Lymphocytes Of Pregnant Women Research Articles

Regulation of the functional activity of peripheral B lymphocytes in pregnant women with a history of recurrent miscarriage

BACKGROUND:The frequency of recurrent miscarriage is up to 5 % of all desired pregnancies and is mainly due to immunological disorders. Dysfunction in the regulation of the functional activity of B lymphocytes is the pathogenetic link in multiple obstetric complications, including habitual miscarriage.
 AIM:The aim of this study was to characterize the regulation of the functional activity of peripheral B lymphocytes in pregnant women with threatened spontaneous miscarriage and a history of habitual miscarriage.
 MATERIALS AND METHODS:We examined 88 women aged 18-40 years at a gestation period of 5-12 weeks. The main group consisted of 36 patients with threatened spontaneous miscarriage at the time of examination and a history of habitual miscarriage. The control group included 28 women with uncomplicated pregnancy. The comparison group consisted of 24 primary pregnant patients with threatened spontaneous abortion at the time of examination. BAFF and APRIL levels in the blood serum were determined by enzyme immunoassay. The content of CD19+BAFFR+B lymphocytes in the lymphocyte gate was evaluated in the peripheral blood by flow cytometry using monoclonal antibodies. Akt mRNA expression was assessed using real-time reverse-transcription quantitative polymerase chain reaction. CD19+В lymphocytes were isolated by direct magnetic separation.
 RESULTS:In the main group, there was an increase in expression of BAFF receptors on peripheral CD19+B lymphocytes and a decrease in the serum BAFF concentration compared to the parameters in the other study groups. We also found a pronounced trend towards a decrease in the serum APRIL level in the main and comparison groups of patients compared to healthy pregnant women. Besides, Akt mRNA expression in peripheral CD19+B lymphocytes was increased in the main group.
 CONCLUSIONS:Threatened habitual abortion is associated with the deficit of the regulatory influence of BAFF and APRIL, which is expressed in the disruption of B cell homeostasis and the weakening of humoral effector mechanisms.

Аналіз показників клітинної та гуморальної ланок імунної системи у вагітних з парвовірусною інфекцією у різні періоди гестації

The article covers the topic Parvovirus infection in pregnant women caused the development of immunopathological reactions. 129 in fected pregnant women and 16 women with physiological pregnancy in the 1st, 2nd and 3rd trimesters were examined. In infected pregnant women, activation of the cellular level of immunity was observed, a significant increase in the number of natural killers in the peripheral blood and an increase in the immunoregulatory index were detected, with greater deviations from the control indicators found in the 1st trimester. In pregnant women, in which clinical complications were noted, the population composition of lymphocytes was characterized by an increased number of NK cells, low infection with the number of cytotoxic CD8+-lymphocytes and a shift in the ratio of CD4+/CD8+totheside, which indicates an increase in helper mechanisms. Increasing the number of T-lymphocytes, T-helpercells, NK/Т cellsandactivated CD25+-lymphocytes in pregnant women with out clinical complications at different periods of pregnancy can contribute to effective elimination of the virus and to prevent severe fetal lesions. Violation of the humoral link of the immune system in the infection of pregnant parvovirus infection was shown in an increase in the serum IgM content in different periods of pregnancy (by 79.1%, 54.2% and 38.5%), and a decrease in IgA levels in the 1st and 2nd trimesters (p>0.05), to a greater extent such changes were found in women with clinical complications of pregnancy and especially in the first trimester. Key words: pregnancy, parvovirusinfection, cellular and humoral immunity.

Oxidative DNA damage in diabetic and mild gestational hyperglycemic pregnant women.

BackgroundPregnant women with mild gestational hyperglycemia present high risk for hypertension, obesity and hyperglycemia, and appeared to reproduce the model of metabolic syndrome in pregnancy, with hyperinsulinemia and insulin resistance. Our clinical studies showed that mild gestational hyperglycemia or gestational diabetes are related to similar adverse maternal and perinatal outcomes. Hyperglycemia and other factors associated with diabetes generate reactive oxygen species that increase DNA damage levels. The aim of this study was to evaluate oxidative DNA damage in lymphocytes of pregnant women with diabetes or mild gestational hyperglycemia.MethodsThe study included 111 pregnant women distributed into three groups based on oral glucose tolerance test (OGTT) and glycemic profiles (GP), as follows: Normal OGTT and GP (control group); Normal OGTT and abnormal GP (mild gestational hyperglycemia group); Abnormal OGTT and GP (diabetic group). Maternal blood samples (5–10 mL) were collected and processed for determination of oxidative DNA damage by the comet assay, using Fpg and Endo III enzymes. Urine samples were also collected for determination of 8-OHdG concentrations by ELISA.ResultsSubjects in the diabetes group presented increased amount of oxidized purines, while mild gestational hyperglycemia women presented with increased oxidized pyrimidines, compared to the control group.ConclusionGestational, overt diabetes and mild gestational hyperglycemia, were all related to increased oxidative DNA damage. Diabetic pregnant women showed increased level of oxidative DNA damage, perhaps mainly due to hyperglycemia. On the other hand, oxidative DNA damage detected in women with mild gestational hyperglycemia might be associated with repercussions from obesity, hypertension and/or insulin resistance. Interestingly, the type of DNA base affected seemed to be dependent on the glycemic profile or oxidative stress.Electronic supplementary materialThe online version of this article (doi:10.1186/1758-5996-7-1) contains supplementary material, which is available to authorized users.

The interaction between air pollution and diet does not influence the DNA damage in lymphocytes of pregnant women

The aim of the study was to evaluate the risk of DNA damage in lymphocytes of pregnant women with respect to hormonal and nutritional status and to air pollution in Lesser Poland. The study was performed on 39 healthy pregnant women. The oxidative DNA damage, alkali-labile sites and uracil in DNA of lymphocytes were measured by using the comet assay. The concentration of 17beta-estradiol, progesterone, DHEA, cholesterol, vitamin B12 and folates were determined. Dietary data were assembled from food diaries. Voivodeship Inspectorate for Environmental Protection in Krakow using automatic pollution monitoring system provided the air pollution information, such as concentrations of PM10, PM2.5, NO, NO2, SO2, CO and O3. Many statistical correlations between DNA damage and air pollutants concentration were found however their biological meaning is still to be explained. It should be taken under consideration, that the protective effect of air pollutants is a result of hormesis, as the measured amounts of air pollutants during the study did not exceed the admissible levels. There was found no diet-and air pollution interaction.

Cigarette smoking during pregnancy: Chromosome translocations and phenotypic susceptibility in mothers and newborns

The effects of maternal cigarette smoking during pregnancy on structural chromosome aberrations were evaluated in peripheral lymphocytes from 239 mothers and their 241 newborns to determine whether smoking during pregnancy, genetic susceptibility, and race are associated with chromosome aberrations including translocations. Demographic information and cigarette smoking data were obtained via questionnaire. There were 119 Caucasian Americans, 118 African Americans, and 2 Asian Americans. The average maternal age was 24.9 ± 5.8 (mean ± S.D.) years. Thirty-nine percent of the Caucasian Americans and 45.4% of the African Americans self-reported that they were active smokers during the index pregnancy. The average number of cigarettes smoked per day was 2.65 ± 5.75 and 1.37 ± 3.17 for Caucasian and African American mothers, respectively. Peripheral blood lymphocytes from the mother and from the fetal side of the placenta were evaluated for chromosome aberrations by whole chromosome painting. Aliquots from the same blood samples were also used to assess genetic susceptibility with an in vitro bleomycin challenge assay. Spontaneous translocation frequencies in both maternal and newborn lymphocytes were not associated with cigarette smoking, socioeconomic status, or education. The absence of a smoking effect may be attributable to the low level of cigarette usage in these subjects. The average bleomycin-induced damage in the maternal and newborn populations was 0.37 ± 0.27 and 0.15 ± 0.14 breaks per cell, respectively, a difference that was highly significant ( p < 0.0001). In newborns there was a positive association between bleomycin sensitivity and the frequencies of aberrations as measured by chromosome painting: p ≤ 0.0007 for dicentrics and fragments, and p ≤ 0.002 for translocations. Caucasian American newborns demonstrated a significant association between dicentrics and fragments as measured by painting, and bleomycin sensitivity ( p ≤ 0.0002), but no such association was observed for African American newborns. The results of this study indicate that while differences were observed between African Americans and Caucasian Americans, race does not appear to be a major contributor to chromosome damage in newborns or their mothers. However, peripheral lymphocytes in pregnant women are more susceptible to genetic damage than peripheral lymphocytes in newborns.

