bluuebunny/crossref_metadata_2025 huggingface dataset:
Pleckstrin-2 Plays an Essential Role in the Pathogenesis of JAK2V617F-Induced Myeloproliferative Neoplasms
Authors: Zhao Baobing , Mei Yang , Sumagin Ronen , Yang Jing , Thorsheim Chelsea , Zhao Liang , Stalker Timothy J. , Wen Jeremy Q. , Crispino John , Abrams Charles S. , Ji Peng
Published: October 2019

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Studies on the pathogenesis of JAK2V617F positive MPNs have primarily focused on deregulation of JAK2 and STAT activities. It remains unclear exactly how JAK2-STAT downstream targets are involved in the development of the MPNs. We previously reported that pleckstrin-2 (Plek2) plays an important role in terminal erythropoiesis in vitro. Here we show that Plek2 is a downstream target of the JAK2-STAT5 pathway in erythroid, megakaryocytic, and granulocytic cells. Plek2 is significantly upregulated by JAK2V617F in both transduced primary mouse hematopoietic cells and in patients with JAK2V617F positive MPNs. Using a JAK2V617F knockin mouse model, we demonstrated that loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including reticulocytosis, thrombocytosis, neutrophilia, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality of JAK2V617F knockin mice (Figure 1A). These phenotypes were also transplantable indicating the role of Plek2 in mediating the pathogenesis of JAK2V617F-induced MPNs is cell-intrinsic (Figure 1B). Our study identifies Plek2 as a novel effector of JAK2-STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs.

Figure 1. Loss of Plek2 Rescues The Lethality of JAK2V617F Knockin Mice. (A) Kaplan-Meier survival analysis of indicated mice. Both males and females were included in each group. JAK2+/+Plek2+/+mice, n=34; JAK2+/+Plek2-/-mice, n=34;JAK2VF/+Plek2+/+ mice, n=36; JAK2VF/+Plek2-/- mice, n=36. (B) Kaplan-Meier survival analysis of the transplanted mice. JAK2+/+Plek2+/+mice, n=10; JAK2+/+Plek2-/-mice, n=10;JAK2VF/+Plek2+/+ mice, n=17; JAK2VF/+Plek2-/- mice, n=17. Figure 1. Loss of Plek2 Rescues The Lethality of JAK2V617F Knockin Mice. (A) Kaplan-Meier survival analysis of indicated mice. Both males and females were included in each group. JAK2+/+Plek2+/+mice, n=34; JAK2+/+Plek2-/-mice, n=34;JAK2VF/+Plek2+/+ mice, n=36; JAK2VF/+Plek2-/- mice, n=36. (B) Kaplan-Meier survival analysis of the transplanted mice. JAK2+/+Plek2+/+mice, n=10; JAK2+/+Plek2-/-mice, n=10;JAK2VF/+Plek2+/+ mice, n=17; JAK2VF/+Plek2-/- mice, n=17.

Disclosures

No relevant conflicts of interest to declare.
Defining Cellular States, Differentiation Trajectories, and Regulatory Networks Through Single Cell Profiling
Authors: Gottgens Bertie
Published: October 2019

Transcription factor proteins have long been recognized as key regulators of blood stem cell function. They are thought to act as components of wider regulatory networks, with combinatorial interactions responsible for directing blood stem cell fate choices. However, the complexity of these networks coupled with the infrequency of stem cells have presented formidable challenges for past research efforts aiming to define blood stem cell regulatory networks. Importantly, recent innovations in single cell genomics and computational network inference technologies offer new opportunities to better understand the regulatory network control of blood cell development. The Göttgens group uses a combination of experimental and computational approaches to study how transcription factor networks control the function of blood stem cells and how mutations that perturb such networks cause leukemia. This integrated approach has resulted in the discovery of new combinatorial interactions between key blood stem cell regulators, as well as experimentally validated computational models for blood stem cells. Current research focuses on single cell genomics of early blood development and the development of computer models to chart the transcriptional landscape of blood stem and progenitor cell differentiation.

Disclosures

No relevant conflicts of interest to declare.
The Mitochondrial Metabolic Checkpoint and Reversing Stem Cell Aging
Authors: Chen Danica
Published: October 2019

Cell cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. Recent advances in hematopoietic stem cell (HSC) biology highlight a mitochondrial metabolic checkpoint that is essential for HSCs to return to the quiescent state. As quiescent HSCs enter the cell cycle, mitochondrial biogenesis is induced, which is associated with increased mitochondrial protein folding stress and mitochondrial oxidative stress. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow HSCs to exit the cell cycle and return to quiescence. Other mitochondrial maintenance mechanisms include mitophagy and asymmetric segregation of aged mitochondria. Because loss of HSC quiescence results in the depletion of the HSC pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in HSCs will increase our understanding of hematopoiesis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions.

Disclosures

No relevant conflicts of interest to declare.
Regulation and Impact of Chromatin Decondensation in Mammalian Cells
Authors: Casellas Rafael
Published: October 2019

50 years ago Vincent Allfrey and colleagues discovered that lymphocyte activation promotes histone acetylation and gene expression. Recent studies clarified that G0 B cells proportionally amplify their transcriptome upon stimulation by a mechanism that involves Myc and TFIIH. We now show that B cell chromatin undergoes massive decondensation by the amplification of histone acetylation, methylation, and nucleosome remodeling. Super-resolution microscopy revealed that histone acetylation triggers rapid spreading of chromatin nanodomains from the periphery to the entire nucleus. Furthermore, nanodomains decompact into single nucleosome fibers by a process that requires Myc and ATP production. Using single molecule tracking, we show that chromatin decompaction increases the dynamics of transcription factor binding. Architecturally, B cell activation mediates a marked shift from long-range to short-range interactions and the formation of loops and contact domains that facilitate interaction between cognate regulatory elements. The data thus reveal the epigenetic and topological features that support transcriptome amplification, and help explain the primed nature of B cells.

Disclosures

No relevant conflicts of interest to declare.
Thrombocythemia and Pulmonary Intra-Alveolar Coagulum in a Young Woman
Authors: LEVINSON B. , JONES R. S. , WINTROBE M. M. , CARTWRIGHT G. E.
Published: October 2019

A description is given of the clinical course and findings at autopsy in a 19-year-old girl with a myeloproliferative syndrome characterized primarily by thrombocythemia. This was associated with an unusual pulmonary disorder in which the alveoli were filled by a non-cellular exudate comprised of carbohydrate, fat and protein which resembled in some respects pneumonia attributed to Pneumocystis carinii. The possible interrelationship of the thrombocythemia and the intra-alveolar coagulum is discussed.
A Comparative Study of Two Regimens of Combination Chemotherapy in Acute Leukemia
Authors: FREI EMIL , HOLLAND JAMES F. , SCHNEIDERMAN MARVIN A. , PINKEL DONALD , SELKIRK GEORGE , FREIREICH EMIL J. , SILVER RICHARD T. , GOLD G. LENNARD , REGELSON WILLIAM
Published: October 2019

A comparative clinical trial of two regimens of combination chemotherapy has been accomplished in acute leukemia by four separate medical and pediatric services. Sixty-five patients were allocated at random to one of two treatment programs. Daily administration of methotrexate with daily 6-mercaptopurine has been compared to methotrexate every third day in the same total dose with daily 6-mercaptopurine.

No difference in frequency of remission, extent of remission or toxicity was observed between the two groups. Among those patients who attained remission status, however, duration of remission (P = .05-.10) and of survival (P = <.05) was longer for the continuous group.

All remissions in children occurred in acute lymphocytic leukemia, whereas all remission in adults were observed in acute myelocytic leukemia. The duration of remissions was somewhat shorter for children with acute lymphocytic leukemia than for adults with acute myelocytic leukemia. The frequency of remission, either partial or complete, was higher in children, however (36 per cent), than in adults (19 per cent), although the confidence limits for each figure overlap.

The duration of acute leukemia in previously untreated patients did not influence response to therapy from the two antimetabolite regimens in this study. In patients who had had prior antimetabolite therapy, however, complete remissions were attained less often than in previously untreated patients.

The toxic manifestations encountered during the administration of these antimetabolites are described. Seventeen deaths occurred during this study, of which 8 occurred in the first 10 days, presumably from leukemia and not drug toxicity. Five patients died with hypoplastic marrows ascribed to drug toxicity.

The toxic manifestations were qualitatively and proportionately the same in patients who attained remission status, and in those patients who failed to remit, but who lived long enough to recognize the onset of remission if it were going to occur. No indication was obtained, therefore, that patients who attained remission were subjected to a greater toxic hazard, in order to achieve the therapeutic benefits observed, than those who did not remit.

The median survival of patients who achieved remission was longer (p <.05) than for patients who did not remit. Since the survival time of remitters from relapse to death was almost identical with the survival time of nonremitters from onset of treatment to death, this difference can be accounted for by the time spent in remission and getting to remission.

The median survival time from symptomatic onset for all children in this study was 12 months, and for adults, 7 months. The median in children is similar to that reported from other clinics. This is evidence that a comparative therapeutic trial in acute leukemia can be accomplished without recognizable compromise of patient welfare.
The Nature and Significance of Megaloblastic Blood Formation
Authors: REISNER EDWARD H.
Published: October 2019

Evidence is presented for a hypothesis that megaloblasts are red blood cell precursors with a prolonged resting phase between mitoses, allowing a longer time for dispersion of the chromatin throughout the nucleus. Anything that retards the rate of cell division can produce cells of similar appearance, but the conditions most conducive to megaloblastic blood formation are states in which vitamin B12 or folic acid are deficient. These two substances function in the synthesis of nucleoproteins as co-enzymes or their precursors concerned with the transfer of one-carbon units. In their absence the synthesis of the extra amounts of deoxyribonucleic acid essential for mitosis can go on but slowly, and the marrow becomes crowded with cells waiting to divide.

The principle source of the extra DNA is cytoplasmic RNA which appears to supply the ribonucleotide precursors of several deoxyribose containing compounds. Most important among these is deoxyuridylic acid which is methylated to form thymidylic acid. The failure of these reactions to occur in the absence of the essential co-enzymes leads to a persistence of RNA in the megaloblastic cytoplasm during the maturation phase, and this is probably responsible for their development into macrocytes.

