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Resume Introduction Les anticoagulants oraux directs sont une alternative recente aux antivitamines K, et peu de donnees sur les patients beneficiant de ces nouveaux traitements existent. Ce travail a pour objectif d’identifier et de decrire le profil des patients traites par un anticoagulant oral direct, admis dans un service d’urgences. Methode L’ensemble des patients, traites par un anticoagulant oral direct, admis aux urgences du CHU de Clermont-Ferrand du 1er janvier 2013 au 31 aout 2013, a ete inclus dans cette etude retrospective et descriptive. Resultats Parmi les 73 patients inclus, 47,9 % etaient traites par dabigatran et 52,1 % par rivaroxaban. Soixante-deux patients avaient une indication pour une fibrillation auriculaire, avec un score de CHADS2 moyen de 2,6 [2,3–3]IC95 %. L’âge moyen etait de 76,4 ans [73,7–79,1]IC95 %. Vingt-neuf patients (39,7 %) avaient au moins une association medicamenteuse connue pour augmenter le risque hemorragique. Le sexe masculin etait significativement superieur chez les patients ayant presente une hemorragie (68,8 versus 38,2 %, p = 0,032). Les patients avec une hemorragie grave avaient une insuffisance renale avec une clairance de la creatinine de 45,2 versus 68,8 mL/min, p = 0,002. Le score de Beyth etait en moyenne significativement plus eleve dans les deux populations : hemorragie (1,8 versus 1,4, p = 0,029) et hemorragie grave (2 versus 1,4, p = 0,016). Conclusion Notre etude a permis d’identifier plusieurs facteurs de risque hemorragique que sont le sexe masculin, un score de Beyth eleve et une clairance de la creatinine diminuee. De plus la population traitee par un nouvel anticoagulant oral admise aux urgences est âgee, avec de nombreuses comorbidites notamment cardiovasculaires et une poly-medication importante.

An audit of the French national health insurance system would be justified by economic considerations alone, but this would risk overlooking the notions of solidarity and freedom to which the French are rightly attached. European comparisons suggest, however, that our system could be made more efficient without undermining public health. The national health insurance system allows each member of the population to receive high-quality medical care. Practitioners have near-total freedom of prescription and practice. Medical care contributes to the ongoing increase in life expectancy, which is currently 73 years and second only to Japan. Healthcare is also a source of a million jobs. Macro-economic spending controls have failed, owing to medical progress and population aging, and also to medical consumerism favored by an unprecedented range of examinations and treatments, the increasing reimbursement of medical care, and the extension of direct payment by the insurer. Many ineffective measures have been implemented, such as tarification according to activity, and hospital certification. Health spending is also increased unnecessarily by bureaucratisation of healthcare spending and the transfer of professionals to posts for which they are not qualified. Some controversial medical prescriptions are not adequately controlled by the health service. Many reforms are based on over-optimistic economic predictions that fail to take related overheads into account. Lobbying by special interests groups undermines reform and the public interest. Too many independent administrative bodies have been created, and many are less efficient than the public structures they replaced. In sum, the French national health insurance system has become less and less efficient over the years.

The ductal epithelium of the semicircular canal forms much of the boundary between the K+-rich luminal fluid and the Na+-rich abluminal fluid. We sought to determine whether the net ion flux producing the apical-to-basal short-circuit current ( I sc) in primary cultures was due to anion secretion and/or cation absorption and under control of receptor agonists. Net fluxes of 22Na, 86Rb, and36Cl demonstrated a basal-to-apical Cl−secretion that was stimulated by isoproterenol. Isoproterenol and norepinephrine increased I sc with an EC50 of 3 and 15 nM, respectively, and isoproterenol increased tissue cAMP of native canals with an EC50 of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had no effect on I sc. Isoproterenol stimulation of I sc and cAMP was inhibited by ICI-118551 (IC50 = 6 μM for I sc) but not by CGP-20712A (1 μM) in primary cultures, and similar results were found in native epithelium. I sc was partially inhibited by basolateral Ba2+ (IC50 = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, and DIDS did not fully fit the profile for CFTR. Our findings show that the canal epithelium contributes to endolymph homeostasis by secretion of Cl− under β2-adrenergic control with cAMP as second messenger, a process that parallels the adrenergic control of K+ secretion by vestibular dark cells. The current work points to one possible etiology of endolymphatic hydrops in Meniere's disease and may provide a basis for intervention.

Direct oral anticoagulants are a recent alternative to vitamin K antagonists but there is a lack of data regarding patients receiving these new types of treatment. The aim of the study was to identify and describe patients receiving direct oral anticoagulants admitted to an emergency unit.All the patients taking direct oral anticoagulants, admitted to the emergency room of the Clermont-Ferrand Hospital from January to August 2013, were included in this retrospective and descriptive study.Among the 73 patients included, 47.9% were treated with dabigatran and 52.1% with rivaroxaban. The indication was stroke prevention in 62 patients with atrial fibrillation whose average CHADS2 score was 2.6 [2.3-3](IC95%). The average age was 76.4 years [73.7-79.1](IC95%). Twenty-nine patients (39.7%) had at least one drug association known for increasing the risk of bleeding. Average scores for bleeding risk were: HAS-BLED 3.1 [2.9-3.3](IC95%) and Beyth 1.5 [1.3-1.6](IC95%). Bleeding patients included a higher percentage of men (68.8 vs. 38.2%, P=0.032). Creatinine clearance was lower in patients with major bleeding (45.2% vs. 68.8 mL/min, P=0.002). The Beyth score was highest in both sub-groups.In our study, we have found that the bleeding risk factors were: male gender, a high Beyth score, and a lowered creatinine clearance. Overall, patients treated with direct oral anticoagulants admitted to the emergency room were old with many co-morbidities, especially cardiovascular conditions; polymedication was frequent.

International audience; The inner ear of mammals contains the vestib-ular apparatus which is involved in the maintenance of posture and balance. The tubular structure of the apparatus is bathed by the potassium-rich endolymph and sodium rich perilymph in the luminal and abluminal compartments , respectively. The luminal compartment is lined by a continuous epithelium with islets of receptor organs, which separates the luminal from the abluminal compartment. The present work focuses on the epitheli-um, without the receptor organs, and shows that it can be reconstituted in culture. The epithelium from 4-day-old Wistar rats was grown on microporous membranes. High transepithelial electrical resistances (4000-6000 Ω·cm 2) were achieved after 4-8 days in culture. The epithelium was characterized by the presence of cytokeratin, ZO-1 protein, occludin, and the presence of tight junctions and kinocilia. The transepithelial resistance of the cell mono-layer withstood endolymph/perilymph dual bathing when the apical pole of the cells was in contact with en-dolymph, but collapsed in the reverse configuration. Weak but statistically highly significant basal to apical rubidium (86 Rb) transport was observed. These findings show that this epithelium maintains its in vivo polarity and could enhance the potassium composition of endo-lymph up to maturity. This new culture model, in which dual bathing is possible, should enable further in vitro studies of the sensory vestibular epithelia.

J. R. Philippot and B. Lebleu acknowledge the Agence Nationale de Recherche sur le SIDA and the Association de Recherche contre le Cancer for support. The authors thank A. Bienveniie from URA-CNRS 1856 (University Montpellier II) for many fruitful discussions during the course of this study. We thank M.-T. Cabanis from the Laboratory of Analytical Chemistry (Faculty of Pharmacy, Montpellier) for titrating calcium. J. P. Bongartz was the recipient of a postdoctoral fellowship from ANRS.The degradation of a drug, its toxicity, the unsuitable target binding or intracellular routing may be circumvented by an efficient drug delivery system. The efficacy of pH-sensitive liposomes is well documented for these purposes: These liposomes have a lipidic composition that makes escape from the endosome and delivery into the cytoplasm possible. The efficacy of pH-sensitive liposomes, comprising dioleoyl phosphatidyl ethanolamine, oleic acid, and cholesterol (DOPE/OA/Chol), loaded with polyrI:polyrC (polyIC), ha...

To evaluate the functional relationship between the calcitonin-gene related peptide (CGRP) receptor in trout gills and guanine nucleotide-binding proteins, we investigated the effect of GTP not only on the CGRP stimulated adenylate cyclase activity but also on the human CGRP binding to trout gill membranes. In the presence of 1 μM GTP, the basal and the CGRP stimulated adenylate cyclase activity were increased by 1.8-fold. In addition, GTP decreased the CGRP binding to gill membranes and accelerated the dissociation of bound labeled hormone. Scatchard analysis of the data revealed that the reduction of human CGRP binding by GTP was mainly due to a decrease in the binding affinity with no significant change in the binding capacity. Thus, the binding of CGRP to fish gill membranes activates adenylate cyclase via a guanine nucleotide dependent mechanism, suggesting the involvement of a guanine nucleotide-binding stimulatory protein in the action of CGRP in fishes.

Recently several groups reported a dramatic improvement of reporter gene transfection efficiency using a fusogenic peptide, derived from the Influenza hemagglutinin envelop protein. This peptide changes conformation at acidic pH and destabilizes the endosomal membranes thus resulting in an increased cytoplasmic gene delivery. We describe the use of a similar fusogenic peptide in order to improve the antiviral potency of antisense oligodeoxynucleotides (anti TAT) and oligophosphorothioates (S-dC28) on de novo HIV infected CEM-SS lymphocytes in serum-free medium. We observed as 5 to 10 fold improvement of the anti HIV activities of the phosphodiester antisense oligonucleotides after chemical coupling to the peptide in a one to one ratio by a disulfide or thioether bond. No toxicities were observed at the effective doses (0.1-1 microM). No sequence specificity was obtained and the fusogenic peptide possessed some antiviral activities on its own (IC50: 6 microM). A S-dC28-peptide disulfide linked conjugate and a streptavidin-peptide-biotinylated S-dC28 adduct showed similar activities as the free S-dC28 oligonucleotide (IC50: 0.1-1 nM). As expected, all the compounds were less potent in the presence of serum but the relative contribution of peptide coupling was maintained.

1. 1. The presence of both salmon calcitonin (CT) and human calcitonin gene-related peptide (CGRP) was investigated in tissues of Palaemon serratus. 2. 2. The variation of these two molecules was studied as a function of the molt cycle. 3. 3. The highest salmon CT concentration was found in the eyestalks and heart whereas CGRP was mainly concentrated in the heart, gills and eyestalks. 4. 4. In the eyestalk, both CT and CGRP-like immunoreactivity were maximal at the B stage. In the haemolymph, the calcium fall observed in the B stage was correlated with the CT increase. No correlation appeared between the circulating calcium and the CGRP concentration.

We have recently identified in medullary thyroid carcinoma the existence of a second calcitonin messenger, generated by a splicing between the 3' coding region of exon 4 and exon 5 of Calc I gene. It differs from the first one in its 3' coding sequence and codes for a calcitonin precursor which generates the same N terminal peptide, calcitonin and a specific 21 amino acid carboxy terminal peptide differing from Katacalcin by its 8 last amino acids. We searched for the expression of this new messenger in normal human thyroid tissue by Northern and by polymerase chain reaction techniques. This second calcitonin messenger was expressed in 4/4 normal thyroids and 4/5 medullary thyroid carcinoma tissue samples. The expression of this second messenger is apparently a common occurrence in C cells whether normal or tumoral.

The calcitonin/calcitonin gene-related peptide gene expresses two different mRNAs by tissue-specific alternative processing. The calcitonin mRNA is produced in thyroid C cells by splicing of the first three exons to the fourth polyadenylated exon. It encodes a protein precursor containing an amino-terminal peptide, calcitonin, and a carboxyl-terminal peptide (CCP I). Calcitonin gene-related peptide (CGRP) mRNA is produced in neuronal cells by splicing of the three common exons to the fifth exon and the polyadenylated sixth exon, leading to the production of a CGRP precursor. Our studies concerning the expression of the calcitonin/CGRP gene in human medullary thyroid carcinoma revealed the presence of a new RNA transcript. Amplification by polymerase chain reaction and direct sequencing showed that the novel transcript is composed of exons 1, 2, and 3, part of exon 4, exon 5, and a polyadenylated exon 6. This transcript contains an open reading frame coding for the known amino-terminal and calcitonin peptides, as well as for a novel calcitonin carboxyl-terminal peptide, CCP II. This third alternative pathway utilizes an internal donor site within the exon coding for calcitonin. The presence of CCP II was demonstrated in plasma and thyroidal tissues of medullary thyroid carcinoma patients, implying that this novel mRNA is actively translated in medullary thyroid carcinoma.

We localized specific binding sites for human calcitonin gene related peptide (hCGRP) in different organs of the trout using labelled human CGRP. Maximal binding was observed in gill and spleen membranes. The binding of 125I-hCGRP was time and temperature dependent. Scatchard analysis of binding data for the spleen and the gills disclosed two binding sites. The constants for the site of high affinity and low capacity (KAM-1 and Bmax (fmol/mg of proteins] were 2.9 x 10(9) for the spleen and 70 and 3.5 x 10(9) for the gill. Salmon calcitonin (sCT) inhibited the binding of 125I-hCGRP to spleen membranes with the same order of potency as hCGRP. In contrast sCT was less effective than hCGRP in suppressing the specific binding of 125I-hCGRP to gill membranes.

Poly(rI:rC) and Ampligen were entrapped in liposomes that were covalently coupled to Protein A, permitting binding to antibodies specific for the major histocompatibility complex-encoded H2K molecule of L929 cells, or to control antibodies. Free and encapsulated polynucleotides were compared for their capacity to stimulate secretion of interferon (IFN) and interleukin-6 (IL-6) and to induce cellular toxicity on L929 cells pretreated with IFN-alpha/beta. Free and encapsulated poly(rI:rC) or Ampligen (poly(rI:rC12-rU] induced similar levels of secretion of IFN over a broad dose range. The activity of the liposome-encapsulated polynucleotides was dependent on its binding to an antibody that permitted cell association and internalization; the same liposomes were inactive in the presence of control antibodies. IL-6 secretion was induced by double-stranded (ds) RNA in a dose-dependent manner, with a significantly greater effect seen for targeted, liposome-encapsulated material. The marked toxicity of targeted poly(rI:rC), as compared to free poly(rI:rC), was confirmed. Encapsulated Ampligen was less toxic than encapsulated Poly(rI:rC).

The evolution of bone changes induced by fluoride after the end of exposure was investigated in lambs. Sodium fluoride (NaF) was given orally at a dose of 3.5 mg/kg per day to 14 animals for 120 days. A group of 7 control and 7 treated lambs was slaughtered at the end of NaF administration (T120) and another group 120 days after the end of NaF exposure (T240). At T120, the bone fluoride content (BFC) was very significantly increased in treated animals. The histomorphometric analysis confirmed that fluoride induces an increase in bone formation (the osteoid perimeter and area were 3-fold and 4.5-fold higher respectively in treated than in control animals). The number of osteoblasts was significantly augmented. Serum osteocalcin level was twice as high in treated animals compared with controls. The bone formation rate at the tissue level (BFR) doubled after treatment, but the apposition rate (Aj.AR) was half that in the control group. The mineralization lag time (Mlt) was 120 days in treated animals compared with 42 days in controls. At T240, BFC had decreased by 50% compared with the level at T120, but it was still significantly higher than in controls. The osteoid and osteoblastic parameters were 2 and 1.3 times higher than in control animals. BFR remained significantly increased in treated animals, but Aj.AR and Mlt were similar in control and treated animals. In conclusion, after 4 months of NaF exposure fluoride induced an increase in osteoblast natality and bone formation at the tissue level, associated with a toxic effect at the individual cell level. Four months after the end of NaF exposure, positive effects on bone formation were still present but the evidence of cellular toxicity had disappeared.

A new calcitonin (CT) immunoradiometric assay, using anti-11-7 and anti-24-32 CT fragment monoclonal antibodies was evaluated and compared to classical RIA. The sensitivity was 2.5 ng/L, the normal basal level (n = 83) was lower than 10 ng/L, the response to pentagastrin stimulation in control subjects was absent in nine and between 10-30 ng/L in nine others. (mean, 15.4). In patients with renal failure the basal level was increased between 10-52 ng/L. In patients with medullary thyroid carcinoma (MTC; n = 28), the basal level was between 189-28,900 ng/L. A pentagastrin test was performed as screening for familial MTC in eight patients with confirmed MTC at subsequent surgery; the calcitonin peak was equal or greater than 38 ng/L. Large differences exist between CT levels measured by RIA and immunoradiometric assay. The latter method provides a greater sensitivity to pentagastrin test and allows a better identification of microcarcinoma in hereditary cases of MTC.

