Mast cell responses to danger signals

Detecting and responding to danger is a paramount function of the immune system. Compounds heralding danger can be divided into two groups: exogenous and endogenous danger signals. The former group consists of conserved microbial structures such as lipopolysaccharide (LPS), while the latter consists of host compounds released or exposed by dead or dying cells as a consequence of trauma, stress or infection. Mast cells are long-lived immune cells present in almost all tissues, and are especially numerous at sites facing the external environment, making them ideal responders to danger signals. The aim of the work presented in this thesis was to investigate mast cell responses to danger signals of exogenous and endogenous origin. In Paper I, we investigated mast cell responses to the exogenous danger signal M-TriDAP, a bacterial peptidoglycan degradation product. We found that cord bloodderived mast cells (CBMCs) express NOD1, the receptor for M-TriDAP. Furthermore, M-TriDAP-treatment of CBMCs resulted in degranulation-independent release of cytokines and chemokines such as TNF, IL-8/CXCL8, MIP-1α/CCL3 and MIP-1β/CCL4. Importantly, we observed an augmented response when M-TriDAP was combined with the TLR4 agonist LPS, indicating cooperation between intracellular and extracellular pattern recognition receptors. In Paper II, we investigated mast cell responses to cell injury by subjecting murine mast cells to the supernatant of fibroblasts rendered necrotic by freeze-thawing. We found that mast cells respond to cell injury in this model by initiating a proinflammatory response, characterized by degranulation-independent release of cytokines and leukotrienes. By using genetically modified mice and molecular inhibitors, we found that the recognition of cell injury was MyD88-, T1/ST2- and p38- dependent. Finally, by using RNA-interference, we could pinpoint IL-33 as the necrotic cell compound that was responsible for the mast cell activation. In Paper III, we investigated responses to IL-33 administration in vivo. Here we found that wild-type C57BL/6 mice respond to intraperitoneal IL-33 administration with neutrophil infiltration. This response was not observed in mast cell-deficient mice but could be restored upon mast cell reconstitution, thus demonstrating a mast cell dependent mechanism. In Paper IV, we investigated the hypothesis that mast cells might function as sensors of damaged epithelia by responding to IL-33 during chronic inflammations of the airways, for instance in asthma. We found that IL-33 is released from necrotic airway epithelial cells and that CBMCs respond to the necrotic supernatant of these cells by secreting IL-5, IL-8/CXCL8, TNF and GM-CSF. However, no release of histamine, LTB4 or PGD2 could be detected. Interestingly, the exact same mediator release pattern was observed when CBMCs were treated with recombinant IL-33, suggesting that IL-33 might be an important factor released by injured airway epithelial cells that activates mast cells. In conclusion, the work presented in this thesis provides further evidence for important roles of mast cells in innate immune responses. The function of mast cells as sensors of cell injury is highlighted; a role that potentially can be either beneficial or detrimental. Finally, novel evidence is provided for the notion that IL-33 is an important danger signal capable of mast cell activation

Mast Cells Respond to Cell Injury through the Recognition of IL-33

Mast cells have been attributed several functions in both health and disease. Mast cell activation and release of inflammatory mediators are associated with the pathogenesis of several diseases, in particular that of allergic diseases. While the notion of mast cells as important, protective sentinel cells is old, this feature of the cell is not well recognized outside the mast cell field. The mast cell is a unique, multifunctional cell of our defense system, with characteristics such as wide-spread tissue distribution, expression of receptors capable of recognizing both endogenous and exogenous agents, and a capability to rapidly respond to triggering factors by selective mediator release. In this review, we discuss the function of mast cells as sentinel cells in the context of cell injury, where mast cells respond by initiating an inflammatory response. In this setting, IL-33 has turned out to be of particular interest. IL-33 is released by necrotic structural cells and is recognized by mast cells via the IL-33 receptor ST2. IL-33 and mast cells probably constitute one important link between cell injury and an inflammatory response that can lead to restoration of tissue function and homeostasis, but might under other circumstances contribute to a vicious circle driving chronic inflammation

The Extended Cleavage Specificity of Human Thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1′-Ser/Ala/Gly/Thr, P2′-not acidic and P3′-Arg. Our analysis also identifies an important role for a P3′-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3′-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200–400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times

