Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

Background: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. Methods: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. Results: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD81 suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)–150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH2/2 or miRNA-1502/2 mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains

Retinoic acid-loading of the major birch pollen allergen Bet v 1 may improve specific allergen immunotherapy: In silico, in vitro and in vivo data in BALB/c mice

5 Pág.This work was supported by the SFB F4606-B28 grant of the Austrian Science Fund FWF.Peer reviewe

Mechanisms Underlying Stage-1 TRPL Channel Translocation in Drosophila Photoreceptors

Background: TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere), TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels. Methodology/Principal Findings: We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content. Conclusions/Significance: Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protei

Targeting prolyl endopeptidase with valproic acid as a potential modulator of neutrophilic inflammation

A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP), has been recently characterized in chronic obstructive pulmonary disease (COPD). This peptide is derived via the proteolytic activity of matrix metalloproteases (MMP's)-8/9 and PE, enzymes produced by neutrophils and present in COPD serum and sputum. Valproic acid (VPA) is an inhibitor of PE and could possibly have an effect on the severity of chronic inflammation. Here the interaction site of VPA to PE and the resulting effect on the secondary structure of PE is investigated. Also, the potential inhibition of PGP-generation by VPA was examined in vitro and in vivo to improve our understanding of the biological role of VPA. UV-visible, fluorescence spectroscopy, CD and NMR were used to determine kinetic information and structural interactions between VPA and PE. In vitro, PGP generation was significantly inhibited by VPA. In vivo, VPA significantly reduced cigarette-smoke induced neutrophil influx. Investigating the molecular interaction between VPA and PE showed that VPA modified the secondary structure of PE, making substrate binding at the catalytic side of PE impossible. Revealing the molecular interaction VPA to PE may lead to a better understanding of the involvement of PE and PGP in inflammatory conditions. In addition, the model of VPA interaction with PE suggests that PE inhibitors have a great potential to serve as therapeutics in inflammatory disorders

NMR spectra of the carbobenzoxy-group of ZPP.

<p>(<b>A</b>) 10 µM ZPP was added to 10 µM rhPE, 10 mM VPA was added to that mixture. A shift of the free ZPP peaks at 7.28 and 7.20 ppm to the left is seen (indicated in the red and green bars respectively). (<b>B</b>) 10 µM ZPP was added to 10 µM rhPE. A shift of the free ZPP peaks at 7.28 and 7.20 ppm to the left is seen. (<b>C</b>) 10 µM ZPP was measured without enzyme. The peaks at 7.28 and 7.20 ppm are the free ZPP fractions. (<b>D</b>) 10 mM VPA was added to 10 µM rhPE, 10 µM ZPP was added to that mixture. Note that the free ZPP peaks at 7.28 and 7.20 ppm do not show a shift to the left; there are no peaks in the highlighted areas.</p

Inhibition of PGP generation by VPA.

<p>(<b>A</b>) Dialyzed Collagen Type I and II were incubated with the lysate of 4.6 * 10⁶ PMN at 37°C for 20 hours to generate PGP. (<b>B</b>) PE activity was measured 30 minutes after incubation of lysate/collagen/VPA mixture and compared to control (no VPA). Data are shown as the mean ± S.E.M. (n = 5–10 per group). Representative of 4 experiments. # p<0.0001 compared to control, $ p<0.01 compared to control, ** p<0.01, ns p>0.05.</p

Effect of VPA on the secondary structure of rhPE.

<p>(<b>A</b>) Three VPA concentrations (6, 12 and 24 mM) were titrated into 2.5 µM rhPE. VPA caused a significant change in the secondary structure of the enzyme. (<b>B</b>) A high concentration of ZPP (1 µM) was added to the 2.5 µM rhPE/24 mM VPA mixture. ZPP did not cause any change in the secondary structure of the enzyme. (<b>C</b>) Adding 1 µM ZPP to 2.5 µM rhPE does not cause any secondary protein structure change. Adding 24 mM VPA on top of that mixture doesn't cause any structure change.</p

VPA decreases cigarette smoke-induced neutrophil influx in BAL fluid of mice.

<p>(<b>A</b>) Total cell numbers, (<b>B</b>) macrophages and (<b>C</b>) neutrophils in the BAL fluid of mice exposed to air or whole body cigarette smoke twice daily during 5 days. The mice received vehicle (PBS) or VPA (100 µg/70 µl PBS) by oropharyngeal aspiration 15 minutes prior to air/smoke exposure. (<b>D</b>) acPGP was measure in the BAL fluid. (<b>E</b>) PE activity was measured in the BAL fluid and compared to control (PBS treated/air exposed mice). N = 5–10 animals per group. Values are expressed as mean+/−S.E.M. **P≤0.01, ***P≤0.001, ns p>0.05.</p