Impact of everolimus plus calcineurin inhibitor on formation of non-HLA antibodies and graft outcomes in kidney transplant recipients: 12-month results from the ATHENA substudy

BackgroundNon-human leukocyte antigen (non-HLA) antibodies including antibodies targeting Angiotensin II type 1 (AT1R) and Endothelin-1 type A (ETAR) receptors represent a topic of interest in kidney transplantation (KTx). This exploratory substudy evaluated the impact of everolimus (EVR) or mycophenolic acid (MPA) in combination with tacrolimus (TAC) or cyclosporine A (CsA) in patients with preformed non-HLA antibodies, potentially associated rejections and/or their impact on renal function over 1 year.MethodsAll eligible patients were randomized (1:1:1) before transplantation to receive either EVR/TAC, EVR/CsA, or MPA/TAC regimen. The effect of these regimens on the formation of non-HLA antibodies within one year post de novo KTx and the association with clinical events was evaluated descriptively in randomized (n = 268) population.ResultsAt Month 12, in EVR/TAC group, higher incidence of patients negative for AT1R- and ETAR-antibodies (82.2% and 76.7%, respectively) was noted, whereas the incidence of AT1R- and ETAR-antibodies positivity (28.1% and 34.7%, respectively) was higher in the MPA/TAC group. Non-HLA antibodies had no influence on clinical outcomes in any treatment group and no graft loss or death was reported.ConclusionsThe studied combinations of immunosuppressants were safe with no influence on clinical outcomes and suggested minimal exposure of calcineurin inhibitors for better patient management.Clinical Trial Registrationhttps://clinicaltrials.gov/ (NCT01843348; EudraCT number: 2011-005238-21)

Physical Model Tests on Spar Buoy for Offshore Floating Wind Energy Conversion

ABSTRACT: The present paper describes the experiences gained from the design methodology and operation of a 3D physical modelexperiment aimed to investigate the dynamic behaviour of a spar buoy floating offshore wind turbine. The physical model consists in a Froude-scaled NREL 5MW reference wind turbine (RWT) supported on the OC3-Hywind floating platform. Experimental tests have been performed at Danish Hydraulic Institute (DHI) offshore wave basin within the European Union-Hydralab+ Initiative, in April 2019. The floating wind turbine model has been subjected to a combination of regular and irregular wave attacks and different wind loads. Measurements of displacements, rotations, accelerations, forces response of the floating model and at the mooring lines have been carried out. First, free decay tests have been analysed to obtain the natural frequency and the modal damping ratios of each degree of freedom governing the offshore. Then, the results concerning regular waves, with orthogonal incidence to the structure, are presented. The results show that most of longitudinal dynamic response occurs at the wave frequency and most of lateral dynamic response occurs at rigid-body frequencies.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 654110, HYDRALAB+

Miz1 Deficiency in the Mammary Gland Causes a Lactation Defect by Attenuated Stat5 Expression and Phosphorylation

Miz1 is a zinc finger transcription factor with an N-terminal POZ domain. Complexes with Myc, Bcl-6 or Gfi-1 repress expression of genes like Cdkn2b (p15(Ink4)) or Cd-kn1a (p21(Cip1)). The role of Miz1 in normal mammary gland development has not been addressed so far. Conditional knockout of the Miz1 POZ domain in luminal cells during pregnancy caused a lactation defect with a transient reduction of glandular tissue, reduced proliferation and attenuated differentiation. This was recapitulated in vitro using mouse mammary gland derived HC11 cells. Further analysis revealed decreased Stat5 activity in Miz1 Delta POZ mammary glands and an attenuated expression of Stat5 targets. Gene expression of the Prolactin receptor (PrlR) and ErbB4, both critical for Stat5 phosphorylation (pStat5) or pStat5 nuclear translocation, was decreased in Miz1 Delta POZ females. Microarray, ChIP-Seq and gene set enrichment analysis revealed a down-regulation of Miz1 target genes being involved in vesicular transport processes. Our data suggest that deranged intracellular transport and localization of PrlR and ErbB4 disrupt the Stat5 signalling pathway in mutant glands and cause the observed lactation phenotype

Protein expression and localization of Prlr and ErbB4.

<p>PrlR (<b>A</b>) and ErbB4 (<b>B</b>) immunofluorescence from control and <i>Miz1</i>Δ<i>POZ</i> mammary gland tissue (lactation day 6). (<b>C</b>) Hypothetical model about the function of Miz1 in the mouse lactating mammary gland. Vesicular transport processes are impaired in <i>Miz1</i>Δ<i>POZ</i> mice due to a decreased gene expression of Miz1 target genes which are involved in the vesicular transport. This causes a reduction of the PrlR and ErbB4 exposure to the plasma membrane, hampering the autoamplifying expression of PrlR. Reduction of PrlR and ErbB4 expression and their diminished availability at the cell surface leads to a decreased amount of phosphorylated Stat5, which is the key regulator during lactation. In consequence, reduced levels of phosphorylated Stat5 dimers (represented by smaller symbol size) cannot adequately activate the transcription of proliferation and differentiation genes in <i>Miz1ΔPOZ</i> glands <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089187#pone.0089187-Hennighausen2" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089187#pone.0089187-Williams1" target="_blank">[53]</a>. Scale bars in A and B: 50 µm.</p

Genes related to vesicular transport processes are bound by Miz1 and down-regulated in <i>Miz1</i>Δ<i>POZ</i> mammary glands.

