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Progression of cystic kidney disease has been linked to activation of the mTORC1 signaling pathway. Yet the utility of mTORC1 inhibitors to treat patients with polycystic kidney disease remains controversial despite promising preclinical data. To define the cell intrinsic role of mTORC1 for cyst development, the mTORC1 subunit gene Raptor was selectively inactivated in kidney tubular cells lacking cilia due to simultaneous deletion of the kinesin family member gene Kif3A. In contrast to a rapid onset of cyst formation and kidney failure in mice with defective ciliogenesis, both kidney function, cyst formation discerned by magnetic resonance imaging and overall survival were strikingly improved in mice additionally lacking Raptor. However, these mice eventually succumbed to cystic kidney disease despite mTORC1 inactivation. In-depth transcriptome analysis revealed the rapid activation of other growth-promoting signaling pathways, overriding the effects of mTORC1 deletion and identified cyclin-dependent kinase (CDK) 4 as an alternate driver of cyst growth. Additional inhibition of CDK4-dependent signaling by the CDK4/6 inhibitor Palbociclib markedly slowed disease progression in mice and human organoid models of polycystic kidney disease and potentiated the effects of mTORC1 deletion/inhibition. Our findings indicate that cystic kidneys rapidly adopt bypass mechanisms typically observed in drug resistant cancers. Thus, future clinical trials need to consider combinatorial or sequential therapies to improve therapeutic efficacy in patients with cystic kidney disease., (Copyright © 2024. Published by Elsevier Inc.)

A limited understanding of the pathology underlying chronic wounds has hindered the development of effective diagnostic markers and pharmaceutical interventions. This study aimed to elucidate the molecular composition of various common chronic ulcer types to facilitate drug discovery strategies. We conducted a comprehensive analysis of leg ulcers (LUs), encompassing venous and arterial ulcers, foot ulcers (FUs), pressure ulcers (PUs), and compared them with surgical wound healing complications (WHCs). To explore the pathophysiological mechanisms and identify similarities or differences within wounds, we dissected wounds into distinct subregions, including the wound bed, border, and peri-wound areas, and compared them against intact skin. By correlating histopathology, RNA sequencing (RNA-Seq), and immunohistochemistry (IHC), we identified unique genes, pathways, and cell type abundance patterns in each wound type and subregion. These correlations aim to aid clinicians in selecting targeted treatment options and informing the design of future preclinical and clinical studies in wound healing. Notably, specific genes, such as PITX1 and UPP1, exhibited exclusive upregulation in LUs and FUs, potentially offering significant benefits to specialists in limb preservation and clinical treatment decisions. In contrast, comparisons between different wound subregions, regardless of wound type, revealed distinct expression profiles. The pleiotropic chemokine-like ligand GPR15L (C10orf99) and transmembrane serine proteases TMPRSS11A/D were significantly upregulated in wound border subregions. Interestingly, WHCs exhibited a nearly identical transcriptome to PUs, indicating clinical relevance. Histological examination revealed blood vessel occlusions with impaired angiogenesis in chronic wounds, alongside elevated expression of genes and immunoreactive markers related to blood vessel and lymphatic epithelial cells in wound bed subregions. Additionally, inflammatory and epithelial markers indicated heightened inflammatory responses in wound bed and border subregions and reduced wound bed epithelialization. In summary, chronic wounds from diverse anatomical sites share common aspects of wound pathophysiology but also exhibit distinct molecular differences. These unique molecular characteristics present promising opportunities for drug discovery and treatment, particularly for patients suffering from chronic wounds. The identified diagnostic markers hold the potential to enhance preclinical and clinical trials in the field of wound healing., (© 2023 Novartis Biomedical Research and The Authors. International Wound Journal published by Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

Data from functional trait databases have been increasingly used to address questions related to plant diversity and trait-environment relationships. However, such databases provide intraspecific data that combine individual records obtained from distinct populations at different sites and, hence, environmental conditions. This prevents distinguishing sources of variation (e.g., genetic-based variation vs. phenotypic plasticity), a necessary condition to test for adaptive processes and other determinants of plant phenotypic diversity. Consequently, individual traits measured under common growing conditions and encompassing within-species variation across the occupied geographic range have the potential to leverage trait databases with valuable data for functional and evolutionary ecology. Here, we recorded 16 functional traits and leaf hyperspectral reflectance (NIRS) data for 721 widely distributed Arabidopsis thaliana natural accessions grown in a common garden experiment. These data records, together with meteorological variables obtained during the experiment, were assembled to create the AraDiv dataset. AraDiv is a comprehensive dataset of A. thaliana's intraspecific variability that can be explored to address questions at the interface of genetics and ecology., (© 2023. The Author(s).)

