25-hydroxyvitamin D sufficiency is associated with lower de novo anti-HLA donor specific antibody and better kidney transplant outcomes.

T-cell mediated rejection (TCMR), de novo anti-HLA donor-specific antibodies (dnDSAs) and ensuing antibody-mediated rejection (ABMR) reduce kidney transplantation (KT) survival. The immunomodulatory effects of 25-hydroxyvitamin D [25(OH)D] could be beneficial for KT outcomes. We aimed to evaluating the association between 25(OH)D levels, the development of dnDSAs, clinical TCMR and ABMR, and graft survival. This single center retrospective study included 253 KT recipients (KTRs) transplanted without preformed DSA between 2010 and 2013. We measured 25(OH)D in successive serum samples: at KT (M0) and M12 for the entire cohort, and additionally at M24 and/or M36 when sera were available. We assessed graft outcomes up to 5 years post-KT. The proportion of KTRs having sufficient 25(OH)D at KT (M0) was high (81.4%) and then dropped at M12 (71.1%). KTRs with sufficient 25(OH)D at M0 experienced less clinical TCMR (HR, 0.41; 95% CI, 0.19-0.88 in multivariate analysis). A sufficient 25(OH)D at M12 was independently associated with a longer dnDSA-free survival (HR, 0.34; 95% CI, 0.17-0.69). There was no association between 25(OH)D and clinical AMBR. Studying the KTRs with 25(OH)D measurements at M12, M24 and M36 (n = 203), we showed that 25(OH)D sufficiency over the 3 first-years post-KT was associated with a longer graft survival in multivariate analyses (HR, 0.39; 95% CI, 0.22-0.70). To our knowledge, this study is the first showing an association between 25(OH)D sufficiency post-KT and dnDSA occurrence in KTRs. Moreover, we reinforce previously published data showing an association between 25(OH)D, TCMR and graft survival in KT.

Acute Effect of a Saffron Extract (Safr'InsideTM) and Its Main Volatile Compound on the Stress Response in Healthy Young Men: A Randomized, Double Blind, Placebo-Controlled, Crossover Study.

According to animal studies, saffron and its main volatile compound safranal may reduce biological and behavioral signs of acute stress. However, little is known about its impact in humans. This study investigated the acute effect of a saffron extract and safranal on the biological and psychological stress responses in healthy men experiencing a laboratory stress procedure. In this double-blind, placebo-controlled, randomized, cross-over study, 19 volunteers aged 18-25 received a single dose of 30 mg saffron extract (Safr'InsideTM), 0.06 mg synthetic safranal, or a placebo on three visits separated by a 28-day washout. Thirteen minutes after administration, participants were exposed to the Maastricht acute stress test (MAST). Salivary cortisol and cortisone were collected from 15 min before the MAST (and pre-dose), 3 min before the MAST, and then 15, 30, 45, 60, and 75 min after the MAST, and stress and anxiety were measured using visual analogic scales. Compared to the placebo, stress and anxiety were significantly toned down after Safranal and Safr'InsideTM administration and coupled with a delay in the times to peak salivary cortisol and cortisone concentrations (p < 0.05). Safr'InsideTM and its volatile compound seem to improve psychological stress response in healthy men after exposure to a lab-based stressor and may modulate the biological stress response.

Memory deficits in a juvenile rat model of type 1 diabetes are due to excess 11ß-HSD1 activity, which is upregulated by high glucose concentrations rather than insulin deficiency.

