1,721,101 research outputs found
Targeted integration of a large transgene cassette by TALEN and ZFN-mediated homologous recombination.
No full textTargeted transgene integration by homologous recombination (HR) represents a promising strategy for gene therapy as it may overcome the issue of insertional mutagenesis associated with retroviral vectors. We recently published the feasibility of using adenoviral vectors (Ad) to package and deliver functional TALEN genes into human cells, demonstrating that Ad-TALEN-mediated transduction results in efficient site-specific DSB formation at the chromosomal safe harbor site AAVS1. Moreover, we demonstrated efficient targeting at AAVS1 in human repopulating epidermal stem cells upon Ad-ZFN cleavage
Inhibition of the mouse double minute 2 protein promotes antileukemia immunity in human acute myeloid leukemia cells
No full textModeling MyD88 deficiency
No full textToll-like receptors (TLRs) are important pathogen recognition receptors, allowing the immune system to recognize and respond to conserved pathogen components. Myeloid differentiation primary response protein 88 (MyD88), is a TLR downstream adaptor protein that is essential for proper functioning of the immune system. MyD88-deficiency is an autosomal recessive disease displaying defective TLR- signaling resulting in high mortality (~50%) due to invasive bacterial disease; disease-causing pathogens are restricted to a small range of bacteria with Streptococcus pneumoniae and Staphylococcus aureus accounting for roughly 2/3 of all serious infections. The phenotype of MyD88-deficiency in mice is surprisingly different: mice are markedly more susceptible to a wider range of pathogens including viruses such as Cytomegalovirus and Herpes simplex virus-1; bacteria such as Listeria monocytogenese and Mycobacteria; parasites such as Toxoplasma gondii; as well as fungal species such as Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus. These significant differences between mouse and human are not well understood and suggest additional redundancies in the innate immune system of humans that are not found in mice. Interrogation of the innate immune system has lagged that of the adaptive immune system for several important reasons including lack of clonality and dramatically increased number of proteins of interest (large number of unique pattern recognition receptors compared to a single T- or B-cell-receptor). These significant differences between humans and mice indicate the need for a disease model in humans. An appropriate disease model requires the use of specialized human immune cells, in this case macrophages, which can be precisely genetically modified. Few existing models of the innate immune system use human primary cell macrophages that are easily genetically modifiable.Such a disease model to further elucidate the role of the central TLR adapter protein, MyD88, was established: fibroblasts were removed from a child with MyD88-deficiency and reprogrammed into induced pluripotent stem cells (iPS cells). A transgenic copy of MyD88 was stably integrated into the genome of the MyD88-deficient iPS cells using the Sleeping Beauty transposon system, creating several iPS cell lines. There are two important splice variants of MyD88—a long version hereafter referred to as MyD88-L, and a short version hereafter referred to as MyD88-S. Signaling through MyD88-L results in NF-kB activation with resulting inflammation, whereas MyD88-S does not. Therefore, iPS cell lines containing MyD88-L, MyD88-S, MyD88E53del (uncorrected), as well as cells derived from a healthy donor, were created. These iPS cells were then differentiated into macrophages, hereafter referred to as iMacs (iPS-derived macrophages), for further analysis. No differences were seen between clones morphologically and by cell marker analysis as well as by several functional tests including phagocytosis and stimulation of reactive oxygen species. Upon stimulation with heat-inactivated Staphylococcus aureus dramatic differences between the clones emerged: the MyD88-deficient macrophages had a significantly reduced cytokine response; the corrected macrophages, however, produced a very similar profile compared to macrophages from a healthy donor. The model shows that a principal problem in MyD88-deficiency is in cytokine signaling. Further, having created this model showed additional insights: iPS cells can be genetically modified using the Sleeping Beauty transposon system. Gene transfer was titrated to allow