Objectives: There is a growing debate surrounding the legalisation of medical assistance in dying (MAID). MAID is currently prohibited by the French law; however, the debate has recently been reinvigorated in France. This study aims to collect opinions of palliative care stakeholders (PCS) regarding the legalisation of MAID and to identify the factors associated with their opinions., Methods: We performed a transversal survey between 26 June 2021 and 25 July 2021, on PCS who were on the French national scientific society for palliative care. Participants were invited by email., Results: 1439 PCS took part and expressed an opinion about the legalisation of MAID. 1053 (69.7%) were against the legalisation of MAID. When forced to choose which option should be privileged if the law had to change, 3.7% favoured euthanasia, 10.1% favoured assisted suicide with provision of lethal drug by a professional, 27.5% favoured assisted suicide with prescription of a lethal drug and 29.5% favoured assisted suicide with provision of a lethal drug by an association. The opinion regarding legalisation of MAID was statistically different depending on the participant profession (p<0.001) and when comparing clinical and non-clinical positions (p<0.001). A quarter of participants (26.7%) believe that legalising MAID might lead them to change their current position., Conclusions: Overall, French palliative care professionals are against a modification of the current legal framework for legalising MAID but some might change their current position if a law was voted. This might destabilise the PCS demography that is already worrying., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Plant-herbivore interactions mediated by plant-plant signalling have been documented in different species but its within-species variability has hardly been quantified. Here, we tested if herbivore foraging activity on plants was influenced by a prior contact with a damaged plant and if the effect of such plant-plant signalling was variable across 113 natural genotypes of Arabidopsis thaliana . We filmed the activity of the generalist herbivore Cornu aspersum during 1 h on two plants differing only in a prior contact with a damaged plant or not. We recorded each snails' first choice, and measured its first duration on a plant, the proportion of time spent on both plants and leaf consumption. Overall, plant-plant signalling modified the foraging activity of herbivores in A. thaliana . On average, snails spent more time and consumed more of plants that experienced a prior contact with a damaged plant. However, the effects of plant-plant signalling on snail behaviour was variable: depending on genotype identity, plant-plant signalling made undamaged plants more repellant or attractive to snails. Genome-wide associations revealed that genes related to stress coping ability and jasmonate pathway were associated to this variation. Together, our findings highlight the adaptive significance of plant-plant signalling for plant-herbivore interactions., Competing Interests: The authors have no conflict of interest to declare., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Objectives: The objective of this multicenter cross-sectional study was to determine predictors of poor glycaemic control in children with type 1 diabetes mellitus (T1DM), particularly with respect to socioeconomic status (SES)., Methods: Our study population consisted of 1154 children who attended T1DM follow-up consultation with a pediatric diabetes specialist. Clinical and demographic data were retrieved retrospectively from patients' records. Individual deprivation was defined by an EPICES (Evaluation of the Deprivation and Inequalities of Health in Healthcare Centers) score ≥ 30. Patients were assigned to quintiles of the European Deprivation Index (EDI) based on their area deprivation scores. We used multivariable linear regression models to detect potential associations between glycaemic control and indicators of low SES., Results: In total, 33% (n = 376) of patients had an EPICES score ≥ 30 and 23% (n = 268) were in the 5th EDI quintile. Multivariable linear regression analysis showed that poor glycaemic control was associated with both individual (β 0.38; 95%CI 0.26-0.5; p < 0.001) and area deprivation (β 0.26; 95%CI 0.08-0.43; p = 0.004). Demographic factors, body mass index (BMI) and insulin regimen were also independently associated with poor glycaemic control (p < 0.001). Interestingly, access to diabetes technologies was not related to SES or either glycaemic control., Conclusion: Low SES is associated with a higher risk of poor glycaemic control, independently of insulin regimen. BMI, age at the time of consultation, duration of diabetes, and insulin regimen. Also have an impact on HbA1c. These parameters need to be considered when developing novel treatment strategies for children with T1DM to better target at-risk patients., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

With advancements in whole slide imaging technology and improved understanding of the features of pathologist workstations required for digital slide evaluation, many institutions are investigating broad digital pathology adoption. The benefits of digital pathology evaluation include remote access to study or diagnostic case materials and integration of analysis and reporting tools. Diagnosis based on whole slide images is established in human medical pathology, and the use of digital pathology in toxicologic pathology is increasing. However, there has not been broad adoption in toxicologic pathology, particularly in the context of regulatory studies, due to lack of precedence. To address this topic, as well as practical aspects, the European Society of Toxicologic Pathology coordinated an expert international workshop to assess current applications and challenges and outline a set of minimal requirements needed to gain future regulatory acceptance for the use of digital toxicologic pathology workflows in research and development, so that toxicologic pathologists can benefit from digital slide technology.

We introduce HistoNet, a deep neural network trained on normal tissue. On 1690 slides with rat tissue samples from 6 preclinical toxicology studies, tissue regions were outlined and annotated by pathologists into 46 different tissue classes. From these annotated regions, we sampled small 224 × 224 pixels images (patches) at 6 different levels of magnification. Using 4 studies as training set and 2 studies as test set, we trained VGG-16, ResNet-50, and Inception-v3 networks separately at each magnification level. Among these model architectures, Inception-v3 and ResNet-50 outperformed VGG-16. Inception-v3 identified the tissue from query images, with an accuracy up to 83.4%. Most misclassifications occurred between histologically similar tissues. Investigation of the features learned by the model (embedding layer) using Uniform Manifold Approximation and Projection revealed not only coherent clusters associated with the individual tissues but also subclusters corresponding to histologically meaningful structures that had not been annotated or trained for. This suggests that the histological representation learned by HistoNet could be useful as the basis of other machine learning algorithms and data mining. Finally, we found that models trained on rat tissues can be used on non-human primate and minipig tissues with minimal retraining.

Several deep learning approaches have been proposed to address the challenges in computational pathology by learning structural details in an unbiased way. Transfer learning allows starting from a learned representation of a pretrained model to be directly used or fine-tuned for a new domain. However, in histopathology, the problem domain is tissue-specific and putting together a labelled data set is challenging. On the other hand, whole slide-level annotations, such as biomarker levels, are much easier to obtain. We compare two pretrained models, one histology-specific and one from ImageNet on various computational pathology tasks. We show that a domain-specific model (HistoNet) contains richer information for biomarker classification, localization of biomarker-relevant morphology within a slide, and the prediction of expert-graded features. We use a weakly supervised approach to discriminate slides based on biomarker level and simultaneously predict which regions contribute to that prediction. We employ multitask learning to show that learned representations correlate with morphological features graded by expert pathologists. All of these results are demonstrated in the context of renal toxicity in a mechanistic study of compound toxicity in rat models. Our results emphasize the importance of histology-specific models and their knowledge representations for solving a wide range of computational pathology tasks.

