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PURPOSE: BRAF(V600E), a major driver of thyroid cancer, evaluated in the context of thyroid hormones and human relaxin. METHODS: Immunohistochemical expression of BRAF(V600E), TSH, TSH receptor (TSHR), T4, T3 receptor (T3R), RLNH2, and its receptor, RXFP1, were evaluated in thyroid tumors from a retrospective U.S. population of 481 cancer cases diagnosed in 1983-2004. RESULTS: BRAF(V600E) was expressed in 52% of all thyroid tumors; expression of other markers ranged from 25% for T4 to 98% for RLNH2. Tumors predominantly exhibited hypothyroid-like conditions characterized by elevated TSH and TSHR and reduced T4. BRAF(V600E) prevalence was significantly higher in tumors expressing TSH, TSHR, T3R, and RXFP1 and lower in tumors expressing T4. The proportion of BRAF(V600E) mutation in classic papillary tumors significantly increased from 56% to 72% over the 21-year period of diagnoses while expression of RXFP1, TSH, TSHR, and T3R decreased in non-tumor. Racial/ethnic differences were observed in thyroid hormone marker expression. Non-tumor expression of TSH, TSHR, and T3R were each associated with shorter overall survival but did not remain significant after adjustment for demographic and clinical factors. CONCLUSIONS: Our study provides the first evidence of the potential interaction of BRAF(V600E) mutation, relaxin, and thyroid hormones in thyroid carcinogenesis. Moreover, our results suggest that hypothyroidism, influenced by RLNH2 activity, may underlie the development of the majority of thyroid cancers and mediate the role of BRAF(V600E) in thyroid carcinogenesis. BRAF(V600E) mutation is increasing in papillary thyroid cancers and may be contributing to the rising incidence of this malignancy

Relaxin, a systemic and placental hormone, has potential roles in fetoplacental growth. Human placenta expresses two RLN genes, RLNH1 and RLNH2. Maternal obesity is common and is associated with abnormal fetal growth. Our aims were to relate systemic and cord blood RLNH2, placental RLNs and their receptor (RXFP1) with fetoplacental growth in context of maternal body mass index, and associations with insulin-like growth factor 2 (IGF2) and vascular endothelial growth factor A (VEGFA) in the same placentas. Systemic, cord blood and placental samples were collected prior to term labor, divided by prepregnancy body mass index: underweight/normal (N = 25) and overweight/obese (N = 44). Blood RLNH2 was measured by ELISA; placental RLNH2, RLNH1, RXFP1, IGF2 and VEGFA were measured by quantitative immunohistochemistry and mRNAs were measured by quantitative reverse transcription PCR. Birthweight increased with systemic RLNH2 only in underweight/normal women (P = 0.036). Syncytiotrophoblast RLNH2 was increased in overweight/obese patients (P = 0.017) and was associated with placental weight in all subjects (P = 0.038). RLNH1 had no associations with birthweight or placental weight, but was associated with increased trophoblast and endothelial IGF2 and VEGFA, due to female fetal sex. Thus, while systemic RLNH2 may be involved in birthweight regulation in underweight/normal women, placental RLNH2 in all subjects may be involved in placental weight. A strong association of trophoblast IGF2 with birthweight and placental weight in overweight/obese women suggests its importance. However, an association of only RLNH1 with placental IGF2 and VEGFA was dependent upon female fetal sex. These results suggest that both systemic and placental RLNs may be associated with fetoplacental growth.

Visfatin/nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in energy metabolism and sirtuins, SIRT1 and SIRT3, which are NAD-dependent deacetylases, are critical for cellular function. All three either regulate or are regulated by intracellular NAD+ levels and therefore available cellular energy, important for placental cell survival and successful pregnancy. This study investigates whether these protective proteins are involved in the placental pathophysiology of pre-eclampsia (PE) and if they are associated with 8-oxo-deoxyguanosine (8OHdG), a marker of oxidative damage or with placental telomere length.Maternal blood and placental samples were collected from 31 patients with PE and 30 controls between 31 and 40 weeks gestation. Quantitative immunohistochemistry was performed on placental specimens for visfatin/Nampt, SIRT1, SIRT3, and nuclear 8OHdG. Plasma visfatin was measured by ELISA and telomere length by Southern blot analysis of telomere restriction fragments.Visfatin/Nampt and SIRT1 in syncytiotrophoblast decreased in PE compared to controls (p 0.0001, p = 0.004 respectively). SIRT3 decreased in PE most significantly at preterm (p = 0.002). 8OHdG was only significantly lower in preterm controls compared to term controls (p = 0.01) and correlated with SIRT1 in all samples (r = 0.27). Telomere length was not different in PE and controls.Decreased visfatin/Nampt, SIRT1 and SIRT3 in syncytiotrophoblast in PE suggests a lack of placental reserve in metabolic energy efficiency, increased inflammation, and lower resistance to environmental stressors. However, there was little effect on nuclear function, or evidence of genomic DNA damage, which would lead to cellular senescence and death.

This study was designed to show whether placental relaxin (RLN), its receptor (RXFP1), or insulin-like peptide 4 (INSL4) might have altered expression in patients with placenta accreta. The baseline expression of their genes through gestation (n = 34) was quantitated in the placental basal plate (BP) and villous trophoblast (TR), and compared to their expression in placenta accreta (n = 6). The proteins were also immunolocalized and quantitated in the accreta tissues. The messenger RNAs (mRNAs) of matrix metalloproteinase 9, -2, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were also measured. Results demonstrated that the BP and TR expressed low levels of RLN/RXFP1 and INSL4 through gestation. In accreta, increased RLN gene and protein in BP were associated with antepartum bleeding whereas INSL4 expression decreased throughout the TR. There were no changes in mRNAs for MMPs, but TIMP-1 was increased only in the invasive TR.

Intrauterine infection frequently leads to preterm birth (PTB), with the pathophysiology involving activation of the innate immune system and its associated inflammatory response. The choriodecidua produces relaxin (RLN) and elevated levels are associated with preterm premature rupture of the fetal membranes. However, it is not increased in bacterially-mediated PTB, but may act as an endogenous sterile inflammatory mediator. Elevated systemic RLN levels from the corpus luteum are also associated with PTB, but the mechanism is unknown. In clinical obstetrics, intrauterine inflammation or infection can coexist with elevated RLN. Therefore, in this study, we further characterized the effects of RLN alone or together with an inflammatory mediator on the production of IL1B, CSF2 (GM-CSF), IL6, IL8 and TNF, from chorionic cytotrophoblasts (CyT), decidual fibroblasts (DF) and stromal cells (DSC), using interleukin-1 beta (IL1B) to mimic sterile inflammation or lipopolysaccharide (LPS) for bacterial infection. Endogenous differences between the cells showed that the CyT expressed more RLN, its receptor RXFP1 and the RXFP1 splice variant D. CyT also showed the most robust cAMP response to RLN with increased IL6 secreted after 4 h, preceded by increased transcription at 1 h, likely due to activation of RXFP1 and cAMP. When all cell types were treated with IL1B and RLN, RLN augmented secretion of IL6 and IL8 from CyT and DF, but not DSC. Similarly, RLN augmented LPS-induced IL6 secretion from CyT and DF. Despite the structural similarity between TLR4 and RXFP1, blocking TLR4 in CyT had no effect on RLN-induced IL6 secretion, suggesting specific activation of RXFP1. Thus, we have shown that in the presence of a low level of intrauterine inflammation/infection, elevated RLN could act on the CyT and DF to augment the inflammatory response, contributing to the pathophysiology of PTB. Summary RLN augments the inflammatory responses induced by IL1B or LPS in chorionic cytotrophoblasts and decidual fibroblasts.

In normal pregnancy, the fetal membranes become increasingly distended towards term and in multifetal gestations they become over-distended. Apoptosis of the amniotic epithelium increases with advancing gestation and may contribute to fetal membrane weakening and rupture. The effects of chronic static stretching for 36 h have been investigated using primary amniotic epithelial cells. Pre-B cell colony-enhancing factor (PBEF) is a stretch-responsive cytokine and expression of its gene, intracellular and secreted protein were all significantly increased by 4 h and its secretion sustained over 36 h, contrasting with the rapid increase and decline in expression of IL-8. Increased expression of SIRT1 and decreased p53 paralleled the changes in PBEF, are known to be responsive to PBEF, and contribute to cell survival. Distension had no effects on proliferation or necrosis but protected the cells from apoptosis, knocking-down PBEF with antisense probes abrogated this protective effect. There was increased immunostaining of PBEF in the compact layer of the amnion in multifetal tissues and significantly fewer apoptotic amniotic epithelial cells. These results show that chronic stretching of the amniotic epithelial cells increases PBEF expression, which protects them from apoptosis.

The human fetal membranes are complex tissues that perform many important functions during gestation. The extracellular matrix provides their strength to withstand the forces directed from the fetus and myometrium. Relaxin is a collagenolytic hormone that causes increased production of the matrix metalloproteinases. Its expression from the decidua is increased in patients with preterm premature rupture of the membranes, and its leucine-rich G receptor 7 is upregulated at preterm. The authors previously showed that relaxin is not involved in the infection-mediated cytokine response, but in the absence of infection, it causes increased secretion of both interleukin -6 and interleukin-8 from the membranes. In this article, the authors propose that relaxin is one of a number of sterile stimuli capable of causing increased proinflammatory cytokines, similar to but less robust than the effects of infection. These probably represent distinct inflammatory pathways involving different intracellular signaling events, which can result in either preterm premature rupture of the membranes or preterm labor. The current challenge is to fully understand these pathways and to clarify their similarities and differences.

Visfatin is both a systemic adipocytokine and the cytosolic enzyme, nicotinamide phosphoribosyl transferase (Nampt). This is a longevity protein, which extends the lifespan of human cells by activating sirtuin 1 (SIRT1). In this study, we sought a role for these proteins in obese pregnant women, who experience more postterm deliveries. Thus, 78 women (26 lean, 24 overweight, and 28 obese) were recruited and maternal blood and placental tissue collected prior to term labor. Plasma levels were measured by enzyme-linked immunosorbent assay and quantitative immunohistochemistry used for placenta. We confirmed maternal plasma interleukin 6 increased according to prepregnancy body mass index (BMI; P < .0001) and showed a linear relationship between BMI and syncytiotrophoblast visfatin/Nampt (P = .021) but not with its levels in maternal plasma. Both systemic and placental visfatin/Nampt were significantly associated with placental SIRT1 levels (P = .028 and .017). Thus, higher visfatin/Nampt may prevent a labor-associated decrease in SIRT1 leading to postterm delivery in obesity.

This study aimed to gain new insights into both systemic and placental leptin and its receptors, with reference to the maternal prepregnancy body mass index (BMI). Thus, 84 women (29 lean, 24 overweight, and 31 obese) were recruited and maternal, cord blood, and placental tissues collected prior to term labor. Plasma levels were measured by enzyme-linked immunosorbent assay and for placenta, immunohistochemistry and messenger RNAs (mRNAs) were quantitated. We confirmed that maternal leptin increased linearly as the soluble receptor decreased with BMI (P = .001). Fetal leptin increased with maternal BMI (P = .02) and birth weight (P = .006) and was higher in female infants (P < .001). Placental mRNA levels of leptin and its receptors showed no change in BMI. However, we show a significant (P = .043) linear increase in leptin in the placental vascular endothelial cells with maternal obesity, while leptin in syncytiotrophoblast showed no statistical change. Leptin receptors localized to syncytiotrophoblast and intravillous macrophages and were unchanged with BMI.

The relaxin receptor has been recently described as a leucine-rich repeat G-protein coupled receptor and designated as LGR7. A closely related receptor, LGR8, is co-expressed by some cells. This study explored the expression of the genes for these receptors in the human fetal membranes and placenta by RT-PCR and the LGR7 protein by immunolocalization. The results showed that LGR7 was well expressed in the fetal membranes, with significantly more in the decidua (p < 0.05) than in the amnion. On the other hand, relatively low levels were expressed in the placenta. The major splice variant of LGR7 was undetectable in either the placenta or fetal membranes. Expression of LGR8 was also below the level of detectability in either tissue. Immunostaining for LGR7 was conducted with antisera to both its endomain and ectodomain, in order to seek evidence for a solubilized ectodomain. However, similar staining patterns were obtained with both antisera, with predominant staining in the cells of the amniotic epithelium, chorionic cytotrophoblast and decidua. Full-thickness fetal membranes from preterm deliveries, before and after labor or after preterm premature rupture of the membrane (PPROM) and labor were collected. In addition, membranes at term, both before and after spontaneous labor were used for analysis of LGR7 gene expression. There was significantly greater LGR7 expressed (p = 0.01) in the preterm period compared to term, indicating a potentially important role for relaxin at this time. There was a marginal decline in LGR7 gene expression after labor and delivery both at preterm and term, which did not reach significance. Immunostaining patterns showed less inter-patient variability than did gene expression, with more intense staining for LGR7 after labor and delivery.

