Breathing allows a multitude of airborne microbes and microbial compounds to access the lung. Constant exposure of the pulmonary microenvironment to immunogenic particles illustrates the need for ...proper control mechanisms ensuring the differentiation between threatening and harmless encounters. Discrimination between live and dead bacteria has been suggested to be such a mechanism. In this study, we performed infection studies of murine precision cut lung slices (PCLS) with live or heat-killed
, in order to investigate the role of viability for induction of an innate immune response. We demonstrate that PCLS induce a robust transcriptomic rewiring upon infection with live but not heat-killed
. Using mutants of the
clinical isolate CHA, we show that the viability status of
is assessed in PCLS by TLR5-independent sensing of flagellin and recognition of the type three secretion system. We further demonstrate that enhanced cytokine expression towards live
is mediated by uptake of viable but not heat-killed bacteria. Finally, by using a combined approach of receptor blockage and genetically modified PCLS we report a redundant involvement of MARCO and CD200R1 in the uptake of live
in PCLS. Altogether, our results show that PCLS adapt the extent of cytokine expression to the viability status of
by specifically internalizing live bacteria.
High expression levels of the calcium-binding proteins S100A8 and S100A9 in myeloid cells in kidney transplant rejections are associated with a favorable outcome. Here we investigated the myeloid ...cell subset expressing these molecules, and their function in inflammatory reactions. Different monocyte subsets were sorted from buffy coats of healthy donors and investigated for S100A8 and S100A9 expression. To characterize S100A9high and S100A9low subsets within the CD14+ classical monocyte subset, intracellular S100A9 staining was combined with flow cytometry (FACS) and qPCR profiling. Furthermore, S100A8 and S100A9 were overexpressed by transfection in primary monocyte-derived macrophages and the THP-1 macrophage cell line to investigate the functional relevance. Expression of S100A8 and S100A9 was primarily found in classical monocytes and to a much lower extent in intermediate and non-classical monocytes. All S100A9+ cells expressed human leukocyte antigen-antigen D related (HLA-DR) on their surface. A small population (<3%) of CD14+ CD11b+ CD33+ HLA-DR− cells, characterized as myeloid derived suppressor cells (MDSCs), also expressed S100A9 to high extent. Overexpression of S100A8 and S00A9 in macrophages led to enhanced extracellular reactive oxygen species (ROS) production, as well as elevated mRNA expression of anti-inflammatory
. The results suggest that the calcium-binding proteins S100A8 and S100A9 in myeloid cells have an immune regulatory effect.
is a major human pathogen causing a variety of diseases ranging from common pharyngitis to life-threatening soft tissue infections and sepsis. Microbial nucleic acids, especially bacterial RNA, have ...recently been recognized as a major group of pathogen-associated molecular patterns (PAMPs) involved in the detection of
via endosomal Toll-like receptors (TLRs)
. However, the individual contribution and cooperation between TLRs as well as cell-type and strain specific differences in dependency on nucleic acid detection during
infection
have not been clarified in detail. Moreover, the role of particularly bacterial RNA for the defense of
infection
remains poorly defined. In this study, we report that in all investigated innate immune cells involved in the resolution of bacterial infections, including murine macrophages, dendritic cells and neutrophils, recognition of
strain ATCC12344 is almost completely dependent on nucleic acid sensing via endosomal TLRs at lower MOIs, whereas at higher MOIs, detection via TLR2 plays an additional, yet redundant role. We further demonstrate that different
strains display a considerable inter-strain variability with respect to their nucleic acid dependent recognition. Moreover, TLR13-dependent recognition of
RNA is largely non-redundant in bone marrow-derived macrophages (BMDMs), but less relevant in neutrophils and bone marrow-derived myeloid dendritic cells (BMDCs) for the induction of an innate immune response
.
, we show that a loss of nucleic acid sensing blunts early recognition of
, leading to a reduced local containment of the bacterial infection with subsequent pronounced systemic inflammation at later time points. Thus, our results argue for a crucial role of nucleic acid sensing via endosomal TLRs in defense of
infection both
and
.
Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a ...hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with ‘internal tandem duplications (FLT3ITD)’ drive production of reactive oxygen in leukemic cells.
FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions.
The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein.
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•Fms-like tyrosine kinase 3 (FLT3) can be activated in intact cells by exposure to hydrogen peroxide.•Signaling of the oncoprotein FLT3ITD (FLT3 with internal tandem duplication) can be attenuated with antioxidants.•FLT3ITD is oxidized at cytoplasmic cysteine residues 695 and 790.•The FLT3ITD cytoplasmic cysteine residues 681, 694, 695, 807, 925, and 945 are essential for cell transformation.•Replacement of cysteine 790 with serine causes FLT3 activation.•The findings indicate the potential of FLT3/FLT3ITD redox regulation through cytoplasmic cysteine residues.
Sachsen ist ein an Fließgewässern reiches Land. Wasserbau- und Gewässerunterhaltung sind in unseren Flüssen regelmäßig erforderlich. Gründe dafür können z. B. Hochwasserschutz, oder der Rückbau von ...Wanderhindernissen sein.
Der Leitfaden informiert über die fischereirechtlichen Bestimmungen, die bei Bau- und Unterhaltungsmaßnahmen in und an Gewässern zu beachten sind. An Hand praktischer Beispiele wird erklärt, wie Bauherren und Gewässerunterhaltungspflichtige negative Effekte auf die Fischartengemeinschaft bei Eingriffen in die Gewässer so weit als möglich minimieren können.
Redaktionsschluss: 30.09.2021
Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not ...available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.
We demonstrate the use of DNA origami to create virus-trapping nanoshells that efficiently neutralize hepatitis B virus (HBV) in cell culture. By modification of the shells with a synthetic ...monoclonal antibody that binds to the HBV envelope, the effective neutralization potency per antibody is increased by approximately 100 times compared to using free antibodies. The improvements in neutralizing the virus are attributed to two factors: first, the shells act as a physical barrier that blocks the virus from interacting with host cells; second, the multivalent binding of the antibodies inside the shells lead to stronger attachment to the trapped virus, a phenomenon known as avidity. Pre-incubation of shells with HBV and simultaneous addition of both components separately to cells lead to comparable levels of neutralization, indicating rapid trapping of the virions by the shells. Our study highlights the potential of the DNA shell system to rationally create antivirals using components that, when used individually, show little to no antiviral effectiveness.
FAM3C/ILEI is an important factor in epithelial-to-mesenchymal transition (EMT) induction, tumor progression and metastasis. Overexpressed in many cancers, elevated ILEI levels and secretion ...correlate with poor patient survival. Although ILEI's causative role in invasive tumor growth and metastasis has been demonstrated in several cellular tumor models, there are no available transgenic mice to study these effects in the context of a complex organism. Here, we describe the generation and initial characterization of a Tet-ON inducible Fam3c/ILEI transgenic mouse strain. We find that ubiquitous induction of ILEI overexpression (R26-ILEI.sup.ind) at weaning age leads to a shortened lifespan, reduced body weight and microcytic hypochromic anemia. The anemia was reversible at a young age within a week upon withdrawal of ILEI induction. Vav1-driven overexpression of the ILEI.sup.ind transgene in all hematopoietic cells (Vav-ILEI.sup.ind) did not render mice anemic or lower overall fitness, demonstrating that no intrinsic mechanisms of erythroid development were dysregulated by ILEI and that hematopoietic ILEI hyperfunction did not contribute to death. Reduced serum iron levels of R26-ILEI.sup.ind mice were indicative for a malfunction in iron uptake or homeostasis. Accordingly, the liver, the main organ of iron metabolism, was severely affected in moribund ILEI overexpressing mice: increased alanine transaminase and aspartate aminotransferase levels indicated liver dysfunction, the liver was reduced in size, showed increased apoptosis, reduced cellular iron content, and had a fibrotic phenotype. These data indicate that high ILEI expression in the liver might reduce hepatoprotection and induce liver fibrosis, which leads to liver dysfunction, disturbed iron metabolism and eventually to death. Overall, we show here that the novel Tet-ON inducible Fam3c/ILEI transgenic mouse strain allows tissue specific timely controlled overexpression of ILEI and thus, will serve as a versatile tool to model the effect of elevated ILEI expression in diverse tissue entities and disease conditions, including cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. ...HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19
IgG
HBsAg
cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2
generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.
In contrast to hypoxia, TLR‐Iigands stabilize HIFlA in a predominantly MYD88‐dependent manner and result in activation of a different set of HIFlA target genes.
HIF1A is a transcription factor that ...plays a central role for the adaptation to tissue hypoxia and for the inflammatory response of myeloid cells, including DCs. HIF1A is stabilized by hypoxia but also by TLR ligands under normoxic conditions. The underlying signaling events leading to the accumulation of HIF1A in the presence of oxygen are still poorly understood. Here, we show that in contrast to hypoxic stabilization of HIF1A, normoxic, TLR‐mediated HIF1A accumulation in DCs follows a different pathway that predominantly requires MYD88‐dependent NF‐κB activity. The TLR‐induced HIF1A controls a subset of proinflammatory genes that are insufficiently induced following hypoxia‐mediated HIF1A induction. Thus, TLR activation and hypoxia stabilize HIF1A via distinct signaling pathways, resulting in differential HIF1A‐dependent gene expression.
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