Therapists and patients perceptions of a mixed reality system designed to improve trunk control and upper extremity function.

A prototype system aimed at improving arm function and trunk control after stroke has been developed that combines mixed-reality (MR) feedback with a mobile seat system (Holoreach). The purpose of this study was to assess the usability of Holoreach in a rehabilitation setting from both the patient and therapist perspective. Ten therapists (eight physiotherapists and two occupational therapists) used the device in their regular therapy programs for fifteen stroke patients with trunk control issues. Each patient received four individual therapy sessions with the device performed under the supervision of the therapist. Therapists and patients kept therapy diaries and used customized questionnaires. At the end of the study two focus groups were conducted to further assess usability. Generally, the prototype system is suitable for training trunk and arm control. The therapists expressed overall positive views on the impact of Holoreach. They characterized it as new, motivating, fresh, joyful, interesting, and exciting. All therapists and 80% of the patients agreed with the statement that training with Holoreach is beneficial for rehabilitation. Nonetheless, improvements are required in the hardware and software, and design. The prototype system contributes at various levels to the rapidly evolving advances in neurorehabilitation, particularly regarding the practical aspect of exercise delivery.

GRP78 as a marker of pre-eclampsia: an exploratory study.

Although the exact mechanisms that lead to shallow invasion or defective trophoblastic differentiation in pre-eclampsia are still unknown, it is widely admitted that the etiology of pre-eclampsia is a defect in trophoblast invasion of the uterine spiral arteries. We have previously observed that the status of a chaperone protein, glucose regulated protein 78 (GRP78) is associated with the invasive properties of cytotrophoblastic cells; we therefore hypothesized that circulating GRP78 could serve as a diagnostic tool in pre-eclampsia. In a prospective case-control study, we quantified GRP78 autoantibodies, complexes of GRP78 with autoantibodies and GRP78 (C-term fragment, N-term fragment and full-length GRP78) by ELISA. Plasma from women diagnosed with pre-eclampsia (n = 16), from women during the first trimester of pregnancy who subsequently developed pre-eclampsia (n = 10) and from healthy pregnant women (controls, n = 58 at term, n = 26 at first trimester) were analysed and compared. We observed no significant difference between pre-eclamptic and healthy pregnant women for autoantibodies-GRP78 complexes or total GRP78 at both first trimester and at delivery. In contrast, the ratio of C-terminal GRP78 over full length GRP78 was significantly different in plasma of pre-eclamptic patients as compared with controls both during first trimester (P < 0.004) and at term (P < 0.0001). Our findings suggest that circulating C-terminal GRP78 reflect the invasive properties of cells, and could be used as a predictive marker for pre-eclampsia early in pregnancy.

IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

Regulation of MMP-9 by p53 in first trimester cytotrophoblastic cells.

BACKGROUND: The matrix metalloproteinase (MMP) family is known to play a key role in tissue remodelling during embryonic development and in pathological conditions, such as cardiovascular disease, arthritis and cancer metastasis. It has been shown previously that p53 regulates positively or negatively the expression of different MMPs. Because of p53 overexpression in trophoblastic cells, and its potential role in regulating MMP-2 and MMP-9 expression in different cell lines, we hypothesized that the expression of MMP-9 could also be regulated by p53 in first trimester cytotrophoblasts (CTB). METHODS AND RESULTS: Transfection experiments in CTB demonstrated that wild-type p53 down-regulates the -670 (P < 0.001) but not the -531 and -90 human MMP-9 promoter/CAT reporter plasmid activity, whereas p53 mutants partially lost this repressive activity. However, endogenous p53 is not able to regulate MMP-9 expression in CTB. The presence of high molecular weight complexes of p53 in CTB suggests a potential mechanism of inactivation of p53 transcriptional activity towards MMPs in these cells. CONCLUSIONS: Although p53 is mutated in trophoblast, it is functionally incompetent towards MMPs in these cells.

Trophoblastic p53 is stabilised by a cis-trans isomerisation necessary for the formation of high molecular weight complexes involving the N-terminus of p53.

