Camilla S. Kronborg

Longitudinal measurement of cytokines in pre-eclamptic and normotensive pregnancies

Objective. To evaluate differences in plasma cytokine levels longitudinally in pre‐eclamptic and normotensive pregnancies. An increased inflammatory response has long been associated with pre‐eclampsia, both early and late in the pre‐eclamptic pregnancy. Design. Blood samples were collected longitudinally during pregnancy from a cohort of 1 631 pregnant women. Thirty‐two women with pre‐eclampsia and 67 normotensive pregnant women were identified from the cohort. Setting. A Danish regional hospital. Samples. Samples were collected from the 18th week of pregnancy until delivery and divided into the following four gestational intervals: <25th week, 26th–29th week, 30th–35th week and >36th week. Methods. Simultaneous measurement of all nine cytokines was done using a capture bead system. Main Outcome Measures. Plasma levels of interleukin (IL)‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, tumor necrosis factor‐α, interferon‐γ and granulocyte macrophage colony‐stimulating factor during pre‐eclamptic and normotensive pregnancies. Results. Pre‐eclampsia was associated with increased tumor necrosis factor‐α between the 26th and 29th week (p=0.0421) and increased IL‐6 after the 36th week (p=0.0044). The other cytokines measured were comparable in the two groups. Conclusions. This large prospective collection of blood samples was undertaken to determine inflammatory status during pre‐eclamptic and normotensive pregnancies. Our results support a tendency towards increased inflammation in pre‐eclampsia, but the measured cytokines are not eligible for prediction, monitoring or diagnosing pre‐eclampsia.

A single rapid point-of-care placental growth factor determination as an aid in the diagnosis of preeclampsia

OBJECTIVES To evaluate the clinical performance of a rapid point-of-care test, Triage PLGF (Alere, San Diego) in the diagnosis of preeclampsia. STUDY DESIGN For the reference range 2212 plasma samples were collected from 595 subjects with normotensive pregnancies, between week 17 of gestation and delivery. In the case-control part, two cohorts of women with preeclampsia (80 women) were matched for maternal age, gestational age (GA) at sampling and parity with normotensive women who delivered at 37weeks or more. RESULTS The areas under the receiver operating characteristic curves (GA<35weeks) were 1.0 and 0.994 (cohort 1 and 2, respectively). The clinical sensitivity of the Triage PLGF test for the pooled GA range of 21⩽GA<35, using a GA dependent cut-off, was 1.0 for both cohorts with specificities of 1.0 and 0.940. CONCLUSIONS The Triage PLGF test distinguishes well between preterm pregnancies with and without preeclampsia.

Folic acid mediates activation of the pro-oncogene STAT3 via the Folate Receptor alpha

The signal transducer and activator of transcription 3 (STAT3) is a well-described pro-oncogene found constitutively activated in several cancer types. Folates are B vitamins that, when taken up by cells through the Reduced Folate Carrier (RFC), are essential for normal cell growth and replication. Many cancer cells overexpress a glycophosphatidylinositol (GPI)-anchored Folate Receptor α (FRα). The function of FRα in cancer cells is still poorly described, and it has been suggested that transport of folate is not its primary function in these cells. We show here that folic acid and folinic acid can activate STAT3 through FRα in a Janus Kinase (JAK)-dependent manner, and we demonstrate that gp130 functions as a transducing receptor for this signalling. Moreover, folic acid can promote dose dependent cell proliferation in FRα-positive HeLa cells, but not in FRα-negative HEK293 cells. After folic acid treatment of HeLa cells, up-regulation of the STAT3 responsive genes Cyclin A2 and Vascular Endothelial Growth Factor (VEGF) were verified by qRT-PCR. The identification of this FRα-STAT3 signal transduction pathway activated by folic and folinic acid contributes to the understanding of the involvement of folic acid in preventing neural tube defects as well as in tumour growth. Previously, the role of folates in these diseases has been attributed to their roles as one-carbon unit donors following endocytosis into the cell. Our finding that folic acid can activate STAT3 via FRα adds complexity to the established roles of B9 vitamins in cancer and neural tube defects.

Serum markers of macrophage activation in pre-eclampsia: no predictive value of soluble CD163 and neopterin

Background. Alternatively activated macrophages expressing the CD163 and CD206 surface receptors are the dominant immune‐cell type found in the placenta. The placental number and distribution of macrophages is altered in pre‐eclampsia, and the generalised inflammatory reaction associated with pre‐eclampsia might lead to shedding of soluble CD163 into the circulation. Methods. Serum samples from 18 women with pre‐eclampsia and 90 normal pregnancies were obtained from a longitudinal study of 955 pregnant women at Randers County Hospital, Denmark. sCD163 and Neopterin were measured by ELISA on samples collected in weeks 18, 28, 32, and 38 of pregnancy. Results. sCD163 levels in pregnancy (2–3 mg/l) were similar to previously measured levels in non‐pregnant women, and did not increase from week 18 to 38. There was a tendency towards higher sCD163 in week 38 in pre‐eclamptic women compared to healthy women. Neopterin increased throughout pregnancy in both healthy (from median 5.4 to 6.7 nmol/l, p<0.0001) and pre‐eclamptic women (from 5.0 to 8.0 nmol/l, p<0.0001), but there were no differences between groups at any time‐point. sCD163 correlated to neopterin in both the control (r = 0.25, p<0.0001) and in the pre‐eclampsia group (r = 0.32, p = 0.011). C‐reactive protein was higher in pre‐eclampsia than in healthy pregnancies by week 38 (159 versus 91 nmol/l, p = 0.0189). Conclusions. The macrophage serum‐markers sCD163 and neopterin are not pre‐symptomatic nor prognostic markers for pre‐eclampsia.

Pre‐symptomatic increase in urine‐orosomucoid excretion in pre‐eclamptic women

Background. Although the pre‐eclamptic pathogenesis begins at least around the 18th week of pregnancy, clinically evident disease often does not appear until the third trimester. This long pre‐symptomatic latency period has led to intensive research for early markers of evolving disease. We evaluated urine excretion and plasma levels of orosomucoid and albumin longitudinally in healthy and pre‐eclamptic pregnancies. Orosomucoid is an acute phase protein involved in inflammation and protection of the endothelium. Methods. From a prospective, longitudinal cohort study consisting of 1,631 women, 32 women developed pre‐eclampsia, and 5 controls for every case of pre‐eclampsia were found. Blood samples were collected 4 times and urine samples 6 times from the 18/19th week and throughout pregnancy. Orosomucoid and albumin in plasma were analysed by standard methods, and in urine by sandwich ELISA. Results. Orosomucoid/creatinin excretion ratio was significantly higher early in pre‐eclamptic pregnancies (from the 20th week of pregnancy, p = 0.0053) compared with healthy pregnancies, the difference increased throughout pregnancy. Albumin/creatinin ratio increased subsequent to the increase in orosomucoid. In the plasma samples, orosomucoid was significantly higher late in pre‐eclamptic pregnancies (≥36th week, p = 0.0275). Conclusions. Pre‐eclampsia is associated with a pre‐symptomatic increase in the urine excretion of orosomucoid, and orosomucoid excretion precedes that of albumin. Orosomucoid excretion can probably be used as a prognostic tool in combination with other screening methods, and seems to be a more sensitive marker for evolving pre‐eclampsia than albumin. Plasma orosomucoid is significantly increased late in pre‐eclampsia. Thus, the increased excretion of orosomucoid must primarily originate from altered renal processing of orosomucoid.

Differential regulation of the interferon induced gene ISG12A by serum from healthy and preeclamptic pregnancies

Endothelial-cell dysfunction is central in the preeclamptic pathogenesis. Several components present in the blood of the preeclamptic mother are capable of mediating this dysfunction. We analyzed the regulation of the ISG12A gene by serum from the third trimester, to elucidate the role of type 1 interferon ISGs late in both healthy and preeclamptic pregnancies. The ISG12A transcription was up-regulated by serum from healthy pregnant women, but not by preeclamptic serum in HeLa and human umbilical vein endothelial cells (HUVEC). However, the ISG12A up-regulation by healthy pregnancy serum was not due to a general type 1 interferon response, since 6-16 and OAS1 were not up-regulated similarly. Also, the up-regulation of ISG12A was independent of the interferon-alpha receptor 2, but dependent on STAT1. Stimulation with folic acid alone or in combination with preeclamptic serum up-regulated ISG12A and 6-16. We conclude that type 1 interferon is not increased in third trimester serum, neither from healthy nor preeclamptic pregnancies. However, since ISG12A mRNA is up-regulated in healthy pregnancies, the ISG12A protein might take part in maintaining endothelial stability, as this function is lacking in preeclamptic pregnancies. Folic acid may ameliorate endothelial cell stability in preeclampsia by up-regulating ISG12A.

Excretion patterns of large and small proteins in pre-eclamptic pregnancies

Objective. Pre‐eclampsia is a serious pregnancy complication causing both fetal and maternal distress. Proteinuria is a diagnostic criterion frequently determined by albuminuria. We determined the protein excretion pattern of additional proteins, immunoglobulin G, transferrin, α1‐microglobulin and β2‐microglobulin, in urine samples collected prospectively during pre‐eclamptic and healthy pregnancies. Design. A prospective cohort study of 1 631 consecutive pregnant women. Setting. A Danish regional hospital. Sample. Thirty‐two women with pre‐eclampsia and 185 healthy control women were identified from the cohort. Urine samples were obtained from the 18th week until delivery and divided into six gestational intervals. Methods. Protein analyses of urine immunoglobulin G, transferrin, α1‐microglobulin and β2‐microglobulin were done with a sandwich ELISA method. Main Outcome Measures. Urine levels of specific proteins during pre‐eclamptic and healthy pregnancies. Results. Immunoglobulin G and transferrin were significantly increased in pre‐eclampsia after the 30th week of pregnancy. α1‐Microglobulin and β2‐microglobulin were differently excreted and found to be higher after the 36th week of pregnancy in pre‐eclampsia, but only α1‐microglobulin increased significantly. Conclusions. Immunoglobulin G, transferrin, α1‐ and β2‐microglobulin excretion patterns indicate initial glomerular damage followed by altered tubular handling of proteins.