Charakterisierung von UNC93B-Toll-like Rezeptor Komplexen und deren Rolle für die angeborene Immunantwort

Toll-like receptors (TLRs) are key players of the innate immune system. Localized at the cell surface or intracellularly, they trigger immune responses upon recognition of pathogenic patterns. After their synthesis in the endoplasmic reticulum (ER), intracellular TLRs 3, 7, and 9 need to move to the endolysosome to meet their ligands. On their way they are escorted by the polytopic membrane protein UNC93B. UNC93B interacts with TLRs 3, 7, and 9 and delivers them from the ER to the endolysosome where they initiate the expression of proinflammatory cytokines and type I interferon (IFN) upon encounter of nucleic acids. In 3d mice, which express a missense mutant of UNC93B (H412R), binding of UNC93B to TLRs is disrupted and UNC93B H412R as well as intracellular TLRs fail to leave the ER. How function and trafficking of UNC93B-TLR complexes is regulated is not entirely understood. A comparison of innate immune cells derived from UNC93B knockout and 3d mice showed that the phenotype of cells from UNC93B knockout mice does not differ from the 3d phenotype. In a proteomics approach, cleft lip and palate transmembrane protein (Clptm), three prime repair exonuclease 1 (Trex1), and the protein tyrosine phosphatase 1B (PTP1B) have been identified as novel binding partners of UNC93B in macrophages. Co-immunoprecipitation experiments verified interactions between UNC93B, the previously uncharacterized protein Clptm, as well as the negative regulator of cytosolic DNA responses Trex1. PTP1B is known to be crucial for intracellular trafficking of receptor tyrosine kinases, but had not been linked to regulation of TLR signaling. Interaction of PTP1B with UNC93B, Clptm, TLR7, and TLR9 was verified by co-immunoprecipitation. The TLR-independent type I IFN response upon infection of bone marrow-derived macrophages with mouse cytomegalovirus, as well as the TLR7- and TLR9-dependent IFNalpha responses in plasmacytoid dendritic cells (pDC) were impaired in cells derived from PTP1B knockout mice compared to wild type cells. Furthermore, PTP1B knockout mice showed significantly reduced IFNalpha and interleukin (IL)-12p40 responses compared to wildtype mice upon stimulation of TLR9 by intravenous administration of CpG oligonucleotides in vivo. This suggests that PTP1B is a critical regulator of the TLR7- and TLR9-dependent IFNalpha response in pDC as well as the TLR-independent type I IFN response in macrophages.Toll-like Rezeptoren (TLRs) sind ein wichtiger Bestandteil des angeborenen Immunsystems. Sie werden an der Zelloberfläche oder intrazellulär exprimiert und lösen bei der Erkennung pathogener Strukturen Immunantworten aus. Nach ihrer Synthese im endoplasmatischen Retikulum müssen die intrazellulären TLRs 3, 7 und 9 ins Endolysosom gelangen, um auf ihre Liganden zu treffen. Das Membranprotein UNC93B interagiert mit den TLRs 3, 7 und 9 und eskortiert sie vom ER zum Endolysosom, wo sie die Expression von proinflammatorischen Zytokinen und Typ I Interferon (IFN) initiieren, wenn sie auf Nukleinsäuren treffen. In 3d Mäusen, die UNC93B mit einer missense-Mutation (H412R) exprimieren, kann UNC93B nicht an die TLRs binden, weshalb UNC93B H412R und intrazelluläre TLRs das ER nicht verlassen können. Wie die Funktion und der Transport von UNC93B-TLR-Komplexen reguliert werden, ist nicht vollständig geklärt. Ein Vergleich von Immunzellen, die aus UNC93B knockout oder 3d Mäusen generiert wurden, zeigte, dass der Phänotyp der UNC93B knockout Zellen sich nicht vom 3d Phänotyp unterscheidet. Cleft lip and palate transmembrane protein (Clptm), three prime repair exonuclease 1 (Trex1) und protein tyrosine phosphatase 1B (PTP1B) wurden mittels eines Proteomik-Ansatzes als neue Bindungspartner von UNC93B identifiziert. Die Interaktion zwischen UNC93B und dem uncharakterisierten Protein Clptm sowie Trex1, einem negativen Regulator der Immunantwort auf zytosolische DNA, wurde mittels Ko-Immunpräzipitation bestätigt. PTP1B ist wichtig für den intrazellulären Transport von Rezeptor-Tyrosinkinasen, wurde aber bisher nicht mit der Regulation von TLRs in Verbindung gebracht. Interaktionen zwischen PTP1B und UNC93B, Clptm, TLR7 und TLR9 wurden mittels Ko-Immunpräzipitation bestätigt. Die TLR-unabhängige Typ I IFN-Antwort nach der Infektion von Makrophagen, die aus PTP1B knockout Mäusen generiert wurden, mit Maus-Cytomegalievirus sowie die TLR7- und TLR9-abhängige IFNalpha-Antwort von plasmazytoiden dendritischen Zellen von PTP1B knockout Mäusen waren im Vergleich zu Wildtyp-Zellen vermindert. Außerdem waren die IFNalpha-Antwort und die Interleukin (IL)-12p40-Antwort nach intravenöser Verabreichung von CpG Oligonukleotiden in vivo in PTP1B knockout Mäusen im Vergleich zum Wildtyp reduziert. Diese Ergebnisse deuten darauf hin, dass PTP1B ein entscheidender Regulator der TLR7- und TLR9-abhängigen IFNalpha-Antwort in pDC sowie der TLR-unabhängigen Typ I IFN-Antwort in Makrophagen ist

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Fully Immunocompetent CD8+ T Lymphocytes Are Present in Autologous Haematopoietic Stem Cell Transplantation Recipients Despite an Ineffectual T-Helper Response

BACKGROUND: Reduced CD4 T lymphocytes counts can be observed in HIV infection and in patients undergoing autologous haematopoietic stem cell transplantation (ASCT). Nevertheless, whereas opportunistic infections (OI) are frequent in HIV-infected individuals with low cell counts, OI are uncommon in ASCT patients. METHODOLOGY/PRINCIPAL FINDINGS: To verify whether this observation could be secondary to intrinsic HIV-correlated T cell defects, we performed in-depth immunologic analyses in 10 patients with comparable CD4 counts in whom lymphopenia was secondary either to HIV-infection or ASCT-associated immunosuppressive therapy and compared them to age-matched healthy subjects. Results showed the presence of profound alterations in CD4+ T lymphocytes in both groups of patients with respect to healthy controls. Thus, a low percentage of CCR7+ CD4+ T cells and a compensative expansion of CD45RA-CCR7- CD4+ T cells, a reduced IL-2/IFN-gamma cytokine production and impaired recall antigens-specific proliferative responses were detected both in ASCT and HIV patients. In stark contrast, profound differences were detected in CD8+ T-cells between the two groups of patients. Thus, mature CD8+ T cell prevailed in ASCT patients in whom significantly lower CD45RA-CCR7- cells, higher CD45RA+CCR7- CD8+ cells, and an expansion of CCR7+CD8+ cells was detected; this resulted in higher IFN-gamma +/TNFalpha production and granzyme CD8+ expression. The presence of strong CD8 T cells mediated immune responses justifies the more favorable clinical outcome of ASCT compared to HIV patients. CONCLUSION/SIGNIFICANCE: These results indicate that CD8 T cells maturation and functions can be observed even in the face of a profound impairment of CD4+ T lymphocytes in ASCT but not in HIV patients. Primary HIV-associated CD8 defects or an imprinting by an intact CD4 T cell system in ASCT could justify these results

VERY HIGH ENERGY γ-RAYS from the UNIVERSE'S MIDDLE AGE: DETECTION of the z = 0.940 BLAZAR PKS 1441+25 with MAGIC

