Application of Peptides Containing the Cleavage Sequence of Pro-TNFα in Assessing TACE Activity of Whole Cells

Tumor necrosis factor-α (TNFα) is presumably shed from cell membranes by TNFα-cleaving enzyme (TACE). The peptides SPLAQAVRSSSR and Dabcyl-LAQAVRSSSR-Edans, each encompassing the cleavage sequence of pro-TNFα recognized by TACE, were applied to intact umbilical vein endothelium (HUVEC), peripheral blood leukocytes (PBL) and the mast cell line HMC-1, which express TACE, to homogenates of rat heart tissue and to membrane and cytoplasmic extracts of PBL. Formation of SPLAQA (specific cleavage) was determined by HPLC, while cleavage (specific plus non-specific) of Dabcyl-TNFα-Edans was followed over time by measuring fluorescence. Participation of TACE was assessed from inhibition due to the drug TAPI-2. Incubation with recombinant human TACE gave specific cleavage, fully inhibitable by TAPI-2 (IC50<0.1 μM). HUVEC rapidly degraded TNFα-peptide, but in a non-specific manner (no SPLAQA detectable) and 50 μM TAPI-2 was without effect. Fluorescence was evoked when Dabcyl- LAQAVRSSSR-Edans was incubated with HMC-1 or PBL and also with cytoplasmic and membrane fractions of lysed PBL, but in no case was there significant inhibition by TAPI-2. However, marginal (10%) inhibition of fluorescence by 50 μM TAPI-2 was observed with homogenized heart tissue. This contained TACE, about 75% of which was without the inhibitory cysteine switch (Western blot). In conclusion, simple peptide analogs of pro-TNFα cannot be employed as substrates for measuring membrane TACE activity, largely due to extensive non-specific proteolytic cleavage by whole cells and cell extracts

Release of TNF-α during myocardial reperfusion depends on oxidative stress and is prevented by mast cell stabilizers

Objectives: Our study sought to elucidate the role of oxidative stress for shedding of tumor necrosis factor-α (TNF-α) and for activating TNF-α-converting enzyme (TACE). Background: TNF-α, a central inflammatory cytokine, is discussed as one of the mediators of reperfusion injury. Shedding of membrane-bound pro-TNF-α is thought to be largely due to TNF-α-converting enzyme (TACE). Methods: Release of TNF-α and TACE dependency were studied in isolated rat hearts and in the human mast cell line HMC-1. Results: In reperfused hearts, interstitial release of TNF-α occurred in two phases (2–10 and >45 min). It depended on the presence of oxygen during reperfusion and was attenuated by reduced glutathione. Infusion of the oxidants H2O2 or HOCl elicited release in non-ischemic hearts. TNF-α release was inhibited in hearts treated with degranulation inhibitors ketotifen or cromoglycate, suggesting mast cells as major source for myocardial TNF-α. This was confirmed by tissue staining. Post-ischemic release of histamine, however, did not parallel that of TNF-α. Heart tissue contained mainly mature TACE. HMC-1 expressed abundant pro-TACE and cleaved the pro-TNF-α-peptide Ac-SPLAQAVRSSSR-NH2. However, cleavage was nonspecific and only partly inhibited by TACE inhibitor TAPI-2 (10–100 μmol/l), while it was stimulated by H2O2 and HOCl and fully blocked by the nonspecific metalloprotease inhibitor o-phenanthroline. Conclusions: The mechanism underlying TNF-α release from post-ischemic myocardium is oxidation-dependent but largely independent of activation of TACE. Mast cell stabilizers may be useful in preventing TNF-α release during reperfusion

15N Fractionation in Star-Forming Regions and Solar System Objects

A central issue for understanding the formation and evolution of matter in the early Solar System is the relationship between the chemical composition of star-forming interstellar clouds and that of primitive Solar System materials. The pristinemolecular content of comets, interplanetary dust particles and carbonaceous chondrites show significant bulk nitrogen isotopic fractionation relative to the solar value, 14N15N 440. In addition, high spatial resolution measurements in primitive materials locally show even more extreme enhancements of 14N15N 100

Template-based geometric transformations of a functionally enriched DMU into FE assembly models