The physiological significance of calcitonin gene-related peptide (CGRP) was investigated by assessing the CGRP stimulated adenylate cyclase activity in various tissues of trout. The highest enzyme concentration was found in gill and stomach membranes. The maximal activity (190% of the basal value) was observed for a concentration of 53.3 nM CGRP I or II. In the presence of 58 nM sCT, the maximal enzyme activity represented 120% of the basal value. No additive effect was observed; this suggests that both CGRP and sCT activities are mediated through the same receptor. The present data are in favour of a role for this neuropeptide operating in branchial cell functions such as calcium transfer from the external to the internal milieu.

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii . The highest concentrations of the peptide were found in gill (1.68 ± 0.09 ng/mg protein) and in intestine (1.06 ± 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 ± 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.

The semicircular canal duct (SCCD) epithelium is a vestibular epithelial domain that was recently shown to actively contribute to endolymph homeostasis by Cl− secretion under control of β2-adrenergic stimulation. By analogy to other Cl− secretory epithelia, we hypothesized that SCCD also provides an active absorptive pathway for Na+ under corticosteroid control. Measurements of short-circuit current ( Isc) demonstrated stimulation (7–24 h) by the glucocorticoids hydrocortisone (EC50 13 nM), corticosterone (33 nM), prednisolone (70 nM), and dexamethasone (13 nM) over physiologically and therapeutically relevant concentrations and its block by amiloride (IC50 470 nM) and benzamil (57 nM), inhibitors of the epithelial sodium channel (ENaC). Isc was also partially inhibited by basolateral ouabain and Ba2+, indicating the participation of Na+-K+-ATPase and a K+ channel in Na+ transport. By contrast, aldosterone stimulated Isc only at unphysiologically high concentrations (EC50 102 nM). The action of all steroids was blocked by mifepristone (RU-486; Kd ∼0.3 nM) but not by spironolactone ( Kd ∼0.7 μM). Expression of mRNA for the α-, β-, and γ-subunits of ENaC was demonstrated in the presence and absence of glucocorticoids. These findings are the first to identify SCCD in the vestibular labyrinth as a site of physiologically significant ENaC-mediated Na+ absorption and osmotically coupled water flux. They further demonstrate regulation of Na+ transport by natural and therapeutic glucocorticoids. The results provide for the first time an understanding of the therapeutic benefit of glucocorticoids in the treatment of Meniere's disease, a condition that is associated with increased luminal fluid volume.

The presence in normal adult man of stem cells sharing the properties of embryonic stem cells opens new avenues for basic and therapeutic research. We describe a stem cell present in normal adult human blood, probably able to give rise to the "reserve" stem cells in charge of repair, present in different organs. These monocytoid circulating cells are able to transdifferentiate into several cell types. In normal man, they are almost quiescent and are strictly controlled by a special subpopulation of T lymphocytes. In diseases such as fibrosis and chondrosarcoma, these cells proliferate and the differentiated cells escape T lymphocyte control. As a consequence, these cells accumulate, giving rise in vitro to a tissue which evoke the lesions characterizing the disorder of the patient, showing spontaneously their pluripotentiality. Neural cell markers are present in this migrating cell, suggesting that pluripotent stem cells present in adult man may derive from the neural crest. These circulating cells could offer a source of stem cells for cellular and gene therapy provided the normal cells could be expanded, their transdifferentiation directed and the control by T lymphocytes maintained.

Stem cells isolated from adult human blood are able to give rise to several different kinds of cell types such as mesenchymal cells, including striated muscle cells, hepatocytes, and endothelial cells. Because independently studied by authors whose interests focused on particular tissue types, these stem cells have been described as different. However, they might well represent one unique population of pluripotent stem cells in homeostatic equilibrium with the ‘reserve’ stem cells buried in organs. In the blood, these stem cells have a monocytic phenotype. In in vitro culture, once they have adhered, they spontaneously differentiate into diverse types of cells reminiscent of embryonic stem cells in culture. Normally, they are almost quiescent cells. But under precise circumstances such as wound-healing, they may proliferate and migrate to the right organ to give rise there to the right type of cells, in order to participate in the repair process. Indeed, such a powerful stem cell needs to be tightly controlled. We illustrate here, by time-lapse videocinematography, how a special subpopulation of T-lymphocytes, for which we coined the name ‘phagic T-lymphocytes’ (PTLs), destroys these stem cells as soon as they differentiate in vitro, i.e., without the purpose of a repair. These stem cells express constitutively HLA-DR molecules and therefore can act as antigen-presenting cells able to activate phagic T-lymphocytes. The targets of these activated phagic T-lymphocytes are the differentiated stem cell themselves. Phagic T-lymphocytes are attracted by the stem cells, circulate around them, then penetrate and circulate inside them until the latter ‘explode’. This mechanism of destruction by phagic T-lymphocytes is unique and seems to be normally restricted to stem cells. It represents a beneficial exception in self-tolerance since it avoids the accumulation of these stem cells out of healing purposes. Interestingly, in disorders such as fibrosis and/or some malignant proliferations, these stem cells proliferate, escape destruction by phagic T-lymphocytes and, as a consequence, accumulate, giving rise to a ‘tissue’ when cultured in vitro.

Summary The neural markers present in the normal circulating monocytoid cells able, in pathological situations, to transdifferentiate into different mesenchymal-type cells, confirm the hypothesis previously raised that these cells derive from the neural crest. In culture, the normal cells display a great plasticity very reminiscent of microglial cells in culture. Almost a quiescent cell in normal individuals, this monocytoid cell shows its division potentialities in pathological situations of fibrosis and cancer (chondrosarcoma) where it is found to spontaneously proliferate. While the normal neofibroblasts are rapidly recognized and destroyed by fibrophagic T-lymphocytes, the pathological cells escape this control and, as a result, they accumulate in vitro giving rise to a tissue sometimes organized as nodules. Although basically the transdifferentiation process is similar in all the pathological situations of fibrosis and cancer studied so far, the end-result phenotype evokes the pathology the patient is suffering from. It evokes osteoblasts in a case of osteomyclosclerosis, chondroidocytes in a case of chondrosarcoma, myelofibroblasts in a case of fibrosis of lung and kidney in a patient under ciclosporine treatment. Hence, this circulating monocytoid cell is a multipotent cell with great division potentiality. These are characteristics of stem/preprogenitor cells. Since this circulating monocytoid cell also bears the neural markers we called it a monocytoid ectomesenchymal stem/preprogenitor cell. Therefore, the existence of an ectomesenchymal system is discussed here. The circulating monocytoid ectomesenchymal stem/preprogenitor cell might be involved in the normal cicatrisation process while the fibrophagic T lymphocytes might be involved in its termination. Impairment of this controlled mechanism might result in the development of fibrosis and/or cancer such as chondrosarcoma in vivo. Interestingly, at least in vitro, proliferation is restricted to the monocytoid cell before transdifferentiation takes place. In this model, fibrosis and cancer might share some common steps going from the proliferation of the monocytoid cells to their transdifferentiation into mesenchymal-type cells and the accumulation of these transdifferentiated cells in the tissues. Then, cancer might be distinguished from fibrosis by the additional acquisition of the ability to proliferate by the transdifferentiated cells. The monocytoid ectomesenchymal stem/preprogenitor cell might also be involved in brain neurodegenerative diseases characterized by an accumulation of microglia. The circulting monocytoid ectomesenchymal stem/preprogenitor cell appears as a target for gene therapy in pathological situations of fibrosis and/or cancer where it proliferates out of control. If the normal cell can be expanded and if its transdifferentiation can be directed, the circulating monocytoid ectomesenchymal stem/preprogenitor cell may become a useful tool for cellular therapy, in case of failure in wound healing and tissue regeneration.

Exposure of humans to cadmium, a common environmental pollutant, is mainly through food intake. However, the mechanisms of intestinal absorption have not been clearly elucidated for this toxic metal ion. In order to investigate the effects of long-term exposure to this metal and the role of metallothioneins in cadmium absorption, we used human-derived Caco-2 cells cultured on porous membrane filters. We first validated this model by quantifying metal uptake and transepithelial transport on control cells and cells adapted to grow for 2 to 5 weeks in the presence of low doses of cadmium in the culture medium. The nontoxic doses of cadmium (0.1, 1.0, and 5 microM), in which Caco-2 cells could be cultured for many passages without deleterious effects, were determined by evaluating transepithelial resistance of the cells and lactate dehydrogenase leakage. After 24 h of 1 microM Cd exposure, intracellular cadmium levels were 3- and 6-fold higher for cells exposed for extended periods to 1 and 5 microM cadmium, respectively, compared to control cells. In control and long-term exposed cells, this accumulation was inhibited by zinc, copper, and pCMBS, but not by verapamil or ouabain. Intracellular metallothionein content was increased 1.5-, 5-, and 12-fold for the cells grown in the presence of 0.1, 1.0, and 5 microM cadmium, respectively, in the culture medium. The amount of metallothionein synthesized and released by the cells was highly correlated with cadmium accumulation and transport. Our results suggest that Caco-2 cell monolayers are a good predictive model for the study of cadmium intestinal absorption following exposure to repeated low doses of cadmium, and confirm the essential role of metallothionein in the regulation of cadmium intestinal absorption.

The high concentrations of molecules immunologically related to salmon calcitonin (CT) and/or to human calcitonin gene-related peptide (CGRP) in the oesophagus of the norway lobster Nephrops norvegicus have been examined. In the present study. We report the purification of these molecules by means of a specific radioimmunoassay for calcitonin and calcitonin gene related peptide. The immunoreactive molecules were tested for their functional similarities with CT and CGRP. This was investigated by measuring their ability to interact with CGRP and CT radioreceptor assays and to stimulate the adenylate cyclase activity in rat liver and kidney membranes, respectively. In addition, the purified product was injected in young rats in order to check for a CT-like biological activity of these molecules. The combination of these tests led us to purify a molecular form of 33 kDa. N-terminal sequence analysis of this protein revealed a considerable homology with the lobster cysteine proteases and the human cathepsin L. Control experiments performed with the highly purified American lobster cysteine protease I showed that crustacean cysteine proteases given in vivo to rats induce a fall in the plasma calcium and phosphate levels. This study therefore adds further documentation for a common ancestral origin of CT, CGRP and the much large cysteine proteases from invertebrates.

The endeavour to enhance gene therapy has led to increased research on the development of simple, efficient and safe delivery systems. This study deals with the use of an artificial cationic lipid on the encapsulation of genetic material in liposomes. The addition of a biologically degradable cationic phospholipid, dipalmitoyl-L-alpha-phosphatidylethanolamine covalently coupled to L-lysine, in a standard liposome formulation allowed us to obtain vesicles with high entrapment of various polynucleotides. Polynucleotide degradation by nucleases is markedly prevented by these liposomes. The preparations were stable in both culture medium and human plasma. This latter finding is consistent with the weak binding of plasma proteins on the liposome surface. The efficiency of this new delivery system was demonstrated in antiviral assays. Finally, these liposomes displayed a relatively low cellular toxicity. All these findings indicate that these cationic vesicles are very suitable for genetic material vehiculation.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, synthesizes large amounts of calcitonin (CT), its biological marker. However, in some cases with a poor prognosis, MTC is associated with low basal CT levels owing to a decrease in the thyroid CT content. Using a murine model of human MTC, we studied the relationships between CT biosynthesis, C cell proliferation, and the circulating CT level during MTC progression. Cell proliferation was revealed by autoradiography of radioactive thymidine incorporation in dividing nuclei, after CT or CT mRNA detection by immunocytochemistry (ICC) or in situ hybridization (ISH). All rat thyroids showed a severe hyperplasia of C cells containing significant amounts of CT and CT mRNA, and a very low mitotic index. Tumours were found in 68% of the thyroids. In the strongly immunoreactive small nodules (ICC+), many labelled nuclei were observed. Subsequently some nodular cells, still containing detectable CT mRNA (ISH+), were not detected by immunocytochemistry (ICC-) owing to a dramatic decrease in secretory granules. Their mitotic index increased, and a rise of the basal CT plasma level was noted. These ISH+, ICC- tumour MTC cells represent a modified aggressive tumour C cell population exhibiting an increased ability to proliferate and were detected by the rise in the basal circulating CT level.

In a preliminary study, ewes received daily oral cadmium chloride administrations and cadmium concentration was measured in blood and tissues. A pharmacokinetic analysis of cadmium disposition was then carried out in ewes administered cadmium chloride iv and, 21 months later, orally in the same ewes. Pharmacokinetic parameters were analysed using a 3-compartment open model. The systemic availability was 0.15-0.5%, the half-life of elimination was 100-150 d, the blood clearance was 0.12-0.16 l.kg-1.d-1 and the steady-state volume of distribution was 17-35 l/kg. Following iv administration cadmium was found in tissues about 2 years later.

A pharmacokinetic analysis of cadmium was carried out by administering cadmium chloride i.v. to 3 ewes. Each received 0.033, 0.1 and 0.33 mg cadmium/kg body weight, with each administration separated by 21 d. Pharmacokinetic parameters were determined using a 3-compartment open model (multiple dosage analysis). The half life of elimination reached 40 to 50 d, the body clearance was 0.3 to 0.4 L/kg/d, and the steady-state volume of distribution was 6 to 28 L/kg. The invariability of the values of the elimination parameters and the good fitting of the mathematical model to the experimental values are in agreement with linear kinetics for cadmium in the ewe.

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons alpha and beta (IFN) genes. With L929 and HeLa cells IFN pretreatment (priming) improves the IFN induction by PolyIC by several orders of magnitude. In the absence of the priming we demonstrate that PolyIC encapsulated into pH-sensitive liposomes (and not into pH-insensitive liposomes) enables L929 cells to secrete IFN efficiently and a low toxicity is observed; on primed cells pH-sensitive liposomes containing PolyIC trigger a high toxicity. With HeLa cells, the absence of the priming PolyIC encapsulated into pH-sensitive liposomes induces weak doses of IFN whereas free PolyIC was ineffective. Our experiments established that a pH drop (from 8 to 5.5) provoked a lipid mixing between pH-sensitive liposomes and cell membranes, likely by a fusion mechanism. Entrapment into pH-sensitive liposomes enhances the effect of PolyIC by several orders of magnitude, which might improve its therapeutic ability as an antitumor or anti-HIV agent.

We have recently identified in medullary thyroid carcinoma the existence of a second calcitonin messenger, generated by a splicing between the 3' coding region of exon 4 and exon 5 of Calc I gene. It differs from the first one in its 3' coding sequence and codes for a calcitonin precursor which generates the same N terminal peptide, calcitonin and a specific 21 amino acid carboxy terminal peptide differing from Katacalcin by its 8 last amino acids. We searched for the expression of this new messenger in normal human thyroid tissue by Northern and by polymerase chain reaction techniques. This second calcitonin messenger was expressed in 4/4 normal thyroids and 4/5 medullary thyroid carcinoma tissue samples. The expression of this second messenger is apparently a common occurrence in C cells whether normal or tumoral.

Previous studies led us to demonstrate in pathological situations that the fibroblast, not the macrophage, was the terminal maturation step of the HLA-DR monocyte and that the entire process came under T-lymphocyte control. Fibrosis which developed under immunosuppressive treatment (cyclosporin) after organ transplantation is an illustration of these in vitro observations. The present in vitro study was undertaken in order to investigate whether or not this transformation process takes place under physiological conditions and if so, the nature of the T-lymphocyte control. We report that normal HLA-DR monocytes/macrophages are able to secrete type 1 collagen and to differentiate into neo-fibroblasts. However, contrarily to what happened in pathology, only a few neo-fibroblasts developed transiently. The addition of conditioned medium (CM) from activated T-lymphocytes greatly enhanced the transformation process. Counteracting this CM effect, cell-to-cell contact between neo-fibroblasts and T-cells resulted in the loss of fibroblastic shape. The 'end-result' macrophage engulfed numerous lymphocytes giving rise to a multinucleated cell. This giant cell no longer adhered to the slide and died. The question is raised as to whether the process observed in vitro is involved in vivo in tissue repair. We also report that HLA-DR monocytes and the neo-fibroblasts which derive from them are able to secrete, in addition to type 1 collagen, a variety of proteins such as uromodulin, amyloid-beta peptide, alpha-fetoprotein and carcinoembryonic antigen. In cystic fibrosis we previously reported a high level of uromodulin production by HLA-DR monocytes differentiating towards the fibroblastic phenotype. Pathologies characterized by excessive production of either alpha-feto-protein, carcinoembryonic antigen, beta-amyloid protein (Alzheimer's disease) should be investigated, taking into account the involvement of HLA-DR monocytes and their possible uncontrolled differentiation into neo-fibroblasts.

Calcitonin and insulin are nowadays the most largely prescribed peptide hormones. It is therefore surprising that for some authors the relevant dosage of calcitonin remains a matter of concern. The dose-effect relationship was established twenty years ago, taking into account the kinetic analysis of calcium metabolism and the clinical data, enabling to assess effective dose of calcitonin. Our clinical experience concerning regularly followed-up patients confirms that the replacement dose of calcitonin is necessary and sufficient for managing the disorders of bone and calcium metabolism. Long-term calcitonin replacement treatment: represents a therapeutic approach preserving the integrity and the sensitivity of the hormone receptors, is devoid of the undesirable effects due to pharmacologic doses of the hormone. Therefore the compliance to the treatment is very high. Replacement therapy should become the rule governing the use of calcitonin.

On April 26, 1986 a major nuclear disaster took place at 1 h 24 min local time, destroying the fourth reactor of the Chernobyl plant. Five years later the consequences of the disaster are still not fully known. Nevertheless the long term future of nuclear energy in the world is uncertain. Questions need to be answered by observing hard facts if emotional attitudes are not to prevail over reality. The reactor and its core were destroyed by an explosion, causing two radioactive jet emissions of iodine 131, followed by caesium 137. Both elements are mainly incorporated in the body via food. The Chernobyl disaster was a consequence of inadequate safety regulations and human error. Enforcement of strict regulations are likely to be highly effective in preventing a further catastrophe. However, governments should consider another possibility. What would be the consequences for public health if a terroristic act deliberately destroyed a nuclear power station?

The kinetics of fluoride was studied in a group of 3 adult ewes given sodium fluoride solutions at 3 dose levels (0.15, 0.375 and 0.75 mg/kg bw). Data were analysed using both compartmental and noncompartmental approaches. A 3-compartment open model was selected to describe the data. Comparison of the different parameters indicated an absence of change with dose level, suggesting that fluoride behaved linearly at the doses under study. The half-life of elimination was 2.57 +/- 1.28 h, the steady-state volume of distribution was 0.26 +/- 0.5 L/kg and the body clearance was 0.105 +/- 0.26 L/kg/h. It was concluded that a single intravascular fluoride administration (bolus) may be used to evaluate oral bioavailability of fluoride in sheep in toxicity studies.

The effects of long-term constant infusion of moderate doses (2-32 ng/h) of salmon calcitonin (sCT) on plasma Ca (and its radionuclide 45Ca), Pi, Mg, and on endogenous rat CT (rCT) metabolism were investigated in the rat. Daily variations were included. 1) The plasma concentrations of Ca and Pi fell and that of Mg increased transiently during infusion, with the duration of responses (1-3 days) depending on the sCT dose. Rats infused with 8 ng/h sCT remained sensitive to CT after 7 and 14 days, as indicated by the effects of minipump removal and of a bolus injection of exogenous sCT on plasma mineral concentrations. 2) In contrast to control rats, the well-established daily variations in plasma Ca and Pi levels were no longer observed after 7 and 14 days of sCT infusion (8 ng/h), but normal variations persisted for plasma Mg, circulating rCT, rCT mRNA, and rCT thyroid content. 3) Statistical analysis of plasma mineral data, collected at five sequential times during days 7 and 14, showed that the means were not significantly different and that the daily variations were essentially identical on days 7 and 14 in control rats. In contrast, the variability of measurements for plasma Ca and Pi, but not for Mg, increased significantly between days 7 and 14 in infused rats, and the mean differences were significantly lower in infused rats on day 7 than in control rats. These results are consistent with a transitory loss of the daily variations for Ca and Pi (day 7) and the later (day 14) spontaneous recovery of some variations in these parameters, although the individuals remain unsynchronized.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin (CT) levels in the ultimobranchial body and in plasma were radioimmunoassayed in rainbow trout, Salmo gairdneri, as a function of the annual cycle. In male and female, there was an important increase in the CT levels in the ultimobranchial body and in plasma. These variations in CT levels in both male and female suggest that sexual maturity influences the synthesis and the secretion of calcitonin in fishes. A positive correlation was observed between plasma and ultimobranchial CT levels and the gonadosomatic index in both sexes, suggesting that CT has a role in the processes involved in gonadal development.

Typical sclerotic lesions in the rabbit aorta can be induced by immunisation with k -elastin [15]. These lesions include fragmentation of the elastic fibers and diffuse calcium deposition, accompanied by an increase of the 5M-guanidinium Cl-soluble (structural glycoprotein-containing) fraction of the media, increase of the hexose and hydroxyproline (collagen) content of the lM MgCl 2 -soluble aorta extract (soluble protein and glycoprotein fraction) and guanidinium-soluble hydroxyproline (polymeric collagen). In animals treated simultaneously with calcitonin, the morphological and most of the biochemical modifications produced by k -elastin immunisation were not observed or lessened. There was much less fragmentation of the elastic lamellae and calcium deposition in the media. Calcitonin administration abolished the increases of the guanidinium-soluble protein fraction, of the glyco-proteins in the MgCl 2 -extracts and of the hydroxyproline (collagen) content in the MgCl 2 and guanidinium extracts. Calcitonin alone produced some modifications in the chemical composition of the aorta, such as a decrease in its elastin content, an increase in the hexose and hexosamine content of all extracts. The distribution of calcium salts in the aorta extracts was also modified by calcitonin administration: the calcium content of the MgCl 2 and guanidinium-extracts decreased and that of the final residue (elastin) increased. The results indicate that calcitonin influences the biosynthetic activity of the smooth muscle cells of the media and exerts a protective action against the biochemical and morphological modifications produced by immun-arteriosclerosis.

A numerical method for estimating accurate first derivative, f ′ ( x ) , of a function, f ( x ) is proposed. This methodology leads to the approximate determination of an optimal step which minimizes rounding and truncation errors. The method described here has been applied to different functions and its performance compared with that of classical methods using a proportional step length and that of Dumontet and Vignes. We show that on a large field of application this algorithm tends to associate the efficiency of the optimal step method with the requisite of a moderate number of evaluations.

