bluuebunny/crossref_metadata_2025 huggingface dataset:
Management of Fetal Alloimmune Thrombocytopenia (AIT): Treatment of 30 Cases in Which the Previous Sibling Suffered an IntraCranial Hemorrhage (ICH).
Authors: Hung Crystal , Wissert Megan , Primiani Andrea , Berkowitz Richard , McFarland Janice G. , Bussel James B.
Published: April 2020

Prevention of fetal-perinatal ICH is the primary goal of antenatal therapy for AIT. When there was an ICH in a prior pregnancy, these fetuses are at virtually 100% risk for a recurrent ICH. Current antenatal treatments given to the mother are intravenous gammaglobulin (IVIG), oral steroids, or both. Two consecutive studies examined risk-based therapy by tailoring therapeutic interventions according to the course of the previous affected sibling with ICH.

Thirty patients with AIT were divided into 4 groups based on the timing of the sibling’s ICH (see Figs. 1 & 2, including abbreviations). In the Extremely High Risk (EHR), Very HR (VHR), HR1, and HR2 arms, an adequate initial response was defined as an Fetal Platelet Count (FPC) ≥30 × 109/L at the time of the first Fetal Blood Sampling (FBS). Repeat FBS was done 3–8 weeks later. If the platelet count did not increase, therapy was intensified (“salvage”) by adding prednisone or additiional IVIG. There were 5 ICHs (see Table), 3 of which were failures of antenatal therapy. One ICH occurred in an EHR patient prior to the use of 2g/kg/wk of IVIG as did one of the 2 ICHs in the HR1 study; both of these hemorrhages occurred on IVIG + pred prior to 30 weeks. Both previous siblings had had early antenatal ICHs. The second HR1 patient with ICH was initially on IVIG alone, added pred when repeat FBS was not possible because of premature contractions, and delivered at 35 weeks with a birth platelet count (BPC) of 14 × 109/L; a Grade I intraventricular ICH was noted on the first day of life. The other 2 ICHs occurred in a VHR infant delivered at 24 weeks gestation with a BPC of 248 × 109/L and in a HR2 35.7 week old infant with a BPC of 137 × 109/L. The effectiveness of the treatment strategy was demonstrated because all groups’ mean BPC was >50 × 109/L; 3 of 4 groups’ mean BPC was >100 × 109/L.

The fact that 20 of 30 of the patients required salvage therapy, and that overall, the BPC was greater than the initial FPC in all arms, attests to the need for monitoring of the FPC and for intensification of therapy in these HR patients. Comparing HR1 to HR2, starting therapy earlier than 20–24 weeks in the HR2 arm appeared to substantially increase the BPC. Antenatal therapy with IVIG 1–2g/kg/wk beginning at approximately 12 weeks gestation was effective in preventing ICH in the highest risk pregnancies in 25 of 30 cases. In the future, treatment may start with IVIG 2g/kg/wk and automatically add prednisone at 24–30 weeks for optimal response. This could also allow avoidance of FBS until at least 32 weeks.

Summary # of Patients # Requiring Salvage Mean FPC at 1st FBS (K) Mean Gestational Age at Birth (weeks) Mean BPC (K) # of ICHs # of Fetal Demises EHR 4 3 23 32.5 155 1 1 VHR 15 11 74.6 33.6 122 2 0 HR1 8 5 21.8 33.1 68.4 2 1 HR2 3 1 54 33.2 122 0 0 Figure Figure
Platelets Mediate Clearance of Lymphocytic Choriomeningitis Virus Infection Preventing Lethal Hemorrhage.
Authors: Iannacone Matteo , Sitia Giovanni , Isogawa Masanori , Whitmire Jason K. , Marchese Patrizia , Chisari Francis V. , Ruggeri Zaverio M. , Guidotti Luca G.
Published: April 2020

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic mouse pathogen of the arenaviridae family. Acute LCMV infection in adult mice has been extensively studied and found to be systemic, essentially asymptomatic and associated with a bone marrow aplasia that produces a transient pancytopenic state. The initial lymphopenia is rapidly reversed, such that within one week of LCMV exposure the mice display lymphocytosis and clear the infection through a response mediated by virus-specific cytotoxic T cells (CTLs). In spite of the thrombocytopenia, the occurrence of hemorrhage in these animals has not been previously investigated, likely because of the lack of overt bleeding symptoms. Other arenaviruses (such as Lassa and Junin) produce systemic infections in humans and cause hemorrhagic diseases that are often lethal. Hemorrhage, mostly mucosal and cutaneous, occurs in the context of profound thrombocytopenia (characteristic of Junin infection) and/or platelet dysfunction (characteristic of Lassa infection), but without disseminated intravascular coagulation (DIC) or other coagulation defects. The pathogenesis of arenavirus infections in humans remains elusive, although disease severity has been associated with the extent of hemorrhage, impaired cellular immunity and lack of viral clearance. We recently showed that platelets may contribute to viral pathogenesis by facilitating the accumulation of virus-specific CTLs at sites of infection. Thus, we reasoned that the thrombocytopenia and/or platelet dysfunction that typify arenavirus infections in humans might not only predispose to the development of hemorrhage but also compromise CTL-mediated viral clearance. Here we report our studies based on the model of LCMV infection in mice. We found that normal inbred mice infected with different isolates of LCMV develop thrombocytopenia associated with decreased platelet function, but show only limited mucosal hemorrhage prior to CD8+ T cell-mediated viral clearance. In contrast, mice depleted of platelets, but not those given anticoagulant treatment, fail to produce a normal CD8+ T cell response or clear LCMV; instead, they develop an interferon (IFN)-α/β-dependent lethal hemorrhagic infection. Transfusion of normal but not activation-blocked platelets into these animals restored the CD8+ T cell responses and allowed clearance of the infection, preventing hemorrhage and death. These results indicate that activated platelets are required for CD8+ T cells to clear LCMV infection and for protecting the host against the induction of an IFN-α/β-dependent, lethal hemorrhagic diathesis.
Lipopolysaccharide Binds Platelet Toll-Like Receptor 4 and Mediates the Activation of Phagocytes in the Reticuloendothelial System (RES): A Novel Mechanism of Host Immunity.
Authors: Semple John W. , Aslam Rukhsana , Speck Edwin R. , Freedman John
Published: April 2020

Toll-like receptors (TLR) comprise a family of transmembrane proteins characterized by multiple copies of leucine-rich repeats in the extracellular domain and an IL-1 receptor motif in their cytoplasmic domain. The TLR family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition and the stimulation of adaptive immune responses. Recently, our laboratory demonstrated that TLR4 expression on platelets was responsible for lipopolysaccharide (LPS)-mediated thrombocytopenia and tumour necrosis factor-α production in vivo (Aslam et al. Blood107:637, 2006). To understand the mechanism of how platelet TLR4 may mediate RES activation, platelets from wild type (WT) and TLR4 knockout mice were incubated with various concentrations of LPS in vitro, washed extensively and transfused into WT mice. Results suggest that only the WT platelets could significantly stimulate TNF-α production in vivo. In vitro flow cytometric analysis of phagocytosis by the monocytic cell line THP-1 demonstrated that platelet TLR4 could efficiently present LPS to THP-1 cells and stimulated them to engulf the LPS-coated platelets. The phagocytosis of the platelets was correlated to elevated levels of intracellular TNF-α production in the THP-1 cells. These results suggest that platelets, via TLR4 expression, can act as initial sentinels of the innate immune system by presenting bacterial products such as LPS to phagocytes of the RES.
Two Mutations (1717 T〉C, GP IX;124del145, GPIb beta) Occur Frequently among Patients with Bernard Soulier Syndrome in India.
Authors: Jacob Rintu Rebecca , Jayandharan G. , Viswabandya Auro , Baidya Shoma , Nair Sukesh C. , George Biju , Mathews Vikram , Chandy Mammen , Srivastava Alok
Published: April 2020

Bernard Soulier syndrome (BSS) is a rare (1:1000000) hereditary bleeding disorder of platelet adhesion, caused by a qualitative or quantitative defect of glycoprotein (GP)Ib/IX/V complex. We report here the molecular basis of BSS in eight unrelated patients from India. The diagnosis was based on low platelet count, presence of giant platelets and aggregometry studies (absence of response to ristocetin) in eight unrelated patients who presented with mucocutaneous bleeding at 17.9 years of age (range:9 months–60yrs). Flow cytometry to assess surface GPIb/IX/V complex using anti CD42b antibodies showed reduced (7.7%–21.1%, n=6) expression. Genomic DNA was screened for mutations in the GPIb alpha, GPIb beta, GP IX genes by a novel PCR and conformation sensitive gel electrophoresis method. Mutations were identified in all the 8 patients. Six different mutations, including missense (n=4) mutations, a deletion and a duplication were identified of which 5 are novel. Three novel missense mutations were identified in a patient (CD42b:21.1%) of which, Tyr492His in GPIb alpha gene was in heterozygosity and the Glu129His, Leu132Pro mutations in GPIb beta was detected in a double homozygous condition. All these substitutions occur at a highly conserved codon in the transmembrane region of GPIb alpha and beta chains, a region critical for anchoring the glycoprotein subunits into the platelet membrane. A novel homozygous deletion of 22 bp in exon 2 of GPIb beta (124del145, p.Arg17CysfsX14) gene was identified in 3 different patients (CD42b: range, 10.6–15.9%). This deletion occurs at a sequence flanking the leucine rich repeat at the NH2-side of GPIb beta protein results in a premature termination of translation (PTC) and a truncated mutant with 30 amino acids. A novel homozygous duplication of 38 nucleotides (2119_2120 dup38) in the transmembrane region resulted in a frameshift (142AlafsX76) in one of the patient. This frameshift predicts termination after the addition of 75 amino acids. A previously reported homozygous transition (1717 T >C) in the GP IX gene resulting in a Cys8Arg substitution was identified in the remaining 3 patients (CD42b:10.02%, 7.7%). The frequent presence of GP IX 1717 T>C and GPIb beta 124del145 in 6 out of 8 (75%) patients studied, suggests that this could be due to a founder effect. This data adds significantly to the mutation database of this condition and will be useful for genetic diagnosis of this condition in India.
The αIIbP258S, a Newly Identified Mutation, and the αIIbG349D Substitutions Cause Glanzmann Thrombasthenia by Impairing αIIb-β3 Association in the Endoplasmic Reticulum.
Authors: Artoni Andrea , Gelain Federica , Afrasiabi Abdolreza , Margaglione Maurizio , Mannucci Pier M.
Published: April 2020

Glanzmann Thrombasthenia is a recessively inherited hemorrhagic disorder caused by the quantitative or qualitative deficiency of integrin αIIbβ3, platelet fibrinogen receptor. We previously analyzed the phenotypic effects of the αIIbG236E substitution, which is located in a strategic point of the highly organized αIIb propeller. We demonstrated that the effect of αIIbG236E is to hamper the association with β3, impairing the correct assembly of the complex. In the present study we analyze the phenotypic effects of two other mutations in the same domain of αIIb. The first mutation, the αIIbP258S substitution, is a newly found missense substitution that was identified during a genetic screening of a cohort of patients with Glanzmann Thrombasthenia coming from Southern Iran. The presence of the mutation was confirmed in several family members affected by Glanzmann Thrombasthenia; no other additional mutations were identified. The effect of the substitution is to eliminate the key proline from blade 4 of αIIb propeller. The second mutation, the αIIbG349D substitution, has been previously described in an Italian patients with Glanzmann Thrombasthenia. It causes a charge substitution in strand D of blade 5 of the propeller. Both substitutions were inserted by site directed mutagenesis in pCDNA3.1 containing normal αIIb cDNA. Human Embryonic Kidney (HEK) cells were then transfected by electroporation with normal or mutated αIIb in conjunction with pCDNA3.1 containing normal β3. After 48 hours cells were analyzed by flow cytometry: the binding of 10E5-FITC (murine anti-human αIIbβ3) in cells expressing αIIbP258Sβ3 or αIIbG349Dβ3 was approximately at the same level of negative controls (cells transfected with αIIb only) and greatly reduced in comparison to the binding to cells transfected with normal αIIbβ3. HEK cells transfected with either αIIbβ3 or αIIbP258Sβ3 or αIIbG349Dβ3 were lysed; cells lysates were analysed by SDS-PAGE electrophoresis and immunoblotting. All the lysates were reacting at the same level with antibodies directed against β3. When immunoblotting was done in non-reducing conditions utilizing an antibody reacting with αIIb (mab1990) a band corresponding to αIIb was present in both lysates, although less intense in cells transfected with mutants αIIb. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbP258Sβ3 or αIIbG349Dβ3 the ratio pro-αIIb: αIIb was 1:1. In conclusion we report the identification of a new mutation responsible for Glanzmann Thrombasthenia located in αIIb propeller. This mutation, in addition with αIIbG349D, causes Glanzmann Thrombasthenia interfering with αIIbβ3 complex formation in the endoplasmic reticulum as shown by the increase amount of pro-αIIb found in cells expressing these mutants.
A Specific Subtype of Sitosterolemia with Macrothrombocytopenia in a Chinese Family.
Authors: Su Yanghua , Wang Zhaoyue , Yang Haiyan , Liu Fang , Bai Xia , Ruan Changgeng
Published: April 2020

Background and Objective. Sitosterolemia is a rare autosomal recessive inherited disorder of lipoprotein metabolism characterized by xanthomas, premature coronary artery disease, arteriosclerosis and increased plasma levels of phytosterols, while hematological abnormalities such as large platelets have seldom been described. Recently, we found a Chinese family with sitosterolemia; three children suffered from sitosterolemia, who presented with obviously large platelets, some as large as erythrocytes. Their platelet volume distribution curves were displaced rightward. The patients’ bleeding time (11,12 and 15 min) was longer than normal range (4–8 min). The platelets could normally aggregate in response to collagen and ADP, but failed to agglutinate in the presence of 1.2-mg/ml ristocetin. Platelet membrane glycoproteins (GP)IIb/IIIa and GPIb were normal. Their parents are consanguineous. In this study, we characterized the clinical, biochemical and molecular genetic features of a Chinese family with sitosterolemia that mainly manifested hematological abnormalities.

Design and Methods. The clinical features of the affected patients in this family were analyzed. Their plasma sterol levels were measured by HPLC method. ABCG5 and ABCG8 genes sequencing searched for the mutation.

Results. The main clinical feature of the three affected patients was macrothrombocytopenia; they had increased plasma sitosterol, stigmasterol and cholestanol levels, but maintained normal cholesterol levels. Sequence analysis of ABCG5 and ABCG8 genes revealed a C to T transition at nucleotide 18802 resulting in a nonsense mutation in ABCG5 exon 1, Gln22X. The affected patients were homozygous whereas their parents and other two family members were heterozygous for this defect. Allele-specific restriction enzyme analysis using BfaIdid not detect this mutation in 70 healthy individual.

Interpretation and Conclusions. A few patients with sitosterolemia may present with obvious macrothrombocytopenia. It suggests that the blood cells could be a “target” in the toxic effect of phytosterols in plasma, the hematological abnormalities might represent a specific subtype of sitosterolemia. Therefore, it is necessary to propose that sitosterolemia should be considered in the differential diagnosis of macrothrombocytopenia. A novel nonsense mutation in ABCG5 was reported.
Scott Syndrome Dogs Demonstrate a Failure of Coated-Platelet Formation.
Authors: Brooks Marjory B. , Catalfamo James L. , Friese Paul , Dale George L.
Published: April 2020

In 2002 Brooks et al. (Blood 99:2434) described a colony of German shepherd dogs (GSD) with a hereditary bleeding diathesis. The affected GSD (aGSD) had normal fluid phase coagulation as well as normal platelet aggregation and secretion, but their platelets failed to generate prothrombinase activity and did not express phosphatidylserine (PS) upon activation with calcium ionophore. These features are characteristic of Scott Syndrome. In the current study, we defined a broader platelet function defect in aGSD, i.e. a failure to produce significant levels of coated-platelets. Coated-platelets, a subclass of activated platelets formed upon dual-agonist activation, express not only surface PS but also retain several alpha-granule proteins, support a robust prothrombinase activity and release microparticles (J. Thromb. Haemost.3:2185, 2005). We activated platelets from control dogs and aGSD with convulxin plus thrombin and performed flow cytometric analyses to detect bound fibrinogen and membrane PS exposure. Post-stimulation, bound fibrinogen was detected on 34.6 ± 8.1 % of control dog platelets (mean ± 1SD; n=5) in contrast to 5.1 ± 2.5 % of aGSD platelets (n=5; p=0.006). In response to the same dual agonists, control dogs had 56.7 ± 10.3 % annexin-positive platelets while aGSD had only 4.7 ± 4.4 % (n=5; p=0.006). Control dogs and aGSD were also tested for their ability to release intracellular calcein upon dual agonist stimulation. As expected from studies with human platelets, control dog platelets released calcein from a sub-population of cells upon dual agonist stimulation; however, aGSD platelets demonstrated minimal loss of calcein [36.6 ± 6.4 % calcein negative control platelets (n=5); 3.5 ± 0.9 % calcein negative aGSD platelets (n=4; p=0.002)]. Additional activation events known to be associated with coated-platelet formation are currently being examined in aGSD to better define the basis of the platelet phenotype. Our results demonstrate that decreased production of coated-platelets is associated with abnormal bleeding and that a single hereditary defect is capable of abolishing prothrombinase activity and coated-platelet formation.
Ubiquitin-Proteolytic Control of HOX Homeodomain Proteins.
Authors: Zhang Jianxuan , Shieh Jae-Hung , Liu Liren , Plasilova Magdalena , Zhang Yue , Morrone Gianni , Moore Malcolm A.S. , Zhou Pengbo
Published: April 2020

