Genetic and environmental risk factors for atherosclerosis regulate transcription of phosphatase and actin regulating gene PHACTR1.

BACKGROUND AND AIMS: Coronary artery disease (CAD) risk is associated with non-coding genetic variants at the phosphatase and actin regulating protein 1(PHACTR1) gene locus. The PHACTR1 gene encodes an actin-binding protein with phosphatase regulating activity. The mechanism whereby PHACTR1 influences CAD risk is unknown. We hypothesized that PHACTR1 would be expressed in human cell types relevant to CAD and regulated by atherogenic or genetic factors. METHODS AND RESULTS: Using immunohistochemistry, we demonstrate that PHACTR1 protein is expressed strongly in human atherosclerotic plaque macrophages, lipid-laden foam cells, adventitial lymphocytes and endothelial cells. Using a combination of genomic analysis and molecular techniques, we demonstrate that PHACTR1 is expressed as multiple previously uncharacterized transcripts in macrophages, foam cells, lymphocytes and endothelial cells. Immunoblotting confirmed a total absence of PHACTR1 in vascular smooth muscle cells. Real-time quantitative PCR showed that PHACTR1 is regulated by atherogenic and inflammatory stimuli. In aortic endothelial cells, oxLDL and TNF-alpha both upregulated an intermediate length transcript. A short transcript expressed only in immune cells was upregulated in macrophages by oxidized low-density lipoprotein, and oxidized phospholipids but suppressed by lipopolysaccharide or TNF-alpha. In primary human macrophages, we identified a novel expression quantitative trait locus (eQTL) specific for this short transcript, whereby the risk allele at CAD risk SNP rs9349379 is associated with reduced PHACTR1 expression, similar to the effect of an inflammatory stimulus. CONCLUSIONS: Our data demonstrate that PHACTR1 is a key atherosclerosis candidate gene since it is regulated by atherogenic stimuli in macrophages and endothelial cells and we identify an effect of the genetic risk variant on PHACTR1 expression in macrophages that is similar to that of an inflammatory stimulus

Suppressor of cytokine signalling protein SOCS3 expression is increased at sites of acute and chronic inflammation.

Treatment of cells with cytokines and growth factors leads to the synthesis of Suppressor of Cytokine Signalling (SOCS) proteins that act as potent negative regulators of signalling via the Jak/STAT pathway. We used immunohistochemistry to identify cells and pathologies where SOCS3 expression might influence acute and chronic inflammatory responses in human tissues. Epitope and GFP tagged SOCS3 fusion proteins were localised predominantly in the nucleus of transfected cells and a validated anti SOCS3 antiserum revealed the expression of SOCS3 in the nucleus and cytoplasm of macrophages, endothelial and epithelial cells in a wide range of normal tissues in tissue microarrays (n = 31 different tissues). Nuclear SOCS3 was only seen in cells expressing a high level of the protein. Comparative immunostaining of acute, chronically and granulomatously inflamed human tissues revealed higher levels of nuclear and cytoplasmic SOCS3 expression in inflamed than in corresponding normal tissues, particularly in recruited leukocyte populations, but also in epithelia. The staining appeared more intense, suggesting higher expression levels, in areas where inflammation was more acute, consistent with the time course of SOCS3 induction described in vitro. Expression of SOCS3 protein by leucocytes and other cell types in tissue sections could be a useful marker of cells undergoing acute or chronic stimulation by cytokines in vivo

Lipid-Induced Epigenomic Changes in Human Macrophages Identify a Coronary Artery Disease-Associated Variant that Regulates \u3cem\u3ePPAP2B\u3c/em\u3e Expression through Altered C/EBP-Beta Binding

Genome-wide association studies (GWAS) have identified over 40 loci that affect risk of coronary artery disease (CAD) and the causal mechanisms at the majority of loci are unknown. Recent studies have suggested that many causal GWAS variants influence disease through altered transcriptional regulation in disease-relevant cell types. We explored changes in transcriptional regulation during a key pathophysiological event in CAD, the environmental lipid-induced transformation of macrophages to lipid-laden foam cells. We used a combination of open chromatin mapping with formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and enhancer and transcription factor mapping using chromatin immuno-precipitation (ChIP-seq) in primary human macrophages before and after exposure to atherogenic oxidized low-density lipoprotein (oxLDL), with resultant foam cell formation. OxLDL-induced foam cell formation was associated with changes in a subset of open chromatin and active enhancer sites that strongly correlated with expression changes of nearby genes. OxLDL-regulated enhancers were enriched for several transcription factors including C/EBP-beta, which has no previously documented role in foam cell formation. OxLDL exposure up-regulated C/EBP-beta expression and increased genomic binding events, most prominently around genes involved in inflammatory response pathways. Variants at CAD-associated loci were significantly and specifically enriched in the subset of chromatin sites altered by oxLDL exposure, including rs72664324 in an oxLDL-induced enhancer at the PPAP2B locus. OxLDL increased C/EBP beta binding to this site and C/EBP beta binding and enhancer activity were stronger with the protective A allele of rs72664324. In addition, expression of the PPAP2B protein product LPP3 was present in foam cells in human atherosclerotic plaques and oxLDL exposure up-regulated LPP3 in macrophages resulting in increased degradation of pro-inflammatory mediators. Our results demonstrate a genetic mechanism contributing to CAD risk at the PPAP2B locus and highlight the value of studying epigenetic changes in disease processes involving pathogenic environmental stimuli

High Prevalence of Pre-Existing Liver Abnormalities Identified Via Autopsies in COVID-19: Identification of a New Silent Risk Factor?

A high prevalence of hepatic pathology (in 17 of 19 cases) was reported in post-mortem (PM) examinations of COVID-19 patients, undertaken between March 2020 and February 2021 by a single autopsy pathologist in two English Coronial jurisdictions. The patients in our cohort demonstrated high levels of recognised COVID-19 risk factors, including hypertension (8/16, 50%), type 2 diabetes mellitus (8/16, 50%) and evidence of arteriopathy 6/16 (38%). Hepatic abnormalities included steatosis (12/19; 63%), moderate to severe venous congestion (5/19; 26%) and cirrhosis (4/19; 21%). A subsequent literature review indicated a significantly increased prevalence of steatosis (49%), venous congestion (34%) and cirrhosis (9.3%) in COVID-19 PM cases, compared with a pre-pandemic PM cohort (33%, 16%, and 2.6%, respectively), likely reflecting an increased mortality risk in SARS-CoV-2 infection for patients with pre-existing liver disease. To corroborate this observation, we retrospectively analysed the admission liver function test (LFT) results of 276 consecutive, anonymised COVID-19 hospital patients in our centre, for whom outcome data were available. Of these patients, 236 (85.5%) had significantly reduced albumin levels at the time of admission to hospital, which was likely indicative of pre-existing chronic liver or renal disease. There was a strong correlation between patient outcome (length of hospital admission or death) and abnormal albumin at the time of hospital admission (p = 0.000012). We discuss potential mechanisms by which our observations of hepatic dysfunction are linked to a risk of COVID-19 mortality, speculating on the importance of recently identified anti-interferon antibodies

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL

DC-SIGN and DC-SIGNR Bind Ebola Glycoproteins and Enhance Infection of Macrophages and Endothelial Cells

AbstractEbola virus exhibits a broad cellular tropism in vitro. In humans and animal models, virus is found in most tissues and organs during the latter stages of infection. In contrast, a more restricted cell and tissue tropism is exhibited early in infection where macrophages, liver, lymph node, and spleen are major initial targets. This indicates that cellular factors other than the broadly expressed virus receptor(s) modulate Ebola virus tropism. Here we demonstrate that the C-type lectins DC-SIGN and DC-SIGNR avidly bind Ebola glycoproteins and greatly enhance transduction of primary cells by Ebola virus pseudotypes and infection by replication-competent Ebola virus. DC-SIGN and DC-SIGNR are expressed in several early targets for Ebola virus infection, including dendritic cells, alveolar macrophages, and sinusoidal endothelial cells in the liver and lymph node. While DC-SIGN and DC-SIGNR do not directly mediate Ebola virus entry, their pattern of expression in vivo and their ability to efficiently capture virus and to enhance infection indicate that these attachment factors can play an important role in Ebola transmission, tissue tropism, and pathogenesis

CD or not CD, that is the question - a digital interobserver agreement study in coeliac disease

OBJECTIVE: Coeliac disease (CD) diagnosis generally depends on histological examination of duodenal biopsies. We present the first study analysing the concordance in examination of duodenal biopsies using digitised whole-slide images (WSIs). We further investigate whether the inclusion of IgA tTG and haemoglobin (Hb) data improves the inter-observer agreement of diagnosis.DESIGN: We undertook a large study of the concordance in histological examination of duodenal biopsies using digitised WSIs in an entirely virtual reporting setting. Our study was organised in two phases: in phase one, 13 pathologists independently classified 100 duodenal biopsies (40 normal; 40 CD; 20 indeterminate enteropathy) in the absence of any clinical or laboratory data. In phase two, the same pathologists examined the (re-anonymised) WSIs with the inclusion of IgA tTG and Hb data.RESULTS: We found the mean probability of two observers agreeing in the absence of additional data to be 0.73 (±0.08) with a corresponding Cohen's kappa of 0.59 (±0.11). We further showed that the inclusion of additional data increased the concordance to 0.80 (±0.06) with a Cohen's kappa coefficient of 0.67 (±0.09).CONCLUSION: We showed that the addition of serological data significantly improves the quality of CD diagnosis. However, the limited inter-observer agreement in CD diagnosis using digitised WSIs, even after the inclusion of IgA tTG and Hb data, indicates the important of interpreting duodenal biopsy in the appropriate clinical context. It further highlights the unmet need for an objective means of reproducible duodenal biopsy diagnosis, such as the automated analysis of WSIs using AI.<br/

Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk

Abstract: Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially