Cost of exome analysis in patients with intellectual disability: a micro-costing study in a French setting.

BACKGROUND: With the development of next generation sequencing technologies in France, exome sequencing (ES) has recently emerged as an opportunity to improve the diagnosis rate of patients presenting an intellectual disability (ID). To help French policy makers determine an adequate tariff for ES, we aimed to assess the unit cost per ES diagnostic test for ID from the preparation of the pre-analytical step until the report writing step and to identify its main cost drivers. METHODS: A micro-costing bottom-up approach was conducted for the year 2018 in a French setting as part of the DISSEQ study, a cost-effectiveness study funded by the Ministry of Health and performed in collaboration with the GAD (Génétique des Anomalies du Développement), a genetic team from the Dijon University Hospital, and a public sequencing platform, the Centre National de Recherche en Génomique Humaine (CNRGH). The analysis was conducted from the point of view of these two ES stakeholders. All of the resources (labor, equipment, disposables and reagents, reusable material) required to analyze blood samples were identified, collected and valued. Several sensitivity analyses were performed. RESULTS: The unit nominal cost per ES diagnostic test for ID was estimated to be €2,019.39. Labor represented 50.7% of the total cost. The analytical step (from the preparation of libraries to the analysis of sequences) represented 88% of the total cost. Sensitivity analyses suggested that a simultaneous price decrease of 20% for the capture kit and 50% for the sequencing support kit led to an estimation of €1,769 per ES diagnostic test for ID. CONCLUSION: This is the first estimation of ES cost to be done in the French setting of ID diagnosis. The estimation is especially influenced by the price of equipment kits, but more generally by the organization of the centers involved in the different steps of the analysis and the time period in which the study was conducted. This information can now be used to define an adequate tariff and assess the efficiency of ES. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT03287206 on September 19, 2017.

Universal test and treat in relation to HIV disease progression: results from a stepped-wedge trial in Eswatini.

OBJECTIVES: Universal test and treat (UTT) is recommended for people living with HIV (PLHIV) to reduce morbidity/mortality and minimize transmission. However, concerns exist that this strategy may lead to more crowded hospitals, longer wait times and poorer service, adversely impacting health outcomes for clients with severe disease. We assessed how UTT was related to markers of disease progression in PLHIV overall and specifically among clients with low CD4 count/high World Health Organization (WHO) stage. METHODS: The analysis was conducted using data from a stepped-wedge trial of UTT in 14 government-managed health facilities in Eswatini from 2014 to 2017. Disease progression was defined as CD4 count falling below 200 cells/µL or baseline value, > 10% weight loss, body mass index (BMI) dropping below 18.5, incident tuberculosis (TB) or HIV-related death; these outcomes also were assessed individually. We assessed multivariate Cox proportional hazard models overall and specifically among clients with CD4 count < 350 cells/µL or WHO stage 3-4 at enrolment. RESULTS: Eight hundred and seven of 3176 clients demonstrated at least one marker of disease progression over 2339 person-years of follow-up. Overall, 62.4% of clients were female; 57.2% were < 35 years old. Compared to clients not exposed to UTT, those exposed to UTT had a lower rate of disease progression overall [adjusted hazard ratio (aHR) 0.60; 95% confidence interval (CI) 0.46-0.78] and a lower rate of CD4 decline (aHR 0.40; 95% CI 0.27-0.58). When the analysis was limited to clients with CD4 count < 350 cells/µL or WHO stage 3-4, UTT was not associated with disease progression (aHR 0.92; 95% CI 0.66-1.29). CONCLUSIONS: UTT reduced HIV disease progression overall and was not detrimental for clients with more severe disease.

Mortality under early access to antiretroviral therapy vs. Eswatini's national standard of care: the MaxART clustered randomized stepped-wedge trial.

