Cytokine secretion in decidual mononuclear cells from term human pregnancy with or without labour: ELISPOT detection of IFN-gamma, IL-4, IL-10, TGF-beta and TNF-alpha.

Cytokines are believed to be important in maintaining pregnancy and in the process of labour induction in humans. The aim of this study was to investigate the secretion of the cytokines interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-10, transforming growth factor-beta (TGF-beta) and tumour necrosis factor-alpha (TNF-alpha) in decidual tissue with or without labour. Decidual tissue was collected from 32 healthy women undergoing elective caesarean sections before the onset of labour (n=17) or after normal vaginal delivery (n=15). Mononuclear cells were analysed for cytokine secretion with ELISPOT. To validate the widely used method of tissue collected at caesarean sections and after vaginal deliveries as a representative of before and after labour, respectively, placenta biopsies were collected from 12 healthy women to study the expression of the prostaglandin pathway enzymes cyclooxygenase-2 (COX-2) and microsomal prostaglandin E(2) synthase (mPGES). Decidual mononuclear cells from term human pregnancy spontaneously secrete IFN-gamma, IL-4, IL-10, TGF-beta and TNF-alpha. No difference was seen in cytokine secretion with or without labour, indicating that decidual leukocytes are not the main cell population responsible for plausible cytokine regulation in the process of termination of pregnancy. Placental tissues obtained after vaginal delivery showed a higher mRNA expression of the prostaglandin regulating molecules COX-2 and mPGES than tissues from caesarean sections before the onset of labour, validating that the model can be used as a representative of the state before and after labour.

Cytokine secretion patterns of NK cells and macrophages in early human pregnancy decidua and blood: implications for suppressor macrophages in decidua.

PROBLEM: Local immune modulation has been shown to be of considerable importance for the maintenance of successful pregnancy. We have previously reported the secretion of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-10 in human decidua from early normal pregnancy. The aim of this study was to investigate the cellular source of cytokine secretion in the decidua, and compare this to secretion patterns in peripheral blood. METHOD OF STUDY: Decidual tissue and peripheral blood was collected from 20 women undergoing surgical abortion during first trimester pregnancy. Monocytes/macrophages and NK cells were enriched by immunomagnetic cell separation and cytokine secretion was detected by enzyme-linked immunosorbent spot-forming cell assay. RESULTS: Decidual and peripheral monocytes/macrophages and NK cells spontaneously secrete IFN-gamma, IL-4 and IL-10. The number of IL-10 secreting cells was significantly higher in decidual macrophages compared with decidual non-monocytic cells as well as compared with blood monocytes/macrophages. These differences were not seen for IFN-gamma or IL-4. CONCLUSIONS: Our results indicate that decidual macrophages subserve important suppressive functions in the pregnant uterus.

Spontaneous secretion of interleukin-4, interleukin-10 and interferon-gamma by first trimester decidual mononuclear cells.

PROBLEM: A T-helper cell type 2 (Th2) cytokine dominated microenvironment has been predicted to be crucial for successful pregnancy. However, little information is available about local cytokine secretion in the human decidua. We determined the spontaneous secretion of interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and IL-10 by decidual mononuclear cells at the single cell level and compared it with their secretion by peripheral blood mononuclear cells (PBMC) in the first trimester of pregnancy. METHODS OF STUDY: The cytokine secretion from decidual and blood cells was detected by a sensitive enzyme-linked immunosorbent spot-forming cell (ELISPOT)-assay. RESULTS: Cells secreting IL-4 (median 153, range 8-530), IL-10 (median 188, range 32-1600) and IFN-gamma (median 123, range 15-1140) were detected in all decidual and blood samples. The cytokine secretion showed a co-linear pattern in both the blood and decidua, i.e. when one cytokine was secreted at high levels, the others followed the trend. No correlation was found between the number of cytokine secreting cells in blood and decidua for any of the cytokines. CONCLUSIONS: Interleukin-4 and IL-10 are locally secreted in the decidua early during normal pregnancy, probably counteracting the fetal rejecting effects of co-expressed IFN-gamma. The cytokine secretion by blood cells does not generally reflect the local secretion pattern during first trimester pregnancy.

Circulating interferon-gamma- and interleukin-4-secreting cells in recurrent spontaneous abortions.