The flat-spectrum radio quasar PKS 1441+25 at a redshift of z = 0.940 is detected between 40 and 250 GeV with a significance of 25.5σ using the MAGIC telescopes. Together with the gravitationally lensed blazar QSO B0218+357 (z = 0.944), PKS 1441+25 is the most distant very high energy (VHE) blazar detected to date. The observations were triggered by an outburst in 2015 April seen at GeV energies with the Large Area Telescope on board Fermi. Multi-wavelength observations suggest a subdivision of the high state into two distinct flux states. In the band covered by MAGIC, the variability timescale is estimated to be 6.4 ±1.9 days. Modeling the broadband spectral energy distribution with an external Compton model, the location of the emitting region is understood as originating in the jet outside the broad-line region (BLR) during the period of high activity, while being partially within the BLR during the period of low (typical) activity. The observed VHE spectrum during the highest activity is used to probe the extragalactic background light at an unprecedented distance scale for ground-based gamma-ray astronomy

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Insights into the high-energy γ-ray emission of Markarian 501 from extensive multifrequency observations in the Fermi era

We report on the γ-ray activity of the blazar Mrk 501 during the first 480 days of Fermi operation. We find that the average Large Area Telescope (LAT) γ-ray spectrum of Mrk 501 can be well described by a single power-law function with a photon index of 1.78 ± 0.03. While we observe relatively mild flux variations with the Fermi-LAT (within less than a factor of two), we detect remarkable spectral variability where the hardest observed spectral index within the LAT energy range is 1.52 ± 0.14, and the softest one is 2.51 ± 0.20. These unexpected spectral changes do not correlate with the measured flux variations above 0.3 GeV. In this paper, we also present the first results from the 4.5 month long multifrequency campaign (2009 March 15-August 1) on Mrk 501, which included the Very Long Baseline Array (VLBA), Swift, RXTE, MAGIC, and VERITAS, the F-GAMMA, GASP-WEBT, and other collaborations and instruments which provided excellent temporal and energy coverage of the source throughout the entire campaign. The extensive radio to TeV data set from this campaign provides us with the most detailed spectral energy distribution yet collected for this source during its relatively low activity. The average spectral energy distribution of Mrk 501 is well described by the standard one-zone synchrotron self-Compton (SSC) model. In the framework of this model, we find that the dominant emission region is characterized by a size ≲0.1 pc (comparable within a factor of few to the size of the partially resolved VLBA core at 15-43 GHz), and that the total jet power (≃1044 erg s-1) constitutes only a small fraction (∼10-3) of the Eddington luminosity. The energy distribution of the freshly accelerated radiating electrons required to fit the time-averaged data has a broken power-law form in the energy range 0.3 GeV-10 TeV, with spectral indices 2.2 and 2.7 below and above the break energy of 20 GeV. We argue that such a form is consistent with a scenario in which the bulk of the energy dissipation within the dominant emission zone of Mrk 501 is due to relativistic, proton-mediated shocks. We find that the ultrarelativistic electrons and mildly relativistic protons within the blazar zone, if comparable in number, are in approximate energy equipartition, with their energy dominating the jet magnetic field energy by about two orders of magnitude. © 2011. The American Astronomical Society

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Endosomal Toll-Like Receptors 7 and 9 Cooperate in Detection of Murine Gammaherpesvirus 68 Infection.

Murine gammaherpesvirus 68 (MHV68) is a small-animal model suitable for study of the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal Toll-like receptor (TLR) escort protein UNC93B, endosomal TLR7, -9, and -13, and cell surface TLR2 in MHV68 detection. We found that the alpha interferon (IFN-α) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to levels in wild type (WT) cells but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7-/-Tlr9-/- double-knockout pDC, the IFN-α response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFN-α response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7-/-Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7-/-Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7-/-Tlr9-/- splenocytes compared to levels in the WT. Our data show that UNC93B-dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.IMPORTANCE The two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), can cause aggressive forms of cancer. These herpesviruses are strictly host specific, and therefore the homolog murine gammaherpesvirus 68 (MHV68) is a widely used model to obtain in vivo insights into the interaction between these two gammaherpesviruses and their host. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells. The innate immune system serves as one of the first lines of host defense, with pattern recognition receptors such as the Toll-like receptors playing a crucial role in mounting a potent antiviral immune response to various pathogens. Here, we shed light on a yet unanticipated role of Toll-like receptor 7 in the recognition of MHV68 in a subset of immune cells called plasmacytoid dendritic cells, as well as on the control of this virus in its host