Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

<p>Abstract</p> <p>Background</p> <p>Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of <it>in vivo </it>expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells.</p> <p>Results</p> <p>We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus.</p> <p>Conclusions</p> <p>These findings indicate that the p53-responsive pCLPG retrovirus did offer expression <it>in vivo </it>and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.</p

Targeting the EIF2AK1 Signaling Pathway Rescues Red Blood Cell Production in SF3B1 Mutant Myelodysplastic Syndromes With Ringed Sideroblasts

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PDE4 inhibition enhances hippocampal synaptic plasticity in vivo and rescues MK801-induced impairment of long-term potentiation and object recognition memory in an animal model of psychosis

Inhibition of phosphodiesterase type 4 (PDE4) by rolipram (4-(3-(cyclopentyloxy)-4-methoxyphenyl)-pyrrolidin-2-one) has been the focus of many behavioral and molecular studies in the recent years. Rolipram exhibits memory-enhancing effects in rodents. In vitro studies have shown that long-term potentiation (LTP), which may comprise a cellular substrate for learning, is also enhanced by rolipram. However, effects have not been assessed in vivo. Rolipram has antipsychotic properties. Psychosis affects cognition and in animal models of psychosis LTP is impaired. In this study, we investigated if PDE4 inhibition improves LTP in healthy animals in vivo and if PDE4 inhibition rescues impaired LTP and prevents object recognition memory deficits in an animal model of psychosis. Recordings were made from the hippocampus of adult, freely behaving Wistar rats. Thirty minutes after treatment with rolipram or vehicle, a tetanus was applied to the medial perforant path to elicit short-term potentiation (STP) in the dentate gyrus. At this time-point, radioimmunoassay revealed that rolipram significantly elevated cyclic adenosine monophosphate levels in the dorsal hippocampus, in line with reports by others that rolipram mediates decreased PDE4 activity. In healthy animals, both intracerebroventricular and subcutaneous treatment with rolipram facilitated STP into LTP, suggesting that PDE4 inhibition may have a permissive role in plasticity mechanisms that are relevant for learning and memory. One week after a single systemic treatment with the irreversible N-methyl--aspartate antagonist, MK801, LTP and object recognition memory were significantly impaired, but could be rescued by PDE4 inhibition. These data suggest that the relief of cognitive disturbances in psychosis models by rolipram may be mediated in part by a rescue of hippocampal LTP

Gene Expression during the Generation and Activation of Mouse Neutrophils: Implication of Novel Functional and Regulatory Pathways

As part of the Immunological Genome Project (ImmGen), gene expression was determined in unstimulated (circulating) mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes), thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1β, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the ImmGen regulatory model provide a basis for additional hypothesis-based research on the importance of changes in gene expression in neutrophils in different conditions

Selective phosphodiesterase inhibitors: a promising target for cognition enhancement

# The Author(s) 2008. This article is published with open access at Springerlink.com Rationale One of the major complaints most people face during aging is an impairment in cognitive functioning. This has a negative impact on the quality of daily life and is even more prominent in patients suffering from neurodegenerative and psychiatric disorders including Alzheimer’s disease, schizophrenia, and depression. So far, the majority of cognition enhancers are generally targeting one particular neurotransmitter system. However, recently phosphodiesterases (PDEs) have gained increased attention as a potential new target for cognition enhancement. Inhibition of PDEs increases the intracellular availability of the second messengers cGMP and/or cAMP. Objective The aim of this review was to provide an overvie

Lineage tracing of Pf4-Cre marks hematopoietic stem cells and their progeny

The development of a megakaryocyte lineage specific Cre deleter, using the Pf4 (CXCL4) promoter (Pf4-Cre), was a significant step forward in the specific analysis of platelet and megakaryocyte cell biology. However, in the present study we have employed a sensitive reporter-based approach to demonstrate that Pf4-Cre also recombines in a significant proportion of both fetal liver and bone marrow hematopoietic stem cells (HSCs), including the most primitive fraction containing the long-term repopulating HSCs. Consequently, we demonstrate that Pf4-Cre activity is not megakaryocyte lineage-specific but extends to other myeloid and lymphoid lineages at significant levels between 15-60%. Finally, we show for the first time that Pf4 transcripts are present in adult HSCs and primitive hematopoietic progenitor cells. These results have fundamental implications for the use of the Pf4-Cre mouse model and for our understanding of a possible role for Pf4 in the development of the hematopoietic lineage

Large-scale exome-wide association analysis identifies loci for White Blood Cell Traits and Pleiotropy with Immune-Mediated Diseases

White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3 UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases