Placental production of progestins is fully effective in villous cytotrophoblasts and increases with the syncytiotrophoblast formation.

Placental syncytiotrophoblast (ST) is considered as the main placental endocrine tissue secreting progesterone, a steroid essential for maintenance of pregnancy. However, each step of progestins production has been poorly investigated in villous cytotrophoblast (VCT) regarding ST formation. We aimed to characterize progestins production during human differentiation of VCT into ST. VCTs were isolated from term placenta and cultivated, with or without forskolin (FSK), to stimulate trophoblast differentiation. Secreted progestins concentrations were determined by immuno-assay and Gas Chromatography-tandem mass spectrometry. Intracellular expression of cholesterol transporter and enzymes involved in steroidogenesis were studied by immunofluorescence, western-blot, and RT-qPCR. Progesterone and pregnenolone are produced by VCT and their secretion increases with VCT differentiation while 17-hydroxyprogesterone concentration remains undetectable. HSD3B1 enzyme expression increases whereas MLN64, the cholesterol placental mitochondrial transporter and P450SCC expressions do not. FSK induces progestins production. Progestins placental synthesis is effective since VCT and increases with ST formation thanks to mitochondria.

Expression of HERV-W Env glycoprotein (syncytin) in the extravillous trophoblast of first trimester human placenta.

Although the extravillous trophoblastic invasion has a critical role in human placental development, nothing is known about HERV-W expression in the extravillous phenotype. The aim of the present study was to localize in first trimester placenta the expression of HERV-W Env glycoprotein and its receptor all along the differentiation pathway of the extravillous phenotype. In addition using an in vitro model of extravillous cytotrophoblastic cell isolation and invasion we investigated the presence of HERV-W transcripts and envelope glycoprotein in cultured extravillous trophoblastic cells. Using monoclonal and polyclonal antibodies, the glycoprotein was immunolocalized in all the cell types of the extravillous phenotype lineage: cytotrophoblastic cells of the column, interstitial extravillous trophoblastic cells, multinucleated giant cells and endovascular trophoblast. Furthermore, using a polyclonal antibody, the D mammalian virus receptor was also localized in the various extravillous trophoblastic phenotypes. In addition, the presence of HERV-W transcripts and protein was demonstrated in cultured extravillous trophoblastic cells. HERV-W Env glycoprotein expressed in villous and extravillous trophoblast can be considered as a specific marker of the human trophoblast.

Large variability of trophoblast gene expression within and between human normal term placentae.

Human placenta extracts are widely used in clinical and fundamental research, particularly to study the hormonal and exchange functions of the placenta. However, very little is known about the distribution of the main hormone mRNAs in the placenta as a whole. Total placenta extracts are heterogeneous in their cellular components, as they contain material of both fetal and maternal origin, and in their structure. Results vary greatly depending upon the location of the biopsy and the number of biopsies performed. We used real-time quantitative RT-PCR to determine whether transcripts corresponding to the main hormones secreted by the human placenta (e.g. hCG, HPL and PGH) are equally distributed within and between term placentae. We also measured cytokeratin 7 transcripts, which are specifically expressed in the trophoblast, and transcripts corresponding to nuclear receptors PPARgamma and RXRalpha. A comparison of the results obtained with 12 different samples from each of four normal term placentae revealed that the amounts of transcripts differ considerably within and between each placenta. This emphasizes the need to study large numbers of samples when looking for significant differences in gene expression.

Impact of trisomy 21 on human trophoblast behaviour and hormonal function.

Although trisomy 21 (T21) is the most frequent genetic abnormality and some maternal serum markers for this fetoplacental aneuploidy are of placental origin, little is known of its impact on placental development. We therefore studied the influence of T21 on trophoblast behaviour. Using cultured cells from 46 human T21 pregnancies, we confirmed the defective morphological and functional differentiation of the villous cytotrophoblast in this setting; indeed, villous cytotrophoblast cells aggregate normally but fuse inefficiently to form the syncytiotrophoblast. This is in part related to the abnormal oxidative status of the T21 cytotrophoblast, characterized by a gene dosage-related increase in SOD-1 (copper-zinc superoxide dismutase) expression and activity. This was associated with a significant (P < 0.01) increase in catalase activity but no significant change in glutathione peroxidase activity. On the basis of these in vitro findings and studies of large panels of maternal serum, we propose a pathophysiological explanation for trisomy 21 maternal serum markers of placental origin.

