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.

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.

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.

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.

[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.

Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells.

Human decidua synthesizes and secretes PRL. We identified the PRL synthesized in endometrial stromal cells and investigated the effect of medroxyprogesterone acetate (MPA), estradiol (E2), porcine relaxin (RLX), and RU486, an antiprogestin, on PRL production by stromal cells from non-pregnant endometrium in primary culture. Stromal cells were isolated from proliferative and secretory endometria and individually cultured in nutrient medium or medium supplemented with different hormone(s). The immunoreactive PRL isolated from culture medium of hormone-stimulated stromal cells was identified and compared to pituitary PRL. Bio-Gel elution pattern and mol wt analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that PRL produced by stromal cells had properties identical to those of pituitary PRL. In addition, PRL mRNA was identified in hormone-stimulated stromal cells using human pituitary PRL cDNA as a hybridization probe. Analysis of mRNA by Northern blotting showed that the size of PRL mRNA isolated from stromal cells was indistinguishable from that of PRL mRNA in human decidua and pituitary tissue. These results indicated that PRL measured in culture medium was synthesized de novo by stromal cells. The PRL content in culture medium was quantitated by RIA. The PRL production rate in stromal cells cultured without hormones ranged from 6-10 ng/day.mg cell protein. After 4-5 days of incubation with RLX or MPA alone, the PRL production rate increased about 2- to 3-fold over the control value. E2 alone had either no effect or slightly decreased the stromal cell PRL production rate. Stromal cells responded to 0.02 microM MPA, and the maximal response was at 0.1-1 microM MPA. A further increase in PRL production was found when stromal cells were treated with a combination of MPA and E2 and MPA, E2 and RLX. In the presence of MPA or MPA and E2, 0.1 ng/ml relaxin increased the PRL production rate. A potent progestin antagonist, RU486, inhibited PRL production in stromal cells treated with MPA, MPA and E2, or MPA and RLX. These results indicate that endometrial PRL production is regulated by the combined effects of steroid hormones (progestin and estrogen) and a peptide hormone (relaxin).

Expression of pro-EPIL peptides encoded by the insulin-like 4 (INSL4) gene in chromosomally abnormal pregnancies.

The recent development of a specific immunoassay based on monoclonal antibodies directed to chain C and chain A of early placenta insulin-like peptide (EPIL) encoded by the INSL4 gene, has made it possible to demonstrate pro-EPIL peptide expression during normal pregnancy. In the present study, we report on the expression of pro-EPIL peptides in chromosomally abnormal pregnancies, namely trisomy 21 and 18. EPIL peptide levels were measured in amniotic fluid (AF) and maternal serum (MS) from pregnancies with trisomy 21 (n=16) or 18 (n=14) and compared to levels detected in AF and MS from 33 chromosomally normal pregnancies between 12 and 32 weeks of gestation. Pro-EPIL peptide levels were significantly higher in amniotic fluids from T21 than in AF from chromosomally normal pregnancies (mean pro-EPIL levels +/- SEM, 449+/-129.2 ng/mL vs 137+/-29.6 ng/mL, P = 0.0195), whereas there was only a trend towards an increase in pro-EPIL peptide levels in maternal serum. In a limited matched gestational age range (15 to 17 weeks), it was confirmed that pro-EPIL peptide levels were significantly higher in AF from T21 pregnancies (644.0+/-155.9 ng/mL, n = 11) than in AF from normal pregnancies (177.8+/-39.0 ng/mL, n = 12; P < 0.0001). Interestingly, the expression patterns of pro-EPIL peptides, human chorionic gonadotropin (hCG) and its free subunits were parallel in T21 pregnancies as recently observed in normal pregnancies. These results are in line with previous observations suggesting that the biosynthesis of both hCG and EPIL follows common regulation pathways.

Human seminal plasma contains a protein that shares physicochemical, immunochemical, and immunosuppressive properties with pregnancy-associated plasma protein-A.

