Tamara I. Pestina

Slug, a highly conserved zinc finger transcriptional repressor, protects hematopoietic progenitor cells from radiation-induced apoptosis in vivo

We show here that a zinc finger transcriptional repressor, Slug, which is aberrantly upregulated by the E2A-HLF oncoprotein in pro-B cell acute leukemia, functions as an antiapoptotic factor in normal hematopoietic progenitor cells. Slug(-/-) mice were much more radiosensitive than wild-type mice, dying earlier and showing accentuated decreases in peripheral blood cell counts, as well as abundant microhemorrhages and widely disseminated bacterial microabscesses throughout the body. Slug expression was detected in diverse subsets of hematopoietic progenitors, but not in more differentiated B and T lymphoid cells, and there was a significant increase in apoptotic (TUNEL-positive) bone marrow progenitor cells in irradiated Slug(-/-) mice compared to wild-type controls. These results implicate Slug in a novel survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage.

Statins protect against fulminant pneumococcal infection and cytolysin toxicity in a mouse model of sickle cell disease

Sickle cell disease (SCD) is characterized by intravascular hemolysis and inflammation coupled to a 400-fold greater incidence of invasive pneumococcal infection resulting in fulminant, lethal pneumococcal sepsis. Mechanistically, invasive infection is facilitated by a proinflammatory state that enhances receptor-mediated endocytosis of pneumococci into epithelial and endothelial cells. As statins reduce chronic inflammation, in addition to their serum cholesterol-lowering effects, we hypothesized that statin therapy might improve the outcome of pneumococcal infection in SCD. In this study, we tested this hypothesis in an experimental SCD mouse model and found that statin therapy prolonged survival following pneumococcal challenge. The protective effect resulted in part from decreased platelet-activating factor receptor expression on endothelia and epithelia, which led to reduced bacterial invasion. An additional protective effect resulted from inhibition of host cell lysis by pneumococcal cholesterol-dependent cytotoxins (CDCs), including pneumolysin. We conclude therefore that statins may be of prophylactic benefit against invasive pneumococcal disease in patients with SCD and, more broadly, in settings of bacterial pathogenesis driven by receptor-mediated endocytosis and the CDC class of toxins produced by Gram-positive invasive bacteria.

The roles of ADP and TXA2 in botrocetin/VWF‐induced aggregation of washed platelets

Summary.  Background: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib‐IX‐V complex initiates a cascade of events leading to αIIbβ3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination‐induced GPIbα‐mediated platelet activation have not been fully described. Methods: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Gαq, or αIIbβ3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, αIIbβ3 and Ca2+ in botrocetin/VWF‐induced signaling. Results: Our data demonstrate that botrocetin/VWF/GPIbα‐mediated agglutination results in calcium‐independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbα‐elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2‐induced αIIbβ3 activation and ADP signaling. TXA2 or ADP can activate αIIbβ3, but both are required for α‐granule secretion and aggregation. Botrocetin/VWF‐induced dense granule secretion is Gαq‐dependent. α‐Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an αIIbβ3‐dependent manner. Conclusions: In contrast to adhesion or shear stress‐induced GPIb‐elicited signaling, agglutination‐elicited GPIb signaling that activates αIIbβ3 requires TXA2. Agglutination‐elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination‐elicited GPIb signaling causes αIIbβ3 activation by a mechanism that is distinct from those used by adhesion, or shear stress‐induced GPIb signaling.

Identification of the Src Family Kinases, Lck and Fgr in Platelets Their Tyrosine Phosphorylation Status and Subcellular Distribution Compared With Other Src Family Members

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.

Amelioration of murine β-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both γ-globin and the MGMT drug-resistance gene

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo.

ABSTRACT The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Δ60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Δ60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.

Hydroxyurea therapy requires HbF induction for clinical benefit in a sickle cell mouse model

Hydroxyurea has proven clinical efficacy in patients with sickle cell disease. Potential mechanisms for the beneficial effects include fetal hemoglobin induction and the reduction of cell adhesive properties, inflammation and hypercoagulability. Using a murine model of sickle cell disease in which fetal hemoglobin induction does not occur, we evaluated whether hydroxyurea administration would still yield improvements in hematologic parameters and reduce end-organ damage. Animals given a maximally tolerated dose of hydroxyurea that resulted in significant reductions in the neutrophil and platelet counts showed no improvement in hemolytic anemia and end-organ damage compared to control mice. In contrast, animals having high levels of fetal hemoglobin due to gene transfer with a γ-globin lentiviral vector showed correction of anemia and organ damage. These data suggest that induction of fetal hemoglobin by hydroxyurea is an essential mechanism for its clinical benefits.

Genomic Analyses of Pneumococci from Children with Sickle Cell Disease Expose Host-Specific Bacterial Adaptations and Deficits in Current Interventions

Sickle cell disease (SCD) patients are at high risk of contracting pneumococcal infection. To address this risk, they receive pneumococcal vaccines, and antibiotic prophylaxis and treatment. To assess the impact of SCD and these interventions on pneumococcal genetic architecture, we examined the genomes of more than 300 pneumococcal isolates from SCD patients over 20 years. Modern SCD strains retained invasive capacity but shifted away from the serotypes used in vaccines. These strains had specific genetic changes related to antibiotic resistance, capsule biosynthesis, metabolism, and metal transport. A murine SCD model coupled with Tn-seq mutagenesis identified 60 noncapsular pneumococcal genes under differential selective pressure in SCD, which correlated with aspects of SCD pathophysiology. Further, virulence determinants in the SCD context were distinct from the general population, and protective capacity of potential antigens was lost over time in SCD. This highlights the importance of understanding bacterial pathogenesis in the context of high-risk individuals.

Hypersusceptibility to invasive pneumococcal infection in experimental sickle cell disease involves platelet-activating factor receptor

Children with sickle cell disease have a 600-fold increased incidence of invasive pneumococcal disease. Platelet-activating factor receptor (PAFr) mediates pneumococcal invasion, and up-regulation of PAFr on chronically activated endothelia could contribute to increased bacterial invasion. Mice transplanted with sickle cell bone marrow developed more extensive infection, and 57% died, compared with 16% of wild-type mice. Histopathological analysis revealed that sickle cell mice expressed significantly more PAFr on endothelia and epithelia. Pharmacological blockade or genetic deletion of PAFr protected sickle cell mice from mortality. We conclude that PAFr plays an important role in hypersusceptibility to pneumococcal infection in sickle cell disease.

AlphaIIbbeta3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause alpha-granule secretion by platelets.

Summary.  The peptide LSARLAF (LSA) causes αIIbβ3‐dependent platelet activation that results in α‐granule secretion and aggregation. LSARLAF‐induced, αIIbβ3‐mediated outside‐in signaling causing α‐granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Gαq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA‐induced αIIbβ3‐mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause α‐granule release by mediating their effects though Gαq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, αIIbβ3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Gαq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Gαq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA‐induced αIIbβ3‐mediated signaling characterized in this study is α‐granule secretion, which provides the fibrinogen required for aggregation of washed platelets.