<p>(<b>A</b>) GSEA analysis comparing the gene expression of wildtype versus <i>Miz1</i>Δ<i>POZ</i> mammary glands. The 100 Miz1 target promoters with the highest tag number were used as the gene set in this analysis. (<b>B</b>) Browser pictures of Miz1 ChIP-Seq profiles at the Miz1 target genes <i>Exoc2</i>, <i>Vamp4</i> and <i>Lrp12.</i> (<b>C</b>) Quantitative RT-PCRs testing the expression of genes indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089187#pone-0089187-t001" target="_blank">Table 1</a>. (<b>D</b>) Electron microscopy showing vesicles with (arrows) and without (asterisks) casein micelles in tissue from control and <i>Miz1</i>Δ<i>POZ</i> animals. (<b>E</b>) Percentage of the two vacuole types in mammary gland epithelial cells of control and <i>Miz1</i>Δ<i>POZ</i> animals from lactation day 10. Data obtained from 4 animals per genotype. Total number of vacuoles counted: ctr, 309–379; ΔPOZ, 339–404. Scale bar in D: 1 µm.</p

Miz1 function on proliferation and apoptosis of mammary gland epithelial cells <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Ki67 immunostaining in control and <i>Miz1</i>Δ<i>POZ</i> lactation day 6 mammary glands. (<b>B</b>) Ki67 labeling index (up) and quantitative RT-PCR (down) obtained from lactation day 6 samples. (<b>C</b>) The expression of potential proliferation regulators like <i>Cdkn1a</i> and <i>Myc</i> was measured by qRT-PCR. (<b>D</b>) The TUNEL assay was performed on tissue from lactation day 1 and lactation day 6, respectively. Quantifications obtained from 20x pictures are shown (at least n = 3 per genotype and time-point). (<b>E</b>) Representative confocal microscopy pictures of acinar structures formed 6, 8 and 10 days after seeding <i>shscr</i> and <i>shMiz1</i> transfected HC11 cells onto Cultrex-coated coverslips. Nuclei were stained with Hoechst (blue), actin filaments with Phalloidin-TRITC (red) and apoptotic cells by cleaved Caspase-3 immunostaining (green). (<b>F</b>) Western Blot showing the knock-down of Miz1 in <i>shMiz1</i> HC11 cells. Numbers indicated are fold changes of band intensities obtained by densitometry (see Materials and Methods). Acinar cell apoptosis and proliferation were quantified by analysis of cleaved caspase-3 (<b>G</b>) and Ki67 (<b>H</b>) positivity in double immunofluorescence confocal microscopy pictures. (<b>I</b>) Quantification of the number of nuclei per acinus in <i>shscr</i> and <i>shMiz1</i> transfected HC11 cells. Data from two independent experiments were merged and the total number of acini analysed is indicated. See Materials and Methods for experimental details. Scale Bar in A: 50 µm; E: 25 µm.</p

GSEA analysis.

<p>ChIP-Seq data from MDA-MB231 cells were combined with microarray expression data obtained from control and <i>Miz1</i>Δ<i>POZ</i> mammary gland tissue at lactation day 6. Listed are genes which show a strong Miz1 binding (>200 binding tags) and a down-regulation in <i>Miz1</i>Δ<i>POZ</i> animals. Genes which are related to vesicular transport processes are highlighted in bold.</p

Stat5 phosphorylation is reduced in <i>Miz1</i>Δ<i>POZ</i> mammary glands and in <i>shMiz1</i> HC11 cells.

<p>(<b>A</b>) Analysis of Stat5a/b mRNA expression in <i>Miz1</i>Δ<i>POZ</i> (ΔPOZ) and control mammary gland tissue (<i>Ctr</i>). (<b>B</b>) Immunoblot analysis of Stat5 in wildtype and <i>Miz1</i>Δ<i>POZ</i> mammary glands. (<b>C</b>) Immunohistochemical staining of pStat5 in the mammary gland tissue from wildtype and <i>Miz1</i>Δ<i>POZ</i> animals. (<b>D</b>) Western blots from HC11 cells stably transfected with a scrambled short hairpin (sh) RNA (shscr) or a Miz1 shRNA (see also Fig. 4<b>E</b> and <b>F</b>). Numbers indicated are fold changes of band intensities obtained by densitometry (see Materials and Methods). Quantitative RT-PCR for the prolactin receptor (<b>E</b>; <i>Prlr</i>), the Supressors of cytokine signalling (<i>Socs</i>) 1, 2 and 3 and caveolin-1 (<b>F</b>; <i>Cav1</i>), and for <i>ErbB4</i> (<b>G</b>). Lactation day 6 samples were used in all <i>in vivo</i> experiments. Scale bar in C: 50 µm.</p

Female mice with a <i>Miz1</i>Δ<i>POZ</i> mammary gland exhibit a lactation defect.

<p>(<b>A</b>) Time course of the body weight of the offspring from control (black, n = 6) and <i>Miz1</i>Δ<i>POZ</i> mothers (blue, n = 6). The number of pups per mother was set to 6 at birth. (<b>B</b>) Size differences in 24-day-old pups did not depend on their gender. (<b>C</b>) Mammary glands from mothers of lactation day 6 were investigated by histology with H & E sections and glands from mothers of lactation day 1 were analysed in whole mounts (<b>D</b>). Morphometric analysis of the adipose tissue content from H & E sections (Lactation day 6) are shown in (<b>E</b>). Note that the difference in the ratio of glandular to adipose tissue is similar during the first and second pregnancies. Scale bar in C: 500 µm.</p