LPS-induced Pellino3 degradation is mediated by p62-dependent autophagy

Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade. Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1. Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism. Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis

Kinetic characterization of selective peroxisome-proliferator-activated receptor gamma modulators in vitro

Background: The ligand-activated transcription factor, peroxisome-proliferator-activated receptor gamma (PPARγ), has been shown to play an essential role in immunosuppression during sepsis. PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion. In the polymicrobial cecum ligation and puncture (CLP) sepsis model in mice, both T-cell-specific gene knockout (Lck-Cre PPARγfl/fl) and systemic pharmacological PPARγ antagonism by GW9662 improved survival. Because GW9662 was only effective when applied 3 hours after CLP, we were interested to extend this time frame. For this reason we characterized the kinetics of SPPARγMs when administered before or in combination with the agonist thiazolidinedione, rosiglitazone. Methods: A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format. Results: Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours. Conclusion: Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis

Attenuated NOX2 expression impairs ROS production during the hypoinflammatory phase of sepsis

Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased. Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS. Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis. Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients

5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells

Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle. Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression. Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells. Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

Leptin restores markers of female fertility in lipodystrophy

Objectives: Female reproductive dysfunction occurs in patients with pathological loss of adipose tissue, i.e. lipodystrophy (LD). However, mechanisms remain largely unclear and treatment effects of adipocyte-derived leptin have not been assessed in LD animals. Methods: In the current study, C57131/6 LD mice on a low-density lipoprotein receptor knockout background were treated with leptin or saline for 8 weeks and compared to non-LD controls. Results: The number of pups born was 37% lower in breeding pairs consisting of LD female mice x non-LD male mice (n = 3.3) compared to LD male mice x non-LD female mice (n = 5.2) (p < 0.05). Mean uterus weight was significantly lower in the saline-treated LD group (18.8 mg) compared to non-LD controls (52.9 mg; p < 0.0001) and increased significantly upon leptin treatment (46.5 mg; p < 0.001). The mean number of corpora lutea per ovary was significantly lower in saline-treated LD animals compared to non-LD controls (p < 0.01) and was restored to non-LD control levels by leptin (p < 0.05). Mechanistically, mRNA expression of ovarian follicle stimulating hormone receptor (p < 0.01) and estrogen receptor beta (p < 0.05), as well as of pituitary luteinizing hormone beta subunit (p < 0.001) and follicle-stimulating hormone beta subunit (p < 0.05), was significantly up regulated in LD mice compared to non-LD controls. In addition, mean time to vaginal opening as a marker of puberty onset was delayed by 12.5 days in LD mice (50.9 days) compared to non-LD controls (38.4 days; p < 0.001). Conclusions: Female LD animals show impaired fertility which is restored by leptin. Future studies should assess leptin as a subfertility treatment in human leptin-deficiency disorders

DataSheet_1_A multi-center interventional study to assess pharmacokinetics, effectiveness, and tolerability of prolonged-release tacrolimus after pediatric kidney transplantation: study protocol for a prospective, open-label, randomized, two-phase, two-sequence, single dose, crossover, phase III b trial.docx