OBJECTIVES: Current WHO guidelines recommend the treatment of all HIV-infected individuals with antiretroviral therapy (ART) to improve survival and quality of life, and decrease infection of others. MaxART is the first implementation trial of this strategy embedded within a government-managed health system, and assesses mortality as a secondary outcome. Because primary findings strongly supported scale-up of the 'treat all' strategy (hereafter Treat All), this analysis examines mortality as an additional indicator of its impact. METHODS: MaxART was conducted in 14 Eswatinian health clinics through a clinic-based stepped-wedge design, by transitioning clinics from then-national standard of care (SoC) to the Treat All intervention. All-cause, disease-related, and HIV-related mortality were analysed using the Cox proportional hazards model, censoring SoC participants at clinic transition. Median follow-up time among study participants was 292 days. There were 36/2034 deaths in SoC (1.77%) and 49/1371 deaths in Treat All (3.57%). RESULTS: Between September 2014 and August 2017, 3405 participants were enrolled. In SoC and Treat All interventions, respectively, the multivariable-adjusted 12-month all-cause mortality rates were 1.42% [95% confidence interval (CI): 0.66-2.17] and 1.60% (95% CI: 0.78-2.40), disease-related mortality rates were 1.02% (95% CI: 0.40-1.64) and 1.10% (95% CI: 0.46-1.73), and HIV-related mortality rates were 1.03% (95% CI: 0.40-1.65) and 0.99% (95% CI: 0.40-1.58). Treat All had no impact on all-cause [hazard ratio (HR) = 1.12, 95% CI: 0.58-2.18, P = 0.73], disease-related (HR = 1.04, 95% CI: 0.52-2.11, P = 0.90), or HIV-related mortality (HR = 0.93, 95% CI: 0.46-1.87, P = 0.83). CONCLUSION: There was no immediate benefit of the Treat All strategy on mortality, nor evidence of harm. Longer follow-up of participants is needed to establish long-term consequences.

[Out of addictions: Alcohol, or alcohol to alcohol]. / Sortir des addictions : à l'alcool, par l'alcool ou vers l'alcool.

Pathways from alcoholism to recovery are documented; less often are those from drug addiction to alcoholism. Biographical approaches allow analyzing how people change their uses and talk about their trajectories of recovery. METHODS: Three hundred and forty-one people (34% women) in the Paris area were questioned on their trajectories with a biographical questionnaire. Some open questions were aimed to understand the connection they made between events in their lives, how recovered they felt and what they considered strengths or obstacles. All the participants had stopped at least one product. Their mean age was 43, and 26% were over 50. STUDY OBJECTIVES: How can the differences between one substance addicts and dual abusers be explained? Can we hypothesize a better result for the patients with a single dependence to alcohol in their lives for the following two reasons? (1) They could really be taken in charge for their alcoholism whereas the dual abusers mostly receive cared for their illicit drug problems with an under estimation of their problem with alcohol. In this case, they turn to alcohol after weaning themselves from their drug dependence so as to return to a social consumption, especially when they are given an opiate treatment. (2) Conversely could we suggest that the dual substance abusers had different trajectories from their childhood (more adverse events, more social difficulties, mental health problems), and that this accumulation explains their skipping from one substance or behaviour to another without any real recovery for decades? RESULTS: All respondents were polydrug users. Eighty-two had been dependent mainly on alcohol. One hundred and twenty-one people had been drug addicts (mostly heroin), which they had stopped on average ten years before the survey. The last group included 138 persons who had been heroin or cocaine addicts and alcoholics in their lives, a third of whom had been dependent on alcohol before their drug addiction (35%), a tenth on both at the same time (10%) and more than half of the users (55%) had turned from drug addiction to alcoholism. The group concerning alcohol dependence includes the oldest participants, on average 49.7, and 55% of them were abstinent at the survey. Conversely, the group "with no alcohol dependence" had mainly turned to opiate treatments. Their histories in dependence and in various social statuses also showed a longer duration out of employment, in sickness or invalidity, or in prison, for the drug dependents as opposed to the "mainly" alcoholics. The population with dual substance abuse experienced twice as many adverse childhood events as the others (P<0.005): it was the case for 19.5% in "mainly alcohol" dependence compared to 38.4% in dual abuse. The recovery capital gave a mean score of 7.56±2.35 (median 7). A score below 6 was considered low. The score was significantly different according to the dependence groups: while 7.3% of "mainly alcohol" dependents had a score below 6, this was the case for 30.4% of the dual group (with alcohol and drugs), and 19% for the "mainly drug dependence" group. Controlling ages, sexes and groups of dependence in a logistic regression, the risk of having a recovery capital below six was more than four times higher for the dual dependents as opposed to the "mainly alcohol" dependents. CONCLUSION: Some people stay for decades in drug addiction centers switching from one dependence to another. Their alcohol drinking should be addressed earlier to prevent them from turning to drinking excessively in order to wean themselves from their drug addiction.

[News Skin lesions : a case report of fixed drug eruption]. / Apparition soudaine de lésions cutanées : à propos d'un cas d'érythème pigmenté fixe.

Fixed drug eruption is an erythematous eruption of one or more centimetric rounded or oval lesions well demarcated, recurrent at the same place and leaving a residual purple pigmentation. Diagnosis is clinical. Skin biopsy is not essential except in doubtful cases (eg bullous drug eruption can simulate Lyell Syndrome or mucosal reminiscent of erythema multiforme). The etiology is almost always drug-induced; rare cases of toxic or food issue were reported. Histopathology is immuno-allergic; recurrences correspond to re-exposure to allergen. There is no specific treatment except stopping the causing drug.

L'érythème pigmenté fixe est une éruption érythémateuse d'une ou plusieurs lésions arrondies ou ovalaires centimétriques bien délimitées, récidivant au même endroit et laissant une pigmentation violacée résiduelle. Le diagnostic est clinique. La biopsie cutanée n'est pas indispensable sauf dans les cas douteux (exemple : érythème pigmenté bulleux pouvant simuler un Syndrome de Lyell ou une atteinte des muqueuses faisant penser à un érythème polymorphe). L'étiologie est presque toujours d'origine médicamenteuse; des rares cas de cause alimentaire ou toxique ont été rapportés. L'histopathologie est donc immuno-allergique ; les récidives correspondent à la réexposition à l'allergène. Il n'existe pas de traitement spécifique hormis l'arrêt du médicament incriminé.

Economic analysis of the costs associated with prematurity from a literature review.

OBJECTIVES: To analyse published cost-of-illness studies that had assessed the cost of prematurity according to gestational age at birth. METHODS: A review of the literature was carried out in March 2011 using the following databases: Medline, ScienceDirect, The Cochrane Library, Econlit and Business Source Premier, and a French Public-Health database. Key-word sequences related to 'prematurity' and 'costs' were considered. Studies that assessed costs according to the gestational age (GA) at the premature birth (<37 weeks of gestation) in industrialized countries and during the last two decades were included. Variations in the reported costs were analysed using a check-list, which allowed the studies to be described according to several methodological and contextual criteria. RESULTS: A total of 18 studies published since 1990 were included. According to these studies, costs were assessed for different follow-up periods (short, medium or long-term), and for different degrees of prematurity (extreme, early, moderate and late). Results showed that whatever the follow-up period, costs correlated inversely with GA. They also showed considerable variability in costs within the same GA group. Differences between studies could be explained by the choices made, concerning i/the study populations, ii/contextual information, iii/and various economic criteria. Despite these variations, a global trend of costs was estimated in the short-term period using mean costs from four American studies that presented similar methodologies. Costs stand at over US$ 100,000 for extreme prematurity, between US$ 40,000 and US$ 100,000 for early prematurity, between US$ 10,000 and US$ 30,000 for moderate prematurity and below US$ 4500 for late prematurity. CONCLUSION: This review underlined not only the clear inverse relationship between costs and GA at birth, but also the difficulty to transfer the results to the French context. It suggests that studies specific to the French health system need to be carried out.

Cost-effectiveness analysis of resorbable biosynthetic mesh in contaminated ventral hernia repair.

