Synergies of THESEUS with the large facilities of the 2030s and guest observer opportunities.

The proposed THESEUS mission will vastly expand the capabilities to monitor the high-energy sky. It will specifically exploit large samples of gamma-ray bursts to probe the early universe back to the first generation of stars, and to advance multi-messenger astrophysics by detecting and localizing the counterparts of gravitational waves and cosmic neutrino sources. The combination and coordination of these activities with multi-wavelength, multi-messenger facilities expected to be operating in the 2030s will open new avenues of exploration in many areas of astrophysics, cosmology and fundamental physics, thus adding considerable strength to the overall scientific impact of THESEUS and these facilities. We discuss here a number of these powerful synergies and guest observer opportunities.

Decrease of calbindin-d28k, calretinin, and parvalbumin by taurine treatment does not induce a major susceptibility to kainic acid.

Taurine, 2-aminoethanesulfonic acid, is present at high concentrations in many invertebrate and vertebrate systems, and it has several biological functions. In addition, it has been related to a neuroprotective role against several diseases, such as epilepsy. It has been reported that taurine induces a decrease of calbindin-D28k, calretinin, and parvalbumin protein levels in the hippocampus 3 days after administration. In the present work we hypothesized that the decrease of these proteins could alter the action of kainic acid (KA) and make mice more susceptible to excitotoxicity. Therefore, we treated mice with taurine and after 3 days treated them with KA. The results showed that taurine pretreatment did not induce a major susceptibility to KA. Moreover, neurodegeneration was reduced in pretreated mice. However, astrogliosis was similar to that observed in mice treated only with KA. The immunohistochemistries for calbindin-D28k, calretinin, and parvalbumin showed that these proteins were reduced as a consequence of KA treatment and of taurine treatment. However, mice pretreated with taurine prior to KA administration presented the same reduction in these proteins as mice treated with only taurine or only KA.

Serum hyperglycosylated human chorionic gonadotropin to predict the gestational outcome in in vitro fertilization/intracytoplasmic sperm injection pregnancies.

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted by the placenta in early pregnancy. Decreased H-hCG levels have been associated with abortion in spontaneous pregnancy. We retrospectively measured H-hCG and dimeric hCG in the sera of 87 in vitro fertilization patients obtained in the 3 weeks following embryo transfer and set the results in relation to pregnancy outcome. H-hCG and dimeric hCG were correlated (r(2) = 0.89), and were significantly decreased in biochemical pregnancy (2 microg/l and 18 IU/l, respectively) compared to early pregnancy loss (22 microg/l and 331 IU/l) and ongoing pregnancy (32 microg/l and 353 IU/l). Only H-hCG tended to discriminate between these last two groups.

[Haemolysis and turbidity influence on three analysis methods of quantitative determination of total and conjugated bilirubin on ADVIA 1650]. / Interférences de l'hémolyse et de la lipémie sur trois méthodes de dosage de la bilirubine totale et conjuguée sur ADVIA 1650.

Plasma bilirubin testing is crucial to prevent the occurrence of neonatal kernicterus. Haemolysis may occur during sampling and interfere with bilirubin determination. Moreover, lipidic infusions may induce plasma lipemia and also interfere with bilirubin measurement. We evaluated the interference of haemolysis and lipemia with three methods of total and direct bilirubin measurement adaptated on an Advia 1650 analyser (Siemens Medical Solutions Diagnostics) : Synermed (Sofibel), Bilirubin 2 (Siemens) and Bilirubin Auto FS (Diasys). The measurement of total bilirubin was little affected by haemolysis with all three methods. The Bilirubin 2 (Siemens) method was the less sensitive to haemolysis even at low bilirubin levels. The measurement of conjugated bilirubin was significantly altered by low heamoglobin concentrations for Bilirubin Auto FS(R) (30 microM or 0,192 g/100 mL haemoglobin) and for Synermed (60 microM or 0,484 g/100 mL haemoglobin). In marked contrast, we found no haemoglobin interference with the Direct Bilirubin 2 reagent which complied with the method validation criteria from the French Society for Biological Chemistry. The lipemia up to 2 g/L of Ivelip did not affect neither the measurement of total bilirubin for all three methods nor the measurement of conjugated bilirubin with the Diasys and Siemens reagents. However, we observed a strong interference starting at 0,5 g/L of Ivelip with the Synermed reagent. Our data suggest that both Siemens and Diasys methods allow to measure accurately total and conjugated bilirubin in hemolytic and lipemic samples, nevertheless, the Siemens methodology is less affected by these interferences.

[Biologic tests for the diagnosis of amniotic fluid embolism]. / Dépistage de l'embolie amniotique: vers un test diagnostique ?

Amniotic fluid embolism is a rare, unpredictable and often lethal complication of pregnancy and childbirth. Because of its variable presentation, an early biologic test would help to establish the diagnosis. We investigated in maternal serum 4 components of amniotic fluid, i.e., alpha-fetoprotein (AFP), l'insuline like growth factor binding protein-1 (IGFBP-1), fetal fibronectin (fFN) and placental alpha1-microglobulin (PAMG-1). On the 6 cesareans controls involved, none of the makers increased after membranes section. PAMG-1 is unsuitable because its detection is always positive or doubtful even in the baseline. On the 7 cases suspected of amniotic fluid embolism, no detectable increase in any of those markers was noted in 3 cases, which is not in favour of this diagnosis. In the remaining cases, IGFBP-1 and fFN became detectable, confirming histological evidences of amniotic fluid embolism in 2 cases. The follow up of those markers in maternal blood confirmed the suspicion of amniotic fluid embolism at 21 wg in one case of ongoing pregnancy. These preliminary results point out the potential interest to assay maternal serum AFP, IGFBP-1 and fFN to confirm amniotic fluid embolism using rapid laboratory tests.

Alpha-1-acid glycoprotein.

Alpha-1-acid glycoprotein (AGP) or orosomucoid (ORM) is a 41-43-kDa glycoprotein with a pI of 2.8-3.8. The peptide moiety is a single chain of 183 amino acids (human) or 187 amino acids (rat) with two and one disulfide bridges in humans and rats,respectively. The carbohydrate content represents 45% of the molecular weight attached in the form of five to six highly sialylated complex-type-N-linked glycans. AGP is one of the major acute phase proteins in humans, rats, mice and other species. As most acute phase proteins, its serum concentration increases in response to systemic tissue injury, inflammation or infection, and these changes in serum protein concentrations have been correlated with increases in hepatic synthesis. Expression of the AGP gene is controlled by a combination of the major regulatory mediators, i.e. glucocorticoids and a cytokine network involving mainly interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF alpha), interleukin-6 and IL-6 related cytokines. It is now well established that the acute phase response may take place in extra-hepatic cell types, and may be regulated by inflammatory mediators as observed in hepatocytes. The biological function of AGP remains unknown; however,a number of activities of possible physiological significance, such as various immunomodulating effects, have been described. AGP also has the ability to bind and to carry numerous basic and neutral lipophilic drugs from endogenous (steroid hormones) and exogenous origin; one to seven binding sites have been described. AGP can also bind acidic drugs such as phenobarbital. The immunomodulatory as well as the binding activities of AGP have been shown to be mostly dependent on carbohydrate composition. Finally, the use of AGP transgenic animals enabled to address in vivo, functionality of responsive elements and tissue specificity, as well as the effects of drugs that bind to AGP and will be an useful tool to determine the physiological role of AGP.

Amyloid and tau pathology of familial Alzheimer's disease APP/PS1 mouse model in a senescence phenotype background (SAMP8).

The amyloid precursor protein/presenilin 1 (APP/PS1) mouse model of Alzheimer's disease (AD) has provided robust neuropathological hallmarks of familial AD-like pattern at early ages, whereas senescence-accelerated mouse prone 8 (SAMP8) has a remarkable early senescence phenotype with pathological similarities to AD. The aim of this study was the investigation and characterization of cognitive and neuropathological AD markers in a novel mouse model that combines the characteristics of the APP/PS1 transgenic mouse model with a senescence-accelerated background of SAMP8 mice. Initially, significant differences were found regarding amyloid plaque formation and cognitive abnormalities. Bearing these facts in mind, we determined a general characterization of the main AD brain molecular markers, such as alterations in amyloid pathway, neuroinflammation, and hyperphosphorylation of tau in these mice along their lifetimes. Results from this analysis revealed that APP/PS1 in SAMP8 background mice showed alterations in the pathways studied in comparison with SAMP8 and APP/PS1, demonstrating that a senescence-accelerated background exacerbated the amyloid pathology and maintained the cognitive dysfunction present in APP/PS1 mice. Changes in tau pathology, including the activity of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 ß (GSK3ß), differs, but not in a parallel manner, with amyloid disturbances.

Glucose inhibits human placental GH secretion, in vitro.

