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     

Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 1

Structure–activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7 nM and functional antagonistic effect of 66 nM in a BRET and 12 nM in a cAMP assay with no functional activity for the other PGD2 DP receptor (27 μM in cAMP).     

Can ant‐eating Zodarion spiders (Araneae: Zodariidae) develop on a diet optimal for euryphagous arthropod predators?

Abstract Little attention is paid to the behavioural and physiological adaptations of ant‐eating predators. It is expected that there should be a strong selection for traits related to prey handling, leading to the evolution of morphological, behavioural and nutritional adaptations. Such adaptations may then entail trade‐offs in handling and utilization of alternative prey. To investigate behavioural as well as nutritional adaptations and the occurrence of the corresponding trade‐offs in two ant‐eating spiders of the genus Zodarion [Zodarion atlanticum Pekár & Cardoso and Zodarion germanicum (C. L. Koch)], spiders are reared on two diets: ants (i.e. their preferred prey) and fruit flies (i.e. an alternative prey that is nutritionally optimal for euryphagous spiders). Food consumption is observed and several fitness‐related life‐history parameters are measured. Although spiders readily accept ants, more than one‐third of 35 spiders refuse to consume fruit flies and starve. Furthermore, severe hunger does not induce these individuals to accept fruit flies. Starving spiders die before moulting to the second stadium. Spiders that eat fruit flies increase only little and slowly in weight, and all of these die during the first two stadia. By contrast, spiders on an ant diet increase dramatically in weight, and develop up to the fourth stadium. These data indicate that fruit flies are not suitable for Zodarion, supporting the hypothesis that there are behavioural and nutritional trade‐offs. Taking into account the results of previous studies, it is suggested that nutritional trade‐offs are generally important for stenophagous spiders.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.     

Whole Genome-Sequencing and Phylogenetic Analysis of a Historical Collection of Bacillus anthracis Strains from Danish Cattle

Bacillus anthracis, the causative agent of anthrax, is known as one of the most genetically monomorphic species. Canonical single-nucleotide polymorphism (SNP) typing and whole-genome sequencing were used to investigate the molecular diversity of eleven B. anthracis strains isolated from cattle in Denmark between 1935 and 1988. Danish strains were assigned into five canSNP groups or lineages, i.e. A.Br.001/002 (n = 4), A.Br.Ames (n = 2), A.Br.008/011 (n = 2), A.Br.005/006 (n = 2) and A.Br.Aust94 (n = 1). The match with the A.Br.Ames lineage is of particular interest as the occurrence of such lineage in Europe is demonstrated for the first time, filling an historical gap within the phylogeography of the lineage. Comparative genome analyses of these strains with 41 isolates from other parts of the world revealed that the two Danish A.Br.008/011 strains were related to the heroin-associated strains responsible for outbreaks of injection anthrax in drug users in Europe. Eight novel diagnostic SNPs that specifically discriminate the different sub-groups of Danish strains were identified and developed into PCR-based genotyping assays.     

Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces.     

Sequence Analysis and Identification of New Isoform of EP4 Receptors in Different Atlantic Salmon Tissues (Salmo salar L.) and Its Role in PGE2 Induced Immunomodulation In Vitro

PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5’ untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.     

Hollow oaks and beetle conservation: the significance of the surroundings

In this study we investigated hollow oaks (Quercus robur, Q. petrea) situated in open landscapes and in forests in Norway in northern Europe, and compared their importance for rare and threatened beetles (Coleoptera). Old, hollow oak trees, both in parks and in forests, were extremely rich in red-listed beetles, and hosted a high proportion of threatened species. The proportion of oak associated species and the mean number of red-listed beetle species per tree was similar in the two site types, but rarefaction showed that for a certain number of individuals, oaks in forests had more threatened and near-threatened species than oaks in parks. The species composition also differed between site types: Park oaks had a higher proportion of species associated with hollows and animal nests, whereas in forests, there was a higher proportion of species depending on dead oak wood in general. Four factors were significant in explaining the richness of red-listed beetles in our study: Tree circumference, cavity decay stage, proportion of oak in the surroundings, and coarse woody debris (CWD) in the surroundings. Forest oaks were smaller, but they still trapped a species richness comparable to that of the larger park oaks—probably a result of high amounts of CWD in the surroundings. We show that oaks in open landscapes and oaks in forest have only partly overlapping beetle assemblages and, thus, cannot be substituted in conservation. Planning for conservation of red-listed beetles associated with this key habitat demands a large scale perspective, both in space and time, as the surroundings have important effects on associated threatened and near threatened species.     

Discovery of novel,orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.