CXCL8((3-73))K11R/G31P antagonizes ligand binding to the neutrophil CXCR1 and CXCR2 receptors and cellular responses to CXCL8/IL-8

We recently reported that CXCL8((3-73))K11R is a high affinity agonist of neutrophil activation and chemotactic responses. In this report we employed CXCL8((3-73))K11R as a template to generate CXCL8/IL-8 analogues with antagonist activities, using site-directed mutagenesis to introduce conservative amino acid substitutions into the first turn within the molecule's beta-pleated sheet region (G31P, P32G) and, in association with these, into the putative receptor-recognition site (T12S, H13F, F17S). We then examined their impact on the analogues' biological activities and found that a G31P substitution rendered CXCL8((3-73))K11R a high affinity antagonist of CXCL8/IL-8. The ranking (in the order of decreasing CXCL8/IL-8 antagonist activities) of the CXCL8((3-73))K11R analogues we generated was, G31P>T12S/G31P>H13F/G31P>T12S/H13F/G31P>P32G approximately T12S/P32G approximately H13F/P32G>T12S/H13F/P32G; CXCL8((3-73))K11R/F17S did not inhibit CXCL8/IL-8-dependent responses. CXCL8((3-73))K11R/G31P had no discernible agonist (beta-glucuronidase release, chemotactic) activity, but at 12.5 ng/ml it bound to purified neutrophils more avidly than did 1.25 microg/ml CXCL8/IL-8. Furthermore, CXCL8((3-73))K11R/G31P competitively antagonized the binding of CXCR1- and CXCR2-specific antibodies to these receptors. Taken together, these data thus provide further impetus to the study of the potential efficacy of CXCL8((3-73))K11R/G31P as a broad-spectrum antagonist of the ELR-CXC chemokines in experimental and clinical settings.     

Butyrate regulates the expression of inflammatory and chemotactic cytokines in human acute leukemic cells during apoptosis

Butyrate is a histone deacetylase inhibitor implicated in many studies as a potential therapy for various forms of cancer. High concentrations of butyrate (>1.5 mM) have been shown to activate apoptosis in several cancer cell lines including prostate, breast, and leukemia. Butyrate is also known to influence multiple signaling pathways that are mediators of cytokine production. The purpose of this study was to evaluate the impact of high concentrations of butyrate on the cancer microenvironment vis-à-vis apoptosis, cellular migration, and capacity to modulate cytokine expression in cancer cells. The results indicate that high concentrations of butyrate induced a 2-fold activation of caspase-3 and reduced cell viability by 60% in U937 leukemia cells. Within 24 h, butyrate significantly decreased the levels of chemokines CCL2 and CCL5 in HL-60 and U937 cells, and decreased CCL5 in THP-1 leukemia cells. Differential effects were observed in treatments with valproic acid for CCL2 and CCL5 indicating butyrate-specificity. Many of the biological effects examined in this study are linked to activation of the AKT and MAPK signaling pathways; therefore, we investigated whether butyrate alters the levels of phosphorylated forms of these signaling proteins and how it correlated with the expression of chemokines. The results show that butyrate may partially regulate CCL5 production via p38 MAPK. The decrease in p-ERK1/2 and p-AKT levels correlated with the decrease in CCL2 production. These data suggest that while promoting apoptosis, butyrate has the potential to influence the cancer microenvironment by inducing differential expression of cytokines.     

Beta-sitosterol and its derivatives repress lipopolysaccharide/d-galactosamine-induced acute hepatic injury by inhibiting the oxidation and inflammation in mice

Beta-sitosterol (Sit) widely exists in natural plants, is classed as phytosterol and known as the “key of life”. Most pharmacological studies and clinical applications are limited because of the fact that Sit is difficult to be solved. Therefore, it is viable to enhance pharmacologic activities of Sit by using its derivatives which can be obtained through the modification of Sit. In this study, 4 kinds of new Sit derivatives were obtained by the esterification reaction. Further, the hepatoprotective effects of Sit and its derivatives were investigated against acute liver injury induced by lipopolysaccharide/d-galactosamine (LPS/GalN) in mice and its mechanism was illustrated by western blot analysis and real-time PCR. The results demonstrated that among its derivatives, 2-naphthoyl Sit ester (Sit-N) (50?mg/kg) showed the strongest activities against acute liver injury. Final experimental results showed that Sit-N significantly decreased the serum activity of aspartate transaminase (AST) and alanine aminotransferase (ALT); Sit-N also markedly reduced tumor necrosis factor (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels. Meanwhile, Sit-N drastically improved the activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT), and suppressed the expression of malondialdehyde (MDA). Results also displayed that over-expression of Toll like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) induced by LPS/Gal N were inhibited by Sit-N. Meanwhile, the expression of nuclear respiratory factor2 (Nrf2) and heme oxygenase-1 (HO-1) were enhanced. The results all above verified the effectiveness of Sit-N against acute liver injury induced by LPS/GalN mediated by TLR4 and Nrf2 pathways.     

