bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Molecular Cloning of SH2- and SH3-Containing Inositol Polyphosphate-5-Phosphatase, Which Is Involved in the Signaling Pathway of Granulocyte-Macrophage Colony-Stimulating Factor, Erythropoietin, and Bcr-Abl
Authors: Odai Hideharu , Sasaki Ko , Iwamatsu Akihiro , Nakamoto Tetsuya , Ueno Hiroo , Yamagata Tetsuya , Mitani Kinuko , Yazaki Yoshio , Hirai Hisamaru
Published: October 2019

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF ). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.
Functional Studies on Platelet Adhesion With Recombinant von Willebrand Factor Type 2B Mutants R543Q and R543W Under Conditions of Flow
Authors: Lankhof Hanneke , Damas Conchi , Schiphorst Marion E. , IJsseldijk Martin J.W. , Bracke Madelon , Sixma Jan J. , Vink Tom , de Groot Philip G.
Published: October 2019

Type 2B von Willebrand disease (vWD) is characterized by the absence of the very high molecular weight von Willebrand factor (vWF ) multimers from plasma, which is caused by spontaneous binding to platelet receptor glycoprotein Ib (GPIb). We studied two mutations in the A1 domain at position 543 in which arginine (R) was replaced by glutamine (Q) or tryptophan (W), respectively. Both mutations were previously identified in vWD type 2B patients. The mutations R543Q and R543W were cloned into a eukaryotic expression vector and subsequently transfected in baby hamster kidney cells overexpressing furin (fur-BHK). Stable cell lines were established by which the mutants were secreted in the cell culture supernatant. The subunit composition and multimeric structure of R543Q and R543W were similar to wild-type (WT) vWF. The mutants showed a spontaneous binding to GPIb. R543Q and R543W showed normal binding to collagen type III or heparin. Both mutants supported platelet adhesion under conditions of flow, usually when preincubated on a collagen type III surface. A low dose (2.5% of the concentration present in normal pooled plasma) of recombinant R543Q or R543W added to normal whole blood inhibited platelet adhesion to collagen type III. No inhibition was found when vWF was used as an adhesive surface. These results indicate that point mutations identified in vWD type 2B cause bleeding symptoms by two mechanisms: (1) the mutants cause platelet aggregation, which in vivo is followed by removal of the aggregates leading to the loss of high molecular weight multimers and thrombocytopenia, (2) on binding to circulating platelets the mutants block platelet adhesion. Relatively few molecules are required for the latter effect.
Human Platelets as a Model for the Binding and Degradation of Thrombopoietin
Authors: Fielder Paul J. , Hass Philip , Nagel Mark , Stefanich Eric , Widmer Ramon , Bennett Gregory L. , Keller Gilbert-André , de Sauvage Frederic J. , Eaton Dan
Published: October 2019

Recent studies have shown that plasma thrombopoietin (TPO) levels appear to be directly regulated by platelet mass and that removal of plasma TPO by platelets via binding to the c-Mpl receptor is involved in the clearance of TPO in rodents. To help elucidate the role of platelets in the clearance of TPO in humans, we studied the in vitro specific binding of recombinant human TPO (rhTPO) to human platelet-rich plasma (PRP), washed platelets (WP), and cloned c-Mpl. Using a four-parameter fit and/or Scatchard analysis, the approximate affinity of rhTPO for its receptor, which was calculated from multiple experiments using different PRP preparations, was between 128 and 846 pmol/L, with ∼25 to 224 receptors per platelet. WP preparations gave an affinity of 260 to 540 pmol/L, with ∼25 to 35 receptors per platelet, and erythropoietin failed to compete with 125I-rhTPO for binding to WP. Binding and dissociation studies conducted with a BiaCore apparatus yielded an affinity of 350 pmol/L for rhTPO binding to cloned c-Mpl receptors. The ability of PRP to bind and degrade 125I-rhTPO was both time- and temperature-dependent and was blocked by the addition of excess cold rhTPO. Analysis of platelet pellets by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 125I-rhTPO was degraded into a major fragment of ∼45 to 50 kD. When 125I-rhTPO was incubated with a platelet homogenate at pH = 7.4, a degradation pattern similar to intact platelets was observed. Together, these data show that human platelets specifically bind rhTPO with high affinity, internalize, and then degrade the rhTPO.
CD6 Ligation Modulates the Bcl-2/Bax Ratio and Protects Chronic Lymphocytic Leukemia B Cells From Apoptosis Induced by Anti-IgM
Authors: Osorio Lyda M. , De Santiago Angelina , Aguilar-SantelisesHå Miguel , Mellstedt kan , Jondal Mikael
Published: October 2019

CD6 and CD5 belong to a scavenger-receptor cysteine-rich (SRCR) super family of membrane glycoproteins that are expressed on chronic lymphocytic leukemia B (B-CLL) cells, normal T cells, and a small subset of normal B cells. CD6 configures in the membrane in relation to the cellular activation level and can act as a coreceptor for T-cell activation. We have examined a group of progressive and nonprogressive B-CLL cells. Most B-CLL cells were positive for CD6 and the expression of CD6 was increased after activation with Staphylococcus aureus Cowan I plus interleukin-2 or 12-O-tetradecanoylphorbol 13-acetate, although anti-CD6 antibodies did not increase proliferative responses to these stimuli. However, anti-CD6 stimulation was found to protect against anti-IgM–induced apoptosis in B-CLL. baxα upregulation and bcl-2 downregulation were found in anti-IgM– and glucocorticoid (GCC)-induced apoptotic cells, respectively. Furthermore, CD6 cross-linking downregulated baxα mRNA levels in anti-IgM–treated cells, resulting in an increased bcl-2/baxα ratio. CD6 activation also prevented bcl-2 mRNA downregulation and apoptosis induced by GCC in one of six GCC-sensitive patients. These data suggest that an interaction between CD6 and its ligand might contribute to B-CLL survival through the modulation of the Bcl-2/Bax ratio.
A Synthetic CD4-CDR3 Peptide Analog Enhances Bone Marrow Engraftment Across Major Histocompatibility Barriers
Authors: Koch Ute , Korngold Robert
Published: October 2019

The efficacy of a synthetic peptide analog mimicking the CDR3-D1 domain of the CD4 molecule was investigated in murine models of allogeneic bone marrow engraftment after transplantation across major histocompatibility complex (MHC) barriers. A single dose of a CD4-CDR3 peptide analog was administered at the time of marrow transplantation to three different allogeneic mouse strain combinations after appropriate sublethal total body irradiation: (1) B10.BR → C57BL/6J (B6), a full allogeneic MHC difference; (2) (B6xDBA/2)F1 → (B6xCBA)F1 , a haploidentical MHC combination; and (3) B6.C-H2bm12 → B6-Ly5.2, involving only a MHC class II difference. Donor-host chimerism was assessed after 1 and 2 months posttransplantation by flow cytometric analysis of spleen and/or lymph node cells. Peptide-treated animals in all three strain combinations exhibited significantly enhanced donor lymphoid engraftment, which was similarly reflected in the total lymphocyte compartment and its T-cell (CD4+, CD8+) and B-cell subsets. In addition, peptide-treated mice in the haploidentical and MHC class II-mismatched strain combinations exhibited prolonged tolerance of both donor and syngeneic host-type tail skin grafts while rejecting third-party allogeneic grafts, thus supporting the reconstitution of immunocompetence in these chimeras. Lymphocytes from the peptide-treated haploidentical chimeric mice also displayed donor-specific tolerance upon stimulation in a one-way mixed lymphocyte reaction. In a 6-day colony-forming unit–granulocyte-macrophage (CFU-GM) assay to quantitate the level of hematopoietic cell engraftment in both the haploidentical and class II-disparate strain combinations, bone marrow cells from the peptide-treated mice exhibited significant increases in CFU-GM compared with the saline-treated control groups. Finally, early multiple treatments with the peptide after transplantation significantly enhanced donor chimerism in donor-presensitized recipient mice across the MHC class II barrier and proved to be significantly more effective than anti-CD4 monoclonal antibody treatment. These results indicate that the structure-based CD4-CDR3 peptide analog may represent a valuable approach to the inhibition of graft rejection after MHC-mismatched bone marrow transplantation.
Hyaluronate-CD44 Interactions Can Induce Murine B-Cell Activation
Authors: Rafi Asimah , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: October 2019

CD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix (ECM). Recent studies have demonstrated that activation through CD44 leads to induction of effector function in T cells and macrophages. In the current study, we investigated whether HA or monoclonal antibodies (MoAbs) against CD44 would induce a proliferative response in mouse lymphocytes. Spleen cells from normal and nude, but not severe combined immunodeficient mice, exhibited strong proliferative responsiveness to stimulation with soluble HA or anti-CD44 MoAbs. Furthermore, purified B cells, but not T cells, were found to respond to HA. HA was unable to stimulate T cells even in the presence of antigen presenting cells (APC) and was unable to act as a costimulus in the presence of mitogenic or submitogenic concentrations of anti-CD3 MoAbs. In contrast, stimulation of B cells with HA in vitro, led to B-cell differentiation as measured by production of IgM antibodies in addition to increased expression of CD44 and decreased levels of CD45R. The fact that the B cells were responding directly to HA through its binding to CD44 and not to any contaminants or endotoxins was demonstrated by the fact that F(ab)2 fragments of anti-CD44 MoAbs or soluble CD44 fusion proteins could significantly inhibit the HA-induced proliferation of B cells. Also, HA-induced proliferation of B cells was not affected by the addition of polymixin B, and B cells from lipopolysaccharide (LPS)-unresponsive C3H/HeJ strain responded strongly to stimulation with HA. Furthermore, HA, but not chondroitin-sulfate, another major component of the ECM, induced B-cell activation. It was also noted that injection of HA intraperitoneally, triggered splenic B cell proliferation in vivo. Together, the current study demonstrates that interaction between HA and CD44 can regulate murine B-cell effector functions and that such interactions may play a critical role during normal or autoimmune responsiveness of B cells.
Identification of the Major Casein Kinase I Phosphorylation Sites on Erythrocyte Band 3
Authors: Wang Cheng C. , Tao Mariano , Wei Tiequan , Low Philip S.
Published: October 2019

Human erythrocyte band 3 is a major substrate of two red blood cell protein kinases, casein kinase I and p72syk protein tyrosine kinase. Although the phosphorylation sites and physiologic consequences of p72syk phosphorylation have been characterized, little is known regarding casein kinase I phosphorylation. In this report, we identify the major phosphorylation site of casein kinase I. Using isolated components, casein kinase I was found to phosphorylate the cytoplasmic domain of band 3 (CDB3), primarily on Thr residues. Classical peptide mapping narrowed the major phosphorylation site to a peptide encompassing residues 24-91. Computer-assisted evaluation of this sequence not only showed two consensus casein kinase I phosphorylation sites, but also provided information on how to proteolytically separate and isolate the candidate sites. Following the suggested protocols, a heptapeptide containing the major phosphorylation site was isolated, subjected to amino acid sequencing, and found to be phosphorylated on Thr 42. A minor phosphorylation site was similarly identified as Ser 303. Because Thr 42 is situated near the binding sites on CDB3 of ankyrin, protein 4.1, protein 4.2, and the glycolytic enzymes, phosphorylation of CDB3 by casein kinase I could conceivably impact erythrocyte structure and/or function.
Lack of Expression of Thy-1 (CD90) on Acute Myeloid Leukemia Cells With Long-Term Proliferative Ability In Vitro and In Vivo
Authors: Blair A. , Hogge D.E. , Ailles L.E. , Lansdorp P.M. , Sutherland H.J.
Published: October 2019

Acute myeloid leukaemia (AML) is thought to be maintained by a small population of leukemic progenitor cells. To define the phenotype of such cells with long-term proliferative capacity in vitro and in vivo, we have used the production of leukemic clonogenic cells (CFU) after 2 to 8 weeks in suspension culture as a measure of these cells in vitro and compared their phenotype with that of cells capable of engrafting nonobese diabetic severe combined immune deficient (NOD/SCID) mice. Leukemic blast peripheral blood cells were evaluated for expression of CD34 and Thy-1 (CD90) antigens. The majority of AML blast cells at diagnosis lacked expression of Thy-1. Most primary CFU-blast and the CFU detected at up to 8 weeks from suspension cultures were CD34+/Thy-1−. AML cells that were capable of engrafting NOD/SCID mice were also found to have the CD34+/Thy-1− phenotype. However, significant engraftment was achieved using both CD34+/Thy-1− and CD34− subfractions from one AML M5 patient. These results suggest that while heterogeneity exists between individual patients, the leukemic progenitor cells that are capable of maintaining the disease in vitro and in vivo differ from normal hematopoietic progenitor cells in their lack of expression of Thy-1.
CIS, a Cytokine Inducible SH2 Protein, Is a Target of the JAK-STAT5 Pathway and Modulates STAT5 Activation
Authors: Matsumoto Akira , Masuhara Masaaki , Mitsui Kaoru , Yokouchi Masahiro , Ohtsubo Motoaki , Misawa Hiroyuki , Miyajima Atsushi , Yoshimura Akihiko
Published: October 2019

We searched for immediate early cytokine responsive genes and isolated a novel gene, CIS (Cytokine Inducible SH2 containing protein) that is induced in hematopoietic cells by a subset of cytokines including interleukin-2 (IL-2), IL-3, and erythropoietin (EPO). The mutant IL-2 receptor that fails to activate STAT5 could not induce CIS, suggesting that STAT5 is involved in the cytokine-inducible expression of CIS. We cloned the 5′-flanking region of the CIS gene and found that about 200 bases upstream of the transcription-initiation site contain four potential STAT5 binding sites (MGF boxes). Luciferase reporter assays showed that these MGF boxes were essential for EPO-dependent promoter activity. Expression of STAT5 and the EPO receptor in HEK293 cells conferred EPO-dependent activation of the CIS promoter. These data indicate that CIS is a target of the JAK-STAT5 pathway of cytokine receptors. CIS contains an SH2 domain and binds to tyrosine-phosphorylated EPO and IL-3 receptors. In HEK293 cells expressing STAT5 and the EPO receptor, EPO-dependent tyrosine phosphorylation of STAT5, as well as EPO-dependent CIS-promoter activation, was suppressed when CIS was coexpressed. Moreover, the induction of oncostatin M, another STAT5 target, as well as the tyrosine-phosphorylation of STAT5, were partially suppressed by CIS expression in Ba/F3 cells. Thus, CIS is a feedback modulator of STAT5; its expression is induced by STAT5 and it negatively modulates STAT5 activation.
Expression of the Id Family Helix-Loop-Helix Regulators During Growth and Development in the Hematopoietic System
Authors: Cooper Cathleen L. , Brady Gerard , Bilia Fillio , Iscove Norman N. , Quesenberry Peter J.
Published: October 2019

To better understand the molecular mechanism(s) by which growth and differentiation of the primitive hematopoietic stem cell is initiated, as well as the means by which the maturing cell can commit to development along a specific cell lineage, we elected to study the Id family of helix-loop-helix (HLH) transcriptional regulators. Some members of the HLH family are expressed in a stage-specific manner during hematopoietic development and can regulate the ability of immature hematopoietic cells to terminally differentiate. None of the four Id family genes were detected in the most primitive progenitors. Id-1 was widely expressed in proliferating bi- and unipotential progenitors, but its expression was downregulated in cells of increasing maturity; conversely, Id-2 and, to a limited extent, Id-3 gene expression increased as cells matured and lost proliferative capacity. Id-2 expression ran counter to that of Id-1 not only during maturation, but during periods of cell growth and arrest as well. This is quite distinct from the nonhematopoietic tissues, in which these two factors are coordinately expressed and suggests that Id-1 and Id-2 might be regulating very different events during hematopoiesis than they regulate in other cell types.
Activation of the Coagulation Cascade in C1-Inhibitor Deficiencies
Authors: Cugno Massimo , Cicardi Marco , Bottasso Bianca , Coppola Raffaella , Paonessa Raffaella , Mannucci Pier Mannuccio , Agostoni Angelo
Published: October 2019

Activation of the contact and complement systems in C1-inhibitor deficiencies is thought to contribute to the pathogenesis of angioedema attacks by releasing kinins. Trigger stimuli of attacks may also activate coagulation. This is particularly important because experimental data suggest that thrombin, the main enzyme of the coagulation cascade, increases vascular permeability and can thus influence edema formation. We have studied 19 patients with hereditary angioedema (HAE) during remission, 5 HAE patients during acute attacks, and 6 patients with acquired angioedema (AAE) during remission and during seven attacks. Thirty normal subjects, matched for sex and age, served as controls. Generation of thrombin was measured by enzyme-linked immunosorbent assay (ELISA) as plasma levels of the prothrombin fragment 1 + 2 (F1 + 2); the initiators of the tissue factor and contact coagulation pathways were investigated by measuring plasma levels of activated factor VII (FVIIa) coagulometrically and activated factor XII (FXIIa) by ELISA. Cleavage of high molecular weight kininogen (HK) was evaluated by immunoblotting analysis. F1 + 2 was slightly increased during remission and further significantly increased during attacks in both HAE (P = .0115) and AAE. FVIIa and FXIIa, normal during remission, increased strikingly during attacks in both HAE (P = .0022 and P = .0044) and AAE. During remission, cleaved HK was normal in HAE and high in AAE; during attacks it increased in HAE (P = .0008) and remained elevated in AAE. Our data indicate that in C1-inhibitor deficient patients there is increased generation of thrombin during attacks, with signs of activation of both the contact and tissue factor coagulation pathways. In conclusion, C1-inhibitor deficiency, whether hereditary or acquired, has demonstrable activation of the coagulation and kinin-forming cascades during attacks and that thrombin should be considered a possible contributing factor in the pathogenesis of edema in HAE and AAE.
A Histomorphometric Comparison of the Effects of Heparin and Low-Molecular-Weight Heparin on Cancellous Bone in Rats
Authors: Muir Jeffrey M. , Hirsh Jack , Weitz Jeffrey I. , Andrew Maureen , Young Edward , Shaughnessy Stephen G.
Published: October 2019

Long-term heparin treatment causes osteoporosis through, an as yet, undefined mechanism. To investigate this phenomenon and to determine the relative benefits of low-molecular-weight heparin (LMWH) use, we treated rats with once daily subcutaneous injections of either unfractionated heparin (1.0 U/g or 0.5 U/g), the LMWH, Tinzaparin (1.0 U/g or 0.5 U/g), or placebo (saline) for a period of 32 days. The effects on bone were then compared both histomorphometrically and biochemically by measuring urinary type I collagen cross-linked pyridinoline (PYD) and serum alkaline phosphatase, markers of bone resorption and formation, respectively. Histomorphometric analysis of the distal third of the right femur, in the region proximal to the epiphyseal growth plate, demonstrated that both heparin and LMWH decrease cancellous bone volume in a dose-dependent fashion, but that heparin causes significantly more cancellous bone loss than does LMWH. Although both heparin and LMWH decrease osteoblast and osteoid surface to a similar extent, only heparin increases osteoclast surface. In support of these histomorphometric findings, biochemical markers of bone turnover demonstrated that both heparin and LMWH treatment produce a dose-dependent decrease in serum alkaline phosphatase, consistent with reduced bone formation, whereas only heparin causes a transient increase in urinary PYD, consistent with an increase in bone resorption. Based on these observations, we conclude that heparin decreases cancellous bone volume both by decreasing the rate of bone formation and increasing the rate of bone resorption. In contrast, LMWH, causes less osteopenia than heparin because it only decreases the rate of bone formation.
A Patient With Type 2N von Willebrand Disease Is Heterozygous for a New Mutation: Gly22Glu. Demonstration of a Defective Expression of the Second Allele by the Use of Monoclonal Antibodies
Authors: Gu J. , Jorieux S. , Lavergne J.M. , Ruan C. , Mazurier C. , Meyer D.
Published: October 2019

We report the case of a Chinese patient who has subnormal von Willebrand factor (vWF ) level and normal vWF multimeric pattern, but a lack of vWF capacity to bind factor VIII (FVIII). Exons 18 to 20 of the patient's vWF gene were analyzed by DGGE and a G2354 → A substitution which changes the encoded amino acid sequence from Gly22 to Glu was identified. The patient is heterozygous for this substitution, creating a unique Sac I restriction site. Recombinant vWF (rvWF ) containing the candidate mutation was transiently expressed in COS-7 cells. It was processed and secreted normally but failed to bind FVIII. FVIII binding ability of hybrid rvWF, obtained by cotransfection of normal and mutated expression vectors and corresponding to a heterozygous genotype, was moderately decreased. To explain this functional discrepancy between patient's plasma vWF and hybrid rvWF, we used anti-vWF monoclonal antibodies (MoAbs) as capture in an enzyme-linked immunosorbent assay test. MoAb 32B12 recognized both wild-type and mutated rvWFs whereas MoAb 418 did not recognize mutated rvWF. Because MoAb 418 also failed to capture the plasma vWF from propositus, it means that his second nonmutated allele is not expressed or expressed at a very low level.
Restricted Use of Cationic Germline VH Gene Segments in Human Rh(D) Red Cell Antibodies
Authors: Boucher Gérard , Broly Hervé , Lemieux Réal
Published: October 2019

The human red cell Rh(D) antigen elicits the production of high-affinity IgG antibodies, which can prevent blood transfusion and cause hemolytic disease of the newborn. It has been known for 20 years that Rh(D) antibodies are among the most positively charged human serum IgGs. Analysis by IEF of 9 human anti-Rh(D) monoclonal antibodies showed that their isoelectric points (pI) (8.3 to 8.6) were also significantly higher than the average pI of serum IgGs (7.0 to 8.5). Sequencing of the anti-Rh(D) H and L chains cDNAs showed a preferential use of VH1 , VH3, JH6, and Vκ1 gene segments. The high pIs in IEF were correlated with a higher number of cationic amino acid residues in the H chain V regions without clustering in the complementary determining region. Computer analysis indicated that the germline VH used in anti-Rh(D) was selected among the most cationic segments available in the human VH repertoire or expressed in normal B cells. These results indicate that the selection of cationic VH segments may be an important early step in the formation of clinically relevant anti-Rh(D) and other red cell antibodies, possibly to facilitate epitope binding in the negatively charged red cell membrane environment.
Prognostic Value of Metaphase-Fluorescence In Situ Hybridization in Follow-up of Patients With Acute Myeloid Leukemia in Remission
Authors: El-Rifai Wa'el , Ruutu Tapani , Elonen Erkki , Volin Liisa , Knuutila Sakari
Published: October 2019

The presence of residual leukemic cells was studied using metaphase-fluorescence in situ hybridization (FISH) in 22 patients with acute myeloid leukemia treated with chemotherapy only or chemotherapy followed by allogeneic bone marrow transplantation. The patients were followed up during their complete remission (CR) for 4 to 108 months (median, 21 months). A total of 88 BM samples was studied. In most of the samples more than 1,000 metaphase cells were analyzed. Residual leukemic cells were detected in 9 of 22 patients (41%). All patients who had an increasing and/or persisting level of abnormal cells in two or more subsequent samples or whose initial samples contained more than 1% of abnormal cells relapsed with one exception, in whom the later subsequent samples showed disappearance of abnormal cells. The time span before the first positive sample seems to be insignificant with regard to the outcome of relapse. Absence or single occurrence of abnormal cells followed by their disappearance was in agreement with CR in all the cases (16 patients). Our results indicate that metaphase-FISH is a reliable tool in the quantitation of residual leukemic cells and provides valuable prognostic information for patients with AML.
Use of Arsenic Trioxide (As2O3 ) in the Treatment of Acute Promyelocytic Leukemia (APL): II. Clinical Efficacy and Pharmacokinetics in Relapsed Patients
Authors: Shen Zhi-Xiang , Chen Guo-Qiang , Ni Jian-Hua , Li Xiu-Shong , Xiong Shu-Min , Qiu Qian-Yao , Zhu Jun , Tang Wei , Sun Guan-Lin , Yang Kan-Qi , Chen Yu , Zhou Li , Fang Zhi-Wen , Wang Yan-Ting , Ma Jun , Zhang Peng , Zhang Ting-Dong , Chen Sai-Juan , Chen Zhu , Wang Zhen-Yi
Published: October 2019

The therapeutic effect of arsenic trioxide (As2O3 ) in the treatment of acute promyelocytic leukemia (APL) was evaluated among 15 APL patients at relapse after all-trans retinoic acid (ATRA) induced and chemotherapy maintained complete remission (CR). As2O3 was administered intravenously at the dose of 10 mg/d. Clinical CR was achieved in nine of 10 (90%) patients treated with As2O3 alone and in the remaining five patients treated by the combination of As2O3 and low-dose chemotherapeutic drugs or ATRA. During the treatment with As2O3 , there was no bone marrow depression and only limited side effects were encountered. Pharmacokinetic studies, which were performed in eight patients, showed that after a peak level of 5.54 μmol/L to 7.30 μmol/L, plasma arsenic was rapidly eliminated, and the continuous administration of As2O3 did not alter its pharmacokinetic behaviors. In addition, increased amounts of arsenic appeared in the urine, with a daily excretion accounting for approximately 1% to 8% of the total daily dose administered. Arsenic contents in hair and nail were increased, and the peak content of arsenic could reach 2.5 to 2.7 μg/g tissue at CR. On the other hand, a decline of the arsenic content in hair and nail was observed after withdrawal of the drug. We conclude that As2O3 treatment is an effective and relatively safe drug in APL patients refractory to ATRA and conventional chemotherapy.
Recurring Structural Chromosome Abnormalities in Peripheral Blood Lymphocytes of Patients With Mycosis Fungoides/Sézary Syndrome
Authors: Thangavelu Maya , Finn William G. , Yelavarthi Krishna K. , Roenigk Henry H. , Samuelson Ellen , Peterson LoAnn , Kuzel Timothy M. , Rosen Steven T.
Published: October 2019

Cytogenetic analysis was performed on peripheral blood lymphocyte cultures from 19 patients with mycosis fungoides (MF )/Sézary syndrome (SS) stimulated with either phytohemagglutinin, a conventional mitogen, or a combination of interleukin-2 (IL-2) plus IL-7. The use of both PHA-stimulated and IL-2 plus IL-7–stimulated cultures enhanced the ability to identify clonal abnormalities. Clonal abnormalities were observed in 11 patients (53%) including one with monosomy for the sex chromosome as the sole abnormality. Five of the 11 patients with clonal abnormalities had normal peripheral white blood cell counts, indicating detectability of clones in the absence of frankly leukemic disease. The presence of clonal abnormalities correlated with advanced stage disease and a significantly reduced survival duration from the time of cytogenetic studies. Clonal abnormalities involving chromosomes 1 and 8 were observed in six cases. In five cases with aberrations of chromosome 1, loss of material involved the region between 1p22 and 1p36. In an additional case, a reciprocal translocation involving 1p33 was observed. Clonal abnormalities involving chromosomes 10 and 17 were observed in 5 cases, clonal abnormalities involving chromosome 2 in 4 cases, and clonal abnormalities involving chromosomes 4, 5, 6, 9, 13, 15, 19, and 20 in 3 cases. In 2 cases a der(8)t(8; 17)(p11; q11) was observed. Regions of the genome that encode T-cell receptors were not involved in abnormalities. The region between 1p22 and 1p36 is identified as a region of the genome that requires detailed analysis toward the identification of potential gene(s) involved in the process of malignant transformation and/or progression in MF/SS.
Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells
Authors: Bellosillo Beatriz , Dalmau Mireia , Colomer Dolors , Gil Joan
Published: October 2019

B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived B lymphocytes that express high levels of Bcl-2. We examined the involvement of CED-3/ICE-like proteases in the apoptosis of B-CLL cells. One of the substrates of these proteases is poly(ADP [adenosine 5′-diphosphate]-ribose) polymerase (PARP). The effect of different factors that induce the apoptosis of B-CLL cells on the proteolytic cleavage of PARP has been studied. Treatment of B-CLL cells with different concentrations of dexamethasone (1 to 1,000 μmol/L) induced in a dose-dependent manner the cleavage of PARP. Dexamethasone induced PARP cleavage after 12 hours of incubation, which was almost complete at 48 hours. PARP cleavage during apoptosis of B-CLL cells was studied in cells from eight patients and a correlation was found between cell viability and the degree of PARP cleavage. Incubation in vitro of B-CLL cells with fludarabine for 48 hours induced PARP cleavage in all the cases studied. Protein kinase C (PKC) activation with 100 nmol/L TPA (12-O-tetradecanoylphorbol 13-acetate) or incubation with interleukin-4 (10 ng/mL) prevented either dexamethasone- or fludarabine-induced proteolysis of PARP. Incubation of B-CLL cells with the CED-3/ICE–like protease inhibitor Z-VAD.fmk inhibited spontaneous and dexamethasone-induced PARP cleavage and DNA fragmentation in a dose-dependent manner. Furthermore, Z-VAD.fmk prevented the cytotoxic effect of dexamethasone. These results indicate that CED-3/ICE–like proteases play an important role in the apoptosis of B-CLL cells.
An Electron Microscope Study of Sectioned Cells of Peripheral Blood and Bone Marrow
Authors: KAUTZ JEAN , DEMARSH Q. B.
Published: October 2019

White blood cells from peripheral blood and bone marrow have been sectioned for study in the electron microscope. Methods of fixation and handling are described. Most of the usual blood cell types have been tentatively identified, and their fine structure is described. The high resolving power of the electron microscope promises to reveal details previously unsuspected, as well as to extend and clarify existing knowledge concerning the cytology of blood cells, both normal and pathologic.

Ultra-thin sectioning, while still a very difficult art, appears to be the best method currently available for visualizing the fine structure of white blood cells, which would otherwise be too thick for penetration by the electron beam. Conditions of satisfactory fixation and dehydration are extremely critical, and care must be exercised in the interpretation of all results in order to separate gross fixation artifacts from the finer precipitation of protoplasmic material which may approximate a true picture of the living cell.
Thalassemia—Hemoglobin C Disease
Authors: ZUELZER WOLF W. , KAPLAN EUGENE
Published: October 2019

An unusual type of severe chronic hypochromic microcytic anemia in a Negro is described and attributed to the interaction of the hemoglobin C gene with the thalassemia gene. The patient and his father were shown to be carriers of the C trait, the mother and several siblings of the thalassemia gene.
Observations on the Nature of the Erythropoietic Serum Factor
Authors: ERSLEV ALLAN J. , LAVIETES PAUL H.
Published: October 2019

Serum and plasma from anemic rabbits were found to contain a factor capable of inducing a reticulocytosis when injected into normal animals. This erythropoietic factor seems to be attached to, or to behave like, a serum albumin, alpha globulin or beta globulin.

The production of the erythropoietic factor was found to be normal in rabbits in which the lymphatic and the hematopoietic tissues had been damaged through the administration of nitrogens mustard.
Differences in Susceptibility of Polymorphonuclear Leukocytes from Several Species to Alteration by Systemic Lupus Erythematosus Serum: Application to a More Sensitive L. E. Phenomenon Test
Authors: CARRERA ANA E. , REID MAY VIRGINIA , KURNICK N. B.
Published: October 2019

Recent investigations have demonstrated that the lupus erythematosus cell phenomenon is influenced, among other things, by intracellular factors. The hypothesis was formulated that leucocytes from different animal species would react with different degrees of intensity to the same L. E. serum. Leucocytes from twelve animal species were tested by counting the number of L. E. cells, "globs" (free homogeneous basophilic masses), droplets and rosettes per 1000 white blood cells and per 1000 P.M.N. after 2 hours’ incubation in the L. E. serum. Controls consisted of similar preparations using normal human serum and homologous plasma. There was a wide range of susceptibility among the various species tested. The chicken and the horse were the most susceptible species, while man fell low in the scale of susceptibility. Classical L. E. cells were produced from leucocytes of most species tested. It is felt that a highly sensitive L. E. test can be performed, using chicken, horse, guinea pig, or dog, instead of human leucocytes.
The Clotting Action of Russell Viper Venom
Authors: RAPAPORT SAMUEL I. , AAS KNUT , OWREN PAUL A.
Published: October 2019

1. It has been shown that Russell viper venom in the presence of lipoid cofactor can clot hemophilia A plasma, hemophilia B plasma and proconvertin deficient plasma within 5 seconds. It is unable to clot proaccelerin deficient plasma rapidly. Thus the clotting behavior of venom-lipoid is similar to convertin.

2. A rapid presumptive test for hypoproconvertinemia is simply to do a Quick clotting time with brain thromboplastin and then with viper venom. Proconvertin deficient plasma will give a prolonged brain thromboplastin time but a normal venom time. Prothrombin or proaccelerin deficient plasma will give long clotting times with both agents.

3. The observation that Russell viper venom acts independently of the proconvertin content of plasma explains the differences between the venom and brain thromboplastins. It also explains the failure of venom to measure the full effect of dicumarol administration.
Combined Inheritance of Purpura Simplex and Ptosis in Four Generations of One Family
Authors: FISHER BEN , ZUCKERMAN GERALD H. , DOUGLASS ROBERT C.
Published: October 2019

Clinical observations are presented on 11 members of a family in which hereditary familial purpura simplex and congenital ptosis coexist in 6. These persons represent 4 generations of dominant genetic transmission of both traits.
The Sickle Cell Trait in Jamaica
Authors: JELLIFFE D. B. , STUART K. L. , WILLS V. G. , JELLIFFE E. F. P.
Published: October 2019

1. A survey was undertaken to determine the incidence of the sickle cell trait among two thousand one hundred and sixteen Jamaican children and adults of different racial groups. The following positives were found: average Jamaican children (Afro-Caucasian mixtures) 5.7 per cent; population of rural Jamaican village 3.9 per cent; Maroon schoolchildren 3.6 per cent; East Indian schoolchildren 0.7 per cent; Chinese children and adults 0 per cent; Caucasian schoolchildren 0 per cent; Caucasaian (Jamaican German) schoolchildren 0 per cent.

2. In the group of one thousand and sixty-seven average Jamaican children, no significant difference could be found between the different age-groups (i.e. infants, younger children, older children), or between the predominantly African and lesser Afro-Caucasian mixtures.

3. The incidence of the sickle cell trait in the other West Indian islands is noted.

4. The possible spread of the sickle cell trait from India to Africa, and its present uneven distribution in Africa is discussed.
The Anemia of Infection
Authors: KRAMMER A. , CARTWRIGHT G. E. , WINTROBE M. M.
Published: October 2019

1. The amount of free coproporphyrin and protoporphyrin its the erythrocytes of twenty normal male subjects was found to be 0.76 ± 0.26 and 31.3 ± 11.9 µg./100 ml. of packed RBC, respectively. In ten normal female subjects the values were 0.71 ± 0.23 and 44.6 ± 19.6 µg./100 ml. of packed RBC, respectively.

2. In eighteen male patients with chronic infection, mean values of 1.53 ± 0.61 µg./100 ml. of packed RBC for free erythrocyte coproporphyrin and 181.2 ± 90.6 µg./100 ml. of packed RBC for free erythrocyte protoporphyrin were found.
Autohemolysis and Other Changes Resulting from the Incubation in Vitro of Red Cells from Patients with Congenital Hemolytic Anemia
Authors: SELWYN J. G. , DACIE J. V.
Published: October 2019

Observations were made on the changes in volume, osmotic fragility, and cation contents of red cells incubated in serum at 37 C. for 24 and 48 hours. The results show that spontaneous autohemolysis is not due to progressive swelling of the cells, but is probably due to degenerative changes in the cell membranes.

On incubation, normal red cells increase in volume during the first 24 hours due to a gain in sodium and water; the cells lose potassium but at a slower rate than they gain sodium. During the second 24 hours of incubation the loss in potassium exceeds the gain in sodium and the cells shrink to near their original volume. These cation changes and the autohemolysis are greatly reduced if glucose is present throughout the 48 hours of incubation.

Red cells from several different types of congenital hemolytic anemia were also studied; important deviations from the normal pattern were observed. In hereditary spherocytosis the rates of autohemolysis, of increase in osmotic fragility, and of potassium loss are greater than normal. The continued presence of glucose during incubation markedly retarded these changes.

In hereditary elliptocytosis trait the red cells behaved normally on incubation. In one case of elliptocytosis with hemolytic anemia, autohemolysis was normal but there was an increased potassium loss. In another patient with hemolytic anemia and increased osmotic fragility autohemolysis was greatly increased. In all these cases of elliptocytosis, glucose reduced the autohemolysis moderately but not to a normal degree.

Four cases of congenital nonspherocytic hemolytic anemia were studied. In two patients (type 1) autohemolysis, osmotic fragility and cation changes on incubation were normal; glucose had a normal effect on the fragility and cation changes, but only slightly reduced the autohemolysis. In the two other patients (type 2) autohemolysis, increase in osmotic fragility, and loss of potassium were markedly increased. Glucose did not retard any of these changes and it was found that the cells were unable to utilize glucose at the normal rate.
The Hematologic Response to Wounding and to Resuscitation Accomplished by Large Transfusions of Stored Blood
Authors: CROSBY WILLIAM H. , HOWARD JOHN M.
Published: October 2019

1. During the third winter campaign in Korea, the hematologic reaction to wounding was studied in thirty-seven casualties at the time of initial resuscitation.

2. Of particular interest was the effect of massive transfusions of stored blood. The results of storage of blood—high plasma hemoglobin and potassium, low labile factor activity, nonviable platelets and leukocytes—had little deleterious effect on patients who received as much as 20 to 30 pints in less than six hours. The loss of transfused red cells because of nonviability was no more than expected.

3. At the time of resuscitation and shortly thereafter, there was a remarkable loss of circulating red cell mass in patients with wounds that involved much tissue destruction. It is believed that the loss was due to hemolysis but the mechanism is unknown. The loss of red cells may be so rapid that a patient with bilateral traumatic amputation of the legs and an adequate hemostasis would become severely anemic if one hesitated to use large, rapid transfusions. Patients with severe shock whose wounds involved less tissue damage (lacerated colon, for example) did not destroy red cells in this fashion. After moderate transfusions such patients often became polycythemic. Transfusions had to be carried out rather gingerly because of a tendency to develop signs of congestion.

4. During the early days of recuperation from severe wounds the patients often tended to become anemic. The anemia appeared to be the result of hemolytic processes plus a relative inhibition of red cell formation.

5. Universal donor blood, group O, was used in all transfusions. In patients who are not group O the massive transfusions resulted in the virtual replacement by red cells of another group. The patient’s plasma sometimes contained antibodies against red cells of his hereditary blood group. Gradual hemolysis of native red cells by transfused antibodies was observed. This was not a clinical hemolytic reaction and did not appear to be detrimental to the patient. The presence of the foreign antibodies made it impossible in some cases to crossmatch the patient with blood of his hereditary group and suggested a source of danger in attempting such transfusions. After transfusion with the universal donor blood has begun, it is recommended that no change be made to blood of another group until at least two weeks have elapsed.

6. No incompatible transfusion reactions were encountered. Several hemoclastic reactions may have been caused by gross contamination of the blood stream from the site of wounds or from the peritoneal cavity.
Increased Growth Promoting But Not Mast Cell Degranulation Potential of a Covalent Dimer of c-Kit Ligand
Authors: Nocka Karl H. , Levine Beth A. , Ko Jone-Lone , Burch Peter M. , Landgraf Bryan E. , Segal Rita , Lobell Robert
Published: October 2019

The native form of soluble c-kit ligand (KL) is a noncovalent dimer. We have isolated a soluble, disulfide-linked dimer of murine KL (KL-CD) by expressing KL in Escherichia coli and refolding the denatured protein under conditions that promote the formation of both noncovalent dimers (KL-NC) and KL-CD. KL-CD exhibits a 10- to 15-fold increase in the ability to stimulate the growth of both the human megakaryocytic cell line MO7e and murine bone marrow-derived mast cells relative to KL-NC. Colony-forming assays of murine bone marrow progenitor cells also reflected this increased potency. However, KL-CD and KL-NC are equally able to prime mast cells for enhanced IgE-dependent degranulation in vitro and activate mast cells in vivo. Improving the growth-promoting activity of KL without changing its mast cell activation potential suggests that KL-CD or a related molecule could be administered in the clinic at doses that stimulate hematopoietic recovery while avoiding significant mast cell activation.