This hypothesis accounts for the occasional presence of megaloblasts in conditions other than pernicious and related deficiency anemias and the occurrence of "macronormoblastic" or "megaloblastoid" marrows in intrauterine life and liver disease. The hypothesis also explains the rapid replacement of megaloblasts by normoblasts in the marrow following specific therapy.
Sickle Cell—Thalassemia Disease in Jamaica
Authors: MACIVER J. E. , WENT L. N. , CHUICKSHANK E. K.
Published: October 2019

Two cases of sickle cell—thalassemia disease are described in young women of mixed Chinese and African parentage. On hemoglobin electrophoresis, a complete suppression of hemoglobin A was found, giving a picture indistinguishable from that seen in sickle cell anemia. The findings in these two cases are contrasted with those in other examples of this disease which we have studied in Jamaica. The importance of these findings in relation to the diagnosis of sickle cell anemia is discussed.
A Circulating Inhibitor (Anti-AcG) Specific for the Labile Factor-V of the Blood-Clotting Mechanism
Authors: FERGUSON JOHN H. , JOHNSTON CHARLES L. , HOWELL DORIS A.
Published: October 2019

Study of the present hemorrhagic disorder establishes, without doubt, the presence of a powerful specific anti-AcG. The deprivation of AcG due to this inhibitor confirms the view that the labile factor plays a key role in the blood clotting system in at least two phases, namely, (1) during thromboplastin generation, and (2) later, during the conversion of prothrombin to thrombin. AcG is an essential component of many special clotting-test systems.
An Unusual Type of Hemoglobinopathy Resembling Sickle Cell-Thalassemia Disease in a Jamaican Family
Authors: WENT L. N. , MACIVER J. E.
Published: October 2019

Three generations of a Jamaican family of African extraction are described, in several members of which an abnormal gene is carried. This gene produces high levels of fetal hemoglobin unassociated with the usual stigmata of thalassemia. It is found in all three generations of the family associated with hemoglobin A only and is also found in at least two members of the family interacting with hemoglobin S. In the latter combination little or no disability results.

The mode of inheritance of this abnormal gene is discussed, and reasons are put forward for a possible protective effect of high fetal hemoglobin levels due to inhibition of sickling.

The findings in the cord blood of the youngest child, including an unusually high percentage of sickling, are discussed, together with follow-up studies to the age of 25 weeks.
Survival Studies of Thalassemic Erythrocytes Transfused into Donors, into Subjects with Thalassemia Minor and into Normal and Splenectomized Subjects
Authors: VULLO C. , TUNIOLI A. M.
Published: October 2019

The analysis of the curves of thalassemic red cell survival studied in children suffering from thalassemia major, in subjects with thalassemia minor, in normal splenectomized subjects and in healthy persons shows:

(a) thalassemic erythrocytes show a marked shortening of life span, with a wide range of half life from case to case;

(b) thalassemic erythrocytes survive longer in patients with thalassemia major and minor than in normal subjects;

(c) the behavior of thalassemic erythrocyte survival curves in the anemic patients and in the normal splenectomized cases is almost similar. On the basis of these results, the authors emphasize that:

(1) valuable data on the seriousness of the hemolytic process in thalassemia major can be drawn only from autotransfusion studies;

(2) in both thalassemia major and minor the spleen has less hemolytic activity on thalassemic red cells than in normal subjects;

(3) since survival curves between anemic and normal subjects differ principally in the initial component where the most abnormal red cells are destroyed by the normal spleen, it is probable that the spleen of Patients with thalassemia major is not so greatly active against these cells.
Characterization and comparison of the red blood cell membrane damage in severe human alpha- and beta-thalassemia
Authors: Advani R , Sorenson S , Shinar E , Lande W , Rachmilewitz E , Schrier SL
Published: October 2019

The aim of the present work was to understand the pathophysiology of the severe human thalassemias as represented by beta-thalassemia intermedia and hemoglobin (Hb) H (alpha-thalassemia) disease. We have previously shown that the material properties of the red blood cell (RBC) and its membrane differ in severe alpha- and beta-thalassemia, and we now show that this difference is probably caused by accumulation of alpha-globin chains at the cytoskeleton in beta-thalassemia, whereas beta-globin chains are associated with the cytoskeleton in alpha- thalassemia. In both alpha- and beta-thalassemia, some of these globin chains have become oxidized as evidenced by loss of the free thiols. Furthermore, there is similar evidence of oxidation of protein 4.1 in beta-thalassemia, whereas beta-spectrin appears to be subject to oxidation in alpha-thalassemia. These observations support the idea that the association of partly oxidized globin chains with the cytoskeleton results in oxidation of adjacent skeletal proteins. The abnormality of protein 4.1 in beta-thalassemia is consistent with a prior observation, and is also in accord with the known importance of protein 4.1 in maintenance of membrane stability, a property that is abnormal in beta-thalassemic membranes.
Nucleolar proteins B23 and C23 as target antigens in chronic graft- versus-host disease
Authors: Wesierska-Gadek J , Penner E , Hitchman E , Kier P , Sauermann G
Published: October 2019