for only 2-3 integration events, which when combined with the EF1α (elongation factor 1 alpha) promoter results in physiological transcription levels. The modified iPS cells could be successfully differentiated into iMacs without silencing of the transgene. This shows both the success of the genetic alteration as well as well as the ability to use the system to answer biological questions: no obvious defects in differentiation in cells with a genetic lesion in MyD88 were seen. Surprisingly, MyD88-deficient iMacs produced more IL-8 compared to other cell lines.While the scientific possibilities of this model are fascinating, the long-term goal is to eventually use gene therapy in the clinic. This thesis shows that it is technically feasible to remove fibroblasts from a patient, reprogram them into iPS cells, repair the genetic lesion, and differentiate the corrected iPS cells into biologically relevant effector cells. In summary, a disease model of MyD88-deficiency led to interesting biological surprises as well as laying the groundwork for bringing this technology into the clinic
Natural history of Pearson syndrome
No full textPS ist eine sehr seltene mitochondriale Erkrankung, die durch SLSMD verursacht wird und durch eine hyporegenerative Anämie im Säuglingsalter gekennzeichnet ist. Ziel dieser Studie war es, die hämatologischen und klinischen Merkmale sowie den natürlichen Verlauf von 25 Patienten mit PS zu beschreiben. Bei den meisten Patienten war Anämie initial das einzige Symptom, das sich im Median im Alter von 5 Monaten entwickelte. Dieses Ergebnis legt nahe, dass bei allen Säuglingen mit hyporegenerativer Anämie aus unbekannter Ursachen, an PS gedacht werden sollte, auch wenn initial keine klinischen Merkmale vorliegen, die auf eine PMD hindeuten. Die typischen Befunde im Knochenmarksausstrich, Vakuolen in erythroiden und myeloischen Vorstufen und der Nachweis von Ringsideroblasten, waren bei den meisten Patienten für die Diagnose PS wegweisend. Die Diagnose wurde außerdem durch den Nachweis von SLSMD bei allen Patienten bestätigt. Interessanterweise wurde eine spontane hämatologische Genesung bei 12 Patienten in einem mittleren Alter von 47 Monaten beschrieben (kumulative Inzidenz: 66 %). Wie erwartet traten im Verlauf der Erkrankung zahlreiche Komplikationen auf, wie Gedeihstörung (n=19), exokrine Pankreasinsuffizienz (n=14), Muskelhypotonie (n=13), Nierentubulopathie (n=8), endokrine Dysfunktion (n=5), kardiale Dysfunktionen (n=5) und Leberdysfunktion (n=5). Die Prognose der Patienten war schlecht, und kein Patient aus der Kohorte hat bis ins Erwachsenenalter überlebt. Die Wahrscheinlichkeit des Gesamtüberlebens lag bei 59 % im Alter von fünf Jahren. Die Hauptursache für die Erkrankung war eine metabolische Azidose. Diese Studie legt nahe, dass ein großer Bedarf an der Entwicklung neuer Therapien besteht, um die Prognose von Patienten mit PS zu verbessern. Einen sehr guten Ansatz könnte hierbei die Gentherapie liefern
Regulation of human early B lymphopoiesis
No full textB lymphocyte development and function are fundamental for the adaptive immune system. B cells act as antigen presenting cells, produce antibodies, induce antibody-dependent cytotoxicity, and secrete cytokines. Disturbance of early B cell development in the bone marrow is associated for instance with increased risk of infections, immune dysregulation, and autoimmunity. Therefore, understanding of the B cell developmental process is crucial for discovery and establishment of better therapies, but also for management of therapy-related adverse effects. Many studies are carried out in mice models, but the knowledge can rarely be directly translated into humans. In this work, we aimed to shed more light on early B lymphopoiesis in humans. We employed high-dimensional flow cytometry and other state-of-the-art technologies (1) to analyze human bone marrow-derived B cell progenitors and (2) to monitor human early B cell development from hematopoietic stem cells using a feeder-free in vitro system.JAK-STAT inhibition is an established therapy regime in autoimmune diseases to dampen JAK-dependent cytokine signaling and inflammation. Early human B cell development is strongly dependent on various cytokines. Therefore, we studied the effect of JAK inhibitor tofacitinib on early B cell development and observed that tofacitinib accelerates and increases early B cell development in vitro. Our findings suggest that JAK inhibition therapy may have unexpected adverse effects in the context of B cell development.Ikaros and Aiolos are important transcription factors during B cell development. Systemic lupus erythematosus and multiple myeloma patients show increased expression of both proteins and are treated with immunomodulatory drugs. Iberdomide is a novel immunomodulatory drug leading to enhanced proteasomal degradation of Ikaros and Aiolos. We investigated iberdomide’s effect on in vitro early B lymphopoiesis and observed that it strongly affects specification and commitment of CD34+ hematopoietic stem cells into early B cell progenitors. However, treatment of already committed progenitors did not affect their development into immature B cells. Our data suggest that the iberdomide treatment interferes with development of distinct early B cell progenitors.Human B cell development has been thought to be largely independent of IL-7 receptor signaling because severe combined immunodeficiency patients with germline mutations in the IL-7Rα chain have relatively normal peripheral blood B cell counts. Analysis of bone marrow-derived early B cell progenitors from two IL-7Rα deficient patients and in vitro development of early B cells in absence of IL-7 allowed us to unravel the role of IL-7 in humans. IL-7 signaling enhances expression of transcription factors that are critical for the function and identity of B cell precursors and induces proliferation of early B progenitors
Engineering an antibody-discernible and functional CD45 variant on hematopoietic stem and progenitor cells via base editing
Full text linkHematopoietic stem cell transplantation (HCT) stands as the unique curative recourse for various hematological malignancies. However, the current standard-of-care relies on untargeted toxic conditioning regimens to deplete malignant cells and remove cells from host’s bone niches. These untargeted regimens limit HCT’s widespread use due to severe off-target toxicity effects. To counteract this roadblock, antigen-specific cell-depleting agents have emerged as promising targeted alternatives. Pairing antigen-specific conditioning regimens with transplantation of depleter-shielded HSPCs has shown promise in the treatment of malignancies characterized by the overexpression of specific cell-surface markers (e.g. CD123 or CD33). Indeed, by rendering transplanted HSPCs resistant to antigen-specific depleters, we can replenish the depleted cell subsets (e.g CD123+ or CD33+ cells) without risking to eradicate them if the need for retreatment arises due to minimal residual disease (MRD) leading to relapse. Nevertheless, focusing solely on CD123 or CD33 limits this strategy to specific cell subtypes. It would therefore be desirable to develop a similar system for a cell surface marker present on all hematopoietic cells, such as CD45. Engineering a CD45 variant shielded from antigen-specific depleters would enable protection of the entire blood system post-HCT while still permitting eradication of all CD45+ cells (e.g. host’s blood cells and hematological cancer cells). In this study, we screened CD45’s extra-cellular domain regions with base editors and generated multiple CD45 protein variants altering the binding of antibodies. We selected the CD45-K352E/G variant profile and improved its base editing rate as it showcased loss of binding of a unique anti-CD45 antibody while still maintaining the surface marker’s expression, stability, and function in human hematopoietic stem and progenitor cells (HSPCs). The resulting loss of antibody binding prompted the modification and humanization of the said anti-CD45 antibody, culminating in the development of an anti-CD45 antibody- drug conjugate (CD45-ADC; CIM053-SG3376). Transplantation of the base edited HSPCs into immunodeficient mice showcased their long-term engraftment and multi-lineage reconstitution ability in sequential host mice. Importantly, in AML mouse models xenografted with HSPCs, administration of the CD45-ADC selectively depleted human leukemia and HSPCs-wt cells while preserving the healthy hematopoietic system derived from the transplanted base edited HSPCs. This approach of generating de novo antigens through gene editing to evade targeted-killing represents a robust strategy for creating cell-specific antigens, address the current limitations of HCT but also suggests broader implications beyond hematological malignancies, offering a promising avenue for future therapeutic development
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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