bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-Inflammatory Effect of Blocking the Interaction between Platelet Glycoprotein Ibα and the Leukocyte Integrin αMβ2.
Authors: Han Qiang , Li Zhijie , Peng Yuandong , Wayne C. , Lopez Jose A.
Published: October 2019

The interaction between the leukocyte integrin Mac-1 (α M β 2) and platelet glycoprotein (GP) Ibα allows leukocytes to firmly adhere to platelet thrombi, enabling their subsequent transmigration into the vessel wall. This interaction is blocked by the GP Ibα monoclonal antibody AP1, which we have previously mapped to the GP Ibα sequence Arg218 to Tyr228. In the current investigation we examined the effectiveness of a GP Ibα-based peptide containing the AP1 epitope in blocking platelet-leukocyte interactions under flow and in an inflammation model based on corneal wound healing. The peptide bound the α M I-domain specifically and saturably and was able to inhibit several GP Ibα-dependent functions under flow:

the adhesion of GP Ib-IX complex-expressing CHO cells to immobilized α M I-domain, the adhesion of THP-1 cells - a monocytic cell line expressing α Mβ 2 - to immobilized glycocalicin (soluble extracellular region of GP Ibα ), adhesion of activated THP-1 cells to surface-adherent GP Ib-IX complex-expressing CHO cells, and the binding of neutrophils to immobilized glycocalicin or a platelet monolayer.

We therefore tested whether the peptide could inhibit inflammatory processes that may be dependent on the interactions between platelets and neutrophils. Neutrophils play an important role in corneal wound healing, but because the cornea is avascular, they must migrate to the wound from the limbal vessels at the periphery of the cornea. Therefore we chose this model of mouse corneal wounding as we have previously demonstrated that antibody-induced platelet depletion markedly reduces neutrophil localization and emigration from the limbal vessels and therefore postulated that platelets may directly facilitate neutrophil emigration. In our model, a 2 mm wound was made at the center of the cornea by removing the epithelium without injuring the stroma. Neutrophil infiltration across the cornea, from periphery to center, was assessed at 12 and 30 hr after the injury. Mice were injected with either AP1 peptide or control peptide (scrambled AP1 peptide) three times at 4 hour intervals beginning immediately after corneal wounding. Mice treated with AP1 peptide showed markedly fewer neutrophils infiltrating all regions of the cornea. These results indicate that the interaction between GP Ibα and Mac-1 is required for efficient leukocyte extravasation. They also suggest that drugs targeting the GP Ibα-α Mβ 2 axis could have anti-inflammatory properties without compromising hemostasis, as the peptide had minimal effects on the interaction of GP Ibα with von Willebrand factor.
Sbds Deficient Neutrophils Exhibit Normal Numbers, Chemotaxis and Phagocytic Functions, but Impaired NADPH Oxidase Activity.
Authors: Zhang Siyi , Sun Chunxian , Zhong Jian , Glogauer Michael , Rommens Johanna M.
Published: October 2019

Shwachman-Diamond syndrome (SDS) is a recessive disease with multi-system involvement, and is characterized by hematological dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. Recurrent infections are a major cause of morbidity for SDS patients, due to neutropenia and proposed defects in neutrophil functions. SDS is caused by mutations in SBDS, a highly conserved gene that has been suggested to be involved in RNA metabolism and/or ribosome biogenesis. A conditional Cre-Lox Sbds allele was generated in the mouse using gene targeting technology, as complete ablation of Sbds has been shown to lead to early embryonic lethality. To investigate neutrophil functions and their specific sensitivity to loss of Sbds, we elected to delete the gene by breeding mice with the conditional allele to mice with Cre recombinase under the control of the lysozyme M promoter. While Sbds was confirmed to be ablated in mature neutrophils in adult offspring mice, their numbers and morphology were found to be normal in peripheral blood. Sbds-ablated neutrophils were also found to have normal chemotatic and chemokinetic abilities, and they demonstrated phagocytic function comparable to neutrophils of control mice. In contrast, NADPH oxidase activity was observed to be greatly reduced in response to phorbol-12-myristate 13-acetate stimulation, while the expression levels and phosphorylation of the NADPH oxidase complex components remained intact. To evaluate the net effect of the loss of Sbds in vivo, clearance of Pseudomonas aeruginosa from infected lungs was investigated, and found to be significantly reduced in mice with Sbds-abated neutrophils compared their wild type littermates. In summary, loss of Sbds does not to affect total peripheral neutrophil numbers directly, or chemotaxis and phagocytic abilities, but was essential for a robust NADPH oxidase response. These findings suggest that neutrophils are compromised with loss of Sbds, but that at least some of the neutrophil deficiencies of SDS patients are likely due to microenvironment deficiencies, or early hematopoiesis abnormalities.
The Neutrophil Specific CD177 Is a Novel Counter-Receptor for Endothelial PECAM-1.
Authors: Sachs Ulrich , Andrei-Selmer Cornelia , Maniar Amudhan , Paddock Cathy , Newman Peter , Chavakis Triantafyllos , Santoso Sentot
Published: October 2019

