Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists

Tobacco smoking has been associated with impaired pulmonary functions and increased incidence of infections; however, mechanisms that underlie these phenomena are poorly understood. In this study, we examined whether smokers' alveolar macrophages (AM) exhibit impaired sensing of bacterial components via TLR2 and TLR4 and determined the effect of smoking on expression levels of TLR2, TLR4 and coreceptors, and activation of signaling intermediates. Smokers' AMs exhibited reduced gene expression and secretion of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and chemokines (RANTES and IL-8) upon stimulation with TLR2 and TLR4 agonists, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride (Pam(3)Cys), and LPS, whereas expression of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist) was not affected. TLR3 activation with polyinosinic-polycytidylic acid led to comparable or even higher cytokine responses in smokers' AMs, indicating that smoking-induced suppression does not affect all TLRs. Comparable expression of cytokines and chemokines was detected in PBMC and purified monocytes obtained from smokers and nonsmokers, demonstrating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible IL-1R-associated kinase-1 (IRAK-1) and p38 phosphorylation and NF-kappaB activation was suppressed in smokers' AMs, whereas TLR2, TLR4, CD14, MD-2 mRNA levels, and TLR4 protein expression were not altered. These data suggest that changes in expression and/or activities of signaling intermediates at the postreceptor level account for smoking-induced immunosuppression. Thus, exposure of AMs to tobacco smoke induces a hyporesponsive state similar to endotoxin tolerance as manifested by inhibited TLR2/4-induced expression of proinflammatory cytokines, chemokines, and impaired activation of IRAK-1, p38, and NF-kappaB, resulting in suppressed expression of proinflammatory mediators.     

Alternative complex formation of the Ca-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis

Intracellular release of calcium ions belongs to the earliest events in cellular stress perception. The molecular mechanisms integrating signals from different environmental cues and translating them into an optimized response are largely unknown. We report here the functional characterization of CIPK1, a protein kinase interacting strongly with the calcium sensors CBL1 and CBL9. Comparison of the expression patterns indicates that the three proteins execute their functions in the same tissues. Physical interaction of CIPK1 with CBL1 and CBL9 targets the kinase to the plasma membrane. We show that, similarly to loss of CBL9 function, mutation of either CBL1 or CIPK1 renders plants hypersensitive to osmotic stress. Remarkably, in contrast to the cbl1 mutant and similarly to the cbl9 mutant, loss of CIPK1 function impairs abscisic acid (ABA) responsiveness. We therefore suggest that, by alternative complex formation with either CBL1 or CBL9, the kinase CIPK1 represents a convergence point for ABA-dependent and ABA-independent stress responses. Based on our genetic, physiological and protein-protein interaction data, we propose a general model for information processing in calcium-regulated signalling networks.     

A long-term porcine model for measurement of gastrointestinal motility

Animal models have become an essential tool in the investigations of gut motility under experimental conditions. To determine the influence of various anaesthetic drugs on the motility pattern of the gastroduodenal tract, a new long-term model has had to be developed for allowing measurements in conscious and unrestrained as well as in sedated and analgosedated pigs. Since mechanical ventilation influences gut motility, it was necessary that this animal model enabled the investigation of the effect of drugs causing sedation and analgosedation during spontaneous breathing. Seven male, castrated pigs, German landrace, 32-40 kg bodyweight (BW) were investigated in this study. After habituation of the pigs to local housing conditions over 5 days, the animals were trained over 4 days to prepare for experimental situations and investigators. Pigs were inserted with a central venous catheter and with percutaneous enterogastrostomy (PEG) under general anaesthesia. Intestinal motility was measured by intraluminal impedancometry. The catheter was introduced over the PEG into the stomach and positioned into the duodenum by duodenoscopy. Measurements were done in conscious, unrestrained pigs and with sedated, and analgosedated animals on subsequent days. The habituation and training of the pigs to the investigators and for the laboratory conditions took between 7 and 9 days. The initial anaesthesia protocol for the instrumentation using remifentanil/propofol led to pyloric spasm and was thus unsuitable for duodenal intubation with an endoscope. In contrast, a combination of ketamine/propofol enabled this procedure. It was practicable to measure gut motility in conscious, unrestrained pigs. Spontaneous breathing was sufficient under propofol sedation and analgosedation using fentanyl-propofol. Systematically local application of polividon iodine in the area of the subcutaneous catheters avoided the necessity of using systemic prophylactic antibiotics. In conclusion, the habituation and training for 9 days enabled the measurement of gut motility by intraluminal impedancometry in conscious pigs. The insertion of the catheter was done during general anaesthesia using a combination of propofol and ketamine. For the future determination of gut motility performed under general anaesthesia, each sedation and analgosedation concept has to be evaluated to see whether it allows spontaneous breathing or whether mechanical ventilation is necessary.     

Oxidative stress is a consequence, not a cause, of aluminum toxicity in the forage legume Lotus corniculatus

? Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. ? Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. ? The exposure of plants to 10 μM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 μM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. ? We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 μM Al.     

Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

Climate–ecosystem modelling made easy: The Land Sites Platform

Dynamic Global Vegetation Models (DGVMs) provide a state-of-the-art process-based approach to study the complex interplay between vegetation and its physical environment. For example, they help to predict how terrestrial plants interact with climate, soils, disturbance and competition for resources. We argue that there is untapped potential for the use of DGVMs in ecological and ecophysiological research. One fundamental barrier to realize this potential is that many researchers with relevant expertize (ecology, plant physiology, soil science, etc.) lack access to the technical resources or awareness of the research potential of DGVMs. Here we present the Land Sites Platform (LSP): new software that facilitates single-site simulations with the Functionally Assembled Terrestrial Ecosystem Simulator, an advanced DGVM coupled with the Community Land Model. The LSP includes a Graphical User Interface and an Application Programming Interface, which improve the user experience and lower the technical thresholds for installing these model architectures and setting up model experiments. The software is distributed via version-controlled containers; researchers and students can run simulations directly on their personal computers or servers, with relatively low hardware requirements, and on different operating systems. Version 1.0 of the LSP supports site-level simulations. We provide input data for 20 established geo-ecological observation sites in Norway and workflows to add generic sites from public global datasets. The LSP makes standard model experiments with default data easily achievable (e.g., for educational or introductory purposes) while retaining flexibility for more advanced scientific uses. We further provide tools to visualize the model input and output, including simple examples to relate predictions to local observations. The LSP improves access to land surface and DGVM modelling as a building block of community cyberinfrastructure that may inspire new avenues for mechanistic ecosystem research across disciplines.     

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.     

Radioimmunoimaging of Liver Metastases with PET Using a 64Cu-Labeled CEA Antibody in Transgenic Mice

Purpose

Colorectal cancer is one of the most common forms of cancer, and the development of novel tools for detection and efficient treatment of metastases is needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (PET) imaging and radioimmunotherapy. Since carcinoembryonic antigen (CEA) is an important target in colorectal cancer, the CEA-specific M5A antibody has been extensively studied in subcutaneous xenograft models; however, the M5A antibody has not yet been tested in advanced models of liver metastases. The aim of this study was to investigate the ⁶⁴Cu-DOTA-labeled M5A antibody using PET in mice bearing CEA-positive liver metastases.

Procedures

Mice were injected intrasplenically with CEA-positive C15A.3 or CEA-negative MC38 cells and underwent micro-computed tomography (micro-CT) to monitor the development of liver metastases. After metastases were detected, PET/MRI scans were performed with ⁶⁴Cu-DOTA-labeled M5A antibodies. H&E staining, immunohistology, and autoradiography were performed to confirm the micro-CT and PET/MRI findings.

Results

PET/MRI showed that M5A uptake was highest in CEA-positive metastases. The %ID/cm³ (16.5%±6.3%) was significantly increased compared to healthy liver tissue (8.6%±0.9%) and to CEA-negative metastases (5.5%±0.6%). The tumor-to-liver ratio of C15A.3 metastases and healthy liver tissue was 1.9±0.7. Autoradiography and immunostaining confirmed the micro-CT and PET/MRI findings.

Conclusion

We show here that the ⁶⁴Cu-DOTA-labeled M5A antibody imaged by PET can detect CEA positive liver metastases and is therefore a potential tool for staging cancer, stratifying the patients or radioimmunotherapy.     

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative,Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing^TM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.     

Toll-like receptor (TLR)2 and TLR4 agonists regulate CCR expression in human monocytic cells

Interactions between proinflammatory and cell maturation signals, and the pathways that regulate leukocyte migration, are of fundamental importance in controlling trafficking and recruitment of leukocytes during the processes of innate and adaptive immunity. We have investigated the molecular mechanisms by which selective Toll-like receptor (TLR)2 and TLR4 agonists regulate expression of CCR1 and CCR2 on primary human monocytes and THP-1 cells, a human monocytic cell line. We found that activation of either TLR2 (by Pam(3)CysSerLys(4)) or TLR4 (by purified LPS) resulted in down-modulation of both CCR1 and CCR2. Further investigation of TLR-induced down-modulation of CCR1 revealed differences in the signaling pathways activated, and chemokines generated, via the two TLR agonists. TLR2 activation caused slower induction of the NF-kappa B and mitogen-activated protein kinase signaling pathways and yet a much enhanced and prolonged macrophage-inflammatory protein 1 alpha (CC chemokine ligand 3) protein production, when compared with TLR4 stimulation. Enhanced macrophage-inflammatory protein 1 alpha production may contribute to the prolonged down-regulation of CCR1 cell surface expression observed in response to the TLR2 agonist, as preventing chemokine generation with the protein synthesis inhibitor cycloheximide, or CCR1 signaling with the receptor antagonist UCB35625, abolished TLR2- and TLR4-induced CCR1 down-modulation. This result suggests an autocrine pathway, whereby TLR activation can induce chemokine production, which then leads to homologous down-regulation of the cognate receptors. This work provides further insights into the mechanisms that regulate leukocyte recruitment and trafficking during TLR-induced inflammatory responses.