Experientia supplementum (2012)最新文献

To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was possible to induce T cell priming to most of the moderate/weak allergens, including lipophilic molecules highly insoluble in culture media. Therefore, the present optimized in vitro human T cell priming assay is a valuable method to detect the sensitizing properties of chemical allergens.

This chapter provides an overview of different methodologies to dissect the ATPase mechanism of motor proteins. The use of ATP is fundamental to how these molecular engines work and how they can use the energy to perform various cellular roles. Rapid reaction and single-molecule techniques will be discussed to monitor reactions in real time through the application of fluorescence intensity, anisotropy and FRET. These approaches utilise fluorescent nucleotides and biosensors. While not every technique may be suitable for your motor protein, the different ways to determine the ATPase mechanism should allow a good evaluation of the kinetic parameters.

Most biochemical processes occur on sub-second time scales. Relaxation and rapid mixing methods allow reactions from microsecond time scales onwards to be monitored in real time. This chapter describes the instrumentation for these techniques and it discusses general topics of sample excitation and signal detection.

Motor proteins convert the chemical energy of adenosine triphosphate (ATP) hydrolysis into directed movement along filamentous tracks, such as DNA, microtubule, and actin. The motile properties of motors are essential to their wide variety of cellular functions, including cargo transport, mitosis, cell motility, nuclear positioning, and ciliogenesis. Detailed understanding of the biophysical mechanisms of motor motility is therefore essential to understanding the physical basis of these processes. In which direction is the motor going? How fast and how far can a single motor walk down its track? How is ATP hydrolysis coupled to directed motion? How do multiple subunits of a motor coordinate with each other during motility? These questions can be addressed directly by tracking motors at a single-molecule level. This chapter will focus on high-resolution fluorescence tracking techniques of the processive cytoskeletal motors: myosins, kinesins, and cytoplasmic dynein. We outline the theoretical and practical considerations for studying these motors in vitro using fluorescence tracking at nanometer precision.

Allergic contact dermatitis is a T cell-mediated skin disease. Many hundreds of organic chemicals and some metal ions are contact sensitizers. They induce an innate inflammatory immune response in the skin that results in the priming of contact sensitizer-specific T cells by dendritic cells in the draining lymph nodes. The factors that determine the strength of this T cell response and thereby define the potency of a contact sensitizer are largely unknown. This chapter highlights different variables such as precursor frequency of antigen-specific T cells, possible bystander activation, and T cell receptor diversity or avidity of the TCR/peptide-MHC interactions, which might impact the quality and strength of T cell responses to contact sensitizers. In addition, different methods available to determine both the frequency of antigen-specific T cells and T cell receptor repertoires are discussed. Identification of the factors determining potency may allow for the development of suitable in vitro assays for potency assessment of contact sensitizers.

Drug hypersensitivity reactions are immune mediated, with T lymphocytes being stimulated by the drugs via their T-cell antigen receptor (TCR). In the nonpathogenic state, the TCR is activated by foreign peptides presented by major histocompatibility complex molecules (pMHC). Foreign pMHC binds with sufficient affinity to TCRαβ and thereby elicits phosphorylation of the cytoplasmic tails of the TCRαβ-associated CD3 subunits. The process is called TCR triggering. In this review, we discuss the current models of TCR triggering and which drug properties are crucial for TCR stimulation. The underlying molecular mechanisms mostly include pMHC-induced exposure of the CD3 cytoplasmic tails or alterations of the kinase-phosphatase equilibrium in the vicinity of CD3. In this review, we also discuss triggering of the TCR by small chemical compounds in context of these general mechanisms.

Myosins are a large superfamily of actin-dependent molecule motors that carry out many functions in cells. Some myosins are cargo carriers that move processively along actin which means that a single molecule of myosin can take many ATP-dependent steps on actin per initial encounter. Other myosins are designed to work in large ensembles such as myosin thick filaments. In vitro motility assays are a powerful method for studying the function of myosins. These assays in general use small amounts of protein, are simple to implement, and can be done on microscopes commonly found in many laboratories. There are two basic versions of the assay which involve different geometries. In the sliding actin in vitro motility assay, myosin molecules are bound to a coverslip surface in a simply constructed microscopic flow chamber. Fluorescently labeled actin filaments are added to the flow chamber in the presence of ATP, and the movement of these actin filaments powered by the surface-bound myosins is observed. This assay has been used widely for a variety of myosins including both processive and non-processive ones. From this assay, one can easily measure the rate at which myosin is translocating actin. The single-molecule motility assay uses an inverted geometry compared to the sliding actin in vitro motility assay. It is most useful for processive myosins. Here, actin filaments are affixed to the coverslip surface. Fluorescently labeled single molecules of myosins (usually ones with processive kinetics) are introduced, and the movement of single molecules along the actin filaments is observed. This assay typically uses total internal reflection fluorescent (TIRF) microscopy to reduce the background signal arising from myosins in solution. From this assay, one can measure the velocity of movement, the frequency of movement, and the run length. If sufficient photons can be collected, one can use Gaussian fitting of the point spread function to determine the position of the labeled myosin to within a few nanometers which allows for measurement of the step size and the stepping kinetics. Together, these two assays are powerful tools to elucidate myosin function.

Small chemical compounds and certain metal ions can activate T cells, resulting in drug hypersensitivity reactions that are a main problem in pharmacology. Mostly, the drugs generate new antigenic epitopes on peptide-major histocompatibility complex (MHC) molecules that are recognized by the T-cell antigen receptor (TCR). In this review we discuss the molecular mechanisms of how the drugs alter self-peptide-MHC, so that neo-antigens are produced. This includes (1) haptens covalently bound to peptides presented by MHC, (2) metal ions and drugs that non-covalently bridge self-pMHC to the TCR, and (3) drugs that allow self-peptides to be presented by MHCs that otherwise are not presented. We also briefly discuss how a second signal-next to the TCR-that naïve T cells require to become activated is generated in the drug hypersensitivity reactions.

In this chapter, we describe experimental techniques used in vitro to illuminate how small teams of motors can work to translocate cargos. We will focus on experiments utilizing in vitro reconstitution, artificial or ex vivo purified cargos, and fluorescence imaging. A number of studies have been able to recapitulate the activities of cargo transport driven by small teams of motors elucidating how multiple motors can work together to transport cargos within the cell. Here, we describe some of the methods employed and highlight important experimental details needed to perform these experiments.

The development of allergic sensitisation by environmental chemicals results in allergic contact dermatitis and highly undesirable morbidity and disability. This form of hypersensitivity is mediated by specific T lymphocytes that recognise the chemical sensitiser bound to self-proteins. Use of deliberate experimental contact sensitisation with dinitrochlorobenzene (DNCB) has been used to investigate the human immune system which exhibits dose-related responses. Many factors contribute to whether sensitisation occurs and the nature and magnitude of the immune response. Chemicals vary in sensitising potency, mainly reflecting their intrinsic protein-binding properties. The amount of sensitiser reaching the immune system is determined by many factors of which the concentration (dose per unit area), the relative lipid solubility and molecular weight are the most critical. Host-related factors contributing to the nature and magnitude of immune responses are mainly genetically determined including gender, age, the biochemical/physical integrity of the epidermal barrier and the quality of the innate and adaptive immune systems. The underlying mechanisms must be elucidated before it will be possible to make reliable predictions of whether a given individual will develop allergic sensitisation by a given chemical.