Pregnancy-associated plasma protein-A (PAPP-A) was detected by RIA in human seminal plasma. This was not due to interference with proteases or binding to seminal plasma proteins, since immunoreactivity was not affected by treatment with protease inhibitors, and the elution of [125I]PAPP-A was not altered by preincubation with seminal plasma. The major component of the seminal plasma PAPP-A coeluted with pure PAPP-A or plasma PAPP-A from pregnant and nonpregnant women. In the RIA, serial dilutions of seminal plasma gave parallel displacement curves to pregnancy plasma. Removing PAPP-A-like material from seminal plasma by adsorbtion to heparin-Sepharose reduced the inhibitory effect of seminal plasma on phytohemagglutinin-induced lymphocyte transformation. The source of seminal plasma PAPP-A-like immunoreactive material remains to be elucidated, but it is unlikely to be the testis, since PAPP-A levels in semen of vasectomized men were similar to those in nonvasectomized men.

Pregnancy-associated plasma protein-A: an inhibitor of the complement system.

Pregnancy-associated plasma protein-A (PAPP-A) was purified to homogenicity from third-trimester pregnancy plasma using ion exchange chromatography and affinity chromatography on con A-Sepharose and anti-human serum-Sepharose. The elution of PAPP-A, alpha 2-macroglobulin, Cl-inhibitor and alpha 1-antitrypsin was followed by immuno-diffusion. the eluates were tested for stimulation or inhibition of complement-induced haemolysis. PAPP-A markedly inhibits the haemolytic activity of complement.

Paracrine and autocrine regulators of trophoblast invasion--a review.

Cytotrophoblastic cells (CTBs) from first trimester placenta follow one of two existing differentiation pathways: villous CTBs (vCTBs) form a monolayer of polarized epithelial stem cells which proliferate and eventually differentiate by fusion to form a syncytiotrophoblast (STB) covering the entire surface of the villus, or they can break through the STB at selected sites (in anchoring villi) to form multilayered columns of non-polarized but invasive CTBs. In vitro, CTBs invade a reconstituted basement membrane, they thus behave like metastatic cells. This invasive behaviour is due to the ability of CTBs to secrete matrix metalloproteinases (MMPs) since tissue inhibitor of MMP (TIMP) inhibits their invasiveness. MMPs are a family of at least 17 human zinc-dependent endopeptidases collectively capable of degrading essentially all components of the extracellular matrix (ECM). Although CTBs behave like metastatic cells, in vivo they are only transiently invasive (first trimester) and their invasion is normally limited only to the endometrium and to the proximal third of the myometrium. This temporal and spatial regulation of trophoblast invasion is believed to be mediated in an autocrine way by trophoblastic factors and in a paracrine way by uterine factors. Several types of regulators have been investigated: hormones, cytokines, growth factors and ECM glycoproteins. This review is not intended to be an exhaustive catalogue of all the potential regulators but is aimed at emphasizing those factors relevant in trophoblast-endometrial interactions.

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.

Immunohistochemical localization of pregnancy-associated plasma protein A in decidua and trophoblast: comparison with human chorionic gonadotrophin and fibrin.

In an immunoperoxidase study, pregnancy-associated placental protein A (PAPP-A) was localized to the cytoplasm of decidual stromal cells, the villous syncytiotrophoblast and on the surface of placental trophoblast. A decidual and a trophoblastic origin is suggested for PAPP-A, and its presence on the surface of the syncytiotrophoblast is interpreted as a possible immunoprotective layer.

Effects of decidua-conditioned medium and insulin-like growth factor binding protein-1 on trophoblastic matrix metalloproteinases and their inhibitors.

