Microbiological water quality and derived health risks from exposure to ornamental water fountains in the city of Hannover.

Ornamental fountains are attractive urban infrastructures helping cities to cope with global warming, as water sprays have great cooling effects due to evaporative properties; however, exposure to microbiologically impaired water from ornamental fountains during recreational activities may result in adverse health outcomes for the exposed population. This study assesses the microbial water quality of four ornamental water fountains (Blätterbrunnen, Körtingbrunnen, Klaus-Bahlsen-Brunnen, and Marstallbrunnen) and performs a quantitative microbial risk assessment (QMRA) for children using Escherichia coli, Enterococci, and Salmonella to quantify the probability of gastrointestinal illnesses and Pseudomonas aeruginosa to quantify the risk of dermal infections. Samples were collected fortnightly in two campaigns in 2020 and 2021 and processed to determine bacterial concentrations. Data on exposure time were obtained during field observations on the selected fountains; a total of 499 people were observed of which 30% were children. Mean bacterial concentrations ranged from 1.6 × 101 to 6.1 × 102 most probable number (MPN)/100 mL for E. coli, 1.2 × 101 -1.2 × 103  MPN/100 mL for Enterococci, 8.6 × 103 -3.1 × 105  CFU/100 mL for Salmonella, and 2.5 × 103 -3.2 × 104  MPN/100 mL for P. aeruginosa. The results of the QMRA study showed that the USEPA illness rate of 36 NEEAR-gastrointestinal illnesses/1000 was exceeded for Enterococci at the Körtingbrunnen, Klaus-Bahlsen-Brunnen, and Marstallbrunnen fountains and for Salmonella and P. aeruginosa at the Körtingbrunnen fountain, suggesting that exposure to microbiologically contaminated water from ornamental fountains may pose a health risk to children. The scenario analysis shows the importance of keeping low bacterial concentrations in ornamental fountains so that the risk of illness/infection to children does not exceed the USEPA illness rate benchmark.

[Disproving the myth of "abscess weather" predisposing to peritonsillitis and peritonsillar abscess]. / Widerlegung des Mythos vom "Abszesswetter", welches das Auftreten von Peritonsillitiden und Peritonsillarabszessen begünstigt.

BACKGROUND: Peritonsillar abscess (PTA) is often seen as a complication of acute tonsillitis and is defined as pus retention between the tonsillar capsule and the peritonsillar tissue. The etiology and pathogenesis have not yet been fully elucidated. A connection between certain weather conditions and temperature fluctuations and the occurrence of abscesses in the head and neck region has been discussed for years. The question here is whether higher temperature fluctuations are predisposing for the formation of abscesses. MATERIALS AND METHODS: A retrospective evaluation of all patients hospitalized with peritonsillitis or PTA in the Department of Otorhinolaryngology of the Klinikum Rechts der Isar of the Technical University of Munich during a period of 10 years (2012-2021) was performed. Each patient was individually correlated with daily temperature data from the statistical meteorological office of the City of Munich. RESULTS: A total of 1450 patients were included, 270 patients (18.62%) with peritonsillitis and 1180 patients (81.38%) with PTA. A correlation between the occurrence of peritonsillitis or PTA and major temperature fluctuations could be excluded in this large patient population. Moreover, a similar frequency of peritonsillitis and PTA was seen throughout the year. CONCLUSION: The myth of a temperature dependence of the development of peritonsillitis or PTA and a so-called abscess weather could be negated in this study.

RESET-PKD: a pilot trial on short-term ketogenic interventions in autosomal dominant polycystic kidney disease.

