Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer ...therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.
•Transcriptome analysis performed on tumor-resident CD4+ Th1, Th17, and Treg cells•Tumor-infiltrating Treg cells are defined by the expression of signature genes•Treg-specific signature genes correlate with patients’ survival in both CRC and NSCLC
Tumor-infiltrating regulatory T cells can suppress effector T cells specific for tumor antigens. De Simone et al. (2016) demonstrate that tumor-infiltrating Treg cells display specific gene signatures that were also validated at the single-cell level. These data can contribute to dissect the molecular networks underlying the biology of tumor-infiltrating Treg cells. As part of the IHEC consortium, this study integrates genetic, epigenetic, and transcriptomic profiling in three immune cell types from nearly 200 people to characterize the distinct and cooperative contributions of diverse genomic inputs to transcriptional variation. Explore the Cell Press IHEC webportal at www.cell.com/consortium/IHEC.
All the different coronavirus SARS-CoV-2 variants isolated so far share the same mechanism of infection mediated by the interaction of their spike (S) glycoprotein with specific residues on their ...cellular receptor: the angiotensin converting enzyme 2 (ACE2). Therefore, the steric hindrance on this cellular receptor created by a bulk macromolecule may represent an effective strategy for the prevention of the viral spreading and the onset of severe forms of Corona Virus disease 19 (COVID-19). Here, we applied a systematic evolution of ligands by exponential enrichment (SELEX) procedure to identify two single strand DNA molecules (aptamers) binding specifically to the region surrounding the K353, the key residue in human ACE2 interacting with the N501 amino acid of the SARS-CoV-2 S. 3D docking in silico experiments and biochemical assays demonstrated that these aptamers bind to this region, efficiently prevent the SARS-CoV-2 S/human ACE2 interaction and the viral infection in the nanomolar range, regardless of the viral variant, thus suggesting the possible clinical development of these aptamers as SARS-CoV-2 infection inhibitors. Our approach brings a significant innovation to the therapeutic paradigm of the SARS-CoV-2 pandemic by protecting the target cell instead of focusing on the virus; this is particularly attractive in light of the increasing number of viral mutants that may potentially escape the currently developed immune-mediated neutralization strategies.
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Novel ssDNA oligos (Aptamers) specifically binding the human ACE2 (hACE2) are able to efficiently prevent the spike/ACE2 interaction.
Background & Aims All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely ...form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture−infectious virus H77S.3. Methods We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Results Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. Conclusions DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis.
Nonalcoholic fatty liver disease (NAFLD) is a rising cause of hepatocellular carcinoma (HCC). We examined whether inherited pathogenic variants in candidate genes (n = 181) were enriched in patients ...with NAFLD-HCC. To this end, we resequenced peripheral blood DNA of 142 NAFLD-HCC, 59 NAFLD with advanced fibrosis, and 50 controls, and considered 404 healthy individuals from 1000 G. Pathogenic variants were defined according to ClinVar, likely pathogenic as rare variants predicted to alter protein activity. In NAFLD-HCC patients, we detected an enrichment in pathogenic (p = 0.024), and likely pathogenic variants (p = 1.9*10
), particularly in APOB (p = 0.047). APOB variants were associated with lower circulating triglycerides and higher HDL cholesterol (p < 0.01). A genetic risk score predicted NAFLD-HCC (OR 4.96, 3.29-7.55; p = 5.1*10
), outperforming the diagnostic accuracy of common genetic risk variants, and of clinical risk factors (p < 0.05). In conclusion, rare pathogenic variants in genes involved in liver disease and cancer predisposition are associated with NAFLD-HCC development.
IL‐10 is an anti‐inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8+ CTL, IL‐10 can promote CTL ...responses. To understand these paradoxic findings, we analyzed the role of IL‐10 produced by human APC subsets in T‐cell responses. IL‐10 production was restricted to CD1c+ DCs and CD14+ monocytes. Interestingly, it was differentially regulated, since R848 induced IL‐10 in DCs, but inhibited IL‐10 in monocytes. Autocrine IL‐10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high‐affinity peptides. Nevertheless, it completely blocked cross‐priming and priming with low‐affinity peptides of a self/tumor‐antigen. IL‐10 also inhibited CD1c+ DC‐induced CD4+ T‐cell priming and enhanced Foxp3 induction, but was insufficient to induce T‐cell IL‐10 production. CD1c+ DC‐derived IL‐10 had also no effect on DC‐induced secondary expansions of memory CTL. However, IL‐15‐driven, TCR‐independent proliferation of memory CTL was enhanced by IL‐10. We conclude that DC‐derived IL‐10 selects high‐affinity CTL upon priming. Moreover, IL‐10 preserves established CTL memory by enhancing IL‐15‐dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL‐10 promotes CTL responses in humans.
Role of IL‐10 in human CD8+ T‐cell (CTL) responses: CD1c+ DC‐derived IL‐10 fine‐tunes DC maturation, and inhibits selectively the priming and cross‐priming of low‐affinity CTL. In addition, TLR‐stimulated CD1c+ DC trans‐present IL‐15, which induces TCR‐independent, “homeostatic” proliferation of memory CTL that is enhanced by IL‐10.
Interferons (IFNs) are key cytokines involved in alerting the immune system to viral infection. After IFN stimulation, cellular transcriptional profile critically changes, leading to the expression ...of several IFN stimulated genes (ISGs) that exert a wide variety of antiviral activities. Despite many ISGs have been already identified, a comprehensive network of coding and non-coding genes with a central role in IFN-response still needs to be elucidated. We performed a global RNA-Seq transcriptome profile of the HCV permissive human hepatoma cell line Huh7.5 and its parental cell line Huh7, upon IFN treatment, to define a network of genes whose coordinated modulation plays a central role in IFN-response. Our study adds molecular actors, coding and non-coding genes, to the complex molecular network underlying IFN-response and shows how systems biology approaches, such as correlation networks, network's topology and gene ontology analyses can be leveraged to this aim.
This work aimed to investigate the ability of different formulations of bioactive glass (BAG)-S53P4 to interfere with bacterial biofilm produced on prosthetic material by methicillin-resistant ...Staphylococcus aureus and multi-drug-resistant Pseudomonas aeruginosa.
Antibiofilm activity of three formulations of bioglass was assessed at different time points through two different analyses: Crystal Violet and confocal laser scanning microscopy assays.
Significant differences in the whole biofilm were observed between BAG-S53P4-treated and control samples, while no marked changes in antibiofilm activity were observed among the tested formulations. Data from colorimetric assay were confirmed by confocal laser scanning microscopy analysis, which evidenced the significant reduction in biomass and a decrease of total cell volume when both S. aureus and P. aeruginosa biofilms were treated with BAG-S53P4.
BAG-S53P4 can be considered as an excellent adjuvant in the treatment of prosthetic infections related to biofilm.
Background & Aims Positive-sense RNA viruses remodel intracellular membranes to generate specialized membrane compartments for viral replication. Several RNA viruses, including poliovirus and ...hepatitis C virus (HCV), require phosphatidylinositol (PI) 4-kinases for their replication. However, it is not known how PI 4-kinases and their product, PI(4)P, facilitate host membrane reorganization and viral replication. In addition, although the HCV replication compartment, known as the membranous web, is believed to be cholesterol enriched, the mechanisms by which this occurs have not been elucidated. We aimed to identify and characterize a PI 4-kinase effector in HCV replication. Methods We used a combination of microscopic and biochemical methods to study HCV replication, web morphology, the distribution of intracellular protein and PI(4)P, along with cholesterol trafficking in HCV-infected cells. PI 4-kinase and oxysterol-binding protein (OSBP) were inhibited using RNA interference or small molecules in cells expressing a full-length genotype 1b replicon or infected with the JFH-1 strain of HCV. Results OSBP was required for HCV replication and membranous web integrity. OSBP was recruited to membranous webs in a PI 4-kinase−dependent manner, and both these factors were found to regulate cholesterol trafficking to the web. We also found OSBP to be required for poliovirus infection but dispensable for dengue virus. Conclusions OSBP is a PI 4-kinase effector in HCV infection, and contributes to the integrity and cholesterol enrichment of the membranous web. OSBP might also be a PI 4-kinase effector in poliovirus infection and could be involved in replication of other viruses that require PI 4-kinases.
Most direct‐acting antivirals (DAAs) that are being developed as therapy against hepatitis C virus target the NS3/4A protease, the NS5A protein, and the NS5B polymerase. The latter enzyme offers ...different target sites: the catalytic domain for nucleos(t)ide analogues as well as a number of allosteric sites for nonnucleos(t)ide inhibitors. Two NS3/4A protease inhibitors have been approved recently, and more than 40 new NS3/4A, NS5A, or NS5B inhibitors are in development. These agents can achieve very high cure rates when combined with pegylated interferon‐β and ribavirin and show promising clinical results when administered in all‐oral combinations. In addition to the more canonical drug targets, new alternative viral targets for small‐molecule drug development are emerging, such as p7 or NS4B and viral entry. Future research will need to define well‐tolerated and cost‐effective DAA combinations that provide the highest rates of viral eradication in all patients (including those with advanced liver disease), the broadest spectrum of action on viral genotypes showing minimal or no clinical resistance, and the shortest treatment duration. (Hepatology 2013)
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