CD177 is a glycosyl-phosphatidylinositol-linked member of the Ly-6 superfamily that is expressed on approximately 50% of human neutrophils, though 3–5% of Caucasians and African Americans are CD177 null. CD177 carries the clinically-important neutrophil antigen, HNA-2a - alloantibodies to which can result in profound neutropenia. Although the role of CD177 in neutrophil function is not known, it has been found to be aberrantly expressed on the neutrophils of most patients suffering from polycythemia vera, and in approximately half of patients with essential thrombocythemia. To further elucidate the function of CD177, we produced a CD177/IgG Fc fusion protein, and used it identify its binding partner on endothelial cells. Flow cytometric analysis revealed that CD177/Fc bound tightly and specifically to human umbilical vein endothelial cells, and an antigen-capture ELISA screen employing a panel of monoclonal antibodies (mAbs) to endothelial cell surface proteins further revealed the target antigen to be the endothelial cell junctional protein, PECAM-1. Surface plasmon resonance analysis showed tight binding between PECAM-1 and CD177, having a Kd of 8.17 x 10−7 M. U937 cells stably transfected with CD177 bound immobilized PECAM-1, and this heterophilic interaction could be blocked by mAbs specific for Ig domains 5 or 6 of the PECAM-1 extracellular domain, or by mAbs specific for CD177. In contrast, no inhibition was observed when mAbs against Ig domains 1 and 2 were applied. The same mAbs were also found to be effective inhibitors of neutrophil transendothelial migration. Taken together, these data establish PECAM-1/CD177 as a previously unrecognized heterophilic receptor/counter-receptor pair that functions in the multi-step adhesion and signaling cascade of events that take place during the inflammatory response.
Diverse Myeloid Lineage Potential Arises in the Yolk Sac of the Mammalian Embryo.
Authors: Palis James , Koniski Anne , Bushnell Timothy P. , McGrath Kathleen E.
Published: October 2019

The emergence of the hematopoietic system in the mammalian embryo is characterized by two temporally overlapping waves of distinct “primitive” and “definitive” erythroid progenitors that peak in the murine yolk sac at E8.5 and E10.5, respectively. We have recently determined that each wave contains megakaryocyte potential (Tober, et al., submitted), but the initial emergence of distinct myeloid lineages is less well understood. Temporal and spatial analysis of hematopoietic progenitors in the murine embryo suggest that the macrophage lineage is associated with both waves, while neutrophil and mast cell lineages are restricted to the second, definitive wave (Palis, et al., Development126:5073, 1999). To better define the emergence of myelopoiesis in the murine embryo, we cultured dissociated E9.5 yolk sac cells in a broad array of cytokines (SCF, IL3, IL5, IL6, IL7, IL9, IL11 and GM-CSF) for seven days and identified morphologically distinguishable macrophages, neutrophils, eosinophils, basophils, and mast cells. The presence of eosinophils was further confirmed by expression of eosinophil peroxidase transcripts. As expected, this multilineage myeloid potential was restricted to c-kit++ cells. However, unlike the bone marrow, these c-kit++ cells in the E9.5 yolk sac were predominately a single population that also express CD41 and Fc gamma receptor (FcγR, CD16/32). FcγR+ cells first emerge in the yolk sac between E8.5 and E9.5 and are not found in the embryo proper until E10.5, when they are restricted to the liver. These findings suggest that the expression of FcγR is restricted in the yolk sac to definitive hematopoiesis and tracks its transition to the fetal liver. While mature definitive erythroid cells first emerge from the fetal liver at E12.5, to our knowledge the onset of granulopoiesis has not been investigated in the early murine embryo. We found approximately 1000 GR1+/Mac1+ cells in the fetal liver at E12.5 and this population expands 100-fold during the next two days of development. These GR1+/Mac1+ cells consisted predominantly of progressively maturing neutrophils. We conclude that the potential to generate multiple myeloid lineages first arises in the yolk sac along with definitive erythroid progenitors and that a robust granulocyte population subsequently emerges in the fetal liver at midgestation.
The Effect of Von Willebrand Factor Glycans on the Interaction with ADAMTS13.
Authors: McKinnon Thomas A.J. , Chion Alain C.K. , Laffan Mike A.
Published: October 2019

Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD 1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD 11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD 3.4nM).

This data demonstrates that the glycan moiety of VWF plays an important role in mediating the interaction with ADAMTS13.
Protein S Down-Regulates Factor VIIIa by Competing with Factor IXa.
Authors: Takeyama Masahiro , Nogami Keiji , Tatsumi Kohei , Fujita Yuri , Tanaka Ichiro , Yoshioka Akira , Shima Midori
Published: October 2019

Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.
Non-Hodgkin Lymphoma B-Cells Induce Intratumoral CD4+CD25− T Cells To Express Foxp3 and Gain Regulatory Function.
Authors: Yang Zhi-Zhang , Novak Anne J. , Witzig Thomas E. , Ansell Stephen M.
Published: October 2019

We have previously shown that CD4+CD25+Foxp3+ regulatory T cells from NHL tumors suppress the function of infiltrating CD4+ T cells and cytolytic CD8+ T cells. Expression of Foxp3 has been demonstrated to be crucial to the development and function of CD4+CD25+ regulatory T cells. However, the mechanistic details that drive development of Foxp3 expression in T cells, in both the normal and malignant scenario, remains to be fully elucidated. Previous studies suggest that Foxp3 expression in CD4+CD25− T cells can be upregulated by tolerizing stimuli such as activation through TCR, corticosteroids, estrogen, and TGF-beta. Because lymphoma B cells have been shown to induce T-cell tolerance, we postulated that lymphoma B cells may play a role in the generation of regulatory T cells by inducing Foxp3 expression in CD4+CD25− T cells.

FoxP3 expression was initially thought to be restricted to CD4+CD25+ regulatory T cell population. However, recent literature suggests that Foxp3 may also be expressed in CD4+CD25− T cells. Using biopsy specimens from patients with B-cell NHL, we found that a subset, 15%, of infiltrating CD4+CD25− T cells express Foxp3 and are capable of suppressing the proliferation and granule production of infiltrating cytotoxic CD8+ T cells. These initial studies suggest that CD4+CD25−Foxp3+ T cells have regulatory function. To explore the underlying mechanism by which Foxp3 expression is regulated, we determined the effect of costimulatory signals on Foxp3 expression in CD4+CD25−Foxp3− T cells. Activation with OKT3/anti-CD28 Ab as well as DC-mediated activation induced Foxp3 expression in a subset of CD4+CD25− T cells. We also found that the presence of lymphoma B cells during activation augmented the induction of Foxp3 expression in CD4+CD25− T cells and that NHL B cell-mediated Foxp3 expression was cell contact-dependent. To better understand the contribution of NHL B cells in Foxp3 expression, we explored the possibility that CD27-CD70 interaction may be involved in Foxp3 expression. Lymphoma B cells express CD70, but not B7-1 and B7-2, which have been shown to be important in protecting tumor cells from lysis and contributing to cancer pathogenesis. Ligation of CD27 by receptor cross-linking enhanced Foxp3 expression in infiltrating CD4+CD25− T cells in B-cell NHL.

Taken together these studies reveal a novel role for NHL B cells in development of regulatory T cells. Our data show that lymphoma B cells induce expression of Foxp3 in infiltrating CD4+CD25− T cells and may result in development of T cells with regulatory function within the tumor microenvironment. Our results also suggest a potential role for CD27-CD70 interactions in this process. The ability of malignant B cells to drive development of regulatory T cells may be one mechanism by which lymphoma B cells protect themselves from anti-tumor immunity. (Supported in part by the Iowa/Mayo Lymphoma SPORE CA97274).
Analysis of Peripheral Blood CD4+/CD25high Treg Cells and FOXP3 mRNA Expression in Patients Treated with Ipilimumab (Monoclonal Human Anti-CTLA4, MDX-010) for Relapse of Malignancy Following Allogeneic Hematopoietic Transplantation.
Authors: Zhou Jiehua , Zhong Rui-kun , Kalcheva Iveta , Medina Bridget , Ball Edward D. , Bashey Asad
Published: October 2019

CTLA-4 is expressed upon activation of T-cells,and serves as an important negative regulator of their effector function. It is also expressed constitutively on CD4+/CD25+ regulatory T cells (Treg), where its function is not clear. Following allogeneic hematopoietic stem cell transplantation (allo-HCT), CTLA-4 function may be involved in suppression of alloreactive T cells that mediate the graft-versus malignancy effect and GVHD. We have studied the administration of a single dose of Ipilimumab (MDX-010), a fully human monoclonal anti-CTLA-4 antibody, in a dose escalation trial in patients with relapse/progression of malignancy following allo-HCT. Here we report effects of ipilimumab on peripheral CD4+/CD25+ cell levels and FOXP3 mRNA expression in these patients. Seventeen patients with a variety of malignancies were enrolled in this study. Ipilimumab was given intravenously at a dose level of 0.1, 0.33, 0.66, 1, and 3mg/kg. The blood samples were obtained prior and after infusion at day 7, 14 and 30. The immunophynotyping of peripheral blood mononuclear cells (PBMC) was analyzed by flow cytometry. CD4+/CD25+ cells from nine patients at day 0, 7, and five normal donors were separated using a Dynal CD4+/CD25+ Treg kit. FOXP3 mRNA expression on CD4+/CD25+ cells were analyzed by a quantitative RT-PCR. Expression level of FOXP3 was normalized to 18S rRNA. Within CD4+ cell population, the percentage of CD4+/CD25high cells was significantly higher in patients at day 0 (11.6±6.7%, n=17), compared with normal donors (3.8±1.6%, n=12; P<0.001). There was no significant change in CD4+/CD25high Treg cells in 17 patients after infusion. However, there was a transient 55±24% decrease of CD4+/CD25high Treg cells in 6 patients at day 7. FOXP3 mRNA expression in CD4+/CD25+ cells was significantly higher in patients at day 0 (2451±1731, n=9) compared with normal donors (918±348, n=5; P<0.001). After ipilimumab infusion, 3/9 patients showed a greater than 50% decrease, 4/9 patients showed no significant change, and 2/9 patients showed a 3–10 fold increase of FOXP3 mRNA expression at day 7. There was no correlation between the dose of ipilimumab and the percentage of CD4+/CD25high cells, or the FOXP3 mRNA expression. We did not observe a correlation of CD4+/CD25high Treg cells and FOXP3 mRNA expression in patients who had a clinical response or immune breakthrough adverse events in response to ipilimumab. These data suggest that in-vivo CTLA-4 blockade does not consistently impact the number of CD4+ CD25high Treg cells and that the clinical effects observed are probably related to the effects of ipilimumab upon activated effector T-cells.
Molecular Dissection of Human Tissue Factor Pre-mRNA Processing: Identification and Characterization of Functional Exonic Splicing Enhancers in Variable Exon 5.
Authors: Tardos Jonathan G. , Bogdanov Vladimir Y.
Published: October 2019

