Functional activation of the formyl peptide receptor by a new endogenous ligand in human lung A549 cells

The formyl peptide receptor (FPR), a heptahelical G protein-coupled receptor on phagocytic leukocytes, can be triggered by bacterially derived oligopeptides of the prototype fMLP. Although FPR expression and activation have been associated with cells of myeloid origin and bacterial inflammation, the receptor has recently been identified in nonmyeloid cells, thus suggesting additional physiological functions and the existence of an endogenous agonist. In this study, we demonstrate the presence and functional activation of the FPR in the human lung cell line A549, which represents an extrahepatic model for the regulation of acute-phase proteins. Activation of the FPR in A549 cells cannot only be triggered by fMLP, but also by an agonistic peptide of the recently identified endogenous FPR ligand, annexin 1. In addition to inducing changes in the F-actin content, annexin 1-mediated triggering of the FPR results in an increased expression of acute-phase proteins. Hence, activation of nonmyeloid FPR by its endogenous ligand annexin 1 could participate in the regulation of acute-phase responses, e.g., during inflammation and/or wound healing.     

Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain

Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.     

Endothelial Rho signaling is required for monocyte transendothelial migration

Bacterial toxins affecting Rho activity in microvascular endothelial cells were employed to elucidate whether endothelial Rho participates in regulating the migration of monocytes across monolayers of cultured endothelial cells. Inactivation of Rho by the Clostridium C3 exoenzyme resulted in an increased adhesion of peripheral blood monocytes to the endothelium and a decreased rate of transendothelial monocyte migration. Cytotoxic necrotizing factor 1-mediated activation of endothelial Rho also reduced the rate of monocyte transmigration, but did not affect monocyte-endothelium adhesion. Thus, efficient leukocyte extravasation requires Rho signaling not only within the migrating leukocytes but also within the endothelial lining of the vessel wall.     

Characterization of a discontinuous epitope on annexin II by site-directed mutagenesis.

Recombinant annexin II mutants were generated to identify amino acids involved in the formation of the discontinuous epitope of the monoclonal antibody H28. Analysis of the various mutant proteins by immunoblotting and enzyme-linked immunosorbent assay revealed that residues Lys27, Arg62, Glu65, and Arg67 are indispensable for H28 reactivity. Residues in equivalent positions are also in close proximity in the recently determined X-ray structure of annexin V, a different member of the same family of Ca2+/lipid-binding proteins. Thus annexins II and V show a similar three-dimensional folding in this region of the molecule. Consequently, the Ca2+ binding sites and the residues phosphorylated by pp60src (Tyr23) and protein kinase C (Ser25) most likely reside on opposite sides of the annexin II molecule.     

Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry

High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats.

In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.     

VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

Blood vessel tubulogenesis requires the formation of stable cell-to-cell contacts and the establishment of apicobasal polarity of vascular endothelial cells. Cell polarity is regulated by highly conserved cell polarity protein complexes such as the Par3-aPKC-Par6 complex and the CRB3-Pals1-PATJ complex, which are expressed by many different cell types and regulate various aspects of cell polarity. Here we describe a functional interaction of VE-cadherin with the cell polarity protein Pals1. Pals1 directly interacts with VE-cadherin through a membrane-proximal motif in the cytoplasmic domain of VE-cadherin. VE-cadherin clusters Pals1 at cell–cell junctions. Mutating the Pals1-binding motif in VE-cadherin abrogates the ability of VE-cadherin to regulate apicobasal polarity and vascular lumen formation. In a similar way, deletion of the Par3-binding motif at the C-terminus of VE-cadherin impairs apicobasal polarity and vascular lumen formation. Our findings indicate that the biological activity of VE-cadherin in regulating endothelial polarity and vascular lumen formation is mediated through its interaction with the two cell polarity proteins Pals1 and Par3.     

Stakeholders’ views on the ethical challenges of pragmatic trials investigating pharmaceutical drugs

BackgroundWe explored the views of key stakeholders to identify the ethical challenges of pragmatic trials investigating pharmaceutical drugs. A secondary aim was to capture stakeholders’ attitudes towards the implementation of pragmatic trials in the drug development process.MethodsWe conducted semistructured, in-depth interviews among individuals from different key stakeholder groups (academia and independent research institutions, the pharmaceutical industry, regulators, Health Technology Assessment (HTA) agencies and patients’ organizations) through telephone or face-to-face sessions. Interviews were structured around the question “what challenges were experienced or perceived during the design, conduct and/or review of pragmatic trials.” Respondents were additionally asked about their views on implementation of pragmatic trials in the drug development process. Thematic analysis was used to identify the ethically relevant features across data sets.ResultsWe interviewed 34 stakeholders in 25 individual sessions and four group sessions. The four perceived challenges of ethical relevance were: (1) less controlled conditions creating safety concerns, (2) comparison with usual care potentially compromising clinical equipoise, (3) tailored or waivers of informed consent affecting patient autonomy, and (4) minimal interference with “real-world” practice reducing the knowledge value of trial results.ConclusionsWe identified stakeholder concerns regarding risk assessment, use of suboptimal usual care as a comparator, tailoring of informed consent procedures and ensuring the social value of pragmatic trials. These concerns increased when respondents were asked about pragmatic trials conducted before market authorization.

Electronic supplementary material

The online version of this article (doi:10.1186/s13063-016-1546-3) contains supplementary material, which is available to authorized users.     

S100-annexin complexes: some insights from structural studies

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex.     

A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR

The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I-mediated antiinflammatory effects.     

Cuauthemone sesquiterpenoids from Blumea alata

The investigation of five Blumea species afforded, in addition to known compounds, several cuauthemone derivatives, most of them being 7,11-epoxides, some thymol derivatives, an eremophilane epoxide and a himachalane diol. From Pterocaulon virgatum a further himachalane derivative was isolated. The structures were elucidated by spectroscopic methods and in part by partial synthesis.