The IL-13/periostin/IL-24 pathway causes epidermal barrier dysfunction in allergic skin inflammation.

BACKGROUND: Barrier dysfunction is an important feature of atopic dermatitis (AD) in which IL-4 and IL-13, signature type 2 cytokines, are involved. Periostin, a matricellular protein induced by IL-4 or IL-13, plays a crucial role in the onset of allergic skin inflammation, including barrier dysfunction. However, it remains elusive how periostin causes barrier dysfunction downstream of the IL-13 signal. METHODS: We systematically identified periostin-dependent expression profile using DNA microarrays. We then investigated whether IL-24 downregulates filaggrin expression downstream of the IL-13 signals and whether IL-13-induced IL-24 expression and IL-24-induced downregulation of filaggrin expression are dependent on the JAK/STAT pathway. To build on the significance of in vitro findings, we investigated expression of IL-24 and activation of STAT3 in mite-treated mice and in AD patients. RESULTS: We identified IL-24 as an IL-13-induced molecule in a periostin-dependent manner. Keratinocytes are the main IL-24-producing tissue-resident cells stimulated by IL-13 in a periostin-dependent manner via STAT6. IL-24 significantly downregulated filaggrin expression via STAT3, contributing to barrier dysfunction downstream of the IL-13/periostin pathway. Wild-type mite-treated mice showed significantly enhanced expression of IL-24 and activation of STAT3 in the epidermis, which disappeared in both STAT6-deficient and periostin-deficient mice, suggesting that these events are downstream of both STAT6 and periostin. Moreover, IL-24 expression was enhanced in the epidermis of skin tissues taken from AD patients. CONCLUSIONS: The IL-13/periostin pathway induces IL-24 production in keratinocytes, playing an important role in barrier dysfunction in AD.

Highly expressed cytoplasmic FcεRIß in human mast cells functions as a negative regulator of the FcRγ-mediated cell activation signal.

BACKGROUND: We recently reported that the interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell (MC) activation and that FcεRIß functions as an amplifier of FcεRI-mediated activation signal. Some of FcεRIß in cytoplasm appeared not to be co-localized with FcεRIα. The function of FcεRIß in the cytoplasm remains unknown. METHODS: The localization of FcεRIß and FcεRIα in giant papillae specimens from patients with allergic keratoconjunctivitis and of FcεRIß, FcεRIα, and Lyn in cultured human MCs was examined using confocal microscopy. FcεRIß was overexpressed using an adenovirus vector system. Mediators were measured by enzyme immunoassays or enzyme-linked immunosorbent assays. RESULTS: In the subepithelial region, FcεRIß was mainly localized in the cell membrane of MCs. In the perivascular region, FcεRIß expression was scattered throughout the cytoplasm and in the cell membrane of MCs. Overexpression of FcεRIß in MCs mainly increased its cytoplasmic expression and slightly up-regulated cell surface FcεRI expression. However, overexpression of FcεRIß in MCs resulted in down-regulation of the tyrosine phosphorylation levels of FcεRIß and Syk and down-regulation of the Ca(2+) influx soon after FcεRI aggregation and then resulted in down-regulation of degranulation, PGD2 synthesis, and production of a set of cytokines. This negative regulatory effect may be due to inhibition of the redistribution of Lyn to small patches within the plasma membrane. CONCLUSION: Cytoplasmic FcεRIß, which is not co-localized with FcεRIα, may function as a negative regulator, as it can capture important signalling molecules such as Lyn.

Comparative in vitro and in vivo antimalarial activity of the indole alkaloids ellipticine, olivacine, cryptolepine and a synthetic cryptolepine analog.

Indole alkaloids ellipticine (1), cryptolepine triflate (2a), rationally designed 11-(4-piperidinamino)cryptolepine hydrogen dichloride (2b) and olivacine (3) (an isomer of 1) were evaluated in vitro against Plasmodium falciparum and in vivo in Plasmodium berghei-infected mice. 1-3 inhibited P. falciparum (IC50≤1.4 µM, order of activity: 2b>1>2a>3). In vitro toxicity to murine macrophages was evaluated and revealed selectivity indices (SI) of 10-12 for 2a and SI>2.8×10² for 1, 2b and 3. 1 administered orally at 50mg/kg/day was highly active against P. berghei (in vivo inhibition compared to untreated control (IVI)=100%, mean survival time (MST)>40 days, comparable activity to chloroquine control). 1 administered orally and subcutaneously was active at 10 mg/kg/day (IVI=70-77%; MST=27-29 days). 3 exhibited high oral activity at ≥50 mg/kg/day (IVI=90-97%, MST=23-27 days). Cryptolepine (2a) administered orally and subcutaneously exhibited moderate activity at 50mg/kg/day (IVI=43-63%, MST=24-25 days). At 50 mg/kg/day, 2b administered subcutaneously was lethal to infected mice (MST=3 days) and moderately active when administered orally (IVI=45-55%, MST=25 days). 1 and 3 are promising compounds for development of antimalarials.

The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell activation.

BACKGROUND: FcεRIß reportedly functions as an amplifier of the FcεRIγ-mediated activation signal using a reconstitution system. However, the amplification mechanisms in human mast cells (MCs) are poorly understood. We previously reported the hyperexpression of FcεRIß of MCs in giant papillae from vernal keratoconjunctivitis patients, compared with that in conjunctivae from nonallergic conjunctivitis patients. Elucidation of the molecular mechanisms of the amplification induced by FcεRIß should provide new targets for novel therapeutic interventions. The aim is to understand in greater details the function of FcεRIß in human MC FcεRI expression and signaling. METHODS: FcεRIß and Lyn expression was reduced using a lentiviral shRNA silencing technique. Localization of Lyn and FcεRIß in cultured MCs was examined by confocal microscopic analysis. Mediators were measured by ELISAs. RESULTS: The diminution of FcεRIß significantly downregulated cell surface FcεRI expression and FcεRI-mediated mediator release/production. The downregulation of FcεRI-mediated degranulation was not only due to the decrease in FcεRI expression. The diminution of FcεRIß inhibited the redistribution of Lyn within the cell membrane following IgE sensitization. The diminution of Lyn in MCs significantly downregulated FcεRI-mediated degranulation. The recombinant cell-penetrating forms of phosphorylated FcεRIß immunoreceptor tyrosine-based activation motif (ITAM) for intracellular delivery disturbed the interaction between Lyn and phosphorylated endogenous FcεRIß ITAM, resulted in inhibiting IgE-dependent histamine release from MCs in vitro and from giant papillae specimens ex vivo. CONCLUSION: The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human MC activation, and specific inhibition of the interaction may represent a new therapeutic strategy for the treatment of human allergic diseases.

Multiple injections of anti-mouse ß2glycoprotein 1 antibody induce FcRγ-dependent fetal growth restriction (FGR) in mice.

OBJECTIVES: The antiphospholipid syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (aPLs), is a leading cause for thromboembolic events, repeated miscarriage, fetal loss and is a major risk factor for fetal growth restriction (FGR) and preeclampsia. In human, anti-ß2 glycoprotein I (aß2GPI) antibody is one of the aPLs and considered to be a specific and important marker for APS. However, pathophysiological changes induced by aß2GPI antibodies in FGR are largely unknown. METHODS: In the present study, we developed a murine FGR model induced by multiple injections of WBCAL-1, a well-characterized mouse aß2GPI monoclonal antibody. RESULTS: Administration of WBCAL-1, but not the isotype control antibody and saline, into pregnant mice specifically decreased the size of fetuses and placentas without affecting the number of delivered pups. Also, a significant increase in urinary albumin and electron microscopic changes, such as splitting layers of basal membranes in the placental labyrinth and rearrangement of pores in glomerular endothelial cells, were observed in WBCAL-1 treated mice. WBCAL-1 injection did not induce any changes in blood pressure and typical parameters of blood thromboembolic symptoms. Furthermore, FcRγ deficiency protected the fetuses from aß2GPI antibody-induced injuries. CONCLUSIONS: Our present findings suggest that proteinuria is a symptom associated with APS-related FGR with placental and renal tissue injuries, and that FcRγ might be a molecular target for prevention of aß2GPI antibody-mediated obstetrical pathologies.