BACKGROUND: The aim of this study was to compare, in terms of cost and serious complications, the use of biosynthetic resorbable parietal mesh with biologic mesh in patients undergoing contaminated ventral hernia repair (modified Ventral Hernia Working Group grade 3). Poly-4-hydroxy-butyrate (P4HB) biosynthetic mesh has rarely been the subject of comparative studies in the context of contamination. Data are required to confirm the effects of a transition from biological mesh to biosynthetic resorbable mesh. PATIENTS AND METHODS: A cost-effectiveness analysis was conducted. It was based on a decision analysis model built with clinical and economic data issued from a before-after study that included 94 patients hospitalized for ventral hernia repair at the University Hospital of Strasbourg (France) from June 2011 to February 2018. The effectiveness endpoint was the number of patients presenting with a serious specific complication or a general complication at 6 months. Data for surgical hospitalization stays, home hospitalizations and ambulatory care costs were included. RESULTS: We found fewer serious complications with biosynthetic mesh: 21% versus 33% with biologic mesh. A cost savings of US $5146 was determined. Deterministic sensitivity analyses and a probabilistic analysis confirmed our findings and the robustness of the model. CONCLUSION: P4HB biosynthetic resorbable mesh appeared to be the most effective and the least costly option. Additional data will be needed to confirm the superiority of biosynthetic mesh in terms of the recurrence risk reduction over a longer period.

Incidental findings in a series of 2500 gene panel tests for a genetic predisposition to cancer: Results and impact on patients.

With next generation sequencing, physicians are faced with more complex and uncertain data, particularly incidental findings (IF). Guidelines for the return of IF have been published by learned societies. However, little is known about how patients are affected by these results in a context of oncogenetic testing. Over 4 years, 2500 patients with an indication for genetic testing underwent a gene cancer panel. If an IF was detected, patients were contacted by a physician/genetic counsellor and invited to take part in a semi-structured interview to assess their understanding of the result, the change in medical care, the psychological impact, and the transmission of results to the family. Fourteen patients (0.56%) were delivered an IF in a cancer predisposition gene (RAD51C, PMS2, SDHC, RET, BRCA2, CHEK2, CDKN2A, CDH1, SUFU). Two patients did not collect the results and another two died before the return of results. Within the 10 patients recontacted, most of them reported surprise at the delivery of IF, but not anxiety. The majority felt they had chosen to obtain the result and enough information to understand it. They all initiated the recommended follow-up and did not regret the procedure. Information regarding the IF was transmitted to their offspring but siblings or second-degree relatives were not consistently informed. No major adverse psychological events were found in our experience. IF will be inherent to the development of sequencing, even for restricted gene panels, so it is important to increase our knowledge on the impact of such results in different contexts.

Pharmacological prevention of sevoflurane- and desflurane-related emergence agitation in children: a meta-analysis of published studies.

BACKGROUND: Emergence agitation (EA) in children is increased after sevoflurane anaesthesia. The efficacy of prophylactic treatment is controversial. The aim of this study was to provide a meta-analysis of the studies of the pharmacological prevention of EA in children. METHODS: A comprehensive literature search was conducted to identify clinical trials that focused on the prevention of EA in children anaesthetized with sevoflurane, desflurane, or both. The data from each trial were combined using the Mantel-Haenszel model to calculate the pooled odds ratio (OR) and 95% confidence interval. I(2) statistics were used to assess statistics heterogeneity and the funnel plot and the Begg-Mazumdar test to assess bias. RESULTS: Thirty-seven articles were found which included a total of 1695 patients in the intervention groups and 1477 in the control ones. Midazolam and 5HT(3) inhibitors were not found to have a protective effect against EA [OR=0.88 (0.44, 1.76); OR=0.39 (0.12, 1.31), respectively], whereas propofol [OR=0.21 (0.16, 0.28)], ketamine [OR=0.28 (0.13, 0.60)], alpha(2)-adrenoceptors [OR=0.23 (0.17, 0.33)], fentanyl [OR=0.31 (0.18, 0.56)], and peroperative analgesia [OR=0.15 (0.07, 0.34)] were all found to have a preventive effect. Subgroup analysis according to the peroperative analgesia given does not affect the results. CONCLUSIONS: This meta-analysis found that propofol, ketamine, fentanyl, and preoperative analgesia had a prophylactic effect in preventing EA. The analgesic properties of these drugs do not seem to have a role in this effect.

[Interest of immunohistochemic markers (Ki67, HMB45, p53) in risk analysis of congenital naevi of little and middle size]. / Intérêt des marqueurs immunohistochimiques (Ki67, HMB45, p53) dans l'analyse de risque des naevi congénitaux de petite et moyenne taille.