Differential Impedance Measurement Method of RFID Transponder Chips at UHF

A novel on-wafer measurement method of RFID transponder chips is presented. A comparison is made between single ended one-port, single ended two-port and differential two-port excitation. The two-port method is a flexible way of measuring chips consisting of both several individuals as well as chip types with different geometries with one and the same probe type. The theory of un-terminating and de-embedding is described and verified by measurements. A qualitative analysis is defined, which explains certain phenomena seen in communication tests performed on RFID protocol level. This is further supported by measurement results from the presented method

Differential Impedance Measurement Method of RFID Transponder Chips at UHF

A novel on-wafer measurement method of RFID transponder chips is presented. A comparison is made between single ended one-port, single ended two-port and differential two-port excitation. The two-port method is a flexible way of measuring chips consisting of both several individuals as well as chip types with different geometries with one and the same probe type. The theory of un-terminating and de-embedding is described and verified by measurements. A qualitative analysis is defined, which explains certain phenomena seen in communication tests performed on RFID protocol level. This is further supported by measurement results from the presented method

Shifted Source Impedance and Nonlinearity Impact on RFID Transponder Communication for Drive-Level Offsets

A measurement and analysis method to quantify the communication quality for ultrahigh-frequency radio frequency identification (RFID) transponder chips under a shifting source environment is presented. A detailed description of theory, setup, and procedure is provided. The differences in on/off impedance (|ΔΓ|-value) of the transponder chip are critical for backscatter modulation and, therefore, the overall communication and transponder response. In the present work, a new quantity which includes the difference in on/off impedance, as well as arbitrary source impedance, is defined as an overall figure of merit. A qualitative analysis, considered as novel in the RFID community, shows the impact a source impedance shift has on the communication quality. The bare chip measurement setup enables an ASK modulated wake up signal, realized as a pulse train. The chip response consisting of modulated impedance states are measured in a pulse profile setup. Results show that different degrees of non-linearity in a transponder chip, depending on vendor and type, will have impact on the communication performance

Differential Transmission Line Loop for RFID Reactive Near-Field Coupling

The differential transmission line loop (DTLL) as an efficient reactive near-field coupling element for programming of radio frequency identification (RFID) inlays is proposed. It is validated, with simulations and measurements, that the differential excitation and the presence of a ground plane are fundamental properties for high performance in coupling while providing high electromagnetic isolation. As a proof of concept, super elliptical loop geometries as coupling elements are investigated for a number of commercially available inlay geometries. From simulations of the DTLL and inlay antenna, coupling efficiencies higher than 80% are predicted. Measurements of the \Delta \Gamma -value indicate a nonlinear input impedance with power, and verify the high values of simulated coupling efficiency. The threshold power levels read from interrogator are below 5 dBm, which are considered as very low in inlay encoding, and hence show that the DTLL is a promising candidate for efficient RFID programming in the reactive near field

Lumped element balun with inherent complex impedance transformation

A novel lumped design approach for complex impedance transforming baluns is presented in this paper. It is shown that a relaxation of symmetry in the T-networks of the out-of-phase-compensated-power-splitter enables complex impedance transformation. Design equations are analytically derived for a total of 4 component values, of which 2 values depend upon the 2 other, which are free variables. The two free component values are used independently for adjustment of input reflection loss, further keeping the balance parameter maximally flat and independent of the load impedance. For Q-values of source and load, not being excessively high, the balun can be realized with only 8 components. A demonstrator is fabricated, transforming 26.9 + j11.1 Ω to 73.8 + j38.6 Ω. An amplitude balance of \ub10.7 dB and phase balance better than \ub15\ub0 is achieved over a 20 % bandwidth. The return loss is higher than 20 dB

Design Equations for Lumped Element Balun With Inherent Complex Impedance Transformation

This paper demonstrates a novel lumped design approach for complex impedance transformation, based on symmetry relaxation in the out-of-phase-compensated-power-splitter. In the modified topology, a total of four component values are used, of which two values analytically depend upon the other two, keeping the balance parameter maximally flat and independent of the source and load impedance, as well as of the balun internal components. From the condition of zero input reflection loss, the two other component values are also determined analytically, hence specifying all component values uniquely. A Monte Carlo sensitivity analysis predicts an amplitude imbalance, which in practice defines the limit in balance operational bandwidth, of better than +/-1 dB, over a 20% bandwidth at 925 MHz operating frequency. Based on the design equations, demonstrators for four different design cases are fabricated, verifying the simulated performance