BackgroundTacrolimus, a calcineurin inhibitor (CNI), is currently the first-line immunosuppressive agent in kidney transplantation. The therapeutic index of tacrolimus is narrow due to due to the substantial impact of minor variations in drug concentration or exposure on clinical outcomes (i.e., nephrotoxicity), and it has a highly variable intra- and inter-individual bioavailability. Non-adherence to immunosuppressants is associated with rejection after kidney transplantation, which is the main cause of long-term graft loss. Once-daily formulations have been shown to significantly improve adherence compared to twice-daily dosing. Envarsus®, the once-daily prolonged-release formulation of tacrolimus, offers the same therapeutic efficacy as the conventional twice-daily immediate-release tacrolimus formulation (Prograf®) with improved bioavailability, a more consistent pharmacokinetic profile, and a reduced peak to trough, which may reduce CNI-related toxicity. Envarsus® has been approved as an immunosuppressive therapy in adults following kidney or liver transplantation but has not yet been approved in children. The objective of this study is to evaluate the pharmacokinetic profile, efficacy, and tolerability of Envarsus® in children and adolescents aged ≥ 8 and ≤ 18 years to assess its potential role as an additional option for immunosuppressive therapy in children after kidney transplantation.Methods/designThe study is designed as a randomized, prospective crossover trial. Each patient undergoes two treatment sequences: sequence 1 includes 4 weeks of Envarsus® and sequence 2 includes 4 weeks of Prograf®. Patients are randomized to either group A (sequence 1, followed by sequence 2) or group B (sequence 2, followed by sequence 1). The primary objective is to assess equivalency between total exposure (of tacrolimus area under the curve concentration (AUC0-24)), immediate-release tacrolimus (Prograf®) therapy, and prolonged-release tacrolimus (Envarsus®) using a daily dose conversion factor of 0.7 for prolonged- versus immediate-release tacrolimus. Secondary objectives are the assessment of pharmacodynamics, pharmacogenetics, adherence, gut microbiome analyses, adverse events (including tacrolimus toxicity and biopsy-proven rejections), biopsy-proven rejections, difference in estimated glomerular filtration rate (eGFR), and occurrence of donor-specific antibodies (DSAs).DiscussionThis study will test the hypothesis that once-daily prolonged-release tacrolimus (Envarsus®) is bioequivalent to twice-daily intermediate-release tacrolimus after pediatric kidney transplantation and may reduce toxicity and facilitate medication adherence. This novel concept may optimize immunosuppressive therapy for more stable graft function and increased graft survival by avoiding T-cell mediated and/or antibody-mediated rejection due to improved adherence. In addition, the study will provide data on the pharmacodynamics and pharmacogenetics of prolonged-release tacrolimus in children and adolescents.Clinical Trial RegistrationEUDRA-CT 2019-003710-13 and ClinicalTrial.gov, identifier NCT06057545.</p

More Judicious Use of Expectant Management for Localized Prostate Cancer during the Last 2 Decades

Urologists have been criticized for overtreating men with low risk prostate cancer and for passively observing older men with higher risk disease. Proponents of active surveillance for low risk disease and critics of watchful waiting for higher risk disease have advocated for more judicious use of observation. Thus, we compared 2 population based cohorts to determine how expectant management has evolved during the last 2 decades. A total of 5,871 men with localized prostate cancer were enrolled in the PCOS (Prostate Cancer Outcomes Study) or the CEASAR (Comparative Effectiveness Analysis of Surgery and Radiation) study. We compared the use of definitive treatment vs expectant management (watchful waiting or active surveillance) across cohorts, focusing on the influence of disease risk, age and comorbidities. Use of watchful waiting or active surveillance was similar in PCOS and CEASAR (14% in each). Compared to the PCOS, more men in the CEASAR study with low risk disease selected watchful waiting or active surveillance (25% vs 15%, respectively), whereas fewer men with intermediate (7% vs 14%) and high risk (3% vs 10%) disease chose watchful waiting or active surveillance (p <0.001 for each). The association of disease risk with watchful waiting or active surveillance was significantly larger in CEASAR than in PCOS (OR 7.3, 95% CI 3.4 to 15.7). Older age was associated with watchful waiting or active surveillance in both cohorts but there was no association between comorbidity and watchful waiting or active surveillance in the CEASAR study. Use of watchful waiting or active surveillance was more aligned with disease risk in CEASAR compared to PCOS, suggesting there has been a pivot from watchful waiting to active surveillance. While older men were more likely to be observed, comorbidity had little, if any, influence

ACR Appropriateness Criteria \u3csup\u3e®\u3c/sup\u3e Radiologic Management of Venous Thromboembolism-Inferior Vena Cava Filters

© 2019 American College of Radiology Venous thromboembolism (VTE)—deep vein thrombosis and pulmonary embolism—is a common cause of morbidity and mortality. The mainstay of VTE prophylaxis and therapy is anticoagulation. In select patients with VTE, inferior vena cava (IVC) filters are used to prevent pulmonary embolism by trapping emboli as they pass from the lower extremity venous system through the IVC. These guidelines review the indications for placement of IVC filters in acute and chronic VTE, as well as the indications for retrieval of implanted IVC filters. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment