bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma Thromboplastin Antecedent (PTA) Deficiency: Clinical, Coagulation, Therapeutic and Hereditary Aspects of a New Hemophilia-like Disease
Authors: ROSENTHAL ROBERT L. , DRESKIN O. HERMAN , ROSENTHAL NATHAN
Published: October 2019

1. An analysis of the original PTA deficient family, including coagulation studies performed upon 13 members comprising 4 generations, has been presented.

2. PTA deficiency is transmitted as an autosomal dominant trait with a probable high degree of penetrance and variable expression of the gene.

3. PTA deficiency can occur its varying degrees ranging from a severe form with prolonged clotting time and markedly abnormal heparin clotting time and prothrombin utilization to a mild form manifesting a normal clotting time and slightly impaired prothrombin utiliztition.

4. Studies on the treatment of PTA deficiency reveal that the defect is corrected by the administration of stored plasma with the effect gradually disappearing over the period of one week.

5. Various properties of PTA are discussed and compared with AHG and PTC.
Splenic Aspiration in Clinical and Experimental Hematology
Authors: WATSON R. JANET , SHAPIRO HENRY D. , ELLISON ROSE RUTH , LICHTMAN HERBERT C.
Published: October 2019

1. The findings obtained by the study of splenic aspirations in 140 patients are presented. Diagnostically they were most helpful in myeloid metaplasia, lymphoma, kala-azar, and Gaucher’s disease.

2. The findings were of particular theoretical interest in multiple myeloma and sickle cell anemia. This method of investigation could be extended to elucidating further the role of the spleen in other diseases.

3. No morbidity was encountered in this series. If certain precautions are observed, splenic puncture is a safe procedure, and should be undertaken in patients with undiagnosed splenomegaly.
Acquired Circulating Anticoagulants in Systemic "Collagen Disease"
Authors: FRICK PAUL G. , WEIMER MARY K.
Published: October 2019

1. A circulating anticoagulant was demonstrated in three patients with "collagen disease." In all cases it was associated with a false positive blood serology and a four plus cephalin cholesterol flocculation.

2. In one instance, the anticoagulant was transferred to a newborn infant where it persisted for seven weeks. The infant also demonstrated the abnormal serologic and turbidimetric tests during the first six months of life.

3. The anticoagulant probably acted as anti-thromboplastin.

4. The occurrence of a clotting inhibitor in association with other manifestations of hypersensitivity and its transplacental transfer strongly suggest that the mechanism of development is immunologic in type.

5. This study included a total of thirty patients with lupus erythematosus disseminatus and associated conditions conventionally grouped under the term "collagen disease." The incidence of circulating anticoagulants was 10 per cent.
Mutations in the Gene for Neutrophil Elastase in Severe Congenital Neutropenia Are Heterozygous and of Male Origin.
Authors: Stein Steven , Paddock Marcia , Bolyard Audrey Anna , Raymond Chris , Olson Maynard , Aprikyan Andrew , Dale David C.
Published: October 2019

Severe congenital neutropenia (SCN) occurs sporadically and as an autosomal-dominant or autosomal-recessive inherited disorder. Most sporadic and autosomal dominant cases are caused by mutations in the gene for neutrophil elastase (NE or ELA2). The gene for this potent serine protease is located on chromosome 19p13.3 and has 5 exons. In collaboration with the Severe Chronic Neutropenia International Registry (SCNIR), we have sequenced the NE gene from blood, bone marrow or other tissues samples for more than 300 patients with the clinical diagnosis of SCN using Big Dye Terminator, version 3.1 and ABI 3700. These studies have included 21 families with 2 or more affected members and 15 families with sporadically occurring SCN, i.e., two healthy parents with normal blood counts having an affected child. In 15 families with sporadic cases of SCN attributable to 13 different mutations in the NE gene, we sought to determine the parent of origin for the NE mutation. The 5 exons of the NE gene were sequenced twice in the parents and the affected child using Big Dye Terminator, version 3.1 and ABI 3700. After confirming the mutation in each patient and the absence of mutations in each parent, PCR was used to scan the adjacent region to the NE gene in the parents and the child to identify polymorphisms that could be used to distinguish between maternally and paternally inherited chromosomes. A fosmid library of 2-4X coverage for each patient’s DNA sample was made which successfully separated the patient’s genome into its constituent’s haplotypes. The fosmids containing the NE gene were identified by PCR and genotyped at the site of the mutant allele and the parent-informative marker. In all cases, the new NE gene mutation in the affected child was on the paternal allele.
Combination of the mTOR Inhibitor Rapamycin and Revlimid™ (CC-5013) Has Synergistic Activity in Multiple Myeloma (MM).
Authors: Raje Noopur , Kumar Shaji , Hideshima Teru , Ishitsuka Kenji , Chauhan Dharminder , Mitsiades Constantine , Podar Klaus , Le Gouill Steven , Richardson Paul , Munshi Nikhil C. , Stirling David I. , Antin Joseph H. , Anderson Kenneth C.
Published: October 2019

Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) mediate growth of MM cells via activation of the mitogen-activated protein kinase (MAPK), JAK2/STAT3, and phosphatidylinositol 3′-kinase/Akt kinase (PI3-K/Akt) signaling cascades. We have previously demonstrated the in vitro and in vivo activity of Revlimid™ (CC-5013), an immunomodulatory analog (IMiD) of thalidomide, in MM. In the present study, we have examined the anti-MM activity of rapamycin, a specific mTOR inhibitor, combined with Revlimid™. This combination was highly synergistic at 0.1nmol/L of rapamycin with 0.1mmol/L of Revlimid™, and remained synergistic at higher concentrations. Based on the Chou-Talalay method, combination indices of < 1 were noted for all dose ranges of Revlimid™ and rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional (doxorubicin, melphalan, dexamethasone) chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by IL-6, IGF-1, or adherence to bone marrow stromal cells (BMSCs). Cytotoxicity triggered by a combination of rapamycin with Revlimid™ resulted in apoptosis of MM cells. Furthermore, differential signaling cascades, including the MAPK and PI3-K/Akt pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies therefore provide the framework for the clinical evaluation of targeted agents like mTOR inhibitors combined with immunomodulatory agents like Revlimid™ to improve patient outcome in MM.
The Natural History of Idiopathic Erythrocythosis: A Cohort Study of 74 Patients.
Authors: Guido Finazzi , Marco Ruggeri , Monica Marconi , Francesco Rodeghiero , Tiziano Barbui
Published: October 2019