The risk to develop melanoma from small or medium size congenital naevus remain controversial. The main goal of the present study was to determine the interest of three immunohistochemical markers (Ki67, HMB45 and p53) in predicting malignant transformation of these congenital naevi and to see if a specific immunohistochemical profile of such transformed naevi can be identified. The markers (Ki67, HMB45 and p53) have been used retrospectively on sections of small or medium size congenital naevi (group NC, n = 15), of melanoma developed on small or medium size congenital naevi (group MNC, n = 15) and of melanoma developed on acquired naevi (group MNA, n = 15). The labelled cells have been counted in different cutaneous layers: junction, superficial dermal layer and deep dermal layer. No reactivity was observed for the three markers in group NC. The percentage of labelled cells was significantly different for the three markers between the group NC and the groups MNC and MNA. There was no difference between the groups MNC and MNA. In the groups MNC and MNA, a gradient in the percentage of labelled cells was observed between superficial and deep layers. These three markers do not differentiate melanoma developed from congenital naevi of small or medium size and melanoma developed from acquired naevi. Moreover, the results suggest that these three markers are useless in predicting the risk of malignant transformation of small or medium size congenital naevi.

Secondary findings from whole-exome/genome sequencing evaluating stakeholder perspectives. A review of the literature.

With the development of next generation sequencing, beyond identifying the cause of manifestations that justified prescription of the test, other information with potential interest for patients and their families, defined as secondary findings (SF), can be provided once patients have given informed consent, in particular when therapeutic and preventive options are available. The disclosure of such findings has caused much debate. The aim of this work was to summarize all opinion-based studies focusing on SF, so as to shed light on the concerns that this question generate. A review of the literature was performed, focusing on all PubMed articles reporting qualitative, quantitative or mixed studies that interviewed healthcare providers, participants, or society regarding this subject. The methodology was carefully analysed, in particular whether or not studies made the distinction between actionable and non-actionable SF, in a clinical or research context. From 2010 to 2016, 39 articles were compiled. A total of 14,868 people were interviewed (1259 participants, 6104 healthcare providers, 7505 representatives of society). When actionable and non-actionable SF were distinguished (20 articles), 92% of respondents were keen to have results regarding actionable SF (participants: 88%, healthcare providers: 86%, society: 97%), against 70% (participants: 83%, healthcare providers: 62%, society: 73%) for non-actionable SF. These percentages were slightly lower in the specific situation of children probands. For respondents, the notion of the «patient's choice¼ is crucial. For healthcare providers, the importance of defining policies for SF among diagnostic lab, learning societies and/or countries is outlined, in particular regarding the content and extension of the list of actionable genes to propose, the modalities of information, and the access to information about adult-onset diseases in minors. However, the existing literature should be taken with caution, since most articles lack a clear definition of SF and actionability, and referred to hypothetical scenarios with limited information to respondents. Studies conducted by multidisciplinary teams involving patients with access to results are sadly lacking, in particular in the medium term after the results have been given. Such studies would feed the debate and make it possible to measure the impact of such findings and their benefit-risk ratio.

[Medical practice assessment: an introduction]. / Introduction à l'évaluation des pratiques professionnelles.

Medical practice assessment is mandatory in France. The goal of this article is to explain to perinatal care providers the concept and the process, which do not seem simple, given the multitude of possible ways to evaluate and validate its medical practices. Concrete examples help to illustrate the process. French regulations now link medical practice assessment with continuing medical education (CME) for physicians. While certification is voluntary, a practice assessment conducted during hospital certification processes and during CME is required for all French physicians.

[Pregnancy and psychoactives substances: prevalence study based on the declared consumption]. / Grossesse et substances psychoactives: étude de prévalence de la consommation déclarée.

OBJECTIVES: Evaluate substance use (tobacco, alcohol, psychotropic drugs, illicit drugs) declared before and during pregnancy. PATIENTS AND METHODS: Two hundred and forty-five pregnant women were interviewed through a self-administered and anonymous questionnaire as they were going to a prenatal consultation in a maternity hospital in the Parisian area. RESULTS: Before pregnancy, 16.3% of women reported smoking and 10.2% carried on smoking during pregnancy. Altogether, 40.8% of women reported alcohol consumption before pregnancy; 25.3% of women had contact with alcohol during pregnancy; 4.5% reported tobacco and alcohol consumption during pregnancy. During the month preceding the study, the consumption of psychotropic drugs (hypnotics, antidepressants or sedatives) was reported by 3.7% of women and that of marijuana by 2.4%. Moreover, the marijuana consumers, who tend to drink alcohol more often, combine important social and familial difficulties and represent a high-risk group. CONCLUSION: Tobacco and alcohol use in this study were lower than in any previously conducted French surveys. Methodological specificities and cultural factors might explain those results. However, this is the first study that asses substance use and marijuana use, in particular, by French pregnant women.

Sinusoidal obstruction syndrome/veno-occlusive disease complication in lymphoma patients treated with oxaliplatin-based regimen: A case series report.

AIM: SOS/VOD is a relevant clinical syndrome that usually appears early after hematopoietic stem cell transplantation. The purpose of this article was to report a case series of SOS/VOD in non-susceptible patients and draw physicians' attention to the plausible relationship between liver injury and oxaliplatin-based chemotherapy, preceding autologous transplantation. METHODS: In this study, we report a case series of SOS/VOD in 4 lymphoma patients following autologous transplantation. The data were collected between July 2013 and November 2015 by analyzing patient's characteristics and outcomes. RESULTS: We noticed 4 severe cases of SOS with unusual presentations in patients who did exhibit few classical risk factors. These patients received R-DHAO before transplantation. CONCLUSIONS: Physicians need to be aware that oxaliplatin-based regimen could contribute to SOS/VOD complications in hematological patients.

[Referential of nursing competencies in the Democratic Republic of Congo within the framework of strengthening human resources for health]. / Référentiel de compétences infirmières en RD du Congo dans le cadre du renforcement des ressources humaines en santé.

This article describes the results of a participatory process aiming to design and develop a referential of competencies for nurses to be used in secondary professional training within the context of the global South, and more specifically in the Democratic Republic of Congo (DRC). Priority focus is given to the presentation of the final validated product. The competencies' referential is accompanied by a training referential as a pre-requisite guide, which outlines the course of study. Only the referential of competencies is described in this article. The result documents, in the first instance, the shape which this referential of competencies takes in reality, from which a school nurse can be brought to mobilise while practicing his/her profession. The referential's basis and presentation is founded on four key skill sets as a starting point. Each of the four key skills were further broken down and divided according to levels of competencies and minimal criteria for perfecting these skills per year of study. The presentation of the referential of competencies ends by regrouping "families of situations" for each year as well as with an adapted glossary. The discussion focuses on the results of the referential of competencies in its context but also on the use which should be made of this tool within the framework of the implementation of the education and training reform for nurses in DRC. Another point is the interest of this approach by skills for various types of professional training in the health sciences.

Soluble and particulate inositol 1,4,5-trisphosphate 5-phosphatases show common antigenic determinants.

Inositol 1,4,5-trisphosphate 5-phosphatase catalyses the dephosphorylation of the phosphate in the 5-position from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. One particulate and two soluble enzymes were previously described in bovine brain. In this study, we have obtained a precipitating antiserum against soluble type I inositol 1,4,5-trisphosphate 5-phosphatase. The particulate, but not the soluble type II enzyme, was immunoprecipitated by the serum. Inositol 1,4,5-trisphosphate 5-phosphatase activity from crude extracts of rat brain, human platelets and rat liver were immunoprecipitated by the same antibodies, suggesting the existence of common antigenic determinant among inositol 1,4,5-trisphosphate 5-phosphatases of diverse sources.

Relative contribution of phosphoinositides and phosphatidylcholine hydrolysis to the actions of carbamylcholine, thyrotropin (TSH), and phorbol esters on dog thyroid slices: regulation of cytidine monophosphate-phosphatidic acid accumulation and phospholipase-D activity. I. Actions of carbamylcholine, calcium ionophores, and TSH.

The actions of carbamylcholine (Cchol), the ionophores A23187 and thapsigargin, and TSH on [3H]cytidine monophosphate-phosphatidic acid ([3H]CAMP-PA) accumulation were studied in prelabeled dog thyroid slices to evaluate phosphatidic acid (PA) generation and inositol recycling by phosphatidylinositol (PtdIns) synthesis. The effects of the same agonists were also measured on phosphatidylbutanol generation in [3H]palmitate- or [3H]myristate-prelabeled slices to assess the activity of phospholipase-D (PLD) and on the effluxes of myo-[3H]inositol and [3H]choline induced by these agents from prelabeled slices. Cchol (10(-6)-10(-4) M) increased inositol phosphate (InsP) generation, with no change in inositol efflux, and contracted the intracellular inositol pool. This suggests a stimulation of PtdIns synthesis as well as hydrolysis. The muscarinic agonist provoked a dramatic accumulation of CMP-PA in the presence of lithium chloride (10 mM), which suggests that when InsP hydrolysis is inhibited, inositol limits the rate of CMP-PA incorporation into PtdIns. Cchol also increased phosphatidylbutanol formation. The latter two actions of Cchol were reproduced by A23187 (10(-5) M) and thapsigargin (2 x 10(-6) M) and were inhibited by calphostin-C, an inhibitor of the regulatory site of protein kinase-C. Cchol also induced increased free choline efflux, with a decreased choline phosphate relative content of the medium. TSH (10 mU/ml) stimulated free inositol efflux and induced a slight and proportional increase in [3H]inositol incorporation in phosphoinositides and InsP. The hormone also increased PA and CMP-PA accumulation exclusively in the presence of the PA phosphatase inhibitor propranolol (10(-4) M), but had no detectable action on PLD activity. None of these effects of TSH was reproduced by forskolin or potentiated by lithium chloride (10 mM). The data demonstrate the existence in thyroid tissue of a PLD-hydrolyzing phosphatidylcholine that was stimulated by Cchol and increased intracellular Ca2+, but not by TSH. The results obtained, besides confirming that TSH does not stimulate PtdInsP2-PLC or affect phosphatidylcholine hydrolysis, suggest that the hormone, instead, stimulates de novo PtdIns synthesis and/or inositol transport. The physiological relevance of these actions of Cchol, increased intracellular Ca2+, and TSH in thyroid metabolism could be related to their divergent effects on thyroid cell metabolism.

Relative contribution of phosphoinositides and phosphatidylcholine hydrolysis to the actions of carbamylcholine, thyrotropin, and phorbol esters on dog thyroid slices: regulation of cytidine monophosphate-phosphatidic acid accumulation and phospholipase-D activity. II. Actions of phorbol esters.

The effects of phorbol dibutyrate (PDBu) on phosphatidylbutanol (PtdBut) generation in [3H]palmitate- or [3H]myristate-prelabeled dog thyroid slices were measured to assess the activity of phospholipase-D (PLD) in the presence or absence of the two inhibitors of protein kinase-C (PKC), staurosporine (STSP) and calphostin-C. The actions of the same agents on [3H]cytidine monophosphate-phosphatidic acid accumulation were also determined to evaluate phosphatidic (PA) generation and inositol recycling to phosphatidylinositol. The effluxes of [3H]choline and [3H]ethanolamine induced by the phorbol ester from prelabeled slices were also evaluated. PDBu (5 x 10(-9) to 5 x 10(-6) M) potently stimulated PLD activity, with a concomitant increase in fatty acids incorporation in phosphatidylcholine (PtdCho). However, under no condition did the phorbol ester result in cytidine monophosphate-phosphatidic acid accumulation. It stimulated the efflux of choline and ethanolamine while decreasing choline and ethanolamine phosphates in the slices and incubation medium. Calphostin-C, inhibiting PKC, decreased PtdBut and PtdCho formation induced by the phorbol ester, as opposed to STSP (5 x 10(-6) M), which did not affect these actions of PDBu and, moreover, reproduced by itself the effects of the phorbol ester on choline efflux and PtdBut generation despite efficient inhibition of other effects of PKC. These data demonstrate the existence in thyroid tissue of a PLD-hydrolyzing PtdCho, which was stimulated by phorbol esters and STSP. They also suggest that the PA formed after PKC stimulation and subsequent PLD activation is channeled toward PtdCho resynthesis when intracellular Ca2+ is not increased, whereas the PA accumulated with a concomitant increase in intracellular Ca2+ is diverted toward phosphatidylinositol synthesis. The physiological relevance of this Ca-independent stimulation of a PKC-coupled PLD in thyroid metabolism could be related to the growth-inducing and dedifferentiating effects of the phorbol esters.

