Modulation of allergen-specific T-cell responses using novel antigen-presenting platforms and transgenic regulatory T-cells

T-Lymphozyten spielen eine wichtige Rolle in der Regulation von allergischen Immunantworten und tragen daher entscheidend sowohl zur Sofort-Typ Reaktion als auch zu den allergischen Spät-Phasen Reaktionen bei. Daher könnten Strategien, die darauf abzielen, die pathologische T Helfer 2 (Th2)- Antwort zu verändern von therapeutischem Wert für allergische Individuen sein. Dennoch fehlen bis heute Modell-Systeme für Allergen-spezifische T-Zell Antworten, die auf human-relevanten Allergenen und den dazugehörigen HLA Restriktionselementen und T-Zell Rezeptoren (TZR) basieren. Daher klonierten und charakterisierten wir solche relevanten Moleküle, die in Antigen-Präsentation und -Erkennung involviert sind (das sind die präsentierenden HLA-Moleküle und die dazu gehörigen TZR α and β Ketten) aus dem Blut von Beifusspollen und Birkenpollen Allergikern. Indem wir 293 Zellen mit den HLA Molekülen, den Allergenen und kostimulatorischen Molekülen transfezierten, und gleichzeitig durch retrovirale Transduktion von humanen T-Zellen mit dem dazugehörigen TZR allergen-spezifische T-Zellen herstellten, gelang es uns, die ‚Allergen-spezifische Synapse’ auf molekularer Ebene zu rekonstruieren. Dieses definierte in vitro Modellsystem erlaubte uns das Verhalten und die Plastizität von allergen-spezifischen T-Zellen nach Stimulation mit unterschiedlichen Konditionen zu untersuchen. Tatsächlich konnten wir zeigen, dass TZR transgene T-Zellen auf die Präsentation des korrekten Peptids und auf kostimulatorische Signale angewiesen waren, um ihre Effektor-Funktion initiieren zu können. In diesem System testeten wir zunächst, ob Virus-ähnlichen Partikeln (‚virus-like particles’; VLP), die wir in 293 Zellen produzierten und mit definierten Zusammensetzungen an HLA/Allergen Komplexen und accessorischen Molekülen ausstatteten, die Kapazität hatten, allergen-spezifische T-Zellen zu modulieren. In diesen Studien konnten wir beobachten, dass allergen-präsentierende VLP perfekt geeignet waren, die Rolle von antigen-präsentierenden Zellen zu übernehmen, und TZR transgene T-Zellen zu expandieren, zu anergisieren oder zu polarisieren, abhängig von der vordefinierten molekularen Ausstattung. Außerdem konnten VLP, die mit dem hochaffinen Interleukin-2 (IL-2) Rezeptor ausgestattet waren, dieses Zytokin in T-Zell Kulturen neutralisieren, womit sie eine wichtige Effektor-Funktion von regulatorischen T-Zellen (Treg) übernehmen konnten. In einem anderen Ansatz verfolgten wir die Hypothese, ob Transfer von Genen, die mit der Funktion von Treg assoziiert sind, uns erlauben würde, allergen-spezifische Treg zu generieren. Dafür verwendeten wir eine multicistronische Expressions Strategie, indem wir die α und β Kette eines Bet v 1 (Birkenpollen-Allergen)-spezifischen TZR über 2A Elemente mit Foxp3, dem Hauptregulator-Gen der Treg Funktion, verbanden. Diese Expressionskassette wurde gemeinsam mit einem IRES-GFP Element in einen retroviralen Expressionsvektor kloniert und in CD4+CD25- T-Zellen transduziert. Diese Foxp3/TZR transgenen T-Zellen zeigten das charakteristische phänotypische Profil von Treg und stark verminderte Proliferation nach allergen-spezifischer und polyklonaler Stimulation, und konnten Bet v 1-spezifische T-Zellen Aktivierungs-abhängig regulieren. Die Etablierung von solchen neuen T-Zell modulierenden Strategien basierend auf artifiziellen Antigen-präsentierenden Plattformen und regulatorischen T-Zellen könnte zur Entwicklung von neuen Therapien für die Behandlung von Allergien, aber auch von Autoimmunerkrankungen und Episoden von Transplantatabstossung, führen.T-lymphocytes play a pivotal role in the orchestration of allergic immune responses and thus critically contribute to immediate phase reactions as well as late phase allergic reactions. Consequently, strategies to modulate the pathological T helper 2 (Th2)-biased response might be of therapeutic benefit for allergic individuals. However, until today model systems for allergic T-cell responses based on human-relevant allergens and the corresponding human restriction elements and T-cell receptors (TCR) are still missing. For that reason we cloned and functionally characterized such relevant molecules involved in antigen-presentation and recognition (i.e. the HLA restriction elements and the corresponding TCR alpha and beta chains) from mugwort pollen and birch pollen allergic individuals. By generation of artificial antigen-presenting ells (APC) based on transfection of 293 cells with the HLA molecules, allergenic peptides and costimulatory molecules in concert with retroviral transduction of human T-cells with corresponding TCR we succeeded in rebuilding the ‘allergen-specific synapse’. This defined in vitro model system allowed us to study signal requirements and plasticity of allergen-specific T-cells. In fact, we could show that TCR transgenic T-cells were strictly dependent on presentation of cognate peptide and costimulatory signals to mount their effector functions. Using this system we tested the capacity of 293 cell-derived virus-like particles (VLP) with distinct compositions of HLA/allergen complexes and accessory molecules to modulate allergen specific T-cell responses. In this context we could demonstrate that allergen-presenting VLP were perfectly suited to assume the role of antigen-presenting cells and to expand, anergize or polarize TCR transgenic T-cells depending on their pre-defined set-up. Similarly, decoration of VLP with the high affinity interleukin-2 receptor allowed for the depletion of IL-2 from T-cell cultures thus mimicking an important effector mechanism of regulatory T-cells (Treg). In a different approach we hypothesized that ectopic expression of transgenes associated with Treg function would enable us to develop also allergen-specific Treg. To that end we applied a multicistronic expression strategy, putting the α and β chains of a Bet v 1 (birch pollen allergen)-specific TCR plus Foxp3, the master regulator of Treg function, along with an IRES-GFP cassette under the control of a retroviral to transduce CD4+CD25- T-cells. These TCR/Foxp3 transgenic T-cells displayed the typical phenotypic profile of Treg, showed hyporesponsiveness in response to polyclonal and allergen-specific stimulation and were able to suppress the effector function of Bet v 1-specific T-cells in an activation-dependent manner. The establishment of such novel T-cell modulatory strategies based on artificial antigen-presenting platforms and regulatory T-cells could possibly lead to novel therapeutic applications in allergic diseases and other immune-mediated disorders