Background and Aims: KRP203 is a potent oral agonist of the sphingosine-1-phosphate receptor subtype 1 that induces the sequestration of peripheral lymphocytes, thereby potentially reducing the number of activated lymphocytes circulating to the gastrointestinal tract., Methods: We conducted a multicentre, double-blind, placebo-controlled, parallel-group, proof-of-concept study to evaluate the efficacy, safety, and tolerability of KRP203 in patients with moderately active 5-aminosalicylate-refractory ulcerative colitis (UC). Patients were randomly assigned to receive 1.2 mg KRP203 or placebo daily for 8 weeks. Primary efficacy variable was clinical remission, defined as partial Mayo Score 0-1 and modified Baron Score 0-1 with rectal bleeding subscore 0., Results: KRP203 was safe and well tolerated overall. The most common adverse events (AEs) were gastrointestinal disorders and headache. Importantly, no KRP203-related cardiac AEs were reported. Total peripheral lymphocytes and selectively affected lymphocyte subtypes decreased, causing marked decreases in naive and central memory CD4+ and CD8+ T cells, and also in B cells. Clinical remission occurred in 2/14 (14%) patients under KRP203, compared with 0/8 (0%) under placebo., Conclusions: Overall, KRP203 was safe and well tolerated by patients with UC. Importantly, no cardiac AEs were reported. Although KRP203 did not meet the minimum clinically relevant threshold for efficacy, the results may suggest that KRP203 treatment is superior to placebo. However, in this small study population, the difference was insignificant. Based on these data, studies with an improved design and a larger population should be considered., Competing Interests: H.H.R. has received expenses from Novartis for attendance of the investigator meeting for this study and has no other conflicts of interest. W.K. has received consultancy fees from Falk, Ferring, Genetic Analysis, Istitut Allergosan, Nikkiso, and Otsuka, and lecturing fees from Abbvie, Ardeypharm, and Tillotts. J.S. has received consultancy fees from Abbvie, Fresenius-Kabi, Immundiagnostik, MSD, Pharmacosmos, Takeda, GI Dynamics, and Vifor; lecturing fees from Abbvie, Falk Foundation, Ferring, Immundiagnostik, MSD, Pharmacosmos, Takeda, Thermofischer, GI Dynamics, and Vifor; and payment for manuscript preparation from Abbvie, Falk Foundation, GI Dynamics, and MSD. P.L.L. has no conflicts of interest to declare. P.J. and P.G. are employees of Novartis. L.C. is an employee and stockholder of Novartis. P.M. is an employee and stockholder of Novartis Institutes of Biomedical Research. F.M. is an employee and stockholder of Novartis Bioventures Ltd. G.R. has consulted to Abbot, Abbvie, Augurix, Boehringer, Calypso, FALK, Ferring, Fisher, Genentech, Essex/MSD, Novartis, Pfizer, Phadia, Roche, UCB, Takeda, Tillots, Vifor, Vital Solutions, and Zeller; received speaker's honoraria from Astra Zeneca, Abbott, Abbvie, FALK, MSD, Phadia, Tillots, UCB, and Vifor; and received educational and research grants from Abbot, Abbvie, Ardeypharm, Augurix, Calypso, Essex/MSD, FALK, Flamentera, Novartis, Roche, Takeda, Tillots, UCB, and Zeller. G.A. has received expenses from Novartis for meetings in connection with this study; consultancy fees from Janssen, Takeda, and Abbvie; and lecturing fees from Abbvie and Pfizer., (Copyright © 2020 by S. Karger AG, Basel.)

The subject of violence in care is regularly covered in scientific literature which puts forward several explanatory scenarios: in patients, violence is thought to be the result of internal and individual factors; while in caregivers, it is linked to external and organisational factors. Often viewed from a negative perspective, violence can also be considered in a positive manner, as a situation analyser., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Severely obese patients undergoing bariatric surgery (BS) are at increased risk for venous thromboembolism (VTE). How standard low molecular weight heparin (LMWH) regimen should be adapted to provide both sufficient efficacy and safety in this setting is unclear. We aimed to compare the influence of four body size descriptors (BSD) on peak anti-Xa levels in BS obese patients receiving LMWH fixed doses to identify which one had the greatest impact. One hundred and thirteen BS obese patients [median body mass index (BMI), 43.3 kg/m 2 (IQR, 40.6-48.7 kg/m 2 )] receiving subcutaneous dalteparin 5000 IU twice daily were included in this prospective monocenter study. Peak steady-state anti-Xa levels were measured peri-operatively following thromboprophylaxis initiation. Only 48% of patients achieved target anti-Xa levels (0.2-0.5 IU/ml). In univariate analysis, age, gender, total body-weight (TBW), lean body-weight (LBW), ideal body-weight (IBW), BMI and estimated glomerural filtration rate (eGFR) were associated with anti-Xa levels. The strongest negative association was observed with LBW (r = -0.56, p < .0001). Receiver operating characteristic curves indicated that among BSD, LBW (cut-off >55.8 kg) had the highest sensitivity (73%) and specificity (69%) to predict sub-prophylactic anti-Xa levels. In multivariate analysis, LBW and eGFR remained associated with anti-Xa levels (β = -0.47 ± 0.08, p < .0001 and β = -0.19 ± 0.08; p = .02, respectively). In BS morbidly obese patients receiving LMWH for thromboprophylaxis after BS, LBW and eGFR are the main determinants of anti-Xa level, and could be proposed in LMWH-based thromboprophylaxis dosing algorithms. The efficacy of a LBW-scale based dosing algorithm for optimal VTE prevention deserves further prospective randomized trials., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Southeast asian ovalocytosis (SAO) is characterized by macro-ovalocytes and ovalo-stomatocytes on blood smear. SAO is common in Malaisia and Papua-New-Guinea where upwards to 40 per cent of the population is affected in some coastal region. Inherited in an autosomal dominant way, illness results from deletion of codons 400-408 in SLC4A1 gene which encodes for band 3 erythrocyte membrane protein. This deletion is responsible for an unusual erythrocyte stiffness and oval shape of the cells on blood smear. Heterozygous carriers are usually asymptomatic whereas homozygous are not viable without an intensive antenatal care. Here, we describe 4 patients diagnosed incidentally by cytogram appearance of the Advia® 2120i (Siemens) representing hemoglobin concentration according to red blood mean cellular volume (GR/VCH).

Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Originally conceptualized as an integrated approach combining conventional toxicology methods with genome-wide expression profiling, toxicogenomics has promised to provide unequivocal relationships between the molecular changes elicited by a compound or a target pathway and the lesions that appear subsequently in the tissues. However, the discipline has only partially delivered on this promise, and the number of publications and submissions related to toxicogenomics is stagnating. The purpose of this article is to outline key factors contributing to a successful implementation of toxicogenomics in the drug discovery and development process. Paradigms and methods of toxicogenomics are briefly reviewed, and the prominence of biostatistics and its limitations in the particular context of nonclinical toxicology studies are discussed. We present specific approaches for pathophysiological contextualization of gene expression data derived from tissues with lesions at variable incidence and severity: "unmixing" (deconvolution) of molecular expression profiles from complex tissues, the invaluable contribution of reference data, the role of establishing causation between expression signals and pathologic changes (phenotypic anchoring), and especially molecular localization. These approaches compensate for the limitations of biostatistical analysis, which in turn, derive from tissue heterogeneity. Finally, impactful applications of toxicogenomics along the drug discovery and development process are exemplified, from the evaluation of potential target toxicities to the selection of candidate compounds and elucidation of the molecular and cellular mechanisms leading to chronic toxicity., (© The Author 2017. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF , activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf - /- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 ( Trp53 ) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large ( Bcl-xL ), Mdm4 , and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

Background: Venous thromboembolism (VTE) is a leading cause of death in obese patients undergoing bariatric surgery (BS), but there is neither consensus nor high-level guidelines yet on VTE prophylaxis in this specific population., Objective: We aimed to evaluate patterns of BS perioperative thromboprophylaxis practices., Setting: French obesity specialized care centers (CSO), which are tertiary care referral hospitals for the most severe cases of obesity METHODS: A detailed questionnaire survey (11 opened, 15 closed questions) investigating their prophylactic schemes of anticoagulation (molecule, dose, weight-adjustment, duration, associated measures, follow-up) was sent to the 37 CSO., Results: Completion rate was 92%. Over 90% of respondents indicated using low molecular weight heparin. Enoxaparin was the most commonly used molecule (89%), twice daily (71%), started mostly 6 hours after BS (74%), whereas fondaparinux (9%), dalteparin (6%), and tinzaparin (6%) were less often prescribed. Dosing varied significantly according to centers from 4000 to 12,000 IU/d, with the most commonly used dose being 8000 IU once daily, 83%, as well as treatment duration (1 week, 9%; 3 weeks, 47%). Half CSO adjusted low molecular weight heparin dose to weight. Biological monitoring was performed in 88%. Only 1 center followed systematically anti-Xa activity. Associated measures such as elastic stoking or intermittent pneumatic compression were used in 32% and 26%, respectively, and both were used in 39%., Conclusion: This study finds significant discrepancies in thromboprophylaxis practices in obese patients undergoing BS, particularly with respect to treatment duration and dose adjustment, highlighting the urgent need for improved implementation of existing clinical practice guidelines in this VTE high-risk population., (Copyright © 2017 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to determine the relative safety of 4 antiviral drugs (telbivudine, tenofovir, adefovir, and entecavir) against hepatitis B virus with respect to kidney function and toxicity in male Sprague Dawley rats. The antiviral drugs were administered once daily for 4 weeks by oral gavage at ∼10 and 25-40 times the human equivalent dose. Main assessments included markers of renal toxicity in urine, magnetic resonance imaging (MRI) of kidney function, histopathology, and electron microscopic examination. Administration of adefovir at 11 and 28 mg/kg for 4 weeks caused functional and morphological kidney alterations in a time- and dose-dependent manner, affecting mainly the proximal tubules and suggesting a mechanism of toxicity related to mitochondrial degeneration/depletion. Of note, the observed adefovir-induced reduction of kidney function was not detected by the standard method of glomerular filtration rate (GFR) measurements (clearance rate of the endogenous marker, creatinine), thereby emphasizing the superiority of MRI in terms of sensitive detection of GFR in rats. For the low dose of 300 mg/kg of tenofovir, minor kidney effects such as nuclear enlargement in the tubular epithelium, and hyaline droplets accumulation were detected, which was also observed for the low dose (11 mg/kg) of adefovir. No assessments could be done at the higher dose of 600/1000 mg/kg tenofovir due to gastrointestinal tract toxicity which prevented treatment of the animals for longer than 1 week. Entecavir at 1 and 3 mg/kg and telbivudine at 600 and 1600 mg/kg caused no toxicologically relevant effects on the kidney., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Antibodies targeting IL-17A or its receptor IL-17RA show unprecedented efficacy in the treatment of autoimmune diseases such as psoriasis. These therapies, by neutralizing critical mediators of immunity, may increase susceptibility to infections. Here, we compared the effect of antibodies neutralizing IL-17A, IL-17F or TNFα on murine host responses to Mycobacterium tuberculosis infection by evaluating lung transcriptomic, microbiological and histological analyses. Coinciding with a significant increase of mycobacterial burden and pathological changes following TNFα blockade, gene array analyses of infected lungs revealed major changes of inflammatory and immune gene expression signatures 4 weeks post-infection. Specifically, gene expression associated with host-pathogen interactions, macrophage recruitment, activation and polarization, host-antimycobacterial activities, immunomodulatory responses, as well as extracellular matrix metallopeptidases, were markedly modulated by TNFα blockade. IL-17A or IL-17F neutralization elicited only mild changes of few genes without impaired host resistance four weeks after M. tuberculosis infection. Further, the absence of both IL-17RA and IL-22 pathways in genetically deficient mice did not profoundly compromise host control of M. tuberculosis over a 6-months period, ruling out potential compensation between these two pathways, while TNFα-deficient mice succumbed rapidly. These data provide experimental confirmation of the low clinical risk of mycobacterial infection under anti-IL-17A therapy, in contrast to anti-TNFα treatment.

Currently, several immunotherapies and BACE (Beta Site APP Cleaving Enzyme) inhibitor approaches are being tested in the clinic for the treatment of Alzheimer's disease. A crucial mechanism-related safety concern is the exacerbation of microhemorrhages, which are already present in the majority of Alzheimer patients. To investigate potential safety liabilities of long-term BACE inhibitor therapy, we used aged amyloid precursor protein (APP) transgenic mice (APP23), which robustly develop cerebral amyloid angiopathy. T2*-weighted magnetic resonance imaging (MRI), a translational method applicable in preclinical and clinical studies, was used for the detection of microhemorrhages throughout the entire brain, with subsequent histological validation. Three-dimensional reconstruction based on in vivo MRI and serial Perls' stained sections demonstrated a one-to-one matching of the lesions thus allowing for their histopathological characterization. MRI detected small Perls' positive areas with a high spatial resolution. Our data demonstrate that volumetric assessment by noninvasive MRI is well suited to monitor cerebral microhemorrhages in vivo. Furthermore, 3 months treatment of aged APP23 with the potent BACE-inhibitor NB-360 did not exacerbate microhemorrhages in contrast to Aβ-antibody β1. These results substantiate the safe use of BACE inhibitors regarding microhemorrhages in long-term clinical studies for the treatment of Alzheimer's disease., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency., (© Society for Leukocyte Biology.)

In this paper, we investigate order-preserving sparse coding for classifying structured data whose atomic features possess ordering relationships. Examples include time sequences where individual frame-wise features are temporally ordered, as well as still images (landscape, street view, etc.) where different regions of the image are spatially ordered. Classification of these structured data is often tackled by first decomposing the input data into individual atomic features, then performing sparse coding or other processing for each atomic feature vector independently, and finally aggregating individual responses to classify the input data. However, this heuristic approach ignores the underlying order of the individual atomic features within the input data, and results in suboptimal discriminative capability. In this work, we introduce an order preserving regularizer which aims to preserve the ordering structure of the reconstruction coefficients within the sparse coding framework. An efficient Nesterov-type smooth approximation method is developed for optimization of the new regularization criterion, with theoretically guaranteed error bound. We perform extensive experiments for time series classification on a synthetic dataset, several machine learning benchmarks, and an RGB-D human activity dataset. We also report experiments for scene classification on a benchmark image dataset. The encoded representation is discriminative and robust, and our classifier outperforms state-of-the-art methods on these tasks.

Sphingosine-1-phosphate (S1P) lyase is considered as a drug target in autoimmune diseases based on the protective effect of reducing activity of the enzyme in animal models of inflammation. Since S1P lyase deficiency in mice causes a severe, lethal phenotype, it was of interest to investigate any pathological alterations associated with only partially reduced activity of S1P lyase as may be encountered upon pharmacological inhibition. Both genetic reduction of S1P lyase activity in mice and inhibition of S1P lyase with a low-molecular-weight compound in rats consistently resulted in podocyte-based kidney toxicity, which is the most severe finding. In addition, skin irritation and platelet activation were observed in both instances. The similarity of the findings in both the genetic model and the pharmacological study supports the value of analyzing inducible partially target-deficient mice for safety assessment. If the findings described in rodents translate to humans, target-related toxicity, particularly podocyte dysfunction, may limit chronic systemic treatment of autoimmune diseases with S1P lyase inhibitors. Furthermore, partial deficiency or inhibition of S1P lyase appears to provide an in vivo rodent model to enable studies on the mechanism of podocyte dysfunction., (© 2015 by The Author(s).)

Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumoral resistance to immune rejection. In humans, constitutive expression of IDO1 has been observed in several tumor types. However, a comprehensive analysis of its expression in normal and tumor tissues is still required to anticipate the risks and potential benefits of IDO1 inhibitors. Using a newly validated monoclonal antibody to human IDO1, we performed an extensive immunohistochemical analysis of IDO1 expression in normal and tumor tissues. In normal tissues, IDO1 was expressed by endothelial cells in the placenta and lung and by epithelial cells in the female genital tract. In lymphoid tissues, IDO1 was expressed in mature dendritic cells with a phenotype (CD83(+), DC-LAMP(+), langerin(-), CD123(-), CD163(-)) distinct from plasmacytoid dendritic cells. Importantly, IDO1-expressing dendritic cells were not enriched in tumor-draining lymph nodes, in contrast with previously reported findings. IDO1-expressing cells were observed in a large fraction (505/866, 58%) of human tumors. They comprised tumor cells, endothelial cells, and stromal cells in proportions that varied depending on the tumor type. Tumors showing the highest proportions of IDO1-immunolabeled samples were carcinomas of the endometrium and cervix, followed by kidney, lung, and colon. This hierarchy of IDO1 expression was confirmed by gene expression data mined from The Cancer Genome Atlas database. Expression of IDO1 may be used to select tumors likely to benefit from targeted therapy with IDO1 inhibitors., (©2014 American Association for Cancer Research.)

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and autophagy. Despite widespread clinical use of mTORC1 inhibitors, the role of mTORC1 in renal tubular function and kidney homeostasis remains elusive. By using constitutive and inducible deletion of conditional Raptor alleles in renal tubular epithelial cells, we discovered that mTORC1 deficiency caused a marked concentrating defect, loss of tubular cells, and slowly progressive renal fibrosis. Transcriptional profiling revealed that mTORC1 maintains renal tubular homeostasis by controlling mitochondrial metabolism and biogenesis as well as transcellular transport processes involved in countercurrent multiplication and urine concentration. Although mTORC2 partially compensated for the loss of mTORC1, exposure to ischemia and reperfusion injury exaggerated the tubular damage in mTORC1-deficient mice and caused pronounced apoptosis, diminished proliferation rates, and delayed recovery. These findings identify mTORC1 as an important regulator of tubular energy metabolism and as a crucial component of ischemic stress responses.

Paracetamol is the commonest cause of acute liver failure in the Western world and biomarkers are needed that report early hepatotoxicity. The liver-enriched microRNA (miRNA), miR-122, is a promising biomarker currently being qualified in humans. For biomarker development and drug toxicity screening, the zebrafish has advantages over rodents; however, blood acquisition in this model remains technically challenging. We developed a method for collecting blood from the adult zebrafish by retro-orbital (RO) bleeding and compared it to the commonly used lateral incision method. The RO technique was more reliable in terms of the blood yield and minimum amount per fish. This new RO technique was used in a zebrafish model of paracetamol toxicity. Paracetamol induced dose-dependent increases in liver cell necrosis, serum alanine transaminase activity, and mortality. In situ hybridization localized expression of miR-122 to the cytoplasm of zebrafish hepatocytes. After collection by RO bleeding, serum miR-122 could be measured and this miRNA was substantially increased by paracetamol 24 h after exposure, an increase that was prevented by delayed (3 h poststart of paracetamol exposure) treatment with acetylcysteine. In summary, collection of blood by RO bleeding facilitated measurement of miR-122 in a zebrafish model of paracetamol hepatotoxicity. The zebrafish represents a new species for measurement of circulating miRNA biomarkers that are translational and can bridge between fish and humans.

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

The purpose of this article is to characterize skin lesions in cynomolgus monkeys following vildagliptin (dipeptidyl peptidase-4 inhibitor) treatment. Oral vildagliptin administration caused dose-dependent and reversible blister formation, peeling and flaking skin, erosions, ulcerations, scabs, and sores involving the extremities at ≥5 mg/kg/day and necrosis of the tail and the pinnae at ≥80 mg/kg/day after 3 weeks of treatment. At the affected sites, the media and the endothelium of dermal arterioles showed hypertrophy/hyperplasia. Skin lesion formation was prevented by elevating ambient temperature. Vildagliptin treatment also produced an increase in blood pressure and heart rate likely via increased sympathetic tone. Following treatment with vildagliptin at 80 mg/kg/day, the recovery time after lowering the temperature in the feet of monkeys and inducing cold stress was prolonged. Ex vivo investigations showed that small digital arteries from skin biopsies of vildagliptin-treated monkeys exhibited an increase in neuropeptide Y-induced vasoconstriction. This finding correlated with a specific increase in NPY and in NPY1 receptors observed in the skin of vildagliptin-treated monkeys. Present data provide evidence that skin effects in monkeys are of vascular origin and that the effects on the NPY system in combination with increased peripheral sympathetic tone play an important pathomechanistic role in the pathogenesis of cutaneous toxicity., (© 2014 by The Author(s).)

The aim of this research is to understand how patients' characteristics increase healthcare providers' perceived workload. Patients' characteristics and dependency, technical and relational complexities of care seem to increase healthcare providers' workload. As workload is multidimensional, we examine which dimensions are affected by patients' characteristics. Our methodology is based on 121 patients assessed with the NASA task load index (NASA-TLX) and a questionnaire filled in by 57 health providers in 2 emergency wards in French hospital settings, to evaluate their attitudes to different patients' characteristics. Our results show that physical demand is the dimension most affected by patients' behaviour and characteristics. Next, we observe that workload increases more due to patients' behaviour than their social characteristics. We propose that a regulation mechanism be taken into account in further research, using methodology based on observations to identify how healthcare providers might adapt their activities to compensate for workload variations caused by patients.

Dipeptidyl peptidase IV (DPP4) is a peptidase whose inhibition is beneficial in Type II diabetes treatment. Several evidences suggest potential implication of DPP4 in skin disorders such as psoriasis, keloids and fibrotic skin diseases where its inhibition could also be beneficial. DPP4 expression in human skin was described mainly in dermal fibroblasts and a subset of keratinocytes in the basal layer. Of importance in the perspective of preclinical experimentation, DPP4 distribution in skin of non-human primate species has not been documented. This report evidences unexpected differences between a set of human and cynomolgus monkey skin samples revealing a major expression of DPP4 in eccrine sweat glands of cynomolgus monkeys but not in humans. This represents a unique distinctive feature compared to the conserved expression of dipeptidyl peptidases 8 and 9 and potential relevant DPP4 substrates such as neuropeptide Y (NPY) and receptors (NPY-receptor 1 and Neurokinin receptor). Finally the observation that cathepsin D, an unrelated protease, shows the opposite expression compared to DPP4 (present in human but not in cynomolgus monkey eccrine sweat glands) could indicate that human eccrine sweat glands evolved a divergent protease repertoire compared to non-human primates. These unexpected differences in the eccrine sweat glands protease repertoire will need to be confirmed extending the analysis to a major number of donors but could imply possible biochemical divergences, reflecting the functional evolution of the glands and the control of their activity. Our findings also demonstrate that non-human primates studies aiming at understanding DPP4 function in skin biology are not readily translatable to human.

Recognizing complex human activities usually requires the detection and modeling of individual visual features and the interactions between them. Current methods only rely on the visual features extracted from 2-D images, and therefore often lead to unreliable salient visual feature detection and inaccurate modeling of the interaction context between individual features. In this paper, we show that these problems can be addressed by combining data from a conventional camera and a depth sensor (e.g., Microsoft Kinect). We propose a novel complex activity recognition and localization framework that effectively fuses information from both grayscale and depth image channels at multiple levels of the video processing pipeline. In the individual visual feature detection level, depth-based filters are applied to the detected human/object rectangles to remove false detections. In the next level of interaction modeling, 3-D spatial and temporal contexts among human subjects or objects are extracted by integrating information from both grayscale and depth images. Depth information is also utilized to distinguish different types of indoor scenes. Finally, a latent structural model is developed to integrate the information from multiple levels of video processing for an activity detection. Extensive experiments on two activity recognition benchmarks (one with depth information) and a challenging grayscale + depth human activity database that contains complex interactions between human-human, human-object, and human-surroundings demonstrate the effectiveness of the proposed multilevel grayscale + depth fusion scheme. Higher recognition and localization accuracies are obtained relative to the previous methods.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Objective: Gain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH., Patients and Methods: The first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca(2+) and to the negative allosteric CASR modulator, NPS2143., Results: Two heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143., Conclusions: The asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.

Introduction: Following a US National Academy of Sciences report in 2007 entitled "Toxicity Testing of the 21st Century: a Vision and a Strategy," significant advances within translational drug safety sciences promise to revolutionize drug discovery and development. The purpose of this review is to outline why investigative safety science is a competitive advantage for the pharmaceutical industry., Areas Covered: The article discusses the essential goals for modern investigative toxicologists including: cross-species target biology; molecular pathways of toxicity; and development of predictive tools, models and biomarkers that allow discovery researchers and clinicians to anticipate safety problems and plan ways to address them, earlier than ever before. Furthermore, the article emphasizes the importance of investigating unanticipated clinical safety signals through a combination of mechanistic preclinical studies and/or molecular characterization of clinical samples from affected organs., Expert Opinion: The traditional boundaries between pharma industry teams focusing on safety/efficacy and preclinical/clinical development are rapidly disappearing in favor of translational safety science-centric organizations with a vision of bringing more effective medicines forward safely and quickly. Comparative biology and mechanistic toxicology approaches facilitate: i) identifying translational safety biomarkers; ii) identifying new drug targets/indications; and iii) mitigating off-target toxicities. These value-adding safety science contributions will change traditional toxicologists from side-effect identifiers to drug development enablers.

Lens explant cultures were used to assess the mechanism of drug-induced cataractogenic potential of NVS001, a peroxisome proliferator-activated receptor delta (PPARδ) agonist, which resulted in cataract in all treated animals during a 13-week rat study. Ciglitazone, a PPARγ agonist and cataractogenic compound, was used as a positive control to validate this model. Rat lenses were extracted and cultured in medium supplemented with antibiotics for 24-h preincubation pretreatment. Lenses showing no signs of damage at the end of the preincubation pretreatment period were randomized into five experimental groups, (1) untreated control, (2) 0.1% dimethyl sulphoxide control, (3) 10μM NVS001, (4) 10μM ciglitazone, and (5) 10μM acetaminophen (negative control). Lenses were treated every 24 h after preincubation pretreatment for up to 48 h. Samples for viability, histology, and gene expression profiling were collected at 4, 24, and 48 h. There was a time-dependent increase in opacity, which correlated to a decrease in viability measured by adenosine triphosphate levels in NVS001 and ciglitazone-treated lenses compared with controls. NVS001 and ciglitazone had comparable cataractogenic effects after 48 h with histology showing rupture of the lens capsule, lens fiber degeneration, cortical lens vacuolation, and lens epithelial degeneration. Furthermore, no changes were seen when lenses were treated with acetaminophen. Gene expression analysis supported oxidative and osmotic stress, along with decreases in membrane and epithelial cell integrity as key factors in NVS001-induced cataracts. This study suggests that in vitro lens cultures can be used to assess cataractogenic potential of PPAR agonists and to study/understand the underlying molecular mechanism of cataractogenesis in rat.

Background: Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor regulating a plethora of detoxifying enzymes and antioxidant genes involved in drug metabolism and defence against oxidative stress. The glucocorticoid receptor (GR) is a ligand-induced transcription factor involved in the regulation of energy supply for metabolic needs to cope with various stressors. GR activity is controlled by glucocorticoids, which are synthesized in the adrenal glands and regenerated mainly in the liver from inactive cortisone by 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1)., Methods and Principal Findings: Using transfected HEK-293 cells and hepatic H4IIE cells we show that glucocorticoids, activated by 11β-HSD1 and acting through GR, suppress the Nrf2-dependent antioxidant response. The expression of the marker genes NQO1, HMOX1 and GST2A was suppressed upon treatment of 11β-HSD1 expressing cells with cortisone, an effect that was reversed by 11β-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H(2)O(2). Moreover, a comparison of gene expression in male and female rats revealed an opposite sexual dimorphism with an inverse relationship between 11β-HSD1 and Nrf2 target gene expression., Conclusions: The results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11β-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific differences in hepatic expression levels of 11β-HSD1 and Nrf2 target genes and the impact of pharmacological inhibition of 11β-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies in vivo.

This article addresses the issue of miscorrelation between hepatic injury biomarkers and histopathological findings in the drug development context. Our studies indicate that the use of toxicogenomics can aid in the drug development decision-making process associated with such miscorrelated data. BLZ945 was developed as a Colony-Stimulating Factor 1 Receptor (CSF-1R) inhibitor. Treatment of BLZ945 in rats and monkeys increased serum alanine aminotransferase (ALT) and aspartate transaminase (AST). However, liver hypertrophy was the only histopathological liver finding in rats, and there was no change in the livers of monkeys. Longer treatment of BLZ945 in rats for 6 weeks caused up to 6-fold elevation of ALT, yet hepatocyte necrosis was not detected microscopically. Toxicogenomic profiling of liver samples demonstrated that the genes associated with early response to liver injury, apoptosis/necrosis, inflammation, oxidative stress, and metabolic enzymes were upregulated. Studies are ongoing to evaluate the mechanisms underlying BL945-induced ALT and AST elevations.

Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and beta2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.

Background: Acute peritonitis is the most frequent complication of peritoneal dialysis (PD). Increased nitric oxide (NO) release by NO synthase (NOS) isoforms has been implicated in acute peritonitis, but the role played by the NOS isoforms expressed in the peritoneum is unknown., Methods: We investigated the structural and functional consequences of acute peritonitis induced by LPS in wild-type (WT) mice versus knockout mice (KO) for the endothelial NOS (eNOS), the inducible NOS (iNOS) or the neuronal NOS (nNOS)., Results: The level of NO metabolites (NOx) in the dialysate was maximal 18 h after LPS injection. LPS induced a significant increase in the transport of small solutes and decreased ultrafiltration in WT mice. These changes, which occurred without vascular proliferation, were paralleled by the upregulation of nNOS and eNOS, and the induction of iNOS. The transport modifications induced by LPS were significantly reversed in eNOS KO mice, but not modified in mice lacking iNOS or nNOS. In contrast, the increase of dialysate NOx was abolished in iNOS KO mice and significantly reduced in eNOS KO mice, but left unchanged in mice lacking nNOS. Mice lacking iNOS also showed more severe inflammatory changes, and a trend towards increased mortality following LPS., Conclusion: These data demonstrate specific roles for NOS isoforms in the peritoneal membrane and suggest that selective eNOS inhibition may improve peritoneal transport during acute peritonitis.

Hypophosphatasia is an inheritable disorder characterised by defective bone mineralisation due to the impaired activity of tissue-non-specific alkaline phosphatase (AP). Clinical presentation ranges from stillbirth without mineralised bone to pathological fractures in late adulthood. During childhood, the main manifestations include rickets, growth delay and dental problems. Fractures and bone pain usually characterise the adult form. A 9-year-old girl was referred for repetitive fractures after minimal trauma. She had normal growth, normal sclerae, no rickets and minimal dental abnormalities. Her sister had also presented fractures. The proband, her sister and mother had low total and bone-specific AP levels and E435K mutation in exon 12 of the liver/bone/kidney AP gene. Low AP levels must lead to genetic analysis. Bone fragility and repetitive fractures may be symptoms of hypophosphatasia in childhood, which must not be neglected. Associated factors such as vitamin D or calcium deficiency must be prevented. In conclusion, hypophosphatasia must not be forgotten as an aetiological factor of repetitive fractures or bone pain in children and AP activity should be checked accurately.