Increased oxidative damage to fibroblasts in skin with and without lesions in psoriasis.

Differences in oxidative damage, as measured by an increase in the carbonylation of macromolecules, were determined in situ with skin biopsies from psoriatic patients and controls. High levels of carbonyl residues were consistently detected in the dermis and never in the epidermis of sections of these skin biopsy samples. The dermis of psoriatic skin without lesions had a higher level of carbonylation than the dermis of normal skin. In this study, we found that there was more oxidative damage in cultured fibroblasts prepared from skin with and without lesions from psoriasis patients than in normal fibroblasts from the skin of age-matched controls. The extent of protein carbonylation in cell extracts was determined by immunoblotting, using an antidinitrophenylhydrazone antibody, and in intact cells was determined by immunocytochemical analysis with the same antibody. The higher level of carbonylation detected was used here as a measure of oxidative stress, and showed that some oxidative damage occurred before the appearance of typical psoriatic plaques. These results suggest that fibroblasts are affected before the onset of psoriasis and that this damage is independent of any inflammatory infiltrate.

In vitro effects of oxygen-derived free radicals on type I and type II cAMP-dependent protein kinases.

Oxygen free radicals may act as second messengers in signal transduction pathways and contribute to inflammatory diseases. We studied the action in vitro of radiolytically generated hydroxyl radicals (.OH) and superoxide radicals (O-2) on the cAMP-dependent protein kinases, I and II (PKAI and -II, respectively). The effects of the gasses O2 and N2O used to produce O-2 or .OH radicals by gamma-radiolysis of the water were also studied. PKAI is more sensitive than PKAII to oxygen gas (10 mM sodium formate) and to hydroxyl and superoxide radicals. Hydroxyl radicals decreased the kinase phosphotransferase activities stimulated either by cAMP or its site-specific analogs for both PKAI and PKAII; however, PKAI was more affected. The binding of [3H]cAMP and of 8-N3-[32P]cAMP to RI regulatory subunits was decreased. .OH caused a loss of tryptophan 260 fluorescence at site A of PKAI and of bityrosine production. Superoxide radicals affected only PKAI. O-2 modified both cAMP-binding sites A and B of the regulatory subunit but had a smaller effect on the catalytic subunit. The catalytic subunit was more sensitive to radicals when free than when part of the holoenzymes during exposure to the oxygen free radicals. These results suggest that oxygen free radicals alter the structure of PKA enzymes. Thus, oxidative modifications may alter key enzymes, including cAMP-dependent protein kinases, in certain pathological states.

Retinoic acid abolishes the calcitonin gene-related peptide autocrine system in F9 teratocarcinoma cells.

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation.

Oxidative modulation of cyclic AMP-dependent protein kinase in human fibroblasts: possible role in psoriasis.

Previous studies have established that cyclic AMP-dependent protein kinase (PKA) activity, as well as 8-azido-[32P]-cAMP binding to the RI and RII regulatory subunits, are decreased in cells from psoriatic patients compared to cells from normal patients. Here we show that the exposure of normal human dermal fibroblasts in culture to hydrogen peroxide and to oxygen free-radical generating systems decreased PKA activity, as well as cyclic AMP binding to the RI and RII regulatory subunits, to levels similar to those observed with psoriatic fibroblasts. Likewise, treatment of normal cytosolic preparations of PKA, as well as purified bovine PKA II, in vitro with free radical generating systems also resulted in decreased PKA activity and 8-azido [32P]-cAMP binding to the RI and RII regulatory subunits. Further, treatment of psoriatic fibroblasts with free radical scavenging agents such as vitamins E and C, and mannitol, and also with superoxide dismutase, restored the ability of RI and RII to bind 8-azido-[32P]-cAMP toward normal levels. Western blot analysis showed that the protein levels of the RI and RII subunits are similar in normal and psoriatic fibroblasts, and that the amounts of RI and RII are not altered by treatment of the cells with free radical-generating systems. These results suggest that oxidative modification may serve as a mechanism to alter PKA activity in human cells, and that an altered oxidative state may be involved in mediating the decrease in PKA activity and cyclic AMP binding noted in cells from psoriatic patients.

Computer-controlled monitoring of bladder pressure in the prevention of 'TUR syndrome': a randomized study of 53 cases.