Previously we observed that sera from recipients of allogeneic bone marrow transplants who developed extensive chronic graft-versus-host disease (GVHD) intensively stained the nucleolar region of target cells in indirect immunofluorescence microscopy. To identify the target antigens, immunoblotting experiments were performed using isolated nuclei, isolated nucleoli, and purified nuclear and nucleolar proteins as the antigen source. The nucleolar phosphoproteins B23 and C23 were identified as the main target antigens. Eleven of 19 extensive chronic GVHD sera reacted with these nucleolar phosphoproteins. In addition, four sera recognized histone H1, and two sera recognized the nuclear lamins A and C. Our patients reacting with the nucleolar proteins had symptoms resembling that of scleroderma or Sjogren's syndrome.
Characterization of the myeloid-specific CD11b promoter
Authors: Pahl HL , Rosmarin AG , Tenen DG
Published: October 2019

The CD11b/CD18 heterodimeric surface antigen is expressed exclusively on human monocytes, macrophages, granulocytes, and natural killer cells. During differentiation of myeloid cell lines, CD11b steady state messenger RNA levels increase significantly; we show here that CD11b transcription rates increase commensurately. A 1.7-kb fragment of CD11b 5′ flanking sequence directs expression of a reporter gene specifically in myeloid cell lines. Deletion analysis localizes elements directing high levels of tissue-specific reporter gene expression to the 412 bp proximal to the transcriptional start site. This sequence contains two consensus binding sites for Sp1, a GATA motif, and a purine-rich sequence that presents potential binding sites for members of the ets family of genes. Analysis of this promoter should result in the isolation of myeloid-specific transcription factors and the development of methods to direct the myeloid-specific expression of heterologous genes.
Hemophilia B Leyden: substitution of thymine for guanine at position - 21 results in a disruption of a hepatocyte nuclear factor 4 binding site in the factor IX promoter
Authors: Reijnen MJ , Peerlinck K , Maasdam D , Bertina RM , Reitsma PH
Published: October 2019

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by an altered developmental expression of blood coagulation factor IX. This form of hemophilia B has been found to be associated with a variety of single point mutations in the factor IX promoter region. We now describe a novel point mutation, T-->G at position -21, in two related patients with the hemophilia B Leyden phenotype. This mutation lies within the factor IX promoter region (-40 to -9) that contains overlapping binding sites for hepatocyte nuclear factor 4 (HNF-4) and androgen receptor. Transient transfection assays in HepG2 cells show that the -21 mutation causes a significant reduction in factor IX promoter activity. Gel mobility shift assays and transient cotransfection experiments revealed that the HNF-4-binding site but not the androgen-responsive element is disrupted by the -21 mutation. A comparison of the -21 mutation with the previously described -20 T-->A mutation (associated with the hemophilia B Leyden phenotype) and -26 G-->C mutation (associated with severe hemophilia B throughout life) was made. It shows that the -21 mutation reduced HNF-4 binding and transactivation to a similar level as the -20 mutation, whereas the -26 mutation completely abolished HNF-4 binding and transactivation. Mobility shift experiments indicate that there was no significant difference in binding affinity of recombinant androgen receptor protein for oligonucleotides containing wild-type and -21 or - 20 mutated DNA. The binding affinity for the oligonucleotide containing the -26 mutation was twofold lower. The results indicate that the disruption of the HNF-4-binding site by the -21 T-->G mutation is the cause of the bleeding disorder in these two patients. This study adds further support for the notion that the recovery from hemophilia at puberty may not only be related to an intact androgen-responsive element but also to the degree of disruption of the HNF-4-binding site.
Regulation of release of hepatocyte growth factor from human promyelocytic leukemia cells, HL-60, by 1,25-dihydroxyvitamin D3, 12-O- tetradecanoylphorbol 13-acetate, and dibutyryl cyclic adenosine monophosphate
Authors: Inaba M , Koyama H , Hino M , Okuno S , Terada M , Nishizawa Y , Nishino T , Morii H
Published: October 2019

Hepatocyte growth factor (HGF) secreted from human promyelocytic leukemia cell line, HL-60, is indistinguishable from HGF in human plasma and its release is significantly stimulated by 12-O- tetradecanoylphorbol-13-acetate (TPA), a differentiation-inducer of HL- 60 cells into monocytes/macrophages (Nishino T et al: Biochem Biophys Res Commun 181:323, 1991). TPA stimulated HGF release from the cells through an activation of C-kinase, but not through a formation of reactive oxygen species. Furthermore, dibutyryl cAMP (dbcAMP), an activator of A-kinase and granulocyte-inducer, also stimulated HGF release. 1,25-Dihydroxyvitamin D3, another monocyte/macrophage-inducer, abated either TPA- or dbcAMP-stimulated synthesis and release of HGF in a dose-dependent manner probably via its nuclear receptor as reflected by vitamin D analog study. The effects of these three reagents on the steady-state levels of HGF mRNA of 6.0 kb corresponded with their effects on its protein levels. Furthermore, a close correlation between intracellular and extracellular HGF levels strongly suggested that these reagents affected HGF release mainly on its synthesis step. Recombinant human HGF significantly stimulated the proliferation and alkaline phosphatase activity of mouse osteoblastic cell line, MC3T3- E1. In summary, HL-60 cells secrete HGF, whose synthesis is specifically regulated by various reagents independent of their differentiation-inducing effects. Because HGF shows a direct effect on osteoblast-like cells, it might be involved in the interaction of bone marrow cells with bone cells.
Rapid induction of mouse virus-like (VL30) element transcripts by erythropoietin in murine erythroid progenitor cells
Authors: Park DJ , Lim RW , Kim HD
Published: October 2019