Patients with absolute erythrocytosis not due to a detectable cause and not fulfilling the criteria for diagnosis of polycythemia vera (PV) are descriptively classified as Idiopathic Erythrocytosis (IE). Based on scanty and retrospective data, this disease is considered to be an heterogeneous entity, including “early” PV, unrecognized secondary erythrocytosis and other miscellaneous conditions. However, appropriate prospective studies to evaluate the natural history of patients with IE are not available. We report here the results of a cohort study of 74 patients with IE (66 males, 8 females, median age 56 years, range 14–82) followed in two Italian institutions. By definition, at baseline all IE patients had increased hematocrit (median 54%, range 48–68%) and increased red blood cell mass (> 25% above mean normal predicted value), but normal leukocyte (median values 8.1 x 109/L, range 2.3–12) and platelet counts (median values 197 x 109/L, range 117–467), as well as normal erythropoietin level, arterial O2 saturation, chest X ray and abdominal ultrasound scanning (i.e. no splenomegaly). Granulocyte PRV-1 expression was also normal in 29 patients (39%) analyzed. At diagnosis, 12 patients (16%) reported a previous history of major thrombosis (7 ischemic cardiopathies, 4 cerebral ischemic events and 1 deep vein thrombosis). All IE patients were treated with phlebotomy to maintain a target hematocrit <45% and 24 patients (32%) were given aspirin, 100 mg/die, for previous thrombosis or microvascular symptoms. No cytotoxic drugs were given. The IE cohort was followed in the outpatient clinic with physical examination and full blood count at least every three months for a median period of 3.5 years (range 1–23). Twentythree patients (31%) were followed for more than 8 years. No patient was lost to follow-up. During the observation period, no disease potentially associated with secondary eryhtrocytosis emerged and no hematological transition into overt PV, myelofibrosis or acute leukemia occurred; two patients had a major thrombotic event (1 cerebral ischemia and 1 deep vein thrombosis) with an estimated incidence of thrombotic complications of 0.8% patient-year. The incidence of thrombosis was significantly lower than observed in 205 patients with overt PV followed during the same period in one of the two institutions (Bergamo, 3.49% patient-year, p<0.05). This study indicates that: a. the natural history of patients with IE, at least in the first years, is characterized by a remarkable and unexpected homogeneity without appearance of overt PV or diseases associated with secondary erythrocythosis; b. the diagnosis of IE identifies a group of absolute erythrocythoses at lower risk of thrombotic complications not requiring cytotoxic drug therapy; c. the diagnostic work-up of patients with absolute erythrocythosis should carefully distinguish IE from PV because the natural history and management of the two diseases is different.
The Hox a Locus Is Strongly Targeted by MMLV in an E2a-PBX1 Induced B-Cell Leukemia Model.
Authors: Bijl Janet J. , Sauvageau Martin , Thompson Alex , Sauvageau Guy
Published: October 2019

We previously developed a B-ALL model from E2a-PBX1 transgenic mice (Bijl et al. Abstract 469 ASH 2003 and paper in preparation). We now exploit this model to identify oncogenic collaborators to E2a-PBX1. To achieve this, 18 newborn E2a-PBX1 transgenic mice and 23 control littermates were intraperitonally injected with MMLV. The occurrence of B-ALL in E2a-PBX1 transgenic animals was significantly accelerated when compared to control littermates (mean survival 162 ± 31 versus 191 ± 50 days, respectively, P=0.03), suggesting the presence of E2a-PBX1 specific collaborators. Inverse PCR was performed on genomic DNA isolated from seven E2a-PBX1 and six control tumors. Seventy-two different retroviral insertion sites were recovered from the tumors. Six common integration sites (CIS) for MMLV were identified; including two novel CIS, i.e. Pde4d, a phosphodiesterase, and a hypothetical phospholipase A930027K05Rik. Strikingly, the Hox a locus was targeted in six of seven E2a-PBX1 tumors, and only one of six control tumors. MMLV integrants in the Hox a locus were detected in a 19kb region located between Hoxa6/Hoxa7 and Hoxa10. Two tumors were targeted twice in this locus. Q-PCR analysis for expression of all Hox a cluster genes identified a significant increase for the Hoxa3 to Hoxa10 genes in the transgenic tumors when compared to the expression in a control MMLV induced B-cell tumor (see table, below). Hoxa7 and Hoxa6 were the most frequently and strongly activated (6/6 and 5/6 transgenic tumors, respectively), showing values of 5700 and 6600 fold over expression in one particular tumor. Interestingly, a Hox a locus insertion in a control tumor (table: last row) did not have any effect on gene activation. In this study we demonstrate a preference for MMLV to target the Hox a locus in an E2a-PBX1context. The resultant gene activation suggests that several members of the Hox a locus are functional collaborators to E2a-PBX1 in the induction of B-cell leukemia.

Fold difference in expression for Hox a cluster genes in Hox a targeted tumors compared to a control tumor.

Insertions a3 a4 a5 a6 a7 a9 a10 a9/a10 −1 1 4 3 138 2 4 a3 17 55 1067 5700 6608 171 62 a10 3 1 11 98 38 3 −4 a9/a10 1 −1 2 21 9 2 −2 a9/a10 2x 13 6 19 221 191 85 2 a7/a9 + a10 8 4 11 66 44 9 149 a10 −2 −6 2 2 −2 −8 −5
GPIbα Dimer Formation and Cell Surface Expression: A Filamin Binding Domain Is Essential for Dimer Formation but Not for Assembly of a GPIb/IX Complex.
Authors: Kanaji Taisuke , Hamasaki Naotaka , Izuhara Kenji , Ware Jerry
Published: October 2019

The hereditary bleeding disorder Bernard-Soulier syndrome (BSS) is caused by an absent or dysfunctional GPIb/IX complex. Lopez et al. have reported that three subunits of the complex (GPIbα, GPIbβ and GPIX) are all necessary for efficient translocation to the cell surface. In contrast, Meyer et al. have demonstrated that GPIbα alone can incorporate into the cell surface. We have established a cell culture system with inducible levels of GPIbβ and GPIX. Using these cell lines, we characterize an intrinsic ability of GPIbα to form dimers and the relevance of specific GPIbα sequences for dimer formation and the assembly of a GPIb/IX complex.

A stable CHO cell line was established that expresses GPIbβ and GPIX under the control of Tet-responsive elements. This cell line was then transfected with WT GPIbα and truncated GPIbα mutants. Expression was monitored by Western blot analysis and flow cytometry.

We observed that expression of GPIbα, by itself, targets GPIbα to the cell surface as a dimer albeit at reduced levels. The GPIbα binding domain for filamin (residues 545–605) is essential for forming a dimer but not for assembly of a GPIb/IX complex. We also observed GPIbα dimer formation in a transgenic mouse that expresses human GPIbα and a mouse chimeric GPIb-IX complex suggesting dimer formation can occur in the platelet. To investigate the role of filamin in the dimerization process, we established a stable cell line deficient in filamin by targeting filamin expression with RNAi. We observed GPIbα dimers in the absence of filamin expression. These results illustrate the importance of expressing all three subunits of the GPIb/IX complex, but also document as intrinsic potential within GPIbα for surface expression via intermolecular homodimerization.
ATP Release from Red Blood Cells Is Regulated by a Negative Feedback Pathway where ADP Acts on P2Y13 Receptors.
Authors: Olsson Martin L. , Wang Lingwei , Olivecrona Goran , Gotberg Matthias , Amisten Stefan , Winzell Maria Sorhede , Erlinge David
Published: October 2019

Background: Red blood cells regulate tissue circulation and O2 delivery by releasing the vasodilator adenosine triphosphate (ATP) in response to hypoxia. When released extracellularly, ATP is rapidly degraded to adenosine diphosphate (ADP) in the circulation by ectonucleotidases. ATP and ADP activate subtypes of the large P2 receptor family (15 subtypes). Here we show that ADP acting on P2Y13 receptors on red blood cells serves as a negative feedback pathway for the inhibition of ATP release.

Methods: mRNA was quantified with real-time PCR. Western blot was used to detect P2 receptors with available antibodies. cAMP levels were determined with an enzyme immunoassay. ATP release was measured in incubated red blood cells using microdialysis and a luciferase assay. In a pig model, catheters were inserted through the carotid artery to place a catheter in the left coronary artery, and through the jugular vein to place a microdialysis probe in the coronary vein. 2-MeSADP was injected in the artery and ATP levels were measured in the coronary vein.

Results: mRNA of the ADP receptor P2Y13 was highly expressed in human red blood cells and reticulocytes, whilst other ADP receptors were not (Fig.1).

Figure Figure

The stable ADP analogue 2-MeSADP decreased ATP release from red blood cells by inhibition of cAMP. The P2Y12 and P2Y13 receptor antagonist AR-C67085 (30 mM), but not the P2Y1 blocker MRS2179, inhibited the effects of 2-MeSADP. At doses where AR-C67085 only blocks P2Y12 (100 nM), it had no effect. AR-C67085 and the nucleotidase apyrase increased cAMP per se, indicating a constant cAMP inhibitory effect of endogenous extracellular ADP. 2-MeSADP reduced plasma ATP concentrations in an in vivo pig model. Furthermore, a missense polymorphism in the coding region of P2Y13 has been found that is in total disequilibrium with 5 polymorphisms in P2Y12 (the important ADP receptor in platelets) forming a haplotype that could contribute to vascular disease.

Conclusion: Our results show that P2Y13 is selectively expressed in human red blood cells. The ATP degradation product ADP inhibited ATP release by acting on this receptor. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation. Because blood consists of approximately 40% red blood cells, containing a 1000-fold higher ATP concentration than plasma (mM vs. uM), even a minor release of ATP from the high intracellular concentrations could have major circulatory effects. A negative system may therefore be of great physiological importance to mitigate ATP release. In addition, this finding could be of interest for efforts to preserve intracellular ATP in red blood cells during storage.
Hypoxic Vasodilation by Red Blood Cells and Impairment in Vascular Disorders.
Authors: Pawloski John R. , McMahon Timothy J. , Ahearne Greg , Piantadosi Claude A. , Singel David J. , Stamler Jonathan S.
Published: October 2019