Azithromycin inhibits IL-1 secretion and non-canonical inflammasome activation

Deregulation of inflammasome activation was recently identified to be involved in the pathogenesis of various inflammatory diseases. Although macrolide antibiotics display well described immunomodulatory properties, presumably involved in their clinical effects, their impact on inflammasome activation has not been investigated. We compared the influence of macrolides on cytokine induction in human monocytes. The role of intracellular azithromycin- accumulation was examined by interference with Ca++-dependent uptake. We have also analysed the signalling cascades involved in inflammasome activation, and substantiated the findings in a murine sepsis model. Azithromycin, but not clarithromycin or roxithromycin, specifically inhibited IL-1α and IL-1β secretion upon LPS stimulation. Interference with Ca++-dependent uptake abolished the cytokine-modulatory effect, suggesting a role of intracellular azithromycin accumulation in the modulatory role of this macrolide. Azithromycin’s inhibiting effects were observed upon LPS, but not upon flagellin, stimulation. Consistent with this observation, we found impaired induction of the LPS-sensing caspase-4 whereas NF-κB signalling was unaffected. Furthermore, azithromycin specifically affected IL-1β levels in a murine endotoxin sepsis model. We provide the first evidence of a differential impact of macrolides on the inflammasome/IL-1β axis, which may be of relevance in inflammasome-driven diseases such as chronic obstructive pulmonary disease or asthma

Testing lupus anticoagulants in a real-life scenario - a retrospective cohort study

Introduction: Lupus anticoagulant (LAC) testing is challenging. Most data are derived from a well-controlled study environment with potential alterations to daily routines. The aim of this retrospective cohort study was to assess the capacity of various LAC screening tests and derived mixing tests to predict a positive result in subsequent confirmation tests in a large cohort of patients. Materials and methods: In 5832 individuals, we retrospectively evaluated the accuracy of the aPTT-A, aPTT-LAscreen, aPTT-FS and dRVVTscreen and of their derived mixing tests in detecting a positive confirmation test result within the same blood specimen. The group differences, degree of correlation and the predictive accuracy of LAC coagulation tests were analysed using the Mann-Whitney U test, the Spearman-rank-correlation and by area under the receiver operating characteristic curve (ROC-AUC) analysis. ROC-AUCs were compared with the Venkatraman´s permutation test. Results: The pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LAscreen showed the best diagnostic accuracy with a ROC-AUC of 0.84 (95% CI: 0.82 – 0.86). No clear advantage of the dRVVT-derived mixing test was detectable when compared to the dRVVTscreen (P = 0.829). Usage of the index of circulating anticoagulant (ICA) did not improve the diagnostic power of respective mixing tests. Conclusions: Among the parameters evaluated, aPTT-LAscreen and derived mixing test parameters were the most accurate tests. In our study cohort, neither other mixing test nor the ICA presented any further advantage in LAC diagnostics

Effect of Topically Administered Chitosan- N

Purpose. The present study was performed to investigate the effect of topically administered chitosan-N-acetylcysteine (C-NAC) on corneal wound healing in a rabbit model. Methods. A total of 20 New Zealand White rabbits were included in the randomized, masked, placebo-controlled experiment. A monocular epithelial debridement was induced by manual scraping under general anesthesia. Animals were randomized to receive either C-NAC two times daily or placebo. Monitoring of corneal wound healing was performed with ultra-high-resolution optical coherence tomography (OCT) and epithelial fluorescein staining. Measurements were done immediately after and up to 72 hours after wound induction. Results. No difference in wound size was found immediately after surgical debridement between the C-NAC group and the placebo group. Wound healing was significantly faster in the C-NAC group compared to the placebo group (p<0.01 for both methods). A good correlation was found between the OCT technique and the epithelial fluorescein staining in terms of wound size (r=0.94). Conclusions. Administration of C-NAC containing eye drops twice daily leads to a faster corneal wound healing in a rabbit model of corneal debridement as compared to placebo. Ultra-high-resolution OCT is considered a noninvasive, dye-free alternative to conventional fluorescein staining in assessing corneal wound healing also in humans

A new investigation of the system NieSn

International audienceThe system NieSn, being one of the key systems for lead-free soldering, has been the subject of intensive investigations in the 1930s and 1940s. The most recent phase diagram assessment as well as various calculations of this system almost exclusively rely on these data. As the assessment seemed to be based on a rather arbitrary selection of experimental data, a new experimental investigation of this system was considered necessary. In this work a new version of the NieSn system was established following a comprehensive survey of existing literature and the investigation of 27 samples. XRD (including high temperature XRD), DTA, EPMA and metallographic examination were performed on these samples annealed at various temperatures. Especially in the region of Ni3Sn considerable differences to existing phase diagram information were recognized. The crystal structure of the Ni3Sn HT-phase was found to be cubic (BiF3-type) by HT-XRD, and the phase transition was determined to be at higher temperature than reported previously. Furthermore a suggestion for the phase transformation in Ni3Sn2 was included. This region of the system was found to be far more complex than hitherto reported, because of the existence of two further incommensurate Ni3Sn2 lowtemperature modifications first discovered by Leineweber et al

PD-1 has a unique capacity to inhibit allergen-specific human CD4+ T cell responses