Production and characterization of a novel monoclonal antibody against progesterone-induced blocking factor (PIBF)

Progesterone-induced blocking factor (PIBF) has been described as an active factor intimately involved in regulation of the immune response in pregnancy. It has been shown that PIBF biased the cytokine balance to Th2-type in pregnancy and inhibited the activity of NK cells. The biological roles of PIBF would be better defined if methods for its detection and measurement in biological fluids are available. However, so far, reliable antibodies have not been developed to be used as specific probes. A monoclonal antibody designated as MAB 3A6 was produced and characterized. MAB 3A6 reacts specifically with PIBF. It can detect this protein in biological fluids when tested by immunoblot and recognizes PIBF expressed on the surface of lymphocytes of pregnant women stimulated in vitro with progesterone. The characteristics of MAB 3A6 makes it the possible basis for development of a clinically applicable assay to assess the presence and concentration of PIBF in biological samples.

The effect of combined therapy with ritodrine, erythromycin and verapamil on the frequency of micronuclei in peripheral blood lymphocytes of pregnant women

The main aim of this study was to investigate the genotoxic effect of combined pharmacotherapy applied in post-operative treatment of cervical cerclage in pregnant women over six days. This study included 19 phenotypically healthy pregnant women in mid-trimester with a diagnosis of cervical insufficiency, mean age 28+/-5.33. The frequency of micronuclei (MN) was estimated in peripheral blood lymphocytes of patients before surgical intervention and after the end of applied pharmacotherapy by application of cytokinesis block micronucleus (CBMN) test. Mean value of baseline MN frequency was 6.84+/-2.91 MN/1000 binucleated cells, and after the end of the applied therapy, 10.32+/-4.27 MN/1000 binucleated cells (P<0.001) were found. The data of cell proliferation index showed that the combined therapy did not induce significant difference in cell kinetics (P>0.05). Our results showed that combined pharmacotherapy treatment over six days significantly increased the frequency of MN in peripheral blood lymphocytes of pregnant women.

Monocytes are progressively activated in the circulation of pregnant women

Pregnancy is characterized by the presence of generalized leukocyte activation. We used flow cytometry to investigate changes in phenotype and intracellular cytokines of circulating granulocytes, monocytes, and T lymphocytes of pregnant women during gestation. We report that peripheral circulation of pregnancy is characterized by an increased percentage of granulocytes and a decrease in lymphocytes. The proportion of monocytes remains stable throughout gestation; however, a progressive up-regulation of surface markers CD11a, CD54, and CD64 was detected. Monocytes also showed higher production of interleukin (IL)-12 and IL-1beta compared with the nonpregnant state, and granulocytes had greater potential to synthesize IL-8. All these changes were particularly marked in late gestation. T lymphocytes did not have any characteristics of the activated state and showed a decreased IL-6 production. These findings demonstrate that activation of maternal monocytes and granulocytes increases during pregnancy and support the idea that pregnancy results in an elevation of the innate immune system and suppression of the adaptive immune system.

Cytokine production in natural killer cells and lymphocytes in pregnant women compared with women in the follicular phase of the ovarian cycle

Objective: To test whether peripheral natural killer (NK) cells, helper T cells, and cytotoxic lymphocytes of pregnant women shift from a type 1 cytokine production toward a type 2 cytokine production as compared with these cells in women in the follicular phase. Design: Prospective study. Setting: Outpatient clinic. Patient(s): Healthy nullipara at 30 weeks’ amenorrhea and healthy nonpregnant women in their follicular phase. Intervention(s): Samples of whole blood were stimulated with phorbol myristate acetate (PMA; Sigma Chemical Co., St. Louis, MO) and Ca-ionophore in the presence of monensin (Sigma). Lymphocytes were stained with α-CD3, α-CD8, and α-interferon gamma (IFN-γ) α-interleukin 2 (IL-2), IL-4, or IL-10. Analysis was performed by flow cytometry. Statistical evaluation was done with the Mann-Whitney U test. Main Outcome Measure(s): Percentage NK cells, helper lymphocytes, and cytotoxic lymphocytes that were producing IFN-γ, IL-2, IL-4, or IL-10. Result(s): There is a statistically significant decrease in the percentage of NK cells, and helper and cytotoxic lymphocytes that produced IFN-γ in pregnant women when compared with women in the follicular phase. There is also a statistically significant decrease in the percentage of helper lymphocytes producing IL-2 in pregnant women compared with nonpregnant women. Conclusion(s): We found a decrease in type 1 cytokine production with no change in type 2 cytokine production after in vitro stimulation of “pregnant” NK cells and lymphocytes as compared with “nonpregnant” NK cells and lymphocytes. We suggest that NK cell and lymphocyte response are shifted away from a type 1 immune response during pregnancy.

IL-12, IL-6 and IFN-gamma production by lymphocytes of pregnant women with rheumatoid arthritis remission during pregnancy.

Rheumatoid arthritis (RA) is an autoimmune disease with progressive activity. The RA remission was observed in women during pregnancy, but the mechanism responsible for remission is hypothetical only and concerns mechanisms of immune regulation such as lymphocyte subpopulations and interleukin production. The lymphocyte subpopulations and interleukin production in vitro in a group of healthy non-pregnant women, healthy pregnant women and pregnant women suffering from RA may help towards a better understanding of regulation of the immune processes. The investigations were performed in trimester III--2 days after delivery and 6 weeks after delivery. Peripheral blood lymphocytes were isolated on Gradisol gradient and analysed immediately or after having been cultured for 72 hours in RPMI medium supplemented with 10% FCS. The cultures were terminated after 72 h, supernatants stored at -72 degrees C for interleukin evaluation. The concentrations of IFN-gamma, IL-2, IL-6, IL-12, TNF-alpha and its soluble receptors R-I, R-II were estimated in non-stimulated and PHA (Sigma, 5 microg/ml) stimulated culture supernatants using ELISA Endogen kits according to the manufacturer's instructions. The general pattern of T cell subpopulation distribution was similar in all analysed groups. Decreased IFN-gamma, IL-12 and increased IL-6 production by lymphocytes after PHA stimulation was found in trimester III in pregnant women with RA as compared to healthy pregnant woman. The obtained results suggest that in pregnant women with RA the TH1 cell response predominates, contrary to healthy pregnant women with TH2 type functional response. These phenomena were not observed after delivery.

Metabolism of progesterone by human lymphocytes: production of neuroactive steroids.

Although it has long been recognized that lymphocytes have the capacity to reduce cortisol at the C3, C5, and C20 positions, the specificity and the physiological variation of these reactions have received little attention. We have shown that such reactions also occur with progesterone. Lymphocytes were isolated from whole blood using Percoll density gradient centrifugation. The cells were incubated for 20 h with tritiated progesterone as radioactive tracer. After extractions into ethyl acetate, the residue was subjected to high performance liquid chromatography, and the radioactivities of the separated compounds were determined. Without cells, 95-97% of the tracer added was recovered in the progesterone peak, while in the presence of 4 x 10(6) lymphocytes, this was reduced to 45-90%. The metabolites obtained included at least 10 different compounds, including those corresponding in their retention times to the neuroactive 5 alpha and 5 beta dihydroprogesterones and their 3 alpha- and 3 beta- tetrahydroprogesterone derivatives. The conversion decreased with the addition of other steroids such as testosterone, cortisol, and corticosterone, suggesting that these steroids are metabolized by the same enzymes. When the cells from two pregnant patients were combined and incubated with tracer, and with and without nonradioactive progesterone, no peaks were detected by two progesterone radioimmunoassays in the absence of added nonradioactive progesterone, while in its presence three peaks corresponding to 5 alpha-dihydroprogesterone, 3 alpha-hydroxy-5 alpha-pregnane-20-dione and 3 beta-hydroxy-5 alpha-pregnane-20-dione eluted before the P peak. Their identities were confirmed using the two different progesterone radioassays that cross-reacted with these metabolites. The highest mean conversion (44.7% +/- 3.2 SE) was found with the lymphocytes of pregnant women and with that of one lactating woman (50%). Conversions by lymphocytes of women in the follicular phase (29.3% +/- 1.3 SE) were significantly lower than those in pregnancy (P = 0.014) but did not differ significantly (P > or = 0.05) from those of women in the luteal phase (22.2% +/- 3.4 SE), those of postmenopausal women (23.5% +/- 4.9 SE), or of men (22.5% +/- 2.4 SE). Lymphocytes appear to provide a hitherto unrecognized but possibly important source of neuroactive steroids.

Up‐Regulated Expression of CD56+, CD56+/ CD16+, and CD19+ Cells in Peripheral Blood Lymphocytes in Pregnant Women With Recurrent Pregnancy Losses

To analyze immunophenotypic profiles of peripheral blood and humoral autoimmune responses in women with a history of recurrent spontaneous abortions (RSA). Peripheral blood lymphocyte subsets by flow cytometry and autoantibodies to phospholipids and nuclear components by ELISA were measured in nonpregnant and pregnant women with RSA of unknown etiology. Thirty-five pregnant and eighty-one nonpregnant women with RSA were studied. Seventeen nonpregnant and twenty-two pregnant normal controls were included. Natural killer (NK) cells (CD56+) were significantly elevated in nonpregnant women with RSA as compared with nonpregnant controls. Pregnant women with RSA demonstrated significantly increased NK (CD56+, CD56+/CD16+) and B cells (CD19+) as compared with pregnant controls. Women who miscarried the index pregnancy demonstrated significantly lower CD3+ cells in comparison with normal controls. Women with RSA and antiphospholipid antibodies showed significantly elevated NK cells when compared with women without antiphospholipid antibodies. Women with autoantibodies to nuclear components demonstrated significantly elevated CD19+/CD5+ cells when compared to women without autoantibodies to nuclear components. Women with RSA demonstrate an abnormal cellular immune response by increasing peripheral natural killer cells and B cells as compared with normal controls.

Suppressed DNA repair capacity of peripheral lymphocytes in pregnant women

The ability to repair potentially carcinogenic lesions was measured for peripheral lymphocytes isolated from women in the first, second and third trimesters of pregnancy and 6 weeks post-partum. Freshly isolated lymphocytes were damaged with 254 nm ultraviolet radiation and allowed to perform repair in their autologous plasma. DNA repair capacity during pregnancy was over 50% lower than that measured 6 weeks post-partum. No significant difference in repair capacity was observed among the three trimesters. Mitogen-stimulated DNA synthesis was also measured for cells cultured in their autologous plasma. There was no significant difference in thymidine incorporation between cells collected during pregnancy (1.02 ± 0.17 cpm/10 6 cells) and cells collected 6 weeks postpartum (1.45 ± 0.94 cpm/10 6 cells). The results are, however, consistent with alterations in the immune response during pregnancy.

Sister chromatid exchanges in the lymphocytes of control women, pregnant women, and women taking oral contraceptives: Effects of cell culture temperature

The incidence of sister chromatid exchange (SCE) was investigated in the lymphocytes of control women, pregnant women, and women using oral contraceptives after culture at 37 degrees C and 40 degrees C. At 37 degrees C, the mean frequency of SCE (MEAN +/- S.E.) was found to be 7.91 +/- 0.30 in pregnant women and 8.53 +/- 0.29 in oral contraceptive users which were significantly higher than the SCE value of 5.56 +/- 0.21 found in control women. Increase in growth temperature to 40 degrees C elevated the SCE frequency to 11.86 +/- 0.44 in pregnant women, 12.76 +/- 0.46 in oral contraceptive users and 7.24 +/- 0.26 in control women. These data indicate that there is a differential induction of SCEs following increased cell culture temperature in the lymphocytes of pregnant women and oral contraceptive users, compared with control women.

Increased rate of E-rosette formation by T lymphocytes of pregnant women who drink ethanol

Ethanol use by pregnant women increased, in a dose-dependent manner, the rate of sheep erythrocyte rosette (E-rosette) formation with T lymphocytes. The time curve for E-rosette formation by T cells from nondrinking subjects was biphasic, with a rapid formation of half the E-rosettes within the first 16 min, followed by a much slower rate for E-rosette formation until the maximal T-cell percentage was reached overnight. For pregnant drinkers, greater than 85% of the E-rosettes formed during the initial rate period, with a concomitant smaller number forming during the overnight incubation. Despite the faster initial rate of E-rosette formation in the drinking subjects, the total percentage T cells was the same for both groups. Other demographic factors, like tobacco or marijuana use, or trimester, did not significantly contribute to the observed differences. An increase in the rate of E rosetting was also obtained by incubating lymphocytes from nondrinkers overnight in physiologically attainable concentrations of ethanol (≤0.1%). These results demonstrate that drinking by pregnant women, even at relatively moderate levels (2 oz/week absolute ethanol), causes alterations in their cellular immune systems. With the ability of ethanol to cross the placental barrier and persist in utero, it is apparent that these levels of ethanol have the potential to affect the developing fetal immune system.

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.

Inability of highly purified preparations of human chorionic gonado-tropin to inhibit the phytohemagglutinin-induced stimulation of lymphocytes

Several investigations, as reviewed by Anderson [ 1] and Billingham [2], were inspired by the fact that the foetus, though possessing different genetic features, is not rejected by the maternal organism like an allograft. Indeed it could be shown, that the maternal organism may specifically be sensitized against placental antigens mediated by the cellular immunity system [3]. An altered reactivity of this system might be one reason, why a rejection does not occur [4,5]. This idea is supported by the observation that the lymphocytes of pregnant women have a diminished non-specific reactivity, as measured by the phytohemagglutinin (PHA) stimulation test [6,7] . One possible explanation for this phenomenon was proposed by different reports. They dealt with an inhibitory effect of human chorionic gonadotropin (hCG) [8-lo] and human chorionic somatomammotropin (hCS) [9] , respectively, on the response of lymphocytes to PHA. These observations might explain the mechanism of acception of the foetus by the maternal organism in connection with the high formation rate of hCG (up to 1 X 106IU/day) during the first trimester of pregnancy. The investigations reported in the literature [9,10] were performed with crude hCG preparations. Therefore, we have studied the effect of highly purified hCG as well as of fractions obtained in the course of hCG purification on lymphocyte stimulation.

Inhibitor of PHA-induced lymphocyte blastformation generated from maternal lymphocytes during early pregnancy.

The supernatant of phytohemagglutinin (PHA)-stimulated lymphocyte culture derived from pregnant women in the first two trimesters inhibited DNA synthesis of normal human lymphocytes after PHA induction. This soluble factor was heat-labile and the same factor could not be found in the supernatant generated from lymphocytes of pregnant women in 3rd trimester as well as from those of the non-pregnant. In contrast, the heatstable factor in the supernatant generated from lymphocytes of non-pregnant women in all trimesters contained an stimulating factor on the DNA synthesis of normal human lymphocytes after PHA induction. An endogenous alternation in lymphocyte competence or an alternation in subpopulation of lymphocytes was suggested at the early stage of pregnancy.