We examined the activation of genes induced by erythropoietin (Epo) in erythroid progenitor cells that were isolated from the spleens of mice infected with anemia-inducing strain of the Friend virus. These erythroid progenitor cells, termed FVA cells, undergo in vitro differentiation to erythrocytes under the influence of Epo within 2 to 3 days. We used a differential hybridization procedure to screen a cDNA library constructed from FVA cells that were treated with Epo 2U/mL in the absence of serum for 2 hours. Of 20,000 recombinant phages, 47 plaques hybridized preferentially to cDNA probe prepared from Epo- stimulated cells. We found at least three different Epo-responsive genes (ERGs) and one of them corresponds to the mouse virus-like (VL30) element, similar to already reported BVL-1. The induction of VL30, which was evident within 30 minutes after Epo exposure, reached a maximum by 1 hour and remained stable for up to 4 hours. The treatment of FVA cells with cycloheximide (CHX) 10 micrograms/mL, which in itself activates the expression of VL30 caused a superinduction of the Epo signal. Changes in intracellular Ca2+ concentrations, either raised by ionomycin or depleted by EGTA, had no effect on the Epo-induced VL30 expression. In addition, protein kinase C (PKC) inhibitors such as staurosporine (3 mumol/L) or H7 (20 mumol/L) and a tyrosine kinase inhibitor, genistein (200 mumol/L), did not inhibit the Epo-induced expression of VL30. TPA (100 ng/mL), a PKC agonist, did not induce VL30 expression. Although the physiologic role of VL30 in the differentiation of erythroid progenitor cells is not known, our findings demonstrate that VL30 is an early ERG, and may be a useful indicator of the initial molecular actions of Epo.
Granzyme B-expressing peripheral T-cell lymphomas: neoplastic equivalents of activated cytotoxic T cells with preference for mucosa- associated lymphoid tissue localization
Authors: de Bruin PC , Kummer JA , van der Valk P , van Heerde P , Kluin PM , Willemze R , Ossenkoppele GJ , Radaszkiewicz T , Meijer CJ
Published: October 2019

T-cell non-Hodgkin's lymphomas can be considered the neoplastic equivalents of immunologically functional, site-restricted T lymphocytes. Little is known about the occurrence and clinical behavior of T-cell lymphomas that are the neoplastic equivalents of different functional T-cell subsets. Here, we investigated the prevalence, preferential site, immunophenotype, and clinical behavior of the neoplastic equivalents of activated cytotoxic T cells (CTLs) in a group of 140 nodal and extranodal T-cell lymphomas. Activated CTLs were shown immunohistochemically with a monoclonal antibody against granzyme B, a major constituent of the cytotoxic granules of activated T cells. Granzyme B-positive T-cell lymphomas were mainly found in mucosa- associated lymphoid tissue (MALT; nose, 63% of the cases; gastrointestinal tract, 46%; and lung, 33%). Granzyme B-positive cases with primary localization in MALT were more often associated with angioinvasion (P = .005), necrosis (P = .002), and histologic characteristics of celiac disease in adjacent mucosa not involved with lymphoma. Eosinophilia was more often observed in granzyme B-negative cases (P = .03). Most cases belonged to the pleomorphic medium- and large-cell group of the Kiel classification. CD30 expression was more often found in granzyme B-positive lymphomas of MALT (P = .04), whereas CD56 expression was exclusively found in nasal granzyme B-positive lymphomas. Immunophenotypically, most of the cases should be considered as neoplastic equivalents of activated CTLs based on the presence of T- cell markers on tumor cells. In two cases of nasal lymphoma, tumor cells probably were the neoplastic counterparts of natural killer cells. The prognosis of the granzyme B-positive gastrointestinal T-cell lymphomas was poor but did not differ from granzyme B-negative gastrointestinal T-cell lymphomas. This indicates that, in peripheral T- cell lymphomas, site of origin is more important as a prognostic parameter than derivation of activated CTLs.
Induction of tyrosine phosphorylation of Vav and expression of Pim-1 correlates with Jak2-mediated growth signaling from the erythropoietin receptor
Authors: Miura O , Miura Y , Nakamura N , Quelle FW , Witthuhn BA , Ihle JN , Aoki N
Published: October 2019

The receptor for erythropoietin (Epo) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, it has been hypothesized that a tyrosine kinase, Jak2, associates with the membrane proximal cytoplasmic region of Epo receptor (EpoR) and mediates the growth signaling from the receptor through tyrosine phosphorylation of cellular substrates. To explore the growth signaling pathways from the EpoR, we analyzed substrates of tyrosine phosphorylation induced by Epo stimulation in cells expressing various mutant EpoRs. The vav proto- oncogene product was found to be tyrosine phosphorylated after Epo stimulation in cells expressing the wild-type EpoR or a truncated receptor, H mutant, that retains the growth signaling function. In these cells, Epo also induced the expression of a serine/threonine kinase, Pim-1. However, Epo stimulation did not have any effect on Vav or Pim-1 in cells expressing a mutant EpoR, PM4 mutant, inactivated by a point mutation, Trp282 to Arg, in the membrane proximal region, which abrogates the interaction with Jak2. On the other hand, both tyrosine phosphorylation of Vav and expression of Pim-1 were observed constitutively in cells expressing a mutant EpoR that is constitutively activated by a point mutation, Arg 129 to Cys, in the extracellular domain. Jak2 was also constitutively tyrosine phosphorylated and activated in cells expressing this mutant, which confirms the crucial role of Jak2 in growth signaling from the EpoR. Taken together, these observations suggest that the tyrosine phosphorylation of Vav and the expression of Pim-1 may play important roles in growth signaling from the EpoR.
The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors
Authors: Gronthos S , Graves SE , Ohta S , Simmons PJ
Published: October 2019

The monoclonal antibody STRO-1 identifies clonogenic bone marrow stromal cell progenitors (fibroblast colony-forming units [CFU-F]) in adult human bone marrow. These STRO-1+ CFU-F have previously been shown to give rise to cells with the phenotype of fibroblasts, adipocytes, and smooth muscle cells. In this study, the osteogenic potential of CFU- F derived from the STRO-1+ fraction of adult human bone marrow was determined. CFU-F were isolated from normal bone marrow aspirates by fluorescence activated cell sorting, based on their expression of the STRO-1 antigen. Osteogenic differentiation was assessed by the induction of alkaline phosphatase expression, by the formation of a mineralized matrix (hydroxyapatite), and by the production of the bone- specific protein osteocalcin. STRO-1+ cells were cultured in the presence of dexamethasone (DEX; 10(-8) mol/L), ascorbic acid 2- phosphate (ASC-2P; 100 mumol/L), and inorganic phosphate (PO4i; 2.9 mmol/L). After 2 weeks of culture, greater than 90% of the cells in each CFU-F colony stained positive for alkaline phosphatase using a monoclonal antibody specific for bone and liver alkaline phosphatase. Alkaline phosphatase activity was confirmed by histochemistry. A mineralized matrix developed in the CFU-F cultures, after 4 weeks of culture in the presence of DEX, ASC-2P, and PO4i. Mineralization was confirmed by both light and electron microscopy. The mineral was identified as hydroxyapatite by electron dispersive x-ray microanalysis and by x-ray diffraction analysis. In replicate cultures, osteocalcin release was shown after exposure of the cells to 1,25-dihydroxyvitamin D3 (10(-7) mol/L) both by radioimmunoassay and Northern blot analysis. This work provides direct evidence that adult human bone marrow-derived CFU-F are capable of differentiating into functional osteoblasts and that osteoprogenitors are present in the STRO-1+ population.
Changes in cytokine secretion patterns of CD4+ T-cell clones in human immunodeficiency virus infection
Authors: Meyaard L , Otto SA , Keet IP , van Lier RA , Miedema F
Published: October 2019

In addition to the loss of CD4+ T cells in later stages of human immunodeficiency virus (HIV) infection, functional defects of Th cells can already be observed in early infection. Decreased interleukin (IL)- 2 and interferon (IFN)-gamma production by CD4+ T cells and diminished delayed type hypersensitivity reactions are indicative for impaired Th1 responses. We studied the cytokine secretion patterns of T-cell clones (TCC) generated by mitogenic stimulation of CD4+ memory T cells. Compared with TCC from HIV-negative controls, TCC isolated from HIV- infected individuals consistently showed increased IL-4 production, often paralleled by increased IL-5 and decreased IFN-gamma production. This resulted in a decreased percentage of Th1 clones with an increase in Th0 clones. To rule out the influence of interindividual differences, we studied two individuals from whom cells were available before and after infection with HIV. Indeed, an increase in Th2 cytokine secretion was observed after HIV-infection. Loss of Th1 and enhanced Th2 responses might further curtail cellular responses resulting in deficiency of cellular immunity in HIV infection.
Molecular analysis of the structure and expression of the RH locus in individuals with D--, Dc-, and DCw- gene complexes
Authors: Cherif-Zahar B , Raynal V , D'Ambrosio AM , Cartron JP , Colin Y
Published: October 2019

Rh blood group antigens of the D, C/c, and E/e series are carried by at least three red cell membrane polypeptides encoded by two highly related genes, RHD and RHCE. Homozygous individuals carrying the D--, Dc-, and DCw- gene complexes are characterized by a total or partial lack of expression of the RHCE-encoded antigens. Analysis of the molecular genetic basis of these rare conditions indicates that complete or partial expression defect of Cc/Ee antigens result from different alterations at the RH locus, but not from gross deletions. No rearrangement or mutation of the RHCE gene could be detected in donors homozygous for the D-- complex, suggesting that the lack of the Cc and Ee antigens might result from a reduced transcriptional activity of the RHCE gene. The Dc- and DCw- gene complexes, however, exhibited an important rearrangement of the RHCE gene. Instead of the normal RHCE gene, both variants carried a hybrid RHCE-D-CE gene in which exons 4 to 9 (Dc- complex) and 2 (or 3) to 9 (DCw- complex) of the RHCE gene, respectively, have been substituted by the equivalent region of the RHD gene. These gene conversion events provide an explanation for the well- described abnormal antigen profiles associated with the Dc- and DCw- complexes, like the increased expression of RhD, the reduced expression of RhC/c or RhCw, and the absence of RhE/e.
Rapid genotyping of the five major platelet alloantigens by reverse dot- blot hybridization
Authors: Bray PF , Jin Y , Kickler T
Published: October 2019