The purpose of this study was to determine whether pre-B-cell colony-enhancing factor is a secreted cytokine in the human amnion and to study its chemotaxic and antiapoptotic properties.Pre-B-cell colony-enhancing factor secretion was studied from amniotic epithelial-like WISH cells and primary amniotic epithelial cells that were seeded on squares of immobilon-P membrane and stimulated with lipopolysaccharide or tumor necrosis factor-alpha, respectively. The pre-B-cell colony-enhancing factor protein was detected both intracellularly and after secretion, as bound to the membrane, by immunostaining and densitometry. Medium and cell lysates that were obtained from WISH cells that were treated with lipopolysaccharide alone or together with a pre-B-cell colony-enhancing factor antisense oligonucleotide to block pre-B-cell colony-enhancing factor translation were also analyzed for secreted pre-B-cell colony-enhancing factor by Western blotting and densitometry. A chemotaxic effect of pre-B-cell colony-enhancing factor on human neutrophils was compared with the chemoattractants interleukin-8 and N-Formyl-Met-Leu-Phe methyl ester in a rapid fluorescence-based neutrophil migration assay. Apoptosis was induced in primary amniotic epithelial cells and fibroblasts by actinomycin D (1 microg/mL); the antiapoptotic effects of pre-B-cell colony-enhancing factor on early apoptosis were measured by the annexin V assay, and the late effects were determined by measurement of nuclear matrix protein in the media.Treatment of amnion cells that adhered to immobilon-P membrane to induce the secretion of pre-B-cell colony-enhancing factor showed significantly (P.05) more pre-B-cell colony-enhancing factor protein surrounding the cells compared with the controls. Although the addition of lipopolysaccharide to cultured WISH cells caused the secretion of pre-B-cell colony-enhancing factor into the medium, co-treatment with an antisense oligonucleotide to pre-B-cell colony-enhancing factor obliterated it. Analysis of the cell lysates showed no significant change, which suggests that most of the pre-B-cell colony-enhancing factor protein had been secreted. No significant chemotaxic effects of pre-B-cell colony-enhancing factor were observed; however, pre-B-cell colony-enhancing factor treatment (100 ng/mL), together with actinomycin D, cancelled the early induction of apoptosis, although there was a dose-dependent and significant late antiapoptotic effect on primary amnion epithelial cells (P.001) and fibroblasts (P.01).Pre-B-cell colony-enhancing factor is a secreted protein from amniotic epithelial cells. Although it had no chemotaxic effects, it was antiapoptotic for both amniotic epithelial cells and fibroblasts and may protect these cells against apoptosis that is induced by chronic distension, labor, or infection.

The study was conducted to determine whether relaxin has a proliferative effect on amniotic epithelial cells and to show that this effect is caused by its stimulation of the insulin-like growth factor-II (IGF-II) gene.Immunolocalization and Northern analysis were used to confirm the expression of IGF-II by the fetal cells in the membranes. Human amniotic epithelial (WISH) cells were treated with doses of IGF-II or human relaxin and their proliferative effects measured. The mechanism of the effect of relaxin on cellular proliferation was studied with the use of an IGF-II-blocking antibody and Northern analysis for IGF-II gene expression after treatment with relaxin. An in vivo correlate was sought by quantitation of relaxin gene expression in 10 fetal membranes from women with normally grown and large for gestational age infants.The amniotic epithelial and cytotrophoblast cells of the fetal membranes expressed IGF-II, as did the amniotic epithelial-like (WISH) cell line. Treatment of WISH cells with IGF-II or relaxin caused a significant (P.03) and dose-related increase in WISH cell proliferation over 5 days. The concurrent treatment with a blocking antibody to IGF-II significantly decreased the proliferative response to IGF-II (P.002) and relaxin (P.002). Treatment with relaxin caused a significant increase (P.003) in the transcription of IGF-II in 24 hours. In fetal membranes, the levels of relaxin gene expression correlated with fetal membrane surface area (r = 0.76) and was significantly greater (P.008) in the membranes from macrosomic infants (4020-4729 g) compared with those normally grown (2855-3830 g).IGF-II and relaxin both caused the proliferation of WISH cells. Concurrent treatment with an IGF-II-blocking antibody abrogated the proliferative effects of both hormones. Relaxin increased the transcription of IGF-II, and its expression levels in the fetal membranes correlated with the membrane surface area as well as neonatal birth weight. These data suggest that relaxin is a growth factor for the fetal membranes.

Objective: Our purpose was to determine whether pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes during normal gestation and parturition in the absence of infection and to show its effects on the expression of interleukin (IL)-6 and IL-8. Study Design: PBEF was immunolocalized in the fetal membranes from early pregnancy, at preterm, and at term. Its expression was quantitated by Northern analysis in separated uninfected amnion, chorion, decidua, and placenta of patients at term before labor and in full-thickness membranes before and after spontaneous labor at preterm and at term. Amnion-like epithelial (WISH) cells and fetal membrane explants were treated with recombinant PBEF (rhPBEF), and the expression of IL-6 and IL-8 was quantitated. Results: PBEF was immunolocalized throughout gestation in the amniotic epithelium and mesenchymal cells as well as the chorionic cytotrophoblast and parietal decidua. Northern analysis showed significantly more (P

Preterm birth (PTB) is a global problem with a high incidence in the developing world. Relaxin (RLN) has classically been associated with parturition, but its role(s) in the human have been difficult to determine. For the first time, we bring together the systemic (ovarian) and autocrine/paracrine (intrauterine) sources of RLN, in an attempt to understand how RLN contributes to PTB in women.

Pre-B-cell colony enhancing factor (PBEF) was first isolated from an activated peripheral blood lymphocyte cDNA library and was found to be involved in the maturation of B-cell precursors. It was subsequently identified as one of the genes upregulated by distending the human fetal membranes in vitro. Here we report on the genomic organization of this gene, which is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA. Neither the gene nor the protein has any homology with other cytokines in any currently available database. The use of two promoters (proximal and distal) may result in differential, tissue specific expression of the PBEF transcripts. The 5'-flanking region lacks the classical sequence motif that would place it with the hematopoietic cytokines; however, it has several putative regulatory elements, suggesting that this gene may be chemically and mechanically responsive to inducers of transcription. The three PBEF mRNA transcripts were observed in both normal and infected human fetal membranes but were significantly upregulated (P

This study was designed to show whether the overexpression of relaxin in the decidua of patients with preterm premature rupture of the membranes is independent of or a consequence of chorioamnionitis.Two experiments were conducted. In the first experiment fetal membranes and decidua were collected from patients with preterm premature rupture of the membranes (n = 17) or preterm labor (n = 17) and were divided according to their degree of histologic infection. Messenger ribonucleic acid was isolated from the tissues and quantitative, sequential Northern analyses were carried out for the expression of human relaxin, interleukin-1beta, interleukin-6, and interleukin-8. The second experiment was aimed at increasing the numbers of messenger ribonucleic acid preparations in the two extreme categories, uninfected and severely infected tissues, with preterm premature rupture of the membranes and preterm labor. Some samples of messenger ribonucleic acid from the first experiment were rerun with the Northern analyses in the second experiment. These repeat samples showed no statistical differences in the results run at different times. Therefore the data from the respective groups of patients in both experiments were pooled for statistical analysis.In both the first experiment and in the pooled data of the two experiments the expression of the relaxin genes was significantly greater (P.005) in the tissues from patients with preterm premature rupture of the membranes compared with those with preterm labor, in the absence of infection. No effect of the level of infection on the expression of relaxin was noted. In contrast, interleukin-6 gene expression was significantly increased (P.05) in severely infected tissues, which was independent of whether the delivery was from preterm premature rupture of the membranes or preterm labor. The expression of the interleukin-1beta and interleukin-8 genes were only marginally increased even in severe infection. Marked patient variability in expression of the interleukin genes, especially in severe infection, was noted.A relaxin-mediated pathway that leads to preterm premature rupture of the membranes may exist independent of infection.

The human fetal membranes are genetically identical to the fetus and form a highly specialized interface between mother and fetus, of considerable significance to the successful maintenance and termination of pregnancy in the higher vertebrates. Additionally, the upright posture of women presents these tissues with a greater mechanical challenge than in other species. The major extracellular matrix components providing tensile strength and elastic recoil are reviewed, as well as the key enzyme, activator/inhibitor system responsible for their remodelling and breakdown. However, this fails to convey the important concept that the matrix components are bound to each other and to the cells involved in their formation and organization. These matrix components are collectively responsible for the biomechanical properties of the tissue, but they must also be considered as dynamic elements of a broader signalling system, which include hormonal autocrine/paracrine systems. A unifying hypothesis is presented, which attempts for the first time to bring these two facets of the matrix together, which permits a potential coordination of local events at the maternal-fetal interface leading to parturition. In order to understand fully both the normal biology and the pathobiology of these tissues, such integration of the cellular and extracellular signalling pathways must be achieved.

Human PRL is synthesized and secreted by the maternal decidua, but not by the chorionic cytotrophoblast of the chorion laeve or the placenta. The sites of action for decidual PRL are currently unknown. Accordingly, Northern analysis and in situ hybridization histochemistry have been used respectively to quantitate and localize the expression of the PRL receptor (PRL-R) gene within the uterus during the peripartal period. Immunocytochemistry and Western blot analysis using an anti-PRL-R antibody (U5) localized the translated protein at the cellular level in the same tissues. As judged by the level of expression of the PRL-R gene and its translated products, the chorionic cytotrophoblast has been shown to be a primary site of action. Novel sites were also shown in the decidua, placental trophoblast, and amniotic epithelium. In situ hybridization was not obtained in the latter despite positive Northern analysis and immunostaining. Western analyses with an antibody (U5) to the extracellular domain of the rat PRL-R detected six major molecular species of 95, 85, 63, less than 63, more than 30, and 30 kDa in cytosol from separated amnion, chorion, and decidua. The two bands at 95 and 85 kDa were approximate values only and represent the mature glycosylated forms of the human PRL-R. The other four major bands were partial degradation products from the PRL-R, showing tissue-specific processing and patient to patient variation related to the spectrum of proteases present in these tissues. The 63- and 30-kDa PRL-R-related proteins were detected in both the cytosol and medium from amnion, chorion, and decidua and were also present in amniotic fluid. The 30-kDa species was equal in size to a recently reported PRL-binding protein in human milk. The release of these two PRL-R-related proteins into amniotic fluid suggests possible functions as binding and or/PRL transport proteins in these tissues. The more than 30-kDa species was detected in high amounts in both cytosol and medium from the decidua, but was absent from amniotic fluid. Further work is required to clarify the structural relationships and potential functions of these immunologically PRL-R-related proteins. This study shows that the PRL-R is widely expressed by both fetal and maternal tissues in late pregnancy. Its increased expression during labor and delivery in the chorion, decidua, and placenta supports an autocrine/paracrine role for decidual PRL in the peripartum.

The two human relaxin genes termed H1 and H2 are expressed in the choriodecidua and placenta and have been proposed to act via specific receptors as local modulators of collagenolysis in the fetal membranes. Such receptors have been inferred, but not demonstrated, from studies of the effect of adding exogenous relaxin to these tissues. Thus conditions were optimized for the binding of 32P-labelled human relaxin H2 to membrane-enriched particulate fractions of human fetal membranes, amnion and chorion, with adhering decidua. The membrane protein concentration was optimal at 250 μg, when incubated at 27 °C for 60 min, at pH 7·5 with Mn2+ and Mg2+ ion concentrations of 2·0 mm. Incubation of membrane particulate fractions with increasing amounts of labelled relaxin H2 suggested the presence of a single class of binding sites with an affinity constant (Ka) of 2·15 nm. The binding was primarily to the chorion and decidua with very little to the amnion layer. The competition for binding of the 32P-labelled human relaxin H2 with unlabelled relaxin H2 gave an IC50 of 28 pm, while an IC50 of 60 pm and 280 pm was obtained for relaxin H1 and porcine relaxin respectively. In contrast, unlabelled guinea-pig relaxin inhibited this binding by only 10% even at a 1000-fold greater concentration than H2, and human recombinant insulin failed to inhibit even at a million-fold concentration of unlabelled relaxin H2. Relaxins H2 and H1 can readily displace the binding of either 32P-labelled human relaxins H1 or H2 and gave very similar displacement curves. The binding affinity of relaxin H2, however, was fivefold higher than that of relaxin H1. These data provide evidence for the presence of a relaxin receptor that may preferentially bind relaxin H2. Journal of Endocrinology (1995) 145, 441–448

The expression of the two human relaxin genes termed H1 and H2 in human reproductive tissues ranges from high to very low copy number depending upon the tissue and reproductive state. The aim of this study was to use two approaches to identify total relaxin transcripts (HI and H2) at the cellular level by using a human relaxin H2 riboprobe and a series of six 48-mer synthetic oligoprobes. The results obtained with both methods were similar in all tissues studied; however, a lower background was achieved with the riboprobe. This was especially noticeable after long exposure times, and a better resolution was generally achieved without clustering of the signals. Treatment of the tissues with proteinase-K failed to increase the sensitivity in any tissue with either probe. The relative levels of expression of the total relaxin gene transcripts was estimated from the different exposure times needed to obtain a good hybridization signal. Thus, the order of expression was: corpus luteum of pregnancycorpus luteum of the cycleplacenta and prostatedecidua parietalis. The results agree well with immunolocalization of the peptide hormone previously performed with both heterologous and homologous relaxin antibodies; the exception was the lack of hybridization signal over the cells of the chorionic cytotrophoblast of the chorion laeve. This suggests that the levels of relaxin gene expression was below the level of detectability with the in situ hybridization technique or that these cells sequester, but do not synthesize, relaxin. Expression in the term placenta varied greatly from tissue to tissue and within any one tissue. A similar variability has been noted for relaxin in this tissue by immunocytochemistry. Methodology for the detection of total relaxin transcripts at the cellular level when expressed in a wide range of copy number will allow the developmental regulation of relaxin gene expression in reproductive and nonreproductive tissues to be visualized.

Immunoreactive relaxin is present in human breast cyst fluid and postpartum milk without concurrent detectable serum levels, suggesting that the breast is a site of relaxin synthesis. Monoclonal and polyclonal antibodies to human relaxin H2 have been used to immunolocalize relaxins in normal, benign and neoplastic breast tissues with the avidin—biotin immunostaining technique. In view of the similarities in amino acid sequence between HI and H2 relaxins, these antibodies to H2 relaxin are likely to detect either or both relaxins present in tissue sections. Staining patterns with these antibodies were identical and showed positive diffuse cytoplasmic staining in normal, lobular and ductal epithelium and in myoepithelial cells in breast tissues from normal prepubertal, cyclic, gestational, lactational and postmenopausal females. Relaxin staining was also present in epithelial and myoepithelial cells of ducts and lobules in benign breast disease as well as in metaplastic epithelium of apocrine microcysts. All breast carcinomas (infiltrating ductal, tubular, medullary, intraductal and infiltrating lobular carcinomas) had strong uniform cytoplasmic staining within the neoplastic epithelial cells. All staining was abolished in normal and neoplastic tissues when the polyclonal antibody was preabsorbed with relaxin. It was necessary to distinguish between the possibilities of relaxins being sequestered by breast tissue and local synthesis. Therefore, the expression of the Hl, H2 or both human relaxin genes in normal and neoplastic breast tissues was studied by the isolation of RNA, synthesis of first strand cDNA and amplification by PCR using primer sets which amplified either both H1 and H2, or specifically only H1 or H2 relaxin. The coamplification of both relaxin genes was verified by Southern analysis, diagnostic restriction enzyme digestion and sequencing. The primer set for H1 relaxin detected H1 gene expression in 1 out of 8 normal and 9 out of 12 neoplastic breast RNA samples. The H2 relaxin gene was found to be expressed in 3 out of 8 of the normal samples but in all 12 of the neoplastic samples, suggesting that this gene is expressed at higher copy number in the neoplastic tissues. This is the first demonstration of the cellular immunolocalization of relaxin and relaxin gene expression in normal and neoplastic breast. This should allow further exploration of relaxin's role(s) in normal breast physiology and in its tumorigenesis.

Relaxin (RLN) is a systemic hormone from the corpus luteum, and its levels remain low during normal human gestation. Indeed, elevation of circulating RLN has long been associated with preterm birth, for which there has been no physiological explanation. Recent studies have shown that RLN suppresses endotoxin-induced cytokine secretion from THP-1 monocytic cells by acting on the glucocorticoid receptor (GR), but its effects on primary macrophages are unknown. Therefore, in the present study, we examined the effects of RLN on cytokine secretion from primary decidual macrophages (DMs) obtained at term before labor. Unlike THP-1 cells, RLN had no effects on the cytokine responses induced by either lipopolysaccharide (LPS) or interleukin (IL) 1B, mimicking infection-induced or sterile inflammation, respectively. However, RLN alone for 4 h significantly decreased (P < 0.05) colony-stimulating factor 2 (CSF2; also known as granulocyte-macrophage colony-stimulating factor) and IL8 but for 24 h significantly increased IL6 (P < 0.01). We show that DMs express both the RLN receptor (RXFP1) and the GR. RLN suppression of CSF2 and IL8 was sensitive to the GR-antagonist mifepristone (RU-486). However, RLN activation of RXFP1 induced a dose-dependent cAMP response, which when mimicked by forskolin also caused significantly increased (P < 0.05) secretion of IL6. Thus, RLN may be anti-inflammatory in DMs via activation of the GR but proinflammatory via activation of RXFP1 and cAMP. In summary, we have shown that RLN targeting DMs may modulate proinflammatory cytokine secretion at the maternal-fetal interface and contribute to the localized inflammatory response associated with parturition in women.

Preterm birth continues to be a growing problem in the USA. Although approximately half of preterm births are caused by intrauterine infection, uterine over-distension is also a cause. In this study we have compared the effects of static stretch, cyclic stretch/release and an inflammatory stimulus alone and in combination on the expression of Pre-B cell colony-enhancing factor (PBEF) and IL-8 in primary amniotic epithelial cells (AEC). We then sought to identify some of the mechanism(s) by which these cells respond to stretching stimuli. We show that cyclic stretch/release is a more robust stimulus for both PBEF and IL-8 than static stretch. Cyclic stretch/release increased both intracellular and secreted PBEF and a combination of both types of stretch was a more robust stimulus to PBEF that IL-8. However, when an inflammatory stimulus (IL-1beta) was added to either kind of stretch, the effect on IL-8 was much greater than that on PBEF. Thus, different kinds of stretch affect the expression of these two cytokines from AEC, but inflammation is a much stronger stimulus of IL-8 than PBEF, agreeing with its primary role as a chemokine. Although the AEC showed morphological signs of increased cellular stress during stretching, blocking reactive oxygen species (ROS) had little effect. However, blocking integrin binding to fibronectin significantly reduced the responses of both PBEF and IL-8 to cyclic stretch/release. The increased PBEF, both intracellularly and secreted, suggests that it functions both to increase the metabolism of the cells, at the same time as stimulating further the cytokine cascade leading to parturition.

The decidua of the human maternal-fetal interface is a local source of intrauterine relaxin, and the choriodecidua expresses its receptor (LGR7). Since these tissues consist of a variety of cells, we sought to identify the primary cell(s) responsible for LGR7 expression and relaxin responsiveness.

In the absence of infection, decidual relaxin (RLN) expression is increased in patients with preterm premature rupture of the membranes (PPROM) resulting in preterm birth, but it is not known whether inflammation stimulates RLN expression or vice versa. This study examined the effect of lipopolysaccharide (LPS) on the expression of RLN mRNA and secreted protein and whether RLN treatment influences secretion of proinflammatory cytokines from the fetal membranes. Explants of human fetal membranes in vitro and rhesus monkey fetal membranes in vivo were treated with LPS, which increased expression of IL-6 but had no effect on RLN. RLN treatment stimulated IL-6 and IL-8 secretion from choriodecidual explants in a subset of patients, as well as from isolated chorionic cytotrophoblast cells but not decidual cells. In vivo results obtained in rhesus monkeys after intra-amniotic infusion of RLN demonstrated increased IL-6 and IL-8 concentrations in amniotic fluid. Our results indicate that increased decidual RLN expression is independent of LPS but may induce a local sterile inflammatory process which potentially contributes to extracellular matrix degradation and weakening of the fetal membranes.

We report here the desensitization and internalization of the relaxin receptor (RXFP1) after agonist activation in both primary human decidual cells and HEK293 cells stably transfected with RXFP1. The importance of beta-arrestin 2 in these processes has also been demonstrated. Thus, in HEK-RXFP1 cells the desensitization of RXFP1 was significantly increased when beta-arrestin 2 was overexpressed. After relaxin activation, beta-arrestin 2 was translocated to the cell membrane and RXFP1 underwent rapid internalization. We have previously shown that RXFP1 forms dimers/oligomers during its biosynthesis and trafficking to the plasma membrane, we now show that internalization of RXFP1 occurs through this dimerization/oligomerization. In nonagonist stimulated cells, it is known that the majority of the RXFP1 is located intracellularly and was confirmed in the cells used here. Constitutive internalization of RXFP1 could account for this and indeed, slow but robust constitutive internalization, which was increased after agonist stimulation was demonstrated. A carboxyl-terminal deleted RXFP1 variant had a similar level of constitutive agonist-independent internalization as the wild-type RXFP1 but lost sensitivity to agonist stimulation. This demonstrated the importance of the carboxyl terminus in agonist-stimulated receptor internalization. These data suggest that the autocrine/paracrine actions of relaxin in the decidua are under additional controls at the level of expression of its receptor on the surface of its target cells.