OBJECTIVE: To evaluate the performance of a computer-controlled monitor of bladder pressure in the prevention of transurethral resection (TUR) syndrome. PATIENTS AND METHODS: The in vitro pressure loss in catheters and endoscopes of different size was measured for irrigant flow rates of 0-500 mL/min to calibrate them before surgery. The calibrations were used in a computerized monitoring system designed to control bladder pressure during TUR of the prostate (TURP). The performance of the system was assessed in a randomized study of 53 patients with a prostate adenoma or carcinoma (Group A, 27 unmonitored patients; Group B, 26 monitored patients). The primary criterion for evaluating the absorption of irrigating fluid was the level of glycine in the blood. RESULTS: When patients with capsule perforation were included in the analysis, there was no statistically significant difference in mean glycine absorption between the groups, although glycine levels were highest in Group A, particularly in those cases with perforation (four in Group A; two in Group B). When the results for patients with capsule perforation were excluded from the analysis, there was a significant difference in the incidence of irrigant absorption between the groups. The extent of absorption was not related to the duration of operation (which was shorter in Group B) nor to the weight of resection chippings. CONCLUSION: The continuous computerized monitoring of bladder pressure during TURP effectively reduced the absorption of irrigant fluid, making the procedure safer for the patient and easier for the surgeon.

Antioxidant enzymes in psoriatic fibroblasts and erythrocytes.

Antioxidant enzyme activities in fibroblasts and erythrocytes prepared from normal and psoriatic patients were measured and compared. The most significant differences were noted in superoxide dismutase (SOD) activities. A dramatic (5.2-fold) increase in Mn-SOD activity along with a lesser (1.8-fold) increase in CuZn-SOD activity was observed in fibroblasts from lesional and nonlesional psoriatic skin. The increase of Mn-SOD activity was correlated with an increase of both protein and mRNA. A slight (1.2-fold) increase in CuZn-SOD activity was also found in psoriatic as compared to normal red blood cells, while Mn-SOD activity was not present in these cells. In contrast, both glutathione peroxidase and catalase activities were only slightly (1.3-fold) increased in psoriatic fibroblasts, with no appreciable change noted in psoriatic erythrocytes. Likewise, glutathione levels were observed to be similar in normal and psoriatic cells. The increases in SOD activities did not appear to correlate with the severity of the disease as expressed by the Psoriatic Area Severity Index score or with plasma inflammatory markers. These results demonstrate that antioxidant enzyme activities, particularly Mn-SOD in fibroblasts and CuZn-SOD in erythrocytes, are significantly elevated in cells from psoriatic patients.

Retinoylation of the type II cAMP-binding regulatory subunit of cAMP-dependent protein kinase is increased in psoriatic human fibroblasts.

Previously, we have reported a defect in the cAMP-dependent protein kinases (cAMP-PK) in psoriatic cells (i.e., a decrease in 8-azido-[32P]cAMP binding to the regulatory subunits and a decrease in phosphotransferase activity) which is rapidly reversed with retinoic acid (RA) treatment of these cells. This led us to examine a possible direct interaction between retinoids and the RI and RII regulatory subunits through retinoylation. Retinoylation of RI and RII present in normal and psoriatic human fibroblasts was analysed by [3H]RA treatment of these cells, followed either by chromatographic separation of the regulatory subunits or by their specific immunoprecipitation. These studies indicated that RI and RII can be retinoylated. [3H]RA labeling of the RII subunit was significantly (P < 0.005) greater in psoriatic fibroblasts (nine subjects; mean 7.47 relative units +/- 1.37 SEM) compared to normal fibroblasts (eight subjects; mean 2.46 relative +/- 0.49 SEM). [3H]RA labeling of and the increase in 8-azido-[32P]-binding to the RI and RII subunit in psoriatic fibroblasts showed a similar time course. This suggests that the rapid effect of retinoic acid treatment to enhance 8-azido-[32P]-cAMP binding to the RI and RII in psoriatic fibroblasts may be due, in part, to covalent modification of the regulatory subunits by retinoylation.

[Severe algoneurodystrophy of the right foot associated with prostatic cancer]. / Algoneurodystrophie sévère du pied droit associée à un cancer prostatique.

The authors report 1 case of particularly severe reflex neurovascular dystrophy whose clinical course was marked by the discovery of a carcinoma of the prostate. There was improvement in the reflex neurovascular dystrophy despite hormonal therapy of the cancer. Reflex neurovascular dystrophy cannot be considered as a form of a paraneoplastic syndrome.

Post-translational abnormality of the type II cyclic AMP-dependent protein kinase in psoriasis: modulation by retinoic acid.

Previously, we have reported a decrease in the binding of a cAMP analog to the regulatory subunits of cAMP-dependent protein kinase (cAMP-PK), as well as a decrease in cAMP-PK activities, in psoriatic cells. Retinoic acid (RA) treatment of these cells can induce an increase in cAMP-PK toward normal levels. To better define the effect of retinoic acid on the cAMP-PK system in psoriatic fibroblasts, Western blot analysis using an RII alpha specific antibody and in vivo phosphorylation experiments were carried out to determine possible changes in the RII regulatory subunit. Our results indicate a decrease in the binding of the cAMP analog 8-azido-[32P]-cAMP with no change in the level of RII protein in psoriatic fibroblasts. In addition, by two-dimensional gel electrophoresis we observed the presence of a phosphorylated form of RII unique to psoriatic cells which is suppressed by RA treatment. This study suggests an altered posttranslational modification of the cAMP-PKII in psoriatic fibroblasts which can be reversed by exposure of these cells to RA.

[Spontaneous retroperitoneal hematoma: a rare cause of hemorrhagic shock]. / Hématome rétropéritonéal spontané: cause rare de choc hémorragique.

A spontaneous retroperitoneal haematoma is an uncommon cause of haemorrhagic shock. We report a case of spontaneous rupture of a renal angiomyolipoma resulting in haemorrhagic shock in a 52-year-old woman. The renal tumor was recognized by sonography and diagnosed by CT-scan. Renal angiography was performed, but embolization was not successful. During the surgical procedure, nephrectomy was required because of persistent bleeding, related to disseminated intravascular coagulation. Outcome was uneventful. Diagnosis and treatment of renal angiomyolipoma are discussed. The Lenk's triad, consisting of acute lumbar pain, symptoms of internal bleeding and lumbar tumefaction, is the usual clinical picture of retroperitoneal haemorrhage. The kidney is the most frequent cause and renal angiomyolipoma is the most frequent benign tumor. Renal angiomyolipoma is either isolated or associated with tuberous sclerosis in up to 20 per cent of patients. Diagnosis is suggested by sonography and confirmed by CT-scan. Renal angiography, performed in haemodynamically stable patients, shows the origin of bleeding and allows embolization. Considering the frequent bilaterality of angiomyolipoma, surgery should be as conservative as possible in order to preserve renal function.

Rapid effect of treatment of psoriatic erythrocytes with the synthetic retinoid acitretin to increase 8-azido cyclic AMP binding to the RI regulatory subunit.

We have recently demonstrated a deficiency in the cyclic adenosine monophosphate (cAMP)-dependent protein kinases (PKA), the intracellular mediator of AMP, in psoriasis. This enzyme defect is expressed in fibroblasts and in red blood cells isolated from psoriatic patients. In these cells, the abnormality noted in cAMP binding to PKA correlates well with the severity of the disease and is corrected by long-term treatment with etretinate. In this study, we determined the effect of oral administration of acitretin in four psoriatic patients on the altered cAMP binding observed with the RI regulatory subunit of PKA in erythrocytes prepared from these patients. Acitretin (30 mg/day) induced a rapid (within 1 h) increase in the ability of the RI regulatory subunit of erythrocytes to bind the 8-azido[32P]cAMP photoaffinity analogue of cAMP. The maximal plateau for this effect of acitretin was observed within 24 h of treatment and preceded the clinical improvement of the disease. The effect of acitretin was dose-dependent, with the maximal response observed at 40 mg acitretin/d. In addition, the rapid exposure (15 min) of erythrocytes isolated from untreated patients exhibiting severe psoriasis to acitretin also promoted an increase in binding of 8-azido[32P]cAMP to the RI cAMP binding protein. Retinoic acid and 13-cis-retinoic acid were as efficient as acitretin in inducing the increase in binding of 8-azido[32P]cAMP to the RI regulatory subunit, whereas arotinoid was without effect. These results suggest that acitretin may act to modify PKA (the RI regulatory subunit) at the post-transcriptional level, and this may reflect, in part, on the mechanism of action of this synthetic retinoid. Further, monitoring this biochemical event may be helpful in determining the choice of retinoid therapy and in the management of its pharmacology.

Calcitonin gene-related peptide: an autocrine growth factor with regulatory activity in vitro.

We show that an autocrine system for calcitonin gene-related peptide (CGRP) exists in F9 teratocarcinoma cells. Synthesis of CGRP by F9 cells was demonstrated by measuring the peptide concentration in cells and medium and by determining specific mRNA in cells. During six days of culture, CGRP secretion did not vary significantly in the medium, while intracellular CGRP and CGRP mRNA levels increased. F9 cells contained a CGRP-sensitive adenylate cyclase system and CGRP increases the accumulation of cAMP in the culture medium. Interestingly affinity purified antibodies against CGRP specifically inhibited growth of F9 cells by 50%. CGRP therefore stimulates F9 cell growth by an autocrine process, suggesting that CGRP may be a growth factor during early embryogenesis.

Calcitonin and calcitonin gene-related peptide are chemotactic for F9 embryonal carcinoma cells.

The chemotactic effect of calcitonin (CT) gene products was tested on F9 teratocarcinoma cells, which are an in vitro model of early embryonic development. CT and CT gene-related peptide (CGRP) induce a significant chemotactic response (chemotactic index, 40-50). The order of potency is: chicken CGRP greater than or equal to salmon CT greater than or equal to human CGRP. Human CT is a less potent chemotactic agent (chemotactic index, 15). Compared to other well known peptides with chemotactic activity, such as platelet-derived growth factor (no activity) and transforming growth factor-beta (chemotactic index, 5), CGRP and CT appear to be very active in attracting F9 cells in the Boyden chamber assay. Interestingly, CT and CGRP exhibit little chemotactic effect toward differentiated teratocarcinoma cells (i.e. retinoic acid-treated F9 cells or parietal endodermal PYS cells). While salmon CT and chicken CGRP activate adenylate cyclase activity in F9 cell membranes by 7- to 8-fold, higher concentrations (greater than 10(-10) M) of these peptides are required to stimulate cAMP formation than are required to mediate the chemotactic effect of these peptides. These data imply the possible involvement of CT gene products in regulating cell migration during early embryonic development.

A cAMP binding abnormality in psoriasis.

In 34 psoriatic patients with various cutaneous manifestations (psoriasis vulgaris, erythroderma psoriaticum, guttate psoriasis), the ability of the RI regulatory subunit of cAMP-dependent protein kinase (PKA) to bind a cAMP analogue (8-azido [32P] cAMP) in erythrocyte membranes was significantly lower than that in 19 normal subjects (mean [SEM] 565 [35] vs 930 [35] fmol/mg protein). This enzyme defect was not found in patients with other forms of dermatitis that can be confused with psoriasis or with other inflammatory diseases. There was a significant negative correlation between the severity of the disease as expressed by the psoriatic area and severity index score and the binding of the cAMP analogue to PKA. A long-term study showed that oral retinoid treatment of psoriatic patients resulted in a correction of the binding defect. Unaffected members of psoriatic families had significantly lower than normal binding of cAMP to PKA (773 [60] fmol/mg protein). This study shows for the first time that in psoriasis a biochemical defect expressed in erythrocytes correlates with the severity of the disease as well as its clinical evolution. These results will be useful in clinical management of psoriatic disease for the choice and follow-up of retinoid therapy.

Effect of retinoic acid on cAMP dependent protein phosphorylation in psoriatic fibroblasts.

Retinoic acid treatment of psoriatic fibroblasts increases the activity of cyclic AMP dependent protein kinase. In this study we report that retinoic acid treatment of cultured psoriatic fibroblasts modifies their subsequent cAMP dependent protein phosphorylation. In the soluble fraction of normal fibroblasts cAMP clearly enhances the in vitro phosphorylation of proteins of MW 37,49,54,56,68,83 kD while retinoic acid treatment of the same cells results in a decrease of the cAMP dependent phosphorylation of the first five of the same proteins. In contrast, in psoriatic fibroblasts from psoriatic patients retinoic acid either has no effect or increases the cAMP dependent phosphorylation of some of these proteins. Moreover the phosphorylation of a protein of MW 54 kD, undetectable in untreated psoriatic cells, is more phosphorylated in the presence of cAMP after retinoic acid treatment. The appearance of this phosphorylated proteins is time dependent and dose dependent upon the addition of retinoic acid. These in vitro phosphorylation results suggest that retinoic acid treatment of psoriatic fibroblasts change the level of cAMP dependent phosphorylation of some cytosolic proteins. These specific phosphorylations could be implicated in a variation of cell functions.