Physiological O2 gradients are principal regulators of blood flow in the microcirculation: position-to-position changes in hemoglobin (Hb) O2 saturation are coupled to regulated vasodilation (“hypoxic vasodilation”). The mechanism by which graded changes in O2 content of blood evoke this response has been a great challenge to understand. A new role for red blood cells (RBCs) in hypoxic dilation of blood vessels and inhibition of platelet activation involving release of nitric oxide (NO) bioactivity is described. We show that NO groups can be transferred within hemoglobin (Hb) from hemes to highly-conserved cysteine thiols (β-Cys93) to form bioactive S-nitrosohemoglobin (SNO-Hb), and that efficient production of SNO-Hb requires selective processing of NO within the β-subunits. Bioactive SNO-Hb is localized primarily to the RBC membrane through interaction with Band 3, the transmembrane anion-exchanger 1 protein (AE1). Upon deoxygenation, transfer of the NO group from β-Cys93 of Hb to a cysteine thiol within AE1 serves the RBC vasodilator activity. In this way, O2 binding in Hb modulates the release of NO bioactivity. We further show that RBC NO bioactivity is inversely proportional to pO2 and impaired in disease. In an aortic ring bioassay sparged with variable concentrations of O2, addition of normal human RBCs elicited graded responses from relaxation at tissue pO2 (~3–7 mm Hg, hypoxic vasodilation), to loss of relaxation and progressively greater contractions at pO2’s of 10–63 mm Hg (hyperoxic vasoconstriction). Notably, RBC SNO-Hb levels and hypoxic vasodilation are impaired in several diseases characterized by vascular dysfunction. For example, in RBCs from patients with pulmonary arterial hypertension (PAH), we found decreased (13% of control) SNO-Hb content (assessed by photolysis-chemiluminescence) and impaired O2-dependent vasodilation (bioassay). RBCs from patients with other ischemic disorders have also been examined: RBCs demonstrate a pathogenesis-based impairment in their ability to mediate hypoxic vasodilation by NO. These results confirm the (patho)physiologic importance of RBC NO, and suggest that RBC dysfunction may contribute to impaired blood flow in diseases of the heart, lung and blood.
Regulation of SCL Expression by the Homeodomain Protein Otx-1 and the Erythroid Transcription Factor GATA-1.
Authors: Martin Richard , Sanguin-Gendreau Virginie , Tremblay Mathieu , Levantini Elena , Magli Christina , Hoang Trang
Published: October 2019

Members of the bicoid homeodomain-containing proteins are important in establishing left-right asymmetry and the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that Otx1, a member of the bicoid homeodomain-containing proteins, is co-expressed with the SCL transcription factor in hematopoietic pluripotent and erythroid progenitor cells and Otx1-deficiency impairs the erythroid compartment in mice, associated with decreased SCL levels. In the present study, we provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. First, we show by chromatin immunoprecipitation that Otx1 and GATA-1 are specifically bound to the SCL proximal promoter in erythroid cells. Second, Otx-1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity depends on the integrity of the proximal GATA site of the SCL promoter 1a. At the molecular level, we show that this synergy occurs via a physical interaction between Otx-1 and GATA-1 in erythroid cells, which maps to the homeodomain of Otx-1. Furthermore, a gain of function of Otx1 in primary hematopoietic cells gives rise to a 6-fold increase in endogenous SCL levels, an increase in TER119-positive erythroid cells and a decrease in the number of CD11b-positive myeloid cells. Finally, a gain of function of SCL rescues the erythroid deficiency in Otx1−/− mice, consistent with the view that SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL operate within the same genetic pathway to specify the erythroid fate during hematopoiesis.
Transgenic Expression of Bach1 Transcription Factor Results in Down-Regulation of the p45 Target Genes in Megakaryocytic Lineage Cells.
Authors: Toki Tsutomu , Katsuoka Fumiki , Kanezaki Rika , Watanabe Seiji , Kamio Takuya , Tandai Satoru , Kurotaki Hidekachi , Yagihashi Soroku , Sun Jiying , Komatsu Norio , Igarashi Kazuhiko , Yamamoto Masayuki , Ito Etsuro
Published: October 2019

Both p45 and BACH transcription factors can form dimers with one of the small Maf proteins, and these heterodimers bind to Maf recognition elements (MARE). MARE is known to act as a critical cis-regulatory element of erythroid and megakaryocytic genes. While detailed analyses of p45 -null mutant mice and small maf compound mutant mice revealed that these factors are both critical for platelet production, the functional contributions of BACH1 and the relationship or redundancy between BACH1 and p45 in megakaryocytes remain to be clarified. To address these issues, we generated transgenic lines of mice bearing human BACH1 cDNA under the control of the GATA-1 locus hematopoietic regulatory domain. The transgenic mouse lines showed significant thrombocytopenia associated with impaired maturation of the megakaryocytes, and they developed myelofibrosis. The megakaryocytes overexpressing the BACH1 transgene exhibited reduced proplatelet formation. Since the phenotype of the BACH1 transgenic mice resembled that of the p45 -deficient mice, we examined the expression of the p45 NF-E2 target genes in the primary megakaryocytes from fetal liver cells of the BACH1 transgenic mice. RT-PCR analyses showed that expression of the hematopoietic-specific ß1-tubulin, thromboxane synthase ( TXAS), and of the 3ß-hydroxy-steroid dehydrogenase genes was significantly downregulated in the megakaryocytes from BACH1 transgenic mice. The TXAS gene is a well-known MARE-dependent gene containing functional MAREs in its promoter and in the second intron. To ask whether BACH1 actually binds to MARE in the megakaryocytic genes, we then performed chromatin immunoprecipitation (ChIP) analysis with a BACH1-specific antibody. A ChIP assay with a human megakaryocytic cell line, UT-7/TPO, demonstrated that BACH1 bound to the promoter and enhancers region in vivo. As expected, co-transfection with BACH1 or Bach1-MafK fusion protein (B1K) expression plasmids repressed the reporter gene activity driven by the TXAS promoter. These findings thus provide evidence that BACH1 acts as a transcriptional repressor in the regulation of MARE-dependent genes in megakaryocytes.
Inflammatory Disease and Abortive Platelet Shedding Caused by a Mutation in a Pivotal Regulator of Actin Dynamics in the redears Mouse.
Authors: Justice Monica J. , Kile Ben T. , Woodward Lanette S.
Published: October 2019