Visfatin, a novel adipokine originally discovered as a pre-B-cell colony enhancing factor, is expressed by amniotic epithelium, cytotrophoblast, and decidua and is over-expressed when fetal membranes are exposed to mechanical stress and/or pro-inflammatory stimuli. Visfatin expression by fetal membranes is dramatically up-regulated after normal spontaneous labor. The aims of this study were to determine if visfatin is detectable in amniotic fluid (AF) and whether its concentration changes with gestational age, spontaneous labor, preterm prelabor rupture of membranes (preterm PROM) and in the presence of microbial invasion of the amniotic cavity (MIAC).In this cross-sectional study, visfatin concentration in AF was determined in patients in the following groups: 1) mid-trimester (n=75); 2) term not in labor (n=27); 3) term in spontaneous labor (n=51); 4) patients with preterm labor with intact membranes (PTL) without MIAC who delivered at term (n=35); 5) patients with PTL without MIAC who delivered preterm (n=52); 6) patients with PTL with MIAC (n=25); 7) women with preterm PROM without MIAC (n=26); and 8) women with preterm PROM with MIAC (n=26). Non-parametric statistics were used for analysis.1) The median AF concentration of visfatin was significantly higher in patients at term than in mid-trimester; 2) Among women with PTL who delivered preterm, the median visfatin concentration was significantly higher in patients with MIAC than those without MIAC; 3) Similarly, patients with PTL and MIAC had a higher median AF visfatin concentration than those with PTL who delivered at term; 4) Among women with preterm PROM, the median AF visfatin concentration was significantly higher in patients with MIAC than those without MIAC.1) Visfatin is a physiologic constituent of AF; 2) The concentration of AF visfatin increases with advancing gestational age; 3) AF visfatin concentration is elevated in patients with MIAC, regardless of the membrane status, suggesting that visfatin participates in the host response against infection.

Immunocytochemistry and Northern analysis were used to show that relaxin is a product of intrauterine tissues of pregnancy. In addition, tissues from a patient without ovaries had similar results on both immunocytochemistry and Northern analysis as tissues from intact patients. The parietal decidua was clearly the major source of relaxin within the uterus and the relaxin mRNA (1.2 kilobases) from this tissue was detected with a 48-mer oligonucleotide probe designed to hybridize with both H1 and H2 relaxin gene transcripts. The mRNA isolated from the placental trophoblast was slightly smaller (1.1 kilobases), and the placental basal plate which has both maternal and fetal cells contained relaxin mRNAs of both sizes. Two monoclonal antibodies (Mabs) raised to synthetic human relaxin (H2) gave different patterns of localization in the fetal membranes, decidua and placenta. One Mab (RLX8) stained the chorionic cytotrophoblast in the fetal membranes and all of the cells in the placental basal plate. The other Mab (RLX6) stained the chorionic cytotrophoblast in some instances and selectively stained the decidua-like cells of the placental syncytiotrophoblast, whereas Mab RLX8 failed to detect this relaxin. Tissues obtained after spontaneous labor and delivery contained significantly less relaxin mRNA than tissues obtained at elective cesarean section without labor, but their hormone contents, as judged by immunocytochemistry, were not different. We conclude that the relaxin gene (H2) is expressed in intrauterine tissues, but that expression and hormone synthesis are not ubiquitous. Whether the relaxin gene H1 is expressed has not been determined.

The relaxin receptor [leucine-rich repeat-containing G protein-coupled receptor 7 (LGR7)] belongs to the leucine-rich repeat containing G protein-coupled receptors subgroup C. Three new LGR7 splice variants have been cloned from the human fetal membranes and shown to be truncated versions of the full-length receptor, encoded by different lengths of the extracellular domain. The expression of their mRNAs has been confirmed by both qualitative and quantitative PCR and shown to be higher in the chorion and decidua before, compared with after, spontaneous labor. When HEK293 cells were transfected with each LGR7 splice variant, their proteins were retained within the endoplasmic reticulum. However, the protein for the shortest variant was also secreted into the medium. We have characterized the intracellular functions and effects of these LGR7 variants on the function of the wild-type (WT)-LGR7. In coexpression studies, each splice variant interacted directly with the WT-LGR7 and exerted a dominant-negative effect on cAMP accumulation by the WT-LGR7 after relaxin treatment. This interaction resulted in the sequestration of the WT-LGR7 inside the cells by down-regulation of its maturation and cell surface delivery. The constitutive homodimerization of WT-LGR7 has been shown here to take place in the endoplasmic reticulum, and the presence of any one of the splice variants decreased this by the formation of heterodimers with the WT-LGR7, supporting the view that homodimerization is a prerequisite for receptor trafficking to the cell surface. These data suggest that the dominant-negative effects of the LGR7 splice variants expressed in the chorion and decidua could be functionally significant in the peripartal period by inhibiting the function of WT-LGR7 and dampening the responsiveness of these tissues to endogenous relaxin.

In view of the important protective role of the fetal membranes, wound sealing, tissue regeneration, or wound healing could be life saving in cases of preterm premature rupture of the membranes. Although many investigators are studying the causes of preterm premature rupture of membranes, the emphasis has not been on the wound healing capacity of the fetal membranes. In this review, the relevant literature on the pathophysiologic condition that leads to preterm premature rupture of membranes will be summarized to emphasize a continuum of events between rupture and repair. We will present the current knowledge on fetal membrane wound healing and discuss the clinical implications of these findings. We will critically discuss recent experimental interventions in women to seal or heal the fetal membranes after preterm premature rupture of membranes.

Relaxin in human pregnancy is both a systemic hormone from the corpus luteum and an autocrine/paracrine hormone at the maternal-fetal interface formed by the decidua/placenta and fetal membranes. We have focused our studies on the autocrine/paracrine roles of relaxin, especially in the preterm premature rupture of the fetal membranes, which causes 30-40% of preterm births. By using different techniques and different tissue collections, our laboratory has shown that expression of the relaxin genes and proteins in the decidua and placenta is increased in patients with preterm premature rupture of the fetal membranes. Relaxin binding and the expression of LGR7 are primarily in the chorion and decidua and are downregulated after spontaneous labor and delivery both at term and preterm. However, expression of LGR7 in the fetal membranes is significantly greater in all clinical situations at preterm than term, suggesting an important role for relaxin in these tissues at that time. The roles of the relaxin system in three potential causes of preterm birth are discussed: in the growth and proliferation of the membranes important for fetal membrane accommodation to fetal and placental growth, in acute infection, and in the inflammatory response leading to the initiation of labor.

Our purpose was to show the effects of pre-B-cell colony-enhancing factor on the genes that are expressed by the human fetal membranes.Explants of fetal membranes (amnion, chorion, and decidua) from three term patients were treated with 100 ng/mL recombinant human pre-B-cell colony-enhancing factor for 4 hours. RNAs were hybridized to gene chips that contained18,000 known genes. One experiment was done in triplicate to assess replication. Data were analyzed to quantitate the signal intensities of each complementary DNA on the array. Confirmation of the results was carried out on tissues from nine other patients by the measurement of the proteins or quantitative real-time reverse transcriptase-polymerase chain reaction.Replication gave92.6% identical results, which showed high method reproducibility. Pre-B-cell colony-enhancing factor treatment caused a significant increase in 103 genes and decrease in 139 genes. Only 8 genes were up-regulated consistently and significantly in all three patients (three key inflammatory cytokines [tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta], four important chemokines [macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, macrophage inflammatory protein-3alpha, and growth-related oncogene-gamma], and prostaglandin-endoperoxide synthase 2). These data were confirmed by the measurement in the media with the use of specific enzyme-linked immunosorbent assays for tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and macrophage inflammatory protein-3alpha and by quantitative real-time reverse transcriptase-polymerase chain reaction for growth-related oncogene-gamma and prostaglandin-endoperoxide synthase 2.Pre-B-cell colony-enhancing factor appears to be at the proximal end of the pathway to labor initiation and may link sterile distention-induced labor with that of infection-induced labor.

