Epigenetic control of TLR4-mediated gene expression induced by bacteria and bacterial cell wall components in human intestinal epithelial cells

Hintergrund: Kommensale Bakterien und bakterielle Zellwandbestandteile können über diverse intrazelluläre Signalwege komplexe Zellreaktionen hervorrufen. Beispielsweise scheinen LPS und probiotische Bakterien DNA-Methylierung zu modifizieren und somit Veränderungen der Genexpression mittels epigenetischer Mechanismen zu steuern. Methoden: In der vorliegenden in vitro Studie wurden intestinale Epithelzellen (Caco-II-Zellen) mit LPS, Flagellin, Lactobacillus rhamnosus GG (LGG) und Lactobacillus delbrueckii (LD) stimuliert und daraufhin die mRNA Level in den Entzündungsprozess involvierter Gene (TNFα, TLR4, p38) und miRNAs (146a, 155) mit Hilfe von q-PCR gemessen. Des Weiteren wurde die DNA-Methylierung von 4 CpG loci in der TNFα-Promotorregion und 4 CpGs des TLR4 Exons mittels Pyrosequenzierung quantitativ bestimmt. Ergebnisse: Stimulation mit LPS, Flagellin, LGG und LD resultierte jeweils in einer Abnahme der mRNA Level von TNFα und TLR4, während p38 mRNA leicht anstieg. Die Behandlung mit Flagellin löste eine 4,42±0,51-fache (p=0,002) Zunahme der miRNA-146a aus, hatte aber keinen Einfluss auf die miRNA-155. Die DNA-Methylierung der vier analysierten TNFα CpG loci bewegte sich zwischen 70-90%. Signifikante Veränderungen der TNFα-Methylierung wurden durch LGG nach 12 Stdn. (+1,07%±0,24, p=0,048), LD nach 24 Stdn. (+1,54%±0,3, p=0,035) und LPS nach 72 Stdn. (-0,58%±0,05, p=0,007) verursacht. TLR4-Methylierungen schwankten zwischen 10-45% in den unterschiedlichen Zellexperimenten. CpG 4 zeigte eine signifikante Zunahme nach 12 Stdn. Behandlung mit LPS (+2,00%±0,42, p=0,041), CpG 3 eine Abnahme nach 72 Stdn. Behandlung mit LGG (-1,77%±0,29, p=0,027) und CpG 2 eine Abnahme nach 12 Stdn. LPS Behandlung (-1,62%±0,26, p=0,024). 12 stündige Stimulation der Zellen mit LGG führte bei TNFα zu einer Zunahme in Methylierung bei gleichzeitiger Abnahme der mRNA Level. Schlussfolgerung: Die Resultate dieser Studie lassen darauf schließen, dass die epigenetische Regulation von TNFα und TLR4 zu der Spezifität der inflammatorischen Reaktionen beiträgt, welche durch Bakterien und deren Zellwandbestandteile ausgelöst werden.Background: Commensal bacterial strains and bacterial cell wall components are proposed to induce differential cell responses regulated by intracellular signalling pathways. Epigenetic modulation of gene expression via DNA methylation was recently discussed to be influenced by LPS and probiotic bacteria. Methods: We analysed the expression of inflammation-relevant genes (TNFα, TLR4, p38) and miRNAs (146a, 155) in caco-II-cells upon stimulation with LPS, flagellin, Lactobacillus rhamnosus GG (LGG) and Lactobacillus delbrueckii (LD) by measuring mRNA levels with q-PCR. Furthermore, DNA methylation of four CpG loci in the TNFα promoter region and four CpGs of the TLR4 exon was measured by using bisulfite-converted DNA for pyrosequencing analysis. Results: LPS, flagellin, LGG and LD each decreased mRNA levels of TNFα and TLR4, while p38 mRNA was slightly increased by each treatment. Flagellin induced miRNA-146a expression by 4.42-fold±0.51 (p=0.002), but did not alter miRNA-155. Methylation of four analysed TNFα CpG loci ranged between 70-90% in un-stimulated cells. Significant changes in TNFα methylation over all four CpGs were caused by LGG after 12h of stimulation (+1.07%±0.24, p=0.048), LD after 24h (+1.54%±0.3, p=0.035) and LPS after 72h (-0.58%±0.05, p=0.007). TLR4 methylation ranged between 10 and 45% in different CpGs and cell culture experiments. CpG 4 was significantly increased by 12h LPS treatment (+2.00%±0.42, p=0.041), CpG 3 was decreased after 72h LGG treatment (-1.77%±0.29, p=0.027) and CpG 2 was also decreased after 12h LPS treatment (-1.62%±0.26, p=0.024). LGG treatment for 12h showed an increase in methylation (+1.07%±0.24, p=0.048) and a decrease in mRNA expression (0.73-fold±0.23, p=0.008) of TNFα. Conclusion: The results indicate, that epigenetic regulation of TNFα and TLR4 contributes to the specificity of inflammatory reactions induced by bacteria and their cell wall components

Cannabinoid Receptor Type-2 in B Cells Is Associated with Tumor Immunity in Melanoma.

Agents targeting the endocannabinoid system (ECS) have gained attention as potential cancer treatments. Given recent evidence that cannabinoid receptor 2 (CB2R) regulates lymphocyte development and inflammation, we performed studies on CB2R in the immune response against melanoma. Analysis of The Cancer Genome Atlas (TCGA) data revealed a strong positive correlation between CB2R expression and survival, as well as B cell infiltration in human melanoma. In a murine melanoma model, CB2R expression reduced the growth of melanoma as well as the B cell frequencies in the tumor microenvironment (TME), compared to CB2R-deficient mice. In depth analysis of tumor-infiltrating B cells using single-cell RNA sequencing suggested a less differentiated phenotype in tumors from Cb2r-/- mice. Thus, in this study, we demonstrate for the first time a protective, B cell-mediated role of CB2R in melanoma. This gained insight might assist in the development of novel, CB2R-targeted cancer therapies

Comparative Structural and Optical Properties of Different Ceria Nanoparticles

Herein a comparative study of five nanocrystalline cerium oxides (CeO2-delta) synthesised by different methods and calcined at 500 degrees C is reported. XRPD analysis showed that stoichiometry parameter delta, crystallite size/strain and lattice constant were only slightly affected by the method utilized. All ceria nanoparticles are nearly spherical in shape with faceted morphology, free of defects and with a relatively uniform size distribution. The average microstrain was found to be approximately 10 times higher than that of bulk counterpart. The absorption edge of nanocrystalline materials was shifted towards a higher wavelengths (red shift) in comparison with bulk counterpart, and band gap values were in the range 2.7-3.24 eV (3.33 eV for bulk counterpart)

BCG hydrogel promotes CTSS-mediated antigen processing and presentation, thereby suppressing metastasis and prolonging survival in melanoma.

BACKGROUND The use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected, and occasionally of distal lesions. However, intralesional-BCG is less effective in patients with visceral metastases and did not significantly improve overall survival. METHODS We generated a novel BCG lysate and developed it into a thermosensitive PLGA-PEG-PLGA hydrogel (BCG hydrogel), which was injected adjacent to the tumor to assess its antitumor effect in syngeneic tumor models (B16F10, MC38). The effect of BCG hydrogel treatment on contralateral tumors, lung metastases, and survival was assessed to evaluate systemic long-term efficacy. Gene expression profiles of tumor-infiltrating immune cells and of tumor-draining lymph nodes from BCG hydrogel-treated mice were analyzed by single-cell RNA sequencing (scRNA-seq) and CD8+ T cell receptor (TCR) repertoire diversity was assessed by TCR-sequencing. To confirm the mechanistic findings, RNA-seq data of biopsies obtained from in-transit cutaneous metastases of patients with melanoma who had received intralesional-BCG therapy were analyzed. RESULTS Here, we show that BCG lysate exhibits enhanced antitumor efficacy compared to live mycobacteria and promotes a proinflammatory tumor microenvironment and M1 macrophage (MΦ) polarization in vivo. The underlying mechanisms of BCG lysate-mediated tumor immunity are dependent on MΦ and dendritic cells (DCs). BCG hydrogel treatment induced systemic immunity in melanoma-bearing mice with suppression of lung metastases and improved survival. Furthermore, BCG hydrogel promoted cathepsin S (CTSS) activity in MΦ and DCs, resulting in enhanced antigen processing and presentation of tumor-associated antigens. Finally, BCG hydrogel treatment was associated with increased frequencies of melanoma-reactive CD8+ T cells. In human patients with melanoma, intralesional-BCG treatment was associated with enhanced M1 MΦ, mature DC, antigen processing and presentation, as well as with increased CTSS expression which positively correlated with patient survival. CONCLUSIONS These findings provide mechanistic insights as well as rationale for the clinical translation of BCG hydrogel as cancer immunotherapy to overcome the current limitations of immunotherapies for the treatment of patients with melanoma

Size effects in the magnetic behaviour of TbAl_2 milled alloys

The study of the magnetic properties depending upon mechanical milling of the ferromagnetic polycrystalline TbAl_2 material is reported. The Rietveld analysis of the X-ray diffraction data reveals a decrease of the grain size down to 14 nm and -0.15 % of variation of the lattice parameter, after 300 hours of milling time. Irreversibility in the zero field cooled - field cooled (ZFC-FC) DC-susceptibility and clear peaks in the AC susceptibility between 5 and 300 K show that the long-range ferromagnetic structure is inhibited in favour of a disordered spin arrangement below 45 K. This glassy behaviour is also deduced from the variation of the irreversibility transition with the field (H^{2/3}) and frequency. The magnetization process of the bulk TbAl_2 is governed by domain wall thermal activation processes. By contrast, in the milled samples, cluster-glass properties arise as a result of cooperative interactions due to the substitutional disorder. The interactions are also influenced by the nanograin structure of the milled alloys, showing a variation of coercivity with the grain size, below the crossover between the multi- and single-domain behaviours.Comment: 23 pages, 11 figures, to appear in J. Phys.: Condens. Ma

Broadband, site selective and time resolved photoluminescence spectroscopic studies of finely size-modulated Y2O3:Eu3+ phosphors synthesized by a complex based precursor solution method

Undoped and Eu3+-doped cubic yttria (Y2O3) nanophosphors of good crystallinity, with selective particle sizes ranging between 6 and 37 nm and showing narrow size distributions, have been synthesized by a complex-based precursor solution method. The systematic size tuning has been evidenced by transmission electron microscopy, X-ray diffraction, and Raman scattering measurements. Furthermore, size-modulated properties of Eu3+ ions have been correlated with the local structure of Eu3+ ion in different sized Y2O3:Eu3+ nanophosphors by means of steady-state and time-resolved site-selective laser spectroscopies. Time-resolved site-selective excitation measurements performed in the 7F0 ¿ 5D0 peaks of the Eu3+ ions at C2 sites have allowed us to conclude that Eu3+ ions close to the nanocrystal surface experience a larger crystal field than those in the nanocrystal core. Under the site-selective excitation in the 7F0 ¿ 5D0 peaks, energy transfer between the sites has also been observed.Authors are grateful to Ministerio de Ciencia e Innovacion of Spain (MICINN) under The National Program of Materials (MAT2010-21270-C04-02/03/04), the Consolider-Ingenio 2010 Program (MALTA CSD2007-0045), Generalitat Valenciana (GVA-ACOMP-2013-012), and to the EU-FEDER Funds for their financial support. F.J.M. and O.G. are grateful to the Vicerrectorado de Investigacion y Desarrollo of the Universitat Politecnica de Valencia (UPV2011-0914 PAID-05-11 and UPV2011-0966 PAID-06-11). S.F.L-L. wishes to thank MICCIN for an FPI grant (BES-2008-003353). Finally, S. R. wishes to thank Universitat Politecnica de Valencia and Universidad de La Laguna for the financial support during her research stays.Ray, S.; León-Luis, SF.; Manjón Herrera, FJ.; Mollar García, MA.; Gomis Hilario, O.; Rodríguez-Mendoza, UR.; Agouram, S.... (2014). Broadband, site selective and time resolved photoluminescence spectroscopic studies of finely size-modulated Y2O3:Eu3+ phosphors synthesized by a complex based precursor solution method. Current Applied Physics. 14(1):72-81. https://doi.org/10.1016/j.cap.2013.07.027S728114

Incorporation and phase separation of Cl in alkaline earth aluminosilicate glasses

Pyrochemical reprocessing of spent nuclear fuels may lead to the generation of chloride containing wastes. 36Cl wastes may also arise from the treatment of irradiated graphite. Such wastes will have limited solubility in the borosilicates currently used for waste vitrification. Despite requiring higher processing temperatures aluminosilicate glasses show promise for this application. In a series of alkaline earth aluminosilicate glasses we demonstrate that chloride solubility is related to the alkaline earth species as follows Sr > Sr+Ba > Ba > Ca > Mg, with the strontium aluminosilicate glass accommodating up to 5.92 at% Cl. Typical chloride retention rates are ~80% of the batched chloride content at 1400ºC. It has also been observed that, when Cl is present in the glass in excess, phase separation firstly occurs as formation of non-Cl crystals (mainly alkaline earth aluminosilicates, with a minority of aluminates); a segregated chloride layer is only formed at higher chlorine loadings. This indicates that chlorine solubility in glass is not only controlled by the capacity of glass network to accommodate Cl– but also by the stability of glass network after Cl– incorporation. In addition, increased incorporation of Cl– in glass results in steadily decreased glass densities and glass transition temperatures

Characterization of a Myeloid Activation Signature That Correlates with Survival in Melanoma Patients

Understanding the cellular interactions within the tumor microenvironment (TME) of melanoma paved the way for novel therapeutic modalities, such as T cell-targeted immune checkpoint inhibitors (ICI). However, only a limited fraction of patients benefits from such therapeutic modalities, highlighting the need for novel predictive and prognostic biomarkers. As myeloid cells orchestrate the tumor-specific immune response and influence the efficacy of ICI, assessing their activation state within the TME is of clinical relevance. Here, we characterized a myeloid activation (MA) signature, comprising the three genes Cxcl11, Gbp1, and Ido1, from gene expression data of human myeloid cells stimulated with poly(I:C) or cGAMP. This MA signature positively correlated to overall survival in melanoma. In addition, increased expression of the MA signature was observed in melanoma patients responding to ICI (anti-PD-1), as compared to non-responders. Furthermore, the MA signature was validated in the murine B16F10 melanoma model where it was induced and associated with decreased tumor growth upon intratumoral administration of poly(I:C) and cGAMP. Finally, we were able to visualize co-expression of the MA signature genes in myeloid cells of human melanoma tissues using RNAscope in situ hybridization. In conclusion, the MA signature indicates the activation state of myeloid cells and represents a prognostic biomarker for the overall survival in melanoma patients