Potential control of Aedes aegypti (Diptera: Culicidae) with Piper aduncum L. (Piperaceae) extracts demonstrated by chromosomal biomarkers and toxic effects on interphase nuclei.

Dillapiol, a phenylpropanoid isolate from essential oils of leaves of Piper aduncum (Piperaceae), has insecticidal, fungicidal and antimicrobial activities. The insecticidal activity of dillapiol was tested in vivo on the larvae and pupae of Aedes aegypti, the mosquito vector of dengue. Specifically, the effect of dillapiol on the formation of micronuclei and chromosome aberrations was analyzed. Dillapiol treatments comprised two concentrations of 200 and 400 micro dissolved in well water, and a pure well water control used to rear four generations of mosquitoes. Micronuclei occurred in mitotic diploid and tetraploid chromosomes of larvae; nuclear abnormalities also occurred in interphase, metaphase, telophase, and single nucleus cells of pupae. Mortality, oviposition, chromosome breakage, and anaphase bridges were significantly greater in the extract treatments than in controls. The genotoxic effects of dillapiol described here suggest that this natural product may be a useful alternative for the control of A. aegypti.

Self-diffusion in a liquid complex plasma.

Self-diffusion has been experimentally studied in a two-dimensional underdamped liquid complex (dusty) plasma. It was found that the self-diffusion coefficient D increases linearly with the temperature T: D/omega(E)a2 = (0.019 +/- 0.007)(T/T(m) - 1), where T(m), omega(E), and a are the melting temperature, the Einstein frequency, and the mean particle separation, respectively. No superdiffusion was observed, whereas a subdiffusion occurred at temperatures close to melting.

Heat transfer in a two-dimensional crystalline complex (dusty) plasma.

Heating and heat transfer were studied in a two-dimensional crystalline complex plasma at the kinetic level. The lattice was formed of microspheres levitated in a plasma sheath. One half of the crystal was heated anisotropically to obtain higher kinetic temperatures in one direction and heat conduction was observed in real time. It was found that the longitudinal phonons conduct heat better than the transverse. The thermometric conductivity coefficient was measured to be 53 mm2/s for longitudinal heating and 30 mm2/s for transverse heating. Heat decay lengths and energy exchange times between the temperature components were determined.

Common and distinct signalling cascades in the production of tumour necrosis factor-alpha and interleukin-13 induced by lipopolysaccharide in RBL-2H3 cells.

BACKGROUND: Activation of mast cells by lipopolysaccharide (LPS) results in the production of TNF-alpha and IL-13. TNF-alpha and IL-13 are key mediators in the development of neutrophilic and allergic inflammation, respectively. LPS-induced TNF-alpha and IL-13 production in mast cells has been reported to be mediated by Toll-like receptor 4 (TLR4) signalling, but differences in signal transduction mechanisms leading to the production of these cytokines are not clearly defined. OBJECTIVE: We investigated the molecular mechanisms responsible for LPS-induced TNF-alpha and IL-13 production in mast cells. METHODS: TNF-alpha and IL-13 production by LPS was assessed by transfecting RBL-2H3 cells with dominant-negative (DN) expression vectors. RESULTS: Transfection of RBL-2H3 cells with plasmids encoding DN mutants of myeloid differentiation protein (MyD88) and TNFR-associated factor (TRAF6) inhibited both LPS-induced TNF-alpha and IL-13 production. IkappaBalpha-DN inhibited LPS-induced production of TNF-alpha, but not IL-13. We also found that inhibition of p38 kinase suppressed both TNF-alpha and IL-13 induction by LPS, and inhibition of JNK reduced IL-13 production, but not TNF-alpha. Furthermore, we found that protein kinase R (PKR) was activated by LPS in these cells. Treatment with 2-aminopurine, a PKR inhibitor, attenuated LPS-induced nuclear factor-kappaB activation and TNF-alpha production, whereas inhibition of PKR had little effect on IL-13 production. CONCLUSION: These findings indicate that the production of TNF-alpha and IL-13 by LPS required TLR4/MyD88/TRAF6 signalling as a common pathway of mast cell-mediated inflammation. We furthermore found that TNF-alpha and IL-13 production were differentially regulated by signalling cascades through PKR and mitogen-activated protein kinases downstream of TRAF6 in mast cells.

Wave spectra in solid and liquid complex (dusty) plasmas.

Spectra of longitudinal and transverse waves were obtained experimentally in liquid and solid two-dimensional complex (dusty) plasmas at different kinetic temperatures. As the temperature increased and the phase state of the plasma changed from solid to liquid, the phonon spectra of both longitudinal and transverse modes broadened (especially at high wave numbers), indicating increased damping. The transverse mode disappeared and a thermal (compressional) mode appeared.

Polarization of wave modes in a two-dimensional hexagonal lattice using a complex (dusty) plasma.

Wave spectra corresponding to the random particle motion in a monolayer Yukawa crystal were studied for various directions of propagation. It was found that there are two wave modes with a polarization alternating between the longitudinal and transverse. In the long-wavelength regime, the modes became purely longitudinal and transverse as was known before. In the short-wavelength regime the spectra strongly depended on the wavelength and the direction of propagation. The results obtained from the experiment, theory, and simulation agreed well with each other.

Nonlinear longitudinal waves in a two-dimensional screened Coulomb crystal.

Nonlinear interactions of longitudinal waves were observed in a two-dimensional plasma crystal, i.e., a lattice composed of highly charged microspheres immersed in a plasma. The waves were launched by radiation pressure of a laser, and wave spectra in omega-k space were analyzed at various amplitudes of waves. At a sufficiently large amplitude of wave, the second and third wave harmonics satisfying a dispersion relation were observed. As the second harmonic propagates from the excitation region, it was amplified for a small distance, and then damped. The experimental results were compared to a nonlinear wave theory and to a molecular dynamic simulation.

Dispersion relations of longitudinal and transverse waves in two-dimensional screened Coulomb crystals.

Dispersion relations of longitudinal and transverse waves in two-dimensional (2D) screened-Coulomb crystals were investigated. The waves were excited in 2D crystals made from complex plasmas, i.e., dusty plasmas, by applying radiation pressure of laser light. The dependencies of the dispersion relation on the shielding parameter, the damping rate, and the wave propagation direction were experimentally measured. The measured dispersion relations agree reasonably with a recently developed theory, and the comparison yields the shielding parameter and the charge on particles.

Phonon spectrum in a plasma crystal.

The Fourier spectra of longitudinal and transverse waves corresponding to random particle motion were measured in a two-dimensional plasma crystal. The crystal was composed of negatively charged microspheres immersed in a plasma at a low gas pressure. The phonons were found to obey a dispersion relation that assumes a Yukawa interparticle potential. The crystal was in a nonthermal equilibrium, nevertheless phonon energies were almost equally distributed with respect to wave number over the entire first Brillouin zone.

Nonlinear compressional pulses in a 2D crystallized dusty plasma.

Compressional pulses were launched in a two-dimensional Yukawa lattice, a hexagonal monolayer of polymer microspheres suspended in a plasma. The pulsed wave was excited by a laser beam, and nonlinear effects were observed for Mach numbers M>0.07 and for variation of particle number density delta(n)/n>0.1, but no steepening of the pulse was detected. The pulse propagation speed was found to be comparable to the sound speed of compressional waves launched with sinusoidal excitation.