The HOX homeodomain proteins are key regulators of hematopoiesis. HOX genes are expressed in primitive hematopoietic cells, and their prompt downregulation is associated with hematopoietic differentiation and maturation. Although transcriptional inactivation of HOX genes during hematopoietic differentiation has been established, little is known about the biochemical mechanisms underlying the subsequent removal of HOX proteins. We have previously shown that the CUL-4A ubiquitin ligase controls the stability of HOXA9 by promoting its ubiquitination and proteasome-dependent degradation. Interfering with CUL-4A biosynthesis results in altered HOXA9 protein levels, which is mirrored by the impairment of myeloid progenitor cells to undergo proper terminal differentiation into granulocytes. Here we show that additional HOX proteins are also subjected to CUL-4A-mediated ubiquitination and destruction. Consistently, the HOX homeodomain, which is highly conserved among all HOX proteins, is responsible for controlling their stability. Silencing of CUL-4A by RNA-mediated interference in human umbilical cord blood CD34+ cells significantly perturbs their self-renewal, expansion, and differentiation properties. These results reveal a novel regulatory mechanism of hematopoiesis by ubiquitin-dependent proteolysis. Recent studies on the biochemical mechanisms underlying HOX ubiquitination and by which leukemogenic HOX fusions evade CUL-4A-dependent degradation will be presented.
Epo Modulation of Cell Cycle Regulatory Genes Via PY343/STAT5 and PI3K Pathways in Primary Erythroblasts.
Authors: Fang Jing , Menon Madhu P. , Sathyanarayana Pradeep , Bogacheva Olga , Bogachev Oleg , Kapelle William , Houde Estelle , Wojchowski Don M.
Published: April 2020

Erythropoietin’s essential role in late erythropoiesis is ascribed to survival effects on colony forming unit-erythroid-derived erythroblasts. However, several studies have pointed to possible Epo regulation of cell cycle factors, and proliferation. To investigate this issue, developmentally-staged KitposCD71high erythroblasts were prepared from murine marrow; were shown to proliferate in response to low-dose Epo (0.1U/ml); and were used in the Affymetrix 430 2.0 array-based discovery of Epo-modulated cell cycle regulatory genes. In keeping with certain prior studies, myelocytomatosis oncogene (c-Myc) was induced while cyclin-dependent kinase inhibitor 1B (p27) was inhibited several-fold. Beyond this, four select cell cycle progression factors were rapidly up-modulated up to five-fold by Epo: nuclear protein 1 (Nupr1), G1 to S phase transition 1 (Gspt1), early growth response 1 (Egr1) and Ngfi-A binding protein 2 (Nab2). B-cell leukemia 6 (Bcl6) in contrast, was down-modulated several-fold. Finally, among cyclins, D2 was up-modulated ~5-fold while G2 was selectively down-modulated 4- to 5-fold (All findings were confirmed by RT-PCR). Studies in erythroblasts expressing knocked-in minimal erythropoietin receptor (EpoR) alleles indicated EpoR proximal tyrosine-343 (PY343) regulation of cyclin D2 induction, and EpoR C-terminal regulation of cyclin G2 inhibition. Despite the capacity of a PY-null EpoR-HM allele to support steady-state erythropoiesis, EpoR-HM erythroblasts proved to be skewed in cell cycle phases, especially in G2/M (0.01% vs ~5% for wt-EpoR and EpoR-H alleles). For cyclin D2, cyclin G2, p27 and Bcl6, in silico models for promoter regulation are outlined which highlight Stat and Forkhead action. In summary, specific new evidence is provided for erythroblast cell cycle entry modulation by Epo (especially as elevated during anemia) via discrete EpoR subdomains, and nine selectively modulated cell cycle regulatory genes.
Endomitotic MKs Undergo Cortical Contraction during Anaphase but Do Not Complete Furrow Ingression.
Authors: Geddis Amy , Fox Norma , Sear Katherine , Tkachenko Eugene , Kaushansky Kenneth
Published: April 2020

Megakaryocytes (MKs) develop high degrees of polyploidy in the course of normal maturation through a process called endomitosis. Previous studies have shown that endomitotic MKs abort anaphase after sister chromatid separation, and studies using fixed cells have shown that a midzone is formed in anaphase but that a well-developed cleavage furrow is not. However, studies in fixed cells may miss transient dynamic events that are critical for evaluating potential mechanisms of failed cytokinesis in endomitosis. Therefore we have used live cell imaging as a tool to further define the ability of primary endomitotic MKs to furrow during anaphase. We imaged mouse marrow MKs expressing tubulin fused to YFP, acquiring brightfield and fluorescent images every 2 minutes. Endomitotic cells were identified by the formation of a multipolar spindle, and anaphase was defined as the point at which separation of spindle poles was visible. The extent of cell shape change following anaphase was determined visually and also by measuring the circularity of the cell, with a perfectly round cell having a circularity value of 1. We found that 56% of endomitotic MKs underwent some shape change (n=25), visible as localized indentation, protrusion or elongation of the cell cortex, with an average reduction in circularity of 0.043 occurring a mean of 14.6 minutes after the initiation of anaphase. After an average of 7.8 minutes the cells generally reverted to a round shape without completing furrow ingression. In contrast, a typical mitotic control cell showed a reduction in circularity of 0.443 occurring 8 minutes after the start of anaphase and resulting in the formation of two cells (see Figure 1, representative cells). To determine if the shape change seen in endomitosis after anaphase is dependent on RhoA, we imaged MKs in the presence of the RhoA inhibitor C3 toxin. Although 2 μg/ml C3 toxin blocked cytokinesis in diploid control cells, it did not measurably alter the frequency (44%, n=16) or degree of shape change (0.039) in endomitotic MKs following anaphase, although treatment did appear to delay shape change (onset occurring a mean of 24.8 min after anaphase) and prolong the period of cortical contractility. Therefore although endomitotic MKs exhibit a phase of increased cortical contractility following anaphase, it is not solely dependent on the activity of RhoA and it does not result in sustained furrow ingression. Live cell imaging represents a powerful new tool for dissecting the mechanisms of endomitosis.

Figure 1. Endomitotic MKs undergo shape change following anaphase Figure 1. Endomitotic MKs undergo shape change following anaphase
Protein Phosphatase 2A Plays an Essential Role in Migration of CD34+ Cells to SDF-1/CXCL12.
Authors: Basu Sunanda , Ray Nicole T. , Atkinson Simon J. , Broxmeyer Hal E.
Published: April 2020

Although cord blood contains significant numbers of hematopoietic stem and progenitor cells (HSPC), its applicability has remained largely in children (1Broxmeyer and Smith, 2004). Homing of hematopoietic stem progenitor cells (HSPC) to bone marrow is a critical determinant for success of transplantation. Enhancement of homing of HSPC from cord blood has the potential to increase the applicability of cord blood transplantation in adults. Therefore, it is important to understand the molecules that underlie directional movement of HSPC. Stromal derived factor-1 (SDF-1)/CXCL12 is the most potent chemoattractant for both mouse and human HSPC and has been shown to play an important role in the homing and retention of HSPC in bone marrow. Earlier studies have shown that PI-3 kinase plays a critical role in chemotaxis by attracting various pleckstin homology (PH) containing domain proteins at the leading edge and spatial localization of PI3Kinase and PTEN is critical for maintaining the leading edge. Controlled activation of Akt/PKB, one of the various PH containg domains, has been described to be required for efficient chemotaxis. In this study we have demonstrated that protein phosphatase 2A (PP2A), a serine-threonine phosphatase, plays an important role in chemotaxis of cord blood CD34+ cells towards SDF-1, primarily by modulating Akt activity. Inhibition of PP2A by okadaic acid (OA) or siRNA impairs chemotaxis; this involved impairment in the ability of CD34+ cells to polarize and reduced speed of movement. This was associated with robust and prolonged Akt phosphorylation. Indeed, over expression of constitutive active Akt in CD34+ cells impaired SDF-1 directed chemotaxis. Co-immunoprecipitation experiments demonstrated increased association of Akt with PP2A following SDF-1 stimulation and this increased association of Akt and PP2A-catalytic subunit was observed at the plasma membrane of SDF-1 stimulated CD34+ cells by confocal microscopy. The importance of PP2A in maintaining a critical level and duration of activated Akt was also supported by our finding that inhibition of PI-3kinase by low dose of LY294002, partially recovered chemotactic activity in CD34+ cells pretreated with OA. Interestingly, glycogen synthase kinase-3 (GSK-3) associated with Akt following SDF-1 stimulation, although it was found to be constitutively associated with PP2A-C. Inhibition of GSK-3 using GSK-3 IX inhibitor impaired chemotaxis, raising the possibility that GSK-3 is a downstream target for Akt in SDF-1 directed chemotaxis. The physiological relevance of our in vitro findings is established from the observation that OA pretreatment impaired engraftment potential of CD34+ cells in NOD-SCID mice. Our findings contribute to the growing understanding of molecules that affect directional movement and may have the potential implications for homing and engraftment of HSPC.
Domain Subclass Conversion Improved Activity of Anti-Mpl Agonist Antibodies in the Form of Whole IgG.
Authors: Kai Masayuki , Motoki Kazuhiro , Yoshida Hideaki , Tsuruhata Kumi , Endo Chisato , Muto Mari , Emuta Chie , Chisaka Yukiko , Shimabe Munetake , Miyazaki Hiroshi , Kataoka Shiro
Published: April 2020

