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The aim of the paper is to prospectively describe early and mid-term outcomes for emergency endovascular aneurysm repair (eEVAR) versus open surgery in acute abdominal aortic aneurysms (aAAAs), both unruptured (symptomatic) and ruptured. We enrolled all consecutive patients treated for aAAA at our center between April 2002 and April 2008. The main outcome parameters were 30-day, 6- and 12-month mortality (all-cause and aneurysm-related). Two hundred forty patients were enrolled in the study. In the unruptured aAAA group (n = 111), 47 (42%) underwent eEVAR. The 30-day, 6- and 12-month mortality rates were 6, 13 and 15% in the eEVAR group versus 11% (NS), 13% (NS) and 16% (NS) in the open group, respectively. In the ruptured aAAA group (n = 129), 25 (19%) underwent eEVAR (mortality rates: 20, 28 and 36%, respectively) compared with 104 (81%) patients who underwent open surgery (mortality rates: 45% (P = 0.021), 60% (P = 0.004) and 63% (P = 0.014), respectively). In conclusion, the present study showed a reduced 30-day, 6- and 12-month mortality of eEVAR compared with open surgery in all patients with aAAA, mainly due to a lower mortality in the ruptured aAAA group. Late aneurysm-related mortality occurred only in the eEVAR group. [ABSTRACT FROM AUTHOR]

BACKGROUND AND AIMS: The role of transforming growth factor beta (TGFβ) in inhibiting T cell function in the normal gut has been studied in animal models. However, the impact of TGFβ inhibition on T cells in the normal human gut remains poorly understood. The effect of TGFβ blockade in normal intestinal biopsies grown ex vivo and lamina propria mononuclear cells (LPMCs) on T-bet, a T-box transcription factor required for T helper cell type (Th)1 differentiation, interferon γ (IFN γ) production, T cell apoptosis and matrix metalloproteinase (MMP)-3 production has therefore been tested. METHODS: TGFβ transcripts were determined by quantitative reverse transcription-PCR in laser-captured gut epithelium and lamina propria. Biopsies and LPMCs were cultured with anti-TGFβ neutralising antibody. After 24 h culture, T-bet was determined by immunoblotting, and T cell apoptosis was assessed by flow cytometry. IFN γ, tumour necrosis factor α (TNFα), interleukin (IL) 2, IL6, IL8, IL10, IL12p70 and IL17 were measured by ELISA. MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were assessed by immunoblotting. RESULTS: A higher number of TGFβ transcripts was found in the lamina propria than in the epithelium in normal gut. T-bet expression was significantly higher in biopsies and LPMCs cultured with anti-TGFβ antibody than in those cultured with control antibody. TGFβ blockade downregulated T cell apoptosis, and induced a significant increase in IFN γ, TNFα, IL2, IL6, IL8 and IL17 production. A higher expression of MMP-3, but not TIMP-1, was observed in the tissue and supernatant of biopsies treated with anti-TGFβ antibody. CONCLUSIONS: The findings support a crucial role for TGFβ in dampening T cell-mediated tissue-damaging responses in the human gut. [ABSTRACT FROM AUTHOR]

Abstract Background Disease severity and functional impairment in patients with intermittent claudication is usually quantified by the measurement of pain-free walking distance (intermittent claudication distance, ICD) and maximal walking distance (absolute claudication distance, ACD). However, the distance at which a patient would prefer to stop because of claudication pain seems a definition that is more correspondent with the actual daily life walking distance. We conducted a study in which the distance a patient prefers to stop was defined as the functional claudication distance (FCD), and estimated the reliability and validity of this measurement. Methods In this clinical validity study we included patients with intermittent claudication, following a supervised exercise therapy program. The first study part consisted of two standardised treadmill tests. During each test ICD, FCD and ACD were determined. Primary endpoint was the reliability as represented by the calculated intra-class correlation coefficients. In the second study part patients performed a standardised treadmill test and filled out the Rand-36 questionnaire. Spearman's rho was calculated to assess validity. Results The intra-class correlation coefficients of ICD, FCD and ACD were 0.940, 0.959, and 0.975 respectively. FCD correlated significantly with five out of nine domains, namely physical function (rho = 0.571), physical role (rho = 0.532), vitality (rho = 0.416), pain (rho = 0.416) and health change (rho = 0.414). Conclusion FCD is a reliable and valid measurement for determining functional capacity in trained patients with intermittent claudication. Furthermore it seems that FCD better reflects the actual functional impairment. In future studies, FCD could be used alongside ICD and ACD.

Abstract Background The ankle brachial index (ABI) is a well-established tool for screening and diagnosis of peripheral arterial disease (PAD). In this study we assessed the validity of ABI determination using a pocket Doppler device compared with automatic vascular laboratory measurement in patients suspected of PAD. Methods Consecutive patients with symptoms of PAD referred for ABI measurement between December 2006 and August 2007 were included. Resting ABI was determined with a pocket Doppler, followed by ABI measurement with automatic vascular laboratory equipment, performed by an experienced vascular technician. The leg with the lowest ABI was used for analysis. Results From 99 patients the mean resting ABI was 0.80 measured with the pocket Doppler and 0.85 measured with vascular laboratory equipment. A Bland-Altman plot demonstrated great correspondence between the two methods. The mean difference between the two methods was 0.05 (P < .001). Multivariate linear regression analysis showed no dependency of the difference on either the average measured ABI or affected or unaffected leg. Conclusion Since the small, albeit statistically significant, difference between the two methods is not clinically relevant, our study demonstrates that ABI measurements with pocket Doppler and vascular laboratory equipment yield comparable results and can replace each other. Results support the use of the pocket Doppler for screening of PAD, allowing initiation of cardiovascular risk factor management in primary care, provided that the equipment operator is experienced.

The upregulation of matrix metalloproteinases (MMPs) in the inflamed gut has mainly been associated with mucosal degradation and ulceration. However, their in vitro capacity to specifically cleave inflammatory mediators indicates that MMPs may also have a profound immunoregulatory impact. In this study, we assessed whether MMPs proteolytically modify intestinal epithelial chemokine signalling. Fully differentiated CaCo-2 cells were grown on filters, stimulated with lL-1β, and exposed basolaterally to nanomolar concentrations of activated MMP-3. Chemotaxis assays of the conditioned media revealed that MMP-3 dosedependently induced the neutrophil, but not monocyte, chemoattractant capacity of CaCo-2 cells. A similar response was obtained when these cells were co-cultured with IL-1β-stimulated colonic fibroblasts (CCD-18co), which expressed various MMPs, including MMP-3, -10, and -12. The addition of doxycyclin, a broad-spectrum MMP inhibitor, disrupted the CaCo-2 chemotactic response. The principal mediator of these protease-related effects was identified as the potent neutrophil chemokine neutrophil activating peptide 2 (NAP-2, CXCL7), a cleavage product of biologically inactive platelet basic protein (PBP). Antibodies against NAP-2 greatly inhibited the MMP-induced chemotactic response, and PBP mRNA and protein was detected in stimulated CaCo-2, but not in CCD-18co cells. Our data suggest that fibroblast-derived MMPs proteolytically activate the neutrophil chemokine NAP-2 from the intestinal epithelium, adding a novel dimension to MMP function and to our understanding of the pathogenesis of intestinal inflammation. [ABSTRACT FROM AUTHOR]

The human TNF/LT locus genes TNF , LTA , and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4 + T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF , LTA , and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays.

Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 μM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

Despite their abundance at gastrointestinal sites, little is known about the role of galectins in gut immune responses. We have therefore investigated the Citrobacter rodentium model of colonic infection and inflammation in Galectin-1 or Galectin-3 null mice. Gal-3 null mice showed a slight delay in colonisation after inoculation with C. rodentium and a slight delay in resolution of infection, associated with delayed T cell, macrophage and dendritic cell infiltration into the gut mucosa. However, Gal-1 null mice also demonstrated reduced T cell and macrophage responses to infection. Despite the reduced T cell and macrophage response in Gal-1 null mice, there was no effect on C. rodentium infection kinetics and pathology. Overall, Gal-1 and Gal-3 play only a minor role in immunity to a gut bacterial pathogen.

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Epigenetics encompasses transient and heritable modifications to DNA and nucleosomes in the native chromatin context. For example, enzymatic addition of chemical moieties to the N-terminal "tails" of histones, particularly acetylation and methylation of lysine residues in the histone tails of H3 and H4, plays a key role in regulation of gene transcription. The modified histones, which are physically associated with gene regulatory regions that typically occur within conserved noncoding sequences, play a functional role in active, poised, or repressed gene transcription. The "histone code" defined by these modifications, along with the chromatin-binding acetylases, deacetylases, methylases, demethylases, and other enzymes that direct modifications resulting in specific patterns of histone modification, shows considerable evolutionary conservation from yeast to humans. Direct modifications at the DNA level, such as cytosine methylation at CpG motifs that represses promoter activity, are another highly conserved epigenetic mechanism of gene regulation. Furthermore, epigenetic modifications at the nucleosome or DNA level can also be coupled with higher-order intra- or interchromosomal interactions that influence the location of regulatory elements and that can place them in an environment of specific nucleoprotein complexes associated with transcription. In the mammalian immune system, epigenetic gene regulation is a crucial mechanism for a range of physiological processes, including the innate host immune response to pathogens and T cell differentiation driven by specific patterns of cytokine gene expression. Here, we will review current findings regarding epigenetic regulation of cytokine genes important in innate and/or adaptive immune responses, with a special focus upon the tumor necrosis factor/lymphotoxin locus and cytokine-driven CD4+ T cell differentiation into the Th1, Th2, and Th17 lineages., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/β-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1β-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1β-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1β-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1β stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1β-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1β-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound repair.

JMJD3 (KDM6B) antagonizes Polycomb silencing by demethylating lysine 27 on histone H3. The interplay of methyltransferases and demethylases at this residue is thought to underlie critical cell fate transitions, and the dynamics of H3K27me3 during neurogenesis posited for JMJD3 a critical role in the acquisition of neural fate. Despite evidence of its involvement in early neural commitment, however, its role in the emergence and maturation of the mammalian CNS remains unknown. Here, we inactivated Jmjd3 in the mouse and found that its loss causes perinatal lethality with the complete and selective disruption of the pre-Bötzinger complex (PBC), the pacemaker of the respiratory rhythm generator. Through genetic and electrophysiological approaches, we show that the enzymatic activity of JMJD3 is selectively required for the maintenance of the PBC and controls critical regulators of PBC activity, uncovering an unanticipated role of this enzyme in the late structuring and function of neuronal networks., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)