A nonlinear model of calcium metabolism in the calcium-deficient rat is pro posed. It takes fully into account the experimental time-courses of plasma calcium and its radionuclide concentrations, circadian variations included. Through its mathematical representation (a set offive first-order differential equations incorporating a nonlinearity representing a second-order autocatalysis), calcium metabolism is considered as a self-organised system and its circadian behaviour is dictated mainly by nonlinear processes involved in bone calcification. From our identification procedure, a probably unique set of parameters is obtained and both temporal stability and the time-course of calcium distribution within the model are studied. The model exhibits a stable limit cycle corresponding to two observed characteristics of calcium metabolism: stability and circadian periodicity. Simulated behaviour of different compartments is discussed in relation to calcium deficiency. In conclusion, this investigation represents the first attempt to elucidate a quantitative study of calcium metabolism, visualised as a self-oscillating system, ie, in a non-steady stable state.

"Sensitive" thyrotropin (TSH), thyroglobulin (TG) and even thyrotropin binding inhibiting immunoglobulins (TBII) assays are now widely available. The objective of the present study was to determine the most accurate of these three parameters to predict the relapse of Graves' disease during the year following treatment discontinuation and to evaluate whether the assay of three markers is able to improve the prediction. TSH, TG and TBII were measured in the sera of 67 Graves' disease patients after at least 12 months of medical treatment. In 52 patients, TBII had also been determined before the beginning of the medical treatment. Under treatment, all the patients were clinically and biologically euthyroid, but in 9 goitrous patients it was impossible to lower the doses of carbimazole without an immediate relapse. The TSH levels of these 9 patients were still low in all cases but one; TG and TBII levels were abnormal in all. In the other 58 patients, the treatment was discontinued; 22 relapsed within one year, more frequently when a goiter was present. The most reliable parameter for the prediction of relapse was found to be TBII, as its specificity was high (94.5%), although its sensitivity was poor (45%); TG was more sensitive (64%) but far less specific (57%); TSH and "initial" TBII appeared to be of a little interest. When TBII was elevated prior to the withdrawal of treatment, the determination of TG was useful: abnormal values of both TBII and TG were always associated with a relapse. When TBII testing was negative, the relapse risk fell to 0.26, and to 0.08 when three criteria were matched: no goiter, negative TBII, normal TG.

Our aim is neither to re-evaluate the term homeostasis, nor to summarize the conventional concepts in the field of calcium metabolism and its regulation, nor even to comment on their advantages and their limitations (excellent recent reviews have been published). This paper is rather a position article and references to the current literature will be made only if they contribute to a better understanding of our proposals; in contrast, emphasis will be placed on a literature which has, until now, remained unfamiliar to the field of calcium metabolism. The text is organized around three related features which are largely dictated by the characteristics of our recently published compartmental self-oscillatory model for rat calcium metabolism: (a) The circadian behavior associated with calcium dynamics in vivo may be viewed as a "key" temporal behavior for investigating the spatiotemporal organization of calcium metabolism in the normal rat. Within the bone, a large part of this circadian behavior should stem from the physico-chemical properties of the transformations of calcium-phosphate associations at the extracellular fluid (ECF)/mature bone interface; (b) an important part of the maintenance of a nearly constant plasma calcium concentration (homeostasis) results from interaction between nonlinear oscillators belonging to both calcium metabolism and calcium-regulating hormones. This implies that: firstly, calcium metabolism, like any biological system, is a complex dynamic system which has evolved over a long period and whose metabolic components--gut, kidney, bone--are organized as dynamic entities, adapted to periodic relationships with the external environment. The intrinsic nature of the circadian behavior of bone calcium efflux proposed here is a sufficient demonstration of this. Secondly, the existence of rhythmic variations in the main calcium regulating hormones, parathyroid hormone (PTH), calcitonin (CT) and vitamin D (VitD), are in agreement with this argument. As developed below, fascinating properties emerge from interaction between oscillators (hormones and target organs) which provide a new perspective on calcium regulation; and (c) one of the striking properties of the kind of nonlinear dynamic system required for this representation of calcium metabolism is that periodicity is only one of many temporal expressions. Thus, qualitative diversity in the temporal expression of calcium metabolism can be expected with varying experimental situations and different modes of temporal regulation of calcium metabolism might be physiologically effective, depending on the species studied.(ABSTRACT TRUNCATED AT 400 WORDS)

p-Hydroxyphenyl propionic resin (Table I, compound 4) was used to prepare the 1–6 protected fragment of LH-RH which was then condensed with “BOP” (benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate) as coupling reagent to the 7–10 residue synthesized on the same resin. Peptidylresin was divided into two aliquots in order to obtain LH-RH after aminolysis and treatment with liquid hydrogen fluoride, LH-RH-COOH after saponification followed by hydrogenation, or treatment with liquid hydrogen fluoride. The resulting hormones were rapidly purified by the sole means of two gel filtrations.

We compared results of five sensitive immunometric assays of serum thyrotropin (TSH) in controls and in different groups of patients with hyperthyroidism, untreated or treated; secondary hypothyroidism; nonthyroidal illness (NTI); or depression; or who were being treated with amiodarone. With most kits, measured TSH concentrations did not overlap between controls and hyperthyroid patients. In untreated secondary hypothyroidism TSH was not always undetectable. Patients with NTI and depression showed many low TSH values, and among these categories of patients, we observed large discrepancies among the kits. This lack of specificity at low concentration means that one cannot assess hyperthyroidism by TSH measurement alone, but it can be used as the first screening test. Similarly, TSH determination cannot be used alone in monitoring therapy (e.g., with carbimazole, thyroxin, amiodarone) to assess the presence of hyperthyroidism. Nonetheless, this assay plays a well-established role in hypothyroidism detection. Four of the five kits were found useful for clinical evaluation, the fifth less so.