PROBLEM: Are recurrent spontaneous abortions (RSAs) associated with deviation of circulating cytokine-secreting cells? METHOD OF STUDY: Interferon (IFN)-gamma- and interleukin (IL)-4 secreting cells were identified by enzyme-linked immunospot (ELISPOT) in blood from 34 women with RSA. Samples were taken before pregnancy and/or pregnancy weeks 7-10, 17-20, and after terminated pregnancy. Eleven healthy primigravidae and 10 non-pregnant women served as controls. RESULTS: No significant difference in numbers of IFN-gamma- and IL-4-secreting cells was noted within the RSA group when abortions and successful pregnancies were compared in samples taken before pregnancy. The number of IFN-gamma- as well as IL-4-secreting cells in pregnancy weeks 17-20 in the RSA group was significantly higher compared with before pregnancy, pregnancy weeks 7-10, and after pregnancy. In samples from non-pregnant women, the number of IFN-gamma- and IL-4-secreting cells was significantly higher in the RSA group compared with controls. CONCLUSIONS: Our results do not indicate a systemic shift in the general balance between T helper 1- and T helper 2-type cytokine pattern. A local shift at the fetomaternal interface seems more probable.

Lymphocyte subsets and mitogen stimulation of blood lymphocytes in preeclampsia.

PROBLEM: The question of whether there are differences in systemic immune reactivity in severe preeclampsia compared with normal pregnancy was addressed. METHOD OF STUDY: During the third trimester, blood samples were taken from 12 pregnant women with severe preeclampsia. Five of the preeclamptic pregnancies were analyzed separately because they were treated with dexamethasone before the blood samples were taken. The seven dexamethasone-treated preeclamptic pregnant women were analyzed and compared with six uncomplicated pregnancies. A control group consisted of 15 nonpregnant females. Lymphocyte subsets were identified by flow cytometry. The function of peripheral blood mononuclear cells (PBMCs) was studied as proliferative responses to mitogens alone and in combination with immunomodulating drugs. RESULTS: An increased number of B lymphocytes (CD19+) (P < 0.05) and natural killer (NK) cells (P < 0.05) was noticed in severe preeclampsia compared with normal pregnancy. The proliferative response of PBMCs in phytohemagglutinin (PHA)-stimulated cultures in autologous serum from patients with severe preeclampsia was reduced (P < 0.05) compared with normal pregnancy. The addition of indomethacin and cimetidine significantly stimulated (P < 0.05) the proliferative responses. The enhancing effect of cimetidine was not found in dexamethasone-treated preeclamptic patients. CONCLUSIONS: The presence of systemic immunosuppression in severe preeclampsia is demonstrated as a reduced proliferative response of PBMCs to PHA, which could be partly restituted by indomethacin or cimetidine, indicating immunosuppressor activity that is mediated by prostaglandin and histamine. Increased levels of B lymphocytes and NK cells were also noticed.

A prospective study on the occurrence of autoantibodies in low-risk pregnancies.

OBJECTIVE: This investigation was done to study the prevalence of anti-nuclear antibodies (ANA), anti-cardiolipin antibodies (aCL), and rheumatoid factor (RF), in presumed healthy women during their pregnancies. STUDY DESIGN: During an 18 month period blood samples were taken in the first, second and third trimester from 1200 pregnant women, representing a low-risk population. Clinical data on the pregnancy outcome were obtained by birth statistics after their deliveries. The diagnoses of preeclampsia, intrauterine growth retardation, fetal death, or abruptio placentae were stated in 57 of these women. An age- and parity-matched control group of 207 women with normal pregnancy outcome was drawn from the same low-risk population (n= 1200). A nonpregnant control group consisted of 157 women. The prevalence of ANA (immunofluorescence microscopy on HEp-2 cells), aCL-immunoglobulin G (enzyme-linked immunosorbent assay), and RF (latex agglutination test) in preeclampsia, intrauterine growth retardation, fetal death, or abruptio placentae were compared to the normal pregnancies, and to the nonpregnant controls. RESULTS: ANA occurred significantly more often (P<0.05) in pregnancies complicated by preeclampsia when compared to normal pregnancies. aCL occurred sparsely in normal as well as complicated pregnancies. RF was infrequently seen among all women in this study. CONCLUSION: An association was noted between the occurrence of ANA and preeclampsia. However, this association was too insensitive to use as a clinical tool.

Increased numbers of circulating interferon-gamma- and interleukin-4-secreting cells during normal pregnancy.

PROBLEM: Systemic immune responses during normal pregnancy are suggested to deviate toward secretion of T helper (Th)2-like cytokines. METHOD OF STUDY: Blood samples from 11 healthy primigravidae in their first, second, and third trimesters, and 8 weeks postpartum were examined. Peripheral blood mononuclear cells were separated and the numbers of interferon (IFN)-gamma- and interleukin (IL)-4-secreting cells were identified by using the enzyme-linked immunospot test. RESULTS: In all three trimesters of pregnancy, and also postpartum, the numbers of IFN-gamma- and IL-4-secreting cells were significantly higher compared with nonpregnant controls (Mann-Whitney; P < 0.001). The numbers of IFN-gamma- and IL-4-secreting cells gradually increased as the pregnancy progressed compared with postpartum (Kruskas-Wallis; P < 0.01 and P < 0.05, for IFN-gamma and IL-4, respectively). The ratios of IFN-gamma/IL-4 during pregnancy and postpartum were unchanged, and also when compared with nonpregnant controls. CONCLUSION: The results indicate a systemic up-regulation of both Th1- and Th2-like immune responses during normal pregnancy.

