EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR

Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, may augment metabolic and vascular actions of insulin. Therefore, we investigated effects of EGCG treatment to simultaneously improve cardiovascular and metabolic function in spontaneously hypertensive rats (SHR; model of metabolic syndrome with hypertension, insulin resistance, and overweight). In acute studies, EGCG (1-100 microM) elicited dose-dependent vasodilation in mesenteric vascular beds (MVB) isolated from SHR ex vivo that was inhibitable by N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase antagonist) or wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor]. In chronic studies, 9-wk-old SHR were treated by gavage for 3 wk with EGCG (200 mg.kg(-1).day(-1)), enalapril (30 mg.kg(-1).day(-1)), or vehicle. A separate group of SHR receiving L-NAME (80 mg/l in drinking water) was treated for 3 wk with either EGCG or vehicle. Vasodilator actions of insulin were significantly improved in MVB from EGCG- or enalapril-treated SHR (when compared with vehicle-treated SHR). Both EGCG and enalapril therapy significantly lowered systolic blood pressure (SBP) in SHR. EGCG therapy of SHR significantly reduced infarct size and improved cardiac function in Langendorff-perfused hearts exposed to ischemia-reperfusion (I/R) injury. In SHR given L-NAME, beneficial effects of EGCG on SBP and I/R were not observed. Both enalapril and EGCG treatment of SHR improved insulin sensitivity and raised plasma adiponectin levels. We conclude that acute actions of EGCG to stimulate production of nitric oxide from endothelium using PI 3-kinase-dependent pathways may explain, in part, beneficial effects of EGCG therapy to simultaneously improve metabolic and cardiovascular pathophysiology in SHR. These findings may be relevant to understanding potential benefits of green tea consumption in patients with the metabolic syndrome.     

Schwartz Formula: Is One k-Coefficient Adequate for All Children?

Background/Objective

Plasma-creatinine-based equations to estimate the glomerular filtration rate are recommended by several clinical guidelines. In 2009, Schwartz et al. adapted the traditional Schwartz equation to children and adolescents but did not find different k-coefficients between children and adolescents (k = 36.5 for all patients). We reevaluated the coefficient of the 2009-Schwartz formula according to sex and age in a pediatric population.

Patients/Methods

We used linear mixed-effects models to reestimate the 2009-Schwartz k-coefficient in 360 consecutive French subjects aged 1 to 18 years referred to a single centre between July 2003 and July 2010 (965 measurements). We assessed the agreement between the estimated glomerular filtration rate obtained with the new formula (called Schwartz-Lyon) and the rate measured by inulin clearance. We then compared this agreement to the one between the measured glomerular filtration rate and 2009-Schwartz formula, first in the French then in a Swedish cohort.

Results

In Schwartz-Lyon formula, k was estimated at 32.5 in boys <13 years and all girls and at 36.5 in boys aged ≥13 years. The performance of this formula was higher than that of 2009-Schwartz formula in children <13 years. This was first supported by a statistically significant reduction of the overestimation of the measured glomerular filtration rate in both cohorts, by better 10% and 30% accuracies, and by a better concordance correlation coefficient.

Conclusions

The performance and simplicity of Schwartz formula are strong arguments for its routine use in children and adolescents. The specific coefficient for children aged <13 years further improves this performance.     

Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival,p53-dependent apoptosis and daunorubicin-induced cytotoxicity

Background

The involvement of protein kinase CK2 in sustaining cancer cell survival could have implications also in the resistance to conventional and unconventional therapies. Moreover, CK2 role in blood tumors is rapidly emerging and this kinase has been recognized as a potential therapeutic target. Phase I clinical trials with the oral small ATP-competitive CK2 inhibitor CX-4945 are currently ongoing in solid tumors and multiple myeloma.

Methods

We have analyzed the expression of CK2 in acute myeloid leukemia and its function in cell growth and in the response to the chemotherapeutic agent daunorubicin We employed acute myeloid leukemia cell lines and primary blasts from patients grouped according to the European LeukemiaNet risk classification. Cell survival, apoptosis and sensitivity to daunorubicin were assessed by different means. p53-dependent CK2-inhibition-induced apoptosis was investigated in p53 wild-type and mutant cells.

Results

CK2α was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34⁺ hematopoietic and bone marrow cells. Inhibition of CK2 with CX-4945, K27 or siRNAs caused a p53-dependent acute myeloid leukemia cell apoptosis. CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin. Baseline and daunorubicin-induced STAT3 activation was hampered upon CK2 blockade.

Conclusions

These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival. CK2 negative regulation of the protein levels of tumor suppressor p53 and activation of the STAT3 anti-apoptotic pathway might antagonize apoptosis and could be involved in acute myeloid leukemia cell resistance to daunorubicin.

     

Development of a statistical model of knee kinetics for applications in pre-clinical testing

Pre-clinical computational testing of total knee replacements (TKRs) often only considers a single patient model with simplified applied loads. In studies of multiple patients, most only take into account geometric differences, especially in studies on the knee. Limited availability of kinetic data means that it is difficult to account for inter-patient variability. Principal component analysis (PCA) based statistical models have been used to capture the variation of a set of data and generate new instances of the data. This study presents a method to create a statistical model of kinetic waveform data. A PCA based statistical model was created of the tibiofemoral joint loads for level gait of preoperative TKR patients using data predicted from a musculoskeletal model. A reconstruction test showed that, using principal components (PCs) representing 95% variance, the median root-mean-squared (RMS) error was <0.1 body weight (BW) for the forces and <0.001 BWm for the moments. Leave-one-out tests were also performed and although the median RMS error increased for each load in comparison to the reconstruction error (maximum was 0.2 BW for the axial force and 0.012 BWm for the varus-valgus moment) these were considered within an acceptable limit. The purpose of creating a statistical model is to be able to sample a large set of data representing a population from a small set of clinical data. Such models can potentially be used in population based studies of TKRs incorporating inter-patient variability.     

Evaluation of the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age: The DESiGN cluster randomised trial

BackgroundAntenatal detection and management of small for gestational age (SGA) is a strategy to reduce stillbirth. Large observational studies provide conflicting results on the effect of the Growth Assessment Protocol (GAP) in relation to detection of SGA and reduction of stillbirth; to the best of our knowledge, there are no reported randomised control trials. Our aim was to determine if GAP improves antenatal detection of SGA compared to standard care.Methods and findingsThis was a pragmatic, superiority, 2-arm, parallel group, open, cluster randomised control trial. Maternity units in England were eligible to participate in the study, except if they had already implemented GAP. All women who gave birth in participating clusters (maternity units) during the year prior to randomisation and during the trial (November 2016 to February 2019) were included. Multiple pregnancies, fetal abnormalities or births before 24⁺¹ weeks were excluded. Clusters were randomised to immediate implementation of GAP, an antenatal care package aimed at improving detection of SGA as a means to reduce the rate of stillbirth, or to standard care. Randomisation by random permutation was stratified by time of study inclusion and cluster size. Data were obtained from hospital electronic records for 12 months prerandomisation, the washout period (interval between randomisation and data collection of outcomes), and the outcome period (last 6 months of the study). The primary outcome was ultrasound detection of SGA (estimated fetal weight <10th centile using customised centiles (intervention) or Hadlock centiles (standard care)) confirmed at birth (birthweight <10th centile by both customised and population centiles). Secondary outcomes were maternal and neonatal outcomes, including induction of labour, gestational age at delivery, mode of birth, neonatal morbidity, and stillbirth/perinatal mortality. A 2-stage cluster–summary statistical approach calculated the absolute difference (intervention minus standard care arm) adjusted using the prerandomisation estimate, maternal age, ethnicity, parity, and randomisation strata. Intervention arm clusters that made no attempt to implement GAP were excluded in modified intention to treat (mITT) analysis; full ITT was also reported. Process evaluation assessed implementation fidelity, reach, dose, acceptability, and feasibility. Seven clusters were randomised to GAP and 6 to standard care. Following exclusions, there were 11,096 births exposed to the intervention (5 clusters) and 13,810 exposed to standard care (6 clusters) during the outcome period (mITT analysis). Age, height, and weight were broadly similar between arms, but there were fewer women: of white ethnicity (56.2% versus 62.7%), and in the least deprived quintile of the Index of Multiple Deprivation (7.5% versus 16.5%) in the intervention arm during the outcome period. Antenatal detection of SGA was 25.9% in the intervention and 27.7% in the standard care arm (adjusted difference 2.2%, 95% confidence interval (CI) −6.4% to 10.7%; p = 0.62). Findings were consistent in full ITT analysis. Fidelity and dose of GAP implementation were variable, while a high proportion (88.7%) of women were reached. Use of routinely collected data is both a strength (cost-efficient) and a limitation (occurrence of missing data); the modest number of clusters limits our ability to study small effect sizes.ConclusionsIn this study, we observed no effect of GAP on antenatal detection of SGA compared to standard care. Given variable implementation observed, future studies should incorporate standardised implementation outcomes such as those reported here to determine generalisability of our findings.Trial registrationThis trial is registered with the ISRCTN registry, ISRCTN67698474.

Matias C Vieira and colleagues evaluate the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age in the DESiGN cluster randomised trial.     

Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

Integrin-linked kinase (ILK) is an intracellular effector of cell–matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing.     

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/Significance

We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.