A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

Background: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. Results: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. Conclusion: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed nanogold-labeled molecules

We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand–receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically fixed samples that reduced auto-nucleation, and a new pre-embedding gold enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies

The Alliance for Cellular Signaling Plasmid Collection: A Flexible Resource for Protein Localization Studies and Signaling Pathway Analysis

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines

Lentiviral Vector Delivery of Human Interleukin-7 (hIL-7) to Human Immune System (HIS) Mice Expands T Lymphocyte Populations

Genetically modified mice carrying engrafted human tissues provide useful models to study human cell biology in physiologically relevant contexts. However, there remain several obstacles limiting the compatibility of human cells within their mouse hosts. Among these is inadequate cross-reactvitiy between certain mouse cytokines and human cellular receptors, depriving the graft of important survival and growth signals. To circumvent this problem, we utilized a lentivirus-based delivery system to express physiologically relevant levels of human interleukin-7 (hIL-7) in Rag2-/-γc-/- mice following a single intravenous injection. hIL-7 promoted homeostatic proliferation of both adoptively transferred and endogenously generated T-cells in Rag2-/-γc-/- Human Immune System (HIS) mice. Interestingly, we found that hIL-7 increased T lymphocyte numbers in the spleens of HIV infected HIS mice without affecting viral load. Taken together, our study unveils a versatile approach to deliver human cytokines to HIS mice, to both improve engraftment and determine the impact of cytokines on human diseases

PD-1 Blockade Expands Intratumoral Memory T Cells

Tumor responses to PD-1 blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, an antibody to PD-1. Biopsies were dissociated and single cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells (moMDSCs) significantly increased in patients’ biopsies taken on treatment. The percentage of cells with a T regulatory phenotype, monocytes, and NK cells did not change while on PD-1 blockade therapy. CD8(+) T memory cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4(+) T effector memory cells significantly decreased on treatment, whereas CD4(+) T effector cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 were detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and MDSCs in tumors, with the CD8(+) T effector memory subset being the major T-cell phenotype expanded in patients with a response to therapy

Classification of current anticancer immunotherapies

© 2014. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and "active" based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.info:eu-repo/semantics/publishedVersio

Law and Grace: a legal and ethical examination

This doctoral dissertation studies the canons and commandments of the Church in relation to the ethical perfection of the faithful. It especially focuses on the presuppositions for the implementation and success of their foundational goal, which is the salvation of mankind and theosis by grace. It emphasizes the cause that led to the failure of the Law of the Old Testament. It was not the nature of the Law and the commandments which resulted from it, but the psychosomatic weakness of human beings to adhere to it. In this way, the good law which was given by God was transformed into a curse. The research particularly emphasizes that the aim of the ethical commandments of the Old and New Testament are the same, that is, the restraint of the faithful from any ethical deviation. Nevertheless, this aim proved that it can be carried out only with the aid of the sanctifying Grace of God, which strengthens a person in his/her spiritual struggle and when his/her psychosomatic strength is insufficient. Also, the therapeutic character of the sacred mysteries is examined. The application of the canons, either with exactness or economy, in the life of the Church and their validity in relation to the pastoral needs of the era in which they were introduced is discussed. The development of the theme in the first two chapters (1. “The Law in the Old Testament and 2. “Jesus: the Fulfillment of the Law) is based in the patristic hermeneutical tradition of biblical passages and in the related more recent bibliography. The third chapter (“A Church of Grace, its Laws and the Spirit of its Canons”) is based in the canonical tradition of the Church up to the 9th century, the interpreters of the canons and contemporary canon law research, while the fourth (“The Divine Grace Offered through the Holy Mysteries and the Canonical and Ethical Presuppositions of Participation of the Faithful”) is supported by the patristic tradition, the canonical tradition of the church and modern studies concerning Christian Ethics. The methodology of the research showed us the indissoluble bond between Canon Law and Ethics, as well as the salvific role of the Grace of God in the mystery of the Church, where salvation is offered freely and human weakness is no longer a barrier for ethical perfection.Η διδακτορική εργασία εξετάζει τους κανόνες και τις διατάξεις της Εκκλησίας σε σχέση με την ηθική τελείωση των πιστών. Ειδικότερα ερευνά τις προϋποθέσεις της εφαρμογής τους και την επιτυχία του θεμελιώδους στόχου τους, που είναι η σωτηρία των ανθρώπων και η κατά χάριν θέωση. Τονίζεται ότι το αίτιο που οδήγησε στην αποτυχία του Νόμου της Π. Διαθήκης, δεν ήταν η φύση τους, αλλά η ψυχοσωματική αδυναμία των ανθρώπων, για να τους τηρήσουν. Με αυτόν τον τρόπο ο αγαθός νόμος που δόθηκε από το Θεό μεταβλήθηκε σε κατάρα. Ειδικότερα τονίζεται ότι ο σκοπός των ηθικών εντολών της Παλαιάς και της Καινής Διαθήκης είναι ο ίδιος, δηλαδή η συγκράτηση των πιστών από οποιαδήποτε ηθική παρεκτροπή. Ωστόσο ο σκοπός αυτός απεδείχθη ότι μπορεί να επιτελεσθεί μόνον με την ενίσχυση της εξαγιαστικής Χάριτος του Θεού, η οποία ενισχύει τον πνευματικό αγώνα του ανθρώπου, του οποίου οι ψυχοσωματικές τους δυνάμεις είναι ανεπαρκείς. Επίσης εξετάζεται ο θεραπευτικός χαρακτήρας των ιερών μυστηρίων, συζητείται η κατ᾿ ακρίβειαν ή κατ᾿ οικονομίαν εφαρμογή των κανόνων στη ζωή της Εκκλησίας, όπως επίσης και η ισχύς τους σε συνάρτηση με τις ανάγκες της εποχής στην οποία θεσπίστηκαν. Η διαπραγμάτευση του θέματος στα δύο πρώτα κεφάλαια της εργασίας (Α’ «Ο Νόμος στην Παλαιά Διαθήκη» και Β’ «Ο Ιησούς το πλήρωμα του Νόμου») βασίστηκε στην πατερική ερμηνευτική παράδοση των βιβλικών κειμένων και στη σχετική νεώτερη βιβλιογραφία. Το τρίτο («Εκκλησία της Χάριτος, η νομοθεσία της και το πνεύμα των κανόνων της») εδράζεται στην κανονική παράδοση της Εκκλησίας μέχρι τον 9ο αιώνα, στους ερμηνευτές των κανόνων και στη σύγχρονη νομοκανονική έρευνα, ενώ το τέταρτο («Η δια των ιερών μυστηρίων προσφερόμενη θεία Χάρις και οι κανονικές και οι ηθικές προϋποθέσεις μετοχής των πιστών») στηρίχθηκε στη Πατερική Παράδοση, στη νομοκανονική παράδοση της Εκκλησίας και στις νεώτερες μελέτες γύρω από τη χριστιανική ηθική. Η μεθοδολογία της έρευνάς μας ανέδειξε τον άρρηκτο δεσμό Κανονικού Δικαίου και Ηθικής, όπως επίσης και τον σωτηριώδη ρόλο της χάριτος του Θεού μέσα στο μυστήριο της Εκκλησίας, όπου η σωτηρία προσφέρεται δωρεάν και οι ανθρώπινες αδυναμίες δεν αποτελούν πλέον εμπόδιο για την ηθική τελείωση