Mouse mutagenesis using forward genetics is valuable as a gene function discovery tool. We are looking for blood defects in a large ENU mutagenesis screen, and have isolated many new mouse mutants that reveal new mechanisms in hematopoiesis. One mutant mouse strain, called redears, is an intriguing model of inflammatory disease and thrombocytopenia. Animals homozygous for the redears (rd) mutation develop spontaneous inflammatory lesions of the ears and tail characterized by neutrophil infiltration and peripheral neutrophila. Unexpectedly, blood platelet numbers are dramatically reduced in rd/rd animals. A thorough analysis of platelet biogenesis shows that the platelet precursor cell, the megakaryocyte, undergoes abnormal maturation, which results in gross morphological abnormalities, increased ploidy and abortive platelet shedding. Here we report a mutation in a novel gene related to the yeast actin-interacting protein Aip1 in rd/rd mice. In yeast, Aip1 interacts with, and increases the activity of cofilin, a key regulator of actin depolymerization. Our data confirm that actin dynamics are dysregulated in rd/rd megakaryocytes and neutrophils. The massive cytoplasmic reorganization that is required for megakaryocyte maturation and platelet shedding has long been assumed to depend on the actin cytoskeleton. Intriguingly, recent studies suggest the process is caspase-dependent, and represents a form of ‘para-apoptosis’. With this in mind, we found that chemotaxis and apoptosis are perturbed in rd/rd neutrophils, suggesting that neutrophils are playing a key role in driving the inflammation. Disrupted actin depolymerization would provide an explanation for chemotactic deficiencies. Further, recent evidence implicating cofilin and other actin regulators in the initiation of apoptosis would suggest that this novel protein may play an essential role in neutrophil cell death. Thus, the redears mouse not only provides the first in vivo demonstration of the critical role of the actin cytoskeleton in megakaryocyte development and platelet production, but also represents a unique reagent to examine the relationship between actin dynamics, cellular maturation, inflammation and apoptosis.

Our ongoing mutagenesis efforts continue to reveal new developmental mechanisms. New mutants, genetic tools, and resources can be found at www.mouse-genome.bcm.tmc.edu
Persistence of GATA1+ Erythroid Progenitors In Vivo in Absence of Zebrafish SCL.
Authors: Juarez Marianne A. , Su Fengyun , Kiel Mark J. , Lyons Susan E.
Published: October 2019

The stem cell leukemia (SCL) gene encodes a bHLH transcription factor essential to the primitive and definitive hematopoietic systems in vertebrates. In the zebrafish, studies of hematopoiesis have shown significant functional conservation with mammals. In order to assess the functions of scl in zebrafish primitive hematopoiesis, zebrafish embryos were analyzed after ablation of Scl protein by morpholino-modified antisense RNA injection. Embryos injected with morpholinos targeting the scl translation initiation site or two independent splice donor sites lacked circulating blood and exhibited cardiac edema by 28 hours post fertilization. RNA in situ hybridization analysis with a panel of hematopoietic-specific genes showed that myeloid lineage development in the anterior lateral mesoderm was absent upstream of draculin and pu.1 expression. In contrast, erythroid development in the posterior lateral mesoderm occurred with little or no functional Scl, as shown by expression of draculin, pu.1, and gata1 in this PLM region. Primitive erythroid cells failed to mature, as evidenced by the absence of circulating cells after 24 hpf. Only upon titration of scl morpholinos to near toxic levels was gata1 reduced or absent in embryos at 21 somites. These findings show that scl plays a vital role in development of all hematopoietic lineages in zebrafish. However, these data reveal differential effects of reduction or loss of Scl on hematopoietic development in the anterior lateral mesoderm versus the posterior lateral mesoderm. The results reveal that reduction or loss of Scl affects erythroid development in the posterior lateral mesoderm or ICM at a progenitor stage of erythroid development, after initiation of gata1 and embryonic globin expression. These data suggest that early erythroid development in the zebrafish requires little or no Scl, while maturation of erythroid progenitors cannot occur in the absence of Scl. Myeloid development in the anterior lateral mesoderm is dependent upon Scl. This embryonic analysis in the zebrafish is the first to demonstrate a transient, in vivo population of erythroid precursors in embryos with severely reduced or absent expression of Scl.
Core Epo Receptor Signaling Circuits in Adult Bone Marrow-Derived Primary Pro-Erythroblasts.
Authors: Wojchowski Don M. , Menon Madhu , Karur Vinit , Vincent Bethany
Published: October 2019

Knock-in experiments have revealed that Epo receptor-dependent steady-state erythropoiesis is supported by a Jak2-coupled cytoplasmic Epo receptor (EpoR) box-1 domain. However, signals that are transduced by this cytoplasmic phosphotyrosine-null EpoR-HM allele (and by a related EpoR-H allele with a restored PY343-STAT5 binding site) are not well understood. To address this basic question, conditions have been optimized for the serum-free expansion of adult bone marrow- derived primary erythroid progenitor cells, and core signaling circuits for EpoR-HM, EpoR-H and wt-EpoR receptor forms have been defined. As an important baseline consideration, each receptor form proved to activate Jak2 at an essentially equivalent time-course and intensity (but EpoR-HM did not detectably activate Stat5, or appreciably stimulate CIS transcription). In addition, EpoR-HM failed to stimulate AKT, and this outcome discounts possible EpoR-HM plus Jak2 coupling to P13K (eg. via IRS-1/2 and/or GAB-1/2). AKT was stimulated at moderate, yet sustained levels via EpoR-H (and a PY343/Stat5 axis). EpoR-HM, however, did couple to p60Src and ERK1, 2. While p60-Src stimulation was somewhat diminished, ERK1/ 2 activation via EpoR-HM was sharp and sustained (as compared directly to EpoR-H and the wt-EpoR). EpoR coupling to apoptotic factors also was assessed. In EpoR-HM erythroblasts, Bcl-xl expression was clearly (and reproducibly) deficient, but occurred at wild-type levels in Stat5-coupled EpoR-H erythroblasts. Bax expression was less affected, but was reproducibly increased 1.5 to 2-fold in both EpoR-HM and EpoR-H erythroblasts. Finally, expression of an under-studied pro-apoptotic DAPK-1 orthologue, DAPK2 (death associated protein kinase-2) was discovered to be markedly elevated in EpoR-HM erythroblasts, but to be repressed to normal levels in EpoR-H cells. With regards to bioactivity, EpoR-HM pro-erythroblasts faltered in their ex vivo development specifically at a transition from a Ter119negCD71low to Ter119posCD71high stage, and were visibly deficient in their levels of hemoglobinization. Overall, these first-time detailed studies of Epo signaling in primary adult pro-erythroblasts from minimal EpoR allele knock-in mice reveal: 1/ that EpoR plus Jak2 dependent erythropoiesis may depend primarily upon enhanced ERK1/2 stimulation (and not upon collateral Stat5 or AKT circuits); 2/ that EpoR-HM retains coupling to p60-Src; 3/ that an EpoR-H/PY343/Stat5 axis (but not an EpoR/Jak2 only axis) can link to AKT; 4/ that EpoR/PY343/Stat5 signals regulate not only Bcl-xl, but also pro-apoptotic DAPK2 expression at a key stage of Epo-dependent pro-erythroblast development; and 5) that EpoR-PY coupled signals contribute in several important ways to red cell development.
Elevated Estimated Pulmonary Arterial Pressures in Patients with Chuvash Polycythemia.
Authors: Gordeuk Victor R. , Sergueeva Adelina I. , Miasnikova Galina Y. , Polyakova Lydia A. , Okhotin Daniel J. , Castro Oswaldo L. , Prchal Josef T.
Published: October 2019

Chuvash polycythemia is characterized by a homozygous 598C>T mutation in VHL and up regulation of HIF-1α during normoxia. Disorders of chronic hypoxia may be complicated by the development of pulmonary hypertension. Because of the up regulation of the hypoxic response in Chuvash polycythemia, we postulated that there may be a tendency to increased pulmonary artery pressures in this condition as well. To test this hypothesis, we analyzed results for Doppler echocardiography in 15 patients with Chuvash polycythemia and 15 Chuvash individuals without polycythemia. The tricuspid regurgitation velocity (TRV) allows estimation of pulmonary artery systolic pressure. A TRV of 2.5 m/sec or higher corresponds to a pulmonary artery systolic pressure of at least 35 mm Hg (normal up to 32 mm Hg), while a TRV of 3.0 m/sec or higher to a pressure of at least 46 mm Hg. The results are summarized in the Table.

Pulmonary artery pressures as estimated by tricuspid regurgitation velocity (TRV) in Chuvash subjects with and without polycythemia

Chuvash polycythemia (n = 15) Controls (n = 15) P Age in years; mean (SD) 35 (17) 35 (17) 1.0 Female sex in no. (%) 8 (53%) 8 (53%) 1.0 Hemoglobin in g/dL; mean (SD) 16.7 (2.3) 13.3 (1.2) <0.001 TRV in m/sec; mean (SD) 2.2 (0.6) 1.4 (0.6) 0.001 TRV > 2.4 m/sec in no. (%) 4 (27%) 0 (0%) 0.1

Most of the patients with Chuvash polycythemia were receiving phlebotomy therapy and therefore many had hemoglobin concentrations in the upper normal range. Four of the patients with Chuvash polycythemia and none of the others had TRV ≥ 2.5 m/sec (range of 2.5 to 3.0), and mean TRVs were significantly higher in the patients with Chuvash polycythemia. Interestingly, the mean ± SD TRV in these 15 patients with Chuvash polycythemia was identical to the mean ± SD TRV that was recently reported in 195 American patients with sickle cell disease (Gladwin et al, NEJM2004;350:886), another hematological condition with a tendency to pulmonary hypertension. While the pulmonary arterial pressures detected so far in Chuvash polycythemia patients are lower than those in patients with primary pulmonary hypertension, our results suggest that pulmonary hypertension may be an unrecognized complication of Chuvash polycythemia.
Serum Level of Interleukin-10 Correlates Well with the Activity of Chronic Graft-Versus-Host Disease and the Responsiveness to Corticosteroid Treatment.
Authors: Yeh Su-Peng , Chiu Chang-Fang , Liao Yu-Min , Lo Wen-Jyi , Liaw Yu-Ching , Lin Chiao-Lin , Chang Jan-Gowth , Hsueh Chung-Tsen
Published: October 2019