Tissue Factor (TF), the regulatory co-factor of FVII/VIIa, is the physiologic trigger of blood coagulation. Two naturally occurring isoforms of TF differ in their structure: the full length TF (flTF) is a membrane-spanning molecule, whereas the recently discovered alternatively spliced TF (asTF), generated by exclusion of exon 5, lacks the transmembrane domain, and is soluble. Biosynthesis of the soluble TF variant appears to be evolutionarily advantageous, as it exists in mice. Both isoforms are present in plasma and produced predominantly by circulating monocytes. The biologic role of asTF is unknown; it has, however, been proposed that precise regulation of the TF isoform ratio in blood is critical to normal hemostasis. This study aims to identify molecular mechanisms governing the fate of variable exon 5 during the processing of the human TF pre-mRNA, and to apply these findings to generate mini-genes expressing altered human flTF/asTF isoform ratios in animal models to elucidate the role of TF isoforms in normal hemostasis and/or development of coagulopathies. In silico analysis of the human TF exon 5 revealed 14 putative exonic splicing enhancer (ESE) motifs for SF2/ASF and SC35, essential SR proteins involved in pre-mRNA splicing. A segment of the TF gene comprising the end of exon 4 through the beginning of exon 6 (including exon 5 and the entire introns 4 and 5) was inserted into the GFP open reading frame of the pGL expression vector. The resultant construct (pGL-hTF) was transfected into a human monocytic cell line (THP-1) and shown to faithfully recapitulate the pattern of TF mRNA isoform production. Site-directed mutagenesis of each putative ESE motif was then performed, and the mRNA isoform levels were assayed by RT-PCR. Removal of four ASF/SF2 sites at positions 100–117 favored exclusion of exon 5. In contrast, removal of either of the two SC35 motifs at positions 33 and 81 favored inclusion of the exon. A pSPL-3b-hTF expression vector, featuring the above mutations in the context of a different promoter and a smaller segment of the TF gene, was generated to confirm these findings: similar shifts in isoform ratio production were observed. Results were further validated by introducing both synergistic and antagonistic double mutations of the functional ESEs in pGL-hTF. Production of the two naturally occurring human TF isoforms appears to be regulated at the post-transcriptional level by SR proteins ASF/SF2 and SC35. Further elucidation of the molecular mechanisms involved in TF isoform production is very likely to provide effective means for creating state-of-the-art animal models to characterize the biologic importance of flTF/asTF ratio in circulating blood. In addition, this study is the first to show that SF2/ASF and SC35 can exert opposite effects on pre-mRNA splicing.
In Vitro and In Vivo Antioxidant Effect of Fermented Papaya Preparation (FPP) on Blood Cells of Beta-Thalassaemia Patients.
Authors: Fibach Eitan , Amer Johnny , Goldfarb Ada , Rachmilewitz Eliezer
Published: October 2019

In sickle cell anemia (SCD) and thalassemia, although the basic lesions are mutations in the globin genes, the pathophysiology involves oxidative stress-mediated cell damage in the bone marrow (ineffective erythropoiesis due to apoptosis of early erythroid precursors) and in the peripheral blood (chronic hemolysis of mature RBC). In addition, some patients develop thromboembolic complications and recurrent bacterial infections, the etiology of which is related at least in part, to documented oxidative stress in platelets and neutrophils (PMN), respectively. To study the presence and the role of oxidative stress in thalassemia and SCD, we adapted flow cytometry techniques for measuring the generation of Reactive Oxygen Species (ROS), the content of reduced glutathione (GSH), membrane lipid peroxidation and externalization of phosphatidylserine (PS) moieties in RBC, platelets and PMN. Cells derived from the peripheral blood of patients with beta-thalassemia major, intermedia or SCD showed increased oxidative status (increased ROS, lipid peroxidation and PS externalization, and decreased GSH) compared with their normal counterparts. Incubating fresh blood samples from patients with thalassemia major and thalassemia intermedia with 10 mg/ml FPP for 16 hours at 37oC reduced the oxidative status of RBC as well as platelets and PMN. Experiments carried out in normal and thalassemic mice (Th3/+, a mouse model of human beta-thalassemia intermedia demonstrated that mice treated for one week with 10 mg/ml FPP (dissolved in the drinking water) had reduced oxidative stress compared to control mice. The in-vivo effect of FPP was tested on 9 patients with beta-thalassemia (6 - major and 3 - intermedia) treated with 3 gr FPP per os three times a day for 12–15 weeks. Following the treatment, the ROS in RBC, platelets and PMN decreased and the GSH increased in all patients (see table). Six of these patients responded by a modest increase in RBC, reticulocytes and hemoglobin levels. These results suggest that FPP may have an important clinical efficacy as an antioxidant in thalassemia and sickle cell anemia.

The in vivo effect of FPP treatment of beta-thalassemia patients Baseline After treatment n Mean ± SE Mean ± SE P-value* * Paired samples t-test RBC 9 324.07 ± 29.19 209.55 ± 23.65 0.001 ROS Platelets 9 223.73 ± 26.49 109.11 ± 8.71 0.001 PMN 9 222.72 ± 46.42 117.61 ± 8.98 0.045 RBC 9 55.37 ± 5.37 94.88 ± 3.71 0.001 GSH Platelets 9 59.41 ± 4.98 97.55 ± 5.26 <0.0001 PMN 9 58.29 ± 5.35 90.06 ± 5.87 0.005
A Threshold Level of Von Willebrand Factor Is Required for Disease Pathogenesis in a Mouse Model of TTP.
Authors: Motto David G. , Zhu Guojing , Wagner Denisa D. , Ginsburg David
Published: October 2019

Thrombotic Thrombocytopenic Purpura (TTP) is a life threatening systemic illness associated with inherited or acquired deficiency of the ADAMTS13 metalloprotease. ADAMTS13 deficiency results in the appearance of ultra-large multimers of von Willebrand Factor (UL-VWF) in the circulation, which are thought to play a central role in the formation of the platelet-rich thrombi which are the pathological hallmark of this disease. Despite the critical role postulated for VWF in TTP pathogenesis, direct experimental evidence in support of this hypothesis is lacking. Recently we demonstrated that a TTP-like syndrome can be induced in ADAMTS13-deficient mice of a mixed CASA/Rk genetic background (Adamts13CASA−/−) by the bacterial agent shigatoxin. As pure CASA/Rk mice exhibit VWF levels 5 to 10-fold higher than most of the common strains of laboratory mice, we hypothesized that increased plasma VWF levels in the Adamts13CASA−/− mice (which ranged from 200–600% of C57BL/6) were responsible for their increased TTP susceptibility. Surprisingly however, plasma VWF levels did not correlate with the degree of thrombocytopenia or hemolytic anemia induced in Adamts13CASA−/− mice by shigatoxin challenge, with mice inheriting the lowest VWF levels having TTP pathology equal to those inheriting the highest levels. To further examine the requirement for VWF in TTP pathogenesis, we generated ADAMTS13-deficient mice on a susceptible CASA/Rk genetic background that were also either haploinsufficient (Vwf+/−) or completely deficient (Vwf−/−) in VWF. Absolute deficiency of VWF resulted in complete protection from shigatoxin-induced thrombocytopenia. However, thrombocytopenia was consistently observed in Vwf+/− mice similar to that previously reported in Adamts13CASA−/− mice with wild-type VWF. Importantly, again no correlation was observed between VWF level and degree of thrombocytopenia among the ADAMTS13-deficient mice that were Vwf haploinsufficient (Vwf+/−), confirming our previous observation. In summary, we now have demonstrated an absolute requirement for VWF in the pathogenesis of TTP in this mouse model system. Together with the observations that increasing VWF levels do not correlate with increasing TTP pathology, these results suggest that a threshold level of VWF is required for induction of TTP, but that further increases of VWF level do not result in worsening disease. Though these findings remain to be confirmed in humans, our data suggest that the wide variation in plasma VWF levels in the human population will not be a significant determinant of TTP susceptibility or disease severity.
Twice-Daily Deferasirox Improves Cardiac Iron Chelation in the Gerbil Model of Iron Cardiomyopathy.
Authors: Otto-Duessel Maya , Aguilar Michelle I. , Nick Hanspeter , Moats Rex , Wood John C.
Published: October 2019

Introduction: Iron cardiomyopathy remains the leading cause of death in thalassemia major patients because effective chelation of cardiac iron is difficult. Cardiac iron stores respond more slowly to chelation therapy and appear to require a sustained gradient of labile iron species between myocytes and serum. We have previously demonstrated the efficacy of once-daily deferasirox in removing stored cardiac iron in the gerbil but changes in cardiac iron were relatively modest compared with hepatic iron. We postulated that twice daily dosing (BID), by sustaining a longer labile iron gradient from myocytes to serum, would produce better cardiac iron chelation than a comparable once daily dose (QD).