Since the molecular cloning of thrombopoietin (TPO), two types of recombinant human TPO, full-length recombinant human TPO (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF), have been tested in a variety of clinical trials. The agents showed clinical efficacy in some cases. However, neutralizing antibodies against endogenous TPO were generated in some healthy volunteers who received PEG-rhMGDF, resulting in thrombocytopenia. Recently, several TPO mimetics are being developed, such as peptide-based molecules, nonpeptidyl small compounds and agonist antibodies. Here we report generation of novel fully human anti-human Mpl agonist antibodies. Two lead monoclonal antibodies MA01 and MA02 were cloned from human antibody-producing mice (KM mice™) immunized with human Mpl-expressing cells. Both MA01 and MA02 were of immunoglobulin gamma 1 (IgG1). These IgG1 agonists promoted proliferation of UT7/TPO cells in vitro, but were >100-fold less effective than TPO. We, therefore, genetically modified MA01 and MA02 in order to enhance the activity and to minimize the effector function. First, constant heavy (CH) region of MA01 was converted to IgG4 (MA01G4) to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). This conversion affected neither binding affinity nor agonistic activity of MA01. Next, hinge region of MA01G4 was changed to human IgG3 hinge (MA01G4344). MA01G4344 showed no change in binding affinity, but showed 10-fold higher activity than original MA01. In the case of MA02, the CH region was converted from G1 to G3344 to generate MA02G3344, in which CH1-hinge region was of IgG3 and CH2-CH3 region was of IgG4. The conversion drastically improved agonistic activity. Although MA02 (G1) activated Jak2 and Erk1/2 but not STAT5 and Akt, MA02G3344 activated all of them. These data suggest that conversion of CH1-hinge region to IgG3 type is essential for agonistic activity of anti-Mpl antibodies. The effective concentration 50 (EC50) values of MA01G4344 and MA02G3344 in UT7/TPO assay were in the range of 0.01–0.1nM. These antibodies stimulated human cord blood-derived CD34+ cells to give rise to CFU-MK colonies without any other supportive cytokines. A single injection of MA01G4344 increased platelet count of human Mpl transgenic mice for over one month. These domain subclass-converted antibodies are expected to provide a new medical option for treatment of chronic thrombocytopenia without losing good properties of natural antibody such as long serum half-life and low immunogenicity.

Figure Figure
Endolyn (CD164) Interacts with CXCR4 and Modulates the CXCL12-Mediated Homing and Migration of Cord Blood CD133+ Hematopoietic Precusor Cells.
Authors: Watt Suzanne M. , Forde Sinead , Tye Brit Jorgensen , Newey Sarah , Roubelakis Maria
Published: April 2020

The sialomucin, endolyn or CD164, has been shown to act as an important regulator in the adhesion of human haemopoietic stem/precursor cells (HPC) to stromal niche cells, while also controlling the entry of primitive human CD34+CD38lo HPC into cycle. Here, we define a novel function for endolyn, by identifying its ability to modulate CD133+ cord blood HPC migration on fibronectin towards CXCL12 in vitro. Interestingly, CD133+ cell migration on fibronectin to CXCL12 was reduced 1) by engaging the functional class II glycosylation-dependent epitope on endolyn with the 103B2/9E10 class II but not N6B6 class III antibody; and 2) by RNAi knockdown of endolyn in both CD133+ HPC and Jurkat cells. The inhibition of migration was more pronounced in the more primitive CD34+CD38lo/− HPC subset than in the CD38+ subset. We show a direct and temporal association of endolyn with the CXCR4 receptor, at the leading edge of CD133+ HPC. When CXCL12 is presented on fibronectin, we first see an upregulation in the association of CXCR4 with the a-4 and a-5 integrins that is closely followed by recruitment of endolyn to this complex. This was confirmed by co-immunoprecipitation studies. Knock-down of endolyn using siRNAs revealed that signaling through CXCR4 via PKC-zeta and Akt pathways was significantly dampened, while leaving MAPK phosphorylation unaffected. Our current studies are aimed at examining the in vivo importance of endolyn in HPC homing to the bone marrow of NOD/SCID mice. Our findings support a novel association between three distinct families of cell surface receptors that regulate both cell migratory and proliferative responses and identify endolyn as a key regulator of CXCR4/CXCL12 function.
Metaphase Cytogenetics Adds to Gene Expression Profiling in the Identification of High Risk Multiple Myeloma.
Authors: Tricot Guido , Zhan Fenghuang , Barlogie Bart , Huang Yongsheng , Sawyer Jeffrey , Shaughnessy John
Published: April 2020

The International Staging System (ISS), based on B2-microglobulin and albumin levels at the time of diagnosis, has now generally been adopted as a new prognostic classification system for multiple myeloma (MM). While readily and widely applicable, ISS does not account for genetic disease features, such as metaphase (CA) and interphase fluorescence in situ hybridization (FISH) cytogenetic abnormalities, which when examined in the context of standard prognostic variables, confer higher hazards of relapse and disease-related death. Recently, gene expression profiling (GEP) uncovered the major prognostic significance for outcome of high expression of CKS1B, a gene mapping to an amplicon at chromosome 1q21. We have performed a comprehensive study of CA, FISH, GEP and ISS staging in 351 newly diagnosed MM patients, treated uniformly on Total Therapy 2. We have analyzed outcome based on a combination of high CKS1B by GEP and CA. GEP-based t(11;14) was prognostically favorable, irrespective of expression of CKS1B and, therefore, was removed from the group of patients with high CKS1B expression. After this adjustment, with the combination of both CA and high CKS1B (approximately 10% of all patients) conferred a very poor outcome with only 24% and 40% of such patients being event-free and/surviving at 3 years, compared with 72% and 84% for the others (p values : <.0001). Such patients fared poorly, irrespective of their ISS stage. Similar prognostic information could be gained by combining CA with FISH-defined amplification of 1q21 and t(11;14). Because of their major prognostic impact, all newly diagnosed patients should be tested for these genetic markers. Novel treatment modalities are justified in the small subgroup of such poor prognosis patients, since they derive only a minor benefit from advances in MM therapy.

CKS1B Q4 + CA (with no CCND1) vs. Others CKS1B Q4 + CA (with no CCND1) vs. Others
GATA-1, Transcriptionally, and Thrombopoietin-Mediated Akt Activation, Post-Translationally, Regulate Continuous Expression of Bcl-xL Protein during Megakaryopoiesis.
Authors: Kozuma Yukinori , Kojima Hiroshi , Yuki Satoshi , Suzuki Hidenori , Nagasawa Toshiro
Published: April 2020

Thrombopoietin (TPO) plays a relevant role for megakaryocyte differentiation from stem cells. One of the important biological activities of TPO is to prevent the apoptosis of megakaryocytic cells. As an anti-apoptotic protein Bcl-xL, which has been proved to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role for megakaryopoiesis. We thus investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early- and mid-stages of the differentiation. Both in vitro in stem cell-derived megakaryocyteic cell culture and in vivo in an animal model injected with anti-platelet antibody, expression of Bcl-xL protein was maintained until platelet-producing stage of the megakaryopoiesis. TPO-depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both stem cell-derived megakaryocytes and TPO-dependent megakaryocytic UT7/TPO cells. As a 12-kD fragment of Bcl-xL appeared by the withdrawal of TPO, we considered that Bcl-xL was cleaved upon TPO-depletion. This cleavage was blocked by a caspase-3-specific inhibitor, suggesting that caspase cleaves Bcl-xL in TPO-depleted megakaryocytes. Furthermore, pretreatment of UT7/TPO cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3K or its downstream signaling molecule inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this possibility, we prepared UT7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. Concerning transcriptional regulation of Bcl-xL, suppression of GATA-1 in UT7/TPO using siRNA caused decreased expression of both c-Mpl and Bcl-xL. Taken together, we conclude that GATA-1 regulates the expression of both c-Mpl and Bcl-xL, and once Bcl-xL is expressed, its protein level is maintained by the TPO-mediated Akt activation.
A Putative Role for VEGFR-1 (FLT-1) in B Cell Commitment and Differentiation.
Authors: Fragoso Rita , Igreja Catia , Appleton Claudia , Henriques Alexandra , Clode Nuno , Wu Yan , Zhu Zhenping , Dias Sergio
Published: April 2020

VEGF and its receptors are expressed in the hematopoietic system. A role for FLT-1 in particular was described in monocyte-macrophage migration and lineage differentiation (Sawano A et al, 2001), megakaryocytes maturation (Casella I et al, 2003) and dendritic cell differentiation (Dikov M et al, 2005). Given that the expression of this receptor in the lymphoid lineage is not known, we to studied FLT-1 expression and a putative function in normal lymphoid progenitors. To address this question we induced in vitro CD34+ cells differentiation into the B cell lineage using a well established assay (on S17 stromal cells). With this approach, we observed that FLT-1 is expressed throughout B cell differentiation increasing along the differentiation process, and reaching its highest at the “immature B cell” stage. We also neutralized FLT-1 during B cell differentiation in vitro. Surprisingly, in the presence of the FLT-1 neutralizing antibody (6.12 monoclonal Ab, from ImClone systems), at the end of the assays (4 different experiments) a significantly higher number of CD19+ cells (mainly immature B cells) were detected. Analyzing some of the transcription factors known to be involved in the commitment and differentiation of lymphoid B cells, we observed that the expression of PU.1, Pax5 and E47 was up-regulated by FLT-1 neutralization. Next, given that FLT-1 function was mainly associated with cell migration, and since it is expressed in B cells that are ready to exit the bone marrow into secondary lymphoid organs, we reasoned that FLT-1 might have a role in B cells exit from the bone marrow. For this purpose, we treated mice with the FLT-1 neutralizing Ab for 3 days and analyzed B cells levels in bone marrow and peripheral blood. FLT-1 neutralization led to a significant decrease (p<0.05) in B cells in the bone marrow and peripheral blood. Taken together, our data supports a clear role for FLT-1 in B cell commitment. To understand if VEGF/PlGF signalling through FLT-1 promotes myeloid differentiation, suppresses B cell differentiation or simply regulates the quiescent state of hematopoietic stem cells, we differentiated in vitro CD34+/FLT-1− cells and CD34+/FLT-1+ cells (10% of CD34+ cells) using the assay described above. Interestingly, CD34+/FLT-1− differentiation in vitro largely promoted B cell differentiation, while CD34+/FLT-1+ cells originated mostly myeloid cell differentiation. We are currently exploiting the molecular basis whereby FLT-1 signalling may impair B cells commitment and possibly promotes myeloid differentiation.
PRV-1 Interacts with the Cytoplasmic Domain of Mpl and Interferes with TPO-Induced Signaling Cascades.
Authors: Johnson Jan , Ghandi Manish , Minear Jennifer N. , Lannutti Brian J.
Published: April 2020

Polycythemia rubra vera-1 (PRV-1) is a member of the uPAR/CD59/Ly6 family of cell surface receptors. An increase in the expression level of PRV-1 mRNA has been shown in patients with polycythemia vera and essential thrombocythemia, however, the ligand and function of PRV-1 remains unclear. Here, we show for the first time that PRV-1 expression alters the TPO-mediated Mpl receptor-signaling pathway. We also report a novel interaction between PRV-1 and the cytoplasmic region of Mpl (121 amino acids) using a yeast two-hybrid screen. Further characterization of this interaction in yeast containing truncated versions of the Mpl receptor cytoplasmic region indicates that the binding of PRV-1 to Mpl is regulated at the distal end of the cytoplasmic region. Deletion analysis utilizing Mpl receptors truncated after 53 (T53) or 69 (T69) cytoplasmic amino acid residues failed to show an association with PRV-1. Further mapping of the cytoplasmic domain of Mpl showed that truncations equal or greater than 111 cytoplasmic residues (T111) restored the interaction with PRV-1. To examine the physiological relevance of this interaction, we co-expressed Mpl and PRV-1 in BaF3 cells. Western blot analysis with an anti-phosphotyrosine antibody on TPO-stimulated BaF3/Mpl/PRV-1 whole cell lysates demonstrated a noticeable change in intracellular phosphorylation when compared to BaF3/Mpl. We found no affect on Jak2 and Mpl tyrosine phosphorylation. In contrast, there were higher levels of tyrosine-phosphorylated STAT5 and STAT3 in BaF3/Mpl cells expressing PRV-1. We then further investigated the effect of PRV-1 expression on MAPK signaling. We found that TPO-stimulation of BaF3/Mpl/PRV-1 cells resulted in a reduction of MAPK phosphorylation compared with cells expressing Mpl alone. These results indicate a novel function for PRV-1 as a signaling molecule in cytokine signaling cascades and may lead to a greater understanding of the mechanism of overexpression of PRV-1 in myeloproliferative disorders.
Telintra™ (TLK199) Enhances Granulocyte Colony-Stimulating Factor Production.
Authors: Stofega Mary R. , Cheng Mingshan , Ma Huiping , Sambucetti Lidia , Keck James G. , Brown Gail L. , Cai Danying
Published: April 2020