Human placenta specifically expresses the GH-V gene leading to the production of placental Growth Hormone (PGH). During pregnancy, PGH levels increase progressively in maternal blood, but its regulation remains unknown. In this study the effect of glucose on PGH secretion by human term placenta was tested, in vitro, by means of two different experimental models: organ culture of villous tissue and primary culture of isolated cytotrophoblasts. PGH was assayed in the culture medium by an immunoradiometric assay using a specific PGH monoclonal antibody. The presence of glucose (25 mmol/L) in the culture medium significantly inhibited (p < 0.001) the secretion of PGH by either placental villous explants or by cultured trophoblast cells. This inhibitory effect of glucose on PGH secretion was dose-dependent. More than 50% inhibition being observed with 5.5 mmol/L. In the same conditions, the daily production of hPL and hCG, were unmodified. Furthermore, the glucose-induced inhibition of PGH secretion was more effective when cultured trophoblast cells are differentiated into syncytiotrophoblast. This study demonstrates, for the first time, that among the gestational polypeptide hormones secreted by the human placenta, only PGH secretion is modulated by glucose, suggesting a key metabolic role for this hormone during pregnancy.

Vitamin A and retinoic acid stimulate within minutes cAMP release and growth hormone secretion in human pituitary cells.

In order to gain a better understanding on the possible role of vitamin A (VA) and retinoic acid (RA) on human growth hormone (GH) secretion, we used the physiological model of pituitary cells perifusion. In perifused cells from pituitary somatotropic adenomas, RA induced within minutes a peak of GH secretion. This effect was dose dependent, maximal effect being observed with 100 nM. The GH release was associated with a discharge of cAMP delayed by 4 to 8 minutes relative to the GH surge. Similar results were obtained after VA stimulation. Our observations provide the first evidence of an action of VA and RA on cAMP production. They suggest a role of RA and VA in the regulation of human GH secretion via the cAMP dependent pathway.

Trophoblast production of a weakly bioactive human chorionic gonadotropin in trisomy 21-affected pregnancy.

Total human chorionic gonadotropin (hCG) is high in maternal serum at 14-18 wk of trisomy 21 (T21)-affected pregnancy, despite low placental hCG synthesis. We sought an explanation for this paradox. We first observed that, in T21-affected pregnancies, maternal serum hCG levels peaked at around 10 wk and then followed the same pattern throughout pregnancy as in controls, albeit at a higher (2.2-fold) level. After delivery, hCG clearance was not significantly different from that in controls. We isolated cytotrophoblasts from 29 T21-affected placentas (12-25 wk) and 13 gestational age-matched control placentas and cultured them for 3 d. In this large series, we confirmed that, in the culture medium of trophoblasts isolated from T21 placentas, hCG secretion was significantly lower (P < 0.003) than in controls, in contrast to the high hCG in maternal serum of the same patients. In T21 cultured trophoblasts, transcripts of sialyltransferase-1 and fucosyltransferase-1 were abnormally high. In corresponding culture medium, hCG was abnormally glycosylated; highly acidic [isoelectric points (pHi) = 4.5] as shown by isoelectric focusing, immunoblotting, and lectin binding; and weakly bioactive (46% of control) as determined using the Leydig cell model. In conclusion, T21 trophoblast cells produced hCG that was weakly bioactive and abnormally glycosylated but whose maternal clearance was unaltered.

Defect of villous cytotrophoblast differentiation into syncytiotrophoblast in Down's syndrome.

The syncytiotrophoblast (ST) is one of the major components of the human placenta, as it is involved in feto-maternal exchanges and the secretion of pregnancy-specific hormones. The aim of this study was to elucidate the formation and function of the ST in trisomy 21 (Down's syndrome). We first used the in vitro model of cytotrophoblast differentiation into ST. Cytotrophoblasts were isolated from 15 trisomy 21-affected placentas (12-35 weeks gestation) and 10 gestational age-matched control placentas. In vitro cytotrophoblasts isolated from normal placenta fused to form the ST. This was associated with an increase in transcript levels and in the secretion of hCG, human placental lactogen, placental GH, and leptin. In trisomy 21-affected placentas, we observed a defect (or a delay) in ST formation and a dramatic decrease in the synthesis and secretion of these hormones compared to those in cultured cells isolated from control age-matched placentas. These results were confirmed by a significant (P < 0.001) decrease in gene expression in total homogenates of trisomy 21-affected placentas compared to controls. These results will be of help in understanding the maternal hormonal markers of fetal trisomy 21 and the consequences of placental defects for fetal development.

N219Y, a new frequent mutation among mut(degree) forms of methylmalonic acidemia in Caucasian patients.

Mutations in the MUT locus encoding for the methylmalonyl-CoA mutase (MCM) apoenzyme are responsible for the mut forms of methylmalonic acidemia (MMA). To date, 49 different mutations have been identified in mut MMA. Only two frequent mutations have been reported in the Japanese population and in African-Americans. Here we report a new missense mutation N219Y (731 A-->T) which we found in five unrelated families of French and Turkish descent. All the patients exhibited a severe mut(degree) phenotype and three of them were homozygotes for N219Y. Direct involvement of the mutation in the loss of enzyme activity was demonstrated by mutagenesis and transient expression study. Mapping of the mutation onto a three-dimensional model of human MCM constructed by homology with the Propionibacterium shermanii enzyme shows that it lies in a highly conserved secondary structure motif and might suggest impaired folding and/or poor stability compatible with the mut(degree) phenotype. Finally, a 1% N219Y carrier frequency was observed in a French anonymous control population. Thus, N219Y is the first frequent mut mutation to be reported in the Caucasian population.

Interleukin 1 beta modulates hepatic synthesis of alpha 1-acid glycoprotein in the fetal rat.

The ability of the fetal rat to respond to interleukin 1 beta (IL1 beta) by expressing alpha 1-acid glycoprotein (AGP) was investigated. Eight and 20 h after injection of 7 ng IL1 beta into 19-day fetuses, liver AGP mRNA increased by a factor of 66 and 82 respectively, while serum AGP levels increased by a factor of 3 and 5. Similar treatment of the mothers altered in the fetuses neither AGP serum levels nor the amount of liver AGP mRNA. The induction of AGP gene expression in the fetal liver in response to IL1 beta was similar to that observed in the adult liver. These results demonstrate that at day 19 the fetal rat liver has acquired a mature acute-phase system.

An enzyme-linked immunoassay for the measurement of rat alpha 1-acid glycoprotein synthesized by cultured hepatocytes.

We have developed a sandwich ELISA to quantify rat alpha 1-acid glycoprotein (AGP). The assay correlated well with RID and the minimum detectable concentration was 1 microgram/l. The assay permits high sensitivity determinations of the rate of synthesis of AGP in vitro. The maximum mean rates observed were 1500 and 1800 ng/24 h/10(6) cells for hepatocytes cultured alone and co-cultured hepatocytes respectively and 39 ng/h/10(6) cells for isolated hepatocytes.

beta-Adrenoceptor blocking drugs and isoprenaline: central effects on cardiovascular parameters.

1. The central hypotensive activity of (+)- and (-)-propranolol (100 microgram), pindolol (100 microgram) and isoprenaline (1 and 4 microgram) injected intracerebroventricularly (i.c.v.) was studied in rats anaesthetized with urethane and chloralose. Blood pressure, cardiac output and heart rate were measured; systolic stroke volume and peripheral vascular resistance were calculated. 2. (+)- and (-)-Propranolol and pindolol induced a fall of blood pressure but (+)-propranolol was less active. The heart rate was reduced more by (-)-propranolol than by (+)-propranolol or (-)-pindolol. The decrease of systolic stroke volume was greater for (-)-propranolol and pindolol than for (+)-propranolol. Peripheral vascular resistance was reduced to the same level but with different time courses, (-)-propranolol having a longer effect than (+)-propranolol and pindolol. 3. Isoprenaline induced a hypotensive effect, while cardiac output and heart rate increased; the systolic stroke volume remained stable but peripheral vascular resistance was significantly decreased. 4. These results suggest that different central regulatory centres are involved in the control of cardiac function and peripheral vascular tone.

Negative correlation between plasma GHRH values and growth velocity in short prepubertal children.

Numerous data suggest that impaired growth hormone secretion in short children is usually related to abnormal regulation of the hormone at the hypothalamic level. In order to improve our understanding of neurohypothalamic dysfunction in short children, we measured basal and peak (after L-dopa stimulation) plasma growth hormone-releasing hormone levels in 43 prepubertal children. Among them, in 23 children suspected of having hypothalamic growth hormone dysregulation, growth hormone-releasing hormone values were significantly higher than those observed in normal short stature children (n = 20), no longer correlated with peak growth hormone following L-dopa, and negatively correlated with growth velocity. This suggests that a predominant inhibitor of growth hormone secretion, such as an increase in somatostatin tone, might be prevalent in a large number of children with partial growth hormone deficiency and suspected hypothalamic growth hormone dysregulation.

Induction of rat alpha-1-acid glycoprotein by phenobarbital is independent of a general acute-phase response.

