Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe
here human tribbles homologues (Htrbs) that control MAPK activity. MAPK ...kinases interact with Trbs and regulate their steady
state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH 2 -terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending
on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase
activation of MAPK.
Aim: To define the contribution made by C reactive protein (CRP) measurement to bacteraemia prediction in adults with medical emergencies in the UK. Methods: This two year cohort study involved 6234 ...patients admitted as emergency cases to the acute medical or infectious diseases services of the Oxford Radcliffe Hospitals, in whom blood cultures were taken on arrival. The main outcome measures were bacteraemia risk associated with admission CRP concentrations, lymphocyte counts, and neutrophil counts. Results: The quantitative associations between CRP concentration, admission lymphocyte count, and neutrophil count were defined. Risk of bacteraemia rose continuously as the CRP increased: no “cutoff” value was evident. Models examining combinations of CRP, neutrophil count, and lymphocyte count were developed and validated using a split sample technique. CRP contributed to a model including lymphocyte and neutrophil counts, but its effect was small. CRP alone performed no better than either a model combining lymphopenia and neutrophilia, or than lymphopenia alone. Conclusions: In patients with acute medical emergencies who are suspected of bacteraemia clinically, CRP concentrations, although associated with bacteraemia, have a limited role in bacteraemia prediction.
Fatal Plasmodium falciparum malaria is accompanied by systemic endothelial activation. To study endothelial activation directly during malaria and sepsis in vivo, the expression of cell adhesion ...molecules on dermal microvascular endothelium was examined in skin biopsies and correlated with plasma levels of soluble (circulating) ICAM-1, E-selectin, and VCAM-1 and the cytokine tumor necrosis factor (TNF)-alpha. Skin biopsies were obtained from 61 cases of severe malaria, 42 cases of uncomplicated malaria, 10 cases of severe systemic sepsis, and 17 uninfected controls. Systemic endothelial activation, represented by the up-regulation of inducible cell adhesion molecules (CAMs) on endothelium and increased levels of soluble CAMs (sCAMs), were seen in both severe and uncomplicated malaria and sepsis when compared with uninfected controls. Plasma levels of sICAM-1, sVCAM-1, and sE-selectin correlated positively with the severity of malaria whereas TNF-alpha was raised nonspecifically in malaria and sepsis. Immunohistochemical evidence of endothelial activation in skin biopsies did not correlate with sCAM levels or disease severity. This indicates a background of systemic endothelial activation, which occurs in both mild and severe malaria and sepsis. The levels of sCAMs in malaria are thus not an accurate reflection of endothelial cell expression of CAMs in a particular vascular bed, and other factors must influence their levels during disease.
Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in infancy with a complex pathology. In adults, the clinical severity of AD has been associated with increases in T helper ...cell type (Th) 2, Th22, and Th17 serum markers, including high levels of CC chemokine ligand (CCL) 17 and CCL22 chemokines.
To explore the possible association between serum chemokine levels and AD severity in infants with moderate-to-severe AD and elevated immunoglobulin E (IgE).
Serum samples (
= 41) obtained from a randomized, double-blind, and clinical dietary intervention study were used to study biomarkers in infants with AD. Baseline- and post-intervention samples (4 months) were used, six chemokines and nine ratios thereof were analyzed using Luminex and correlated to AD severity. In the initial study, the infants were randomized to receive extensively hydrolyzed whey-based formula without (control) or with short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (9:1) and
M-16V (active).
31 Infants up to 11 months of age, with an objective-SCORAD score (oSCORAD) ≥ 20 and elevated total-IgE and/or specific-IgE levels were included. In time, the median oSCORAD decreased in both groups by -8 (control,
< 0.05; active,
< 0.01). Irrespective of dietary intervention, several changes in Th2 chemokines (CCL17 and CCL22), inflammatory chemokine (CCL20), and the Th1 chemokine, CXC chemokine ligand (CXCL) 9, were detected over time. Overall CCL17 correlated to oSCORAD (
= 0.446,
< 0.01). After 4 months of dietary intervention, CXCL9 was higher (
< 0.01) in the active group compared with control active, 2.33 (1.99-2.89); controls, 1.95 (1.77-2.43) log 10 median (range). In addition, a reduction in Th2/Th1 chemokine ratios for CCL17/CXCL9, CCL22/CXCL9, CCL20/CXCL10, and CCL20/CXCL11 was detected associated with the active intervention.
While this study is small and exploratory in nature, these data contribute to immune biomarker profiling and understanding of AD in infants.
The accumulation of DNA sequence information from large-scale genomic and random library sequencing projects is leading to the rapid identification of many putative genes, virtual transcripts and ...ESTs of unknown function. There is therefore an increasing need for high throughput, sensitive and robust methods for identification and characterisation of genes, and/or their products, based on function. We describe a high throughput functional expression screen based on semi-quantitative analysis of enhanced green fluorescent protein expression in single cells by confocal microscopy. The assay was implemented in a micro-scale format, requiring around 10
4 cells/test. The system was validated by co-transfection of a series of cDNAs encoding pro-inflammatory cytokine intracellular signal mediators with a d2EGFP reporter containing a cytokine responsive promoter. The majority of the test plasmids gave a detectable signal above background at a pool size of 250–500. Replicate tests indicate that the assay is reproducible at this pool size. At this level we demonstrate that large (>10
6 transformants) libraries can be feasibly screened.