International audiencePre-processing of CAD models derived from Digital Mock-Ups (DMUs) into finite element (FE) models is usually completed after many tedious tasks of model preparation and shape transformations. It is highly valuable for simulation engineers to automate time-consuming sequences of assembly preparation processes. Here, it is proposed to use an enriched DMU with geometric interfaces between components (contacts and interferences) and functional properties. Then, the key concept of template-based transformation can connect to assembly functions to locate consistent sets of components in the DMU. Subsequently, sets of shape transformations feed the template content to adapt components to FE requirements. To precisely monitor the friction areas and the mesh around bolts, the template creates sub-domains into their tightened components and preserves the consistency of geometric interfaces for the mesh generation purposes. From a user-selected assembly function, the method is able to robustly identify, locate and transform groups of components while preserving the consistency of the assembly needed for FE models. To enlarge the scope of the template in the assembly function taxonomy, it is shown how the concept of dependent function enforces the geometric and functional consistency of the transformed assembly. To demonstrate the proposed approach, a business oriented prototype processes bolted junctions of aeronautical structures

Birch pollen induces toll-like receptor 4-dependent dendritic cell activation favoring t cell responses

Seasonal exposure to birch pollen (BP) is a major cause of pollinosis. The specific role of Toll-like receptor 4 (TLR4) in BP-induced allergic inflammation and the identification of key factors in birch pollen extracts (BPE) initiating this process remain to be explored. This study aimed to examine (i) the importance of TLR4 for dendritic cell (DC) activation by BPE, (ii) the extent of the contribution of BPE-derived lipopolysaccharide (LPS) and other potential TLR4 adjuvant(s) in BPE, and (iii) the relevance of the TLR4-dependent activation of BPE-stimulated DCs in the initiation of an adaptive immune response. In vitro, activation of murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs by BPE or the equivalent LPS (nLPS) was analyzed by flow cytometry. Polymyxin B (PMB), a TLR4 antagonist and TLR4-deficient BMDCs were used to investigate the TLR4 signaling in DC activation. The immunostimulatory activity of BPE was compared to protein-/lipid-depleted BPE-fractions. In co-cultures of BPE-pulsed BMDCs and Bet v 1-specific hybridoma T cells, the influence of the TLR4-dependent DC activation on T cell activation was analyzed. In vivo immunization of IL-4 reporter mice was conducted to study BPE-induced Th2 polarization upon PMB pre-treatment. Murine and human DC activation induced by either BPE or nLPS was inhibited by the TLR4 antagonist or by PMB, and abrogated in TLR4-deficient BMDCs compared to wild-type BMDCs. The lipid-free but not the protein-free fraction showed a reduced capacity to activate the TLR4 signaling and murine DCs. In human DCs, nLPS only partially reproduced the BPE-induced activation intensity. BPE-primed BMDCs efficiently stimulated T cell activation, which was repressed by the TLR4 antagonist or PMB, and the addition of nLPS to Bet v 1 did not reproduce the effect of BPE. In vivo, immunization with BPE induced a significant Th2 polarization, whereas administration of BPE pre-incubated with PMB showed a decreased tendency. These findings suggest that TLR4 is a major pathway by which BPE triggers DC activation that is involved in the initiation of adaptive immune responses. Further characterization of these BP-derived TLR4 adjuvants could provide new candidates for therapeutic strategies targeting specific mechanisms in BP-induced allergic inflammation

Initiating pollen sensitization: complex source, complex mechanisms

The mechanisms involved in the induction of allergic sensitization by pollen are not fully understood. Within the last few decades, findings from epidemiological and experimental studies support the notion that allergic sensitization is not only dependent on the genetics of the host and environmental factors, but also on intrinsic features of the allergenic source itself. In this review, we summarize the current concepts and newest advances in research focusing on the initial mechanisms inducing pollen sensitization. Pollen allergens are embedded in a complex and heterogeneous matrix composed of a myriad of bioactive molecules that are co-delivered during the allergic sensitization. Surprisingly, several purified allergens were shown to lack inherent sensitizing potential. Thus, growing evidence supports an essential role of pollen-derived components co-delivered with the allergens in the initiation of allergic sensitization. The pollen matrix, which is composed by intrinsic molecules (e.g.proteins, metabolites, lipids, carbohydrates) and extrinsic compounds (e.g.viruses, particles from air pollutants, pollen-linked microbiome), provide a specific context for the allergen and has been proposed as a determinant of Th2 polarization. In addition, the involvement of various pattern recognition receptors (PRRs), secreted alarmins, innate immune cells, and the dependency of DCs in driving pollen-induced Th2 inflammatory processes suggest that allergic sensitization to pollen most likely results from particular combinations of pollen-specific signals rather than from a common determinant of allergenicity. The exact identification and characterization of such pollen-derived Th2-polarizing molecules should provide mechanistic insights into Th2 polarization and pave the way for novel preventive and therapeutic strategies against pollen allergies