Ex vivo Analyse antigen-spezifischer CD4+ T-Zell-Antworten im Infektionsmodell der murinen Listeriose

Antigen-reaktive CD4+ T-Zellen spielen eine wichtige Rolle bei der Entwicklung schützender Immunität. So sind Kenntnisse über die Bildung und Erhaltung CD4+ T-Zellen möglicherweise bedeutend für die Entwicklung verbesserter Impfstoffe und neuer Therapieansätze bei akuten und chronischen Infektionserkrankungen. Mit dieser Arbeit wurde eine genaue Charakterisierung CD4+ T-Zell-Antworten im Vergleich zu CD8+ T-Zellen am Infektionsmodell der murinen Listeriose vorgenommen. So konnten erstmals wichtige Unterschiede zwischen den beiden T-Zell-Kompartimenten beschrieben werden, insbesondere die Beobachtung, dass - im Gegensatz zu CD8+ T-Zellen - CD4+ Effektor-Gedächtniszellen nach bakterieller Infektion kontinuierlich im gesamten Organismus über die Zeit abnehmen

Advances in Automated Generation of Convolutional Neural Networks from Synthetic Data in Industrial Environments

The usage of convolutional neural networks has revolutionized data processing and its application in the industry during the last few years. Especially detection in images, a historically hard task to automate is now available on every smart phone. Nonetheless, this technology has not yet spread in the industry of car production, where lots of visual tests and quality checks are still performed manually. Even though the vision capabilities convolutional neural networks can give machines are already respectable, they still need well prepared training data that is costly and time-consuming to produce. This paper describes our effort to test and improve a system to automatically synthesize training images. This existing system renders computer aided design models into scenes and out of that produces realistic images and corresponding labels. Two new models, Single Shot Detector and RetinaNet are retrained under the use of distractors and then tested against each other. The better performing RetinaNet is then tested for performance under training with a variety of datasets from different domains in order to observe the models strength and weakness under domain shifts. These domains are real photographs, rendered models and images of objects cut and pasted into different backgrounds. The results show that the model trained with a mixture of all domains performs best

Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

BACKGROUND: Transforming growth factor-βs (TGF-βs), bone morphogenetic proteins (BMPs) and activins are important regulators of developmental cell growth and differentiation. Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors. RESULTS: There are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules. We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (associated molecule with the SH3 domain of STAM), an adapter shown to be involved in BMP signaling. Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in Jun kinase activation domain binding protein and proteasomal subunits) domain at the carboxy-terminus. We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-β-dependent promoters. Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2. We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-β signaling. Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-β. CONCLUSIONS: This report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-β signaling pathway. Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-β signaling pathway

Combustion behavior of single iron particles-part I:An experimental study in a drop-tube furnace under high heating rates and high temperatures

Micrometric spherical particles of iron in two narrow size ranges of (38–45) µm and (45–53) µm were injected in a bench scale, transparent drop-tube furnace (DTF), electrically heated to 1400 K. Upon experiencing high heating rates (104–105 K/s) the iron particles ignited and burned. Their combustion behavior was monitored pyrometrically and cinematographically at three different oxygen mole fractions (21%, 50% and 100%) in nitrogen. The results revealed that iron particles ignited readily and exhibited a bright stage of combustion followed by a dimmer stage. There was evidence of formation of envelope micro-flames around iron particles (nanometric particle mantles) during the bright stage of combustion. As the burning iron particles fell by gravity in the DTF, contrails of these fine particles formed in their wakes. Peak temperatures of the envelope flames were in the range of 2500 K in air, climbing to 2800 K in either 50% or 100% O2. Total luminous combustion durations of particles, in the aforesaid size ranges, were in the range of 40–65 ms. Combustion products were bimodal in size distribution, consisting of micrometric black magnetite particles (Fe3O4), of sizes similar to the iron particle precursors, and reddish nanometric iron oxide particles consisting mostly of hematite (Fe2O3).</p

Flow Cytometric Analyses of Lymphocyte Markers in Immune Oncology: A Comprehensive Guidance for Validation Practice According to Laws and Standards

Many anticancer therapies such as antibody-based therapies, cellular therapeutics (e.g., genetically modified cells, regulators of cytokine signaling, and signal transduction), and other biologically tailored interventions strongly influence the immune system and require tools for research, diagnosis, and monitoring. In flow cytometry, in vitro diagnostic (IVD) test kits that have been compiled and validated by the manufacturer are not available for all requirements. Laboratories are therefore usually dependent onmodifying commercially available assays or, most often, developing them to meet clinical needs. However, both variants must then undergo full validation to fulfill the IVD regulatory requirements. Flow cytometric immunophenotyping is a multiparametric analysis of parameters, some of which have to be repeatedly adjusted; that must be considered when developing specific antibody panels. Careful adjustments of general rules are required to meet legal and regulatory requirements in the analysis of these assays. Here, we describe the relevant regulatory framework for flow cytometry-based assays and describe methods for the introduction of new antibody combinations into routine work including development of performance specifications, validation, and statistical methodology for design and analysis of the experiments. The aim is to increase reliability, efficiency, and auditability after the introduction of in-house-developed flow cytometry assays