Current evidence suggest the interaction between subset of T cells, the T help1 (Th1) and T help2 (Th2), plays an important role in the pathogenesis of GVHD. Cytokines, either produced by Th1/Th2 or other cells, are important regulators in the whole process and will define the polarization of Th1 or Th2 from naïve T cell. In this study, we used ELISA (R&D, Minneapolis, MN, US) to monitor the serum level of Interleukin-4 (IL-4), IL-10, IL-12, and interferon-γ (INF-γ) in patients receiving allogeneic transplant weekly from day 0 to at least day 200. From Jan. 2003, consecutive 20 patients were enrolled. The GVHD prophylaxis consisted of Cyclosporine-A and short course Methotrexate. Of the 20 patients, 2 had grade 3/4 acute GVHD (aGVHD) and 7 had extensive chronic GVHD (cGVHD). The serum levels of IL-4 and IL-12 were below the detectable level (0.13pg/ml and 0.5pg/ml respectively) in most occasions, even during the period of GVHD. Nevertheless, the serum level of IL-10 correlated well with the activity of cGVHD and we used data gathered from 5 patients to demonstrate this good correlation in figure 1 and 2. Patient 1 (open square, figure 1) had no GVHD and the IL-10 levels were below 5pg/ml during the whole period of follow-up. Patient 2 (solid triangle, figure 1) and 3 (open triangle, figure 2) had de novo cGVHD. The IL-10 level increased gradually when cGVHD developed, however, both clinical manifestations of cGVHD and serum IL-10 level decreased rapidly after the administration of Prednisolone (black bar on figure 1 and 2, with the thickness indicating the relative dosage of Prednisolone). Patient 4 (open circle, figure 2) had grade 2 aGVHD that resolved quickly after Prednisolone treatment. However, IL-10 level increased gradually after discontinuing Prednisolone and cGVHD developed subsequently. After adding Prednisolone again, cGVHD improved and IL-10 decreased to undetectable level rapidly. Patient 5 (solid square, figure 2) had steroid-refractory cGVHD. The IL-10 level kept above 5pg/ml for more than 1 month despite Prednisolone 1mg/kg/day giving at the same time and the patient eventually needed further salvage treatment (thalidomide). The serum INF-γ were undetectable in most occasions. It became detectable during period of cGVHD in patients 3, 4 and 5 but not patient 2. In conclusion, post-transplant serum IL-10 level correlates well with the clinical activity of cGVHD as well as the responsiveness to corticosteroid treatment. This novel finding will allow the functional evaluation of individuals’ immune system after allogeneic transplant and should give insight for the adjustment of immunosuppression necessary for cGVHD control.

Figure Figure Figure Figure
Treatment with Hydroxyurea in Sickle Cell/ ß Thalassemia: A Long-Term Experience.
Authors: Maggio Aurelio , Rigano Paolo , Renda Disma
Published: October 2019

Sickle cell/ß-Thalassemia is a common disease in areas where ß-Thal and ßS genes are endemic, like in Sicily. In the current study we evaluated clinical and hematological data of Sicilian patients with Sickle cell/ß-Thalassemia treated with Hydroxyurea (HU). The endpoint of the study was to evaluate the efficacy of HU in terms of reduction of sickle cell crises after 2 years of treatment in comparison with the 2 years before. Moreover, we evaluated the outcome after long-term treatment. Fortytwo patients (18 males, mean age 36, range 18–53) were treated with HU (mean dosage 15 mg/kg, range 10–30) for an average 6.6 years follow-up (range 3–9 years). Twentytwo were ß0/ßS and 20 ß+/ßS genotype. All had 3 or more crises in the year before starting HU. We observed a significant reduction in sickle cell crises (7.8 ± 6.9 crises per year versus 0.9 ± 1.8 per year, P < 0.0001), hospitalizations (2.5 ± 2.9 per year versus 0.3 ± 1.5, P < 0.0001), and days in hospital (22.4 ± 21.9 per year versus 1.2 ± 2.3, P < 0.0001). Altogether, there was a 86% reduction in vasoocclusive events in comparison with the 2 years before (P < 0.001). Moreover, there was a significant increase of MCV (71.4 versus 97.5fl, P < 0.0001), HbF (7.5 versus 25.2 %, P < 0.0001), and decrease of WBC (11.4 versus 9.2 109/L P < 0.01) and reticulocytes (14.1 versus 10.2%, P< 0.01). Finally, redution of hyperdense cells and increase of erithropoyetin were seen. After a mean follow-up of 6.6 years, 39 patients are alive. Three died (2 end stage HCV related liver disesases, 1 bleeding after ERCP). Nine of the 40 alive patients developed complications: 1 acute chest syndrome, 2 strokes, 2 myocardial infarctions, 4 bone necrosis. Brain MRI of 15 patients after and during a mean of 6.9 years of HU treatment showed 2 new onset strokes, 1 of which in a patient with a previous stroke. Moreover, 4 patients developed new onset asymptomatic ischemic brain lesions. In every case there had been a significant reduction of sickle cell crises. There were two cases of cancer, occurring in two patients who were brother and sister: lung cancer in the former, a proeviously heavy smoker, breast cancer in the latter. No further serious adverse events were seen. Five patients with iron overload were treated with Deferiprone: no drug interaction with HU was noticed. Our study confirms that HU is effective in reducing clinical relevant crises of patients with Sickle cell/ß-Thalassemia. However, our preliminary data suggest that chronic organ damages are not prevented by HU. Safety has to be assessed by more prolonged studies.
Upregulation of Endogenous Erythropoietin (EPO) in Healthy Subjects by Inhibition of Hypoxia Inducible Factor (HIF) Prolyl Hydroxylase.
Authors: Urquilla P. , Fong A. , Oksanen S. , Leigh S. , Turtle E. , Flippin L. , Brenner M. , Muthukrishnan E. , Fourney P. , Lin A. , Yeowell D. , Molineaux C.
Published: October 2019