Paternal leukocytes selectively increase secretion of IL-4 in peripheral blood during normal pregnancies: demonstrated by a novel one-way MLC measuring cytokine secretion.

PROBLEM: It has been proposed that immune responses in normal pregnancy are Th2-like, thereby protecting the fetus and placenta from being rejected. Some studies have shown Th2-deviated systemic responses to different antigens and mitogens. The aim of this study was to demonstrate the specific T cell cytokine responses directed toward paternal histocompatibility leukocyte antigen (HLA), because this is the most prominent target for rejection of the feto-placental unit. METHOD OF STUDY: A novel one-way mixed leukocyte culture (MLC) combined with the detection of cytokine secretion with a sensitive ELISPOT assay was developed. Peripheral blood from 11 pregnant women was investigated with respect to allo-reactivity toward paternal leukocytes and pooled leukocytes from unrelated blood donors. This was done at three different occasions during pregnancy and 8 weeks after delivery. Nine age-matched non-pregnant women served as controls. RESULTS: In the second and third trimesters of pregnancy significantly larger numbers of IL-4-secreting cells (Th2) were induced by paternal leukocytes as compared to unrelated leukocytes. CONCLUSIONS: The findings indicate a selective immune deviation toward Th2, which may protect the fetus from rejection and thus may be an important homeostatic mechanism in normal pregnancies.

Clinical consequences of anal sphincter rupture during vaginal delivery.

BACKGROUND: Rupture of the anal sphincters at childbirth is considered rare in obstetric literature. Long-term effects are sparingly mentioned. In clinical practice, however, it is not uncommon to meet women with anal incontinence. The aim of our study was to record the incidence and to evaluate the consequences of rupture of the anal sphincter at childbirth. STUDY DESIGN: Fifty-one consecutive women with primarily sutured anal sphincter rupture and 31 women without anal sphincter rupture were prospectively studied after vaginal delivery. All were assessed clinically at 3 days, 6 weeks, and 6 months after delivery. After 6 months, all women underwent anorectal manometry and answered a questionnaire about incontinence, social function, and general health. RESULTS: The overall incidence of sphincter rupture was 2.4 percent. Significantly lower values were found for maximum anal squeeze pressure and squeeze pressure area 6 months postpartum in the women with sphincter rupture compared with those without rupture. The resting pressures did not differ between groups. Approximately 40 percent of the women in both groups had noted some fecal incontinence by 6 months postpartum. Symptoms were significantly more severe in patients with sphincter rupture. CONCLUSIONS: Anal sphincter rupture was 2.4 times as common as reported in Swedish birth statistics. The high incidence of fecal incontinence by 6 months postpartum in all women is surprising and deserves further investigation, specifically regarding occult sphincter rupture.

Lymphocyte subsets and mitogen stimulation of blood lymphocytes in normal pregnancy.

PROBLEM: In normal pregnancy the maternal immune system should be directed towards tolerance or suppression in order not to reject the partly foreign feto-placental unit. The aim of this investigation was to find hallmarks of systemic immunosuppression during normal pregnancy. METHODS: Five healthy primigravidae were examined during pregnancy and postpartum with flow cytometric analysis to define T and B lymphocyte subsets in peripheral blood. In addition, we studied the proliferative response of lymphocytes to mitogens or interleukin-2 (IL-2) alone or in combination with immunomodulating drugs or interleukin-4 (IL-4). The results were compared to healthy, non-pregnant women. RESULTS: During pregnancy and early puerperium we noted an immune balance in favour of suppression, as measured by increased numbers of T "helper/suppressor" (CD4+CD45RA+) and "suppressor"/effector T cells (CD8+S6F1-), and decreased numbers of T "helper/inducer" (CD4+CD29+), T "helper/memory" (CD4+CD45RO+), killer/effector T cells (CD8+S6F1+), and Natural Killer cells (CD56+), as well as decreased numbers of activated lymphocytes expressing IL-2 receptor (CD25+) and T cells expressing HLA-DR (HLA-DR+CD3+). During pregnancy, lymphocyte proliferation was impaired in autologous serum with concanavalin A (ConA), phytohemagglutinin (PHA), or IL-2. A difference in proliferative response to PHA or IL-2 between cultures with AB serum and autologous serum is suggestive of an immunosuppressor factor in serum during pregnancy. Indomethacin significantly increased lymphocyte proliferation in autologous serum with ConA, indicating PGE2 mediated suppressor activity during pregnancy. Chlorambucil and cimetidine modulated the proliferative response to ConA, indicating an alkylating agent sensitive and a histamine dependent suppressor activity during pregnancy. CONCLUSIONS: During normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present.

Lymphocyte subsets and autoantibodies in pregnancies complicated by placental disorders.

PROBLEM: The survival of the fetoplacental unit might partly depend on down-regulation of rejection reactions. Pathological maternal cellular immune response mechanisms could therefore be of pathogenic importance in pregnancies complicated by placental disorders. METHOD: A flow-cytometric analysis of T-cell subsets and B-cell, as well as serological tests for anticardiolipin antibodies (aCL), antinuclear antibodies (ANA), and rheumatoid factor (RF) were done on 90 women with complicated pregnancies. The results were compared with that of nonpregnant women (n = 5), and normal pregnant women (n = 5) in the third trimester. RESULTS: Two women, suffering from severe preeclampsia and eclampsia respectively, had aCL on 12 and 13 units, respectively. ANA occurred in 11 patients with moderate and severe preeclampsia and intrauterine growth retardation. All women had negative RF tests. Within the CD4 T-helper subpopulations, the proportion of suppressor/inducer T-cell population (CD4+CD45RA+) significantly increased, while the memory and helper/inducer T-cells (CD4+CD45RO+ and CD4+CD29+) significantly decreased during normal pregnancy compared to nonpregnant controls. This deviation of CD4 subpopulations was not found, or was less pronounced, in complicated pregnancies. The proportion of cytotoxic T cells (CD8+S6F1+) was significantly reduced during normal pregnancy. This reduction was less pronounced in complicated pregnancies. CONCLUSION: Systemic immunological deviations toward suppression or decreased activity of the immunological response as seen in normal pregnancies, was not observed in preeclampsia, intrauterine growth retardation, intrauterine fetal death, and abruptio placentae. This lack of suppression or increased T-cell activity may have a primary pathogenic role in some women with a complicated pregnancy, or it may be secondary to the placental disorders.

An open prospective randomized study of dinoproston and gemeprost in second trimester legal abortions.

The aim of this open prospective randomized study was to compare two non-invasive methods for second trimester abortion using gemeprost pessaries (Cervagem) and dinoproston gel (Cerviprost) concerning effectiveness, time for abortion, consumption of analgesics, infection-rate and side-effects. Forty women admitted for second trimester legal abortion were allocated to treatment with either gemeprost (20) intravaginally or dinoproston (20) intracervically. All patients were pre-treated with a 3 mm in diameter Lamicel tent applied intracervically for about four hours. The success-rate was 95% for gemeprost and 75% for dinoproston within approximately 48 hours. The median abortion time calculated from the insertion of the Lamicel tent for the successful cases was 22 h 0 min for gemeprost and 24 h 5 min for dinoproston (not significant). The shortest abortion time was found among parous women in the Cervagem group. The difference between Cerviprost and Cervagem in parous women was statistically significant. For nonparous women there were no significant differences in abortion time between the two regimes. No significant difference was found in the demand of Pethidin and the infection-rate between the two groups. No major side effects of the treatment were found. Even if no significant difference in successful abortions was found, probably due to the small patient material, Cervagem seems to be the most appropriate of the two non-invasive methods, because of a 95 per cent success-rate within 48 hours, but also due to its simplicity in design.

Lymphedema of legs and truncus as a complication of cancer colli uteri and treatment.

A case of secondary lymphedema of legs and truncus due to cancer colli uteri and treatment is presented. Differential diagnosis and treatment models are suggested.

Alteration of steroid hormone sensitivity during the cultivation of human mammary carcinoma cells.

During early cultivation steps of the newly derived and karyotyped human mammary carcinoma line EFM-19, the cells developed faster growth rates and became increasingly less responsive to the presence of serum in the culture medium. No drastic alterations of the morphology and of the karyotype were observed, and carcinoembryogenic antigen remained expressed during the course of the cultivation. In experimental incubations at various time intervals after the explantation, the cell proliferation was analyzed for dose-dependent effects of estradiol, cortisol, progesterone, and testosterone. After 16 wk of cultivation of the stock culture in the presence of estradiol, the cells had acquired a distinct sensitivity to estradiol resulting in permanent growth enhancement. The withdrawal of cortisol from the medium of the stock culture subsequently provoked the loss of the initially noted stimulation of the proliferation by cortisol. The stimulatory effect of progesterone on the proliferation was reversed to inhibition when the stock culture was deprived of cortisol in the growth medium. The results indicate that the choice of steroid hormones in the stock culture medium was determining the quality of the cellular growth responses.