AIMS/HYPOTHESIS: Children with diabetes may display cognitive alterations although vascular disorders have not yet appeared. Variations in glucose levels together with relative insulin deficiency in treated type 1 diabetes have been reported to impact brain function indirectly through dysregulation of the hypothalamus-pituitary-adrenal axis. We have recently shown that enhancement of glucocorticoid levels in children with type 1 diabetes is dependent not only on glucocorticoid secretion but also on glucocorticoid tissue concentrations, which is linked to 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity. Hypothalamus-pituitary-adrenal axis dysfunction and memory alteration were further dissected in a juvenile rat model of diabetes showing that excess 11ß-HSD1 activity within the hippocampus is associated with hippocampal-dependent memory deficits. Here, to investigate the causal relationships between diabetes, 11ß-HSD1 activity and hippocampus-dependent memory deficits, we evaluated the beneficial effect of 11ß-HSD1 inhibition on hippocampal-related memory in juvenile diabetic rats. We also examined whether diabetes-associated enhancement of hippocampal 11ß-HSD1 activity is due to an increase in brain glucose concentrations and/or a decrease in insulin signalling. METHODS: Diabetes was induced in juvenile rats by daily i.p. injection of streptozotocin for 2 consecutive days. Inhibition of 11ß-HSD1 was obtained by administrating the compound UE2316 twice daily by gavage for 3 weeks, after which hippocampal-dependent object location memory was assessed. Hippocampal 11ß-HSD1 activity was estimated by the ratio of corticosterone/dehydrocorticosterone measured by LC/MS. Regulation of 11ß-HSD1 activity in response to changes in glucose or insulin levels was determined ex vivo on acute brain hippocampal slices. The insulin regulation of 11ß-HSD1 was further examined in vivo using virally mediated knockdown of insulin receptor expression specifically in the hippocampus. RESULTS: Our data show that inhibiting 11ß-HSD1 activity prevents hippocampal-related memory deficits in diabetic juvenile rats. A significant increase (53.0±9.9%) in hippocampal 11ß-HSD1 activity was found in hippocampal slices incubated in high glucose conditions (13.9 mmol/l) vs normal glucose conditions (2.8 mmol/l) without insulin. However, 11ß-HSD1 activity was not affected by variations in insulin concentration either in the hippocampal slices or after a decrease in hippocampal insulin receptor expression. CONCLUSIONS/INTERPRETATION: Together, these data demonstrate that an increase in 11ß-HSD1 activity contributes to memory deficits observed in juvenile diabetic rats and that an excess of hippocampal 11ß-HSD1 activity stems from high glucose levels rather than insulin deficiency. 11ß-HSD1 might be a therapeutic target for treating cognitive impairments associated with diabetes.

Consensus statement by the French Society of Endocrinology (SFE) and French Society of Pediatric Endocrinology & Diabetology (SFEDP) on diagnosis of Cushing's syndrome.

Cushing's syndrome is defined by prolonged exposure to glucocorticoids, leading to excess morbidity and mortality. Diagnosis of this rare pathology is difficult due to the low specificity of the clinical signs, the variable severity of the clinical presentation, and the difficulties of interpretation associated with the diagnostic methods. The present consensus paper by 38 experts of the French Society of Endocrinology and the French Society of Pediatric Endocrinology and Diabetology aimed firstly to detail the circumstances suggesting diagnosis and the biologic diagnosis tools and their interpretation for positive diagnosis and for etiologic diagnosis according to ACTH-independent and -dependent mechanisms. Secondly, situations making diagnosis complex (pregnancy, intense hypercortisolism, fluctuating Cushing's syndrome, pediatric forms and genetically determined forms) were detailed. Lastly, methods of surveillance and diagnosis of recurrence were dealt with in the final section.

Altered Cortisol Metabolism Increases Nocturnal Cortisol Bioavailability in Prepubertal Children With Type 1 Diabetes Mellitus.

Objective: Disturbances in the activity of the hypothalamus-pituitary-adrenal axis could lead to functional alterations in the brain of diabetes patients. In a later perspective of investigating the link between the activity of the hypothalamus-pituitary-adrenal axis and the developing brain in children with diabetes, we assessed here nocturnal cortisol metabolism in prepubertal children with type 1 diabetes mellitus (T1DM). Methods: Prepubertal patients (aged 6-12 years) diagnosed with T1DM at least 1 year previously were recruited, along with matched controls. Nocturnal urine samples were collected, with saliva samples taken at awakening and 30 minutes after awakening. All samples were collected at home over 5 consecutive days with no detectable nocturnal hypoglycaemia. The State-Trait Anxiety Inventory (trait scale only) and Child Depression Inventory were also completed. Glucocorticoid metabolites in the urine, salivary cortisol (sF) and cortisone (sE) were measured by liquid chromatography-tandem mass spectrometry. Metabolic data were analysed by logistic regression, adjusting for sex, age, BMI and trait anxiety score. Results: Urine glucocorticoid metabolites were significantly lower in T1DM patients compared to controls. 11ß-hydroxysteroid dehydrogenase type 1 activity was significantly higher, while 11ß-hydroxysteroid dehydrogenase type 2, 5(α+ß)-reductase and 5α-reductase levels were all lower, in T1DM patients compared to controls. There was a significant group difference in delta sE level but not in delta sF level between the time of awakening and 30 minutes thereafter. Conclusions: Our findings suggest that altered nocturnal cortisol metabolism and morning HPA axis hyperactivity in children with T1DM leads to greater cortisol bioavailability and lower cortisol production as a compensatory effect. This altered nocturnal glucocorticoid metabolism when cortisol production is physiologically reduced and this HPA axis hyperactivity question their impact on brain functioning.

[About a case of macro-FSH analytical interference]. / Une grosse FSH qui trompe énormément : à propos d'un cas de macro-FSH.

An 18-year-old woman was referred by her GP to the endocrinology department of the University Hospital of Bordeaux on suspicion of premature ovarian failure because of a disorder of the menstrual cycle and pathological results of biological exploration of the gonadotropic axis. Repeatedly-found elevated concentrations of FSH contrasted with a normal concentration of LH leading to a hypothesis of ovarian failure. However, different investigations favoured an analytical interference. The presence of heterophilic antibodies or anti-mouse antibodies (HAMA) was unlikely but, finally, a complex combining FSH and autoantibody (called macro-FSH) was evidenced.

Hair cortisol and cortisone measurements for the diagnosis of overt and mild Cushing's syndrome.

OBJECTIVE: Hair cortisol (HF) and cortisone (HE) measurements reflect tissular exposure to cortisol over months and are increased in overt Cushing's syndrome (CS). No data is available in mild CS. We compared the diagnostic performance of HF and HE between patients with overt or mild CS. DESIGN: Single centre retrospective study. METHODS: HF&HE were measured by LC-MS/MS in 48 consecutive adult females with Cushing's disease (CD), ectopic ACTH syndrome, secreting adenomas and carcinomas, and adrenal incidentalomas. All had impaired dexamethasone suppression tests. Overt CS (n = 25) was diagnosed in front of specific symptoms, a mean UFC (>1.5 ULN) and increased midnight serum cortisol or salivary cortisol. Mild CS (n = 23) was diagnosed in patients lacking specific symptoms and displaying at least one additional biological abnormality including mildly increased UFC (≤1.5 ULN), increased midnight serum cortisol or salivary cortisol and suppressed plasma ACTH in patients with adrenal tumours. In this study, 84 healthy subjects and obese patients served as controls. RESULTS: HF and HE showed roughly similar performance in overt CS (92 and 100% sensitivity, 91 and 99% specificity, respectively). HF and HE were lower in mild CS but higher than in controls (P < 0.01). HE was correlated with midnight serum cortisol (P < 0.02) and volume of adrenal incidentalomas (P < 0.04) but not with UFC. HF and HE had 59% and 68% sensitivity, and 79 and 94% specificity, respectively, for the diagnosis of mild CS. Contrary to UFC, both HF and HE were in the range of overt CS in 11/23 patients with mild CS. Patients with mild CS and increased HE required more antihypertensive treatments and showed worser lipid profiles than patients with normal HE. CONCLUSIONS: HF and HE measurement performed better in overt than in mild CS but is a useful adjunct to diagnose mild CS and to identify adrenocortical incidentalomas responsible for excessive cortisol exposure.

Non-invasive Diagnostic Strategy in ACTH-dependent Cushing's Syndrome.

CONTEXT: Inferior petrosal sinus sampling (IPSS) is used to diagnose Cushing's disease (CD) when dexamethasone-suppression and CRH tests, and pituitary magnetic resonance imaging (MRI), are negative or give discordant results. However, IPSS is an invasive procedure and its availability is limited. OBJECTIVE: To test a noninvasive diagnostic strategy associated with 100% positive predictive value (PPV) for CD. DESIGN: Retrospective study. SETTING: Two university hospitals. PATIENTS: A total of 167 patients with CD and 27 patients with ectopic ACTH-syndrome investigated between 2001 and 2016. MAIN OUTCOME MEASURE(S): Performance of a strategy involving the CRH and desmopressin tests with pituitary MRI followed by thin-slice whole-body computed tomography (CT) scan in patients with inconclusive results. RESULTS: Using thresholds of a cortisol increase > 17% with an ACTH increase > 37% during the CRH test and a cortisol increase > 18% with an ACTH increase > 33% during the desmopressin test, the combination of both tests gave 73% sensitivity and 98% PPV of CD. The sensitivity and PPV for pituitary MRI were 71% and 99%, respectively. CT scan identified 67% EAS at presentation with no false-positives. The PPV for CD was 100% in patients with positive responses to both tests, with negative pituitary MRI and CT scan. The Negative Predictive Value was 100% in patients with negative responses to both tests, with negative pituitary MRI and positive CT scan. Using this strategy, IPPS could have been avoided in 47% of patients in whom it is currently recommended. CONCLUSIONS: In conjunction with expert radiologic interpretation, the non-invasive algorithm studied significantly reduces the need for IPSS in the investigation of ACTH-dependent Cushing's syndrome.

What do you need to know about mass spectrometry? A brief guide for endocrinologists.

In routine hormonology, liquid chromatography mass spectrometry (LCMS) is now an established technique for androgen, urinary cortisol and metanephrine assay. It has the undeniable advantage of great analytical specificity, but with sensitivity that clearly depends on financial investment in a very high-end spectrometer. We describe the general principles of LCMS and the routine applications so far developed in hormonology. The purpose is to familiarise endocrinologists with the techniques under development and their pros and cons.

[What to do in the event of a suspicion of analytical interference during an immunoassay?] / Conduite à tenir devant une suspicion d'interférence analytique lors d'un immunodosage.

Identifying analytical interference is a challenge for the medical biologist in providing advice to the prescriber. Indeed, these analytical interferences often have deleterious consequences on the care of patients. Understanding their mechanisms and mastering corrective procedures is essential to limit these management errors. Faced with the many questions from clinicians in current practice, we propose an algorithm for managing a sample when interference is suspected.

Effects of Saffron Extract Supplementation on Mood, Well-Being, and Response to a Psychosocial Stressor in Healthy Adults: A Randomized, Double-Blind, Parallel Group, Clinical Trial.

Anxiety, stress, and low mood are closely related and may contribute to depressive symptoms. Among non-pharmacological solutions to improve subclinical mood symptoms and resilience to stress, natural products such as saffron-identified as promising following preliminary beneficial effects in major depressive disorder-represent a relevant strategy. This study aimed to assess the efficacy of 8 weeks' supplementation with 30 mg standardized saffron extract on emotional well-being in healthy adults with subclinical feelings of low mood and anxiety and/or stress and evaluate the acute effect of saffron in response to a lab-based psychosocial stressor. The study adopted a double-blind, randomized, parallel groups design in which 56 healthy male and female individuals (18-54 years) received either a saffron extract or a placebo for 8 weeks. Chronic effects of saffron on subjective anxiety, stress, and depressive feelings were assessed using a questionnaire battery [including Profile of Mood State-2, (POMS)] and acute effects in response to a lab-based psychosocial stressor were measured through psychological and physiological parameters. Urinary crocetin levels were quantified. Participants who received the saffron extract reported reduced depression scores and improved social relationships at the end of the study. Urinary crocetin levels increased significantly with saffron supplementation and were correlated with change in depression scores. The typical stress-induced decrease in heart rate variability (HRV) during exposure to the stressor was attenuated following acute saffron intake. Saffron extract appears to improve subclinical depressive symptoms in healthy individuals and may contribute to increased resilience against the development of stress-related psychiatric disorders. Clinical trials number: NCT03639831.

Hormonal Status and Cognitivo-Emotional Profile in Real-Life Patients With Neuropathic Pain: A Case Control Study.

BACKGROUND: The specific impact of neuropathic pain and recommended neuropathic pain treatments on the hormonal and immune status of patients has been so far poorly explored. This study aimed at studying, in real life, the hypothalamic-pituitary-adrenal axis and the cytokine profile of patients with neuropathic pain. It also explored their links with cognition, emotion, quality of life, and drug treatment. METHODS: This prospective study (clinicaltrials.gov NCT01543425) included 60 patients with neuropathic pain and 60 age- and gender-matched healthy volunteers after obtaining signatures of informed consent. A number of parameters were measured: adrenocorticotropic hormone, cortisol, cortisol awakening response, dehydroepiandrosterone sulphate, sex hormone binding globulin, testosterone, 17-ß-estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, cytokines, brain-derived neurotrophic factor, and vitamin D. Psychological parameters were assessed by questionnaires. RESULTS: Patients with neuropathic pain had lower levels of adrenocorticotropic hormone (P = 0.009) and dehydroepiandrosterone sulphate (P < 0.001) than controls, and the cortisol awakening response was impaired. Patients were more depressed and anxious (P < 0.001) and had a diminished quality of life (P < 0.001), which was influenced by cytokines (P = 0.0067) and testosterone (P = 0.028). Antidepressants and antiepileptics appeared to interfere with testosterone and cognitivo-emotional domains. CONCLUSION: An impairment of the hormonal status and of the immune system was observed in patients. It identified testosterone as a potential pivotal mediator between antidepressants/antiepileptics and quality of life. Further studies must address the exact impact of different types of drugs on central effects, of gender differences, and of the immune system of neuropathic pain.

Immunotherapy-induced hypothyroidism A report of melanoma treated by ipilimumab and nivolumab.

We report the case of a patient treated by ipilimumab and nivolumab for a metastatic melanoma. After a mild clinical thyroiditis and a transient biological hyperthyroidism she rapidly demonstrated a peripheral hypothyroidism with appearance of antibodies against thyroperoxidase and thyroglobulin.

Insulin treatment partially prevents cognitive and hippocampal alterations as well as glucocorticoid dysregulation in early-onset insulin-deficient diabetic rats.

The diagnosis of Type 1 Diabetes (T1D) in ever younger children led us to question the impact of insulin deficiency or chronic hyperglycemia on cerebral development and memory performances. Here, we sought abnormalities in these traits in a model of streptozotocin-induced diabetes in juvenile rats treated or not by insulin. We made the assumption that such alterations would be related, at least in part, to excessive glucocorticoid exposition in hippocampal neurons. We have compared 3 groups of juvenile rats: controls, untreated diabetics and insulin-treated diabetics. Diabetes was induced by streptozotocin (65 mg/kg IP/day, 2 consecutive days), at postnatal days 21 and 22 and a subcutaneous pellet delivering 2 U of insulin/day was implanted in treated diabetic rats 3 days later. Three weeks after diabetes induction, cognitive performances (Y maze, object location and recognition tests), in vivo brain structure (brain volume and water diffusion by structural magnetic resonance imaging), and hippocampal neurogenesis (immunohistochemical labeling) measurements were undertaken. Corticosterone levels were evaluated in plasma under basal and stress conditions, and within hippocampus together with 11ß-dehydrocorticosterone to assess 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) activity. The comparison of the three experimental groups revealed that, compared to controls, untreated diabetic rats showed decreased cognitive performances in Y-maze and object location test (p < 0.05), decreased brain and hippocampal microstructure (p < 0.05), and decreased maturation and survival of hippocampal newborn neurons (p < 0.05). These alterations were associated with increased plasma corticosterone at the baseline nadir of its secretion (p < 0.001) and during the recovery phase following a restraint stress (p < 0.001), as well as increased hippocampal corticosterone levels (p < 0.01) and 11ß-HSD1 activity (p < 0.05). As untreated diabetic rats, insulin-treated diabetic rats displayed decreased brain volume and water diffusion (p < 0.05 compared to controls) and intermediate memory performances and hippocampal neurogenesis (p value not significant compared to either controls or untreated diabetics). Moreover, they were similar to controls for basal plasma and hippocampal corticosterone and 11ß-HSD1 activity but show increased plasma corticosterone during the recovery phase following a restraint stress similar to untreated diabetics (p < 0.001 compared to controls). Thus, insulin did not completely prevent several hippocampal-dependent behavioral and structural alterations induced by diabetes in juvenile rats which may relate to the higher cognitive difficulties encountered in T1D children compared to non-diabetic controls. Although insulin restored basal corticosterone and 11ß-HSD1 activity (in hippocampus and plasma), the negative feedback regulation of corticosterone secretion after stress was still impaired in insulin-treated diabetic rats. Further characterization of insulin control on glucocorticoid regulation and availability within hippocampus is awaited.

LC-MSMS assays of urinary cortisol, a comparison between four in-house assays.

BACKGROUND: Twenty-four hour urinary free cortisol (UFC) determination can be used for screening and follow-up of Cushing syndrome (CS). As immunoassay methods lack specificity for UFC measurement, the use of high-performance liquid chromatography coupled to mass spectrometer (LC-MSMS) is recommended. The aim of our study was to compare UFC results using four LC-MSMS methods performed in four independent laboratories in order to evaluate interlaboratory agreement. METHODS: Frozen aliquots of 24-h urine samples (78 healthy volunteers and 20 patients with CS) were sent to four different laboratories for analysis. Following liquid-liquid or solid-liquid extraction, UFC were determined using four different LC-MSMS assay. RESULTS: UFC intra- and interassays variation coefficients were lower than 10% for each centre. External quality control results were not significantly different. UFC normal ranges (established from healthy volunteers) were 17-126, 15-134, 12-118 and 27-157 nmol/day, respectively. Classification of UFC from healthy volunteers and patients with CS using a 95th percentile threshold was similar. However, for extreme UFC values (<50 or >270 nmol/day), negative or positive bias was noted. CONCLUSIONS: Even for highly specific methods such as LC-MSMS, variations of results can be found depending on analytical process. Validation of LC-MSMS methods including determination of the reference range is essential.

Investigating the association of vitamin D with blood pressure and the renin-angiotensin-aldosterone system in hypertensive subjects: a cross-sectional prospective study.

The hypothesis that vitamin D (25(OH)D) insufficiency plays a role in occurring of various disease has led to a rise in requests of dosages and to an increase of health-care costs. 25(OH)D insufficiency is associated with increased risk of cardiovascular disease and hypertension in many studies. Animal studies demonstrated that 25(OH)D insufficiency activates renin angiotensin system but corresponding humans data are limited. The aim of the study was to document relationship between 25(OH)D, blood pressure, and renin angiotensin system in hypertensive subjects. In all, 248 hypertensive individuals, 46.8 years (±14), were hospitalized for an etiological assessment of hypertension in this cross-sectional study over two calendar years. 25(OH)D, plasma renin activity, and aldosterone were determined in stringent conditions and blood pressure was measure. Statistical analyses were carried out to analyze the association between 25(OH)D, blood pressure, and renin angiotensin system using linear and logistic regressions with adjustments on relevant variables. In all, 80% of the studied population had a 25(OH)D insufficiency. There were no significant association between 25(OH)D and levels of systolic or diastolic blood pressure, plasma renin activity, and aldosterone whatever the statiscal method used after adjustment. 25(OH)D is not associated with blood pressure and renin angiontensin component in hypertensive subjects. These results corroborate the interventional studies which are for a large majority negatives. A new definition of the 25(OH)D insufficiency in general population is necessary.

Vitamin A, endocrine tissues and hormones: interplay and interactions.

Vitamin A (retinol) is a micronutrient critical for cell proliferation and differentiation. In adults, vitamin A and metabolites such as retinoic acid (RA) play major roles in vision, immune and brain functions, and tissue remodelling and metabolism. This review presents the physiological interactions of retinoids and endocrine tissues and hormonal systems. Two endocrine systems have been particularly studied. In the pituitary, retinoids targets the corticotrophs with a possible therapeutic use in corticotropinomas. In the thyroid, retinoids interfere with iodine metabolism and vitamin A deficiency aggravates thyroid dysfunction caused by iodine-deficient diets. Retinoids use in thyroid cancer appears less promising than expected. Recent and still controversial studies investigated the relations between retinoids and metabolic syndrome. Indeed, retinoids contribute to pancreatic development and modify fat and glucose metabolism. However, more detailed studies are needed before planning any therapeutic use. Finally, retinoids probably play more minor roles in adrenal and gonads development and function apart from their major effects on spermatogenesis.

Urinary sampling for 5HIAA and metanephrines determination: revisiting the recommendations.

CONTEXT: Biogenic amines such as 5-hydroxy-indole acetic acid (5HIAA) the main metabolite of serotonin or metanephrines (catecholamines metabolites) are used as biomarkers of neuroendocrine tumours. OBJECTIVE: To re-evaluate the recommendations for urinary sampling (preservatives, diet, drugs, etc.) as many of the reported analytical interferences supporting these recommendations are related to obsolete assays. METHODS: Bibliographic analysis of old and modern assays concerning preservation, extraction, assay and interferences. RESULTS: 5HIAA may degrade as soon as urine is excreted. Thus, acids as preservatives (hydrochloric or acetic acid) have to be immediately added. Care should be taken not to decrease the pH under 2. Urine preservative for metanephrine assays is not mandatory. Diets including serotonin-, tryptophan- and dopamine-rich foods have to be avoided depending on the biomarkers investigated (bananas, plantain, nuts, etc.). Tryptophan-rich over-the-counter formulas have to be prohibited when 5HIAA has to be assayed. Acetaminophen may interfere with electrochemical detection depending on high-pressure liquid chromatography (HPLC) parameters. No interference is known with mass spectrometric assays but with the one described for metanephrines determination. Some drugs interfere however with serotonin and catecholamines secretion and/or metabolism (monoamine oxidase inhibitors, serotonin or dopamine recapture inhibitors, etc.). CONCLUSION: Revisited recommendations are provided for the diet, the drugs and the preservatives before HPLC coupled with electrochemical and mass spectrometry assays.

Nasal airway epithelial cell IL-6 and FKBP51 gene expression and steroid sensitivity in asthmatic children.

BACKGROUND: Many asthmatic patients exhibit uncontrolled asthma despite high-dose inhaled corticosteroids (ICS). Airway epithelial cells (AEC) have distinct activation profiles that can influence ICS response. OBJECTIVES: A pilot study to identify gene expression markers of AEC dysfunction and markers of corticosteroid sensitivity in asthmatic and non-asthmatic control children, for comparison with published reports in adults. METHODS: AEC were obtained by nasal brushings and primary submerged cultures, and incubated in control conditions or in the presence of 10 ng/ml TNFalpha, 10-8M dexamethasone, or both. RT-PCR-based expression of FKBP51 (a steroid hormone receptor signalling regulator), NF-kB, IL-6, LIF (an IL-6 family neurotrophic cytokine), serpinB2 (which inhibits plasminogen activation and promotes fibrin deposition) and porin (a marker of mitochondrial mass) were determined. RESULTS: 6 patients without asthma (median age 11yr; min-max: 7-13), 8 with controlled asthma (11yr, 7-13; median daily fluticasone dose = 100 µg), and 4 with uncontrolled asthma (12yr, 7-14; 1000 µg fluticasone daily) were included. Baseline expression of LIF mRNA was significantly increased in uncontrolled vs controlled asthmatic children. TNFalpha significantly increased LIF expression in uncontrolled asthma. A similar trend was observed regarding IL-6. Dexamethasone significantly upregulated FKBP51 expression in all groups but the response was blunted in asthmatic children. No significant upregulation was identified regarding NF-kB, serpinB2 and porin. CONCLUSION: LIF and FKBP51 expression in epithelial cells were the most interesting markers of AEC dysfunction/response to corticosteroid treatment.

Retinoic acid increases glucocorticoid receptor phosphorylation via cyclin-dependent kinase 5.

Glucocorticoid receptor (GR) function is modulated by phosphorylation. As retinoic acid (RA) can activate some cytoplasmic kinases able to phosphorylate GR, we investigated whether RA could modulate GR phosphorylation in neuronal cells in a context of long-term glucocorticoid exposure. A 4-day treatment of dexamethasone (Dex) plus RA, showed that RA potentiated the (Dex)-induced phosphorylation on GR Serine 220 (pSer220GR) in the nucleus of a hippocampal HT22 cell line. This treatment increased the cytoplasmic ratio of p35/p25 proteins, which are major CDK5 cofactors. Roscovitine, a pharmacological CDK5 inhibitor, or a siRNA against CDK5 prevented RA potentiation of GR phosphorylation. Furthermore, roscovitine counter-acted the effect of RA on GR sensitive target proteins such as BDNF or tissue-transglutaminase. These data help understanding the interaction between RA- and glucocorticoid-signalling pathways, both of which have strong influences on the adult brain.