P53 is a transcription factor also called the "cellular gatekeeper of genome" because it can induce cell cycle arrest in G1, apoptosis or affect DNA replication in response to DNA damage. Wild type p53 is localised in both the cytoplasm and nucleus of first trimester trophoblastic cells (CTB). Immunoblotting of CTB with different p53 antibodies led us to suggest that the N-terminus of p53 could be involved in the formation of high molecular weight complexes (HMWC), leading to the stabilisation of p53 in these cells. Here, we demonstrate that the N-terminus of p53 is involved in the formation of HMWC. Post-translational modifications of p53 seem to be responsible for its stabilisation and inactivation in CTB. We demonstrate that cis-trans isomerisation of proteins by the prolyl isomerase Pin1 is indispensable for the formation of these HMWC and stabilisation of p53. In contrast to observations made in other cells, in CTB, interaction of Pin1 and p53 does not involve phosphorylation of residues ser33, thr81 and ser315 of p53; on the contrary, phosphorylation of p53 leads to the rapid disappearance of some HMWC and destabilises p53. Moreover, decreasing HMWC or inhibiting Pin1 activity increases p53 activity towards its target genes MMP-9 and MMP-2, thus confirming the role of Pin-1 and these HMWC in the regulation of trophoblast invasiveness.

Status of p53 in first-trimester cytotrophoblastic cells.

p53 has been called the cellular gatekeeper of the genome because it can induce cell-cycle arrest in G1, apoptosis or affect DNA replication in response to DNA damage. As p53 has been observed in first-trimester cytotrophoblastic cells (CTB), but its expression in normal cells is generally not detectable because of its short half-life, p53 could play an important role in cellular differentiation and/or in the control of the invasion of trophoblastic cells; therefore, p53 status was investigated in these cells. Using different antibodies recognizing different epitopes of p53 protein, abundant p53 expression was observed both in nuclear and in cytoplasmic compartments of first-trimester CTB. Whereas p53 was detected in the nuclei of few trophoblastic cells with an antibody recognizing the N-terminal epitope of the protein, high expression level of p53 in the cytoplasm of CTB was detected with an antibody recognizing the middle part of p53. The lack of immunoreactivity of p53 with antibodies recognizing the epitopes located at the N-terminus of p53 and the high level of p53 protein observed in the cytoplasm of CTB suggest that the N-terminus of p53 is involved in the formation of complexes. These cytoplasmic complexes were detected under non-reducing conditions in western blot analysis and had apparent molecular weights (MW) of 195, 167 or 125 kDa. These complexes could prolong the half-life of p53 in the cytoplasm of CTBs. By contrast, in the nuclei of CTBs, p53 seems to be present as a tetramer.

Can we improve the diagnosis of rupture of membranes? The value of insulin-like growth factor binding protein-1.

Our objective was to assess the value of insulin-like growth factor binding protein-1 (IGFBP-1) and other tests for the diagnosis of rupture of the membranes (ROM). We included 49 women with suspected ROM. The gold standard for membranes status was defined based on clinical examination, ultrasonography, tests results (except IGFBP-1) and labour information. Sensitivity, specificity, positive predictive value and negative predictive value of each test were as follows, respectively: IGFBP-1 (86, 74, 73 and 87%); bromothymol (64, 100, 100 and 77%); fern test (62, 96, 93 and 75%) and ultrasonography (19, 100, 100 and 61%). The detection of IGFBP-1 in vaginal secretions has high sensitivity for the diagnosis of ROM.

Expression of matrix metalloproteinase-2 and mutant p53 is increased in hydatidiform mole as compared with normal placenta.

Matrix metalloproteinases (MMPs) are group of enzymes thought to play an important role in trophoblastic and tumor invasion. The aim of our study was to investigate the trophoblastic expression of MMPs and p53 in normal trophoblast and hydatidiform moles (HM). Paraffin sections of 45 specimens, including 14 complete hydatidiform moles (CM), 15 partial hydatidiform moles (PM), 8 atypical partial hydatidiform moles (aPM), and 8 controls were selected. Classification of HM was established on histologic criteria and supported by the DNA ploidy results. Tissue sections from each case were immunostained with monoclonal antibodies, cytokeratin-7, MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, and p53 wild type (p53wt) and mutant types (mutp53). Staining for cytokeratin-7 revealed a positive reaction in 93% of the samples. MMP-2 was mainly expressed in the syncytiotrophoblast of HM and found in 62% of aPM, 60% PM, and 93% CM. The mutp53 was mainly and focally expressed in syncytiotrophoblastic cells and was found in 63% of aPM, 80% PM, and 93% CM. Expression of MMP-2 and mutp53 was both significantly greater in HM vs control group (P < 0.05) and greater in CM vs PM and aPM (P < 0.05). No significant difference was observed for cytokeratin-7, MMP-9, TIMP-1, and p53wt between the HM subgroups and between HM and control group. MMP-2 and mutp53 are overexpressed in HM as compared with normal trophoblast and might participate in the invasive behavior of the HM.

Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

[Identification of the intercycle FSH signal in urine samples (uFSH)]. / Identification du signal FSH intercycle par la mesure de la FSH urinaire (FSHu).

OBJECTIVE: The intercycle FSH signal that initiates follicular recruitment and marks the functional onset of the menstrual cycle is of small amplitude and while it commonly occurs on cycle day 3, this often varies. Hence, its identification and measurement in serum (sFSH) requires serial daily samplings. We attempted to determine whether urine measurements of FSH (uFSH) could offer a non-invasive alternative, using a model where the intercycle FSH signal is controlled by timely use of exogenous E2. PATIENTS AND METHODS: Pilot prospective trial in 21 infertile women having received E2, from day 25 of the previous cycle until the 1st Friday after menses. Blood and first void urine samples were collected, starting on the last day of E2 (baseline) for assessing FSH and creatinin. A sonogram was performed for identification of maturing follicles (>12 mm). RESULTS: uFSH and uFSH/Cr showed good correlation with sFSH (R = 0.52 and 0.63, P < 0.0001 and P < 0.0001, respectively). In 15/21 patients who had an intercycle sFSH elevation, this was confirmed by uFSH elevation, both occurring within 2-4 days after stopping E2. In all these women, the sonogram showed evidence of impending ovulation. The amplitude of the uFSH signal was on average 3 times higher than its sFSH counterpart. In 6/21 women, no intercycle FSH elevation was detected and no ovulation occurred. DISCUSSION AND CONCLUSION: Our results show that the intercycle FSH signal can easily be identified and measured in urine. This novel approach permits more precise assessments of ovarian physiology than with blood measurements.

Metalloproteinases and human placental invasiveness.

Matrix metalloproteinases (MMPs) are the main mediators of extracellular matrix degradation. This confers an important role to these enzymes in the invasion of the trophoblast cells where their expression are spatiotemporally regulated. The regulation of MMPs activity is complex and is established at different levels. Their expressions depend on various cis-elements in their gene promoter, and are induced or repressed by various soluble factors. After expression and secretion, the proMMPs must be activated through an activation network. Then, the enzyme activity is regulated by inhibition or stabilization. In this review, we shall focus on the expression, the role in invasion and the regulation of MMPs in the human placenta.

[Update endocrinology: Thyroid sonography]. / Update Endokrinologie: Sonographie der Schilddrüse.

Thyroid ultrasound has developed to be an important diagnostic method. Thyroid and extrathyroid diseases can be differentiated. Ultrasonography shows an exact image of thyroid structures in which typical images for some pathological entities can be observed. Clinical experience with thyroid diseases is essential when using thyroid ultrasonography. Thyroid ultrasonography does not correlate with histopathologic findings very well. Therefore fine needle aspiration biopsy should be used without hesitation. Solitary thyroid nodules larger than 1-1.5 cm in diameter or other potentially malignant structures must be investigated by a fine needle aspiration biopsy. Cytopathologic results of a fine needle aspiration biopsy have a high accuracy, but follicular neoplasms need a histopathologic examination to discriminate between an adenoma and a follicular carcinoma. Colour-flow Doppler ultrasonography gives helpful information about the blood circulation but is not necessary for a reliable diagnostic work up of thyroid diseases. Clinical findings, laboratory results, ultrasonography and fine needle aspiration biopsy can be used alone or in combination, depending on the indication observed, to help find the accurate diagnosis.

Differential expression of the nm23 genes in the developing human trophoblast.

NDP kinases are the non-specific enzymes which catalyse the synthesis of the NTPs through a transfer reaction using ATP as phosphoryl donor. In addition to their enzymatic activity, they display other not yet explained functions related to cell growth, differentiation and apoptosis, embryonic development, tumour progression and metastasis. In this study, the expression patterns of the three highly related NDP kinases A, B and C isoforms were investigated in the developing human trophoblast. Both NDP kinase A and B were found to be primarily present in the villous and extravillous cytotrophoblasts, while NDP kinase C was found almost exclusively in the syncytiotrophoblast layer. This suggests that NDP kinase A and B could be a marker for the mononuclear stage of differentiation of villous trophoblasts, while NDP kinase C could be a marker of the syncytiotrophoblast layer.

Possible interactions between leptin, gonadotrophin-releasing hormone (GnRH-I and II) and human chorionic gonadotrophin (hCG).

Leptin is a metabolic signal to the reproductive axis, where it increases the plasma levels of luteinising hormone (LH) and follicle stimulating hormone (FSH). Since the placental regulation of human chorionic gonadotrophin (hCG) mimics that of the pituitary LH, we undertook this study to see if leptin could be involved in the secretion and synthesis of hCG in first-trimester trophoblast. We incubated cytotrophoblastic cells (CTB) with GnRH-I or GnRH-II, for 4 or 48 h and collected the media at different times thereafter. GnRH-II was more potent than GnRH-I when incubated for 4 h with CTB. Leptin secretion, as measured at 4 h, was significantly stimulated by GnRH-II. When measured at 24 h leptin values were also increased as compared to controls. Neither GnRH-I, nor GnRH-II had any effect on leptin secretion when incubated for 48 h with CTB. Leptin was also added to perifused placental explants, and samples (in which hCG was measured) were collected every 3 min. Leptin significantly stimulated hCG secretion by explants and induced a pulse of hCG immediately (within 6 min) after its injection, increasing significantly the area under the curve (P=0.04) and the amplitude (P=0.02) of hCG pulses. We conclude that GnRH-II is more effective than GnRH-I in stimulating leptin secretion. This difference could be explained by the existence of two different types of placental GnRH receptors or two different pathways of GnRH degradation. Furthermore, we observe that leptin has a significant stimulatory effect on hCG pulsatility.

Modulation of placental vascular endothelial growth factor by leptin and hCG.

Vascular endothelial growth factor (VEGF) has been identified as an endothelium-specific mitogen and inducer of angiogenesis and endothelial cell survival. Leptin and hCG have also been suggested as possible regulators of angiogenesis in various models. In-vivo and in-vitro assays revealed that leptin has an angiogenic activity and that the vascular endothelium is a target for leptin. Thus, we hypothesized that products of cytotrophoblastic cells may play a role in placental angiogenesis and we therefore investigated the effects of leptin and hCG on cytotrophoblast VEGF secretion. We incubated cytotrophoblastic cells (CTB) with recombinant human leptin (rhLept) (0-4 pg/ml) or hCG (0-30000 IU/ml) for 4 h. rhLept significantly stimulated hCG (P = 0.0045) and decreased VEGF release (P = 0.0008) by CTB in a concentration-dependent manner. On the other hand, increasing concentrations of hCG (0-30000 IU/ml), induced a significant inhibition of leptin secretion (P = 0.0028) and a marked dose-dependent stimulation of VEGF(165) secretion (P < 0.0001). We observed an increase of >1000-fold in basal trophoblastic VEGF secretion with physiological concentrations of hCG in vitro. An inhibitory effect of hCG on trophoblastic leptin secretion was also observed, suggesting that hCG might exert a possible negative feedback on trophoblastic release of leptin. We hypothesize that trophoblastic products such as hCG and leptin are probably involved in the control of VEGF secretion at the maternal-fetal interface.

Regulation of trophoblastic gelatinases by proto-oncogenes.