Cord Blood Vα24-Vβ11+ Natural Killer T Cells Display a Th2-Chemokine Receptor Profile and Cytokine Responses

Background: The fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT) cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24(+)Vβ11(+) NKT cells expressing Th1/Th2-related chemokine receptors (CKR) were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR(+) subsets. Results: Cord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3+ and CCR5+ cord blood NKT cells (Th1-related) were present at lower percentages. Within CD4negCD8neg (DN) NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3+, CCR4+, CCR5+, CCR6+, CCR7+, CCR8+ and CXCR4+ DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3+, CCR6+ and CCR8+ cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR) transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells. Conclusions: Despite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens

High frequencies of PMN-MDSCs are associated with low suppressive capacity in advanced stages of HIV-1 infection

Background Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are an immature cell type that inhibits the effector functions of T lymphocytes in chronic HIV infection. A well-known immunological feature of the disease course is the development of immune exhaustion, which is correlated with excessive immune activation in late-stage disease. Here, we hypothesized that immune exhaustion would also affect PMN-MDSCs in late-stage HIV-1 infection. Methods We evaluated untreated chronically HIV-infected patients (progressors, n = 10) and control groups (controllers, patients with non-small cell lung carcinoma and healthy controls, n = 16) with regard to levels of PMN-MDSCs and their inhibitory potential. Additionally, we studied CD8 T cell effector functions (interferon-gamma, TNF alpha, IL-2 and CD107) and parameters of CD8 T cell activation (CD38 and HLA-DR) and exhaustion (PD-1 and LAG-3) by flow cytometry. Plasma inflammation markers analyzed here were IL-6, IL-8, soluble CD14, highly sensitive CRP, and cystatin C. Results Coincubation experiments with isolated PMN-MDSCs led to a significant inhibition of CD8 T cell proliferation (p < 0.0001), with a significant correlation between PMN-MDSC frequency and suppressive capacity: the higher the frequency of PMN-MDSCs was, the lower the suppressive capacity (rho = 0.51, p = 0.0082). Stratifying all study subjects into subgroups with PMN-MDSC frequencies above or below 2.5% resulted in a significantly increased suppressive capacity in patients with frequencies below 2.5% (p = 0.021). While there was no correlation with the cellular activation markers CD38 and HLA-DR, high IL-8 levels were significantly associated with high PMN-MDSC frequencies (rho = 0.52, p = 0.0074) and low suppressive capacity (rho = 0.47, p = 0.019). Conclusions In this study, we demonstrate for the first time that PMN-MDSCs show limited effector functions in advanced disease stages of HIV infection. The hyperactive immune state is associated with this loss of function. However, we show an association with the proinflammatory cytokine IL-8, which is an important factor for the migration and adhesion of polymorphonuclear cells

Myeloid Zinc Finger 1 (Mzf1) Differentially Modulates Murine Cardiogenesis by Interacting with an Nkx2.5 Cardiac Enhancer

Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations

The 14-bp deletion polymorphism in the HLA-G gene displays significant differences between ulcerative colitis and Crohn's disease and is associated with ileocecal resection in Crohn's disease

HLA-G is a non-classical MHC class Ib molecule predominantly expressed in cytotrophoblasts and under pathological conditions also in chronically inflamed and in malignant tissues. Recently an increased expression of HLA-G was found in ulcerative colitis (UC), but not in Crohn's disease (CD). The HLA-G gene is located in IBD3, a linkage region for inflammatory bowel disease (IBD). A 14-bp deletion polymorphism (Del+/Del−) within exon 8 of the HLA-G gene might influence transcription activity and is therefore of potential functional relevance. To investigate whether the 14-bp deletion polymorphism is associated with IBD, 371 patients with CD, 257 patients with UC and 739 controls were genotyped. The heterozygous genotype (P = 0.031) and the Del+ phenotype (P = 0.038) were significantly increased, whereas the homozygous Del− phenotype (P = 0.038) was significantly decreased in UC when compared with CD. Thus, the 14-bp deletion polymorphism within the HLA-G gene displayed significant differences between UC and CD. Moreover, a significant increase of the Del+ allele (P = 0.002) and the Del+/Del+ genotype (P = 0.013) and a consecutive decrease of the Del−/− genotype (P = 0.024) were observed in those CD cases positive for ileocecal resection. Thus, a potential effect of the HLA-G gene in IBD may affect both UC and CD. Other polymorphisms linked to the 14-bp deletion polymorphism might also contribute to immunopathogenesis. As there are several partly functional polymorphisms within the promoter region potentially influencing HLA-G expression, further studies in IBD are necessary in the context of differential expression of HLA-G between UC and C

TAMEP are brain tumor parenchymal cells controlling neoplastic angiogenesis and progression

Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor parenchymal cells may promote specific phases of disease progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Notably, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell population, by conditional Sox2-knockout, drastically reduced glioblastoma vascularization and size. Hence, TAMEP emerge as a tumor parenchymal component with a strong impact on glioblastoma progression