The aim of this paper is to describe the design features and practicalities of conducting a cluster randomized trial of obesity management in primary care. The aim of the trial is to assess the effectiveness of an obesity management educational intervention delivered to staff within primary care practices (unit of randomization) in terms of change in body weight of their patients (unit of analysis) at one year. The design features which merit particular attention in this cluster randomized trial include standardization of intervention, sample size considerations, recruitment of patients prior to randomization of practices, method of randomization to balance control and intervention practices with respect to practice and patient level characteristics, and blinding of outcome assessment. The practical problems (and our solutions) associated with implementing these design features, particularly those that result in a time delay between baseline data collection, randomization and intervention, are discussed.

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions. Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.

At the 1999 annual meeting of the Society for the Study of Reproduction there were three speakers in the minisymposium entitled "I've got to get out of here: fetal-maternal interactions involved in parturition". The primary focus was on research progress in understanding the mechanisms involved in human parturition. Although the title of the symposium emphasized the need to "get out", there was considerable emphasis on understanding the problem of "getting out too early" or preterm birth. While preterm birth is unusual in most species, it is of major clinical importance in the human. The data presented by one of the speakers is reviewed here with a focus on preterm labor and preterm premature rupture of the fetal membranes as mechanisms involved in the diverse pathology of preterm birth.

We report rates and causes of death for a cohort of 246 eating-disordered women and provide descriptive information on their eating disorder and comorbid diagnoses.Data on mortality were collected as part of a longitudinal study of anorexia nervosa and bulimia nervosa, now in its 11th year. Other data sources included death certificates, autopsy reports, relative interviews, and a National Death Index search.Seven deaths have occurred during the study, all among anorexic subjects with a history of binging and purging and with comorbid Axis I disorders. The crude mortality rate was 5.1%. The standardized mortality ratios for death (9.6) and suicide (58.1) were significantly elevated (p. 001).Anorexia nervosa is associated with a substantial risk of death and suicide. Features correlated with fatal outcome are longer duration of illness, binging and purging, comorbid substance abuse, and comorbid affective disorders.

This study investigates both the impact of eating disorders (ED) on pregnancy outcome and the impact of pregnancy on cognitive and behavioral symptoms of EDs.Data on pregnancy outcome (live birth [LB], therapeutic abortion [TAB], and spontaneous abortion [SAB]) and ED symptomatology were collected as part of a large, prospective longitudinal study of anorexia nervosa (AN) and bulimia nervosa (BN). Data were gathered using a semistructured interview administered every 6 months to 246 subjects.We identified 54 women who reported 82 pregnancies (46 LB, 25 TAB, and 11 SAB). Pregnancy outcome was not significantly related to any of the clinical variables studied. Women with BN showed a significant decrease in the severity of their ED symptoms during pregnancy, and this decrease was sustained through 9 months postpartum. Women with AN also demonstrated a significant reduction in ED symptoms, however, these symptoms returned to prepregnancy levels by 6 months postpartum.Our prospective findings reveal an elevated TAB rate for ED women along with a general reduction in the severity of ED symptoms during pregnancy.

This study was undertaken to determine which genes were up-regulated by acute distention in an amniotic epithelial cell line and in human fetal membranes.WISH cells, a human amniotic epithelial cell line, were grown on silicone elastomer sheets coated with extracellular matrix and reproducibly distended by 40% in a novel device for 4 hours. Differential gene expression was analyzed by means of suppression subtractive hybridization. Expression of the identified genes was then quantitated by Northern blot analysis in fetal membrane explants after distention in the same device for 4 hours. The effect of distention on apoptosis of the cells and tissue samples was concomitantly studied by means of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method.The genes for interleukin 8 and pre-B-cell colony-enhancing factor were found to be up-regulated in both the WISH cells and the distended fetal membranes. The apoptotic index values in both the cells and the tissue samples were unaffected by distention.Acute distention induces the up-regulation of interleukin 8 and pre-B-cell colony-enhancing factor in both WISH cells and human fetal membranes and does not cause apoptosis.

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.

Objective : To determine whether suppression subtractive hybridization can detect genes in fetal membranes that are upregulated by infection, preterm premature rupture of membranes (PROM), or labor. Methods : Using suppression subtractive hybridization, messenger RNAs from a preterm fetal membrane obtained at cesarean delivery without labor (control) were subtracted from a pool of messenger RNAs of three patients with preterm PROM and vaginal delivery. Eight candidate genes identified as upregulated were quantitated by Northern analysis in each of the tissues and in additional patient subgroups. Results : Eight differentially upregulated genes were identified Inpreterm labor with PROM. Four of the genes are known to be involved in the response to inflammation or infection, and subsequent histologic examination showed one of the preterm PROM tissues to be infected. F-actin capping protein and chitinase precursor, not previously known to be involved in infection, were also upregulated in the infected tissue from preterm PROM. Northern blots using additional subgroups of patients showed that a regulatory G-protein signaling protein gene was significantly upregulated at term by labor in addition to significant upregulation of interleukin-8. There was a strong correlation between the gene expression for complement factor-B and duration of membrane rupture in the patients with preterm PROM. Conclusion : Two novel genes potentially involved in the response to inflammation or infection have been identified. A regulatory G-protein signaling protein and interleukin-8 gene expression were upregulated by labor. Complement factor-B gene expression was directly related to the duration of membrane rupture.

The purpose of the study was to investigate whether the thermal environment in which babies slept before developing haemorrhagic shock encephalopathy syndrome (HSES) diVered from that of other babies. Data were collected by standardised interview from parents of 31 babies who had had HSES before the age of 7 months and compared with equivalent data for 124 control babies, with matching for outside temperature on the relevant night and for age. Multivariate analysis showed a strong association between HSES and covering of the baby’s head by bedding, the odds ratio being 30.7 (95% confidence interval, 2.5 to 384). There were weaker associations with other aspects of the thermal environment. This suggests a link between HSES and some cases of cot death, supports the suggestion that HSES may be caused by overheating, and reinforces advice that babies should be placed to sleep in such a way that they are less likely to become totally covered. (Arch Dis Child 1999;81:155‐158)

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.

Nine healthy volunteers were studied to validate the reproducibility of the Doppler perfusion index--the ratio of hepatic arterial to total liver blood flow--and to evaluate the method of its derivation and the influence of the variable parameters necessary for its calculation. Wide intraobserver variability was observed, and Doppler perfusion index values were consistently outside the previously reported normal range.

Background—Most ulcers are caused, one can deduce, by Helicobcter pylori or by use of non-steroidal anti-inflammatory drugs (NSAIDs). Whether both together are worse than one alone is something that is quite unknown.Aim—To study both factors in order to see whether they interact together positively.Method—A case control study of ulcer bleeding in elderly patients chosen without weeding.Results—NSAID usage increased risk substantially. So did H pylori infection (but relative risk less than three). Neither seemed to interact. Their actions were discretely intact.Conclusion—H pylori effects ulcer bleeding in an adverse manner but does not make the risk of NSAIDs worse.

To calculate the incidence of type 1 diabetes in Scottish children aged less than 15 years between 1984 and 1993; to examine changes in incidence; and to calculate the prevalence of diabetes at the end of this period.Three data sources were used to construct the Scottish Study Group for the Care of Young Diabetics register: active reporting of all new cases; reports from the Scottish Morbidity Register 1; and local registers.All children resident in Scotland diagnosed with primary insulin dependent diabetes mellitus when less than 15 years of age between 1984 and 1993.Annual incidence and prevalence rate for Scotland; time trend in incidence over the 10 years; differences in incidence between the three different age groups; and completeness of the register.The average annual incidence for Scotland was 23.9/100,000 children. The prevalence rate was 1.5/1000 in 1993. A total of 2326 cases was identified from the three sources. Capture-recapture analysis suggests a case ascertainment of 98.6%. The annual incidence rates increased at a rate of 2% each year (rate ratio = 1.02, 95% confidence interval (CI) 1.01 to 1.03). The incidence was higher in boys than girls (rate ratio = 1.08, 95% CI 1.00 to 1.18), and the incidence rates increased with age: 15.3/100,000/year for age 0-4 years, 24.4/ 100,000/year for age 5-9 years, and 31.9/ 100,000/year for age 10-14 years.The incidence of type 1 diabetes in Scotland is increasing and the prevalence is relatively high. These findings have important implications for health service resource allocation. The Scottish Study Group for the Care of Young Diabetics' register provides a base for monitoring and research.

The decidua and placenta synthesize the human relaxins, termed H1 and H2, believed to be involved in collagen remodeling in the amnion and chorion in an autocrine/paracrine manner. The developmental regulation of the relaxin genes was quantitated in normal pregnancy by in situ hybridization histochemistry with six 48-mer oligonucleotide probes that detect both relaxin genes. A significant increase in relaxin expression occurred in both decidua (p0.01) and placenta (p0.05) at 12.5-14.4 wk gestation, with the mean peak value in the placenta more than double that of the decidua, suggesting a coordinate regulation of the relaxin genes. At term after spontaneous labor and delivery, a marginal increase in both decidual and placental relaxin gene expression occurred. Given these normal data, three abnormal preterm situations were investigated: 1) premature uterine contractions without prior rupture of the membranes, 2) premature rupture of the fetal membranes (PPROM), 3) cesarean section for medical reasons with intact membranes and no uterine contractions. Tissues showing intrauterine infection were eliminated. Significantly more relaxin was expressed in the preterm decidua from patients with PPROM when compared to patients in group 1 (p0.02) or group 3 (p0.008). These data were confirmed by Northern analysis with a relaxin cRNA probe. The placental tissues after PPROM also had a significantly higher and a uniform overexpression of relaxin in the placental syncytiotrophoblast. Tissues collected at term, in comparison, showed no such increases in decidua or placenta.

The human fetal membranes provide a sterile biomechanical container which adjust by growth to mid-pregnancy to the increase in fetal size, and by elasticity to the forceful movements of the fetus. The molecular basis for this elasticity is not known, yet reduced elasticity may lead to their premature rupture and preterm birth, a major problem in perinatal medicine. Classically, elastin confers the property of elastic recoil to elastic fibres which are assembled from a family of tropoelastin precursors. These are covalently cross-linked to form insoluble elastin by formation of desmosine and isodesmosine, catalysed by the enzyme lysyl oxidase. The amnion, chorion and decidua were shown by Northern analysis and RT-PCR to contain detectable levels of tropoelastin mRNA and the mRNA encoding lysyl oxidase. The proteins encoded by these mRNAs were also identified by Western blotting and immunolocalization. Further, insoluble elastin was extracted from the human fetal membranes and shown by comparison to elastin preparations from other elastic tissues to have a reasonable desmosine content. Finally, scanning electron microscopy confirmed the presence of multiple layers of an apparently very thin elastic system in this tissue. This biochemical and histopathologic study has demonstrated therefore that the human fetal membranes synthesize and deposit a novel elastic fibre. The presence of such an elastic system in these tissues provides, for the first time, a probable molecular basis for the elastic properties of this tissue.

Interstitial collagen types I and III are the predominant collagens in the amniotic and chorionic connective tissues. However, this matrix also contains proteoglycans, fibronectin, laminin, and elastin, which together with the collagens may undergo partial degradation prior to fetal membrane rupture at term. In this study, stromelysin (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were immunolocalized in fetal membranes obtained at term prior to labor. MMP-3 stained the cells of the amniotic epithelium, fibroblasts and macrophages of the amniotic and chorionic matrix, and those of the chorionic cytotrophoblast; there was no staining in the maternal decidua. TIMP-1 showed a similar staining pattern, except that the staining was darker in some amniotic epithelial cells and was present in the maternal decidua. The maternal decidua produces the two human relaxins H1 and H2; the latter, when incubated with explants of human fetal membranes, caused a dose-dependent and significant increase in expression of the MMP-3 gene and its secreted protein into the media. A significant effect of relaxin H2 on 92-kDa gelatinase (MMP-9) gene expression was also shown--an effect requiring poly(A)+ RNA rather than total RNA. Both relaxin H1 and H2 caused a significant increase in secretion of MMP-9 protein and its enzyme activity in the media. The magnitude of the effects of the two relaxins was similar, in contrast to findings from other biological studies in which relaxin H2 was shown to be more active. Neither of the relaxins had any effect on 72-kDa gelatinase (MMP-2) activity or on the TIMP-1 protein or its activity. This study suggests that local relaxins may be involved in the degradation of the complex fetal membrane extracellular matrix and may cause activation of an enzyme cascade resulting in fully activated MMP-9. Such effects could be important in the degradative pathways occurring in the amnion and chorion in the peripartal period.

Decidual and placental relaxins have been proposed as autocrine/ paracrine hormones in the remodeling of collagen in the amnion and chorion in the last weeks of pregnancy. The matrix metalloproteinase-1 (MMP-1) is a key enzyme in the degradation of the interstitial collagens which predominate in the fetal membranes. Distribution of the MMP-1 gene and of the MMP-1 protein was shown by in situ hybridization and immunolocalization, respectively, in amnion, chorion, and decidua collected from patients before the onset of spontaneous labor. The distribution of MMP-1 in the chorionic cytotrophoblast and decidua coincided with that of the human relaxin receptor, detected by tissue section autoradiography in tissues collected at the same stage of pregnancy. Fetal membrane explants were used to study the effect of exogenous human relaxin H2. These responded by a dose-dependent increase in expression of the MMP-1 gene, in its secreted protein, and in its enzyme activity in the medium. A similar dose-dependent increase in the tissue plasminogen activator (tPA) gene and protein upon exposure of the explants to relaxin H2 suggested a coordinated cascade system, resulting in increases in secreted activities of MMP-1, MMP-3 (stromelysin), and MMP-9 (gelatinase B). There was no effect on the genes or proteins for MMP-2 (gelatinase A) or tissue inhibitor of metalloproteinase-1 (TIMP-1), showing the specificity of the response. This coordinated regulation by relaxin H2 of tPA, MMP-1, MMP-3, and MMP-9 would result in more complete degradation of the fetal membrane extracellular matrix components.