Mutations in the transcriptional regulator MeCP2 severely impact key cellular and molecular signatures of human astrocytes during maturation

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Urocortin 3 expression at baseline and during inflammation in the colon: Corticotropin releasing factor receptors cross-talk

Urocortins (Ucn1-3), members of the corticotropin-releasing factor (CRF) family of neuropeptides, are emerging as potent immunomodulators. Localized, cellular expression of Ucn1 and Ucn2, but not Ucn3, has been demonstrated during inflammation. Here, we investigated the role of Ucn3 in a rat model of Crohn's colitis and the relative contribution of CRF receptors (CRF₁ and CRF₂) in regulating Ucn3 expression at baseline and during inflammation. Ucn3 mRNA and peptide were ubiquitously expressed throughout the GI tract in naïve rats. Ucn3 immunoreactivity was seen in epithelial cells and myenteric neurons. On day 1 of colitis, Ucn3 mRNA levels decreased by 80% and did not recover to baseline even by day 9. Next, we ascertained pro- or anti-inflammatory actions of Ucn3 during colitis. Surprisingly, unlike observed anti-inflammatory actions of Ucn1, exogenous Ucn3 did not alter histopathological outcomes during colitis and neither did it alter levels of pro-inflammatory cytokines IL-6 and TNF-α. At baseline, colon-specific knockdown of CRF₁, but not CRF₂ decreased Ucn3 mRNA by 78%, whereas during colitis, Ucn3 mRNA levels increased after CRF₁ knockdown. In cultured cells, co-expression of CRF₁ + CRF₂ attenuated Ucn3-stimulated intracellular Ca²⁺ peak by 48% as compared to cells expressing CRF₂ alone. Phosphorylation of p38 kinase increased by 250% during colitis and was significantly attenuated after Ucn3 administration. Thus, our results suggest that a balanced and coordinated expression of CRF receptors is required for proper regulation of Ucn3 at baseline and during inflammation.     

Altered apolipoprotein A-V expression during the acute phase response is independent of plasma triglyceride levels in mice and humans

Plasma triglyceride (TG) levels are altered during the acute phase response (APR). Plasma levels of the recently discovered apolipoprotein A-V (apoA-V) are inversely associated with plasma TG. The aim of this study was to investigate the change of apoA-V plasma levels and hepatic apoA-V expression during the APR in relation to plasma TG. During human APR plasma apoA-V was decreased as were plasma TG (each P<0.01). Also early in the course of the murine APR plasma apoA-V levels and hepatic apoA-V expression were decreased and changed in the same direction as plasma TG. Treatment of HepG2 cells with TNF-alpha and IL-1beta decreased apoA-V mRNA levels early by 42% and 55%, respectively (each P<0.001). However, in promoter/reporter assays the human apoA-V promoter was unresponsive to proinflammatory cytokines. Instead, we demonstrate that a significant decrease in apoA-V mRNA stability in response to treatment with TNF-alpha and IL-1beta is the underlying basis of decreased apoA-V expression during the APR (P<0.05). These data demonstrate that (i) apoA-V expression decreases early during the APR due to changes in mRNA stability, and (ii) during the APR apoA-V is not inversely related to plasma TG levels in mice and humans, thereby identifying a relevant pathophysiological setting, in which the previously reported close inverse association between these parameters does not hold true.     

NAD+-dependent sirtuin 1 and 6 proteins coordinate a switch from glucose to fatty acid oxidation during the acute inflammatory response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.     

Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT‐8 cells by a proteomic approach. HCT‐8 cells were incubated with interleukin 1β, tumor necrosis factor‐α, and interferon‐γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC‐ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT‐8 cells and in 2‐4‐6‐trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS‐regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.     

Interleukin-13 secretion by normal and posttransplant T lymphocytes; in vitro studies of cellular immune responses in the presence of acute leukaemia blast cells

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4⁺ and CD8⁺ TCRαβ⁺ T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin+IL-2+ B lymphocyte accessory cells). The CD4⁺ cells showed significantly higher IL-13 levels than the CD8⁺ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon γ levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells. Received: 24 April 1997 / Accepted: 24 July 1997     

The involvement of nitric oxide,nitric oxide synthase,neutrophil elastase,myeloperoxidase and proinflammatory cytokines in the acute lung injury caused by phorbol myristate acetate

Phorbol myristate acetate (PMA) causes acute lung injury (ALI). The present study was designed to elucidate the role of nitric oxide (NO), inducible NO synthase (iNOS), neutrophil elastase (NE) and other mediators in the ALI caused by PMA. In isolated rat’s lungs, PMA at various doses (1, 2 and 4 μg/g lung weight) was added into the lung perfusate. Vehicle group received dimethyl sulfoxide (the solvent for PMA) 100 μg/g. We measured the lung weight changes, pulmonary arterial pressure, capillary filtration coefficient, exhaled NO, protein concentration in bronchoalveolar lavage (PCBAL) and Evan blue dye leakage. Nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and myeloperoxidase (MPO) in lung perfusate were determined. Histopathological examination was performed. We detected the iNOS mRNA expression in lung tissue. PMA caused dose-dependent increases in variables for lung changes, and nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and MPO in lung perfusate. The pathology was characterized by alveolar hemorrhagic edema with inflammatory cell infiltration. Scanning electron microscopy revealed endothelial damage. PMA upregulated the expression of iNOS mRNA. Our results suggest that neutrophil activation by PMA causes release of NE, upregulation of iNOS and a series of inflammatory responses leading to endothelial damage and ALI.     

Experimental acute hypoxia in healthy subjects: evaluation of systolic and diastolic function of the left ventricle at rest and during exercise using echocardiography

To clarify whether or not systolic and diastolic function of the human left ventricle (LV) were decreased during acute hypoxia, at rest and with exercise, 14 healthy male volunteers [age 25.9 (SD 3.0) years, height 182.9 (SD 7.1) cm, body mass 75.9 (SD 6.9)kg] were examined using M-mode and 2D-mode echocardiography to determine the systolic LV function as well as Doppler-echocardiography for the assessment of diastolic LV function on 2 separate test days. In random order, the subjects breathed either air on 1 day (N) or a gas mixture with reduced oxygen content on the other (H; oxygen fraction in inspired gas 0.14). Measurements on either day were made at rest, several times during incremental cycle exercise in a supine position (6-min increments of 50 W, maximal load 150 W) and in 6th min of recovery. Corresponding measurements during N and H were compared statistically. Arterial O₂ tension (P _aO₂) was normal on N-day. All subjects showed a marked acute hypoxia at rest [P _aO₂, 54.5 (SD 4.6) mmHg], during exercise and recovery on H-day. The latter was associated with tachycardia compared to N-day. All echocardiographic measurements at rest were within the limits of normal values on both test days. Ejection time, end-systolic and end-diastolic left ventricular dimensions as well as the thickness of left posterior wall and of interventricular septum showed no statistically significant influence of H either at rest or during exercise. Stroke volume and cardiac output were always higher on H-day, which could be attributed to a slight reduction in end-systolic volume with unaffected end-diastolic volume as well as to increased heart rates. Among the indices of systolic LV function the fractions of thickening in the left ventricular posterior wall and interventricular septum showed no differences between H and N at rest or during exercise. However, fibre shortening, ejection fraction and mean circumferential fibre shortening were increased on H-day on all occasions. The mitral-valve-Doppler ratio, the index of diastolic LV function, was decreased with H at rest, showed a more pronounced reduction during exercise and was still lower in 6th min of recovery compared to N-day. It was concluded that with acute hypoxia of the severity applied in this study left ventricular systolic function in our healthy subjects showed a pronounced improvement and left ventricular diastolic function was reduced, both at rest and with exercise.     

The utility of inflammation and platelet biomarkers in patients with acute coronary syndromes

Introduction

Thrombotic and inflammatory mechanisms are involved in the pathophysiology of acute coronary syndrome (ACS). The aim of the study was the evaluation of inflammation (white blood cells count/WBC, C-reactive protein/CRP, interleukin-6/IL-6) and platelet (platelet count/PLT, mean platelet volume/MPV, large platelet/LPLT, beta-thromboglobulin/β-TG) biomarkers in the groups of ACS patients depending on the severity of signs and symptoms and compared to controls without coronary artery disease.

Changes in the content of glycogen and its metabolites during acute exposure ofAnabas testudineus (Bloch) to furadan

Significant differences were observed in glycogen metabolism ofAnabas testudineus exposed to an acute lethal (1.56 mg/litre) and a sublethal (0.56 mg/litre) concentration of furadan. At sublethal concentration, the muscle glycogen which was utilized during the early periods of exposure, was replenished in the later period of exposure and at 120 h, the muscle glycogen levels were higher than the control. At higher concentration, the liver glycogen levels showed an increase presumably at the expense of fuel reserves of the muscle.     

Down-regulation of cyclic nucleotide phosphodiesterase PDE1A is the key event of p73 and UHRF1 deregulation in thymoquinone-induced acute lymphoblastic leukemia cell apoptosis

Thymoquinone (TQ), the active principle of Nigella sativa black seeds, has anti-proliferative properties on numerous cancer cell types. Others and we have previously reported that TQ acts as agent that triggers cell cycle arrest and apoptosis through either a p53- or p73-dependent pathway. However, the immediate targets recruited upon TQ-induced cytotoxicity have not yet been clearly identified. We therefore asked whether cyclic nucleotide phosphodiesterases (PDEs) could be involved in TQ-triggered pro-apoptotic reactivity; PDEs are regulators of intracellular levels of cyclic nucleotides and therefore can modulate cAMP and cGMP-dependent cell death pathways. Our results showed that TQ specifically repressed PDE1A expression in the acute lymphoblastic leukemia Jurkat cell line. This effect is concomitant with the previously described sequential deregulation of the expression of the tumor suppressor protein p73 and the epigenetic integrator UHRF1 (Ubiquitin-like, PHD Ring Finger 1). Interestingly, RNA-interference knock-down of PDE1A expression as well as decreased PDE1A expression induced growth inhibition of Jurkat cells, cell cycle arrest and apoptosis through an activation of p73 and a repression of UHRF1. Conversely, PDE1A re-expression counteracted the cellular pro-apoptotic effects of TQ in association with a p73 repression and UHRF1 re-expression. Altogether, our results show that TQ induced an initial down-regulation of PDE1A with a subsequent down-regulation of UHRF1 via a p73-dependent mechanism. This study further proposes that PDE1A might be involved in the epigenetic code inheritance by regulating, via p73, the epigenetic integrator UHRF1. Our findings also suggest that a forced inhibition of PDE1A expression might be a new therapeutic strategy for the management of acute lymphoblastic leukemia.     

Association between public media and trends in new acute coronary syndrome presentations during the first COVID‑19 wave in the Netherlands

BackgroundWe aimed to evaluate the association between public media and trends in new presentations of acute coronary syndrome (ACS) during the first wave of the coronavirus disease 2019 (COVID‑19) in the Netherlands.MethodsNew ACS presentations per week in 73 hospitals during the first half of 2019 and 2020 were retrieved from the national organisation Dutch Hospital Data and incidence rates were calculated. Stratified analyses were performed by region, type of ACS and patient characteristics.ResultsAfter the first confirmed COVID‑19 case and during lockdown, numbers declined by up to 41% (95% confidence interval (CI): 36–47%) compared to 2019. This reduction was more pronounced for non-ST-segment elevation myocardial infarction (NSTEMI) (48%; 95% CI: 39–55%) and unstable angina (UA; 50%; 95% CI: 40–59%) than for STEMI (34%; 95% CI: 23–43%). There was no association between ACS and COVID‑19 incidence rate per region. After the steep decline, a public campaign encouraged patients not to postpone hospital visits. Numbers then increased, without a rebound effect. Trends were similar irrespective of sex, age or socio-economic status. During the outbreak, compared to coronary artery bypass graft procedures, relatively more (acute) percutaneous coronary interventions for NSTEMI and UA were performed.ConclusionNew ACS presentations decreased by up to 41%. Lockdown measures and public campaigns, rather than COVID‑19 incidence, were associated with significant changes in new ACS presentations. Even though causality cannot be established, this emphasises the role of the public media and healthcare organisations in informing patients to prevent underdiagnoses of ACS and associated health damage.Supplementary InformationThe online version of this article (10.1007/s12471-021-01603-5) contains supplementary material, which is available to authorized users.     

Soluble Fas/Apo-1 (CD95) levels during T cell activation in the presence of acute myelogenous leukemia accessory cells; contributions from local release and variations in systemic levels

Fas (CD95/Apo-1) exists both in membrane-bound and in biologically active soluble (s) forms. Ligation of membrane-expressed Fas can induce apoptosis, and Fas-mediated signaling seems to be involved in T-cell-induced apoptosis of human acute myelogenous leukemia (AML) blasts. The local release of sFas by AML blasts may then function as a protective mechanism by competing with membrane-bound Fas for binding sites on the common Fas ligand (FasL). sFas was released by AML blasts during in vitro culture, and this release was modulated by several cytokines that can be secreted by activated T cells. Increased levels of sFas could be detected during in vitro activation of T cells in the presence of native AML accessory cells, and this was observed both for (i) mitogenic activation of CD4⁺ and CD8⁺ T cell clones derived from acute leukemia patients with therapy-induced leukopenia and (ii) allostimulated activation of T cells derived from normal donors. However, local in vivo levels of sFas will also be influenced by variations in systemic levels. High serum levels of sFas were detected in acute leukemia patients during chemotherapy-induced cytopenia, but these levels decreased during complicating bacterial infections. In contrast, serum levels of sFasL were normal in leukopenic patients. The present results support the hypothesis that local release of sFas can function as a protective mechanism against AML-reactive T cells, but the effects of this local release are, in addition, modulated by variations in systemic levels of sFas (but not sFasL). Received: 9 March 2000 / Accepted: 25 May 2000