Ultrastructure of the Membrana Limitans Interna after Dye-Assisted Membrane Peeling

The purpose of this study was to investigate the ultrastructure of the membrana limitans interna (internal limiting membrane, ILM) and to evaluate alterations to the retinal cell layers after membrane peeling with vital dyes. Twenty-five patients (25 eyes) who underwent macular hole surgery were included, whereby 12 indocyanine green (ICG)- and 13 brilliant blue G (BBG)-stained ILM were analyzed using light, transmission electron and scanning electron microscopy. Retinal cell fragments on the ILM were identified in both groups using immunohistochemistry. Comparing ICG- and BBG-stained membranes, larger cellular fragments were observed at a higher frequency in the BBG group. Thereby, the findings indicate that ICG permits an enhanced separation of the ILM from the underlying retina with less mechanical destruction. A possible explanation might be seen in the known photosensitivity of ICG, which induces a stiffening and shrinkage of the ILM but also generates retinal toxic metabolite

Immunological Memory Transferred with CD4 T Cells Specific for Tuberculosis Antigens Ag85B-TB10.4: Persisting Antigen Enhances Protection

BACKGROUND:High levels of death and morbidity worldwide caused by tuberculosis has stimulated efforts to develop a new vaccine to replace BCG. A number of Mycobacterium tuberculosis (Mtb)-specific antigens have been synthesised as recombinant subunit vaccines for clinical evaluation. Recently a fusion protein of TB antigen Ag85B combined with a second immunodominant TB antigen TB10.4 was emulsified with a novel non-phospholipid-based liposomal adjuvant to produce a new subunit vaccine, investigated here. Currently, there is no consensus as to whether or not long-term T cell memory depends on a source of persisting antigen. To explore this and questions regarding lifespan, phenotype and cytokine patterns of CD4 memory T cells, we developed an animal model in which vaccine-induced CD4 memory T cells could transfer immunity to irradiated recipients. METHODOLOGY/PRINCIPAL FINDINGS:The transfer of protective immunity using Ag85B-TB10.4-specific, CD45RB(low) CD62L(low) CD4 T cells was assessed in sub-lethally irradiated recipients following challenge with live BCG, used here as a surrogate for virulent Mtb. Donor T cells also carried an allotype marker allowing us to monitor numbers of antigen-specific, cytokine-producing CD4 T cells in recipients. The results showed that both Ag85B-TB10.4 and BCG vaccination induced immunity that could be transferred with a single injection of 3x10(6) CD4 T cells. Ten times fewer numbers of CD4 T cells (0.3x10(6)) from donors immunised with Ag85B-TB10.4 vaccine alone, transferred equivalent protection. CD4 T cells from donors primed by BCG and boosted with the vaccine similarly transferred protective immunity. When BCG challenge was delayed for 1 or 2 months after transfer (a test of memory T cell survival) recipients remained protected. Importantly, recipients that contained persisting antigen, either live BCG or inert vaccine, showed significantly higher levels of protection (p<0.01). Overall the numbers of IFN-gamma-producing CD4 T cells were poorly correlated with levels of protection. CONCLUSIONS/SIGNIFICANCE:The Ag85B-TB10.4 vaccine, with or without BCG-priming, generated TB-specific CD4 T cells that transferred protective immunity in mice challenged with BCG. The level of protection was enhanced in recipients containing a residual source of specific antigen that could be either viable or inert

The free volume of poly(vinyl methylether) as computed in a wide temperature range and at length scales up to the nanoregion

14 páginas, 12 figuras.In the present work, we focus on the free volume evaluations from different points of view, including the aspect of probe sizes, temperature, and cavity threshold. The free volume structure is analyzed on structures of poly(vinyl methylether) prepared by fully atomistic molecular dynamics. At first, the temperature behavior of an overall free volume and a free volume separated into individual cavities is shown. The origin of large free volume cavities is explained. A complex view on the cavity number is provided, while a complicated behavior previously observed is now explained. The number of large cavities remained almost constant with the temperature. Oppositely, the number of small cavities related to the atomic packing changes with temperature in a distinct way for glassy and supercooled regions. The cavity number maxima determine a percolation threshold according to percolation theory. The change in polymer properties with temperature can be related to a percolation of the free volume according to the free volume theory, when proper probe radii ∼0.8 Å are used for its observation. A construction of probabilistic distribution of free volume sizes is suggested. The free volume distributions reported here are bimodal. The bimodal character is explained by two different packings—atomic and segmental—forming a prepeak and a main peak on the distribution. Further attention is dedicated to comparisons of the computed free volume sizes and the ortho-positronium (o-Ps) lifetimes. The prepeak of the free volume distribution is probably unseen by o-Ps because of a cavity threshold limit. The effect of the shape factor on the computed o-Ps lifetimes is tested. The quasicavities obtained by redistributing the free volume maintain the ratio of the main dimensions with temperature. Finally, novel data on the cavity environment are provided, while it is suggested how these can be useful with the recent developments in the positron annihilation methods. The coordination number of large cavities with the polymer segments is around 1, as predicted in the free volume theory. Similarly to the percolation and the cavity number, the coordination number exhibits a change when explored by a suitable probe radius ∼0.8 Å. The insightful visualizations showed properties of interest investigated within the actual work.This work was supported by Project No. MAT2007– 63681 (Spanish Ministry of Education) and Grant No. IT- 436–07 (Basque Government). Support from Spanish Ministry of Education Grant No. CSD2006-53 is also acknowledged.Peer reviewe

Random Migration and Signal Integration Promote Rapid and Robust T Cell Recruitment

To fight infections, rare T cells must quickly home to appropriate lymph nodes (LNs), and reliably localize the antigen (Ag) within them. The first challenge calls for rapid trafficking between LNs, whereas the second may require extensive search within each LN. Here we combine simulations and experimental data to investigate which features of random T cell migration within and between LNs allow meeting these two conflicting demands. Our model indicates that integrating signals from multiple random encounters with Ag-presenting cells permits reliable detection of even low-dose Ag, and predicts a kinetic feature of cognate T cell arrest in LNs that we confirm using intravital two-photon data. Furthermore, we obtain the most reliable retention if T cells transit through LNs stochastically, which may explain the long and widely distributed LN dwell times observed in vivo. Finally, we demonstrate that random migration, both between and within LNs, allows recruiting the majority of cognate precursors within a few days for various realistic infection scenarios. Thus, the combination of two-scale stochastic migration and signal integration is an efficient and robust strategy for T cell immune surveillance

Epithelial Membrane Protein 2 Suppresses Non-Small Cell Lung Cancer Cell Growth by Inhibition of MAPK Pathway

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs

MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors

Circadian Clocks in Mouse and Human CD4+ T Cells

Though it has been shown that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression by qPCR in unstimulated CD4+ T cells and immune responses of PMA/ionomycin stimulated CD4+ T cells by FACS analysis purified from blood of healthy subjects at different time points throughout the day. Molecular clock as well as immune function was further analyzed in unstimulated T cells which were cultured in serum-free medium with circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, IL-2, IL-4, IFN-γ production and CD40L expression in freshly isolated CD4+ T cells. Further analysis of IFN-γ and CD40L in cultivated T cells revealed that these parameters remain rhythmic in vitro. Moreover, circadian luciferase reporter activity in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed regulation of the NF-κB pathway as a candidate mechanism mediating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic CD4+ T cell immune responses

Mechanistic insights into p53‐regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells

Late‐stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post‐translational modifications (PTMs). While the relevance of p53 C‐terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild‐type p53 or p53‐negative human CRC cells, cells with acetylation‐defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase‐1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan‐treated p53‐positive CRC cells. This specifically relies on the C‐terminal acetylation of p53 by CREB‐binding protein/p300 and the presence of C‐terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C‐terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53‐proficient CRC