The regulatory role of in vitro decidualized stromal cells (DESCM) and their main secretory product insulin-like growth factor binding protein-1 (IGFBP-1) was studied on the secretion of trophoblastic gelatinases and tissue inhibitor of metalloproteinase (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DESCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. TIMP-1, hCG, and fetal fibronectin (fFN) were measured by commercially available immunoassays. DESCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9, TIMP-1 and fFN. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1, had no effect on MMP-2 , MMP-9 or fFN but inhibited hCG. It is concluded that a factor secreted by decidual cells inhibits the gelatinolytic property of trophoblast by increasing TIMP-1. Other decidual factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does override the inhibitory effect of TIMP-1. Since in contrast to DESCM, IGFBP-1 increases the total gelatinolytic activity of CTB, it cannot be the primary active decidual factor regulating the proteolytic activity of CTB. The possibility of an integrin-mediated effect of IGFBP-1 on CTB is discussed.

Pregnancy-associated plasma protein A (PAPP-A) specifically inhibits the third component of human complement (C3).

Pregnancy-associated plasma protein A (PAPP-A), a macromolecular glycoprotein associated with pregnancy, was shown to inhibit complement-induced haemolysis and to bind heparin reversibly. Because of the inhibitory effects of heparin on the complement cascade it was not clear if the inhibition of complement activity observed with PAPP-A (isolated from heparin plasma) was attributable to the heparin moiety bound to PAPP-A. This work demonstrates that heparin exerts an inhibitory effect on complement activity but that heparin-free PAPP-A is also inhibitory. PAPP-A specifically inhibits C3 by binding to this complement subcomponent and not by inhibiting C3 convertase as demonstrated for C3 inactivator.

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro.

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-microm pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of alpha5 and alpha6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly (P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG (P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl cAMP (db cAMP) and the cAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell UEG-3) invasion. The collagenolytic activity of trophoblastic cells (EG-3) was increased by hCG stimulation. No changes were shown in the expression of alpha5 and alpha6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.

Expression of stromelysin-3 in the human placenta and placental bed.

Human placentation is mediated by fetal trophoblastic cells which penetrate into the decidualized uterine endometrium. Trophoblast invasion requires the precisely regulated secretion of specific proteinases able to degrade the endometrial basement membranes and extracellular matrix. To document further the involvement of these proteinases during human placentation, we evaluated in vivo the expression of stromelysin-3, a member of the metalloproteinase family, during the first and third trimesters of pregnancy, by means of immunohistochemistry, in situ hybridization and Northern blot analysis. Human extravillous trophoblasts invading the maternal decidua produced stromelysin-3 during both, the first and third trimesters of pregnancy, but to a lesser extent during the latter. In floating villi, stromelysin-3 expression was restricted to the syncytiotrophoblasts that line intervillous vascular spaces. In conclusion, stromelysin-3 is expressed by differentiated, non-proliferative villous and extravillous trophoblastic cells in early and late placental beds and villi, and its pattern of expression evolves during pregnancy. Our observations suggest that stromelysin-3 could play a role in human placentation.

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.

Involvement of trophoblast in embryo implantation: regulation by paracrine factors.

In order to investigate the regulatory role of only one endometrial cell type on trophoblastic invasion, we explored the effects of culture medium conditioned by in vitro decidualised stromal cells (DCM) and of insulin-like growth factor binding protein-1 (IGFBP-1, the main secretory product of decidual cells) on the trophoblastic secretion of gelatinases and tissue inhibitor of metalloproteinases (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. Tissue inhibitor of metalloproteinases (TIMP-1) was measured by a commercially available immunoassay. DCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9 and TIMP-1. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1 and had no effect on MMP-2 and MMP-9. We conclude that a factor secreted by decidual cells (possibly TGFbeta) inhibits the total gelatinolytic activity of trophoblast by increasing TIMP-1 but other factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does not shift the equilibrium between the gelatinases and TIMP-1 in favour of the gelatinases. In contrast to DCM, IGFBP-1 increases the total gelatinolytic activity probably by stimulating another gelatinase (stromelysin-1?) as MMP-2 and MMP 9 are unchanged by IGFBP-1. The possibility of an integrin mediated effect of IGFBP-1 on CTB is discussed.