Surface molecules essential for positive selection are retained but interfered in thymic epithelial cells after monolayer culture.

It has been reported that the three-dimensional structure of thymic epithelial cells (TECs) is responsible for thymic positive selection but that this ability disappears when TECs are cultured in monolayer. These results have supported the hypothesis that certain TEC-specific molecules are extinguished during monolayer culture. In this study, using MHC class II-restricted T-cell receptor transgenic mice, we demonstrated that preselected CD4(+)8(+) (DP) thymocytes were inhibited from developing into CD4(+)8(-) (CD4SP) cells in reaggregate thymus organ culture with monolayer-cultured TECs, but this inhibition was removed when TECs were cultured in monolayer with protein synthesis inhibitor or when the cultured TECs were treated with fixative. These results seem to be inconsistent with the previous hypothesis and indicate that monolayer culture allows TECs to retain the surface molecules necessary for positive selection but interferes with their function, which must be sustained for three dimensional structure.

Laser-excited mach cones in a dusty plasma crystal

Experimental studies of the formation and structure of Mach cones in a plasma crystal are presented. Plasma crystals are ordered structures of charged microspheres trapped in the sheath of an rf discharge plasma. Using a monolayer crystal with a hexagonal lattice, Mach cones were excited by the radiation pressure of a focused laser beam. The beam was swept at a supersonic speed through the crystal, in a controlled and repeatable manner. A multiple Mach cone structure was observed, with at least three distinct Mach cones. The Mach angle relation was verified over a wide range of Mach numbers, for both the first and second cones. The sound speed, measured from the first Mach angle, was found to increase with the particle number density. Two methods of determining the particle charge and screening distance are developed, making use of the sound speed and an assumption of a Yukawa interparticle potential. Molecular-dynamics simulations of the experiment were carried out, using a monolayer of particles interacting through a Yukawa potential, and these show close agreement with the experiment.

Transverse waves in a two-dimensional screened-coulomb crystal (Dusty plasma)

Transverse shear waves were observed experimentally in a two-dimensional screened Coulomb crystal. They were excited by applying a chopped laser beam to a 2D dusty plasma, i.e., a monolayer of charged microspheres levitated in a plasma. Measurements of the dispersion relation reveal an acoustic, i.e., nondispersive, character over the entire range of wave numbers measured, 0.2

Molecular basis for functional maturation of thymocytes: increase in c-fos translation with positive selection.

In the process of positive selection, immature CD4+8+ double positive (DP) thymocytes expressing TCR reactive to self-MHC by appropriate avidity develop into mature thymocytes. Positive selection involves not only down-regulation of either CD4 or CD8 but also acquisition of immunocompetent potential such as cell proliferation and cytokine production. To understand the molecular basis for such functional maturation during the positive selection process, we examined whether nonselected DP, selected DP, and CD4+8- single positive thymocytes possess the activation potential for signaling pathways from mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) to AP-1. In response to stimulation, a marked induction of c-Fos protein expression as well as cell proliferation is detected only in CD4+8- single positive cells but not in selected and nonselected DP cells, though mitogen-activated protein kinase activities and c-fos transcripts are equally induced. In the presence of proteasome inhibitors, c-Fos protein became detectable in selected DP cells but still not in nonselected DP cells, suggesting that DP cells receiving positive selection signals acquire the capacity to translate the c-fos gene, but it may not be sufficiently high to overcome the degradation of c-Fos protein. These data indicate that the translating ability of the c-fos gene is up-regulated in the thymic positive selection process, from nonselected DP to CD4+8- single positive cells through positively selected DP cells. The distinguished responsiveness to stimulation in thymocytes with and without positive selection may be a result in part of the distinct regulation of the c-fos gene at the translational level.