Dietary salt intake in chronic kidney disease: recent studies and their practical implications.

Epidemiologic studies in the general population show that the level of dietary salt intake is associated with increases in blood pressure (BP), cardiovascular events, and mortality. According to trial data, reducing salt intake lowers the incidence of these 3 outcomes. On the basis of this evidence, the World Health Organization and other bodies recommend restricting salt intake. The association of salt intake with BP and cardiovascular disease has also been seen in chronic kidney disease (CKD), and trials of salt reduction in CKD have shown benefit, reflected by reduced BP and a lower rate of cardiovascular events. However, these trials have typically used resource­intensive approaches to dietary salt reduction that are not suitable for routine clinical care, and salt intake typically remains high in people with CKD. The OxSalt care bundle is a low­cost intervention that was demonstrated in the OxCKD1 trial to help people with CKD lower their salt intake, and could be applied in routine clinical practice.

Impact of the COVID-19 pandemic on emergency department attendances and acute medical admissions.

BACKGROUND: To better understand the impact of the COVID-19 pandemic on hospital healthcare, we studied activity in the emergency department (ED) and acute medicine department of a major UK hospital. METHODS: Electronic patient records for all adult patients attending ED (n = 243,667) or acute medicine (n = 82,899) during the pandemic (2020-2021) and prior year (2019) were analysed and compared. We studied parameters including severity, primary diagnoses, co-morbidity, admission rate, length of stay, bed occupancy, and mortality, with a focus on non-COVID-19 diseases. RESULTS: During the first wave of the pandemic, daily ED attendance fell by 37%, medical admissions by 30% and medical bed occupancy by 27%, but all returned to normal within a year. ED attendances and medical admissions fell across all age ranges; the greatest reductions were seen for younger adults in ED attendances, but in older adults for medical admissions. Compared to non-COVID-19 pandemic admissions, COVID-19 admissions were enriched for minority ethnic groups, for dementia, obesity and diabetes, but had lower rates of malignancy. Compared to the pre-pandemic period, non-COVID-19 pandemic admissions had more hypertension, cerebrovascular disease, liver disease, and obesity. There were fewer low severity ED attendances during the pandemic and fewer medical admissions across all severity categories. There were fewer ED attendances with common non-respiratory illnesses including cardiac diagnoses, but no change in cardiac arrests. COVID-19 was the commonest diagnosis amongst medical admissions during the first wave and there were fewer diagnoses of pneumonia, myocardial infarction, heart failure, cellulitis, chronic obstructive pulmonary disease, urinary tract infection and other sepsis, but not stroke. Levels had rebounded by a year later with a trend to higher levels of stroke than before the pandemic. During the pandemic first wave, 7-day mortality was increased for ED attendances, but not for non-COVID-19 medical admissions. CONCLUSIONS: Reduced ED attendances in the first wave of the pandemic suggest opportunities for reducing low severity presentations to ED in the future, but also raise the possibility of harm from delayed or missed care. Reassuringly, recent rises in attendance and admissions indicate that any deterrent effect of the pandemic on attendance is diminishing.

Long-term health outcomes of people with reduced kidney function in the UK: A modelling study using population health data.

BACKGROUND: People with reduced kidney function have increased cardiovascular disease (CVD) risk. We present a policy model that simulates individuals' long-term health outcomes and costs to inform strategies to reduce risks of kidney and CVDs in this population. METHODS AND FINDINGS: We used a United Kingdom primary healthcare database, the Clinical Practice Research Datalink (CPRD), linked with secondary healthcare and mortality data, to derive an open 2005-2013 cohort of adults (≥18 years of age) with reduced kidney function (≥2 measures of estimated glomerular filtration rate [eGFR] <90 mL/min/1.73 m2 ≥90 days apart). Data on individuals' sociodemographic and clinical characteristics at entry and outcomes (first occurrences of stroke, myocardial infarction (MI), and hospitalisation for heart failure; annual kidney disease stages; and cardiovascular and nonvascular deaths) during follow-up were extracted. The cohort was used to estimate risk equations for outcomes and develop a chronic kidney disease-cardiovascular disease (CKD-CVD) health outcomes model, a Markov state transition model simulating individuals' long-term outcomes, healthcare costs, and quality of life based on their characteristics at entry. Model-simulated cumulative risks of outcomes were compared with respective observed risks using a split-sample approach. To illustrate model value, we assess the benefits of partial (i.e., at 2013 levels) and optimal (i.e., fully compliant with clinical guidelines in 2019) use of cardioprotective medications. The cohort included 1.1 million individuals with reduced kidney function (median follow-up 4.9 years, 45% men, 19% with CVD, and 74% with only mildly decreased eGFR of 60-89 mL/min/1.73 m2 at entry). Age, kidney function status, and CVD events were the key determinants of subsequent morbidity and mortality. The model-simulated cumulative disease risks corresponded well to observed risks in participant categories by eGFR level. Without the use of cardioprotective medications, for 60- to 69-year-old individuals with mildly decreased eGFR (60-89 mL/min/1.73 m2), the model projected a further 22.1 (95% confidence interval [CI] 21.8-22.3) years of life if without previous CVD and 18.6 (18.2-18.9) years if with CVD. Cardioprotective medication use at 2013 levels (29%-44% of indicated individuals without CVD; 64%-76% of those with CVD) was projected to increase their life expectancy by 0.19 (0.14-0.23) and 0.90 (0.50-1.21) years, respectively. At optimal cardioprotective medication use, the projected health gains in these individuals increased by further 0.33 (0.25-0.40) and 0.37 (0.20-0.50) years, respectively. Limitations include risk factor measurements from the UK routine primary care database and limited albuminuria measurements. CONCLUSIONS: The CKD-CVD policy model is a novel resource for projecting long-term health outcomes and assessing treatment strategies in people with reduced kidney function. The model indicates clear survival benefits with cardioprotective treatments in this population and scope for further benefits if use of these treatments is optimised.

Process of care and activity in a clinically inclusive ambulatory emergency care unit: progressive effect over time on clinical outcomes and acute medical admissions.

Clinically relevant outcomes for same-day emergency care provided by ambulatory emergency care units (AECs) are largely unknown. We report the activity and outcomes for a large UK adult AEC operating an ambulatory-care-by-default model without specific exclusion criteria. The AEC consultant triaged all acute medical referrals to either the AEC or the standard non-ambulatory 'take' pathway during AEC opening hours. The proportion of acute medical referrals seen in the AEC increased to 42% (mean 700 referrals seen per month) in the last 6 months of the study and numbers seen in the non-ambulatory pathway fell. The most common diagnoses were for chest pain, pneumonia, cellulitis, heart failure and urinary system disorders. Seventy-four point eight per cent of patients completed their care in a single visit. In the last calendar year, the conversion rate from AEC to inpatient admission was 12%, and the 30-day readmission rate was 6.9% and 18% for the AEC and non-ambulatory pathways, respectively. Across the whole study period, the 30-day mortality was 1.6% and 6.9% for the AEC and non-ambulatory pathway, respectively. This ambulatory approach is safe and effective.

Hierarchical Modular DNA Assembly Using MetClo.

DNA assembly methods are essential for multiple applications including synthetic biology. We recently developed MetClo, a method that uses a single type IIS restriction enzyme for hierarchical modular DNA assembly. This offers great flexibility in the design of the assembly experiment and simplicity of execution. Here we describe a protocol for hierarchical assembly of large DNA constructs from modular DNA parts using the MetClo vector set, a set of assembly vectors designed for the MetClo method.

Cytoplasmic Citrate Flux Modulates the Immune Stimulatory NKG2D Ligand MICA in Cancer Cells.

Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N-glycosylation gene N-acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition.

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression.

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8+ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.

Effects of antihypertensives, lipid-modifying drugs, glycaemic control drugs and sodium bicarbonate on the progression of stages 3 and 4 chronic kidney disease in adults: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the effects of drug interventions that may modify the progression of chronic kidney disease (CKD) in adults with CKD stages 3 and 4. DESIGN: Systematic review and meta-analysis. METHODS: Searching MEDLINE, EMBASE, Database of Abstracts of Reviews of Effects, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, International Clinical Trials Registry Platform, Health Technology Assessment, Science Citation Index, Social Sciences Citation Index, Conference Proceedings Citation Index and Clinical Trials Register, from March 1999 to July 2018, we identified randomised controlled trials (RCTs) of drugs for hypertension, lipid modification, glycaemic control and sodium bicarbonate, compared with placebo, no drug or a drug from another class, in ≥40 adults with CKD stages 3 and/or 4, with at least 2 years of follow-up and reporting renal function (primary outcome), proteinuria, adverse events, maintenance dialysis, transplantation, cardiovascular events, cardiovascular mortality or all-cause mortality. Two reviewers independently screened citations and extracted data. For continuous outcomes, we used the ratio of means (ROM) at the end of the trial in random-effects meta-analyses. We assessed methodological quality with the Cochrane Risk of Bias Tool and confidence in the evidence using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework. RESULTS: We included 35 RCTs and over 51 000 patients. Data were limited, and heterogeneity varied. Final renal function (estimated glomerular filtration rate) was 6% higher in those taking glycaemic control drugs (ROM 1.06, 95% CI 1.02 to 1.10, I2=0%, low GRADE confidence) and 4% higher in those taking lipid-modifying drugs (ROM 1.04, 95% CI 1.00 to 1.08, I2=88%, very low GRADE confidence). For RCTs of antihypertensive drugs, there were no significant differences in renal function. Treatment with lipid-modifying drugs led to a 36% reduction in cardiovascular disease and 26% reduction in all-cause mortality. CONCLUSIONS: Glycaemic control and lipid-modifying drugs may slow the progression of CKD, but we found no pooled evidence of benefit nor harm from antihypertensive drugs. However, given the data limitations, further research is needed to confirm these findings. PROSPERO REGISTRATION NUMBER: CRD42015017501.

A retrospective analysis of outcomes in low- and intermediate-high-risk pulmonary embolism patients managed on an ambulatory medical unit in the UK.

Pulmonary embolism (PE) is common and guidelines recommend outpatient care only for PE patients with low predicted mortality. Outcomes for patients with intermediate-to-high predicted mortality managed as outpatients are unknown. Electronic records were analysed for adults with PE managed on our ambulatory care unit over 2 years. Patients were stratified into low or intermediate-to-high mortality risk groups using the Pulmonary Embolism Severity Index (PESI). Primary outcomes were the proportion of patients ambulated, 30-day all-cause mortality, 30-day PE-specific mortality and 30-day re-admission rate. Of 199 PE patients, 74% were ambulated and at 30 days, all-cause mortality was 2% (four out of 199) and PE-specific mortality was 1% (two out of 199). Ambulated patients had lower PESI scores, better vital signs and lower troponin levels (morning attendance favoured ambulation). Over a third of ambulated patients had an intermediate-to-high risk PESI score but their all-cause mortality rate was low at 1.9% (one out of 52). In patients with intermediate-to-high risk, oxygen saturation was higher and pulse rate lower in those who were ambulated. Re-admission rate did not differ between ambulated and admitted patients. Two-thirds of patients with intermediate-to-high risk PE were ambulated and their mortality rate remained low. It is possible for selected patients with intermediate-to-high risk PESI scores to be safely ambulated.

[Spanish adaptation of the free OxMaR software for minimization and randomization of clinical studies]. / Versión en español del software gratuito OxMaR para minimización y aleatorización de estudios clínicos.

Randomized clinical trials provide the highest level of scientific evidence. The method used for randomization should make the group to which each case will be assigned unpredictable and facilitate the concealment of the randomization sequence. Centralized methods, generally implemented with computer support, are considered the safest to avoid biases. The OxMaR system, acronym for Oxford Minimization and Randomization, was published as free and open source software in 2014. It works online in a web environment and allows simple randomization and adaptive assignment through minimization. We present a Spanish version developed in collaboration with the author of the original English version. The system has been modified to work on low cost shared web servers and also to allow the concealment of the randomization sequence.

MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme.

Efficient DNA assembly is of great value in biological research and biotechnology. Type IIS restriction enzyme-based assembly systems allow assembly of multiple DNA fragments in a one-pot reaction. However, large DNA fragments can only be assembled by alternating use of two or more type IIS restriction enzymes in a multi-step approach. Here, we present MetClo, a DNA assembly method that uses only a single type IIS restriction enzyme for hierarchical DNA assembly. The method is based on in vivo methylation-mediated on/off switching of type IIS restriction enzyme recognition sites that overlap with site-specific methylase recognition sequences. We have developed practical MetClo systems for the type IIS enzymes BsaI, BpiI and LguI, and demonstrated hierarchical assembly of large DNA fragments up to 218 kb. The MetClo approach substantially reduces the need to remove internal restriction sites from components to be assembled. The use of a single type IIS enzyme throughout the different stages of DNA assembly allows novel and powerful design schemes for rapid large-scale hierarchical DNA assembly. The BsaI-based MetClo system is backward-compatible with component libraries of most of the existing type IIS restriction enzyme-based assembly systems, and has potential to become a standard for modular DNA assembly.

Intragenic transcriptional interference regulates the human immune ligand MICA.

Many human genes have tandem promoters driving overlapping transcription, but the value of this distributed promoter configuration is generally unclear. Here we show that MICA, a gene encoding a ligand for the activating immune receptor NKG2D, contains a conserved upstream promoter that expresses a noncoding transcript. Transcription from the upstream promoter represses the downstream standard promoter activity in cis through transcriptional interference. The effect of transcriptional interference depends on the strength of transcription from the upstream promoter and can be described quantitatively by a simple reciprocal repressor function. Transcriptional interference coincides with recruitment at the standard downstream promoter of the FACT histone chaperone complex, which is involved in nucleosomal remodelling during transcription. The mechanism is invoked in the regulation of MICA expression by the physiological inputs interferon-γ and interleukin-4 that act on the upstream promoter. Genome-wide analysis indicates that transcriptional interference between tandem intragenic promoters may constitute a general mechanism with widespread importance in human transcriptional regulation.

Purine nucleotide metabolism regulates expression of the human immune ligand MICA.

Expression of the cell-surface glycoprotein MHC class I polypeptide-related sequence A (MICA) is induced in dangerous, abnormal, or "stressed" cells, including cancer cells, virus-infected cells, and rapidly proliferating cells. MICA is recognized by the activating immune cell receptor natural killer group 2D (NKG2D), providing a mechanism by which immune cells can identify and potentially eliminate pathological cells. Immune recognition through NKG2D is implicated in cancer, atherosclerosis, transplant rejection, and inflammatory diseases, such as rheumatoid arthritis. Despite the wide range of potential therapeutic applications of MICA manipulation, the factors that control MICA expression are unclear. Here we use metabolic interventions and metabolomic analyses to show that the transition from quiescent cellular metabolism to a "Warburg" or biosynthetic metabolic state induces MICA expression. Specifically, we show that glucose transport into the cell and active glycolytic metabolism are necessary to up-regulate MICA expression. Active purine synthesis is necessary to support this effect of glucose, and increases in purine nucleotide levels are sufficient to induce MICA expression. Metabolic induction of MICA expression directly influences NKG2D-dependent cytotoxicity by immune cells. These findings support a model of MICA regulation whereby the purine metabolic activity of individual cells is reflected by cell-surface MICA expression and is the subject of surveillance by NKG2D receptor-expressing immune cells.

Requirement for cystatin C testing in chronic kidney disease: a retrospective population-based study.

BACKGROUND: Creatinine-based estimated glomerular filtration rate (eGFR) determines chronic kidney disease (CKD) stage, but underestimates renal function. The 2014 updated guidance from the National Institute for Health and Care Excellence (NICE) recommends that GPs reduce overdiagnosis of CKD stage 3a (eGFR 45-60 ml/min/1.73 m2) by using the renal biomarker cystatin C. AIM: To determine the population requirement for cystatin C testing, compared with current national availability of the assay. DESIGN AND SETTING: Retrospective study of primary care laboratory requests in Oxfordshire, England. METHOD: The first creatinine results from tests ordered in primary care over a 6-year period (2008-2014) in a population of 600 000 in Oxfordshire were analysed and the number of patients with CKD stage 3a without proteinuria (who, in accordance with NICE guidance, required cystatin C) was determined. A conservative estimate of the national need was provided by scaling the population of Oxfordshire to the national population (CKD prevalence in the county is below the national average). Cystatin C assay availability was determined using national databases of laboratory assay provision. RESULTS: From a population of 600 000, there were 22 240 individuals with stable stage 3a CKD and no proteinuria. As the population of Oxfordshire equates to 1% of the UK population, there is an initial requirement for at least 2 million people to have their CKD status determined with cystatin C testing. Eight laboratories (2.1% of UK laboratories) reported cystatin C assay provision. CONCLUSION: There is a substantial gap between cystatin C assay requirements in primary care and national assay provision. This is a major barrier to implementing NICE guidance.

Global Prevalence of Chronic Kidney Disease - A Systematic Review and Meta-Analysis.

Chronic kidney disease (CKD) is a global health burden with a high economic cost to health systems and is an independent risk factor for cardiovascular disease (CVD). All stages of CKD are associated with increased risks of cardiovascular morbidity, premature mortality, and/or decreased quality of life. CKD is usually asymptomatic until later stages and accurate prevalence data are lacking. Thus we sought to determine the prevalence of CKD globally, by stage, geographical location, gender and age. A systematic review and meta-analysis of observational studies estimating CKD prevalence in general populations was conducted through literature searches in 8 databases. We assessed pooled data using a random effects model. Of 5,842 potential articles, 100 studies of diverse quality were included, comprising 6,908,440 patients. Global mean(95%CI) CKD prevalence of 5 stages 13·4%(11·7-15·1%), and stages 3-5 was 10·6%(9·2-12·2%). Weighting by study quality did not affect prevalence estimates. CKD prevalence by stage was Stage-1 (eGFR>90+ACR>30): 3·5% (2·8-4·2%); Stage-2 (eGFR 60-89+ACR>30): 3·9% (2·7-5·3%); Stage-3 (eGFR 30-59): 7·6% (6·4-8·9%); Stage-4 = (eGFR 29-15): 0·4% (0·3-0·5%); and Stage-5 (eGFR<15): 0·1% (0·1-0·1%). CKD has a high global prevalence with a consistent estimated global CKD prevalence of between 11 to 13% with the majority stage 3. Future research should evaluate intervention strategies deliverable at scale to delay the progression of CKD and improve CVD outcomes.

What is the relationship between renal function and visit-to-visit blood pressure variability in primary care? Retrospective cohort study from routinely collected healthcare data.

OBJECTIVE: To determine the relationship between renal function and visit-to-visit blood pressure (BP) variability in a cohort of primary care patients. DESIGN: Retrospective cohort study from routinely collected healthcare data. SETTING: Primary care in Nijmegen, the Netherlands, from 2007 to 2012. PARTICIPANTS: 19 175 patients who had a measure of renal function, and 7 separate visits with BP readings in the primary care record. OUTCOME MEASURES: Visit-to-visit variability in systolic BP, calculated from the first 7 office measurements, including SD, successive variation, absolute real variation and metrics of variability shown to be independent of mean. Multiple linear regression was used to analyse the influence of estimated glomerular filtration rate (eGFR) on BP variability measures with adjustment for age, sex, diabetes, mean BP, proteinuria, cardiovascular disease, time interval between measures and antihypertensive use. RESULTS: In the patient cohort, 57% were women, mean (SD) age was 65.5 (12.3) years, mean (SD) eGFR was 75.6 (18.0) mL/min/1.73m(2) and SD systolic BP 148.3 (21.4) mm Hg. All BP variability measures were negatively correlated with eGFR and positively correlated with age. However, multiple linear regressions demonstrated consistent, small magnitude negative relationships between eGFR and all measures of BP variability adjusting for confounding variables. CONCLUSIONS: Worsening renal function is associated with small increases in measures of visit-to-visit BP variability after adjustment for confounding factors. This is seen across the spectrum of renal function in the population, and provides a mechanism whereby chronic kidney disease may raise the risk of cardiovascular events.

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.

MICA Expression Is Regulated by Cell Adhesion and Contact in a FAK/Src-Dependent Manner.

MICA is a major ligand for the NKG2D immune receptor, which plays a key role in activating natural killer (NK) cells and cytotoxic T cells. We analyzed NKG2D ligand expression on a range of cell types and could demonstrate that MICA expression levels were closely linked to cellular growth mode. While the expression of other NKG2D ligands was largely independent of cell growth mode, MICA expression was mainly found on cells cultured as adherent cells. In addition, MICA surface expression was reduced through increase in cell-cell contact or loss of cell-matrix adherence. Furthermore, we found that the reduction in MICA expression was modulated by focal adhesion kinase (FAK)/Src signaling and associated with increased susceptibility to NK cell-mediated killing. While the mechanisms of tumor immune evasion are not fully understood, the reduction of MICA expression following loss of attachment poises a potential way by which metastasizing tumor cells avoid immune detection. The role of FAK/Src in this process indicates a potential therapeutic approach to modulate MICA expression and immune recognition of tumor cells during metastasis.

Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that regulates PPAP2B 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.