Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASpI294T was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and I healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+)T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation

Effects of a high-dose 24-h infusion of tranexamic acid on death and thromboembolic events in patients with acute gastrointestinal bleeding (HALT-IT): an international randomised, double-blind, placebo-controlled trial

Background: Tranexamic acid reduces surgical bleeding and reduces death due to bleeding in patients with trauma. Meta-analyses of small trials show that tranexamic acid might decrease deaths from gastrointestinal bleeding. We aimed to assess the effects of tranexamic acid in patients with gastrointestinal bleeding. Methods: We did an international, multicentre, randomised, placebo-controlled trial in 164 hospitals in 15 countries. Patients were enrolled if the responsible clinician was uncertain whether to use tranexamic acid, were aged above the minimum age considered an adult in their country (either aged 16 years and older or aged 18 years and older), and had significant (defined as at risk of bleeding to death) upper or lower gastrointestinal bleeding. Patients were randomly assigned by selection of a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients received either a loading dose of 1 g tranexamic acid, which was added to 100 mL infusion bag of 0·9% sodium chloride and infused by slow intravenous injection over 10 min, followed by a maintenance dose of 3 g tranexamic acid added to 1 L of any isotonic intravenous solution and infused at 125 mg/h for 24 h, or placebo (sodium chloride 0·9%). Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was death due to bleeding within 5 days of randomisation; analysis excluded patients who received neither dose of the allocated treatment and those for whom outcome data on death were unavailable. This trial was registered with Current Controlled Trials, ISRCTN11225767, and ClinicalTrials.gov, NCT01658124. Findings: Between July 4, 2013, and June 21, 2019, we randomly allocated 12 009 patients to receive tranexamic acid (5994, 49·9%) or matching placebo (6015, 50·1%), of whom 11 952 (99·5%) received the first dose of the allocated treatment. Death due to bleeding within 5 days of randomisation occurred in 222 (4%) of 5956 patients in the tranexamic acid group and in 226 (4%) of 5981 patients in the placebo group (risk ratio [RR] 0·99, 95% CI 0·82–1·18). Arterial thromboembolic events (myocardial infarction or stroke) were similar in the tranexamic acid group and placebo group (42 [0·7%] of 5952 vs 46 [0·8%] of 5977; 0·92; 0·60 to 1·39). Venous thromboembolic events (deep vein thrombosis or pulmonary embolism) were higher in tranexamic acid group than in the placebo group (48 [0·8%] of 5952 vs 26 [0·4%] of 5977; RR 1·85; 95% CI 1·15 to 2·98). Interpretation: We found that tranexamic acid did not reduce death from gastrointestinal bleeding. On the basis of our results, tranexamic acid should not be used for the treatment of gastrointestinal bleeding outside the context of a randomised trial

Differential regulation of T cell receptor gamma genes in immature thymocyte populations

Immature thymocytes that lack both Lyt-2 (CD8) and L3T4 (CD4) expression can respond rapidly to stimulation with phorbol ester and calcium ionophore by expressing some gene products characteristic of mature, activated T cells. Here we studied the effect of such short-term stimulation on the number of copies per cell of RNA for components of the T cell receptor complex. Although, upon stimulation, mRNAs for T cell receptor β chain accumulated to higher levels, the cells did not rapidly increase their expression of α-chain transcripts from rearranged or germ-line genes. Transcripts from the C_γ1 (Cγ13.4) and Cγ2 (Cγ10.5) genes were differentially regulated. The rarer Cγ1 transcripts were strongly induced, while the initially abundant C_γ2 transcripts showed a modest decrease in transcripts per cell within 24 h. Thus, the ratio of these two transcripts could be shifted dramatically prior to any significant change in the cellular composition of the population. These results suggest regulatory processes that may contribute to the observed expression of γ products in vitro or in normal development

Activation of T cell antigen receptor alpha- and beta-chain genes in the thymus: implications for the lineages of developing cortical thymocytes

Mammalian T lymphocytes mature in the thymus through a series of differentiation events that involve both rapid proliferation and extensive cell death. The mechanisms that govern these processes are currently unknown; however, both mitogenesis and death affect particular subpopulations of cells, suggesting the selective amplification and destruction of specific T cell clones. In mature peripheral T cells, proliferation is most commonly triggered by the recognition of antigen through the T cell antigen receptor complex. If antigen recognition also controls proliferation in the thymus, the differential expression of antigen receptor genes during maturation could play some role in determining the fate of developing T cells. In this study, we examined the expression of the alpha- and beta-chain genes of the T cell antigen receptor in different subpopulations of adult thymocytes. We compared two postmitotic populations--one that appears committed to die and one that appears mature--and several blast cell populations that are enriched for precursors of one or another presumptive lineage. We have found that Lyt-2-, L3T4- precursor thymocytes express much lower levels of both alpha- and beta-chain mRNA than the cells likely to be their immediate descendents. Furthermore, our results show that the cells of the major cortical lineage, which have at least a 95% probability of death, nevertheless express high levels of mature mRNA encoding both the alpha- and the beta-chains of the T cell antigen receptor. These results have important implications for the mechanisms involved in the overproduction and elimination of this major class of T lymphocyte

Differential regulation of T cell receptor gamma genes in immature thymocyte populations

Immature thymocytes that lack both Lyt-2 (CD8) and L3T4 (CD4) expression can respond rapidly to stimulation with phorbol ester and calcium ionophore by expressing some gene products characteristic of mature, activated T cells. Here we studied the effect of such short-term stimulation on the number of copies per cell of RNA for components of the T cell receptor complex. Although, upon stimulation, mRNAs for T cell receptor β chain accumulated to higher levels, the cells did not rapidly increase their expression of α-chain transcripts from rearranged or germ-line genes. Transcripts from the C_γ1 (Cγ13.4) and Cγ2 (Cγ10.5) genes were differentially regulated. The rarer Cγ1 transcripts were strongly induced, while the initially abundant C_γ2 transcripts showed a modest decrease in transcripts per cell within 24 h. Thus, the ratio of these two transcripts could be shifted dramatically prior to any significant change in the cellular composition of the population. These results suggest regulatory processes that may contribute to the observed expression of γ products in vitro or in normal development

Enhancer-deleted retroviral vectors restore high levels of superoxide generation in a mouse model of CGD

BACKGROUND: Retroviral vectors possess many advantages for use in gene therapy protocols, especially within the haematopoietic system. A number of attendant problems, however, still limit their safety in clinical application. The effects of the enhancer present in the retroviral long terminal repeat (LTR) are a major concern for the clinical usage of such vectors, as they can exert a powerful regulatory influence on the genes that surround them. METHODS: To improve the safety and widen the applicability of retroviral vectors for use in gene therapy we have developed an enhancer-deleted (Delta-LTR) retroviral vector that retained high titre and demonstrated transcriptional activity in myeloid cells. RESULTS: When used to correct a mouse model of autosomal recessive chronic granulomatous disease, the Delta-LTR vectors gave acceptable levels of gene transfer to mouse bone marrow cells. Evidence for a slight preferential expression in myeloid cells was obtained with all the vectors studied. Nitroblue tetrazolium assay of superoxide generation in mouse bone marrow derived haematopoietic colonies revealed that transduction with Delta-LTR vectors could restore functional NADPH oxidase to cells from these animals. Superoxide assay of peripheral blood confirmed that, although relatively low numbers of cells were transduced, the Delta-LTR vector was capable of reconstituting very high levels of oxidase activity, comparable to that obtained from normal cells. CONCLUSIONS: The Delta-LTR vector described here could provide the basis for a new generation of retroviral vectors with improved safety