Amino acid substitutions in platelet membrane glycoproteins result in alloantigens. Identifying these polymorphisms is important in alloimmune-mediated platelet disorders. Immunophenotyping platelet antigens can be limited by the unavailability of specific antisera. The goal of this work was to identify human platelet antigen genotypes in individuals using a technique that would circumvent the limitations of immunophenotyping and be clinically applicable. We have successfully applied the reverse dot-blot (RDB) technique to the genotyping of the five major human platelet alloantigen systems. Allele-specific oligonucleotides (ASOs) representing each allele of these alloantigens were covalently linked to a filter. Biotinylated oligonucleotides flanking the polymorphic sequences in genomic DNA were used to amplify genomic DNA by the polymerase chain reaction (PCR), and these products were hybridized to the filters containing the ASOs. Reactivity was detected with a chromogenic substrate. This nonradioactive methodology identifies all 15 possible genotypes in a well-defined control group of individuals and requires only two PCR reactions per patient sample. RDB analysis was used to successfully genotype women and family members with neonatal alloimmune thrombocytopenia and with posttransfusion purpura and to prenatally genotype the amniocytes from a fetus at risk for thrombocytopenia. The RDB methodology is specific, sensitive, and rapid and should enhance our ability to accurately diagnose disorders of alloimmune platelet destruction.
Inhibiting interleukin-1 and tumor necrosis factor-alpha does not reduce induction of plasminogen activator inhibitor type-1 by endotoxin in rats in vivo
Authors: Emeis JJ , Hoekzema R , de Vos AF
Published: October 2019

In experimental animals and humans, intravenous (i.v.) injection of endotoxin induces large increases in circulating plasminogen activator inhibitor type-1 (PAI-1), a major inhibitor of blood fibrinolysis. A similar increase is seen after the injection of interleukin-1 (IL-1) or of tumor necrosis factor-alpha (TNF-alpha), suggesting that these cytokines mediate the induction, by endotoxin, of PAI-1. To test this hypothesis we pretreated rats, before i.v. endotoxin, with compounds that inhibit the formation of cytokines (pentoxifylline; dexamethasone), or with compounds that inhibit the action of these cytokines (anti-TNF antiserum for TNF-alpha; IL-1 receptor antagonist for IL-1). None of these pretreatments affected the induction of PAI-1 synthesis by endotoxin. However, pretreatment did reduce the endotoxin- induced increase in plasma tPA antigen concentration. Thus, the data suggest that, in rats in vivo, TNF-alpha and IL-1 are not significantly involved in the induction of PAI-1 by endotoxin.
Sequential analysis of 43 patients with non-Hodgkin's lymphoma: clinical correlations with cytogenetic, histologic, immunophenotyping, and molecular studies
Authors: Whang-Peng J , Knutsen T , Jaffe ES , Steinberg SM , Raffeld M , Zhao WP , Duffey P , Condron K , Yano T , Longo DL
Published: October 2019

Few reports correlating specific cytogenetic abnormalities with distinct subtypes of lymphoma have performed serial studies at diagnosis and at tumor recurrence or progression. In our file of 325 cytogenetically analyzed non-Hodgkin's lymphoma (NHL) patients studied over the past decade, 43 had serial biopsies, 39 of whom had at least two successful preparations; of the 43, nine had one and 32 had two or more cytogenetically abnormal specimens. In this study, we correlated cytogenetic, histopathologic, molecular, and clinical parameters. Patients with low-grade lymphomas were as likely as patients with intermediate- or high-grade lymphomas to acquire new chromosomal abnormalities with time (16 of 23 patients as compared with 7 of 16; P2 = .11, chi 2 test). In four patients, originally diagnosed indolent disease progressed to aggressive disease; all had t(14;18), all gained additional chromosomal abnormalities with disease progression, and three of the four expressed abnormalities associated with disease progression and/or short survival: der(18), +7, and/or +12. Cytogenetic results from early disease were compared with those obtained later in disease: in the t(14;18) group, the most common abnormalities were +7 (eight patients) and der(18) (five patients), both seen later in disease. The most common abnormalities in patients without t(14;18) were 6q deletions; they were seen in both early and late disease and were associated with significantly shorter survivals (P2 = .0014) compared with all patients without 6q deletions. Secondary chromosomal abnormalities, observed after at least one previous abnormal study, were seen in 19 of 22 t(14;18) patients and in 11 of 21 patients without t(14;18) and were associated with a poor survival (P2 = .13) compared with patients without any secondary chromosomal abnormalities. Chromosome 1 abnormalities were seen in almost half of the patients and were observed in initial specimens and early in disease as well as late in disease and as secondary abnormalities; 1q involvement was more frequent than 1p (15 versus eight patients) and was significantly associated with poor survival only in patients with intermediate-/high- grade disease; the most common breakpoints were 1q21-q22 (nine patients) and 1p36 (six patients). Breakpoints at 2q21 and 3q27-q29 were limited to patients with t(14;18) and were almost exclusively secondary in nature. Molecular studies in 24 of our patients showed discrepancies with the cytogenetic results in only three patients: two had t(14;18) but no molecular rearrangements while two patients had no visible t(14;18) but were positive for major breakpoint region (MBR) rearrangement. The presence of MBR or minor breakpoint cluster (MCR) rearrangement had no apparent effect on survival.(ABSTRACT TRUNCATED AT 400 WORDS)
Long-term outcome of aplastic anemia in adults treated with antithymocyte globulin: comparison with bone marrow transplantation
Authors: Paquette RL , Tebyani N , Frane M , Ireland P , Ho WG , Champlin RE , Nimer SD
Published: October 2019

The outcome of 155 adult aplastic anemia (AA) patients treated with antithymocyte globulin (ATG, Upjohn, Kalamazoo, MI) at University of California, Los Angeles from 1977 to 1988 was evaluated. The median survival of the 146 patients who did not undergo bone marrow transplantation was 5.6 years, with 49% +/- 4% surviving more than 6 years. The most important predictor of survival was positive response to ATG (P < 0.001), which was observed in 48% of patients. Among pretreatment variables, disease severity was the best predictor of survival. Patients with moderate AA (MAA) had significantly better survival than those with severe (SAA) or very severe (VSAA) disease (P = 0.04). The 6-year actuarial survival rates of the three groups were 71% +/- 9%, 48% +/- 7% and 38% +/- 7%, respectively. Cox regression analysis found disease severity to be the only pretreatment variable significantly associated with survival (P = .02). Patient age, sex, disease etiology, concurrent treatment with androgens, or duration of ATG therapy were not associated with differences in survival or response to ATG. Late clonal hematologic complications (ie, myelodysplasia, acute myelogenous leukemia) were observed in 5 of the 77 patients followed for more than 2 years after ATG treatment. In addition, one case of non-Hodgkin's lymphoma and three solid tumors occurred in the ATG-treated patients. The survival of 56 ATG-treated patients with SAA or VSAA between the ages of 16 and 43 did not differ significantly from that of 55 adult AA patients who underwent bone marrow transplant (BMT) during the same time period (P = 0.6). However, 6-year survival rates improved from 43% for patients transplanted before 1984, to 72% for those who underwent BMT between 1984 and 1989. In contrast, there was no difference in the survival rates of patients treated with ATG during these two time periods (46% v 45%, respectively). The results suggest a superior long-term outcome for adult patients with SAA treated with BMT rather than with ATG alone, using current protocols.
Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells
Authors: Fackler MJ , Krause DS , Smith OM , Civin CI , May WS
Published: October 2019

CD34 is expressed on human and murine hematopoietic stem and progenitor cells and its clinical usefulness for isolation of stem/progenitor cells has been well established. Although expression of CD34 is regulated in a developmental stage-specific manner, the function of CD34 is not known. Recently we have shown that both a full-length and truncated form of CD34 protein is expressed by hematopoietic cells (Blood 84:691, 1994). To test whether failure to suppress either form of CD34 could affect terminal myeloid differentiation, we constitutively expressed these CD34 proteins in murine M1 myeloid leukemia cells, which can be terminally differentiated to macrophages by treatment with interleukin-6 of leukemia inhibitory factor. Surprisingly our results show that forced expression of the full-length but not the truncated form of CD34 impedes terminal differentiation by these agents. Because the difference between the two forms of CD34 protein resides in the length of their respective cytoplasmic tail domains, our findings strongly suggest that the cytoplasmic domain region of full-length CD34 is responsible for the observed maturation arrest phenotype. These findings suggest a potential negative regulatory role for full-length CD34 in hematopoietic cell differentiation and may explain, at least in part, the block in maturation observed in CD34+ acute myeloid leukemia.
A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia
Authors: Gordon MS , Nemunaitis J , Hoffman R , Paquette RL , Rosenfeld C , Manfreda S , Isaacs R , Nimer SD
Published: October 2019

To evaluate the hematologic effects of recombinant human interleukin-6 (rhIL-6, Escherichia coli, SDZ ILS 969, IL-6), and determine its toxicity profile, we performed a phase I trial of IL-6 in 22 patients with various myelodysplastic syndromes (MDS), platelet counts < 100,000/microL, and < 5% bone marrow (BM) blasts. Patients received one of four doses of IL-6 (1.0, 2.5, 3.75, and 5.0 micrograms/kg/d) as a subcutaneous injection on day 1, followed by a 7-day wash-out period, and then 28 days of IL-6 therapy. Dose-limiting toxicities of fatigue, fever, and elevated alkaline phosphatase were seen at 5.0 micrograms/kg/d; the maximum tolerated dose was 3.75 micrograms/kg/d. All patients experienced at least grade II fever and all had an increase in acute phase proteins. Eight patients (36%) experienced at least a transient improvement in platelet counts; three fulfilled the criteria for response, whereas five others had clinically significant increases that failed to meet response criteria. Various IL-6-related toxicities prevented more than three patients from receiving maintenance therapy. Two of the three patients who received maintenance IL-6 therapy had a persistent increase in platelet counts, during 3 and 12 months of IL-6 therapy, respectively. Laboratory studies indicated that IL-6 increased the frequency of higher ploidy megakaryocytes but did not significantly increase the number of assayable megakaryocytic progenitor cells, suggesting that IL-6 acts as a maturational agent rather than a megakaryocyte colony-stimulating factor. Although IL-6 therapy can promote thrombopoiesis in some MDS patients, its limited activity and significant therapy-related toxicity preclude its use as a single agent in this patient population. Further studies, combining low doses of IL-6 with other hematopoietic growth factors, are underway.