Methods: Twenty four eight-to-ten week old female Mongolian gerbils underwent iron dextran loading for 10 weeks, followed by a 1 week iron equilibration period. Animals were divided into 3 treatment groups of 8 animals apiece and treated with deferasirox 100 mg/kg/day as a single dose (n=8), deferasirox 100 mg/kg/day divided BID (n=8), or sham chelation (n=8) for a total of 12 weeks. Following euthanasia, organs were harvested for quantitative iron determination and tissue histology.

Results: All of the animals tolerated the 10-week loading and 12-week chelation with no adverse events. After the 22-week period, hepatic iron content was 17.7 ± 4.8 mg and 21.1 ± 5.3 mg in the QD and BID groups, compared with 43.1 ± 5.3 mg in the sham-chelated animals (p < 0.0001 sham versus treatment, p=0.2 between groups). Corresponding cardiac iron content was 0.19 ± 0.04 mg, 0.16 ± 0.04 mg and 0.23 ± 0.04 mg respectively. These changes were significant in the BID group (p< 0.006 versus sham). While BID versus QD cardiac iron contents were not statistically different, treatment groups were clearly distinct when viewed as a scattergram of heart and liver iron content (Figure 1). The BID treated animals exhibited lower cardiac iron for any given hepatic iron content. This is best observed by comparing the ratio of cardiac to hepatic iron content (Figure 2, which was 0.78% in the BID group versus 1.11% in the QD treated animals (p=0.0007).

Discussion: In Mongolian gerbils, deferasirox effectively removed liver and cardiac iron with either BID or QD dosing. BID dosing changed the relative cardiac and liver iron chelation profile compared with QD dosing, trading improvements in cardiac iron elimination for a modest decrease in hepatic chelation. Caution should be exercised in extrapolating these results to humans, however clinical studies of BID administration should be considered for patients requiring high chelator dosing or who have high cardiac iron burden.

Figure 1 Figure 1. Figure 2 Figure 2.
A C-Terminal Exosite of von Willebrand Factor Domain A2 Interacts with ADAMTS13 Spacer Domain To Accelerate Substrate Cleavage.
Authors: Gao Weiqiang , Anderson Patricia J. , Majerus Elaine M. , Tuley Elodee A. , Sadler J. Evan
Published: October 2019

ADAMTS13 is a plasma metalloproteinase that limits platelet-rich thrombi by cleaving von Willebrand factor (VWF) at the Tyr1605-Met1606 bond in the A2 domain. A minimal substrate that consists of GST linked to VWF residues Asp1596-Arg1668 with a C-terminal 6×His tag (GST-VWF73) is cleaved rapidly by plasma ADAMTS13. Further removal of Glu1660-Arg1668, which comprises a predicted C-terminal α-helix (GST-VWF64) markedly reduced the rate of cleavage, suggesting this helix comprises an exosite for substrate recognition. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of GST-VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. After removal of the GST moiety by proteolysis, the kinetic constants for cleavage of VWF73 and VWF64 were determined for several recombinant ADAMTS13 variants using a quantitative MALDI-MS assay (Table). Comparison of the specificity constants (kcat/Km) shows that ADAMTS13 truncated after the spacer domain (construct MDTCS) cleaved VWF73 ~20-fold faster than did a similar enzyme without the spacer domain (construct MDTC). In contrast, both MDTCS and MDTC cleaved VWF64 slowly at a rate similar to the cleavage of VWF73 by MDTC. Most of the variation in cleavage rates was explained by differences in Km, suggesting that the spacer domain recognizes an exosite at the C-terminus of VWF73 that is missing from VWF64. Cleavage of VWF73 yields a C-terminal product (cVWF63). Purified cVWF63 (7.5 μM) inhibited MDTCS activity toward VWF73 or VWF64 (1.5 μM) by »90%, but did not inhibit MDTC, suggesting that the exosite of VWF73 or cVWF63 interacts directly with the spacer domain. Moreover, cVWF63 inhibited the cleavage of multimeric VWF by full-length ADAMTS13 and MDTCS up to 80%, but did not inhibit MDTC. The selective effects of deleting the ADAMTS13 spacer domain and Glu1660-Arg1668 of the VWF domain A2 suggest that the C-terminal exosite of the VWF A2 domain accelerates substrate cleavage by binding specifically to the ADAMTS13 spacer domain.

Table Kinetics studies of VWF73 and VWF64 cleavage by ADAMTS13 and its truncations VWF73 VWF64 Enzyme K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) FL-ADAMTS13 1.7±0.4 1.3±0.1 7.5±2.0 37.7±12.8 1.9±0.4 0.5±0.3 MDTCS 0.8±0.2 1.7±0.1 20.5±6.6 5.5±1.4 0.6±0.1 1.0±0.3 MDTC 16.0±4.5 1.8±0.3 1.1±0.5 17.9±6.3 1.6±0.3 0.9±0.5
The Role of Survivin in Angiogenesis during Zebrafish Embryonic Development.
Authors: Leung Anskar Y.H. , Ma Alvin C.H. , Meng Anming , Verfaillie Catherine M. , Liang Raymond
Published: October 2019

We investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5′ untranslated region (UTR) (SurUTR) and sequences flanking the initiation codon (SurATG) of zebrafish survivin-1 gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:GFP) embryos. In embryos co-injected with SurUTR and SurATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of SurUTR and SurATG-MOs was demonstrated by translation inhibition of co-injected 5′UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Hematopoietic cell development at 18 hpf, before the onset of circulation, was unaffected in the survivin MO injected embryos. In conclusion, survivin is involved in angiogenesis during zebrafish development and is under VEGF regulation.
NOTCH1-Induced T-Cell Leukemia in Transgenic Zebrafish.
Authors: Chen Jihua , Jette Cicely , Kanki John P. , Aster Jon , Thomas Look A. , Griffin James D.
Published: October 2019

Activating mutations in the NOTCH1 gene have been found in about 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL). In order to study the molecular mechanisms by which altered Notch signaling induces leukemia, a zebrafish model of NOTCH1-induced T-cell leukemia was generated using TAN-1, the NOTCH1 oncogene generated by the t(7;9)(q34;q34.3) chromosome translocation associated with human T-ALL. Seven of sixteen mosaic fish developed a T cell lymphoproliferative disease at about 5 months. These neoplastic cells extensively invaded tissues throughout the fish and caused an aggressive and lethal leukemia when transplanted into irradiated recipient fish. A stable transgenic fish line was then generated, which also develops leukemia, but with a longer latency for leukemia onset. This longer latency allowed crosses to be done to evaluate potential genetic interactions between NOTCH1 and other T-ALL oncogenes. Interestingly, LMO2 did not cooperate with NOTCH1 to induce T-ALL, while bcl2 had dramatic effects on latency and progression of T-ALL in this zebrafish model. These results suggest that the transforming functions of NOTCH1 and LMO2 may be redundant, while the functions of NOTCH1 and bcl2 are highly complementary. The ability of this model to detect a strong interaction between NOTCH1 and bcl2 suggests that genetic modifier screens have a high likelihood of revealing other genes that can cooperate with NOTCH1 to induce T-ALL.
Relationship between mTOR-Mediated Upregulation of Glycolysis and Chemosensitivity of ALL Blasts.
Authors: Frolova Olga , Buglio Daniela , Samudio Ismael , McQueen Teresa , Kantarjian Hagop , Weiser Mary A. , Andreeff Michael , Konopleva Marina
Published: October 2019

Cancer cells exhibit a high rate of glycolysis, which leads to a stringent dependence on extracellular glucose for maintaining intracellular ATP levels. Leukemic cells infiltrating bone marrow have been shown to be markedly hypoxic compared to normal bone marrow cells, and stimulation of glycolysis is thought to be an adaptive mechanism to hypoxia. The serine/threonine kinase AKT promotes continued cell growth and metabolism by increasing glucose uptake, stimulating glycolysis and ATP production, at least in part via mTOR-dependent stabilization of HIF1-α. The purpose of this study was to investigate the role of mTOR signaling in the regulation of glycolytic activity of leukemic cells growing under high glucose or hypoxic conditions. Culture of Jurkat cells or primary ALL lymphoblasts in hypoxia (1% O2) resulted in robust induction of HIF-1α protein level which was significantly higher in high glucose conditions. This induction was blocked by inhibition of mTOR signaling, in association with decreased HK-2 mRNA and Glut-1 mRNA/protein expression. Likewise, hypoxia induced lactic acid production, a surrogate marker of glycolysis, and this induction was prevented by RAD001 (p=0.01). This data suggest that PI3K/AKT/mTOR pathway critically controls HIF-1α-dependent upregulation of glucose transport and glycolysis under in leukemic cells growing under pro-glycolytic conditions. We next examined the relationship between enhanced glycolytic activity and chemosensitivity to Doxorubicin. Doxorubicin (25ng/ml) efficiently inhibited growth of Jurkat cells under normoxic conditions supplemented with 4 mM glucose (equivalent of 72mg/dl). In contrast, Doxorubicin was approximately 50% less effective when cells were cultured either in high (14mM, equivalent of 252mg/dl) glucose or under hypoxia with low- or high-glucose supplementation. Inhibition of mTOR signaling strikingly enhanced effects of Doxorubicin resulting in complete inhibition of growth, and this effect was apparent only under high glucose conditions. We next examined effects of RAD001 in samples from 11 patients’ samples from newly diagnosed B-ALL patients growing under high/low glucose in hypoxic or normoxic conditions. RAD001 alone did not induce apoptosis in ALL cells (p>0.05). In contrast, inhibition of mTOR signaling significantly enhanced Doxorubicin-induced apoptosis in 8 samples, with highest sensitization observed in high glucose environment (specific apoptosis, Doxorubicin alone, 5.6%, RAD001 7.7%, RAD001+Doxorubicin 22.4%, p<0.02). In conclusion, blockade of mTOR-dependent HIF-1α and its downstream target Glut-1 efficiently promotes chemosensitivity of ALL cells under pro-glycolytic conditions. Hence, mTOR inhibition or blockade of HIF-1α-mediated glycolysis may play an important role in chemosensitization and improved outcomes in ALL.
A Phase II Study of Nilotinib, a Novel Tyrosine Kinase Inhibitor Administered to Imatinib Resistant or Intolerant Patients with Chronic Myelogenous Leukemia (CML) in Blast Crisis (BC) or Relapsed/Refractory Ph+ Acute Lymphoblastic Leukemia (ALL).
Authors: Ottmann Oliver , Kantarjian Hagop , Larson Richard , le Courte Philipp , Baccarani Michele , Rafferty Teresa , Weitzman Aaron , Giles Francis
Published: October 2019