During the first trimester of pregnancy, certain cytotrophoblastic cells (CTB) of anchoring villi invade the underlying decidua. Regulation of this invasive behaviour depends on cytokines and growth factors secreted by decidua and trophoblast, which modulate metalloproteinase (MMP) secretion of CTB. Since MMP-9 expression by CTB is a prerequisite for matrigel invasion and since the promoter region of the MMP-9 gene contains two AP-1 binding sites, we hypothesized, that transient activation of c-jun and c-fos oncogenes (which bind to form AP-1) by tumour necrosis factor (TNFalpha), or the phorbol ester TPA will promote the invasive phenotype of CTB and induce the production of MMP-9.TNFalpha or TPA when added to primary cultures of CTB increase MMP-9 activity and MMP-9 mRNA. This effect is inhibited by cycloheximide indicating the necessity of protein synthesis. TPA or TNFalpha induces also the binding of nuclear proteins (extracted from treated CTB) to a radiolabelled oligonucleotide corresponding to the consensus sequence of the TPA responsive element. Antibodies to Jun and Fos can displace this binding. Transient transfection of antisense mRNA to jun or fos into CTB inhibits the immunoreactivity and gelatinolytic activity of MMP-9. We conclude that AP-1 is necessary but may not be sufficient for transactivation of the MMP-9 gene in human CTB.

Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models.

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.

Endocrine, paracrine and autocrine regulation of trophoblastic metalloproteinases.

Gelatinase A and B (MMP-2 and MMP-9) are secreted by cytotrophoblast (CTB); these enzymes digest the major constituents of the endometrial extracellular matrices (ECM). Direct evidence links the expression of MMPs to the metastatic phenotype of tumour cells and tissue inhibitor of metalloproteinases (TIMP) to the inhibition of metastatisation. Gelatinase B (MMP-9, and no other MMP) has been shown in vitro to mediate CTB invasion. ECM components are known to influence adhesion, spreading, migration and differentiation of cells through specific cell surface receptors called integrins. While CTB migrate from the villous into the decidua they modulate their integrin repertoire, secrete MMP-9 and acquire the capacity to digest their environment. Although CTB behave like metastatic cells, in vivo they are only transiently invasive (first trimester) and their invasion is essentially limited to the endometrium and to the proximal myometrium. This temporal and spatial regulation seems to be mediated in a paracrine way by uterine factors and in an autocrine way by trophoblastic factors. We investigated the effects of endometrial regulators such as leukaemia inhibitory factor (LIF), tumour necrosis factor (TNF), transforming growth factor beta (TGFb), interleukin-1 and 6 (IL-1, IL-6) and insulin-like growth factor binding protein-1 (IGFBP-1) as well as trophoblastic factors such as hCG and leptin. All these factors markedly influenced the secretion and/or activation of MMP-2 and MMP-9. Most cytokines influence cell behaviour by modulating phosphorylation of transcription factors. Among these we identified two oncogene products (Jun and Fos) which were activated by TNF or phorbol esters and which promoted the synthesis of MMP-9. We conclude that decidual and trophoblastic products are autocrine or paracrine regulators of trophoblastic invasion of the endometrium and that some of these products act by activating the transcription of early response genes such as transcription factors.

Effects of leptin, interleukin-1alpha, interleukin-6, and transforming growth factor-beta on markers of trophoblast invasive phenotype: integrins and metalloproteinases.

Phenotypic changes of integrin and metalloproteinase secretion of the invasive human cytotrophoblast are regulated by cytokines and growth factors, but how this occurs is not completely understood. We used 24-h cytotrophoblast cultures from first trimester pregnancies to investigate the effects of leptin and cytokines on the expression of the alpha2, alpha5, and alpha6 integrin subunits and on the activity of metalloproteinase-2 (gelatinase A) and metalloproteinase-9 (gelatinase B). The alpha2 subunit was marginally upregulated by leptin and interleukin-1alpha (IL-1alpha). All compounds tested upregulated, in some degree, the alpha5 expression. The a6 integrin subunit was massively upregulated, by leptin, interleukins, and transforming growth factor-beta. None of the factors tested affected metalloproteinase-2 activity, but the activity of metalloproteinase-9 was upregulated by leptin and IL-1alpha. In conclusion, leptin and IL-1alpha actively induce some of the changes that cytotrophoblasts undergo to achieve a more invasive phenotype. A novel role for leptin is proposed during early pregnancy: leptin might be an autocrine/paracrine regulator of cytotrophoblast invasiveness during implantation and placentation.