T lymphocytes have a crucial role in initiating and promoting type I allergies. Their responses are tightly regulated by numerous activating and inhibitory signals provided by APCs. Here we have addressed the role of the major coinhibitory receptors PD-1, CTLA-4, BTLA and LAG-3 in allergen-specific CD4+ T cell responses. PBMCs of healthy individuals and 41 patients allergic to house dust mites, birch, grass or mugwort pollen were stimulated with allergenic extracts and expression of coinhibitory receptors on responding CD4+ T cells was assessed. Blocking antibodies to PD-1, CTLA-4, BTLA and LAG-3 were used to evaluate the role of coinhibitory pathways. Allergen-specific CD4+ T cells showed strong upregulation of PD-1, LAG-3 and CTLA-4 upon stimulation, whereas BTLA was downregulated. Blockade of PD-1 strongly enhanced proliferation and cytokine production (IL-10; TH1 cytokines IFN-, TNF-; TH2 cytokines IL-5, IL-13) of allergen-specific CD4+ T cells derived from allergic as well as non-allergic individuals. BTLA blockade enhanced proliferation but not cytokine production in response to house dust mite extract. Blocking LAG-3 was ineffective and surprisingly, we observed reduced proliferation and cytokine production in presence of a CTLA-4 antibody. Our results point to a unique potency of PD-1 pathways to dampen allergen-specific human T cells.(VLID)472519

Scientific Reports / Azithromycin inhibits IL-1 secretion and non-canonical inflammasome activation

Deregulation of inflammasome activation was recently identified to be involved in the pathogenesis of various inflammatory diseases. Although macrolide antibiotics display well described immunomodulatory properties, presumably involved in their clinical effects, their impact on inflammasome activation has not been investigated. We compared the influence of macrolides on cytokine induction in human monocytes. The role of intracellular azithromycin-accumulation was examined by interference with Ca++-dependent uptake. We have also analysed the signalling cascades involved in inflammasome activation and substantiated the findings in a murine sepsis model. Azithromycin, but not clarithromycin or roxithromycin, specifically inhibited IL-1 and IL-1 secretion upon LPS stimulation. Interference with Ca++-dependent uptake abolished the cytokine-modulatory effect, suggesting a role of intracellular azithromycin accumulation in the modulatory role of this macrolide. Azithromycins inhibiting effects were observed upon LPS, but not upon flagellin, stimulation. Consistent with this observation, we found impaired induction of the LPS-sensing caspase-4 whereas NF-B signalling was unaffected. Furthermore, azithromycin specifically affected IL-1 levels in a murine endotoxin sepsis model. We provide the first evidence of a differential impact of macrolides on the inflammasome/IL-1 axis, which may be of relevance in inflammasome-driven diseases such as chronic obstructive pulmonary disease or asthma.(VLID)491092

Evaluation of the Septifast MGrade Test on Standard Care Wards--A Cohort Study.

BACKGROUND:The immediate need for appropriate antimicrobial therapy in septic patients requires the detection of the causative pathogen in a timely and reliable manner. In this study, the real-time PCR Septifast MGrade test was evaluated in adult patients meeting the systemic inflammatory response syndrome (SIRS) criteria that were treated at standard care wards. METHODS:Patients with clinical suspected infection, drawn blood cultures (BC), the Septifast M(Grade) test (SF) and sepsis biomarkers were prospectively screened for fulfillment of SIRS criteria and evaluated using the criteria of the European Centre of Disease Control (ECDC) for infection point prevalence studies. RESULTS:In total, 220 patients with SIRS were prospectively enrolled, including 56 patients with detection of bacteria in the blood (incidence: 25.5%). BC analysis resulted in 75.0% sensitivity (95% confidence interval, CI: 61.6%- 85.6%) with 97.6% specificity (CI: 93.9%- 99.3%) for detecting bacteria in the blood. In comparison to BC, SF presented with 80.4% sensitivity (CI: 67.6%- 89.8%) and with 97.6% specificity (CI: 93.9%- 99.3%). BC and SF analysis yielded comparable ROC-AUCs (0.86, 0.89), which did not differ significantly (p = 0.558). A trend of a shorter time-to-positivity of BC analysis was not seen in bacteremic patients with a positive SF test than those with a negative test result. Sepsis biomarkers, including PCT, IL-6 or CRP, did not help to explain discordant test results for BC and SF. CONCLUSION:Since negative results do not exclude bacteremia, the Septifast M(Grade) test is not suited to replacing BC, but it is a valuable tool with which to complement BC for faster detection of pathogens