Nilotinib is a highly selective, aminopyrimidine which is 30-fold more potent in vitro than imatinib and active against 32/33 imatinib resistant Bcr-Abl mutations. This open-label study was designed to evaluate the safety and efficacy of Nilotinib administered at a dose of 400 mg bid by hematologic/cytogenetic response (HR/CyR) rates in imatinib resistant or intolerant BC patients or patients with relapsed/refractory Ph+ALL. Preliminary data are presented for 96 BC and 34 Ph+ALL patients. The median age for the Ph+ALL patients was 47 years (range 26 to 75) and for the BC patients 54 years (range 19 to 78). There were 16 males and 18 females with Ph+ALL and 60 males and 36 females with BC. Chromosomal abnormalities other than Ph+ were noted in 12 (35%) and 48 (50%) of BC patients. Extramedullary involvement was present in 3 (9%) Ph+ALL patients and 35 (37%) of BC patients. Treatment is ongoing for 8 (24%) of Ph+ALL patients and 31 (32%) of BC patients. The majority of discontinuations were due to disease progression and occurred in 18 (53%) BC patients and 41 (43%) Ph+ALL. CHR was reported in 12 (13%) BC patients, marrow responses in 6 (6%), return to chronic phase and stable disease in 17 (18%) patients each. Complete responses were reported in 2 (6%) Ph+ALL 1 relapsed/refractory and 1 patient with minimal residual disease). The most frequent Grade 3 or 4 adverse events occurring in patients with Ph+ALL were thrombocytopenia in 3 (9%) patients, and 2 (6%) patients each had neutropenia, blood bilirubin increased, ALT elevation and bone pain. The most frequent Grade 3 or 4 adverse events occurring in patients with BC were thrombocytopenia 23 (24%), neutropenia 17 (18%), and anemia in 9 (9%) of patients. Nilotinib has clinical activity and an acceptable safety and tolerability profile in pts with imatinib resistant or intolerant BC and relapsed/refractory Ph+ ALL patients.
In Vivo Control of Acute Lymphoblastic Leukemia by Immunostimulatory CpG Oligonucleotides.
Authors: Reid Gregor S.D. , Fujii Hisaki , Trudeau Jacqueline , Hall Junior , Fish Jonathan D. , Teachey David T. , Brown Valerie I. , Schultz Kirk R. , Grupp Stephan A.
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While current therapy is effective for the majority of children, relapsed ALL remains the fifth most common pediatric malignancy and responds very poorly to treatment. In addition, about 50% of ALL cases occur in adults, and while remission is achieved in many patients the majority of these will succumb to relapsed disease. In most cases relapse is treated with more intensive chemotherapy or hematopoietic cell transplantation, therapies that are associated with significant toxicity. In both patient populations, therefore, there is an urgent need for novel approaches to inhibit disease progression. Immunostimulatory DNA oligonucleotides containing CpG motifs (CpG ODN) have been shown to induce strong anti-tumor immune activity in a variety of model systems and are currently being evaluated in clinical trials. We have previously reported that CpG ODN stimulation of ALL cells alters their antigen presentation ability and enhances Th1 responses by allogeneic T cells. In this study, using NOD-SCID mice bearing xenografts of primary human ALL or human ALL cell lines, we demonstrate that CpG ODN stimulate significant immune activity against ALL cells in vivo. A single intravenous injection of 300micrograms Class B CpG ODN induced a significant reduction in percentage of human ALL cells in peripheral blood (P<0.0001), spleen (P=0.0036) and liver (P=0.0004), but not bone marrow (P=0.254), compared to PBS treatment of mice. The reduced leukemia burden in the spleens of CpG ODN treated mice correlated with a significantly higher percentage of Annexin-V positive ALL cells (P=0.008 for PBS vs CpG; P=0.016 for CpG vs control ODN), indicating the induction of leukemia cell death in these mice. In addition, repeated administration of CpG ODN mediated continued disease control, and significantly improved survival of mice with established primary sample or cell line derived human ALL (P=0.002). The death of leukemia cells in vivo was independent of the ability of ALL cells to respond directly to CpG ODN, and correlated with the production of significant amounts of interferon-alpha, interferon-gamma and IL-12 in treated mice. Cell depletion studies implicate natural killer cells in the CpG ODN induced killing of human ALL. Preliminary results obtained using the Emu-ret transgenic mouse model of B cell leukemia indicate that CpG ODN treatment also induces killing of syngeneic leukemia cells, and work is ongoing to identify the immune mechanisms mediating this activity. Based on these results, we hypothesize that CpG ODN will have considerable potential as a novel agent for the prevention of ALL progression.