High-altitude hypoxia stimulates erythropoiesis in anemic hemodialysis patients. Similarly, intermittent exposure to hypobaric hypoxia elevates EPO and stimulates erythropoiesis in normal subjects. HIF is a transcription factor that mediates the body’s response to hypoxia. The stability and activity of HIF are regulated by HIF Prolyl Hydroxylase (HIF-PH). Inhibition of HIF-PH results in the accumulation of HIF leading to the upregulation of erythropoietic genes. A FibroGen HIF-PH inhibitor, FG-2216, is an orally active small molecule with appropriate pharmacokinetic and pharmacodynamic activity for testing in humans. In animal studies, inhibitors of HIF-PH induce erythropoietic changes qualitatively similar to the effects of intermittent exposure to high altitude. Phase 1 studies were initiated in healthy male subjects to determine the safety, tolerability, pharmacokinetics, and biologic activity of FG-2216 dosed orally (0.3 to 20 mg/kg). Serum levels of FG-2216 increased in a dose-dependent fashion; its elimination was characterized by a half-life ~ of 14 hr. Dose-dependent elevation in serum EPO levels was observed with a minimum effective dose of 6 mg/kg. FG-2216 was subsequently administered using a schedule of 2 or 3 times a week for three weeks at doses of 10 and 20 mg/kg or placebo. Increased EPO was observed after each dose of FG-2216. There was no decrement in EPO response to subsequent doses, indicating a resetting of the EPO response during the dosing interval. An increase in reticulocytes and soluble transferrin receptor was accompanied by a modest increase in hematocrit and hemoglobin above normal values at baseline. A total of 54 subjects received FG-2216, which was well tolerated. There were no serious adverse events or dose-limiting toxicities. Adverse events possibly related to FG-2216 were mild and subsided with continued administration. The data provide first proof of concept for upregulation of endogenous EPO and erythropoietic responses in humans by a specific HIF-PH inhibitor.
Early In Vivo Depletion of Suicide Gene-Modified T-Lymphocytes Transplanted in Conjunction with CD34+ Enriched Blood Stem Cells May Be Associated with an Increased Risk of Secondary Graft Failure.
Authors: Fehse Boris , Ayuk Francis A. , Kröger Nicolaus , Fang Lubin , Kühlcke Klaus , Heinzelmann Marion , Zabelina Tatjana , Fauser Axel A. , Zander Axel R.
Published: October 2019

Introduction of a “suicide gene” into donor T-lymphocytes is a promising strategy to prevent severe graft-versus-host disease (GvHD) in the setting of allogeneic blood stem cell transplantation (B-SCT). We initiated a clinical phase I/II-study combining CD34-enriched peripheral B-SCT and Herpes simplex virus thymidine kinase- (HSV-tk) transduced donor T-cell infusions at days 0 and (optional) 60. Before our study was put on hold (after the leukemia cases in the French X-SCID trial) three patients were treated within the protocol each receiving >4x106/kg highly enriched CD34+ and appr. 5x106/kg suicide gene-modified (SGM) donor T-cells. All patients showed fast donor cell engraftment. Patient 2 (without signs of GvHD) received two SGM T-cell infusions and is well >2.5 years thereafter. Patient 1, showed an early (days 11–13) strong increase of SGM cell-counts peaking at 7.2x107/l, but did not develop GvHD. Unexpectedly, his blood was completely cleared from SGM T-lymphocytes a few days later. This patient received a second dose of SGM T-cells (day 65), which vanished within 2 days strongly indicative of their immune rejection. Indeed, MLR data confirmed anti-SGM reactivity of this patient’s PBL possibly related to an HSV reactivation during transplantation. Patient 3 showed a similar early sharp rise in SGM T-cells (days 14–17), in his case associated with acute skin GvHD grade II. Treatment with ganciclovir led to complete resolution of GvHD and rapid disappearance of SGM donor T-cells. Importantly, both patients 1 and 3 developed secondary graft failure at days 156 and 119. In summary, we confirmed previous reports that the use of SGM T-lymphocytes may allow control of acute GVHD. At the same time, we made the worrying observation that early total in vivo depletion of SGM donor T-cells may be associated with an increased risk of transplant rejection. This suggests that minimum numbers of donor CD3+ cells are required post-transplant not only to facilitate engraftment, but also to prevent late rejection.
Inducible Caspase 9 as a Safety Switch in Genetically Modified Cytotoxic T Cells.
Authors: Straathof Karin C. , Pule Martin A. , Yotnda Patricia , Dotti Gianpietro , Vanin Elio F. , Brenner Malcolm K. , Heslop Helen E. , Spencer David M. , Rooney Cliona M.
Published: October 2019

T cell therapy has proven effective to prevent and treat viral infections and immunogenic tumors. Application of this strategy to less immunogenic tumors that employ tumor evasion mechanisms may require genetic modification of the infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves have tempered enthusiasm. A safe and efficient means of destroying aberrant cells in vivo would ameliorate these concerns. The xenogeneic Herpes Simplex Virus I-derived thymidine kinase gene has been used as a suicide strategy for this purpose, but its immunogenicity results in immune elimination of the infused T cells and precludes administration of anti-viral drug ganciclovir for other indications. We adapted an alternative strategy using inducible human death molecules. Human caspase 9 was fused to a modified FK506 binding protein that allows for conditional dimerization using a chemical inducer of dimerization (CID). Administration of this small molecule pharmaceutical that has proven safe in healthy volunteers (Iuliucci et al, J Clin Pharmacol 2001, 41:870–879), activates the caspase molecule resulting in apoptosis of the cell. This system consists of human gene products with low potential immunogenicity. We chose caspase 9 as the death molecule since it is highly sensitive to dimerization and acts late in the apoptosis pathway, thereby reducing the risk of impairment of its function by cellular inhibitors of apoptosis. However, only low-level expression of the full-length inducible Caspase 9 (iCasp9) construct was compatible with survival of CTL. Deletion of the caspase recruitment domain (CARD) and removal of one of the two FKBPs allowed expression of iCasp9 in >70% of primary human T cells after a single transduction with a retrovirus vector. In vitro a single 10 nM dose of CID induced apoptosis in > 99% of T cells sorted for high transgene expression. A similarly high percentage of iCasp9-expressing T cells underwent apoptosis after CID administration in vivo in a mouse SCID human xenograft model. This functional activity is a log greater than previously available self-destruction systems. Importantly, even without sorting, activation of iCasp9 when co-expressed with transgenic IL-12 (a cytokine with potent anti-tumor activity) resulted in elimination of all IL-12 secreting cells. Unlike inducible Fas, iCasp9 maintained its function in MT-2 cells that over-express the anti-apoptotic molecule bclxl, demonstrating the importance of using a death molecule that acts downstream of the death inhibitors. Since iCasp9 does not impair T cell phenotype or function, these characteristics favor incorporation of inducible caspase 9 as safety feature in human T cell therapies.