Partial hepatectomy leads to an orchestrated regenerative response, activating a cascade of cell signaling events necessary for cell cycle progression and proliferation of hepatocytes. However, the ...identity of the humoral factors that trigger the activation of these pathways in the concerted regenerative response in hepatocytes remains elusive. In recent years, extracellular ATP has emerged as a rapidly acting signaling molecule that influences a variety of liver functions, but its role in hepatocyte growth and regeneration is unknown. In this study, we sought to determine if purinergic signaling can lead to the activation of c‐jun N‐terminal kinase (JNK), a known central player in hepatocyte proliferation and liver regeneration. Hepatocyte treatment with ATPγS, a nonhydrolyzable ATP analog, recapitulated early signaling events associated with liver regeneration—that is, rapid and transient activation of JNK signaling, induction of immediate early genes c‐fos and c‐jun, and activator protein‐1 (AP‐1) DNA‐binding activity. The rank order of agonist preference, UTP>ATP>ATPγS, suggests that the effects of extracellular ATP is mediated through the activation of P2Y2 receptors in hepatocytes. ATPγS treatment alone and in combination with epidermal growth factor (EGF) substantially increased cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expression and hepatocyte proliferation in vitro. Extracellular ATP as low as 10 nM was sufficient to potentiate EGF‐induced cyclin D1 expression. Infusion of ATP by way of the portal vein directly activated hepatic JNK signaling, while infusion of a P2 purinergic receptor antagonist prior to partial hepatectomy inhibited JNK activation. In conclusion, extracellular ATP is a hepatic mitogen that can activate JNK signaling and hepatocyte proliferation in vitro and initiate JNK signaling in regenerating liver in vivo. These findings have implications for enhancing our understanding of novel factors involved in the initiation of regeneration, liver growth, and development. (HEPATOLOGY 2004;39:393–402.)
Forebrain neurons have weak intrinsic antioxidant defences compared with astrocytes, but the molecular basis and purpose of this is poorly understood. We show that early in mouse cortical neuronal ...development in vitro and in vivo, expression of the master-regulator of antioxidant genes, transcription factor NF-E2-related-factor-2 (Nrf2), is repressed by epigenetic inactivation of its promoter. Consequently, in contrast to astrocytes or young neurons, maturing neurons possess negligible Nrf2-dependent antioxidant defences, and exhibit no transcriptional responses to Nrf2 activators, or to ablation of Nrf2's inhibitor Keap1. Neuronal Nrf2 inactivation seems to be required for proper development: in maturing neurons, ectopic Nrf2 expression inhibits neurite outgrowth and aborization, and electrophysiological maturation, including synaptogenesis. These defects arise because Nrf2 activity buffers neuronal redox status, inhibiting maturation processes dependent on redox-sensitive JNK and Wnt pathways. Thus, developmental epigenetic Nrf2 repression weakens neuronal antioxidant defences but is necessary to create an environment that supports neuronal development.
Multiple cellular proteins have been identified as participating in Toll/interleukin-1 receptor-mediated inflammatory gene expression. The continuing isolation of novel components, based on sequence ...similarities, protein-protein interactions and protein purification, suggests that many elements of this signalling network remain to be identified. We report here the development of a high-throughput functional screening platform and its application for the identification of components of inflammatory signalling networks. Our results enable us to estimate that 100-150 gene products are involved in controlling the transcription of the human interleukin 8 gene. The approach, which is simple and robust, constitutes a general method for mapping signal transduction systems and for rapid isolation of a large number of signalling components based on the control of pathways leading to regulation of gene expression.
Sustained inflammatory responses are central to the development and progression of chronic diseases, including atherosclerosis and rheumatoid arthritis. A large number of stimuli initiate ...inflammation by acting on Toll–Interleukin-1 related (TIR) domain containing receptors, producing multiple second messengers and thence large scale transcriptional changes. The mechanism by which this activation occurs is complex, and the continuing isolation of novel pathway components, mostly based on sequence similarities and protein–protein interaction studies, suggests that many elements of the TIR-initiated signalling network remain to be identified. Here we use a new technique, allowing identification of components based on function. We report the performance of the screen, our identification of human
tribbles as a novel protein family regulating inflammatory signalling networks, and the detection of ten other components with poorly characterized roles in inflammatory signalling pathways. In total, we have identified 28 signalling molecules of diverse molecular mechanism by screening 11% of a cDNA library for the ability to modulation expression of human IL-8, and other molecules remain to be followed up. The results suggest that the number of human genes involved in IL-8 induction pathways exceed 100. The isolation of signalling components by the approach we describe allows detection of new classes of signalling components independent of existing techniques for doing so; it is simple and robust, and constitutes a general method for mapping signal transduction systems controlling gene expression.
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