Screening for pre-eclampsia with competing-risks model using placental growth factor measurement in blood samples collected before 11 weeks' gestation.

OBJECTIVES: To describe the distributional properties and assess the performance of placental growth factor (PlGF) measured in blood samples collected before 11 weeks' gestation in the prediction of pre-eclampsia (PE). METHODS: The study population consisted of pregnant women included in the Pre-eclampsia Screening in Denmark (PRESIDE) study with a PlGF measurement from the routine combined first-trimester screening (cFTS) blood sample collected at 8-14 weeks' gestation. PRESIDE was a prospective multicenter study investigating the predictive performance of the Fetal Medicine Foundation (FMF) first-trimester screening algorithm for PE in a Danish population. In the current study, serum concentration of PlGF in the cFTS blood samples was analyzed in batches between January and June 2021. RESULTS: A total of 8386 pregnant women were included. The incidence of PE was 0.7% at < 37 weeks' gestation and 3.0% at ≥ 37 weeks. In blood samples collected at 10 weeks' gestation, PlGF multiples of the median (MoM) were significantly lower in pregnancies with preterm PE < 37 weeks compared to unaffected pregnancies. However, PlGF MoM did not differ significantly between pregnancies with PE and unaffected pregnancies in samples collected before 10 weeks' gestation. CONCLUSIONS: The gestational-age range for PlGF sampling may be expanded from 11-14 to 10-14 weeks when assessing the risk for PE using the FMF first-trimester screening model. There is little evidence to support the use of PlGF in blood samples collected before 10 weeks' gestation. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Pre-eclampsia screening in Denmark (PRESIDE): national validation study.

OBJECTIVES: To investigate the predictive performance of the Fetal Medicine Foundation (FMF) first-trimester screening algorithm for pre-eclampsia in a Danish population and compare screening performance with that of the current Danish strategy, which is based on maternal risk factors. METHODS: This was a prospective study of women with a singleton pregnancy attending for their first-trimester ultrasound scan and screening for aneuploidies at six Danish university hospitals between May 2019 and December 2020. Prenatal data on maternal characteristics and medical history were recorded, and measurements of mean arterial pressure (MAP), uterine artery pulsatility index (UtA-PI), serum pregnancy-associated plasma protein-A (PAPP-A) and serum placental growth factor (PlGF) were collected without performing a risk assessment for pre-eclampsia. Information on acetylsalicylic acid use was recorded. After delivery, pregnancy outcome, including gestational age at delivery and pre-eclampsia diagnosis, was recorded. Pre-eclampsia risk assessment for each woman was calculated blinded to outcome using the FMF screening algorithm following adjustment to the Danish population. Detection rates (DRs) of the FMF algorithm were calculated for a fixed screen-positive rate (SPR) of 10% and for the SPR achieved in the current Danish screening. RESULTS: A total of 8783 pregnant women were included, with a median age of 30.8 (interquartile range (IQR), 28.1-33.9) years. The majority were white (95%), naturally conceiving (90%), non-smokers (97%) and had no family history of pre-eclampsia (96%). The median body mass index was 23.4 (IQR, 21.2-26.6) kg/m2 . A complete risk assessment including maternal characteristics, MAP, UtA-PI, PlGF and PAPP-A was available for 8156 women (92.9%). In these women, UtA-PI was measured bilaterally with a median value of 1.58 (IQR, 1.27-1.94) and the median resting MAP of 80.5 (IQR, 76.1-85.4) mmHg in two consecutive measurements. Among these, 303 (3.7%) developed pre-eclampsia, including 55 (0.7%) cases of pre-eclampsia with delivery < 37 weeks of gestation and 16 (0.2%) cases of pre-eclampsia with delivery < 34 weeks. At a SPR of 10%, combined screening using the FMF algorithm based on maternal characteristics, MAP, UtA-PI, PlGF and PAPP-A had a DR of 77.4% (95% CI, 57.6-97.2%) for pre-eclampsia with delivery < 34 weeks, 66.8% (95% CI, 54.4-79.1%) for pre-eclampsia with delivery < 37 weeks and 44.1% (95% CI, 38.5-49.7%) for pre-eclampsia with delivery at any gestational age. The current Danish screening strategy using maternal risk factors detected 25.0% of women with pre-eclampsia with delivery < 34 weeks and 19.6% of women with pre-eclampsia with delivery < 37 weeks at a SPR of 3.4%. When applying the FMF algorithm including maternal characteristics, MAP, UtA-PI and PlGF at the fixed SPR of 3.4%, the DRs were 60.5% (95% CI, 36.9-84.1%) for PE with delivery < 34 weeks and 45.2% (95% CI, 32.0-58.5%) for PE with delivery < 37 weeks. CONCLUSION: In this large Danish multicenter study, the FMF algorithm based on maternal characteristics, MAP, UtA-PI, PlGF and PAPP-A predicted 77.4% of cases with pre-eclampsia with delivery < 34 weeks and 66.8% of cases with pre-eclampsia with delivery < 37 weeks of gestation at a SPR of 10%, suggesting that the performance of the algorithm in a Danish cohort matches that in other populations. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Tamoxifen-independent Cre-activity in SMMHC-CreER T2 mice.

Background and aims: Recent technological advances have established vascular smooth muscle cells (SMCs) as central players in atherosclerosis. Increasingly complex genetic mouse models have unveiled that 30-70% of cells in experimentally induced atherosclerotic lesions derive from a handful of medial SMCs, and that these can adopt a broad range of plaque cell phenotypes. Most of these models are based on the SMMHC-CreER T2 mouse line as Cre-driver. Importantly, Cre-activation can be controlled in time (by administration of tamoxifen, TAM), which is critical to avoid unwanted effects of premature recombination events. The aim of this study was to scrutinize an unexpected observation of TAM-independent Cre-activity in this mouse line. Methods: Cre-activity was assessed by PCR in tissues from SMMHC-CreER T2 mice crossed with mice homozygous for loxP-flanked (floxed) exon 4 of Ccn2 (our gene-of-interest), and Ccn2 protein was measured in aortas by targeted mass spectrometry. Results: We observed spontaneous near-complete excision of floxed Ccn2 in aortas from adult mice that were not treated with TAM. As a result, Ccn2 protein was significantly reduced in aortas from these mice, but not to the same extent as TAM-treated littermates. Remarkably, most of the excision was completed in 4-week-old mice. Excision was Cre-dependent, as knockout bands were negligible in heart and liver (dominated by non-SMCs) of these mice, and undetectable in the aorta in the absence of Cre. Conclusion: Our observations warrant caution, and we advocate inclusion of appropriate controls (i.e., TAM-untreated mice) in future studies.

Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/-, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

The brown bear as a translational model for sedentary lifestyle-related diseases.

Sedentary lifestyle accelerates biological ageing, is a major risk factor for developing metabolic syndrome and is associated with cardiovascular disease, diabetes mellitus, kidney failure, sarcopenia and osteoporosis. In contrast to the linear path to worsening health in humans with metabolic syndrome, brown bears have developed a circular metabolic plasticity enabling these animals to tolerate obesity and a 'sedentary lifestyle' during hibernation and exit the den metabolically healthy in spring. Bears are close to humans physiology wise, much closer than rodents, the preferred experimental animals in medical research, and may better serve as translational model to develop treatments for lifestyle-related diseases. In this review, aspects of brown bear hibernation survival strategies are outlined and conceivable experimental strategies to learn from bears are described.

Antimicrobial peptide CAP18 and its effect on Yersinia ruckeri infections in rainbow trout Oncorhynchus mykiss (Walbaum): comparing administration by injection and oral routes.

The antimicrobial peptide CAP18 has been demonstrated to have a strong in vitro bactericidal effect on Yersinia ruckeri, but its activity in vivo has not been described. In this work, we investigated whether CAP18 protects rainbow trout Oncorhynchus mykiss (Walbaum) against enteric red mouth disease caused by this pathogen either following i.p. injection or by oral administration (in feed). It was found that injection of CAP18 into juvenile rainbow trout before exposure to Y. ruckeri was associated with lowered mortality compared to non-medicated fish although it was less effective than the conventional antibiotic oxolinic acid. Oral administration of CAP18 to trout did not prevent infection. The proteolytic effect of secretions on the peptide CAP18 in the fish gastrointestinal tract is suggested to account for the inferior effect of oral administration.

A robust immunoassay for pregnancy-associated plasma protein-A2 based on analysis of circulating antigen: establishment of normal ranges in pregnancy.

Pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, two homologous metzincin metalloproteases, are both tightly linked to regulation within the insulin-like growth factor (IGF) system because of their specific cleavage of IGF binding proteins. Recent studies suggest that PAPP-A may be involved in clinical conditions related to unwanted cellular growth, and the circulating levels of PAPP-A is an established biomarker in prenatal screening for chromosomal abnormalities. Microarray data indicate that PAPP-A2 has potential as a biomarker for pre-eclampsia. However, well-characterized immunological methods of quantification are not available. We therefore developed monoclonal antibodies against recombinant PAPP-A2. The antibodies were epitope mapped against recombinantly expressed chimeras between PAPP-A2 and PAPP-A. Furthermore, circulating PAPP-A2 was immunoaffinity purified and characterized by sequence analysis and mass spectrometry. Unlike PAPP-A, PAPP-A2 is a noncovalent dimer in which each subunit of 1558 amino acids originates from all of the 22 predicted coding exons. A previously hypothesized variant (PAPP-E) does not exist, but low amounts of a C-terminally truncated PAPP-A2 variant was detected. A sensitive and robust ELISA for full-length PAPP-A2 was developed and used to establish normal ranges of PAPP-A2 through pregnancy. The functional sensitivity of this ELISA at 20% CV was 0.08 ng/ml, and the serum concentration of PAPP-A2 was found to increase during pregnancy in agreement with placental synthesis. The existence of this assay will enable an assessment of the biomarker potential of PAPP-A2 in pre-eclampsia as well as other clinical conditions.

Reduced muscle activation during exercise related to brain oxygenation and metabolism in humans.

Maximal exercise may be limited by central fatigue defined as an inability of the central nervous system to fully recruit the involved muscles. This study evaluated whether a reduction in the cerebral oxygen-to-carbohydrate index (OCI) and in the cerebral mitochondrial oxygen tension relate to the ability to generate a maximal voluntary contraction and to the transcranial magnetic stimulated force generation. To determine the role of a reduced OCI and in central fatigue, 16 males performed low intensity, maximal intensity and hypoxic cycling exercise. Exercise fatigue was evaluated by ratings of perceived exertion (RPE), arm maximal voluntary force (MVC), and voluntary activation of elbow flexor muscles assessed with transcranial magnetic stimulation. Low intensity exercise did not produce any indication of central fatigue or marked cerebral metabolic deviations. Exercise in hypoxia (0.10) reduced cerebral oxygen delivery 25% and decreased 11+/-4 mmHg (P<0.001) together with OCI (6.2+/-0.7 to 4.8+/-0.5, P<0.001). RPE increased while MVC and voluntary activation were reduced (P<0.05). During maximal exercise declined 8+/-4 mmHg (P<0.05) and OCI to 3.8+/-0.5 (P<0.001). RPE was 18.5, and MVC and voluntary activation were reduced (P<0.05). We observed no signs of muscular fatigue in the elbow flexors and all control MVCs were similar to resting values. Exhaustive exercise provoked cerebral deoxygenation, metabolic changes and indices of fatigue similar to those observed during exercise in hypoxia indicating that reduced cerebral oxygenation may play a role in the development of central fatigue and may be an exercise capacity limiting factor.

Tissue microarrays compared with whole sections and biochemical analyses. A subgroup analysis of DBCG 82 b&c.

INTRODUCTION: The tissue microarray (TMA) technique comprises the potential of significantly reducing time and tissue spent on slicing and performing immunohistochemical (IHC) stainings of paraffin-embedded tumor tissue. Tissue heterogeneity is an argument against using TMAs, which has been dealt with by increasing the size and number of cores punched from each tumor. No consensus exists on the most optimal size, number, and position of TMA cores in the donor paraffin block and no information exist regarding agreement between TMA cores from two different paraffin blocks from the same tumor or between TMA cores and biochemical analyses. PATIENTS AND METHODS: A central and a peripheral 1mm core and a whole section from each of 54 paraffin blocks from 27 breast cancers included in a one-institution cohort, and a single 1mm central TMA core, from each breast tumor from 1000 patients included in the DBCG82 b&c trials, were IHC stained for ER, PgR and HER2. In addition, ER and PgR were measured in the DBCG82 b&c trials by a biochemical analysis. Statistical analyses included Kappa statistics, Kaplan-Meier survival curves, Log-rank tests, and Cox regression hazards analyses. RESULTS AND CONCLUSION: IHC stainings for ER, PgR, and HER2 showed a substantial agreement between a single 1mm TMA core and the corresponding whole section, between central and peripheral cores, and between cores from two different paraffin blocks from the same tumor. In addition, a fine agreement was found for ER and PgR between IHC stainings of TMA cores and biochemical analyses. Divergence between IHC and biochemical analyses was predominantly due to the chosen thresholds. IHC staining of one 1mm core from each tumor revealed a significant independent prognostic value of PgR and HER2 on overall survival. In conclusion, IHC stainings for ER, PgR, and HER2 of just a single 1mm TMA core seems to be sufficient, as no significant heterogeneity was noticed.

Impact of BCL2 and p53 on postmastectomy radiotherapy response in high-risk breast cancer. A subgroup analysis of DBCG82 b&c.

PURPOSE: To examine p53 and BCL2 expression in high-risk breast cancer patients randomized to postmastectomy radiotherapy (PMRT). PATIENTS AND METHODS: The present analysis included 1 000 of 3 083 high-risk breast cancer patients randomly assigned to PMRT in the DBCG82 b&c studies. Tissue microarray sections were stained with immunohistochemistry for p53 and BCL2. Median potential follow-up was 17 years. Clinical endpoints were locoregional recurrence (LRR), distant metastases (DM), overall mortality, and overall survival (OS). Statistical analyses included Kappa statistics, chi(2) or exact tests, Kaplan-Meier probability plots, Log-rank test, and Cox univariate and multivariate regression analyses. RESULTS: p53 accumulation was not significantly associated with increased overall mortality, DM or LRR probability in univariate or multivariate Cox regression analyses. Kaplan-Meier probability plots showed reduced OS and improved DM and LRR probabilities after PMRT within subgroups of both p53 negative and p53 positive patients. Negative BCL2 expression was significantly associated with increased overall mortality, DM and LRR probability in multivariate Cox regression analyses. Kaplan-Meier probability plots showed a significantly improved overall survival after PMRT for the BCL2 positive subgroup, whereas practically no survival improvement was seen after PMRT for the BCL2 negative subgroup. In multivariate analysis of OS, however, no significant interaction was found between BCL2 and randomization status. Significant reductions in LRR probability after PMRT were recorded within both the BCL2 positive and BCL2 negative subgroups. CONCLUSION: p53 was not associated with survival after radiotherapy in high-risk breast cancer, but BCL2 might be.

Expression of membrane progesterone receptors on human T lymphocytes and Jurkat cells and activation of G-proteins by progesterone.

Although there is significant evidence for progesterone's role as an immunomodulator, nuclear progesterone receptors have not been consistently identified in immune cells. Recently, three new putative membrane progesterone receptors (mPRs), mPRalpha, mPRbeta, and mPRgamma have been described. The objective of this study was to examine whether mPRs are expressed in peripheral blood leukocytes (PBLs) in women of reproductive age, and to further characterize them in T lymphocytes and immortalized T cells (Jurkat cells). Transcripts for mPRalpha and mPRbeta but not mPRgamma, were detected by RT-PCR in PBLs, T lymphocytes, and Jurkat cells. Western blot analysis showed the presence of the mPRalpha and mPRbeta proteins on cell membranes of T lymphocytes and Jurkat cells. Expression of the mPRalpha mRNA was upregulated in the luteal phase of the menstrual cycle in cluster of differentiation (CD)8+, but not in CD4+, T lymphocytes. Radioreceptor assays revealed specific [(3)H]progesterone binding to T- and Jurkat cell membranes (K(d) 4.25 nM) characteristic of steroid membrane receptors. Progesterone activated an inhibitory G-protein (G(i)), suggesting that mPRs are coupled to G(i) in Jurkat cells. These results suggest a potential novel mechanism for progesterone's immunoregulatory function through activation of mPRs.

Slow spontaneous [Ca2+] i oscillations reflect nucleotide release from renal epithelia.

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca(2+)](i) increases in renal epithelia. Spontaneous [Ca(2+)](i) increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 +/- 6.7% (n = 23) of the cells showed spontaneous [Ca(2+)](i) increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca(2+)](i) oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca(2+)](i) oscillatory activity. The association between spontaneous [Ca(2+)](i) increases and nucleotide signalling was further tested in 132-1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca(2+)](i) increases. Transfection with either hP2Y(6) or hP2Y(2) receptors revealed a striking degree of oscillations. Similar spontaneous [Ca(2+)](i) increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y(2) knock out mice (0.050 +/- 0.020 events per second, n = 8) compared to the wild type (0.147 +/- 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca(2+)](i) oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.

