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Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.
Background/Aims: Middle East respiratory syndrome coronavirus (MERS-CoV) and Marburg virus (MARV) are among the World Health Organization’s top 8 emerging pathogens. Both zoonoses share nonspecific early symptoms, a high lethality rate, and a reduced number of specific treatment options. Therefore, we evaluated extracorporeal virus and glycoprotein (GP) elimination by lectin affinity plasmapheresis (LAP).
Methods: For both MERS-CoV (pseudovirus) as well as MARV (GPs), 4 LAP devices (Mini Hemopurifiers, Aethlon Medical, San Diego, CA, USA) and 4 negative controls were tested. Samples were collected every 30 min and analyzed for reduction in virus infectivity by a flow cytometry-based infectivity assay (MERS-CoV) and in soluble GP content (MARV) by an immunoassay.
Results: The experiments show a time-dependent clearance of MERS-CoV of up to 80% within 3 h (pseudovirus). Up to 70% of MARV-soluble GPs were eliminated at the same time. Substantial saturation of the binding resins was detected within the first treatment hour.
Conclusion: MERS-CoV (pseudovirus) and MARV soluble GPs are eliminated by LAP in vitro. Considering the high lethality and missing established treatment options, LAP should be evaluated in vivo. Especially early initiation, continuous therapy, and timed cartridge exchanges could be of importance.
Our understanding of human biology and disease is based on the last millennia’s gain of knowledge, which has been exponentially accelerated since the invention of optical and "biochemical" microscopes like transcriptomics and other omics technologies.
In order to broaden our knowledge of an important human transcription factor, T-Cell Acute Lymphocytic Leukemia 1 (TAL1), some of these technologies were used.
TAL1’s gene or promoter structure is altered in about 20-30% of T-ALL. In addition, there is an increase in TAL1 expression in ca. 60% of pediatric and about 45% of adult T-ALL. Physiologically, TAL1 is an indispensable factor in hematopoiesis: in the murine knockout model, blood cells vanish in the early embryonic period. In addition, the TF is also relevant in adult erythropoiesis.
Accordingly, the identification of novel TAL1 target genes was significant both for clinical reasons and in order to understand the hematopoietic functions.
We performend a combined RNA- and ChIPseq approach. After a lentiviral mediated knockdown in K562 cells RNAseq was performed using the Illumina high-throughput method. Overall, the RNAseq yielded one billion good quality sequencing fragments. They made identification of up- and downregulated transcripts as well as associated biological processes, cellular components, molecular function and dominant KEGG signaling pathways possible. Furthermore, more than 2-fold altered coding transcripts and lncRNA were analyzed for relevant TAL1-binding in the transcription start area. There were 3205 significantly altered coding transcripts and 5136 significantly altered lncRNA. By integrating an Encode TAL1-ChIPseq in K562 cells (using a cutoff fold change of 2x) a relevant TAL1 binding could be detected with 71 coding and 416 lncRNA genes.
The combination of RNA- and ChIPseq yields a wealth of relevant results. Accordingly, TAL1 has complex pro- and anti-malignant effects in all areas of oncogenesis like described by Hanahan and Weinberg. Various interactions with target genes and signaling cascades in inter alia proliferation (e.g. HEMGN, MYC, AHI1, YPEL3, BTG2), angiogenesis (e.g. EGFL7, LTBP3), apoptosis (e.g. BCL3, BCL2A1, BMF), immune evasion (e.g. CMTM6) and inflammation (e.g. IL23 and PTGS1) have been revealed, thus complementing the knowledge about pro- and anti-oncogenic effects of TAL1. In addition, it was possible to identify target genes relevant for erythropoiesis and possible osteogenesis. Concerning lncRNA, interesting potential effectors have been identified. However, they still need to be functionally characterized. Relating the results to Virchow’s first description of leukemia as "white blood" the role of TAL1 in leukemia’s genesis but also in erythropoiesis has been confirmed and extended, thus contributing to explain Virchow’s observation: "...therefore, when I speak of white blood, I mean in fact a blood in which the proportion between the red and colorless (in white) blood corpuscles is reversed ...” (Virchow R. Weisses Blut. Frorieps Notizen 1845;36:151-156).
Stem cell-based therapies require cells with a maximum regenerative capacity in order to support regeneration after tissue injury and organ failure. Optimization of this regenerative potential of mesenchymal stromal/stem cells (MSC) or their conditioned medium by in vitro preconditioning regimens are considered to be a promising strategy to improve the release of regenerative factors. In the present study, MSC were isolated from inguinal adipose tissue (mASC) from C57BL/6 mice, cultured, and characterized. Then, mASC were either preconditioned by incubation in a hypoxic environment (0.5% O2), or in normoxia in the presence of murine epidermal growth factor (EGF) or tumor necrosis factor α (TNFα) for 48 h. Protein expression was measured by a commercially available array. Selected factors were verified by PCR analysis. The expression of 83 out of 308 proteins (26.9%) assayed was found to be increased after preconditioning with TNFα, whereas the expression of 61 (19.8%) and 70 (22.7%) proteins was increased after incubation with EGF or in hypoxia, respectively. Furthermore, we showed the proliferation-promoting effects of the preconditioned culture supernatants on injured epithelial cells in vitro. Our findings indicate that each preconditioning regimen tested induced an individual expression profile with a wide variety of factors, including several growth factors and cytokines, and therefore may enhance the regenerative potential of mASC for cell-based therapies.