In congenital hyperinsulinism of infancy (CHI), the loss of K-ATP channels (composed of Kir6.2 and SUR1 subunits) in beta cells induces permanent insulin secretion and severe hypoglycaemia. By contrast, Sur1 ( -/- ) mice do not present such defects. We have investigated the impact of Sur1 gene inactivation on mouse islet cell morphology, structure and basic physiology. Pancreata were collected from young, adult and old wild-type (WT) and Sur1 ( -/- ) mice. After immunostaining for hormone, the total endocrine tissue, cell proportion, cell size and intra-insular distribution, hormone content and Glut-2 expression were quantified by morphometry. Basic physiological parameters were also measured. In young Sur1 ( -/- ) mice, the total endocrine tissue and proportion of beta cells were higher (P<0.05) than in WT mice, whereas the proportion of delta cells was lower (P<0.01). In old Sur1 ( -/- ) mice, alpha cells were frequently located in the central regions of islets (unlike WT islets) and their proportion was increased (P<0.05). Glut-2 protein and mRNA levels were lower in old Sur1 ( -/- ) islets (P<0.02). Insulinaemia, fasting insulin and glucagon contents were equivalent in both groups of pancreata. Thus, the islets of Sur1 ( -/- ) mice present morphological modifications that have not been described in CHI and that might reflect an adaptive mechanism controlling insulin secretion in these mice.

Background/aims: Endometrial cells are chronically exposed to iron due to cyclic menstrual bleeding. Iron induces expression of adhesion molecules in endothelial cells. The purpose of this study was to investigate iron incorporation by human endometrial cells and to test whether iron may stimulate expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1., Methods: Endometrial stromal and epithelial cells were cultured in medium alone or supplemented with INF-gamma or transferrin (Tf). Iron incorporation by cells was quantified by densitometry of ferritin immunostaining. ICAM-1 and VCAM-1 expression were evaluated at the transcriptional level by real-time RT-PCR. Membrane-bound and soluble protein levels of ICAM-1 were measured by quantitative immunohistochemistry and ELISA, respectively., Results: Tf induced a significant increase in ferritin immunostaining in both endometrial cell types. Endometrial cells treated with INF-gamma expressed more ICAM-1 and VCAM-1 than untreated cells. By contrast, Tf treatment did not alter ICAM-1 and VCAM-1 expression in cultured endometrial cells., Conclusions: Endometrial cells are able to incorporate iron from Tf and to metabolize it to ferritin. Iron, unlike interferon-gamma, does not appear to be involved in the regulation of ICAM-1 and VCAM-1 expression in cultured endometrial cells.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death, and its incidence is increasing worldwide. Due to the known risk factors (mainly hepatitis B and C viruses), we believe there is a rationale for a chemopreventive approach to treat HCC. Here, based on described in vitro data, we evaluated the preventive effects of lanreotide, a somatostatin analog, on the induction of early carcinogenic events. We monitored preneoplastic foci induced by a two-stage initiation/promotion model of hepatocarcinogenesis in male Wistar rats, using diethylnitrosamine and 2-acetylaminofluorene. Lanreotide was given starting the day after the first diethylnitrosamine injection. By quantitative morphometry, we showed that lanreotide significantly decreases the size of induced preneoplastic foci. Analysis of proliferation and apoptosis assessed by immunohistochemistry, showed decreased proliferation and increased cell death in rats treated with lanreotide. As these events were associated with a significant decreased expression of the cell cycle regulator cyclin D1 and an increased expression of the cyclin-dependent kinase inhibitor p27(kip1) compared to the non-treated group, it is tempting to speculate that these factors are involved in the favorable effect of lanreotide. In conclusion, lanreotide significantly decreases early carcinogenic transformation in a two-step rat model. As lanreotide has a low toxicity profile, we believe it would be interesting to evaluate its effect in chemoprevention of HCC.

The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.

Na+/H+ exchangers (NHE) constitute a family of membrane antiporters that contribute to the regulation of cellular pH and volume in many tissues, including pancreatic islets. We investigated the molecular identity of NHE in rodent and human endocrine pancreas, and determined its cellular and subcellular localization. NHE1 was the most abundantly expressed isoform in rat islets, and was also expressed in mouse and human islets. By western blot, an antiserum raised against the C-terminus end of NHE1 confirmed the presence of a ~100 kDa protein corresponding to NHE1 in islets and unexpectedly unveiled the existence of a ~65 kDa cross-reactive NHE1-related protein. By immunohistochemistry, the antiserum labelled the membranes of pancreatic acini and ducts, but also diffusely stained the cytoplasm of insulin, glucagon and somatostatin cells as well as endocrine cells of the adrenal medulla. Electron microscopy localized the NHE1 immunoreactivity in the membrane of secretory granules, an unexpected finding supported by a decrease in immunohistochemical signal in degranulated beta-cells. Islets of Slc9A1(swe/swe) mice, which lack full NHE1 protein, were found to express an mRNA corresponding to the 3' end of NHE1 as well as the ~65 kDa protein. They still showed the cytoplasmic labelling but no plasma membrane was stained. We conclude that both the full-length and the shorter-splice variant of NHE1 are expressed in all cell types of the endocrine pancreas and in the adrenal medulla of rodents and humans. The complete protein is addressed to the plasma membrane and the shorter one to the membrane of secretory granules where its function remains to be established.

Purpose: To assess whether hepatic fibrosis is associated with a restriction in the diffusion of water that can be analyzed with diffusion-weighted MR imaging (DWI) of the liver., Materials and Methods: DWI was performed in 10 normal rats and 15 rats with liver fibrosis. Echo-planar DWI was performed in the living rats at 1.5 T and repeated immediately after the animals were killed. Afterwards the livers were explanted, fixed in Bouin solution, and imaged with a DW spin-echo sequence at 4.7 T. Fibrosis was quantified by densitometry on Sirius red-stained histological sections., Results: In living rats the apparent diffusion coefficient (ADC) decreased with the severity of liver fibrosis (controls: 1535 +/- 294 mm(2)/second; CCl(4) (5 weeks) 1129 +/- 273 mm(2)/second; CCl(4) (9 weeks): 943 +/- 132 mm(2)/second; P = 0.002). An inverse correlation between ADC and liver fibrosis volume density was observed (r = -0.712, P < 0.001). In contrast, these findings were not observed in the rats after they were killed or in the fixated livers., Conclusion: Decreased ADC correlated with increased liver fibrosis in living rats, but not after death. These results suggest that restricted water diffusion cannot be assessed by DWI in liver fibrosis. Other factors, such as a decrease of perfusion, may explain the decrease of the hepatic ADC measured in vivo in rats with liver fibrosis.