Phenobarbital (PB) induces transcription of the alpha 1-acid glycoprotein (AGP) gene, one of the major positive acute-phase proteins, the expression of which is controlled by a specific combination of glucocorticoids and cytokines. This raises questions as to the involvement of glucocorticoids and cytokine pathways in the PB-mediated effect on AGP gene expression. We found that the pattern of whole-serum proteins in PB-treated rats differed markedly from that observed during a typical acute inflammatory response (in turpentine-treated rats): levels of some positive acute-phase proteins (APP) increased slightly (alpha 1-acid glycoprotein, haptoglobin, hemopexin and T-kininogen), while levels of alpha 2 macroglobulin, the most sensitive marker of the acute-phase reaction, decreased. Among the negative APP, neither albumin nor prealbumin decreased while CBG increased. The cytokines involved in AGP gene regulation (mainly IL1, IL6 and TNF alpha) do not therefore seem to mediate the effect of PB on acute-phase protein expression. Glucocorticoid involvement is also ruled out by the observed enhancement of the effect of PB on AGP expression in adrenalectomized animals. Our results suggest that phenobarbital acts on AGP expression by a mechanism independent of the inflammatory pathway.

Expression of pregnancy-associated plasma protein-A (PAPP-A) during human villous trophoblast differentiation in vitro.

Pregnancy-associated placental protein-A (PAPP-A), first isolated from maternal serum, has been identified as a metalloprotease cleaving insulin-like growth factor binding protein-4 (IGFBP-4). The source of PAPP-A during pregnancy is unclear. We therefore investigated PAPP-A expression during in vitro human villous cytotrophoblast cell (CT) differentiation into syncytiotrophoblast (ST). CT were isolated from normal first trimester, second trimester and term placentae (n=10) and cultured to form ST. PAPP-A mRNA was quantified by real-time PCR, and PAPP-A protein expression was studied by immunocytochemistry and TRACE technology with specific monoclonal antibodies. PAPP-A mRNA expression in total placental extracts increased during the course of pregnancy. PAPP-A protein was detected in the cytoplasm of both CT and ST. ST formation in vitro was associated with a 19-fold increase in PAPP-A mRNA expression and an 8-fold increase in PAPP-A secretion into the culture medium. No significant difference in PAPP-A production was observed between cultured cells isolated from early and term placentae. In conclusion, PAPP-A production in vitro, is associated to the differentiation of villous cytotrophoblast cells into syncytiotrophoblast, independently of the age of gestation.

Nuclear retinoic acid receptor characterization in cultured human trophoblast cells: effect of retinoic acid on epidermal growth factor receptor expression.

Vitamin A is an important factor during gestation and its metabolite, retinoic acid (RA), is a potent teratogen. However, RA action on the placenta is still poorly understood. In this study we analysed the presence of RARs and RXRs in human trophoblastic cells. We determined that RAR alpha was the more expressed form in term placenta, and that RAR beta was induced by RA treatment. Then we analysed RA effects on endocrine activities and on epidermal growth factor (EGF) receptor expression. We found that RA decreased 125I-labeled EGF binding and EGF-dependent phosphorylation. Furthermore, RA treatment led to a concentration-dependent decrease in the amount of EGFR protein expression. This treatment also decreased EGF receptor mRNA levels, suggesting transcriptional regulation of the EGF receptor. Thus we demonstrated that RA could interact with feto-placental development by modulating trophoblast EGF receptors expression, probably via its nuclear receptors.

Effects of calcitonin on human trophoblastic cells in culture: absence of autocrine control.

The purpose of this work was to investigate the effects of calcitonin (CT) on trophoblastic cells with respect to cAMP levels and human chorionic gonadotrophin (hCG) secretion in cultured cells from first-trimester and term placentas and in a choriocarcinoma cell line (JEG-3). The expression of the CT gene was investigated to elucidate a putative autocrine control of CT during pregnancy. The addition of salmon CT (10(-10) M and above) resulted in concentration-dependent increases in cAMP secretion by normal trophoblastic cells from term and first-trimester placentas. Moreover, CT was found to increase cAMP secretion preferentially in completely differentiated cells, i.e. after 4-7 days in culture. Addition to the culture medium of JEG-3 cells slightly increased cAMP secretion only at a concentration of 10(-8) M. The basal level of hCG in the medium was found to be higher in the first-trimester than in the term trophoblast culture, but salmon CT induced an increase in hCG secretion by term placenta cells only. CT gene expression in our experimental model was investigated to elucidate a putative autocrine control of CT action during pregnancy. It was not found to be expressed in syncytiotrophoblast cells from either first-trimester or term placenta cells by the method used. Our data demonstrate the absence of autocrine control of CT effects in trophoblastic cells, and suggest that CT is likely to exert its effect preferentially on differentiated cells.