BACKGROUND: Ketogenic dietary interventions (KDI) have been shown to be effective in animal models of polycystic kidney disease (PKD), but data from clinical trials are lacking. METHODS: Ten autosomal dominant PKD (ADPKD) patients with rapid disease progression were enrolled at visit V1 and initially maintained a carbohydrate-rich diet. At V2, patients entered one of the two KDI arms: a 3-day water fast (WF) or a 14-day ketogenic diet (KD). At V3, they resumed their normal diet for 3-6 weeks until V4. At each visit, magnetic resonance imaging kidney and liver volumetry was performed. Ketone bodies were evaluated to assess metabolic efficacy and questionnaires were used to determine feasibility. RESULTS: All participants [KD n = 5, WF n = 5; age 39.8 ± 11.6 years; estimated glomerular filtration rate 82 ± 23.5 mL/min/1.73 m2; total kidney volume (TKV) 2224 ± 1156 mL] were classified as Mayo Class 1C-1E. Acetone levels in breath and beta-hydroxybutyrate (BHB) blood levels increased in both study arms (V1 to V2 average acetone: 2.7 ± 1.2 p.p.m., V2 to V3: 22.8 ± 11.9 p.p.m., P = .0006; V1 to V2 average BHB: 0.22 ± 0.08 mmol/L, V2 to V3: 1.88 ± 0.93 mmol/L, P = .0008). Nine of 10 patients reached a ketogenic state and 9/10 evaluated KDIs as feasible. TKV did not change during this trial. However, we found a significant impact on total liver volume (ΔTLV V2 to V3: -7.7%, P = .01), mediated by changes in its non-cystic fraction. CONCLUSIONS: RESET-PKD demonstrates that short-term KDIs potently induce ketogenesis and are feasible for ADPKD patients in daily life. While TLV quickly changed upon the onset of ketogenesis, changes in TKV may require longer-term interventions.

Obstruction level associated with outcome in hypoglossal nerve stimulation.

PURPOSE: Selective hypoglossal nerve stimulation (sHNS) constitutes an effective surgical alternative for patients with obstructive sleep apnea (OSA). sHNS results in tongue protrusion and consecutive alleviation of obstructions at the tongue base level (lower obstructions). Furthermore, obstructions at the soft palate level (upper obstructions) may be prevented through palatoglossal coupling as seen on sleep endoscopy. However, it has not been studied if the distribution of obstruction level during a whole night measurement is a relevant factor for the treatment outcome. METHODS: Obstruction levels were measured with a manometry system during a whole night of sleep in 26 patients with OSA (f = 1, m = 25; age 59.4 ± 11.3; BMI = 29.6 ± 3.6) either before (n = 9) or after sHNS implantation (n = 12). Five patients received a measurement before and after implantation. Obstructions were categorized into velar (soft palate and above), infravelar (below soft palate), and multilevel obstructions. An association between obstruction level and treatment outcome was calculated. RESULTS: The mean distribution of preoperative obstruction level could be divided into the following: 38% velar, 46% multilevel, and 16% infravelar obstructions. Patients with a good treatment response (defined as AHI < 15/h and AHI reduction of 50%) had fewer preoperative velar obstructions compared to non-responder (17% vs. 54%, p-value = 0.006). In patients measured after sHNS implantation, a significantly higher rate of multilevel obstructions per hour was measured in non-responders (p-value = 0.012). CONCLUSIONS: Selective hypoglossal nerve stimulation was more effective in patients with fewer obstructions at the soft palate level. Manometry may be a complementary diagnostic procedure for the selection of patients for HNS.

Multidirectional activity of bakuchiol against cellular mechanisms of facial ageing - Experimental evidence for a holistic treatment approach.

OBJECTIVE: Skin ageing is a multifactorial process involving formation of reactive oxygen species, consecutive inflammation with reduced epidermal and dermal cell viability and resulting damage to the extracellular matrix. Effective dermocosmetic treatment modalities should ideally address these hallmarks in a holistic approach. Here, we determined the corresponding activity profile of bakuchiol, a plant-derived meroterpene, in an array of in vitro, ex vivo and in vivo studies and compared it to retinol, currently considered as gold standard in topical antiageing cosmetics. METHODS: The antioxidative capacity and power of bakuchiol and retinol were analysed by measuring 2,2'-diphenyl-1-picrylhydrazyl (DPPH) reduction via its absorption decay and electron spin resonance spectroscopy, respectively. Effects on prostaglandin E2 (PGE2), macrophage migration inhibitory factor (MIF), fibroblast growth factor 7 (FGF7), collagen type I and VII (COL1A1, COL7A1), fibronectin (FN) levels as well as the metabolization of water-soluble tetrazolium 1 (WST-1) were determined in human dermal fibroblasts. Epidermal regeneration was assessed utilizing an in vitro wound healing model. FN protein levels were analysed ex vivo after treatment with a formulation containing bakuchiol, retinol or vehicle using suction blister fluid. Skin condition improvement was determined in vivo in a split-face comparison study after application of bakuchiol or vehicle. RESULTS: In contrast to retinol, bakuchiol demonstrated high antioxidative efficacy. Levels of PGE2 and MIF were significantly decreased by both bakuchiol and retinol. Bakuchiol but not retinol significantly increased FGF7 protein levels. WST-1 metabolization levels were significantly augmented by bakuchiol and retinol. Bakuchiol and retinol application led to a significant augmentation of COL1A1, COL7A1 and FN protein levels. Wounds supplemented with bakuchiol but not retinol displayed a significant increase in epidermis regeneration. Clinically, areas treated with a bakuchiol-containing formulation showed a statistically significant increase in FN protein values after a 4-week application compared to untreated areas and areas treated with vehicle. CONCLUSION: These data provide evidence for the multidirectional efficacy of bakuchiol against cellular hallmarks of skin ageing. Its activity profile shares some common features with retinol but demonstrates several hitherto unknown biopositive effects in our studies, namely stimulation of the critical extracellular matrix component FN, and accelerated epidermal regeneration and wound healing.

OBJECTIF: le vieillissement de la peau est un processus multifactoriel impliquant la formation de dérivés réactifs de l'oxygène, une inflammation consécutive qui entraîne une viabilité réduite des cellules du derme et de l'épiderme, et endommage la matrice extracellulaire. Pour être efficaces, les traitements dermocosmétiques devraient dans l'idéal traiter ces caractéristiques selon une approche holistique. Ici, nous avons déterminé le profil d'activité correspondant du bakuchiol, un méroterpène d'origine végétale, dans une série d'études in vitro, ex vivo et in vivo, et l'avons comparé au rétinol, qui est aujourd'hui considéré comme la référence parmi les cosmétiques anti-âge topiques. MÉTHODES: la capacité antioxydante et la puissance du bakuchiol et du rétinol ont été analysées en mesurant la réduction du 2,2-diphényl-1-picrylhydrazyl (DPPH) selon sa décroissance par absorption et à l'aide d'une spectroscopie par résonance magnétique électronique, respectivement. Les effets sur la prostaglandine E2 (PGE2), le facteur inhibiteur de la migration (FIM) des macrophages, le facteur de croissance des fibroblastes 7 (FGF7), le collagène de type I et VII (COL1A1, COL7A1), les taux de fibronectine (FN), ainsi que la métabolisation du tétrazolium 1 soluble dans l'eau (WST-1) ont été déterminés dans des fibroblastes dermiques humains. La régénération épidermique a été évaluée à l'aide d'un modèle de cicatrisation des plaies in vitro. Les taux de fibronectine ont été analysés ex vivo après un traitement avec une formulation contenant du bakuchiol, du rétinol ou un excipient à l'aide d'un liquide d'aspiration sous forme de vésicules. L'amélioration de l'état de la peau a été déterminée in vivo dans une étude de comparaison en hémiface après l'application de bakuchiol ou d'un excipient. RÉSULTATS: Contrairement au rétinol, le bakuchiol s'est avéré présenter une efficacité antioxydante élevée. Les taux de PGE2 et de FIM ont significativement diminué avec le bakuchiol et le rétinol. L'application de bakuchiol s'est accompagnée d'une augmentation significative des taux de protéine FGF7, mais pas celle de rétinol. Les taux de métabolisation du WST-1 ont augmenté de façon significative avec le bakuchiol et le rétinol. L'application de bakuchiol et de rétinol a entraîné une augmentation significative des taux de protéines COL1A1, COL7A1 et fibronectine. Les plaies supplémentées en bakuchiol, mais pas en rétinol, ont montré une augmentation significative de la régénération épidermique. Sur le plan clinique, les zones traitées avec une formulation contenant du bakuchiol ont montré une augmentation statistiquement significative des taux de fibronectine après une application de 4 semaines par rapport aux zones non traitées et aux zones traitées avec un excipient. CONCLUSION: ces données fournissent des preuves de l'efficacité multidirectionnelle du bakuchiol contre les caractéristiques cellulaires du vieillissement de la peau. Son profil d'activité partage certaines caractéristiques communes avec le rétinol, mais démontre plusieurs effets biopositifs jusqu'alors inconnus dans nos études : la stimulation de la fibronectine, composante essentielle de la matrice extracellulaire, et une régénération épidermique et une cicatrisation accélérée des plaies.

Perception of challenges in management of neurological cases in the emergency room.

OBJECTIVE: To investigate emergency clinicians' comfort level in assessing neurological emergencies and to identify opportunities to foster enhanced training of clinical neurology in the emergency room. DESIGN: Internet-based survey. SETTING: University teaching hospitals and private referral centers. SUBJECTS: One hundred and ninety-two emergency and critical care specialists and resident trainees (ECC) and 104 neurology specialists and resident trainees (NEUR) in clinical practice. INTERVENTIONS: An internet-based survey was distributed via veterinary professional organizations' listserves and message boards and responses were collected between March and April 2020. ECC completed a survey evaluating stress levels associated with neurological emergencies, confidence with neurological examinations, and neuroanatomical localization. NEUR completed a similar survey to report their perception of their ECC colleagues' confidence in the assessment of neurological cases. Chi-square and Mann-Whitney U-tests were used to compare categorical responses and confidence scores between groups. P < 0.002 was considered significant. MEASUREMENTS AND MAIN RESULTS: Fifty-two percent of ECC found neurological emergencies slightly challenging, whereas 85% of NEUR found them moderately to extremely challenging for ECC (P < 0.0001). ECC's median self-reported confidence score in performing a neurologic examination on a scale of 0-100 was 75 (interquartile range [IQR], 27), while NEUR reported a median ECC confidence of 44 (IQR, 25; P < 0.0001). Median self-reported ECC confidence in localizing intracranial, spinal, and neuromuscular disease was 67 (IQR, 40), 88 (IQR, 21), and 60 (IQR, 37), respectively, which was significantly higher than median NEUR-reported ECC confidence of 35 (IQR, 38), 51 (IQR, 31), and 18 (IQR, 20), respectively (all P < 0.0001). Following case transfer, 34% of ECC received NEUR feedback in >75% of cases. CONCLUSIONS: Noticeable discrepancies between ECC and NEUR perceptions of ECC clinical confidence were seen, while no firm evidence of neurophobia could be inferred. Improvements in interdepartmental communication and teaching of clinical neurology may be warranted.

Feasibility and impact of ketogenic dietary interventions in polycystic kidney disease: KETO-ADPKD-a randomized controlled trial.

Ketogenic dietary interventions (KDIs) are beneficial in animal models of autosomal-dominant polycystic kidney disease (ADPKD). KETO-ADPKD, an exploratory, randomized, controlled trial, is intended to provide clinical translation of these findings (NCT04680780). Sixty-six patients were randomized to a KDI arm (ketogenic diet [KD] or water fasting [WF]) or the control group. Both interventions induce significant ketogenesis on the basis of blood and breath acetone measurements. Ninety-five percent (KD) and 85% (WF) report the diet as feasible. KD leads to significant reductions in body fat and liver volume. Additionally, KD is associated with reduced kidney volume (not reaching statistical significance). Interestingly, the KD group exhibits improved kidney function at the end of treatment, while the control and WF groups show a progressive decline, as is typical in ADPKD. Safety-relevant events are largely mild, expected (initial flu-like symptoms associated with KD), and transient. Safety assessment is complemented by nuclear magnetic resonance (NMR) lipid profile analyses.

Ketogenic dietary interventions in autosomal dominant polycystic kidney disease-a retrospective case series study: first insights into feasibility, safety and effects.

Background: Our laboratory published the first evidence that nutritional ketosis, induced by a ketogenic diet (KD) or time-restricted diet (TRD), ameliorates disease progression in polycystic kidney disease (PKD) animal models. We reasoned that, due to their frequent use for numerous health benefits, some autosomal dominant PKD (ADPKD) patients may already have had experience with ketogenic dietary interventions (KDIs). This retrospective case series study is designed to collect the first real-life observations of ADPKD patients about safety, feasibility and possible benefits of KDIs in ADPKD as part of a translational project pipeline. Methods: Patients with ADPKD who had already used KDIs were recruited to retrospectively collect observational and medical data about beneficial or adverse effects and the feasibility and safety of KDIs in questionnaire-based interviews. Results: A total of 131 ADPKD patients took part in this study. About 74 executed a KD and 52 a TRD for 6 months on average. A total of 86% of participants reported that KDIs had improved their overall health, 67% described improvements in ADPKD-associated health issues, 90% observed significant weight loss, 64% of participants with hypertension reported improvements in blood pressure, 66% noticed adverse effects that are frequently observed with KDIs, 22 participants reported safety concerns like hyperlipidemia, 45 participants reported slight improvements in estimated glomerular filtration rate and 92% experienced KDIs as feasible while 53% reported breaks during their diet. Conclusions: Our preliminary data indicate that KDIs may be safe, feasible and potentially beneficial for ADPKD patients, highlighting that prospective clinical trials are warranted to confirm these results in a controlled setting and elucidate the impact of KDIs specifically on kidney function and cyst progression.

Automated Kidney and Liver Segmentation in MR Images in Patients with Autosomal Dominant Polycystic Kidney Disease: A Multicenter Study.

Background: Imaging-based total kidney volume (TKV) and total liver volume (TLV) are major prognostic factors in autosomal dominant polycystic kidney disease (ADPKD) and end points for clinical trials. However, volumetry is time consuming and reader dependent in clinical practice. Our aim was to develop a fully automated method for joint kidney and liver segmentation in magnetic resonance imaging (MRI) and to evaluate its performance in a multisequence, multicenter setting. Methods: The convolutional neural network was trained on a large multicenter dataset consisting of 992 MRI scans of 327 patients. Manual segmentation delivered ground-truth labels. The model's performance was evaluated in a separate test dataset of 93 patients (350 MRI scans) as well as a heterogeneous external dataset of 831 MRI scans from 323 patients. Results: The segmentation model yielded excellent performance, achieving a median per study Dice coefficient of 0.92-0.97 for the kidneys and 0.96 for the liver. Automatically computed TKV correlated highly with manual measurements (intraclass correlation coefficient [ICC]: 0.996-0.999) with low bias and high precision (-0.2%±4% for axial images and 0.5%±4% for coronal images). TLV estimation showed an ICC of 0.999 and bias/precision of -0.5%±3%. For the external dataset, the automated TKV demonstrated bias and precision of -1%±7%. Conclusions: Our deep learning model enabled accurate segmentation of kidneys and liver and objective assessment of TKV and TLV. Importantly, this approach was validated with axial and coronal MRI scans from 40 different scanners, making implementation in clinical routine care feasible.Clinical Trial registry name and registration number: The German ADPKD Tolvaptan Treatment Registry (AD[H]PKD), NCT02497521.

Chondrocytes From Osteoarthritic and Chondrocalcinosis Cartilage Represent Different Phenotypes.

Basic calcium phosphate (BCP)-based calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease and hypertrophic differentiation of chondrocytes. Chondrocalcinosis (CC) is associated with calcium pyrophosphate dihydrate (CPPD) deposition disease in the joint inducing OA-like symptoms. There is only little knowledge about the effect of CPPD crystals on chondrocytes and the signaling pathways involved in their generation. The aim of this study was to investigate the chondrocyte phenotype in CC cartilage and the effect of CPPD crystals on chondrocytes. Cartilage samples of patients with CC, patients with severe OA, and healthy donors were included in this study. The presence of CC was evaluated using standard X-ray pictures, as well as von Kossa staining of cartilage sections. OA severity was evaluated using the Chambers Score on cartilage sections, as well as the radiological Kellgren-Lawrence Score. Patients with radiologically detectable CC presented calcification mainly on the cartilage surface, whereas OA patients showed calcification mainly in the pericellular matrix of hypertrophic chondrocytes. OA cartilage exhibited increased levels of collagen X and matrix metalloproteinase 13 (MMP13) compared with CC and healthy cartilage. This observation was confirmed by qRT-PCR using cartilage samples. No relevant influence of CPPD crystals on hypertrophic marker genes was observed in vitro, whereas BCP crystals significantly induced hypertrophic differentiation of chondrocytes. Interestingly, we observed an increased expression of p16 and p21 in cartilage samples of CC patients compared with OA patients and healthy controls, indicating cellular senescence. To investigate whether CPPD crystals were sufficient to induce senescence, we incubated chondrocytes with BCP and CPPD crystals and quantified senescence using ß-gal staining. No significant difference was observed for the staining, but an increase of p16 expression was observed after 10 days of culture. Primary chondrocytes from CC patients produced CPPD crystals in culture. This phenotype was stabilized by mitomycin C-induced senescence. Healthy and OA chondrocytes did not exhibit this phenotype. BCP and CPPD crystals seem to be associated with two different chondrocyte phenotypes. Whereas BCP deposition is associated with chondrocyte hypertrophy, CPPD deposition is associated with cellular senescence.