TLK199 is a novel glutathione analog inhibitor of the enzyme GST P1-1. GST P1-1 has been shown to be a negative regulator of c-Jun N-terminal kinase, which has been implicated in the control of cellular growth and differentiation. Studies in rodents have shown that TLK199 treatment increases the levels of circulating white blood cells and stimulates the production of CFU-GM in bone marrow. In a rodent model of 5-FU-induced neutropenia, treatment with TLK199 accelerated the recovery of neutrophil levels at a rate similar to that observed with G-CSF. In a recently reported Phase 2 clinical trial in patients with refractory MDS involving multilineage blood cell dysfunction, treatment with TLK199 resulted in a 65% Hematological Improvement of neutrophils, erythrocytes and/or platelets, confirming the myelorestorative effect of TLK199 in humans. The present study was designed to determine if the myelostimulative effects of TLK199 on neutrophils and their precursors are due in part to increased levels of endogenous G-CSF. TLK199 was tested in normal and neutropenic rodents for its effects on G-CSF production. A single administration of TLK199 (p.o. or i.p.) to normal mice resulted in up to 500% increase in G-CSF serum levels. In a rat model of 5-FU-induced neutropenia, TLK199 administration accelerated the recovery of circulating neutrophils, which corresponded to maximal G-CSF serum levels being observed earlier than vehicle control. The ability of TLK199 to enhance G-CSF production in primary human bone marrow stromal cells was also examined. Treatment of these cells with TLK199 potentiated IL-1b-induced production of G-CSF, GM-CSF and IL-6 from 200 to 400%. These data suggest that the enhancement of G-CSF production may contribute to the accelerated recovery of neutrophils observed with TLK199 treatment under neutropenic conditions. Together with the results from the Phase 2 clinical trial in MDS, these data support further clinical assessment of TLK199 treatment for chemotherapy-induced cytopenia.
Polymorphisms in Human Homeobox HLX1 and DNA Repair RAD51 Genes Increase the Risk of Therapy-Related Acute Myeloid Leukemia.
Authors: Jawad Mays , Seedhouse Claire , Russell Nigel H. , Plumb Mark
Published: April 2020

It is generally accepted that genetic low penetrance susceptibility and resistance genes within the general population make a significant contribution to therapy-related acute myeloid leukaemia (t-AML) risk. The risk of malignant transformation is defined by the number of mutations required and the mutation rate, but one additional theoretical risk factor is the target cell frequency, as this defines the number of genomes at risk. Whilst the genetic control of target cell frequency is unknown in humans, a leading candidate gene is the HLX1 homeobox transcription factor gene. HLX1 is specifically expressed on CD34+ cells and plays a vital role in hemopoietic development. In addition the HLX1 gene lies within a proposed human t-AML susceptibility locus on chromosome 1, as defined by murine radiation-induced AML genetic studies. We have examined the distribution of a C/T-3′UTR polymorphism in HLX1 in 189 AML patients, including 42 cases of t-AML, and 169 control subjects. The presence of a variant HLX1-3′UTR T allele resulted in a significant increase in the risk of t-AML (OR= 3.36, 95% CI 1.65–6.84); to our knowledge, this is the first time that a gene which is implicated in target cell biology has been associated with an increased risk of malignant transformation. In addition we examined polymorphisms in genes involved in DNA repair (RAD51 and XRCC2), carcinogen detoxification (HYL1) and methionine metabolism (MTHFR and MS). There was no difference in the distribution of the XRCC2, HYL1, MTHFR or MS polymorphisms in the control and AML cohorts studied. The RAD51 homologous recombination DNA repair gene polymorphism (135G/C-5′UTR) has been shown to result in enhanced promoter activity with consequent elevated mRNA expression and this polymorphism has previously been demonstrated to increase t-AML risk (OR=2.66, 95% CI 1.17–6.02). When combined analysis was performed on RAD51 (135G/C- 5′ UTR) and HLX1 (C/T -3′UTR) a 9.5-fold increase in the risk of t-AML was associated with the presence of variant alleles of both genes (OR= 9.50, 95% CI 2.22–40.64). The synergistic genetic interaction between the HLX1-C/T (3′- UTR) and RAD51-135 G/C polymorphisms demonstrates that target cell biology together with an increased homologous recombination DNA repair capacity, significantly increases the risk of t-AML. We suggest that this is because the HLX1-C/T (3′- UTR) polymorphism results in a higher number of stem cells; hence during genotoxic therapy (for a primary malignancy) there is an increased target cell number for genotoxic damage and potential malignant transformation. An optimum repair system is necessary to ‘deal’ with this burden of repair, however, the presence of altered repair capacity due to the RAD51-135C/T polymorphism may result in mis-repair and together with lack of apoptosis (due to a over-efficient repair system) will encourage the perpetuation of the oncogenic transformation and AML susceptibility.