Regulation and metabolic role of phospholipase D activity in human thyroid and cultured dog thyrocytes.

The actions of TSH, ATP, the ionophore A23187, the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin, and phorbol dibutyrate (PDBu) on 3H-cytidine-monophosphate phosphatidic acid (3H-CMP-PA) accumulation were studied in human thyroid slices to evaluate PA generation and inositol recycling towards phosphatidyl-inositol synthesis. The effects of the same agonists also were measured on phosphatidylbutanol (PtdBut) generation in 3H-palmitate or 3H-myristate prelabeled slices to assess the activity of phospholipase D (PLD). The phospholipid target of this PLD was determined on 3H-choline prelabeled human thyroid slices by measuring 3H-choline release in incubation medium and slices and 3H-choline incorporation in phospholipids. TSH (10 U/L) stimulated 3H-CMP-PA accumulation in an LiCl-and propranolol-insensitive way, as well as 2H-fatty acids incorporation into PA, diacylglycerol, and phosphatidylcholine (PtdCho) with on evidence of dose-dependent effects and had no detectable action on PLD activity. The effects of TSH were not reproduced by Bu2cAMP or forskolin. Thapsigargin and A23187 both increased CMP-PA accumulation and PtdBut generation, whereas ATP only stimulated PLD activity. The phorbol ester PDBu (5 x 10(-7) mol/L) increased PtdBut formation and 3-H-fatty acid incorporation into PtdCho, but had no effect on CMP-PA generation. Staurosporine (STSP) (5 x 10(-6) mol/L), a nonspecific inhibitor of protein kinase C, unexpectedly reproduced the effects of PDBu. The increase of 3H-choline in slices' supernatant and the decrease of 3H-choline-labeled PtdCho induced by PDBu, ATP, thapsigargin, and STSP indicate that the activated PLD hydrolyzed PtdCho. We suggest that the PA generation induced by PLD stimulation could contribute to the stimulated H2O2 formation and iodide organification observed with the agonists inducing PtdBut accumulation. Indeed, Bu2cAMP and forskolin, known to decrease iodide organification in human thyroid, inhibited the PLD stimulation induced by ATP and PDBu. In cultured dog thyrocytes, phorbol esters, and STSP induced DNA synthesis and dedifferentiation, whereas thapsigargin inhibited TSH-induced growth and killed phorbol esters stimulated cells, suggesting a positive role of PLD stimulation towards dedifferentiated growth and of simultaneously raised [Ca2+)i and stimulated protein kinase C-PLD towards growth arrest and cellular death.

Pharmacokinetics of folinic acid and 5-methyltetrahydrofolic metabolite after repeated oral administration of calcium folinate following methotrexate treatment.

The pharmacokinetic profiles of folinic acid (FA) and its active metabolite, 5-methyltetrahydrofolic acid, were studied after oral administration of decreasing doses of calcium folinate during 37 courses of high and intermediate dose methotrexate treatment in 25 lymphoma patients. FA was administered at a dose of 6 x 50 mg in 15 courses, 6 x 25 mg in seven courses, 6 x 15 mg in 10 courses and 6 x 7.5 mg in 5 courses. FA, 5-methyltetrahydrofolic acid, methotrexate and 70H-methotrexate were assayed simultaneously by high performance liquid chromatography. When FA was administered at doses between 50 and 15 mg, maximum concentrations of both the drug and its metabolite were always obtained after 1 to 2 h and remained stable. The same was true for the equilibrium concentration of the two products at doses over 15 mg. These findings suggest saturation of absorption and metabolism of folinic acid at doses over 15 mg.