Hepatic vein tumor thrombosis in patients with hepatocellular carcinoma: Prevalence and clinical significance.

BACKGROUND: There is strong evidence that portal vein tumor thrombosis (PVTT) is associated with poor survival in patients with hepatocellular carcinoma (HCC). However, data regarding the clinical significance of hepatic vein tumor thrombosis (HVTT) is rare, particularly in Western patients. OBJECTIVE: To determine the HVTT prevalence in a Western patient population and its impact on survival. METHODS: We included 1310 patients with HCC treated in our tertiary referral center between January 2005 and December 2016. HVTT and PVTT were diagnosed with contrast-enhanced cross-sectional imaging. Overall survival (OS) was calculated starting from the initial HCC diagnosis, and in a second step, starting from the first appearance of vascular invasion. RESULTS: We observed macrovascular invasion (MVI) in 519 patients who suffered from either isolated HVTT (n = 40), isolated PVTT (n = 352), or both combined (HVTT + PVTT) (n = 127). Calculated from the initial HCC diagnosis, the median OS for patients with isolated HVTT was significantly shorter than that of patients without MVI (13.3 vs. 32.5 months, p < 0.001). Calculated from the first appearance of MVI, the median OS was similar among patients with isolated HVTT (6.5 months), isolated PVTT (5 months), and HVTT + PVTT (5 months). Multivariate analysis confirmed HVTT as an independent risk factor for poor survival. CONCLUSIONS: HVTT may be more common than typically reported. In most patients, it was accompanied by PVTT. Isolated HVTT occurred less frequently and later than isolated PVTT; however, once developed, it had the same deleterious impact on survival. Therefore, patients with HVTT should be classified as advanced stage of HCC.

Hypoglossal nerve stimulation therapy does not alter tongue protrusion strength and fatigability in obstructive sleep apnea.

STUDY OBJECTIVES: Hypoglossal nerve stimulation (HNS) is an effective surgical alternative for patients with obstructive sleep apnea (OSA). HNS therapy relies on the stimulation of the hypoglossal nerve to open the upper airways. This stimulation could lead to alterations in tongue strength and fatigability, which could alter treatment outcome over time. The aim of the study was to investigate whether HNS alters tongue strength and fatigability. METHODS: Tongue protrusion strength (peak pressure in kPa) and fatigability (time to task failure during 50% of peak pressure contraction) were measured with a pressure transducer at least 2 months after HNS implantation (n = 30). These results were compared to a group of patients with OSA (n = 38) and a non-OSA control group (n = 35). RESULTS: Median tongue protrusion strength was lower (54.7 [43.8, 63.0] versus 60.7 [53.7, 66.0] kPa, P = .013) and fatigue occurred more quickly (21.3 [17.4, 26.3] versus 26.0 [19.3, 31.3] seconds, P = .017) in the patients with OSA compared to the non-OSA control group. In multiple regression analysis, age was a significant factor for tongue strength and diagnosis of OSA for tongue fatigability. Tongue strength and fatigability did not differ between patients with OSA with conservative therapy or observation versus after HNS implantation (51.8 [41.3, 63.4] versus 56.3 [45.0, 62.3] kPa, P = .502; 20.8 [16.3, 26.2] versus 21.8 [18.3, 26.8] seconds, P = .418). CONCLUSIONS: Tongue strength decreases with age. Tongue fatigability is more pronounced in people with OSA. However, approximately 1.5 years of HNS therapy on average does not alter tongue strength or fatigability compared to an OSA control group. CLINICAL TRIAL REGISTRATION: Registry: ClinicalTrials.gov; Title: Change in Tongue Strength and Fatigue After Upper Airway Stimulation Therapy; Identifier: NCT03980158.

Neuronal effects of thyroid hormone metabolites.

Thyroid hormones and their metabolites are active regulators of gene expression, mitochondrial function and various other physiological actions in different organs and tissues. These actions are mediated by a spatio-temporal regulation of thyroid hormones and metabolites within a target cell. This spatio-temporal resolution as well as classical and non-classical actions of thyroid hormones and metabolites is accomplished and regulated on multiple levels as uptake, local activation and signaling of thyroid hormones. In this review, we will give an overview of the systems involved in regulating the presence and activity of thyroid hormones and their metabolites within the brain, specifically in neurons. While a wealth of data on thyroxin (T4) and 3,5,3'-triiodothyronine (T3) in the brain has been generated, research into the presence of action of other thyroid hormone metabolites is still sparse and requires further investigations.

Silychristin, a Flavonolignan Derived From the Milk Thistle, Is a Potent Inhibitor of the Thyroid Hormone Transporter MCT8.

Thyroid hormones (THs) are charged and iodinated amino acid derivatives that need to pass the cell membrane facilitated by thyroid hormone transmembrane transporters (THTT) to exert their biological function. The importance of functional THTT is affirmed by the devastating effects of mutations in the human monocarboxylate transporter (MCT) 8, leading to a severe form of psychomotor retardation. Modulation of THTT function by pharmacological or environmental compounds might disturb TH action on a tissue-specific level. Therefore, it is important to identify compounds with relevant environmental exposure and THTT-modulating activity. Based on a nonradioactive TH uptake assay, we performed a screening of 13 chemicals, suspicious for TH receptor interaction, to test their potential effects on THTT in MCT8-overexpressing MDCK1-cells. We identified silymarin, an extract of the milk thistle, to be a potent inhibitor of T3 uptake by MCT8. Because silymarin is a complex mixture of flavonolignan substances, we further tested its individual components and identified silychristin as the most effective one with an IC50 of approximately 100 nM. The measured IC50 value is at least 1 order of magnitude below those of other known THTT inhibitors. This finding was confirmed by T3 uptake in primary murine astrocytes expressing endogenous Mct8 but not in MCT10-overexpressing MDCK1-cells, indicating a remarkable specificity of the inhibitor toward MCT8. Because silymarin is a frequently used adjuvant therapeutic for hepatitis C infection and chronic liver disease, our observations raise questions regarding its safety with respect to unwanted effects on the TH axis.

Ring Finger Protein 11 Inhibits Melanocortin 3 and 4 Receptor Signaling.

Intact melanocortin signaling via the G protein-coupled receptors (GPCRs), melanocortin receptor 4 (MC4R), and melanocortin receptor 3 (MC3R) is crucial for body weight maintenance. So far, no connection between melanocortin signaling and hypothalamic inflammation has been reported. Using a bimolecular fluorescence complementation library screen, we identified a new interaction partner for these receptors, ring finger protein 11 (RNF11). RNF11 participates in the constitution of the A20 complex that is involved in reduction of tumor necrosis factor α (TNFα)-induced NFκB signaling, an important pathway in hypothalamic inflammation. Mice treated with high-fat diet (HFD) for 3 days demonstrated a trend toward an increase in hypothalamic Rnf11 expression, as shown for other inflammatory markers under HFD. Furthermore, Gs-mediated signaling of MC3/4R was demonstrated to be strongly reduced to 20-40% by co-expression of RNF11 despite unchanged total receptor expression. Cell surface expression was not affected for MC3R but resulted in a significant reduction of MC4R to 61% by co-expression with RNF11. Mechanisms linking HFD, inflammation, and metabolism remain partially understood. In this study, a new axis between signaling of specific body weight regulating GPCRs and factors involved in hypothalamic inflammation is suggested.

High T3, Low T4 Serum Levels in Mct8 Deficiency Are Not Caused by Increased Hepatic Conversion through Type I Deiodinase.

BACKGROUND: The Allan-Herndon-Dudley syndrome is a severe psychomotor retardation accompanied by specific changes in circulating thyroid hormone levels (high T3, low T4). These are caused by mutations in the thyroid hormone transmembrane transport protein monocarboxylate transporter 8 (MCT8). OBJECTIVE: To test the hypothesis that circulating low T4 and high T3 levels are caused by enhanced conversion of T4 via increased activity of hepatic type I deiodinase (Dio1). METHODS: We crossed mice deficient in Mct8 with mice lacking Dio1 activity in hepatocytes. Translation of the selenoenzyme Dio1 was abrogated by hepatocyte-specific inactivation of selenoprotein biosynthesis. RESULTS: Inactivation of Dio1 activity in the livers of global Mct8-deficient mice does not restore normal circulating thyroid hormone levels. CONCLUSIONS: Our data suggest that although hepatic Dio1 activity is increased in Mct8-deficient mice, it does not cause the observed abnormal circulating thyroid hormone levels. Since global inactivation of Dio1 in Mct8-deficient mice does normalize circulating thyroid hormone levels, the underlying mechanism and relevant tissues involved remain to be elucidated.

A Nonradioactive Uptake Assay for Rapid Analysis of Thyroid Hormone Transporter Function.

Thyroid hormones (TH) are actively taken up into target cells via TH-transmembrane transporters (THTT). Their activity and expression patterns define a layer of endocrine regulation that is poorly understood. Therefore, THTT are potential targets for interfering agents (endocrine disruptors) as well as for pharmacological interventions. Inactivating mutations have been identified as the underlying cause of heritable diseases (monocarboxylate transporter 8-associated Allan-Herndon-Dudley syndrome) and might also define a class of subclinical TH insensitivity. As a basic tool to solve questions regarding THTT substrate specificity, activation or inactivation by compounds and functional changes from mutations, uptake assays with radiolabeled tracers are standard. Due to the need for radioactive isotopes, this technique is limited to screening of labelled substrates and disadvantageous regarding handling, setup, and regulatory issues. To overcome these hurdles, we developed an uptake assay protocol using nonradioactive ligands. In brief, uptake of nonradioactive iodine-containing substrate molecules was monitored via Sandell-Kolthoff reaction. The novel assay was designed to the common microtiter plate layout. As a prove-of-principle, we measured TH uptake by monocarboxylate transporter 8-transfected MDCK1 cells. Titrations with bromosulphthalein as an example for inhibitor screening setups and a side-by-side comparison with the radioactive method prove this assay to be reliable, sensitive, and convenient. Furthermore, the method was applicable on primary murine astrocytes, which enables high-throughput screening studies on in vitro model systems with physiological transporter regulation. Due to its design, it is applicable for high-throughput screening of modulatory compounds, but it is also a safe, inexpensive and an easily accessible method for functional testing of THTT in basic science.

The orphan receptor Gpr83 regulates systemic energy metabolism via ghrelin-dependent and ghrelin-independent mechanisms.

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways.

Establishment of a mouse model with misregulated chromosome condensation due to defective Mcph1 function.

Mutations in the human gene MCPH1 cause primary microcephaly associated with a unique cellular phenotype with premature chromosome condensation (PCC) in early G2 phase and delayed decondensation post-mitosis (PCC syndrome). The gene encodes the BRCT-domain containing protein microcephalin/BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage. We report here on the first mouse model of impaired Mcph1-function. The model was established based on an embryonic stem cell line from BayGenomics (RR0608) containing a gene trap in intron 12 of the Mcph1 gene deleting the C-terminal BRCT-domain of the protein. Although residual wild type allele can be detected by quantitative real-time PCR cell cultures generated from mouse tissues bearing the homozygous gene trap mutation display the cellular phenotype of misregulated chromosome condensation that is characteristic for the human disorder, confirming defective Mcph1 function due to the gene trap mutation. While surprisingly the DNA damage response (formation of repair foci, chromosomal breakage, and G2/M checkpoint function after irradiation) appears to be largely normal in cell cultures derived from Mcph1(gt/gt) mice, the overall survival rates of the Mcph1(gt/gt) animals are significantly reduced compared to wild type and heterozygous mice. However, we could not detect clear signs of premature malignant disease development due to the perturbed Mcph1 function. Moreover, the animals show no obvious physical phenotype and no reduced fertility. Body and brain size are within the range of wild type controls. Gene expression on RNA and protein level did not reveal any specific pattern of differentially regulated genes. To the best of our knowledge this represents the first mammalian transgenic model displaying a defect in mitotic chromosome condensation and is also the first mouse model for impaired Mcph1-function.