsequently delivering at term expressed significantly higher levels of biologically active components, including Fas ligand (FasL) and HLA-DR, than those from pregnancies delivering preterm. Standardizing for protein concentrations, exosomes from term-delivering pregnancies exhibited greater suppression of CD3-zeta and JAK3 than those delivering preterm. The suppression of CD3-zeta and JAK3 correlated with exosome expression levels of FasL (r2= 0.92 and r2= 0.938, respectively). Fractionation of exosomes from term-delivering pregnancies by continuously eluting electrophoresis indicated that intact 42 kD FasL and an unidentified 24-kDa protein were associated with CD3-zeta suppression. Our results demonstrated that exosomes from pregnancies ultimately delivering at term are present at significantly greater concentrations than those from pregnancies delivering preterm; however, exosomes from term-delivering pregnancies also exhibit significantly greater suppression of CD3-zeta and JAK3.     

Impairment of endothelial function in women with a history of preeclampsia: an indicator of cardiovascular risk

Preeclampsia is a disorder of pregnancy diagnosed by gestational hypertension and proteinuria. Epidemiological evidence suggests that women who experience preeclampsia are at a greater risk of hypertension and heart disease later in life compared with women who had normal pregnancies. Our objective was to determine whether endothelial function is impaired in postpartum women with a history of preeclampsia in their first pregnancy. We measured forearm blood flow (FBF) by venous occlusion plethysmography in 50 healthy women: 16 with prior preeclampsia, 14 with a prior normotensive pregnancy, and 20 never pregnant controls. The postpartum women participated 6-12 mo after delivery. Heart rate (HR) and blood pressure (BP) were concurrently monitored on the contralateral arm. Hemodynamic variables were assessed at baseline and during a mental stress test known to elicit endothelium-dependent vasodilatation. We found that baseline FBF, HR, systolic BP, and diastolic BP did not significantly differ among the groups, whereas mean arterial pressure in the preeclamptic group was greater than that of the normal pregnancy group (P = 0.03). Stress-induced FBF (percent change over baseline) was reduced in the preeclamptic group compared with both the normal pregnancy and never pregnant groups (P = 0.06) and was significantly attenuated compared with women with prior normal pregnancies (91% vs. 147%, P = 0.006). These data demonstrate that women with a history of preeclampsia exhibit impaired endothelial function up to 1 yr postpartum. This observation may explain their increased risk for hypertension and cardiovascular disease.     

Immune complexes bind preferentially to Fc gamma RIIA (CD32) on apoptotic neutrophils, leading to augmented phagocytosis by macrophages and release of proinflammatory cytokines

Many human inflammatory diseases are associated with tissue deposition of immune complexes and influx of neutrophils. We show that immune complexes bind preferentially to apoptotic neutrophils via FcgammaRIIA (CD32) and that increased binding is associated with clustering of immune complexes on the plasma membrane of the apoptotic cell. Phagocytosis of immune complex-opsonized apoptotic neutrophils by human macrophages was substantially enhanced (4.4-fold increase compared with control apoptotic neutrophils) and stimulated macrophages to release the proinflammatory cytokines TNF-alpha and IL-6. Immune complexes may perturb the normal pathways for clearance of apoptotic neutrophils by augmenting their clearance at the price of proinflammatory cytokine release. This represents a novel mechanism by which immune complexes may modulate the resolution of inflammation.     

Low serum selenium concentration and glutathione peroxidase activity in intrahepatic cholestasis of pregnancy.

Serum selenium concentrations were found to be significantly lower in women with intrahepatic cholestasis of pregnancy than in women with normal pregnancies during the last trimester of pregnancy and post partum. The activity of the selenoenzyme glutathione peroxidase had a significant positive correlation with selenium concentration and it was also significantly lower in women with the disease. These findings suggest that selenium deficiency and reduced glutathione peroxidase activity are associated with the aetiopathogenesis of intrahepatic cholestasis of pregnancy.     

Maternal serum progesterone, 17 beta-estradiol and estriol are increased in pregnancies which follow treatment with human menopausal gonadotropins: effects of multiple gestation and maternal endocrine status

A high incidence of premature labor, incompetent cervix and fetal wastage occurs in multiple gestations which follow treatment with human menopausal gonadotropins (HMG). In order to determine the effect of treatment with HMG on hormone secretion in human pregnancy, progesterone (PROG), 17 beta-estradiol (E2), estriol (E3) and human chorionic gonadotropin (hCG) were determined by radioimmunoassay in 341 serum specimens from 229 normal singleton pregnancies and in 79 serum specimens from 20 pregnancies following induction of ovulation with HMG in women with either hypothalamic amenorrhea (HA) or the polycystic ovary syndrome (PCO). Fitting equations were found for the log transformed normal values and the residuals were obtained by subtraction of the predicted normal values from the log transformed values observed in the HMG pregnancies. In pregnancies which followed treatment with HMG, PROG and E2 were initially elevated above normal. As pregnancy progressed, the deviation from normal became proportionately less. PROG (P less than 0.025) was lower and E2 (P less than 0.025) and E3 (P less than 0.05) were higher in PCO pregnancies than in HA pregnancies. Multiple gestation produced increases in PROG (P less than 0.005), E2 (P less than 0.005) and E3 (P less than 0.001) in comparison to singleton pregnancies.     

Trisomic pregnancy and earlier age at menopause

We tested the hypothesis that the connection between advanced maternal age and autosomal trisomy reflects the diminution of the oocyte pool with age. Because menopause occurs when the number of oocytes falls below some threshold, our hypothesis is that menopause occurs at an earlier age among women with trisomic pregnancies than it does among women with chromosomally normal pregnancies. To determine their menstrual status, we interviewed women from our previous study of karyotyped spontaneous abortions who, in 1993, were age >/=44 years. Premenopausal women completed interviews every 4-5 mo, until menopause or until the study ended in 1997. The primary analyses compare 111 women whose index pregnancy was a trisomic spontaneous abortion with two groups: women whose index pregnancy was a chromosomally normal loss (n=157) and women whose index pregnancy was a chromosomally normal birth (n=226). We used a parametric logistic survival analysis to compare median ages at menopause. The estimated median age at menopause was 0.96 years earlier (95% confidence interval -0.18 to 2.10) among women with trisomic losses than it was among women with chromosomally normal losses and chromosomally normal births combined. Results were unaltered by adjustment for education, ethnicity, and cigarette smoking. Our results support the hypothesis that trisomy risk is increased with decreased numbers of oocytes. Decreased numbers may indicate accelerated oocyte atresia or fewer oocytes formed during fetal development.     

Urinary Estriol Estimations in Normal and Abnormal Pregnancies

Urinary estriol estimations from 24-hour urine specimens were studied in 65 women in the course of normal pregnancies and compared to 18 analyses in women in whom fetal death had occurred. There is a significant difference in the levels of estriol excretion between these two categories. Serial studies were carried out on patients whose pregnancies ended in normal delivery and in patients whose pregnancies were complicated by eclampsia and by hypertension of various degrees of severity. A definite correlation between urinary estriol excretion and the health of the fetus in utero was found. The information that these estimations provide can be of assistance to the clinician in the management of selected patients where the fetus is in danger prior to delivery.     

Nitric oxide synthesis in placenta is increased in intrauterine growth restriction and fetal hypoxia

In order to study the possible role of nitric oxide (NO) in the human placenta, we measured the concentration of its stable metabolite nitrite (NO2-) in the placentas of women with normal pregnancies and those from pregnancies complicated by intrauterine growth restriction (IUGR) with or without fetal hypoxia. We have measured nitrites by the Griess reaction in 15 placentas from IUGR pregnancies and 12 controls. Cerebroumbilical ratio (C:U) was recorded by color Doppler ultrasound and values below 1 were considered to be a predictor for fetal hypoxia. NO2- levels measured in pathological placentas were increased for at least 93% as compared to control. Subjects from pregnancies complicated by IUGR and fetal hypoxia had increased NO2- as compared to the placentas from pregnancies with IUGR and normal fetal oxygenation. NO production in placenta is increased in pregnancies with IUGR. This effect is more pronounced in those with compromised fetal oxygenation.     

The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans. Complement- and erythrocyte complement receptor 1-dependent mechanisms

The role of complement and its receptor on erythrocytes (CR1) in the physiologic elimination of large immune complexes from the circulation of humans was assessed. Large radiolabeled soluble tetanus toxoid- anti-tetanus toxoid complexes were injected i.v. into three normal individuals and three patients with SLE. These complexes were prepared in antibody excess and were 45S in size, fixed C and bound to E CR1 in vitro. The percentage of complexes bound in vitro was directly proportional to CR1 number/E in four normal subjects and three SLE patients. After i.v. injection into normal subjects, complexes were cleared rapidly, with a monoexponential rate constant (10.3 to 11% complexes cleared/min). In the SLE patients, clearance was best explained by two phases: the first occurred within the first minute indicating immediate trapping of a fraction of the complexes (19.5 to 25.3% of injected complexes trapped), the second was monoexponential and was similar to the normal range. A large fraction of complexes bound within the first minute to E in vivo; the percentage of binding was variable, ranging from 16.3% to 71.5% and was related to E CR1 number. In a second study complexes were injected that had been attached to autologous E by opsonization with C in vitro. Their elimination was similarly monoexponential, except in one SLE patient in whom there was significant initial trapping (30.9%). A fraction of these complexes were released from E within the first minute, the percentage release being greatest in the patient with the lowest CR1 number (81.4%). E bearing immune complexes remained in the circulation and were not transiently sequestered in the liver or spleen. This is the first study of the clearance of soluble immune complexes in vivo in humans and shows that C and CR1 on E participate in immune complex clearance reactions, and that abnormal clearance can be detected in the form of rapid removal of immune complexes from the circulation.