Risk of radiation-induced subcutaneous fibrosis in relation to single nucleotide polymorphisms in TGFB1, SOD2, XRCC1, XRCC3, APEX and ATM--a study based on DNA from formalin fixed paraffin embedded tissue samples.

PURPOSE: In two previously published studies, associations with risk of radiation-induced subcutaneous fibrosis were found for single nucleotide polymorphisms (SNP) in TGFB1 (transforming growth factor beta 1 gene), XRCC1 (X-ray repair cross-complementing 1 gene), XRCC3 (X-ray repair cross-complementing 3 gene), SOD2 (manganese superoxide dismutase gene) and ATM (gene of ataxia telangiectasia mutated). The present study was conducted to seek a confirmation of these findings. MATERIALS AND METHODS: Like the 41 patients previously investigated, the 120 subjects included in the present study were accrued from a historical cohort of 319 post-mastectomy radiotherapy patients. All patients received hypo-fractionated radiotherapy. The TGFB1 position--509, codons 10 and 25, XRCC1 codons 194, 280 and 399, XRCC3 codon 241, SOD2 codon 16, ATM codon 1853 and APEX (apurinic/apyrimidinic exonuclease gene) codon 148 polymorphisms were assessed based on archival histological material. Differences in fibrosis risk were quantified from dose-response assessments. RESULTS: For none of the investigated polymorphisms, significant associations with risk of subcutaneous fibrosis were observed. A detailed analysis did not reveal any obvious explanation for the discrepancy between the previous and the present study. CONCLUSION: The previously observed associations with risk of radiation-induced subcutaneous fibrosis could not be replicated in the present study. Further studies are needed to elucidate the influence of genetic variation upon normal tissue radiosensitivity.

Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.

Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE-->ESE, ESE-->MSE, and MSE-->LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy specimens) from subjects who are as normal as possible in a human study and including those with unknown histology, can be classified by their molecular signatures and correspond to known phases of the menstrual cycle with identical results using two independent analytical methods. Also, the results enable global identification of biological processes and molecular mechanisms that occur dynamically in the endometrium in the changing steroid hormone milieu across the menstrual cycle in normo-ovulatory women. The results underscore the potential of gene expression profiling for developing molecular diagnostics of endometrial normalcy and abnormalities and identifying molecular targets for therapeutic purposes in endometrial disorders.

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles.

In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work, we showed that follicular fluid from late dominant equine follicles (35 mm diameter) contains a proteolytic activity against IGFBP-2. First follicular fluid from dominant follicles contained lower levels of native IGFBP-2 than the corresponding serum, as assessed by Western ligand blotting. In contrast, immunoblotting experiments showed much higher levels of a 12 kDa proteolytic fragment in dominant follicular fluid than in the serum. Moreover, equine dominant follicular fluid was able to induce proteolysis of exogenous recombinant bovine (rb)IGFBP-2, this degradation being dose-dependently enhanced by IGFs. The proteolytic activity against IGFBP-2 in equine follicles was partially immunoneutralized by a polyclonal antibody raised against human PAPP-A. Moreover, cleavage of rbIGFBP-2 by equine follicular fluid was dose-dependently inhibited by a peptide derived from the heparin-binding domain of IGFBP-5, as well as by peptides derived from other heparin-binding domain-containing proteins such as connective tissue growth factor, vitronectin and heparin-interacting protein, previously shown to inhibit PAPP-A. Finally, the proteolytic activity was very low in subordinate follicles, was high in both early (25 mm diameter) and late (35 mm diameter) dominant follicles, and was slightly lower in preovulatory follicles recovered 35 h after human chorionic gonadotropin (hCG) treatment.Overall, these data show that in the equine ovary, the selection of dominant follicles is associated with an increase of the proteolytic degradation of IGFBP-2 by PAPP-A, as for IGFBP-4, and potentially other protease(s), probably contributing to the increase in IGF bioavailability. In atretic subordinate follicles, the decrease in the proteolytic degradation of IGFBP-2, probably due in part to a direct inhibition by peptides containing heparin-binding domains, contributes to the increase in IGFBP-2 levels and the decrease in IGF bioavailability. The expression of PAPP-A and IGFBP-2 mRNA during folliculogenesis remain to be investigated in the mare.

Screening for Down's syndrome in early and late first and second trimester using six maternal serum markers.

The efficiency of six maternal serum markers for Down's syndrome (DS), alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), free beta-hCG, pregnancy-associated plasma protein-A (PAPP-A), the proform of eosinophil major basic protein (ProMBP), pregnancy-specific-beta-1-glycoprotein (SP(1)), and combinations thereof, was examined. Discriminant analysis in 156 DS pregnancies and 546 controls defined three effective combinations of serum marker logMoMs (multiples of the median in control samples) in three gestational age windows, i.e. Index I (weeks 7-9) = 0.52 logMoM ProMBP + 0.28 logMoM PAPP-A - logMoM SP(1); Index II (weeks 10-12) = 1.94 logMoM free beta-hCG - logMoM SP(1), and Index III (weeks 15-19) = 0.78 logMoM free beta-hCG + 1.12 logMoM ProMBP - logMoM AFP. The estimated detection rates of indices and age for a false-positive rate (FPR) of 5% were 73% for Index I, 69% for Index II, and 60% for Index III. Including the ultrasound marker nuchal translucency, using a DS at term risk of 1 : 400 as cut-off, the detection rates of the indices increased to 86, 83, and 82% for FPRs of 4.3, 4.1, and 5.8%, respectively. The indices are promising markers for screening for DS.

A simple method to test if the internal mammary lymph nodes are covered by the wide tangent technique in radiotherapy for high-risk breast cancer.

AIM: It is often complicated to include the internal mammary lymph nodes in the radiation field after breast-conserving therapy. Using the wide tangent technique the internal mammary lymph nodes are generally presumed to be included if the medial tangential field border is placed 3 cm across the midline. The current study was designed to test the validity of this assumption, and if possible, to correct the wide tangents without using computed tomography (CT) scanning. PATIENTS AND METHODS: Twenty-one consecutive, high-risk, post-lumpectomy patients were included. An arrangement of three copper wires was mounted in wax placed perpendicular to the skin surface at the ipsilateral border of sternum at intercostal spaces 2 to 4. During a standard simulation for wide tangents, it was examined if the length of the copper wires projected beneath the skin surface (representing the depth of the internal mammary lymph nodes, measured by ultrasound) were included in the wide tangent fields. RESULTS: In only one patient were the internal mammary lymph nodes covered by the wide tangent technique. In 14 of the remaining 20 patients the lateral tangential field border was subsequently moved in the posterior direction, and the internal mammary lymph nodes could be included without unacceptable normal tissue involvement. In the last six patients the irradiated heart and lung volumes exceeded acceptable tolerance levels with this correction, and these patients were referred for three-dimensional CT dose planning. CONCLUSION: The presented simple technique may be helpful if CT scanning is not available. In all other cases CT-based dose plan should ideally be used as a standard in the planning of radiotherapy after breast-conserving surgery to assure optimal inclusion of the relevant target, and to avoid irradiation of large volumes of critical normal tissue.

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation.

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.

Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development.

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Pregnancy-associated plasma protein A as a marker of acute coronary syndromes.

BACKGROUND: Circulating markers indicating the instability of atherosclerotic plaques could have diagnostic value in unstable angina or acute myocardial infarction. We evaluated pregnancy-associated plasma protein A (PAPP-A), a potentially proatherosclerotic metalloproteinase, as a marker of acute coronary syndromes. METHODS: We examined the level of expression of PAPP-A in eight culprit unstable coronary plaques and four stable plaques from eight patients who had died suddenly of cardiac causes. We also measured circulating levels of PAPP-A, C-reactive protein, and insulin-like growth factor I (IGF-I) in 17 patients with acute myocardial infarction, 20 with unstable angina, 19 with stable angina, and 13 controls without atherosclerosis. RESULTS: PAPP-A was abundantly expressed in plaque cells and extracellular matrix of ruptured and eroded unstable plaques, but not in stable plaques. Circulating PAPP-A levels were significantly higher in patients with unstable angina or acute myocardial infarction than in patients with stable angina and controls (P<0.001). A PAPP-A threshold value of 10 mlU per liter identified patients who had acute coronary syndromes with a sensitivity of 89.2 percent and a specificity of 81.3 percent. PAPP-A levels correlated with levels of C-reactive protein and free IGF-I, but not with markers of myocardial injury (troponin I and the MB isoform of creatine kinase). CONCLUSIONS: PAPP-A is present in unstable plaques, and circulating levels are elevated in acute coronary syndromes; these increased levels may reflect the instability of atherosclerotic plaques. PAPP-A is a new candidate marker of unstable angina and acute myocardial infarction.